1 Nikogore

Zerumbone Synthesis Essay

Abstract

Almost 25 centuries ago, Hippocrates, the father of medicine, proclaimed “Let food be thy medicine and medicine be thy food.” Exploring the association between diet and health continues today. For example, we now know that as many as 35% of all cancers can be prevented by dietary changes. Carcinogenesis is a multistep process involving the transformation, survival, proliferation, invasion, angiogenesis, and metastasis of the tumor and may take up to 30 years. The pathways associated with this process have been linked to chronic inflammation, a major mediator of tumor progression. The human body consists of about 13 trillion cells, almost all of which are turned over within 100 days, indicating that 70,000 cells undergo apoptosis every minute. Thus, apoptosis/cell death is a normal physiological process, and it is rare that a lack of apoptosis kills the patient. Almost 90% of all deaths due to cancer are linked to metastasis of the tumor. How our diet can prevent cancer is the focus of this review. Specifically, we will discuss how nutraceuticals, such as allicin, apigenin, berberine, butein, caffeic acid, capsaicin, catechin gallate, celastrol, curcumin, epigallocatechin gallate, fisetin, flavopiridol, gambogic acid, genistein, plumbagin, quercetin, resveratrol, sanguinarine, silibinin, sulforaphane, taxol, γ-tocotrienol, and zerumbone, derived from spices, legumes, fruits, nuts, and vegetables, can modulate inflammatory pathways and thus affect the survival, proliferation, invasion, angiogenesis, and metastasis of the tumor. Various cell signaling pathways that are modulated by these agents will also be discussed.

Keywords: Inflammation, NF-κB, Nutraceuticals, Therapeutics, Tumorigenesis

1 Introduction

Tumor formation in humans is a multistage process involving a series of events and generally occurs over an extended period. During this process, accumulation of genetic and epigenetic alterations leads to the progressive transformation of a normal cell into a malignant cell. Cancer cells acquire several abilities that most healthy cells do not possess: they become resistant to growth inhibition, proliferate without dependence on growth factors, replicate without limit, evade apoptosis, and invade, metastasize, and support angiogenesis [1]. Although the mechanisms by which cancer cells acquire these capabilities vary considerably among the various types of tumors, most of the physiological changes associated with these mechanisms involve alteration of signal transduction pathways. During the past quarter century, researchers’ understanding of the proteins involved in the various steps of tumor cell development has grown, providing opportunities for identifying new targets for therapeutic development (Fig. 1).

Fig. 1

Progression of tumor cell development involves survival, proliferation, invasion, angiogenesis, and metastasis. NF-κB activation regulates tumor cell development by targeting one or more steps in the pathway. Carcinogens activate NF-κB,...

Despite the development of these new therapies, however, cancer remains the second-leading cause of death in the USA and accounts for nearly one in every four deaths. The American Cancer Society estimates that 569,490 Americans will die of cancer in 2010 (www.cancer.org/docroot/stt/stt_0.asp). It is now believed that 90–95% of all cancers are attributed to lifestyle, with the remaining 5–10% attributed to faulty genes [2]. In 2010, for example, about 171,000 cancer deaths will be caused by tobacco use alone. In addition, one third of all cancer deaths in America are attributed to poor nutrition, physical inactivity, overweight, and obesity [3].

Multiple epidemiological and animal studies have shown that consumption of foods rich in fruits and vegetables decreased the occurrence of cancers [4-8]. Almost 30 years ago, Professors Doll and Peto, after conducting an epidemiological study for the World Health Organization, suggested that appropriate nutrition could prevent approximately 35% of cancer deaths and that up to 90% of certain cancers could be avoided by dietary enhancement [9, 10]. A recent elegant review by Chan and Giovannucci [11] provided an overview of the epidemiological evidence supporting the roles of diet, lifestyle, and medication in reducing the risk of colorectal cancer. Similarly, a wealth of information is available, implicating dietary agents in cancers of the skin [12], prostate [13, 14], breast [15], lung [16, 17], and gastrointestinal tract [18]. These studies suggest that much of the suffering and death from cancer could be prevented by consuming a healthy diet, reducing tobacco use, performing regular physical activity, and maintaining an optimal body weight.

It is now clear that cancerous phenotypes result from the dysregulation of more than 500 genes at multiple steps in cell signaling pathways [19, 20]. This indicates that inhibition of a single gene product or cell signaling pathway is unlikely to prevent or treat cancer. However, most current anticancer therapies are based on the modulation of a single target. The ineffective, unsafe, and expensive monotargeted therapies have led to a lack of faith in these approaches. Therefore, the current paradigm for cancer treatment is either to combine several monotargeted drugs or to design drugs that modulate multiple targets. As a result, pharmaceutical companies have been increasingly interested in developing multitargeted therapies. Many plant-derived dietary agents, called nutraceuticals, have multitargeting properties. In addition, these products are less expensive, safer, and more readily available than are synthetic agents [19]. Some nutraceuticals are currently in clinical trials (www.clinicaltrials.gov), but others have already been approved for human use [21-23].

A nutraceutical (a term formed by combining the words “nutrition” and “pharmaceutical”) is simply any substance considered to be a food or part of a food that provides medical and health benefits [23, 24]. The term nutraceutical was coined by Stephen DeFelice in 1989 [23, 25]. During the past decade, a number of nutraceuticals have been identified from natural sources, some of which are shown in Fig. 2. Nutraceuticals are chemically diverse (Fig. 3) and target various steps in tumor cell development (Fig. 4; Table 1).

Fig. 2

Common sources of nutraceuticals, which include spices, legumes, fruits, nuts, and vegetables

Fig. 3

Chemical structure of nutraceuticals

Fig. 4

Targets of nutraceuticals during tumor progression. Nutraceuticals can target survival, proliferation, invasion, angiogenesis, and metastasis steps and can influence various steps of tumor cell development by targeting one or more molecules of inflammation...

Table 1

Sources of nutraceuticals and their molecular target linked to cancer

Because of the vast number of nutraceuticals identified to date, we cannot discuss all of them. We will therefore focus on some of the more promising nutraceuticals in this review, including allicin, apigenin, berberine, butein, caffeic acid, capsaicin, catechin gallate, celastrol, curcumin, epigallocatechin gallate (EGCG), fisetin, flavopiridol, gambogic acid, genistein, plumbagin, quercetin, resveratrol, sanguinarine, silibinin, sulforaphane, taxol, γ-tocotrienol, and zerumbone, in the context of five specific processes of tumorigenesis: survival, proliferation, invasion, angiogenesis, and metastasis. Since chronic inflammation is one of the major mediators of tumor progression and nuclear factor-κB (NF-κB) is one of the major inflammatory transcription factors involved in the regulation of various steps of tumor cell development, we will also discuss how nutraceuticals can modulate NF-κB and can thus affect survival, proliferation, invasion, angiogenesis, and metastasis of the tumor.

2 Regulation of inflammatory pathways by nutraceuticals

During the past two decades, much evidence has emerged, indicating that, at the molecular level, most chronic diseases, including cancer, are caused by a dysregulated inflammatory response [26]. One of the most important links between inflammation and cancer is proinflammatory transcription factor NF-κB. NF-κB is a ubiquitous and evolutionarily conserved transcription factor that regulates the expression of genes involved in the transformation, survival, proliferation, invasion, angiogenesis, and metastasis of tumor cells (Fig. 1).

The first clue linking NF-κB to cancer was the realization that c-rel, which is the cellular homolog of the v-rel oncogene, encodes a NF-κB subunit and that all of these proteins share the same DNA binding domain, the Rel homology domain [27]. Constitutively active NF-κB has now been identified in tissues of most cancer patients, including those with leukemia and lymphoma and cancers of the prostate, breast, oral cavity, liver, pancreas, colon, and ovary [26].

In its resting stage, NF-κB resides in the cytoplasm as a heterotrimer consisting of p50, p65, and the inhibitory subunit IκBα [28]. On activation, the IκBα protein undergoes phosphorylation, ubiquitination, and degradation. p50 and p65 are then released, are translocated to the nucleus, bind specific DNA sequences present in the promoters of various genes, and initiate their transcription. A number of proteins are involved in the NF-κB signaling pathway. Because of the relevance of the NF-κB signaling pathway in cancer, this pathway has been proven to be an attractive target for therapeutic development. More than 700 inhibitors of the NF-κB activation pathway have been reported, including antioxidants, peptides, small RNA/DNA, microbial and viral proteins, small molecules, and engineered dominant-negative or constitutively active polypeptides [29].

During the past two decades, our laboratory and other researchers’ laboratories have shown that nutraceuticals can exert anticancer activity by suppressing the NF-κB signaling pathway. Curcumin, derived from the ancient Indian medicine turmeric, is a widely studied nutraceutical. When human colonic epithelial cells were pretreated with curcumin, inhibition in tumor necrosis factor (TNF)-α-induced cyclooxygenase 2 (COX-2) gene transcription and NF-κB activation was observed [30]. Curcumin inhibited IκB degradation through downregulation of NF-κB-inducing kinase and IκB kinase (IKK). Curcumin has also been reported to suppress the TNF-α-induced nuclear translocation and DNA binding of NF-κB in a human myeloid leukemia cell line through suppression of IκBα phosphorylation and subsequent degradation [31]. Curcumin has been shown to inhibit IκBα phosphorylation in human multiple myeloma cells [32] and murine melanoma cells [33] through suppression of IKK activity, which contributed to its antiproliferative, proapoptotic, and antimetastatic activities. Recently, we showed that curcumin has the potential to sensitize human colorectal cancer to capecitabine by modulation of cyclin-D1, COX-2, matrix metalloproteinase (MMP)-9, vascular endothelial growth factor (VEGF), and CXC chemokine receptor 4 (CXCR4) expression in an orthotopic mouse model. This was accompanied by inhibition in NF-κB activation [34].

Guggulsterone, obtained from the Commiphora mukul tree, suppresses NF-κB activation through inhibition of IKK-dependent IκBα degradation [35]. Resveratrol, a phytoalexin present in grapes, was shown to induce apoptosis and suppress constitutive NF-κB in rat and human pancreatic carcinoma cell lines [36]. Mammary tumors isolated from rats treated with resveratrol displayed reduced expression of COX-2 and MMP-9, accompanied by reduced NF-κB activation [37]. Treatment of human breast cancer MCF-7 cells with resveratrol also suppressed NF-κB activation and cell proliferation [37]. Capsaicin, a major ingredient of the pepper, has shown chemopreventive and chemoprotective effects [38-42]. Topical application of capsaicin has been associated with inhibition in phorbol 12-myristate 13-acetate (PMA)-induced mouse skin tumor formation and NF-κB activation [43]. The inhibitory effect of capsaicin on NF-κB activation was attributed to blockage of IκBα degradation and NF-κB translocation into the nucleus.

Caffeic acid phenethyl ester has been shown to suppress NF-κB activation by suppressing the binding of the p50–p65 complex directly to DNA [44], whereas both sanguinarine and emodin act by blocking the degradation of IκBα. Alkaloid sanguinarine can prevent phosphorylation and degradation of IκBα in response to TNF, phorbol ester, interleukin (IL)-1, or okadaic acid stimulation [45]. Similar to sanguinarine, emodin inhibits TNF-dependent IκBα degradation [46]. Recently, emodin was shown to oxidize the redox-sensitive site on NF-κB and prevented NF-κB binding to target DNA in HeLa cells, which was associated with a reduction in tumor size [47].

EGCG, an antioxidant found in green tea, has been shown to suppress malignant transformation in a 12-O-tetradecanoylphorbol-13-acetate-stimulated mouse epidermal JB6 cell line, which is mediated by blocking NF-κB activation [48]. EGCG treatment of human epidermal keratinocytes resulted in significant inhibition of ultraviolet-B-induced activation of IKKα, phosphorylation, and subsequent degradation of IκBα and nuclear translocation of p65 [49]. More recently, EGCG was found to abrogate p300-induced p65 acetylation in vitro and in vivo, to increase the level of cytosolic IκBα, and to suppress TNF-α-induced NF-κB activation. Furthermore, EGCG treatment inhibited the acetylation of p65 and the expression of NF-κB target genes in response to diverse stimuli [50]. Another nutraceutical, gallic acid, obtained from natural products such as gallnuts, sumac, oak bark, and green tea, was recently reported to possess anti-histone acetyltransferase activity, thus showing the potential to downregulate NF-κB activation [51]. Anacardic acid, derived from traditional medicinal plants, can also inhibit NF-κB activation by inhibiting p65 acetylation [52].

Thus, nutraceuticals may block one or more steps in the NF-κB signaling pathway, such as the inhibition of IKK activity, IκBα phosphorylation, p65 nuclear translocation, p65 acetylation, and p65 DNA binding. Some nutraceuticals that have the potential to suppress NF-κB activation are shown in Fig. 1. NF-κB can be activated by various carcinogens, some of which are also shown in Fig. 1.

3 Regulation of tumor cell development by nutraceuticals

3.1 Regulation of tumor cell survival by nutraceuticals

Under normal physiological conditions, the human body maintains homeostasis by eliminating unwanted, damaged, aged, and misplaced cells. Homeostasis is carried out in a genetically programmed manner by a process referred to as apoptosis (programmed cell death) [53-55]. Cancer cells are able to evade apoptosis and grow in a rapid and uncontrolled manner. One of the most important ways by which cancer cells have gained this ability is through mutation in the p53 tumor suppressor gene. Without a functional p53 gene, cells lack the DNA-damage-sensing capability that would normally induce the apoptotic cascade. A complex set of proteins, including caspases, proapoptotic and antiapoptotic B cell lymphoma (Bcl)-2 family proteins, cytochrome c, and apoptotic protease activating factor (Apaf)-1, execute apoptosis either by an intrinsic or extrinsic pathway. The intrinsic pathway is mitochondria dependent, whereas the extrinsic pathway is triggered by death receptors (DRs).

Some antiapoptotic proteins such as Bcl-2 and B cell lymphoma extra large (Bcl-xL) [56] and survivin [57] are overexpressed in a wide variety of cancers. Therefore, selective downregulation of antiapoptotic proteins and upregulation of proapoptotic proteins and p53 in cancer cells offer promising therapeutic interventions for cancer treatment. A number of nutraceuticals have shown potential against tumor cell survival by inducing apoptosis with use of various mechanisms in multiple types of cancer cells (Table 2).

Table 2

Effect of nutraceuticals on tumor cell survival

Some of the most common ways that nutraceuticals inhibit survival of tumor cells is by activating caspases, inducing proapoptotic proteins, and downregulating antiapoptotic proteins. Acetoxychavicol acetate, for example, a tropical ginger compound, decreased cell viability in breast-carcinoma-derived MCF-7 and MDA-MB-231 cells through a casp-3-dependent increase in apoptosis [58]. In a recent study, berberine induced apoptosis that was associated with reduction in mitochondrial membrane potential and changes in the Bcl-2-associated X protein (Bax)/Bcl-2 ratio [59]. Berberine also induced casp-3, casp-8, and casp-9 activation and the release of cytochrome c from mitochondria through generation of reactive oxygen species (ROS) [59]. Katiyar et al. [60] showed that berberine can induce apoptosis in A549 and H1299 human lung cancer cells that correlated with disruption of mitochondrial membrane potential, reduction in Bcl-2 and Bcl-xL levels, and increased Bax, Bcl-2 homologous antagonist/killer (Bak), and casp-3 activation.

Flavopiridol, a semisynthetic flavone, was shown to enhance TNF-induced apoptosis through activation of the bid-cytochrome–casp-9–casp-3 pathway in human myeloid cells. This induced apoptosis was associated with inhibited AKT8 virus oncogene cellular homolog (AKT) activation and inhibited expression of various antiapoptotic proteins such as inhibitor of apoptosis protein (IAP)-1, IAP-2, X-chromosome-linked IAP (XIAP), Bcl-2, and Bcl-xL [61]. Gu et al. [62] showed that gambogic acid can induce apoptosis in MCF-7 cancer cells through upregulation of p53 and downregulation of Bcl-2. In human malignant melanoma A375 cells, gambogic acid induced apoptosis that was associated with increased Bax expression and decreased Bcl-2 expression [63]. Garcinol was shown to induce apoptosis through inhibition of tyrosine phosphorylation of focal adhesion kinase (FAK) and downregulation of Rous sarcoma oncogene cellular homolog (Src), extra-cellular signal-regulated kinase (ERK), and AKT survival signaling in human colorectal cancer cell line HT-29 [64]. Indole-3-carbinol induced apoptosis through activation of p53 and cleavage of casp-3, casp-8, and casp-9 in lung cancer A549 cells [65].

Resveratrol induced apoptosis in human multidrug-resistant SPC-A-1/CDDP cells associated with downregulation in survivin [66]. Sanguinarine sensitized human gastric adenocarcinoma AGS cells to TNF-related apoptosis-inducing ligand (TRAIL)-induced apoptosis via downregulation of AKT and activation of casp-3 [67]. In MDA-MB-231 human breast carcinoma cells, sanguinarine induced apoptosis through mediation of ROS production, decrease in mitochondrial membrane potential, release of cytochrome c, activation of casp-3 and casp-9, and downregulation of antiapoptosis proteins XIAP and cIAP-1 [68]. Human leukemia U937 cells, when treated with sanguinarine, induced apoptosis through upregulation of Bax, induction of caspase activation, and downregulation of Bcl-2 [69]. Curcumin, the major polyphenol present in turmeric, is a potent inducer of apoptosis in cancer cells. Curcumin induces upregulation of proapoptotic proteins such as Bax, Bcl-2-interacting mediator of cell death (Bim), Bak, p53 upregulated modulator of apoptosis (Puma), and PhoRbol-12-myristate-13-acetate-induced protein 1 (Noxa) and downregulation of the antiapoptotic proteins Bcl-2 and Bcl-xL [70, 71].

Embelin was shown to enhance TRAIL-mediated apoptosis in malignant glioma cells by downregulation of the short isoform of FLICE/caspase-8 inhibitory protein [72]. Xanthohumol (XN), a chalcone, enhanced TRAIL-induced apoptosis in prostate cancer cells [73]. In human colon cancer cells, XN induced apoptosis through upregulation of casp-3, casp-8, and casp-9 activation and downregulation in Bcl-2 expression [74].

Transcription factor specificity proteins (Sp), including Sp1, Sp3, and Sp4, are known to regulate survivin and are required for survival of tumor cells. Betulinic acid, a pentacyclic triterpene, was recently shown to decrease expression of survivin and induce apoptosis in LNCaP prostate cancer cells through targeted degradation of Sp proteins [75].

Some nutraceuticals have been shown to induce apoptosis through upregulation of DRs. Oleandrin sensitized lung cancer cells to TRAIL-induced apoptosis through upregulation of DR4 and DR5 [76]. We recently showed that garcinol can sensitize human colon cancer cells to TRAIL-induced apoptosis through induction of DR4 and DR5 [77]. Capsaicin was shown to sensitize malignant glioma cells to TRAIL-mediated apoptosis via DR5 upregulation and survivin downregulation [78]. Similarly, celastrol potentiated TRAIL-induced apoptosis through downregulation of cell survival proteins and upregulation of DR4 and DR5 in human breast cancer cells [79]. Enhancement in TRAIL-induced apoptosis was recently observed in human colon cancer cells by zerumbone. This was mediated through upregulation of DR4 and DR5 and generation of ROS [80]. In another study, zerumbone triggered apoptotic events independent of functional p53 in liver cancer cells through upregulation of Bax and downregulation of Bcl-2 [81].

Insulin-like growth factor I receptor (IGFIR) has emerged as a key therapeutic target in many human malignancies, including childhood cancers such as Ewing family tumors (EFT). EGCG was found to inhibit survival of EFT through inhibition of IGFIR activity, induction of apoptosis through upregulation of Bax, and decreased expression of Bcl-2, Bcl-XL, and myeloid cell leukemia (Mcl)-1 proteins [82]. Induction of DNA damage and apoptosis in human ovarian cancer cells by genistein, a predominant isoflavone present in soybeans, was mediated through phosphorylation and activation of p53 and a decrease in the ratio of Bcl-2/Bax, Bcl-xL/Bax, and phosphorylated AKT levels [83]. Silymarin inhibited survival of hepatocellular carcinoma HepG2 cells by inducing apoptosis and facilitating cytochrome c release, upregulating proapoptotic proteins, and downregulating antiapoptotic proteins [84].

Some nutraceuticals have the potential to inhibit survival of tumor cells through mediation of the signal transducers and activators of transcription protein (STAT)-3 pathway. Muto et al. [85] showed that emodin can induce apoptosis in human myeloid cells through the elimination of Mcl-1. Emodin inhibited IL-6-induced activation of Janus-activated kinase 2 (JAK2) and phosphorylation of STAT-3; it also triggered casp-3 and casp-9 activation. Induction of apoptosis by emodin was almost abrogated in Mcl-1-overexpressing myeloma cells. These observations indicated that emodin can induce apoptosis in myeloid cells via downregulation of Mcl-1. Capsaicin has been reported to induce apoptosis in multiple myeloid cells through downregulation of STAT-3-regulated expression of Bcl-2, Bcl-xL, and survivin [86]. Adult T cell leukemia is an aggressive malignancy of peripheral T cells infected with human T cell leukemia virus type 1 (HTLV-1). Deguelin was shown to induce apoptosis in HTLV-1-transformed T cells via inhibition of survivin expression and STAT-3 phosphorylation through the ubiquitin/proteasome pathway [87]. In our laboratory, deguelin induced apoptosis in various cancer cells through the downregulation of antiapoptotic gene products [88].

Most nutraceuticals target by inhibiting NF-κB activation, thereby inhibiting NF-κB-regulated antiapoptotic proteins. Acetoxychavicol acetate inhibited cellular growth of multiple myeloma cells in vivo and in vitro through induction of apoptosis, activation of casp-8, inactivation of NF-κB, and downregulation of antiapoptotic proteins [89]. Garcinol induced apoptosis in human breast cancer MCF-7 and MDA-MB-231 cells through caspase activation and downregulation of NF-κB-regulated genes [90]. Plumbagin induced apoptosis with concomitant inactivation of Bcl-2 and the DNA binding activity of NF-κB in breast cancer cells [91]. In non-small-cell lung cancer, plumbagin induced apoptosis through mediation of c-Jun N-terminal kinase (JNK) and the casp-3 pathway [92]. In addition, Murtaza et al. [93] demonstrated that fisetin can induce apoptosis in chemoresistant human pancreatic PaC AsPC-1 cells through suppression of DR3-mediated NF-κB activation. Sulforaphane inhibited survival of orthotopically implanted PC-3 tumors through upregulation of DR4, DR5, Bax, and Bak and inhibition of NF-κB, phosphoinositide 3-kinase (PI3K)/AKT, and mitogen-activated protein kinase (MAPK)/ERK kinase (MEK) activation pathways [94].

We have identified a number of nutraceuticals from natural sources that target one or more steps in the NF-κB activation pathway to sensitize and induce apoptosis in a variety of cancer cells. The most popular among these are acetoxychavicol acetate [95], evodiamine [96], noscapine [97], indirubin [98], isodeoxyelephantopin [99], anacardic acid [52], coronarin D [100], thymoquinone [101], γ-tocotrienol [102], β-escin [103], and withanolides [104].

3.2 Regulation of tumor cell proliferation by nutraceuticals

Dysregulated proliferation is one of the major characteristics of tumorigenesis. In normal cells, proliferation is regulated by a delicate balance between growth signals and antigrowth signals. Cancer cells, however, acquire the ability to generate their own growth signals and become insensitive to antigrowth signals [1]. Their growth is controlled by cell cycle regulators at the G1/S-phase boundary, in the S phase, and during the G2/M phases of the cell cycle. A precise set of proteins called cyclins and cyclin-dependent kinases (CDKs) control the progression of cell cycle events. Whereas cyclin binding is required for CDK activity, CDK inhibitors (CKIs) such as p21 and p27 prevent CDK activity and prevent cell cycle progression. The G1-to-S-phase transition also requires cellular v-myc myelocytomatosis viral oncogene homolog (c-Myc), and inhibition of c-Myc expression leads to growth arrest [105]. Deregulated expression of c-Myc has been implicated in a number of human malignancies [106, 107]. The expression of c-Myc in turn is regulated by cdc25, a phosphatase that activates CDKs.

The well-characterized tumor suppressor p53 has been implicated in controlling the G1-to-S-phase transition and in blocking cell cycle progression at the G1 phase in response to DNA damage [108]. A number of genes controlling cell cycle progression, including the CKI p21, are transcribed in a p53-dependent manner [109, 110]. Rb is a tumor suppressor retinoblastoma protein that, like p53, functions as a negative regulator of cell growth [111]. Rb inactivation or deletion has been found in many cancers, including retinoblastomas and carcinomas of the lung, breast, bladder, and prostate. By binding to and inhibiting transcription factors such as elongation 2 factor (E2F), which are necessary for S-phase entry, Rb is believed to inhibit cell cycle progression [112]. On the other hand, phosphorylation of Rb (pRb) by CDK/cyclin complexes results in the release of active E2F species to stimulate the transcription of genes involved in DNA synthesis and S-phase progression [113-115]. COX-2, an inducible prostaglandin endoperoxide synthase 2, has been linked with tumor cell proliferation. It can be rapidly induced by growth factors, cytokines, and tumor promoters and is associated with inflammation [116-119]. Reports have demonstrated increased amounts of COX-2 in both premalignant and malignant tissues [120, 121].

Currently, a number of inhibitors based on cell cycle regulators, including nutraceuticals, are being developed as therapeutic intervention for cancer prevention. Nutraceuticals have been shown to have potential in cancer prevention for halting cell cycle progression by targeting one or more steps (Table 3) in the cell cycle. Most nutraceuticals prevent the transition of cancer cells from the G1 to S phase. Some of these nutraceuticals act through p53 and some through Rb. Acetyl-keto-beta-boswellic acid was shown to arrest colon cancer cells at the G1 phase, which was associated with decreases in cyclin-D1, cyclin-E, CDK-2, CDK-4, and pRb and an increase in p21 [122]. In Ehrlich ascites tumor cells, acetoxychavicol acetate was shown to stimulate the accumulation of tumor cells in the G1 phase of the cell cycle, which was accompanied by a decrease in pRb and an increase in Rb [123]. β-Escin, a triterpene saponin, induced cell cycle arrest at the G1/S phase by inducing p21 and reducing pRb in a p53-independent manner in HT-29 human colon cancer cells [124]. In gastric cancer cells, curcumin was shown to suppress the transition of cells from the G1 to S phase, which was accompanied by a decrease in cyclin-D1 and p21-activated kinase 1 activity [125].

Table 3

Effect of nutraceuticals on tumor cell proliferation

Deguelin exhibited an antiproliferative effect in breast cancer cells by arresting cells at the S phase [126]. Emodin showed antiproliferative activity through a p53- and p21-dependent pathway and arrested liver cancer HepG2 cells in the G1 phase [127]. Fisetin was shown to arrest prostate cancer LNCaP cells at the G1 phase, which was associated with a decrease in cyclin-D1, cyclin-D2, and cyclin-E and their activating partners CDK-2, CDK-4, and CDK-6 and with the induction of p21 and p27 [128].

The effect of piceatannol on the proliferation of DU145 human prostate cancer cells was investigated. Piceatannol caused cells to accumulate in the G1 phase and was associated with a decrease in cyclin-A, cyclin-D1, CDK-2, and CDK-4 [129]. Another nutraceutical, silibinin, caused lung cancer cells to accumulate at the G1 phase, which correlated with decreased CDK-2 and CDK-4 activities [130]. Silymarin arrested hepatocellular carcinoma HepG2 cells at the G1 phase, concomitant to a reduction in β-catenin, cyclin-D1, c-Myc, and proliferating cell nuclear antigen [84]. Thymoquinone, a component of Nigella sativa, was shown to abrogate the progression of prostate cancer cells from the G1 to S phase. These effects correlated with upregulation in p21 and p27 and downregulation in androgen receptor and E2F-1 [131]. Quercetin also induced cell cycle arrest at the G1 phase by elevating p53, p21, and p27 in a human hepatoma cell line in vitro [132]. Sulforaphane was shown to suppress proliferation of epithelial ovarian cancer cells through G1 cell cycle arrest, reduction in pRb and free E2F-1, and increase in Rb [133].

Some nutraceuticals prevent tumor cell proliferation by preventing transitions from the G2 to M phase. Butein was shown to inhibit cell growth in human hepatoma cancer cell lines—HepG2 and Hep3B—by inducing G2/M phase arrest. This inhibition in cell growth was associated with increased phosphorylation of ataxia telangiectasia mutated (ATM), checkpoint kinase (Chk)-1, and Chk-2, and reduction in cell division cycle 25 homolog c (cdc25c) levels. The inhibition in cell growth was also correlated with ROS generation and JNK activation [134]. Celasterol was shown to inhibit cell proliferation in C6 glioma cells by arresting the cells at the G2/M phase through upregulation of p21 and p27 and downregulation of CDK-2 [135]. Evodiamine exhibited antiproliferative activity by arresting human thyroid ARO cancer cells at the G2/M phase, which was associated with decreased expression of cdc2-p15 [136].

Recently, an ataxia telangiectasia and Rad3-related protein–Chk-1-mediated DNA damage response was shown to trigger p53/p21activation and G2/M arrest in HepG2 and A549 cells in response to gambogic acid treatment [137]. Betulinic acid evoked an increase in the G2/M phase population and a decrease in the S-phase population in human gastric adenocarcinoma cells. This correlated with a decrease in Hiwi and its downstream target cyclin-B1 [138]. Zerumbone was shown to suppress proliferation of leukemic NB4 cells by inducing G2/M cell cycle arrest, decreasing cyclin-B1 expression, and phosphorylating ATM/Chk-1/Chk-2 and cdc25c [139].

Berberine exhibited antiproliferative activity against human osteosarcoma cells by inducing cell cycle arrest at the G1 and G2/M phases. Whereas induction of G1 arrest was accompanied by p53-dependent upregulation of p21, G2/M arrest occurred regardless of p53 status [140]. Guggulsterone was shown to suppress the proliferation of cancer cells through inhibition of DNA synthesis and induction of cell cycle arrest in the S phase; these effects were mediated through downregulation of cyclin-D1 and cdc2 and upregulation of p21 and p27 [141].

NF-κB has been shown to bind to the promoter of genes involved in cellular proliferation. A few nutraceuticals target one or more steps in NF-κB activation to regulate tumor cell proliferation. With use of an orthotopic murine model of ovarian cancer, curcumin was shown to inhibit tumor growth that correlated with inhibition in NF-κB and a STAT-3 activation pathway [142]. In another study, curcumin exhibited antiproliferative activity in association with decreased expression of cyclin-D1 and CDK-4 in breast cancer cell lines MDA-MB-231 and BT-483 [143]. Fisetin, a naturally occurring flavonoid, was shown to downregulate COX-2 expression and to inhibit prostaglandin E2 secretion in HT29 human colon cancer cells; this correlated with decreased activity in wint signaling through downregulation of β-catenin, inhibition in epidermal growth factor receptor activity, activation of NF-κB, and subsequent decrease in cyclin-D1 expression [144].

We have identified a number of nutraceuticals with the potential to inhibit proliferation of cancer cells through inhibition of the NF-κB activation pathway and NF-κB-dependent gene products involved in proliferation such as c-Myc, COX-2, and cyclin-D1. Some of these nutraceuticals are flavopiridol [61], anacardic acid [52], coronarin D [100], diosgenin [145], isodeoxyelephantopin [99], morin [146], noscapine [97], pinitol [147], and ursolic acid [148].

S-phase kinase-associated protein 2 (Skp2), an F-box protein with an NF-κB binding site in its promoter, has been implicated in the degradation of p21 and p27. Recently, Tubocapsanolide A, a bioactive withanolide, was shown to induce G1 growth arrest in A549, H358, and H226 human lung cancer cells. The antiproliferative effects of Tubocapsanolide A were mediated through inhibition of binding of the RelA subunit of NF-κB to Skp2, inhibition of Skp2 expression, and upregulation of p21 and p27 [149].

The antiproliferative activity of capsaicin correlated with decreased expression of E2F-responsive proliferative genes such as cyclin-E, thymidylate synthase, cdc25A, and cdc6 in small-cell lung cancer [150]. Gossypol was shown to inhibit the growth of MAT-LyLu prostate cancer cells by arresting the cells at the G0/G1 phase and downregulating cyclin-D1, CDK-4, and pRb expression. These effects of gossypol were associated with modulation of transforming growth factor β-1 and AKT signaling [151].

Genistein has been shown to inhibit the growth of several cancer cells [152-157]. In breast cancer and melanoma cells, genistein induced G2/M cell cycle arrest [157, 158]. Although most studies indicated that genistein causes G2/M arrest, some showed that genistein could also arrest mouse fibroblast and melanoma cells at the G0/G1 phase of the cell cycle [159]. In addition, genistein was shown to halt cell growth by upregulating p21 in various cancer cells [160-163]. Touny and Banerjee [164] reported the involvement of upstream kinases myelin transcription factor 1 (Myt-1) and Wee-1 in the transcriptional repression of cyclin-B1 and activation of p21in prostate cancer cells. They found that genistein treatment increased Myt-1 levels and decreased Wee-1 phosphorylation, providing new insight into the possible mechanism of genistein-induced G2/M arrest.

3.3 Regulation of tumor cell invasion by nutraceuticals

Tumor cell invasion and metastasis are interrelated processes involving cell growth, cell adhesion, cell migration, and proteolytic degradation of tissue barriers such as the extracellular matrix and basement membrane. Several proteolytic enzymes, including MMPs (chiefly MMP-2 and MMP-9) [165, 166] and intercellular adhesion molecule (ICAM; chiefly ICAM-1), participate in the degradation of these barriers [167, 168]. A number of studies in lung, colon, breast, and pancreatic carcinomas have demonstrated overexpression of MMPs in malignant tissues compared with adjacent normal tissues [169-176]. Apart from MMPs, cysteine proteases [177] and serine proteases [178] such as urokinase-type plasminogen activator (u-PA) have also been involved in the invasion and metastasis of cancer cells. Since both u-PA and u-PA receptor (u-PAR) contain binding sites for NF-κB and activator protein (AP)-1 in their promoter regions [179-181], inhibition of these transcription factors will eventually result in the inhibition of u-PA–u-PAR complex and subsequent suppression of invasive behavior.

A wide variety of nutraceuticals derived from natural sources has been shown to inhibit tumor cell invasion and metastasis by targeting one or more molecules (Table 4). Allicin inhibited TNF-α-induced ICAM-1 expression in human umbilical endothelial cells (ECs) [182]. S-Allylcysteine and S-allylmercaptocysteine, obtained from garlic, suppressed the invasion ability of androgen-independent invasive prostate cancer cells [183] through restoration of E-cadherin expression. Allyl isothiocyanate (AITC) suppressed MMP-2 and MMP-9 at both protein and mRNA levels in human hepatoma SK-Hep1 cells in vitro [184]. Apigenin plays an important role in inhibiting the adhesion and motility of breast cancer cells through mediation of the HER2–HER3–PI3K–AKT pathway [185]. Apigenin inhibited metastasis of lung melanoma cells by inhibiting vascular cell adhesion molecule 1 (VCAM-1) expression in a dose-dependent manner [186].

Table 4

Effect of nutraceuticals on tumor cell invasion

Ezrin is highly expressed in metastatic tumors and is involved in filopodia formation as well as promotion of tumor metastasis. Berberine, an alkaloid, was recently shown to inhibit invasion and motility in nasopharyngeal carcinoma cell line 5-8F through repression of ezrin phosphorylation at Thr567 by Rho kinase and inhibition in filopodia formation [187]. Berberine has also been reported to suppress in vitro migration and invasion of human SCC-4 tongue squamous cancer cells through inhibition of FAK, IKK, NF-κB, u-PA, and MMP-2 and MMP-9 [59].

Increasing evidence has shown that epithelial–mesenchymal transition plays a critical role in tumor cell metastasis. Butein, a polyphenolic compound obtained from stem bark of cashews, was recently shown to inhibit migration and invasion through the ERK-1/ERK-2 and NF-κB signaling pathways in human bladder cancer cells. The inhibitory effect of butein was associated with the reversal of epithelial–mesenchymal transition [188]. We have shown that butein can inhibit TNF-α-induced invasion in human lung adenocarcinoma H1299 cells, which was associated with inhibition in NF-κB activation and downregulation in MMP-9 [189].

Caffeic acid had a strong inhibitory effect on MMP-9 activity in nonspecific cell types in vitro [190]. Capsaicin significantly inhibited the migration of highly metastatic B16-F10 melanoma cells through inhibition of the PI3K/AKT/rat sarcoma (Ras)-related C3 botulinum toxin substrate 1 signaling pathway [191]. Carnosol reduced MMP-9 levels in mouse melanoma cells in vitro through downregulation of NF-κB and AP-1 [192]. β-Carotene inhibited the invasion of rat ascites hepatoma AH109A cells in a dose-dependent manner by acting as ROS quenchers [193]. Catechin gallate, a phenolic compound obtained from the red pine, inhibited the invasion and migration of SK-Hep-1 human hepatocellular carcinoma cells, which strongly correlated with reduced expression of MMP-2 and MMP-9 [194]. Celastrol, a quinone methide triterpene from the medicinal plant Tripterygium wilfordii, exerted potent antimetastatic activity both in vitro and in vivo [195] through p38 MAPK, suppression of β-1 integrin ligand affinity, focal adhesion formation, reduced phosphorylation of FAK, and inhibition of cell–extracellular matrix adhesion of human lung cancer 95-D and mouse melanoma B16-F10 cells. Crocetin was shown to suppress ICAM-1 and MMPs in bovine endothelial cells [196].

Curcumin exerted a dose- and time-dependent inhibitory effect on the invasion and migration of mouse–rat hybrid retina ganglion cells (N18) in vitro [197]. This inhibited invasion was associated with downregulation of PKC, FAK, NF-κB p65, Rho A, MMP-2, and MMP-9. In Hep2 human laryngeal cancer cells, curcumin inhibited tumor cell invasion and metastasis that were associated with downregulated MMP-2 expression and reduced activity and expression of integrin receptors, FAK, and membrane-type 1 MMP [198]. Diallyl disulfide inhibited the activation of MMP-2 and MMP-9 in human umbilical vein endothelial cells (HUVECs) in vitro [199].

The chemokine receptor CXCR4, with its unique ligand CXC chemokine ligand 12 (CXCL12), is required for metastasis of breast cancer cells [200, 201]. 3, 3’-Diindolylmethane showed antimetastatic ability in MCF-7 and MDA-MB-231 breast cancer cells by lowering CXCR4 and CXCL12 levels [200, 201]. With the use of androgen-insensitive prostate cancer (DU-145) cells, Vayalil and Katiyar [202] showed that EGCG can inhibit fibroblast-conditioned medium-induced production of pro and active forms of MMP-2 and MMP-9. Nuclear localization of NF-κB, as well as MMP-9 expression and invasion, was suppressed in lung carcinoma cells treated with EGCG [203].

Takada et al. [96] recently showed that evodiamine can inhibit TNF-induced invasion in human lung adenocarcinoma H1299 cells through inhibition in NF-κB activation and downregulation in MMP-9. The antimetastatic potential of fisetin was mediated through inhibition of phosphorylation of ERK-1/ERK-2 and downregulation in expression of MMP-2 and u-PA in A549 cells [204].

c-erbB-2 is a key molecule for breast cancer metastasis, and overexpression of c-erbB-2 has been correlated with increased MMP secretion and metastatic potential in breast cancer cells [205]. Flavopiridol was found to inhibit the secretion of MMP-2 and MMP-9 in the breast cancer cells. Inhibition in MMP secretion was associated with significant downregulation of c-erbB-2 and inhibition of cell invasion [206]. Ganoderic acids isolated from Ganoderma lucidum suppressed invasive behavior of breast cancer cells by inhibiting AP-1 and NF-κB activity, resulting in inhibition of u-PA secretion [207]. Genistein inhibited cell adhesion to vitronectin and cell migration of invasive breast cancer cells by inhibiting the transcriptional activity of AP-1 and NF-κB, resulting in the suppression of u-PA secretion from cancer cells [208]. [6]-Gingerol inhibited cell adhesion, invasion, motility, and activities of MMP-2 and MMP-9 in human breast cancer cell lines in vitro [209]. Indole-3-carbinol suppressed the 17-β-estradiol-stimulated migration and invasion in estrogen-responsive MCF-7 cells. The suppressed invasion was associated with an increase in invasion suppressor molecules, E-cadherin, and α-, β-, and γ-catenin [210].

Lycopene, a dietary constituent present in tomatoes, red fruits, and vegetables, was recently shown to suppress migration and invasion of hepatoma cell line SK-Hep-1, which was associated with upregulation of a metastasis suppressor gene, nm23-H1 [211]. Myricetin inhibited MMP-2 expression and enzyme activity in colorectal carcinoma cells in vitro [212]. Piperine inhibited MMP production in melanoma cells in vitro, preventing collagen matrix invasion in a dose-dependent manner [213]. Quercetin decreased expression of MMP-2 and MMP-9 in a dose-dependent manner in PC-3 prostate cancer cells in vitro [214]. Resveratrol reduced the migratory and invasive abilities of A549 lung cancer cells and was associated with inhibition of NF-κB activation and expression of MMP-2 and MMP-9 [215]. Sanguinarine inhibited invasiveness of MDA-MB-231 human breast carcinoma cells by decreasing the activities of MMP-2 and MMP-9 [67]. Silibinin, a flavonolignan, inhibited invasion and motility of SCC-4 tongue cancer and A459 lung cancer cells by down-regulating MMP-2 and u-PA and upregulating tissue inhibitor of metalloproteinase (TIMP)-2 and PAI-1 expression [216, 217]. Recently, Lee et al. [218] reported that silibinin reduced PMA-induced invasion of MCF-7 cells through specific inhibition of AP-1-dependent MMP-9 expression. Sulforaphane inhibited the activation of MMPs, thereby inhibiting lung metastasis induced by melanoma cells in mice [219].

The invasion and metastatic capacities of SGC-7901 gastric adenocarcinoma cells and their correlation with antimetastatic mechanisms induced by γ-tocotrienol were explored. Cell attachment was decreased by the γ-tocotrienol, which was associated with decreased MMP-2 and MMP-9 expression and upregulation of TIMP-1 and TIMP-2 [220]. Ursolic acid has been reported to reduce IL-1β- or TNF-α-induced rat C6 glioma cell invasion through downregulation of NF-κB activation and MMP-9 expression [221]. Zerumbone downregulated expression of CXCR4 on HER2-overexpressing breast cancer cells in a dose- and time-dependent manner. Suppression of CXCR4 expression by zerumbone correlated with the inhibition of CXCL12-induced invasion of both breast and pancreatic cancer cells [222]. In another study, zerumbone suppressed TNF-induced NF-κB activation and NF-κB-mediated MMP-9 expression that correlated with inhibition in tumor cell invasion [223].

3.4 Regulation of tumor cell angiogenesis by nutraceuticals

Angiogenesis, the process during which new blood vessels are formed from preexisting ones, can be classified as either physiological or pathological. Physiological angiogenesis provides a driving force for organ development in ontogeny, is necessary for ovulation, and is a prerequisite for wound healing; pathological angiogenesis occurs during tumor growth at primary and metastatic sites [224]. The angiogenic cascade during tumor development consists of the release of angiogenic factors, binding of angiogenic factors to receptors on ECs, EC activation, degradation of the basement membrane by proteases, and migration and proliferation of ECs. Adhesion molecules then help to pull the sprouting blood vessels forward, and ECs are finally organized into a network of new blood vessels [225]. The signaling pathway governing tumor angiogenesis is exceedingly complex, involving various angiogenic mediators. The major signaling mediators include VEGF, platelet-derived growth factor, fibroblast growth factors (FGFs), epidermal growth factor, ephrins, angiopoietins, endothelins, integrins, cadherins, and notch [226].

Since the role of angiogenesis in tumor development was first revealed [227], a number of antiangiogenic compounds have been developed, including bevacizumab (Avastin), sunitinib (SUTENT), sorafenib (Nexavar), cediranib maleate (Recentin), and pazopanib [226]. Many nutraceuticals have shown angiogenesis-modulating properties by targeting one or more steps in the signaling pathway (Table 5).

Table 5

Effect of nutraceuticals on tumor cell angiogenesis

Alliin showed potential to inhibit FGF-2-induced human EC tube formation and angiogenesis in a chick chorioallantoic membrane (CAM) model. Alliin also inhibited VEGF-induced angiogenesis in the CAM model [228]. In a C57BL/6 mouse model bearing B16-F10 melanoma cells, AITC inhibited NO synthesis and TNF-α production, which correlated with inhibited angiogenesis [229]. Recently, the antiangiogenic effect of AITC was investigated in Swiss albino mice into which Ehrlich ascites tumor cells were transplanted [230]. AITC significantly reduced vessel sprouting and exhibited potent antiangiogenic activity that was associated with significant reduction in VEGF expression.

Fang et al. [231] showed that apigenin can inhibit expression of hypoxia-inducible factor 1 (HIF-1) and VEGF in various types of cancer cells under normoxic and hypoxic conditions; this inhibition was associated with significant inhibition in tumor angiogenesis. In another study, caffeic acid suppressed STAT-3-mediated HIF-1 and VEGF expression, which correlated with inhibited vascularization and angiogenesis in mice bearing Caki-I human renal carcinoma cells [232].

Capsaicin has been shown to inhibit in vitro and in vivo angiogenesis. In vitro, capsaicin inhibited VEGF-induced capillary-like tube formation of primary cultured human ECs. It also inhibited VEGF-induced vessel sprouting in a rat aortic ring assay, VEGF-induced vessel formation in a mouse Matrigel plug assay, and VEGF-induced p38 MAPK, p125 (FAK), and AKT activation [233].

Curcumin was found to completely prevent induction of VEGF synthesis in microvascular ECs stimulated with glycation end products, which was mediated by downregulation of NF-κB and AP-1 activity [234]. Curcumin also inhibited angiogenesis through mediation of angiopoietins 1 and 2, HIF-1, and heme oxygenase 1 in cancer cells [235].

Diallyl sulfide reduced the serum level of VEGF in C57BL/6 mice bearing B16-F10 melanoma cells [199]. EGCG inhibited production of VEGF and IL-8 from normal human keratinocytes [236-238]. In human colon cancer cells, EGCG attenuated VEGF production through inhibition of ERK-1 and ERK-2 kinases [239]. Recently, EGCG inhibited ephrin-A1-mediated EC migration as well as tumor angiogenesis through inhibition in ERK-1/ERK-2 activation [240].

Flavopiridol decreased hypoxia-mediated HIF-1α expression, VEGF secretion, and tumor cell migration in human U87MG and T98G glioma cell lines [241]. These in vitro data were correlated with reduced vascularity of intracranial syngeneic GL261 gliomas in animals treated with flavopiridol. Gambogic acid inhibited activation of VEGF receptor 2 and of downstream kinases such as c-Src, FAK, and AKT and inhibited angiogenesis in HUVECs and human prostate cancer cells (PC3) [242]. Genistein suppressed VEGF and FGF-2 expression and inhibited tyrosine kinase phosphorylation and activation of AKT and NF-κB, resulting in inhibition of angiogenesis in renal cell carcinoma [243-246]. [6]-Gingerol, in response to VEGF, blocked capillary-like tube formation and strongly inhibited both sprouting of ECs in rat aorta and formation of new blood vessels in mouse cornea [247].

Luteolin inhibited VEGF-induced survival and proliferation of HUVECs through PI3K/AKT-dependent pathways [248]. Perillyl alcohol decreased the release of VEGF from cancer cells and stimulated expression of Ang2 by ECs, indicating that it might suppress neovascularization and induce vessel regression [249]. In another study, quercetin inhibited hypoxia-induced VEGF expression in NCI-H157 cells, which correlated with suppression in STAT-3 tyrosine phosphorylation, suggesting that inhibition of STAT-3 function may play a role in angiogenesis inhibition [250].

Resveratrol is able to suppress the growth of new blood vessels in animals. It directly inhibits capillary endothelial cell growth and blocks both VEGF- and FGF-receptor-mediated angiogenic responses through inhibition of phosphorylation of MAPK in ECs [251]. Rosmarinic acid (RA), a water-soluble polyphenolic compound, reduced the intracellular ROS level, H2O2-dependent VEGF expression, and IL-8 release of ECs. These activities were related to the antiangiogenic potential of RA [252]. Sanguinarine exhibited antiangiogenic activity by directly suppressing the proliferative effect of VEGF on ECs; this effect was mediated through downregulation of VEGF-induced AKT activation [253].

The in vivo efficacy of silibinin against human colorectal carcinoma HT29 xenograft growth in mice was investigated recently. Silibinin administration was associated with antiangiogenic activities in nude mice, resulting in downregulation of nitric oxide synthase, COX, HIF-1α, and VEGF expression [254]. In human prostate cancer cells, sulforaphane inhibited NF-κB-regulated VEGF expression in vitro [255]. In HUVECs, sulforaphane inhibited angiogenesis through activation of forkhead homeobox type O transcription factors and inhibition of MEK/ERK and PI3K/AKT pathways [256]. In human leukemic cell lines, taxol, obtained from the bark of Taxus brevifolia, showed antiangiogenic activity by inhibiting VEGF production and HIF-α expression [257]. In a human gastric adenocarcinoma SGC-7901 cell line, γ-tocotrienol inhibited cobalt(II) chloride-induced accumulation of HIF-1α and paracrine secretion of VEGF. A decrease in VEGF secretion was associated with decreased activation of ERK-1/ERK-2 [258]. The antiangiogenic activity of ursolic acid was shown recently; ursolic acid inhibited capillary formation in C57BL/6 mice bearing B16-F10 melanoma cells. Levels of serum VEGF, NO, and proinflammatory cytokines were significantly reduced in ursolic-acid-treated animals compared with those in control animals [259]. Vanillin, a food flavoring agent, suppressed hepatocyte-growth-factor-induced tumor cell angiogenesis in a mouse model that was mediated through inhibition of PI3K/AKT signaling and VEGF expression [260].

4 Summary, conclusions, and future perspective

Abstract

Background

Zingiber zerumbet Smith is a perennial herb, broadly distributed in many tropical areas. In Malaysia, it’s locally known among the Malay people as “lempoyang” and its rhizomes, particularly, is widely used in traditional medicine for the treatment of peptic ulcer disease beyond other gastric disorders.

Aim of the study

The aim of the current study is to evaluate the gastroprotective effect of zerumbone, the main bioactive compound of Zingiber zerumbet rhizome, against ethanol-induced gastric ulcer model in rats.

Materials and Methods

Rats were pre-treated with zerumbone and subsequently exposed to acute gastric ulcer induced by absolute ethanol administration. Following treatment, gastric juice acidity, ulcer index, mucus content, histological analysis (HE and PAS), immunohistochemical localization for HSP-70, prostaglandin E2 synthesis (PGE2), non-protein sulfhydryl gastric content (NP-SH), reduced glutathione level (GSH), and malondialdehyde level (MDA) were evaluated in ethanol-induced ulcer in vivo. Ferric reducing antioxidant power assay (FRAP) and anti-H. pylori activity were investigated in vitro.

Results

The results showed that the intragastric administration of zerumbone protected the gastric mucosa from the aggressive effect of ethanol-induced gastric ulcer, coincided with reduced submucosal edema and leukocyte infiltration. This observed gastroprotective effect of zerumbone was accompanied with a significant (p <0.05) effect of the compound to restore the lowered NP-SH and GSH levels, and to reduce the elevated MDA level into the gastric homogenate. Moreover, the compound induced HSP-70 up-regulation into the gastric tissue. Furthermore, zerumbone significantly (p <0.05) enhanced mucus production, showed intense PAS stain and maintained PG content near to the normal level. The compound exhibited antisecretory activity and an interesting minimum inhibitory concentration (MIC) against H. pylori strain.

Conclusion

The results of the present study revealed that zerumbone promotes ulcer protection, which might be attributed to the maintenance of mucus integrity, antioxidant activity, and HSP-70 induction. Zerumbone also exhibited antibacterial action against H. pylori.

Citation: Sidahmed HMA, Hashim NM, Abdulla MA, Ali HM, Mohan S, Abdelwahab SI, et al. (2015) Antisecretory, Gastroprotective, Antioxidant and Anti-Helicobcter Pylori Activity of Zerumbone from Zingiber Zerumbet (L.) Smith. PLoS ONE 10(3): e0121060. https://doi.org/10.1371/journal.pone.0121060

Academic Editor: Gianfranco Pintus, University of Sassari, ITALY

Received: November 21, 2014; Accepted: January 28, 2015; Published: March 23, 2015

Copyright: © 2015 Sidahmed et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Data Availability: All relevant data are within the paper.

Funding: The authors would like to express their extreme thankfulness and gratefulness to University of Malaya (PG151-2012B) and the Ministry of Higher Education Malaysia under High Impact Research grant (UM-MOHE UM.C/625/1/HIR/MOHE/SC/09) for providing financial funds to perform this work. Also, the authors would like to express their utmost gratitude and appreciation to the late Prof. Datuk Dr. A. Hamid A. Hadi for his help and support throughout this study. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Gastric ulcer is the most common digestive system disease affecting a lot of people worldwide and has sparked medical and global economic interest. Many factors are involved in the development of gastric ulcer, such as stress, habit of smoking, nutritional deficiency, ingestion of non-steroidal anti-inflammatory drugs, hereditary predisposition and infection by H. pylori [1]. Although there are many drugs currently used in the clinical field to manage gastric ulcer disease, the majority of them exhibit several adverse reactions. Thus, there is need to find out more effective and less toxic antiulcer agents [2]. The high focus on the herbal medicines and the pharmacological activities of their bioactive compounds has resulted in the discovery of numerous natural drugs or herbal extracts [3]. In particular, a large number of medicinal plants demonstrated anti-ulcer properties [4], with quite less cost and a wide range of safety margin[5].

The Zingiberaceae plant family is most abundant in Southeast Asia, and it is widely used in traditional medicine [6]. Zingiber zerumbet Smith is one of the Zingiberaceae species, characterized by its significant economic outcome, since it used as a spice and as traditional medicine [7]. The plant is widespread in several tropical countries such as India, Bangladesh, Malaysia, Nepal, and Sri Lanka [8]. The rhizomes of Zingiber zerumbet have been intensively studied and found to exhibit a wide range of pharmacological activities such as antipyretic, analgesic properties, anti-inflammatory, chemo-preventive activities [9], antinociceptive, antiulcer, antioxidant, anticancer, antimicrobial, antihyperglycemic, antiallergic and antiplatelet activities [3]. In Malaysia, Zingiber zerumbet is locally called “lempoyang” and the rhizomes of the plant are widely used as traditional medicine for the treatment of peptic ulcers [8, 9], stomach ache, diarrhoea, asthma, rheumatism and as an anti-inflammatory [3].

Zerumbone, a monocyclic sesquiterpene compound (2,6,10-cy-cloundecatrien-1-one, 2,6,9,9-tetramethyl-,(E,E,E)-), was reported as the predominant bioactive compound from the rhizomes of Zingiber zerumbet [7, 10]. Earlier toxicity study on zerumbone determined its LD 50 value is 1.84 g/kg [11]. Although, zerumbone showed selective cytotoxic activity towards certain cancer cell lines, however, it has no or less effect on normal cell line [7]. Many studies have been performed to elucidate the biological activities of zerumbone, demonstrated many pharmacological activities such as antinociciptive, anti-inflammatory, antitumor, antiproliferative and antiplatelet aggregation [3]. However, there was no report or investigation on its effect on gastric ulcer. Thus, in our continuous search for a natural antiulcer compound from Malaysian herbal medicines, we isolated zerumbone from the rhizomes of Zingiber zerumbet for gastroprotective study.

Many experimental gastric ulcer models have been created to examine and identify the causes of gastric mucosal injuries, among them the ethanol ulcer model. Ethanol-induced gastric tissue damage in experimental animals is the most common ulcer model, since it penetrates easily and rapidly into the gastric mucosa, mediating various pathological events result in ulcer formation. [12]. Therefore, ethanol ulcer model is the ideal ulcer model and extensively have been utilized for the assessment of new antiulcer compounds [13].

Despite the traditional use of Zingiber zerumbet as antiulcer medicinal plant, and to the best of our knowledge, there was no such data among the extensive search on this valuable rhizome to show it is gastroprotective mechanisms. Thus, this study was conducted to provide a scientific base for the use of Zingiber zerumbet rhizomes and to illustrate the possible mechanism(s) that might be involved in the antiulcer action of its main bioactive constituent, zerumbone.

Materials and Methods

Plant material and isolation of zerumbone compound

Zerumbone (Fig. 1) was isolated from the rhizome of Zingiber zerumbet (Voucher No. ZZ-2009-127) deposited at the Herbarium of the Laboratory of Natural Products, IBS, University Putra Malaysia, UPM Serdang, Malaysia. Pure zerumbone crystals were prepared according to the method reported earlier [7]. The purity of zerumbone compound was identified using HPLC and LC–MS, respectively.

Drugs and chemicals

Omeprazole, griess reagent, TPTZ and DTNB were purchased from Sigma-Aldrich Chemical Co. Kuala Lumpur, Malaysia. ketamine and xylazine were purchased from Pet Arcade Co, Kuala Lumpur, Malaysia. All other used chemicals and reagents were of analytical grade.

Animals

Disease-free Sprague-Dawley male rats (225–250 ± 5g) and their standard commercial feed pellets were purchased from the Experimental Animal Unit, University of Malaya, Faculty of Medicine, Institutional Animal care And Use Committee (FOM IACUC), Kuala Lumpur, Malaysia. All animals were kept under constant environmental temperature of 22°C, with 12 hrs light/dark cycles and free access to feed and distilled water. Rats were caged in groups of 2–3 each and left for one week as acclimatization period.

Ethic statement

This study was presented to the institutional ethical review board (FOM IACUC) for approval, and the approval was granted (2013-10-8/FAR/R/HMAS). All in vivo experimental procedures were performed in sterile condition in the Experimental Animal Unit of FOM IACUC following their guidelines. All animals received humane care, according to the criteria outlined in the “Guide for the Care and use of Laboratory Animals” prepared by the National Academy of Sciences and published by the National Institute of Health, USA.

Antisecretory study

The effect of zerumbone on gastric acid output was determined following the recommended method [14]. Sprague Dawley rats were assigned equally into four groups (n = 6). After 24 hrs fasting, the animals anesthetized using ketamine 50 mg/kg and xylazine 5 mg/kg then their abdomen was open, the stomach was exposed and the pylorus was ligated. Immediately after pylorus ligature, animals were received the treatments intraduodenally (5mL/kg b.w) according to the following grouping:

  • Group-1 control (5% Tween 80 v/v)
  • Group-2 standard (omeprazole 30mg/kg)
  • Group-3 zerumbone (5 mg/kg)
  • Group-4 zerumbone (10 mg/kg)

Following treatment, the abdomen was then sutured. The surgery procedure was accomplished without any unintended deaths of animals. After four hours of continuous observation and monitoring, all animals sacrificed using the CO2 chamber, the abdomen was opened to place another ligature at the oesophageal end, then all the stomachs were removed immediately and the gastric content was collected into tubes and centrifuged at 2000 ×g for 5 min and the gastric secretion volume (mL) was determined. The pH value of the gastric juice was recorded using a digital pH meter. The total acidity of the gastric juice was determined by titrating with 0.01 N sodium hydroxide using phenolphthalein as indicator. One mL of the gastric juice was transferred into 100 mL conical flask, 2 or 3 drops of phenolphthalein solution were added and the titration was preceded until a definite pink color appeared. The total volume of alkali added was noted. The total acidity (expressed as mEq/l) was calculated using the following formula:

Gastroprotective study

The gastroprotective effect of zerumbone was determined against ethanol ulcer model. Sprague Dawley rats were divided randomly into five groups (n = 6). Overnight fasted animals were treated orally (5mL/kg b.w) as follows:

  • Group-1 normal control (5% Tween 80 v/v)
  • Group-2 ulcer control (5% Tween 80 v/v)
  • Group-3 standard (omeprazole 20mg/kg)
  • Group-4 zerumbone (5 mg/kg)
  • Group-5 zerumbone (10 mg/kg)

One hour later, all groups, except for group-1, were received absolute ethanol (5mL/kg) [15]. After one hour of continuous observation and monitoring, all animal anetheized using Ketamine 50 mg/kg and xylazine 5 mg/kg prior to collect the blood samples via cardiac puncture into tubes. The blood samples then centrifuged for 10 min at 11,940 x g to get pure serum which was stored at −80°C prior to use later for biochemical analysis [16]. After that, all the rats sacrificed using the CO2 chamber. The stomach was immediately removed, opened along the greater curvature, washed by iced cold phosphate buffer solution (PBS), photographed by an inverted microscope with digital camera (BX60 Olympus) and subjected for the determination of gastric ulcer area (mm2) using the image J software [17]. The gastroprotective assessment was displayed as an inhibition percentage (I %) calculated by the following formula described by [18].

Thereafter, each gastric tissue was cut into pieces and processed for further histological and mechanistic studies.

Measurement of gastric mucus content and serum biochemical parameters

Each Stomach was opened along the greater curvature and washed with iced cold phosphate buffer solution (PBS). Using a glass slide, the surface of the gastric mucosa was gently scraped off and the collected mucus was weighted using a precise electronic balance [19]. The collected animal serum samples were analyzed at University Malaya Medical Centre using Hitachi Auto-analyzer to evaluate changes in serum biomarkers.

Histological evaluation

For histological evaluation, a small fragment from each stomach was fixed in 10% buffered formalin solution, Followed by tissue dehydration with alcohol and xylene. After the dehydration, each sample was embedded in paraffin wax, sectioned on slides at 5 μm with a Leica rotation microtome. Group of slides were stained with hematoxylin and eosin (H&E) according to the recommended method [20].

Lesion scoring system

Histopathological analysis was made under an inverted digital camera BX60 Olympus and the image J software was used to characterize the histopathological alteration in the damaged area [17]. The results were graded according to the literature scoring system [21]. The microscopic scoring was as follows: epithelial cell loss (score: 0–3), edema in the submucosa (score: 0–4), hemorrhagic damage (score: 0–4), and the presence of inflammatory cells (score: 0–3). The maximum total score was estimated up to 14. The evaluation performed by a pathologist who was unaware and blinded to the treatment.

PAS staining

in oreder to assess the effect of the compound on mucosal glycoprotein’s production, group of the prepared tissue sections were stained with commercial periodic acid Schiff base (PAS) following the manufacture instruction (Sigma Aldrich, Malaysia, Periodic Acid-Schiff (PAS) Kit). The positive glycoprotein site will appear as magenta color. Image J software was used for the determination of the positively stained area (μm2) [17].

Immunohistochemistry

Immunostaining was performed using DAKO ARK (Animal Research Kit), Peroxidase (DAKO, Carpinteria, CA, USA), to investigate the immunohistochemical localization of heat shock protein-70 (HSP70 Mouse monoclonal antibody, IgG2b, 200μg/mL-1:100). The protein was purchased from Santa Cruz Biotechnology, Inc., California, USA. The positive antigen site will appear as brown color. Image J software was used for the determination of the positively stained area (μm2) [17].

Preparation of gastric tissue homogenate

Small fragments of each stomachs were weighted and homogenized (10% w/v) in 0.1 mol/l PBS containing mammalian protease inhibitor cocktail. The homogenates were then centrifuged at 10000 x g for 15 min at 4°C. The clear supernatant was aliquot and stored in −80 o C prior to quantify the biochemical parameters in the gastric tissue homogenate: GSH, MDA, NP-SH and PG.

Glutathione (GSH) levels

GSH content into the gastric homogenate (GSH nmol/g tissue) was estimated using Ellman procedure [22]. Aliquot from the prepared tissue homogenate was allowed to react with 5,5-ditiobis-2-nitrobenzoic acid (DTNB) and the absorbance was read on a spectrophotometer at 412 nm.

Thiobarbituric acid reactive substance assay

Thiobarbituric acid reactive substance (TBARS) assay was used to estimate gastric content of MDA, where MDA is a direct index of lipid peroxidation level [23]. In brief and according to the recommended method [24], the gastric homogenate was added to a 0.126 mL solution containing 26 mM thiobarbituric acid, 0.26 M HCL, 15% trichloroacetic acid and 0.02% butaylated hydroxyltoluene. The mixture was incubated in a water bath at 95°C for 1 h. After cooling, the mixture was centrifuged at 3000 g for 10 min. The absorbance was read in a spectrophotometer at 532 nm and the results were expressed in μmol/g tissue MDA. Tetramethoxy propane was used as standard.

Estimation of nonprotein sulfhydryls

Gastric Mucosal nonprotein sulfhydryls (NP-SH) (μmol/g of tissue) was measured according to the recommended method of [25]. In brief, Aliquot of 5ml of the stomach homogenate was mixed with a solution containing 4 ml of distilled water and 1 ml of 50% Trichloroacetic acid. The mixture was vortex for 15 min and centrifuged at 3000× g. 2 ml of supernatant was mixed with 4 ml of 0.4 M Tris Buffer at pH 8.9; 0.1 ml of DTNB (5,5 dithio-bis-(2-nitrobenzoic acid)) was added and the mixture was shaken. The Absorbance was recorded within 5 min after DTNB addition at 412 nm against a reagent blank with no homogenate.

Estimation of prostaglandin E 2

Sample from the oxyntic gland area was taken by biopsy (about 100 mg), immediately from each animal of the treated groups after they were sacrificed to determine the mucosal generation of PGE2 by competitive enzyme immunoassay using enzyme immunoassay kit for prostaglandin estimation (Cayman Chemicals). The gastric mucosa of different treated groups was excised and homogenized in an ice-cold Tris/HCl buffer containing 50 mM Tris/HCl (pH 7.4), 100 mM sodium chloride, 1 mM calcium chloride, 1 mg/mL D-glucose and 28 μM indomethacin according to the method of [26]. The protein concentration of the homogenate was measured by the method of [27]. Homogenate was centrifuged at 12,000×g for 30 min at 4°C for the determination of PGE2 concentration. The supernatant was transferred in separate vial and kept at − 70°C until assayed. The concentration of PGE2 present in the supernatant was measured in duplicates with PGE2 enzyme immunoassay kit. The assay was performed in a total volume of 150 μL with the following components being added in 50 μL volumes: standards or homogenate, enzymatic tracer and specific antiserum. After 1 h incubation at room temperature on shaker, the plates were washed and 200 μL of Ellman's reagents were dispensed into each well. After 1 h, the absorbance at 412 nm for each well was measured. Results were expressed as ng of PGE2 per mg of protein.

Ferric-reducing antioxidant power (FRAP) assay

To describe the total antioxidant activity of zerumbone, the Ferric-reducing antioxidant power (FRAP) assay was accomplished following the procedure described by [28]. In Brief, FRAP reagent was prepared freshly from acetate buffer (pH 3.6), 10 mM TPTZ (2,4,6-Tri(2-pyridyl)-s-triazine) solution in 40 mM HCl and 20 mM iron (III) chloride solution in proportions of 10:1:1 (v/v), respectively. 50 μl of the compound were added to 1.5 ml of the FRAP reagent in the dark. 4 min later the absorbance was recorded at 593 nm. The standard curve was constructed linear (R2 = 0.9723) using iron (II) sulfate solution (100–1000 μM) and the results were expressed as μM Fe (II)/g dry weight of the compound.

In vitro anti-Helicobacter pylori activity

Two H. pylori strains NCTC 11637 (American Type Culture Collection ATCC 43504) and J99 (ATCC 700824) were cultured with brain heart infusion broth (BHI; Oxoid) supplemented with 10% horse serum (Invitrogen) incubated at 37°C in a humidified CO2 incubator (Forma Steri-Cycle) for 3 days. Minimum inhibitory concentration (MIC) was determined by a modified microtiter broth dilution method on sterile 96-well Polypropylene microtitre plates with round-bottom wells (Eppendorf). Briefly, zerumbone was dissolved and diluted in 5% DMSO to give a 10x working stock solution. H. pylori was diluted to a final concentration of 2 x 10^6 CFU/mL in culture medium. Aliquots of 10μL of zerumbone were added to 90μL of H. pylori in a well of the microtitre plate. Concentration of the compound ranged from 31.25 to 250 μg/mL. The microtiter plate was incubated for 3 days in a CO2 incubator. The plate was examined visually and measured using a microplate reader (Varioskan Flash) at 600 nm to determine the lowest concentration showing complete growth inhibition, which was recorded as the MIC. Minimum bactericidal concentration (MBC) as the lowest concentration without growth on a chocolate agar plate supplemented with 7% lysed horse blood. Wells containing H. pylori with 10 μL of 5% DMSO and BHI medium containing 250 μg/mL zerumbone were used as control and blanks respectively. The results were recorded in accordance with the Clinical and Laboratory Standards Institute [29].

Statistical analysis

The statistical differences between groups were determined according to SPSS version 16.0 using ordinary one-way ANOVA followed by Dunnetts multiple comparison tests. Analysis and graphs were prepared with GraphPad Prism version 5.02 for Windows, GraphPad Software, San Diego California USA, www.graphpad.com. All tests were performed at least in triplicates, and the values were represented as mean ± S.E.M (standard error of the mean). A value of P <0.05 was considered significant denoted by (*).

Results

Effect of zerumbone on gastric acid secretion

Intraduodenal administration of zerumbone at doses of 5 and 10 mg/kg b.w and omeprazole at 30 mg/kg to the experimental rats, immediately after pylorus ligature, were significantly (p <0.05) reduced the acid output of the gastric content secreted during a period of 4 h. Table 1 shows the statistical significant differences between treatment groups on gastric acid secretion compared to the control.

Gross evaluation

The results showed that animal pre-treated with zerumbone or omeprazole were considerably reduced ulcer area formation compared to the ulcer control group. Omeprazole at 20mg/kg b.w and zerumbone at doses of 5 and 10 mg/kg b.w were significantly (p <0.05) inhibited ulcer formation by 76.77%, 75.59% and 88.75%, respectively as shown in Table 2. Gross observation showed that zerumbone pretreated groups (Fig. 2D and 2E) or omeprazole group (Fig. 2C) were considerably have less gastric lesions to the gastric mucosa compared to the ulcer control group; where ethanol-induced intense gastric mucosal damage in the form of an elongated band of hemorrhages (Fig. 2B).

Fig 2. Gross evaluation.

Macroscopic appearance of the stomach of experimental rats submitted to ethanol-induced gastric ulcer in different groups showed that the normal control group exhibited an intact stomach without lesion (group A), ulcer control group showed extensive lesions to the gastric mucosa appears as elongated bands of hemorrhage (white arrow) (group B), reference control group pretreated with omeprazole at 20 mg/kg showed mild injuries to the gastric mucosa as opposed to group B (group C). Whereas, rats pretreated with zerumbone at 5 and 10 mg/kg, (group D and E, respectively) showed moderate to slight injuries to the gastric mucosa as opposed to group B (magnification: 1.8 x). (n = 6).

https://doi.org/10.1371/journal.pone.0121060.g002

Gastric mucus content and biochemical analysis

The ulcer control group showed the lowest content of gastric mucus; while zerumbone-pretreated groups were significantly (p <0.05) increased the mucus production compared to the ulcer control group (Table 2). On the other hand, the serum analysis showed that the ulcer control group had increased levels of liver enzymes (AST and ALT). However, zerumbone pretreatment significantly (p <0.05) reduced the elevated such parameters (Table 2).

Histological evaluation

Histological observation to the ulcer control group stained by H&E showed extensive gastric lesions, submucosal edema and leukocytes infiltration (Fig. 3B). However, zerumbone pretreated groups have relatively better protection as seen by decreasing ulcer area, reduction or complete absence of edema and leukocytes infiltration and flattening of mucosal fold was also observed (Fig. 3D and 3E, respectively). Further, the histological evaluation of H &E-stained gastric mucosa in rat pretreated with the studied doses of zerumbone against ethanol ulceration was interpreted as lesion score using image J software for each group in Table 3.

Fig 3. Histological evaluations.

Results showed the histological appearance of the gastric mucosa of the experimental rats in the normal control group displayed normal arrangement of the gastric epithelium and gastric gland in the mucosal region (group A). Histological appearance of the ulcer control group showed extensive disruption to the surface epithelium and hemorrhagic necrosis, penetrating deeply into the gastric mucosa (white arrow), and extensive edema and leukocyte infiltration in the submucosa (black arrow) (group B). However, the rats pretreated with omeprazole at 20 mg/kg (group C) or that pretreated with zerumbone at 5 and 10 mg/kg (group D and E, respectively) showed the improved histological appearance compared to the user control (H & E stain: 20x). (n = 6).

https://doi.org/10.1371/journal.pone.0121060.g003

Pas staining

PAS staining showed that, omeprazole pretreatment at 20 mg/kg and zerumbone pretreatment at 5 and 10 mg/kg were resulted in the expansion of a substantially continuous PAS-positive mucous gel layer that lining the entire gastric mucosal surface observed as magenta colour (Fig. 4C, 4D and 4E, respectively). However, stomachs of animals in the ulcer control group didn’t exhibit this magenta color of PAS stain, indicating the deleterious effect of ethanol on gastric mucus (Fig. 4B). The histological evaluation of PAS-stain was Further, interpreted as positively stained area (μm2) using image J software for each group in Table 4. Thess findings denote the potential effect of zerumbone to conserve the gastric mucus against the deleterious effect of ethanol.

Fig 4. PAS staining.

Results showed that the histological appearance of the gastric mucosa of the normal control group displayed ordinary gastric mucus content as appeared by the faint PAS stain to the mucus cells (group A). In contrast, ulcer control group showed the absence of the PAS stain and the complete depletion of the mucous layer (group B). Omeprazole pretreatment at 20 mg/kg showed intense PAS stain noted as a bright purple color in the mucus cells lining the gastric pits, due to the carbohydrate-rich and viscous mucus they secrete (group C). Zerumbone pretreatment at 5 and 10 mg/kg (group D and E, respectively) have gradually increased in the mucosal secretion of the gastric glands. The black arrow indicates the glycoprotein appear as magenta color (PAS stain: 20x), (n = 6).

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Immunhistochemical

Immunohistochemical staining showed the overexpression of HSP-70 proteins in the gastric tissue of animals pretreated with the study doses of zerumbone appeared by the intense brown color of the positively stained antigen (Fig. 5C and 5D), while the ulcer control group didn’t activate HSP-70 at all (Fig. 5A). The immunoreactivity of HSP70 was interpreted as a positive stained area (μm2) using image J software for each group in Table 5. The results indicates that the HSP-70 expression in zerumbone-pretreated group might be contributed to its observed gastroprotection effect.

Fig 5. Immunohistochemical localization of Hsp-70 protein.

The results showed the insignificant HSP70 expression in the gastric tissue of rat in the ulcer control group (group A). However, microscopic observation detected overexpression of HSP70 protein in the gastric tissue of rats pretreated with omeprazole at 20 mg/kg (group B) and zerumbone at 5 and 10 mg/kg (group C and D, respectively). The antigen site appears as a brown color (IHC stain: 20x), (n = 6).

https://doi.org/10.1371/journal.pone.0121060.g005

Effect of zerumbone on PGE2 synthesis

In this study, as displayed in Fig. 6A, zerumbone at both study doses (5 and 10 mg/kg) was significantly (p < 0.05) still able to preserve a high PGE2 level despite administration of ethanol, when compared to the ulcer control group, where ethanol produced a sharp drop in PGE2 level. This outcome indicates the likely implication of PGE2 in the protective action of zerumbone against ethanol ulceration.

Fig 6. Gastric homogenate contents.

The results showed the effect of zerumbone on gastric tissue homogenate contents of (A) prostaglandin E2 (PGE2), (B) non-protein sulfhydryl compound (NP-SH), (C) glutathione (GSH) and (D) malondialdehyde (MDA) levels. Compared to the ulcer control group, zerumbone pre-treatment significantly (p <0.05) increased the level of PGE2, NP-SH, and GSH. Meanwhile the level of MDA was decreased. (*) Indicate the significant differences between treatment groups at (p <0.05) compared to the ulcer control group. The results are expressed as mean ± SEM, (n = 6) by ordinary one-way ANOVA with Dunnett’s multiple comparison tests using Graph Pad Prism version 5.

https://doi.org/10.1371/journal.pone.0121060.g006

Effect of zerumbone on NP-SH compounds level

The level of the NP-SH was decreased subsequently to the intragastric administration of ethanol without treatment in the ulcer control group. In contrast, zerumbone pretreatment at 10 mg/kg significantly (p <0.05) elevated NP-SH level in the pretreated group compared to the ulcer control group. However, zerumbone at 5 mg/kg produced weaker effects on the increased in NP-SH level (Fig. 6B). The results indicate the probable involvement of NP-SH gastric content in the gastroprotection effect of zerumbone in this study.

Effect of zerumbone on GSH level

The GSH level was significantly decreased subsequently to the intragastric administration of ethanol without treatment in the ulcer control group than all the pre-treated groups. However, zerumbone pretreatment significantly (p <0.05) restored the depleted GSH level compared to the ulcer group (Fig. 6C). The results point to the possible involvement of this endogenous antioxidant in the experimental gastroprotective effect of zerumbone.

Effects of zerumbone on lipid peroxidation

MDA is used as an indicator of lipid peroxidation [23]. The ulcer control group showed the higher MDA level than the other pretreated groups. However, zerumbone pretreatment significantly (p <0.05) decreased gastric MDA level compared to the ulcer group (Fig. 6D). The results showed the efficacy of zerumbone to enhance cellular antioxidant system, evidenced by the reduced level of lipid peroxidation, which may implicate in its gastroprotection action.

In vitro antioxidant evaluation of zerumbone

Given that Zerumbone demonstrated antioxidant activity evidenced by increased GSH level and inhibiting TBARS formation, FRAP assay was performed to evaluate the free radical scavenging activity of the tested compound. Zerumbone exhibited FRAP value of 58.3 ± 2.08 and the result was well compared with ascorbic acid showed 215.5 ± 3.11. Thus, it seems that the antioxidant activity of zerumbone is through the enhancement of the cellular antioxidant pathway.

In vitro anti-Helicobacter pylori activity

In our continuous investigation for the possible mechanisms underlying the observed gastroprotective effect of zerumbone in this study, the microtiter dilution method was performed to examine the antibacterial action of zerumbone against H. pylori. Zerumbone represents respective MIC value of 250 μg/ml against two H. pylori strains; H. pylori NCTC11637 and H. pylori J99.

Discussion

In this study, zerumbone from Zingiber zerumbet demonstrated gastroprotective efficacy against ethanol ulcer model in rats. The pretreatment by the intragastric administration of zerumbone at 5 and 10 mg/kg was efficiently protected the gastric mucosa from the damaging effect of ethanol in a dose dependent manner and the following discussion is to identify the possible mechanism(s) involved.

It's well know that peptic ulcer disease arises from the imbalance between the mucosal protective factors and the aggressive factors [30] and many gastroprotective agents were found to improve the cellular integrated work to increase mucosal resistance or to decrease the aggressive factors [31]. Nowadays, the therapeutic strategy for peptic ulcer treatment is focused on either the suppression of gastric acid secretion or the enhancement of gastro-protective factors [32]. Thus, we assessed the effect of zerumbone on in vivo gastric acid secretion against the pylorus ligature model in rats. The model of pylorus ligature is charackterized by producing a surge in the gastric acid secretion and stasis of acid. Therefore, the pylorus ligature model is an ideal and common method used to investigate the possible alterations in the gastric acid parameters. [33]. In the current search, the intradudenal administration of zerumbone was found to reduce the volume and total acidity of gastric juice induced by the pylorus ligature. These alterations might probably be due to the antisecretory activity of zerumbone.

Ethanol ulcer model is the widely used in vivo experiment to assess the gastroprotective activity of different agents from botanical resources [5]. It was found that ethanol-induced gastric injuries by direct and indirect toxic effect through different pathophysiological pathways [34]. Experimentally, the intragastric administration of ethanol was recognized to produce elongated bands of hemorrhagic, extended submucosal edema, mucosal crumbliness, inflammatory cells infiltration and epithelial cell loss [35]. In our study, zerumbone was administered orally into rats and this pretreatment protect the gastric mucosa from the deleterious effect of ethanol in a dose-dependent manner and the results were well confirmed by the light microscopy of the histopathological examination.

It was evidenced that reactive oxygen species (ROS)are implicated in the pathogenesis of ethanol-induced gastric mucosal injury [35]. ROS have an essential physiological role in the cellular homeostasis. ROS are unpaired molecules generated as a normal products during the mitochondrial respiration and from the peroxisomes to catalyze different redox reactions within the living organisms. In some cases, ROS production is increasing for a defensive purpose in response to certain external stimuli, harmful diet and human disease [36]. Normally these produced ROS are neutralized by endogenous antioxidant cellular system such as glutathione (GSH) and superoxide dismutase (SOD). However, oxidative stress status will occur when ROS production accumulated and exceeded over the cellular antioxidant system or when the defensive system is not functioning well to neutralize those oxidants [37]. Consequently, oxidative stress can cause lipid peroxidation, cellular death and tissue damage [38]. Oxidative stress was recognized as one of the major pathogenic causes, which concerned with the induction and the aggravation of gastric ulcer [39]. It was established that oxidative stress and diminished of endogenous antioxidant molecules are implicated in the direct and immediate deleterious effect of ethanol-induced gastric mucosal damage [40]. Non-protein sulphydryl compounds (NP-SH) are one of the most important protective factors against oxidative stress induced gastric ulcer by detracting the deliberated ROS in different experimental models such as in the ethanol gastric ulcer model [40]. Thus, high gastric content of NP-SH was found to protect gastric mucosa from the deleterious effect of ethanol [41]. Previous studies showed that ethanol administration is accompanied by a drop of endogenous sulfhydryl compounds, particularly and the most importantly glutathione (GSH) [42]. Subsequently, the low gastric GSH level increased the rate of lipid peroxidation, which mediate gastric tissue damage [40]. Malondialdehyde (MDA) is the main end product of lipid peroxidation. Thus, measurement of gastric MDA level can estimate indirectly the level of lipid peroxidation [43]. Thus, considering the role of NP-SH and GSH as an endogenous antioxidants providing a cellular protection against oxidative damage and the significance of MDA as a lipid peroxidation marker, we evaluated the effect of zerumbone on NP-SH, GSH and MDA level in gastric tissue homogenate. The results showed that zerumbone was significantly restored the depleted NP-SH and GSH level and decreased MDA level due to ethanol administration, as opposed to the ulcer control group. These findings suggest the possible efficacy of zerumbone to enhance cellular antioxidant system, which may consider one of its gastroprotective pathways.

It is well known the essential role of heat shock protein (HSPs) as gastroprotective factors against various stimuli [44]. HSPs are functioning as stressor proteins; HSP70s in particular are the chief stressor proteins expressed to confirm cellular protection as they refold or getting rid of the damaged proteins [45]. They up-regulated in response to various internal or external stimuli such as oxidative stress among others. Many pure natural compounds were reported to demonstrate cytoprotection effect against oxidative damage due to their activities as HSPs inducers [46]. Evidences showed that many nontoxic HSP-inducers are beneficial to provide cellular protection against gastric ulcer [47]. In our study, zerumbone pretreatment significantly induced HSP70 expression in the ulcerated gastric tissue of the experimental animals, indicating the possible participation of HSP70 in the observed gastroprotection effect of zerumbone.

For more investigation, we assessed the antioxidant activity of zerumbone compound by it is in vitro effect in FRAP assay. The FRAP assay was commonly used to evaluate the antioxidant activity of different medicinal agents [48, 49]. Practically, the antioxidant compound served as a reducing agent by donating a hydrogen atom. Thus, the reducing capacity of an agent is a remarkable indicator of its antioxidant power [50, 51]. In the current study, zerumbone showed insignificantly reducing activity. Thus it could be hypothesized that zerumbone might promote gastroprotection activity possibly through indirect and cellular antioxidant pathway.

It is well known the fundamental role of prostaglandins (PGs), particularly PGE 2 and PGI 2, in modulating the integrity of gastric mucosal layers and a variety of cytoprotective factors [52, 53]. PGs plays a significant part in resisting gastric mucosal injury as they control gastric acid secretion, enhances gastric mucus and bicarbonate production [54], increases mucosal blood flow and prohibits the diffusion of ulcerative agents into the gastric mucosa [55]. Earlier studies demonstrated the gastroprotective action of PG against ethanol-induced gastric damage [56]. So, as to investigate if this defensive factor is concern in the gastroprotection promoted by zerumbone, PGE 2 enzyme immune assay was performed. The results showed that zerumbone significantly preserved the gastric mucosal content of PGE 2, indicating the possible involvement of PGs in zerumbone- mediate gastroprotection against ethanol ulceration.

The stomach is always exposed to harmful endogenous and exogenous substances. Therefore, it possesses many defenses mechanisms to protect itself from damaging and extensive injury [57]. One of these gastroprotective mechanisms, and the most importantly, the gastric mucus layer, which is served as the first defensive line and physical barrier against the caustic effect of gastric acid secretion due to it is viscous, elastic, adherent and transparent characteristics [58]. In addition, gastric mucus has scavenging activity against ROS, thus provide antioxidant protection to the whole gastrointestinal tract [59]. It was reported earlier that ethanol ulcer model is associated with diminished of the mucus barrier and bicarbonate secretion [60]. Practically, periodic acid-schiff (PAS) staining is the widely used histochemical procedure to investigate the presence of glycoproteins. The periodic acid oxidizes the diol functional groups in the mucus, result in the formation of aldehydes, which in turn react with the schiff base reagent which precipitate a purple-magenta color [61]. In our work, we found that the administration of ethanol in rats diminished the gastric mucus layer. Nevertheless, pretreatment with zerumbone was significantly prevented a decrease in mucus production, as established by the observed intense magenta color, which signify the defensive effect of zerumbone on gastric mucus layer. These results demonstrated the essential role of mucus as a defensive factor in the observed gastroprotection promoted by zerumbone. Taken into account the stimulatory effect of NP-SH [62] and PGE 2 [54] on character and gastric mucus synthesis, we hypothesized that those factors may be implicated and elucidated the positive impact of zerumbone on maintaining gastric mucus production.

H. pylori is the most prevalent bacterial infection, affecting approximately 50% of the population around the world. H. pylori is widely considered as the major causative factors in the pathogenesis of peptic ulcer disease [63]. The bacterium is gram-negative pathogen, characterized by the flagellated and spiral-shaped, can colonize in the gastric mucus layer and adhere to the epithelium, where it obtained its supplements. The microscopic examination on the infected individuals showed that H. pylori generated gastric mucosal damage what is most distinguished by the infiltration of chronic inflammatory cells [64]. The current therapeutic regimen for H. pylori treatment consists of triple medicines; two antibiotics (amoxicillin and clarithromycin or metronidazole) plus a proton pump inhibitor or bismuth. Although this protocol verifies high efficacy in H. pylori eradication, it demonstrates some obstacles such as antimicrobial resistance and the less convenience usage to the patients [65]. For these reasons, there is increasing need to explore new antimicrobial agents with high efficacy to overcome the mentioned drawbacks of the current regimen. Many researches were conducted to discover potent antimicrobial agents against

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