Abstract
Organosulfur compounds (OSCs) are a group of small molecules commonly present in Allium vegetables, such as garlic, onions chives, and shallots that have garnered scientific interest for their noted health benefits. OSCs have been evaluated for their potential to prevent or treat major diseases including cancer. Epidemiological evidence of inverse association between increased intake of Allium vegetables and cancer risk is now substantiated by animal studies wherein true causal relationships between OSCs and cancer prevention have been found. This chapter summarizes the chemistry, metabolism, and bioavailability of commonly studied OSCs and the latest developments regarding their anticarcinogenic effects in cell culture and animal models. Data pertinent to clinical trials assessing safety and anticancer efficacy of OSCs are also discussed.
Keywords
- Prostate Cancer Cell
- Proliferate Cell Nuclear Antigen
- Reactive Oxygen Species Generation
- Aberrant Crypt Focus
- Aristolochic Acid
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.
1 Introduction
Beneficial properties of Allium vegetables, especially garlic, have been recognized for thousands of years. It has been suggested that garlic can enhance stamina, physical strength, and increase longevity, in addition to functioning as an analgesic, antimicrobial, and antiseptic (Bianchini and Vainio 2001). Together with other types of Allium vegetables such as onions, shallots, leeks, and chives, garlic is one of the most commonly consumed foods. Each garlic clove weighs roughly 2–4 g and the average intake varies considerably from individual to individual. In the United States, the average intake of garlic has been estimated to be 0.6 g per week or less, while in other countries, e.g., China, the intake may be close to 20 g or more (reviewed by Milner 2004).
Published data provides undeniable evidence for health-promoting effects of Allium vegetables and their constituents. It has been well documented that Allium vegetables and their constituents can reduce the risk of major chronic diseases, including cardiovascular diseases, diabetes, and cancer, in addition to their immunomodulatory benefits, protection against infections, and anti-aging effects (Agarwal 1996; Milner 2001; Rahman 2001; Powolny et al. 2011). Studies focusing on garlic and its OSCs in the past three decades have provided a substantial body of evidence demonstrating their ability to prevent or treat various diseases. Several investigations focusing on pharmacological effects of Allium vegetables and related OSCs have documented an inverse association with disease development. The anticancer properties of Allium vegetables are well supported by epidemiological studies (Shukla and Kalra 2007). Even though the mechanistic details associated with health benefits of OSCs remain partially unknown, the usage of garlic and its products continues to grow. This chapter first introduces the constituents of garlic, including metabolites produced after disruption of its cellular integrity, their molecular mechanisms of anticancer action, and preclinical and clinical evidence for their safety and efficacy. The chapter then concludes with summary and future directions.
2 Chemistry and Metabolism
The primary sulfur-containing compounds in intact garlic are γ-glutamyl-S-alk(en)yl-l-cysteines that are hydrolyzed or oxidized to S-allylcysteine (SAC) and S-alk(en)yl-l-cysteine sulfoxide(alliin) (Fig. 1a). When garlic is processed by crushing or chewing, compounds in the intact garlic are converted into several volatile OSCs within a short period of time. Allinase, a key vacuolar enzyme, is responsible for the conversion of alliin into allicin. The transiently formed and highly unstable compound, allicin (Fig. 1b) is converted into various OSCs including diallyl sulfide (DAS), diallyl disulfide (DADS), diallyl trisulfide (DATS), ajoene, and dithiins (Higdon 2007; Block et al. 1984; Block 1992; Amagase et al. (review) 2001).
3 Pharmacological Attributes of OSCs in Relation to Anticancer Effects
3.1 Induction of Apoptosis
The process of apoptosis is highly dysregulated in cancer cells contributing to abnormal cell growth, thus leading to increased tumor burden. OSCs have the potential of inducing apoptosis and contributing to the growth suppression of cancer cells. Sundaram and Milner (1996a, b) showed induction of apoptosis by DADS in colon cancer cells. Further studies focused on elucidation of the mechanistic details of apoptosis induction by OSCs have revealed the involvement of Bcl-2 class proteins. For example, apoptosis induction by DATS was more pronounced in prostate cancer cells PC-3 and DU-145 compared to DAS and DADS and that the induction of apoptosis was correlated with a decrease in Bcl-2 levels and reduced Bcl2:Bax interaction activating the mitochondria-mediated intrinsic pathway (Xiao et al. 2004). In the same study, it was also identified that DATS-induced hyperphosphorylation of Bcl-2 was mediated in part by c-Jun N-terminal kinases (JNK) and ERK1/2. Subsequent studies from the same group showed that DATS induces apoptosis in LNCaP prostate cancer cells by increasing Bak protein levels and decreasing Bcl-2 and Bcl-xL protein levels (Kim et al. 2007). However, ectopic expression of Bcl-2 conferred protection against DATS-induced apoptosis only in PC-3 cells and not LNCaP cells (Xiao et al. 2004; Kim et al. 2007). It was reasoned that the genotypic differences between these cells, including their p53 status and androgen responsiveness, were responsible for the observed differential effect. Similarly, in other cancer models such as lung cancer, neuroblastoma, breast cancer, and skin cancer, OSCs were shown to increase the ratio of Bax/Bcl-2, upregulating Bax protein levels and decreasing the levels of Bcl-2 and Bcl-xL proteins (Hong et al. 2000; Karmakar et al. 2007; Nakagawa et al. 2001; Li et al. 2002a, b; Wang et al. 2010, 2012a, b).
It is interesting to point out that OSCs have little or no effect on normal cells, although the mechanism underlying their selectivity for cancer cells is not completely understood. For example, the PrEC normal prostate epithelial cells were more resistant to DATS-induced apoptosis than prostate cancer cells (Kim et al. 2007). Similarly, DAS or DADS administration induced apoptosis in SH-SY5Y neuroblastoma cells and did not impact the viability of primary neurons (Karmakar et al. 2007). In a different study, ajoene caused apoptotic cell death in human leukemia cells but had no effect on normal peripheral mononuclear blood cells (Dirsch et al. 1998).
3.2 Modulation of Carcinogen Metabolism
Multiple studies indicate that chemopreventive effect of OSCs is at least in part due to their ability to inhibit the activation of carcinogens and/or increase detoxification of the activated metabolites. N-nitrosodimethylamine (NDMA) is a by-product of industrial processes, while 4-(methylnitrosamino)1-(3-pyridyl)-1-butanone (NKK) is a carcinogen found in tobacco smoke. Their toxicity and carcinogenicity is dependent upon activation by the Phase 1 drug-metabolizing enzyme, CYP 2E1. Early studies utilizing in vitro cell culture and in vivo animal models revealed that OSC treatment prevented cytotoxicity and tumor formation induced by NDMA and NKK (Hong et al. 1992). Furthermore, it was suggested that OSCs inhibit P450 2E1 by both competitive inhibition and suicide inactivation (Brady et al. 1991). OSCs may also protect against acetaminophen toxicity in mice via inhibition of CYP2E1 (Wang et al. 1996). More recent evidence confirmed that oral OSC treatment at 100–400 mg/kg depressed CYP2E1 activity in male Sprague–Dawley rats in a dose-dependent manner. This correlated with statistically significant reductions in serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activity following treatment with the hepatotoxicant thioacetamide (Kim et al. 2014), indicating a reduction in liver damage.
Experimental evidence suggests that OSCs may also enhance the detoxification of carcinogens via induction of Phase 2 drug-metabolizing enzymes. One recent study confirmed that oil-soluble garlic compounds activate metabolizing enzymes that detoxify carcinogens. This resulted in a reduction of the development of mammary cancer in animals and suppression of growth of human breast cancer cells in culture (Tsubura et al. 2011). Earlier studies demonstrated the ability of OSCs to prevent benzo(a)pyrene (BP)-induced for stomach tumorigenesis in mice. The authors attributed this to increased expression of NAD(P)H:quinone oxidoreductase (NQO), an enzyme implicated in the detoxification of activated quinone metabolites of BP (Singh et al. 1998). Munday and Munday (2001) documented increases in the activity of the Phase II enzymes NQO and glutathione-S-transferase (GST) in rat tissues following oral doses of both DADS and DATS. OSCs enhanced glutathione content of intestinal mucosa and liver in irradiated Swiss albino mice (Chittezhath and Kuttan 2006). Oil-soluble OSCs induced GST phosphorylation via activation of c-Jun NH2-terminal kinase (JNK) in neuroblastoma cells (Filomeni et al. 2003). Studies have confirmed activation of GST by JNK in normal rat liver cells (Tsai et al. 2011).
In summary, OSCs act as a double-edged sword in chemoprevention by inhibiting carcinogen activation by Phase 1 enzymes while simultaneously enhancing detoxification of activated carcinogenic intermediates via induction of Phase 2 enzymes (Herman-Antosiewicz et al. 2007).
3.3 Inhibition of Cell Cycle Progression
Numerous studies have demonstrated the antiproliferative effects of DATS in a variety of cancer cell types, including liver, gastric, colon, prostate, lung, bladder, and skin cancer cells. The DATS appear to induce cell cycle arrest in the G2/M phase; however, the mechanism by which this occurs may be cell-type specific (for review see Antony and Singh 2011; Yi and Su 2013). In colon cancer cells, DADS-induced G2/M phase arrest was associated with hyperphosphorylation and decreased expression of cell division cycle 25C phosphatase (Cdc25C), inhibition of cdc2 kinase activation, and decreased formation of cdc2/cyclin B1 complex formation (Knowles and Milner 2000). Similarly, Xiao et al. (2005) demonstrated destruction and hyperphosphorylation of Cdc25C and inhibition of cdc2/cyclin B1 kinase activity in prostate cancer cells upon treatment with DATS. This effect was dependent upon generation of reactive oxygen species (ROS). Wang et al. (2010) demonstrated increased ROS generation in DATS-treated skin cancer cells. ROS production associated with G2/M arrest was also seen in neuroblastoma (Filomeni et al. 2003), colon (Song et al. 2009), and lung (Wu et al. 2009) cancer cells.
A role for checkpoint 1 kinase (Chk1) in OSC-mediated cell cycle arrest was also proposed. Chk1 is normally activated in response to DNA damage and results in cell cycle arrest prior to DNA repair or apoptosis. It has been found that SATS-induced mitotic arrest is dependent upon activation of Chk1 in prostate cancer cells and gastric cancer cells (Herman-Antosiewicz and Singh 2005; Ling et al. 2010).
Cell cycle arrest in liver tumor cells was associated with decreased cyclin-dependent kinase 7 protein levels and increased cyclin B1 protein levels (Wu et al. 2004). OSC-induced mitotic arrest in human leukemic cells was attributed to activation and nuclear translocation of nuclear factor-κB (NF-κB) followed by NF-κB binding to the promoter region of cyclin-dependent kinase inhibitor 1 (p21) (Dasgupta and Bandyopadhyay 2013). OSC induced upregulation of p21 was also demonstrated in a study utilizing colon cancer cells (Liao et al. 2009). Multiple studies have associated mitogen-activated protein kinase (MAPK) activation with G2/M arrest. DADS have been shown to trigger cell cycle arrest in both colon cancer (Knowles and Milner 2003) and lung carcinoma cells (Hui et al. 2008). In both studies, this was associated with increased activity of extracellular signal-regulated kinase (ERK). In contrast, DADS-mediated arrest in gastric tumor cells was associated with upregulation of p38 MAPK (Yuan et al. 2004). Finally, several studies have identified OSC binding sites on components of tumor cell cytoskeleton and correlated morphological changes to the cytoskeleton with G2/M arrest (Hosono et al. 2005; Xiao et al. 2005; Aquilano et al. 2010).
Cell cycle arrest, if sustained, provides a powerful preventative mechanism to the growth of tumors both in vitro and in vivo. In some of the studies cited above, cell cycle arrest was transient, but was then followed by apoptosis (Xiao et al. 2005; Song et al. 2009; Aquilano et al. 2010; Dasgupta and Bandyopadhyay 2013). Regardless of the mechanism by which it occurs, cell cycle arrest appears to be a common pharmacological effect of OSCs in cancer cells.
3.4 Induction of Reactive Oxygen Species
Accumulating evidence suggests a role for reactive oxygen species (ROS) in cancer cell apoptosis by OSCs. DADS-induced apoptosis in SH-SY5Y neuroblastoma cells was associated with ROS generation (Karmakar et al. 2007). Likewise, ajoene-induced apoptosis in human promyelocleukemic cells was linked to ROS and activation of NF-κB (Dirsch et al. 1998). Studies involving prostate cancer cells (Kim et al. 2007), basal cell carcinoma (Wang et al. 2012a, b), MCF-7 breast cancer cells (Na et al. 2012), and leukemia cells (Choi and Park 2012) also reported that DATS-induced apoptosis was mediated through ROS generation.
The precise mechanism by which DATS causes ROS generation is not fully understood, but some advances in this context are worthy of discussion. For example, apoptosis induction and cell cycle arrest by DADS and DATS were shown to be mediated through increased intracellular calcium (Park et al. 2002; Wang et al. 2010). Cell cycle arrest and apoptosis induction by DATS in prostate cancer cells was shown to be mediated by ROS, generated through increase in labile iron due to proteasomal degradation of ferritin (Antosiewicz et al. 2006). Wang et al. (2010) showed that DATS-dependent calcium increase in basal cell carcinoma (BCC) cells was accompanied by intracellular ROS generation. The ROS generation by OSCs was also linked with the generation of hydrogen sulfide (H2S) where it was shown that OSCs can be converted to H2S in human red blood cells (Benavides et al. 2007). More recently, it was shown that DATS-induced H2S is associated with the generation of ROS and activation of mitochondria-mediated apoptosis pathway in human breast cancer MCF-7 cells (Na et al. 2012). Based on these studies, it is evident that OSC-dependent ROS generation is crucial for their anticancer effects.
3.5 Inhibition of Angiogenesis and Cell Invasion
Inhibition of angiogenesis, which is required for tumor growth, is another major effect of some OSCs. Mousa and colleagues demonstrated the anti-angiogenic potential of alliin as it inhibited the tube formation, dependent on fibroblast growth factor-2 and vascular endothelial growth factor (VEGF) both in human endothelial cells and in a chick chorioallantoic membrane assay (Mousa and Mousa 2005). In the same study, the authors also showed that the anti-angiogenic potential of alliin, which was in part mediated by increase in cellular nitric oxide and p53 protein expression, increased in the presence of vitamin C and vitamin E. The DATS-associated anti-angiogenic properties were examined in human umbilical vein endothelial cells (HUVEC). DATS was shown to inhibit formation of capillary-like tube structure and migration by HUVECs. Mechanistic details revealed suppression of VEGF secretion, downregulation of VEGF receptor-2 protein level, and inactivation of Akt upon treatment with DATS (Xiao et al. 2006a, b).
The effect of OSCs on migration and invasion of cancer cells has also been studied. Ajoene administration inhibited B16/BL6 melanoma cell adhesion to LEC1 cells in vitro and also significantly inhibited lung metastasis in C57BL/6 mice injected with B16/Bl6 melanoma cells (Taylor et al. 2006). Similarly, DADS treatment resulted in the inhibition of HUVEC cell proliferation and activity of matrix metalloprotease-2 (MMP-2) and MMP-9 (Meyer et al. 2004). The DATS administration in a transgenic mouse model of prostate cancer (Transgenic Adenocarcinoma of Mouse Prostate; TRAMP) not only prevented the development of poorly differentiated prostate cancer but also inhibited pulmonary metastasis multiplicity (Singh et al. 2008). In a study using osteosarcoma cells, DATS was shown to exhibit antitumor activity by targeting Notch1 signaling and inhibiting cell invasion and angiogenesis partly through downregulation of VEGF, MMP-2, and MMP-9 (Li et al. 2013). DAS, DADS, and DATS were shown to inhibit migration, invasion, and angiogenesis. For example, exposure of human colon cancer Colo 205 cells to all three OSCs resulted in inhibition of PI3K, Ras, MEKK3, MKK7, ERK1/2, JNK1/2, and p38 which correlated with the inhibition of MMP-2, -7, and -9, essential for cell migration and invasion (Lai et al. 2013). DATS was also shown to inhibit mRNA and protein levels of VEGF and MMP-2, -7, and -9 in human colon cancer HT29 cells (Lai et al. 2015). In addition to reducing MMP levels, inhibitory effects of DADS in Colo 205 cells and LNCaP cells were also found to be associated with reduced levels of proteins associated with tight junctions functionality [Lai et al. (2013), Shin et al. (2010), for review see Yi and Su (2013)].
3.6 Immunomodulation
Immunomodulatory effects of garlic and its OSCs have been reported (for a review, see Schafer and Kaschula 2014). Studies have identified that garlic and its constituents can effectively strengthen the host immune system within the tumor against the immunosuppressive activity of an emerging tumor (Schafer and Kaschula 2014). Aged garlic extract along with an anticancer agent suppressed tumor growth of sarcoma-180 and Lewis Lung carcinoma LL/2 cells injected in mice by inducing cellular immune response through the activation of NK cells and cytotoxic T cells (Kyo et al. 1998). In addition, aged garlic extract was also found to stimulate lymphocyte proliferation, macrophages phagocytosis, and lymphocyte infiltration into tumors and enhanced NK cell number and activity (Schafer and Kaschula 2014). A recent study showed that intra-tumor inoculation of a protein fraction of fresh garlic bulbs was more efficient than garlic extract in infiltrating the tumor with CD8+ T cells (Ebrahimi et al. 2013). DADS downregulated the levels of CCL-2 (an important chemokine which favors tumor cell migration and expansion) induced by TNF-α in MDA-MB 231 breast cancer cells (Bauer et al. 2014). It was reasoned that CCL-2 release by breast cancer cells may be regulated by pro-inflammatory cytokines through NF-κB or ERK.
Even though studies have identified conflicting evidence (for a review, see Schafer and Kaschula 2014) with the immunomodulation properties of OSCs, it is noteworthy to mention that immunomodulatory effects of OSCs may contribute to their overall anticancer activity.
3.7 Modulation of Histone Acetylation
Balance between histone acetylation and deacetylation is crucial for regulation of gene expression essential for normal cellular processes. However, this balance is often lost in cancer cells, favoring their uncontrolled growth and progression. Discovery of agents that can either block the activity of histone deacetylases (HDACs) or promote the histone acetylation activity is crucial in combating cancer development. There are examples of clinical success of this approach (vorinostat). Studies have shown that garlic and its compounds have the potential of both increasing histone acetylation and inhibiting HDAC activity (for reviews, see Druense-Pecollo and Latino-Martel 2011; Yi and Su 2013). For example, DADS caused an increase in the acetylation of histones H3 and H4 in DS19 and K562 human leukemic cells. Acetylation was also induced in rat hepatoma and human breast cancer cells by DADS and its metabolite, allylmercaptan. Moreover, in the same study it was shown that allylmercaptan was more potent in inhibiting HDAC compared to DADS (Lea et al. 1999). The induction of histone acetylation by S-allylmercaptocysteine (SAMC), allicin, and DADS in various cancer models was also shown (Lea and Randolph 2001; Lea et al. 2001). DADS was also shown to promote cellular accumulation in G2/M phase by decreasing HDAC activity and increasing histone H3 and H4 acetylation, thus causing an increase in p21 mRNA and protein levels in colon cancer CaCo-2 and HT29 cells (Druesne et al. 2004). A recent study showed that DATS-induced acetylation of histones H3 and H4 and inhibition of HDAC activity contributed to the inhibition of glioblastoma xenograft growth (Wallace et al. 2013).
4 Preclinical In Vivo Evidence for Chemopreventive Effects of OSCs
A wealth of literature exists characterizing effects of Allium-derived OSCs on prevention of cancer initiation and promotion. The primary compounds investigated include the oil-soluble DATS, DADS, DAS, and ajoene, and the major water-soluble component SAC. With the exception of ajoene, these compounds have received roughly equal attention in the literature.
4.1 DATS
Although other OSCs have received significant attention for their chemopreventive ability against carcinogens, the majority of preclinical studies with DATS used rodents that either received a cancerous cell implant or were genetically susceptible to cancer. The major cancers investigated with DATS include lung, colon, liver, prostate, skin, and breast, with a predominance of prostate cancer studies. These studies were initiated in 2005 (Table 1).
Female BALB/c nude mice with human lung adenocarcinoma cell (A549) xenografts showed significantly retarded tumor growth with no apparent side effects when given a DATS oral gavage of 0.6 nmol every other day for 30 days (Li et al. 2012). Singh et al. (2008) studied the ability of oral DATS (1 and 2 mg/day, thrice weekly for 13 weeks) to inhibit lung metastasis from prostate cancer in TRAMP mice. DATS administration did not reduce the incidence of metastasis, but multiplicity was lower in the treatment group compared with control.
Female nude mice subcutaneously implanted with human colon cancer cells (HCT-15) exhibited a marked reduction in tumor volume relative to control after 25 days of DATS treatment at 6 mg/kg IV every 3 days initiated 7 days after xenograft implantation (Hosono et al. 2005). Statistical significance was not reported, but average tumor volume was less in the treatment group versus control mice. Wu et al. (2011) found that female BALB/c mice with mouse colon carcinoma cell (CT-26) allografts had significantly reduced tumor volumes and weights when administered DATS at 50 mg/kg by oral gavage every 4 days starting 4 weeks prior to cell inoculation.
In a unique study by Zhang et al. (2007), polybutylcyanoacrylate nanoparticles containing DATS were tested for 2 weeks of treatment against orthotopically transplanted HepG2 liver cancer cells. The subcutaneously grown tumors were subsequently implanted under the envelope of the liver in BALB/c nude mice. IV DATS or DATS-filled nanoparticles were injected every other day for 14 days at 1.5 mg/kg. The DATS nanoparticles were markedly more liver targeted than DATS alone, which showed predominantly renal localization. Both formulations showed significant spleen distribution. Moreover, DATS nanoparticles retarded growth of liver tumors more than DATS with no weight loss. To determine a molecular basis for the result, PCNA and Bcl-2 proteins were assessed. Both were downregulated in the tumors from DATS nanoparticle-treated mice compared with control tumors.
In preclinical prostate cancer studies on DATS, investigators mostly employed the TRAMP model or grown xenografts with PC-3 cell implantation. Studies with TRAMP mice focused on changes in protein expression of cell growth and apoptosis related proteins, while the follow-up ones directly assessed effectiveness against tumor growth. Kim et al. (2011) found that DATS given at 2 mg/day thrice weekly for 13 weeks caused significant downregulation of XIAP while inducing survivin protein in TRAMP mice. Ectopic expression of XIAP partially reversed DATS-induced apoptosis in prostate cancer cells, strengthening the contention that DATS-induced XIAP downregulation is important for induction of apoptosis. Stan and Singh (2009) demonstrated that the same dose and schedule of DATS suppressed androgen receptor (AR) protein expression in poorly differentiated prostate cancer in TRAMP mice. Therefore, there is evidence indicating that DATS can both trigger apoptosis and attenuate prostate cancer-promoting receptor expression.
Oral gavage of DATS in TRAMP mice at either 1 or 2 mg/day, three times a week for 13 weeks, significantly inhibited progression to poorly differentiated prostate cancer, and showed a trend toward a reduction in prostate weight, albeit not significant (Singh et al. 2008). Dorsolateral prostate showed reduced cell proliferation from DATS treatment and induction of cyclinB1 and securin protein. DATS did not increase apoptosis or affect angiogenesis. In Chandra-Kuntal and Singh (2010), mice from the same study were further assessed. The reduction in poorly differentiated prostate cancer cells from the 2 mg DATS treatment group in Singh et al. (2008) correlated with a decrease in phosphorylated STAT3, an oncogenic protein. However, forced expression of STAT3 in prostate cancer cells did not attenuate the response to DATS, indicating that inhibition of STAT3 activation is not required for the preventive effect of DATS.
Xiao et al. 2006a, b found that oral DATS at 6 μmol thrice weekly significantly slowed tumor growth in male nude mice subcutaneously implanted with PC-3 cells. Tumors were 2/3 smaller in treated mice in 20 days vs. untreated mice. DATS caused more apoptotic bodies and increased expression of Bax and Bak (pro-apoptotic proteins). However, DATS did not inhibit angiogenesis, and was apparently not toxic, as demonstrated by lack of weight loss. Shankar et al. (2008) approached PC-3 cell implantation differently by implanting directly into the prostate rather than studying the cells’ subcutaneous growth. They found that oral DATS given every day 5 days a week at 40 mg/kg inhibited growth of the prostate cancer implant vs. control. When DATS was combined with TRAIL (tumor necrosis factor-related apoptosis-inducing ligand), the combination was even more effective at growth inhibition than DATS. TRAIL-R1/DR4 and TRAIL-R2/DR5 protein induction, caspase-8 activation, and apoptosis induction were also greater with the combination than either agent alone. DATS also inhibited angiogenesis and metastasis-related protein expression and Akt and NF-κB activation, but again did so more strongly when combined with TRAIL (Shankar et al. 2008).
The two-stage murine model of skin cancer with DMBA (dimethylbenz[a]anthracene) as the initiator and TPA (tetradecanoylphorbol-13-acetate) as the promoter was employed to test the cancer protective effect of DATS (Shrotriya et al. 2010). Topical DATS at 25 μmol 30 min before TPA application was effective in suppressing TPA-induced COX-2 expression. COX-2 suppression appeared to have been caused by a DATS-triggered reduction in DNA binding of activator protein 1 (AP-1), a transcription factor for COX-2. DATS also reduced JNK and Akt activation, and moreover, reduced the incidence and multiplicity of skin papillomas. COX-2 has been linked with skin cancer through its inflammatory role in the body, thus providing a mechanistic explanation for the reduced papilloma formation.
Na et al. (2012) employed BALB/c mice implanted with human breast cancer cells (MCF-7) directly into the thoracic area and promoted with estradiol-releasing implanted devices. Oral DATS at 5 μmol/kg twice weekly for 1 month inhibited xenograft growth.
Cell studies indicated that DATS induced apoptosis that was dependent on JNK. DATS increased DNA binding of AP-1. This may appear to be in contrast to the skin cancer study by Shrotriya et al. (2010) with respect to AP-1 effects; however, in Shrotriya et al. (2010), DATS’s effects on AP-1 DNA binding were assessed in the context of TPA-promoted skin cancer, whereas in Na et al. (2012) DATS’s effects on AP-1 DNA binding were assessed in the presence of estradiol-promoted breast cancer. Thus, DATS effects on AP-1 are likely context/environment dependent.
4.2 DADS
DADS have been assessed in treatment or prevention of cancers of the skin, mammary tissue, liver, colon, kidney, and stomach, in addition to leukemia. Earlier studies generally employed cancer initiators and promoters (two-stage model), whereas later studies relied heavily on xenografts of various cancer cell types (Table 2).
Dwivedi et al. (1992) employed the SENCAR mice, which are sensitive to two-stage skin cancer induction. Topical DADS (1 mg) 30 min prior to DMBA initiation and 30 min before each recurrent TPA administration inhibited skin papilloma formation from the 9th week of promotion. Survival of mice was also increased after DADS treatment. The mechanism of chemoprevention by DADS in this study is unclear, but authors postulated the modulation of bioactivation of DMBA by DADS.
Ip et al. (1992) assessed effects of DADS against DMBA-induced mammary tumors in rats. Oral DADS at 1.8 mmol/kg given 96, 48, and 24 h before intragastric DMBA (10 mg) reduced mammary tumor incidence by 61 % and total tumor yield by 56 %. DAS also significantly reduced both incidence and yield, but was weaker than DADS. Suzui et al. (1997) tested DADS against 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine-induced mammary tumors in rats. DADS at 200 ppm in the diet significantly reduced the total number of mammary tumors per rat by 63 % and trended toward a reduction in tumor incidence.
Sundaram and Milner (1996a, b) used a human colon tumor cell line, HCT-15, to study anticancer effects of DADS. Intraperitoneal or intragastric DADS treatment (1 mg thrice weekly) reduced tumor volume without apparent toxicity. The intraperitoneal route was more effective (69 % reduction in volume) than the later. DADS was as effective as 5-fluorouracil (5-FU), a well-established antineoplastic drug. Interestingly, concomitant DADS and 5-FU was no more effective than either agent alone, but DADS reduced 5-FU toxicity. This finding indicated that DADS might inhibit the pharmacological action of 5-FU or perhaps affect its bioavailability. Liao et al. (2007) tested efficacy against SW480 colon cancer cells implanted subcutaneously in BALB/C nude mice and found that intraperitoneal DADS (30 mg/kg) inhibited tumor growth and reduced proliferating cell nuclear antigen (PCNA) expression.
Xiang et al. (2005) implanted human gastric cancer cells, MGC803, subcutaneously into nude BALB/c mice. Three different doses of DADS, 50, 100, and 200 mg/kg intraperitoneally thrice weekly reduced tumor weight compared to control by 27.8 %, 66.1 %, and 73 %, respectively. This dose–response profile may indicate that a ceiling effect will be exhibited by DADS at least with respect to this tumor type. Subsequently, Tang et al. (2013) used coadministration of DADS and a microRNA (miR-200b and miR-22) that is upregulated in response to DADS treatment in MGC803 cells. The combination treatment enhanced the effects of DADS against the subcutaneously implanted cancer cells in nude BALB/c mice.
Takahashi et al. (1992) aimed for a comprehensive approach by sequentially introducing carcinogens to male F344 rats that targeted different organs. N-diethylnitrosamine was one of the primary carcinogens used in the study. DADS were administered, after initiation, at a dose of 200 mg/kg thrice weekly for 6–24 weeks depending on the organ under investigation. DADS significantly inhibited colon adenoma and renal nephroblastoma; however, although the related garlic constituent, DAS, trended toward inhibiting colon adenoma, significance was not achieved. Moreover, DAS did not appear to exhibit any effect on renal cancer. Perhaps as important as determining therapeutic targets for DADS is the identification of cancers that do not respond to DADS. In this study, 100 % of animals acquired alveolar hyperplasia, but neither DADS nor DAS exhibited any chemopreventive ability in lung. In addition, urinary bladder cell hyperplasia was not significantly inhibited by DADS or DAS. It is possible that the statistical power of the study was a limiting factor, particularly because only 35 % of animals exhibited bladder hyperplasia. This would require highly consistent results to recognize a true difference between treatment groups. Other cancers were relatively rare in the study, precluding any assessment of DADS or DAS efficacy. It is worth noting that DAS-treated animals exhibited induction of placental glutathione-S-transferase (GST-P), an enzyme considered to be indicative of a pre-neoplastic state. Thus, this study suggests that DAS may promote liver cancer. Unfortunately, later studies were not designed to assess and confirm this hepatic finding, as they focused on different organs.
The majority of studies with DADS are aimed at specific organs or tissues; however, Singh et al. (1996) took a different approach. They used an H-ras oncogene-transformed fibroblast cell line (NIH 3T3) in xenografts in nude BALB/c mice. When cells were implanted subcutaneously, oral administration of DADS delayed and inhibited tumor growth, which correlated with a reduction in membrane association of p21H-ras. Interestingly, DADS also inhibited hepatic and tumoral 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase, an enzyme important in prenylation, and thus anchoring of proteins, e.g., Ras, in the plasma membrane. Therefore, the investigation indicates that DADS may reduce extracellular growth signal sensitivity in cancer cells.
4.3 DAS
Studies on DAS in relation to cancer began in the late 1980s. It has commonly been studied for its effects on skin cancer, but has also been assessed for neoplasms of the esophagus, stomach, mammary tissue, liver, colon, and kidney (Table 3). One of the first studies on DAS, by Wargovich et al. (1988), focused on esophageal cancer initiated by N-nitrosomethylbenzylamine (NMBA) in rats. DAS, when given at 200 mg/kg orally 3 h prior to NMBA (3 and 5 mg/kg), inhibited DNA damage by up to 64 % and inhibited tumor formation completely with high statistical significance. DAS inhibited gastric and hepatic metabolism of NMBA, thus indicating that DAS could prevent the formation of DNA-alkylating metabolites. However, whether DAS inhibited esophageal cancer by inhibiting metabolism of NMBA is unknown, as this mechanism would suggest that the carcinogenic metabolite(s) of NMBA can target esophageal cells via the basolateral aspect after reaching the systemic circulation. In a later study by Wargovich et al. (1992), DAS again showed a significant ability to suppress initiation of esophageal cancer by NMBA; however, a dose dependence was clearly observed. That is, 10 mg/kg DAS gavage weekly for 5 weeks (3 h prior to NMBA) failed to inhibit esophageal cancer or papillomas, while 100 mg/kg weekly reduced tumor incidence by 34 %, tumor multiplicity by 53 %, papilloma multiplicity by 42 %, and squamous cell carcinoma by 88 %, all statistically significantly. A very important distinction was made in this study, as when DAS, 200 mg/kg weekly, was given after NMBA treatment it was completely ineffective at preventing tumors or papillomas.
As described in the previous section, Dwivedi et al. (1992) employed the SENCAR mouse model to test DAS in parallel with DADS. When given topically, either DAS or DADS, 1 mg, 30 min prior to DMBA and 30 min before each TPA administration, inhibited skin papilloma formation from the 9th week of promotion and increased survival. As described previously, the authors suggested the possibility of modified bioactivation of DMBA by DAS and DADS as the mechanism of chemoprevention. Several later studies continued with the assessment of DAS against chemically induced skin cancer. Singh and Shukla (1998a) also used the two-stage model of skin cancer (DMBA then TPA). In the study, topical DAS (250 μg thrice weekly for 3 weeks) given prior to the topical initiator, DMBA, but not prior to each promoter application, significantly delayed the onset of tumors and reduced the total number of tumors by 81 % compared to vehicle control. When 250 μg of DAS was given topically 1 h prior to each topical promoter treatment, but not prior to initiation with DMBA, the total number of tumors was reduced by 76 %. Singh and Shukla (1998b) tested DAS against skin cancer induced by DMBA or benzo[a]pyrene (BaP) without the use of a promotion phase. Topical DAS given 1 h prior to topical DMBA 3 times per week for 28 weeks significantly reduced the multiplicity of skin tumors in female Swiss albino mice. When DAS was given 1 h after DMBA, the chemopreventive effect remained, but was somewhat attenuated. DAS also inhibited BaP-induced skin tumors, but in contrast to DMBA experiments, it trended toward a stronger chemopreventive potential when applied after BaP. The reason for this contrast with respect to timing-dependent efficacy of DAS against these two different carcinogens remains unknown. Some plausible factors to consider are potential differences in the transdermal permeation rates of the carcinogens or in the time delay before toxicodynamic effects are exhibited. In a more recent study, George et al. (2011) found that topical DAS (250 μg thrice weekly) delayed onset and incidence of skin tumors in male BALB/c mice by 55 % and 45 %, respectively, when started in parallel with a standard two-stage skin cancer carcinogen treatment (DMBA and TPA). Efficacy was increased (84 % tumor reduction) when DAS was combined with pomegranate fruit extract (PFE) in drinking water. PFE, incidentally, showed significant efficacy by itself. From the molecular aspect, DAS reduced expression of phospho-ERK1/2, phospho-JNK1, and phospho-IκB-α, and reduced activity of NF-κB and IKK-α. Again, when DAS was combined with PFE, the effect on all of these molecular indicators was stronger. Thus, this report demonstrated that DAS and DAS + PFE (DAS < DAS + PFE) may work to inhibit a common cell proliferation pathway in skin cancer.
In a study described in the previous section, Ip et al. (1992) assessed DAS and DADS against DMBA-induced mammary tumors in rats. Gastric gavage of DAS at 1.8 mmol/kg given 96, 48, and 24 h before intragastric DMBA (10 mg) significantly reduced mammary tumor incidence by 39 % and total tumor yield by 41 %. DADS showed superior efficacy to DAS in both measures.
The details of a study by Takahashi et al. (1992) comparing DAS and DADS in liver, colon, and renal cancer, induced by various carcinogens, have also been described above. Briefly, DAS had a tendency to inhibit colon adenoma, but failed to reach significance. Further, DAS failed to reduce renal cancer, alveolar hyperplasia, or bladder cell hyperplasia, and induced a marker of pre-neoplasia, namely GST-P, in liver. In a different study, Surh et al. (1995) showed that 250 μM DAS inhibited N-demethylation of NDMA in rat liver extract by 27 %, indicating that at least some metabolic activity was affected by DAS. Thus, whether DAS is hepatoprotective or detrimental in the presence of a carcinogen very likely depends on the metabolic fate of the specific carcinogen in question, and how DAS influences that fate. Further, whether DAS alone increases the risk of liver cancer remains an important unanswered question.
Hadjiolov et al. (1993) deviated to some extent from the commonly studied carcinogens when assessing DAS against aristolochic acid (AA)-induced gastric cancer. AA is a potent carcinogen found in some herbal preparations that is capable of inducing a variety of tumors in several organs. AA induced stomach, bladder, and thymus tumors after 12 weeks of exposure in male BD-6 rats. Intragastric DAS (150 mg/kg) reduced the incidence of stomach carcinomas and sarcomas from 45 % to 10 % when given 4 h before AA, and from 45 % to 0 % when given twice, 24 and 4 h, before AA. DAS decreased gastric DNA damage, but did not appear to reduce bladder cell hyperplasia. The total tumor burden was reduced from 60 % to 10 % with single administration of DAS, and from 60 % to 0 % with double administration of DAS prior to AA.
4.4 Ajoene
Of all major OSCs in garlic, ajoene has received the least attention in animal studies. To date, such studies have involved skin cancer, melanoma, hepatocarcinoma, and an ascites-derived sarcoma (Table 4). Li et al. (Li et al. 2002a, b) studied one stereoisomer, Z-ajoene, against subcutaneously implanted sarcoma 180 (an ascites/peritoneal-derived mouse cancer) and mouse hepatocarcinoma 22. Z-Ajoene, 8 mg/kg IP daily, significantly reduced sarcoma growth by 32 %, and 4 mg/kg IP daily significantly reduced hepatocarcinoma growth by 42 %. Nishikawa et al. (2002) employed the two-stage model of skin cancer with DMBA and TPA in ICR mice wherein they demonstrated that 250 μg topical ajoene, given 1 h before each TPA treatment, reduced the multiplicity of skin cancer to 4.9 % of that observed in ajoene-naïve mice at 18 weeks. Taylor et al. (2006) used the C57BL/6 B16/BL6 mouse melanoma model to show that 25 mg/kg of IP ajoene given every other day reduced primary tumor growth and lung metastases.
4.5 SAC
As one of the major water-soluble constituents of garlic, SAC has been studied quite extensively in animal models for its chemopreventive properties (Table 5). In animal models, it has extensive oral bioavailability and is excreted variably depending on the species. Although in rats it is significantly renally eliminated as the N-acetyl-S-allylcysteine (NSAC) metabolite, mice show less of this metabolite, and more of the parent compound, in the urine (Nagae et al. 1994). In contrast, dogs show very little renal elimination of both compounds (Nagae et al. 1994). SAC and NSAC, of course, bear some resemblance to N-acetylcysteine (NAC), the antidote for acetaminophen (paracetamol) toxicity. It is therefore worth noting that NAC has also received some attention for its potential as a more general hepatoprotective compound, as it is able to bolster the glutathione-based detoxification/conjugation system. NAC is thus likely useful also in instances without acetaminophen exposure. This information may be helpful to consider when interpreting studies on SAC.
Studies with SAC have involved cancers of the GI tract, including the oral cavity, stomach, liver, and colon, and mammary and prostate tumors. One of the first studies on SAC in cancer was reported by Hatono et al. (1996). In the study they used dimethylhydrazine (DMH) as both an initiator and promoter of colon cancer in male Fischer-344 rats. When SAC was administered via diet at 0.125 or 0.25 g/kg of food starting one week prior to initiation with DMH, and continuously thereafter, aberrant crypt foci (precursors of colon cancer) were reduced by 33 % and 54 %, respectively. In contrast, when SAC was initiated 2 weeks after starting DMH it had no effect on aberrant crypt foci. When SAC was given at 1.8 mmol/kg/day to the rats, a significant increase in glutathione-S-transferase (GST) activity was seen in the liver, early and middle small intestine, and colon. Further investigation revealed an increase in GST protein expression. Thus, it is probable that SAC is chemopreventive via bolstering detoxification systems in the GI tract and liver. Whether SAC can also act like NAC to increase glutathione stores is unknown, but being devoid of a protonated sulfur may preclude it from having this role.
Velmurugan et al. (2003) assessed SAC against gastric cancer in rats induced by serial administration of N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) and saturated sodium chloride (S-NaCl). Intragastric SAC at 200 mg/kg thrice weekly, starting the day after initiating MNNG, suppressed the incidence of gastric tumors and increased the antioxidant status in the stomach, blood, and liver. When the gastric glutathione/GST system was assessed, it was found that SAC alone or MNNG + S-NaCl alone increased GSH levels and GST activity; however, MNNG + S-NaCl did so to a significantly greater extent. Moreover, MNNG + S-NaCl given concomitantly with SAC yielded the highest increase in GSH concentration and GST activity in the stomach. Therefore, the earlier study by P et al. demonstrating that SAC can induce GST in the liver substantiates these results in stomach. That MNNG + S-NaCl alone increased gastric GST activity likely reflects an “attempt” to protect gastric tissue by fortifying its antioxidant capacity. Again, exactly how SAC triggers GST upregulation is unknown. A subsequent study by Velmurugan et al. (2005) again used intragastric SAC against MNNG + S-NaCl, both given in the same frequency and sequence as in the prior study; however, the dose of SAC was 100 mg/kg thrice weekly instead of 200 mg/kg. This study also found SAC to be chemopreventive against gastric tumors, but efficacy was significantly greater when SAC was combined with thrice-weekly lycopene (1.25 mg/kg), the primary chromophore-bearing molecule in red tomatoes. From a mechanistic aspect, SAC appeared to have reversed the anti-apoptotic effect of MNNG + S-NaCl in gastric tissue. For example, Bcl-2 overexpression was attenuated by SAC to an extent that approached control levels. There was also a trend for SAC to increase caspase 3 activity that had been reduced by MNNG + S-NaCl, but this was not significant until SAC was combined with lycopene. The expression and functional changes were demonstrated in gastric tissue; however, whether the tissue originated from tumors or unaltered stomach is unclear.
Sundaresan and Subramanian (2003) studied liver cancer in rats induced by a combination of N-nitrosodiethylamine (NDEA) and carbon tetrachloride (CCl4). The carcinogens produced tumors in 6 of 6 rats, but oral gavage of SAC (200 mg/kg given on alternate days than carcinogens) prevented tumors completely (0 of 6) and elevated the antioxidant status. Although circulating levels of reduced glutathione (GSH) were depleted by the carcinogen mix by approximately 50 %, SAC significantly reversed this effect, with GSH returning to 87 % of normal. Moreover, when SAC was given in the absence of carcinogens, the GSH level rose above the control concentration to 116 %. Interestingly, when the same group later assessed changes in liver GSH in response to the same carcinogen (NDEA), their results contrasted somewhat with blood GSH (Sundaresan and Subramanian 2008). That is, whereas NDEA depleted blood GSH, it increased hepatic GSH. However, in both blood and liver, SAC alone increased GSH, and, in addition, GSH was greater in NDEA + SAC vs. NDEA alone in both studies. Sundaresan and Subramanian (2008) also found that hepatic GST activity differed in treatment groups. Specifically, NDEA increased GST activity, which was further increased by adding SAC. SAC alone also significantly increased GST activity vs. control, but to a lesser extent than NDEA or NDEA + SAC. Finally, when Sundaresan and Subramanian (2008) evaluated two additional major detoxification enzymes in the liver, namely superoxide dismutase (SOD) and catalase (CAT), they found that they differed somewhat in response to NDEA and SAC compared to GST. In particular, SOD and CAT activities were reduced by NDEA, an effect that was partially reversed by SAC. Further, SAC alone was able to increase SOD and CAT activities, thus exhibiting some consistency with its effects on GST. In a more recent study, Ng et al. (2012) used male BALB/c nude mice with an orthotopic xenograft of a human metastatic hepatocellular carcinoma cell line (MHCC97L-luc). The cells were grown subcutaneously and then implanted into the liver where they were monitored with an in vivo imaging system. SAC, given at 1 mg/kg/day, did not reduce incidence of established tumors, but significantly reduced tumor volume (74 % reduction). SAC trended toward reducing lung metastases (i.e., 87.5 % without SAC, 37.5 % with SAC), but did not reach statistical significance unless combined with the anticancer/DNA-binding drug cisplatin. In vitro, albeit at very high concentrations (i.e., 5 to 40 mM), SAC dose dependently induced apoptosis and necrosis with an accompanying stimulation of caspases 3 and 9 and suppression of Bcl-2, Bcl-xL, and PCNA. E-cadherin expression was also increased, which corresponded to impaired migration in vitro. Therefore, perhaps surprisingly, SAC showed in vivo efficacy against liver cancer at blood concentrations that were likely at least 100 times lower than those shown to trigger cell death in vitro. Without knowing the hepatic concentration of SAC, or the potency of its metabolite(s), these in vivo results are somewhat difficult to compare and contrast with the in vitro results.
Pai et al. (2012) assessed SAC against a human oral cancer cell line subcutaneous xenograft, CAL-27, in BALB/c nude mice. SAC dose dependently inhibited subcutaneous tumor growth and suppressed phosphorylation of Akt, mTOR, inhibitor of κ-B-α, and ERK1/2. SAC reduced expression of cyclin D1 protein, cyclooxygenase-2 (COX-2), vimentin, and NF-κB-65 (RelA), and increased expression of cell cycle inhibitor p16Ink4. Thus, SAC exhibited a broad scope of antiproliferative activity at the molecular level.
Cohen et al. (1999) assessed SAC against N-methylnitrosourea (NMU)-induced mammary tumors in rats. Dietary SAC (2,000 ppm or approximately 167 mg/kg/day) initiated 7 days before NMU and continued for 18 weeks after NMU was ineffective at preventing tumors. The authors reported that the blood SAC concentration was below the limit of detection of the HPLC method used, but analysis of metabolites was not performed. The authors suggested that the lack of efficacy, which contrasted with other studies on SAC, may be due to nonlinear dose effects. However, this is the only study assessing SAC against NMU-induced mammary tumors. Therefore, given the considerably high dose, and the fact that the majority of other SAC studies were on the GI tract (prior to complete metabolism), the results may indeed be accurate in this unique study.
Although prostate cancer has been studied quite extensively with some organosulfur compounds, e.g., DATS, SAC has received little attention. Chu et al. (2006) used nude mice implanted subcutaneously with a human androgen-independent prostate cancer cell line, CWR22R. SAC, 1000 mg/kg intraperitoneally daily for 7 weeks, inhibited tumor growth significantly by 62.4 % with no apparent toxicity. Serum PSA declined significantly by about 50 % compared to untreated xenograft-bearing mice. E-cadherin and γ-catenin expression wes restored, indicating an inhibition of invasiveness by SAC. Apoptosis increased with a significant reduction in Bcl-2 and an increase in cleaved caspase-3 in response to SAC.
5 Clinical Trials with Allium Vegetables as a Source of OSCs
Clinical validation of the positive results from cell culture and animal models was undertaken in a few population-based studies which investigated the protective benefits of Allium vegetables and their constituents. To date, most of the clinical trials looked into the chemopreventive properties of Allium vegetables on various cancers particularly those of the digestive tract. In the Prostate, Lung, Colorectal, and Ovarian Screening (PLCO) Trial from 1993 to 2001, men and women were screened for colorectal cancer where 29,413 control subjects were compared to 3057 cases with at least one adenoma of the distal large bowel to investigate the association of fruit and vegetable intake and the risk of colorectal adenoma development. The study concluded that diets high in fruits and dark-green vegetables, deep yellow vegetables, and garlic and onions are associated with decreased risk of colorectal adenomas (Millen et al. 2007). In a small double-blind randomized clinical trial which studied the use of high-dose aged garlic extracts (AGE) to reduce the number of colorectal adenomas, 37 patients were randomly assigned to receive either high (2.4 mL/day) or low (0.16 mL/day) dose of AGE after adenomas larger than 5 mm were removed by polypectomy. It was identified that the number of colorectal adenomas in the control group increased steadily, but patients who received high dose had significant reductions in size and number after one year of treatment (Tanaka et al. 2006). Association of aged garlic extract or oil and gastric cancer was studied in a factorial double-blind, placebo-controlled trial, the Shandong Intervention Trial (Ma et al. 2012). 3365 participants with Helicobacter pylori, associated with increased gastric lesions and cancer, were either treated with two weeks of antibiotics or long-term administration (7.3 years) of aged garlic extract and oil, or a vitamin supplement. A 14.7 year follow-up showed a non-statistically significant reduction in gastric cancer incidence and mortality with garlic and vitamin treatment groups (Ma et al. 2012). Data from the European Prospective Investigation into Cancer and Nutrition study were also used to study gastric cancer (GC) and adenocarcinomas of the esophagus (ADO). Baseline data was collected in 521,457 men and women in ten European countries. After an average of 6.5 years follow-up, 330 gastric cancer and 65 adenocarcinoma of the esophagus cases were diagnosed and used as the case subjects. Although there was no reduction in ADO, there was a borderline significant negative association between onion and garlic intake and intestinal gastric cancer risk (Millen et al. 2007).
Clinical trials also looked at the effect of organosulfur compounds in cancers affecting women. Data from the European Prospective Investigation into Cancer and Nutrition study was used to study the effect of fruit and vegetable consumption on the risk of epithelial ovarian cancer. 325,640 female participants with no incidence of cancer were interviewed and reassessed after an average of 6.3 years. There were 581 cases of primary invasive epithelial ovarian cancer after this time period. A high consumption of garlic and onion had a borderline significant reduction of cancer risk (Schulz et al. 2005). Data from the Shanghai Women’s Health Study (SWHS) and Shanghai Men’s Health Study (SMHS) were used to study the relationship between food groups and liver cancer risk. A total of 132,837 Chinese men and women were surveyed about their lifestyle and nutritional habits. After a follow-up of 10.9 years (SWHS) or 5.5 years (SMHS), 267 liver cancer cases were diagnosed in the two years following study enrollment. High intake of Allium vegetables such as garlic was shown to be associated with a significantly decreased liver cancer risk (Zhang et al. 2013).
An Italian case–control study looked at the relationship between onion and garlic intake and endometrial cancer. 454 endometrial cancer cases and 908 controls were interviewed after being admitted to the same hospitals for a wide spectrum of acute, non-neoplastic conditions. The consumption of garlic showed a nonsignificant decrease in risk at low levels and significant decrease in risk of endometrial cancer at high levels of garlic consumption (Galeone et al. 2008). A hospital-based study in Northwest China studied the effects of various foods on the risk of developing multiple myeloma (MM). The study consisted of 220 confirmed MM cases and 220 individually matched patient controls. High shallot and garlic intake was significantly associated with a reduced risk of multiple MM (Wang et al. 2012a, b). A similar hospital-based case–control study which looked at the effect of diet on prostate cancer concluded a borderline risk reduction in those subjects who consumed more than 5.5 g of garlic a week (Salem et al. 2011).
6 Concluding Remarks and Future Perspectives
At this juncture, significant evidence has mounted to support the use of Allium vegetables and their constituents in cancer chemoprevention. That is not to say that regular use of Allium-derived OSCs will protect against all cancers or at all times, but rather that an overall protective influence appears to be observed with regular exposure to these compounds. As can be gleaned from the preceding discussion, compounds in intact garlic exhibit quite a complex series of chemical modifications when sliced, chewed, or pulverized. Mechanistic, preclinical, and clinical studies on these compounds have provided us with a wealth of information. There is significant evidence demonstrating that these compounds can induce apoptosis and inhibit cell cycle progression in neoplastic cells, perhaps selectively over normal cells. The molecular mediators of these actions remain an intense area of investigation and a subject of controversy. For example, it remains unknown whether the MAPK pathway is upregulated or downregulated by OSCs to prevent cancer, as results have been conflicting. Similarly, although production of ROS has gained support as a therapeutic mechanism of action of some OSCs, at least one OSC, namely SAC, appears to induce a systemic antioxidant state. These discrepancies in mechanistic details must be resolved; however, they cannot overshadow the clear evidence of the benefits of OSCs. In contrast to the aforementioned controversy, a quite consistent mechanistic explanation for the protection against carcinogens, by OSCs, is the upregulation of very important detoxification systems within various tissues. For example, in both cell work and animal studies, OSCs from garlic consistently upregulate both GST expression and activity, and increase the abundance of its co-substrate, namely, reduced glutathione (GSH). Furthermore, various OSCs exhibit a complementary effect on Phase I-metabolizing enzymes. That is, they can inhibit formation of toxic carcinogen metabolites via inhibiting CYP 2E1. These beneficial effects can be extrapolated to suggest that garlic-derived OSCs likely provide a general hepatoprotection and reduce the toxicity of noncarcinogenic chemicals (e.g., acetaminophen). Other putative anticancer effects of OSCs, such as immunomodulation and anti-angiogenesis, remain controversial, but evidence continues to accumulate in these areas.
The antineoplastic potential of OSCs is exhibited quite extensively in animal models. A variety of cancers have been significantly suppressed in rodents, either at the initiation stage, the promotion stage, or both. In brief, DATS has shown utility against colon, prostate, liver, skin, and breast neoplasms. DADS exhibited efficacy against gastric and colon cancers in particular, and also some utility against skin and other cancers. DAS has shown particular efficacy against carcinogen-induced skin cancer as well as various gastrointestinal-type cancers. Ajoene has not received much attention, but may be effective against skin and hepatic cancers. As the major water-soluble constituent of garlic, SAC has demonstrated utility against carcinogen-induced and implanted liver tumors, as well as efficacy in the majority of the gastrointestinal tract. Prospective clinical trials and epidemiological studies on garlic and onions have become more common starting in 2005. The summary from these investigations is that garlic is likely beneficial in cancer chemoprevention. Some studies have shown a statistically significant protective action of garlic, while others have shown a promising trend in this direction. It appears that achieving statistical significance can be challenging at times, particularly because the incidence of some of the cancers studied was quite low during the duration of the studies. Achieving greater statistical power in future studies will undoubtedly reveal a more precise therapeutic role for Allium vegetables in chemoprevention. Finally, clinical investigations have largely used total garlic, rather than the isolated OSCs assessed in animal studies. This informational gap must be closed to facilitate confident and meaningful decisions in the future.
Abbreviations
- 5-FU:
-
5-Fluorouracil
- AA:
-
Aristolochic acid
- ALT:
-
Alanine aminotransferase
- AST:
-
Aspartate aminotransferase
- B[a]P:
-
Benzo[a]pyrene
- CA:
-
Cancer
- CCl4:
-
Carbon tetrachloride
- COX-2:
-
Cyclooxygenase 2
- CYP 2E1:
-
Cytochrome P450 isoenzyme 2E1
- DADS:
-
Diallyl disulfide
- DAS:
-
Diallyl sulfide
- DATS:
-
Diallyl trisulfide
- DMBA:
-
7,12-Dimethylbenz[a]anthracene
- DMH:
-
Dimethylhydrazine
- DR4 and DR5:
-
Death receptor
- ERK:
-
Extracellular signal-regulated kinase
- GST:
-
Glutathione-S-transferase
- HDACs:
-
Histone deacetylases
- HMG-CoA:
-
3-Hydroxy-3-methyl-glutaryl-Coenzyme A
- HUVEC:
-
Human umbilical vein endothelial cells
- i.p.:
-
Intraperitoneal
- JNK:
-
c-Jun N-terminal kinases
- MNNG:
-
Methylnitronitrosoguanidine
- MMP:
-
Matrix metalloprotease
- NAC:
-
N-acetyl-l-cysteine
- NDEA:
-
N-nitrosodiethylamine
- NDMA:
-
N-nitrosodimethylamine
- NDN:
-
N-diethylnitrosamine
- NMBA:
-
N-nitrosomethylbenzylamine
- NF-κB:
-
Nuclear factor-κB
- NKK:
-
(methylnitrosamino)1-(3-pyridyl)-1-butanone
- NMU:
-
N-methylnitrosourea
- OSCs:
-
Organosulfur compounds
- PAH:
-
Polycyclic aromatic hydrocarbon
- PBS:
-
Phosphate-buffered saline
- PCNA:
-
Proliferating cell nuclear antigen
- PFE:
-
Pomegranate fruit extract
- PK:
-
Pharmacokinetic
- ppm:
-
Parts per million
- PSA:
-
Prostate-specific antigen
- pSTAT3:
-
Phosphorylated signal transducer and activator of transcription 3
- q3d:
-
Every 3 days
- q4d:
-
Every 4 days
- qod:
-
Every other day
- qd:
-
Every day
- ROS:
-
Reactive Oxygen Species
- s.c.:
-
Subcutaneous
- SAC:
-
S-allylcysteine
- SAMC:
-
S-allylmercaptocysteine
- S-NaCl:
-
Saturated sodium chloride
- TPA:
-
12-O-tetradecanoylphorbol 13-acetate
- TRAIL:
-
Tumor necrosis factor-related apoptosis-inducing ligand
- TRAMP:
-
Transgenic adenoma of a mouse prostate
- VC:
-
Vinyl carbamate
- XIAP:
-
X-linked inhibitor of apoptosis protein
- Z-ajoene:
-
Z isomer of ajoene
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Bommareddy, A., VanWert, A.L., McCune, D.F., Brozena, S.L., Witczak, Z., Singh, S.V. (2016). The Role of Organosulfur Compounds Derived From Allium Vegetables in Cancer Prevention and Therapy. In: Ullah, M., Ahmad, A. (eds) Critical Dietary Factors in Cancer Chemoprevention. Springer, Cham. https://doi.org/10.1007/978-3-319-21461-0_6
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