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Chemopreventive Potential of Chlorophyllin: A Review of the Mechanisms of Action and Molecular Targets a
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Siddavaram Nagini , Fabrizio Palitti & Adayapalam T. Natarajan
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Department of Biochemistry and Biotechnology, Faculty of Science, Annamalai University, Tamil Nadu, India b
Department of Ecological and Biological Sciences, University of Tuscia, Viterbo, Italy Published online: 04 Feb 2015.
Click for updates To cite this article: Siddavaram Nagini, Fabrizio Palitti & Adayapalam T. Natarajan (2015) Chemopreventive Potential of Chlorophyllin: A Review of the Mechanisms of Action and Molecular Targets, Nutrition and Cancer, 67:2, 203-211, DOI: 10.1080/01635581.2015.990573 To link to this article: http://dx.doi.org/10.1080/01635581.2015.990573
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Nutrition and Cancer, 67(2), 203–211 Copyright Ó 2015, Taylor & Francis Group, LLC ISSN: 0163-5581 print / 1532-7914 online DOI: 10.1080/01635581.2015.990573
Chemopreventive Potential of Chlorophyllin: A Review of the Mechanisms of Action and Molecular Targets Siddavaram Nagini Department of Biochemistry and Biotechnology, Faculty of Science, Annamalai University, Tamil Nadu, India
Fabrizio Palitti and Adayapalam T. Natarajan
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Department of Ecological and Biological Sciences, University of Tuscia, Viterbo, Italy
Chlorophyllin (CHL), a water soluble semisynthetic derivative of the ubiquitous plant pigment chlorophyll used as a food additive, is recognized to confer a wide range of health benefits. CHL has been shown to exhibit potent antigenotoxic, anti-oxidant, and anticancer effects. Numerous experimental and epidemiological studies have demonstrated that dietary supplementation of CHL lowers the risk of cancer. CHL inhibits cancer initiation and progression by targeting multiple molecules and pathways involved in the metabolism of carcinogens, cell cycle progression, apoptosis evasion, invasion, and angiogenesis. The modulatory effects of CHL on the hallmark capabilities of cancer appear to be mediated via abrogation of key oncogenic signal transduction pathways such as nuclear factor kappa B, Wnt/b-catenin, and phosphatidylinositol-3-kinase/Akt signaling. This review provides insights into the molecular mechanisms of the anticancer effects of dietary CHL.
INTRODUCTION Epidemiological studies related to human health and the importance of nutrition have created increased interest in the prevention of age-related diseases such as cancer. Human diet contains a diverse array of food constituents exhibiting protective effects against a wide range of carcinogens. The protective elements in the diet that prevent cancer include selenium, folic acid, vitamin B12, vitamin D, chlorophyll, and antioxidants such as the carotenoids. A balanced diet with these elements in appropriate quantities could significantly reduce the risk of cancer (1,2). Of late, chlorophyllin (CHL), a water soluble, semisynthetic food-grade derivative of the ubiquitous plant pigment chlorophyll has attracted the focus of attention as a potential anticancer agent. CHL is obtained by the alkaline hydrolysis Submitted 22 April 2014; accepted in final form 29 October 2014. Address correspondence to Siddavaram Nagini, Department of Biochemistry and Biotechnology, Faculty of Science, Annamalai University, Annamalainagar-608 002, Tamil Nadu, India. Phone: C91-4144239842. Fax: C91-4144-238145/238080. E-mail: s_nagini@yahoo. com; [email protected]
of chlorophyll and reacts readily with environmental mutagens and carcinogens. CHL has been used as a medicine for more than 50 years without any adverse effects. The U.S. Food and Drug Administration (FDA) has approved over the counter use of CHL as an internal deodorant in doses up to 300 mg/day. CHL is used for controlling body, fecal, and urinary odor in geriatric patients and as an accelerant in wound healing. CHL is also used as a food coloring agent and a health food additive in cakes, beverages, sweets, and ice cream (3,4). Several in vitro and in vivo studies have demonstrated the antigenotoxic, antioxidative, and anticarcinogenic activities of CHL against numerous carcinogens (5–7). This review summarizes molecular mechanisms underlying the anticancer activity of CHL.
CHEMISTRY Chlorophyll contains 4 pyrrole nitrogen rings bonded to a central magnesium atom, and a fifth ring containing carbon atoms and a long phytol tail. The phytol tail of chlorophyll confers hydrophobicity and limits binding efficacy with carcinogens and mutagens. The predominant structural feature that differentiates CHL from chlorophyll (Fig. 1) is the replacement of magnesium ion by copper, methyl, and phytyl ester groups by sodium and potassium, and absence of the phytol tail. The antigenotoxic and anticancer effects of CHL are believed to be mediated via the porphyrin ring either by scavenging free radicals or by forming complexes with planar carcinogens (8,9).
ANTIGENOTOXIC EFFECTS The antigenotoxic effects of CHL have been mainly attributed to its ability to form complexes with polycyclic planar structures of mutagens (10). CHL has been documented to exhibit strong antimutagenic effects against a wide range of mutagens including aflatoxin B1 (AFB1), polycyclic aromatic hydrocarbons (PAHs), heterocyclic amines, and g-radiation 203
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have a protective effect at toxic concentrations of DMBA and to increase the cytotoxicity of DMBA thereby affecting cell proliferation. Co-administration of CHL with anticancer agents such as oxaliplatin was shown to potentially inhibit tumorigenicity with reduced genotoxicity (20). CHL was found to interact with the intercalating agent acridine (ICR 192) and reduce its binding with DNA (21).
ANTICANCER EFFECTS Several in vitro, in vivo, and epidemiological studies have demonstrated the anticancer activities of CHL.
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FIG. 1. Structure of chlorophyllin.
(5,10–12]. In Drosophila melanogaster, CHL suppressed the activity of monofunctional alkylating agents such as nitrosamines and nitrosoureas (13). Recently, Pimentel et al. (14) reported that an increase in the copper content of sodium-copper CHL resulted in a greater inhibition of genetic damage induced by gamma rays in somatic cells of Drosophila. Lagerqvist et al. (11) demonstrated that CHL exerts potent antimutagenic effects against PAH diol epoxides derived from B[a]P, dibenzo[a,h]anthracene (DBA), dibenzo[a,l]pyrene (DBP), and benzo[c]phenanthrene (BPh) in Chinese hamster V79 cells. Administration of CHL was also found to attenuate the formation of DNA adducts in normal human mammary epithelial cells exposed to B[a]P (15). CHL exerted strong anticlastogenic activity against several classes of chemicals including cesium chloride, mercuric chloride, cobalt chloride, chromium (VI) oxide, N-nitroso-N-ethyl urea, and urethane in mammalian cells (16). CHL was found to reduce chromosomal aberrations induced by ethyl methane sulfonate in cultured mammalian cells during S and G2/S phase of the cell cycle as well as the formation of micronuclei (MN) and bone marrow toxicity in sodium nitrite treated mice (17,18). Oral administration of CHL was shown to protect against induction of DNA single strand breaks and MN in B[a] P treated C57BL/6J mice (19). Recently, Grossi et al. (12) evaluated the anticlastogenic effects of CHL on N-methyl-N’-nitro-N-nitrosoguanidine (MNNG) and 7,12-dimethylbenz[a]anthracene (DMBA)induced frequencies of MN in human HepG2 cells in vitro and mouse polychromatic erythrocytes in vivo. CHL was found to have a protective effect against MNNG in vitro when the treatment was given simultaneously, suggesting a direct interaction between MNNG and CHL, whereas with DMBA, CHL significantly reduced the frequencies of induced MN when administered prior to and after carcinogen treatment. In vivo, CHL did not exert any protective effects against either MNNG or DMBA induced MN. However, CHL treatment was found to
In Vitro Studies A number of in vitro studies have analyzed the antiproliferative effects of CHL on various transformed cell lines. CHL was shown to retard the growth and proliferation of MCF-7 breast carcinoma cells by deactivating the extracellular signalregulated kinases (ERKs) and inducing apoptosis (22). Chimploy et al. (23) demonstrated that CHL treatment induced Sphase cell cycle arrest in human colon cancer cells (HCT116) in a p53-independent manner by reducing the expression of DNA synthesis and repair enzymes, ribonucleotide reductase subunits (R1 and R2), and G1/S checkpoint controls. CHL is reported to inhibit malignant transformation induced by transbenzo(a)pyrene-trans-7,8-dihydrodiol-9,10-epoxide (transBPDE) in 16HBE human bronchial epithelial cell line (24,25). CHL was shown to inhibit the growth of HCT116 colon cancer cells by inducing apoptosis (26). It was also demonstrated that CHL induces photocytotoxicity and cell death in T24 and 5637 human bladder cancer cells (27). CHL e4 and CHL f were shown to act as photosensitizers and induce apoptotic cell death in 5637 and T24 human bladder cancer cells (27,28). Konıckova et al. (29) reported the antiproliferative effects of CHL from Spirulina platensis against pancreatic cancer cells in vitro.
In Vivo Studies The first in vivo anticancer activity of CHL was reported in the rainbow trout model by Dashwood et al. (10) who showed a significant reduction in AFB1-induced DNA adducts and hepatic tumors following dietary supplementation of CHL. CHL was shown to attenuate dibenzo(def,p)chrysene and AFB1-induced liver carcinogenesis by reducing tumor incidence, tumor multiplicity, DNA adduct formation and by modulating genes involved in apoptosis, cell proliferation, and invasion in the rainbow trout model (30,31). Co-administration of CHL with probiotic fermented milk was also found to offer significant protection against AFB1-induced hepatocellular carcinoma (5). CHL was also reported to inhibit AFB1, 2amino-3-methylimidazo[4,5-f]quinoline, and DBP-induced
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multiorgan carcinogenesis in the rat and rainbow trout models (32–35). CHL was shown to inhibit the development of PhIPinduced rat mammary tumors by reducing the metabolic activation of PhIP (36). CHL was demonstrated to inhibit the development of DMBA-induced mouse skin papillomas and hamster buccal pouch carcinomas (37–39). CHL administration has also been reported to offer significant protection against DBP-initiated transplacental carcinogenesis (40). In a recent report, we demonstrated inhibition of MNNG induced rat forestomach carcinogenesis by dietary supplementation of CHL (41). Although several studies have demonstrated the anticancer effects of CHL when administered both prior to and simultaneously with the carcinogen, a few studies have reported lack of preventive or therapeutic efficacy with CHL supplementation. Wang et al. (42) demonstrated that dietary CHL administration did not offer protection against 1,2-dimethylhydrazine (DMH)-induced small intestinal and colon carcinogenesis. Orner et al. (43) reported that post-initiation treatment with CHL did not exert any significant effects on AFB1-induced preneoplastic foci in the liver and colon. However, Blum et al. (44) showed that postinitiation treatment with higher concentrations of CHL offers significant protection against DMHinduced rat colon carcinogenesis.
Epidemiological Studies A double-blinded placebo-controlled intervention trial conducted by Egner et al. (45) in Qidong, People’s Republic of China revealed that CHL consumption for over a period of 4 mo led to 50% reduction in the urinary excretion of AFB1-DNA adducts among individuals at high risk for the development of hepatocellular carcinoma. In another randomized double-blinded clinical trial, copper chlorine e(4) ethyl ester and copper chlorine e(4) derivatives were identified in the serum of study participants, suggesting that these 2 components may be involved in preventing carcinogen absorption from the gut and for the chemoprotective effects of CHL (46). An unblinded cross-over study conducted by Jubert et al. (47) among 4 human volunteers demonstrated rapid uptake and urinary elimination of AFB1. Treatment with chlorophyll and CHL significantly impeded AFB1 absorption in these subjects suggesting that coconsumption may limit the bioavailability of ingested aflatoxin in humans. More recently, Shaughnessy et al. (48) conducted a crossover design study to explore the effects of CHL administration on fried meat associated carcinogenesis, which revealed that CHL supplementation to individuals consuming high-temperature meat diet containing high levels of heterocyclic amines reduced the levels of hydrolyzed and unhydrolyzed urinary and faecal mutagens. In addition, CHL administration also reduced DNA damage in the target colorectal cells.
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MECHANISM UNDERLYING ANTICANCER EFFECTS Whole genomic microarray analysis of the changes in gene expression pattern revealed that dietary supplementation of CHL significantly modulates the expression profiles of 104 genes that are predominantly associated with cell adhesion, cell–cell communication, invasion, and transforming growth factor beta (TGFb) signaling in DMBA-induced hamster buccal pouch carcinogenesis model (39). Studies by John et al. (49) have also indicated that CHL administration to B[a]P treated NHMECs modulated the expression of 30 genes most of which were associated with xenobiotic metabolism, cell signaling, cell motility, cell proliferation, cell cycle control, metabolism, DNA repair, and apoptosis pathways. Proteomic analysis by MALDI-TOF-TOF-MS following addition of sodium iron CHL to human hepatoma 3B cells identified 32 threefold differentially expressed proteins that are involved in cell proliferation, apoptosis, antioxidant function, and signal transduction (50). Table 1 summarizes the anticancer effects of CHL. A detailed discussion on the modulatory effects of CHL on various oncogenic signaling pathways and processes ensues. Xenobiotic-Metabolizing Enzymes and Antioxidants A large proportion of environmental carcinogens such as PAHs are metabolically activated to form highly reactive electrophilic intermediates that bind covalently to DNA to form adducts that are normally repaired by DNA repair enzymes. Failure to repair these adducts before replication causes mutations eventually culminating in neoplastic transformation. The probability that a carcinogen will reach a target cell and cause DNA damage is dependent upon the balance between carcinogen activation catalyzed by phase I cytochrome P450 (CYP) enzymes and detoxification accomplished by the Phase 2 enzymes predominantly glutathione S-transferases, key regulators of various cytoprotective antioxidant, and detoxifying enzymes (51). Coordinated induction of phase II detoxifying enzymes, antioxidants, and DNA repair genes by the transcription factor nuclear factor erythroid-2 related factor-2 (Nrf2) serves to eliminate toxic electrophilic intermediates and reactive oxygen species generated by the metabolism of xenobiotics thereby preventing malignant transformation [52]. In a recent study, we demonstrated a reduced expression of phase I CYP enzymes with decreased formation of 8hydroxy-2’-deoxyguanosine, an indicator of oxidative stress-induced DNA damage following dietary supplementation of CHL in the DMBA-induced hamster buccal pouch carcinogenesis model [53]. This was associated with upregulation of the Phase 2 detoxification enzymes glutathione S-transferases and NAD(P)H:quinone oxidoreductase 1, the antioxidant enzymes superoxide dismutase, catalase, and glutathione peroxidase, and the DNA repair enzymes 8oxoguanine glycosylase 1, xeroderma pigmentosum, and xray repair cross complementing group 1. Analysis of the
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TABLE 1 Summary of the anticancer effects of chlorophyllin Type of study In vitro
Cell line/animal tumor model/ human subjects B(a)P exposed normal mammary epithelial cells (NHMECs)
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Human bladder cancer cells Human breast cancer cells, MCF-7
Human bronchial epithelial cells, 16HBE Human colon cancer cells, HCT116
In vivo
AFB1-induced rat hepatocarcinogenesis /multi-organ carcinogenesis
AFB1 and dibenzo(def,p)chryseneinduced rainbow trout liver carcinogenesis DBP-induced multiorgan carcinogenesis DMBA-induced hamster buccal pouch carcinogenesis
DMBA-induced mouse skin papillomagenesis MNNG-induced forestomach carcinogenesis
Mechanisms
Reference(s)
Altered expression of genes involved in cell proliferation, apoptosis, and signaling pathways Reduced CYP1A1 and CYP1B1 expression Decreased BPdG adduct Apoptosis induction Cell cycle arrest at G0/G1 phase Deactivation of extracellular signal regulated kinases Downreguation of cyclin D1 and Bcl-2 Downregulation of E-cadherin, cyclin D1 and cyclin E expression Induction of S-phase cell cycle arrest Downregulation of DNA repair and synthesis enzymes Increased E-cadherin and alkaline phosphatase expression Augmentation of b-catenin accumulation in plasma membrane Upregulation of bid, caspase-8 and caspase-3 Complex -mediated reduction of carcinogen uptake Decreased AFB1-DNA adduct formation Reducing lipid peroxidation Enhancing antioxidant enzymes Modulation of c-myc, bax, bcl-2, cyclin-D1, p53 and rasp-21 Inhibition of carcinogen - DNA adduct formation
(24,65)
Inhibition of DBP-DNA adduct formation
(44–47) (39)
(42) (40,43)
(13,45,48)
(46, 47)
(49,55)
Attenuation of NF-kB, Wnt/b-catenin, PI3K/Akt, and TGF-b signaling Activation of Nrf2/Keap-1 signaling Downregulation of CYP and 8-OHdG expression Upregulatiion of antioxidant, detoxification and DNA repair enzymes Induction of intrinsic apoptosis Modulation of genes associated with cell adhesion, cell-cell communication and angiogenesis Increased GST and GSH activity Altered expression of biotransformation enzymes Inhibition of TGFb signaling
(55,56,69)
(54) (61) (70)
(continued on next page)
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TABLE 1 Summary of the anticancer effects of chlorophyllin (Continued) Type of study
Cell line/animal tumor model/ human subjects
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DMH-induced rat colon carcinogenesis IQ-induced rat multi-organ carcinogenesis IQ and DMH-induced rat colon carcinogenesis
Human intervention
PhIP-induced rat mammary carcinogenesis Individuals at high risk for hepatocellular carcinoma Healthy volunteers Healthy volunteers
Mechanisms Downregulation of COX-2 expression Inhibition of NF-kB signaling Inhibition of IQ-DNA adduct formation Downregulation of Ctnnb1 and APC mutations Reduced b-catenin and c-jun expression Decreased cell proliferation Increased apoptosis Inhibition of PhIP-DNA binding
Reference(s)
(50,51) (47,60)
(53)
Increased urinary elimination of aflatoxin B1
(61)
Reduced urinary excretion of aflatoxin-DNA adduct Diminished excretion of hydrolysed and unhydrolyzed urinary and faecal mutagenicity Inhibition of DNA damage
(63) (64)
AFB1 D aflatoxin B1; B[a]P D benzo[a]pyrene; DMBA D 7,12-dimethylbenz[a]anthracene; DMH D 1,2-dimethylhydrazine; IQ D 2-amino-3-methylimidazo [4,5-f]quinolone; MNNG D N-methyl-N’-nitro-N-nitrosoguanidine; NF-kB D nuclear factor kappa B; Nrf2 D nuclear factor erythroid-2 related factor-2; PI3K D phosphatidylinositol-3-kinase; TGFb D transforming growth factor beta.
mechanism underlying the protective effects of CHL revealed nuclear translocation of Nrf2 with downregulation of its negative regulator, Keap-1, providing substantial evidence that CHL drives Nrf2/Keap1 signaling with consequent transactivation of genes involved in promoting DMBA detoxification and protection against DNA damage. In human umbilical vein endothelial cells, CHL was reported to confer protection against oxidative stress by inducing the Nrf2 signaling pathway (54,55). These studies provide a mechanistic basis for the potent antioxidant function of CHL. CHL is documented to confer a higher degree of protection against free radicals than other known antioxidants such as ascorbic acid and glutathione (56). Several studies have demonstrated the antioxidant activity of CHL against a wide range of oxidant species including superoxide anion radical (O2─), hydroxyl radicals (OH), deoxyribose peroxyl radicals (ROO), ABTS radicals (2,2’-azino-bis(3-ethylbenzthia-zoline-6-sulphonic acid), singlet oxygen (1O2), and peroxynitrite. The high reaction rate constant and formation of face-to-face complexes between CHL and carcinogens have been suggested to contribute to the antioxidant activity of CHL (56–59). The mechanisms underlying the radioprotective and anticancer effects of CHL include free radical scavenging, decreased lipid and protein oxidation, restoration of enzymic antioxidants, and enhanced DNA repair (57,60).
Effects on Cell Proliferation and Differentiation Studies on a panel of cancer cell lines have provided evidence that the antiproliferative effects of CHL are mediated by inducing G0/G1 or S phase cell cycle arrest (22–26,30). Chiu et al. (22) demonstrated that CHL treatment inhibits the proliferation of MCF-7 breast cancer cells by deactivating ERKs, a subfamily of mitogen activated protein kinases that play a key role in cell survival, proliferation, and differentiation. This was associated with depletion of cyclin D1 and increase in cells in the G0/G1 phase of the cell cycle. CHL treatment was shown to inhibit trans-BPDE induced malignant transformation in 16HBE human bronchial epithelial cells by reducing the expression of E-cadherin and cyclins (24,25). CHL administration was also reported to promote cell differentiation in HCT116 colon cancer cells by modulating the expression of E-cadherin and alkaline phosphatase (26). Effects on Apoptosis Apoptosis, a form of programmed cell death, plays a crucial role in maintaining tissue homeostasis. Endogenous apoptotic death machinery is principally activated via 2 major signaling pathways, the extrinsic and the intrinsic pathway that converge at the activation of caspases to cleave substrates vital for cell survival (61,62). Several phytochemicals induce apoptosis via the intrinsic pathway by promoting mitochondrial outer membrane permeabilization with release of intermembrane space
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proteins such as secondary mitochondria-derived activator of caspase (Smac)/direct inhibitor of apoptosis protein (IAP) binding protein with low pI (DIABLO) and cytochrome C into the cytosol. Members of the Bcl-2 and IAP family proteins function as key regulators of intrinsic apoptosis. In a recent study, we have shown that dietary CHL supplementation induces intrinsic apoptosis in a hamster model of oral oncogenesis by modulating the expression of pro- and antiapoptotic members of the Bcl-2 family suggesting that CHL promotes Bax oligomerization and mitochondrial outer membrane permeabilization. CHL was also shown to induce the efflux of the apoptogenic molecules cytochrome C and Smac/DIABLO into the cytosol, whilst simultaneously downregulating the expression of optic atrophy 1, a profusion dynamin-related protein of the inner mitochondrial membrane that modifies the morphology of the mitochondrial cristae. Furthermore, CHL supplementation activated the caspase cascade and induced cleavage of the DNA repair enzyme polyADP ribose polymerase. CHL was found to enforce nuclear localization of the IAP protein survivin, an event that facilitates apoptosis by inhibiting caspases and inducing p53 and Bax (38). These findings are substantiated by reports of CHL-induced apoptosis induction in cancer cells in vitro and in animal tumor models in vivo (26,32,35,63). Thus, it appears that CHL promotes apoptosis by modulating the expression of a multitude of molecules involved in the apoptotic signaling cascade.
Effects on Matrix Invasion and Angiogenesis Invasion and angiogenesis, hallmark capabilities of malignant tumors play a central role in promoting cancer progression and metastasis. Tumor invasion is regulated by a delicate balance between matrix metalloproteinases (MMPs) that catalyze proteolytic degradation of the extracellular matrix and their inhibitors that control extracellular matrix breakdown (64,65). MMP activation is also recognized to trigger angiogenic signaling through the release of several proangiogenic factors that promote neovascularization such as hypoxiainducible factor 1a (HIF-1a), vascular endothelial growth factor (VEGF), platelet-derived growth factor, and interleukins [66]. CHL treatment was found to downregulate the expression of interleukin 1 beta, a proinflammatory cytokine with a proangiogenic role in human mammary cell lines exposed to B[a]P (15). Studies from our laboratory have demonstrated that CHL administration inhibits tumor invasion and angiogenesis during DMBA-induced hamster buccal pouch carcinogenesis by upregulating tissue inhibitor of MMP-2 expression, and downregulating MMP-2, MMP-9, HIF-1a, VEGF, and VEGF receptor R2 (39,67). Accumulating evidence indicates that the modulatory effects of CHL on cell proliferation, apoptosis evasion, invasion, and angiogenesis are mediated by abrogation of key oncogenic signal transduction pathways such as nuclear factor kappa B (NF-kB), Wnt/b-catenin, and
phosphatidylinositol-3-kinase/protein kinase B (Akt) PI3K/ Akt signaling (38,67,68).
Modulation of Oncogenic Transcription Factors and Signaling Pathways NF-kB, a prosurvival transcription factor, is localized in the cytoplasm as a heterodimer of p50 and p65, sequestered by inhibitor of kappa B (IkB). Phosphorylation of IkB by IkB kinase beta (IKKb) followed by ubiquitination and proteasomal degradation releases the NF-kB heterodimer, which subsequently translocates to the nucleus, binds to kB elements in DNA, and regulates the expression of about 500 genes involved in cell proliferation, apoptosis, invasion, metastasis, and angiogenesis (69). CHL supplementation was shown to inhibit the development of colonic neoplasms in DMH-treated mice by selectively inhibiting the NF-kB signaling pathway (70). CHL was also found to prevent the activation of NF-kB and its binding to the kB elements in DNA in lipopolysaccharide-stimulated murine splenic mononuclear cells and RAW 264.7 and to restore IkB-a in the cytosolic extract of lipopolysaccharidetreated cells (71). A study from this laboratory demonstrated that dietary CHL prevents the development of DMBA-induced hamster buccal pouch carcinomas by inactivating NF-kB signaling via inhibition of IKKb mediated phosphorylation and degradation of IkB, thereby impairing the nuclear translocation of NF-kB and blocking transactivation of genes involved in malignant transformation and progression (38). Aberrant activation of TGFb signaling stimulates tumor progression by facilitating acquisition of the essential hallmark capabilities of cancer cells (72). Recently, we reported that dietary CHL inhibits the development of MNNG-induced forestomach carcinomas by downregulating the expression of TGFb RI, TGFb RII, and Smad 2 and 4 and upregulating Smad 7, thereby abrogating canonical TGFb signalling. Furthermore, attenuation of TGFb signaling by CHL also blocked cell proliferation, angiogenesis, invasion, and metastasis, and induced mitochondria mediated cell death (41). The canonical Wnt/b-catenin signaling pathway that plays a key role in the regulation of various cellular processes such as cell proliferation, apoptosis, epithelial-mesenchymal transition, and angiogenesis is one of the prominent pathways influenced by NF-kB (73). Dietary supplementation of CHL was shown to block the prosurvival Wnt signaling pathway by downregulating PI3K/Akt with consequent inhibition of glycogen synthase kinase-3b phosphorylation thereby preventing nuclear translocation of b-catenin (67). These findings are substantiated by Carter et al. (74) who reported that CHL limits tumor invasion and metastasis by facilitating b-catenin trafficking away from the nucleus into the plasma membrane via the Hakai pathway. We demonstrated a positive correlation between blockade of PI3K/Akt and Wnt/b-catenin signaling by CHL and inhibition of tumor angiogenesis in the hamster
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buccal pouch carcinogenesis model. In particular, CHLinduced downregulation of PI3K/Akt could inactivate HIF-1a, a master regulatory transcriptional factor and enhancer of VEGF signaling (67). CONCLUSION CHL has gained considerable attention in recent years owing to its high safety and efficacy without any adverse side effects. Numerous studies in a panel of human cancer cell lines and in a variety of experimental animal models have revealed that CHL influences multiple molecules and pathways involved in the metabolism of carcinogens, antioxidant defenses, cell proliferation, apoptosis, invasion, and angiogenesis to exert its chemopreventive effects. Thus dietary phytochemicals such as CHL that affect multiple signal transduction pathways involved in cancer initiation and progression hold promise as ideal candidates for cancer chemoprevention and therapy. However, caution is still warranted for clinical application, and extensive investigations on optimal dose, metabolism, bioavailability, tissue distribution, pharmacokinetics, interference with endogenous metabolic pathways, and crosstalk between signaling circuits are necessary before therapeutic utilization of CHL. ACKNOWLEDGMENT The authors thank P. Thiyagarajan for help with the illustrations. FUNDING Financial assistance from the Department of Biotechnology, New Delhi, India and European Commission FUNCFOOD (FP7-KBBE-3-245030) under the 7th Framework of the Indo-EU Joint Collaborative Project on FUNCFOOD is gratefully acknowledged. REFERENCES 1. Riboli E: The role of metabolic carcinogenesis in cancer causation and prevention: evidence from the European Prospective Investigation into Cancer and Nutrition. Cancer Treat Res 159, 3–20, 2014. 2. Gonzalez-Vallinas M, Gonzalez-Castejon M, Rodrıguez-Casado A, and Ramırez de Molina A: Dietary phytochemicals in cancer prevention and therapy: a complementary approach with promising perspectives. Nutr Rev 71, 585–599, 2013. 3. PDR for Nutritional Supplements: In Chlorophyll/Chlorophyllin, Hendler SS and Rorvick,D (ed.). Montvale, NJ: Medical Economics Company, 2001, pp. 88–90. 4. Tumolo T and Lanfer-Marquez U: Copper chlorophyllin: a food colorant with bioactive properties? Food Res Int 46, 451–459, 2012. 5. Ferguson LR, Philpott M, and Karunasinghe N: Dietary cancer and prevention using antimutagens. Toxicology 198, 147–159, 2004. 6. Williams DE: The rainbow trout liver cancer model: response to environmental chemicals and studies on promotion and chemoprevention. Comp Biochem Physiol C Toxicol Pharmacol 155, 121–127, 2012.
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Nutrition and Cancer Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/hnuc20
Chemopreventive Potential of Chlorophyllin: A Review of the Mechanisms of Action and Molecular Targets a
b
Siddavaram Nagini , Fabrizio Palitti & Adayapalam T. Natarajan
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Department of Biochemistry and Biotechnology, Faculty of Science, Annamalai University, Tamil Nadu, India b
Department of Ecological and Biological Sciences, University of Tuscia, Viterbo, Italy Published online: 04 Feb 2015.
Click for updates To cite this article: Siddavaram Nagini, Fabrizio Palitti & Adayapalam T. Natarajan (2015) Chemopreventive Potential of Chlorophyllin: A Review of the Mechanisms of Action and Molecular Targets, Nutrition and Cancer, 67:2, 203-211, DOI: 10.1080/01635581.2015.990573 To link to this article: http://dx.doi.org/10.1080/01635581.2015.990573
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Nutrition and Cancer, 67(2), 203–211 Copyright Ó 2015, Taylor & Francis Group, LLC ISSN: 0163-5581 print / 1532-7914 online DOI: 10.1080/01635581.2015.990573
Chemopreventive Potential of Chlorophyllin: A Review of the Mechanisms of Action and Molecular Targets Siddavaram Nagini Department of Biochemistry and Biotechnology, Faculty of Science, Annamalai University, Tamil Nadu, India
Fabrizio Palitti and Adayapalam T. Natarajan
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Department of Ecological and Biological Sciences, University of Tuscia, Viterbo, Italy
Chlorophyllin (CHL), a water soluble semisynthetic derivative of the ubiquitous plant pigment chlorophyll used as a food additive, is recognized to confer a wide range of health benefits. CHL has been shown to exhibit potent antigenotoxic, anti-oxidant, and anticancer effects. Numerous experimental and epidemiological studies have demonstrated that dietary supplementation of CHL lowers the risk of cancer. CHL inhibits cancer initiation and progression by targeting multiple molecules and pathways involved in the metabolism of carcinogens, cell cycle progression, apoptosis evasion, invasion, and angiogenesis. The modulatory effects of CHL on the hallmark capabilities of cancer appear to be mediated via abrogation of key oncogenic signal transduction pathways such as nuclear factor kappa B, Wnt/b-catenin, and phosphatidylinositol-3-kinase/Akt signaling. This review provides insights into the molecular mechanisms of the anticancer effects of dietary CHL.
INTRODUCTION Epidemiological studies related to human health and the importance of nutrition have created increased interest in the prevention of age-related diseases such as cancer. Human diet contains a diverse array of food constituents exhibiting protective effects against a wide range of carcinogens. The protective elements in the diet that prevent cancer include selenium, folic acid, vitamin B12, vitamin D, chlorophyll, and antioxidants such as the carotenoids. A balanced diet with these elements in appropriate quantities could significantly reduce the risk of cancer (1,2). Of late, chlorophyllin (CHL), a water soluble, semisynthetic food-grade derivative of the ubiquitous plant pigment chlorophyll has attracted the focus of attention as a potential anticancer agent. CHL is obtained by the alkaline hydrolysis Submitted 22 April 2014; accepted in final form 29 October 2014. Address correspondence to Siddavaram Nagini, Department of Biochemistry and Biotechnology, Faculty of Science, Annamalai University, Annamalainagar-608 002, Tamil Nadu, India. Phone: C91-4144239842. Fax: C91-4144-238145/238080. E-mail: s_nagini@yahoo. com; [email protected]
of chlorophyll and reacts readily with environmental mutagens and carcinogens. CHL has been used as a medicine for more than 50 years without any adverse effects. The U.S. Food and Drug Administration (FDA) has approved over the counter use of CHL as an internal deodorant in doses up to 300 mg/day. CHL is used for controlling body, fecal, and urinary odor in geriatric patients and as an accelerant in wound healing. CHL is also used as a food coloring agent and a health food additive in cakes, beverages, sweets, and ice cream (3,4). Several in vitro and in vivo studies have demonstrated the antigenotoxic, antioxidative, and anticarcinogenic activities of CHL against numerous carcinogens (5–7). This review summarizes molecular mechanisms underlying the anticancer activity of CHL.
CHEMISTRY Chlorophyll contains 4 pyrrole nitrogen rings bonded to a central magnesium atom, and a fifth ring containing carbon atoms and a long phytol tail. The phytol tail of chlorophyll confers hydrophobicity and limits binding efficacy with carcinogens and mutagens. The predominant structural feature that differentiates CHL from chlorophyll (Fig. 1) is the replacement of magnesium ion by copper, methyl, and phytyl ester groups by sodium and potassium, and absence of the phytol tail. The antigenotoxic and anticancer effects of CHL are believed to be mediated via the porphyrin ring either by scavenging free radicals or by forming complexes with planar carcinogens (8,9).
ANTIGENOTOXIC EFFECTS The antigenotoxic effects of CHL have been mainly attributed to its ability to form complexes with polycyclic planar structures of mutagens (10). CHL has been documented to exhibit strong antimutagenic effects against a wide range of mutagens including aflatoxin B1 (AFB1), polycyclic aromatic hydrocarbons (PAHs), heterocyclic amines, and g-radiation 203
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have a protective effect at toxic concentrations of DMBA and to increase the cytotoxicity of DMBA thereby affecting cell proliferation. Co-administration of CHL with anticancer agents such as oxaliplatin was shown to potentially inhibit tumorigenicity with reduced genotoxicity (20). CHL was found to interact with the intercalating agent acridine (ICR 192) and reduce its binding with DNA (21).
ANTICANCER EFFECTS Several in vitro, in vivo, and epidemiological studies have demonstrated the anticancer activities of CHL.
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FIG. 1. Structure of chlorophyllin.
(5,10–12]. In Drosophila melanogaster, CHL suppressed the activity of monofunctional alkylating agents such as nitrosamines and nitrosoureas (13). Recently, Pimentel et al. (14) reported that an increase in the copper content of sodium-copper CHL resulted in a greater inhibition of genetic damage induced by gamma rays in somatic cells of Drosophila. Lagerqvist et al. (11) demonstrated that CHL exerts potent antimutagenic effects against PAH diol epoxides derived from B[a]P, dibenzo[a,h]anthracene (DBA), dibenzo[a,l]pyrene (DBP), and benzo[c]phenanthrene (BPh) in Chinese hamster V79 cells. Administration of CHL was also found to attenuate the formation of DNA adducts in normal human mammary epithelial cells exposed to B[a]P (15). CHL exerted strong anticlastogenic activity against several classes of chemicals including cesium chloride, mercuric chloride, cobalt chloride, chromium (VI) oxide, N-nitroso-N-ethyl urea, and urethane in mammalian cells (16). CHL was found to reduce chromosomal aberrations induced by ethyl methane sulfonate in cultured mammalian cells during S and G2/S phase of the cell cycle as well as the formation of micronuclei (MN) and bone marrow toxicity in sodium nitrite treated mice (17,18). Oral administration of CHL was shown to protect against induction of DNA single strand breaks and MN in B[a] P treated C57BL/6J mice (19). Recently, Grossi et al. (12) evaluated the anticlastogenic effects of CHL on N-methyl-N’-nitro-N-nitrosoguanidine (MNNG) and 7,12-dimethylbenz[a]anthracene (DMBA)induced frequencies of MN in human HepG2 cells in vitro and mouse polychromatic erythrocytes in vivo. CHL was found to have a protective effect against MNNG in vitro when the treatment was given simultaneously, suggesting a direct interaction between MNNG and CHL, whereas with DMBA, CHL significantly reduced the frequencies of induced MN when administered prior to and after carcinogen treatment. In vivo, CHL did not exert any protective effects against either MNNG or DMBA induced MN. However, CHL treatment was found to
In Vitro Studies A number of in vitro studies have analyzed the antiproliferative effects of CHL on various transformed cell lines. CHL was shown to retard the growth and proliferation of MCF-7 breast carcinoma cells by deactivating the extracellular signalregulated kinases (ERKs) and inducing apoptosis (22). Chimploy et al. (23) demonstrated that CHL treatment induced Sphase cell cycle arrest in human colon cancer cells (HCT116) in a p53-independent manner by reducing the expression of DNA synthesis and repair enzymes, ribonucleotide reductase subunits (R1 and R2), and G1/S checkpoint controls. CHL is reported to inhibit malignant transformation induced by transbenzo(a)pyrene-trans-7,8-dihydrodiol-9,10-epoxide (transBPDE) in 16HBE human bronchial epithelial cell line (24,25). CHL was shown to inhibit the growth of HCT116 colon cancer cells by inducing apoptosis (26). It was also demonstrated that CHL induces photocytotoxicity and cell death in T24 and 5637 human bladder cancer cells (27). CHL e4 and CHL f were shown to act as photosensitizers and induce apoptotic cell death in 5637 and T24 human bladder cancer cells (27,28). Konıckova et al. (29) reported the antiproliferative effects of CHL from Spirulina platensis against pancreatic cancer cells in vitro.
In Vivo Studies The first in vivo anticancer activity of CHL was reported in the rainbow trout model by Dashwood et al. (10) who showed a significant reduction in AFB1-induced DNA adducts and hepatic tumors following dietary supplementation of CHL. CHL was shown to attenuate dibenzo(def,p)chrysene and AFB1-induced liver carcinogenesis by reducing tumor incidence, tumor multiplicity, DNA adduct formation and by modulating genes involved in apoptosis, cell proliferation, and invasion in the rainbow trout model (30,31). Co-administration of CHL with probiotic fermented milk was also found to offer significant protection against AFB1-induced hepatocellular carcinoma (5). CHL was also reported to inhibit AFB1, 2amino-3-methylimidazo[4,5-f]quinoline, and DBP-induced
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multiorgan carcinogenesis in the rat and rainbow trout models (32–35). CHL was shown to inhibit the development of PhIPinduced rat mammary tumors by reducing the metabolic activation of PhIP (36). CHL was demonstrated to inhibit the development of DMBA-induced mouse skin papillomas and hamster buccal pouch carcinomas (37–39). CHL administration has also been reported to offer significant protection against DBP-initiated transplacental carcinogenesis (40). In a recent report, we demonstrated inhibition of MNNG induced rat forestomach carcinogenesis by dietary supplementation of CHL (41). Although several studies have demonstrated the anticancer effects of CHL when administered both prior to and simultaneously with the carcinogen, a few studies have reported lack of preventive or therapeutic efficacy with CHL supplementation. Wang et al. (42) demonstrated that dietary CHL administration did not offer protection against 1,2-dimethylhydrazine (DMH)-induced small intestinal and colon carcinogenesis. Orner et al. (43) reported that post-initiation treatment with CHL did not exert any significant effects on AFB1-induced preneoplastic foci in the liver and colon. However, Blum et al. (44) showed that postinitiation treatment with higher concentrations of CHL offers significant protection against DMHinduced rat colon carcinogenesis.
Epidemiological Studies A double-blinded placebo-controlled intervention trial conducted by Egner et al. (45) in Qidong, People’s Republic of China revealed that CHL consumption for over a period of 4 mo led to 50% reduction in the urinary excretion of AFB1-DNA adducts among individuals at high risk for the development of hepatocellular carcinoma. In another randomized double-blinded clinical trial, copper chlorine e(4) ethyl ester and copper chlorine e(4) derivatives were identified in the serum of study participants, suggesting that these 2 components may be involved in preventing carcinogen absorption from the gut and for the chemoprotective effects of CHL (46). An unblinded cross-over study conducted by Jubert et al. (47) among 4 human volunteers demonstrated rapid uptake and urinary elimination of AFB1. Treatment with chlorophyll and CHL significantly impeded AFB1 absorption in these subjects suggesting that coconsumption may limit the bioavailability of ingested aflatoxin in humans. More recently, Shaughnessy et al. (48) conducted a crossover design study to explore the effects of CHL administration on fried meat associated carcinogenesis, which revealed that CHL supplementation to individuals consuming high-temperature meat diet containing high levels of heterocyclic amines reduced the levels of hydrolyzed and unhydrolyzed urinary and faecal mutagens. In addition, CHL administration also reduced DNA damage in the target colorectal cells.
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MECHANISM UNDERLYING ANTICANCER EFFECTS Whole genomic microarray analysis of the changes in gene expression pattern revealed that dietary supplementation of CHL significantly modulates the expression profiles of 104 genes that are predominantly associated with cell adhesion, cell–cell communication, invasion, and transforming growth factor beta (TGFb) signaling in DMBA-induced hamster buccal pouch carcinogenesis model (39). Studies by John et al. (49) have also indicated that CHL administration to B[a]P treated NHMECs modulated the expression of 30 genes most of which were associated with xenobiotic metabolism, cell signaling, cell motility, cell proliferation, cell cycle control, metabolism, DNA repair, and apoptosis pathways. Proteomic analysis by MALDI-TOF-TOF-MS following addition of sodium iron CHL to human hepatoma 3B cells identified 32 threefold differentially expressed proteins that are involved in cell proliferation, apoptosis, antioxidant function, and signal transduction (50). Table 1 summarizes the anticancer effects of CHL. A detailed discussion on the modulatory effects of CHL on various oncogenic signaling pathways and processes ensues. Xenobiotic-Metabolizing Enzymes and Antioxidants A large proportion of environmental carcinogens such as PAHs are metabolically activated to form highly reactive electrophilic intermediates that bind covalently to DNA to form adducts that are normally repaired by DNA repair enzymes. Failure to repair these adducts before replication causes mutations eventually culminating in neoplastic transformation. The probability that a carcinogen will reach a target cell and cause DNA damage is dependent upon the balance between carcinogen activation catalyzed by phase I cytochrome P450 (CYP) enzymes and detoxification accomplished by the Phase 2 enzymes predominantly glutathione S-transferases, key regulators of various cytoprotective antioxidant, and detoxifying enzymes (51). Coordinated induction of phase II detoxifying enzymes, antioxidants, and DNA repair genes by the transcription factor nuclear factor erythroid-2 related factor-2 (Nrf2) serves to eliminate toxic electrophilic intermediates and reactive oxygen species generated by the metabolism of xenobiotics thereby preventing malignant transformation [52]. In a recent study, we demonstrated a reduced expression of phase I CYP enzymes with decreased formation of 8hydroxy-2’-deoxyguanosine, an indicator of oxidative stress-induced DNA damage following dietary supplementation of CHL in the DMBA-induced hamster buccal pouch carcinogenesis model [53]. This was associated with upregulation of the Phase 2 detoxification enzymes glutathione S-transferases and NAD(P)H:quinone oxidoreductase 1, the antioxidant enzymes superoxide dismutase, catalase, and glutathione peroxidase, and the DNA repair enzymes 8oxoguanine glycosylase 1, xeroderma pigmentosum, and xray repair cross complementing group 1. Analysis of the
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TABLE 1 Summary of the anticancer effects of chlorophyllin Type of study In vitro
Cell line/animal tumor model/ human subjects B(a)P exposed normal mammary epithelial cells (NHMECs)
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Human bladder cancer cells Human breast cancer cells, MCF-7
Human bronchial epithelial cells, 16HBE Human colon cancer cells, HCT116
In vivo
AFB1-induced rat hepatocarcinogenesis /multi-organ carcinogenesis
AFB1 and dibenzo(def,p)chryseneinduced rainbow trout liver carcinogenesis DBP-induced multiorgan carcinogenesis DMBA-induced hamster buccal pouch carcinogenesis
DMBA-induced mouse skin papillomagenesis MNNG-induced forestomach carcinogenesis
Mechanisms
Reference(s)
Altered expression of genes involved in cell proliferation, apoptosis, and signaling pathways Reduced CYP1A1 and CYP1B1 expression Decreased BPdG adduct Apoptosis induction Cell cycle arrest at G0/G1 phase Deactivation of extracellular signal regulated kinases Downreguation of cyclin D1 and Bcl-2 Downregulation of E-cadherin, cyclin D1 and cyclin E expression Induction of S-phase cell cycle arrest Downregulation of DNA repair and synthesis enzymes Increased E-cadherin and alkaline phosphatase expression Augmentation of b-catenin accumulation in plasma membrane Upregulation of bid, caspase-8 and caspase-3 Complex -mediated reduction of carcinogen uptake Decreased AFB1-DNA adduct formation Reducing lipid peroxidation Enhancing antioxidant enzymes Modulation of c-myc, bax, bcl-2, cyclin-D1, p53 and rasp-21 Inhibition of carcinogen - DNA adduct formation
(24,65)
Inhibition of DBP-DNA adduct formation
(44–47) (39)
(42) (40,43)
(13,45,48)
(46, 47)
(49,55)
Attenuation of NF-kB, Wnt/b-catenin, PI3K/Akt, and TGF-b signaling Activation of Nrf2/Keap-1 signaling Downregulation of CYP and 8-OHdG expression Upregulatiion of antioxidant, detoxification and DNA repair enzymes Induction of intrinsic apoptosis Modulation of genes associated with cell adhesion, cell-cell communication and angiogenesis Increased GST and GSH activity Altered expression of biotransformation enzymes Inhibition of TGFb signaling
(55,56,69)
(54) (61) (70)
(continued on next page)
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TABLE 1 Summary of the anticancer effects of chlorophyllin (Continued) Type of study
Cell line/animal tumor model/ human subjects
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DMH-induced rat colon carcinogenesis IQ-induced rat multi-organ carcinogenesis IQ and DMH-induced rat colon carcinogenesis
Human intervention
PhIP-induced rat mammary carcinogenesis Individuals at high risk for hepatocellular carcinoma Healthy volunteers Healthy volunteers
Mechanisms Downregulation of COX-2 expression Inhibition of NF-kB signaling Inhibition of IQ-DNA adduct formation Downregulation of Ctnnb1 and APC mutations Reduced b-catenin and c-jun expression Decreased cell proliferation Increased apoptosis Inhibition of PhIP-DNA binding
Reference(s)
(50,51) (47,60)
(53)
Increased urinary elimination of aflatoxin B1
(61)
Reduced urinary excretion of aflatoxin-DNA adduct Diminished excretion of hydrolysed and unhydrolyzed urinary and faecal mutagenicity Inhibition of DNA damage
(63) (64)
AFB1 D aflatoxin B1; B[a]P D benzo[a]pyrene; DMBA D 7,12-dimethylbenz[a]anthracene; DMH D 1,2-dimethylhydrazine; IQ D 2-amino-3-methylimidazo [4,5-f]quinolone; MNNG D N-methyl-N’-nitro-N-nitrosoguanidine; NF-kB D nuclear factor kappa B; Nrf2 D nuclear factor erythroid-2 related factor-2; PI3K D phosphatidylinositol-3-kinase; TGFb D transforming growth factor beta.
mechanism underlying the protective effects of CHL revealed nuclear translocation of Nrf2 with downregulation of its negative regulator, Keap-1, providing substantial evidence that CHL drives Nrf2/Keap1 signaling with consequent transactivation of genes involved in promoting DMBA detoxification and protection against DNA damage. In human umbilical vein endothelial cells, CHL was reported to confer protection against oxidative stress by inducing the Nrf2 signaling pathway (54,55). These studies provide a mechanistic basis for the potent antioxidant function of CHL. CHL is documented to confer a higher degree of protection against free radicals than other known antioxidants such as ascorbic acid and glutathione (56). Several studies have demonstrated the antioxidant activity of CHL against a wide range of oxidant species including superoxide anion radical (O2─), hydroxyl radicals (OH), deoxyribose peroxyl radicals (ROO), ABTS radicals (2,2’-azino-bis(3-ethylbenzthia-zoline-6-sulphonic acid), singlet oxygen (1O2), and peroxynitrite. The high reaction rate constant and formation of face-to-face complexes between CHL and carcinogens have been suggested to contribute to the antioxidant activity of CHL (56–59). The mechanisms underlying the radioprotective and anticancer effects of CHL include free radical scavenging, decreased lipid and protein oxidation, restoration of enzymic antioxidants, and enhanced DNA repair (57,60).
Effects on Cell Proliferation and Differentiation Studies on a panel of cancer cell lines have provided evidence that the antiproliferative effects of CHL are mediated by inducing G0/G1 or S phase cell cycle arrest (22–26,30). Chiu et al. (22) demonstrated that CHL treatment inhibits the proliferation of MCF-7 breast cancer cells by deactivating ERKs, a subfamily of mitogen activated protein kinases that play a key role in cell survival, proliferation, and differentiation. This was associated with depletion of cyclin D1 and increase in cells in the G0/G1 phase of the cell cycle. CHL treatment was shown to inhibit trans-BPDE induced malignant transformation in 16HBE human bronchial epithelial cells by reducing the expression of E-cadherin and cyclins (24,25). CHL administration was also reported to promote cell differentiation in HCT116 colon cancer cells by modulating the expression of E-cadherin and alkaline phosphatase (26). Effects on Apoptosis Apoptosis, a form of programmed cell death, plays a crucial role in maintaining tissue homeostasis. Endogenous apoptotic death machinery is principally activated via 2 major signaling pathways, the extrinsic and the intrinsic pathway that converge at the activation of caspases to cleave substrates vital for cell survival (61,62). Several phytochemicals induce apoptosis via the intrinsic pathway by promoting mitochondrial outer membrane permeabilization with release of intermembrane space
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proteins such as secondary mitochondria-derived activator of caspase (Smac)/direct inhibitor of apoptosis protein (IAP) binding protein with low pI (DIABLO) and cytochrome C into the cytosol. Members of the Bcl-2 and IAP family proteins function as key regulators of intrinsic apoptosis. In a recent study, we have shown that dietary CHL supplementation induces intrinsic apoptosis in a hamster model of oral oncogenesis by modulating the expression of pro- and antiapoptotic members of the Bcl-2 family suggesting that CHL promotes Bax oligomerization and mitochondrial outer membrane permeabilization. CHL was also shown to induce the efflux of the apoptogenic molecules cytochrome C and Smac/DIABLO into the cytosol, whilst simultaneously downregulating the expression of optic atrophy 1, a profusion dynamin-related protein of the inner mitochondrial membrane that modifies the morphology of the mitochondrial cristae. Furthermore, CHL supplementation activated the caspase cascade and induced cleavage of the DNA repair enzyme polyADP ribose polymerase. CHL was found to enforce nuclear localization of the IAP protein survivin, an event that facilitates apoptosis by inhibiting caspases and inducing p53 and Bax (38). These findings are substantiated by reports of CHL-induced apoptosis induction in cancer cells in vitro and in animal tumor models in vivo (26,32,35,63). Thus, it appears that CHL promotes apoptosis by modulating the expression of a multitude of molecules involved in the apoptotic signaling cascade.
Effects on Matrix Invasion and Angiogenesis Invasion and angiogenesis, hallmark capabilities of malignant tumors play a central role in promoting cancer progression and metastasis. Tumor invasion is regulated by a delicate balance between matrix metalloproteinases (MMPs) that catalyze proteolytic degradation of the extracellular matrix and their inhibitors that control extracellular matrix breakdown (64,65). MMP activation is also recognized to trigger angiogenic signaling through the release of several proangiogenic factors that promote neovascularization such as hypoxiainducible factor 1a (HIF-1a), vascular endothelial growth factor (VEGF), platelet-derived growth factor, and interleukins [66]. CHL treatment was found to downregulate the expression of interleukin 1 beta, a proinflammatory cytokine with a proangiogenic role in human mammary cell lines exposed to B[a]P (15). Studies from our laboratory have demonstrated that CHL administration inhibits tumor invasion and angiogenesis during DMBA-induced hamster buccal pouch carcinogenesis by upregulating tissue inhibitor of MMP-2 expression, and downregulating MMP-2, MMP-9, HIF-1a, VEGF, and VEGF receptor R2 (39,67). Accumulating evidence indicates that the modulatory effects of CHL on cell proliferation, apoptosis evasion, invasion, and angiogenesis are mediated by abrogation of key oncogenic signal transduction pathways such as nuclear factor kappa B (NF-kB), Wnt/b-catenin, and
phosphatidylinositol-3-kinase/protein kinase B (Akt) PI3K/ Akt signaling (38,67,68).
Modulation of Oncogenic Transcription Factors and Signaling Pathways NF-kB, a prosurvival transcription factor, is localized in the cytoplasm as a heterodimer of p50 and p65, sequestered by inhibitor of kappa B (IkB). Phosphorylation of IkB by IkB kinase beta (IKKb) followed by ubiquitination and proteasomal degradation releases the NF-kB heterodimer, which subsequently translocates to the nucleus, binds to kB elements in DNA, and regulates the expression of about 500 genes involved in cell proliferation, apoptosis, invasion, metastasis, and angiogenesis (69). CHL supplementation was shown to inhibit the development of colonic neoplasms in DMH-treated mice by selectively inhibiting the NF-kB signaling pathway (70). CHL was also found to prevent the activation of NF-kB and its binding to the kB elements in DNA in lipopolysaccharide-stimulated murine splenic mononuclear cells and RAW 264.7 and to restore IkB-a in the cytosolic extract of lipopolysaccharidetreated cells (71). A study from this laboratory demonstrated that dietary CHL prevents the development of DMBA-induced hamster buccal pouch carcinomas by inactivating NF-kB signaling via inhibition of IKKb mediated phosphorylation and degradation of IkB, thereby impairing the nuclear translocation of NF-kB and blocking transactivation of genes involved in malignant transformation and progression (38). Aberrant activation of TGFb signaling stimulates tumor progression by facilitating acquisition of the essential hallmark capabilities of cancer cells (72). Recently, we reported that dietary CHL inhibits the development of MNNG-induced forestomach carcinomas by downregulating the expression of TGFb RI, TGFb RII, and Smad 2 and 4 and upregulating Smad 7, thereby abrogating canonical TGFb signalling. Furthermore, attenuation of TGFb signaling by CHL also blocked cell proliferation, angiogenesis, invasion, and metastasis, and induced mitochondria mediated cell death (41). The canonical Wnt/b-catenin signaling pathway that plays a key role in the regulation of various cellular processes such as cell proliferation, apoptosis, epithelial-mesenchymal transition, and angiogenesis is one of the prominent pathways influenced by NF-kB (73). Dietary supplementation of CHL was shown to block the prosurvival Wnt signaling pathway by downregulating PI3K/Akt with consequent inhibition of glycogen synthase kinase-3b phosphorylation thereby preventing nuclear translocation of b-catenin (67). These findings are substantiated by Carter et al. (74) who reported that CHL limits tumor invasion and metastasis by facilitating b-catenin trafficking away from the nucleus into the plasma membrane via the Hakai pathway. We demonstrated a positive correlation between blockade of PI3K/Akt and Wnt/b-catenin signaling by CHL and inhibition of tumor angiogenesis in the hamster
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buccal pouch carcinogenesis model. In particular, CHLinduced downregulation of PI3K/Akt could inactivate HIF-1a, a master regulatory transcriptional factor and enhancer of VEGF signaling (67). CONCLUSION CHL has gained considerable attention in recent years owing to its high safety and efficacy without any adverse side effects. Numerous studies in a panel of human cancer cell lines and in a variety of experimental animal models have revealed that CHL influences multiple molecules and pathways involved in the metabolism of carcinogens, antioxidant defenses, cell proliferation, apoptosis, invasion, and angiogenesis to exert its chemopreventive effects. Thus dietary phytochemicals such as CHL that affect multiple signal transduction pathways involved in cancer initiation and progression hold promise as ideal candidates for cancer chemoprevention and therapy. However, caution is still warranted for clinical application, and extensive investigations on optimal dose, metabolism, bioavailability, tissue distribution, pharmacokinetics, interference with endogenous metabolic pathways, and crosstalk between signaling circuits are necessary before therapeutic utilization of CHL. ACKNOWLEDGMENT The authors thank P. Thiyagarajan for help with the illustrations. FUNDING Financial assistance from the Department of Biotechnology, New Delhi, India and European Commission FUNCFOOD (FP7-KBBE-3-245030) under the 7th Framework of the Indo-EU Joint Collaborative Project on FUNCFOOD is gratefully acknowledged. REFERENCES 1. Riboli E: The role of metabolic carcinogenesis in cancer causation and prevention: evidence from the European Prospective Investigation into Cancer and Nutrition. Cancer Treat Res 159, 3–20, 2014. 2. Gonzalez-Vallinas M, Gonzalez-Castejon M, Rodrıguez-Casado A, and Ramırez de Molina A: Dietary phytochemicals in cancer prevention and therapy: a complementary approach with promising perspectives. Nutr Rev 71, 585–599, 2013. 3. PDR for Nutritional Supplements: In Chlorophyll/Chlorophyllin, Hendler SS and Rorvick,D (ed.). Montvale, NJ: Medical Economics Company, 2001, pp. 88–90. 4. Tumolo T and Lanfer-Marquez U: Copper chlorophyllin: a food colorant with bioactive properties? Food Res Int 46, 451–459, 2012. 5. Ferguson LR, Philpott M, and Karunasinghe N: Dietary cancer and prevention using antimutagens. Toxicology 198, 147–159, 2004. 6. Williams DE: The rainbow trout liver cancer model: response to environmental chemicals and studies on promotion and chemoprevention. Comp Biochem Physiol C Toxicol Pharmacol 155, 121–127, 2012.
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