Effects of Sanguinarine and Chelerythrine on the Cell Cycle and Apoptosis

OBJECTIVES: This review summarizes the involvement of sanguinarine and chelerythrine in cell cycle regulation and cell death in various cell lines. It is focused on their potential in the treatment of cancer. METHODS: We conducted a search of PubMed, ScienceDirect and Medline for papers on the molecular mechanisms of the biological activity of sanguinarine and chelerythrine published mainly from 1995 to 2006. RESULTS AND CONCLUSIONS: Our analysis of the published studies suggested that these alkaloids are not only good candidates for chemotherapeutic regimens but may also contribute to the development of successful immune therapies of some carcinomas due to their apoptotic potential. However, the complete signalling cascade in which sanguinarine and chelerythrine treatment induces apoptotic cell death is not yet understood. Overall, the results of recent studies suggest that sanguinarine and chelerythrine may be useful as agents in the management of cancer.

From the chemistry point of view, both alkaloids interconvert between the cationic vs. neutral form (i.e.hydroxide adduct or pseudobase).They penetrate the cell membrane in the form of nonpolar pseudobase 3 .The iminium bond, C 6 =N + in the cationic form is susceptible to nucleophilic attack and plays a key role in inhibition of SH-proteins 4 .The binding of SA and CHE with human serum albumin and L-cysteine is radically weaker at pH 5.0 than at pH 7.4.This observation is the basis for the conclusion 5 that the neutral form (pseudobase) of SA and CHE can interact with proteins, it is involved in the interactions with cellular biomacromolecules and may elicit a cytotoxic response 6 .SA and CHE forms in vitro DNA adducts via modification of the C (11) =C (12) bond (calf thymus DNA, rat hepatic microsomes, NADPH, pH 7.4) detectable by 32 P-postlabelling 7 .
QBA fractions from S. canadensis (SANGUINARIA) and M. cordata (SANGUIRITRIN) are used in toothpastes and mouthwashes as antiplaque agents.SANGUIRITRIN is applied as a veterinary preparation for mastoiditis in cows 8 .The QBA fraction from the part of M. cordata, containing SA and CHE is an active component of the preparation Sangrovit ® as an additive to animal feeds.
Despite the above, SA, dihydrosanguinarine (DHSA) and CHE are considered to be the toxic components of the Argemone mexicana seed oil 9 .Several studies have suggested that singlet oxygen and hydroxyl radicals are involved in argemone oil toxicity.The role of QBA in the genesis of epidemic dropsy syndrome (A.mexicana poisoning) has not been explicated to date 10 , although SAmediated DNA damage in vitro 11 (Fig. 1) and in vivo [12][13][14] has been published.In studies focused on SA-mediated DNA damage in mice, the minimum genotoxically ef-Scheme 1.
fective SA concentration (10 mg/kg body weight) was found after administration of a single intraperitoneal dose.The authors referred to benz[c]acridine (3,4-benzacridine) as the principal toxic agent.This compound has been considered as the only metabolic product of SA biotransformation 15,16 .Psotová et al. failed to confirm the formation of benz[c]acridine as a metabolite of SA in rats after administration of a single dose of SA (10 mg.kg -1 ) (ref. 17 ).DHSA was always found simultaneously with SA in the plasma and liver samples and its concentration was higher than SA.It is hypothesized that formation of DHSA is the first step in SA detoxification in rats.The same SA detoxification system was also found in a plant tissue culture (Eschscholzia californica) (ref. 18).The reduction of SA to the DHSA form probably starts in the gastrointestinal tract by intestinal reductases 19 .After absorption of both substances, SA is further reduced by nonspecific cytosolic and microsomal reductases 17 .Vecera et al. reported that SA was distributed to all rat organs using [ 3 H]-SA and nearly 98% of orally administered SA was excreted in the faeces 20 .
This aside, the biological activities of SA and CHE are of particular interest in molecular biology and medicine.This review summarizes recent in vitro studies focused on the molecular mechanisms of SA and CHE effects on programmed cell death in a variety of human cell line models.

Effects on programmed cell death
In the last few years, clinical trials using plant-derived drugs for prevention and/or treatment of tumors have become increasingly widespread in cancer therapy.Search for novel agents designed to induce cell cycle arrest and apoptosis in cancer cells is being seriously pursued.QBAs affect eukaryotic cells in many ways and several cellular targets for their action have recently been established.The interaction of compounds with organelles and other intracellular targets depends on both cellular permeability and intracellular distribution 21 .In recent years, QBAs (especially SA) have gained increasing attention as potential agents in the treatment of cancer.SA and CHE have been reported to exert cell growth-inhibitory effects via the induction of apoptosis in a variety of cancer cells [22][23][24][25] .The ability of tumor cells to evade apoptosis plays a significant role in their resistance to conventional therapeutic regimens 26 .Apoptosis is a term used to describe the terminal morphological and biochemical events seen in programmed cell death (PCD) (ref. 27).Cell death as well as cell proliferation and differentiation are important for homeostasis.Cells undergoing PCD are characterized by morphological changes, including cellular shrinkage, plasma and nuclear membrane blebbing, organelle relocalization and compaction, nuclear DNA condensation with or without fragmentation, and hypersegmentation of nuclear chromatin of irregular size [28][29][30] .These hypersegmented nuclear structures may then bud from the rapidly blebbing cell surface to form "apoptotic bodies" 31 .Three mechanisms are known to be involved in the apoptotic process: (i) a receptor-ligand mediated mechanism, (ii) a mitochondrial pathway and (iii) a mechanism in which the endoplasmic reticulum plays a central role.All three mechanisms activate caspases which are responsible for the characteristic morphological changes observed during apoptosis 32 .Cell death marked by cellular swelling should be called oncosis.Necrosis is the cell death as a result of direct injury, usually beginning at the cell surface.Necrotic cells exhibit early lysis of the plasma membrane before any significant alterations in nuclear morphology.The surface features of necrosis are also very different from those of apoptosis.Necrotic cells swell and lyse, whereas apoptotic cells show intense cell surface zeiotic blebbing 31 .The genetic basis for apoptosis implies that cell death, like any other metabolic or developmental program, can be disrupted by mutation.In fact, defects in apoptotic pathways are now thought to contribute to a number of human diseases, ranging from neurodegenerative disorders to malignancy 32 .A number of studies have revealed a high frequency of apoptosis in spontaneously regressing tumors and in tumors treated with cytotoxic anticancer agents 33 .There is a premise that anticancer agents induce apoptotic cell death and this implies that cellular responses occurring after the drug-target interaction can have impact on drug-induced cell death 34 .

SA/CHE-mediated activation of caspase-3 and depletion of GSH
The caspase-cascade system plays a vital role in the induction, transduction and amplification of intracellular apoptotic signals.Caspases, closely associated with apoptosis, are aspartate-specific cysteine proteases and members of the interleukin-1β-converting enzyme family.ii) cell cycle arrest 22 ; iii) the mitochondrial damage 25 .
The activation and function of caspases, involved in the delicate caspase-cascade system, are regulated by various kinds of molecules, such as the inhibitor of apoptosis protein, Bcl-2 family proteins, Ca 2+ , and calpain 45 .Generally, there are two pathways through which the caspase family proteases can be activated: (i) one is the death signalinduced, death receptor-mediated pathway; (ii) the other is the stress-induced, mitochondrion-mediated pathway.Not all caspases are involved in apoptosis.The caspases that have been well described are caspase-3, -6, -7, -8, and caspase-9 (ref. 46,47 .Caspase-3 is a key factor in apoptosis execution.The proactive form, procaspase-3, can be activated by caspase-3, -8, -9, -10, cysteine protease protein of molecular mass 32 kDa (CPP32), granzyme B (Gran B), Effects of sanguinarine and chelerythrine on the cell cycle and apoptosis and others.The downstream substrates of caspase-3 include procaspase-3, procaspase-6, procaspase-9, DNAprotein kinase (DNA-PK), protein kinase C (PKCγ), poly(ADP-ribose) polymerase (PARP), D4-GDP-dissociation inhibitor for Rho family GTPases (D4-GDI), and various components of the cytoskeleton.The mitochondrial damage causes the release of cytochrome c and activation of Apaf-1 and thereby initiating activation of the caspase machinery that, in fact, is believed to be the execution phase of apoptosis 48 .SA treatment (0.1, 0.25, 0.5, 1 and 2 μM) was found to result in increased levels of cytochrome c and Apaf-1 and in a significant increase in the active form of caspase-3, caspase-7, caspase-8, and caspase-9 in a dose-dependent manner in human immortalized HaCaT keratinocytes 25 .This process was accompanied by cleavage of the caspase-3 substrate PARP and subsequent DNA degradation and apoptotic death.These results clearly suggest the role of mitochondrial events in SA-mediated apoptosis of HaCaT keratinocytes.Treatment with SA (2.1 μM and 4.2 μM) for 4 h induced apoptosis displayed by cell surface blebbing and caspase-3 activation was confirmed by induction of proteolytic cleavage of PARP in human papillomavirus (HPV) type 16-immortalized endocervical cells 38 .On the other hand, concentrations of SA (8.5 μM and 16.9 μM) caused oncosis characterized by cell swelling, vacuolation, enhanced permeabilization of cytoplasmatic membrane and inactivation of caspase-3 (ref. 38).A similar effect of both alkaloids, SA and CHE, was found on a primary human uveal melanoma OCM-1 cell line 39 .At lower concentrations (2.7 and 10 μM) of SA or CHE, apoptosis was found in the cells, whereas for higher doses of alkaloids (20 μM) necrosis was the dominant form of cell death.SA treatment (IC 50 0.9-3.3μM) notably inhibited the growth of human melanoma M4Beu, breast cancer MCF-7 and prostate cancer PC-3 cell lines without difference in cytotoxicity against normal human fibroblasts and cancer cells 36 .This effect was related to a rapid apoptotic response subsequent to GSH depletion and caspase-3/7 activation.SA-mediated caspase-3 activation was also seen in a human erythroleukemia K 562 cell line in the apoptosis-inducing concentration (4 μM) (ref. 41).

SA-induced targeting of Bcl-2 protein family
Members of the Bcl-2 protein family regulate and execute many cell intrinsic apoptotic pathways, including those arising from dysregulated expression of cellular oncogenes.The Bcl-2 family members can be divided into three subfamilies classified according to their content of Bcl-2 homology (BH) domains: (i) the anti-apoptotic members (Bcl-2, Bcl-X L , Mcl-1, Bcl-1-w, Ced-9, etc.) share all four BH domains; (ii) the multi-BH domain pro-apoptotic members (Bax, Bak, Bok, Bcl-X S ) contain BH1-3 domains; (iii) a diverse group of loosely related pro-apoptotic proteins (Bid, Bik, Bad, Bim, PUMA, NOXA, etc.) only have a BH3 domain.
In human leukaemia JM1 and K 562 cell lines SAinduced apoptosis with classic morphologic changes was observed at low concentrations (4 μM) and cell death comparable to oncosis was found at high concentrations (34 μM) (ref. 40).SA-treated K 562 cells showed a significant increase in expression of the pro-apoptotic Bax protein during apoptosis.It has been reported that SA probably targets a very efficient up-stream Bax activation system 40 .Treatment of human neuroblastoma SH-SY5Y cells with CHE (2.5 and 5 μM) for 16 h induced a substantial decrease in mitochondrial membrane potential and triggered apoptosis through a mechanism involving direct targeting of Bcl-2 family proteins 43 .CHE was found to act as an antagonist to the anti-apoptotic protein Bcl-X L (ref. 43) and to inhibit protein kinase C (ref. 49).Both SA and CHE were also shown to inhibit anti-apoptotic mitogen-activated protein kinase phosphatase 50 .The effect of SA treatment (0.1-2 μM for 24 h) was also evaluated on the levels of Bax and Bcl-2 protein expression in immortalized human keratinocytes (HaCaT) (ref. 25).SA treatment of HaCaT keratinocytes resulted in significant decrease in the levels of anti-apoptotic Bcl-2 protein and a dose-dependent increase in the pro-apoptotic Bax protein.SA caused apoptotic death of HaCaT cells by the release of cytochrome c from mitochondria into the cytosol and the activation of Apaf-1 in the cytosol.

SA-mediated alterations in cell cycle
Cell-cycle progression relies on activation of cyclins and cyclin-dependent kinases (CDKs), which successively act together in G1 to initiate the S phase and in G2 to initiate mitosis.To prevent abnormal proliferation, cyclin-CDK complexes are precisely regulated by two families of cell cycle inhibitors that block their catalytic activity 51 .Since uncontrolled cyclin-dependent kinase activity is often the cause of human cancer, their function is tightly regulated by cell-cycle inhibitors such as the p21 Waf1/Cip1 and p27 Kip1 proteins.Following anti-mitogenic signals or DNA damage, p21 Waf1/Cip1 and p27 Kip1 bind to cyclin-CDK complexes to inhibit their catalytic activity and induce cell-cycle arrest 52 .Adhami et al. 22 focused on the antiproliferative effects of SA on growth/proliferation of human prostate cancer cells and the involvement of cell cycle regulatory events as the mechanism of this response.SA treatment (0.2-2 μM for 24 h) in both cancer cell lines resulted in (i) significant induction of CDK inhibitors p21 Waf1/Cip1 and p27 Kip1 in total levels; (ii) down-regulation of cyclin D 1 , D 2 and E; (iii) and down-regulation of CDK 2, 4, and 6.SA imparted cell cycle dysregulation in both androgen-sensitive (LNCaP) and androgen-insensitive (DU-145) human prostate carcinoma cells via up-regulation of CDK inhibitors involved in G0/G1 progression of cell cycle.The authors found that SA caused cell cycle blockade and apoptosis of prostate cancer cells irrespective of their androgen status.
Holy et al. 37 investigated the responses of cell cycle regulatory molecules to SA (5-10 μM), using immunocytochemical methods that visualized cyclin D 1 and topoisomerase II behavior in MCF-7 breast cancer cell line.SA-mediated cellular events resulted in extended cell cycle arrest in G0/G1 and inhibition of cell proliferation.The growth inhibition was accompanied by a striking re-localization of cyclin D 1 and topoisomerase II from the nucleus to the cytoplasm.SA was incorporated by cells and concentrated in the nucleus within minutes, where it rapidly inhibited DNA synthesis.This model suggested that SA may help suppress malignant cell growth at subapoptotic concentrations.
Recent study analyzed the antiproliferative activity of SA in relation to its effects on mitosis and microtubule assembly.SA did not cause the arrest of cell cycle progression at mitosis but it induced aggregation of tubulin in the presence of microtubule-associated proteins.The binding of SA to tubulin induced conformational changes in tubulin 53 .One of the major obstacles to effective drug action is the efflux of drugs after their entry into cells by protein pumps such as P-glycoprotein and multiple resistance protein 1(ref. 54).As SA binds tightly to tubulin, it may be difficult for the efflux machinery to pump out drugs.Thus, the tight binding of SA to tubulin may be beneficial in cancer chemotherapy 53 .

SA-mediated activation of NF-κB
NF-κB is a nuclear transcription factor that regulates expression of a large number of genes involved in the regulation of apoptosis, tumorigenesis, inflammation, atherosclerosis, viral replication and many autoimmune diseases 55 .NF-κB is activated by a variety of stimuli that include cytokines, their receptors, growth factors, cell adhesion proteins, lymphokines, radiation, pharmacologic agents, and stress.In its inactive form, NF-κB is sequestered in the cytoplasm, bound inhibitor proteins of the IκB family.The various stimuli that activate NF-κB cause phosphorylation of IκB, which is followed by its ubiquinitation and degradation.This results in exposure of the nuclear localization signals on NF-κB subunits and the subsequent translocation of the molecule to the nucleus.In the nucleus, NF-κB binds with the consensus sequence of various genes and thus activates their transcription 56 .
SA has been found to be a potential candidate for intervening in NF-κB-dependent pathological responses 57 .Treatment of human myeloid ML-1a cells with tumor necrosis factor rapidly activated NF-κB and this activation was completely suppressed by SA in a dose-and time-dependent manner.SA did not inhibit the binding of NF-κB protein to the DNA but inhibited the pathway leading to NF-κB activation.Chelerythrine had no effect on NF-κB activation

CHE-/SA-mediated apoptosis induced by generation of reactive oxygen species
Reactive oxygen species (ROS) production is known to play an essential role in mediating cytochrome c release and subsequent apoptosis in cardiac myocytes 58 .It was demonstrated that antioxidant N-acetyl-L-cysteine (NAC) could significantly inhibit the release of cytochrome c.
Yamamoto et al. investigated that CHE, inhibitor of PKC, rapidly and potently induced cell death in neonatal rat cardiac myocytes 44 .It was found that generation of ROS rather than inhibition of PKC critically mediated CHE-induced rapid myocyte apoptosis.Cardiac myocyte death by CHE at 6-30 μM was accompanied by many characteristic findings of apoptosis, including pyknosis and cell shrinkage, posphatidyl serine externalization and activation of caspases.Another unique aspect of CHEinduced cell death was the fact that the morphological changes and myocyte death were strictly concentration-dependent.Typical cardiac myocyte apoptosis was induced at concentrations within a narrow range (between 6 and 30 μM), which was commonly used to block activities of PKC in cardiac myocytes.CHE caused cytochrome c release from mitochondria and this was significantly inhibited in the presence of NAC, suggesting that ROS mediate CHE-induced cytochrome c release.CHE induced cardiac myocyte apoptosis more rapidly than exogenously applied H 2 O 2 .These results suggest that CHE rapidly induced cardiac myocyte apoptosis and that ROS production, possibly H 2 O 2 , and subsequent cytochrome c release from mitochondria play an important role in mediating CHE-induced rapid cardiac myocyte apoptosis.CHE was shown to be a useful positive control for determination of cardiac myocyte apoptosis and a model for explaining the signalling mechanism including potent apoptosis in cardiac myocytes 44 .
It has been reported that SA induced apoptosis which depends on the production of nitric oxide (NO) and superoxide radicals in prostate cancer cells 35 .This study was focused on cyclooxygenase-2 (COX-2) expression which may rescue prostate cancer cells from SA-induced apoptosis by a mechanism involving inhibition of NO synthase (NOS) activity.These findings support the suggestion that coadministration of COX-2 inhibitors with SA may be developed as a strategy in the management of prostate cancer 35 .

CONCLUSION
Successful resolution to the design of agents that effectively induce tumor regression lies, at least in part, in overcoming the inherent resistance in many transformed cells to undergo apoptosis.Owing to their apoptotic potential these alkaloids are hypothesized to be not only good candidates for chemotherapeutic regimens, but may also contribute to the development of successful immune therapies of some carcinomas.SA itself or some of its derivatives might be good candidates as pro-apoptotic drugs for cancer therapy.However, the complete signalling cascade by which SA and/or CHE treatment induces apoptotic cell death is not yet elucidated.Overall, the results of recent studies suggest that SA and CHE could be developed as agents in the management of cancer.