Studies On The Cell Cycle Arrest And Apoptosis Induced By Tetrazolium Violet In C6 And C127 Cancer Cells

Background:Cancer remains one of the leading causes of death throughout the world, according to the analysis of WHO, although great efforts have been made. In the last decade of 20^th century, many new approaches to control malignant cancer have been developed, which includes conventional methods consisting of surgery, radiotherapy, and chemotherapy, and the lately used immunotherapy and gene therapy. Surgery, although being most commonly used in clinical practice, could not get rid of residue cells mat are often seen in the advanced tumors. And, conventional chemotherapy or radiotherapy are strictly limited for that it does not have a satisfying site targeting ability, which is associated with a higher risk of unacceptable toxicity to normal tissues. Therefore, development of the therapeutic agents mat may specifically target the cancer cells while leaving normal counterparts spared, and sensitize tumor cells to chemotherapeutic agents has long been one of the main aims of oncologists and other researchers in the fields. Recently, Kong et al found that tetrazolium violet could inhibit the growth of tumor of various kinds, but the detailed biochemical processes are unclear.Growing evidence indicated mat more and more small-molecule agents exert their tumor-killing actions through, at least partially, inducing cell cycle arrest and/or apoptosis. A successful cell division cycle requires the orderly and unidirectional transition from one cell cycle phase to the next The regulatory circuits consist of a series of checkpoints, which ensures mat passages could be allowed only aftercompletion of the related critical cell cycle events. Passage of the cell cycle checkpoints ultimately requires the activation of intracellular enzymes known as cyclin-dependent kinases (CDKs). These kinases are positively regulated by cyclins (A, B, D, and E) and are negatively regulated by CDKIs (the Cip/Kip family and the INK4 family). Therefore, the inhibition of CDKs and their up-regulator in malignant cells provides a new strategy in the fight against cancer.Apoptosis is a crucial biological process that prevents uncontrolled cell proliferation and eliminates harmful cells. Also, apoptosis is an important phenomenon in cytotoxicity induced by anticancer drugs. Even though studies on apoptosis have been extensive, the mechanism of apoptosis remains controversial. Here, we review the current status of the molecular mechanisms of anticancer drug-induced apoptosis in order to assess the contribution of molecular-level analysis to cancer chemotherapy and tumor biology. It is apparent that the molecular mechanisms by which anticancer drugs induce apoptosis are mediated by two major pathways: the intrinsic pathway, in which mitochondria plays a central role, and the extrinsic pathway, in which cell plasma membrane death receptors act as the starting point of the apoptotic process. The mitochondria-dependent pathway is regulated by Bcl-2 family proteins and involves the release of cytochrome c, Smac/DIABLO, AIF and Endo G. The release of cytochrome c, Smac/DIABLO, AIF and Endo G are the central gate in turning on/off apoptosis, and are regulated by the interaction of proapoptotic proteins, including Bid, Bax and Bak, and antiapoptotic proteins including Bcl-2 and Bcl-X(L), and a specific class of inhibitors of apoptosis proteins (IAPs) including Akt, and survivin. In this study, we investigated the relationship between apoptosis mediated by mitochondria and the cancer cells treatment TV. In the death receptor pathway, death receptors are activated by binding to their cognate ligand. For example, Fas and FasL can active FADD, then bind to the receptor's intracellular domain called the death-inducing signaling complex (DISC). Once bound, FADD recruits procaspase-8 and propaspase-10 using the death effecter domains (DEDs), which binds to the adaptor molecule, and along with the receptor, where caspase-8 and caspase-10 are activated.Activated caspase-8 and caspase-10 subsequently cleave and activate down-stream caspases, such as caspase-3,-6,-7 and trigger apoptosis, and activated caspase-8 also cleave and actived Bid, which can crosstalk between extrinsic pathway and the intrinsic pathway. Several reports have suggested that anticancer drugs kill susceptible cells by inducing expression of death receptor ligands, especially Fas ligand (FasL). Caspases (cysteine dependent aspartate-specific proteases) are organized in a cascade with initiating (upstream or apical) caspases, such as, caspase-2, caspase-8, caspase-9, and caspase-10, being responsible for activating the executing (effecter or downstream) caspases, such as, caspase-3, caspase-6, caspase-7. In both the intrinsic and extrinsic pathway, activation of caspases play indispensable role, which are responsible directly or indirectly for the morphologic and biochemical events that characterize the apoptotic cell. Numerous proteins that regulate these cell death proteases have been discovered, including proteins belonging to the Bcl-2, inhibitor of apoptosis, caspase-associated recruitment domain, death domain, and death effector domain families.The cell cycle and apoptosis are intimately related, as evidenced by the central role of p53, both in cell cycle arrest and in the induction of apoptosis. The p53 tumor suppressor, which functions as a transcription factor, plays a crucial role in preventing the onset of cancer and in determining the response of tumor cells to therapy. It restricts cellular growth by inducing senescence, cell cycle arrest (at Gl and/or G2 phase) or apoptosis. Intriguingly, p53 can intervene at every major step in apoptotic pathways: from extrinsic death receptor signaling, through the convergent pathway component Bid, to the intrinsic mitochondria pathways involving apoptosome formation, and culminating in direct caspase activation. Many of these effects are mediated through the activation of specific p53-target genes. In addition, p53 is able to activate apoptotic pathways by transcription independent mechanisms.Based on above considerations, we investigated the role of TV in regulation of cell cycle and apoptosis in brain tumor C6 and breast cancer C127 cells, and stressedanalysis the relationship between p53 expression and cell cycle arrest and apoptotic pathway mediated by TV. In addition, we also studied the role of TV in regulation of caspases activities, and the apoptotic pathway mediated by death receptor and mitochondria in C6 and C127 cells treatment with TV.Objectives:The overall objective of this proposal is to investigate the in vitro effects of a new anticancer agent, tetrazolium violet (TV or Cocid-1), on the growth of C6 and C127 tumor cells, the in vivo effects of TV on the growth of glioma established subcutaneously in rats, the possible mechanism of TV in inhibiting tumor cells growth, and advantages of TV as a potential anticancer agent.Methods:Effect of TV on cell proliferationIn the in vitro studies:Viable cells were determined by cell counting assay, using a hemocytometer.Viability was accomplished by MTT assay.Cell proliferation was quantified by Brdu incorporation assay.In the in vivo studies:Effect of TV on the growth of glioma established subcutaneously in rats was evaluatedby measuring the volume of tumor mass with a digimatic calliper.Detection of cell apoptosis1. DNA fragmentation was determined by TUNEL labeling2. Fragmentation of nuclei and chromatin condensation was detected using H3328 staining3. Membrane blebbing was detected by using morphologic observation assay4. Exteriorization of phosphatidylserine was detected by Annexin V-FITC/PI binding5. The ratio of apoptosis and/or necrosis was determined by Annexin V-FITC/PIbinding and LDH assay.For further differentiation of apoptosis and necrosis, a series of experiments, including the use of trypan blue exclusion assay, Annexin V-FITC/PI assay and LDH release assay, were performed.Cell cycle analysisFlow cytometry was used to test the effects of the TV on cell cycle.Effects of TV on the expression of proteinsDetection of protein expression was performed using immunofluorescence assay or/and by ELISA assay or/and by western blotting.Activity of caspasesColorimetric assay and fluorescent assay were used to detect the activities of caspases.Results:1. TV was effective in inhibiting the growth of the tested tumor cells (C127 and C6) in a dose and time-dependent manner, and was effective in inhibit the growth of C6 established subcutaneously in rat.2. Flow cytometry analysis demonstrated that after exposing to TV for 24-48h, most of C6 cells was blocked in Go/Gi phase in a time-dependent manner, but M phase was not changed obviously, and C127 cells was arrested in Gi/Sand G2/M phase, respectively, depending on the time.3. Morphological changes of apoptosis such as cell shrinkage and membrane blebbing were observed in C6 cells following TV treatment for 12 h, then being followed by more apoptotic bodies with dramatic cell detachment after 48 h. In C127 cells, there were many vacuoles around nuclear but no specificmorphological changes of apoptosis were observed following TV treatment for 24 h. After 48 h, only a few of apoptotic bodies with dramatic cell detachment were detected.4. In H33258 assay, nuclear fragmentation, chromatin condensation and apoptotic body were observed in C6 cells and C127 cells following treatment with TV at given concentration for 24-48 h.5. Using DeadEnd? Colorimetric TUNEL System, nuclear fragmentation and intranucleosomal DNA fragmentation were detected in C6 cells and C127 cells after being treated with TV at given concentration for 24-48 h.6. Tested by trypan blue exclusion assay, Annexin V-FITC/PI assay and LDH release assay, TV inhibited C6 cell growth mainly through inducing apoptosis rather than necrosis. For C127 cells, lower dose of TV caused death of cells mainly in the form of apoptosis. Once the concentration of TV was increased above lOuM, necrosis became major form of cell death.7. Immunofluorescence staining demonstrated that in C6 cells and C127 cells, treatment with TV for 24 h dramatically up-regulated the relative quantity of p53. significantly reduced the number of PCNA-positive cells. And treatment with TV for 24 h significantly increased Bax expression, slightly increased Bcl-2 level, and dramatically elevated the Bax/Bcl-2 ratio in C6 cells, but not in C127 cells.8. ELSIA assay demonstrated that treatment of C6 cells and C127 cells with TV for 6, 12, 24, 36 and 48 h dramatically up-regulated the expressions of p53, p21, Fas and FasL, and might result in a p53-dependent up-regulation in p21, Fas and FasL expression.9. Western blot analysis demonstrated that treatment with TV increased the protein levels of CDK2 and pRb (Gl/S and G2/M arrest regulatory proteins) as well as p53 and p21 (CIP1/WAF1) but not those of p27, Cyclin E and Rb.10. Caspases assay indicated that in C6 cells, TV-induced apoptosis involved caspase-3, caspaes-8 and caspase-9, and in C127 cell, only caspase-8 and caspase-3 were observed in initiating cell apoptosis.Conclusions:1. TV could significantly inhibit the growth of rumor cells, with the morphological changes and mechanisms varying with the tested cell types (C6 cells and C127 cells).2. TV inhibited C6 cell growth mainly through inducing apoptosis rather than necrosis. In C127 cells, the forms of cell death varied with TV concentration. Lower doses of TV (<10 uM) resulted mainly in apoptosis, otherwise (more than lOuM), necrosis.3. TV induced apoptosis in C6 cells and C127 cells through up-regulating of p53, the expression of p21 and PCNA, inducing cell cycle arrest.4. TV induced C6 cell arrest at Gl/S and C127 cell arrest at Gl/S and G2/M, which were associated with a decrease in the expressions of CDK2 and pRb proteins and up-regulations of p53 and CDK inhibitor p21(Cipl), but not with p27(Kipl).5. TV might induce apoptosis in C6 cells at several levels through the activation of apoptotic cascades mitochondria /Fas/caspase-8 caspase-9 and caspase-3 and through triggering the p53-associated intrinsic pathway and extrinsic pathway, and induce apoptosis in C127 cells through the activation of apoptosis cascades Fas/caspase-8 and caspase-3, and in p53-associated pathway.