| Background and Objective :The universality of resistance to chemotherapeutic drugs is a great problem in clinical anticancer treatment. Cytotoxins from Cobra venom, also called cardiotoxins, is toxic protein isolated from cobra snake venom. Cytotoxins have an alternative lethal effect on various cancer cells. Cytotoxins play an important role in anticancer research of snake venom. Present studies indicated that the cobra cytotoxins had an anticancer effect probably through plasma membranous action, mitochondrial way or lysosomal cell death way. Cytotoxin 1 (CTX1) is a polypeptide isolated and purified from Naja atra Cantor venom. Since the effect of CTX1 on cancer cells has not been studied, the aim of our study is to identify the anticancer effect and related mechanism of CTX1. The contents of our study include: the impact of CTX1 on cell viability of cell lines in vitro and survival period of mice bearing P388 ascitic tumor; the impact of CTX1 on cancer cell death; the impact of CTX1 on mitochondrial membrane potential and apoptotic protein Caspase 8, Caspase 9; the impact of CTX1 on lysosomal membrane permeabilization and Cathepsin B; the effect of caspase family inhibitor Z-VAD-FMK and Cathepsin B inhibitor CA-074 Me on CTX1-induced cancer cell death.Methods:1 Assay on anticancer effect of CTX11.1 Cell relative viability was measured using an MTS method on various cells (MCF-7, H22, K562, P388 and 16HBE).1.2 Assessment on survival period in KMF mice bearing murine leukemia P388 cells. 2 The impact of CTX1 on cancer cell death2.1 Cell death was analyzed by flow cytometry after AnnexinV- FITC and PI double staining.2.2 Cell death in live culture system was observed by fluorescence microscopy (Annexin V-FITC and PI double staining for MCF-7 cells; PI staining for K562 cells).2.3 The Lactate Dehydrogenase(LDH) activity was assayed.3 Mitochondrial membrane potential was measured by flow cytometry after Rhodamine 123 staining.4 The impact of CTX1 on lysosomal membrane permeabilization and Cathepsin B activity4.1 Lysosomes were visualized with a Fluorescence Microscope or Confocal Laser Scanning Microscope after LysoTracker Red DND-99 or LysoTracker Green DND-26 staining.4.2 Cathepsin B activity was measured using a Pan-wavelength microplate reader after treatment with Cathepsin B Activity Assay Kit.5 Protein expression level of GAPDH, Caspase 8, Caspase 9 and Cathepsin B were assayed by Western Blotting.Results:1 The impact of CTX1 on cell viability of cell lines in vitro and survival period of mice bearing P388 ascitic tumor1.1 MTS assay was used to detect the cell viability: CTX1 significantly suppressed the relative viability of cells (MCF-7, H22, K562, P388 and 16HBE) in dose-dependent and time- dependent manner (P<0.05). The inhibitory effect of CTX1 on 16HBE cell was more weak than the indicated cancer cells. These showed the cytotoxic activity of CTX1 was selective on cancer cells. The median inhibitory concentrations (IC50) of CTX1 on different cancer cells were different which indicated CTX1 had selective inhibitory effect on different cancer cells.1.2 CTX1 (1/3LD50,1.40 mg/kg/day)treatment greatly prolonged the survival period and cumulative survival rate of mice bearing P388 ascitic tumor. Differences between CTX1 and control groups were considered significant (P<0.05).2 The impact of CTX1 on cancer cell death2.1 Cell death was detected by flow cytometry (FCM): CTX1 induced K562 and P388 cancer cell death in dose-dependent and time- dependent manner. CTX1 mainly caused the late phase of apoptosis and necrosis while the early phase of apoptosis was not obvious.2.2 Cell death was observed using fluorescence microscopy:A majority of K562/MCF-7 cells in the control group were not stained by PI, they had normal size, the cytomembrane was smooth, and the condition was good. The PI-stained cells (dead or dying cells) elevated markedly when the concentration of CTX1 increased or the time prolonged. In the experiment of MCF-7, the CTX1-treated cells are mostly not stained with Annexin V-FITC, this showed CTX1 didn't induce the early phase of apoptosis.2.3 The LDH activity of P388 cell culture system elevated markedly when the concentration of CTX1 increased.3 The impact of CTX1 on mitochondrial membrane potential and apoptotic protein Caspase 8, Caspase 93.1 CTX1 led to loss of mitochondrial membrane potential:The P388 cells were detected by FCM with Rhodamine 123 staining, the fluorescence intensity of CTX1-treated cells was significantly weaker than the cells in control group. The percentage of P388 cells with low fluorescence increased in a dose-dependent method when the concentration of CTX1 elevated (P<0.05).3.2 The results of Western Blotting showed CTX1 did not cause obvious change of protein expression levels of GAPDH, Caspase 8 and Caspase 9.4 The impact of CTX1 on lysosomal membrane permeabilization and Cathepsin B4.1 The fluorescence intensity of CTX1-treated P388 cells was significantly weaker than the cells in control group after lysosomal staining. The decrease of fluorescence in MCF-7 or P388 cells reflecting a loss of lysosomal membrane integrity. CTX1 induced lysosomal membrane permeabilization. 4.2 CTX1 triggers Cathepsin B activation:P388 cells were treatedt for 12 h, the CB activity (A.U./mg of protein) in control group, BSA (4μg/ml) group,CTX1 (2, 4μg/ml) were respectively (8.49±0.18), (8.09±0.14), (25.83±2.54), (25.17±4.25). CB activity in CTX1-treated cells was about three fold of control. Differences between CTX1 and control groups were considered significant (P<0.05).4.3 The result of Western Blotting showed CTX1 did not cause obvious change of protein expression level of Cathepsin B.5 Effect of Z-VAD-FMK and CA-074 Me on CTX1-induced cancer cell death5.1 P388 cells were incubated with Z-VAD-FMK (10μM, the pan-caspase inhibitor) or CA-074 Me (7.5μM, the CB inhibitor) or the combination of both for 1 h prior to CTX1 (4μg/ml) treatment for 12 h, and cell relative viability was assessed with the MTS method. The results showed Z-VAD-FMK and CA-074 Me could both increase the relative viability CTX1-treated P388 cells.5.2 P388 cells were treated as 5.1, cell death was analyzed by flow cytometry after AnnexinV- FITC and PI double staining. The results showed Z-VAD-FMK and CA-074 Me could both decrease CTX1-induced P388 cell death.5.3 Cells were treated as 5.1, cell death was observed using fluorescence microscopy:CA-074 Me could decrease CTX1-induced MCF-7 cell death, and Z-VAD-FMK did not decrease CTX1-induced MCF-7 cell death.Conclusion:1 CTX1 possessed an anticancer effect in vovo and in vitro, had selective inhibitory effect on different cancer cells in dose-dependent and time-dependent manner.2 CTX1 induced apoptosis and necrosis of cancer cells.3 CTX1 mainly caused necrosis of cancer cells, the probable mechanism included lysosomal membrane permeabilization and Cathepsin B activation. |
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