Antitumor Activity Of PAC-1 In Combination With MCP In Colon Cancer Cell Lines

Backgroundcolon cancer is a common malignant tumor. In our country, the morbidity of colrectal cancer is rising quickly in recent years. In spite of the advances of treatment for colorectal cancer, the five-year survival rate have not enhanced significantly.Chemotherapy is one important step fellowing the surgical operation. The efficacy of chemotherapy has been severely hinderned by the chemoresistance and non-specific toxicity.The molecular targeted therapy is aimed at the key molecule in tumor developing process, suppressed cancer cell's growth and proliferation by Blocking cell signalling , controlling its gene expression and the change of biology behavior,or through Forcing tumor angiogenesis, it plays a positive role of antitumor. Beacaus of it better molecular selectivities, molecular targeted therapy can highly-efficient killing cancer cells selectively, and reducing the normal tissue's damage. At present,the clinical efficacy of molecular targeted therapy is not completely satisfied, because all the drug targets are focus on upstream of apoptotic cascade, in which the upstream proapoptotic signal is notproperly transmitted to activate the executioner caspases. Therefore , seeking for the targeted drugs which could specificity induced tumour cell to death and has little toxicity-side effect target has become the hot off the press.Procaspase activating compound one (PAC-1), which was found by Hergenrother and his colleagues at the University of Illinois, through screening more than 20,000 structurally diverse compounds, can activate procaspase-3 into caspase-3,and induce apoptosis. Modified citrus pectin was extracted from the natural pectin .It is one kind of polysaccharides production,the galactose is the main ingredients. In recent years, more researches find that Modified citrus pectin has effectivities in antitumor.Despite it has proved that PAC-1 could efficiently induce cell to death,it targeted at the key executioner protein caspase-3 which is in the downstream of apoptotic cascade. Modified citrus pectin is the competitive inhibitor of Galectin-3'in vivo. Galectin-3 and Bcl-2 have the obvious sequence similarity, in the carboxyl terminal both of them have NWGR motif which is essential for Bcl-2 to inhibiting apoptosis. Bcl-2 is the inhibitor of apoptosis protein, it acting on endogenous apoptosis cascade.It has been reported that PAC-1 and Modified citrus pectin have the antitumor activity through inhibiting the key proteins or key ways during apoptosis. Up today, there are no research deal with the inhibition of colon cancer cells growth and apoptosis by PAC-1 combined with MCP. According to: caspase-3 is the key executioner protein, Bcl-2 adjustandcontrol apoptosis by regulating the release of Cyt-c. By inference, enhanced the effect of PAC-1 in combination with Modified citrus pectin on cancer cell apoptosis and proliferation by activate caspase-3 directly and inhibit the activity of Bcl-2.Objective:The point of penetration of this project is induce cancer cell to apoptosis,in vitro to study: 1)the effect of PAC-1 on human colorectal cancer cell apoptosis and proliferation, explore the anticancer mechanisms through the protein expression of caspase-3,8,9,PARP,bcl-2 and galectin-3. 2) in vitro investigation of the enhanced effect of PAC-1 plus Modified citrus pectin in colorectal cancer cell.Methods:1,Cell lines: human colorectal cancer cell lines SW116,HCT116,HT-29,SW620 and SW480.2,MTT analysis of different concentration of PAC-1 induce human colorectal cancer cells death after 72 hours and calculate IC50,then select the sensitive and insensitive cell to PAC-1.3,MTT analysis of the enhanced effect of 5mg/ml Modified citrus pectin on PAC-1 to induce the sensitive and insensitive colorectal cancer cell to death.4,Observed the change of colorectal cancer cells after 24 hours exposed to PAC-1 by microscope.5,Flow cytometric analysis of the effect of PAC-1 and IC25,IC50,IC75 concentrations of PAC-1 plus 5mg/ml of Modified citrus pectin on apoptosis and cell cycle status.6,Western bloting anlysis the expression levels chang of PAC-1/ PAC-1 plus Modified citrus pectin on apoptosis key protein caspase-3,8,9,PARP and bcl-2.7,Uses statistics software SPSS13.0 to carry on the statistics analysis; mean values between two groups use two independent sample T-test variance, Many group of mean value comparisons use the One Way ANOVA, P<0.05 has the significance difference, all measurement data expressed by x±s.Results:1,PAC-1 induced significant cell death depended on the exposure dose in all tested cancer cells at micro mole levels.The IC50 values for different ,cell lines were follows: SW116:22.75±8.70umol/L,HCT116:2.98±1.08 umol/L,HT-29:11.27±2.24 umol/L,SW620 : 3.65±0.21 umol/L和SW480 : 4.08±1.12 umol/L.Because the high concentration is unable to dissolve, the MTT assay about Modified citrus pectin is unable to complete done.2,For further experiments, chosing the sensitive cell SW116 and insensitive cell SW620 to PAC-1;3,Because the high concentration of Modified citrus pectin is unable to dissolve, 5mg/ml is chosed for joint action. CI of IC25 concentration of PAC-1+5mg/ml of Modified citrus pectin is 1.54638; CI of IC50 concentration of PAC-1+5mg/ml of Modified citrus pectin is 0.57996; CI of IC75 concentration of PAC-1+5mg/ml of Modified citrus pectin is 0.80478. 4,Cells treated by low concentration of PAC-1(IC25) show visible apoptosis characters after 2 hours, apoptosis which depended on the exposure dose and time is characterized by marked changes in celluar morphology ,incuding chromatin cindenstion,membrance blebbing, nuclear breakdown,and the appearance of membrance-associated apoptotic bodies. This result is coincide with the MTT experimental result.5,Flow cytometric analysis:cells were treated with IC25,IC50 and IC75 PAC-1 for 4,8,24,48 hours.In comparison with the untreated cells,low concentration of PAC-1(IC25) shows slightly induced apoptosis. PAC-1 at IC50 and IC75 significantly enhanced apoptpsis after 4 hours treatment on SW116 and SW620 cells.6,Flow cytometric analysis:the treatment of SW116 and SW620 cells with PAC-1(IC25,IC50,IC75) in conbination with Modified citrus pectin(5mg/ml)significantly enhanced apoptpsis after the same time, compaired with the cells treated with PAC-1.7,PAC-1 alone or combined with Modified citrus pectin can increase the cell number of G0/G1 period and decrease the cell number of G2/M period,blocking the cell cycle,this effect depends on the exposure dose and time.8,Western bloting results showed that the SW116 and SW620 cells treated with PAC-1 at IC50 after 4,8,24,48 hours resulted in the activation of caspase-3, PARP and Rock-1 cleavage,which phenomenon is depended on the exposure dose and time.The activation of caspase-8,9 were not obvious. In comparison PAC-1(IC50) plus Modified citrus pectin(5mg/ml)resulted in the activation of caspase-3,9,PARP and Rock-1 cleavage to a more degree. PAC-1 plus Modified citrus pectin induced significant expression of proapoptotic protein Bax and a significantly decrease of antiapoptotic protein bcl-2 was detected in SW116 and SW620 cells,the ratio of Bax/bcl-2 is raise.Conclusions:1,PAC-1 was very effective as a single agent in killing 5 human colorectal cancer cell lines. 2,Low cincentration of PAC-1 in combination with Modified citrus pectin has antagonistic effect on induced human colorectal cancer cells apoptosis and proliferation;middle and high cincentration of PAC-1 in combination with Modified citrus pectin has Synergy effect on human colorectal cancer cells'apoptosis and proliferation.The best killing effect on human colorectal cancer cells is middle cincentration of PAC-1 plus Modified citrus pectin.3,PAC-1 alon or in combination with Modified citrus pectin significantly increase the activation of caspase-3 and PARP cleavage, the activation of caspase-8,9 were not obvious. So PAC-1 directly activated procaspase-3 to caspase-3 in vitro and induces apoptosis in cancerous cells.Mitochondrial pathway may play a critical role in PAC-1 plus Modified citrus pectin induced cell death.4,PAC-1 alon or in combination with Modified citrus pectin can prolonged Cell proliferation.