Study Of The Synergistic Function Of Ras And Its Common Mutants With P53 To Chemosensitivity Of Colorectal Cancer Cells

Objective Ras is a proto-oncogene, whose mutation is largely found in multiple tumor cells. The frequency of Ras mutation in human Colorectal Cancer is found to be above 30% and is associated with colorectal cancer occurrence, development and efficacy prediction of targeted therapies. Mutated Ras is activated to be an oncogene with its structure changed. Then multiple downstream signal pathways are activated to be involved with tumor invasion and metastasis. P53 gene is the earliest found tumor suppressor gene, which participates in cell cycle regulation, cell apoptosis, senescent and et al. p53 is also found to have close relationship with maintain of DNA stability, DNA repair. In recently years, p53 has been used in clinical therapy as a gene therapy target by increase p53 gene expression level and repairation of p53 function. It has been found in many studies that there is complicated synergistic effect between tumor suppressor p53 and oncogene Ras. Therefore, in the drug therapy of colorectal cancer with high mutation frequency of Ras, the co-relationship between p53 and Ras mutations should be deeply investigated by in vivo and in vitro experiments. This study used colorectal cancer cell model to investigate the effect of synergistic between p53 and Ras and its common mutants on drug sensitivity of colorectal cancer cells to OXA, by over expression of p53 and Ras and its mutants. This study will provide reliable experimental data for improvement drug therapeutic effect and development new assistant therapy methods under high frequency of Ras mutation circumstances.Methods The first part of the experiment: Tumor and para-tumor normal tissues were collected from 67 colorectal cancer patients. Total m RNA was extracted and reversed transcripted, then amplified K-Ras and p53 gene. Mutations of K-Ras and p53 were analyzed by direct sequencing;Comparisons of the differences between tumor tissues and para-tumor tissues were calculated using the chi-square test, Correlations between K-Ras mutations and p53 mutations were analyzed using a Spearman’s rank test. The second part: HCT 116 p53-/-cell line was cultured and divided into 6 groups: 1) Ras single transfected group; 2) Ras V12 signle transfected group; 3) Ras N17 signle transfected group; 4) Ras+Ad-p53 transfected group; 5) Ras V12+Ad-p53 transfected group; 6) Ras N17+Ad-p53 transfected group; HCT116cells were cultured to density 60%, transfected with indicated plasmids and Adenovirus for 24 hrs, then treated with OXA for 12 hrs. Cell apoptosis was analyzed by Calcein/PI Fluorescence in situ analysis; MTT method was used to analyze cell survival rates under OXA treatment; Total RNA was extracted, reversed transcripted. Apoptosis related gene Bax and Bcl-2 m RNA transcription levels were analyzed by Realtime PCR; Total protein were extracted. Expression of Bax, Bcl-2, p53, Ras and their related proteins under treatment of OXA were analyzed by Western Blotting.Results 1. Paired tumor tissues and para-tumor normal tissues from 67 colorectal cancer patients were collected. Total m RNA was extracted and reverse transcripted. K-Ras and p53 gene were amplified and gene mutations were detected by direct sequencing. It was found that, K-Ras 12 amino acid residue in tumor tissues 25.37%(17/67) mutated the most frequently. K-Ras 12 amino acid mutation was much lower in para-tumor tissues14.9%(10/67)(p<0.01)than in tumor tissues. Mutation of K-Ras 13 amino acid residue was also high. Frequency of K-Ras 13 amino acid residue were 10.45%(7/67)and 2.99%(2/67)(p<0.01)in tumor and para-tumor tissues. Mutation of K-Ras 17 amino acid residue was the lowest. Only 1 patient was found to harbor No.17 acid residue mutation in tumor and para-tumor tissues(p>0.05). The rate of p53 236 point mutation was 25.37%(17/67)in tumor tissues, which was higher than in para-tumor tissues 14.9%(10/67)(p<0.01). Frequency of p53 249 point mutation were 20.9%(14/67)and 11.94%(8/67)(p<0.05)in tumor and para-tumor tissues. No significance relationship was found between Ras mutation and p53 mutation analyzed by using Spearman’s rank test. 2. By using Calcein-AM/PI staining, the apoptosis rates of Ras transfected group, Ras V12 transfected group, Ras N17 transfected group was found to be(8.3±1.5)%,(16.8±1.7)%,(4.4±1.6)%. When p53 was absent, the apoptosis of Ras V12 transfected groups(16.8±1.7)% were significantly higher than Ras( 8.3±1.5)%(p <0.01)and Ras N17 single transfected groups(4.4±1.6)%(p <0.01). This suggested that under the influence of L-OHP, Ras V12 mutations could significantly increase the rate of apoptosis. While apoptosis rates of Ras+Ad-p53 groups, Ras V12+Ad-p53 groups, Ras N17+Ad-p53 groups was(13.5±1.9)%,(25.9±2.2)%,(7.6±1.2)%, it showed that apoptosis of three p53-Ad treated groups increased significantly. Apoptosis of Ras V12 single transfected group(25.9±2.2)% was the highest and Ras N17 transfected was the lowest (7.6±1.2)%. This indicated that under the effect of L-OHP drugs, Ras and p53 had a synergistic effect of enhancing apoptosis. 3. To confirm the results of Calcein-AM / PI, Cell viability was detected by MTT assay. It was found that the average OD values were 0.875、0.769、0.647、0.482、0.316、0.247 in Ras single transfected groups, Ras V12 single transfected groups, Ras N17 single transfected groups, Ras+Ad-p53 groups, Ras V12+Ad-p53 groups and Ras N17+Ad-p53 groups. Cell survival rates was 93.3%, 82.0%, 69.0%, 51.4%, 33.7% and 26.3% seperately. 4. The expression level of apoptotic protein Bax of three p53-Ad treated groups detected by Western Blotting increased significantly, which confirmed that the Ras and p53 synergistically enhanced the pro-apoptosis function. But the results were opposite to Bcl-2 and Bax; When p53 was over expressed, the expression level of Bax in Ras V12 transfected group(25.9±2.2)% was the highest and Ras N17 transfected group was the lowest(7.6±1.2)%, which was also opposite to Bcl-2 and Bax. 5. Finally, Real-time PCR showed, whether p53 exists or not, the expression level of Bax m RNA in Ras V12 transfected group was significantly higher than Ras N17 transfected group, and three p53-Ad treated groups increased significantly.Conclusion 1. In patients with colorectal cancer, K-Ras and p53 gene mutation rate of tumor tissues is higher than the para-tumor normal tissues, which further confirmed the mutation and tumorigenesis of these two genes are closely related; 2. Under the treatment of Oxaliplatin, over expression of Ras mutants could affect cell apoptosis; 3. p53 had synergy with Ras; Over expression of p53 could increase pro-apoptosis function of Ras and increase cells drug sensitivity.