Ginsenoside Rg3 Sensitizes Colon Cancer Cells To DNA Damage Reagents By Inhibiting WEE1 Kinase Expression

BackgroundColorectal cancer is one of the most common gastrointestinal malignant cancer, its incidence and mortality are the third and second place of all malignant tumors respectively. Rg3 is the composed active ingredient that extracted from ginseng, it can inhibit cell proliferation, migration and invasion, and promote apoptosis. WEE1 could regulate cell cycle checkpoint of G2, its expression could affect cytotoxicity of DNA damage drugs, such as cisplatin, 5-fluorouracil, paclitaxel and so on. Rg3 could synergistic sensitization cisplatin and 5- fluorouracil for cell proliferation of inhibition effect in gastric cacner and ovarian cancer. However, the relationship of Rg3, DNA damage drugs and WEE1 expression have not been reported. In this study, we would investigate the relationship of the three factors in colorectal cancer, and probed into the molecular mechanism.Part I Ginsenoside Rg3 sensitizes colon cancer cells to DNA damage reagents by inhibiting WEE1 kinase expression ObjectiveTo explore whether Rg3 could effect the sensitivity of DNA damage drugs in cell proliferation and demonstrated the molecular mechanism. Methods1. In order to discuss the affection of Rg3 sensitizing DNA damage drugs in colorectal cancer cell proliferation. The CCK-8 assay was used to detect cell proliferation. we detected the affection of different concentration of Rg3 combined with different concentration of cisplatin and 5-FU on colorectal cancer cell proliferation.2. In order to explore the potential molecular mechanism of Rg3 inhibited cell proliferation, we detected the m RNA level of WEE1 after treating with Rg3;3. To explore the function of WEE1 on cell death of Rg3 synergy DNA damage drugs. Firstly, we overexpressed WEE1 by plasmid in colorectal cancer cell lines Wi Dr,SW948 and COLO205; secondly, we detected cell proliferate viability by CCK-8 assay after treating cells with different transfection concentration of pc DNA-WEE1 combined with 10μM/L cisplatin +75μM/L Rg3 or 3μM/L 5-FU +50μM/L Rg3. Results1. cisplatin and 5-FU could inhibit Wi Dr, SW948 and COLO205 cell proliferation in a dose-dependent fashion; after treating cells with the same concentration of cisplation or 5-FU, with the increase application of Rg3, the cell proliferate activity gradually decreased;2. With the increase concentration of Rg3, the expression of WEE1 gradually decreased in Wi Dr,SW948 and COLO205 cells; after treating cell with Rg3 in the concentration of 50μM/L, the expression of WEE1 gradually reduced in the three colorectal cancer cell lines over time;3. The expression of WEE1 was upregulated after the transfection of pc DNA-WEE1 plasmid; after treating cells with cisplatin(10μM/L) combined with Rg3(75μM/L) or 5-FU(3μM/L) combined with Rg3(50μM/L), with the increase of pc DNA-WEE1 transfected concentration, the proliferated activity was gradually increased in Wi Dr,SW948 and COLO205 cells Conclusion1. Ginsenoside Rg3 sensitizes colon cancer cells to DNA damage reagents(cisplatin and 5-FU);2. Rg3 decreased WEE1 expression in dose- and time-dependent fashion;3. Overexpression of WEE1 in colon cancer cell lines suppressed the sensitization effect of ginsenoside Rg3 to cisplatin and 5-FUPart II Positive expression of WEE1 predicts a worse prognosis in colorectal cancer ObjectiveTo explore the relationship of WEE1 expression and clinical characteristic and prognosis in colorectal cancer Methods1. In order to discuss the expression of WEE1 in tumor or adjacent normal tissue of colorectal cancer, we used Western blot and q RT-PCR to detect WEE1 expression in tumor or adjacent normal tissues;2. In order to explore the relationship of WEE1 expression and the charactaristic of colorectal caner patients. We used Chi-squared test to detect the relationship of negative or positive WEE1 expression and age, gender, tumor location, tumor size, tumor classification, lymph node metastasis, distant metastasis,Peritoneal metastasis, Duke’s staging, T staging, N staging, M staging and TNM staging;3. Kaplan–Meier survival curves were constructed and the log-rank test was performed for univariate analysis. Multivariate analysis was performed using Cox’s proportional hazards model. Results1. We first determined the WEE1 protein expression status in CRC tissues by western blot and q RT-PCR in a cohort of 33 patients. We found that the level of WEE1 protein and m RNA is higher in tumor tissue than that in adjacent normal tissues.;2. By IHC assay, the positive WEE1 expression was found in 86 patients and negative in 56 patients;3.The positive WEE1 expression is correlated with tumor classification(P=0.033), N staging(P=0.005), TNM staging(P=0.009), Duke’s staging(P=0.001) and lymph node metastasis(P=0.006);4. The positive WEE1 expression predicted worse Overall survival(OS) and Disease-free survival(DFS); univarite analysis indicated that tumor size(HR=0.5, 95% Confidential interval 0.263-0.949,P=0.034),TNM staging(HR=1.402,95%CI 0.934-6.176,P=0.012),M staging(HR=1.562,95%CI 0.231-4.685,P=0.002), liver metastasis( HR=3.462, 95%CI 1.265-10.256, P=0.014) and WEE1 positive expression(HR=7.608,95%CI 3.019-19.185,P<0.001)were significantly with prognosis; multivariate Cox regression analysis was also performed to evaluate the potential of TNM staging(HR=2.899,95%CI 2.072-8.056,P<0.001)and WEE1 positive expression(HR=6.533,95%CI 2.93-14.566,P <0.001)as independent predictor of OS in CRC patients.5. WEE1 expression was not correlated with OS and DFS in I staging colorectal cancer patients; however, in II and III staging patients, WEE1 positive expression predicted worse OS and DFS. ConclusionWEE1 was upregulated in tumor tissues of colorectal cancer and the positive expression predicts worse prognosis in colorectal cancer patients;WEE1 positive expression and TNM staging were independent predictor of OS in CRC patients;...