Regulatory Effects Of Src On ERK Signal Transduction Pathways In Cervical Cancer Cells

Objetive:The occurrence and development of cervical cancer had close relationship with abnormal expression of related factors in signal transduction pathway. c-Src protein has been over-expressed and activated in many types of human cancer including cervical cancer, and influenced the development and prognosis of the disease. Src can activate Ras/Src-Raf-MEK-ERK signal transduction pathways, thus mediating and adjusting a variety of biological activity of tumor cells, but whether Src mediate activation of ERK signal transduction pathways, thus influenced cervical cancer cells biological characteristics were still unknown. This study adopted vitro cell experiment and adopted Src intervention to evaluate the regulation effect of Src on extracellular signal-regulated kinase 1/2(ERK 1/2), nuclear transcription factor c-Fos and c-Jun, and trans-factors hn RNPE1 in ERK signal transduction pathway, to explore whether Src can mediate the activation of ERK signal transduction pathways in cervical cancer.Methods:Human cervical cancer cell lines Hela(HPV-positive) and C33A(HPV-negative) at logarithmic phase of growth were exposed to Src kinase inhibitor-PP2 at the concentrations of IC50, and form the PP2-treated group as the experimental group and the control group.Cellular proliferation and viability were measured by cell count and Cell Counting Kit-8(CCK-8). Fluorescence-activated cell sorting(FACS) was conducted to analyze cell cycle and apoptosis. Real-time PCR and Western blot assay were used to evaluate the expression of Src, extracellular signal-regulated kinase 1/2(ERK 1/2), nuclear transcription factors c-Fos, c-Jun, nuclear transduction regulatory factor-hn RNP E1 in gene transcription and translation levels. All analyses were performed and analysed with SPSS statistical software(version 17.0).Results:1. Evaluation of PP2 inhibition effect on Hela and C33 A cells:(1)According to theCCK-8 results, 20μM of PP2 as IC50, 24 h after passaged for Hela, and 12μM of PP2 as IC50, 24 h after passaged for C33 A was was chosed as the most appropriate conditions.(2)PP2 significantly down-regulated p-Src protein in Hela and C33 A, Hela decreased by29.47%(t=4.547, P=0.045), while C33 A decreased by 33.33%(t=5.354, P=0.033). In brief,we had chieved satisfactory intervention results based on the above restrain condition. In addition, p-Src decreased in Hela was almost the same as in C33 A with no significant difference. Meanwhile, Src in Hela and C33 A decreased by 33.56% and 29.47% due to Src inhibition, respectively, but the difference had no statistical significance(t=1.662, P=0.238;t=3.233, P=0.082).(3) PP2 up-regulated Src m RNA in both Hela and C33 A, but the difference was only significant in Hela, in addition, Src m RNA in Hela increased obviously more than in C33 A due to Src inhibition(t=13.909, P<0.001).2. Influence of Src on cervical cancer cell lines general biological characteristics.(1) Influence of Src on Cervical cancer cell morphology and cells activity. Untreated cells were round and large, with a bright cytoplasm and good refraction, while PP2-treated cells were round with evidence of cell shrinkage and a translucent cytoplasm. PP2-treated cells exhibited typical apoptotic features, such as membrane blebbing and cell shrinkage.These results suggest that PP2 might have induced apoptosis in Hela and C33 A. Before Src inhibition, Hela and C33 A achieved growth peak in 72 h after inoculation, except 24 hours,Hela growed faster than C33 A with statistical difference. Living cells of inhibitor group continued to decrease, and the difference was statistically significant compared with the control group. At each time point, the inhibition rate in Hela and C33 A had not statistically difference, indicating PP2 had a similar inhibition effect on cell growth for Hela and C33 A.(2) Src induced cervical cell proliferation and inhibited cell apoptosis. Before Src inhibition, the percentage of G0/G1 was higher in C33 A than in Hela, while the percentage of S and G2/M was lower than in Hela. FACS showed that C33 A has a lower cell proliferation index PI with the number of 51.50±2.40 than Hela with 59.70±1.48, while C33 A has a higher apoptosis rate with 6.90±1.50 than Hela with 3.74±1.11. After Src inhibition, G1 phase increased, while G2/M and S phase decreased, meanwhile PI decreased in both Hela and C33 A with significant difference. In the inhibition group,C33 A still has a higher apoptosis rate of 43.04±0.33 than Hela with 14.50±1.86, and the difference was statistically significant. In comparison, G2/M decreased more in Hela than in C33 A, while G0/G1 and S had no significant difference in both Hela and C33 A. PI ofHela decreased almost the same as C33A(P>0.05), while the apoptosis rate increased less than C33 A with statistical difference.3. Effect of Src on the gene expression of ERK 1/2, c-Fos, c-Jun, hn RNP E1.(1) PP2 up-regulated the ERK 1, ERK 2, c-Fos, c-Jun gene m RNA expression in both Hela and C33 A, except ERK1 and ERK2 m RNA increased in C33 A had no statistically difference, the other gene m RNA all increased with statistical difference. hn RNP E1 m RNA and protein decreased due to Src inhibition. In comparison, the m RNA of ERK1,c-Fos and c-Jun increased more in Hela than in C33 A with statistical difference, but hn RNP E1 m RNA changed almost the same in two kinds of cervical cancer cell lines due to Src inhibition.(2) PP2 down-regulated series proteins involved in ERK signal transduction pathway.In detail, in both Hela and C33 A cells, ERK 1/2, p-ERK 1/2, p-c-Fos, hn RNP E1 proteins decreased, while c-Jun and p-c-Jun proteins increased with statistically difference. In comparison, p-Src, p-ERK 1/2 and c-Fos proteins decreased more in C33 A than in Hela,c-Jun protein and p-c-Jun protein increased lower in C33 A than in Hela with statistical difference, meanwhile p-c-Fos and hn RNP E1 protein were almost the same in Hela and C33 A.Conclusions:1. Inhibition Src kinase activity down-regulated p-Src proteins in both Hela and C33 A,indicating that the target of PP2 was p-Src protein, while the Src phosphorylation is vital step for ERK signaling pathways activation. Src m RNA increased after Src inhibition,indicating that there may exist other signal factors regulating Src gene m RNA levels.Considering that Src m RNA in Hela(HPV-positive) increased more than in C33A(HPV-negative) due to Src inhibition, while p-Src in Hela decreased less than in C33 A, prompting that HPV may assist Src activation in inducing the malignant transformation of cervical cancer cells.2. Src can regulate cell cycle, induced cervical cancer cell growth and inhibited cell apoptosis. After Src inhibition, the viability of cervical cancer cells decreased, and PI decreased, meanwhile, the apoptosis rate obviously increased, indicating that the activity of Src kinase closely associated with cervical cancer general biological characteristics,down-regulated of Src kinase activity may interfere cell phase enter S from G0/G1 phase,inhibited cell proliferation and accelerating cell apoptosis.3. Src can regulate series of key factors involved in ERK signal transduction pathway such as Src, ERK 1/2, c-Jun and c-Fos gene and hn RNP E1 expression, thus influenced cervical biological functions. Considering ERK1, ERK2, c-Fos and c-Jun m RNA increased,hn RNP E1 m RNA decreased, meanwhile p-Src, ERK 1/2, p-ERK 1/2, hn RNP E1 and p-c-Fos protein decreased due to Src inhibition, indicating that Src regulate key signal factors in ERK signal transduction pathway such as Src, ERK 1/2, c-Fos, hn RNP E1 gene expression, mainly in protein levels, leading to malignant transformation of cervical cancer,this result suggests Src can serve as a potential carrier of targeted therapy of cervical cancer.The related gene m RNA increased after Src inhibition, suggesting there may exist negative feedback regulation triggered by decreased of p-Src levels. c-Jun m RNA, c-Jun protein and p-c-Jun protein increased after Src inhibition, indicating that other signal factors may involve in activating of c-Jun gene expression, or there may exist many complex regulating mechanisms involved in ERK signal transduction pathway, further research were needed to figure out the possible mechanism. We also found that c-Jun differs from c-Fos gene,appeared with ascension of m RNA and protein, which indicates that AP-1(which was formed by c-Jun interacts with Fos or other Jun proteins) may be a double-edged sword in tumorigenesis or canceration.4. PI in Hela decreased almost the same as C33 A, while the AI in Hela increased less than in C33 A due to Src inhibition. By comparison, p-Src, p-ERK 1/2, c-Fos protein in C33 A decreased more than in Hela, indicating HPV infection might have a synergic action with Src activation in activating ERK signal transduction pathway and inducing malignant transformation of cervical cancer. To confirm whether the difference was due to HPV infection, we need to add related intervention experiment targeting HPV E6/E7 protein and Src intervention in the same cell.