Study Of Inhibitory Effects Of Combining Application Of Ginsenoside Rg3 And IRESSA On A431 Cell Proliferation

OBJECTIVE:Ginsenoside Rg3 is a Chinese herbal drug monomer microselected from the Red ginseng. Many reports confirm that Rg3 has medicinal effects, such as anti-tumorigenesis and anti-tumor metastasis. Epidermal growth factor receptor (EGFR) is a member of ErbB family. It's an important transmembrane receptor. Activated EGFR induced by one ligand starts intracellular signal cascade. The subsequent signal stimulates transcription factors and finally influences cellular responses, including proliferation, apoptosis, migration and adhesion. IRESSA, a selective epidermal growth factor receptor tyrosine kinase inhibitor, it is used to cure the non-small cell lung cancer and other malignant tumors in clinical treatment. According to the literature reports, the single agent model can not meet the clinical needs, combination with other chemotherapy drugs is a hot topic. This study was to assess the effects of IRESSA in combination with Ginsenoside Rg3 on cell proliferation in A431 cell line.MATERIALS AND METHODS:A431 cells were cultured in vitro. The cells were grouped as followed: control group, Ginsenoside Rg3 treated group, IRESSA treated group and combined Ginsenoside Rg3 and IRESSA treated group. The inhibitory rate was detected by MTT in A431 after the treatment; To detect the expression of proliferating cell nuclear antigen (PCNA) in each group by Western Blot; To detect the expression of FUT4 in each group by RT-PCR; to detect the expression of p-EGFR in each group by Western Blot.RESULTS : 1. Inhibitory effect of Ginsenoside Rg3 (40μg/ml), IRESSA (0.025μmol/l) and combining Ginsenoside Rg3 and IRESSA on proliferation of A431 cells by MTT. The cell growth speed of each group was slower than the control group, and the combining Ginsenoside Rg3 and IRESSA treated group was the slowest. The inhibition rate in Ginsenoside Rg3 treated group was 20.92%, in IRESSA treated group was 23.47%, and in combined Ginsenoside Rg3 and IRESSA treated group was 35.71% after 24 h. The inhibition rate in Ginsenoside Rg3 treated group was 25.00%, in IRESSA treated group was 32.98%, and in combined Ginsenoside Rg3 and IRESSA treated group was 44.37% after 48 h. The inhibition rate in Ginsenoside Rg3 treated group was 42.10%, in IRESSA treated group was 48.58%, and in combined Ginsenoside Rg3 and IRESSA treated group was 55.48% after 72 h. The inhibition rate in Ginsenoside Rg3 treated group was 57.55%, in IRESSA treated group was 65.86%, and in combined Ginsenoside Rg3 and IRESSA treated group was 70.52% after 96 h(p<0.05).2. Under an inverted microscope, we observed that the cells were full, irregular polygon, adherent to the culture plate in control group. The cells were slightly rounded, apoptotic and atropous in experiment groups. Compared with other groups, the obvious morphological changes occurred in the combined Ginsenoside Rg3 and IRESSA treated group.3. The expression of PCNA by Western Blot analysis. A431 cells were treated differently with control group (culture medium only), Ginsenoside Rg3 treated group(320μg/ml), IRESSA treated group(1μmol/l)and combined Ginsenoside Rg3 and IRESSA treated group after 72 h. The results showed that the inhibitory effect on PCNA was different among the four groups, the effect of combined Ginsenoside Rg3 and IRESSA treated group was the most obviously(p<0.05).4. The expression of FUT4 genes in RT-PCR. The results showed that the inhibition on FUT4 expression was different among the four groups, and the effect in combined Ginsenoside Rg3 and IRESSA treated group was the most obviously(p<0.05).5. The expression of p-EGFR was detected by Western Blot. The results showed that the activation of p-EGFR was different among the four groups, the effect in combined Ginsenoside Rg3 and IRESSA treated group was the most obviously(p<0.05).Conclusion: Combinied Ginsenoside Rg3 and IRESSA treatment can inhibit the A431 cell proliferation. The effect was more significant when the cells were treated with combined Ginsenoside Rg3 and IRESSA. The expression of both p-EGFR was decreased and further study is needed.