Effect and mechanism of rapamycin on proliferation and apoptosis of human lung cancer cells


Wenning Cao, Lan Ma

Abstract


The purpose of this study was to investigate the effects of RAPA on the proliferation and the expression of p53, Bcl-2 and Bax proteins in cultured human small cell lung cancer (NCI-H446) cells, and to explore the possible mechanism of RAPA-treated NCI-H446 cells with different concentrations of RAPA-treated NCI-H446 cells. The proliferation of NCI-H446 cells in all groups was assayed by the CCK-8 method. FITC-Annexin V/PI double staining method was used to determine the apoptosis of NCI-H446 cells. The immunohistochemical SP method was used to detect the expression of p53, Bcl-2 and Bax. Expression of p53, Bcl-2 and Bax mRNA was detected by RT-PCR. The results showed that, after 48h treatment, the proliferation of NCI-H446 cells treated with 5ng/mL, 10ng/mL and 15ng/mL RAPA decreased significantly (P < 0.05) and the proliferation inhibition rate increased significantly (P < 0.05) compared with the control group, and the proliferation inhibition rate had a dose-dependent relationship with RAPA. Compared with the control group, the apoptosis rate of NCI-H446 cells treated with 5ng/mL, 10ng/mL and 15ng/mL RAPA increased significantly (P < 0.05), and there was a dose-dependent relationship between the apoptosis rate and RAPA. The expression of Bcl-2 protein and mRNA was higher in the control group, while the expression of p53 and Bax protein and mRNA was lower. The expression of Bcl-2 protein and mRNA decreased and the expression of p53 and Bax protein and mRNA increased gradually with the increase of concentration and the prolongation of action time in 5ng/mL, 10ng/mL and 15ng/mL RAPA groups. In the control group, the intracellular Ca2+ concentration was constant, and there was no significant change with time; while in the 5ng / mL, 10ng / mL, and 15ng / mL RAPA group, the intracellular Ca2+ concentration in the RAPA group increased significantly after 12 h of administration (P <0.05); After that, with the prolonged action time of the medicine, the intracellular Ca2+ concentration in the 5ng / mL, 10ng / mL, and 15ng / mL RAPA group decreased, but at 72h, the effect was 5ng / mL, 10ng / mL, and 15ng / mL RAPA. The intracellular Ca2+ fluorescence intensity in the group was still significantly higher than that in the control group (P <0.05). In conclusion, RAPA can induce apoptosis of NCI-H446 cells by down-regulating Bcl-2 gene expression, up-regulating P53 and Bax gene expression, and increasing intracellular Ca2+ concentration and its apoptosis induction effect have timeliness and dose-effect.

Keywords


Lung cancer; Rapamycin; Cell proliferation; Apoptosis; Bcl-2; p53; Bax.

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