Evaluation of antagonistic effects of metformin with Cisplatin in gastric cancer cells

Abstract

Metformin has recently been introduced as an anti-cancer agent. In this study, we evaluated the effect of metformin and metformin/cisplatin on human gastric MKN-45 cell line. When we used metformin alone, it could inhibit proliferation and induce apoptosis, but it diminish anti-proliferative effects of cisplatin when they are used in combination. Further, we checked mRNA levels of survivin, mTOR, and Akt by real-time PCR. When MKN-45 cells were treated with metformin/cisplatin, the expression of survivin and mTOR were increased. The antagonistic effect of metformin on cisplatin could be through survivin and mTOR signaling pathways. Our results also suggest that interfering effect of metformin on cisplatin may be also through upregulation of Akt. Regarding the pivotal role of Akt in drug resistance, it may be reasonable to conclude that the antagonistic effect of metformin on cisplatin effect may be through this central mediator of drug resistance. Taken together, it seems that metformin is not a good option for sensitizing MKN-45 cell line to cisplatin, and in co-prescription of metformin and cisplatin in gastric cancer patients who suffer diabetes type 2, it should be highly cared.

Keywords: Akt; Cisplatin; Gastric cancer; Metformin; Survivin; mTOR.

Figure 1

Evaluation of metformin, cisplatin and metformin /cisplatin on the viability of human adenocarcinoma cell lines MKN-45. The cell line was treated with different concentrations of metformin and/or cisplatin for 48 and 72 hrs and their viability was assessed using MTT assay. Results are expressed as a percentage of viability compared to the untreated control and are presented as mean±SD from three independent experiments (P<0.05 by one-way variance analysis)

Figure 2

Effect of metformin, cisplatin and metformin/cisplatin on apoptosis in MKN-45 cells. A) Hoechst 33342 stains the condensed chromatin in apoptotic cells more brightly than the chromatin in normal cells and Propidium Iodide (PI) is only permeate to dead cells (FL2: PI, FL6: Hoechst 33342). As shown in figure, there is significant change in the apoptosis rate of metformin, cisplatin and metformin/cisplatin treated MKN-45 cells compared with the control. B) Quantified values of apoptosis in MKN-45 cell line treated with metformin, cisplatin and metformin/cisplatin. As shown in this figure, the cisplatin-induced apoptosis at concentration of 5 μM is significantly higher than the combination of metformin 10 /cisplatin 5. Statistically different values of ^*P<0.05 and ^**P<0.01 are determined compared with the control

Figure 3

Effect of metformin, cisplatin and metformin/cisplatin on transcriptional levels of the survivin gene measured by real-time PCR. Data are shown as fold change in relative expression compared with HPRT1 on the basis of comparative Ct (2^-ΔΔCt) method. As shown in fig, treatment with metformin or cisplatin for 48 h significantly reduces expression of survivin. However, metformin/cisplatin leads to increased expression of survivin compared to treatment with cisplatin. Values are shown as mean ± SD. Statistically different values of ^*P<0.05 and ^**P<0.01 are determined compared with the control

Figure 4

Effect of metformin, cisplatin and metformin/cisplatin on transcriptional levels of the Akt gene measured by real-time PCR. As shown in figure, when the cell line was treated with metformin/cisplatin, the expression of Akt is significantly increased compared to drugs separately. Values are shown as mean ± SD. Statistically different values of ^*P<0.05 and ^**P<0.01 are determined compared with the control

Figure 5

Effect of metformin, cisplatin and metformin/cisplatin on transcriptional levels of the mTOR gene measured by real-time PCR. Treatment with metformin/cisplatin results inan increase in the expression of mTOR compared to treatment with cisplatin. Values are shown as mean±SD. Statistically different values of ^*P<0.05 and ^**P<0.01 are determined compared with the control