An in vitro study on the effects of the combination of salinomycin with cisplatin on human gastric cancer cells

Abstract

The present study aimed to investigate the anticancer effects of cisplatin (DDP) combined with salinomycin (SAL) on the gastric cancer cell line SGC‑7901, as well as to explore the mechanisms underlying their actions. An MTT assay was used to evaluate the inhibitory effects of SAL, DDP and their combination on gastric cancer cell proliferation. Morphological alterations of cancer cells following treatment were observed under an inverted phase‑contrast microscope and a fluorescence microscope. Cell cycle progression and apoptosis were analyzed using flow cytometry. The expression of nuclear factor (NF)‑κB p65 and Fas protein ligand (L) in cancer cells was assessed using immunocytochemistry. The present results demonstrated that the combination of SAL and DDP significantly inhibited the proliferation (P<0.05) and altered the morphological characteristics of SGC‑7901 cells, thus suggesting that SAL may enhance the susceptibility of gastric cancer cells to DDP. In addition, treatment with a combination of SAL and DDP resulted in S phase‑arrest and increased the apoptotic rate of SGC‑7901 cells. Furthermore, marked FasL upregulation and NF‑κB p65 downregulation were observed in cancer cells treated with the combination of SAL and DDP. The results of the present study demonstrated that the combination of SAL and DDP induced the apoptosis of human gastric cancer cells, and suggested that the underlying mechanism may involve the upregulation of FasL and downregulation of NF‑κB p65.

Figure 1.

Morphological alterations of SGC-7901 cells following treatment with SAL, DDP or a combination of SAL and DDP. (A) Control cells received no treatment. (B) Cells were treated with 6 µmol/l DDP for 48 h. (C) Cells were treated with 8 µmol/l SAL for 48 h. (D) Cells were treated with a combination of 8 µmol/SAL and 6 µmol/l DDP for 48 h. Arrows indicate shrinkage of nuclear membranes. Magnification, ×100. SAL, salinomycin; DDP, cisplatin.

Figure 2.

Morphological alterations of apoptotic SGC-7901 cells treated with SAL, DDP or a combination of SAL and DDP were observed under a fluorescence microscope. Cells were treated with 6 µmol/l DDP, 8 µmol/l SAL, or 8 µmol/l SAL combined with 6 µmol/l DDP for 48 h. Control cells received no treatment. Subsequently, cells were stained with PI and AO. Arrows indicate condensed DNA and apoptotic bodies. Magnification, ×100. SAL, salinomycin; DDP, cisplatin; PI, propidium iodide; AO, acridine orange.

Figure 3.

Effects of SAL, DDP or their combination on cell cycle distribution of SGC-7901 cells. Cells were treated with 6 µmol/l DDP, 8 µmol/l SAL, or 8 µmol/l SAL combined with 6 µmol/l DDP for 48 h. Control cells received no treatment. Subsequently, flow cytometry was used for cell cycle analysis. Representative and quantitative results are presented. (A) Control cells. (B) Cells treated with 6 µmol/l DDP. (C) Cells treated with 8 µmol/l SAL. (D) Cells treated with a combination of 8 µmol/ SAL and 6 µmol/l DDP. (E) Percentage of cells in S phase. Data are expressed as the mean ± standard deviation. *P<0.05 compared with the control group; ^#P<0.05 compared with the DDP or SAL groups. SAL, salinomycin; DDP, cisplatin.

Figure 4.

Apoptotic rates of SGC-7901 cells following treatment with SAL, DDP or a combination of SAL and DDP. Cells were treated with 6 µmol/l DDP, 8 µmol/l SAL, or 8 µmol/l SAL combined with 6 µmol/l DDP for 48 h. Control cells received no treatment. Subsequently, flow cytometry was used to assess cellular apoptosis. Representative and quantitative results are presented. (A) Control cells. (B) Cells treated with 6 µmol/l DDP. (C) Cells treated with 8 µmol/l SAL. (D) Cells treated with a combination of 8 µmol/ SAL and 6 µmol/l DDP. (E) Apoptotic rate of cells in the various treatment groups. Data are expressed as the mean ± standard deviation. *P<0.05 compared with the control group; ^#P<0.05 compared with the DDP or SAL groups. SAL, salinomycin; DDP, cisplatin; PI, propidium iodide.

Figure 5.

NF-κB p65 expression in SGC-7901 cells following treatment with SAL, DDP or a combination of SAL and DDP for 48 h. (A) As a negative control, untreated cells were incubated with 1X PBS, instead of primary antibody. (B-E) Cells were incubated with anti-NF-κB p65 primary antibody. (B) Control cells received no treatment. (C) Cells were treated with 6 µmol/l DDP. (D) Cells were treated with 8 µmol/l SAL. (E) Cells were treated with a combination of 8 µmol/SAL and 6 µmol/l DDP. Magnification, ×100. NF, nuclear factor; SAL, salinomycin; DDP, cisplatin.

Figure 6.

FasL expression in SGC-7901 cells following treatment with SAL, DDP or a combination of SAL and DDP for 48 h. (A) As a negative control, untreated cells were incubated with 1X PBS, instead of primary antibody. (B-E) Cells were incubated with anti-FasL primary antibody. (B) Control cells received no treatment. (C) Cells were treated with 6 µmol/l DDP. (D) Cells were treated with 8 µmol/l SAL. (E) Cells were treated with a combination of 8 µmol/SAL and 6 µmol/l DDP. Magnification, ×100. FasL, Fas protein ligand; SAL, salinomycin; DDP, cisplatin.