Sichong formula inhibits the proliferation and migration of human gastric cancer cells

Abstract

Purpose: Traditional Chinese medicine (TCM) has gained increasing attention for the treatment of multiple chronic diseases, such as cancer. Here we aim to identify the antitumor activity of Sichong formula, a novel TCM, in human gastric cancer cells and investigate the underlying mechanisms.

Methods: The AGS and MKN45 gastric cancer cells were treated with Sichong formula at different concentrations. The proliferation rates were tested by CCK-8 and colony formation assays. Cell migration and invasion were tested by scratch and transwell assays. Gelatin zymography was used to detect the matrix metalloproteinase 9 (MMP9) activity in cell suspendents. Cell apoptosis was analyzed by Annexin V/PI staining and flow cytometry. The expression of interest proteins was tested by Western blot.

Results: Cell proliferation analysis indicated that Sichong formula inhibited cell viability of AGS and MKN45 cells in a dose- and time-dependent manner. The IC50 values were 240 μg/mL and 200 μg/mL for AGS and MKN45 cells, respectively. Furthermore, we found that Sichong formula could inhibit the invasion and migration of gastric cancer cells, which might be mediated by the downregulation of MMP9 activity. Flow cytometry results indicated that Sichong formula induced apoptosis in gastric cancer cells through upregulation of Bax/Bcl2 ratio and activation of caspase cascade. The results from Western blot indicated that Sichong formula resulted in cell autophagy and inactivation of AKT signaling pathway.

Conclusion: Our data suggest that Sichong formula inhibits the proliferation and migration and induces apoptosis in human gastric cancer cells. The inhibitory effect of Sichong formula was, at least partly, mediated by cell autophagy and AKT pathway.

Keywords: MMP9; apoptosis; invasion; migration.

Figure 1

Sichong formula inhibited gastric cancer cell proliferation in a dose- and time-dependent manner. A concentration gradient of Sichong formula (0 μg/mL, 5 μg/mL, 10 μg/mL, 20 μg/mL, 40 μg/mL, 80 μg/mL, 120 μg/mL, 160 μg/mL, 200 μg/mL, 300 μg/mL, 400 μg/mL, and 500 μg/mL) was used to incubate with (A) AGS and (B) MKN45 gastric cancer cells for 48 hrs. Cell viability was detected by a CCK-8 assay. The calculated IC50 values were 240 μg/mL and 200 μg/mL for AGS and MKN45 cells, respectively. (C) AGS and (D) MKN45 cells were treated with 80 μg/mL and cell viability was detected at 0 hr, 24 hrs, 48 hrs, and 72 hrs. (E) Clone formation ability of AGS and MKN45 cells treated with Sichong formula (80 μg/mL) was tested. All experiments were performed at 3 times. *P<0.05.

Figure 2

Sichong formula treatment inhibited the invasion and migration abilities of gastric cancer cells in transwell assays. (A) and (D) Theinvasion and migration abilities of human AGS and MKN45 cells were detected by transwell assays, and cells were imaged under a light microscope. (B, C) and (E, F) The statistical results of invaded or migrated cells. The treatment regimen of Sichong formula was 80 μg/mL for 48 hrs. All experiments were performed at 3 times. *P<0.05.

Figure 3

Sichong formula treatment inhibited the wound healing of human gastric cancer cells. (A) and (B) Cell migration was detected in AGS and MKN45 cells using a scratch assay. (C) The activity of MMP9 was detected by gelatin zymography. The treatment regimen of Sichong formula was 80 μg/mL for 48 hrs. All experiments were performed at 3 times. *P<0.05. Abbreviation: MMP9, matrix metalloproteinase 9.

Figure 4

Sichong formula treatment induced apoptosis in gastric cancer cells through regulating Bcl2/Bax and caspase cascade. (A) and (B) cell apoptosis of AGS and MKN45 cells were analyzed by flow cytometry and Flowjo software. (C) Apoptosis-related proteins Bcl2, Bax, and C-Caspase 3 were detected by Western blot. The treatment regimen of Sichong formula was 80 μg/mL for 48 hrs. (D) cell apoptosis of GES-1 cells was analyzed by flow cytometry and Flowjo software. All experiments were performed at 3 times. *P<0.05.

Figure 5

Sichong formula treatment induced cell autophagy and inhibited the activation of AKT signaling pathway. (A) Autophagy-related biomarkers LC3, Beclin-1 and P62 were detected in AGS and MKN45 cells using Western blot after treatment of Sichong formula. (B) AKT signaling pathway members AKT, phosphorylated AKT (p-AKT) and Cyclin D1 were detected in AGS and MKN45 cells using Western blot after treatment of Sichong formula. The treatment regimen of Sichong formula was 80 μg/mL for 48 hrs. All experiments were performed at 3 times. *P<0.05.