Indirubin suppresses ovarian cancer cell viabilities through the STAT3 signaling pathway

Abstract

Background: Indirubin is the active component of Danggui Longhui Wan, a traditional Chinese medicine formulation. Due to its anti-inflammation and anti-tumor effects, indirubin has been widely used for the treatment of inflammation, cancer, and other chronic disease. Herein, we aimed to investigate the role and mechanism of indirubin in human ovarian cancer cell proliferation.

Materials and methods: The cell viability was determined by Cell Counting Kit-8 and colony formation assays by treatment with different dosages of indirubin over 72 hours. Apoptosis was examined by flow cytometry with fluorescein isothiocyanate Annexin V Apoptosis Detection Kit. Western blot assay was finally applied to analyze the expression of cancer-related STAT3 pathway and its downstream proteins.

Results: Indirubin was found to significantly inhibit cell viability and induce apoptosis in 2 human ovarian cancer cell lines. Mechanistic studies revealed that indirubin treatment led to reduced levels of phosphorylated-STAT3, thus repressing the downstream pro-survival proteins and elevating pro-apoptosis ones.

Conclusion: Our study provided the evidence for anti-survival activity of indirubin by inhibiting cell viability and inducing apoptosis in human ovarian cancer cells, which involved impaired STAT3 signaling pathway. Our findings further support indirubin as a potential drug candidate against human ovarian cancer.

Keywords: STAT3 signaling; cell viability; indirubin; ovarian cancer.

Figure 1

Indirubin inhibited cell viability in ovarian cancer cells. Notes: (A) A2780 and OVCAR3 cells were incubated with indirubin at different concentrations (0, 0.5, 1, 2, 5, 10, and 20 µM) for 72 hours. (B, C) A2780 and OVCAR3 cells were exposed to indirubin (2 and 5 µM), respectively, for different time points (0, 24, 48, and 72 hours). Cell viability was measured using CCK-8 assays. (D) Colony formation assay of A2780 and OVCAR3 cells was treated with indirubin (2 and 5 µM), respectively. The right panel shows the quantitative results. Each experiment was performed in triplicate independently. The data are presented as mean ± SD. *P<0.05, **P<0.01, ***P<0.001 vs control group. Abbreviation: CCK-8, Cell Counting Kit-8.

Figure 2

Indirubin induced apoptosis in ovarian cancer cells. Notes: A2780 (A) and OVCAR3 (B) cells were treated with indirubin at different concentrations (0, 0.5, 1, 2, 5, 10, and 20 µM) for 72 hours. Flow cytometry analysis showed indirubin induced higher apoptosis with increasing doses of indirubin. (C) Early and late apoptotic events of A2780 cells treated without or with different dose of indirubin were detected by flow cytometry. Top left quadrant indicates non-apoptotic cells; top right quadrant represents late apoptosis events; bottom right quadrant represents early apoptosis cells; and bottom left quadrant represents living cells. Each experiment was performed in triplicate independently. The data are presented as mean ± SD. **P<0.01, ***P<0.001 vs control group.

Figure 3

Indirubin-mediated expression of survival-related protein altered. Notes: (A) Western blot assay showed that indirubin downregulated the phosphorylated level of STAT3 and the expression of survival related protein Cyclin D1, C-myc with increasing concentrations in A2780 and OVCAR3 cells. Each experiment was performed in triplicate independently. (B) Quantitative results shown by ImageJ software. **P<0.01; ***P<0.001.

Figure 4

Indirubin-mediated expression of apoptosis-related protein altered. Notes: (A) Western blot assay showed that indirubin downregulated the expression of anti-apoptosis protein Bcl-xL and upregulated pro-apoptosis protein Bax, cleaved caspase 3 with increasing concentrations in A2780 and OVCAR3 cells. Each experiment was performed in triplicate independently. (B) Quantitative results shown by ImageJ software. *P<0.05; **P<0.01; ***P<0.001.

Figure 5

Colivelin could partly reverse the indirubin-induced suppression of cell proliferation and phosphorylation of Stat3. Notes: (A) A2780 cells were pretreated with 0.5 µM colivelin (Stat3 activator) or DMSO for 1 hour and followed by treatment with or without 5 µM indirubin. Seventy-two hours later, the cells were harvested for Western blot assay. (B) A2780 cells were pretreated with 0.5 µM colivelin or DMSO for 1 hour and followed by treatment with or without 5 µM indirubin. Seventy-two hours later, the cells were harvested for flow cytometry analysis. (C) OVCAR3 cells were pretreated with 0.5 µM colivelin or DMSO for 1 hour and followed by treatment with or without 5 µM indirubin. Seventy-two hours later, the cells were harvested for Western blot assay. (D) OVCAR3 cells were exposed to 5 µM indirubin with or without 0.5 µM colivelin for 72 hours. Cell viability was measured by CCK-8 assays. The data are presented as mean ± SD. *P<0.05, **P<0.01, ***P<0.001 vs control group. Abbreviation: NS, not significant.