Rhein shows potent efficacy against non-small-cell lung cancer through inhibiting the STAT3 pathway

Abstract

Background: Non-small-cell lung cancer (NSCLC) comprises about 85% of all lung cancers and is usually diagnosed at an advanced stage with poor prognosis. The IL-6/STAT3 signaling pathway plays a pivotal role in NSCLC biology. Rhein is a lipophilic anthraquinone extensively found in medicinal herbs. Emerging evidence suggests that Rhein has significant antitumor effects, supporting the potential uses of Rhein as an antitumor agent.

Methods: Cell viability and colony formation were performed to examine Rhein's potent anti-proliferative effect in human NSCLC cell lines PC-9, H460 and A549. Flow cytometry-based assay was employed to study whether Rhein could affect cell apoptosis and cycle. The expression level of P-STAT3, apoptosis and cycle-related proteins Bcl-2, Bax, MDM2, CDC2, P53 and CyclinB1 were detected by Western blotting. The xenograft models were used to evaluate the in vivo effect of Rhein.

Results: We found that Rhein could significantly reduce the viability and stimulate apoptosis in human NSCLC cells in a dose-dependent manner. Western blot analysis results suggested that the antitumor effect of Rhein might be mediated via STAT3 inhibition. Rhein upregulated the expression of the proapoptotic protein Bax and downregulated the expression of the antiapoptotic protein Bcl-2. In addition, Rhein induced the arrest of NSCLC cells in the G2/M phase of the cell cycle and dose dependently inhibited the expression of cycle-related proteins. The Rhein also inhibited tumor growth in H460 xenograft models.

Conclusion: Rhein shows potent efficacy against NSCLC through inhibiting the STAT3 pathway. Our results also suggest that Rhein has a promising potential to be used as a novel antitumor agent for the treatment of NSCLC.

Keywords: EGFR; NSCLC; Rhein; STAT3; apoptosis; diacerein; inhibitor.

Conflict of interest statement

Disclosure The authors report no conflicts of interest in this work.

Figures

Figure 1

Rhein inhibits viability and colony formation of NSCLC cells. Notes: (A) Chemical structure of Rhein and Diacerein. (B) Effects of Rhein and Diacerein inhibit cell viability and colony formation of PC-9. Cells viability: PC-9 cell lines were treated with various concentrations of Rhein and Diacerein for 48 hours, and were analyzed by MTT assay and the IC50 values were calculated. Colony formation: PC-9 cells were treated with a concentration gradient of Rhein and Diacerein for 24 hours and incubated for 1 week; then, the clones were fixed with ice-cold methanol and stained with crystal violet. (C) Effects of Rhein and Diacerein inhibit cell viability and colony formation of H460. (D) Effects of Rhein and Diacerein inhibit cell viability and colony formation of A549. Abbreviation: NSCLC, non-small-cell lung cancer.

Figure 2

Western blot analysis of P-STAT3 in PC-9, A549 and H460 cells. Notes: (A) H460 cell lines were treated with a concentration gradient of Rhein and Diacerein. The expression of P-STAT3 was found by Western blot analysis. (B) PC-9 cell lines were treated with a concentration gradient of Rhein and Diacerein. The expression of P-STAT3 was found by Western blot analysis. (C) A549 cell lines were treated with a concentration gradient of Rhein and Diacerein. The expression of P-STAT3 was found by Western blot analysis. (D) H460 cells were transfected with luciferase reporter gene plasmid and treated with Rhein for 24 hours. The results were normalized to the Renilla luciferase activity. The bars indicate the mean ± SD. Statistically significant differences (Student’s t-test), *P<0.05; **P<0.01; ***P<0.001. (E) The STAT3 plasmid was transfected into H460 cells and then treated with Rhein for 24 hours. Cells were then lysed and subjected to immunoblotting with indicated antibodies. GAPDH was used as a loading control.

Figure 3

Rhein induced apoptosis of NSCLC cells. Notes: (A) PC-9 cell lines were treated with a concentration gradient of Rhein for 48 hours and stained with Annexin V and propidium iodide. (B) PC-9 cell lines were treated with a concentration gradient of Diacerein for 48 hours and stained with Annexin V and propidium iodide. (C) H460 cell lines were treated with a concentration gradient of Rhein for 48 hours and stained with Annexin V and propidium iodide. (D) H460 cell lines were treated with a concentration gradient of Diacerein for 48 hours and stained with Annexin V and propidium iodide. Representative histograms from flow cytometry analysis in the two human NSCLC cells treated with various concentrations of Rhein and Diacerein. Assays were performed in triplicate. *P<0.05, **P<0.01, and ***P<0.001. (E) Western blot analysis of apoptosis-related proteins. The expression of Bcl-2 and Bax in PC-9 cells. (F) The expression of Bcl-2 and Bax in H460 cells. Abbreviations: NSCLC, non-small-cell lung cancer.

Figure 4

Rhein induces G2/M cell cycle arrest in human NSCLC cell lines. Notes: (A) Induction of cell cycle arrest in PC-9 and H460 cells analyzed by flow cytometry after treatment with a concentration gradient of Rhein for 24 hours. Representative histograms from flow cytometry analysis in the two human NSCLC cells treated with various concentrations of Rhein. Assays were performed in triplicate. (B) Western blot analysis of cycle-related proteins MDM2, CDC2, Cyclin B1 and P53 in PC-9. (C) Western blot analysis of cycle-related proteins MDM2, CDC2, Cyclin B1 and P53 in H460 cells. Abbreviation: NSCLC, non-small-cell lung cancer.

Figure 5

Antitumor activity of Rhein in H460 xenograft animal model. Notes: (A) The result of tumor volume. (B) The tumor weight (n=6). (C) Representative images of the tumor tissue in control and treatment groups (n=6). (D) The tumor tissues were extracted and a Western blot assay was performed. (E) Mice body weight. (F) Kidneys, livers, lungs and hearts tissues from three groups were sectioned at 5 μm and the slides were stained with H&E. All images were obtained by microscope with 20× magnification. **P<0.01****P<0.0001.