Rhein sensitizes human colorectal cancer cells to EGFR inhibitors by inhibiting STAT3 pathway

Abstract

Background: Activation of epidermal growth factor receptor (EGFR) has been reported in a variety of cancer types, including colorectal cancer (CRC), and represents a potential chemotherapeutic drug target. EGFR tyrosine kinase inhibitors (EGFR-TKIs) have been increasingly applied in the clinical treatment of CRC, but development of drug resistance during the treatment has greatly limited their application. Signal transducer and activator of transcription 3 (STAT3) and its mediated signal transduction pathway play an important role in the occurrence, development and metastasis of CRC, and are related to the development of EGFR-TKI resistance in CRC.

Methods: Cell viability, colony formation and cellular morphology were examined to evaluate the potent antiproliferative effect of the STAT3 inhibitor napabucasin, LY5 and rhein on the human CRC cell lines HCT116, SW620, RKO and DLD-1. Flow cytometry-based analysis was employed to determine whether rhein can affect the cell cycle and apoptosis. The expression level of phosphorylated STAT3 (P-STAT3), and cell cycle- and apoptosis-related proteins BCL2, CDC2 BAX, Cyclin D1 and Cyclin B1 were detected by Western blot analysis.

Results: This study revealed that rhein can significantly reduce cell viability and stimulate apoptosis in human CRC cells in a dose-dependent manner. In addition, rhein induced cell cycle arrest at the G2/M phase in CRC cells and dose-dependently inhibited the expression of cell cycle-related proteins. Additionally, it was found that napabucasin, LY5 and rhein considerably sensitized cells to the EGFR-TKI erlotinib, thus suppressing CRC cell proliferation. Rhein also inhibited the phosphorylation of its downstream target STAT3. Inhibition of STAT3 and EGFR phosphorylation was also observed after treatment with a combination of rhein and EGFR inhibitors.

Conclusion: This study confirmed the synergistic effect of STAT3 inhibitor and EGFR inhibitor in CRC cell lines. Additionally, we found that rhein sensitizes human CRC cells to EGFR-TKIs by inhibiting STAT3 pathway. When combined with EGFR-TKIs, rhein may be a novel STAT3 inhibitor in CRC.

Keywords: EGFR; STAT3; colorectal cancer; rhein; tyrosine kinase inhibitor.

Figure 1

The combined effect of LY5 and Erlotinib on CRC cell proliferation measured by the MTT assay. (A) HCT116 cells were treated, in combination or separately with the STAT3 small molecule inhibitor LY5 and/or EGFR small molecule inhibitor erlotinib at different concentrations for 72 h. (B) DLD-1 cells were treated in combination or separately with LY5 and/or erlotinib at different concentrations for 48 h. (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001). Abbreviations: DMSO, dimethyl sulfoxide; L, LY5; E, erlotinib; CRC, colorectal cancer; EGFR, epidermal growth factor receptor.

Figure 2

The combined effect of STAT3 inhibitors and erlotinib on CRC cell migration by wound healing assay. (A) HCT116 cells were treated, in combination or separately, with EGFR small molecule inhibitor erlotinib (Er, 2 μM) and/or STAT3 small molecule inhibitor L (LY5, 2 μM) and allowed to migrate into the scratched area for 24 h. (B) DLD-1 cells were treated, in combination or separately, with Er (2 μM) and/or L (1 μM) and allowed to migrate into the scratched area for 24 h. (C) RKO cells were treated, in combination or separately, with Er (2 μM) and/or napabucasin (1 μM) and allowed to migrate into the scratched area for 24 h. Wound healing assay for migration was conducted by scratching the cells with a yellow tip when cells grew into a monolayer. (D) RKO cells were treated, in combination or separately, with Er (2 μM) and/or napabucasin (1 μM) to verify the effect on MMP-2 protein. Data were obtained from 3 independent experiments. (*P<0.05, **P<0.01, ****P<0.0001). Abbreviations: CRC, colorectal cancer; EGFR, epidermal growth factor receptor.

Figure 3

Rhein sensitizes human CRC cells to EGFR inhibitors by inhibiting STAT3 pathway. (A) HCT116 and SW620 cells were seeded in a 96-well plate at a density of 5,000 cells per well and then cultured for 24 h. The cells were treated with rhein at the indicated concentrations. After a 48 h treatment, cell proliferation in each group was measured by the MTT assay, the IC50 is indicated. (B) HCT116 cells were treated with rhein (50 μM) for different periods of time (0, 3, 6, 9, 12 and 24 h). Total protein was extracted, and the expression levels of P-EGFR, EGFR, P-STAT3, STAT3 and GAPDH proteins were measured by Western blot analysis. (C) Western blot analysis results from P-EGFR were calculated. (D) Western blot analysis results from P-STAT3 were calculated. (E) HCT116 cells were treated with rhein (0，15，30，50 μM) for 24 hrs. Total protein was extracted, and the expression levels of P-JAK2, JAK and GAPDH proteins were measured by Western blot analysis. (F) HCT116 cells were treated with rhein (0，15，30，50 μM) for 24 hrs. And cells were treated with IL-6 for 30 mins，the expression levels of P-STAT3, STAT3 and GAPDH proteins were measured by Western blot analysis. (G) Western blot analysis results from P-STAT3 were calculated. (H) The combination effect of rhein and erlotinib on cell viability (***P<0.001, ****P<0.0001). Abbreviations: CRC, colorectal cancer; EGFR, epidermal growth factor receptor.

Figure 4

Induction of apoptosis in CRC cells by the combination of rhein and erlotinib. (A) HCT116 and SW620 cells were treated, in combination or separately, with erlotinib (Er; 10 μM) and/or rhein (Rh; 100 μM) for 24 h. The level of apoptosis was evaluated by analyzing Annexin V and PI staining, by flow cytometry analysis. (B) A statistical diagram of cell apoptosis (*P<0.05, ***P<0.001). Abbreviation: CRC, colorectal cancer.

Figure 5

Rhein (100 μM) and erlotinib (10 μM), in combination or separately, cause cell cycle arrest in colon cancer cells. (A) Flow cytometry analysis of two different colon cancer cell lines, HCT116 and SW620. (B) A statistical diagram of cell cycle arrest by flow cytometry analysis. (**P<0.01, ***P<0.001).

Figure 6

Rhein and erlotinib efficiently synergistically suppresses phosphorylation of STAT3 and EGFR. (A) Morphological changes of HCT116 colon cancer cells observed by light microscopy after drug treatment. (B) Colony formation assay of HCT116 cells. (C) HCT116 cells were treated, in combination or separately, with erlotinib (Er; 10 μM) and/or rhein (Rh; 50 μM) for 24 h. P-STAT3, P-EGFR, STAT3 and EGFR were detected by Western blot analysis. GAPDH was used as the control protein. (D) HCT116 cells were treated, in combination or separately, with erlotinib (10 μM) and/or rhein (50 μM) for 24 h. CDC2, CyclinB1，CyclinD1，BCL2 and BAX were detected by Western blot analysis, GAPDH was used as the control protein, ****P<0.0001.