α‑hederin overcomes hypoxia‑mediated drug resistance in colorectal cancer by inhibiting the AKT/Bcl2 pathway

Abstract

Currently, chemoresistance is a major challenge that directly affects the prognosis of patients with colorectal cancer (CRC). In addition, hypoxia is associated with poor prognosis and therapeutic resistance in patients with cancer. Accumulating evidence has shown that α‑hederin has significant antitumour effects and that α‑hederin can inhibit hypoxia‑mediated drug resistance in CRC; however, the underlying mechanism remains unclear. In the present study, viability and proliferation assays were used to evaluate the effect of α‑hederin on the drug resistance of CRC cells under hypoxia. Sequencing analysis and apoptosis assays were used to determine the effect of α‑hederin on apoptosis under hypoxia. Western blot analysis and reverse transcription‑quantitative PCR were used to measure apoptosis‑related protein and mRNA expression levels. Furthermore, different mouse models were established to study the effect of α‑hederin on hypoxia‑mediated CRC drug resistance in vivo. In the present study, the high expression of Bcl2 in hypoxic CRC cells was revealed to be a key factor in their drug resistance, whereas α‑hederin inhibited the expression of Bcl2 by reducing AKT phosphorylation in vitro and in vivo, and promoted the apoptosis of CRC cells under hypoxia. By contrast, overexpression of AKT reversed the effect of α‑hederin on CRC cell apoptosis under hypoxia. Taken together, these results suggested that α‑hederin may overcome hypoxia‑mediated drug resistance in CRC by inhibiting the AKT/Bcl2 pathway. In the future, α‑hederin may be used as a novel adjuvant for reversing drug resistance in CRC.

Keywords: Bcl2; CRC; chemoresistance; hypoxia; α‑hederin.

Figure 1

α-hederin overcomes hypoxia-mediated drug resistance in colorectal cancer cells. (A) Effect of α-hederin on the viability of different types of cells under normoxia and hypoxia. (B) Colony formation assay showed the effect of α-hederin on the proliferation of different types of cells under normoxia and hypoxia. (C) BrdU experiment showed the effect of α-hederin on the proliferation of different types of cells under normoxia and hypoxia. Data are presented as the mean ± SD. Compared to control group treated with no drugs, ^*,#P<0.05, ^**,##P<0.01 vs. control group (0 µM).

Figure 2

Apoptotic pathway serves a key role in the effects of α-hederin on overcoming hypoxia-mediated resistance in CRC cells. (A) Differentially expressed genes were observed in a volcano plot. (B) Enrichment analysis of GO terms. (C) Enrichment analysis of KEGG pathways. (D) Gene expression profiling was conducted to observe the changes in related genes after α-hederin treatment of CRC cells under hypoxia. (E) Reverse transcription-quantitative PCR was used to measure the expression levels of Bcl2 and Bcl-xL in CRC cells treated with α-hederin under hypoxia. Data are presented as the mean ± SD. ^*P<0.05, ^**P<0.01. CRC, colorectal cancer; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes.

Figure 3

α-hederin overcomes drug resistance in CRC cells under hypoxic conditions by promoting apoptosis. (A) Flow cytometry and (B) JC-1 immunofluorescence assays were used to evaluate the apoptosis of hypoxic CRC cells treated with α-hederin. (C) ELISA was used to observe the activities of cytochrome c and Caspase-3 after α-hederin treatment. (D) Western blot analysis was used to measure the expression levels of apoptosis-related proteins after α-hederin treatment of CRC cells under hypoxia; β-actin blot is the loading control blot for all of the proteins. Data are presented as the mean ± SD. ^*P<0.05, ^**P<0.01 vs. vehicle. CRC, colorectal cancer; PI, propidium iodide.

Figure 4

AKT/Bcl2 signalling pathway is a key mechanism by which α-hederin overcomes hypoxia-mediated resistance in CRC cells. (A) Treatment of hypoxic CRC cells with α-hederin affected the expression of related proteins, as determined by western blot analysis, β-actin blot is the loading control blot for all of the proteins. (B) Reverse transcription-quantitative PCR was used to measure the expression levels of HIF-1α, Bcl2 and Bcl-xL after α-hederin treatment of hypoxic CRC cells. After OE of AKT, the effect of α-hederin treatment on the (C) protein and (D) mRNA expression levels of Bcl2 and Bcl-xL in hypoxic CRC cells was observed. (E and F) After OE of AKT, the apoptosis of hypoxic CRC cells treated with α-hederin was evaluated. After OE of AKT, the levels of (G) cytochrome c and (H) Caspase 3 were observed after α-hederin treatment of hypoxic CRC cells. Data are presented as the mean ± SD. ^*P<0.05, ^**P<0.01 vs. vehicle or as indicated. CRC, colorectal cancer; OE, overexpression; p-, phosphorylated; PI, propidium iodide.

Figure 5

α-hederin overcomes hypoxia-mediated resistance in colorectal cancer cells via the AKT/Bcl2 pathway in vivo. (A) Xenograft tumour growth curves. (B) Representative images of the immunohistochemical staining of Ki67, Bcl2, Bcl-xL, p-AKT and HIF-1α in tissues and the evaluation of apoptosis in each group by TUNEL. Scale bars, 50 µm. (C) Images and weights of tumours. (D) Reverse transcription-quantitative PCR was used to measure the expression levels of Bcl2 and Bcl-xL in tissues. (E) Mouse weight curves after treatment in the CRC xenograft model. (F) Detection of changes in blood biochemical indicators in mice. (G) Haematoxylin and eosin staining to observe organ damage (magnification, x100). Data are presented as the mean ± SD. ^*P<0.05, ^**P<0.01 vs. hypoxia; ^#P<0.05, ^##P<0.01 vs. normoxia. ALT, alanine aminotransferase; AST, aspartate aminotransferase; N.S., not significant; RBC, red blood cell; PLT, platelets; WBC, white blood cell.

Figure 6

α-hederin overcomes hypoxia-mediated resistance by reducing Bcl2 and Bcl-xL expression in PDX mouse models. (A) Immunohistochemical analysis of HIF-1α and anti-apoptotic proteins in clinical tissue samples. (B) Immunohistochemical score was used to analyse the correlation between HIF-1α expression and anti-apoptotic protein expression. (C) The relationship between HIF-1α expression levels and immunohistochemical score of anti-apoptotic proteins is shown. (D and E) PDX growth curves in mice. (F and G) Images and weights of tumours. RT-qPCR was used to measure the expression levels of Bcl2 and Bcl-xL in tissues. Representative images of IHC of Ki67, Bcl2, Bcl-xL, p-AKT and HIF-1α in tissues and evaluation of apoptosis in each group by TUNEL. Scale bars, 50 µm. Data are presented as the mean ± SD. ^*P<0.05, ^**P<0.01 vs. vehicle or as indicated. IHC, immunohistochemistry; PDX, patient-derived xenograft.