Melittin as an Activator of the Autophagy and Unfolded Protein Response Pathways in Colorectal HCT116 Cell Line

Abstract

Background: The potential anticancer effect of melittin has motivated scientists to find its exact molecular mechanism of action. There are few data on the effect of melittin on the UPR and autophagy as two critical pathways involved in tumorigenesis of colorectal and drug resistance. This study aimed to investigate the effect of melittin on these pathways in the colorectal cancer (CRC) HCT116 cells.

Methods: MTT method was carried out to assess the cytotoxicity of melittin on the HCT116 cell line for 24, 48, and 72 h. After selecting the optimal concentrations and treatment times, the gene expression of autophagy flux markers (LC3-βII and P62) and UPR markers (CHOP and XBP-1s) were determined using qRT-PCR. The protein level of autophagy initiation marker (Beclin1) was also determined by Western blotting.

Results: MTT assay showed a cytotoxic effect of melittin on the HCT116 cells. The increase in LC3-βII and decrease in P62 mRNA expression levels, along with the elevation in the Beclin1 protein level, indicated the stimulatory role of melittin on the autophagy. Melittin also significantly enhanced the CHOP and XBP-1s expressions at mRNA level, suggesting the positive role of the melittin on the UPR activation.

Conclusion: This study shows that UPR and autophagy can potentially be considered as two key signaling pathways in tumorigenesis, which can be targeted by the BV melittin in the HCT116 cells. Further in vivo evaluations are recommended to verify the obtained results.

Keywords: Bee venom; Colorectal neoplasms; Unfolded protein response; Autophagy.

Fig. 1

Effect of melittin on the growth of HCT116 colon cancer cell line. The cells were exposed to the different concentrations of melittin for 24 h (A), 48 h (B), and 72 h (C). The cell viability was assessed by MTT assay. Data are reported as mean ± SD of three independent assays (n = 3; ^*p < 0.05; ^**p < 0.01; ^***p < 0. 001 compared with control (untreated HCT116) cells

Fig. 2

Induction of the gene expression of LC3-βII and P62 and protein expression of Beclin1 by melittin. The relative mRNA expression levels are presented for LC3-βII (A) and P62 (B), and protein expression level is presented for Beclin1 (c) in HCT116 cells for 24 h. Data are reported as mean ± SD of three independent assays (n = 3; ^***p < 0.001)

Fig. 3

Increased expression levels of CHOP and XBP-1s genes by melittin. The relative mRNA expression levels are presented for XBP-1s (A) and CHOP (b) in HCT116 cells for 24 h. Data are reported as mean ± SD of three independent assays (n = 3; ^***p < 0.001 compared with control)