High glucose inhibits autophagy and promotes the proliferation and metastasis of colorectal cancer through the PI3K/AKT/mTOR pathway

Abstract

Background: Colorectal cancer (CRC) ranks among the most prevalent malignancies worldwide, characterized by its complex etiology and slow research progress. Diabetes, as an independent risk factor for CRC, has been widely certified. Consequently, this study centers on elucidating the intricacies of CRC cells initiation and progression within a high-glucose environment.

Methods: A battery of assays was employed to assess the proliferation and metastasis of CRC cells cultured under varying glucose concentrations. Optimal glucose levels conducive to cells' proliferation and migration were identified. Western blot analyses were conducted to evaluate alterations in apoptosis, autophagy, and EMT-related proteins in CRC cells under high-glucose conditions. The expression of PI3K/AKT/mTOR pathway-associated proteins was assessed using western blot. The effect of high glucose on xenograft growth was investigated in vivo by MC38 cells, and changes in inflammatory factors (IL-4, IL-13, TNF-α, IL-5, and IL-12) were measured via serum ELISA.

Results: Our experiments demonstrated that elevated glucose concentrations promoted both the proliferation and migration of CRC cells; the most favorable glucose dose is 20 mM. Western blot analyses revealed a decrease in apoptotic proteins, such as Bim, Bax, and caspase-3 with increasing glucose levels. Concurrently, the expression of EMT-related proteins, including N-cadherin, vimentin, ZEB1, and MMP9, increased. High-glucose cultured cells exhibited elevated levels of PI3K/AKT/mTOR pathway proteins. In the xenograft model, tumor cells stimulated by high glucose exhibited accelerated growth, larger tumor volumes, and heightened KI67 expression of immunohistochemistry. ELISA experiments revealed higher expression of IL-4 and IL-13 and lower expression of TNF-α and IL-5 in the serum of high-glucose-stimulated mice.

Conclusion: The most favorable dose and time for tumor cells proliferation and migration is 20 mM, 48 h. High glucose fosters CRC cell proliferation and migration while suppressing autophagy through the activation of the PI3K/AKT/mTOR pathway.

Keywords: PI3K; autophagy; colorectal cancer; high‐glucose.

FIGURE 1

High glucose promotes tumor cell proliferation. (A) The activity of tumor cells stimulated with different glucose concentrations by CCK‐8. Top: HCT116, Bottom: DLD1. (B) Colony formation assay of HCT116 which cultured with different glucose concentrations. (C) Colony formation assay of DLD1 which cultured with different glucose concentrations.

FIGURE 2

High glucose promotes tumor cell migration. (A) Wound healing assay in HCT116 cells under different glucose concentrations. (B) Wound healing assay in DLD1 cells under different glucose concentrations. (C) Transwell migration experiment in HCT116 cells under different glucose concentrations. (D) Transwell migration experiment in DLD1 cells under different glucose concentrations. (E) Invasion assay of HCT116 cells under different glucose concentrations. (F) Invasion assay of DLD1 cells under different glucose concentrations. (G, H) Statistical graphs of wound healing assay for HCT116 and DLD1 cells. (I, J) Statistical graphs of transwell migration assay for HCT116 and DLD1 cells. (K, L) Statistical graphs of invasion assay for HCT116 and DLD1 cells. The values are presented as the mean ± SD of three independent experiments, *p < 0.05, vs. 0 mM 24 h, #p < 0.05, ##p < 0.01 vs. 0 mM 48 h.

FIGURE 3

High glucose suppresses apoptosis‐related protein expression and promotes EMT‐related protein expression. (A) The expression of apoptosis‐related factors inhibited by high glucose was detected by western blot in HCT116 cells. (B) The expression of apoptosis‐related factors inhibited by high glucose was detected by western blot in DLD1 cells. (C) The expression of EMT‐related protein promoted by high glucose was detected by western blot in HCT116 cells. (D) The expression of EMT‐related protein promoted by high glucose was detected by western blot in DLD1 cells. The values are presented as the mean ± SD of three independent experiments, *p < 0.05, **p < 0.01 vs. 0 mM.

FIGURE 4

High glucose regulates apoptosis by inhibiting autophagy. (A) The expression of autophagy‐related proteins inhibited by high glucose was detected by western blot in HCT116 cells. (B) The expression of autophagy‐related proteins inhibited by high glucose was detected by western blot in DLD1 cells. (C) Transfection of HCT116 cells with Ad‐mCherry‐GFP‐LC3B. (D) The expression of autophagy and apoptosis‐related proteins was detected by western blot in HCT116 cells with CQ. (E) The expression of autophagy and apoptosis‐related proteins was detected by western blot in DLD1 cells with CQ. The values are presented as the mean ± SD of three independent experiments, *p < 0.05, vs. 0 mM, #p < 0.05, vs. 0 mM + CQ.

FIGURE 5

High glucose enhances the expression of PI3K/AKT/mTOR pathway proteins. (A) The PI3K/AKT/mTOR pathway protein was activated by high glucose in HCT116 cells by western blot. (B) The PI3K/AKT/mTOR pathway protein was activated by high glucose in DLD1 cells by western blot. (C) The inhibitor (LY294002) restored activation of the PI3K/AKT/mTOR pathway in HCT116 cells by western blot. (D) The inhibitor (LY294002) restored activation of the PI3K/AKT/mTOR pathway in DLD1 cells by western blot; the values are presented as the mean ± SD of three independent experiments, *p < 0.05, vs. 0 mM; #p < 0.05, vs. 20 mM.

FIGURE 6

Xenograft growth promoted by high glucose. (A) Effects of high glucose on the tumorigenic ability of MC38 cells. (B) Tumor weight (P < 0.05). (C) Tumor growth curve (P < 0.05). (D) Higher expression of KI67 protein detected by IHC in tumor tissue of high‐glucose group (400×). (E) The expression of autophagy‐related proteins inhibited by high glucose was detected by western blot in tumor tissues. (F–J) relevant inflammatory factors detected by ELISA (IL‐4, IL‐13, TNF‐α, IL‐5, IL‐12), *p < 0.05, vs. the control group.

FIGURE 7

Schematic diagram showing the mechanism by which high glucose promotes CRC progression.