Role of endoplasmic reticulum stress in apoptosis induced by HK2 inhibitor and its potential as a new drug combination strategy

Abstract

Compared with normal cells, tumor cells mainly obtain energy through aerobic glycolysis. Hexokinase 2 (HK2) plays a key role in the regulation of tumor cell aerobic glycolysis, and targeting HK2 has become a new strategy for cancer treatment. However, little is known about the role of HK2 in colon cancer and the regulation of its targeted inhibitors. In this study, we found that the expression of HK2 in colorectal cancer tissues was significantly higher than that in adjacent tissues, and the expression level of HK2 in metastatic colorectal cancer was further increased. Meanwhile, the expression level of HK2 was closely related to clinical TNM stage and outcome of colorectal cancer patients. We provide here evidence that HK2 inhibitor 3-Bromopyruvate acid (3-BP) can significantly inhibit the survival and proliferation of colon cancer cells, and induce apoptosis through mitochondrial apoptosis signaling pathway. In addition, we found that 3-BP can also induce endoplasmic reticulum stress in colon cancer cells, the mechanism may be through the increase of intracellular calcium concentration. In vitro and in vivo experiments showed that inhibition of endoplasmic reticulum stress could further increase the proliferation inhibition and apoptosis induced by 3-BP. Collectively, our results show that HK2 is highly expressed in colorectal cancer. 3-BP, an inhibitor of HK2, can induce apoptosis and endoplasmic reticulum stress in colon cancer cells. Endoplasmic reticulum stress plays a protective role in cell death induced by 3-BP. This result suggested that targeting HK2 and endoplasmic reticulum stress may be a valuable strategy in targeted and combination therapy of colon cancer.

Keywords: 3-Bromopyruvate acid; Colon cancer; Endoplasmic reticulum stress; Hexokinase 2·apoptosis.

Fig. 1

Upregulation of HK2 was associated with the poor clinical outcome. A IHC detected the HK2 protein expression in colorectal cancer and paired adjacent normal tissues. B IHC detected the HK2 protein expression in colorectal cancer with or without lymph node metastasis. C Kaplan–Meier analysis of the HK2 expression on overall survival of colorectal cancer patients. D Kaplan–Meier analysis of the HK2 expression on overall survival of colorectal cancer patients with lymph node metastasis

Fig. 2

HK2 inhibitors inhibited the proliferation, survival, and induced apoptosis in colon cancer cells. A The cell viability of HCT116 and RKO was detected by MTT assay for 24 h. B The cell proliferation of HCT116 and RKO was detected by colony-forming assay. C Flow cytometry detected the apoptotic rate of HCT116 and RKO induced by 3-BP for 12 h. D Western blotting detected the expression of cleaved caspase-3 of HCT116 and RKO induced by 3-BP for 24 h (*p < 0.05, **p < 0.01 vs control group)

Fig. 3

HK2 inhibitor induced colon cancer cell death through mitochondrial apoptosis signaling pathway. A Flow cytometry detected the changes of mitochondrial membrane potential of RKO and HCT116 induced by 3-BP for 12 h. B, C Western blotting detected the expression of Bax, Bcl-2, and cytochrome C of RKO and HCT116 induced by 3-BP for 24 h (*p < 0.05, **p < 0.01, ***p < 0.001 vs control group)

Fig. 4

3-BP–induced endoplasmic reticulum stress in colon cancer cells. A, C Immunofluorescence analysis examined the distribution of PDI protein treatment with 3-BP in RKO and HCT116. B, D Western blotting detected the expression of Bip, PDI, ATF4, eIf-2a, and p-eIf-2a of RKO and HCT116 induced by 3-BP (*p < 0.05, **p < 0.01 vs control group)

Fig. 5

3-BP increased the cytoplasmic calcium concentration of colon cancer cells. A, B Fluorescence microscope detected the cytoplasmic calcium concentration of RKO and HCT116 induced by 3-BP

Fig. 6

Inhibition of endoplasmic reticulum stress increased 3-BP–induced cell death in colon cancer cells. A The cell viability was measured of RKO and HCT116 treated by 3-BP with or without endoplasmic reticulum stress inhibitor 4-PBA and TUDCA for 24 h. B, C Flow cytometry detected the apoptotic rate of RKO and HCT116 treated by 3-BP with or without 4-PBA and TUDCA for 12 h. D, E Western blotting detected the expression of cleaved caspase-3 of RKO and HCT116 treated by 3-BP with or without 4-PBA and TUDCA for 24 h (*p < 0.05 vs control group, ^#p < 0.05 vs 3-BP group)

Fig. 7

3-BP and combination with TUDCA suppressed colon cancer growth in nude mice. A HCT116 xenografts were divided into 4 groups, control, 3-BP (15 mg/kg), TUDCA (3 mg/kg), and 3-BP + TUDCA. On day 15 post first injection, xenograft tissues were removed and photographed. B The weight of tumor tissue in each group was measured. C, D In the course of drug action, tumor volume and animal weight were measured (*p < 0.05, **p < 0.01, ***p < 0.001)

Fig. 8

Schematic diagram demonstrating the potential mechanism of 3-BP induction of ER-stress and apoptosis of mitochondrial pathway in colon cancer cells. 3-BP can inhibit the proliferation of colon cancer cells by inhibiting the activity of HK2 kinase. In addition, 3-BP can induce apoptosis and death in colon cancer cells by activating mitochondrial apoptosis signal pathway. At the same time, 3-BP can induce endoplasmic reticulum stress by affecting the concentration of endoplasmic reticulum calcium, and the occurrence of endoplasmic reticulum stress alleviates the apoptosis induced by 3-BP. Combined with endoplasmic reticulum stress inhibitor can further increase the apoptosis of colon cancer cells induced by 3-BP