| Background Breast cancer is one of the most common female cancers in the world, with the high morbidity and high motality. With the change of the people's life style, type 2 diabetes mellitus also has a high incidence worldwide, as a serious threat to people's health. Both breast cancer and type 2 diabetes share similar etiology and signal pathways, including high BMI, obesity, sedentary lifestyle and biological factors, such as hyperinsulinemia and hyperglycemia. A growing number of studies indicated that type 2 diabetes was closely related to the prognosis of breast cancer, type 2 diabetes significantly increased the incidence of breast cancer, therefore, diabetes mellitus is also an independent prognostic risk factors of breast cancer. What'more, the mortality rate of patients with breast cancer combined with type 2 diabetes is higher than the patients without diabetes. With the microenvironment of high blood glucose, tumor cells may increase the utilization of blood glucose to promote the development of tumor, at the same time, the tumor cells under the condition of low oxygen and low nutrition, low pH environment, endoplasmic reticulum stress can be activated within the tumor cells, using unfolded proteins reaction to promote the development of tumor. We hypothesized that under the environment of the high blood glucose, the endoplasmic reticulum stress may be induced in the tumor cells to promote tumor cell proliferation, migration and invasion. Blood glucose fluctuation is an independent risk factors for the prognosis of patients with diabetes, the greater the fluctuation of blood sugar, the higher the incidence of chronic complications of diabetes, the worse prognosis. Preliminary experimental results show that high volatility rather than a continuous high blood glucose can promote the endoplasmic reticulum stress.This article explores the effect of proliferation, migration and invasion in breast cancer cell by intermittent high glucose via endoplasmic reticulum stress.Methods1. Western blot was performed to detect the expression of CHOP and GRP78 in human breast cancer cell MCF-7 after treated with glucose of 5.5mmol/L?11mmol/L and 22mmol/L for 2 days?4 days?6 days. After treated with glucose of 22mmol/L for 6 days, the proliferation of the MCF-7 cells was observed by MTT assay, the apoptosis of the cells was tested by TUNEL assay, the ability of migration and the invasion was analysed by Wound healing and Transwell assay. To figure out the effect of endoplasmic reticulum stress, after treasted with 4-PBA(the inhibition of ERS), and then MTT and TUNEL assays were implemented to measure cell proliferation and apoptosis, wound healing assay and transwell assays were used to measure the ability of cell migration and invasion.2. After treated with 4-PBA(the inhibition of ERS), RT-PCR and Western blot were performed to detect the expression of CHOP?GRP78?PTP1B and STAT3 in human breast cancer MCF-7, meanwhile, the enzyme activity of PTP1B was analysed by protease peptidase activity. To confirm that the PTP1B was specifically activated by intermittent high glucose, CinnGEL 2Me was used to block the activity of PTP1B.Western blot was to detect the expressions of CHOP?GRP78?PTP1B and STAT3, and then MTT and TUNEL assays were implemented to measure cell proliferation and apoptosis, wound healing assay and transwell assays were used to measure the ability of cell migration and invasion. PTP1B shRNA lentivirus was used for knocking down PTP1B to further testify this effect. And then Western blot was used to detect the expression of CHOP?GRP78?PTP1B and STAT3, and the proliferation of the MCF-7 cells was observed by MTT assay, the apoptosis of the cells was tested by TUNEL assay, the ability of migration and the invasion was analysed by Wound healing and Transwell assay.Results1. The overall effects of the intermittent high glucose on ER stress in MCF-7 cells was assessed using the western blots. The results demonstrate that intermittent high glucose induces the expression of CHOP and GRP78 in a dose and gradient-dependent manner, with maximal expression observed with a duration of 6 days. The intermittent high glucose promotes MCF-7 cells viability, while an ER stress inhibitor 4-PBA triggered the intermittent high glucose-induced MCF-7 cell apoptosis. After 6 days the wound was almost covered due to the influx of highly migratory cells in the intermittent high glucose groups, whereas 4-PBA treated cells remained close to the initial state. Compared with the control, a dose-dependent augment in the number of invasive cells was seen in the intermittent high glucose groups. Meanwhile,4-PBA treated cells respectively reduced.2. Western blotting assay and RT-PCR were also used to find out the mechanism of the intermittent high glucose induced MCF-7 cells viability, migration, invasion. The expressions of GRP78 and CHOP were increased in the intermittent high glucose groups and decreased by 4-PBA. Simultaneously, the expressions of PTP1B and u-STAT3 increased in the intermittent high glucose groups and decreased by 4-PBA. But the phosphorylation of STAT3 decreased in the intermittent high glucose groups and increased by 4-PBA. PTP1B enzyme activity also increased in the intermittent high glucose groups and decreased by 4-PBA. The expression of GRP78 mRNA and CHOP mRNA were increased in the intermittent high glucose groups and decreased by 4-PBA. Simultaneously, the expression of PTP1B mRNA increased in the intermittent high glucose groups and decreased by 4-PBA. The expression of STAT3 mRNA only increased in 22 mM glucose group,whereas no significant impact by 4-PBA. The PTP1B inhibitor(CinnGEL 2Me) reduced MCF-7 cells viability and triggered MCF-7 cell apoptosis under the intermittent high glucose condition. Meanwhile, after 6 days the wound was almost covered due to the influx of highly migratory cells in the intermittent high glucose groups, whereas CinnGEL 2Me-treated cells remained close to the initial state. Compared with the control, a dose-dependent augment in the number of invasive cells was seen in the intermittent high glucose groups, whereas CinnGEL 2Me-treated cells respectively reduced. PTP1B enzyme activity was also increased in the intermittent high glucose groups and decreased by CinnGEL 2Me. The expressions of PTP1B, u-STAT3 and CCL5 increased in the intermittent high glucose groups and decreased by CinnGEL 2Me. But the phosphorylation of STAT3 was decreased in the intermittent high glucose groups and increased by CinnGEL 2Me. PTPIB shRNA lentivirus was used for knocking down PTP1B to further testify this effect. The PTP1B shRNA similarly reduced MCF-7 cells viability, triggered apoptosis, inhibited cells migration and invasion under the intermittent high glucose condition. Meanwhile, the expression of PTP1B, u-STAT3 and CCL5 were also increased in the intermittent high glucose groups and decreased by PTP1B shRNA. But the phosphorylation of STAT3 was decreased in the intermittent high glucose groups and increased by PTP1B shRNA.Conclusion (1) Endoplasmic reticulum stress can be activated with breast cancer cell MCF-7 by intermittent high glucose in a concentration-dependent and time-dependent manner. (2) Intermittent high glucose induced the promoted proliferation, migration and invasion, and suppressed apoptosis in breast cancer cell MCF-7 via endoplasmic reticulum stres. ER stress inhibitor 4-PBA relief the MCF-7 cells proliferation, migration and invasion. (3) Intermittent high glucose increased activity of PTP1B. (4) Specific PTP1B inhibitor and PTP1B/shRNA both slowed down the intermittent high glucose induced MCF-7 cell growth, migration and invasion. (5) Specific PTP1B inhibitor and PTP1B/shRNA both significantly suppressed the expression of key factors through the PTP1B/STAT3 signal pathway. |
PDF Full Text Request