| Aim:The incidence of breast cancer has been increased year by year, and it has become one of the most common female malignancy. With changing lifestyles, the incidence of diabetes is also on the rise. Type2diabetes (T2DM)and breast cancer share some common risk factors such as obesity and age. Studies have showen that people with T2DM have a higher risk for breast cancer than the normal population. T2DM can also promote the progression of breast cancer, resulting in higher death rates. Epidemiological studies have shown that T2DM and breast cancer are closely related. Tumor cells may improve the utilization of serum glucose to promote cell proliferation, migration and invasion because of the higher serum glucose in T2DM patients. A large number of misfolded or unfolded protein will be produced because of the high metabolic state of tumor cells, and these proteins maybe degradated through ubiquitin proteasome pathway (UPP). We speculated that protein degradation accelerated by activating the UPS in high serum glucose condition, thus ensuring tumor cells vigorous growth. This article will explore whether the PKCδ-dependent ubiquitin proteasome system activation plays an important role in high glucose-induced breast cancer cell growth and metastasis.Methods:(1) To investigate the effects of high glucose on breast cancer cell MCF-7, MTT and TUNEL assays were performed to measure cell proliferation and apoptosis, wound healin and transwell assays were used to measure the cell migration and invasion.(2) To figure out whether glucose regulated UPS activity, we assessed proteasome peptidase activity to measure the UPS activity in MCF-7cells that had been treated with different glucose conditions. To confirm that the UPS was specifically activated by high glucose, MCF-7cells were coinfected with RFP and GFPu adenoviruses followed by incubation in the presence of different concentration of glucose. Western blots were performed to detect RFP, GFPu protein expression. After pretreatment with UPS inhibitor bortezomib, MTT and TUNEL assays were performed to measure cell proliferation and apoptosis, wound healin and transwell assays were used to measure the cell migration and invasion.(3) To elucidate the mechanism by which UPS activity was upregulated, Phosphorylation of PKC8and PKC8were detected by western blot using specific antibodies. Peptide assay was used to measure proteasomal peptidase activity of MCF-7cells after been pretreated with PKC8specific inhibitor rottlerin. To confirm the effects of PKCδ on UPS further, we investigated whether PKC8activity affected expression of specific proteasome substrate GFPu. RFP and GFPu were detected by western blot in different conditions after coinfecting MCF-7cells with adenovirus/RFP and adenovirus/GFPu. For PKC8knockdown, MCF-7cells was infected with lentivirus carrying PKC8/shRNA and western blot were used to detect RFP, GFPu, PKCδ. To confirm the functional consequences of PKC8inhibition, the growth, migration, and invasion of cell were assessed by MTT, TUNEL, wound healing, and transwell assays, respectively.Results:(1) High glucose enhanced proteasome activity, accompanied with the promoted proliferation, migration and invasion, and suppressed apoptosis in human breast cancer MCF-7cells.(2) Proteasome inhibitor bortezomib (BZM) mitigated high glucose-induced MCF-7cells growth and invasion.(3) High glucose increased phosphorylation of protein kinase C delta (PKCδ).(4) Specific PKC8inhibitor rottlerin attenuated high glucose-stimulated cancer cell growth and invasion.(5) Inhibition of PKCδ by both rottlerin and PKC8/shRNA significantly suppressed high glucose-induced proteasome activity.Conclusions:Our results demonstrated for the first time that PKCδ-dependent UPS activation upregulated MCF-7cell growth and invasion after high glucose treatment. Characterizing the mechanism by which PKC8regulated the UPS and its pathophysiological consequences will provide important information regarding breast cancer diagnosis and therapy. |
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