Mechanism Research On Inhibition Of High Glucose-induced Human Breast Cancer Cell Growth By Wogonin

Breast cancer is the most common malignancy of women worldwide. The American Cancer Society (ACS) annual statistics in 2015 shows that new cases of breast cancer in women continued to hold the first place, while the mortality rate by cancer-related deaths ranks second. As the tumor cells take advantage of nutrition to maintain their proliferation from glucose, hyperglycemia provides an excellent environment to breast cancer cell for survival and growth. High glucose in vivo is an important factor to promote tumor cell proliferation, differentiation and migration, and hyperglycemia or diabetes increase the risk of morbidity and mortality of breast cancer. Hyperglycemia or diabetes with breast cancer has been a serious problem, which threat to human health, economic, social and family, and become a heavy financial and mental stress. Therefore, clarifying the relationship of development, mechanisms and therapeutic targets between hyperglycemia or diabetes and breast cancer, and looking for a relatively safe and effective treatment for the disease have the extremely vital clinical significance.In recent years, the application of traditional Chinese medicine in tumor has become a hot topic. Wogonin, belonging to the flavonoids, is one of the active ingredients of traditional Chinese herbal medicine skullcap. It is reported that flavonoids have a variety of pharmacological effects, including antioxidant, anti-inflammatory, anti-viral, anti-tumor and anti-allergic activity. Many studies confirmed the anti-tumor activity of Wogonin in vitro, including its inhibition of myeloid leukemia cells, primary liver cancer cells, breast cancer cells, murine sarcoma cells, lung cancer cells, prostate cancer cells and bladder cancer cells. Interestingly, wogonin did not induce apoptosis of the peripheral blood mononuclear cells and fibroblasts.At present, there are conflicting reports about mechanism of inhibiting human breast cancer by Wogonin in domestic and foreign literature. Particularly, Wogonin inhibition of high glucose-induced breast cancer cells has not been reported. Combined with the existing reports and preliminary results of this study, we simulated high glucose in vitro, compared the developing of breast cancer cells between normal glucose concentration and high, and then observe the inhibition to breast cancer cells under different environments in vitro by Wogonin. We reasonable hypothesis that wogonin can activate one or more subtype of MAPKs to promote its phosphorylation through MAPK signaling pathways, thereby inhibite high glucose-induced human breast cancer cell proliferation, invasion and metastasis.This study consists of two parts:Part 1:The effect of high glucose on human breast cancer MCF-7 cellsObjective:To study the high glucose effect on human breast cancer MCF-7 cell viability, migration and invasion, and explore its possible mechanism.Methods:The relative activity of MCF-7 cells on different concentrations of glucose was detected by MTT assay. The invasion ability of MCF-7 cells on different concentrations of glucose was detected by Transwell experiment. Observe the migration of MCF-7 cells on different concentrations of glucose by wound healing experiment. The phenomenon of apoptosis were observed and analyzed under different concentrations of glucose by TUNEL and DAPI. Western-blotting technology was used to detect the expression of total and phosphorylated proteins of AKT, PKCδ, and subtypes of MAPKs. Finally the data were analyzed by medical statistical software.Results:(1) Contrast with the group of normal concentration (5.5mM) glucose, the number of MCF-7 cells on high concentrations (11mM,22mM) glucose group measured by MTT was obviously increased. The longer the cells are exposed to glucose, the more the relative cells number. The difference was statistically significant (p<0.05, 0.01). (2) Contrast with the group of normal concentration (5.5mM) glucose, the number of MCF-7 cells invasion through the basement membrane to lower chamber on high concentrations (11mM,22mM) glucose group measured by Transwell was obviously increased, and the difference was statistically significant (p<0.05,0.01). (3) Contrast with the group of normal concentration (5.5mM) glucose, the number of MCF-7 cells in healing area on high concentration (11mM,22mM) glucose group by wound healing was obviously increased, the difference was statistically significant (p< 0.05,0.01). (4) Contrast with the group of normal concentration (5.5mM) glucose, TUNEL method showed red fluorescence of MCF-7 cells decreased, and DAPI method showed blue fluorescence increased on high concentration (11mM,22mM) glucose group, the difference was statistically significant (p<0.05,0.01).(5) Western-blotting detect that the expression of phosphorylated AKT and PKCδ increased whereas phosphorylated P38MAPK decreased on high concentration (11mM,22mM) glucose group compared with the control group, and the difference was statistically significant ( p<0.05).Conclusion:(1) In human breast cancer MCF-7 cells, high glucose can improve viability, invasion, migration, and inhibit apoptosis. (2) high glucose can promote tumor growth and progression through activating phosphorylation of AKT and PKCδ, while inhibiting phosphorylation of P38MAPK.Part 2:Mechanism of Inhibition high glucose-induced MCF-7 cell growth by WogoninObjective:To study the mechanism of inhibition high glucose-induced MCF-7 cell viability, invasion and migration, and promoting apoptosis by Wogonin.Methods:The relative activity of MCF-7 cells on different concentrations glucose homeostasis after wogonin treatment was measured by MTT assay. The invasive ability was measured by Transwell assay. Wound healing was used to observe migration of MCF-7 cells on different concentrations of glucose homeostasis after wogonin treatment. TUNEL and DAPI were used to observe and analyze apoptosis of MCF-7 cells on different concentrations of glucose homeostasis after wogonin treatment. The expression of total and phosphorylated protein of AKT, PKCδ and MAPKs subtypes of MCF-7 cells on different concentrations of glucose homeostasis after wogonin treatment were measured by Western-blotting. To further prove that P38MAPK signaling pathway participate in anti-tumor effect by wogonin, we use P-P38 inhibitor SB203580 and P38shRNA, and repeat all of experiments and observe the results. The data were analyzed by medical statistical software.Results:(1) Compared with untreated cells, MTT assay measured that cell viability obviously reduced on high glucose (11mM,22mM) environment after wogonin treatment by a concentration-dependent manner, the inhibition rate was 45.1% and 29.1% respectively, and the higher the concentration of glucose, the more obvious inhibition in cells. The the difference was statistically significant (p<0.05,0.01). (2) Compared with untreated cells, Transwell assay testes that the number of MCF-7 cells, which through from media intrusion into the small chamber reduced on high glucose (llmM,22mM) environment after wogonin treatment, the difference was statistically significant (p<0.05). (3) compared with untreated cells, wound healing tested that MCF-7 cells migrate close to the initial state on high glucose (11mM,22mM) environment after wogonin treatment, the number of migration cells reduced obviously, the difference was statistically significant (p<0.05). (4) Compared with untreated cells, TUNEL method showed red fluorescence of MCF-7 cells increased obviously, and DAPI method showed blue fluorescence decreased on high glucose (11mM,22mM) environment after wogonin treatment, the difference was statistically significant (p< 0.01). (5) compared with untreated cells, Western-blotting detected that the level of phosphorylated AKT and PKCδ decreased while phosphorylated P38 increased on high glucose (11mM,22mM) environment after wogonin treatment, the difference was statistically significant (p<0.05). (6)After addition P-P38 inhibitors SB203580, on high glucose (11mM,22mM) environment after wogonin treatment, the expression of phosphorylated P38 decreased, the relative activity, invasion and migration of MCF-7 cells increased, while apoptosis decreased, the difference was statistically significant (p <0.05,0.01). (7) Compared with the control group, After addition P38shRNA, on high glucose (11mM,22mM) environment after wogonin treatment, the ration of phosphorylated P38/total P38 decreased, the relative activity, invasion and migration of MCF-7 cells increased, while apoptosis decreased, the difference was statistically significant (p<0.05,0.01).Conclusion:Wogonin can inhibit the effect of high glucose-induced MCF-7 cell survival, migration and invasion, and promote apoptosis through inhibiting phosphorylation of AKT and PKCδ, and activating phosphorylation of P38MAPK.