The functional role of the EZH2 gene in controlling breast cancer stem cells.

PURPOSE: Breast cancer is caused by rare populations of self-renewing cancer stem cells that might also play a role in tumor relapse. Genes that regulate cancer stem cells are, therefore, of great interest in controlling cancer. EZH2 gene expression is reported to be elevated during breast cancer progression and it plays a role in expanding breast stem cell populations. In the current study, we analyzed the correlation between the silencing effect of EZH2 and breast cancer stem cell expansion. METHODS: We used CD44+/CD24-/low cells to develop initial-, moderate-, and advanced-stage breast cancers in female NOD/SCID mice. Immunohistochemistry and western blotting were used to study the expression of aldehyde dehydrogenase 1 (ALDH1) and EZH2 in different stages of breast cancer. RESULTS: Histology showed that as tumors progressed, the pathological condition changed exhibiting enlarged nuclei, higher cell proliferation, and more invasive cells. In EZH2-silenced mice histopathology also showed enlarged cell nucleus, lesion formation and cell aggregation. Immunohistochemistry and western blotting analyses of EZH2 and ALDH1 demonstrated elevated expression as tumors progressed to the next level. Interestingly, the expression of ALDH1 in EZH2-silenced breast cancer tissue showed prolonged overexpression. CONCLUSIONS: We conclude that the normal expression of EZH2 in cancer tissue controls cancer stem cell expansion, because it is highly elevated in EZH2-silencing cancer tissue.

Expression of SOX11 and HER2 and their association with recurrent breast cancer.

BACKGROUND: Recurrent breast cancer occurs as a result of divergent gene expression in response to therapeutic intervention. A recent report showed that SOX11, an embryogenic mammary transcription factor, is overexpressed in breast cancer. HER2 is also dysregulated in breast cancer stem cells; however, the relative expression of these two genes in recurrent breast cancer has not been investigated. METHODS: Mouse models of mild and advanced stage recurrent breast cancer were developed via implantation of different doses of 4T1 Luc2GFP cells. The cellular morphology of normal and recurrent breast cancer tissues was analyzed using standard histological methods. SOX11, HER2, and ALDH1 expression levels were analyzed via immunohistochemistry and western blotting. RESULTS: Histological analyses revealed that treatment with doxorubicin limited mild recurrent cancer but was ineffective against advanced stage recurrent cancers, as evidenced by increased cell proliferation. SOX11 was consistently overexpressed in mild and advanced stage breast cancers treated with doxorubicin, relative to HER2, which exhibited reduced expression in response to doxorubicin treatment in both mild and advanced stage recurrent breast cancer. In advanced stage recurrent breast cancer, SOX11 expression was more readily observed across the cell surface and was correlated with the overexpression of the breast cancer stem cell marker ALDH1. CONCLUSIONS: These results show that SOX11 expression was directly associated with breast cancer stem cell populations. In contrast, HER2 expression was strongly associated with drug treatment effects, but was not correlated with breast cancer stem cell survival in recurrent breast cancer.

Puerarin prevents diabetic cardiomyopathy in vivo and in vitro by inhibition of inflammation.

Diabetic cardiomyopathy (DCM) is one of the chief diabetes mellitus complications. Inflammation factors may be one reason for the damage from DM. The purpose of this research is to study the potential protective effects of puerarin on DM and the possible mechanisms of action related to NF-κB signal pathway. Following administration of puerarin to the disease model rat, several changes were observed including the changes of serum biochemical index, improved diastolic dysfunction, and enhanced endogenous antioxidant enzymes activities, further NF-κB signaling activation. Puerarin showed cardio-protective effects on DCM by inhibiting inflammation, and it might be a potential candidate for the treatment of DCM.

[The dynamic expression changes of myocardium p-p38MAPK, NF-κB and COX-2 in rats after an exhausted exercise].

OBJECTIVE: To observe the dynamic expression changes of p38 mitogen-activated protein kinase (p38MAPK), nucler facter kappa B (NF-κB) and cyclooxygenase-2 (COX-2) in myocardial tissue after an exhausted exercise and study the impact of p38MAPK, NF-κB and COX-2 on its myocardial damage. METHODS: Sixty Wister male rats were randomly divided into the control group (n = 10) and the exhausted exercise group (n = 50). Then the exhausted exercise group was further divided into 5 subgroups, namely 0 h, 3 h, 6 h, 12 h, 24 h after an exhausted exercise (n = 10). The myocardial injury animal model was set up by using an exhausted swimming exercise and the expression of p-p38MAPK, NF-κB and COX-2 were examined by Western blot. RESULTS: Compared with the control group, the expression of p-p38MAPK were increased significantly (P < 0.01) in all the groups and the 3 h group was the highest( P < 0.01); The expression of NF-κB were increased significantly (P < 0.05) in all the groups but 0 h P > 0.05) and the 6h group was increased significantly compared with the other groups( P < 0.05); The expression of COX-2 were increased significantly( P < 0.05) in all the groups but 0 h and the 24 h groups was increased significantly compared with the other groups(P < 0.05). CONCLUSION: p38MAPK was activated in an acute exhausted exercise, p-p38MAPK may play an important role in modulating NF-κB and COX-2 expression and mediating the exhausted exercise induced myocardial damage.

Methylamine irisolidone, a novel compound, increases total ATPase activity and inhibits apoptosis in vivo and in vitro.

We propose to further research the protective effect of MMI on myocardium ischemic rat model and H9c2 cells that underwent cell apoptosis induced by hypoxia. We established the myocardium ischemic rat model via the cardiac surgical procedures in vivo and treated the model rats with different concentration of MMI. In vitro, with the pretreatment of MMI for 12 h in the model of Na2S2O4-induced hypoxia injury, the H9c2 cells viability was determined by MTT assay. We found that MMI had significantly improved cardiac function of the myocardial ischemia, and significantly decreased the reactive oxygen species level. The expression of P53, Bcl-2, Bax, and caspase-9 was also induced by MMI. In vitro study revealed a concentration-dependent increase in cell viability associated with MMI pretreatment. Annexin V-FITC and PI staining results showed that MMI had a preventive effect on hypoxia-induced apoptosis in H9c2 cells. MMI also inhibited the mitochondrial membrane potential decrease and increased total ATPase activity during hypoxia in H9c2 cells. In conclusion, MMI can enhance the cardiac function in myocardial ischemic rat and increase cell viability and attenuate the apoptosis in H9c2 cells induced by hypoxia, which was associated with inhibiting MMP decreasion and increasing total ATPase activity.

Prognostic effect analysis of molecular subtype on young breast cancer patients.

OBJECTIVE: To make a prognostic effect analysis of molecular subtype on young breast cancer patients. METHODS: Totally 187 cases of young breast cancer patients less than 40 years old treated in Obstetrics and Gynecology Hospital of Fudan University between June 2005 and June 2011 were included in our study. We described their clinical-pathological characteristics, disease-free survival (DFS) rate, and overall survival (OS) rate after a median follow-up period of 61 months. The factors associated with prognosis were also evaluated by univariate and multivariate analyses. RESULTS: All patients were premenopausal, with an average age of 35.36±3.88 years old. The mean tumor size was 2.43±1.53 cm. Eighty-one cases had lymph node metastasis (43.3%), 126 cases had lymphovascular invasion (67.4%), and 125 cases had histological grade III (66.8%) disease. Twenty-seven cases (14.4%) were Luminal A subtype, 99 cases (52.9%) were Luminal B subtype, 29 cases (15.5%) were human epidermal growth factor receptor 2 (HER-2) overexpression subtype, while 32 cases (17.1%) were triple negative breast cancer (TNBC) subtype according to 2013 St Gallen expert consensus. One hundred and thirty-five cases underwent mastectomy whereas 52 cases had breast-conserving surgery. One hundred and seventy-eight cases underwent adjuvant or neoadjuvant chemotherapy. Recurrence or metastasis occurred in 29 cases, 13 of which died. The 5-year DFS and OS rates were 84% and 92%. Multivariate analysis showed that nodal status (P=0.041) and molecular subtype (P=0.037) were both independent prognostic factors of DFS, while nodal status (P=0.037) and TNBC subtype (P=0.048) were both independent prognostic factors of OS. CONCLUSIONS: Molecular subtype is an independent prognostic factor of young breast cancer patients. TNBC has a high risk of relapse and death.

Activation of Wnt/ß-catenin/GSK3ß signaling during the development of diabetic cardiomyopathy.

BACKGROUND: As Wnt/ß-catenin/glycogen synthase kinase 3ß (GSK3ß) signaling has been implicated in myocardial injury and diabetic cardiomyopathy (DCM) is a major part of diabetic cardiovascular complications, we therefore investigated the alterations of Wnt/ß-catenin/GSK3ß signaling during the development of DCM. METHODS: The rat model of diabetes mellitus (DM) was established using a single intraperitoneal injection of streptozotocin (STZ, 60 mg/kg). The alterations of Wnt/ß-catenin/GSK3ß signaling were determined 4, 8, and 12 weeks following DM using Western blotting, immunohistochemistry, and quantitative real-time reverse transcriptase polymerase chain reaction. Cardiac pathology changes were evaluated using hematoxylin and eosin, Masson trichromatic, and terminal dUTP nick-end labeling staining. RESULTS: Histological analyses revealed that DM induced significant myocardial injury and progressive cardiomyocyte apoptosis. The protein and mRNA levels of Wnt2, ß-catenin, and c-Myc were progressively increased 4, 8, and 12 weeks following DM. The expression of T-cell factor 4 and phosphorylated of GSK3ß on Ser9 were progressively increased. However, the expression of the endogenous Wnt inhibitor Dickkopf-1 was increased after STZ injection and then decreased as DCM developed. CONCLUSION: Wnt/ß-catenin/GSK3ß signaling pathway is activated in the development of DCM. Further investigation into the role of Wnt signaling during DCM will functionally find novel therapeutic target for DCM.

[The dynamic change of serum CK, CK-MB and myocardium histomorphology after exhausted exercise in rats].

OBJECTIVE: To study the dynamic changes of serum CK, CK-MB and myocardium histomorphology in different time periods after single bout and repeated exhausted exercise in rats. METHODS: The animal models of myocardial injury were established by exhausted swimming. Creatine kinase (CK), creatine kinase mass (CK-MB) activities in serum were measured immediately at 3, 6, 12, 24, 48 and 96 hours after exhausted exercise, and the dynamic changes of myocardial histopathology were examined. RESULTS: The CK, CK-MB activities were significantly increased immediately at 3, 6, 12 hours and peaked at 6 hours after single bout of exhausted exercise, meantime the degree of inflammatory cell infiltrate and strong acidophil staining were gradually increased in myocardium of rat, and the myocardial injury was most severe at 12 hours. After 1-week consecutive daily exhausted swimming, CK, CK-MB in serum were obviously increased immediately at, 3, 6, 12, 48 and 96 hours postexercise and peaked immediately and at 96 hours respectively postexercise. There were different degrees of myocardial injury in different time of recovery phase, and was most severe at 48 hours postexercise. CONCLUSION: The myocardial injury was induced by excessive exercise and/or exhausted exercise, and the resulting delayed-onset myocardial injury was further certified.

[Protective effect of puerarin on stress-induced gastric mucosal injury in rats].

OBJECTIVE: To study the protective effect of puerarin on stress-induced gastric mucosal injury in rats. METHOD: The model of gastric ulcer was established by restraint plus water-immersion stress in rats. Gastric motility was monitored by the method of "Gas Balloon". Gastric mucosal blood flow was recorded by laser-Doppler flowmetry. Colorimetric method was used to determine the content of NO and ET in gastric mucosal tissue. Meantime the pathologic changes of gastric mucosal was examined. RESULT: Puerarin could significantly attenuated gastric mucosal damage induced by water-immersion stress, inhibited gastric motility, specially decreased the index of gastric motility and percentage of gastric contraction time and numbers of violent contraction. The gastric mucosal blood flow and NO level in gastric mucosal were enhanced, while ET level was reduced by puerarin. The degree of tissue damage in gastric mucosal was also significantly attenuated after administration fo puerarin. CONCLUSION: Puerarin exerts a significant protective effect on water-immersion stress-induced gastric mucosal damage by relaxing the vessels, increasing NO level in gastric mucosal, increasing regional gastric mucosal blood flow and inhibiting gastric motility.