[Hirsutine induces apoptosis of human breast cancer MDA-MB-231 cells through mitochondrial pathway].

The aim of this study was to investigate the effects of hirsutine on apoptosis of breast cancer cells and its possible mechanism. The MCF-10A, MCF-7 and MDA-MB-231 cells were treated with hirsutine at different concentrations for 48 h or incubated with 160 µmol/L hirsutine for 24, 48, and 72 h. The MCF-10A cell line is a non-tumorigenic epithelial cell line, and the MCF-7 and MDA-MB-231 are human breast adenocarcinoma cell lines. CCK-8 assay was employed to detect the cell viability. Flow cytometry was used to assay the apoptosis and mitochondrial membrane potential (MMP). The protein expressions of Bcl-2, Bax, cleaved-caspase 9, cleaved-caspase 3 and cytochrome C (Cyt C) in the MDA-MB-231 cells were detected by Western blotting. The results showed that hirsutine remarkably reduced the viability of MCF-7 and MDA-MB-231 cells in a time- and dose-dependent manner (P < 0.05) with IC50 values of 447.79 and 179.06 µmol/L, respectively. In the MDA-MB-231 cells, hirsutine induced apoptosis and depolarization of MMP (P < 0.05), released Cyt C from mitochondria (P < 0.05), and activated caspase 9 and caspase 3 (P < 0.05). However, these effects induced by hirsutine were all inhibited by cyclosporin A (CsA) (P < 0.05), a specific inhibitor of mitochondrial permeability transition pore (MPTP). In addition, hirsutine down-regulated the protein level of Bcl-2 and up-regulated the protein level of Bax (P < 0.05). These results suggest that hirsutine may induce apoptosis of human breast cancer MDA-MB-231 cells through decreasing the ratio of Bcl-2 to Bax, opening MPTP, releasing Cyt C from mitochondria, and activating caspase 9 and caspase 3.

Luteolin Modulates SERCA2a Leading to Attenuation of Myocardial Ischemia/ Reperfusion Injury via Sumoylation at Lysine 585 in Mice.

BACKGROUND/AIMS: The myocardial sarcoplasmic reticulum calcium ATPase (SERCA2a) is a pivotal pump responsible for calcium cycling in cardiomyocytes. The present study investigated the effect of luteolin (Lut) on restoring SERCA2a protein level and stability reduced by myocardial ischemia/reperfusion (I/R) injury. We verified a hypothesis that Lut protected against myocardial I/R injury by regulating SERCA2a SUMOylation. METHODS: The hemodynamic data, myocardial infarct size of intact hearts, apoptotic analysis, mitochondrial membrane potential (ΔΨm), the level of SERCA2a SUMOylation, and the activity and expression of SERCA2a were examined in vivo and in vitro to clarify the cardioprotective effects of Lut after SUMO1 was knocked down or over-expressed. The putative SUMO conjugation sites in mouse SERCA2a were investigated as the possible regulatory mechanism of Lut. RESULTS: Initially, we found that Lut reversed the SUMOylation and stability of SERCA2a as well as the expression of SUMO1, which were reduced by I/R injury in vitro. Furthermore, Lut increased the expression and activity of SERCA2a partly through SUMO1, thus improving ΔΨm and reducing apoptotic cells in vitro and promoting the recovery of heart function and reducing infarct size in vivo. We also demonstrated that SUMO acceptor sites in mouse SERCA2a involving lysine 585, 480 and 571. Among the three acceptor sites, Lut enhanced SERCA2a stability via lysine 585. CONCLUSIONS: Our results suggest that Lut regulates SERCA2a through SUMOylation at lysine 585 to attenuate myocardial I/R injury.

Effect of MicroRNA-30e on the Behavior of Vascular Smooth Muscle Cells via Targeting Ubiquitin-Conjugating Enzyme E2I.

BACKGROUND: Many microRNAs (miRNAs) have recently been shown to demonstrate critical roles in differentiation, proliferation and migration of vascular smooth muscle cells (VSMCs).Methods and Results:In this study, a certain amount of miRNA expression in VSMCs was evaluated by real-time polymerase chain reaction, and it was found that microRNA-30e (miR-30e) was expressed more strongly than other common vascular well-expressed miRNAs in vitro. Subsequently, both a gain and loss of function study was performed in vitro and in vivo. It was found that miR-30e in VSMCs was strongly downregulated concomitantly with stimulation, and miR-30e inhibited VSMCs proliferation and migration both in vitro and in vivo. Furthermore, ubiquitin-conjugating enzyme E2I (Ube2i) was identified as the target gene of endogenous miR-30e by luciferase reporter assay, and it was confirmed that overexpression of miR-30e significantly reduced Ube2i and inhibited the phenotypic switch of VSMCs. Knockdown of Ube2i had an influence over the proliferation and migration of cultured VSMCs, as same as the miR-30e mimic did. Overexpression of miR-30e induced the apoptosis of VSMCs and deregulated the protein expression of IkBα, which is crucial for the NFκB signal pathway. CONCLUSIONS: The results of this study indicated that miR-30e in VSMCs exerted an anti-atherosclerosis effect via inhibiting proliferation and migration, and promoting apoptosis of VSMCs. More specifically, it was demonstrated that miR-30e exhibited these effects on VSMCs partially through targeting Ube2i and downregulating the IκBα/NFκB signaling pathway.

Outer Balloon Ligation Increases Success Rate of Ischemia-Reperfusion Injury Model in Mice.

BACKGROUND: Coronary artery disease is a growing public health problem and a major cause of morbidity and mortality. Experimental animal models provide valuable tools for studying myocardial ischemia reperfusion (I/R) injury in vivo. OBJECTIVE: The purpose of this study was to describe a new method (outer balloon ligation) to induce myocardial I/R injury in mice. METHODS: Ninety-nine male C57BL/6J mice were randomly divided into three groups: sham group, classic method group (I/R-C) and the new method group (I/R-N). The surgical procedure and recovery time were recorded. The levels of TNF-α, IL-6, cTnT and LDH were detected by ELISA kits. Hematoxylin-eosin staining was applied to assess neutrophil infiltration. Moreover, surgical survival, myocardial infarction areas, and cardiac function measurements were also recorded. RESULTS: The reperfusion operation time in the I/R-N group were markedly less than the I/R-C group (14.73±2.86 vs. 168.60±33.01 sec, p <0.0001). Similarly, the recovery time in I/R-N group was shorter than the I/R-C group (45.39±15.39 vs. 101.70±19.33 min, p <0.0001). The levels of TNF-α and IL-6 in I/R-N group were also markedly lower than in I/R-C group (136.5±22.21 vs. 170.5±24.79 pg/ml, p <0.05 and 100.3±23.74 vs. 144.40±22.24 pg/ml, p <0.001). Compared I/R-N group with I/R-C group, the levels of neutrophil infiltration, cTnT and LDH had no significant differences. Surgical survival rate was 96.7% in the I/R-N group, which was significantly improved compared to the rate of 80% in the I/R-C group. However, there were no significant differences in the areas of myocardial infarction and cardiac function between the two groups. CONCLUSIONS: Compared with the classic method, our new method of inducing myocardial I/R injury has higher efficiency and less tissue damage in mice, but achieves the same modeling effects.

Influence of lymphatic endothelial cells on proliferation and invasiveness of esophageal carcinoma cells in vitro and lymphangiogenesis in vivo.

The aim of the study was to investigate the interaction between esophageal carcinoma cells with different differentiation degree and esophageal carcinoma-related lymphatic endothelial cells. Different lymphatic endothelial cell conditioned mediums were used to cultivate well-differentiated esophageal carcinoma EC9706 cells and poorly differentiated esophageal carcinoma KYSE150 cells, and immunocytochemistry and Western blot analyses were applied to detect the expression of MMP-9 protein and TIMP-2 protein in each group; in situ hybridization and RT-PCR methods were used to detect the expression of MMP-9 and TIMP-2 mRNA in each group; CCK-8 method was used to detect cell proliferation in each group; and transwell method was utilized to detect cell invasiveness in each group. Through constructing the transplanted tumor model of esophageal carcinoma of nude mice, the D2-40 and LYVE-1 immunohistochemical staining was performed on transplanted tumors and surrounding tissues, lymphatic microvessels were marked, and lymphatic microvessel density (LMVD) was measured. The expression of MMP-9 protein and mRNA in experimental group was significantly higher than that in control groups (P < 0.05); TIMP-2 protein and mRNA expression in experimental group was significantly lower than that in control groups (P < 0.05); cell proliferation ability and invasiveness ability in experimental group were significantly higher than those in control groups (P < 0.05); LMVD-marked D2-40 and LMVD-marked LYVE-1 of transplanted tumor tissue in the experimental group were significantly higher than those in control groups (P < 0.05). The esophageal squamous carcinoma-related lymphatic microvessel could promote the proliferation and invasive ability of esophageal squamous carcinoma cells in vitro. It had different effects on esophageal carcinoma cells with different differentiation degree and had more obvious influence on poorly differentiated esophageal carcinoma cells, which may be related to the up-regulated MMP-2 expression and down-regulated TIMP-2 expression of esophageal carcinoma cells. The esophageal squamous carcinoma-related lymphatic microvessel endothelial cells could promote the growth of esophageal carcinoma-transplanted tumor of nude mice and lymphangiogenesis.