[The preliminary research on the gap junction mechanisms for synergistic effects of liuwei di-huang pill containing serum on suicide gene therapy of melanoma].

OBJECTIVE: To study the gap junction mechanisms for synergistic effects of liuwei di-huang pill (LDP) containing serum on HSV-tk/GCV suicide gene therapy of mouse malignant melanoma B16 cells. METHODS: The LDP containing serum (2.5% LDP serum group, 5.0% LDP serum group, and 10.0% LDP serum group) and the control serum group were set up. The effects of LDP on mRNA expressions of Cx26 and Cx43 in mouse malignant melanoma B16 cells were detected by RT-PCR assay. The effects of LDP on protein expressions of Cx26 and Cx43 in B16 cells were detected using Western blot and indirect immunofluorescence assay. The interference efficiencies of Cx26-309, Cx26-337, Cx26-367, Cx26-2098 SiRNA on Cx26 gene in B16 cells were detected using RT-RCR technique. Cx26-2098 SiRNA, due to the optimal interference efficiency, was chosen to interfere Cx26 gene, and the bystander effect of LDP + HSV-tk/GCV was observed. The effects of the gap junctional intercellular communication (GJIC) inhibitor glycyrrhetinic acid on the killing of LDP + HSV-tk/GCV system to B16 cells were detected by MTT assay. In this experiment, the control serum group, 2.5% LDP serum group, 5. 0% LDP serum group, 10.0% LDP serum group, the control serum combined GCV group, 2.5% LDP serum combined GCV group, 5.0% LDP serum combined GCV group, 10.0% LDP serum combined GCV group, 2.5% LDP serum combined GCV + glycyrrhetinic acid group, 5.0% LDP serum combined GCV + glycyrrhetinic acid group, 10.0% LDP serum combined GCV + glycyrrhetinic acid group were set up. The final concentration of GCV was 20 micromol/L. The final concentration of glycyrrhetinic acid was 50 micromol/L. RESULTS: LDP containing serum could increase the protein and mRNA expressions of Cx43 in B16 cell in a dose-dependent manner. It had bi-directional regulation on the Cx26 protein and mRNA expressions. The low dose LDP had inhibition while high dose LDP could up-regulate its expression. After SiRNA interfered Cx26 gene, there was no obvious change in the bystander effect of LDP combined suicide gene therapy between before and after interference. There was significant reduction in the inhibition rate between before (48.75%, 59.39%, and 69.28%) and after blockage (29.14%, 38.71%, and 58.13%) of glycyrrhetinic acid in the 2. 5%, 5.0%, 10.0% LDP serum combined GCV groups (P <0.01). CONCLUSION: The synergistic effects of LDP containing serum on HSV-tk/GCV suicide gene therapy in mouse malignant melanoma B16 cells were correlated with the gap junction mechanisms, which might be achieved through increasing the expressions of Cx26 and Cx43.

Induction of apoptosis by evodiamine involves both activation of mitotic arrest and mitotic slippage.

Evodiamine (Evo) is an indole quinazoline alkaloid isolated from the fruit of Evodia rutaecarpa Bentham. Previous studies have shown that Evo exhibits anti-proliferative anti-tumor activities in several cancer types, but its target(s) and underlying mechanism(s) of action remain unclear. In the present study, we sought to establish a cell synchronization model in order to examine the anti-proliferative and apoptotic mechanisms of Evo in the human gastric cancer cell line SGC-7901. In addition, we transfected these cells with full-length or non-degradable (ND) cyclinB1 to evaluate the relationship between the induction of apoptosis and activation of mitotic arrest and mitotic slippage by Evo. Our results demonstrated that Evo markedly inhibited cell growth and was cytotoxic to SGC-7901 cells. Furthermore, transient Evo treatment (<16 h) caused reversible mitotic arrest, but sustained mitotic arrest was required to initiate apoptosis. The time required to reverse the apoptotic effects of Evo was between 16 and 20 h. We also demonstrated that promotion of mitotic slippage by a CDK1 inhibitor enhanced apoptosis. Furthermore, we evaluated the effect of delaying mitotic slippage by overexpressing ND cyclinB1, which delayed apoptosis. In conclusion, these results indicate that Evo-induced apoptosis is associated with mitotic arrest and subsequent mitotic slippage, which may underlie the actions of Evo in the treatment and prevention of cancer.

[Effect of sinomenine on tumor necrosis factor-alpha and nuclear factor-kappaB in the heterotopic tissue in rats with endometriosis].

OBJECTIVE: To investigate the effect of sinomenine on the level of tumor necrosis factor-alpha (TNF-alpha) and nuclear factor-kappaB (NF-kappaB) and in the heterotopic tissue in rats with endometriosis. METHODS: The rats with endometriosis were divided into sinomenine lavage group, blank control group, model group and danazol group, and the levels of TNF-alpha and NF-kappaB in the heterotopic tissues of the rats were detected with immunohistochemistry. RESULTS: Sinomenine lavage and danazol treatment both significantly decreased the levels of levels of TNF-alpha and NF-kappaB in the heterotopic tissues of the rats as compared with the model group (P<0.05), and lesions were significantly smaller in sinomenine lavage group than in danazol group. CONCLUSION: Sinomenine can inhibit the production and activity of TNF-alpha and NF-kappaB to suppress the adhesion, implantation, infiltration and growth of the endometrial cells in the rat model of endometriosis.