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Activation and proliferation of hepatic stellate cells (HSC) play an important role in the progress of liver fibrosis. HSC activation occurs in response to inflammatory cytokines, cellular interactions with immune cells, and morphogenetic signals. The literature hints to a role of the adaptor protein MyD88 in fibrosis. Although curcumin has been shown to exert inhibitory effects on the proliferation of HSC in vitro , its influence on the MyD88 pathway in HSC has remained unclear. Here, we investigated whether curcumin accelerates apoptosis of HSC through the MyD88 pathway. HSC (rat HSC T6) were divided into a control group, MyD88 small interfering RNA (siRNA) group, curcumin group, and curcumin + MyD88 siRNA group. The MyD88 siRNA groups were exposed to siRNA for 48 h. The curcumin groups were cultured in the presence of curcumin for 24 h. Apoptosis was detected by flow cytometry. For Toll-like receptor (TLR) 2 and 4 as well as MyD88 and the dependent factors NF- κ B, TNF- α , and IL-1 β , mRNA expression was detected by reverse transcription polymerase chain reaction (RT-PCR). For MyD88, protein expression was further observed by Western Blot. Both curcumin and MyD88 siRNA inhibited the mRNA expression of MyD88 pathway-related effectors (TLR2, TLR4, NF- κ B, TNF- α , IL-1 β ) in HSC. Furthermore, both treatments reduced the expression of MyD88 protein in HSC and promoted their apoptosis. These effects were more obvious in the curcumin + MyD88 siRNA group. This study demonstrates that curcumin promotes apoptosis of activated HSC by inhibiting the expression of cytokines related to the MyD88 pathway. It elucidates the possible mechanisms of curcumin in inducing apoptosis of HSC through the MyD88 pathway., Competing Interests: Conflict of Interest: The authors declare no conflict of interest., (Georg Thieme Verlag KG Stuttgart · New York.)

Autophagy is a metabolic process that is important in fibrogenesis, in which cellular components are degraded by lysosomal machinery. Transforming growth factor β1 (TGF‑β1) is a potent fibrogenic cytokine involved in liver fibrosis; however, it remains elusive whether autophagy is regulated by TGF‑β1 in this process. In the present study, the function of TGF‑β1‑mediated autophagy in the proliferation and apoptosis of hepatic stellate cells (HSCs) was investigated. A rat HSC cell line (HSC‑T6) was incubated with or without TGF‑β1 followed by bafilomycin A1, and microtubule-associated proteins 1A/1B light chain 3 (LC3) small interfering (si)RNA was used to inhibit autophagy in order to assess the association between TGF‑β1 and autophagy. HSC‑T6 cell transient transfection was accomplished with a pLVX‑AcGFP‑N1‑rLC3B‑encoding plasmid. An MTS assay and flow cytometry were utilized to detect proliferation and apoptosis of HSC‑T6 cells. Quantitative polymerase chain reaction, immunofluorescence and western blot analysis were used to detect the presence of activation markers. Proliferation was increased and apoptosis was reduced in HSC‑T6 cells treated with TGF‑β1 compared with cells subjected to serum deprivation. However, when HSC‑T6 cells were treated with bafilomycin A1 and LC3 siRNA, increased apoptosis and reduced proliferation were observed. In addition, protein and mRNA expression levels of the autophagy marker LC3 were significantly increased. GFP‑LC3 punctate markings were more prolific following TGF‑β1 treatment of HSC‑T6 cells, indicating that TGF‑β1 may rescue HSC‑T6 cells from serum deprivation and reduce apoptosis via autophagy induction. The present study elucidated the possible functions of TGF‑β1‑mediated autophagy in the pathological process of liver fibrosis.

This study was designed to investigate the prophylactic effects and the mechanisms of curcumin on liver fibrosis in rats. Liver fibrosis was induced in 72 Sprague Dawley rats by intraperitoneal injection of carbon tetrachloride. Rats were divided into control, liver fibrosis, high, medium, and low dose curcumin (200, 100, and 50 mg kg(-1), respectively), and colchicine (0.1 mg kg(-1)) groups. After 8 weeks of treatment, histopathological examination was performed on hepatic tissues, and liver fibrosis was graded. Hepatic stellate cells activity was examined by smooth muscle alpha-actin immunohistochemistry staining, and apoptosis was detected by terminal deoxynucleotidyl transferase dUTP nick-end labeling. The liver fibrosis score in the high, medium, and low dose curcumin group (5.79 +/- 1.80, 8.58 +/- 3.34, and 9.58 +/- 3.32, respectively) and the colchicine group (4.91 +/- 1.28) was significantly lower than in the fibrosis group (20.40 +/- 3.38, P < 0.01). The ratio of activated hepatic stellate cells in the three curcumin groups (0.97 +/- 0.69, 2.06 +/- 0.58, and 3.49 +/- 1.03, respectively) and the colchicine group (0.78 +/- 0.31) was significantly lower than in the fibrosis group (6.08 +/- 1.13, P < 0.05). The apoptosis index in the three curcumin groups (0.57 +/- 0.21, 0.37 +/- 0.22, and 0.34 +/- 0.21, respectively) was higher than in the fibrosis (0.09 +/- 0.09, P < 0.05) or the colchicine group (0.16 +/- 0.19, P < 0.05). Curcumin prevents carbon tetrachloride-induced liver fibrosis in rats. The prevention of liver fibrosis may be due to the inhibition of the activation of hepatic stellate cells and induction of their apoptosis.

Purpose: The cross-reacting material 197 (CRM197) is a mutation of the diphtheria toxin. The protein of CRM197 was used successfully for the therapy of various tumors in the recent studies. In this study, the recombinant adenoviruses containing the CRM197gene(AdCRM197) were used to enhance the cellar toxicity of gemcitabine in human glioma U87, U251, and H4 cells.Procedures: MTT assay and flow cytometric analysis were performed to test the apoptosis of the U87, U251 and H4 cells with the combined treatment of AdCRM197 plus gemcitabine. Western blotting analyses were carried out to detect the cell apoptosis of the mitochondrial pathway. And the xenograft nude mice were used to observe the enhanced antitumor effect of AdCRM197 in vivo.Results: AdCRM197 sensitizes human glioma cells to gemcitabine in vitro by the mitochondrial pathway. Tumor volume was inhibited and survival time was prolonged in the U251 or U87 xenografted nude mice with gemcitabine plus AdCRM197. The enhanced antitumor effect of AdCRM197 was also detected by the immunohistochemical analyses and TUNEL staining.Conclusion: The authors found that AdCRM197 sensitized the human glioma to gemcitabine not only in vitro but also in vivo. They provide the first evidence that adenovirus-mediated CRM197 may be a potential chemosensitizing agent for the treatment of cancer. The diphtheria toxin is of great toxicity that even one molecule of diphtheria toxin is enough to kill one cell. However, because of the high toxicity, the diphtheria toxin would kill the packing cells when it is being packaged into the recombinant viruses. Therefore, the diphtheria toxin is hard to be used in the gene therapy for virus vectors. The cross-reacting material 197 (CRM197) is a mutation of the diphtheria toxin. Unlike DTA, CRM197 exhibit a weak toxicity. The week toxicity of CRM197 is a good feature for the virus packaging. In the present study, we used a recombinant adenovirus which carried a CRM197 gene (AdCRM197) to enhance the cellar toxicity of gemcitabine in human glioma cells. [ABSTRACT FROM AUTHOR]

In order to evaluate the potential application value of cidofovir (CDV) in the prevention of human papillomavirus (HPV) infection and treatment of cervical cancer, the inhibitory effect of CDV on the proliferation of HPV 18-positive HeLa cells in cervical cancer was preliminarily investigated, using cisplatin (DDP) as a positive control. An MTT assay was used to analyze the effects of CDV and DDP on HeLa cell proliferation. In addition, clone formation assay and Giemsa staining were used to examine the extent of HeLa cell apoptosis caused by CDV and DDP. Flow cytometry was also used to detect the shape and size of apoptotic cells following propidium iodide staining, while western blot analysis identified the expression levels of of E6 and p53 proteins in HeLa cells. A cell climbing immunofluorescence technique was used to locate the subcellular position of p53 in HeLa cells. The results demonstrated that CDV and DDP inhibited the proliferation of HeLa cells in a concentration- and time-dependent manner. Flow cytometry showed that CDV and DDP treatments resulted in cell arrest in the S-phase, and triggered programmed cell death. Furthermore, western blot analysis revealed that CDV and DDP inhibited E6 protein expression and activated p53 expression in HeLa cells. Finally, the immunofluorescence results indicated that CDV and DDP inhibited the nuclear export of p53 by E6 protein, which is required for degradation of endogenous p53 by MDM2 and human papilloma virus E6. In conclusion, CDV and DDP inhibited HeLa cell proliferation in a concentration- and time-dependent manner, reduced the expression of E6 protein, and reinstated p53 protein activity. Thus, CDV regulates cell cycle arrest and apoptosis, and may be a potential cervical cancer therapeutic strategy. [ABSTRACT FROM AUTHOR]