Therapeutic Actions of the Chinese Herbal Formulae with Cold and Heat Properties and Their Effects on Ultrastructures of Synoviocytes in Rats of the Collagen-Induced Arthritis

The therapeutic actions of Qing Luo Yin (QLY清络饮) with heat property and Wen Luo Yin (WLY温络饮) with cold property on pain, swelling of the ankle, arthritis index and ultrastructures of synoviocytes were compared in rats of type II collagen-induced arthritis (CIA), with tripterygium glycosidorum (TG) used as control. The results indicated that both QLY and WLY could reduce pain, swelling of the ankle and the arthritis index of CIA, and QLY had better effects in reducing the swelling of the ankle and controlling the secondary pathological lesions as compared with WLY. Investigation on the ultrastructures of synoviocytes indicated that both QLY and WLY could reduce the number of Golgi apparatus, rough surface endoplasmic reticulum, dense bodies, matrix filaments and vacuoles so as to suppress the excessive secretion of synoviocytes in rats of CIA.

Recombinant vascular basement-membrane-derived multifunctional peptide inhibits angiogenesis and growth of hepatocellular carcinoma

AIM： To investigate the anti-angiogenic and antitumor activities of recombinant vascular basement membrane-derived multifunctional peptide （rVBMDMP） in hepatocellular carcinoma （HCC）. METHODS： HepG2, Bel-7402, Hep-3B, HUVE-12 and L-02 cell lines were cultured in vitro and the inhibitory effect of rVBMDMP on proliferation of cells was detected by MTT assay. The in vivo antitumor efficacy of rVBMDMP on HCC was assessed by HepG2 xenografts in nude mice. Distribution of rVBMDMP, mechanism by which the growth of HepG2 xenografts is inhibited, and microvessel area were observed by proliferating cell nuclear antigen （PCNA） and CD31 immunohistochemistry. RESULTS： MTT assay showed that rVBMDMP markedly inhibited the proliferation of human HCC （HepG2, Bel-7402, Hep-3B） cells and human umbilical vein endothelial （HUVE-12） cells in a dose-dependent manner, with little effect on the growth of L-02 cells. When the ICs0 was 4.68, 7.65, 8.96, 11.65 and 64.82 μmol/L, respectively, the potency of rVBMDMP to HepG2 cells was similar to 5-fluorouracil （5-FU） with an IC50 of 4.59 μmol/L. The selective index of cytotoxicity to HepG2 cells of rVBMDMP was 13.8 （64.82/4.68）, which was higher than that of 5-FU [SI was 1.9 （8.94/4.59）]. The VEGF-targeted recombinant humanized monoclonal antibody bevacizumab （100 mg/L） did not affect the proliferation of HepG2, Bel-7402, Hep-3B and L-02 cells, but the growth inhibitory rate of bevacizumab （100 mg/L） to HUVE-12 cells was 87.6% ± 8.2%. AIternis diebus intraperitoneal injection of rVBMDMP suppressed the growth of HepG2 xenografts in a dose-dependent manner, rVBMDMP （1, 3, 10 mg/kg） decreased the tumor weight by 12.6%, 55.9% and 79.7%, respectively, compared with the vehicle control. Immunohistochemical staining of rVBMDMP showed that the positive area rates （2.2% ± 0.73%, 4.5%± 1.3% and 11.5% ±3.8%） in rVBMDMP treated group （1, 3, 10 mg/kg） were significantly higher than that （0.13% ± 0.04%） in the control group （P 〈 0.01）. The positive area rates （19.0% ± 5.7%, 12.2% ± 3.5% and 5.2% ±1.6% ） of PCNA in rVBMDMP treated group （1, 3, 10 mg/kg） were significantly lower than that （29.5% ± 9.4%） in the control group （P 〈 0.05）. rVBMDMP at doses of 1, 3 and 10 mg/kg significantly reduced the tumor microvessel area levels （0.26%± 0.07%, 0.12% ± 0.03% and 0.05% ± 0.01% vs 0.45% ± 0.15%） in HepG2 xenografts （P 〈 0.01）, as assessed by CD31 staining. CONCLUSION： rVBMDMP has effective and unique anti-tumor properties, and is a promising candidate for the development of anti-tumor drugs.

7-difluoromethoxyl-5,4'-di-n-octylgenistein inhibits growth of gastric cancer cells through downregulating forkhead box M1

AIM: To investigate whether the 7-difluoromethoxyl-5, 4'-di-n-octylgenistein (DFOG), a novel synthetic genistein analogue, affects the growth of gastric cancer cells and its mechanisms. METHODS: A series of genistein analogues were prepared by difluoromethylation and alkylation, and human gastric cancer cell lines AGS and SGC-7901 cultured in vitro were treated with various concentrations of genistein and genistein analogues. The cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The cells were incubated by DFOG at different concentrations. The growth inhibitory effects were evaluated using MTT and clonogenic assay. The distribution of the phase in cell cycle was analyzed using flow cytometric analysis with propidium iodide staining. The expression of the transcription factor forkhead box M1 (FOXM1) was analyzed by reverse transcription-polymerase chain reaction and Western blotting. The expression levelsof CDK1, Cdc25B, cyclin B and p27KIP1 protein were detected using Western blotting. RESULTS: Nine of the genistein analogues had more effective antitumor activity than genistein. Among the tested analogues, DFOG possessed the strongest activity against AGS and SGC-7901 cells in vitro. DFOG significantly inhibited the cell viability and colony formation of AGS and SGC-7901 cells. Moreover, DFOG efficaciously arrested the cell cycle in G2/M phase. DFOG decreased the expression of FOXM1 and its downstream genes, such as CDK1, Cdc25B, cyclin B, and increased p27KIP1 at protein levels. Knockdown of FOXM1 by small interfering RNA before DFOG treatment resulted in enhanced cell growth inhibition in AGS cells. Up-regulation of FOXM1 by cDNA transfection attenuated DFOG-induced cell growth inhibition in AGS cells. CONCLUSION: DFOG inhibits the growth of human gastric cancer cells by down-regulating the FOXM1 expression.

Casticin Attenuates Stemness in Cervical Cancer Stem-Like Cells by Regulating Activity and Expression of DNMT1

Objective:To explore whether casticin(CAS)suppresses stemness in cancer stem-like cells(CSLCs)obtained from human cervical cancer(CCSLCs)and the underlying mechanism.Methods:Spheres from He La and Ca Ski cells were used as CCSLCs.DNA methyltransferase 1(DNMT1)activity and m RNA levels,self-renewal capability(Nanog and Sox2),and cancer stem cell markers(CD133 and CD44)were detected by a colorimetric DNMT activity/inhibition assay kit,quantitative real-time reverse transcription-polymerase chain reaction,sphere and colony formation assays,and immunoblot,respectively.Knockdown and overexpression of DNMT1 by transfection with sh RNA and c DNA,respectively,were performed to explore the mechanism for action of CAS(0,10,30,and 100 nmol/L).Results:DNMT1 activity was increased in CCSLCs compared with He La and Ca Ski cells(P<0.05).In addition,He La-derived CCSLCs transfected with DNMT1 sh RNA showed reduced sphere and colony formation abilities,and lower CD133,CD44,Nanog and Sox2 protein expressions(P<0.05).Conversely,overexpression of DNMT1 in He La cells exhibited the oppositive effects.Furthermore,CAS significantly reduced DNMT1 activity and transcription levels as well as stemness in He La-derived CCSLCs(P<0.05).Interestingly,DNMT1 knockdown enhanced the inhibitory effect of CAS on stemness.As expected,DNMT1 overexpression reversed the inhibitory effect of CAS on stemness in He La cells.Conclusion:CAS effectively inhibits stemness in CCSLCs through suppression of DNMT1 activation,suggesting that CAS acts as a promising preventive and therapeutic candidate in cervical cancer.