ABSTRACT
Aim To determine whether 8-bromo-7-me-thoxychrysin ( BrMC) inhibits in vitro carcinogenicity via up-regulating miR-519d expression and down-regu-lating Twist1 expression in liver cancer stem-like cells ( LCSLCs) derived from SMMC-7721 cell line. Meth-ods The second generation spheroids derived from SMMC-7721 cell line were obtained by sphere-forming assay and were considered as LCSLCs . Then LCSLCs were treated with various concentrations ( 1.0, 3.0, 10.0 μmol·L-1) of BrMC. The expression level of miR-519d was detected using real-time PCR. And in vitro carcinogenicity was investigated by sphere-forming assay and clone-forming assay in agar. The transcrip-tional activity and protein expression of Twist1 were an-alyzed using luciferase reporter assay and Western blot. Moreover, the molecular mechanism of BrMC was elucidated via miR-519 mimic transfection and Twist1 gene transduction, respectively. Results Compared with SMMC-7721 cells, miR-519d-3p was low-ex-pressed and Twist1 was over expressed in LCSLCs. And the sphere-forming ratio and the clone-forming ra-tio decreased by treatment with BrMC ( 1.0, 3.0, 10.0 μmol·L-1) in a dose-dependent manner. Fur-thermore, luciferase reporter assay demonstrated miR-519d could directly target the 3′ untranslated region of Twist1 mRNA and regulate protein expression. miR-519d mimic enhanced the effects of BrMC (3.0 μmol ·L-1) . However, Twist1 gene transduction effective-ly reversed the effects of BrMC ( 3.0 μmol·L-1) . Conclusion BrMC inhibits in vivo carcinogenicity via regulating miR-519/Twist1 signal axis in LCSLCs de-rived from SMMC-7721 cell line.
ABSTRACT
Aim To investigate whether 8-bromo-7-methoxychrysin ( BrMC ) can reverse the epithelial-mesenchymal transition ( EMT) in sphere-forming cells (SFCs) derived from cervical cancer SiHa cell line. Methods Human cervical cancer SiHa cell line was cultured in vitro and then SFCs were obtained from Si-Ha cell line using suspension culture with the stem-condition culture system. After treatment with BrMC for 72 h, the morphology of SFCs of SiHa cell line was observed via monolayer adherence culture. The expres-sion levels of E-cadherin, N-cadherin and Twist1 pro-teins were analyzed using western blot assay. Results SFCs of SiHa cell line presented the mesenchyme-like( spindle-shaped) cell shape and exhibited reduced E-cadherin and elevated N-cadherin protein expres-sion, compared with the parental cells. After treatment with BrMC(1. 0 μmol·L-1 ) for 72 h, SFCs of SiHa cell line changed from spindle-shaped cells in to poly-gon shaped cells under monolayer adherence culture. After treatment with BrMC for 24 h, the expression lev-els of E-cadherin protein were up-regulated and the ex-pression levels of N-cadherin and Twist1 proteins were decreased in SFCs derived from SiHa cell line. Con-clusion BrMC effectively reverses EMT of SFCs de-rived from cervical cancer SiHa cell line, and its mechanism is involved in down-regulating the expres-sion of Twist protein.
ABSTRACT
Aim To investigate the effect of 8-bromo-7-methoxychrysin(BrMChR) on apoptosis of human gastric carcinoma SGC-7901 cell line.Methods SGC-7901 cells were cultured and the inhibitory effect of BrMChR on the proliferation of SGC-7901 cell line was measured by MTT assay.The apoptosis of SGC-7901 cells induced by BrMChR was analyzed using flow cytometry (FCM) with PI staining.DNA ladder bands were observed by DNA agarose gel electrophoresis.Results MTT assay suggested that BrMChR markedly inhibited the proliferation of SGC-7901 cells in a concentration-dependent manner,its IC50 was 2.6 ?mol?L-1,the potency of BrMChR was 8 times more than that of lead compound,chrysin (ChR,IC50 was 16.5 ?mol?L-1),and was 3 times more than that of 5-fluorouracil (5-FU,IC50 was 7.7 ?mol?L-1). FCM with PI staining indicated the apoptotic rate of SGC-7901 cell line treated with BrMChR (1.25,5.00,20.00 ?mol?L-1) for 48 h was (19.8%?0.2%),(36.8%?1.9%) and (45.5%?3.5%) respectively and the rate treated with BrMChR (1.25 ?mol?L-1) was higher than that treated with ChR (20.00 ?mol?L-1,12.9%?1.5%). DNA agarose gel electrophoresis showed that when treated with BrMChR(20.00 ?mol?L-1) for 48 h,the gene DNA of SGC-7901 cells presented typical DNA ladder bands which could attenuated by the effect of pre-incubation of GW9662(10.00 ?mol?L-1),a blocker of PPAR?.Conclusion BrMChR might induce apoptosis of SGC-7901 cell line partially by activating PPAR?.