Xiaocheng Cao

Casticin induces ovarian cancer cell apoptosis by repressing FoxM1 through the activation of FOXO3a

Casticin, a polymethoxyflavone, is reported to have anticancer activities. The aim of the present study was to examine the molecular mechanisms by which casticin induces apoptosis in ovarian cancer cells. The human ovarian cancer cell lines SKOV3 and A2780 were cultured in vitro. Various molecular techniques, including histone/DNA enzyme-linked immunosorbent assay (ELISA), reverse transcription polymerase chain reaction (RT-PCR), western blot analysis and gene transfection, were used to assess the expression of FOXO3a and forkhead box protein M1 (FoxM1) in casticin-treated ovarian cancer cell lines. Casticin-induced apoptotic cell death was accompanied by the activation of transcription factor FOXO3a, with a concomitant decrease in the expression levels of FoxM1 and its downstream target factors, namely survivin and polo-like kinase 1 (PLK1), and an increase in p27KIP1. A small inhibitory RNA (siRNA) knockout of FoxM1 potentiated casticin-induced apoptosis in ovarian cancer cells. Silencing FOXO3a expression using siRNA increased FoxM1 expression levels and clearly attenuated the induction of apoptosis by casticin treatment. These results show that casticin-induced apoptosis in ovarian cancer may be caused by the activation of FOXO3a, leading to FoxM1 inhibition.

Regulation of the FOXO3a/Bim signaling pathway by 5,7-dihydroxy-8-nitrochrysin in MDA-MB-453 breast cancer cells

We previously demonstrated that 5,7-dihydroxy-8-nitrochrysin (NOC), a novel synthetic chrysin analog, preferentially inhibits HER-2/neu-overexpressing MDA-MB-453 breast cancer cell growth by inducing apoptosis; however, the precise molecular mechanism was unclear. In this study, we demonstrated that NOC significantly induces apoptosis of MDA-MB-453 cells and that this is primarily mediated through a mitochondrial death cascade. This was presented as a loss of mitochondrial membrane potential, release of cytochrome c and activation of caspase-9. NOC induces a significant increase in levels of the BH3-only protein Bim. Small interfering RNA-mediated knockdown of Bim markedly attenuated NOC-induced apoptosis. An upstream transcriptional regulator of Bim, forkhead box O3a transcription factor (FOXO3a), experienced a decrease in phosphorylation and nuclear translocation. Silencing of FOXO3a resulted in a marked attenuation in the expression of Bim, as well as protection against NOC-mediated apoptosis. Furthermore, NOC-induced activation and nuclear localization of FOXO3a was associated with reduced levels of Akt phosphorylation. These results suggest that NOC induces apoptosis in MDA-MB-453 human breast cancer cells via caspase activation and modulation of the Akt/FOXO3a pathway.

Casticin inhibits epithelial-mesenchymal transition of liver cancer stem cells of the SMMC-7721 cell line through downregulating Twist.

The existence of cancer stem cells (CSCs) is central to the pathogenesis and therapeutic target of human hepatocellular carcinoma. The aim of this study was to investigate the effects of casticin on epithelial-mesenchymal transition (EMT) of liver cancer stem cells (LCSCs) derived from the SMMC-7721 cell line. Our results demonstrated that CD133+ sphere-forming cells (SFCs) sorted from the SMMC-7721 cell line not only possessed a higher capacity to form tumor spheroids in vitro, but also had a greater potential to form tumors when implanted in Balb/c-nu mice, indicating that CD133+ SFCs possessed similar traits to LCSCs. Casticin increased the expression levels of E-cadherin and decreased those of N-cadherin in LCSCs. Treatment of LCSCs with casticin for 48 h also decreased the levels of the EMT-associated transcription factor, Twist. Overexpression of Twist attenuated the casticin-induced regulation of E-cadherin and N-cadherin protein expression, as well as the EMT capacity of LCSCs. In conclusion, CD133+ SFCs of the SMMC-7721 cell line may represent a subpopulation of LCSCs with the characteristics of EMT. Furthermore, casticin targeted LCSCs through the inhibition of EMT by downregulating Twist.

Casticin inhibits self-renewal of liver cancer stem cells from the MHCC97 cell line

Casticin exerts anticarcinogenic activity in several types of cancers, including human hepatocellular carcinoma (HCC). The aim of the present study was to investigate the effects of casticin, which is derived from Fructus Viticis Simplicifoliae, on the self-renewal capacity of liver cancer stem cells (LCSCs) derived from the HCC MHCC97 cell line. The present study demonstrated that casticin significantly inhibited the proliferation of LCSCs from the MHCC97 cell line in a dose-dependent manner (P<0.05), the half maximal inhibitory concentration of the parental cells and LCSCs was 17.9 and 0.5 μmol/l, respectively. Furthermore, casticin reduced the sphere-forming capacity of LCSCs and downregulated β-catenin protein expression in a concentration-dependent manner. Lithium chloride, an agonist known to activate the Wnt/β-catenin signaling pathway, attenuated the casticin-induced downregulation of β-catenin protein expression and inhibited the self-renewal capacity. To the best of our knowledge, the present study is the first to demonstrate that casticin effectively eradicates LCSCs and β-catenin was identified as the potential target. Thus, casticin may offer a novel therapeutic approach for the treatment of HCC.

A candidate Chinese medicine preparation-Fructus Viticis Total Flavonoids inhibits stem-like characteristics of lung cancer stem-like cells

BackgroundCancer stem cells (CSCs) are considered as the origin of tumor relapse. Here, we investigated the effects of Fructus Viticis total flavonoids (FVTF) on the characteristics of lung cancer stem-like cells (LCSLCs) derived from human small cell lung cancer NCI-H446 cell line and its potential mechanism.MethodsHuman small cell lung cancer NCI-H446 cell line was cultured in vitro. The CD133+ cells were sorted from NCI-H446 cell line by magnetic separation.The suspended culture with stem cell-conditioned medium was used to amplify CD133+ sphere-forming cells (SFCs). The stem cell characteristics of CD133+ SFCs were evaluated using cell self-renewal capacity by tumor sphere formation assay, migration and invasion capacity by Transwell assay, tumorigenicity by xenograft model in nude mouse and cancer stem cell markers expression levels by western blot. The effects of FVTF on the properties of LCSLCs were examined by tumorsphere formation assay and transwell chamber assay. The expression level of p-Akt was determined by western blot analysis.ResultCD133+ SFCs derived from human small cell lung cancer NCI-H446 cells exhibited stemness properties of tumorsphere formation and tumorigenesis capacity comparing to the parental cells. FVTF relative selectively inhibited the proliferation of LCSLCs, suppressed tumor sphere forming capacity and migration and invasion of LCSLCs, and down-regulated the protein expression of stem cell markers (CD133, CD44 and ALDH1), self-renewal associated transcription factors (Bmi1, Nanog and OCT4) and invasion associated transcription factors (Twist1 and Snail1) in a dose-dependent manner. Moreover, we found that FVTF treatment could significantly decrease the phosphorylation level of Akt in LCSLCs. Meanwhile, LY294002 and FVTF synergistically inhibited the characteristics of LCSLCs.ConclusionFVTF inhibits the characteristics of LCSLCs through down-regulating expression of p-Akt.

8-Bromo-7-methoxychrysin-blocked STAT3/Twist axis inhibits the stemness of cancer stem cell-like cell originated from SMMC-7721 cells

Signal transducer and activator of transcription 3 (STAT3) is a member of the family of latent cytoplasmic transcriptional factors that could regulate cell proliferation, survival, and development. It has been reported that Twist is a target gene of STAT3, and STAT3/Twist signaling plays an important role in regulating cancer progress. Here, to explore whether 8-bromo-7-methoxychrysin (BrMC) inhibits liver cancer stem-like cell (LCSLC) properties via disrupting STAT3/Twist signaling, we cultured SMMC-7721 cells in vitro, and evaluated the effects of BrMC on the stemness of spheroids by determining the sphere-forming capability and migration. The sphere formation assay results showed a concentration-dependent decrease of sphere-forming capacity in LCSLCs (P < 0.05) treated with different concentrations of BrMC. Wound-healing assays results demonstrated a concentration-dependent decline in cell migration of LCSLCs treated with different concentrations of BrMC. In addition, CD133, CD44, and ALDH1 levels were decreased in LCSLCs treated with BrMC. Treatment with different concentrations of BrMC also reduced the expressions of p-STAT3 and Twist1 proteins. The effect of BrMC was substantially enhanced by co-treatment with JSI-124, a specific inhibitor of STAT3. Ectopic expression of Twist1 attenuated the inhibitory effects of BrMC on sphere formation, migration, and expression of the markers in LCSLCs. However, it had no affect on p-STAT3 expression in LCSLCs. These results demonstrated that BrMC inhibits the stemness of LCSLCs originated from SMMC-7721 cell line by inhibiting STAT3/Twist signal axis.

Upregulation of FoxM1 by MnSOD Overexpression Contributes to Cancer Stem-Like Cell Characteristics in the Lung Cancer H460 Cell Line

Manganese superoxide dismutase promotes migration and invasion in lung cancer cells via upregulation of the transcription factor forkhead box M1. Here, we assessed whether upregulation of forkhead box M1 by manganese superoxide dismutase overexpression mediates the acquisition of cancer stem-like cell characteristics in non-small cell lung cancer H460 cells. The second-generation spheroids from H460 cells were used as lung cancer stem-like cells. The levels of manganese superoxide dismutase, forkhead box M1, stemness markers (CD133, CD44, and ALDH1), and transcription factors (Bmi1, Nanog, and Sox2) were analyzed by Western blot. Sphere formation in vitro and carcinogenicity of lung cancer stem-like cells were evaluated by spheroid formation assay and limited dilution xenograft assays. Knockdown or overexpression of manganese superoxide dismutase or/and forkhead box M1 by transduction with short hairpin RNA(shRNA) or complementary DNA were performed for mechanistic studies. We showed that manganese superoxide dismutase and forkhead box M1 amounts as well as the expression levels of stemness markers and transcription factors sphere formation in vitro, and carcinogenicity of lung cancer stem-like cells were higher than in monolayer cells. Lung cancer stem-like cells transduced with manganese superoxide dismutase shRNA or FoxM1 shRNA exhibited decreased sphere formation and lower amounts of stemness markers and transcription factors. Overexpression of manganese superoxide dismutase or FoxM1 in H460 cells resulted in elevated sphere formation rates and protein levels of stemness markers and transcription factors. Meanwhile, manganese superoxide dismutase knockdown or overexpression accordingly altered forkhead box M1 levels. However, forkhead box M1 knockdown or overexpression had no effect on manganese superoxide dismutase levels but inhibited or promoted lung cancer stem-like cell functions. Interestingly, forkhead box M1 overexpression alleviated the inhibitory effects of manganese superoxide dismutase knockdown in lung cancer stem-like cells. In a panel of non-small cell lung cancer cells, including H441, H1299, and H358 cells, compared to the respective monolayer counterparts, the expression levels of manganese superoxide dismutase and forkhead box M1 were elevated in the corresponding spheroids. These findings revealed the role of forkhead box M1 upregulation by manganese superoxide dismutase overexpression in maintaining lung cancer stem-like cell properties. Therefore, inhibition of forkhead box M1 upregulation by manganese superoxide dismutase overexpression may represent an effective therapeutic strategy for non-small cell lung cancer.

Co-culture of ovarian cancer stem-like cells with macrophages induced SKOV3 cells stemness via IL-8/STAT3 signaling

Among recent concepts in the cancer biology field, the tumor microenvironment is highly associated with cancer stem cells, and plays a key role in tumor progression. This study aimed to explore the mechanism that the stemness induction of SKOV3 cell line by macrophages derived from THP-1 cells, which was co-cultured with SKOV3-derived ovarian cancer stem-like cells (OCSLCs). Sphere formation, soft agar colony formation, and expression levels of CD133 and CD44 were assessed to reflect OCSLC properties. ELISA was used to evaluate secretion profile changes in macrophages co-cultured with or without SKOV3-derived OCSLCs. For mechanistic evaluation, rhIL-8, IL-8 neutralizing antibody (IL-8 Ab), signal transducer and activator of transcription 3 (STAT3) shRNA and STAT3 cDNA were used. The results showed that IL-10, VEGF, MMP-9, IL-8 secretion and CD163 and STAT3 expression levels in macrophages co-cultured with OCSLCs were increased compared with those from THP-1 cells, while IL-12 and NO amounts were significantly reduced, reflecting M2 macrophage polarization. Addition of rhIL-8 to THP-1 cell conditioned media promoted M2 macrophage polarization and stemness in SKOV3 cells, which were suppressed by IL-8 Ab addition to co-culture conditioned media. Consistently, overexpression of STAT3 induced M2 macrophage polarization and stemness in SKOV3 cells, which were inhibited by STAT3 knockdown in macrophages from THP-1 cells. Importantly, STAT3 overexpression rescued the effects of IL-8 Ab on M2 macrophage polarization and stemness in SKOV3 cells. These results suggested that stemness induction in SKOV3 cells by macrophages co-cultured with SKOV3-derived OCSLCs involved IL-8/STAT3 signaling.