8-bromo-7-methoxychrysin suppress stemness of SMMC-7721 cells induced by co-culture of liver cancer stem-like cells with hepatic stellate cells.

BACKGROUND: Our previous works have demonstrated that 8-bromo-7-methoxychrysin suppressed stemness of human hepatocellular carcinoma (HCC) cell line SMMC-7721 induced by condition medium from hepatic stellate cell line LX-2 that was activated by liver cancer stem-like cells (LCSCs). However, whether and whereby BrMC inhibits the stemness induced by co-culture of LCSCs and LX-2 cells remains to be investigated. METHODS: The second-generation spheres by sphere culture were identified and used as SMMC-7721-and MHCC97H-derived LCSLCs. SMMC-7721-and MHCC97-derived LCSCs/LX-2 cells transwell co-culture system was treated with BrMC and its lead compound chrysin. The concentrations of IL-6, IL-8, HGF and PDGF in condition medium from co-culture were measured by enzyme-linked immunosorbent assay (ELISA). The stemness of SMMC-7721 cells was evaluated by sphere formation assay and western blot analysis for expression levels of cancer stem cell markers (CD133 and CD44).The expression levels of cancer-associated fibroblast markers (FAP-α and α-SMA) were employed to evaluate pathologic activation of LX-2 cells. Addition of IL-6 and/or HGF or deletion of IL-6 and/or HGF was conducted to investigate the mechanisms for BrMC and chrysin treatment in SMMC-7721-derived LCSLCs co-cultured with LX-2cells. RESULTS: The co-culture of LCSLCs with LX-2 cells increased sphere formation capability as well as expression of CD133 and CD44 in SMMC-7721 cells, meanwhile, upregulated expression of FAP-α in LX-2 cells. ELISA indicated that the concentrations of IL-6 and HGF were significantly elevated in Co-CM than that of condition media from co-cultured SMMC-7721 cells/LX-2 cells. Treatment of BrMC and chrysin with co-cultures of SMMC-7721- and MHCC97H-derived LCSLCs and LX-2 cells effectively inhibited the above responses. Moreover, addition of IL-6 and/or HGF induced stemness of SMMC-7721 cells and activation of LX-2 cells, conversely, deletion of IL-6 and/or HGF suppressed those. Furthermore, the inhibitory effects of BrMC and chrysin on stemness of SMMC-7721 cells and activation of LX-2 cells were attenuated by addition of IL-6 or HGF, and enhanced by deletion of IL-6 or HGF. CONCLUSIONS: Our results suggest IL-6 and HGF may be the key communication molecules for the interaction between LCSLCs and HSCs, and BrMC and chrysin could block these effects and be the novel therapeutic candidates for HCC management.

5-allyl-7-gen-difluoromethoxychrysin enhances TRAIL-induced apoptosis in human lung carcinoma A549 cells.

BACKGROUND: 5-allyl-7-gen-difluoromethoxychrysin (AFMC) is a novel synthetic analogue of chrysin that has been reported to inhibit proliferation in various cancer cell lines. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising anti-cancer agent. METHODS: The cytotoxicity of A549 and WI-38 cells were determined using colorimetry. Apoptosis was detected by flow cytometry (FCM) after propidium iodide (PI) fluorescence staining and agarose gel electrophoresis. Caspase activities were evaluated using enzyme-linked immunosorbent assay (ELISA).The expressions of DR4 and DR5 were analyzed using FCM and western blot. RESULTS: Subtoxic concentrations of AFMC sensitize human non-small cell lung cancer (NSCLC) A549 cells to TRAIL-mediated apoptosis. Combined treatment of A549 cells with AFMC and TRAIL significantly activated caspase-3, -8 and -9. The caspase-3 inhibitor zDEVD-fmk and the caspase-8 inhibitor zIETD-fmk blocked the apoptosis of A549 cells induced by co-treatment with AFMC and TRAIL. In addition, we found that treatment of A549 cells with AFMC significantly induced the expression of death receptor 5 (DR5). AFMC-mediated sensitization of A549 cells to TRAIL was efficiently reduced by administration of a blocking antibody or small interfering RNAs against DR5. AFMC also caused increase of the Sub-G1 cells by TRAIL treatment and increased the expression levels of DR5 in other NSCLC H460 and H157 cell lines. In contrast, AFMC-mediated induction of DR5 expression was not observed in human embryo lung WI-38 cells, and AFMC did not sensitize WI-38 cells to TRAIL-induced apoptosis. CONCLUSIONS: AFMC synergistically enhances TRAIL-mediated apoptosis in NSCLC cells through up-regulating DR5 expression.

Apigenin inhibits HeLa sphere-forming cells through inactivation of casein kinase 2α.

The protein kinase casein kinase 2 (CK2) has been implicated in stem cell maintenance and its aberrant activation has been demonstrated in several types of cancer, including cervical cancer. In the present study, it was demonstrated that the sphere-forming cells (SFCs) of HeLa cell lines exhibited self-renewal capacity, indicating that they possessed the properties of cervical cancer stem-like cells. HeLa-derived SFCs exhibited a higher level of CK2α protein, compared with the parental cells. Apigenin, a dietary flavonoid, led to a dose-dependent inhibition of the self-renewal capacity and the protein expression of CK2α in HeLa-derived SFCs. Furthermore, forced overexpression of CK2α resulted in a decrease in the inhibition of CK2α expression and the self-renewal capacity induced by apigenin in HeLa-derived SFCs. These results suggested that apigenin inhibits the self-renewal capacity of HeLa-derived SFCs through downregulation of CK2α expression.

Inhibition of cell growth by BrMC through inactivation of Akt in HER-2/neu-overexpressing breast cancer cells.

We previously reported that chrysin (ChR) and its analogs induced cell cycle arrest and apoptosis in human estrogen receptor-positive/-negative breast cancer cells. However, it was unknown whether 8-bromo-7-methoxychrysin (BrMC), a novel synthetic ChR analog, inhibited the cell growth of human epidermal growth factor receptor 2 (HER-2)/neu-overexpressing breast cancers. In the present study, it was demonstrated that BrMC preferentially inhibited the cell viability of HER-2/neu-overexpressing MDA-MB-453 and BT-474 cells. Western blot analysis revealed that HER-2/neu expression and tyrosine phosphorylation were inhibited by BrMC in a concentration-dependent manner; whereas the proteasome inhibitor, MG-132, significantly prevented BrMC-induced HER-2/neu depletion and cell death in MDA-MB-453 cells. This directly indicated that BrMC-induced HER-2/neu depletion and cell growth inhibition was mediated by a proteasomal pathway. BrMC significantly downregulated the expression of cyclin D1, cyclin E and CDK4, followed by the suppression of protein kinase B phosphorylation and downstream effectors, GSK-3ß and ß-catenin. A colony formation assay also confirmed the growth-inhibitory effects of BrMC. Thus, these findings clearly demonstrate the anticancer activity of BrMC against human HER-2/neu-overexpressing breast cancer cells. Thus, these findings clearly demonstrate the anticancer activity of BrMC against human HER 2/neu-overexpressing breast cancer cells, and highlight BrMC as a promising candidate for breast cancer therapy.

7-difluoromethoxyl-5,4'-di-n-octyl genistein inhibits ovarian cancer stem cell characteristics through the downregulation of FOXM1.

7-Difluoromethoxyl-5,4'-di-n-octylgenistein (DFOG) is a novel synthetic genistein analogue that possesses anti-cancer activity in a variety of cancers, including ovarian cancer. The objective of the present study was to investigate whether DFOG inhibits the self-renewal capacity of ovarian cancer stem-like cells (OCSLCs) and to identify its potential mechanism of action. It was found that the sphere-forming cells (SFCs) of the SKOV3 cell line exhibited a self-renewal capacity and high tumorigenicity, indicating that they possessed the properties of ovarian cancer stem cells (OCSCs). It was also shown for the first time that DFOG preferentially inhibited proliferation, self-renewal capacity and expression of stem cell markers [cluster of differentiation (CD)133, CD44 and aldehyde dehydrogenase 1 (ALDH1)] in the SFCs derived from the SKOV3 cells. These effects were accompanied by the downregulation of forkhead box M1 (FOXM1) expression. Overexpression of FOXM1 rescued the DFOG-induced downregulation of FOXM1, CD133, CD44 and ALDH1 protein expression. It also inhibited the self-renewal capacity of the SFCs derived from the SKOV3 cells. Thus, DFOG appears to inhibit the characteristics of OCSLCs by downregulating FOXM1 expression.

Casticin induces breast cancer cell apoptosis by inhibiting the expression of forkhead box protein M1.

Casticin is an active ingredient derived from Fructus Viticis, a traditional Chinese medicine. This study aimed to investigate the role of forkhead box O3 (FOXO3a) in breast cancer cells and examine the regulatory mechanisms of FOXO3a in response to casticin treatment of the cells by ELISA, flow cytometry, small interfering RNA (siRNA) transfection and western blot analysis. Casticin treatment induced apoptosis and reduced the expression of the transcription factor forkhead box protein M1 (FOXM1). In addition, FOXM1 repression induced by casticin treatment was associated with the activation of FOXO3a via increased dephosphorylation. Notably, silencing FOXO3a expression by siRNA-mediated gene knockdown attenuated casticin-mediated apoptosis. Collectively, these findings suggest that FOXO3a is a critical mediator of the inhibitory effects of casticin on apoptosis in breast cancer cells.

8-bromo-7-methoxychrysin inhibits properties of liver cancer stem cells via downregulation of ß-catenin.

AIM: To evaluate whether 8-bromo-7-methoxychrysin (BrMC), a synthetic analogue of chrysin, inhibits the properties of cancer stem cells derived from the human liver cancer MHCC97 cell line and to determine the potential mechanisms. METHODS: CD133(+) cells were sorted from the MHCC97 cell line by magnetic activated cell sorting, and amplified in stem cell-conditioned medium to obtain the enriched CD133(+) sphere forming cells (SFCs). The stem cell properties of CD133(+) SFCs were validated by the tumorsphere formation assay in vitro and the xenograft nude mouse model in vivo, and termed liver cancer stem cells (LCSCs). The effects of BrMC on LCSCs in vitro were evaluated by MTT assay, tumorsphere formation assay and transwell chamber assay. The effects of BrMC on LCSCs in vivo were determined using a primary and secondary xenograft model in Balb/c-nu mice. Expressions of the stem cell markers, epithelial-mesenchymal transition (EMT) markers and ß-catenin protein were analyzed by western blotting or immunohistochemical analysis. RESULTS: CD133(+) SFCs exhibited stem-like cell properties of tumorsphere formation and tumorigenesis capacity in contrast to the parental MHCC97 cells. We found that BrMC preferentially inhibited proliferation and self-renewal of LCSCs (P < 0.05). Furthermore, BrMC significantly suppressed EMT and invasion of LCSCs. Moreover, BrMC could efficaciously eliminate LCSCs in vivo. Interestingly, we showed that BrMC decreased the expression of ß-catenin in LCSCs. Silencing of ß-catenin by small interfering RNA could synergize the inhibition of self-renewal of LCSCs induced by BrMC, while Wnt3a treatment antagonized the inhibitory effects of BrMC. CONCLUSION: BrMC can inhibit the functions and characteristics of LCSCs derived from the liver cancer MHCC97 cell line through downregulation of ß-catenin expression.

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 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.

8-bromo-5-hydroxy-7-methoxychrysin targeting for inhibition of the properties of liver cancer stem cells by modulation of Twist signaling.

Emerging evidence has suggested that cancer stem cells with expression of surface biomarkers including CD133 and CD44 have more aggressive biological behavior, including epithelial-mesenchymal transition (EMT), which are closely related to invasion. The upregulation and nuclear relocation of the EMT regulator Twist1 have been implicated in the tumor invasion and metastasis of human hepatocellular carcinoma (HCC). In this study, we aimed to isolate and characterize a small population of CD133+ cells that existed in the HCC cell line SMMC-7721 by MACS and investigated the possible roles of 8-bromo-7-methoxychrysin (BrMC), a synthetic analogue of chrysin, in inhibiting the properties of CD133+ sphere-forming cells (SFCs) derived from the HCC cell line SMMC-7721, namely liver cancer stem cells (LCSCs). Based on the data, BrMC inhibited the proliferation, self-renewal and invasion of LCSCs in vitro and in vivo, downregulated the expression of the LCSC biomarkers CD133 and CD44 and induced EMT by downregulating the expression of Twist and ß-catenin in LCSCs. BrMC potentiated the inhibition of LCSCs self-renewal after reduction of twist protein levels, which was attenuated when twist was overexpressed. This study not only provides an important experimental and theoretical basis for investigation of BrMC in LCSCs, but also helps in the development of effective therapeutic medicine for HCC.

Casticin induces caspase-mediated apoptosis via activation of mitochondrial pathway and upregulation of DR5 in human lung cancer cells.

OBJECTIVE: To assess if casticin induces caspase-mediated apoptosis via activation of mitochondrial pathway and upregulation of DR5 in human lung cancer cells. METHODS: Human non-small-cell lung carcinoma cell lines H460, A549 and H157 were cultured in vitro. The cytotoxic activities were determined using MTT assay. The apoptotic cells death was examined by flow cytometry using PI staining and DNA agarose gel electrophoresis. The activities of caspase-3, -8 and -9 were measured via ELISA. Cellular fractionation was determined by flow cytometry to assess release of cytochrome c and the mitochondrial transmembrane potential. Bcl-2/Bcl-XL/XIAP/Bid/DR5 and DR4 proteins were analyzed using western blot. RESULTS: The concentrations required for a 50% decrease in cell growth (IC(50)) ranged from 1.8 to 3.2 µM. Casticin induced rapid apoptosis and triggered a series of effects associated with apoptosis by way of mitochondrial pathway, including the depolarization of the mitochondrial membrane, release of cytochrome c from mitochondria, activation of procaspase-9 and -3, and increase of DNA fragments. Moreover, the pan caspase inhibitor zVAD-FMK and the caspase-3 inhibitor zDEVD-FMK suppressed casticin-induced apoptosis. In addition, casticin induced XIAP and Bcl-XL down-regulation, Bax upregulation and Bid clearage. In H157 cell line, casticin increased expression of DR5 at protein levels but not affect the expression of DR4. The pretreatment with DR5/Fc chimera protein effectively attenuated casticin-induced apoptosis in H157 cells. No correlation was found between cell sensitivity to casticin and that to p53 status, suggesting that casticin induce a p53-independent apoptosis. CONCLUSIONS: Our results demonstrate that casticin induces caspase-mediated apoptosis via activation of mitochondrial pathway and upregulation of DR5 in human lung cancer cells.

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 levels of CDK1, Cdc25B, cyclin B and p27(KIP1) 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 p27(KIP1) 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.