Jianguo Cao

Inhibition of proliferation and induction of G1-phase cell-cycle arrest by dFMGEN, a novel genistein derivative, in lung carcinoma A549 cells.

Genistein (GEN) is a molecule of great interest as a potent chemopreventive agent against atherosclerosis and cancer. However, the bioavailability of GEN is very low in vivo. Our previous study showed that a GEN derivative, 7-difluoromethyl-5,4′-dimethoxygenistein (dFMGEN) has a better bioavailability than GEN in vivo. In this study, we further evaluated the efficacy of dFMGEN as a candidate for cancer therapy. We demonstrated that dFMGEN treatment decreased the viability of A549 cells in a concentration- and time-dependent manner and induced cell-cycle arrest at the G1 phase. G1 phase arrest was correlated with a significant reduction of Cdk4 and cyclin D1 protein level. Further studies showed that cyclin-dependent kinase (Cdk)4 and cyclin D1 protein-level decrease was caused by Cdk inhibitors p15, p21, and p27 level increase, and decreased protein level directly suppressed Rb protein phosphorylation and E2F-1 expression, then cell-cycle progression was arrested. Finally, we also found that dFMGEN has a dosage effect in suppressing tumor growth in vivo, and that dFMGEN was well tolerated by animals. In summary, our results suggest that dFMGEN has therapeutic potential for the treatment of human lung cancer.

Reversal of liver cancer-associated stellate cell-induced stem-like characteristics in SMMC-7721 cells by 8-bromo-7-methoxychrysin via inhibiting STAT3 activation

Hepatic stellate cells (HSCs) that are activated by human hepatocellular carcinoma (HCC) cells secrete a variety of cytokines, which are the main component of the HCC microenvironment. We aimed to determine whether 8-bromo-7-methoxychrysin (BrMC) could interfere in cross-talk of the human hepatic stellate cell line LX-2 and liver cancer stem-like cells (LCSLCs) to inhibit the characteristics of LCSLCs endowed with the capacity of sustaining human hepatocellular carcinoma (HCC) self-renewal and progression, and to identify its potential mechanism of action. We found that the levels of fibroblast activation protein (FAP) were augmented in LX-2 cells treated with the conditioned medium of LCSLCs (LCSLC-CM) compared to those cultured with routine medium, indicating that the LCSLC-CM can activate LX-2 cells to become liver cancer-associated stellate cells (LCAHSCs). Furthermore, sphere forming capability of SMMC-7721 cells was enhanced and stem cell-related protein expression was significantly increased following treatment with the conditioned medium of LCAHSCs (LCAHSC-CM). Moreover, the level of p-STAT3 was increased in LX-2 cells treated with LCSLC-CM and BrMC reduced expression of p-STAT3. Combination of BrMC and the selective inhibitor of STAT3 cucurbitacin I (JSI-124) synergistically suppressed the LCSLC characteristics in SMMC-7721 cells. Collectively, our data showed that BrMC inhibited the interaction between LX-2 cells and HCC-derived CSCs, and did so potentially through modulation of the STAT3 pathway. Future therapeutic strategies employing anti-CSC therapy should confirm the potential of cucurbitacin I (JSI-124) and BrMC as potent therapeutic agents.

Inhibition of growth and motility of human A549 lung carcinoma cells by a recombined vascular basement membrane derived peptide.

We have previously constructed a recombined vascular basement membrane derived multifunctional peptide (rVBMDMP) which can inhibit tumor growth. The aim of this study is to explore the effects and mechanisms of rVBMDMP on growth and motility/invasion in human A549 lung carcinoma cells. The effect of rVBMDMP on A549 cell viability was determined by MTT assay while the motility/invasion was measured by scratch and transwell assays. Molecules that responded to rVBMDMP treatment of A549 cells were explored using the high-throughput Cancer Pathway Finder cDNA Microarray. We identified 16 genes that were up-regulated, including GZMA, ITG alphaV, MCAM and Kiss1 and 21 genes that were down-regulated, including uPA, uPAR, CDC25A, IGF1 and FGF2. Selective differentially expressed genes were further analyzed by real-time quantitative PCR and Western blot analysis. These findings contribute to the understanding of the molecular mechanisms mediating rVBMDMP action, and suggest that rVBMDMP is a promising novel agent for the treatment of human lung carcinoma.

8-bromo-7-methoxychrysin Reversed M2 Polarization of Tumor-associated Macrophages Induced by Liver Cancer Stem-like Cells

BACKGROUND Hepatocellular carcinoma (HCC) is related to chronic liver inflammation. M2 polarization of tumor-associated macrophages (TAMs) in the tumor microenvironment promotes liver cancer stem-like cell (LCSLC) self-renewal capability and carcinogenicity. Therefore, reversing M2 polarization of TAMs could be an effective approach to cure HCC. OBJECTIVE To evaluate whether 8-bromo-7-methoxychrysin (BrMC) has an effect on M2 polarization of TAMs. METHOD LCSLC and conditional medium were obtained by sphere forming assay. Identification of LCSLC were analyzed by sphere forming, wound-healing and invasion assay. TAM and effects of BrMC on it were validated by immunofluorescence staining, ELISA and griess assay. Expressions of cancer stem cell and macrophage marker were analyzed by western blotting. RESULTS Our results showed that BrMC significantly suppressed the expression of the M2 macrophage marker CD163. Furthermore, BrMC influenced the secretion profile of cytokines of TAMs. Mechanistically, BrMC reversed M2 polarization of TAMs due to inhibition of NF-κB activation. CONCLUSION BrMC may be a potentially novel flavonoid agent that can be applied for disrupting the interaction of LCSLCs and TAMs.

Effects of VBMDMP on the reversal of cisplatin resistance in human lung cancer A549/DDP cells

Tumor drug resistance is a major obstacle to cancer chemotherapy. We previously constructed a fusion protein based on two tumstatin-derived sequences named recombinant VBMDM (rVBMDMP). We preliminarily confirmed its inhibition of HUVEC and colon cancer cell growth. The present study further systematically observed the inhibitory effect of rVBMDMP on lung cancer cell growth and analyzed a possible mechanism to provide a theoretical basis for the development of new antitumor protein drugs. The effect of rVBMDMP on human lung adenocarcinoma (A549) and cisplatin-resistant human lung adenocarcinoma (A549/DDP) cell proliferation was evaluated by MTS assay. Hoechst 33342 staining performed together with fluorescence microscopy and immunoblot analysis were used to examine the effects of rVBMDMP on the apoptosis of A549/DDP cells. A protein phosphorylation chip was used to identify changes in rVBMDMP-induced signaling protein phosphorylation. Changes in the phosphatidylinositol 3 kinase (PI3K)/Akt signal transduction pathway and expression of multidrug resistance protein (MRP-2)-related molecules following rVBMDMP treatment in A549/DDP cells were evaluated by western blot analysis. A lung cancer xenograft model was used to evaluate the reversal effect of rVBMDMP on drug-resistance of A549/DDP cell tumors to cisplatin in vivo. The results demonstrated that rVBMDMP increased the phosphorylation of 79 signaling proteins, including focal adhesion kinase (FAK), caspase-6, Fas, FasL and FAF1 and downregulated 30 signaling proteins, including integrin αV, integrin β3, PI3K/Akt, NF-κB and MRP-2 compared with the controls. rVBMDMP also increased the sensitivity of A549 and A549/DDP cells to cisplatin and directly induced apoptosis, which may be related to MRP-2 and Bcl-2 downregulation. The effects of growth inhibition and apoptosis induction of rVBMDMP on A549/DDP cells may be related to the inhibition of integrin αVβ3 and PI3K/Akt protein phosphorylation. Finally, we observed an increase in cancer cell sensitivity to cisplatin by rVBMDMP using the A549/DDP cell xenograft model in nude mice. Our study suggests that rVBMDMP may be an effective potential chemotherapy sensitizer and may be a viable drug candidate in anticancer therapies.

Synergistic inhibition of characteristics of liver cancer stem-like cells with a combination of sorafenib and 8-bromo-7-methoxychrysin in SMMC-7721 cell line

Sorafenib, a multi-kinase inhibitor, has shown its promising antitumor effect in a series of clinical trials, and has been approved as the current standard treatment for advanced hepatocellular carcinoma (HCC). 8-Bromo‑7-methoxychrysin (BrMC) is a novel chrysin synthetic analogue that has been reported to inhibit the growth of various tumor cells and possess properties for targeting liver cancer stem cells (LCSCs) . The present study investigated the synergistic targeting effects on the properties of liver cancer stem-like cells (LCSLCs) by a combination of sorafenib and BrMC in SMMC-7721 cell line. We also investigated whether this effect involves regulation of HIF-1α, Twist and NF-κB protein. We found that the sphere-forming cells (SFCs) from the SMMC‑7721 cells possessed the properties of LCSLCs. Sorafenib diminished the self-renewal capacity and downregulated the expression of stem cell biomarkers (CD133, CD44 and ALDH1) in a dose-dependent manner, while BrMC cooperated with sorafenib to strengthen this inhibition. Moreover, the combination of sorafenib and BrMC led to a remarkable decrease in the cellular migration and invasion, the downregulation of N-cadherin protein and upregulation of E-cadherin protein, and increase of cell apoptosis in LCSLCs. BrMC has a remarkable antagonistic effect on the upregulation of protein expression and DNA binding activity of NF-κB (p65) induced by sorafenib. In addition, our results indicated that the synergistic inhibition of sorafenib and BrMC on the characteristics of LCSLCs involves the downregulated expression of HIF-1α and EMT regulator Twist1. Collectively, the combination therapy of sorafenib and BrMC could be a new and promising therapeutic approach in 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.

Casticin inhibits the epithelial-mesenchymal transition in ovarian carcinoma via the hedgehog signaling pathway

Casticin inhibits migration, invasion and induced apoptosis in numerous cancer cells; however, the Hedgehog (Hh) signaling pathway is a key factor in the epithelial-mesenchymal transition (EMT). The present study aimed to assess whether casticin affects the expression of members of the Hh signaling pathway and EMT effectors in ovarian carcinoma. The ovarian cancer SKOV3 cell line was incubated in the presence of various concentrations of casticin or cyclopamine. Next, the expression levels of the main Hh signaling effector glioma-associated oncogene-1 (Gli-1) and EMT-associated factors [Twist-related protein 1 (Twist1), E-cadherin and N-cadherin] were determined by western blotting and reverse transcription-quantitative polymerase chain reaction. Cell proliferation and growth were assessed using MTT and soft agar assays; cell migration and invasion was evaluated using an in vitro migration assay and a transwell invasion assay, respectively. Compared with control group values, Gli-1, Twist1 and N-cadherin expression levels were reduced, whereas E-cadherin levels were increased in the casticin- and cyclopamine-treated groups. Incubation with casticin or cyclopamine resulted in markedly reduced SKOV3 cell viability, migration and invasion, in a dose-dependent manner. To the best of our knowledge, the findings of the present study indicated for first time that casticin may inhibit EMT via Hh signaling in vitro, reducing the migratory ability of ovarian cancer cells.

Lx2-32c inhibits the formation of mammosphere from MDA-MB-231 cells and induces apoptosis involving in down-regulating FoxM1

Cancer stem cells (CSCs) are a subset of cancer cells which have self-renewal ability and exist in various tumors. Inhibition of CSCs self-renewal is considered as a new method for tumor therapy. A novel semi-synthetic taxane analogue, Lx2-32c, could overcome drug resistance in various cancer cell lines. In this study, it was found that Lx2-32c inhibited the proliferation and mammosphere formation of MDA-MB-231-derived cancer stem cell-like cells (MCSCLCs) and induced apoptosis, as well as down-regulated the expression of FoxM1 and CD44 in MCSCLCs. Simultaneously, it was proved that Lx2-32c combined with thiostreption, a FoxM1 inhibitor inhibited proliferation and mammosphere formation of MCSCLCs and induced apoptosis to a more extent than Lx2-32c alone; thiostreption could also enhance the effect of Lx2-32c of reduction of the expression of FoxM1 and CD44. All of these results indicated that Lx2-32c is a novel semi-synthetic taxane analogue which inhibits the self-renewal of MCSCLCs cells and induces apoptosis involving in down-regulating FoxM1.