Bai-Cheng He

The PTEN/PI3K/Akt and Wnt/β-catenin signaling pathways are involved in the inhibitory effect of resveratrol on human colon cancer cell proliferation

Colon cancer is one of the most common malignancies and the treatments for colon cancer have been developed substantially in the last decades, but there is still a great clinical need to explore new treatment regimens due to the undesirable prognosis. In this investigation, we demonstrated the anti-proliferative and apoptosis-inducing activities of resveratrol (Res) in human colon cancer cells, and the possible mechanisms underlying these effects. We used crystal violet staining, flow cytometry and western blotting to validate the anti-proliferative and apoptosis-inducing effects of Res on HCT116 cells. A xenograft tumor model was used to confirm the anti-proliferative effects of Res. We employed polymerase chain reaction, western blotting, recombinant adenovirus and luciferase reporter assay to explore the possible mechanism(s) of action. We found that Res inhibits significantly the proliferation and promotes apoptosis in HCT116 cells, as well as inhibits the xenograft tumor growth of colon cancer. Res upregulates the expression of phosphatase and tensin homolog (PTEN) and decreases the phosphorylation of Akt1/2. The exogenous expression of PTEN inhibits the PI3K/Akt signal and promotes the anti-proliferative effects of Res in HCT116 cells, while knockdown of PTEN increases PI3K/Akt signal but reduces the anti-proliferative function of Res. The protein and mRNA expression of β-catenin are all decreased by Res concentration-dependently. Thus, our findings strongly suggest that the anti-proliferative effects of Res in human colon cancer cells may be mediated by regulating separately the PTEN/PI3K/Akt and Wnt/β-catenin signaling.

BMP9 and COX-2 form an important regulatory loop in BMP9-induced osteogenic differentiation of mesenchymal stem cells

Mesenchymal stem cells (MSCs) can self-renew and differentiate into osteogenic, chondrogenic, adipogenic and myogenic lineages. Its reported that bone morphogenetic protein 9 (BMP9) is one of the most potent osteogenic BMPs to initiate the commitment of MSCs to osteoblast lineage. Cyclooxygenase-2 (COX-2) is critical for bone fracture healing and osteogenic differentiation in MSCs. However, the relationship between COX-2 and BMP9 in osteogenesis remains unknown. Herein, we investigate the role of COX-2 in BMP9-induced osteogenesis in MSCs. We demonstrate that COX-2 is up-regulated as a target of BMP9 in MSCs. Both COX-2 inhibitor (NS-398) and COX-2 knockdown siRNAs can effectively decrease alkaline phosphatase (ALP) activities induced by BMP9 in MSCs. NS-398 also down-regulates BMP9-induced expression of osteopontin and osteocalcin, so does the matrix mineralization. The in vivo studies indicate that knockdown of COX-2 attenuates BMP9-induced ectopic bone formation. In perinatal limb culture assay, NS-398 is shown to reduce the hypertropic chondrocyte zone and ossification induced by BMP9. Mechanistically, knockdown of COX-2 significantly inhibits the BMP9 up-regulated expression of Runx2 and Dlx-5 in MSCs, which can be rescued by exogenous expression of COX-2. Furthermore, knockdown of COX-2 apparently reduces BMP9 induced BMPR-Smad reporter activity, the phosphorylation of Smad1/5/8, and the expression of Smad6 and Smad7 in MSCs. NS-398 blocks the expression of BMP9 mediated by BMP9 recombinant adenovirus. Taken together, our findings suggest that COX-2 plays an important role in BMP9 induced osteogenic differentiation in MSCs; BMP9 and COX-2 may form an important regulatory loop to orchestrate the osteogenic differentiation in MSCs.

All-trans retinoic acid modulates bone morphogenic protein 9-induced osteogenesis and adipogenesis of preadipocytes through BMP/Smad and Wnt/β-catenin signaling pathways.

It is known that excessive adipogenesis contributes to osteoporosis, suggesting that trans-differentiation of adipogenic committed preadipocytes into osteoblasts may be a potential therapeutical approach for osteoporosis. We explored whether bone morphogenic protein 9 (BMP9) could induce 3T3-L1 preadipocytes to trans-differentiate into osteoblasts. BMP9 effectively increased expression of osteogenic markers and promoted mineralization in preadipocytes. However, BMP9 also led to adipogenic differentiation of preadipocytes, as evidenced by increased lipid accumulation and up-regulation of adipogenic transcription factors. In order to regulate the switch between osetogenesis and adipogenesis, we evaluated the effect of all-trans retinoic acid (ATRA) on BMP9-induced differentiation of preadipocytes. We found that ATRA enhanced BMP9-induced osteogenic differentiation and blocked BMP9-induced adipogenic differentiation both in vitro and in vivo. Mechanistically, ATRA was shown to elevate BMP9 expression and activate BMP/Smad signaling. Additionally, BMP9 and ATRA exerted a synergistic effect on activation of Wnt/β-catenin signaling. Knockdown of β-catenin abolished the stimulatory effect of ATRA on BMP9-induced alkaline phosphatase activity and reversed the inhibitory effect of ATRA on BMP9-induced adipogenesis in preadipocytes. Furthermore, ATRA and BMP9 synergistically repressed glycogen synthase kinase 3β (GSK3β) activity and promoted Akt phosphorylation, and inhibited expression of phosphatase and tensin homologue deleted on chromosome 10 (PTEN) that antagonizes phosphatidylinositol-3-kinase (PI3K) function, suggesting that Wnt/β-catenin signaling was activated at least partly through PI3K/Akt/GSK3β pathway. Collectively, ATRA mediated BMP9-induced osteogenic or adipogenic differentiation of 3T3-L1 preadipocytes by BMP/Smad and Wnt/β-catenin signaling. The combination of BMP9 and ATRA may be explored as an effective therapeutic strategy for osteoporosis.

The role of COX-2 in mediating the effect of PTEN on BMP9 induced osteogenic differentiation in mouse embryonic fibroblasts.

Mouse embryonic fibroblasts (MEFs) are multi-potent progenitor cells (MPCs), can differentiate into different lineages, such as osteogenic, and adipogenic. PTEN, a tumor suppressor, may be involved in regulating bone development through interacting with COX-2. BMP9, the most potent osteogenic BMPs, can up-regulate COX-2 in MPCs. Whether PTEN is involved in BMP9 induced osteogenic differentiation in MPCs remains unknown. The goal of this investigation is to identify the effect of PTEN on BMP9-induced osteogenic differentiation in MPCs and dissect the possible mechanism underlay this. We found that BMP9 down-regulates PTEN, and PTEN inhibitor (VO) effectively increases different osteogenic markers induced by BMP9 in MEFs. Exogenous expression of PTEN inhibits BMP9 induced ectopic bone formation apparently. Mechanistically, we found that VO can enhance BMP9 induced BMPs/Smads signaling prominently without no substantial effects on cell cycle. Further analysis indicates that VO can promote BMP9-induced expression of COX-2 in MEFs, which can be eliminated by PI3K inhibitor. Additionally, COX-2 knockdown abolishes the effect of VO on BMP9-induced ALP activities in MEFs. Our findings suggest that PTEN plays an important role in regulating BMP9 induced osteogenic differentiation in MPCs, which may be mediated by PTEN/PI3K/Akt signaling to modulate the expression of COX-2.

The role of IGFBP-5 in mediating the anti-proliferation effect of tetrandrine in human colon cancer cells

Colon cancer is one of the most common malignancies, causes considerable morbidity and mortality. The current treatment for colon cancer is more modest than had been hoped. There is an urgent clinical need to explore new agents or adjuvants for colon cancer treatment. Natural products and their derivates act as one of the major source for anticancer agent. In the present study, we investigated the anti-proliferation and chemoprevention effects of tetrandrine (Tet) on colon cancer cells to uncover the possible molecular basis of this effect. We found that Tet can inhibit proliferation and induce apoptosis in LoVo cells. With dimethylhydrazine (DMH) and dextran sodium sulfate (DSS) induced colon cancer model, we found that Tet can prevent or inhibit DMH plus DSS induced aberrant crypt foci (ACF) and colon cancer formation, as well as suppress tumor growth in the xenograft colon cancer model. Tet can downregulate the expression of IGFBP-5 in LoVo cells. Exogenous expression of IGFBP-5 can attenuate the anti-cancer activity of Tet, while IGFBP-5 knockdown potentiates this effect of Tet on LoVo cells. Tet can inhibit Wnt/β-catenin signaling transduction, which can be partly reversed by exogenous expression of IGFBP-5, but is enhanced by IGFBP-5 knockdown. Our results demonstrated that the anticancer activity of Tet in colon cancer cells may be mediated partly by downregulating the expression of IGFBP-5, thus inactivating Wnt/β-catenin signaling transduction.

Oridonin inhibits the proliferation of human osteosarcoma cells by suppressing Wnt/β-catenin signaling

It has been reported that oridonin (ORI) can inhibit proliferation and induce apoptosis in various types of cancer cell lines. However, the exact mechanism for this function remains unclear. In this study, we investigated the proliferation inhibitory effect of ORI on human osteosarcoma (OS) 143B cells and dissected the possible molecular mechanism(s) underlying this effect. We demonstrated that ORI can inhibit proliferation, induce apoptosis and arrest the cell cycle in 143B cells. Using luciferase reporter assay, we found that the Wnt/β-catenin signaling was inhibited in 143B cells by ORI. Accordingly, the total protein levels and nuclear translocation of β-catenin were reduced by ORI treatment. ORI increased glycogen synthase kinase 3β (GSK3β) activity and upregulated Dickkopf-1 (Dkk-1) expression. We found that Dkk-1 overexpression or β-catenin knockdown can potentiate the proliferation inhibitory effect of ORI in 143B cells, while β-catenin overexpression attenuated this effect. Using the xenograft tumor model of human OS, we demonstrated that ORI effectively inhibited the growth of tumors. Histological examination showed that ORI inhibited cancer cell proliferation, decreased the expression of PNCA and β-catenin. Our findings suggest that ORI can inhibit 143B OS cell proliferation by downregulating Wnt/β-catenin signal transduction, which may be mediated by upregulating the Dkk-1 expression and/or enhancing the function of GSK3β. Therefore, ORI can be potentially used as an effective adjuvant agent for the clinical management of OS.

BMP9/p38 MAPK is essential for the antiproliferative effect of resveratrol on human colon cancer.

Colon cancer is one of the most common malignancies of the digestive system. Although more effective therapeutic strategies have been developed in the last decades, there is still a great clinical need to explore new treatment regimens for colon cancer due to the undesirable prognosis. In the present study, we investigated the anticancer activity of resveratrol (Res) in human colon cancer cells, and the possible mechanism underlying this effect. We employed crystal violet staining, flow cytometry and western blotting to test the antiproliferation- and apoptosis-inducing effects of Res in LoVo cells. A xenograft tumor model was also introduced to confirm the in vivo anticancer effect of Res. Using PCR, western blotting, a recombinant adenovirus and a specific inhibitor of p38 MAPK or bone morphogenetic protein receptor (BMPR) to explore the possible molecular mechanisms. We found that Res markedly inhibited the proliferation and promoted the apoptosis of LoVo cells, and suppressed the in vivo tumor growth of colon cancer. Res substantially upregulated the expression of bone morphogenetic protein 9 (BMP9). Exogenous expression of BMP9 enhanced the anticancer effect of Res in LoVo cells, while BMP9 knockdown partly reduced this activity. Res increased the activation of p38 MAPK, which was enhanced by the exogenous expression of BMP9. The anticancer activity of Res, or Res combined with BMP9, was reduced partly by the p38 MAPK inhibitor. The BMPR inhibitor almost abolished the Res-induced activation of p38 MAPK, and attenuated the antiproliferative effect of Res in the LoVo cells. Our findings strongly suggest that the anticancer effect of Res in human colon cancer cells may be partly mediated by upregulation of BMP9 to activate p38 MAPK in a BMPR-dependent manner.

TGF-β1/PTEN/PI3K signaling plays a critical role in the anti-proliferation effect of tetrandrine in human colon cancer cells

The diagnosis and treatment for colon cancer have been greatly developed, but the prognosis remains unsatisfactory. There is still a great clinical need to explore new efficacious drugs for colon cancer treatment. Tetrandrine (Tet) is a bis-benzylisoquinoline alkaloid. It has been shown that Tet may be a potential candidate for cancer treatment, but the explicit mechanism underlying this activity remains unclear. In this study, we investigated the anticancer activity of Tet in human colon cancer cells and dissected the possible mechanism. With cell viability assay and flow cytometry analysis, we confirmed that Tet can effectively inhibit the proliferation and induce apoptosis in HCT116 cells. Mechanically, we found that Tet greatly increases the mRNA and protein level of TGF-β1 in HCT116 cells. Exogenous TGF-β1 enhances the anti-proliferation and apoptosis inducing effect of Tet in HCT116 cells, which has been partly reversed by TGF-β1 inhibitor. Tet decreases the phosphorylation of Akt1/2/3 in HCT116 cells. This effect can be enhanced by exogenous TGF-β1, but partly reversed by TGF-β1 inhibitor. Tet exhibits no effect on total level of PTEN, but decreases the phosphorylation of PTEN; exogenous TGF-β1 enhances the effect of Tet on decreasing the phosphorylation of PTEN, which was partly reversed by TGF-β1 inhibitor. Our findings suggested that Tet may be a promising candidate for colon cancer treatment, and the anticancer activity may be mediated by inactivating PI3K/Akt signaling through upregulating TGF-β1 to decrease the phosphorylation of PTEN.

Follicle‐Stimulating Hormone β‐Subunit Potentiates Bone Morphogenetic Protein 9‐Induced Osteogenic Differentiation in Mouse Embryonic Fibroblasts

Postmenopausal osteoporosis (PMOP)‐related fractures usually result in morbidity and mortality in aging women, so it remains a global public health concern, and new effective safe treatments are urgently needed recently. Efficient osteogenesis from mesenchymal stem cells (MSCs) would have the clinical application potential in treating multiple osteal disorders. Follicle‐stimulating hormone (FSH), a pituitary glycoprotein hormone highly associated with menopausal bone turnover, whose peculiar part of receptor binding is follicle‐stimulating hormone β‐subunit (FSHβ). Bone morphogenetic protein 9 (BMP9), a potent osteogenic factor, can up‐regulate FSHβ in mouse embryonic fibroblasts (MEFs). However, it is unclear, whether extrapituitary FSHβ affects BMP9‐induced osteogenesis in MEFs. In this study, we investigated the role of FSHβ in BMP9‐induced osteogenesis in MEFs. We found that exogenous expression of FSHβ significantly increased BMP9‐induced alkaline phosphatase activity (ALP), the expression of osteogenic transcriptional factors, Runx2 and Osx, and the established late osteogenic markers, osteopontin (OPN) and osteocalcin (OCN), so does the ectopic bone formation. Mechanistically, FSHβ dramatically enhanced BMP9‐induced BMP/Smad signal transduction, presenting the augment phosphorylation of Smad1/5/8, whereas treatment with anti‐FSHβ antibodies suppressed these effects. An adenylate cyclase inhibitor obviously suppressed ALP and BMP/Smad signal transduction induced by BMP9 or the combination of BMP9 and FSHβ in MEFs. Collectively, our findings suggested that FSHβ may promote BMP9‐induced activation of BMP/Smad signaling through a FSH/FSH receptor (FSHR)/cAMP dependent pathway in MEFs partly. J. Cell. Biochem. 118: 1792–1802, 2017.

Resveratrol inactivates PI3K/Akt signaling through upregulating BMP7 in human colon cancer cells

Colon cancer is common worldwide and accounts for the significant cancer related morbidity and mortality in patients. Although extensive advancement has been made in colon cancer treatment and diagnosis in the last decades, there is still a giant gap between the clinical expectation. It has been reported that resveratrol (Res) may be a potential candidate for cancer treatment. However, the specific mechanism underlying this activity remains unclear. In this study, we investigated the anticancer activity of Res in human colon cancer cells, and unveiled the possible mechanism for this effect. With cell viability, flow cytometry, PCR and western blot analysis, we demonstrated the efficacious anticancer activity of Res in HCT116 cells. Mechanically, we found that Res greatly upregulates BMP7 in HCT116 cells. Exogenous BMP7 enhances the anticancer effect of Res in HCT116 cells, which was almost reversed by the BMP7 specific antibody. Res does not activate the BMPs/Smads signaling, but decreases the phosphorylation of Akt1/2/3 substantially in HCT116 cells. Exogenous BMP7 enhances the inhibitory effect of Res on the phosphorylation of Akt1/2/3, while BMP7 immunodepletion reverses this effect notably. Res markedly decreases the phosphorylation of PTEN, which can be enhanced by exogenous BMP7 but partly reversed by the BMP7 antibody. Our findings suggested that Res may be a promising candidate for colon cancer treatment, and the anticancer activity may be mediated by inactivating PI3K/Akt signaling through upregulating BMP7 to decrease, at least, the phosphorylation of PTEN.