Chun-Han Hou

Bone morphogenetic protein-2 enhances the motility of chondrosarcoma cells via activation of matrix metalloproteinase-13

Chondrosarcoma is a low-grade sarcoma characterized by developing metastases and high local recurrence rate. Bone morphogenetic protein-2 (BMP-2) plays an essential role in tumor progression and metastasis. Here we found that BMP-2 induced the migration of human chondrosarcoma cells (JJ012 cells). BMP-2 also increased the secretion of metalloproteinase-13 (MMP-13) in JJ012 cells, as shown by reverse transcriptase-polymerase chain reaction, western blot and zymographic analysis. The MMP-13 small interfering RNA inhibited the BMP-2-induced MMP-13 expression and thereby significantly inhibited the BMP-2-induced cell migration. Furthermore, phosphatidylinositol 3-kinase inhibitor (PI3K; Ly294002) or Akt inhibitor suppressed BMP-2-induced MMP-13 mRNA expression. Transient transfection with dominant negative p85 and Akt mutant also showed that the PI3K/Akt signaling pathway was involved in BMP-2-induced MMP-13 expression. In addition, AP-1 decoy oligodeoxynucleotide also suppressed the MMP-13 promoter activity enhanced by BMP-2. Moreover, BMP-2 increased the binding of c-Fos and c-Jun to the AP-1 element on the MMP-13 promoter. Taken together, our results indicated that BMP-2 enhanced the invasiveness of chondrosarcoma cells by increasing MMP-13 expression through the PI3K, Akt, c-Fos/c-Jun and AP-1 signal transduction pathway.

WISP-1 increases MMP-2 expression and cell motility in human chondrosarcoma cells

Chondrosarcoma is a type of highly malignant tumor with a potent capacity to invade locally and cause distant metastasis. Chondrosarcoma shows a predilection for metastasis to the lungs. WISP-1 is a cysteine-rich protein that belongs to the CCN (Cyr61, CTGF, Nov) family of matricellular proteins. However, the effect of WISP-1 on migration activity in human chondrosarcoma cells is mostly unknown. Here we found that WISP-1 increased the migration and expression of matrix metalloproteinase (MMP)-2 in human chondrosarcoma cells (JJ012 cells). We also found that human chondrosarcoma tissues had significant expression of the WISP-1 which was higher than that in normal cartilage. α5β1 monoclonal antibody and MAPK kinase (MEK) inhibitors (PD98059 and U0126) inhibited the WISP-1-induced increase of the migration and MMP-2 up-regulation of chondrosarcoma cells. WISP-1 stimulation increased the phosphorylation of focal adhesion kinase (FAK), MEK and extracellular signal-regulated kinase (ERK). In addition, NF-κB inhibitors also suppressed the cell migration and MMP-2 expression enhanced by WISP-1. Moreover, WISP-1 increased NF-κB luciferase activity and binding of p65 to the NF-κB element on the MMP-2 promoter. Taken together, our results indicated that WISP-1 enhances the migration of chondrosarcoma cells by increasing MMP-2 expression through the α5β1 integrin receptor, FAK, MEK, ERK, p65 and NF-κB signal transduction pathway.

Cyr61 promotes epithelial-mesenchymal transition and tumor metastasis of osteosarcoma by Raf-1/MEK/ERK/Elk-1/TWIST-1 signaling pathway

BackgroundOsteosarcoma is the most common primary malignant tumor in children and young adults, and its treatment requires effective therapeutic approaches because of a high mortality rate for lung metastasis. Epithelial to mesenchymal transition (EMT) has received considerable attention as a conceptual paradigm for explaining the invasive and metastatic behavior during cancer progression. The cysteine-rich angiogenic inducer 61 (Cyr61) gene, a member of the CCN gene family, is responsible for the secretion of Cyr61, a matrix-associated protein that is involved in several cellular functions. A previous study showed that Cyr61 expression is related to osteosarcoma progression. In addition, Cyr61 could promote cell migration and metastasis in osteosarcoma. However, discussions on the molecular mechanism involved in Cyr61-regulated metastasis in osteosarcoma is poorly discussed.ResultsWe determined that the expression level of Cyr61 induced cell migration ability in osteosarcoma cells. The Cyr61 protein promoted the mesenchymal transition of osteosarcoma cells by upregulating mesenchymal markers (TWIST-1 and N-cadherin) and inhibiting the epithelial marker (E-cadherin). Moreover, the Cyr61-induced cell migration was mediated by EMT. The Cyr61 protein elicited a signaling cascade that included αvβ5 integrin, Raf-1, mitogen-activated protein kinase (MEK), extracellular signal-regulated kinase (ERK), and Elk-1. The reagent or gene knockdown of these signaling proteins could inhibit Cyr61-promoted EMT in osteosarcoma. Finally, the knockdown of Cyr61 expression obviously inhibited cell migration and repressed mesenchymal phenotypes, reducing lung metastasis.ConclusionOur results indicate that Cyr61 promotes the EMT of osteosarcoma cells by regulating EMT markers via a signal transduction pathway that involves αvβ5 integrin, Raf-1, MEK, ERK, and Elk-1.

CCN4 induces IL-6 production through αvβ5 receptor, PI3K, Akt, and NF-κB singling pathway in human synovial fibroblasts

IntroductionOsteoarthritis (OA) is the most common degenerative joint disease that is involved in the degradation of articular cartilage. The exact etiology of OA is not completely understood. CCN4 is related to up-regulation in the cartilage of patients with osteoarthritis. Previous studies have shown that CCN4 might be associated with the pathogenesis of OA, but the exact signaling pathways in CCN4-mediated IL-6 expression in synovial fibroblasts (SF) are largely unknown. Therefore, we explored the intracellular signaling pathway involved in CCN4-induced IL-6 production in human synovial fibroblast cells.MethodsCCN4-induced IL-6 production was assessed with quantitative real-time qPCR and ELISA. The mechanisms of action of CCN4 in different signaling pathways were studied by using Western blotting. Neutralizing antibodies of integrin were used to block the integrin signaling pathway. Luciferase assays were used to study IL-6 and NF-κB promoter activity. Immunocytochemistry was used to examine the translocation activity of p65.ResultsOsteoarthritis synovial fibroblasts (OASFs) showed significant expression of CCN4 and the expression was higher than in normal SFs. OASF stimulation with CCN4 induced concentration- and time-dependent increases in IL-6 production. Pretreatment of OASFs with αvβ5 but not α5β1 and αvβ3 integrin antibodies reduced CCN4-induced IL-6 production. CCN4-mediated IL-6 production was attenuated by PI3K inhibitor (LY294002 and Wortmannin), Akt inhibitor (Akti), and NF-κB inhibitor (PDTC and TPCK). Stimulation of cells with CCN4 also increased PI3K, Akt, and NF-κB activation.ConclusionsOur results suggest that CCN4 activates αvβ5 integrin, PI3K, Akt, and NF-κB pathways, leading to up-regulation of IL-6 production. According to our results, CCN4 may be an appropriate target for drug intervention in OA in the future.

HMGB-1 induces IL-6 production in human synovial fibroblasts through c-Src, Akt and NF-κB pathways.

High mobility group box chromosomal protein 1 (HMGB‐1) is a widely studied, ubiquitous nuclear protein that is present in eukaryotic cells, and plays a crucial role in inflammatory response. However, the effects of HMGB‐1 on human synovial fibroblasts are largely unknown. In this study, we investigated the intracellular signaling pathway involved in HMGB‐1‐induced IL‐6 production in human synovial fibroblast cells. HMGB‐1 caused concentration‐ and time‐dependent increases in IL‐6 production. HMGB‐1‐mediated IL‐6 production was attenuated by receptor for advanced glycation end products (RAGE) monoclonal antibody (Ab) or siRNA. Pretreatment with c‐Src inhibitor (PP2), Akt inhibitor and NF‐κB inhibitor (pyrrolidine dithiocarbamate and L‐1‐tosylamido‐2‐phenylenylethyl chloromethyl ketone) also inhibited the potentiating action of HMGB‐1. Stimulation of cells with HMGB‐1 increased the c‐Src and Akt phosphorylation. HMGB‐1 increased the accumulation of p‐p65 in the nucleus, as well as NF‐κB luciferase activity. HMGB‐1‐mediated increase of NF‐κB luciferase activity was inhibited by RAGE Ab, PP2 and Akt inhibitor or RAGE siRNA, or c‐Src and Akt mutant. Our results suggest that HMGB‐1‐increased IL‐6 production in human synovial fibroblasts via the RAGE receptor, c‐Src, Akt, p65, and NF‐κB signaling pathways. J. Cell. Physiol. 226: 2006–2015, 2011.

AMP-activated protein kinase is involved in COX-2 expression in response to ultrasound in cultured osteoblasts

It has been shown that ultrasound (US) stimulation accelerates fracture healing in the animal models and in clinical studies. Cyclooxygenase-2 (COX-2) is a crucial mediator in mechanically induced bone formation. AMP-activated protein kinase (AMPK) has reported to sense and regulate the cellular energy status in various cell types. Here we found that US-mediated COX-2 expression was attenuated by LKB1 and AMPKalpha1 small interference RNA (siRNA) in human osteoblasts. Pretreatment of osteoblasts with AMPK inhibitor (araA and compound C), p38 inhibitor (SB203580), NF-kappaB inhibitor (PDTC), IkappaB protease inhibitor (TPCK) and NF-kappaB inhibitor peptide also inhibited the potentiating action of US. US increased the kinase activity and phosphorylation of LKB1, AMPK and p38. Stimulation of osteoblasts with US activated IkappaB kinase alpha/beta (IKKalpha/beta), IkappaBalpha phosphorylation, IkappaBalpha degradation, p65 phosphorylation at Ser(276), p65 and p50 translocation from the cytosol to the nucleus, and kappaB-luciferase activity. US-mediated an increase of IKKalpha/beta activity, kappaB-luciferase activity and p65 and p50 binding to the NF-kappaB element was inhibited by araA, SB203580 and LKB1 siRNA. Our results suggest that US increased COX-2 expression in osteoblasts via the LKB1/AMPKalpha1/p38/IKKalphabeta and NF-kappaB signaling pathway.

Ultrasound Stimulates NF-κB Activation and iNOS Expression Via the Ras/Raf/MEK/ERK Signaling Pathway in Cultured Preosteoblasts

It has been shown that ultrasound (US) stimulation accelerates fracture healing in the animal models and non‐operatively clinical uses. Nitric oxide (NO) is a crucial early mediator in mechanically induced bone formation. Here we found that US‐mediated inducible nitric oxide synthase (iNOS) expression was attenuated by Ras inhibitor (manumycin A), Raf‐1 inhibitor (GW5074), MEK inhibitor (PD98059), NF‐κB inhibitor (PDTC), and IκB protease inhibitor (TPCK). US‐induced Ras activation was inhibited by manumycin A. Raf‐1 phosphorylation at Ser338 by US was inhibited by manumycin A and GW5074. US‐induced MEK and ERK activation was inhibited by manumycin A, GW5074, and PD98059. Stimulation of preosteoblasts with US activated IκB kinase α/β (IKK α/β), IκBαphosphorylation, p65 phosphorylation at Ser276, p65, and p50 translocation from the cytosol to the nucleus, and κB‐luciferase activity. US‐mediated an increase of IKK α/β, IκBα, and p65 phosphorylation, κB‐luciferase activity and p65 and p50 binding to the NF‐κB element was inhibited by manumycin A, GW5074, and PD98059. Our results suggest that US increased iNOS expression in preosteoblasts via the Ras/Raf‐1/MEK/ERK/IKKαβ and NF‐κB signaling pathways. J. Cell. Physiol. 220: 196–203, 2009.

Ultrasound increased BMP‐2 expression via PI3K, Akt, c‐Fos/c‐Jun, and AP‐1 pathways in cultured osteoblasts

It has been shown that ultrasound (US) stimulation accelerates fracture healing in the animal models and in clinical studies. Bone morphogenetic protein (BMP) is a crucial mediator in bone formation during fracture healing. Here we found that US stimulation increased BMP‐2 expression but not other BMPs. US induced BMP‐2 transcription is mediated by AP‐1 element but not estrogen receptor response element and GC‐rich Sp1 response element. Pretreatment of osteoblasts with phosphatidylinositol 3‐kinase (PI3K) inhibitor (Ly294002) and Akt inhibitor inhibited the potentiating action of US; these results were further substantiated by transfecting with the dominant negative mutants of p85 and Akt. US stimulation increased the phosphorylation of p85 subunit of PI3K and serine 473 of Akt. Transfection of osteoblasts with c‐Fos and c‐Jun antisense oligonucleotide also reduced US‐increased BMP‐2 expression. US‐increased the binding of c‐Fos and c‐Jun to the AP‐1 element on the BMP‐2 promoter and the enhancement of AP‐1 luciferase activity was inhibited by Ly294002 and Akt inhibitor. Our results suggest that US increased BMP‐2 expression in osteoblasts via the PI3K, Akt, c‐Fos/c‐Jun, and AP‐1 signaling pathway. J. Cell. Biochem. 106: 7–15, 2009.

Hyperthermia Induces Apoptosis through Endoplasmic Reticulum and Reactive Oxygen Species in Human Osteosarcoma Cells

Osteosarcoma (OS) is a relatively rare form of cancer, but OS is the most commonly diagnosed bone cancer in children and adolescents. Chemotherapy has side effects and induces drug resistance in OS. Since an effective adjuvant therapy was insufficient for treating OS, researching novel and adequate remedies is critical. Hyperthermia can induce cell death in various cancer cells, and thus, in this study, we investigated the anticancer method of hyperthermia in human OS (U-2 OS) cells. Treatment at 43 °C for 60 min induced apoptosis in human OS cell lines, but not in primary bone cells. Furthermore, hyperthermia was associated with increases of intracellular reactive oxygen species (ROS) and caspase-3 activation in U-2 OS cells. Mitochondrial dysfunction was followed by the release of cytochrome c from the mitochondria, and was accompanied by decreased anti-apoptotic Bcl-2 and Bcl-xL, and increased pro-apoptotic proteins Bak and Bax. Hyperthermia triggered endoplasmic reticulum (ER) stress, which was characterized by changes in cytosolic calcium levels, as well as increased calpain expression and activity. In addition, cells treated with calcium chelator (BAPTA-AM) blocked hyperthermia-induced cell apoptosis in U-2 OS cells. In conclusion, hyperthermia induced cell apoptosis substantially via the ROS, ER stress, mitochondria, and caspase pathways. Thus, hyperthermia may be a novel anticancer method for treating OS.

The Metastasectomy and Timing of Pulmonary Metastases on the Outcome of Osteosarcoma Patients

Background The author intended to clarify the therapeutic effect and prognostic factors of metastasectomy and timing of pulmonary metastases in osteosarcoma patents. Methods Data was obtained retrospectively on all consecutive osteosarcoma patients from 1985 to 2005 in authors institute. Fifty-two patients with pulmonary nodules were identified, including 24 patients undergoing pulmonary metastasectomy treatment. These patients were categorized into four groups: group 1, patients with lung metastases at the initial presentation; group 2, lung metastases identified during the period of pre-operative chemotherapy; group 3, lung metastases identified during period of the post-operative chemotherapy; group 4, lung metastases identified after therapy for the primary osteosarcoma completed. Results In our study, the 2-, 3-, and 5-year overall survival rates for 52 patients were 49%, 39% and 20%. The 2-year overall survival rates were 18% for group 1, 32% for group 3, and 70% for group 4 (p < 0.001). The 5-year overall survival rate was 34% for group 4. Patients who underwent metastesectomy showed a better survival outcome as compared with the patients not undergoing metastasectomy (p = 0.003). The 2-year and 5-year overall survival rates of only one lung metastatic nodule were 62% and 50%, and for initially multiple lung metastatic nodules, 45% and 5%, respectively. In addition, the patients presented with lung metastases had a worse prognosis as compared with those without initial lung metastases (p = 0.0001). Conclusions The patients having single metastatic nodule showed a better prognosis than those with multiple lung nodules. Furthermore, those patients who underwent metastasectomy survived longer than those not undergoing metastasectomy. Patients who had late metastases after complete chemotherapy had a better prognosis; whereas those who had metastases identified at the initial presentation predicted a poor prognosis.