Shun-Fu Chang

Glucose adsorption to chitosan membranes increases proliferation of human chondrocyte via mammalian target of rapamycin complex 1 and sterol regulatory element‐binding protein‐1 signaling

Osteoarthritis (OA) is currently still an irreversible degenerative disease of the articular cartilage. Recent, dextrose (d‐glucose) intraarticular injection prolotherapy for OA patients has been reported to benefit the chondrogenic stimulation of damaged cartilage. However, the detailed mechanism of glucoses effect on cartilage repair remains unclear. Chitosan, a naturally derived polysaccharide, has recently been investigated as a surgical or dental dressing to control breeding. Therefore, in this study, glucose was adsorbed to chitosan membranes (CTS‐Glc), and the study aimed to investigate whether CTS‐Glc complex membranes could regulate the proliferation of human OA chondrocytes and to explore the underlying mechanism. Human OA and SW1353 chondrocytes were used in this study. The experiments involving the transfection of cells used SW1353 chondrocytes. A specific inhibitor and siRNAs were used to investigate the mechanism underlying the CTS‐Glc‐regulated proliferation of human chondrocytes. We found that CTS‐Glc significantly increased the proliferation of both human OA and SW1353 chondrocytes comparable to glucose‐ or chitosan‐only stimulation. The role of mammalian target of rapamycin complex 1 (mTORC1) signaling, including mTOR, raptor, and S6k proteins, has been demonstrated in the regulation of CTS‐Glc‐increased human chondrocyte proliferation. mTORC1 signaling increased the expression levels of maturated SREBP‐1 and FASN and then induced the expressions of cell cycle regulators, that is, cyclin D, cyclin‐dependent kinase‐4 and ‐6 in human chondrocytes. This study elucidates the detailed mechanism behind the effect of CTS‐Glc complex membranes in promoting chondrocyte proliferation and proposes a possible clinical application of the CTS‐Glc complex in the dextrose intraarticular injection of OA prolotherapy in the future to attenuate the pain and discomfort of OA patients.

Upregulation of Bone Morphogenetic Protein-2 Synthesis and Consequent Collagen II Expression in Leptin-stimulated Human Chondrocytes.

Bone morphogenetic proteins (BMPs) play positive roles in cartilage development, but they can barely be detected in healthy articular cartilage. However, recent evidence has indicated that BMPs could be detected in osteoarthritic and damaged cartilage and their precise roles have not been well defined. Extremely high amounts of leptin have been reported in obese individuals, which can be associated with osteoarthritis (OA) development. The aim of this study was to investigate whether BMPs could be induced in human primary chondrocytes during leptin-stimulated OA development and the underlying mechanism. We found that expression of BMP-2 mRNA, but not BMP-4, BMP-6, or BMP-7 mRNA, could be increased in human primary chondrocytes under leptin stimulation. Moreover, this BMP-2 induction was mediated through transcription factor-signal transducer and activator of transcription (STAT) 3 activation via JAK2-ERK1/2-induced Ser727-phosphorylation. Of note, histone deacetylases (HDACs) 3 and 4 were both involved in modulating leptin-induced BMP-2 mRNA expression through different pathways: HDAC3, but not HDAC4, associated with STAT3 to form a complex. Our results further demonstrated that the role of BMP-2 induction under leptin stimulation is to increase collagen II expression. The findings in this study provide new insights into the regulatory mechanism of BMP-2 induction in leptin-stimulated chondrocytes and suggest that BMP-2 may play a reparative role in regulating leptin-induced OA development.

Expression of Beclin Family Proteins Is Associated with Tumor Progression in Oral Cancer

Background Beclin 1 and Beclin 2 are autophagy-related proteins that show similar amino acid sequences and domain structures. Beclin 1 established the first connection between autophagy and cancer. However, the role of Beclin 2 in cancer is unclear. The aims of this study were to analyze Beclin 1 and Beclin 2 expressions in oral cancer tissues and in cell lines, and to evaluate their possible roles in cancer progression. Methods We investigated Beclin 1 and Beclin 2 expressions by immunohistochemistry in 195 cases of oral cancer. The prognostic roles of Beclin 1 and Beclin 2 were analyzed statistically. In vitro, overexpression and knockdown of Beclin proteins were performed on an oral cancer cell line, SAS. The immunofluorescence and autophagy flux assays confirmed that Beclin proteins were involved in autophagy. The impacts of Beclin 1 and Beclin 2 on autophagy and tumor growth were evaluated by conversion of LC3-I to LC3-II and by clonogenic assays, respectively. Results Oral cancer tissues exhibited aberrant expressions of Beclin 1 and Beclin 2. The cytoplasmic Beclin 1 and Beclin 2 expressions were unrelated in oral cancer tissues. In survival analyses, high cytoplasmic Beclin 1 expression was associated with low disease specific survival, and negative nuclear Beclin 1 expression was associated with high recurrent free survival. Patients with either high or low cytoplasmic Beclin 2 expression had significantly lower overall survival and disease specific survival rates than those with moderate expression. In oral cancer cells, overexpression of either Beclin 1 or Beclin 2 led to autophagy activation and increased clonogenic survival; knockdown of Beclin 2 impaired autophagy and increased clonogenic survival. Conclusions Our results indicated that distinct patterns of Beclin 1 and Beclin 2 were associated with aggressive clinical outcomes. Beclin 1 overexpression, as well as Beclin 2 overexpression and depletion, contributed to tumor growth. These findings suggest Beclin proteins are associated with tumorigenesis.

Low Shear Stress Attenuates COX-2 Expression Induced by Resistin in Human Osteoarthritic Chondrocytes†

Low shear stress has been proposed to play a reparative role in modulating cartilage homeostasis. Recently, epidemiological studies have found a positive correlation between the resistin level in serum and synovial fluid and osteoarthritis (OA) severity in patients. However, the effect of moderate shear stress on the catabolic stimulation of resistin in OA chondrocytes remains unclear. Hence, this study was to investigate whether low shear stress could regulate resistin‐induced catabolic cyclooxygenase (COX)‐2 expression in human OA chondrocytes and the underlying mechanism. Human OA chondrocytes and SW1353 chondrosarcoma cells were used in this study. Two modes of low shear stress (2 dyn/cm2), pre‐shear and post‐shear, were applied to the chondrocytes. A specific activator and siRNAs were used to investigate the mechanism of low shear stress‐regulated COX‐2 expression of resistin induction. We found that human OA chondrocytes exposed to different modes of low shear stress elicit an opposite effect on resistin‐induced COX‐2 expression: pre‐shear for a short duration attenuates the resistin effect by inhibiting the transcription factor nuclear factor (NF)‐κB‐p65 subunit and the cAMP response element binding protein; however, post‐shear over a longer duration enhances the resistin effect by activating only the NF‐κB‐p65 subunit. Moreover, our results demonstrated that the regulation of both shear modes in resistin‐stimulated COX‐2 expression occurs through increasing AMP‐activated protein kinase activation and then sirtuin 1 expression. This study elucidates the detailed mechanism of low shear stress regulating the resistin‐induced catabolic COX‐2 expression and indicates a possible reparative role of moderate shear force in resistin‐stimulated OA development. J. Cell. Physiol. 232: 1448–1457, 2017.

Fulvic Acid Attenuates Resistin-Induced Adhesion of HCT-116 Colorectal Cancer Cells to Endothelial Cells

A high level of serum resistin has recently been found in patients with a number of cancers, including colorectal cancer (CRC). Hence, resistin may play a role in CRC development. Fulvic acid (FA), a class of humic substances, possesses pharmacological properties. However, the effect of FA on cancer pathophysiology remains unclear. The aim of this study was to investigate the effect of resistin on the endothelial adhesion of CRC and to determine whether FA elicits an antagonistic mechanism to neutralize this resistin effect. Human HCT-116 (p53-negative) and SW-48 (p53-positive) CRC cells and human umbilical vein endothelial cells (HUVECs) were used in the experiments. Treatment of both HCT-116 and SW-48 cells with resistin increases the adhesion of both cells to HUVECs. This result indicated that p53 may not regulate this resistin effect. A mechanistic study in HCT-116 cells further showed that this resistin effect occurs via the activation of NF-κB and the expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1). Co-treating cells with both FA and resistin revealed that FA significantly attenuated the resistin-increased NF-κB activation and ICAM-1/VCAM-1 expression and the consequent adhesion of HCT-116 cells to HUVECs. These results demonstrate the role of resistin in promoting HCT-116 cell adhesion to HUVECs and indicate that FA might be a potential candidate for the inhibition of the endothelial adhesion of CRC in response to resistin.

Regulation of Calcification in Human Aortic Smooth Muscle Cells Infected with High-glucose-treated Porphyromonas Gingivalis†

Porphyromonas (P.) gingivalis infection leading to the periodontitis has been associated with the development of systemic diseases, including cardiovascular diseases and diabetes. However, the effect of a high concentration of glucose (HG) on the invasion efficiency of P. gingivalis and the consequent modulation of pathogenesis in vascular cells, especially in the vascular smooth muscle cells (VSMCs), remains unclear. Hence, the aim of this study was to investigate whether treating P. gingivalis with HG could change its invasion capability and result in VSMC calcification and the underlying mechanism. Human aortic SMCs (HASMCs) and P. gingivalis strain CCUG25226 were used in this study. We found that HGPg infection of HASMCs could initiate the HASMC calcification by stimulating the autocrine regulation of bone morphogenetic protein (BMP) 4 in HASMCs. The upregulation of BMP4 expression in HASMCs was mediated by toll‐like receptor 4 and ERK1/2‐p38 signaling after P. gingivalis infection. Moreover, the autocrine action of BMP4 in HGPg infection‐initiated HASMC calcification upregulated BMP4‐specific downstream smad1/5/8‐runx2 signaling to increase the expressions of bone‐related matrix proteins, that is, osteopontin, osteocalcin, and alkaline phosphatase. This study elucidates the detailed mechanism of HGPg infection‐initiated calcification of HASMCs and indicates a possible therapeutic role of BMP4 in P. gingivalis infection‐associated vascular calcification.

Metformin increases the cytotoxicity of oxaliplatin in human DLD-1 colorectal cancer cells through down-regulating HMGB1 expression

Colorectal cancer (CRC) is the fourth most common cause of cancer death worldwide. Chemotherapy has been the major strategy for treating patients with advanced CRC. Oxaliplatin (OXA) is used as both an adjuvant and neoadjuvant anticancer agent available to treat advanced CRC. High‐mobility group box 1 protein (HMGB1) is a critical regulator of cell death and survival. HMGB1 overexpression has been shown to be resistant to cytotoxic agents. In addition, Metformin, a widely used drug for diabetes, has emerged as a potential anticancer agent. In this study, we examined whether HMGB1 plays a role in the OXA‐ and/or metformin‐induced cytotoxic effect on CRC cells. The results showed that treatment with OXA increased HMGB1 expression in the ERK1/2‐ and Akt‐dependent manners in DLD‐1 cells. HMGB1 gene knockdown enhanced the cytotoxicity and cell growth inhibition of OXA. Moreover, OXA‐increased HMGB1 expression was by inducing NF‐κB‐DNA‐binding activity to in DLD‐1 cells. Compared to a single agent, OXA combined with metformin administration resulted in cytotoxicity and cell growth inhibition synergistically, accompanied with reduced HMGB1 level. These findings may have implications for the rational design of future drug regimens incorporating OXA and metformin for the treatment of CRC.

2 dyn/cm2 shear force upregulates kruppel-like factor 4 expression in human chondrocytes to inhibit the interleukin-1β-activated nuclear factor-κB: CHANG et al.

The shear force effect on human chondrocytes is time and magnitude dependent. Recently, kruppel‐like factor (KLF) 4 has been identified as a pleiotropic protein and its activity in cells is dependent on different stimuli and/or cell types. The role of KLF4 in chondrocytes is still unclear and there has been no report determining whether shear force regulates KLF4 levels in chondrocytes. Hence, this study was carried out to investigate the role of KLF4 in human chondrocytes under shear force stimulation and the underlying mechanism. Human primary and SW1353 chondrocytes were used in this study. The shear forces at 2, 5, or 15 dyn/cm2 intensity were applied to both types of human chondrocytes. The specific small interfering RNAs, activators, and inhibitors were used to study the detailed mechanism of shear force. The presented results showed that 2, but not 5 and 15, dyn/cm2 shear force increases KLF4 expression in human primary and SW1353 chondrocytes. Extracellular signal‐regulated kinase 5 induced peroxisome proliferator‐activated receptor γ transcription activity to increase KLF4 transcription. Moreover, the KLF4 induction in human chondrocytes in response to 2 dyn/cm2 shear force could attenuate interleukin (IL)‐1β‐stimulated nuclear factor‐κB activation. These results elucidate the role of KLF4 in antagonizing the effect of IL‐1β in human chondrocytes under 2 dyn/cm2 shear force stimulation and provide a possible mechanism to demonstrate the protection of moderate forces or exercises in cartilage.

Leptin induces MMP1/13 and ADAMTS 4 expressions through bone morphogenetic protein-2 autocrine effect in human chondrocytes

The induction of bone morphogenetic protein (BMP)2 in injured and arthritis articular cartilage has been proposed, but the precise mechanism has not been clearly clarified. Our previous study has found that leptin could stimulate the BMP2 autocrine effect to increase the anabolic collagen II expression when it initiates the catabolic response in human chondrocytes. It has been suggested that this BMP2 autocrine effect contributes to a reparative role in leptin‐stimulated human chondrocytes. In this study, we further determined whether this BMP2 autocrine effect also affect the expressions of catabolic matrix metalloproteinases (MMPs) and a disintegrin and metalloproteinase with thrombospondin motif (ADAMTS). Human primary and SW1353 chondrocytes were used in this study. It was shown that leptin could induce the expressions of MMP1, 3, and 13 and ADAMTS4 and 5 in both human primary and SW1353 chondrocytes. Leptin‐increased MMP1/13 (not MMP3) and ADAMTS4 (not ADAMTS5) expressions were affected by the leptin‐upregulated BMP2 and its specific downstream Smad1/5 signaling. Moreover, both HDAC3 and 4 are involved in regulating leptin‐induced BMP2 upregulation and then affect MMP1 and 13 and ADAMTS4 expression. Both HDAC3 and 4 also affect leptin‐increased MMP3 mRNA expression but not through BMP2 autocrine effect of leptin induction. Our results further elucidated the role of BMP2 autocrine effect in matrix‐degrading enzymes expressions under leptin stimulation. The findings in this study provide new insights into the possible mechanism of BMP2 induction in leptin‐stimulated chondrocytes and in leptin‐induced OA development.

Effect of 5-fluorouracil on excision repair cross-complementing 1 expression and consequent cytotoxicity regulation in human gastric cancer cells: LIU et al

Gastric cancer is the third leading cause of cancer mortality all over the world. The combination therapy of surgery with chemotherapy, that is, 5‐fluorouracil (5‐FU) and platinum‐containing anticancer drugs, is becoming a current clinical strategy for patients with gastric cancer because of the lower curative rate and higher cancer recurrence rate of patients treated with only surgery. However, the development of drug resistance in cancer cells is still the most challenge in clinical chemotherapy. Excision repair cross‐complementing 1 (ERCC1), an essential member of nucleotide excision repair system, recently has been suggested to be a predictive biomarker of treatment evaluation and might affect the outcomes of chemotherapy. Thus, this study was aimed to investigate whether ERCC1 expression could be regulated, and its role in gastric cancer cells treated with 5‐FU and the underlying mechanism. Human AGS gastric cancer cells were used in this study. It was shown that ERCC1 expression could be upregulated in AGS cells treated with 5‐FU and this upregulation could subsequently attenuate the cytotoxicity of 5‐FU in AGS cells. Moreover, 5‐FU–upregulated ERCC1 expression was regulated by extracellular signal‐regulated kinase (ERK) 1/2 and p38 signaling through activating the transcription factor c‐jun/activator protein (AP)‐1. These results indicated the role of ERCC1 in the development of drug resistance to 5‐FU in AGS cells. The mechanism elucidation concerning the ERK1/2 and p38 kinases and transcription factor c‐jun/AP‐1 might contribute another idea to the development of chemotherapy strategy for the gastric cancers in the future.