• Tissue Engineering and Regenerative Medicine
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• v.15 no.6
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• pp.793-801
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• 2018

BACKGROUND: The aim of this study was to evaluate the combined effect of low-level laser treatment (LLLT) and recombinant human bone morphological protein-2 (rhBMP-2) applied to hypoxic-cultured MC3T3-E1 osteoblastic cells and to determine possible signaling pathways underlying differentiation and mineralization of osteoblasts under hypoxia. METHODS: MC3T3-E1 cells were cultured under 1% oxygen tension for 72 h. Cell cultures were divided into four groups: normoxia control, low-level laser (LLL) alone, rhBMP-2 combined with LLLT, and rhBMP-2 under hypoxia. Laser irradiation was applied at 0, 24, and 48 h. Cells were treated with rhBMP-2 at 50 ng/mL. Alkaline phosphatase activity was measured at 3, 7, and 14 days to evaluate osteoblastic differentiation. Cell mineralization was determined with Alizarin red S staining at 7 and 14 days. Western blot assays were performed to evaluate whether p38/protein kinase D (PKD) signaling was involved. RESULTS: The results indicate that LLLT and rhBMP-2 synergistically increased alkaline phosphatase (ALP) activity and mineralization. Western blot analyses showed that expression of type I collagen, runt-related transcription factor 2 (RUNX2), and Osterix (Osx), increased and expression of hypoxia-inducible factor 1-alpha ($HIF-1{\alpha}$), decreased more in the LLLT and rhBMP-2 combined group than in the rhBMP-2 or LLL alone groups. Moreover, LLLT and rhBMP-2 stimulated p38 phosphorylation and rhBMP-2 and LLLT increased Prkd1 phosphorylation. CONCLUSION: Combined treatment with rhBMP-2 and LLL induced differentiation and mineralization of hypoxic-cultured MC3T3-E1 osteoblasts by activating p38/PKD signaling in vitro.

• Asian Pacific Journal of Cancer Prevention
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• v.14 no.6
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• pp.3625-3630
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• 2013

Reactive oxygen species (ROS) are known to promote mesothelial carcinogenesis that is closely associated with asbestos fibers and inflammation. Epithelial to mesenchymal cell transition (EMT) is an important process involved in the progression of tumors, providing cancer cells with aggressiveness. The present study was performed to determine if EMT is induced by $H_2O_2$ in human malignant mesothelioma (HMM) cells. Cultured HMM cells were treated with $H_2O_2$, followed by measuring expression levels of EMT-related genes and proteins. Immunohistochemically, TWIST1 expression was confined to sarcomatous cells in HMM tissues, but not in epithelioid cells. Treatment of HMM cells with $H_2O_2$ promoted EMT, as indicated by increased expression levels of vimentin, SLUG and TWIST1, and decreased E-cadherin expression. Expression of stemness genes such as OCT4, SOX2 and NANOG was also significantly increased by treatment of HMM cells with $H_2O_2$. Alteration of these genes was mediated via activation of hypoxia inducible factor 1 alpha (HIF-$1{\alpha}$) and transforming growth factor beta 1 (TGF-${\beta}1$). Considering that treatment with $H_2O_2$ results in excess ROS, the present study suggests that oxidative stress may play a critical role in HMM carcinogenesis by promoting EMT processes and enhancing the expression of stemness genes.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.11
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• pp.4637-4642
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• 2014

Background: Hypoxia-inducible factor $1{\alpha}$ (HIF-$1{\alpha}$) plays an important role in regulating cell survival and angiogenesis, which are critical for tumor growth and metastasis. Genetic variations of HIF1A have been shown to influence the susceptibility to many kinds of human tumors. Increased expression of HIF-$1{\alpha}$ has also been demonstrated to be involved in tumor progression. However, the prognostic value of single nucleotide polymorphisms (SNPs) inthe HIF1A gene remains to be determined in most cancer types, including colorectal cancer (CRC). In this study, we sought to investigate the predictive role of HIF1A SNPs in prognosis of CRC patients and efficacy of chemotherapy. Materials and Methods: We genotyped two functional SNPs in HIF1A gene using the Sequenom iPLEX genotyping system and then assessed their associations with clinicopathological parameters and clinical outcomes of 697 CRC patients receiving radical surgery using Cox logistic regression model and Kaplan Meier curves. Results: Generally, no significant association was found between these 2 SNPs and clinical outcomes of CRC. In stratified analysis of subgroup without adjuvant chemotherapy, patients carrying CT/TT genotypes of rs2057482 exhibited a borderline significant association with better overall survival when compared with those carrying CC genotype [Hazard ratio (HR), 0.47; 95% confidence interval (95% CI): 0.29-0.76; P < 0.01]. Moreover, significant protective effects on CRC outcomes conferred by adjuvant chemotherapy were exclusively observed in patients carrying CC genotype of rs2057482 and in those carrying AC/CC genotype of rs2301113. Conclusions: Genetic variations in HIF1A gene may modulate the efficacy of adjuvant chemotherapy after surgery in CRC patients.

• Journal of Gastric Cancer
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• v.11 no.1
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• pp.16-22
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• 2011

Purpose: Eupatilin is an antioxidative flavone and a phytopharmaceutical derived from Artemisia asiatica. It has been reported to possess anti-tumor activity in some types of cancer including gastric cancer. Eupatilin may modulate the angiogenesis pathway which is part of anti-inflammatory effect demonstrated in gastric mucosal injury models. Here we investigated the anti-tumor effects of eupatilin on gastric cancer cells and elucidated the potential underlying mechanism whereby eupatilin suppresses angiogenesis and tumor growth. Materials and Methods: The impact of eupatilin on the expression of angiogenesis pathway proteins was assessed using western blots in MKN45 cells. Using a chromatin immunoprecipitation assay, we tested whether eupatilin affects the recruitment of signal transducer and activator of transcription 3 (STAT3), aryl hydrocarbon receptor nuclear translocator (ARNT) and hypoxia-inducible factor-$1{\alpha}$ (HIF-$1{\alpha}$) to the human VEGF promoter. To investigate the effect of eupatilin on vasculogenesis, tube formation assays were conducted using human umbilical vein endothelial cells (HUVECs). The effect of eupatilin on tumor suppression in mouse xenografts was assessed. Results: Eupatilin significantly reduced VEGF, ARNT and STAT3 expression prominently under hypoxic conditions. The recruitment of STAT3, ARNT and HIF-$1{\alpha}$ to the VEGF promoter was inhibited by eupatilin treatment. HUVECs produced much foreshortened and severely broken tubes with eupatilin treatment. In addition, eupatilin effectively reduced tumor growth in a mouse xenograft model. Conclusions: Our results indicate that eupatilin inhibits angiogenesis in gastric cancer cells by blocking STAT3 and VEGF expression, suggesting its therapeutic potential in the treatment of gastric cancer.

• Radiation Oncology Journal
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• v.34 no.3
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• pp.230-238
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• 2016

Purpose: Hypoxia can impair the therapeutic efficacy of radiotherapy (RT). Therefore, a new strategy is necessary for enhancing the response to RT. In this study, we investigated whether the combination of nanoparticles and RT is effective in eliminating the radioresistance of hypoxic tumors. Materials and Methods: Gold nanoparticles (GNPs) consisting of a silica core with a gold shell were used. CT26 colon cancer mouse model was developed to study whether the combination of RT and GNPs reduced hypoxia-induced radioresistance. Hypoxia inducible $factor-1{\alpha}$ ($HIF-1{\alpha}$) was used as a hypoxia marker. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining were conducted to evaluate cell death. Results: Hypoxic tumor cells had an impaired response to RT. GNPs combined with RT enhanced anti-tumor effect in hypoxic tumor compared with RT alone. The combination of GNPs and RT decreased tumor cell viability compare to RT alone in vitro. Under hypoxia, tumors treated with GNPs + RT showed a higher response than that shown by tumors treated with RT alone. When a reactive oxygen species (ROS) scavenger was added, the enhanced antitumor effect of GNPs + RT was diminished. Conclusion: In the present study, hypoxic tumors treated with GNPs + RT showed favorable responses, which might be attributable to the ROS production induced by GNPs + RT. Taken together, GNPs combined with RT seems to be potential modality for enhancing the response to RT in hypoxic tumors.

• Tuberculosis and Respiratory Diseases
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• v.57 no.1
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• pp.37-46
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• 2004

Background : Lung pericytes are important constituent cells of blood-air barrier in pulmonary microvasculature. These cells take part in the control of vascular contractility and permeability. In this study, it was hypothesized that change of lung pericytes might be attributable to pathologic change in microvasculature in acute lung injury. The purpose of this study was how hypoxia change proliferation and genetic expression in lung pericytes. Methods : From the lungs of several Sprague-Dawley rats, performed the primary culture of lung pericytes and subculture. Characteristics of lung pericytes were confirmed with stellate shape in light microscopy and immunocytochemistry. 2% concentration of oxygen and $200{\mu}M$ $CoCl_2$ were treated to cells. Tryphan blue method and reverse transcription-polymerase chain reaction were done. Results : 1. We established methodology for primary culture of lung pericytes. 2. Hypoxia inhibited cellular proliferation in pericytes. 3. Hypoxia could markedly induce vascular endothelial growth factor(VEGF) and smad-2. 4. Hypoxia-inducible factor-$1{\alpha}$(HIF-$1{\alpha}$) was also induced by 2% oxygen. Conclusion : Viability of lung pericytes are inhibited by hypoxia. Hypoxia can stimulate expression of hypoxia-responsive genes. Pericytic change may be contributed to dysfunction of alveolar-capillary barrier in various pulmonary disorders.

• The Journal of Internal Korean Medicine
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• v.34 no.2
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• pp.178-191
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• 2013

Objectives : In this study, we made an effort to investigate the protective mechanism of Ukgan-san (UGS) extracts on hypoxia-induced C6 glial cell death. Methods : The cell viability was assessed by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MMT) assay and cell morphological changes were analysed with microscope after staining with crystal violet (CV). Reactive oxygen species (ROS) formation was assessed by flow cytometer after staining with 2'7'-dichlorofluorescein diacetate (DCF-DA). We also analyzed expression of hypoxia-inducible factor-1 alpha (HIF-$1{\alpha}$) and p53, processing of procaspase-3 and procyclic acidic repetitive protein (PARP) by western blot method. Results : We estimated the elevated cell viability by UGS extract on $CoCl_2$-induced C6 glial cells. UGS attenuated $CoCl_2$-induced ROS formation in C6 glial cells and also showed a protective activity compared to antioxidants and exhibited abrogation of LDH-released by $CoCl_2$. UGS suppressed the typical apoptotic cell death markers, caspase-3 and PARP activation. UGS inhibited $CoCl_2$-induced HIF-1${\alpha}$ expression which is known as a major regulator for hypoxia-induced cell death, and suppressed p53 expression. Conclusions : These results suggest that UGS extract contains protective constituents for hypoxia-induced C6 glial cell death.

• Food Science and Biotechnology
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• v.18 no.6
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• pp.1423-1429
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• 2009

Rhizopus oryzae KSD-815 was isolated from nuruk that has been used to make Korean traditional wines. This study was performed to investigate the effect of cultures of R. oryzae KSD-815 on cardiovascular disorders and cancer metastasis. Firstly, these cultures were sequentially fractionationed with n-hexane (TAHe), ethylacetate (TAE), n-butanol (TAB), and $H_2O$ (TAW). The TAE inhibited the activity of angiotensin-converting enzyme (ACE) and TAB suppressed platelet aggregation in vitro. TAE and TAB inhibited cell motility of human breast cancer cells. Furthermore, TAW interrupted the formation of neovasculature and tube-like structure, and down-regulated the expression of angiogenic factors, basic fibroblast growth factor (bFGF), tumor necrosis factor-$\alpha$ (TNF-$\alpha$), and hypoxia-inducible factor-$1{\alpha}$ (HIF-$1{\alpha}$) in breast cancer cells. These results indicated that cultures of R. oryzae KSD-815 display the inhibitory activities on hypertension, platelet aggregation, and metastasis, and suggest that these cultures might be further probed for the purposes as therapeutic agents or dietary supplements.

• International Journal of Oral Biology
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• v.38 no.3
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• pp.111-119
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• 2013

Objective. To investigate the effects of the hypoxia inducible factor-1 (HIF-1) activation-mimicking agent cobalt chloride ($CoCl_2$) on the osteogenic differentiation of human mesenchymal stem cells (hMSCs) and elucidate the underlying molecular mechanisms. Study design. The dose and exposure periods for $CoCl_2$ in hMSCs were optimized by cell viability assays. After confirmation of $CoCl_2$-induced HIF-$1{\alpha}$ and vascular endothelial growth factor expression in these cells by RT-PCR, the effects of temporary preconditioning with $CoCl_2$ on hMSC osteogenic differentiation were evaluated by RT-PCR analysis of osteogenic gene expression, an alkaline phosphatase (ALP) activity assay and by alizarin red S staining. Results. Variable $CoCl_2$ dosages (up to $500{\mu}M$) and exposure times (up to 7 days) on hMSC had little effect on hMSC survival. After $CoCl_2$ treatment of hMSCs at $100{\mu}M$ for 24 or 48 hours, followed by culture in osteogenic differentiating media, several osteogenic markers such as Runx-2, osteocalcin and osteopontin, bone sialoprotein mRNA expression level were found to be up-regulated. Moreover, ALP activity was increased in these treated cells in which an accelerated osteogenic capacity was also verified by alizarin red S staining. Conclusions. The osteogenic differentiation potential of hMSCs could be preserved and even enhanced by $CoCl_2$ treatment.

• Asian Pacific Journal of Cancer Prevention
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• v.16 no.6
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• pp.2569-2574
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• 2015

Necroptosis, also known as "programmed necrosis", has emerged as a critical factor in a variety of pathological and physiological processes and is considered a cell type-specific tightly regulated process with mechanisms that may vary rather greatly due to the change of cell line. Here we used HT-29, a human colon cancer cell line, to establish a necroptosis model and elucidate associated mechanisms. We discovered that cobalt chloride, a reagent that could induce hypoxia-inducible $factor-1{\alpha}(HIF1{\alpha})$ expression and therefore mimic the hypoxic microenvironment of tumor tissue in some aspects induces necroptosis in HT-29 cells when caspase activity is compromised. On the other hand, apoptosis appears to be the predominant death form when caspases are functioning normally. HT-29 cells demonstrated significantly increased RIPK1, RIPK3 and MLKL expression in response to cobalt chloride plus z-VAD treatment, which was accompanied by drastically increased $IL1{\alpha}$ and IL6 expression, substantiating the notion that necrosis can induce profound immune reactions. The RIPK1 kinase inhibitor necrostatin-1 and the ROS scavenger NAC each could prevent necrosis in HT-29 cells and the efficiency was enhanced by combined treatment. Thus by building up a necroptosis model in human colon cancer cells, we uncovered that mechanically RIP kinases collaborate with ROS during necrosis promoted by cobalt chloride plus z-VAD, which leads to inflammation. Necroptosis may present a new target for therapeutic intervention in cancer cells that are resistant to apoptotic cell death.