• Molecules and Cells
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• 제43권11호
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• pp.935-944
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• 2020

Aryl hydrocarbon receptor nuclear translocator (ARNT) plays an essential role in maintaining cellular homeostasis in response to environmental stress. Under conditions of hypoxia or xenobiotic exposure, ARNT regulates the subset of genes involved in adaptive responses, by forming heterodimers with hypoxia-inducible transcription factors (HIF1α and HIF2α) or aryl hydrocarbon receptor (AhR). Here, we have shown that ARNT interacts with DDB1 and CUL4-associated factor 15 (DCAF15), and the aryl sulfonamides, indisulam and E7820, induce its proteasomal degradation through Cullin-RING finger ligase 4 containing DCAF15 (CRL4^DCAF15) E3 ligase. Moreover, the two known neo-substrates of aryl sulfonamide, RNA-binding motif protein 39 (RBM39) and RNA-binding motif protein 23 (RBM23), are not required for ARNT degradation. In line with this finding, aryl sulfonamides inhibited the transcriptional activities of HIFs and AhR associated with ARNT. Our results collectively support novel regulatory roles of aryl sulfonamides in both hypoxic and xenobiotic responses.

• International Journal of Oral Biology
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• 제38권3호
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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.

• 한방재활의학과학회지
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• 제15권1호
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• pp.25-37
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• 2005

목적 : 진피(Citus Unshiu, CU)는 실험적으로 항산화, 항알러지, 항종양 효과 등이 있다고 보고되고 있다. 본 연구에서는 비만세포에서 PMA와 A23187에 의하여 유도된 싸이토카인의 증가에 대한 진피 추출물의 억제 효과와 그 기전을 알아보고자 한다. 방법 : PMA+A23187에 의한 싸이토카인 생성에 대한 진피 추출물의 억제효과 기전을 조사하기 위하여 진피를 처리한 후 $IL-1{\beta}$, IL-8, $TNF-{\alpha}$의 생산 및 혈관내피성장인자 (VEGF), 과립구 대식구 군집 자극 인자 (GM-CSF), 저산소 유도 인자(HIF-1)의 발현을 조사하였다. 결과 : 실험에서 PMA와 A23187을 처리한 경우 대조군에 비하여 $IL-1{\beta}$, IL-8, $TNF-{\alpha}$의 생산을 유의하게 증가시켰으나 진피 추출물을 처리한 군에서는 유의하게 억제되었다. 특히 $IL-1{\beta}$의 생성은 $103.7{\pm}5%$, IL-8 생성은 $34.6{\pm}7%$, $TNF-{\alpha}$의 생성은 $85.9{\pm}4.5%$ 억제되었다. (P<0.05). 또한 진피 추출물은 PMA와 A23187에 의하여 증가한 VEGF, GM-CSF, $HIF-1{\alpha}$의 발현 증가를 억제하였다 (약 90.9%의 VEGF, 61.6%의 GM-CSF). 결론 : 이러한 결과는 진피가 염증반응에 대한 HIF-1의 억제자로 작용할 수 있으면, 진피가 비만세포 매개성 염증 질환 치료제의 후보 물질이 될 것으로 사료된다.

• Radiation Oncology Journal
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• 제34권3호
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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.

• Tissue Engineering and Regenerative Medicine
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• 제15권6호
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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.

• Molecules and Cells
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• 제38권6호
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• pp.528-534
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• 2015

Hypoxia-inducible factor (HIF) is a key regulator of tumor growth and angiogenesis. Recent studies have shown that, BIX01294, a G9a histone methyltransferase (HMT)-specific inhibitor, induces apoptosis and inhibits the proliferation, migration, and invasion of cancer cells. However, not many studies have investigated whether inhibition of G9a HMT can modulate HIF-$1{\alpha}$ stability and angiogenesis. Here, we show that BIX01294 dose-dependently decreases levels of HIF-$1{\alpha}$ in HepG2 human hepatocellular carcinoma cells. The half-life of HIF-$1{\alpha}$, expression of proline hydroxylase 2 (PHD2), hydroxylated HIF-$1{\alpha}$ and von Hippel-Lindau protein (pVHL) under hypoxic conditions were decreased by BIX01294. The mRNA expression and secretion of vascular endothelial growth factor (VEGF) were also significantly reduced by BIX01294 under hypoxic conditions in HepG2 cells. BIX01294 remarkably decreased angiogenic activity induced by VEGF in vitro, ex vivo, and in vivo, as demonstrated by assays using human umbilical vein endothelial cells (HUVECs), mouse aortic rings, and chick chorioallantoic membranes (CAMs), respectively. Furthermore, BIX01294 suppressed VEGF-induced matrix metalloproteinase 2 (MMP2) activity and inhibited VEGF-induced phosphorylation of VEGF receptor 2 (VEGFR-2), focal adhesion kinase (FAK), and paxillin in HUVECs. In addition, BIX01294 inhibited VEGF-induced formation of actin cytoskeletal stress fibers. In conclusion, we demonstrated that BIX01294 inhibits HIF-$1{\alpha}$ stability and VEGF-induced angiogenesis through the VEGFR-2 signaling pathway and actin cytoskeletal remodeling, indicating a promising approach for developing novel therapeutics to stop tumor progression.

• 생명과학회지
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• 제22권1호
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• pp.41-48
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• 2012

봉독은 동양의학에서 관절염, 류마티즘 및 각종 암을 포함하여 다양한 질병을 치료하기 위하여 이용되었다. 최근 봉독의 신생혈관 억제효과에 대한 연구가 보고되었으나 정확한 분자메커니즘에 대해서는 보고가 미흡하다. 따라서, 본 연구는 봉독이 인간결장암세포인 HCT116세포에서 신생혈관생성과 종양진행에 중요한 역할을 하는 HIF-$1{\alpha}$와 VEGF 발현 억제효과를 조사하였다. 그 결과 봉독은 $CoCl_2$로 유도한 저산소 상태에서 VEGF와, HIF-$1{\alpha}$의 발현을 감소시키며 HIF-$1{\alpha}$의 promoter 영역인 HRE 활성을 억제하였다. 이러한 봉독의 HIF-$1{\alpha}$ 발현억제효과는 ERK1/2의 인산화 조절을 통한 것이며, 봉독은 p38, JNK, AKT의 인산화에는 영향을 끼치지 않았다. 또한 봉독의 효과를 나타내는 단일물질 탐색을 위해 봉독의 생리활성 물질로 알려진 아파민과 멜리틴을 조사한 결과, HIF-$1{\alpha}$와 VEGF 억제효과는 아파민에 기인하는 것이라고 예상 할 수 있었다. 이와 같은 결과를 통하여 본 연구에서는 봉독의 혈관신생 억제에 대한 새로운 신호전달기전 및 인간 결장암세포 전이 억제제로서의 잠재성을 확인하였다.

• Asian Pacific Journal of Cancer Prevention
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• 제13권11호
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• pp.5363-5369
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• 2012

High-risk human papillomavirus (HPV) especially HPV-16 and HPV-18 types are speculated to be important risk factors in non-smoking associated lung cancer in Asia. Increasing evidence has demonstrated that HPV oncoproteins may contribute to lung tumorigenesis and cell transformation. Importantly, HPV 16/18 E6 and E7 oncoproteins can mediate expression of multiple target genes and proteins, such as p53/pRb, VEGF, HIF-$1{\alpha}$, cIAP-2, and hTERT, and contribute to cell proliferation, angiogenesis and cell immortalization through different signaling pathways in lung cancer. This article provides an overview of experiment data on HPV-associated lung cancer, describes the main targets on which HPV E6/E7 oncoproteins act, and further discusses the potential signaling pathways in which HPV E6/E7 oncoproteins are involved. In addition, we also raise questions regarding existing problems with the study of HPV-associated lung cancer.

• Asian Pacific Journal of Cancer Prevention
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• 제15권11호
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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.

• Tuberculosis and Respiratory Diseases
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• 제57권1호
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• pp.37-46
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• 2004

연구 배경 : 허파혈관주위세포는 허파미세혈관에서 혈액공기 장벽을 이루고 있는 중요한 세포이다. 이 세포는 생리학적으로 혈류와 혈관의 투과성을 조절하는 기능이 있다. 본 연구는 급성폐손상/급성호흡곤란증후군에서 혈관 과투과성 및 개형에 혈관주위세포의 변화가 중요한 역할을 할 것으로 보고 시작하게 되었다. 흰쥐로 부터 일차 배양한 허파혈관주위세포에 저산소 상태를 만들었을 때, 세포의 생존능에 미치는 영향과 저산소증에 의해 유도되는 유전자의 발현을 살펴보았다. 방 법 : 흰쥐로부터 허파혈관주위세포를 일차 배양 및 계대 배양하였다. 광학 현미경 및 세포 면역 화학 염색으로 세포를 확인하였다. 2% $O_2$의 세포 배양기와 $200{\mu}M$ $CoCl_2$를 처리하였다. 세포의 증식은 tryphan blue 염색 후 세포수를 세는 방법을 택하였다. 유전자 발현은 역전사 중합 효소 연쇄반응을 이용하였다. 결 과 : 1. 흰쥐로부터 허파혈관주위세포를 성공적으로 일차 배양 및 계대 배양할 수 있었다. 2. 2% $O_2$나 $CoCl_2$에서 혈관주위세포는 24시간, 36시간, 48시간에 증식이 억제되었다. 3. 허파혈관주위세포에 저산소 상태의 자극을 주면 VEGF와 smad-2의 발현이 현저하게 증가하였다. 4. 허파혈관주위세포의 HIF$1{\alpha}$, COX-2는 저산소상태에서 VEGF, smad-2에 비해 발현의 변화가 현저하지 않았다. 결 론 : 허파혈관주위세포를 일차 배양함으로써 허파꽈리혈관장벽의 연구를 위한 한 모델을 만들었고, 저산소 상태에서 증식의 억제와 유전자 발현의 변화를 살펴봄으로써 허파혈관손상의 기전을 규명하는데 향후 도움이 될 것으로 보인다.