• Asian Pacific Journal of Cancer Prevention
• /
• 제15권12호
• /
• pp.5043-5047
• /
• 2014

Nowadays increasing effectiveness in cancer therapy and investigation of formation of new strategies that enhance antiproliferative activity against target organs has become a subject of interest. Although the molecular mechanisms of apoptosis can not be fully explained, it is known that cell suicide program existing in their memory genetically is activated by pathophysiological conditions and events such as oxidative stress. Low pressure (hypobaric) conditions that create hypoxia promote apoptosis by inhibiting cell cycling. In this study, determination of the effects of fractional hypobaric applications at different times on HeLa cells at cellular and molecular levels were targeted. Experiments were carried out under hypobaric conditions (35.2 kPa) in a specially designed hypobaric cabin including 2% $O_2$ and 98% N. Application of fractional hypobaric conditions was repeated two times for 3 hours with an interval of 24 hours. At the end of the implementation period cells were allowed to incubate for 24 hours for activation of repair mechanisms. Cell kinetic parameters such as growth rate (MTT) and apoptotic index were used in determination of the effect of hypobaric conditions on HeLa cells. Also in our study expression levels of the Bcl-2 gene family that have regulatory roles in apoptosis were determined by the RT-PCR technique to evaluate molecular mechanisms. The results showed that antiproliferative effect of hypobaric conditions on HeLa cells started three hours from the time of application and increased depending on the period of exposure. While there was a significant decrease in growth rate values, there was a significant increase in apoptotic index values (p<0.01). Also molecular studies showed that hypobaric conditions caused a significant increase in expression level of proapoptotic gene Bax and significant decrease in antiapoptotic Bfl-1. Consequently fractional application of hypobaric conditions on HeLa cell cultures increased both antiproliferative and apoptotic effects and these effects were triggered by the Bax gene.

• Journal of Radiation Protection and Research
• /
• 제43권2호
• /
• pp.66-74
• /
• 2018

Background: Tumor response to anticancer therapies can much be influenced by microenvironmental factors. In this study, we determined the effect of these microenvironmental factors on DNA methylation using A549 human lung adenocarcinoma cell line. Materials and Methods: We subjected A549 cells to various conditions mimicking tumor microenvironment including hypoxia, acidosis (sodium lactate), oxidative stress ($H_2O_2$), bystander effect (supernatant from doxorubicin (Dox)-treated or irradiated cells), and immune cell infiltration (supernatant from THP-1 or Jurkat T cells). Genomic DNA was isolated from these cells and analyzed for DNA methylation. Clonogenic cell survival, gene expression, and metabolism were analyzed in cells treated with some of these conditions. Results and Discussion: We found that DNA methylation level was significantly decreased in A549 cells treated with conditioned media from Dox-treated cells or Jurkat T cells, or sodium lactate, indicating an active transcription. To determine whether the decreased DNA methylation affects radiation sensitivity, we exposed cells to these conditions followed by 6 Gy irradiation and found that cell survival was significantly increased by sodium lactate while it was decreased by conditioned media from Dox-treated cells. We further observed that cells treated with conditioned media from Dox-treated cells exhibited significant changes in expression of genes including BAX and FAS (involved in apoptosis), NADPH dehydrogenase (mitochondria), EGFR (cellular survival) and RAD51 (DNA damage repair) while sodium lactate increased cellular metabolism rather than changing the gene expression. Conclusion: Our results suggest that various tumor microenvironmental factors can differentially influence DNA methylation and hence radiosensitivity and gene expression in A549 cancer cells.

• Clinical and Experimental Pediatrics
• /
• 제50권7호
• /
• pp.686-693
• /
• 2007

목 적 : Mycophenolate mofetil (MMF)의 활성 대사산물인 (MPA는 IMPDH의 잠재적인 반응 억제제로써 새로운 면역치료제로 사용되고 있다. 이러한 MPA는 신경계에서 흥분독성 손상 후 뇌세포를 보호하고, 미세아교세포에서는 세포사멸사(apoptosis)를 유도하지만, 저산소성 허혈성 뇌질환에서 MPA의 효과는 아직 알려지지 않아, 본 연구에서 Rice-Vannucci 모델을 이용한 신생 백서의 저산소성 허혈성 뇌 손상과 저산소 상태의 태아 백서 뇌세포 배양에서 MPA의 뇌보호 효과를 알아보고자 실험하였다. 방 법 : 생후 7일된 백서의 좌측 총 경동맥을 결찰한 후 저산소 (8% $O_2$) 상태에서 2시간 노출하여, 저산소성 허혈성 뇌 손상을 유발하고 뇌 손상 전후에 MPA(10 mg/kg)를 투여하여 대조군과 비교하였다. 또한, 재태기간 18일된 태아 백서의 대뇌피질 세포를 배양하여 1% $O_2$ 배양기에서 저산소 상태로 세포손상을 유도하여 저산소군, 손상 전후 MPA 투여군($10{\mu}g/mL$)으로 나누어 정상산소군과 비교하였다. 세포사멸사와의 관련을 알아보기 위해서 Bcl-2, Bax, caspase-3 항체로 western blotting하였고 Bcl-2, Bax, caspase-3 primer를 이용하여 real-time PCR을 하였다. 결 과 : 형태학적으로 H&E 염색상 MPA를 투여한 군에서 뇌 보호 효과를 보였다. Western blotting과 real-time PCR을 이용한 저산소성 허혈성 뇌손상 동물 모델뿐만 아니라 저산소 상태로 태아 백서 뇌세포 배양 실험에서도 MPA 투여한 경우 caspase-3의 발현과 Bax/Bcl-2의 비율이 감소함을 보였다. 결 론 : 본 연구에서 MPA가 anti-apoptosis 작용을 통하여 주산기 저산소성 허혈성 뇌 손상에 뇌보호 역할을 하는 것을 알 수 있었고 향후 신생아 저산소성 허혈성 뇌병증의 치료에 임상적 적용이 가능하리라 생각된다.

• Clinical and Experimental Pediatrics
• /
• 제49권3호
• /
• pp.317-325
• /
• 2006

목 적 : 주산기 저산소성 허혈성 뇌손상의 병태 생리에서 nitric oxide(NO)가 급성 저산소성 허혈(hypoxia-ischemia, HI) 후 재산소-재관류기(reoxygenation-reperfusion, RR)에 대뇌의 혈역학 및 에너지 대사에 미치는 영향을 규명하기 위하여, NO 합성 억제제인 NG-monomethyl-L-arginine(L-NMMA)와 NO 합성 촉진제인 L-arginine(L-Arg) 투여를 통하여 뇌신경 세포에 어떠한 영향을 주는지 알아보고자 하였다. 방 법 : 생후 3일 이내의 신생자돈 28마리를 대상으로 무작위로 나누어, Sham 처치만 받은 정상 대조군(n=9), HI와 RR만 유발한 실험 대조군(n=7), HI 이후 RR 직전에 L-NMMA 투여군(n=6)과 L-arginine 투여군(n=6) 등 4군으로 구분하였다. 실험은 ether을 흡입 시킨 후 thiopental을 정주하고, 기관 삽관 후 인공호흡기 등의 처지를 끝낸 후, HI를 유발하기 위하여 실험군에서 수술 겸자로 양측 경동맥을 폐쇄한 후 8% 산소로 30분간 흡입하였고, RR을 시행하기 위하여 경동맥 폐색을 풀고 흡입 산소농도를 60%로 올려 1시간까지 투여하면서 관찰하였다. 생리적 변수로 혈압과 동맥혈 가스 소견을 관찰하였고, 뇌의 혈역학적 변화와 에너지 상태는 near infrared spectroscopy(NIRS)를 이용하여 대뇌의 산화 헤모글로빈($HbO_2$), 환원헤모글로빈(Hb), 환산 헤모글로빈(HbD), 싸이토크롬 $aa_3$(Cyt $aa_3$) 등을 지속적으로 관찰하여 비교하였다. 또한 실험 종료 시 얻은 뇌조직에서 $Na^+$, $K^+$-ATPase의 활성도 및 지질 대사산물인 conjugated dienes, 고에너지 인분자인 ATP(adeninetriphosphate)와 phosphocreatine(PCr)을 비교하였다. 결 과 : 생리적 변수의 변화에서는 실험군 모두에서 정상 대조군에 비하여 혈압, 동맥혈 산소 분압, pH, base excess 등이 유의하게 감소하였고(P<0.05), 젖산은 유의하게 증가하였다(P<0.05). L-NMMA와 L-Arg군에서 실험 대조군과 유의한 차이는 없었다. 실험군에서 RR 1시간 후 pH를 제외한 혈압, 동맥혈 산소 분압, base excess 등의 이상소견은 모두 기저치로 회복되었고, 실험군간에 유의한 차이가 없었다. NIRS 소견에서 $HbO_2$와 HbD는 HI 동안 정상 대조군에 비하여 실험군 모두에서 유의하게 감소하였으나(P<0.05), RR 직후 기저치로 회복되었으며, $HbO_2$는 RR 40분 이후 정상 대조군에 비해 유의하게 감소하였다(P<0.05). Hb은 정상 대조군을 제외한 모든 실험군에서 HI 동안 유의하게 증가하였다가(P<0.05), RR 직후 기저치로 회복되었다. 산화 Cyt $aa_3$는 HI 동안 실험군 모두에서 감소하는 경향을 보였고, RR 이후 다시 증가하였다. 정상 대조군과 각 실험군간에 유의한 차이는 없었다. 뇌의 $Na^+$, $K^+$-ATPase 활성도와 conjugated dienes은 실험군 모두에서 정상 대조군(제1군)에 비하여 유의하게 감소하였다(P<0.05). 뇌의 ATP, phosphocreatine은 실험군 모두에서 정상 대조군과 차이가 없었고, 또한 실험군간에도 유의한 차이가 없었다. 결 론 : 신생 자돈에서 급성 저산소성 허혈 이후 재산소-재관류기 동안 NO 합성 억제제인 L-NMMA나 NO 생성 촉진제인 L-arginine이 뇌 혈역학이나 뇌의 에너지 대사에는 특별한 변화를 일으키지 않았다. 따라서 급성 저산소성 허혈성 뇌손상에서 재산소화 재관류기 초기에는 NO가 뇌손상의 주요한 기전으로 작용하지 않을 것으로 사료된다. 또한 뇌혈역학 및 생화학적 검사 결과 등에서 급성기에는 에너지 부전 상태가 주요한 세포손상 기전이 아니고, 이온 농도의 변화에 의한 뇌부종, 산소유리기에 의한 뇌세포 손상이 저산소성 허혈성 뇌손상의 급성기에 주로 작용하는 뇌세포 손상의 주요 기전임을 시사한다. 따라서 NO 생성 억제제 혹은 생성 전구물질인 L-Arg은 뇌신경 세포 보호 효과를 보이지 않아 급성 주산기 가사의 치료제로서 제한이 됨을 알 수 있었다. 그러나 좀 더 명확한 효과를 보기 위하여 선택적 억제제의 사용, 제제의 용량 및 투여시기, 손상 후 좀더 긴 시간 이후의 변화에 대한 연구가 필요하다.

• Asian Pacific Journal of Cancer Prevention
• /
• 제15권11호
• /
• pp.4637-4642
• /
• 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 Korean Neurosurgical Society
• /
• 제29권8호
• /
• pp.1008-1018
• /
• 2000

Objective : Glutamate induced excitotoxicity is one of the leading causes of cell death under pathologic condition. However, there is controversy whether excitotoxicity may also participate in the neuronal death under low intensity insult such as simple hypoxia or hypoglycemia. To investigate the role of NMDA receptor in low intensity insult, we chose anoxia as the method of injury and used organotypically cultured hippocampal slice as the material of experiment. Materials & Methods : The hippocampal slices cultured for 2-3 weeks were exposed to 60 minutes of complete oxygen deprivation(anoxia). Neuronal death was assessed with Sytox stain. Corrected optical density of fluorescence in gray scale, used as cellular death indicator, was obtained from pictures taken at 24 and 48 hours following the insult. The well-known in vivo phenomenon of regional difference in susceptibility of hippocampal sub-fields to ischemic insult was reproduced in HOSC(hippocampal organotypic slice culture) by complete oxygen deprivation injury. Results : $CA_1$ was the most vulnerable to complete oxygen deprivation in hippocampus while $CA_3$ was resistant. Oxygen deprivation for 10 and 20 minutes with glucose(6.5g/l) present was insufficient to induce neuronal death in the cultured hippocampal slice. However, after 30 minutes exposure under anoxic condition, neuronal death was able to be detected in the center of $CA_1$ area. The intensity and area of fluorescence indicating cell death correlated with the duration of oxygen deprivation. NMDA receptor and non-NMDA receptor blocking with MK-801(30 & $60{\mu}M$) and CNQX($100{\mu}M$) did not provide cellular protection to HOSC against damage induced by oxygen deprivation, but increased intracellular calcium buffering capacity with BAPTA-AM($10{\mu}M$) was effective in preventing neuronal death (p=0.01, Student's t-test). Cycloheximide($1{\mu}g/ml$, $10{\mu}g/ml$) provided no protection to HOSC against insult of complete oxygen deprivation for 60 minutes and combined therapy of MK-801(30 & $60{\mu}M$) and cycloheximide(1 & $10{\mu}g/ml$) was also ineffective in preventing neuronal death. Conclusion : The results of this study show that the another mechanism not associated with glutamate receptor(NMDA & non NMDA) may play major role in cell death mechanisms induced by complete oxygen deprivation and increased intracellular calcium during anoxia may participate in the neuronal death mechanism of oxygen deprivation. Further investigation of the calcium entry channel activated during oxygen deprivation is necessary to understand the neuronal death of anoxia.

• 생명과학회지
• /
• 제19권7호
• /
• pp.905-916
• /
• 2009

본 연구는 저산소증에서 반하가 대뇌신경세포의 유전자 표현에 미치는 영향을 알아보기 위하여 배양한 $E_{18}$의 흰쥐 대뇌 신경세포를 반하로 처리하고, 저산소증을 유도한 후 microarray 기법으로 유전자 표현 변화를 조사하였다. Microarray 결과 tubb5, tgfa, ptpn11, n-ras, pdgfa 등 세포의 성장 분화에 관여하는 유전자들의 표현이 증가하였으며, 세포 자연사를 억제하는 mcl-1 유전자의 표현 또한 증가하였다. 한편 세포 자연사를 유도하는 tieg 유전자는 표현이 감소하였다(Fig. 3). 반하에 의하여 수많은 유전자의 표현이 변화되었고, 세포사를 촉진하는 유전자의 표현이 크게 증가되는 경우(예, alox12, faf1)도 있어 본 연구결과만으로 일반적인 결론을 유도하기는 어려웠다. 그러나 대략적으로 반하는 저산소증에서 주로 세포의 성장과 분화를 유지하고, 세포 자연사를 방지하는 유전자들의 표현을 증가시켜 신경 세포사를 보호하는 것으로 이해된다.

• Journal of Korean Neurosurgical Society
• /
• 제67권4호
• /
• pp.418-430
• /
• 2024

Objective : Isoflurane, a widely used common inhalational anesthetic agent, can induce brain toxicity. The challenge lies in protecting neurologically compromised patients from neurotoxic anesthetics. Choline alfoscerate (L-α-Glycerophosphorylcholine, α-GPC) is recognized for its neuroprotective properties against oxidative stress and inflammation, but its optimal therapeutic window and indications are still under investigation. This study explores the impact of α-GPC on human astrocytes, the most abundant cells in the brain that protect against oxidative stress, under isoflurane exposure. Methods : This study was designed to examine changes in factors related to isoflurane-induced toxicity following α-GPC administration. Primary human astrocytes were pretreated with varying doses of α-GPC (ranging from 0.1 to 10.0 µM) for 24 hours prior to 2.5% isoflurane exposure. In vitro analysis of cell morphology, water-soluble tetrazolium salt-1 assay, quantitative real-time polymerase chain reaction, proteome profiler array, and transcriptome sequencing were conducted. Results : A significant morphological damage to human astrocytes was observed in the group that had been pretreated with 10.0 mM of α-GPC and exposed to 2.5% isoflurane. A decrease in cell viability was identified in the group pretreated with 10.0 µM of α-GPC and exposed to 2.5% isoflurane compared to the group exposed only to 2.5% isoflurane. Quantitative real-time polymerase chain reaction revealed that mRNA expression of heme-oxygenase 1 and hypoxia-inducible factor-1α, which were reduced by isoflurane, was further suppressed by 10.0 µM α-GPC pretreatment. The proteome profiler array demonstrated that α-GPC pretreatment influenced a variety of factors associated with apoptosis induced by oxidative stress. Additionally, transcriptome sequencing identified pathways significantly related to changes in isoflurane-induced toxicity caused by α-GPC pretreatment. Conclusion : The findings suggest that α-GPC pretreatment could potentially enhance the vulnerability of primary human astrocytes to isoflurane-induced toxicity by diminishing the expression of antioxidant factors, potentially leading to amplified cell damage.

• Asian Pacific Journal of Cancer Prevention
• /
• 제14권6호
• /
• pp.3625-3630
• /
• 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.

• Molecular & Cellular Toxicology
• /
• 제4권2호
• /
• pp.144-149
• /
• 2008

Despite versatile activity (cancericidal, antimicrobial, hypoxia inducible factor (HIF) inhibition, immune deactivation of DNA bis-intercalation agent, echinomycin, its specific mechanism has been elusive. Of these novel mechanisms, we reported that using human colon cancer cells (HT-29), apoptotic machinery induced by echinomycin might be dependent of caspase-3 pathway. Despite a partial enlightenment of prototypic signal path triggered by echinomycin, the role of Bcl-2 in this signaling pathway is unclear. To address this issue, we explored whether or not echinomycin would overcome the anti-apoptotic impact of Bcl-2 in HT-29 cells by the controlled Bcl-2 overexpression. Prior to this proof, we confirmed that echinomycin induces mitochondrial depolarization, then triggering the mitochondrial pathway of apoptosis with an involvement of upstream cas-pases-3. Transiently transfection with inactive Bax-DNA failed to prevent echinomycin-induced apoptosis in HT-29 cells. To dissect the role of Bcl-2 in echinomycin-induced apoptosis, HT-29 cells were transiently transfected with Bcl-2 DNA for overexpression and then treated with echinomycin for 24h. Combined analyses of DNA fragmentation and flow cytometric analysis clearly verified that echinomycin-induced apoptosis was drastically attenuated by Bcl-2 overexpression, whereas a control vector rarely affected echinomycin-induced apoptosis. Collectively, these data verify that Bcl-2 regulates echinomycin-induced apoptosis in HT-29 cells. To my knowledge, this is the first evidence that of diverse, structured minor groove binders (MGB), the prototypic echinomycin might control the apoptotic signaling via Bcl-2-mitochondrial pathway.