대장암 세포에서 Mad1이 저산소 자극으로 유도되는 Doxorubicin 내성에 미치는 영향

학위논문 (석사)-- 서울대학교 대학원 : 의과학과, 2012. 2. 박종완.Cancer cells acquire resistance to chemotherapy under hypoxia. Hypoxia-inducible factors (HIFs) play major roles in altered oxygen milieus, and are known to associate with the hypoxic acquisition of drug resistance. Yet, it is uncertain which molecules mediate such the resistance. We profiled gene expression in colon cancer cells and found the hypoxic induction of MAX dimerization protein 1 (Mad1), which antagonizes the oncogenic action of MYC by competing for MAX binding. In the present study, we examined if Mad1 contributes to the hypoxia-induced chemoresistance in colon cancer cells. The Mad1 expression was confirmed by RT-PCR and western blotting. The hypoxic induction of Mad1 was attenuated by HIF-1α siRNAs, but less by HIF-2α siRNAs. A hypoxia response element was identified in the proximal region of the Mad1 promoter and its transcriptional activity was dependent on HIF-1α. To investigate how hypoxic cells acquire drug resistance, we treated doxorubicin to HCT116 cells under normoxic or hypoxic conditions, and found that doxorubicin -treated cells in hypoxia survived more than those in normoxia. It is well known that doxorubicin stimulates the generation of mitochondrial ROS, and thereby kills cancer cells. The doxorubicin-induced ROS production was significantly reduced under hypoxia, which was reversed by Mad1 knock-down. Also, the Mad1 knock-down reactivated the caspase-9/caspase-3/PARP apoptotic pathway in hypoxia. Mad1 inhibits the Myc-driven mitochondrial biogenesis, which was presented as mitochondrial enlargement on electron micrograph. When Mad1 was knocked-down, mitochondria was not enlarged even in hypoxia, suggesting that Mad1 determines the hypoxic suppression of mitochondrial activity. Summarizing, hypoxia-induced Mad1 lowers the doxorubicin production of mitochondrial ROS, and subsequently contributes to the tumor resistance to doxorubicin. Therefore, Mad1 could be a potential target for sensitizing cancer cells to redox-cycling drugs like doxorubicin.암세포는 저산소 환경에서 항암제에 대한 저항성을 획득한다. 저산소 유도 인자(HIFs)는 저산소 환경에서 세포의 적응과 생존에 주요한 역할을 한다고 알려져 있으며, 저산소와 연관된 항암제의 내성의 획득에도 관련이 있다고 여겨진다. 이에 저산소 환경에서 발현이 증가하는 유전자들을 대장암 세포주를 이용하여 확인하였고, MAX dimerizing protein 1 (Mad1)이라고 불리는 Myc 길항체가 저산소 조건에서 유도됨을 발견하였다. 다음으로 Mad1의 발현이 대장암 세포에서 저산소에 의해 유도되는 항암제 내성에 기여하는 지를 알아보고자 하였다. Mad1의 발현은 RT-PCR과 Western blotting을 통해 확인하였다. 저산소 조건에서 증가된 Mad1의 증가는 HIF-1α siRNA에 의해 감소하였고, HIF-2α siRNA에 의해서는 HIF-1α siRNA보다 감소 효과가 적었다. Mad1의 promoter에 저산소 반응 요소(hypoxia response element, HRE)가 존재함을 확인하였고, 이 HRE는 HIF-1α에 의해 활성화되었다. 저산소 암세포가 어떻게 항암제 내성을 얻게 되는 지 알아보기 위해, HCT116 대장암 세포에 doxorubicin을 처치하였을 때, 정상 산소 환경보다 저산소 환경에서 생존력이 우수함을 확인하였다. 또한 doxorubicin에 의한 ROS의 생산은 저산소에 의해 유의미하게 감소하였으며, Mad1을 감소시켰을 때 다시 이러한 감소가 회복되었다. 특히 Mad1의 억제는 저산소에서 caspase-9/ caspase-3/PARP로 이어지는 세포사멸과정 (apoptotic pathway)을 재활성화시켰다. Mad1이 Myc에 의해 촉진되는 미토콘드리아 생합성을 억제시키고 있음을 전자현미경에서 미토콘드리아의 크기 증가를 통해 확인하였다. Mad1의 발현을 감소시키는 경우 저산소 환경에서조차 미토콘드리아 크기가 증가하지 않았고, 이는 저산소에 의한 미토콘드리아 활성의 감소가 Mad1에 의해 결정됨을 시사한다. 이상의 결과를 정리하면, 저산소에 유도되는 Mad1은 doxorubicin에 의한 미토콘드리아 ROS를 낮추고, 결국 doxorubicin에 대한 암세포의 내성을 유도한다. 따라서 Mad1은 doxorubicin과 같이 산화-환원 순환을 이용한 항암제에 대한 저산소 내성을 극복할 수 있는 좋은 치료 타겟이 될 수 있다.Maste

Urine 5-Eicosatetraenoic Acids as Diagnostic Markers for Obstructive Sleep Apnea

Early detection of obstructive sleep apnea (OSA) is needed to reduce cardiovascular sequelae and mortality. Full-night polysomnography has been used for diagnosing OSA, but it is too expensive and inconvenient for patients to handle. Metabolome-wide analyses were performed to find and validate surrogate markers for OSA. We further investigated the mechanism underlying hypoxic induction of the markers in human cells and mice. Arachidonic acid derivatives 5-HETE and 5-oxoETE were detected in urine samples. The levels (mean ± SD, ng per mg creatinine) of 5-HETE and 5-oxoETE were 56.4 ± 26.2 and 46.9 ± 18.4 in OSA patients, respectively, which were significantly higher than those in controls (22.5 ± 4.6 and 18.7 ± 3.6). Both levels correlated with the apnea-hypopnea index and the lowest oxygen saturation on polysomnography. After the treatment with the continuous positive airway pressure, the metabolite levels were significantly reduced compared with those before the treatment. In human mononuclear cells subjected to intermittent hypoxia, 5-HETE and 5-oxoETE productions were induced by hypoxia-inducible factor 1 and glutathione peroxidase. When mice were exposed to intermittent hypoxia, 5-HETE and 5-oxoETE were excreted more in urine. They were identified and verified as new OSA markers reflecting hypoxic stress. The OSA markers could be used for OSA diagnosis and therapeutic evaluation

Epigenetic priming targets tumor heterogeneity to shift transcriptomic phenotype of pancreatic ductal adenocarcinoma towards a Vitamin D susceptible state

Abstract As a highly heterogeneous tumor, pancreatic ductal adenocarcinoma (PDAC) exhibits non-uniform responses to therapies across subtypes. Overcoming therapeutic resistance stemming from this heterogeneity remains a significant challenge. Here, we report that Vitamin D-resistant PDAC cells hijacked Vitamin D signaling to promote tumor progression, whereas epigenetic priming with glyceryl triacetate (GTA) and 5-Aza-2′-deoxycytidine (5-Aza) overcame Vitamin D resistance and shifted the transcriptomic phenotype of PDAC toward a Vitamin D-susceptible state. Increasing overall H3K27 acetylation with GTA and reducing overall DNA methylation with 5-Aza not only elevated the Vitamin D receptor (VDR) expression but also reprogrammed the Vitamin D-responsive genes. Consequently, Vitamin D inhibited cell viability and migration in the epigenetically primed PDAC cells by activating genes involved in apoptosis as well as genes involved in negative regulation of cell proliferation and migration, while the opposite effect of Vitamin D was observed in unprimed cells. Studies in genetically engineered mouse PDAC cells further validated the effects of epigenetic priming for enhancing the anti-tumor activity of Vitamin D. Using gain- and loss-of-function experiments, we further demonstrated that VDR expression was necessary but not sufficient for activating the favorable transcriptomic phenotype in respond to Vitamin D treatment in PDAC, highlighting that both the VDR and Vitamin D-responsive genes were prerequisites for Vitamin D response. These data reveal a previously undefined mechanism in which epigenetic state orchestrates the expression of both VDR and Vitamin D-responsive genes and determines the therapeutic response to Vitamin D in PDAC