Demethylation of HIN-1 reverses paclitaxel-resistance of ovarian clear cell carcinoma through the AKT-mTOR signaling pathway.
BACKGROUND
Methylation of HIN-1 is associated with poor outcomes in patients with ovarian clear cell carcinoma (OCCC), which is regarded to be an aggressive, chemo-resistant histological subtype. This study aimed to evaluate whether 5-aza-2-deoxycytidine (5-aza-2-dC) can reverse methylation of the HIN-1 gene to restore chemo-sensitivity of OCCC and the possible mechanism.


METHODS
In vitro flow cytometric analysis and evaluation of caspase-3/7 activity of paclitaxel-sensitive and resistant OCCC cell lines were performed. Methylation status and expression changes of HIN-1 in the OCCC cell lines treated with 5-aza-2-dC were evaluated, and immunohistochemical staining of HIN-1 in OCCC tissues was performed. In vivo tumor growth with or without 5-aza-2-dC treatment was analyzed, and Western blotting of AKT-mTOR signaling-related molecules was performed.


RESULTS
G2-M phase arrest was absent in paclitaxel-resistant OCCC cells after treatment with the cytotoxic drug. The caspase activities of the chemo-resistant OCCC cells were lower than those of the chemo-sensitive OCCC cells when treated with paclitaxel. Methylation of HIN-1 was noted in paclitaxel-resistant OCCC cell lines and cancerous tissues. 5-aza-2-dC reversed the methylation of HIN-1, re-activated the expression of HIN-1, and then suppressed the in vivo tumor growth of paclitaxel-resistant OCCC cells. Immunoblotting revealed that phospho-AKT473 and phospho-mTOR were significantly increased in HIN-1-methylated paclitaxel-resistant OCCC cell lines. However, the expressions of phospho-AKT at Ser473 and Thr308 and phospho-mTOR decreased in the OCCC cells with a high expression of HIN-1.


CONCLUSIONS
Demethylating agents can restore the HIN-1 expression in paclitaxel-resistant OCCC cells through the HIN-1-AKT-mTOR signaling pathway to inhibit tumor growth.
Gynecologic Cancer Center, Department of Obstetrics and Gynecology, Cathay General Hospital, Taipei, Taiwan. cmho@cgh.org.tw.|School of Medicine, Fu Jen Catholic University, Hsinchuang, New Taipei City, Taiwan. cmho@cgh.org.tw.|School of Medicine, Taipei Medical University, Taipei, Taiwan. cmho@cgh.org.tw.
Chih-Ming
CM
Ho
Department of Medical Research, Cathay General Hospital, Sijhih, New Taipei, Taiwan. aaronhuang@cgh.org.tw.|Department of Biochemistry, National Defense Medical Center, Taipei, Taiwan. aaronhuang@cgh.org.tw.
Chi-Jung
CJ
Huang
Department of Pathology, Cathay General Hospital, Taipei, Taiwan. drshhuang@gmail.com.
Shih-Hung
SH
Huang
Graduate Institute of Medical Sciences, School of Medicine, Taipei Medical University, Taipei, Taiwan. cmbsfc21@tmu.edu.tw.
Shwu-Fen
SF
Chang
Department of Obstetrics and Gynecology, National Taiwan, University Hospital, Taipei, Taiwan. wenfangcheng@yahoo.com.|Graduate Institute of Oncology, National Taiwan, University Hospital, Taipei, Taiwan. wenfangcheng@yahoo.com.|Graduate Institute of Clinical Medicine, College of Medicine, National Taiwan, University, Taipei, Taiwan. wenfangcheng@yahoo.com.
Wen-Fang
WF
Cheng
D018262:Adenocarcinoma, Clear Cell / Q000188:drug therapy / Q000378:metabolism*; D000328:Adult; D000368:Aged; D000818:Animals; D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology / Q000627:therapeutic use; D045744:Cell Line, Tumor; D016207:Cytokines / Q000096:biosynthesis*; D019175:DNA Methylation / Q000187:drug effects / Q000502:physiology; D019008:Drug Resistance, Neoplasm / Q000187:drug effects / Q000502:physiology; D005260:Female; D006801:Humans; D051379:Mice; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D008875:Middle Aged; D010051:Ovarian Neoplasms / Q000188:drug therapy / Q000378:metabolism*; D017239:Paclitaxel / Q000494:pharmacology / Q000627:therapeutic use*; D051057:Proto-Oncogene Proteins c-akt / Q000096:biosynthesis*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D058570:TOR Serine-Threonine Kinases / Q000096:biosynthesis*; D025521:Tumor Suppressor Proteins / Q000096:biosynthesis*
D000972:Antineoplastic Agents, Phytogenic; D016207:Cytokines; C434732:SCGB3A1 protein, human; D025521:Tumor Suppressor Proteins; C546842:MTOR protein, human; D051057:Proto-Oncogene Proteins c-akt; D058570:TOR Serine-Threonine Kinases; D017239:Paclitaxel