Coupled down-regulation of mTOR and telomerase activity during fluorouracil-induced apoptosis of hepatocarcinoma cells.
BACKGROUND
Hepatocellular carcinoma (HCC) is the most invasive and frequently diagnosed malignancy and the second leading cause of cancer death in many regions of Asia. The PI3K/Akt/mTOR signal pathway is involved in multiple cellular functions including proliferation, differentiation, tumorigenesis, and apoptosis. Up-regulation of telomerase activity is thought to be a critical step leading to cell transformation.


METHODS
This study investigated changes in mTOR pathway and telomerase activity in hepatocarcinoma cell line SMMC-7721 treated with chemotherapeutic agent 5-fluorouracil (5-Fu). We detected apoptosis of hepatocarcinoma cells by TUNEL assay. Telomerase activity, hTERT transcription level and p- p70 S6k was demonstrated by the telomeric repeat amplification protocol and silver staining assay, Dual-Luciferase Reporter Assay and Western blot analysis respectively.


RESULTS
Treating SMMC-7721 cells with 5-Fu leads to apoptosis of the cells, and reduction in telomerase activity, as well as a dramatic reduction in the activated form of p70 S6 kinase, a mTOR substrate. The 5-Fu treatment nearly abolishes transcription of hTERT (the major component of telomerase) mRNA. Treating SMMC-7721 cells with Rapamycin, a specific mTOR inhibitor, significantly reduce hTERT protein level but did not affect hTERT transcription. 5-Fu and rapamycin were synergistic in regards to down-regulation of telomerase activity in hepatocarcinoma cells.


CONCLUSIONS
These results suggest that chemotherapeutic agent 5-Fu may down-regulate telomerase activity at both transcriptional level and PI3K/Akt/mTOR pathway-dependent post-transcriptional level to facilitate hepatocellular carcinoma cell apoptosis.
Bu
Xinxin
X
Tumor Immunology and Gene Therapy Center, Eastern Hepatobiliary Hospital, Second Military Medical Universisty, 225 Changhai Road, Shanghai 200438, China. jesscica123@126.com
Jia
Fengqi
F
Wang
Weifeng
W
Guo
Xianling
X
Wu
Mengchao
M
Wei
Lixin
L
D017209:Apoptosis; D006528:Carcinoma, Hepatocellular / Q000378:metabolism* / Q000473:pathology*; D045744:Cell Line, Tumor; D015536:Down-Regulation; D005472:Fluorouracil / Q000494:pharmacology*; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D020287:In Situ Nick-End Labeling; D008113:Liver Neoplasms / Q000378:metabolism* / Q000473:pathology*; D011494:Protein Kinases / Q000096:biosynthesis*; D015398:Signal Transduction; D020123:Sirolimus / Q000494:pharmacology; D058570:TOR Serine-Threonine Kinases; D019098:Telomerase / Q000096:biosynthesis* / Q000502:physiology; D014158:Transcription, Genetic
D011494:Protein Kinases; C546842:MTOR protein, human; D058570:TOR Serine-Threonine Kinases; D019098:Telomerase; D005472:Fluorouracil; D020123:Sirolimus