[The killing effect and bystander effect of linamarase/linamarin suicide gene system on human hepatocellular carcinoma cell line HepG2 in vitro].
OBJECTIVE
To investigate the killing effect of linamarase/linamarin (lis/lin) system on hepatocellular carcinoma cell line HepG2 in vitro.


METHODS
A cDNA library was built from RNA of cassava by RT-PCR, then the linamarase gene was amplified from it by PCR and cloned into the eukaryotic expression vector plasmid pEGFP-N1, which made up the recombinant plasmid pEGFP-N1-lis. The human HCC cells HepG2 were transfected with the recombinant plasmid mediated by electroporation and screened by G418 to yield the positive clone which was termed HepG2/lis. The expression of lis was confirmed by fluorescent staining, RT-PCR and Western blot. The killing effect and bystander effect of linamarin with different concentrations on HepG2 was detected by MTT.


RESULTS
RT-PCR confirmed the expression of lis gene in HepG2 and Western blot analysis confirmed existence of lis-EGFP fusion protein in HepG2. Linamarin in low concentration had shown notable cytotoxic effect on HepG2/lis. When HepG2/lis cells were mixed with parental HepG2 cells at a ratio of 10:90 and cultivated in 500 mg/L lin medium, significant bystander effect was observed in vitro.


CONCLUSIONS
The linamarase/linamarin suicide gene system has strong killing effect and bystander effect on HCCs with the concentration of 500 mg/L lin.
Chen
Ming-hao
MH
Department of Hepatobilliary Surgery, Xijing Hospital, Fourth Military Medical University, Xi'an 710032, China. kuakee@fmmu.edu.cn
Ma
Jun
J
Li
Hai-min
HM
Li
Jun
J
Li
Ren
R
Zhang
Fu-qin
FQ
Dou
Ke-feng
KF
D015153:Blotting, Western; D024201:Bystander Effect / Q000502:physiology*; D006528:Carcinoma, Hepatocellular / Q000628:therapy*; D041722:Genes, Transgenic, Suicide / Q000235:genetics / Q000502:physiology*; D015316:Genetic Therapy / Q000379:methods; D056945:Hep G2 Cells; D006801:Humans; D008113:Liver Neoplasms / Q000628:therapy*; D009570:Nitriles / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D001617:beta-Glucosidase / Q000235:genetics / Q000502:physiology*
D009570:Nitriles; C021514:cyanogenic beta-glucosidase; D001617:beta-Glucosidase; C005091:linamarin