[Study on apoptosis of oral squamous carcinoma cell line KB and KBv200 induced by survivin RNAi].
OBJECTIVE
Survivin, a member of IAPs (inhibition of apoptosis proteins) family, has been revealed by many studies to inhibit the activation of caspase-3, caspase-7 and caspase-9, and thus increase the anti-apoptotic effect of cancer cells, which subsequently makes the cells to become resistant to chemotherapy in cancer treatment. Survivin is expressed in human oral squamous carcinoma cell line KB and its multiple-drug resistant (MDR) cell line KBv200. This study was to compare the apoptosis induced by silencing survivin gene between these two squamous cell lines.


METHODS
Mu6/survivin plasmid which expressed shRNA against survivin was transfected into both KB and KBv200 cells to inhibit the expression of survivin gene. Survivin mRNA and protein expression was detected by semi-quantitative RT-PCR and flow cytometry (FCM), respectively. Cell apoptotic rate was measured by flow cytometry after PI staining, and apoptotic morphology of cells was observed under a fluorescent microscope after Hoechst33258 staining. Activation of caspase-3 was measured by colorimetric assay. MTT was used to evaluate the growth inhibition of tumor cells.


RESULTS
Expression of survivin, which was higher in KBv200 at mRNA and protein level, was detected in both KB and KBv200. After 48 h transfection with mu6/survivin plasmid, the expression level of survivin mRNA in KB and KBv200 was reduced by (61.98+/-8.12)% and (59.29+/-6.32)%; and that of survivin protein was decreased by (44.25+/-5.26)% and (38.76+/-4.31)% compared with the control group. Moreover, KB and KBv200 cells exhibited typical apoptotic cell morphology by Hoechst 33258 staining. Apoptosis, which was higher in KB cells, was increased significantly at 24, 48, 72 h after the transfection of mu6/surviving in KB and KBv200 cells, and reached the peak at 48 h. Similarly, activation of caspase-3 was elevated significantly, and reached the peak at 48 h. MTT assay revealed the inhibitory rates were (33.04+/-2.16)%, (56.25+/-3.32)% and (58.26+/-5.14)% in KB cells, and (35.23+/-3.42)%, (44.90+/-4.12)% and (44.19+/-4.37)% in KBv200 cells at 24, 48, 72 h after the transfection, respectively. Growth rate was lower in KB cells at 48, 72 h after the transfection compared with KBv200 cells.


CONCLUSIONS
RNA interference could silence the expression of survivin, which significantly induces apoptosis not only in KB cells, but also in drug resistant KBv200 cells.
Yan
Lin
L
State Key Laboratory of Oncology in South China, Guangzhou, Guangdong, 510060, P. R. China.
Chen
Wei-Qiang
WQ
Liang
Yong-Ju
YJ
Dai
Chun-Ling
CL
Wang
Xiao-Hong
XH
Shi
Zhi
Z
Chen
Li-Ming
LM
Fu
Li-Wu
LW
D000970:Antineoplastic Agents / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D053148:Caspase 3 / Q000378:metabolism; D049109:Cell Proliferation / Q000187:drug effects; D018432:Drug Resistance, Multiple; D019008:Drug Resistance, Neoplasm; D005434:Flow Cytometry; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D051034:Inhibitor of Apoptosis Proteins; D007624:KB Cells; D008869:Microtubule-Associated Proteins / Q000096:biosynthesis* / Q000235:genetics; D010957:Plasmids; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism; D000077022:Survivin; D014162:Transfection
D000970:Antineoplastic Agents; C107489:BIRC5 protein, human; D051034:Inhibitor of Apoptosis Proteins; D008869:Microtubule-Associated Proteins; D012333:RNA, Messenger; D000077022:Survivin; D053148:Caspase 3