[Effects of silencing H-ras gene by RNA interference on cetuximab-sensitivity of cetuximab-resistant human nasopharyngeal carcinoma cells].
OBJECTIVE
To explore the changes of the sensitivity of cetuximab-resistant human nasopharyngeal carcinoma (hNPC) cells 5-8F/Erbitux to cetuximab by silencing H-ras gene with RNA interference (RNAi).


METHODS
The 5-8F/Erbitux cells were induced by stepwise exposure to increasing doses of cetuximab. Western blot was conducted to detect the protein levels of H-ras and K-ras. Real-time PCR was employed to detect the expression of H-ras and K-ras. H-ras-shRNA plasmids (shRNA vector carrying the H-ras gene) were constructed and transferred into 5-8F/Erbitux cells. The gene and protein expression levels of H-ras and the changes of the sensitivity of 5-8F/Erbitux cells to cetuximab after transfection were measured, respectively.


RESULTS
After treatment with cetuximab for 3 and 5 days, the resistance index (RI) of the 5-8F/Erbitux cells was 1.2 and 1.1, and the protein levels of H-ras and K-ras in 5-8F/Erbitux cells were 0.798 +/- 0.019 and 0.190 +/- 0.011, respectively, significantly higher than that in the 5-8F cells (P<0.001). The gene expressions of H-ras and K-ras in 5-8F/Erbitux cells were 1.260 +/- 0.114 and 0.850 +/- 0.006, respectively. Compared with 5-8F cells, the former was higher (P = 0.016) and the latter was lower (P = 0.000). After transfection with H-ras-shRNA plasmid, the 5-8F/Erbitux cells showed reduced levels of H-ras gene and protein, and the cell apoptosis and inhibition rates increased significantly (P<0.001).


CONCLUSIONS
H-ras siRNA can reverse cetuximab-resistance of 5-8F/Erbitux cells through down-regulation of H-ras gene expression, indicating that the generation of cetuximab-resistance in 5-8F/Erbitux cells is associated with amplification and overexpression of the H-ras gene.
Cancer Center, Nanfang Hospital, The Southern Medical University, Guangzhou 510515, China.
Qiang
Q
Zuo
Rong-Cheng
RC
Luo
D000911:Antibodies, Monoclonal / Q000494:pharmacology*; D061067:Antibodies, Monoclonal, Humanized; D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis; D002277:Carcinoma; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D000068818:Cetuximab; D019008:Drug Resistance, Neoplasm; D015972:Gene Expression Regulation, Neoplastic; D011905:Genes, ras; D006801:Humans; D000077274:Nasopharyngeal Carcinoma; D009303:Nasopharyngeal Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D010957:Plasmids; D016283:Proto-Oncogene Proteins p21(ras) / Q000378:metabolism*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics; D014162:Transfection
D000911:Antibodies, Monoclonal; D061067:Antibodies, Monoclonal, Humanized; D000970:Antineoplastic Agents; D034741:RNA, Small Interfering; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras); D000068818:Cetuximab