Class III beta-tubulin mediates sensitivity to chemotherapeutic drugs in non small cell lung cancer.
First line therapy for non-small cell lung carcinoma (NSCLC) commonly includes combination therapy with a tubulin-binding agent (TBA) and a DNA-damaging agent. TBAs suppress microtubule dynamics by binding to the beta-tubulin subunit of alpha/beta-tubulin, inducing mitotic arrest and apoptosis. Up-regulation of class III beta-tubulin (betaIII-tubulin) has been implicated in clinical resistance in NSCLC, ovarian and breast tumors treated in combination with a TBA and DNA-damaging agent. To investigate the functional significance of betaIII-tubulin in resistance to both these classes of agents, small interfering RNA (siRNA) was used to silence the expression of this isotype in two NSCLC cell lines, NCI-H460 and Calu-6. Reverse transcription-PCR and immunoblotting showed that betaIII-siRNA potently inhibited the expression of betaIII-tubulin, without affecting the expression of other major beta-tubulin isotypes. Clonogenic assays showed that betaIII-siRNA cells were significantly more sensitive to TBAs, paclitaxel, vincristine, and vinorelbine, and for the first time, DNA-damaging agents, cisplatin, doxorubicin, and etoposide compared with controls. Cell cycle analysis of H460 betaIII-siRNA cells showed reduced accumulation at the G(2)-M boundary and an increase in the sub-G(1) population in response to TBA treatment compared with control cells. Importantly, betaIII-siRNA cells displayed a significant dose-dependent increase in Annexin V staining when treated with either paclitaxel or cisplatin, compared with controls. These findings have revealed a novel role for betaIII-tubulin in mediating response to both TBA and DNA-damaging agent therapy and may have important implications for improving the targeting and treatment of drug-refractory NSCLC.
Gan
Pei Pei
PP
Children's Cancer Institute Australia for Medical Research, Randwick, New South Wales, Australia.
Pasquier
Eddy
E
Kavallaris
Maria
M
D002289:Carcinoma, Non-Small-Cell Lung / Q000188:drug therapy* / Q000235:genetics / Q000378:metabolism*; D002455:Cell Division / Q000187:drug effects; D045744:Cell Line, Tumor; D002945:Cisplatin / Q000494:pharmacology; D004249:DNA Damage; D016195:G2 Phase / Q000187:drug effects; D020868:Gene Silencing; D006801:Humans; D008175:Lung Neoplasms / Q000188:drug therapy* / Q000235:genetics / Q000378:metabolism; D017239:Paclitaxel / Q000494:pharmacology; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics; D014162:Transfection; D014404:Tubulin / Q000096:biosynthesis* / Q000172:deficiency / Q000235:genetics; D014750:Vincristine / Q000494:pharmacology
D012333:RNA, Messenger; D034741:RNA, Small Interfering; C498585:TUBB3 protein, human; D014404:Tubulin; D014750:Vincristine; D017239:Paclitaxel; D002945:Cisplatin