Determinants of differential doxorubicin sensitivity between SCLC and NSCLC.
Doxorubicin (DOX) transport activity of Ral-interacting protein (RLIP76) in non-small cell lung cancer (NSCLC) is approximately twice that of in small cell lung cancer (SCLC). Since protein-kinase-C (PKC)alpha mediated phosphorylation of RLIP76 causes doubling of the specific activity of RLIP76, and NSCLC cells are known to have greater PKCalpha activity, we examined the contribution of PKC mediated phosphorylation of RLIP76 towards intrinsic DOX-resistance in human NSCLC. Expression of a deletion mutant RLIP76(delPKCalpha-sites) followed by depletion of the wild-type RLIP76 using a siRNA targeted at one of the deleted regions resulted in generation of cells expressing only the mutant protein, which could not be phosphorylated by PKCalpha. DOX-transport activity of the mutant RLIP76 purified from NSCLC and SCLC was similar and comparable to that of RLIP76 purified from the wild-type SCLC. However, this activity was significantly lower than that of RLIP76 purified from the wild-type NSCLC. After siRNA mediated depletion of PKCalpha, DOX-transport activities of RLIP76 purified from SCLC and NSCLC were indistinguishable. Depletion of PKCalpha inhibited the growth of NSCLC more than SCLC cells (70+/-3% vs. 43+/-5%, respectively). PKCalpha-depletion lowered the IC(50) of NSCLC cell lines for DOX to the same level as that observed for SCLC. RLIP76(-/-) mouse embryonic fibroblasts (MEFs) were significantly more sensitive to DOX as compared with RLIP76(+/+) MEFs (IC(50) 25 vs. 125nM, respectively). However, PKCalpha-depletion did not affect DOX-cytotoxicity towards RLIP76(-/-) MEFs, as opposed to RLIP76(+/+) MEFs which were sensitized by 2.2-fold. These results demonstrate that RLIP76 is a primary determinant of DOX-resistance, and that PKCalpha mediated accumulation defect and DOX-resistance in NSCLC is primarily due to differential phosphorylation of RLIP76 in SCLC and NSCLC.
Singhal
Sharad S
SS
Department of Chemistry and Biochemistry, University of Texas at Arlington, 401 W, 3rd Street, CPB RM # 312, Arlington, TX 76019-0065, USA.
Wickramarachchi
Dilki
D
Singhal
Jyotsana
J
Yadav
Sushma
S
Awasthi
Yogesh C
YC
Awasthi
Sanjay
S
D018528:ATP-Binding Cassette Transporters / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D000903:Antibiotics, Antineoplastic / Q000494:pharmacology* / Q000627:therapeutic use; D001693:Biological Transport, Active / Q000187:drug effects / Q000235:genetics; D002289:Carcinoma, Non-Small-Cell Lung / Q000188:drug therapy / Q000235:genetics* / Q000378:metabolism; D018288:Carcinoma, Small Cell / Q000188:drug therapy / Q000235:genetics* / Q000378:metabolism; D045744:Cell Line, Tumor; D004317:Doxorubicin / Q000494:pharmacology* / Q000627:therapeutic use; D019008:Drug Resistance, Neoplasm / Q000187:drug effects / Q000235:genetics*; D004622:Embryo, Mammalian / Q000166:cytology / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects / Q000235:genetics; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D020690:GTPase-Activating Proteins / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008175:Lung Neoplasms / Q000188:drug therapy / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D010766:Phosphorylation / Q000187:drug effects; D051571:Protein Kinase C-alpha / Q000378:metabolism; D011499:Protein Processing, Post-Translational / Q000187:drug effects / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics / Q000494:pharmacology; D017384:Sequence Deletion / Q000235:genetics
D018528:ATP-Binding Cassette Transporters; D000903:Antibiotics, Antineoplastic; D020690:GTPase-Activating Proteins; C095637:RALBP1 protein, human; D034741:RNA, Small Interfering; D004317:Doxorubicin; D051571:Protein Kinase C-alpha