Nicotine inhibits apoptosis induced by chemotherapeutic drugs by up-regulating XIAP and survivin

Abstract

Non-small cell lung cancer (NSCLC) demonstrates a strong etiologic association with smoking. Although nicotine is not carcinogenic, it can induce cell proliferation and angiogenesis and suppress apoptosis induced by certain agents. Here we show that nicotine inhibits apoptosis induced by the drugs gemcitabine, cisplatin, and taxol, which are used to treat NSCLCs. This protection correlated with the induction of XIAP and survivin by nicotine in a panel of human NSCLC cell lines, and depletion of XIAP and survivin ablated the protective effects of nicotine. The antiapoptotic effects of nicotine were mediated by dihydro beta-erythroidine-sensitive alpha3-containing nicotinic acetylcholine receptors and required the Akt pathway. Chromatin immunoprecipitation assays demonstrated that nicotine stimulation caused an increased recruitment of E2F1 and concomitant dissociation of retinoblastoma tumor suppressor protein (Rb) from survivin promoter in A549 cells. Moreover, ablation of E2F1 levels caused abrogation of the protective effects of nicotine against cisplatin-induced apoptosis in A549 cells whereas ablation of signal transducer and activator of transcription 3 levels had no effect. These studies suggest that exposure to nicotine might negatively impact the apoptotic potential of chemotherapeutic drugs and that survivin and XIAP play a key role in the antiapoptotic activity of nicotine.

Fig. 1.

Nicotine inhibits drug-induced apoptosis in lung cancer cells. (a) RT-PCR showing the expression of nAChR subunits in A549, NCI-H23, and H1299 NSCLC cells. cDNA from human aortic endothelial cells was the positive control; PCR for GAPDH was used as the loading control. (b) nAChR protein expression in A549, NCI-H23, and H1299 cells as seen by Western blotting. Lysates from HEK293 cells were used as the negative control. (c) Nicotine inhibits apoptosis induced by chemotherapeutic drugs. Quiescent A549, NCI-H23, and H1299 cells were treated with 20 μM gemcitabine, cisplatin, or taxol; the presence of 1 μM nicotine inhibited apoptosis, as seen in a TUNEL assay. (d) A similar experiment as in c where apoptosis in A549 cells was assessed by PARP cleavage; although there was a significant amount of PARP cleavage in cells treated with the drugs, it was greatly reduced when nicotine was present. Induction of p53 and p21/Waf1/CIP1 by the drugs was also inhibited by nicotine.

Fig. 2.

Nicotine induces XIAP and survivin. (a) Western blot analysis showing that nicotine induces XIAP and survivin levels in A549 cells but not the levels of c-IAP-1 and c-IAP-2. (b) Nicotine up-regulates the levels of XIAP and survivin in a dose-dependent manner in A549 cells. (c) A Western blot showing the induction of XIAP and survivin by nicotine in A549, NCI-H23, and H1299 cells. (d) Nicotine induces XIAP and survivin in cells treated with chemotherapeutic drugs. Quiescent A549, NCI-H23, and H1299 cells were treated with 20 μM gemcitabine, cisplatin, or taxol in the presence or absence of 1 μM nicotine. Nicotine enhanced the levels of XIAP and survivin irrespective of whether drugs are present. (e) The antiapoptotic effect of nicotine could be abrogated by siRNAs to XIAP and survivin but not by nontargeting control siRNAs. The siRNAs were transfected individually or in combination; at 18 h after transfection, the cells were rendered quiescent and treated with gemcitabine, cisplatin, or taxol in the presence or absence of 1 μM nicotine for 36 h. Apoptosis was measured by using a TUNEL assay. (f) Nicotine-mediated inhibition of apoptosis is mediated by nAChRs on A549 cells. Apoptosis induced by gemcitabine, cisplatin, and taxol was inhibited by 1 μM nicotine; this inhibition could be prevented by hexamethonium bromide but not by atropine, as measured by PARP cleavage. Induction of XIAP and survivin by nicotine was also prevented by hexamethonium bromide. (g) DhβE, an antagonist of α3/β2- and α4/β2-subunits, prevented nicotine-mediated inhibition of apoptosis as well as induction of XIAP and survivin in A549 cells. α-Lobeline, an antagonist of α4/β2, had no effect, suggesting that α3/β2-subunits play the predominant role.

Fig. 3.

Mechanism of nicotine-mediated induction of XIAP and survivin. (a) Semiquantitative RT-PCR showing the induction of survivin, but not XIAP mRNA, by 1 μM nicotine. cDNA prepared from starved and nicotine-stimulated A549 cells was examined by RT-PCR for XIAP and survivin; PCR for actin was done as a loading control. (b) Akt pathway is involved in nicotine-mediated inhibition of apoptosis. A 10 μM concentration of LY290042 ablated nicotine-induced inhibition of apoptosis in A549 cells as seen in a TUNEL assay; induction of XIAP and survivin was inhibited as well (c). (d) Ubiquitination of XIAP is induced by gemcitabine, cisplatin, and taxol but inhibited by nicotine. Quiescent A549 cells were treated with 20 μM gemcitabine, 20 μM cisplatin, or 20 μM taxol in the presence or absence of 1 μM nicotine for 36 h. Cell lysates were immunoprecipitated with a polyclonal anti-XIAP antibody, and the immunoprecipitates were probed by using an anti-ubiquitin monoclonal antibody by Western blot analysis. (e) The IP/Western blot experiment was performed in the reverse fashion by immunoprecipitating the indicated lysates by a ubiquitin monoclonal antibody and examining the presence of XIAP in the immunoprecipitates by immunoblotting. (f) Western blots demonstrating the selective ablation of E2F1 and Stat3 levels in A549 cells by specific siRNAs. A nontargeting siRNA served as control for all experiments. Eighteen hours after transfection, the cells were rendered quiescent and treated with 1 μM nicotine for 36 h. E2F1–siRNA abrogated nicotine-mediated induction of survivin, whereas levels of XIAP and Stat1 remained unchanged. In contrast, Stat3-siRNA did not affect the expression of either survivin or XIAP. (g) The protective effect of nicotine on cisplatin-induced apoptosis could be abrogated by siRNA to E2F1, but not by Stat3-siRNA or a nontargeting control-siRNA. A549 cells were transfected with the indicated siRNAs as described in the text. Eighteen hours after transfection, the cells were rendered quiescent and subsequently treated with 20 μM cisplatin for 36 h, in the presence or absence of 1 μM nicotine, and apoptosis was measured by TUNEL assay. (h) Chromatin IP assays showing the differential occupancy of E2F1, Rb and Stat3 on the survivin promoter. The stimulation of A549 cells with nicotine leads to increased E2F1 binding and concomitant dissociation of Rb from the proximal site (region +38 to +43) of the survivin promoter. In contrast, Stat3 was not found to be associated with either proximal (+38 to +43) or distal (−806 to −800) sites of the survivin promoter in either quiescent or nicotine-stimulated A549 cells.