In this study, we demonstrated that nicotine inhibits chemotherapy-induced apoptosis by the modulation of the mitochondrial death pathway in lung cancer cells. Nicotine was noted to inhibit cisplatin- and etoposide-induced apoptosis in A549 cells. Nicotine prevented chemotherapy-induced reduction of mitochondrial membrane potential, activation of caspase-9, and translocation of Bax to the mitochondria. Akt mediated phosphorylation of proapoptotic protein Bad, and up-regulation of antiapoptotic protein, XIAP, was seen in cells exposed to nicotine. Furthermore, differential activation of MAPK was found to play a role in mediating chemotherapy-induced apoptosis, as well as antiapoptotic effects of nicotine in these cells. MEK1 mediated the antiapoptotic effects of nicotine, whereas MEK2 mediated chemotherapy-induced apoptosis. Regulation of the mitochondrial signaling pathway appeared to be critically involved in mediating the effects of nicotine as well as chemotherapy in lung cancer cells. Using the mitochondrial anion channel inhibitor, DIDS, we demonstrated that the antiapoptotic effects of nicotine against TNF-α–induced apoptosis are mediated by the mitochondrial signaling pathway in lung cancer cells. Furthermore, using A549-ρ0 cells that lack mitochondrial DNA and functional electron transport, we demonstrated that the intact mitochondria play a central role in mediating antiapoptotic effects of nicotine in these cells. The mitochondrial regulation of nicotine imposes an important mechanism that can critically impair the treatment of lung cancer, as activation of the mitochondrial death pathway is one of the most common mechanisms by which many cancer-therapeutic agents, including drugs, radiation, and heat treatments, induce apoptosis in the tumor cells.
The biological effects of nicotine are mediated by nicotine acetylcholine receptors. High-affinity nAChRs are widely expressed in both human lung cancer cells and normal lung cells (8
). Dasgupta and colleagues and others (10
) recently demonstrated that human non–small cell lung cancer cells (A549, H23, H441, and H1299) show abundant expression of α3-, α4-, α5-, α7-, and α10-nAChRs subunits. The antiapoptotic effects of nicotine were observed in A549, H441, and H23 non–small cell lung cancer cells in our study. Multiple signaling pathways have been implicated in mediating effects of nicotine via nAChRs in various cells. Nicotine is known to activate intracellular Ca2+
release and modulate Raf/MEK/extracellular signal–regulated kinases, protein kinase C, Akt, NF-κB, XIAP, Bcl2, and c-Myc–dependent cell survival and proliferation (21
). Our findings corroborate these previously published data showing that MAPK and Akt pathways mediate antiapoptotic effects of nicotine in these cells. We also found that the differential regulation of MAPKs mediated the effects of nicotine and chemotherapy; MEK1 mediated the cell survival effects induced by nicotine, whereas MEK2 mediated chemotherapy-induced apoptosis. Nicotine induces Akt-mediated up-regulation of antiapoptotic protein, XIAP, which contributes to nicotine-mediated inhibition of apoptosis. Although, nicotine-induced activation of various kinases (MAPK, Akt, protein kinase C, etc.) via nAChRs are known to regulate downstream mitochondrial signaling, nicotine has also been reported to cause neuroprotective effects directly through the interactions with mitochondria, independent of the nAChRs (24
). Thus, intact mitochondria and mitochondrial signaling are critical in mediating antiapoptotic effects of nicotine, as shown in our study.
Several cancer-therapeutic agents, including drugs, radiation, and heat treatments, appear to kill tumor cells by inducing apoptosis by activation of the mitochondrial death pathway (6
). Therefore, nicotine modulation of mitochondrial signaling at multiple levels of the signaling cascade appears to be imperative in the prevention of chemotherapy-induced apoptosis in lung cancer. Oxidative stress induced by chemotherapy agents activates the intrinsic or mitochondrial death pathway, resulting in permeabilization and ΨΔm, which follows the release of various proapoptotic mediators, such as cytochrome c, caspase-9, and apoptosis-inducing factor (24
). Our data demonstrate that nicotine prevents chemotherapy-induced reduction in mitochondrial membrane potential and blocks caspase-9 activation in A549 cells. The translocation and localization of proapoptotic proteins, Bax, Bak, and Bid, from the cytosol to the mitochondria, is required to induce cell death by activation of the mitochondrial apoptotic pathway (20
). Consistent with these previous reports, we found that chemotherapy induces mitochondrial translocation of the proapoptotic protein, Bax; however, nicotine blocks these effects (20
). Nicotine induces Akt-mediated phosphorylation of proapoptotic protein, Bad, thus preventing downstream activation of the mitochondrial signaling pathway (14
). The mitochondrial electron transport chain generates ROS, which are then transported into the cytoplasm through voltage-dependent anion channels. The mitochondrial anion channel inhibitor, DIDS, protects cells from oxidative injury by blocking the egress of mitochondrial ROS into the cytoplasm (27
). Inhibition of superoxide release by DIDS from the mitochondrial matrix prevents mitochondrial damage by blocking the ΨΔm and depolarization of the mitochondria. Cormier and colleagues and others (24
) recently reported that nicotine significantly decreases superoxide anion generation in brain mitochondria by causing a direct effect of nicotine on the mitochondria respiratory chain, independent of its receptor. In our study, we found that DIDS blocked the antiapoptotic effects of nicotine, whereas it did not modulate TNF-α–induced apoptosis, suggesting the role of mitochondrial signaling in mediating antiapoptotic effect of nicotine in lung cancer cells. In addition, antiapoptotic effects of nicotine, in part, were mediated by Akt signaling pathway in these cells.
Furthermore, we found that intact mitochondria and mitochondrial function are critical in mediating antiapoptotic effects of nicotine as well as chemotherapy-induced apoptosis in A549 cells. To determine the role of mitochondria, we generated A549-ρ0 cells that lack mitochondrial DNA by slow chemical elimination of mitochondrial DNA by the ethidium bromide technique (15
). These cells showed substantial decreases in ROS production. The lack of normal oxidative phosphorylation in these cells was confirmed by comparing antimycin-A–induced ROS formation in A549 and A549-ρ0 cells by using dichlorofluorescein assay (15
). The mitochondria-deprived cells demonstrated a relative resistance to cisplatin-induced apoptosis as compared with wild-type A549 cells, and showed a modest decrease in ΔΨm on exposure to cisplatin, whereas nicotine failed to show any antiapoptotic effects or modulate the ΔΨm induced by cisplatin in A549-ρ0 cells. Moreover, addition of Akt inhibitor did not alter these results.
These data suggest that intact mitochondria play a critical role in mediating antiapoptotic effects of nicotine as well as chemotherapy-induced apoptosis in lung cancer cells. A hypothetical model based on our results, showing the mechanisms underlying antiapoptotic effects of nicotine via modulation of mitochondrial signaling in A549 cells, is shown in .
Schematic diagram of nicotine-mediated mitochondrial signaling in the prevention of chemotherapy-induced apoptosis in lung cancer. ERK = extracellular signal–regulated kinase.
In summary, we show that nicotine prevents chemotherapy-induced apoptosis in human lung cancer cells by modulating the mitochondrial signaling pathway. These effects of nicotine are critically important, especially in patients undergoing treatment of lung cancer, as activation of the mitochondrial death pathway is one of the most common mechanisms by which many cancer-therapeutic agents induce apoptosis in tumor cells. Our findings are consistent with clinical studies showing that patients who continue to smoke have worse survival, possibly due to tumor progression and resistance to cancer therapy (2
). We speculate that active smoking, as well as nicotine supplementation, may reduce the response to chemotherapeutic agents. Strategies aimed at understanding nicotine-mediated signaling may facilitate the development of improved therapies for lung cancer.