Enormous efforts have been devoted by various research groups to identify the role of nicotine in the process of carcinogenesis by tobacco. It has been implicated as a potential risk factor in the proliferation of various cancer cells [14
] and has been shown to induce enhanced tissue perfusion and angiogenesis [5
]. In the present study, we have convincingly shown that nicotine induces proliferation in lung cancer cells, especially in those with impaired p53 status. It also induces an array of antiapoptotic factors in these cells. Although nicotine has been shown to induce proliferation in lung cancer cells [4
], the regulatory role of p53 on nicotine-induced proliferation has not yet been addressed. We observed a significant increase in the proliferation of lung cancer cells lacking p53. This established a ground for us to evaluate the effect of p53 in the induction of various survival signals by nicotine.
The relation between NF-κB and p53 is controversial. Conceptually, over-expression of NF-κB is linked to the development and progression of various tumors [43
] and inhibition of NF-κB function allegedly suppress tumorigenesis and leads to the regression of tumors. It has been documented that both NF-κB and p53 compete for the transcriptional co-activators [28
]. Recent reports suggest that the presence of p53 prevents NF-κB activation though, the mechanism is not clear. However, contradicting these observations, p53-induced NF-κB activation has also been reported [44
]. It has also been shown that inhibition of p53 does not inhibit NF-κB function [27
In our present study we observed a strong activation of NF-κB by nicotine in the p53 null H1299 cell line, while no significant activation of the same was noted in the p53 proficient A549 cells, at any of the concentrations studied. Zhang et al
] have reported that pre-treatment with 100 μM nicotine up-regulate both mRNA and protein level expression of NF-κB in A549. However, supporting our observation, Moodie et al
] have reported that cigarette smoke condensate, in which nicotine is one of the major components (0.3 mg/cigarette), failed to induce NF-κB in A549 cells.
A cross-talk between Akt and p53 acting as cell control machinery for switching between survival and death has been reported. Akt activation has also been reported in p53 null cells [27
]. In our study, 10-9
M - 10-8
M nicotine induced phosphorylation of Akt in H1299 cells. In contrast, a 100 fold higher concentration of nicotine was required to produce similar effects in A549. Several reports indicate interdependence between NF-κB and Akt [26
]. However, Akt independent activation of NF-κB has also been reported [30
]. In the present study we noted a strong phosphorylation of Akt by nicotine in A549 cells, although it failed to induce NF-κB in A549. This indicates that, nicotine-induced Akt phosphorylation is independent of NF-κB activation status of the cell.
No clear consensus between p53 and COX-2 exists in the literature. While some studies show that p53 up-regulates COX-2 [46
], others show that p53 down-regulates it [47
]. We observed induction of COX-2 by nicotine in A549 cells though it failed to induce NF-κB, strengthening the notion that nicotine-induced COX-2 activation in A549 cells is independent of NF-κB. Even though COX-2 is generally considered as an NF-κB dependent gene [26
], there are studies which report the activation of COX-2 independent of NF-κB [48
]. In parallel, we observed an NF-κB and p53 independent induction of Cyclin D1 by nicotine, though the expression profile was less in A549 cells. Cyclin D1 is also considered as an NF-κB dependent gene [49
] although, recent studies implicate Cyclin D1 expression through pathways independent of NF-κB [32
IAP family of proteins is another essential target of NF-κB [50
]. The present study as well as a previous study by Dasgupta et al
], have noticed over-expression of IAPs by nicotine in A549 and H1299 cells. Of interest, this is the first study reporting the difference in the activation pattern of IAPs between both these cell lines. Bcl2 is another molecule which has a key role in regulating nicotine-induced survival signaling and has often been considered as NF-κB dependent. Interestingly, we observed Bcl2 up-regulation in both the cell lines, though the pattern of up-regulation varied between cell lines. In rapport to our observation, a novel nicotine-stimulated survival pathway that involves Bcl2 phosphorylation through MAPK pathway has been reported earlier [10
Nicotine also induces p53 [25
] which, often regulates the phosphorylation pattern of MAPKs [36
]. On the contrary, another study reports that MAPK activation occurs only in cells with functional p53 [52
], indicating a reciprocal interaction between p53 signaling pathway and MAPK pathway. We observed a discrete difference in the phosphorylation pattern of MAPKs in both the cells. The involvement of p53 was further confirmed when the phosphorylation pattern was reversed when p53 was inactivated in A549 and introduced in H1299 cells.
AP-1 is a transcriptional regulator and phosphorylation of MAPKs leads to nuclear translocation of AP-1 [53
]. Although NF-κB and AP-1 are regulated by different mechanisms, several studies indicate that they are activated simultaneously [54
]. Nicotine induced nuclear translocation of AP-1 in both the cell lines, even though it failed to induce NF-κB in A549. However, as in the case of MAPKs, depending on the p53 status of the cell line, there was a significant change in the activation pattern of AP-1. In support of the involvement of p53 in AP1 activation, the nuclear translocation pattern of AP-1 was reversed in A549-p53DN and H1299-p53WT cells.
The ability of nicotine to enhance adherence-independent proliferation of tumor cells is well documented [55
]. We observed a marked difference in the number and size of the clones between both H1299 and A549 cells treated with nicotine, which was strongly corroborating within the concentration range of nicotine at which the survival signals are activated in both the cells, implicating a regulatory role of these survival signals on nicotine-induced enhancement of clonogenic potential. As there was a drastic reduction in the number of clones on treatment with curcumin in both the cells, it is also evident that curcumin inhibits the effect of nicotine by down-regulating these survival signals. Nicotine has a high toxicity in comparison to many other alkaloids such as cocaine and higher doses of nicotine have been reported to be lethal (0.5-1.0 mg/kg for adult humans, and 10 mg for children) [56
]. In our study, we also observed that all the survival signals induced by nicotine are abrogated at higher concentrations suggesting that there may be a balance between the pro-survival and anti-survival signals induced by this compound at different concentrations. However the exact concentration at which this switching over occurs is still unclear as we observed the cessation of various survival signals at different concentrations of nicotine.
Activation of almost all the survival signals investigated in this study is involved in the process of lung cancer progression. It has also been shown that p53 and p21 negatively regulate these survival signals [28
]. Induction of p53 and its downstream target p21 by nicotine has been correlated to the inhibition of cell proliferation by nicotine [25
]. We observed a strong induction of p53 as well as p21 by lower concentrations of nicotine in A549 cells which may be the reason for the absence of proliferative signals at this concentration in these cells. A strong induction of p21 was noted in response to nicotine in H1299 cells transfected with wild type p53 as well. But we did not observe any change in the expression of p53 in A549-p53DN cells. This is expected since p53 is non-functional in these cells, and hence cannot regulate the expression of p21. Similarly, no difference in expression of p21 in response to nicotine treatment was noted in H1299 cells as well as A549-p53DN cells, again confirming the role of p53 in regulating the nicotine-induced signaling events. Hence, we hypothesize that the induction of p53 and p21 by lower concentrations of nicotine in A549 cells may be one of the reasons why nicotine failed to induce these survival signals at these concentrations in A549, though further studies are required to confirm this hypothesis.
Our hypothesis that p53 is the key regulator of nicotine-induced survival signaling in lung cancer cells, was further authenticated in two more lung cancer cells with mutant p53 status. Lower concentrations of nicotine induced proliferation in these cells and pre-treatment with curcumin prevented it and, as in H1299, lower concentrations of nicotine induced NF-κB nuclear translocation in these cells too. In addition, when p53 was silenced in A549 cells by p53 siRNA, as in A549-p53 DN cells, nicotine induced a strong nuclear translocation of NF-κB, while co-transfection of these cells with WT p53 completely inhibited the same, substantiating the regulatory role of p53 in nicotine signaling.
The data obtained from our study is very significant because the average plasma level concentration of nicotine in a typical pack per day smoker is between 20-40 mg [57
]. Studies have indicated that chemotherapeutic drugs like cisplatin, which induce p53 in cells with a wild-type p53 gene can induce apoptosis in these cells while cells with mutated p53 are unaffected [58
]. Supporting this observation, Dasgupta et al
] have shown that nicotine prevents the cisplatin-induced apoptosis in A549 cells with active p53. We also disclose a detrimental role of nicotine in lung cancer patients with mutant p53 status. Hence our study has proved beyond doubt that p53 has a significant role in regulating the survival signals induced by nicotine in lung cancer cells. It is also clear that curcumin down-regulates these survival signals induced by nicotine in both the cells, independent of their p53 status.