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Cancer Cell Biology
Nicotinic modulation of therapeutic response in vitro and in vivo
Article first published online: 16 APR 2012
Copyright © 2012 UICC
International Journal of Cancer
Volume 131, Issue 11, pages 2519–2527, 1 December 2012
How to Cite
Warren, G. W., Romano, M. A., Kudrimoti, M. R., Randall, M. E., McGarry, R. C., Singh, A. K. and Rangnekar, V. M. (2012), Nicotinic modulation of therapeutic response in vitro and in vivo. Int. J. Cancer, 131: 2519–2527. doi: 10.1002/ijc.27556
- Issue published online: 25 SEP 2012
- Article first published online: 16 APR 2012
- Accepted manuscript online: 24 MAR 2012 03:54AM EST
- Manuscript Accepted: 13 MAR 2012
- Manuscript Received: 6 OCT 2011
- American Cancer Society. Grant Number: MRSG-11-031-01-CCE
- American Society of Clinical Oncology Foundation (Conquer Cancer Foundation)
- Young Investigators Award
- hypoxia-inducible factor 1-alpha (HIF-1α);
- lung cancer;
Tobacco use significantly increases the risk of developing cancer. Moreover, there is growing evidence that tobacco use decreases survival in cancer patients. Nicotine, a systemically available component of tobacco, is associated with tumor promotion and decreased apoptosis in cell culture; however, the role of nicotine on response to radiotherapy (RT) or chemoradiotherapy (CRT) in vivo has not been evaluated. Our study evaluated the effects of nicotine administration on cancer cell survival in cell culture and mouse models. Nicotine increased survival in two cell lines following RT in vitro. Nicotine administration in mice during fractionated RT or CRT increased xenograft regrowth as compared to RT or CRT alone. Nicotine increased hypoxia-inducible factor 1-alpha (HIF-1α) expression in tumor xenografts without altering expression of carbonic-anhydrase, a clinical marker of tumor hypoxia. The effects of nicotine on HIF-1α expression were transient, returning to baseline levels within 2-3 days after nicotine removal. Further mechanistic studies indicated that inhibition of phosphoinositide-3-kinase (PI3K) prevented nicotine-mediated increases in HIF-1α expression as well as the prosurvival effects of nicotine on RT. These findings imply that during tobacco use, nicotine may function as a systemic agent through acute and reversible regulation of HIF-1α expression and a decreased therapeutic response.
Tobacco use is the most significant preventable risk factor for the development of lung cancer and continued tobacco use during cancer treatment is associated with poor therapeutic outcomes.1–3 Analysis of the effects of tobacco use in cancer patients demonstrates that smoking decreases survival in both tobacco-related and nontobacco-related cancers.4 Importantly, the effects of tobacco use appear to be systemic rather than localized and thus supporting the role of a systemically available agent as a mediator of the adverse effects of tobacco.
Nicotine is a systemically available agent of tobacco that is currently advocated as a clinical standard of care for smoking cessation.5 As one of more than 7,000 compounds in tobacco smoke, nicotine has the capacity to penetrate all tissues in the body and bind to a spectrum of nicotinic acetylcholine receptors (nAChR) present on both normal (noncancerous) and cancerous tissue. Nicotinic activation of nAChRs in cancerous tissue leads to broad downstream activation of several tumor promoting proteins including activation of the phosphoinositide kinase-3 (PI3K)-Akt cascade as well as the Ras-Raf-MEK-ERK1/2 cascade, resulting in increased proliferation, angiogenesis, invasion, metastasis and decreased apoptosis.6–8 In parallel, nicotine administration has been shown to decrease the cytotoxic effects of chemotherapy and radiotherapy (RT) in vitro9–12; however, no studies have evaluated the effects of nicotine on therapeutic response in vivo.
Separate studies demonstrate that nicotine administration significantly increases hypoxia inducible factor-1 alpha (HIF-1α) in vitro.13 Traditionally, HIF-1α is induced by hypoxia and responsible for activation pathways to stimulate angiogenesis.13 Clinically, hypoxia and HIF-1α expression in patient tumors have been associated with poor therapeutic outcomes in cancer patients.14–16 Although data demonstrate that nicotine increases HIF-1α expression in vitro,17 there are no clinical correlates of the effects of nicotine on tumor hypoxia, HIF-1α expression or therapeutic response. The purpose of our study was to evaluate the effects of nicotine administration on therapeutic response and to relate response to the effects of nicotine on tumor hypoxia and HIF-1α expression.
Material and Methods
Human H460 and A549 lung cancer cells were purchased through American Type Culture Collection (ATCC, Manassas, VA). Cells were maintained in RPMI-1640 (Invitrogen, Grand Island, NY) or Eagles minimal essential medium (EMEM, ATCC, Manassas, VA) media containing 10% fetal bovine serum (FBS, Sigma-Aldrich, St. Louis, MO). All chemicals were obtained through Sigma-Aldrich (St. Louis, MO) except LY294002 and PD98059 which were obtained through Calbiochem-EMD (Gibbstown, NJ). All antibodies were obtained through Santa Cruz Biotechnology (Santa Cruz, CA) except p-Akt (Ser-473, Cell Signaling Technology) and HIF-1α (Novus Biologicals, Littleton, CO). Secondary antibodies were obtained through GE-Amersham (Piscataway, NJ). Male athymic nude Foxn1nu mice were obtained through Harlan Labs (Indianapolis, IN). All animal procedures were conducted according to the protocols approved by the Institutional Animal Care and Use Committee of the University of Kentucky and Roswell Park Cancer Institute.
In vitro survival
Cells were maintained in media containing 10% FBS at 37°C in a humidified atmosphere of 5% CO2 and all procedures were performed in cells with <20 passages after purchase from ATCC. Clonogenic survival assays were performed to assess the cytotoxic effects of treatment with RT. Cells were treated with dose-escalated nicotine (0, 1, 2.5 and 5 μM) 2 hr prior to treatment with dose-escalated RT (0, 2, 4 and 6 Gy using 120 kVp X-rays) delivered using a Faxitron RX650 (Lincolnshire, IL). Cells were allowed to grow for between 12 and 14 days and individual colonies were counted using colony definition based on at least 50 cells in a colony. Comparisons were made between nicotine- and nonnicotine-treated groups.
To test the effects of specific inhibitors on nicotine-mediated decreases in therapeutic response, inhibitors were administered 1 hr prior to nicotine treatment and 3 hr prior to treatment with RT. Specific inhibitors included: α-bungarotoxin (0.5 μM) as an inhibitor of the α7-nAChR, LY294002 (5 μM) as a PI3K inhibitor, and PD98059 (5 μM) as a MEK inhibitor. Colonies were performed as described above and comparisons were made between nonnicotine treated and nicotine with inhibitor-treated groups.
Cell lysates were prepared from H460 cells treated as indicated. The cells were washed with ice cold 1× phosphate-buffered saline (PBS) and lysed in buffer containing the following: 50 mM Tris, pH 8.0, 150 mM NaCl, 1% Triton X-100, 0.1% SDS and protease and phosphatase inhibitors. Cell lysates were cleared by centrifugation at 14,000 rpm for 10 min at 4°C, and protein concentrations were determined by BCA Assay (Pierce). Aliquots of equivalent protein concentration with SDS-PAGE sample buffer were incubated for 4 min at 100°C. Lysates were electrophoresed on 7.5, 10 or 4–15% polyacrylamide gels (BioRad) and transferred to polyvinylidene difluoride membrane (Millipore, Bedford, MA) overnight at 4°C. Membranes were blocked for 1 hr at room temperature with either 5% BSA in Tris-buffered saline (TBS) containing 0.1% Tween-20 (TTBS) or 5% nonfat dry milk in TTBS according to vendor recommendation. The membranes were then incubated overnight at 4°C in either the 5% BSA or the 5% nonfat dry milk in TTBS with the indicated antibodies at 1:1,000 dilutions (pAkt [Ser-473] from Cell Signaling Technology; MMP-2, actin and HIF-1α from Santa Cruz). Membranes were washed three times with TTBS for 5 min each and incubated with either donkey anti-rabbit (1:2,000) or anti-mouse (1:5,000) secondary antibody conjugated to horseradish peroxidase (Amersham Pharmacia Biotech, Piscataway, NJ). Visualization of the protein bands was performed using the enhanced chemiluminescence plus kit as recommended by the manufacturer (Perkin-Elmer, Boston, MA).
In Vivo tumor regrowth assays
To test the effects of nicotine on tumor regrowth, human H460 lung cancer xenografts were generated in male Foxn1nu athymic nude mice. Mice received xenografts at 8–10 weeks of age and approximately 25–28 g in weight. Cells were cultured as described above, trypsinized, quenched with media containing 10% FBS and centrifuged. Media containing FBS was aspirated and cells were washed twice with ice-cold media without FBS. Cells were resuspended in media without FBS and mice were innoculated with 1.5 million H460 cells suspended in 50 uL of media to form single xenografts in the right rear flank.
Tumors were allowed to form and measured daily until tumors reached 5 mm in maximal dimension. Prior dose escalation experiments demonstrated that the maximum tolerated dose of nicotine for this experiment was 60 μg/mouse (data not shown). When cells reached 5 mm in maximal dimension, mice were then randomized to one of the three nicotine treatment groups: N0 (no nicotine) animals were treated with saline control injections every other day for 6 days, NS (short-term nicotine) animals were treated with nicotine (60 μg) subcutaneously every other day × 6 days and NL (long-term nicotine) were treated with nicotine subcutaneously (60 μg) every other day until endpoint defined as tumor growth to 15 mm in greatest linear dimension or a maximum of 28 days of growth after reaching 5 mm in greatest dimension. Subcutaneous nicotine injections were preferred over oral administration because mice treated with cisplatin experienced decreased oral intake. Consequently, subcutaneous administration ensured consistent nicotine delivery. Mice weighed between 30 and 38 g at 28 days after starting treatment.
Within each nicotine treatment group, mice were further randomized to treatment with observation, RT (3 Gy daily × 5 days delivered using a clinical Therapax orthovoltage irradiator, 150 kVp filtered X-rays with a 1-cm applicator prescribed to deliver 3 Gy daily to 5 mm depth), or concurrent chemoradiotherapy (RT + cisplatin 3 mg/kg daily IP × 5 days). Consequently, there were nine randomization groups (n = 10 mice per group). Tumor volumes were estimated using orthogonal measurements every other day until endpoint and volume was defined as L × W × W/2 (where L = largest dimension and W = lesser dimension orthogonal to L). Comparisons were made between tumor volumes in non-nicotine and nicotine-treated groups.
Tumor xenografts were allowed to grow to 5 mm in maximal dimension and mice were randomized to N0, NS or NL as described above. NS-treated animals received 60 μg nicotine subcutaneously every other day for 6 days followed by observation and NL received 60 μg nicotine subcutaneously every other day until tumors were explanted at 15 mm growth in the maximal dimension. Tumors (at 15 mm maximal dimension) were explanted and immediately fixed in 4% paraformaldehyde for 48 hr. Tumors were then paraffin embedded and 5 μm sections were prepared to evaluate the changes in histological protein expression. Immunohistochemical HIF-1α expression was analyzed according to the methods described by Chintala et al.18 and Vaughan et al.19 Briefly, the procedure is a multilayer technique with antigen retrieval using Target Retrieval Solution (TRS, Dako, Carpinteria, CA) in a pressure cooker (Cell Marque, Rocklin, CA) according to the manufacturer's protocol. A biotin blocking kit (Dako) was used to block endogenous biotin and 0.4 μg/mL primary monoclonal mouse anti-human anti-HIF-1α (Novus Biologicals) diluted in 2.5% goat serum in PBS with 0.05% Tween (PBS-T) applied at 4°C overnight. Sections were then incubated with goat vs. mouse biotinylated secondary antibody (Jackson ImmunoResearch Labs, West Grove, PA) for 15 min followed by Elite ABC reagent (Vector Labs, Burlingame, CA) for 20 min. Sections were exposed for 10 min to a 1:35 dilution of amplification reagent (Catalyzed Signal Amplification System, Dako) in PBS-T followed by 20 min exposure to streptavidin conjugated to horseradish peroxidase (Zymed/Invitrogen, San Francisco, CA) visualization using chromogen DAB (Dako) for 1 min. Steps were separated by rinsing with PBS-T followed by 0.03% casein for 5 min. Duplicate slides were treated with a mouse IgG2b isotype match as a negative control and a human squamous cell A253 xenograft was used as a positive control. Expression of HIF-1α manifests as a dark nuclear stain. Slides were then counterstained with hematoxylin, dehydrated, cleared and coverslipped.
Areas of hypoxia were identified immunohistochemically by staining for carbonic-anhydrase (CAIX). Sections (5 μm) were cut, placed on charged slides and dried in a 60°C oven for 1 hr. Room temperature slides were deparaffinized in three changes of xylene and rehydrated using graded alcohols. Endogenous peroxidase was quenched with aqueous 3% hydrogen peroxide for 10 min and washed with PBS-T. Antigen retrieval was performed in citrate buffer (pH 6.0) in a microwave for 10 min and slides were loaded on a DAKO autostainer with the following program: casein 0.03% (in PBS-T) is used to block for 30 min, blown off and the primary antibody CAIX (Santa Cruz, Santa Cruz, CA) is applied at 1:50 dilution to slides for 60 min. An isotype-matched control (rabbit IgG, 4 μg/mL) was used on a duplicate slide in place of the primary antibody as a negative control. A PBS-T wash was followed by rabbit Envision + polymer (Dako) for 30 min. PBS-T was used as a wash and the chromagen DAB + (Dako) was applied for 10 min (color reaction product—brown). Slides are then counterstained with hematoxylin, dehydrated, cleared and coverslipped. Adjacent tumor slices were stained for HIF-1α as shown in Figure 3. By analyzing consecutive tumor slices, areas of hypoxia were compared to HIF-1α expression.
Statistical comparisons were made for colony survival and tumor regrowth comparing nicotine treatment vs. non-nicotine treatment using t-tests with significance noted at p < 0.05.
In vitro cell survival
Preliminary data using water-soluble tetrazolium assays demonstrated that nicotine increased survival following RT at doses between 1 and 5 μM with a decreased protective effect at 10 μM and a cytotoxic effect at 20–100 μM (data not shown). Higher doses of nicotine exceeding 100 μM were not tested. Using this preliminary data, 1–5 μM nicotine was used for the remainder of treatments in cell culture experiments. As shown in Figure 1, nicotine significantly increased cell survival at single fraction RT doses in both H460 and A549 cell lines. Nicotine conferred a consistent average magnitude of 34% survival advantage regardless of RT dose or cell line (range, 18–37%, p < 0.05).
Nicotine and tumor regrowth
Using human H460 lung cancer xenografts in athymic nude mice, the effects of nicotine on fractionated RT and CRT were evaluated. Both short-term nicotine (NS) and long-term nicotine (NL) had no effect on overall tumor growth as compared to non-nicotine-treated controls (N0, Fig. 2a). Endpoint (maximal dimension, 15 mm) was reached for all tumors at a median of 9 days after initiating treatment (range, 8–11 days).
In xenograft bearing nude mice treatment with fractionated RT or CRT significantly reduced tumor regrowth in a highly reproducible manner (Fig. 2b). Long-term nicotine (NL treated with nicotine 60 μg subcutaneously every other day delivered during RT/CRT and continuing until tumors were explanted at 28 days) significantly increased tumor regrowth in tumors treated with RT (3 Gy daily for 5 days, Fig. 2c) and CRT (RT with concurrent cisplatin 3 mg/kg IP daily for 5 days, Fig. 2d) as compared to non-nicotine-treated controls (N0 treated with RT/CRT alone). Specifically, average tumor regrowth at 28 days was 199 mm3 in RT-N0 animals vs. 356 mm3 in RT-NL animals (p < 0.05) and 103 mm3 in CRT-N0 animals vs. 170 mm3 in CRT-NL animals (p < 0.05). These data suggest that long-term nicotine may decrease therapeutic response, but continued nicotine administration after completion of RT/CRT also suggests that increased tumor regrowth is due to increased proliferation after RT/CRT.
The potential proliferative effects of nicotine have been shown in prior studies.6, 7 To differentiate the effects of nicotine on therapeutic response as compared to proliferation, a second short-term nicotine administration group was included (NS, treated with nicotine 60 μg subcutaneously every other day only during RT/CRT). Long-term nicotine administration (NL) provided information on the effects of nicotine for therapeutic response and potentially proliferation after completion of RT/CRT. Short-term nicotine administration (NS) eliminated the potential proliferative effects of nicotine after completion of RT/CRT and would evaluate the effects of nicotine on therapeutic response. Data demonstrate that short-term nicotine (NS) significantly increased tumor regrowth with a volume of 328 mm3 at 28 days for RT (p < 0.05 vs. N0-treated mice, Fig. 2c) and 178 mm3 at 28 days for CRT (p < 0.05 vs. N0-treated mice, Fig. 2d). As shown in Figures 2c and 2d, tumor regrowth curves for long-term nicotine (NL) and short-term nicotine (NS) were very similar and not statistically significantly different. Collectively, these data support that in tumors treated with RT/CRT, nicotine increases tumor regrowth specifically through a decreased therapeutic response during RT/CRT rather than through a change in proliferation after completion of RT/CRT.
In vivo data supported a nicotine-mediated decrease in the therapeutic response to RT and CRT rather than a proliferative effect after completion of RT/CRT. Given data demonstrating that nicotine increases HIF-1α in vitro17 and clinical studies implicating HIF-1α as a poor prognostic factor for cancer treatment outcomes,14–16 the effect of nicotine administration on in vivo HIF-1α expression was evaluated in tumor specimens. As shown in Figure 3a, HIF-1α expression in tumors was significantly increased in NL-treated animals, but had nearly returned to normal in NS treated animals where nicotine removal had occurred 2–3 days prior to tumor explant, suggesting that nicotine-induced HIF-1α expression may be transient and reversible. To evaluate the effect of nicotine administration on tumor hypoxia as related to HIF-1α expression, adjacent slices of tumor tissue were stained for CAIX (carbonic anhydrase) and HIF-1α to assess direct correlations between HIF-1α expression and a clinical marker of hypoxia (CAIX) in the same tumor microenvironment. As shown in Figure 3b, nicotine had no significant effect on CAIX expression. Findings dichotomizing HIF-1α expression from CAIX expression were repeated in five separate animals (Fig. 3b), suggesting that nicotine acutely and reversibly altered HIF-1α expression with no apparent effect on CAIX expression. Quantification of HIF-1α and CAIX is summarized in Table 1, demonstrating a significantly increase expression ratio for HIF-1α:CAIX in tumors treated with long-term nicotine (NL) as compared to controls (NO), but no difference was noted in short-term nicotine.
PI3K, RT response and HIF-1α expression
Nicotine administration activates a broad spectrum of tumor promoting pathways, but prior data support PI3K as a potential mediator of the effects of nicotine on therapeutic response. Other significant pathways identified in a preliminary analysis of nicotine-mediated signal transduction8 were also inhibited. Independent dose escalation of α-bungarotoxin (BTX, an α7-nAChR inhibitor), PD98059 (PD, a MEK inhibitor) and LY294002 (LY, a PI3K inhibitor) in combination with RT demonstrates that 0.5 μM BTX, 5 μM PD or 5 μM LY had no sensitizing effect on RT in A549 or H460 cells (data not shown). As shown in Figure 4a, inhibition of PI3K prevented nicotine-mediated increases in survival following RT in both A549 and H460 cells. In both H460 and A549 cells, inhibition of the α7-nAChR with α-BTX prevented nicotine-mediated changes in survival following RT, but MEK inhibition with PD98059 (PD) prevented only changes in A549 cells. However, inhibition of PI3K with LY294002 (LY) prevented nicotine-mediated increases in p-Akt (Ser-473), MMP2 and HIF-1α (Fig. 4b). These data suggest that PI3K may be a common critical mechanism to nicotine-induced changes in p-Akt, MMP2, HIF-1α and response to RT.
This is the first study to demonstrate that nicotine can decrease the effectiveness of RT and CRT in vivo. Importantly, data demonstrate that nicotine specifically during RT/CRT is important for altering therapeutic response. Further analysis demonstrates that nicotine appears to increase HIF-1α expression in vivo with no change in a clinical marker of tumor hypoxia (immunohistochemical CAIX expression). Data demonstrate that the effects of nicotine on HIF-1α expression appear acute and reversible. Inhibition of PI3K appears to prevent the effects of nicotine on modulating HIF-1α expression and response to RT, suggesting that PI3K is an important mediator of the effects of nicotine on therapeutic response.
In our study, the effect of nicotine on RT was the primary focus, but the in vivo effect of nicotine on concurrent CRT was also analyzed to determine reproducibility and provide a similar treatment paradigm comparable to treatments in clinical populations. Data demonstrate that nicotine decreased the effectiveness of RT in vitro and in vivo. Moreover, nicotine decreased the effectiveness of CRT in vivo. The in vivo effects of nicotine on therapeutic response are a significant extension of prior in vitro observations and support nicotine as an important systemically available component of tobacco for decreasing the efficacy of cancer treatments. Importantly, the observation that short-term nicotine (NS) produced similar tumor regrowth curves as long-term nicotine (NL) further suggests that nicotine exposure specifically during treatment is the critical determinant of therapeutic outcome. The potential impact of nicotine on therapeutic response rather than on proliferative response in this model is further emphasized by the observation that nicotine significantly increased tumor regrowth following RT and CRT (Figs. 2c and 2d) with no apparent effect on tumor growth in the absence of RT or CRT (Fig. 2a).
Nicotine is a systemically available compound of tobacco that has been shown to increase proliferation, migration, angiogenesis and decrease the effectiveness of chemotherapy and RT in vitro.6–12 Systemically expressed nAChRs appear to modulate many of the effects of nicotine, though β-adrenergic receptors also appear to modulate some of the tumor promoting pathways of nicotine.20 The α7-nAChR has been shown to be important for modulating the effects of nicotine in cancer cells thereby suggesting that the α7-nAChR may be a potential therapeutic target.6 One potential confounder of targeting systemic α7-nAChRs in clinical populations is severe toxicity. For example, α-BTX, a specific α7-nAChR inhibitor, is a potent neuromuscular inhibitor that leads to paralysis and respiratory failure in humans. As a consequence, alternative therapeutic strategies may be required to prevent the adverse effects of inhibiting the α7-nAChR. In our study, inhibition of PI3K prevented the effects of nicotine on RT response and induction of p-Akt, MMP-2 and HIF-1α. Others have also shown that inhibition of PI3K prevents many of the effects of nicotine on modulating tumor-promoting activities such as proliferation, angiogenesis and response to chemotherapy.6, 7 Data suggest that PI3K is an important mediator of the effects of nicotine on HIF-1α expression.17 In our study, inhibition of PI3K prevented nicotine-mediated changes in HIF-1α and RT response in vitro. As a result, inhibition of PI3K may provide a therapeutic target to prevent the systemic effects of tobacco in cancer treatment populations.
Several clinical studies demonstrate that HIF-1α is associated with a poor prognosis and therapeutic resistance in several cancer systems including lung and head and neck cancer.14–16 Traditionally, HIF-1α expression coincides with hypoxia and induction of HIF-1α promotes angiogenesis to overcome the effects of hypoxia13; however, other factors can also stimulate HIF-1α expression including acute nicotine administration.17 Recent data demonstrate that HIF-1α appears to be important for radiation response in vivo through paracrine signaling interactions between tumors and stromal vasculature.21 Data reported herein demonstrate that nicotine increases HIF-1α expression in an acute and reversible manner coinciding with the period of RT response in vivo. Importantly, HIF-1α expression appears to be modulated within a specific tumor microenvironment rather than diffusely throughout the tumor. Comparison of a clinical marker for tumor hypoxia (CA-IX) with HIF-1α expression further suggests that HIF-1α expression appears to be regulated by nicotine independently of tumor hypoxia. Collectively, these data suggest that nicotine-mediated activation of PI3K leads to downstream induction of HIF-1α and decreased response to RT in a specific subpopulation of tumor cells. These data also suggest that nicotine may confound the relationship between clinical markers of hypoxia and HIF-1α expression.
An important consequence of these data is the potential that some prognostic biomarker expression (such as with HIF-1α) may be significantly altered with the addition of tobacco products. Currently, there are no clinical studies that have reported the prognostic utility of HIF-1α expression with concomitant assessment of tobacco use at the time of tissue acquisition. Data herein suggest that nicotine is a systemically available component of tobacco that may acutely and reversibly alter tumor HIF-1α expression. As a result, including accurate real-time assessments of tobacco use may be important for considering the prognostic utility of HIF-1α or other potential cancer biomarkers based on protein expression.
Notably, the dose of nicotine used in these experiments is high. The purpose in delivering a high dose of nicotine in our study was to achieve as high an intratumoral concentration as possible. The high concentrations were of particular importance because lung cancer cells in the lungs of cancer patients should experience some of the highest concentration of nicotine in the body owing to the direct contact with nicotine in cigarette smoke. Recent data estimate an approximate 0.9–1 mg of nicotine inhaled per cigarette.22, 23 As a consequence, a one pack per day smoker (20 cigarettes per day) should inhale approximately 18 mg of nicotine per day. An average lung weighs approximately 1–1.2 kg (or 2–2.4 kg for two lungs in a human), resulting in the delivery of approximately 18 mg of nicotine to 2–2.4 kg of lung tissue (or 7.5–9 mg of nicotine/kg of lung tissue) delivered each day in a one pack per day smoker. Thus, the authors felt that the rationale for a high-dose nicotine administration model was justified.
One may conclude that these data argue against nicotine replacement therapy (NRT) in smokers owing to the adverse tumor-promoting effects of nicotine.6–12, 20 On the other hand, NRT can be useful to replace cravings in smokers and is well known to enhance tobacco cessation efforts.5 Moreover, NRT allows for the elimination of thousands of other chemicals present in cigarette smoke.23 Data suggest that nicotine content is estimated at 1 mg for inhaled tobacco smoke, but that considerable variability may occur in actual nicotine content absorbed by smokers.22 Standard prescribing guidelines for NRT generally consist of up to 96 mg daily (4 mg/piece of gum up to 24 pieces per day) for heavy smokers with at least a 25 cigarette per day habit. For an average of 70-kg male, this equates to a maximum recommended clinical intake 1.37 mg/kg/day. With an average starting weight of 25 g in our study, 60 μg of nicotine delivered every other day results in a 2.4 mg/kg dose every other day or a 1.2 mg/kg average daily dose. However, in our study where nicotine was administered as a bolus every other day results in a markedly different blood concentration profile as compared to repeated lower dose administrations associated with standard NRT. For this reason, additional data with a more representative nicotine administration schedule are necessary to further evaluate the potential impact of these results in patients using NRT.
In summary, our study demonstrates that nicotine is a systemically available component of tobacco that may decrease therapeutic response and may acutely modulate potential cancer biomarkers such as HIF-1α. Data suggest that nicotine exposure specifically during treatment is a critical determinant of therapeutic response. Collectively, these data suggest that the systemic effects of tobacco products may be an important factor to consider in cancer care owing to potential alterations in biomarker expression and decreases in therapeutic response to conventional cancer treatment.
- 5Treating tobacco use and dependence: 2008 update. Rockville (MD): U.S. Department of Health and Human Services, Public Health Service; 2008 May, 257.
- 23U.S. Department of Health and Human Services. How Tobacco Smoke Causes Disease: The Biology and Behavioral Basis for Smoking-Attributable Disease: A Report of the Surgeon General. Atlanta, GA: U.S. Department of Health and Human Services, Centers for Disease Control and Prevention, National Center for Chronic Disease Prevention and Health Promotion, Office on Smoking and Health, 2010.