The Influence of Nicotine on Lung Tumor Growth, Cancer Chemotherapy, and Chemotherapy-Induced Peripheral Neuropathy

S. Lauren Kyte; David A. Gewirtz

doi:10.1124/jpet.118.249359

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TABLE 1

Lung cancer cell lines grouped by species and lung cancer type

The cell lines indicated as primary were derived from human lung cancer tissue samples and not purchased commercially.

Species	Lung Cancer Type	Lung Cancer Cell Lines
Human	Nonsmall cell lung cancer	A549, H23, H157, H358, H460, H1299, H1703, H1975, H5800, PC9, 11–18
	Small cell lung cancer	DMS-53, H446, N417, N592
	Adenocarcinoma	HCC827, T1 (primary), 201T (primary)
	Bronchoalveolar carcinoma	H1650
	Papillary adenocarcinoma	H441
	Squamous cell carcinoma	SW900
Mouse	Lewis lung carcinoma	LLC
Mouse	Adenocarcinoma	LKR, Line1

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TABLE 2

In vitro effects of nicotine on lung cancer

Lung Cancer Cell Line	Nicotine (μM)	Duration of Treatment	Serum Concentration	Cellular Response (Assay)	Result (Relative to Control)	Reference
14 SCLC and NSCLC lines	0.1–1	5 days	10%	Viability (MTT)	No effect	Maneckjee and Minna (1990)
H460, H157	0.1–1	7 days	10%	Viability (MTT)	No effect	Chen et al. (2002)
201T	1	48 h	10%	Viability (MTS)	No effect	Carlisle et al. (2007)
H460	0.1, 1	5 days	10%	Viability (Cell Titer-Glo)	20%, 25% increase^*	Zheng et al. 2007)
A549	1	24 h	10%	Viability (MTT)	20% increase^*	Zhang et al. (2009)
A549		24 h	10%	Growth ([³H]-thymidine)	50% increase^*	Zhang et al. (2009)
A549, H1299	0.1, 1	72 h	Not indicated	Viability (MTT)	H1299: 20%, 5% increase^†	Puliyappadamba et al. (2010)
		72 h		Viability (MTT)	A549: 10%, 15% increase^†
		72 h		Growth ([³H]-thymidine)	H1299: 15%, 5% increase^†
		72 h		Growth ([³H]-thymidine)	A549: 20%, 10% increase^†
		Previously treated for 72 h, then seeded		Proliferation (colony formation)	A549: 175% increase (1 μM)^†
H441, H1299	1	30 min or 7 day^{^a}	10%	Viability (MTT)	100%, 75% increase (30 min),^*	Al-Wadei et al. (2012)
H441, H1299	1	30 min or 7 day^{^a}	10%	Viability (MTT)	375%, 250% increase (7 days)^*	Al-Wadei et al. (2012)
H446	0.1–1	12–72 h	10%	Viability (MTT)	8, 5% increase at 12 h (0.1, 0.25 μM),^† no effect at 24–48 h,	Zeng et al. (2012)
H446	0.1–1	12–72 h	10%	Viability (MTT)	8% decrease at 72 h (0.5, 1 μM)^†	Zeng et al. (2012)
A549	1	3–5 days	10%	Viability (MTT)	40%–80% increase^*	Wu et al. (2013)
A549	1	24 h	10%	Invasion (Boyden)	60% increase^*	Wu et al. (2013)
A549	0.1, 1	24 h	10%	Viability (MTS)	40%, 55% decrease^*	Gao et al. (2016)
LKR, H5800	1	2 wk^{^b}	10%	Proliferation (colony formation)	13%, 24% increase^†	Nishioka et al. (2010)
SW900	1	24 h	Not indicated	Proliferation (cell counting)	275% increase^*	Chernyavsky et al. (2015)
A549	1	24 h	10%	Invasion (Transwell)	7% increase	Sun and Ma (2015)
A549	1	8 or 24 h	10%	Migration (wound healing)	10% increase (8 h), 28% increase (24 h)^*	Sun and Ma (2015)
A549, H460, LLC, T1	0.1–1	24 h	10%	Viability (MTS, MTT)	No effect	Kyte et al. (2018)
A549, H460	1	48–96 h		Viability (MTS, MTT)	No effect
	1	48 h		Proliferation (cell counting)	No effect
	1	24 h		Proliferation (colony formation)	No effect
A549	0.5, 1	16 h	10%	Angiogenesis (HIF-1α)	350%, 750% increase^*	Zhang et al. (2007)
				Angiogenesis (VEGF)	14% increase (0.5 μM),
				Angiogenesis (VEGF)	43% increase (1 μM)^*
A549, H1299, H1975	0.1, 1	24 h	10%	Viability (MTT)	A549: 39, 52% increase^*	Ma et al. (2014)
					H1299: 13% increase (0.1 μM),
					20% increase (1 μM)^*
					H1975: 30% increase (0.1 μM),
					52% increase (1 μM)^*
A549	0.1–1	16 h		Angiogenesis (HIF-1α)	20%–40% increase (0.1, 0.5 μM),
A549	0.1–1			Angiogenesis (HIF-1α)	100% increase (1 μM)^*
A549	0.1–1	16 h		Angiogenesis (VEGF)	75%, 125% increase (0.1, 0.5 μM),
A549	0.1–1			Angiogenesis (VEGF)	175% increase (1 μM)^*

HIF-1α, hypoxia-inducible factor 1-α; LLC, Lewis lung carcinoma; MTS, (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium); MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; SCLC, small cell lung cancer; T1, primary human lung carcinoma; VEGF, vascular endothelial growth factor.
↵^a Nicotine was replaced every 24 h.
↵^b Nicotine was replenished every 4 days.
↵* Statistically significant.
↵^† Statistical significance not indicated.

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TABLE 3

In vitro effects of nicotine on lung cancer under nonphysiologic conditions and/or with nonpharmacological concentrations of nicotine

Lung Cancer Cell Line	Nicotine	Duration of Treatment	Serum Concentration	Cellular Response (Assay)	Result (Relative to Control)	Reference
H460, H157	0.01–	7 days	10%	Viability (MTT)	H460: 5% increase (10, 100 μM), 5% decrease (1 mM)	Chen et al. (2002)
H460, H157	1 mM	7 days	10%	Viability (MTT)	H157: 5% decrease (10 μM), 5% increase (0.1–1 mM)	Chen et al. (2002)
201T	10 μM	48 h	10%	Viability (MTS)	No effect	Carlisle et al. (2007)
H460	10 nM, 0.01–1 mM	5 days	10%	Viability (Cell Titer-Glo)	12.5%–50% increase	Zheng et al. (2007)
H460	10 nM, 0.01–1 mM	5 days	10%	Viability (Cell Titer-Glo)	(10 nM, 10–100 μM),^* no effect (1 mM)	Zheng et al. (2007)
A549, H1299	1 nM to 10 mM	72 h	Not indicated	Viability (MTT)	A549: 5%–18% increase (1 nM to 10 μM), no effect (100 μM), 5%–40% decrease (1–10 mM)^†	Puliyappadamba et al. (2010)
					H1299: 10%–30% increase (1–100 nM), no effect (1–100 μM),
					40%–80% decrease (1–10 mM)^†
H446	2.5–15 μM	12–72 h	10%	Viability (MTT)	0%–85% decrease^†	Zeng et al. (2012)
A549	0.01, 10 μM	24 h	10%	Viability (MTS)	No effect (0.01 μM),	Gao et al. (2016)
A549	0.01, 10 μM	24 h	10%	Viability (MTS)	75% decrease (10 μM)^*	Gao et al. (2016)
A549, H1975	10 nM to 100 μM	48 h	0% for 72 h, then treated	Viability (MTS)	A549: 12.5% increase (50 nM to 100 μM),^* H1975: no effect	Mucchietto et al. (2017)
A549, H1975	10 nM to 100 μM	48 h	0% for 72 h, then treated	Proliferation (cell counting)	A549: 33%–66% increase,^* H1975: no effect	Mucchietto et al. (2017)
A549	0.5–10 μM	72 h	0%	Growth (BrdU)	0%–9% increase	Jarzynka et al. (2006)
Line1	1 μM	18 h	0% for 72 h, then treated	Growth (BrdU)	180% increase^†	Davis et al. (2009)
LKR	1 μM	24 h	0.2% for 24 h, then treated	Growth ([³H]-thymidine)	200% increase^†	Nishioka et al. (2010)
A549, H1299	1 nM to 100 μM	24 h	Not indicated	Growth ([³H]-thymidine)	5%–20% increase (1 nM to 1 μM),^†	Puliyappadamba et al. (2010)
A549, H1299	1 nM to 100 μM	24 h	Not indicated	Growth ([³H]-thymidine)	5%–20% decrease (10–100 μM)^†	Puliyappadamba et al. (2010)
A549	1 μM	18 h	0% for 36 h, then treated	Growth (BrdU)	150% increase^*	Dasgupta et al. (2011)
A549, H1650	1 μM	18 h	0% for 24 h, then treated	Growth (BrdU)	175%–180% increase^†	Pillai et al. (2011)
A549, H1650	1 μM	24 h	0% for 24 h, then treated	Invasion (Boyden)	90%–100% increase^†	Pillai et al. (2011)
A549, H1650	1 μM	18 h	0% for 24 h, then treated	Growth (BrdU)	75%, 100% increase^†	Nair et al. (2014)
A549, H1650	1 μM	24 h	0% for 24 h, then treated	Invasion (Boyden)	75%, 150% increase^†	Nair et al. (2014)
LLC	1 pM to 100 μM	Not indicated	0.1%	Proliferation (cell counting)	No effect	Heeschen et al. (2001)
H157, H1703	100 nM	3 days^{^a}	0.1%	Proliferation (cell counting)	50%–95% increase^*	Tsurutani et al. (2005)
H1299	10 nM	Previously treated for 72 h, then seeded	Not indicated	Proliferation (colony formation)	150% increase^†	Puliyappadamba et al. (2010)
A549	0.01–10 μM	18 h	0% (before and during treatment)	Invasion (Boyden)	10% decrease (10 nM), 50%–160% increase (0.1–1 μM), 90% increase (10 μM)^†	Dasgupta et al. (2009)
A549	0.01–10 μM	24 h	0% (during treatment)	Migration (wound healing)	10%–100% increase (0.01–1 μM), 25% increase (10 μM)^†	Dasgupta et al. (2009)
N417	500 μM	Previously treated for 7 days, then seeded	10%	Proliferation (colony formation)	130% increase^*	Martínez-García et al. (2010)
N417	500 μM	Previously treated for 7 days, then seeded	0.5%	Migration (Transwell)	55% increase^*	Martínez-García et al. (2010)
A549, H1299	0.1–1 μM	36 h	0% for 24 h, treated, then seeded	Proliferation (cell counting)	50%–200% increase^*	Liu et al. (2015)
				Migration (wound healing)	30% increase^*
				Invasion (Transwell)	20% increase^*
A549, H1650, H1975, H23, H358	1 μM	24 h	0% for 36 h, then treated	Invasion (Boyden)	120%–430% increase^*	Pillai et al. (2015)
A549, H1299	1 μM,	48 h	0% for 12 h, then treated	Viability (CCK-8)	25%, 40% increase^*	Gong et al. (2014)
	10 nM	48 h		Invasion (Transwell)	75% increase^*
		48 h		Migration (wound healing)	25%, 30% increase^*
		72 h		Migration (wound healing)	25%, 30% increase^*
A549, H460, LLC, T1	5, 10 μM	24 h	10%	Viability (MTS, MTT)	No effect	Kyte et al. (2018)
A549, H460	1 μM	48–96 h	0%–5%	Viability (MTS, MTT)	A549: no effect, H460: 25% increase with 0% serum at 96 h^*	Kyte et al. (2018)
A549	5 μM	48 h	10%	Invasion (QCM)	950% increase^*	Zhang et al. (2007)
	5, 10 μM	16 h		Angiogenesis (HIF-1α)	1000%, 1100% increase^*
	5, 10 μM	16 h		Angiogenesis (VEGF)	130%, 170% increase^*
A549, H1299, H1975	10, 50 μM	24 h	10%	Viability (MTT)	A549: 40% increase (10 μM),^* no effect (50 μM)	Ma et al. (2014)
A549, H1299, H1975	10, 50 μM	24 h		Viability (MTT)	H1299: 13%, 14% increase, H1975: 65% increase (10 μM),^* 40% increase (50 μM)
A549	5 μM	16 h		Angiogenesis (HIF-1α)	25% increase
A549	5 μM	16 h		Angiogenesis (VEGF)	150% increase
A549	5 μM	36 h	10%	Invasion (Transwell)	230% increase^*	Shi et al. (2015)
		16 h		Angiogenesis (VEGF protein, mRNA)	25% increase,^* 700% increase^*
		16 h		Angiogenesis (HIF-1α mRNA)	100% increase^*

BrdU, bromodeoxyuridine; CCK-8, cell counting kit-8; HIF-1α, hypoxia-inducible factor 1-α; LLC, Lewis lung carcinoma; MTS, (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium); MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; QCM, QCM (TM) collagen-based cell invasion assay; T1, primary human lung carcinoma; VEGF, vascular endothelial growth factor.
↵^a Nicotine was replaced every 24 h.
↵* Statistically significant.
↵^† Statistical significance not indicated.

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TABLE 4

In vitro effects of nicotine in combination with chemotherapy on lung cancer

Lung Cancer Cell Line	Nicotine	Chemotherapy	Duration of Treatment	Serum Concentration	Cellular Response (Assay)	Result (Relative to Chemotherapy Alone)	Reference
A549	1 μM	Cisplatin 40 μM	24 h	10%	Apoptosis (annexin V)	30% decrease^†	Jin et al. (2004)
A549, H157	1 μM	Cisplatin 40 μM	6%–48 h	10%	Apoptosis (annexin V)	0%–40% decrease^†	Xin and Deng (2005)
A549, H157	1 μM	Cisplatin 40 μM	24 h	10%	Apoptosis (annexin V)	40% decrease^†	Xin and Deng (2005)
LKR	1 μM	Cisplatin 5 μM	Nicotine for 1 h, then cisplatin for 24 h	10%	Apoptosis (sub-G1)	20% decrease^†	Nishioka et al. (2010)
LKR	1 μM	Cisplatin 5 μM	Nicotine for 1 wk, then cisplatin for 24 h	10%	Apoptosis (sub-G1)	5% decrease^†	Nishioka et al. (2010)
H446	0.1–1 μM	Cisplatin 10 μM	12–72 h	10%	Viability (MTT)	13%–20% increase^†	Zeng et al. (2012)
			36 h		Apoptosis (AV/PI)	No effect (0.1–0.5 μM),
			36 h		Apoptosis (AV/PI)	15% decrease (1 μM)^*
H5800, LKR	0.5 μM	Cisplatin 0.6 μM	Nicotine for 24 h, then cotreatment of 48 h	10%	Apoptosis (annexin V)	60% decrease^*	Nishioka et al. (2014)
A549	1 μM	Cisplatin 20 μM	Nicotine for 24 h, then cisplatin for 24 h	10%	Apoptosis (AV/PI)	40% decrease^*	Liu et al. (2015)
A549	1 μM	Cisplatin 35 μM	Nicotine for 24 h, then cotreatment of 24 h	10%	Viability (MTT)	25% increase^*	Zhang et al. (2009)
		Etoposide 20 μM			Viability (MTT)	35% increase^*
		Cisplatin 35 μM			Apoptosis (DNA fragmentation ELISA)	35% decrease^*
		Etoposide 20 μM			Apoptosis (DNA fragmentation ELISA)	20% decrease^*
H1299	1 μM	Cisplatin 40 μM	96 h	10%	Apoptosis (annexin V)	40% decrease^*	Zhao et al. (2009)
H1299	1 μM	Etoposide 40 μM	96 h	10%	Apoptosis (annexin V)	30% decrease^*	Zhao et al. (2009)
A549	1 μM	Doxorubicin 10 μM	Nicotine for 1 h, then cotreatment of 48 h	10%	Viability (XTT)	25% increase^*	Nakada et al. (2012)
A549	1 μM	Doxorubicin 10 μM	Nicotine for 1 h, then cotreatment of 48 h	10%	Apoptosis (caspase-Glo 3/7)	300% decrease^*	Nakada et al. (2012)
PC9, HCC827	1 μM	Erlotinib 1 nM to 5 μM	72 h	10%	Viability (MTS)	IC₅₀ 31 nM → 43 nM (PC9),^* IC₅₀ 46 nM → 140 nM	Li et al. (2015)
201T	1 μM	Gefitinib 35 μM	48 h	10%	Viability (MTS)	30% increase	Carlisle et al. (2007)
PC9, 11–18	1 μM	Gefitinib 5 nM to 50 μM	72 h	10%	Viability (MTT)	IC₅₀ 24 nM → 22 nM, 0.35 μM → 0.33 μM	Togashi et al. (2015)
PC9, 11–18	1 μM	Gefitinib 5 nM to 50 μM	Nicotine for 3 mo, then cotreatment of 72 h	10%	Viability (MTT)	IC₅₀ 24 nM → 76 nM,^* 0.35 μM → 1.09 μM^*	Togashi et al. (2015)
A549, H460	1 μM	Paclitaxel 50 nM	Paclitaxel for 24 h, 24-h drug-free, nicotine for 24 h	10%	Proliferation (colony formation)	No effect	Kyte et al. (2018)
		Paclitaxel 50 nM	Nicotine for 24 h, then 24-h cotreatment		Proliferation (cell counting)	No effect
		Paclitaxel 100 nM	48 h		Apoptosis (AV/PI)	No effect
		Paclitaxel 100 nM	48 h		Apoptosis (sub-G1)	No effect

AV/PI, annexin V/propidium iodide; ELISA, enzyme-linked immunosorbent assay; MTS, (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium); MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; XTT, 2,3-Bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide inner salt.
↵^† Statistical significance not indicated.
↵* Statistically significant.

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TABLE 5

In vitro effects of nicotine in combination with chemotherapy on lung cancer under nonphysiologic conditions and/or with nonpharmacological concentrations of nicotine

Lung Cancer Cell Line	Nicotine	Chemotherapy	Duration of Treatment	Serum Concentration	Cellular Response (Assay)	Result (Relative to Chemotherapy Alone)	Reference
A549	1 μM	Cisplatin 20 μM	24 h	0% for 36 h, then treated	Apoptosis (TUNEL)	40% decrease^*	Dasgupta et al. (2011)
H446	2.5–15 μM	Cisplatin 10 μM	12–72 h	10%	Viability (MTT)	10%–20% increase (2.5 μM),	Zeng et al. (2012)
			12–72 h		Viability (MTT)	0%–50% decrease (5–15 μM)^†
			36 h		Apoptosis (AV/PI)	25%–50% decrease^*
A549, H1299, H23	1 μM	Cisplatin 20 μM	36 h	0%	Apoptosis (TUNEL)	20%–40% decrease^†	Dasgupta et al. (2006)
		Gemcitabine 20 μM				20%–25% decrease^†
		Paclitaxel 20 μM				25%–50% decrease^†
N417	Previous nicotine exposure (500 μM for 7 days)	Cisplatin (5–100 μM)	48 h	10%	Viability (MTT)	50% increase^*	Martínez-García et al. (2010)
		Etoposide (5–100 μM)				50% increase^*
		Mitomycin (5–50 μM)				IC₅₀ 10 μM → 20 μM^*
		Paclitaxel (5–100 μM)				IC₅₀ 35 μM → 70 μM^*
201T	10 μM	Gefitinib 35 μM	48 h	10%	Viability (MTS)	47% increase (10 μM)^*	Carlisle et al. (2007)
A549	1 μM	Gemcitabine 10 μM	36 h	0% for 24 h, then treated	Apoptosis (TUNEL)	20% decrease^*	Guo et al. (2013)
H157, H1703	10 μM	Paclitaxel 100 nM	48 h	0.1%	Apoptosis (sub-G1)	8% decrease^*	Tsurutani et al. (2005)
H157, H1703	10 μM	Etoposide 100 μM	48 h	0.1%	Apoptosis (sub-G1)	15% decrease^*	Tsurutani et al. (2005)

AV/PI, annexin V/propidium iodide; MTS, (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium); MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; TUNEL, terminal deoxynucleotidyl transferase dUTP nick-end labeling.
↵* Statistically significant.
↵^† Statistical significance not indicated.

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TABLE 6

In vivo effects of nicotine on lung cancer

Lung Cancer Model	Mouse Strain	Nicotine Dose, Route of Administration	Duration of Treatment	Tumor Measurement	Result (Relative to Control)	Reference
N592	Nude	20 or 200 μg/d, s.c. (osmotic minipump)	14 days	Volume	No effect	Pratesi et al. (1996)
N417 (nicotine-treated, 500 μM for 7 days)	Nude	—	—	Volume	100% increase^*	Martínez-García et al. (2010)
N417 (nicotine-treated, 500 μM for 7 days)	Nude	—	—	Growth (Ki-67⁺)	30% increase	Martínez-García et al. (2010)
DMS-53	Nude	24 mg/kg per day, s.c. (osmotic minipump)	1 mo	Volume	250% increase^*	Improgo et al. (2013)
DMS-53	Nude	24 mg/kg per day, s.c. (osmotic minipump)	1 mo	Weight	380% increase^*	Improgo et al. (2013)
A549	Nude, ovariectomized	200 μg/ml in drinking water	38 days	Volume	20% increase	Jarzynka et al. (2006)
				Growth (Ki-67⁺)	300% increase^*
				Microvascular density	80% increase
H460	Foxn1^nu	60 μg, s.c., every other day	6 or 28 days	Volume	No effect	Warren et al. (2012)
				Angiogenesis (HIF-1α)	75% increase (acute),
				Angiogenesis (HIF-1α)	1300% increase (chronic)^*
A549	SCID-Beige	i.p., every other day (dose not indicated)	7 wk	Size (luminescence)	120% increase^*	Pillai et al. (2015)
A549	SCID-Beige	i.p., every other day (dose not indicated)	7 wk	Lung metastasis	75% increase^†	Pillai et al. (2015)
A549	Nude BALB/c	1 μM in drinking water	20 days	Volume	88% increase^†	Liu et al. (2015)
A549	Nude BALB/c	1 μM in drinking water	20 days	Weight	185% increase^*	Liu et al. (2015)
A549 (nicotine-treated, 5 μM)	Nude BALB/c	—	—	Angiogenesis (hemoglobin)	170% increase^*	Shi et al. (2015)
PC9	BALB/cAJc1-nu/nu	0.6 mg/kg, i.v., 5×/wk or 100 μg/ml in drinking water, then combination with erlotinib (100 mg/kg, p.o.)	Nicotine for 18 days	Volume	24% and 39% increase for i.v. and p.o., respectively^*	Li et al. (2015)
PC9	BALB/cAJc1-nu/nu		Nicotine + Erlotinib for 10 days	Volume	200% and 300% increase for i.v. Nic + ER and p.o. Nic + ER, respectively, compared with ER alone^*	Li et al. (2015)
Line1	BALB/c	1 mg/kg, i.p., 3×/wk	2 wk	Volume	225% increase^*	Davis et al. (2009)
				Tumor recurrence	200% increase^*
				Lung metastasis	700% increase^*
		25 mg/kg per day via transdermal patch		Volume	65% increase^*
		25 mg/kg per day via transdermal patch		Lung metastasis	230% increase^*
LLC	C57BL/6J	100 μg/ml in drinking water	16 days	Volume	100% increase^*	Heeschen et al. (2001)
LLC	C57BL/6	100 μg/ml in drinking water	14 days	Volume	75% increase^*	Nakada et al. (2012)
LLC	C57BL/6J	24 mg/kg per day, s.c. (osmotic minipump)	7 days	Volume	No effect	Kyte et al. (2018)
NNK, i.p.	A/J	1 mg/kg, i.p., 3×/wk	4 wk	Area	135% increase^†	Davis et al. (2009)
NNK, i.p.	A/J	1 mg/kg, i.p., 3×/wk	4 wk	Lung metastasis	60% increase^*	Davis et al. (2009)
NNK, i.p.	Ab6F1 (A/J × C57BL/6J)	100 μg/ml in drinking water	12 wk	Multiplicity	No effect	Maier et al. (2011)
				Volume	No effect
				Incidence	35% increase
				Growth (Ki-67⁺)	No effect
NNK, i.p.	A/J	200 μg/ml in drinking water	2, 44, or 46 wk	Volume	No effect	Murphy et al. (2011)
				Multiplicity	No effect
				Incidence	No effect
NNK, i.p.	A/J	1 mg/kg, i.p.,	10 wk	Incidence	125% increase^*	Iskandar et al. (2013)
NNK, i.p.	A/J	3× per week	10 wk	Volume	80% increase^*	Iskandar et al. (2013)
Spontaneous tumor	Kras^LA2/+ C57BL/6J	100 μg/ml in drinking water	6 wk	Multiplicity	No effect	Maier et al. (2011)
Spontaneous tumor	Kras^LA2/+ C57BL/6J	100 μg/ml in drinking water	6 wk	Growth (Ki-67⁺)	No effect	Maier et al. (2011)

HIF-1α, hypoxia-inducible factor 1-α; i.p., intraperitoneal; LLC, Lewis lung carcinoma; NNK, nicotine-derived nitrosamine ketone; p.o., oral.
↵* Statistically significant.
↵^† Statistical significance not indicated.