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Role of lycopene in smoke-promoted chronic obstructive pulmonary disease and lung

carcinogenesis

Jelena Mustra Rakic, Xiang-Dong Wang

PII: S0003-9861(20)30448-3
DOI: https://doi.org/10.1016/j.abb.2020.108439
Reference: YABBI 108439

To appear in: Archives of Biochemistry and Biophysics

Received Date: 22 April 2020

Revised Date: 20 May 2020
Accepted Date: 27 May 2020

Please cite this article as: J.M. Rakic, X.-D. Wang, Role of lycopene in smoke-promoted chronic
obstructive pulmonary disease and lung carcinogenesis, Archives of Biochemistry and Biophysics
(2020), doi: https://doi.org/10.1016/j.abb.2020.108439.

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© 2020 Published by Elsevier Inc.

1 Role of lycopene in smoke-promoted chronic obstructive pulmonary

2 disease and lung carcinogenesis

3 Jelena Mustra Rakicab, Xiang-Dong Wangab*

a
4 Nutrition and Cancer Biology Lab, Jean Mayer USDA-Human Nutrition Research

5 Center on Aging (HNRCA) at Tufts University, Boston, MA, USA.

b
6 Biochemical and Molecular Nutrition Program, Friedman School of Nutrition and Policy,

7 Tufts University, Boston, MA, USA

9 *To whom correspondence should be addressed:

10 Nutrition and Cancer Biology Laboratory

11 JM USDA-HNRCA at Tufts University

12 711 Washington Street, Room 514

13 Boston, MA 02111, USA

14 Phone: 617-556-3130; Fax: 617-556-3344

15 Email: xiang-dong.wang@tufts.edu

16

1
17 Electronic Word Count: 5744

18 Total Number of Figures: 2

19 Total Number of Tables: 1

20 Running Title: Lycopene prevents COPD and lung cancer

21 Funding: supported by the US Department of Agriculture grant [1950-51000-074S] and

22 NIFA/AFRI grant [67017-26363]. Any opinions, findings, conclusion, or

23 recommendations expressed in this publication are those of the author(s) and do not

24 necessarily reflect the views of the U.S. Department of Agriculture.

25 Declaration of competing: Jelena Mustra Rakic and Xiang-Dong Wang, no conflicts of

26 interest.

27 Abbreviations: COPD, chronic obstructive pulmonary disease; ATI: alveolar type I;

28 ATII: alveolar type II; CS, cigarette smoke; NNK, 4-(methylnitrosamino)-1-(3-pyridyl)-1-

29 butanone; PAH: polycyclic aromatic hydrocarbons; NNN: nitrosamines N-

30 nitrosonornicotine; nAChR: nicotinic acetylcholine receptor; RCT, reverse cholesterol

31 transport; ABC: ATP-binding cassette; LXRα: liver X Receptor alpha; PPARα:

32 peroxisome proliferator-activated receptor alpha; SR-B1: scavenger receptor B1; BCO1:

33 β-carotene 15,15′ −oxygenase; BCO2: β-carotene 9′,10′ −oxygenase; SNPs: single

34 nucleotide polymorphisms.

35

36

37

38

2
39 Abstract

40 Chronic obstructive pulmonary disease (COPD) and lung cancer are a major cause of

41 morbidity and mortality worldwide, with cigarette smoking being the single most

42 important risk factor for both. Emerging evidence indicate alterations in reverse

43 cholesterol transport-mediated removal of excess cholesterol from lung, and

44 intracellular cholesterol overload to be involved in smoke-promoted COPD and lung

45 cancer development. Since there are currently few effective treatments for COPD and

46 lung cancer, it is important to identify food-derived, biologically active compounds, which

47 can protect against COPD and lung cancer development. High intake of the carotenoid

48 lycopene, as one of phytochemicals, is associated with a decreased risk of chronic lung

49 lesions. This review article summarizes and discusses epidemiologic evidence, in vitro

50 and in vivo studies regarding the prevention of smoke-promoted COPD and lung

51 carcinogenesis through dietary lycopene as an effective intervention strategy. We focus

52 on the recent research implying that lycopene preventive effect is through targeting the

53 main genes involved in reverse cholesterol transport. This review also indicates gaps in

54 knowledge about the function of lycopene against COPD and lung cancer, offering

55 directions for further research.

56 Keywords: lycopene, smoke, COPD, lung cancer, reverse cholesterol transport

57

58

59

60

61

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62 1. Introduction

63 Strong relationship between smoking and chronic lung diseases, such as chronic

64 obstructive pulmonary disease (COPD) and lung cancer, has been well established [1-

65 4]. Dietary factors are broadly believed to be critical in prevention of many chronic

66 diseases, including chronic lung diseases [5-7]. Randomized clinical trials found high-

67 dose β-carotene supplementation to be ineffective in reducing lung cancer risk in

68 smokers [8, 9], switching the focus of research to other fruits and vegetables rich in

69 carotenoids, such as tomato, which high consumption has been associated with

70 decreased risk of lung cancer [10, 11]. Emerging evidence suggests that tomato

71 carotenoid, lycopene, can prevent smoke-promoted COPD and lung cancer [12].

72 Lycopene is among six most abundant carotenoids present in human plasma [13].

73 Smokers have lower levels of lycopene [14, 15], which makes this carotenoid

74 additionally interesting as intervention strategy against smoke-related diseases.

75 Pulmonary cholesterol overload, due to altered reverse cholesterol transport (RCT), has

76 been recognized as one of the critical pathways in chronic inflammation, leading to

77 COPD and lung cancer [16-20]. In recent years, we gained greater knowledge about

78 lycopene function on critical genes of reverse cholesterol transport [12, 21, 22]. In this

79 review we concentrate on lycopene beneficial effect in COPD and lung carcinogenesis.

80 We also focus on reverse cholesterol transport as a common underlying mechanism of

81 lycopene biological function against COPD and lung cancer.

82 2. Cigarette smoking and lung lesions

83 Despite the well-publicized adverse effects of tobacco smoking, approximately one

84 billion people smoke worldwide, with global prevalence of tobacco smoking being 23%,

4
 85 regional prevalence ranging from 20% in Caribbean and North America to 45% in

86 Eastern and South-eastern Asia [23, 24]. On average, there are four times more

87 smokers among men than women, except for most parts of Europe, where the

88 difference is much less [24, 25]. According to the World Health Organization, tobacco

89 smoking causes approximately 6 million premature deaths worldwide; including that of

90 people exposed to second-hand smoke [4, 26]. In addition to being a risk factor for

91 many chronic diseases and premature deaths, tobacco smoking is classified as a group

92 1 carcinogen by the International Agency for Research on Cancer, the World Health

93 Organization’s agency [24]. Likewise, cigarette smoke (CS), which is derived from

94 combustion of tobacco in cigarettes, contains thousands of chemicals, with more than

95 70 being carcinogens [1]. The human lungs are particularly vulnerable to oxidative

96 damage from cigarette smoke exposure. Among carcinogens present in cigarette

97 smoke, 20 compounds are known to be lung carcinogens, including polycyclic aromatic

98 hydrocarbons (PAH), 4-(Methylnitrosamino)-1-(3-Pyridyl)-1- Butanone (NNK),

99 nitrosamines N-nitrosonornicotine (NNN), volatiles such as 1,3-butadiene and ethylene

100 oxide, metals such as cadmium, and the radioactive compound 210Po [1]. PAH, NNK,

101 and NNN are considered to be the most potent carcinogens cigarette smoke known to

102 create DNA adducts and mutations promoting carcinogenesis [1, 25]. Nicotine is

103 generally considered addictive, but is a non-carcinogenic chemical found in smoke that

104 stimulates reward system by binding to nicotinic acetylcholine receptor (nAChR) in the

105 central nervous system. However, binding of nicotine to nAChR can activate pathways

106 involved in carcinogenesis [27]. Furthermore, while cotinine is a major metabolite of

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107 nicotine, nicotine can be metabolized to human carcinogens, NNK and NNN [28],

108 supporting its involvement in adverse health outcomes.

109 Recent reports showed COPD became the third most common cause of death globally

110 in 2010 [29, 30]. COPD is a progressive and irreversible chronic lung disease that

111 includes two main types, emphysema and chronic bronchitis, which can be present

112 individually or concurrently [30]. Emphysema is alveolar enlargement and destruction

113 [31]. Chronic bronchitis is characterized by airflow obstruction with accumulation of

114 inflammation and mucus production [30]. There are several pharmacological and non-

115 pharmacological interventions used in management of COPD exacerbation; however,

116 there is no agent that can reverse the existing disease [32, 33]. Cigarette smoking is the

117 leading cause of COPD [34]. Data from animal models suggest excessive and

118 prolonged inflammation to be the main driving force for development of smoke-induced

119 COPD and other lung lesions [1, 12, 16, 31, 35-39]. Indeed, being a concentrated

120 source of reactive oxidative species (ROS), CS is known to induce oxidative damage

121 leading to prolonged innate and adaptive immune inflammatory response, which is

122 amplified in those smokers who develop severe COPD [36, 39, 40].

123 Lung cancer is the most common cause of death among all cancer deaths with a five-

124 year survival rate being roughly 16% [41, 42]. The major risk factor for lung cancer is

125 cigarette smoking with 90% of all lung cancers being attributed to smoking [1]. COPD

126 patients have up to a five times higher risk of being diagnosed with lung cancer [3, 43],

127 indicating that COPD and lung cancer development may share a common mechanism.

128 While many mechanisms are proposed in smoke-promoted lung carcinogenesis, anti-

6
129 inflammatory agents have been shown to protect against lung cancer in vivo [1], and

130 thus inflammation is viewed as a potential chemopreventive target.

131 Excessive inflammation is a driving force in COPD and cancer development [16, 37, 44-

132 46]. Combustion of cigarettes is known to induce production of ample reactive ROS or

133 reactive nitrogen species (RNS) that activate lung epithelial cells and immune cells

134 present in lungs, such as macrophages and neutrophils. Activated cells release pro-

135 inflammatory cytokines and chemokines leading to increased immune cell infiltration

136 and eventually chronic inflammation in lungs [2, 45, 47, 48]. Given that immune cells

137 release oxidants that can initiate signal transduction pathways leading to genomic

138 alterations of epithelial cells and oxidative DNA damage, impaired DNA repairs,

139 inhibition of apoptosis, and activation of proto-oncogenes [45], the accumulation of

140 immune cells in lung may explain how chronic inflammation facilitates COPD and

141 cancer development.

142 3. Cholesterol homeostasis

143 Cholesterol is a lipophilic molecule, which serves as a precursor in synthesis of vitamin

144 D and steroid hormones. Furthermore, cholesterol is used for bile acid synthesis, which

145 facilitates digestion of fats and absorption of fat-soluble vitamins in the intestine [49, 50].

146 Cholesterol is also a building block of cell membrane bilayers and its homeostasis is

147 crucial for cell morphology and function [51-53]. Humans acquire most of the cholesterol

148 endogenously, and approximately 25% from diet [54]. Additionally, body levels of

149 cholesterol are usually kept constant by regulation of cholesterol uptake, biosynthesis,

150 transportation, metabolism, and excretion [55, 56]. For example, cholesterol

151 biosynthesis can be increased or decreased depending on the availability of the cellular

7
152 cholesterol. One of the crucial pathways that mediates removal of excess cholesterol

153 from peripheral tissues, such as lungs and macrophages, and transport it back to the

154 liver for the reuse by the body or excretion from the body by conversion to bile acids

155 keeping cholesterol equilibrium, is reverse cholesterol transport (RCT) (Fig. 1) [57].

156 Liver X receptor (LXR) is a nuclear hormone receptor serving as a sensor of whole body

157 cholesterol. It regulates expression of many genes involved in RCT, including ABCA1

158 and ABCG1, ATP-binding cassette (ABC) transporters, involved in cholesterol cell efflux

159 [58, 59]. Cholesterol is a lipophilic molecule that requires a transporter in the blood.

160 Lipoproteins are cholesterol carriers, with a lipid center and hydrophilic surface, allowing

161 them to travel in the blood. There are several types of lipoproteins, from high-density

162 lipoproteins (HDL), intermediate-density lipoproteins (IDL), low-density lipoproteins

163 (LDL), to very low-density lipoproteins (VLDL). Transport of excess cholesterol from

164 peripheral tissues is carried throughout the body on HDLs. Excess cholesterol is

165 shuttled to nascent and mature HDL by previously mentioned ABCA1/G1 transporters.

166 Once cholesterol is incorporated in HDL, it can be delivered to the liver by binding to a

167 scavenger receptor B1 (SR-B1) [52]. Also, cholesterol can be transferred from HDL to

168 VLDL, and eventually LDL. Circulating LDL cholesterol is mainly metabolized after

169 binding to hepatic LDL-R, implying involvement of LDL-R in RCT [60, 61]. In addition to

170 LXR, ABCA1 expression can be regulated by peroxisome proliferator-activated receptor

171 (PPARα) which also belongs to nuclear hormone receptor family, but serves rather as a

172 fatty acid sensor [62]. PPARα is known to regulate the expression of SR-B1, a mediator

173 of selective uptake of HDL cholesterol ester, which is highly expressed in the liver [63].

174 Furthermore, a lipoprotein essential for HDL transport in the blood, apo-A1, is also a

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175 PPARα target gene [64]. Although the important role of RCT has been recognized in

176 many chronic diseases, its role in pulmonary disease is only starting to be appreciated

177 [65].

178 4. Chemistry and metabolism of tomato lycopene

179 A tomato is edible, red fruit that belongs to the Solanaceae family, with Solanum

180 lycopersicum being the only domesticated species [66]. However, tomato is often

181 classified as vegetable, especially for the culinary and nutritional purposes [67]. The

182 United States is among the world's greatest producers of tomatoes. In the United

183 States, tomatoes are one of the most consumed vegetables, together with potatoes,

184 lettuce and onions [67]. The average tomato consumption is 8 kg per capita in the

185 United States, with ~ 25% being in a raw form, and ~ 75% in processed forms, such as

186 tomato sauces, purees, canned tomatoes, ketchup and juice [66, 67]. Tomatoes are a

187 valuable source of many bioactive components, but they are best known as a source of

188 carotenoid lycopene [67].

189 There are more than 700 carotenoids present in nature, with only 50 of them found in

190 human diet [68]. The most common carotenoids found in human plasma are: β-

191 carotene, α-carotene, β-cryptoxanthin, lutein, zeaxanthin, and lycopene [13, 68].

192 Lycopene is a naturally occurring carotenoid responsible for the red color of fruits and

193 vegetables [13]. The key sources of lycopene are tomatoes and processed tomato

194 products, which are usually a more concentrated source of lycopene than fresh

195 tomatoes [69]. In addition to tomatoes, other good lycopene sources include pink

196 grapefruit, watermelon, papaya, pink guava, and apricot (Table 1.) [69, 70]. The

197 average intake of lycopene varies depending on the region, ranging from ~1 mg/day in

9
198 the United Kingdom to ~10 mg/day in the Unites States [69]. Lycopene can be found in

199 carotenoid-protein or crystalline forms in chloroplasts and/or chromoplasts of the fresh

200 food, which highly affects its bioavailability. Indeed, data have shown higher availability

201 of lycopene in tomatoes that were thermally processed than from fresh tomatoes [71].

202 Food analysis reported 9.9 – 13.44 mg lycopene/100 g of ketchup, and 0.88 – 7.74 mg

203 lycopene/100 g of fresh tomatoes, depending on tomato variety, maturity, and

204 environmental conditions [69]. Additionally, lycopene bioavailability depends on diet.

205 Given that lycopene is a lipid-soluble compound, consuming lycopene with fats

206 increases lycopene bioavailability [72]. Data from eighteen publications identified

207 lycopene concentration in different tissues to be as following: testis (15.21 nmol/g),

208 adrenals (14.49 nmol/g), the liver (3.55 nmol/g), adipose (0.94 nmol/g), prostate (0.48

209 nmol/g), lung (0.44 nmol/g), kidney (0.42 nmol/g) colon (0.42 nmol/g), heart (0.35

210 nmol/g), skin (0.33 nmol/g) thyroid (0.29 nmol/g), ovary (0.28 nmol/g), breast (0.15

211 nmol/g) [72]. Plasma concentration of lycopene is 0.60 μmol/L, which is ~ 6% of the

212 whole body lycopene [72].

213 Lycopene is considered a non-provitamin A carotenoid, because it lacks vitamin A

214 activity. Lycopene is predominantly cleaved excentrically by mitochondrial β-carotene

215 9′,10′ -oxygenase (BCO2) at the 9’,10’ double bond, which yields apo−10’−lycopenoids,

216 with apo−10’–lycopenal being the primary cleavage product that can be reduced to

217 apo−10’−lycopenol or oxidized into apo-10’-lycopenoic acid (ALA) [13]. The BCO2

218 preferentially cleaves cis-lycopene isomers, which are dominant form of lycopene in

219 plasma and tissues [13, 72]. In ferrets, supplementation with all-trans-lycopene, a form

220 found in food, for 9 weeks resulted in plasma cis-lycopene accumulation more than

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221 50%, mainly 5-cis isomer [14], which is consistent with data from human studies

222 reporting cis-isomers to be 50–79% of total lycopene in plasma [73]. Among several

223 metabolites detected in lung tissue of the ferrets, apo-10′-lycopenol was identified at

224 concentration of 8 ± 3 pmol/g lung tissue [74]. The expression of BCO2 mRNA in a lung

225 of ferret was up-regulated 4-fold by lycopene supplementation, compared to animals not

226 receiving lycopene supplementation [74]. However, apo-10′-lycopenal or ALA were not

227 detected, suggesting that apo-10′- lycopenal, the primary cleavage product, might be a

228 short-lived intermediate product that can be reduced to apo-10′-lycopenol, and ALA may

229 be present at too low concentration to be detected. However, emerging data indicated

230 that ALA may be a very potent chemopreventive agent [75-77], which will be discussed

231 later in this review. β-Carotene 15,15′-oxygenase (BCO1) cleaves pro-vitamin A

232 carotenoids, such as beta-carotene (β-carotene) and beta-cryptoxanthin (BCX), at the

233 central carbon 15,15’ double bond generating vitamin A [13]. Lycopene can be cleaved

234 by BCO1 but with much lower activity than pro-vitamin A carotenoids [13]. Also, it is

235 important to mention that lycopene can yield other cleavage products when exposed to

236 oxygen or other chemical and physical factors due to conjugated double bonds in the

237 carbon chain. Indeed, many studies demonstrated formation of oxidative metabolites of

238 lycopene, such as apo–lycopenal and apo–carotendials, in both in vitro and in vivo

239 systems [78, 79]; apo-6′-,8′-,10-′,12′-, and 14′-lycopenal were detected in the plasma of

240 individuals who consumed tomato products [80]. The identification of lycopene

241 metabolites brings into question whether lycopene metabolites may contribute to the

242 biological functions attributed to lycopene.

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243 Recent data suggested that single nucleotide polymorphisms (SNPs) in BCO1 and/or

244 BCO2 enzymes alter carotenoids levels in the body [81-83]. Indeed, groups of subjects

245 bearing different alleles in single nucleotide polymorphisms located in or near genes

246 involved in carotenoid metabolism, including BCO2, display different blood and/or tissue

247 concentrations of carotenoids [83]. Further, two BCO1 SNPs, rs6564851 and

248 rs12934922, were found to explain 5.6% of the variation in plasma lycopene

249 concentrations [82], though BCO1 is not a dominant cleavage enzyme of lycopene.

250 These findings may explain variation in epidemiological reports regarding lycopene and

251 lung cancer, and imply on importance of assessing individual genotype in controlled

252 clinical trials. Beside the effect on carotenoids body level, emerging evidence suggest

253 carotenoid cleavage enzymes have broader roles than cleavage of carotenoids, and

254 may play a part in diseases development, including carcinogenesis [81, 84]. Particularly,

255 BCO2 status was explored in terms of prostate cancer suppressor. Gong et al. [84]

256 found that restoration of BCO2 gene expression, which was suppressed during prostate

257 carcinogenesis, inhibited androgen-resistant prostate cancer cell growth and tumor

258 formation, independent of BCO2 enzymatic role in lycopene metabolism; further,

259 upregulation of BCO2 expression blocked nuclear factor kappa B activity, which is

260 known to induce the expression of various pro-inflammatory genes contributing to the

261 pro-tumorigenic microenvironment, suggesting an important role of BCO2 in cancer

262 development. Interestingly, in androgen-sensitive prostate cancer cells lycopene

263 treatment inhibited cancer cell growth and upregulated BCO2 expression, implying that

264 protective effect of lycopene may be due to its modulatory effect on BCO2 expression

265 [84]. Given that androgen-sensitive is an early stage, and androgen-resistant more

12
266 aggressive form of the prostate cancer indicates that lycopene may serve as a

267 chemopreventive agent.

268 5. Preventive effects of tomato lycopene against smoke-promoted COPD and lung

269 carcinogenesis

270 Observational studies reported that people eating more fruits and vegetables, which are

271 rich in β-carotene, had lower rates of lung cancer [69, 85]. However, disappointing

272 outcomes of the Alpha-Tocopherol, Beta-Carotene Cancer Prevention (ATBC) trial and

273 the Beta-Carotene and the Retinol Efficacy Trial (CARET) [8, 9], where β-carotene

274 supplementation resulted in either no beneficial effect among healthy men or harmful

275 effect in lung of smokers, discouraged the use of high-dose β-carotene in smoking

276 population. Indeed, an intake of 20 mg of β-carotene per day in the ATBC trial, and 30

277 mg of β-carotene in the CARET trial, which is roughly 10 and 15 times higher than the

278 average intake of β-carotene in the United States, has been shown to increase the lung

279 cancer risk in smokers [8, 9]. Findings from these two large clinical trials shifted the

280 research focus to other components of fruits and vegetables. Population-based studies

281 found stronger inverse association between tomato intake and lung cancer risk than

282 with β-carotene, suggesting tomato as a food with potential anticancer properties [10,

283 11]. Review of epidemiological evidence reported inverse association between tomato-

284 rich diet or blood lycopene level and the risk of the various cancers, with the evidence

285 for the benefits being among the strongest for the lung cancer [6, 86, 87]. Furthermore,

286 tomato extract inhibited induced lung toxicity by preventing inflammation and

287 macrophages infiltration in experimental study [88].

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288 Lycopene is the most abundant carotenoid in tomatoes [89]. Several reviews addressed

289 the preventive role of lycopene against lung cancer, including experimental and

290 observational studies [90], so we will concentrate on the work related to effect of

291 lycopene and its metabolite ALA on smoke-induced/promoted lung cancer and COPD.

292 Both, lycopene intake and lycopene serum levels have been associated with decreased

293 risk of lung cancer, while serum lycopene has been positively associated with lung

294 function in COPD patients [7, 91]. The Nurses' Health Study (NHS) and the Health

295 Professionals Follow-Up Study (HPFS), which together included more than 10 000

296 people, explored the association between intake of individual carotenoids and lung

297 cancer risk by smoking status [6]. Pooled analyses of these two prospective US studies

298 found a significant 32% decrease in lung cancer risk for the high versus low intake of

299 diet high in variety of carotenoids, but there was considerable confounding by smoking

300 status for individual carotenoids. Specifically, there was a strong inverse association

301 between α-carotene intake and lung cancer risk in never smokers, whereas other

302 carotenoids showed non-significant inverse association [6]. In current smokers, a

303 significant 37% lower risk of lung cancer was observed for the top compared with the

304 bottom quintile of lycopene intake, with tomato sauce being the main source of lycopene

305 [6]. Furthermore, when pooled analyses accounted for the lycopene bioavailability in

306 food, the association between the bioavailability-adjusted lycopene score and the risk of

307 lung cancer was stronger in each cohort than when using estimates of lycopene intake

308 based on the nutritional database [6]. Pooled analysis of seven cohort studies also

309 reported association between high lycopene intake and a lower risk of lung cancer in

310 current smokers, but the association was marginal (P = 0.06). Possibly, the low

14
311 correlation coefficient between estimated lycopene intake and actual blood lycopene

312 level may explain why they missed to confirm significant association [92]. When

313 exploring the effect of lycopene on lung cancer risk, blood lycopene would be an

314 appropriate measure due to variability in bioavailability of lycopene from food,

315 depending on cooking methods and presence of other nutrients, as well as variation

316 among individuals in lycopene bioavailability. Regarding COPD, the protective effect of

317 lycopene was supported in cross-sectional study exploring the association between

318 antioxidant nutrients and lung function. In particular, the study reported positive

319 association between serum lycopene and forced expiratory volume in the first second

320 (FEV1%) and forced vital capacity (FVC%) in patients with chronic airflow limitation,

321 including COPD patients [7].

322 The anti-carcinogenic effect of lycopene has been shown in many cancer cell types,

323 including lung cancer cells [93, 94]. Although there is no strong evidence on the

324 beneficial effect of lycopene against smoke-induced carcinogenesis in vitro, one study

325 reported growth-inhibitory effect of lycopene against squamous carcinoma cell [94], one

326 of the most common types of cancer in smokers [42]. Several animal studies, including

327 mice and ferrets, reported preventive role of lycopene against smoke-induced/promoted

328 lung carcinogenesis and COPD [12, 14, 46, 95]. Also, lycopene metabolites have been

329 found to inhibit lung cancer cell growth [76, 77], suggesting that chemopreventive effect

330 of lycopene may be mediated by its metabolites. Indeed, in the A/J mouse model, Lian

331 et al. demonstrated lycopene metabolite ALA to be an effective chemopreventive agent

332 against tobacco carcinogen, NNK, induced carcinogenesis in lungs [77]. ALA inhibited

333 the growth of normal human bronchial epithelial (NHBE) cells, immortalized human

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334 bronchial epithelial (BEAS-2B) cells and non-small cell lung cancer (A549) cells ; These

335 cells has shown a dose-dependent and time-dependent increase in the accumulation of

336 nuclear factor erythroid 2–related factor 2 protein, induced mRNA expression of heme

337 oxygenase-1, as well as inhibition of both endogenous ROS production and H2O2-

338 induced oxidative damage, following ALA treatment. Furthermore, Cheng et al.

339 demonstrated inhibitory effect of ALA on migration, invasion and angiogenesis in BEAS-

340 2B and A549 cell lines [75]. These in vitro studies provided a mechanistic understanding

341 for the chemopreventive effect of lycopene metabolites against lung cancer

342 development in animal models in vivo. While there is no study comparing the beneficial

343 effect of lycopene and its metabolites against smoke-induced carcinogenesis, both,

344 lycopene and its metabolites, are detected and quantified in human plasma and ferret

345 lung [73, 74, 80], indicating that chemopreventive effect may be result of their

346 distinguished and/or synergistic role in the body. This notion was supported by previous

347 studies demonstrating that lycopene and ALA inhibited steatosis and liver cancer in

348 BCO2 knock out mice through different mechanisms [96, 97]. ALA reduced hepatic total

349 cholesterol and elevated cholesterol efflux genes cytochrome P450 family 7A1 and

350 ABCG5 [97]. These ALA-mediated effects were not mimicked by lycopene-

351 supplementation. Intriguingly, lycopene induced PPARα and PPARγ-related genes in

352 mesenteric adipose tissue that increases mitochondrial uncoupling (cell death–inducing

353 DNA fragmentation factor, α subunit-like effector a, PR domain-containing 16,

354 uncoupling protein 3) [97]. Clearly, further investigation to compare the cancer

355 preventive effects of the lycopene metabolites and lycopene itself against COPD and

356 lung cancer through differential mechanisms is needed.

16
357 Lycopene administration alleviated emphysema, common feature of COPD, in mouse

358 model [95]. Liu et al. demonstrated that daily lycopene supplementation with 1.1 mg/kg

359 and 4.3 mg/kg of lycopene inhibited smoke-induced lung squamous metaplasia in

360 ferrets [14]. The doses are equivalent to a lycopene intake of ~15 and ~60 mg/d in

361 humans, which are ~1.5 and ~6 times respectively higher than the average intake of

362 lycopene in the United States [14]. Very recently, we showed that lycopene feeding

363 inhibited smoke/NNK-promoted COPD and lung carcinogenesis in the same model [12].

364 Indeed, smoke exposure for 22 weeks resulted in emphysema and chronic bronchitis,

365 both features of COPD, which were prevented by lycopene feeding. In the same study,

366 lycopene inhibited smoke-promoted pulmonary preneoplastic lesions, such

367 as squamous metaplasia and atypical adenomatous hyperplasia [12]. Doses of

368 lycopene used in the study are equivalent to ~30 and ~90 mg of daily lycopene intake in

369 humans, which are roughly 3 to 9 times higher than the average intake of lycopene in

370 the United States [14]. A ferret model is particularly valuable in preclinical studies,

371 because ferrets mimic human conditions in terms of lycopene absorption, tissue

372 distribution and concentrations, and metabolism [12, 14, 98, 99]. For example, the

373 concentration of lycopene in ferret lung (342 nmol/kg) [14], following lycopene intake

374 equivalent to ~15 mg/d of lycopene in humans, was within the range of lung lycopene

375 concentration in humans (100 – 500 nmol/kg) [100]. Interestingly, when ferrets were

376 exposed to smoke, lycopene levels decreased for 90% in lung and 40% in plasma [14],

377 which is in line with the data from National Health and Nutrition Examination Survey III

378 reporting lower serum level of lycopene in male smokers compared with male non-

379 smokers [15]. Furthermore, harmful effect of pharmacological β-carotene doses in

17
380 cigarette smokers described in the ATBC and CARET clinical trials was explored in the

381 same model [101]. High doses of β-carotene supplementation in smoking environment

382 have been shown to form an undesirable polar metabolites [102], which could promote

383 cancer development by induction of carcinogen-bioactivating enzymes, such as

384 pulmonary cytochrome P450 (CYPs). CYP1A1/1A2 are known to participate in the

385 metabolic activation of many CS carcinogens, such as PAH, NNK and NNN [103, 104];

386 further CYP1A1/1A2 upregulation and activation predispose individual to cancer risk

387 [105, 106]. Interestingly, Paolini et al. [105] found that high-dose β-carotene alone

388 significantly increased CYP1A1/1A2. In ferrets, high β-carotene intake combined with

389 CS exposure was found to enhance catabolism of retinoic acid into polar metabolites 4-

390 oxo-RA and 18-hydroxy-RA via upregulation of pulmonary CYP 1A1/1A2 resulting in

391 carcinogenesis [101]. Because retinoic acid is required for normal epithelial cell growth

392 and regulation through its nuclear receptors [107], enhanced RA catabolism may

393 contribute to lung carcinogenesis. These findings suggest that increased lung cancer

394 risk in smokers after supplementation with pharmacological doses of β-carotene could

395 be result of both, RA catabolism and boosted expression of CYP1A1/1A2.

396 Nevertheless, it is important to mention that enhanced carcinogenesis in smoke-

397 exposed ferrets was associated with pulmonary β-carotene concentration of 26 μmol/kg

398 in animals fed with equivalent to 30 mg β-carotene/d in humans [101]. When ferrets

399 were fed with 60 mg lycopene/d equivalent, the lycopene concentration in lung tissue

400 was 1.2 μmol/kg and inhibited the smoke-induced development of lung carcinogenesis

401 [14], implying that adverse effect of β-carotene may be due to excessive β-carotene

402 accumulated in lung tissue.

18
403 6. Lycopene suppresses smoke-promoted COPD and lung cancer by restoring

404 RCT

405 The importance of functional ABC transporters in protection against pulmonary

406 inflammation and disease has been suggested only recently [58]. The major cells found

407 in lung are alveolar type I (ATI), alveolar type II (ATII), endothelial, interstitial, and

408 macrophages [48]. Transporters, ABCA1 and ABCG1, are expressed in lungs, with

409 ABCA1 being primarily expressed in ATI, ATII, and alveolar macrophages in lungs [108,

410 109]. Loss of Abcg1 in mice resulted in pulmonary cholesterol deposition and chronic

411 inflammation characterized by substantial infiltrates of macrophages and leucocytes in

412 lungs [18]. Furthermore, mice lacking Abca1 transporter had increased cholesterol

413 concentrations in lungs and macrophages accompanied by altered pulmonary structure

414 and respiratory distress with shallow breathing [19]. Overall, altered lung morphology

415 characterized by progression of inflammation, alveolar macrophage enlargement and

416 ATII pneumocyte hyperplasia has been shown in ABC transporter knock out mice [16,

417 18, 19, 58, 110], implying the critical role of RCT transporters and pulmonary cholesterol

418 level in chronic inflammation and lung lesions development [18, 19, 110]. Further,

419 upregulation of ABCA1/G1 leading to recovery of cholesterol efflux has been shown to

420 prevent CS-induced emphysema in vitro and in mice, as well as to have anti-cancer

421 activity in vitro [16, 111].

422 Cholesterol overload has been shown to promote an inflammatory response in vitro and

423 in vivo [17]. Different mechanisms for cholesterol’s effect on inflammation have been

424 proposed, from cholesterol altering lipid raft integrity in plasma membranes and

425 enhancing response to toll-like receptor ligands, such as endotoxins [17, 112] to a more

19
426 recent concept that cholesterol accumulation leads to formation of cholesterol crystals

427 promoting nucleotide-binding oligomerization domain 3 (NLRP3) inflammasome

428 activation [113, 114]. Indeed, cholesterol crystals activated NLRP3 leading to pro-

429 inflammatory cytokine release in macrophages [115].

430 Early studies from the last century have reported adverse effects of smoking on serum

431 lipoprotein levels, including decrease of HDL, important lipoprotein involved in RCT and

432 removal of excess cholesterol from peripheral tissue [116]. In normal conditions,

433 cholesterol overload would lead to synthesis of oxysterol, the known ligands of LXRα.

434 Oxysterol accumulation would activate LXRα, leading to increased expression of its

435 target genes, ABCA1/G1, followed by greater efflux of cholesterol and restored

436 cholesterol homeostasis in the lungs [17]. However, in vitro and more recent in vivo

437 studies have shown a modulatory effect of different cigarette smoke treatments or NNK

438 on nuclear hormone receptors, LXRα and PPARα, and their target genes involved in

439 RCT [117-120]. Indeed, Sonett et al. [16] confirmed that CS exposure was able to

440 downregulate RCT transporters, ABCA1/G1, resulting in cholesterol overload and

441 inflammation in the lungs of mice, demonstrating altered RCT-mediated cholesterol

442 levels as a major player in smoke-promoted lung inflammation. Furthermore, analysis of

443 human lung samples showed altered gene expression of many RCT-related genes in

444 smokers relative to non-smokers, as well as in COPD patients relative to non-COPD

445 patients [16, 52]. In smokers, ABCA1 expression was downregulated; expressions of

446 both transporters, ABC1 and ABCG1, were decreased in COPD patients, who were

447 mostly smokers, compared to the non-COPD patients [16, 52]. However, the exact

448 mechanism through which CS downregulates PPARα, LXRα and ABCA1/G1 is

20
449 unknown. Dysregulation of microRNAs (miRNAs) by cigarette smoke has been

450 associated with expression of genes involved in inflammatory signaling and oxidative

451 stress [121, 122]. Possibly downregulation of PPARα/LXRα and ABCA1/G1 may be

452 explained by smoke-induced increased expression of miRNAs (miRNA-21, miRNA-155,

453 miRNA-20) [123], which are known to target PPARα/LXRα and ABCA1/G1 [124, 125].

454 Accumulating evidence from in vitro and in vivo studies have demonstrated lycopene

455 involvement in many biological processes and proposed several mechanisms of the

456 lycopene protective effect in the body. These include affecting redox activity [126],

457 induction of phase II enzymes [76], inhibition of cell proliferation and/or apoptosis

458 induction [21, 94], inhibition of invasion and metastasis [75, 127], regulating gene

459 expression via transcription factors [19, 79, 80], anti-inflammatory effect [89, 128-130],

460 as well as hypocholesterolemic effect [75, 97, 131, 132]. Here we reviewed most recent

461 studies on importance of lycopene modulatory effect on reverse cholesterol metabolism

462 in smoke-promoted COPD and lung carcinogenesis.

463 Experimental studies provided evidence that lycopene can attenuate smoke-promoted

464 inflammation in lungs [12, 46, 99]. In mice, acute and chronic exposure to cigarette

465 smoke resulted in increased inflammation, which was reduced by lycopene treatment at

466 25 or 50 mg/kg/day [46, 95]. Further, in ferrets, lycopene feeding equivalent to ~30 and

467 ~90 mg of lycopene daily in humans dose-dependently decreased the magnitude of

468 peribronchial/bronchiolar infiltrates of inflammatory cells and alveolar septal infiltrates of

469 inflammatory cells [12]. Lycopene has been shown to modulate cholesterol pathways in

470 cell culture, animal models and humans [75, 97, 131, 132]. Specifically, lycopene has

471 been reported to maintain cholesterol homeostasis by acting via transcription factors,

21
472 such as nuclear hormone receptors involved in the RCT [12, 21, 22, 75, 133]. In the

473 androgen-dependent human prostate cancer (LNCaP) cells, Yang et al. demonstrated

474 that lycopene treatment (2.5 − 10 μM) increased the expression of LXRα at 24 and 48 h

475 and the expression of its target gene ABCA1 at 96 h, suggesting LXR-dependent

476 increase of the ABCA1 expression after lycopene treatment. An increase of LXR and

477 ABCA1 expression resulted in decrease of the intracellular total cholesterol levels and

478 proliferation inhibition of the androgen-dependent human prostate cancer cells; the

479 effects were reversed by the LXRα knockdown [21]. Further, in human macrophages

480 Palozza et al. showed that lycopene (0.5–2 μM) dose-dependently reduced intracellular

481 total cholesterol, which was associated with increased expression of LXRα and ABCA1

482 [22]. In addition, lycopene and its metabolites were found to modulate expression of

483 PPARs [12, 21, 75]. Upregulation of PPARs enhances RCT [12, 134-136]. Indeed,

484 PPARα and PPARγ activators induced the expression of ABCA1 gene and cholesterol

485 efflux through stimulatory action on LXRα expression in macrophages; further, also in

486 macrophages, Nakaya et al. confirmed that the PPARα agonist increased cholesterol

487 efflux and ABCA1/G1 expression in the LXRα-dependent manner [134]. In previously

488 mentioned studies by Yang et al. and Palozza et al. lycopene treatment also

489 upregulated PPARγ in human prostate cancer cells and macrophages [21, 22].

490 Interestingly, lycopene metabolite ALA dose-dependently increased the mRNA

491 expression and protein levels of PPARγ in human THLE-2 liver cells [75], indicating that

492 preventive effect of lycopene on smoke-induced lung lesion may be, at least in part, due

493 to lycopene metabolites. In the ferret model, lycopene feeding equivalent to ~30 and

494 ~90 mg of lycopene intake in humans increased mRNA and protein expression of

22
495 PPARα and LXRα, reversing the effect of smoke in lungs. This was accompanied by

496 upregulation of ABCA1/G1 expression and decrease in lung cholesterol accumulation,

497 providing the first evidence linking a protective role of dietary lycopene against smoke-

498 promoted COPD and preneoplastic lesions to RCT-mediated cholesterol accumulation

499 in lungs [12]. Collectively, lycopene is a potential dietary factor that can upregulate main

500 genes involved in RCT-mediated removal of excess cholesterol from the lung, thereby

501 contributing to the amelioration of inflammation, resulting in prevention of smoke-

502 promoted COPD and lung carcinogenesis (Fig. 2.).

503 7. Conclusion and future directions

504 Observational and experimental evidence imply that dietary lycopene may prevent

505 smoke-promoted COPD and lung carcinogenesis. Recently, RCT has been shown to

506 play an important role in smoke-promoted chronic inflammation. We reviewed the most

507 recent studies regarding preventive effect of lycopene against smoke-promoted COPD

508 and lung cancer, and the importance of the RCT-mediated cholesterol overload in these

509 diseases. This review reveals several research directions that can be considered in

510 expanding the area of lycopene beneficial effect on smoke-related lung diseases. CS

511 has been shown to downregulate mRNA expression of PPARα, LXRα and its target

512 genes, ABCA1/G1 resulting in cholesterol overload and inflammation in lungs [12].

513 Several studies reported upregulation of miRNAs that target PPARα, LXRα and

514 ABCA1/G1 after smoke exposure [55]. However, whether this is the mechanism causing

515 dysfunctional RCT in smoking environment is yet to be elucidated. Future studies

516 should utilize in vitro (e.g. human bronchial epithelial cell line) studies to explore the role

23
517 of the miRNAs in CS-induced downregulation of PPARα, LXRα and ABCA1/G1. Next,

518 dietary lycopene consistently has been shown to have a protective effect against

519 smoke-promoted lung lesions. However, given the well-known unexpected outcome of

520 the ATBC and CARET clinical trials, where carotenoid consumption, precisely β-

521 carotene intake, increased the risk for lung cancer [9], additional research using human

522 data should be done. Technology development and genome wide association studies

523 can help us to explore genetic variants, such as SNPs, and the effect they have on

524 health, as well as the interaction of lycopene with these genetic variants. BCO1 and

525 BCO2 polymorphisms have been shown to affect lycopene levels in plasma. Still, to

526 what extent they affect lycopene accumulation in the body, and what are their roles

527 beyond being cleavage enzyme are still to be elucidated. Association between the

528 lycopene protective effect on smoke-promoted lung and RCT-mediated cholesterol

529 accumulation in lungs has been reported [12]. It would be interesting to explore

530 causality of the RCT in lycopene protective effect against CS-promoted COPD and lung

531 carcinogenesis in the cell culture [e.g. utilizing human bronchial epithelial cell line, and

532 human monocytic leukemia cells to explore protective role of lycopene against

533 palmitate-induced CS-promoted inflammation, with or without addition of the PPARα

534 and LXRα agonist/antagonists (such as fenobibrate/GW6471, T0901317/GGPP

535 respectively). Additionally, given that lycopene metabolite ALA inhibited tobacco

536 carcinogen, NNK, -induced lung carcinogenesis in mice, and was able to upregulate

537 PPARγ in vitro, it would be interesting to explore whether ALA inhibits the CS-promoted

538 lung lesions, and whether it shares the same RCT mechanism with lycopene. Ferret

539 studies demonstrated BCX, a pro-vitamin A carotenoid, to prevent smoke induced

24
540 inflammation and squamous metaplasia in lungs [45]. We know from the ATBC and the

541 CARET trials that supra-physiological doses of β-carotene have a pro-carcinogenic

542 effect in smokers [9]. Likewise, many studies reported adverse effects of excessive

543 single nutrient intake [137]. Thus, future studies should explore how combining of the

544 nutrients affects disease outcomes, such as lung and liver lesions. It would be exciting

545 to observe whether a combination of carotenoids, such as lycopene and BCX, would

546 have a synergistic protective effect against smoke-related COPD, lung carcinogenesis

547 and what are the underlying mechanisms involved. Additionally, it is worth mentioning

548 that consumption of carotenoids from range of fruit and vegetables would be in line with

549 recommendations provided by the Dietary Guidelines for Americans advising eating

550 variety of fruits and vegetables.

551

552

553

554

555

556

557

558

559

560

561

562

25
563 Tables

564 Table 1. Lycopene content in different dietary sources. Adapted from the references

565 [69, 70].

Lycopene content of common fruits and vegetables

Lycopene (mg/100g of wet basis) Sources

0.7−20 Fresh tomatoes

2.3−7.2 Fresh watermelon

2.0−5.3 Fresh papaya

5.2−5.5 Pink guava

0.4−3.4 Pink grapefruit

0.01−0.05 Apricot

Lycopene content of various tomato products

Lycopene (mg/100 g) ± SD Sources

17.23 ± 2.18 Ketchup

15.99 ± 0.9 Spaghetti sauce

55.45 ± 4.33 Tomato paste

17.98 ± 1.47 Tomato sauce

566

567

568

569

570

571

26
572 Figures

573 Figure 1. Excess cholesterol removed from peripheral tissue by RCT. RCT prevents

574 cholesterol overload in peripheral tissue, such as lungs and macrophages. ABCA1 and

575 ABCG1 transporters, which are highly expressed in lungs, allow removal of excess

576 cholesterol from lungs by facilitating its transport to nascent/mature HDL, which then

577 can be uptaken by SR-B1 receptor in liver. Cholesterol carried on HDL can also be

578 exchanged to VLDL, and later LDL, which can be taken by liver through LDL-R.

579 Depending on cholesterol status in liver, cholesterol will be reused and returned to body

580 or excreted from the body in a form of a bile acid. RCT: reverse cholesterol transport;

581 ABCA1: ATP-binding cassette A1; ABCG1: ATP-binding cassette G1; HDL: high-density

582 lipoprotein; VLDL: very low-density lipoprotein; LDL: low-density lipoprotein; SR-B1:

583 scavenger receptor B1; LDL-R: low-density lipoprotein receptor.

27
584
585 Figure 2. Lycopene protective effect against smoke-promoted COPD and preneoplastic

586 lesions is associated with RCT-mediated restored pulmonary cholesterol homeostasis.

587 Dietary lycopene significantly upregulated mRNA expression of RCT genes, PPARα,

588 LXRα and its target genes ABCA1/ABCG1 transporters leading to decrease of

589 cholesterol pulmonary levels and inflammation, preventing COPD and lung cancer;

590 RXR: retinoid X receptor; PPARE: peroxisome proliferator hormone response elements

591 PPARα: peroxisome proliferator-activated receptor alpha; NLRP3: nucleotide-binding

592 oligomerization domain 3; LXRα: liver X receptor alpha; LXRE, LXR-responsive

593 element; ABCA1: ATP-binding cassette A1; ABCG1: ATP-binding cassette G1.

594

28
595

596

597 Declaration of competing

598 The authors declare that there are no conflicts of interest.

599 Acknowledgments

600 This work was supported by the US Department of Agriculture grant [1950-51000-074S]

601 and NIFA/AFRI grant [67017-26363]. Any opinions, findings, conclusion, or

602 recommendations expressed in this publication are those of the author(s) and do not

603 necessarily reflect the views of the U.S. Department of Agriculture.

29
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