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Keywords:

  • lung cancer;
  • β-carotene;
  • molecular markers;
  • proliferating cellular nuclear antigen

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Conflict of Interest Disclosures
  7. References

BACKGROUND:

β-Carotene supplementation showed neither benefit nor harm among apparently healthy physicians (all men) in the Physicians' Health Study (PHS) trial. The objective of the current investigation was to evaluate how long-term β-carotene supplementation affects molecular markers of lung carcinogenesis in the PHS.

METHODS:

The protein levels of total p53, cyclin D1, proliferating cellular nuclear antigen (PCNA), retinoic acid receptor β (RARβ), and cytochrome p450 enzyme 1A1 (CYP1A1) were measured using the immunohistochemical method in 40 available archival lung tissue samples from patients who were diagnosed with lung cancer in the PHS. The protein levels of these markers were compared by category of β-carotene treatment assignment and other characteristics using unconditional logistic regression models.

RESULTS:

The positivity for total p53, RARβ, cyclin D1, and PCNA was nonsignificantly lower among lung cancer patients who were assigned to receive β-carotene than those who were assigned to receive β-carotene placebo. There was a borderline significant difference in CYP1A1 positivity with an OR of 0.2 (95% confidence interval, 0.2-1.1; P = .06) in a comparison of men who received β-carotene and men who received β-carotene placebo.

CONCLUSIONS:

The 50-mg β-carotene supplementation on alternate days had no significant influence on molecular markers of lung carcinogenesis that were evaluated in the PHS. This finding provides mechanistic support for the main PHS trial results of β-carotene, which demonstrated no benefit or harm to the risk of developing lung cancer. Cancer 2009. © 2009 American Cancer Society.

β-Carotene can block certain carcinogenic processes and inhibit tumor cell growth in experimental studies.1 Observational studies have indicated that higher intakes or blood levels of β-carotene are associated with a reduced risk of lung cancer.2-5 However, the results from 3 large randomized trials indicated that β-carotene supplementation produced neither benefit nor harm on the incidence of lung cancer among apparently healthy physicians (all men) in the Physicians' Health Study (PHS) (50 mg β-carotene on alternate days for 12 years)6 but increased the risk of lung cancer among smokers in the Alpha-Tocopherol, Beta-Carotene (ATBC) Cancer Prevention Study (20 mg β-carotene daily for 6.1 years)7 and among smokers and asbestos workers in the Beta-Carotene and Retinol Efficacy Trial (CARET) (a combination of 30 mg β-carotene and 25,000 IU retinyl palmitate daily for 4 years).8

The p53 tumor suppressor gene plays a pivotal role in the balance of cell proliferation and apoptosis, in the cellular response to various cellular stresses, and in suppressing lung carcinogenesis.9 The structural changes of the p53 protein induced by p53 gene mutations, which frequently are observed in lung cancer,9 enable the mutant protein to become more stable, resulting in p53 accumulation.10 Loss of p53 function is an early event of lung carcinogenesis.9 Retinoic acid derived from either vitamin A or β-carotene acts on normal bronchial epithelium by inducing mucous and blocking squamous differentiation.11 Because squamous metaplasia occurs during the early stages of lung carcinogenesis, perturbations in retinoid signaling may contribute to lung carcinogenesis.12, 13 Retinoic acid receptor β (RARβ) is a retinoic acid-responsive gene. Cytochrome P450 1A1 enzyme (CYP1A1), a phase I metabolizing enzyme that is involved in the bioactivation of carcinogenic tobacco products like polycyclic aromatic hydrocarbons, has been strongly implicated in lung cancer.14

Findings from our studies in ferrets suggest that high-dose β-carotene and cigarette smoke exposure enhance retinoic acid catabolism through the induction of CYP1A1 and CYP1A2 in the lungs.15 In addition, high-dose β-carotene and smoke exposure increased levels of total p53, cyclin D1, and proliferating cellular nuclear antigen (PCNA) and squamous metaplasia but decreased RARβ in the lung tissue of ferrets; whereas low-dose β-carotene attenuated the smoke-induced p53 levels and slightly decreased squamous metaplasia but did not significantly affect cyclin D1, PCNA, or RARβ.16, 17 Because little is known about how long-term β-carotene supplementation affects molecular markers in human lungs, we measured and compared the protein levels of total p53, RARβ, cyclin D1, PCNA, and CYP1A1 in archival lung tissues from patients who were diagnosed with lung cancer and received β-carotene or β-carotene placebo in the PHS.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Conflict of Interest Disclosures
  7. References

Study Population

The first PHS was a randomized trial of a 2 × 2 factorial design of aspirin (350 mg) and β-carotene (50 mg) on alternate days in the primary prevention of cancer and cardiovascular disease among men in the United States (22,071 physicians ages 40 to 84 years at randomization).6 The trial ended on December 31, 1995. More than 7000 of these physicians, along with 7000 new physicians, currently are taking part in the second PHS, which is testing β-carotene, vitamin C, vitamin E, and a multivitamin in the primary prevention of chronic diseases.

Participants were sent yearly questionnaires to ascertain their compliance to study treatment assignments and endpoints of interests. Whenever a report of cancer was made, the medical records, including the pathology report, were sought. We were able to obtain records for >95% of the reported cases. Physicians who were blinded to the treatment assignment and other exposures reviewed medical records. We also extracted information on histology; the presence or absence of metastases; and the location, size, and grade of tumors at diagnosis, which were classified according to the Manual of Tumor Nomenclature and Coding by the American Cancer Society (1968 edition).

Deaths were ascertained through repeated questionnaire mailings, followed by telephone calls, and supplemented by searches of the National Death Index. We obtained death certificates and medical records to assign the cause of death. Follow-up for nonfatal outcomes was >97% complete; and, for mortality, it was 100% complete.

Archival formalin-fixed, paraffin-embedded lung tissue blocks were sought from the 194 surgery and biopsy cases of lung cancer diagnosed. Blocks from a total of 40 patients with available adequate specimens were processed for routine hematoxylin and eosin-stained slides and unstained slides. One patient was excluded from the analysis because of a prior diagnosis of prostate cancer before β-carotene randomization. The 39 cases in this analysis were similar to the total lung cancer cases in the PHS in term of patient and tumor characteristics. Written informed consent was obtained from participants, and the Human Subjects Research Committee at Brigham and Women's Hospital approved the study.

Immunohistochemical Assays

Unstained slides (4 μm thick) from the lung tissue blocks were immunostained for p53, RARβ, cyclin D1, PCNA, and CYP1A1 using the standard avidin-biotin complex immunoperoxidase method (Vectastain ABC-Elite; Vector Laboratories, Burlingame, Calif) as described previously.18 Primary antibodies had dilutions of 1:100 for p53 and CYP1A1, 1:50 for RARβ and cyclin D1, and 1:200 for PCNA. Antibodies against p53, RARβ, cyclin D1, and PCNA were purchased from Santa Cruz Biotechnology (Santa Cruz, Calif). Antibody against CYP1A1 was purchased from ABR-Affinity BioReagents Inc. (Golden, Colo).

Microscopic Evaluation

The sections were examined under light microscopy by 2 independent investigators who were blinded to treatment groups. Representative areas of each section were selected, and 2000 tumor cells were counted under higher magnification (×400) for a total of 10 fields in these areas, and immunoreactivity was quantified based on the percentage of positive tumor cells among a total of 2000 cells.

When the tumor cell nuclei were stained dark brown, the cells were classified as positive for p53, cyclin D1, and PCNA. For RARβ, when the tumor cell perinuclei were stained with a defiantly brown color, the cells were classified as positive. For CYP1A1, the cells were classified as positive if the tumor cell cytoplasm was stained with brown color above cytoplasmic background. Representative immunohistochemical staining for each molecular marker is shown in Figure 1. Immunoreactivity of molecular markers for each section was classified further as positive or negative using the cutoff points for immunohistochemical assays for lung cancer samples in previous studies. For p53, cyclin D1, and PCNA, sections were classified as positive when ≥10%,19 ≥5%,20 and ≥20% (approximate median value)20 of the 2000 tumor cells showed nuclear staining, respectively. RARβ was classified as positive when ≥5% of 2000 tumor cells showed perinuclei staining,21 and CYP1A1 was classified as positive when ≥10% of 2000 tumor cells showed cytoplasm staining.22

thumbnail image

Figure 1. Representative immunohistochemical staining for lung total p53, retinoic acid receptor β (RARβ), cyclin D1, proliferating cellular nuclear antigen (PCNA), and cytochrome p450 enzyme 1A1 (CYP1A1) (the top photomicrographs show adenocarcinoma, and the bottom photomicrographs show squamous cell carcinoma).

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Statistical Analysis

First, we compared the distribution of baseline risk factors for lung cancer, including age, smoking status, and randomized aspirin treatment assignment, by randomized β-carotene treatment assignment to assess their potential for confounding. We also compared the distribution of age at diagnosis and lung tumor characteristics between the β-carotene group and the β-carotene placebo group. Statistical significance between groups was tested by using Wilcoxon rank-sum test for continuous variables and the Mantel-Haenszel test for categorical variables.

The odds ratios (ORs) and 95% confidence intervals (CIs) for having tumors that were positive for each lung molecular marker according to the category of randomized β-carotene treatment assignment, age at enrollment or diagnosis, smoking status, and tumor characteristics were calculated using unconditional logistic regression models with adjustments for age at enrollment (in years) and smoking status (never, past, or current). In a separate analysis, we also controlled for randomized aspirin treatment assignment in multivariate models. The software package SAS version 9.1 (SAS Institute Inc., Cary, NC) was used for all analyses. All P values were 2-sided at a significance level of α = .05 (P ≤ .05).

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Conflict of Interest Disclosures
  7. References

The median age at enrollment was 57.5 years (Table 1). Of the 39 patients with lung cancer, 20.5% were never smokers, 33.3% were past smokers, and 46.2% were current smokers. In addition, 10.3% were diagnosed with small cell lung cancers, 82% were diagnosed with nonsmall cell lung cancers, and 7.7% were diagnosed with other cancers (mesothelioma). Among nonsmall cell lung cancers, adenocarcinoma (53.8%) was the most frequently diagnosed lung cancer, squamous cell carcinoma (20.5%) was the second, and large cell carcinoma (5.1%) and combined squamous cell carcinoma and adenocarcinoma (2.6%) were relatively rare. The average age at diagnosis was 71.1 years. When diagnosed, more than half of lung cancers were poorly differentiated or undifferentiated (51.3%) or had distant metastasis (61.5%). Baseline age, smoking status, randomized aspirin treatment assignment, age at diagnosis, and lung tumor characteristics were distributed similarly between the β-carotene group and the β-carotene placebo group (Table 1).

Table 1. Characteristics of the 39 Lung Cancer Patients in The Physicians' Health Study
 No. of Patients (%) 
CharacteristicAll PatientsNon-β-Carotene Groupβ-Carotene GroupP*
  • *

    The Wilcoxon rank-sum test was used for continuous variables, and the Mantel-Haenszel test was used for categorical variables.

All patients39 (100)20 (51.3)19 (48.7) 
Median age at enrollment [range], y57.5 [42.3-82.3]59.6 [42.3-68.6]56 [46.2-82.3].22
 Age at enrollment, y   .37
  ≤6026 (66.7)12 (46.2)14 (53.8) 
  >6013 (33.3)8 (61.5)5 (38.5) 
Smoking status   .39
  Never8 (20.5)6 (75)2 (25) 
  Past13 (33.3)5 (38.5)8 (61.5) 
  Current18 (46.2)9 (50)9 (50) 
Randomized to aspirin   .87
  No20 (51.3)10 (50)10 (50) 
  Yes19 (48.7)10 (52.6)9 (47.4) 
Median age at diagnosis [range], y71.1 [52.5-90.6]70.7 [52.5-83.3]71.1 [59.8-90.6].91
 Age at diagnosis, y   .87
  ≤7019 (48.7)10 (52.6)9 (47.4) 
  >7020 (51.3)10 (50)10 (50) 
Tumor type   .58
  Small cell carcinoma4 (10.3)2 (50)2 (50) 
  Nonsmall cell carcinoma    
  Adenocarcinoma21 (53.8)10 (47.6)11 (52.4) 
  Squamous cell carcinoma8 (20.5)4 (50)4 (50) 
  Large cell carcinoma2 (5.1)2 (100)0 (0) 
  Combined squamous/adenocarcinoma1 (2.6)0 (0)1 (100) 
  Others3 (7.7)2 (66.7)1 (33.3) 
Histologic grading/differentiation   .18
  Well/moderately8 (20.5)3 (37.5)5 (62.5) 
  Poorly14 (35.9)6 (42.9)8 (57.1) 
  Undifferentiated6 (15.4)4 (66.7)2 (33.3) 
  Missing11 (28.2)7 (63.6)4 (36.4) 
Distant metastasis   .13
  No15 (38.5)10 (66.7)5 (33.3) 
  Yes24 (61.5)10 (41.7)14 (58.3) 

The positivity for total p53 and RARβ was nonsignificantly lower among lung cancer patients who were assigned to received β-carotene than among patients who were assigned to receive β-carotene placebo (Table 2). Total p53 positivity also was less common among patients who were smokers, who were younger at baseline or at diagnosis, who had undifferentiated tumors, or who had distant metastasis; these differences were not statistically significant except for age at baseline (P = .01). Conversely, RARβ positivity tended to be more common among smokers, among those who were younger at baseline or at diagnosis, among those with poorly or undifferentiated tumors, and among those with distant metastasis; none of these differences were statistically significant.

Table 2. Positivity, Odds Ratios, and 95% Confidence Intervals for the Expression of Lung P53 and Retinoic Acid Receptor β by β-Carotene Treatment and Other Characteristics
 No. of Patients (%)  No. of Patients (%)  
VariableP53 PositiveP53 NegativeOR (95% CI)*PRARβ PositiveRARβ NegativeOR (95% CI)*P
  • RARβ indicates retinoic acid receptor β; OR, odds ratio; CI, confidence interval.

  • *

    Adjusted for age at enrollment and smoking status.

All patients22 (56.4)17 (43.6)  20 (51.3)19 (48.7)  
Randomized to β-carotene        
 No13 (65)7 (35)1.0 (Referent) 11 (55)9 (45)1.0 (Referent) 
 Yes9 (47.4)10 (52.6)0.8 (0.2-3.4).719 (47.4)10 (52.6)0.5 (0.1-2.2).38
Smoking status        
 Never6 (75)2 (25)1.0 (Referent) 2 (25)6 (75)1.0 (Referent) 
 Past7 (53.8)6 (46.2)0.3 (0.0-2.2).227 (53.8)6 (46.2)3.5 (0.5-24.2).21
 Current9 (50)9 (50)0.3 (0.0-2.3).2511 (61.1)7 (38.9)4.7 (0.7-30.5).10
β-Carotene/smoking        
 Non-β-carotene/never-smoking4 (66.7)2 (33.3)1.0 (Referent) 2 (33.3)4 (66.7)1.0 (Referent) 
 β-Carotene/never-smoking2 (10)0 (0) 0 (0)2 (100) 
 Non-β-carotene/past-smoking3 (60)2 (40)0.5 (0.0-6.6).573 (60)2 (40)2.9 (0.2-35.3).41
 β-Carotene/past-smoking4 (50)4 (50)0.3 (0.0-3.3).334 (50)4 (50)1.9 (0.2-18.0).57
 Non-β-carotene/current-smoking6 (66.7)3 (33.3)0.6 (0.1-6.0).636 (66.7)3 (33.3)3.8 (0.4-36.4).25
 β-Carotene/current-smoking3 (33.3)6 (66.7)0.3 (0.0-2.9).305 (55.6)4 (44.4)2.6 (0.3-22.3).39
Age at enrollment, y        
 ≤6011 (42.3)15 (57.7)1.0 (Referent) 15 (57.7)11 (42.3)1.0 (Referent) 
 >6011 (84.6)2 (15.4)9.6 (1.6-58.1).015 (38.5)8 (61.5)0.4 (0.1-1.7).21
Age at diagnosis, y        
 ≤709 (47.4)10 (52.6)1.0 (Referent) 12 (63.2)7 (36.8)1.0 (Referent) 
 >7013 (65)7 (35)2.1 (0.6-8.0).268 (40)12 (60)0.4 (0.1-1.4).14
Tumor type        
 Small cell carcinoma0 (0)4 (100) 1 (25)3 (75)0.2 (0.0-2.5).20
 Adenocarcinoma15 (71.4)6 (28.6)1.0 (Referent) 10 (47.6)11 (52.4)1.0 (Referent) 
 Squamous cell carcinoma5 (62.5)3 (37.5)0.6 (0.1-4.3).594 (50)4 (50)0.5 (0.1-3.1).42
 Large cell carcinoma1 (50)1 (50)0.3 (0.0-8.9).492 (100)0 (0) 
 Combined squamous/adenocarcinoma0 (0)1 (100) 1 (100)0 (0) 
 Others1 (33.3)2 (66.7)0.1 (0.0-2.0).122 (66.7)1 (33.3) 
Histologic grading/differentiation        
 Well/moderately5 (62.5)3 (37.5)1.0 (Referent) 3 (37.5)5 (62.5)1.0 (Referent) 
 Poorly8 (57.1)6 (42.9)0.8 (0.1-6.8).858 (57.1)6 (42.9)2.7 (0.4-19.1).33
 Undifferentiated1 (20)5 (80)0.2 (0.0-3.4).263 (50)3 (50)1.9 (0.2-22.1).61
 Missing8 (72.7)3 (27.3)1.7 (0.2-14.4).656 (54.5)5 (45.5)2.7 (0.4-19.7).34
Distant metastasis        
 No11 (73.3)4 (26.7)1.0 (Referent) 6 (40)9 (60)1.0 (Referent) 
 Yes11 (45.8)13 (54.2)0.4 (0.1-2.0).2714 (58.3)10 (41.7)1.6 (0.4-6.8).55

The positivity for cyclin D1 or PCNA (cell proliferation indices) also was nonsignificantly lower among lung cancer patients who were assigned to receive β-carotene than among those who were assigned to receive β-carotene placebo (Table 3). The positivity for cyclin D1 or PCNA was less common among patients who were older at diagnosis, but the difference was statistically significant only for PCNA (P = .03). However, the positivity for cyclin D1 or PCNA tended to be more common among patients who had poorly differentiated or undifferentiated tumors and patients who had distant metastasis (Table 3). There were no meaningful differences in the positivity for cyclin D1 or PCNA by smoking status, age at enrollment, or tumor type.

Table 3. Positivity, Odds Ratios, and 95% Confidence Intervals for the Expression of Lung Cyclin D1 and Proliferating Cellular Nuclear Antigen by β-Carotene Treatment and Other Characteristics
 No. of Patients (%)  No. of Patients (%)  
VariableCyclin D1 PositiveCyclin D1 NegativeOR (95% CI)*PPCNA PositivePCNA NegativeOR (95% CI)*P
  • Or indicates odds ratio; CI, confidence interval; PCNA, proliferating cellular nuclear antigen.

  • *

    Adjusted for age at enrollment and smoking status.

All patients24 (61.5)15 (38.5)  24 (61.5)15 (38.5)  
Randomized to β-carotene        
 No13 (65)7 (35)1.0 (Referent) 13 (65)7 (35)1.0 (Referent) 
 Yes11 (57.9)8 (42.1)0.7 (0.2-2.8).6411 (57.9)8 (42.1)0.7 (0.2-2.9).64
Smoking status        
 Never5 (62.5)3 (37.5)1.0 (Referent) 5 (62.5)3 (37.5)1.0 (Referent) 
 Past8 (61.5)5 (38.5)1.0 (0.2-6.0).979 (69.2)4 (30.8)1.3 (0.2-8.4).79
 Current11 (61.1)7 (38.9)0.9 (0.2-5.2).9410 (55.6)8 (44.4)0.8 (0.1-4.2).76
β-Carotene/smoking        
 Non-β-carotene/never-smoking4 (66.7)2 (33.3)1.0 (Referent) 3 (50)3 (50)1.0 (Referent) 
 β-Carotene/never-smoking1 (50)1 (50)0.4 (0.0-13.3).642 (100)0 (0) 
 Non-β-carotene/past-smoking4 (80)1 (20)1.9 (0.1-31.3).665 (100)0 (0) 
 β-Carotene/past-smoking4 (50)4 (50)0.5 (0.1-4.5).514 (50)4 (50)1.0 (0.1-8.5).97
 Non-β-carotene/current-smoking5 (55.6)4 (44.4)0.6 (0.1-5.4).635 (55.6)4 (44.4)1.2 (0.1-10.3).88
 β-Carotene/current-smoking6 (66.7)3 (33.3)1.0 (0.1-9.4).985 (55.6)4 (44.4)1.3 (0.2-10.3).82
Age at enrollment, y        
 ≤6016 (61.5)10 (38.5)1.0 (Referent) 15 (57.7)11 (42.3)1.0 (Referent) 
 >608 (61.5)5 (38.5)1.0 (0.3-4.0)>.999 (69.2)4 (30.8)1.5 (0.4-6.5)0.56
Age at diagnosis, y        
 ≤7014 (73.7)5 (26.3)1.0 (Referent) 15 (78.9)4 (21.1)1.0 (Referent) 
 >7010 (50)10 (50)0.4 (0.1-1.4).139 (45)11 (55)0.2 (0.1-0.9).03
Tumor type        
 Small cell carcinoma2 (50)2 (50)0.8 (0.1-7.9).842 (50)2 (50)0.8 (0.1-8.8).87
 Adenocarcinoma12 (57.1)9 (42.9)1.0 (Referent) 14 (66.7)7 (33.3)1.0 (Referent) 
 Squamous cell carcinoma5 (62.5)3 (37.5)1.3 (0.2-8.0).804 (50)4 (50)0.6 (0.1-3.7).54
 Large cell carcinoma2 (100)0 (0) 2 (100)0 (0) 
 Combined squamous/adenocarcinoma1 (100)0 (0) 1 (100)0 (0) 
 Others2 (66.7)1 (33.3)1.6 (0.1-23.3).751 (33.3)2 (66.7)0.2 (0.0-3.2).26
Histologic grading/differentiation        
 Well/moderately3 (37.5)5 (62.5)1.0 (Referent) 4 (50)4 (50)1.0 (Referent) 
 Poorly11 (78.6)3 (21.4)8.4 (1.0-69.9).0511 (78.6)3 (21.4)7.4 (0.8-66.4).07
 Undifferentiated4 (66.7)2 (33.3)5.0 (0.4-62.9).214 (66.7)2 (33.3)5.2 (0.4-70.5).21
 Missing6 (54.5)5 (45.5)2.4 (0.3-17.1).385 (45.5)6 (54.5)1.2 (0.2-8.3).88
Distant metastasis        
 No7 (46.7)8 (53.3)1.0 (Referent) 7 (46.7)8 (53.3)1.0 (Referent) 
 Yes17 (70.8)7 (29.2)3.5 (0.8-16.2).1017 (70.8)7 (29.2)3.7 (0.8-17.2).10

CYP1A1 positivity was marginally significantly lower among lung cancer patients who were assigned to receive β-carotene than among those who were assigned to receive β-carotene placebo with an OR of 0.2 (95% CI, 0.1-1.1; P = .06) (Table 4). CYP1A1 positivity also was less common among patients who were diagnosed at an older age (P = .13), but it was more common among patients who had poorly differentiated (P = .04) or undifferentiated (P = .07) tumors and patients who had distant metastasis (P = .15).

Table 4. Positivity, Odds Ratios, and 95% Confidence Intervals for the Expression of Lung Cytochrome p450 Enzyme 1A1 by β-Carotene Treatment and Other Characteristics
 No. of Patients (%)  
VariableCYP1A1 PositiveCYP1A1 NegativeOR (95% CI)*P
  • CYP1A1 indicates cytochrome p450 enzyme 1A1; OR, odds ratio; CI, confidence interval.

  • *

    Adjusted for age at enrollment and smoking status.

All patients18 (46.2)21 (53.9)  
Randomized to β-carotene    
 No12 (60)8 (40)1.0 (Referent) 
 Yes6 (31.6)13 (68.4)0.2 (0.1-1.1).06
Smoking status    
 Never4 (50)4 (50)1.0 (Referent) 
 Past8 (61.5)5 (38.5)1.5 (0.3-9.1).66
 Current6 (33.3)12 (66.7)0.5 (0.1-2.8).44
β-Carotene/smoking    
 Non-β-carotene/never-smoking3 (50)3 (50)1.0 (Referent) 
 β-Carotene/never-smoking1 (50)1 (50)0.8 (0.0-23.5).90
 Non-β-carotene/past-smoking5 (100)0 (0) 
 β-Carotene/past-smoking3 (37.5)5 (62.5)0.5 (0.1-5).58
 Non-β-carotene/current-smoking4 (44.4)5 (55.6)0.7 (0.1-6.1).75
 β-Carotene/current-smoking2 (22.2)7 (77.8)0.3 (0.0-2.9).30
Age at enrollment, y    
 ≤6011 (42.3)15 (57.7)1.0 (Referent) 
 >607 (53.9)6 (46.2)1.4 (0.3-5.6).65
Age at diagnosis, y    
 ≤7011 (57.9)8 (42.1)1.0 (Referent) 
 >707 (35)13 (65)0.4 (0.1-1.4).13
Tumor type    
 Small cell carcinoma1 (25)3 (75)0.8 (0.1-12.2).86
 Adenocarcinoma10 (47.6)11 (52.4)1.0 (Referent) 
 Squamous cell carcinoma3 (37.5)5 (62.5)0.9 (0.1-7.0).94
 Large cell carcinoma2 (100)0 (0) 
 Combined squamous/adenocarcinoma1 (100)0 (0) 
 Others1 (33.3)2 (66.7)0.4 (0.0-6.3).53
Histologic grading/differentiation    
 Well/moderately differentiated2 (25)6 (75)1.0 (Referent) 
 Poorly differentiated8 (57.1)6 (42.9)13.3 (1.1-156.3).04
 Undifferentiated3 (50)3 (50)14.7 (0.8-269.7).07
 Missing5 (45.5)6 (54.5)5.1 (0.5-52.9).17
Distant metastasis    
 No5 (33.3)10 (66.7)1.0 (Referent) 
 Yes13 (54.2)11 (45.8)3.2 (0.7-15.5).15

With regard to randomized aspirin treatment, there were no differences in the positivity for p53 (OR, 1.0; 95% CI, 0.2-4.0), PCNA (OR, 1.1; 95% CI, 0.3-4.2), or CYP1A1 (OR, 1.1; 95% CI, 0.3-4.4) between individuals who were assigned to receive aspirin versus aspirin placebo; however, the positivity for RARβ (OR, 0.7; 95% CI, 0.2-2.7) and cyclin D1 (OR, 0.5; 95% CI, 0.1-1.8) was nonsignificantly lower among those who were assigned to receive aspirin. The results for p53, RARβ, cyclin D1, PCNA, and CYP1A1 according to randomized β-carotene treatment did not change appreciably after also controlling for randomized aspirin treatment assignment (data not shown).

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Conflict of Interest Disclosures
  7. References

In the PHS main trial, the parent study of this investigation of lung tissue molecular markers, the 50-mg β-carotene treatment on alternate days provided no benefit or harm on lung cancer development.6 The relative risks of lung cancer by randomized β-carotene assignment in the PHS, which had 11%, 39%, and 50% of current, past, and nonsmokers at baseline, respectively, were 0.90 (95% CI, 0.58-1.40) for current smokers, 1.00 (95% CI, 0.62-1.61) for former smokers, and 0.78 (95% CI, 0.34-1.79) for nonsmokers.6 In the current study, which involved lung cancer patients within the PHS, the positivity for total p53, RARβ, cyclin D1, PCNA, and CYP1A1 was nonsignificantly lower in patients who were assigned to receive β-carotene than those who were assigned to receive β-carotene placebo. The results of lung tissue molecular markers by randomized β-carotene supplementation were not affected by randomized aspirin treatment assignment. The nonsignificant results from lung tissue molecular markers are consistent with the main PHS trial result of β-carotene on lung cancer risk.

By using the ferret animal model, we previously provided the first in vivo evidence that high-dose β-carotene (equivalent to the 30 mg daily dose of β-carotene used in the CARET trial), cigarette smoke exposure, and their combination substantially increased protein levels of total p53, which represents p53 accumulation.17 By contrast, low-dose β-carotene (equivalent to the 6 mg daily dose of β-carotene attainable from a diet high in fruits and vegetables) had no influence on total p53 in nonsmoke-exposed ferrets but reduced total p53 induced by cigarette smoke exposure in ferrets.17 In addition, high-dose β-carotene and smoke exposure increased levels of cyclin D1 and PCNA and increased squamous metaplasia in the lung tissue of ferrets, whereas low-dose β-carotene had no potentially detrimental effects and even slightly decreased cell proliferation or squamous metaplasia induced by cigarette smoke in ferrets.16 When combined with β-tocopherol and ascorbic acid, both doses of β-carotene reduced cigarette smoke-induced squamous metaplasia and restored retinoic acid concentrations in ferrets.23 Combined β-carotene, α-tocopherol, and ascorbic acid also prevented cigarette smoke-induced and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced up-regulation of p53 and decreased both preneoplastic and neoplastic lesions in ferrets.23

Because malfunction of p53 may result in increased cell proliferation and, ultimately, tumor development and progression, elevated levels of p53 accumulation may serve as a marker for monitoring genotoxic effects and tumorigenesis. A nonsignificantly lower positivity for total p53, cyclin D1, and PCNA levels in the β-carotene group observed in lung cancer patients in the PHS is consistent with the results of the low-dose β-carotene group from the in vivo ferret animal model studies.16 In the PHS, blood β-carotene levels were 4 times higher in the β-carotene group than in the β-carotene placebo group (1.2 mg/L vs 0.3 mg/L).6 Similarly, blood β-carotene levels also were approximately 4 times higher in the low-dose β-carotene group than in the control group in the ferret study (25 ± 5 nmol/L vs 7 ± 3 nmol/L).16 In the CARET and ATBC studies, 2 trials that demonstrated a harmful effect of β-carotene supplementation on lung cancer, there were approximately 12 times and 17 times differences in blood β-carotene levels between the β-carotene group and the β-carotene placebo group, respectively (2.1 mg/L vs 0.18 mg/L in the CARET study8 and 3.0 mg/L vs 0.18 mg/L in the ATBC study7), It is noteworthy that the enhancement of smoke-induced lung lesions was observed in the ferrets with blood β-carotene levels that were also 17 times higher than those in the control group.16 Among 3 large, randomized β-carotene trials, the PHS had much higher levels of blood β-carotene in the β-carotene placebo group compared with the levels for those groups in the CARET and ATBC studies (0.3 mg/L vs 0.18 mg/L vs 0.18 mg/L, respectively) but had much lower levels of blood β-carotene in the β-carotene group (1.2 mg/L vs 2.1 mg/L vs 3.0 mg/L, respectively).6-8

CYP1A1, which is a phase I metabolizing enzyme, is expressed preferentially in the lung,24 where it is inducible and converts procarcinogens into highly reactive intermediates that bind to DNA, forming adducts.14 It has been demonstrated that high-dose β-carotene with or without smoke exposure induces CYP1A1 in the lung,15, 25 which leads to enhanced retinoic acid catabolism, resulting in decreased retinoic acid levels and diminished retinoid signaling in animal models.16, 26 Previous data in rats suggest that β-apo-8′-carotenal, an excentric cleavage product of β-carotene, but not intact β-carotene, stimulates the induction of CYP1A1.27 Our previous studies in ferrets demonstrated that the formation of β-apo-carotenals and other oxidative excentric cleavage products of β-carotene was enhanced by smoke exposure,26 indicating that β-carotene is unstable in the free radical-rich environment of the lungs in smokers. Thus, the induction of CYP1A1 by oxidative cleavage products of β-carotene, high-dose β-carotene, or smoke exposure in the lung may bioactivate carcinogens and abolish retinoic acid, thereby enhancing lung carcinogenesis. In addition, these oxidative excentric cleavage metabolites of β-carotene themselves may be involved directly in the carcinogenic process.28 In the current study, the 50-mg β-carotene supplementation on alternate days in the PHS lowered lung CYP1A1 levels, suggesting that this regimen may confer some protection against lung carcinogenesis at molecular levels. These data also suggest that the results of β-carotene trials may be related to the doses of β-carotene that were used and/or instability of the β-carotene molecule in the lungs of cigarette smokers, which are rich in free radicals.

RARβ messenger RNA was undetectable by in situ hybridization in approximately half of nonsmall cell lung cancers.29 Restoration of RARβ2 in a RARβ-negative lung cancer cell line also reportedly inhibited tumorigenicity in nude mice.30 In addition, 9-cis-retinoic acid inhibited lung carcinogenesis in the A/J mouse model, which was accompanied by increased expression of RARβ.31 These data suggest that loss of RARβ is associated with lung carcinogenesis. Treatments with 9-cis-retinoic acid in former smokers up-regulated RARβ expression in the bronchial epithelium but had no significant effect on squamous metaplasia.32 Strong RARβ expression also has been associated with a significantly worse outcome in patients with early-stage nonsmall cell lung cancers.33 In the current study, RARβ positivity was nonsignificantly lower among men who were assigned to receive β-carotene than among those who were assigned to receive the placebo. In the ferret study, RARβ levels in lung tissue did not change in the low-dose β-carotene group16 but were down-regulated in the high-dose β-carotene groups (alone or with smoke exposure).16, 26 Future studies to illustrate the role of RARβ in lung carcinogenesis are warranted.

In summary, our data suggest that 50-mg β-carotene supplementation on alternate days had no significant influence on the molecular markers of lung carcinogenesis that we evaluated in the PHS. This finding provides mechanistic support for the main PHS trial results of β-carotene, which indicated no benefit or harm on the risk of lung cancer.

Conflict of Interest Disclosures

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Conflict of Interest Disclosures
  7. References

Supported by grants CA34944, CA40360, CA097193, HL26490, and HL34595 from the National Institutes of Health and by an investigator-initiated research grant from the BASF-The Chemical Company.

The authors made no other disclosures.

References

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Conflict of Interest Disclosures
  7. References
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