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I I I I I I I I I I I I I I I I I I THE EFFECT OF BETA-CAROTENE ON LUNG CANCER Lai Bai-tang and Wan Hui Beijing Tuberculosis and Thoracic Tumor Research Institute, Beijing, China Introduction As a pro-vitamin A, beta-carotene is present in abundance in green peppers, carrots, and pumpkins. A re-examination of evidence from prospective and case-control studies allowed Peto et al. to hypothesize in 1981 that dietary beta-carotene (B-C) has a preventive role against cancer. More recently, studies on beta-carotene in rats and mice have shown that beta-carotene has a protective action against tumors induced by different carcinogens at various sites including the skin, oral cavity, salivary gland, colon and bladder. Results from epidemiologic studies have also indicated that the concentration of beta-carotene in the serum of patients suffering from lung cancer is much lower than normal persons. The relative risk of lung cancer among subjects with low beta-carotene intake was significantly elevated. The purpose of this study was to investigate the effect of beta-carotene on lung cancer in vivo and in vitro. Materials and Methods 1. The Effect Of Beta-Carotene On Colony Forming Ability Of Lung Cancer Cells. The human large lung cancer cell line 801 was obtained from Hospital 301 in Beijing and was maintained in glass culture bottles containing Dubecco's Minimal Essential Medium (DMEM) and 10% newborn calf serum. The 801 cells were plated in polystyrene petri dishes at a density of 500 cells per dish and then cultured in a CO2 incubator. Beta-carotene was dissolved in dimethylsulfoxide (DMSO) and diluted to 3.125 µg/ml and 6.25 µg/ml. The two different concentrations of beta-carotene were then added into the dish-plated cells. After treating cells for 24, 48, 72 hr, the medium was changed to one without beta-carotene. After 14 days the dishes were stained with Giemsa stain. Colonies containing more than 10 cells were counted. The colony forming unit (CFE) was defined by the equation: CFE= No. of colonies formed x 100% No. of cells plated (500) ~ 0 ~ i -4 00 W ~ W A I

I 2. The Effect Of Beta-Carotene On Spontaneous Metastasis In Mice With Adenocarcinoma. LA795 mice with lung adenocarcinoma were purchased from Tanzing Medical Pharmaceutical Institute. Tumors were taken from LA795 mice to make a Ix107/ml cell suspension. Two-tenths of a ml cell suspension (2 x 106 cells) were transplanted into T739 mice using the sub-cutaneous route. Tumors were taken from the mice when they had grown to 1 cm in size. The "treated" mice had been on diets containing B-C (25 mg/100g) for 2 weeks before injection of the tumor cells and continued on the same diet for 2 weeks after the resection of tumors. In order to compare the treatment group with controls, the lungs were taken from the mice four weeks after tumor resection. The metastatic lesions in the lungs were counted under a steromicroscope, and the percentage of decrease in the number of lesions in the treatment group was calculated. 3. The Effect Of Beta-Carotene On Synthesis Of DNA and RNA In Human Lung Cancer Cells And Lymphocytes. A 1 x 105/ml suspension of 801 cells was plated into 96 wells in polystyrene plates. Each well contained 1 ml suspension of 801 cells. After 24 hr, the culture medium was replaced by a medium containing 25 µg/ml beta-carotene using dimethyl sulfoxide (DMSO) as a solvent control, [3H]-thymidine (Tdr) or [3H]-uridine (Urd) (1 µCi/ml) was added into each well for two hr. The cpm (counts per minute) value of cells in each well was determined with a 210G scintillation counter. The average value of the counts in wells was considered as the value of a group. The incorporation of label into human lymphocytes was counted using the same method. 4. The Effect Of Beta-Carotene On Expression Of RAS Gene In Cells This study utilized the Neo-ras cell line with a high expression of the ras gene (a 3T9 cell line transfected with ras genes obtained from Australia Biological Institute). 1 x 104 cells/ml were plated into 24 polystyrene dishes. Each well contained 1 ml medium (1 x 104 cells). On the next day, the medium was replaced by one containing beta-carotene (12.5 µg/ml) using DMSO as a solvent control. After 24 hr, the coverglasses (with cells) were removed, dried in air, and fixed with cold acetone. The cells were stained with monoclonal antibody against p21 II-ras-horseradish-peroxidase RAM-IgD and examined under a microscope. Results 1. The Effect Of Beta-Carotene On Colony Forming Ability Of Lung Cancer Cells The inhibitory action of beta-carotene at different concentrations (6.25 µg/nil and 3.125 µg/ml), and different treatment time (24, 48, 72 hr) on colony formation of 801 cells was investigated. The CFE of 801 cells exposed to beta-carotene at a concentration of 6.25 µg/ml for 24 hr was inhibited significantly. (Table 1). - 2 - I I I ' I I I I I 1 I I I t

I I I I I I I I I I I I I I The inhibitory effect increased with the time that the cells were exposed to beta-carotene. Beta- carotene at 12.5 µg/ml was shown to completely inhibit CFE. The results indicated that beta-carotene has a concentration-and-time-dependent inhibitory effect on growth of lung cancer cells. 2. The Inhibitory Effect Of Beta-Carotene On Spontaneous Metastasis Of Murine Pulmonary Adenocarcinoma. TA795 murine adenocarcinoma used in our experiment is a tumor with high malignancy. It is very likely to metastasize to the lungs of inbred T739 mice. We counted metastatic lesions in the lungs of the mice fed B-C and in control groups. The results are shown in Table 2. When the metastatic lesions in lungs of mice in the B-C group were compared with the controls, a 42-68% decrease in spontaneous lung metastasis of LA795 murine pulmonary adenocarcinoma was observed in the treated group (p<0.01). 3. The Effect Of Beta-Carotene On DNA And RNA Synthesis In Lung Cancer Cells. Table 3 shows the results of three separate experiments with similar results. The incorporation of 3H-thymidine (TDR) or 3H-uridine (UDR) in the "B-C-treated" cells was significantly decreased, (p<0.05). Compared to controls there was no inhibitory effect of B-C on the synthesis of DNA and RNA in human lymphocytes (Table 4). 4. The Inhibitory Effects Of Beta Carotene On RAS Gene Expression In Lung Cancer Cells. The Neo-ras cells (with high expression ras genes) were stained by monoclonal antibody against p21H-ras-HRP bound to RAM-IgG. There were many dark-blue precipitates under the membrane of the Neo-ras cells. When the cells were exposed to beta-carotene for 24 hr, the dark-blue precipitates were apparently decreased. This suggests that expression of the ras gene in Neo-ras was inhibited by B-C. Discussion In recent years, it has been reported that beta-carotene inhibits the development of animal tumors induced by many carcinogens. A relationship between beta-carotene concentration in serum and the relative risk for lung cancer has been suggested by epidemiologic studies. Stabelins et al. surveyed 2,874 men from 1971 to 1978 and measured beta-carotene in their serum. Among the 533 mortalities in the 12 year study, 204 died of cancer (lung cancer 68, stomach cancer 30, colon cancer 17, all other malignancies 99). Interestingly, the mean concentration of B-C in the serum of men who died from cancer was significantly lower than in the survivors. The mean beta-carotene concentration in the serum of 2341 survivors was 0.428 µmol/L, but was 0.217 µmol/L in 68 lung cancer cases, 0.274 µmol/L in 20 stomach cancers, and 0.342 µmol/L for all the other cancer groups. The relative risk for lung cancer of subjects with low beta-carotene (less than 0.23 µmol/L) was significantly elevated (p < 0.05). In fact, when B-C in serum is lower than 0.28 µmol/L, the incidence of cancer will collectively increase by 1.74- 2.26 times. The present study provided evidence for an inhibitory effect of B-C on human lung cancer in vivo N 0 and in vitro. Joel et al. found an inhibitory effect of B-C on cancer cells at high concentrations in vitro. co ~ ~ 00 - 3 - W ~ W M I

I When lung cancer cell line SK-MES was exposed to beta-carotene at 78 µmol/L or 300 µmol/L, a 70- 80% decrease in cell density was noted. In our experiments, we have observed a lower concentration of beta-carotene to exert an inhibitory effect on lung cancer cells. When 801 cells were exposed to beta- carotene at 6.25 µg/ml, the ability of the cells to form colonies was inhibited. Complete inhibition was seen at 12.5 µg/ml. LA795 murine lung adenocarcinoma is a highly malignant lung cancer. When LA795 tumors were transplanted into T739 inbred mice by subcutaneous, muscular or peritoneal injection, cells of the tumor could spontaneously metastasize to the lung. In our experiments, when T739 tumors grew to 1 cm diameter and were then resected, a 42-68% (p<0.01) decrease in spontaneous lung metastasis was observed in mice fed a diet supplemented with beta-carotene (25 mg/100 diet). From these results, it can be hypothesized that beta-carotene can be used to prevent relapse and metastasis in postoperative patients with lung cancer. Santamaria et al. used beta-carotene to prevent relapse of 15 cases with cancer after operation. Results show that survival of patients with cancer (including lung cancer, colon cancer, bladder cancer, head and neck cancer) was longer in those who used B-C. The mechanism of action of beta-carotene on cancer cells has been studied. Okuzumi found that in cells exposed to 10 µg/nil beta-carotene for 4 hr, N-myc gene expression was decreased. Our studies show that beta-carotene at 12.5 µg/ml can decrease Ras gene expression which in turn raises the possibility that p21, product of the Ras gene, is associated with lung cancer cell multiplication. Another possible mechanism of the action of B-C, suggested by Soda et al., is the stimulation of the immune system. Volunteers given beta-carotene (180 ml/day) for two weeks were found to have elevated okT4, okT3 lymphocytes as well as an increase in serum beta-carotene. In our studies we also observed an increase in a T4/T8 ratio in the serum of volunteers taking B-C. Conclusion 1. Beta-carotene at a concentration of 6.25 µg/ml was shown to inhibit significantly the colony forming efficiency (CFE) of cultured human lung cancer 801 cells. Complete inhibition occurred at 12.5 µg/ml. 2. A 42-68 % decrease in spontaneous lung metastasis of TA739 murine pulmonary adenocarcinoma on T730 inbred mice was observed when the mice were fed a diet with beta-carotene (25 mg/100g diet) 3. The synthesis of DNA and RNA in 801 cells was decreased (p <0.05) after treating the cells with beta-carotene for 24 hr. 4. Expression of p21 Ras gene in Neo-ras cells was inhibited by beta-carotene. 00 ~ ~ - 4 - rp W I I ' I I I I I 1 , I I I I I I

I ' I I I I I I I I I I I I , Table 1. The Effect of Beta-carotene on Colony Forming Ability of Lung Cancer Cells (colony numbers/dish) Dose (µgtml) . . . . Treatment 3.125 6.23 . . - A2.5 Tmte (hr) . . . .. . DM50 CFE 13-C CFE DMSO CFE B-C - CEE- . DMSO .:.~ CFE. B-. CF6: Control (9b) (%) (%) (%) . (%) . C.. t%F . 2A 44 42 41 15 36 0 41 (43.3) 0.6 47 (42.0) 05 40 (39.0) 7.9 17(14) 2,8 31 (30.0) 6.1 0 0 (0) 45 39 38 t0 39 0 48 55 45 51 27 39 0 52 (53) 10.6 51 (50.3) 10 45 (47) 9.4 26 (25.7) 5.l 33 (31.3) 6.2 0 0 (0) 52 55 45 24 32 0 72 55 62 47 8 20 0 42 (50.7) 11.34 59 (54.7) 10.9 45 (44.7) 8.9 8(8.7) 1.7 15 (18.5) 3.8 0 0 (0) 55 43 42 10 20 0 O for mean colony numbers of each dish Table 2. The Inbibitory Effects of Beta-carotene on Spontaneous Lung Metastasis in T739 Mice Experiment Group Nmnber Body Lung Nmnber Mean . ~ Decrease . of Wcight Weight of Nmnb[33 i peftewa8e Animals Animals of of wl[b metzstatie poststatie wmors/tota) - tuwors (%). mice I DMSO 17 27.2 0.34 14/17 35.2 B-C 17 27.3 0.34 13/17 11.2° 68 II DMSO 24 24.2 0.19 24/24 50.1 B-C 24 24.1 0.26 20/24 29° 42 ~ a p<0.01 N O i V CO W ~ C.f CO I

I Table 3. The Effects of Beta-carotene on DNA and RNA Synthesis in Lung Cancer Cell DNA Mean ineotpomtion on , , - RNA Mean 5ncerpottitlort ,~ . cpm3N-thymidine(Tdr) cpm3N-ruidine(Udr) Fipcriment DM50 comrol . B-C. ' DMSO cunttul .. i1-C . 1 100 78 118 78 128 (1l0) 90 (873) I52 (154) 64(81) 102 94 162 102 II 55 50 173 103 73 (67.3) 56 (53) 158 (I71) 108 (105) 63 50 183 104 [II 72 56 236 194 72(70) 62(50) 258 (246) 104 (178) 60 58 248 178 O for mean cpm of groups Table 4. Effect of Beta-carotene on DNA Synthesis of DNA in Isolated Lymphocytes Incorparatlan of mcan 3A tbymitline (RIr) Expuimant Db151)cbnorol -&C . 1 72 102 88(81) 90(82) 82 62 78 74 II 120 170 312 (249) 3l4 (248) 318 258 I11 170 164 188 (100) 188 (172) 140 166 O for mean cpm of groups N ~ 3 v ~ - 6 - ~ W I 1 I I I I I I 1 I I I I I I I