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THE EFFECT OF BETA-CAROTENE ON LUNG CANCER Lai Bai-tang, Wang Hui. Beijing Thoracic Tumor Research Institute, Beijing, China 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 far lower that of norma]. persons. The relative risk of lung cancer among subjects with low beta-carotene was significantly elevated. The purpose of this study was to investigate the effect of beta-carotene on lung cancer in vivo and in vitro. MATERIAL AND METHODS 1. THE EFFECT OF BETA-CAROTENE ON COLONY FORMING ABILITY OF LUNG CANCER CELL LINE. The human large cell lung cancer line 801 was obtained from Hospital 301 in Beijing and was maintained in glass culture bottles containing 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. The beta-carotene was dissolved in DMSO and diluted to 6.25 Ag/ml and 3.125 µg/ml. The two different concentrations of beta-carotene were then added into the dish-plated cells. After treating cells for 24, 48, 72 hours, the medium was changed to one without beta-carotene. After 14 days the dishes were stained with Giemsa. Colonies containing more than 10 cells were counted, Number of colony formed x 100% CFE = Number of cells plated (500) 2. THE EFFECT OF BETA-CAROTENE ON SPONTANEOUS METASTASIS IN MICE WITH ADENOCARCINOMA. LA795 mice with lung adenocarcinoma were purchased from Tanzing Medical Pharmacy Institute. Tumors were taken from LA795 mice to make 1 x 107/ml cell suspension. The 0.2 ml cell suspension (2 x 106 cells) was transplanted into the T739 mice by 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 µg/100g) from 2 weeks before transplanting the tumor cells to 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 the steromicroscope, and the percentage of decrease in the treatment group was calculated. - 2 -

3. THE EFFECT OF BETA-CAROTENE ON SYNTHESIS OF DNA AND RNA IN lIUMAN LUNG CANCER CI;I,i,S AND LYMPHOCYTES. A 1 x 105/mi suspension of 801 cells was planted into 96 wells in polystyrene plates. Each well contained 1 ml suspension of 801 cells. After 24 hours, the medium was replaced by one containing 25 µg/ml beta-carotene and DMSO as a solvent control, [3H]-hymidine (Tdr) or [3H]-uridine (Urd) (1 µCi/ml) was added into each well for two hours. The Cpm (counts per minute) value of cells in each well was determined with a-21oG Scintillation counter. The average value of the wells was regarded as the value of a group. Using the same method, human lymphocytes were counted. 4. THE EFFECT OF BETA-CAROTENE ON EXPRESSION OF RAS GENE IN CELLS This study utilized the Neo-ras cell line with high expression ras genes (a 3T9 cell line transfected with ras genes obtained from Australia Biological Institute). 1 x 104/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) and by DMSO as a solvent control. After 24 hours, the coverglasses (with cells) were removed to dry in air, and fixed with cold acetone. The cells were stained with monoclonal antibody against p21 II-ras-horseradish-peroxidase RAM- IgD and examined by microscopy. - 3 -

i RESULT 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 Ag/ml and 3.125 µg/ml), and different treatment time (24, 48, 72 hours) on colony formation of 801 cells was investigated. The CFE of 801 cells exposed to beta-carotene at concentration of 6.25 µg/ml for 24 hours was inhibited significantly. Table1 1 shows results of the inhibitory effect of beta-carotene on colony forming efficiency for 801 cells. 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 inhibiting effect on lung cancer depending on concentration and time that the cells are exposed to beta-carotene. 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 under the steromicroscopy. Table 2 shows the results. We compared the metastatic lesions in lungs of mice in the B-C group with those in the control one. A 42-68% (p<0.01) decrease in spontaneous lung metastasis of LA795 murine pulmonary adenocarcinoma was observed - 4 -

when T739 inbred strain mice were fed beta-carotene (25 mg/100g) diets. The action of the beta-carotene against metastasis of cancer cells in the mice was demonstrated. 3. THE EFFECT OF BETA-CAROTENE ON ABILITY OF DNA AND RNA SYNTHESIS OF LUNG CANCER CELLS. Table 3 shows that the three experiments have the same results. Incorporation rate of 3H-thymidine (TDR) or 3H-uridine (UDR) in the cells exposed to B-C was apparently decreased (p<0.05), but this decrease did not appear in human lymphocytes. The results indicated that the ability the synthesize DNA and RNA in lung cancer cells was inhibited by beta-carotene. There was no inhibitory effect 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 p2lH-ras-HRP bound to RAM- IgGw There were many dark-blue precipitates under the membrane of the Neo-ras cells. When the cells were exposed to beta-carotene for 24 hours, the dark-blue precipitates were apparently decreased. This suggests that expression of the ras gene in Neo-ras was inhibited by B-C. - 5 -

DISCUSSION In recent years, it has been shown that beta-carotene inhibits the development of animal tumors induced by many carcinogens. In human epidemiological research, it has been proven that there is a relationship between beta-carotene concentration in serum and the relative risk for lung cancer. Stabelins et al surveyed 2,874 men from 1971 to 1878 and measured beta-carotene in their serum during the 12 years of observation, 533 men died, 204 of cancer (lung cancer 68, stomach cancer 30, colon cancer 17, all other malignancies 99). 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 2421 survivors was 0.428 4mol/1. Serum B-C was 0.217 Amol/l in 68 lung cancers. 0.274 4mol/1 in 20 stomach cancers, and 0.342 µmol/1 for all the other cancers group. The relative risk for lung cancer of stabjects with low beta-carotene (less than 0.23 4mol/1) was significantly elevated (p<0.05). If B-C in serum is lower than 0.28 µmol/1, the incidence of cancer will increase by 1.74 2.26 times. The present study proved an inhibitory action of B-C on human lung cancer in vivo and in vitro. Joel at al. found an inhibitory effect of B-C on cancer cells at high concentrations in vitro. When lung cancer cell line SK-MES was exposed to beta-carotene at 78 µmol/l or 300 '.imol/1, a 70-80% decrease in cell density was noted. In our experiments, we have observed the effect of beta- carotene un/una cancer cells at a lower concentration. When 8o1 - 6 -

cells were exposed to beta-carotene at 6.25 gg/ml, colony forming efficiency was inhibited. Complete inhibition was seen at 12.5 µg/ml. This indicates that beta-carotene at low concentration inhibits multiplication of lung cancer (PH). LA795 murine lung adenocarcinoma is a highly malignant lung cancer. When LA795 tumor is transplanted into T739 inbred mouse by subcutaneous, muscular or peritoneal injection, cells of the tumor can 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, when mice were fed a diet with beta-carotene (25 mg/100 diet). From the results, we think that it is possible to use beta- carotene 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 beta-carotene at 10 µg/ml by 4 hours, N-myc gene expression was decreased. The author thought that this was an important mechanism against multiplication of cancer cells. In our experiment it was shown that beta-carotene at 12.5 gg/ml can decrease expression of P21 protein of the Ras gene. We think perhaps a decrease in P21 in - 7 -

cells is associated with inhibition of cell multiplication. Another possible mechanisms of action is stimulation of the immune system as suggested by Soda et.al. Alexander gave volunteers beta-carotene (180 ml/day). After two weeks, okT4, okT3 lymphocytes and serum of beta-carotene were increased. Among 5 volunteers taking beta-carotene 30 mg/per day, three had an increase in okT4 cells, and four had an increase in the ratio. It is indicated that beta-carotene has effects on the distribution of sub-populations of lymphocytes. In our experiment, we have also observed a T4/T8 rate increase in the serum of volunteers taken B- C. From our experiment we know beta-carotene at 12.5 µg/ml can inhibit synthesis of [)N11 and RNA in co118, but it has no eftact on lymphocyte. Thus, beta-carotene could be used to lower the incidence of lung cancer and to prevent replace and metastasis in postoperative lung cancer patients. CONCLUSION 1. Beta-carotene at a concentration of 6.25 gg/ml was shown to inhibit significantly the colony forming efficiency (CFE) of cultured human lung cancer 801 cell line. 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 r.cg/100g diet) - 8 -

3. The synthesis of DNA and RNA in 801 cells was decreased (p<0.05) after treating the cells by beta-carotene for 24 hours. 4. Expression of P21 Ras genes in Neo-ras cells was inhibited by beta-carotene.

TABLE 1 THE EFFECT OF BETA-CAROTENE ON COLONY FORMING ABILITY OF LUNG CANCER CELLS (COLONY NUMBERS/DISH) Dose ({t/ml ) treatment time (hr) 3.125 6.25 12.5 DMSO control CFE (X) B-C CFE (X) DMSO CFE (X) B-C CFE (X) DMSO CFE (X) B-C CFE (X) 24 44 42 41 15 36 0 41(43.3) 0.6 47(42.0) 0.5 40(39.0) 7.9 17(14) 2.8 31(30.0) 6.1 0(0) 0 45 39 38 10 39 0 48 55 45 51 27 39 0 52(53) 10.6 51(50.3) 10 45(47) 9.4 26(25.7) 5.1 33(31.3) 6.2 0(0) 0 52 55 45 24 32 0 72 55 62 47 8 20 0 (50.7) 11.34 59(54.7) 10.9 45(44.7) 8.9 8(8.7) 1.7 15(19.5) 3.8 0(0) 0 55 43 42 10 20 0 0 for mean colony numbers of each dish