Anti-proliferative Action Of Dehydroepiandrosterone On Human Cancer Cell Lines And Its Molecular Mechanism

Recently, official statistical report issued by Ministry of Health show: in our country, there are 1,600,000 new cases of malignant tumors and about 1,300,000 deaths per year. Of average five deaths,one succumb to malignant tumor. In every 200 families, there is one subjected to the strike of malignant tumours development or death. Only as far as Beijing be concerned, there are about 12,000 new cases and nearly 10,000 deaths per year. among them,male malignant tumours in first five mortality are lung cancer, liver cancer, gastric cancer, esophageal carcinoma,and intestinal cancer in turns. Female malignant tumours in first five mortality are lung cancer, liver cancer, gastric cancer, breast cancer, and esophageal carcinoma in turns. With the increace of population quantity and the acceleration of population aging, the morbidity and mortality of tumors present a upgrade tendency. Therefore, prophylaxis and therapy of tumors are constantly focuses in medical science territory of our country. Dehydroepiandrosterone (DHEA) is a 19-carbon endogenous steroid prehormone, major secretary product of the human adrenal cortex. DHEAS is sulfate metabolite of DHEA, which convert to androgen-testosterone, estrogen-estrone, and corticosteroid hormone, so it is considered an important precursor of steroid hormone. DHEA itself may also have extensive biologic activities. The serum level of DHEA and DHEAS different with age and sex in human, Serum DHEAS levels are very high during newborn period, the mean is 3490ng/ml; they peak in female during the ages of 15-19 years, the mean is 2479ng/ml. the levels decrease gradually with aging in both sexes .at 70 years of age, DHEAS concentrations are only 670ng/ml in male and 450ng/ml in female, the levels are only 1/3 of those in young adults or more less. In the 1980s, some researcher found that the reduced serous level of DHEA was negative correlated with carcinogenesis rate; they suggested that the reduction of serous DHEA concentrations is one of the tumorigenic risk factors .In addition, age-associated serous DHEA concentrations changes also accompany with increased morbility of cardiovascular disease and decreased organism immunological function. Thus DHEA was considered to have a anti-aging effect, accordingly leading to the DHEA and DHEAS research upsurge in the 1990s. Researches show that reduction of serous DHEA concentrations prone to development breast cancer and bladder carcinoma. Long-term supplying DHEA has obvious chemoprevention action on a variety of animal tumor models, such as breast cancer, hepatoma, colon cancer, and lung cancer in rats. However, its molecular mechanisms of action are not complete understanding, to further identify the molecular mechanisms of DHEA and the actions of its metabolites, we take human hepatoblastoma cell line (HepG2) and human colonic adenocarcinoma cell line (HT-29) as study models in vitro, rats transplatnted Morris hepatomas as bearing cancer models in vivo, researched the inhibitory action of DHEA and DHEAS on tumor growth and its mechanism of action. DHEA inhibit tumor growth (1)Experiments in vitro: We analyzed the Anti-proliferative activities of DHEA on HepG2 and HT-29 cells by MTT assay and BrdU assay. with the increasing of pharmacal concentration (1,10,50,100,200μΜ),the percentages of HepG2 and HT-29 survival cells are 92.7±0.93%,84.7±1.23%,62.4 ±0.83%,42.2 ±0.63%,50.7 ±0.23% and 92.5 ±0.35%,89.5 ±0.69%,80.5 ±1.12%,65.8 ±0.94%,70.6 ±0.63%, respectively. The results suggest that with the increasing of pharmacal concentration, the Anti-proliferative activities present increasing tendency. Glucose-6-phosphate dehydrogenase (G6PD), an initial enzyme of phospho-pentose metabolism, directly determines the levels of cellular DNA anabolic metabolism. For this reason, we further analyzed G6PD activities when above-mentioned tumour cells are incubated by DHEA for 24hr, then we found that the G6PD activities of the two tumour cells both obviously decrease , which show positive correlation with cell proliferation(r=0.959,n=6,P<0.05). qualities of DHEA converted from effective inhibiting concentrations are 100μΜ=28.84mg·L-1,200μΜ=57.68mg·L-1,they demonstrate that DHEA inhibiting tumor growth reaches mildigram order of magnitude.These results conclude: DHEA itself is not a antineoplastic chemotherapy medicine apparently; but DHEA interfere G6PD activities obviously, and inhibit cellular DNA synthesis, these suggest that DHEA can act as a supplementary medication in tumor therapies. (2)Experiments in vivo: we administer 600mg·kg-1 of DHEA to the rats of bearing Morris hepatomas model everyday. after 4 weeks , the tumor weights of DHEA treated group (8.31±0.61 g)were obviously less than those of the control(14.57±0.56 g), the inhibitory rate reaches 43%(P<0.05) .The results conform to those of the experiments in vitro, under the large dose conditions, DHEA showed inhibitory action on tumors. Meanwhile, model rats had not adverse reactions, the serum density indexes of Total-cholesterol, TG and HDL–C all did not present anomal-change, and critical organs had not structural desease by pathological assay. Conclusion: DHEA can act as a co-medicine in tumor therapies, and also can beused for prophylaxis before tumors development. Tumour cell apoptosis and cell cycle blockage induced by DHEA Annexin V is a phospholipid-conjugated protein. Phospholipids extroverted at earlier period of apoptosis can specifically bind Annexin V. Therefore fluorescent labelling AnnexinV and propidine iodide (PI) incorporation assays becomes a sensitive index to detect the early apoptotic cells. our results show that When the cells were treated with 100μΜof DHEA, the percentage of apoptotic HepG2 cells reaches the highest value (18.26±2.56%); using PI labelling to detect cell cycle, at 100μΜof DHEA, the percentage of HepG2 cells in G0/G1 stage reaches 82.4±1.9% while that of the control group is only 48.6±1.2%; the percentage of HT-29 cells in G0/G1 stage is much higher to 90.3±2.7% while that of the control group is only 59.1±1.2%. Conclusion: DHEA can induce HepG2 cells apoptosis and result in G0/G1 stage blockage of HepG2 cells and HT-29 cells. Phosphorylation of Akt was blocked by DHEA Phosphatidylinositol3-kinase(PI3K)is a important member of growth factor receptors superfamily , participating intracellular signal transduction. Activated PI3K act on phosphatidylinositol-3,4-diphosphate located on cytomembrane , which is converted to phosphatidylinositol-3,4,5-triphosphare, then activate serine/threonine kinase Akt (also called protein kinase B,PKB) by activated phosphoinositol-dependent protien kinase PDK, the activated PI3K through the activation of proteins or enzymes such as Caspase-9,FKHR,Bad,and GSK-3 by Ser473 and/or Thr308 phosphorylation promotes cell growth and suppresses apoptosis. PTEN (phosphatase and tensin homology detected on chromosome ten) catalyze the dephosphorylation of tyrosine and serine/ threonine, then depress PI3K activities indirectly, its mechanisms of actioncorrelate with PI3K/Akt signaling pathway. therefore, we investigate the decrease of Thr308 ,Ser473 phosphorylation and the increase of PTEN phosphorylation after treated by DHEA , model in vitro was analyzed by Western blot assay , and bearing cancer in vivo was analyzed by Western blot and immunohistochemistry assay .The resultes show that DHEA regulating the phosphorylation of Akt and PTEN directly result in tumour cells apoptosis. In the research , applying a classical inhibitor(LY294002)and an activator(HGF)of PI3K ,we found that after treated by LY with DHEA or not , the Thr308and Ser473 protein levels both have significant decrease . The results of DHEA and HGF used together show that the phosphorylation levels of Akt protein have no obviously difference from those of using HGF alone, they both can not cut down the phosphorylation levels of Akt. Conclusion: PI3K and Akt present separating phenomenon, it indicates that Akt also has other signaling pathway except for phosphorylation of PI3K, they are a important finding and innovative point of my investigation Applying DHEA in vivo improve the immunological function of bearing cancer animals We detected CD3,CD4,CD8 in spleens of rats , and found that after implanted Morris hepatomas , the immunological functions of bearing cancer Buffalo rats descend to (56.46±6.12)%,(46.55±3.76)% and (30.22±6.40)%,respectively ; in bearing cancer rats treated by DHEA , tumor weights lost and CD3,CD4,CD8 recover to normal control levels for (74.97 ±8.29)%,(59.46±4.37)%和(34.02±8.56)%,respectively . DHEA can regulate and recover the decreased immunological function of bearing cancer rats, as for whether tumor weight lost directly correlate to immunological function recovery is still a question.