| BackgroundLeptin is an important proteohormoneis with multi-functions acting on multi- target organs. its main physiological functions include①inhibiting ingestion and increasing energy expenditure;②regulating growth and development;③regulating inflammatory reaction and immune function;④promoting the growth of epithelial cells and blood vessels;⑤regulating neuroendocrine;⑥protecting the digestive functions;⑦maintaining normal metabolism of blood lipid etc. In addition, it takes part in reproduction, blood pressure, hemopoiesis, fetus's growth, endocrine regulation and other physiological functions. In recent years, it has been gradually found that leptin has close relationship with the development of many kinds of malignant tumors. leptin mainly derives from white adipose tissue and it conducts its biological effects through combination with leptin receptor. its main physiological functions includes suppressing appetite, reducing energy intake, increasing energy expenditure, inhibiting fat synthesis, regulating fat deposition etc. High fat diets can increase leptin secretion. Serum leptin levels can reflect the quantity of fat cells and adipose tissue volume.Leptin is also called ob protein. active Leptin with the molecular weight of 16ku is composed of 146 amino acid residue which is derived from a kind of secretory protein with 167 amino acid expressed by obesity gene. Its main function is to adjust the energy balance, is to maintain normal weight essential hormone. Leptin with main function of regulating energy balance is a kind of basic hormone for maintaining normal weight. Since Zhang first found leptin gene, numerous studies have been conducted on leptin's biological effects. In recent years, the relationship between leptin and the development of tumors also has been gradually studied. Especially leptin can stimulate the proliferation of various tumor cells whose mechanism is probably related to increasing tumor cells'ability to invade the basement membrane and promoting angiogenesis within tumor. So leptin is regarded as a new kind of tumor growth promoting factor. The experiment shows leptin and leptin receptor are expressed in tumor tissues and tumor cell lines of breast cancer, lung adenocarcinoma and gastrointestinal tumors.Prostate cancer is a kind of disease of senile diseases which rarely onsets in patients before 50, and 90% of patients with prostate cancer suffer death after 65, and medianage is 77. There is a great diversity regarding its morbidity and mortality worldwide. In the United States, prostate cancer incidence occupies the first place during male tumors and its mortality ranks only second to lung cancer. The place with the lowest incidence and mortality of prostate cancer which is 2/10 million is Shanhai, China,as 1/60 of that in the USA. Prostate cancer incidence in our country is lower, merely 2.41/10 million. But as the development of the aging of the population and the increasing of obese people increased, the incidence of prostate cancer has significantly increased recently. In our country prostate cancer incidence in male malignant urological and reproductive tumors is in the third place. So prostate cancer is quietly affected the males'quality of life and life expectancy over the age of 50 of our country. Survey from epidemiological researchers found that the development of prostate cancer was affected by many factors, such as: the increase of androgens content, obesity, aging, smoking or drinking, familial inheritance, and other environmental factors and genetic factors. So far, specific pathogenes of prostate cancer is still not clear. But what is sure that the development of prostate cancer has a close relationship with obesity. Clinically, androgen blocker is the most effective treatment of prostate cancer. Unfortunately, prostate cancer cells of about 80 percent develop androgen-independent growth after androgen blocking therapy of 12-18 months. androgen deprivation therapy loses its effect and tumor continues to grow and deterioration until the patients died from the disease. The development of androgen independent prostate cancer is the most tough problem. Mechanism of AIPC is research hotspot recently. Through the above analysis, it was concluded that how to delay the transition of prostate cancer from androgen dependent prostate cancer to androgen independent prostate caneer, and explore the treatment of androgen dependent prostate cancer have become the present focus in the research of prostate cancer.At present, LNCaP,DU- 145 and PC-3, three types of cell lines, have been widely used in the basic research of prostate cancer, of which LNCaP is cell line of androgen dependence prostate cancer, and DU- 145 and PC - 3 are those of androgen independent prostate cancer.It was found that JAK2 - STAT3 is the main pathways of signal transmission for leptin. Through coupling and cytoplasmic tyrosine kinase JAK activating, Leptin receptor realizes signal transduction. Tt was observed that VEGF, TGF-β1 and bFGF of androgen independent prostate cancer cells in vitro excessively express beacause of stimulation from leptin, and migration activity of prostate cancer cell also increases accordingly, which showed that the leptin plays a role in promoting proliferation of androgen independent prostate cancer cell and inducing cell migration.It was shown that AG490, JAK enzyme inhibitors, can significantly inhibit cancer cell proliferation due to leptin by blocking STAT3 phosphorylation, which shows that signal transduction channel of JAK2-STAT3 plays an important role in tumor cell proliferation stimulated by leptin. Relevant research of leptin plays a role in the development of prostate cancer through JAK2 - STAT3 is less at home and abroad. So the experiment determines DU- 145 proliferation in leptin of different concentrations by MTT to detect secreting function of prostate cancer cells in the stimulation from leptin of different concentrations. In the experiment, it was preliminaryly discussed the function of leptin in promoting proliferation of prostate cancer cell DU– 145, according to mechanism of AG490 blocking JAK2 - STAT3 signal transduction channel.Purpose:This experiment is aimed at exploring the condition of non-hormone dependent prostate cancer cells with different concentrations of leptin and the JAK enzyme inhibitor, and to observe the growth of prostate cancer cells and related gene expression under the intervention of leptin, and to provide further clues for prostate cancer pathogenesis in the clinical application.Methods:1. 1. By in vitro culture method, choose prostate cancer cells DU- 145 of non-hormone dependence from Chinese academy of medical sciences tumor cell s bank as the experimental seed cells.2. Conduct observation and identification on the prostate cancer cells cultivated.3. Cultivate the prostate cancer cells with better grouth condition and divided randomly into 15 groups added into 1640 nutrient mediums with leptin of different concentration of 0 ng/ml, 1 ng/ml, 10 ng/ml, 100ng/ml, 1000ng/ml respectively. Cultivate the cells with certain concentration of leptin for 12h, 24h and 48h respectively.4. By MTT method, the activity of prostate cancer cells DU-145 cultured in vitro were determined in the above 15 groups with different concentrations of leptin.5. Divide prostate cancer cells of better growth condition randomly into four groups treated with 1640 nutrient solution with leptin of 100ng/mL, leptin of 100 ng/mL + AG490 20 umol/L, leptin of 100 ng/mL + AG490 50 umol/L, and leptin of 100 ng/mL + AG490 100 umol/L respectively for 24h. 6. By MTT method, determine the inhibition function of AG490 on DU-145 proliferation of leptin in the above 4 groups with different concentration of AG490.7. Through flow cytometry instrument, detect cell proliferation of prostate cancer cell DU– 145 in the intervention of leptin with differentconcentrations. 8. The results were conducted statistical analysis with software SPSS13.0.Results:1. Through the cell morphology observation, it is confirmed that the cell from Chinese academy of medical sciences tumor cells bank is prostate cancer cell DU-145.2. MTT method shows: in determination of leptin intervention each cell activity obviously better than the blank group, at 0 100ng/mL thin qualities concentration range, thin quality quantity chroma is taller, the more obvious cell proliferation, When thin qualities concentration to 100ng/mL, its most prominent, promote value-added effect in the 100-1000ng/mL, within the scope of thin qualities, heighten of chroma of cell proliferation easing, cell proliferation difference was statistically significant (P > 0.05).3. Prostate cancer cells was impacted by different concentrations of AG490 which has inhibition function on proliferation-promoting effect of leptin on DU-145 cell. Through MTT method, AG490 with concentration increased, its inhibition function was gradually strengthened 24h later. Through comparation between each group with different concentrations, there is statistically significant difference (P < 0.05).4. Results through flow cytometry determination show: cell proliferation in each group with leptin intervention was obviously better than that of blank group. The number of cells increases gradually with leptin concentrations increasing and cell PI value was the highest when leptin concentration reaches to 1000 ng/ml. After AG490 was put in, cell PI value gradually reduced as AG490 concentration heightened. Compared with other groups, the difference is also significant (P < 0.05).Conclusion:The leptin can promote proliferation, cytoactive and their related gene expression of prostatic cancer cell DU-145 which is one kind of nonhormone dependent cell. With leptin concentration increasing, the condition of prostatic cancer cell in proliferation and cytoactive is increased. While the enhanced effect will be obviously inhibited due to AG490. Thus, it is concluded that leptin plays an important role in the development of prostate cancer which may be realized through JAK2 - STAT3 signal transduction channel. |
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