The Research Of Parathyroid Hormone-related Protein Varying With Sex And Sex Hormones Status In Nonsmall Cell Lung Cancer

[Background and Objective]Lung cancer is a common malignancy tumor. In recent years, the incidence rate is rising. The common type is non-small cell lung cancer (NSCLC). The pathogenesis, development and prognosis is varying with sex. Women who are smokers have an increased susceptibility to lung cancer compared with men who are smokers. However, female is a favorable prognostic factor after diagnosis; it was demonstrated that women have a significantly longer survival than men do at all stages.The level of gastrin-releasing peptide in lung cancer (including parathyroid hormone-related protein, PTHrP) is varying by gender. PTHrP is expressed in two-thirds of NSCLC patients. Does this factor slow the growth of lung cancer in women? So the female patients have a better prognosis than male patients do. It is very important to confirm whether PTHrP is an independent prognostic indicator of lung cancer.In addition to cause malignant hypercalcemia, PTHrP can regulate the rate of proliferation and apoptosis of cancer cells. It also regulates the expression of matrix metalloproteinases and hinders tumor angiogenesis. So it can regulate the progression of tumor and change the sensitivity of tumor to the treatment, which influences different aspects of cancer pathogenesis. Some researchers have observed that PTHrP affected tumor progression in several animal cancer models. A large number of study has evaluated that PTHrP is the main cause of hypercalcemia in lung cancer, but there was little work to reveal the relationship between tumor progress, survival or prognosis and PTHrP in lung cancer. Therefore, the objective of this study is to know whether sex hormones regulate the secretion of PTHrP, whether the expression of PTHrP is related with the survival rate and sex of NSCLC patients.[Methods]Part I The experiment of cytobiologyExperimental cell lines:Human lung adenocarcinoma A549cell line, Human lung squamous cell carcinoma YTMLC-90cell line. Positive control cell lines:Human prostate carcinoma LNCaP cell line(positive cell lines of androgen receptor expression), Human breast carcinoma MCF7cell line(positive cell lines of estrogen receptor expression).1. Cell recovery, culture and passage2. The expression of estrogen receptor ER-α,ER-β, androgen receptor AR, PTHrPmRNA and PTHrP was detected in experimental cell lines and positive control cell lines with RT-PCR detection analysis and Western Blot analysis.3. A549, YTMLC-90cells were grown in6-well plates for48hours. Then cells were cultured with17β-estradiol. testosterone together for48hours, which were from0.5nM to lOnM. PTHrPmRNA expression of A549. YTMLC-90cells was detected with real-time quantitative PCR analysis.Part II The experiment of animals1. Transplanted cells:A549, YTMLC-90cells that were cultured to logarithmic phase2. Modeling animals:Take female and male Nu/Nu (SPF grade) nude mice which were4-6weeks of age. including70females,30males, a total of100mice. According to different experimental treatments, female nude mice were randomly divided into groupA,groupB. group C,group D, male nude mice were randomly divided into group E and group F.The female experimental group (GroupA. a total of20):10nude mice were inoculated A549cells, the other10were inoculated YTMLC-90cells subcutaneously in the right shoulder, while testosterone (0.2mg/day) was injected into the left hind limb for28days. The female experimental control group (GroupB. a total of20):10nude mice were inoculated A549cells, the other10were inoculated YTMLC-90cells subcutaneously in the right shoulder. The female placebo control group (GroupC, a total of20):10nude mice were inoculated A549cells, the other10were inoculated YTMLC-90cells subcutaneously in the right shoulder, while normal saline (0.9%NS,0.2mg/day) was injected into the left hind limb for28days. The female blank control group (GroupD, a total of10):Nude mice were not given any experimental treatment.The male experimental control group (GroupE, a total of20):10nude mice were inoculated A549cells. the other10were inoculated YTMLC-90cells subcutaneously in the right shoulder. The male blank control group (GroupF. a total of10):Nude mice were not given any experimental treatment.After nude mice were killed,tumor tissues and lung tissues were used to do HE staining for pathological diagnosis and immunohistochemical analysis for PTHrP expression. Then we measured serum testosterone, serum interleukin-8(IL-8), tumor homogenate protein content of total protein and detected expression of PTHrP mRNA and PTHrP.Part III Clinical study125cases of non-small cell lung cancer patients, male73. female52The inclusion criteria:Patients had complete clinical data who underwent lung resection and were confirmed histopathologically. The exclusion criteria:Patients had chemotherapy, radiotherapy, immunotherapy and other treatments before surgery. Patients had other organ tumors. Patients had immune system diseases. Patients had other chronic wasting disease.1. Three specimens included cancer tissues, non-cancerous adjacent lung tissues and distant normal lung tissues. Distant normal lung tissues:distance more than5cm from the edge of lung carcinoma tissues; Non-cancerous adjacent lung tissues:distance between cancer tissues and distant normal lung tissues.2. One specimen was fixed with10%formaldehyde for pathological diagnosis and immunohistochemical analysis for PTHrP expression. The other sample was used to analyze PTHrPmRNA expression with Real-time quantitative PCR analysis.3. Follow up:125male and female NSCLC patients were followed up. To analyze the relevance of the expression of PTHrP and prognosis.[Results]Lung cancer model in nude mice1.76of80nude mice (95%) which were transplanted with A549, YTMLC-90cells developed parenchymal lung carcinomas. The tumors contained pleomorphic cells that had distinct heteromorphosis and poor differentiation. With the help of the special immunohistochemistry marker. we confirmed adenocarcinoma and squamous carcinoma in specimens of nude mice.2. The expression of PTHrP and PTHrPmRNA in tissues of nude mice with tumors was higher than one in nude mice without tumors with immunohistochemical analysis. Western Blot analysis and real-time quantitative PCR.3. Tumor burden of nude mice which were injected with A549, YTMLC-90cells (tumor volume, tumor weight, tumor homogenate protein content of total protein, serum IL-8levels):Tumor burden of male nude mice were larger than one of female nude mice. Tumor burden of female nude mice which were injected with testosterone were larger than one of female nude mice which were injected with normal saline. The expression of ER, AR, PTHrPmRNA, PTHrP in A549, YTMLC-90lung carcinoma cell lines and LNCaP, MCF7positive control cell lines1. ER-β,AR were expressed in A549, YTMLC-90lung carcinoma cell lines and LNCaP. MCF7positive control cell lines. Neither cell line expressed ER-a.2. PTHrPmRNA,PTHrP were expressed in A549. YTMLC-90lung carcinoma cell lines and LNCaP. MCF7positive control cell lines.Sex hormones regulate the secretion of PTHrP1. PTHrPmRNA expression of females nude mice was higher than one of males nude mice.2. PTHrPmRNA expression of females nude mice with testosterone injection was lower than one of females nude mice with normal saline injection. 3. PTHrP and PTHrPmRNA expression of women patients' tumor samples were higher than one of men patients'tumor samples.Androgen plays a negative regulatory role for PTHrP1. Experimental control groupB,group E and blank control groupD,groupF:The tumor total protein, serum IL-8levels in male mice (group E) were significantly greater than ones were in female mice (groupB)(ρ<0.05); Tumor total protein, serum1L-8levels in female (groupD) and male mice (groupF) of blank control group were close to zero.2. Experimental group A and placebo control group C:The serum testosterone levels of the experimental group were higher than ones of the placebo control group. PTHrP and PTHrPmRNA expression of the experimental group was lower than one of the placebo control group.The tumor burden of the experimental group was larger than one of the placebo control group.3. A549,YTMLC-90cells were treated with different concentrations of17β-estradiol and testosterone for48hours. The expression of PTHrPmRNA in A549,YTMLC-90cells which were given estradiol was not changed, while the expression of PTHrPmRNA in A549. YTMLC-90cells which were given testosterone was significantly reduced. The relationship between PTHrP expression in NSCLC patients and their survivalIn NSCLC patients. PTHrP and PTHrPmRNA expression was higher in the tumor tissues, female patients. The prognosis of female patient had correlation with PTHrP expression. Women patients whose PTHrP expression was positive had a higher survival rate. But the prognosis of male patients had nothing to do with PTHrP expression.[Conclusions]1. Estrogen had no effect on PTHrPmRNA expression of human lung adenocarcinoma A549cells, human lung squamous cell carcinoma YTMLC-90cells. Androgen reduced PTHrPmRNA expression of cells and played a negative regulatory role.2. The relationship between the expression of PTHrP. PTHrPmRNA and tumor burden was negative correlation in nude mice with tumor. Tumor burden was smaller when the expression of PTHrPand PTHrPmRNA was higher. Tumor burden was greater when the expression of PTHrP and PTHrPmRNA was lower. 3. The prognosis of female NSCLC patients had correlation with the expression of PTHrP. But the prognosis of male NSCLC patients had nothing to do with the expression of PTHrP. Female patients whose PTHrP expression was positive had longer survival than male patients whose PTHrP expression was positive do. PTHrP expression of female NSCLC patients was a survival advantage predictor.