The Influence And Mechanism Of Cisplatin On Autophagy Of Endometrial Cancer

The biological carcinogenesis of endometrial cancer is a very complex process, yet the exact pathogenesis remains unclear. Researches indicated that the mutation and deactivation of certain cancer suppressor genes and abnormal activation of oncogenes might be playing important roles in occurrence and development of endometrial cancer. Finding effective anticancer methods to restore early changes of cellular components in occurrence stage of endometrial cancer, and to eliminate mutated or damaged organelles after the formation of endometrial cancer, have always been research hotspots in recent years.Autophagy occupies important role in normal cell’s development and responses to changing environmental stimuli, as well as in occurrence and development of tumors. In the evolution process of lives, autophagy is an old biological phenomenon widely existing in plants and other lower life forms, and is an important way for living organisms to degrade intracellular proteins, complete organelle transformation and maintain stable internal environments, as well as one of the measures to eliminate cancer cells in mammals. Under normal physiological conditions, autophagy is desirable for cells to maintain homeostasis, this is because autophagy is a conservative metabolic process induced by hunger, hypoxia, hyperpyrexia or drugs, and can degrade long-period protein organelles and cytoplasm, making it an essential pathway to maintain cellular homeostasis. When stress occurs, autophagy prevents accumulation of damaged toxic or carcinogenic proteins and organelles and inhibits cell canceration, however, once tumors have formed, autophagy will provide cancer cells with richer nutrients to promote tumor growth. Hence, autophagy plays a two-sided role in occurrence and development of tumors.There are currently 3 types of autophagy based on the occurrence process: macroautophagy, microautophagy and chaperone-mediated autophagy. Macroautophagy, which is the so-called autophagy, refers to the process that aged or damaged cytoplasmic proteins or organelles are transported into lysosomes in the form of vesicles for digestion and degradation. Microautophagy refers to the process that lysosomes actively and directly devour cytoplasmic constituents. Chaperone-mediated autophagy refers to the process that certain molecular chaperones (e.g.:hsp70) helping with transposition of unfolded proteins into lysosomes. The commonly mentioned term of autophagy generally refers to macroautophagy. Autophagy is a biological process involving plenty of small molecule proteins. Human autophagy has been clearly and extensively studied through transmission electron microscope (TEM) observation, the indispensable microtubule-associated protein light chain 3 required to initiate autophagy is also commonly utilized for autophagosome detection.To maintain a stable environment inside cells, the base level of autophagy is maintained at a relatively low level under normal conditions, and can be rapidly up-regulated under certain "dangerous" conditions, such as hunger, cell reconstruction, growth hormone deficiency, and excessive damaged organelles or metabolic wastes inside cells. Mammalian target of rapamycin (mTOR) pathway is an important regulatory factor in cell growth and proliferation. A great number of studies showed that mTOR signaling pathway dysregulation is closely associated with cell proliferation. The homologous gene of which in human is FRAP1 (FK506 binding protein 12-rapamycin associated protein 1), a serine/threonine protein kinase. mTOR is an indispensable process in cell proliferation and can receive multiple upstream signals such as Class I PI3K, IGF-1/2, MAPK, and is also able to detect nutrient and energy changes. Overexpression of mTOR pathway has been detected in many tumors. The mTOR pathway is overexpressed that can be found in many tumors. There is phosphatidylinositol 3-kinase (MAPK), protein kinase B (AKT) and mitogen-activated protein kinase in a variety of tumor cells, including endometrial cancer. The activation of these enzymes can promote tumor cell proliferation, progression of the disease. The signaling pathways involved in cell proliferation, differentiation, and apoptosis regulating glucose transport and other cell functions. Increased PI3K activity often associated with a variety of cancers.Insulin-like growth factor-1 (IGF-1) is the most common autophagy agonist.Autophagy is a considerably complex biological process strictly regulated by a series of related genes. Autophagy dysregulation is closely associated with the occurrence and development of endometrial cancer. Cisplatin is a first-line chemotherapy drug for endometrial cancer, whether it is affecting autophagy of endometrial cancer cells and related mechanisms are yet to be illustrated. As a sensor of amino acids, energy and nutritional status, mTOR may induce changes of its own activity by activating or suppressing certain upstream signal factors, and at the same time adjust formation of downstream autophagy compounds to exert direct regulatory effect on autophagy. Therefore, clarifying the role of mTOR pathway in the mechanism of cisplatin on endometrial cancer autophagy will help to analyze and treat endometrial cancer in molecular level.In our study, the relation between cisplatin and autophagy in endometrial cancer and the related mechanisms were studied. It was planned to cultivate Ishikawa endometrial cancer cells in vitro, which were then treated with cisplatin at different concentrations for different time lengths, and the cell activity was measured using MTS assay. The concentration gradients were 10,20,40 and 80μg/mL, and each concentration was carried out for 5 time lengths of 0,12,24,48 and 72 h. The IC50 dose was then identified and drug concentrations for the dose of cisplatin and time lengths in the next step were determined.20μg/mL and 0,12 and 24 h were selected for grouping, TEM was used to observe the formation of autophagic vacuoles, fluorescence microscope was applied to observe fluorescence aggregation of GFP-LC3 so as to determine whether cisplatin can induce autophagy in endometrial cancer cells, Western blot was performed to detect accumulations of PI3K, AKT and mTOR proteins in mTOR pathway. In order to further clarify the role of mTOR pathway in cisplatin-induced autophagy of endometrial cancer cells, IGF-1 (PI3K agonist, 100ng/ml) was co-cultivated with cisplatin (20μg/mL,24 h), then TEM was employed to observe the formation of autophagic vacuoles, fluorescence microscope was adopted to observe fluorescence aggregation of GFP-LC3 in plasmids to detect autophagy. The influence and related mechanisms of cisplatin on autophagy of endometrial cancer cells were illustrated in this study, which has provided a new target and laid an experimental foundation for the treatment of endometrial cancer.Background and Objection:Backgrounds1. The incidence of endometrial cancer is growing worldwidelyEndometrial cancer is an epithelial malignancy arises from endometrium, is one of the three most common malignancies in women’s genital tract, and is most common in elderly women. With the extension of human longevity, growing obese population and increased use of endogenous and exogenous hormones, the incidence of endometrial cancer is growing worldwidely, and has replaced cervical cancer to be the most common malignancy in women’s genital tract. Most patients are already in late stage when diagnosed, and it also has poor prognosis with an approximately 30% five-year survival rate, while early stage adenocarcinoma of endometrium has good surgical effect and an over 90% five-year survival rate, thus it is extremely important to diagnose early stage endometrial cancer late stage endometrial cancer patients and find appropriate chemotherapy regimens. As present, researchers have focused on investigating the mechanisms of chemotherapy drugs on endometrial cancer from molecular biology aspect.2. Autophagy is closely associated with the occurrence and development of endometrial cancer.Autophagy is also known as type II programmed cell death, which is a series of biochemical processes of cell "self-digestion" manifested by appearance of double-membrane vesicles known as autophagosomes in cytoplasm that isolate long-lived proteins and organelles from the rest of the cell, and is the formation of autophagic vacuoles with independent double-membrane structure in cells under physiological or pathological effects (such as nutrient deficiency, hyperpyrexia, hypoxia, drugs, etc.). Autophagy can promote survival of tumor cells, but also can inhibit tumorigenesis. Studies have shown that as precancerous lesion, endometrial cells with atypical hyperplasia have higher autophagic activity than normal endometrial cells, whereas the autophagic activity of adenocarcinoma is significantly lower. As a protection mechanism of body, increased activity of autophagy may cause negative balance of related proteins, thus inhibiting malignant changes of cells. However, when autophagy fails to restore such abnormal differentiations, the abnormal differentiations will further accumulate and eventually cause cell canceration.3. Cisplatin is a first-line chemotherapy drug for endometrial cancer, thus it is of great significance to further understand the relationship, between autophagy and cisplatin after it has been applied on endometrial cancer cells.Cisplatin is a platinum-containing chemotherapy drug with extensive clinical applications, it is a non-cycle-dependent chemotherapy drug and plays an important role in chemotherapy of endometrial cancer. Hence, it is absolutely necessary to further understand the mechanism of cisplatin’s effect on endometrial cancer cells. It was discovered by recent studies on cell death that, in addition to apoptosis, programmed cell death also includes autophagic cell death and necroptosis, this has provided new ideas for tumor treatments. In different tissues and cell research, the functions of autophagy cisplatin-induced was different.In hepatoma cell Huh-7 and HepG2,down-regulated miR-199a-5p thereby activating Atg7 could enhance autophagy, and found that the tumor cells via autophagy by inducing cancer cell resistance. However, in the study of laryngeal carcinoma cell Hep-2, the use of 3-MA or knock down Beclin-1 inhibition of autophagy, can enhance cisplatin-induced apoptosis. Autophagy in cancer therapy is very complex role, and the role of different mechanisms in different tumor medicament may be different, so understanding the relationship between cisplatin in endometrial cancer and autophagy and its mechanism is essential.4. PI3K/Akt/mTOR signaling pathway activation were associated with poor prognosis and development of endometrial cancer.Mammalian target of rapamycin (mTOR) is the PI3K/AKT pathway downstream molecule. As a conserved serine/threonine protein kinase, it is associated with cell growth, proliferation, survival, and autophagy. mTOR signaling pathway is associated with the regulation of protein synthesis, cell cycle,which is essential to initiatioh signal required by the GO/G1 phase into the S phase of cell division and regulation of anabolic center. The study suggested that PI3K/Akt/ mTOR signaling pathway activation were associated with poor prognosis and development of endometrial cancer. The activation of PI3K/Akt/mTOR signaling pathway and endometrial cancer gene changes also were related to the progress of the tumor. The study found that the P13K enhanced catalytic activity, activating PI3K/Akt pathway, ultimately promote cancerous cells. Endometrial carcinoma in PI3K/Akt/mTOR signaling pathways of excessive expression, promote cells continue to eventually arise.ObjectionAutophagy is a new target of tumor treatment, however, many of its molecular mechanisms are yet to be further illustrated. In this study, whether cisplatin can cause autophagy in endometrial cancer was analyzed, and related mechanisms were revealed, so as to provide experimental bases to further perfect the relational theory of autophagic mechanism of cisplatin’s therapeutic effect in endometrial cancer treatment.Methods and materialsIshikawa cells were cultivated in vitro and treated with cisplatin at different concentrations for different time lengths, and the cell activity was measured using MTS assay. The concentration gradients were 10,20,40 and 80μg/mL, and each concentration was carried out for 0,12,24,48 and 72 h. The IC50 dose was then identified and drug concentrations and treatment time in the next step were determined. Based on MTS results,20μg/mL and 0,12 and 24 h were selected, TEM was used to observe the formation of autophagic vacuoles, fluorescence microscope was used to observe the fluorescence aggregation of green fluorescent protein and microtubule associated protein 1 light chain 3 fusionprotein (GFP-LC3), so as to detect whether cisplatin can induce autophagy in Ishikawa endometrial cancer cells. Western blot was performed to detect accumulations of PI3K, AKT and mTOR proteins in mTOR pathway. In order to further clarify the role of mTOR pathway in cisplatin-induced autophagy of endometrial cancer cells, IGF-1(100ng/ml,24h) was co-cultivated with cisplatin (20μg/mL,24 h), then TEM was utilized to observe the formation of autophagic vacuoles, fluorescence microscope was employed to observe fluorescence aggregation of GFP-LC3 to detect autophagy.Statistical analysisAll data were presented as mean±standard deviation or mean±standard error, SPSS 19.0 software was employed for statistical analysis, analysis of variance (ANOVA) and paired t test were performed, P<0.05 suggested a statistical difference.Results:Influence of cisplatin on Ishikawa cell proliferationMTS results showed that Ishikawa cells are sensitive to cisplatin,10μg/mL cisplatin had inhibitory effect on Ishikawa cell proliferation, after treated with 10 μg/mL cisplatin for 0,24,48 and 72 h, the proliferation inhibition rates were 18.27±2.25,24.88±3.58,38.27±5.34 and 45.43±4.2512, respectively. With the increase of treatment time and concentration, the inhibitory effect on cell proliferation significantly increased (P<0.01). In 20μg/mL cisplatin treatment group, cell proliferation inhibition rates of all treatment times were all below 50%, where as in 40 and 80μg/mL cisplatin treatment groups, cell proliferation inhibition rates of all treatment times were all above 50%. Based on the results of MTS assay,20μg/mL cisplatin treatment for 12 h and 24 h were selected as concentration and treatment times for following experiments.Effect of cisplatin on Ishikawa cell autophagyFormation of autophagosomes in Ishikawa cells treated with 20μg/mL cisplatin for 12 h and 24 h were observed using TEM. After being cultivated for 12 h after 20 μg/mL cisplatin treatment, Ishikawa cells were observed with pyknotic chromatin and double-membrane autophagosome,s inside cells. After being cultivated for 24 h after 20μg/mL cisplatin treatment, Ishikawa cells were observed with pyknotic nuclei and obvious vacuoles. Compared with control group, cells in 20μg/mL cisplatin 12 h treatment group had increased number of autophagosomes; and cells in 20μg/mL cisplatin 24 h treatment group had increased number of autophagosomes comparing with that of 20μg/mL cisplatin 12 h treatment group.Confocal immunofluorescence microscopy was applied to detect expression of autophagy-related protein LC3, and results showed that LC3 expression level was higher in 20μg/mL cisplatin 12 h treatment group than that in control group, and higher in 20μg/mL cisplatin 24 h treatment group than that in 20μg/mL cisplatin 12 h treatment group.Inhibitory effect of cisplatin on mTOR signaling pathwayIn order to detect the mechanism of cisplatin promoting Ishikawa cell autophagy, Western blot was performed to detect expressions of PI3K p85, phosphorylated AKT1, total AKT1, phosphorylated mTOR and total mTOR proteins. As shown by the results, after being treated with 20μg/mL cisplatin for 24 h, expression levels of phosphorylated AKT1, phosphorylated mTOR and PI3K p85 significantly decreased, while expression levels of total AKT1 and total mTOR showed no significant variations.Reversion of cisplatin’s effect on autophagy of endometrial cancer cells by co-cultivation of IGF-1 and cisplatinIn order to further confirm that cisplatin causes Ishikawa cell autophagy by inhibiting mTOR signaling pathway, three groups were set, which were control group, cisplatin group (20μg/mL,24 h) and co-cultivation group (100 ng/mL IGF-1 and 20 μg/mL cisplatin,24 h). TEM was employed to detect autophagosomes, Western blot was performed to detect LC3 protein expression. As shown by the results, TEM-detected autophagosomes and LC3 protein expression in co-cultivation group were both less than that in cisplatin group, but more than that in control group.Conclusion1.10μg/mL cisplatin has an inhibitory effect on proliferation of endometrial cancer cells in a time-and dose-dependent manner;2. Cisplatin has a promoting effect on autophagy of endometrial cancer cells;3. Cisplatin promotes autophagy in endometrial cancer cells by inhibiting mTOR signaling pathway.