Cellular Autophagic Capacity Changes During The Tumorigenesis And Development Of Endometrioid Adenocarcinoma

BackgroundThe endometrioid adenocarcinoma, whose glands resemble those of the normal endometrium, accounts for 80%~90% of endometrial carcinomas. The etiology of endometrioid adenocarcinoma is not fully understood, but mutation of the tumor suppressor gene PTEN is considered to be one of the most frequent molecular events.Autophagy is a dynamic process in which intracellular isolation membrane sequesters cytoplasm and organelles to form a double-membrane, spheric autophagosome, the autopahgsome then fuses with the lysosome into a single-memebrane, spheric autolysosome, where the cytoplasm or the organelles are degraded by the hydrolases and the resulting macromolecules are recycled by the cell. Autophagy, a protein degradation mechanism that ubiquitously conserved in all eukaryotes, regulates the cell proliferation together with protein synthesis. Autophagy is used to recycle the cytoplasm and remove excess or defectiveorgandies to maitain the cellular homeostasis.Autophagy, as a physiological process in cells, is under strict regulation. While the tumor suppressor gene PTEN positively regulates autophagy by inhibiting the PI3K-Akt pathway, Beclin 1 positively regulates autophagy by forming III class PI3K complex which is important for the formation of autophagosome. The change of the cellular autophagic capacity is considered to be closely related to the tumorigenesis. Autophagic cell death is type II programmed cell death, which is different from apoptosis and caspase-independent. Deprivation of nutrition, oxidative stress and drugs can induce autophagic cell death, which is characterized by the appearance of double- or multiple-membrane cytoplasmic vesicles engulfing cytoplasm and organelles such as mitochondria and endoplasmic reticulum.The transmission electron microscope has been used to study the morphology of autophagosome and autolysosome. Microtubule-associated protein 1 light chain 3 (LC3), mainly located in the membrane of autophagosome, is a specific protein marker of autophagosome. Cathepsin D is an aspartyl endopeptidase in lysosome, which represents most of the endopeptidase activity of lysosome, can reflect the degradative capacity of autolysosome.The loss of PTEN expression is closely related to the tumorigenesis of endometrioid adenocarcinoma, and researches in other tumors have found that PTEN positively regulates autophagy. But the change of cellular autophagic capacity during the tumorigenesis and development of endometrioid adenocarcinoma has't been reported so far.ObjectiveThis study demonstrates the change of cellular autophagic capacity during the tumorigenesis and development of endometrioid adenocarcinoma by studying the protein expression of LC3 and Cathepsin D in normal proliferative endometria,endometrial hyperplasia and endometrioid adenocarcinomas, and their associations with clinical-pathologic parameters in endometrioid adenocarcinomas.Meterials and Methods1. The transmission electron microscope was employed to observe the morphology of autophagosome and autolysosome on a normal endometrium.2. Immunohistochemistry was employed to assess the protein expression of LC3 and Cathepsin D in 12 cases of normal proliferative endometria, 12 cases of endometrial hyperplasia and 51 cases of endometrioid adenocarcinomas. The relationship between protein expression and clinical-pathologic parameters in endometrioid adenocarcinomas was studied.3. Western blot was employed to assess the protein expression of LC3 and Cathepsin D in 13 cases of normal proliferative endometria and 16 cases of endometrioid adenocarcinomas.4. The SPSS 12.0 software was employed to analyse the data.Results1. While the autophagosome is spheric and near the nuclear, whose electron density is equal to the cytoplasm, the autolysosme is a vesicle, whose electron density is a little higher than the cytoplasm.2. Results of the immunohistochemistry of LC3 and Cathepsin D1) Both LC3 and Cathepsin D reside in the cytoplasm.2) The staining intensity of LC3 in endometrioid adenocarcinomas was significantly lower than that in normal proliferative endometria and endometrial hyperplasia (Z = -2.902, P =0.004;Z = -3.518, P =0.000).3) The staining intensity of LC3 in endometrioid adenocarcinomas was significantly inversely correlated with the histological grade and the surgical-pathological stage (r = -0.390, P = 0.005;r = -0.312, P= 0.026). Thestaining intensity of LC3 in G2 and G3 is significantly lower than that in Gi (Z = -2.594, P = 0.009), and the staining intensity of LC3 in III~IV group is significantly lower than that in I-—II group (Z = -2.208, P = 0.027).4) The expression of Cathepsin D in endometrioid adenocarcinomas was significantly lower than that in normal proliferative endometria (P = 0.013). But no significance was found in the subgroups of the endometrioid adenocarcinomas (P > 0.05).3. Results of the western blot of LC3 and Cathepsin D1) Both the mean levels of LC3 and Cathepsin D in endometrioid adenocarcinomas are significantly lower than those in normal proliferative endometria (t=2.442, P =0.021;*=3.805,P =0.001).2) In detail, while the mean levels of 48kDa band of Cathepsin D in endometrioid adenocarcinomas is significantly lower than that in normal proliferative endometria (f=4.060, P =0.000), the mean levels of 34kDa band is not the same case (J=l .854, P =0.075).Conclusions1. Not only the formation of autophagosome but also the degradative capacity of lysosome are downregulated in endometrioid adenocarcinomas.2. That the cellular autophagic capacity is downregulated from normal proliferative endometria to endometrioid adenocarcinomas indicates that autophagy might play an important role during the tumorigenesis of endometrioid adenocarcinoma.3. That the cellular autophagic capacity is inversely correlated with the surgical-pathological stage indicates that autophagy might also play an important role during the development of endometrioid adenocarcinoma.