| BackgroundProstate cancer is not only one of the most common urological malignant tumors in men but also the second cause of male cancer-related death. The incidence of prostate cancer in America is higher than lung cancer so it becomes the highest incidence of male malignant tumors. Researches showed that the number of patient diagnosized with prostate cancer in China especially in the economically developed area is growing gradually that may be related to westernized dietary patterns in China. In future, the incidence of prostate cancer is likely to become the No.l, seriously influencing the health of the male citizens in China. Due to the lack of obvious symptoms of prostate cancer at early stage, it is easy to be ignored and missed the best treatment opportunity. At that time, patients at adveanced stage may already have local or distant metastasis and treatments for these patients included endocrine therapy, chemotherapy, or palliative care, etc. Up to date, endocrine therapy is the first choice for them. However, the disease will develop into hormone-refractory prostate cancer at later stage.Castration-resistant prostate cancer refers to the stage that prostat cancer keeps progress despite receiving the continuous androgen deprivation therapy (ADT). It should meet the following conditions:(1) the level of serum testosterone reached the level of castration (50 ng/dl, or 1.7 nmol/L); (2) the interval of 1 week,3 consecutive PSA rise and the lowest value rised more than 50%. At that time, the endocrine treatment is invalid. The current study suggests the development of hormone-dependent prostate cancer to castration-resistant prostate cancer may be associated with the following mechanisms:(1) the existence of the prostate cancer stem cells; (2) apoptosis related gene and the abnormal expression of tumor suppressor genes and pathways; (3) ligands independent androgen receptor activation; (4) the androgen receptor hypersensitivity; (5) the androgen receptor mutations. But so far, the complete mechanisms are poor. In recent years, more and more studies show that other unknown mechanisms may be existed in the whole transformation process, especially the extracellular mechanisms.For cells, prostate cancer develops from hormone dependence to castration resistant is a long process. During the process, change not only ouucrs in the cell but also in the environment around the tumors. And the environment around the tumors is also known as tumor microenvironment, referred to the internal environment at which tumor develops and progresses. The tumor microenvironment is a complex integrated system, which contains the tumor cells, pluripotent stromal cells or mesenchymal stem cells, fibroblasts and capillary, epithelial progenitor cells, and a variety of immune cells and cytokines. Tumor microenvironment could not only provide the "fertile soil" for the growth of tumor, but also induce tumor progression, cause persistent tumor cell genetic and phenotypic changes, make it has the ability of growth advantage and adapt to the change of the environment. At present, large number of studies have found that the tumor microenvironment can promote many kinds of tumors including prostate cancer to proliferate, survival and induce angiogenesis as well as inhibiting the immune system and escape from immune detection. Tumor microenvironment also promotes invasion and metastasis by activation of immune cellsThe current researches about tumor microenvironment mainly focus on extracellular vesicles. Extracellular vesicles are another way of communication between cells except for cytokines. Extracellular vesicles are lipid bilayer-enclosed vesicles,30～2000 um in diameter, containing proteins, lipids and nucleic acids. Studies have shown that extracellular vesicle is not only involved in normal physiological regulatory activities, but also participates in a variety of pathological processes including cancer, inflammatory disease, autoimmune and neural degenerative diseases. The extracellular vesicles derived from tumor cells closely associated with tumor progression, involved in regulating tumor cell growth, invasion, metastasis and angiogenesis. On the basis of biogenesis, extracellular vesicles are categorized into exosomes and microvesicles. Because the exosomes were synthesized in the cytoplasm and rich in active proteins, lipids and nucleic acid, they play a pivotal role in cells interaction. Studies have confirmed that the secretion of exosomes is closely related to the progression of prostate cancer, including chemotherapy resistance, immune-suppression, proliferation, apoptosis, and angiogenesis. However, there are no studies reported whether exosomes is involved in the process from hormone-dependent progress to castration-resistance or not.In this study, we firstly explored the functions of the tumor microenvironment in the emergence of castration resistance in prostate cancer. Then we extracted the exosomes from hormone-independent prostate cancer cells and added into the hormone-dependent prostate cancer cells, and we observed the influence of exosomes on the emergence of castration resistance of prostate cancer in vitro and in vivo. Finally, we use the microarrays and LC-MS/MS to explore the mechanisms. Our study was aim to find the way to delay the progress to castration-resistant prostate cancer, and provide a new direction for further treatment intervention.Objective1. To investigate the role of tumor microenvironment on androgen-dependent prostate cancer progression to castration-resistant prostate cancer.2. To testify the role of the exsomes on androgen-dependent prostate cancer progression to castration-resistant prostate cancer3. To explore the mechanisms that exosomes involved in emergence of castration resistance.Methods1. LNCAP cells co-culture with PC-3 cells promotes LNCAP cells progress to castration resistance.(1) LNCAP cells were placed in the upper chamber while PC-3 cells were placed in the lower chamber. Two types of cells co-culture in the Transwell system. After 4 weeks, the cells were used for subsequent analysis.(2) MTS for detecting the proliferative ability of LNCAP cells after co-culture with PC-3 cells:Digesting and counting cells, assigning to 96-well plates with each hole 1 ×104 cells/100ul. We Add the MTS at each time point (Oh,24h,48h,72h and 96h). After 4 hours incubation, we collected the data of OD490 by microplate reader.(3) Colony formation assay for detecting the colony formation ability of LNCAP cells after co-culture with PC-3 cells:Digesting and counting cells.1000 cells were placed in a fresh six-well plate and maintained in RMPI-1640 medium containing 10% FBS for two weeks and colonies were fixed and stained with 0.1% crystal violet and counted.(4) Western Blot for detecting the protein expressions of AR and PSA after co-culture: Extracting the total protein from the cells in each group, running protein SDS electrophoresis and analyzing AR and PSA protein expression before and after co-culture.(5) Quantitive RT-PCR for detecting the mRNA expressions of AR and PSA after co-culture:Extracting the RNA and reverse transcription into cDNA. Mix the cDNA, primers, SYBR Green and DEPC H2O together according to certain proportion and then the quantitative analysis was performed.2. PC-3 cells promote LNCAP cells progress to castration resistance by secretion of exosomes.(1) PC-3 cells exosomes or LNCAP cells exosomes were extracted by Exoquick-TC kit. After the content of exosomes was determined by BCS methods, exososmes were added in the cells. The rest of the exosomes were saved in the-80 ℃.(2) Western Blot for identifying the surface markers (TSG101, Alix and HSP70) of exosomes:Extracting the total protein from the cells in each group, running protein SDS electrophoresis and analyzing TSG101, Alix and HSP70 protein expression.(3) Transmission electron microscope for observing the shape of exosomes:Exosome preparation 5μl was placed on 200 mesh copper grids. Grids were stained with 20μl 2.0% phosphotungstic acid for 5 min and allowed to dry. A transmission electron microscope was used to view samples.(4) MTS for detecting the proliferative ability of LNCAP cells added with PC-3 cells exosomes:Digesting and counting cells, assigning to 96-well plates with each hole 1 x 104 cells/100ul. We Add the MTS at each time point (Oh,24h,48h,72h and 96h). After 4 hours incubation, we collected the data of OD490 by microplate reader.(5) Colony formation assay for detecting the colony formation ability of LNCAP cells added with PC-3 cells exosomes:Digesting and counting cells.1000 cells were placed in a fresh six-well plate and maintained in RMPI-1640 medium containing 10% FBS for two weeks and colonies were fixed and stained with 0.1% crystal violet and counted.(6) Flow cytometry for detecting the cell cycle of LNCAP cells added with PC-3 cells exosomes:Digesting and counting cells, and PBS resuspension. Cells were fixed with 70% iced ethanol at 4℃ overnight. On the next day, the supernatant was removed by centrifuge. Cell pellets were added with 50μl RNA enzymes and 450μl PI staining fluid. The samples were analyzed in 4h.(7) NOD/SCID mice tumorigenicity experiment for detecting the tumorigenicity of LNCAP cells added with PC-3 cells exosomes:culture cells, subcutaneous injection and draw the growth curve.(8) Western Blot for detecting the protein expressions of AR and PSA after adding with exosomes:Extracting the total protein from the cells in each group, running protein SDS electrophoresis and analyzing AR and PSA protein expression before and after adding with exosomes.(9) Quantitive RT-PCR for detecting the mRNA expressions of AR and PSA after adding with exosomes:Extracting the RNA and reverse transcription into cDNA. Mix the cDNA, primers, SYBR Green and DEPC H2O together according to certain proportion and then the quantitative analysis was performed.3. The mechanisms of exosomes derived from PC-3 cells promoting LNCAP cells progress to castration resistance.(1) The differential expression analysis of the LNCAP cells treated with and without PC-3 cells exosomes was performed using the AFFYMETRIX microarray and analyzed by GO and KEGG pathway databases.(2) Western Blot for detecting the protein expressions of HMOX1:Extracting the total protein from the cells in each group, running protein SDS electrophoresis and analyzing HMOX1 protein expression.(3) Quantitive RT-PCR for detecting the mRNA expressions of HMOX1:Extracting the RNA and reverse transcription into cDNA. Mix the cDNA, primers, SYBR Green and DEPC H2O together according to certain proportion and then the quantitative analysis was performed.(4) Immunohistochemistry for detecting the protein expression of HMOX1 in prostate cancer tissues.(5) Adding hemin or siRNA targeting for HMOX1 to up-regulate or down-regualte the expression in LNCAP cells. Western Blot and Quantitive RT-PCR were performed to verify the efficiency.(6) Quantitive RT-PCR for detecting the mRNA expressions of HMOX1 after adding with hemin or siRNA:Extracting the RNA and reverse transcription into cDNA. Mix the cDNA, primers, SYBR Green and DEPC H2O together according to certain proportion and then the quantitative analysis was performed.(7) Western Blot for detecting the protein expressions of HMOX1 after adding with hemin or siRNA:Extracting the total protein from the cells in each group, running protein SDS electrophoresis and analyzing HMOX1 protein expression.(8) MTS for detecting the proliferative ability of LNCAP cells after up-regualting or down-regulating the HMOX1:Digesting and counting cells, assigning to 96-well plates with each hole 1 x 104 cells/100ul. We Add the MTS at each time point (Oh, 24h,48h,72h and 96h). After 4 hours incubation, we collected the data of OD490 by microplate reader.(9) Colony formation assay for detecting the colony formation ability of LNCAP cells after up-regulating or down-regulating the HMOX1:Digesting and counting cells. 1000 cells were placed in a fresh six-well plate and maintained in RMPI-1640 medium containing 10% FBS for two weeks and colonies were fixed and stained with 0.1% crystal violet and counted.4. To explore the components of PC-3 cells exosomes promoting the emergence of castration resistance. (1) The differential proteins analysis of the LNCAP exosomes or PC-3 exosomes was performed using the LC-MS/MS and analyzed by GO and KEGG pathway databases.Results1. LNCAP cells co-culture with PC-3 cells promotes LNCAP cells progress to castration resistance.(1) Representative images showed that the morphology of LNCAP cells changed significantly after co-culture with PC-3 cells with stronger cell mobility, spindle morphology and less cytoplasmic.(2) The results of MTS showed that there was no significant difference of cell proliferation rate between LNCAP cells co-culture with PC-3 cells and normal LNCAP cells under normal condition; under the condition of castration, the cell proliferation rate of LNCAP cells co-culture with PC-3 cells was significantly higher than normal LNCAP cells on day 3 to 5.(3) The results of colony formation showed that the colony formation rate of LNCAP cells co-culture with PC-3 cells was significantly higher than normal LNCAP cells no matter under the normal condition or castration condition. Moreover, under the condition of castration, the difference between the two groups was more obvious.(4) The results of quantitative PCR showed that the mRNA expressions of AR and its downstream genes CDK1, CDK2 and GRBE1 were obviously decreased in LNCAP cells co-culture with PC-3 cells than normal LNCAP cells. In addition, after co-culture with PC-3 cells, the sensitivity of LNCAP cells to androgen is reduced.(5) The results of Western Blot showed that the protein expressions of AR and PSA in LNCAP cells were significantly decreased after co-culture with PC-3 cells.2. PC-3 cells promote LNCAP cells progress to castration resistance by secretion of exosomes.(1) The results of transmission electron microscope showed that the exosomes derived from PC-3 cells or LNCAP cells had typical characteristics of exosomes and 50～100 nm in diameter.(2) The results of Western Blot showed that the exosomes from two PCa cell lines expressed the specific surface markers of exosomes (TSG101, Alix and HSP70) but not GAPDH.(3) The results of MTS showed that the cell proliferation rate of LNCAP cells added with PC-3 cells exosomes was significantly higher than normal LNCAP cells no matter under the normal condition or castration condition. Moreover, under the condition of castration, the difference between the two groups was more obvious.(4) The results of colony formation showed that the colony formation rate of LNCAP cells added with PC-3 cells exosomes was significantly higher than normal LNCAP cells no matter under the normal condition or castration condition. Moreover, under the condition of castration, the difference between the two groups was more obvious.(5) The results of cell cycle analysis showed that the S phase rate of LNCAP cells added with PC-3 cells exosomes was significantly lower than normal LNCAP cells no matter under the normal condition or castration condition.(6) The results of NOD/SCID mice tumorigenicity experiment showed that the tumorigenicity of LNCAP cells treated with PC-3 cells exosomes was significantly increased compared with normal LNCAP cells under the normal condition. Importantly, under the castration condition, normal LNCAP cells were unable to form tumors whereas LNCAP cells treated with PC-3 exosomes were still able to develop tumors.(7) The results of quantitative PCR showed that the mRNA expression of AR was obviously decreased in LNCAP cells treated with PC-3 cells exosomes than normal LNCAP cells. In addition, after treated with PC-3 cells exosomes, the sensitivity of LNCAP cells to androgen is reduced.(8) The results of Western Blot showed that the protein expressions of AR and PSA in LNCAP cells were significantly decreased after treated with PC-3 cells exosomes.3. The mechanisms of exosomes derived from PC-3 cells promoting LNCAP cells progress to castration resistance.(1) The results of microarray showed that there were 72 genes with at least a 1.5-fold difference of mRNA expression in LNCAP cells treated with PC-3 exosomes as compared to normal LNCAP cells.(2) The results of quantitative PCR showed that the mRNA level of HMOX1 in LNCAP cells rised significantly after treated with PC-3 exosomes.(3) The results of Western Blot showed that the mRNA level of HMOX1 in LNCAP cells rised significantly after treated with PC-3 exosomes.(4) Immunohistochemistry results showed that the expression of HMOX1 was much higher in androgen-indepent prostate cancer than in hormone-dependent prostate.(5) The results of Western Blot and qRT-PCR showed hemin or siRNA against HMOX1 can effectively up-regulate or down-regulate the HMOX1 expression, respectively.(6) The results of MTS showed that, under the normal condition, there was no significant difference between normal LNCAP cells or treated with hemin. But in the absence of androgen, up-regulating the expression of HMOX1 can promote LNCAP cell proliferation; when the HMOX1 expression was blocked, the proliferation of LNCAP cells was inhibited under the normal condition. And in the absence of androgens, all groups of LNCAP cells were unable to grow.(7) The results of colony formation showed that, under the normal condition, there was no significant difference between normal LNCAP cells or treated with hemin. But in the absence of androgen, normal LNCAP cells can not form colonies; when the HMOX1 expression was blocked, there was no significant difference among groups. And in the absence of androgens, all groups of LNCAP cells were unable to form colonies.4. To explore the components of PC-3 cells exosomes promoting the emergence of castration resistance.(1) The results of LC-MS/MS illustrated that there were 2815 protein exist in PC-3 exosomes while there were 2810 protein exists in LNCAP exosomes. There were 2810 proteins exist in both two groups and there were five proteins only exist in the PC-3 exosomes. The number of protein content change≥ 1.5 fold was 1342. Compared with the PC-3 exososmes, the up-regulated proteins and down-regulated proteins were 677 and 665, respectively.Conclusion1. The proliferation and colony formation ability of LNCAP cells were increased under the castration condition after LNCAP cells co-culture with PC-3 cells.2. The proliferation and colony formation ability of LNCAP cells were increased under the castration condition after LNCAP cells treated with PC-3 cells exosomes.3. PC-3 exosomes promote LNCAP cells progress to castration resistance partially via up-regulating the expression of HMOX1. |
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