Background - Prostate cancer (PCa) is the most common malignancy in men. After initial response to androgen deprivation therapy, PCa can evolve to a castration-resistant phenotype, for which therapeutic options are poorly effective. In the tumor microenvironment, sources of cholesterol, reactive oxygen species (ROS) and pro-inflammatory molecules can favor cancer cell proliferation. Indeed, cells need cholesterol to proliferate, as structural and functional component of cell membranes and as substrate for the synthesis of hormones and oxysterols. In addition, recent evidence suggests that oxidative stress can play a role in the pathogenesis and the progression of prostate cancer (PCa). Aim - Since HDL are known to promote cell cholesterol efflux and to exert antioxidant activities, aim of the study was to investigate whether HDL can reduce cholesterol content and oxidative stress in PCa cells, thus modulating their proliferation. Methods - Androgen-sensitive and -resistant PCa cell lines (LNCaP and PC-3, respectively) were tested and compared with non tumor prostate epithelial cells (PNT2). Lipoproteins were isolated by ultracentrifugation from the plasma of healthy volunteers. ROS levels and cell cholesterol content were assessed by fluorescence. Oxidative stress was induced by H2O2 and cell cholesterol content was increased by incubation with LDL. The expression of proteins involved in cell cholesterol metabolism was assessed by by RT-PCR, western blotting or immunofluorescence. Cell proliferation was assessed by cell count, cell cycle analysis and colony formation assay. Bortezomib was used to inhibit the proteasome. Results - HDL significantly reduced basal and H2O2-induced oxidative stress in normal, androgen-sensitive and -resistant PCa cell lines. The androgen receptor, SRBI, ABCA1 and G1 transporters were not involved. Consequently, HDL completely blunted H2O2-induced increase of cell proliferation, through their capacity to prevent the H2O2-induced shift of cell cycle distribution towards G2/M phase. Cholesterol homeostasis is altered in PCa cells. PCa cells, that needs of cholesterol, mainly rely on lipoprotein uptake than intracellular synthesis of it. In LNCaP cells, the incubation with LDL increased cholesterol content and cell proliferation. The concomitant incubation with HDL promoted cell cholesterol reduction and blunted LDLinduced cell proliferation. On the contrary, in PC-3 cells HDL caused a further increase of cholesterol content and lost their ability to limit LDL-induced cell proliferation. In PC-3 cells, ABCA1 mRNA levels were elevated, but the protein was hardly detectable in the absence of pathogenic mutations in the ABCA1 gene. The treatment with the proteasome inhibitor bortezomib restored ABCA1 expression and consequently the ability of HDL to reduce cell cholesterol content and LDL-induced proliferation. Conclusions - Collectively, HDL can inhibit the proliferation of androgen-sensitive and castration-resistant PCa cell lines induced by oxidative stress and LDL. In castration-resistant PCa cells, HDL prevented LDLinduced cell proliferation only after the restoration of ABCA1 expression through proteasome inhibition.

THE ANTITUMORAL ROLE OF HDL: IN VITRO STUDIES ON PROSTATE CANCER CELL LINES / E. Giorgio ; tutor: M.Gomaraschi ; coordinator: A. L. Catapano. - : . Dipartimento di Scienze Farmacologiche e Biomolecolari, 2021 Jan 13. ((33. ciclo, Anno Accademico 2020.

THE ANTITUMORAL ROLE OF HDL: IN VITRO STUDIES ON PROSTATE CANCER CELL LINES

E. Giorgio
2021-01-13

Abstract

Background - Prostate cancer (PCa) is the most common malignancy in men. After initial response to androgen deprivation therapy, PCa can evolve to a castration-resistant phenotype, for which therapeutic options are poorly effective. In the tumor microenvironment, sources of cholesterol, reactive oxygen species (ROS) and pro-inflammatory molecules can favor cancer cell proliferation. Indeed, cells need cholesterol to proliferate, as structural and functional component of cell membranes and as substrate for the synthesis of hormones and oxysterols. In addition, recent evidence suggests that oxidative stress can play a role in the pathogenesis and the progression of prostate cancer (PCa). Aim - Since HDL are known to promote cell cholesterol efflux and to exert antioxidant activities, aim of the study was to investigate whether HDL can reduce cholesterol content and oxidative stress in PCa cells, thus modulating their proliferation. Methods - Androgen-sensitive and -resistant PCa cell lines (LNCaP and PC-3, respectively) were tested and compared with non tumor prostate epithelial cells (PNT2). Lipoproteins were isolated by ultracentrifugation from the plasma of healthy volunteers. ROS levels and cell cholesterol content were assessed by fluorescence. Oxidative stress was induced by H2O2 and cell cholesterol content was increased by incubation with LDL. The expression of proteins involved in cell cholesterol metabolism was assessed by by RT-PCR, western blotting or immunofluorescence. Cell proliferation was assessed by cell count, cell cycle analysis and colony formation assay. Bortezomib was used to inhibit the proteasome. Results - HDL significantly reduced basal and H2O2-induced oxidative stress in normal, androgen-sensitive and -resistant PCa cell lines. The androgen receptor, SRBI, ABCA1 and G1 transporters were not involved. Consequently, HDL completely blunted H2O2-induced increase of cell proliferation, through their capacity to prevent the H2O2-induced shift of cell cycle distribution towards G2/M phase. Cholesterol homeostasis is altered in PCa cells. PCa cells, that needs of cholesterol, mainly rely on lipoprotein uptake than intracellular synthesis of it. In LNCaP cells, the incubation with LDL increased cholesterol content and cell proliferation. The concomitant incubation with HDL promoted cell cholesterol reduction and blunted LDLinduced cell proliferation. On the contrary, in PC-3 cells HDL caused a further increase of cholesterol content and lost their ability to limit LDL-induced cell proliferation. In PC-3 cells, ABCA1 mRNA levels were elevated, but the protein was hardly detectable in the absence of pathogenic mutations in the ABCA1 gene. The treatment with the proteasome inhibitor bortezomib restored ABCA1 expression and consequently the ability of HDL to reduce cell cholesterol content and LDL-induced proliferation. Conclusions - Collectively, HDL can inhibit the proliferation of androgen-sensitive and castration-resistant PCa cell lines induced by oxidative stress and LDL. In castration-resistant PCa cells, HDL prevented LDLinduced cell proliferation only after the restoration of ABCA1 expression through proteasome inhibition.
GOMARASCHI, MONICA
CATAPANO, ALBERICO LUIGI
HDL; Prostate Cancer; Proliferation
Settore BIO/14 - Farmacologia
THE ANTITUMORAL ROLE OF HDL: IN VITRO STUDIES ON PROSTATE CANCER CELL LINES / E. Giorgio ; tutor: M.Gomaraschi ; coordinator: A. L. Catapano. - : . Dipartimento di Scienze Farmacologiche e Biomolecolari, 2021 Jan 13. ((33. ciclo, Anno Accademico 2020.
Doctoral Thesis
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/2434/798690
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