Investigating the superoxide mediated survival pathway in the prostate cancer cell line LNCaP

INVESTIGATING THE SUPEROXIDE MEDIATED SURVIVAL PATHWAY IN THE PROSTATE CANCER CELL LINE LNCAP GOH SHIJIE B.Sci (Hons), NUS A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF BIOCHEMISTRY NATIONAL UNIVERSITY OF SINGAPORE 2012 i Acknowledgements I would like to express my heartfelt gratitude to my supervisor and mentor, A/P Marie-Véronique Clément, for her constant guidance, encouragement and advice over the past nine years It has been a long journey since the day I stepped into her office, a hapless kid who knew nothing about science Through her infectious enthusiasm and patient guidance, I had developed a strong interest for research and eventually decided to pursue this Ph.D However, I could not have possibly completed the work in this thesis without her constant encouragement and stimulating discussions I also wish to thank my lab colleagues and friends for their help; be it in the form of encouragement, advice, reagents or just a listening ear I would like to specifically thank Ms Teong Huey Fern, Dr Sharon Lim and Dr Michelle Chang Ker Xing who took me under their wings and taught me many things despite their busy schedules I would also like to thank Mr Ping Yueh Shyang for the insightful discussions and very enjoyable collaboration experience Last but not least, I would like to thank my dearest wife, Ms Adeline Tan, for her support and belief in me I really appreciate her kind understanding and accommodation to my irregular lab working hours ii Contents Acknowledgements ii Contents iii Summary vi List of Figures viii Abbreviations x CHAPTER 1: INTRODUCTION 1.1 CANCER BIOLOGY 1.2 SURVIVAL PATHWAYS 1.2.1 Growth factor signaling 1.2.2 PI3K-Akt signaling pathway 1.3 APOPTOSIS 1.3.1 The extrinsic and intrinsic pathway 1.3.2 MOMP and Bcl-2 family proteins 1.3.3 Regulation of BH3-only proteins 10 1.3.4 Models of Bax/Bak activation 13 1.4 Cancer cell metabolism and ph regulation 19 1.4.1 Cancer cell metabolism and glycolysis 19 1.4.2 Regulation of intracellular pH 20 1.5 ROS SIGNALING AND CANCER 22 1.5.1 Sources of intracellular ROS 22 1.5.2 ROS chemistry 23 1.5.3 Antioxidant defence 24 1.5.4 ROS as signaling components 25 1.6 PROSTATE CANCER 26 1.6.1 Prostate cancer and androgen receptor 27 1.6.2 Prostate cancer and PTEN 27 1.6.3 Oxidative stress in prostate cancer 28 1.6.4 The LNCaP model 29 1.7 AIM OF STUDY 32 CHAPTER 2: MATERIALS AND METHODS 33 iii 2.1 MATERIALS 33 2.1.1 Chemicals 33 2.1.2 Cell culture 34 2.1.3 Drug treatments 34 2.1.4 Antibodies 35 2.2 METHODS 36 2.2.1 Bax activation assay (saponin) 36 2.2.2 Bax activation assay (Leucoperm) 36 2.2.3 Caspase activity 37 2.2.4 Determination of intracellular pH, NHE activity and proton affinity 38 2.2.5 Bad phosphorylation ELISA assay 39 2.2.6 Kinase assay for Bad phosphorylation 40 2.2.7 Mitochondrial subcellular fractionation 41 2.2.8 SDS-PAGE and Western blotting 41 2.2.9 Gene knockdown by RNA interference 43 2.2.10 Measurement of intracellular H2O2 level 44 2.2.11 Measurement of intracellular O2˙ˉ level 44 2.2.12 Protein concentration determination 45 2.2.13 Propidium iodide staining for DNA fragmentation 45 2.2.14 RNA isolation and PCR 46 2.2.15 Statistical analysis 47 CHAPTER 3: RESULTS 48 3.1 MECHANISM OF LY294002 INDUCED APOPTOSIS 48 3.1.1 Bad dephosphorylation is essential for apoptosis 49 3.1.2 Bax is required for LY294002 induced apoptosis 53 3.1.3 Bak activation is required for apoptosis 58 3.1.4 Bcl-xL downregulation is required for apoptosis 62 3.2 SERUM PREVENTS LY294002 INDUCED APOPTOSIS 66 3.2.1 Serum promotes overexpression of Bcl-xL 71 3.2.2 Serum maintains Bad phosphorylation 73 3.2.3 Serum prevents Bax activation and translocation induced by LY294002 75 3.3 SUPEROXIDE REGULATION OF CELL SURVIVAL 79 iv 3.3.1 Superoxide reduction results in loss of serum protection against LY294002 induced apoptosis 82 3.3.2 Serum and superoxide are distinct pathways 86 3.3.3 Superoxide promotes Bcl-xL expression 88 3.3.4 Superoxide maintains Bad phosphorylation 90 3.3.5 Superoxide prevents Bax activation 112 CHAPTER 4: DISCUSSION 148 4.1 SUPEROXIDE MAINTAINS PI3K-AKT INDEPENDENT SURVIVAL IN LNCAP CELLS 148 4.1.1 EGF, R1881 and serum prevents LY294002 induced in LNCaP cells 148 4.1.2 Superoxide promotes survival independently from PI3K-Akt pathway 149 4.2 SUPEROXIDE MAINTENANCE OF BAD PHOSPHORYLATION IS PIM-1 MEDIATED 151 4.3 PIM-1 ACTIVITY CAN BE REGULATED BY SUPEROXIDE 153 4.3.1 Pim-1 half life and stability are increased in LNCaP cells 154 4.3.2 Redox regulation of Pim-1 activity 154 4.4 AKT SILENCING CAN DECREASE BAD SER75 PHOSPHORYLATION 156 4.5 NON-PH REGULATION FUNCTIONS OF NHE 157 4.6 MAINTENANCE OF NHE-2 FUNCTION IS ESSENTIAL FOR PREVENTION OF BAX ACTIVATION 158 4.7 PIM-1 MEDIATED REGULATION OF NHE-2 159 4.8 SUPEROXIDE IS AN IMPORTANT MEDIATOR OF LNCAP SURVIVAL 160 4.9 POTENTIAL APPLICATIONS 161 4.10 CONCLUSION 163 References 165 Appendices 188 v Summary Prostate cancer is the cancerous development of the prostate, and develops over several years with little or no clinical symptoms Hence, the detection and diagnosis of prostate cancer usually occurs in the late metastatic stage, resulting in poor prognosis One of the most common mutations found in prostate cancer is the inactivation mutation of PTEN This leads to the constitutive activation of PI3K-Akt signaling, conferring prostate cancer cells the ability to survive without external mitogenic signals However, current monotherapies targeting the PI3K-Akt survival pathway remain ineffective, suggesting that there exists an alternate PI3K-Akt independent survival pathway in prostate cancer cells Increasingly, cancer progression and aggressiveness have been found to correlate positively with mild but higher than normal oxidative stress, which has been shown to enhance cancer cell survivability and chemoresistance More importantly, the investigation of redox signaling in prostate cancer cells has identified the superoxide anion (O2˙ˉ) as the key reactive oxygen species in enhancing cell survival In this study, we provide evidence for the role of O2˙ˉ in the activation of the PI3K-Akt independent survival signaling in LNCaP, the most widely used in vitro model for prostate cancer LNCaP cells are able to survive and grow in the absence of growth factors, but undergo apoptosis upon the shutting down of the PI3K-Akt pathway by LY294002 However, EGF, R1881 and serum were shown to protect LNCaP cells from LY294002 induced apoptosis, by maintaining Bad phosphorylation and/or upregulating Bcl-xL expression In this study, the roles of the Bcl-2 family proteins were investigated and a set of parameters were defined It was found that vi LNCaP survival was enhanced by maintaining Bad phosphorylation at serine 75, upregulating Bcl-xL expression and preventing Bax/Bak translocation and activation Superoxide was shown in this study to be able to protect LNCaP cells against LY294002 induced apoptosis This was achieved by preventing Bax/Bak activation via the defined parameters: maintaining Bad serine 75 phosphorylation, increasing Bcl-xL expression and preventing Bax activation We also show evidence that Pim-1 was the main effector in O2˙ˉ signaling; maintaining Bad serine 75 phosphorylation This was consistent with reports of Pim-1 being a prognostic marker in prostate cancer Also, we have demonstrated for the first time that NHE-2 is required for Bax activation in LNCaP cells, and that NHE-2 mediated Bax activation is prevented by O2˙ˉ signaling In summary, this study has highlighted the crucial role of O2˙ˉ in the maintenance of the PI3K-Akt independent survival pathway in LNCaP cells vii List of Figures Figure 1: The hallmarks of cancer Figure 2: The Bcl-2 protein family Figure 3: BH3-only proteins can engage apoptosis via many different cellular processes 11 Figure 4: Direct activation and displacement model 16 Figure 5: Bcl-xL dependent Bax retrotranslocation 18 Figure 6: Graphical representation of the NHE-1 protein 21 Figure 7: Production of ROS in cells 23 Figure 8: Mechanisms of survival enhancement in LNCaP 31 Figure 9: Role of Bad in LY294002 induced cell death 52 Figure 10: LY294002 treatment results in increased Bax translocation and activation 55 Figure 11: Bax is required in LY294002 induced cell death 57 Figure 12: Bax and Bak are involved in LY294002 induced apoptosis 61 Figure 13: Bcl-xL downregulation is required for LY294002 induced apoptosis 63 Figure 14: Mechanism of LY294002 induced apoptosis 65 Figure 15: Serum prevents LY294002 induced apoptosis 67 Figure 16: Serum rescues LY294002 induced cell death in LNCaP cells 70 Figure 17: Serum can increase Bcl-xL expression 72 Figure 18: Serum maintains Bad phosphorylation 74 Figure 19: Serum prevents Bax activation and translocation induced by LY294002 77 Figure 20: Serum prevents apoptosis in LNCaP cells 78 Figure 21: Decrease in superoxide can bypass serum protection 81 Figure 22: Superoxide reduction results in loss of serum protection against LY294002 induced apoptosis 85 Figure 23: Superoxide and serum are distinct pathways 87 Figure 24: Superoxide promotes Bcl-xL expression 89 Figure 25: Superoxide maintains Bad phosphorylation 93 Figure 26: Pim-1 can phosphorylate Bad at serine 75 in LNCaP 96 Figure 27: DPI, quercetagetin and removal of serum can lower phosphorylated Bad levels 103 Figure 28: Dephosphorylation of Bad by quercetagetin bypasses serum protection 107 Figure 29: Inhibition of Pim-1 by quercetagetin in the absence of Akt results in high caspase activity 111 Figure 30: DPI sensitizes LNCaP cells to cell death by causing Bax conformational change 114 Figure 31: DPI induced intracellular acidification can be rescued by DDC 119 Figure 32: Inhibition of NHE by EiPa results in intracellular acidification and lowers pH at which NHE begins proton extrusion 121 viii Figure 33: Inhibition of NHE by EiPa removes protective effect of serum against LY294002 induced cell death 124 Figure 34: EiPa treatment results in increased Bax activation 127 Figure 35: Inhibition of NHE-1 by cariporide results in intracellular acidification but is unable to sensitize cells to LY294002 induced cell death 130 Figure 36: NHE isoform expression in LNCaP 132 Figure 37: Loss of NHEs and results in intracellular acidification 135 Figure 38: Loss of NHE-2 results in increased Bax activation 138 Figure 39: NHE-2 prevents Bax activation and intracellular acidification 140 Figure 40: Cytoplasmic C-terminus amino acid sequences of NHE isoforms expressed in LNCaP 142 Figure 41: Pim-1 inhibition of quercetagetin has no effect on pH and NHE activity 143 Figure 42: NHE-2 plays a more important role in the regulation of pH in LNCaP than NHE-1 146 Figure 43: Death circuitry in LNCaP 147 ix Abbreviations BCECF-CM 2’,7’-bis(2-carboxyethyl)-5-(and-6)-carboxyfluorescein BSA Bovine serum albumin CM-H2DCFDA 5-(and-6)-chloromethyl-2’,7’-dichlorofluorescin diacetate DDC Diethyldithiocarbamate DMSO Dimethylsulfoxide 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component of a survival pathway activated by docetaxel and promotes survival of docetaxel-treated prostate cancer cells J Biol Chem 283(30):20635-20644 Zhang B, Zhang Y and Shacter E (2004) Rac1 inhibits apoptosis in human lymphoma cells by stimulating Bad phosphorylation on Ser-75 Mol Cell Biol 24(14):6205-6214 187 Appendices 188 189 ... result in cell death The delicate redox balance is kept in check, and is often the deciding factor in determining cell fate in these cancer cells 1.6 PROSTATE CANCER Prostate cancer, the cancerous... classified under the extrinsic and intrinsic pathway The extrinsic pathway involves the direct transduction of an external signal that activates apoptosis The intrinsic pathway is mediated by the mitochondria,... of the proteins are designated and the bold lines define the BH domains ‘TM’ marks the hydrophobic transmembrane domain Bcl-2 proteins play an important role in determining cell fate and survival,