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研究生:林育嘉
研究生(外文):Yu-Chia Lin
論文名稱:探討P2X7受器相關及非相關機制對於Neuro-2a神經母細胞瘤的細胞存活的調控
論文名稱(外文):Examine the P2X7 receptor-dependent and -independent regulation of cell survival of Neuro-2a neuroblastoma cells
指導教授:孫興祥
指導教授(外文):Synthia H. Sun
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:神經科學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
畢業學年度:97
語文別:英文
論文頁數:53
中文關鍵詞:嘌呤類受器神經母細胞瘤
外文關鍵詞:P2X7Neuro-2aneuroblastomaATPpurinergic receptoroATP
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研究指出,在腫瘤外的細胞間隙有高濃度的ATP存在,故推測在腫瘤細胞的生理功能上需要ATP調控的訊息傳遞路徑。P2X7受器是低親合性的嘌呤類受器,且在某些癌症細胞上有較高的表現,顯示其與腫瘤細胞關係密切。因此,在本篇研究中我們想要研究P2X7受器在Neuro-2a神經母細胞瘤上對於其增生、細胞週期及死亡所扮演的角色。此外,維他命A酸(retinoic acid, RA)是維他命A的代謝物,且常被用來作為分化神經母細胞瘤的趨化因子。在我們之前的研究發現維他命A酸誘導神經分化的作用可能是透過降低P2X7受器表現。故在本篇論文中也將更深入地探討維他命A酸造成P2X7受器表現量降低的機制。我們研究發現水解細胞外的ATP、用拮抗劑或RNA干擾去抑制P2X7受器都會明顯地造成細胞數目下降,顯示有內生性活化的P2X7受器在維持細胞族群的大小。然而,藉由鈣離子載體ionomycin提升細胞內鈣離子濃度並無法回復抑制P2X7受器後所造成的細胞數目減少,顯示此作用可能與P2X7受器誘導的鈣離子入流無關。利用BrdU嵌入試驗(BrdU incorporation assay)發現P2X7受器的拮抗劑oATP但非BBG會抑制Neuro-2a細胞的增生。此外,我們利用流式細胞儀分析發現oATP但非BBG會改變細胞週期。顯示oATP對細胞增生及細胞週期造成的影響並非經由P2X7受器。有趣的是,利用oATP或siRNA抑制P2X7受器會造成明顯的細胞死亡,顯示P2X7受器會支持Neuro-2a細胞的存活。此外,oATP也會造成人類神經母細胞瘤SH-SY5Y細胞的細胞活性下降。我們利用即時聚合酶連鎖反應(real-time PCR)發現RA會促進P2X7受器的轉錄。活化RAR而非RXR會降低P2X7受器的蛋白質表現量,表示RAR訊息傳遞路徑可能參與在RA降低P2X7受器蛋白質表現量的過程中。此外,抑制ERK活化無法回復RA造成的P2X7受器表現量降低,顯示ERK/MAPK路徑與RA降低P2X7受器蛋白質表現量的過程無關。總括而言,我們的結果指出P2X7受器會支持Neuro-2a神經母細胞瘤的細胞存活。此外,RA會藉由一RAR活化相關而ERK/MAPK路徑無關的非轉錄調控來讓P2X7受器表現量下降。
Recent studies indicated that ATP is concentrated in tumor-built microenvironment up to hundreds micromolar range and P2X7 receptor (P2X7R), a low affinity ATP receptor, has high expression level in some of the tumor cells suggesting the ATP-based signaling pathways play roles in tumor physiology. Thus, in the present study, we examined the role of P2X7R in growth, cell cycle profile and cell survival of Neuro-2a neuroblastoma cells. Moreover, retinoic acid (RA), a bioactive metabolite of vitamin A, is considered as a potential agent for differentiation neuroblastoma cells. In our previous studies, the RA-induced Neuro-2a differentiation was associated with decreases in P2X7R expression. Therefore, in the present study, we also investigated the mechanisms involved in RA-decreased P2X7R expression of Neuro-2a cells. Our results revealed that hydrolysis of extracellular ATP, or inhibition by antagonist (oATP and BBG) or siRNA knockdown the expression of P2X7R significantly reduced cell number suggesting that the basal activation of P2X7R was important in maintaining cell viability. However, elevation of intracellular calcium by ionomycin could not restore the oATP-decreased cell viability suggesting that the oATP effect might be mediated through a mechanism other than the P2X7R-induced Ca2+ influx. By using BrdU incorporation assay, we found that oATP but not BBG attenuated cell proliferation. By using flow cytometry, we also found that oATP but not BBG altered cell cycle progression. Thus the oATP altered cell proliferation and cell cycle progression may be mediated through a P2X7R-independent mechanism. Interestingly, either inhibition or knockdown P2X7R induced a dramatic cell death, indicating that P2X7R was important in supporting cell survival of Neuro-2a cells. In addition, oATP also decreased cell viability in human SH-SY5Y neuronblastoma cell-line. To elucidate the possible mechanism involving in RA-decreased P2X7R expression, we conducted real-time PCR. Our results revealed that RA enhanced the transcription of P2X7R. Activation of RAR but not RXR decreased P2X7R protein expression, indicating RAR pathway might be involved in RA-decreased P2X7R process. In addition, inhibition of MAPK activation did not rescue the RA-decreased P2X7R expression, suggesting that the RA-decreased P2X7R expression might be mediated through an ERK/MAPK independent pathway. In conclusion, our data indicated that the basal activation of P2X7R was required in maintaining cell population of Neuro-2a murine neuroblastoma cells. Furthermore, we demonstrated that RA down-regulated P2X7R expression was mediated through a RAR-involved but ERK/MAPK independent and non-transcriptional mechanism.
Abstract 4
中文摘要 5
Chapter 1. Introduction 6
1.1 ATP and Purinergic Receptors 6
1.2 Purinergic Signaling in Tumor Microenvironment 6
1.3 P2X7 Receptor 7
1.3.1 Receptor Properties and its Signaling Complex 7
1.3.2 Functional Roles of P2X7R in the Nervous System 8
1.3.3 To be a Potential Biomarker in Cancer Diagnosis 9
1.4 Retinoic Acid (RA) 9
1.5 Specific Aims 10
Chapter 2. Materials and Methods 12
2.1 Materials 12
2.2 Neuro-2a cell culture 12
2.3 RA application 12
2.4 Western blotting analysis 13
2.5 Immunocytochemistry 13
2.6 Calcium imaging 14
2.7 Knockdown of P2X7 receptors by siRNA targeting 14
2.8 Cell viability assay 15
2.9 BrdU incorporation assay 15
2.10 Cell cycle studies 15
2.11 LDH release detection assay 16
2.12 Real-time PCR 16
2.13 Data analysis 16
Chapter 3. Results 17
3.1 Naïve Neuro-2a neuroblastoma cells express functional P2X7 receptors. 17
3.2 P2X7 receptor abolishment decreases cell number in naïve Neuro-2a cells. 17
3.3 ATP-hydrolyzing enzyme, apyrase, reduces cell number in naïve Neuro-2a cells. 18
3.4 Over-activation of P2X7R doesn’t promote cell proliferation in naïve Neuro-2a cells. 19
3.5 Calcium signaling is not involved in the P2X7R-decreased cell viability. 19
3.6 Examination of the mechanisms involved in the P2X7R-maintained cell number -- the cell proliferation. 20
3.7 Examination of the mechanisms involved in the P2X7R-maintained cell number --- the cell cycle progression. 21
3.8 Examination of the mechanisms involved in the P2X7R-maintained cell number --- the cell survival. 21
3.9 Examination of P2X7 receptors in human SH-SY5Y neuroblastoma cell-line. 22
3.10 Retinoic acid time-dependently reduces P2X7R protein expression. 23
3.11 Retinoic acid increases but not decreases P2X7R mRNA expression. 23
3.12 RAR activation may be involved in RA-decreased P2X7R expression. 23
3.13 RA-decreased P2X7R expression is ERK/MAPK pathway independent. 24
Chapter 4. Discussion 25
4.1 P2X7 receptors maintain cell viability through calcium independent mechanisms. 25
4.2 P2X7 receptors support proliferation in various cell types. 25
4.3 Periodate oxidized-ATP alters cell cycle progression through a P2X7R independent mechanism. 25
4.4 P2X7R plays a supportive but not cytolytic role in Neuro-2a neuroblastoma cells. 26
4.5 Periodate oxidized-ATP reduced cell viability through both P2X7R-dependent and independent mechanisms in Neuro-2a cells. 26
4.6 RA induces P2X7R decrease through a post-transcriptional regulation. 27
4.7 The significance of our research to the treatment of neuroblastoma. 27
Figures 29
Figure 1. Neuro-2a neuroblastoma cells express functional P2X7 receptors. 29
Figure 2. Antagonists of P2X7 receptor reduce cell viability and cell number in naïve Neuro-2a cells. 30
Figure 3. P2X7R down-regulation by siRNA reduces cell viability and cell number in naïve Neuro-2a cells. 31
Figure 4. ATP-hydrolyzing enzyme, apyrase, reduces cell viability and cell number in naïve Neuro-2a cells. 32
Figure 5. Stimulation by P2X7R agonist BzATP doesn’t affect cell viability. 33
Figure 6. Elevation of intracellular calcium by ionomycin can not restore oATP- or siRNA-decreased cell viability. 34
Figure 7. P2X7R antagonist oATP but not BBG attenuates cell proliferation. 35
Figure 8. P2X7R antagonist oATP but not BBG alters cell cycle progression. 36
Figure 9. BzATP competition fails to restore the oATP-induced cell cycle alternation. 37
Figure 10. P2X7R knockdown by siRNA doesn’t affect cell cycle progression. 38
Figure 11. P2X7R antagonist oATP but not BBG increases LDH release. 39
Figure 12. P2X7R knockdown by siRNA increases LDH release. 40
Figure 13. P2X7R knockdown by siRNA increases caspase3 activation. 41
Figure 14. P2X7R antagonist oATP but not BBG reduces cell viability in human SH-SY5Y neuroblastoma cells. 42
Figure 15. Retinoic acid time-dependently reduced P2X7R protein expression. 43
Figure 16. Retinoic acid promotes P2X7 receptor mRNA expression. 44
Figure 17. RA and RAR-specific agonist TTNPB decrease P2X7R protein expression in Neuro-2a cells. 45
Figure 18. Activation of ERK1/2 is not involved in the RA-decreased P2X7R expression. 46
Table 47
Reference 48
Adinolfi E, Kim M, Young MT, Di Virgilio F and Surprenant A (2003) Tyrosine phosphorylation of HSP90 within the P2X7 receptor complex negatively regulates P2X7 receptors. J Biol Chem. 278, 37344-37351.

Amstrup J and Novak I. (2003) P2X7 receptor activates extracellular signal-regulated kinases ERK1 and ERK2 independently of Ca2+ influx. Biochem J. 374, 51-61.

Armstrong JN, Brust TB, Lewis RG and MacVicar BA (2002) Activation of presynaptic P2X7-like receptors depresses mossy fiber-CA3 synaptic transmission through p38 mitogen-activated protein kinase. J Neurosci. 22, 5938-5945.

Ballerini P, Rathbone MP, Di Iorio P, Renzetti A, Giuliani P, D'Alimonte I, Trubiani O, Caciagli F and Ciccarelli R (1996) Rat astroglial P2Z (P2X7) receptors regulate intracellular calcium and purine release. Neuroreport. 7, 2533-2537.

Baricordi OR, Melchiorri L, Adinolfi E, Falzoni S, Chiozzi P, Buell G and Di Virgilio F. (1999) Increased proliferation rate of lymphoid cells transfected with the P2X(7) ATP receptor. J Biol Chem. 274, 33206-33208.

Beigi RD, Kertesy SB, Aquilina G, Dubyak GR. (2003) Oxidized ATP (oATP) attenuates proinflammatory signaling via P2 receptor-independent mechanisms. Br J Pharmacol. 140:507-519.

Bianco F, Ceruti S, Colombo A, Fumagalli M, Ferrari D, Pizzirani C, Matteoli M, Di Virgilio F, Abbracchio MP and Verderio C (2006) A role for P2X7 in microglial proliferation. J Neurochem. 99, 745-758.

Brändle U, Kohler K and Wheeler-Schilling TH (1998) Expression of the P2X7-receptor subunit in neurons of the rat retina. Brain Res Mol Brain Res. 62, 106-109.

Cañón E, Cosgaya JM, Scsucova S and Aranda A. (2004) Rapid effects of retinoic acid on CREB and ERK phosphorylation in neuronal cells. Mol Biol Cell. 15, 5583-5592.

Clagett-Dame M, McNeill EM and Muley PD. (2006) Role of all-trans retinoic acid in neurite outgrowth and axonal elongation. J Neurobiol. 66, 739-756.

Di Virgilio F. (2003) Novel data point to a broader mechanism of action of oxidized ATP: the P2X7 receptor is not the only target. Br J Pharmacol. 140, 441-443.

Duan S, Anderson CM, Keung EC, Chen Y, Chen Y and Swanson RA (2003) P2X7 receptor-mediated release of excitatory amino acids from astrocytes. J Neurosci. 23, 1320-1328.

Ferrari D, Los M, Bauer MK, Vandenabeele P, Wesselborg S and Schulze-Osthoff K. (1999) P2Z purinoreceptor ligation induces activation of caspases with distinct roles in apoptotic and necrotic alterations of cell death. FEBS Lett. 447, 71-75.

Franke H and Illes P (2006) Involvement of P2 receptors in the growth and survival of neurons in the CNS. Pharmacol Ther. 109, 297 – 324.

Guo C, Masin M, Qureshi OS and Murrell-Lagnado RD (2007) Evidence for functional P2X4/P2X7 heteromeric receptors. Mol Pharmacol.72, 1447-1456.

Ishola TA and Chung DH. (2007) Neuroblastoma. Surg Oncol. 16, 149-156.

Jacques-Silva MC, Rodnight R, Lenz G, Liao Z, Kong Q, Tran M, Kang Y, Gonzalez FA, Weisman GA and Neary JT. (2004) P2X7 receptors stimulate AKT phosphorylation in astrocytes. Br J Pharmacol. 141, 1106-1117.

Kim M, Jiang LH, Wilson HL, North RA and Surprenant A (2001a) Proteomic and functional evidence for P2X7 receptor signaling complex. EMBO J. 20, 6347-6358.

Kim M, Spelta V, Sim J, North RA and Surprenant A (2001b) Differential assembly of rat purinergic P2X7 receptor in immune cells of the brain and periphery. J Biol Chem. 276, 23262-23267.

Kong Q, Wang M, Liao Z, Camden JM, Yu S, Simonyi A, Sun GY, Gonzalez FA, Erb L, Seye CI and Weisman GA (2005) P2X7 nucleotide receptors mediate caspase-8/9/3-dependent apoptosis in rat primary cortical neurons. Purinergic Signal. 1, 337–347.

Kukley M, Stausberg P, Adelmann G, Chessell IP and Dietrich D (2004) Ecto- nucleotidases and nucleoside transporters mediate activation of adenosine receptors on hippocampal mossy fibers by P2X7 receptor agonist 2’-3’-O-(4-Benzoylbenzoyl)-ATP. J Neurosci. 24, 7128-7139.

Larsson KP, Hansen AJ and Dissing S. (2002) The human SH-SY5Y neuroblastoma cell-line expresses a functional P2X7 purinoceptor that modulates voltage-dependent Ca2+ channel function. J Neurochem. 83, 285-298.

Lin JH, Takano T, Arcuino G, Wang X, Hu F, Darzynkiewicz Z, Nunes M, Goldman SA and Nedergaard M. (2007) Purinergic signaling regulates neural progenitor cell expansion and neurogenesis. Dev Biol. 302, 356-366.

Li X, Qi X, Zhou L, Catera D, Rote NS, Potashkin J, Abdul-Karim FW and Gorodeski GI. (2007) Decreased expression of P2X7 in endometrial epithelial pre-cancerous and cancer cells. Gynecol Oncol. 106, 233-243.

Li X, Zhou L, Feng YH, Abdul-Karim FW and Gorodeski GI. (2006) The P2X7 receptor: a novel biomarker of uterine epithelial cancers. Cancer Epidemiol Biomarkers Prev. 15, 1906-1913.

Maden M. (2002) Retinoid signalling in the development of the central nervous system. Nat Rev Neurosci. 3, 843-853.

Miloso M, Villa D, Crimi M, Galbiati S, Donzelli E, Nicolini G and Tredici G. (2004) Retinoic acid-induced neuritogenesis of human neuroblastoma SH-SY5Y cells is ERK independent and PKC dependent. J Neurosci Res. 75, 241-252.

Ohta A, Gorelik E, Prasad SJ, Ronchese F, Lukashev D, Wong MK, Huang X, Caldwell S, Liu K, Smith P, Chen JF, Jackson EK, Apasov S, Abrams S and Sitkovsky M. (2006) A2A adenosine receptor protects tumors from antitumor T cells. Proc Natl Acad Sci. 103, 13132-13137.

Panner A, Cribbs LL, Zainelli GM, Origitano TC, Singh S and Wurster RD. (2005) Variation of T-type calcium channel protein expression affects cell division of cultured tumor cells. Cell Calcium. 37, 105-119.

Panner A and Wurster RD. (2006) T-type calcium channels and tumor proliferation. Cell Calcium. 40, 253-259.

Papp L, Vizi ES and Sperlágh B. (2007) P2X7 receptor mediated phosphorylation of p38MAP kinase in the hippocampus. Biochem Biophys Res Commun. 355, 568-574.

Pellegatti P, Raffaghello L, Bianchi G, Piccardi F, Pistoia V and Di Virgilio F. (2008) Increased level of extracellular ATP at tumor sites: in vivo imaging with plasma membrane luciferase. PLoS ONE. 3, e2599.

Ryu JK, Choi HB, Hatori K, Heisel RL, Pelech SL, McLarnon JG and Kim SU. (2003) Adenosine triphosphate induces proliferation of human neural stem cells: Role of calcium and p70 ribosomal protein S6 kinase. J Neurosci Res. 72, 352-362.

Schug TT, Berry DC, Shaw NS, Travis SN and Noy N. (2007) Opposing effects of retinoic acid on cell growth result from alternate activation of two different nuclear receptors. Cell. 129, 723-733.

Schüle R, Rangarajan P, Yang N, Kliewer S, Ransone LJ, Bolado J, Verma IM and Evans RM. (1991) Retinoic acid is a negative regulator of AP-1-responsive genes. Proc Natl Acad Sci 88, 6092-6096.

Smart ML, Gu B, Panchal RG, Wiley J, Cromer B, Williams DA and Petrou S. (2003) P2X7 receptor cell surface expression and cytolytic pore formation are regulated by a distal C-terminal region. J Biol Chem. 278, 8853-8860.

Solini A, Cuccato S, Ferrari D, Santini E, Gulinelli S, Callegari MG, Dardano A, Faviana P, Madec S, Di Virgilio F and Monzani F. (2008) Increased P2X7 receptor expression and function in thyroid papillary cancer: a new potential marker of the disease? Endocrinology. 149, 389-396.

Sperlágh B and Illes P (2007) Purinergic modulation of microglial cell activation. Purinergic Signal. 3, 117-127.

Sperlágh B, Köfalvi A, Deuchars J, Atkinson L, Milligan CJ, Buckley NJ and Vizi ES (2002) Involvement of P2X7 receptors in the regulation of neurotransmitter release in the rat hippocampus. J Neurochem. 81, 1196-1211.

Sperlágh B, Vizi ES, Wirkner K and Illes P (2006) P2X7 receptors in the nervous system. Prog Neurobiol. 78, 327-346.

Surprenant A, Rassendren F, Kawashima E, North RA and Buell G (1996) The cytolytic P2Z receptor for extracellular ATP identified as a P2X receptor (P2X7). Science 272, 735-738.

Tayama Y, Kawahara H, Minami R, Shimada M and Yokosawa H. (2007) Association of Rpn10 with high molecular weight complex is enhanced during retinoic acid-induced differentiation of neuroblastoma cells. Mol Cell Biochem. 306, 53-57.

Wang CM, Chang YY, Kuo JS and Sun SH (2002) Activation of P2X7 receptors induced [3H] GABA release from the RBA-2 type-2 astrocyte cell line through a Cl-/HCO3- dependent mechanism. Glia 37, 8–18.

Zhang X, Zhang M, Laties AM and Mitchell CH (2005) Stimulation of P2X7 receptors elevates Ca2+ and kills retinal ganglion cells. Invest Ophthalmol Vis Sci. 46, 2183-2191.
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