臺灣博碩士論文加值系統

順鉑 (順一雙氨雙氯鉑抗癌藥，cisplatin)是一種鉑製劑的抗癌藥物，可用來治療多種癌症，然而癌細胞對其易產生抗藥性的特質是降低成功治療的一大因素。這篇文章中，使用對順鉑感性（cisplatin-susceptible; NTUB1）與抗性(cisplatin-resistant; NTUB1/P)的成對人類尿路上皮癌細胞株（human urothelial carcinoma cell line）來研究順鉑所造成細胞毒性與癌細胞對其產生抗性的機制。首先，我們發現順鉑無法在抗性細胞株NTUB1/P內增加與細胞凋亡相關蛋白BAX/BCL-2間之比值、細胞內活性氧化物（reactive oxygen species;ROS）之生成、與粒腺體質量。同時在細胞處理過順鉑後，具抗性的NTUB1/P細胞株內累積鉑（Platinum）的含量較少，順鉑吸收的減少是為其主因。沒有證據顯示液態式內吞（Fluid-phase endocytosis），caveolar所媒介的內吞（caveolar-mediated endocytosis,），與第一型銅輸送蛋白(copper transporter 1; CTR1)參與鉑的吸收。針對Aquaporins，鈉鉀ATPase（Na+，K+-ATPase）和鉀離子通道(potassium channels) 的則抑制可以減少在感性細胞株NTUB1內鉑的累積，由此推測這三個通道有助於吸收順鉑進入細胞內。然而增加抗性細胞株NTUB1/P細胞內鉑的含量並不能增加順鉑對其產生的毒性。由此可以推測在NTUB1/P細胞株的細胞內，較少的鉑含量並不是其抗性產生的主因，本研究的其他證據顯示抗細胞凋零蛋白BCL-2、第一型血紅素氧化酶（heme oxygenase-1; HO-1）與調節細胞週期的p16的過度表現才是造成人類尿路上皮癌細胞株對順鉑產生抗性的主要原因。本研究亦發現NTUB1/P細胞株對三氧化二砷 （arsenic trioxide; ATO）有交叉抗性的產生，其細胞內砷含量較感性NTUB1細胞株要少，此是因為其有較少的aquaporin-3與較高的多重抗藥性蛋白(multidrug-resistant protein; MRP)表現。

Cisplatin is a platinum-based chemotherapy drug used to treat various types of cancers. However, the development of resistance to cisplatin is a major obstacle for successful treatment. In this study, an isogenic pair of cisplatin-susceptible (NTUB1) and –resistant (NTUB1/P) human urothelial carcinoma cell lines was used to elucidate the mechanism of cisplatin-induced cytotoxicity and resistance. First, cisplatin failed to increase BAX/BCL-2 ratio, reactive oxygen species (ROS) generation, and mitochondrial mass in NTUB1/P cells. Second, the significantly lower intracellular platinum concentration, which resulted from the decreased cisplatin uptake, was found in NTUB1/P cells. Fluid-phase endocytosis, caveolar-mediated endocytosis, or copper transporter 1 (CTR1) did not involve in platinum uptake in NTUB1 cells. Inhibition of aquaporins, Na+,K+-ATPase, and potassium channels decreased platinum accumulation in NTUB1 cells, and it suggested these three channels mediated platinum uptake. However, the enhancement of intracellular platinum concentration did not increase the susceptibility of NTUB1/P cells to cisplatin treatment. This indicated that reduction of intracellular platinum accumulation was not the account for the development of cisplatin resistance here. Instead, the over expression of anti-apoptotic Bcl-2, anti-oxidative heme oxygenase-1 (HO-1) and cell cycle regulator p16 seemed to be more important for the gaining of cisplatin resistance in these human urothelial carcinoma cells. Meanwhile, NTUB1/P cells were also cross-resistant to arsenic trioxide (ATO), and lower arsenic accumulation was because of lower expression of aquaporin-3 (AQP3) and higher expression of multidrug-resistant protein (MRP).

I. Introduction……………………………………………………………..……...…..….. 1
I.1. Bladder cancer and cisplatin treatment………………… …………………….. 1
I.2. Drug action of cisplatin……………………………………………….……..… 2
I.3. Molecular Bases of Cisplatin Resistance…………………………………….... 5
I.4. Specific aims ...…………………………………………………………………. 8

II. Materials and methods…………………………………………………………..…. 9
II.1. Cell culture ……………………………………………………………………. 9
II.2. Cytotoxicity assay …………………………………………………………….. 9
II.3. Apoptosis assay ………………………………………………………………… 9
II.4. Protein extraction and Western blot analysis ………………………………….. 9
II.5. Analysis of reactive oxygen species (ROS) generation ……………………….. 10
II.6. Determination of mitochondrial mass ………………………………….……… 10
II.7. Intracellular platinum and arsenic accumulation measurement ……………….. 11
II.8. Fluid-phase endocytosis assay ………………………………………………… 11
II.9. Microarray analysis ……………………………………………………………. 11
II.10. Preparation of cisplatin liposomes …………………………………………… 12
II.11. Transfection of siRNA ………………………………………………………. 12
II.12. RNA isolation and RT-PCR …………………………………………………. 13
II.13. Statistical analyses ……………………………………………………………. 13
III. Results………………………………………………………………....................... 13
III.1. The mechanisms of cisplatin-induced cytotoxicity…………………………… 13
III.1.1. NTUB1 cells were more sensitive to cisplatin probably due to the
apoptotic effect after cisplatin treatment ……………………………… 13
III.1.2. Pro-apoptotic BAX and anti-apoptotic BCL-2 protein were
over-expressed in NTUB1/P cells. Cisplatin up-regulated
pro-apoptotic Bax protein expression and increased BAX/BCL-2 ratio
in NTUB1 cells………………………………………………... ..….… 14
III.1.3. Reactive oxygen species (ROS) was induced by cisplatin, and
played a role in cisplatin-induced cytotoxicity in NTUB1 cells…….. 15
III.1.4. Caspase-dependent pathways were involved in cisplatin-induced
cytotoxicity in NTUB1 cells…………………………………………. 15
III.1.5. Changes of mitochondrial mass was involved in cisplatin-induced cytotoxicity in NTUB1 cells ………………………………………….. 16
III.1.6. N-nitroso propoxur caused higher cytotoxicity in NTUB1/P than
NTUB1 cells, and increased BAX/BCL-2 ratio, ROS generation, and mitochondrial mass in NTUB1/P cells ………………………………… 16
III.2. The mechanisms of cisplatin resistance in NTUB1/P cells…………………... 17
III.2.1. Lower intracellular platinum accumulation in NTUB1/P than NTUB1
cells ………………………………………………………………… 17
III.2.2. Fluid-phase endocytosis was not different in NTUB1 and NTUB1/P
cells ………………………………………………………………….. 17
III.2.3. Microarray analysis of endocytosis-related proteins on NTUB1 and
NTUB1/P cells……………………………………………………….. 18
III.2.4. Caveolin gene knockdown did not affect cisplatin-mediated
cytotoxicity and intracellular platinum accumulation in NTUB1 cells.. 19
III.2.5. Microarray analysis of several channels, pumps, or transporters on
NTUB1 and NTUB1/P cells ………………………………………….. 19
III.2.6. The role of CTR1 in cisplatin resistance in NTUB1/P cells………….. 20
III.2.7. The role of aquaporins, Na+,K+-ATPase, and potassium channels in
platinum accumulation in NTUB1 cells……………………………… 21
III.2.8. The increase in intracellular platinum accumulation in NTUB1/P
cells did not cause the same cytotoxic effect as seen in NTUB1 cells. 23
III.2.9. Effect of cisplatin liposome on platinum sensitivity in NTUB1 and
NTUB1/P cells……………………………………………………….. 24
III.2.10. BCL-2 and HO-1 were upregulated in NTUB1/P cells. Knockdown
of Bcl-2 or HO-1 sensitized the NTUB1/P cells to cisplatin treatment. 25
III.2.11. p16 were upregulated in NTUB1/P cells. Knockdown of p16
sensitized the NTUB1/P cells to cisplatin treatment…………………. 26
III.2.12. Knockdown of BCL-2, HO-1 and p16 sensitized the NTUB1/P cells
to cisplatin liposome treatment………………………………………. 27
III.2.13. The roles of CYR61 and MAGE1 on cisplatin resistance in
NTUB1/P cells……………………………………………………… 27
III.3. Cross-resistant to arsenic trioxide (ATO) in NTUB1/P cells…......................... 28
III.3.1. NTUB1/P was cross-resistant to ATO……………………………… 28
III.3.2. ATO did not affect BAX/BCL-2 ratio, ROS induction and
mitochondrial mass change in NTUB1/P cells………………………… 29
III.3.3. Lower intracellular arsenic accumulation in NTUB1/P than NTUB1
Cells………………………………………………………………….. 29
III.3.4. Caveolin gene knockdown did not affect ATO-mediated cytotoxicity
and intracellular arsenic accumulation in NTUB1 cells ……………… 30
III.3.5. The role of aquaporins in arsenic accumulation in NTUB1 cells …….. 30
III.3.6. The role of MRP1/2 in arsenic accumulation in NTUB1 and NTUB1/P
cells…………………………………………………………………….. 30

IV. Discussion…………………………………………………………………………. 31
IV.1.The mechanisms of cisplatin-induced cytotoxicity …………………………….. 31
IV.2. The mechanisms of cisplatin resistance in NTUB1/P cells ……………………. 33
IV.3. Cross-resistant to ATO in NTUB1/P cells …………………………………..…… 36
IV.4. Future direction …………………………………………………………………. 37

V. Conclusion ……………………………………………………………………...… 37

VI. References ………………………………………………………………………… 37

VII. Appendix …………………………………………………………………………….. 94

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