臺灣博碩士論文加值系統

一、 SAHA結合PARP抑制劑提供新的肝癌組合型治療方法
基因體不穩定(genomic instability)在2011年被歸類於十大癌症共同特徵之一。因為大量的DNA受損最終導致細胞死亡，以DNA修復系統為標靶的抑制劑可被用於抗癌藥物開發。Suberoylanilide hydroxamic acid (SAHA)是histone deacetylases (HDACs)的抑制劑，現今已進入臨床試驗第二、三期，用於治療多種癌症，但對於SAHA誘發細胞死亡的機制至今仍未完全釐清。先前的實驗結果顯示促進reactive oxygen species (ROS)的釋放與引發雙股DNA斷裂，可能是SAHA造成計劃性細胞凋亡(apoptosis)的原因之一。當細胞內DNA受損時，腫瘤抑制蛋白p53會活化其下游的訊息傳遞路徑，藉由啟動DNA修復或計劃性細胞凋亡避免DNA受損之細胞癌化。根據我們實驗室先前的研究結果，有TP53基因突變和PARP4表現量下降的肝癌病人往往伴隨著基因體不穩定標記—第四號染色體長臂(chromosome 4q)的異質性缺失(loss of heterozygosity, LOH)，暗示肝癌細胞對於引起DNA受損藥物的敏感度可能與細胞內TP53的狀態(status)和PARP4的表現量有關。3-Aminobenzamide (3-ABA)是一種泛效性的PARP抑制劑，可藉由影響PARP1/2活性，降低細胞對於DNA單股斷裂的修復能力，因此我們使用3-ABA加上SAHA同時抑制DNA單股和雙股斷裂的修復系統，以達到毒殺帶有TP53突變肝癌細胞的目的。當癌細胞同時接受SAHA和3-ABA時，DNA雙股斷裂的標記—γ-H2AX和計劃性細胞凋亡的標記—經剪切的caspase 3/PARP1都顯著增加，但減弱肝癌細胞內生性TP53的表現量或穩定轉染突變的TP53則會提高細胞對SAHA及3-ABA的抗性。我們也觀察到，癌細胞對於SAHA與3-ABA的毒性效果包括細胞週期停滯和細胞死亡，取決於本身的遺傳背景。3-ABA增強由SAHA引起的CDKN1A基因向上調控只見於HepG2細胞株；而在Hep3B細胞株內表現量顯著增強的TXNIP基因，可以導致ROS的釋出和DNA雙股斷裂。當SAHA/3-ABA結合ROS抑制劑N-acetylcysteine時，由其引發未摺疊蛋白反應(unfolded protein response, UPR)下游基因GRP94和CHOP的向上調控受到抑制，細胞死亡程度隨之降低。此外，我們也結合SAHA和另一種PARP抑制劑rucaparib處理癌細胞，結果未影響DNA雙股斷裂程度但伴隨著增加p21的向上調控，更顯著地引發癌細胞死亡。因此，我們的實驗結果顯示由SAHA結合PARP抑制劑所造成的肝癌細胞死亡，是經由多方途徑和影響因子所調控。此外，我們建議可運用不同的SAHA與PARP抑制劑組合，考量肝癌病人的TP53狀態和PARP4表現量的變異性，設計適合病患的個人化醫療。

二、 PARP4蛋白的定性分析
Poly(ADP-ribose) polymerase family, member 4 (PARP4)蛋白質催化細胞核與細胞質裡蛋白質的PARsylation反應。過去研究顯示，PARsylation修飾後的蛋白質參與在偵測DNA受損、修復及與細胞死亡有關的訊息傳遞路徑，因此我們想探討PARP家族中的PARP4是否具有維持基因體完整性(genomic integrity)之功能。當細胞處理引起DNA雙股斷裂的試劑後，PARP4從細胞質移動至細胞核內，並且在HeLa細胞處理H2O2後，PARP4與DNA雙股斷裂標記—γ-H2AX在細胞中有相似的分布(co-localization)；而HEK293T細胞處理etoposide後，PARP4與PAR在細胞中有相似的分布。值得注意的是，PARP4進核的現象發生於4μM SAHA處理後兩小時，是一個早期發生的反應。我們也發現減弱癌細胞中PARP4的表現量會增加細胞在SAHA/3-ABA的處理下DNA雙股斷裂和細胞死亡的程度，並且SAHA向下調控PARP4 mRNA和蛋白質的表現量，而PARP抑制劑作用在PARP家族上，此結果顯示SAHA及3-ABA共同促進細胞死亡可能透過調控PARP4的表現量和活性。綜合以上的實驗結果，我們支持PARP4在維持基因體的穩定上扮演重要的角色，並且建議PARP4可以做為設計抗癌藥物的標靶。

I. Combination therapy of SAHA and PARP inhibitors in HCC treatment
Genomic instability is one of the cancer hallmarks. Because severe DNA damage results in cell death, inhibitors of the DNA repair system can be used as anticancer drugs. An inhibitor of histone deacetylases (HDACs) - suberoylanilide hydroxamic acid (SAHA), is being evaluated for phase2/3 clinical trial to treat several types of cancers. Mechanism of SAHA-induced cell death is not totally clear, but reactive oxygen species (ROS) release and double strand break (DSB) may play a role in apoptosis of SAHA-treated cells. Upon DNA damage, p53 activates its downstream target genes and triggers DNA repair or apoptosis. In our previous study, mutation of TP53 and down-regulation of PARP4 were found to be associated with chromosome 4q loss of heterozygosity in hepatocellular carcinoma (HCC), implicating that sensitivity to DNA-damaging agents in HCC cells might depend on the TP53 status and PARP4 expression level. A pan-PARP inhibitor 3-aminobenzamide (3-ABA), known to inhibit the single strand break repairing system by targeting PARP1/2, was chosen as a candidate to be combined with SAHA for treating HCC cells with TP53 mutation. As a result of impaired DNA repair, DSB and apoptosis were enhanced, as measured by γ-H2AX staining and caspase 3/PARP1 cleavage, respectively. Sensitivity to SAHA and 3-ABA was reduced in TP53 knocked down or mutant TP53 stably transfected HepG2 cells. Depending on genetic background, SAHA/3-ABA-induced cytotoxicity was variable and could be attributed to cell cycle arrest and cell death. In HepG2 cells, SAHA-induced up-regulation of CDKN1A was enhanced by 3-ABA. In Hep3B cells, TXNIP level was further elevated by 3-ABA, contributing to ROS release and DSB. Consistently, N-acetylcysteine, a ROS inhibitor, reduced the expression of the targeted genes, GRP94 and CHOP, of unfolded protein response, and blunted SAHA/3-ABA induced cell death. Another PARP inhibitor rucaparib, when combining with SAHA, induced prominent cell death, and it was accompanied by up-regulation of p21 (WAF1/CIP). Unlike 3-ABA, rucaparib did not enhance SAHA-induced DSB. Thus our results support that multiple pathways and effectors are involved in SAHA-induced cancer cell death, and, contingent on TP53, a new strategy of therapeutic development can be designed by adding PARP inhibitors, to achieve personalized medicine for HCC.

II. Cellular characterization of PARP4
Poly(ADP-ribose) polymerase family, member 4 (PARP4) catalyzes poly(ADP-ribosyl)ation PARsylation of numerous proteins localized in cytosol and nuclei. In previous studies, PARsylation of targeted proteins were known to be involved in regulating DNA damage detection, DNA repair, and cell death pathways. To investigate whether PARP4 also participates in the maintenance of genome integrity, subcellular localization of PARP4 was analyzed. Nuclear translocation of PARP4 was observed with DSBs induced by various DNA-damaging agents. In H2O2-treated HeLa cells, PARP4 localized with γ-H2AX, a DSB marker, while in etoposide-treated HEK293T cells, co-localization of PARP4 and PAR was observed. Notably, PARP4 nuclear localization is an early event and it became detectable by fluorescence microscopy two hours after addition of 4 μM SAHA. PARP4 knockdown enhanced DSBs and cell death in SAHA/3-ABA-treated cancer cells. Finally, SAHA targeted on PARP4 by down-regulation of mRNA and protein expression, suggesting that enhancement of cell death by combining SAHA with PARP inhibitors might work through their effects on the PARP4 level and DNA repair. Taken together, our data support that PARP4 may function in the maintenance of genome integrity, and it could be served as a target to design anticancer drugs.

Abbreviations.....................................i
Chinese Abstract................................iii
English Abstract..................................v
Table of Contents...............................vii
1. Introduction.................................1
1.1 Hepatocellular carcinoma (HCC)...............1
1.2 Genomic instability in cancer................6
1.3 Histone deacetylase (HDAC) inhibitors........8
1.4. Poly(ADP-ribose) polymerases (PARPs)........10
2. Materials and methods.......................14
2.1 Cell culture and transfection...............14
2.2 RNA interference............................14
2.3 Generation of mutant TP53 stable clones.....14
2.4 Single agent cytotoxicity assays and combination drug treatment...................................15
2.5 Immunofluorescence and confocal microscopy..15
2.6 Western blotting............................15
2.7 RT-qPCR.....................................16
2.8 Flow cytometry..............................16
2.9 Statistical analysis........................17
3. Results
Part I. Combination therapy of SAHA and PARP inhibitors in HCC treatment.................................18
3.1. 3-ABA enhanced SAHA-induced DSBs in HCC cell lines............................................18
3.2. TP53 deficiency reduced sensitivity to SAHA was compensated by co-treating with 3-ABA in HCC cells............................................18
3.3 Exogenous p53 mutants affected sensitivity to SAHA and/or 3-ABA in TP53-WT HepG2 cells..............19
3.4. Inducing mixed cell death by SAHA and/or 3-ABA in HCC cells with different genetic backgrounds.....20
3.5. SAHA induced cell cycle arrest in HCC cells............................................21
3.6. 3-ABA enhanced SAHA-induced ROS accumulations in HCC cells........................................23
3.7. A ROS inhibitor, NAC, reduced 3-ABA enhanced DSBs and apoptosis in SAHA-treated HCC cells..........24
3.8. The unfolded protein response (UPR) mediated SAHA-triggered apoptosis..............................24
3.9. Find other combinations of inhibitors enhancing cell death mediate through simultaneously targeted on the DSB repair system and PARP family in HCC cells...25
3.10. Combination of SAHA with a PARP inhibitor, rucaparib, may be a potent strategy for treating various cancers..........................................26
3.11. Mechanisms involved in rucaparib enhancing SAHA-reduced cell viability...........................27
4. Results
Part II. Cellular characterization of PARP4......28
4.1. Nuclear translocations of PARP4 in the DNA-damaging agents-treated cancer cells......................29
4.2. Co-localizations of nuclear translocated PARP4 with γ-H2AX or PAR....................................31
4.3. PARP4 knockdown increased DSBs in SAHA- or ETO-treated cells....................................31
4.4. Increasing susceptibility to SAHA and PARP inhibitors in PARP4 knocked down cancer cells....32
4.5. Mechanisms involved in SAHA and/or PARP inhibitors triggered cell death in the PARP4 knocked down cancer cells............................................32
5. Discussion.................................35
5.1. Functions of 3-ABA in enhancing SAHA-reduced cell viability to HCC cells...........................36
5.2. Effects of the TP53 status in cancer cells sensitizing to SAHA, 3-ABA, and rucaparib........40
5.3. PARP4 may function in the DNA damage response and serve as a drug in cancer therapy................42
5.4 Rucaparib versus 3-ABA......................43
6. References..................................46
7. Tables......................................49
Table 1. Rucaparib enhanced cell death in the SAHA-treated cancer cells.............................49
Primers list.....................................50
8. Figures and Figure legends..................51
Part I. Combination therapy of SAHA and 3-ABA in HCC treatment........................................51
Part II. Cellular characterization of PARP4......79
9. Appendices..................................95

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