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系統識別號 U0026-1009201202385600
論文名稱(中文) 過度表現α-catulin促進卵巢癌的紫杉醇抗藥性
論文名稱(英文) Overexpression of α-catulin promotes taxol resistance in ovarian cancer
校院名稱 成功大學
系所名稱(中) 口腔醫學研究所
系所名稱(英) Institute of Oral Medicine
學年度 100
學期 2
出版年 101
研究生(中文) 江宜眞
研究生(英文) Yi-Chen Chiang
學號 t46991034
學位類別 碩士
語文別 英文
論文頁數 41頁
口試委員 指導教授-陳玉玲
口試委員-洪澤民
口試委員-謝達斌
口試委員-許耿福
中文關鍵字 α-catulin  RAD50  DNA 傷害反應  癌症幹細胞  太平洋紫杉醇 
英文關鍵字 α-catulin  RAD50  DNA damage response  Cancer stem cell  paclitaxel 
學科別分類
中文摘要 α-catulin 為82kDa α-catenin相關的蛋白質,在過往的研究中認為α-catulin為一致癌基因。α-catulin主要藉由調控NF-κB反應途徑,調節Rho的訊號傳遞途徑增強腫瘤轉移能力。然而,在過往文獻上,對於α-catulin如何調控癌症的生成的資訊還是不多。卵巢癌的發生率在台灣女性中約占5%,但卻是女性癌症死亡率之首,而造成卵巢癌死亡率如此高的原因之一是因為卵巢癌病人對於各種藥物化學治療的反應不佳,並且易有復發情形。因此若能進一步了解卵巢癌的復發機制將有助於改善臨床病徵。 我們發現,在四十位卵巢癌的病人中,α-catulin的表現量和腫瘤的大小(p=0.0385)、腫瘤的復發有關(p=0.0221),和腫瘤的種類、病人的存活與否無關。此外,在我們之前的研究中發現過度表現α-catulin的卵巢癌細胞株A2780,會增加太平洋紫杉醇(Taxol)的耐受性,但不影響對Cisplatin的耐受性。同時,我們也利用微陣列晶片技術分析了在正常細胞中α-catulin和DNA傷害反應基因的關係,發現當抑制α-catulin時,數種DNA傷害反應基因表現量會下降;相反地,當我們過度表現α-catulin時,這些DNA傷害反應基因的表現量則會上升。 此外,將A2780處理太平洋紫杉醇時,ATM,RAD50和BRCA1會下降。因此我們認為在卵巢癌中,α-catulin可能會藉由調控DNA傷害反應基因,進而促使對太平洋紫杉醇的耐受性增加。我們也進一步發現在過度表現α-catulin的細胞株,處理Taxol後,其DNA傷害反應基因RAD50的表現量較不會減少。因此α-catulin可能是藉由減少太平洋紫杉醇所造成的RAD50的抑制,進而增加耐受性。另一方面,過度表現α-catulin時,細胞內的數種癌症幹細胞標記也有上升的情形。但是藉由腫瘤球體(Tumor-sphere assay)實驗中可以發現具有癌症幹細胞特性的細胞中α-catulin 的表現量沒有比較高,因此α-catulin 可能不是藉由癌症幹細胞的特性增加太平洋紫杉醇的耐受性。 卵巢癌的復發為現今治療上急需解決的問題,我們的研究提供了一個治療上的標的,可以進一步研究,以期做為未來標靶治療的方向。
英文摘要 α-Catulin is an 82 kDa α-catenin related protein, and is reported to be an oncogene. α-Catulin can regulate the NF-κB pathway and modulate the Rho pathway signaling to enhance tumor metastatic ability. However, there is little information available in literature about the mechanism of α-catulin regulated cancer progression. Ovarian cancer (OVCa) accounts for 5% of all cancers among female in Taiwan, and they are the cause of more deaths than any other female genital tract cancer. The dismal prognosis of OVCa is mainly due to the high recurrence rates. Therefore, better understand of ovarian cancer recurrence will led to improve the molecular therapeutics and clinical outcome. In this study, we showed that α-catulin is positively correlated with tumor size (p=0.0385) and incidence of tumor recurrence (p=0.0221) in OVCa (n=40). In contrast, the expression level of α-Catulin is no difference between serous type and clear cell type OVCa and is not correlated with patient survival, either. In addition, overexpression of α-catulin enhances paclitaxel resistance in OVCa cell line A2780, but it didn’t affect cisplatin resistance. DDR genes expression decreased after paclitaxel treating in parental A2780 cells. Microarray analyses showed that several genes involved in DNA damage response (DDR) could be altered in both α-catulin-overexpressed and α-catulin-silenced cancer cells implying that DDR pathway may be important in paclitaxel resistance of OVCa. We found that α-catulin overexpression prevents the decrease of RAD50, a DDR gene, by paclitaxel treatments in OVCa cells. Furthermore, overexpression of α-catulin also increases several cancer stem cell marker genes. However, α-catulin expression did not increase in tumor sphere cells which are considered as stem-like cells. In conclusion, α-catulin may increase paclitaxel resistance by upregulated DNA damage response and prevented RAD50 declined. Our study may provide a new target therapy against ovarian cancer recurrence.
論文目次 Contents
Abstract in Chinese I
Abstract III
Acknowledgement V
Contents VII
Abbreviation X
Introduction 1
1. General introduction of ovarian cancer 1
2. Recurrence of ovarian cancer 1
3. General introduction of paclitaxel 2
4. General introduction of α-catulin 3
5. DNA damage response 4
6. General introduction of cancer stem cell 4
Rational and Specific Aims 6
Materials and methods 7
Reagents and Equipments 7
1. Clinical sample 7
2. RNA extraction. 7
3. Quantitative Real-time PCR (qRT-PCR) analysis 7
4. Cell Culture 8
5. Transfection of plasmid 8
6. Drug treatment 9
7. WST1 cell viability assay 9
8. Production and infection of recombinant lentiviral particles 9
9. Western blot analysis 10
Results 12
1. Expression of α-catulin was positively correlated with recurrence and tumor stage of ovarian cancer. 12
2. Overexpression of α-catulin didn’t increase cisplatin resistance. 12
3. Overexpression of α-catulin didn’t alter paclitaxel-induced cell cycle arrest. 13
4. Overexpression of α-catulin didn’t alter paclitaxel-induced cell cycle arrest. 13
5 α-Catulin prevents paclitaxel-induced decreases of DNA damage response genes in ovarian cancer cells. 14
6 α-Catulin expression level is correlated with OCT4 expression level in ovarian cancer cell lines. 15
Discussion 17
1. Clinical evidence of α-catulin in recurrence of ovarian cancer 17
2. Drug resistance is mainly reason to tumor relapse. 17
3. Overexpression of α-catulin leads paclitaxel resistance is a potential mechanism of recurrent ovarian cells. 18
4. Cancer stem cell is not mainly mechanism of α-catulin induced paclitaxel. 20
Conclusion 21
Reference 22
Table 28
Figure 29
Figure 1. Expression profile of α-catulin in ovarian cancer cell lines. 29
Figure 2. Overexpression of α-catulin promotes paclitaxel resistance in ovarian cancer cells. 30
Figure 3. Overexpression of α-catulin didn’t alter paclitaxel induced cell cycle arrest. 31
Figure 4. Overexpressions of α-catulin up-regulated DNA damage response related genes. 32
Figure 5. Overexpression of α-catulin prevented RAD50 down-regulation after Taxol treating. 33
Figure 6. Endogenous expression of α-catulin and cancer stem cell marker genes. 34
Figure 7. Overexpression of α-catulin promotes cancer stem cell marker expresson 35
Figure 8. Cancer stem cell marker genes expression in A2780CP70 adherent and sphere cells. 36
Figure 9. A diagram elucidated endogenous α-catulin modulated the viability of ovarian cancer cells to paclitaxle drugs by up-regulating DDR genes. 37
Appendix 38
Appendix 1. Reagents 38
Appendix 2. Equipments 40
Autobiography 41
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