Thioredoxin 與化療藥物治療後產生癌細胞抗藥性之關聯性

Abstract

研究背景 神經纖維母細胞乃常見好發於兒童之惡性腫瘤，死亡率極高，且經治療後，仍常見復發及轉移之現象 (Marvis, 2010)。根據癌症幹細胞之理論，認為癌症之所以常見復發及轉移之現象，是由於腫瘤裡面存在著癌症幹細胞，此類細胞在經由化療藥物處理下可以繼續存活 (Jordan, Guzman, and Noble, 2007)。 而根據本研究團隊於2002年所做之實驗發現 (Andoh , Chock , and Chiueh, 2001)，經由pre-conditioned之SH-SY5Y cells (N cells) 會表現CD-133 細胞表面抗原及Trx，相較於wild type control cells (D cells) N cells可形成一顆腫瘤及轉移。 D cells之Trx表現量較低並且對於外界氧化壓力較敏感而較容易造成細胞凋亡之現象。 研究目的 希望了解Thioredoxin 表現量是否影響癌症幹細胞與化療藥物抗藥性之間的關係，並了解Trx之redox cycling與化療藥物抗藥性之關係。 研究方法 希望藉由Western blotting來了解在control D cell及pre-conditioned N cell 其Thioredoxin表現量的差異。並藉由Hoechst 33258 細胞核染劑及Trypan blue exclusion assay 來了解在加入etoposide (0 to 83 μM) 後，兩株細胞細胞凋亡與存活率之差異。藉由螢光顯微鏡觀察觀察control D cell 在加入etoposide (0 to 83 μM) 作用24小時後，細胞型態變化情形。使用DNCB (50 μM) 做為 Trx之redox cycling的抑制劑。 結果 此次實驗首先比較pre-conditioned N cells 與control D cells 之Trx表現量之差異，結果顯示N cells之thioredoxin表現量確實較D cells高3倍。接著進行etoposide加藥實驗，經實驗計算發現，pre-conditioned N cells要達到與control D cells相同之 a細胞凋亡比率，需27倍的藥物劑量。再者加入DNCB (50 μM)抑制Trx之redox cycling，可發現經DNCB處理之 N cells 其細胞凋亡之比率與control D cells相近。此結果顯示thioredoxin表現量差異的確會影響N cells之存活率，並且影響etoposide所產生之細胞凋亡導致細胞對化療藥物產生抗藥性之現象。另外在進行加藥實驗時發現，D cells 在高劑量化療藥物 (83 μM etoposide) 作用時，經由顯微鏡觀察，發現其細胞型態改變且與 N cell之型態相似，並且Trx表現量上升。因此推測可能在化療藥物作用下，normal cancer cell 會經由刺激而突變或是篩選成與癌症幹細胞相似之細胞。 結論 Trx表現量確實會避免癌症細胞對化療藥物etoposide所導致之細胞凋亡產生抗藥性。並且control D cells 在etoposide作用之下，Trx表現量及細胞型態皆有改變。Trx之redox cycling在化療藥物產生抗藥性中扮演重要的角色。

Backgrounds Neuroblastoma is a kind of children cancer that has a relatively high mortality, recurrence and metastasis; drug resistance is frequently seen after the treatment with chemotherapy (Marvis, 2010). According to the theory of cancer stem cell (CSC), the reason why cancer usually recurs and metastasizes is due to the CSC exists in the tumor which can even survive and grow into a new tumor after chemotherapy (Jordan, Guzman, and Noble, 2007). After a new discovery of our team (Andoh, Chock, and Chiueh, 2002), a CD-133 positive and Trx-expressed CSC-like cells (N cells) were isolated from the pre-conditioned human SH-SY5Y cells (unpublished observations of Chiueh et al). N cells produced a solid tumor but not by its wild type control cells (D cells) in nude mice. D cells are associated with low Trx expression and also sensitive to oxidative injury and related apoptotic cell death.

Objective To understand whether Trx expression affects the relationship between the preconditioned N cells and resistance to chemotherapy; to investigate the redox cycling role of Trx in mediating chemo-resistance Methods Trx expression in control D cells and pre-conditioned N cells were measured by the Western blotting. Etoposide (0 to 83 μM)-induced apoptotic changes and cell viability in D and N cells were assayed by Hoechst 33258 nuclear staining and Trypan blue exclusion assay, respectively. The redox cycling of Trx was blocked by a Trx reductase inhibitor 1-Chloro-2,4-dinitrobenzene (DNCB (50 μM) in N cells. Cell morphology in control D cells after etoposide treatment (0 to 83 μM) for 24 hours was imaged by using fluorescence microscopy. Results First, this experiment was to compare the Trx expression of the pre-conditioned N cells with the control D cells. The result indicates that the Trx expression of N cells was 3 times greater than D cells. Etoposide-induced apoptosis in the pre-conditioned N cells were less than that in the control D cells by 27-fold. Next, after the co-treatment of N cells with 50 μM DNCB to block the redox cycling of Trx, the apoptosis cell percentage of DNCB-treated N cells was close to the control D cells. The present results show that the elevation of the Trx expression improved not only the survival rate of N cells, but also induced chemo-resistance reflected by a reduction in the apoptotic cell death caused by etoposide. In addition, following the etoposide (83 μM) treatment, the phenotype of the D cells was changed to N cell morphology with a concurrent increase in Trx levels. This phenomenon infers that the normal cancer cell may be mutated and altered into CSC-like cells under the stimulation of chemopherapy. Conclusions The expression of Trx can prevent cancer cells such as N cells from apoptotic cell death caused by etoposide. The Trx expression and the cell morphology were altered after the treatment of etoposide in the control D cells. The redox cycling of Trx in human neuroblastoma cancer cells may play a critical role in the development of chemo-resistance.