併用Doxorubicin或Paclitaxel與反譯寡核苷酸微脂粒對白血病細胞之療效探討

白血病為國內外的高發性惡性腫瘤之一，可分為急性、慢性的淋巴球白血病與骨髓性白血病。癌症的發生可能和癌細胞之程式性死亡－細胞凋亡(apoptosis)無法正常進行有關。細胞凋亡又稱為細胞程式死亡，為一受到許多因子調節之複雜過程，其目的為移除生物體所不需要或是已失去生理活性之細胞。Bcl-2蛋白質家族為調控細胞凋亡的因子之一，其中的Bcl-2蛋白之作用為抑制細胞凋亡，許多癌細胞Bcl-2基因過度表現，細胞凋亡無法正常發生，導致癌細胞過度增生，如果能夠降低癌細胞之Bcl-2蛋白質的表現，即可誘導癌細胞之細胞凋亡發生。反譯治療(antisense therapy)是將特定基因材料，如反譯寡核苷酸(oligodeoxynucleotides, ODN)導入細胞以減少其不正常基因之表現。本實驗所使用之反譯寡核苷酸為G3139，是一種對Bcl-2 mRNA有專一性的反譯寡核苷酸，使細胞Bcl-2蛋白質的表現減少。　本實驗之目的為藉由利用G3139微脂粒以增加化療藥物：doxorubicin或paclitaxel之抗癌效果。實驗中所使用的微脂粒由DC-Chol/egg-PC/PEG-DSPE 以22.5:76:1.5 mol%與G3139所組成，本實驗所使用之細胞株包含：K562、NCI-H929及CCRF-CEM。在細胞攝入的實驗部分，利用流式細胞儀以及螢光顯微鏡來觀察及定量細胞對於G3139微脂粒之攝入能力。同時利用西方墨點法來測定Bcl-2的蛋白質表現量。最後觀察在給藥之後24及48小時之細胞存活率，檢視藥物之細胞毒殺能力。 G3139微脂粒之粒徑大小平均為148.9±2.5 nm；在4℃之環境下儲存，在七週內其粒徑沒有明顯之改變；包覆ODN 的能力約為85.5±5.3%，表面電荷為17.5±1.3mV。實驗結果顯示，隨著投與之G3139濃度提升細胞之螢光表現也較強，表示G3139可以有效地將被導入細胞中，然而和未被包覆的G3139相比，經微脂粒乘載之G3139並沒有顯著增加G3139之攝入；同時在給予1 μM G3139微脂粒24小時之後可以發現Bcl-2蛋白質之表現有些許之下降。最後在細胞存活性試驗觀察到不論是在24小時或是48小時之組別中，高濃度的G3139微脂粒皆有較強的細胞毒殺能力。不同之細胞株對於化療藥物之敏感性也有不同，其中K562對於此兩種化療藥物皆有著較強的敏感性；而併用G3139微脂粒之後可增加paclitaxel對於NCI-H929之毒殺能力，但對於K562則無太大影響。此結果可能和細胞本身之敏感性有關，未來可以繼續朝著不同藥物對於不同細胞株之不同毒殺能力繼續探討。

關鍵字

反譯寡核苷酸；微脂粒； Bcl-2 ； G3139

並列摘要

Leukemia is one of the high incidence domestic malignancies. Leukemia can be divided into acute and chronic lymphocytic leukemia and acute and chronic myelogenous leukemia. Cancer may arise from the dysfunction in the apoptotic pathway. Apoptosis, or programmed cell death, is a highly regulated process that allows a cell to self-degrade in order for the body to eliminate unwanted or dysfunctional cells. One of the apoptosis-regulator is Bcl-2 family and Bcl-2, a member of this family, is an important anti-apoptotic protein in regulating the apoptosis pathway. Downregulation of Bcl-2 may be able to induce apoptosis, and further increase cell death in tumor cells. Antisense therapy is a strategy of anticancer therapy designed to introduce genetic material, antisense oligodeoxynuclotides (ODNs), into cells to downregulate abnormal genes. G3139, an antisense ODN designed to specifically bind to Bcl-2 mRNA and further downregulate Bcl-2 protein expression, was used in this study. The aim of this study was to enhance the anticancer effect of chemotherapeutic agents, Doxorubicin or Paclitaxel, using G3139-containing liposomes. The liposomes are composed of DC-Chol / egg-PC/ PEG-DSPE (22.5:76:1.5 mol%) and G3139. Leukemia-related cell lines, K562, NCI-H929 and CCRF-CEM, were used in this study. Cellular uptake of different formulations of liposomal G3139 was observed by flow cytometer and fluorescent microscope. In the meantime, the Bcl-2 protein level was evaluated by western blot to confirm whether G3139 work or not in selective cancer cells. Cell viability was evaluated by MTS assay in 24 and 48hr. G3139-containing liposomes had a mean diameter of 148.9±2.5 nm. No significant particle size changes were observed for 7 weeks at 4℃. Encapsulation efficiency of ODNs in the liposomes was 85.5±5.3% and the zeta-potential was 17.5±1.3mV.The results in cellular uptake studies demonstrated that G3139 can be delivered into cells as higher concentration of G3139-liposomes exhibited higher fluorescence intensity. However, no significant difference was observed between free G3139 and G3139-liposomes. Slightly higher Bcl-2 downregulation effect was observed in cells treated with 1 ?嵱 G3139-liposomes at 24 hr. In cytotoxicity studies, cells treated with higher concentration of G3139 showed higher cytoxiticity in both time points. Three cell lines used in the current study exhibited different sensitivity to chemotherapeutic agents, as K562 had lowest IC50 in both drugs.G3139-liposomes could enhance the cytotoxic effect to paclitaxel in NCI-H929 cells but not in K562 cells. This may be due to the difference in sensitivity of chemotherapeutic agents in these cell lines. Further studies are needed to confirm this effect.

並列關鍵字

antisense oligodeoxynuclotides ； liposome ； Bcl-2 ； G3139

參考文獻

1. Pooley, R.J., Clinical hematology atlas. Arch Pathol Lab Med, 1999. 123(11): p. 1125.

2. Henderson, L.M. and J.B. Chappel, NADPH oxidase of neutrophils. Biochim Biophys Acta, 1996. 1273(2): p. 87-107.

3. Kakizuka, A., et al., Chromosomal translocation t(15;17) in human acute promyelocytic leukemia fuses RAR alpha with a novel putative transcription factor, PML. Cell, 1991. 66(4): p. 663-74.

4. Landis, S.H., et al., Cancer statistics, 1998. CA Cancer J Clin, 1998. 48(1): p. 6-29.

6. Hoelzer, D.F., Therapy of the newly diagnosed adult with acute lymphoblastic leukemia. Hematol Oncol Clin North Am, 1993. 7(1): p. 139-60.

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併用Doxorubicin或Paclitaxel與反譯寡核苷酸微脂粒對白血病細胞之療效探討

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