利用反譯寡核苷酸微脂粒– G3139提升Paclitaxel或Doxorubicin於癌細胞之敏感性

Using liposomal antisense oligonucleotide – G3139 to sensitize paclitaxel or doxorubicin in cancer cell lines

成恆宇(Hang-Yu Cheng)

指導教授 ： 邱士娟

臺北醫學大學/藥學院/藥學研究所/碩士(2010年)

https://doi.org/10.6831/TMU.2010.00180

摘要

反譯治療是先進的癌症基因治療策略。反譯寡核苷酸(ODN)主要是由短鏈的核苷酸合成，可藉由與特定的mRNA 序列互補雜合，進而干擾基因的表現，並抑制蛋白質的合成。
細胞凋亡是引起細胞死亡的主要生理機制之一，對於癌症的治療有著重要的影響。細胞凋亡在體內由許多蛋白所調控，例如:BCl-2。Bcl-2 為抑制細胞凋亡的因子，可藉由數種途徑抑制細胞凋亡。許多癌細胞的異常增生及對傳統癌症治療的抗藥性都與Bcl-2 的過度表現有關。因此，藉由一反譯寡核苷酸 (G3139) 抑制Bcl-2 的表現，進而增加癌症治療的療效，成為了可期望的治療策略之一。
因為反譯寡核苷酸為帶負電之高分子量物質，使其不易穿透細胞膜，且其在體內易被酵素、核酸酶破壞。將G3139 以陽離子脂質載體包覆，可改善反譯寡核苷酸的傳遞。本實驗之目的為將反譯寡核苷酸以陽離子脂質載體包覆，並探討其與paclitaxel 或doxorubicin併用於兩種血液癌症之細胞效果。
本實驗之微脂粒由DC-Chol/egg-PC/PEG-DSPE 以22.5:76:1.5 mol% 組成，其粒徑大小平均為 215.8±5.3 nm，包覆ODN 的能力可達58.61±5.38%。為了增加標靶運輸的效果，可將運鐵蛋白當作配位體經後嵌入法修飾微脂粒表面。
在細胞存活實驗中，將paclitaxel 或doxorubicin 分別加入K562、以及NCI-H929兩種不同的癌細胞，另外給予反譯寡核苷酸微脂粒或經運鐵蛋白修飾之反譯寡核苷酸微脂粒。經24、48、72 或96小時於37°C、5% CO2培養後，最後藉由加入MTS，測定其吸光值來觀察細胞的存活率，並以此推估藥物治療的IC50。
在doxorubicin的給藥實驗裡，對於K562細胞，1 μM G3139微脂粒在給藥後72小時明顯提升了細胞對化療藥物的敏感性，而3 μM G3139微脂粒則於給藥後48小時即有此效果；於NCI-H929的細胞中，1 μM 及3μM G3139微脂粒分別於給藥後48及24小時即明顯提升了癌細胞對化療藥物的敏感性。在paclitaxel的給藥實驗中，在兩種濃度之G3139微脂粒給藥後 ，K562及NCI-H929癌細胞分別於48及24小時即有類似的併用效果。然而，經運鐵蛋白修飾之G3139微脂粒其效果並無顯著的不同。
總結以上結果，G3139微脂粒及運鐵蛋白修飾之G3139微脂粒均可以有效的提升K562及NCI-H929對paclitaxel或doxorubicin的敏感性，但兩者之間尚無顯著差異。

關鍵字

Bcl-2 ； 反譯寡核苷酸 ； 微脂粒 ； 標靶運輸 ； 運鐵蛋白

並列摘要

Antisense therapy is a novel strategy in cancer gene therapy. Antisense oligonucleotides (ODN) are short and synthetic fragments of nucleic acids designed to modulate gene expression and inhibit encoded protein production by specifically hybridize to the target complementary mRNA.
Apoptosis, or programmed cell death, plays an important role in cancer therapy. Bcl-2 is one of the anti-apoptotic proteins. Over-expression of Bcl-2 results in proliferation of cancer cells and resistance of traditional cancer therapy. Therefore, down-regulating the Bcl-2 expression by ODN is a promising way to improve cancer therapy.
G3139 is an antisense oligonucleotide designed to specifically bind to human bcl-2 mRNA sequence and then decrease bcl-2 protein translation. However, ODN poorly passes cell membrane due to its negative charges and high molecular weight. Besides, ODN is also easily destroyed by enzymes and nucleases in vivo. Encapsulating ODN in cationic lipid vesicles is an attractive approach to improve antisense delivery. Addition of targeting moiety such as conjugating holo-transferrin to ODN liposomes may further improves the delivery strategy.
This study was aimed to encapsulate ODN in lipid vesicles and compare the effect of paclitaxel or doxorubicin combined with ODN liposome or transferrin-ODN (Tf-ODN) liposomes in two hematological cancer cell lines. The lipid composition used in this study was DC-Chol/egg-PC/PEG-DSPE (22.5:76:1.5 mol%). Liposomes containing G3139 had a mean diameter of 218.7±5.3 nm. Encapsulation efficiency of ODN in the liposomes was 58.61±5.38%. In order to enhance the targeting effect, 0.5 mol% transferrin (Tf)-PEG-DSPE was inserted into the lipid bilayer by post-insertion.
Cell survival assay was applied to investigate the sensitizing effects of paclitaxel or doxorubicin with ODN liposomes or Tf-ODN liposomes. Two cancer cell lines (K562 and NCI-H929) was used in this study. Cells were incubated with various concentrations of paclitaxel or doxorubicin combined with ODN liposomes or Tf-ODN liposomes at 37°C for 24, 48, 72 or 96 hours. The IC50 of treatment was determined from optical density after MTS reagent was added.
In doxorubicin group, 1 μM G3139 liposome sensitized K562 cells to lower concentration of doxorubicin after 72 h treatment, and 3 μM G3139 liposomes revealed the similar effect in K562 cells after 48 h. Besides, 1 μM and 3 μM G3139 liposomes sensitized NCI-H929 cells to lower concentration of doxorubicin after 48 h and 24 h treatment. On the other hand, in paclitaxel group, this combined effect was also observed in both concentrations of G3139 liposomes after 48 h treatment in K562 cells and 24 h in NCI-H929 cells. However, the combined effect had no difference between G3139 liposomes and Tf-G3139 liposomes.
In conclusion, G3139 liposomes could sensitize both K562 and NCI-H929 cells to lower concentration of doxorubicin or paclitaxel whereas Tf-G3139 liposomes did not reveals better effect.

並列關鍵字

Bcl-2 ； antisense oligonucleotide ； liposome ； transferrin

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