State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu, China.
J Cancer Res Clin Oncol. 2010 Aug;136(8):1229-42. doi: 10.1007/s00432-010-0773-3. Epub 2010 Feb 7.
Focal adhesion kinase (FAK) is a non-receptor protein tyrosine kinase implicated in cancer cell survival, proliferation, and in various steps in the metastatic cascade. In the present study, we took advantage of a cationic liposome as gene carrier and targeted FAK function through both in vitro and in vivo approaches.
We utilized a plasmid-encoded hairpin RNA targeting the human FAK mRNA (pGensil2-shRNA/FAK), as a means to inhibit FAK expression for evaluating its anti-tumor effect in vitro and in vivo. Human MDA-MB-435S breast cancer cells were transfected with pGensil2-shRNA/FAK and examined for apoptosis by propidium iodide staining, DNA ladder, and flow cytometric analysis. For in vivo study, subcutaneous breast carcinomatosis models in nude mice were established to evaluate the therapeutic potential of pGensil2-shRNA/FAK. Assessments of proliferation (Ki-67), apoptosis (TUNEL) and angiogenesis (CD31) were done using immunohistochemical analysis.
Transcripts expressed from plasmid both in vitro and in vivo were identified by northern blot analysis. pGensil2-shRNA/FAK effectively down-regulated the expression of FAK as demonstrated in vitro by real time RT-PCR and western blot analysis, whereas by real time RT-PCR and IHC staining of MDA-MB-435S tumors growing subcutaneously. Breast cancer cells lacking FAK expression undergo apoptosis in vitro. Systemic delivery of cationic liposome-complexed plasmids targeting FAK, resulted in the diminishment of subcutaneous tumor growth beyond the effects observed with liposomes carrying a non-specific shRNA. This diminishment in growth was associated with elevated levels of apoptosis (TUNEL staining), decreased cell proliferation (Ki-67 staining) and diminished endothelial cell density (CD31 staining).
These results indicate that the systemic delivery of plasmid DNA targeting FAK function using cationic liposome as a gene carrier, represents a promising avenue for breast cancer therapy.
黏着斑激酶（FAK）是一种非受体酪氨酸蛋白激酶，参与癌细胞的存活、增殖以及转移级联的多个步骤。在本研究中，我们利用阳离子脂质体作为基因载体，并通过体外和体内方法靶向 FAK 功能。
我们利用针对人 FAK mRNA 的质粒编码发夹 RNA（pGensil2-shRNA/FAK），作为抑制 FAK 表达的手段，用于评估其在体外和体内的抗肿瘤作用。将 pGensil2-shRNA/FAK 转染入人 MDA-MB-435S 乳腺癌细胞，通过碘化丙啶染色、DNA 梯状带和流式细胞术分析评估细胞凋亡。对于体内研究，建立裸鼠皮下乳腺癌转移模型，以评估 pGensil2-shRNA/FAK 的治疗潜力。通过免疫组织化学分析评估增殖（Ki-67）、凋亡（TUNEL）和血管生成（CD31）。
通过Northern blot 分析鉴定了体内外质粒表达的转录物。pGensil2-shRNA/FAK 有效地下调了 FAK 的表达，这在体外通过实时 RT-PCR 和 Western blot 分析得到证实，而在皮下生长的 MDA-MB-435S 肿瘤的实时 RT-PCR 和 IHC 染色中也得到证实。缺乏 FAK 表达的乳腺癌细胞在体外发生凋亡。靶向 FAK 的阳离子脂质体复合物质粒的全身递送导致皮下肿瘤生长的减少，超过了携带非特异性 shRNA 的脂质体的作用。这种生长减少与凋亡增加（TUNEL 染色）、细胞增殖减少（Ki-67 染色）和内皮细胞密度降低（CD31 染色）相关。
这些结果表明，使用阳离子脂质体作为基因载体，系统递送针对 FAK 功能的质粒 DNA，为乳腺癌治疗提供了一种有前途的途径。
