Molecular Targeting of 15-Lipoxygenase-1 in Colon Cancer

描述(由申请者提供)：细胞凋亡是开发更好的结直肠癌治疗方法的目标。我们发现：(A)亚油酸的产物通过15-1环氧合酶-1(15-LOX-1)、13-S-羟基十八碳二烯酸(13-S-HODE)恢复结直肠癌细胞的凋亡；(B)13-S-HODE和15-LOX-1在人结直肠癌中表达下调；(C)非甾体抗炎药恢复15-LOX-1在结直肠癌细胞中的表达，诱导细胞凋亡，抑制肿瘤发生。人端粒酶逆转录酶(HTERT)启动子可以在肿瘤细胞中选择性表达基因。这项研究验证了以下假设：通过腺病毒递送系统，在hTERT启动子控制下选择性地恢复15-LOX-1在结直肠癌细胞中的表达，将重建结直肠癌细胞的凋亡并抑制肿瘤的发生。目的：1.为了确定端粒酶逆转录酶介导的15-LOX-1在腺病毒载体中的表达是否通过15-LOX-1的表达而在结直肠癌细胞中重建凋亡，我们将在hTERT启动子的控制下将表达15-LOX-1的重组腺病毒载体(Ad-15-LOX-1)转染结直肠癌细胞系，并在体外检测15-LOX-1的表达、13-S-HODE水平和凋亡率。目的：通过腺病毒介导的hTERT启动子调控15-LOX-1基因的表达，通过瘤内注射将15-LOX-1基因导入人结直肠癌细胞裸鼠皮下移植瘤中，观察15-LOX-1的表达对结直肠癌细胞成瘤率和细胞凋亡率的影响。目的：为确定hTERT-15-LOX-1腺病毒系统给药是否选择性地在肿瘤细胞中表达15-LOX-1抑制肿瘤生长，我们将在荷人结肠癌细胞肝转移的裸鼠体内静脉注射Ad-15-LOX-1，并评价15-LOX-1表达对肿瘤生长的影响。目的：为探讨15-LOX-1是否下调Bcl2诱导结直肠癌细胞凋亡，我们将检测(A)Ad-15-LOX-1对结直肠癌细胞Bcl2表达的影响，以及(B)过表达Bcl2是否抑制了15-LOX-1诱导的细胞凋亡。证实肿瘤选择性靶向15-LOX-1抑制肿瘤形成的可行性将为未来基于分子靶向15-LOX-1的临床前和临床治疗策略的发展铺平道路

DESCRIPTION (provided by applicant): Apoptosis is being targeted to develop better therapies for colorectal cancer. We have found that (a) linoleic acid's product through 15-1ipoxygenase-1 (15-LOX-1), 13-S-hydroxyoctadecadienoic acid (13-S-HODE), restores apoptosis in colorectal cancer cells, (b) 13-S-HODE and 15-LOX-1 are downregulated in human colorectal cancer, and (c) NSAIDs restore 15-LOX-1 expression in human colorectal cancer cells to induce apoptosis and inhibit tumorigenesis. Human telomerase reverse transcriptase (hTERT) promoter can selectively express genes in cancer cells. The proposed research tests the following hypothesis: Selective restoration of 15-LOX-1 expression under the control of hTERT promoter in colorectal cancer cells via an adenoviral delivery system will reestablish apoptosis and inhibit tumorigenesis in colorectal cancer cells. Specific Aims: Aim 1: To determine whether hTERT-driven 15-LOX-1 expression via an adenoviral vector reestablishes apoptosis through the expression of 15-LOX-1 in colorectal cancer cells, we will transfect colorectal cancer cell lines with an adenoviral vector (Ad-15-LOX-1) that expresses 15-LOX-1 under the control of the hTERT promoter and will measure 15-LOX-1 expression, 13-S-HODE levels, and apoptosis rates in vitro. Aim 2: To assess whether 15-LOX-1 expression driven by hTERT promoter through an adenoviral transfection system suppresses colorectal tumorigenesis in vivo, we will transfect Ad-15-LOX-1 by intratumoral injections into subcutaneous xenografts of human colorectal cancer cells in nude mice and assess the effects of 15-LOX-1 expression on tumorigenesis and apoptosis rates. Aim 3: To determine whether the systemic administration of hTERT-15-LOX-1 adenovirus selectively expresses 15-LOX-1 in tumor cells suppressing tumor growth, we will administer Ad-15-LOX-1 intravenously to nude mice carrying human colon cancer cell liver metastases and evaluate the 15-LOX-1 expression effects on tumor growth. Aim 4: To determine whether 15-LOX-1 downregulates Bcl-2 to induce apoptosis in colorectal cancer cells, we will examine (a) the effects of transfecting Ad-15-LOX-1 into colorectal cancer cells on Bcl-2 expression and (b) if overexpressing Bcl-2 inhibits 15-LOX-1 induced apoptosis. Confirming the feasibility of tumor selective targeting of 15-LOX-1 to inhibit tumorigenesis will pave the way for future preclinical and clinical development of therapeutic strategies based on molecularly targeting 15-LOX-1