Therapeutic molecular targeting of 15-lipoxygenase-1 in colon cancer

Abstract

Molecular targeting for apoptosis induction is being developed for better treatment of cancer. Downregulation of 15-lipoxygenase-1 (15-LOX-1) is linked to colorectal tumorigenesis. Re-expression of 15-LOX-1 in cancer cells by pharmaceutical agents induces apoptosis. Antitumorigenic agents can also induce apoptosis via other molecular targets. Whether restoring 15-LOX-1 expression in cancer cells is therapeutically sufficient to inhibit colonic tumorigenesis remains unknown. We tested this question using an adenoviral delivery system to express 15-LOX-1 in in vitro and in vivo models of colon cancer. We found that (i) the adenoviral vector 5/3 fiber modification enhanced 15-LOX-1 gene transduction in various colorectal cancer cell lines, (ii) the adenoviral vector delivery restored 15-LOX-1 expression and enzymatic activity to therapeutic levels in colon cancer cell lines, and (iii) 15-LOX-1 expression downregulated the expression of the antiapoptotic proteins X-linked inhibitor of apoptosis protein (XIAP) and BcL-XL, activated caspase-3, triggered apoptosis, and inhibited cancer cell survival in vitro and the growth of colon cancer xenografts in vivo. Thus, selective molecular targeting of 15-LOX-1 expression is sufficient to re-establish apoptosis in colon cancer cells and inhibit tumorigenesis. These data provide the rationale for further development of therapeutic strategies to target 15-LOX-1 molecularly for treating colonic tumorigenesis.

Figure 1

The effects of adenoviral 5/3 fiber modification on 15-LOX-1 transduction. (a) Schematic representation of the modified adenoviral vector with 5/3 chimeric fiber. (b) Transduction of 15-LOX-1 via modified Ad-5/3 vector vs. unmodified Ad-5 vector. Caco-2, LoVo, HCT-116, HT-29, and LS174 colon cancer cells were transfected with 500 viral particles per cell of unmodified adenoviral vector that expresses 15-LOX-1 (Ad-5-15-LOX-1), the modified Ad-5/3-15-LOX-1, and the modified Ad-5/3-luciferase (Ad-5/3-Luc), or were left untransfected (Mock). Cells were harvested, and total RNA was extracted and processed for 15-LOX-1 measurements by quantitative real-time PCR. The relative expression levels were calculated as the values relative to that of the calibrator sample (Caco-2 cells treated with NaBT for 48 hours). Values shown are the means ± SDs of triplicate experiments. *P < 0.0001, Ad-5/3-15-LOX-1 versus Ad-5/3-Luc, Ad-5-15-LOX-1, and Mock.

Figure 2

15-LOX-1 transduction by adenoviral vector transfection in colon cancer cells. (a and b) 15-LOX-1 mRNA expression by adenoviral transfection into colon cancer cells. LoVo (a) and HT-29 (b) colon cancer cell lines were transfected with either the modified Ad-5/3-15-LOX-1 vector or with the control vector, Ad-5/3-luciferase (Ad-5/3-Luc). Cells were harvested 24 hours after transfection, and 15-LOX-1 mRNA was measured with real-time PCR. The relative expression levels were calculated as the values relative to that of a calibrator sample (Caco-2 cells treated with NaBT for 48 hours). Values shown are the means ± SDs of triplicate measurements. *P < 0.0001, Ad-5/3-15-LOX-1 versus Ad-5/3-Luc. (c and d) 15-LOX-1 protein expression by adenoviral transfection into colon cancer cells. LoVo (c) and HT-29 (d) cells were transfected with either Ad-5/3-15-LOX-1 or Ad-5/3-Luc, and cells were harvested 48 hours later and processed for 15-LOX-1 protein expression by Western blotting. + represents a positive control for 15-LOX-1. (e and f) Enzymatic activity of the 15-LOX-1 protein expressed by Ad-5/3-15-LOX-1 into colon cancer cells. LoVo (e) and HT-29 (f) cells were transfected with either Ad-5/3-15-LOX-1 or Ad-5/3-Luc, and cells were harvested 48 hours later and processed for 13-S-HODE, the major enzymatic product of 15-LOX-1. Values shown are the means ± SDs of triplicate measurements. *P < 0.0001 Ad-5/3-15-LOX-1 versus Ad-5/3-Luc.

Figure 3

Effects of 15-LOX-1 expression by adenoviral vector on cell survival and apoptosis induction in cancer cells. (a) Kinetics of effects of 15-LOX-1 expression on colon cancer cell survival. HCT-116 colon cancer cell lines were transfected with either Ad-5/3-15-LOX-1 or with the control vector Ad-5/3-luciferase (Ad-5/3-Luc) at a concentration of 150 viral particles/cell. Cell survival was measured using sulforhodamine B at 0, 24, 48, 72, and 96 hours. Results are presented as the ratios of Ad-5/3-15-LOX-1– or Ad-5/3-Luc–transfected cells to untransfected cells (survival proportion). Values shown are the means ± SDs of six replicate experiments. (be) Effects of 15-LOX-1 expression via adenoviral vector on apoptosis in colon cancer cells. (b) Effects of 15-LOX-1 expression on XIAP and BcL-XL expression in colon cancer cells. HCT-116 cells were transfected with either Ad-5/3-15-LOX-1 or Ad-5/3-Luc (100 viral particles/cell), and cells were harvested 24 and 48 hours later and processed for XIAP and BcL-XL expression by Western blotting. (c) Effect of 15-LOX-1 expression on apoptosis, as measured by level of caspase-3 activity. HCT-116 cells were transfected with Ad-5/3-15-LOX-1 or Ad-5/3-Luc (100 viral particles/cell). Cells were harvested 48 hours later and processed for measuring caspase-3 enzymatic activity. Values shown are the means ± SDs of triplicate experiments. *P < 0.0001 Ad-5/3-15-LOX-1 versus Ad-5/3-Luc and Mock. (d) Effect of 15-LOX-1 expression on apoptosis measured as floating cell ratio. HCT-116 cells were transfected with Ad-5/3-15-LOX-1 or Ad-5/3-Luc in the indicated concentrations, and the floating and attached cells were counted 72 hours later. The proportion of floating cells to total cells was calculated as a measure of apoptosis. Values shown are the means ± SDs of triplicate experiments. *P < 0.0001 Ad-5/3-15-LOX-1 versus Ad-5/3-Luc. (e) Effect of 15-LOX-1 expression on apoptosis as measured by DNA laddering. HCT-116 cells were transfected with Ad-5/3-15-LOX-1 or Ad-5/3-Luc, and cells were harvested 72 hours later and processed for DNA laddering assays. The lanes are numbered with the concentration of viral particles/cell for each vector. The DNA laddering pattern typical of apoptosis was observed for Ad-5/3-15-LOX-1 in various concentrations but not for Ad-5/3-Luc.

Figure 4

15-LOX-1 expression via an adenoviral vector inhibits colonic tumorigenesis in vivo. (a) 15-LOX-1 expression via the Ad-5/3-15-LOX-1 adenoviral vector into LoVo colon cancer xenografts. Nude mice with LoVo colon cancer cell xenografts were killed 3 days after injection of Ad-5/3-15-LOX-1 adenoviral vector (Ad-5/3-15-LOX-1), Ad-5/3-luciferase adenoviral vector (Ad-5/3-Luc), or PBS. Xenografts were harvested and then processed for 15-LOX-1 Western blotting. + represents a positive control for 15-LOX-1. (b) Effects of 15-LOX-1 expression on growth of LoVo xenografts in nude mice. Xenografts of LoVo colon cancer cells in 35 mice divided into three groups were injected with PBS (control) or Ad-5/3-Luc or Ad-5/3-15-LOX-1 adenoviral vectors. A growth-curve model was used to fit the animal data and assess whether 15-LOX-1 expression affected tumor growth using a log-linear mixed-effect model with treatments, time after injection, and their interaction as the fixed effects and the animal as a random effect. Values shown are the means ± SEs. Repeated experiments showed similar results. (c) This photograph of representative mice treated as described in panel b was taken on day 44 from the time of adenoviral vectors or PBS injection. Mice are labeled PBS (control), Luc (Ad-5/3-Luc adenoviral vector), and 15-LOX (Ad-5/3-15-LOX-1 adenoviral vector). Complete xenograft regression was noted in only the Ad-5/3-15-LOX-1 adenoviral vector group (in 5 of 24 xenografts).