Lovastatin enhances adenovirus-mediated TRAIL induced apoptosis by depleting cholesterol of lipid rafts and affecting CAR and death receptor expression of prostate cancer cells

Abstract

Oncolytic adenovirus and apoptosis inducer TRAIL are promising cancer therapies. Their antitumor efficacy, when used as single agents, is limited. Oncolytic adenoviruses have low infection activity, and cancer cells develop resistance to TRAIL-induced apoptosis. Here, we explored combining prostate-restricted replication competent adenovirus-mediated TRAIL (PRRA-TRAIL) with lovastatin, a commonly used cholesterol-lowering drug, as a potential therapy for advanced prostate cancer (PCa). Lovastatin significantly enhanced the efficacy of PRRA-TRAIL by promoting the in vivo tumor suppression, and the in vitro cell killing and apoptosis induction, via integration of multiple molecular mechanisms. Lovastatin enhanced PRRA replication and virus-delivered transgene expression by increasing the expression levels of CAR and integrins, which are critical for adenovirus 5 binding and internalization. Lovastatin enhanced TRAIL-induced apoptosis by increasing death receptor DR4 expression. These multiple effects of lovastatin on CAR, integrins and DR4 expression were closely associated with cholesterol-depletion in lipid rafts. These studies, for the first time, show correlations between cholesterol/lipid rafts, oncolytic adenovirus infection efficiency and the antitumor efficacy of TRAIL at the cellular level. This work enhances our understanding of the molecular mechanisms that support use of lovastatin, in combination with PRRA-TRAIL, as a candidate strategy to treat human refractory prostate cancer in the future.

Figure 1. Lovastatin significantly enhanced tumor suppression efficacy of oncolytic adenovirus constructs, AdE4 and AdE4-TRAIL

A. Structure diagram of prostate cancer-specific replicative adenovirus AdE4, AdE4-TRAIL, AdE4-Luc (AdE4 vector encoding a luciferase gene) and AdE4(ΔTATA) (a replication-deficient adenovirus due to the deletion of TATA box of Ad E1a gene). B. CWR22rv xenografts were established in athymic nude mice. Mice were randomized 3 weeks after cell inoculation (8 mice/group, 1 tumor xenograft/ mouse). Tumors were treated with lovastatin at 15 mg/kg/day, i.p. for 7 consecutive days), or AdE4 (2 ×10⁷ IFU in 100mL 1×PBS i.t.), AdE4-TRAIL (2 ×10⁷ IFU in 100mL 1×PBS i.t.), or lovastatin (i.p.) combined with AdE4 (i.t.), or lovastatin (i.p.) combined with AdE4-TRAIL (i.t.), for up to 28 days. Tumor size was measured on day 3 (D3), and then once every 7 days until 28 days (D28), and expressed as the average per group (n=8 xenografts/group),. C. Representative photographs of tumor xenografts (arrows) in 2 of the mice treated with AdE4-TRAIL (top) or lovastatin plus AdE4-TRAIL (bottom). On day 28, gross tumor size is much less in mice treated with lovastatin plus AdE4-TRAIL compared to those in mice treated with AdE4-TRAIL alone. D. Photomicrographs of immunohistochemistry of virus (Ad E1a) in tissue sections of xenografts after 28 days of treatment. The number of positive cells was counted in 10 randomly selected vision fields (original magnification: ×200) for each tissue section; the average of 3 tissue sections was used to represent each tumor. Lovastatin significantly enhanced virus infection inside tumor tissues (see graph below photomicrograph). E. Photomicrographs of tissue sections of xenografts showing fluorescence-labeled TUNEL+ apoptotic cells in tumor xenografts. The green fluorescent cells were counted in 10 randomly selected vision fields (original magnification: ×200) for each tissue section; the average of 3 tissue sections was used to represent each tumor. Lovastatin significantly enhanced AdE4-TRAIL-induced apoptosis inside tumor tissues (see graph below photomicrograph).

Figure 1. Lovastatin significantly enhanced tumor suppression efficacy of oncolytic adenovirus constructs, AdE4 and AdE4-TRAIL

A. Structure diagram of prostate cancer-specific replicative adenovirus AdE4, AdE4-TRAIL, AdE4-Luc (AdE4 vector encoding a luciferase gene) and AdE4(ΔTATA) (a replication-deficient adenovirus due to the deletion of TATA box of Ad E1a gene). B. CWR22rv xenografts were established in athymic nude mice. Mice were randomized 3 weeks after cell inoculation (8 mice/group, 1 tumor xenograft/ mouse). Tumors were treated with lovastatin at 15 mg/kg/day, i.p. for 7 consecutive days), or AdE4 (2 ×10⁷ IFU in 100mL 1×PBS i.t.), AdE4-TRAIL (2 ×10⁷ IFU in 100mL 1×PBS i.t.), or lovastatin (i.p.) combined with AdE4 (i.t.), or lovastatin (i.p.) combined with AdE4-TRAIL (i.t.), for up to 28 days. Tumor size was measured on day 3 (D3), and then once every 7 days until 28 days (D28), and expressed as the average per group (n=8 xenografts/group),. C. Representative photographs of tumor xenografts (arrows) in 2 of the mice treated with AdE4-TRAIL (top) or lovastatin plus AdE4-TRAIL (bottom). On day 28, gross tumor size is much less in mice treated with lovastatin plus AdE4-TRAIL compared to those in mice treated with AdE4-TRAIL alone. D. Photomicrographs of immunohistochemistry of virus (Ad E1a) in tissue sections of xenografts after 28 days of treatment. The number of positive cells was counted in 10 randomly selected vision fields (original magnification: ×200) for each tissue section; the average of 3 tissue sections was used to represent each tumor. Lovastatin significantly enhanced virus infection inside tumor tissues (see graph below photomicrograph). E. Photomicrographs of tissue sections of xenografts showing fluorescence-labeled TUNEL+ apoptotic cells in tumor xenografts. The green fluorescent cells were counted in 10 randomly selected vision fields (original magnification: ×200) for each tissue section; the average of 3 tissue sections was used to represent each tumor. Lovastatin significantly enhanced AdE4-TRAIL-induced apoptosis inside tumor tissues (see graph below photomicrograph).

Figure 2. Lovastatin significantly enhanced induction of cell killing and apoptosis by AdE4 and AdE4-TRAIL

CWR22rv and C4-2 cells were treated with lovastatin (5μM for CWR22rv and 2.5μM for C4-2) for 16 hours, followed by virus infection of AdE4(ΔTATA)(replication-deficient adenovirus), AdE4 or AdE4-TRAIL (100 vp/cell). A. The cells were stained with crystal violet, and the optical density was analyzed at OD490, at day 5 after virus infection. An index of relative cell killing activity for each treatment was calculated by comparing the treatment groups to control (PBS+DMSO). B. The percent of apoptotic cells was calculated at 48 hours after virus infection, as described in the Methods.

Figure 3. Lovastatin significantly enhanced AdE4-delivered transgene expression

CWR22rv and C4-2 cells were treated with vehicle (DMSO) or lovastatin at 10 μM for 16 hours. Then cells were infected with 100 vp/cell of AdE4 (A.) or AdE4-Luc (B.) or AdE4-TRAIL (C.) for the next 48 hours. AdE4-infected green fluorescent cells were monitored by FACS (A.); luciferase activity was measured by a luminometer (B.); TRAIL protein expression was assessed by western blotting (C.), all as described in Methods.

Figure 4. Lovastatin significantly enhanced adenoviral binding, internalization and intercellular trafficking to the nuclei

A. CWR22rv cells were treated with vehicle (DMSO) or lovastatin at 10 μM for 16 hours, then exposed to 5000 vp/cell of AdE4 at 4ºC for 60 minutes. The unbound viral particles were removed; DNA of bound virus particles was processed for analysis of adenovirus E1a copy number by quantitative PCR assay. B. After viral binding, cells were allowed to internalize virus particles at 37ºC for other 30 minutes. The attached but uninternalized viral particles were removed, and the DNA of internalized adenoviral particles for adenoviral E1a copy number using quantitative PCR. C. Nuclear DNA was separated, and adenovirus E1a copy number inside the nuclei was analyzed by quantitative PCR. D. Screen views of virus trafficking in individual cells, as monitored by the Amnis Imagestream^X cell analyzer. Adenoviral particles were labeled with Alexa Fluor® 488 dye. CWR22rv cells were treated with vehicle (DMSO) or lovastatin at 10 μM for 16 hours, and then exposed to virus at 5000 vp/cell of dye-labeled AdE4 for 60 minutes at 37ºC, to allow virus internalization; and then for 30 minutes, to allow for virus trafficking to the nuclei. Nuclei were stained with DAPI. Cells and viral particles were monitored using INSPIRE™ software, and the co-localization data of viral particles and nuclei were analyzed using IDEAS software. Three representative AdE4-treated single cells are shown in the left panel, and 3 representative AdE4 plus lovastatin-treated single cells are shown in the right panel. BF: brightfield showing the cell morphology; DAPI: nuclei; virus: dye-stained viral particles; DAPI/virus: co-localization of virus and nuclei showing viruses inside the nuclei; BF/virus: co-localization of virus and cell showing viruses inside cells.

Figure 5. Lovastatin induced cell apoptosis, and sensitized cancer cells to TRAIL-induced apoptosis

A. Representative cells to enable visualization and overlays of lipid rafts/cholesterol in non-malignant and cancer cell lines, using confocal microscopy. Non-malignant cells PZ-HPV-7 and MCF10A and prostate cancer cells C4-2, PC-3 and LNCaP were labeled with Alexa red Fluo555/565 – CTXB (lipid rafts, glycolipoprotein microdomains, GM) and green filipin (cholesterol, CH) and analyzed by confocal microscopy. B. The correlation of level of lipid rafts/cholesterol and the sensitivity of cells to lovastatin-induced apoptosis. Serum-starved cells were treated with lovastatin at 10 μM or DSMO control for 16 hours, with triplicate-wells for each cell line. Apoptotic cells were stained with Annexin V-FITC and PI, and detected by flow cytometry. Data were expressed as the ratio of lovastatin-treated cells to control. C. The effect of lovastatin on the level of cholesterol in the lipid rafts and apoptosis in prostate cancer cells. Serum-starved cells were treated with DSMO (control) or lovastatin at 10 μM for 16 hours, and then incubated with or without 500 μM cholesterol for 2 hours. Cells were stained with CTXB-Alexa 555/558 (GM) and filipin (CH), and monitored by confocal microscopy. The cells were processed for Annexin V-FITC and PI staining, and the percent apoptotic cells analyzed by flow cytometry. Each experiment was replicated 3 times. D. Prostate cancer cells are resistant to TRAIL-induced apoptosis. Prostate cancer cells LNCap, C4-2, CWR22rv, PC-3, DU145, non-malignant prostate epithelial cells PZ-HPV-7, keratinocytes, non small lung adenocarcinoma cells A549 and colon cancer Lovo cells were treated with TRAIL protein for 24 hours at a range of doses (n=5 for each cell line). Cell viability was measured by MTT assay at 72 hours after drug treatment. E. Lovastatin significantly enhanced TRAIL-induced apoptosis in prostate cancer cells, but not in normal cells. LNCaP, C4-2, PC-3, PZ-HPV-7 and keratinocytes were treated with lovastatin at 10 μM for 16 hours, before being treated with or without TRAIL(200 ng/mL) for 24 hours (n=4/group). The cells were processed for Annexin V-FITC and PI staining, and the percent apoptotic cells analyzed by flow cytometry.

Figure 6

A to D. Lovastatin increased protein expression of selected cell receptors, which included CAR and integrin αv, β1 and β3, and TRAIL receptors DR4. CWR22rv, C4-2 or PZ-HPV-7 cells were treated with DSMO (control) or lovastatin at 10 μM for 16 hours. Protein expression of integrin β1 and β3 were determined in CWR22rv cells by flow cytometry (A, B); expression of CAR, integrin αν, DR4, DR5 and cleaved caspase 3 were determined in CWR22rv and C4-2 cells by western blotting (C, D). Protein expression of CAR in PZ-HPV-7 cells is shown in C. E. and F. Lovastatin significantly enhanced CAR and DR4 expression inside xenograft tumor tissues of mice after 28 days of treatment. CAR and DR4 expression in tissue sections of xenografts were evaluated by immunohistochemical staining. The number of positive cells was counted in 10 randomly selected vision fields (×40) for each tissue section; 3 tissue sections were averaged to represent each tumor.

Figure 6

A to D. Lovastatin increased protein expression of selected cell receptors, which included CAR and integrin αv, β1 and β3, and TRAIL receptors DR4. CWR22rv, C4-2 or PZ-HPV-7 cells were treated with DSMO (control) or lovastatin at 10 μM for 16 hours. Protein expression of integrin β1 and β3 were determined in CWR22rv cells by flow cytometry (A, B); expression of CAR, integrin αν, DR4, DR5 and cleaved caspase 3 were determined in CWR22rv and C4-2 cells by western blotting (C, D). Protein expression of CAR in PZ-HPV-7 cells is shown in C. E. and F. Lovastatin significantly enhanced CAR and DR4 expression inside xenograft tumor tissues of mice after 28 days of treatment. CAR and DR4 expression in tissue sections of xenografts were evaluated by immunohistochemical staining. The number of positive cells was counted in 10 randomly selected vision fields (×40) for each tissue section; 3 tissue sections were averaged to represent each tumor.