Down-regulation of multiple cell survival proteins in head and neck cancer cells by an apoptogenic mutant of adenovirus type 5

Abstract

Head and neck squamous cell carcinomas (HNSCC) are one of the leading causes of cancer deaths world wide. Up-regulation of the epidermal growth factor receptor (EGFR) and BCL-2 family anti-apoptosis proteins in these cancers is linked to aggressive tumor growth, metastasis and chemoresistance. Infection of two HNSCC cell lines, SCC25 and CAL27 by an Ad5 mutant (lp11w) defective in coding for the viral anti-apoptosis protein, E1B-19K efficiently induced apoptotic cell death. In cells infected with lp11w there was a dramatic down-regulation of EGFR by apoptosis-dependent and -independent mechanisms. The levels of the anti-apoptotic proteins BCL-2, BCL-xL and MCL-1 were also down-regulated in lp11w-infected cells compared to uninfected or Ad5-RM infected cells. Infection with lp11w also enhanced sensitivity of the HNSCC cells to the chemotherapeutic drug cisplatin. Our results suggest that adenoviral vectors defective in E1B-19K would be valuable for efficient down-regulation of cell survival proteins and EGFR in epithelial cancers and could be exploited as oncolytic agents to treat HNSCCs.

Figure 1. Effect of Ad5 mutants on cell lysis

(A). Diagrammatic illustration of Ad5 mutants. The top panel shows the organization of the E1A region and mutants in the E1A region. The bottom panel shows mutants in E1B, E3 and ‘I’-leader regions. The nucleotide positions corresponding to various mutations are indicated. (B). Effect of Ad5 mutants on cell viability. SCC25 cells in 96-well plates were infected with indicated viruses at various MOIs (PFU/cell), and cell viability was determined by the MTS assay 144 hr after infection. (C) Viral spread assay. SCC25 cells in 24-well plates were infected with Ad5-RM or lp11w at various MOIs and stained with crystal violet 144 hr after infection. (D). Replication of Ad5-RM and lp11w. The relative viral genome copy number was determined by real-time PCR analysis 144 hr after infection of SSC25 cells with Ad5-RM or lp11w.

Figure 1. Effect of Ad5 mutants on cell lysis

(A). Diagrammatic illustration of Ad5 mutants. The top panel shows the organization of the E1A region and mutants in the E1A region. The bottom panel shows mutants in E1B, E3 and ‘I’-leader regions. The nucleotide positions corresponding to various mutations are indicated. (B). Effect of Ad5 mutants on cell viability. SCC25 cells in 96-well plates were infected with indicated viruses at various MOIs (PFU/cell), and cell viability was determined by the MTS assay 144 hr after infection. (C) Viral spread assay. SCC25 cells in 24-well plates were infected with Ad5-RM or lp11w at various MOIs and stained with crystal violet 144 hr after infection. (D). Replication of Ad5-RM and lp11w. The relative viral genome copy number was determined by real-time PCR analysis 144 hr after infection of SSC25 cells with Ad5-RM or lp11w.

Figure 2. Apoptotic activity of lp11w

(A). Effect of Ad5 mutants on caspase activation. SCC25 cells were infected with indicated mutants and proteolytic processing of pro-caspase 3 and PARP were determined by western blot analysis. (B). Ultra-structure of infected cells. Thin sections of Ad-infected cells were analyzed by light microscopy (top panel) and by electron microscopy (bottom two panels). Apoptotic cells in the top panel of lp11w-infected samples are pointed out by arrows. (C). Flow cytometric analysis of annexin V-staining patterns of Ad-infected cells. The quantification of annexin V-positive and propidium iodide-positive cells is shown in the bar diagram.

Figure 2. Apoptotic activity of lp11w

(A). Effect of Ad5 mutants on caspase activation. SCC25 cells were infected with indicated mutants and proteolytic processing of pro-caspase 3 and PARP were determined by western blot analysis. (B). Ultra-structure of infected cells. Thin sections of Ad-infected cells were analyzed by light microscopy (top panel) and by electron microscopy (bottom two panels). Apoptotic cells in the top panel of lp11w-infected samples are pointed out by arrows. (C). Flow cytometric analysis of annexin V-staining patterns of Ad-infected cells. The quantification of annexin V-positive and propidium iodide-positive cells is shown in the bar diagram.

Figure 2. Apoptotic activity of lp11w

(A). Effect of Ad5 mutants on caspase activation. SCC25 cells were infected with indicated mutants and proteolytic processing of pro-caspase 3 and PARP were determined by western blot analysis. (B). Ultra-structure of infected cells. Thin sections of Ad-infected cells were analyzed by light microscopy (top panel) and by electron microscopy (bottom two panels). Apoptotic cells in the top panel of lp11w-infected samples are pointed out by arrows. (C). Flow cytometric analysis of annexin V-staining patterns of Ad-infected cells. The quantification of annexin V-positive and propidium iodide-positive cells is shown in the bar diagram.

Figure 3. Effect of Ad-infection on anti-apoptotic and pro-apoptotic proteins

(A). Western blot analysis of Ad-infected SCC25 cells. Western-blot analyses were carried out at 15 or 20 hr post infection. (B). Western-blot analysis of Ad-infected CAL27 cells.

Figure 4. Apoptosis-dependent clearance of EGFR

(A). CAL27 cells were infected with indicated viruses and incubated with zVAD-fmk (50 µM) or with vehicle and the effects on EGFR and PARP were determined by western blot analysis at 20 hr post-infection. (B). CAL27 cells were infected with indicated mutants and recombinants (top) and the effects on EGFR and PARP were determined by western blot analysis.

Figure 5. Immunofluorescence analysis of EGFR expression in Ad-infected cells

CAL27 (A) or SCC25(B) cells were infected with indicated viruses and indirect immunofluorescence analysis and confocal microscopy were carried out cells fixed at 20 hr post-infection.

Figure 6. Effect of Ad-infection on cisplatin toxicity

CAL27 (A) and SCC25 (B) cells were infected with dl312 or Ad5-RM or lp11w at an MOI of 10, treated with indicated concentrations of cisplatin at 24 hr post infection and cell viability was determined at 24 hr after drug treatment by the MTS assay. The experiments were repeated at least three times independently and the data are means ± SD. Statistical differences were assessed using two-way ANOVA followed by a Bonferroni post hoc test, where appropriate, using GraphPad Prism 5 software. P-value was determined by comparing dl 312 vs Ad5-RM, dl 312 vs lp11w and Ad5-RM vs lp11w and also with all the cisplatin concentrations. Statistical significance was accepted at a value of *P<0.05, **P<0.01 and ***P<0.001.