Oncolytic adenoviral mutants with E1B19K gene deletions enhance gemcitabine-induced apoptosis in pancreatic carcinoma cells and anti-tumor efficacy in vivo

Abstract

Purpose: Pancreatic adenocarcinoma is a rapidly progressive malignancy that is highly resistant to current chemotherapeutic modalities and almost uniformly fatal. We show that a novel targeting strategy combining oncolytic adenoviral mutants with the standard cytotoxic treatment, gemcitabine, can markedly improve the anticancer potency.

Experimental design: Adenoviral mutants with the E1B19K gene deleted with and without E3B gene expression (AdDeltaE1B19K and dl337 mutants, respectively) were assessed for synergistic interactions in combination with gemcitabine. Cell viability, mechanism of cell death, and antitumor efficacy in vivo were determined in the pancreatic carcinoma cells PT45 and Suit2, normal human bronchial epithelial cells, and in PT45 xenografts.

Results: The DeltaE1B19K-deleted mutants synergized with gemcitabine to selectively kill cultured pancreatic cancer cells and xenografts in vivo with no effect in normal cells. The corresponding wild-type virus (Ad5) stimulated drug-induced cell killing to a lesser degree. Gemcitabine blocked replication of all viruses despite the enhanced cell killing activity due to gemcitabine-induced delay in G1/S-cell cycle progression, with repression of cyclin E and cdc25A, which was not abrogated by viral E1A-expression. Synergistic cell death occurred through enhancement of gemcitabine-induced apoptosis in the presence of both AdDeltaE1B19K and dl337 mutants, shown by increased cell membrane fragmentation, caspase-3 activation, and mitochondrial dysfunction.

Conclusions: Our data suggest that oncolytic mutants lacking the antiapoptotic E1B19K gene can improve efficacy of DNA-damaging drugs such as gemcitabine through convergence on cellular apoptosis pathways. These findings imply that less toxic doses than currently practiced in the clinic could efficiently target pancreatic adenocarcinomas when combined with adenoviral mutants.

Fig. 1

Low doses of gemcitabine sensitize pancreatic cancer but not normal cells to E1B19K-deleted mutants. A, viruses used in the study with the respective deletions indicated. PT45 (B) and Suit2 (C) cells were infected with viruses and treated with 5 nmol/L (black) or 10 nmol/L (grey) gemcitabine. D, normal NHBE cells infected with mutants and treated with 5 nmol/L (black), 10 nmol/L (grey) or10 μmol/L (white) gemcitabine. Cells were analyzed for viability with the MTS assay 3 d after treatment; EC₅₀ values were calculated and presented as percentages of virus-treated compared with control cells, as described in Materials and Methods. Data are the averages of three to four experiments ± SE; *P < 0.05 and **P < 0.01for B and C, and D is the average of one representative study in triplicate ± SE with *P < 0.05 compared with virus-treated cells.

Fig. 2

Gemcitabine prevents viral replication in PT45 and Suit2 pancreatic cell lines and in normal cells. A, replication in PT45 and (B) Suit 2 cells was analyzed after 48 h (grey) and 72 h (black) of treatment with 10 nmol/L gemcitabine, and (C) NHBE cells after 48 h and 72 h of treatment with viruses alone (black or grey) and in combination with gemcitabine at 10 nmol/L (striped) and 10 μmol/L (crossed).Virus and drug were added simultaneously and both cells and media were analyzed byTCID₅₀ assays. The data are from a representative of three experiments. The * indicates that replication levels were below the detection limit of the assay, < 1pfu/cell. D, QPCR of viral DNA in PT45 cells 24 and 48 h after infection.Viral DNA copies were determined after treatment with 10 nmol/L (striped) and 10 μmol/L (crossed) gemcitabine or with the Ad5ΔE1B19K virus alone (black). Gene amplification was normalized to input virus (3 h after infection; n = 3).

Fig. 3

Early and late viral genes are expressed in PT45 cells infected with viral mutants in the presence of gemcitabine. A, immunoblot of cells infected with Ad5, Ad5ΔE1B19K, dl309, or dl337 and treated with 10 nmol/L gemcitabine. Cell extracts were prepared 48 h after infection and 20 μg of protein loaded in each lane for detection of E1A expression. B, cells infected with Ad5wt and Ad5ΔE1B19K harvested 24 to 72 h after infection and analyzed for E1A and hexon expression (upper panel). Lower panel, light microscopy of NHBE cells grown on glass slides, infected with AdΔ19K in the presence of 10 nmol/L gemcitabine, stained for E1A expression at 24 to 72 h, magnification 200. C, quantitative reverse transcription-PCR of E1A and (D) penton mRNA levels in response to gemcitabine 24, 48, and 72 h after treatment. Standard curves were prepared for each gene and results were normalized to 18S RNA in every sample, as described in Materials and Methods. Data are representative of two to three experiments.

Fig. 4

Cell cycle analysis of PT45 cells in the presence of viral mutants and gemcitabine. A, flow cytometric analysis of cells treated with gemcitabine at 10 nmol/L, the Ad5ΔE1B19K mutant alone or in combination. Cells were fixed and stained with propidium iodide 24, 48, 72, and 96 h after treatment. B, histograms illustrating the cell cycle-phase distribution in percentages in response to combination treatments with 10 nmol/L or 10 μmol/L gemcitabine and the ΔE1B19K mutant 96 h after treatment; sub-G₁ (black), G₁ (grey), S (white), and G₂/M (top light grey). C, immunoblot of changes in expression levels of cell cycle related proteins in response to mutants and gemcitabine (10 nmol/L) combinations. Cells were harvested 72 h after treatment. D, cells were serum-starved and treated with aphidocholine (5 μg/mL) for 24 and 48 h followed by viral DNA (hexon) amplification (left panel); right, the corresponding cell cycle diagram 24 h after addition of aphidocholine. Gene amplification was normalized to input virus (3 h after infection); data are representative of three studies.

Fig. 5

Combination treatment enhances gemcitabine-induced apoptotic cell death in PT45 cells. A, PT45 cells were treated with 100 ppc of each viral mutant with and without 10 nmol/L gemcitabine for 72 h and analyzed for cell death using an Annexin Vantibody and propidium iodide. Lower black bars, annexin positive only; upper grey bars, both annexin- and propidium iodide–positive cells; dashed lines, predicted additive value of total apoptotic cells (annexin positive ± propidium iodide staining). B, cells were treated as in A and analyzed by a specific antibody for active caspase-3 by flow cytometry, 72 h after treatment initiation. Dashed lines, predicted additive values of combination treatments. Insert, immunoblot of inactive procaspase-3 (34 kD) and the active cleaved caspase-3 subunits (17 and 20 kD) at the same time point. C, changes in mitochondrial membrane potential (Δψ) determined by tetramethylrhodamine ethyl ester perchlorate (TMRE) staining and cytometry analysis, expressed as % of live cells with unchanged Δψ (TMRE-positive only). Cells were treated with 10 μmol/L gemcitabine alone or in combination with 100 ppc of each viral mutant. Staurosporin (St)-treated cells (3 μmol/L) were included as a reference for mitochondrial dysfunction (left panel). Right panel, Δψ in response to combinations of mutant or Ad5 with gemcitabine (10 nmol/L) at 96 h in PT45 and Suit2 cells. Representative data from four to six studies. D, dose response to Ad5ΔE1B19K alone and in combination with 10 nmol/L gemcitabine (G) with and without the pan-caspase inhibitor zVAD-fmk (Z) at 25 μmol/L. Cell death was determined by MTS assay (left panel). Right, inhibition of cell death by zVAD-fmk (Z) at 5 and 25 μmol/L in gemcitabine-treated (20 nmol/L) and untreated control cells (Ctrl; 25 μmol/L Z). Data are representative of three experiments with triplicate samples for each study in A, B, and D.

Fig. 6

Combinations of low doses of the ΔE1B19K-deleted mutant and gemcitabine inhibit tumor progression. A, animals with PT45 s.c. tumors were treated with the indicated doses (mock = E1A-deleted virus) and tumor growth was monitored. ** P < 0.01 and *P < 0.05 for combination treatments compared with either single-agent treatment and *P < 0.05 for each treatment compared with mock-treated animals. Significance determined by one-way ANOVA analysis. B, Kaplan-Meier curves for the indicated treatment groups. All combinations were significantly different from single-agent treatments with **P < 0.05. The percentages of mice free from tumor progression (tumor volume < 500 μL) at each time point were estimated using the Kaplan-Meier method, 6 to 10 animals per group. C, immunohistochemistry of representative tumor sections from the studies with viruses at 1 10⁶ vp and gemcitabine at 2.5 mg/kg.Three tumors were evaluated from each treatment group; representative micrographs at 100 and 200 magnification for hexon and E1A, respectively.