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. Author manuscript; available in PMC: 2009 Sep 15.
Published in final edited form as: Cancer Res. 2008 Sep 3;68(18):7439–7447. doi: 10.1158/0008-5472.CAN-08-0072

Mechanism of In Vitro Pancreatic Cancer Cell Growth Inhibition by mda-7/IL-24 and Perillyl Alcohol

Irina V Lebedeva 1, Zhao-zhong Su 1,6, Nichollaq Vozhilla 1,6, Lejuan Chatman 1, Devanand Sarkar 1,2,6, Paul Dent 3, Mohammad Athar 4, Paul B Fisher 1,2,5,6,*
PMCID: PMC2596728  NIHMSID: NIHMS64697  PMID: 18768668

Abstract

The death rate for pancreatic cancer approximates the number of new cases each year and when diagnosed current therapeutic regimens provide little benefit in extending patient survival. These dire statistics necessitate the development of enhanced single or combinatorial therapies to decrease the pathogenesis of this invariably fatal disease. Melanoma differentiation associated gene-7/interleukin-24 (mda-7/IL-24) is a potent cancer gene therapeutic because of its broad-spectrum cancer-specific apoptosis-inducing properties as well as its multi-pronged indirect anti-tumor activities. However, pancreatic cancer cells demonstrate inherent resistance to mda-7/IL-24 that is caused by a block of translation of mda-7/IL-24 mRNA in these tumor cells. We now reveal that a dietary agent perillyl alcohol (POH) in combination with Ad.mda-7 efficiently reverses the mda-7/IL-24 ‘protein translational block' by inducing reactive oxygen species thereby resulting in MDA-7/IL-24 protein production, growth suppression and apoptosis. Pharmacological inhibitor and siRNA studies identify xanthine oxidase as a major source of superoxide radical production causing these toxic effects. Since both POH and Ad.mda-7 are being evaluated in clinical trials, combining a dietary agent and a virally delivered therapeutic cytokine provide an innovative approach for potentially treating human pancreatic cancer.

Keywords: mda-7/IL-24, POH, reactive oxygen species, cancer-selective apoptosis, xanthine oxidase

Introduction

Pancreatic ductal adenocarcinoma is the predominant form of pancreatic cancer and one of the most lethal and aggressive human malignancies. Approximately 37,000 new cases are diagnosed annually in the United States alone, which is virtually the same number of deaths reported to occur every year due to metastatic complications (an NCI's estimate shows this number to be 33,370 for 2007). Meaningful therapy in the form of surgical resection is feasible only with early-stage detection, but this applies to just <20% of patients and results in a 5-yr survival of <20% (1, 2). For the vast majority of patients, the overall 5-yr survival is <5%, which is attributable to multiple factors, including the plethora of molecular genetic changes contributing to the cellular phenotype of pancreatic neoplasms that are believed to produce resistance to chemotherapy and radiation therapy. This medical problem is further compounded by the lack of therapeutic approaches targeting metastatic disease. Considering these terrible statistics, it is imperative to develop rational molecular target-based preventative and therapeutic strategies for this consistently fatal disease.

Using subtraction hybridization, we discovered genes, originally called melanoma differentiation-associated (mda), which encode molecules that are now better defined in terms of their physiological and pathological importance and have been shown to play crucial roles in cell cycle control, metastasis, senescence, differentiation, innate immunity and apoptosis (3). One of these originally novel genes, mda-7, displayed an inverse relationship with the pathogenesis and progression of melanoma (4). We showed that mda-7 manifests tumor suppressor-like functions in vitro and in vivo in athymic human tumor xenograft animal models (5). Ectopic expression of mda-7 (by transfection or by adenovirus transduction) exerts potent growth-suppressive and apoptosis-inducing effects, not only in human melanoma cells, but also in a wide spectrum of human cancer cells, including malignant glioma, osteosarcoma, mesothelioma and carcinomas of the breast, cervix, colon, lung, ovary and prostate (reviewed in (6-8)). Based on structure, chromosomal location and biological/biochemical properties, mda-7 has now been classified as a novel member of the IL-10 gene family, IL-24 (6-8). Data from multiple laboratories including our own indicate that mda-7/IL-24, a secreted cytokine, which also manifests “bystander” anti-tumor activity, can retard tumor growth by impinging on several critical signaling pathways resulting in tumor apoptosis as well as by inhibiting tumor angiogenesis and modulating immune responses (8-10). Remarkably, in the context of normal cells/tissues, no such growth suppressive or cytotoxic effects are evident (reviewed in (6-9)). Considering these intriguing differential properties in tumor versus normal cells, mda-7/IL-24 was evaluated for its in vivo efficacy employing a number of human tumor xenograft murine models. These results confirmed potent selective anti-tumor activity of this cytokine and prompted testing (using a replication incompetent adenovirus, Ad.mda-7 - INGN 241) in patients with advanced carcinomas and melanoma (11-13). A recent Phase I clinical trial was highly promising confirming the retention of tumor-specific activity in patients. More impressively, mda-7/IL-24 was well tolerated and showed no adverse effects in these patient populations (11-13). Based on these data, investigations are now in progress to further evaluate the potential therapeutic applications of this novel cytokine for cancer gene therapy in Phase II clinical trials.

Although effective in virtually all human tumor cells tested, pancreatic cancer cells represent an enigma, being inherently resistant to Ad.mda-7-based therapy (14, 15). Transfection or adenovirus transduction of mda-7/IL-24 under conditions in which growth suppression and apoptosis occur in various other human tumor cells was without activity (14, 15). An interesting mechanism underlies this resistance, limited conversion of mda-7/IL-24 mRNA into protein in pancreatic tumor cells due to a ‘protein translational block' targeting this mRNA (14, 15). This ‘protein translational block' in mutant K-ras pancreatic cancer cells could be reversed by ablating K-ras expression as well as by inhibiting its downstream target, extracellular regulated kinase 1/2 (ERK1/2), resulting in growth arrest and apoptosis in pancreatic cancer cells in an analogous manner as observed in other permissive cancer cells (14, 15). We also discovered that augmented generation of reactive oxygen species (ROS) could restore mda-7/IL-24 mRNA translation in pancreatic cancer cells irrespective of their K-ras status (16). Although of potential clinical interest, many agents that could be used with Ad.mda-7 for gene therapy of pancreatic carcinomas also manifest non-specific toxicity, thereby limiting their utility.

We hypothesized that specific non-toxic dietary agent(s) with ROS-inducing properties might be agents of choice that would complement Ad.mda-7 action providing a safe combination for human use with potential to abolish the pathogenesis of pancreatic cancer. We presently evaluated perillyl alcohol (POH), a dietary monoterpene present in a variety of plants, including citrus plants, for potential synergy of action with Ad.mda-7. Burke et al. showed that dietary monoterpenes, farnesol, geraniol and POH, block growth of transplanted PC-1 hamster pancreatic adenocarcinomas in Syrian Golden hamsters potentially by their ability to inhibit the prenylation of growth-regulatory proteins other than K-Ras, including H-Ras (17, 18). Karlson et al. demonstrated that POH is effective in inhibiting the growth of pancreatic tumor cells harboring activated ras oncogenes, acting in a Ras-independent manner (19). Stark et al. (20) reported chemotherapeutic effects of POH on pancreatic cancer. They found that POH reduces the growth of hamster pancreatic tumors to less than half that of controls. Moreover, 16% of perillyl alcohol-treated pancreatic tumors completely regressed, whereas no control tumors regressed (20)._Published data suggests that POH blocks a number of important signaling pathways that include ras and ERK in addition to nuclear factor kappa B (NF-κB) (21-24). POH also prevents the isoprenylation of the Ras family of small GTPase proteins (25, 26). Additionally, the pharmacokinetics of POH has been defined in both murine models and humans (27, 28), and Phase I and II clinical trials have been conducted which reveal that this dietary agent is well tolerated (23, 29). Employing a battery of human pancreatic carcinoma cells in culture, we document that the combination of POH and Ad.mda-7 efficiently reversed the mda-7/IL-24 ‘protein translational block' in pancreatic cancer cells by generating ROS, via induction of xanthine oxidase (XO), resulting in pancreatic cancer growth suppression and apoptosis. These studies elucidate a new combinatorial approach for pancreatic cancer involving a dietary supplement and a viral gene therapy that may show promise for treating this devastating cancer.

Materials and Methods

Cell Lines

AsPC-1, MIA PaCa-2, PANC-1 and BxPC-3 pancreatic carcinoma cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA). The human immortalized pancreatic mesenchymal cell line LT2 was purchased from Chemicon (Temecula, CA) and cultured according to the manufacturer's instructions. Stable clones of PANC-1, and MIA PaCa-2 pancreatic carcinoma cells expressing mda-7/IL-24 mRNA were obtained by transfection of the corresponding cells with an mda-7/IL-24 expression vector and selection of clones in hygromycin as described previously (16). Northern and Western blotting, respectively, confirmed expression of mda-7/IL-24 mRNA in these clones, without detectable protein.

Virus Construction, Purification and Infectivity Assays

The recombinant replication-defective Ad.mda-7 virus was created in two steps as described previously (5) and plaque purified by standard procedures. Cells were infected with 100 pfu/cell of Ad.vec or Ad.mda-7 viruses (50 pfu/cell of each virus) and analyzed as described.

RNA Isolation and Northern Blot Assays

Total RNA was extracted from cells using the Qiagen RNeasy mini kit (Qiagen, Hiden, Germany) according to the manufacturer's protocol and was used for Northern blotting as previously described (5, 15, 30).

Purification of polysomes, RNA extraction and Northern blot analysis

Polysomes were purified essentially as described previously (31). Cells (2 × 106) were infected with Ad, and 48 h later the cells were harvested in 500 μl Buffer A (200 mM Tris-HCl pH 8.5, 50 mM KCl, 25 mM MgCl2, 2mM EGTA, 100 μg/ml heparin, 2% polyoxyethylene 10-tridecyl ether and 1% sodium deoxycholate supplemented with Complete Mini protease inhibitor cocktail and RNase inhibitor, Invitrogen, Carlsbad, CA) and centrifuged at 12,000 rpm for 10 min at 4°C to clear cell debris. The supernatant was loaded on top of a 10−50% sucrose gradient prepared in Buffer B (50 mM Tris-HCl, 25 mM KCl and 10 mM MgCl2) and was centrifuged at 40,000 rpm for 1 h at 4°C. Fractions of 500 μl were collected, the O.D. at 260 nm was monitored and polysome fractions were identified (typically fractions 10−20). RNA was extracted from each fraction with Qiagen RNeasy mini kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol and Northern blotting was performed as described using a radio-labeled mda-7/IL-24 cDNA probe.

MTT Viability Assays

Cell viability was assessed by MTT assays as described previously (32).

Annexin V Binding Assays

Cells were trypsinized, washed once with complete medium and PBS, resuspended in 0.5 ml of binding buffer containing 2.5 mM CaCl2 and stained with allophycocyanin (APC)-labeled Annexin-V and propidium iodide (PI) (BD Biosciences, Palo-Alto, CA) for 15 min at RT. Flow cytometry assays were performed immediately after staining using FACS Calibur (Becton-Dickinson, Mountain View, CA). Data were analyzed using CellQuest software, version 3.1 (Becton Dickinson).

Preparation of Cell Extracts and Western Blotting Analysis

Cells were washed 2X with cold PBS and lysed on ice for 30 min in 100 μl of cold RIPA buffer [50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.1% SDS, 1% NP-40, and 0.5 % sodium deoxycholate] with freshly added 0.1 mg/ml phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate, and 1 mg/ml aprotinin. Aliquots of cell extracts containing 20−50 μg of total protein were resolved in 12% SDS-PAGE and transferred to Immobilon-P PVDF membranes (Millipore Corp., Bedford, MA). Filters were blocked and stained with appropriate antibodies as described in the Results section. ECL was performed according to the manufacturer's recommendation. For all Western blots, equal loading of protein was verified by re-blotting of membranes for EF-1α protein.

Assessment of ROS Production

To determine ROS production, cells were stained with 2.5 μM hydroethidine (HE) or 5 μM 2’,7’-dichlorodihydrofluorescein diacetate (DCFH-DA) in PBS for 30 min at 37°C in the dark (16, 33). Immediately after staining, cells were analyzed by flow cytometry (FACS Calibur, Becton-Dickinson, Mountain View, CA), and data were analyzed using CellQuest software, version 3.1 (Becton Dickinson). For inhibition experiments, N-acetyl-L-cysteine (NAC) or allopurinol (AP) (both from Sigma) was added 2 h prior to infection with Ad.mda-7. In all cases, cells were gated to exclude cell debris.

Small interfering RNA (siRNA) experiments

Adherent cells were transfected with siRNA using Lipofectamine™ 2000 reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. Briefly, cationic lipid complexes were formed with 600 pmol of RNA and 30 μl of Lipofectamine™ 2000 reagent in 3 ml of OPTI-MEM® I Reduced Serum Medium (Invitrogen, Carlsbad, CA) and added to 10-cm dishes containing 15 ml of complete medium (final concentration of siRNA was 40 nM). Cells were maintained in culture for 24 h, and infected with Ad.vec or Ad.mda-7 (50 pfu/cell) followed with the addition of 200 μM of POH or 0.03% of DMSO (vehicle) 2 h later. Next day, cells were trypsinized, counted and re-plated for MTT, ROS, Annexin V binding and Western blot assays. SiRNA specific for XO (accession number NM_00379) and control scrambled siRNA were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA).

Statistical Analysis

All of the experiments were performed at least three times. Results are expressed as mean ± S.E. Statistical comparisons were made using an unpaired two-tailed Student's t-test. A P <0.05 was considered significant.

Results

Combinatorial treatment with Ad.mda-7 and POH induces growth inhibition and MDA-7/IL-24 protein production in pancreatic cancer cell lines in vitro by generation of ROS

We employed established pancreatic cancer cell lines, AsPC-1, MIA PaCa-2, and PANC-1 (mutant K-ras) and BxPC-3 (wild type K-ras), as well as normal immortal pancreatic mesenchymal cells (LT2), to investigate cancer-specific growth inhibitory properties of combinatorial treatment with Ad.mda-7 and POH. A non-toxic concentration of POH (200 μM) was chosen, which might be clinically achievable in patients, to evaluate a combinatorial effect of POH and Ad.mda-7. Multiple pharmacokinetic studies indicate that 140 − 500 μM of perillic acid and 10 − 40 μM of dihidroperillic acid, the two main metabolites of POH, are found in plasma and urine after oral administration of POH-containing capsules (34). Since the growth inhibition effect of mda-7/IL-24 is mediated by generation of ROS we also included pre-treatment with a non-toxic dose of antioxidant N-acetyl-L-cysteine in these studies. In a 6-day assay, POH or Ad.mda-7 alone had no discernible effect on any cell type while their combination significantly inhibited growth of pancreatic cancer cells, PANC-1 and BxPC-3 irrespective of their K-ras status, with no growth inhibitory effect on LT2 cells (Fig. 1A). Pre-treatment with NAC significantly protected PANC-1 and BxPC-3 cells from growth inhibition by this combinatorial treatment. Similar findings were also observed in AsPC-1 and MIA PaCa-2 pancreatic carcinoma cells (data not shown).

Figure 1.

Figure 1

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Combination of Ad.mda-7 with POH efficiently inhibits in vitro growth of pancreatic cancer cells irrespective of their K-ras genotype, without affecting immortal normal human cells by generation of ROS. A, Viability of LT2 cells and PANC-1 and BxPC-3 pancreatic cancer cells was measured by MTT assays 6 days post-treatment. Cells were pre-treated with NAC prior to the indicated Ad infection. Asterisks denote statistically significant values, p<0.001. B. Cells were treated as in A and Western blot assays were performed 48 h post infection for the indicated proteins.

In normal cells, such as LT2, MDA-7/IL-24 protein expression could be detected upon Ad.mda-7 infection alone and simultaneous treatment with POH did not significantly alter the level of MDA-7/IL-24 protein (Fig. 1B). However, MDA-7/IL-24 protein expression was detected in PANC-1 and BxPC-3 pancreatic cancer cells receiving a combinatorial treatment of POH and Ad.mda-7, but not either agent alone, indicating that POH treatment overrides the intrinsic ‘translational block of mda-7/IL-24 mRNA' observed in pancreatic cancer cells (14, 15) (Fig. 1B). These results were similar in pancreatic carcinoma cells irrespective of carrying wild type or mutant K-ras and our treatment protocol did not alter the expression level of p21 K-RAS (data not shown). Pretreatment with NAC completely nullified MDA-7/IL-24 protein expression in both LT2 and pancreatic cancer cells, indicating that ROS production plays a fundamental role in generation of MDA-7/IL-24 protein (Fig. 1B).

MDA-7/IL-24 protein expression and apoptosis induction in pancreatic cancer cells after combinatorial treatment of Ad.mda-7 and POH are dependent on xanthine oxidase-mediated ROS production

As a corollary to our results obtained for cell viability, we found that a combinatorial treatment with POH and Ad.mda-7-induced significant apoptosis (30−40%) in pancreatic carcinoma cells whereas single treatment with these agents alone did not manifest apoptosis (Fig. 2A; results are shown for PANC-1 cells and similar results were observed in AsPC-1, MIA PaCa-2 and BxPC-3 cells). Similar findings were obtained when apoptosis was evaluated by two different assays, Annexin V binding assay and FACS analysis of PI-stained cells counting sub-G0 population (the data of the latter studies not shown). Apoptosis induction by Ad.mda-7 and POH treatment could be effectively inhibited by treatment with NAC and AP, a pharmacological inhibitor of ROS-generating enzyme xanthine oxidase (XO) (Fig. 2A). No apoptosis was observed in LT2 cells treated with a single agent or their combinations (data not shown).

Figure 2.

Figure 2

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Combination of Ad.mda-7 with POH induces apoptosis in pancreatic cancer cells in vitro by a ROS-dependent mechanism that involves XO. PANC-1 cells were infected with Ad.mda-7 or Ad.vec (100 pfu/cell) and treated with DMSO (vehicle) or POH alone or pretreated with NAC (10 mM) or with allopurinol (100 μM). Annexin V binding assay (A), ROS detection (B) and Western blot analysis (C) were done 24 h after treatment. Average results of at least three independent experiments ± S.E. are presented. Qualitatively similar results were observed in AsPC-1 and BxPC-3 cells (presented as Supplemental Figure).

ROS production was measured after different treatment protocols employing two fluorescent dyes, DCFH-DA and HE, which enabled us to distinguish between different types of ROS (35). Following POH + Ad.mda-7 treatment HE-stained cells showed fluorescence indicating that superoxide anions/hydroxyl radicals are generated (Fig. 2B). However, no significant increase in fluorescent staining was detected with DCFH-DA, which mainly detects hydrogen peroxide/nitric oxide generation (data not shown). NAC or AP pre-treatment completely abolished ROS generation by the combinatorial treatment of POH and Ad.mda-7 (Fig. 2B). Similar to NAC, as shown in Fig. 1B, AP pre-treatment completely abrogated generation of MDA-7/IL-24 protein after POH and Ad.mda-7 treatment (Fig. 2C) confirming the critical role of XO in ROS generation, MDA-7/IL-24 protein production and apoptosis induction following the combinatorial treatment.

Involvement of XO was further confirmed by genetic approaches, employing siRNA targeting XO (Fig. 3). In this experiment, cells treated with scrambled siRNA were used as a control. Western blot assays confirmed an efficient down-regulation of XO protein following XO siRNA transfection, but not following control scrambled siRNA transfection (Fig. 3A). XO siRNA transfected PANC-1 cells, when treated with Ad.mda-7 and POH in combination, did not show MDA-7/IL-24 protein expression (Fig. 3A), decreased viability, ROS production or apoptosis induction (Fig. 3B). Western blot analysis also demonstrated that a combination treatment with Ad.mda-7 and POH down-regulated anti-apoptotic Bcl-2 and Bcl-xL proteins. These changes are eliminated by treatment with siRNA targeting XO (Fig. 3C). Similar results were obtained in AsPC-1 and BxPC-3 pancreatic carcinoma cell lines (data not shown).

Figure 3.

Figure 3

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Down-regulation of XO expression using siRNA transfection inhibits ROS production and subsequent apoptosis induction in PANC-1 cells following treatment with the combination of Ad.mda-7/IL-24 and POH. PANC-1 cells were transfected with scrambled control siRNA or XO siRNA and infected the next day with 100 pfu/cell of Ad.vec or Ad.mda-7/IL-24. POH or vehicle (DMSO) was added to the cells 2 h post-infection. The next day, cells were trypsinized and re-plated for different assays as described in Materials and Methods. MTT assays were performed on day 6. (A, C) Western blot analysis, (B) MTT assays (top panel, average for three different experiments ± S.E. are shown), ROS detection (middle panel) and apoptosis (bottom panel) assays were performed 24 h post-infection. Average results of at least three independent experiments for ROS and apoptosis detection are presented, and S.D. did not exceed 5%. Asterisks denote statistically significant values, p<0.001. Similar results were observed in AsPC-1 and BxPC-3 cells.

Single treatment of stable clones of pancreatic cancer cells expressing mda-7/IL-24 mRNA with POH induces growth suppression and apoptosis that correlates with ROS production and MDA-7/IL-24 protein production and involves XO

Further support for the efficacy of the combination of mda-7/IL-24 and POH in suppressing growth and induction of apoptosis in pancreatic cancer cells was provided by studies employing PANC-1 clones, PANC M8 and PANC M14, and MIA PaCa-2 clones, PaCa M1 and PaCa M2, stably expressing mda-7/IL-24 mRNA (Fig. 4). Despite stable expression of mda-7/IL-24 mRNA, these clones of pancreatic cancer cells do not express detectable MDA-7/IL-24 protein (16). In these mda-7/IL-24 mRNA expressing clones, treatment with only POH, at concentrations not affecting growth of parental PANC-1 or MIA PaCa-2 cells induced growth suppression and a loss of viability (Fig. 4A). A potential role of ROS production in mediating these effects in mda-7/IL-24 mRNA-expressing clones was confirmed by the fact that the antioxidant NAC inhibited apoptosis induction and ROS production in these POH-treated pancreatic tumor cells (Fig. 4B). Inhibition of XO by AP also abrogated POH-induced apoptosis induction, ROS generation and MDA-7/IL-24 protein production in PANC M8 cells (Fig. 4C). Similarly, XO siRNA also inhibited MDA-7/IL-24 protein expression, decreased viability, ROS production, apoptosis induction, and associated with apoptosis changed the levels of Bcl-2 and Bcl-xL proteins (Figs. 3 and 5).

Figure 4.

Figure 4

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Single treatment with POH induces growth suppression, MDA-7/IL-24 protein production and apoptosis in stable clones of pancreatic cancer cells expressing mda-7/IL-24 mRNA by a ROS-dependent mechanism. Cells were treated with POH alone or pretreated for 2 h with NAC and treated with POH. Cell viability was analyzed by MTT assays at day 6 (A). Annexin V binding assays (top panel) and ROS detection (bottom panel) assays (B) were done after 24 h. PANCm8, a stable clone of PANC-1 cells, was untreated or pretreated for 2 h with NAC (10 mM) or with AP (100 μM) and then treated with POH (200 μM) or vehicle (DMSO). Annexin V binding assays (left panel), ROS detection (right panel) assays (C) and Western blot analysis (D) were performed 24 h after POH treatment. Average results of at least three independent experiments for ROS and apoptosis detection are presented, and S.D. did not exceed 5%. Asterisks denote statistically significant values, p<0.001.

Figure 5.

Figure 5

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siRNA-mediated down-regulation of XO expression prevents ROS production and apoptosis induction upon POH treatment in PANCm8 cells. Cells were transfected with scrambled control or XO siRNA and POH or DMSO were added the next day. Western blot analysis (A, C) was performed 24 h post-treatment with POH. Viability of the cells was assessed by MTT assays (B, top) on day 6. ROS detection assays (B, middle) and Annexin V binding assays (B, bottom) were performed 24 h post-treatment with POH. Average results of at least three independent experiments for ROS and apoptosis detection are presented, and S.D. did not exceed 5%.

Combination treatment of pancreatic cancer cells with POH and Ad.mda-7 facilitates association of mda-7/IL-24 mRNA with polysomes

Translational regulation requires mRNA association with polysomes. We, therefore, tested whether treatment with POH and Ad.mda-7 facilitates association of mda-7/IL-24 mRNA with polysomes resulting in the generation of MDA-7/IL-24 protein. AsPC-1 pancreatic carcinoma cells were infected with Ad.mda-7 (50 pfu/cell) and then treated with vehicle (DMSO) or POH (200 μM). Polysomal fractions were isolated from the cell lysates and analyzed by Northern blotting using an mda-7/IL-24 cDNA probe. We confirmed that in the presence of POH, mda-7/IL-24 mRNA is associated with polysomes to a significantly greater extent when compared to vehicle treatment (Fig. 6A).

Figure 6.

Figure 6

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POH treatment facilitates mda-7/IL-24 mRNA association with polysomes and a hypothetical model of POH plus Ad.mda-7 induction of apoptosis in pancreatic cancer cells. (A) AsPC-1 cells were infected with Ad.mda-7/IL-24 and treated with DMSO (vehicle) or POH (200 μM). Forty-eight h later, polysomal fractions were isolated and analyzed by Northern blot as described in Materials and Methods. The numbers represent fraction numbers with fractions 10 to 20 representing the polysomes. (B) Proposed model of POH plus Ad.mda-7 apoptosis-inducing activity in pancreatic carcinoma cells. Infection of Ad.mda-7 in pancreatic carcinoma cells results in mda-7/IL-24 mRNA, but no protein or inhibition of growth or induction of apoptosis. POH treatment of Ad.mda-7-infected pancreatic carcinoma cells results in mda-7/IL-24 mRNA association with polysomes and production of MDA-7/IL-24 protein. This combination treatment results in induction of XO and ROS, which mediates apoptosis.

Discussion

Despite aggressive therapeutic approaches, resistance of pancreatic cancer to established treatment regimens still constitutes a major problem in providing long-term survival benefit to these patients. In this investigation, we demonstrate a remarkable synergism between the action of a dietary agent, POH, and a gene therapy, mda-7/IL-24, in inhibiting pancreatic cancer growth in vitro. Our data demonstrates that non-toxic doses of POH sensitize resistant pancreatic carcinoma cells, but not normal immortal pancreatic mesenchymal cells, to mda-7/IL-24-mediated gene therapy. This novel strategy to selectively kill pancreatic carcinoma cells by mda-7/IL-24 remains equally effective for various types of pancreatic tumor cells including those harboring both wild type and mutant K-ras genotypes. We also elucidate a primary mechanism invoking this enhanced sensitivity to the combinatorial effect of Ad.mda-7/IL-24 + POH that involves elevated ROS production that reverses the ‘protein translational block' in mda-7/IL-24 mRNA conversion into protein by facilitating mRNA association with polysomes that ultimately culminates in apoptosis of pancreatic cancer cells. This combinatorial effect did not involve a change in K-ras protein levels. Interestingly, our results also demonstrated that generation of oxidative stress is required for this process as pretreatment of pancreatic carcinoma cells with antioxidants such as NAC blocked both MDA-7/IL-24 protein expression and apoptosis.

The precise mechanism by which POH can promote the production of ROS remains to be determined. Multiple sources of ROS production are activated in response to treatment with various chemoprevention and chemotherapeutic agents (36). However, in this investigation we demonstrate that XO serves as a major source of ROS production in this system. Employing a genetic approach using siRNA we show that abrogating XO protein levels in these cells reduces ROS production, MDA-7/IL-24 protein expression, apoptosis and associated with apoptosis changes in expression levels of anti-apoptotic Bcl-2 and Bcl-xL proteins. Similar results were obtained using a pharmacological approach in which pancreatic cancer cells were treated with a dose of AP, a small molecular weight inhibitor of XO, which functions as a specific scavenger of either superoxide or hydroxyl radicals (37). It is well established that XO is an important source of ROS, which generates superoxide and hydroxyl radicals in the cytoplasm (38). Our results also show that these ROS target mitochondria leading to its membrane depolarization, and a subsequent drop in membrane potential proceeded by down-regulation of the anti-apoptotic proteins Bcl-2 and Bcl-xL (Fig. 3). These results provide evidence that activation of XO by POH is a crucial step required for effective Ad.mda-7 gene therapy of pancreatic cancer. However, once MDA-7/IL-24 protein is generated in pancreatic cancer cells, the subsequent mechanism(s) of elimination of cancer cells may remain essentially identical to those observed in other cancer-types such as melanoma and prostate cancer (30, 32, 33, 39), i.e., direct apoptosis-inducing effects and indirect ‘bystander’ anti-tumor effects, including inhibition of angiogenesis and immune modulation.

It is well-known that treatments of pancreatic cancer cells with agents that increase generation of ROS induce oxidative stress, augment cell killing, and inhibit tumor growth (40-44). However, similar treatment regimens in normal control cells exhibit significantly less oxidative stress as well as associated physiological and pathophysiological responses. These results are substantiated by the observations in the present study where we did not observe comparable damage in immortalized control cells of different origins, including those of pancreas. This differential effect on cancer versus normal cells is widely reported for many other agents that only affect cancer cells sparing normal cell populations (45, 46). We hypothesize that POH at low doses induces short-term initial ROS production by XO, but this burst of ROS production is not sufficient by itself to induce apoptosis in pancreatic cancer cells. However, this initial release of ROS might be sufficient to abrogate the ‘protein translational block' thereby resulting in association of increased amounts of mda-7/IL-24 mRNA with polysomes and consequently MDA-7/IL-24 protein production. This latter event may then promote a feedback loop resulting in a more significant and intense release of ROS that triggers mitochondrial depolarization, mitochondrial potential drop and the apoptotic cascade (Fig. 6B). It is possible that treatment with POH could also inactivate the GSH anti-oxidant system, which is involved in scavenging of superoxides (47, 48). This possibility is supported by the protective effects observed with NAC and Tiron in this study, which might restore the GSH levels in combination treated cells. Additional studies are ongoing to experimentally validate this hypothesis.

In summary, we describe a novel approach of combining a dietary agent with gene therapy as a potent inhibitor of pancreatic cancer cell growth and survival. The safety of both POH and Ad.mda-7 has been confirmed by clinical trials and studies in animal models also reveal that this combination profoundly inhibits pancreatic cancer xenografts in nude mice (unpublished data). These exciting observations pave the way for future clinical evaluation of POH and Ad.mda-7 combination therapy in the near future to determine if this approach has efficacy in patients with pancreatic cancer.

Acknowledgements

The present study was supported in part by NIH grants R01 CA097318 and R01 CA098712, and the Samuel Waxman Cancer Research Foundation. We thank Dr. Xiuwei Tang for assistance with and suggestions relative to K-ras biochemical studies. PBF is the Thelma Neumeyer Corman Chair in Cancer Research and a SWCRF Investigator.

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