Walter RJ, et al. Newcastle Disease Virus LaSota Strain Kills Human Pancreatic Cancer Cells in Vitro with High Selectivity. JOP. J Pancreas (Online) 2012 Jan 10; 13(1):45-53.

ORIGINAL ARTICLE

 

JOP. J Pancreas (Online) 2012 Jan 10; 13(1):45-53.

 

 

Newcastle Disease Virus LaSota Strain Kills Human Pancreatic Cancer Cells in Vitro with High Selectivity

 

 

Robert J Walter, Bashar M Attar, Asad Rafiq, Sooraj Tejaswi, Megan Delimata

 

 

Division of Gastroenterology and Hepatology, Cook County - John H. Stroger Hospital. Chicago, IL, USA

 

 

ABSTRACT

Context Pancreatic cancer is highly resistant to treatment. Previously, we showed that Newcastle disease virus (NDV) strain 73-T was highly cytotoxic to a range of tumor types in vitro and in vivo but the effects of NDV on pancreatic tumors are unknown. We determined the cytotoxicity of the lentogenic LaSota strain of NDV (NDV-LS) toward 7 different human pancreatic tumor cell lines and 4 normal human cell lines (keratinocytes, fibroblasts, pancreatic ductal cells, and vascular endothelial cells). Methods Cytotoxicity assays used serially diluted NDV incubated for 96 hours post-infection. Cells were fixed, stained, and minimum cytotoxic plaque forming unit (PFU) doses were determined (n=10-24/cell line). Results Normal cells were killed only by high doses of NDV-LS. The cytotoxic doses for pancreatic ductal cells, fibroblasts, and vascular endothelial cells were 729, 626, and 1,217 plaque forming units, respectively. In contrast, most pancreatic cancer cells were killed by much lower doses. The doses for PL45, Panc 10.05, PANC-1, BxPC3, SU.86.86, Capan-1 and CFPAC-1 were 0.15, 0.41, 0.43, 0.55, 1.30, 17.1 and 153 plaque forming units, respectively. Conclusions Most pancreatic tumor cells were more than 700 times more sensitive to NDV-LS killing than normal cells. Such avirulent, lentogenic NDV strains may have therapeutic potential in the treatment of pancreatic cancers.

 

 

INTRODUCTION

 

Pancreatic cancer has proven to be highly resistant to treatment. At present, the 5-year survival after diagnosis of pancreatic cancer is very low, about 4%. Clearly, novel treatment methods are needed. In previous studies, we showed that the mesogenic Newcastle disease virus (NDV) strain 73-T was highly cytotoxic to a variety of tumor cells both in vitro and in vivo but caused relatively little damage to normal cells [1, 2, 3, 4, 5]. However, very little is known about the direct effects of virulent or avirulent NDV strains on human pancreatic tumors.

NDV is an enveloped negative-sense single-strand RNA virus in the family Paramyxoviridae and genus Rubulavirus [6]. Its genomic RNA contains 6 genes which encode 8 proteins [7]. It has been studied for many years due to its ability to kill a variety of types of human tumors with high potency and specificity. This selectivity is this thought to arise from the weak endogenous interferon response in tumor cells as compared to normal cells [8, 9], but NDV also acts as an immune stimulatory adjuvant in vivo. Although it can cause high morbidity and mortality in avian species, NDV has few harmful effects in humans except for self-limiting conjunctivitis and mild to moderate flu-like symptoms [9]. The virus also exhibits a low rate of spontaneous mutation, low levels of recombination or antigenic drift, and does not become integrated into host DNA [10, 11]. These features make NDV a particularly promising candidate for tumor therapy.

Native NDV strains have been employed in animals and in patients as anti-tumor agents in 3 different modalities: 1) injection of infectious virus; 2) administration of virus infected oncolysate; or 3) infected whole cell vaccines. For direct cytolysis, live native mesogenic or velogenic NDV is added to cell cultures or injected into subjects where it infects and replicates in tumor cells which subsequently undergo apoptosis and lysis. Lentogenic NDV strains such as LaSota (LS), Hitchner-B1, or Ulster are seldom used in this way because they are considered to be non-lytic and thus less likely to have direct cytolytic effects. Krishnamurthy et al. [12] showed that NDV-LS replicated efficiently in 4 different tumor cell types in vitro but did not describe any cytopathic effects. As a vaccine, lentogenic strains such as NDV-Ulster are often used as adjuvants to stimulate an immune response against tumor cells or antigens. This method does not require NDV replication in tumor cells or direct viral induced lysis of target cells [13, 14].

The binding of NDV to target cells does not seem to require a specific receptor, instead ubiquitous sialic acid moieties on the cell surface serve as binding sites. NDV cytotoxicity appears to hinge upon the formation of multinucleated syncytia [5]. NDV fusogenicity, both for virus entry into the cell and for syncytia formation, involves both the fusion (F) and hemagglutinin-neuraminidase (HN) viral transmembrane proteins [15, 16, 17]. However, NDV virulence in chicken, which is highly dependent on F protein primary structure, is not the only or even the main determinant of NDV fusogenicity and cytotoxicity in mammalian tumor cells. As a result, the range of viral strain infectivity in chicken (i.e., lentogenic, mesogenic, velogenic) may not be as relevant to direct oncolytic potential of NDV strains in human tumors [18].

The direct cytolytic effects of mesogenic and velogenic NDV strains (e.g., 73-T, Beaudette C, Italien, Roakin, MTH-68) have been reported previously [5, 14, 19, 20, 21, 22, 23] but such highly infectious strains are problematic in clinical use due to possible unintentional release of highly infectious virus into the environment. As a result, avirulent lentogenic strains may be better alternatives for future clinical use. We evaluated the direct cytotoxicity of the lentogenic LaSota (LS) strain of NDV toward normal human cell lines including primary keratinocytes (HEKn), fibroblasts (HuFbs), immortalized pancreatic ductal cells (HPDE), and vascular endothelial cells (HUVEC). We compared this to their cytolytic effects on several different human pancreatic tumor cell lines (PANC-1, PL45, Panc 10.05, CFPAC-1, Capan-1, SU.86.86 and BxPC3).

 

MATERIALS AND METHODS

 

Virus Preparation and Cell Lines

 

NDV LaSota strain was a kind gift from Dr. R Iorio (University of Massachusetts, Worcester, MA, USA). This stock was amplified by passage through 10-day-old chick embryos. Three to four days after inoculation with 10,000 plaque forming units (PFU) of NDV, allantoic fluid was removed from the eggs aseptically and centrifuged at 13,000 g for 10 min to remove debris. Supernatants were divided and stored frozen at -80°C until use.

NDV stock was quantified using plaque assays as described previously [1, 2] and by hemagglutination (HA) assays. For plaque assays, monolayers of spontaneously transformed embryonic chicken fibroblasts (UMNSAH/DF-1) were cultured in 48-well plates with DMEM plus 10% fetal calf serum until confluence was attained. Serial 5-fold or 10-fold dilutions of virus-containing stocks were added to monolayers and, after 4 days of incubation at 37°C in a 5% CO2 incubator, cell monolayers were fixed with 100% methanol and stained with 0.2% crystal violet [1, 2]. For time course studies of cytotoxicity, cells were incubated with virus for 1, 2, 3, 4, 5, or 6 days after which time monolayers were fixed and stained. One plaque forming unit (PFU) was defined as the amount of NDV required to kill all cells in a well containing confluent chicken fibroblasts after 4 days of incubation.

 

Hemagglutination (HA) Assay

 

The HA titer of NDV suspensions was determined by end point dilution of erythrocyte agglutination. Chicken erythrocytes (Rockland Biologicals, Gilbertsville, PA, USA) in Alsever’s solution were washed 3 times in PBS and resuspended at a concentration of 5x107 cells/mL. Briefly, PBS (25 µL) was added to duplicate sets of wells in 96-well round bottom microtiter plates and 1/10 diluted NDV-LS in allantoic fluid (25 µL) was placed into the first pair of wells and then diluted by 2-fold serial dilutions. Next, 25 µL of PBS were added to each well. Finally, 25 µL of RBCs were added, plates were incubated at either room temperature or 4°C for 60 min. Plates were then assessed for hemagglutination and photographed [24, 25].

 

Cell Lines and Culture Conditions

 

Spontaneously transformed embryonic chicken fibroblasts (UMNSAH/DF-1, ATCC, Manassas, VA, USA) were grown in DMEM plus 10% fetal calf serum (FCS). Normal primary human keratinocytes derived from preputial skin of neonatal males (HEKn; Clonetics, San Diego, CA, USA) were used at passages ranging from 4 to 15. They were grown in EpiLife with calcium, bovine pituitary extract, and EGF. Normal primary human fibroblasts derived from preputial skin of adolescent males (HuFb), were adapted to culture, used at passages ranging from 7 to 20, and grown in DMEM plus 10% FCS. Normal human vascular endothelial cells (HUVEC) were maintained in Medium 200 plus low serum growth supplement (LSGS; Invitrogen, Carlsbad, CA, USA) and used at early passages. The immortalized human pancreatic ductal cell line, HPDE6-E6E7-c7 (HPDE), was the kind gift of Dr. MS Tsao (Ontario Cancer Institute, Toronto, Canada). This cell line was originally derived from human pancreatic ductal epithelium that had been transfected in vitro with the E6 and E7 genes from human papilloma virus 16 effectively immortalizing the cell line. These cells were shown to be phenotypically and functionally very similar to normal pancreatic ductal epithelium and, in terms of gene expression, were also similar to normal pancreatic epithelium [26, 27].

PANC-1 and SU.86.86 pancreatic epithelial carcinoma, PL45 and CFPAC-1 pancreatic ductal adenocarcinoma, and BxPC3, Capan-1, and Panc 10.05 pancreatic adenocarcinoma were obtained from ATCC (Manassas, VA, USA). PANC-1 and PL45 were cultured in DMEM with 10% FCS. SU.86.86 and BxPC3 were grown in RPMI-1640 with 10% FCS. Panc 10.05 cells were grown in RPMI-1640 containing 1 mM pyruvate, 0.23 U/mL human insulin, and 15% FCS. CFPAC-1 and Capan-1, both of which express CFTR, the cystic fibrosis transmembrane regulator, were grown in Iscove DMEM with 10% or 20% FCS, respectively. All media contained 50 units/mL penicillin and 50 µg/mL streptomycin sulfate.

 

Cytotoxicity Assays

 

To determine the optimal duration for cytotoxicity assays using mammalian cells, the time course for the effect was evaluated. Cells were exposed to NDV-LS for 1 to 6 days and cytotoxicity was assessed each day as described below. Simple cytotoxicity assays were performed as described previously with minor variations [1]. Briefly, each cell line was plated into 48-well plates with fully supplemented media. When the cells had grown to confluence, medium was aspirated and DMEM was added to all wells. NDV was then added and serial 5-fold or 10-fold dilutions were performed. After allowing 60 min for virus adhesion at 37°C, media containing non-adherent virus was aspirated and replaced with fresh DMEM supplemented with antibiotics. After 1-6 days of incubation at 37°C in an atmosphere of 5% CO2 plus 95% air, culture medium was removed, cell monolayers were fixed with 100% methanol and stained with 0.2% crystal violet in 50% methanol.

Acetylated trypsin (2.5 µg/mL, final) was added to the culture medium for many experiments during the 4 day post-infection incubation period. Acetylated trypsin is stable for extended periods in culture media at physiological temperatures and is capable of activating any NDV-LS virus progeny that may be released from infected cells [25]. The concentration of acetylated trypsin was determined in preliminary experiments using a range of trypsin concentrations. The highest trypsin concentration at which no effects on culture morphology or cell survival occurred was chosen for all subsequent experiments (2.5 µg/mL, final; data not shown). Human keratinocytes were exquisitely sensitive to trypsin such that any trace of this enzyme in the media proved to be cytotoxic.

 

STATISTICS

 

For each experiment, the lowest virus dose that still resulted in the lysis of most or all cells in a given well was recorded. This ‘minimum cytotoxic dose’ was then used to characterize each cell line and virus strain. Between 10 and 24 repetitions (n) of these cytotoxicity determinations were performed for each cell type. Non-parametric statistics were applied. Wilcoxon matched-pairs tests were used to compare the effects of acetylated trypsin (AT) on cytotoxicity and Kruskal-Wallis ANOVA was used to evaluate the differences between CF and non-CF patient derived pancreatic cancer cell lines.

Due to the prevailing hypothesis that the presence of AT or some other protease is necessary for full activity or cytotoxicity of NDV, one-tailed tests were used for evaluating the effects of AT while two-tailed tests were chosen for the other analyses. Descriptive statistics (mean±SEM) were obtained and groups were compared by using the Kruskal-Wallis ANOVA with Dunn’s multiple comparison tests using Prism 3.03 software (GraphPad, San Diego, CA, USA). P values less than 0.05 were considered statistically significant.

 

RESULTS

 

Hemagglutination Assays and PFU Determination in Diploid Chicken Fibroblasts

 

Hemagglutination assays showed that the NDV-LS stock suspension contained 102,400 to 204,800 HA units/mL (Figure 1). Confluent chicken fibroblasts exposed to NDV-LS serially diluted by factors of 5 or 10 and then incubated for 4 days showed complete cytotoxicity even at extreme dilutions of stock virus suspension. The greatest dilution at which complete killing occurred was used to establish PFU equal to 1 (n=22). This dilution corresponded to 1.9 to 3.9 E-3 HA units of NDV-LS stock.

 

 

Figure 1. Photograph of a hemagglutinin assay plate for NDV-LS. The HA titer was determined as described in the Methods section. Two-fold serial dilutions of NDV-LS stock were added to the wells in duplicate in a microtiter plate. The greatest dilution in which erythrocyte agglutination still occurred was either 1/2,560 or 1/5,120. This represents 102,400-204,800 HA units/mL.

 

 

Time Course of NDV-LS Cytotoxicity

 

Time course studies (n=6) showed that most cell killing occurred by day 4 of exposure to NDV-LS for SU.86.86 and BxPC3 cells (Figure 2). Other cell lines responded similarly (data not shown), so 4 days post-exposure was used as the terminus for all subsequent experiments.

 

 

Figure 2. Time course for cytotoxicity caused by NDV-LS in SU.86.86 and BxPC3 pancreatic cancer cells. Most cell killing induced by NDV-LS occurred by day 4 post-infection in both cell lines.

 

 

Acetylated Trypsin Increased NDV-LS Cytotoxicity

 

For most pancreatic cancer cell lines, the inclusion of acetylated trypsin in culture media decreased the amount of NDV required for cytotoxicity by factors of 1.3- to 5.6-fold although extensive killing occurred in the absence of added acetylated trypsin (Figure 3ab). On average, with acetylated trypsin the minimum cytotoxic dose of NDV-LS in all pancreatic cancer lines studied was decreased by half (P<0.001, Wilcoxon matched-pairs test). Similarly, the cytotoxic dose with or without acetylated trypsin in normal control lines was decreased by 64% (P<0.001, Wilcoxon matched-pairs test).

 

 

Figure 3. The minimum cytotoxic dose of NDV-LS required for normal human cells (a.) or pancreatic cancer cells (b.) cultured with (+) or without (-) acetylated trypsin (AT). The bars represent means±SEM. With acetylated trypsin, the minimum cytotoxic dose of NDV-LS in all pancreatic cancer lines studied was decreased by half (P<0.001, Wilcoxon matched-pairs test). Similarly, the cytotoxic dose with or without acetylated trypsin in normal control lines was decreased by 64% (P<0.001, Wilcoxon matched-pairs test). Without acetylated trypsin, HPDE or HuFb or HPDE or HEKn cells (n=10, 15, 11, 10, respectively) were significantly less sensitive to NDV-LS cytotoxicity as compared to Panc 10.05, PL45, PANC-1, SU.86.86, or BxPC3 cells (P<0.01, Kruskal-Wallis with Dunn’s multiple comparison tests; n=16, 14, 24, 15, 12, respectively).

 

 

NDV-LS Was Cytotoxic for Normal Diploid Human Cells Only at High Doses

 

NDV-LS was cytotoxic toward normal human cells (Figures 4 and 5) but only at relatively high PFU levels (with acetylated trypsin: 252 to 729; without acetylated trypsin: 1,058 to 2,173). Without acetylated trypsin, normal diploid HuFb and HEKn cultures required very high levels of NDV-LS (mean PFU±SEM: 2,173±359 and 1,771±439, respectively) to induce complete cytotoxicity. Without acetylated trypsin, HPDE and HUVEC cells also required high NDV-LS doses (PFU±SEM: 1,058±194 and 1,217±209, respectively) for cytotoxicity. With acetylated trypsin, complete cytotoxicity was seen in HPDE, HuFb, and HUVEC cells with 729±255, 626±143, and 252±71 PFU, respectively. UV-irradiated NDV-LS was cytotoxic for normal and pancreatic cancer lines only at PFU levels exceeding 50,000 (Figure 5).

 

 

Figure 4. Scatterplot of NDV-LS cytotoxicity in normal control human cell lines in the presence of acetylated trypsin (n ranged from 10 to 19). The mean minimum cytotoxic doses for each cell type were high (ranging from 252 to 729) and are shown as horizontal lines here. HEKn cells were killed by acetylated trypsin alone so NDV cytotoxicity could not be determined in its presence.

 

 

Figure 5. Photographs of multiwell plates stained with crystal violet showing NDV-LS cytotoxicity in normal human cell lines. In duplicate rows of wells, unstained wells represent those in which total cytotoxicity and lysis had occurred. The far left well in each row was a negative control that received no NDV. Asterisks (*) mark the wells where the minimum cytotoxic dose of NDV was seen for HuFbs, HEKn, HUVEC, HPDE cultures. Acetylated trypsin had been present in all wells shown except those containing HEKn cells. Even at 50,000 PFU, UV-inactivated NDV-LS had little effect on HUVEC cell viability.

 

 

NDV-LS Was Cytotoxic for Most Pancreatic Tumor Cell Lines at Low Doses

 

NDV-LS was cytotoxic toward most pancreatic cancer cell lines (Figures 6 and 7) at much lower PFU levels (with acetylated trypsin, ranging from 0.15 to 1.3) than those seen for normal cells. With acetylated trypsin, means±SEM for minimum cytotoxic NDV dose with Panc 10.05, PL45, PANC-1, SU86.86, and BxPC3 were 0.41±0.13, 0.15±0.03, 0.43±0.13, 1.30±0.61, and 0.55±0.24, respectively. Without acetylated trypsin, these values were somewhat higher at 0.70±0.16, 0.19±0.04, 0.74±0.16, 3.4±0.94, and 3.1±1.0, respectively.

With acetylated trypsin, HPDE or HuFb cells were significantly less sensitive (P<0.001, Kruskal-Wallis with Dunn’s multiple comparison tests) to killing than were Panc 10.05, PL45, PANC-1, SU.86.86, or BxPC3 cells. HUVEC cells were also significantly less sensitive (P<0.01 Kruskal-Wallis with Dunn’s multiple comparison tests) to killing than were Panc 10.05, PANC-1, or PL45 cells. Without acetylated trypsin, HPDE or HuFb or HPDE or HEKn cells were significantly less sensitive to NDV-LS cytotoxicity as compared to Panc 10.05, PL45, PANC-1, SU.86.86, or BxPC3 cells (P<0.01, Kruskal-Wallis with Dunn’s multiple comparison tests).

Pancreatic cancer cell lines derived from cystic fibrosis patients (Capan-1 and CFPAC-1) were noticeably less sensitive to killing by NDV-LS. The mean minimum PFU±SEM for these cell types was 17±7 and 153±24, respectively with acetylated trypsin and 15±3 and 422±111, respectively without acetylated trypsin. Tumor lines derived from patients with cystic fibrosis were significantly less sensitive (P<0.001, Kruskal-Wallis test) to killing by NDV-LS than were other pancreatic tumor lines either with or without acetylated trypsin.

 

 

Figure 6. Scatterplot of NDV-LS cytotoxicity in human pancreatic cancer cell lines in the presence of acetylated trypsin (n=13 to 17). The mean minimum cytotoxic doses for each cell type were low (ranging from 0.15 to 152) and are shown as horizontal lines here. Tumor lines derived from patients with cystic fibrosis (CFPAC-1 and Capan-1) were significantly less sensitive (P<0.001, Kruskal-Wallis test) to killing by NDV-LS than were other pancreatic tumor lines either with or without acetylated trypsin. With acetylated trypsin, HPDE or HuFb cells were significantly less sensitive (P<0.001, Kruskal-Wallis with Dunn’s multiple comparison tests) to killing than were Panc 10.05, PL45, PANC-1, SU.86.86, or BxPC3 cells. HUVEC cells were also significantly less sensitive (P<0.01 Kruskal-Wallis with Dunn’s multiple comparison tests) to killing than were Panc 10.05, PANC-1, or PL45 cells. Without acetylated trypsin, HPDE or HuFb or HPDE or HEKn cells were significantly less sensitive to NDV-LS cytotoxicity as compared to Panc 10.05, PL45, PANC-1, SU.86.86, or BxPC3 cells (P<0.01, Kruskal-Wallis with Dunn’s multiple comparison tests).

 

 

Figure 7. Photographs of multiwell plates stained with crystal violet showing NDV-LS cytotoxicity in pancreatic cancer cell lines. Unstained wells represent those in which total cytotoxicity and lysis had occurred. The far left well in each row was a negative control that received no NDV. Asterisks (*) mark the wells where the minimum cytotoxic dose of NDV was seen for PANC-1, BxPC3, SU.86.86, PL45, Capan-1, Panc 10.05, and CFPAC-1 cultures. Acetylated trypsin had been present in all wells shown. At 50,000 PFU, UV-inactivated NDV-LS had little effect on PL45 cell viability.

 

 

DISCUSSION

 

Normal human cells were killed only by relatively high doses of NDV-LS. This was seen in normal diploid human keratinocytes, fibroblasts, and vascular endothelial cells as well as pancreatic ductal epithelial cells. In contrast, all non-CF pancreatic cancer cell types studied here were killed by much lower NDV doses ranging from 194- to 11,437-fold less virus. In contrast, pancreatic tumor cell lines derived from patients with CF showed significantly elevated resistance to NDV-LS cytotoxicity (ranging from 1.6- to 148-fold less virus than normal cells) compared to other pancreatic cancer cell lines. This resistance may reflect membrane related changes due to mutations in the CF transmembrane conductance regulator gene or alterations in the intracellular ionic milieu induced by the regulator. This resistance to NDV killing exhibited by CF patient-derived tumors may be an important consideration in future clinical trials involving NDV.

UV-inactivated NDV was mildly cytotoxic for tumor cells, but only at very high doses (PFU greater than 50,000). A similar effect has been described previously for 73-T and other strains of NDV [1, 2, 3, 5, 28]. UV-inactivated NDV is still capable of binding to cells and, when present in large amounts, will promote cell fusion and the formation of multinucleated cells which subsequently die. Exogenously added acetylated trypsin increased the cytotoxicity of NDV-LS by factors of 1.3- to 5.6-fold, but very high levels of cytotoxicity for pancreatic tumor cells were seen in its absence. This suggests that NDV-LS may replicate to yield infectious virus in the absence of acetylated trypsin or that the fully activated form of the virus is not necessary to achieve potent cytotoxicity in pancreatic tumors. Viral activation could be accomplished by endogenous pancreatic enzymes that cleave the viral F protein to generate the highly infectious form of the F protein. Finally, the rate of cell proliferation or doubling time for each cell line was unrelated to the amount of NDV-LS required for cytotoxicity (Figure 8).

 

 

Figure 8. Minimum cytotoxic dose of NDV-LS for each pancreatic cancer cell type plotted against the doubling time for each cell type. There is no relationship apparent between the doubling time and the sensitivity of these cell types to NDV killing.

 

 

Several NDV strains (MTH-68, 73-T, Ulster, PV701, HUJ) have been shown to be cytotoxic for a range of classes of human tumors and, in clinical studies, some have shown promise for treating a variety of tumor types. Strain MTH-68 has been shown to have beneficial effects in glioma, astrocytoma, and various advanced cancers [29, 30, 31], 73-T in sarcomas, carcinomas, and melanomas [1, 2, 3, 4, 5, 14, 20, 32, 33, 34]; PV701 in various advanced solid tumors [9, 35, 36, 37], HUJ in glioblastoma and lung tumors [38, 39, 40], and Ulster strain in melanoma, breast, and gastrointestinal tumors [41, 42, 43, 44]. Some of these NDV strains have been used primarily as immune adjuvants (Ulster by Schirrmacher et al. [41, 42, 43, 45]; 73-T by Cassel et al. [33, 34, 46]; MTH-68 by Csatary et al. [30, 47]). On the other hand, some of these strains can exert effects via direct cytolytic activity toward tumor cells (e.g., 73-T [1, 2, 3, 5, 19, 20, 48]; PV701 [9, 35, 36]; HUJ [39, 40]; MTH-68 [49, 50, 51]).

However, the susceptibility of pancreatic tumors or tumor cells to NDV has been studied only to a very limited extent. In a phase I clinical trial using PV701, Pecora et al. [35] studied 9 primary pancreatic carcinoma patients of which 1 or 2 showed measurable tumor size reductions. Zamarin et al. [52] showed that the lentogenic Hitchner-B1 strain of NDV could cause a 50% decrease in cell survival in PANC-1 cells but no decrease in MIA PaCa-2 cell survival after 3 days in vitro. In 2007, Fabian et al. [53] showed that the mesogenic NDV strain MTH-68/H was highly cytotoxic for PANC-1 cells. Schirrmacher et al. [13] also reported that Ulster strain could infect and replicate in 2 established human pancreatic cancer cell lines and in more than 10 primary tumor explants in vitro and Jarahian et al. [54] found that PANC-1 cells infected with NDV-Ulster were killed more efficiently by NK cells. In 2003, Liang et al. [55] reported disease stabilization in one patient with pancreatic head cancer treated with NDV-LaSota IV strain as a vaccine. These reports offer some optimism regarding the potential for NDV efficacy in the treatment of pancreatic neoplasms.

Lentogenic NDV strains such as NDV-LS have been studied mainly as immune adjuvants in infected tumor cell vaccines or oncolysates rather than for any direct tumor cytotoxicity [41, 42, 43, 44, 45, 55, 56, 57, 58]. It is often assumed that these lentogenic strains would have poor tumor cytotoxicity due to their low infectivity and lysogenicity in chickens. This low infectivity is determined by the primary amino acid sequence of the F protein of NDV which contains few basic amino acid residues in the critical 395-403 positions. Such lentogenic strains must be activated by exogenous trypsin-like proteases such as those found in allantoic fluid or in the gastrointestinal tract to obtain infectious virus in chickens [59, 60]. Thus, activated NDV-LS is expected to undergo only a single round of infection in human tumor cells unless appropriate protease activation of progeny virus particles occurs. If this had been to occur, increased virus infectivity of otherwise weakly infectious virus might be elicited.

We have shown that even lentogenic NDV-LS, which is poorly infectious in chicken, is highly cytolytic for human pancreatic tumor cells and highly specific for tumor versus normal human cells. Further, the inclusion of acetylated trypsin together with the NDV-LS resulted in a modest but significant increase in cytotoxicity suggesting that NDV-LS is highly cytotoxic to pancreatic tumor cells even in the absence of F protein activation by exogenous trypsin. In chicken, NDV may be viscerotropic or neurotropic depending on the strain [61, 62, 63]. Most lentogenic strains are viscerotropic having a marked propensity to infect enteric organs. The Hitchner-B1 and LaSota strains are often used as bird vaccines to protect against velogenic strains of NDV and typically proliferate most efficiently in the respiratory and gastrointestinal tracts [16, 64]. This may be related to the presence of proteases in these locations that cleave the NDV fusion protein thereby increasing the infectivity of the virus [65, 66]. This predilection for the GI tract, the potential for NDV-LS activation there, and its high level of differential cytotoxicity toward pancreatic tumor cells in vitro may make this lentogenic strain of NDV particularly useful in the treatment of pancreatic cancer.

 

 

Received November 12th, 2011 - Accepted December 12th, 2011

 

Key words La Sota vaccine; Neoplasms; Newcastle disease virus; Newcastle disease virus vaccine MTH-68-H; Oncolytic Virotherapy; Oncolytic Viruses; Pancreatic Neoplasms; Serine Proteases; Trypsin; V protein, Paramyxovirus; Vaccines; Viral Fusion Proteins

 

Abbreviations AT acetylated trypsin; FCS: fetal calf serum; HA: hemagglutinin; HEKn: human keratinocyte; HN: hemagglutinin-neuraminidase; HPDE: human pancreatic ductal epithelial cell; HuFb: human fibroblast; HUVEC: human vascular endothelial cell; NDV: Newcastle disease virus; NDV-LS, NDV-La Sota strain; PFU, plaque forming unit

 

Acknowledgments The authors thank Drs. M Peeples and R Iorio for helpful discussions and for initial stock samples of NDV-LS strain. The authors also wish to thank Dr. MS Tsao for providing the immortalized human pancreatic ductal cells, HPDE6-E6E7-c7. Parts of this study were published previously in abstract form in Gastroenterology 2010 138:S451-3 [67, 68, 69]

 

Financial support This study was supported by the Division of Gastroenterology and Hepatology, Cook County - John H Stroger Hospital

 

Conflict of interest The authors have no potential conflict of interest

 

Correspondence
Robert J Walter
Division of Gastroenterology and Hepatology, Rm 1413
John Stroger Hospital of Cook County
1900 West Polk St.
Chicago, IL 60612
USA
Phone: +1-312.864.0578
Fax: +1-312.864.9624
E-mail: rwalter@rush.edu

 

 

References

1.    Lorence RM, Katubig BB, Reichard KW, Reyes HM, Phuangsab A, Sassetti MD, Walter RJ, Peeples ME. Complete regression of human fibrosarcoma xenografts after local Newcastle disease virus therapy. Cancer Res 1994; 54:6017-6021. [PMID 7954437]

2.    Lorence RM, Reichard KW, Katubig BB, Reyes HM, Phuangsab A, Mitchell BR, Cascino CJ, Walter RJ, Peeples ME. Complete regression of human neuroblastoma xenografts in athymic mice after local Newcastle disease virus therapy. J Natl Cancer Inst 1994; 86:1228-1233. [PMID 8040891]

3.    Phuangsab A, Lorence RM, Reichard KW, Peeples ME, Walter RJ. Newcastle disease virus therapy of human tumor xenografts: antitumor effects of local or systemic administration. Cancer Lett 2001; 172:27-36. [PMID 11595126]

4.    Reichard KW, Lorence RM, Katubig BB, Peeples ME, Reyes HM. Retinoic acid enhances killing of neuroblastoma cells by Newcastle disease virus. J Pediatr Surg 1993; 28:1221-1225. [PMID 8263678]

5.    Reichard KW, Lorence RM, Cascino CJ, Peeples ME, Walter RJ, Fernando MB, Reyes HM, Greager JA. Newcastle disease virus selectively kills human tumor cells. J Surg Res 1992; 52:448-453. [PMID 1619912]

6.    de Leeuw OS, Koch G, Hartog L, Ravenshorst N, Peeters BP. Virulence of Newcastle disease virus is determined by the cleavage site of the fusion protein and by both the stem region and globular head of the haemagglutinin-neuraminidase protein. J Gen Virol 2005; 86:1759-1769. [PMID 15914855]

7.    Steward M, Vipond IB, Millar NS, Emmerson PT. RNA editing in Newcastle disease virus. J Gen Virol 1993; 74 ( Pt 12):2539-2547. [PMID 8277263]

8.    Stojdl DF, Lichty B, Knowles S, Marius R, Atkins H, Sonenberg N, Bell JC. Exploiting tumor-specific defects in the interferon pathway with a previously unknown oncolytic virus. Nat Med 2000; 6:821-825. [PMID 10888934]

9.    Lorence RM, Roberts MS, O'Neil JD, Groene WS, Miller JA, Mueller SN, Bamat MK. Phase 1 clinical experience using intravenous administration of PV701, an oncolytic Newcastle disease virus. Curr Cancer Drug Targets 2007; 7:157-167. [PMID 17346107]

10. Toyoda T, Sakaguchi T, Hirota H, Gotoh B, Kuma K, Miyata T, Nagai Y. Newcastle disease virus evolution. II. Lack of gene recombination in generating virulent and avirulent strains. Virology 1989; 169:273-282. [PMID 2705298]

11. Sakaguchi T, Toyoda T, Gotoh B, Inocencio NM, Kuma K, Miyata T, Nagai Y. Newcastle disease virus evolution. I. Multiple lineages defined by sequence variability of the hemagglutinin-neuraminidase gene. Virology 1989; 169:260-272. [PMID 2705297]

12. Krishnamurthy S, Takimoto T, Scroggs RA, Portner A. Differentially regulated interferon response determines the outcome of Newcastle disease virus infection in normal and tumor cell lines. J Virol 2006; 80:5145-5155. [PMID 16698995]

13. Schirrmacher V, Haas C, Bonifer R, Ahlert T, Gerhards R, Ertel C. Human tumor cell modification by virus infection: an efficient and safe way to produce cancer vaccine with pleiotropic immune stimulatory properties when using Newcastle disease virus. Gene Ther 1999; 6:63-73. [PMID 10341877]

14. Schirrmacher V, Griesbach A, Ahlert T. Antitumor effects of Newcastle Disease Virus in vivo: local versus systemic effects. Int J Oncol 2001; 18:945-952. [PMID 11295039]

15. Sergel T, McGinnes LW, Morrison TG. The fusion promotion activity of the NDV HN protein does not correlate with neuraminidase activity. Virology 1993; 196:831-834. [PMID 8372451]

16. Sergel T, McGinnes LW, Peeples ME, Morrison TG. The attachment function of the Newcastle disease virus hemagglutinin-neuraminidase protein can be separated from fusion promotion by mutation. Virology 1993; 193:717-726. [PMID 8384752]

17. Sergel TA, McGinnes LW, Morrison TG. A single amino acid change in the Newcastle disease virus fusion protein alters the requirement for HN protein in fusion. J Virol 2000; 74:5101-5107. [PMID 10799584]

18. Hanson RP, Brandly CA. Identification of vaccine strains of Newcastle disease virus. Science 1955; 122:156-157. [PMID 14396376]

19. Lorence RM, Rood PA, Kelley KW. Newcastle disease virus as an antineoplastic agent: induction of tumor necrosis factor-alpha and augmentation of its cytotoxicity. J Natl Cancer Inst 1988; 80:1305-1312. [PMID 2459402]

20. Cassel WA, Garrett RE. Newcastle disease virus as an antineoplastic agent. Cancer 1965; 18:863-868. [PMID 14308233]

21. Bar-Eli N, Giloh H, Schlesinger M, Zakay-Rones Z. Preferential cytotoxic effect of Newcastle disease virus on lymphoma cells. J Cancer Res Clin Oncol 1996; 122:409-415. [PMID 8690751]

22. Ahlert T, Schirrmacher V. Isolation of a human melanoma adapted Newcastle disease virus mutant with highly selective replication patterns. Cancer Res 1990; 50:5962-5968. [PMID 2203523]

23. Elankumaran S, Chavan V, Qiao D, Shobana R, Moorkanat G, Biswas M, Samal SK. Type I interferon-sensitive recombinant Newcastle disease virus for oncolytic virotherapy. J Virol 2010; 84:3835-3844. [PMID 20147405]

24. McGinnes LW, Pantua H, Reitter J, Morrison TG. Newcastle disease virus: propagation, quantification, and storage. Curr Protoc Microbiol 2006; Chapter 15:Unit. [PMID 18770579]

25. McGinnes LW, Pantua H, Laliberte JP, Gravel KA, Jain S, Morrison TG. Assembly and biological and immunological properties of Newcastle disease virus-like particles. J Virol 2010; 84:4513-4523. [PMID 20181713]

26. Furukawa T, Duguid WP, Rosenberg L, Viallet J, Galloway DA, Tsao MS. Long-term culture and immortalization of epithelial cells from normal adult human pancreatic ducts transfected by the E6E7 gene of human papilloma virus 16. Am J Pathol 1996; 148:1763-1770. [PMID 8669463]

27. Ouyang H, Mou L, Luk C, Liu N, Karaskova J, Squire J, Tsao MS. Immortal human pancreatic duct epithelial cell lines with near normal genotype and phenotype. Am J Pathol 2000; 157:1623-1631. [PMID 11073822]

28. Schirrmacher V, Jurianz K, Roth C, Griesbach A, Bonifer R, Zawatzky R. Tumor stimulator cell modification by infection with Newcastle Disease Virus: analysis of effects and mechanism in MLTC-CML cultures. Int J Oncol 1999; 14:205-215. [PMID 9917494]

29. Csatary LK, Eckhardt S, Bukosza I, Czegledi F, Fenyvesi C, Gergely P, Bodey B, Csatary CM. Attenuated veterinary virus vaccine for the treatment of cancer. Cancer Detect Prev 1993; 17:619-627. [PMID 8275514]

30. Csatary LK, Bakacs T. Use of Newcastle disease virus vaccine (MTH-68/H) in a patient with high-grade glioblastoma. JAMA 1999; 281:1588-1589. [PMID 10235150]

31. Csatary LK, Moss RW, Beuth J, Torocsik B, Szeberenyi J, Bakacs T. Beneficial treatment of patients with advanced cancer using a Newcastle disease virus vaccine (MTH-68/H). Anticancer Res 1999; 19:635-638. [PMID 10216468]

32. Zorn U, Dallmann I, Grosse J, Kirchner H, Poliwoda H, Atzpodien J. Induction of cytokines and cytotoxicity against tumor cells by Newcastle disease virus. Cancer Biother 1994; 9:225-235. [PMID 7820184]

33. Murray DR, Cassel WA, Torbin AH, Olkowski ZL, Moore ME. Viral oncolysate in the management of malignant melanoma. II. Clinical studies. Cancer 1977; 40:680-686. [PMID 196740]

34. Cassel WA, Murray DR. A ten-year follow-up on stage II malignant melanoma patients treated postsurgically with Newcastle disease virus oncolysate. Med Oncol Tumor Pharmacother 1992; 9:169-171. [PMID 1342060]

35. Pecora AL, Rizvi N, Cohen GI, Meropol NJ, Sterman D, Marshall JL, Goldberg S, Gross P, O'Neil JD, Groene WS, Roberts MS, Rabin H, Bamat MK, Lorence RM. Phase I trial of intravenous administration of PV701, an oncolytic virus, in patients with advanced solid cancers. J Clin Oncol 2002; 20:2251-2266. [PMID 11980996]

36. Lorence RM, Pecora AL, Major PP, Hotte SJ, Laurie SA, Roberts MS, Groene WS, Bamat MK. Overview of phase I studies of intravenous administration of PV701, an oncolytic virus. Curr Opin Mol Ther 2003; 5:618-624. [PMID 14755888]

37. Roberts MS, Lorence RM, Groene WS, Bamat MK. Naturally oncolytic viruses. Curr Opin Mol Ther 2006; 8:314-321. [PMID 16955694]

38. Freeman AI, Zakay-Rones Z, Gomori JM, Linetsky E, Rasooly L, Greenbaum E, et al. Phase I/II trial of intravenous NDV-HUJ oncolytic virus in recurrent glioblastoma multiforme. Mol Ther 2006; 13:221-228. [PMID 16257582]

39. Yaacov B, Eliahoo E, Lazar I, Ben-Shlomo M, Greenbaum I, Panet A, Zakay-Rones Z. Selective oncolytic effect of an attenuated Newcastle disease virus (NDV-HUJ) in lung tumors. Cancer Gene Ther 2008; 15:795-807. [PMID 18535620]

40. Lazar I, Yaacov B, Shiloach T, Eliahoo E, Kadouri L, Lotem M, et al. The oncolytic activity of Newcastle disease virus NDV-HUJ on chemoresistant primary melanoma cells is dependent on the proapoptotic activity of the inhibitor of apoptosis protein Livin. J Virol 2010; 84:639-646. [PMID 19864394]

41. Liebrich W, Schlag P, Manasterski M, Lehner B, Stohr M, Moller P, Schirrmacher V. In vitro and clinical characterisation of a Newcastle disease virus-modified autologous tumour cell vaccine for treatment of colorectal cancer patients. Eur J Cancer 1991; 27:703-710. [PMID 1829908]

42. Ockert D, Schirrmacher V, Beck N, Stoelben E, Ahlert T, Flechtenmacher J, et al. Newcastle disease virus-infected intact autologous tumor cell vaccine for adjuvant active specific immunotherapy of resected colorectal carcinoma. Clin Cancer Res 1996; 2:21-28. [PMID 9816085]

43. Ahlert T, Sauerbrei W, Bastert G, Ruhland S, Bartik B, Simiantonaki N, et al. Tumor-cell number and viability as quality and efficacy parameters of autologous virus-modified cancer vaccines in patients with breast or ovarian cancer. J Clin Oncol 1997; 15:1354-1366. [PMID 9193327]

44. Pomer S, Schirrmacher V, Thiele R, Lohrke H, Brkovic D, Staehler G. Tumor response and 4 year survival-data of patients with advanced renal-cell carcinoma treated with autologous tumor vaccine and subcutaneous R-IL-2 and IFN-alpha(2b). Int J Oncol 1995; 6:947-954. [PMID 21556623]

45. Steiner HH, Bonsanto MM, Beckhove P, Brysch M, Geletneky K, Ahmadi R, et al. Antitumor vaccination of patients with glioblastoma multiforme: a pilot study to assess feasibility, safety, and clinical benefit. J Clin Oncol 2004; 22:4272-4281. [PMID 15452186]

46. Cassel WA, Murray DR, Phillips HS. A phase II study on the postsurgical management of Stage II malignant melanoma with a Newcastle disease virus oncolysate. Cancer 1983; 52:856-860. [PMID 6871827]

47. Csatary LK, Telegdy L, Gergely P, Bodey B, Bakacs T. Preliminary report of a controlled trial of MTH-68/B virus vaccine treatment in acute B and C hepatitis: a phase II study. Anticancer Res 1998; 18:1279-1282. [PMID 9615801]

48. Cassel WA, Garrett RE. Tumor immunity after viral oncolysis. J Bacteriol 1966; 92:792. [PMID 4288498]

49. Fabian Z, Torocsik B, Kiss K, Csatary LK, Bodey B, Tigyi J, et al. Induction of apoptosis by a Newcastle disease virus vaccine (MTH-68/H) in PC12 rat phaeochromocytoma cells. Anticancer Res 2001; 21:125-135. [PMID 11299726]

50. Csatary LK, Gosztonyi G, Szeberenyi J, Fabian Z, Liszka V, Bodey B, Csatary CM. MTH-68/H oncolytic viral treatment in human high-grade gliomas. J Neurooncol 2004; 67:83-93. [PMID 15072452]

51. Hrabak A, Csuka I, Bajor T, Csatary LK. The cytotoxic anti-tumor effect of MTH-68/H, a live attenuated Newcastle disease virus is mediated by the induction of nitric oxide synthesis in rat peritoneal macrophages in vitro. Cancer Lett 2006; 231:279-289. [PMID 16399229]

52. Zamarin D, Martinez-Sobrido L, Kelly K, Mansour M, Sheng G, Vigil A, et al. Enhancement of oncolytic properties of recombinant Newcastle disease virus through antagonism of cellular innate immune responses. Mol Ther 2009; 17:697-706. [PMID 19209145]

53. Fabian Z, Csatary CM, Szeberenyi J, Csatary LK. p53-independent endoplasmic reticulum stress-mediated cytotoxicity of a Newcastle disease virus strain in tumor cell lines. J Virol 2007; 81:2817-2830. [PMID 17215292]

54. Jarahian M, Watzl C, Fournier P, Arnold A, Djandji D, Zahedi S, et al. Activation of natural killer cells by Newcastle disease virus hemagglutinin-neuraminidase. J Virol 2009; 83:8108-8121. [PMID 19515783]

55. Liang W, Wang H, Sun TM, Yao WQ, Chen LL, Jin Y, et al. Application of autologous tumor cell vaccine and NDV vaccine in treatment of tumors of digestive tract. World J Gastroenterol 2003; 9:495-498. [PMID 12632504]

56. Bohle W, Schlag P, Liebrich W, Hohenberger P, Manasterski M, Moller P, Schirrmacher V. Postoperative active specific immunization in colorectal cancer patients with virus-modified autologous tumor-cell vaccine. First clinical results with tumor-cell vaccines modified with live but avirulent Newcastle disease virus. Cancer 1990; 66:1517-1523. [PMID 2208003]

57. Schlag P, Manasterski M, Gerneth T, Hohenberger P, Dueck M, Herfarth C, et al. Active specific immunotherapy with Newcastle-disease-virus-modified autologous tumor cells following resection of liver metastases in colorectal cancer. First evaluation of clinical response of a phase II-trial. Cancer Immunol Immunother 1992; 35:325-330. [PMID 1394336]

58. Schirrmacher V, Fournier P. Newcastle disease virus: a promising vector for viral therapy, immune therapy, and gene therapy of cancer. Methods Mol Biol 2009; 542:565-605. [PMID 19565923]

59. Gotoh B, Ogasawara T, Toyoda T, Inocencio NM, Hamaguchi M, Nagai Y. An endoprotease homologous to the blood clotting factor X as a determinant of viral tropism in chick embryo. EMBO J 1990; 9:4189-4195. [PMID 2174359]

60. Nagai Y. Protease-dependent virus tropism and pathogenicity. Trends Microbiol 1993; 1:81-87. [PMID 8143121]

61. Alexander DJ. Gordon Memorial Lecture. Newcastle disease. Br Poult Sci 2001; 42:5-22. [PMID 11337967]

62. Gotoh B, Ohnishi Y, Inocencio NM, Esaki E, Nakayama K, Barr PJ, et al. Mammalian subtilisin-related proteinases in cleavage activation of the paramyxovirus fusion glycoprotein: superiority of furin/PACE to PC2 or PC1/PC3. J Virol 1992; 66:6391-6397. [PMID 1404596]

63. Nagai Y. Virus activation by host proteinases. A pivotal role in the spread of infection, tissue tropism and pathogenicity. Microbiol Immunol 1995; 39:1-9. [PMID 7783672]

64. Ogasawara T, Gotoh B, Suzuki H, Asaka J, Shimokata K, Rott R, Nagai Y. Expression of factor X and its significance for the determination of paramyxovirus tropism in the chick embryo. EMBO J 1992; 11:467-472. [PMID 1371460]

65. Swayne DE, King DJ. Avian influenza and Newcastle disease. J Am Vet Med Assoc 2003; 222:1534-1540. [PMID 12784958]

66. Fujii Y, Sakaguchi T, Kiyotani K, Yoshida T. Comparison of substrate specificities against the fusion glycoprotein of virulent Newcastle disease virus between a chick embryo fibroblast processing protease and mammalian subtilisin-like proteases. Microbiol Immunol 1999; 43:133-140. [PMID 10229267]

67. Attar BM, Walter RJ, Delimata M, Tejaswi SLK, Rafiq A. Exogenously added trypsin is not required for high in vitro cytotoxicity of Newcastle disease virus strain LaSota in pancreatic tumor cells. Gastroenterology 2010; 138(Suppl 1):s451-2.

68. Walter RJ, Attar BM, Rafiq A, Delimata M, Tejaswi SLK. Three avirulent, lentogenic strains of Newcastle disease virus are cytotoxic for human pancreatic tumor cells in vitro. Gastroenterology 2010; 138(Suppl 1):s452.

69. Walter RJ, Attar BM, Tejaswi SLK, Rafiq A, Delimata M. Newcastle disease virus LaSota strain kills human pancreatic cancer tumor cells in vitro with high selectivity. Gastroenterology 2010; 138(Suppl 1):s453.

 

 

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