Reduced Newcastle disease virus-induced oncolysis in a subpopulation of cisplatin-resistant MCF7 cells is associated with survivin stabilization

MCF7 cell line was employed as a model to study NDV oncolytic activity in CDDP-sensitive versus CDDP-resistant breast cancer cells. During the CDDP treatment, surviving MCF7 cells showed an average of 2-fold increase in size compared to the control untreated MCF7 (Figure 1A). There was no change in the DNA content of the enlarged cells (data not shown). During trypsinization, it took a longer time for them to be dislodged suggesting a stronger attachment to flask surfaces. During the 30-day maintenance in drug-free media, the surviving cells' morphology reverted back to almost similar to the parental MCF7 (Figure 1B, right panel). Prior to infection experiments, their level of resistance to CDDP was determined using the MTT assay. The resulting IC₅₀ for both MCF7 and MCF7-CR was found to be 130.7 μM and 163.4 μM, respectively (Figure 1B). The newly established MCF7-CR showed 25% increase in resistance towards CDDP compared to the parental MCF-7. The resistant factor of MCF7-CR was calculated as 1.3.

To investigate the effects of NDV-AF2240 cytotoxicity on MCF7 and MCF7-CR, the cells were infected with the virus at 1.0 MOI and subjected to a flowcytometric analysis at 4 different (12, 24, 48 and 96) hpi. Mock-infected cells were used as controls. For both cell lines, percent viability appeared to decrease over time (Figure 2). Even though the MCF7-CR is more resistant to CDDP than its parental MCF-7, these cells are still susceptible to NDV oncolysis. A particularly drastic decrease in cell viability was observed during the first 12 hpi, followed by a gradual decrease for the rest of the infection period. At 24 hpi, more viable cells were observed in the infected MCF7-CR compared to the MCF7. Higher viability of MCF7-CR than MCF7 was maintained until the end of the experiment. From 12 hpi to 24 hpi, percent viability in infected MCF7-CR did not change suggesting a degree of cellular resistancy towards NDV cytotoxicity in the surviving cell population.

To examine whether the different pattern of cell death observed in the infected MCF7 versus MCF7-CR cells also affected NDV viral protein synthesis, total cell lysates were then subjected to SDS-PAGE and immunoblotting analyses. Mock-infected cells were used as controls. At 0 hpi, no viral protein was detected on the membrane for both the MCF7 and MCF7-CR samples (Figure 3A). After 12 hpi, several bands representing different viral proteins were observed on the membrane. This observation suggested that NDV protein synthesis occurred at high efficiency within the first 12 hours of infection. As the infection time progressed, the intensity of virus protein bands started to differ between the MCF7 and MCF7-CR.

As our objective was to study how well NDV can infect cisplatin-resistant breast cancer cells, we used cisplatin sensitive MCF7 as the benchmark. Based on the protein band intensity at 12 hpi, the MCF7-CR appeared to contain more viral proteins than MCF7 (Figure 3A). To confirm this observation, we probed the samples with a monoclonal antibody against the nucleocapsid protein (NP) of NDV. The NP protein is the most abundant protein in the virus which makes it easier to detect and analyse [17]. To investigate quantitative difference between the band intensities, the NP blot was analyzed using the ImageJ software and the intensity values were normalized against the β-actin loading control. The plotted values showed that the amount of the NP protein inside MCF7 is lower compared to MCF7-CR and their intensity differences started to increase as time progressed (Figure 3B). These results suggest that in the infected MCF7-CR, NDV protein synthesis occurred at higher level compared to the parental MCF7 cells.

We have shown that NDV protein level was higher in the infected MCF7-CR compared to MCF7. To test whether this increase in viral protein correlates with an increase in viral progeny production, we performed a plaque assay using culture media obtained from the infected cells at 12, 24, 48 and 96 hpi. At all the time points tested, virus yield in the MCF7-CR were always higher than MCF7 (Figure 4). However, the difference was statistically significant only at 24, 48 and 96 hpi. This increase was in line with the amount of viral protein detected in the cells (Figure 3). It is interesting to note that at 24 hpi, virus yield in MCF7-CR was markedly increased compared to the other samples. This sudden jump in virus production correlated with reduced NDV-induced oncolysis (Figure 2B) and increased viral protein level (Figure 3B).

Survivin, a member of the IAP family, is highly expressed in most cancers. It has been shown to be upregulated during virus infection to assist virus production [18, 19]. To test whether survivin level is affected by NDV infection particularly in MCF7-CR, we performed immunoblot studies on all the infected and mock-infected MCF7 and MCF7-CR samples. Since survivin upregulation is known to be a factor in cisplatin resistance [10, 20, 21], we used CDDP as a positive control in this study. Exposure of MCF7-CR but not MCF7 led to increased survivin level in the cells (data not shown). These results confirmed that our MCF7-CR responded the same way to CDDP as other previously reported cisplatin-resistant MCF7 cell lines (22). When MCF7-CR and MCF7 cells were infected with NDV, their survivin levels increased beginning immediately after the adsorption period (considered as 0 hpi in this study; Figure 5A). In certain viral infections, survivin expression was increased and it is shown to be involved in facilitating virus production [22]. At later time points after the infection, the level of survivin began to decrease. When the survivin bands were quantitated and plotted, a dramatic decrease was noted at 24 hpi in MCF7 compared to its level at 12 hpi (Figure 5B). In MCF7-CR on the other hand, the survivin level, despite being lower than MCF7, was retained. Hence, there is no significant change in MCF7-CR survivin level from 12 to 24 hpi. This retention of survivin level in MCF7-CR may contribute towards the reduced cell death (Figure 2) and increased virus production (Figure 4) at 24 hpi.

Estrogen-dependent MCF7 cells were obtained from the ATCC (HTB-22) and maintained in DMEM media supplemented with 10% fetal bovine serum and 1% antibiotic/antimycotic. The media, serum and antibiotic/antimycotic was purchased from PAA Laboratories: Pasching, Austria. A humidified atmosphere of 37°C and 5% CO₂ was provided for cell growth. Cisplatin-resistant MCF-7 (MCF7-CR) was established from the surviving population of the parental MCF7 breast cancer cells (ATCC) following a cisplatin treatment. The treatment was performed as a seven-cyclic 24 h exposure to a final concentration of 50 mM cisplatin (Sigma-Aldrich: St. Louis, MO). Following the treatment, surviving cells were maintained for another 30 days in drug-free media. MCF-7 and MCF-7 CR cells were routinely tested for the presence of Mycoplasma to avoid contamination.

Cells were plated in triplicates into 96-well tissue culture plates at a concentration of 1 × 10⁴ cells per well in 200 μl of culture media. Cisplatin was added at a final concentration in the range of 0 – 200 μM 24 h post-plating and the cells were incubated for 24 h in 5% CO₂ at 37°C. 200 μl of MTT reagent (Sigma Aldrich: St. Louis, MO; final concentration of 0.5 mg/ml in DMEM) was added to each well for 4 h incubation at 37°C, followed by addition of 100 μl of DMSO (Sigma Aldrich: St. Louis, MO). Absorbance was read at an excitation wavelength of 595 nm on an ELISA microplate reader (Model 550; BioRAD: Philadelphia, PA). Cell survival was measured as a percentage of surviving cell over untreated control cells. For both cell lines the concentration of cisplatin which resulted in 50% growth inhibition (IC₅₀) was determined.

Exponentially growing MCF-7 and MCF-7 CR cells were seeded in T-75 flasks at a concentration of 3 × 10⁶ cells per flask. The cells were then infected with NDV at 1 multiplicity of infection (MOI). After 1 h, virus-containing medium was removed and fresh growth medium was introduced into each flask. The cell culture was incubated at 37°C and 5% CO₂ and harvested at different time points. Uninfected cells were used as controls.

Cell pellets were harvested by lysis in 1× RIPA buffer (Pierce Biotechnology: Rockford, IL) containing protease inhibitor cocktail (Roche Diagnostics: Mannheim, Germany) and quantitated using the BCA Protein Assay kit (Pierce Biotechnology: Rockford, IL). Equal amount of protein samples were added to 6× sodium dodecyl sulfate loading buffer (1 μl loading buffer: 5 μl protein sample). The sample mixtures were boiled for 5 min before being resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) to separate the proteins on the basis of their molecular weights. After electrophoresis, the proteins were transferred to a polyvinylidene fluoride (PVDF) transfer membrane by electroblotting, and probed for survivin and NDV proteins using an anti-survivin rabbit mAb (Cell Signaling Technology: Danvers, MA), anti-actin (Sigma Aldrich: St. Louis, MO), anti-NDV and anti-NP. The blot was developed and individual bands were quantitated and normalized to the β-actin control using the ImageJ software (Wayne Rasband, NIH: Bethesda, MD).

A monolayer of SW620 cells was prepared in wells of a 6-well plate. 10-fold dilutions of virus stock were prepared and 1 ml aliquots were inoculated onto the cell monolayers. The plate was incubated at 37°C and 5% CO₂ for 1 h. After the incubation period, the monolayers were covered with a nutrient medium containing agar and the plate was incubated for 6 days. The monolayers were fixed with 1% formaldehyde in 0.15 N NaCl and stained with neutral red for overnight. The plate was washed and dried for plaque observation.

Statistical analyses were performed using the GraphPad Prism 5 (GraphPad Software, Inc., La Jolla, CA). Student's t-test was used to evaluate the significance of differences between experimental groups. Statistically-significant differences were defined as p values less than 0.05.