In Vitro Synergistic Enhancement of Newcastle Disease Virus to 5-Fluorouracil Cytotoxicity against Tumor Cells

Background: Chemotherapy is one of the antitumor therapies used worldwide in spite of its serious side effects and unsatisfactory results. Many attempts have been made to increase its activity and reduce its toxicity. 5-Fluorouracil (5-FU) is still a widely-used chemotherapeutic agent, especially in combination with other chemotherapies. Combination therapy seems to be the best option for targeting tumor cells by different mechanisms. Virotherapy is a promising agent for fighting cancer because of its safety and selectivity. Newcastle disease virus is safe, and it selectively targets tumor cells. We previously demonstrated that Newcastle disease virus (NDV) could be used to augment other chemotherapeutic agents and reduce their toxicity by halving the administered dose and replacing the eliminated chemotherapeutic agents with the Newcastle disease virus; the same antitumor activity was maintained. Methods: In the current work, we tested this hypothesis on different tumor cell lines. We used the non-virulent LaSota strain of NDV in combination with 5-FU, and we measured the cytotoxicity effect. We evaluated this combination using Chou–Talalay analysis. Results: NDV was synergistic with 5-FU at low doses when used as a combination therapy on different cancer cells, and there were very mild effects on non-cancer cells. Conclusion: The combination of a virulent, non-pathogenic NDV–LaSota strain with a standard chemotherapeutic agent, 5-FU, has a synergistic effect on different tumor cells in vitro, suggesting this combination could be an important new adjuvant therapy for treating cancer.


Introduction
The mainstays of treatment for advanced cancers are chemotherapy and radiotherapy. However, they are limited due to the resistance of tumor cells to these agents, as well as their narrow therapeutic index. Therefore, combination therapies were invented to overcome cancer cell resistance and to increase the anti-tumor effect while considering the toxicity for normal tissue [1]. 5-Fluorouracil is an important chemotherapeutic agent for many solid tumors, particularly gastrointestinal, brain, and head and neck malignancies. 5-FU has also been actively investigated during the last 40 years for many tumors. However, the role of systemic 5-FU in cancer therapy has been limited by the fact that dose-limiting side effects (myelosuppression and stomatitis) are usually reached before evidence of antitumor response [2,3]. Antitumor chemotherapeutic agents, such as 5-fluorouracil, are toxic to the small intestine and make it dysfunctional [4]. Effective antitumor

Virus
The lentogenic virulent strain of NDV (LaSota) was obtained from Al-Kindy Company for veterinarian vaccines (Baghdad, Iraq). A stock of infectious virus was propagated in embryonated chicken eggs, harvested from allantoic fluid, and purified from debris by centrifugation (3000 rpm, 30 min, 4˝C). NDV was quantified using a hemagglutination test in which one hemagglutination unit (HAU) is defined as the smallest virus concentration leading to visible chicken erythrocyte agglutination.

Chemotherapeutic Agent
5-FU (5-Fluorouracil)-SP Pharmaceuticals (Albuquerque, NM, USA) were purchased from the Radiation and Atomic Medicine Hospital (Baghdad, Iraq). This agent was diluted with a medium without calf bovine serum just before use for in vitro studies.

Combination Cytotoxicity Assays
To determine the cytotoxic effect of NDV and 5-FU in combination treatment, the MTT cell viability assay was conducted on 96-well plates (Becton, Dickinson, Franklin Lakes, NJ, USA). Hep-2, RD, AMN3, and Vero cells were seeded at 1ˆ10 4 cells/well. After 24 h. or a confluent monolayer was achieved, cells were treated with the virus alone (infected with NDV at 128 HAU with two-fold serial dilutions), the drug alone (the chemotherapeutic agent 5-FU at 5 µg in two-fold serial dilutions to 0.039 µg/mL), or a combination of the two (virus + 5-FU in two-fold serial dilutions). The procedure of adding these therapeutic agents involved addition of the virus for 2 h at room temperature to allow for viral attachment and penetration. Afterwards, cells were washed with PBS and serial dilutions of the drug were added to the infected and non-infected cells. Cell viability was measured after 72 h of infection by removing the medium, adding 28 µL of 2 mg/mL solution of MTT (Sigma-Aldrich, St. Louis, MO, USA) and incubating the cells for 1.5 h at 37˝C. After removing the MTT solution, the crystals remaining in the wells were solubilized by the addition of 130 µL of DMSO (Dimethyl Sulphoxide) (BDH, London, UK) followed by 37˝C incubation for 15 min with shaking [20]. The absorbency was determined on a microplate reader (Organon Teknika Reader 230S, Salzburg, Austria) at 492 nm (test wavelength); the assay was performed in triplicate. The inhibition rate of cell growth (the percentage of cytotoxicity) was calculated as (A´B)/Aˆ100, where A is the mean optical density of untreated wells, and B is the optical density of treated wells. The LC50 is the lowest concentration that kills 50% of the cells [21]. Each experiment was repeated at least three times in triplicate.

Chou-Talalay Analysis
The median effect doses (ED50) were calculated for the drug and NDV for each cell line. For synergism determination, NDV and 5-FU were studied as a non-constant ratio. To analyze the combination of NDV and 5-FU, Chou-Talalay combination indices (CI) were calculated using CompuSyn software (Combo Syn, Inc., Paramus, NJ, USA). Non-fixed ratios of NDV and chemotherapeutics, as well as mutually exclusive equations, were used to determine the CIs. A CI between 0.9 and 1.1 is considered additive, whereas CI < 0.9 and CI > 1.1 indicate synergism and antagonism, respectively [22,23].

Combination Chemotherapy and Viral Cytotoxicity in Vitro
To study the potential interaction between NDV and chemotherapy in vitro, the effectiveness of the combined treatment for several concentrations of 5-FU with NDV at various hemagglutination conditions was evaluated in the Hep-2, RD, AMN3, and Vero cell lines. Cells were treated with NDV and with 5-FU or with the combination of NDV and 5-FU. The cell viability was determined after 72 h using the MTT assay.
In RD, Rhabdomyosarcoma, combination therapy had significant 71.6% cytotoxicity (PR = 29%) (p: 0.0001) at 0.625 µg/mL 5-FU and 16 HAU NDV. NDV treatment alone showed 64.1% cytotoxicity (PR = 35.9%) (p: 0.0001) at 16 HAU, and 1.25 µg/mL 5-FU therapy alone also showed a significant cytotoxic effect of 53.8% (PR = 46.2%) (p: 0.0001), which is less than the combination of NDV and half the dose of 5-FU. Combination therapy at 0.312 µg/mL 5-FU and 8HAU showed 49.7% G.I. (p: 0.002), whereas the growth inhibition effect of chemotherapy alone with 5-FU at two-fold dose (0.625 µg/mL) was 39.7% G.I. (PR = 60.3%) (p: 0.1) (Figure 2a). Data were further analyzed using Chou-Talalay equations and the dose-oriented isobologram technique. There was synergism between NDV and 5-FU at 50% growth inhibition doses, as represented in Figure 2b   In AMN3, mouse mammary adenocarcinoma cell line combination therapy was effective (p: 0.006) and had a similar effect as with 5-FU alone at two-fold doses. There were no significant differences between combination with half-dose of chemotherapy compared with 5-FU alone. These results show that NDV could compensate for the reduction in the 5-FU doses even though there was no significant effect of NDV treatment on tumor cells alone at any of the concentration (Figure 3a).  In AMN3, mouse mammary adenocarcinoma cell line combination therapy was effective (p: 0.006) and had a similar effect as with 5-FU alone at two-fold doses. There were no significant differences between combination with half-dose of chemotherapy compared with 5-FU alone. These results show that NDV could compensate for the reduction in the 5-FU doses even though there was no significant effect of NDV treatment on tumor cells alone at any of the concentration (Figure 3a).    In AMN3, mouse mammary adenocarcinoma cell line combination therapy was effective (p: 0.006) and had a similar effect as with 5-FU alone at two-fold doses. There were no significant differences between combination with half-dose of chemotherapy compared with 5-FU alone. These results show that NDV could compensate for the reduction in the 5-FU doses even though there was no significant effect of NDV treatment on tumor cells alone at any of the concentration (Figure 3a).    To study the effect of combination treatment on non-cancer cells, the Vero monkey kidney transformed cell line was used. Generally, most of the concentrations used alone or in combination lacked significant differences ( Figure 4). As there was no significant cytotoxic effect, there was no need to do Chou-Talalay equation.   To study the effect of combination treatment on non-cancer cells, the Vero monkey kidney transformed cell line was used. Generally, most of the concentrations used alone or in combination lacked significant differences ( Figure 4). As there was no significant cytotoxic effect, there was no need to do Chou-Talalay equation.   To study the effect of combination treatment on non-cancer cells, the Vero monkey kidney transformed cell line was used. Generally, most of the concentrations used alone or in combination lacked significant differences ( Figure 4). As there was no significant cytotoxic effect, there was no need to do Chou-Talalay equation.

Discussion
The primary objective of this study was to determine if we can augment cancer chemotherapy by virotherapy. Furthermore, we sought to determine if we can reduce the toxicity of cancer chemotherapeutic agents by reducing the administered dose of 5-FU and replacing it with Newcastle disease virus therapy, while maintaining the same, or more, anti-tumor activity and overcoming resistance to chemotherapy.
Based on our results in four different cell lines, the lentogenic NDV strain (LaSota used as live vaccines against NDV) exhibited oncolytic activity on three tumor cell lines and to a lower degree on the transformed cell line (Vero). Previous studies have shown that a virulent NDV strain is oncolytic [14,24]. The NDV LaSota strain showed anti-lymphoma activity both in vitro and in vivo [25]. Furthermore, Walter et al. [26] showed that the Newcastle Disease Virus LaSota strain kills human pancreatic cancer cells in vitro with 700-fold higher selectivity than normal cells.
Fabian et al. [27] used an attenuated pathogenic NDV MTH-68/H strain, which was originally a vaccine strain. This strain showed antitumor activity both in vitro and in vivo in a clinical trial [28,29]. Moreover, Pecorna et al. [30] used a naturally attenuated strain of NDV (PV701) that exhibits a broad range of oncolytic activity against human tumors in vitro; they introduced the strain into clinical trials. Schirrmacher et al. [14] used a lentogenic virulent Ulster strain and found that infection of cancer cells by non-lytic non-virulent NDV Ulster strain (30 HU/107 cells) eventually causes tumor cell death in vitro, and it also has replication selectivity in tumor cells [31].
The combination of NDV and 5-FU showed greater cytotoxic efficacy than NDV alone or at a two-fold dose of 5-FU alone. The effect appears to be synergistic according to Chou-Talalay analysis. The mechanism(s) of synergistic activity for the combination of 5-FU with NDV is unknown, but we propose a few hypotheses. First, NDV may augment the anti-tumor activity of 5-FU by increasing the cellular sensitivity to chemotherapeutic agents, and this enhanced sensitivity is partially caused by the induction of apoptosis in response to virulent NDV strains [32]. Second, a synergistic dose of 5-FU may augment the viral replication, as suggested by many studies on oncolytic viruses [19,33]. Each agent may work independently on different cell populations, but this is unlikely to be the case here. In addition, virotherapy with NDV may complement the anti-tumor activity of 5-FU, which selectively targets tumor cell populations that are resistant to chemotherapy. This may be of important value because most human tumors consist of a mixture of cells that have a different genetic makeup. Heterogeneity in the tumor cell populations may be the major reason most monotherapies fail to achieve complete tumor remission [34]. Moreover, one of the objectives of this study was to reduce the toxic side effects of chemotherapy in cancer patients. This can be achieved by reducing the administered dose while maintaining the same or stronger antitumor activity. The current experimental results support this claim, but in vivo evaluation is needed.
Several characteristics of the NDV LaSota strain that are favorable for human use include the genetic stability of the vaccine strains, absence of genetic recombination, lack of antigenic drift, and lack of observed human to human transmission [35,36]. The Newcastle disease virus has been safely administered to humans in clinical trials; additionally, accidental exposure in farmers is reported to induce only self-limiting conjunctivitis [28,35,37]. While NDV is safe and lacks toxicity, 5-FU causes myelosuppression and stomatitis before achieving an antitumor response [2].

Conclusions
A virulent, non-pathogenic NDV LaSota strain, in combination with a standard chemotherapeutic agent, 5-FU, has a synergistic effect in vitro on different tumor cells, suggesting this approach could be an important new adjuvant therapy for treating cancer.