Abstract:
A method for treating human tumor cells to induce apoptotic cell death thereof includes the step of infecting the tumor cells with a combination of the Herefordshire strain of Newcastle Disease Virus and a chemotherapeutic agent. The range of concentrations of chemotherapeutic agent/Herefordshire strain is in the range of 100/1 to 1/1. Illustrative chemotherapeutic agents include cisplatin, methotrexate, vincristine, bleomycin and dacarbazine.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS  
       [0001]     This is a non-provisional application based upon U.S. provisional application Ser. No. 60/524,726, filed Nov. 25, 2003, now pending. 
     
    
     FIELD OF THE INVENTION  
       [0002]     The present invention relates to a method for treating human tumor cells to induce apoptotic cell death thereof with a Newcastle Disease Virus (NDV) strain and, more particularly, to a method for treating human tumor cells with a combination of a Newcastle Disease Virus strain and a chemotherapeutic agent.  
       BACKGROUND OF THE INVENTION  
       [0003]     It has already been demonstrated that the viral vaccine known as MTH-68/H, developed by United Cancer Research Institute (Ft. Lauderdale, Fla.) and available from UCRI Hungary Ltd. of Budapest, Hungary, containing highly purified, attenuated, mesogenic Herefordshire Newcastle Disease virus strain (hereinafter “Herefordshire strain”), has significant oncolytic capacity. The strain is nonpathogenic in humans and was found to have antineoplastic effects in patients with certain therapy resistant tumors, such as glioblastoma, colorectal cancer, melanoma and hematological malignancies. This oncolytic effect is, at least in part, due to its direct cytotoxicity. Cell death caused by this strain of Newcastle Disease Virus comes in the form of apoptosis. As used herein, the vaccine designation “MTH-68/H” refers to the aforementioned viral vaccine containing highly purified, attenuated Herefordshire strain.  
         [0004]     Notwithstanding the acknowledged oncolytic effect of this Newcastle Disease viral strain it is believed that it can be a still more effective therapeutic agent against human tumor cells when used in combination with other oncolytic agents and that the combination will demonstrate a synergistic cytotoxicity which is more effective than either agent alone  
       SUMMARY OF THE INVENTION  
       [0005]     It is, therefore, a primary object of the present invention to characterize the oncolytic capacity of a purified, attenuated Herefordshire strain.  
         [0006]     It is also an object of the present invention to demonstrate the effect of the Herefordshire strain on cell lines originating from human tumors.  
         [0007]     It is another object of the present invention to demonstrate the cytotoxic effect of the Herefordshire strain in combination with chemotherapeutic agents in cell lines originating from human tumors.  
         [0008]     The foregoing and other objects are achieved in accordance with the present invention by providing a method for treating human tumor cells to induce apoptotic cell death thereof comprising the step of infecting the tumor cells with the Herefordshire strain.  
         [0009]     In another aspect of the present invention there is provided another method for treating human tumor cells to induce apoptotic cell death thereof comprising the steps of infecting the tumor cells with a combination of the Herefordshire strain and a chemotherapeutic agent.  
         [0010]     In still another aspect of the present invention, the chemotherapeutic agents which evidence a synergistic cytotoxic effect, in combination with Herefordshire strain, on human tumor cells include: cisplatin, methotrexate, vincristine, bleomycin and dacarbazine.  
         [0011]     In yet another aspect of the present invention, the ratio of chemotherapeutic agent to Herefordshire strain in the combination is in the range of 100:1 to 1:1.  
     
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0012]      FIG. 1  is a graphical representation of the cytotoxicity of MTH-68/H on control cells.  
         [0013]      FIG. 2  is a graphical representation of the cytotoxicity of MTH-68/H on melanoma cell lines.  
         [0014]      FIG. 3  is a graphical representation of the cytotoxicity of MTH-68/H on human colorectal cancer cell lines.  
         [0015]      FIG. 4  is a graphical representation of the cytotoxicity of MTH-68/H on human prostate cancer cell lines.  
         [0016]      FIG. 5  is a graphical representation of the cytotoxicity of MTH-68/H on human pancreas cancer cell lines.  
         [0017]      FIG. 6  is a graphical representation of the cytotoxicity of MTH-68/H on human lung cancer cells.  
         [0018]      FIG. 7  is a graphical representation of the cytotoxicity of MTH-68/H on human astrocytoma cells.  
         [0019]      FIG. 8  is a graphical representation of the cytotoxicity of MTH-68/H on human A431 cancer cells.  
         [0020]      FIG. 9  is a graphical representation of various NDV preparations on PANC-1 cells.  
         [0021]      FIG. 10  is a graphical representation of various NDV preparations on HeLa cells.  
         [0022]      FIG. 11  is a graphical representation of the cytotoxicity of the MTH-68/H/cisplatin combination on NCI-H460 cells.  
         [0023]      FIG. 12  is a graphical representation of the cytotoxicity of the MTH-68/H/methotrexate combination on NCI-H460 cells.  
         [0024]      FIG. 13  is a graphical representation of the cytotoxicity of the MTH-68/H/bleomycin combination on NCI-H460 cells.  
         [0025]      FIG. 14  is a graphical representation of the cytotoxicity of the MTH-68/H/vincristine combination on HCT-116 cells.  
         [0026]      FIG. 15  is a graphical representation of the cytotoxicity of the MTH-68/H/bleomycin combination on HCT-116 cells.  
         [0027]      FIG. 16  is a graphical representation of the cytotoxicity of the MTH-68/H/dacarbazine combination on PC-3 cells.  
         [0028]      FIG. 17  is a graphical representation of the cytotoxicity of the MTH-68/H/bleomycin combination on HeLa cells.  
         [0029]      FIG. 18  is a graphical representation of the cytotoxicity of the MTH-68/H/bleomycin combination on HT-29 cells.  
         [0030]      FIG. 19  is a graphical representation of the cytotoxicity of the MTH-68/H/chlorpromazine combination on PC-12 cells.  
     
    
     DESCRIPTION OF THE PREFERRED EMBODIMENT  
       [0031]     To demonstrate the cytotoxicity of the Herefordshire strain and the synergistic cytoxicity of combination of the Herefordshire strain with chemotherapeutic agents, several studies were conducted on various human cell lines. The main features of the cell lines used in these studies are summarized in Table I. The cell lines were cultured in media described in Table I. Cultures were infected with freshly suspended batches of virus preparations.  
         [0032]     The following Newcastle disease virus strains were utilized:  
         [0033]     Herefordshire Strain  
         [0034]     The H (Herefordshire) strain of Newcastle Disease Virus was used in the form of the vaccine product MTH-68/H, obtained from UCRI Hungary Limited. The titre of the vaccine was 10 8.3  EID in one ml. The vaccine was stored at −20° C. and protected from light. The lyophilized vaccine was dissolved in 1 ml sterile saline immediately prior to use.  
         [0035]     LaSota  
         [0036]     LaSota is an avirulent (lentogenic) ND vaccine virus strain. The titre of the vaccine was approximately 10 9 -10 10  particles/ml. The vaccine was stored at −80° C.  
         [0037]     Vitayest  
         [0038]     Vitapest is an avirulent lentogenic ND vaccine virus strain. The titre of the vaccine was approximately 10 9  particles/ml. The vaccine was stored at −80° C.  
         [0039]     The following procedures were employed:  
         [0040]     Cell Proliferation Assay  
         [0041]     Proliferation and viability of cell lines under various experimental conditions  
                                                                                                         TABLE I                           Cell lines used in this study                Species                           of       Cell line   origin   Tissue of origin   Comment   Culture medium   Source                    Non-cancerous cell lines            NIH 3T3   mouse   normal fibroblast   —   DMEM,   ATCC                       10% calf serum       Rat-1   rat   normal fibroblast   —   DMEM,   ATCC                       10% calf serum       CHO   hamster   ovarian cells   —   DMEM,   from J. Szekeres                       20% FBS           human   foreskin fibroblast   primary culture   DMEM,   from G. Sáfrány                       20% FBS            Cancer cell lines            PC12   rat   phaeochromocytoma   —   DMEM,   from G. M.                       10% horse serum,   Cooper                       5% FBS       PC12-   rat   phaeochromocytoma   expresses   DMEM,   from M. Pap       dn-p53           dominant   10% horse serum,                   negative p53   5% FBS       PC12-   rat   phaeochromocytoma   overexpresses   DMEM,   from Zs. Fábián       p53 +             wt-p53   10% horse serum,                       5% FBS       HeLa   human   cervix   low p53   DMEM,   ATCC               adenocarcinoma   expression   10% FBS       MCF-7   human   breast   p53-positive   DMEM,   ATCC               adenocarcinoma       10% FBS       293T   human   kidney   transformed   DMEM,   ATCC                   with adenovirus   10% FBS                   5 DNA       Cos-7   African   kidney   SV40-   DMEM,   ATCC           green       transformed   10% FBS           monkey       PANC-1   human   pancreas epitheloid       RPMI1640   from Schering               carcinoma       10% FBS                       supplemented                       with non-essential                       amiono acids and                       Na-pyruvate       DU 145   human   prostate carcinoma   brain metastasis   DMEM Ham′F12   from Schering                       10% FBS       NCI-   human   large cell lung cancer   positive for c-   DMEM Ham′F12   from Schering       H460           myb, v-fes, v-   10% FBS                   fms, c-raf 1, Ha-                   ras, Ki-ras and                   N-ras mRNA       HT-29   human   colorectal cancer   p53 mutation,   DMEM Ham′F12   from Schering                   truncated c-Met   10% FBS       PC-3   human   prostate   bone metastasis   DMEM Ham′F12   from Schering               adenocarcinoma       10% FBS       B16   mouse   melanoma       DMEM,   from J. Szekeres                       10% FBS       HCT-116   human   colorectal cancer   activated ras   RPMI1640   from Schering                       5% FBS       U373   human   astrocytoma       DMEM,   from G. Sáfrány                       10% FBS       HT-25   human   colorectal cancer       DMEM Ham′F12   from J. Timár                       10% FBS       HT-199   human   melanoma   truncated c-Met   DMEM Ham′F12   from J. Timár                       10% FBS       WM983B   human   melanoma   truncated c-Met   DMEM Ham′F12   from J. Timár                       10% FBS       HT-168-   human   melanoma   truncated c-Met   DMEM Ham′F12   from J. Timár       M1               10% FBS       A431   human   epithelial cancer   HPV +     DMEM Ham′F12   from J. Timár                   low p53   5% FBS                  
 
 were analyzed using the WST-1 kit of Roche Molecular Biochemicals following the manufacturers instructions. Optimal cell culture and assay conditions were determined in preliminary experiments. 1-4×10 4  cells/well were seeded in standard culture medium in 24-well plates. Cultures were infected with the virus preparations at different titres (ranging from 100/1 to 1/100 cell/particle ratios) for 72 hours. WST-1 assays were performed for 120 minutes and light absorption (A 440 ) of media were taken in 96-well plates using an ELISA reader. 
 
         [0042]     No-treatment and anisomycin-treated (1 μg/ml) cultures were used for negative and ctytotoxicity-positive controls, respectively.  
         [0043]     Analysis of Virus Replication  
         [0044]     Cells were cultured in 1 ml standard medium (see Table I) at a density of 4×10 4  cells/well in 24-well dishes. Cells were infected with MTH-68/H, La Sota or Vitapest NDV strains at various cell/particle ratios. Incubations were performed for 72 hours, media were harvested and stored at −80° C. until titration. No treatment and anisomycin (1 μg/ml) treatment were used as controls.  
         [0045]     Detection of DNA Fragmentation  
         [0046]     2-5×10 6  cells were cultured in DMEM (Dulbecco&#39;s modified Eagle medium) containing serum for 24 hours. Treatments were carried out as indicated in the legends of each of the Figures. Four positive control samples were incubated for 24 hours in serum-free DMEM or with anisomycin (1 μg/ml); for negative control they were kept in high-serum DMEM. After incubation for the time periods indicated in the Figures, cells were collected by scraping them into their own medium and then centrifuged at 1000 rpm for 5 minutes. The soluble DNA of these cells was extracted by the following method. Collected cells were solubilized on ice in extraction solution containing 0.5% Triton X-100, 5 mM TRIS pH 7.4, 5 mM EDTA for 20 minutes. Soluble DNA in the supernatant rsulting from centrifugation at 13500 rpm for 20 minutes at 4° C. was extracted with phenol/chloroform, chloroform, and finally precipitated with ethanol. The precipitates were treated with DNase free RNase A (Sigma-Aldrich, Steinheim, Germany (2 mg/ml) at 37° C. for 1 hour. DNA fragments were separated by electrophoresis in 1.8% agarose gels, and visualized on a UV transilluminator after staining the gel with SYBR Gold (Molecular Probes, Eugene, Oreg.).  
         [0047]     Western Blot Analysis  
         [0048]     Immunoblot analysis using antibodies against proteins indicated was performed as described by the manufacturers Cell Signaling (Beverly, Mass.) and Transduction Labs.  
         [0049]     Protein concentrations were determined using the Bio-Rad Protein DC assay, and equivalent amounts of protein were resolved by SDS polyacrylamide gel electrophoresis using either 12% or 16% polyacrylamide gel. The proteins were transferred to an ECL membrane (Amersham Pharmacia Biotech AB., Uppsala, Sweden). Immune complexes were visualized using an enhanced chemiluminescence detection kit (Amersham Pharmacia Biotech AB) following the manufacturer&#39;s instructions. The following antibodies were used: Cleaved Caspase-3 (Rat specific), Cleaved Caspase-9 (Rat specific) from Cell Signaling (Beverly, Mass.) and PK R from Transduction Labs.  
         [0050]     Electrophoretic Mobility Shift Assay (EMSA)  
         [0051]     Nuclear extracts were prepared as described by Xu &amp; Cooper in “Identification of a candidate c-mos repressor that restricts transcription of germ cell-specific genes”; Mol Cell Biol 1995; 15: 5369-5375. All subsequent steps were performed at 4° C. Cell pellets were washed twice in ice cold phosphate-buffered saline (1× PBS) and resuspended in 10 volumes of buffer containing 10 mM HEPES pH 7.9, 1.5 mM MgCl 2 , 10 mM KCl, 0.5 mM dithiothreitol (DTT), protease inhibitors (Complete, Mini EDTA-free tablets, Boehringer Mannheim), phosphatase inhibitors (Phosphatase Inhibitor Cocktail, Sigma) and placed on ice for 10 minutes. After vigorous vortexing, nuclei were collected by centrifugation in a microcentrifuge and resuspended in 2 volumes of buffer containing 20 mM HEPES pH 7.9,25% glycerol, 420 mM NaCl, 1.5 mM MgCl 2 , 0.2 mM EDTA, 0.5 mM DTT, protease inhibitors, phosphatase inhibitors and placed on ice for 20 minutes. After centrifugation in a microcentrifuge, the supernatants were saved, aliquoted and stored at −80° C. Protein concentrations were determined with the Bio-Rad Protein Assay Kit (Coomassie Brilliant Blue dye).  
         [0052]     5′-end labeling of oligonucleotides was performed using [γ- 32 P]-ATP and T4 polynucleotide kinase (Amersham Pharmacia Biotech Inc.) according to the manufacturer&#39;s protocol. After reconstitution of Ready-To-Go T4 polynucleotide kinase by adding 25 μl water and incubation at room termperature for 2-5 minutes, 5-10 pmol of 5′-ends of oligonucleotide, 22 μl water and 2 μl of [γ- 32 P]-ATP (3000 Ci/mmol, 10 μCI/μl) were added, mixed gently and incubated at 37° C. for 30 minutes. The reaction was stopped by adding 5 μl of 250 mM EDTA. Labelled oligonucleotides were collected by Spin Column 10 (Sigma).  
         [0053]     The protein-DNA binding reaction was performed as follows: 10-20 μg nuclear proteins were mixed with 1 μg poly(dI-dC), 100 ng nonspecific single-stranded oligonucleotide and 4 μl buffer containing 10 mM HEPES pH 7.5, 10% glycerol, 1 mM EDTA, 100 mM NaCl. Sufficient amount of distilled water was added to bring the reaction volume to 18 μl. After 15 minutes incubation at room temperature the mixture was completed with 2 μl, approximately 100 000 cpm of  32 P-labelled oligonucleotide (total reaction volume was 20 μl) and incubation at room temperature was continued for another 30 minutes.  
         [0054]     DNA-protein complexes were electrophoresed in 5% non-denaturing polyacrylamide gel (5 ml 30% acrylamide-bisacrylamide mixture, 2.5 ml 10× Tris Base, Borate, EDTA buffer pH 8.3, 17.5 ml distilled water, 20 μl TEMED, 50 μl 25% ammonium per sulphate) using the Tris Base, Borate, EDTA buffer system (pH 8.3) for 2.5 h at 200V. Gels were dried and analyzed by a Cyclone Phosphorlmager system (Packard Instrument Co. Inc., Meriden, Conn.).  
         [0055]     With reference to  FIGS. 1-8  and Table II there can be seen the results obtained by infecting various tumor cell lines with the Herefordshire strain utilized in the form of the MTH-68/H vaccine.  
         [0056]     WST-1 Proliferation Assays  
         [0057]     Control and tumor cell lines were tested for MTH-68/H cytotoxicity using the WST-1 kit. The results are summarized in Table II. Human fibroblasts were completely resistant to MTH-68/H even at very high virus titers (800 particles for 1 cell, see  FIG. 1 ). This resistance was probably not caused by the high concentration of serum (20% FBS) used to grow the cells, since the presence of serum did not inhibit the cytotoxic effect of MTH-68/H on three tumor cell lines tested (PANC-1, HeLa, MCF-7). In contrast, Chinese hamster ovary cells (CHO cell line) displayed moderate sensitivity to MTH-68/H, comparable to certain tumor cell lines (See  FIG. 1  and Table II).  
         [0058]     Melanoma Cell Lines  
         [0059]     All three human melanoma cell lines tested (HT-199, WM983B and HT168-M1) are highly sensitive to MTH-68/H. See  FIG. 2  and Table II.  
         [0060]     Human Colorectal Cell Lines  
         [0061]     All three human colorectal cancer cell lines tested are sensitive to MTH-68/H (HT-29&gt;HCT-116&gt;HT-25). See  FIG. 3  and Table II.  
         [0062]     Human Prostate Cancer Cell Lines  
         [0063]     Both cell lines tested are sensitive to MTH-68/H (PC3&gt;DU-145). See  FIG. 4  and Table II.  
         [0064]     Human Pancreas Cancer Cell Line  
         [0065]     The PANC-1 cell line is one of the most MTH-68/H sensitive cell lines. See  FIG. 5  and Table II.  
         [0066]     Human Large Cell Lung Cancer Cell Line  
         [0067]     The NCI-H460 cell line is quite sensitive to MTH-68/H cytotoxicity. See  FIG. 6  and Table II.  
         [0068]     Human Astrocytoma Cell Line  
         [0069]     U373 cells have moderate sensitivity to MTH-68/H. See  FIG. 7  and Table II.  
         [0070]     A431 Human Carcinoma Cell Line  
         [0071]     The A431 human epithelial cancer cell line is moderately sensitive to MTH-68/H. See  FIG. 8  and Table II.  
         [0072]     To provide a basis for comparison, the NDV strains LaSota and Vitapest were also tested for their oncolytic potential. Liquid, unpurified batches of MTH-68/H, LaSota and Vitapest preparations that were isolated under identical conditions were tested on human tumor cells and compared. The preparations had the following approximate titers:  
                                                           MTH-68/H   10 8.8     particles/ml           LaSota   10 9 -10 10     particles/ml           Vitapest   10 9     particles/ml                      
 
         [0073]     The fresh virus preparations were tested on PANC-1(see  FIG. 9 ) and HeLa cells (see  FIG. 10 ). On both cell lines all three NDV preparations were found to be cytotoxic, but MTH-68/H was 10 3 -10 4  times more effective than LaSota or Vitapest.  
                                                                                             TABLE II                           The cytotoxicity of MTH-68/H in various cell lines                    MTH-68/H                       titer causing               50%   Semiquantitative               cytotoxicity*   assessment of       Cell line   Source   (cell/particle)   cytotoxicity   Experiment                    Non-cancerous cell lines            Rat-1   normal rat fibroblasts       &lt;1/100   −   #32       NIH3T3   normal mouse fibroblasts       &lt;1/100   −   #34       CHO   chinese hamster ovary     10/1-1/1   ++   #66, #68           human fibroblasts       &lt;1/800   −   #86            Cancer cell lines            PC12   rat pheochromocytoma    1/10   +   #45       HeLa   human cervical cancer   &gt;100/1      ++++   #18       MCF-7   human breast cancer    1/10   +   #19       293T   adenovirus-transformed   &gt;100/1      ++++   #20           human kidney       Cos-7   SV40-transformed   1/1   ++   #22           monkey kidney       PANC-1   human pancreas cancer   &gt;100/1      ++++   #80       DU 145   human prostate cancer      5/1-1/1   ++   #81       NC1-H460   human large cell lung      50/1-10/1   +++   #82           cancer       HT-29   human colorectal cancer   10/1    ++   #83       PC-3   human prostate cancer      50/1-10/1   +++   #84       B16   mouse melanoma       1/10-1/50   +   #54 #58       HCT-116   human colorectal cancer     10/1-5/1   ++   #100, #105,                       #106       U373   astrocytoma   1/5   +   #107       HT-25   human colorectal cancer   5/1   ++   #116       HT-199   human melanoma    &gt;10/1         +++   #116       WM 983B   human melanoma    &gt;10/1         +++   #119       HT168-M1   human melanoma   5/1   ++   #119       A431   human epithelial cancer   5/1   ++   #119                 *Control: 0% cytotoxicity; anisomycin (1 μg/ml): 100% cytotoxicity.             
 
 Synergism Between MTH-68/H and Chemotherapeutics 
 
         [0074]     A potential clinical application of MTH-68/H is its use in combination with other therapeutic regimens, especially chemotherapeutic treatments, to increase efficacy and reduce toxicity. Therefore, several cytostatic agents were tested in combination with MTH-68/H on various tumor cell lines. The highest nontoxic concentrations of the drugs for each cell line were determined in preliminary experiments, and then these concentrations were used in combination with MTH-68/H to demonstrate synergy. The results of these tests are summarized in Table III. Graphical representations of the cytotoxicity of MTH-68/H/chemotherapeutic agent combinations on human tumor cell lines are shown in  FIGS. 11-18 . Each of these Figures shows the cytoxicity of the chemotherapeutic agent alone, of chemotherapeutic agent/MTH-68/H combinations in ranges from 100/1 to 1/1 and of MTH-68/H alone. In each case, it can be seen that the cytotoxicity of the combination was better than each agent alone, demonstrating the synergy of their combination.  
         [0075]     Interestingly, when similar tests were conducted using MTH-68/H and chlorpromazine on PC12, MCF-7, B16, CHO, 293T and HeLa cells, no significant synergy between chlorpromazine and MTH-68/H was observed. See Table III and  FIG. 19 .  
         [0076]     While the present invention has been described in terms of specific embodiments thereof, it will be understood that no limitations are intended to the details of the disclosed methods other than as defined in the appended claims.  
                                                     TABLE III                           Cytotoxicity of Chemotherapeutic/MTH-68/H combinations in Various Cell Lines            MTH-68/H +   Cisplatin   Methotrexate   Vincristine   5-Fluorouracil   Chlorpromazine   Dacarbazine   BCNU   Bleomycin               PC12   ++               −   +                   # 46               # 50   #52       MCF-7   ++   −   +   −   +   −   +           # 47   # 47   # 47   #47   # 75   # 103   # 103                   # 103       B16           ++       −   −                   # 58       # 73   # 54                           # 64   # 56                           # 65       CHO                   +-                           # 66                           # 72       293T   ++   −   +   +   −       −   −           # 101   # 101   # 101   # 101   # 67       # 92   # 93       HeLa   +           +   −   −   −   ++           # 98           # 98   # 74   # 125   # 94   # 95       HCT-116   +       ++           +   +   ++           # 105       # 106           # 105   # 105   # 106       Panc-1   −           −       −   −           # 125           # 109       # 125   # 109       HT-29   −   −   +   −       −       ++           # 117   # 122   # 117   # 122       # 122       # 117       NCI-H460   ++   ++   −   +       −   −   ++           # 118   # 126   # 118   # 126       # 126   # 126   # 126                   # 126       PC-3                       ++   −                                   # 124       DU-145   −   +   −           +       −           # 124       # 124                   # 124                 − no synergy            + weak synergy            ++ significant synergy