USE OF MEVALONATE METABOLIC PATHWAY INHIBITOR AND ALPHAVIRUS IN PREPARING ANTI-TUMOR DRUG

Abstract

Disclosed is the use of a mevalonate metabolic pathway inhibitor and an alphavirus in preparing an anti-tumor drug. The mevalonate metabolic pathway inhibitor can be used in preparing an alphavirus anti-tumor synergist. Disclosed are a pharmaceutical composition containing the mevalonate metabolic pathway inhibitor and the alphavirus, a drug kit containing the mevalonate metabolic pathway inhibitor and the alphavirus, and the use of the mevalonate metabolic pathway inhibitor and the alphavirus in treating tumors, especially those that are insensitive to the alphavirus.

Claims

1-10. (canceled)

11. A method of treating a human subject with tumor, comprising administering to the subject an alphavirus and a mevalonate metabolic pathway inhibitor.

12. The method of claim 11, wherein the mevalonate metabolic pathway inhibitor comprises a substance that inhibits a pathway initiated from acetoacetyl-CoA up to farnesyl pyrophosphate, and/or a protein farnesyl modification pathway inhibitor, and/or a geranylgeranylation modification pathway inhibitor.

13. The method of claim 11, wherein the mevalonate metabolic pathway inhibitor comprises a substance that inhibits the activity or formation of any one or more of acetoacetyl-CoA, acetyl-CoA, 3-hydroxy-3-methylglutaryl-CoA, mevalonate, phosphomevalonate, pyrophosphomevalonate, isopentenylpyrophosphate, dimethylacryldiphosphate, geranylpyrophosphate, and farnesylpyrophosphate, and/or a substance that inhibits the activity or formation of any one or more of HMG-CoA synthase, HMG-CoA reductase, mevalonate kinase, phosphomevalonate kinase, mevalonate diphosphate decarboxylase, and farnesyl diphosphate synthase.

14. The method of claim 11, wherein the mevalonate metabolic pathway inhibitor comprises a substance that inhibits activity of HMG-CoA reductase, or a substance that degrades HMG-CoA reductase, or a genetic tool that decreases the HMG-CoA reductase level, or any combination thereof; and/or a substance that inhibits activity of farnesyltransferase, or a substance that degrades farnesyltransferase, or a genetic tool that reduces farnesyltransferase levels; and/or a substance that inhibits activity of geranylgeranyltransferase, or a substance that degrades geranylgeranyltransferase, or a genetic tool that reduces geranylgeranyltransferase levels, or any combination thereof.

15. The method of claim 11, wherein the mevalonate metabolic pathway inhibitor comprises an HMG-CoA reductase inhibitor.

16. The method of claim 15, wherein the HMG-CoA reductase inhibitor comprises a statin compound.

17. The method of claim 15, wherein the HMG-CoA reductase inhibitor comprises a compound selected from at least one of pravastatin, fluvastatin, lovastatin, simvastatin, atorvastatin, cerivastatin, rosuvastatin, and pitavastatin calcium, or a derivative thereof having an HMG-CoA reductase inhibitory effect, or a pharmaceutically acceptable salt, solvate, tautomer, or isomer thereof

18. The method of claim 11, wherein the mevalonate metabolic pathway inhibitor comprises a farnesyltransferase inhibitor.

19. The method of claim 18, wherein the farnesyltransferase inhibitor is selected from one or more of quinolinone, benzodiazepine, and arylpyrrole; and/or the farnesyltransferase inhibitor is selected from at least one of Tipifarnib, FTI277, L-70472, J-104135, A-166120, and manumycin, or a derivative thereof having a farnesyltransferase inhibitory effect, or a pharmaceutically acceptable salt, solvate, tautomer, or isomer; and/or the farnesyltransferase inhibitor is selected from at least one of CVFM and CIFM.

20. The method of claim 18, wherein the farnesyltransferase inhibitor is a farnesyltransferase subunit FNTB inhibitor.

21. The method of claim 11, wherein the mevalonate metabolic pathway inhibitor comprises a nucleotide consisting of at least one of the sequence of: TABLE-US-00008 SEQIDNo:1:AACCCAAUGCCCAUGUUCCdTdT; SEQIDNo:2:ACGACTCGGTGGAAACAGT; and SEQIDNo:3:CGAGTTCTTTCACCTACTA.

22. The method of claim 11, wherein the mevalonate metabolic pathway inhibitor comprises at least one of Tipifarnib, FTI277, fluvastatin and atorvastatin, or a derivative thereof having a mevalonate metabolic pathway inhibitory effect, or a pharmaceutically acceptable salt, solvate, tautomer, or isomer thereof.

23. The method of claim 11, wherein the mevalonate metabolic pathway inhibitor comprises a geranylgeranyltransferase inhibitor.

24. The method of claim 11, wherein the mevalonate metabolic pathway inhibitor comprises a geranylgeranyl pyrophosphate inhibitor; and/or a geranylgeranyl diphosphate synthase 1 inhibitor; and/or Rab geranylgeranyltransferase subunit beta inhibitor.

25. The method of claim 11, wherein the mevalonate metabolic pathway inhibitor comprises at least one selected from a group consisting of an antibody, antibody functional fragment, peptide and peptoids; and/or at least one selected from a group consisting of gene interference material, gene editing material, gene silencing material and gene knockout material; and/or at least one selected from a group consisting of DNA, RNA, PNA and DNA-RNA-hybrid; and/or at least one selected from a group consisting of siRNA, dsRNA, miRNA, shRNA and ribozyme.

26. The method of claim 11, wherein the alphavirus is selected from at least one of M1 virus and Getah virus.

27. The method of claim 11, wherein a nucleotide sequence of the alphavirus has at least 97.8% homology to a nucleotide sequence of M1 virus deposited with Accession No. CCTCC V201423.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0078] FIG. 1 siRNA targeting 3-hydroxy-3-methylglutaryl-CoA reductase HMGCR and M1 virus significantly increase the morphological lesions of human intestinal cancer and pancreatic cancer cell strains;

[0079] FIG. 2 Treatment of siRNA targeting 3-hydroxy-3-methylglutaryl-CoA reductase HMGCR combined with M1 virus significantly reduces the survival rate of the human intestinal cell carcinoma strain.

[0080] FIG. 3 The statin and M1 virus significantly increase morphological lesions of human intestinal cell carcinoma strains and promote replication of M1 virus; wherein A shows a phase difference diagram of the human intestinal cell carcinoma strain after fluvastatin and M1 treatment; and B shows fluvastatin and atorvastatin promote the expression of M1 virus protein.

[0081] FIG. 4 Treatment of siRNA targeting subunit FNTB of farnesyltransferase combined with M1 virus significantly increases morphological lesions in the human intestinal cell carcinoma strain and pancreatic cancer cell strain.

[0082] FIG. 5 The farnesyltransferase inhibitors Tipifarnib, FTI277 and M1 significantly increase morphological lesions in the human intestinal cell carcinoma strain and pancreatic cancer cell strain.

[0083] FIG. 6 Treatment of the farnesyltransferase inhibitor Tipifarnib combined with M1 virus significantly reduce the survival of the human intestinal cell carcinoma strain and pancreatic cancer cell strain.

[0084] FIG. 7 Interfering the upstream pathway and four branches of downstream pathway of the mevalonate pathway to screen that blocking of farnesylation and geranylgeranylation pathways in downstream branches promotes replication of M1 virus.

[0085] FIG. 8 The farnesyltransferase inhibitor Tipifarnib combined with M1 virus significantly inhibited the growth of transplanted tumors of the human intestinal cell carcinoma strain and pancreatic cell carcinoma strain; wherein A shows a growth curve of the transplanted tumor of the human intestinal cell carcinoma strain; and B shows a growth curve of the transplanted tumor of the human pancreatic cell carcinoma strain.

[0086] FIG. 9. Mevalonic acid metabolic pathway diagram

[0087] The full names corresponding to the English abbreviations for the enzymes referred to in FIG. 9 are as follows:

[0088] HMGCR: 3 -hydroxy-3-methylglutaryl-CoA reductase

[0089] HMGCS1: 3-hydroxy-3-methylglutaryl-CoA synthase 1 HM

[0090] MVK: mevalonate kinase

[0091] PMVK: phosphomevalonate kinase

[0092] MVD: mevalonate diphosphate decarboxylase

[0093] IDI1: isopentenyl-diphosphate delta isomerase 1

[0094] FDPS: farnesyl diphosphate synthase

[0095] GGPS1: geranylgeranyl diphosphate synthase 1

[0096] DHCR 7: 7-dehydrocholesterol reductase

[0097] RAGBBTB: Rab geranylgeranyltransferase subunit beta

[0098] PGGT1B: protein geranylgeranyltransferase type I subunit beta

[0099] FNTB: farnesyltransferase, CAAX box, beta

[0100] SQLE: squalene epoxidase

DETAILED DESCRIPTION

[0101] The following embodiments further illustrate the present disclosure, but the embodiments of the present disclosure are not limited to the following examples. Any equivalent changes or modifications made in accordance with the principles or concepts of the present disclosure should be regarded as the scope of protection of the present disclosure.

[0102] Without being specifically indicated, the materials and experimental methods employed in the present disclosure are conventional materials and methods.

[0103] The term selected from in the specification is used in connection with a selected object and is to be understood as, for example: x is selected from: A, B, C, . . . E or X is selected from one or more of A, B, C, . . . and E, and the like, are understood to mean that X comprises one, or any combination of two, or any combination of more of A, B, C, . . . E. It is not excluded that X also includes some other class of substances.

[0104] In addition to the specific enzyme inhibitors mentioned above, the inhibitors of the present disclosure may be selected from specific enzyme inhibitors already known in the art, or substances found to have specific enzyme inhibition after subsequent studies. For example, with respect to farnesyltransferase inhibitors, the farnesyltransferase inhibitors of the present disclosure may also be selected from farnesyltransferase inhibitors known in the art, or substances found to have farnesyltransferase inhibitory effects upon subsequent studies. The same holds true for HMG-CoA reductase inhibitors, geranylgeranyltransferase or other specific enzyme inhibitors.

[0105] The enzymes involved in the various mevalonate metabolic pathways exemplified in the present disclosure are as follows, along with their known sequences (reported in NCBI, below are NCBI Gene IDs).

[0106] HMGCR: 3-hydroxy-3-methylglutaryl-CoA reductase ID:3156

[0107] HMGCS1: 3-hydroxy-3-methylglutaryl-CoA synthase 1 HM ID:3157

[0108] MVK: mevalonate kinase ID:4598

[0109] PMVK: phosphomevalonate kinase ID:10654

[0110] MVD: mevalonate diphosphate decarboxylase ID:4597

[0111] IDI1: isopentenyl-diphosphate delta isomerase 1 ID:3422

[0112] FDPS: farnesyl diphosphate synthase ID:2224

[0113] GGPS1: geranylgeranyl diphosphate synthase 1 ID:9453

[0114] DHCR7: 7-dehydrocholesterol reductase ID:1717

[0115] RAGBBTB: Rab geranylgeranyltransferase subunit beta ID:5876

[0116] PGGT1B: protein geranylgeranyltransferase type I subunit beta ID:5229

[0117] FNTB: farnesyltransferase, CAAX box, beta ID:2342

[0118] SQLE: squalene epoxidase ID:6713

[0119] Of course, the above sequences are not intended to be limiting. Since it is not excluded that are newly discovered and perform similar functions, or other analogs and the like, which may vary in amino acid sequence or nucleotide sequence, for example, proteins with a sequence identity of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or at least 99.5%, or at least 99.8%, they may be subsequently found to achieve similar functions, which belong to the above-mentioned analogs. Inhibitors designed for them are also within the scope of protection of the present disclosure.

EXAMPLE 1 TREATMENT OF siRNA TARGETING 3-HYDROXY-3-METHYLGLUTARYL CoA REDUCTASE (HMGCR) COMBINED WITH M1 VIRUS SIGNIFICANTLY INCREASED MORPHOLOGICAL LESIONS IN HUMAN INTESTINAL CANCER AND PANCREATIC CANCER CELL STRAINS

Materials

[0120] Human intestinal cell carcinoma strain HCT-116 (purchased from the Cell Bank of the Chinese Academy of Sciences), human pancreatic cancer cells Capan-1 (purchased from ATCC), SW 1990 (purchased from ATCC), M1 virus (Accession number CCTCC V201423), high glucose DMEM medium (purchased from Corning), and inverted phase contrast microscope.

Method

[0121] The cells were inoculated into a 35 mm culture dish, cultured until the confluence degree of the cells reaches 60% and subjected to the following interference treatment: firstly, a Lipofectamine RNAiMAX solution was prepared with Opti-MEM by diluting according to 2 L: 198 L per culture dish and uniformly mixing; secondly, an siRNA solution was prepared with Opti-MEM by diluting according to 1.8 L: 198 L per culture dish, wherein the final concentration of siRNA was 25 nM, and mixing gently; finally, the diluted Lipofectamine RNAiMAX and siRNA were mixed, and stood for 15 minutes at room temperature; the mixed solution was added into a culture dish containing 1.5 mL of serum-free culture medium; and the medium was changed to a complete medium after 24 hours, and infected with M1 virus (1MOI). The changes in cell morphology were observed after 48 h under the inverted phase contrast microscope.

[0122] The sequence of the siRNA is as follows:

TABLE-US-00003 SEQIDNo:1:AACCCAAUGCCCAUGUUCCdTdT

Result

[0123] As shown in FIG. 1, colorectal cancer cells HCT-116, pancreatic cancer cells Capan-1 and SW 1990 cells grew well adherently under the treatment of disordered interfering fragments; the interfered HMGCR did not affect the morphology of tumor cells; in the case of infection with M1 virus alone, a small number of cells were observed under the microscope to be shrunk, rounded, and enhanced in refractive index; however, after 48 hours of M1 infection, cells in the HMGCR interference group obviously showed a large number of dead cells with enhanced refractive index, floating or adhering to the culture dish.

EXAMPLE 2 TREATMENT OF siRNA TARGETING 3-HYDROXY-3-METHYLGLUTARYL-CoA REDUCTASE HMGCR COMBINED WITH M1 VIRUS SIGNIFICANTLY REDUCED THE SURVIVAL RATE OF HUMAN INTESTINAL CANCER CELL STRAINS

Materials

[0124] Human intestinal cell carcinoma strain HCT-116 (purchased from the Cell Bank of the Chinese Academy of Sciences), M1 virus (Accession number CCTCC V201423), and high glucose DMEM medium (purchased from Corning).

Method

[0125] a) Cell culture: human intestinal cell carcinoma strain HCT-116 was grown in a DMEM complete medium containing 10% FBS, 100 U/ml penicillin and 0.1 mg/ml streptomycin; all cell strains were cultured in a constant temperature closed incubator at 5% CO.sub.2, 37 C. (95% RH) and observed by the inverted microscope. Cells were passaged once for approximately 2-3 days and the cells in logarithmic growth phase were used for formal experiments.

[0126] b) The cells were inoculated into a 24-well plate at 30,000 cells/well; the cells were infected with M1 virus (MOI=1) after 24 hours of interference treatment with siRNA targeting HMGCR; 72 hours after infection, the cell survival rate was detected by MTT assay, which comprises the following steps: MTT solution was added at 100 L/well; after incubation for 3 hours at 37 C., the supernatant was aspirated off and DMSO solution was added at 1 mL/well; after shaking well, the plate was placed in a microplate reader and the absorbance was measured at 570 nm.

Result

[0127] As shown in FIG. 2, infection with M1 virus alone caused about 20% cell death in HCT-116 cells; however, infection with M1 virus can cause more than 70% cell death following interfering the HMG-CoA reductase gene (HMGCR).

[0128] The sequence of the siRNA is as follows:

TABLE-US-00004 SEQIDNo:1:AACCCAAUGCCCAUGUUCCdTdT

EXAMPLE 3 THE STATIN AND M1 VIRUS SIGNIFICANTLY INCREASED MORPHOLOGICAL LESIONS OF HUMAN INTESTINAL CELL CARCINOMA STRAINS AND PROMOTED REPLICATION OF M1 VIRUS

Materials

[0129] Human intestinal cell carcinoma strain HCT-116 (purchased from the Cell Bank of the Chinese Academy of Sciences), M1 virus (Accession number CCTCC V201423), high glucose DMEM medium (purchased from Corning), and inverted phase contrast microscope.

Method

[0130] a) Cell culture: human intestinal cell carcinoma strain HCT-116 was grown in a DMEM complete medium containing 10% FBS, 100 U/ml penicillin and 0.1 mg/ml streptomycin; all cell strains were cultured in a constant temperature closed incubator at 5% CO.sub.2, 37 C. (95% RH) and observed by the inverted microscope. Cells were passaged once for approximately 2-3 days and the cells in logarithmic growth phase were used for formal experiments.

[0131] b) Cell treatment and morphological observation: cells in logarithmic phase growth were selected, prepared as a cell suspension in DMEM complete medium (containing 10% fetal bovine serum, and 1% double antibody) and inoculated into a 24-well plate at a density of 410.sup.5/well. Cells were infected with M1 virus (MOI=1), treated with M1 virus (MOI=1) combined with fluvastatin or atorvastatin (1 and 10 M), and cellular proteins were harvested after 24 hours and immunoblotted.

[0132] c) Cells were inoculated into a 24-well plate at 30,000 cells/well; after the cells were treated with fluvastatin 2 (M), they were infected with M1 virus (MOI=1); and the changes of cell morphology were observed under the inverted phase contrast microscope after 48 hours.

Result

[0133] FIG. 3A shows that M1 alone had no significant effect on cell morphology, fluvastatin alone induced lesions in a small number of cells, and when the cells were treated with the two drugs at the same time, significant lesions in cells occur; as shown in FIG. 3B, M1 viral proteins were slightly up-regulated at a drug concentration of 1 M; when the concentration of statins was increased to 10 M, the expression of M1 viral proteins (structural protein El and non-structural protein NS3) were significantly up-regulated compared to the drug-free group. The result demonstrated that statins promote expression of M1 viral proteins.

EXAMPLE 4 TREATMENT OF siRNA TARGETING SUBUNIT FNTB OF FARNESYLTRANSFERASE COMBINED WITH M1 VIRUS SIGNIFICANTLY INCREASED MORPHOLOGICAL LESIONS IN HUMAN INTESTINAL AND PANCREATIC CANCER CELL STRAINS

Materials

[0134] Human intestinal cell carcinoma strain HCT-116 (purchased from the Cell Bank of the Chinese Academy of Sciences), human pancreatic cell carcinoma strain Capan-1 (purchased from ATCC), SW 1990 (purchased from ATCC), M1 virus (Accession number CCTCC V201423), high glucose DMEM medium (purchased from Corning), and inverted phase contrast microscope.

Method

[0135] The cells were inoculated into a 35 mm culture dish, cultured until the confluence degree of the cells reaches 60% and subjected to the following interference treatment: firstly, a Lipofectamine RNAiMAX solution was prepared with Opti-MEM by diluting according to 2 L: 198 L per culture dish and uniformly mixing; secondly, an siRNA solution was prepared with Opti-MEM by diluting according to 1.8 L: 198 L per culture dish, wherein the final siRNA concentration was 10 or 2 nM, and mixing gently; finally, the diluted Lipofectamine RNAiMAX and siRNA were mixed, and stood for 15 minutes at room temperature; the mixed solution was added into a culture dish containing 1.5 mL of serum-free culture medium; and the medium was changed to a complete medium after 24 hours, and infected with M1 virus (1MOI). The changes in cell morphology were observed after 48 h under the inverted phase contrast microscope.

[0136] The sequence of the siRNA is as follows:

TABLE-US-00005 SEQIDNo:2:ACGACTCGGTGGAAACAGT SEQIDNo:3:CGAGTTCTTTCACCTACTA

Result

[0137] As shown in FIG. 4, the colorectal cancer cells HCT-116, pancreatic cancer cells Capan-1 and SW1990 cells grew well adherently under the treatment of disordered interfering fragments; the interfered FNTB did not affect the morphology of tumor cells; in the case of infection with M1 virus alone, a small number of cells were observed under the microscope to be shrunk, rounded, and enhanced in refractive index; however, after 48 hours of M1 infection, cells in the FNTB interference group obviously showed a large number of dead cells with enhanced refractive index, floating or adhering to the culture dish.

EXAMPLE 5 THE FARNESYLTRANSFERASE INHIBITORS TIPIFARNIB, FTI277 and M1 VIRUS SIGNFICANTLY INCREASED MORPHOLOGICAL LESIONS OF HUMAN INTESTINAL CELL CARCINOMA STRAINS

Materials

[0138] Human intestinal cell carcinoma strain HCT-116 (purchased from the Cell Bank of the Chinese Academy of Sciences), M1 virus (Accession number CCTCC V201423), high glucose DMEM medium (purchased from Corning), and inverted phase contrast microscope.

Method

[0139] a) Cell culture: human intestinal cell carcinoma strain HCT-116 was grown in a DMEM complete medium containing 10% FBS, 100 U/ml penicillin and 0.1 mg/ml streptomycin; all cell strains were cultured in 5% CO2, 37 C. incubator (95% RH) and observed by the inverted microscope. Cells were passaged once for approximately 2-3 days and the cells in logarithmic growth phase were used for formal experiments.

[0140] b) Cell treatment and morphological observation: cells in logarithmic phase growth were selected, prepared as a cell suspension in DMEM complete medium (containing 10% fetal bovine serum, and 1% double antibody) and inoculated into a 24-well plate at a density of 2.510.sup.4/well. Cells were treated with Tipifarnib (1, 0.1 M) alone, FTI277 (10, 1 M) alone, M1 virus (MOI=1) infection, M1 virus (MOI=1) combined with Tipifarnib (1, 0.1 M), M1 virus (MOI=1) combined with FTI277 (10, 1 M), with no addition of M1 virus, FTI277 and Tipifarnib as a control, and the cell morphology changes were observed under the inverted phase contrast microscope after 48 hours.

Result

[0141] As shown in FIG. 5, the morphology of the cells was observed under the phase contrast microscope, HCT-116 cells were monolayer adherently grown, and the cells were closely arranged with a consistent phenotype. After 48 h of treatment with FTI1277 or Tipifarnib (50 nM) and M1 virus (MOI=1), the morphology of the cells changed significantly. Compared with the control group cells, M1 solely treatment group and other solely treatment groups, the number of cells in the combined treatment group decreased significantly, the cell body contracted into globular shape, and the refractive index increased significantly, and death-like lesions are showed.

EXAMPLE 6 TREATMENT OF THE FARNESYLTRANSFERASE INHIBITOR TIPIFARNIB COMBINED WITH M1 VIRUS SIGNIFICANTLY REDUCED THE SURVIVAL OF HUMAN INTESTINAL CANCER AND PANCREATIC CANCER CELL STRAINS

Materials

[0142] Human intestinal cell carcinoma strain HCT-116 (purchased from the Cell Bank of the Chinese Academy of Sciences), human pancreatic carcinoma cell strain SW1990 (purchased from ATCC), human normal liver cell strain L-02 (purchased from the Cell Bank of the Chinese Academy of Sciences), M1 virus (accession number CCTCC V201423), high-glucose DMEM medium (purchased from Corning), and automatic enzyme-linked detection microplate reader.

Method

[0143] a) Cells inoculation, and administration treatment: cells in logarithmic phase growth were selected, prepared as a cell suspension in DMEM complete medium (containing 10% fetal bovine serum, and 1% double-antibody) and inoculated into 96-well plates at a density of 410.sup.3/well. After 12 hours, the cells were seen completely adherent and divided into control group, Tipifarnib alone group, M1 infection group and Tipifarnib/M1 combination group. The dosages used were: M1 virus (MOI =1) infected cells: Tipifarnib (50 nM).

[0144] B) Reaction of MTT with intracellular succinate dehydrogenase: after 48 h of culture, 20 l of MTT (5 mg/ml) was added to each well and the incubation was continued for 4 h, at which point microscopic examination revealed the formation of granular blue-violet formazan crystals in living cells.

[0145] c) Dissolution of formazan particles: the supernatant was carefully aspirated off, DMSO 100 l/well was added to dissolve the crystals formed, shaken on a microshaker for 5 min, and the optical density (OD) of each well was measured on the enzyme-linked detector at a wavelength of 570 nm. Each group of experiments was repeated 3 times. Cell viability=OD of drug treated group/OD of control group100%.

Result

[0146] As shown in FIG. 6, M1 virus alone treatment had a small survival rate inhibition effect on tumor cell HCT-116 as the tumor cell survival rate reached 97.0%, the tumor cell survival rate of the 50 nM Tipifarnib treatment group still reached 80%, however, when the 50 nM Tipifarnib was used combined with M1 virus of the same MOI (Tipifarnib+M1), the tumor cell survival rate was greatly reduced to 38%; M1 virus alone treatment had a small survival rate inhibition effect on tumor cell SW190 as the tumor cell survival rate reached 90%, the tumor cell survival rate of the 50 nM Tipifarnib treatment group still reached 90%, however, when the 50 nM Tipifarnib was used combined with M1 virus of the same MOI (Tipifarnib+M1), the tumor cell survival rate was greatly reduced to about 40%; and the combined treatment had no obvious killing effect on L02 cells. It is thus demonstrated at the cellular level that the mevalonate metabolic pathway protein inhibitor can enhance the oncolytic effect of M1 virus without killing normal cells.

EXAMPLE 7 INTEFERING THE UPSTREAM PATHWAY AND FOUR BRANCHES OF DOWNSTREAM OFO THE MEVALONATE PATHWAY AND SCREENINGN FARNESYLATION AND GERANYLGERANYLATION PATHWAYS IN DOWNSTREAM BRANCHES TO BE BLOCKED TO PROMOTE REPLICATION OF M1 VIRUS

Materials

[0147] Human intestinal cell carcinoma strain HCT-116 (purchased from the Cell Bank of the Chinese Academy of Sciences), M1 virus (Accession number CCTCC V201423), high glucose DMEM medium (purchased from Corning), and inverted phase contrast microscope.

Method

[0148] The cells were inoculated into a 35 mm culture dish, cultured until the confluence degree of the cells reaches 60% and subjected to the following interference treatment: firstly, a Lipofectamine RNAiMAX solution was prepared with Opti-MEM by diluting according to 2 L: 198 L per culture dish and uniformly mixing; secondly, an siRNA solution was prepared with Opti-MEM by diluting according to 1.8 L: 198 L per culture dish, wherein the final concentration of siRNA interfering HMGCR, SQLE, FNTB, PGGT 1B and RABGGTB (specifically see FIG. 9 and the description of the Figures) were 25, 25, 4, 20 and 20 nM respectively, and mixing gently; finally, the diluted Lipofectamine RNAiMAX and siRNA were mixed, and stood for 15 minutes at room temperature; the mixed solution was added into a culture dish containing 1.5 mL of serum-free culture medium; and the medium was changed to a complete medium after 24 hours, and infected with M1 virus (1MOI). Cell lysates were collected after 24 hours and immunoblotted.

[0149] The siRNAs are as follows:

[0150] Gene for interfering HMG-CoA reductase (HMGCR)

TABLE-US-00006 SEQIDNo:1:AACCCAAUGCCCAUGUUCCdTdT

[0151] Interfering farnesyltransferase subunit FNTB

TABLE-US-00007 SEQIDNo:2:ACGACTCGGTGGAAACAGT SEQIDNo:3:CGAGTTCTTTCACCTACTA

[0152] Interfering Rab geranylgeranyltransferase subunit beta

[0153] SEQ ID No: 4 SASI-HS01-00112524 (purchased from Sigma)

[0154] Result: the expression of structural protein E1 and nonstructural protein NS3 of the virus increased significantly after the HMGCR, the farnesyltransferase subunit FNTB and the geranylgeranyltransferase RABGGTB thereof were intefered, whereas interfering the cholesterol synthesis pathways SQLE and PGGT1B had no significant effect on the viral proteins (FIG. 7).

EXAMPLE 8 TIPIFARNIB COMBINED WITH M1 VIRUS SIGNIFICANTLY INHIBITED THE GROWTH OF XENOGRAFTS OF HUMAN INTESTINAL AND PANCREATIC CELL CARCINOMA STRAINS

Materials

[0155] M1 virus (Accession number CCTCC V201423), human hepatoma cell strain HCT-116 (purchased from ATCC), human pancreatic cancer cell strain SW 1990 (purchased from ATCC), and 4 week old female BALB/c nude mice.

Method

[0156] This experiment used a randomized, single-blind design. 510.sup.6 HCT-116 or SW 1990 cells were injected subcutaneously into the dorsal flank of the 4 week old BALB/c nude mouse.

[0157] When the tumor size reached 50 mm3, the mice were divided into three groups, including untreated control group, Tipifarnib alone group (500 g/kg/d by intraperitoneal injection), M1 infection alone group (210.sup.9 PFU/kg/d by tail vein injection of M1 virus) and Tipifarnib/M1 combined group (same dose of Tipifarnib and M1 virus), and treated with 6 consecutive injections. The length, width and weight of the tumor were measured every two days, and the volume of the tumor was measured according to the formula (lengthwidth.sup.2)/2. One way ANOVA was performed after measuring tumor volume, ***indicates p<0.001.

Result

[0158] As shown in FIG. 8, tumor volumes in two tumor cell transplanted tumor animals with pathological tumor dissection showed that treatment with Tipifarnib alone and M1 alone resulted in only a slight reduction in tumor volume compared to the control group, whereas treatment with Tipifarnib/M1 in combination resulted in a significant reduction in tumor volume, and one way ANOVA showed statistical differences.

[0159] The described embodiments of the present disclosure are merely illustrative examples, and the embodiments of the present disclosure are not limited to the above, and any other changes, modifications, substitutions, combinations, and simplifications that may be made without departing from the spirit and principles of the present disclosure are intended to be equivalent and fall within the scope of the present disclosure.

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