Application of genetically engineered bacteria VNP20009-M in the preparation of drugs for preventing and treating cancer metastasis

Abstract

The present invention discloses an application of a genetically engineered bacterium VNP20009-M of attenuated Salmonella typhimurium in the preparation of a drug for preventing and treating post-metastatic cancer. The genetically engineered bacterium VNP20009-M has tumor targeting and significant effects of inhibiting metastasis and growth on cancer cells, and can be used for the preparation of a drug for preventing and treating post-metastatic tumors.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to application of genetically engineered bacteria VNP20009-M in preparing medicines for preventing and treating cancer. Background Art

[0002] Malignant tumors are a major disease that threatens human health and patient survival in today's society. According to statistics from the World Health Organization (WHO), in 2012, there were about 14 million new cancer patients and about 8.2 million deaths from cancer worldwide. According to data released by the my country Cancer Registry Center in 2012, there were about 3.5 million new cancer cases in China each year, and about 2.5 million deaths. Among them, tumor metastasis is the main cause of death in cancer patients. Tumor metastasis is a complex, multi-step biological process. When cancer cells in a tumor patient develop to a certain extent, they will be transferred to distant sites through the circulatory system and survive, forming tumors of the same type as the primary tumor. Metastasis, as the main malignant manifestation of tumors, seriously affects the efficacy and prognosis of tumor patients. When the primary tumor is clinically diagnosed, about 50% of patients have already developed metastasis. Tumor metastasis is the biggest killer threatening the survival of tumor patients and the biggest bottleneck in tumor treatment. In today's wide variety of anti-cancer drug market, most of the first-line chemotherapy drugs in clinical practice (such as doxorubicin, doxorubicin, paclitaxel, etc.) act on the survival, proliferation, and vascular survival of tumors. Drugs that specifically target tumor metastasis are relatively rare, and the few anti-tumor metastasis drugs available have limited efficacy due to their large side effects and patient drug resistance. Therefore, anti-tumor metastasis drugs have long been in a state of severe shortage in the market, and the development of drugs for the treatment of tumor metastasis is of great practical significance.

[0003] Histone methyltransferase EZH2 is an active component of histone methyltransferase in the polycomb repressive complex 2 (PRC2). It participates in chromatin condensation by adding three methyl groups to lysine 27 (H3K27) of histone 3, thereby inhibiting the transcription of related genes (such as tumor suppressor genes). Studies have found that EZH2 and histone H3K27 methylation are closely related to cancer. EZH2 was first found to be highly expressed in lymphoma, metastatic prostate cancer and breast cancer, and is associated with the infiltration of breast cancer. In addition, EZH2 is overexpressed in many human malignant tumors such as lung cancer, lymphoma, leukemia, pancreatic cancer, cervical cancer, intestinal cancer, liver cancer, gastric cancer, melanoma, kidney cancer, bladder cancer, etc., and its expression level is significantly increased in metastatic tumors, which is closely related to the poor prognosis of cancer patients. Preclinical models of drugs targeting EZH2 have shown that they can inhibit the progression of brain cancer and prostate cancer. Therefore, EZH2 can be used as a potential drug target for the treatment of cancer metastasis, by reducing the expression and activity of EZH2, thereby reducing histone methylation and enhancing the expression of tumor suppressor genes to treat tumors.

[0004] With the development of targeted and genetic engineering technologies for bacteria and viruses, research on bacterial treatment of tumors has increased rapidly since the mid-1990s. Researchers have found that Salmonella typhimurium can be used as a good gene carrier, which can kill tumor cells in mice in a targeted and effective manner. Salmonella is a group of Gram-negative, invasive intracellular facultative anaerobes that parasitize the intestines of humans and animals. VNP20009 is an attenuated Salmonella typhimurium strain with msb B and pur I genes deleted. It is genetically stable and sensitive to antibiotics. The msb B gene is necessary for lipid acylation to form endotoxins. Its deletion prevents the A end of lipids from being acylated, reducing toxicity; the pur I gene is involved in purine metabolism. Its deletion requires exogenous adenine for bacterial reproduction. VNP20009 also reduces the tumor necrosis factor (TNF) produced by the body itself, thereby reducing inflammatory responses. Therefore, its low pathogenicity improves the safety of clinical treatment. VNP20009 has been widely used in cancer research. It can act on a variety of mouse solid tumor models, including melanoma, lung cancer, colon cancer, breast cancer, and kidney cancer. A major advantage of VNP20009 as a tumor gene therapy vector is that it can be highly targeted and accumulated in the tumor site. In mouse models of various solid tumors, researchers found that the number of VNP20009 in the tumor was 200 to 1000 times higher than that in major organs such as the liver. VNP20009 can preferentially accumulate and multiply in the hypoxic necrotic area of ​​tumor tissue. The bacterial amplification generation in tumor tissue is significantly higher than that in normal tissue in the same period of time, making it possible for attenuated Salmonella to become a new anti-tumor agent and a carrier for tumor targeted therapy. Possible mechanisms by which Salmonella slows tumor growth include: nutrients required for tumor growth are consumed by bacteria, enzymes produced by bacteria, such as asparaginase, can deplete essential amino acids for tumor growth; local toxins secreted by bacteria into the extracellular microenvironment or tumor necrosis factor α produced can affect tumor angiogenesis; in addition, nonspecific inflammatory responses at the site of bacterial growth can potentially activate anti-tumor T cells. However, the inhibitory effect of VNP20009 on tumor metastasis has not yet been found.

[0005] In order to maintain their high proliferation rate, tumor cells need sufficient nutrition. In addition to sugar, the demand for methionine (Met), glutamine, arginine, etc. is particularly large. Studies have confirmed that methionine dependence is a common feature of most tumor cells, such as breast cancer, prostate cancer, lung cancer, colon cancer, kidney cancer, bladder cancer, melanoma, glioma, etc., while normal cells do not have Met dependence. Some in vivo and in vitro experiments have successively confirmed that directly consuming a methionine-deficient diet can delay the proliferation of tumor cells. However, if Met is lacking or insufficient in the diet for a long time, it will cause malnutrition and metabolic disorders in the body, and it can also aggravate cancer due to the long-term hypomethylation of DNA. Then, by specifically decomposing Met through methioninase (L-methioninase), thereby reducing methionine in the body, it can more effectively inhibit the growth of tumor cells or make them disappear. However, since mammals do not express methioninase themselves, the exogenous administration method has certain side effects and often causes immune responses in the body. Methionine is an essential amino acid. Under the catalysis of methionine adenosyltransferase, S-adenosylmethionine (SAM) is generated. SAM, also known as active methionine, is the most important direct methyl donor in the body and participates in the methyl transfer catalytic reaction of various substances such as DNA and protein in the body. Among them, EZH2 can transfer the active methyl of SAM to specific amino acids of histones, thereby participating in the epigenetic modification of chromosomes and inhibiting the transcription of related genes. We have previously prepared attenuated Salmonella VNP20009-M expressing methioninase, which can promote cell apoptosis in breast cancer, pancreatic cancer, and prostate cancer, and has a good anti-tumor effect. We have submitted patent applications (patent numbers 201310062253.7, 201310688936.3, and 201410183149.8), of which 201310062253.7 has been authorized. Methionine enzyme decomposition reduces the methionine content in the body, which can not only promote the apoptosis of tumor cells, but also has the potential to effectively inhibit the activity of EZH2 by reducing the SAM content, thereby inhibiting tumor metastasis. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide an application of the genetically engineered bacteria VNP20009-M of attenuated Salmonella typhimurium in the preparation of biological medicines for preventing and treating cancer metastasis.

[0007] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0008] The invention discloses an application of genetically engineered bacteria VNP20009-M in the preparation of drugs for preventing and treating post-metastatic cancer, wherein the genetically engineered bacteria is attenuated Salmonella typhimurium VNP20009 cloned with L-methioninase gene.

[0009] The genetically engineered bacterium VNP20009-M is preferably attenuated Salmonella typhimurium VNP20009 carrying a plasmid, wherein the L-methioninase gene is cloned on the plasmid.

[0010] The genetically engineered bacteria VNP20009-M is constructed according to the following method: the L-methioninase gene is subcloned into a plasmid to obtain an L-methioninase expression plasmid, and the L-methioninase expression plasmid is electroporated into attenuated Salmonella typhimurium VNP20009.

[0011] Wherein, the plasmid includes but is not limited to pSVSPORT plasmid, pTrc99A plasmid, pcDNA3.1 plasmid, pBR322 plasmid or pET23a plasmid.

[0012] Most preferably, in the process of constructing the genetically engineered bacterium VNP20009-M, when the pSVSPORT plasmid is selected, the L-methioninase gene is subcloned into the plasmid to obtain the L-methioninase expression plasmid, and then the L-methioninase expression plasmid is electroporated into the attenuated Salmonella typhimurium VNP20009 to obtain the genetically engineered bacterium.

[0013] The electroconversion conditions are as follows: voltage 2400V, resistance 400Ω, capacitance 25μF, and discharge time 4ms.

[0014] The cancer includes, but is not limited to, lung cancer, breast cancer, prostate cancer, liver cancer, pancreatic cancer, colon cancer, rectal cancer, stomach cancer, esophageal cancer, laryngeal cancer, leukemia, lymphoma, melanoma, uterine cancer, ovarian cancer, skin cancer, bronchial cancer, bronchiolar cancer, urethral cancer, kidney cancer, oral cancer, vaginal cancer, bile duct cancer, bladder cancer or nasopharyngeal cancer.

[0015] The above-mentioned cancer prevention and treatment administration methods can be administered through various routes, including but not limited to: oral administration, local administration, injection administration (including but not limited to intravenous, peritoneal, subcutaneous, intramuscular, intratumoral administration), etc.

[0016] The invention discloses an application of a genetically engineered bacterium VNP20009-M of attenuated Salmonella typhimurium in preparing a histone methyltransferase EZH2 inhibitor. The genetically engineered bacterium is an attenuated Salmonella typhimurium VNP20009 cloned with an L-methioninase gene.

[0017] The genetically engineered bacterium VNP20009-M is preferably attenuated Salmonella typhimurium VNP20009 carrying a plasmid, wherein the L-methioninase gene is cloned on the plasmid.

[0018] The L-methioninase and the genetically engineered bacteria VNP20009-M are constructed according to the following method: the L-methioninase gene is subcloned into a plasmid to obtain an L-methioninase expression plasmid, the L-methioninase expression plasmid is transformed into Escherichia coli for purification to obtain methioninase protein, or is electro-transformed into attenuated Salmonella typhimurium VNP20009 to obtain VNP20009-M.

[0019] Wherein, the plasmid includes but is not limited to pSVSPORT plasmid, pTrc99A plasmid, pcDNA3.1 plasmid, pBR322 plasmid or pET23a plasmid.

[0020] Most preferably, in the process of constructing the genetically engineered bacterium VNP20009-M, when the pSVSPORT plasmid is selected, the L-methioninase gene is subcloned into the plasmid through the Kpn I and Hind III restriction sites to obtain the L-methioninase expression plasmid, and then the L-methioninase expression plasmid is electroporated into the attenuated Salmonella typhimurium VNP20009 to obtain the genetically engineered bacterium.

[0021] The electroconversion conditions are as follows: voltage 2400V, resistance 400Ω, capacitance 25μF, and discharge time 4ms.

[0022] The above-mentioned histone methyltransferase EZH2 inhibitor can be administered through various routes, including but not limited to: oral, topical, injection (including but not limited to intravenous, peritoneal, subcutaneous, intramuscular, intratumoral administration), etc.

[0023] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0024] (1) The genetically engineered bacterium VNP20009-M not only has anti-tumor effects, but can also effectively inhibit tumor metastasis.

[0025] (2) The attenuated Salmonella typhimurium (VNP20009-M) expressing methioninase provided by the present invention inhibits the expression and activity of methyltransferase EZH2, becomes an inhibitor of methyltransferase EZH2, reduces the methylation level of histone H3K27, and thus inhibits the invasion and metastasis of tumors. It has broad prospects for application in the prevention and treatment of malignant tumors, especially the metastasis of cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is the 1% agarose gel electrophoresis diagram of plasmid pSVSPORT-L-methioninase digestion identification.

[0027] Figure 2 This is a diagram showing the expression results of methioninase identified by Western blot.

[0028] Figure 3 This is a graph showing the results of detecting methioninase activity in Salmonella.

[0029] Figure 4 Transwells detection showed that the genetically engineered bacteria VNP20009-M inhibited the invasion of prostate cancer cells (***, p<0.001).

[0030] Figure 5 Transwells assay showed that methioninase inhibited the invasion of prostate cancer cells (***, p<0.001).

[0031] Figure 6 This is the result of Western blot identification of EZH2 and H3K27me3 expression in prostate cancer cell PC-3 cells after high expression of methioninase.

[0032] Figure 7 This is the result of quantitative analysis of free methionine content in prostate cancer tumor tissue by high performance liquid chromatography-tandem mass spectrometry (*, p<0.05).

[0033] Figure 8 This is the result of quantitative analysis of adenosylmethionine (SAM) content in prostate cancer tumor tissue by high performance liquid chromatography-tandem mass spectrometry (**, p<0.01, *, p<0.05).

[0034] Fig. 9 This is a graph showing the effect of Salmonella injection on the body weight of nude mice.

[0035] Fig.10 The number of intravenous administrations is 2*10 5 In vivo imaging of prostate cancer signal changes over time after CFU Salmonella.

[0036] Fig.11 The number of intravenous administrations is 2*10 5 Prostate cancer signal change curve after CFU Salmonella.

[0037] Fig.12 The number of intravenous administrations is 2*10 5 Figure 3. Tumor size of the spine of prostate cancer mice 55 days after CFU Salmonella.

[0038] Fig.13 The number of intravenous administrations is 2*10 5 Results of HE pathological sections of the spine of mice with prostate cancer 55 days after CFU of Salmonella.

[0039] Fig.14 The number of intravenous administrations is 2*10 5 Results of HE pathological sections of the testis and epididymis of prostate cancer mice 55 days after CFU Salmonella.

[0040] Fig.15 The number of intravenous administrations is 2*10 6 In vivo imaging of pancreatic cancer signals over time was used to observe the changes in pancreatic cancer signals after CFU Salmonella infection.

[0041] Fig.16 The number of intravenous administrations is 2*10 5 In vivo imaging of CFU Salmonella to observe changes in breast cancer signals over time.

[0042] Fig.17 The number of intravenous administrations is 2*10 5 Signal change curve of breast cancer mice after CFU Salmonella infection.

[0043] Fig.18 The number of intravenous administrations is 2*10 6 HE pathological section results of the lungs of mice with breast cancer after CFU Salmonella infection. The arrows indicate the invasion of breast cancer tumors in the lung tissues of mice.

[0044] Fig.19 The number of intravenous administrations is 2*10 6 Survival rate in mouse lung cancer model after CFU Salmonella.

[0045] Fig. 20 The number of intravenous administrations is 2*10 6 The results of mouse lung tumors after CFU Salmonella infection. The arrows indicate the mouse lung tumors.

[0046] Fig.21 The number of intravenous administrations is 2*10 5 Figure 3. Tumor size results of the mouse model of liver cancer after CFU Salmonella.

[0047] Fig. 22 The number of intravenous administrations is 2*10 5 Lung pathological section results of the CFU Salmonella post-liver cancer mouse model. The arrows indicate the invasion of liver cancer tumors in the lung tissue of the mouse. DETAILED DESCRIPTION

[0048] The present invention can be better understood according to the following examples. However, it is easy for those skilled in the art to understand that the contents described in the examples are only used to illustrate the present invention, and should not and will not limit the present invention described in detail in the claims.

[0049] Example 1: Construction of genetically engineered bacteria.

[0050] (1) Construction of a plasmid expressing the L-methioninase gene.

[0051] First, synthesize the L-methioninase (GenBank: L43133.1) gene and subclone it into the pUC57 plasmid (GenScript), and then subclone it into the pSVSPORT plasmid (Invitrogen) through the Kpn I and Hind III restriction sites to obtain the pSVSPORT-L-methioninase expression plasmid. The specific construction process is as follows:

[0052] The pSVSPORT plasmid was double-digested with Kpn I and Hind III. The digestion system was: 2 μg plasmid DNA, 3 μL 10× buffer, 1.5 μL Kpn I enzyme, 1.5 μL Hind III enzyme, and ddH 2 The volume was made up to 30 μL with 1% HO and incubated at 37°C for 3 h. The enzyme digestion system was then separated by electrophoresis in a 1% agarose gel, a 4.1 kb DNA band was cut out, and the DNA was purified using a gel recovery and purification kit.

[0053] The DNA fragment encoding the L-methioninase region was obtained by total gene synthesis and subcloned into the pUC57 plasmid (GenScript). The fragment was double-digested with Kpn I and Hind III. The digestion system was as follows: 3 μg plasmid DNA, 3 μL 10× buffer, 1.5 μL Kpn I enzyme, 1.5 μL Hind III enzyme, and ddH 2 Add 0 to make up the volume to 30 μL and incubate at 37°C for 3 h. Then separate the enzyme digestion system by electrophoresis in 1% agarose gel, cut out a 1.2 kb DNA band, and purify the DNA using a gel recovery purification kit.

[0054] pSVSPORT (Kpn I / Hind III) and the L-methioninase coding region DNA fragment (Kpn I / HindIII) were ligated. 2 μL of vector, 6 μL of insert fragment, and 1 μL of T4 DNA ligase were added to the ligation reaction and incubated at 16°C for 16 h.

[0055] Transform the ligation product into competent cells of E.coli DH5α (Takara). Take a tube of 50μL of DH5α competent cells and place it on ice. After it melts, add 5μL of the above ligation product to it, flick it to mix, and then incubate on ice for 30min; heat shock at 42℃ for 60s, and then stand on ice for 2min; add 500μL of LB liquid culture medium without resistance, shake and culture at 37℃ for 1h, then spread it on the LB culture medium plate containing ampicillin resistance and culture overnight.

[0056] After the clones grow out, pick a single clone colony and put it into 3 mL of LB culture medium containing ampicillin, culture it at 37℃ with shaking for 16 hours, extract the plasmid DNA, and identify it with Kpn I and Hind III restriction enzymes. Two DNA bands of 4.1 kb and 1.2 kb can be obtained in the positive clones, such as Figure 1 As shown. Sequencing was then performed to further confirm that the sequence of the positive clone was completely correct.

[0057] (2) Constructing VNP20009 bacteria carrying a plasmid and VNP20009 bacteria carrying a plasmid cloned with the gene for L-methioninase.

[0058] The pSVSPORT and pSVSPORT-L-methioninase expression plasmids were electroporated into the VNP20009 strain (YS1646, ATCC No. 202165), respectively, and named VNP20009-V and VNP20009-M. The specific construction process is as follows:

[0059] Place the competent bacteria VNP20009 on ice, wait for it to melt, move it into the pre-cooled electroporation cup, add 2μL of plasmid, flick to mix, and incubate on ice for 1min. Place the electroporation cup in the electroporator, and set the conditions to 2400V voltage, 400Ω resistance, 25μF capacitance, and 4ms discharge time. Immediately after the electroporation, add 1mL of SOC medium and mix gently. Incubate at 37℃ with shaking for 1h; use a pipette to blow the bacterial precipitate evenly and spread it on the LB-O medium plate containing ampicillin resistance. Then place the plate in a 37℃ incubator for 16h. After VNP20009-V and VNP20009-M were cultured with LB-O, the plasmid was extracted and the enzyme digestion was correct.

[0060] Take 1×10 8Proteins were extracted from Salmonella using protein lysis buffer, and subjected to 10% SDS-PAGE electrophoresis. The membranes were then transferred to PVDF membranes in a constant pressure ice bath. After BSA blocking at room temperature for 1 hour, the membranes were rinsed with TBST for 3×5 minutes, and rabbit anti-L-methioninase antibody (1:1000) was added. The membranes were incubated at 4°C overnight. The membranes were rinsed with TBST for 3 times, each for 5 minutes, and then HRP-labeled anti-rabbit secondary antibody (1:10000) was added. The membranes were incubated at room temperature for 1 hour, and then rinsed with TBST for 3 times, each for 5 minutes. The membranes were developed using the ECL chemiluminescence method. The results are shown in Figure 2. Figure 2 As shown, there is a specific band at a molecular weight of about 43kD, indicating that the expression level of L-methioninase in VNP20009-M is significantly increased compared with VNP20009 and VNP20009-V.

[0061] L-methionine and pyridoxal were mixed with VNP20009-V and VNP20009-M respectively, incubated at 37°C for 10 min, terminated with 50% trichloroacetic acid, centrifuged and collected the supernatant, mixed thoroughly with 3-methyl-2-benzothiazolinone hydrazone hydrochloride hydrate (MBTH), incubated at 50°C for 30 min, and the absorbance at 320 nm was measured. The amount of enzyme that catalyzes the conversion of 1 μmol α-ketobutyrate per minute was defined as 1 enzyme activity unit. The results showed ( Figure 3 ), the methioninase activity of Salmonella VNP20009-M was 10 times higher than that of VNP20009-V.

[0062] Example 2: Anti-tumor metastasis effect of genetically engineered bacteria VNP20009-M.

[0063] 1. Effect of genetically engineered bacteria VNP20009-M on the invasive ability of prostate cancer cells. Prostate cancer cells PC-3 (androgen-independent) in the logarithmic growth phase were inoculated in a 6-well plate. When the cell density grew to about 70%-80%, 1*10 6 VNP20009-V and VNP20009-M were added to each well, and a blank control group was set up. After the bacteria and cells were co-cultured for 30 min, they were washed 5 times with PBS, and then the cells were digested with trypsin, collected by centrifugation, counted, and counted according to 5*10 4 / Chamber: Add cells to a transwell chamber covered with matrigel gel. Use serum-free culture medium in the upper chamber and culture medium containing 20% ​​fetal bovine serum in the lower chamber. Both PBS and culture medium contain 12μg / mL penicillin, 20μg / mL streptomycin, and 10μg / mL kanamycin. After 36 hours of cell culture, crystal violet staining was performed, and the cells that penetrated the chamber were photographed and counted. The counting results are shown in Figure 4, ***P<0.001. The results showed that after VNP20009-M was co-cultured with prostate cancer cells, the number of cells that could pass through the chamber was significantly reduced compared with the PBS group and the VNP20009-M group, indicating that VNP20009-M had a significant inhibitory effect on the invasion ability of tumor cells.

[0064] 2. Effect of methioninase on the invasion ability of prostate cancer cells. Prostate cancer cells PC-3 in the logarithmic growth phase were inoculated in a 6-well plate. When the cell density grew to about 70%-80%, Roche transfection reagent was used to transfect the empty vector and the plasmid with high expression of methioninase, and a blank control group was set up. 24 hours after transfection, a transwell invasion experiment was performed, and the operation method was the same as above. The results are as follows Figure 5 As shown in the figure, after high expression of methionine, the number of prostate cancer cells passing through the chamber decreased, indicating that the cell invasion ability decreased. Total cell protein was extracted 48 hours after transfection, and western blot was performed to detect histone methyltransferase EZH2. Nuclear protein was extracted 72 hours after transfection, and western blot was performed to detect the downstream target molecule of EZH2, histone H3K27. The results are shown in the figure. Figure 6 As shown, after high expression of methioninease, the expression of methyltransferase EZH2 decreased and the methylation level of histone H3K27 decreased, that is, the activity of EZH2 was significantly reduced.

[0065] 3. Subcutaneous injection 2*10 6 Personal prostate cancer cell PC-3. When the tumor volume reaches 0.1-0.2cm 3 The tumor-bearing nude mice were randomly divided into PBS group, VNP20009-V group and VNP20009-M group. 6 The dose of CFU / mouse was administered by intratumoral injection, and the control group was injected with the same volume of PBS. On the 10th day after administration, the nude mouse tumors were removed, the tumor tissue proteins were extracted, and the free methionine content and adenosylmethionine content in the tumor tissue were quantitatively analyzed using high-performance liquid chromatography-tandem mass spectrometry. The results showed that after administration of Salmonella VNP20009-M, the free methionine content in the tumor tissue was significantly reduced, about 3 / 4 of the PBS group and VNP20009-V group ( Figure 7 ), and the content of adenosylmethionine was also significantly reduced, about 2 / 3 of that in the PBS group and the VNP20009-V group ( Figure 8 ). These results suggest that VNP20009-M can be used as a good EZH2 inhibitor, which can effectively reduce the content of methionine and adenosylmethionine in tissues and effectively inhibit the activity of EZH2 by reducing the content of SAM.

[0066] The above experiments suggest that after VNP20009-M invades tumor cells, it expresses methioninease, which may reduce the expression and activity of histone methyltransferase EZH2, thereby reducing the invasion and metastasis ability of tumor cells.

[0067] 4. Construct a tumor cell line expressing luciferase. Take 293FT cells in good condition and in the logarithmic growth phase and inoculate them into six-well plates and culture them overnight at 37°C and 5% CO. 2 The next day, 293FT cells were transfected with Roche transfection reagent, and 1.5 μg PLJM1-luc, 1.125 μg psPAX2, and 0.375 μg PMD2.G plasmid were co-transfected into each well. 48 hours after transfection, the cell culture supernatant was collected, centrifuged at 4°C, 3000 r / min for 5 minutes, and the supernatant was stored at -80°C, which is the luciferase lentivirus. Prostate cancer cells PC-3 and pancreatic cancer cells CFPAC-1 were inoculated in 6-well plates, and 1 ml (about 1.0×10 8 Infection titer) luciferase lentivirus and polybrene at a final concentration of 8μg / ml. After 24 hours, puromycin was added to the culture medium for selection, and the medium was changed every 2 days. After 2 weeks, tumor cell lines PC-3-luc and CFPAC-luc that stably expressed luciferase were obtained.

[0068] 5. Culture PC-3-luc cells in F-12K medium containing 10% fetal bovine serum at a rate of 5×10 5 The cells were injected into nude mice through the tail vein. The tumor-bearing nude mice were randomly divided into PBS group, VNP20009-V group and VNP20009-M group, with 10 mice in each group.

[0069] 6. Use LB-O to culture VNP20009-V and VNP20009-M. When OD ≈ 0.6, collect the cells and resuspend them in PBS at 2 × 10 5 The mice were injected with the same volume of PBS as the control group. After administration, the activity, food intake and body weight of the nude mice were observed. Fig. 9 As shown in the figure, the weight of mice was not affected after the injection of bacteria. There was no abnormality in the feeding and feces of nude mice, indicating that VNP20009-V and VNP20009-M had no obvious toxicity to nude mice.

[0070] 7. The metastasis and growth of mouse tumors were observed using a small animal in vivo imaging system (Caliper) at 1, 11, 24, 44, and 56 days after administration. Each mouse was intraperitoneally injected with 150 μg / kg D-luciferin potassium, which reacted with the luciferase expressed by tumor cells to produce optical signals. The images were displayed using an instrument to show the location and number of tumor cells. The results are shown in Figure 2. Fig.10 As shown in the figure, one day after administration, the tumor signal intensity of each group was basically the same. However, starting from the 11th day after administration, strong signals appeared in the spine and epididymis of mice in the PBS group and VNP20009-V group, and the whole body signals of these two groups of mice increased linearly over time ( Fig.11 ). From the 45th day, soybean-sized protrusions were observed on the spines of mice in the PBS group. They were hard and had poor mobility. After dissection, tumor masses were found on the spines of nude mice. The metastatic tumors were gray-white and protruded on the surface of the lumbar vertebrae in a mass shape. The diameter was about 1 cm ( Fig.12 The signal intensity of tumor cells in the VNP20009-M group was significantly lower than that in the PBS and VNP20009-V groups, and the light signal was almost undetectable in some mice. Fig.13 ) Consistent with the results of small animal in vivo imaging, the spinal medullary cavity of mice in the PBS group and the VNP20009-V group contained densely packed undifferentiated epithelial tumor cells, which were disorderly arranged, with obvious heteromorphism, large and darkly stained nuclei, and polymorphic and uneven chromatin in the nuclei. Some tumor cells had destroyed the spinal bone. At the same time, a large number of tumor cells were found infiltrating the testicles and epididymis of the two groups of mice ( Fig.14 ). However, only a small amount of tumor cells were observed in the spine, testicles, and epididymis of mice in the VNP20009-M group, and even no tumor cells were observed in some mice. In addition, no obvious tumor infiltration was observed in the lungs, livers, kidneys and other tissues of mice in each group. These results show that Salmonella VNP20009-M can significantly inhibit the metastasis and growth of prostate cancer tumors to bones.

[0071] 8. The operation method is the same as above 5, 6 and 7. The same number of pancreatic cancer cells CFPAC-luc were injected into the tail vein. On the third day after modeling, 2*10 6 The dose of CFU / mouse was administered by tail vein injection, and the control group was injected with the same volume of PBS. Similarly, in vivo imaging was performed regularly to observe tumor metastasis. The results showed that both the PBS group and the VNP20009-V group showed strong fluorescent signals, mainly in the lungs. However, no signal was observed in the VNP20009-M group ( Fig.15 ). These results suggest that Salmonella VNP20009-M can also effectively inhibit the metastasis and growth of pancreatic cancer tumors.

[0072] 9. The operation method is the same as above 5, 6 and 7. Inject 1.25*10 5 breast cancer cells MDA-MB-231-luc. On the third day after modeling, 2*10 5The dose of CFU / mouse was administered by tail vein injection, and the control group was injected with the same volume of PBS. Similarly, in vivo imaging was performed regularly to observe tumor metastasis. The results showed that both the PBS group and the VNP20009-V group showed strong fluorescent signals, mainly located in the lungs, and the signal intensity increased significantly over time ( Fig.16 , Fig.17 ). The pathological section results of mice were consistent with the results of in vivo imaging. The lungs of the PBS and VNP20009-V groups were infiltrated with tumor cells. These cells had large and darkly stained nuclei and obvious heteromorphism ( Fig.18 Arrows indicate tumor cells). In the VNP20009-M group, almost no signal was observed, and the pathological sections of the lungs showed that the number of tumor cells was significantly reduced, and even no tumors were observed. In addition, the spine of the mice in the PBS group also had tumor metastasis, and no abnormalities were found in other tissues. These results also suggest that Salmonella VNP20009-M can also effectively inhibit the metastasis and growth of breast cancer tumors.

[0073] 10. The operation method is the same as above 5 and 6. Inject 5*10 5 On the third day after modeling, 2*10 6 The dose of CFU / mouse was administered by tail vein injection, and the control group was injected with the same volume of PBS. After administration, the weight of the mice was measured regularly, the condition of the mice was observed, and the time of death of the mice was recorded. The results showed that all the mice in the PBS group died after 38 days, 40% of the mice in the VNP20009 group survived, and 60% of the mice in the VNP20009-M group survived. The preliminary results of the survival rate experiment showed that VNP20009-M can significantly prolong the survival of tumor-bearing mice ( Fig.19 ). After the mice were dissected, it was found that the mice in the PBS group and the VNP20009-V group had severe lung tumors, while the metastatic lesions in the lungs of the mice in the VNP20009-M group were significantly smaller and less in number than those in the control group ( Fig. 20 ). These results indicate that Salmonella VNP20009-M can also effectively inhibit the growth and metastasis of lung cancer tumors.

[0074] 11. Subcutaneous injection 2*10 6 On the third day after modeling, the tumor-bearing nude mice were randomly divided into PBS group, VNP20009-V group and VNP20009-M group. 5 The dose of CFU / mouse was administered by tail vein injection, and the control group was injected with the same volume of PBS. The results showed that the tumors of the mice in the control group grew normally and rapidly, but after administration of Salmonella VNP20009-M, the tumor growth stagnated and the tumors disappeared in some mice ( Fig.21). Pathological sections of the mouse lungs showed that the lungs of the PBS and VNP20009-V groups were infiltrated with tumor cells, and the nuclei of these cells were large and darkly stained, with obvious heteromorphism. Fig. 22 The arrows indicate tumor cells. However, the number of tumor cells in the lungs of the VNP20009-M group was significantly reduced, and even no tumors were observed. These results also suggest that Salmonella VNP20009-M can also effectively inhibit the metastasis and growth of liver cancer tumors.

[0075] The above animal experiments further illustrate that the attenuated Salmonella typhimurium VNP20009-M used in the present invention can inhibit tumor metastasis by inhibiting the expression and activity of EZH2, and has a good anti-tumor effect.

[0076] The present invention shows that attenuated Salmonella typhimurium VNP20009-M can be used to prepare drugs for treating anti-tumor metastasis. In addition to prostate cancer, breast cancer, pancreatic cancer, lung cancer and liver cancer, other tumors include liver cancer, colon cancer, rectal cancer, gastric cancer, esophageal cancer, laryngeal cancer, leukemia, lymphoma, melanoma, uterine cancer, ovarian cancer, skin cancer. The metastasis of bronchial cancer, bronchiolar cancer, urethral cancer, kidney cancer, oral cancer, vaginal cancer, bile duct cancer, bladder cancer, nasopharyngeal cancer and various other cancers may all be effective. The above plasmid is not limited to the pSVSPORT plasmid, and the pTrc99A plasmid, pcDNA3.1 plasmid, pBR322 plasmid or pET23a plasmid and the above plasmid cloned with L-methioninase gene also have similar effects.

Claims

1. Use of genetically engineered bacteria VNP20009-M in the preparation of histone methyltransferase EZH2 inhibitors, wherein the genetically engineered bacteria VNP20009-M is attenuated Salmonella typhimurium VNP20009 cloned with L-methioninase gene.

2. The use according to claim 1, characterized in that: The genetically engineered bacteria VNP20009-M is an attenuated Salmonella typhimurium VNP20009 carrying a plasmid, wherein the L-methioninase gene is cloned on the plasmid.

3. The use according to claim 2, characterized in that: The genetically engineered bacteria VNP20009-M is constructed according to the following method: the L-methioninase gene is subcloned into a plasmid to obtain an L-methioninase expression plasmid, and the L-methioninase expression plasmid is electroporated into attenuated Salmonella typhimurium VNP20009.

4. The use according to claim 2 or 3, characterized in that: The plasmid includes but is not limited to pSVSPORT plasmid, pTrc99A plasmid, pcDNA3.1 plasmid, pBR322 plasmid or pET23a plasmid.

5. The use according to claim 1, characterized in that: The inhibitor is administered orally, topically or by injection.

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