CN110638839A

CN110638839A - Application of recombinant salmonella VNP20009-M in preparation of medicine for treating osteosarcoma

Info

Publication number: CN110638839A
Application number: CN201911046352.XA
Authority: CN
Inventors: 赵子建; 张馨丹; 李芳红; 赵正刚; 李美蓉; 萧耿苗
Original assignee: Guangdong University of Technology
Current assignee: Guangdong University of Technology
Priority date: 2019-10-30
Filing date: 2019-10-30
Publication date: 2020-01-03

Abstract

The invention relates to application of a genetically engineered bacterium VNP20009-M in preparation of a medicine for treating osteosarcoma, which comprises induction of apoptosis, inhibition of cell proliferation and migration, and belongs to the field of tumor biology. The present invention utilizes attenuated salmonella as a vector carrying a gene for methionine enzyme in order to specifically deplete methionine in osteosarcoma to inhibit the growth of osteosarcoma. The genetically engineered bacterium VNP20009-M can obviously inhibit the proliferation of tumor cells and the growth of osteosarcoma in vivo, indicates that the genetically engineered bacterium VNP20009-M is expected to become an important target for anti-osteosarcoma proliferation research, provides a foundation for preparing anti-osteosarcoma proliferation medicines, has good development and application prospects, and has potential clinical application values. The administration mode of the genetically engineered bacteria is preferably intratumoral injection.

Description

Application of recombinant salmonella VNP20009-M in preparation of medicine for treating osteosarcoma

Technical Field

The invention relates to application of a genetically engineered bacterium VNP20009-M in preparation of a medicine for treating osteosarcoma, which comprises induction of apoptosis, inhibition of cell proliferation and migration, and belongs to the field of tumor biology.

Background

Osteosarcoma (OS) is one of the most common malignant bone tumors, the incidence rate of the osteosarcoma is the first of primary bone tumors, and the osteosarcoma is better to be found in teenagers of 0-24 years old, and the osteosarcoma is the next of the old, wherein the incidence rate and the incidence age of the teenagers are relatively stable, and the incidence rate and the incidence age of the old are relatively large in variation. Amputation was the most common method of treating OS before the 70's of the 20 th century, but the 5-year survival rate of OS patients treated with this method has not exceeded 20%. With the progress of surgical operation technology, the proposal of new adjuvant chemotherapy concept and the improvement of the treatment level of postoperative chemoradiotherapy, the traditional amputation is gradually replaced by the combination of new adjuvant chemotherapy and limb protection operation, and the survival rate is obviously improved. However, in the study of the last 30 years, OS patients are treated by the combination of neoadjuvant chemotherapy and limb-protecting surgery, and the 5-year survival rate is about 70 percent and is not changed significantly. Despite increasing the duration of chemotherapy, increasing the dose of chemotherapeutic drugs, or the use of immunotherapy, patient improvement remains limited. In recent years, new methods for treating OS are continuously appeared, and new ideas are provided for treating OS by using new methods such as immunotherapy, TSC (TSC-induced systemic hepatitis C) pathway therapy, targeted therapy, gene therapy and the like, but the new ideas are still required to completely overcome the new ideas of scientific researchers and clinicians of various countries in the world. Therefore, the new drugs for treating osteosarcoma are still in the market shortage state, and the development of the new drugs has extremely important practical significance.

Salmonella is a group of gram-negative, invasive, intracellular facultative anaerobes that parasitize in the gut of humans and animals. VNP20009 is an attenuated salmonella typhimurium strain with msbB and purI gene deletion, and is stable in heredity and sensitive to antibiotics. The msbB gene is necessary for the acylation of lipid to endotoxin, and the deletion of the msbB gene prevents the acylation of the tail end of the lipid A, thereby reducing the toxicity; the purI gene is involved in purine metabolism, and its deletion requires exogenous gonadal purine for bacterial reproduction. VNP20009 also reduces Tumor Necrosis Factor (TNF) produced by the auto-induced body, thereby reducing inflammatory responses. Thus, its low pathogenicity enhances safety for clinical treatment. VNP20009 has been widely used in cancer research and it can act on a variety of mouse solid tumor models, including melanoma, lung, colon, breast, kidney. One major advantage of VNP20009 as a tumor gene therapy vector is its ability to highly target the accumulation at the tumor site. Researchers found that the number of VNP20009 in tumors was 200-to l 000-fold higher than that in major organs such as liver in mouse models of various solid tumors. VNP20009 can be preferentially gathered and propagated in the hypoxic necrosis area of tumor tissue, and the amplification times of bacteria in the tumor tissue are obviously higher than those of normal tissue in the same time, so that attenuated salmonella becomes a novel anti-tumor preparation and a carrier for tumor targeted therapy. Possible mechanisms by which salmonella cause a reduction in tumor growth include: nutrients required for tumor growth are consumed by bacteria, and enzymes produced by the bacteria, such as asparaginase, can exhaust amino acids essential for tumor growth; local toxins secreted by bacteria into the extracellular microenvironment or produced tumor necrosis factor alpha can affect tumor angiogenesis; in addition, non-specific inflammatory reactions at the site of bacterial growth can potentially activate anti-tumor T cells.

Tumor cells require sufficient nutrients to maintain their high proliferation rate, and in addition to sugars, Methionine (Met), glutamine, arginine, and the like are required in particularly large amounts. Studies have shown that Met dependence is a common feature of most tumor cells, such as breast, lung, colon, kidney, bladder, melanoma, glioma, etc., while normal cells do not have Met dependence. Several in vivo and in vitro experiments have successively demonstrated that the direct consumption of methionine-deficient diets can delay the proliferation of tumor cells. However, long-term deficiency or insufficiency of Met in the diet can cause malnutrition and metabolic disturbance of the body, and can also aggravate canceration due to the fact that DNA is in a hypomethylation state for a long time. Then, by specifically decomposing Met by methioninase (L-methioninase), thereby reducing methionine levels in vivo, tumor cell growth can be more effectively inhibited or regressed. Animal experiments have demonstrated that intraperitoneal injection of methioninase can inhibit the growth of sarcoma of gytian and lung tumors in nude mice. Clinical trials have shown that methioninase can significantly reduce the methionine content in plasma by intravenous injection of methioninase every 24h for four patients with breast, lung, kidney and lymphoma tumors, respectively. However, since mammals do not express methioninase, exogenous administration has a certain side effect and often causes immune reaction in the body.

Disclosure of Invention

The invention aims to solve the technical problem of providing the application of the genetically engineered bacterium VNP20009-M in preparing a medicament for treating osteosarcoma, which is specifically shown in the aspects of inhibiting the proliferation and migration of the genetically engineered bacterium and inducing the apoptosis of osteosarcoma cells.

In order to solve the technical problems, the invention adopts the following technical scheme:

the application of the genetically engineered bacterium VNP20009-M in preparing the medicine for inhibiting the proliferation and migration of osteosarcoma and inducing the apoptosis of osteosarcoma cells is within the protection range of the invention.

The invention utilizes in vitro tumor cell culture and in vivo nude mouse subcutaneous tumor, and researches the pathophysiology effect of VNP20009-M on osteosarcoma cells through cell proliferation experiments, cell migration experiments and tumor volume observation by intratumoral injection. The experiment proves that: the genetic engineering bacteria VNP20009-M and osteosarcoma cells can be co-cultured to remarkably induce apoptosis; the over-expression of methioninase can obviously inhibit the proliferative capacity of human osteosarcoma cells MNNG-HOS; obviously reducing the migration capability of MNNG-HOS, U2OS and SaoS-2; VNP20009-M treatment can obviously inhibit the growth of subcutaneous tumor-bearing model tumor.

The genetically engineered bacterium VNP20009-M is derived from the attenuated salmonella typhimurium of the invention patent and the application of the genetically engineered bacterium thereof in preparing a medicament for treating pancreatic cancer, and has the patent number of 201310688936.3.

Has the advantages that: the present invention utilizes attenuated salmonella as a vector carrying a gene for methionine enzyme in order to specifically deplete methionine in osteosarcoma to inhibit the growth of osteosarcoma. The genetically engineered bacterium VNP20009-M can obviously inhibit the proliferation of tumor cells and the growth of osteosarcoma in vivo, indicates that the genetically engineered bacterium VNP20009-M is expected to become an important target for anti-osteosarcoma proliferation research, provides a foundation for preparing anti-osteosarcoma proliferation medicines, has good development and application prospects, and has potential clinical application values. The administration mode of the genetically engineered bacteria is preferably intratumoral injection.

Drawings

FIG. 1 is a schematic diagram of the induction of apoptosis of genetically engineered bacteria VNP20009-M, PBS in control group and bacteria control group after co-culture with osteosarcoma cells. All data are expressed as mean ± standard deviation. (n-3), P <0.05, P <0.01, P <0.001and P <0.0001, all compared to the solvent control.

FIG. 2 is a graph showing the ability of cells to migrate after overexpression of methioninase by U2OS, Saos-2, MNNG-HOS. (n ═ 3),. P <0.05,. P <0.01,. P <0.001 and. P <0.0001 were compared to solvent control B.

Fig. 3 is a schematic representation of the inhibition of proliferation of osteosarcoma cells by overexpressing the methioninase genes (n ═ 3),. P <0.05,. P <0.01,. P <0.001 and. P <0.0001 compared to solvent control B.

FIG. 4 is a schematic diagram showing the growth of a nude mouse subcutaneous osteosarcoma planttumor after treatment by genetically engineered bacteria VNP 20009-M. All data are expressed as mean ± standard deviation. (n-3), P <0.05, P <0.01, P <0.001and P <0.0001, all compared to the solvent control.

Detailed Description

In order to make the present invention more clear and intuitive for those skilled in the art, the present invention will be further described with reference to the accompanying drawings.

Example 1

Flow cytometry for detecting induction effect of VNP20009-M on osteosarcoma apoptosis

1) Collecting MNNG-HOS osteosarcoma cells in logarithmic growth phase at 2 x 10⁵Each cell/ml is inoculated in 6-well culture plates, and each well is 2 ml;

2) after the plates are paved, respectively adding bacteria with experimental concentration, wherein the ratio of bacteria to cells is 1:50, setting a PBS control group and a bacteria control group, and continuously culturing the cells for 4 hours;

3) after 4h the cells were harvested, washed 2 times with cold PBS and prepared 1X 10 with 1. multidentatingbuffer⁶one/mL cell suspension, 100u L in flow tube, according to the kit instructions add 5 u L7 AA-D and 5 u L PE staining, gently vortex cells, room temperature light-proof incubation 15min, then 400u L BindingBuffer in the tube, within 1h for flow cell detection, FlowJoV10 analysis software analysis results.

The results are shown in FIG. 1: VNP20009-M can remarkably induce MNNG-HOS osteosarcoma cell apoptosis.

Example 2

After overexpression of methioninase by U2OS, Saos-2, MNNG-HOS, the cell migration ability was examined.

1) Single cell suspensions were prepared from plasmid transfected cells MNNG-HOS, U2OS, Saos-2 at 2 x 10 per well⁵200ul of each was inoculated on a transwell chamber. Totally divided into 3 groups, namely a complete control group A, a plasmid transfection control group B and a plasmid transfection group C;

2) a medium content of 20% FBS acts as a chemoattractant in the lower chamber. Non-invasive cells were removed with a cotton swab. The invading cells were stained with crystal violet and microscopic images were taken to quantify the invading cells.

The results are shown in FIG. 2: the migratory capacity of osteosarcoma cells is significantly reduced. CON is complete control group, EGFP is plasmid control group, MEGL is plasmid group carrying methioninase gene (experimental group).

Example 3

The CCK8 method measures the effect of overexpression of the methionine enzyme gene on osteosarcoma cell proliferation.

1) Single cell suspensions were prepared from MNNG-HOS cells in logarithmic growth phase. 2ml of cell suspension (containing 2X 10 cells) per well⁵Individual cells) were inoculated in 6-well plates and divided into 3 groups in total, namely a complete control group A, a plasmid transfection control group B and a plasmid transfection group C;

2) after the plates are paved, adding the liposome, the mixed solution of the liposome and the control plasmid and the mixed solution of the liposome and the plasmid carrying the methioninase into each component respectively, and continuously culturing the cells for 24 hours;

3) the cells after plasmid transfection MNNG-HOS are taken to prepare single cell suspension, and 100ul of cell suspension (containing 2 multiplied by 10) is added in each well³Individual cells) are inoculated in a 96-well plate and divided into 3 groups in total, namely a complete control group A, a plasmid transfection control group B and a plasmid transfection group C; and each large group is provided with 3 detection holes of 24h,48h,72h and 96 h.

3) Adding 10ul of CCK-8 solution into each hole for 24h,48h,72h and 96h respectively to avoid generating bubbles;

4) the cells were incubated for an additional 1-2 hours, the plates were removed and the absorbance at 450uM was measured using a microplate reader.

The results are shown in FIG. 3: the proliferative capacity of the osteosarcoma cells MNNG-HOS was significantly reduced at various time points.

Experimental example 4

And (3) constructing a nude mouse subcutaneous tumor-bearing model, and observing the treatment effect of VNP20009-M on solid tumors.

1) Single cell suspensions were prepared from MNNG-HOS cells in logarithmic growth phase. Osteosarcoma cells MNNG-HOS were cultured in DMEM medium containing 10% fetal bovine serum in a cell count of 4.5X 10⁶One was inoculated subcutaneously in the right axilla of nude mice. Every 2 to 3 days, the state of the mice was observed, and the tumor size (volume: 0.52 × length × width) was measured with a vernier caliper²)。

2) Grouping and dosing regimen for laboratory animals

When the tumor volume reaches 60-80 mm³When the tumor-bearing nude mice are divided into 3 groups (n is 8) randomly, the tumor-bearing nude mice are administrated once by adopting an intratumoral injection mode, PBS with the same volume is injected into a control group, the weight of the mice is continuously measured, the activity state of the mice is observed, the size of the tumors of the mice is measured by a vernier caliper regularly, the tumor volume is calculated, and the growth curve of the tumors is drawn.

(1) PBS group: an equal volume of PBS was given intratumorally as a blank control.

(2) Drug control group: 10 μ l of 2X 106 CFU/VNP 20009 was administered intratumorally once.

(3) VNP20009-M group: 10 μ l of 2X 106 CFU/SGN 1 were administered intratumorally once.

3) Determination of body weight in tumor-bearing mice

And measuring and recording the body weight of the tumor-bearing mice every 2 to 3 days.

The results are shown in FIG. 4: the tumor becomes smaller obviously, and VNP20009-M inhibits the growth of subcutaneous tumor obviously.

The research results show that the gene engineering bacterium VNP20009-M adopted by the invention can inhibit the proliferation and migration of osteosarcoma cells; inhibiting the growth of osteosarcoma in vivo; has good anti-tumor effect.

The embodiments described above are presented to enable those skilled in the art to make and use the invention. It will be readily apparent to those skilled in the art that various modifications to these embodiments may be made, and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the embodiments described herein, and those skilled in the art should make improvements and modifications to the present invention based on the disclosure of the present invention within the protection scope of the present invention.

Claims

1. Application of recombinant salmonella VNP20009-M in preparation of drugs for inducing osteosarcoma apoptosis.

2. Application of recombinant salmonella VNP20009-M in preparing medicine for inhibiting osteosarcoma cell proliferation is provided.

3. Application of recombinant salmonella VNP20009-M in preparation of drugs for inhibiting osteosarcoma cell migration.

4. The use according to any one of claims 1 to 3, wherein the administration is by intratumoral injection.