Use of glutamine metabolism inhibitor v-9302 in the preparation of a drug for treating diseases caused by arsenic-induced cell malignant transformation

Abstract

The application relates to application of a glutamine metabolism inhibitor V-9302 in preparation of a drug for treating diseases caused by cell malignant transformation of arsenic. The application is characterized in that the glutamine metabolism inhibitor V-9302 is used to inhibit the cell malignant transformation caused by arsenic, the malignant transformation phenotype of cells is reversed after the glutamine metabolism is inhibited, the cell doubling time is increased, the migration rate and the soft agar colony formation rate are reduced, and it is proved that the enhancement of the glutamine metabolism promotes the cell malignant transformation mediated by the synthesis of glutathione.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to the application of a glutamine metabolism inhibitor, V-9302, in the preparation of a drug for treating arsenic-induced malignant cell transformation. Background Technology

[0002] Arsenic is a metalloid that is ubiquitous in the environment. Exposure to unsafe levels of arsenic usually occurs through the consumption of contaminated food or drinking water, especially over a long period of time.

[0003] Currently, the World Health Organization (WHO) and the Environmental Protection Agency (EPA) recommend a limit of 10 µg / L (10 ppb) for arsenic levels in drinking water. However, many countries, including China, Bangladesh, India, Mexico, Chile, Argentina, the United States, Vietnam, and Canada, have reported arsenic concentrations far exceeding this limit, ranging from 50 to over 1000 µg / L. People with long-term exposure to this range of arsenic have a significant risk of developing various diseases, including skin cancer, lung cancer, and cardiovascular disease. The carcinogenic potential of arsenic has been well-established in epidemiological studies and numerous experimental models. Inorganic arsenic is classified as a Group 1 carcinogen by the International Agency for Research on Cancer (IARC) and is currently ranked first on the priority list of substances proposed by the Astronomical Registry of Toxic Substances and Diseases (ASTDR). In vitro malignant transformation cell models of arsenic are commonly used to study the carcinogenic mechanisms of arsenic. Studies by Erik JTokar et al. have shown that long-term exposure of human epithelial stem cells to 5.0 mM inorganic arsenic for 18 weeks induces a malignant tumor stem cell-like phenotype, characterized by high invasiveness, loss of contact inhibition, and high secretion of matrix metalloproteinase-9 (MMP-9). Research by Yan-hong Cui et al. found that long-term treatment of HaCaT cells with 100 nM arsenic downregulated m6A methylation, inducing malignant transformation. Research by Yuan-lin Qi et al. showed that long-term treatment of BEAS-2B cells with 0.25 µM sodium arsenite induced malignant transformation. In recent years, public awareness of health issues has gradually increased, and research on the hazards of low-dose environmental pollutants has also grown annually. One case-control study on low-concentration arsenic, despite the concentration of inorganic arsenic in drinking water (<5 µg / L) being below the standard limit or arsenic levels reported in other highly polluted areas and neighboring East Asian countries, still influenced the prevalence of non-melanoma skin cancer in the population.

[0004] Cancer cells' dependence on glutamine metabolism makes them an attractive target for anticancer therapy. Currently, several chemical drugs target glutamine metabolism, focusing on the pathway from glutamine transport in cells to its conversion to α-ketoglutarate. Due to the overexpression of GLS in various cancers, several drugs have targeted GLS and demonstrated tumor-specific anti-proliferative effects, such as CB-839 and BPTES. CB-839 has entered clinical trials. Meanwhile, given the high expression of the glutamine transporter ASCT2 in various tumors, numerous compounds target ASCT2 for inhibition, such as GPNA, V-9302, MEDI 7247, and Benzylserine. However, studies on glutamine metabolism inhibitors in basal cell carcinoma or squamous cell carcinoma of the skin have not been reported. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides the application of glutamine metabolism inhibitor V-9302 in the preparation of a drug for treating arsenic-induced malignant cell transformation.

[0006] The first objective of this invention is to provide the use of glutamine metabolism inhibitor V-9302 in the preparation of a drug for treating arsenic-induced malignant cell transformation.

[0007] In one embodiment of the present invention, the disease is basal cell carcinoma or squamous cell carcinoma of the skin, and is used in a medicament.

[0008] In one embodiment of the present invention, the cells are selected from HaCaT.

[0009] In one embodiment of the invention, the drug further includes a pharmaceutically or pharmacologically acceptable carrier.

[0010] In one embodiment of the present invention, the carrier is selected from one or more of the following: disintegrant, diluent, lubricant, adhesive, humectant, flavoring agent, suspending agent, surfactant, and preservative.

[0011] In one embodiment of the present invention, the dosage form of the drug is tablets, capsules, soft capsules, granules, pills, oral liquids, emulsions, dry suspensions, dry extracts, or injections.

[0012] A second object of the present invention is to provide a pharmaceutical composition comprising the aforementioned pharmaceutical composition.

[0013] A third object of the present invention is to provide a kit comprising the aforementioned drug and the aforementioned drug composition.

[0014] A fourth object of the present invention is to provide the use of the said drug, the said pharmaceutical composition, and the said kit in the preparation of a drug for treating arsenic-induced malignant cell transformation.

[0015] In one embodiment of the present invention, the disease is basal cell carcinoma or squamous cell carcinoma of the skin, and is used in a medicament.

[0016] The technical solution of the present invention has the following advantages compared with the prior art:

[0017] This invention inhibits arsenic-induced malignant transformation of cells through treatment with the glutamine metabolism inhibitor V-9302. The malignant transformation phenotype was reversed after glutamine metabolism was inhibited, and cell doubling time, migration rate, and soft agar colony formation rate were increased, indicating that enhanced glutamine metabolism mediates glutathione synthesis and promotes malignant transformation. The HaCaT cell malignant transformation model simulates the development and progression of basal cell carcinoma and squamous cell carcinoma of the skin. Summary of the Invention

[0018] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings, wherein...

[0019] Figures 1-3 This invention describes the changes in indicators of NaAsO2-induced malignant transformation of HaCaT cells.

[0020] Figure 4 This diagram shows the changes in ASCT2 and GCLC protein expression in T-HaCaT cells after treatment with V-9302 according to the present invention. In the figure: Figure A shows the ASCT2 and GCLC protein bands; Figure B shows the quantitative analysis of ASCT2 protein; and Figure C shows the quantitative analysis of GCLC protein. * P <0.05: The difference was statistically significant compared with the normal passaged control group cells; # P <0.05: The difference was statistically significant compared with the solvent control group.

[0021] Figure 5 This figure shows the changes in the levels of glutamine metabolites after T-HaCaT cells were treated with V-9302 according to the present invention. In the figure: after T-HaCaT cells were treated with V-9302: Figure A represents the glutamine utilization rate of cells, Figure B represents the glutamate content of cells, Figure C represents the glycine content of cells, Figure D represents the cysteine ​​content of cells, and Figure E represents the total GSH content of cells. * P <0.05: The difference was statistically significant compared with the normal passaged control group cells; #P <0.05: The difference was statistically significant compared with the solvent control group.

[0022] Figure 6 This figure shows the changes in malignant phenotype-related indicators after T-HaCaT cells were treated with V-9302 according to the present invention. In the figure: After V-9302 treatment of T-HaCaT cells: Figure A shows the change in cell doubling time; Figures B and C show the changes in cell migration ability; Figures D and E show the changes in cell colony formation ability on whole agar. * P <0.05: The difference was statistically significant compared with the normal passaged control group cells; # P <0.05: The difference was statistically significant compared with the solvent control group. Detailed Implementation

[0023] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0024] (I) Model Establishment

[0025] (1) The establishment of the HaCaT cell malignancy model by treating HaCaT cells with 0.1 µM NaAsO2 is as follows:

[0026] HaCaT cells were treated with 0.0 or 0.1 µM NaAsO2 for up to 35 passages. Cell morphology was observed using an inverted microscope. The results are as follows: Figure 1 As shown in -A, the cell morphology of the 35th generation infected group changed compared to the passaged control group. Specifically, the cell volume decreased, the cell boundaries became less clear, the cell arrangement became disordered, and the cell morphology became irregular polygonal, with an increased frequency of multinucleated giant cells. Changes in doubling time at generations 21, 28, and 35 were also examined, and the results are as follows: Figure 1 As shown in -B, the cell doubling time of the 21st and 28th generation virus-treated groups was not significantly different from that of the passage control group, while the cell doubling time of the 35th generation virus-treated group was significantly reduced. P <0.05). Cell migration ability, such as Figure 2 As shown in -C and 3-E, the 0.1 µM NaAsO2 treated group had a longer cell migration distance at passage 35 compared to the passaged control group, and the calculated cell migration rate was also significantly higher than that of the passaged control group; at the same time, the number of cells in the 0.1 µM NaAsO2 treated group that crossed the transwell at passage 35 was significantly increased. P <0.05). Soft agar colony-forming ability as... Figure 2As shown in Figure D, the number of soft agar clones in the 0.1 µM NaAsO2 treatment group increased after 35 passages, and the clone formation rate was significantly higher than that of the passaged control group. P <0.05). Cell invasion ability, such as Figure 3 As shown in Figure -F, the number of cells that passed through a matrix gel-treated transwell after 35 passages in the 0.1 µM NaAsO2 treatment group was significantly increased. P <0.05).

[0027] (2) After HaCaT cells were treated with 0.1 μM NaAsO2 for 35 generations, as described in Part II, the cells underwent malignant transformation, characterized by changes in cell morphology, enhanced soft agar colony formation ability, significantly shortened cell doubling time, and significantly increased cell migration and invasion abilities. Therefore, the cells that underwent malignant transformation at this time were defined as HaCaT cells that underwent malignant transformation with 0.1 μM NaAsO2 (T-HaCaT).

[0028] (ii) All reagents and materials used in this invention are commercially available products.

[0029] (1) The HaCaT cell line was purchased from the China Center for Type Culture Collection;

[0030] (2) V-9302 (HY-112683, MCE, USA), and other materials involved are commercially available products.

[0031] Example 1: Treatment of T-HaCaT cells with V-9302 drug

[0032] 1) Seeding: Normal passaged control cells and T-HaCaT cells were digested and collected to prepare a cell suspension. The required number of cells per well in a six-well plate was 2 × 10⁶ cells. 5 The cells are then seeded into plates. After the cells have reached 70-80% confluence in the six-well plates, further drug treatment is performed.

[0033] 2) Dosing: The treatment group and the control group are as shown in the table below:

[0034] 1-HaCaT

[0035] 2,3,4-T-HaCaT.

[0036]

[0037] 3) After drug treatment, continue culturing the cells for 24 hours, and then collect the cells to detect subsequent indicators.

[0038] Example 2: Changes in GCLC and ASCT2 proteins in T-HaCaT cells after treatment with the glutamine metabolism inhibitor V-9302.

[0039] (I) Western blot detection of protein expression

[0040] (1) SDS-PAGE gel preparation

[0041] Following the instructions of the Yamei gel preparation kit, add the liquids from the kit sequentially to prepare the lower gel (separating gel) and the upper gel (stacking gel). When preparing the upper gel, insert a 10-well comb to form lanes. After the gel solidifies, subsequent electrophoresis can be performed.

[0042] (2) Electrophoresis

[0043] After calculating the loading volume of each group of samples based on the volume determined by BCA concentration, the samples are added to the lanes of the upper gel in a specific order. Care should be taken to prevent sample overflow from the lanes, which could cause errors in the loading volume. Simultaneously, 8 μL of protein marker should be added to the lanes on both sides of the sample edge. After all samples have been loaded, 1× electrophoresis buffer is added to the electrophoresis tank, the power is turned on, and electrophoresis is performed. Generally, electrophoresis is performed at a constant voltage of 75 V for 45 minutes, then adjusted to 120 V and continued until the end.

[0044] (3) Transfer membrane

[0045] The 1× transfer buffer should be prepared in advance and refrigerated. Cut the PVDF membrane to the same size as the prepared gel, and pre-soak it in anhydrous methanol for 1 min to activate the PVDF membrane. Simultaneously, immerse the "transfer sandwich clamp" in the 1× transfer buffer, placing the gel and membrane in a "black gel, white membrane" configuration, ensuring no air bubbles exist between them. Press the transfer clamp firmly into the transfer tank, add the 1× transfer buffer, and transfer at a constant current of 200 mA for 120 min.

[0046] (4) Closed

[0047] After the transfer is complete, remove the PVDF membrane from the "transfer sandwich clamp" and place it in the blocking solution. Incubate for 60 min at room temperature on a shaker.

[0048] (5) Primary antibody incubation

[0049] Dilute the antibody with primary antibody diluent according to the dilution ratio specified in the antibody's instructions. Cut a PVDF membrane to obtain a corresponding target protein band based on the molecular weight indicated by the protein marker and the molecular weight of the target protein. Incubate the target protein band overnight in an incubator containing the appropriate antibody diluent at 4ºC with gentle shaking on a shaker.

[0050] (6) Washing

[0051] Take the incubation box that has been incubated overnight to recover the primary antibody, and wash the membrane 5 times with 1×TBST, 5 min each time.

[0052] (7) Secondary antibody incubation

[0053] The secondary antibody was diluted with 1×TBST according to the manufacturer's instructions (1:1000). The washed protein bands were then incubated in the secondary antibody at room temperature for 60 min.

[0054] (8) Washing

[0055] Wash the membrane 4-5 times with 1×TBST, 5 minutes each time.

[0056] (9) Development

[0057] Prepare the developing solution according to the ratio of solution A to solution B = 1:1, mix well, and then drop it evenly onto the PVDF membrane. At the same time, quickly place the protein band into the gel imaging instrument, adjust the parameters, and expose it.

[0058] (10) Data Analysis

[0059] The exposure results were analyzed for grayscale values ​​using ImageJ software, and the results were statistically processed. Changes in GCLC and ASCT2 protein levels after treatment of T-HaCaT cells with the glutamine metabolism inhibitor V-9302: Results are shown below. Figure 4 As shown, the expression of GCLC and ASCT2 proteins in the T-HaCaT cell group was significantly increased compared with that in the passaged control group ( P <0.05%, after using the glutamine metabolism inhibitor V-9302 on T-HaCaT cells, GCLC expression did not change compared with the solvent control group, while ASCT2 protein expression was significantly reduced. P <0.05). The results suggest that ASCT2, as a target protein of V-9302, has been successfully inhibited.

[0060] (II) GSH and GSSG Testing

[0061] 1) Prepare the following reagents according to the kit instructions:

[0062]

[0063] 2) Cell Collection: Cells from key monitoring points in the normal passaged control group and the 0.1 μM arsenic-treated group were collected. After washing the cells once with PBS, the cells were collected into centrifuge tubes and centrifuged at 1,000 g, discarding the supernatant. 30 μL of protein removal reagent M was added to every 10 μL of cell pellet (the weight of the 10 μL pellet can be estimated using the weight of the cell pellet; the weight of the cell pellet should be measured separately for the centrifuge tubes before and after cell collection). The samples underwent two rapid freeze-thaw cycles (liquid nitrogen and a 37°C water bath) and were then incubated on ice for 5 min. Afterward, the samples were centrifuged at 4°C, 10,000 g for 10 min, and the supernatant was collected for subsequent analysis.

[0064] 3) Measurement of GSSG in samples: Take a portion of the sample and add diluted GSH removal auxiliary solution and removal working solution, as shown in the table below:

[0065]

[0066] 4) Add each component according to the table below:

[0067]

[0068] 5) The absorbance values ​​of each group were measured using a microplate reader at a wavelength of 412 nm. The results of changes in the content of glutamine metabolites after treatment of T-HaCaT cells with the glutamine metabolism inhibitor V-9302 are shown in [the table below]. Figure 5 .like Figure 5 As shown, the glutamine utilization rate and the levels of intracellular glutamate, glycine, cysteine, and total GSH in the T-HaCaT cell group were significantly higher than those in the passaged control group. P <0.05%, after using the glutamine metabolism inhibitor V-9302 on T-HaCaT cells, compared with the solvent control group, the glutamine utilization rate and the contents of glutamate, glycine, cysteine ​​and total GSH in the cells were significantly decreased. P <0.05). The results suggest that V-9302 treatment inhibited glutamine metabolism in cells, and its mediated glutathione synthesis was also suppressed.

[0069] Example 3: Cell doubling time detection

[0070] 1) After digesting four groups of cells—HaCaT cells, T-HaCaT cells, T-HaCaT cells + DMSO, and T-HaCaT cells + V-9302 + DMSO—cell suspensions were prepared and counted. Each group was seeded into 24-well plates with 10,000 cells per well.

[0071] 2) Every 24 hours, cells from 3 wells in each group were collected and counted separately, with each well counted three times. Counting was performed for two consecutive days. The calculation formula was: TD = (t × lg2) / (lg Nh - lg Ni). Experimental results are shown below. Figure 6 .

[0072] Example 4: Cell Scratch Assay

[0073] After digesting four groups of cells—HaCaT cells, T-HaCaT cells, T-HaCaT cells + DMSO, and T-HaCaT cells + V-9302 + DMSO—the cells were collected, counted, and the cell counts were standardized. Then, each group of cells was seeded into a 6-well plate with 3 replicates per group. The plates were then placed in an incubator for further culture.

[0074] 1) HaCaT cells from passage 35 of the normal passage control group and the 0.1 μM NaAsO2 treatment group were collected as cell suspensions, seeded into plates, and the cell count was adjusted to ensure that the number of cells per well of a six-well plate was 2.5 × 10⁶. 5 Each group was set up with three replicates to ensure that the cells could cover the bottom of the dish after 24 hours of culture.

[0075] 2) After incubating the six-well plate in an incubator for 24 h, use a 200 μL pipette tip perpendicular to the bottom of the six-well plate to perform a scratching operation. The scratching force should be consistent and the scratch lines should be smooth straight lines.

[0076] 3) After the scratching is completed, discard the culture medium with a pipette, add PBS to gently wash the cells and aspirate as much PBS as possible, add serum-free culture medium, and observe and photograph the scratches on the well plate with an inverted microscope. Then continue to incubate in an incubator.

[0077] 4) After 48 h of culture, observation and photography were performed under an inverted microscope. Three fields of view of the scratch at the center of each well were selected for photography, and ImageJ software was used for quantitative analysis of the scratch images.

[0078] 5) Calculate cell migration rate based on software analysis data. Cell migration rate indicates the speed at which cells migrate. Cell migration rate = (g0 - gt) / g0, where g0 represents the distance measured in the "exposed" area of ​​the scratch when the scratch image is taken immediately after scratching, and gt represents the distance measured in the "exposed" area of ​​the scratch when the scratch image is taken 48 hours after scratching.

[0079] Example 5: Soft agar cloning experiment

[0080] 1) Prepare a 1.4% agarose solution using double-distilled water and agarose powder, and autoclave it before incubating it in a 42°C water bath for a certain period of time. Mix the 1.4% agarose solution with an equal volume of 2×DMEM complete culture medium to obtain a 0.7% agarose solution. Pipette 2 mL of the 0.7% agarose solution into a 35 mm diameter petri dish (add slowly to prevent the formation of air bubbles), and allow it to solidify at room temperature to obtain the bottom layer gel.

[0081] 2) Collect HaCaT cells, T-HaCaT cells, T-HaCaT cells + DMSO, and T-HaCaT cells + V-9302 + DMSO4 groups of cells. Mix an equal volume of cell suspension containing 5000 cells with an equal volume of 0.7% agarose solution, mixing slowly and avoiding the formation of bubbles, to obtain a 0.35% agarose cell suspension.

[0082] 3) Slowly add 1.5 mL of 0.35% agarose cell suspension to the bottom layer gel. After solidification, the top layer gel is obtained.

[0083] 4) After the upper gel solidifies, add 1 mL of DMEM high-glucose complete medium to the wells. Place the plate in an incubator and continue culturing for about 4 weeks (the medium needs to be changed every two weeks).

[0084] 5) Observe under an inverted microscope and count the clonal colonies with a diameter greater than 50 μm.

[0085] 6) Soft agar colony formation rate = number of colonies / number of inoculated cells.

[0086] Example 6: Statistical Analysis.

[0087] All experimental data are expressed as mean ± standard deviation. SPSS 19.0 statistical software was used for data analysis. Between-group comparisons were performed using the homogeneity of variance test and one-way ANOVA. For pairwise comparisons, the Student-Newman-Keuls (SNK) test was used if variances were homogeneous; otherwise, the Games-Howell test was used. The significance level was set at α = 0.05. P <0.05 indicates a statistically significant difference. Statistical charts were generated using Graphpad Prism 8 software. Changes in malignant phenotype-related indicators after treatment of T-HaCaT cells with the glutamine metabolism inhibitor V-9302.

[0088] The results are as follows Figure 6 As shown, the cell doubling time of the T-HaCaT cell group was significantly lower than that of the passaged control group, while the cell doubling time of the V-9302-treated T-HaCaT cell group was increased and significantly higher than that of the solvent control group.P <0.05). Changes in cell migration ability, such as Figure 6 As shown in Figure B, the cell migration rate of the T-HaCaT cell group was significantly higher than that of the passaged control group, while the cell migration rate of the V-9302-treated T-HaCaT cell group was significantly lower than that of the solvent control group. P <0.05). This suggests that V-9302 treatment inhibited cell migration. Figure 6 As shown in Figure C, T-HaCaT cells formed a large number of distinct clonal colonies in soft agar. Furthermore, the colony formation rate of T-HaCaT cells in soft agar was higher than that of the passaged control group. After treatment with V-9302, the number of clonal colonies formed in soft agar decreased, and the colony formation rate in soft agar was significantly lower than that in the solvent control group. P <0.05). The above results suggest that V-9302 treatment inhibited the malignant transformation of cells, as evidenced by the reversal of the malignant transformation phenotype after the inhibition of glutamine metabolism, and the enhanced glutamine metabolism mediating glutathione synthesis and promoting the malignant transformation of cells.

[0089] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. Use of glutamine metabolism inhibitor V-9302 in the manufacture of a medicament for treating a disease of arsenic-induced malignant transformation of HaCaT cells, which is skin basal cell carcinoma or skin squamous cell carcinoma.

2. Use according to claim 1, characterized in that, The medicament further comprises a pharmaceutically or pharmacologically acceptable carrier.

3. Use according to claim 2, characterized in that, The carrier is selected from one or more of disintegrants, diluents, lubricants, binders, humectants, flavoring agents, suspending agents, surfactants and preservatives.

4. Use according to claim 1, characterized in that, The dosage form of the medicament is tablet, capsule, granule, pill, oral solution, emulsion, dry suspension, dry extract or injection.

5. Use of a kit in the manufacture of a medicament for treating a disease of arsenic-induced malignant transformation of HaCaT cells, which is skin basal cell carcinoma or skin squamous cell carcinoma, the kit comprising glutamine metabolism inhibitor V-9302.

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