Application of SIRT5 inhibitor 1 in treatment of Philadelphia chromosome positive acute lymphocytic leukemia

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to application of SIRT5inhibitor 1 in treatment of Philadelphia chromosome positive acute lymphocytic leukemia. The invention proves that the SIRT5inhibitor 1 can be used for specifically inhibiting the expression of the SIRT5 of the Ph + ALL cell; it is found for the first time that the SIRT5inhibitor 1 can weaken the Ph + ALL cell proliferation capacity, the cell cycle is retarded in the G2 / M period, and the apoptosis level is remarkably increased; it is found that the SIRT5inhibitor 1 enables the mitochondrial membrane potential level of the Ph + ALL cells to be obviously reduced, respiratory chain electron leakage is caused, reactive oxygen species (ROS) in the cells is increased, and then cell death is promoted; when the NAC is used for resisting the ROS effect, the effect of the SIRT5inhibitor 1 for causing apoptosis of the Ph + ALL cells is reversed, and it is further proved that the SIRT5inhibitor 1 promotes cell death by increasing the ROS in the Ph + ALL cells. The invention aims to provide a new strategy and method for treating Ph + ALL.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical research, and in particular relates to application of SIRT5 inhibitor 1 in treating Philadelphia chromosome-positive acute lymphoblastic leukemia. Background Art

[0002] Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ALL) is a malignant proliferative disease originating from hematopoietic stem / progenitor cells. Its typical feature is the chromosomal translocation t(9;22)(q34;q11), which leads to the fusion of the q34 region of the BCR gene and the q11 region of the ABL1 gene, forming the BCR::ABL1 fusion gene, which is the key pathogenic gene of Ph+ALL and has potential tyrosine kinase activity, promoting the malignant proliferation and survival of lymphocytes, leading to the development of leukemia. Currently, the main treatment for Ph+ALL is TKIs combined with chemotherapy, which can significantly improve the prognosis of patients. However, Ph+ALL has a high relapse rate, a short disease-free survival time and a poor prognosis. For relapsed or refractory Ph+ALL, clinical trials and new drug research are urgently needed to provide new treatment hope.

[0003] We performed proteomic sequencing and KEGG pathway enrichment analysis on Ph+ALL and other leukemia cells and found that many of the top 20 major enriched pathways involved metabolism. At the same time, bioinformatics was used to analyze the differentially expressed molecules related to energy metabolism in SUP-B15 and other leukemia cell lines, among which the differential expression level of SIRT5 was the most significant. In addition, it has been reported in the literature that in the mitochondria where SIRT3-5 proteins are located, a variety of metabolic processes including glucose metabolism can occur (see DeBerardinis, RJ & Keshari, KRMetabolic analysis as a driver for discovery, diagnosis, and therapy. Cell 185, 2678–2689 (2022).). After testing and verifying the mRNA and protein levels of SIRT5 molecules in various leukemia cells, it was found that SIRT5 was indeed more expressed in Ph+ALL. Therefore, we used SIRT5 inhibitor 1 to specifically inhibit the expression of SIRT5. Tests confirmed that SIRT5inhibitor 1 can specifically inhibit the expression of SIRT5 in Ph+ALL cells. Therefore, we further explored the effects of SIRT5inhibitor 1 on the proliferation, cycle, and apoptosis of Ph+ALL cells, as well as the related mechanisms, in order to provide a basis and experimental basis for SIRT5inhibitor 1 as a new option for the treatment of Ph+ALL. Summary of the invention

[0004] In order to solve the above technical problems, the present invention provides a new strategy to enhance the therapeutic effect of Ph+ALL.

[0005] The purpose of the present invention is achieved through the following technical solutions:

[0006] The embodiments of the present invention provide an experimental verification of the use of SIRT5 inhibitor 1 in the treatment of Philadelphia chromosome-positive acute lymphoblastic leukemia.

[0007] Furthermore, the drug is compound 49.

[0008] Furthermore, the drug is used for Ph+ALL.

[0009] Furthermore, the drug can weaken the proliferation ability of Ph+ALL cells, block the cell cycle at the G2 / M phase, and promote cell death by significantly increasing the intracellular ROS level.

[0010] Compared with the prior art, the present invention has the following advantages:

[0011] (1) The present invention discovered the use of SIRT5 inhibitor 1 in the treatment of Ph+ALL, which provides an experimental basis for the use of SIRT5 molecules as drug targets for the relief and / or treatment of Ph+ALL and has high clinical translation value.

[0012] (2) The present invention first discovered that SIRT5 inhibitor 1 can weaken the proliferation ability of Ph+ALL cells, block the cell cycle at the G2 / M phase, and significantly increase the apoptosis level. It was also found that SIRT5 inhibitor 1 significantly reduced the mitochondrial membrane potential level of Ph+ALL cells, causing respiratory chain electron leakage to increase intracellular ROS, thereby promoting cell death; when NAC was used to resist the action of ROS, the effect of SIRT5 inhibitor 1 on Ph+ALL cell apoptosis was reversed. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 It was shown that SIRT5 inhibitor 1 significantly inhibited the expression of SIRT5 molecules;

[0014] Figure 2 It was shown that SIRT5 inhibitor 1 inhibited the proliferation and cell cycle of Ph+ALL cells;

[0015] Figure 3 It was shown that SIRT5 inhibitor 1 increased the apoptosis level of Ph+ALL cells;

[0016] Figure 4 It was shown that after treatment with SIRT5 inhibitor 1, the mitochondrial membrane potential of Ph+ALL cells decreased, the ROS level increased, and apoptosis increased. DETAILED DESCRIPTION

[0017] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are exemplary only and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the scope of protection of the present invention.

[0018] The specific test process will be given below.

[0019] 1. SIRT5 specific inhibitor SIRT5 inhibitor 1 specifically inhibits the expression of SIRT5: Western blot experiments confirmed that SIRT5inhibitor 1 specifically inhibits the expression of SIRT5 in a concentration-dependent manner;

[0020] 2. Detect the effect of SIRT5 inhibitor 1 on the proliferation and cycle of Ph+ALL cells: First, use CCK-8 and clone formation experiments to detect the effect of SIRT5inhibitor 1 on the proliferation ability of Ph+ALL cells. Then, use flow cytometry to detect the cycle changes of Ph+ALL cells after treatment with SIRT5 inhibitor 1; finally, use Western Blot to detect the expression levels of apoptosis and cycle-related molecules;

[0021] 3. Detection of mitochondrial membrane potential, intracellular ROS level and apoptosis level of Ph+ALL cells after inhibition of SIRT5: First, K562 and SUP-B15 were treated with SIRT5 inhibitor1; then, the mitochondrial membrane potential detection kit (JC-1) and flow cytometry were used to detect the changes in mitochondrial membrane potential level and ROS level. Next, the antioxidant NAC was used to antagonize the effect of ROS, and the apoptosis level was detected by flow cytometry.

[0022] 4. Experimental Methods

[0023] (1) The method for culturing leukemia cells in the embodiment of the present invention:

[0024] K562 and SUP-B15 cell lines were cultured in RPMI 1640 medium containing 10% fetal bovine serum in an atmosphere of 5% CO. 2 and a constant temperature environment of 37°C. Observe the growth status of cells every day, and replace the culture medium or perform cell passage in time. Collect cells in the logarithmic growth phase for subsequent experimental studies.

[0025] (2) The method of proteomic sequencing and bioinformatics technology KEGG pathway enrichment analysis in the embodiment of the present invention:

[0026] First, the samples were lysed to extract proteins and their concentrations were determined. Then, trypsin was used to digest the proteins according to the filter-assisted sample preparation (FASP) procedure. After that, the digested protein fragments and their characteristics were detected by liquid chromatography-mass spectrometry. Finally, expression fold difference analysis and GO and KEGG functional enrichment analysis were performed using bioinformatics methods.

[0027] (3) Method for detecting protein expression level by Western blotting in the embodiment of the present invention:

[0028] 1) Extract total protein: Collect cells in good condition into a centrifuge tube, centrifuge at 1000 rpm for 5 min, discard the supernatant; wash 2-3 times with 1 ml of pre-cooled PBS. Add an appropriate amount of freshly prepared protein lysis buffer (RIPA: PMSF: NaF: Na 3VO 4 =100:1:1:1), let stand on ice for 5 minutes, then shake vigorously for 30 seconds, vortexing every 5 minutes. After complete lysis, centrifuge at 4°C and 13000rpm for 25 minutes. Transfer the supernatant to another clean EP tube and detect the protein concentration by BCA method. Add 1 / 4 volume of 5×SDS loading buffer, mix well, denature in a 100°C metal bath for 10 minutes, and store in a -40°C refrigerator for later use.

[0029] 2) Gel preparation and electrophoresis: Prepare 10% separation gel, pour the gel, add 1ml of anhydrous ethanol to press the gel, let it stand at room temperature for 30 minutes to fully solidify, pour out the ethanol and wipe it with filter paper, place the comb teeth, prepare 5% concentrated gel, slowly add it to the upper layer of the separation gel, let it stand at room temperature for 30 minutes to solidify. Calculate the sample loading amount according to the measured protein concentration. Choose two-step electrophoresis: the first step is 80V, 100mA electrophoresis for 30 minutes; the second step is 120V, 100mA electrophoresis for 90 minutes.

[0030] 3) Transfer: First, according to the molecular weight of the protein to be tested, cut a polyvinylidene fluoride (PVDF) membrane of appropriate size and soak it in methanol for 30 seconds, wash it with distilled water for 2 minutes, and then soak the PVDF membrane and filter paper in pre-cooled wet transfer solution. According to the molecular weight of the target protein, cut the gel and soak it in pre-cooled wet transfer solution, and finally place the sponge, filter paper, PVDF membrane, gel, filter paper and sponge in order from the positive electrode to the negative electrode. Transfer the membrane with a constant current electrophoresis of 220mA, and the transfer time is determined by the molecular weight of the protein.

[0031] 4) Blocking: After transfer, block with 5% skimmed milk powder on a shaker at room temperature for 2 h.

[0032] 5) Antibody incubation: Place the membrane on a wax plate, add the primary antibody working solution pre-diluted in a certain ratio, evenly cover the PVDF membrane, and incubate overnight at 4°C; after recovering the primary antibody working solution, wash the PVDF membrane in TBST for 3 times, 10 min / time; finally, add the secondary antibody labeled with horseradish peroxidase diluted in a certain ratio to the PVDF membrane, incubate at room temperature for 1.5 h, and wash 3 times with TBST, 10 min / time.

[0033] 6) Color development and imaging: In a dark room, place the PVDF membrane on a wax plate, add the prepared chemiluminescent reagent (liquid A:liquid B = 1:1) to cover the PVDF membrane, and display the image in an imaging system.

[0034] (4) The specific method of detecting the degree of inhibition of cell proliferation after treatment with different concentrations of SIRT5 inhibitor 1 using the Cell Counting Kit-8 (CCK-8) in the present invention is as follows:

[0035] K562 and SUP-B15 cells in the logarithmic growth phase were collected and seeded in 96-well plates at a density of 4000 cells per well. Different concentrations of SIRT5 inhibitor 1 solution (0 μM, 2 μM, 4 μM, 8 μM, 10 μM) were added, and 1640 medium containing 10% FBS was supplemented to a final volume of 100 μl per well. The plates were placed at 37°C and 5% CO. 2 The cells were cultured continuously in an incubator. 10 μl of CCK-8 reagent was added to each well at 0 h, 24 h, and 48 h, respectively. The cells were placed in a 37°C incubator and incubated in the dark for 2 h. The absorbance was detected at a wavelength of 450 nm using an enzyme-labeled instrument.

[0036] (5) The method for detecting the mitochondrial membrane potential of cells treated with SIRT5 inhibitor 1 by the mitochondrial membrane potential detection kit in the embodiment of the present invention:

[0037] Collect cells from each group by centrifugation, discard the supernatant, and resuspend in 0.5 ml of cell culture medium. Add 0.5 ml of JC-1 staining working solution to the cell suspension, invert several times to mix, and incubate at 37°C for 20 minutes. After the incubation, centrifuge at 600g for 3-4 minutes at 4°C to precipitate the cells, and discard the supernatant. Wash twice with JC-1 staining buffer (1X), resuspend the cells with an appropriate amount of JC-1 staining buffer (1X), and observe with a fluorescence microscope or laser confocal microscope.

[0038] (6) Method for flow cytometry detection of apoptosis level, cycle and ROS level of cells after inhibition of SIRT5 in the embodiment of the present invention:

[0039] 1) Cell apoptosis: Collect cells from each group by centrifugation at 1000 rpm for 5 min, wash the cells with 1 ml of pre-cooled PBS, repeat twice. In the experiment of using NAC to antagonize ROS, first add 10nM NAC to treat the cells for 1 hour, then add SIRT5 inhibitor 1 and culture for 48 hours. Collect cells from each group and wash them with 1 ml of pre-cooled PBS, repeat three times, 500 rpm, 5 min; take 5×10 5 Resuspend the cells in PBS at 300g for 5 min, discard the supernatant and add 500μL diluted 1×AnnexinVBinding Buffer working solution to resuspend the cells; add 5μL Annexin V-APC and 5μL DAPI staining solution to the cell suspension, gently vortex to mix, incubate at room temperature in the dark for 15 min, and detect immediately after the reaction is completed.

[0040] 2) Cell cycle: Collect cells from each group by centrifugation at 3000 rpm for 5 min, wash the cells with 1 ml of pre-cooled PBS, repeat twice, resuspend the cells with 75% ethanol solution, and fix them in a 4°C refrigerator overnight; discard the supernatant, add 100 μL RNase A, and incubate at 37°C in the dark for 30 min; discard the supernatant, add 400 μL PI, and incubate at 4°C in the dark for 30 min. After the reaction is completed, immediately detect on the machine.

[0041] 3) ROS level: Collect cells from each group by centrifugation at 1000 rpm for 5 min, wash the cells with 1 ml of pre-cooled PBS, repeat twice, and transfer to a new EP tube; dilute the DCFH-DA probe with serum-free culture medium at a ratio of 1:1000, add 1 ml of the diluted probe solution to each EP tube to resuspend the cell pellet, incubate at 37°C for 30 min, and gently invert up and down every 5 min to mix; place the EP tube in a low-temperature centrifuge, centrifuge at 1000 rpm for 5 min, wash with 1 ml of pre-cooled PBS, repeat twice to remove unbound DCFH-DA probe; resuspend the cells with 500 μl of PBS, and analyze by flow cytometry.

[0042] 5. Experimental Results

[0043] (1) SIRT5 inhibitor 1 specifically inhibits the expression of SIRT5

[0044] Figure 1 Western blotting experiments were used to confirm that SIRT5 inhibitor 1 specifically inhibited the expression of SIRT5 in a concentration-dependent manner. (2) SIRT5 inhibitor 1 treatment weakened the proliferation ability of Ph+ALL cells and arrested their cell cycle.

[0045] Figure 2 A and B are the results of clone formation experiment and CCK-8 experiment. After inhibiting SIRT5, the proliferation ability of K562 cells did not change significantly, while the proliferation ability of SUP-B15 cells decreased significantly. Figure 2 C is the result of flow cytometry detection of cell cycle. After inhibiting SIRT5, the cell cycle of SUP-B15 cells was mainly blocked in the G2 / M phase; while the cell cycle of K562 cells showed no obvious change. Figure 2 D is the Western blotting experiment to detect the expression levels of cycle-related molecules. The results showed that after treatment with SIRT5 inhibitor 1, the expression of CyclinB1 in SUP-B15 cells was significantly downregulated, and the levels of cycle substrate-related molecules p21 and p27 were significantly upregulated, while there was no significant change in K562 cells. Figure 3Flow cytometry analysis showed that the apoptosis level of SUP-B15 cells increased after treatment with SIRT5 inhibitor 1, while the changes in K562 cells were not significant. The above research results confirmed that inhibition of SIRT5 weakened the proliferation ability of SUP-B15 cells, increased apoptosis, and arrested the cell cycle at the G2 / M phase, which indicates that high expression of SIRT5 can promote the tumor biological characteristics of SUP-B15 cells and is of great significance for SUP-B15 cells to maintain their malignant phenotype.

[0046] (3) After treatment with SIRT5 inhibitor 1, mitochondrial membrane potential of Ph+ALL cells decreased, ROS levels increased, and apoptosis increased

[0047] Figure 4 A shows that after SUP-B15 cells were treated with SIRT5 inhibitor 1 for 48 hours, the red fluorescence of mitochondria turned into green fluorescence, indicating that the mitochondrial membrane potential level of SUP-B15 cells decreased significantly, while the mitochondrial membrane potential of K562 cells did not change significantly. Figure 4 B is the ROS level detection result, which shows that compared with K562 cells, the ROS level of SUP-B15 cells was significantly increased after treatment with SIRT5 inhibitor1. Figure 4 CD is to use the antioxidant NAC to antagonize the effect of ROS, and then use flow cytometry to detect the level of cell apoptosis. The results showed that after SUP-B15 and K562 cells were treated with NAC and SIRT5 inhibitor1, the effect of SIRT5 inhibitor 1 killing SUP-B15 cells could be partially antagonized, but there was no obvious effect on K562 cells. The above results show that the highly expressed SIRT5 in SUP-B15 inhibits the generation of ROS and inhibits cell apoptosis, thereby enhancing cell survival.

[0048] The embodiments described in the present invention are only preferred specific implementation schemes of the present invention and are not used to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. Application of SIRT5 inhibitor 1 in the treatment of Philadelphia chromosome-positive acute lymphoblastic leukemia.

2. The use according to claim 1, characterized in that The drug is compound 49.

3. The use according to claim 1, characterized in that The medicine is used for treating Philadelphia chromosome positive acute lymphoblastic leukemia (Ph+ALL).

4. The use according to claim 1, characterized in that: The drug has the following effects in Ph+ALL: (1) Compound 49 was used to inhibit the expression of SIRT5 in Ph+ALL cells; (2) Compound 49 can weaken the proliferation ability of Ph+ALL cells and arrest the cell cycle at the G2 / M phase; (3) Compound 49 can significantly reduce the △φm level of Ph+ALL cells, causing electron leakage and increasing intracellular ROS, thereby promoting cell death.

Images