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

Abstract

The present invention belongs to the field of biomedicine technology, and specifically relates to the use of SIRT5 inhibitor 1 in the treatment of Philadelphia chromosome-positive acute lymphoblastic leukemia. The present invention confirms that SIRT5 inhibitor 1 can specifically inhibit the expression of SIRT5 in Ph+ALL cells; and for the first time finds that SIRT5 inhibitor 1 can weaken the proliferation ability of Ph+ALL cells, arrest the cell cycle at the G2 / M phase, and significantly increase the level of apoptosis; and finds that SIRT5 inhibitor 1 significantly reduces the mitochondrial membrane potential level of Ph+ALL cells, causing respiratory chain electron leakage, leading to an increase in intracellular reactive oxygen species (ROS), thereby promoting cell death; when NAC is used to resist the action of ROS, the effect of SIRT5 inhibitor 1 on Ph+ALL cell apoptosis is reversed, further confirming that SIRT5 inhibitor 1 promotes cell death by increasing ROS in Ph+ALL cells. The purpose of the present invention is to provide a new strategy and method for treating Ph+ALL.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical research technology, and particularly relates to the use 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 disorder originating from hematopoietic stem / progenitor cells. Its hallmark feature is the t(9;22)(q34;q11) chromosomal translocation, which results in fusion of the q34 region of the BCR gene with the q11 region of the ABL1 gene, forming the BCR::ABL1 fusion gene. This gene is a key pathogenic gene in Ph+ALL and possesses potent tyrosine kinase activity, promoting the malignant proliferation and survival of lymphocytes and leading to the development of leukemia. Currently, the mainstay of treatment for Ph+ALL is TKIs combined with chemotherapy, which can significantly improve patient outcomes. However, Ph+ALL is associated with a high relapse rate, short disease-free survival, and a poor prognosis. Clinical trials and new drug development are urgently needed to provide new therapeutic opportunities for relapsed or refractory Ph+ALL.

[0003] We performed proteomic sequencing and KEGG pathway enrichment analysis on Ph+ALL and other leukemia cells and found that several of the top 20 enriched pathways involved metabolism. Bioinformatics analysis also identified differentially expressed molecules related to energy metabolism in SUP-B15 and other leukemia cell lines, with SIRT5 showing the most significant differential expression. Furthermore, literature has reported that mitochondria, where SIRT3-5 proteins reside, can host a variety of metabolic processes, including glucose metabolism (see DeBerardinis, RJ & Keshari, KR. Metabolic analysis as a driver for discovery, diagnosis, and therapy. Cell 185, 2678–2689 (2022)). Detection of SIRT5 mRNA and protein levels in various leukemia cells confirmed that SIRT5 expression is indeed elevated in Ph+ALL. Therefore, we used SIRT5 inhibitor 1 to specifically inhibit the expression of SIRT5. Testing 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 foundation 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, arrest 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 application of SIRT5 inhibitor 1 in the treatment of Ph+ALL, providing an experimental basis for the application 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, arrest 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, leading to an increase in intracellular ROS, and thus 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 of Ph+ALL cells and arrested their cell cycle;

[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 with reference to specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are merely exemplary 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 solutions of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, and such modifications and replacements fall within the scope of protection of the present invention.

[0018] The specific test process is given below.

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

[0020] 2. Detect the effect of SIRT5 inhibitor 1 on the proliferation and cell cycle of Ph+ALL cells: First, CCK-8 and colony formation assays were used to detect the effect of SIRT5 inhibitor 1 on the proliferation of Ph+ALL cells. Then, flow cytometry was used to detect changes in the cell cycle of Ph+ALL cells after treatment with SIRT5 inhibitor 1. Finally, Western blot was used to detect the expression levels of apoptosis- and cell cycle-related molecules.

[0021] 3. Detection of mitochondrial membrane potential, intracellular ROS levels, and apoptosis in Ph+ ALL cells after SIRT5 inhibition: First, K562 and SUP-B15 cells were treated with SIRT5 inhibitor 1. Then, changes in mitochondrial membrane potential and ROS levels were measured using a mitochondrial membrane potential assay kit (JC-1) and flow cytometry, respectively. Next, the antioxidant NAC was used to antagonize ROS, and apoptosis was measured using flow cytometry.

[0022] 4. Experimental Methods

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

[0024] K562 and SUP-B15 cell lines were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum at 37°C and 5% CO2. Cell growth was monitored daily, and the medium was changed or cells were passaged as needed. Cells in the logarithmic growth phase were harvested for subsequent experimental studies.

[0025] (2) Methods of proteomic sequencing and KEGG pathway enrichment analysis using bioinformatics technology in the embodiments of the present invention:

[0026] First, the samples were lysed to extract proteins, and their concentrations were determined. Next, the proteins were digested using trypsin using the Filter-Assisted Sample Preparation (FASP) procedure. The digested protein fragments were then analyzed and characterized using liquid chromatography-mass spectrometry. Finally, bioinformatics methods were used to analyze fold-difference expression and functional enrichment of GO and KEGG genes.

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

[0028] 1) Total protein extraction: Collect healthy cells into a centrifuge tube, centrifuge at 1000 rpm for 5 minutes, and discard the supernatant. Wash the pellet two to three times with 1 ml of pre-chilled PBS. Add an appropriate amount of freshly prepared protein lysis buffer (RIPA:PMSF:NaF:Na3VO4 = 100:1:1:1) depending on the pellet volume. Incubate on ice for 5 minutes, then shake vigorously for 30 seconds, vortexing every 5 minutes. Once lysis is complete, centrifuge at 13,000 rpm for 25 minutes at 4°C. Transfer the supernatant to a clean EP tube and determine protein concentration using the BCA assay. Add 1 / 4 volume of 5× SDS loading buffer, mix thoroughly, and denature the pellet in a 100°C metal bath for 10 minutes. Store in a refrigerator at -40°C until ready for use.

[0029] 2) Gel Preparation and Electrophoresis: Prepare 10% separating gel, pour the gel, add 1 ml of anhydrous ethanol to press the gel, and let it stand at room temperature for 30 minutes to fully solidify. Pour off the ethanol and wipe dry with filter paper. Place the comb in place, prepare 5% stacking gel, and slowly add it to the top of the separating gel. Let it stand at room temperature for 30 minutes to solidify. Calculate the sample loading amount based on the measured protein concentration. Use a two-step electrophoresis method: the first step is 80V, 100mA electrophoresis for 30 minutes; the second step is 120V, 100mA electrophoresis for 90 minutes.

[0030] 3) Transfer: First, cut a polyvinylidene fluoride (PVDF) membrane of appropriate size based on the molecular weight of the protein to be tested and soak it in methanol for 30 seconds. Rinse with distilled water for 2 minutes. Then, soak the PVDF membrane and filter paper in pre-chilled transfer buffer. Cut a gel according to the molecular weight of the target protein and soak it in pre-chilled transfer buffer. Finally, place a sponge, filter paper, PVDF membrane, gel, filter paper, and sponge in the order of positive to negative. Transfer the membrane using a constant current of 220 mA. The transfer time is determined by the molecular weight of the protein.

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

[0032] 5) Antibody Incubation: Place the membrane on a wax plate and add pre-diluted primary antibody working solution to evenly cover the PVDF membrane. Incubate at 4°C overnight. After recovering the primary antibody working solution, wash the PVDF membrane three times in TBST (10 min each time). Finally, add horseradish peroxidase-labeled secondary antibody diluted in a certain ratio to the PVDF membrane. Incubate at room temperature for 1.5 h and wash three times in TBST (10 min each time).

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

[0034] (4) Specific method for 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 examples of the present invention:

[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 culture medium containing 10% FBS was supplemented to a final volume of 100 μl per well. The cells were cultured continuously in a 37°C, 5% CO2 incubator. 10 μl of CCK-8 reagent was added to each well at 0 h, 24 h, and 48 h, respectively. The cells were incubated in a 37°C incubator in the dark for 2 h, and the absorbance was detected using a microplate reader at a wavelength of 450 nm.

[0036] (5) Method for detecting the mitochondrial membrane potential of cells treated with SIRT5 inhibitor 1 using 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 incubation, pellet the cells by centrifugation at 600g for 3-4 minutes at 4°C and discard the supernatant. Wash twice with JC-1 staining buffer (1X), resuspend the cells in an appropriate amount of JC-1 staining buffer (1X), and observe under a fluorescence microscope or laser confocal microscopy.

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

[0039] 1) Cell apoptosis: Cells from each group were collected by centrifugation at 1000 rpm for 5 minutes and washed twice with 1 ml of pre-chilled PBS. In the experiment of using NAC to antagonize ROS, cells were first treated with 10 nM NAC for 1 hour, and then SIRT5 inhibitor 1 was added and cultured for 48 hours. Cells from each treated group were collected and washed three times with 1 ml of pre-chilled PBS at 500 rpm for 5 minutes. 5 × 10 5 Resuspend the cells in PBS at 300g for 5 min, discard the supernatant and add 500μL of diluted 1× Annexin V Binding Buffer to resuspend the cells; add 5μL of Annexin V-APC and 5μL of 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: Cells from each group were collected by centrifugation at 3000 rpm for 5 min, washed twice with 1 ml of pre-chilled PBS, resuspended in 75% ethanol, and fixed overnight in a 4°C refrigerator. The supernatant was discarded, 100 μL of RNase A was added, and the cells were incubated at 37°C in the dark for 30 min. The supernatant was discarded, 400 μL of PI was added, and the cells were incubated at 4°C in the dark for 30 min. After the reaction was completed, the cells were immediately analyzed.

[0041] 3) ROS levels: Cells from each group were collected by centrifugation at 1000 rpm for 5 min, washed twice with 1 ml of pre-chilled PBS, and transferred to new EP tubes. The DCFH-DA probe was diluted with serum-free medium at a ratio of 1:1000, and 1 ml of the diluted probe solution was added to each EP tube to resuspend the cell pellet. The tubes were incubated at 37°C for 30 min, gently mixing by inverting every 5 min. The EP tubes were placed in a refrigerated centrifuge and centrifuged at 1000 rpm for 5 min. The cells were washed twice with 1 ml of pre-chilled PBS to remove unbound DCFH-DA probe. The cells were resuspended in 500 μl of PBS and analyzed by flow cytometry.

[0042] 5. Experimental Results

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

[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 assay and CCK-8 assay. 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 arrested at the G2 / M phase; while the cell cycle of K562 cells showed no obvious change. Figure 2 D shows Western blotting assays to detect the expression levels of cycle-related molecules. The results showed that treatment with SIRT5 inhibitor 1 significantly downregulated the expression of CyclinB1 in SUP-B15 cells, while significantly upregulated the levels of cycle substrate-related molecules p21 and p27, whereas no significant changes were observed in K562 cells. Figure 3Flow cytometry analysis revealed that treatment with SIRT5 inhibitor 1 increased apoptosis in SUP-B15 cells, while no significant changes were observed in K562 cells. These findings confirm that SIRT5 inhibition weakens SUP-B15 cell proliferation, increases apoptosis, and arrests the cell cycle at the G2 / M phase. This suggests that high SIRT5 expression can promote the tumor biology of SUP-B15 cells and is crucial for maintaining their malignant phenotype.

[0046] (3) After treatment with SIRT5 inhibitor 1, the mitochondrial membrane potential of Ph+ALL cells decreased, the ROS level 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 to green fluorescence, indicating that the mitochondrial membrane potential level of SUP-B15 cells was significantly decreased, 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 in SUP-B15 cells was significantly increased after treatment with SIRT5 inhibitor1. Figure 4 CD used the antioxidant NAC to antagonize ROS, followed by flow cytometry analysis of cell apoptosis. The results showed that co-treatment of SUP-B15 and K562 cells with NAC and SIRT5 inhibitor 1 partially antagonized the killing effect of SIRT5 inhibitor 1 on SUP-B15 cells, but had no significant effect on K562 cells. These results suggest that SIRT5, which is highly expressed in SUP-B15 cells, inhibits ROS production and apoptosis, thereby enhancing cell survival.

[0048] The embodiments described in the present invention are only preferred specific implementation plans of the present invention and are not intended 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 scope of protection of the present invention.

Claims

1. Application of SIRT5 inhibitor 1 in the preparation of drugs for the treatment of Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ALL).

2. The use according to claim 1, characterized in that SIRT5 inhibitor 1 is compound 49.

3. The use according to claim 1, characterized in that SIRT5 inhibitor 1 has the following effects in Ph+ALL: (1) SIRT5 inhibitor 1 is used to inhibit the expression of SIRT5 in Ph+ ALL cells; (2) SIRT5 inhibitor 1 weakened the proliferation ability of Ph+ALL cells and arrested their cell cycle at the G2 / M phase; (3) SIRT5 inhibitor 1 reduces the △φm level of Ph+ALL cells, causing electron leakage and increasing intracellular ROS, thereby promoting cell death.

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