Combination and application of anti-tumor drugs with PARG inhibitors

Abstract

This invention discloses an antitumor drug combination and its application in conjunction with a PARG inhibitor. Through natural product library screening and CCK-8 assays, this invention found that PARG knockout gastric cancer cells exhibited significantly enhanced sensitivity to ginsenoside CK. Further colony formation assays showed that PARG knockout significantly enhanced the inhibitory effect of ginsenoside CK on gastric cancer cell proliferation. In in vivo experiments, using a CDX model, it was verified that PARG knockout significantly enhanced the antitumor activity of ginsenoside CK against gastric cancer. These findings provide important experimental evidence for the clinical application of PARG as a therapeutic target for gastric cancer and ginsenoside CK as a potential therapeutic drug. This invention also found that the combination of PARG inhibitors and ginsenoside CK has a better killing effect on various other cancer cells, indicating that this enhanced effect is broad-spectrum.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the combination and application of anti-tumor drugs in combination with PARG inhibitors. Background Technology

[0002] Gastric cancer is one of the most common malignant tumors with high incidence and mortality rates worldwide. Despite some progress in diagnosis and treatment in recent years, gastric cancer treatment still faces many challenges and limitations. The main reasons for the difficulty in early diagnosis of gastric cancer are the lack of obvious early symptoms, low screening coverage, and the lack of effective biomarkers. In recent years, advancements in radiotherapy, chemotherapy, and neoadjuvant therapy have significantly improved the overall survival rate of gastric cancer patients; however, the benefits of these treatments remain limited, and drug resistance and tolerance are frequently observed. Overall, the treatment options for gastric cancer are limited, and the development of new therapeutic drugs is urgently needed.

[0003] Targeting DNA damage repair pathways may offer potential treatment strategies for gastric cancer patients. Poly(ADP-ribose) polymerase (PARP) is an important protein in DNA damage repair. PARP inhibitors represent a novel approach for targeting DNA repair mechanisms. Since PARG protein plays a similar role to PARP protein in the DNA damage repair pathway, PARG has the potential to become a new target for gastric cancer treatment.

[0004] Existing PARG inhibitors (PARGi) are divided into those derived from natural product extracts and those from small molecules. Existing small molecule inhibitors mainly include: poly(ADP-ribose) analogue-adenosine diphosphate (hydroxymethyl)pyrrolidone diol (ADP-HPD), compound N-bis-(3-phenylpropyl)9-oxo-fluorene-2,7-diamide (GPI16552), rhodamine-based PARG inhibitors (RBPIs), PDD00017273, COH34, JA2131, IDE161, etc.

[0005] For example, invention application CN117486874A discloses a benzo[a]ary aromatic ring compound, its pharmaceutical composition and its application, wherein the pharmaceutical composition is used in the prevention and / or treatment of cancer or PARG-related diseases, and the cancer is preferably ovarian cancer, breast cancer, pancreatic cancer, prostate cancer or gastric cancer.

[0006] Currently, the efficacy of PARG inhibitors as monotherapy for gastric cancer remains limited. Therefore, screening for other drugs that can be used in combination with PARG inhibitors to improve the treatment effect of gastric cancer is still of practical significance.

[0007] Ginsenoside CK (G CK), CAS No.: 39262-14-1, Molecular Formula: C 36 H 62 O8.

[0008] Ginsenoside CK exhibits pharmacological activities in areas such as anti-tumor, liver protection, and anti-inflammation. For example, invention application CN116763799A discloses the use of ginsenoside CK in the preparation of products that inhibit the proliferation of ovarian cancer cells, showing that ginsenoside CK can inhibit the proliferation of sensitive and drug-resistant ovarian cancer cells. Summary of the Invention

[0009] To address the aforementioned shortcomings in the prior art, this invention provides an anti-tumor drug combination and its application in conjunction with a PARG inhibitor.

[0010] This invention, through screening over 800 drugs from a natural product library, discovered that ginsenoside compound K (GCK) has a significant killing effect on PARG-KO gastric cancer cells. Therefore, we considered it as a candidate drug for combination with PARG inhibitors, and further evaluated its killing effect on PARG-KO gastric cancer cells and xenografts through in vivo and in vitro experiments, providing a new approach for gastric cancer treatment. Therefore, the combination of PARG inhibitors and ginsenoside K represents a novel potential clinical treatment strategy.

[0011] This invention first provides an anti-tumor drug combination in combination with a PARG inhibitor, including a PARG inhibitor and ginsenoside CK.

[0012] Preferably, the PARG inhibitor is at least one of the following: 6'-thiomethylxanthine derivatives JA2131, ADP-HPD, GPI16552, RBPIs, PDD00017273, COH34, JA2131, and IDE161.

[0013] More preferably, the PARG inhibitor is a 6'-thiomethylxanthine derivative, JA2131. The structural formula of JA2131 is shown in Formula 1:

[0014] Formula 1.

[0015] The structural formula of ginsenoside CK (G CK) is shown in Formula 2:

[0016] Equation 2.

[0017] Preferably, the concentration of PARG inhibitor is 20-80 μM; the concentration of ginsenoside CK is 20-80 μM.

[0018] This invention also provides the application of an antitumor drug combination in combination with a PARG inhibitor in the preparation of anticancer drugs. The antitumor drug combination in combination with a PARG inhibitor includes a PARG inhibitor and ginsenoside CK.

[0019] Preferably, the PARG inhibitor is at least one of the following: 6'-thiomethylxanthine derivatives JA2131, ADP-HPD, GPI16552, RBPIs, PDD00017273, COH34, JA2131, and IDE161.

[0020] More preferably, the PARG inhibitor is a 6'-thiomethylxanthine derivative JA2131.

[0021] Preferably, the cancer type is lung cancer, stomach cancer cells, colon cancer cells, liver cancer, esophageal cancer, or pancreatic cancer.

[0022] More preferably, lung cancer is lung adenocarcinoma, gastric cancer is gastric adenocarcinoma, and esophageal cancer is esophageal squamous cell carcinoma.

[0023] Preferably, the concentration of PARG inhibitor is 20-80 μM; the concentration of ginsenoside CK is 20-80 μM.

[0024] Compared with the prior art, the beneficial effects of the present invention are as follows.

[0025] This invention, through screening a natural product library and conducting CCK-8 assays, revealed that PARG knockout gastric cancer cells exhibited significantly enhanced sensitivity to ginsenoside CK. Further clonogenic assays showed that PARG knockout significantly enhanced the inhibitory effect of ginsenoside CK on gastric cancer cell proliferation. In in vivo experiments, using a CDX model, it was verified that PARG knockout significantly enhanced the antitumor activity of ginsenoside CK against gastric cancer. These findings provide important experimental evidence for the clinical application of PARG as a therapeutic target for gastric cancer and ginsenoside CK as a potential therapeutic agent.

[0026] This invention has found that the combined use of PARG inhibitors and ginsenoside CK also has a better killing effect on a variety of other cancer cells, indicating that this enhancement is broad-spectrum. Attached Figure Description

[0027] Figure 1 To screen for potential drugs that may be used in combination with PARG inhibitors. Figure 1 In this context, A represents cell plating, drug treatment, and CCK8 assay for cell viability. Figure 1In the diagram, B represents the drug library required for drug screening, and the point indicated by the arrow is ginsenoside CK.

[0028] Figure 2 This study investigated the effects of combined use of the PARG inhibitor (JA2131) and ginsenoside CK (GCK) on the antitumor activity of gastric cancer in a tumor CDX model, and examined the immunohistochemical staining of tumor tissues treated with GCK. Among these findings, Figure 2 In the diagram, A represents the tumor of the HGC27 xenograft model after G CK treatment; Figure 2 In the figure, B represents the tumor weight statistics of the HGC27 xenograft model after G CK treatment; Figure 2 In the figure, C represents the tumor volume growth curve of the HGC27 xenograft model after G CK treatment. Figure 2 In the diagram, D represents the immunohistochemical staining images of Tunel (TUNEL), Ki-67, and H&E in tumor tissue. Scale bar: 50 μm. * represents p < 0.05, *** represents p < 0.001, **** represents p < 0.0001, ns represents p > 0.05, and n = 5.

[0029] Figure 3 The effect of combined use of PARG inhibitor (JA2131, represented by PARGi in the figure) and ginsenoside CK (G CK) on cell proliferation activity. Figure 3 In the figure, A represents the effect of the checkerboard assay on the combined use of PARG inhibitor (JA2131) and GCK on the viability of HGC27 cells. Figure 3 In the figure, B represents the effect of the combined use of PARG inhibitor (JA2131) and GCK on cell viability as detected by the MTS assay. Figure 3 C in the figure represents a statistical bar chart of HGC27 cell colony formation under the combined action of PARG inhibitor (JA2131) and GCK in a colony formation assay. Figure 3 In the figure, D represents the mode of cell death of HGC27 cells under the combined action of PARG inhibitor (JA2131) and GCK as detected by the MTS assay. **** represents p<0.0001, *** represents p<0.001, ns represents P>0.05, and n=3.

[0030] Figure 4 The effect of combined use of the PARG inhibitor (JA2131, represented by PARGi in the figure) and GCK on the activity of tumor cells in different cancers. Figure 4In the figures, A and F represent the effects of the combined use of the PARG inhibitor (JA2131) and GCK on the activity of human lung adenocarcinoma A549, human gastric adenocarcinoma AGS, human colon cancer HCT116, human liver cancer HepG2, human esophageal squamous cell carcinoma KYSE150, and human pancreatic cancer PANC-1 cells, respectively. **** represents p<0.0001, *** represents p<0.001, n=3. Detailed Implementation

[0031] Example 1: Drug screening to identify potential drugs that may be used in combination with PARG inhibitors

[0032] Figure 1 In the diagram, A represents the experimental steps for drug screening, including cell plating, drug treatment, and CCK8 assay for cell viability.

[0033] (1) Construction of PARG knockout cell lines

[0034] First, a knockout plasmid sgRNA fragment was designed based on the CRISPR-Cas9 system, and the corresponding targets are shown in Table 1. The target fragment sgRNA was then incorporated into the CRISPR-Cas9 system to construct a CRISPR-Cas9 plasmid for PARG knockout.

[0035] HEK293T cells in good condition were seeded in 10cm culture dishes. When the cell density reached 80%, the cells were washed twice with preheated PBS and then replaced with DMEM medium without penicillin-streptomycin. Cell transfection: The transfection solution was prepared according to the system in Table 2 using Lipofectamine 3000 transfection reagent. The solution from group B was slowly added dropwise to the solution from group A, and after incubation at room temperature for 10 min, it was added to HEK293T cell culture dishes and incubated at 37℃ for 10 h. The cells were then washed twice with preheated PBS and replaced with DMEM medium without penicillin-streptomycin. After 48 h of culture, the culture medium was collected, centrifuged at 3000 rpm for 10 min to remove cell debris, and the supernatant was filtered through a 0.45 μm filter into sterile 15 mL centrifuge tubes to obtain the target virus solution.

[0036] HGC27 and AGS cells in logarithmic growth phase were digested and seeded into 6-well plates. When the cell density reached 40%-50%, viral medium (1 mL medium + 1 mL virus solution + 1 μL polybrene) was added for infection for 24 h. After infection, the cell status was observed, and the medium was replaced with one without antibiotics, followed by selection using puromycin-containing medium. Gene knockout efficiency was verified by Western blotting, and the cells were passaged.

[0037] Table 1 PARG sgRNA primers

[0038]

[0039] Table 2 Lipofectamine 3000 Transfection System

[0040]

[0041] The PARG knockout cell line obtained using PARG-sg1 was named KO1 cells, and the PARG knockout cell line obtained using PARG-sg2 was named KO2 cells.

[0042] (2) Cell seeding: HGC27-WT (wild-type human gastric cancer cells), KO1 cells and KO2 cells were seeded into 96-well plates at 2000 cells per well and cultured overnight.

[0043] (3) Drug treatment: Using the constructed natural product library (more than 800 drugs, purchased from Targetmol, #L6000), the cells in the 96-well plates were treated with drugs for 48 h according to the predetermined experimental design. Cell viability was then detected using a CCK-8 assay kit: the target cells were digested and counted, and 2 × 10⁶ cells were taken from each well. 3 HGC27-WT, KO1, and KO2 cells were seeded at 200 μL per well in a 96-well plate. The plates were incubated at 37°C with 5% CO2 for 1–5 days. The culture medium was discarded daily, and fresh medium containing 10% CCK-8 was added, followed by incubation for another 2 hours. After incubation, the absorbance at 450 nm was measured using a microplate reader. Cell viability was calculated using the following formula:

[0044] Survival rate = (OD experimental group - OD blank group) / (OD control group - OD blank group) × 100%.

[0045] Use GraphPad Prism 9 software to process data, calculate survival rates, or plot survival curves and calculate IC50 values.

[0046] (4) Data Processing and Analysis: After obtaining the survival rate data of HGC27-WT and KO1 cells, the difference between the two is calculated, i.e., [WT (survival rate) - KO (survival rate)]. For example... Figure 1 As shown in B, by analyzing the difference data, drugs that are more sensitive to PARG knockout gastric cancer cells were screened out. The survival rate of wild-type and PARG knockout HGC27 cells treated with the drugs was detected by the CCK8 assay. It was found that the cell survival rate was lower after using G CK (ginsenoside CK), indicating that G CK has good anti-tumor activity, which provides a reference for subsequent research.

[0047] Example 2: Study on the effect of GCK on the antitumor activity of PARG-KO (PARG gene knockout) gastric cancer cells using a tumor CDX model.

[0048] The effect of PARG knockout enhancing GCK on the antitumor activity of gastric cancer was further investigated using an HGC27 cell xenograft model with PARG-WT (wild-type PARG gene) and KO1 (PARG gene knockout using CRISPR-Cas9 gene editing technology) (as shown in Example 1). Four groups were set up: WT group (wild-type group), PARG-KO group (PARG gene knockout group), WT+GCK group (wild-type with added GCK), and PARG-KO+GCK group (PARG gene knockout with added GCK), with 5 mice in each group. When the average tumor diameter exceeded 4 mm, intraperitoneal injection of the drug was initiated at a dose of 30 mg / kg, administered every 3 days. Mouse weight and tumor diameter were measured.

[0049] By day 18, compared with the WT group, the tumor size in the PARG-KO group and the G CK treatment group was reduced, and the tumor size in the PARG-KO+G CK group was significantly smaller than that in the other three groups. Figure 2 (A) Based on the measured tumor volume growth curves, it was found that the tumor weight decreased in the PARG-KO group and the WT+G CK group compared to WT; the tumor weight in the PARG-KO+G CK group was the smallest, significantly lower than the other three groups (A). Figure 2 (B in the text); Compared with the WT group, the tumor growth rate of the PARG-KO group and the WT+G CK group was reduced, and the tumor growth rate of PARG-KO under G CK treatment was more significantly inhibited ( Figure 2 C in the text). Immunohistochemical staining results of TUNEL, Ki67 and H&E markers in tumor tissue showed ( Figure 2 In the D group, the WT group had the highest number of Ki67-positive cells, indicating the strongest tumor proliferation ability; while the PARG-KO+G CK group had the lowest number of Ki67-positive cells, indicating the weakest tumor proliferation ability. Furthermore, the WT group had the lowest TUNEL positivity rate, suggesting the strongest anti-apoptotic ability of its tumors; in contrast, the PARG-KO+G CK group had the highest TUNEL positivity rate, indicating the weakest anti-apoptotic ability of its tumors.

[0050] In summary, the in vivo CDX model and immunohistochemical staining results of tumor tissue show that the use of G CK can enhance the anti-tumor activity against PARG-KO gastric cancer cells, inhibit tumor proliferation, and promote tumor apoptosis.

[0051] Example 3: In vitro experimental study on the anti-proliferative activity of the PARG inhibitor (JA2131) and GCK in combination against gastric cancer.

[0052] (1) In order to investigate the cell killing effect of the combined use of PARG inhibitor (JA2131) and G CK, the killing effect of different concentrations of PARG inhibitor (JA2131) and G CK alone or in combination on HGC27 cells was first detected by the drug combination checkerboard method, and the cell survival rate was detected by the SRB method.

[0053] After digesting the target cells into a cell suspension, cell counting was performed, with 3×10⁶ cells being counted. 3 HGC27 cells were seeded per well into 96-well plates. PARG inhibitor (JA2131) concentrations of 80, 60, 40, 20, 10, 5, 1, and 0 μM were added sequentially to rows 2-7 of the 96-well plate. GC-CK concentrations of 80, 60, 40, 20, 10, 5, 1, and 0 μM were added sequentially to columns 2-8 of the 96-well plate. A control group (cells only, no drug) and a blank group (cells only, no drug) were also set up. 50 µL of ice-cold 10% TCA (trichloroacetic acid) was added to each well and gently mixed. The plates were fixed at 4°C for 30-60 minutes. The cells were slowly washed 5 times with water and air-dried. 100 µL of 0.4% SRB (sulfonylrhodamine B) solution (dissolved in 1% glacial acetic acid) was added to each well and incubated at room temperature in the dark for 30 minutes. Each well was quickly rinsed 4-5 times with 1% glacial acetic acid to remove unbound dye and air-dried again. Add 200 µL of Tris buffer to each well and shake to dissolve the bound SRB dye. Measure the absorbance of each well at 560 nm using a microplate reader.

[0054] Figure 3 The results of the checkerboard assay in Part A showed that the cell survival rate of HGC27 cells treated with the combined PARG inhibitor (JA2131) and GCK was lower than that treated with either drug alone. The combined treatment with PARG inhibitor (JA2131) and GCK had a synthetic lethal effect on HGC27 cells.

[0055] (2) After confirming the effect of the combination of PARG inhibitor (JA2131) and G CK through the checkerboard method experiment, this application further determined the effect of PARG inhibitor on cell activity under the combined action of G CK through the MTS experiment (cell viability assay).

[0056] Digest and count the target cells, then take 2 × 10⁻⁶ cells. 3HGC27 cells were seeded into 96-well plates. The plates were incubated at 37°C with 5% CO2 for 24 hours until the cells were stable and adhered. After adding the drug, incubation continued for 24-72 hours. Then, 20 µL of MTS solution was added to each well, gently agitated, and the plates were returned to the incubator for another 1-4 hours in the dark. The absorbance (OD490) was read at 490 nm using a microplate reader. Cell viability was calculated using the following formula: Viability = (OD experimental group - OD blank group) / (OD control group - OD blank group) × 100%.

[0057] The results are as follows Figure 3 As shown in Figure B, HGC27 cell survival was significantly reduced after co-treatment with 20 μM PARG inhibitor (JA2131) and 20 μM G CK. Through MTS experiments, this application found that the combined use of PARG inhibitor (JA2131) and G CK had a better killing effect on tumor cells.

[0058] (3) In order to investigate the effect of the combined use of PARG inhibitor (JA2131) and G CK on cell proliferation, HGC27 cells were evaluated in vitro using a colony formation assay.

[0059] Cells in good condition and in the logarithmic growth phase were seeded into 6-well plates (500 cells per well) and incubated at 37°C with 5% CO2, with 3 replicates per group. After the cells formed a clear colony, the culture medium was discarded, the cells were washed once with PBS, and fixed with 4% paraformaldehyde for 15 min. After washing again with PBS, the cells were stained with 0.1% crystal violet solution for 15 min. After staining, the 6-well plates were scanned to obtain images of colony formation, and analyzed using Image software. The cell colony count was normalized to the untreated control group.

[0060] The results of clonogenesis studies showed that the number of cell clones was significantly lower when PARG inhibitor (JA2131) and GCK were used in combination than when PARG inhibitor (JA2131) and GCK were used alone, and the difference was statistically significant. Figure 3 (C)

[0061] (4) Subsequently, the death mode of HGC27 cells under the combined action of PARG inhibitor (JA2131) and G CK was detected by MTS experiment.

[0062] The results are as follows Figure 3The results showed that, compared with the group treated with the PARG inhibitor (JA2131) and GCK, the addition of the apoptosis inhibitor (Z-VAD-FMK) to the combined treatment with PARG inhibitor (JA2131) and GCK significantly rescued cell death and increased cell survival. However, no similar effects were observed with the addition of the pyroptosis inhibitor (VX765), the necroptosis inhibitor (Nec-1), or the ferroptosis inhibitor (Ferr-1). These results indicate that the PARG inhibitor (JA2131) enhances the killing effect of GCK on tumor cells by promoting apoptosis, further revealing the important role of PARG in regulating apoptosis.

[0063] Example 4: In vitro experimental study on the antiproliferative activity of PARG inhibitor (JA2131) and GCK in combination in different cancer types

[0064] Drug combination experiments were conducted on human lung adenocarcinoma cells A549, human gastric adenocarcinoma cells AGS, human colon cancer cells HCT116, human liver cancer cells HepG2, human esophageal squamous cell carcinoma cells KYSE150, and human pancreatic cancer cells PANC-1.

[0065] The target cells were digested and counted, and 2 × 10⁻⁶ cells were taken from each cell. 3 A549 cells (human lung adenocarcinoma cells), AGS cells (human gastric adenocarcinoma cells), HCT116 cells (human colon cancer cells), HepG2 cells (human liver cancer cells), KYSE150 cells (human esophageal squamous cell carcinoma cells), and PANC-1 cells (human pancreatic cancer cells) were seeded into 96-well plates. After drug addition, the plates were incubated at 37°C with 5% CO2 for 48 hours. The culture medium in the 96-well plates was then discarded, and fresh medium containing 10% CCK-8 was added to each well, followed by incubation for another 0.5–4 hours. After incubation, the absorbance of each well at 450 nm was measured using a microplate reader. Cell viability was calculated using the following equation: Viability = (OD experimental group - OD blank group) / (OD control group - OD blank group) × 100%. GraphPad Prism 9 software was used to process the experimental data and calculate cell viability.

[0066] The results are as follows Figure 4 As shown, co-treatment with 20 μM PARG inhibitor (JA2131) and 20 μM G CK significantly reduced the survival rate of different cancer cells. Through this experiment, this application found that the combined use of PARG inhibitor (JA2131) and G CK has a better killing effect on tumor cells of different cancer types.

Claims

1. A combination of antitumor drugs in conjunction with PARG inhibitors, characterized in that, Including PARG inhibitors and ginsenoside CK; The PARG inhibitor is a 6'-thiomethylxanthine derivative, JA2131.

2. The antitumor drug combination with a PARG inhibitor according to claim 1, characterized in that, The concentration of PARG inhibitors used is 20-80 μM; the concentration of ginsenoside CK used is 20-80 μM.

3. The application of antitumor drug combinations in conjunction with PARG inhibitors in the preparation of anticancer drugs, characterized in that, Anti-tumor drug combinations that combine PARG inhibitors include PARG inhibitors and ginsenoside CK; The PARG inhibitor is a 6'-thiomethylxanthine derivative JA2131; The cancer types are lung cancer, stomach cancer, colon cancer, liver cancer, esophageal cancer, or pancreatic cancer; Lung cancer is lung adenocarcinoma, stomach cancer is gastric adenocarcinoma, and esophageal cancer is esophageal squamous cell carcinoma.

4. The application according to claim 3, characterized in that, The concentration of PARG inhibitors used is 20-80 μM; the concentration of ginsenoside CK used is 20-80 μM.

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