A composition for inhibiting proliferation of gastric cancer cells and application thereof

Abstract

The application discloses a kind of compositions for inhibiting proliferation of gastric cancer cells and application thereof, it is related to the field of biological medicine.The composition includes H3B-120 and PRT4165.Experiments have confirmed that the composition has synergistic effect in inhibiting gastric cancer cells, and the inhibition rate of the proliferation of gastric cancer cells can reach more than 80%.Clone formation experiment proves that H3B-120 and PRT4165 combined scheme can almost completely block long-term clone proliferation of tumor cells.The flow cytometry apoptosis detection result shows that the total apoptosis ratio of gastric cancer cells is increased to more than 70% after H3B-120 and PRT4165 combined treatment, and the pro-apoptotic ability is greatly enhanced.The composition provided by the application relies on the synergistic effect of double drugs to strengthen the growth inhibition and apoptosis induction effect on tumor cells, and can also reduce the single drug concentration, has the potential to reduce drug toxicity, provides a new idea for the treatment of gastric cancer.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a composition for inhibiting the proliferation of gastric cancer cells and its application. Background Technology

[0002] Gastric cancer is a common malignant tumor of the digestive system. In most cases, the disease has progressed to the middle or late stages at the time of diagnosis, making surgical cure impossible. Clinically, systemic chemotherapy is the primary treatment intervention. While current mainstream chemotherapy drugs can control lesion progression to a certain extent, they have significant limitations: chemotherapy drugs lack tumor cell specificity and indiscriminately kill actively proliferating normal cells, easily causing adverse reactions such as gastrointestinal damage and suppression of hematopoietic function. Some patients cannot complete a full treatment cycle due to insufficient tolerance. Furthermore, continuous chemotherapy gradually induces multidrug resistance in tumors, significantly reducing the tumor-suppressive effect of subsequent drugs and making it difficult to sustainably delay disease progression. Therefore, there is an urgent clinical need for safe, efficient, and novel intervention methods.

[0003] Related molecular biology studies have confirmed that the abnormally high expression of various specific protein molecules is closely related to the malignant proliferation and invasion process of gastric cancer. These specific molecules are only highly expressed in tumor lesion tissues and are almost not expressed in normal tissues surrounding the lesion. The molecular expression level is positively correlated with the pathological grade of the lesion and the risk of malignant development, and has the potential to be used as targeted intervention targets. Small molecule inhibitors developed against the above-mentioned specific targets can specifically downregulate the expression of the corresponding target proteins, and only act on tumor cells carrying abnormally high expression targets. Compared with traditional chemotherapy drugs, they can reduce damage to normal tissues and represent an important research direction for optimizing tumor treatment plans.

[0004] Current research largely focuses on inhibiting single targets, and using a single-target inhibitor alone can only slightly reduce tumor cell proliferation, resulting in limited overall tumor-suppressing effects. There is an urgent need to develop targeted drug combinations that can simultaneously act on two types of oncogenic targets and exhibit synergistic tumor-suppressing activity. These combinations would enhance the efficiency of tumor cell inhibition through synergistic effects, while simultaneously reducing the dosage of individual drugs and minimizing potential long-term toxic side effects. This would provide a novel treatment option for gastric cancer lesions exhibiting high expression of both targets. Summary of the Invention

[0005] The purpose of this invention is to provide a composition for inhibiting the proliferation of gastric cancer cells and its application, thereby addressing the problems existing in the prior art. The composition provided by this invention can synergistically inhibit the proliferation of gastric cancer cells, offering a new approach to the treatment of gastric cancer.

[0006] To achieve the above objectives, the present invention provides the following solution: The present invention provides a composition for inhibiting the proliferation of gastric cancer cells, comprising H3B-120 and PRT4165.

[0007] The present invention also provides the use of the above-described composition in the preparation of a medicament for treating gastric cancer.

[0008] Furthermore, the gastric cancer mentioned is gastric adenocarcinoma.

[0009] Furthermore, the drug includes pharmaceutically acceptable excipients.

[0010] Furthermore, the excipients include fillers, binders, disintegrants, emulsifiers, flavoring agents, preservatives, or colorants.

[0011] The present invention also provides a medicament for treating gastric cancer, wherein the active ingredient comprises the above-described composition.

[0012] Furthermore, the drug also includes pharmaceutically acceptable excipients.

[0013] Furthermore, the excipients include fillers, binders, disintegrants, emulsifiers, flavoring agents, preservatives, or colorants.

[0014] The present invention discloses the following technical effects: This invention develops a composition for inhibiting the proliferation of gastric cancer cells, comprising H3B-120 and PRT4165, whose combined use produces a significant synergistic anti-tumor effect. CCK-8 assays showed that this composition inhibited the proliferation of gastric cancer cells by over 80%. ZIP quantitative synergistic model analysis revealed a synergistic effect between H3B-120 and PRT4165 in inhibiting gastric cancer cells. Clonogenic assays demonstrated that the combination of H3B-120 and PRT4165 nearly completely blocked the long-term clonal proliferation of tumor cells. Flow cytometry apoptosis detection results showed that the total apoptosis rate of gastric cancer cells increased to over 70% after combined treatment with H3B-120 and PRT4165, significantly enhancing the pro-apoptotic ability. The composition provided by this invention enhances the growth inhibition and apoptosis induction effects on tumor cells through the synergistic effect of the two drugs, and can also reduce the concentration of single-drug administration, possessing the potential to reduce drug toxicity and side effects, providing a new and highly effective targeted combination therapy for the treatment of gastric cancer. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0016] Figure 1The dose-response curve of PRT4165 on 23132 / 87 cells; Figure 2 The dose-response curve of PRT4165 on SGC-7901 cells; Figure 3 The dose-response curve of H3B-120 on 23132 / 87 cells; Figure 4 The dose-response curve of H3B-120 on SGC-7901 cells; Figure 5 The diagram shows the synergistic analysis of H3B-120 and PRT4165 in SGC-7901 cells; where A is the dose-response matrix and B is the ZIP synergistic score distribution. Figure 6 The diagram shows the synergistic analysis of H3B-120 and PRT4165 in 23132 / 87 cells. In the diagram, A is the dose-response matrix and B is the ZIP synergistic score distribution. Figure 7 Microscopic images showing the effects of crystal violet staining on the colony formation of two types of gastric cancer cells, using H3B-120 and PRT4165 alone and in combination. Detailed Implementation

[0017] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0018] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0019] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0020] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0021] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0022] Terminology Explanation: PRT4165 is an effective inhibitor of PRC1-mediated H2A ubiquitination. It regulates histone H2A monoubiquitination at lysine 119 by inhibiting the E3 ubiquitin ligase activity of the PRC1 core components RING1A and RING1B. PRT4165 has the CAS number 31083-55-3 and the molecular formula C2. 15 H9NO2, structural formula: .

[0023] H3B-120 is a highly selective, competitive, and allosteric inhibitor of carbamoyl phosphate synthase 1 (CPS1). The CAS number for H3B-120 is 2194903-42-7, and its molecular formula is C2. 19 H 24 N4O2S, structural formula is: .

[0024] The ZIP quantitative synergistic model is a classic computational model for quantitatively assessing the interaction of two drugs in combination. It integrates the core assumptions of the Bliss independent effect and the Loewe additive model. Based on the fitting of a four-parameter logarithmic logistic function to the dose-response curve of a single drug, it fixes the concentration of one drug and fits the Hill equation of the other drug. The mean of the fit is taken as the theoretical expected tumor-suppressing effect when there is no interaction. By comparing the difference between the actual inhibitory effect and the theoretical expected effect, the degree of synergy / antagonism between drugs is quantified. It can output a continuous synergistic score matrix under the full concentration gradient, which is suitable for the quantitative analysis of cell viability detection data of two-drug gradient combination, and provides statistical quantitative basis for the interaction effect of drug combination.

[0025] The ZIP synergy score, a quantification index of the difference calculated by the ZIP model, is used to determine the type of interaction between two drugs in combination. (1) ZIP synergy score > 10: The actual tumor suppression effect of the combination is higher than the theoretical expectation without interaction, and it is judged as a synergistic effect. The larger the value, the higher the synergistic effect. (2) -10≤ZIP synergy score≤10: The combined effect is consistent with the theoretical effect of single drug superposition, and there is no interaction between the two drugs. It is judged as an additive effect. (3) ZIP synergy score < -10: The combined effect is lower than the theoretical expectation and is judged as antagonistic.

[0026] Example 1 I. Experimental Materials 1.1 Cells Gastric adenocarcinoma cell lines 23132 / 87 and SGC-7901 were both purchased from Nanjing Kebai Biotechnology Co., Ltd.

[0027] 1.2 Experimental Reagents Drugs: H3B-120 and PRT4165 were both prepared into stock solutions by dissolving in dimethyl sulfoxide (DMSO). During the experiment, they were diluted to the corresponding working concentrations. DMSO was used as a blank control.

[0028] II. Experimental Methods 2.1 Detection of CCK-8 cell viability and synergistic effect of dual-drug therapy (1) Cell seeding: After aspirating and centrifuging, discard the supernatant, add 1 mL of culture medium to resuspend the cells, mix well, and then add 10 μL of the cell suspension to a counting chamber to count the number of cells. After dilution, add 3×10⁻⁶ cells to each well of a 96-well plate. 3 Prepare 6 replicates for each treatment group using 100 μL of cells. After seeding all groups of cells into the plate, add an extra ring of 100 μL PBS around the outermost cells to help keep the cells moist and avoid affecting cell growth.

[0029] (2) Drug treatment: 24 h after cell seeding, the cells were treated with drugs for 48 h (PRT4165: PRC1 inhibitor; H3B-120: CPS1 inhibitor).

[0030] (3) CCK-8 treatment: Add 100 μL of CCK-8 mixture to each well (the CCK-8 reagent and serum-free culture medium are prepared at a volume ratio of 1:9), mix well, and incubate at 37°C for 1.5 h.

[0031] (4) Result determination: After the reaction was completed, the 96-well plate was wrapped with tin foil to protect it from light and the absorbance value at 450 nm was measured.

[0032] Calculate the inhibition rate using the following formula: Inhibition rate (%) = [1 - (OD value of experimental group - OD value of blank well) / (OD value of control group - OD value of blank well)] × 100%.

[0033] Graphpad Prism was used to plot dose-response curves; the concentration and inhibition rate data of the two drugs were imported into the SynergyFinder online platform to quantitatively analyze and visualize the synergistic effect of the two drugs, and to screen the optimal synergistic dosing concentration.

[0034] 2.2 Crystal Violet Colony Formation Experiment 1500 gastric cancer cells were seeded in each well of a 6-well plate and cultured adherently for 24 h. After 24 h, the cells were divided into four groups: DMSO control group, H3B-120 single drug group, PRT4165 single drug group, and dual drug combination group (H3B-120+PRT4165, abbreviated as H3B+PRT). The specific drug treatment is shown in Table 1. After 14 days of continuous culture, the cells were washed with PBS, fixed, stained with crystal violet, and photographed under a microscope to compare the cell clonal proliferation capacity of each group.

[0035] Table 1 Experimental groups for crystal violet colony formation experiment

[0036] 2.3 Flow cytometry detection of apoptosis 3 × 10⁶ seeds per well in a 6-well plate 5 Cells were cultured adherently for 24 h and then divided into groups for drug administration: DMSO control, H3B-120 single drug, PRT4165 single drug, and dual drug combination group (H3B-120+PRT4165, abbreviated as H3B+PRT). The specific drug administration treatment is the same as in Table 1. After 48 h of drug treatment, all cells were digested and collected. The cells were stained with Annexin V / 7-AAD apoptosis kit, and the apoptosis ratio of each group was detected by flow cytometry within 1 h. The differences in apoptosis induction between the single drug and combination groups were statistically analyzed and compared.

[0037] 2.4 Cell resuscitation and passage methods 2.4.1 Cell resuscitation (1) Melting: After removing 23132 / 87 cells and SGC-7901 cells from liquid nitrogen, quickly place them in a 37°C water bath. Remove the cells after 90 seconds. During this time, the cells need to be shaken continuously to ensure that the cells are heated and melted evenly.

[0038] (2) Centrifugation: Transfer the cells from the cryopreservation tube to a 15 mL centrifuge tube using a 1 mL pipette, add 5 mL of complete culture medium, and then place the tube in a centrifuge and centrifuge at 1000 rpm for 5 min to collect the cells (to remove dimethyl sulfoxide from the cryopreservation cell solution).

[0039] (3) Cell culture: Take out the 15 mL tube after centrifugation, discard the supernatant, add 5 mL of complete culture medium, gently pipette to mix, and aspirate into a T25 culture flask. Then shake the culture flask about 10 times in a figure-eight pattern to ensure uniform cell growth.

[0040] 2.4.2 Cell passage (1) Centrifugation: After the liquid in the T25 culture flask turns yellow or the growth density reaches about 80%, it is passaged. Use a 10 mL pipette to aspirate the yellow cell suspension in the T25 culture flask and place it in a 15 mL centrifuge tube. Centrifuge at 1000 rpm for 5 min.

[0041] (2) Resuspension: Take out 15 mL centrifuge tubes from the centrifuge, discard the old culture medium with a pipette, add 10 mL of complete culture medium, and then gently mix with a pipette.

[0042] (3) Culture: After mixing, the cells are evenly divided into two T25 culture flasks. The date, cell name, generation number and other information are written on the flask. Shake the culture flask about 10 times in a figure-eight pattern to ensure uniform cell growth. Then place it in a 37℃, 5% carbon dioxide incubator for culture.

[0043] 2.5 Statistical Analysis The independent samples t-test was used to compare the differences between the control group and each experimental group. P< A value of 0.05 is considered statistically significant.

[0044] III. Experimental Results 1. Synergistic inhibitory effect of PRT4165 and H3B-120 in gastric cancer cells This invention uses the CCK8 assay to test the inhibitory effects of PRT4165 monotherapy, H3B-120 monotherapy, and the combination of PRT4165 and H3B-120 on gastric cancer cells. The test results for the two monotherapy drugs are shown below. Figures 1-4 This invention visualizes the results of dual-drug combination therapy using the Synergy Fingder online platform, based on CCK8 experimental test results. The results are shown below. Figures 5-6The results showed that in SCG-7901 cells, the combination of H3B-120 (160 μM) and PRT4165 (77.6 nM) achieved the best inhibitory effect (80.6%); in 23132 / 87 cells, the combination of H3B-120 (80 μM) and PRT4165 (31.4 nM) achieved the best inhibitory effect (80.8%). The ZIP quantitative synergistic model showed that in SCG-7901 cells, the global average synergistic score was 13.72 (P<0.001). The surface area of ​​the medium-to-high concentration combination region was significantly raised, corresponding to a high ZIP synergistic score. Only a few green negative values ​​(weak antagonism, negligible) appeared in the very low concentration combination region. The synergistic scores of H3B-120 and PRT4165 were greater than 10 in the range of (50-160) μM: (15-150) nM. In 23132 / 87 cells, the global average synergistic score was 16 (P<0.001). The surface area of ​​the medium-to-high concentration combination region was significantly raised, corresponding to a high ZIP synergistic score. No synergistic scores below 0 were observed. The synergistic scores of H3B-120 and PRT4165 were greater than 10 in the range of (40-80) μM: (15-55) nM.

[0045] 2. Effects of PRT4165 and H3B-120 on the proliferation of gastric cancer cells In this invention, 1500 cells were evenly seeded in each well of a six-well plate. After 24 hours, drugs were added for treatment. Depending on whether it was an experimental group or a control group, dimethyl sulfoxide, H3B-120, PRT4165, or a combination of two drugs (H3B-120 + PRT4165) were added. After 14 days of culture, the culture medium was discarded, the cells were washed, stained with crystal violet, and photographed. The results are shown below. Figure 7 .

[0046] like Figure 7 As shown, the DMSO control group exhibited a large number of dense, large cell clones with high clonal density and deep staining, indicating vigorous cell proliferation. Treatment with H3B-120 or PRT4165 alone reduced the number of clones in both types of gastric cancer cells to some extent, with smaller clones and lighter staining, demonstrating that single-drug therapy only mildly inhibited the proliferation of gastric cancer cell clones. In contrast, the combined H3B-120 and PRT4165 group showed extremely few cell clones, with only scattered small cell colonies and almost no formation of large, intact clones. Comparison of the four staining images revealed that the combined use of these two drugs significantly inhibited the proliferation of SGC-7901 and 23132 / 87 gastric cancer cell clones compared to single inhibitors, demonstrating that the combination of these two drugs synergistically inhibits the long-term proliferation of gastric cancer cells.

[0047] 3. Effects of PRT4165 and H3B-120 on apoptosis in gastric cancer cells In this invention, each of the six-well plates is uniformly seeded with 3×10⁻⁶ seeds. 5 Cells were treated with drugs 24 h later. Depending on whether it was an experimental group or a control group, dimethyl sulfoxide, H3B-120, PRT4165, or a combination of two drugs (H3B-120+PRT4165) were added. After 48 h of drug treatment, the cells were digested and apoptosis staining was performed. The results of flow cytometry are shown in Table 2.

[0048] Table 2 Flow cytometry results

[0049] As shown in Table 2, in SGC-7901 cells, the total apoptosis rate in the DMSO control group was only 15.72%; treatment with H3B-120 and PRT4165 alone slightly increased the apoptosis rate, with limited apoptosis-inducing effects; combined treatment with H3B-120 and PRT4165 significantly increased the total apoptosis rate to 75.09%, with a significantly higher proportion of apoptotic cells than the control group and the single-drug groups. In 23132 / 87 cells, the apoptosis rate in the DMSO control group was 13.10%; both single drugs only slightly increased the apoptosis level; combined treatment with H3B-120 and PRT4165 resulted in a total apoptosis rate of 71.10%, with a significant increase in apoptosis.

[0050] The results showed that PRT4165 combined with H3B-120 could synergistically induce apoptosis in gastric cancer cells, and the pro-apoptotic effect was far superior to that of the two inhibitors used alone.

[0051] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A composition for inhibiting the proliferation of gastric cancer cells, characterized in that, Including H3B-120 and PRT4165.

2. The use of the composition as described in claim 1 in the preparation of a medicament for treating gastric cancer.

3. The application as described in claim 2, characterized in that, The stomach cancer mentioned is gastric adenocarcinoma.

4. The application as described in claim 2, characterized in that, The drug includes pharmaceutically acceptable excipients.

5. The application as described in claim 4, characterized in that, The excipients include fillers, binders, disintegrants, emulsifiers, flavoring agents, preservatives, or colorants.

6. A drug for treating gastric cancer, characterized in that, The active ingredient includes the composition of claim 1.

7. The drug as described in claim 6, characterized in that, The drug also includes pharmaceutically acceptable excipients.

8. The drug as described in claim 7, characterized in that, The excipients include fillers, binders, disintegrants, emulsifiers, flavoring agents, preservatives, or colorants.

Images