CN115350176B

CN115350176B - Gastric cancer tumor cell and gastric cancer tumor stem cell therapeutic drug and application thereof

Info

Publication number: CN115350176B
Application number: CN202210825914.6A
Authority: CN
Inventors: 陈铁; 植韵诗; 孙雅欣; 沈小芳; 张景宜; 李晰; 赵倩倩
Original assignee: Shenzhen University
Current assignee: Shenzhen University
Priority date: 2022-07-14
Filing date: 2022-07-14
Publication date: 2023-10-13
Anticipated expiration: 2042-07-14
Also published as: CN115350176A

Abstract

The application provides a gastric cancer tumor cell and a gastric cancer tumor stem cell therapeutic drug and application thereof, and the drug BF4 (0.0625 mu M to 50 mu M), BF4-2 (0.0625 mu M to 10 mu M), BF4-3 (5 mu M to 10 mu M), BF4-4 (1 mu M to 10 mu M) and BF4-5 (0.25 mu M to 10 mu M) can block proliferation of gastric cancer cell lines and tumor stem cells derived from gastric cancer patients, promote apoptosis, have obvious concentration dependence and the target protein of BF4 action is CPT2.

Description

Gastric cancer tumor cell and gastric cancer tumor stem cell therapeutic drug and application thereof

Technical Field

The application belongs to the field of biological medicines, and in particular relates to a novel gastric cancer tumor cell and a gastric cancer tumor stem cell therapeutic drug.

Background

As can be seen from the statistical data results published by the international cancer research Institute (IARC) in 2020, the newly increased number of cases of gastric cancer is 1089103 worldwide, which is the fifth most frequent cancer in the world; the number of newly added death cases is 768793, and the death rate is ranked fourth. Gastric cancer accounts for 5.6% of all cancer cases, which accounts for 7.7% of all deaths. Gastric cancer incidence rates are up to 7.1% among male cancer patients, fourth among all male cancer patients, and mortality rates are up to 9.1%, fourth among male cancer mortality rates; whereas in female cancer patients the incidence is relatively low, about 4.0%, in the seventh place; mortality in female cancer patients is relatively high, about 6.0%, and is ranked fifth. Stomach cancer is highly pronounced in south america, east asia and eastern europe in terms of the affected areas. The cause of gastric cancer is numerous and its exact mechanism is not yet fully known, but helicobacter pylori is the first major risk factor for gastric cancer. Therefore, it is important to timely screen, diagnose and reasonably and effectively treat the patients with the disease. However, in some countries, particularly countries with lower incidence of gastric cancer (including the united states), such screening strategies are considered to be too costly and not necessary. Moreover, in china, more than 80% of patients are already at advanced stages of gastric cancer at the time of diagnosis, so that many may miss the best opportunity to radical resection therapy or may increase the risk of postoperative metastasis and recurrence. It follows that gastric cancer remains a health problem that all humans need to face together.

According to the 2019 edition of stomach cancer diagnosis and treatment guide of the clinical oncology society of China, the current treatment scheme of stomach cancer in China is generally as follows: early gastric cancer treatment is generally based on endoscopic surgical resection (emr+esd); the middle stage is mainly resected by gastric surgery, and is assisted by chemotherapy; advanced stage is mainly assisted by chemotherapy/radiotherapy, and assisted by gastric surgical excision. 1. The second-line chemotherapeutics are 5-FU+metal platinum and docetaxel+irinotecan/taxol respectively. The three-wire is the targeting drug, and trastuzumab and apatinib are the main drugs. It can be seen that chemotherapy still plays a very important role in the treatment of gastric cancer. In the practical application process, the medicines are often combined, so that more remarkable treatment effect can be obtained. Although these drugs have been shown to have clinically desirable therapeutic effects, toxic side effects (e.g., loss of appetite, nausea, vomiting, diarrhea, etc.) often also cause additional pain to the patient during the course of treatment, reducing the quality of life of the patient. It is therefore imperative to find more new, efficient and low-toxicity chemotherapeutics to extend the survival of patients, particularly patients with advanced gastric cancer, to improve the quality of life of patients and to reduce the economic burden on patients.

The currently developed targeted drugs mainly comprise three types of protein kinase inhibitors, immune test point inhibitors and tumor stem cell pathway inhibitors, and the tumor stem cell pathway inhibitors are the most interesting in the research and development of the currently targeted anticancer drugs. In 1977, the concept of tumor stem cells was first proposed-tumor stem cells are self-renewing cells with long-term proliferation differentiation potential, and gene expression profiles demonstrated that tumor stem cells are closer to embryonic stem cells than adult stem cells. In recent years, the research on the resistance of tumor cells to chemotherapy, tumor recurrence and metastasis has considered that very few tumor stem cells existing in tumors can achieve the aim by activating survival promotion or anti-apoptosis pathways, over-expressing drug efflux pumps, improving DNA repair capacity and the like after being stimulated by the outside. Therefore, only if the molecular mechanism of the gastric cancer stem cells is fully known, a new drug treatment target point is found, and the gastric cancer can be effectively treated by combining a standard chemotherapy scheme and an anti-tumor stem cell therapy, and the efficiency of traditional chemotherapy can be effectively improved and the drug resistance can be reduced.

The applicant successfully separates and identifies gastric cancer tumor stem cells at home and abroad in 2011, and provides a solid cell model for the research and development of subsequent targeted drugs. In the early work, the inventor uses gastric cancer tumor stem cells as a model and uses a drug library (663 compounds) to perform activity screening. Surprisingly, the inventor finds that BF4 can inhibit the growth of gastric cancer cell lines and promote the rounding death of the gastric cancer cell lines; can also inhibit the growth of gastric cancer tumor stem cells, promote the rounding death of the gastric cancer tumor stem cells, and has shorter onset time, lower onset concentration and better cancer cell killing effect compared with 5-Fu. The experimental result of nude mice tumor-bearing shows that BF4 can obviously inhibit the growth of transplanted tumor derived from gastric cancer tumor cells; and the effect is better than that of 5-Fu.

In the preliminary anti-cancer mechanism research, the inventor finds that BF4 promotes apoptosis of gastric cancer cells, and the target protein acted by BF4 is CPT2, but the specific mechanism of the action is not clear, and further intensive research and exploration are needed. Through the research of deep systems, a new drug action target point or a new molecular mechanism of gastric cancer cells can be discovered, simultaneously, BF4 is hopefully developed into a new gastric cancer therapeutic drug, and becomes a new 5-Fu substitute chemotherapeutic drug, thereby providing a new method for gastric cancer treatment, driving the new development and utilization of the novel 5-Fu substitute chemotherapeutic drug, developing a new drug with higher efficiency and lower toxicity, and being expected to bring better social effect and higher economic value.

Disclosure of Invention

In order to solve the technical problems, the application provides application of BF4 and salts thereof in preparing medicaments for treating gastric cancer tumors and gastric cancer tumor stem cells, wherein the BF4 has the structural formula as follows:

the application also provides application of BF4 analogue BF4-2 in preparing medicaments for treating gastric cancer tumors and gastric cancer tumor stem cells, and the structural formula is as follows:

the application also provides application of BF4 analogue BF4-3 in preparing medicaments for treating gastric cancer tumors and gastric cancer tumor stem cells, and the structural formula is as follows:

the application also provides application of BF4 analogue BF4-4 in preparing medicaments for treating gastric cancer tumors and gastric cancer tumor stem cells, and the structural formula is as follows:

the application also provides application of BF4 analogue BF4-5 in preparing medicaments for treating gastric cancer tumors and gastric cancer tumor stem cells, and the structural formula is as follows:

among them, BF4 and its analogues are purchased from Shanghai Tao Shu biotechnology limited.

Preferably, in the present application, the tumor is one or more of esophageal cancer, gastric cancer, colorectal cancer, lung cancer and brain cancer.

Preferably, the medicament obtained by the application is injection, spray or needle powder.

The application aims to solve the problems of toxic and side effects of the traditional chemotherapeutics and the high price of the targeted drugs, a new drug for treating gastric cancer cells and tumor stem cells is found from drug-like compounds, the drug has better effect, low toxicity and lower onset concentration than the clinically used drug 5FU, the onset time is earlier than the clinically used drug 5FU, and the effect of BF4 (CAS number: 592514-16-4) in tumor treatment is disclosed for the first time at home and abroad.

The medicine of the application has higher effect concentration on killing normal skin mucosa cells than gastric cancer tumor cells, has smaller cytotoxin effect, has high-efficiency killing effect on gastric cancer cell lines and tumor stem cells of gastric cancer patients, and overcomes the defect that the traditional medicine can not kill the tumor stem cells.

The gastric cancer stem cells in the application are obtained by autonomous separation by the inventor: chen T, yang K, yu J, et al identification and expansion of cancer stem cells in tumor tissues and peripheral blood derived from gastric adenocarcinoma patients [ J ]. Cell research.2012,22 (1): 248-258.

The medicaments BF4 (0.0625 mu M to 50 mu M), BF4-2 (0.0625 mu M to 10 mu M), BF4-3 (5 mu M to 10 mu M), BF4-4 (1 mu M to 10 mu M) and BF4-5 (0.25 mu M to 10 mu M) can block proliferation of gastric cancer cell lines and tumor stem cells derived from gastric cancer patients, promote apoptosis and have obvious concentration dependence.

The application protects the treatment of BF4 (CAS No. 592514-16-4) and its analogues in the field of gastric cancer, as well as the treatment in other tumors.

The cancer stem cells of the stomach cancer patient used in the application can be more similar to the cancer cell state of the tumor patient.

BF4 (CAS No. 592514-16-4) and analogues thereof and BF4 salts and analogues thereof in the present application are used in tumor therapy.

The application also provides a gastric cancer tumor cell and a gastric cancer tumor stem cell therapeutic drug, and the target protein of the drug is CPT2.

Correspondingly, the application provides application of the target protein CPT2 in treating gastric cancer tumor cells and gastric cancer tumor stem cells.

Drawings

Fig. 1: chemical information of BF4 and analogs thereof. Including code, chemical structure, CAS number, chemical formula, and molecular weight.

Fig. 2: effects of BF4 and 5FU on various gastric cancer cell viability. The X axis is drug concentration, the Y axis is cell viability; gastric cancer cell lines (HGC 27, AGS, MGC 803), gastric cancer tumor stem cells (GT 112, GT 0603), normal skin mucosa cells (Hacat).

Fig. 3: effect of BF4 and its analogues on various gastric cancer cell viability. The X axis is drug concentration, the Y axis is cell viability; gastric cancer cell lines (HGC 27, AGS, MGC 803), gastric cancer tumor stem cells (GT 112, GT 0603), normal skin mucosa cells (Hacat).

Fig. 4: morphological changes of 5FU (0 μm, 0.03125 μm, 0.0625 μm, 0.125 μm, 0.25 μm, 0.5 μm, 1 μm, 2 μm, 5 μm, 10 μm) on gastric cancer cell lines (HGC 27, AGS, MGC 803) and gastric cancer tumor stem cells (GT 112, GT 0603) under an inverted microscope.

Fig. 5: morphological changes of BF4 and its analogues (0. Mu.M, 0.03125. Mu.M, 0.0625. Mu.M, 0.125. Mu.M, 0.25. Mu.M, 0.5. Mu.M, 1. Mu.M, 2. Mu.M, 5. Mu.M, 10. Mu.M) on gastric cancer cell line HGC27 under an inverted microscope.

Fig. 6: morphological changes of BF4 and its analogues (0. Mu.M, 0.03125. Mu.M, 0.0625. Mu.M, 0.125. Mu.M, 0.25. Mu.M, 0.5. Mu.M, 1. Mu.M, 2. Mu.M, 5. Mu.M, 10. Mu.M) on gastric cancer cell line GT0603 under an inverted microscope.

Fig. 7: morphological changes of BF4 and its analogues (0. Mu.M, 0.03125. Mu.M, 0.0625. Mu.M, 0.125. Mu.M, 0.25. Mu.M, 0.5. Mu.M, 1. Mu.M, 2. Mu.M, 5. Mu.M, 10. Mu.M) to gastric cancer cell line MGC803 under an inverted microscope.

Fig. 8: morphological changes of BF4 and its analogues (0. Mu.M, 0.03125. Mu.M, 0.0625. Mu.M, 0.125. Mu.M, 0.25. Mu.M, 0.5. Mu.M, 1. Mu.M, 2. Mu.M, 5. Mu.M, 10. Mu.M) to gastric cancer cell lines AGS under an inverted microscope.

Fig. 9: morphological changes of BF4 and its analogues (0. Mu.M, 0.03125. Mu.M, 0.0625. Mu.M, 0.125. Mu.M, 0.25. Mu.M, 0.5. Mu.M, 1. Mu.M, 2. Mu.M, 5. Mu.M, 10. Mu.M) on gastric cancer cell line GT112 under an inverted microscope.

Fig. 10: morphological changes of BF4 and 5FU (0. Mu.M, 0.03125. Mu.M, 0.0625. Mu.M, 0.125. Mu.M, 0.25. Mu.M, 0.5. Mu.M, 1. Mu.M, 2. Mu.M, 5. Mu.M, 10. Mu.M) on gastric cancer stem cells GT112, GT0603 under an inverted microscope (48 h).

Fig. 11: morphological changes of BF4 and 5FU (0. Mu.M, 0.03125. Mu.M, 0.0625. Mu.M, 0.125. Mu.M, 0.25. Mu.M, 0.5. Mu.M, 1. Mu.M, 2. Mu.M, 5. Mu.M, 10. Mu.M) on gastric cancer stem cells GT112, GT0603 under an inverted microscope (72 h).

Fig. 12: the cloning result of the flat plate shows that BF4 and 5FU obviously inhibit the survival and proliferation of gastric cancer cells; and (3) performing BF4 concentration gradient treatment on GT0603 and GT112 gastric cancer tumor stem cells and HGC27, AGS and MGC803 gastric cancer cell lines for 48 hours, replacing fresh complete culture medium without medicines, culturing for one week to macroscopic cell mass, fixing methanol, staining with 5% crystal violet, counting clone numbers, and performing statistical analysis.

Fig. 13: and (3) carrying out mass spectrometry on the adhesive tape in the red square frame by using the magnetic beads to pull down silver-stained strips after proteins, and further carrying out Western Blot verification on the mass spectrometry result, so that obvious up-regulation of CPT2 of the BF4-Botin group is found.

Fig. 14: BF4 inhibits growth of human gastric cancer tumor cell HGC27 in BALB/c-nu nude mice. A. Control and experimental groups of nude mice were compared every three days in weight. B. Comparison of the body weights of the nude mice in the control group and the experimental group. C. Comparison of tumor sizes in control and experimental groups. D. Tumor volume comparison E for experimental and control groups, tumor weight comparison for experimental and control groups.

Fig. 15: HE staining analysis results of nude mice heart, liver, spleen, lung, kidney, small intestine, tumor.

Detailed Description

Preferred embodiments of the present application will be described in further detail below with reference to the attached drawings:

1. method of

1.1CCK8 colorimetry for determining IC of BF4, BF4 analogue and 5FU ₅₀ Value, drawing a dose-response curve and an aging curve

In vitro amplifying two gastric cancer tumor stem cells (in situ tumor stem cells GT0603 and GT 112), two gastric cancer tumor cell lines (HGC 27, AGS and MGC 803) and a gastric mucosa normal cell line (Hacat), and taking cells in logarithmic growth phase for digestion, counting and resuspension. The cell resuspension is divided into three groups of zeroing group, control group and experimental group, and the control group and experimental group are respectively 5.5×10 per hole ³ Individual cells were seeded in 96-well plates and zeroed groups were filled with equal amounts of medium without cells, 100 μl of medium per well. 3 compound holes are arranged at the same drug concentration, and 9 drug concentrations are arranged in total. Three drug treatment times were set for each cell line, 24h, 48h, 72h, respectively. The cells were inoculated for 24 hours and then treated with drugs at concentrations of 0. Mu.M, 0.03125. Mu.M, 0.0625. Mu.M, 0.125. Mu.M, 0.25. Mu.M, 0.5. Mu.M, 1. Mu.M, 2. Mu.M, 5. Mu.M, 10. Mu.M, and 20. Mu.M, respectively; equal amounts of solvent DMSO were added to the control and zeroed groups, respectively.

After 24h, 48h, 72h of drug treatment, 10 μl CCK8 reagent (slowly add along the wall, avoiding air bubbles) was added to each well using a multichannel pipette. The 96-well plate with CCK8 reagent added is placed in an incubator for incubation. After one half hour incubation, cell viability was measured at 450nm using an enzyme-labeled instrument, and cell viability values were expressed as OD means. After the OD value of the zeroing group is deducted, respectively drawing a concentration-survival rate quantitative effect fitting curve and an aging effect fitting curve of each cell line, and calculating the IC of BF4 on different cells at different treatment times ₅₀ The value is used for determining the effect of BF4 on normal cells of skin, gastric cancer tumor stem cells and gastric cancer tumor cell lines in vitro.

1.2 morphological observations

The gastric cancer tumor stem cells (in situ tumor stem cells GT0603, GT 112), two gastric cancer tumor cell lines (HGC 27, AGS, MGC 803) were subjected to cell digestion, centrifugation, counting, resuspension, inoculation, and treatment with drugs BF4, BF4 analog and 5FU (drug concentrations of 0. Mu.M, 0.03125. Mu.M, 0.0625. Mu.M, 0.125. Mu.M, 0.25. Mu.M, 0.5. Mu.M, 1. Mu.M, 2. Mu.M, 5. Mu.M, 10. Mu.M, 3 duplicate wells were provided for each drug concentration, drug treatment time of 48 h) respectively, and then subjected to observation of changes in cell morphology in an inverted microscope, and the inverted microscope was adjusted, and cells of the control group and experimental group having drug concentrations of 0.03125. Mu.M, 0.0625. Mu.M, 0.125. Mu.M, 0.25. Mu.M, 1. Mu.M, 2. Mu.M, 5. Mu.M, and 10. Mu.M were randomly selected for recording the photographed data.

1.3 plate cloning experiments to evaluate the anti-tumor cell proliferation Capacity of BF4 and 5FU

In the experiment, gastric cancer tumor stem cells (in-situ tumor stem cells GT0603 and GT 112) in logarithmic growth phase and gastric cancer tumor cell lines (HGC 27, AGS and MGC 803) are respectively inoculated in 24 pore plates, and are divided into a dosing group and a control group, wherein each group comprises 3 compound pores and 500 cells in each pore. The BF4 is added after being cultured for 24 hours in an incubator, and the dosage concentration is divided into three groups of low, medium and high, namely 0 mu M (equal amount of DMSO is added), 0.03125 mu M, 0.0625 mu M and 0.125 mu M respectively; the 5FU administration concentration was divided into three groups of low, medium and high, namely 0. Mu.M (equivalent DMSO was added), 0.5. Mu.M, 1. Mu.M and 2. Mu.M, and the culture was continued for 48 hours after administration. After 48h fresh complete medium without drug was changed and cultivation continued for a week until macroscopic cell mass was reached.

After macroscopic cell clusters were cultured, the upper medium was carefully aspirated, and after three washes with pre-chilled PBS, the PBS was discarded. 0.5ml of methanol was slowly added to each well along the wall, and after being placed on a shaking table and slowly shaken for 15 minutes, the methanol was discarded. 0.5ml of crystal violet dye was slowly added to each well along the wall, and after 20 minutes of dyeing with slow shaking on a shaker, the crystal violet dye was discarded. And (3) slowly washing off crystal violet dye solution by flowing water, putting the crystal violet dye solution in a fume hood for airing, and evaluating the influence of BF4 on the survival and proliferation capacity of gastric cancer tumor stem cells according to the number of cell aggregates.

1.4Pulldown experiments BF4 binding proteins

In the experiment, gastric cancer tumor cell lines HGC27 in logarithmic growth phase are respectively inoculated in 10cm ² Culture dish placed in incubatorAfter 24h of culture, 50 mu M of Botin and 50 mu M of BF4-Botin are respectively used as a control group and a dosing group, the supernatant is sucked away 24h after the dosing, 4ml of PBS is added for washing twice, RIRA lysate containing protease inhibitor and phosphatase inhibitor is added to prepare a protein sample, the protein content is measured by a BCA method, the BF4-Botin is combined by streptavidin coupled magnetic beads, a target protein is pulled down, the size and position of the protein band are further determined by gel electrophoresis, and then protein mass spectrometry analysis is carried out to further confirm target protein.

1.5 construction of tumor-bearing nude mouse model and detection of BF4 in vivo activity

1.5.1 treatment of gastric cancer tumor cells HGC27

In the experiment, gastric cancer tumor cells HGC27 are selected as the transplanted tumor construction cells. Inoculating gastric cancer tumor cell HGC27 at 10cm ² In the culture dish, when the monolayer grows to be fully covered to 90% of the bottom of the whole culture dish, namely, when the cells are in logarithmic growth phase, removing the upper layer culture medium, pre-cooling PBS, washing, discarding the PBS, adding 1ml of pancreatin to digest the cells into single cells, and immediately stopping digestion by using the culture medium containing serum. Put into a centrifuge for centrifugation at 800rpm/3min. Discarding the upper medium of the centrifuge tube, adding pre-cooled PBS for resuspension, and diluting to 2×10 per 0.2ml ⁵ Cell suspensions of individual cells.

1.5.2 construction of tumor-bearing nude mouse model

BALB/c-nu nude mice are divided into an experimental group, a control group and a positive control group, wherein the experimental group comprises 5 nude mice, the control group comprises 5 nude mice, and the positive control group comprises 5 nude mice. Placing the above prepared cell suspension in ice box, and taking into animal room, and injecting into armpit skin with 6-gauge needle syringe, each injection being 0.15ml and containing about 2×10 ⁶ Individual cells. Before injection, the armpit injection part is wiped and disinfected by using 75% alcohol, and the air in the needle head is removed; the needle is penetrated forward by about 1cm from the needle inserting position during inoculation and then injection is carried out; after the injection is completed, the needle head is slowly withdrawn, so that liquid leakage is avoided. Mice were observed daily for status and tumor growth.

1.5.3BF4 in vivo Activity assay

Determining success of vaccinationThereafter, 10mg/kg of BF4 was administered by intraperitoneal injection in the experimental group, physiological saline was administered by intraperitoneal injection in the control group, and 15mg/kg of 5-FU was administered by intraperitoneal injection in the positive control group. Nude mice body weight and tumor volume were measured and recorded periodically. When the tumor of the nude mice in the control group grows to 1.5cm long, CO is adopted ₂ Killing nude mice, dissecting and stripping tumor tissues, weighing and recording tumor body weight, drawing a survival curve, and carrying out statistical analysis to determine the effect of BF4 in vivo on gastric cancer tumor stem cells.

Taking out heart, liver, spleen, lung, kidney, small intestine, tumor, etc. of nude mice, formalin-fixing, paraffin embedding and HE staining analysis, and exploring whether BF4 has serious side effect on nude mice, damaging the organ function of nude mice, etc.

1.6 statistical treatment

Data are presented as mean ± Standard Deviation (SD) for each experimental result. The t-test was used to determine statistical significance differences, with significant differences in data results when P values were < 0.05. All line and bar graphs in this experiment were plotted using GraphPad Prism 9.0 software.

2. Experimental results

2.1CCK8 in vitro colorimetric determination shows that BF4 has good killing effect on gastric cancer cells, the onset of action concentration is lower than that of 5FU, and the onset of action time is earlier than that of 5 FU.

As shown in fig. 2 and 3, after the drug BF4 is used to treat gastric cancer tumor stem cells GT0603 and GT112, gastric cancer cells HGC27, AGS and MGC803 and a normal skin mucosa cell Hacat at different concentrations and for different times, the killing effect of each cell is different. Wherein, the killing effect of the gastric cancer cell strain MGC803 is most obvious, and HGC27 and AGS are secondary; the tumor stem cells GT0603 and GT112 show obvious time dependence and dose dependence, and the better the killing effect is along with the increase of the concentration of the medicine and the increase of the treatment time; although the killing effect on the normal skin mucosa cells Hacat shows concentration dependence and time dependence, the IC is realized in three treatment time of the experiment ₅₀ The values were far greater than other cell lines. From this, it was determined for the subsequent experiments by the inventors that the basic experimental conditions were that the drug treatment concentrations were 0. Mu.M, respectively (additionEquivalent DMSO), 0.03125. Mu.M, 0.0625. Mu.M, 0.125. Mu.M, treatment time of 48h.

2.2 observation of the effects of BF4 on cells under an inverted microscope

As shown in fig. 4 to 9, gastric cancer cells GT112, MGC803, HGC27, AGS and GT0603 were treated with drugs BF4 of 0.03125 μm, 0.0625 μm, 0.125 μm, 0.25 μm, 0.5 μm, 1 μm, 2 μm, 5 μm, 10 μm for 48 hours, and then the morphological changes of the cells under the drug treatment were observed under an inverted microscope, which intuitively reflected the inhibition of gastric cancer cell proliferation by drug BF 4. The cells of the control group grew vigorously, had good extensibility, were closely packed, were substantially irregularly polygonal in shape (MGC 803 is relatively regular), were full, and had fewer floating cells. The cells of the administration group have the advantages that the proliferation speed of the cells is reduced along with the increase of the concentration of BF4, the gaps among the cells are obviously enlarged, the cell bodies are gradually shrunken and rounded, the cells fall off from a culture dish and float on the surface of a culture medium, the rounded cells are very much, smooth and transparent, the normal cell morphology is lost, the BF4 has the inhibition effect on the growth of five gastric cancer cells, and the result is basically consistent with a CCK8 experiment.

As shown in fig. 10 and 11, gastric cancer tumor stem cells GT112 and GT0603 were treated with drug BF4 of 0.03125 μm, 0.0625 μm, 0.125 μm, 0.25 μm, 0.5 μm, 1 μm, 2 μm, 5 μm, 10 μm for 48 hours, and then observed under an inverted microscope for morphological changes of the cells under the drug treatment, visually reflecting the inhibition of proliferation of gastric cancer tumor stem cells by drug BF 4. The control group had good cell growth status, and the cell spheres were smooth and clear spheres with no or few individual cells. The cells of the administration group start to be rough along with the increase of the concentration and the administration time of BF4, the cell balls are ulcerated into single cells, the cell balls are dull and gradually shrink and round, and the normal tumor stem cell morphology is lost, so that BF4 has the inhibition effect on the growth of gastric cancer tumor stem cells.

The plate cloning result shows that BF4 remarkably inhibits the survival and proliferation of gastric cancer cells, the effective concentration is lower than that of 5FU, and after treatment of gastric cancer tumor stem cells GT0603 and GT112 and gastric cancer cells HGC27 and AGS and MGC803 for 48 hours by using BF4 with different concentrations, fresh complete culture medium without medicines is replaced for culturing for one week until cell aggregates are visible to naked eyes. Each cell line showed a different degree of drug concentration dependence, with the increase in drug concentration, its clonal proliferation capacity was reduced, and cell clumps were fewer or even absent. Wherein, when the drug reaches 0.0625 mu M, the gastric cancer cell strains HGC27, AGS, GT0603 and GT112 have almost no cell survival, while other cell strains have less cell survival and small cell mass. This is substantially in line with the CCK8 experimental results, as shown in fig. 12.

2.3BF4 is capable of specifically binding to CPT2 protein

After treating gastric cancer cell line HGC27 with 50. Mu.M of Botin and 50. Mu.M of BF4-Botin for 24 hours, collecting cell extract protein, combining BF4-Botin with streptavidin-coupled magnetic beads, pulling down target protein, and performing gel electrophoresis to obtain the result shown in FIG. 13. Protein mass spectrometry is carried out on the gel point (namely red frame part) of 55-100kDa in the cleavage map, the mass spectrum result is analyzed, and by combining with the analysis of cell morphological change, the size of the cleaved protein band, the total number of the enriched proteins and the like, 4 proteins (CPNE 3, CPT2 and SACM1L, PDCD IP) are found to have obvious differences, and then Western Blot gel electrophoresis analysis is carried out, so that only CPT2 has obvious binding specificity and CPT2 in BF4-Botin group is obviously up-regulated. From this, the basic conclusion is drawn: BF4 specifically binds to CPT2 protein, causing its downstream changes, which in turn promote cell death, as shown in fig. 13.

2.5BF4 in vivo inhibition of growth of nude mice transplanted tumor of human gastric cancer tumor cells

In BF4 in vivo activity assay, BALB/c-nu nude mice were divided into three groups. Wherein 5BF4 experimental groups; 5FU experimental group 5; control group 5. In the 5FU experimental group, 15mg/kg of 5FU is given to BALB/c-nu nude mice in the abdominal cavity after successful construction of the transplanted tumor; in the BF4 experimental group, after the transplanted tumor is constructed successfully, 10mg/kg of BF4 is given to the BALB/c-nu nude mice in the abdominal cavity, and the weight and the volume of the tumor-bearing tumors are found to be obviously lower than those of the 5FU experimental group and the control group, which shows that the BF4 can inhibit the growth of human gastric cancer tumor cells HGC27 in vivo, as shown in figure 14.

HE staining analysis, found that the heart, liver, spleen, lung, kidney, small intestine of BF4 experimental group was morphologically unchanged from the control group; compared with the control group, the tumors of the BF4 experimental group have obvious cell damage due to the nuclear deletion of tumor cells, which shows that BF4 has no serious side effect on nude mice, does not damage the organ function of the nude mice and can specifically kill the tumor cells, as shown in figure 15.

3. Conclusion(s)

BF4 (CAS number: 592514-16-4) and analogues thereof BF4-2 (CAS number: 149550-68-5), BF4-3 (CAS number: 860787-52-6), BF4-4 (CAS number: 171286-13-8) and BF4-5 (CAS number: 303139-57-3) not only can inhibit the growth of gastric cancer cell lines and promote the apoptosis thereof; BF4 (CAS number: 592514-16-4) also inhibits the growth of gastric cancer tumor stem cells and promotes apoptosis thereof; BF4 is injected into the tumor-bearing nude mice by the abdominal cavity, the BF4 can obviously inhibit the growth of the transplanted tumor of the human gastric cancer tumor cells and has no obvious toxic or side effect. Pull down experiments and Western Blot results show that BF4 can be specifically combined with CPT2 protein; the mechanism of action of BF4 (CAS number 592514-16-4) in the treatment of gastric cancer tumor cells may be associated with the inhibition of CPT2.

Therefore, BF4 (CAS number: 592514-16-4) and its analogues BF4-2 (CAS number: 149550-68-5), BF4-3 (CAS number: 860787-52-6), BF4-4 (CAS number: 171286-13-8), BF4-5 (CAS number: 303139-57-3) can be used as a novel therapeutic agent for gastric cancer tumor cells and gastric cancer tumor stem cells.

The foregoing is a further detailed description of the application in connection with the preferred embodiments, and it is not intended that the application be limited to the specific embodiments described. It will be apparent to those skilled in the art that several simple deductions or substitutions may be made without departing from the spirit of the application, and these should be considered to be within the scope of the application.

Claims

1. The application of BF4 and the salt thereof in preparing medicaments for treating gastric cancer and tumor has the structure as follows:

。

2. the application of BF4-2 and its salt in preparing medicines for treating gastric cancer and tumor is characterized in that the structural formula of BF4-2 is as follows:

。

3. application of BF4-3 and its salt in preparing medicines for treating gastric cancer and tumor, the structural formula of BF4-3 is as follows:

。

4. application of BF4-4 and salts thereof in preparing medicaments for treating gastric cancer tumors, wherein the structural formula of BF4-4 is as follows:

。

5. application of BF4-5 and salts thereof in preparing medicaments for treating gastric cancer tumors, wherein the structural formula of BF4-5 is as follows:

。

6. the use according to claim 1, wherein BF4 is capable of blocking proliferation of gastric cancer cell lines at a concentration of 0.0625 to 50 μm.

7. The use according to claim 2, wherein BF4-2 is capable of blocking proliferation of gastric cancer cell lines at a concentration of 0.0625 to 10 μm.

8. The use according to claim 3, wherein BF4-3 is capable of blocking proliferation of gastric cancer cell lines at a concentration of 5 to 10 μm.

9. The use according to claim 4, wherein BF4-4 is capable of blocking proliferation of gastric cancer cell lines at 1 to 10 μm.

10. The use according to claim 5, wherein BF4-5 is capable of blocking proliferation of gastric cancer cell lines at 0.25 to 10 μm.