Application of clofampol in preparation of medicine for treating gastric cancer

Abstract

The invention relates to an application of clofampol in preparation of a medicine for treating gastric cancer, and relates to the technical field of medicines. According to the invention, the new application of the clofampol in preparing the medicine for treating the gastric cancer is provided for the first time, and the medicine exerts the tumor inhibition effect through targeted inhibition of the cell viability of the gastric cancer stem cells and induction of necrotic apoptosis of the gastric cancer stem cells. Besides, the invention provides the application of the combination of the clofampol and the chemotherapeutic drug 5-Fu in preparing the drug for treating the gastric cancer for the first time, so that the use concentration of the chemotherapeutic drug 5-Fu can be reduced, the side effect of the chemotherapeutic drug 5-Fu can be reduced, and the lethality of the chemotherapeutic drug to gastric cancer cells can be greatly enhanced by the combination of the clofampol and the chemotherapeutic drug 5-Fu; the targeting function of the medicine on tumor cells is improved, so that an effective chemotherapy regimen is provided for treating the gastric cancer, the physical and psychological burden of a patient is relieved, the living quality of the patient is improved, and remarkable social benefits and economic benefits are achieved.

Description

Technical Field

[0001] The invention relates to the technical field of medicine, and in particular to the application of clofocol in preparing medicine for treating gastric cancer. Background Art

[0002] Gastric cancer is currently the fourth most common tumor in the world and the third most common cause of cancer-related death in the world. Gastric cancer is more common in men aged 40 to 60, and is relatively rare in women.

[0003] As we all know, the occurrence and development of gastric cancer is a process of gradual accumulation of multiple steps and multiple factors. More than half of patients are caused by Helicobacter pylori infection. In addition to the common Helicobacter pylori infection factors, various external and internal factors such as living environment, eating habits and geography can cause gastric cancer. At present, the causes of gastric cancer and the specific molecular mechanisms of the disease are not very clear, and research on the occurrence and development of gastric cancer is still ongoing.

[0004] At present, the treatment of gastric cancer is still mainly surgical resection and chemotherapy. With the gradual improvement of medical technology, new surgical methods and chemotherapy drugs for the treatment of gastric cancer continue to emerge, and the technical level of doctors has significantly improved. Although gastric cancer cannot be completely cured, the survival time and quality of life of patients have been greatly improved. In particular, the introduction of new treatment instruments and the use of chemotherapy drugs can reduce the growth and metastasis of tumor cells in patients while minimizing the damage to normal tissues and cells, thus greatly reducing the pain of patients during and after surgery. However, for patients with advanced gastric cancer, the crazy growth and metastasis of tumor cells cause the malignancy of gastric cancer to be extremely high and progress rapidly. In the end, patients usually die due to factors such as repeated occurrence of tumors, metastasis to other tissues and organs, and drug resistance caused by long-term use of chemotherapy drugs.

[0005] Gastric cancer is difficult to treat mainly because of the lack of early diagnostic markers and the frequent discovery of gastric cancer in the late stage. Therefore, the molecular and cellular basis of the occurrence and development of gastric cancer needs to be further studied in order to provide the best treatment strategy. In recent years, some tumor genes, tumor suppressor genes and small molecule inhibitors have been increasing in the study of gastric cancer. For example, P53, KRAS and SEMAPHORIN3E have been found to have potential carcinogenic or tumor suppressor effects. Small molecule inhibitors developed for tumor-related genes and signaling pathways have also been used in clinical practice. For example, apatinib is a small molecule inhibitor of vascular endothelial growth factor receptor 2, which is currently in clinical phase II and III clinical trials. With the continuous deepening of research, more drugs for gastric cancer treatment have been discovered, providing new methods and strategies for early diagnosis and subsequent treatment of gastric cancer.

[0006] With the continuous deepening of the study of tumor heterogeneity, the traditional clonal evolution theory is challenged by the emerging tumor stem cell theory. The tumor stem cell theory believes that there is a group of cells in the tumor that have self-renewal, multidirectional differentiation and high tumorigenicity. This small group of cells is the root cause of tumor heterogeneity. Many studies have shown that because tumor stem cells have strong self-renewal and multidirectional differentiation abilities, they play a vital role in maintaining the malignant proliferation of tumors, cell invasion and metastasis, tumor resistance and recurrence. So far, the function of tumor stem cells has been well explained in tumors such as leukemia, breast cancer, colorectal cancer, and prostate cancer.

[0007] In recent years, a large number of experiments have confirmed the existence of gastric cancer stem cells (GCSCs) and their key role in the occurrence, development, drug resistance and recurrence of gastric cancer. On the one hand, GCSCs have the characteristics of gastric cancer cells, such as unlimited proliferation, invasion and metastasis. At the same time, they also have self-renewal and multidirectional differentiation potentials similar to stem cells. Under appropriate conditions, they can differentiate into early progenitor cells and adult tumor cells, while adult tumor cells eventually die after limited proliferation and differentiation. Zhang et al. sorted CD44 + CD24 + The cells were injected into mice and the results showed that as low as 200 CD44 + CD24 + The cells can form tumors in mice, and this group of cells can also be isolated from tumor tissues, suggesting that GCSCs have the functions of self-renewal and multipotential differentiation. Ohkuma's team found that CD71 - Gastric cancer cells can form tumors in mice, and CD71 + The cell population could not form tumors in mice, and CD71 -The cell population has strong invasion and metastasis capabilities and resistance to chemotherapy drugs. Further studies have confirmed that gastric cancer stem cells have strong invasion and metastasis capabilities both in vivo and in vitro, thus accelerating the occurrence and development of gastric cancer. The main reason for gastric cancer recurrence is that gastric cancer patients develop resistance to chemotherapy drugs, which makes it impossible to prevent the continued malignant proliferation of tumor cells. Conventional chemotherapy drugs mainly act on tumor cells in the cell cycle process, while GCSCs are usually in a G0 resting state, and some GCSCs undergo gene mutations and DNA damage and repair, so they are less sensitive to conventional chemotherapy drugs, leading to clinical drug resistance and late recurrence in gastric cancer patients. Therefore, the existence of GCSCs is considered to be the potential driving force for the occurrence and development of gastric cancer and the main source of drug resistance and recurrence. Targeting the various biological properties of GCSCs, exploring their potential molecular mechanisms and developing new therapeutic drugs have become research hotspots in this field. However, the current research on GCSCs at home and abroad is not in-depth, and there are still many problems and challenges that need to be solved. In recent years, many studies at home and abroad have reported drugs that can target cancer stem cells, including some natural drug molecules and drugs that have been used clinically. For example, compound extracts composed of Huoxue Huayu Recipe, the effective components of Chongshengsuiyin, Xishu, Donglingcao and Baihuashediancao all have an inhibitory effect on the growth of tumor stem cells. In addition, metformin, a drug used to treat diabetes, and sorafenib, a drug used to treat liver cancer, have a good effect in inhibiting tumor growth, but their targeting function and safety cannot be proven, so the effect is very limited.

[0008] In summary, the current treatment of gastric cancer is mainly based on surgical resection and chemotherapy. Patients with advanced gastric cancer usually choose surgical treatment and adjuvant chemotherapy. This method not only increases the physical pain of patients, but also the mental and material pressure it brings cannot be underestimated. There is still a risk of recurrence and metastasis in the future. Therefore, the exploration and use of new targeted drugs can reduce the damage to normal tissues and cells to the greatest extent while reducing the growth and metastasis of tumor cells in patients, thereby greatly reducing the pain of patients during and after surgery, and even helping patients avoid the risk of surgery. However, conventional chemotherapy drugs mainly act on tumor cells in the cell cycle process, while gastric cancer stem cells are usually in the G0 resting state, and some gastric cancer stem cells undergo gene mutations and DNA damage and repair, so they are less sensitive to conventional chemotherapy drugs, resulting in clinical drug resistance and late recurrence in gastric cancer patients. Several drugs that can target tumor stem cells reported in recent years can inhibit tumor growth to a certain extent, but these drugs cannot play their function of targeting tumor stem cells under the premise of ensuring safety, so the effect is limited. Summary of the invention

[0009] In view of this, the present invention provides an application of clofoctol in the preparation of a drug for treating gastric cancer. The main purpose is to propose for the first time that clofoctol (CFT) can target and inhibit the cell viability of gastric cancer stem cells and induce their necroptosis, thereby being used as a new application for the preparation of anti-gastric cancer drugs.

[0010] In order to achieve the above object, the present invention mainly provides the following technical solutions:

[0011] On the one hand, the embodiments of the present invention provide the use of clofocol in the preparation of a drug for treating gastric cancer.

[0012] Preferably, the drug for treating gastric cancer is a targeted drug for treating gastric cancer.

[0013] On the other hand, an embodiment of the present invention proposes the use of clofocol in the preparation of a drug for targeted inhibition of gastric cancer stem cells.

[0014] Preferably, the drug for treating gastric cancer and the drug for targeted inhibition of gastric cancer stem cells can both target and inhibit the cell activity of gastric cancer stem cells and target and induce apoptosis of gastric cancer stem cells.

[0015] Preferably, the apoptosis of gastric cancer stem cells is necrotizing apoptosis of gastric cancer stem cells.

[0016] On the other hand, the present invention provides an application of clofocol and 5-fluorouracil (abbreviated as 5-Fu) in the preparation of a drug for treating gastric cancer. Preferably, the drug for treating gastric cancer is a targeted drug for treating gastric cancer.

[0017] Preferably, the mass concentration ratio of chloroform to 5-fluorouracil (5-Fu) is 2.5:1 to 5:1.

[0018] Preferably, the drug for treating gastric cancer is a chemotherapy drug.

[0019] On the other hand, an embodiment of the present invention provides a drug for treating gastric cancer, wherein the drug for treating gastric cancer includes clofocalone; preferably, the drug for treating gastric cancer is a targeted drug for treating gastric cancer; preferably, the drug for treating gastric cancer can target the inhibition of the cell activity of gastric cancer stem cells and target the promotion of apoptosis of gastric cancer stem cells; preferably, the drug for treating gastric cancer also includes 5-fluorouracil (abbreviated as 5-Fu). Preferably, the mass concentration ratio of clofocalone and 5-fluorouracil (5-Fu) is 2.5:1 to 5:1. Preferably, the drug for treating gastric cancer is administered intraperitoneally.

[0020] Compared with the prior art, the use of clofocol in the preparation of a drug for treating gastric cancer has at least the following beneficial effects:

[0021] On the one hand, the present invention proposes for the first time a new use of clofocalone in the preparation of a drug for treating gastric cancer, which exerts its tumor-suppressing effect by inhibiting the cell viability of gastric cancer stem cells in a targeted manner and inducing their necroptosis. At the same time, the present invention also proposes a new use of clofocalone in the preparation of a drug for inhibiting gastric cancer stem cells in a targeted manner. Here, clofocalone used as a drug can exert its function of targeting tumor stem cells while ensuring safety.

[0022] On the other hand, the present invention proposes for the first time the combined use of clofocol and the chemotherapy drug 5-Fu, which can not only reduce the concentration of the chemotherapy drug 5-Fu and reduce its side effects, but also the combined use of the two can greatly enhance the lethality of the chemotherapy drug to gastric cancer cells and improve the targeting function of the drug to tumor cells, thereby providing a more effective chemotherapy regimen for the treatment of gastric cancer, reducing the physical and mental burden of patients, and improving the quality of life of patients, with significant social and economic benefits.

[0023] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention and implement it according to the contents of the specification, the following is a detailed description of the preferred embodiments of the present invention in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The effect of chlorfenapyr on the cell activity of gastric cancer stem cells; Figure 1 Figure A is a graph showing the cell viability of gastric cancer stem cells inhibited by clofodine (CFT) at different concentrations (0.01uM, 0.1uM, 1uM, 10uM and 100uM); the types of gastric cancer stem cells include: two stem cell models induced by gastric cancer cell lines (MGC-803GCSCs and SGC-7901GCSCs) and an organoid model isolated from tumor tissue of a gastric cancer patient (GC1 ODs). Figure 1 Figure B shows the effect of clofoate phenol (CFT) on the cell viability of two common gastric cancer cell lines including MGC-803 and SGC-7901, a primary gastric cancer cell line GC1 isolated from gastric cancer patient tissue, and a gastric mucosal epithelial cell line (GES-1). Figure 1 Figure C shows the effects of three concentrations of clofocol on the cell activity of gastric cancer stem cells (MGC-803GCSCs, SGC-7901GCSCs, GC1 ODs) at different times. Figure 1 The control group in Figure C is the PBS-treated group.

[0025] Figure 2Figure A shows the cell apoptosis of three gastric cancer stem cell models, including two gastric cancer cell line-induced stem cell models (MGC-803GCSCs and SGC-7901GCSCs) and an organoid model isolated from tumor tissue of gastric cancer patients (GC1 ODs). Figure 2 Figure B shows the early and late apoptosis ratios of three stem cell models after cells were treated with clofolate (CFT). * indicates that there is a statistical difference between the treatment group and the control group. In addition, Figure 2 The CTL controls in panels A and B are blank controls. Figure 2 In Figure A, "comp-FIC-A" and "comp-pl-A" refer to the names of two fluorescent dyes.

[0026] Figure 3 The effect of clofocol on the tumor formation ability in mice. Figure 3 Figure A is a graph showing the changes in the body weight of mice in the treatment group and the control group. Figure B is a graph showing the trend of tumor volume changes over time in the treatment group and the control group (CTL). Figure C is a comparison and statistical graph of tumor size between the treatment group and the control group. * indicates that there is a statistical difference between the treatment group and the control group.

[0027] Figure 4 The mechanism of clofocol-induced apoptosis of gastric cancer stem cells is necroptosis. Figure 4 Figure A shows the changing trend of tumor necrosis factor (TNF) in gastric cancer stem cells after clofocaltol treatment. Figure B shows the effect of clofocaltol on the expression of key proteins in the process of necroptosis.

[0028] Figure 5 The effect of clofocol combined with 5-Fu on cell viability and apoptosis of gastric cancer cells. Figure 5 Figure A shows the effects of the control group (CTL), 5-Fu, and clofocol combined with 5-Fu on the cell viability of two gastric cancer stem cell models (MGC-803GCSCs and SGC-7901GCSCs) and a gastric mucosal epithelial cell line (GES-1). Figure 5 Figure B shows the apoptosis effects of control (CTL), 5-Fu, and clofoscarpine combined with 5-Fu on two gastric cancer stem cell models (MGC-803GCSCs and SGC-7901GCSCs). Figure 5 In Figure A, "comp-FIC-A" and "comp-pl-A" refer to the names of two fluorescent dyes.

[0029] Figure 6 This is a schematic diagram of the principle of using clofocol in combination with low-concentration chemotherapy drug 5-fluorouracil to treat gastric cancer. DETAILED DESCRIPTION

[0030] In order to further explain the technical means and effects adopted by the present invention to achieve the predetermined invention purpose, the specific implementation methods, structures, features and effects of the present invention application are described in detail below in conjunction with the accompanying drawings and preferred embodiments. In the following description, different "one embodiment" or "embodiment" does not necessarily refer to the same embodiment. In addition, specific features, structures, or characteristics in one or more embodiments may be combined in any suitable form.

[0031] Regarding the terms "gastric cancer stem cells" and "necroptosis" mentioned in the present invention, the explanations are as follows:

[0032] Gastric cancer stem cells: a group of potential malignant cells in gastric cancer that have unlimited proliferation and differentiation capabilities and can induce the occurrence, development, drug resistance and recurrence of gastric cancer; compared with ordinary gastric cancer cells, this type of cell is more malignant and, like "seeds", has a high tolerance to adverse environments and huge malignant potential. At present, there are two main ways to separate gastric cancer stem cells. One is to separate based on the surface markers of gastric cancer stem cells; but since the research on the surface markers of gastric cancer stem cells is still immature and inconvenient to operate, it is less used. The other is to construct a stem cell model with gastric cancer stem cell characteristics by inducing tumor cells to form spheres. This method is simple and easy to operate, and many studies have confirmed that this model can be used as a cell model of gastric cancer stem cells. The three gastric cancer stem cell models used in this patent are induced by tumor cell sphere culture, including gastric cancer stem cells induced by two gastric cancer cell lines MGC-803 and SGC-7901 (MGC-803GCSCs and SGC-7901GCSCs) and a gastric cancer cell derived from gastric cancer patient tissue, induced by 3D culture (GC1 ODs).

[0033] Necroptosis: It is the passive death of cells due to infection or damage. The process is inflammatory and involves a large number of inflammatory factors. Necroptosis has its own special signal transduction pathway, such as the TNF / RIPK1 / RIPK3 / MKL signal transduction pathway.

[0034] Clofoctol (CFT) is an antibacterial drug, mainly used to treat infections of the upper respiratory tract and the ear, nose and throat caused by Gram-positive bacteria. It is widely used in France and Italy, and was first approved for marketing in France. In addition, the prior art also suggests that clofoctol has an effect on glioma and lymphocyte cell lines. However, the prior art does not have any reports or research suggesting that clofoctol inhibits gastric cancer.

[0035] The inventors of the present invention have discovered for the first time that clofocol has a specific inhibitory effect on gastric cancer stem cells (MGC-803GCSCs and SGC-7901GCSCs) enriched in two human gastric cancer cell lines, gastric cancer organoids (GC1 ODs) from a gastric cancer patient, and a normal gastric mucosal epithelial cell line (GES-1) at the cellular level. The inventors of the present invention have found through various cell biological methods that clofocol can specifically induce apoptosis of gastric cancer stem cells without affecting normal gastric mucosal epithelial cells, and have further verified it in animals. In addition, the apoptosis process induced by clofocol is necroptosis mediated by TNF. In addition, the inventors of the present invention have found through research that the combined use of clofocol and low-concentration chemotherapy drug 5-Fu can enhance the killing effect of chemotherapy drugs on gastric cancer cells. This shows that clofocol is likely to become one of the more potential drugs for treating gastric cancer in the future.

[0036] Based on the above research of the inventor, the present invention proposes a new use of clofocaltol for preparing a drug for treating gastric cancer, and clarifies that the drug exerts its tumor-suppressing effect by inhibiting the cell viability of gastric cancer stem cells and inducing their necroptosis. Furthermore, the present invention also proposes that clofocaltol is used in combination with the chemotherapy drug 5-Fu (fluorouracil 5-Fu), which can not only reduce the concentration of the chemotherapy drug and reduce its side effects, but also the combined use of the two can greatly enhance the lethality of the chemotherapy drug to gastric cancer cells and improve the drug's targeting function on tumor cells, thereby providing a more effective chemotherapy regimen for the treatment of gastric cancer, reducing the patient's physical and mental burden, and improving the patient's quality of life, with significant social and economic benefits.

[0037] The present invention is further described in detail below through specific experimental examples:

[0038] Example 1

[0039] This example mainly verifies the effect of clofocol on the cell activity of gastric cancer stem cells, and specifically conducts a cell proliferation (CCK8) experiment, wherein the experimental process is as follows:

[0040] Cells in logarithmic growth phase were collected and 5×10 3 Add cells / well to a flat-bottom 96-well plate for culture, set drug concentration gradient and time gradient; take out the 96-well plate to be measured, add 10ul CCK8 to each well, and culture in an incubator away from light for 1-3h; take out the cell culture plate, and measure the absorbance of each well at 490nm on an ELISA reader, which can indirectly reflect the viability of the cells.

[0041] The results of the cell proliferation (CCK8) experiment showed that different concentrations (0.01uM, 0.1uM, 1uM, 10uM, 100uM) of clofofurol (CFT) could significantly inhibit the cell viability of gastric cancer stem cells (MGC-803GCSCs, SGC-7901GCSCs, GC1 ODs), and the most significant effect was observed at a concentration of 10uM (see Figure 1 In addition, CFT had no significant effect on the cell viability of common gastric cancer cell lines, nor did it affect the cell viability of normal gastric epithelial cell line (GES-1) (see Figure 1 (as shown in Figure B).

[0042] To further verify the above experimental results, the inventors used 3 concentrations (1uM, 10uM and 100uM) of clofofurol (CFT) to treat gastric cancer stem cells (MGC-803GCSCs, SGC-7901GCSCs, GC1 ODs), and then observed the cell viability of gastric cancer stem cells at different time points (3h, 6h, 9h, 12h, 24h and 48h). Figure 1 As shown in C, with the increase of clofoate phenol (CFT) concentration, the cell viability of gastric cancer stem cells gradually decreased, and the performance was most significant at 48h.

[0043] This indicates that CFT can target and affect the cell viability of gastric cancer stem cells, rather than normal gastric mucosal epithelial cells and common gastric cancer cell lines.

[0044] In summary, this example illustrates the targeting effect of clofocaltol on gastric cancer stem cells from the perspective of cell proliferation; the cell viability experiment is mainly used to prove the effect of different concentrations of clofocaltol on the cell viability of gastric cancer stem cells, and the effect of clofocaltol on normal gastric mucosal epithelial cells GES-1 and common gastric cancer cell lines is observed to prove its targeting effect.

[0045] Example 2

[0046] This embodiment mainly verifies the effect of chlorformin on apoptosis of gastric cancer stem cells. The main experimental process is as follows: Annexin V:FITC Apoptosis Detection Kit (BD Biosciences, USA) kit is used to perform flow cytometry detection of cell apoptosis by BD FACSCanto II Flow cytometry System (BD Biosciences, USA). Annexin V is one of the sensitive indicators for detecting cell apoptosis, and the change of the plasma membrane is one of the early changes when cells undergo apoptosis. When cells undergo apoptosis, the membrane phosphatidylserine (PS) turns from the inside of the plasma membrane to the outside. Annexin V has a high affinity for phosphatidylserine, and thus binds to the phosphatidylserine exposed on the outside of the cell. PI is a nucleic acid dye that cannot penetrate the intact cell membrane, but in the middle and late stages of apoptosis and necrotic cells, it can enter the cell and bind to DNA. Therefore, Annexin V is often used in combination with nucleic acid dyes for identifying cell life and death to distinguish early apoptotic cells, late apoptotic cells and dead cells. The specific process is as follows: trypsinize cells, prepare single cell suspensions, and count cells. Pipette 1×106 cells into a 5ml flow cytometry tube; Pipette 100μl of cell suspension into a new flow cytometry tube, add 5μl FITC-Annexin V and 5μl PI respectively. Mix gently and react at room temperature in the dark for 15min; add 400μl 1×BindingBuffer to each tube and detect on the machine within 1h. Calculate the proportion of early apoptosis and mid-late apoptosis respectively, and compare and analyze the differences among the groups.

[0047] Experimental results: CFT can significantly increase the apoptosis rate of gastric cancer stem cells (see Figure 2 Figure A and Figure B in Figure 1). Among them, Figure 2 Figure A shows the apoptosis of three gastric cancer stem cell models, including two gastric cancer cell line-induced stem cell models (MGC-803GCSCs and SGC-7901GCSCs) and an organoid model isolated from tumor tissue of a gastric cancer patient (GC1 ODs). Figure 2 As shown in the results of Figure B, after the cells were treated with CFT, the early and late apoptosis rates of the three stem cell models were as high as 25%, which was significantly higher than that of the control group. Therefore, it can be seen that CFT can significantly induce apoptosis of gastric cancer stem cells.

[0048] In summary, this example illustrates the targeting effect of clofocaltol on gastric cancer stem cells from the perspective of cell apoptosis; the apoptosis of three types of gastric cancer stem cells was detected mainly by flow cytometry, proving the targeted pro-apoptotic effect of clofocaltol on gastric cancer stem cells.

[0049] Example 3

[0050] This example mainly verifies the effect of clofocol on the tumor formation ability in mice.

[0051] This example illustrates the effect of clofocaltol on gastric cancer stem cells from the perspective of tumor formation in mice. Specifically, gastric cancer stem cells (SGC-7901GCSCs) were implanted subcutaneously in mice, and after tumors grew in mice, the control group (CTL) was intraperitoneally injected with PBS, and the treatment group (CFT) was injected with clofocaltol every day, and then the body weight and tumor volume of the mice were measured every other day, and statistical analysis was performed.

[0052] like Figure 3 As shown in Figures A and B, there was no significant difference in the body weight of the two groups of mice, but over time, the tumor volume of the mice in the clofocol-treated group was significantly reduced.

[0053] Two weeks later, the tumors of the mice were removed and the sizes of the tumors in the two groups were counted. Figure 3 As shown in Figure C, it can be seen that chlorfenapyr can significantly reduce the tumor tissue induced by gastric cancer stem cells, further indicating that chlorfenapyr has a significant growth inhibitory effect on gastric cancer stem cells.

[0054] In summary, this example illustrates the targeting effect of clofocaltol on gastric cancer stem cells from the perspective of tumor formation in mice; it mainly constructs a subcutaneous tumor model of gastric cancer stem cells in mice, and then injects clofocaltol intraperitoneally to observe its effect on tumor formation and apoptosis of tumor cells.

[0055] Example 4

[0056] This example mainly verifies that clofocol induces necroptosis of gastric cancer stem cells.

[0057] This example reveals that the form of gastric cancer stem cell death induced by clofocol is a special form of necroptosis, which is manifested by a significant increase in tumor necrosis factor (TNF) and the activation of the RIPK1 / RIPK3 / MLKL signaling pathway. The specific process is as follows: Collect cells from the treatment group and the control group, and then use the QIAGEN kit to extract RNA from the cells, the steps are as follows:

[0058] (1) Before the experiment, add the corresponding volume of anhydrous ethanol to Buffer RWT and RPE according to the instructions of the kit.

[0059] (2) Collect the cell precipitate to be extracted, wash it three times with 1× PBS, scrape it with a cell scraper, centrifuge it, discard the supernatant, add an appropriate amount of TRizol lysis buffer, or take out the cell lysis sample frozen at -80℃ and thaw it on ice.

[0060] (3) Add chloroform equivalent to one fifth of the volume of TRizol lysis buffer (100 μl corresponds to 500 μl) and vortex to mix for 15 seconds.

[0061] (4) Incubate at room temperature for 2-3 min and centrifuge at 12,000 g, 4°C for 15 min.

[0062] (5) Carefully aspirate the upper clear supernatant and place it in a new 1.5 ml EP tube. Then add 1.5 times the volume of anhydrous ethanol and vortex to mix.

[0063] (6) Each time, aspirate 700 μl of sample and add it to the RNeasy Mini column. Place it in a 2 ml collection tube and cap it tightly. Centrifuge at 8000 g for 15 s at room temperature. Discard the filtrate. If there is any remaining sample, repeat the centrifugation once.

[0064] (7) Add 700 μl of RWT buffer to the column, cap tightly, centrifuge at 8000 g for 15 s, and discard the filtrate.

[0065] (8) Add 500 μl of RPE buffer to the column, cap tightly, centrifuge at 8000 g for 15 s, and discard the filtrate.

[0066] (9) Add 500 μl of RPE buffer to the column, cap tightly, centrifuge at 8000 g for 2 min, and discard the filtrate.

[0067] (10) Place the filter column in a new 2 ml collection tube and centrifuge the empty column at 8000 g for 1 min to dry the column.

[0068] (11) Place the filter column in a new 1.5 ml EP tube, add 50 μl RNase-free water, close the lid, centrifuge at 8000 g for 1 min at room temperature, and collect the filtrate.

[0069] (12) The OD260 / OD280 of the RNA sample was measured (1.8-2.0) using a Nanodrop Spectrophotometer (NanoDrop 2000c, Thermo Fisher Scientific, USA) and stored at -80°C or in liquid nitrogen.

[0070] Then, the fluorescence quantitative PCR reaction was carried out, and the operation process was as follows:

[0071] (1) Reverse transcription PCR (PrimeScript TM RT Master Mix, Takara, Japan), prepare the reverse transcription reaction system on ice according to the kit instructions, see Table 1 for details, as follows:

[0072] Table 1

[0073] 5×PrimeScript RT Master Mix 2μl Total RNA 500ng <![CDATA[RNase Free ddH2O]]> up to 10μl

[0074] The reverse transcription reaction was carried out in a common PCR instrument under the following conditions: 37°C for 15 min; 85°C for 5 s for reverse transcriptase inactivation; and then stored at 4°C.

[0075] (2) Prepare the reaction system shown in Table 2 according to the kit instructions:

[0076] Table 2

[0077]

[0078]

[0079] (3) PCR reaction conditions: pre-denaturation at 95°C / 30 s; PCR reaction at 95°C / 5 s, 60°C / 30 s, for 40 cycles; melting curve at 95°C / 15 s, 60°C / 60 s, and 95°C / 15 s.

[0080] (4) Analysis method: After the reaction system was shaken and mixed using an oscillator, the liquid on the tube wall was removed by centrifugation at 3000 rpm for 5 min, and the amplification reaction was performed using the 7900HT Real-Time PCR System (Applied Biosystems, USA). According to the instrument operation instructions, 5 replicate wells were set for each group, and all experiments were repeated 3 times to ensure the reliability of the results. The housekeeping gene Actin was used as the internal control of PCR. Finally, the formula 2 was used. -ΔΔCt Comparison of gene expression differences among different groups was performed.

[0081] like Figure 4 As shown, after the gastric cancer stem cell model induced by gastric cancer cell lines MGC-803 and SGC-7901 was treated with clofocalone, the expression of intracellular TNF was significantly upregulated, and the expression of phosphorylated RIPK1, phosphorylated RIPK3 and phosphorylated MLKL in the RIPK1 / RIPK3 / MLKL signaling pathway was significantly upregulated; therefore, clofocalone can induce necroptosis of gastric cancer stem cells.

[0082] In summary, this example illustrates from the mechanism that the apoptosis of gastric cancer stem cells induced by chlorfenapyr is not a general form of apoptosis, but an inflammatory apoptosis process, namely, necroptosis; the expression levels of key proteins and genes in the necroptosis of gastric cancer stem cells induced by chlorfenapyr were verified by protein immunoblotting and fluorescence quantitative PCR methods, proving that the apoptosis of gastric cancer stem cells induced by chlorfenapyr is necroptosis.

[0083] Example 5

[0084] This example mainly verifies the effect of clofocol combined with 5-Fu on cell viability and apoptosis of gastric cancer cells.

[0085] To further explore the clinical application value of clofocaltol, we will combine low-concentration chemotherapy drugs 5-Fu and clofocaltol to act on gastric cancer cells, and observe the effect of their combined use on the growth of gastric cancer cells. The specific process is as follows: First, gastric cancer stem cells and normal gastric mucosal epithelial cell lines are cultured in a culture plate, and then 5-Fu and clofocaltol are added to treat the cells respectively, and then CCK8 experiments are performed. The specific operation is shown in Example 1; the cell apoptosis experiment is shown in the example.

[0086] like Figure 5 As shown in Figure A, 5-Fu alone has a slight inhibitory effect on the proliferation of two types of gastric cancer cells, while the combination of the two drugs significantly inhibits the growth of gastric cancer cells, but has basically no significant effect on the cell viability of normal gastric mucosal epithelial cells.

[0087] At the same time, this example also tested the effect of the combination of the two drugs on gastric cancer cell apoptosis. The results showed that the combination of clofocol and 5-Fu can significantly increase the apoptosis rate of cells (see Figure 5 Figure B in the figure).

[0088] It can be seen that chloroform can significantly enhance the killing ability of the chemotherapy drug 5-Fu on gastric cancer cells. The combination of the two drugs is expected to become a new idea for the clinical treatment of gastric cancer.

[0089] Figure 6 This is a schematic diagram of the principle of using clofocol in combination with low-concentration chemotherapy drug 5-fluorouracil to treat gastric cancer. Figure 6 It can be seen that clofocol can induce apoptosis of gastric cancer stem cells through the tumor necrosis factor-mediated necroptosis mechanism, but has no effect on normal cells; in addition, clofocol combined with low-concentration chemotherapy drug 5-fluorouracil can significantly enhance the killing effect of chemotherapy drugs on gastric cancer cells.

[0090] In summary, the chemotherapy drug 5-Fu has a strong killing effect on the cell viability of gastric cancer cells, but high concentrations of 5-Fu usually bring very painful clinical side effects to patients. Therefore, this example uses a low concentration of 5-Fu combined with clofocol to detect the effect of the combined drug on the cell viability and apoptosis of gastric cancer cells by cell viability and flow cytometry. The advantages of using the combination of two drugs in this embodiment are: on the one hand, chemotherapy drugs have a strong killing ability on gastric cancer cells, but at the same time they will also cause irreversible damage to normal gastric mucosal cells; therefore, the low concentration of chemotherapy drug 5-Fu used in this embodiment not only partially retains the killing effect of chemotherapy drugs on gastric cancer cells, but more importantly, greatly reduces its damage to normal gastric mucosal cells; on the other hand, the drug focused on in this embodiment, chlorfluanidine, has no effect on the cell viability of ordinary gastric cancer cells and normal gastric mucosal cells, but exerts its tumor-suppressing effect by targeting the necroptosis of gastric cancer stem cells, and gastric cancer stem cells are a group of cells with potential carcinogenicity and recurrence in gastric cancer, and conventional chemotherapy drugs have no killing effect on this group of cells; chlorfluanidine well compensates for the killing effect of chemotherapy drugs on gastric cancer stem cells, so that the combination of the two drugs can play the greatest role in gastric cancer and can also minimize the damage to normal gastric mucosal cells.

[0091] Here, the present invention verifies through the above embodiments that: chlorfenapyr can target the effect of inhibiting the cell activity of gastric cancer stem cells, chlorfenapyr can target the induction of apoptosis of gastric cancer stem cells, chlorfenapyr can inhibit the ability of tumor formation in mice, chlorfenapyr can induce necroptosis of gastric cancer stem cells, and chlorfenapyr combined with 5-Fu can enhance the proliferation inhibition and apoptosis-promoting effect of chemotherapy drugs on gastric cancer cells. It can be seen that chlorfenapyr can play its role in treating gastric cancer by inducing necroptosis of gastric cancer stem cells and inhibiting the growth of tumors under the skin of mice. Chlorfenapyr combined with 5-fluorouracil (5-Fu), a commonly used chemotherapy drug in clinical practice, can significantly increase the killing ability of chemotherapy drugs on gastric cancer cells. Although the gastric cancer cell model used in the above embodiments includes MGC-803 and SGC-7901 gastric cancer cell lines, and primary gastric cancer cells separated from a gastric cancer patient, it is a gastric cancer stem cell model induced by the above three cells, but it is not limited to this.

[0092] In summary, the present invention proposes for the first time a new use of clofocalone for preparing a drug for treating gastric cancer, and the drug exerts its tumor-suppressing effect by inhibiting the cell viability of gastric cancer stem cells in a targeted manner and inducing their necroptosis. In addition, the present invention proposes for the first time the use of clofocalone in combination with the chemotherapy drug 5-Fu in preparing a drug for treating gastric cancer, which can not only reduce the concentration of the chemotherapy drug 5-Fu and reduce its side effects, but also the combined use of the two can greatly enhance the lethality of the chemotherapy drug to gastric cancer cells and improve the drug's targeting function on tumor cells, thereby providing a more effective chemotherapy regimen for the treatment of gastric cancer, reducing the physical and mental burden of patients, and improving the quality of life of patients, with significant social and economic benefits.

[0093] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Any simple modification, equivalent change and modification made to the above embodiment according to the technical essence of the present invention still falls within the scope of the technical solution of the present invention.

Claims

1. The application of clofocol in the preparation of medicines for treating gastric cancer.

2. Application of clofocol in the preparation of drugs for targeted inhibition of gastric cancer stem cells.

3. The use according to claim 1 or 2, characterized in that: The drug for treating gastric cancer and the drug for targeted inhibition of gastric cancer stem cells can both targetedly inhibit the cell activity of gastric cancer stem cells and targetedly induce apoptosis of gastric cancer stem cells.

4. The use according to claim 3, characterized in that: The gastric cancer stem cell apoptosis is necrotizing apoptosis of gastric cancer stem cells.

5. Application of clofocol and 5-fluorouracil in the preparation of medicines for treating gastric cancer.

6. The use according to claim 5, characterized in that: The mass concentration ratio of chloroform to 5-fluorouracil is 2.5:1 to 5:

1.

7. A drug for treating gastric cancer, characterized in that: The medicine for treating gastric cancer includes clofocalone.

8. The drug for treating gastric cancer according to claim 7, characterized in that: The drug for treating gastric cancer can target and inhibit the cell activity of gastric cancer stem cells and target and induce apoptosis of gastric cancer stem cells.

9. The drug for treating gastric cancer according to claim 7 or 8, characterized in that: The drug for treating gastric cancer also includes 5-fluorouracil; preferably, the mass concentration ratio of clofocol to 5-fluorouracil is 2.5:1 to 5:

1.

10. The drug for treating gastric cancer according to any one of claims 7 to 9, characterized in that: The drug for treating gastric cancer is administered intraperitoneally.

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