Application of TROP2 inhibitor in preparation of ferroptosis sensitizer and / or inducer and pharmaceutical composition for enhancing ferroptosis

Abstract

The invention provides an application of a TROP2 inhibitor in preparation of a ferroptosis sensitizer and / or inducer, and a pharmaceutical composition for enhancing ferroptosis. The pharmaceutical composition comprises a ferroptosis inducer and a TROP2 inhibitor. According to the application, the effect of TROP2 in regulation and control of lung cancer cell ferroptosis is found for the first time through experiments, based on the discovery, a TROP2 inhibitor and a ferroptosis inducer (such as RSL3) are jointly applied, and the result shows that the sensitivity of lung cancer cells to RSL3-induced ferroptosis can be enhanced by inhibiting TROP2, so that the effect of jointly promoting cell ferroptosis and further inhibiting tumor development is achieved; and a new effective scheme is expected to be provided for tumor treatment.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine. More specifically, the present invention relates to the use of a TROP2 inhibitor in the preparation of a ferroptosis sensitizer and / or inducer, and a pharmaceutical composition for enhancing ferroptosis. Background Art

[0002] Lung cancer is one of the malignant tumors with the highest incidence and mortality rates globally, and its pathogenesis is complex and involves multiple genetic and environmental factors. Due to the lack of obvious early symptoms of lung cancer, most patients are diagnosed at an advanced stage, and the treatment options mainly include surgery, chemotherapy, radiotherapy, targeted therapy, and immunotherapy, etc. Although molecular targeted drugs and immunotherapy have significantly improved the prognosis of some patients in recent years, the overall five-year survival rate is still low, and there is an urgent need to further study new diagnostic techniques and treatment strategies to improve the early detection rate and treatment effect of lung cancer.

[0003] Ferroptosis is a form of programmed cell death discovered in recent years that is mediated by iron ions and characterized by the accumulation of lipid peroxidation. It was first proposed by Dixon et al. in 2012. The proposal of this concept broadens the cognitive boundary of the mechanism of cell death, which is significantly different from traditional forms of apoptosis, necrosis, and autophagy. Its occurrence is closely related to the inactivation of glutathione peroxidase 4 (GPX4) and the excessive accumulation of intracellular reactive oxygen species (ROS). However, some studies have reported that the insufficient level of intracellular ROS and the unsatisfactory accumulation of drugs in lung cancer lesions hinder the efficacy of ferroptosis therapy. Therefore, although the potential of ferroptosis in the treatment of some malignant tumors has gradually emerged, its specific molecular mechanism has not been fully elucidated, especially the mechanism of action in the tumor microenvironment and heterogeneous tumors remains to be further studied.

[0004] TROP2 (Trophoblast cell-surface antigen 2) is a transmembrane glycoprotein belonging to the EpCAM family. It was initially discovered in placental trophoblast cells and is widely expressed in a variety of normal epithelial tissues and tumor tissues. After reviewing domestic and foreign literature and data, there is no relevant report on the application of TROP2 in ferroptosis. Summary of the Invention

[0005] The purpose of the present invention is to provide the use of a TROP2 inhibitor in the preparation of a ferroptosis sensitizer and / or inducer, and a pharmaceutical composition for enhancing ferroptosis, so as to improve the sensitivity of tumors (such as lung cancer) to ferroptosis therapy and enhance the anti-tumor effect.

[0006] Based on the above, the first object of the present invention is to provide the use of a TROP2 inhibitor in the preparation of a ferroptosis sensitizer and / or inducer.

[0007] Preferably, the TROP2 inhibitor includes any one or a combination of multiple ones of: siRNA of the encoding gene of TROP2 or a gene knockout vector, or a protein antibody of TROP2, or other inhibitors capable of inhibiting the expression of TROP2.

[0008] Preferably, the TROP2 inhibitor is siRNA, and the nucleotide sequence of the siRNA is as shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0009] Preferably, when the TROP2 inhibitor is prepared as a ferroptosis sensitizer, it is used in combination with a ferroptosis inducer to enhance the ferroptosis effect, and the ferroptosis inducer includes at least one of RSL3, Erastin or ML162.

[0010] The second object of the present invention is to provide a pharmaceutical composition for enhancing ferroptosis, and the pharmaceutical composition includes: a ferroptosis inducer and a TROP2 inhibitor.

[0011] Preferably, the TROP2 inhibitor is siRNA, and the nucleotide sequence of the siRNA is as shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0012] Preferably, the ferroptosis inducer includes at least one of RSL3, Erastin or ML162.

[0013] Preferably, the pharmaceutical composition further includes a pharmaceutically acceptable carrier.

[0014] The third object of the present invention is to provide the use of the foregoing pharmaceutical composition in the preparation of an anti-tumor drug.

[0015] Preferably, the tumor includes lung cancer.

[0016] Compared with the prior art, the beneficial effects of the present invention at least include:

[0017] The present invention for the first time discovers the role of TROP2 in regulating ferroptosis of lung cancer cells through experiments, and based on this discovery, combines the TROP2 inhibitor and a ferroptosis inducer (such as RSL3). The results show that inhibiting TROP2 can enhance the sensitivity of lung cancer cells to ferroptosis induced by RSL3, thereby achieving the effect of jointly promoting cell ferroptosis and then inhibiting tumor development, and is expected to provide a new effective solution for tumor treatment. Description of the Drawings

[0018] Figure 1 It shows that interfering with TROP2 inhibits the growth of lung cancer cells; wherein:

[0019] A shows the detection results of TROP2 expression levels in lung cancer cells after transfection with TROP2-si#1 and TROP2-si#2;

[0020] B shows the detection results of cell viability in lung cancer cells after transfection with TROP2-si#1 and TROP2-si#2;

[0021] C shows the cell clone proliferation of lung cancer cells after transfection with TROP2-si#1 and TROP2-si#2.

[0022] Figure 2 It shows the exploration results of the mechanism of interfering with TROP2 to inhibit the growth of lung cancer cells; among them:

[0023] A shows the results of transcriptome sequencing analysis of lung cancer cells with / without TROP2 interference;

[0024] B shows the detection results of MDA levels in lung cancer cells after transfection with TROP2-si#1 and TROP2-si#2;

[0025] C shows the detection results of GSH levels in lung cancer cells after transfection with TROP2-si#1 and TROP2-si#2;

[0026] D shows the result diagram of observing the cell morphology of each group by transmission electron microscopy.

[0027] Figure 3 It shows the result of inhibiting TROP2 expression to enhance RSL3-induced ferroptosis in lung cancer cells; among them:

[0028] A shows the detection results of cell viability of each group of cells;

[0029] B shows the detection results of MDA levels of each group of cells;

[0030] C shows the detection results of GSH levels of each group of cells.

[0031] Figure 4 It shows that RSL3 and TROP2 interference significantly inhibit the growth of PC9 tumors in nude mice; among them:

[0032] A shows the photos of the final tumor tissues of each group of mice;

[0033] B shows the weights of the final tumor tissues of each group of mice;

[0034] C shows the growth curves of the tumor volumes of each group of mice. Detailed implementation mode

[0035] The technical solution of the present invention will be further described below in conjunction with the drawings and embodiments.

[0036] Term Explanation

[0037] MDA (malondialdehyde) is one of the products of lipid peroxidation. An increase in its level usually indicates that lipid peroxidation has occurred within cells, which is an important hallmark of ferroptosis. During ferroptosis, iron ions within cells react with hydrogen peroxide to generate hydroxyl radicals (·OH), which in turn trigger lipid peroxidation, leading to the accumulation of oxidation products such as MDA. Therefore, by detecting the level of MDA, the degree of ferroptosis can be indirectly evaluated.

[0038] GSH (glutathione) is an important antioxidant that can neutralize free radicals within cells and protect cells from oxidative stress damage. During ferroptosis, the level of GSH changes, usually manifested as the consumption of GSH or the inhibition of its synthesis. By detecting the level of GSH, the changes in the antioxidant capacity within cells can be understood, thereby evaluating the degree of ferroptosis. In addition, the change in GSH synthesis is also one of the important hallmarks of ferroptosis.

[0039] As described in the background art, although the potential of ferroptosis in the treatment of malignant tumors is gradually emerging, due to significant differences in the tumor microenvironment, tumor heterogeneity, etc. among different types of tumors, the ferroptosis efficacy of different types of tumors varies greatly, and the ferroptosis efficacy of some malignant tumors (such as lung cancer) is not satisfactory.

[0040] To solve the above technical problems, the present invention has conducted a large number of experiments and studies. Unexpectedly, it was found that when using siRNA to interfere with the expression of TROP2 in lung cancer cells, it would lead to a slowdown in the growth of lung cancer cells and an increase in the ROS level. After sequencing, it was found that it had a high correlation with ferroptosis. Further in vitro and in vivo experiments confirmed that TROP2 interference could increase the sensitivity of lung cancer cells to ferroptosis treatment, promote ferroptosis of lung cancer cells, and ultimately significantly improve the anti-tumor effect.

[0041] Based on the above, the present invention first provides the use of a TROP2 inhibitor in the preparation of a ferroptosis sensitizer and / or a ferroptosis inducer. When the TROP2 inhibitor is prepared as a ferroptosis sensitizer, it is used in combination with a ferroptosis inducer to enhance the ferroptosis effect.

[0042] In some embodiments, the TROP2 inhibitor includes: siRNA of the coding gene of TROP2 or a gene knockout vector, or a protein antibody of TROP2, or any one or a combination of multiple other inhibitors that can inhibit the expression of TROP2. As an example, the TROP2 inhibitor is siRNA, and the nucleotide sequence of the siRNA is as shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0043] In some embodiments, the ferroptosis inducer includes at least one of RSL3, Erastin, or ML162.

[0044] On the other hand, the present invention also provides a pharmaceutical composition for enhancing ferroptosis, which comprises: a ferroptosis inducer and a TROP2 inhibitor.

[0045] In some embodiments, the TROP2 inhibitor is siRNA, and the nucleotide sequence of the siRNA is as shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0046] In some embodiments, the ferroptosis inducer includes at least one of RSL3, Erastin, or ML162. Among them, RSL3 (RAS Selective Lethal 3) is a commonly used ferroptosis inducer. By directly inhibiting the activity of GPX4, it triggers the accumulation of lipid peroxidation and ultimately leads to ferroptosis. When RSL3 inhibits GPX4, the intracellular antioxidant defense is weakened, the level of lipid peroxides increases, resulting in damage to the cell membrane integrity and inducing ferroptosis.

[0047] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. "Pharmaceutically acceptable carrier" refers to a carrier for administering therapeutic agents, including various excipients and diluents. This term refers to such pharmaceutical carriers: they are not necessarily active ingredients themselves and have no excessive toxicity after administration. Suitable pharmaceutically acceptable carriers are well-known to those of ordinary skill in the art. A full description of pharmaceutically acceptable carriers can be found in Remington’s Pharmaceutical Sciences. Pharmaceutically acceptable carriers in the composition may contain liquids, such as water, phosphate buffer solution, ringer solution, physiological saline, balanced salt solution, glycerol, or sorbitol, etc. Additionally, there may also be auxiliary substances in these carriers, such as lubricants, glidants, wetting agents or emulsifiers, pH buffering substances, and stabilizers, such as albumin.

[0048] On the other hand, the present invention also provides the application of the aforementioned pharmaceutical composition in the preparation of anti-tumor drugs. In some embodiments, the tumor includes lung cancer.

[0049] The following will be combined with the attached Figures 1 to 4 And specific embodiments to describe in detail the experimental exploration process of the present invention.

[0050] (1) Exploration of the mechanism of interfering with TROP2 to inhibit the growth of lung cancer cells

[0051] 1. Interfering with TROP2 expression can inhibit the growth of lung cancer cells

[0052] In this invention, lung cancer cells PC9 and H292 purchased from ATCC were co-cultured with siRNA transfected to interfere with TROP2 respectively to observe the effect of interfering with TROP2 on the growth of lung cancer cells. Meanwhile, si-CON was included as an experimental control.

[0053] Two siRNAs interfering with TROP2 were designed, denoted as TROP2-si#1 and TROP2-si#2 respectively. The specific sequences are as follows:

[0054] The nucleotide sequence of TROP2-si#1 is: 5’-CGCUCAUCUAUUACCUGGA-3’ (SEQ ID NO: 1);

[0055] The nucleotide sequence of TROP2-si#2 is: 5’-CGUGGACAACGAUGGCCUCUA-3’ (SEQ ID NO: 2).

[0056] The experimental operation process is as follows:

[0057] (1) 18 - 24 hours before lentiviral transfection, lung cancer cells were seeded into 96-well plates so that the cell confluence at the time of transfection was 30% - 50%.

[0058] (2) The siRNA (i.e., TROP2-si#1 or TROP2-si#2) was diluted in an EP tube containing 250 μL of Opti-MEM medium and gently mixed evenly. In another EP tube, Lipofectamine 2000 was also diluted with 250 μL of Opti-MEM medium. The concentration ratio of the diluted Lipofectamine 2000 to siRNA (pmol) was 1:1. After dilution, it was incubated at room temperature for 5 min; after incubation, the diluted siRNA was gently mixed with the diluted Lipofectamine 2000, and the resulting mixture was incubated at room temperature for 20 min.

[0059] (3) 500 μL of the incubated mixture was added to the wells containing cells and medium, gently shaken crosswise to mix evenly, and then cultured in a 37 °C, CO2 incubator for 24 h. Among them, the complete medium could be replaced 4 - 6 h after the start of transfection. When replacing, some cells could be collected, lysed with RIPA lysis buffer, and after BCA quantification, immunoblotting was used to detect the effect of siRNA interfering with TROP2. The results are as Figure 1 shown in A. Both TROP2-si#1 and TROP2-si#2 could significantly reduce the expression level of TROP2 in the two lung cancer cells, and the interference effect on H292 cells was particularly significant.

[0060] The remaining cells were plated in a 96-well plate at a density of 3000 cells per well. Six replicates were set for the control group and the two experimental groups respectively. At 1, 2, and 3 days, CCK8 solution was added to detect the absorbance at 450 nm to evaluate the effect of siRNA interfering with TROP2 on cell growth. The results are as Figure 1 shown in B. Compared with the control group (si-CON), the growth of cells in the experimental groups was significantly inhibited.

[0061] In addition, the transfected and cultured cells were plated in a six-well plate at a density of 1000 cells per well. The culture medium was changed regularly. After 10 - 14 days of culture, colony formation occurred. The well plate was gently washed with 1×PBS, fixed with 4% paraformaldehyde, stained with 0.1% crystal violet, and photographed. The results are as Figure 1 shown in C. Compared with the control group, after inhibiting the expression of TROP2, the proliferation of cells was significantly inhibited.

[0062] 2. Interfering with the expression of TROP2 can promote ferroptosis of lung cancer cells

[0063] Based on the above research, after further collecting cells from each group, extracting RNA and performing transcriptome sequencing, it was found that the slowdown of cell growth after TROP2 depletion was highly correlated with ferroptosis. Therefore, it was speculated that TROP2 interference was associated with cell ferroptosis, and relevant verification experiments were carried out on this speculation. It was proved by using a kit to detect the changes in the levels of GSH and MDA in PC9 and H292 cells after TROP2 interference, and using an electron microscope to observe the changes in cell structure. In this example, the lentivirus (i.e., shTROP2) was packaged with the sequence of TROP2-si#1 to achieve the long-term interference effect of TROP2 in PC9 and H292 cells. The experimental process and results are as follows:

[0064] (1) Collect the cells after control (i.e., shCON treatment) and TROP2 interference treatment (i.e., the experimental group, shTROP2 treatment). Resuspend 10 million cells in 100 μL of PBS. After ultrasonic treatment, centrifuge at 8000 g for 15 minutes, and take the supernatant for subsequent MDA detection. Treat the samples according to the kit instructions. Incubate the supernatant in a 95℃ metal bath for 1 hour and then cool on ice. Subsequently, centrifuge at 8000 g for 10 minutes. Take 200 μL of the supernatant and put it into a transparent 96-well plate. Use an enzyme-linked immunosorbent assay (ELISA) reader to detect the absorbance at 532 nm. When performing data analysis, measure the protein concentration of the samples and calculate the MDA concentration using the standard curve method.

[0065] (2) Collect the control and TROP2 - interfered cells. Resuspend every 1 million cells in 300 μL of PBS, and perform ultrasonic treatment at 4°C with a frequency of 25 KHz for 30 seconds, with a 20 - second interval, and a total duration of 30 minutes. Subsequently, centrifuge the cells at 8000 g for 10 minutes. Collect the supernatant, mix it with a protein - removing reagent, and centrifuge again. Obtain the supernatant for subsequent reduced glutathione detection. Mix the sample with the working solution of the detection probe and add it to a 96 - well plate. After incubating at room temperature for 5 minutes, use an enzyme - linked immunosorbent assay (ELISA) reader to detect the absorbance at 412 nm. Make a standard curve to calculate the concentration of GSH.

[0066] (3) Collect the control and TROP2 - interfered cells, fix them with an electron microscopy fixative for more than 24 hours, and then fix them with 1% osmium tetroxide prepared with 0.1 M phosphate - buffered saline PB (pH 7.4) in the dark at room temperature for 2 hours. After dehydration with a gradient of ethanol, infiltrate and embed them with resin blocks. Cut the resin blocks into 1.5 - μm semi - thin sections with a semi - thin microtome, stain them with toluidine blue, and localize them under an optical microscope. Cut the resin blocks into 60 ultra - thin sections of 80 nm with an ultra - thin microtome, stain them, and observe and take pictures under a transmission electron microscope.

[0067] The results are as Figure 2 shown. Analysis of the transcriptome sequencing results shows that the slowdown of cell growth after TROP2 depletion is significantly correlated with the ferroptosis pathway ( Figure 2 as shown in A). Compared with the control group, the MDA concentration in the experimental group cells is significantly increased ( Figure 2 as shown in B), the GSH concentration is significantly decreased ( Figure 2 as shown in C), and TEM shows that after TROP2 inhibition, the mitochondria in the cells become smaller, the membrane density increases, and the cristae decrease or even disappear ( Figure 2 as shown in D). These results indicate that inhibiting the expression of TROP2 can promote ferroptosis in lung cancer cells and exacerbate the degree of cell ferroptosis.

[0068] 3. Inhibiting TROP2 expression can enhance RSL3 - induced ferroptosis in lung cancer cells

[0069] Based on the above research, the present invention designs an experiment to jointly use a TROP2 inhibitor (in this example, lentivirus packaged with the TROP2 - si#1 sequence, i.e., shTROP2) and a ferroptosis inducer (RSL3 in this example) in order to make them play a synergistic role, enhance the ferroptosis of tumor cells, and thus improve the anti - tumor effect. The specific experimental process includes:

[0070] After using lentivirus to interfere with TROP2 in lung cancer cells (PC9 or H292) (MOI (Multiplicity of Infection) = 10), they were divided into three groups and the following related treatments were carried out: RSL3 group, adding RSL3 to make its final concentration 5 μM; RSL3+Fer group, adding RSL3 (final concentration 5 μM) and ferroptosis inhibitor Fer1 (final concentration 5 μM); DMSO group, adding the same volume of DMSO. It should be noted that lung cancer cells without TROP2 interference treatment were also included and subjected to the above three groups of treatments. 24 hours later, the cells of each group were seeded into 96-well plates, with 8000 cells per well and 6 replicates per well.

[0071] 24 hours after seeding, 10 μL of CCK8 reagent was added, and after incubating in the incubator for two hours, the absorbance at 450 μm was measured to evaluate the cell viability of each group of cells. The results are as Figure 3 shown in A of the figure. Interference with TROP2 increased the sensitivity of tumor cells to RSL3, and the use of Fer1 could partially restore cell viability.

[0072] In addition, the levels of GSH and MDA in each group of cells were also detected, and the detection method referred to the experimental content in the relevant part above. The results are as Figure 3 shown in B and C of the figure. Compared with the control group (shCON group), after treatment with RSL3 alone (shCON+RSL3 group) or only interference with TROP2 (shTROP2 group), the MDA level in the cells increased and the GSH level decreased, indicating that when the two were treated alone, they could cause ferroptosis of the cells, but the effect of the combined treatment (shTROP2+RSL3 group) was better, and it could exert a significant synergistic effect, significantly enhancing the degree of ferroptosis of lung cancer cells. The above results indicate that inhibiting TROP2 expression has an enhancing effect on RSL3-induced ferroptosis.

[0073] 4. RSL3 and TROP2 interference significantly inhibit the growth of PC9 tumors in nude mice

[0074] To further prove the regulatory effects of RSL3 and TROP2 on tumors, relevant evaluations were carried out through animal experiments. Specifically, it includes:

[0075] PC9 cells with 60% density were used in the experiment. They were infected with a control lentivirus (shCON) and an interfering lentivirus (shTROP2) with MOI (Multiplicity of Infection) = 10, and 5 μg / mL Polybrene was added to enhance the infection efficiency. After infection, the cells were amplified to a density of 80 - 90%, digested with trypsin, washed with pre-cooled PBS, and then resuspended in PBS to form a suspension of 6×106 cells / 100 μL. Twenty-four nude mice aged 6 - 8 weeks were randomly divided into two groups (n = 12 / group), and shCON or shTROP2-infected cells (100 μL / mouse) were subcutaneously injected into the back. When the tumors were visible to the naked eye, the nude mice treated with shCON injection were further divided into a control group (shCON) and an RSL3 treatment group (shCON + RSL3), and the nude mice treated with shTROP2 injection were divided into a TROP2 interference group (shTROP2) and a TROP2 interference + RSL3 group (n = 6 / group) for subsequent intervention. Among them, RSL3 was dissolved in DMSO, and the RSL3 treatment group and the TROP2 interference + RSL3 treatment group were intraperitoneally injected with an RSL3 solution at a concentration of 5 mg / kg every other day for 13 days. The body weight and tumor volume of the mice were measured regularly. After 13 days, the mice were sacrificed, and the tumors were dissected and photographed.

[0076] The results are as Figure 4 shown in A - C of the figure. Compared with the mice treated with RSL3 alone or TROP2 interference, the final tumor volume and weight of the mice in the TROP2 interference + RSL3 treatment group were significantly smaller than those in the other three groups, indicating that inhibiting TROP2 expression has an enhancing effect on RSL3-induced ferroptosis.

[0077] In summary, through experiments, the present invention first discovered the role of TROP2 in regulating ferroptosis of lung cancer cells. Based on this discovery, a combination of a TROP2 inhibitor and a ferroptosis inducer (such as RSL3) was applied. The results showed that inhibiting TROP2 can enhance the sensitivity of lung cancer cells to RSL3-induced ferroptosis, thereby achieving the effect of jointly promoting cellular ferroptosis and inhibiting tumor development, and is expected to provide a new effective solution for tumor treatment.

[0078] Although the content of the present invention has been described in detail through the above preferred embodiments, it should be recognized that the above description should not be considered as a limitation of the present invention. After those skilled in the art have read the above content, various modifications and substitutions of the present invention will be obvious. Therefore, the protection scope of the present invention should be defined by the appended claims.

Claims

1. Use of a TROP2 inhibitor in the preparation of a ferroptosis sensitizer and / or inducer.

2. The application according to claim 1, characterized in that, The TROP2 inhibitor includes: any one or a combination of more of siRNA or a gene knockout vector of the coding gene of TROP2, or a protein antibody of TROP2, or other inhibitors capable of inhibiting the expression of TROP2.

3. The application according to claim 2, characterized in that, The TROP2 inhibitor is siRNA, and the nucleotide sequence of the siRNA is as shown in SEQ ID NO: 1 or SEQ ID NO:

2.

4. The application according to claim 1, wherein When the TROP2 inhibitor is prepared as a ferroptosis sensitizer, it is used in combination with a ferroptosis inducer to enhance the ferroptosis effect, and the ferroptosis inducer includes at least one of RSL3, Erastin or ML162.

5. A pharmaceutical composition for enhancing ferroptosis, characterized in that, The pharmaceutical composition includes: a ferroptosis inducer and a TROP2 inhibitor.

6. The pharmaceutical composition according to claim 5, characterized in that, The TROP2 inhibitor is siRNA, and the nucleotide sequence of the siRNA is as shown in SEQ ID NO: 1 or SEQ ID NO:

2.

7. The pharmaceutical composition according to claim 5, characterized in that, The ferroptosis inducer includes at least one of RSL3, Erastin or ML162.

8. The pharmaceutical composition according to claim 5, wherein The pharmaceutical composition further includes a pharmaceutically acceptable carrier.

9. Use of the pharmaceutical composition according to any one of claims 5-8 in the preparation of an anti-tumor drug.

10. The application according to claim 9, characterized in that, The tumor includes lung cancer.

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