Experimental method of plumbagin in medicine for resisting non-small cell lung cancer

Abstract

The invention relates to the technical field of medicines, and discloses an experimental method of plumbagin in anti-non-small cell lung cancer drugs, the drugs comprise effective plumbagin and pharmaceutically acceptable carriers, and can induce expression of GPX4, so that the anti-non-small cell lung cancer drugs are obtained. The generation of ROS in non-small cell lung cancer subjects can be improved, the expression of STING and TBK1 phosphorylated proteins in the non-small cell lung cancer subjects can be increased, and meanwhile, the anti-tumor effect can be effectively improved when the compound is used together with a PD-1 inhibitor. A new drug treatment strategy is provided for clinical treatment of the non-small cell lung cancer, and theoretical and experimental foundations are laid for development of innovative drugs with proprietary intellectual property rights.

Description

Technical Field

[0001] The invention relates to the technical field of medicines, in particular to an experimental method for plumbagin in an anti-non-small cell lung cancer drug. Background Art

[0002] The high prevalence of non-small cell lung cancer (NSCLC) and its resistance to immunotherapy pose a major challenge to cancer treatment. However, increasing evidence suggests that new immunotherapy approaches, such as adoptive cell therapy, immune checkpoint inhibitors (ICIs), and cancer vaccines, hold promise in addressing this challenge. ICIs work by inhibiting or downregulating the programmed cell death 1 (PD-1) protein on the surface of CD8+ T cells and regulatory T cells (Tregs), thereby inducing an anti-tumor response. However, only a small fraction of NSCLC patients respond to immunotherapy during treatment, and even among responders, resistance may develop. It is important to investigate whether the presence of activated CD8+ T cells in tumors significantly promotes the treatment of NSCLC. In addition, the presence of activated dendritic cells (DCs) and the expression of CD8+ T cells within tumors and in the peritumoral stroma have also been shown to be associated with a positive prognostic impact in the treatment of NSCLC. Promoting immune cell infiltration and improving the tumor microenvironment are expected to enhance the efficacy of ICIs.

[0003] Ferroptosis is a form of cell death characterized by the accumulation of iron ions, reactive oxygen species (ROS), lipid peroxidation (LPO), glutathione depletion (GSH), and changes in mitochondrial structure. In recent years, it has gained attention as a potential target for cancer therapy in recent years. Studies have shown that CD8+ T lymphocytes can utilize the cytokine IFN-γ to induce ferroptosis in tumor cells in vivo. In addition, triggering ferroptosis in tumor cells has been found to improve the efficacy of immunotherapy for cancer. These results suggest that inducing ferroptosis in tumor cells can improve their therapeutic efficacy. There is increasing evidence for the interaction between ferroptosis and stimulation of interferon genes (STING) signaling in cancer therapy. Ferroptosis has been identified as promoting oxidative damage to intracellular DNA, leading to accumulation of double-stranded DNA (dsDNA), which is further engulfed or translocated to the cytoplasm by DCs and macrophages. This in turn activates the STING pathway, accompanied by elevated IFN-γ, TNF-α, and GzmB, which belongs to the type I interferon family and has anti-tumor effects. In addition, STING pathway activation can also promote the activation of dendritic cells infiltrating CD8+ T lymphocytes into the tumor microenvironment. Therefore, it remains to be explored whether ferroptosis can further activate the STING pathway of innate immune cells to enhance the efficacy of ICIs in NSCLC.

[0004] Ferroptosis and STING signaling may provide new insights into improving immunotherapy outcomes in the treatment of non-small cell lung cancer. Plumbagin (PLB) is a novel compound extracted from the root of Plumbaginaceae, which has long been recognized for its efficacy in traditional Chinese medicine for the treatment of various diseases, such as eczema, inflammatory diseases, abdominal pain, and amenorrhea. PLB has shown anti-tumor biological effects. However, the effect of PLB on ferroptosis and its specific mechanism are still unclear, and its potential synergistic effects in the treatment of non-small cell lung cancer remain unknown.

[0005] Therefore, we proposed an experimental method for plumbagin in anti-non-small cell lung cancer drugs to solve the problems raised above. Summary of the invention

[0006] The purpose of the present invention is to provide an experimental method for plumbagin in anti-non-small cell lung cancer drugs to solve the problem that the above background technology proposes that the effect of PLB on ferroptosis and its specific mechanism are still unclear in the process of treating non-small cell lung cancer, and its potential synergistic effect still remains unknown.

[0007] To achieve the above purpose, the present invention provides the following technical solutions: an experimental method for plumbagin in anti-non-small cell lung cancer drugs, including the use of 5-hydroxy-2-methylnaphthalene-1,4-dione in the preparation of anti-non-small cell lung cancer drugs.

[0008] Furthermore, the drug comprises an effective dose of 5-hydroxy-2-methylnaphthalene-1,4-dione and a pharmaceutically acceptable carrier.

[0009] Furthermore, the pharmaceutically acceptable carrier includes fillers, diluents, binders, disintegrants, emulsifiers and drug carriers without toxic side effects.

[0010] Furthermore, the drug is prepared into a pharmaceutically acceptable dosage form.

[0011] Furthermore, the dosage forms include tablets, drops, and capsules.

[0012] Furthermore, the drug is prepared in the form of a single-dose drug.

[0013] Furthermore, the single-dose drug includes 5-hydroxy-2-methylnaphthalene-1,4-dione.

[0014] Furthermore, the drug can downregulate the expression of ferroptosis-related proteins FTH1, Nrf2, Slc7a11 and GPX4 in subjects with non-small cell lung cancer.

[0015] Furthermore, the indicated drugs were able to upregulate ROS generation in subjects with non-small cell lung cancer.

[0016] Furthermore, the drug can increase the expression of H-H2AX protein.

[0017] Compared with the prior art, the present invention has the following beneficial effects: At present, the treatment drugs for non-small cell lung cancer are mainly Western medicine, targeted drugs and PD-1 immune checkpoint inhibitors, which are limited in treatment effect, response rate and toxic side effects. The present invention found that Rhe has the ability to effectively inhibit the proliferation of non-small cell lung cancer, which provides a theoretical basis for exploring low-toxic, high-efficiency and multi-target Chinese medicine monomers against non-small cell lung cancer, and has important clinical significance for the treatment of non-small cell lung cancer. This will provide new drug treatment targets and new treatment strategies for the clinical treatment of non-small cell lung cancer, and also lay a theoretical and experimental foundation for the development of innovative drugs with independent intellectual property rights. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is a schematic diagram of Embodiment 1 of the present invention; Figure 2 It is a schematic diagram of the second embodiment of the present invention; Figure 3 It is a schematic diagram of Embodiment 3 of the present invention; Figure 4 It is a schematic diagram of Embodiment 4 of the present invention; Figure 5 This is a schematic diagram of Embodiment 5 of the present invention. DETAILED DESCRIPTION

[0019] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0020] Example 1: Please refer to Figure 1 ; In this embodiment: the chemical structure of PLB is as follows: Figure 1 As shown in A. Figure 1 B shows the curves of the survival rate of all NSCLC cell lines after PLB treatment as a function of dose and time, as well as the IC50 of PLB in different cancer cells. In addition, dose-dependent inhibition of clonal growth was observed in H358 and A549 cell lines ( Figure 1 CD) Example 2: Please refer to Figure 2 ; In this example: LPO levels in NSCLC cells increased significantly after PLB treatment. In addition, we detected the expression of ferroptosis-related proteins and found that the levels of negative regulatory proteins such as glutaminase, FTH1, Nrf2, Slc7a11 and GPX4 were significantly decreased ( Figure 2 B). In addition, PLB also leads to increased iron ion levels. Ferroptosis inhibitors DFO and Fer-1 can prevent PLB-induced downregulation of FTH1, Nrf2, Slc7a11, and GPX4 in A549 and H358 cells, thereby preventing ferroptosis ( Figure 2 Importantly, DFO or Fe-1 partially attenuated PLB-induced cell death in non-small cell lung cancer cells ( Figure 2 FG).

[0021] Example 3: Please refer to Figure 3 ; In this example: PLB treatment can upregulate the expression of γ-H2AX protein, a DNA damage marker, in non-small cell lung cancer cells. This effect is due to the significant reduction of ferroptosis after pretreatment with DFO or Fe-1, indicating that PLB-induced ferroptosis leads to DNA oxidative damage in non-small cell lung cancer cells ( Figure 3 AB). Ferroptosis substances released by cytoplasmic dsDNA from dying tumor cells can activate cGAS-STING signaling when engulfed by DCs and macrophages. To investigate its effect on immune cell activation, we collected supernatants from tumor cells co-cultured with PLBs and then co-incubated with macrophages to measure the expression of M1 and M2 markers. The results showed an upregulation of M1 markers, indicating a shift toward M1 polarization ( Figure 3 C). In addition, plb-treated tumor supernatants promoted DC maturation ( Figure 3 D). In addition, downstream proteins of the STING pathway, including phos-STING, phos-TBK1, and phos-IRF3, were expressed in RAW264.7 cells ( Figure 3 E). Meanwhile, CCL5, CXCL10, and IFN-β increased, further confirming the activation of the STING pathway in APCs. The results showed that plb-mediated ferroptosis triggered DNA damage in NSCLC cells, promoted macrophage polarization, and activated the STING pathway in RAW264.7 cells.

[0022] Example 4: Please refer to Figure 4 ; In this example: In vivo study, Lewis lung cancer model was established to observe the anti-tumor effect of PLB in vivo. The results showed that the mice had a longer survival time compared with other groups ( Figure 4A). In contrast, PLB treatment showed no effect, further confirming that PLB was harmless to mice as evidenced by organ index and organ HE data. In addition, after PLB treatment, the tumor volume and weight of mice gradually decreased 10% compared with the control group ( Figure 4 BC). Antitumor effect DFO significantly eliminated PLB, indicating that PLB inhibited tumor growth and may be related to ferroptosis. In addition, co-treatment with DFO rescued GPX4 levels in tumor tissues with reduced PLB, indicating that PLB triggered ferroptosis ( Figure 4 DE). Subsequently, we examined the immune status induced by PLB. PLB treatment increased immune infiltration, MHC-2+CD11B+DCs and increased M1-like macrophages and infiltrating CD8+ T cells in tumors ( Figure 4 FH).

[0023] Example 5: Please refer to Figure 5 ; In this example, the potential synergistic effect of PLB combined with PD-1 monoclonal antibody in the treatment of non-small cell lung cancer was studied in vivo. Then, we explored the potential of PLB to enhance the anti-tumor activity of anti-PD-1 monoclonal antibody in non-small cell lung cancer. Figure 5 Schematic diagram of A. Figure 5 B combined therapy has a better survival rate and no obvious side effects ( Figure 5 BC). In addition, PLB, anti-PD-1 mAb and their combination can reduce tumor weight and size. Compared with the PBS group, tumor growth was significantly reduced in the combination group. Figure 5 DE). The combination therapy also increased the frequency and number of DCs and macrophages. Further analysis showed an increase in the level of M1-like macrophages and a decrease in M2-like macrophages in the combination group compared with anti-PD-1 mAb alone ( Figure 2 ). The results showed that the combined treatment promoted the polarization of macrophages M2 to M1. In addition, compared with anti-anti-PD-1 mAb or anti-anti-PD-1 mAb monoclonal antibody, the combined treatment promoted the infiltration of CD8+ T cells and produced more TNF-α, GzmB and IFN-γ in the tumor than the PLB group alone ( Figure 5 F).

[0024] The contents not described in detail in this specification belong to the prior art known to professional and technical personnel in this field.

[0025] Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for those skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to make equivalent substitutions for some of the technical features therein. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. An experimental method for plumbagin in anti-non-small cell lung cancer drugs, characterized in that: The medicine comprises plumbagin and a pharmaceutically acceptable carrier.

2. The experimental method of plumbagin in anti-non-small cell lung cancer drugs according to claim 1, characterized in that: The medicine comprises an effective dose of 5-hydroxy-2-methylnaphthalene-1,4-dione and a pharmaceutically acceptable carrier.

3. The experimental method of plumbagin in anti-non-small cell lung cancer drugs according to claim 2, characterized in that: The pharmaceutically acceptable carriers include fillers, diluents, adhesives and drug-carrying carriers without toxic or side effects.

4. The experimental method of plumbagin in anti-non-small cell lung cancer drugs according to claims 1-3, characterized in that: The drug is prepared into a pharmaceutically acceptable dosage form.

5. The experimental method of plumbagin in anti-non-small cell lung cancer drugs according to claim 4, characterized in that: The dosage forms include tablets, drops, and capsule preparations.

6. The experimental method of plumbagin in anti-non-small cell lung cancer drugs according to claim 5, characterized in that: The drug is prepared in the form of a single-dose drug.

7. The experimental method of plumbagin in anti-non-small cell lung cancer drugs according to claim 6, characterized in that: The drug is a single dose of plumbagin.

8. The experimental method of plumbagin in anti-non-small cell lung cancer drugs according to claim 7, characterized in that: The drug is able to downregulate GPX4 and protein expression in subjects with non-small cell lung cancer.

9. The experimental method of plumbagin in anti-non-small cell lung cancer drugs according to claim 8, characterized in that: The drug is capable of upregulating the production of ROS in subjects with non-small cell lung cancer.

10. The experimental method of plumbagin in anti-non-small cell lung cancer drugs according to claim 9, characterized in that: The drug can upregulate changes in the immune microenvironment of subjects with non-small cell lung cancer and effectively kill tumor cells.

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