Application of FOSL1 gene in the preparation of drugs that induce copper death in tumor cells

Abstract

This invention relates to the field of biomedical technology and provides an application of the FOSL1 gene in the preparation of a drug that induces copper death in tumor cells, wherein the tumor is non-small cell lung cancer. This invention specifically induces mitochondrial damage and triggers copper death by targeting and regulating FOSL1 gene expression. Based on the differential expression and regulatory role of FOSL1 in radiation-resistant NSCLC cells, this invention clarifies the core mechanism by which FOSL1 mediates mitochondrial damage, triggers copper death, and sensitizes radiotherapy. It provides a drug that can induce copper death in radiation-resistant NSCLC cells and a precise, safe, and efficient strategy for reversing tumor cell radiation resistance.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to the application of the FOSL1 gene in the preparation of drugs that induce copper death in tumor cells. Background Technology

[0002] Lung cancer has the leading cause of death among human malignant tumors, with non-small cell lung cancer (NSCLC) accounting for approximately 80% of all lung cancer cases. In NSCLC patients, radiotherapy or concurrent chemoradiotherapy has become a core treatment approach. However, during treatment, tumor cells can acquire radiation resistance, leading to local control failure and affecting treatment efficacy. The factors contributing to radiation resistance are complex and multifaceted, primarily including hypoxia, abnormalities in radiation resistance-related signal transduction pathways, and abnormal expression of genes related to DNA damage repair responses.

[0003] Copper death is a process caused by copper ions (Cu) 2+ A novel programmed cell death mechanism triggered by overload may offer new insights into reversing radiation resistance. The mechanism of copper death involves copper ions specifically binding to acylated proteins in the mitochondrial tricarboxylic acid (TCA) cycle, inducing oligomerization of toxic proteins and collapse of the mitochondrial respiratory chain, ultimately leading to cell death. Copper death is independent of the traditional apoptosis / necrosis pathway and theoretically could circumvent common apoptosis resistance mechanisms in tumor cells.

[0004] Although progress has been made in the study of the mechanisms of radiation resistance in NSCLC, the current technology still faces the following key bottlenecks: existing copper death induction strategies (such as copper-loaded drugs) lack specific molecular targets for radiation-resistant cells, resulting in unclear mechanisms of action and uncontrollable efficacy; traditional copper-loaded drugs induce death through exogenous copper ion overload, which can easily disrupt the body's copper homeostasis and induce systemic adverse reactions such as liver / neurotoxicity, thus limiting their clinical applicability; single interventions in pathways such as hypoxia or DNA repair are insufficient to overcome the multi-mechanism synergistic effect of radiation resistance and cannot circumvent the apoptosis / necrosis resistance mechanisms of tumor cells.

[0005] Therefore, there is an urgent need to find a target and a new drug that can induce copper death in radiation-resistant NSCLC cells in order to improve the prognosis of patients with radiation-resistant lung cancer and to provide new alternative strategies for the treatment of clinically refractory NSCLC. Summary of the Invention

[0006] The purpose of this invention is to provide the application of the FOSL1 gene in the preparation of drugs that induce copper death in tumor cells, thereby addressing the problems raised in the background art.

[0007] To address the above problems, the present invention provides an application of the FOSL1 gene in the preparation of drugs that induce copper death in tumor cells.

[0008] Furthermore, the tumor is lung cancer.

[0009] Furthermore, the tumor is non-small cell lung cancer.

[0010] Furthermore, by targeting and regulating FOSL1 gene expression, mitochondrial damage can be specifically induced, thereby triggering copper death.

[0011] Another object of the present invention is to provide the application of the FOSL1 gene in the preparation of a drug for reversing the radiation resistance of tumor cells.

[0012] Another object of the present invention is to provide a drug for inducing copper death in tumor cells, comprising a pharmaceutically acceptable vector and including a FOSL1 overexpression plasmid.

[0013] Furthermore, the method for preparing the drug includes the following steps:

[0014] A microemulsion system was obtained by adding NaOH aqueous solution to a mixed solution of ethanol, oleic acid and n-hexane.

[0015] Cu(NO3)2·3H2O and 1,3,5-benzenetricarboxylic acid were added to the microemulsion system and reacted to obtain Cu-MOF;

[0016] Cu-MOF was modified with DSPE PEG2000 to obtain nanoparticles;

[0017] The drug was obtained by co-incubating the nanoparticles with the FOSL1 overexpression plasmid.

[0018] This invention, based on the differential expression and regulatory role of FOSL1 in radiation-resistant NSCLC cells, clarifies the core mechanism by which FOSL1 mediates mitochondrial damage, triggers copper death, and enhances radiosensitization. It provides a drug that can induce copper death in radiation-resistant NSCLC cells and a precise, safe, and efficient strategy for reversing tumor cell radiation resistance. Specifically, by targeting and regulating FOSL1 gene expression, exogenous copper ion introduction can be avoided, eliminating the risk of copper metabolism disorder at its source. By activating copper death independent of traditional death pathways, mitochondrial function and DNA repair capacity in radiation-resistant cells are simultaneously disrupted, providing important guidance for the clinical treatment of radiation-resistant lung cancer. The drug provided by this invention, through the above mechanisms, effectively improves the prognosis of patients with radiation-resistant lung cancer and provides a new alternative strategy for the treatment of clinically refractory NSCLC. Attached Figure Description

[0019] Figure 1 The figure shows the results of differential gene enrichment analysis in cells from different treatment groups (A549-RR group and A549 group).

[0020] Figure 2 The figure shows the results of differential gene enrichment analysis in cells from different treatment groups (A549-RR group and A549-RR+ES group).

[0021] Figure 3 This diagram shows the gene expression results enriched in copper death-related pathways.

[0022] Figure 4 The diagram shows a comparison of differentially expressed genes in cells from different treatment groups. In the diagram, A is a comparison of differentially expressed genes between the A549-RR group and the A549 group; B is a comparison of differentially expressed genes between the A549-RR group and the A549-RR+ES group.

[0023] Figure 5 Figure 1 shows the results of measuring copper ion concentration (A) and cell death rate (B) after trypan blue staining under different treatment conditions (n=3, *P<0.05, **P<0.01).

[0024] Figure 6 This is a schematic diagram of the synthesis process of nanomedicines provided in an embodiment of the present invention.

[0025] Figure 7 Figures show the characterization results of the nanomedicine provided in the embodiments of the present invention; in the figures, a is a Cu-MOF transmission electron microscope image; b is the CMDP transmission electron microscope mapping of each element; c and d are the XRD and XPS images showing the characteristic peak distribution of each element in Cu-MOF, respectively; e is the CMDP Fourier transform infrared spectroscopy (FTIR) result; f and g are the CMDP hydration particle size distribution and potential change diagrams, respectively; h is the transmission electron microscope image of CMDP degradation at different pH levels; i is the ICP (inductively coupled plasma) analysis of copper ion release from CMDP at different pH levels; j is the ESR showing the ·OH characteristic peak in CMDP; k and l are the results and quantification diagrams of GSH consumption detected by ultraviolet spectrophotometer.

[0026] Figure 8 Figures show the results of cell experiments in different groups; in the figures, a is the detection result of the colony-forming ability of CMDP on A549-RR; b is the live and dead staining of A549-RR after CMDP treatment; c is the flow cytometry detection result; d is the ROS level after CMDP treatment of A549-RR; e is the mitochondrial ROS generation; f is the mitochondrial membrane potential level; g is the change in mitochondrial calcium ion concentration.

[0027] Figure 9Figures show the in vivo experimental results of different groups of animals; in the figures, a is a photograph of A549-RR cell xenograft tumors in different groups of SCID mice treated with different methods; b is a comparison of body weight changes in different groups of A549-RR tumor-bearing SCID mice; c is a comparison of tumor volume in different groups of A549-RR cell xenograft tumor-bearing SCID mice; d is a comparison of H&E staining, immunohistochemical changes in FOSL1 and Ki67 proteins in A549-RR cell xenograft tumor tissues of SCID mice after different methods of treatment. Detailed Implementation

[0028] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0029] Based on the "apoptosis / necrosis resistance" phenotype exhibited by radiation-resistant NSCLC cells, this invention discovers for the first time that the transcription factor FOSL1 (FOS Like 1) shows significantly differential expression in radiation-resistant model cells under the influence of copper death inducers. FOSL1 is a key target regulating the copper death process in radiation-resistant NSCLC cells; targeted regulation of FOSL1 can specifically induce mitochondrial damage (including membrane potential collapse and respiratory chain complex dysfunction), thereby triggering copper death. Based on the above findings, this invention proposes a novel treatment strategy: by designing drugs that can target and regulate FOSL1, copper death can be specifically induced in radiation-resistant NSCLC cells.

[0030] Specifically, in one embodiment of the present invention, the application of the FOSL1 gene in the preparation of a drug that induces copper death in tumor cells is provided. By targeting and regulating the expression of the FOSL1 gene, mitochondrial damage is specifically induced, thereby triggering copper death in tumor cells such as NSCLC. Through this mechanism, the radiation resistance of tumor cells such as NSCLC can be reversed.

[0031] In another embodiment of the invention, a drug for inducing copper death in tumor cells and reversing radiation resistance in tumor cells is also provided, comprising a pharmaceutically acceptable vector and including a FOSL1 overexpression plasmid.

[0032] Specifically, the preparation method of the above-mentioned drug includes the following steps:

[0033] S1. Add 0.5-1 mL of NaOH aqueous solution (0.1-0.15 M) to a mixed solution of 1-1.5 mL ethanol, 0.2-0.4 mL oleic acid and 0.15-0.2 mL n-hexane, and stir and mix at 45-55 °C to obtain a constant temperature microemulsion system.

[0034] S2. Add 8-12 mg of Cu(NO3)2·3H2O and 8-12 mg of 1,3,5-benzenetricarboxylic acid to the above microemulsion system, heat to 65-75℃ for reaction, and then centrifuge and wash to obtain Cu-MOF;

[0035] S3. Modify the above Cu-MOF with DSPE PEG2000 to obtain nanoparticles; specifically, disperse DSPE-PEG2000 in sterile enzyme-free water, disperse 10-15 mg of the obtained Cu-MOF in 2 mL of DSPE-PEG2000 solution with a concentration of 0.8-1.2 mg / mL, and stir vigorously overnight to carry out modification.

[0036] S4. The above nanoparticles are co-incubated with the FOSL1 overexpression plasmid for 10-15 hours to obtain the above drug.

[0037] The preparation method of the FOSL1 overexpression plasmid is as follows: A large-scale amplification of the commercially available FOSL1 plasmid (HumanpLenti-HA-FOSL1-puro) was performed. First, positive clones were picked, and single colonies were picked from the transformed LB plates and inoculated into 3-5 mL of LB liquid medium (with Amp 100 μg / mL). Then, the culture was incubated at 37℃ with shaking at 200-250 rpm for 12-16 hours (until OD600≈2.0, and the medium became turbid). The harvested bacterial culture was centrifuged (4000 rpm, 10 minutes, 4℃) to collect the bacterial cells, and the supernatant was discarded. The plasmid was purified using a commercially available plasmid large-scale extraction kit according to the detailed procedure in the instructions to obtain the FOSL1 overexpression plasmid, and the concentration (Nanodrop) and purity (A260 / A280≈1.8) were determined.

[0038] Example 1: Constructing a radiation resistance model for lung cancer A549 cells. Parental A549 cells in logarithmic growth phase were irradiated with 6 Gy (1.02 Gy / min) and then cultured. When the cells reached the end of logarithmic growth phase again, they were passaged, and the same dose was irradiated again the following day. This process was repeated for a total dose of 30 Gy over 5 irradiations. The surviving cells were then monocloned to obtain a cell clone named A549-RR.

[0039] In A549-RR cells, the copper death inducer elesclomol (ES) was added, and three groups were established: A549 (untreated A549 cells), A549-RR (irradiated A549-RR cells), and A549-RR+ES (A549-RR cells treated with ES). High-throughput transcriptome sequencing and differentially expressed gene enrichment analysis revealed that differentially expressed genes in A549-RR cells compared to A549 cells were enriched in pathways closely related to copper death. After adding the copper death inducer ES to radiation-resistant cells (A549-RR+ES), the expression of genes enriched in pathways related to copper death mechanisms was reversed (e.g., ...). Figure 1 and Figure 2 (As shown). Compared with the A549-RR group, the parental A549 group showed significantly different expression of genes enriched in copper death-related pathways, and gene expression was reversed after the addition of a copper death inducer, including the FOSL1 gene (e.g., Figure 3 (As shown).

[0040] Example 2: High-throughput sequencing of A549, A549-RR, and A549-RR+ES cells from Example 1 was performed using qPCR. The top nine differentially expressed genes in the sequencing results were verified to confirm the reliability of the sequencing results. The target gene FOSL1 (e.g., Figure 4 (As shown in A and B). Additionally, FOSL1 siRNA fragments were synthesized using existing techniques, and the changes in copper death after FOSL1 knockdown in A549 and A549-RR+ES cells were detected by measuring copper ion concentration and using trypan blue staining. The results are as follows: Figure 5 As shown in Figures A and B; the results showed that, compared with normal A549-RR+ES cells (ES group) and A549 cells (control group), knocking down FOSL1 in A549-RR+ES cells (siFOSL1+ES group) reduced copper ion concentration and cell death, confirming that FOSL1 is a key target for copper death in A549 and A549-RR cells.

[0041] Example 3: As Figure 6 As shown, based on the fact that FOSL1 is a key gene for inducing copper death in cells, this embodiment provides a nanomedicine for inducing copper death in tumor cells and reversing radiation resistance in tumor cells. Its preparation method includes the following steps:

[0042] S1. Add 0.8 mL of NaOH aqueous solution (0.125 M) to a mixed solution of 1.2 mL ethanol, 0.3 mL oleic acid and 0.17 mL n-hexane, and stir at 50 °C to obtain a constant temperature microemulsion system.

[0043] S2. Add 10 mg of Cu(NO3)2·3H2O (dissolved in 0.2 mL of deionized water before adding) and 10 mg of 1,3,5-benzenetricarboxylic acid (dissolved in a mixture of 0.13 mL of deionized water and 0.17 mL of ethanol before adding) to the above microemulsion system, heat to 70 °C, and maintain the reaction for 2 hours with continuous stirring. Then collect the product by centrifugation and wash it three times with a mixture of cyclohexane and anhydrous ethanol to obtain Cu-MOF.

[0044] S3. Modify the above Cu-MOF with DSPE PEG2000 to obtain CMD nanoparticles; specifically, disperse DSPE-PEG2000 in sterile enzyme-free water, disperse 12 mg of the obtained Cu-MOF in 2 mL of DSPE-PEG2000 solution with a concentration of 1 mg / mL, and stir vigorously overnight for modification.

[0045] S4. Co-incubate the above-mentioned nanoparticles CMD with the FOSL1 overexpression plasmid for 12 hours to obtain the nanomedicine CMDP.

[0046] The structure and properties of the nanomedicine CMDP prepared above were characterized, and the results are as follows: Figure 7 As shown in the image, the transmission electron microscopy (TEM) images reveal that the Cu-MOF nanoparticles exhibit a spherical morphology (e.g., ...). Figure 7 As shown in a and b); the X-ray diffraction (XRD) pattern shows multiple diffraction peaks (e.g., as shown in a and b); Figure 7 As shown in c), this indicates that the obtained nanomedicine is a crystalline material, rather than a completely amorphous one; X-ray photoelectron spectroscopy (XPS) analysis confirmed the presence of Cu, C, N, P, and O elements (e.g., ...) in the CMDP nanomedicine. Figure 7 (As shown in d). Fourier transform infrared spectroscopy (FT-IR) results demonstrate that after modification with DSPE-PEG2000, the 2880 cm⁻¹... -1 and 1100cm -1 Significant new peaks appeared at 1240 cm⁻¹, corresponding to ν(CH) and ν(COC) of the PEG chain, respectively, confirming successful encapsulation. -1 The ν(P=O) peak further confirms the presence of DSPE phospholipids. The characteristic carboxylic acid peak of Cu-MOF is 1602 / 1398 cm⁻¹. -1 The reduced strength suggests that the PEG layer partially obscures the MOF surface. After loading the FOSL1 plasmid, the strength at 1220 cm⁻¹... -1 The peak enhancement at 1650 cm⁻¹ confirms the presence of the DNA phosphate backbone. -1 The weak peaks may be attributed to nucleic acid base vibrations. The shift of the Cu-O vibration peak to lower wavenumbers indicates that the plasmid interacts with the Cu on the MOF surface via phosphate groups. 2+Interactions. Furthermore, the broadening of the ν(COC) peak of PEG suggests that the plasmid and PEG chain are bound via hydrogen bonds (e.g., Figure 7 As shown in e). When the hydrated particle size and zeta potential of the CMDP nanomedicine were measured at each stage of synthesis, its diameter changed from 184 nm to 220 nm, and the zeta potential value changed from negative to positive. Compared with Cu-MOF nanoparticles, this further proves that DSPE-PEG2000 has successfully coated the surface of the nanoparticles, and the FOSL1 overexpression plasmid has been successfully loaded (as shown in e). Figure 7 (as shown in f and g). When CMDP nanomedicines were exposed to solutions with pH values ​​of 7.4 and 6.4, they maintained their intact material morphology. However, when exposed to a solution with pH value of 5.4 for the same period, the material degraded and lost its original morphology. This phenomenon is attributed to the inherent instability of the coordination bonds within the CMDP nanomedicines (e.g., ...). Figure 7 (as shown in h and i). Furthermore, CMDP nanomedicines exhibit more dramatic GSH consumption under both GSH and hydrogen peroxide conditions, possibly due to the Cu+ / Cu ratio. 2+ It can react with GSH and hydrogen peroxide, leading to the rapid consumption of GSH (e.g. Figure 7 (k and l), and generate reactive oxygen species (such as k and l). Figure 7 As shown in j), the above results demonstrate that the embodiments of the present invention successfully synthesized the nanomedicine CMDP with copper death induction activity.

[0047] Example 4: Cell experiments were conducted according to the following groups: -IR was the no-radiation treatment group, +IR was the radiation treatment group; the Con group was the control group and received no treatment; FOSL1 OE The first group consisted of FOSL1 gene overexpression plasmids, i.e., commercially available FOSL1 overexpression plasmids; the second group consisted of empty copper-based nanomaterials without FOSL1 overexpression plasmids, i.e., the CMD nanoparticles prepared in Example 3; and the third group consisted of nanomedicines loaded with FOSL1 overexpression plasmids, i.e., the CMDP nanomedicines prepared in Example 3. Experimental results are as follows: Figure 8 As shown.

[0048] Clonalization experiments showed -IR (Con group, FOSL1) OE Group, CMD group, CMDP group) and +IR (Con group, FOSL1) OE (Groups 1, 2, 3, and 4) Under the same dose of irradiation, cell colony formation was significantly reduced in the CMD and CMDP groups (e.g., group 3, 2, and 3). Figure 8 (as shown in a). Furthermore, PI staining experiments showed that the +IR CMDP group had the highest number of cell deaths compared to other groups (e.g., as shown in a). Figure 8(As shown in b). Flow cytometry was used to detect cell apoptosis, and it was found that the +IR CMDP group had the highest cell death rate, further confirming the killing effect of CMDP on tumor cells (e.g., ...). Figure 8 (as shown in c).

[0049] The ability of CMDPs to generate ROS in tumor cells was detected using DCFH-DA as a fluorescent probe. Compared with other groups, the +IR CMDP group produced the most ROS (e.g., ...). Figure 8 As shown in d). The accumulation of intracellular mitochondrial ROS was further confirmed by detection using the mitochondrial-specific probe MitoSOX Red (e.g., as shown in d). Figure 8 As shown in e), consistent with the above results, it proves that after CMDP enters tumor cells, irradiation can induce the production of a large amount of reactive oxygen species and cause mitochondrial damage.

[0050] This invention uses JC-1 to detect mitochondrial membrane potential. It was found that compared to other groups, the +IRCMDP group showed the largest decrease in membrane potential, confirming that the +IRCMDP group causes severe mitochondrial damage (e.g., Figure 8 (as shown in f). Simultaneously, Hoechst 33342 and Rhod-2 AM were used to detect the fluorescence intensity of mitochondrial calcium ions, revealing that the fluorescence intensity of Rhod-2 AM increased in the +IR CMDP nanoparticle group (as shown in f). Figure 8 (As shown in g). The above results all demonstrate that under radiation induction, CMDP can induce mitochondrial damage and affect mitochondrial function, and its tumor-killing effect is significantly better than that of the -IR group and the radiotherapy-only group.

[0051] Example 5: The tumor-killing effect of the nanomedicine CMDP prepared in Example 3 was verified through in vivo experiments. Specifically, 6-week-old female SCID mice were housed in isolators. Standard feed was sterilized by cobalt source irradiation, and bedding and drinking water were sterilized by high temperature and high pressure. Similar to Example 4, the experiment was divided into 8 groups: -IR (Con group, FOSL1 group) OE Group, CMD group, CMDP group) and +IR (Con group, FOSL1) OE Groups G1, G2, G3, G4, G5, G6, G7, and G8 (CMD, CMDP, and G8) were designated as G1, G2, G3, G4, G5, G6, G7, and G8, respectively, to establish a stable FOSL1 cell model. Cells from different treatment groups were subcutaneously seeded into the lateral aspect of the right leg of mice, and tumor growth was observed and recorded daily until the tumor reached 200 mm. 3 At that time, local irradiation of 20 Gy was administered at a dose rate of 2.0 Gy / min. After 14 days of continuous observation, subcutaneous tumors were removed for subsequent functional experiments (such as...). Figure 9 As shown in a). The results showed that after treatment in different groups, the body weight of mice did not change significantly (as shown in a). Figure 9(As shown in b). Subcutaneous tumors were measured and photographed, and it was found that the +IR CMDP group had the smallest tumor volume in mice after irradiation compared with other treatment groups (e.g., as shown in b). Figure 9 (As shown in c). Using HE staining of tumor tissue and immunohistochemical staining, compared with other treatment groups, the +IR CMDP group showed reduced tumor cell nuclear volume, denser and more deeply stained chromatin, and fragmented nuclear chromatin after treatment. Immunohistochemistry indicated a gradual decrease in Ki-67, while FOSL1 expression remained high in the +IR CMDP group (as shown in c). Figure 9 (As shown in d). The above mouse experimental results demonstrate the tumor-killing effect of the nanomedicine CMDP.

[0052] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. The application of the human FOSL1 gene in the preparation of a drug that induces copper death in radiation-resistant non-small cell lung cancer cells, characterized in that, By targeting and overexpressing the human FOSL1 gene, mitochondrial damage is specifically induced, thereby triggering copper death.

2. Application of human FOSL1 gene overexpression plasmid in the preparation of drugs for reversing radiation resistance in radiation-resistant non-small cell lung cancer cells.

3. A drug for inducing copper death in radiation-resistant non-small cell lung cancer cells, comprising a pharmaceutically acceptable carrier, characterized in that, It also includes human FOSL1 overexpression plasmids.

4. The drug for inducing copper death in radiation-resistant non-small cell lung cancer cells according to claim 3, characterized in that, The method for preparing the drug includes the following steps: A microemulsion system was obtained by adding NaOH aqueous solution to a mixed solution of ethanol, oleic acid and n-hexane. Cu(NO3)2·3H2O and 1,3,5-benzenetricarboxylic acid were added to the microemulsion system and reacted to obtain Cu-MOF; Cu-MOF was modified with DSPE PEG2000 to obtain nanoparticles; The drug was obtained by co-incubating nanoparticles with human FOSL1 overexpression plasmid.

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