Application of disulfiram in reducing drug resistance of BRAF mutant melanoma targeted drug and preparation

Abstract

The invention discloses application of disulfiram in reduction of drug resistance of a BRAF mutant melanoma targeted drug and a preparation, relates to the technical field of medicines, and provides a new way and means for reduction of drug resistance of a BRAF inhibitor in treatment of BRAF mutant melanoma.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to the use of disulfiram in preparing a preparation for alleviating or reducing the drug resistance of BRAF mutant melanoma targeted drugs and a preparation thereof. Background Art

[0002] Among the known melanoma pathogenic genes, BRAF is the most common mutation driver gene, which confers a more aggressive phenotype to tumor cells by activating the MAPK pathway. Compared with non-BRAF mutant melanoma patients, most patients with BRAF mutations have a poor prognosis. Therefore, targeted therapy with BRAFi (such as vemurafenib and dabrafenib) has brought significant clinical benefits to cases carrying BRAF V600 E / K / R / D monomers. However, soon after the application of BRAF inhibitors, more than 50% of patients develop resistance, even in patients with high initial treatment responses. Given that reactivation of MAPK signaling is the main cause of BRAFi resistance, a dual combination of MEK inhibitors and BRAF inhibitors was developed to overcome this situation. Unfortunately, although this approach does delay the development of resistance, about 70% of patients still experience disease progression within 2 years. Therefore, there is still a need to develop new strategies other than MAPK pathway inhibition to overcome BRAFi resistance.

[0003] Disulfiram (DSF) is a member of the dithiocarbamate family. It is widely used in clinical anti-alcoholism drugs and is currently considered an effective anticancer drug for gastric cancer, thyroid cancer, liver cancer and breast cancer. There are also literatures that record that disulfiram has a therapeutic effect on non-BRAF mutant melanoma, but there are still few studies on disulfiram in the field of BRAF mutant melanoma. The research on disulfiram is mainly used as a drug for the treatment of melanoma. There are no reports on the development of other applications of disulfiram in the treatment of BRAF mutant melanoma. At present, the use of BRAFi inhibitors in the treatment of BRAF melanoma is a mainstream and efficient treatment method in clinical practice. Therefore, it is urgent to explore a method to alleviate or reduce the resistance of BRAFi inhibitors in the treatment of BRAF mutant melanoma. Summary of the invention

[0004] In view of this, the purpose of the present invention is to provide the use of disulfiram in the preparation of a preparation for alleviating or reducing the drug resistance of BRAF mutant melanoma targeted drugs and its preparation, so as to provide a new approach and means for reducing the drug resistance of BRAF melanoma treated with BRAFi inhibitors.

[0005] The present invention solves the above technical problems by the following technical means:

[0006] Disulfiram in reducing resistance to targeted drugs in BRAF-mutant melanoma.

[0007] Furthermore, the BRAF mutant melanoma targeted drug includes a BRAFi inhibitor.

[0008] Furthermore, the BRAFi inhibitors include vemurafenib, dabrafenib, and Encorafenib.

[0009] Furthermore, the BRAF mutant melanoma includes BRAF V600 E, BRAF V600K, BRAF V600R, and BRAFV600D mutant melanoma.

[0010] The present invention also discloses the use of disulfiram in preparing a preparation for reducing the drug resistance of targeted drugs of BRAF mutant melanoma.

[0011] Furthermore, the preparation raw materials include disulfiram.

[0012] Furthermore, the preparation is used in combination with a BRAFi inhibitor to reduce drug resistance of the BRAFi inhibitor, and the disulfiram reduces drug resistance of the BRAFi inhibitor.

[0013] Beneficial effects of the present invention:

[0014] The present invention discloses that DSF reduces the drug resistance of BRAFi inhibitors through TXNIP-TRX interaction, so disulfiram can be used to reduce or alleviate the drug resistance of BRAFi inhibitors used in the treatment of BRAF mutant melanoma. It has high clinical significance for patients with BRAF mutant melanoma who are no longer suitable for BRAFi inhibitors to treat, so the combined use of DSF and BRAFi inhibitors may be a promising strategy to overcome BRAFi resistance. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 :

[0016] A. Cytotoxicity test results of drug-resistant cells produced after culturing 451Lu and UACC62 parental cells;

[0017] B. Survival rate results of BRAF-resistant cells in Vem group, DSF+Vem group, and DSF+VemTTM group;

[0018] C. Viability results of BRAF-resistant cells in Vem group, DSF+Vem group, and DSF+Vem+ZVF+DBF group;

[0019] D. Survival rate results of BRAF-resistant cells in Vem group, DSF+Vem group, and DSF+Vem+NEC-1 group;

[0020] E. Survival rate results of BRAF-resistant cells in Vem group, DSF+Vem group, and DSF+Vem+FER-1 group;

[0021] F. Schematic diagram of the treatment regimen of nude mice bearing 451Lu-R tumors using vemurafenib, DSF, and TTM.

[0022] Figure 2 :

[0023] G. Images of ex vivo tumors in mice receiving indicated treatments;

[0024] H. Calculation results of tumor volume in Vem group, DSF+Vem group, and DSF+VemTTM group;

[0025] I. Calculation results of tumor weights of Vem group, DSF+Vem group, and DSF+VemTTM group;

[0026] (Data are mean ± SD of three replicates. P values ​​were calculated by two-tailed Student's t-test. *P < 0.05, **P < 0.01, ***P < 0.001)

[0027] Figure 3 :

[0028] J. WB analysis results of Caspase 3 expression level after DSF treatment;

[0029] (Data are mean ± SD of three replicates. P values ​​were calculated by two-tailed Student's t-test. *P < 0.05, **P < 0.01, ***P < 0.001)

[0030] Figure 4 :

[0031] A. RNA sequencing (RNA-seq) analysis of gene enrichment pathways in the Vem group and the Vem+DSF group;

[0032] B. Transmission electron microscopy (TEM) analysis of mitochondrial morphology in the Vem group and the Vem+DSF group;

[0033] C. Results of mitochondrial membrane potential (MMP) after immunofluorescence staining analysis of BRAF-resistant cells in Vem group and Vem+DSF group;

[0034] D. Seahorse detection method analysis of mitochondrial oxidative phosphorylation and glycolysis results of BRAF-resistant cells in Vem group and Vem+DSF group;

[0035] E. Seahorse detection method analysis of glycolysis results of BRAF-resistant cells in Vem group and Vem+DSF group;

[0036] (Data are mean ± SD of three replicates. P values ​​were calculated by two-tailed Student's t-test. *P < 0.05, **P < 0.01, ***P < 0.001)

[0037] Figure 5 :

[0038] A. Immunofluorescence staining analysis of Mt-ROS levels in anti-BRAF cells treated with Vem, DSF+Vem, and DSF+Vem+Mito-TEMPO groups;

[0039] B. CCK-8 detection of cell viability results in Vem group, DSF+Vem group, and DSF+Vem+Mito-TEMPO group;

[0040] C. TEM analysis of the changes in mitochondrial morphology in the Vem group, DSF+Vem group, and DSF+Vem+Mito-TEMPO group;

[0041] D. Immunofluorescence staining analysis of MMP levels in the Vem group, DSF+Vem group, and DSF+Vem+Mito-TEMPO group;

[0042] E. The mitochondrial oxidative phosphorylation profiles after treatment in the Vem group, DSF+Vem group, and DSF+Vem+Mito-TEMPO group were analyzed using the Seahorse assay;

[0043] (Data are mean ± SD of three replicates. P values ​​were calculated by two-tailed Student's t-test. *P < 0.05, **P < 0.01, ***P < 0.001)

[0044] Figure 6 :

[0045] A. Flow cytometry analysis of mt-ROS levels in BRAF-resistant cells in the Vem group, DSF+Vem group, and DSF+VemTTM group;

[0046] B. Immunofluorescence staining analysis of mt-ROS levels in BRAF-resistant cells in the Vem group, DSF+Vem group, and DSF+VemTTM group;

[0047] C. TEM analysis of mitochondrial morphology of BRAF-resistant cells in Vem group, DSF+Vem group, and DSF+VemTTM group;

[0048] D. Immunofluorescence staining analysis of MMP levels in BRAF-resistant cells in the Vem group, DSF+Vem group, and DSF+VemTTM group;

[0049] E. Result of Seahorse detection method for mitochondrial oxidative phosphorylation of BRAFi-resistant cells in Vem group, DSF+Vem group, and DSF+VemTTM group;

[0050] F: Plate cloning test results;

[0051] (Data are mean ± SD of three replicates. P values ​​were calculated by two-tailed Student's t test. *P < 0.05, **P < 0.01, ***P < 0.001.)

[0052] Figure 7 :

[0053] A. Results of RNA-seq analysis of genes with significant differences between the Vem group and the Vem+DSF group;

[0054] B. Western blotting (WB) analysis of TXNIP expression levels in BRAFi-resistant cells in the Vem group, DSF+Vem group, and DSF+Vem+TTM group;

[0055] C. CCK-8 assay results of cell viability of BRAFi-resistant cells after positive control and negative control were treated with Vem and DSF+Vem, respectively;

[0056] D. TEM detection of mitochondrial morphology of BRAF-resistant cells after positive control and negative control were treated with Vem and DSF+Vem respectively;

[0057] E. The results of immunofluorescence staining to detect the MMP levels of BRAF-resistant cells in positive control and negative control after Vem treatment and DSF+Vem treatment respectively;

[0058] F. The results of Seahorse detection of mitochondrial oxidative phosphorylation in BRAF-resistant cells after positive control and negative control were treated with Vem and DSF+Vem respectively;

[0059] G. Flow cytometry results of detecting mt-ROS levels in BRAF-resistant cells treated with Vem and DSF+Vem for positive control and negative control, respectively;

[0060] (P values ​​were calculated by two-tailed Student's t-test. *P<0.05, **P<0.01, ***P<0.001)

[0061] Figure 8 :

[0062] H. In vitro tumor images of nude mice transfected with 451Lu-R tumors in negative control (NC) and positive control with TXNIP knockout (ko-TXNIP) after treatment with Vem or Vem+DSF;

[0063] I. The tumor volume results of positive control and negative control after Vem treatment and DSF+Vem treatment respectively;

[0064] J. Result graph of tumor weight of positive control and negative control after treatment with Vem and DSF+Vem respectively;

[0065] (P values ​​were calculated by two-tailed Student's t test. *P<0.05, **P<0.01, ***P<0.001.)

[0066] Fig. 9 :

[0067] A. Result diagram of proteins interacting with TXNIP by data independent acquisition (DIA) quantitative proteomics analysis;

[0068] B. CO-IP analysis of the interaction between TXNIP and TRX2 in BRAF-resistant cells after treatment with Vem group and Vem+DSF group;

[0069] C. CCK-8 detection of BRAF-resistant cell viability in Vem group, Vem+DSF group, and DSF+Vem+TXNIP-IN-1 group;

[0070] D. TEM analysis of mitochondrial morphology of BRAF-resistant cells in Vem group, Vem+DSF group, and DSF+Vem+TXNIP-IN-1 group;

[0071] E. Immunofluorescence analysis of MMP levels in BRAF-resistant cells in the Vem group, Vem+DSF group, and DSF+Vem+TXNIP-IN-1 group;

[0072] F. Seahorse assay of mitochondrial oxidative phosphorylation in BRAF-resistant cells in Vem group, Vem+DSF group, and DSF+Vem+TXNIP-IN-1 group;

[0073] G. Flow cytometry analysis of mt-ROS levels in BRAF-resistant cells in the Vem group, Vem+DSF group, and DSF+Vem+TXNIP-IN-1 group.

[0074] (P values ​​were calculated by two-tailed Student's t test. *P<0.05, **P<0.01, ***P<0.001.) DETAILED DESCRIPTION

[0075] The present invention will be described in detail below with reference to the embodiments and accompanying drawings:

[0076] Example 1: Disulfiram inhibits the growth of BRAFi-resistant melanoma

[0077] Two melanoma cell lines carrying BRAF V600E mutation, 451Lu and UACC62, were used to generate vemurafenib-resistant (VR) cells. Cytotoxicity experiments were then performed to verify that disulfiram (DSF) had the effect of inhibiting the growth of BRAFi-resistant melanoma.

[0078] In the in vitro experiments, the concentration of disulfiram used was 1.5uM and the concentration of vemurafenib used was 5uM.

[0079] 451Lu and UACC 62 cells (referred to as 451Lu-P and UACC 62139P) were cultured at 5×10 3 The cells were seeded at a density of 10 cells / well in a 96-well plate and continuously exposed to increasing concentrations of vemurafenib (Vem) (0.2, 0.5, 1, 2, and 5 μM). The sensitivity of melanoma cells to vemurafenib was evaluated by determining the half-maximal inhibitory concentration (IC 50). After 16 weeks, two BRAF-resistant cell lines were successfully established, named vemurafenib-resistant 451Lu cells (451Lu-R) and vemurafenib-resistant UACC 62 cells (UACC 62-R), and maintained in medium containing 5 μM vemurafenib.

[0080] The results were obtained by performing IC50 assay on vemurafenib-resistant melanoma cells pre-cultured with different vemurafenib for 48 hours and 451Lu and UACC62 parental cells. Figure 1 As shown in A, the values ​​obtained are: the IC50 value of 451Lu-R (451Lu-resistant cells) is 97.99 μM, and the IC50 value of 451Lu-P is 1.805 μM;

[0081] The IC50 value of UACC62-R (UACC62 resistant cells) was 52.06 μM, and the IC50 value of UACC62-P was 52.06 μM.

[0082] BRAF-resistant cells (451Lu-R, UACC62-R) were treated with 20 μM tetrathiomolybdate (TTM) overnight and then treated with 1.5 μM DSF + 5 μM Vem to obtain the DSF + Vem + TTM group. The survival rate was compared between the Vem group treated with 5 μM Vem and the DSF + Vem group treated with 1.5 μM DSF + 5 μM Vem. Figure 1 As shown in B.

[0083] BRAF-resistant cells were pretreated with 30 μM apoptosis inhibitor ZVF (Z-VAD-FMK) and 50 μM apoptosis inhibitor DBF (D-Boc-FMK), and then treated with 1.5 μM DSF + 5 μM Vem to obtain the DSF + Vem + ZVF + DBF group. The cell viability results were compared between the Vem group and the DSF + Vem group. Figure 1 As shown in C.

[0084] BRAF-resistant cells were pretreated with 20 μM necroptosis inhibitor NEC-1 (Necrostatin-1), and then treated with 1.5 μM DSF + 5 μM Vem to obtain the DSF + Vem + NEC-1 group. The survival rate of resistant cells was obtained by comparing the Vem group and the DSF + Vem group. Figure 1 As shown in D.

[0085] BRAF-resistant cells were pretreated with 10 μM ferroptosis inhibitor FER-1 (ferrostatin-1), and then treated with 1.5 μM DSF + 5 μM Vem to obtain the DSF + Vem + FER-1 group. The survival rate of the resistant cells was compared between the Vem group and the DSF + Vem group. Figure 1 As shown in E.

[0086] Result analysis:

[0087] The combination of DSF+Vem significantly inhibited the cell viability of BRAF-resistant cells, and the plate cloning experiment further confirmed the growth inhibitory effect of DSF on BRAF-resistant cells. When BRAF-resistant cells were co-treated with inhibitors of known cell death pathways while using DSF, tetrathiomolybdate (TTM) was found to significantly abolish DSF-induced cell death, while apoptosis inhibitors (Z-VAD-FMK and Boc-D-FMK), necroptosis inhibitor (NEC-1) and ferroptosis inhibitor (FER-1) failed to reverse the cytotoxicity, indicating that the efficacy of cell death caused by DSF is largely mediated by increasing intracellular copper accumulation.

[0088] Example 2: Animal Experiment

[0089] Based on the above conclusions, further animal mouse experiments were conducted.

[0090] Six-week-old female BALB / c nude mice were used for animal experiments. This animal experiment was approved by the Animal Care and Use Committee of the Animal Facility of the Fourth Military Medical University.

[0091] To evaluate the efficacy of disulfiram (DSF) + vemurafenib in the treatment of BRAFi-resistant melanoma, 1×10 7 451Lu-R melanoma cells were subcutaneously injected into BALB / c nude mice to establish a subcutaneous melanoma tumor model in mice.

[0092] When the tumor volume reached 50 cubic millimeters (7 days after the injection of drug-resistant cells), drug treatment was carried out. The mice were injected with Vem, DSF, and TTM drugs once every two days for a total of 14 days (7 times). The specific concentrations used each time were as follows:

[0093] Vem group: 30 mg / kg vemurafenib;

[0094] Vem+DSF group: 30mg / kg vemurafenib + 60mg / kg disulfiram (DSF);

[0095] Vem+DSF+TTM group: 30 mg / kg vemurafenib + 60 mg / kg disulfiram (DSF) + 0.3 mg / kg tetrathiomolybdate (TTM);

[0096] Mice were sacrificed 14 days after receiving the indicated treatments, and tumor tissues were harvested, weighed, and photographed. Figure 2 G. Figure 2 H. Figure 2 As shown in I.

[0097] analyze Figure 2 The results of GI showed that compared with other groups, the tumor growth of mice was significantly inhibited after treatment with Vem+DSF, and its volume and weight were significantly reduced compared with the other two groups. This shows that DSF can significantly reduce the drug resistance of BRAFi inhibitors.

[0098] Embodiment 3:

[0099] Given that MEK activation is responsible for most BRAFi resistance, we investigated whether the inhibitory effect of DSF was associated with inhibition of the MEK pathway. The expression level of Caspase 3 (a hallmark protein of apoptosis) was detected after MEK activity was inhibited using the MEK inhibitor Trametinib (Tra). The concentrations of Vem, DSF, and Tra were 5 μM, 1.5 μM, and 2 nM, respectively.

[0100] Western blot (WB) showed that, unlike the combination with the MEK inhibitor Trametinib (Tra), the combination with DSF did not affect ERK phosphorylation in BRAFi-resistant melanoma cells. Figure 3 As shown in Figure 6J, we further performed WB analysis to confirm that there was no change in the expression level of Caspase 3, indicating that the inhibitory effect of DSF was independent of the inhibition of the MEK pathway.

[0101] Embodiment 4:

[0102] 451Lu-R BRAF-resistant cells were treated with 5μM Vem and 5μM Vem+1.5μM DSF, respectively. RNA sequencing (RNA-seq) was then used to compare the expression transcripts between the BRAF-resistant cells treated with the Vem group and the Vem+DSF group. The results were as follows: Figure 4 As shown in A: According to gene ontology (GO) analysis, the differentially expressed genes are enriched in the gene sets related to mitochondrial structure and function. Copper is not only required for the assembly of mitochondrial copper enzymes, but also participates in mitochondrial function and dynamics.

[0103] Based on the above conclusions:

[0104] Transmission electron microscopy (TEM) was used to analyze mitochondria. Figure 4 B: In BRAF-resistant cells treated with Vem+DSF, transmission electron microscopy (TEM) showed swollen mitochondria with vacuoles and / or abnormal cristae.

[0105] The results of flow cytometry and confocal laser scanning microscopy using JC-1 dye were shown in Figure 4 C: In BRAF-resistant cells treated with Vem+DSF, mitochondrial membrane potential (MMP) was significantly impaired.

[0106] The results of the ATP content test are as follows Figure 4 D: Decreased ATP production in cells treated with Vem+DSF.

[0107] The results obtained by the Seahorse test are as follows Figure 4E: The combined use of DSF+Vem significantly inhibited mitochondrial oxidative respiration of VR cells, while the extracellular acidification rate (ECAR) was not affected.

[0108] Therefore, it can be seen that DSF reverses resistance to BRAFi by inducing mitochondrial dysfunction.

[0109] Embodiment 5:

[0110] We further explored how DSF reverses resistance to BRAFi by inducing mitochondrial dysfunction.

[0111] The anti-BRAF cells were pretreated with 10 μM Mito-TEMPO (a mitochondrial targeted antioxidant that can specifically remove reactive oxygen species in mitochondria) overnight to remove mitochondrial oxygen active mt-ROS, and then treated with 1.5 μM DSF+5 μM Vem to obtain the DSF+Vem+Mito-TEMPO group. The Vem group and the DSF+Vem group were compared, and the Mt-ROS level was analyzed by immunofluorescence staining. The results are as follows: Figure 5 As shown in A;

[0112] Cell viability was determined using CCK-8 and plate cloning, and the results were as follows Figure 5 As shown in B;

[0113] The results of TEM analysis of mitochondrial morphology were as follows: Figure 5 As shown in C;

[0114] The MMP levels were analyzed by immunofluorescence staining. Figure 5 As shown in D;

[0115] The results of analyzing mitochondrial oxidative phosphorylation using the Seahorse assay were as follows: Figure 5 As shown in E.

[0116] The analysis results show that:

[0117] The cytotoxicity of oxidative stress on BRAFi-resistant melanoma cells was determined. Flow cytometry and Mito-SOX staining revealed excessive accumulation of mt-ROS in resistant cells exposed to Vem+DSF. CCK-8 and plate cloning assays demonstrated that Mito-TEMPO largely rescued the growth inhibition of DSF+Vem. TEM photography, MMP assay, Seahorse assay, and ATP content detection revealed that pretreatment with Mito-TEMPO restored DSF-induced mitochondrial dysfunction. In summary, the above results indicate that DSF causes mitochondrial dysfunction by inducing oxidative stress damage.

[0118] Embodiment 6:

[0119] BRAF-resistant cells were pretreated with TTM to deprive the culture system of copper, and then the mitochondrial structure and function were evaluated.

[0120] After pre-treating BRAF-resistant cells with 20 μM TTM, the pre-treated BRAF-resistant cells were treated with 1.5 μM DSF and 5 μM Vem to obtain the DSF+Vem+TTM group. The Vem group and the DSF+Vem group were compared, and the mt-ROS level of BRAF-resistant cells was analyzed by flow cytometry. The results are shown in 6A.

[0121] Immunofluorescence staining was used to show the mt-ROS levels in BRAF-resistant cells, and the results are shown in 6B;

[0122] TEM was used to analyze the changes in mitochondrial morphology, and the results were as follows: Figure 6 As shown in C;

[0123] Immunofluorescence staining was used to show the MMP levels, and the results were as follows Figure 6 As shown in D;

[0124] The Seahorse assay was used to analyze mitochondrial oxidative phosphorylation, and the results were as follows: Figure 6 As shown in E.

[0125] The analysis results show that:

[0126] First, flow cytometry and confocal microscopy showed that TTM significantly reduced the elevated mitochondrial oxidative stress induced by DSF in resistant cells. Second, TEM showed that when exposed to Vem+DSF, pretreatment with TTM maintained the normal structure of mitochondria, characterized by uniform electron density and clear straight cristae of mitochondrial cristae. Third, cells pretreated with TTM showed cellular activity against DSF-induced MMP damage and mitochondrial oxidative respiration impairment. Finally, we demonstrated by plate cloning assay ( Figure 6 F), TTM can restore the proliferation activity of resistant cells treated with DSF+Vem. Collectively, the above findings confirm that mitochondrial dysfunction caused by Vem+DSF through induction of oxidative stress can be restored by copper deprivation.

[0127] Embodiment 7:

[0128] RNA sequencing data were used to identify potential key genes mediating DSF-induced oxidative damage in the Vem group and the Vem+DSF group. Figure 7 As shown in A, thioredoxin-interacting protein (TXNIP), which is closely related to the intracellular ROS level, was selected as a candidate gene among the genes with the most significant changes in the Vem+DSF combination.

[0129] Compared with the DSF+Vem+TTM group treated with TTM, the expression of TXNIP was confirmed by western blotting and immunofluorescence staining (results are shown in Figure 7 B).

[0130] To further verify the key role of TXNIP in DSF-induced mitochondrial dysfunction, a TXNIP knockout resistant melanoma cell model was established using CRISPR-Cas9 technology, and a negative control (NC) without TXNIP gene knockout and a positive control (ko-TXNIP) with TXNIP gene knockout were obtained. Then, the cells were treated with Vem and DSF+Vem and the following experiments were performed:

[0131] CCK-8 and plate cloning assays were used to show the cell viability of BRAF-resistant cells, and the results were as follows Figure 7 As shown in C;

[0132] The morphology of mitochondria after stimulation was analyzed by TEM, and the results were as follows: Figure 7 As shown in D;

[0133] Immunofluorescence staining was used to detect the MMP levels in BRAF-resistant cells. Figure 7 As shown in E;

[0134] The results of using Seahorse to detect mitochondrial oxidative phosphorylation in BRAF-resistant cells are as follows: Figure 7 As shown in F;

[0135] Flow cytometry was used to detect the level of mt-ROS in BRAF-resistant cells, and the results were as follows: Figure 7 As shown in G.

[0136] Result analysis:

[0137] CCK-8 and plate cloning assays showed that the inhibitory effect of the Vem+DSF combination on drug-resistant cells was significantly reversed after TXNIP knockout, indicating the key role of TXNIP in DSF-induced cytotoxicity. TEM images showed that TXNIP knockout eliminated the damage to mitochondrial structure caused by the Vem+DSF combination. In addition, DSF-induced MMP damage and mitochondrial oxidative respiration damage were restored by TXNIP knockout. We believe that TXNIP knockout can restore mitochondrial function by reducing mt-ROS, and flow cytometry and immunofluorescence detection have verified that in the process of DSF-induced BRAF inhibitor-resistant cell death: DSF increases mitochondrial ROS by increasing TXNIP, leading to mitochondrial dysfunction and further leading to the death of drug-resistant cells.

[0138] Embodiment 8:

[0139] Based on the conclusion of Example 7, the mouse model validation was continued.

[0140] Six-week-old female BALB / c nude mice were used for the animal experiments. This animal experiment was approved by the Animal Care and Use Committee of the Animal Facility of the Fourth Military Medical University.

[0141] To evaluate the key role of TXNIP+DSF+Vem in the combined treatment of BRAFi-resistant melanoma, 1×10 7 451Lu-R NC / 451Lu-R ko-TXNIP melanoma cells were subcutaneously injected into BALB / c nude mice to establish negative control (NC) and TXNIP gene knockout (ko-TXNIP) mouse melanoma subcutaneous tumor models.

[0142] When the tumor volume reached 50 cubic millimeters (7 days after the injection of drug-resistant cells), drug treatment was carried out. The mice were injected with Vem and DSF drugs once every two days for a total of 14 days (7 times), as follows:

[0143] Vem group: 30 mg / kg vemurafenib;

[0144] Vem+DSF group: 30mg / kg vemurafenib + 60mg / kg disulfiram (DSF);

[0145] Mice were sacrificed 14 days after receiving the indicated treatments, and tumor tissues were harvested, weighed, and photographed. Figure 8 H. Figure 8 I. Figure 8 As shown in J.

[0146] analyze Figure 8 HJ's results showed that in the nude mouse model, the combination of Vem+DSF failed to inhibit the growth of resistant cells with TXNIP knockout. In the BRAFi-resistant melanoma nude mouse model with TXNIP knockout, DSF combined with Vem could not inhibit the growth of its tumor. This further proved through in vivo animal experiments that TXNIP is the key molecule that induces the death of BRAFi-resistant cells by DSF combined with Vem.

[0147] Embodiment 9:

[0148] The TXNIP protein interaction network was constructed for the Vem group and the Vem+DSF group using the STRING database (http: / / www.string-db.org), and proteins interacting with TXNIP were obtained (DIA) for quantitative proteomics analysis. Thioredoxin (TXN / TRX) was identified as a key mediator (e.g. Fig. 9 A).

[0149] Co-immunoprecipitation (CO-IP) was used to analyze the interaction between TXNIP and TRX2 in BRAF-resistant cells in the Vem group and the Vem+DSF group. Fig. 9 (B) Results confirm the enhanced interaction of TXNIP with TRX2 after DSF stimulation.

[0150] BRAF-resistant cells were pretreated overnight (8 h) with 10 μM TXNIP-IN-1 (a recently developed inhibitor of TXNIP-TRX complex formation) to obtain the DSF+Vem+TXNIP-IN-1 group. The Vem group and the Vem+DSF group were compared. CCK-8 was used to detect the viability of BRAF-resistant cells. The results were as follows: Fig. 9 As shown in C;

[0151] TEM was used to analyze the mitochondrial morphology of BRAF-resistant cells, and the results were as follows: Fig. 9 As shown in D;

[0152] Immunofluorescence analysis of MMP levels in BRAF-resistant cells revealed the following results: Fig. 9 As shown in E;

[0153] The Seahorse assay was used to analyze the mitochondrial oxidative phosphorylation process in BRAF-resistant cells, and the results were as follows Fig. 9 As shown in F;

[0154] Flow cytometry was used to analyze the mt-ROS levels in BRAF-resistant cells, and the results were as follows: Fig. 9 As shown in G.

[0155] The analysis results show that:

[0156] The restorative effect of TXNIP-IN-1 on DSF-induced growth inhibition was confirmed by CCK-8 assay and clone formation. Pretreatment with TXNIP-IN-1 largely maintained the normal structure and function of mitochondria exposed to Vem+DSF. Importantly, TXNIP-IN-1 also protected BRAF-resistant cells from oxidative damage caused by Vem+DSF. These results suggest that TXNIP mediates mitochondrial oxidative damage by interacting with TRX 2.

[0157] In general, the conclusions of the above examples demonstrate that DSF exhibits a significant inhibitory effect on BRAFi-resistant melanoma cells and can significantly inhibit tumor growth in mice that have become resistant to BRAF inhibitors, indicating that the combination of DSF and BRAF inhibitors may be a promising strategy to overcome BRAFi resistance.

[0158] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the present invention. Although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the purpose and scope of the technical solutions of the present invention, which should be included in the scope of the claims of the present invention. The techniques, shapes, and structural parts not described in detail in the present invention are all known technologies.

Claims

1. Application of disulfiram in reducing the resistance of targeted drugs in BRAF mutant melanoma.

2. The use according to claim 1, characterized in that: The BRAF mutant melanoma targeted drug includes a BRAFi inhibitor.

3. The use according to claim 2, characterized in that: The BRAFi inhibitors include vemurafenib, dabrafenib, and Encorafenib.

4. The use according to claim 3, characterized in that: The BRAF mutant melanoma includes BRAF V600 E, BRAF V600K, BRAF V600R, and BRAF V600D mutant melanoma.

5. Application of disulfiram in the preparation of preparations for reducing the resistance of targeted drugs in BRAF mutant melanoma.

6. A preparation for reducing the drug resistance of BRAF mutant melanoma targeted drugs, characterized in that: The preparation raw materials include disulfiram.

7. The preparation according to claim 6, characterized in that The formulation is used in combination with a BRAFi inhibitor when reducing drug resistance of the BRAFi inhibitor, and the disulfiram reduces drug resistance of the BRAFi inhibitor.

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