阅读说明：本技术 化合物ngsc12在药物制备中的应用 (Application of compound NGSC12 in preparation of medicines ) 是由 王学宝 叶发青 岳卫霞 郑楠 鲁颖 孙豆豆 韩怿农 于 2021-09-24 设计创作，主要内容包括：本发明公开了一种NGSC12在制备逆转肿瘤细胞耐药性药物中的应用,涉及肿瘤细胞耐药性治疗的技术领域。本发明所述NGSC12与紫杉醇联合用于制备逆转肿瘤紫杉醇耐药性的药物。研究发现,NGSC12能显著降低P-gp的表达上调和JAK2/STAT3通路的磷酸化,NGSC12与化疗药物紫杉醇联用能够显著提高耐药细胞对化疗药物的敏感性,从而逆转肿瘤耐药,大大提高疗效。这些结果表明NGSC12联用紫杉醇,能有效增强紫杉醇对耐受性肿瘤的治疗效果。临床发现许多恶性肿瘤细胞对紫杉醇具有耐药性,因此NGSC12有较好的开发价值和应用前景。(The invention discloses an application of NGSC12 in preparation of a drug for reversing drug resistance of tumor cells, and relates to the technical field of drug resistance treatment of tumor cells. The NGSC12 and paclitaxel are combined to prepare a drug for reversing the drug resistance of tumor paclitaxel. Researches show that NGSC12 can remarkably reduce the expression up-regulation of P-gp and the phosphorylation of JAK2/STAT3 pathway, and the combined use of NGSC12 and chemotherapeutic drug taxol can remarkably improve the sensitivity of drug-resistant cells to chemotherapeutic drugs, thereby reversing tumor drug resistance and greatly improving the curative effect. These results indicate that NGSC12 can effectively enhance the therapeutic effect of paclitaxel on resistant tumors when used in combination with paclitaxel. Clinical application shows that many malignant tumor cells have drug resistance to taxol, so NGSC12 has good development value and application prospect.)

1. Use of NGSC12 in the manufacture of a medicament for the treatment of cancer;

the tumor cells in the cancer are drug-resistant tumor cells;

the structural formula of the NGSC12 is as follows:

2. the use of NGSC12 in the preparation of a medicament according to claim 1, wherein said NGSC12 or medicament reverses drug resistance in drug resistant tumor cells by targeting the JAK2/STAT3 signal axis.

3. The use of NGSC12 in the manufacture of a medicament according to claim 1, wherein said NGSC12 or medicament is for inhibiting the expression of the drug-resistant protein P-gp in drug-resistant tumor cells.

4. The use of NGSC12 in the preparation of a medicament according to any one of claims 1 to 3, wherein the NGSC12 or the medicament is used for overcoming the drug resistance of tumor cells to paclitaxel.

5. The use of NGSC12 in the preparation of a medicament for reversing tumor cell resistance according to claim 4, wherein the tumors comprise all tumors undergoing chemotherapy with paclitaxel or paclitaxel prodrugs, such as lung cancer, breast cancer, pancreatic cancer, and gastric cancer.

6. The use of NGSC12 in the preparation of a medicament for reversing drug resistance in tumor cells according to claim 5, wherein said tumor is a breast cancer resistant to paclitaxel.

7. The use of NGSC12 in the preparation of a medicament for reversing drug resistance in tumor cells according to claim 4, wherein the concentration of NGSC12 is in the range of 0.1-25 μ M.

8. The pharmaceutical composition is characterized by comprising an active ingredient and a pharmaceutical excipient, wherein the active ingredient consists of NGSC12 and paclitaxel.

9. The pharmaceutical composition of claim 8, wherein the molar ratio of NGSC12 to paclitaxel is 10: 0.1-50.

10. The pharmaceutical composition of claim 8 or 9, wherein the NGSC12 is present in the composition at a concentration ranging from 300nM to 1300nM, and the paclitaxel is present in the composition at a concentration ranging from 100nM to 700 nM.

Technical Field

The invention relates to a new medical application of NGSC12, in particular to an application of NGSC12 in reversing drug resistance of tumor paclitaxel, belonging to the technical field of drug resistance treatment of tumor cells.

Background

Breast Cancer (BC) is one of the most common cancers, and epidemiological statistics show that by 2030, the global BC burden is expected to increase by 60%, with more than 220 million new cases, and more than 110 million death pathologies. Triple Negative Breast Cancer (TNBC) is the most aggressive histological subtype of breast cancer and is characterized by short response to chemotherapy, poor overall survival and the lack of effective targeted therapies, whereas non-targeted chemotherapy is the primary means of treatment. Current treatment strategies for breast cancer include surgical resection, radiation therapy, and especially chemotherapy. However, for triple negative breast cancer patients, chemotherapy and radiation therapy can cause severe side effects and reduce the quality of life of the patients, while long-term use of chemotherapeutic drugs and radiation therapy can result in patients with acquired resistance and resistance to radiation therapy. Paclitaxel is a diterpenoid compound isolated from the bark of yew, and can inhibit normal cell division by acting on microtubule/tubulin system, induce apoptosis, inhibit the progression of various tumors including triple negative breast cancer, and gradually become the first-line drug for treating patients with advanced triple negative breast cancer and unresectable breast cancer. However, many patients with triple negative breast cancer do not respond to initial chemotherapy with paclitaxel or acquired resistance and cross-resistance occur in subsequent treatments, resulting in poor prognosis and higher mortality in clinical patients, and therefore, there is an urgent need to develop new therapies, potentiators or adjuvant combination therapies for triple negative breast cancer to improve cure rate.

A plurality of researches show that the over-expression of P-glycoprotein (P-glycoprotein, P-gp) is one of the most common and important factors for generating drug resistance of tumor cells to paclitaxel, the P-gp is a unique ATP dependent membrane transporter, two P-gp genes exist in a human body, namely MDR1 and MDR2, the function of MDR2 is not determined, and the over-expression of MDR1 often causes the drug resistance of the tumor cells.

Niclosamide is an FDA approved anthelmintic that has been reused for the treatment of a variety of diseases, including viral and microbial infections, diabetes and parkinson's disease. Niclosamide can cause cytoplasmic acidification, which has potential role in down-regulating mammalian rapamycin complex 1(mTORC1) and STAT3 signaling pathways. Recent studies indicate that niclosamide can exert good anticancer activity in various human malignancies, for example, redox-sensitive nanoparticles based on xylan-lipoic acid conjugate can be used for targeted drug delivery of niclosamide to exert effect in ovarian cancer, a novel STAT3 inhibitor modified based on niclosamide can enhance anticancer effect of chemotherapeutic drugs on colorectal cancer, and niclosamide can exert anticancer activity by reactivating protein phosphatase tumor suppressor 2A (CDKN2A) in non-small cell lung cancer (NSCLC). In the early stage of the research, the pharmaceutical chemistry subject group analyzes and synthesizes a JAK2/STAT3 pathway inhibitor NGSC12 by performing structure optimization on niclosamide (which is suitable for resisting intestinal worms and is also a STAT3 inhibitor with better activity due to poor water solubility and low in vivo bioavailability), wherein the formula is as follows: tert-butyl4- (4-chloro-3-sulfamoylbenzoylamino) benzoate, its english-named: tert-butyl4- (4-chloro-3-sulfoarylbenzamido) benzoate.

Disclosure of Invention

The invention aims to find a new tumor treatment way targeting JAK2/STAT3, and provides application of NGSC12 in preparation of drugs for reversing drug resistance of tumor cells. NGSC12 reduces the expression of drug-resistant related protein P-gp (P glycoprotein) in drug-resistant tumor cells, and can reverse tumor drug resistance by combining with anticancer drugs, thereby greatly improving the curative effect.

The invention provides application of NGSC12 in preparation of a drug for reversing drug resistance of tumor cells, wherein NGSC12 and paclitaxel are combined for preparing the drug.

In particular, the NGSC12 is used for inhibiting the expression of a drug-resistant protein P-gp (P glycoprotein) in drug-resistant tumor cells.

Wherein the NGSC12 reverses resistance of tumor cells to paclitaxel.

Preferably, the tumor includes lung cancer, breast cancer, pancreatic cancer, gastric cancer, and the like.

The invention also provides a pharmaceutical composition, which comprises an active ingredient and a pharmaceutic adjuvant, wherein the active ingredient consists of NGSC12 and paclitaxel.

Preferably, the molar ratio of NGSC12 to paclitaxel is 10: 0.1-50.

Further preferably, the concentration range of the NGSC12 in the composition is 300-1300nM, and the concentration range of the paclitaxel in the composition is 100-700 nM; most preferably, the concentration of NGSC12 in the composition is 700nM and the concentration of paclitaxel in the composition is in the range of 250 nM.

Experiments on chemotherapy-resistant triple-negative breast cancer cells (the triple-negative breast cancer cells are MDA-MB-231 cells, and chemotherapeutic drugs are paclitaxel) show that NGSC12 can inhibit the in vivo and in vitro activity of MDA-MB-231/DR cells by inhibiting the expression up-regulation of P-gp and the phosphorylation of JAK2/STAT3 pathway, and reverse the paclitaxel resistance of MDA-MB-231/DR. NGSC12 is combined with paclitaxel to change the growth of chemotherapy-resistant triple-negative breast cancer cells by accelerating apoptosis, inhibiting cell proliferation, inhibiting cell invasion and migration and the like.

Drawings

FIG. 1 shows the establishment of drug resistant triple negative breast cancer cell lines, where ((A) the phosphorylation activation of JAK2/STAT3 signal pathway in parental cells and drug resistant cell lines by Western blotting analysis, (B) the quantitative analysis of P-JAK2/JAK2, (C) the quantitative analysis of P-STAT3/STAT3, and (D) the expression of P-gp in parental cells and drug resistant cell lines by Western blotting analysis.

FIG. 2 shows the effect of NGSC12 on the activation of JAK2/STAT3 and P-gp expression in MDA-MB-231/DR cells, wherein (A) the phosphorylation activation of JAK2/STAT3 signaling pathway in MDA-MB-231/DR cells after treatment with NGSC12 (0-4. mu.M) at different concentrations was analyzed by Western blotting, (B) the quantitative analysis of JAK2/STAT3 pathway-related proteins, (C) the expression of P-gp in MDA-MB-231/DR cells after treatment with NGSC12 (0-4. mu.M) at different concentrations was analyzed by Western blotting, and (D) the quantitative analysis of P-gp expression.

FIG. 3 is a graph of the effect of NGSC12 on MDA-MB-231/DR cell proliferation and apoptosis, where (A) the effect of NGSC12 administered alone or in combination on MDA-MB-231/DR cell proliferation is measured by MTT, (B) the effect of NGSC12 administered alone or in combination on MDA-MB-231/DR cell apoptosis is measured by flow cytometry, (C) the effect of NGSC12 administered alone or in combination on apoptosis and proliferation-related protein expression in MDA-MB-231/DR cells is measured by Western blotting, and (D) the quantitative analysis of apoptosis and proliferation-related protein expression in MDA-MB-231/DR cells.

Fig. 4 shows the effect of NGSC12 on MDA-MB-231/DR cell migration and invasion activity, where (a) cell invasion (panel X200) after NGSC12 treatment and combination was examined by Transwell, (B) cell migration (panel X200) after control, NGSC12 treatment and combination was examined by scratch assay, (C) cell migration and invasion-associated protein expression after control, NGSC12 treatment and combination was examined by western blotting.

Detailed Description

The experimental methods used in the following examples are conventional methods unless otherwise specified, and materials, reagents and the like used therein are commercially available without otherwise specified.

Example 1 establishment of a Taxol-resistant triple-negative breast cancer cell line

(1) Establishment of taxol-resistant triple-negative breast cancer cell line

MDA-MB-231, MDA-MB-436 and MDA-MB-468 cells were treated with increasing concentrations of paclitaxel (300nM to 5 μ M) for 8 months to establish drug resistant cells. Starting with 1/2 pairs of the IC50 concentrations of the parent MDA-MB-231, MDA-MB-436 and MDA-MB-468 cells for paclitaxel (approximately 100nM), 100nM paclitaxel was added to MDA-MB-231 cells and allowed to grow to 80% confluence. Then, the passage was performed periodically, and the concentration of paclitaxel was increased at each passage until the maximum concentration reached 5. mu.M.

(2) Detecting the effect of paclitaxel and NGSC12 with different concentrations on the proliferation of triple negative breast cancer cells/drug-resistant triple negative breast cancer cell lines

The half inhibitory concentrations of paclitaxel on MDA-MB-231/DR and MDA-MB-231/WT cells, MDA-MB-436/DR and MDA-MB-436/WT cells, and MDA-MB-468/DR and MDA-MB-4681/WT cells were determined using the MTT method (IC 50). Will be about 7X 10³The individual cells were seeded in 96-well plates and treated with paclitaxel (300 nM-6. mu.M) at different concentrations, all at 37 ℃ with 5% CO₂Incubate for 72h, then discard old culture medium, add MTT assay reagents, incubate at room temperature for 3h, then add 50 g/. mu.L of solubilization solution per well to determine activity. Use ofThe absorbance of the solution was measured at 490nm by the Discover microplate reader.

TABLE 1 IC of Taxol on triple negative breast cancer cells/drug resistant triple negative breast cancer cells₅₀

TABLE 2 IC of NGSC12 on triple negative breast cancer cells/drug resistant triple negative breast cancer cells₅₀

The experiment was repeated 3 times and the results were averaged ± standard deviation. The establishment of the triple-negative mammary cell strain after paclitaxel resistance is verified through MTT analysis, and the result shows that compared with the prior resistance, the sensitivity of the triple-negative mammary cell after paclitaxel resistance to paclitaxel is remarkably reduced, the IC50 is remarkably increased, and the calculation of the index of the drug-resistant cell shows that the drug-resistant index of MAD-MB-231 and MAD-MB-468 to paclitaxel is 13.2, and the drug-resistant index of MAD-MB-436 to paclitaxel is 13.8, and in addition, compared with the prior resistance, the IC50 of NGSC12 to parent cells is increased, but the difference is not remarkable.

Example 2 Western blot analysis of the effects of NGSC12 on paclitaxel-resistant MDA-MB-231 cell-associated protein expression

After different treatments, the cells were collected by digestion with trypsin without EDTA, washed twice with PBS and collected at 5X 10⁵Adding the cell into a lysis solution (PMSF, cocktai, TritonTMX100 and NaVO3 are added into a basic lysis solution), and then cracking for 35-40 min on ice; after lysis, the cells were centrifuged at 12000rpm at 4 ℃ for 15min and the protein concentration was measured using a Byunnan BCA protein concentration measurement kit, the protein samples were mixed with loading buffers and treated at 100 ℃ for 5min, 100. mu.g of total cellular protein was loaded to each well, and the protein was isolated at 25mA for 240 min. Soaking the PVDF membrane in methanol for 2min, opening a membrane transferring clamp, laying a wet sponge pad on a black surface representing a negative electrode, adding 2 layers of wet filter paper, carefully laying SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) glue on the filter paper, and covering the PVDF membrane with a proper size on the surface; then covering two layers of moist filter paper, then padding a moist spongy cushion, closing the red positive plate, clamping, inserting the red positive plate into a corresponding position of a film transferring groove, transferring the film at the temperature of 4 ℃ at 100V/100mA, sealing the film for 2 hours at the room temperature by adopting 5% skimmed milk powder and TBST after the film is transferred, and then reacting with a corresponding primary antibody: vimentin primary antibody (1:500), Snail primary antibody (1:500), E-cadherin primary antibody (1:2000), N-cadherin primary antibody (1:1000), P-gp primary antibody (1:500), Bax primary antibody (1:500), Bad primary antibody (1:200), C-myc primary antibody (1:500), Bcl-2 primary antibody (1:500), GAPDH primary antibody (1:500) were shaken overnight on a shaker, the overnight hybridized membrane was taken out, put into a membrane washing cassette containing a sufficient volume of TBST, washed on a shaker 10min at a time, 3 times continuously, incubated for 1h at room temperature with horseradish peroxidase-labeled immunoglobulin conjugate-rabbit anti-mouse IgG secondary antibody (1:500), the membrane was washed with TBST, laid flat on a dry plane, freshly prepared ECL Reagent was dropped onto a membrane surface with a pencil-labeled pencil, reacted for 5min, and then excess ECL Reagent was sucked off as much as possible at the edge of the membrane, the film is coatedThe signal was collected in a chemiluminescence apparatus, Image analysis was performed by Image and normalization of protein expression was performed with GAPDH expression as an internal control. The experiment was repeated 3-4 times.

Example 3 NGSC12 Combined with paclitaxel for MDA-MB-231/DR cell Combination Index (CI) analysis

The MTT method is used for detecting the effect of NGSC12 on cell proliferation inhibition by single use and combination with paclitaxel, and CompuSyn software is used for analysis and processing, wherein the CompuSyn software is drug combination effect software developed aiming at Chou-Talalay median pharmacodynamic method mathematical model, the given CI can reflect that the drug combination is additive effect, synergistic effect or antagonistic effect, and the method is the most approved method for quantitatively analyzing the drug synergistic effect at present. The CI values obtained by the five groups of combined administration with different concentrations in the experimental design are all less than 1, which indicates that the medicines have synergistic effect; where the second group had a paclitaxel concentration of 250nM and NGSC12 a concentration of 700nM with a minimum CI of 0.57, see Table 1 below for the concentrations used in the combinations referred to.

TABLE 1 Combination Index (CI) for paclitaxel and NGSC12 combinations

Note: CI <1 indicates synergy; CI ═ 1 indicates additive effects; CI >1 indicates antagonism.

Example 4 NGSC12 inhibits MDA-MB-231/DR cell proliferation and promotes its apoptotic Effect

After different treatments, the cells were collected by digestion with trypsin without EDTA, washed twice with PBS and collected at 5X 10⁵Adding 500 mu L of annexin V Binding Buffer into the cells, adding 5 mu L of annexin V-FITC, uniformly mixing, adding 5 mu L of LPropidium Iodide, uniformly mixing, reacting at room temperature in a dark place for 10min, and detecting the apoptosis condition by using a flow cytometer (Ex 488 nm; Em 530 nm).

The experiment is repeated for 3-4 times, and experimental data are analyzed by Flowjo 10 software. The results are shown in fig. 3, MDA-MB-231/DR is treated at a concentration (250nM) far lower than the paclitaxel IC50 value and the effect of the combination is good as a control group, and the MTT analysis verifies the effect of NGSC12 on the proliferation activity of MDA-MB-231/DR, and the results show that the proliferation activity of MDA-MB-231/DR in NGSC12 group is obviously inhibited and the apoptosis is obviously increased compared with the control group of MDA-MB-231/DR cells, and further the western blot analysis shows that the expression levels of cell proliferation related protein (Cmy-c) and anti-apoptosis related protein (Bcl-2) in NGSC12 group of cells are obviously reduced and the expression of apoptosis related protein (Bax, Bad) is obviously increased compared with the control group of MDA-MB-231/DR cells.

Compared with the NGSC12 group, after the cells are treated by the combination group (paclitaxel 250nM + NGSC 12700 nM, synergy group), the MDA-MB-231/DR proliferation activity is further inhibited, the apoptosis is obviously increased (figure 3A-3B), the expression level of cell proliferation related protein (Cmy-C) and anti-apoptosis related protein (Bcl-2) is obviously reduced, the expression level of apoptosis related protein (Bax, Bad) is obviously increased (figure 3C-3D), and the difference has statistical significance (P is less than 0.05).

Example 5 NGSC12 inhibits MDA-MB-231/DR cell invasion Activity

And (3) putting the matrix gel frozen and stored at the temperature of-20 ℃ in a refrigerator for melting. Diluting the matrigel and the serum-free culture solution according to the proportion of 1:5, uniformly mixing, and placing on ice for later use. In the sterile operation box, the Transwell chamber package is opened, the sterile operation is put into a 24-pore plate, 200 mu L of diluted matrigel solution is sucked and uniformly spread on the upper chamber surface of the chamber, the speed is low, the generation of bubbles is avoided, and the incubation is carried out in a cell culture box for 30 min. After the matrigel was solidified, 500. mu.L of culture medium containing 10% FBS as a chemotactic factor was added to the wells of the 24-well plate to induce cell membrane penetration. The cells after 24h of different treatments were washed twice with PBS, resuspended in serum-free medium and adjusted to a cell density of 1X 10⁵200 mu L of cell suspension is added into the upper chamber of the 24-hole culture plate, 600 mu L of complete culture solution is added into the lower chamber, and the culture plate is placed in a cell culture box for culturing for 36 hours. The chamber was removed, the culture medium in the chamber was aspirated, and the cells on the upper chamber were wiped off with a cotton swab. Care should be taken not to apply too much force to avoid damaging the bottom of the chamber. Moving the chamber to 100% methanol, fixing at room temperature for 20min, taking out the chamber, washing with PBS, staining with crystal violet solution for 5min, washing the chamber with PBS, and soaking in wet cotton rodThe cells on the membrane surface of the bottom of the upper chamber were wiped off by the heart, and after the chamber was completely air-dried, 5 fields were randomly selected, counted by an olympics inverted microscope, and photographed.

The experiment is repeated for 3-4 times, and experimental data are analyzed by Flowjo 10 software. As shown in FIG. 4, the effect of NGSC12 on the invasion activity of MDA-MB-231/DR was confirmed by Transwell experimental analysis, and the result showed that the invasion activity of MDA-MB-231/DR in NGSC12 group was significantly inhibited compared with that of the control group (FIG. 4A).

Example 6 NGSC12 inhibits MDA-MB-231/DR cell migration Activity

After 24h of different treatment, the cells were washed twice with PBS and resuspended in serum-free medium, the cell density was adjusted to 1X 10⁵About 5X 10 per well⁵After the cells are incubated overnight, after the cells adhere to the wall and grow full, a scratch is drawn by a microsyringe gun head perpendicular to the bottom of the 6-hole plate and the marking line, attention is paid to uniformity, and the width of the scratch is ensured to be consistent. The culture medium and the exfoliated cells were aspirated, washed with PBS, and cultured again after changing the medium. Respectively taking pictures 0h and 30h after the scratch, measuring the width of the scratch by using Photoshop software, and calculating the healing rate of the scratch by representing the migration capacity according to the following calculation method: (scratch width of 0 h-scratch width of 30 h)/scratch width of 0 h.

The influence of NGSC12 on MDA-MB-231/DR migration activity is verified by scratch experiment analysis, and the result shows that the migration activity of MDA-MB-231/DR in NGSC12 group is obviously inhibited compared with the cells in a control group (FIG. 4B), and further Western blot analysis shows that the expression level of cell invasion related proteins (N-cadherin, Vimentin, Snail) in the cells in NGSC12 group is obviously reduced and the expression of E-cadherin is obviously increased compared with the cells in MDA-MB-231/DR in the control group (FIG. 4C).

11 statistical analysis

The results of this study are expressed as mean ± Standard Deviation (SD) and were statistically analyzed using Graphpad 6.0 statistical software. Differences between the two groups were tested using the t test and comparisons of differences between groups were tested using one-way analysis of variance (ANOVA) and Dunnett's multiple comparisons. P values <0.05 were considered statistically significant.