Application of HSF1A in preparation of medicine for resisting lung cancer metastasis

Abstract

The invention discloses application of HSF1A in preparation of a medicine for resisting lung cancer metastasis, and relates to the technical field of anti-tumor medicines. It is found for the first time that HSF1A can be used for inhibiting non-small cell lung cancer metastasis. The HSF1A is specifically combined with a CCT1 subunit ATPase structural domain of a TRIC / CCT compound, so that the protein folding function of the TRIC / CCT compound is interfered, and three key transfer promoting signal channels including YAP, STAT3 and mTOR are inhibited at the same time. In a pulmonary metastatic tumor model constructed by mouse tail intravenous injection, the formation of pulmonary metastatic tumor is obviously reduced by the HSF1A, and the fact that the HSF1A also has strong anti-metastatic capability in an in-vivo environment is proved; the safety is high, and the development and utilization prospects are good. The invention not only provides a new application of the HSF1A, but also provides a new thought and method for adjuvant therapy of cancers.

Description

Technical Field

[0001] The present invention belongs to the technical field of anti-tumor medicine, and in particular relates to an application of HSF1A in the preparation of an anti-lung cancer metastasis drug. Background Art

[0002] Malignant tumor metastasis is the most serious challenge facing clinical treatment, accounting for approximately 90% of cancer-related deaths. Lung cancer, due to its highly invasive nature and propensity to metastasize, has a five-year survival rate of less than 20%. Despite significant progress in the clinical application of targeted therapies (such as EGFR-TKIs and ALK inhibitors) and immune checkpoint inhibitors (such as PD-1 / PD-L1 antibodies) in recent years, genomic instability, epigenetic heterogeneity, and microenvironmental adaptive resistance in tumor cells still lead to ultimate treatment failure in over 60% of patients. This situation urgently requires the development of new targeted drugs targeting key regulatory nodes of tumor metastasis.

[0003] Recent studies have revealed that the molecular chaperone system plays a pivotal regulatory role in the tumor metastasis cascade. Among them, TRiC / CCT (TCP-1 ring complex / chaperone protein containing TCP-1 subunits) is a class II molecular chaperone unique to eukaryotic cells. Its unique double-ring structure (two back-to-back octameric rings composed of CCT1-8 subunits) not only participates in the folding process of approximately 10% of intracellular proteins (including cytoskeletal proteins such as Actin and Tubulin), but also precisely regulates the functional activation of multiple key tumor proteins (such as STAT3 signaling protein, mTOR pathway regulatory protein Raptor and mLST8, etc.). Notably, the subunits of the TRiC / CCT complex are significantly overexpressed in a variety of malignant tumors (such as non-small cell lung cancer and triple-negative breast cancer) and are closely associated with poor patient prognosis. These characteristics make it a highly promising anti-tumor therapeutic target.

[0004] Current anti-tumor strategies targeting TRiC / CCT fall into two main categories: one, represented by peptide inhibitors such as CT20p, which bind to the apical domain of the CCT2 subunit with high affinity and competitively block substrate protein recognition and loading, leading to protein misfolding and accumulation, thereby activating the endoplasmic reticulum stress-induced apoptosis pathway. The major drawback of CT20p is its poor in vivo stability and the risk of immunogenicity. The other, represented by small molecule inhibitors such as HSF1A, specifically bind to the ATPase domain of CCT1 and disrupt the protein folding function of the TRiC / CCT complex by competitively inhibiting ATP hydrolysis. Although this compound has been shown to alleviate proteotoxic stress in neurodegenerative diseases by activating heat shock transcription factor 1 (HSF1) and exhibit significant cardioprotective effects, its therapeutic potential in oncology, particularly against metastasis, remains largely unexplored. Summary of the Invention

[0005] The present invention aims to provide an application of HSF1A in the preparation of an anti-lung cancer metastasis drug to address the above-mentioned deficiencies in the prior art.

[0006] To achieve the above objectives, the present invention provides the following technical solution: Use of the HSF1A and the pharmaceutically acceptable carrier of HSF1A described in the present invention in the preparation of an anti-tumor metastasis drug, wherein the structural formula of the HSF1A is as follows:

[0007]

[0008] Preferably, the drug is HSF1A or a preparation prepared by adding pharmaceutical excipients to HSF1A.

[0009] Furthermore, the pharmaceutical excipients include one or more of diluents, excipients, fillers, adhesives, wetting agents, disintegrants, absorption enhancers, surfactants, adsorption carriers, and lubricants.

[0010] Furthermore, the dosage form of the preparation includes tablets, capsules, oral liquids, injections or powder injections.

[0011] Preferably, the HSF1A inhibits the metastasis of lung cancer through the YAP signaling pathway, the STAT3 signaling pathway and the mTOR signaling pathway.

[0012] Furthermore, HSF1A inhibits the metastasis of lung cancer through the YAP signaling pathway by upregulating p-YAPS127.

[0013] Furthermore, the HSF1A inhibits the metastasis of lung cancer through the STAT3 signaling pathway by downregulating p-STAT3Y705.

[0014] Further, the HSF1A inhibits lung cancer metastasis through the mTOR signaling pathway, which is achieved by down-regulating p-4E-BP1T37 / 46 and p-S6K1T389.

[0015] Preferably, the lung cancer is non-small cell lung cancer.

[0016] Compared with the prior art, the beneficial effects of the present application are embodied in:

[0017] (1) HSF1A specifically binds to the CCT1 subunit ATPase domain of the TRiC / CCT complex, not only interfering with its protein folding function, but also inhibiting the YAP, STAT3 and mTOR three key pro-metastasis signaling pathways. HSF1A significantly up-regulates p-YAP S127, down-regulates p-STAT3 Y705 and the phosphorylation levels of p-4E-BP1 and p-S6K1, which are effect molecules of the mTOR pathway. Compared with the existing polypeptide inhibitor (such as CT20p) that only targets the specific binding of the CCT2 subunit to its specific substrate, HSF1A acts on the core ATP hydrolysis mechanism of TRiC / CCT, more comprehensively destroys the function of the complex, and avoids the compensatory activation that may be triggered by single subunit inhibition. It solves the problems of insufficient targeting specificity and unclear mechanism in the prior art.

[0018] (2) HSF1A as a small molecule compound overcomes the defects of low oral bioavailability and instability in vivo of polypeptide inhibitors, can be administered by intraperitoneal injection, and is compatible with long-term administration after optimization of the solvent system (DMSO / corn oil, <5% DMSO). According to the migration characteristics of different lung cancer cell lines (such as H1299, A549 and 889DTC), the optimal action concentration is 50-100 μM, which provides a new strategy for the development of anti-tumor metastasis drugs. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 is a molecular structure diagram of HSF1A;

[0020] Figure 2 is a Transwell experiment for verifying that HSF1A inhibits the migration of lung cancer cells H1299;

[0021] Figure 3 is a relative migration statistics of the Transwell experiment for verifying that HSF1A inhibits the migration of lung cancer cells H1299;

[0022] Figure 4 is a Transwell experiment for verifying that HSF1A inhibits the migration of lung cancer cells A549;

[0023] Figure 5To verify the relative migration statistics of Transwell assay in which HSF1A inhibits the migration of lung cancer A549 cells;

[0024] Figure 6 Transwell assay to verify that HSF1A inhibits the migration of lung cancer cells 889DTC;

[0025] Figure 7 To verify the relative migration statistics of Transwell assay in which HSF1A inhibits the migration of lung cancer cells 889DTC;

[0026] Figure 8 To identify the key signaling pathways by which HSF1A inhibits H1299 cell migration using immunoblotting technology;

[0027] Figure 9 To identify the key signaling pathways by which HSF1A inhibits A549 cell migration using immunoblotting technology;

[0028] Figure 10 To identify the key signaling pathway by which HSF1A inhibits 889DTC cell migration using immunoblotting technology;

[0029] Figure 11 This is a statistical chart of weight changes in mice with lung cancer metastasis model;

[0030] Figure 12 Fluorescence microscopy was used to detect lung metastasis in mice.

[0031] Figure 13 HE staining of lung tissue of mice with lung cancer metastasis;

[0032] Figure 14 Statistical chart of the number of lung metastases in mice. DETAILED DESCRIPTION

[0033] The following examples are only used to more clearly illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention.

[0034] The main compounds used in the examples and comparative examples were all commercially available products and were not subjected to any further purification treatment.

[0035] Human embryonic kidney cells HEK293T and human non-small cell lung cancer cells (H1299 and A549) were purchased from the Cell Bank of Type Culture Collection, Chinese Academy of Sciences; highly metastatic mouse lung adenocarcinoma cells 889DTC were donated by the Winslow Laboratory of Stanford University.

[0036] All cells were cultured in a constant temperature incubator at 37°C and 5% CO2. The specific culture medium is as follows:

[0037] ①HEK293T cells: cultured in DMEM medium containing 10% fetal bovine serum (FBS);

[0038] ②H1299, A549 and 889DTC cells: cultured in RPMI-1640 medium containing 10% FBS.

[0039] Example 1

[0040] Transwell assays were performed to verify the effect of HSF1A on the migration of different lung cancer cells, including the following steps:

[0041] HSF1A (MedChemExpress) was dissolved in DMSO to obtain solutions with concentrations of 50 mM and 100 mM, respectively. H1299, A549, and 889DTC lung cancer cells were cultured to a confluence of 80% and treated with 0 μM (same volume of DMSO), 50 μM, and 100 μM concentrations for 18 hours. 600 μL of serum-free medium was added to the lower chamber of a 24-well plate, and 100 μL of serum-free medium was added to the upper chamber of a Transwell (Corning, pore size 8 μm). The cells were placed in a 37°C, 5% CO2 incubator for 2 hours to eliminate the effects of membrane surface tension on cell migration. The drug-treated cells were digested with 0.05% trypsin, centrifuged at 800 g for 5 minutes, collected, washed twice with PBS to completely remove residual serum, and resuspended in serum-free medium to adjust the cell concentration to 4 × 10 5 cells / mL. Take 100μL of cell suspension and inoculate it into the upper chamber of Transwell. Add 600μL of culture medium containing 15% fetal bovine serum as chemoattractant to the lower chamber of 24-well plate. During operation, be careful to avoid bubbles between the liquids in the upper and lower chambers to ensure the consistency of migration direction. H1299 cells were cultured at 37℃ for 7h, A549 cells were cultured at 37℃ for 24h, and 889DTC cells were cultured at 37℃ for 18h. The number of cells that passed through the lower surface of the upper chamber was counted. The results of H1299 cell Transwell experiment are shown in the figure. Figure 2 As shown, the relative migration statistics are as follows Figure 3 As shown; A549 cell Transwell assay results are shown Figure 4 As shown, the relative migration statistics are as follows Figure 5 As shown; 889DTC cell Transwell assay results are shown Figure 6 As shown, the relative migration statistics are as follows Figure 7 shown.

[0042] The experimental results showed that HSF1A treatment significantly inhibited the migration of three lung cancer cell lines compared to the control group in a dose-dependent manner: the number of migrating cells in the 50μM HSF1A treatment group was 50±5.1% (H1299), 49.4.7±4.6% (A549), and 22±2.4% (889DTC) of the control group, respectively. The inhibitory effect was even more pronounced in the 100μM treatment group, with the number of migrating cells further reduced to 34.5±3.7% (H1299), 37.4±3.5% (A549), and 11±1.9% (889DTC) of the control group. The differences between the groups were statistically significant (P<0.0001). These results fully confirm that HSF1A can effectively inhibit the migration activity of lung cancer cells in vitro, and its inhibitory effect is enhanced in a concentration-dependent manner, providing an important experimental basis for subsequent studies of its anti-tumor metastasis effects.

[0043] Example 2

[0044] Immunoblotting technology was used to identify the inhibitory effect of HSF1A on key pro-metastatic signaling pathways in tumor cells, including the following steps:

[0045] H1299, A549, and 889DTC lung cancer cells were cultured to 80% confluence and treated with 0 μM (control group), 50 μM, and 100 μM HSF1A for 18 h, respectively. The cells were gently scraped with a cell scraper and centrifuged at 400 g for 5 min to collect the cell pellet. RIPA lysis buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) containing protease and phosphatase inhibitors was added and lysed on ice for 20 min, vortexing three times during the process. The cells were centrifuged at 16,000 g at 4°C for 15 min, and the supernatant was collected. The protein concentration was determined by BCA assay. 20-40 μg of protein was mixed with loading buffer and denatured at 95°C for 5-10 min. The protein was analyzed by SurePAGE. TM Precast gradient gel (Nanjing GenScript) was used, 15-30 μg of protein was loaded into each well, and electrophoresis was performed at 120V constant voltage for 90 min. The PVDF membrane was activated with methanol for 10 s, equilibrated with transfer buffer, and transferred at a constant current of 400 mA for 30 min. The membrane was rinsed once with TBST, blocked with 5% skim milk at room temperature for 30 min, incubated with primary antibody at 4°C overnight, incubated with HRP-labeled secondary antibody of the corresponding species at room temperature for 1 h, and washed three times with TBST, each for 10 min. The membrane was developed using an enhanced ECL chemiluminescence kit, and images were acquired using ImageLab software. The regulatory effects of HSF1A on phosphorylated YAP (S127), phosphorylated STAT3 (Y705), and downstream effector molecules of the mTOR pathway, phosphorylated 4E-BP1 (T37 / 46) and phosphorylated S6K1 (T389), were detected. The results are shown in Figure 3. Figure 8 (H1299 cells), Figure 9 (A549 cells) and Figure 10 (889DTC cells) are shown.

[0046] The experimental results show that in the three lung cancer cells, the regulation results of HSF1A on the three pathway proteins are consistent; in the YAP pathway, HSF1A dose-dependently up-regulates the phosphorylation of YAP(S127); in the STAT3 pathway, HSF1A significantly reduces the phosphorylation of STAT3(Y705); and in the mTOR pathway, HSF1A significantly inhibits the phosphorylation of 4E-BP1(T37 / 46) and S6K1(T389). These results suggest that HSF1A may inhibit the activation of multiple pro-metastatic signaling pathways by interfering with the function of TRiC / CCT to exert its biological effect of inhibiting tumor cell metastasis.

[0047] Example 3

[0048] The animal experiment confirmed the in vivo pharmacodynamic evaluation of HSF1A in inhibiting lung cancer metastasis, including the following steps:

[0049] In this study, the 889DTC cell line stably expressing tdTomato red fluorescent protein was used to establish a lung cancer metastasis model. Male BALB / c nude mice (6-8 weeks old, SPF level, body weight 18-22 g) were used for the experiment after adaptive feeding for 3 days in a constant temperature and humidity SPF level barrier system. 1x10 5 6tumor cells (200 μL sterile PBS suspension) were injected through the tail vein. The mice were closely observed for 30 min after inoculation to ensure no acute embolism reaction. The experimental animals were randomly divided into 3 groups (n=6 / group): control group (normal saline + 5% DMSO / corn oil solvent), low-dose group (37.5 mg / kg HSF1A), and high-dose group (75 mg / kg HSF1A). HSF1A was first dissolved with DMSO to obtain a 150 mg / mL stock solution, which was stored at -80°C in the dark, and the shelf life was not more than 6 months; an appropriate amount of the stock solution was diluted with medical-grade corn oil to the working concentration (7.5 mg / mL and 3.75 mg / mL), which was prepared immediately before use. The dosing regimen was as follows: the animals were injected intraperitoneally (10 mL / kg) at a fixed time (9:00-10:00 AM) every day for 6 consecutive days starting on the day of cell inoculation. The injection sites were alternately selected on the left and right lower abdomen to avoid local irritation. The body weight of the mice was measured every other day during the experiment to assess drug toxicity, and the results are shown in Figure 11

[0050] The animals were sacrificed 15 days after inoculation, and euthanasia was performed by CO2 asphyxiation followed by cervical dislocation. After the lungs were completely removed, they were first rinsed with PBS, and then fluorescent microscopy was performed to detect the red fluorescently labeled metastatic foci, and the results are shown in Figure 12 ​To further confirm the metastasis, lung tissue was fixed with 4% paraformaldehyde for 24 hours, then embedded in paraffin, serially sectioned (4 μm thickness) and stained with HE. Figure 13 As shown, the number of lung metastases in mice was counted, and the results were as follows Figure 14 shown.

[0051] Based on the fact that HSF1A can inhibit the migration of tumor cells in vitro and the activity of multiple intracellular signaling pathways, this example further systematically evaluated the in vivo anti-metastatic activity and safety of HSF1A by establishing a nude mouse metastasis model of 889DTC lung cancer cells stably expressing tdTomato red fluorescent protein. Figure 12 As shown in the table 1, the number of lung metastases in the HSF1A-treated group was significantly reduced in a dose-dependent manner compared with the control group. The number of metastatic foci was shown in the table 1 below. Only 2 of the 6 mice in the high-dose group had lung metastases, with an inhibition rate of 77.0%, P < 0.001. The HE staining results are shown in the table 1. Figure 13 As shown in Figure 3, the number of lung metastases in the HSF1A-treated group was significantly smaller than that in the control group, and the relationship was dose-dependent. Figure 11 The body weight fluctuations of mice in each group were less than 10%, and no obvious toxic reactions were observed. Figure 14 As shown, the number of lung metastases in the HSF1A-treated group was significantly lower than in the control group, and this was dose-dependent, with higher doses resulting in fewer metastatic tumors. These data confirm that HSF1A can effectively inhibit lung cancer metastasis in vivo while maintaining a good safety profile, providing important experimental evidence for the development of anti-metastasis drugs.

[0052] Table 1: Statistics of mouse lung cancer metastasis rate

[0053] Group Transfer rate Ctrl 100％(6 / 6) HSF1A-37.5mg / kg 83.3％(5 / 6) HSF1A-75mg / kg 33.3％(2 / 6)

[0054] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the technical principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A use of HSF1A in the preparation of an anti-lung cancer metastasis drug, characterized in that: The structural formula of HSF1A is shown below:

2. The use of HSF1A according to claim 1 in the preparation of an anti-lung cancer metastasis drug, characterized in that: The drug is HSF1A or a preparation prepared by adding pharmaceutical excipients to HSF1A.

3. The use of HSF1A in the preparation of an anti-lung cancer metastasis drug according to claim 2, characterized in that: The pharmaceutical excipients include one or more of diluents, excipients, fillers, adhesives, wetting agents, disintegrants, absorption promoters, surfactants, adsorption carriers, and lubricants.

4. The use of HSF1A in the preparation of an anti-lung cancer metastasis drug according to claim 2, characterized in that: The dosage forms of the preparation include tablets, capsules, oral liquids, injections or powder injections.

5. The use of HSF1A in the preparation of an anti-lung cancer metastasis drug according to claim 1, characterized in that: The HSF1A inhibits the metastasis of lung cancer through the YAP signaling pathway, STAT3 signaling pathway and mTOR signaling pathway.

6. The use of HSF1A in the preparation of an anti-lung cancer metastasis drug according to claim 5, characterized in that: HSF1A inhibits the metastasis of lung cancer through the YAP signaling pathway by upregulating p-YAP S127.

7. The use of HSF1A in the preparation of an anti-lung cancer metastasis drug according to claim 5, characterized in that: The HSF1A inhibits the metastasis of lung cancer through the STAT3 signaling pathway by downregulating p-STAT3Y705.

8. The use of HSF1A in the preparation of an anti-lung cancer metastasis drug according to claim 5, characterized in that: The HSF1A inhibits the metastasis of lung cancer through the mTOR signaling pathway by downregulating p-4E-BP1T37 / 46 and p-S6K1 T389.

9. The use of HSF1A in the preparation of an anti-lung cancer metastasis drug according to claim 1, characterized in that: The lung cancer is non-small cell lung cancer.

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