CN111686248B - Medicine for treating cancer - Google Patents

Abstract

The invention discloses a medicine for treating cancer. The invention finds that the expression of PRMT1 is obviously increased in cancer tissues relative to normal tissues, is directly related to the malignancy degree of tumors and worse prognosis, and researches on the PRMT 1-mediated methylation of HSP70 and the influence and the fundamental mechanism of the methylation on the treatment effect of cancer cells are carried out. The invention finds that the overexpression of PRMT1 increases the arginine methylation of HSP 70. Its methylation protects cancer cells from apoptosis caused by various cellular stress and treatment by enhancing binding of AU-rich elements in HSP70 and BCL-2mRNA 3' -UTR and increasing BCL-2mRNA stability, and correspondingly increasing BCL-2 protein expression. The pharmacotherapeutic antibody and the small molecule inhibitor provided by the invention can block the arginine methylation site of the heat shock protein, and can reduce the survival capability of cancer cells and increase the treatment effect.

Description

Medicine for treating cancer

Technical Field

The invention relates to the technical field of medicaments, in particular to a medicament for treating cancer.

Background

In clinical practice, the inherent and acquired resistance or tolerance of malignancies to treatment modalities is a key challenge, resulting in existing treatment strategies with little impact on overall survival. Alterations in the balance of pro-apoptotic and anti-apoptotic signals within cancer cells are associated with therapeutic resistance, cancer progression and metastasis, which are major causes of cancer-related death.

70kDa heat shock protein (HSP70s) is an evolutionarily conserved family of proteins, ATP-dependent chaperones. HSP70 is encoded by the HSPA gene family, with 13 HSP70 family members in mammals. HSP70 is a major member of this family and plays a key role in promoting cell survival under a variety of stress conditions, including cytotoxic chemotherapy. HSP70 is a classical chaperone protein with highly conserved domains that facilitate protein folding. It is directly combined with several important proteins, and these proteins are involved in endogenous and exogenous apoptosis pathways, so that it can inhibit cell death and provide survival advantage for tumor cell. In addition to its typical function as a chaperone, HSP70 has RNA binding capability and can bind to and stabilize specific mRNA molecules containing AU-rich elements (AREs) in the 3 '-untranslated region (3' -UTR). HSP70 RNA binding activity is independent of its chaperone function.

The function of HSP70 is regulated by a variety of post-translational modifications, such as phosphorylation, acetylation, malonylation, ubiquitination, and methylation. For the first time, it was found that methylation of the HSP70 protein is the conserved K561 residue, and 3 methylation of this lysine residue alters the affinity for α -synuclein. Recently, it has been shown in the literature that CARM1/PRMT 4-mediated methylation of R469 affects the binding of HSP70 and TFIIH, and thus the activation of RAR beta 2 gene.

Disclosure of Invention

In order to overcome the defects in the prior art, the invention aims to provide a medicament for treating cancer.

The invention aims to provide a medicament for inhibiting the proliferation, invasion and migration of malignant tumors aiming at poor curative effect of the malignant tumors. The invention aims to provide a medicament containing an arginine methylated heat shock protein HSP70 antibody or a small molecule inhibitor aiming at the inherent or acquired resistance or tolerance of malignant tumor to a treatment mode, so as to inhibit the resistance or tolerance of malignant tumor to treatment and improve the curative effect.

The medicine provided by the invention can reduce the binding affinity of heat shock protein and BCL-2mRNA 3' -UTR, reduce the stability of BCL-2mRNA, reduce the amount of BCL-2 protein in tumor cells, improve the sensitivity of the tumor cells to chemotherapy or radiotherapy and ensure that the tumor cells are subjected to apoptosis.

The purpose of the invention is realized by at least one of the following technical solutions.

The invention provides a medicine for treating cancer, which comprises an antibody of arginine methylated heat shock protein HSP70, a polypeptide fragment of arginine methylated heat shock protein HSP70 or a small molecule inhibitor of arginine methylated heat shock protein HSP 70.

Further, the antibody of the arginine methylated heat shock protein HSP70 is an antibody blocking arginine methylated or unmethylated amino acid residues of the heat shock protein.

Further, the antibody of the arginine methylated heat shock protein HSP70 is an antibody blocking the 416 th and 447 th arginine residues of HSP 70.

Further, the small molecule inhibitor of the arginine methylated heat shock protein HSP70 is a small molecule inhibiting methylated or unmethylated arginine residues of heat shock proteins. The small molecule inhibitor is a natural or artificially designed small molecule

Further, the polypeptide fragment of the arginine methylated heat shock protein HSP70 is a polypeptide fragment containing the 416 th arginine residue of human HSP70, a polypeptide fragment containing the 447 th arginine residue of human HSP70 or a combination of the two.

The sequence of the polypeptide fragment of the 416 th arginine residue (R416) of the arginine methylated heat shock protein HSP70 is GGVMTALIKRNSTIPTKQTQ; the sequence of the polypeptide fragment of the 447 th arginine residue (R447) of the arginine methylated heat shock protein HSP70 is GVLIQVYEGERAMTKDNNLL. The combined polypeptide fragment has the sequence of GGVMTALIKRNSTIPTKQTQIFTTYSDNQPGVLIQVYEGERAMTKDNNLL.

The heat shock protein HSP70 is a substrate of protein arginine methyltransferase 1(PRMT1), and PRMT1 catalyzes the methylation of heat shock protein HSP70 arginine. The sites of the PRMT1 catalyzing the methylation of the arginine of the heat shock protein HSP70 are arginine residues (R416 and R447) at positions 416 and 447.

The medicine for treating cancer provided by the invention can inhibit methylation of arginine residues (R416 and R447) at 416 th and 447 th sites of the HSP70, reduce the binding affinity of the HSP70 and BCL-2mRNA 3' -UTR, reduce the stability of the BCL-2mRNA, inhibit the proliferation, migration and invasion of malignant tumor cells and promote the apoptosis of the malignant tumor cells. The medicine provided by the invention can be used for inhibiting the resistance or tolerance of tumors to chemotherapeutic drugs and improving the curative effect of tumor treatment methods. The tumor treatment method comprises a treatment method such as gemcitabine and the like and radiation treatment.

The medicine provided by the invention can treat malignant tumors such as pancreatic duct adenocarcinoma, gastric cancer and the like, and can also treat inflammatory diseases such as Crohn's disease, ulcerative colitis and the like.

The invention discovers for the first time that the combination of HSP70 and RNA is directly related to the treatment tolerance of cancer cells and depends on the methylation of protein arginine of HSP 70. The findings of the present invention further reveal that inhibition of the PRMT1-HSP70-BCL-2 signaling axis can decrease cancer cell viability and increase therapeutic efficacy. Thus, targeted inhibition of this signaling axis represents a new therapeutic strategy and protocol for malignancy.

Compared with the prior art, the invention has the following advantages and beneficial effects:

the drug provided by the invention has the advantages that the arginine methylation heat shock protein HSP70 antibody and the small molecule inhibitor are used for sealing the arginine methylation site of the heat shock protein, so that the apoptosis of malignant tumor cells is promoted, and the proliferation, migration and invasion of the malignant tumor cells are inhibited; secondly, the medicine provided by the invention comprises an arginine methylated heat shock protein HSP70 antibody and a small molecule inhibitor, can be combined with a tumor treatment method, improves the sensitivity of malignant tumor cells to chemotherapy and radiotherapy, and solves the resistance or tolerance of the malignant tumor cells to radiotherapy and chemotherapy.

Drawings

FIG. 1-A is a graph showing the results of volume change over time in a mouse pancreatic ductal adenocarcinoma (pancreatic cancer) transplant tumor model;

FIG. 1-B is a graph showing the results of mass change over time in a mouse model of pancreatic ductal adenocarcinoma (pancreatic cancer) transplantable tumor;

FIG. 1-C is a graph of the results of wound healing and invasion experiments with the knockdown of PRMT1 in MDA28 cells with high expression of protein arginine methyltransferase 1(PRMT1) and the overexpression of PRMT1 in PANC-1 cells with low expression of PRMT 1;

FIG. 1-D is a graph of the quantitative results of the calculated cell migration and invasion capacities of cells by means of Transwell experiments and counting, in which PRMT1 was knocked down in MDA28 cells highly expressed in protein arginine methyltransferase 1(PRMT 1);

FIG. 1-E is a graph of the quantitative results of the calculated cell migration and invasion capacities of cells overexpressing PRMT1 in PANC-1 cells with low expression of protein arginine methyltransferase 1(PRMT1) by Transwell experiments and counting;

FIG. 2-A is a graph showing the results of PRMT1 methylated HSP 70;

FIG. 2-B is a graph showing the results of verifying whether the arginine residue of HSP70 is a PRMT1 methylation site;

FIG. 3-A is a graph showing the effect of methylation of two arginine residues R416 and R447 of HSP70 on BCL-2mRNA expression levels;

FIG. 3-B is a graph showing the results of increased expression of BCL-2 by recombinant Wild Type (WT) in HSP 70-deficient cells;

FIG. 3-C is a graph of the results of HSP70 methylation affecting the stability of BCL-2 mRNA;

FIG. 3-D is a graph showing the results of a ribonucleoprotein immunoprecipitation (RNP-IP) experiment in HSP70 knock-out MiaPaCa-2 cells;

FIG. 3-E is a graph of the results of quantifying FIG. 3-D using imaging software;

FIG. 3-F is a graph showing the results of a ribonucleoprotein immunoprecipitation (RNP-IP) experiment in HSP70 knockout HEK-293 cells;

FIG. 3-G is a graph showing the results of direct binding of HSP70 to AREs of BCL-2 mRNA;

FIG. 4-A is a graph showing the results of methylation of HSP70 to protect pancreatic cancer cells against Etoposide;

FIG. 4-B is a graph showing that methylation of HSP70 protects pancreatic cancer cells against oxidative stress (H)₂O₂) A graph of results of (1);

FIG. 4-C is a graph showing the results of HSP70 methylation in counterintuitive deficiency in pancreatic cancer cells;

FIG. 4-D is a graph of the results of wild-type HSP70 protecting cells from apoptosis, while hypomethylated HSP70 did not protect against stress-induced apoptosis;

FIG. 4-E is a graph showing the results of wild-type HSP70 inhibiting apoptosis;

FIG. 5-A is a graph of the results of experiments in which arginine methylation at specific sites affected HSP70 function in gemcitabine resistance;

FIG. 5-B is a graph of cell growth curves treated with different concentrations of gemcitabine after transfection of wild type HSP70 with a MiaPaCa-2 cell clone (KO-16) of a wild type HSP70 Knockout (KO);

FIG. 5-C is a plot of cell growth curves treated with different concentrations of gemcitabine after transfection of wild type HSP70 with a MiaPaCa-2 cell clone of wild type HSP70 Knockout (KO);

FIG. 5-D is a graph showing the results of over-expression of PRMT1 in FG cells;

FIG. 5-E is a graph showing the results of siRNA knockdown of PRMT1 in FG cells;

FIG. 5-F is a graph of the results of treatment of MiaPaCa-2 cells with DB75(5 μm), gemcitabine (20 μm), or a combination of the two for 48 hours;

FIG. 6-A is a graph showing the effect of resistance of a polypeptide fragment comprising human HSP70 arginine residue 416, 447 or a combination thereof to MiaPaCa-2 cells;

FIG. 6-B is a graph showing the effect of antibodies specifically blocking the 416 th and 447 th arginine residues of human HSP70 on the resistance of MiaPaCa-2 cells.

Detailed Description

The following examples are presented to further illustrate the practice of the invention, but the practice and protection of the invention is not limited thereto. It is noted that the processes described below, if not specifically described in detail, are all realizable or understandable by those skilled in the art with reference to the prior art. The reagents or apparatus used are not indicated to the manufacturer, and are considered to be conventional products available by commercial purchase.

All human pancreatic adenocarcinoma cell lines, HPNE cells and HEK293T cells were purchased from American Type Culture Collection, unless otherwise specified. MDA28 and MDA48 pancreatic adenocarcinoma cell lines were donated by professor pyrograph of MD anderson cancer center, texas university, usa. The generation of FG human pancreatic adenocarcinoma cell lines has been reported (Vezeridis et al, PMID 2296181). All cell lines were incubated at 37 ℃ in 5% CO₂The single-layer adherent is added with 10% fetal calf serum, sodium pyruvate, non-essential amino acid, l-glutamine, penicillin/streptomycin and vitamin solution.

anti-PRMT 1(2449S) antibody was purchased from Cell Signaling Technology; anti-PRMT 5(07-405) antibody was purchased from EMD Millipore; anti-HA (TA100012) and anti-Myc (TA150121) antibodies were purchased from OriGene Technologies. anti-HSP 70 antibody (ADI-SPA-810) was purchased from enzor biosciences. Anti-HSC70(sc-7298), Anti-GRP78(sc-376768), Anti-GRP75(sc-13967), Anti-GST (sc-138), Anti- β -actin (sc-47778) and Anti-BCL-2(sc-7382) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz Biotechnology). Anti- α -tubulin (MABT205) and Anti-asymmetric dimethylarginine antibody, ASYM24, were purchased from EMD Millipore Corporation. Anti-asymmetric demethylation of histone H4R3 antibody was purchased from Active Motif. Specific targeting siRNA from SANTA Cruz or siRNA of PRMT1 (SiPRMT1) sequence 5'-GCCAACAAGUUAGACCACG-3', synthesized by Sigma-Aldrich; the siRNA (SiHSP70) sequence of HSP70 is 5'-CGGUGGUGCAGUCGGACAUGA-3'. Real-time PCR and reverse transcription PCR primers are shown in Table 1. PRMT 1-specific inhibitors DB75 and TC-E5003 were purchased from Tocris Bioscience, Inc. Etoposide and H₂O₂Purchased from Sigma-Aldrich. Gemcitabine hydrochloride was purchased from United States Pharmacopeia (USP).

PRMT1 transcript variant 1 plasmid (over-expression PRMT1 plasmid) was purchased from OriGene Technologies and the ORF was subcloned into the backbone of the pCMV6-AC-HA-His vector and carries a C-terminal HA-His tag. HSPA1A (HSP70), HSPA5(GRP-78), HSPA8(HSC70), HSPA9(GRP-75) plasmids were purchased from OriGene Technologies, and the backbone vector was pCMV6-Entry with a C-terminal Myc-DDK tag. HSP70 and PRMT1 deletion mutant plasmids were generated by Herculase II fusion enzyme (Agilent Technologies) PCR and inserted into either the original vector or pGEX-KG vector for bacterial expression. Arginine-alanine (R-A) mutant HSP70 plasmids including 416, 447, 458 single R-A mutants, 416, 447 double R-A (2RA) and 3R-A (3RA) plasmids. The GFP-PRMT1 and GST-PRMT1 plasmids were from the American Md Anderson cancer center, McMark, Bedford, Dr.

Female athymic nude mice and C57BL/6 mice were purchased from Jackson laboratories. Mice were used at 8 weeks of age. All animals were housed in a university animal facility approved by the state-of-the-art laboratory animal care assessment and certification agency under current regulations and standards.

The first embodiment is as follows: mouse pancreatic ductal adenocarcinoma (pancreatic cancer) transplantable tumor experiments.

In xenograft tumor model, mouse pancreatic ductal adenocarcinoma (pancreatic cancer) cells (1 × 10)⁶) Resuspended in 0.1mL of Hank's buffer without calcium and magnesium and injected subcutaneously into the flanks of nude mice. The length and width of the tumor were measured twice weekly with calipers. The PRMT1 inhibitor DB75 was injected i.p. into mice twice a week for 3 weeks. The low dose is 5mg/kg and the high dose is 20 mg/kg. The vehicle solution was used as a control. Tumor-bearing mice were euthanized at designated time points either near death or post-inoculation, and tumors were excised and weighed. Swelling and swelling treating medicineTumor volume (mm)³) The calculation formula is that the short diameter is 2 multiplied by the long diameter/2.

The experimental results are shown in FIG. 1-A and FIG. 1-B. FIG. 1-A, which depicts a mouse pancreatic ductal adenocarcinoma (pancreatic cancer) transplant tumor model, mice receiving low (5mg/kg) and high (20mg/kg) protein arginine methyltransferase inhibitor DB75 treatment twice weekly, after 21 days experiment, tumor volume changes, Ctrl in FIG. 1-A representing a control group; low indicates Low dose group; high indicates the High dose group. FIG. 1-B, which depicts a mouse pancreatic ductal adenocarcinoma (pancreatic cancer) transplant tumor model, mice transplanted tumor weight twice weekly after 21 days experiment completion receiving low (5mg/kg) and high (20mg/kg) protein arginine methyltransferase inhibitor DB75 treatment. The measurement results of the morphology, the tumor volume and the tumor weight of the transplanted tumor show that the PRMT1 inhibitor DB75 can inhibit the growth of the tumor of a mouse, and the inhibition effect is positively correlated with the DB75 concentration.

Example two: migration and invasion experiments of human pancreatic ductal carcinoma cells.

Day before transfection, 5X 10⁵MDA28 and PANC-1 cells were seeded in 6-well plates in 1.5mL of cell culture medium containing 10% FBS. The number of cells used for initial seeding was chosen so that cell confluence reached 70-90% within 24 hours. Add 100pmol siRNA (or 2. mu.g DNA) to 250. mu.l Opti-MEM and mix gently; mix lipofectamin2000 reagent well, dilute 5 μ l lipofectamin2000 with 250 μ l Opti-MEM, mix gently, stand 5 minutes at room temperature; mixing the diluted siRNA \ DNA and lipofectamin 2000; gently mix and leave at room temperature for 20 minutes to allow formation of siRNA/lipofectamin (or DNA/lipofectamin) complexes. Add 500. mu.l siRNA/lipofectamine (or DNA/lipofectamine) complexes to the wells of the corresponding plates containing cells and medium and gently shake the plate back and forth. Transferring SiPRMT1 into MDA28 cells, using siCtrl as a control, transferring pHA-PRMT1 into PANC-1 cells, using pcDNA3.1 as a control, performing cell scratching the cells the next day after transfection, washing the cells with PBS 3 times, adding 1ml of cell culture medium (10% fetal bovine serum plus 90% DMEM culture medium plus 1% penicillin streptomycin); the cells were cultured at 37 ℃ in 5% carbon dioxide for 12 hours and photographed. Scratch widths were measured at 0 and 12 hours。

30ug of Martrigel gel was applied to the Transwell chamber and 20 million of each of the above transfected MDA28 cells and PANC-1 cells were added and placed in culture; after culturing for 48 hours under the condition of 37 ℃ and 5% carbon dioxide, the filter membrane is fixed by ethanol, hematoxylin and eosin are stained, and the number of cells passing through Martrigel is counted by photographing and observing. The magnification is 200 times.

The results are shown in FIGS. 1-C, 1-D and 1-E. FIG. 1-C depicts the knockdown of PRMT1 in MDA28 cells with high expression of protein arginine methyltransferase 1(PRMT1), the overexpression of PRMT1 in PANC-1 cells with low expression of PRMT1, and the ability of cell invasion observed by wound healing and invasion experiments. FIG. 1-D depicts the quantitative results of the calculated ability of cells to migrate and invade by means of Transwell experiments and counting the knockdown of PRMT1 in MDA28 cells highly expressed in protein arginine methyltransferase 1(PRMT 1). FIGS. 1-E depict the quantification of the ability of cells to migrate and invade calculated by Transwell experiments and counting of overexpression of PRMT1 in PANC-1 cells with low expression of the protein arginine methyltransferase 1(PRMT 1). After the expression of PRMT1 is knocked down by siRNA, the migration and invasion capacity of human pancreatic ductal carcinoma cell MDA28 with high expression of PRMT1 is inhibited; after PRMT1 is over-expressed in human pancreatic ductal carcinoma cells PANC-1 with low expression of PRMT1, the migration and invasion capabilities of the cells are enhanced, which indicates that PRMT1 has the capability of promoting the invasion and migration of tumor cells.

Example three: immunoprecipitation experiments

Using the transfection method described in example two, the PRMT overexpression plasmid was transfected in mouse pancreatic ductal carcinoma cells, pcDNA3.1 as a negative control, and 36 hours after transfection, the culture was decanted and the flask was inverted over absorbent paper to blot the absorbent paper dry of the culture. 1ml of 4 ℃ pre-cooled PBS (0.01M pH7.2-7.3) was added to each well of cells. Cells were lysed with the addition of protease inhibitor Pierce IP lysis buffer (Thermo Fisher Scientific). The cell lysates were incubated with HSP70 antibody overnight (12 hours) on a rotator at 4 ℃. Protein A/G + agarose (Santa Cruz) was then added and incubated for a further 2 hours at 4 ℃. Followed by 4 washes with PBST plus protease inhibitor, followed by 2 XSDS-PAGEBoiling for 5 minutes, the protein complex is released from the agarose. The protein concentration of whole cell lysates was determined by Coomassie blue emission, adjusted to the same protein concentration for each sample and loaded uniformly, and standard Western blotting procedures were performed to detect methylated HSP70 protein using ASYM24 antibody and total HSP70 protein using HSP70 antibody. Use of Pierce^TMThe Enhanced Chemiluminescence (ECL) system detects protein bands. Quantifying the Western blot result by ImageJ software; the expression level of beta-Actin is standardized. Under a single blot, its ratio to internal control is expressed as a fold change in its expression.

The results are shown in FIG. 2-A. FIG. 2-A depicts that PRMT1 can methylate HSP 70. The methylation level of HSP70 was detected with an antibody specific for methylated arginine (ASYM24), and when PRMT1 was overexpressed in pancreatic cancer cells, the result showed that the methylation level of HSP70 was increased. By immunoprecipitation experiments, it was first demonstrated that PRMT1 and HSP70 are able to form a complex, and further that the level of methylation of HSP70 in the PRMT1/HSP70 complex is elevated. Indicating that PRMT1 is able to catalyze methylation of HSP 70.

Example four: in vitro methylation experiments.

Peptide fragments of R416, R447 and R458 of 19 amino acids in length and corresponding methylation site mutant peptides R416A, R447A and R458A were synthesized for methylation experiments in vitro. A mature peptide containing three GAR repeats (R3) was used as a positive control. The synthetic peptide or recombinant protein was mixed with 2. mu.g of PRMT1 recombinant protein (Origene Technologies), 2. mu. Ci³H-labeled S-adenosylmethionine (SAM) (Perkin Elmer) was added to 20. mu.l of methylation buffer (50mM Tris. HCl, pH8.0,150mM NaCl,1mM EDTA) for incubation, and the reaction was incubated at 30 ℃ for 3H. The reaction was measured by Liquid Scintillation Counting (LSC). A small circular nitrocellulose membrane was dropped on 10. mu.l of the mixture, rinsed three times with methylation buffer, then the membrane was air dried, immersed in ScintiVerse II Cocktail (Thermo Fisher Scientific), placed in a glass scintillation vial, and measured with a liquid scintillation counter (Beckman Coulter). Decomposition Per Minute (DPM) of each peptide was calculated as final DPM ═ DPM (wild type) -DPM (mutant).

The results are shown in FIG. 2-B. FIG. 2-B depicts the arginine residue of HSP70 as a PRMT1 methylation site. The invention synthesizes peptide segments of R416, R447 and R458 with the length of 19 amino acids and corresponding methylation site mutation peptides of R416A, R447A and R458A, and the peptide segments are used for in vitro methylation experiments. A mature peptide containing three GAR repeats (R3) was used as a positive control. When 3H-labeled S-adenosylmethionine (SAM) is added as a methyl donor, PRMT1 can methylate the R416 and R447 sites instead of the R458 site.

Example five: structural analysis of PRMT1/HSP70 complex protein

Since the crystal structure of human PRMT1 does not exist at present, the invention firstly establishes a homologous model of human PRMT1 and methyl donor SAM in a complex based on the crystal structure of rat PRMT1, and the model has high sequence consistency with human PRMT 1. The initial structure of HSP70 peptides comprising R416, R447 and R458 was taken from the PDB structure 4PO 2. Flexible protein peptide docking was performed using Z-dock and Cluspro2.0 webserver, and the optimal pose was retained for further study. The target arginine residues R416 and R447 are apparently located deep in the pocket formed by PRMT1 protein Glu152, Tyr156, Glu161 residues and the ligand SAM, between chain β 4 and helix α D. The guanidine groups of the target arginines R416 and R447 form salt bridges with E152 and E161, respectively. In addition, the aromatic group of Tyr156 is parallel to the methylene group of the target arginine. These interactions contribute to the stable binding of the targeted arginine residue to the enzyme site, which is consistent with the results of the in vitro methylation experiments of the present invention, suggesting that both sites are methylated by PRMT 1.

Example six: the regulation of BCL-2 expression by methylation of arginine sites at R416 and R447 sites of HSP70 was demonstrated.

siHSP70, pHSP70Mut and pHSP70WT were transferred into MDA28 cells by the transfection method described in example two, siCtrl and pCDAN3.1 were used as controls, and then culture was continued for 36-48 hours, and according to the standard sampling and Western Blotting method described in example three, HSP70 antibody was used to detect the expression level of HSP70 protein, BCL-2 antibody was used to detect the expression level of BCL-2 protein, and β -Actin was used as an internal reference.

pHSP70Mut and pHSP70WT were transferred into HSP70 knock-out cell clones KO-16, KO-19, KO-31 and KO-37 cells, respectively, by the transfection method described in example two, pCDAN3.1 was used as a control, and then treated with gemcitabine (20. mu.M) for further culturing for 36 to 48 hours, and HSP70 protein expression level was measured using HSP70 antibody, BCL-2 protein expression level was measured using BCL-2 antibody, and Tubulin was used as an internal reference, according to the standard sampling and Western Blotting method described in example three.

The results are shown in FIG. 3-A and FIG. 3-B. FIG. 3-A depicts the effect of methylation of two arginine residues R416 and R447 of HSP70 on BCL-2mRNA expression levels. FIG. 3-B further depicts that recombinant Wild Type (WT), but not R416A and R447A double mutant HSP70(Mut), increased BCL-2 expression in HSP 70-deficient cells. HSP70 effect of methylation of two arginine residues R416 and R447 on BCL-2mRNA expression levels. First, by altering HSP70 expression in MDA28 cells, it was found that down-regulation of HSP70 by siHSP70 significantly reduced BCL-2 expression, while wild-type HSP70(WT) increased BCL-2 protein levels (fig. 3-a). Further, recombinant Wild Type (WT), but not R416A and R447A double mutant HSP70(Mut), increased BCL-2 expression in HSP 70-deficient cells (fig. 3-B).

Example seven: mRNA half-life experiments.

By the transfection method described in example two, the wild-type HSP70 expression vector HSP70WT and the two HSP70 arginine residues R416A and R447A mutant expression vectors were transferred into mouse pancreatic cancer cells, cultured for 36 hours, and treated with actinomycin-d (ActD) for 4 hours to avoid initiating the ActD-based apoptosis program. Total RNA was extracted using RNeasy Mini Kit (QIAGEN) at 0, 0.5, 1, 2, 3 and 4 hours after ActD treatment. The above RNA samples were analyzed for BCL-2 and GAPDH mRNA expression using the multiplex qRT-PCR method described above, with an average of three replicates for each data point. + -. SD. Transcript levels were normalized to GAPDH levels. Nonlinear regression was performed using GraphPad Prism6 software to calculate the first order decay constant (k) and corresponding mRNA half-life to give ln2/k mRNA half-life (t 1/2).

The results are shown in FIG. 3-C. FIG. 3-C depicts whether HSP70 methylation affects the stability of BCL-2 mRNA. BCL-2mRNA decayed slowly in wild-type HSP70 transfected cells with a half-life of 5.07 ± 0.36 hours, whereas BCL-2 transcripts decayed faster in mutant HSP70 transfected cells (t1/2 ═ 3.02 ± 0.29 hours). Indicating that HSP70 methylation affects the stability of BCL-2 mRNA.

Example eight: ribonucleoprotein immunoprecipitation (RNP-RIP).

Wild-type and mutant Myc-tagged HSP70 plasmids and Myc control plasmids were transfected into HSP70 knockout Mia PaCa-2 cells, respectively, by the transfection methods described in example seven. After 36 hours of incubation, ribonucleoprotein complexes comprising Myc-HSP70 were purified from cell lysates using anti-Myc antibody-conjugated magnetic beads. The magnetic beads were washed with lysis buffer to which an RNase inhibitor was added, and then RNA molecules in the complex were extracted using RNeasy Mini Kit (QIAGEN). Using SuperScript^TMIV First-Strand Synthesis System (Invitrogen), mRNA was reverse transcribed into cDNA. The PCR product was amplified by 30 cycles of PCR reaction, separated on 2% agarose gel, stained by EB, and photographed under UV light. BCL-2, VEGFA and GAPDH are detected in a sample as internal references, the sequences of primer pairs are shown in a table 1, and the sequences of peptide fragments are shown in a table 2.

TABLE 1 primers

Primer name	Sequence (5 '-3')
		BCL-2 3′-UTR primer 1forward	CTGGTGGGAGCTTGCATCAC
BCL-2 3′-UTR primer 1reverse	TCTTAAACAGCCTGCAGCTTTG
		BCL-2 3′-UTR primer 2forward	CGCAGAACCTGCCTGTGTCC
BCL-2 3′-UTR primer 2reverse	CATTGCCTCTCCTCACGTTCC
		VEGFA
3′-UTR primer forward	TCTACCTCCACCATGCCAAGTG
		VEGFA
3′-UTR primer reverse	GGGTCTCGATTGGATGGCAG
		GAPDH mRNA primer forward	CTCTGCTCCTCCTGTTCGACAG
GSPDH mRNA primer reverse	CAATACGACCAAATCCGTTGACT

TABLE 2 peptide fragments

The results are shown in FIG. 3-D, FIG. 3-E and FIG. 3-F. FIG. 3-D depicts ribonucleoprotein immunoprecipitation (RNP-IP) experiments in HSP70 knock-out MiaPaCa-2 cells. FIG. 3-E depicts the results of quantifying FIG. 3-D using imaging software. FIG. 3-F depicts the results of ribonucleoprotein immunoprecipitation (RNP-IP) experiments in HSP70 knock-out HEK-293 cells. Wherein lanes 2, 6, 10 of FIG. 3D show BCL-2 primer pair 1 product, lanes 3, 11 show BCL-2 primer pair 2 product, lanes 4, 8, 12 show VEGFA primer pair product, and lanes 5, 9, 13 show GAPDH primer pair product. The first lane is a100 bp DNA ladder. Wild-type HSP70 binds more BCL-2mRNA than mutant HSP70, given the same amount of mRNA and protein input. VEGFA mRNA is also more abundant in wild type than mutant HSP70, where VEGFA mRNA serves as a positive control because HSP70 has been previously reported to bind VEGFA mRNA.

Example nine: electrophoretic migration transfer experiment

To describe the direct binding of HSP70 to AREs of BCL-2mRNA, electrophoretic migration transfer experiments were performed using purified wild-type and mutant HSP70 proteins and biotin-labeled RNA probes.

Specifically, the binding of Myc-Hsp70 protein to an RNA substrate was qualitatively assessed using the LightShiftTM chemiluminescent RNA EMSA kit (Thermo Fisher Scientific) according to the manufacturer's instructions. Briefly, biotin-labeled BCL-23 ' -UTR RNA fragments or UA-enriched positive control fragments (5'-gugauuauuuauuauuuauuuauuauuuauuuauuuag-3') were incubated with recombinant proteins in binding buffer at 25 ℃ for 30 minutes. The reaction was then subjected to 5% polyacrylamide gel electrophoresis and then transferred to a positively charged nylon membrane. After cross-linking the RNA to the membrane, biotin-labeled RNA was detected by streptavidin-horseradish peroxidase (HRP) coupling and chemiluminescence.

The results are shown in FIGS. 3-G. FIG. 3-G depicts the direct binding of HSP70 to AREs of BCL-2 mRNA. Wild type HSP70 has higher binding affinity to the ARE-containing RNA probe than mutant HSP 70.

Example ten: effect of drug concentration on cell survival.

Gene-specific knockout cell lines were generated using the Crisp/cas9 system. The first 100bp coding sequence of the human HSPA1A/B gene was analyzed by using an online CRISPR design tool (http:// CRISPR. mit. edu /) for leader design. The targeting sequence of HSPA1A/B is 5'-atggccaaagccgcggcgat-3' in the forward direction and 5'-atcgccgcggctttggccat-3' in the reverse direction. Random leader sequences not directed against any known human genes were used as controls, forward 5'-ccgccccgagttcaaggtggagcg-3', reverse 5'-cgctccaccttgaactcggggcgg-3'. The forward and reverse oligonucleotides were annealed to each other, inserted into the vector pSpCas9(BB) -2A-Puro (Addgene #62988), and digested with BbsI restriction enzyme. The recombinant plasmid was transfected into MIAPaCa-2 cells, and the cells were treated with puromycin (1. mu.g/ml) for 48-72 hours. After puromycin selection, single cell clones were seeded onto 96-well plates using flow cytometry. After culturing for 10-14 days, specific gene deletion of each cell was detected by Western blot and genomic DNA sequencing. Two HSP70 knock-out cell clones (KO-16 and KO-20) were obtained.

Using the cell transfection method of example two, pcDNA3.1, SHP70 wild-type overexpression vectors pHSP70WT, R416A and R447A double mutant HSP70 overexpression vector pHSP70Mut were transformed into HSP70 knock-out cell clones KO-16 and KO-20, respectively. Cells were seeded in 96-well plates at 3000 cells per well 36 hours after transfection. Different dilution gradients of Etoposide or H₂O₂Addition to DMEM medium or glucose deprivation, 2% FBS addition, 48 hours of incubation followed by Cell Counting Kit-8(CCK-8, Dojindo Molecular Technologies) to determine Cell viability and IC50 for each drug was calculated using Prism GraphPad 6 software. The viability of the control group was determined as 100% and the concentration of each drug was determined 3 times.

The results are shown in FIG. 4-A, FIG. 4-B and FIG. 4-C. FIG. 4-A depicts HSP70 methylation conferring resistance to Etoposide on pancreatic cancer cells. FIG. 4-B depicts HSP70 methylation conferring resistance to oxidative stress (H) in pancreatic cancer cells₂O₂). FIG. 4-C depicts HSP70 methylation enables pancreatic cancer cells to counteract auxotrophy (glucose deprivation). Wild-type HSP70 has a significantly better protective function against various survival stresses than mutant HSP70

Example eleven: flow cytometry.

Using the ten cells of example, pcDNA3.1, SHP70 wild-type overexpression vectors pHSP70WT, R416A and R447A double mutant HSP70 overexpression vector pHSP70Mut were transferred into HSP70 knock-out cell clones KO-16 and KO-20, respectively, the cells were treated with 20. mu.M gemcitabine, the cells transfected with pcDNA3.1 were treated with solvent as a blank, and the cells were treated for 24 hours, and then apoptotic cells were analyzed by Annexin v/PI staining and flow cytometry.

The results are shown in FIG. 4-D. Fig. 4-D depicts that wild-type HSP70 protects cells from apoptosis, while hypomethylated HSP70 has no protective effect on stress-induced apoptosis. After 20 μ M gemcitabine treatment, wild type HSP70 had a stronger anti-apoptotic capacity than the 416A and R447A double mutant HSP70 and pcdna3.1 control group.

Example twelve: apoptosis detection

The ten cells of the example, namely the wild type overexpression vectors pHSP70WT, R416A and R447A of SHP70, the double mutant HSP70 overexpression vector pHSP70Mut transfected with pcDNA3.1 and SHP70 were transferred into HSP70 knock-out cell clones KO-16 and KO-20, respectively, the cells were treated with 20. mu.M gemcitabine, the cells transfected with pcDNA3.1 were treated with a solvent as a blank, and after 24 hours of treatment, cleavage of caspase-9 was detected by the Western blot method described in the third example. Caspase-9 antibody (1: 1000 dilution) was used.

The results are shown in FIGS. 4-E. The results in FIG. 4-E depict Caspase-9 lysis in cells over-expressed by wild-type and R-A mutant HSP70 after treatment with solvent (negative control) and gemcitabine, as detected by WersternBlot, demonstrating that wild-type HSP70 inhibits apoptosis and R-A mutant HSP70 attenuates this protection. After 20 μ M gemcitabine treatment, caspase-9, a marker of apoptosis, was less cleaved in cells expressing wild-type HSP70, and further wild-type HSP70 had stronger anti-apoptotic ability than 416A and R447A double mutant HSP70 and pcdna3.1 control group.

Example thirteen: cell growth experiments

KO-20, KO-41, 8902 were cloned from HSP70 knock-out (KO) Mia PaCa-2 cells by transfecting a wild-type HSP70 expression vector (HSP70-wt), HSP70 single mutant (R416A) or double mutant (HSP70-Mut) expression vector, respectively, by the three transfection methods described in the examples, and inoculating cells into 96-well plates, 3000 cells per well, culturing for 3-4 days under normal culture conditions, and measuring relative cell growth daily using the CCK-8 method described in example four (expressed as "OD" value).

Using the transfection method described in example three, 2 HSP70 knockout cell clones (KO-16 and KO-20) were transfected with pcDNA3.0 control plasmid or wild-type HSP70 expression plasmid (HSP70WT), respectively. And the cells were seeded in 96-well plates at 3000 cells per well, gemcitabine-containing medium was added to each well, a concentration gradient (specific concentration gradient) was set, and after 24 or 48 hours of treatment, three more wells per treatment were set, and the cell inhibition rate was measured using the CCK8 method described in example four.

HSP70 knockout Mia PaCa-2 cells were transfected with Wild Type (WT), single methylation site mutants (R416A or R447A) or 416 and 447 double site mutant (Mut) HSP70 plasmids using the transfection method described in example three. Cell growth inhibition by gemcitabine was determined by determining cell viability using the CCK8 method described in example four. sgCtrl served as control group. P values were determined by one-way anova.

PRMT1 overexpression vectors pPRMT1 and siPRMT1 were transfected into FG cells using the transfection method described in example three, allowed to overexpress or knock out again in FG cells, and the cells were seeded into 96-well plates, 3000 cells per well, gemcitabine-containing medium per empty, concentration gradients (specific concentration gradients) were set, three replicate wells per treatment were set 24 or 48 hours after treatment, and the cell inhibition rate was determined using the CCK8 method described in example four.

MiaPaCa-2 cells were seeded into 96-well plates at 3000 cells per well, cultured overnight (12 hours) normally, and after the cells were attached, treated with DB75 (5. mu.M), gemcitabine (20. mu.M), or a combination of both for 48 hours, and the number of viable cells was determined by the CCK-8 method described in example four.

The results of the experiment are shown in FIG. 5-A, FIG. 5-B, FIG. 5-C, FIG. 5-D, FIG. 5-E and FIG. 5-F: arginine methylation at specific sites affected HSP70 function in gemcitabine resistance. The present invention transfects two single arginine-alanine mutants, R416A and R447A, and a double mutant (Mut) into HSP 70-deficient cells. Wild-type HSP70 slightly promoted cell growth (fig. 5-a), and also restored levels of drug resistance similar to the parent MiaPaCa-2 cell (fig. 5-B), whereas single mutants of R416A and R447A were only able to rescue about half of the drug resistance level, and the double-mutated protein was not able to rescue drug resistance (fig. 5-C). Because these two arginine residues are the methylation sites of PRMT1, the present invention manipulates the expression of PRMT1 in FG cells and assesses its resistance to gemcitabine. As expected, overexpression of PRMT1 increased gemcitabine resistance, while down-regulation of PRMT1 decreased gemcitabine resistance in FG cells (fig. 5-D, fig. 5-E). The PRMT 1-specific inhibitor DB75 also sensitised pancreatic cancer cells to gemcitabine-induced cell death (fig. 5-F), Veh in fig. 5-F representing drug solvent; DB75 represents the protein arginine methyltransferase inhibitor DB 75; GEM represents gemcitabine. These results indicate that PRMT 1-mediated methylation of HSP70 arginines 416 and 447 is critical for drug resistance in cancer cells.

Example fourteen: the polypeptide and the antibody inhibit the drug resistance of tumor cells.

Synthesizing a peptide segment containing 20 amino acids of human HSP70 arginine 416 and 447 residues and a peptide segment containing 50 amino acids of arginine 416 and 447 residues.

Polyclonal antibodies against HSP70 arginine 416 and 447 residues were prepared using the synthetic peptide fragments described above for future use.

The MiaPaCa-2 cells are inoculated into a 96-well plate, 5000 cells per well are added under the cell culture condition in the second example, when the cells are adhered to the wall, the polypeptide or the antibody is respectively added into the cells, the final concentration of the polypeptide and the antibody is 10 mu mol, gemcitabine with the final concentration of 20 mu M is added after 24 hours of treatment, the culture is continued, and the cell growth condition is detected by using CCK 8.

The results are shown in FIG. 6-A, and the inhibition of gemcitabine by CCK8 was calculated by examining cell proliferation of MiaPaCa-2 cells 48 hours after gemcitabine treatment (20. mu.M), and the polypeptides R416, R447 and the polypeptide R416+ R447 were all able to increase the proliferation of tumor cells by gemcitabine. The same effect was observed with antibodies directed against HSP70 arginine 416 and 447 residues, and the results are shown in FIG. 6-B, where Mock is a blank control, ab-R416 is an antibody blocking the R416 arginine residue, ab-R447 is an antibody blocking the R447 arginine residue, cell proliferation was examined by the CCK8 method to generate growth curves showing that ab-R416 and ab-R447 have increased gemcitabine inhibition of tumor cell growth.

The above examples are only preferred embodiments of the present invention, which are intended to be illustrative and not limiting, and those skilled in the art should understand that they can make various changes, substitutions and alterations without departing from the spirit and scope of the invention.

Sequence listing

<110> university of southern China's science

<120> a medicine for treating cancer

<160> 16

<170> SIPOSequenceListing 1.0

<210> 1

<211> 20

<212> DNA

<213> BCL-2

<400> 1

ctggtgggag cttgcatcac 20

<210> 2

<211> 22

<212> DNA

<213> BCL-2

<400> 2

tcttaaacag cctgcagctt tg 22

<210> 3

<211> 20

<212> DNA

<213> BCL-2

<400> 3

cgcagaacct gcctgtgtcc 20

<210> 4

<211> 21

<212> DNA

<213> BCL-2

<400> 4

cattgcctct cctcacgttc c 21

<210> 5

<211> 22

<212> DNA

<213> VEGFA

<400> 5

tctacctcca ccatgccaag tg 22

<210> 6

<211> 20

<212> DNA

<213> VEGFA

<400> 6

gggtctcgat tggatggcag 20

<210> 7

<211> 22

<212> DNA

<213> GAPDH

<400> 7

ctctgctcct cctgttcgac ag 22

<210> 8

<211> 23

<212> DNA

<213> GAPDH

<400> 8

caatacgacc aaatccgttg act 23

<210> 9

<211> 19

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 9

Gly Gly Arg Gly Gly Phe Gly Gly Arg Gly Gly Phe Gly Gly Arg Gly

1 5 10 15

Gly Phe Gly

<210> 10

<211> 19

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 10

Gly Gly Ala Gly Gly Phe Gly Gly Ala Gly Gly Phe Gly Gly Ala Gly

1 5 10 15

Gly Phe Gly

<210> 11

<211> 19

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 11

Gly Val Met Thr Ala Leu Ile Lys Arg Asn Ser Thr Ile Pro Thr Lys

1 5 10 15

Gln Thr Gln

<210> 12

<211> 19

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 12

Gly Val Met Thr Ala Leu Ile Lys Ala Asn Ser Thr Ile Pro Thr Lys

1 5 10 15

Gln Thr Gln

<210> 13

<211> 20

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 13

Val Leu Ile Gln Val Tyr Glu Gly Glu Arg Ala Met Thr Lys Asp Asn

1 5 10 15

Asn Leu Leu Gly

20

<210> 14

<211> 20

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 14

Val Leu Ile Gln Val Tyr Glu Gly Glu Ala Ala Met Thr Lys Asp Asn

1 5 10 15

Asn Leu Leu Gly

20

<210> 15

<211> 20

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 15

Met Thr Lys Asp Asn Asn Leu Leu Gly Arg Phe Glu Leu Ser Gly Ile

1 5 10 15

Pro Pro Ala Pro

20

<210> 16

<211> 20

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 16

Met Thr Lys Asp Asn Asn Leu Leu Gly Ala Phe Glu Leu Ser Gly Ile

1 5 10 15

Pro Pro Ala Pro

20

Claims (4)

1. A medicament for treating cancer, which comprises a polypeptide fragment of an arginine methylated heat shock protein HSP70, wherein the polypeptide fragment is a polypeptide fragment containing an arginine residue at 416 th position of HSP70, a polypeptide fragment containing an arginine residue at 447 th position of HSP70 or a polypeptide fragment of a combination of the two, and the medicament inhibits methylation of arginine at 416 th and/or 447 th positions of HSP 70.

2. The drug for treating cancer as claimed in claim 1, wherein the peptide fragment of arginine residue 416 of the arginine methylated heat shock protein HSP70 has the sequence GGVMTALIKRNSTIPTKQTQ.

3. The drug for treating cancer as claimed in claim 1, wherein the sequence of the polypeptide fragment of arginine residue 447 in the protein HSP70 is GVLIQVYEGERAMTKDNNLL.

4. The drug for treating cancer according to claim 1, wherein the sequence of the combined polypeptide fragment is GGVMTALIKRNSTIPTKQTQIFTTYSDNQPGVLIQVYEGERAMTKDNNLL.

Images