CN116549487A - Method for inducing G1/S-specific cyclin-D1 type ubiquitination, application and preparation - Google Patents
Method for inducing G1/S-specific cyclin-D1 type ubiquitination, application and preparation Download PDFInfo
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- CN116549487A CN116549487A CN202310307392.5A CN202310307392A CN116549487A CN 116549487 A CN116549487 A CN 116549487A CN 202310307392 A CN202310307392 A CN 202310307392A CN 116549487 A CN116549487 A CN 116549487A
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K33/00—Medicinal preparations containing inorganic active ingredients
- A61K33/24—Heavy metals; Compounds thereof
- A61K33/36—Arsenic; Compounds thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
- A61K31/425—Thiazoles
- A61K31/428—Thiazoles condensed with carbocyclic rings
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
- A61P35/02—Antineoplastic agents specific for leukemia
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- Health & Medical Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Pharmacology & Pharmacy (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Chemical & Material Sciences (AREA)
- Epidemiology (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Organic Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Inorganic Chemistry (AREA)
- Hematology (AREA)
- Oncology (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention is suitable for the field of antitumor drugs, and provides a method for inducing G1/S-specific cyclin-D1 type ubiquitination, wherein an environment in which G1/S-specific cyclin-D1 exists contains SUMO protein, the SUMO protein is induced to modify the G1/S-specific cyclin-D1, and the G1/S-specific cyclin-D1 degradation and apoptosis of tumor cells are promoted. The arsenical agent and the derivatives thereof and SUMO agonist N106 induce G1/S-specific cyclin-D1 type ubiquitination, improve the degradation efficiency of the G1/S-specific cyclin-D1, facilitate degradation by proteasome, reduce the accumulation amount and promote apoptosis of tumor cells.
Description
The application is a divisional application of patent application No. 202111284748.5, which is filed on 11/01/2021 and has the invention name of a method, application and preparation for inducing G1/S-specific cyclin-D1 type ubiquitination.
Technical Field
The invention belongs to the field of antitumor drugs, and relates to a method for inducing G1/S-specific cyclin-D1 ubiquitination, application and a preparation.
Background
Proteins associated with cell cycle regulation are found when uncoordinated growth and proliferation of tumors are observed, and it is thought that tumors have a close relationship with cell cycle regulation disorders. G1/S-specific cyclin-D1 is one of the cell cycle regulators that is abnormally high expressed in all types of tumor cells. Thus, inhibition of high expression and abnormal accumulation of G1/S-specific cyclin-D1 is an important approach to the treatment of cancer. The effect of treating the mantle cell lymphoma can be achieved by inhibiting the high expression of the G1/S-specific cyclin-D1 in the mantle cell lymphoma. In the prior art, G1/S-specific cyclin-D1 degradation may be partially induced by phosphorylation of the ubiquitin/protease system, but G1/S-specific cyclin-D1 degradation is not efficient.
Small ubiquitin-like modifier (SUMO) is a protein similar to ubiquitin protein found in recent years, and is used for modifying substrate protein. In the prior art, more than 200 substrate proteins can be modified by SUMO, and the substrate proteins have wide application in the field of modification of the substrate proteins due to diversity of the substrate proteins modified by SUMO. However, methods and formulations for modifying G1/S-specific cyclin-D1 and reducing its expression have not been disclosed.
In view of the above-mentioned drawbacks of the prior art, the present invention provides a method, use and formulation for inducing G1/S-specific cyclin-D1-like ubiquitination.
Disclosure of Invention
The embodiment of the invention aims to provide a method, application and preparation for inducing G1/S-specific cyclin-D1 type ubiquitination, and aims to solve the problem of low efficiency of G1/S-specific cyclin-D1 degradation by using SUMO to induce G1/S-specific cyclin-D1 type ubiquitination.
The embodiment of the invention is realized by a method for inducing G1/S-specific cyclin-D1 type ubiquitination, wherein the environment where the G1/S-specific cyclin-D1 exists contains SUMO protein, the SUMO protein is induced to modify the G1/S-specific cyclin-D1, and the G1/S-specific cyclin-D1 degradation is promoted.
Further, the G1/S-specific cyclin-D1 is modified by inducing the SUMO protein with an arsenical and/or arsenical derivative.
Further, the SUMO protein is one or more of SUMO-1, SUMO-2 and SUMO-3 proteins.
Further, ubc9E2 binding enzyme is also present in the environment where the G1/S-specific cyclin-D1 is present.
Further, the Ubc9E2 binding enzyme couples the G1/S-specific cyclin-D1 and SUMO proteins via isopeptide bonds.
Further, the arsenical derivative comprises an arsenical derivative selected from disodium hydrogen arsenite (Na 2 HAsO 3 ) And its hydrate, potassium arsenate (H) 2 AsKO 4 ) One or more of Carbarsone (Carbarsone) and nifedipine (Nitarsone).
Further, the G1/S-specific cyclin-D1 is modified by induction of the SUMO protein with SUMO agonist N106.
The G1/S-specific cyclin-D1 is cyclin in mantle cell lymphoma, leukemia, breast cancer, colorectal cancer, bladder cancer, parathyroid tumor, melanoma, lung cancer, prostate cancer or osteosarcoma lesion cells.
It is another object of an embodiment of the present invention to provide the use of a combination of an arsenical agent and/or arsenical derivative with SUMO agonist N106 for the manufacture of a medicament for the treatment of tumors.
Further, the arsenical derivative is selected from one or more of disodium arsenite and its hydrate, potassium arsenate, carbarsone and nifenanic acid.
It is another object of embodiments of the present invention to provide a formulation for inducing ubiquitination of G1/S-specific cyclin-D1, the formulation comprising one or more of an arsenical agent, an arsenical derivative and a SUMO agonist N106.
The arsenical agent and the derivatives thereof and N106 induce G1/S-specific cyclin-D1 type ubiquitination, improve the degradation efficiency of the G1/S-specific cyclin-D1, facilitate degradation by a proteasome and reduce the accumulation amount thereof.
Drawings
In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings that are required to be used in the description of the embodiments of the present application will be briefly described below.
FIG. 1 is a chemical structural formula of Arsenic Trioxide (ATO), an arsenic derivative and N106 provided by the present invention;
FIG. 2 is a graph of cell transfection provided in an embodiment of the present invention;
FIG. 3 is a graph of lysine mutant plasmids constructed and transfected into ψ -K-x-D/E as provided in the examples of the present invention;
FIG. 4 is a graph of ATO-promoted ubiquitination-like spectrum provided by an embodiment of the present invention;
FIG. 5 is a graph of N106 and arsenical and derivatives thereof promoting ubiquitination of the class provided by the examples of the present invention;
FIG. 6 is a graph of an in vitro decay analysis of cells provided by an embodiment of the present invention;
FIG. 7 is a graph of a cell viability assay provided by an embodiment of the present invention;
FIG. 8 is a graph of a biological activity test provided in an embodiment of the present invention.
Detailed Description
In order to make the technical problems, technical schemes and beneficial effects to be solved more clear, the invention is further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
Like ubiquitination, modification of the substrate by SUMO is a multi-step enzymatic reaction. The SUMO precursor cleaves several amino acids at the carboxy terminus (C-terminus) under the action of a specific protease (senhrin/SUMO-specific protease, SENP), thereby exposing the glycylglycine residue to form the mature SUMO. First, the C-terminal glycine of mature SUMO is linked to the cysteine residue of the E1 activating enzyme (SUMO-activating enzyme) by a thioester bond. SUMO is then transferred to the cysteine residue of the E2-binding enzyme (SUMO-conjugating enzyme). Only one E2 binding enzyme, ubc9, has been found. Ubc9 can directly recognize the substrate, thereby eventually coupling SUMO to lysine residues of the substrate protein via isopeptide bonds. In some cases, E3 ligase (SUMO E3 ligase) may enhance Ubc9 efficiency and specificity of transferring SUMO to the substrate protein. In mammals, four SUMO genes, SUMO-1, SUMO-2, SUMO-3 and SUMO-4, respectively, have been found. There are 6 dessumoylases in humans, a specific protease known as SENP. SENP1 and SENP2 may shear the first three types of SUMO, while SENP3, SENP5, and SENP6 prefer to shear SUMO-2 and SUMO-3.
SUMO agonist N106
(4-Methoxy-N- [5- (4-methoxyphenyl) -1,3, 4-oxazol-2-yl-2-benzothiazolami ne, CAS: 862974-25-2), the effect of promoting ubiquitination-like modification is achieved mainly by activating the E1 activating enzyme of SUMO.
Arsenic Trioxide (ATO) is the main component of the arsenical agent and is clinically applied to chemotherapy for treating acute promyelocytic leukemia. The main action mechanism of arsenic trioxide for treating acute promyelocytic leukemia is to promote ubiquitin-like modification (SUMO) of oncoprotein PML, namely, arsenic trioxide is directly combined with cysteine in a zinc finger structure at the end of oncoprotein PML to induce conformational change and multimerization of protein, and then SUMO modification and ubiquitination modification are carried out to be degraded by proteasome.
The invention provides a method for inducing G1/S-specific cyclin-D1 type ubiquitination, which improves the degradation efficiency of G1/S-specific cyclin-D1, is favorable for degradation by a proteasome and reduces the accumulation amount of the cyclin-D1 in cells.
In the method for inducing G1/S-specific cyclin-D1 type ubiquitination, the environment where G1/S-specific cyclin-D1 exists contains SUMO protein, the SUMO protein is induced to modify G1/S-specific cyclin-D1, and G1/S-specific cyclin-D1 degradation is promoted.
The arsenical agent, the arsenical agent derivative and the SUMO agonist N106 induce G1/S-specific cyclin-D1 ubiquitination, so that degradation by a proteasome is facilitated, and the degradation efficiency of the G1/S-specific cyclin-D1 is improved.
In one embodiment, the modification of G1/S-specific cyclin-D1 is induced by an arsenical and/or arsenical derivative.
Further, the SUMO protein is one or more of SUMO-1, SUMO-2 and SUMO-3 proteins.
Further, ubc9E2 binding enzyme is also present in the environment where the G1/S-specific cyclin-D1 is present.
Further, the Ubc9E2 binding enzyme couples the G1/S-specific cyclin-D1 and SUMO proteins via isopeptide bonds.
Further, the arsenical derivative comprises an arsenical derivative selected from disodium hydrogen arsenite (Na 2 HAsO 3 ) And its hydrate, potassium arsenate (H) 2 AsKO 4 ) One or more of Carbarsone (Carbarsone) and nifedipine (Nitarsone). Referring to fig. 1, fig. 1 shows chemical structural formulas of Arsenic Trioxide (ATO), an arsenical derivative and N106 according to the present invention.
In another embodiment, the SUMO agonist N106 is used to induce modification of G1/S-specific cyclin-D1 by the SUMO protein.
In the above examples, the G1/S-specific cyclin-D1 is a cyclin in mantle cell lymphoma, leukemia, breast cancer, colorectal cancer, bladder cancer, parathyroid tumor, melanoma, lung cancer, prostate cancer or osteosarcoma lesion cells.
The invention also provides the use of a combination of an arsenical agent and/or an arsenical derivative and a SUMO agonist N106 in the manufacture of a medicament for the treatment of a tumour.
Further, the arsenical derivative is selected from one or more of disodium arsenite and its hydrate, potassium arsenate, carbarsone and nifenanic acid.
In the above examples, the G1/S-specific cyclin-D1 is a cyclin in mantle cell lymphoma, leukemia, breast cancer, colorectal cancer, bladder cancer, parathyroid tumor, melanoma, lung cancer, prostate cancer or osteosarcoma lesion cells.
The invention also provides a formulation for inducing G1/S-specific cyclin-D1-like ubiquitination, the formulation comprising one or more of an arsenical agent, an arsenical derivative and a SUMO agonist N106.
The invention is further illustrated by the following specific experimental examples.
Example 1 cell culture
Human colon cancer HCT-116 cells and human embryonic kidney 293 (HEK 293) cells were cultured in 96-well plates to which Dulbecco's Modified Eagle's Medium (DMEM) and fetal bovine serum at 10% by volume of the medium were added. At 37 ℃ CO 2 Culturing in an incubator at a concentration of less than 5%. Human lichen lymphoma JeKo-1 cells were cultured in 96-well plates with RPMI-1640 medium added to the plates. At 37 ℃ CO 2 Culturing in an incubator at a concentration of less than 5%.
EXAMPLE 2 cell transfection
(1) Later on in log growth, HCT-116 cells from example 1 were collected. DNA from the G1/S-specific cyclin-D1 plasmid was transiently transferred into HCT-116 cells in 6 cm dishes using lipidated amine 3000. Empty vectors were used to keep the total amount of transfected DNA plasmid per group unchanged. The Flag-EGFP plasmid was co-controlled as an internal control to evaluate transfection efficiency. Cell extracts G1/S-specific cyclin-D1 proteins were collected and subjected to immunoblotting and Immunoprecipitation (IP) detection 24 hours after transfection.
(2) Transfecting Ubc9 plasmid on the basis of step (1)
Collecting the cells transfected with the G1/S-specific cyclin-D1 plasmid in step (1), and performing transfection of Ubc9 plasmid in the same manner as in step (1). Cell extracts G1/S-specific cyclin-D1 proteins were collected and subjected to immunoblotting and Immunoprecipitation (IP) detection 24 hours after transfection.
(3) Transfecting SUMO-1, SUMO-2, SUMO-3 or SENP1 plasmid on the basis of step (2)
The Ubc9 plasmid-transfected cells in step (2) were collected and the transfection of SUMO-1, SUMO-2, SUMO-3 or SENP1 plasmid was performed in the same manner as in step (1). Cell extracts G1/S-specific cyclin-D1 proteins were collected and subjected to immunoblotting and Immunoprecipitation (IP) detection 24 hours after transfection.
(4) Adding an MG132 protease inhibitor on the basis of the step (2)
Collecting the cells transfected with Ubc9 plasmid in (2), and culturing HCT-116 cells by adding MG132 proteasome inhibitor into the selective medium. Cell extracts G1/S-specific cyclin-D1 proteins were collected and subjected to immunoblotting and Immunoprecipitation (IP) detection 24 hours after transfection.
Results: referring to FIG. 2, the +in FIG. 2 indicates that the corresponding plasmid or enzyme is positive, the-indicates that the corresponding plasmid or enzyme is negative, and the corresponding protein content is provided. In FIG. a there are four sets of bands, each set of bands showing, from left to right, the first column of cells transfected with the G1/S-specific cyclin-D1 plasmid and protein expression of G1/S-specific cyclin-D1, the second column of cells transfected with Ubc9/SUMO-1/2/3 plasmid or the G1/S-specific cyclin-D1 plasmid with only trace amounts of G1/S-specific cyclin-D1 protein accumulation, the third column of cells transfected with the G1/S-specific cyclin-D1 plasmid, ubc9/SUMO-1/2/3 plasmid and SENP1 plasmid, the G1/S-specific cyclin-D1 plasmid with significant accumulation of G1/S-specific cyclin-D1 and Ubc9/SUMO-1/2/3 plasmid with addition of MG132 proteasome inhibitor, and the fourth column of G1/S-specific cyclin-D1 protein with significant accumulation.
After HEK293 cells are transfected with the G1/S-specific cyclin-D1 plasmid, the expression level of the G1/S-specific cyclin-D1 protein in the cells is up-regulated. SUMO-1/2/3 or Ubc9 plasmid is advantageous for decomposition by inducing SUMO formation of G1/S-specific cyclin-D1 protein, accumulation of G1/S-specific cyclin-D1 protein in cells is reduced, and SUMO formation of G1/S-specific cyclin-D1 protein induced by Ubc9 or SUMO-1/2/3 can be inhibited when SENP1 plasmid having reverse SUMO formation is present, decomposition of G1/S-specific cyclin-D1 protein is not facilitated, and accumulation amount is increased. Ubc9 and SUMO-1/2/3 plasmids are capable of producing an enzyme that induces SUMO formation of the G1/S-specific cyclin-D1 protein, effecting SUMO formation of the G1/S-specific cyclin-D1 protein, and reducing the activities of Ubc9 and SUMO-1/2/3 enzymes in the presence of the MG132 proteasome inhibitor, thereby inhibiting SUMO formation of the G1/S-specific cyclin-D1 protein.
EXAMPLE 3 construction and transfection of lysine mutant plasmid in ψ -K-x-D/E
The pcDNA G1/S-specific cyclin-D1 HA original plasmid (# 166130) was purchased on Addgene, and the G1/S-specific cyclin-D1 HA plasmid was one of the G1/S-specific cyclin-D1 plasmids. The K (lysine) points of different sites of the original plasmid of G1/S-specific cyclin-D1 HA are mutated into R (arginine) by synthesizing primers requiring mutation sites by using a site directed mutagenesis kit (Agilent, california, USA) kit, so that the G1/S-specific cyclin-D1 obtained by expressing the mutated G1/S-specific cyclin-D1 HA plasmid can participate in the combination of the K at the 48, 72,149,238,269 sites of SUMO. The Ubc9 plasmid can induce SUMO of G1/S-specific cyclin-D1 protein to promote decomposition. The K site of the SUMO is subjected to sequential mutation corresponding to the K site of the G1/S-specific cyclin-D1 HA original plasmid, and whether the G1/S-specific cyclin-D1 HA original plasmid after the mutation of the K site can be subjected to SUMO conversion by the G1/S-specific cyclin-D1 is detected.
The mutant plasmid transfected cells were performed as follows:
the density of the planted cells is about 70-80%, and the serum-free culture medium is replaced; 15 μl lipofectamine3000 reagent was dissolved in 150 μl of 4 serum-free medium (lipofectamine reagent to serum-free medium volume ratio 1:10); dissolving 14 mu l of small stem or plasmid in 700ul of serum-free culture medium (DNA concentration is 0.5-5 mu g/. Mu.l); adding the culture medium dissolved with DNA into the culture medium dissolved with lipofectamine reagent; standing at room temperature for 5min; adding the DNA-lipid mixture into a cell culture dish, and adding 5% CO at 37deg.C 2 Culturing in a cell culture box under the concentration condition for 6 hours, and then replacing a serum normal culture medium; the reaction is carried out after 24 to 72 hoursAnd (5) one-step experimental detection analysis.
Results: referring to FIG. 3, the +in FIG. 3 indicates positive for the corresponding plasmid or enzyme, negative for the corresponding plasmid or enzyme, and the content of the corresponding protein in the strip.
The sequence aag for coding the K (lysine) at 149 th is mutated into cgg, the amino acid coded by the cgg is R (arginine), the consensus motif ψ -K-x-D/E of the modified tetrapeptides of the SUMO protein is not satisfied any more, and as a result, the G1/S-specific cyclin-D1 cannot be ubiquitinated by the SUMO protein, so that the G1/S-specific cyclin-D1 specific SUMO site is positioned at the K at 149 th.
Example 4ATO promotes ubiquitination of class
A conventional six-well plate has about 5 x 10 per well 6 The cell amount, the volume of the medium per well was 2ml, the ATO concentration added per well was 2.5. Mu.M (i.e., 2.5. Mu. Mol/L), and the protease inhibitor MG132 concentration was 10. Mu.M (i.e., 10. Mu. Mol/L). After 2.5. Mu.M ATO and 10. Mu.M MG132 treatment of mantle cell lymphoma Jeko-1 cells for 2h, the suspension cells were collected by centrifugation, and the total cell proteins were extracted by adding RIPA lysis and buffer. Homogenizing and incubating cells in RIPA lysis and buffer, with the addition of 1mM PMSF, 1mM Na to the RIPA lysis and buffer 3 VO 4 And proteasome inhibitor cocktail MG 132. 13000 g RIPA lysis and buffer were centrifuged at 4℃for 15 min and total protein was collected. All proteins associated with SUMO binding were then "dragged" out of the total protein with beads conjugated with SUMO-2/3 antibody, followed by WB immunoblotting of these proteins, and SUMO-specific G1/S-specific cyclin-D1 protein content was detected with G1/S-specific cyclin-D1 antibody.
Results: referring to fig. 4, the left side of the spectrum represents the molecular weight of the protein, and the right side represents the types of proteins corresponding to different protein bands. The upper + indicates positive for the corresponding plasmid or enzyme, -indicates negative for the corresponding plasmid or enzyme. The content of the proteins corresponding to the bands in the spectrogram. The upper longer band represents the total protein amount, and the lower shorter band represents the G1/S-specific cyclin-D1 content of the total protein.
As a result, as shown in FIG. 4, the accumulation of the enzyme inducing ubiquitination of G1/S-specific cyclin-D1 type was significantly increased and the accumulation of G1/S-specific cyclin-D1 protein was significantly decreased after ATO treatment.
EXAMPLE 5N106 and arsenical and derivatives thereof promote ubiquitination of the class
A conventional six-well plate has about 5 x 10 per well 6 Cell mass, medium volume per well of 2ml, ATO at a concentration of 2.5. Mu.M, N106 at 50. Mu.M or HAsNa at 500. Mu.M was added per well 2 O 3 ·7H 2 O、H 2 AsKO 4 One of Carbarsone, nitarsone. The ATO treatment time is 4 hours; HAsNa 2 O 3 ·7H 2 O、H 2 AsKO 4 The treatment times of Carbarsone, nitarsone and N106 were 12h. After treatment, the suspension cells were collected by centrifugation, and the total cell proteins were extracted by adding RIPA lysis and buffer. Homogenizing and incubating cells in RIPA lysis and buffer, with the addition of 1mM PMSF, 1mM Na to the RIPA lysis and buffer 3 VO 4 And proteasome inhibitor cocktail MG 132. 13000 g RIPA lysis and buffer were centrifuged at 4℃for 15 min and total protein was collected. Conventional WB assays were then performed for G1/S-specific cyclin-D1 expression, α -Tublin as a control.
Results: referring to fig. 5, the substances for promoting ubiquitination are added above the spectrogram, and the bands in the spectrogram correspond to the protein content.
Any substance of N106, an arsenical agent and derivatives thereof can promote the ubiquitination of the G1/S-specific cyclin-D1 protein, which is beneficial to the degradation of the G1/S-specific cyclin-D1 protein, and the accumulation of the G1/S-specific cyclin-D1 protein is reduced. Among them, the addition of ATO, carbarsone, nitarsone or N106 is superior.
EXAMPLE 6 in vitro decay analysis of cells
24 hours after transfection of the G1/S-specific cyclin-D1 plasmid-transfected cells obtained in step (1) in example 2, the cells were divided into 8 plates of 10 cm, each with a cell number of approximately 13.7X10 6 . With 2.5. Mu.M ATO, 50. Mu.M N106 or 500. Mu.M HAsNa, respectively 2 O 3 ·7H 2 O、H 2 AsKO 4 Treatment with one of Carbarsone, nitarsone, culturing for 12 hr, and detecting G1/S-specificity by flow cytometryDecay of cyclin-D1 protein.
Results: referring to fig. 6, Q4 in fig. 6: (annexin V-FITC) -/PI-, the cells of this region are living cells; q3: (annexin V-FITC) +/PI-, the cells of this region are early apoptotic cells; q2 (annexin V+FITC) +/PI+, the cells in this region are late apoptotic cells; q1: (annexin V-FITC) -/PI+, the cells in this region are necrotic cells. Apoptosis rate is the sum of Q2 and Q3 quadrant percentages.
Compared with the control group, through 2.5. Mu.M ATO, 50. Mu.M N106 or 500. Mu.MHAsNa 2 O 3 ·7H 2 O、H 2 AsKO 4 The apoptosis biomarker PI-annexin V positive rate of the mantle cell lymphoma Jeko-1 cells treated by any one of Carbarsone, nitarsone is obviously increased, and the apoptosis rate is up-regulated. Description of N106, ATO or its derivative HAsNa 2 O 3 ·7H 2 O，H 2 AsKO 4 Any substance in Carbarsone, nitarsone can promote SUMO of G1/S-specific cyclin-D1 protein and promote apoptosis of cells.
EXAMPLE 7 cell viability assay
24 hours after transfection of the G1/S-specific cyclin-D1 plasmid-transfected cells obtained in step (1) in example 2, the cells were divided into 8 plates of 10 cm, each with a cell number of approximately 13.7X10 6 . Treated with 0, 1, 2.5, 10. Mu.M ATO, respectively, and incubated for 12 hours after treatment, cells were collected for 1, 6, 24, and 48 hours and incubated with 80. Mu.g/ml cyclic amide in conventional medium. Cell viability was measured using CCK8 kit.
Results: referring to fig. 7, 3 replicates were performed for each ATO concentration treated cell at each time point in fig. a. Panel b is absorbance at various time points.
In FIG. a, the mortality of cells treated with ATO at different concentrations after 6h was up-regulated; in panel b, the absorbance of ATO-treated cells was small compared to the control group, indicating that the activity of ATO-treated Jeko-1 cells decreased with the increase of the treatment time.
Example 8 biological Activity test
For immunodeficient mice (strain)Transplantation tumor test for mantle cell lymphoma for NOD-Prkdcem26 IL2rgem 26/Gpt), transplantation Jeko-1 cell mass of 5×10 6 When the transplanted tumor reaches 1cm 3 Thereafter, the intratumoral injection treatment with ATO (3.2 mg/kg) was performed three times a week with physiological saline for the control group.
Results: referring to fig. 8, the tumors of the immunodeficient mice treated with ATO injection in fig. a are significantly smaller than those of the immunodeficient mice injected with physiological saline.
Figure b, tumor of immunodeficient mice stripped out for comparison, tumor of immunodeficient mice treated with ATO injection was significantly smaller than tumor of immunodeficient mice injected with physiological saline.
Panel c shows the tumor volumes of immunodeficient mice at various time points, and it can be seen that after 10 days, the tumor volumes of immunodeficient mice treated with ATO (3.2 mg/kg) intratumoral injection were significantly smaller than those of immunodeficient mice injected with physiological saline, and the volume difference was gradually increased with the lapse of time.
Panel d shows tumor weights of immunodeficient mice from the experimental and control groups measured by dissecting tumor tissue. The tumor weight of the immunodeficient mice treated with ATO injection was significantly smaller than that of the immunodeficient mice injected with physiological saline.
ATO can target and locate in tumor tissue to accelerate apoptosis of tumor cells and influence growth of tumor cells.
The results of the above specific examples demonstrate that N106, arsenicals, and arsenical derivatives target the SUMO site of tumor cells, induce G1/S-specific cyclin-D1 SUMO, promote decomposition of G1/S-specific cyclin-D1, reduce the accumulation of G1/S-specific cyclin-D1, and promote apoptosis of tumor cells. Wherein the arsenic-treated cells induce an increase in the accumulation of G1/S-specific cyclin-D1 SUMO-forming enzyme.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, and alternatives falling within the spirit and principles of the invention.
Claims (9)
1. Use of an arsenical agent and/or a combination of an arsenical derivative and SUMO agonist N106 for the manufacture of a medicament for inducing ubiquitination of G1/S-specific cyclin-D1 type.
2. Use of an arsenical agent and/or a combination of an arsenical agent derivative and SUMO agonist N106 according to claim 1 for the manufacture of a medicament for inducing G1/S-specific cyclin-D1-like ubiquitination, characterized in that the environment in which G1/S-specific cyclin-D1 is present comprises SUMO protein, and that the arsenical agent and/or arsenical agent derivative and SUMO agonist N106 achieve G1/S-specific cyclin-D1-like ubiquitination by inducing SUMO protein modification of G1/S-specific cyclin-D1 protein.
3. Use of an arsenical and/or arsenical derivative according to claim 1 in combination with SUMO agonist N106 for the manufacture of a medicament for inducing ubiquitination of G1/S-specific cyclin-D1 class, wherein the arsenical derivative is selected from one or more of disodium arsenite and hydrates thereof, potassium arsenate, carbarsone and nifenanic acid.
4. Use of an arsenical agent and/or a combination of an arsenical agent derivative and SUMO agonist N106 according to claim 2 for the manufacture of a medicament for inducing ubiquitination of G1/S-specific cyclin-D1 class, wherein the SUMO protein is one or more of SUMO-1, SUMO-2 and SUMO-3 proteins.
5. Use of an arsenical agent and/or a combination of an arsenical agent derivative and SUMO agonist N106 according to claim 2 for the manufacture of a medicament for inducing ubiquitination of G1/S-specific cyclin-D1, characterized in that Ubc9E2 binding enzyme is also present in the environment in which G1/S-specific cyclin-D1 is present.
6. Use of an arsenical agent and/or a combination of an arsenical agent derivative and SUMO agonist N106 according to claim 5 for the manufacture of a medicament for inducing ubiquitination of G1/S-specific cyclin-D1 class, wherein the Ubc9E2 binding enzyme couples the G1/S-specific cyclin-D1 and SUMO proteins via an isopeptide bond.
7. Use of an arsenical agent and/or a combination of an arsenical agent derivative and SUMO agonist N106 according to claim 1 for the manufacture of a medicament for inducing ubiquitination of G1/S-specific cyclin-D1 class, wherein the G1/S-specific cyclin-D1 is a cyclin in mantle cell lymphoma, leukemia, breast cancer, colorectal cancer, bladder cancer, parathyroid tumor, melanoma, lung cancer, prostate cancer or osteosarcoma lesion cells.
8. Use of an arsenical agent and/or a combination of an arsenical agent derivative and SUMO agonist N106 according to claim 1 or 2 for the manufacture of a medicament for inducing ubiquitination of G1/S-specific cyclin-D1, characterized in that the G1/S-specific cyclin-D1-specific ubiquitination site is K at position 149 of G1/S-specific cyclin-D1.
9. Use of an arsenical agent and/or a combination of an arsenical agent derivative and SUMO agonist N106 according to claim 3 for the manufacture of a medicament for inducing ubiquitination of G1/S-specific cyclin-D1 class, wherein the arsenical agent has an action concentration of 1 to 10 μm, preferably 2.2 μm, the disodium arsenite and hydrates thereof, the potassium arsenate, the carbaarsine or nifenanic acid has an action concentration of 500 μm, and the SUMO agonist N106 has an action concentration of 50 μm.
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