CN104997776B - Ni(1-oxidopyridine-2-thione)2Application - Google Patents
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Abstract
The invention discloses Ni (1-oxidopyridine-2-thione)2Application and Ni (1-oxidopyridine-2-thione) in preparation prevention and treatment tumour medicine2As application of the deubiquitination enzyme inhibitor in the drug that preparation prevents and treats inflammation, tissue ischemia-reperfusion injury, myocardial hypertrophy disease.It additionally provides with Ni (1-oxidopyridine-2-thione)2For the anti-curing oncoma of active constituent, inflammation, tissue ischemia-reperfusion injury, myocardial hypertrophy disease drug, provide more choices for clinic.
Description
Technical field
Originally bright to be related to field of pharmaceutical chemistry technology, in particular to a kind of deubiquitination enzyme inhibitor Ni (1-
oxidopyridine-2-thione)2New opplication in medicine.
Background technique
It is well known that protein is the necessary material for forming cell, it is the movable main undertaker of cell function, regulates and controls egg
The update of white matter is one of basic link of cell metabolism.Intracellular about 30% newly synthesized protein can in 10 minutes quilt
Degradation (Schubert et al., 2000) rapidly.The protein of intracellular this high speed, which updates, needs a kind of special system
Accurately regulate and control which albumen need to be degraded which be retained.Ubiquitin-proteasome system is exactly such a regulatory protein
The important system of matter degradation and function.
Ubiquitin-proteasome system (Ubiquitin-proteasome system, UPS) is recently extremely concerned tune
It controls protein degradation and participates in the important system of intracellular a variety of life processes, UPS is made of ubiquitin and proteasome, and ubiquitin is
One contains the small molecule of 76 amino acid, can be identified and be degraded by proteasome by the protein of ubiquitin tag；Proteasome
It is then the cylindric macromolecular being responsible for the degradation of proteins into amino acid fragment.It is more than into the cell 80% protein is logical
Cross UPS degradation, such as degrade intracellular false folding or impaired protein, Cell cycle regulatory proteins, oncogene, tumour suppression
The factor processed；Regulation antigen submission and transcription factor activity etc. (Coux et al., 1996；Hershko&Ciechanover,
1998)。
Ubiquitin-proteasome system protein degradation matter mainly includes two steps: ubiquitin starts enzyme by a series of ubiquitin
Poly-ubiquitin chains more on target protein marker and the degradation of 26S proteasome are marked to the target protein of upper ubiquitin chain.Ubiquitination process is
State is reversible, can be removed by a kind of enzyme-specific for being referred to as deubiquitinating enzymes (DUBs) by the albumen of ubiquitination.The mankind
About 100 DUBs of genome encoding, are broadly divided into 2 major class: ubiquitin-specific proteases (USPs),
Ubiquitin carboxy-terminal hydrolases (UCHs), wherein USPs accounts for the overwhelming majority (Fraile et
al.,2012；Fortelny et al.,2014).Deubiquitinating enzymes (DUBs) are the important sets in Ubiquitin-proteasome system
At part, it has mediated ubiquitin or ubiquitin chain to remove from target protein, is the key link that UPS is functioned.It is intracellular many
DUBs participates in and has adjusted the life processes such as cell cycle, apoptosis and transcription, closely related with the occurrence and development of tumour.
Tumour cell updates metabolism fastly, and UPS is in relative activation state, therefore the anti-tumor drug pair of targeting proteins enzyme body
Tumour cell has selectivity to become the popular target spot of oncotherapy.Bortezomib (bortezomib), trade name ten thousand
Jade-like stone (Velcade) is a kind of targeting in the inhibitor of 20S proteasome, has been approved by the fda in the United States for clinical treatment
The diseases such as Huppert's disease and lymthoma, and achieve good curative effect.Since most DUBs are cysteine class eggs
White enzyme, the cysteine residues of active site and a variety of electrophilic reagents have very high reactivity, relative to E3 ligase, with
DUBs is that target spot has more druggability.Relative to 20S proteasome inhibitor, most of DUBs inhibitor is just for special part
Deubiquitinating enzymes, therefore DUBs inhibitor is more selective.There is document report deubiquitination enzyme inhibitor to exist in succession recent years
Anti-tumor aspect shows remarkable result (referring to Linder S. etc., Nat Med 2011； 17(12):1636-1640；
Chauhan D, etc. Cancer Cell 2012；22(3):345–358；Liu NN, etc. Scientific Reports
2014；4:5240；Liu NN, etc. Oncotarget 2014；5 (14): 5453-5471) in conclusion deubiquitinating enzymes press down
System has good potential applicability in clinical practice in terms of oncotherapy.
Summary of the invention
The object of the present invention is to provide a kind of deubiquitination enzyme inhibitor Ni (1-oxidopyridine-2-thione)2?
New opplication in medicine.
Realize that the technical solution of above-mentioned purpose is as follows.
Ni(1-oxidopyridine-2-thione)2Abbreviation NiPT, structural formula are as follows.
Ni(1-oxidopyridine-2-thione)2Application in preparation prevention and treatment tumour medicine.
The tumour is leukaemia in one of the embodiments,.
Ni(1-oxidopyridine-2-thione)2As deubiquitination enzyme inhibitor in preparation prevention and treatment inflammation, tissue
Ischemia reperfusion injury, myocardial hypertrophy disease drug in application.
It is a further object of the present invention to provide a kind of drugs of anti-curing oncoma.
Specific technical solution is as follows.
The active ingredient of the drug includes Ni (1-oxidopyridine-2-thione)2。
It is a further object of the present invention to provide a kind of prevention and treatment inflammation, tissue ischemia-reperfusion injury, myocardial hypertrophy disease
Drug.
Specific technical solution is as follows.
It is a kind of prevention and treatment inflammation, tissue ischemia-reperfusion injury, myocardial hypertrophy disease drug, the drug activity at
Part includes Ni (1-oxidopyridine-2-thione)2。
Present invention discover that NiPT has apparent inhibiting effect, especially 26S proteasome correlation to go general deubiquitinating enzymes
Elementization enzyme (USP14/UCHL5).As metal complex, the anticancer drugs such as NiPT and cis-platinum are different, and NiPT does not have DNA molecular
Have an impact, does not cause DNA damage effect.
Present invention discover that cytotoxic effect NiPT universal in cancerous cell line, and also have to the cell line of cisplatin resistance
Effect.Moreover, NiPT has therapeutic effect to in-vivo tumour, especially there is Selective toxicity to leukaemia cell.The present invention
It provides more choices for clinically anti-curing oncoma, inflammation, tissue ischemia-reperfusion injury, myocardial hypertrophy.
Detailed description of the invention
Fig. 1 is the variation of ubiquitin protein amount in NiPT induction tumour cell.
Fig. 2 is the influence of NiPT specific substrate GFPu and ubiquitin protein exogenous to proteasome.
Fig. 3 is NiPT on the active influence of 20S proteasome proteolytic.
Fig. 4 is NiPT active to deubiquitinating enzymes especially 26S proteasome deubiquitinating enzymes (USP14/UCHL5)
It influences.
The case where Fig. 5 is NiPT and DNA molecular interaction.
Fig. 6 is influence of the NiPT in tumour cell to DNA damage effector molecule.
Fig. 7 is influence of the NiPT to kinds of tumor cells vigor.
Fig. 8 is influence of the NiPT to non-transformed cell (normal cell) vigor.
Fig. 9 is the Apoptosis of NiPT induction and the expression of caspase GAP-associated protein GAP.
Figure 10 is NiPT protease inhibition body function and the precedence relationship that induces cell apoptosis.
Figure 11 is influence of the EDTA to NiPT the ubiquitin protein aggregation and Apoptosis induced.
Figure 12 is influence of the NiPT to leukaemia leukoprotease body function and Apoptosis.
Figure 13 is influence of the NiPT to human peripheral blood single nucleus cell proteasome function and Apoptosis.
Figure 14 is influence of the NiPT to the inhibition situation of transplanted tumor in nude mice (A549) and to ubiquitin protein in tumor tissues.
Figure 15 is influence of the NiPT to the inhibition situation of transplanted tumor in nude mice (K562) and to ubiquitin protein in tumor tissues.
Figure 16 is the high-efficient liquid phase chromatogram and hydrogen spectrogram for synthesizing NiPT.
Specific embodiment
In the present embodiment mode, inventor has found that NiPT has apparent inhibiting effect, especially 26S egg to deubiquitinating enzymes
White enzyme body correlation deubiquitinating enzymes (USP14/UCHL5).Ni (1-oxidopyridine-2-thione) of the present invention2?
Application in preparation prevention and treatment tumour medicine.On the other hand, Ni (1-oxidopyridine-2-thione)2As removing ubiquitin
Change application of the enzyme inhibitor in the drug that preparation prevents and treats inflammation, tissue ischemia-reperfusion injury, myocardial hypertrophy disease
The preparation method of NiPT, bibliography, the specific steps are as follows:
Into the 100mL four-hole boiling flask equipped with dropping funel and thermometer, be added 6g sodium pyrithione and 10mL go from
Sub- water.The pH value for adjusting reaction mixture is 8.5, is stirred and heated to 70~75 DEG C, the nickel sulfate of dropwise addition 30% is water-soluble in 1h
Liquid 4.7g, with the addition of nickel sulfate, the pH value of reaction mixture is gradually decreased, and 10% sodium hydroxide is added dropwise and adjusts and to pH is
8.5.Insulation reaction 1h, it is cooling.Obtained precipitating is washed with distilled water 3 times, with 25mL ethanol washing 3 times, is dried at 80 DEG C,
Obtain the Ni (1-oxidopyridine-2-thione) of brown2.(referring to HOSSEINI SM, KAUFMAN C W, HOBBS
P, et al,US,5650095[P],1997-07-22).It is identified by nuclear magnetic resonance spectroscopy and efficient liquid-phase chromatography method, really
Recognizing the compound is Ni (1-oxidopyridine-2-thione)2.As a result referring to Figure 16 (a) (b).
In order to better illustrate the present invention, it is easy to understand technical solution of the present invention, combines legend by the following examples
The present invention is further elaborated, but is not used in the limitation present invention.
Embodiment 1:NiPT induces the variation of ubiquitin protein amount in tumour cell.
After being respectively acting on A549 and A459/DDP cell 12 with the NiPT (1.25,5,7.510 μM) of various concentration, receive
Collect cell extraction total protein, be NiPT (0.5,1.0,2.0,4.0 μM) with the concentration on K562 cell, carries out Western
Blot immunoblotting detects the expression of Ub-Prs, K48-, p27, PARP.The result shows that the obvious protease inhibition body of NiPT
Function.As a result referring to Fig. 1.
Western Blot method: gel required for 1 configuration SDS-PAGE.It is separated first, in accordance with recipe configuration lower layer
Glue is slowly added in the layer glass clamping plate being equipped with after configuration is good, and appropriate double steam water seal crimpings are added；It is separated to lower layer
After gelling is solid, the upper water in upper step is outwelled, comb is plugged after configured concentration glue is added, waits for quietly solidifying.2 by extraction
It after albumen is denatured by boiling, is added in gel pore according to protein concentration with identical sample size, starting electrophoresis, (concentration glue 85V, divides
From glue 100V), stop electrophoresis after a certain period of time, it will be on protein delivery to pvdf membrane using wet turn of method.After 3 transferring films,
PVDF film is taken out, 1h is closed using 5% skimmed milk power room temperature.After 4 closings, washed film 3 times, each 5min with PBST,
A certain amount of configured primary antibody is added, is incubated at room temperature 1-2h.After 5 primary antibodies are incubated for, washed film 3 times, each 5min with PBST,
A certain amount of secondary antibody is added, is incubated at room temperature 1-2h.It after 6 secondary antibodies are incubated for, is washed film 3 times, each 5min, is developed with PBST.
The influence of embodiment 2:NiPT specific substrate GFPu and ubiquitin protein exogenous to proteasome
After handling GFPu-HEK-293 cell (stable transfection GFPu) 12h with the NiPT of various concentration, cell extraction is collected
Total protein, carries out Western Blot immunoblotting (referring to embodiment 1) or fluorescence microscope is taken pictures in situ, detection Ub-Prs,
The expression of GFPu.The result shows that the obvious protease inhibition body function of NiPT.As a result referring to fig. 2.
Embodiment 3:NiPT is on the active influence of 20S proteasome proteolytic
The NiPT of various concentration acts on the 20S proteasome of purifying, and Chymotrypsin-like is added and digests substrate
Suc-LLVY-aminomethycoumarin (AMC), the fluorescence that multi-function microplate reader (350/438nm) detects AMC release are strong
Degree.The result shows that NiPT is on the Chymotrypsin-like enzymolysis activity of purifying 20S proteasome without influence.As a result referring to
Fig. 3 (a).
After handling A549, A459/DDP, K562 cell 4h with the NiPT of various concentration, collects cell extraction and crack liquid eggs
It is white, Chymotrypsin-like enzymatic hydrolysis substrate Suc-LLVY-aminomethycoumarin (AMC), multifunctional enzyme mark is added
Instrument (350/438nm) detects the fluorescence intensity of AMC release.The result shows that NiPT is to intracellular 20S proteasome
Chymotrypsin-like enzymolysis activity is without influence.As a result referring to Fig. 3 (b) (c) (d).
Embodiment 4:NiPT is to deubiquitinating enzymes especially 26S proteasome deubiquitinating enzymes (USP14/UCHL5) activity
Influence
Binding ability and the mode of action of the computer molecular docking technology to NiPT and 26S proteasome USP14, UCHL5
Carry out simulation and forecast.Experimental applications software Discovery Studio 2.0 carries out specific molecular docking, specific experiment method
Are as follows: receptor prepares: protein structures (come from PDB), delete unnecessary molecule, add polarity hydrogen to receptor；Ligand is quasi-
It is standby: the processing of energy minimum is carried out (in view of in physiological conditions, NiPT (L1) can be dissociated, so ginseng to ligand molecular
It is NiPT ion (L2) with the molecule docked, then carries out molecular docking with CDOCKER.As a result referring to fig. 4 (a).
Using purifying 26S (a) and donor of the untreated cell pyrolysis liquid (b) as DUBs, NiPT (0.5,1.0,
2.0 μM) act on albumen 30min after, Ub-AMC (400nM) substrate as DUBs (USP class/UCH class) is added, it is multi-functional
The every 2min detection of microplate reader (350/438nm) is primary, observes the active dynamic change of DUB.NiPT can inhibit thin as the result is shown
The activity of deubiquitinating enzymes intracellular especially 26S proteasome deubiquitinating enzymes (USP14/UCHL5).As a result referring to fig. 4 (b)
(c)(d)。
UbVS is cysteine class DUBs specific inhibitor, and NiPT contestable antagonism UbVS is to 26S proteasome
The inhibiting effect of DUBs.It may determine that the combination situation of NiPT and UCHL5 and USP14 by detecting HA-UbVS.This experiment is used
The NiPT of various concentration acts on purifying 26S proteasome 30min, and wide spectrum DUBs inhibitor NEM is as positive control；Later plus
Enter HA-UbVS (250ng) reaction 30min, it is then horizontal using Western blot method (referring to embodiment 1) detection HA.Knot
Fruit shows that with UCHL5 and USP14 covalent bond can occur for NiPT.As a result referring to fig. 4 (e).
After acting on purifying 26S proteasome 30min with the NiPT (5,50 μM) of various concentration, ubiquitin tetramer chain is added
Substrate of the Ub4 (K48-linked) as DUBs (USP14/UCHL5/POH1), using using Western blot method (referring to
Embodiment 1) detection Ub4 cracking situation.NiPT can inhibit to dose-dependant the activity of 26S proteasome DUBs as the result is shown.Knot
Fruit is referring to fig. 4 (f).
The case where embodiment 5:NiPT and DNA molecular interact
NiPT (the 1.93*10 of fixed concentration-5M is dissolved in DMSO), gradually increase DNA solubility [0 (a), 1.42 (b),
2.85 (c), 4.27 (d) * 10-5M is dissolved in Tris-HCl buffer, 0.05M, pH=7.40], to contain comparable sodium DMSO's
Tris-HCl buffer solution is reference, scans absorption spectrum of the different mixed systems within the scope of 200-600nm.The result shows that
After mixing with DNA, absorption spectrum of the NiPT within the scope of 300-500nm does not change, thus it is speculated that does not have between NiPT and DNA
It interacts.As a result referring to Fig. 5 (a).
Concentration [c (EB)=2 μM, c (DNA)=1.4*10 of fixed EB-DNA system-5M is dissolved in Tris-HCl buffer,
0.05M, pH=7.40], PtPT [1.5 (A), 3.8 (B), 7.2 (C) * 10 of various concentration are added thereto-5M], excitation wavelength
514nm scans its fluorescence spectrum.The concentration of contained DMSO is identical in solution.The result shows that NiPT is added, EB-DNA system
Fluorescence intensity and peak position are without apparent variation.Speculate that the binding pattern between NiPT and DNA may not be common intercalation knot
It closes.As a result referring to Fig. 5 (b).
Influence of the embodiment 6:NiPT in tumour cell to DNA damage effector molecule
Respectively use CDDP (2.5uM), NiPT (1.25uM) handle A549 cell 3h, 6h, 12h after, by cell with 3% it is more
It is cleaned after polyformaldehyde is fixed, wears film with X-100 containing 1%Triton and 0.5%NP-40,5% BAS closing is added after cleaning,
It sucks BSA and primary antibody (anti-γ-H2AX antibody) is added dropwise, after being incubated for 1-2h, 0.1%NP-40 is washed 3 times, and secondary antibody is added dropwise, it is incubated for 1-2h,
0.1%NP-40 is washed 3 times, and dropwise addition DAPI develops a film after being protected from light 20min and mounting, fluorescence microscope are taken pictures.The result shows that NiPT is not
Cause the expression of intracellular DNA damage marker γ-H2AX.As a result referring to Fig. 6 (a).
After handling K562 cell 6h with the NiPT (0.25,0.5,1.0uM) and CDDP (2.5,5,10uM) of various concentration,
Collect cell extraction total protein；Concentration on A549 cell is then: NiPT (1.25,2.5,5.0uM) and CDDP (2.5,
5,10uM).Using Western Blot method detect DNA damage GAP-associated protein GAP γ-H2AX, p-ATM, p-chk2, p-chk1
With the expression of PARP.The result shows that PtPT does not generate DNA damage effect with concentration increase in tumour cell.As a result
Referring to Fig. 6 (b).
After NiPT (2.5uM) and CDDP (2.5uM) acts on A549 cell 6h, 12h, 18h, the total egg of cell extraction is collected
It is white, using Western Blot method detect DNA damage GAP-associated protein GAP γ-H2AX, p-ATM, p-chk2, p-chk1 and
The expression of PARP.The result shows that PtPT does not generate DNA damage effect as time went in tumour cell.As a result join
See Fig. 6 (c).
Influence of the embodiment 7:NiPT to kinds of tumor cells vigor
(1.25,2.5,5.0,10,20 μM) processing U266, K562 of NiPT and CDDP of various concentration, A549/DDP,
After A549, SMMC-7721 cell 48, MTS solution is added and is incubated for 1-4h, microplate reader (490nM) detects its absorbance value.As a result
It has been shown that, compared to CDDP, NiPT can obviously inhibit the vigor of kinds of tumor cells, and be in dose-dependence.As a result referring to figure
7。
Influence of the embodiment 8:NiPT to non-transformed cell (normal cell) vigor
After (1.25,2.5,5.0,10,20 μM) processing LO2,16HBE cells 48 of NiPT and CDDP of various concentration, it is added
MTS solution is incubated for 1-4h, and microplate reader (490nM) detects its absorbance value.The results show that suppression of the NiPT to non-transformed cell vigor
It makes weak compared with CDDP.As a result referring to Fig. 8.
With (5.0,10,20 μM) processing K562 cells of PtPT (2.5,5.0,10 μM) and CDDP of various concentration for 24 hours after,
The bis- transfection reagents of AnnexinV-FITC/PI, flow cytomery Apoptosis situation is added.The left side is statistic histogram.As a result
Show PtPT can dose-dependant induction K562 Apoptosis and effect be substantially better than CDDP.As a result referring to Fig. 8.
The Apoptosis of embodiment 9:NiPT induction and the expression of caspase GAP-associated protein GAP
With (0.25,0.5,1.0 μM) processing K562 cell of NiPT of various concentration for 24 hours after, AnnexinV-FITC/ is added
The bis- transfection reagents of PI, flow cytomery Apoptosis situation.The left side is statistic histogram.The result shows that NiPT can dosage according to
Rely induction K562 Apoptosis.As a result referring to Fig. 9 (c).
After various concentration NiPT (0.25,0.5,1.0 μM) acts on K562 cell for 24 hours, cell extraction total protein is collected；With
(0.5 μM) processing K562 cell 12h of NiPT, for 24 hours, after 36h, collect cell extraction total protein.Then Western Blot is carried out
Immunoblotting (referring to embodiment 1) detects the expression of caspase-3, -8, -9 and PARP and its activated form.As a result table
Bright, NiPT can induce tumour cell PARP cutting and caspase activation.As a result referring to Fig. 9 (a).
With (0.25,0.5,1.0 μM) processing K562 cell of NiPT of various concentration for 24 hours after, rhodamine dyeing is added
Reagent, flow cytomery mitochondrial membrane potential situation.The left side is statistic histogram.The result shows that NiPT can dose-dependant
Cause the decline of K562 mitochondrial membrane potential in anoxic.As a result referring to Fig. 9 (b).
Precedence relationship embodiment 10:NiPT protease inhibition body function and induced cell apoptosis
After handling A549, A549/DDP, K562 cell respectively with NiPT (5 μM), at the appointed time (6h, 12h, 18h,
Cell extraction total protein is collected for 24 hours), is carried out Western Blot immunoblotting (referring to embodiment 1), detection Ub-Prs, K48-,
The expression of p27, PARP.The result shows that NiPT causes the cutting of PARP after protease inhibition body function.As a result referring to
Figure 10.
Influence of the embodiment 11:EDTA to NiPT the ubiquitin protein aggregation and Apoptosis induced
After (5 μM) joint EDTA (100 μM) of NiPT (5 μM) or NiPT act on A549 cell 12h, it is total to collect cell extraction
Albumen carries out Western Blot immunoblotting (referring to embodiment 1), detects the expression of Ub-Prs, PARP.As a result table
Bright, the cutting of the ubiquitin protein aggregation and PARP of NiPT induction can be reversed in EDTA.As a result referring to Figure 11 (a).
After (5 μM) joint EDTA (100 μM) of NiPT (5 μM) or NiPT act on K562 cell 12h, AnnexinV- is added
The bis- transfection reagents of FITC/PI, flow cytomery Apoptosis situation.The left side is statistic histogram.The result shows that EDTA is reversible
Turn the Apoptosis of NiPT induction.As a result referring to Figure 11 (b).
Influence of the embodiment 12:NiPT to leukaemia leukoprotease body function and Apoptosis
In the case where informed consent, 6 normal persons (Nm) and 6 leukemia patient (Pt) blood are extracted as sample,
It is added after handling cell for 24 hours with the NiPT of various concentration by the mononuclearcell in lymphocyte separation medium separating sample
MTS solution is incubated for 1-4h, and microplate reader detects its absorbance value at 490nM.Scatter plot is done with IC50 value.The results show that
NiPT has selectivity to leukaemia cell.As a result referring to Figure 12 (a).
Leukemia patient (#2, ALL) cell isolates mononuclearcell by lymphocyte separation medium, with various concentration
(0.25,0.5,1.0 μM) processing cell of NiPT for 24 hours after, be added the bis- transfection reagents of AnnexinV-FITC/PI, flow cytometer examine
Survey Apoptosis situation.The left side is statistic histogram.The result shows that NiPT can dose-dependant induced apoptosis in leukemia cell lines.Knot
Fruit is referring to Figure 12 (b).
Leukemia patient (#1, CML/#3, AML) cell isolates mononuclearcell by lymphocyte separation medium, with not
With concentration NiPT (0.25,0.5,1.0 μM), CDDP (10 μM) and Vel (50nM) processing cell for 24 hours after, be added
The bis- transfection reagents of AnnexinV-FITC/PI, fluorescence microscope are taken pictures in situ.The result shows that NiPT can dose-dependant inducing leukemia
Apoptosis.As a result referring to Figure 12 (c).
Leukemia patient (#2, #4, #7) cell isolates mononuclearcell by lymphocyte separation medium, uses various concentration
(0.5,1.0,2.0 μM) processing cell of NiPT for 24 hours after, collect cell extraction total protein, pass through Western Blot method (ginseng
See embodiment 1) detection Ub-Prs, K48- and PARP expression.The result shows that NiPT can inhibit leukaemia cell's protease
Body function and induced apoptosis in leukemia cell lines.As a result referring to Figure 12 (d).
Influence of the embodiment 13:NiPT to human peripheral blood single nucleus cell proteasome function and Apoptosis
Normal human peripheral blood (#2) isolates mononuclearcell by lymphocyte separation medium, with the NiPT of various concentration
After (0.25,0.5,1.0 μM), CDDP (10 μM) and Vel (50nM) handle cell for 24 hours, the bis- dyes of AnnexinV-FITC/PI are added
Reagent, fluorescence microscope are taken pictures in situ.The result shows that NiPT induces human peripheral blood single nucleus cell apoptosis energy compared with CDDP and Vel
Power is weak, has cell selective.As a result referring to Figure 13 (a).
Normal human peripheral blood (#3, #6) isolates mononuclearcell by lymphocyte separation medium, with various concentration
After NiPT (0.25,0.5,1.0 μM), CDDP (10 μM) and Vel (50nM) handle cell for 24 hours, pass through Western Blot method
Detect the expression of Ub-Prs, K48- and PARP.The result shows that NiPT is to human peripheral blood single nucleus cell proteasome function
Inhibit weaker, there is cell selective.As a result referring to Figure 13 (b).
Inhibition situation of the embodiment 14:NiPT to transplanted tumor in nude mice (A549) and the shadow to ubiquitin protein in tumor tissues
It rings
By A549 cell suspension (5 × 106/ ml, serum-free RPMI1640 culture medium) inoculate it is naked in 12 BALB/c
On the outside of mouse (18-20g is purchased from Guangdong Province's animal center) back, it is randomly divided into 2 groups (control group, NiPT groups), every group 6, abdominal cavity
It is administered (NiPT 5mg/kg/day), records nude mice weight daily, record nude mouse tumor volume every other day, continue 15 days.Tumour is big
It is small, tumor weight, nude mice weight, tumor volume change.The results show that NiPT can significantly inhibit the life of transplanted tumor in nude mice (A549)
It is long.As a result referring to Figure 14 (a) (b) (c) (d).
The nude mice of A549 cell is inoculated with after intraperitoneal administration, 2 groups (control group, NiPT group) transplanting tumor tissues are taken, using exempting from
Epidemic disease group method detects the expression of Ub-Prs, K48-, p21 and Cleaved-caspase3 in tumor tissues.As a result table
Bright, NiPT can cause the aggregation of ubiquitin protein matter in A549 tumor tissues.As a result referring to Figure 14 (e).
Inhibition situation of the embodiment 15:NiPT to transplanted tumor in nude mice (K562) and the shadow to ubiquitin protein in tumor tissues
It rings
By K562 cell suspension (5 × 106/ ml, serum-free RPMI1640 culture medium) inoculate it is naked in 12 BALB/c
On the outside of the back mouse (18-20g, be purchased from Guangdong Province's animal center), be randomly divided into 2 groups (control group, NiPT groups), concrete scheme referring to
Embodiment 14.The results show that NiPT can significantly inhibit the growth of transplanted tumor in nude mice (K562) and cause nude mice K562 tumor tissues
The aggregation of middle ubiquitin protein matter.As a result referring to Figure 15.
Each technical characteristic of embodiment described above can be combined arbitrarily, for simplicity of description, not to above-mentioned reality
It applies all possible combination of each technical characteristic in example to be all described, as long as however, the combination of these technical characteristics is not deposited
In contradiction, all should be considered as described in this specification.
The embodiments described above only express several embodiments of the present invention, and the description thereof is more specific and detailed, but simultaneously
It cannot therefore be construed as limiting the scope of the patent.It should be pointed out that coming for those of ordinary skill in the art
It says, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to protection of the invention
Range.Therefore, the scope of protection of the patent of the invention shall be subject to the appended claims.
Claims (1)
1. the following Ni of structural formula (1-oxidopyridine-2-thione)2Application in preparation prevention and treatment tumour medicine
The tumour is leukaemia.
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| CN108938630B (en) * | 2017-05-22 | 2020-03-03 | 广州医科大学 | New application of 2-mercaptopyridine-N-oxide gold salt compound |
| CN108295091A (en) * | 2018-02-26 | 2018-07-20 | 广州医科大学 | The application of AgDT compounds |
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Non-Patent Citations (4)
| Title |
|---|
| Cu(II)/Ni(II)-2-巯基吡啶-N-氧化物的制备及表征;陈颂仪等;《中国药科大学学报》;19951231;第26卷(第1期);62-64 * |
| Nickel Complexes for Robust Light-Driven and Electrocatalytic Hydrogen Production from Water;Amit Das, et al.;《ACS Catal》;20150114;1397-1406 * |
| The cytotoxicity of copper(II) complexes of heterocyclic thiosemicarbazones and 2-substituted pyridine N-oxides;Douglas X West, et al.;《Anti-Cancer Drugs》;19931231;第4卷;241-249 * |
| 陵水暗罗枝、叶中的巯基吡啶类化合物及其细胞毒活性研究;吕子明等;《中国中药杂志》;20100131;第35卷(第1期);53-57 * |
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