CN103961711A - Synergetic Application of nicotinamide phosphoribosyltransferase (NAMPT) depressor and NQO1 substrate to treatment of non-small cell lung cancer - Google Patents
Synergetic Application of nicotinamide phosphoribosyltransferase (NAMPT) depressor and NQO1 substrate to treatment of non-small cell lung cancer Download PDFInfo
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- CN103961711A CN103961711A CN201410222694.3A CN201410222694A CN103961711A CN 103961711 A CN103961711 A CN 103961711A CN 201410222694 A CN201410222694 A CN 201410222694A CN 103961711 A CN103961711 A CN 103961711A
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Abstract
The invention relates to a combined drug, comprising a certain amount of nicotinamide phosphoribosyltransferase (NAMPT) depressor and a certain amount of NQO1 substrate. The NAMPT depressor and the NQO1 substrate have synergistic effects in the anti-tumor process, and the action of the composition in the case of combined utilization of the NAMPT depressor and the NQO1 substrate is superior to the superposition when all the drugs are independently used. The combined drug can be applied to treatment of the non-small cell lung cancer and other cancers. Therefore, a novel treatment method for treating the non-small cell lung cancer is provided by the invention.
Description
Technical field
The present invention relates to natural medicine field and pharmaceutical field, be specifically related to NAMPT inhibitor is combined the medicine of preparation treatment tumor purposes with NQO1 substrate.More specifically, relate to the purposes of NAMPT inhibitor and NQO1 substrate Synergistic treatment nonsmall-cell lung cancer.
Background technology
Nonsmall-cell lung cancer is the malignant tumor that M & M is the highest, in the cause of death being caused by tumor, ranked first position.There is no at present the Therapeutic Method of effective low toxicity.
The nicotinamide adenine dinucleotide nicotinamide adenine dinucleotide NAD+ that abridges.Can in oxidation-reduction process, from substrate, accept a hydrogen atom and an electronics, become reduced form, in the electron transfer process of respiratory chain, play core pivotal role.NAD+ has also obtained broad research in the effect that maintains the aspects such as cell metabolic activity, cellular stress opposing and life in recent years.For example, NAD+ can be used as ADP-ribosyl donor and participates in the activation process of DNA repairase PARP, and then affects DNA damage and reply; Also can participate in transcriptional control as the active necessary coenzyme of deacetylase SIRT1 performance, stress resist and the process such as cell differentiation.In cell, the regulation and control of NAD+ level are to determining that cell fate plays an important role.NAD+ is synthetic to be divided into de novo synthesis and to remedy synthetic two paths, and wherein salvage route is the main source of NAD+ in cell.Nicotinamide phosphoribosyl transferase (NAMPT) is being controlled NAD+ and is being remedied in synthetic path niacin amide (NAM) to the conversion of NAD+, for remedying synthetic rate-limiting enzyme.The existing part of NAMPT inhibitor based on lack performance antitumaous effect by NAD+ enters clinical trial.CN103347860A expresses the method for the disease of NAMPT during disclosing and having suppressed the compound of NAMPT activity, the compositions that contains this compound and treat disease.CN103384668A discloses compound for suppressing NAMPT and compositions and synthetic, application and antidote.FK866 is the effective inhibitor of NAMPT of finding the earliest, suppresses constant K
ifor nmol rank.The tests such as Tie-qiang Bi and Xiang-ming Che show, FK866 is less in non-tumor cell toxic, can suppress kinds of tumor cells growth, and non-evident effect.
Tanshinone ⅡA and β-lapachol are NQO1 targeted drug, via NQO1 metabolism and bring into play antitumor action.
Tanshinone ⅡA structural formula is:
Molecular formula is C
19h
18o
3.
β-lapachol structural formula is:
Molecular formula is C
15h
16o
3.
CN1264580 discloses TANSHINONES in the new purposes of preparing in medicine for treating tumor thing, in animal body, experiment and clinical trial show that TANSHINONES has the good result of killing tumor cell, inducing tumor cell differentiation and inducing apoptosis of tumour cell, and toxic and side effects is little, have a good application prospect.CN103006669A discloses tanshinone ⅡA mediation FOXO1 Dependent antineoplastic mechanism, relates generally to tanshinone ⅡA by the cells apoptosis of FOXO1 Dependent induction Non-small cell lung carcinoma cell line A549.The people such as Huang X have opened altogether β-lapachol NQO1 dependency in A549 cell and have caused also characteristic (the Huang X of inducing apoptosis of tumour cell of NAD+ downward in 2012, DongY, Bey EA, Kilgore JA, Bair JS, Li LS.An NQO1substratewith potent antitumor activity that selectively kills by PARP1-induced programmed necrosis.Cancer Res.2012Jun15; 72 (12): 3038-47.)
Summary of the invention
The object of this invention is to provide the drug combination method of oncotherapy safely and effectively, adopt NAMPT inhibitor and NQO1 substrate combination medicine, to reach the effect of collaborative onset.
NQO1 substrate of the present invention comprises tanshinone ⅡA and β-lapachol.NAMPT inhibitor refers to material or the medicine that can suppress NAMPT activity, blocking-up NAD+ generation.The selected NAMPT inhibitor of the present invention is FK866.In invention, explain NAMPT inhibitor and NQO1 substrate and there is synergism in induction Non-small cell lung carcinoma apoptosis process.The demonstration of MTT experimental result, NAMPT inhibitor FK866 can increase the weight of NQO1 substrate tanshinone ⅡA and the cytotoxicity of β-lapachol to A549 cell.LC-MS
nnAD after detection administration
+level, finds that FK866 combined effect can significantly aggravate the NAD that tanshinone ⅡA and β-lapachol cause
+level reduces.The active test experience result of SIRT1 shows, FK866 can increase the weight of tanshinone ⅡA and the inhibition of β-lapachol to SIRT1 activity.Western Blot experimental result shows that FK866 can strengthen Ac-FOXO1 activation and the PARP1 activation of tanshinone ⅡA and the induction of β-lapachol, the inhibition of aggravation to SIRT1 albumen.
Brief description of the drawings
Fig. 1: tanshinone ⅡA and β-lapachol time dependence raise NAD
+salvage pathway synzyme NAMPT expresses
Fig. 2: A, FK866 increases the cytotoxicity of tanshinone ⅡA to A549 cell; B, FK866 increases the cytotoxicity of β-lapachol to A549 cell
Fig. 3: FK866 can increase the weight of the NAD that tanshinone ⅡA and β-lapachol cause
+level reduces
Fig. 4: FK866 can increase the weight of tanshinone ⅡA and the inhibition of β-lapachol to SIRT1 activity
The Ac-FOXO1 that Fig. 5: FK866 can strengthen tanshinone ⅡA and the induction of β-lapachol activates and PARP1 activation, aggravates the two inhibition to SIRT1 albumen.
Detailed description of the invention
Following embodiment, sets forth anticancer usage of the present invention by mechanism, but does not represent embodiment limitation of the present invention.
Embodiment 1.PCR experiment
Experiment material:
Non-small cell lung carcinoma cell A549 cell is purchased from ATCC, in 37 DEG C, and 5%CO
2cellar culture under condition, culture medium is the RPMI-1640 (Gibco) containing 10% calf serum (PAA); Hyclone (Fetal bovine serum) is purchased from Hyclone (Logan, Utah, USA); Trypsin Trypsin) be purchased from Amersco (Solon, Ohio, USA); Penicillin, streptomycin, dimethyl disulfide are all purchased from Nanjing Sheng Xing bio-engineering corporation (Jiangsu) for diphenyl four thiazoles (MTT), GAPDH antibody; Trizol total RNA extraction reagent, RT-PCR test kit, Real-time PCR Master Mix (SYBR Green) are purchased from Takara (Dalian is precious biological).
Experimental technique:
A549 cell is inoculated in 6 orifice plates, administration tanshinone ⅡA 40 μ M, or β-lapachol 20 μ M (2h withdrawal) processing time be respectively 0h, 2h, 4h, 8h, 12h, 24h; .Every 1 × 10
6in cell, add 1mL Trizol, blow to liquid clarification and acellular agglomerate and be transferred in 1.5mL EP pipe with 1mL liquid-transfering gun, extract total RNA according to the operation of Takara total RNA extraction reagent box, and it is quantitative to carry out RNA.Be cDNA according to RT-PCR test kit by mRNA reverse transcription, require to carry out PCR test operation according to ThermalCycler DiceTM Real Time System (TaKaRa Code:TPS00) operation instructions.Quantitative PCR reaction condition:
Stage1: denaturation: 95 DEG C, 30sec
Stage2:PCR reaction: 95 DEG C, 5sec; Tm, 15sec; 72 DEG C, 30sec; 45Cycles
Stage3: melt curve analysis analysis: 95 DEG C of 15sec; 70 DEG C of 15sec.
Adopt Livak method semi-quantitative analysis RT-PCR testing result.
Embodiment 2.MTT experiment
A549 cell is inoculated in 96 orifice plates, and incubation growth to 80% is full, gives FK86610nM, gives variable concentrations tanshinone ⅡA (0,0.4,1,2,4,10,20,40 μ M) and process 72h after 1 hour; Or change without medicine culture medium culturing to 24h after β-lapachol (0,1.25,2.5,5,10,20 μ M) processing 2h.Change in serum-free medium every hole and add 20 μ L (5mg/mL) MTT, put temperature in CO2 gas incubator and remove MTT solution after incubating 4h, add dissolving crystallized (the 37 DEG C of shaking tables of DMSO150 μ L, 50r/min), after 10min, take out to be placed in microplate reader (measuring wavelength 570nm, reference wavelength 630nm) and measure its absorbance.Administration group cell survival rate calculates with following formula:
Survival rate=processed group absorbance/matched group absorbance * 100%
Embodiment 3.LC-MS
ndetect NAD in cell
+level
A549 cell is seeded to 6 orifice plates, and incubation growth to 80% is full, gives FK86610nM, gives 40 μ M tanshinone ⅡAs and process 48 hours after 1 hour; Or 20 μ M β-lapachol is processed 24h.Discard culture medium, normal saline swings washes cell, every hole adds 1ml80% to contain 50ng/ml2-chlorine adenosine (interior mark) methanol, place 20 minutes for-80 DEG C, ice bath scrapes cell proceed to EP pipe, ultrasonication, centrifugal 5 minutes of 14000rpm, supernatant proceeds in new EP pipe, volatilizes 100 μ l ultra-pure waters redissolution sample introductions.
Chromatographic condition: chromatographic column is amide post (3x100mm i.d., 5 μ m, Chrom-Matrix Inc), 40 DEG C of column temperatures.Mobile phase: water (A) is the aqueous solution containing 5mM ammonium acetate, organic facies (B) is methanol, column temperature is 40 DEG C, flow velocity is 0.2mL/min, adopts gradient elution mode to carry out chromatographic isolation, and gradient arranges as follows: 0.50-2.5min80% (B), 2.5-4min80%-30% (B), 4-6.5min30% (B), 6.5-7.5min30-80% (B), 7.5-15.0min80% (B); Sampling volume is 10 μ L.
Mass spectrum condition: with electric spray ion source (ESI), setting source parameter is respectively: spray voltage (IonSpray Voltage/IS) 4500V, assisted gas 1 (Ion Source Gas1/GS1, N2) 12Arb, assisted gas 2 (Ion Source Gas2/GS2, N2) 50Arb, 500 DEG C of assisted gas heating-up temperatures (Temperature/TEM), gas curtain gas (Curtain Gas/CUR) 30Arb, collision gas (Collision Gas/CAD, N2) 4Pa.Select multiple ion reaction monitoring (MRM) under positive ion mode (Positive), setting Q0 entrance voltage (Entrance Potential/EP) is 10V, and Q2 outlet voltage (Collision CellExit Potential/CXP) is 15V.The MRM parameter of NAD+ is: parent ion (Q1Mass) is 664.300Da, daughter ion (Q3Mass) is 136.300Da, removing a bunch voltage (Declustering Potential/DP) is 95V, and collision voltage (Collision Energy/CE) is 60eV.The MRM parameter of NADH is: parent ion (Q1Mass) is 666.300Da, daughter ion (Q3Mass) is 649.200Da, removing a bunch voltage (Declustering Potential/DP) is 93V, and collision voltage (Collision Energy/CE) is 23eV.The MRM parameter of interior mark (IS) is: parent ion (Q1Mass) is 302.200Da, daughter ion (Q3Mass) is 169.900Da, removing a bunch voltage (Declustering Potential/DP) is 70V, and collision voltage (Collision Energy/CE) is 25eV.NAD+, NADH appearance time is 3.23min, inside marking peak time is 3.41min.
Embodiment 4.SIRT1 is active to be detected
The active detection of SIRT1 illustrates and tests according to fluorescence SIRT1 detection kit (Sigma CS1040).
Cell extracts through lysate, ultrasonication, and centrifuging and taking supernatant 20 μ L add black surround clear bottom 96 orifice plates, with 10 μ L fluorescence SIRT1 substrates and 5 μ LNAD
+solution mixes, incubated at room 30 minutes.Add 5 μ L nitrite ions to hatch 10 minutes.Fluorescence microplate reader reads the fluorescence absorbance of excitation wavelength 340nm, emission wavelength 430nm.
Taking fluorescence standard preparation abscissa in test kit as fluorogenic substrate concentration, vertical coordinate is the standard curve of absorbance, experimental group numerical value is to represent matched group percent.
Embodiment 5.Western Blot experiment
A549 cell is inoculated in 6 orifice plates, and incubation growth to 80% is full, gives FK86610nM, gives 40 μ M tanshinone ⅡAs and process 48 hours after 1 hour; Or 20 μ M β-lapachol is processed 24h.PBS washed cell once after, be placed on slab, cell is scraped to collection with cell sleaker, the centrifugal cell precipitation that obtains of 8000rpm × 1min, in every 20 μ L cell packs, add 100 μ LRIPA cell pyrolysis liquids, on ice after cracking 30min, ultrasonication 5 times, each 2s, 15000rpm × 10min is centrifugal, and the supernatant that obtains is the total protein sample extracting.BCA method is measured protein content.Protein sample adds sample-loading buffer in 3: 1 by volume and boils 5min.Preparation 8%SDS-PAGE, carries out electrophoretic separation to sample.After electrophoretic separation, the electrotransfer that wets, goes to pvdf membrane by albumen.Pvdf membrane, sealing after 1h containing in the TBST solution of 5% defatted milk powder, is hatched primary antibodie for 4 DEG C and is spent the night, corresponding two anti-incubated at room 1h, and ECL develops, the imaging of Bio-Rad gel imaging instrument.
Claims (6)
1. a combination medicine, comprises NAMPT inhibitor and NQO1 substrate and optionally can comprise one or more pharmaceutically suitable carrier.
2. combination medicine claimed in claim 1, is characterized in that this combination medicine comprises the pharmaceutical composition that contains NAMPT inhibitor and NQO1 substrate and optional one or more pharmaceutically suitable carrier of associating.
3.NAMPT inhibitor and NQO1 substrate have the purposes of antitumor action medicine in preparation.
4.NAMPT inhibitor is in the purposes that is used for the treatment of nonsmall-cell lung cancer or other cancers for the preparation of administration together with NQO1 substrate.
5.NQO1 substrate is in the purposes that is used for the treatment of nonsmall-cell lung cancer or other cancers for the preparation of administration together with NAMPT inhibitor.
6. be used for the treatment of a method for nonsmall-cell lung cancer, the method comprises the NQO1 substrate and the treatment of NAD+ synthetic inhibitor combination medicine that use effective dose.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104524579A (en) * | 2014-11-26 | 2015-04-22 | 中国药科大学 | Denovo synthesis inhibitor with NQO1 targeted drug as NAD+ and application of denovo synthesis inhibitor to tumor resistance |
| WO2023246464A1 (en) * | 2022-06-24 | 2023-12-28 | 澳门大学 | Use of cryptotanshinone substance and combined composition thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102869261A (en) * | 2010-03-01 | 2013-01-09 | 瑞科西有限公司 | Compounds and therapeutic uses thereof |
| CN103006669A (en) * | 2013-01-04 | 2013-04-03 | 中国药科大学 | Application of tanshinone IIA to giving play to anti-tumor effect mechanism via FOXO1 pathway |
| US20130317027A1 (en) * | 2010-03-01 | 2013-11-28 | Myrexis, Inc. | Compounds and therapeutic uses thereof |
-
2014
- 2014-05-23 CN CN201410222694.3A patent/CN103961711A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102869261A (en) * | 2010-03-01 | 2013-01-09 | 瑞科西有限公司 | Compounds and therapeutic uses thereof |
| US20130317027A1 (en) * | 2010-03-01 | 2013-11-28 | Myrexis, Inc. | Compounds and therapeutic uses thereof |
| CN103006669A (en) * | 2013-01-04 | 2013-04-03 | 中国药科大学 | Application of tanshinone IIA to giving play to anti-tumor effect mechanism via FOXO1 pathway |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104524579A (en) * | 2014-11-26 | 2015-04-22 | 中国药科大学 | Denovo synthesis inhibitor with NQO1 targeted drug as NAD+ and application of denovo synthesis inhibitor to tumor resistance |
| WO2023246464A1 (en) * | 2022-06-24 | 2023-12-28 | 澳门大学 | Use of cryptotanshinone substance and combined composition thereof |
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