CN109061185A - A method of copper carrier antitumor and anticancer agent is designed based on redox cycle strategy - Google Patents

A method of copper carrier antitumor and anticancer agent is designed based on redox cycle strategy Download PDF

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CN109061185A
CN109061185A CN201810925317.4A CN201810925317A CN109061185A CN 109061185 A CN109061185 A CN 109061185A CN 201810925317 A CN201810925317 A CN 201810925317A CN 109061185 A CN109061185 A CN 109061185A
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张军民
房建国
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Lanzhou University
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Abstract

The purpose of the present invention is to provide a kind of methods based on redox cycle strategy design copper carrier antitumor and anticancer agent.Utilize the special redox character of cancer cell, in the cell under the participation of two antioxidant systems of thioredoxin and glutathione, dexterously using intracellular small molecule mercaptan, copper carrier and copper ion is triangular reacts to each other, achievees the purpose that carrier is split in the cell, regeneration, delivers copper ion again.The present invention is compared with the traditional method, the efficiency with very high transport copper ion, and is had a extensive future.

Description

A method of copper carrier antitumor and anticancer agent is designed based on redox cycle strategy
Technical field
The present invention relates to chemical medicines, are carried in particular to one kind based on redox cycle strategy design copper The method of body antitumor and anticancer agent.
Background technique
Compared with normal cell, cancer cell show increased active oxygen (ROS) level to maintain its malignant phenotype to increase, To rely more on the mechanism of " redox adaptation ", i.e., ROS level is maintained to (Nat in the range of allowing its cheat death Rev Drug Discov., 2013,12 (12): 931-947).Based on such biochemical differences, provided for the design of antitumor and anticancer agent Brand-new method.
Copper level in cancer cell significantly increase compared with normal cell (Cancer Treat Rev., 2009,35 (1): 32-46), when intracellular copper concentration is increased, the therapeutic window between normal cell and cancer cell makes copper ion carrier anti- Cancer drug develops into possible (Chem Rev., 2014,114 (1): 815-62).Importantly, due to clinical mainstream anticancer The problems such as toxic side effect and drug resistance of drug platinum class, so that replacing the platinum in platinum medicine to become metal kind anti-cancer drugs with copper One of the trend of object research and development (Metallomics, 2015,7 (11): 1459-1476).Based on this, using special in cancer cell Copper level design antitumor and anticancer agent become inevitable.
Has the report using both the above strategy development anticancer drug at present, but special in cancer cell there has been no utilizing Redox environment and external copper ion between the renewable carrier of reaction designing, thus reach can recycle delivery copper ion The method of purpose.More importantly existing copper will be avoided by developing the method for this redox cycle strategy design copper carrier Copper ion is unidirectionally brought into intracellular drawback by carrier, is had the necessity of reality and is had a extensive future.
Summary of the invention
Method of the invention is a kind of based on redox cycle strategy design copper load the technical problem to be solved is that providing The method of body antitumor and anticancer agent.
To solve the above problems, of the present invention a kind of based on redox cycle strategy design copper carrier antitumor and anticancer agent The mentality of designing of method are as follows: using the special redox character of cancer cell, thioredoxin (Trx) and paddy in the cell Under the participation of the sweet peptide of Guang (GSH) two antioxidant systems, dexterously using intracellular small molecule mercaptan, copper carrier and copper from Son is triangular to be reacted to each other, and achievees the purpose that carrier splits, regenerates, reruns copper ion in the cell.
To solve the above problems, of the present invention a kind of based on redox cycle strategy design copper carrier antitumor and anticancer agent Method, the specific steps are as follows:
(1) it is based on the above mentality of designing, design can deliver the copper ion, control molecule that can not deliver copper ion, intracellular Molecule after dismantling, specific molecular structure are as follows:
(2) three above molecule cooperates with the investigation to cancer cell toxicity with copper ion.
(3) measurement of cellular redox state.
(4) in extracellular fluid carrier molecule concentration measurement, regenerate under confirmation carrier Redox Condition in the cell.
The method of the present invention is compared with the prior art (Fig. 1):
Art methods (Figure 1A): most of copper carriers and copper ion form complex compound (Eq.1 in Figure 1A) and make copper Ions across cell membranes.Since the intracellular concentration of copper is lower, copper complex is dissociated to passively release copper ion (in Figure 1A Eq.2).However, once intracellular content of copper ion reaches certain level, the reversible reaction Eq.2 of equation becomes advantageous. Obviously, it is this delivery copper ion method to copper from the efficiency of conveying by the inherent balance constant of carrier and intracellular copper Limitation.
The method of the present invention (Figure 1B): carrier is coordinated with copper ion first and passes through cell membrane, enters in carrier-copper complex After cell, intracellular environment can destroy the structure of carrier to discharge copper ion (Eq.2 in Figure 1B).In addition, carrier can be with (Eq.3 in Figure 1B) is regenerated under the conditions of certain cells, reaches circulation delivery copper ion, it is clear that this method obviously mentions The high conevying efficiency of copper.
The design method of reproducible copper ion carrier of the present invention has novelty well, in anticancer drug Design aspect has broad application prospects, and mentality of designing involved in this method also has on design new copper ionophore There is good application.
Detailed description of the invention
Specific embodiments of the present invention will be described in further detail with reference to the accompanying drawing.But this should not be interpreted as Limitation to the method for the present invention.
Fig. 1 is that the method for the present invention is schemed compared with the prior art.
Fig. 2 is DPY by Cu2+It is loaded into cell.It (A) is DPY and CuCl2After acting on HepG2 cell, intracellular copper ion is dense Degree has regular hour dependence, and (B) is DPY, DPH, DMe and CuCl2After acting on HepG2 cell for 24 hours, intracellular copper The variation of ion concentration, (C) are DPY, DPH, DMe and CuCl2After acting on HeLa cell for 24 hours, the change of intracellular copper ion concentration Change.
Fig. 3 is the variation of intracellular sulfhydryl levels.It (A-C) is DPY, DPH, DMe and CuCl respectively2Act on HepG2 cell The variation diagram of 6h (A), 12 h (B) and for 24 hours (C) intracellular sulfhydryl levels afterwards.It (D) is DPY, DPH, DMe and CuCl2Act on HeLa The variation diagram of cell intracellular sulfhydryl levels after for 24 hours.
Fig. 4 is the situation of change of intracellular Trx system.(A) and (B) is DPY and CuCl respectively2After acting on HepG2 cell, The active variation of intracellular TrxR and Trx, (C) and (D) is DPY and CuCl respectively2After acting on HeLa cell, intracellular TrxR With the active variation of Trx, (E) is the intracellular TrxR1 protein expression situation of HepG2, and (F) is the intracellular Trx1 albumen table of HepG2 Up to situation, (G) is the measurement of the intracellular Trx redox state of HepG2, and (H) is quantified to (G) figure.
Fig. 5 is the situation of change of intracellular GSH system.It (A) is DPY and CuCl2After acting on HepG2 and HeLa cell, carefully The variation of GR intracellular, (B) are DPY and CuCl2After acting on HepG2 cell, the situation of change of intracellular GSSG and total GSH.
Fig. 6 is DPY and CuCl2After acting on HepG2 cell, intracellular ROS situation of change.It (A) is that probe DCFH-DA is surveyed For fixed cell imaging as a result, (B) is the flow cytometer measurement result of probe DCFH-DA measurement, (C) is the quantitative knot to (B) Fruit.
Specific embodiment
Embodiment 1: a method of copper carrier antitumor and anticancer agent, including following step are designed based on redox cycle strategy It is rapid:
(1) intracellular copper ion concentration measurement.
Intracellular copper ion concentration increases, and illustrates that DPY can deliver copper ion and enter into the cell.
The present invention has investigated DPY and CuCl2Intracellular copper ion intake after collective effect tumour cell, if DPY As copper ion carrier, by Cu2+It is loaded into cell, then intracellular Cu2+Content will increase.We set up four groups respectively Test: blank control group；20μM CuCl2Group；20μM CuCl2With 20 μM of DPY collective effect groups；20μM CuCl2With 20 μM DPH collective effect group.The above grouping drug is separately added into HepG2 cell, after acting on 0,0.5,1,3,6,12,24 and 48h, Cell quantification is collected, intracellular content of copper ion is detected with inductively coupled plasma atomic emission spectrometry (ICP-AES) Variation, as shown in Figure 2 A, with blank control group, DPH compared with Cu collective effect group, independent Cu group, DPY and Cu collective effect With the increase of action time, intracellular copper content obviously increases group, and there are time dependences, is almost saturated for 24 hours, Further illustrating DPY can be Cu2+It brings into the cell, and there are time dependences.Fig. 2 B and C are specified above respectively Drug concentration acts on HepG2 and HeLa cell content of copper ion variation diagram intracellular after for 24 hours, as shown in Fig. 2 B, for For HepG2, the intracellular copper content of blank group (Control group) is 0.26 ± 0.10, the intracellular copper content of independent Cu group Be 0.92 ± 0.31, DPH and the intracellular copper content of Cu collective effect group be 1.18 ± 0.14, and DPY and Cu collective effect group Intracellular copper content is 13.59 ± 0.93, and as a result obviously numerical value of three groups than front is high, and same result is in HeLa cell Inside also occur, as shown in Figure 2 C, the above medicine group to HeLa cytosis for 24 hours after, DPY and Cu collective effect group as the result is shown Intracellular copper content is significantly raised, sufficiently illustrates DPY as Cu2+Carrier, can be by Cu2+It is loaded into cell, to make The copper content obtained into the cell is significantly raised.
The operating method of example 1:
HepG2 or HeLa cell (1-3 × 106) triplicate kind in 100mm culture dish overnight it is adherent, add prescribed concentration Drug, act on specified time, collect cell.Portion is used for protein quantification；Other two parts are detected for copper ion concentration: sample Product are dissolved with concentrated nitric acid, detect copper content with inductively coupled plasma atomic emission spectrometry (ICP-AES).
Embodiment 2: a method of copper carrier antitumor and anticancer agent, including following step are designed based on redox cycle strategy It is rapid:
(1) intracellular copper ion concentration measurement.
Intracellular copper ion concentration increases, and illustrates that DPY can deliver copper ion and enter into the cell with embodiment 1.
(2) intracellular total sulfhydryl levels measurement.
Intracellular sulfhydryl levels decline, illustrates that small molecule mercaptan participates in this circular response.
As described in Example 1, copper ion can be loaded into cell by DPY.In the cell under the action of reproducibility thiol molecule, Cu can be released2+, then the content of intracellular small molecule sulfydryl necessarily declines, so the present invention has detected intracellular total mercapto Base molecular level change, as shown in figures 3 a-c, respectively with 20 μM of CuCl2, 20 μM of DPY and 20 μM of CuCl2With 20 μM of DPY Collective effect HepG2 cell 6h (Fig. 3 A), 12h (Fig. 3 B) or for 24 hours (Fig. 3 C), it is simultaneously quantitative to collect cell, measures intracellular total mercapto Base, as the result is shown individual CuCl2Intracellular sulfydryl is had little effect with DPY, but the two collective effect can be bright Intracellular total sulfydryl is reduced aobviously, and there is time dependence；The phenomenon similarly occurs in HeLa cell, and 20 μM CuCl2With 20 μM of DPY collective effect HeLa cells for 24 hours after, intracellular total sulfydryl declines (Fig. 3 D).Illustrate DPY Cu2+It carries After entering cell, with it redox reaction occurs for intracellular thiol molecule, so that intracellular total sulfhydryl levels decline.
Above example has absolutely proved that the carrier needs the participation of intracellular sulfydryl to the release that copper ion delivers, exactly sharp With the reproducibility of sulfydryl, just making this method is occurred based on intracellular redox cycle.
2Cu2++2RSH→2Cu++RSSR+2H+
The operating method of example 2:
Intracellular total detection of SH, is detected using DTNB method.
1), the drafting of standard curve
The GSH stock solution of 10mM is diluted to 0.01,0.05,0.1,0.2,0.5,1 and 2mM with RIPA buffer respectively Normal concentration, respectively plus the 6M guanidine hydrochloride of the normal concentration of 10 μ l and 90 μ l DTNB containing 1mM are into 96 orifice plates, slight to shake It is dynamic, absorbance value at 412nm, using GSH concentration as abscissa, OD are measured after reacting at room temperature 5min412For ordinate mapping, must mark Directrix curve equation are as follows: y=0.309x-0.000501, R2=0.9998.
2), specific measurement
1-3×106Cell (according to cell category) kind is adherent overnight in 100mm culture dish, adds the drug of prescribed concentration, Specified time is acted on, collects and cell and is washed 2 times with PBS, with RIPA buffer lytic cell on ice, Bradford standard measure Intracellular protein concentration dilutes unified protein concentration (preferably operation is all diluted to same concentration every time, such as 2 μ g/ μ l).
Total detection of SH in lysate: the 6M guanidine hydrochloride of 10 μ l of cell pyrolysis liquid and 90 μ l DTNB containing 1mM to 96 orifice plates In, it is slight to shake, absorbance value at 412nm is measured after reacting at room temperature 5min.
100 μ l cell pyrolysis liquids: being divided into two pipes of each 50 μ l by total detection of SH in albumen, and a pipe surveys total mercapto in lysate Base, another 50 μ l lysate of pipe add -20 DEG C of acetone being pre-chilled of 150 μ l, and 40min is placed at -20 DEG C and is sunk to albumen It forming sediment, supernatant is abandoned in centrifugation, and it is quantitative with 50 μ l RIPA buffer soluble proteins, it is other to be same as above, measure OD412。
Total sulfydryl amount is calculated as the calibration curve of standard by GSH, is that unit indicates with " mmol/mg protein ", It is compareed with Control group to observe the lifting of total sulfydryl.
Embodiment 3: a method of copper carrier antitumor and anticancer agent, including following step are designed based on redox cycle strategy It is rapid:
(1) intracellular copper ion concentration measurement.
Intracellular copper ion concentration increases, and illustrates that DPY can deliver copper ion and enter into the cell with embodiment 1.
(2) intracellular total sulfhydryl levels measurement.
Intracellular sulfhydryl levels decline, illustrates that small molecule mercaptan participates in this circular response, with embodiment 2.
(3) intracellular thioredoxin system situation of change measurement.
Intracellular thioredoxin system changes, and further illustrates that reproducibility sulfydryl and oxidative stress participate in this and follow Ring reaction.
Intracellular thioredoxin (Trx) and glutathione (GSH) system, which is two, important maintains intracellular oxygen The system for changing reduction balance, since DPY is Cu2+After being loaded into cell, intracellular total sulfydryl is on a declining curve, then to cell Whether interior antioxidant system Trx system has an impact, and the following present invention has detected intracellular Trx system change situation.
Firstly, the present invention has detected 20 μM of CuCl2, 20 μM of DPY and 20 μM of CuCl2With 20 μM of DPY collective effects After HepG2 cell, intracellular TrxR and the active variation of Trx, as illustrated in figures 4 a andb, individual CuCl2With DPY to thin TrxR and Trx activity intracellular has little effect, but the two collective effect can significantly reduce intracellular TrxR and Trx Activity, it is active to Trx to inhibit the inhibition for comparing enzyme stronger, illustrate that the Trx of reduced form may take part in Cu2+Redox Reaction.Result above equally occurs in HeLa cell, the two collective effect for 24 hours after, intracellular TrxR and Trx activity is bright Aobvious decline (Fig. 4 C and D).DPY is further illustrated Cu2+After being loaded into tumour cell, the Trx of intracellular reduced form may join With Cu2+Redox reaction, in addition, the two collective effect also inhibits the activity of TrxR, so that Cellular Oxidation type Trx cannot be converted to the Trx of reduced form well, and intracellular redox equilibrium is destroyed.
Secondly, the present invention has detected the expression of intracellular TrxR and Trx protein content, 20 μM of CuCl2、20μM DPY And 20 μM of CuCl2With 20 μM of DPY collective effect HepG2 cells for 24 hours after, collect cell, utilize polypropylene after cracking is quantitative Acrylamide gel electrophoresis (Polyacrylamide gelelectrophoresis, PAGE) detects protein expression situation, as a result shows Show either independent role or the two collective effect, on the expressing quantity of intracellular TrxR1 without influencing (figure 4E), but individual CuCl2HepG2 cell is acted on DPY, the content of Trx1 is almost unchanged, but after the two collective effect, The content of Trx1 significantly reduces, and there are concentration dependant (Fig. 4 F), illustrates DPY by Cu2+Be loaded into cell after, may with it is intracellular Trx occur redox reaction, cause the expressing quantity of Trx1 to reduce.Since the activity and albumen table of intracellular Trx It is reduced up to amount, then whether the redox state of intracellular Trx can change, so finally we have detected drug After acting on HepG2 cell for 24 hours, the redox state of intracellular Trx changes.As shown in Figure 4 G, for no drug effect The amount of reduction-state Trx1 of HepG2 cell (normal group) be significantly higher than oxidation state, but when both collective effect after, cell The Trx1 amount of internal oxidition state is significantly raised, and the Trx1 of reduction-state is nearly no detectable, individual DPY and CuCl2Effect HepG2 cell, and normally organize identical, intracellular Trx1 mainly exists with reduction-state, little to impact cell.Illustrate DPY and CuCl2After collective effect HepG2 cell, intracellular Trx redox state is changed, mainly with the shape of oxidation state Formula exists.Fig. 4 H more can to the quantization of reduction-state Trx1 and oxidation state the Trx1 ratio of normal group and the two collective effect group After intuitively illustrating the two collective effect cell, intracellular more normal group of Trx state is had changed a lot, and improves The accumulation of Cellular Oxidation state Trx.On the whole, after the two collective effect HepG2 cell, intracellular Trx system is destroyed Balance.
The redox of the copper ion that carrier is brought into and intracellular Trx system is dexterously utilized in the inventive method, So that the important oxidation-reduction system in external copper ion and body cell interacts, change Cellular Oxidation also Original state delivers copper ion to regeneration carrier loop.
The operating method of example 3:
Intracellular TrxR determination of activity, by 0.5-2 × 106A cell (according to cell category) kind is in the culture dish of 100mm In, when the adherent density of cell is 70% or so, the trial drug effect specified time of various concentration is added, is drawn in drug Enter same amount of DMSO as control experiment, collects cell and washed 2 times with PBS.With RIPA buffer (50mM Tris-HCl PH 7.5,2mM EDTA, 0.5% dexycholate, 150mM NaCl, 1%Triton X-100,0.1%SDS, 1mM Na3VO4It is extracted 30 minutes on ice with 1mM PMSF), and primary, the total protein in extraction cell that was vortexed every 5 minutes.Pass through Bradford standard measure protein concentration.The cell of 20 μ g (protein quantification is generally added 10 μ l to 2 μ g/ μ l) total protein will be contained Extract with containing 100mM Tris-HCl (pH 7.6), 0.3mM insulin, 660 μM of NADPH and 3mM EDTA mixed liquor (MM) and 15 μM of E.coli Trx mixing, every 50 μ l of hole total volume, and being incubated for 30 minutes at 37 DEG C.Every hole is added The 6M guanidine hydrochloride (pH 8.0) of 200 μ l DTNB containing 1mM terminates reaction.With other all mixtures in addition to adding E.coli Trx For blank sample, the absorbance at 412nm is measured, the absorbance of blank sample is subtracted from the absorbance of sample, is obtained each Opposite TrxR activity in sample is used and introduces same amount of DMSO in drug as control, same amount of DMSO is added and is compareed In experiment, the activity of TrxR is indicated with the percentage with control group in cell.
Intracellular Trx determination of activity, by 0.5-2 × 106A cell (according to cell category) kind is in the culture dish of 100mm In, when the adherent density of cell is 70% or so, the trial drug effect specified time of various concentration is added, is drawn in drug Enter same amount of DMSO as control experiment, collects cell and washed 2 times with PBS.With RIPA buffer (50mM Tris-HCl PH 7.5,2mM EDTA, 0.5% dexycholate, 150mM NaCl, 1%Triton X-100,0.1%SDS, 1mM Na3VO4It is extracted 30 minutes on ice with 1mM PMSF), and primary, the total protein in extraction cell that was vortexed every 5 minutes.Pass through Bradford standard measure protein concentration.The cell of 20 μ g (protein quantification is generally added 10 μ l to 2 μ g/ μ l) total protein will be contained Lysate with containing 100mM Tris-HCl (pH 7.6), 0.3mM insulin, 660 μM of NADPH and 3mM EDTA mixed liquor (MM) and 170nM recombinant rat TrxR1 is mixed, every 50 μ l of hole total volume, and is incubated for 30 minutes at 37 DEG C.Every hole is added The 6M guanidine hydrochloride (pH 8.0) of 200 μ l DTNB containing 1mM terminates reaction.With other all mixed in addition to adding recombinant rat TrxR1 Conjunction object is blank sample, measures the absorbance at 412nm, the absorbance of blank sample is subtracted from the absorbance of sample, is obtained Opposite Trx activity in each sample is used and introduces same amount of DMSO in drug as control, by same amount of DMSO addition pair According to the facts testing, the activity of Trx is indicated with the percentage with control group in cell.
The method that PAO-sepharose method detects intracellular Trx redox state develops (Biochem by us Pharmacol, 2016,102:34-44).Concrete operation method: 1), prescribed concentration drug and 0.1% DMSO function cells For 24 hours or 48h, collect cell, immediately lytic cell and quantitative, general protein concentration it is quantitative between 4-6 μ g/ μ l (according to cell Depending on type, parallel test several times is preferably fixed consistent)；2) surely measured 100 μ l of cell pyrolysis liquid, is respectively taken to set three control groups, It is respectively as follows: without any processing group of lysate, Diamide oxidation (5mM) group and TCEP reduction (5mM) group, specifically to above 100 μ l cell pyrolysis liquids are incubated for 30 minutes at 37 DEG C with Diamide and TCEP, to reach complete oxidation and reduction；3), will The PAO-Sapharose of 20% ethyl alcohol storage takes 500 μ l (how much choosing with specific reference to sample) to be washed three times with TE, spare.4), Medicine group, control group is taken to determine each 100 μ l of measured lysate (4-6 μ g/ μ l) respectively, respectively plus the PAO- of equivalent Sapharose is incubated for 30 minutes at room temperature or 37 DEG C, primary every vibration in 5 minutes, centrifugation repeatedly loading with Accelerating reduction state Trx and PAO-Sapharose is acted on)；5) it, is centrifuged, takes 100 μ l of supernatant respectively, be oxidation state Trx；6), PAO- is washed with TE Sapharose to remove remaining oxidation state Trx, is added containing Unithiol (DMPS) (20mM) twice 100 μ l of TE buffer is incubated for 30 minutes at room temperature or 37 DEG C, and primary every vibration in 5 minutes, loading makes reduction-state repeatedly for centrifugation Trx taken off from PAO-Sapharose, be centrifuged, take supernatant, be reduction-state Trx, it is spare；7), add 4 × loading, 100 DEG C of albuminates；8), by 15% SDS-PAGE and Western Blot testing inspection.
Western Blot method can refer to Cell Signaling Technology (CST) operation,www.cellsignal.com/WBProtocol.Concrete operations are by 1-3 × 106Cell kind is stayed overnight in 100mm culture dish It is adherent, add the drug of prescribed concentration, act on specified time, collects cell and washed 2 times with PBS, with Bradford standard measure cell Interior protein concentration, dilutes unified protein concentration, and upper equivalent sample is used in the SDS-PAGE of reduction, transfer protein to pvdf membrane The TBST of 5% skimmed milk power is closed 1 hour, and 4 DEG C of primary antibody overnight incubations, TBST is washed 3 times, and secondary antibody is incubated at room temperature 1 hour, TBST washing, imaging are that control quantifies destination protein using ImageJ with Actin.
Embodiment 4: a method of copper carrier antitumor and anticancer agent, including following step are designed based on redox cycle strategy It is rapid:
(1) intracellular copper ion concentration measurement.
Intracellular copper ion concentration increases, and illustrates that DPY can deliver copper ion and enter into the cell with embodiment 1.
(2) intracellular total sulfhydryl levels measurement.
Intracellular sulfhydryl levels decline, illustrates that small molecule mercaptan participates in this circular response, with embodiment 2.
(3) intracellular thioredoxin system situation of change measurement.
Intracellular thioredoxin system changes, and further illustrates that reproducibility sulfydryl and oxidative stress participate in this and follow Ring reaction, with embodiment 3.
(4) intracellular glutathione system situation of change measurement.
Intracellular glutathione system changes, and further illustrates that reproducibility sulfydryl and oxidative stress participate in this circulation Reaction.
For intracellular Antioxidative Defense System in addition to Trx system, GSH system is also a critically important oxidant defense system System, the following present invention have detected when DPY is Cu2+After being loaded into cell, intracellular GSH system change situation.
20 μM of CuCl is had detected first2, 20 μM of DPY and 20 μM of CuCl2With 20 μM of DPY collective effect cell After for 24 hours, the activity of intracellular GR, as shown in Figure 5A, and for HepG2 and HeLa cell, individual CuCl2With DPY to thin Almost without influence, but after the two collective effect, intracellular GR activity is decreased obviously GR activity intracellular.
Since the two collective effect can inhibit intracellular GR activity, then the GSH/ of measure of cell internal oxidition reduction balance GSSG ratio is also changed certainly, so after then having detected drug effect HepG2 cell for 24 hours, intracellular GSH water Flat variation.As shown in Figure 5 B, individual 20 μM of CuCl2Compared with the control group with 20 μM of DPY groups, total GSH and GSSG are basic There is no variation, also there is no variations substantially for the ratio of GSSG and total GSH；20μM CuCl2Make jointly with 20 μM of DPY Compared with the control group with group, total GSH is slightly increased, and GSSG raising is more obvious, and close to 5 times of control group, GSSG is accounted for always The ratio of GSH also sharply increases, and close to 5 times of control group, this may be since cupric (Cu (II)) is reduced the GSH of type It is reduced into monovalence copper (Cu (I)), is accumulated in the cell with the addition forms of Cu (I)-GSSG, is relatively compareed so as to cause GSSG Group sharply increases.
The redox of the copper ion that carrier is brought into and intracellular GSH system is dexterously utilized in the inventive method, So that the important oxidation-reduction system in external copper ion and body cell interacts, carrier is regenerated cyclically to deliver Copper ion.
The operating method of example 4:
Intracellular GSH assay
1), the drafting of GSH standard curve:
The GSH stock solution of 10mM is diluted to the GSH working solution of 1mM with KPE lysate, with its configure 0,1.25,2.5, 5,10,15 and 30 μM of GSH, it is stand-by as normal concentration.
On 96 orifice plates, it is separately added into the above normal concentration of 20 μ l, parallel 2 holes of each concentration, following every hole point 120 μ l DTNB and GR mixed liquors (DTNB 0.33mg/ml, GR 1.67U/ml) is not added；Last every hole is added 60 μ l's NADPH (NADPH 0.67mg/ml) carries out kinetic determination immediately, and every 10s reads a data, surveys 12 points, i.e. measurement altogether 2min.Standard curve is done to OD value with the time.Equation is as follows: y=0.0104x+0.0068, R2The unit of=0.999, x are μ M。
2), the drafting of GSSG standard curve:
The GSSG (molecular weight: 612.6g/mol) of 5mg is weighed, 816 μ l tri-distilled waters are added, are prepared into the stock solution of 10mM (method refers to Beyotime Assay Kit specification) is stand-by.
The GSSG stock solution of 10mM is diluted to the GSSG working solution of 1mM with KPE lysate, with its configure 0,0.625, 1.25,2.5,5,10 and 15 μM of GSSG is protected from light respectively to the divinyl pyridine that 2 μ l are added in its every 100 μ l at 25 DEG C It is incubated for 1h, the triethanolamine of 6 μ l is then added, places 10min.It is other ibid to operate.Obtain standard curve are as follows: y=0.016x+ 0.0056, R2=0.999.
3) the intracellular GSH content after drug effect, is measured
1-3×106Cell (according to cell category) kind is adherent overnight in 100mm culture dish, adds the drug of prescribed concentration, Specified time is acted on, cell is collected and is washed 2 times with PBS, uses KPE buffer (0.1%Triton X-100 and 0.6% sulphur immediately Base salicylic acid is in the 0.1M kaliumphosphate buffer containing 5mM EDTA, pH 7.5) lytic cell on ultrasonic cell disrupte instrument Solution is collected supernatant at 4 DEG C with 3000 g (or 13000rpm) centrifugation 5 minutes immediately, used by (be placed in and carry out on ice) Bradford standard measure protein concentration.In order to measure total glutathione (general protein concentration quantitative in 1-2 μ g/ μ l), to every The solution (120 μ l) containing 0.33mg/ml DTNB and 1.66U/ml GR is added in a sample (20 μ l).Then 60 μ l are added NADPH (0.66mg/ml), measure the absorbance value variation at the place 412nm immediately, every 10s measurement once, 2min in total.In order to GSSG content (general protein concentration is quantitative in 4-6 μ g/ μ l) is measured, is measured after deriving GSH with 2- vinylpyridine.By 2 μ l's 2- vinylpyridine is added in 100 μ l cell lysates, is protected from light 1h after mixing at room temperature, is fully apparent from lysate The GSH of middle reduced form is added the triethanolamine of 6 μ l, is protected from light 10min, with GSSG in the measurement detection lysate of total GSH Content,.The amount that GSSG is subtracted from total glutathione just obtains GSH content.The unit of GSH content nmol/mg protein It indicates.
Intracellular GR determination of activity, concrete operations: 1-3 × 106Cell (according to cell category) is planted in 100mm culture dish It is adherent overnight, add the drug of prescribed concentration, act on specified time, collects cell and washed 2 times with PBS, existed with RIPA buffer Lytic cell on ice, Bradford standard measure intracellular protein concentration dilute unified protein concentration.200mM phosphate will be contained The mixed liquor of (pH 7.0), 2mM EDTA, 20mM GSSG and 2mM NADPH in 96 orifice plates, are added in addition and contain 80 μ g (generally handles Protein quantification adds 20 μ l to 4 μ g/ μ l) cell pyrolysis liquid of total protein triggers reaction, use H2O supplements volume to every hole total volume 100 μ l measure the reduction of initial 3 minutes absorbances at 340nm with microplate reader to measure GR activity.In addition to adding GSSG Other all mixtures are blank sample, similarly measure the decreasing value of preceding 3 minutes absorbances at 340nm, decline from sample sets The slope value subtracted subtracts the slope value of blank sample, obtains the opposite GR activity in each sample, same amount of DMSO is added In control experiment, the activity of GR is indicated with the percentage with control group in cell.
Embodiment 5: a method of copper carrier antitumor and anticancer agent, including following step are designed based on redox cycle strategy It is rapid:
(1) intracellular copper ion concentration measurement.
Intracellular copper ion concentration increases, and illustrates that DPY can deliver copper ion and enter into the cell with embodiment 1.
(2) intracellular total sulfhydryl levels measurement.
Intracellular sulfhydryl levels decline, illustrates that small molecule mercaptan participates in this circular response, with embodiment 2.
(3) intracellular thioredoxin system situation of change measurement.
Intracellular thioredoxin system changes, and further illustrates that reproducibility sulfydryl and oxidative stress participate in this and follow Ring reaction, with embodiment 3.
(4) intracellular glutathione system situation of change measurement.
Intracellular glutathione system changes, and further illustrates that reproducibility sulfydryl and oxidative stress participate in this circulation Reaction, with embodiment 4.
(5) intracellular ROS level measurement.
Intracellular ROS level increases, after illustrating that copper ion enters cell, stimulation Cellular Oxidation stress generation, then Redox reaction occurs really in the cell for secondary proof.
DPY is Cu2+After being loaded into cell, intracellular available copper is increased, intracellular redox equilibrium is caused to be lost It adjusts, antioxidant system is destroyed, and so as to cause a large amount of ROS to generate, the present invention has detected DPY Cu2+It is loaded into cell Afterwards, intracellular ROS variation.Firstly, with 20 μM of CuCl2, 20 μM DPY and 20 μM of CuCl2DPY with 20 μM makees jointly After HepG2 cell 2h, intracellular ROS is detected by cell imaging with ROS detection probe DCFH-DA, find 20 μM of DPY with 20μM CuCl2Collective effect HepG2 cell 2h, can produce ROS, and individually DPY and individual CuCl2But it cannot generate ROS (Fig. 6 A)；In addition, the present invention passes through flow cytometer after being dyed with DCFH-DA in order to further verify to result above The generation of intracellular ROS is had detected, is detected after same drug effect 2h, it is as shown in Figure 6B, consistent with result above, individually DPY and individual CuCl2ROS can not be generated, and the two collective effect can generate a large amount of ROS, specifically quantitatively see figure 5C.DPY is by Cu2+Bring intracellular, Cu into2+Redox reaction may occur with intracellular small molecule sulfydryl and generate Cu+, Cu+It can It can be in O2Under the action of generate superoxide anion.
2Cu2++2RS-→2Cu++RSSR (1)
Cu++O2→Cu2++O2 -. (2)
RS-+O2→RSSR+O2 -. (3)
It is worth noting that, according to equation (3), carrier molecule can be again in the above complicated redox reaction It is raw, redox reaction special in external copper and cancer cell is dexterously utilized and cyclically delivers copper ion.
The operating method of example 5:
DCFH-DA detects intracellular ROS, DCFH-DA be it is a kind of itself there is no fluorescent penetrant dyestuff, when entering cell Afterwards, DCFH is generated by intracellular esterase hydrolyzed, and DCFH cannot penetrating cell membrane, largely accumulated in intracellular, It generates the DCF for having fluorescence with intracellular ROS, by detecting the fluorescence of DCF, can reflect intracellular ROS indirectly It is horizontal.
DHE detects intracellular ROS, and DHE (Dihydroethidium, dihydro second ingot), is to predominantly detect intracellular ROS, special It is not the fluorescence probe of the intracellular superoxide anion level of detection, after it enters cell, with intracellular superoxide anion Dehydrogenation is acted on, to generate Ethidium, this substance can combine with intracellular DNA or RNA and generate red fluorescence.
Concrete operations: by every hole 1-5 × 105Kind is in 12 orifice plates for a cell (choosing number according to cell category), to thin The drug (suspension cell can directly dosing object) that various concentration is added after born of the same parents are adherent is incubated for specified time at 37 DEG C, replaces DCFH-DA or DHE (10 μM) is added under the conditions of being protected from light and continues to be incubated for 30 minutes for the culture medium of serum-free.Before cell imaging With PBS rinsing cell three times to remove remaining DCFH-DA or DHE, uses and introduce same amount of DMSO in drug as to according to the facts It tests, cell imaging or flow cytomery, the level of ROS is judged according to the power of green fluorescence or red fluorescence.
Embodiment 6: a method of copper carrier antitumor and anticancer agent, including following step are designed based on redox cycle strategy It is rapid:
(1) intracellular copper ion concentration measurement.
Intracellular copper ion concentration increases, and illustrates that DPY can deliver copper ion and enter into the cell with embodiment 1.
(2) intracellular total sulfhydryl levels measurement.
Intracellular sulfhydryl levels decline, illustrates that small molecule mercaptan participates in this circular response, with embodiment 2.
(3) intracellular thioredoxin system situation of change measurement.
Intracellular thioredoxin system changes, and further illustrates that reproducibility sulfydryl and oxidative stress participate in this and follow Ring reaction, with embodiment 3.
(4) intracellular glutathione system situation of change measurement.
Intracellular glutathione system changes, and further illustrates that reproducibility sulfydryl and oxidative stress participate in this circulation Reaction, with embodiment 4.
(5) intracellular ROS level measurement.
Intracellular ROS level increases, after illustrating that copper ion enters cell, stimulation Cellular Oxidation stress generation, then Redox reaction occurs really in the cell for secondary proof, with embodiment 5.
(6) measurement of extracellular fluid DPY concentration.
The increase of extracellular fluid DPY concentration, explanation has newly-generated DPY again, can recycle delivery copper ion
The present invention detects DPY and CuCl by liquid phase tandem mass spectrum (LC-MS/MS) method2It is trained after collective effect HepG2 cell The variation of DPY concentration in base is supported, to investigate release Cu2+Whether newly-generated DPY can be from going in extracellular fluid into the cell afterwards It goes.With 100 μM of DPY and 20 μM of CuCl2Collective effect HepG2 cell 0,1,2,4,8,12 and for 24 hours detects in culture medium DPY concentration, as shown in table 1, the concentration of DPY in the medium first reduces to be increased afterwards, illustrates DPY by Cu2+Delivery to intracellular, Cu is released under the action of thiol molecule in the cell2+, and a series of redox reactions occur, newly-generated DPY again can be with Operating constantly delivers Cu to extracellular fluid by way of circulation2+, so that the balance of cellular redox system is upset, Variation in current embodiment (3) and (4) out.
The inventive method is dexterously utilized DPY carrier and copper ion is loaded into cell, generates DMe while discharging copper ion Molecule can produce new DPY under the oxidizing condition of DMe in the cell, to achieve the purpose that circulation delivery copper ion.
The variation of DPY concentration in 1 different time points extracellular fluid of table
*,P<0.05vs.4h.
The operating method of example 6:
LC-MS/MS detects DPY concentration, and the standard DPY series of 0,0.5,1,2.5,5,10 and 25 μ g/ml of configuration makees standard Curve obtains Y=1.71x-2.16, R2=0.9939.
By 1 × 106A HepG2 cell kind overnight adherent (after 12h), adds 100 μM of CuCl in 60mm culture dish2With 20 μM DPY, collect 60 μ l culture mediums respectively at 0,1,2,4,8,12,24,36 and 48h, each point adds 60 μ l again and newly cultivate Base, it is quantitative according to standard curve with the DPY concentration of LC-MS/MS detection different time points.

Claims (10)

1. a kind of method based on redox cycle strategy design copper carrier antitumor and anticancer agent, it is characterised in that carrier can be by two Valence copper ion (Cu2+) delivery to intracellular, discharges Cu in the cell2+, and new carrier can be generated simultaneously, it transports with continuing cycling through Carry Cu2+。
2. according to claim 1, a method of copper carrier antitumor and anticancer agent is designed based on redox cycle strategy, including Following steps:
(1) measurement of intracellular copper ion concentration.
(2) measurement of intracellular total sulfhydryl levels.
(3) intracellular thioredoxin system (Trx) situation of change measures.
(4) intracellular glutathione system (GSH) situation of change measures.
(5) intracellular ROS level measurement.
(6) measurement of extracellular fluid DPY concentration.
3. according to claim 1 with 2 it is described it is a kind of based on redox cycle strategy design copper carrier antitumor and anticancer agent methods, Be characterized in that: the intracellular copper ion concentration in the step (1) increases.
4. according to claim 1 with 2 it is described it is a kind of based on redox cycle strategy design copper carrier antitumor and anticancer agent methods, It is characterized in that: intracellular total sulfhydryl levels decline in the step (2).
5. according to claim 1 with 2 it is described it is a kind of based on redox cycle strategy design copper carrier antitumor and anticancer agent methods, Be characterized in that: the intracellular thioredoxin system in the step (3) changes, and Trx reduced activity, protein expression subtracts It is few, and show oxidation state.
6. according to claim 1 with 2 it is described it is a kind of based on redox cycle strategy design copper carrier antitumor and anticancer agent methods, Be characterized in that: the intracellular glutathione system in the step (4) changes, and GSH, which shows oxidation state, to be increased.
7. according to claim 1 with 2 it is described it is a kind of based on redox cycle strategy design copper carrier antitumor and anticancer agent methods, Be characterized in that: the intracellular intracellular ROS level in the step (5) increases.
8. according to claim 1 with 2 it is described it is a kind of based on redox cycle strategy design copper carrier antitumor and anticancer agent methods, Be characterized in that: the DPY concentration of the extracellular fluid in the step (6) increases.
9. new copper carrier caused by design method according to claim 1.
10. the application that design method according to claim 1 generates new support.
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