CN108635354A - Methanesulfomide type small molecular inhibitor application in preparation of anti-tumor drugs - Google Patents
Methanesulfomide type small molecular inhibitor application in preparation of anti-tumor drugs Download PDFInfo
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Abstract
5 application in preparations of anti-tumor drugs of Methanesulfomide type small molecular inhibitor DHW, belong to biomedicine technical field.The present invention is that the drug DHW 5 for effectively inhibiting growth of tumour cell is filtered out from the micromolecular inhibitor library of methylsulfonyl class, it can effectively inhibit the proliferation of Cervical Cancer HeLa Cells, breast cancer BT549 cells, Non-small Cell Lung Cancer A 549, oophoroma SKOV 3 cell and stomach cancer cell SGC 7901,503nhibiting concentration is below 9.0uM, especially best to the inhibiting effect of breast cancer BT549 cells.The inhibitor can target PI3K kinases, block the conduction of PI3K signal paths, cause the endogenous pathway of Cycle Arrest and mitochondria mediation to induce tumour cell that apoptosis occurs.The inhibitor of the present invention can be used for preparing anti-tumor drug, and development for the anti-tumor small molecular drug of kinase inhibitor class and exploitation provide new thinking and mechanism.
Description
Technical field
The invention belongs to biomedicine technical fields, and in particular to a kind of Methanesulfomide type small molecular inhibitor DHW-5 exists
Prepare the application in antitumor drug.
Background technology
Tumour is to threaten the important killer of human health and life, and incidence and lethality occupy the head of various diseases
Position.Currently, clinically used treatment means have operative treatment, chemotherapy, radiotherapy and biotherapy, but clinical treatment tumour is used at present
Most of drug there is a series of toxic side effects such as very strong immunosupress, damage whole body internal organs.Therefore, there is an urgent need to
Research and develop the novel drug with tumour-specific.
Phosphatidylinositol-3-kinase (Phosphoinositide 3kinase, PI3K) is a kind of phosphokinase, it can
Intracellular signal is integrated and be translated into signal in growth factor, cell factor and other environment, to the more of cell
Kind vital movement is regulated and controled, including cell growth, proliferation, metabolism, survival and protein synthesis etc..Currently, having a large amount of
Document report, the abnormal activation of the PI3K signal paths and generation of tumour, transfer and drug resistance have very close pass
System, it is more common especially in breast cancer cell.It is newest that some researches show that the activation rate of PI3K is up in breast cancer cell
70%, and in some breast cancer cells, the high activity state of PI3K signal paths directly reduces chemotherapy and HER2 targetings
The effect for the treatment of.Therefore, the research of PI3K signal pathway inhibitors has become the hot spot studied at present.
PI3K inhibitor can inhibit the activation of protein kinase, and the signal path to be participated in it blocks, most
Induction inhibits the drug of growth of tumour cell eventually.Although at present it is existing targeting PI3K micromolecular inhibitor enter preclinical phase and
Clinical experimental stage, but the R and D of PI3K inhibitor also have the prodigious rising space, it would be desirable to by rationally setting
The micromolecular compound of novel targeted PI3K is counted and optimizes, these micromolecular compounds can effectively reduce the intracellular activation of PI3K
Level realizes the purpose of specific killing tumour.
Invention content
The purpose of the present invention is a kind of effective tumor inhibitor is filtered out from Methanesulfomide type small molecular inhibitor library
DHW-5 has found that it has the function of inhibiting kinds of tumor cells proliferation, the especially inhibited proliferation in BT549 cells more
Significantly.The drug can target PI3K, block the conduction of PI3K signal paths, to induce BT549 apoptosis and thin
Born of the same parents' Cycle Arrest.Based on above discovery, the object of the present invention is to provide Methanesulfomide type small molecular inhibitor DHW-5 to prepare
Application in antitumor drug, and then provide new think of for the development and exploitation of the anti-tumor small molecular drug of kinase inhibitor class
Road and mechanism.
Methanesulfomide type small molecular inhibitor DHW-5 of the present invention, chemical structural formula are as follows：
Application of the Methanesulfomide type small molecular inhibitor of the present invention in preparing anti-tumor drug, the drug
Active constituent be Methanesulfomide type small molecular inhibitor DHW-5, and further include pharmaceutically receptible auxiliary material.
Methanesulfomide type small molecular inhibitor DHW-5 of the present invention, can：
(1) inhibit Cervical Cancer HeLa Cells, breast cancer BT549 cells, Non-small Cell Lung Cancer A 549, oophoroma
The proliferation of SKOV-3 cells and stomach cancer cell SGC-7901,503nhibiting concentration are below 10uM, especially to breast cancer BT549 cells
Inhibiting effect it is best；
(2) migration and the colony Forming ability of BT549 cells are reduced；
(3) with 933 4 Lys of PI3K catalytic subunits 802, Tyr 836, Val 851 and Asp acid residues sites
The structural region of composition combines；
(4) PI3K kinases is targeted, the phosphorylation level of intracellular PI3K downstream effects object AKT is reduced, so that under
The conduction of trip signal path changes, including up-regulation period GAP-associated protein GAP p21 and activation Caspase-3 albumen, Qian Zheke
To cause the cell generation G1 phases to be blocked, the latter's inducing cell apoptosis.
The present invention outstanding feature be：The Methanesulfomide type small molecular inhibitor DHW-5 of the present invention can target PI3K
Kinases has good antitumor activity, has better anticancer effect especially in breast cancer cell.Therefore, of the invention
Can be used for preparing anti-tumor drug, and development for the anti-tumor small molecular drug of kinase inhibitor class and exploitation provide it is new
Thinking and mechanism.
Description of the drawings
Fig. 1：After a concentration of 50.0 μM of Methanesulfomide type small molecular inhibitor handles BT549 cells and HeLa cells for 24 hours
The survival rate column diagram of cell；
Fig. 2：Respectively for 24 hours with 0.0 (i.e. Control), 1.0,2.5,5.0 μM of DHW-5 processing BT549 cells,
The fluorescent microscopy images of Transwell experiment detection cell migration abilities；
Fig. 3：Respectively for 24 hours with 0.0 (i.e. Control), 1.0,2.5,5.0 μM of DHW-5 processing BT549 cells, colony shape
It takes pictures figure (A) and quantitative column diagram (B) at the fluorescence microscope of experiment；
Fig. 4：Respectively for 24 hours with 0.0 (i.e. Control), 1.0,2.5,5.0,10.0 μM of DHW-5 processing BT549 cells, stream
The Apoptosis scatter plot of formula cell art detection；
Fig. 5：Respectively for 24 hours with 0.0 (i.e. Control), 1.0,2.5,5.0 μM of DHW-5 processing BT549 cells, DAPI dyes
The fluorescent microscopy images for the apoptotic body that color detection generates into the cell；
Fig. 6：Respectively for 24 hours with 0.0 (i.e. Control), 1.0,2.5,5.0 μM of DHW-5 processing BT549 cells, streaming is thin
The peak figure of the cell cycle of born of the same parents' art detection；
Fig. 7：The bioinformatics simulation drawing of Autodock computer softwares；(A) DHW-5 and PI3K catalytic subunits phase interaction
Use simulation drawing；(B) hydrophobic binding pocket of DHW-5 and PI3K catalytic subunits generates interaction simulation drawing；(C) DHW-5 and egg
The interaction of hydrogen bond site simulation drawing of white kinases.
Fig. 8：Influence diagrams of the DHW-5 to BT549 intracellular protein expressions；(A-B) DHW-5 is to the intracellular eggs of BT549
The Western blot figures that white kinases and its phosphorylation level influence；(C) DHW-5 is to BT549 cyclin expressions
The Western blot figures of influence；(D) Western that DHW-5 influences BT549 cell death related protein expressions
Blot schemes.
Specific implementation mode
The present invention is further described in detail With reference to embodiment, the embodiment provided is only for explaining
The bright present invention, the range being not intended to be limiting of the invention.
The configuration of 1 drug of embodiment
Methanesulfomide type small molecular inhibitor DHW-5 (is combined into) solid powder by Shenyang Pharmaceutical University Song Hongrui professor's projects
End is dissolved with suitable dimethyl sulfoxide (DMSO) (DMSO), is obtained the drug solution of a concentration of 5mM, then (is purchased from 1640 culture mediums
Gibco companies) by 100 times of drug dilution, 50 μM of drug solution is obtained, the primary dcreening operation concentration using the concentration as drug, other
Concentration carries out the dilution of smaller concentration with 1640 culture mediums (being purchased from Gibco companies) again on the basis of the concentration.
Recovery, culture and the passage of 2 cell of embodiment
After taking out the cryopreservation tube for preserving BT549 cells in liquid nitrogen container, it is immediately placed in the beaker equipped with 37 DEG C of warm water,
After the cell suspension in cryopreservation tube melts, cryopreservation tube is transferred in superclean bench rapidly, then by the cell after thawing
Suspension is slowly added into the centrifuge tube equipped with phosphate buffer (PBS), and 1500rpm centrifuges 3min.After discarding centrifugation
Cell precipitation is resuspended with 1640 culture mediums (being purchased from Gibco companies) of the 1mL fetal calf serums for containing 10% (V/V) for supernatant,
Cell after resuspension is added in the culture dish of a diameter of 10cm containing 1640 culture mediums of 10mL (being purchased from Gibco companies),
Then under the conditions of cell being placed in 37 DEG C, contain 5% (V/V) CO2Cell incubator in cultivated.When cell growth extremely
When the density of 80%-90%, old 1640 culture medium (being purchased from Gibco companies) is discarded, then washed and discarded with PBS, be then added
The pancreatin of 2mL is placed in 37 DEG C, 5% (V/V) CO2Cell incubator in digest 2min, to every ware cell be added 3mL 1640
Culture medium (being purchased from Gibco companies) terminates pancreatin digestion, blows and beats attached cell with pipettor and dissipates, and cell is transferred to centrifugation
Guan Zhong, 1500rpm centrifuge 3min.Cell after centrifugation aseptically abandons supernatant, (public purchased from Gibco with 1640 culture mediums
Department) cell precipitation is resuspended, the ratio that 1640 culture mediums of 3mL (being purchased from Gibco companies) are added according to a culture dish passes cell
In generation, under the conditions of cell is finally placed in 37 DEG C, contains 5% (V/V) CO2Cell incubator in cultivated, obtain adherent
Cell.
The preliminary screening and IC of 3 Methanesulfomide type small molecular inhibitor active anticancer of embodiment50The detection of value
Active anticancer and IC50The detection of value is the method using MTT.
Testing principle：Mtt assay is also known as MTT colorimetric methods, is a kind of method of detection cell survival and growth.MTT can be by
The succinate dehydrogenase contained in living cells mitochondria restores empurpled first a ceremonial jade-ladle, used in libation particle, and this particle is can not to be dissolved in water
, but the mitochondria of dead cell can not but be restored, DMSO can dissolve first a ceremonial jade-ladle, used in libation particle, with microplate reader in 492nm wavelength
Lower measurement its light absorption value, i.e. OD values, can reflect cell quantity indirectly.Thus, we can pass through the variation pair of the reaction color
The existence of cell and death condition are evaluated.
Specific steps：
(1) the adherent cell that embodiment 2 obtains is digested and collected first, and the cell of collection is carried out with blood counting chamber
It counts, finally according to 7 × 103The quantity in a/hole spreads 96 orifice plates, with isometric PBS polishings around the aperture of cell.
(2) 96 orifice plates of above-mentioned inoculating cell are placed in 37 DEG C, 5% (V/V) CO2Cell incubator in cultivate.
(3) after the cell cultivated is adherent, 1640 culture mediums (being purchased from Gibco companies) being sucked out in hole, then into every hole
Be added containing 5.0,10.0,15.0,20.0,25.0,1640 culture mediums of 30.0uM Methanesulfomide type small molecular inhibitors (are purchased from
Gibco companies), per 200 μ L of hole, do three groups it is parallel, be that 1640 trainings are only added in cell if negative control group, that is, Control groups
Base (being purchased from Gibco companies) is supported, Methanesulfomide type small molecular inhibitor is not contained.
(4) in 37 DEG C, 5% (V/V) CO2Cell incubator culture for 24 hours after, to every hole be added 15 μ L MTT solution,
37 DEG C be protected from light under the conditions of reaction 4h after, abandon supernatant, then per hole be added 150 μ L DMSO, be put into microplate reader and shake
3min。
(5) wavelength for selecting 492nm, detects absorbance, i.e. OD values.The survival of cell is finally calculated according to the OD values in each hole
Rate and inhibiting rate, calculation formula are as follows：
Survival rate=experimental group OD average values/control group OD average value × 100%
Inhibiting rate=(control group OD average values-experimental group OD average values)/control group OD average value × 100%
According to cell inhibitory rate experimental result, IC can be calculated using Graphpad Prism softwares50Value.
The preliminary screening of Methanesulfomide type small molecular inhibitor active anticancer is the human breast carcinoma for selecting this laboratory to preserve
Cell line BT549 cells and Human cervical cancer cell lines HeLa cells.Methanesulfomide type small molecular inhibitor is carried out using mtt assay
Toxicity assessment.It was found that DHW-1,5,51 can effectively inhibit the growth of BT549 and HeLa cells, and proliferation inhibition rate is 90%
Left and right.And other drugs (DHW-3,6,7,13,50,52,53,54,55,56,74) same concentrations and in action time to this
Two kinds of cell lines are without apparent inhibition (Fig. 1, abscissa：Methanesulfomide type small molecular inhibitor, Control are represented
Negative control group, 1 represents DHW-1, and 3 represent DHW-3, other numbers and so on；Ordinate：The survival rate of cell).In this hair
In bright, further investigated mainly is carried out to the antitumor activity of DHW-5 and mechanism.
IC50Value refers to the 503nhibiting concentration of measured drug, i.e., certain certain density drug-induced tumour cell is dead
50% is died, which is known as 50% inhibition concentration i.e. IC50Value.IC50The measurement of value is 9 plants of people's cancers for selecting this laboratory to preserve
Cell line.For it is above-mentioned filter out effective inhibit the micromolecular inhibitor DHW-5 of growth of tumour cell with containing serum
1640 culture mediums (being purchased from Gibco companies) are diluted to 5.0,10.0,15.0,20.0,25.0,30.0 μM, act on 9 plants of cancer cells
24h.By cell growth inhibition assay as a result, it has been found that DHW-5 pairs of 9 plants of tumour cells have different cytotoxicities, wherein right
The toxicity of BT549 cells is maximum, i.e. DHW-5 is best to the proliferation inhibiting effect of BT549 cells, IC50Value is about 3.6uM (tables
1)。
Table 1：ICs of the DHW-5 in different cancerous cell lines50Value
4 Methanesulfomide type small molecular inhibitor DHW-5 of embodiment reduces the transfer ability research of BT549 cells
The transfer ability of detection cell is tested using Transwell.
Testing principle：Transwell experiments are to measure a kind of method of growth of tumour cell and movement.The experimental technique
Main material is the cells Transwell.The cells Transwell are the structures of a cup-shaped with permeability, are put it into thin
In born of the same parents' culture plate, upper interior deserves to be called room, claims lower room, to hold culture supernatants in upper chamber, lower layer's training is held in lower room in culture plate
Nutrient solution, levels culture solution are separated by with polycarbonate membrane.By cell kind in upper interior, since polycarbonate membrane has permeability,
Ingredient in lower layer's culture solution can influence indoor cell, so as to study the ingredient in lower layer's culture solution to tumour
The influence of cell migration.
Specific steps：
(1) the adherent BT549 cell pancreatin that embodiment 2 obtains is digested and is collected, after counting, according to 2 ×
104The cell quantity in/hole is inoculated in six orifice plates, and 1640 culture mediums (be purchased from Gibco) of 2mL are added per hole, be placed in 37 DEG C,
5% (V/V) CO2Cell incubator in overnight incubation to exponential phase.
(2) according to the good various concentration of beforehand dilution (0.0,1.0,2.5,5.0uM) drug be added in six orifice plates, set
In 37 DEG C, 5% (V/V) CO2Cell incubator in cultivate for 24 hours.
(3) it collects cell and counts, according to 2 × 103Cell is added to Transwell upper chambers by the quantity in/hole, in upper chamber
1640 culture mediums (be purchased from Gibco company) of the 200 μ L without serum are added, and the ox blood that quality final concentration of 1% is added is pure
Brotein equilibrium osmotic pressure, room is 600 μ L normally 1640 culture mediums containing serum (being purchased from Gibco companies) under Transwell.
(4) culture plate is put in 37 DEG C, 5% (V/V) CO2Cell incubator for 24 hours after, cell is taken out, it is light with cotton swab
The cell of the light cell for removing upper chamber upper surface, small chamber lower surface fixes 20min using 75% (V/V) ice ethyl alcohol of precooling.
(5) cell is cleaned twice with cold PBS, it is 0.1% violet staining 20min finally to use mass concentration.
(6) it cleans cell twice with PBS, takes pictures under fluorescence microscope.
Respectively with 0.0 (i.e. Control), 1.0,2.5,5.0 μM of Methanesulfomide type small molecular inhibitor DHW-5 processing
BT549 cells for 24 hours, observe influence of the drug to cell migration ability.The results are shown in Figure 2, compared with the control group, drug-treated
Cell migration ability afterwards obviously weakens, and with the increase of drug concentration, and this function and effect are more apparent.It can be with from figure
Find out, when drug dose reaches 5.0 μM, BT549 cells can hardly penetrate film.
5 Methanesulfomide type small molecular inhibitor DHW-5 of embodiment reduces the Colony forming ability of BT549 cells
The Colony forming ability of detection cell is tested using Colony forming.
Testing principle：Colony refers to being proliferated the cell mass formed by an ancester cell in culture medium in vitro, and tumour is thin
Born of the same parents can infinite multiplication, with Colony forming ability.
Determination step：
(1) the adherent BT549 cells that embodiment 2 obtains are digested and are collected through pancreatin, and the cell of collection is laid on six orifice plates
In, it is incubated overnight.
(2) various concentration (0.0,1.0,2.5,5.0uM) drug-treated for 24 hours after, digest and collect cell, taken after counting
A certain amount of cell (3000-5000/hole) is added in six orifice plates, is placed in 37 DEG C, 5% (V/V) CO2Cell incubator in
Culture.
(3) 1640 culture medium (being purchased from Gibco companies), continuous culture two weeks are changed weekly.
(4) 1640 culture mediums (being purchased from Gibco companies) are discarded, cell 20min is fixed with ice ethyl alcohol.
(5) cleaned twice with PBS, then with mass concentration be 0.1% violet staining 20min.
(6) it is cleaned twice with PBS, fluorescence microscope is taken pictures.
(7) the dissolving crystallized purple of ethyl alcohol of 1mL 75% (V/V) is added into six orifice plates for clapped photograph, crystal violet is collected in
In centrifuge tube.
(8) absorption value under 578nm wavelength is detected with microplate reader, quantitative analysis is carried out to Colony forming ability.
Compared with control group, that is, Control, the Colony forming ability of agent-feeding treatment group cell obviously weakens.In drug concentration
When being 5.0 μM, tumour cell almost loses the ability of Colony forming.According to quantitative analysis as a result, drug concentration point
When Wei not be 2.5 μM and 5.0 μM, the inhibiting rate of soft agar clonogenic assay ability be respectively up to 55.63% and 25.67%, statistics
Analysis shows that significant difference (Fig. 3).Therefore, which, which fully demonstrates this drug, has significantly antitumor activity.
6 Methanesulfomide type small molecular inhibitor DHW-5 induction BT549 apoptosis researchs of embodiment
Apoptosis is detected using the bis- dye methods of Annexin V-FITC/PI.
Testing principle：In normal cell, phosphatidylserine (PS) is only distributed in the inside of cell membrane lipid bilayer,
During early apoptosis occurs for cell, the phosphatidyl serine (PS) existed only in originally on the inside of plasma membrane can turn up, and
Annexin V can combine closely with PS, be marked by carrying out fluorescein (FITC) to Annxin V, by what is be marked
Annexin V can be combined with the PS on the outside of viable apoptotic cell film, be used to detect the cell of early apoptosis.Propidium iodide
(Propidium Iodide, PI) can be combined with intracellular nucleic acid, but cannot be entered inside normal cell, and
It can enter and the intracellular of middle and advanced stage apoptosis has occurred, so the experiment is exactly mainly by the way that Annexin V- are used in combination
Cell in different apoptosis periods, can be detected by two kinds of dyestuffs of FITC and PI.The cell of early apoptosis mainly shows
Mono- positive for Annexin V-FITC, downright bad cells show is PI mono- positive, and the cell of late apoptic is then shown as pair
It is positive.
It is as follows：
(1) the BT549 cell pancreatin that embodiment 2 obtains is digested and is collected, after counting, according to 2 × 104/ hole
Cell quantity be inoculated in six orifice plates, 1640 culture mediums (be purchased from Gibco companies) of 2mL are added per hole, be placed in 37 DEG C, 5%
(V/V)CO2Cell incubator in overnight incubation to exponential phase.
(2) after above-mentioned cell culture for 24 hours, per hole be added 2mL it is sterile PBS cleaning, will contain different diluted concentrations (0.0,
1.0,2.5,5.0,10.0uM) 1640 culture mediums (being purchased from Gibco companies) of DHW-5 be added in six orifice plates, mark,
Six orifice plates are placed in 37 DEG C, 5% (V/V) CO2Cell incubator in for 24 hours.
(3) six orifice plates are taken out, washs cell twice with PBS, pancreatin digests and collects cell.
(4) cell count and collection 1 × 106400uL combination buffer suspension cells are added in a cell.
(5) after 5 μ L Annexin V-FITC mixings are added into the system of above-mentioned 400uL, it is placed on the place's of being protected from light incubation
20min。
(6) 5 μ L PI, mixing are added into above-mentioned reaction system, room temperatures are protected from light, are incubated 5min.
(7) combination buffer that 100 μ L are added into above-mentioned reaction system is made into the system of 500 μ L, is sieved with 200 mesh
In net filtration to streaming pipe, is measured and analyzed with flow cytometer in 1h.
The results are shown in Figure 4 for flow cytomery Apoptosis, and DHW-5 can obviously induce BT549 cells to generate
Apoptosis, and with the increase of drug concentration, the ratio of Apoptosis shows the trend obviously increased；With 1.0 μM of drug
Apoptosis situation is compared after processing, and the ratio for the cell middle and advanced stage apoptosis that 10.0 μM of drug-treateds are crossed significantly increases.As it can be seen that
DHW-5 can induce tumour cell to generate apoptosis, and with the increase of drug concentration, and tumour cell is from early apoptosis to late period
The ratio of apoptosis conversion is also increasing.
7 Methanesulfomide type small molecular inhibitor DHW-5 inductions BT549 of embodiment generates apoptotic body into the cell
The method that the detection of cytomorphology is dyed using DAPI.
Measuring principle is as follows：The chemical name of DAPI is 4 ', 6- diamidinos -2-phenylindone (4 ', 6-diamidino-2-
Phenylindole), which can be combined with the DNA of intracellular generates very strong fluorescence signal, to the DNA of cell into
Blue-fluorescence is presented in fluorescence microscopy microscopic observation in line flag, the nucleus being marked by DAPI.
Concrete operation step：
(1) adherent BT549 cells are digested and is collected through pancreatin, the cell of collection is counted with blood counting chamber, according to 2
×104The quantity in/hole is laid in six orifice plates, and 2mL 1640 culture mediums (being purchased from Gibco companies) are added per hole, be placed in 37 DEG C, 5%
(V/V)CO2Cell incubator in overnight incubation.
(2) after above-mentioned cell adherent growth, be added different operating concentration (0.0,1.0,2.5,5.0,10.0uM)
Culture plate is placed in 37 DEG C, 5% (V/V) CO by DHW-52Cell incubator in cultivate for 24 hours.
(3) supernatant in six orifice plates is discarded, the ethyl alcohol of 75% (V/V) of 4 DEG C of precoolings of 1mL is added per hole, is placed in 4 DEG C of items
10min is fixed under part.
(4) it is washed twice with PBS, the last DAPI methanol solutions that a concentration of 0.1 μ g/mL of 300 μ L are added per hole, room temperature
Under the conditions of, dye 10min.
(5) PBS cleans cell twice, and fluorescence microscope is simultaneously taken pictures.
It is that fluorescence microscope is taken pictures the results show that compared with negative control group i.e. Control, the BT549 after agent-feeding treatment
Apparent DNA break, i.e. apoptotic body are generated into the cell, and as drug concentration increases, this phenomenon is more apparent (Fig. 5).This
The result of study of project is consistent with the result of front flow cytomery Apoptosis, and all demonstrating drug can be particularly evident
Promotion apoptosis of tumor cells.
The 8 Methanesulfomide type small molecular inhibitor DHW-5 induction BT549 cells generation G1 phases of embodiment block
It is detected using flow cytometer cell cycle.
Measuring principle：According to the difference of the periodic characteristics of cell cycle events, the cell cycle by man-made division be G1,
S, G2 the and M phases.PI dyestuffs can be combined with nucleic acid DNA, in conjunction with amount and DNA content direct proportionality, it is thin by streaming
Born of the same parents' art can detect the fluorescence signal intensity of dyestuff, to the content of the intracellular DNA of indirect determination.And intracellular DNA content
Difference shows the cell cycle phase that cell is in different phase.
Concrete operation step is as follows：
(1) adherent BT549 cells are digested and is collected through pancreatin, the cell of collection is counted with blood counting chamber, according to 2
×104The quantity in/hole is laid in six orifice plates, and 1640 culture mediums (being purchased from Gibco companies) of 2mL are added per hole, six orifice plates are set
In 37 DEG C, 5% (V/V) CO2Cell incubator in cultivate.
(2) after cell adherent growth, 1640 culture mediums (being purchased from Gibco) in six orifice plates are discarded, and want sterile PBS
Wash twice, according to beforehand dilution various concentration (0.0,1.0,2.5,5.0uM) drug be added in six orifice plates, be placed in 37
DEG C, 5% (V/V) CO2Cell incubator in cultivate for 24 hours.
(3) after cell culture for 24 hours afterwards discard 1640 culture mediums (be purchased from Gibco) containing drug, wash cell two with PBS
It is secondary, adherent cell dissociation is got off with pancreatin and is collected into centrifuge tube, 1500rpm centrifuges 3min, discards supernatant.
(4) primary, the 1500rpm that washs cell precipitation with PBS, centrifuges 3min, abandons supernatant.The 100 μ L precoolings of each sample
PBS be resuspended, then add the ethyl alcohol of 300 μ L precoolings, gently after mixing, be put into -20 DEG C of refrigerators.
(5) after 2h, the cell fixed through ethyl alcohol is taken out, 1500rpm centrifuges 3min, abandons supernatant.
(6) it is cleaned once with the PBS of 100 μ L precoolings, 1500rpm centrifuges 3min, abandons supernatant.Wait for ethyl alcohol volatilization completely with
Afterwards, the PBS of 200 μ L room temperature of each sample is resuspended.
(7) the 10mg/mL RNaseA that 15 μ L are added are incubated 30min after being uniformly mixed under 37 DEG C of water bath conditions.
(8) the PI dye liquors of 200 μ L are added into each sample, can be used to stream measuring after the filtering of 200 mesh screens.
From the result of flow cytomery cell cycle it can be found that Methanesulfomide type small molecular inhibitor DHW-5 is lured
The cell-cycle arrest that BT549 cells produce the significant G1 phases is led, and with the increase of drug concentration, the G1 phases block more
Obviously.When drug concentration reaches 5.0 μM, 55.93% when the ratio of the G1 phases of cell is by not dosing increases 80.7%,
The result of study shows that drug-induced BT549 cells generation G1 phases block (Fig. 6).
9 molecular docking of embodiment simulates Methanesulfomide type small molecular inhibitor DHW-5 and interacts with intracellular kinases
It is carried out using Autodock bioinformatics softwares.
Measuring principle：Molecular docking is simulated according to ligand and receptor acting " lock-key principle " between ligand and receptor
Interaction.Ligand and acceptor interaction are the processes of molecular recognition, include mainly electrostatic interaction, hydrogen bond action, hydrophobic
Effect, model ylid bloom action etc..In recent years, molecular docking method has become a technology in area of computer aided drug research field.
Assay method：Utilize Autodock bioinformatics softwares simulation Methanesulfomide type small molecular inhibitor DHW-5's
The interaction of 20 kinds of closely related protein kinases occurs with tumour for chemical constitution.
The results are shown in Table 2 for molecular docking simulation, by comparing freely combining between DHW-5 and variety classes kinases
Can, it finds freely to combine energy minimum between the drug and 5KAE i.e. PI3K kinases, it is the tightest to show that the drug is combined with PI3K
Close, thus result can speculate that the possible action target spot of the drug is PI3K.
Table 2：20 kinds are freely combined energy between kinases and DHW-5
By the way that the drug and PI3K kinases are further carried out molecular docking, as a result show the drug mainly with PI3K kinases
Catalytic subunit interaction, tetra- amino acid residues of LYS802, TYR836, VAL851 and ASP933 with PI3K catalytic subunits
There are interaction of hydrogen bond (Fig. 7).Therefore, DHW-5 is mainly four amino of the catalytic subunit by hydrogen bond action and PI3K
Sour residue interaction, and then the catalytic activity of PI3K kinases can be inhibited.
Influences of the 10 Methanesulfomide type small molecular inhibitor DHW-5 of embodiment to BT549 intracellular protein expressions is ground
Study carefully
Some and the cell growth that intracellular is had detected using the technological means of protein immunoblot (Western blot) are increased
Grow the expression of GAP-associated protein GAP.
Measuring principle：It is that the cell or tissue gross protein after electrophoretic separation is transferred to solid support NC films from gel
Or on pvdf membrane, a kind of protein detection techniques of detection of specific antibody specific antigen are then used, by analyzing coloring
Position obtains the information of specific protein expression in the cell or tissue analyzed with color depth.
It is as follows：
(1) adherent BT549 cells are digested and is collected through pancreatin, the cell of collection is counted with blood counting chamber, according to 7
×104The quantity in/hole is laid in six orifice plates, and 1640 culture mediums (being purchased from Gibco) of 2mL are added per hole, are placed in 37 DEG C, 5% (V/
V)CO2Cell incubator in cultivate.
(2) after cell culture to adherent growth, 1640 culture mediums (being purchased from Gibco) in six orifice plates is discarded, are used in combination
PBS is washed twice, and is added in 1640 culture mediums containing drug (being purchased from Gibco) to six orifice plates, is placed in 37 DEG C, 5% (V/V)
CO2Cell incubator in cultivate the required time.
(3) 1640 culture mediums (being purchased from Gibco companies) in six orifice plates are sucked out, it is heavy that the PBS being pre-chilled with 4 DEG C washs cell
It forms sediment twice, 100uL cell pyrolysis liquids RIPA is added in each sample, and (protease inhibitors containing 1uL, is purchased from lysate
Beijing Ding Guo companies), this process whole process carries out on ice.
(4) after cell completely cracking, under the conditions of 4 DEG C, 12000rpm centrifuges 10min, and gentle aspiration albumen supernatant turns
It moves on in new centrifuge tube, and marks.
(5) experimental method of BCA protein quantifications is used, the albumen concentration of each sample is according to minimum protein sample concentration
It is diluted, finally makes the albumen concentration of sample consistent.
(6) be added into each sample 5 × SDS loading buffer (sample-loading buffer, ingredient include glycerine, SDS,
DTT, Tris-base, bromophenol blue) 100 DEG C of water-baths are put into, boil 10min.
(7) after sample cooling, the SDS-PAGE electrophoresis of 12% (V/V), the loading of unified all samples are ready for
Volume.
(8) electrophoresis tank concentration glue uses 80V constant pressures, separation gel to use 120V constant pressures, wait for bromophenol blue frontal migration to gel
Stop electrophoresis when bottom, prepares transferring film.
(9) using wet turn of method transferring film, using 0.45 μm of pvdf membrane, which needs before the use in methanol solution
Film is dipped at least 30min in transfer liquid by middle activation 1min later, same to need in advance the sponge needed for transferring film, filter paper
30min is impregnated in transfer liquid.
(10) gel run is taken out, according to the sequence of sponge-filter paper-gel-pvdf membrane-filter paper-sponge, is turned in wet method
It moves in instrument and places, prepare electricity and turn, electricity turns condition and is traditionally arranged to be constant current 250mA, and electricity turns 2h in 4 DEG C of ice baths.
(11) PBST (a kind of cleaning solution, configured by PBS and Tween-80) solution and skimmed milk power is used to be configured to matter
The confining liquid for measuring a concentration of 3%, pvdf membrane is taken out, and is cut out film according to the molecular weight of required albumen, and be put into confining liquid, is waited for film
After all soaking, at least 1.5h is closed.
(12) primary antibody is incubated, the primary antibody solution of target protein specificity is prepared with PBS buffer solution, by the pvdf membrane and one after closing
Anti- 4 DEG C overnight.
(13) pvdf membrane is washed 3 times with PBST buffer solutions, each 10min.
(14) secondary antibody is incubated, secondary antibody diluent is identical as primary antibody diluent preparation method, is incubated at room temperature 45min.
(15) pvdf membrane is washed 3 times with PBST buffer solutions, each 5min.
(16) by after cleaning pvdf membrane take out, ECL luminescent solutions are equably added dropwise on film, with gel imaging instrument into
Row exposure.
To probe into whether the drug causes BT549 intracellular protein kinases and its phosphorylation level to change, use is dense
Degree handles 4h, 15h and for 24 hours respectively for 10 μM of drug to BT549 cells, and the result of Western blot is shown in Fig. 8, with control group
I.e. Control is compared, and medicine group can significantly reduce the expression of the phosphorylation of the downstreams intracellular PI3K albumen AKT, but right
The expression of total AKT albumen does not influence.And action time is longer, and inhibition is more apparent.It is continuing with 5 μM, 10 μ
For 24 hours, the result of detection is consistent with above-mentioned experimental result, DHW- for the drug effect BT549 cells of M and 20 μM of three kinds of various concentration
5 can effectively inhibit the phosphorylation level of the intracellular AKT of BT549, including two phosphorylation sites of Ser473 and Thr308, and
The concentration of drug is higher, and the expression of phosphorylation AKT is lower (Fig. 8 A-8B).We can speculate that DHW-5 may as a result,
It is the inhibitor of AKT upstream proteins PI3K.
The molecular mechanism of action that the G1 phases block is generated in order to probe into the drug-induced tumour cell, by drug-treated
BT549 cells can raise the expression with period relevant albumen p21, and with the increase of drug concentration, and effect is just more aobvious
It writes (Fig. 8 C).As it can be seen that DHW-5 may be by up-regulated expression p21 albumen and by cell-cycle arrest in the G1 phases.
In order to illustrate the mechanism of action that the drug-induced cell generates apoptosis from the level of protein molecular, drug concentration is used
Respectively 5 μM and 10 μM effect BT549 cells are for 24 hours.The results show that when drug concentration is 10 μM, the table of Pro-caspase 3
Apparent downward has occurred up to level, it should be the result shows that DHW-5 has activated the relevant 3 (figures of PROTEIN C aspase of Apoptosis
8D).Speculated according to above-mentioned experimental result, DHW-5 can make tumour by the Caspase mediated by mitochondria the approach relied on
Apoptosis.
Claims (3)
1. Methanesulfomide type small molecular inhibitor DHW-5 application in preparations of anti-tumor drugs, the following institute of structural formula of DHW-5
Show,
2. Methanesulfomide type small molecular inhibitor DHW-5 application in preparations of anti-tumor drugs as described in claim 1,
It is characterized in that：The tumour is Cervical Cancer HeLa Cells, breast cancer BT549 cells, Non-small Cell Lung Cancer A 549, ovary
Cancer SKOV-3 cells or stomach cancer cell SGC-7901.
3. Methanesulfomide type small molecular inhibitor DHW-5 application in preparations of anti-tumor drugs as described in claim 1,
It is characterized in that：The active constituent of drug is Methanesulfomide type small molecular inhibitor DHW-5 described in claim 1, further includes pharmacy
Upper receptible auxiliary material.
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| CN111208283A (en) * | 2018-11-21 | 2020-05-29 | 中国科学院上海生命科学研究院 | Synergistic tumor inhibiting composition and application thereof |
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| CN111445961B (en) * | 2020-04-02 | 2024-03-26 | 辽宁大学 | Application of anthocyanin screened based on molecular simulation technology in inhibiting malignant biological behaviors of colon cancer cells |
| CN111529527A (en) * | 2020-06-17 | 2020-08-14 | 西南医科大学 | Application of benzamide compound in IAP protein inhibitor and preparation of antitumor drug |
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| CN112791088A (en) * | 2021-01-19 | 2021-05-14 | 浙江大学 | Application of natural antitumor active substance |
| CN112791088B (en) * | 2021-01-19 | 2022-11-01 | 浙江大学 | Application of natural antitumor active substance |
| CN113476455A (en) * | 2021-08-18 | 2021-10-08 | 佛山市妇幼保健院 | Application of formamide derivative CP068 in preparation of antitumor drugs |
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