CN106727586A

CN106727586A - A kind of new opplication of Artesunate

Info

Publication number: CN106727586A
Application number: CN201611106935.3A
Authority: CN
Inventors: 马虎; 周建国; 张钰; 柏玉举; 吕水萍; 刘大海; 岳国军; 石磊; 张廷友; 沈刚
Original assignee: Affiliated Hospital of Zunyi Medical University
Current assignee: Affiliated Hospital of Zunyi Medical University
Priority date: 2016-12-06
Filing date: 2016-12-06
Publication date: 2017-05-31

Abstract

The present invention provides a kind of new opplication of Artesunate, and Artesunate is used to reverse adenocarcinoma of lung Tarceva resistance, or Artesunate and Tarceva drug combination.Resistance is eventually produced to solve existing molecular targeted agents, its problem clinically applied is limited.The invention belongs to lung cancer therapy field.

Description

A kind of new opplication of Artesunate

Technical field

Adenocarcinoma of lung Tarceva resistance is reversed the present invention relates to one kind, belongs to lung cancer therapy field.

Background technology

Lung cancer is to cause cancer-related death chief reason, and the treatment for appearing as lung cancer of molecular targeted therapy brings New hope, it has clear and definite action site, and adverse reaction is small, determined curative effect.The shortcoming of molecular targeted agents is it It is expensive, can not fully erased focus, and eventually produce resistance, this point limits its application clinically..

The content of the invention

It is an object of the invention to：A kind of new opplication of Artesunate is provided, it is final to solve existing molecular targeted agents Resistance can be produced, its problem clinically applied is limited.

To solve the above problems, intend using the new opplication of such a Artesunate, Artesunate is used to reverse adenocarcinoma of lung Tarceva resistance, further, Artesunate and Tarceva drug combination.

Artesunate is used for the reverse research method of adenocarcinoma of lung Tarceva resistance：

ART is studied the external anti-tumor activity of human lung adenocarcinoma PC9 cells：Culture PC9 cell lines, add various concentrations ART, with cell Counting Kit-8 (cck-8) detect PC9 cells proliferation rate, Real-time RT-PCR and The expression of western-blot detection EGFR, IGF1R, AXL and Akt mRNA, albumen and apoptosis-related protein；

The culture of Tarceva secondary resistance cell line and the detection of drug resistance：The increasing of PC9/ER is detected with cck-8 methods Grow rate, calculate resistance multiple, Real-time RT-PCR and western-blot detection EGFR, IGF1R, AXL, Akt mRNA and The expression of albumen；

Experiment in vitro checking ART reverses effect and the mechanism of adenocarcinoma of lung ER resistances：Added in PC9/ER cell lines different ART, ER or drug combination of concentration, and with LY294002 as positive control, proliferation rate, Real-time RT- are surveyed with cck-8 methods The expression of PCR and western-blot detection EGFR, IGF1R, AXL and Akt m RNA and albumen；

By transplanted tumor in nude mice experiment in vivo verify ART reverse adenocarcinoma of lung ER resistances effect and mechanism, Tarceva after Hair property medicine-resistant cell line abbreviation PC9/ER, Tarceva abbreviation ER, Artesunate abbreviation ART.

Compared with prior art, Artesunate of the present invention can effectively reverse adenocarcinoma of lung Tarceva resistance, coordinate Lung cancer for prognosis can effectively be improved after Tarceva, solve the problems, such as that molecular targeted agents can produce resistance well, entered The application clinically of one step propulsion molecular targeted agents.

Brief description of the drawings

Cell quantity and morphologic change after various concentrations Artesunate treatment PC9 cells under Fig. 1 light microscopes (x200), wherein：

A：0.1%DMSO control groups； B：25 μm of ol/L groups of Artesunate

C：Artesunate 50mol/L groups； D：Artesunate 100mol/L groups；

Inhibited proliferation of Fig. 2 Artesunates to PC9 cells；

After Fig. 3 Artesunates treatment 48h in cell EGFR, IGF1R, AXL and Akt mRNA relative expression, note：*p< 0.05vs control**p<0.01vs control；

After Fig. 4 Artesunates treatment 48h in PC9 cells EGFR, IGF1R, AXL and Akt and its phosphorylation expression；

After Fig. 5 Artesunates treatment 48h in PC9 cells apoptosis-related protein expression；

Fig. 6 Artesunates are to PC9 cells and the inhibited proliferation of PC9/ER cells；

Fig. 7 different experiments groups act on the protein expression and phosphorylation level situation of change of IGF1R and AXL after PC9/ER cells；

Fig. 8 different experiments groups act on the protein expression and phosphorylation level situation of change of Akt after PC9/ER cells；

Fig. 9 different experiments groups act on the expression of apoptosis-related protein and phosphorylation level situation of change after PC9/ER cells；

Figure 10 different experiments group nude mice body weight situations of change；

Figure 11 different experiments group nude mice tumor volume situations of change, note：*p<0.05vs Con groups n=5.

Specific embodiment

To make the object, technical solutions and advantages of the present invention clearer, invention will be described in further detail below.

Experimental example 1：

Artesunate is studied the external anti-tumor activity of human lung adenocarcinoma PC9 cells

1 materials and methods

1.1 major experimental materials

1.1.1 cell line

Human lung adenocarcinoma PC9 cells are given by the entire PLA of new bridge hospital of Third Military Medical University institute of oncology professor Chen Zhengtang.

1.1.2 main agents

1.1.3 main material equipment

1.2 experimental techniques

1.2.1 the recovery of cell, cultivate, pass on and freeze

The recovery of tumour cell and culture

The cryopreservation tube that will be equipped with human lung adenocarcinoma PC9 cell lines is taken out from liquid nitrogen container, and 37 DEG C -40 DEG C are put into rapidly equipped with warm water Beaker in quickly rock, the cell liquid for freezing quickly is dissolved in 30s, cell is transferred in sterile centrifugation tube, add RPMI-1640 nutrient solutions ((FBS) containing 10% hyclone) 5ml, cell suspension is softly blown and beaten into by cell, in centrifuge from The heart (1000rpm is centrifuged 3-5min), sucks supernatant, the 5ml of RPMI-1640 nutrient solutions (containing 10%FBS) is added, by cell Piping and druming is planted in 25ml blake bottles after mixing, and is placed in 37 DEG C, 5%CO₂Cultivated in the cell incubation case of saturated humidity, incubated overnight It is in adherent growth after visible cell is observed under inverted microscope, sucks supernatant, washed 2-3 times with PBS solution, adds RPMI-1640 nutrient solutions (containing 10%FBS) 5ml.In observation of cell form under inverted microscope, liquid is changed in good time.

The passage of tumour cell

In observation of cell form under inverted microscope, see that cell is covered with left to the 80-90% of Tissue Culture Flask bottom of bottle volume Can be passed on when right, it would be desirable to which the human lung adenocarcinoma PC9 cells of passage are placed in super-clean bench, outwell the old nutrient solution in blake bottle, Washed 2-3 times with PBS solution, with the 0.25% trypsase 1-2ml vitellophags containing 0.025%EDTA, cover bottle cap rearmounted In observation of cell under inverted microscope, a small amount of fresh medium containing 10%FBS is rapidly added when cell withdraws projection change bowlder, The cell for having digested softly is blown and beaten repeatedly to disengage it from cultivating bottle wall, cell is transferred in a new centrifuge tube, in centrifuge Centrifugation (1000rpm, 3-5min), at this moment can see that there is one layer of white precipitate centrifuge tube bottom, the cell being as collected into, carefully Supernatant is abandoned in suction, is added a certain amount of fresh culture (the big bottles of 7-10ml/, 3-5ml/ bottles) containing 10%FBS and is made carefully Born of the same parents' suspension, gently piping and druming is mixed, and is then dispensed into again in new blake bottle, is placed in 37 DEG C, 5%CO₂The cell of saturated humidity is incubated Educate culture in case.Fresh medium was changed after passage per 2-3 days, using exponential phase cell as experimental subjects.

Tumour cell freezes

Selection is in the human lung adenocarcinoma PC9 cells of exponential phase, is most better than and freezes previous evening and change liquid, then with containing The 0.25% trypsase 1-2ml vitellophags of 0.025%EDTA, in being observed under inverted microscope, become when cell withdraws projection Bowlder is rapidly added a small amount of fresh medium containing 10%FBS, the cell for having digested softly is blown and beaten repeatedly and disengages it from blake bottle Wall, and cell suspension is dispersed into, then cell is transferred in new centrifuge tube, (1000rpm is centrifuged 3- for centrifugation in centrifuge 5min), at this moment can see that there is one layer of white precipitate centrifuge tube bottom, the cell being as collected into, careful suction abandons supernatant, then adds Enter the DMEM high glucose mediums containing 10%DMSO and 30% hyclone, be transferred in 2ml cryopreservation tubes after mixing cell, according to -4 30min is placed in DEG C refrigerator, 2h is placed in -20 DEG C of refrigerators, after -80 DEG C of sequential processes of refrigerator overnight, in moving into liquid nitrogen container It is long-term to preserve.

1.2.2 cell count

Cell counting count board and cover glass are wiped with 75% absolute ethyl alcohol, is dried standby.Cover glass is covered after drying On the counting chamber of blood counting chamber.One bottle of human lung adenocarcinoma PC9 cell is taken, 0.25% tryptose containing 0.025%EDTA is added Enzyme 1-2ml, is observed under inverted microscope, and a small amount of fresh cultured containing 10%FBS is rapidly added when cell withdraws projection change bowlder Liquid, softly blows and beats the cell for having digested and disengages it from cultivating bottle wall, and be dispersed into single cell suspension repeatedly.Then will be single with pipette tips Cell suspension suctions out a little (about 200 μ l), is added dropwise at cover glass edge, makes sky of the single cell suspension full of cover glass and tally Between gap.In being examined under inverted microscope and calculate four TCSs of block plaid, the principle that the cell of line ball is followed It is to disregard a lower and meter left side on meter to disregard the right side.Then calculated by formula：Cell number/ml：Four cell number sum/4 × 10 of big lattice⁴ × extension rate.Count 3 times respectively, average.

1.2.3 cck-8 detects inhibited proliferations of the ART to human lung adenocarcinoma PC9 cells

The PC9 cells of exponential phase are pressed into each hole 5 × 10³The density of individual cell is seeded in 96 orifice plates, is per hole 100 μ l, put 37 DEG C, 5%CO₂After cultivating 24h in incubator, ART is added its final concentration is respectively 0 μm of ol/L, 6.25 μm of ol/ L, 12.5 μm of ol/L, 25 μm of ol/L, 50 μm of ol/L, 100 μm of μ l of ol/L final volumes 150, and zeroing group, negative control are set (0.1%DMSO), blank control group, each concentration repeat 3 holes.Respectively after culture 24h, 48h, 72h, careful suction abandons supernatant, The RPMI-1640 fresh cultures without FBS are added per hole, then adds 10 μ l cck8 solution (to try not to produce gas per hole Bubble, in order to avoid the reading of influence OD values), 96 orifice plates are placed in 37 DEG C, 5%CO₂Continue to be incubated 1-4h (preferably consistent) in incubator Afterwards, with absorbance (OD) value at ELIASA Detection wavelength 490nm.

Cell proliferation rate (%)=[A (intervention)-A (blank)]/[A (feminine gender)-A (blank)] × 100

A (intervention)：The absorbance of cell+nutrient solution+cck-8 solution+drug solution；

A (blank)：The absorbance of nutrient solution+cck-8 solution；

A (feminine gender)：The absorbance of cell+nutrient solution+cck-8 solution；

Cell inhibitory rate (%)=1- cell proliferation rates (%)

1.2.4 Real-time RT-PCR detect the expression of intracellular mRNA

The extraction of RNA

Growth selection is in good condition, exponential phase PC9 cells, by 5 × 10⁵It is individual that cell/bottle is inoculated in 75ml is thin In born of the same parents' blake bottle.Drug-treated is separately added into after 24h, experimental group is ART (25 μM, 100 μM), and 0.1%DMSO solution is in addition Control group.After drug-treated, 37 DEG C, 5%CO are placed in₂, 48h is cultivated in the cell incubation case of saturated humidity.By in blake bottle Old nutrient solution is blotted only, then is cleaned 2-3 times with the PBS solution of precooling, finally blots the PBS solution in net Tissue Culture Flask.

1ml Trizol are added in every bottle of cell, nothing is transferred to after standing about 3-5min on ice, fully being blown and beaten with suction pipe In EP pipes (1.5ml) of enzyme.

The chloroform of 1/5 volume is subsequently adding, mixing of turning upside down places 10min after 37 DEG C.In low-temperature and high-speed centrifuge Middle centrifugation (4 DEG C, 12000g, 15min), carefully takes out EP pipes from centrifuge, and liquid is divided into three layers (upper strata is after centrifugation RNA, middle level is DNA and bottom is protein), it is careful to draw upper strata to another new EP pipes.

Isometric isopropanol is added, turn upside down mixing EP liquid in pipe, 37 DEG C of placement 10min.In low-temperature and high-speed from (4 DEG C, 12000g, 10min) are centrifuged in scheming, careful suction abandons supernatant.

Lentamente 75% ethanol 1mL is added along tube wall.In (4 DEG C, 1000g, 5min) are centrifuged on centrifuge, careful suction is abandoned Clear liquid, places empty dry by the sediment in EP pipes.

Often pipe adds 20 μ L DEPC solution (depending on precipitation capacity), after being completely dissolved, -80 DEG C of preservations.Then with ultraviolet point OD value of the light photometric determination total serum IgE at 260nm and 280nm, calculates concentration and purity.

Reverse transcription

Carried out using Dalian TaKaRa reverse transcription reagent box, reaction condition is as follows：

Being placed in after aforesaid liquid is mixed on Bio Rad-PCR reaction instrument carries out reverse transcription reaction (37 DEG C of 30min), then Inactivation reverse transcriptase (85 DEG C of 5s).

Real-time fluorescence quantitative PCR

Real-time quantitative PCR detection is carried out using Bio-Rad systems

Reaction system：The μ L of cumulative volume 20, set three multiple holes.

Amplification curve condition：95 DEG C of 30 seconds (predegeneration) → 95 DEG C (40 circulations) in 5 seconds → 60 DEG C 40 seconds (amplified reactions).

Solubility curve condition：72℃.

MRNA primers

Table 1 is used for the primer of Real-time RT-PCR

Interpretation of result processes (relative quantification method)：

A.Ct average=(Ct1+Ct2+Ct3)/3 (repeating pipe)；

B.dCt=Ct average- medians；

C. expression=2 ∧ (- dCt) of gene；

D. the expression of the expression/reference gene of relative quantification=genes of interest

1.2.5 Western blot detect the expression of intracellular protein

Take the people's lung after experimental group (final concentration of 25 μM, 100 μM of ART) and control group (0.1%DMSO solution) treatment 48h Old nutrient solution is abandoned in gland cancer PC9 cells, suction, is cleaned 2-3 times with the PBS solution of precooling, adds RIPA lysates (to be mixed with protease Inhibitor and inhibitors of phosphatases) the abundant cell lysis of 1ml, in 30min is stood on ice, cell is gently scraped off culture bottle wall, It is transferred in EP pipes, in centrifugation (4 DEG C, 12000g, 20min) on low-temperature and high-speed centrifuge.

A small amount of supernatant is taken, BCA methods determine protein concentration.Albumen is adjusted to same concentration, by 1:4 ratios addition 5 × on Sample buffer solution, boils 5min, and sample is carefully added into the sample well of polyacrylamide gel after after thoroughly cooling.

Electrophoresis：Polyacrylamide gel electrophoresis (polyacrlamine gel electrophoresis, PAGE) is carried out to turn Film (wet turn)：Transferring film is carried out using the wet systems that transfer from one department to another of Bio-Rad, through the transfer of 150V (time of transferring film is determined by molecular size range) Afterwards；Washed with TBS 3 times, every time 10 minutes.

Closing：TBS containing 5% skimmed milk power is closed 1 hour.

Primary antibody is incubated：After skimmed milk power closing, cleaned with TBST 3 times, 10 minutes/time, add primary antibody, -4 DEG C of refrigerators are abundant Overnight incubation.

Secondary antibody is incubated：TBST rinse 3 times, 10 minutes every time, add horseradish peroxidase-labeled specific secondary antibody (with 1:5000 dilution proportions) in, it is positioned on shaking table, normal temperature is incubated 1 hour.

TBST is washed 3 times, every time 10 minutes.

Detection：Detected with ECL chemoluminescence methods, then carried out gel imaging instrument scanning.

1.3 statistical methods

Every group of experiment in triplicate, statistical analysis is carried out with SPSS20.0 statistical softwares to obtained experimental data, is owned Data are represented with mean ± standard deviation (mean ± SD), using one-way analysis of variance analysis group difference.P<0.05 thinks It is variant, P<0.01 thinks there is significant difference.

2 results

2.1 ART are on human lung adenocarcinoma PC9 cell quantities and morphologic influence

PC9 cells are acted on the ART of final concentration of 25 μm of ol/L, 50 μm of ol/L and 100 μm of ol/L, in optics after 48h Basis of microscopic observation, is compared with control group (0.1%DMSO), and the cell quantity of each medication group is significantly reduced, and cellular morphology is by short Fusiformis gradually becomes irregular shape or triangle, and with the increase of dosage, quantity is reduced must be become apparent from.(see Fig. 1)

2.2 influence using cck-8 methods detection ART to PC9 ability of cell proliferation

PC9 is acted on final concentration of 6.25 μm of ol/L, 12.5 μm of ol/L, 25 μm of ol/L, 50 μm of ol/L and 100 μm of ol/L Cell, determines the OD values of 490nm wavelength after 24h, 48h and 72h, as a result show, the ART of various concentrations size is to PC9 cells Multiplication capacity has an impact, and has dose dependent, and the IC50 for being computed 48h is 32.14 ± 4.69 μm of ol/L.(be shown in Table 2 and Fig. 2)

The sweet wormwood of table 2Amber ester pairPC9 cell inhibitory effect effects

Group difference^*P ＜ 0.05:Group difference #p ＜ 0.05

Influences of 2.3 ART to EGFR, IGF1R, AXL and AKT mRNA expression in adenocarcinoma of lung PC9 cells

Experimental group (ART25 μm of ol/L and 100 μm of ol/L) and control group (0.1%DMSO solution) effect 48h are collected respectively PC9 cells afterwards, extract row Real-time RT-PCR detections after total serum IgE, by Bio-Rad systems obtain intracellular EGFR, The Relative copy number of IGF1R, AXL and Akt gene.Compare with control group, PC9 intracellular EGFR mRNA after experimental group treatment Expression quantity increase compared with control group, and the expression quantity of IGF1R, AXL and Akt mRNA then decreases, and there is also dose-dependant Property, dosage is higher, the more notable (P of difference<0.01).(see Fig. 3)

The influence that 2.4 ART are expressed adenocarcinoma of lung PC9 cell EGFR, IGF1R, AXL and Akt albumen and apoptosis-related protein

2.4.1 ART can lower the phosphorylation level of PC9 cells AXL and Akt

Experimental group (ART 25 μm of ol/L and 100 μm of ol/L) and control group (0.1%DMSO solution) effect 48h are collected respectively PC9 cells afterwards, extract after total protein with Western blot detect GAP-associated protein GAP EGFR, p-EGFR, IGF1R, p-IGFIR, The change of AXL, p-AXL, Akt, p-Akt expression.It can be seen that intracellular EGFR, p-EGFR expression quantity of PC9 is more right after ART effects 48h Unobvious according to group change, the expression quantity of IGF1R, p-IGF1R, AXL, p-AXL, Akt, p-Akt albumen has difference compared with control group The reduction of degree, and become apparent with the expression reduction of phosphorylation level.(see Fig. 4)

2.3.2 expression of the ART to PC9 cell death related proteins has an impact

ART25 μm of ol/L and 100 μm of ol/L of collection is acted on 48 hours and the cell of 0.1%DMSO control groups is carried out respectively The expression of Western blot quantitative determination apoptosis-related proteins.It was found that ART acts on Caspase-3, Active after 48h The expression of caspase-3, PARP, Cleaved PARP has different degrees of rise.(see Fig. 5)

Experimental example 2：

Artesunate reverses the experiment in vitro of PC9/ER cell Tarceva resistances

1 materials and methods

1.1 major experimental materials

1.1.1 main agents

LY294002 is configured to 20nmol/L mother liquors (being dissolved with DMSO) first, is preserved for a long time in -20 DEG C of refrigerators, working solution Empirically it is required with fresh cell culture fluid with preceding being prepared.

1.2 experimental techniques

1.2.1 cell recovery, the experimental technique cultivated, pass on and freeze are the same.

1.2.2 the influence using cck8 kits detection ART to PC9/ER ability of cell proliferation.

By the PC9 cells and PC9/ER cells of exponential phase with every hole 5 × 10³The density of individual cell, is inoculated in 96 holes Plate, per the μ l of hole 100, is placed in 37 DEG C, 5%CO₂After cultivating 24h in incubator, ART is added its final concentration is respectively 6.25 μ Mol/L, 12.5 μm of ol/L, 25 μm of ol/L, 50 μm of ol/L, 100 μm of μ l of ol/L volumes 150, are detected with cck8 methods.

1.2.3 Western blot detect the table of intracellular EGFR, IGF1R, AXL and Akt albumen and apoptosis-related protein Up to situation of change

Collect respectively through the mono- medicine groups of ART (10 μm of ol/L), the mono- medicine groups of ER (5 μm of ol/L), ART joint ER groups, LY294002 PC9 cells and PC9/ER cells in exponential phase in positive controls, negative control group, using Western Blot realities Test detection ART and combine ER medications to PC9 cells and PC9/ER cell EGFR, IGF1R, AXL and Akt albumen and apoptosis-related protein Influence.

1.3 statistical analysis：

2 results

2.1 influence using cck-8 methods detection ART to PC9 cells and PC9/ER ability of cell proliferation

Distinguished with the ART of final concentration of 6.25 μm of ol/L, 12.5 μm of ol/L, 25 μm of ol/L, 50 μm of ol/L and 100 μm of ol/L PC9 cells and PC9/ER cells are acted on, the OD values of 490nm wavelength are determined after 48h, as a result shown, ART pairs of various concentrations The multiplication capacity of PC9 cells and PC9/ER cells has an impact, and has dose dependent, and drug concentration is higher, and inhibiting rate is higher, Wherein PC9 cells are 28.80 ± 2.84 μm of ol/L to the IC50 of ART, and PC9/ER cells are 25.78 ± 5.03 μ to the IC50 of ART Mol/L, difference is not statistically significant.(see Fig. 6 and Biao 3)

IC50 the and IC10 values of the PC9 cells of table 3 and PC9/ER cells to Artesunate

2.2 ART and influence of the ER Combination interventions to adenocarcinoma of lung PC9/ER ability of cell proliferation

PC9/ER cells are respectively acting on ER Combination interventions with the mono- medicines of ER and ART, the OD of 490nm wavelength is determined after 48h Value, and calculate IC50.Result shows that PC9/ER cells are 4.363 μm of ol/L to the IC50 of the mono- medicines of ER, and PC9/ER cells are to ART It is 2.488 μm of ol/L with the IC50 of ER Combination interventions.(being shown in Table 4)

The PC9/ER cells of table 4 are to the mono- medicines of ER and the IC50 values of ART and ER Combination interventions

Reversing drug resistance multiple IC50 (ER)/IC50 (ER+ART)=1.75

2.3 ART and influence of the ER Combination interventions to adenocarcinoma of lung PC9/ER cell IGF1R and AXL protein expression situations

2.3.1 ART can lower the protein expression and phosphorylation level of PC9/ER cells IGF1R and AXL with ER drug combinations Situation.

The mono- medicine groups of ART, the mono- medicine groups of ER, ART joint ER intervention groups are collected respectively acts on 48 hours and 0.1%DMSO control groups PC9/ER cells, extract total protein after with Western blot detect IGF1R, p-IGF1R, AXL, p-AXL expression. It can be seen that ART joint ER intervention groups IGF1R, p-IGF1R, the expression of AXL, p-AXL albumen have different degrees of drop compared with control group It is low, and Expression of phosphorylated level reduction become apparent from.(see Fig. 7)

2.3.2 ART can lower the protein expression and phosphorylation level situation of PC9/ER cells Akt with ER Combination interventions.

The mono- medicine groups of ER, the mono- medicine groups of ART, ART joint ER intervention groups, ER is collected respectively to combine LY294002 (10 μm of ol/L) and do Pre- group, ER+ART and LY294002 intervention groups act on the PC9/ER cells of 48 hours and 0.1%DMSO control groups, extract total protein The expression of Akt and p-Akt is detected with Western blot afterwards.It can be seen that the mono- medicine groups of ART, ER joint ART medication group p-Akt eggs White expression has different degrees of reduction compared with control group, compares with PI3K inhibitor LY294002 drug combination groups without substantially poor It is different.(see Fig. 8)

2.3 ART and influence of the ER Combination interventions to adenocarcinoma of lung PC9/ER cell death related protein expressions

The mono- medicine groups of ER, the mono- medicine groups of ART, ART joint ER intervention groups are collected respectively acts on 48 hours and 0.1%DMSO control groups PC9/ER cells, extract after total protein with Western blot detect Caspase-3, Active caspase-3, PARP, The expression of Cleaved PARP.Thus it is clear that ART joints ER intervention groups Caspase-3, Active caspase-3, PARP, The expression of Cleaved PARP albumen has different degrees of rise compared with control group, and Expression of phosphorylated level is raised and become apparent from. (see Fig. 9)

Experimental example 3

Artesunate reverses the experiment in vivo of PC9/ER cell Tarceva resistances

1 experimental technique

The foundation of 1.1 experimental animal tumor models

The PC9/ER cell numbers bottle of exponential phase is selected, then with the 0.25% trypsase 1- containing 0.025%EDTA 2ml vitellophags, in being observed under inverted microscope, the new of 10%FBS are contained when cell withdraws projection change bowlder and is rapidly added on a small quantity Fresh nutrient solution, softly blows and beats the cell for having digested and disengages it from cultivating bottle wall repeatedly, and is dispersed into cell suspension, then by cell It is transferred in new centrifuge tube, in (1000rpm, 5min) is centrifuged on centrifuge, by the white cell precipitation PBS solution of gained Cleaning 2-3 times, is counted with cell counting count board, and cell concentration then is adjusted into 1 × 10 with fresh medium (being free of FBS)⁷/ Ml, is aseptically immediately sent to SPF grades of animal feeding room.In the right side armpit hypodermic injection 1 × 10 of every nude mice⁷/ml PC9/ER cells, there is obvious skin mound, as inject successfully in every injection 0.1ml, observable injection site, extraction rapidly Syringe needle, skin of sterilizing.Continuous Observation has 48 nude mices (52 nude mices, dead 4 altogether) rice occur at the armpit of right side after 1 week The big lesser tubercle of grain, illustrates that model in nude mice is successfully established.Continue to cultivate and observe.

1.2 experiment packets

48 nude mices are randomly divided into 6 groups, every group 8.

Experiment packet is：Negative control group

The mono- medicine groups of ART

The mono- medicine groups of ER

ER+ART Combination intervention groups

ER+LY294002 Combination intervention groups

ER+ART+LY294002 intervention groups

1.3 medications

Treat that tumor average diameter reaches 100mm³After start gavage treatment, once a day, saline control group give 0.5ml without Bacterium physiological saline gavage.

1.4 antitumor actions are observed

Observe nude mice once within every 2 days after administration, main contents observe tumour to weigh nude mice body weight and measurement gross tumor volume The major diameter (Length) and minor axis (width) of measurement knurl body are needed during volume, gross tumor volume (v), v=(length is finally calculated ×width)/2.14d after medication, wherein 5 tumor bearing nude mices are put to death with cervical dislocation method, are weighed, relatively more each experimental group nude mice The situation of change of body weight.Then knurl body is completely stripped, observation knurl body color, shape, coating and surrounding wetting situation.Every group remains Remaining nude mice continues to observe to natural death, has indifference to observe its Overall survival.

1.5 immunofluorescences

1st, frozen section is fixed：Frozen section takes out rewarming from refrigerator, dries moisture, and cold acetone fixes 10min, treats third Ketone it is completely dry after rocked on decolorization swinging table in PBS (PH7.4) washing 3 times, each 5min.

2nd, antigen retrieval：Histotomy is placed in the reparation box for filling with EDTA antigen retrievals buffer solution (PH8.0) in microwave Antigen retrieval is carried out in stove.Low fiery 10min, should prevent buffer solution excessive vaporization during this, be sure not dry plate.Will after natural cooling Slide is placed in PBS (PH7.4) and washing 3 times, each 5min is rocked on decolorization swinging table.(liquid and Repair strength are repaired according to group Knit to determine)

3rd, BSA closings:Section is drawn a circle (prevent antibody from flowing away) with groupization pen after slightly drying around tissue, is added dropwise in circle With 3%BSA uniform fold tissues, room temperature closing 30min.

4th, primary antibody is added：Confining liquid is gently got rid of, the primary antibody that PBS is prepared by a certain percentage is added dropwise in section, section keeps flat In 4 DEG C of overnight incubations in wet box.(wet box is interior plus a small amount of water prevents antibody from evaporating)

5th, secondary antibody is added：Slide is placed in PBS (PH7.4) and washing 3 times, each 5min is rocked on decolorization swinging table.Section is slightly The secondary antibody that kind corresponding to primary antibody is added dropwise after drying in circle covers tissue, lucifuge incubation at room temperature 50min.

6th, DAPI redyes nucleus：Slide is placed in PBS (PH7.4) and washing 3 times is rocked on decolorization swinging table, every time 5min.Section is added dropwise DAPI dye liquors, lucifuge incubation at room temperature 10min in circle after slightly drying.

7th, mounting：Slide is placed in PBS (PH7.4) and washing 3 times, each 5min is rocked on decolorization swinging table.Section is slightly got rid of With anti-fluorescent quenching mountant mounting after dry.

8th, microscopy is taken pictures：Cut into slices in being observed under Nikon inverted fluorescence microscope and gather image.(burst of ultraviolel wavelength 330- 380nm, launch wavelength 420nm；FITC green glows excitation wavelength 465-495nm, launch wavelength 515-555nm；CY3 feux rouges excitation waves 510-560 long, launch wavelength 590nm)

9th, result interpretation：It is blueness under ultraviolet exciting that DAPI dyes the nucleus for coming, and positive expression is corresponding fluorescence The feux rouges or green glow of element mark

1.6 statistical analysis

2 results

Nude mice in the experiment of 2.1 nude mice lotus knurls and into knurl position

PC9/ER cells are inoculated with nude mice by subcutaneous, about 1 week after inoculation, the big brief summary of millet appearance can be touched right side armpit is subcutaneous Section, gradually increases later, and transplanting success rate is 98% (47/48).

2.2 nude mice changes of weight

After each experimental group inoculation PC9/ER cells 7d, the Tumor formation of tumor bearing nude mice is observed, tumor average diameter to be measured reaches 100mm³After start gavage treatment.Once a day, body weight of every 2 days of period weighing, counts each group nude mice after Continuous Observation 14d Body weight, as a result show, very much not, toxicity is not significantly increased each group nude mice body weight amplitude of variation after showing drug combination.(see Figure 10)

2.3 tumor volumes change

After model of nude mice bearing tumor is successfully established, tumor average diameter to be measured reaches 100mm³After start gavage treatment.Daily Once, tumor volume of every 2 days of period measurement, unifies to put to death nude mice with cervical dislocation after medication 14d, peels off knurl body, system The tumor volume of measurement in meter 14d, tumorous size has differences in finding different experiments group, and each experimental group tumor volume is more right Reduced according to group.(see Figure 11)

Knurl body tissue in the experiment of 2.4 nude mice lotus knurls

Each experimental group in unifying to put to death nude mice with cervical dislocation after gavage treatment 14d, then completely strips knurl body, Tumorous size is different during each experimental group can be observed, and the knurl body of the visible tumor bearing nude mice of naked eyes is in nodositas, and coating is mostly more complete, Being in canescence not to surrounding wetting, knurl body, without the performance such as hemorrhagic necrosis stove more.

The protein expression of Akt and p-Akt in 2.5 Immunofluorescence test nude mice knurl body tissues

After model of nude mice bearing tumor is successfully established, tumor average diameter to be measured reaches 100mm³After start gavage treatment.Daily Once, unify to put to death nude mice with cervical dislocation after medication 14d, peel off knurl body, -80 DEG C of refrigerator frosts are immediately placed in, using ice Freeze the protein expression of Akt and p-Akt in the Immunofluorescence test nude mouse tissue of section, as a result show the mono- medicines of ART and drug combination The protein expression of the internal Akt and p-Akt of group is relative to be lowered.

Discuss

Non-small cell lung cancer (NSCLC) is most common Lung Cancer Types, accounts for 80% or so of all lung cancer, although Synthetic (operation, chemotherapy and radiation) is treated to have been developed, but 5 years survival rates of Patients with Non-small-cell Lung are only left 17% It is right^[22].Current clinical research shows that EGF-R ELISA (EGFR) tyrosine kinase inhibitor (TKI) is replaced as Ji is non- Buddhist nun and Tarceva can significantly improve the Overall survival of EGFR genetic mutation (such as 19 exons mutations) cancer patient.However, Most of tumours can all produce EGFR-TKI resistances after (median time 8-12 month) after a while, and EGFR-TKIs is obtained Property resistance is typically inevitable, which has limited its clinical practice.However, the mechanism of EGFR-TKIs acquired resistances is not yet It is fully apparent from.

The influence topmost reason of molecular targeted therapy clinical practice is acquired resistance, causes EGFR-TKI acquired resistance to The mechanism of medicine has a lot, in addition to EGFR mutation and MET proto-oncogenes are expanded, it is also recently found that the machine of other acquired resistances System, such as the activation of by-passing signal (such as c-Met, HGF, AXL)^[23], downstream signal exception (as K-RAS mutation, PTEN Missing), the apoptotic pathways (such as BCL2-like11/BIM polymorphisms missing) of EGFR-TKI mediations and histology change. Therefore we should actively find and develop that Synergistic action is strong, toxic and side effect is small, can reverse small molecule targeted therapy resistance Medicine, is the direction of domestic and foreign scholars research

Research in recent years finds that effective anti-knurl composition is extracted and found from Chinese herbal medicine has turned into the uncommon of oncotherapy Hope and new way.Wherein artemisinin-based drug is the focus of research, has many researchs to antineoplastic point of Artesunate therein Handset system is inquired into, and current research has proven to Artesunate and PI3K/Akt, NF- κ B, MAPK, PKC, STATs etc. are believed Number path has inhibitory action, but specific mechanism is unclear.The result of study of this seminar early stage shows Artesunate energy Suppress growth and the propagation of human pulmonary epithelial cells with concentration and time dependence, and can dose-dependently induce thin Born of the same parents' apoptosis.

Based on background above, we are further inquired into the antitumor mechanism of ART, are detected using cck-8 first ART treatment human lung adenocarcinoma PC9 cell 24h, 48h, 72h of various concentrations, as a result find ART equally can with concentration and time according to Property ground is relied to suppress PC9 cell growths and propagation, this shows that ART has CDCC to adenocarcinoma of lung, and it is thin that it can slow down tumour The propagation of born of the same parents.And the result of qRT-PCR and Western Blot finds, after being processed through ART, intracellular IGF1R, AXL and Akt MRNA and protein level have different degrees of downward, while the protein expression of its activated form p-IGF1R, p-AXL and p-Akt Downward becomes apparent from.And intracellular apoptosis-related protein Caspase-3, Active caspase-3, PARP, Cleaved PARP is equal In the presence of different degrees of rise, wherein Active caspase-3 and Cleaved PARP raise more obvious.These result tables Influences of the bright ART to PC9 ability of cell proliferation may be by suppressing EGFR by-passing signals (IGF1R, AXL) and downstream signal is logical The activation on road (PI3K/Akt) realizes, may be relevant with the apoptosis of further induction PC9 cells.

The activation of by-passing signal is to cause one of important mechanisms of secondary resistance.The research display transmembrane protein of the past IGF1R can bypass the action target spot of EGFR-TKI with the PI3K/Akt signal paths in alternative activation downstream, reach cell and continue Propagation and the effect of anti-apoptotic, cause EGFR-TKI to fail to respond to any medical treatment, so as to produce acquired resistance^[24-25].Also research shows AXL plays an important role in the cellular activities such as cell propagation, migration, diffusion, anti-apoptotic, EMT^[26], and under AXL Trip molecule has a lot, including PLC γ, MMP-9, SOCS, PI3K/Akt, ERK1/2, SRC etc.^[27-28].Sawu etc. is in stomach cancer MKN7 Found in cell line, Gas6/AXL signal pathways can start downstream PI3K/Akt signal paths, prevent cell startup program Death program, suppresses apoptosis in gastric cancer^[29-30].By-passing signal IGF1R and AXL common downstream signaling pathway is PI3K/Akt Signal path, the signal path is the important path of cell survival, and the Akt of activation activates or suppress downstream by phosphorylation Target protein Bad, Caspase 9, IKK, mTOR and p21Clip etc., and then adjust propagation, differentiation, apoptosis and the migration of cell. Akt as PI3K/Akt signal paths hinge, the overexpression in kinds of tumors, prognosis with tumour is into negative correlation.P-Akt makees It is the activated form of Akt, the propagation of cell can be promoted, suppresses the apoptosis of cell, and then promote transfer, the angiogenesis of tumour. Its unique biological function determines its important function in tumour^[31].Research also shows PI3K/Akt signal paths Overactivity can cause EGFR-TKI resistances, and suppressing PI3K/Akt signal paths can make cell line regain medicaments insensitive Property^[32]。

Therefore, it is presumed that, Artesunate can be by suppressing EGFR by-passing signals IGF1R, AXL and downstream signaling pathway The activation of PI3K/Akt makes acquired resistance cell again sensitive to molecular targeted agents.In order to verify our supposition, we By building PC9/ER resistant models, there is the situation of acquired resistance in simulation clinic EGFR-TKIs, carry out improving grinding for resistance Study carefully.

We pass through heavy dose of Tarceva constant impingement PC9 cells first, obtain the resistance to Tarceva of Secondary cases thin Born of the same parents system PC9/ER cells, drug resistance is detected with cck-8, and resistance multiple is 11.94 times, points out to be successfully established resistance to Tarceva PC9/ER cells.Then by Real-time RT-PCR and Western Blot find PC9/ER cells in exist IGF1R, The overexpression of AXL and Akt, as a result points out the acquired resistance of PC9 cells with the activation of EGFR by-passing signals (such as IGF1R, AXL) It is relevant, it is consistent with current result of study.

Then we further study pharmacological actions of the ART to PC9/ER cells, and the result display ART of cck-8 can be same The propagation of PC9/ER cells, and IC50 and PC9 cell no significant differences can be suppressed, it is resistance in the absence of intersecting between prompting ART and ER The property of medicine.Next we have detected the inhibitory action of the mono- medicines of ER and joint ART and ER to PC9/ER cells, hair with cck-8 methods again PC9/ER cells IC50 is lower after existing Combination intervention, illustrates there is reversing drug resistance by drug combination that (reversal index is 1.75 Times), also illustrate that Artesunate can make PC9/ER cells recover the sensitiveness to ER again.And the result table of Western Blot Bright PC9/ER intracellular IGF1R, AXL and Akt protein level has different degrees of downward, especially shows its phosphorylation water On flat, wherein Combination intervention group is lowered more obvious.And apoptosis-related protein Caspase-3, Active caspase-3, PARP, Cleaved PARP have different degrees of rise.In testing in vivo, it has been found that the mono- medicines of ART and drug combination The protein expression of group internal IGF1R, AXL and Akt is lowered, but drug combination does not increase its toxic and side effect.These Result shows that ART can suppress the propagation of PC9/ER cells really, and perhaps the mono- medicines of ART and ART and ER drug combinations can play Improve resistance, PC9/ER cell lines is regained the pharmacological action of drug susceptibility.Its mechanism may make by suppressing EGFR The activation of by-passing signal (IGF1R, AXL) and downstream signaling pathway (PI3K/Akt) and the apoptosis of induction PC9/ER cells are realized 's.

In sum, the study find that, IGF1R, AXL and its downstream signaling pathway (PI3K/Akt) take part in NSCLC's Generation evolution, and ART can be by suppressing the activation and rise of IGF1R, AXL and its downstream signaling pathway (PI3K/Akt) Apoptosis-related protein plays antineoplastic action, and it is logical also to suppress IGF1R, AXL and its downstream signal in mdr cell The activation on road (PI3K/Akt) and apoptosis-induced, the Suppressive effect of enhancing Tarceva, make the cell of resistance to Tarceva again to strategic point Lip river is sensitive for Buddhist nun.This research provides new foundation for Artesunate as candidate anti-tumor medicine, improves patients with lung cancer prognosis One new thinking is provided and brings new hope to patients with lung cancer.

Full text conclusion

1.ART can suppress the propagation of human lung adenocarcinoma PC9 cells, and its mechanism may be believed with inducing cell apoptosis and suppression EGFR The activation of number path is relevant.

2.ART can reverse the resistance of PC9/ER, its mechanism be probably with suppress EGFR by-passing signals paths (IGF1R, AXL the activation) with downstream signaling pathway (PI3K/Akt) is relevant.

Claims

1. a kind of new opplication of Artesunate, it is characterised in that：The Artesunate is used to reverse adenocarcinoma of lung Tarceva resistance.

2. the new opplication of Artesunate according to claim 1, it is characterised in that：Artesunate and Tarceva joint are used Medicine.