CN106727603A - Application of the DEMETHYLZEYLASTERAL in the medicine for preparing treatment cancer of pancreas - Google Patents
Application of the DEMETHYLZEYLASTERAL in the medicine for preparing treatment cancer of pancreas Download PDFInfo
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Abstract
The present invention relates to application of the DEMETHYLZEYLASTERAL in the medicine for preparing treatment cancer of pancreas.There is significant lethal effect to human pancreatic cancer cell present invention demonstrates compound DEMETHYLZEYLASTERAL, with induced tumor cell-cycle arrest in the G0/G1 phases, and anti-pancreatic cancer effect can be played with the dependent cell apoptosis of Caspase 3 by the way that Induces Autophagy is dead；During with gemcitabine drug combination, DEMETHYLZEYLASTERAL can significantly reduce the IC50 of gemcitabine, and drug combination has more preferable inhibitory action to human pancreatic cancer cell；DEMETHYLZEYLASTERAL can promote gemcitabine antitumous effect with Induces Autophagy death during low concentration, and DEMETHYLZEYLASTERAL mainly promotes gemcitabine chemotherapy effect by promoting Apoptosis during high concentration.Based on this, DEMETHYLZEYLASTERAL can be used to prepare the medicine and gemcitabine drug combination for the treatment of cancer of pancreas, or for studying cell autophagy or Apoptosis mechanism.
Description
Technical field
The present invention relates to pharmaceutical technology field, specifically, it is related to DEMETHYLZEYLASTERAL preparing the medicine for the treatment of cancer of pancreas
Application in thing.
Background technology
In situation is raised year by year, in the U.S., its death toll occupies the 4th to the incidence of disease of cancer of pancreas in all tumours, suffers from
The overall 5 years survival rates of person are 8% or so, and recent decades are without significantly improving.With gemcitabine (Gemcitabine) as basic scheme
Though chemotherapy can to a certain extent improve the prognosis of Pancreas cancer patients, effective percentage is still unsatisfactory, and patient life
Quality is substantially reduced.Though combined chemotherapy and targeted therapy scheme have trial more, overall treatment effect is not good enough, and efficiently high poison,
Compare with gemcitabine list medicine, the limited advantages of drug combination.Multidisciplinary synthesis are advocated currently for advanced pancreatic cancer patient
Treatment, but the biological characteristics due to tumour in itself, the sensitiveness to chemicotherapy are poor, and the effect for improving prognosis is very limited.
Therefore, it is very necessary to explore the new treatment of pancreatic cancer medicine of research and development.
DEMETHYLZEYLASTERAL (demethyzelyasteral, abbreviation ZST93) molecular formula C29H36O6, molecular weight 480.59,
CAS 107316-88-1, chemical structural formula is as shown in Figure 1A.
《Fudan University》Academic dissertation " Primary Study of DEMETHYLZEYLASTERAL extracorporeal anti-tumor function " is disclosed within 2009,
DEMETHYLZEYLASTERAL has been inquired into T-24 human bladder cancer cells, SPC-A1 human lung carcinoma cells, SW620 human colon cancer cells, 7860
The external life of the different tumor cell lines of people's clear cell carcinoma of kidney cell, BEL7404 human liver cancer cells, 6 kinds of 7901 gastric carcinoma cells
Inhibitory action long, it was demonstrated that DEMETHYLZEYLASTERAL is respectively provided with certain growth inhibition effect to the tumor cell line of 6 kinds of separate sources,
And in time and concentration dependent, wherein T-24 and 7901 cell lines are most sensitive to medicine, SPC-A1 and 7860 least sensitive；
DEMETHYLZEYLASTERAL has also been inquired into the growth inhibition effect of Patients with Urinary System Tumors cell Du-145, PC-3, EJ cell line and right
The influence that its cell cycle changes, it was demonstrated that DEMETHYLZEYLASTERAL can significantly inhibit the growth in vitro of Du-145, PC-3, EJ cell,
Its mechanism may be related in the G0/G1 phases to by cell block.
However, at present DEMETHYLZEYLASTERAL to the therapeutic action of human pancreas cancer there is not yet document report.
The content of the invention
The purpose of the present invention is directed to deficiency of the prior art, there is provided the new application of DEMETHYLZEYLASTERAL.
In the first aspect of the present invention, there is provided DEMETHYLZEYLASTERAL is in the medicine for preparing prevention or treatment cancer of pancreas
Using.
In the second aspect of the present invention, there is provided DEMETHYLZEYLASTERAL is preparing the anti-pancreatic cancer effect of enhancing gemcitabine
Application in medicine.
In the third aspect of the present invention, there is provided a kind of pharmaceutical composition of anti-pancreatic cancer, with DEMETHYLZEYLASTERAL and Ji
His shore of west is active component, and comprising pharmaceutically acceptable carrier.
Described pharmaceutically acceptable carrier, including excipient, such as starch, water；Lubricant, such as magnesium stearate；Disintegration
Agent, such as microcrystalline cellulose；Filler, such as lactose；Binding agent, such as pregelatinized starch, dextrin, using method well known in the art
Various formulations are made, oral or injection is taken in such as tablet, capsule, granule, solution, syrup, injection, transfusion
Methods of administration such as (including intravenous injection, drip-feed, intramuscular injection, hypodermic injections) carries out the treatment of anti-pancreatic cancer.
In the fourth aspect of the present invention, there is provided DEMETHYLZEYLASTERAL suppresses the medicine of human pancreatic cancer cell propagation preparing
Or the application in reagent.
Described human pancreatic cancer cell is human pancreatic cancer cell T3M4, Colo-357, Capan-1, ASPC-1, MIA
PaCa-2 or PANC-1.
In the fifth aspect of the present invention, there is provided application of the DEMETHYLZEYLASTERAL in reagent preparation, described reagent is used
In：
A) expression of Cyclin D1 and Cyclin A2 is suppressed；
B) inducing pancreatic cancer cell-apoptosis；
C) Caspase-3 is activated；Or
D) inducing pancreatic cancer cell autophagy.
The invention has the advantages that：Have to human pancreatic cancer cell present invention demonstrates compound DEMETHYLZEYLASTERAL significant
Lethal effect, can be with induced tumor cell-cycle arrest in the G0/G1 phases, and by the way that Induces Autophagy is dead and Caspase-
3 dependent cell apoptosis play anti-pancreatic cancer effect；During with gemcitabine drug combination, DEMETHYLZEYLASTERAL can be significantly reduced
The IC50 of gemcitabine, drug combination has more preferable inhibitory action to human pancreatic cancer cell；DEMETHYLZEYLASTERAL during low concentration
Gemcitabine antitumous effect can be promoted with Induces Autophagy death, and DEMETHYLZEYLASTERAL mainly passes through during high concentration
Apoptosis is promoted to promote gemcitabine chemotherapy effect.Based on this, DEMETHYLZEYLASTERAL can be used to prepare the medicine for the treatment of cancer of pancreas
Thing and gemcitabine drug combination, or for studying cell autophagy or Apoptosis mechanism.
Brief description of the drawings
Fig. 1：ZST93 chemical structural formulas and the Proliferation Ability of ZST93 inductions.(A) chemical structural formula of ZST93；(B)CCK-
8 methods detect six kinds of human pancreatic cancer cell inhibiting rates；(C) CCK-8 methods detection ZST93 treatment 24h, 48h, 72h, MIA PaCa-2 suppressions
Rate processed；(D)MIA PaCa-2 IC50；(E) CCK-8 methods detection ZST93 treatment 24h, 48h, 72h, PANC-1 inhibiting rates；(F)
PANC-1 IC50.**P<0.01, * * * P<0.001, * * * * P<0.0001.
Fig. 2：The cell-cycle arrest of ZST93 inductions.(A) after flow cytometer detection ZST93 treatment 72h, MIA PaCa-2 cells
Cycle；(B) after flow cytometer detection ZST93 treatment 72h, the PANC-1 cell cycles；(C) Real-time PCR detections ZST93 treatment
After 72h, MIA PaCa-2 Cyclin D1 mRNA expressions；(D) after Real-time PCR detections ZST93 treatment 72h,
PANC-1 Cyclin D1 mRNA expressions；(E) after Real-time PCR detections ZST93 treatment 72h, MIA PaCa-2
Cyclin A2 mRNA expressions；(F) after Real-time PCR detections ZST93 treatment 72h, PANC-1 Cyclin A2
MRNA expressions.*P<0.05, * * P<0.01, * * * P<0.001, * * * * P<0.0001.
Fig. 3~18：Influences of the ZST93 to human pancreatic cancer cell autophagy and the crosstalk relation with Apoptosis.(Fig. 3)
After (Fig. 4) flow cytometer detection ZST93 joint Z-VAD-FMK treatment 72h, MIA PaCa-2 Apoptosis ratios；(Fig. 5) (Fig. 6) flows
After formula detection ZST93 joint Z-VAD-FMK treatment 72h, PANC-1 Apoptosis ratios；(Fig. 7) CCK-8 methods detection ZST93 connection
After closing Z-VAD-FMK treatment 72h, MIA PaCa-2 inhibiting rates；The detection ZST93 joint Z-VAD-FMK treatment of (Fig. 8) CCK-8 methods
After 72h, PANC-1 inhibiting rates；(Fig. 9) Western blot detection ZST93 treatment 72h after, MIA PaCa-2 Caspas-3 and
LC3 expressions；(Figure 10) MIA PaCa-2 LC3II/LC3I relative expression levels；(Figure 11) Western blot are detected
After ZST93 treatment 72h, PANC-1 Caspas-3 and LC3 expressions；(Figure 12) PANC-1 LC3II/LC3I relative expression's water
It is flat；After (Figure 13) CCK-8 methods detection ZST93 joint 3-MA treatment 72h, MIA PaCa-2 inhibiting rates；(Figure 14) CCK-8 methods are detected
After ZST93 joint 3-MA treatment 72h, PANC-1 inhibiting rates；(Figure 15) (Figure 16) flow cytometer detection ZST93 joint 3-MA treatment 72h
Afterwards, MIA PaCa-2 Apoptosis ratio；After (Figure 17) (Figure 18) flow cytometer detection ZST93 joint 3-MA treatment 72h, PANC-1 is thin
Born of the same parents' apoptosis ratio.*P<0.05, * * P<0.01, * * * P<0.001, * * * * P<0.0001.
Figure 19~30：Influences of the ZST93 to pancreatic cancer cell gemcitabine sensitiveness.(Figure 19) CCK-8 methods are detected
After Gemcitabine joint ZST93 treatment 72h, MIA PaCa-2 inhibiting rates；(Figure 20) MIA PaCa-2 IC50；(Figure 21)
After CCK-8 methods detection Gemcitabine joint ZST93 treatment 72h, PANC-1 inhibiting rates；(Figure 22) PANC-1IC50；(Figure 23)
After Western blot detection Gemcitabine joint ZST93 treatment 72h, MIA PaCa-2 Caspas-3 and LC3 expression water
It is flat；(Figure 24) MIA PACa-2 LC3II/LC3I relative expression levels；(Figure 25) Western blot detect Gemcitabine
After joint ZST93 treatment 72h, PANC-1 Caspas-3 and LC3b expressions；(Figure 26) PANC-1 LC3II/LC3I are relative
Expression；After (Figure 27) (Figure 28) flow cytometer detection Gemcitabine joint ZST93,3-MA treatment 72h, MIA PaCa-2 are thin
Born of the same parents' apoptosis ratio；After (Figure 29) (Figure 30) flow cytometer detection Gemcitabine joint ZST93,3MA treatment 72h, PANC-1 cells wither
Die ratio.*P<0.05, * * P<0.01, * * * P<0.001, * * * * P<0.0001.
Figure 31~44：Proliferation Abilities and Chemosensitizing effect of the In vivo study ZST93 to human pancreas cancer.(Figure 31) (Figure 38)
Every group of photo of tumor tissues；(Figure 32) (Figure 39) every five days measurement transplantable tumor volumes, and draw tumour growth curve；(Figure 33)
(Figure 40) every group of quality of tumor tissues；(Figure 34) (Figure 41) TUNEL detects tumor tissues level of apoptosis；(Figure 35) (Figure 42) is every
The apoptosis rate of group tumor tissues；(Figure 36) (Figure 43) SABC detects tumor tissues Ki-67 expressions；(Figure 37) (Figure 44)
Every group of Ki-67 positive rate of tumor tissues.*P<0.05, * * P<0.01, * * * P<0.001, * * * * P<0.0001(vs
control).#P<0.05, ###P<0.001, ####P<0.0001(vs gemcitabine).
Specific embodiment
The specific embodiment that the present invention is provided is elaborated below in conjunction with the accompanying drawings.
The preparation of the DEMETHYLZEYLASTERAL of embodiment 1
1st, preparation method
Velamen of Tripterygium wilfordii is crushed, and is put back into stream pot, adds 1.5 times of ethyl acetate, is heated to reflux 3 hours, is repeated 2 times,
Acetic acid ethyl acetate extract is obtained, solvent is reclaimed, dry extract is obtained, yield is 8.25%, taking the silica gel of 7.5 times of amounts of medicinal extract carries out post layer
Analysis, using n-hexane/acetone gradient elution, obtains DEMETHYLZEYLASTERAL (abbreviation ZST93) crude product.Acetone recrystallization, crystallization are used again
Put in 60 DEG C of vacuum drying ovens and dry 4 hours, obtain ZST93 yellow crystal of the purity more than 99%.
2nd, physicochemical characteristicses
Prepared ZST93 is yellow powder or crystal, odorless, and fusing point is：248℃.Molecular formula：C29H36O6, molecular weight
For：480.59, structural formula is as shown in Figure 1A.
TLC is checked：Contain catechol structure in ZST93, with strong reproducibility, can show green with ferric chloride reaction
Color.Ultraviolet-visible spectrophotometry is determined absorption maximum at 246nm.
The purposes research of the DEMETHYLZEYLASTERAL of embodiment 2
1st, material and method
1.1 materials
The ZST93 of purity prepared by embodiment 1 more than 99%.
Human pancreatic cancer cell T3M4, Colo-357, Capan-1, ASPC-1, MIA PaCa-2 and PANC-1 are purchased from
ATCC。
Cell Counting kit-8 purchased from Dojindo Molecular Technologies Inc. (Kumamoto,
Japan)；ReverTraQPCR RT kit are purchased from TOYOBO (Tokyo, Japan)；SYBR Green PCR Master
Mix is purchased from TOYOBO (Tokyo, Japan)；Cell apoptosis detection kit purchased from BD Biosciences (San Jose, CA,
USA)；Tunel original positions apoptosis detection kit is purchased from KeyGENBioTECH (Nanjing, China).
DMEM culture mediums, RPMI-1640 culture mediums, hyclone are purchased from Gibco (Grand Island, NY, USA)；3-
MA, Z-VAD-FMK are purchased from Selleckchem (Houston, TX, USA)；Anti- β-actin antibody are purchased from MBL
Technologies (Woburn, MA, USA)；Anti-cleaved caspase-3antibody, Anti-LC3antibody are purchased
From Cell Signaling (Danvers, MA, USA)；Anti-caspase-3antibody, Anti-Ki-67antibody are purchased
From Abcam (Cambridge, MA, USA)；Hydrochloride for injection gemcitabine is purchased from LILLY companies of the U.S.；6 week old BALB/c nude mices
Purchased from Laboratory Animal Science portion of Department Of Medicine, Peking University, 16~20g of quality.
1.2 methods
1.2.1 cell culture
Human pancreatic cancer cell T3M4, Colo-357, Capan-1, ASPC-1, MIA PaCa-2 and PANC-1 are incubated at and contain
In DMEM the or RIMP-1640 nutrient solutions of 10% hyclone, 100U/mL penicillin and 100 μ g/mL streptomysins, it is placed in containing 5%
CO2, being cultivated in 37 DEG C of cell culture incubators of constant temperature and humidity, every 2~3d changes liquid 1 time.Cell monolayer adherent growth, to 70%~
Trypsin Induced passage during 80% fusion.
1.2.2 cell survival assay
The cell in exponential phase is collected, with every hole 5 × 103Individual cell is inoculated into 96 orifice plates, after cell attachment, point
Not Jia Ru debita spissitudo gradient gemcitabine or (and) ZST93, every group of hole of repetition 3, while setting blank zeroing group (not refinement
Born of the same parents add the PBS of equivalent) and control group (0.1%DMSO solution).After medicine effect 72h, Cell Counting are used
Kit-8 detects cell viability.Each hole adds 0.01mL CCK-8,37 DEG C, 5%CO2Continue to cultivate 1~3h in cell culture incubator,
ELIASA surveys each hole 450nm absorbances (A) value.Inhibiting rate (%)=100%- (experimental group A values-blank zeroing A values)/(control
Group A values-blank zeroing A values) × 100%.
1.2.3WesternBlot
UV Absorption method determines protein concentration.After albuminous degeneration, 10% sodium dodecyl (SDS) polyacrylamide coagulates
Gel electrophoresis, after go on nitrocellulose filter, 5% skimmed milk power TBST solution closing 2h, plus 4 DEG C of primary antibody overnight after, secondary antibody room
Temperature is incubated 1h, and ELC chemical methods light.
1.2.4Real-time PCR
Trizol methods extract human pancreatic cancer cell total serum IgE.Use ReverTraQPCR RT kit are inverted
Record, Real-time PCR detections are carried out using SYBR Green PCR Master Mix.With GAPDH as internal reference, each gene
MRNA expressions are with (2-△△CT) represent.Primer sequence is as follows：GAPDH, 5'-ACGGATTTGGTCGTATTGGG-3'(SEQ
ID NO.1), 5'-TGATTTTGGAGGGATCTCGC-3'(SEQ ID NO.2)；Cyclin D1,
5'-ACGAAGGTCTGCGCGTGTT-3'(SEQ ID NO.3), 5'-CCGCTGGCCATGAACTACCT-3'(SEQ
ID NO.4)；Cyclin A2,5'-CAGTGTGAAGATGCCCTGGCTT-3'(SEQ ID NO.5),
5'-CAAGGATGGCCCGCATACTGTTA-3'(SEQ ID NO.6)。
1.2.5 Flow Cytometry detects the cell cycle
In order that cell cycle synchronization, collects the human pancreatic cancer cell in exponential phase, free serum culture 24h.Receive
Cell after collection free serum culture 24h, is inoculated in 6 orifice plates, and after cell attachment, experimental group cell adds the ZST93 of various concentrations,
Using the culture medium containing 0.1%DMSO as control, after culture 72h, each group cell each 1 × 10 is collected6It is individual, phosphate buffer
(PBS) wash 2 times, the fixed 24h of 70% 4 DEG C of ethanol.Containing 50 μ g/ml propidium iodide (PI) and 100 μ g/ml
The PBS solution room temperature lucifuge of RNase is incubated 30min.The U.S. BD FACSCalibur flow cytomery cell cycles.
1.2.6 Flow Cytometry detects Level of Apoptosis
The cell in exponential phase is collected, 6 orifice plates are inoculated in, after cell attachment, experimental group cell adds different dense
The gemcitabine of degree or (and) ZST93, using the culture medium containing 0.1%DMSO as control, after culture 72h, use Apoptosis
Detection kit detects Apoptosis ratio.Collect cell and washed 2 times with phosphate buffer (PBS), Binding buffer solutions
Suspension cell simultaneously adjusts born of the same parents' concentration for 1 × 106Individual/ml.Take and add 5 μ l Annexin V-FITC and 5 μ in 100 μ l cell suspensions
L PI dye liquors, mix, and room temperature lucifuge is incubated 15min, is subsequently adding 400 μ l Binding buffer solutions, in 1h, using U.S. BD
FACSCalibur flow cytomery Apoptosis.
1.2.7 Nude Mouse Model
6 week old BALB/c nude mices, 16~20g of body weight, digital random method is divided into 6 groups, every group 6, each group body weight, sex,
Week old balanced proportion.1×107The individual MIA PaCa-2 cells in exponential phase are suspended in 200 μ l PBS, are expelled to 4
All BALB/c nude mices armpits, set up human pancreas cancer Nude Mouse Model, routine observation growth of transplanted human situation.The 15th after inoculation
It rises, and gemcitabine group gives intraperitoneal injection gemcitabine (50mg/kg, 1 time/3 days) and the ethanol water (1 of oral administration gavage 5%
Times/day), median dose ZST93 groups give oral administration gavage ZST93 (160mg/kg, 1 times/day) and intraperitoneal injection PBS (1 time/3
My god), low dosage ZST93 groups give oral administration gavage ZST93 (80mg/kg, 1 times/day) and intraperitoneal injection PBS (1 time/3 days), agent high
Amount ZST93 groups give oral administration gavage ZST93 (200mg/kg, 1 times/day) and intraperitoneal injection PBS (1 time/3 days), drug combination group
Give intraperitoneal injection gemcitabine (50mg/kg, 1 time/3 days) and oral ZST93 (160mg/kg, 1 times/day), control group gives
The PBS (1 time/3 days) and the ethanol water of oral administration gavage same volume 5% (1 times/day) of intraperitoneal injection same volume.Periodic measurement is moved
Plant the major diameter (a) of knurl tubercle, footpath (b) wide, by V=(a × b2The formula of)/2 calculates transplantable tumor volume.After 45 days (after administration 30 days)
Nude mice is put to death, excised tumor is weighed.
1.2.8 SABC
FFPE after the paraformaldehyde of nude mice model tumor tissue 4% is fixed.After dewaxing hydration, antigen retrieval is carried out, it
Section is immersed in 0.3% hydrogen peroxide 15 minutes afterwards, 4 DEG C of overnight incubations of Ki-67 antibody, HRP secondary antibodies are incubated.Seen under microscope
Examine, randomly select 5 visuals field, calculate Ki-67 positive rates.
1.2.9Tunel test
A transplanted tumor in nude mice tumor tissues part is fixed into rear FFPE with 4% paraformaldehyde.Withered using Tunel original positions
Detection kit of dying is operated according to product description.Observation by light microscope, randomly selects 5 visuals field, calculates apoptosis rate.
1.2.10 statistical analysis
Analyzed using the statistical softwares of SPSS 13.0, all data mean ± standard deviations represent, group difference relatively makes
Checked with independent sample mean t.P<0.05 thinks that difference is statistically significant.
2nd, result
2.1 pairs of inhibited proliferations of human pancreatic cancer cell
It is to detect that ZST93, to the inhibited proliferation of human pancreatic cancer cell, from 6 kinds of human pancreatic cancer cells, adds respectively
Plus the ZST93 of various concentrations is processed 72 hours, CCK-8 methods detection inhibiting rate.Result of study shows, different human pancreatic cancer cells
It is more sensitive (Figure 1B) to ZST93, and as the inhibiting rate that increases of ZST93 concentration gradually increases, ZST93 is pointed out for people's pancreas
The inhibited proliferation of adenocarcinoma cell has concentration dependent.
Select the human pancreatic cancer cell MIA PaCa-2 and the human pancreatic cancer cell of relative insensitivity to ZST93 rdativery sensitives
PANC-1 carries out follow-up study.MIAPaCa-2 and PANC-1 add the ZST93 of various concentrations respectively, respectively process 24 hours, 48
Hour, 72 hours, CCK-8 methods detection inhibiting rate.Result of study shows that 24h, 48h, 72h are equal to MIA PaCa-2 for ZST93 effects
With certain inhibitory action (Fig. 1 C), with the extension of ZST93 action times, IC50 is gradually reduced (Fig. 1 D), points out ZST93
There is time dependence to the inhibited proliferation of MIA PaCa-2.ZST93 has identical to the inhibited proliferation of PANC-1
Result (Fig. 1 E and 1F).
Influences and molecular mechanism of 2.2 ZST93 to the human pancreatic cancer cell cycle
Compare influences of the ZST93 to the human pancreatic cancer cell cycle.The ZST93 treatment 72h of various concentrations, control group addition
0.1% DMSO, streaming method detection cell cycle.Result of study shows, with increasing for ZST93 concentration, human pancreatic cancer cell
It is that MIA PaCa-2 and PANC-1 cell cycle distributions are significantly changed, ratio shared by the cell of G0/G1 phases gradually rises (figure
2A and 2B).Further to study the mechanism of ZST93 cell cycle regulations, Real-time PCR methods have detected various concentrations
The expression of Cyclin D1 and Cyclin A2mRNA after ZST93 treatment 72h.Result of study shows, Cyclin D1 and
Cyclin A2mRNA expression is gradually reduced (Fig. 2 C~F).The above results show, growth inhibitions of the ZST93 to human pancreatic cancer cell
Effect has concentration dependent, while the target spot for also having pointed out ZST93 to act on is likely located at G1 → S turning points, ZST93 is by suppression
The expression of Cyclin D1 processed and Cyclin A2, make the cell cycle stop at the GO/G1 phases cannot be introduced into DNA synthesis the phase, so as to press down
Cell propagation processed.
The influence of the 2.3 pairs of human pancreatic cancer cell autophagy and the crosstalk relation with Apoptosis
Caspase-3 inhibitor Z-VAD-FMK can suppress Caspase-3 activationinduced apoptosis, recover
Cell viability.Various concentrations ZST93 be have detected to human pancreatic cancer cell level of apoptosis, inhibiting rate and Caspase-3 expressions
Influence.Result of study shows that high concentration ZST93 is activated in can dramatically increasing human pancreas cancer MIA PaCa-2 and PANC-1 cells
The expression of type Capase-3, inducing cell apoptosis；But only slightly increase the expression water of activated form Capase-3 during low concentration
It is flat, fail to cause level of apoptosis to significantly change；Caspase-3 inhibitor Z-VAD-FMK can suppress ZST93 inductions
Apoptosis, reduces inhibiting rate (Fig. 3~12).The above results show that high concentration ZST93 can be induced by activating Caspase-3
Human pancreatic cancer cell apoptotic death.
3-MA is a kind of autophagy inhibitor, can suppress cell autophagy.LC3 is the mark of autophagy, when autophagy occurs,
Kytoplasm LC3-I changes into film combination LC3-II, LC3II/LC3I and can be used to assess the quantity of autophagosome.Result of study shows
Show, low concentration ZST93 can induce cell autophagy, but with the increase of ZST93 concentration, autophagy level weakens (Fig. 9~12).3-MA
After suppressing cell autophagy level, tumor cell activity is not recovered, and Level of Apoptosis can on the contrary dramatically increased, ZST93
The Apoptosis of induction can strengthen (Figure 13~18) by 3-MA.The result shows the ZST93 of various concentrations is respectively by luring
Guided cell autophagy death (during low concentration) and apoptotic death (during high concentration) play antitumor action in pancreatic cancer cell,
The pancreatic cancer cell Death Mechanism of ZST93 inductions includes autophagy death and apoptotic death, and intersects in the presence of certain between the two
Dialogue mechanism.
The influence of the 2.4 pairs of pancreatic cancer cell gemcitabine sensitiveness and molecular mechanism
Inhibitory action of the ZST93 to human pancreatic cancer cell is combined using CCK-8 methods detection gemcitabine.Result of study table
Bright, drug combination has more preferable inhibitory action (Figure 19) to MIA PaCa-2.During gemcitabine treatment 72h, MIA PaCa-2
IC50 be 4.10 ± 0.22ng/ml, gemcitabine combine 0.1 μ g/ml ZST93 can make IC50 be reduced to 2.60 ±
0.03ng/ml, gemcitabine combines 0.5 μ g/ml ZST93 can make IC50 be reduced to 1.13 ± 0.09ng/ml (Figure 20).Connection
Sharing medicine has identical inhibitory action (Figure 21) to PANC-1, and during gemcitabine treatment 72h, the IC50 of PANC-1 is 10.56
± 2.93 μ g/ml, gemcitabine combines 0.5 μ g/ml ZST93 can make IC50 be reduced to 0.76 ± 0.07 μ g/ml, Ji Xita
Combine 5 μ g/ml ZST93 so that IC50 is reduced to 0.06 ± 0.02 μ g/ml (Figure 22) in shore.The result shows various concentrations
The ZST93 of (low concentration, high concentration) can significantly increase sensitiveness of the pancreatic carcinoma to gemcitabine chemotherapy, and its IC50 is equal
It is remarkably decreased.
Next, have detected the autophagy and apoptosis of ZST93 inductions to human pancreatic cancer cell gemcitabine chemosensitivity
Influence.Result of study shows that gemcitabine mainly plays antitumor activity by inducing cell apoptosis.When with low concentration ZST93
During use in conjunction, the human pancreatic cancer cell level of apoptosis of gemcitabine induction is used alone less than gemcitabine, and cell autophagy
Level is dramatically increased, and illustrates that tumour cell occurs autophagy dead；During gemcitabine joint high concentration ZST93, human pancreas cancer is thin
Born of the same parents' level of apoptosis is used alone higher than gemcitabine, and the increase of cell autophagy level is not obvious, illustrates that tumour cell mainly occurs
Apoptotic death.Autophagy inhibitor 3-MA can improve the Apoptosis induced during the ZST93 joint gemcitabines of various concentrations
Level, further enhancing anti-pancreatic cancer effect (Figure 23~30) of ZST93 joint gemcitabines.These results indicate that ZST93
Sensitiveness of the pancreatic cancer cell to gemcitabine chemotherapy can be promoted by two kinds of approach of Induces Autophagy and apoptosis respectively, this two
There is certain crosstalk mechanism in kind cell death mechanism.
Proliferation Abilities and Chemosensitizing effect of the 2.5 In vivo study ZST93 to human pancreas cancer
In order to detect inhibitory action of the internal ZST93 to human pancreas cancer, from human pancreatic cancer cell MIA PaCa-2, build
Nude Mouse Model, carries out In vivo study.Nude mice is randomized into six groups：Control group, median dose ZST93 groups, low dosage
ZST93 groups, high dose ZST93 groups, gemcitabine group, joint group.Periodic measurement transplantable tumor volume, transplantable tumor is opened after being inoculated with 15 days
Begin to be administered, administration puts to death nude mice after 30 days, and excised tumor is weighed, Tunel methods detection tumor death level, Immunohistochemical Method detection
Tumour Ki-67 expressions.Result of study shows, ZST93 can suppress tumour growth, and into dose dependent (Figure 31~
33)；Tunel experiments show, ZST93 can be with inducing apoptosis of tumour cell, and into dose dependent (Figure 34 and 35)；SABC
Experiment shows, ZST93 can suppress Ki-67 expression, and into dose dependent (Figure 36 and 37).Meanwhile, result of study is also indicated that,
Gemcitabine joint ZST93 is used alone (Figure 38~40), use in conjunction to the inhibition of human pancreas cancer better than gemcitabine
Group apoptosis rate is (Figure 41 and 42) higher, and Ki-67 expressions are lower (Figure 43 and 44).
3rd, conclusion
Compound ZST93 has significant lethal effect to human pancreatic cancer cell, can be with induced tumor cell-cycle arrest
In the G0/G1 phases, and anti-pancreatic cancer effect is played with Caspase-3 dependent cells apoptosis by the way that Induces Autophagy is dead；
During with gemcitabine drug combination, ZST93 can significantly reduce the IC50 of gemcitabine, and drug combination has to human pancreatic cancer cell
There is more preferable inhibitory action；ZST93 can promote gemcitabine antitumous effect with Induces Autophagy death during low concentration,
And ZST93 mainly promotes gemcitabine chemotherapy effect by promoting Apoptosis during high concentration.
The above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
Member, on the premise of the inventive method is not departed from, can also make some improvement and supplement, and these are improved and supplement also should be regarded as
Protection scope of the present invention.
SEQUENCE LISTING
<110>Zhongshan Hospital Attached to Fudan Univ
Peking University First Hospital
<120>Application of the DEMETHYLZEYLASTERAL in the medicine for preparing treatment cancer of pancreas
<130> /
<160> 6
<170> PatentIn version 3.3
<210> 1
<211> 20
<212> DNA
<213>Artificial sequence
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tgattttgga gggatctcgc 20
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<213>Artificial sequence
<400> 3
acgaaggtct gcgcgtgtt 19
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<211> 20
<212> DNA
<213>Artificial sequence
<400> 4
ccgctggcca tgaactacct 20
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<212> DNA
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Claims (6)
1. application of the DEMETHYLZEYLASTERAL in the medicine for preparing prevention or treatment cancer of pancreas.
2. application of the DEMETHYLZEYLASTERAL in the medicine for preparing the anti-pancreatic cancer effect of enhancing gemcitabine.
3. a kind of pharmaceutical composition of anti-pancreatic cancer, it is characterised in that with DEMETHYLZEYLASTERAL and gemcitabine as active component,
And comprising pharmaceutically acceptable carrier.
4. application of the DEMETHYLZEYLASTERAL in the medicine or reagent that suppress human pancreatic cancer cell propagation is prepared.
5. application according to claim 4, it is characterised in that described human pancreatic cancer cell is human pancreatic cancer cell
T3M4, Colo-357, Capan-1, ASPC-1, MIA PaCa-2 or PANC-1.
6. application of the DEMETHYLZEYLASTERAL in reagent preparation, it is characterised in that described reagent is used for：
A) expression of Cyclin D1 and Cyclin A2 is suppressed；
B) inducing pancreatic cancer cell-apoptosis；
C) Caspase-3 is activated；Or
D) inducing pancreatic cancer cell autophagy.
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| CN114767692A (en) * | 2022-04-15 | 2022-07-22 | 中国科学院化学研究所 | Combined medicine of demethylzelaronal and metformin |
| CN114767869A (en) * | 2022-04-15 | 2022-07-22 | 中国科学院化学研究所 | Combined medicine of demethyleulasialdehyde and src inhibitor |
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| CN102114065A (en) * | 2011-01-28 | 2011-07-06 | 河南中医学院 | Longhairy antenoron herb and common threewingnut root Chinese medicinal composition with effect of preventing and treating cancer and application thereof |
| CN105497041A (en) * | 2015-12-17 | 2016-04-20 | 中国科学院上海有机化学研究所 | Application of pentacyclic triterpene compound and medicine composition |
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| CN114767692A (en) * | 2022-04-15 | 2022-07-22 | 中国科学院化学研究所 | Combined medicine of demethylzelaronal and metformin |
| CN114767869A (en) * | 2022-04-15 | 2022-07-22 | 中国科学院化学研究所 | Combined medicine of demethyleulasialdehyde and src inhibitor |
| CN114767692B (en) * | 2022-04-15 | 2024-07-16 | 中国科学院化学研究所 | Combined medicine of norzeranal and metformin |
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