CN109602752B - Triptolide is in induction cancer cell autophagy and hdac inhibitor is cooperateed with to treat the application in tumour - Google Patents
Triptolide is in induction cancer cell autophagy and hdac inhibitor is cooperateed with to treat the application in tumour Download PDFInfo
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- CN109602752B CN109602752B CN201910030895.6A CN201910030895A CN109602752B CN 109602752 B CN109602752 B CN 109602752B CN 201910030895 A CN201910030895 A CN 201910030895A CN 109602752 B CN109602752 B CN 109602752B
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- A61K31/585—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids containing heterocyclic rings, e.g. danazol, stanozolol, pancuronium or digitogenin containing lactone rings, e.g. oxandrolone, bufalin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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Abstract
Application of the triptolide in induction cancer cell autophagy.Experiments prove that Thunder God A prime can effectively cause the generation of cancer cell autophagy and apoptosis, while confirming that cell autophagy caused by inhibiting triptolide can promote the death of the apoptosis and cancer cell of cancer cell；Triptolide can promote the generation of ROS and the activation of JNK, reduce the expression of p-Akt and p-mTOR, the expression of the multilevel major protein for changing autophagy and apoptosis primary signal pathways ROS/JNK and Akt/mTOR, cause the generation of autophagy and apoptosis, to influence the activity of cancer cell, brain glioblastoma cell is can be improved for the sensibility of hdac inhibitor SAHA in triptolide.
Description
Technical field
The present invention relates to cell autophagies and apoptosis field, more particularly, to triptolide in induction cancer cell autophagy
On effect.
Background technique
Glioma (Glioma) is most common, most fatal one of primary malignancy brain tumor, have invasion it is strong, into
The features such as exhibition is rapid and easy to recur.Clinically the standard treatments of glioma are to be aided with radiotherapy based on surgical operation at present
With the comprehensive treatment of First-line chemotherapy medicine Temozolomide (TMZ).However, intrinsic and acquired resistance of its curative effect by TMZ
Limitation, and general reaction is poor.Therefore, there is an urgent need to a kind of replacement therapy drugs to effectively treat glioma.
Triptolide (Triptolide) is isolated first Diterpenoid epoxide lactone from tripterygium wilfordii, is Thunder God
The main component of rattan bioactivity.Triptolide has extensive bioactivity, and such as anti-inflammatory, immunosupress is practised contraception, anti-angiogenic
Generation and anticancer etc..It is current studies have shown that triptolide have in terms for the treatment of certain diseases associated with inflammation and tumour it is potential
Advantage, many corresponding molecular targets have been reported.2007, native compound triptolide, duplicated rattan A prime, capsicum
Element, curcumin and qinghaosu are referred to as most possibly developed into the model of modern medicines by Cell magazine.More and more research tables
Bright, triptolide is the broad-spectrum anti-cancer drug with strong activity, and triptolide can almost be killed from prostate,
All cancer cells of colon, mammary gland, blood and kidney etc., currently, some derivatives of triptolide come into clinic and comment
The valence stage.Previously some researches show that triptolide can inhibit the proliferation of glioma in vivo and in vitro, but its molecular mechanism is still
It is unclear.
Apoptosis (apoptosis), also known as I type apoptosis (PCD) are highly conserved in a kind of evolution
Height adjustment cell death mode.Apoptosis is to the development of the multicellular organism including tumour and stable state to Guan Chong
It wants.
Although apoptosis is the main mechanism during cancer chemotherapy, another adjusting machine for being known as autophagy is had recently emerged
System.Autophagy is that cellular content is transmitted to the mistake that lysosome is degraded by a kind of autophagosome structure for being known as duplicature
Journey.However, autophagy is different in the different phase of tumor development and the effect between individual cells and cancerous tissue.Mesh
Before, due to double action of the autophagy between cell survival and cell death, effect of the autophagy in antitumor is still that order is ground
The person of studying carefully feel confused with it is contradictory.In addition, the relationship between autophagy and apoptosis is also not very clear.In tumour cell, autophagy can be with
Inhibit cell death caused by apoptosis, can also act synergistically with apoptosis, collectively promote the death of cancer cell.Therefore, more
It is extremely important and necessary for understanding function of the autophagy in tumour cell.
Active oxygen (ROS) is the active form of oxygen, when be in reduced levels when, ROS be to cell it is beneficial, however, ROS
Build up the generation that can promote tumour.Mitochondria is the main place for generating ROS.JNK signal path is that ROS signal is logical
One of numerous downstreams cascade on road, it is closely related with cell Proliferation, differentiation, mitosis, cell survival and death.Akt/mTOR
Signal path is one of normal cell and tumor cell survival, growth, proliferation and the main adjusting approach of protein synthesis.Before
Research shows that Akt/mTOR access participate in cell autophagy and apoptosis process.
Drug combination is the hot spot in oncotherapy especially chemotherapy, for overcoming the drug resistance of tumour, reduces the secondary work of poison
With.In this field, the inhibitor SAHA of HDAC has been FDA approval listing for treating the drug of solid tumor, but it is controlled
Therapeutic effect is not good enough.
Summary of the invention
The present invention provides a kind of drug that can promote brain glioblastoma cell apoptosis and autophagy generation: triptolide.The medicine
Object by influencing the expression of ROS/JNK and Akt/mTOR signal path GAP-associated protein GAP, so as to cause brain glioblastoma cell apoptosis and
The generation of autophagy.
To achieve the above object, application of the triptolide in the drug in preparation induction cancer cell autophagy.
The drug is to the phosphorylation water that the inducing action of cancer cell autophagy is by the generation and JNK albumen that enhance ROS
It is flat, inhibit the expression of p-Akt and p-mTOR albumen to play a role.
The cancer cell is brain glioblastoma cell.
Inducement in the cancer cell autophagy process is to enhance the generation of ROS and the table of p-JNK albumen in cancer cell
It reaches, inhibits the expression of p-Akt and p-mTOR albumen.
A kind of triptolide is provided again according to demand in preparation ROS or p-JNK protein activator, p-Akt or p-mTOR
Application in inhibitor.
A kind of triptolide is provided again according to demand in the research preparation of ROS, p-JNK, p-Akt or p-mTOR albumen
On application
There is provided again according to demand a kind of triptolide research ROS, p-JNK, p-Akt or p-mTOR albumen and it
Substrate protein biological function on application.
A kind of application of triptolide in the starting agent of preparation autophagy or/and inducer is provided again according to demand.
The cancer cell is brain glioblastoma cell.
According to demand, the present invention also provides the pharmaceutical compositions of the inhibitor of triptolide and HDAC composition, for making
The drug of standby treatment tumour, wherein the hdac inhibitor is preferably SAHA；The tumour is preferably glioma.Its
In, the weight ratio of the inhibitor of triptolide and HDAC is 1:1-100, preferably 1:1-10.
It is an advantage of the current invention that
1, the effect and the two that present invention firstly discloses triptolide in terms of cancer cell specific induction of apoptosis and autophagy
Between relationship, by induce autophagocytosis, help cancer cell return its normal condition, inhibit the generation of apoptosis.
Triptolide and inhibitors of apoptosis share the death that can promote cancer cell.
2, Triptolide can be by promoting ROS/JNK and Akt/mTOR signal path being inhibited to realize it to apoptosis and oneself
The effect bitten.Specifically, triptolide can promote the generation of ROS and then activate JNK albumen, promote ROS/JNK signal
Access, while the expression of p-Akt and p-mTOR albumen can be inhibited, negative interaction is further influenced in Akt/mTOR signal path
Apoptosis and autophagy.
3, as a kind of drug, triptolide can with targeting act on cancer cell, effectively control cancer cell and lure
The apoptosis of cancer cell is led, and on normal cell without influence.
4, as a kind of cooperative drug, present invention discover that identical rattan A prime can cooperate with SAHA, brain glioblastoma cell is improved
For the sensibility of SAHA.
Detailed description of the invention
Fig. 1: Triptolide chemical structural formula
Fig. 2: the triptolide of various concentration acts on brain glioblastoma cell system U251, U87-MG and C6 and normal star
After shape spongiocyte Astrocyte different time, CCK8 detects the variation of cell activity.
After Fig. 3: the triptolide of various concentration acts on brain glioblastoma cell system U251, U87-MG and C6 for 24 hours, light
Learn the variation of micro- sem observation cell quantity and form.
Fig. 4: the triptolide of various concentration acts on brain glioblastoma cell system U251, U87-MG and C6 for 24 hours,
After hoechst 33242 contaminates nucleus, the form of fluorescence microscope nucleus detects the generation of apoptosis.
Fig. 5: the triptolide of various concentration acts on brain glioblastoma cell system U251, U87-MG and C6 for 24 hours, FITC
The bis- dye method detections of Annexin V/PI are extracellularly in the apoptosis of approach.
Fig. 6: the triptolide of various concentration acts on brain glioblastoma cell system U251, U87-MG and C6 for 24 hours, JC-1
Kit detects into the cell in the generation of approach apoptosis.
Fig. 7: various concentration triptolide acts on brain glioblastoma cell system U251, U87-MG and C6 for 24 hours or 300nM
Triptolide act on three kinds of cell line different times after, Western blotting detect apoptosis-related protein
The variation of Cleaved PARP and Cleaved-caspase 3,8,9.
Fig. 8: U251 cell transient transfection mRFP-EGFP-LC3B plasmid, triptolide individually or triptolide and from
After biting inhibitor 3-MA collective effect transfection cell, the formation of confocal microscopy autophagosome.
Fig. 9: various concentration triptolide acts on brain glioblastoma cell system U251, U87-MG and C6 for 24 hours or 300nM
Triptolide act on three kinds of cell line different times after, autophagy GAP-associated protein GAP LC3 II, p62 and Beclin-1 variation
Western blotting result.
Figure 10: autophagy inhibitor 3-MA or after inhibitors of apoptosis z-VAD and triptolide be jointly processed by cell, CCK8 inspection
Survey the variation of cell activity.
After Figure 11: Triptolide and autophagy inhibitor 3-MA is jointly processed by cell, Apoptosis by Flow Cytometry
Variation.
After Figure 12: Triptolide and autophagy inhibitor 3-MA or inhibitors of apoptosis z-VAD is shared, autophagy is related to apoptosis
Albumen LC3 II, p62, Beclin-1, cleaved-PARP, the Western that cleaved-caspase 3,8 and 9 changes
Blotting result
Figure 13: NAC (ROS inhibitor) three kinds of cell line 2h of advanced processing are used, then are acted on the triptolide of 300nM
After cell 12h, the generation of ROS is detected by fluorescence microscope with DCFH-DA probe.
Figure 14: NAC (ROS inhibitor) three kinds of cell line 2h of advanced processing are used, then are acted on the triptolide of 300nM
After cell 12h, pass through the generation of flow cytomery ROS with DCFH-DA probe.
Figure 15: NAC (ROS inhibitor) three kinds of cell line 2h of advanced processing are used, then are acted on the triptolide of 300nM
Cell for 24 hours after, CCK8 detect cell activity.
Figure 16；After Triptolide and ROS inhibitor NAC is shared, the Western of autophagy and apoptosis-related protein expression
Blotting result.
Figure 17: SP (jnk inhibitor) advanced processing U251 cell 2h is used, then is acted on carefully with the triptolide of 300nM
After born of the same parents 12h, the generation of Flow cytometry ROS.
Figure 18: various concentration triptolide acts on U251 cell and acts on cell with the triptolide of 300nM for 24 hours
After different time, JNK in cell, Akt, the Western blotting knot of mTOR albumen and its phosphorylated protein changes of contents
Fruit.
Figure 19: Triptolide and after jnk inhibitor SP or Akt inhibitor MK or mTOR inhibitors Ra share, autophagy phase
Close the Western blotting result of albumen and apoptosis-related protein variation.
Figure 20: Triptolide and after jnk inhibitor SP or Akt inhibitor MK or mTOR inhibitors Ra share, CCK8 inspection
Survey the variation of cell activity.
Figure 21: in nude mice brain glioblastoma cell model, influence of the various concentration triptolide to gross tumor volume.
Figure 22: Triptolide to the western blotting result of the intracorporal GAP-associated protein GAP of mouse.
Figure 23: Triptolide to the immunohistochemistry and immunofluorescence results of tumor tissues index of correlation.
Figure 24: Triptolide to the mechanism of action figure of brain glioblastoma cell.
Specific embodiment
Below in conjunction with the drawings and specific embodiments, the present invention is further explained.These embodiments are merely to illustrate the present invention
Rather than it limits the scope of the invention.In the following examples, the experimental methods for specific conditions are not specified, usually according to conventional strip
Part or according to the normal condition proposed by manufacturer.Unless otherwise defined, all professional and scientific terms as used herein and sheet
Meaning known to the skilled person of field is identical.In addition, any method similar to or equal to what is recorded and material all may be used
Applied in the method for the present invention.
Experimental method used in following embodiments is conventional method unless otherwise specified.Material used, reagent
Deng being commercially available unless otherwise specified.
The effect that 1 Triptolide of embodiment inhibits brain glioblastoma cell to grow in vitro
One, brain glioblastoma cell strain in vitro culture
Cell used in this experiment is human glioma cell U251, U87-MG and mouse Glial cells C6, uses addition
The MEM culture medium of 10% fetal calf serum, carries out cell culture in 37 DEG C, 5%CO2 incubator, grows to cell and accounts for culture
When ware bottom 70~80%, digests and pass on using 0.1% pancreas enzyme -EDTA.
Two, the detection of cell activity
This experiment inhibits brain glioblastoma cell to grow using CCK8 method and fluorescence microscope to triptolide (Fig. 1)
Effect detected.Triptolide is dissolved in DMSO and is made into 100 μM of concentrates, -20 DEG C of preservations, when use is distinguished dilute
It is interpreted as 30,100,300,1000nM.After cell spreads 96 orifice plates (2500-5000 cells/well) 24, various concentration is added
After triptolide different time, cell activity is detected with CCK8 method.After effect for 24 hours, cellular morphology is observed with optical microscopy
With the variation of quantity.
Experimental result: the results show that triptolide can effectively inhibit the growth of brain glioblastoma cell, to normal star
The poor activity of shape colloid.(Fig. 2 and Fig. 3)
2 Triptolide of embodiment can cause in brain glioblastoma cell in the generation with external approach apoptosis.
This experiment use fluorescence microscope, flow cytometer and Western Blotting method detection into the cell with it is external
The generation of apoptosis.
One, the variation of fluorescence microscope karyomorphism
Experimental method: cell spreads 96 orifice plates (2500-5000 cells/well), and second day at the triptolide of 300nM
After managing cell for 24 hours, nucleus is contaminated with hoechst 33242, passes through the variation of fluorescence microscope karyomorphism.
Experimental result: the results show that increasing with triptolide concentration, there is different degrees of dye in three kinds of cells
Chromatin condensation, marginalisation, the typical apoptosis form (Fig. 4) such as nuclear membrane cracking and apoptotic body.
Two, the generation of flow cytomery apoptosis
Experimental method: it by cell dissociation, is centrifuged and is resuspended in corresponding culture solution, with 2.5X 105The density kind in/hole
It plants in 6 orifice plates, is placed in 37 DEG C, 5%CO2It is incubated overnight in incubator.It second day, is handled with the triptolide of various concentration
Cell for 24 hours after, the generation of Apoptosis is detected with the bis- dye methods of FITC Annexin V/PI and JC-1 kit respectively.
Experimental result: the bis- dye methods of FITC Annexin V/PI show, with the raising of triptolide concentration, cell
Apoptosis ratio increases (Fig. 5).Show extracellularly in the generation of approach apoptosis.JC-1 is the results show that triptolide can cause
JC-1 red fluorescence/green fluorescence ratio decline (Fig. 6), shows the decline of mitochondrial membrane potential, is that inherent approach apoptosis is early
The index of phase.
Three, the generation of Western Blotting method detection Apoptosis
After cell spreads 6 orifice plates for 24 hours, for 24 hours or 300nM is used with the triptolide of various concentration processing cell
After triptolide handles cell different time, cell is collected, extracts albumen, detects apoptosis phase with Western Blotting method
Close the variation of albumen.
Experimental result: the results show that triptoldie can cause inherent approach and external approach apoptosis-related protein table
The variation reached.
3 Laser Scanning Confocal Microscope of embodiment detects triptolide to the inducing action of brain glioblastoma cell autophagy
Experimental method: this experiment using Laser Scanning Confocal Microscope to the autophagy of the triptolide brain glioblastoma cell induced into
Row detection.The bis- luciferase plasmids of 0.8ug mRFP-EGFP-LC3 are transiently transfected into U251 cell with liposome lip2000 2.0ul
In, for marking and tracking the variation of LC3 and autophagy stream.Wherein GFP is acid-sensitive type GFP albumen, and mRFP is stable
Luciferase expression group, is not influenced by the external world.It after entering second stage due to autophagosome, is merged, is formed certainly with lysosome
Bite lysosome.Autophagy lysosome can cause PH to decline due to the acidic environment inside lysosome, and GFP is quenched, therefore, GFP's
Weaken the smooth degree that can indicate that autophagy lysosome is formed, GFP is fewer, then circulates from autophagosome to the autophagy lysosome stage
It is more smooth.Conversely, autophagosome and lysosome fusion are suppressed, autophagy lysosome process is obstructed.MRFP is stable expression always
, thus journey can be flowed into evaluate autophagy by the bright spot ratio of GFP and mRFP.After transfection for 24 hours, with 3-MA pretreatment cell
2h, then cell is handled for 24 hours with the triptolide of 300nM, it is placed under Laser Scanning Confocal Microscope and observes and take pictures.
Experimental result: can see by the result that Laser Scanning Confocal Microscope takes, and triptolide can be induced effectively carefully
The bright spot of the generation of born of the same parents' autophagy, red fluorescence and green fluorescence increases.When triptolide and 3-MA are shared, this effect
Weaken.(Fig. 8) illustrates, triptolide can effectively facilitate the generation of autophagosome.
4 LC3 protein labeling method of embodiment detects triptolide to the inducing action of brain glioblastoma cell autophagy:
LC3 is the marker protein that cell autophagy is formed, and has two kinds of forms of LC3I and LC3II.When no autophagy occurs,
The albumen exists in the form of LC3I in cytoplasm, and when autophagy is formed, endochylema type LC3 (i.e. LC3-I) can digest a bit of polypeptide,
It is changed into (autophagosome) membranous type (i.e. LC3-II), the size of LC3-II/I ratio can estimate the height of autophagy level.Beclin-1
It is starting albumen when autophagy occurs for cell, p62 is a critical proteins of autophagy lysosomal degradation autophagy substrate.
Experimental method: by cell seeding in 6 orifice plates, 37 DEG C are placed in, 5%CO2It is incubated overnight in incubator.Second day,
Cell is divided into three groups, first group of removal original culture medium is added fresh configuration, contains 30,100,300,1000nM
Continue culture in the culture medium of triptolide for 24 hours；Fresh configuration is added after second group of removal culture medium, contains 300nM's
Continue to cultivate in culture medium, terminates culture respectively at the 6 of culture, 12,24,36h.It is total that cell is extracted immediately after cell termination culture
Albumen, Western Blotting method detect the variation of the expression quantity of above-mentioned autophagy GAP-associated protein GAP.
Experimental result: Western Blotting is the results show that in three kinds of cell lines, the table of Beclin-1 and LC3II
Raising trend is extended with the time up to increasing for triptolide activity.Meanwhile the expression of P62 with
Triptolide activity increases and reduces (Fig. 9).Illustrate that triptolide can cause the generation of glioma autophagy.
The influence that autophagy caused by 5 triptolide of embodiment grows brain glioblastoma cell
Experimental method: by cell seeding in 96 orifice plates, 37 DEG C are placed in, 5%CO2It is incubated overnight in incubator.Second day,
After pre-processing U251 cell with the 3-MA of 3mM, cell is handled for 24 hours with the triptolide of 300nM, with CCK8 method detection cell
Survival rate.
Experimental result: the results show that after using autophagy inhibitor 3-MA, the survival rate of cell is decreased obviously.Explanation
Cell autophagy caused by triptolide may play a part of cell and promote survival.(Figure 10)
The relationship of autophagy and apoptosis caused by 6 Triptolide of embodiment.
Experimental method: by cell seeding in 6 orifice plates, 37 DEG C are placed in, 5%CO2It is incubated overnight in incubator.Second day,
Former culture medium is removed, after 3-MA or 30 μM of 3mM of z-VAD pretreatment cell 2h, is handled with the triptolide of 300nM thin
Born of the same parents are for 24 hours.After cell terminates culture, it is divided into two groups.First group of collection cell, with the variation of flow cytomery cell autophagy.
Second group is extracted total protein of cell immediately, and Western Blotting method detects the table of autophagy GAP-associated protein GAP and apoptosis-related protein
Up to the variation of amount.
Experimental result: flow cytometer detection the results show that when using 3-MA inhibit autophagy after, the apoptosis rate of cell obviously increases
(Figure 11)；After Western Blotting is the results show that inhibit autophagy using 3-MA, the expression quantity of apoptosis-related protein is increased,
After inhibiting apoptosis using z-VAD, the expression of autophagy GAP-associated protein GAP equally increases (Figure 12).To sum up the result shows that,
Apoptosis caused by triptolide and autophagy can mutually inhibit.
The generation of 7 ROS of embodiment is that triptolide causes apoptosis and autophagy necessary.
One, Triptolide can cause the generation of ROS
Experimental method: by cell seeding in 6 orifice plates, 37 DEG C is placed in, is incubated overnight in 5%CO2 incubator.Second day,
After NAC (ROS inhibitor) pretreatment cell 2h, 300nM triptolide is added and continues after cultivating 12h, is visited with DCFH-DA
Needle passes through the generation of fluorescence microscope and flow cytomery ROS respectively.
Experimental result: the triptolide as the result is shown of fluorescence microscope and flow cytometry can promote the generation of ROS,
NAC can inhibit this effect of triptolide.(Figure 13 and Figure 14)
Two, the generation of cell death and apoptosis and autophagy caused by the adjustable triptolide of ROS.
Experimental method: U251 cell NAC pre-processes 2h, then with after triptolide processing for 24 hours, is detected respectively with CCK8
The activity of cell, with the variation of Western Blotting detection autophagy and apoptosis-related protein.
Experimental result: the results show that after NAC and triptolide are shared, compared to triptolide, the work of cell is applied alone
Property increase (Figure 15), the expression of autophagy and apoptosis-related protein declines (Figure 16).
The generation of 8 ROS of embodiment can cause the activation of JNK, and triptolide causes activation and the Akt and mTOR of JNK
Inhibition.
One, the generation of ROS can cause the activation of JNK
Experimental method: by cell seeding in 6 orifice plates, 37 DEG C are placed in, 5%CO2It is incubated overnight in incubator.Second day,
After SP (jnk inhibitor) pretreatment cell 2h, 300nM triptolide is added and continues after cultivating 12h, with DCFH-DA probe
Pass through the generation of flow cytomery ROS.
Experimental result: when triptolide and SP are jointly processed by cell, what the generation of ROS increased becomes apparent from (Figure 17),
And NAC can inhibit the phosphorylation level (Figure 16) of JNK, show that JNK may be the downstream molecules of ROS, ROS may promote JNK
Activation.
Two, Triptolide causes the activation of JNK and the inhibition of Akt and mTOR.
Experimental method: by cell seeding in 6 orifice plates, 37 DEG C are placed in, 5%CO2It is incubated overnight in incubator.Second day,
Remove culture medium after fresh configuration is added, contain 30,100,300,1000nM triptolide culture for 24 hours, or remove training
The culture medium containing 300nM of fresh configuration is added after supporting base, continues culture 3 respectively, 6,12, terminate culture afterwards for 24 hours.Cell is whole
After only cultivating, cell is collected, detects related pathways JNK, the variation of Akt and mTOR index expression with Western Blotting.
Experimental result: Western Blotting the results show that with triptolide activity increase and the time
Extension, the phosphorylation level of JNK increases, and the phosphorylation level of Akt and mTOR is then suppressed (Figure 18).These the result shows that
Triptolide may have activated ROS/JNK signal path, it is suppressed that Akt/mTOR signal path.
Cell death caused by the 9 adjustable triptolide of JNK and Akt/mTOR signal path of embodiment and apoptosis and
The generation of autophagy.
Experimental method: by cell seeding in 6 orifice plates, 37 DEG C are placed in, 5%CO2It is incubated overnight in incubator.Second day,
It removes former culture medium and is added 300nM's after pre-processing 2h with SP, MK (Akt inhibitor) or Ra (mTOR inhibitors)
Triptolide's continues culture for 24 hours.After cell terminates culture, autophagy index of correlation is detected with Western Blotting respectively
The variation of LC3 II, Beclin1 and p62 expression, with the variation of CCK8 detection cell activity.
Experimental result: SP can inhibit the generation of apoptosis caused by triptolide and autophagy, and triptolide is inhibited to draw
The cell death risen.MK and Ra then has opposite effect (Figure 19).It further demonstrates that, triptolide is by activating ROS/
JNK signal path inhibits Akt/mTOR signal path to cause the autophagy of glioma cell.
The internal verifying of 10 triptolide of embodiment effect.
Experimental method: by 3.5X105U251 cell injects the caudate nucleus of BALB/c nude mice by stereotaxic instrument, causes naked
The Brain Glioma Model of mouse.After the success of 7 days models, be injected intraperitoneally respectively big (0.4mg/kg), in (0.2mg/kg), small
The triptolide and Temozolomide (20mg/kg) of (0.1mg/kg) dosage.Once every other day, co-injection 7 times.The 14th day after administration
Materials calculate the volume of tumour by nuclear-magnetism, with Western Blotting, immunofluorescence and immunohistochemistry detection autophagy, wither
Die the expression of GAP-associated protein GAP and related pathways albumen.
Experimental result:, can be with the results show that triptolide can inhibit the growth (Figure 21) of internal brain glioblastoma cell
The variation for causing apoptosis and autophagy index of correlation, causes the generation of apoptosis and autophagy, and the phosphorylation level of JNK increases, Akt and
The phosphorylation level of mTOR reduces (Figure 22 and Figure 23).Show that triptolide can cause the autophagy of brain glioblastoma cell, it is main
If inhibiting Akt/mTOR signal path to realize by activation JNK signal path.It is consistent with Vitro Experimental Results.
Embodiment 11 duplicates rattan A prime and SAHA synergistic treatment glioma
Based on the discovery of embodiment 1-10, triptolide has by promoting ROS/JNK and inhibiting Akt/mTOR signal
Access realizes its effect to apoptosis and autophagy；Further, triptolide has human glioma cells system U251 and U87
There is apparent apoptosis-induced effect；Therefore we have further investigated triptolide and colloid tumor medicine known in the art
Drug combination.Based on the result of study of cell mechanism experiment, we are using triptolide 0.2ug/ml as basic concentration
Carry out drug combination research.
Experimental method: it by U251 cell dissociation, is centrifuged and is resuspended in corresponding culture solution, with 1X 105The density in/hole
It plants in 96 orifice plates, is placed in 37 DEG C, 5%CO2It is incubated overnight in incubator.Second day, cell is grouped, first group of removal is former
Fresh configuration is added as a control group in culture medium, and culture medium of other each groups addition containing various concentration drug continues to cultivate
24h。
Each group administration concentration is as follows:
After cell terminates culture, cell is collected, using mtt assay measurement group of cells activity and calculates inhibiting rate (inhibiting rate
=(control group OD value-administration group OD value)/control group OD value × 100%).
Synergistic effect calculation method: determined that (referring to non-patent literature: Dai Tijun, which merges, to be used using the equal q value method of Nintaus
Quantitative analysis [J] Chinese Pharmacological Bulletin of medicine, 1998 (5): 479-480.), q value is acquired by formula below: q=PA+B/
(PA+PB-PA×PB).P in formulaA、PBAnd PA+BRespectively A medicine group, B medicine group and two medicine combination group treatment rates.Q < 1 illustrates that two medicines close
With rear generation antagonism；Q > 1 illustrates to generate synergistic effect after two medicines share, and q=1 illustrates to generate to be added after two medicines share to make
With.
Experimental result:
| Inhibiting rate (%) | Q value | |
| Group 1 | 41.3±2.9 | - |
| Group 2 | 1.2±0.6 | - |
| Group 3 | 6.9±1.8 | - |
| Group 4 | 29.7±3.1 | - |
| Group 5 | 48.4±3.5 | - |
| Group 6 | 42.7±3.3 | 1.02 |
| Group 7 | 66.8±4.1 | 1.48 |
| Group 8 | 84.3±3.7 | 1.43 |
| Group 9 | 88.1±4.2 | 1.26 |
It can be seen from the results above that triptolide and Vorinostat ratio are only that performance is simple both in 1:0.1
Summation action, and ratio shows as obvious coordinate repression in 1:1-100, wherein best with 1:1 and 1:10 effect.It mentions
Show that triptolide and Vorinostat drug combination produce apparent synergistic effect, optimal proportion in terms for the treatment of glioma
Between 1:1-10.For similar result in U87 cell line, we have been similarly observed that similar result.
In addition, we have investigated triptolide and Temozolomide, chloroethene using same method in U251 cell line
The drug combination effect of base nitroso ureas, Etoposide, vincristine or bevacizumab, Temozolomide, chlorethylnitrosourea,
Etoposide, bevacizumab and vincristine do not find to generate synergistic effect with triptolide.
Claims (3)
1. the application of triptolide and hdac inhibitor preparation synergistic treatment tumour medicine, the hdac inhibitor are Fu Li
Promise he, the tumour be glioma.
2. application described in claim 1, wherein the weight ratio of the triptolide and Vorinostat is 1:1-100.
3. application as claimed in claim 2, wherein the weight ratio of the triptolide and Vorinostat is 1:1-10.
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