CN105943527A - Applications of autophagy inhibitors in enhancing tumor cell growth inhibiting effect under hypoxia condition of drugs prepared by adopting CH282-5 - Google Patents
Applications of autophagy inhibitors in enhancing tumor cell growth inhibiting effect under hypoxia condition of drugs prepared by adopting CH282-5 Download PDFInfo
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Abstract
The invention provides applications of autophagy inhibitors in enhancing the tumor cell growth inhibiting effect under the hypoxia condition of drugs prepared by adopting CH282-5. The invention relates to the research on anti-cancer drugs, provides the thought of enhancing the inhibiting effect of CH282-5 for a colon cancer cell line under the hypoxia condition by adopting the autophagy inhibitors (3-MA and CQ), and provides the thought for treating the solid tumor under the hypoxia microenvironment. The result shows that the two autophagy inhibitors have the effects of enhancing the tumor growth inhibiting effect of CH282-5 under the hypoxia condition to different extents. To sum up, the research preliminarily shows that under the hypoxia condition, the enhancement of autophagy reduces the effect of CH282-5 in inhibiting the tumor cell growth. The applications can be taken as the earlier-stage foundation of other in-vivo researches, and one possibility for the resistance mechanisms of other anticancer compounds under the hypoxia tumor microenvironment is also provided.
Description
Technical field
The present invention relates to a kind of autophagy inhibitor enhancing CH282-5 under preparing hypoxia condition, suppress growth of tumour cell medicine
Application in thing.
Background technology
Colorectal cancer is one of current modal disease, and it is straight that the annual whole world has about 1,200,000 patients to be diagnosed as knot
Intestinal cancer, and have more than 600,000 patients and directly or indirectly die from colorectal cancer.There were significant differences for its sickness rate in each department,
This has close ties with so-called western life.The sickness rate of male's colorectal cancer is higher than women.Additionally, the morbidity of colorectal cancer
Rate can increase along with the increase at age, and the Colorectal Cancer the median age of such as developed country is 70 years old.And for knot
The Drug therapy of intestinal cancer, is but limited to a variety of causes including tumor microenvironment, does not has significance curative effect to improve.
Tumor microenvironment is the interior environment that tumor is residing in its generating process, by tumor cell itself, Interstitial cell, micro-
Blood vessel, tissue fluid and a small amount of infiltrating cells etc. collectively constitute.In the generating process of entity tumor, although tumor has and persistently lures
Send out the ability of angiogenesis and form abundant capillary network, but tumor cell oxygen consumption is higher has exceeded what blood capillary can be provided by
Oxygen amount, this often leads to be the microenvironment of a kind of hypoxia inside entity tumor.It is true that in most of entity tumors, all deposit
It is environment at hypoxia, on the one hand, hypoxia microenvironment can stimulate including VEGF, PDGF
The secretion of angiogenesis factor, promote angiogenesis；On the other hand, hypoxia can promote the autophagy of tumor cell further,
The aggravation tumor cell opposing to chemotherapeutics, and promote the transfer of tumor cell.
Cell autophagy (autophagy) is intracellular impaired, degeneration or the protein of aging and organelle to be transported
Lysosome carries out the process of digestion degraded, and under normal physiological conditions, cell autophagy is beneficial to cell and keeps homeostasis.But swollen
In oncocyte, autophagy provides more rich nutrition for cancerous cell, promotes tumor growth.In the treatment of most of solid tumors, wither
Tolerance of dying is the important mechanisms producing tumor drug resistance, and cell autophagy can prevent the apoptosis that antineoplastic agent is induced, and promotes tumor drug resistance.
Summary of the invention
It is an object of the invention to provide a kind of autophagy inhibitor enhancing CH282-5 under preparing hypoxia condition, suppress tumor cell
Application in growth medicine.
Compound used therefor CH282-5 of the present invention is Chinese Academy of Sciences's Shanghai organic chemistry institute synthesis, and it is sent out in advance biology
Bright having applied for a patent, the patent No.: 201310182988.3, the present invention is follow-up discovery, still mainly uses colon cancer cell
System (HCT116, HT29).Present invention primarily contemplates the medicine site that plays a role is inside tumor, however solid tumor internal be hypoxia
Environment, so detection medicine (1% O under low-oxygen environment2) anticancer effect be also one of which important process.
The present invention, by with reference to Nature protocol, is made hypoxia and cultivates equipment, then ensured by Anaerobic indicator
It is low-oxygen environment in cultivating equipment.
Respectively under the conditions of hypoxia condition and normal oxygen, processing colon carcinoma cell line HCT116 and HT29 with CH282-5,72 is little
Shi Hou, uses MTS method to measure suppression ratio, and under the conditions of finding to compare normal oxygen, under hypoxia condition, the inhibitory action of CH282-5 substantially drops
Low.
Respectively under the conditions of hypoxia condition and normal oxygen, processing colon carcinoma cell line HCT116 and HT29 with CH282-5,24 is little
Shi Hou, uses Western blot to detect two kinds of conventional autophagy marks (LC3-I, P62), under finding that LC3-I and P62 is substantially
Adjust, show that autophagy strengthens.
Under the conditions of hypoxia and normal oxygen, by using two kinds of conventional autophagy inhibitors (3-MA, CQ) with CH282-5 process
Suppress autophagy while colon carcinoma cell line HCT116 and HT29, after 72 hours, use MTS method to measure suppression ratio, compare and do not make
Use autophagy inhibitor group, use the inhibitory action of the CH282-5 of autophagy inhibitor group substantially to reduce.
Conclusion: for CH282-5, the enhancing of autophagy is to cause it to suppress under low oxygen conditions under growth of tumour cell ability
The reason of fall.And by using autophagy inhibitor (3-MA, CQ) CH282-5 can be strengthened under hypoxia condition to colon carcinoma cell line
The inhibitory action of (HCT116, HT29).
The enforcement step of the present invention is as described below:
The first step, with reference to Nature protocol, makes hypoxia and cultivates equipment, then ensure to cultivate dress by Anaerobic indicator
Standby interior for low-oxygen environment.
Second step, MTS method measures the inhibitory action of CH282-5 under the conditions of hypoxia and normal oxygen.
3rd step, detects autophagy mark LC3-I and P62 by Western blot.
4th step, uses two kinds of conventional autophagy inhibitors (3-MA, CQ), detects whether after suppression autophagy, CH282-5
The inhibitory action to growth of tumour cell can be recovered under low oxygen conditions.
The present invention relates to the research of cancer therapy drug, and propose to use autophagy inhibitor (3-MA, CQ) to strengthen CH282-5 low
Inhibitory action to colon carcinoma cell line under the conditions of oxygen, proposes the thinking that a kind for the treatment of is in the solid tumor of hypoxia microenvironment.
Accompanying drawing explanation
Fig. 1 CH282-5 is at 1% O2With 20% O2Impact on HCT116, HT29 cell survival rate under the conditions of two kinds.MTS
Method, is measured absorbance (A by microplate reader490nm), calculate suppression ratio.
Fig. 2 1% O2On P62, LC3-I and LC3-II albumen in the impact of HCT116 and HT29 cell inner expression.
Western blot method, CH282-5 processes 24 hours.
Fig. 3 3-MA/CQ strengthens CH282-5 at 1% O2Under the conditions of inhibitory action to HCT116 and HT29.
* in figureP＜ 0.05.
Detailed description of the invention
The present invention is expanded on further below in conjunction with instantiation.These examples are used merely to explain the present invention, rather than
Limit the scope of the present invention.The experimental technique of unreceipted specific experiment condition in following Examples, generally according to normal condition.
Embodiment one: hypoxia cultivates equipment preparation, measures CH282-5 to two kinds of colons under the conditions of hypoxia and normal oxygen two kinds
The inhibitory action of cancerous cell line.
(1% O of mixed gas used by the present invention2、5% CO2、94% N2) purchased from Chinese Academy of Sciences's Shanghai organic chemistry institute.Training
Support bottle to be reequiped by lucite bottle, before using, detect its sealing property.Manufacturing process is with reference to Nature Protocols.Carry out
Before cell is cultivated, in Anaerobic indicator (Qingdao Hai Bo Bioisystech Co., Ltd) detection culture bottle, whether can keep low
Oxygen environment, to characterize being successful of hypoxia cultivation equipment.
Material therefor of the present invention includes following material, people source colon carcinoma cell line (HCT116, HT29), and DMEM height sugar is cultivated
Base (Sai Mo flies company, the U.S.), hyclone (FBS, Gibco company, the U.S.), penicillin and streptomycin (PS, Invitrogen
Company, the U.S.), used medium of the present invention preparation ratio is: 89%DMEM+10%FBS+1%PS, the most all claims the training of this configuration ratio
Foster base is culture medium.Hypoxia condition of culture is 37 ° of C, 5% CO2、1% O2, normal oxygen condition of culture is 37 ° of C, 5% CO2、20% O2。
This case method, it is standby that MTS reagent is sub-packed in 1.5 mL EP pipes.Cell is inoculated in 96 orifice plates, and HCT116 cell is
3000 cells/well, HT29 cell is 5000 cells/well, 3 multiple holes.Every kind of cell is divided into two groups of (20% O2、1% O2) mistake
Night cultivates；Suck old culture medium gently, add culture medium containing different CH282-5 concentration (Concentraton gradient: 0 μM, 5 μMs, 10
μM, 20 μMs, 40 μMs), cultivate 72 hours, suck old culture medium gently, add 200 μ L(20 μ L MTS+180 μ L cultivate
Base) to hatch 4 hours, microplate reader detects each hole absorbance (A490 nm).
This example is that MTS method measures variable concentrations gradient CH282-5 at 1% O2With 20% O2Under the conditions of to colon cancer tumours
The suppression situation of cell line.See Fig. 1 and Biao 1.
Result shows, at 1% O CH282-5 concentration is relatively low when2With 20% O2Under the conditions of two kinds, cell survival rate phase
With, show that hypoxia itself cell growth will not have inhibitory action, along with raising 20% O of CH282-5 concentration2The cell of group is deposited
Motility rate is decreased obviously, and 1% O2The cell survival rate of group but declines slowly, and CH282-5 concentration is difference when 10 μMs and 20 μMs
The most notable, but tend to again equal along with the concentration of CH282-5 further improves two groups of cell survival rates, now hypoxia condition
Protective effect to cell is broken by the CH282-5 of high concentration completely.So analyzing this type of result, we need to select suitably
CH282-5 concentration, in this example, we select CH282-5 concentration to be 10 μMs or 20 μMs to carry out statistical analysis, obtain notable
Sex differernce.
Additionally this example is by being calculated CH282-5 at 1% O2With 20% O2Under the conditions of to 3 class tumor cell lines
IC50, result shows 1% O2Under the conditions of IC50 compare 20% O2IC50 increase at least 1.5 times.Find with reference to other document,
Under low oxygen conditions, the IC50 of cancer therapy drug typically has the raising of about 2 times.
Embodiment two: detection conventional autophagy mark P62, LC3-I and LC3-II
Autophagy reduces the behind reason that cancer therapy drug plays a role often, and tumor cell often passes through under low-oxygen environment
Autophagy provides more nutrient substance for himself, and then we detect 1% O by Western blot2With 20% O2Under conditions of
P62 and LC3-I, the expression of LC3-II.
This example material therefor is, P62 antibody (Cell Signaling Technology company, the U.S.), LC3 I/II
Antibody, β-Actin antibody (R&D Systems company, the U.S.).Two kinds of colon cancer cell HCT116 and HT29 are respectively classified into 4 groups:
(1) 20% O2；(2) 20% O2+20 μM CH282-5；(3) 1% O2；(4) 1% O2+20 μM CH282-5.CH282-5's is dense
It is to be determined by embodiment one that degree selects 20 μMs.The process time of 4 groups is 24 hours, is put in by cell the most pre-after 24 hours
Cold, collect cell, cell lysis with scraper plate, add the protease inhibitor PMSF of 1%, centrifugal, remove supernatant, add loading
Buffer, 100 ° of C boil 10 minutes, every hole loading 20 μ L, and overnight, two is anti-with horseradish peroxidase, secretly in an anti-closing
Develop in room.See Fig. 2 and Biao 2.
This sample result shows at 1% O2Under conditions of the P62 band of two kinds of cell lines all have and substantially weaken.HCT116 and
The LC3-I of HT29 cell line has more apparent thin out.In the autophagy generating process of tumor cell, P62 can bound substrates transport
Degrade to autophagy lysosome, cause P62 protein level to reduce；Autophagy lysosome forming process LC3-I can be changed into LC3-
II, and LC3-II can be attached to the lysosomal surface of autophagy.When autophagy strengthens, whole circulation meeting acceleration, to decompose more
Thing the most to be decomposed.
From this example, under hypoxia condition, the autophagy of HCT116 and HT29 is remarkably reinforced.This causes the most exactly
The reason that under hypoxia condition, CH282-5 suppression growth of tumour cell effect declines.
Embodiment three: detect whether two kinds of conventional autophagy inhibitors (CQ, 3-MA) can recover CH282-5 at hypoxia condition
Under the inhibitory action low to colon carcinoma cell line
It is same set of equipment that hypoxia used by this example cultivates equipment with embodiment one.
The used colon carcinoma cell line of this example is still HCT116, HT29.
Two kinds of autophagy inhibitor concentration used by this example select with reference to the early stage patent of CH282-5, higher concentration from
Bite inhibitor itself and cell can be produced toxicity, and 5 μMs can preferably be suppressed autophagy that cell the most also will not produce substantially poison
Property.
From above example one and example two, the effect of CH282-5 suppression growth of tumour cell is bright under low oxygen conditions
Aobvious reduction, autophagy is remarkably reinforced, therefore it is presumed that CH282-5 suppresses the obvious reason reduced of effect of growth of tumour cell very
It is probably autophagy to strengthen.Then we select two kinds of conventional autophagy inhibitors (3-MA, CQ), the generation of suppression autophagy, Jin Erguan
Whether the effect of the suppression growth of tumour cell examining CH282-5 can return to normal oxygen level.
This example will be tested according to packet once:
HCT116+3-MA:20% O2、20% O2+5 μM 3-MA、1% O2、1% O2+5 μM 3-MA
HCT116+CQ:20% O2、20% O2+5 μM CQ、1% O2、1% O2+5 μM CQ
HT29+3-MA:20% O2、20% O2+5 μM 3-MA、1% O2、1% O2+5 μM 3-MA
HT29+CQ:20% O2、20% O2+5 μM CQ、1% O2、1% O2+5 μM CQ
All process above each group with the CH282-5 of 0 μM, 5 μMs, 10 μMs, 20 μMs, 40 μMs Concentraton gradient.
Two kinds of colon carcinoma cell lines of HCT116, HT29 respectively with 3000/hole, 5000/hole, 3 multiple holes.Overnight incubation,
After cell attachment, add CH282-5 and the autophagy inhibitor of respective concentration according to above packet, be then placed in respective concentration
Oxygen cultivate equipment in cultivate 72 hours.
After 72 hours, suck old culture medium gently, add 200 μ L(20 μ L MTS+180 μ L culture medium) to hatch 4 little
Time, microplate reader detection each hole absorbance (A490 nm).
Seeing Fig. 3, this sample result shows, by 20% O2Group, 20% O2+ 5 μMs of 3-MA groups and 20% O2+ 5 μMs of CQ groups
Compare, it has been found that 5 μMs of 3-MA itself to two kinds of colon carcinoma cell lines all without obvious Developing restraint effect, and 5 μMs of CQ phases
Than 5 μMs of 3-MA, two kinds of colon carcinoma cell lines are had Developing restraint effect slightly, but will not produce relatively for interpretation of result
Big impact.By the analysis of example one, we select when CH282-5 concentration is 10 μMs or 20 μMs, are estimated at hypoxia bar
Under part, can autophagy inhibitor recover the CH282-5 suppression efficiency to tumor cell line.Result shows, no matter HCT116 or
HT29, compares 1% O2Group, 1% O2+ 5 μMs of CQ groups all have and significantly strengthen inhibitory action.
Although the present invention describes specific example, but having is a little obvious to those skilled in the art
The present invention can be made various changes and changes the most under the premise without departing from the spirit and scope of the present invention.Therefore, institute
Attached claim covers all these change within the scope of the present invention.
Claims (1)
1. an autophagy inhibitor strengthens CH282-5 and suppresses the application in growth of tumour cell medicine under preparing hypoxia condition.
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