CN106822085A - Express purposes of the oncolytic adenovirus joint Quercetin of TRAIL in liver cancer cancer cell multiplication is suppressed - Google Patents
Express purposes of the oncolytic adenovirus joint Quercetin of TRAIL in liver cancer cancer cell multiplication is suppressed Download PDFInfo
- Publication number
- CN106822085A CN106822085A CN201611171522.3A CN201611171522A CN106822085A CN 106822085 A CN106822085 A CN 106822085A CN 201611171522 A CN201611171522 A CN 201611171522A CN 106822085 A CN106822085 A CN 106822085A
- Authority
- CN
- China
- Prior art keywords
- trail
- quercetin
- oncolytic adenovirus
- medicine box
- pharmaceutical composition
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
- A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/47—Quinolines; Isoquinolines
- A61K31/49—Cinchonan derivatives, e.g. quinine
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/19—Cytokines; Lymphokines; Interferons
- A61K38/191—Tumor necrosis factors [TNF], e.g. lymphotoxin [LT], i.e. TNF-beta
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
- A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
- A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
- A61K48/0025—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
- A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Zoology (AREA)
- Gastroenterology & Hepatology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Immunology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The present invention relates to the invention belongs to field of biological pharmacy, and in particular to purposes of the oncolytic adenovirus joint Quercetin of expression TRAIL in liver cancer cancer cell multiplication is suppressed.Specifically, oncolytic adenovirus the present invention relates to contain expression TRAIL have the treatment or inhibitory action that cooperate with to the propagation of liver cancer or HCC with the combination of Quercetin and other drugs, and then for the propagation for the treatment of liver cancer or suppression HCC provides new composition or medicine box.
Description
Technical field
The invention belongs to field of biological pharmacy, and in particular to the oncolytic adenovirus joint Quercetin of expression TRAIL is suppressing
Purposes in liver cancer cancer cell multiplication.
Background technology
Hepatocellular carcinoma (HCC) is one of most common malignant tumour, and it is listed in global 5th most common cancer, and year is dead
Rate die more than 500,000.
PCT/US2014/033675 is disclosed for treating the method for including the various cancers of primary and secondary carcinoma of liver
And composition, while being related to purposes of the quinacrine in the manufacture of the medicament for intractable liver cancer.
PCT/US2008/081645 provides the method for treating liver cancer.These methods include giving comprising the few core of modification
The compound of thuja acid, wherein modified oligonucleotide targeting miRNA.The invention also provides the composition for treating liver cancer.
This based composition includes the compound comprising modified oligonucleotide, wherein modified oligonucleotide targeting miRNA.The invention is sent out
Existing some miRNA overexpressions in liver cancer (such as hepatocellular carcinoma), therefore be selected as being targetted by modified oligonucleotide.This
Outward, the invention also found some miRNA in the overexpression in the hepatocellular carcinoma cells of dioxin, so be selected as by
Modified oligonucleotide is targetted.
201080047449.3 disclose by effective dosage be named as ITE for aryl hydrocarbon (Ah) acceptor
(AhR) one of endogenic ligand or its analog (active component) PCI or eradicate cancer to the subject with cancer
The method of disease.By the blood level of active component described in subject after measurement administration, its effective dose and administration frequency are determined
Rate.Will using carrier system prepare active component partly, enteral ground or parenterally apply to subject.The preparation
Medicine can also be administered together with one or more other novel remedies for cancer.After the subject breaks away from cancer, there is provided maintain
It is administered to ensure to eradicate cancer.Subject wherein with cancers such as liver cancer preferably receives treatment.
Adenovirus (Ad) is found by Wallace Rowe and its colleague in nineteen fifty-three first.Due to the Ad in tissue cultures
CPE, was used to treat cervix cancer in clinical experimental study in the virus in 1956.In past 20 years,
Used using oncolytic virus and produced excellent therapeutic efficiency.Most of clinical and preclinical studies concentrate on oncolytic virus and repair
Decorations, to improve, tumour transduction targeting, tumour-specific are replicated, intra-tumor is propagated and antiviral and anti-tumor immune response tune
Save and be transferred to foreign gene.The modification of oncolytic virus and the hybridization engineering of oncolytic virus are the especially promising for the treatment of of cancer
New method.Using the virus therapy of oncolytic Ad due to its high titre, insert the ability of the therapeutic gene of large-size and dividing
Split has extensive clinical practice with the transduction efficiency high in Unseparated Cell.Importantly, when oncolytic Ad is replicated, it
Not by its genome conformity to host；Therefore, they do not induce carcinogenic related mutagenesis.These unique functions cause oncolytic
Ad can turn into efficient gene carrier, have in terms of gene delivery optimal security (and the oncolytic virus of correlation, it is such as molten
Knurl sex reversal record virus, slow virus are compared with adeno-associated virus).
At present, the oncolytic virus different more than 12 kinds carries out the Phase I clinical trial for different type cancer.Oncolytic
Property Ad be to concentrate first and most the oncolytic virus of research so far, H101 (mutant of missing E1B55K genes) is in
Food and Drug Administration of state (CFDA) ratifies for treating head and neck cancer (Oncorine, the double prestige Biotech in Shanghai).Former
In research, we use virus therapy strategy Development cancer target gene (CTGVT), and generate a new E1B55K gene
Missing oncolytic adenovirus ZD55-TRAIL, it carries tumor necrosin relative death inducing ligand (TRAIL), and the part is swollen
The member of tumor necrosis factor superfamily simultaneously thinks that it is the new candidate of anticancer therapy.TRAIL can be various swollen with selective induction
The apoptosis of oncocyte, the toxicity to surrounding normal cell is negligible.Selectivity of the TRAIL in tumour growth is suppressed is
Show in vitro and in vivo.By contrast, TRAIL can cause the base of antagonism dead signal path with activating transcription factor NF- κ B
The transcription of cause.Unfortunately, due to this opposite or adjustment effect that TRAIL is played, although its have it is noticeable anti-swollen
Tumor activity, but many cancer cells display that the resistance to TRAIL.TRAIL is participated in by this negative anti-of the apoptotic signal of NF- κ B
The actual mechanism for presenting regulation is not yet illustrated completely.Fortunately, it has recently been demonstrated that by suppressing survival-signal and propagation base
The expression of cause activates the expression of apoptosis-associated genes simultaneously, and the therapeutic alliance with TRAIL and other drugs or chemical substance can be assisted
With the cell death or Apoptosis mechanism that increase in various tumour cells.
Quercetin (3,3', 4', 5,7- pentahydroxyflavones) is the main meals flavones found in various fruits and vegetables
Alcohol, including onion, apple skin, lettuce, cauliflower, capsicum, celery and unsweetened cocoa.Quercetin have anti-oxidant, anti-inflammatory,
Anti-angiogenesis, antiproliferative and rush apoptosis property, therefore, can be to the formation of anticancer and progress.According to these effects, by it
To cause the ability of cancer cell death, Quercetin causes cell viability to the selective modification signal transduction path related to carcinogenesis
Reduction, also, induce many cancer cell (including mammary gland, colon, lung, ovary and prostate cancer) apoptosis.
The content of the invention
The present inventor proves that ZD55-TRAIL and Quercetin can act synergistically kill in vitro and in vivo first
HCC cells.Additionally, our result of study shows that Quercetin significantly inhibits the NF κ B paths activated by ZD55-TRAIL so that
Apoptotic signal is dominant.We show that the therapeutic alliance of ZD55-TRAIL and Quercetin is a new practicality in current research
Therapeutic strategy.
In order to preferably cure liver cancer or for treatment liver cancer provides another feasible selection, the prognosis of raising patient or
Quality of life, it was found by the inventors of the present invention that the combination of the oncolytic adenovirus and Quercetin of expression TRAIL is thin to liver cancer or liver cancer
The propagation of born of the same parents has treatment or the inhibitory action of collaboration, and then for the propagation for the treatment of liver cancer or suppression HCC is provided newly
Therapy or means.
The present invention suppresses hepatoma cell proliferation function influence and enters using the oncolytic adenovirus and Quercetin joint of expression TRAIL
Row is external and experiment in vitro, and being as a result displayed in both the oncolytic adenovirus and Quercetin of expression TRAIL use in conjunction has collaboration
Suppress the effect of hepatoma cell proliferation.
The oncolytic adenovirus of expression TRAIL of the present invention can be the science oncolytic adenovirus for carrying trail dna
ZD55-TRAIL(Han,X.,et al.,Synergistic combination of histone deacetylase
inhibitor suberoylanilide hydroxamic acid and oncolytic adenovirus ZD55-TRAIL
as a therapy against cervical cancer.Mol Med Rep,2015.12(1):P.435-41.), it has
Tumour replicates specificity, to normal cytotoxic.The oncolytic adenovirus genome structure for expressing TRAIL is shown in Figure 1A.
Therefore, one aspect of the present invention provides the oncolytic adenovirus for expressing TRAIL in preparation for being treated in subject
Purposes in the pharmaceutical composition or medicine box of liver cancer or suppression hepatoma cell proliferation, wherein described pharmaceutical composition or medicine box contains
Express the oncolytic adenovirus and Quercetin of TRAIL, optionally, described pharmaceutical composition or medicine box also containing for treat liver cancer or
Suppress the other drugs active component of hepatoma cell proliferation, such as gemcitabine, Plerixafor (AMD3100) or taxol etc..
Optionally, the oncolytic adenovirus and Quercetin of the expression TRAIL in described pharmaceutical composition or medicine box are respectively in separate appearance
The oncolytic adenovirus and Quercetin that TRAIL is deposited or expressed in device are deposited in same container.
The invention provides liver cancer or the method for suppressing hepatoma cell proliferation is treated in subject, it includes being received to described
Examination person simultaneously or sequentially applies the oncolytic adenovirus and Quercetin of expression TRAIL.Optionally, in the method for the invention, can be with
Simultaneously or sequentially apply other treatment liver cancer or suppress the active constituents of medicine of hepatoma cell proliferation, such as gemcitabine,
Plerixafor (AMD3100) or taxol etc.；Or simultaneously or sequentially apply quinindium.
For subject of the present invention, it is preferably mammal, is more preferably people.
For the oncolytic adenovirus and Quercetin of expression TRAIL of the invention, those skilled in the art can make to it
Any modification, on condition that the modification does not negatively affect its activity.For example, can be modified compound or be loaded in other
On carrier, to improve its half-life period in vivo；Or can be connected with known penetrating peptide, to promote the compounds of this invention
Transdermal absorption crosses blood-brain barrier etc..In a word, those skilled in the art can carry out various modifications to compound of the invention
With improve delivery efficiency or for other purposes and keep its activity.For the modification of virus, for example, those skilled in the art can
To carry out genetic modification, so that the active component of the other resistance cancers of its expression.This kind of modification is also in the scope of the present invention
Within.
The oncolytic adenovirus and Quercetin of the expression TRAIL as active component of the invention can be together with can pharmaceutically connect
The carrier received is used together.In addition to the active ingredient (s, the method for the present invention, purposes and product can also be included and suitably pharmaceutically may be used
The carrier of receiving, including promote active component to be processed into the excipient of preparation (being for example suitable for the preparation injected or be transfused) and help
Agent.
The preparation for being suitable to inject or be transfused may include that aqueous and non-aqueous sterile injection liquid and aqueous and non-aqueous sterile are mixed
Suspension, the aseptic parenteral solution optionally comprising antioxidant, buffer, bacteriostatic agent and can make preparation and purpose recipient
The isobaric solute of blood, the sterile suspensions may include suspending agent and thickener.The preparation may be present in UD or
In multi-dose container, the ampoule of such as sealing, and freeze-dried (lyophilized) condition can be stored in, immediately using it is preceding only
Need to add sterile liquid carrier, such as water for injection.
Active component of the invention optionally can be combined with solid excipient, and optionally grinds resulting mixing
Thing, and when needing, after suitable auxiliary agent is added, the mixture of particle is processed, to obtain required formulation.Suitable excipient
Particularly filler is for example sugared, including lactose, sucrose, mannitol or D-sorbite；Cellulose or starch formulation, gelatin, yellow alpine yarrow
Glue, methylcellulose, hydroxypropyl methyl cellulose, sodium carboxymethylcellulose and/or polyvinylpyrrolidone (PVP).When needing,
Disintegrant, such as PVPP, agar or alginic acid or its salt such as sodium alginate can be added.
The amount of the oncolytic adenovirus and Quercetin of applying expression TRAIL in the present invention can be in energy Synergistic treatment subject
Any amount of liver cancer or suppression hepatoma cell proliferation, it can be comparable to about 0.2-25mg Quercetins, preferably 0.02-30mg Mongolian oaks
The dosage of the oncolytic adenovirus of Pi Su and 1-100MOI expression TRAIL.It is highly preferred that dosage unit includes the quercitrin of about 1-5mg
Element and 20-60MOI express the oncolytic adenovirus of TRAIL.Most preferably, dosage unit includes the Quercetin and 40- of about 2-3mg
50MOI expresses the oncolytic adenovirus of TRAIL.The measure of effective dose in the ability of those skilled in the art, in particular according to this
Under the enlightenment of the disclosure that text is provided.
According to the present invention, pharmaceutical product (medicine, medicament) of the invention or pharmaceutical composition can be with any effective doses
Using administration subject.Preferably, pharmaceutical product (medicine, medicament or medicine box) of the invention or pharmaceutical composition can be with multiple
Dosage is administered, such as from about 2 to about 15 times dosage, more preferably from about 4-10 times dosage, most preferably from about 6 times dosage.Especially excellent
The embodiment of choosing, in administration process, with every three weeks administration frequencies about once by pharmaceutical product of the invention (medicine, medicine
Agent) or pharmaceutical composition be administered to subject, for example inject, infusion or oral.In particularly preferred embodiment, it is logical to be administered
Cross intravenous injection and apply Quercetin, the oncolytic adenovirus of TRAIL are expressed by intraperitoneal injection.
It should be understood that pharmaceutical product (medicine, medicament or medicine box) of the invention or pharmaceutical composition can be by for by appointing
The mode of any suitable of the suitable approach administration of meaning is prepared.
The dosage unit of pharmaceutical product (medicine, medicament) of the invention or pharmaceutical composition is received based on conventional being administered
Examination person.For example, dosage unit can be administered more than once a day, once in a week, monthly etc..Dosage unit can be with two
It is administered based on times/week, i.e., twice a week, for example once every three days.
As it is used herein, "comprising" with " including ", " containing " or " being characterised by " it is synonymous, and in being included in
Or opening, and it is not excluded for the other element do not stated or method and step.Term "comprising" any table herein
State, particularly describing the method for the present invention, purposes or during product, it is thus understood that including substantially by the component or element or
Those products, method and purposes that step is constituted and is made up of the component or element or step.The sheet of description exemplified here
Invention suitably can be in the situation in the absence of any one or more of element not specifically disclosed herein, one or more limitation
Under put into practice.
Can be included in pharmaceutical product (such as pharmaceutical composition or medicine box) of the invention and be related to the specification of the pharmaceutical product,
And the specification can contain following content：Indication (such as liver cancer), application dosage (such as above-mentioned exemplarily illustrate)
And issuable side effect etc..
The term and statement for having used herein are used as descriptively rather than restrictive term, and in this kind of term and statement
Use in be not expected to exclude shown in and the feature or part thereof any equivalent, it is appreciated that various modifications are in request
It is possible in the scope of the present invention of protection.Although it is therefore understood that the present invention passed through preferred embodiment and optionally
Feature is specifically disclosed, but those skilled in the art can be using the modification and transformation of concept disclosed herein, and such modification
It is considered as in the scope of the present invention for such as being defined by accessory claim with change.
To be illustrated more clearly that the present invention, it is described in detail in conjunction with following examples, but these embodiments are only
To exemplary description of the invention, the limitation to the application is should not be construed as.
Brief description of the drawings
Fig. 1：The sign of ZD55-TRAIL.(A) schematic structure of ZD55-TRAIL.In ZD55-TRAIL, E1B 55-
KDa genes are replaced by the expression cassette of SV40polyA and TRAIL.(B) ZD55-TRAIL is identified by western engram analysis.Point
Not Yong Quercetin (10 μM), ZD55-TRAIL (2MOI) or Quercetin (10 μM) and ZD55-TRAIL (2MOI) to process HuH-7 thin
Born of the same parents.After 48 hours, cell lysate is prepared for analyzing the expression of TRAIL and E1A albumen.The cell of simulated infection is used as right
According to.GAPDH is used as protein loading control.
Fig. 2：The HCC cell growth inhibitions of Quercetin enhancing ZD55-TRAIL mediations.(A) HCC cell lines HepG2, HuH-7
It is small with ZD55-TRAIL (1,2,5,10MOI), Quercetin (5,10,25,50 μM) or combined treatment 48 respectively with SMMC-7721
When.The figure illustrates three example results of independent experiment.By MTT evaluation of measuring cell viabilities.(B)ZD55-TRAIL
Cooperative effect of the joint Quercetin to HCC cells.Analyzed by combinatorial index (CIN) and quantified, and it is right to be expressed as 1g (CIN)
Impacted ratio.In the case where that may be calculated, the confidential interval of display 95%.
Fig. 3：The HCC Apoptosis of Quercetin enhancing ZD55-TRAIL α inductions.(A) dye using Hoechst 33342
Detection apoptosis.By HuH-7 bed boards in 96 orifice plates and with ZD55-TRAIL (2MOI), Quercetin (10 μM) or ZD55-TRAIL
(10 μM) infection of (2MOI) plus Quercetin.In order to determine apoptosis degree, HuH- is processed with Hoechst 33342 within 72 hours after treatment
7 cells 30 minutes, then observe under inverted fluorescence microscope.Red arrow represents positive apoptotic cells.Original magnification
It is 400.(B) HuH-7 cells are processed as follows：ZD55-TRAIL (2MOI), Quercetin (10 μM) or ZD55-TRAIL (2MOI) add
Quercetin (10 μM).Untreated cell is used as control.After 48 hours, by Flow Cytometry Assay apoptosis.(C) ZD55- is used
TRAIL (2MOI), Quercetin (10 μM) or ZD55-TRAIL (2MOI) plus (10 μM) of Quercetin infection HuH-7 cells 48 hours.
Full cell extract is prepared, and carries out Western Western blottings to detect the activation of caspase approach.Use the GAPDH of expression
As loading control.
Fig. 4：Quercetin suppresses the activation of NF- κ B in the HCC cells that ZD55-TRAIL is induced.Use ZD55-TRAIL
(2MOI), Quercetin (10 μM) or (10 μM) treatment HuH-7 cells of ZD55-TRAIL (2MOI) plus Quercetin.After 48 hours, receive
Collection cell lysate simultaneously carries out western blot measure to check the change of I κ B α, p65 and p50.GAPDH using expression makees
It is loading control.
Fig. 5：The internal cooperative effect of ZD55-TRAIL and Quercetin.(A) different time measures gross tumor volume after the treatment.
Data are expressed as average value ± SD (n=6).*, P<0.01.(B) the figure illustrates the final time point the (the 55th in execution mouse
My god) each group is to the inhibitory action of tumour growth.
Specific embodiment
The present invention is further described in conjunction with the accompanying drawings and embodiments.
Material used and method are as follows in the embodiment of present invention.
Cell line and virus
Polymerase chain reaction SMMC-7721, HepG2 and HuH-7 are purchased from Chinese Academy of Sciences's Cell Culture Collection
Cell bank (CBTCCAS, Chinese Shanghai), and cultivate Dulbecco improvement Eagle culture mediums (DMEM, GIBCO,
Carlsbad, CA), it is supplemented with 10% heat-inactivated hyclone (FBS, GIBCO, Carlsbad, CA).By cell at 37 DEG C
Under in 5%CO2Incubated in humidified incubator.The structure of recombination oncolytic adenovirus ZD55-TRAIL and production are as previously described.Pass through
Infection HEK293 cells carry out the amplification of recombined adhenovirus.
The CTA of In Vitro Synergistic Effects and quantitative analysis
3- (4,5- dimethylthiazole -2- bases) -2,5- diphenyltetrazolium bromides (MTT) are purchased from Sigma-Aldrich (St
Louis, MO).Cell viability is obtained as described previously by MTT measure.In short, by 1 × 104Individual SMMC-7721, HepG2
Or HuH-7 cells are seeded in 96 orifice plates, and ZD55-TRAIL and the Quercetin treatment with prescribed concentration.48 hours after treatment,
To adding 10 μ L MTT solution (5g/L) in each hole, and it is incubated 4 hours at 37 DEG C.Using DNA enzymatic mark instrument (Tecan,
Maennedorf, Switzerland) measurement 570nm at absorbance.
Hoechst 33342 is dyeed
Hoechst 33342 is dyeed for detecting that apoptosis changes.HuH-7 cells respectively use ZD55-TRAIL, Quercetin or
The combined treatment of ZD55-TRAIL and Quercetin.After treatment 72 hours, by Hoechst 33342 (1mg/ml, the Sigma- of 5 μ L
Aldrich, St Louis, MO) add cell 30 minutes, and observe result under inverted fluorescence microscope.Untreated cell
With comparing.
Flow cytometry
With ZD55-TRAIL (2MOI), Quercetin (10 μM) or ZD55-TRAIL (2MOI) and (10 μM) treatment of Quercetin
HuH-7 cells.After 48 hours, according to specification, dye individually detection using the double dyeing of Annexin V-FITC and PI or PI and wither
Die cell.Using flow cytometry (FACStar cytofluorometer, BD Biosciences) inspection Apoptosis and carefully
Born of the same parents' cycle progress.
Western blot analysis
Collect HuH-7 cells and washed twice with PBS, then cracked in RIPA buffer solutions.By by cell extract
PVDF (polyvinylidene fluoride) films are loaded on 12%SDS- polyacrylamide gels and are subsequently transferred to separate protein.So
Film is incubated with the primary antibody and dilution for the following factor afterwards：Adenovirus -5E1A (1:1000), TRAIL (1:1000),
capase-9(1:1000), caspase-3 (1:1000), PARP (1:1000), GAPDH (1:1000), p65 (1:500), p60
(1:500) with I κ B α (1:500).Secondary antibody used and its dilution are anti-rabbit (1:5000) with anti-mouse (1:5000).Anti- gland
The antibody of virus -5E1A, Capase-9, Caspase-3, PARP and GAPDH is purchased from Santa Cruz Biotechnology
(Santa Cruz, CA).For p65, the antibody of p60 and I κ B α is available from Cell Signaling (Danvers, MA).
Zoopery
The license of zoopery is authorized by the animal protection of China and using the committee.Animal and experiment are according to said mechanism
Standard carry out.The male BALB/C nude mices of 5 week old are purchased from Chinese Shanghai Chinese Academy of Sciences Shanghai Experimental Animal Center.By HuH-
7 cell subcutaneous injections are in the right flank abdomen of male nude mouse.Per weekly check mouse three times observing tumor development.Once hypodermic tumour
Develop and reach about 100-150mm3, nude mice is randomly divided into four groups (every group of 6 mouse).Then, respectively with ZD55-TRAIL,
Quercetin, and described viral and described medicine combination or PBS injection mouse.ZD55-TRAIL is applied in intra-tumoral injection mode
(1×109Plaque-forming unit-PFU, every mouse), at the same by with the dosage of 150mg/kg body weight to being applied in Mouse Stomach
With Quercetin or by injection or by being administered in stomach 100 μ lPBS mediums are applied to parallel control.It is every using slide measure
3 days measurements length of tumor and width (mm), and calculate gross tumor volume using following formula：Gross tumor volume (mm3)=(A × B2)/2, wherein
A and B represent length and width respectively.Then tumor growth curve is obtained according to cubing.
Statistical analysis
The significant property of experimental statistics is expressed as average value ± standard deviation (SD), and by the softwares of Graph Pad 6.0
(GraphPad Software, San Diego, CA) is analyzed.Student t inspections are carried out to determine the significant property of statistics.P values<0.05
It is considered to have statistical significance.
Embodiment 1：The HCC cell growth inhibitions of Quercetin enhancing ZD55-TRAIL mediations.
By the gene constructed oncolytic adenovirus carrier ZD55 of the E1B 55-kDa of deleted adenovirus 5；It can be many swollen
Copy choice in oncocyte.Based on cancer target gene-Viro-Therapy, we pack therapeutic gene using ZD55
TRAIL simultaneously obtains new recombination oncolytic adenovirus ZD55-TRAIL (Figure 1A).In order to verify the sign of ZD55-TRAIL, we make
The protein expression of adenovirus E 1 A and therapeutic gene TRAIL is detected with western engram analysis.With no ZD55-TRAIL
The compared with control cells for the treatment of is compared, and there is E1A in the HuH-7 cells for adding Quercetin to infect with ZD55-TRAIL and ZD55-TRAIL
With the strongly expressed of TRAIL, show ZD55-TRAIL can in HCC cells high level copy choice Figure 1B).
In order to assess the cell death whether Quercetin strengthens the HCC cells of ZD55-TRAIL inductions, such as material and method
Described in carry out MTT measure.HCC cell lines are infected with the combination of single ZD55-TRAIL, single Quercetin or both
SMMC-7721, HepG2 and HuH-7.Result shows, compared with control cell lines, the combined treatment of ZD55-TRAIL and Quercetin
Cause to act on (Fig. 2) to the enhanced tumor-killing of HCC.
Embodiment 2：Quercetin enhancing ZD55-TRAIL induction HCC Apoptosis.
Hoechst 33342 is then carried out to dye to determine with ZD55-TRAIL plus Quercetin, ZD55-TRAIL or independent
Quercetin treatment HuH-7 cells morphological change.Result is shown in Fig. 3 A.This proves, is individually processed with ZD55-TRAIL
Compare, obvious apoptosis is caused with the coprocessing of Quercetin, it is characterised in that it was observed that Chromatin condensation, karyorrhexis and apoptosis are small
Body.For the effect of the apoptosis that quantitative Quercetin and ZD55-TRAIL are induced, annexin-V- fluorescein isothiocyanates are used
(FITC)/PI is double to be dyeed to analyze Apoptosis (Fig. 3 B).Result shows, is jointly processed by with Quercetin with ZD55-TRAIL
HuH-7 apoptosis rates are 58.9%, the twice (27.4%) that almost ZD55-TRAIL is individually processed.
Western blot analysis prove the ability that ZD55-TRAIL has activation caspase dependence paths, including
The activation and the cutting of PARP of caspase-9, caspase-3.This unique effect can be by with Quercetin and ZD55-
TRAIL co-therapies are further enhanced (Fig. 3 C).
Embodiment 3：Quercetin suppresses the activation of NF- κ B in the HCC cells that ZD55-TRAIL is induced
Suppress NF- κ B dependent transcriptions and then make HCC cells sensitive to ZD55-TRAIL to prove that Quercetin has
Ability, we determine described to process the expression of I κ B α, p65 and p50 that causes and change using Western blotting.As shown in figure 4,
The expression of the reduction of IkBa, the p65 and p50 induction more independent than ZD55-TRAIL in the therapeutic alliance of Quercetin and ZD55-TRAIL
It is more significant, it shows that Quercetin can reduce the NF- κ B activation of ZD55-TRAIL mediations really, and by promoting ZD55-
The apoptosis of TRAIL inductions reduces its transcriptional activity (Fig. 4).
Embodiment 4：The HCC Tumor growth inhibitions of ZD55-TRAIL- mediations in Quercetin reinforcement
In order to test the therapeutic effect of Quercetin and the ZD55-TRAIL treatment of combination in vivo, using using HuH-7 cells
What the HCC Tumor Xenograft Models of foundation had a single and therapeutic alliance treatment carries out zoopery.Draw tumour growth bent
Line with compare 49 days observe during its antitumor efficacy difference.As shown in the figure.As shown in Figure 5A, with receive PBS injection
Those are compared, the independent Quercetin of receiving, the mean tumour volume of the animal of the intra-tumoral injection of ZD55-TRAIL and therapeutic alliance
Significant reduction.As expected, combined therapy is treated than Quercetin (P=0.001) and single ZD55-TRAIL (P=0.002)
More effectively.Additionally, and carrier, individually treatment is compared for Quercetin or ZD55-TRAIL, and Quercetin and combining for ZD55-TRAIL are controlled
Treatment causes the animal survival rate (Fig. 5 B) for improving.
Embodiment 5：ZD55-TRAIL, Quercetin and quinindium three significantly inhibit liver cancer cell growth in vivo
The female BAl BIc of 4-5 week old/c nude mices are purchased from Chinese Academy of Sciences's Shanghai Experimental Animal Center (Shanghai, China).It is all
Experimental implementation meet requirement of the experimental animal in welfare and ethics.By HuH-7 cells (2X106, in PBS) and naked
The right side hypodermic injection of mouse.Once hypodermic tumour size reaches 100-150mm3, nude mice is randomly divided into 3 groups (every group 8).So
Combination and Quercetin, the ZD55-TRAIL and quinindium (quinidine, Quin) three of Quercetin and ZD55-TRAIL are applied afterwards
The combination of person.ZD55-TRAIL(1X109PFU/ mouse) by the way of intra-tumoral injection, and Quercetin and quinindium are with 5mg/
The amount intraperitoneal injection of Kg body weight, 100 μ l PBS used as control, inject every other day by continuous injection three times.Since injection, every 5 days
Measure tumor size and the survival of nude mice is counted within the time of 75 days.
As a result, it was confirmed that when the 50th day Quercetin and ZD55-TRAIL combination and Quercetin, ZD55-TRAIL
The lotus knurl block of nude mice can be substantially eliminated with the combination of quinindium, but the 75th day at the end of experiment, Quercetin and ZD55-
The combination of TRAIL causes that the survival rate of nude mice is 62.5% (3 nude mice death), and Quercetin, ZD55-TRAIL and quinindium
The nude mice survival rate of the combination of three is 87.5% (1 nude mice death).
Although with above embodiments describing the present invention, it should be appreciated that before without departing substantially from spirit of the invention
Put, the present invention can further be modified and changed, and these modification and variation belong to protection scope of the present invention it
It is interior.
Claims (10)
1. the oncolytic adenovirus of expression TRAIL are being prepared for treating liver cancer in subject or suppressing the medicine of hepatoma cell proliferation
Purposes in compositions or medicine box, wherein described pharmaceutical composition or medicine box contain the oncolytic adenovirus and Mongolian oak of expression TRAIL
Skin element and pharmaceutically acceptable carrier.
2. purposes according to claim 1, wherein described pharmaceutical composition or medicine box are also containing for treating liver cancer or suppression
The other drugs active component of hepatoma cell proliferation processed.
3. purposes according to claim 2, wherein described other drugs active component is such as gemcitabine, Plerixafor
Or taxol.
4. the purposes according to any one of claim 1-3, wherein the expression in described pharmaceutical composition or medicine box
The oncolytic adenovirus and Mongolian oak of TRAIL are deposited or expressed to the oncolytic adenovirus and Quercetin of TRAIL in separate container respectively
Pi Su is deposited in same container.
5. the purposes according to any one of claim 1-3, wherein also containing Kui in described pharmaceutical composition or medicine box
Buddhist nun's fourth.
6. the purposes of quinindium according to claim 5, wherein carry Quercetin and quinindium is deposited in same container, and
The oncolytic adenovirus for expressing TRAIL are deposited in another container.
7. the oncolytic adenovirus of expression TRAIL, Quercetin and quinindium are being prepared for treating liver cancer or suppression in subject
Purposes in the pharmaceutical composition or medicine box of hepatoma cell proliferation.
8. expression TRAIL oncolytic adenovirus prepare in subject raise HCC in Caspase-3,
Purposes in the pharmaceutical composition or medicine box of the expression of Caspase-9 and/or PARP, wherein described pharmaceutical composition or medicine
Box contains the oncolytic adenovirus and Quercetin of expression TRAIL.
9. purposes according to claim 8, wherein described pharmaceutical composition or medicine box also contains quinindium.
10. purposes according to claim 9, the oncolytic adenopathy of the expression TRAIL in described pharmaceutical composition or medicine box
Storage or the storage in same container in separate container respectively of poison, Quercetin and quinindium.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611171522.3A CN106822085B (en) | 2016-12-17 | 2016-12-17 | Application of oncolytic adenovirus expressing TRAIL and quercetin in inhibition of liver cancer cell proliferation |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611171522.3A CN106822085B (en) | 2016-12-17 | 2016-12-17 | Application of oncolytic adenovirus expressing TRAIL and quercetin in inhibition of liver cancer cell proliferation |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106822085A true CN106822085A (en) | 2017-06-13 |
| CN106822085B CN106822085B (en) | 2020-03-17 |
Family
ID=59139484
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201611171522.3A Expired - Fee Related CN106822085B (en) | 2016-12-17 | 2016-12-17 | Application of oncolytic adenovirus expressing TRAIL and quercetin in inhibition of liver cancer cell proliferation |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106822085B (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1173817A (en) * | 1995-10-26 | 1998-02-18 | 巴克·诺顿药物有限公司 | Method, compsns and kits for increasing the oral bioavilability of pharmaceuticalagents |
| CN101982168A (en) * | 2010-11-02 | 2011-03-02 | 山东大学 | Quercetin nano-micelle preparation and preparation method thereof |
| CN102229962A (en) * | 2011-05-26 | 2011-11-02 | 陕西师范大学 | Oncolytic adenovirus vector for modifying and expressing two exogenous genes by fibrin, construction method and application of vector |
| CN102229961A (en) * | 2011-05-26 | 2011-11-02 | 陕西师范大学 | Conditionally replicating oncolytic adenoviral vector used for expressing two exogenous genes and modified by small peptide, construction method and application thereof |
| CN102796709A (en) * | 2011-05-27 | 2012-11-28 | 中国科学院上海生命科学研究院 | Liver cancer-specific gene-virus and application thereof |
-
2016
- 2016-12-17 CN CN201611171522.3A patent/CN106822085B/en not_active Expired - Fee Related
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1173817A (en) * | 1995-10-26 | 1998-02-18 | 巴克·诺顿药物有限公司 | Method, compsns and kits for increasing the oral bioavilability of pharmaceuticalagents |
| CN101982168A (en) * | 2010-11-02 | 2011-03-02 | 山东大学 | Quercetin nano-micelle preparation and preparation method thereof |
| CN102229962A (en) * | 2011-05-26 | 2011-11-02 | 陕西师范大学 | Oncolytic adenovirus vector for modifying and expressing two exogenous genes by fibrin, construction method and application of vector |
| CN102229961A (en) * | 2011-05-26 | 2011-11-02 | 陕西师范大学 | Conditionally replicating oncolytic adenoviral vector used for expressing two exogenous genes and modified by small peptide, construction method and application thereof |
| CN102796709A (en) * | 2011-05-27 | 2012-11-28 | 中国科学院上海生命科学研究院 | Liver cancer-specific gene-virus and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| QIUWEI PAN: "Synergistic induction of tumor cell death by combining cisplatin with an oncolytic adenovirus carrying TRAIL", 《MOL CELL BIOCHEM》 * |
| X LIU 等: "Gene-viro-therapy targeting liver cancer by a dual-regulated oncolytic adenoviral vector harboring IL-24 and TRAIL", 《CCANCER GENE THERAPY》 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106822085B (en) | 2020-03-17 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Heo et al. | Sequential therapy with JX-594, a targeted oncolytic poxvirus, followed by sorafenib in hepatocellular carcinoma: preclinical and clinical demonstration of combination efficacy | |
| AU2011229490B2 (en) | Cancer therapy with a parvovirus combined with an HDAC inhibitor | |
| TWI732025B (en) | Application of Bcl-XL inhibitor and oncolytic virus in the preparation of anti-tumor drugs | |
| CN110022867A (en) | High mobility group protein BOX I mutant | |
| CN102357104B (en) | The application be combined in therapeutic treatment of myxoma virus and rapamycin | |
| Ottolino-Perry et al. | Oncolytic vaccinia virus synergizes with irinotecan in colorectal cancer | |
| JP7312695B2 (en) | Oncolytic virus targeting STAT3 | |
| US8377450B2 (en) | Clone of Newcastle disease virus, its manufacture and its application in the medical treatment of cancer | |
| De Silva et al. | Double trouble for tumours: exploiting the tumour microenvironment to enhance anticancer effect of oncolytic viruses | |
| CN104398526B (en) | The application of triptolide and Celastrol in antineoplastic is prepared | |
| KR20080039344A (en) | Cancer treatment using viruses, fluoropyrimidines and camptothecins | |
| CN117257782B (en) | Application of melitracin in reversing Oritinib resistance | |
| Cai et al. | Novel recombinant coxsackievirus B3 with genetically inserted basic peptide elicits robust antitumor activity against lung cancer | |
| CN109793727A (en) | A kind of pharmaceutical composition and its application of effective anti-malignant tumor | |
| CN106822085A (en) | Express purposes of the oncolytic adenovirus joint Quercetin of TRAIL in liver cancer cancer cell multiplication is suppressed | |
| CN109568313A (en) | A kind of antineoplastic combination drug and its preparing the application in anticancer drug | |
| Shi et al. | Synergistic antitumor effect of combining metronomic chemotherapy with adoptive cell immunotherapy in nude mice | |
| CN106955292A (en) | A kind of pharmaceutical composition and purposes for treating the cancer of the esophagus | |
| CN107661324B (en) | Application of the Propafenone in preparation treatment oesophagus cancer drug | |
| CN105233285B (en) | The use in conjunction of Epac direct or indirect agonist and oncolytic virus | |
| CN112999235B (en) | Application of glycosyl polyether compound in preparing anti-paramyxovirus or anti-enterovirus medicines | |
| US9566307B2 (en) | Pharmaceutical composition | |
| Chaurasiya et al. | An oncolytic poxvirus encoding hNIS, shows antitumor efficacy and allows tumor imaging in a liver cancer model | |
| US11617729B2 (en) | Uses of guanidine hydrochloride as a drug for treating cancers/tumors | |
| WO2022127788A1 (en) | Application of lenvatinib and aurora-a kinase inhibitor in preparing cancer-inhibiting drugs |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20200317 Termination date: 20201217 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |