CN108888754A - A kind of anti-tumor compositions and its application containing ubenimex and trail protein - Google Patents

A kind of anti-tumor compositions and its application containing ubenimex and trail protein Download PDF

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CN108888754A
CN108888754A CN201810897991.6A CN201810897991A CN108888754A CN 108888754 A CN108888754 A CN 108888754A CN 201810897991 A CN201810897991 A CN 201810897991A CN 108888754 A CN108888754 A CN 108888754A
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trail
ubenimex
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tumor
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陈淑珍
倪隽
王晓飞
商悦
李毅
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Institute of Medicinal Biotechnology of CAMS
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Abstract

The present invention provides a kind of anti-tumor compositions containing ubenimex and trail protein and its applications, belong to pharmaceutical technology field.Anti-tumor compositions provided by the invention containing ubenimex and trail protein include ubenimex solution and trail protein solution；For ubenimex solution using NaOH solution as solvent, the concentration of the ubenimex solution is 0.01~0.1mg/ml；For trail protein solution using PBS buffer solution as solvent, the concentration of the trail protein solution is 0.039~5 μm of ol/L.The present invention also provides anti-tumor compositions application in preparations of anti-tumor drugs.Experiment in vivo and experiment in vitro show：When ubenimex and trail protein drug combination, CDI value < 1 illustrates that composition provided by the invention has the function of synergistic antitumor, has extremely significant therapeutic effect than single drug effect.

Description

A kind of anti-tumor compositions and its application containing ubenimex and trail protein
Technical field
The invention belongs to pharmaceutical technology fields, and in particular to a kind of antineoplastic combinations containing ubenimex and trail protein Object and its application.
Background technique
Cancer is primarily assaulting fortified position one of problem of facing of human health, though tumour correlative study in recent years achieve it is certain Progress, but at present treating cancer still with operation excision based on, supplemented by radiotherapy and embolic chemotherapy.Therapeutic efficiency is low, toxic side effect Greatly, the key factor for influencing current cancer therapies effect is still the problems such as high recurrence rate.In order to improve curative effect, it is secondary anti-to reduce poison Answer, find safely and effectively treatment method it is extremely urgent, wherein the combination therapy means of the high-efficiency low-toxicity based on existing drug by Gradually as the focus of the area research and breach.
TRAIL (tumornecrosis factor-related apoptosis inducing ligand), i.e. tumour Necrosin relative death inducing ligand, is a kind of cell factor of body itself synthesis, its soluble form can with death by Body interacts to form tripolymer, recruitment death domain connection albumen (Fas associated death domain, FADD) and pro-caspase8/10 forms dead inducement signal compound (death-inducing signaling Complex, DISC), after pro-caspase8/10 is sheared activation, and then specific inducing apoptosis of tumour cell.Due at certain There is drug resistance in a little oncotherapies, is at present stopped at for the clinical research of TRAIL clinical II phase, which has limited TRAIL eggs The white application in treatment tumour.
Bestatin, i.e. ubenimex, be 1976 by Japanese scholars Mei Ze shore husband from the netted strepto- of olive A kind of low molecule dipeptide compound being isolated in the culture solution of (streptomy ces olivoreticuli), chemical name For N- [(2S, 3R) -3- amino -2- hydroxyl -4- benzene butyryl]-L-Leu, entitled hundred scholar of domestic goods is glad, belongs to national two classes New drug, it is used as a kind of immunomodulator on Present clinical, is had and is promoted NK cell function, increases the function such as IL-8 activity Energy.Specific inhibitor of the Bestatin as Aminopeptidase N (aminopeptidase N, also known as CD13) has certain resist Function of tumor：On the one hand tumour is inhibited by immunological regulation indirectly, on the other hand by inhibiting CD13 to play anti-tumor effect.To the greatest extent Pipe Bestatin embodies certain antitumous effect in terms of suppressing growth of tumour cell, inhibiting, But mechanism of action not yet fully states.Bestatin independent medication at present, limited efficacy is ineffective for treatment of cancer, this Greatly limit application of the Bestatin in treatment tumour.
Summary of the invention
In view of this, the purpose of the present invention is to provide a kind of anti-tumor compositions containing ubenimex and trail protein And its application, the anti-tumor compositions can improve extremely significantly drug effect.
In order to achieve the above-mentioned object of the invention, the present invention provides following technical scheme：
The present invention provides a kind of anti-tumor compositions containing ubenimex and trail protein, including ubenimex solution With trail protein solution；
For the ubenimex solution using NaOH solution as solvent, the concentration of the ubenimex solution is 0.01~0.1mg/ ml；
For the trail protein solution using PBS buffer solution as solvent, the concentration of the trail protein solution is 0.039~5 μ mol/L。
Preferably, the concentration of the ubenimex solution is 0.03~0.08mg/ml.
Preferably, the concentration of the NaOH solution is 0.1mol/L.
Preferably, the pH value of the ubenimex solution is 8.0.
Preferably, the concentration of the trail protein solution is 0.1~2.5 μm of ol/L.
Preferably, the concentration of the PBS buffer solution is 0.01mol/L.
The present invention provides the anti-tumor compositions application in preparations of anti-tumor drugs.
Preferably, the effective concentration of ubenimex is 2mg/ml in the drug.
Preferably, the effective concentration of trail protein is 1mg/ml in the drug.
Preferably, the anti-tumor drug lowers the expression and/or suppression of P-Erk albumen by induced tumor Apoptosis Cell migration processed plays a role.
The present invention provides a kind of anti-tumor compositions containing ubenimex and trail protein, Bestatin can significantly increase The cells apoptosis of strong TRAIL induction, Bestatin can also be remarkably reinforced TRAIL and make to the inhibition of tumor cell migration ability With, at the same Bestatin and TRAIL be used in combination it is also related with P-Erk protein expression level downward.Ubenimex and TRAIL Albumen has apparent collaboration tumor-inhibiting action.Experiment in vivo and experiment in vitro show：Using CDI value as both evaluations drug phase When the index of interaction, ubenimex and trail protein drug combination, CDI value < 1 illustrates composition tool provided by the invention There is synergistic antitumor, there is extremely significant therapeutic effect than single drug effect.
The present invention provides the anti-tumor compositions application in preparations of anti-tumor drugs.The present invention will be described anti-swollen Tumor composition, which is applied to, to be prepared in anti-tumor drug, and the drug effect of anti-tumor drug can be greatly improved, to improve oncotherapy In effect.
Detailed description of the invention
Fig. 1 is Bestatin and TRAIL individually and drug combination is to 3 kinds of growth of tumour cell inhibited proliferations, wherein： Fig. 1-a be TRAIL purity and size qualification result, 1：Trail protein；2：anti-TRAIL Westernblot；Fig. 1-b is not With concentration Bestatin independent medication to 3 kinds of growth of tumour cell inhibited proliferations；Fig. 1-c is that various concentration TRAIL is independent And its respectively with 0.01mg/ml, 0.1mg/ml Bestatin drug combination is to 3 kinds of growth of tumour cell inhibited proliferations；Figure 1-c-1 is A549 cell survival rate；Fig. 1-c-2 is A549 cell CDI Data-Statistics figure；Fig. 1-c-3 is HT1080 cell survival rate； Fig. 1-c-4 is HT1080 cell CDI Data-Statistics figure；Fig. 1-c-5 is PANC-1 cell survival rate；Fig. 1-c-6 is PANC-1 cell CDI Data-Statistics figure；
Fig. 2 be Bestatin and TRAIL individually and drug combination inducing apoptosis of tumour cell effect, wherein：Fig. 2-a is AnnexinV-FITC/PI apoptosis detection kit combination flow cytometer carries out apoptosis of tumor cells detection, * P<0.05 and * * P <0.01 represents the inspection with control group comparison,##P<0.01 represents the inspection with the mono- medicine group comparison of TRAIL, and Fig. 2-a-1 is A549 Cell streaming figure, Fig. 2-a-2 are A549 apoptosis rate statistical chart, and Fig. 2-a-3 is HT1080 cell streaming figure, and Fig. 2-a-4 is HT1080 apoptosis rate statistical chart, Fig. 2-a-5 are PANC-1 cell streaming figure, and Fig. 2-a-6 is PANC-1 apoptosis rate system Meter figure；Fig. 2-b is that Westernblot detection cell death related protein includes 8 albumen of PARP, caspase 3 and caspase Expression variation；
Fig. 3 is influence of the P-Erk albumen in Bestatin and TRAIL synergistic antitumor effect, wherein：Fig. 3-a is Westernblot detects P-Erk protein expression level in 3 kinds of tumour cells and animal tumor tissue, and Fig. 3-a-1 is A549 cell The expression of middle P-Erk albumen, Fig. 3-a-2 are the expression of P-Erk albumen in HT1080 cell, and Fig. 3-a-3 is PANC- The expression of P-Erk albumen in 1 cell, Fig. 3-a-4 are the expression of P-Erk albumen in animal tumor tissue；Fig. 3-b is Mtt assay detects P-Erk and combines the inhibition active influence of growth and proliferation of cell, * P with TRAIL in Bestatin<0.05, * * P< 0.01, Fig. 3-b-1 is A549 cell survival rate, and Fig. 3-b-2 is HT1080 cell survival rate；
Fig. 4 is Transwell experimental analysis Bestatin and influence (40 of the TRAIL drug combination to cell migration ability ×), * * P<0.01 represents the inspection with control group comparison,##P<0.01 represents the inspection with the mono- medicine group comparison of TRAIL, and Fig. 4-1 is The A549 cell migration ability mirror following figure, Fig. 4-2 are A549 cell migration ability statistical chart, and Fig. 4-3 is HT1080 cell migration energy The power mirror following figure, Fig. 4-4 are HT1080 cell migration ability statistical chart；
Fig. 5 is the inhibitory effect of Bestatin and TRAIL drug combination to HT1080 transplanted tumor in nude mice, wherein：Fig. 5-a is HT1080 transplantable tumor nude mice weight change curve (n=6)；Fig. 5-b be Bestatin and TRAIL individually and drug combination pair The inhibiting effect (n=6) of HT1080 transplanted tumor in nude mice growth, * * P<0.01；Fig. 5-c is 4 groups of tumor mass weight (n=6), * * P< 0.01 represents the inspection with control group comparison,##P<0.01 represents the inspection with the mono- medicine group comparison of TRAIL；Fig. 5-d is naked from four groups The HT1080 tumor mass photo that removing obtains in mouse body；Fig. 5-e is the pathology of the nude mouse tumor based on H&E decoration method and major organs It learns and checks；
Fig. 6 is that mtt assay detects the influence that Bestatin and DCA drug combination are proliferated growth of tumour cell in comparative example 1.
Specific embodiment
The present invention provides a kind of anti-tumor compositions containing ubenimex and trail protein, including ubenimex solution With trail protein solution；For the ubenimex solution using NaOH solution as solvent, the concentration of the ubenimex solution is 0.01 ~0.1mg/ml；For the trail protein solution using PBS buffer solution as solvent, the concentration of the trail protein solution is 0.039 ~5 μm of ol/L.
Anti-tumor compositions provided by the invention include ubenimex solution.The concentration of the ubenimex solution is preferably 0.03~0.08mg/ml, more preferably 0.05mg/ml.The pH value of the ubenimex solution is preferably 8.0.The NaOH is molten The concentration of liquid is preferably 0.1mol/L.The present invention is not particularly limited the source of the Bestatin (i.e. ubenimex), adopts With ubenimex known in the art.In embodiments of the present invention, Bestatin is ground purchased from Chinese food drug assay Study carefully institute, is white powder, molecular weight 308D, 100mg/ bottle, purity 99.5%.The preparation method of the ubenimex solution is excellent Choosing includes the following steps：After ubenimex powder is dissolved with 0.1mol/LNaOH solution, pH to 8.0 is adjusted with HCl solution, is prepared At the stock solution of 20mg/ml, it is placed in -20 DEG C of preservations after packing, when use dilutes.
Anti-tumor compositions provided by the invention include trail protein solution.The concentration of the trail protein solution is preferred For 0.1~2.5 μm of ol/L, more preferably 0.2~0.7 μm of ol/L, most preferably 0.5 μm of ol/L.The concentration of the PBS buffer solution Preferably 0.01mol/L.The preparation method of the trail protein discloses in the prior art, is specifically shown in the prior art (weight The group apoptosis induction ligand related preparation method of human soluble tumor necrosis factor, open (bulletin) number:CN1696304A, it is open (bulletin) day：2005.11.16).The preparation method of trail protein solution preferably includes following steps：With 0.01mol/L PBS It is configured to the stock solution of 44.38 μm of ol/L for solvent, then with 0.22 μm of membrane filtration, is placed in -20 DEG C of preservations after packing.
The present invention provides the anti-tumor compositions application in preparations of anti-tumor drugs.
In the present invention, the effective concentration concentration of ubenimex is 2mg/ml in the drug.TRAIL egg in the drug White effective concentration is preferably 1mg/ml.Ubenimex solution and the independent packing and storing of trail protein solution, make in the drug Used time is added simultaneously.The dosage form of the anti-tumor drug is preferably injection.The preparation method of injection of the present invention does not have It is specifically limited, using the preparation method of injection known in the art.
In the present invention, the anti-tumor drug preferably passes through induced tumor Apoptosis, lowers the expression of P-Erk albumen And/or cell migration is inhibited to play antitumor action.Experiment in vivo and experiment in vitro show：Ubenimex and trail protein connection When sharing medicine, CDI value < 1 illustrates that composition provided by the invention has the function of synergistic antitumor, has than single drug effect There is extremely significant therapeutic effect.
Below with reference to embodiment to a kind of anti-tumor compositions containing ubenimex and trail protein provided by the invention and Its application is described in detail, but they cannot be interpreted as limiting the scope of the present invention.
Experimental material explanation
1. cell strain
Human lung carcinoma cell line A549, human fibrosarcoma cell's strain HT1080 freeze from this laboratory passage, with containing matter The 1640 culture medium culture of RPMI of 10% fetal calf serum of concentration is measured, human pancreas cancer cell strain PANC-1 comes from this laboratory passage It freezes, with the DMEM culture medium culture containing 10% fetal calf serum of mass concentration.Cell is digested with 0.25% pancreas enzyme -EDTA Passage, is incubated at 5%CO2, in the incubator under the conditions of 37 DEG C.
2. cell culture material
1640 culture medium of RPMI, DMEM culture medium, pancreatin and 0.01M PBS (1 ×) solution are purchased from U.S. Hyclone Company is placed in 4 DEG C of preservations.Fetal calf serum is purchased from U.S. GIBCO company, and 56 DEG C of inactivation 30min are placed in -20 DEG C of preservations after packing.
3. drug and albumen
Bestatin (i.e. ubenimex) be purchased from National Institute for Food and Drugs Control, be white powder, molecular weight 308D, 100mg/ bottles, purity 99.5% adjusts pH to 8.0 or so with HCl solution, prepares after the dissolution of 0.1mol/L NaOH solution At the stock solution of 20mg/ml, it is placed in -20 DEG C of preservations after packing.
The trail protein stock solution that 0.01M PBS is that solvent is configured to 44.38 μM, after 0.22 μm of membrane filtration packing It is placed in -20 DEG C of preservations.
4. main agents and antibody
MTT is purchased from Beijing Suo Laibao Science and Technology Ltd；DMSO is that domestic analysis is pure；anti-p44/42MAPK(Erk 1/ 2), anti-Phospho-p44/42MAPK (Erk 1/2) and TPA (12-O-Tetradecanoylphorbol-13- Acetate) it is purchased from U.S. CST company；PD98059 is purchased from U.S. MCE company；β-tubulin mouse monoclonal antibody and β-Actin mouse monoclonal antibody Purchased from Bo Aoruijing (Beijing) development in science and technology Co., Ltd；HRP label mountain sheep anti-mouse igg and HRP label goat anti-rabbit igg are purchased from Bioisystech Co., Ltd of Zhong Shan Golden Bridge；AnnexinV-FITC/PI apoptosis detection kit is purchased from the biological section of connection in the friendship of Beijing Skill Co., Ltd.The cell Transwell is purchased from U.S. Corning company.
Embodiment 1
Bestatin causes cell survival rate to decline with TRAIL drug combination, makees compared with independent medication in significant collaboration With
Mtt assay detection Bestatin and TRAIL is individually and drug combination acts on 3 kinds of Cytostatic to tumor cell
A549, HT1080 and PANC-1 cell of logarithmic growth phase grow speed according to different cells after pancreatin digests Degree, is seeded in 96 orifice plates with 2000~5000/hole density, is placed in 37 DEG C of incubator cultures for 24 hours, will after cell is adherent The drug of various concentration is added in 96 orifice plates (100 hole μ l/) for Bestatin and TRAIL proportional diluted：A549:Bestatin 0~200nmol/L of 0.01mg/ml or 0.1mg/ml, TRAIL；HT1080：Bestatin 0.01mg/ml or 0.1mg/ml, 0~5 μm of ol/L of TRAIL；PANC-1：Bestatin 0.01mg/ml or 0.1mg/ml, 0~5 μm of ol/L of TRAIL；(joint is used When medicine, two medicines are corresponding while being added).3 multiple holes are arranged in each concentration, and set up control group and blank group, are placed in 37 DEG C of cultures After case culture 48h, MTT 20 μ l, the 37 DEG C of incubator culture 4h of 5mg/ml are added in every hole, are inhaled and are abandoned supernatant, and 200 μ l are added in every hole DMSO, room temperature low speed shake 10min, measure light absorption value at 570nm with microplate reader.By formula：Survival rate=(AT-AB)/ (AC-AB) × 100% calculating cell survival rate, (wherein AT, AC, AB respectively represent dosing group, control group, blank group and are averaged A570 Value), using adding consistency as abscissa, cell survival rate is that ordinate draws concentration-response curve.CDI value=AB/A × B is (wherein AB represents OD value/control group OD value of two medicine joint groups, and A represents OD value/control group OD value of A medicine group, and B represents the OD of B medicine group Value/control group OD value).
CDI value (coefficient ofdrug interaction, drug interaction coefficient) is both special evaluations The common counter of drug interaction, if CDI value is less than 1：In synergistic effect (non-compounding obtains, i.e. the non-1+1=2 of function and effect, But 1+1 > 2)；If CDI value is greater than 1：In antagonism (non-1+1=2, but 1+1 ﹤ 2).
MTT experiment result is as shown in Figure 1.When the two drug combination, in A549 tumour cell, in Bestatin 0.01mg/ Under the conditions of ml and 0.1mg/ml administration, when TRAIL activity is 50~200nmol/L, CDI value is respectively less than 1；It is thin in HT1080 In born of the same parents, under the conditions of Bestatin 0.01mg/ml and 0.1mg/ml administration, TRAIL activity is 0.039~5 μm of ol/L When, CDI value is respectively less than 1；In PANC-1 cell, under the conditions of Bestatin 0.01mg/ml and 0.1mg/ml administration, TRAIL When activity is 0.039~5 μm of ol/L, CDI value is respectively less than 1 respectively.And in this 3 kinds of tumours of A549, HT1080 and PANC-1 In cell, within the scope of comparable sodium after TRAIL or Bestatin independent medication, cell is grown by a degree of suppression System, but inhibitory effect is not obvious.The above results illustrate that compared with independent medication group, 0.039~5 μm of ol/L concentration TRAIL divides Not and after 0.01mg/ml, 0.1mg/ml Bestatin drug combination, cell survival rate is decreased obviously, cell growth inhibition It significantly increases, drug synergism is extremely obvious.
Embodiment 2
Bestatin and TRAIL drug combination lead to Apoptosis effect, in significant synergistic effect compared with independent medication
1. the double dye method detection Bestatin and TRAIL of flow cytometry are individually and drug combination inducing apoptosis of tumour cell is imitated It answers
A549, HT1080 and PANC-1 cell of logarithmic growth phase grow speed after pancreatin digests, according to different cells Degree, with 1.5 × 105~2.5 × 105A/hole density is seeded in 6 orifice plates, after being placed in 37 DEG C of incubator cultures cell being adherent for 24 hours, Drug is added in 6 orifice plates by following concentration, A549：Bestatin0.1mg/ml, TRAIL 25nmol/L；HT1080： Bestatin 0.1mg/ml, TRAIL 80nmol/L；PANC-1：Bestatin 0.1mg/ml, TRAIL 80nmol/L；(connection When sharing medicine, two medicines are corresponding while being added), after effect for 24 hours, cell suspension is collected into 15ml centrifuge tube after pancreatin digests Interior, 1000rpm is centrifuged 3min, inhales and abandons supernatant, with being centrifuged again after PBS rinse, inhales and abandons supernatant, every pipe be added 100 μ l 1 × Annexin V Binding Solution, 5 μ l Annexin V-FITC and 10 μ l PI Solution conjugates, room temperature are quiet After setting 15min, every pipe adds 400 μ l 1 × AnnexinVBinding Solution, is carried out in 1h with flow cytometer Measurement carries software FACScan and analyzes data result.Apoptosis rate=early apoptosis of cells %+ cell late apoptic %. (wherein AB represents the apoptosis rate of two medicine joint groups to CDI value=AB/A × B, and A represents the apoptosis rate of A medicine group, and B represents B The apoptosis rate of medicine group).
Streaming result is as shown in Fig. 2-a, in A549 cell, with TRAIL (25nmol/L) independent medication (42.31 ± 2.52) % and Bestatin (0.1mg/ml) independent medication (2.30 ± 0.30) % are compared, Apoptosis after the two drug combination Rate significantly rises (64.08 ± 4.71) %, and CDI value is 0.64；It is independent with TRAIL (80nmol/L) in HT1080 cell Medication (17.51 ± 4.02) % and Bestatin (0.1mg/ml) independent medication (10.76 ± 1.48) % is compared, and the two joint is used Apoptosis rate significantly rises (40.15 ± 4.03) % after medicine, and CDI value is 0.81；Equally, in PANC-1 cell, with TRAIL (80nmol/L) independent medication (18.89 ± 1.35) % and Bestatin (0.1mg/ml) independent medication (6.08 ± 1.09) % is compared, and apoptosis rate also significantly rises (46.19 ± 8.45) % after the two drug combination, and CDI value is 0.71. The above results explanation, in this 3 kinds of tumour cells of A549, HT1080 and PANC-1, TRAIL independent medication can be to a certain extent Apoptosis is induced, and after the two drug combination, Bestatin can be obviously promoted the Apoptosis effect of TRAIL generation, effect Effect highly significant.
2.Westernblot detects the variation of cell death related protein expression
A549, HT1080 and PANC-1 cell of logarithmic growth phase grow speed after pancreatin digests, according to different cells Degree, with 1.5 × 105~2.5 × 105A/hole density is seeded in 6 orifice plates, after being placed in 37 DEG C of incubator cultures cell being adherent for 24 hours, A549 in 6 orifice plates is added by following concentration in drug：Bestatin 0.1mg/ml, TRAIL 10nmol/L；HT1080： Bestatin 0.1mg/ml, TRAIL 80nmol/L；PANC-1：Bestatin 0.1mg/ml, TRAIL 80nmol/L；(connection When sharing medicine, two medicines are corresponding while being added), cell is collected in effect afterwards for 24 hours, appropriate RIPA lysate (containing 1%PMSF) is added, On ice after crack protein 30min, extremely with BCA kit measurement protein concentration, progress SDS-PAGE electrophoresis and by protein delivery On pvdf membrane, 1h is closed at room temperature using 5% skim milk, is added by the diluted related primary antibody of corresponding proportion, 4 DEG C of low speed shakes It swings overnight；Corresponding secondary antibody is added after 3 times for TBST rinse 5min/ times, is incubated at room temperature 1h, TBST rinse 5min/ time, general after 3 times Millipore company shines detection liquid A and B by 1:1 mixing, dropwise addition develop the color on pvdf membrane, and gel imaging system is taken pictures.
Westernblot result is as shown in Fig. 2-b, in this 3 kinds of tumour cells of A549, HT1080 and PANC-1, with list Private medicine group is compared, Bestatin and Cleaved-PARP, Cleaved caspase-8 and Cleaved after TRAIL drug combination The up-regulation of caspase-3 protein expression level illustrates that Bestatin enhances the swollen of TRAIL induction by caspase Dependent Apoptosis of tumor effect, this is consistent with streaming result in Fig. 2-a, and TRAIL can be significantly increased by further illustrating Bestatin The cells apoptosis of induction produces synergistic antitumor effect after the two drug combination.
Embodiment 3
The downward of P-Erk plays a significant role in Bestatin and TRAIL Combinative antitumor efficacy
1.Westernblot detects P-Erk protein expression level in tumour cell
A549, HT1080 and PANC-1 cell of logarithmic growth phase grow speed according to different cells after pancreatin digests Degree, with 1.5 × 105~2.5 × 105A/hole density is seeded in 6 orifice plates, after being placed in 37 DEG C of incubator cultures cell being adherent for 24 hours, Drug is added in 6 orifice plates by following concentration：A549：Bestatin 1,2,4mg/ml；HT1080：Bestatin 0.1,0.5, 1mg/ml；PANC-1：Bestatin 0.5,1,2mg/ml.Cell is collected after acting on the corresponding time, appropriate RIPA lysate is added (containing 1%PMSF) on ice after crack protein 30min, with BCA kit measurement protein concentration, carries out SDS-PAGE electrophoresis simultaneously By on protein delivery to pvdf membrane, 1h is closed at room temperature using 5% skim milk, is added and presses corresponding proportion diluted related one Anti-, 4 DEG C of low speed concussions are overnight；Corresponding secondary antibody is added after 3 times for TBST rinse 5min/ times, is incubated at room temperature 1h, TBST rinse Millipore company is shone into detection liquid A and B by 1 after 5min/ times, 3 times:1 mixing, dropwise addition develop the color on pvdf membrane, coagulate Glue imaging system is taken pictures.Bestatin dosage is 20mg/kg in zoopery, and gastric infusion stops after every successive administration 5 times 2 days.22nd day execution nude mice, and dissect and obtain tumor tissues, after RIPA lysate (containing 1%PMSF) homogenate is added, step It is identical as cell protein.
Westernblot result is as shown in Fig. 3-a, in 4h, 8h, 12h and time point for 24 hours, after Bestatin is added, P-Erk protein expression level is lowered in this 3 kinds of tumour cells of A549, HT1080 and PANC-1；After Bestatin 12h is administered, 3 P-Erk protein expression level declines with the increase of Bestatin drug concentration in kind tumour cell；HT1080 nude mice model In tumor model, after Bestatin is administered, P-Erk protein expression level is lowered in tumor tissues, shows that Bestatin and TRAIL joins Close antitumor action or related to the downward of P-Erk protein expression level.
2.MTT method detects P-Erk and combines the inhibition active influence of growth and proliferation of cell with TRAIL in Bestatin
A549, HT1080 and PANC-1 cell of logarithmic growth phase grow speed after pancreatin digests, according to different cells Degree, is seeded in 96 orifice plates with 2000~5000/hole density, after being placed in 37 DEG C of incubator cultures cell being adherent for 24 hours, by Erk Inhibitor PD98059 (final concentration of 20 μm of ol/L) shifts to an earlier date 6h addition, and Erk activator TPA (final concentration of 200nM) shifts to an earlier date 0.5h After addition, the TRAL albumen of various concentration is added in 96 orifice plates (100 hole μ l/)：A549：TRAIL 10nmol/L；HT1080： TRAIL 80nmol/L.3 multiple holes are arranged in each concentration, and set up control group and blank group.It is placed in 37 DEG C of incubator culture 48h Afterwards, MTT 20 μ l, the 37 DEG C of incubator culture 4h of 5mg/ml are added in every hole, inhale and abandon supernatant, and 200 μ l DMSO, room temperature is added in every hole Low speed shakes 10min, and light absorption value is measured at 570nm with microplate reader.By formula survival rate=(AT-AB)/(AC-AB) × (wherein AT, AC, AB respectively represent dosing group, control group, blank group and are averaged A 100% calculating cell survival rate570Value), with dosing Concentration is abscissa, and cell survival rate is that ordinate draws concentration-response curve.
MTT experiment result is as shown in Fig. 3-b, and in A549 cell and HT1080 cell, TRAIL itself is to two kinds of tumours Cell has a degree of growing multiplication inhibiting effect, but effect is not obvious, and it is common with Erk inhibitor PD98059 After effect, cell survival rate is decreased obviously, and after adding Erk activator TPA, which is relieved.The above results are said Bright PD98059 can significantly increase TRAIL to the growing multiplication inhibiting effect of cell, and TPA can partially reverse this effect, in conjunction with figure It is important that 3-aWesternblot result can be shown that the downward of P-Erk plays in Bestatin and TRAIL Combinative antitumor efficacy Effect.
Embodiment 4
Bestatin and TRAIL drug combination inhibit the external transfer ability of tumour cell, in significant compared with independent medication Synergistic effect
Transwell experimental analysis Bestatin and influence of the TRAIL drug combination to cell migration ability
It is separately added into the serum free medium of 100 μ l and 600 μ l in the cell Transwell and mistress, is placed in 37 DEG C of trainings Support case pre-balance 2h.It will be centrifuged after the digestion of tumour cell pancreatin, and obtain that cell will be resuspended with serum free medium after cell precipitation, Carry out cell count.It inhales and abandons indoor equilibrium liquid, different according to different cell migration abilities, adjustment cell density is 2~5 × 105 A/ml is added to 100 μ l cell suspension of cell, and 600 culture medium of the μ l containing 20%FBS of outer interior addition is careful not in cell Nesting is lower to generate bubble.It is separately added into various concentration drug：A549：Bestatin 0.1mg/ml, TRAIL4nmol/L； HT1080：Bestatin 0.1mg/ml, TRAIL 20nmol/L；(when drug combination, two medicines are corresponding while being added).It is placed in 37 After DEG C incubator culture for 24 hours, takes out upper chamber and be immersed in PBS and washes 1 time, be put into the methanol of -20 DEG C of pre-coolings and fix 15-30min, then Take out be immersed in 3 times are washed in PBS after be put into dyeing liquor of the 600 μ l containing 0.1% crystal violet and dye 15min and use cotton after PBS washes 3 times Label wipe the cell that residual liquid and inside are not shifted inside and outside nested film away, take pictures in microscopically observation.To objectively evaluate cell Migration situation carefully cuts film along nested film edge with blade, is put into 96 orifice plates, and the acetic acid of 150 μ l 33% is added in every hole, Room temperature low speed shakes 10min, sufficiently dissolves in hole and carefully takes out film after crystal violet, measures light absorption value at 570nm with microplate reader. By formula：A570(%control)=AT/AC × 100% (wherein AT, AC respectively represent dosing group, control group is averaged A570Value), Numerical value is lower, and to represent cell movement transfer ability weaker.(wherein AB represents the A of two medicine joint groups to CDI value=AB/A × B570(% Control), A represents the A of A medicine group570(%control), B represent the A of B medicine group570(%control)).
Transwell experimental result is as shown in Fig. 4-1~Fig. 4-4, independent with TRAIL (4nmol/L) in A549 cell Medication (92.43 ± 6.07) % and Bestatin (0.1mg/ml) independent medication (95.69 ± 5.32) % is compared, and the two joint is used A after medicine570(%control) is remarkably decreased (65.71 ± 3.82) %, and CDI value is 0.74；In HT1080 cell, with TRAIL (20nmol/L) independent medication (82.59 ± 2.45) % and Bestatin (0.1mg/ml) independent medication (90.20 ± 2.46) % is compared, A after the two drug combination570(%control) is remarkably decreased (49.22 ± 4.22) %, and CDI value is 0.66.The above results illustrate in both tumour cells of A549 and HT1080, after TRAIL or Bestatin are administered alone, Cell migration ability is by a degree of inhibition, but effect is not obvious.And when the two drug combination, cell migration ability by To significantly inhibiting, illustrate that TRAIL can be remarkably reinforced to the inhibiting effect of Tumor Cell Migration transfer ability in Bestatin.
Embodiment 5
Experiment in vivo verifies Bestatin and TRAIL drug combination to the inhibiting effect of HT1080 transplanted tumor in nude mice, with list Private medicine is compared in significant synergistic effect
HT1080 cell in good condition is passed on into amplification, after pancreatin digests, cell density is adjusted to 4 with PBS × 107A/ml.From female BAl BIc/c nude mice of magnificent Fukang company purchase 18~20g of weight, in the subcutaneous every inoculation of nude mice armpit 200 μ l cell suspensions.Be grouped at random according to nude mice weight, every group 6, be divided into 4 groups, respectively control group, TRAIL group, Bestatin group, TRAIL and Bestatin joint group.Wherein TRAIL dosage be 10mg/kg, tail vein injection 1 week 1 time, Totally 3 times；Bestatin dosage is 20mg/kg, and gastric infusion stops 2 days after every successive administration 5 times.Record is primary within every two days The major diameter of nude mice weight and tumour and wide diameter, observe the nude mice state of mind.Nude mice is put to death at the 22nd day, dissection obtains main dirty Device, and solution cuts tumor mass simultaneously, is processed into pathological section, dyes through hematoxylin-eosin (H&E), Leica image analysis system is seen It examines and takes pictures.Tumor mass volume is calculated (a represents tumour major diameter, and b represents the wide diameter of tumour) according to formula V=ab2/2, draws nude mice Tumor growth curve and changes of weight curve graph.(wherein AB represents the average knurl weight of two medicine joint groups/right to CDI value=AB/A × B According to a group average knurl weight, A represents average knurl weight/control group average knurl weight of A medicine group, and B represents average knurl weight/control group of B medicine group Average knurl weight).
For results of animal as shown in figure 5, each group nude mice is in good condition, each main organs morphological function is normal, does not occur Death, changes of weight section (Fig. 5-a, Fig. 5-e) within 10% illustrate drug dose used within nude mice tolerance range； And from the point of view of Fig. 5-b tumor mass volume change, TRAIL and Bestatin drug combination group can significantly inhibit HT1080 transplanted tumor in nude mice Growth；Compared with TRAIL independent medication group (1.16 ± 0.24) g and Bestatin independent medication group (1.10 ± 0.26) g, connection Sharing medicine group tumor mass weight significantly reduces (0.46 ± 0.08) g (Fig. 5-c)；By observation each group tumor mass shape size it can also be seen that The tumor mass of TRAIL and Bestatin joint group is significantly smaller (Fig. 5-d)；And it is found after observing tumor tissues, joint group There is more necrotic zone (Fig. 5-e)；In addition, being computed, it is mono- that the tumour inhibiting rate (64.62%) of joint group is significantly stronger than TRAIL Private medicine group (10.98%) and Bestatin independent medication group (15.71%), and CDI value is 0.47 (CDI<0.7 represents significantly Synergistic effect).When the above results synthesis shows TRAIL (10mg/kg) and Bestatin (20mg/kg) independent medication, have no bright Aobvious tumor killing effect, and occur apparent collaboration tumor-inhibiting action after the two drug combination, pole is obtained in Transplanted tumor model Its significant therapeutic effect.
Comparative example 1
DCA (sodium dichloroacetate), i.e. dichloroacetate sodium, are a kind of inhibition of pyruvic dehydrogenase kinase Agent is often used for treating the metabolic-related disorders such as metabolic mitochondrial disease.More and more documents show since it can be indirect Pyruvic dehydrogenase is activated, causes tricarboxylic acid cycle and oxidative phosphorylation to increase, and then can induce apoptosis of tumor cells, but have Body mechanism of action not yet illustrates completely.The present invention provides as follows in order to illustrate the creativeness of the technical solution of the present invention program Description of test.
The influence that mtt assay detection Bestatin and DCA drug combination are proliferated growth of tumour cell
The HCT116 colon cancer cell of logarithmic growth phase is seeded to 96 holes after pancreatin digests with 3000/hole density In plate, it is placed in 37 DEG C of incubator cultures for 24 hours, after cell is adherent, Bestatin and DCA is diluted in proportion, drug is by following dense Degree is added in 96 orifice plates (100 hole μ l/)：(when drug combination, two medicines are corresponding same by Bestatin 0.01~5mg/ml, DCA 10mM When be added).3 multiple holes are arranged in each concentration, and set up control group and blank group, after being placed in 37 DEG C of incubator culture 48h, every hole MTT 20 μ l, the 37 DEG C of incubator culture 4h of 5mg/ml are added, inhales and abandons supernatant, 200 μ l DMSO, the shake of room temperature low speed is added in every hole 10min is swung, measures light absorption value at 570nm with microplate reader.By formula：Survival rate=(AT-AB)/(AC-AB) × 100% is calculated (wherein AT, AC, AB respectively represent dosing group, control group, blank group and are averaged A cell survival rate570Value), it is cross with adding consistency Coordinate, cell survival rate are that ordinate draws concentration-response curve.(wherein AB represents two medicine joint groups to CDI value=AB/A × B OD value/control group OD value, A represent OD value/control group OD value of A medicine group, and B represents OD value/control group OD value of B medicine group).
After MTT experiment result in tumour cell as shown in fig. 6, be individually added into Bestatin (0.01~5mg/ml), carefully Intracellular growth proliferation receives a degree of inhibition, while after adding DCA (10mM), cell survival rate and no significant difference, And CDI value does not have changing rule, is not less than 1.Illustrate compared with independent medication group, both Bestatin and DCA drug combination Growth of tumour cell proliferation is not influenced substantially afterwards, is more acted on without synergistic antitumor.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered It is considered as protection scope of the present invention.

Claims (10)

1. a kind of anti-tumor compositions containing ubenimex and trail protein, which is characterized in that including ubenimex solution and Trail protein solution；
For the ubenimex solution using NaOH solution as solvent, the concentration of the ubenimex solution is 0.01~0.1mg/ml；
For the trail protein solution using PBS buffer solution as solvent, the concentration of the trail protein solution is 0.039~5 μm of ol/ L。
2. anti-tumor compositions according to claim 1, which is characterized in that the concentration of the ubenimex solution is 0.03 ~0.08mg/ml.
3. anti-tumor compositions according to claim 1 or 2, which is characterized in that the concentration of the NaOH solution is 0.1mol/L。
4. anti-tumor compositions according to claim 1 or 2, which is characterized in that the pH value of the ubenimex solution is 8.0。
5. anti-tumor compositions according to claim 1, which is characterized in that the concentration of the trail protein solution is 0.1 ~2.5 μm of ol/L.
6. anti-tumor compositions according to claim 1 or 5, which is characterized in that the concentration of the PBS buffer solution is 0.01mol/L。
7. anti-tumor compositions application in preparation of anti-tumor drugs described in claim 1~6 any one.
8. application according to claim 7, which is characterized in that the effective concentration of ubenimex is 2mg/ in the drug ml。
9. application according to claim 7, which is characterized in that the effective concentration of trail protein is 1mg/ in the drug ml。
10. according to application described in claim 7~9 any one, which is characterized in that the anti-tumor drug is swollen by inducing Apoptosis of tumor lowers the expression of P-Erk albumen and/or cell migration is inhibited to play a role.
CN201810897991.6A 2018-08-08 2018-08-08 A kind of anti-tumor compositions and its application containing ubenimex and trail protein Withdrawn CN108888754A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113876958A (en) * 2021-06-18 2022-01-04 中国医学科学院医药生物技术研究所 Pharmaceutical composition for treating cancer and application thereof

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113876958A (en) * 2021-06-18 2022-01-04 中国医学科学院医药生物技术研究所 Pharmaceutical composition for treating cancer and application thereof
CN113876958B (en) * 2021-06-18 2023-05-12 中国医学科学院医药生物技术研究所 Pharmaceutical composition for treating cancer and application thereof

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