CN108938606B - Avobenzone application in preparation of anti-tumor drugs - Google Patents

Abstract

The present invention provides Avobenzone application in preparations of anti-tumor drugs.Present invention firstly discovers that the interaction that Avobenzone can be destroyed significantly between two protein of PHB and c-Raf1 then inhibits growth of tumour cell to destroy tumour cell signal path；The specificity of Avobenzone target tumor is high, and side effect is smaller and side effect counter-measure is more clear.Due to and non-targeted individual gene or protein, Avobenzone will not generate Sustained drug pressure to specific molecular in cancer cell or single protein and its be caused to generate mutation, greatly reduce the appearance of clinical drug-resistant.The present invention further passes through molecular biology experiments and cytological experiments and confirms that Avobenzone can significantly inhibit the transfer of colon cancer cell for the first time, extends the application range of Avobenzone, improves its application value, have important practical significance to oncotherapy.

Description

Avobenzone application in preparation of anti-tumor drugs

Technical field

The invention belongs to pharmaceutical technology fields, in particular to Avobenzone application in preparation of anti-tumor drugs.

Background technique

Colon cancer is common malignant tumour, and producing cause is more complicated, is still treated at present without special targeted drug The type cancer.Operation excision, radiation and chemotherapy are still three big means for the treatment of of colon cancer, however traditional therapeutic strategies exist The disadvantages of period is long, side effect is big, easy to recur.Originally the targeted drug such as Gefitinib of exploitation for lung cancer therapy etc. is in clinic On be also used as the treatment of colon cancer, however the anticancer drug of this target individual gene or albumen is at one section of clinical use Between after often will appear drug resistance phenomenon.Therefore obtaining safe and efficient, lasting colon cancer targeted drug has urgent reality meaning Justice.

Existing chemotherapeutics such as has biggish poison secondary along platinum medicine because of kill tumour cell that cannot be specific It acts on and easy to recur.Existing antineoplastic target drug is mostly the drug of the single oncogene of target or single protein, such Drug can specially recognizing tumor cells, there is preferable therapeutic effect, the targeted drug of target single protein molecule is such as lucky It is non-that the types of cancer such as treatment lung cancer are often used to for Buddhist nun, Tarceva etc..Although such targeted drug can also be used to treatment knot Intestinal cancer, but target gene is easy to produce medicament-resistant mutation under the continuous action pressure of such drug, the tumour cell funeral after mutation It has lost to the sensibility of drug and no longer by Drug inhibition, has caused tumor recurrence.Frequent gene mutation makes in colon cancer cell There is drug resistance at medication 2~3 months or so in patient, i.e., existing targeted drug, which cannot continue to work, inhibits tumor development.Cause This develops new colon cancer target agent and has important practical significance.

Avobenzone (Avobenzone) is the small of the uvioresistant irradiation of U.S. Food and Drug Administration (FDA) approval Molecular drug, the substance can be decomposed under ultraviolet irradiation, therefore be one of the main components of sun-proof substance, and the substance is Large-scale application is in clinical and market.But in addition to this sun-proof function, other effectiveness such as treatment of cancer of Avobenzone is simultaneously Have no direct research or report, the report about Avobenzone prevention cutaneum carcinoma is mainly from molecular biology in the prior art Angle has inquired into Avobenzone and has decomposed the ultraviolet luminous energy of counteracting under ultraviolet light irradiation in the principle i.e. drug in terms of preventing cutaneum carcinoma Amount is to reduce injury of the ultraviolet light to skin, however there is no verifying Avobenzones to the cutaneum carcinoma having already appeared for the document Therapeutic effect, also not from the research of cell biology angle and verify the drug to the meaning of skin cancer treatment (Reis JS, Corrêa MA,Chung MC,Dos Santos JL.Synthesis,antioxidant and photoprotection activities of hybrid derivatives useful to prevent skin cancer.Bioorg Med Chem 2014；22(9):2733-8.).

Currently, there is not yet the prior art report the influence that Avobenzone grow cancer cell (especially colon cancer cell) and Effect.

Summary of the invention

The purpose of the present invention is to overcome the shortcomings of the existing technology and deficiency, provides Avobenzone and is preparing anti-tumor drug In application.

The purpose of the invention is achieved by the following technical solution:

Avobenzone application in preparation of anti-tumor drugs.

The effective concentration of the Avobenzone is preferably 1~10 μM.

The anti-tumor drug contains one or more pharmaceutically acceptable carriers or auxiliary material.

The auxiliary material be preferably sustained release agent, excipient, filler, adhesive, wetting agent, disintegrating agent, sorbefacient, At least one of surfactant or lubricant.

The anti-tumor drug can be prepared into various dosage forms using the conventional method of this field, such as capsule, pill, piece The dosage form of the oral administrations such as agent, oral solution, granule, tincture.

The tumour is preferably colon cancer.

It works different from traditional antineoplastic target drug using individual gene or protein as target spot, it is provided by the invention Colon cancer targeted drug Avobenzone is using the interaction between the protein in tumour cell signal path as target spot, this is just Utmostly avoid the appearance of drug resistance.Specifically, mainly by destroying between two protein of PHB and c-Raf1 It interacts and works, destroy entire signal path to play, then generate the effect for inhibiting colon metastasis of cancer.

The present invention has the following advantages and effects with respect to the prior art:

1. present invention firstly discovers that Avobenzone can significantly destroy the phase interaction between two protein of PHB and c-Raf1 With, and confirm that Avobenzone can significantly inhibit colon cancer cell for the first time by molecular biology experiments and cytological experiments Transfer, extends the application range of Avobenzone, improves its application value.

2. the specificity of Avobenzone target tumor is high, and side effect is smaller and side effect is answered compared with classic chemotherapy drug It is more clear to measure.

3. compared with existing tumor-targeting drug, Avobenzone and non-targeted individual gene or protein, but target In the interaction of two protein, Sustained drug pressure will not be generated to specific molecular in cancer cell or single protein and led It causes it to generate mutation, greatly reduces the appearance of clinical drug-resistant.

4. the treatment of colon cancer, Avobenzone provided by the present invention are still used for without special tumor-targeting drug at present It has important practical significance to the inhibiting effect of colon metastasis of cancer.

Detailed description of the invention

Fig. 1 is the operating procedure flow chart of superELISA in embodiment 1.

Fig. 2 is the absorbance value result analysis chart of different small-molecule drugs in embodiment 1.

Fig. 3 is the co-immunoprecipitation experiment result figure of embodiment 2.

Fig. 4 is the protein immunoblotting experimental result picture of embodiment 3.

Fig. 5 is the cell invasion analysis of experimental results figure of embodiment 4.

Specific embodiment

Present invention will now be described in further detail with reference to the embodiments and the accompanying drawings, but embodiments of the present invention are unlimited In this.

Embodiment 1 passes through the antitumor mechanism of superELISA technical research Avobenzone

One, the preparation of fusion protein PHB1-his, C-Raf1-gst

(1) expression and purification of PHB1-his fusion protein

1. constructing recombinant expression carrier pET-his: according to ncbi database source of people PHB1 protein gene sequence, (NCBI is logged in Number: NM_001281496.1), the primer of coding PHB1 gene is designed and synthesized using Primer Premier 5.0, at both ends Double enzyme site is added, and (upstream termination restriction enzyme site is BamH1, and downstream end restriction enzyme site is EcoR1, is purchased from TAKARA Company), PCR amplification goes out target gene band.

2. detecting PCR product with 1% agarose gel electrophoresis, DNA QIAquick Gel Extraction Kit (the limited public affairs of Tiangeng biochemical technology are utilized Department, article No.: DP214) recovery purifying target fragment.Double digestion target gene fragment and empty carrier pET (green cloud are carried out with restriction endonuclease Its biotech company, article No. D2902), take target gene fragment 470ng and empty carrier 200ng to carry out digestion respectively, condition is 37 DEG C of digestion 4h；The digestion products for mixing target gene and empty carrier later carry out DNA purification and recovery (50 μ L of total volume), take pure Change 35 μ L of recovery product and mixes+8 μ L ddH of 2 μ L T4DNA ligase (Takara company)+5 μ L buffer₂O；Take 10 μ L connections Product is converted to E.coli Top10 competent cell, and Jing Kana resistant panel screens positive recombinant, carries out bacterium colony PCR to it Verifying and DNA sequencing verifying compare analysis sequencing result through NCBI Blast.Plasmid is placed in -20 DEG C of long-term preservations, and bacterial strain is added 15% glycerol is stored in -80 DEG C.

3. the inducing expression of fusion protein: correct Top10-pET-PHB1 will be sequenced and be inoculated in containing ammonia benzyl resistance In the LB culture medium of (100ng/ μ L), 37 DEG C of shaken cultivations are stayed overnight.Bacterium solution is accessed the strain of activation in 1:100 ratio by next day Fresh culture expands culture, 37 DEG C with 220rpm shaken cultivation to OD600=0.6~0.8 when, isopropylthio half is added Lactoside (IPTG), the final concentration of 0.5mM of IPTG, same condition collect thallus, 4700rpm after continuing 4~6h of inducing expression In 4 DEG C of centrifugation 45min, thallus is resuspended for 1 × PBS buffer solution and thalline were collected by centrifugation again.By the thallus being collected into liquid nitrogen and 37 DEG C multigelation 3 times, then ice-bath ultrasonic 30min (5s is opened, 5s close) to bacterium solution becomes that clarification is bright, and ultrasonication object is in 4 DEG C It is centrifuged 30min with 10000rpm, abandons precipitating, the aperture 0.22mm membrane filtration supernatant, supernatant was used for column purification.Take a small amount of supernatant (about 15 μ L) uses 12%SDS-PAGE detection fusion protein expression situation.

4. the Ni-NTA affinitive layer purification column using QIAGEN company purifies recombinant protein.Specific steps are as follows:

(1) after mixing Ni-NTA resin, 2mL upper prop is taken, 30min is stored at room temperature.

(2) with 10 times of column volumes sterilizing ultrapure water ddH₂O cleans pillar.

(3) with the equilibration buffer pillar of 10 times of column volumes.

(4) the supernatant sample liquid of collection is slowly fitted into chromatographic column.

(5) resin is rinsed with the equilibrium liquid of 10 times of column volumes and collect loading efflux；

(6) it uses respectively and contains 10mM imidazoles, 25mM imidazoles, 50mM imidazoles, the buffer of 100mM imidazoles and 500mM imidazoles Nickel column is eluted, collects eluent respectively.It is examined with expression quantity and purity of the 12%SDS-PAGE to PHB1-his fusion protein It surveys.

(7) respectively with the equilibration buffer of 10 times of column volumes (20mM sodium phosphate dodecahydrate, 500mM NaCl, pH 7.0 ~7.5) and 10 times of column volume ddH₂O cleans pillar, and 20% ethyl alcohol of 5mL, 4 DEG C of preservations are added into pillar.

The concentration of 5.KCTD12-his fusion protein: using 10KD super filter tube (Millipore, article No.: ACS501012) into The concentration of row fusion protein, 5000rpm centrifugation, 4 DEG C of ultrafiltration to 500 μ L use 20mM Tris-HCl (NaCl containing 100mM) later Rinse concentrate three times, and repeated centrifugation is three times.The protein concentrate of acquisition uses BCA protein concentration detection kit (ThermoFisher, article No.: 23227) measuring concentration and is placed on -80 DEG C of preservations.

(2) expression and purification of C-Raf1-gst fusion protein

1. constructing recombinant expression carrier pGEX-4T-1-C-Raf1-gst: according to ncbi database source of people C-Raf1 albumen base Because of sequence (NCBI accession number: NM_001354689.1), coding C-Raf1 is designed and synthesized using Primer Premier 5.0 The primer of gene, double enzyme site is added at both ends, and (upstream termination restriction enzyme site is BamH1, and downstream end restriction enzyme site is EcoR1 is purchased from TAKARA company), PCR amplification.

2. detecting PCR product with 1% agarose gel electrophoresis, DNA QIAquick Gel Extraction Kit (the limited public affairs of Tiangeng biochemical technology are utilized Department, article No.: DP214) recovery purifying target fragment.Target gene fragment 680ng and empty carrier 200ng is taken to carry out digestion respectively, Condition are as follows: 37 DEG C of digestion 4h；The digestion products for mixing target gene and empty carrier later carry out DNA purification and recovery (50 μ of total volume L), 35 μ L of purification and recovery product is taken to mix+8 μ L ddH of 2 μ L T4DNA ligase (Takara company)+5 μ L buffer₂O, mixing Uniformly overnight in 16 DEG C of connections；10 μ L connection products are taken to convert to 21 competent cell of E.coli BL, through ammonia benzyl resistant panel Positive recombinant is screened, bacterium colony PCR verifying and DNA sequencing verifying are carried out to it, compares analysis sequencing result through NCBI Blast. Plasmid is placed in -20 DEG C of long-term preservations, and bacterial strain is added 15% glycerol and is stored in -80 DEG C.

3. the inducing expression of fusion protein: correct BL 21-PGEX-4T-1-C-Raf1 will be sequenced and be inoculated in containing ammonia benzyl In the LB culture medium of resistance (100ng/ μ L), 37 DEG C of shaken cultivations are stayed overnight.Next day is by the strain of activation in 1:100 ratio by bacterium solution Access fresh culture and expand culture, 37 DEG C with 220rpm shaken cultivation to OD600=0.6~0.8 when, IPTG is added to end Concentration is 0.5mM, and same condition continues to collect thallus after 4~6h of inducing expression, 4700rpm in 4 DEG C of centrifugation 45min, 1 × Thallus is resuspended for PBS buffer solution and thalline were collected by centrifugation again.By the thallus being collected into liquid nitrogen and 37 DEG C multigelation 3 times, so Ice-bath ultrasonic 30min (5s is opened, and 5s is closed) is bright to bacterium solution change clarification afterwards, and ultrasonication object is centrifuged in 4 DEG C with 10000rpm 30min, abandons precipitating, and supernatant was used for column purification.Take a small amount of supernatant (about 15 μ L) with 12%SDS-PAGE detection fusion albumen table Up to situation.

4. the GST affinitive layer purification column using GE company purifies recombinant protein, specific steps are as follows:

(1) after mixing GST affinity chromatography resin, 2mL upper prop is taken, 30min is stored at room temperature.

(2) with 10 times of column volumes sterilizing ultrapure water dd H₂O cleans pillar.

(3) pillar is balanced with the Binding buffer (1 × PBS, pH 7.4) of 10 times of column volumes.

(4) the supernatant sample liquid of collection is slowly fitted into chromatographic column.

(5) lower foreign protein is washed with the Binding buffer of 10 times of column volumes.

(6) it is eluted with the Elution buffer of 5 times of column volumes (10mM glutathione+50mM Tris-HCl), collects stream Liquid out, i.e. C-Raf1-GST fusion protein.It is carried out with content and purity of the 12%SDS-PAGE to C-Raf1-GST fusion protein Detection.

(7) with the Binding buffer and 10 times of column volume ddH of 10 times of column volumes₂O cleans pillar, is added into pillar 20% ethyl alcohol of 5mL, 4 DEG C of preservations.

5. the concentration of fusion protein: carrying out fusion protein using 10KD super filter tube (Millipore, article No.: ACS501012) Concentration, 5000rpm centrifugation, 4 DEG C of ultrafiltration to 500 μ L rinse concentrate three times using 1 × PBS later, and repeated centrifugation is three times. Using BCA protein concentration detection kit, (article No.: 23227) ThermoFisher measures concentration and puts the protein concentrate of acquisition It sets and is saved at -80 DEG C.

Two, setting superELISA is tested

Associated operating steps flow chart is as shown in Figure 1.

1. coating: carrying out first layer antibody gst-tag antibody (article No.: 66001- according to the method for coating of ELISA 2-Ig, be purchased from Proteintech Group) coating, using common ELISA coating buffer (be purchased from NeoBioscience, article No.: NBC01 antibody) is formulated as the antibody diluent that concentration is 1ng/ μ L, 100 μ L antibody are added in the hole of each 96 hole elisa plate Dilution, 4 DEG C of coatings are overnight.

2. closing: using phosphate buffer (PBS) (12 hypophosphite monohydrate of PBS:4g NaCl, 0.1g KCl, 1.815g Disodium hydrogen, 0.12g dipotassium hydrogen phosphate, is dissolved in the ultrapure water of 400mL, is settled to 500mL use) cleaning elisa plate 1 time 5 Minute；300 μ L 5% (w/v) bovine serum albumin(BSA)s (BSA) (PBS preparation) are added in every hole；37 DEG C are closed 2 hours.

3. protein C-the Raf1-gst of purifying is added: 1 μ g purifying protein is mixed in advance and is diluted in 200 μ L PBS, The C-Raf1-gst albumen after dilution is added according to 1 hole μ g/, the protein concentration in final each hole is 5 μ g/mL, and room temperature is slow It shakes 5 hours；It is cleaned elisa plate 5 times, every time 5 minutes according to above-mentioned cleaning method later.

4. the protein PHB1-his of purifying is added: 1 μ g purifying protein being mixed in advance and is diluted in 200 μ L PBS, is pressed The PHB1-his albumen after dilution is added according to 1 hole μ g/, the protein concentration in final each hole is 5 μ g/mL；37 DEG C of standings are incubated It educates 3~4 hours；It is cleaned elisa plate 5 times, every time 5 minutes according to above-mentioned cleaning method later.The medicine that will be calculated at the same time Object is added in reacting hole, makes final concentration of 10 μM of drug.

5. his-tag antibody (article No.: ab9108 is purchased from Abcam) antibody is added: according to 1:2000 ratio, by 1 μ G antibody dilutes in 2000 μ L 5%BSA, obtains antibody diluent, and 100 μ L antibody diluents are added in every hole, and incubation at room temperature 2 is small When or 4 DEG C overnight incubation.Note: gst-tag antibody antibody sources attribute need to be with his-tag antibody antibody category Property it is different, in the present embodiment, gst-tag antibody is that mouse is anti-, and his-tag antibody is rabbit-anti.

6. washing Excess antibody: cleaning method is as above, cleans 5 times, every time 5 minutes.

7. being incubated for secondary antibody: will be in conjunction with the secondary antibody goat with horseradish peroxidase of his-tag antibody primary antibody Anti-rabbit IgG HRP (article No.: ab6721；Purchased from Abcam) according to mass volume ratio 1:2000,1 μ g of antibody is diluted to 2000 μ L 5%BSA in, obtain antibody diluent, every hole is added 100 μ L antibody diluents and is incubated for.

8. colour developing: the common ELISA developing solution TMB of 100 μ L is added in every hole, and (article No.: TMS.12 is purchased from NeoBioscience), place 1 minute for 37 DEG C, immediately with 50 μ L terminate liquids (article No.: EST001 is purchased from NeoBioscience) Color development stopping.

9. reading numerical values: carrying out dual wavelength measurement using spectrophotometer after color stability, counted according to OD450-OD630 Calculation obtains the absorbance value in each hole, and then analyzes drug effectiveness.

Medicament sources for this experiment screening make in what U.S. Food and Drug Administration (FDA) ratified for clinic Small-molecule drug library (FDA-approved Drug Library is purchased from Selleck, article No.: L1300).In the present embodiment Research screening is carried out to 88 small-molecule drugs altogether, the batch small-molecule drug is in company with the PHB1-his protein one purified It rises and is added in reaction system (i.e. above-mentioned steps (4)).

Three, the selection result is analyzed

Being studied from FDA small-molecule drug library using superELISA technology being capable of specificity inhibition PHB and c-Raf1 phase The small-molecule drug of interaction.It shares 88 small-molecule drugs and participates in research, as a result as shown in Fig. 2, horizontal line, which refers to, only carries out albumen Matter interaction, not positive controls of agent-feeding treatment more than horizontal line and horizontal line refer to the drug for not occurring inhibitory effect, horizontal line with Under point out the drug for having showed inhibitory effect.Making comparisons when analysis with the control group on same elisa plate, (absorbance value is close to zero Several groups be negative control group, one on horizontal line group be positive controls).Wherein Avobenzone is mutual to PHB-c-Raf 1 The inhibition of effect is the most obvious (at arrow direction).

According to the literature, the interaction of PHB and c-Raf1 is to inhibit the two an important factor for leading to tumor progression Interaction has inhibits cancer metastasis and cultivation effect (Chiu CF, Ho MY, Peng JM, Hung SW, Lee well WH,Liang CM,et al.Raf activation by Ras and promotion of cellular metastasis require phosphorylation of prohibitin in the raft domain of the plasma membrane.Oncogene 2013；32 (6): 777-87., Doudican NA, Orlow SJ.Inhibition of the CRAF/prohibitin interaction reverses CRAF-dependent resistance to vemurafenib.Oncogene 2017；36(3):423-428.).

The present embodiment passes through superELISA the experiment proves that Avobenzone specific can inhibit PHB and c-Raf1 mutual Effect.

2 co-immunoprecipitation experiment of embodiment

Co-immunoprecipitation experiment (Co-IP) is to examine the important means that interacts in aleuroplast, in order to further study The anticancer mechanism and effect of Avobenzone are verified, the present embodiment carries out co-immunoprecipitation experiment (Co-IP) to the work of Avobenzone With further being studied.Co-IP specific steps are as follows:

(1) in advance by 2 groups, every group of 1,000,000 colon cancer HCT116 cells are layered in Tissue Culture Dish, and are separately added into DMSO and Avobenzone.DMSO is added according to 1 μ L:1000 μ L DMEM culture liquid proportional, and Avobenzone is dilute using DMEM culture solution It releases, ultimate density is 10 μM；

Culture processing cell 24 hours, clean cell twice with PBS later, each culture dish adds 1mL under (2) 37 DEG C of environment 0.25% trypsin digestion cell makes its culture dish wall that falls off；It is then terminated and is digested with every hole 5mL complete culture solution, 300g revolving speed is normal Temperature centrifugation 3 minutes is collected cell and is resuspended with 5mL PBS, repeated centrifugation 1 time.

(3) cleaned colon cancer cell is collected into the EP pipe of 1.5mL.Configured lysate is added, this cracking Liquid is that (article No.: P0013 is purchased from the green skies to western the and IP lysate containing 1%PMSF, 1%PI and inhibitors of phosphatases Biotech company), it cracks on ice, every 5min turns upside down several times, soft, it cannot be vortexed, after cracking 30min, 4 DEG C of centrifugation 30min of 13200rpm.

(4) it takes supernatant to manage to new EP, surveys protein concentration using BCA method, it is each subsequent set of to take 1mg protein progress Co-IP anti- It answers.Every group of 30 μ L of addition Protein A/G agarose and rabbit igg (article No.: 10284-1-AP；Purchased from Proteintech) 1 μ G, 4 DEG C of rotations are incubated for 1 hour.Subsequent 4 DEG C, 2500rpm is centrifuged 5min, takes supernatant protein sample into new EP pipe.This step is to go Except in sample can in next step when PHB1 antibody incubation non-specific binding protein.

(5) 2 μ g rabbit igg antibody are added in the control histone sample of DMSO processing, add in the albumen sample of Avobenzone processing Enter 2 μ g PHB1 antibody (article No.s: 2426s；Purchased from Cell Signaling Technology) it is used as experimental group, 4 DEG C of rotations are incubated Educate 16~18h.

(6) 30 μ L Protein A/G agarose beads are added in every group of sample, 4 DEG C of rotations are incubated for 4h.

(7) 2500rpm is centrifuged 5 minutes, removes supernatant；Soft rinse beads is washed with western and IP lysate, later 2500rpm centrifugation 5 minutes, in triplicate.Remove supernatant, every group of 30 μ L SDS lysate (article No.: P0013G of addition；It is purchased from Green skies biotech company).Be vortexed concussion one minute, boiling water 10min.Supernatant is carried out SDS-PAGE electrophoresis point by centrifugation From and western blot analyze Co-IP effect.

Western blot step bibliography: how is the quantitative proteomics research that protein updates in the cell cycle Gui Wei master thesis).

As a result as shown in figure 3, Co-IP proves that Avobenzone can significantly destroy the effect of PHB1-C-Raf1.

The present embodiment proves that Avobenzone can significantly inhibit the mutual of PHB1 and C-Raf1 by molecular biology method Effect, can destroy signal path relevant to cancer metastasis in colon cancer cell, it was demonstrated that Avobenzone is thin to colon cancer The influence of born of the same parents' signal path.

The research of embodiment 3MEK activation level

Protein MEK is the downstream molecules of PHB-c-Raf1 signal path, by the activation level, that is, phosphorylation for detecting MEK State (pMEK) can confirm whether the interaction between PHB and c-Raf1 is destroyed.The reduction of pMEK level also implies that The transfer ability of colon cancer cell declines.

The activation level of MEK is detected by the method for protein immunoblotting (western blot), the specific steps are as follows:

1. colon cancer cell HCT116 or colon cancer cell RKO (being purchased from ATCC, article No. CRL-2577) are layered on six orifice plates In, every 500,000 cell of hole.Add DMSO (control group) and each 2 μ L of Avobenzone (experimental group) into culture hole respectively, each hole adds Enter 2000 μ L of DMEM complete culture solution, wherein final concentration of 10 μM of Avobenzone.Culture solution is mixed, 37 DEG C of cultures handle cell 48 hours.

2.PBS cleans cell twice, and each hole adds 0.25% trypsin digestion cell of 0.5mL to make its culture dish wall that falls off；With It is terminated and is digested with every hole 1mL complete culture solution afterwards, cell suspension is collected into 1.5mL EP pipe, and 300g revolving speed room temperature is centrifuged 3 points Clock is collected cell and is resuspended with 1mL PBS, repeated centrifugation 1 time.

3. removing supernatant, the configured 50 μ L of lysate of every group of addition, this lysate is to contain 1%PMSF, 1%PI and phosphorus SDS cell pyrolysis liquid (the article No.: P0013G of sour enzyme inhibitor；Purchased from green skies biotech company).It cracks on ice, every 5min The concussion that is vortexed is primary, after cracking 30min, 4 DEG C of centrifugation 30min of 13200rpm.

4. surveying protein concentration using BCA method, and every group of 30 μ g albumen is respectively taken to carry out western blot experiment, specific steps With reference to: the quantitative proteomics that protein updates in the cell cycle study what Gui Wei master thesis of.This experiment difference The antibody used is MEK, and the corresponding primary antibody of pMEK, Actin is purchased from Cell Signaling Technology company, article No. Respectively 4694S, 9154S, 3700S.All primary antibody concentration are that (1 μ g protein is diluted in 2000 μ L 5%BSA to 1:2000 In).

5.WB tests to obtain the variation of MEK activation level in (experimental group) colon cancer cell after Avobenzone is handled, the change Change is embodied by the height of pMEK level.

Protein immunoblotting experimental result is as shown in figure 4, show in the colon cancer cell of Avobenzone (Avo) processing MEK phosphorylation (pMEK) is horizontal to be reduced, and is further confirmed Avobenzone and is inhibited interaction between PHB and c-Raf1.

4 cell invasion test research of embodiment

1. HCT116 colon carcinoma cell line (article No.: CCL-247 is purchased from American Type Culture collection warehousing ATCC) is taped against In six orifice plates, 500,000, every hole cell.Preparation original concentration is 10mM Avobenzone, and when dosing is thin according to+2000 μ L of 2 μ L drug The Avobenzone of the ratio of born of the same parents' culture solution, 10 μM of the drug ultimate density for touching cell, 10 μM of concentration handles colon cancer Cell 48 hours, trypsin digestion cell is used later, and cell is resuspended with serum-free medium and counts preparation cell invasion experiment.With The cell experiment group of DMSO processing is as negative control group (NC).

2. cell prepares: first clean and sterile cell being placed in the hole of dedicated 24 orifice plate of Transwell, it is ensured that put It is flat；5 μ L matrigels are uniformly mixed with 95 μ L pre-cooling serum-free medium later and are carefully taped against in cell upper chamber, are stored at room temperature 30 Solution is sopped up after minute, matrigel can form colloid thin layer in cell upper chamber at this time, to simulate the matrix such as vascular wall in human body Class formation.

3. 600 μ L complete mediums are gently added by hole wall on 24 orifice plates, guarantee that cell counterdie and culture medium sufficiently connect Touching obstructs without bubble, then cell suspension is mixed well, and gently drops evenly in each cell isometric equal number of 200 μ L cell suspensions, it is ensured that cell is spread uniform.

4. the tissue culture plate planted is fixed, 37 DEG C are placed on, 5%CO₂It is cultivated 48 hours in incubator.

5. cell is fixed: taking out cell, lightly twice of rinse in PBS adds the fixed counterdie cell 30min of methanol.

6. violet staining: being washed twice with PBS, methanol is removed, with 0.1% violet staining 20min.

7. cleaning observation: washing away the crystal violet on cell with PBS or pure water, and wet cotton swab dabs off cell upper layer The cell of film, and wash clean are not worn.

8. taking pictures: the cell cleaned up is dried, 24 clean orifice plates is placed in, is observed under inverted fluorescence microscope, And it takes pictures in 5 visuals field (upper and lower, left and right, in) that same position is chosen in each cell；

9. statistical analysis: calculating the cell on photo using software I mageJ or calculate its area coverage, counted Analysis.

As a result as shown in figure 5, Avobenzone processing cell (experimental group) and DMSO handle control group between cell It compares, invasive ability has dropped 4 times, shows that Avobenzone can significantly inhibit the transfer of colon cancer cell.

Above embodiments prove that Avobenzone can significantly destroy the interaction between protein PHB and c-Raf1, and Therefore the transfer of inhibition colon cancer cell that can be potent, has important clinical value and commercial value.

The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment Limitation, other any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention, It should be equivalent substitute mode, be included within the scope of the present invention.

Claims (7)

1. Avobenzone application in preparation of anti-tumor drugs, it is characterised in that:

The tumour is colon cancer.

2. Avobenzone application in preparation of anti-tumor drugs according to claim 1, it is characterised in that:

The effective concentration of the Avobenzone is 1~10 μM.

3. Avobenzone application in preparation of anti-tumor drugs according to claim 1, it is characterised in that:

The anti-tumor drug contains one or more pharmaceutically acceptable carriers or auxiliary material.

4. Avobenzone application in preparation of anti-tumor drugs according to claim 3, it is characterised in that:

The auxiliary material is sustained release agent, excipient, filler, adhesive, wetting agent, disintegrating agent, sorbefacient, surface-active At least one of agent or lubricant.

5. Avobenzone application in preparation of anti-tumor drugs according to claim 1, it is characterised in that:

The anti-tumor drug can be prepared into pharmaceutical preparation using the conventional method of this field.

6. Avobenzone application in preparation of anti-tumor drugs according to claim 5, it is characterised in that:

The pharmaceutical preparation is oral drug preparation.

7. Avobenzone application in preparation of anti-tumor drugs according to claim 6, it is characterised in that:

The oral drug preparation is capsule, pill, tablet, oral solution, granule, tincture.