CN103304635B - Application of a kind of cyclic peptide compound antitumor and preparation method thereof - Google Patents
Application of a kind of cyclic peptide compound antitumor and preparation method thereof Download PDFInfo
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Abstract
The invention discloses application of cyclic peptide compound antitumor and preparation method thereof, belong to biomedicine technical field, cyclic peptide Compound C P-1 or CP-2 has anticancer to grow and the function of propagation, regulation and control Cancer Cell cycle and regulation and control Cancer Cell cycle protein expression, thus can be applied to and prepare cancer therapy drug and/or healthcare products.The invention still further relates to the preparation method of cyclic peptide Compound C P-1 and CP-2, take streptomycete as bacterial strain, optimization culture fermentation condition, fermentation after product is separated through silicagel column and reverse phase silica gel post after treatment, finally by preparative chromatography post, be separated and obtain the activated cyclic peptide compound of tool, present method compared with prior art, simple, environmental friendliness, cost are low, output is high, are a kind of methods of effective raising microorganism active secondary metabolite output.
Description
Technical field
The invention belongs to biomedicine technical field, be specifically related to application of a kind of cyclic peptide compound antitumor and preparation method thereof.
Background technology
Cancer is the first killer of human health, global pathogenesis of cancer numbers in 2008 and death toll are respectively 1,266 ten thousand and 7,560,000, estimating 2015 will have 1,500 ten thousand new cases, and M & M in the trend risen year by year, and mainly concentrates on developing country.
For most cancer patients, still effective medicine is lacked at present, the existing antineoplastic chemotherapy medicine applied clinically, though some has certain curative effect, but be mostly cytotoxic drug, inhibition tumor cell is value-added while, also the propagation of organism normal cell can be suppressed, as hair follicle cell and medullary cell etc., and produce certain side effect, the side effect of some chemotherapeutics is quite large, even threat to life, make patient very painful, this limits the clinical application of chemotherapeutics on the one hand, also be difficult to obtain due result for the treatment of on the other hand.However, the medicine of effective treatment urinary system cancer is also still lacked at present clinically, as the medicine etc. of against kidney cancer.In addition, the development of medical skill in decades just improves the result for the treatment of of early-stage cancer, remains helpless for terminal cancer.
Therefore, research and develop high-efficiency low-toxicity, cost is low, targeting is high antitumor drug especially against kidney cancer new drug be current problem demanding prompt solution.The cyclic peptide compound also not having bibliographical information to obtain from streptomycete (Streptomyces hawaiiensis NRRL15010) at present through inspection information has anti-tumor activity, the especially activity of against kidney cancer.
Summary of the invention
The object of the present invention is to provide application of a kind of cyclic peptide compound antitumor and preparation method thereof, present method cost is low, output is high, simple, environmental friendliness, the cyclic peptide antitumor activity of compound prepared is high, thus contributes to playing a role preventing and treating in process of cancer.
The present invention is achieved through the following technical solutions:
Cyclic peptide Compound C P-1 or CP-2 is preparing the application in antitumor drug and/or healthcare products, wherein, described cyclic peptide Compound C P-1 and the chemical structural formula of CP-2 as follows:
The application of cyclic peptide Compound C P-1 or CP-2 in preparation against kidney cancer medicine and/or healthcare products.
Described against kidney cancer medicine and/or healthcare products are the medicine and/or the healthcare products that suppress human renal carcinoma cell growth and propagation.
Described against kidney cancer medicine and/or healthcare products are medicine and/or the healthcare products in regulation and control kidney cancer cell cycle.
Described against kidney cancer medicine and/or healthcare products are the medicine and/or the healthcare products that make kidney cancer cell stop at the G1 phase.
Described against kidney cancer medicine and/or healthcare products are the medicine and/or healthcare products that suppress p-Erk, c-fos, cyclinD1, CDK4 and PCNA to express.
A preparation method for cyclic peptide compound, comprises the following steps:
1) be inoculated in by streptomycete NRRL15010 in the Preliminary fermentation liquid after sterilizing, at 25 ~ 35 DEG C, under 200 ~ 400r/min, fermentation culture 5 ~ 7 days, obtains zymocyte liquid; Described Preliminary fermentation liquid, in massfraction, comprises the D-Glucose of 0.3 ~ 0.8%, the yeast extract of 0.3 ~ 0.6%, the malt extract of 0.5 ~ 1.0%, the calcium carbonate of 0.02 ~ 0.10%, the D101 macroporous adsorbent resin of 1 ~ 3%, and surplus is water;
2) filtered by zymocyte liquid, obtain filtrate and resin and mycelial mixture, be extracted with ethyl acetate filtrate for several times, combined ethyl acetate part, obtains acetic acid ethyl acetate extract;
At room temperature, with ethyl acetate, methyl alcohol cold soaking extraction resin and mycelial mixture several respectively, united extraction liquid, obtains acetic acid ethyl acetate extract and methanol extract liquid respectively;
3) acetic acid ethyl acetate extract and acetic acid ethyl acetate extract are merged, be evaporated to dry, obtain the first medicinal extract; Methanol extract liquid is evaporated to dry, obtains the second medicinal extract; First medicinal extract and the second medicinal extract are merged, obtains total medicinal extract;
4) by total medicinal extract through column chromatography purification, be separated obtain cyclic peptide Compound C P-1 and CP-2.
Step 2) described in the number of times being extracted with ethyl acetate filtrate be 2 ~ 4 times, and the volume ratio of each extraction ethyl acetate used and filtrate is 1 ~ 3:1;
Described use ethyl acetate, the methyl alcohol number of times that cold soaking extracts resin and mycelial mixture is respectively 2 ~ 5 times, the time that each cold soaking extracts is 1 ~ 24h, and each cold soaking extracts ethyl acetate used, the amount of methyl alcohol is resin and 1 ~ 5 times of mycelial volume of mixture.
Column chromatography purification described in step 4) comprises three phases:
First stage, by total medicinal extract loading to silica gel column chromatography, using chloroform-methanol system as elutriant, (100:1) ~ (1:1) carries out gradient elution by volume, every 1500ml collects a stream part, and carrying out TLC detection to effluent liquid, is that stream part of (50:1) ~ (20:1) wash-out merges by volume by chloroform and methyl alcohol, normal pressure or evaporated under reduced pressure solvent, obtain crossing post part for the first time;
Subordinate phase: first time is crossed post part, be splined on reversed-phase silica gel chromatography post, using methanol-water system as elutriant, (1:100) ~ (100:1) carries out gradient elution by volume, every 800ml collects a stream part, and carrying out TLC detection to effluent liquid, is that stream part of (70:30) ~ (80:20) wash-out merges by volume by chloroform and methyl alcohol, normal pressure or evaporated under reduced pressure solvent, obtain second time and cross post part;
Phase III, second time is crossed post part, is splined on high performance liquid chromatography separator column, be separated and obtain cyclic peptide Compound C P-1 and CP-2.
Described high performance liquid chromatography separator column is Shimadzu shimpac ODS20 × 250mm, it is under 298nm that high performance liquid chromatography is separated, using methanol-water system or acetonitrile-water system as moving phase, isocratic elution is carried out by 8ml/min, in described methanol-water system, methyl alcohol and water volume ratio are 73:27, and in acetonitrile-water system, the volume ratio of acetonitrile and water is 68:32.
Compared with prior art, the present invention has following useful technique effect:
Unrestricted, the propagation that has no limits of cancer cells are the reasons causing body cell growing multiplication mechanism severe disorder, the invention discloses cyclic peptide Compound C P-1, CP-2, human renal carcinoma cell growth, kidney cancer cell Clone formation and kidney cancer cell cycle can be suppressed, thus reduce kidney cancer cell propagation, diffusion.
By the survival rate that MTT experiment confirms cyclic peptide Compound C P-1, CP-2 significantly can reduce kidney cancer cell; Can kidney cancer cell be significantly suppressed to be bred by cloning efficiency experiment confirmation cyclic peptide Compound C P-1, CP-2; The kidney cancer cell cycle can be regulated and controled by flow cytometry apoptosis rate experiment confirmation cyclic peptide Compound C P-1, CP-2, the expression of associated protein in the kidney cancer cell cycle can be suppressed again by protein immunoblot experiment confirmation cyclic peptide Compound C P-1, CP-2, have found the action target spot of CP-1, so cyclic peptide Compound C P-1 and CP-2 can be applied to and prepare antitumor drug and/or healthcare products.
The present invention prepares the method for cyclic peptide Compound C P-1, CP-2, with streptomycete (Streptomyceshawaiiensis NRRL15010) for bacterial strain, optimization culture fermentation condition, fermentation after product is separated through silicagel column and reverse phase silica gel post after treatment, finally by preparative chromatography post, separation obtains the activated cyclic peptide Compound C P-1 and CP-2 of tool, present method compared with prior art, simple, environmental friendliness, cost are low, output is high, are a kind of methods of effective raising microorganism active secondary metabolite output.
Accompanying drawing explanation
Fig. 1 is the ESI mass spectrum of cyclic peptide Compound C P-1 prepared by the present invention;
Fig. 2 is the ESI mass spectrum of cyclic peptide Compound C P-2 prepared by the present invention;
Fig. 3 is the HPLC color atlas of cyclic peptide compound prepared by the present invention;
Fig. 4 detects cyclic peptide Compound C P-1, CP-2 prepared by the present invention to the vitality test result of three kinds of kidney cancer cells with mtt assay; Wherein, (a) and (b) are CP-1, CP-2 vitality test result to 786-O, and (c), (d) are for CP-1, CP-2 are to the vitality test result of 769-P, and (e), (f) are for CP-1, CP-2 are to the vitality test result of ACHN;
Fig. 5 is that the cyclic peptide Compound C P-1, the CP-2 that prepare with flow cytomery the present invention are to the cell cycle regulating result of two kinds of kidney cancer cell 786-O and 769-P;
Fig. 6 is that the present invention the cyclic peptide Compound C P-1, the CP-2 that prepare affect result to three kinds of kidney cancer cell 786-O, 769-P and ACHN cloning efficiencies;
Fig. 7 is that the cyclic peptide Compound C P-1 for preparing of the present invention affects result to protein expression relevant to the cell cycle in 786-O with 769-P two kinds of kidney cancer cells.
Embodiment
Below in conjunction with specific embodiment and accompanying drawing, the present invention is described in further detail, and the explanation of the invention is not limited.
1, the preparation of cyclic peptide Compound C P-1 and CP-2:
Embodiment 1
1) be inoculated in the Preliminary fermentation liquid after sterilizing by streptomycete (Streptomyces hawaiiensis NRRL15010), at 30 DEG C, shaking table speed is under the condition of 300 turns/min, and fermentation culture 5 ~ 7 days, obtains zymocyte liquid;
Wherein, described initial incubation liquid, in massfraction, its composition comprises: 0.5%D-glucose, 0.5% yeast extract, 0.8% malt extract, 0.05% calcium carbonate, 1%D101 macroporous adsorbent resin, and surplus is water; The present invention streptomycete (Streptomyces hawaiiensis NRRL15010) used is purchased from ATCC.
2) by the zymocyte liquid 5 layers of filtered through gauze after cultivation, obtain filtrate, resin and mycelial mixture respectively, be extracted with ethyl acetate filtrate 3 times, the volume ratio of ethyl acetate used and filtrate is 1:1; At room temperature, with ethyl acetate, methyl alcohol respectively cold soaking extract resin and mycelial mixture 3 times, each 12h, merge and extract the acetic acid ethyl acetate extract and methanol extract liquid that obtain at every turn, during described cold soaking extracts, the consumption of ethyl acetate, methyl alcohol is respectively 1 ~ 5 times of resin and mycelial volume of mixture;
3) extracting solution of acetic acid ethyl acetate extract and ethyl acetate is merged, be evaporated to dry, obtain the first medicinal extract, methanol extract liquid is evaporated to dry, obtain the second medicinal extract, the first medicinal extract and the second medicinal extract are merged, obtains total medicinal extract;
4) by total medicinal extract loading to silica gel column chromatography, using chloroform-methanol system as elutriant, (100:1) ~ (1:1) carries out gradient elution by volume, under chloroform and methyl alcohol volume ratio are the gradient of 50:1,20:1,5:1 and 1:1, each gradient elution 3 ~ 4 retention volume, every 1500ml collects a stream part, detects through TLC, merge composition basic phase homogeneous turbulence part, obtain 8 stream parts altogether; Wherein, just chloroform and methyl alcohol are that stream part of (50:1) ~ (20:1) wash-out merges by volume, normal pressure or evaporated under reduced pressure solvent, obtain first time to cross post part;
First time is crossed post part, be splined on reversed-phase silica gel chromatography post, using methanol-water system as elutriant, (1:100) ~ (100:1) carries out gradient elution by volume, under chloroform and methyl alcohol volume ratio are the gradient of 50:50,60:40,70:30,80:20 and 90:10, and each gradient elution 3 ~ 4 retention volume, every 800ml collects a stream part, detect through TLC, merge composition basic phase homogeneous turbulence part, obtain 6 stream parts altogether; Be that stream part of (70:30) ~ (80:20) wash-out merges by volume by chloroform and methyl alcohol, normal pressure or evaporated under reduced pressure solvent, obtained second time and crossed post part;
5) second time is crossed post part, be splined on high performance liquid chromatography separator column, be separated and obtain cyclic peptide Compound C P-1 and CP-2.
Separation condition is: Shimadzu LC-6AD semi-preparative liquid chromatography instrument, differential refraction detector and UV-detector, chromatographic column is Shimadzu shimpac ODS20 × 250mm, flow velocity is 8ml/min, ultraviolet detection wavelength is 298nm, carry out isocratic elution using methanol-water or acetonitrile-water system as moving phase, in described methanol-water system, methyl alcohol and water volume ratio are 73:27, and in acetonitrile-water system, the volume ratio of acetonitrile and water is 68:32.
Show see Fig. 3, HPLC preparative chromatography figure, at 20 minutes and about 16 minutes, occurred that interarea amasss peak respectively, collection is separated the monomeric compound obtained afterwards and is respectively: CP-1 and CP-2.
2, structural characterization is carried out to being separated the monomeric compound obtained
1) carry out structural characterization to CP-1, analytical results is as follows:
CP-1, pale yellow powder, IR (KBr), cm
-1: 3310,1748,1668,1649,1523.UV λ
max(nm): 298.MS result is see Fig. 1, and display molecular weight is 718, and molecular formula is C
38h
50n
6o
8, hydrogen spectrum prompt for cyclic peptide compound, hydrogen modal data and ownership as follows:
1h-NMR (400MHz, CDCl
3): methylproline hydrogen signal, 4.46 (1H, dd, α-H), 2.06 (1H, m, β-H), 1.81 (1H, m, β-H), 2.36 (1H, m, γ-H), 0.98 (3H, d, γ-CH
3), 3.47 (1H, dd, δ-H), 3.08 (1H, dd, δ-H); Glycine signal, 8.46 (1H, brs, NH), 4.88 (1H, q, α-H), 1.36 (3H, d, α-CH
3); Sarcosine signal, 4.75 (1H, m, α-H), 1.50 (3H, d, α-CH
3), 2.82 (3H, s, N-CH
3); Proline(Pro) signal, 5.27 (1H, dd, α-H); 2.35 (1H, m, β-H), 1.97 (1H; m, β-H), 2.16 (1H, m; γ-H), 1.98 (1H, m, γ-H); 3.74 (1H, m, δ-H); 3.52 (1H, m, δ-H); Serine signal, 5.82 (1H, brs, NH), 4.49 (1H, dd, α-H), 4.84 (1H, dd, β-H), 3.54 (1H, dd, β-H); Phenylalanine signal, 6.69 (1H, brs, NH), 4.61 (1H, dd, α-H); 2.99 (2H, m, β-H), 7.15 (2H, m, 2 '-H, 6 '-H); 7.28 (2H, m, 3 '-H, 5 '-H), 7.18 (1H, m, 4 '-H); Signal on unsaturated fatty acids, 6.26,7.30,6.26,6.49,6.13,5.88,1.80. is according to characterization data, and determine that CP-1 is cyclic peptide compound, be made up of 6 amino acid, have a unsaturated fatty acids side chain, its structure is:
2) carry out structural characterization to CP-2, analytical results is as follows:
CP-2: pale yellow powder, IR (KBr), cm
-1: 3280,1728,1640,1599,798.UV λ
max(nm): 298.MS result is see Fig. 2, and display molecular weight is 704, and molecular formula is C
37h
48n
6o
8, hydrogen spectrum prompt for cyclic peptide compound, hydrogen modal data and ownership as follows: 1H-NMR (400MHz, CDCl
3): proline(Pro) hydrogen signal, 4.45 (1H, dd, α-H), 2.16 (1H, m, β-H), 1.98 (1H, m, β-H), 1.98 (1H, m, γ-H), 1.88 (1H, d, γ-CH
3), 3.61 (1H, dd, δ-H), 3.29 (1H, dd, δ-H); Glycine signal, 8.56 (1H, brs, NH), 4.88 (1H, q, α-H), 1.37 (3H, d, α-CH
3); Sarcosine signal, 4.79 (1H, m, α-H), 1.51 (3H, d, α-CH
3), 2.84 (3H, s, N-CH
3); Proline(Pro) signal, 5.17 (1H, dd, α-H); 2.35 (1H, m, β-H), 1.98 (1H; m, β-H), 2.16 (1H, m; γ-H), 1.98 (1H, m, γ-H); 3.74 (1H, m, δ-H); 3.59 (1H, m, δ-H); Serine signal, 6.82 (1H, brs, NH), 4.51 (1H, dd, α-H), 4.84 (1H, dd, β-H), 3.58 (1H, dd, β-H); Phenylalanine signal, 6.29 (1H, brs, NH), 4.65 (1H, dd, α-H); 2.96 (2H, m, β-H), 7.15 (2H, m, 2 '-H, 6 '-H); 7.29 (2H, m, 3 '-H, 5 '-H), 7.21 (1H, m, 4 '-H); Signal on unsaturated fatty acids, 6.25,7.30,6.26,6.48,6.13,5.88,1.79. is according to characterization data, and determine that CP-2 is cyclic peptide compound, be made up of 6 amino acid, have a unsaturated fatty acids side chain, its structure is:
Embodiment 2
1) be inoculated in the Preliminary fermentation liquid after sterilizing by streptomycete (Streptomyces hawaiiensis NRRL15010), at 25 DEG C, shaking table speed is under the condition of 400 turns/min, and fermentation culture 5 days, obtains zymocyte liquid;
Wherein, described initial incubation liquid, in massfraction, its composition comprises: 0.3%D-glucose, 0.3% yeast extract, 0.8% malt extract, 0.02% calcium carbonate, the D101 macroporous adsorbent resin of 1%, and surplus is water; The present invention streptomycete (Streptomyces hawaiiensis NRRL15010) used is purchased from ATCC.
2) by the zymocyte liquid 5 layers of filtered through gauze after cultivation, obtain filtrate, resin and mycelial mixture respectively, be extracted with ethyl acetate filtrate 3 times, the volume ratio of ethyl acetate used and filtrate is 2:1; At room temperature, with ethyl acetate, methyl alcohol respectively cold soaking extract resin and mycelial mixture 4 times, each 16h, merge respectively and extract the acetic acid ethyl acetate extract and methanol extract liquid that obtain at every turn, in described cold soaking extraction process, the amount of each extraction ethyl acetate used, methyl alcohol is respectively 3 times of resin and mycelial volume of mixture;
3) extracting solution of acetic acid ethyl acetate extract and ethyl acetate is merged, be evaporated to dry, obtain the first medicinal extract, methanol extract liquid is evaporated to dry, obtain the second medicinal extract, the first medicinal extract and the second medicinal extract are merged, obtains total medicinal extract;
4) by total medicinal extract through column chromatography purification, purification condition with embodiment 1, be separated obtain cyclic peptide Compound C P-1 and CP-2.
Embodiment 3
1) be inoculated in the Preliminary fermentation liquid after sterilizing by streptomycete (Streptomyces hawaiiensis NRRL15010), at 35 DEG C, shaking table speed is under the condition of 200 turns/min, and fermentation culture 7 days, obtains zymocyte liquid;
Wherein, described initial incubation liquid, in massfraction, its composition comprises: 0.6%D-glucose, 0.6% yeast extract, 0.5% malt extract, 0.02% calcium carbonate, 2%D101 macroporous adsorbent resin, and surplus is water; The present invention streptomycete (Streptomyces hawaiiensis NRRL15010) used is purchased from ATCC.
2) by the zymocyte liquid 5 layers of filtered through gauze after cultivation, obtain filtrate, resin and mycelial mixture respectively, be extracted with ethyl acetate filtrate 2 times, the volume ratio of ethyl acetate used and filtrate is 3:1; At room temperature, with ethyl acetate, methyl alcohol respectively cold soaking extract resin and mycelial mixture 3 times, each 24h, merge and extract the acetic acid ethyl acetate extract and methanol extract liquid that obtain at every turn, in described cold soaking extraction process, the amount of each extraction ethyl acetate used, methyl alcohol is respectively 2 times of resin and mycelial volume of mixture;
3) extracting solution of acetic acid ethyl acetate extract and ethyl acetate is merged, be evaporated to dry, obtain the first medicinal extract, methanol extract liquid is evaporated to dry, obtain the second medicinal extract, the first medicinal extract and the second medicinal extract are merged, obtains total medicinal extract;
4) by total medicinal extract through column chromatography purification, purification condition with embodiment 1, be separated obtain cyclic peptide Compound C P-1 and CP-2.
Embodiment 4
1) be inoculated in the Preliminary fermentation liquid after sterilizing by streptomycete (Streptomyces hawaiiensis NRRL15010), at 30 DEG C, shaking table speed is under the condition of 350 turns/min, and fermentation culture 6 days, obtains zymocyte liquid;
Wherein, described initial incubation liquid (for the nutrient solution that the present invention optimizes), in massfraction, its composition comprises: 0.8%D-glucose, 0.3% yeast extract, 1.0% malt extract, 0.10% calcium carbonate, the D101 macroporous adsorbent resin of 3%, and surplus is water; The present invention streptomycete (Streptomyceshawaiiensis NRRL15010) used is purchased from ATCC.
2) by the zymocyte liquid 5 layers of filtered through gauze after cultivation, obtain filtrate, resin and mycelial mixture respectively, be extracted with ethyl acetate filtrate 4 times, the volume ratio of ethyl acetate used and filtrate is 1:1; At room temperature, with ethyl acetate, methyl alcohol respectively cold soaking extract resin and mycelial mixture 5 times, each 1h, merge and extract the acetic acid ethyl acetate extract and methanol extract liquid that obtain at every turn, in described cold soaking extraction process, the amount of each extraction ethyl acetate used, methyl alcohol is respectively 1 times of resin and mycelial volume of mixture;
3) extracting solution of acetic acid ethyl acetate extract and ethyl acetate is merged, be evaporated to dry, obtain the first medicinal extract, methanol extract liquid is evaporated to dry, obtain the second medicinal extract, the first medicinal extract and the second medicinal extract are merged, obtains total medicinal extract;
4) by total medicinal extract through column chromatography purification, purification condition with embodiment 1, be separated obtain cyclic peptide Compound C P-1 and CP-2.
Embodiment 5
1) streptomycete (Streptomyces hawaiiensis NRRL15010) being inoculated in the cumulative volume after sterilizing is in 20L Preliminary fermentation liquid, and at 30 DEG C, shaking table speed is under the condition of 300 turns/min, and fermentation culture 5 ~ 6 days, obtains fermented liquid;
Wherein, described initial incubation liquid (for the nutrient solution that the present invention optimizes), in massfraction, its composition comprises: 0.8%D-glucose, 0.5% yeast extract, 0.6% malt extract, 0.02% calcium carbonate, the D101 macroporous adsorbent resin of 1%, and surplus is water; The present invention streptomycete (Streptomyceshawaiiensis NRRL15010) used is purchased from ATCC.
2) by the zymocyte liquid 5 layers of filtered through gauze after cultivation, obtain filtrate, resin and mycelial mixture respectively, be extracted with ethyl acetate filtrate 2 ~ 4 times, the volume ratio of ethyl acetate used and filtrate is 1:1; At room temperature, with ethyl acetate, methyl alcohol respectively cold soaking extract resin and mycelial mixture 5 times, each 5h, merge respectively and extract the acetic acid ethyl acetate extract and methanol extract liquid that obtain at every turn, during described cold soaking extracts, the consumption of ethyl acetate, methyl alcohol is respectively 5 times of resin and mycelial volume of mixture;
4) by total medicinal extract through column chromatography purification, purification condition with embodiment 1, be separated obtain cyclic peptide Compound C P-1 and CP-2.
3, the anti-tumor activity experiment of cyclic peptide Compound C P-1 and CP-2
1) cell cultures and Pharmaceutical formulations:
By human renal carcinoma cell line 786-O, 769-P and ACHN cell is inoculated in porous plate and (is purchased from American Type Culture Collection), with the RPMI-1640 substratum (being purchased from Gibco company of the U.S.) containing 10% foetal calf serum (being purchased from HyClone company of the U.S.) at 5%CO
2cultivate different time for 37 DEG C in incubator, 0.25% pancreatin (being purchased from Sigma Co., USA) digests and goes down to posterity.
With the storing solution that cyclic peptide Compound C P-1 and CP-2 is made into 20mM by DMSO, keep in Dark Place in-20 DEG C, stand-by.During MTT experiment, with containing 10%(volumetric concentration) the RPMI-1640 substratum of the foetal calf serum working concentration that it is diluted to further 1,10,20,50,100 μM adds corresponding experimental port.
2) mtt assay detection ring peptides CP-1, CP-2 is to the restraining effect of human renal carcinoma cell growth
Experimental technique: experiment is divided into sample sets and control group;
Sample sets: CP-1 or CP-2 adding 1,10,20,50,100 μM respectively;
Control group: with the RPMI-1640 nutrient solution of 10% foetal calf serum containing same concentrations DMSO in 1 μM of CP-1 or CP-2 diluent.
To take the logarithm 786-O in vegetative period, 769-P and ACHN cell, respectively with 200 μ L, 4 × 10
3individual cells/well is inoculated in 96 orifice plates, respectively establishes 5 multiple holes.After dosing 48, after 72h, discard original fluid, the 5mg/ml MTT(that every hole adds serum-free medium and 20 μ L is purchased from Sigma company) mixing solutions 200 μ L, cultivates 4h, abandon supernatant, every hole adds 150 μ L DMSO, and vibration 10min, makes crystallisate fully dissolve.
Detect the absorbancy of each hole 490nm by microplate reader, and calculate cell survival rate:
Cell survival rate (%)=(experimental group A
490mean value/blank group A
490mean value) × 100%.Take drug level as X-coordinate, cell survival rate (%) value is that ordinate zou draws cell growth curve.
Inhibiting rate (%)=(1-treatment group OD value/blank group OD value) × 100%)
Interpretation:
See Fig. 4, from MTT interpretation of result display, CP-1 and CP-2 all has obvious growth in vitro restraining effect to 786-O, 769-P and ACHN cell.CP-1 and CP-2 of different concns acts on 786-O, after 769-P and ACHN cell, when medicine CP-1 and CP-2 concentration are less than 10 μMs, CP-1 and CP-2 is to 786-O, 769-P and ACHN cell growth inhibition is not obvious, after medicine CP-1 and CP-2 concentration are greater than 20 μMs, along with the rising of drug level and the prolongation of action time, 786-O, 769-P and ACHN cell survival rate significantly declines.
Along with the increase of concentration, CP-1 and CP-2 is to three kinds of kidney cancer cell 786-O, and the inhibiting rate of 769-P and ACHN also increases; Along with the increase of action time, except to 769-P extracellular, the inhibiting rate of CP-1 and CP-2 to other two kinds of kidney cancer cell 786-O and ACHN also increases.CP-1 is to three kinds of kidney cancer cell effects IC of 48 hours
50value is respectively: 30,25 and 50 μMs, acts on the IC of 72 hours
50value is respectively: 20,30 and 22 μMs; CP-2 is to three kinds of kidney cancer cell effects IC of 48 hours
50value is respectively: 70,65 and 73 μMs, acts on the IC of 72 hours
50value is respectively: 40,65 and 70 μMs.As can be seen here, the restraining effect of CP-1 is stronger.
3) cyclic peptide Compound C P-1, CP-2 are on the impact of kidney cancer cell Clone formation
Cell clone refers to the cell colony that individual cells is formed by mitotic division, and colony's dependency and the multiplication capacity of its Forming ability and tumour cell are closely related.The one-tenth knurl ability of the large I reflection tumour cell of clonality and grade malignancy are also the good index evaluating unicellular survival and propagation.
Experimental technique: respectively 786-O, 769-P with ACHN trysinization also blows and beats into individual cells, be suspended in perfect medium, cell suspension is carried out gradient dilution, is inoculated in 24 orifice plates, inoculum density is respectively 150 μ L/ holes, respectively establish 3 parallel holes, every hole adds CP-1 or CP-2, and final concentration is 50 μMs, by cell in CO
2cultivate in incubator after 5 days and abandon nutrient solution, wash 2 times with PBS, use 1% violet staining after 4% paraformaldehyde is fixing, flowing water slowly washes away staining fluid, dry air, scanning Tissue Culture Plate.
Interpretation:
See Fig. 6, can find out, utilize cyclic peptide Compound C P-1, to act on the kidney cancer cell cloning efficiency after 5 days be zero to CP-2, and control group cloning efficiency is 100%, therefore CP-1 and CP-2 effectively can suppress 786-O, the ability of 769-P and ACHN cell clonal formation, confirms the restraining effect that cyclic peptide Compound C P-1 and CP-2 breeds kidney cancer cell further.
4) flow cytomery cyclic peptide Compound C P-1, CP-2 are on the impact in kidney cancer cell cycle
Experimental technique: with 50 μMs of CP-1 and CP-2 process 786-O, 769-P and ACHN cell, after 48h, collecting cell, washs 2 times with PBS, and adding volume fraction is 70% ethanol, is put-20 DEG C of internal fixtion, spends the night.After taking out centrifuge washing, PBS washs 2 times, adds 150 μ L RNA enzyme (RNase), after putting 37 DEG C of incubation 30min, then adds the mixing of 10mg/mL150 μ L PI dye liquor, puts room temperature, after lucifuge 30min, with the distribution of flow cytomery cell cycle.
Interpretation:
See Fig. 5, for cyclic peptide Compound C P-1, CP-2 are to the cell cycle analysis result of two kinds of kidney cancer cell 786-O and 769-P, the ratio of the G0/G1 phase cell after CP-1 and CP-2 process all significantly raises, the ratio of S phase and G2/M phase cell all reduces simultaneously, has no obvious apoptotic peak (Sub G0) and occurs.CP-1 and CP-2 is under same concentrations effect, and after after CP-1 process 786-O cell, G0/G1 phase cell proportion can reach 85.03%, CP-2 process 786-O cell, G0/G1 phase cell proportion reaches 64.80%.After after CP-1 process 769-P cell, G0/G1 phase cell proportion reaches 80.67%, CP-2 process 769-P cell, G0/G1 phase cell proportion has 61.84%.
After CP-1 and CP-2 process, the cell of two kinds of kidney cancer cell 786-O and 769-P mainly stops at the G1 phase, especially stronger with the effect of CP-1, as in 786-O cell, compared with the percentage of cells being in the G1 phase 51.4% with control group, the percentage of cells stopping at the G1 phase after CP-1 process is 80.1%; In 769-P cell, the percentage of cells that control group and CP-1 treatment group are in the G1 phase is respectively 54.4% and 80.0%, visible, and these two compounds are mainly worked in the G1 phase by the T suppression cell cycle.
5) protein immunoblot experiment, CP-1 is on the impact of kidney cancer cell cycle correlative protein expression
Experimental technique: collect respectively with the 786-O after 50 μMs of CP-1 process 48h, 769-P and ACHN cell, obtain total protein with RIPA cell pyrolysis liquid lysing cell, Bradford colorimetric determination.Prepare 12% polyacrylamide gel (SDS-PAGE), by every hole 30 μ g albumen loading, under 120V voltage after protein isolate, then adopt 25V voltage effect 12h to go to wet for the albumen on gel on nitrocellulose filter.After 5% skim-milk room temperature closes 1h, add T-Erk(respectively and win gloomy 1:200 difficult to understand), p-Erk(wins gloomy 1:200 difficult to understand), c-fos(Santa Cruz1:400), CyclinD1(Santa Cruz1:200), CDK4(Santa Cruz1:400), PCNA(SCT1:2000) become 1:10000 with Haikang on GAPDH(), 4 DEG C of refrigerator overnight incubation, after the abundant rinsing of TBST 5 times, add fluorescence two anti-igg (LI-COR company) room temperature lucifuge and hatch 1h, TBST rinsing 5 times, scans with Odyssey Dual band IR laser imaging system (LI-COR company).Adopt GAPDH as internal reference in experiment, scanning gained image adopts QuantityOne software to do gray-scale value analysis, and with the relative level of the ratio protein expression of GAPDH phase.
Interpretation:
See Fig. 7, as can be seen from band of expression, 786-O, 769-P cell compares with blank group, DMSO group through the medicine group of CP-1 process, and CP-1 can significantly lower cyclinD1, CDK4 protein expression.Detect the expression of DNA synthesis associated protein PCNA, 786-O, 769-P cell is after CP-1 process, and PCNA is equal down-regulated expression compared with control group simultaneously.The expression of cyclin cyclinD1 plays a significant role from the G0 phase at cell to the process of S phase, the expression of sense cycle albumen cyclinD1 upstream modulin, finds that p-Erk and c-fos protein expression level all significantly declines.
In sum, utilize streptomycete (Streptomyces hawaiiensis NRRL15010) as bacterial strain, culture condition is optimized, by MTT experiment, cyclic peptide compound monomer CP-1 and CP-2 be isolated to, confirms that cyclic peptide compound significantly can reduce the survival rate of kidney cancer cell; Confirm that cyclic peptide compound can significantly suppress kidney cancer cell to be bred by cloning efficiency experiment; Confirm that cyclic peptide compound can regulate and control the kidney cancer cell cycle by the experiment of flow cytometry apoptosis rate, confirm that cyclic peptide compound can suppress the expression of associated protein in the kidney cancer cell cycle by protein immunoblot experiment again, have found the action target spot of CP-1, so cyclic peptide Compound C P-1 and CP-2 can be applied to and prepare antitumor drug and/or healthcare products.
Claims (4)
1. the application of cyclic peptide Compound C P-1 or CP-2 in preparation against kidney cancer medicine and/or healthcare products, described against kidney cancer medicine and/or healthcare products are the medicine and/or the healthcare products that suppress human renal carcinoma cell growth and propagation; Wherein, described cyclic peptide Compound C P-1 and the chemical structural formula of CP-2 as follows:
2. apply as claimed in claim 1, it is characterized in that, described against kidney cancer medicine and/or healthcare products are medicine and/or the healthcare products in regulation and control kidney cancer cell cycle.
3. apply as claimed in claim 2, it is characterized in that, described against kidney cancer medicine and/or healthcare products are the medicine and/or the healthcare products that make kidney cancer cell stop at the G1 phase.
4. apply as claimed in claim 1, it is characterized in that, described against kidney cancer medicine and/or healthcare products are the medicine and/or healthcare products that suppress p-Erk, c-fos, cyclinD1, CDK4 and PCNA to express.
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