CN1748674A - Natural abscisic acid is used to prepare the new purposes of tumor cell " differentiating inducer " medicine - Google Patents
Natural abscisic acid is used to prepare the new purposes of tumor cell " differentiating inducer " medicine Download PDFInfo
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- CN1748674A CN1748674A CNA2004100406825A CN200410040682A CN1748674A CN 1748674 A CN1748674 A CN 1748674A CN A2004100406825 A CNA2004100406825 A CN A2004100406825A CN 200410040682 A CN200410040682 A CN 200410040682A CN 1748674 A CN1748674 A CN 1748674A
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Abstract
The present invention relates to a kind of new purposes of natural materials, be specifically related to the new purposes of natural abscisic acid.At present tumor incidence rate height, the invention discloses the new purposes that a kind of natural abscisic acid is used to prepare tumor cell " differentiating inducer " medicine, promptly adopt 5g/L-1 * 10
-6Advantages such as propagation and inducing tumor cell that the g/L natural abscisic acid can effectively suppress tumor cell transform to normal cell, and the present invention has simple to operate, and is effective, and toxic and side effects is little.
Description
One. affiliated field:
The present invention relates to a kind of new purposes of natural materials, be specifically related to the new purposes of natural abscisic acid.
Two, background technology:
For a long time, people have carried out unremitting struggle with the tumor of one of principal disease that threatens people ' s health, and operation, radiotherapy, chemotherapy become three big means of oncotherapy.But these three kinds of means toxic and side effects are big, poor selectivity.In recent years, tumor induction and differentiation therapy are extensively carried out all over the world, have successively held 4 symposiums in the world, and the treatment of the induction of tumor cell has been pushed to from the basis clinical, thereby opened up new way for tumor treatment.
A large amount of studies confirm that, uses some chemical substances malignant cell is reversed, and breaks up to normal cell; These can make malignant cell to the material that normal cell transforms, and are called as " differentiating inducer ".Under " differentiating inducer " effect, the tumor cell that dedifferentes can be induced to the differentiation of normal cell direction, thereby shows Normocellular biological property, even can be transformed into normal cell fully.Different being of this " induction treatment " and traditional treatment measure maximum is not by killing and wounding oncocyte, reaches the purpose of treatment tumor but be converted to normal cell by the guiding malignant cell.
The spontaneous differentiation of tumor cell reaches the discovery of being induced differentiation under the effect of some chemical substance, makes people strive to find the differentiating inducer of tumor cell.Since 1971 find that dimethyl sulfoxine (DMSO) can make the differentiation of Frind erythroleukemia cell, found that successively kinds of tumor cells can be induced differentiation by some chemical compound.As: suppress the propagation of retinoblastoma cell under r-interferon 100-1000 μ/ml concentration and induce differentiation; Ganglioside GM
3Can inducing early, children's grain leukemia HL-60 cell breaks up to mononuclear cell; Butanoic acid induces the K562 cell to break up to the erythrocyte direction under 1400 μ M concentration; Hexamethylene bisacetamide (HMBA) is 5 * 10
-3Can induce the Frind erythroleukemia cell differentiation more than 90% under the M concentration; Ginsenoside Rh
2Have the function of obvious inducing mouse B16 cell differentiation, cell is blocked at G
1Phase and can not breeding; R8923 mainly is made up of hexamethylene ethylene lateral chain and polar group, inducing early, children's grain leukemia HL-60 cell breaks up to ripe granulocyte, the U937 cell is broken up to macrophage, and nearest clinical research proves that retinoic acid has obvious curative effects to acute promyelocytic leukemic.
Natural abscisic acid [(+)-cis, trans-Abscisic Acid, ABA] be a kind of plant endogenous degeneration-resistant material, play important effect in the growth of plant, in growing, its micro-natural being present in the various plants, comprise various cereal crops, vegetable, melon and fruit, nut etc. are mainly obtained by fungus scale fermenting and producing at present.The molecular formula of ABA is C
15H
20O
4, molecular weight 264.33, its chemical name is: 5-(1 '-hydroxyl-2 ', 6 ", 6 "-trimethyl-4 '-oxo-2 '-cyclohexene-1 '-methyl)-3-methyl-2-is suitable-and 4-is anti--pentadienoic acid, chemical structural formula is as follows:
ABA be a kind of be the sesquiterpene carboxylic acid of ultimate unit with the isoprene, very similar to the retinoic acid chemical constitution, all be to constitute by cyclohexene, side chain and a polar group; ABA is a kind of derivant of carotenoid, but carotenoid metabolism in human body is a retinoic acid.As everyone knows, R8923 is human the most effective, the most deep class differentiating inducer of finding of Study on mechanism, but owing to obvious toxic and side effects has limited its Application and Development.ABA has high similarity with retinoic acid on 26S Proteasome Structure and Function, and proves through the toxicology test of strictness, at maximal dose (SD rat LD
50＞5000mg/Kg) time,, thereby be expected to become effective differentiating inducer for the treatment of human malignancies to biological and environment is without any side effects.
Three, summary of the invention:
We find that in research process natural abscisic acid has the function of differentiating inducer to kinds of tumor cells, promptly can induce malignant cell to transform to normal cell, so finished the present invention.
The objective of the invention is to disclose the new purposes that a kind of natural abscisic acid is used to prepare tumor cell " differentiating inducer " medicine.
Natural abscisic acid is used to prepare effective working concentration of tumor cell " differentiating inducer " medicine, generally in 5g/L-1 * 10
-6G/L is preferably 2g/L-1 * 10
-3G/L, the best is 1g/L-5 * 10
-2G/L can be by being lower than under 50 ℃ the temperature preparation of conventional formulation method; The medicine of its preparation can be peroral dosage form, exterior-applied formulation and instillation dosage form, and model of action can be induced differentiation in vivo for live body injection or for oral administration; Also can external application be coated with outward etc. or be used for tumor cell tissue and cultivate, carry out external evoked differentiation.
Studies show that ABA can make cancerous cell change to the normal cell direction, its mechanism of action is: ABA makes a large amount of propagation phase cells be stuck in the S phase, makes it realize cell division and has suppressed the propagation of tumor cell, inducing apoptosis of tumour cell simultaneously effectively; ABA can act on tumor cell inner cell framing structure, changes the malignant proliferation ability of cancerous cell, induces it to develop to the normal cell direction, plays the effect of anticancer propagation; Use Research of Animal Model for Study, find that ABA can suppress the generation of tumor cancer week blood vessel preferably, thereby play the effect that suppresses tumor growth; Find that simultaneously ABA can reduce the expression of tumor cell Ki67, its proliferation activity is lowered, confirm that therefore ABA has the interior inducing tumor cell of body transformed and suppressed effect from its propagation to normal cell.
The present invention has the following advantages:
1, induction is effective, energy good restraining cancerous cell and growth of tumor;
2, natural abscisic acid belongs to natural drug, and little to normal cell injury, toxic and side effects is very little;
3, easy to use, simple to operate.
Four, the specific embodiment:
Embodiment 1: natural abscisic acid is studied the induction of prostate gland cancer cell:
The carcinoma of prostate DU-145 cell that will be in exponential phase is divided into two groups of experimental group, matched groups, and experimental group adds 20ul2 * 10
-2G/L ABA (being dissolved in dehydrated alcohol) makes that the ABA final concentration is I0 in the culture fluid
-2G/L, matched group adds the 20ul dehydrated alcohol, and the medium and small bovine serum concentration of culture fluid is 130mI/L, the conventional method cultured cell.Routine replacement culture fluid behind the dosing 48h, (ABA concentration is I0 to keep identical drug level
-2G/L).In the incubation, the inversion optical microscope dynamic observes the change on the DU-145 cellular morphology.
The result:
Cellular morphology and Ultrastructural observation: the DU-145 cell of centrifugal collection cultivation and ABA effect 36h, 0.01molL
-1The PH7.4PBS washed twice is fixed with 3% glutaraldehyde, after osmic acid is fixing, and dehydration, embedding, ultrathin section, uranium acetate and lead citrate dyeing, the change of transmission electron microscope observation tumor cell form and structure.Found that it is not good that growth conditions appears in tumor cell, the karyopycnosis phenomenon increases, and organelle quantity reduces, and tonofibril disappears, and it is big that cell compartment becomes, and phenomenon such as the acellular vitellarium of strip occurs.Cellular control unit is evenly distributed, and size is consistent, shows no obvious abnormalities.
Flow cytometer detects and analyzes: behind the ABA effect carcinoma of prostate DU-145 cell 72h, collect the experimental group and the cellular control unit of tumor cell, with 0.01molL
-1The PH7.4PBS washed twice is fixed with 70% ice ethanol, places 4 ℃ of refrigerators, recentrifuge before detecting is abandoned supernatant, the PBS washed twice, 4 ℃ of following bromination third ingot dyeing 30min are with variation, analysis of cells apoptosis and the cell cycle race conditio of cells were tested by flow cytometry dna content.
ABA effect back tumor cell flow cytometer testing result shows: behind the experimental group ABA effect DU-145 cell 72h, the DU-145 cell detects through flow cytometer and typical hypodiploid " apoptosis small peak " occurs, the apoptosis of tumor cells rate is up to 32%, a large amount of propagation phase cells are stuck in the S phase, make it can't realize cell division, the carrying out of blocking-up cell cycle, thereby the propagation of inhibition DU-145 cell, inducing tumor cell generation apoptosis.Matched group flow cytometer testing result Non Apparent Abnormality is found.
Embodiment 2: natural abscisic acid is studied the induction of children's grain leukemia HL-60 cell morning:
Children's grain leukemia HL-60 cell morning that will be in exponential phase is divided into two groups of experimental group, matched groups, and experimental group adds 10ul 5 * 10
-1G/L ABA (being dissolved in dehydrated alcohol) makes that the ABA final concentration is 1 * 10 in the culture fluid
-1G/L, matched group adds the 10ul dehydrated alcohol, and the medium and small bovine serum concentration of culture fluid is 130mI/L, the conventional method cultured cell.Routine replacement culture fluid behind the dosing 48h, (ABA concentration is 1 * 10 to keep identical drug level
-1G/L).In the incubation, transmission electron microscope dynamic observes the change on the HL-60 cellular morphology.
The HL-60 cell of centrifugal collection cultivation of cellular morphology and Ultrastructural observation and ABA effect 24h is used 0.01molL
-1The PH7.4PBS washed twice is fixed with 3% glutaraldehyde, after osmic acid is fixing, and dehydration, embedding, ultrathin section, uranium acetate and lead citrate dyeing, the change of transmission electron microscope observation tumor cell form and structure.
Behind the ABA effect HL-60 cell 24h, the microvillus of transmission electron microscope observation tumor cells showed nuclear membrane disappears, and organelle quantity reduces, and tonofibril disappears, and may alleviate relevant with the tumor cell malignant phenotype; And typical apoptosis ultrastructure feature appears in the part tumor cell.And chromatin is evenly distributed in the corresponding cellular control unit nuclear of Electron microscope showed, and kernel exists, and shows no obvious abnormalities.
Flow cytometer detects and analyzes HL-60 experimental group and the matched group tumor cell of collecting ABA effect 12h and 24h, with 0.01molL
-1The PH7.4PBS washed twice is fixed with 70% ice ethanol, places 4 ℃ of refrigerators, recentrifuge before detecting is abandoned supernatant, the PBS washed twice, 4 ℃ of following bromination third ingot dyeing 30min are with variation, analysis of cells apoptosis and the cell cycle race conditio of cells were tested by flow cytometry dna content.
Testing result shows: behind the ABA effect HL-60 cell, can be synthetic by the DNA that influences tumor cell, and make a large amount of propagation phase cells be stuck in the S phase, make it can't realize cell division, the carrying out of blocking-up cell cycle, thereby the propagation of inhibition cell; Simultaneously, by raising the cAMP/PKA approach, the inducing cancer cell differentiation enters programmed cell death, effectively inducing tumor cell generation apoptosis.The equal Non Apparent Abnormality of matched group flow cytometer testing result is found.
Embodiment 3.
3. natural abscisic acid becomes the influence of tumor ability to transplanted tumor in nude mice
1). material
(1) 3 the week age Balb/c.nu nude mouse, about the about 20g of average weight, be male.
(2) nude mice SPF raises and uses barrier system.
(3) human mouth cancer Tca8113 cell strain 2.
2) method
2.1) the inoculating cell preparation
Cultivate the Tca8113 cell to exponential phase according to test one method, the tumor cell of adherent growth is made it to take off wall, dispersion with Digestive system, with serum-free medium centrifuge washing 2 times, living cell counting is adjusted into 2 * 10 with PBS liquid
7/ ml cell suspension.Extract an amount of tumor cell suspension inoculation nude mice with syringe with No. 6 syringe needles, 1 position of every animal inoculation (being inoculated in the oxter of nude mice), each position injection 0.2ml tumor cell suspension contains living cells 4 * 10
6
2.2) drug treating
When treating that tumor proliferation can know that extremely touching tumor ties (tumor knot Φ 2-4mm), animal is divided into matched group and treatment group at random, 3 of matched groups, 4 of treatment groups.The grouping administration: ABA treatment group, through intraperitoneal injection, 2 times/week, 20mg/kg/ time; Matched group, PBS liquid replaces ABA, and medication is identical with experimental group.In 4 weeks of successive administration, totally 8 times, take off neck and put to death nude mice, termination test.Cut after the tumor piece weighs, tumor tissues is divided into several piece, use formalin fixed person, be used for HE dyeing histological observation, diagnosis and SABC and detect; With the fixing person of 3% glutaraldehyde, be used for the transmission electron microscope observing ultrastructural change.
2.3) calculating inhibition rate of tumor growth (%)
Put to death animal and carry out the dissection of tumor body, it is heavy that electronic balance (precision 0.0001g) takes by weighing and write down tumor, calculates tumour inhibiting rate, and formula is as follows:
The average tumor of average tumor weight/matched group heavy * 100% is organized in the average tumor weight-treatment of inhibition rate of tumor growth (%)=matched group
2.4) histological observation
Observe tumor tissue histological characteristic and cancer week blood vessel situation of change
3) result
(1). the histological observation result
In the nude mice tumor tissue piece, cell characteristics and archeocyte strain are in full accord, can clarify a diagnosis to be the transplanted tumor due to people's oral cancer Tca8113 cell strain.In organizing in matched group tumor week, have a large amount of new vesselses to form, experimental group is then than the obvious minimizing of matched group.
(2). when experiment finishes, find that the metastatic tumor tumor piece of ABA experimental group animal is significantly less than the tumor piece (Fig. 4-1, Fig. 4-2) of PBS control animals.4 transplanted tumor in nude mice tumors of experimental group heavily are respectively: 1.9890g, 2.3515g, 2.4053g, 2.6804g, and average tumor heavily is 2.3565g; 3 nude mice tumors of PBS matched group heavily are respectively 4.5420g, 4.6053g, 4.8734g, and average tumor heavily is 4.6736g.The average tumor of experimental group animal is heavy obviously heavy greater than the average tumor of control animals, and credit is analysed by statistics, and difference between the two has bright significance (P＜0.05); Calculating ABA is 49.6% to the growth inhibition ratio of transplanted tumor.
(3). be covered with the sepia positive particle in the nucleus of a large amount of cells of matched group, the distribution of particles inequality, nucleus is deep mixed, has the cell more than 2/3 to be the Ki67 positive expression in each high power field approximately; In each high power field of experimental group, 1/4 the nucleus of only having an appointment presents the sepia positive expression, and positive particle distributes consistent.
Claims (5)
1, natural abscisic acid is used to prepare the new purposes of tumor cell " differentiating inducer " medicine.
2, natural abscisic acid according to claim 1 is used to prepare the new purposes of tumor cell " differentiating inducer " medicine, it is characterized by: effective working concentration of natural abscisic acid is generally in 5g/L-1 * 10 in the tumor cell of preparation " differentiating inducer " medicine
-6G/L.
3, natural abscisic acid according to claim 2 is used to prepare the new purposes of tumor cell " differentiating inducer " medicine, it is characterized by: effective working concentration of natural abscisic acid is preferably 2g/L-1 * 10 in the tumor cell of preparation " differentiating inducer " medicine
-3G/L, the best is 1g/L-5 * 10
-2G/L.
4, natural abscisic acid according to claim 1 is used to prepare the new purposes of tumor cell " differentiating inducer " medicine, it is characterized by: the medicine of its preparation can be peroral dosage form, exterior-applied formulation and instillation dosage form.
5, natural abscisic acid according to claim 1 is used to prepare the new purposes of tumor cell " differentiating inducer " medicine, it is characterized by: be lower than under 50 ℃ the temperature preparation of conventional formulation method.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8389024B2 (en) | 2005-06-22 | 2013-03-05 | Gonzalo Romero M. | Abscisic acid against cancer |
| JP2013542925A (en) * | 2010-09-17 | 2013-11-28 | バレント・バイオサイエンシーズ・コーポレイション | Pharmaceutical and dietary supplement abscisic acid compositions |
| WO2019053673A1 (en) | 2017-09-18 | 2019-03-21 | Euromed, S.A. | Method for preparing a botanical extract of abscisic acid |
-
2004
- 2004-09-14 CN CNA2004100406825A patent/CN1748674A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8389024B2 (en) | 2005-06-22 | 2013-03-05 | Gonzalo Romero M. | Abscisic acid against cancer |
| JP2013542925A (en) * | 2010-09-17 | 2013-11-28 | バレント・バイオサイエンシーズ・コーポレイション | Pharmaceutical and dietary supplement abscisic acid compositions |
| EP2616059A4 (en) * | 2010-09-17 | 2014-03-26 | Valent Biosciences Corp | Pharmaceutical and nutraceutical compositions of abscisic acid |
| WO2019053673A1 (en) | 2017-09-18 | 2019-03-21 | Euromed, S.A. | Method for preparing a botanical extract of abscisic acid |
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