CN105194550A - Compound traditional Chinese medicine composition and application thereof to resistance to prostate cancer - Google Patents

Compound traditional Chinese medicine composition and application thereof to resistance to prostate cancer Download PDF

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CN105194550A
CN105194550A CN201510664970.6A CN201510664970A CN105194550A CN 105194550 A CN105194550 A CN 105194550A CN 201510664970 A CN201510664970 A CN 201510664970A CN 105194550 A CN105194550 A CN 105194550A
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CN105194550B (en
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易正芳
胡美纯
刘明耀
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East China Normal University
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Abstract

The invention discloses a compound traditional Chinese medicine composition and application thereof to resistance to prostate cancer. The compound traditional Chinese medicine composition is mainly prepared from the following traditional Chinese medicine raw materials in parts by weight: 4-12 parts of bark of ailanthus altissima, 12-30 parts of spreading hedyotis herb, 3-15 arts of curcuma zedoary, 15-30 parts of salvia miltiorrhiza, 5-15 parts of selfheal and 4-12 parts of liquorice. According to the compound traditional Chinese medicine composition having a prostate cancer-resistant effect, a raw material composition formula is selected according to the traditional Chinese medicine theory and pathogenesis of prostate cancer, all components are scientific and reasonable in ratio, and pharmacological experiment and preclinical experiment results show that the compound traditional Chinese medicine composition provided by the invention has a good prostate cancer-resistant effect, not only can obviously restrain the proliferation and growth of the prostate cancer, but also can restrain migration, invasion and transfer of the prostate cancer, obviously improve the survival quality of tumor-bearing mice, and improve all immune indexes of the tumor-bearing mice, is convenient and rapid in administration route, has no obvious adverse effects, can be developed to be a new-generation prostate cancer-resistant drug and has high potential of becoming a good prostate cancer treatment prescription.

Description

A kind of Traditional Chinese medicine compound composition and the application in anti-prostate cancer thereof
Technical field
The present invention relates to medical art, be specifically related to a kind of Traditional Chinese medicine compound composition and preparing the application in anti-prostate cancer.
Background technology
Carcinoma of prostate (Prostatecancer, PCa), as the modal malignant tumor of male reproductive system, is a kind of disease being apt to occur in elderly men, and its sickness rate increases with the age and increases.The sickness rate of carcinoma of prostate distributes very inconsistent in worldwide.There are more than 90 ten thousand new cases in the annual whole world, and wherein 260,000 multiple-cases are dead because of the deterioration of carcinoma of prostate.The sickness rate of American-European countries's carcinoma of prostate is apparently higher than area, Asia and Africa.In the U.S., carcinoma of prostate accounts for first of American male tumor incidence, the second of fatality rate.In China, the sickness rate of carcinoma of prostate increased 70% more in the past, occupy the first place of China's male reproductive system Incidence rate of increase, sickness rate is increased to 7.9 people/100,000 male demographic of 2005 from 1.71 people/100,000 male demographic of 1993, and rises in the growth rate with annual 10%.According to the bibliographical information of " Journal of Clinical Oncology " the 04th phase in 2013 about Chinese carcinoma of prostate disease currency and Analysis on Epidemic Trend, the sickness rate of China's carcinoma of prostate presents obvious sustainable growth trend, and carcinoma of prostate is just becoming the urinary system malignant tumor having a strong impact on China's men's health.Coming 10 years, along with Chinese society ageing phenomenon is on the rise, the sickness rate of China's carcinoma of prostate is maybe by increase by more than 1 times, and the sickness rate of carcinoma of prostate may enter peak period.The clinical manifestation of carcinoma of prostate is common is dysuria, in incrementally occurring the symptoms such as frequent micturition, urgent micturition, hematuria and with the continuous pain at pain when urinating or burning sensation and back of the body bottom, upper leg portion or pelvis place.Prostate cancer hour without any clinical manifestation, often to occur that metastasis symptom is medical reason the earliest, is therefore depended merely on clinical symptoms and is difficult to early diagnosis.Because carcinoma of prostate is longer for incubation period, be difficult to discover, during discovery, generally develop into advanced prostate cancer, cause serious adverse consequences to patient.
The effect of Chinese medicine prevention tumor is already by clinical confirmation.The traditional Chinese medical science is to the treatment of carcinoma of prostate, and " General Treatise on the Cause and Symptoms of Diseases " as far back as Han dynasty Zhang Zhongjing " Medical Treasures of the Golden Chamber " and Sui Dynasty's Chao Yuanfang (550-630 A.D.) just touches upon, described in ancient Chinese medicine doctor respectively has thereafter.According to main clinical manifestation and the sign of primary disease, carcinoma of prostate is often included into " stranguria ", " hematuria ", " gathering ", “ mass in the abdomen by Chinese medicine " etc. disease category.Kidney qi virtual loss, blood stasis lose essence, build up the part of the body cavity below the umbilicus, housing the bladder, kidneys and bowels is the main etiology and pathogenesis of carcinoma of prostate.The Jing-Yue Complete Works of Ming Dynasty's Zhang Jingyue has pointed out the method for the treatment of such as heat-clearing and toxic substances removing, blood circulation promoting and blood stasis dispelling, hardness softening and stasis-eliminating, warming YANG to promote diuresis more all sidedly.Modern medicine man determines determination for the treatment of based on pathogenesis obtained through differentiation of symptoms and signs in a large amount of clinical practice, attack evil righting distinguish the pattern of controlling, in cooperation operation, radiotherapy, chemotherapy, endocrine therapy process, play potentiation, reduce toxic reaction and untoward reaction, the clinical practice of determination for the treatment of based on pathogenesis obtained through differentiation of symptoms and signs carcinoma of prostate is extensive.For the etiology and pathogenesis that carcinoma of prostate is total, damp invasion of lower energizer, phlegm and blood stasis, the hepatic and renal YIN deficiency, QI and blood deficiency four kinds of pattern of syndrome adopt by clinical research.Many scholars use the folk prescriptions such as some clinical proved recipes and compound treatment or combine western medical treatment carcinoma of prostate, all can improve curative effect and the quality of life of patient to a certain extent, mitigation symptoms, improve muscle power, reduce serum PSA (Prostatespecificantigen, PSA) level, occur the time that androgen independence is resisted after postponing anti-androgen therapy, the quality of life of patient is able to obvious improvement.The research of bibliographical information Traditional Chinese Medicine Function for cell strain in the research of carcinoma of prostate is in recent years more, new mechanism of action, adjustment path are constantly set forth, deepen the mechanism of Chinese medicine carcinoma of prostate to a certain extent, specify that the curative effect of Chinese medicine carcinoma of prostate.Patients with terminal loses operation, radiotherapy, chemotherapeutic machine meeting, and simple Chinese medicine palliates the agonizing sufferings to reach, and extends life cycle, improves the object of quality of life.Chinese medicine compound, because of its significant clinical efficacy, plays the effect that can not be substituted in the treatment of many cancers such as carcinoma of prostate, at raising patients ' life quality, delays to have huge application potential and development prospect in neoplasm metastasis and progress.Seek effective, low toxicity Chinese medicine, particularly centering advanced refractory patients with prostate cancer, there is far-reaching clinical meaning.Therefore necessary under the guidance of traditional Chinese medicine theory, research and develop that a kind of anti-prostate cancer is effective, at the bottom of untoward reaction, reasonable price, the compound Chinese medicinal preparation easily applied.
Growth and the transfer of carcinoma of prostate depend on androgen closely.Androgenic secretion about 90% comes from testis, and about 10% comes from adrenal gland.Enter cell after androgen (as dihydrotestosterone, Dihydrotestosterone, DHT) is secreted to combine with androgen receptor (Androgenreceptor, AR).Androgen receptor is a kind of important nuclear factor, regulates and controls numerous gene expression and the propagation of prostate gland cancer cell.After androgen is combined with androgen receptor, androgen receptor enters nucleus promotor gene and expresses (as the important indicator of examination carcinoma of prostate---PSA), thus promotes growth and the transfer of carcinoma of prostate.Therefore, emasculation hormone therapy (comprising castration and Testectomy and Drug inhibition androgen levels) has obvious curative effects for most of carcinoma of prostate.But emasculation hormone therapy generally only can maintain 1.5 to 4.0 years to the control of tumor, nearly all patients with prostate cancer finally all transfers Androgen Independent Prostate Cancer (Androgen-independentprostatecancer to, AIPC), and then develop into hormone-refractory prostate cancer (Hormonerefractoryprostatecancer, HRPC), also castration-resistant prostate cancer (Castrationresistantprostatecancer, CRPC) is claimed.Now, emasculation hormone therapy also just loses effect.CRPC is the major reason causing prostate cancer patient's death.The generator of CRPC is shaped with a variety of, comprises androgen receptor sudden change, expression of androgen receptor disappears and the cell mass amplification etc. of androgen insensitivity under low androgen milieu.In other words, although emasculation hormone therapy does not have effect, but androgen receptor plays important role too in most of castration-resistant prostate cancer, so androgen receptor is the important target spot of the general carcinoma of prostate for the treatment of and castration-resistant prostate cancer at present.
Current global medical circle does not still have a kind of medicine can cure carcinoma of prostate separately.The essential therapeutic arsenals of carcinoma of prostate comprises operation, radiotherapy, chemotherapy and Biotherapeutics, not only patient will experience a narrow escape from death pain, household economy pressure is huge, debt-ridden, and people are least receptible to be final after ill relatives bear considerable distress or will to experience the hardship of life and death departure.Food and drug administration (Foodanddrugadministration, FDA) medicine of the most newly approved treatment CRPC comprises abiraterone (Abiraterone) and MDV3100 (Enzalutamide), the former suppresses androgenic synthesis, the latter is the antagonist of androgen receptor, these two kinds of medicines are all for androgen receptor signal path, significant curative effect is obtained in the treatment of castration-resistant prostate cancer, but after being through above-mentioned endocrine therapy and targeting androgen receptor Drug therapy, patient still can produce drug resistance, therapeutic effect is very limited, and somewhat expensive, the ability to shoulder economically of patient is poor.In addition, the side effect caused by chemicotherapy is also the bottleneck of clinical treatment.As one of the important means of carcinoma of prostate Comprehensive Treatment, Chinese medicine, because of its significant clinical efficacy, plays the effect that can not be substituted in the treatment of many cancers such as carcinoma of prostate.The Chinese material medicine resource abundant in conjunction with China's reserves and time-honored Chinese medical theory, develop for carcinoma of prostate drug resistance, the newtype drug combination that has China's independent intellectual property right has very great clinical meaning to treatment carcinoma of prostate.
Summary of the invention
Technical problem to be solved by this invention is to overcome the deficiencies in the prior art, provide a kind of evident in efficacy, toxic and side effects is little, the Traditional Chinese medicine compound composition with anti-prostate cancer effect of taking convenience, preparation method and the application before the treatment in row adenocarcinoma field thereof.
The present invention proposes. a kind of Traditional Chinese medicine compound composition, comprises the Chinese crude drug of following parts by weight: ailanthus altissima (mill.) swingle skin 4 ~ 12 parts, Herba Hedyotidis Diffusae 12 ~ 30 parts, Rhizoma Curcumae 3 ~ 15 parts, Radix Salviae Miltiorrhizae 15 ~ 30 parts, Spica Prunellae 5 ~ 15 parts, 4 ~ 12 parts, Radix Glycyrrhizae.
Wherein, preferably, described Traditional Chinese medicine compound composition, comprises the Chinese crude drug of following parts by weight: ailanthus altissima (mill.) swingle skin 6 ~ 9 parts, Herba Hedyotidis Diffusae 15 ~ 24 parts, Rhizoma Curcumae 4 ~ 12 parts, Radix Salviae Miltiorrhizae 18 ~ 21 parts, Spica Prunellae 6 ~ 10 parts, 6 ~ 9 parts, Radix Glycyrrhizae.
The invention allows for the preparation method of Traditional Chinese medicine compound composition described above, comprise the following steps:
(1) soak: get Six-element Chinese crude drug described in claim 1 or 2, add distilled water immersion, obtain crude drug distilled water mixture;
(2) decoct: the crude drug distilled water mixture described in step (1) is decocted, obtains Chinese medicine crude extract;
(3) concentrated: Chinese medicine crude extract step (2) prepared concentrates, and obtains the concentrated liquid of tradition Chinese medicine;
(4) degerming: to get the concentrated liquid of tradition Chinese medicine that step (3) prepares degerming by filtering with microporous membrane, namely obtain described Traditional Chinese medicine compound composition.
The invention allows for the application described Traditional Chinese medicine compound composition being applied to preparation treatment malignant tumor medicine.
Wherein, described malignant tumor is that the abnormal amplification of androgen receptor and sudden change cause, and comprises carcinoma of prostate, breast carcinoma, pulmonary carcinoma, colon cancer, the brain cancer, skin carcinoma, bladder cancer, renal carcinoma.
Present invention further proposes the purposes that described Traditional Chinese medicine compound composition is applied to preparation treatment carcinoma of prostate medicine.
Present invention further proposes described Traditional Chinese medicine compound composition and be applied to the purposes of apoptosis suppressing the growth of prostate gland cancer cell, propagation, transfer, migration, invasion and attack and promotion prostate gland cancer cell.
Wherein, described carcinoma of prostate is castration-resistant prostate cancer or androgen-dependent prostate cancer.
Present invention further proposes the purposes of the transcriptional activity of the transcriptional activity of the androgen receptor described Traditional Chinese medicine compound composition being applied to the induction of vitro inhibition dihydrotestosterone and the androgen receptor of disappearance ligand domain, or for vitro inhibition estrogen receptor activity.
The invention allows for the medicine described Traditional Chinese medicine compound composition and pharmaceutically acceptable carrier being made injectable fluid, aerosol, emulsifiable paste, gel, pill, syrup, transdermal patch, capsule, microcapsule, tablet, granule or oral liquid formulation.
Traditional Chinese medicine compound composition of the present invention can be used alone as medicine or with other drug conbined usage.
The invention allows for described Traditional Chinese medicine compound composition, for the purposes of the propagation of the Prostatic cancer cell lines 22RV1 of vitro inhibition androgen receptor sudden change, the propagation of suppression androgen-dependent prostate JEG-3 LNCaP.
The invention allows for described Traditional Chinese medicine compound composition, for the purposes of the transfer of vitro inhibition prostate gland cancer cell, or the purposes of the migration of vitro inhibition prostate gland cancer cell PC-3 and LNCaP and 22RV1, or the purposes of the transcriptional activity of the androgen receptor of the transcriptional activity of the androgen receptor of vitro inhibition dihydrotestosterone induction and disappearance ligand domain, or vitro inhibition estrogen receptor activity, suppress prostate carcinoma cell growth, the purposes of the apoptosis of migration and invasion and promotion prostate gland cancer cell, or the purposes of androgen receptor mutant clone 22RV1 cell proliferation that vitro inhibition bicalutamide or MDV3100 fail to respond to any medical treatment.
The invention allows for described Traditional Chinese medicine compound composition, the propagation of the Prostatic cancer cell lines 22RV1 suddenlyd change for suppressing androgen receptor in body, suppress the propagation of androgen-dependent prostate JEG-3 LNCaP, or for suppressing the transfer of prostate gland cancer cell in body, or for suppressing growth and the transfer of castration-resistant prostate cancer in vivo, or for suppressing estrogen receptor activity, suppress prostate carcinoma cell growth, the apoptosis of migration and invasion and promotion prostate gland cancer cell, or the androgen receptor mutant clone 22RV1 cell proliferation for suppressing bicalutamide or MDV3100 to fail to respond to any medical treatment, or for suppressing the growth of prostate gland cancer cell 22RV1 in Mice Body, or for suppressing the transfer of prostate gland cancer cell 22RV1 in Mice Body.
The invention allows for the application of being increased in the medicine of relevant malignancy disease for the preparation for the treatment of and androgen receptor climate abnormality by described Traditional Chinese medicine compound composition.
Wherein, described malignant tumor is the application in the propagation of the abnormal amplification of androgen receptor and the malignant cell caused that suddenlys change, growth, migration and infiltration; Wherein, described malignant tumor comprises carcinoma of prostate, breast carcinoma, pulmonary carcinoma, colon cancer, the brain cancer, skin carcinoma, bladder cancer, renal carcinoma.
The Traditional Chinese medicine compound composition that the present invention mentions, is made up of the Chinese crude drug of following parts by weight: ailanthus altissima (mill.) swingle skin 4 ~ 12 parts, Herba Hedyotidis Diffusae 12 ~ 30 parts, Rhizoma Curcumae 3 ~ 15 parts, Radix Salviae Miltiorrhizae 15 ~ 30 parts, Spica Prunellae 5 ~ 15 parts, 4 ~ 12 parts, Radix Glycyrrhizae.
Wherein, preferably, described Traditional Chinese medicine compound composition, is made up of the Chinese crude drug of following parts by weight: ailanthus altissima (mill.) swingle skin 6 ~ 9 parts, Herba Hedyotidis Diffusae 15 ~ 24 parts, Rhizoma Curcumae 4 ~ 12 parts, Radix Salviae Miltiorrhizae 18 ~ 21 parts, Spica Prunellae 6 ~ 10 parts, 6 ~ 9 parts, Radix Glycyrrhizae.
In the present invention's application, the Traditional Chinese medicine compound composition provided suppresses the transcriptional activity of androgen receptor.The Traditional Chinese medicine compound composition provided suppresses the propagation of the Prostatic cancer cell lines of androgen receptor sudden change.The propagation of its Prostatic cancer cell lines 22RV1 suppressing androgen receptor to suddenly change in vitro and in vivo, the propagation of suppression androgen-dependent prostate JEG-3 LNCaP.
In the present invention's application, the Traditional Chinese medicine compound composition provided suppresses the transfer of prostate gland cancer cell in vitro and in vivo.
In the present invention's application, the Traditional Chinese medicine compound composition provided suppresses transcriptional activity and the prostate carcinoma cell growth of androgen receptor.
In the present invention's application, the Traditional Chinese medicine compound composition provided suppresses the prostate gland cancer cell propagation of the primary separation of patient's tumor tissues source property.
In the present invention's application, the Traditional Chinese medicine compound composition provided suppresses the Clone formation of prostate gland cancer cell.
In the present invention's application, the Traditional Chinese medicine compound composition induced androgen receptor provided expresses the apoptosis of positive cell strain LNCaP and 22RV1.
In the present invention's application, the Traditional Chinese medicine compound composition provided suppresses growth and the transfer of castration-resistant prostate cancer cell in mouse in vivo models.
In the present invention's application, the androgen receptor mutant clone 22RV1 cell proliferation that the Traditional Chinese medicine compound composition provided suppresses bicalutamide or MDV3100 to fail to respond to any medical treatment.
In the present invention's application, transfer in the body of the Traditional Chinese medicine compound composition vitro inhibition ptostate cancer PC 3 cell line provided and the migration of LNCaP cell and 22RV1 cell.
In the present invention's application, the Traditional Chinese medicine compound composition provided suppresses the growth of the Prostatic cancer cell lines of Prostatic cancer cell lines LNCaP, 22RV1, PC-3 and the primary separation of patient's tumor tissues source property.
In the present invention's application, Traditional Chinese medicine compound composition is provided to produce inhibiting application and method to androgen receptor.The Traditional Chinese medicine compound composition provided is by blocking the protein expression level of androgen receptor thus significantly lowering the transcriptional activity of androgen receptor and suppress its downstream gene expression.
In the present invention's application, the activity of the luciferase reporter gene that the Traditional Chinese medicine compound composition provided suppresses androgen receptor to regulate and control in vitro, can the propagation of strong inhibition prostate gland cancer cell and migration in vitro and in vivo, and can effectively block androgen receptor signal path.
In the present invention's application, the Traditional Chinese medicine compound composition provided efficiently can not only suppress the propagation of androgen-dependent prostate JEG-3 LNCaP, and the Prostatic cancer cell lines 22RV1 that androgen receptor can be suppressed to suddenly change (the sudden change androgen receptor of this cell strain expression sustained activation, such as, and to clinical medicine androgen receptor antagonists, bicalutamide and MDV3100 etc. have resistance) propagation.In addition, the present invention demonstrates the transfer of provided Traditional Chinese medicine compound composition suppression carcinoma of prostate from vitro and in vivo.Further experiment proves that the Prostatic cancer cell lines of expressing androgen receptor has good sensitivity to Traditional Chinese medicine compound composition provided by the present invention.This Traditional Chinese medicine compound composition suppresses the protein expression level of androgen receptor and then inhibits the transcriptional activity of androgen receptor and the growth of prostate gland cancer cell.Traditional Chinese medicine compound composition provided by the present invention not only can treat early stage androgen-dependent prostate cancer, also can treat castration-resistant prostate cancer in late period.
The application with the Traditional Chinese medicine compound composition of anti-prostate cancer effect provided by the invention, can make the medicine of injectable fluid, aerosol, emulsifiable paste, gel, pill, syrup, transdermal patch, capsule, microcapsule, tablet, granule or oral liquid formulation by the extract of Traditional Chinese medicine compound composition and pharmaceutically acceptable carrier.
Having the Traditional Chinese medicine compound composition of anti-prostate cancer effect and carrier lactose or corn starch when the Traditional Chinese medicine compound composition with anti-prostate cancer effect provided by the invention makes tablet, magnesium stearate lubricant is added when needing, mix homogeneously, is then pressed into tablet.There is the Traditional Chinese medicine compound composition of anti-prostate cancer effect and carrier lactose or corn starch mix homogeneously, granulate when the Traditional Chinese medicine compound composition with anti-prostate cancer effect provided by the invention makes capsule, then encapsulatedly make capsule.When granule made by the compound recipe with anti-prostate cancer effect provided by the invention, there is the Traditional Chinese medicine compound composition of anti-prostate cancer effect and diluent lactose or corn starch mix homogeneously, granulate, dry, make granule.
The invention also discloses the application that the provided Traditional Chinese medicine compound composition with anti-prostate cancer effect or its pharmaceutically acceptable pharmaceutical preparation are increased in the medicine of relevant malignancy disease to androgen receptor climate abnormality in preparation treatment.
In order to realize above object, the Traditional Chinese medicine compound composition with anti-prostate cancer effect provided by the invention, it is made up of the raw material of following parts by weight: ailanthus altissima (mill.) swingle skin 4 ~ 12 parts, Herba Hedyotidis Diffusae 12 ~ 30 parts, Rhizoma Curcumae 3 ~ 15 parts, Radix Salviae Miltiorrhizae 15 ~ 30 parts, Spica Prunellae 5 ~ 15 parts, 4 ~ 12 parts, Radix Glycyrrhizae.
Preferably, above-described a kind of Traditional Chinese medicine compound composition with anti-prostate cancer effect, it is made up of the raw material of following parts by weight: ailanthus altissima (mill.) swingle skin 6 ~ 9 parts, Herba Hedyotidis Diffusae 15 ~ 24 parts, Rhizoma Curcumae 4 ~ 12 parts, Radix Salviae Miltiorrhizae 18 ~ 21 parts, Spica Prunellae 6 ~ 10 parts, 6 ~ 9 parts, Radix Glycyrrhizae.
The present invention is under the guidance of theory of Chinese medical science, dialectically to treat, in conjunction with pathogeny and the disease symptom of carcinoma of prostate, with the right supporting anticancer wholistic therapy theory of the benefiting QI and nourishing blood of Chinese traditional treatment carcinoma of prostate, strong kidney toxin expelling, eliminating stasis to stop pain, promoting blood circulation and hemostasis, endocrine regulation, inducing diuresis for treating stranguria syndrome, repercussive eliminating stagnation for the rule for the treatment of carries out composition of prescription.Based on the above rule for the treatment of, through lot of documents retrieval and experimental verification, filter out a kind of Traditional Chinese medicine compound composition with anti-prostate cancer effect of the present invention, selected ailanthus altissima (mill.) swingle skin, Herba Hedyotidis Diffusae, Rhizoma Curcumae, Radix Salviae Miltiorrhizae, Spica Prunellae, Radix Glycyrrhizae 6 taste Chinese medicine carries out prescription: ailanthus altissima (mill.) swingle skin in side, bitter in the mouth, puckery, cold in nature, the merit of tool heat clearing and damp drying, hemostasis with astringents, containing antineoplastic component.Herba Hedyotidis Diffusae, bitter in the mouth, sweet, cold in nature, heat-clearing and toxic substances removing, dampness removing is treating stranguria, has stronger antipyretic and antidote functions, is usually used in the cards such as pyretic toxicity, the effective ingredient such as its oleanolic acid contained, triterpenic acid, Coumarins and polysaccharide and effective site all have antitumor action, and wherein triterpenes antitumous effect is the strongest.Share with ailanthus altissima (mill.) swingle skin and bring out the best in each other, effect high-tensile strength is grand, and the two is monarch drug in side altogether.Rhizoma Curcumae, bitter in the mouth is warm in nature, and the merit of blood circulation promoting and blood stasis dispelling, reducing swelling and alleviating pain is distinguished, promoting flow of QI and blood, stopping pain and expelling blood stasis, and effective ingredient Oleum Curcumae is generally acknowledged anticancer good medicine.Radix Salviae Miltiorrhizae, bitter in the mouth, cold nature, blood circulation promoting and blood stasis dispelling, nourishing blood to tranquillize the mind, removing heat from blood eliminating carbuncle, main containing fat-soluble Diterpenoids from bulbus and water miscible phenolic acid components.Diterpenoids from bulbus tanshinone IIA reports maximum anti-tumor active ingredients in current Radix Salviae Miltiorrhizae.Rhizoma Curcumae and Radix Salviae Miltiorrhizae all have effect of eliminating stasis to stop pain, nourishing blood and promoting blood circulation, are ministerial drug altogether.Spica Prunellae, bitter in the mouth, pungent, cold in nature, be conventional antipyretic, there is effect of liver heat removing and eyesight improving, powder detoxifcation; The merit of its hard masses softening and resolving in this side is obvious, and playing and assist a ruler in governing a country monarch drug softening tumor locus get rid of the effect of carbuncle poison further, is adjuvant drug.Radix Glycyrrhizae, sweet in the mouth, property is put down, invigorating the spleen and replenishing QI, heat-clearing and toxic substances removing, coordinating the actions of various ingredients in a prescription, mainly playing and promotes body's immunity adjustment, recovering the effect of healthy energy, for making medicine in the party.The strict compatibility of the monarch principles of formulating prescriptions pressed by 6 taste medicines, plays effect of benefiting QI and nourishing blood, strong kidney toxin expelling, eliminating stasis to stop pain, promoting blood circulation and hemostasis, endocrine regulation, inducing diuresis for treating stranguria syndrome, repercussive eliminating stagnation altogether.
Traditional Chinese medicine compound composition preparation method of the present invention: the Traditional Chinese medicine compound composition every agent side medicine Six-element Chinese crude drug crude drug dry product some grams getting above-mentioned parts by weight, adds 10 ~ 15 times of volume distilled water immersions 1 ~ 3 hour of Traditional Chinese medicine compound composition medical material amount; Decoct in automatic decocting device for herbal medicine, decocting number of times is 1 to 3 time, decocts 0.5 ~ 1 hour at every turn; The Chinese medicine crude extract prepared evaporation and concentration to final volume of uncapping on automatic decocting device for herbal medicine is 5 ~ 10% of primary extract volume; Chinese medicine decoction mother solution is obtained after obtained the concentrated liquid of tradition Chinese medicine is degerming by 0.22 micrometer Millipore membrane filtration, for subsequent use in 4 degrees Celsius of Refrigerator stores.
The Traditional Chinese medicine compound composition with anti-prostate cancer effect provided by the invention compared with prior art has following advantage:
(1) Traditional Chinese medicine compound composition with anti-prostate cancer effect provided by the invention, choose material combination according to Chinese medical theory and carcinoma of prostate pathogenesis to make, each component proportion is rigorous, heresy is just taken into account, monarch, lifting sink-float, play heat-clearing and toxic substances removing altogether, promoting flow of QI and blood, inducing diuresis for treating stranguria syndrome, hard masses softening and resolving, disappear silt pain relieving, the merit of benefiting QI and nourishing blood, be can treat damp and hot accumulate long-pending, the good recipe of junction type carcinoma of prostate in blood stasis, obviously can not only suppress prostate cancer growth and the transfer of tumor-bearing mice, improve life quality and immune function, extend life cycle, improve biochemical indicator, reduce focus scope, with the medication of chemotherapeutics cyclophosphamide combined, there is the effect of efficacy enhancing and toxicity reducing, and have no adverse reaction, show effect of good anticancer righting.
(2) Traditional Chinese medicine compound composition with anti-prostate cancer effect provided by the invention, by lowering the protein level of androgen receptor, suppress the expression of its downstream gene, block androgen receptor signal path, thus realizing the object suppressing carcinoma of prostate, curative effect of medication is definite, clear mechanism, make new breakthroughs in prostate cancer therapy, the field blank of China's basic dependence on import of current prostate cancer therapy medication and independent intellectual property right medicine scarcity can be filled up to a certain extent.
(3) Traditional Chinese medicine compound composition with anti-prostate cancer effect provided by the invention, preparation method cost is low, environmental protection, can realize industrialized great production.
(4) Traditional Chinese medicine compound composition with anti-prostate cancer effect provided by the invention, can be prepared into various dosage form with pharmaceutically acceptable carrier, facilitate clinical taking, and is expected to be developed to new antiprostate cancer.
Term and definition
Castration-resistant prostate cancer (CRPC): prostate cancer patient is recurred after castration (Testectomy or androgen deprivation reduce androgen), the carcinoma of prostate that castration is invalid is called castration-resistant prostate cancer, and this carcinoma of prostate is the maximum killer threatening patients with prostate cancer.
Prostate gland cancer cell PC-3: the differentiation degree separated from human prostata cancer Bone tumour tumor is low, the prostate gland cancer cell of androgen independence, do not express endogenic androgen receptor, there is moderate metastatic potential, show significant cell carcinoma feature, be widely used in the research of the carcinoma of prostate of hormone refractory.
Prostate gland cancer cell LNCaP: set up the Metastatic Lymph Nodes specimen from patients with prostate cancer in 1980 by Horoszewicz etc., remain the feature of prostate cancer tumor cells and early differentiation function thereof, there is significant epithelial cell characteristic, expressing androgen receptor, is the prostate gland cancer cell typically with androgen-dependent.
Prostate gland cancer cell 22RV1: be by the human prostata cancer epithelial cell line carrying out in androgen-dependent prostate cancer CWR22 cell xenograft to mice being separated after continuous passage.This expression of cell lines prostate specific antigen, depends on androgenic stimulation and grows, having very strong one-tenth tumor ability in nude mice.
Cell proliferation: cell produces new individuality and the process of offspring in the mode of division.Unicellular organism, produces new individuality in fissional mode.Multicellular organism, produces new cell in fissional mode, is used for cell old and feeble and dead in added body; Meanwhile, multicellular organism can by a germ cell, through division and the differentiation of cell, finally develops into new many cells individual.By cell division, the hereditary material that can will copy, is assigned to fifty-fifty in two daughter cells and goes.Cell proliferation is the basis of biology growing, growth, breeding and heredity, is the important vital signs of organism.
Apoptosis: be maintain homeostasis, orderly, programmed cell death autonomous by the cell of Gene Handling.Apoptosis relates to the effect of the activation of series of genes, expression and regulation and control etc., is a kind of process of cell death initiatively striven for for adapting to living environment.
Cell migration: to creep also referred to as cell, signaling or cell movement is the movement that phalangeal cell produces after receiving migration signal or experiencing the gradient of Cucumber.Cell migration be cell head pseudopodium extension, new stick foundation, cyton afterbody is contracted in alternation procedure on space-time.Cell migration is one of Normocellular basic function, and being the physiological process that body normal growth is grown, is also the ubiquitous a kind of forms of motion of living cells.All cell migration is related in the process such as fetal development, angiogenesis, wound healing, immunoreation, inflammatory reaction, atherosclerosis, cancer metastasis.
Cell invasion: tumor cell passes through far-end transfer is carried out in basement membrane migration ability to interstitial or its hetero-organization.Basement membrane is a kind of extracellular matrix of specialization, is the physical barriers that must pass through in Nasopharyngeal neoplasms process.The constituent of basement membrane is by interacting with cell surface receptor integrin.Tumor cell destroys the organizational structure of basement membrane by secreting substrate degradation enzyme, regulate the expression of extracellular matrix receptor integrin simultaneously, creates favorable conditions for adhering to through basement membrane with at target organ, breeding.
Tumor growth: tumor cell causes because of division and proliferation tumor in situ volume to increase and invades surrounding tissue gap, lymphatic vessel or Ink vessel transfusing, without obvious boundary together with contiguous normal structure compact siro spinning technology.
Neoplasm metastasis: tumor cell invades lymphatic vessel, blood vessel or body cavity position by its original site, takes the process of another position or organ continued growth formation and primary tumo(u)r same-type tumor with blood flow, Lymph flow to.
Prostate cancer growth and transfer: in the process that carcinoma of prostate cancerates, the ability due to tumor cell proliferation and migration, invasion and attack strengthens and causes the apoptosis rate of tumor cell group body dynamics, tumor cell, tumor-blood-vessel growth, tumor evolution and the heterogeneous process worsened.Carcinoma of prostate becomes in the process of growth with transfer is more and more rich in aggressive, finally causes tumor growth to accelerate, produces the infiltration of surrounding tissue and far-end transfer.
Androgen receptor (AR): androgen receptor belongs to the steroid receptor in nuclear receptor superfamily.Human androgen receptor is a single copy gene, molecular weight is about 110 kilodaltons, generally be made up of four domains: N holds transcriptional activation domain (N-terminaldomain, NTD), DNA land (DNAbindingdomain, DBD), hinge region and ligand binding domain (Ligandbindingdomain, LBD).The generation of androgen receptor and carcinoma of prostate develops closely related.Androgen is as after dihydrotestosterone (Dihydrotestosterone, DHT) is combined with androgen receptor, and androgen receptor can enter nucleus and combines with transcription activating domain thus start downstream gene expression.Androgen receptor antagonists such as bicalutamide (Bicalutamide) and the MDV3100 (Enzalutamide) of current Clinical practice combine with the ligand binding domain of androgen receptor (LBD) and inhibit its transcriptional activity.
The transcriptional activity of androgen receptor: androgen receptor is the typical nuclear receptor transcription factor, by mediating the expression of the multiple eukaryotic gene of androgenic acting regulatory.Its concrete function is regulate and control transcribing and expression of its downstream target gene by the mode of transcriptional activation, under ligand dependent approach and ligand-independent approach, all can play transcriptional activity.
The androgen receptor of disappearance ligand domain: androgen receptor LBD district is made up of 12 sections of discontinuous α spirals, forms a ligand binding pocket, has the function of binding partner.Androgen receptor may lack LBD district when suddenling change, and the androgen receptor having lacked LBD district has the composition transcriptional activation function identical with complete androgen receptor.
Luciferase reporter gene detects (Luciferaseassay): luciferase (Luciferase, Luc) reporter gene detection system is for substrate is to detect the active a kind of reporting system of LUC Photinus pyralis LUC Photinus pyralis FL (Fireflyluciferase) with fluorescein (Luciferin).Luciferase can be oxidized to Oxy-luciferin by catalysis Luciferin, in the process of Luciferin oxidation, can send bioluminescence (Bioluminescence).Then Chemiluminescence Apparatus (Luminometer) or liquid flashing determining instrument also can be claimed to measure the bioluminescence discharged in Luciferin oxidizing process by fluor tester.Fluorescein and this bioluminescence system of luciferase, can detect the expression of gene extremely sensitive, efficiently, is to detect transcription factor and the interactional a kind of detection method of genes of interest promoter region DNA.
Accompanying drawing explanation
Figure 1 shows that the inhibition of Traditional Chinese medicine compound composition of the present invention to each strain prostate gland cancer cell proliferation activity.
Fig. 1 (A) represents the suppression that Traditional Chinese medicine compound composition of the present invention is bred each strain prostate gland cancer cell and the selectivity effect on normal prostatic epithelium cell.
Fig. 1 (B) represents that the proliferation inhibiting effect of Traditional Chinese medicine compound composition of the present invention to LNCaP cell is time gradient and Concentraton gradient dependency.
Fig. 1 (C) represents that the proliferation inhibiting effect of Traditional Chinese medicine compound composition of the present invention to 22RV1 cell is time gradient and Concentraton gradient dependency.
Fig. 1 (D) represents that the proliferation inhibiting effect of Traditional Chinese medicine compound composition of the present invention to PC-3 cell is time gradient and Concentraton gradient dependency.
Fig. 1 (E) represents that the proliferation inhibiting effect of Traditional Chinese medicine compound composition of the present invention to the prostate gland cancer cell of the primary separation of patient's tumor tissues source property is time gradient and Concentraton gradient dependency.
Figure 2 shows that the inhibition of Traditional Chinese medicine compound composition of the present invention to the Clone formation of each strain prostate gland cancer cell.
Fig. 2 left side represents Traditional Chinese medicine compound composition of the present invention respectively to the white light photo of the Clone formation inhibition of LNCaP, 22RV1.
Traditional Chinese medicine compound composition of the present invention is represented respectively to the statistical result of the Clone formation inhibition of LNCaP, 22RV1 on the right of Fig. 2.
Figure 3 shows that the apoptosis-induced effect of Traditional Chinese medicine compound composition of the present invention to prostate gland cancer cell.
Fig. 3 (A) represents the apoptosis-induced effect of Traditional Chinese medicine compound composition of the present invention to LNCaP cell.
Fig. 3 (B) represents the apoptosis-induced effect of Traditional Chinese medicine compound composition of the present invention to 22RV1 cell.
Fig. 3 (C) represents the statistical result of Traditional Chinese medicine compound composition of the present invention to the apoptosis-induced effect of LNCaP, 22RV1.
Figure 4 shows that the inhibition of Traditional Chinese medicine compound composition of the present invention to migration of prostate cancer cells, invasion and attack.
Fig. 4 represents the inhibitory action of Traditional Chinese medicine compound composition of the present invention to PC-3 cell lateral transfer process.
The inhibitory action of Traditional Chinese medicine compound composition of the present invention to LNCaP cell vertical migration process is represented in Fig. 4.
The inhibitory action of Traditional Chinese medicine compound composition of the present invention to 22RV1 cell-invasion process is represented under Fig. 4.
Figure 5 shows that Traditional Chinese medicine compound composition of the present invention is by affecting androgen receptor signal path thus the molecule mechanism of suppression carcinoma of prostate.
Fig. 5 (A) represents the inhibition that Traditional Chinese medicine compound composition of the present invention is expressed the luciferase reporter gene detecting androgen receptor transcription activity in LNCaP cell under dihydrotestosterone (DHT) induction.
Fig. 5 (B) represents the inhibition that Traditional Chinese medicine compound composition of the present invention is expressed the luciferase reporter gene detecting androgen receptor transcription activity in 22RV1 cell under dihydrotestosterone (DHT) induction.
Fig. 5 (C) represents the inhibition that Traditional Chinese medicine compound composition of the present invention is expressed the luciferase reporter gene that in PC-3 cell, external source transfected wild-type androgen receptor (AR) detects androgen receptor transcription activity afterwards under dihydrotestosterone (DHT) induction.
Fig. 5 (D) represents that Traditional Chinese medicine compound composition of the present invention does not affect the transcriptional level of androgen receptor (AR) gene self in LNCaP cell.
Fig. 5 (E) represents that Traditional Chinese medicine compound composition of the present invention lowers the expression of androgen receptor (AR) albumen in LNCaP cell.
Fig. 5 (F) represents that Traditional Chinese medicine compound composition of the present invention suppresses androgen receptor (AR) to its three classical downstream target gene TMPRSS2, the transcriptional activation of FKBP5, PSA.
Figure 6 shows that Traditional Chinese medicine compound composition of the present invention suppresses the growth of 22RV1 xenograft tumours in nude mice by subcutaneous lotus tumor body in animal model.
Fig. 6 (A) represents the white light photo of Traditional Chinese medicine compound composition of the present invention to the gross tumor volume inhibition peeled off in 22RV1 xenograft tumor growth model.
Fig. 6 (B) represents the statistical result of Traditional Chinese medicine compound composition of the present invention to the inhibition that gross tumor volume in 22RV1 xenograft tumor growth model increases with natural law.
Fig. 6 (C) represents the white light photo of Traditional Chinese medicine compound composition of the present invention to the tumor weight inhibition peeled off in 22RV1 xenograft tumor growth model.
Figure 7 shows that Traditional Chinese medicine compound composition of the present invention suppresses growth and the transfer of 22RV1-luc xenograft tumours in nude mice original position xenograft tumor growth transfer body in animal model.
Fig. 7 (A) represents the living imaging result of Traditional Chinese medicine compound composition of the present invention to tumor fluorescence intensity inhibition in 22RV1-luc xenograft tumor growth metastasis model.
Fig. 7 (B) represents that Traditional Chinese medicine compound composition of the present invention forms the statistical result of the inhibition of Nodules to whole body internal organs to 22RV1-luc xenograft tumor growth metastasis model situ neoplasm metastasis.
Fig. 7 (C) represents the statistical result of Traditional Chinese medicine compound composition of the present invention to the inhibition that general tumour fluorescence intensity in 22RV1-luc xenograft tumor growth metastasis model increases with natural law.
Fig. 7 (D) represents Traditional Chinese medicine compound composition of the present invention impact on nude mice body weight under gastric infusion mode.
Detailed description of the invention
In conjunction with following specific embodiments and the drawings, technology of the present invention is described in further detail.Protection content of the present invention is not limited to following examples.Under the spirit and scope not deviating from inventive concept, the change that those skilled in the art can expect and advantage are all included in the present invention, and are protection domain with appending claims.Concrete material proportion, process conditions and result thereof described by embodiment only for illustration of the present invention, and should can not limit the present invention described in detail in claims yet.Implement process of the present invention, condition, reagent, experimental technique etc., except the following content mentioned specially, be universal knowledege and the common practise of this area, the present invention is not particularly limited content.
Embodiment 1: the preparation of Traditional Chinese medicine compound composition of the present invention
There is in the present embodiment the Traditional Chinese medicine compound composition of anti-prostate cancer effect, be made up of following raw material: get ailanthus altissima (mill.) swingle skin 9 parts, Herba Hedyotidis Diffusae 30 parts, Rhizoma Curcumae 15 parts, Radix Salviae Miltiorrhizae 30 parts, Spica Prunellae 15 parts, 9 parts, Radix Glycyrrhizae, adds 15 times of volume distilled water immersions 3 hours of Traditional Chinese medicine compound composition medical material amount; Decoct in automatic decocting device for herbal medicine, decocting number of times is 2 times, each decoction 1 hour; The Chinese medicine crude extract prepared uncaps evaporation and concentration to final volume simmer down to 70 milliliters on automatic decocting device for herbal medicine; The concentrated Chinese medicine decoction mother solution final concentration obtained is about 1.5 grams of crude drug/milliliters.Chinese medicine decoction mother solution is obtained after obtained the concentrated liquid of tradition Chinese medicine is degerming by 0.22 micrometer Millipore membrane filtration, for subsequent use in 4 degrees Celsius of Refrigerator stores.
The Traditional Chinese medicine compound composition adopted in following examples 2-7 is prepared by embodiment 1
Embodiment 2: Traditional Chinese medicine compound composition of the present invention is to the proliferation activity of different prostate gland cancer cell, the inhibitory action of Clone formation and the apoptosis-induced effect to different prostate gland cancer cell
Technical method:
1. the cultivation of cell
Cell (LNCaP, 22RV1, PC-3, PNT1A) used in this experiment is all purchased from Chinese Academy of Sciences's Shanghai cell bank; Another strain cell comes from the prostate gland cancer cell (PCa primary cell) of the primary separation of Changhai Hospital, Shanghai City's prostate cancer patient's tumor tissues source property, and cell culture is in 37 degrees Celsius of constant incubator (humidity 95%, CO 2concentration 5%) in, culture medium is the RPMI-1640 (Gibco) containing 10% hyclone (Front).
2.SRB (Sulforhodamine) method measures cell proliferation
Different Prostatic cancer cell lines is with 5 × 10 3individual/hole density is seeded to 96 orifice plates (Corning), and after 24 hours, add variable concentrations Traditional Chinese medicine compound composition of the present invention, matched group adds the PBS of equivalent, establishes 6 multiple holes for each group.Drug treating, after 48 hours, adds pre-cooled TCA (trichloroacetic acid, 50%, w/v) and hatches 60 minutes fixed cells in 4 degrees Celsius.After fixing, running water 5 times, air-dry.Every hole adds 50 microlitre SRB dye liquors (4%, w/v), incubated at room dyeing in 10 minutes.By dye liquor sucking-off, every hole adds 1% acetic acid 100 microlitre and washes 5 times, removes non-combination dye.After air-dry, it is 10 mMs of Tris solution 100 microlitres that every hole adds concentration, and the SRB dyestuff combined is dissolved in concussion.96 orifice plates are placed in microplate reader (SPECTRAMAX190), under 515 nano wave lengths, measure OD value.Statistical analysis Traditional Chinese medicine compound composition is for the impact of cell proliferation level.For detecting Traditional Chinese medicine compound composition of the present invention to the inhibitory action of Prostatic cancer cell lines whether in Concentraton gradient dependency and time Concentraton gradient dependency, inoculate different Prostatic cancer cell lines with 5 × 10 3individual/hole density to 96 orifice plate, after 24 hours, add variable concentrations Traditional Chinese medicine compound composition of the present invention, matched group adds the PBS of equivalent, establishes 6 multiple holes for each group.Continue cultivation 24 hours respectively, after 48 hours and 72 hours, detect the impact of Traditional Chinese medicine compound composition for cell proliferation level by above-mentioned same method.
3. colony formation
Clone forming method (Colonyformation) can be used to detect tumor cell external formed clone propagation with become tumor ability.Tumor cell can form macroscopic solid tumor by autotomy propagation is final in vivo, when testing in vitro, can reflect that it generates the power of solid tumor ability in vivo according to the clonality of tumor cell.Be linked in 6 orifice plates by 1000 prostate gland cancer cells, add variable concentrations Traditional Chinese medicine compound composition of the present invention, fixed by cell, then use violet staining after one week with paraformaldehyde after cell attachment, take pictures and add up clone's number, often group experiment in triplicate.
4. Apoptosis by Flow Cytometry
In normal cell, Phosphatidylserine (Phosphatidylserine, PS) is only distributed in the inner side of cell membrane lipid bilayer, and early stage at apoptosis, and the Phosphatidylserine (PS) in cell membrane is turned on one's side laterally by adipose membrane.The Ca of AnnexinV to be a kind of molecular weight be 35 ~ 36kD 2+dependency cardiolipin binding protein, has high affinity with Phosphatidylserine, therefore is combined with the after birth of the early stage cell of apoptosis by the Phosphatidylserine exposed outside cell.Therefore AnnexinV is taken as one of sensitive indexes detecting early apoptosis of cells.AnnexinV is carried out fluorescein (EGFP, FITC etc.) labelling, using the AnnexinV that marked as fluorescent probe, utilize fluorescence microscope or flow cytometer can detect apoptotic generation.Propidium iodide (Propidiumiodide, PI) is a kind of nucleic acid dye, and it can not through complete cell membrane, but to the cell of apoptosis middle and advanced stage and dead cell, and PI can permeate through cell membranes and nucleus is incarnadined.Therefore AnnexinV and PI coupling is used, just the cell differentiation being in different apoptosis period can be come.Concrete operation step is as follows: different prostate gland cancer cell after 36 hours, collects suspension cell centrifugal (2000 revs/min, centrifugal 5 minutes) through the medicinal liquid process of Traditional Chinese medicine compound composition variable concentrations of the present invention; The collected by trypsinisation of attached cell not containing EDTA; 1 ~ 5 × 10 are collected with PBS washed cell secondary (2000 revs/min, centrifugal 5 minutes) 5cell; Add the Bindingbuffer suspension cell of 500 microlitres; After adding 5 microlitre AnnexinV-FTIC mixings, add 5 microlitre PI, mixing; Room temperature, lucifuge, reaction 5 ~ 15 minutes; In 1 hour, carry out the upper machine testing of flow cytometer.
Experimental result:
As shown in Figure 1, Traditional Chinese medicine compound composition of the present invention is to each strain prostatitis cancerous cell, and the propagation comprising the primary prostate gland cancer cell in tissue of patient source has very significant inhibition, and its 48 hours half-inhibition concentrations are about 5 mcg/ml.Traditional Chinese medicine compound composition of the present invention except the primary prostate gland cancer cell to conventional Prostatic cancer cell lines and tissue of patient source proliferation inhibiting effect significantly except, although to normal prostate epithelial cell strain PNT1A in higher concentrations (100 mcg/ml) also have slight suppression, but inhibition is very little at low concentrations, demonstrate it to having good selectivity between normal cell and prostate gland cancer cell.Meanwhile, Traditional Chinese medicine compound composition of the present invention presents Concentraton gradient dependency and time gradient dependency to the inhibition that each strain prostate gland cancer cell is bred.In addition, as shown in Figure 2, the Clone formation of the Traditional Chinese medicine compound composition of the present invention also each strain prostate gland cancer cell of strong inhibition.Fig. 3 shows Traditional Chinese medicine compound composition of the present invention and significantly promotes prostate gland cancer cell LNCaP, the apoptosis of 22RV1.The potential drug that experimental result points out Traditional Chinese medicine compound composition of the present invention can grow as anti-prostate cancer.
Embodiment 3: Traditional Chinese medicine compound composition of the present invention is to the inhibitory action of migration of prostate cancer cells, invasive ability
Technical method:
1. line migration experiment
For whether research Traditional Chinese medicine compound composition of the present invention suppresses the lateral transfer of prostate gland cancer cell, PC-3 cell is seeded to 6 orifice plates, at 37 degrees Celsius of 5%CO 2cellar culture 24 hours in incubator, grows to 100% completely to cell.Change serum-free medium, continue cultivation 12 hours.In the culture hole covering with cell, carry out cut with the sterilizing rifle head of 200 microlitres, wash cell twice with PBS after cut, the cell floated is sucked, in every hole, add 1 milliliter of complete medium.Respectively to adding variable concentrations Traditional Chinese medicine compound composition in cell culture well, culture plate is put into CO 2incubator, 37 degrees Celsius are continued cellar culture 24 hours.The situation that basis of microscopic observation cell moves to dashed part, takes pictures.The migration of statistical analysis various dose medicine group enters the cell quantity of scribe area, determines the impact of medicine on cell migration ability.
2.Transwell cell migration experiment
The permeable polycarbonate film spread bottom Transwell cell there is the micropore of a large amount of diameter 8 microns, during experiment, Transwell cell is put into 24 orifice plates, each hole of 24 orifice plates is divided into upper and lower two Room by polycarbonate film respectively, the little indoor of Transwell deserve to be called room, claim lower room, upper indoor splendid attire culture supernatants in culture plate, lower indoor splendid attire lower floor culture fluid, levels culture fluid is separated by with polycarbonate membrane.Cell is seeded in upper indoor, because polycarbonate membrane has permeability, composition in lower floor's culture fluid can be induced to the cell downward room migration of indoor, makes cell move to face, lower room from the face, upper room of thin film, and therefore this experiment is the Classic Experiments of research cell movement.For whether research Traditional Chinese medicine compound composition of the present invention suppresses the vertical migration of prostate gland cancer cell, utilize Transwell cell, access excessive LNCaP cell in upper room, this like cell will move room downwards; Add the Traditional Chinese medicine compound composition of variable concentrations, statistics of taking pictures after 12 hours cell migration rate simultaneously.
3.Transwell method detects cell invasion
Transwell cell is put into 24 orifice plates, upper indoor splendid attire culture supernatants, lower indoor splendid attire lower floor culture fluid, levels culture fluid is separated by with polycarbonate membrane, on Transwell cell, room spreads one deck collagen stroma Matrigel in advance, then by cell inoculation wherein.Because polycarbonate membrane has permeability, the composition in lower floor's culture fluid can induce the cell of indoor to move downwards, makes cell move to face, lower room from the face, upper room of thin film.But, tumor cell to move to lower room, the various chemical dissolution collagen stromas that namely must first utilize self to discharge, again by dissolving the cavity motion, this process, similar to tumor cell impregnation process in vivo, can be used for reflecting the invasiveness ability of tumor cell.Therefore this experiment is the Classic Experiments of research cell invasion.Take the logarithm the tumor cell of trophophase with 5 × 10 4individual/hole is inoculated in the upper room of Transwell cell, and dosing group adds the Traditional Chinese medicine compound composition of respective concentration respectively, and matched group adds the PBS of equivalent.Respectively complete medium is added respectively in lower room.37 degrees Celsius of 5%CO 2cultivate 12 hours in incubator.Take out Transwell cell, dip in the upper room one side of water wiping Transwell cell with cotton swab, the cell not wearing film is wiped.Transwell cell room temperature in 4% paraformaldehyde is fixed 10 minutes, uses tap water through 1% violet staining after 10 minutes.Take pictures under microscope, to count in every hole the cell number in 5 visuals field up and down.Often organize and on average establish 3 multiple holes.Statistical various dose Traditional Chinese medicine compound composition dosing group wear theca cell quantity, determine the impact of Traditional Chinese medicine compound composition on cell invasion ability.
Experimental result:
As shown in Figure 4, Traditional Chinese medicine compound composition of the present invention at lower doses (2.5 mcg/ml) just effectively can suppress the invasive ability of the lateral transfer of PC-3, the vertical migration of LNCaP and 22RV1 to result.Fig. 4 left side is the experiment white light photographic result of three kinds of cells respectively, is the statistical result of three kinds of cells on the right of Fig. 4 respectively.The potential drug that experimental result points out Traditional Chinese medicine compound composition of the present invention can shift as anti-prostate cancer.
Embodiment 4: the transcriptional activity of Traditional Chinese medicine compound composition vitro inhibition androgen receptor of the present invention
Technical method: luciferase reporter gene detects
This tests plasmid used is MMTV-LUC and Renila.MMTV-LUC is the reporter plasmid with androgen receptor transcription active element (ARresponseelements) and luciferase, this plasmid expression can be started after androgen receptor is combined with androgen and go out luciferase, luciferase can send fluorescence by its substrate luciferin of catalysis, just can be detected the degree of Androgen Receptor Activation by the intensity detecting fluorescence; Renila internal reference reporter gene, with the promoter region of renilla luciferase, can express renilla luciferase, and can determine that whether the transfection efficiency often organizing cell is consistent by this plasmid, androgen does not affect Renila internal reference reporter plasmid.Androgen receptor (Androgenreceptor can be suppressed for tentatively probing into Traditional Chinese medicine compound composition of the present invention, AR) transcriptional activity, we use liposome 2000 (Lipofectamine2000Reagent) transfection MMTV-LUC and Renila two kinds of plasmids respectively in Prostatic cancer cell lines 22RV1 and LNCaP, enter external source wild type AR in addition at the PC-3 transit cell of not expressing AR; Cell transfecting adds androgen dihydrotestosterone (Dihydrotestosterone, DHT) and activates MMTV-LUC reporter gene expression LUC Photinus pyralis LUC Photinus pyralis FL after 24 hours; Variable concentrations Traditional Chinese medicine compound composition is added again while adding DHT, process cell lysis after 12 hours and measure the luciferase expression intensity of two kinds of reporter genes, the final result activity of MMTV-LUC represents compared with the activity of internal reference plasmid Renila, and often group experiment in triplicate.
Experimental result:
As shown in Fig. 5 A, 5B, 5C, Traditional Chinese medicine compound composition of the present invention dose-dependently can both reduce the activity of the MMTV-LUC reporter gene that androgen (DHT) is induced in different Prostatic cancer cell lines, and just can reach half suppression ratio (IC at low concentration (about 2.5 mcg/ml) 50), prove that Traditional Chinese medicine compound composition of the present invention can the transcriptional activity of vitro inhibition androgen receptor.
Embodiment 5: Traditional Chinese medicine compound composition of the present invention significantly lowers the transcriptional level of androgen receptor protein level and target gene thereof
Technical method:
1. polymerase chain reaction (Polymerasechainreaction, PCR) experiment
LNCaP cell is after the process of variable concentrations Traditional Chinese medicine compound composition, and with TRIzol separation and Extraction RNA, RNA obtains cDNA after reverse transcription, by the mrna expression level of AR Auele Specific Primer by reverse transcriptional PCR (RT-PCR) qualitative detection AR gene; Detected the expression of the mRNA of three by fluorescence real-time quantitative PCR (Q-PCR) with different AR downstream target gene Auele Specific Primers (TMPRSS2, FKBP5 and PSA).
2. immunoblotting (Westernblot) experiment
LNCaP cell is after adding the process of variable concentrations Traditional Chinese medicine compound composition, cell extracts albumen through cracking, albumen uses polyacrylamide gel electrophoresis separating sample proteins after denatured by boiling, then transfer on cellulose nitrate film, first the antibody incubation two hours of androgen receptor AR is used, hatching one hour with resisting with fluorescently-labeled two again, finally detecting the impact of Traditional Chinese medicine compound composition of the present invention on AR protein expression level with LicorOdyssey Dual band IR laser imaging system.
Experimental result:
As shown in fig. 5e, immunoblot experiment (Westernblot) proves that Traditional Chinese medicine compound composition of the present invention significantly can suppress the protein expression level of androgen receptor; Along with the increase of adding consistency, the expressing quantity of AR obviously reduces thereupon, points out the inhibitory action of Traditional Chinese medicine compound composition of the present invention to prostate cancer tumor cells propagation and migration may realize by suppressing androgen receptor signal path.RT-PCR experiment finds that Traditional Chinese medicine compound composition of the present invention does not affect the mRNA level in-site (Fig. 5 D) of AR under finite concentration, but significantly suppress the transcriptional activation (Fig. 5 F) of its downstream target gene.The transcriptional level of three classics representative target gene TMPRSS2, FKBP5 and PSA in Q-PCR experiment display AR downstream is all subject to the remarkable suppression of Traditional Chinese medicine compound composition of the present invention.Above experimental result demonstrates the suppression of Traditional Chinese medicine compound composition of the present invention to prostate cancer growth and transfer from multiple angle and realizes by suppressing androgen receptor signal path active.
Embodiment 6: the inhibition of Traditional Chinese medicine compound composition of the present invention in nude mice carcinoma of prostate subcutaneous lotus tumor growth model and carcinoma of prostate growth in situ metastasis model
Technical method:
1. Traditional Chinese medicine compound composition of the present invention is to the inhibitory action of nude mice carcinoma of prostate subcutaneous lotus tumor growth model
Human Prostate Cancer Cells 22RV1 is utilized to set up nude mice carcinoma of prostate subcutaneous lotus tumor growth model.By 5,000,000 22RV1 cell subcutaneous injection to male immunization deficient mice (BLAB/c-nude in 7 week age, nude mice) right side dorsal sc, when Subcutaneous tumor grows to about 100 cubic millimeters, mice is divided into 8 groups (mean tumour volume is identical), be followed successively by model group, matched group, Chinese medicine compound (not containing ailanthus altissima (mill.) swingle skin) group, Chinese medicine compound low dose group, Chinese medicine compound high dose group, FUFANG KUSHEN ZHUSHEYE group, cyclophosphamide (Cyclophosphamide, CTX) group, CTX+ Chinese medicine compound high dose group.Route of administration and dosage are as described below successively: model group mice is left intact, control group mice gives the PBS gavage (0.1 milliliter) of equivalent every day, Chinese medicine compound (not containing ailanthus altissima (mill.) swingle skin) organizes the Chinese medicine compound (0.1 milliliter not containing ailanthus altissima (mill.) swingle skin that mice gives equivalent every day, concrete decocting method is consistent with Chinese medicine compound of the present invention, do not add ailanthus altissima (mill.) swingle skin crude drug, corresponding minimizing concentrates final volume, actual dose 15 grams/kg/day), Chinese medicine compound low dose group mice gives the Chinese medicine compound mother solution gavage (0.05 milliliter of 1/2 amount every day, actual dose 7.5 grams/kg/day), Chinese medicine compound high dose group mice gives Chinese medicine compound mother solution gavage (0.1 milliliter every day, mother liquid concentration 1.5 grams of crude drug/milliliters, actual dose 15 grams/kg/day), FUFANG KUSHEN ZHUSHEYE group mice gives 0.1 milliliter of FUFANG KUSHEN ZHUSHEYE lumbar injection (actual dose 0.6 gram/kg/day) every day, CTX group mice gives 0.05 milliliter of CTX lumbar injection (actual dose 0.05 gram/kg/day) every day, CTX+ Chinese medicine compound high dose group mice gives 0.05 milliliter of CTX lumbar injection and 0.1 milliliter of Chinese medicine compound mother solution gavage (actual dose 0.05 gram/kg/day+15 grams/kg/day) every day.The gross tumor volume of a subcutaneous lotus tumor of mouse back is measured, according to formula volume=length × wide every 2 days 2× 0.52 calculates, statistics gross tumor volume.A Mouse Weight is measured every 2 days.Dispenser put to death mice after 14 days, got Subcutaneous tumor, took pictures.
2. Traditional Chinese medicine compound composition of the present invention is to the inhibitory action of nude mice carcinoma of prostate original position lotus tumor growth metastasis model
Metastatic prostate cancer cell 22RV1 is proceeded to LUC Photinus pyralis LUC Photinus pyralis FL (Luciferase) expression plasmid (this plasmid is with G418 resistance screening labelling), and surely turn cell line with G418 screening, surely turning cell can expressing luciferase, fluorescence can be sent after luciferase contacts with its substrate luciferin, the position of the scope of fluorescence and intensity and tumor and being proportionate property of volume, so namely can detect quantity and the position of tumor cell by living animal imaging system.What build surely turns cell line called after 22RV1-luc, the mixing of 22RV1-luc cell matrigel is expelled to mouse prostate dorsal part leaf, after tumor xenograft success, growth and the transfer case of the 22RV1-luc cell in Mice Body can be detected by bioluminescence imaging technology.Specific operation process is: after male immunization deficient mice anesthesia in 7 week age, row abdominal cavity opening is dissected, expose prostate, mouse prostate dorsal part leaf is expelled to by after 500,000 22RV1-luc cell matrigel mixings, living imaging systems axiol-ogy fluorescent value is begun through and by mice random packet and administration from the 0th day, administering mode is identical with previous zoopery, detects once and adds up the growth curve chart that fluorescence intensity increases with natural law, measure a Mouse Weight every 2 days every 3 days.Terminate experiment when experiment proceeds to the 15th day, put to death mice, peel off mice organs statistics metastatic nodules.
Experimental result:
As shown in Figure 6, in nude mice carcinoma of prostate subcutaneous lotus tumor growth model, the mice Subcutaneous tumor size of 8 groups obviously variant (Fig. 6 A) for result.Fig. 6 B is shown the mensuration increased along with experiment natural law by gross tumor volume and shows, model group and matched group gross tumor volume are maximum, and do not have significant difference between these two groups, illustrates that modeling successfully.The tumor final volume that Chinese medicine compound (not containing ailanthus altissima (mill.) swingle skin) is organized has slight minimizing relative to matched group, certain effect is described, but effect is not too obvious; The experimental group of effect more not containing ailanthus altissima (mill.) swingle skin of Chinese medicine compound low dose group is effective, but still not too remarkable; Chinese medicine compound high dose group effect is comparatively remarkable, illustrates that Chinese medicine compound of the present invention effectively can suppress the growth of prostate cancer; FUFANG KUSHEN ZHUSHEYE group, as the Chinese medicine compound positive control of patent medicine, also show the effect of good Tumor suppression in this experiment, but from experimental result, inhibition is strong not as good as the inhibition of Chinese medicine compound of the present invention; CTX group, as the positive control of classic chemotherapy medicine, has obvious inhibition to tumor; CTX+ Chinese medicine compound high dose group is the most obvious to the effect of tumor suppression, is the group that in all experimental grouies, gross tumor volume is minimum, illustrates that Traditional Chinese medicine compound composition of the present invention can strengthen the tumor killing effect of classic chemotherapy medicine.Similar experimental result (Fig. 6 C) be also illustrate that to the weight statistics peeling off tumor.
In addition, as shown in Figure 7, in nude mice carcinoma of prostate original position lotus tumor growth metastasis model, Fig. 7 A is medication therapeutic effect figure (n >=5) to mouse prostate tumor in situ after 14 days, to be taken pictures picture by living animal imaging system, in figure, colored region represents fluorescence signal, and show that this region has tumor cell to assemble, it is larger that fluorescence intensity more deeply feels bright tumor.Experimental result and previous zooperal result similar, Chinese medicine compound high dose group effect is more remarkable, illustrate that Traditional Chinese medicine compound composition of the present invention effectively can suppress growth (Fig. 7 C) and the transfer (Fig. 7 B) of prostate cancer, and the more positive Chinese medicine compound of this inhibition---FUFANG KUSHEN ZHUSHEYE is eager to excel, suitable with the inhibition of classic chemotherapy medicine CTX; The Traditional Chinese medicine compound composition process of each group does not obviously affect (Fig. 7 D) to Mice Body weight average above, and preliminary explanation Traditional Chinese medicine compound composition of the present invention is less to the toxic and side effects of body under effective dose; The lower Mouse Weight of CTX process declines to some extent.In conjunction with the experimental result of Mouse Weight, certain toxicity is there is to body in CTX as a chemotherapeutics, cause the slight decline of Mouse Weight, and compared with the Mouse Weight of CTX group, when Traditional Chinese medicine compound composition of the present invention and CTX drug combination, the most significant in all groups to the inhibition of tumor growth and transfer, and Mouse Weight is recovered (Fig. 7 D), illustrate that Traditional Chinese medicine compound composition of the present invention can not only strengthen the tumor killing effect of classic chemotherapy medicine, the damage to body caused because of the toxicity of classic chemotherapy medicine can also be improved, thus prove that Traditional Chinese medicine compound composition of the present invention has the effect to classic chemotherapy drug-enhancer attenuation.
Embodiment 7: the efficacy enhancing and toxicity reducing effect of Traditional Chinese medicine compound composition of the present invention on tumor bearing nude mice tumor suppression and the impact of immune function
Technical method:
1. Traditional Chinese medicine compound composition of the present invention is on the impact of nude mice index and spleen index
Observing model group, the nude mice life quality of matched group and administration group and animation different after, especially there is pachylosis in various degree in chemotherapeutics CTX group tumor-bearing mice, lethargy, slack idle dynamic, the states such as bradykinesia loses weight, then skin is smooth for Chinese medicine compound drug combination group mice of the present invention, spirit is good, movable sensitive, Normal-weight, tumor in situ block is less, therefore infer that the efficacy enhancing and toxicity reducing effect of Traditional Chinese medicine compound composition of the present invention to chemotherapeutics is likely by improving immune function of mice, improve every immune indexes to realize.Therefore, by the nude mice of embodiment 6 situ tumor growth metastasis model in experiment end after death, weigh spleen weight and body weight, calculate index and spleen index.
2. Traditional Chinese medicine compound composition of the present invention affects nude mouse serum leukocyte count object
By the nude mice of embodiment 6 situ tumor growth metastasis model in experiment end after death, eye socket gets blood, and accurately to suck blood 20 microlitres with rifle suction pipe, rifle head outer wall depletion of blood, puts into blood gently rapidly and fill 380 microlitre leukocyte diluents, shake up.By the method for red blood cell count(RBC), by leukocyte suspension instillation counting chamber, leave standstill 2 to 3 minutes, counting after leukocyte sinks.Under low power lens, leukocyte is rounded, starches bright, and core is atropurpureus, slightly refractive power, and feature can be distinguished with impurity phase whereby.Sum × 500 of a number of white blood cells=5 large lattice, each group difference of statistics after counting.
3. Traditional Chinese medicine compound composition of the present invention is to the impact of the T cell proliferation response that concanavalin A (ConcanamycinA, ConA) is induced
By the nude mice of embodiment 6 situ tumor growth metastasis model in experiment end after death, get after spleen is weighed and put into the pre-prepd test tube that pre-cooling PBS is housed, spleen in test tube and washing liquid are poured in plate, spleen is put into cellular filter tweezers and extrudes spleen, obtain cell suspension.Afterwards cell suspension is collected in test tube, 1500 revs/min, centrifugal 6 minutes, outwell washing liquid, add erythrocyte cracked liquid cracking 3 minutes, to be seenly after liquid color shoals, add washing liquid PBS again, with membrane filtration in another test tube, 1500 revs/min again, centrifugal 6 minutes, outwell washing liquid and cell culture fluid, adding ConA (working concentration 5 mcg/ml) stimulates T lymphocytosis, be placed in 37 degrees Celsius of constant incubator (humidity 95%, CO 2concentration 5%) cultivate 72 hours.Srb assay measures the absorbance (A value) of T cell thus detects its proliferative conditions in microplate reader.
Experimental result:
Result is as shown in table 1, the impact of the T cell proliferation response that the efficacy enhancing and toxicity reducing effect of Traditional Chinese medicine compound composition of the present invention to tumor bearing nude mice tumor suppression and the Three Represents index to tumor bearing nude mice immunologic function---index and spleen index, number of white blood cells and concanavalin A (ConcanamycinA, ConA) are induced all has significant difference.
Table 1 shows the statistical result of three typical index of immune function of mice.Each administration group of Traditional Chinese medicine compound composition compared with model group matched group, three indexs there are no significant difference.Three indexs of CTX group comparatively model group matched group are compared, all there is significant difference, again demonstrate the toxicity of chemotherapeutics CTX to body, certain damage is had to immune system, and Traditional Chinese medicine compound composition of the present invention is with it after drug combination, obviously can recover its toxic action that mice is caused, significantly improve the life quality of tumor-bearing mice.The results show, compared with CTX, Traditional Chinese medicine compound composition toxic and side effects of the present invention is little, can not cause atrophy of immune organ, and with CTX coupling, there is obvious efficacy enhancing and toxicity reducing effect, point out the anti-tumor activity of Traditional Chinese medicine compound composition of the present invention and immunoregulation effect to have direct dependency.In sum, Traditional Chinese medicine compound composition of the present invention not only has the function suppressing prostate cancer growth and transfer, the tumor killing effect of classic chemotherapy medicine can also be strengthened, improve the damage to body caused because of the toxicity of classic chemotherapy medicine, thus prove that Traditional Chinese medicine compound composition of the present invention has the effect to classic chemotherapy drug-enhancer attenuation.
The efficacy enhancing and toxicity reducing effect of table 1 Traditional Chinese medicine compound composition of the present invention on tumor bearing nude mice tumor suppression and the impact of immune function
Note: compare with model group matched group, * P < 0.05, * * P < 0.01
Compare with CTX group, ^^P < 0.01
Embodiment 8: the checking of Traditional Chinese medicine compound composition preferred version of the present invention
In order to verify the preferred effect of each component proportion of Traditional Chinese medicine compound composition of the present invention, take the Six-element Chinese crude drug dry product of different ratio as shown in table 2 combination, make Chinese medicine decoction according to the method in embodiment 1 and carry out the parallel laboratory test of embodiment 2-7 with the Traditional Chinese medicine compound composition in embodiment 1, find that Traditional Chinese medicine compound composition 8a-8g drug effects different in the present embodiment is the same with the Traditional Chinese medicine compound composition in embodiment 1, all can reach the same technique effect of embodiment 2-7, therefore demonstrate the preferred version of each component proportion in Traditional Chinese medicine compound composition of the present invention.
The Traditional Chinese medicine compound composition of table 2 different ratio
above-described embodiment, only in order to technical conceive of the present invention and feature are described, its object is to those skilled in the art can be understood content of the present invention and implement according to this, can not limit the scope of the invention with this.Every equivalence done by the essence of content of the present invention changes or modifies, and all should be encompassed in scope.

Claims (15)

1. a Traditional Chinese medicine compound composition, is characterized in that, comprises the Chinese crude drug of following parts by weight: ailanthus altissima (mill.) swingle skin 4 ~ 12 parts, Herba Hedyotidis Diffusae 12 ~ 30 parts, Rhizoma Curcumae 3 ~ 15 parts, Radix Salviae Miltiorrhizae 15 ~ 30 parts, Spica Prunellae 5 ~ 15 parts, 4 ~ 12 parts, Radix Glycyrrhizae.
2. Traditional Chinese medicine compound composition according to claim 1, is characterized in that, comprises the Chinese crude drug of following parts by weight: ailanthus altissima (mill.) swingle skin 6 ~ 9 parts, Herba Hedyotidis Diffusae 15 ~ 24 parts, Rhizoma Curcumae 4 ~ 12 parts, Radix Salviae Miltiorrhizae 18 ~ 21 parts, Spica Prunellae 6 ~ 10 parts, 6 ~ 9 parts, Radix Glycyrrhizae.
3., as the preparation method of the Traditional Chinese medicine compound composition of claim 1 or 2, it is characterized in that, comprise the following steps:
(1) soak: get Six-element Chinese crude drug described in claim 1 or 2, add distilled water immersion, obtain crude drug distilled water mixture;
(2) decoct: the crude drug distilled water mixture described in step (1) is decocted, obtains Chinese medicine crude extract;
(3) concentrated: Chinese medicine crude extract step (2) prepared concentrates, and obtains the concentrated liquid of tradition Chinese medicine;
(4) degerming: to get the concentrated liquid of tradition Chinese medicine that step (3) prepares degerming by filtering with microporous membrane, namely obtain described Traditional Chinese medicine compound composition.
4. Traditional Chinese medicine compound composition according to claim 1 is applied to the application of preparation treatment malignant tumor medicine.
5. application according to claim 4, is characterized in that, described malignant tumor comprises carcinoma of prostate, breast carcinoma, pulmonary carcinoma, colon cancer, the brain cancer, skin carcinoma, bladder cancer, renal carcinoma.
6. Traditional Chinese medicine compound composition according to claim 1 is applied to the application of preparation treatment by the medicine of the propagation of the abnormal amplification of androgen receptor and the malignant cell caused that suddenlys change, growth, migration and infiltration.
7. Traditional Chinese medicine compound composition as claimed in claim 1 is applied to the purposes of preparation treatment carcinoma of prostate medicine.
8. Traditional Chinese medicine compound composition according to claim 1 is applied to the purposes of apoptosis suppressing the growth of prostate gland cancer cell, propagation, transfer, migration, invasion and attack and promotion prostate gland cancer cell.
9. purposes as claimed in claim 7, it is characterized in that, described carcinoma of prostate is castration-resistant prostate cancer or androgen-dependent prostate cancer.
10. Traditional Chinese medicine compound composition according to claim 1 is applied to the purposes of the transcriptional activity of the transcriptional activity of the androgen receptor of vitro inhibition dihydrotestosterone induction and the androgen receptor of disappearance ligand domain, or for vitro inhibition estrogen receptor activity.
Traditional Chinese medicine compound composition according to claim 1 is applied to the propagation of Prostatic cancer cell lines 22RV1 of vitro inhibition androgen receptor sudden change, the purposes of the propagation of suppression androgen-dependent prostate JEG-3 LNCaP by 11..
12. purposes Traditional Chinese medicine compound composition according to claim 1 being applied to the migration of vitro inhibition prostate gland cancer cell PC-3 and LNCaP and 22RV1, or the purposes of androgen receptor mutant clone 22RV1 cell proliferation that vitro inhibition bicalutamide or MDV3100 fail to respond to any medical treatment.
Traditional Chinese medicine compound composition according to claim 1 is applied in body the transfer suppressing prostate gland cancer cell by 13., or suppresses growth and the transfer of castration-resistant prostate cancer in body, or in Mice Body, suppress growth or the transfer of prostate gland cancer cell 22RV1.
14. purposes as described in claim 4-13, it is characterized in that, described Traditional Chinese medicine compound composition and pharmaceutically acceptable carrier are made the medicine of injectable fluid, aerosol, emulsifiable paste, gel, pill, syrup, transdermal patch, capsule, microcapsule, tablet, granule or oral liquid formulation.
15. application according to claim 4-13, is characterized in that, described Traditional Chinese medicine compound composition is used alone or with other drug conbined usage.
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