WO2017063562A1 - 一种中药复方组合物及其在抗前列腺癌中的应用 - Google Patents

一种中药复方组合物及其在抗前列腺癌中的应用 Download PDF

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WO2017063562A1
WO2017063562A1 PCT/CN2016/101970 CN2016101970W WO2017063562A1 WO 2017063562 A1 WO2017063562 A1 WO 2017063562A1 CN 2016101970 W CN2016101970 W CN 2016101970W WO 2017063562 A1 WO2017063562 A1 WO 2017063562A1
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chinese medicine
traditional chinese
prostate cancer
parts
cells
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PCT/CN2016/101970
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English (en)
French (fr)
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易正芳
胡美纯
刘明耀
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华东师范大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/88—Liliopsida (monocotyledons)
    • A61K36/906—Zingiberaceae (Ginger family)
    • A61K36/9066—Curcuma, e.g. common turmeric, East Indian arrowroot or mango ginger

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  • the invention relates to the technical field of medicine, in particular to a traditional Chinese medicine compound composition and application thereof in preparing anti-prostate cancer.
  • Prostate cancer the most common malignant tumor in the male reproductive system, is a disease that occurs in older men, and its incidence increases with age.
  • the incidence of prostate cancer is very inconsistent throughout the world. There are more than 900,000 new cases worldwide each year, of which more than 260,000 have died of prostate cancer.
  • the incidence of prostate cancer in Europe and the United States is significantly higher than in Asia and Africa.
  • prostate cancer accounts for the first place in the incidence of cancer in the United States and the second in the mortality rate.
  • the incidence of prostate cancer has increased by 70% compared with the past, ranking first in the incidence of malignant tumor growth in male reproductive system in China. The incidence rate increased from 1.71/100,000 males in 1993 to 7.9 in 2005.
  • Prostate cancer is becoming a urinary artery that seriously affects the health of men in China. Systemic malignancy.
  • the incidence of prostate cancer in China may more than double, and the incidence of prostate cancer may enter a peak period.
  • the clinical manifestations of prostate cancer are often dysuria, with progressive urinary frequency, urgency, hematuria and other symptoms accompanied by pain or burning sensation during urination and continuous pain in the lower back, upper thigh or pelvis.
  • the growth and metastasis of prostate cancer is closely dependent on androgens. About 90% of androgen secretion comes from the testes, and about 10% comes from the adrenal glands. Androgen (such as dihydrotestosterone, DHT) is secreted into the cell and can bind to the Androgen receptor (AR).
  • the androgen receptor is an important nuclear transcription factor that regulates numerous gene expression and proliferation of prostate cancer cells. When androgen binds to the androgen receptor, the androgen receptor enters the nucleus to initiate gene expression (such as screening for an important indicator of prostate cancer - PSA), thereby promoting prostate cancer growth and metastasis.
  • de-androgen therapy including castration treatment to remove testes and drugs to inhibit androgen levels
  • de-androgen therapy has a significant effect on most prostate cancers.
  • the control of tumors by androgen therapy can only be maintained for 1.5 to 4.0 years.
  • AIPC Androgen-independent prostate cancer
  • HRPC Hormone refractory prostate cancer
  • CRPC Castration resistant prostate cancer
  • prostate cancer There is no single drug in the global medical community that can cure prostate cancer alone.
  • the main treatments for prostate cancer include surgery, radiotherapy, chemotherapy and biological therapy. Not only do patients have to experience the pain of nine deaths, the family economic pressure is huge, and the debt is very heavy. The most unacceptable thing is that the affected relatives endure great pain and finally have to Experience the bitterness of life and death.
  • the latest approved drugs for the treatment of CRPC by the Food and Drug Administration (FDA) include Abiraterone and MDV3100 (Enzalutamide), the former inhibiting the synthesis of androgen, the latter being the androgen receptor.
  • Antagonists both of which target the androgen receptor signaling pathway, have achieved significant results in the treatment of castration-resistant prostate cancer, but after the above-mentioned endocrine therapy and targeted androgen receptor therapy, the patient remains It will produce drug resistance, the treatment effect is very limited, and the cost is high, and the patient's economic affordability is poor. In addition, caused by radiotherapy and chemotherapy The side effects are also the bottleneck of clinical treatment.
  • Chinese medicine plays an irreplaceable role in the treatment of many cancers such as prostate cancer because of its remarkable clinical efficacy. Combined with China's abundant Chinese herbal medicine resources and long-established theory of traditional Chinese medicine, the development of a new drug combination for drug resistance against prostate cancer and possessing China's independent intellectual property rights has great clinical significance for the treatment of prostate cancer.
  • the technical problem to be solved by the present invention is to overcome the deficiencies of the prior art, and to provide a traditional Chinese medicine compound composition with anti-prostate cancer effect, which has remarkable curative effect, small toxic and side effects, and convenient taking, and a preparation method thereof and the same in the field of treating prostate cancer. application.
  • the invention provides a traditional Chinese medicine compound composition, comprising the following parts by weight of Chinese medicinal materials: 4-12 parts of skunk skin, 12-30 parts of Hedyotis diffusa, 3-15 parts of medlar, 15-30 parts of Danshen, summer 5 to 15 parts of dry grass and 4 to 12 parts of licorice.
  • the traditional Chinese medicine compound composition comprises the following parts by weight of Chinese medicinal materials: 6-9 parts of skunk skin, 15-24 parts of Hedyotis diffusa, 4-12 parts of medlar, and 18-21 parts of Danshen, summer. 6 to 10 parts of dry grass and 6 to 9 parts of licorice.
  • the invention also proposes a preparation method of the traditional Chinese medicine compound composition as described above, comprising the following steps:
  • the traditional Chinese medicine concentrate prepared in the step (3) is sterilized by filtration through a microporous membrane to obtain the traditional Chinese medicine compound composition.
  • the invention also proposes the application of the traditional Chinese medicine compound composition to the preparation of a medicament for treating malignant tumors.
  • the malignant tumor is caused by abnormal amplification and mutation of androgen receptor, including prostate cancer, breast cancer, lung cancer, colon cancer, brain cancer, skin cancer, bladder cancer, and kidney cancer.
  • the invention further provides the use of the traditional Chinese medicine compound composition for preparing a medicament for treating prostate cancer.
  • the invention further provides the use of the traditional Chinese medicine compound composition for inhibiting growth, proliferation, metastasis, migration, invasion and promotion of apoptosis of prostate cancer cells.
  • prostate cancer is castration-resistant prostate cancer or androgen-dependent prostate cancer.
  • the present invention further provides the use of the traditional Chinese medicine combination composition for inhibiting the transcriptional activity of androgen receptor induced by dihydrotestosterone in vitro and the transcriptional activity of the androgen receptor lacking the ligand domain, or for inhibiting in vitro Androgen receptor activity.
  • the invention also provides that the traditional Chinese medicine compound composition and the pharmaceutically acceptable carrier are made into an injectable fluid, an aerosol, a cream, a gel, a pill, a syrup, a transdermal patch, a capsule, A drug in the form of a microcapsule, a tablet, a granule or an oral solution.
  • the traditional Chinese medicine composition of the present invention can be used alone as a drug or in combination with other drugs.
  • the present invention also provides the use of the above-mentioned traditional Chinese medicine composition for the proliferation of prostate cancer cell line 22RV1 which inhibits androgen receptor mutation in vitro, and inhibits proliferation of androgen-dependent prostate cancer cell line LNCaP.
  • the invention also provides the use of the traditional Chinese medicine compound composition for inhibiting metastasis of prostate cancer cells in vitro, or inhibiting migration of prostate cancer cells PC-3 and LNCaP and 22RV1 in vitro, or inhibiting dihydrotestosterone in vitro.
  • Use of the induced androgen receptor transcriptional activity and deletion of the transcriptional activity of the androgen receptor of the ligand domain, or inhibition of androgen receptor activity in vitro, inhibition of prostate cancer cell growth, migration and invasion, and promotion of prostate cancer cells Use of Apoptosis, or Inhibition of Proliferation of Androgen Receptor Mutant Cell Line 22RV1 Cells Inhibited by Bicalutamide or MDV3100 In Vitro
  • the present invention also provides the use of the traditional Chinese medicine combination composition for proliferating a prostate cancer cell line 22RV1 which inhibits androgen receptor mutation in vivo, inhibits proliferation of androgen-dependent prostate cancer cell line LNCaP, or is used for in vivo inhibition.
  • Metastasis of prostate cancer cells or for inhibiting the growth and metastasis of castration-resistant prostate cancer in vivo, or for inhibiting androgen receptor activity, inhibiting prostate cancer cell growth, migration and invasion, and promoting apoptosis of prostate cancer cells , or to inhibit the proliferation of androgen receptor-mutant cell line 22RV1 cells that are ineffective in bicalutamide or MDV3100 treatment, or to inhibit the growth of prostate cancer cell 22RV1 in mice, or to inhibit prostate cancer in mice Transfer of cells 22RV1.
  • the invention also proposes the use of the traditional Chinese medicine compound composition for the preparation of a medicament for treating a malignant tumor disease associated with androgen receptor mutation and abnormal amplification.
  • the malignant tumor is used in the proliferation, growth, migration and infiltration of malignant tumor cells caused by abnormal amplification and mutation of the androgen receptor; wherein the malignant tumor includes prostate cancer, breast cancer, lung cancer, colon cancer , brain cancer, skin cancer, bladder cancer, kidney cancer.
  • the traditional Chinese medicine compound composition mentioned in the present invention is prepared from the following parts by weight of Chinese medicinal materials: 4 to 12 parts of skunk skin, 12 to 30 parts of Hedyotis diffusa, 3 to 15 parts of medlar, and 15 to 30 parts of Danshen, summer. 5 to 15 parts of dry grass and 4 to 12 parts of licorice.
  • the traditional Chinese medicine compound composition is prepared from the following parts by weight of Chinese medicinal materials: 6-9 parts of skunk skin, 15-24 parts of Hedyotis diffusa, 4-12 parts of medlar, and 18-21 parts of Danshen. , Prunella vulgaris 6 to 10 parts, licorice 6 to 9 parts.
  • the provided traditional Chinese medicine combination composition inhibits the transcriptional activity of the androgen receptor.
  • the provided traditional Chinese medicine combination composition inhibits proliferation of an androgen receptor-mutated prostate cancer cell line. It inhibits the proliferation of the androgen receptor-mutant prostate cancer cell line 22RV1 in vitro and in vivo, and inhibits the proliferation of the androgen-dependent prostate cancer cell line LNCaP.
  • the provided traditional Chinese medicine compound composition inhibits the metastasis of prostate cancer cells in vitro and in vivo.
  • the traditional Chinese medicine combination composition inhibits the transcriptional activity of the androgen receptor and the growth of prostate cancer cells.
  • the provided traditional Chinese medicine compound composition inhibits proliferation of prostate cancer cells derived from primary tumor tissue of a patient.
  • the traditional Chinese medicine combination composition inhibits colony formation of prostate cancer cells.
  • the provided traditional Chinese medicine compound composition induces apoptosis of the androgen receptor-positive cell lines LNCaP and 22RV1.
  • the provided traditional Chinese medicine combination composition inhibits the growth and metastasis of castration-resistant prostate cancer cells in a mouse in vivo model.
  • the provided traditional Chinese medicine compound composition inhibits the proliferation of the androgen receptor mutant cell line 22RV1 cells which is ineffective in the treatment with bicalutamide or MDV3100.
  • the provided traditional Chinese medicine combination object inhibits migration of prostate cancer cell lines PC-3 and LNCaP cells and in vivo transfer of 22RV1 cells.
  • the provided traditional Chinese medicine compound composition inhibits the growth of prostate cancer cell lines LNCaP, 22RV1, PC-3 and prostate cancer cell lines derived from primary tumor tissues of patients.
  • an application and a method for inhibiting androgen receptor production by a traditional Chinese medicine compound composition are provided.
  • the provided traditional Chinese medicine combination composition significantly down-regulates the androgen receptor transcriptional activity and inhibits downstream gene expression by blocking the protein expression level of the androgen receptor.
  • the traditional Chinese medicine compound composition can inhibit the activity of the androgen receptor-regulated luciferase reporter gene in vitro, and can strongly inhibit the proliferation and migration of prostate cancer cells in vitro and in vivo, and can effectively block the male Hormone receptor signaling pathway.
  • the traditional Chinese medicine compound composition can not only effectively inhibit the proliferation of the androgen-dependent prostate cancer cell line LNCaP, but also inhibit the androgen receptor-mutated prostate cancer cell line 22RV1 (the cell line expression is continuously activated). Mutation of the androgen receptor, and proliferation of clinical drug androgen receptor antagonists, such as bicalutamide and MDV3100. Furthermore, the present invention demonstrates in vitro and in vivo that the provided traditional Chinese medicine combination composition inhibits metastasis of prostate cancer. Further experiments demonstrated that the prostate cancer cell line expressing the androgen receptor has better sensitivity to the traditional Chinese medicine compound composition provided by the present invention.
  • the Chinese herbal compound composition inhibits the protein expression level of the androgen receptor and thereby inhibits the transcriptional activity of the androgen receptor and the growth of prostate cancer cells.
  • the traditional Chinese medicine compound composition provided by the invention can not only treat early androgen-dependent prostate cancer, but also treat advanced castration-resistant prostate cancer.
  • the application of the traditional Chinese medicine compound composition with anti-prostate cancer effect provided by the invention can be made into an injectable fluid, an aerosol, a cream, a gel, and an extract of the traditional Chinese medicine compound composition and a pharmaceutically acceptable carrier. Pills, syrups, transdermal patches, capsules, microcapsules, tablets, granules or oral liquid dosage forms.
  • the traditional Chinese medicine compound composition having anti-prostate cancer effect provided by the invention has a traditional Chinese medicine compound composition having anti-prostate cancer effect and a carrier lactose or corn starch, and if necessary, a lubricant magnesium stearate is added and uniformly mixed. It is then compressed into tablets.
  • the traditional Chinese medicine compound composition with anti-prostate cancer effect provided by the invention is made into a capsule, the traditional Chinese medicine compound composition having anti-prostate cancer effect and the carrier lactose or corn starch are uniformly mixed, granulated, and then encapsulated to form a capsule.
  • the traditional Chinese medicine compound composition having the anti-prostate cancer effect and the diluent lactose or corn starch are uniformly mixed, granulated, and dried to prepare granules.
  • the invention also discloses the application of the provided traditional Chinese medicine compound composition with anti-prostate cancer effect or a pharmaceutically acceptable pharmaceutical preparation thereof for preparing a medicament for treating malignant tumor diseases associated with androgen receptor mutation and abnormal amplification. .
  • the present invention provides a traditional Chinese medicine compound composition having anti-prostate cancer effect, which is prepared from the following parts by weight: 4 to 12 parts of skunk, 12 to 30 parts of Hedyotis diffusa, and 3 ⁇ 15 parts, 15 to 30 parts of Salvia miltiorrhiza, 5 to 15 parts of Prunella vulgaris, 4 to 12 parts of licorice.
  • the above-mentioned traditional Chinese medicine compound composition having anti-prostate cancer effect is prepared from the following parts by weight of raw materials: 6-9 parts of skunk skin, 15-24 parts of Hedyotis diffusa, and scorpion 4 to 12 parts, 18 to 21 parts of Salvia miltiorrhiza, 6 to 10 parts of Prunella vulgaris, and 6 to 9 parts of licorice.
  • the invention combines dialectical treatment, combined with the pathogenesis and symptoms of prostate cancer, and treats prostate cancer with Yiqi Yangxue, Jianshen Detoxification, Sanyu Pain Relief, Promoting Blood and Hemostasis, Regulating Endocrine and Diuretic
  • the overall treatment concept of “positive and anti-cancer” for leaching and eliminating phlegm and blood stasis is the compatibility of the treatment.
  • a Chinese herbal compound composition with anti-prostate cancer effect according to the present invention is selected, and the selected skunk skin, Hedyotis diffusa, Rhizoma Curcumae, Salvia miltiorrhiza, Prunella vulgaris, Licorice 6-flavor Chinese medicine is used in the prescription: Fangzhong skunk skin, taste bitter, phlegm, cold, with heat and dampness, blood stasis and blood stasis, contains anti-tumor ingredients.
  • Hedyotis diffusa has a strong heat-clearing and detoxifying effect, often used in heat poisoning, etc. It contains oleanolic acid, triterpenic acid, and fragrant beans.
  • the active ingredients and effective parts such as hormones and polysaccharides have anti-tumor effects, and triterpenoids have the strongest anti-tumor effect. It is combined with the skunk skin to complement each other, and the effect is high, and both of them are Fang Zhongjun. It is a well-recognized anti-cancer medicine. It is a kind of anti-cancer medicine. It is a kind of anti-cancer medicine. It is a kind of anti-cancer medicine.
  • Salvia miltiorrhiza bitter taste, slightly cold, blood circulation, nourishing blood and soothe the nerves, cooling blood and eliminating phlegm, mainly contains fat-soluble diterpenoids and water-soluble phenolic acid.
  • the diterpenoid component tanshinone IIA is currently the most reported anti-tumor active ingredient in Salvia miltiorrhiza. Both phlegm and salvia have the effects of dilating phlegm and relieving pain, promoting blood circulation and nourishing blood.
  • Prunella vulgaris, bitter, spicy, cold is a commonly used heat-clearing drug, has the effect of clearing the liver and clearing the eye, dispersing and detoxifying; in this side, the work of softening and stagnating is obvious, and the auxiliary drug is further softened and eliminated.
  • scorpion venom is adjuvant. Licorice, sweet, flat, spleen and qi, clearing away heat and detoxifying, reconciling various medicines, in this side mainly promotes the adjustment of the body's immune function and restores the role of righteousness.
  • the preparation method of the traditional Chinese medicine compound composition of the invention comprises: taking the above-mentioned parts by weight of the traditional Chinese medicine compound composition, a plurality of grams of the traditional Chinese medicine of the Chinese herbal medicine, and adding the medicinal material of the traditional Chinese medicine compound composition to soak for 1 to 3 hours in 10 to 15 volumes of distilled water.
  • Decoction in Chinese medicine automatic decoction the number of boiling times is 1 to 3 times, each boiling for 0.5 to 1 hour;
  • the prepared crude extract of traditional Chinese medicine is opened on the automatic decoction of Chinese medicine and evaporated to the final volume. 5 ⁇ 10% of the volume of the initial extract;
  • the obtained traditional Chinese medicine concentrate is filtered through a 0.22 micron microfiltration membrane to obtain a mother liquor decoction mother liquor, and stored in a refrigerator at 4 degrees Celsius for use.
  • the traditional Chinese medicine compound composition with anti-prostate cancer effect provided by the invention is prepared according to the theory of traditional Chinese medicine and the pathogenesis of prostate cancer, and the distribution ratio of each group is stricter, the evil is taking care of both, the monarch and the ambassador, the ups and downs, the ensemble Clearing away heat and detoxifying, qi and activating blood, diuretic Tonglin, softening and dispersing, eliminating silt and relieving pain, and replenishing qi and nourishing blood is a good treatment for moist heat accumulation and blood stasis type prostate cancer, which can not only significantly inhibit tumor growth.
  • Prostate cancer growth and metastasis in mice improve quality of life and immune function, prolong survival, improve biochemical indicators, narrow the scope of the lesion, and synergistically with the chemotherapy drug cyclophosphamide, and no adverse reactions, showing A very good anti-cancer effect.
  • the traditional Chinese medicine compound composition with anti-prostate cancer effect provided by the invention can inhibit the expression of the downstream gene by blocking the protein level of the androgen receptor, and block the androgen receptor signaling pathway, thereby realizing the inhibition of prostate cancer.
  • the drug has a definite curative effect and a clear mechanism of action. It has made new breakthroughs in the treatment of prostate cancer, which can fill the gap in the field of prostate cancer treatment drugs that rely on imports and lack of proprietary intellectual property drugs.
  • the traditional Chinese medicine compound composition with anti-prostate cancer effect provided by the invention has low preparation cost and environmental protection, and can realize industrialized large-scale production.
  • the traditional Chinese medicine compound composition having anti-prostate cancer effect provided by the present invention can be prepared into various dosage forms together with a pharmaceutically acceptable carrier, and is convenient for clinical administration, and is expected to be developed into a new anti-prostate cancer drug.
  • Castration-resistant prostate cancer a patient with prostate cancer who has relapsed after castration (removal of testes or chemical castration to reduce androgen), and castration-ineffective prostate cancer called castration-resistant prostate cancer. Cancer is the biggest killer of patients with prostate cancer.
  • Prostate cancer cell line PC-3 a low degree of differentiation, androgen-independent prostate cancer cells isolated from human prostate cancer bone metastasis tumors, do not express endogenous androgen receptors, have moderate metastatic potential, It exhibits remarkable epithelial cell carcinoma characteristics and is widely used in the study of androgen-resistant prostate cancer.
  • Prostate cancer cell LNCaP established by Horuszewicz et al in 1980 from lymph node metastasis specimens of prostate cancer patients, retaining the characteristics of prostate cancer tumor cytology and its early differentiation function, with significant epithelial cell characteristics, androgen expression
  • the receptor is a typical prostate cancer cell with androgen dependence.
  • Prostate cancer cell 22RV1 is xenografted into androgen-dependent prostate cancer CWR22 cells A human prostate cancer epithelial cell line isolated after serial passage. This cell line expresses prostate specific antigen, which grows depending on the stimulation of androgen, and has a strong tumorigenic ability in nude mice.
  • Cell proliferation The process by which cells produce new individuals and offspring in a split manner. Single-celled organisms that produce new individuals in a manner that divides cells. Multicellular organisms, which produce new cells in the form of cell division to supplement the cells of aging and death in the body; at the same time, multicellular organisms can be transformed into a new multicellular cell by a fertilized egg, through cell division and differentiation. individual. Through cell division, the replicated genetic material can be evenly distributed into two daughter cells. Cell proliferation is the basis of organism growth, development, reproduction and inheritance, and is an important life feature of organisms.
  • Apoptosis In order to maintain homeostasis, cells controlled by genes are autonomous, ordered, and programmed to die. Apoptosis involves the activation, expression and regulation of a series of genes, and is a cell death process that is actively pursued to adapt to the living environment.
  • Cell migration Also known as cell crawling, cell migration, or cell movement, is the movement of a cell after it receives a migration signal or senses a gradient of certain substances.
  • Cell migration is an extension of the cell's head pseudopod, new adhesion establishment, and cell body tail contraction in space and time.
  • Cell migration is one of the basic functions of normal cells, a physiological process of normal growth and development of the body, and a form of exercise that is prevalent in living cells.
  • Cell migration is involved in embryonic development, angiogenesis, wound healing, immune response, inflammatory response, atherosclerosis, cancer metastasis, and the like.
  • the basement membrane is a specialized extracellular matrix that is a physical barrier that must be crossed during tumor cell metastasis.
  • the composition of the basement membrane interacts with the cell surface receptor integrin. Tumor cells destroy the tissue structure of the basement membrane by secreting matrix degrading enzymes, and regulate the expression of the extracellular matrix receptor integrin, which creates favorable conditions for the adhesion and proliferation of the target membrane through the basement membrane.
  • Tumor growth Tumor cells are enlarged due to dysplasia and invade the surrounding tissue space, lymphatic vessels or blood vessels, and are closely connected with adjacent normal tissues without obvious boundaries.
  • Tumor metastasis The process in which a tumor cell invades a lymphatic vessel, a blood vessel, or a body cavity from its primary site, and carries it along with blood flow, lymphatic flow to another site, or the organ continues to grow to form a tumor of the same type as the primary tumor.
  • Prostate cancer growth and metastasis In the process of malignant transformation of prostate cancer, tumor cell population dynamics, tumor cell apoptosis rate, tumor angiogenesis, tumor progression and heterogeneity deterioration due to enhanced tumor cell proliferation and migration and invasion the process of. Prostate cancer becomes more and more aggressive during growth and metastasis, eventually leading to accelerated tumor growth, infiltration of peripheral tissues and distant metastasis.
  • the androgen receptor belongs to the steroid receptor in the nuclear receptor superfamily.
  • the human androgen receptor is a single-copy gene with a molecular weight of approximately 110 kilodaltons and generally consists of four domains: the N-terminal domain (NTD) and the DNA binding domain (DNA binding domain, DBD), hinge region and Ligand binding domain (LBD).
  • Androgen receptors are closely related to the development of prostate cancer. Androgen For example, when Dihydrotestosterone (DHT) binds to androgen receptor, the androgen receptor can enter the nucleus and bind to the transcriptional activation domain to initiate downstream gene expression.
  • DHT Dihydrotestosterone
  • MDV3100 Enzalutamide
  • the androgen receptor is a typical nuclear receptor transcription factor that regulates the expression of various eukaryotic genes by mediating the action of androgens. Its specific function is to regulate the transcription and expression of its downstream target genes by transcriptional activation, and to exert transcriptional activity under both ligand-dependent pathway and ligand-independent pathway.
  • the Androgen Receptor LBD Region consists of 12 discrete alpha helices that form a ligand binding pocket with the ability to bind to a ligand.
  • the androgen receptor may lack the LBD region in the case of mutation, and the androgen receptor lacking the LBD region has the same constitutive transcriptional activation function as the intact androgen receptor.
  • Luciferase assay The Luciferase (Luc) reporter assay system uses Luciferin as a substrate to detect the activity of Firefly luciferase. Luciferase catalyzes the oxidation of Luciferin to Oxy-luciferin, which emits bioluminescence during Luciferin oxidation. The bioluminescence released during the oxidation of Luciferin can then be determined by a fluorometer, also known as a Luminometer or a liquid scintillation meter. The bioluminescent system of fluorescein and luciferase can detect the expression of genes extremely sensitively and efficiently, and is a detection method for detecting the interaction between transcription factors and the promoter region of the target gene.
  • Fig. 1 is a graph showing the inhibitory effect of the traditional Chinese medicine composition on the proliferative activity of prostate cancer cells of each strain.
  • Fig. 1(A) shows the inhibition of proliferation of prostate cancer cells of each strain and the selective effect on normal prostate epithelial cells by the traditional Chinese medicine composition of the present invention.
  • Fig. 1(B) shows the time-dependent and concentration-dependent gradient-dependent effect of the composition of the traditional Chinese medicine compound on the proliferation of LNCaP cells.
  • Fig. 1(C) shows the time-dependent and concentration-dependent gradient-dependent effect of the Chinese herbal composition of the present invention on the proliferation inhibition of 22RV1 cells.
  • Fig. 1(D) shows the time-dependent and concentration-dependent gradient-dependent effect of the traditional Chinese medicine compound composition on the proliferation of PC-3 cells.
  • Fig. 1(E) shows the time-dependent and concentration-dependent gradient-dependent effect of the combination of the traditional Chinese medicine composition of the present invention on the proliferation of prostate cancer cells derived from a patient's tumor tissue.
  • Fig. 2 is a graph showing the inhibitory effect of the Chinese herbal composition of the present invention on the colony formation of prostate cancer cells of each strain.
  • Fig. 2 shows the white light which inhibits the formation of LNCaP and 22RV1 by the Chinese herbal compound composition of the present invention. photo.
  • Fig. 2 shows the statistical results of the inhibitory effect of the traditional Chinese medicine composition of the present invention on the colony formation of LNCaP and 22RV1, respectively.
  • Fig. 3 is a graph showing the effect of the Chinese herbal compound composition on apoptosis induction of prostate cancer cells.
  • Fig. 3(A) shows the apoptosis-inducing effect of the traditional Chinese medicine composition of the present invention on LNCaP cells.
  • Fig. 3 (B) shows the apoptosis-inducing effect of the traditional Chinese medicine composition of the present invention on 22RV1 cells.
  • Fig. 3(C) shows the statistical results of the induction of apoptosis of LNCaP, 22RV1 by the traditional Chinese medicine composition of the present invention.
  • Fig. 4 is a graph showing the inhibitory effect of the traditional Chinese medicine composition on the migration and invasion of prostate cancer cells.
  • Fig. 4 shows the inhibitory effect of the traditional Chinese medicine composition of the present invention on the lateral migration process of PC-3 cells.
  • Fig. 4 shows the inhibitory effect of the traditional Chinese medicine composition of the present invention on the longitudinal migration process of LNCaP cells.
  • Figure 4 shows the inhibitory effect of the Chinese herbal composition of the present invention on the invasion process of 22RV1 cells.
  • Fig. 5 is a view showing the molecular mechanism of the traditional Chinese medicine composition for inhibiting prostate cancer by affecting the androgen receptor signaling pathway.
  • Fig. 5(A) shows the inhibitory effect of the traditional Chinese medicine composition of the present invention on the expression of the luciferase reporter gene for detecting androgen receptor transcriptional activity induced by dihydrotestosterone (DHT) in LNCaP cells.
  • DHT dihydrotestosterone
  • Fig. 5(B) shows the inhibitory effect of the traditional Chinese medicine composition of the present invention on the expression of the luciferase reporter gene for detecting androgen receptor transcriptional activity induced by dihydrotestosterone (DHT) in 22RV1 cells.
  • DHT dihydrotestosterone
  • Figure 5 (C) shows the fluorescence of the Chinese herbal compound composition for detecting androgen receptor transcriptional activity after exogenous transfection of wild-type androgen receptor (AR) in PC-3 cells induced by dihydrotestosterone (DHT).
  • the enzyme inhibits the inhibitory effect of gene expression.
  • Fig. 5(D) shows that the Chinese herbal composition of the present invention does not affect the transcription level of the androgen receptor (AR) gene itself in LNCaP cells.
  • Fig. 5(E) shows that the Chinese herbal compound composition of the present invention down-regulates the expression level of androgen receptor (AR) protein in LNCaP cells.
  • AR androgen receptor
  • Figure 5 (F) shows that the Chinese herbal compound composition of the present invention inhibits the transcriptional activation of the androgen receptor (AR) for its three classical downstream target genes TMPRSS2, FKBP5, PSA.
  • AR androgen receptor
  • Figure 6 is a graph showing the inhibition of the growth of 22RV1 xenograft tumors in a subcutaneous tumor-bearing animal model of nude mice according to the present invention.
  • Fig. 6(A) is a white light photograph showing the effect of the combination of the traditional Chinese medicine composition of the present invention on the tumor volume inhibiting effect in the 22RV1 xenograft tumor growth model.
  • Fig. 6(B) is a graph showing the statistical results of the inhibitory effect of the Chinese herbal compound composition of the present invention on the tumor volume increase with the number of days in the 22RV1 xenograft tumor growth model.
  • Fig. 6(C) is a white light photograph showing the tumor weight inhibiting effect of the Chinese herbal compound composition of the present invention on the peeling of the 22RV1 xenograft tumor growth model.
  • Figure 7 is a graph showing the inhibition of growth and metastasis of 22RV1-luc xenograft tumors in an in vivo animal model of in situ xenograft tumor growth and metastasis in nude mice of the present invention.
  • Fig. 7 (A) shows the results of in vivo imaging of the inhibitory effect of the Chinese herbal compound composition of the present invention on the tumor fluorescence intensity in the 22RV1-luc xenograft tumor growth and metastasis model.
  • Fig. 7(B) is a graph showing the statistical results of the inhibitory effect of the Chinese herbal compound composition of the present invention on the in situ tumor metastasis to the whole body organ-forming metastatic nodules in the 22RV1-luc xenograft tumor growth and metastasis model.
  • Fig. 7(C) is a graph showing the statistical results of the inhibitory effect of the Chinese herbal compound composition of the present invention on the increase in the fluorescence intensity of the whole body tumor with the increase in the number of days in the 22RV1-luc xenograft tumor growth and metastasis model.
  • Fig. 7(D) shows the effect of the traditional Chinese medicine composition of the present invention on the body weight of nude mice under the mode of intragastric administration.
  • the traditional Chinese medicine compound composition having anti-prostate cancer effect in the present embodiment is made of the following raw materials: 9 parts of skunk skin, 30 parts of Hedyotis diffusa, 15 parts of medlar, 30 parts of Salvia miltiorrhiza, 15 parts of Prunella vulgaris, 9 parts of licorice Soaked in 15 times volume of distilled water of Chinese herbal compound composition for 3 hours; decoction in Chinese medicine automatic decoction, the number of boiling times is 2 times, each boiling for 1 hour; the prepared crude extract of traditional Chinese medicine in traditional Chinese medicine
  • the automatic decoction device was evaporated and concentrated to a final volume of 70 ml; the final concentration of the concentrated Chinese herbal decoction mother liquor was about 1.5 g crude drug/ml.
  • the obtained traditional Chinese medicine concentrate was filtered through a 0.22 micron microfiltration membrane to obtain a mother liquor decoction mother liquid, and stored in a refrigerator at 4 degrees Celsius for use.
  • the cells used in this experiment were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences; the other cell was derived from prostate cancer cells derived from the primary tumor of prostate cancer patients in Shanghai Changhai Hospital.
  • Primary PCa cells were cultured in a 37 ° C incubator (95% humidity, 5% CO 2 concentration) in RPMI-1640 (Gibco) containing 10% fetal bovine serum (Front).
  • SRB sulfonyl rhodamine
  • Different prostate cancer cell lines were inoculated into 96-well plates (Corning) at a density of 5 ⁇ 10 3 cells/well. After 24 hours, different concentrations of the traditional Chinese medicine compound composition of the present invention were added, and the control group was added with an equal amount of PBS, and each group was set to 6 Multiple holes. After 48 hours of drug treatment, cells were fixed by adding pre-cooled TCA (trichloroacetic acid, 50%, w/v) for 60 minutes at 4 degrees Celsius. After fixing, rinse the running water 5 times and air dry. 50 ⁇ l of SRB stain (4%, w/v) was added to each well and stained for 10 minutes at room temperature.
  • TCA trichloroacetic acid
  • the dye solution was aspirated and washed with 100 ⁇ l of 1% acetic acid per well for 5 times to remove unbound dye. After air drying, 100 microliters of a 10 millimolar Tris solution was added to each well, and the bound SRB dye was dissolved by shaking. The 96-well plate was placed in a microplate reader (SPECTRA MAX 190) and the OD value was measured at a wavelength of 515 nm. The effect of the Chinese herbal compound composition on the level of cell proliferation was statistically analyzed.
  • Colony formation can be used to detect the proliferation and tumorigenic ability of tumor cells to form clones in vitro.
  • Tumor cells can proliferate by themselves to form macroscopic tumors that are visible to the naked eye.
  • the ability of tumor cells to form solid tumors can be reflected in the ability of tumor cells to form solid tumors.
  • 1000 prostate cancer cells were inserted into 6-well plates. After the cells were attached, different concentrations of the traditional Chinese medicine compound composition were added. After one week, the cells were fixed with paraformaldehyde, then stained with crystal violet, photographed and counted. Each set of experiments was repeated three times.
  • phosphatidylserine In normal cells, phosphatidylserine (PS) is only distributed inside the lipid bilayer of the cell membrane, and in the early stage of apoptosis, phosphatidylserine (PS) in the cell membrane is turned from the inside of the lipid membrane to the outside.
  • Annexin V is a Ca 2+ -dependent phospholipid binding protein with a molecular weight of 35 to 36 kD. It has a high affinity with phosphatidylserine, so it can bind to the cell membrane of early apoptosis cells through phosphatidylserine exposed on the outside of the cell. Therefore, Annexin V is used as one of the sensitive indicators for detecting early apoptosis of cells.
  • Annexin V was labeled with fluorescein (EGFP, FITC, etc.), and labeled Annexin V was used as a fluorescent probe, and apoptosis was detected by fluorescence microscopy or flow cytometry.
  • Propidium iodide (PI) is a nucleic acid dye that does not penetrate the intact cell membrane, but for advanced and late apoptotic cells, PI can pass through the cell membrane to stain the nucleus. Therefore, by matching AnnexinV with PI, cells in different stages of apoptosis can be distinguished.
  • the specific steps are as follows: different prostate cancer cells are treated with different concentrations of the drug solution of the traditional Chinese medicine compound composition for 36 hours, and then the suspension cells are collected for centrifugation (2000 rpm, centrifugation for 5 minutes); the adherent cells are EDTA-free pancreas Enzyme digestion was collected; cells were washed twice with PBS (2000 rpm, centrifugation for 5 minutes) to collect 1 to 5 ⁇ 10 5 cells; 500 ⁇ l of Bindingbuffer suspension cells were added; 5 ⁇ L of Annexin V-FTIC was added and mixed. Add 5 ⁇ l of PI and mix; room temperature, protected from light, and react for 5 to 15 minutes; perform on-line detection by flow cytometry within 1 hour.
  • the traditional Chinese medicine compound composition has a significant inhibitory effect on the proliferation of the progenitor cancer cells of each strain, including the primary prostate cancer cells derived from the patient tissue, and the half-time inhibitory concentration is about 5 ⁇ g in 48 hours.
  • the traditional Chinese medicine composition of the present invention has a significant inhibitory effect on the proliferation of the conventional prostate cancer cell line and the primary prostate cancer cell derived from the patient tissue, and the normal prostate epithelial cell line PNT1A has a high concentration (100 ⁇ g/ml). Slight inhibition, but at low concentrations, the inhibitory effect is small, showing a good selectivity between normal cells and prostate cancer cells.
  • the inhibitory effect of the traditional Chinese medicine compound composition on the proliferation of prostate cancer cells of each strain is in a concentration gradient-dependent and time-gradient-dependent manner.
  • the traditional Chinese medicine composition of the present invention also strongly inhibits the colony formation of prostate cancer cells of each strain.
  • Figure 3 shows that the Chinese herbal composition of the present invention significantly promotes apoptosis of prostate cancer cells LNCaP, 22RV1. The experimental results suggest that the traditional Chinese medicine composition of the present invention can be used as a potential drug against prostate cancer growth.
  • Example 3 Inhibition of migration and invasion ability of prostate cancer cells by the Chinese herbal compound composition of the present invention
  • PC-3 cells were seeded into 6-well plates and cultured in a 37 ° C 5% CO 2 incubator for 24 hours until the cells were 100% full. .
  • the serum-free medium was replaced and incubation was continued for 12 hours.
  • a 200 ⁇ l sterilized tip was used for scratching, and after scratching, the cells were washed twice with PBS, and the floated cells were aspirated, and 1 ml of complete medium was added to each well.
  • Different concentrations of the traditional Chinese medicine compound composition were separately added to the cell culture wells, and the culture plate was placed in a CO 2 incubator, and the conventional culture was continued at 37 ° C for 24 hours. Under the microscope, observe the movement of the cells to the underlined portion and take a picture. The number of cells migrated into the streaked area by different doses of the drug group was statistically analyzed to determine the effect of the drug on cell migration ability.
  • the permeable polycarbonate film on the bottom of the Transwell chamber has a large number of micropores with a diameter of 8 microns.
  • the Transwell chamber is placed in a 24-well plate, and the polycarbonate film divides each hole of the 24-well plate into upper holes. , the next two rooms, Transwell
  • the small chamber is called the upper chamber
  • the lower chamber is called the lower chamber
  • the upper chamber is filled with the upper culture medium
  • the lower chamber is filled with the lower culture medium
  • the upper and lower culture liquids are separated by a polycarbonate membrane.
  • the components in the lower culture medium can induce the cells in the upper chamber to migrate to the lower chamber due to the permeability of the polycarbonate membrane, so that the cells move from the upper chamber surface to the lower chamber surface of the membrane, thus
  • This experiment is a classic experiment to study cell movement.
  • a Transwell chamber is used to access an excess of LNCaP cells in the upper chamber, so that the cells migrate to the lower chamber; and different concentrations of the traditional Chinese medicine compound composition are added. Photocell migration was measured after 12 hours.
  • the Transwell chamber is placed in a 24-well plate.
  • the upper chamber contains the upper culture medium
  • the lower chamber contains the lower culture medium.
  • the upper and lower culture solutions are separated by a polycarbonate membrane.
  • the upper chamber of the Transwell chamber is pre-plated with a collagen matrix Matrigel, and then the cells are placed. Inoculate it. Due to the permeability of the polycarbonate membrane, the components in the lower culture medium can induce the cells in the upper chamber to migrate downward, causing the cells to move from the upper chamber surface to the lower chamber surface of the membrane.
  • tumor cells are to move to the lower chamber, they must first dissolve the collagen matrix by using various chemical substances released by themselves, and then move through the dissolved cavity. This process is similar to the infiltration process of tumor cells in the body.
  • This experiment is a classic experiment to study cell invasion.
  • the tumor cells in the logarithmic growth phase were inoculated into the upper chamber of the Transwell chamber at 5 ⁇ 10 4 cells/well.
  • the drug-added group was added with the corresponding concentration of the traditional Chinese medicine compound composition, and the control group was added with the same amount of PBS.
  • Complete medium was added to each of the lower chambers. Incubate for 12 hours in a 37 ° C 5% CO 2 incubator. Remove the Transwell chamber, wipe the upper chamber side of the Transwell chamber with a cotton swab, and wipe off the unpenetrated cells.
  • the Transwell chamber was fixed in 4% paraformaldehyde for 10 minutes at room temperature, stained with 1% crystal violet for 10 minutes, and rinsed with tap water. Photographs were taken under a microscope, and the number of cells in five fields in the upper, lower, left, and right sides of each well was counted. There are an average of 3 duplicate holes in each group. The number of transmembrane cells in different doses of Chinese herbal compound composition plus drug group was statistically compared to determine the effect of Chinese herbal compound composition on cell invasion ability.
  • the traditional Chinese medicine composition of the present invention can effectively inhibit the lateral migration of PC-3, the longitudinal migration of LNCaP and the invasion ability of 22RV1 at a lower dose (2.5 ⁇ g/ml).
  • the left side of Figure 4 is the experimental white light photo results of the three cells, and the right side of Figure 4 is the statistical results of the three cells.
  • the experimental results suggest that the traditional Chinese medicine compound composition can be used as a potential drug against prostate cancer metastasis.
  • Example 4 Inhibition of androgen receptor transcriptional activity by the combination of the traditional Chinese medicine compound of the present invention
  • MMTV-LUC is a reporter gene plasmid with androgen receptor transcriptional activation element (AR response elements) and luciferase.
  • the androgen receptor can bind to androgen to activate the plasmid to express luciferase.
  • Luciferase can be used. It catalyzes the fluorescence of its substrate fluorescein, and detects the degree of androgen receptor activation by detecting the intensity of fluorescence; the Renila internal reference reporter gene carries the promoter region of Renilla luciferase, which can express Renilla luciferase.
  • This plasmid can determine whether the transfection efficiency of each group of cells is consistent, and androgen has no effect on the Renila internal reference reporter plasmid.
  • Chinese herbal compound composition of the present invention can inhibit the transcriptional activity of androgen receptor (AR)
  • the Chinese herbal compound composition of the present invention can dose-dependently reduce androgen (DHT)-induced MMTV-LUC reporter gene activity in different prostate cancer cell lines, and at a low concentration ( A half-inhibition rate (IC 50 ) was achieved at about 2.5 ⁇ g/ml, demonstrating that the Chinese herbal composition of the present invention can inhibit the transcriptional activity of the androgen receptor in vitro.
  • DHT androgen
  • IC 50 A half-inhibition rate
  • Example 5 The traditional Chinese medicine compound composition significantly down-regulates androgen receptor protein levels and the transcription level of its target gene
  • LNCaP cells were isolated and extracted with TRIzol.
  • the RNA was reverse transcribed to obtain cDNA.
  • the mRNA expression level of AR gene was qualitatively detected by reverse transcription-PCR (RT-PCR) using AR-specific primers.
  • the mRNA expression levels of the three were detected by fluorescent real-time quantitative PCR (Q-PCR) using different AR downstream target gene-specific primers (TMPRSS2, FKBP5 and PSA).
  • LNCaP cells were treated with different concentrations of traditional Chinese medicine compound composition
  • the cells were extracted by cleavage, the protein was boiled and denatured, and the protein sample was separated by polyacrylamide gel electrophoresis, and then transferred to nitrocellulose membrane, first with androgen receptor.
  • the antibody of AR was incubated for two hours, and then incubated with a fluorescently labeled secondary antibody for one hour.
  • the effect of the composition of the traditional Chinese medicine compound of the present invention on the expression of AR protein was examined using a Licor Odyssey two-color infrared laser imaging system.
  • Fig. 5E Western blot showed that the traditional Chinese medicine compound composition can significantly inhibit the protein expression level of androgen receptor; with the increase of drug concentration, the protein expression of AR is significantly reduced. It is suggested that the inhibitory effect of the traditional Chinese medicine compound composition on proliferation and migration of prostate cancer tumor cells may be achieved by inhibiting the androgen receptor signaling pathway.
  • the RT-PCR experiment found that the Chinese herbal compound composition of the present invention did not affect the mRNA level of AR at a certain concentration (Fig. 5D), but significantly inhibited the transcriptional activation of its downstream target gene (Fig. 5F).
  • Example 6 Inhibitory effect of the traditional Chinese medicine compound composition on subcutaneous tumor growth model of prostate cancer in nude mice and in situ growth and metastasis model of prostate cancer
  • a human prostate cancer cell 22RV1 was used to establish a subcutaneous tumor growth model of nude mice.
  • Five million 22RV1 cells were subcutaneously injected into the right dorsal back of 7-week-old male immunodeficient mice (BLAB/c-nude, nude mice).
  • the subcutaneous tumors grew to about 100 mm3, the mice were divided into 8 groups. (The average tumor volume is the same), followed by model group, control group, Chinese medicine compound (excluding skunk skin) group, Chinese medicine compound low-dose group, Chinese medicine compound high-dose group, compound Kushen injection group, cyclophosphamide (Cyclophosphamide, CTX) group, CTX+ Chinese medicine compound high dose group.
  • mice in the model group were not treated at all, and the mice in the control group were given an equal amount of PBS per day (0.1 ml), and the mice in the traditional Chinese medicine compound (excluding the skunk skin) group were given daily.
  • the body weight of the mice was measured every 2 days. The mice were sacrificed 14 days after the application, subcutaneous tumors were taken and photographed.
  • Stable cells can express luciferase. Fluorescein will fluoresce when contacted with its substrate fluorescein. The range and intensity of fluorescence are positive with the location and volume of the tumor. Correlation, such that the number and location of tumor cells can be detected by the animal in vivo imaging system.
  • the constructed stable cell line was named 22RV1-luc, and 22RV1-luc cells were mixed with Matrigel and injected into the dorsal lobe of mouse prostate.
  • 22RV1 in mice can be detected by in vivo imaging.
  • - Growth and metastasis of luc cells The specific operation procedure was as follows: 7-week-old male immunodeficient mice were anesthetized, and the abdominal cavity was dissected to expose the prostate. 500,000 22RV1-luc cells were mixed with Matrigel and injected into the dorsal lobe of the prostate. The fluorescence value was detected by the living imaging system at 0 days, and the mice were randomly grouped and administered. The administration method was the same as that of the previous animal experiment, and the growth curve was measured every 3 days and the fluorescence intensity increased with the number of days. The body weight of the mice was measured every 2 days. The experiment was terminated on the 15th day of the experiment, the mice were sacrificed, and the number of statistical metastatic nodules in the organs of the mice was stripped.
  • Fig. 6 shows that the tumor volume increased with the increase of the experimental days, and the tumor volume of the model group and the control group was the largest, and there was no significant difference between the two groups, indicating that the modeling was successful.
  • the final volume of the tumor in the traditional Chinese medicine compound (excluding skunk skin) group was slightly reduced compared with the control group, indicating that there was a certain effect, but the effect was not obvious; the effect of the low-dose group of Chinese medicine compound was lower than that without skunk skin.
  • the effect of the group was good, but it was still not significant; the high-dose group of Chinese medicine compound was more effective, indicating that the traditional Chinese medicine compound of the present invention can effectively inhibit the growth of prostate cancer tumor; the compound Kushen injection group is a traditional Chinese medicine compound positive control. In this experiment, it also showed a good effect of inhibiting tumors, but from the experimental results, the inhibitory effect was not as strong as that of the traditional Chinese medicine compound of the present invention; the CTX group was used as a positive control for traditional chemotherapy drugs, and the tumor was obvious.
  • FIG. 7A is a therapeutic effect diagram (n ⁇ 5) of the mouse prostate orthotopic tumor after 14 days of administration, through the living body imaging system of the animal. Photographing the picture, the colored area in the figure represents the fluorescent signal, indicating that there is tumor cell aggregation in this area, and the deeper the fluorescence intensity, the larger the tumor.
  • the experimental results are similar to those of the previous animal experiment.
  • the Chinese medicine compound high-dose group is more effective, indicating that the traditional Chinese medicine compound composition of the present invention can effectively inhibit the growth of prostate cancer tumors (Fig. 7C) and metastasis (Fig.
  • the combination of the traditional Chinese medicine composition of the present invention and CTX has the most significant inhibitory effect on tumor growth and metastasis in all groups, and the body weight of the mouse is restored (Fig. 7D), indicating the traditional Chinese medicine of the present invention.
  • the compound composition can not only enhance the anti-tumor effect of the traditional chemotherapeutic drugs, but also improve the damage to the body caused by the toxicity of the traditional chemotherapeutic drugs, thereby demonstrating that the traditional Chinese medicine compound composition of the present invention has the effect of attenuating and attenuating the traditional chemotherapeutic drugs. effect.
  • Example 7 Effect of Chinese herbal compound composition on synergistic and attenuating effects and immune function indexes of tumor-bearing nude mice
  • the control group and the drug-administered group, especially the tumor-bearing mice in the chemotherapy drug CTX group showed different degrees of skin roughness, apathy, laziness, and unresponsiveness.
  • the invention further reduces the state, and the traditional Chinese medicine compound combination group of the invention has smooth skin, good spirit, sensitive activity, normal body weight and small in situ tumor mass, so it is speculated that the traditional Chinese medicine compound composition of the invention increases the chemotherapy drug.
  • the effect of attenuating the effect may be achieved by improving the immune function of the mouse and improving various immune indexes. Therefore, after the nude mice in the in situ tumor growth metastasis model of Example 6 were sacrificed at the end of the experiment, the spleen weight and body weight were weighed, and the spleen index was calculated.
  • the spleens were weighed and placed in a pre-prepared test tube containing pre-cooled PBS, and the spleens and washings in the test tubes were poured into the dishes.
  • the spleen was placed in a cell strainer and the spleen was squeezed with forceps to obtain a cell suspension.
  • the cell suspension was collected into a test tube, centrifuged at 1500 rpm for 6 minutes, and the washing solution was drained, and the red blood cell lysate was lysed for 3 minutes. After the liquid color became light, the washing liquid PBS was added, and the membrane was filtered.
  • Table 1 shows the statistical results of three typical indicators of mouse immune function. There were no significant differences in the three indicators between the respective administration groups of the Chinese herbal compound composition and the control group of the model group. Compared with the model group, the three indicators in the CTX group showed significant differences. The toxicity of the chemotherapy drug CTX to the body was again verified, and the immune system was damaged. The traditional Chinese medicine compound composition of the present invention was combined with the drug. After that, it can significantly restore its toxic effects on mice, and significantly improve the quality of life of tumor-bearing mice.
  • the traditional Chinese medicine compound composition of the invention has small toxic and side effects, does not cause atrophy of the immune organs, and has obvious synergistic and attenuating effects in combination with CTX, suggesting the resistance of the traditional Chinese medicine compound composition of the present invention.
  • the traditional Chinese medicine compound composition of the invention not only has the function of inhibiting the growth and metastasis of prostate cancer, but also enhances the anti-tumor effect of the traditional chemotherapy drug, and improves the damage to the body caused by the toxicity of the traditional chemotherapy drug, thereby It is proved that the traditional Chinese medicine compound composition of the invention has the effect of increasing efficiency and attenuation of traditional chemotherapeutic drugs.
  • Table 1 Effect of Chinese herbal compound composition on synergistic and attenuating effects and immune function indexes of tumor-bearing nude mice
  • Example 8 Verification of the preferred scheme of the traditional Chinese medicine compound composition of the present invention
  • Example 2 In order to verify the preferred effect of the distribution ratio of each group of the traditional Chinese medicine compound composition of the present invention, the dried Liuyi Chinese herbal medicines of different ratios as shown in Table 2 were weighed, and the traditional Chinese medicine decoction was prepared according to the method of Example 1 and implemented.
  • the Chinese medicine compound composition of Example 1 was subjected to the parallel experiment of Examples 2-7, and it was found that the different Chinese medicine compound compositions 8a-8g in the present example were as effective as the Chinese medicine compound composition in Example 1, and were all implemented.

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Abstract

一种具有抗前列腺癌作用的中药复方组合物及其应用，该中药复方组合物包括下列重量份数的中药原料：臭椿皮4～12份，白花蛇舌草12～30份，莪术3～15份，丹参15～30份，夏枯草5～15份，甘草4～12份。该具有抗前列腺癌作用的中药复方组合物，根据中医药理论和前列腺癌的发病机制选取原料配伍组方，各组份配比科学合理，药理实验和临床前实验结果表明该中药复方组合物具有良好的抗前列腺癌的作用，不仅能显著抑制前列腺癌的增殖和生长，更能抑制前列腺癌的迁移、侵袭和转移，明显提高荷瘤小鼠的生存质量，改善荷瘤小鼠的各项免疫指标，且给药途径方便简捷，无明显不良反应，可被开发成新一代抗前列腺癌的药物，有望成为治疗前列腺癌的良方。

Description

一种中药复方组合物及其在抗前列腺癌中的应用 技术领域
本发明涉及医药技术领域，具体涉及一种中药复方组合物及其在制备抗前列腺癌中的应用。
背景技术
前列腺癌(Prostate cancer，PCa)作为男性生殖系统最常见的恶性肿瘤，是一种好发于老年男性的疾病，其发病率随年龄增加而增长。前列腺癌的发病率在全世界范围内分布很不一致。每年全球有90多万新发病例，其中26万多病例因前列腺癌的恶化而死亡。欧美国家前列腺癌的发病率明显高于亚非地区。在美国，前列腺癌占美国男性肿瘤发病率的第一位、致死率的第二位。在我国，前列腺癌的发病率较以往增长了70％，居我国男性生殖系统恶性肿瘤发病增长率的首位，发病率从1993年的1.71人/10万男性人口增加到2005年的7.9人/10万男性人口，并且还在以每年10％的增长速度攀升。根据《临床肿瘤学杂志》2013年第04期关于中国前列腺癌发病现状和流行趋势分析的文献报道，我国前列腺癌的发病率呈现明显的持续增长趋势，前列腺癌正成为严重影响我国男性健康的泌尿系统恶性肿瘤。未来10年，随着我国社会老龄化现象日趋严重，我国前列腺癌的发病率或将增加1倍以上，前列腺癌的发病率可能会进入高峰期。前列腺癌的临床表现常见为排尿困难，呈渐进性地出现尿频、尿急、血尿等症状并伴随排尿时的疼痛感或灼烧感及背下部、大腿上部或骨盆处的连续疼痛。前列腺癌肿瘤小时无任何临床表现，常以出现转移症状为最早的就诊原因，因此单靠临床症状很难早期诊断。由于前列腺癌潜伏期较长，很难察觉，发现时一般已经发展为晚期前列腺癌，给患者造成严重不良后果。
中医药防治肿瘤的效果早已被临床证实。中医对前列腺癌的治疗，早在汉代张仲景《金匮要略》及隋朝巢元方的《诸病源候论》就已论及，其后历代医家各有所述。根据本病的主要临床表现及体征，中医学往往将前列腺癌归入“淋证”、“尿血”、“积聚”、“癥瘕”等疾病范畴。肾气亏虚、瘀血败精、聚积下焦是前列腺癌主要的病因病机。明代张景岳的《景岳全书》较全面地提示了清热解毒、活血祛瘀、软坚消结、温阳利水等治法。近代医学家在大量临床实践中确定了辨证施治、攻邪扶正的辨治模式，在配合手术、放疗、化疗、内分泌治疗过程中起增效、减少毒性反应和不良反应的作用，辨证论治前列腺癌临床应用广泛。针对前列腺癌总的病因病机，湿热下注、痰瘀互结、肝肾阴虚、气血两虚四种证型已被临床科研所采用。诸多学者运用一些临床验方等单方和复方治疗或联合西医治疗前列腺癌，均可在一定程度上提高患者的疗效及生活质量，减轻症状，改善体力，降低血清前列腺特异性抗原(Prostate specific antigen，PSA)的水平，延迟抗雄激素治疗后出现雄激素非依赖性抵抗的 时间，患者的生活质量得以明显改善。近几年文献报道中医药作用于前列腺癌的研究中对于细胞株的研究较多，新的作用机制、调节通路不断被阐述，在一定程度上深化了中医药治疗前列腺癌的机理，明确了中医药治疗前列腺癌的疗效。晚期患者失去手术、放疗、化疗机会，单纯用中医药治疗以达到减轻痛苦，延长生存期，提高生命质量的目的。中药复方因其显著的临床疗效，在前列腺癌等诸多癌症的治疗中发挥着无可替代的作用，在提高患者生活质量、延缓肿瘤转移与进展方面具有巨大的应用潜力与发展前景。寻求有效、低毒的中医药，特别是对中晚期难治性前列腺癌患者而言，具有深远的临床意义。因此很有必要在传统中医药理论的指导下，研究开发出一种抗前列腺癌效果好、不良反应底、价格合理、易推广应用的中药复方制剂。
前列腺癌的生长和转移密切依赖于雄激素。雄激素的分泌约90％来自于睾丸，约10％来自于肾上腺。雄激素(如双氢睾酮，Dihydrotestosterone，DHT)被分泌后进入细胞可以与雄激素受体(Androgen receptor，AR)结合。雄激素受体是一种重要的核转录因子，调控众多的基因表达和前列腺癌细胞的增殖。当雄激素与雄激素受体结合后，雄激素受体进入细胞核启动基因表达(如筛查前列腺癌的重要指标——PSA)，从而促进前列腺癌的生长和转移。因此，去雄激素治疗(包括去势治疗即切除睾丸和药物抑制雄激素水平)对于大多数前列腺癌有明显疗效。但去雄激素治疗对肿瘤的控制一般仅能维持1.5到4.0年，几乎所有前列腺癌患者最终均转为雄激素非依赖性前列腺癌(Androgen-independent prostate cancer,AIPC)，进而发展为激素难治性前列腺癌(Hormone refractory prostate cancer,HRPC)，也称去势抵抗性前列腺癌(Castration resistant prostate cancer,CRPC)。此时，去雄激素治疗也就失去了效果。CRPC是导致前列腺癌病人死亡的重要原因。CRPC的发生机制有很多种，包括雄激素受体突变、雄激素受体表达消失以及在低雄激素环境下雄激素不敏感的细胞群扩增等。换言之，虽然去雄激素治疗没有效果，但是在大多数去势抵抗性前列腺癌中雄激素受体也同样扮演着重要的角色，所以雄激素受体是目前治疗一般前列腺癌和去势抵抗性前列腺癌的重要靶点。
当今全球医疗界尚没有一种药物能单独治愈前列腺癌。前列腺癌的主要治疗手段包括手术、放疗、化疗及生物治疗，不仅患者要经历九死一生之痛，家庭经济压力巨大，负债累累，人们最不能接受的是患病亲人承受了极大痛苦后最终还是要经历生死离别之苦。美国食品和药物管理局(Food and drug administration，FDA)最新批准的治疗CRPC的药物包括阿比特龙(Abiraterone)和MDV3100(Enzalutamide)，前者是抑制雄激素的合成，后者是雄激素受体的拮抗剂，这两种药物都是针对雄激素受体信号通路，在去势抵抗性前列腺癌的治疗方面取得显著的疗效，但是经过上述内分泌治疗及靶向雄激素受体药物治疗后，病人依然会产生药物抵抗，治疗效果十分有限，且费用昂贵，病人的经济承受能力差。此外，放化疗所引起 的副作用也是临床治疗的瓶颈。作为前列腺癌综合治疗的重要手段之一，中医药因其显著的临床疗效，在前列腺癌等诸多癌症的治疗中发挥着无可替代的作用。结合我国蕴藏量丰富的中药材资源及历史悠久的中医药理论，开发出针对前列腺癌药物抵抗的、拥有我国自主知识产权的新型药物组合对治疗前列腺癌具有十分重大的临床意义。
发明内容
本发明所要解决的技术问题在于克服现有技术的不足，提供一种疗效显著、毒副作用小、服用方便的具有抗前列腺癌作用的中药复方组合物、制备方法及其在治疗前列腺癌领域中的应用。
本发明提出了.一种中药复方组合物，包括下列重量份数的中药材：臭椿皮4～12份，白花蛇舌草12～30份，莪术3～15份，丹参15～30份，夏枯草5～15份，甘草4～12份。
其中，优选地，所述中药复方组合物，包括下列重量份数的中药材：臭椿皮6～9份，白花蛇舌草15～24份，莪术4～12份，丹参18～21份，夏枯草6～10份，甘草6～9份。
本发明还提出了如上所述中药复方组合物的制备方法，包括以下步骤：
(1)浸泡：取权利要求1或2所述六味中药材，加入蒸馏水浸泡，得到生药蒸馏水混合物；
(2)煎煮：将步骤(1)所述的生药蒸馏水混合物进行煎煮，得到中药粗提物；
(3)浓缩：将步骤(2)制备得到的中药粗提物浓缩，得到中药浓缩液；
(4)除菌：取步骤(3)制备得到的中药浓缩液通过微孔滤膜过滤除菌，即得到所述中药复方组合物。
本发明还提出了将所述中药复方组合物应用于制备治疗恶性肿瘤药物的应用。
其中，所述恶性肿瘤为雄激素受体异常扩增和突变导致，包括前列腺癌、乳腺癌、肺癌、结肠癌、脑癌、皮肤癌、膀胱癌、肾癌。
本发明进一步提出了所述的中药复方组合物应用于制备治疗前列腺癌药物的用途。
本发明进一步提出了所述的中药复方组合物应用于抑制前列腺癌细胞的生长、增殖、转移、迁移、侵袭和促进前列腺癌细胞的凋亡的用途。
其中，所述前列腺癌为去势抵抗性前列腺癌或雄激素依赖性前列腺癌。
本发明进一步提出了将所述中药复方组合物应用于体外抑制双氢睾酮诱导的雄激素受体的转录活性以及缺失配体结构域的雄激素受体的转录活性的用途，或用于体外抑制雄激素受体活性。
本发明还提出了将所述的中药复方组合物和药学上可接受的载体制成可注射流体、气雾剂、乳膏、凝胶剂、丸剂、糖浆剂、透皮贴剂、胶囊剂、微囊、片剂、颗粒剂或口服液剂型的药物。
本发明的中药复方组合物可以作为药物单独使用或与其他药物联合使用。
本发明还提出了将所述的中药复方组合物，用于体外抑制雄激素受体突变的前列腺癌细胞株22RV1的增殖、抑制雄激素依赖性前列腺癌细胞株LNCaP的增殖的用途。
本发明还提出了将所述的中药复方组合物，用于体外抑制前列腺癌细胞的转移的用途，或体外抑制前列腺癌细胞PC-3和LNCaP以及22RV1的迁移的用途，或体外抑制双氢睾酮诱导的雄激素受体的转录活性以及缺失配体结构域的雄激素受体的转录活性的用途，或体外抑制雄激素受体活性、抑制前列腺癌细胞生长、迁移和侵袭以及促进前列腺癌细胞的凋亡的用途，或体外抑制比卡鲁胺或MDV3100治疗无效的雄激素受体突变细胞株22RV1细胞增殖的用途。
本发明还提出了将所述的中药复方组合物，用于体内抑制雄激素受体突变的前列腺癌细胞株22RV1的增殖、抑制雄激素依赖性前列腺癌细胞株LNCaP的增殖，或用于体内抑制前列腺癌细胞的转移，或用于在体内抑制去势抵抗性前列腺癌的生长和转移，或用于抑制雄激素受体活性、抑制前列腺癌细胞生长、迁移和侵袭以及促进前列腺癌细胞的凋亡，或用于抑制比卡鲁胺或MDV3100治疗无效的雄激素受体突变细胞株22RV1细胞增殖，或用于在小鼠体内抑制前列腺癌细胞22RV1的生长，或用于在小鼠体内抑制前列腺癌细胞22RV1的转移。
本发明还提出了将所述的中药复方组合物用于制备治疗与雄激素受体突变和异常扩增相关的恶性肿瘤疾病的药物中的应用。
其中，所述恶性肿瘤为雄激素受体异常扩增和突变导致的恶性肿瘤细胞的增殖、生长、迁移和浸润中的应用；其中，所述恶性肿瘤包括前列腺癌、乳腺癌、肺癌、结肠癌、脑癌、皮肤癌、膀胱癌、肾癌。
本发明提及的中药复方组合物，由下列重量份数的中药材制成：臭椿皮4～12份，白花蛇舌草12～30份，莪术3～15份，丹参15～30份，夏枯草5～15份，甘草4～12份。
其中，优选地，所述中药复方组合物，由下列重量份数的中药材制成：臭椿皮6～9份，白花蛇舌草15～24份，莪术4～12份，丹参18～21份，夏枯草6～10份，甘草6～9份。
本发明应用中，所提供的中药复方组合物抑制雄激素受体的转录活性。所提供的中药复方组合物抑制雄激素受体突变的前列腺癌细胞株的增殖。其在体外和体内抑制雄激素受体突变的前列腺癌细胞株22RV1的增殖、抑制雄激素依赖性前列腺癌细胞株LNCaP的增殖。
本发明应用中，所提供的中药复方组合物在体外和体内抑制前列腺癌细胞的转移。
本发明应用中，所提供的中药复方组合物抑制雄激素受体的转录活性和前列腺癌细胞生长。
本发明应用中，所提供的中药复方组合物抑制病人肿瘤组织源性原代分离的前列腺癌细胞增殖。
本发明应用中，所提供的中药复方组合物抑制前列腺癌细胞的克隆形成。
本发明应用中，所提供的中药复方组合物诱导雄激素受体表达阳性的细胞株LNCaP及22RV1的凋亡。
本发明应用中，所提供的中药复方组合物在小鼠体内模型中抑制去势抵抗性前列腺癌细胞的生长和转移。
本发明应用中，所提供的中药复方组合物抑制比卡鲁胺或MDV3100治疗无效的雄激素受体突变细胞株22RV1细胞增殖。
本发明应用中，所提供的中药复方组合物体外抑制前列腺癌细胞株PC-3和LNCaP细胞的迁移及22RV1细胞的体内转移。
本发明应用中，所提供的中药复方组合物抑制前列腺癌细胞株LNCaP、22RV1、PC-3及病人肿瘤组织源性原代分离的前列腺癌细胞株的生长。
本发明应用中，提供中药复方组合物对雄激素受体产生抑制作用的应用及方法。所提供的中药复方组合物通过阻断雄激素受体的蛋白表达水平从而显著下调雄激素受体的转录活性并抑制其下游基因表达。
本发明应用中，所提供的中药复方组合物在体外抑制雄激素受体调控的荧光素酶报告基因的活性，能够在体外和体内强烈抑制前列腺癌细胞的增殖和迁移，并且能够有效阻断雄激素受体信号通路。
本发明应用中，所提供的中药复方组合物不仅能高效抑制雄激素依赖性前列腺癌细胞株LNCaP的增殖，而且能够抑制雄激素受体突变的前列腺癌细胞株22RV1(该细胞株表达持续激活的突变雄激素受体，且对临床药物雄激素受体拮抗剂，例如比卡鲁胺和MDV3100等具有抗性)的增殖。此外，本发明从体外和体内证明了所提供的中药复方组合物抑制前列腺癌的转移。进一步实验证明表达雄激素受体的前列腺癌细胞株对本发明所提供的中药复方组合物具有较好的敏感性。该中药复方组合物抑制雄激素受体的蛋白表达水平进而抑制了雄激素受体的转录活性和前列腺癌细胞的生长。本发明所提供的中药复方组合物不仅可以治疗早期雄激素依赖性前列腺癌，也可以治疗晚期去势抵抗性前列腺癌。
本发明提供的具有抗前列腺癌作用的中药复方组合物的应用，可以将中药复方组合物的提取物和药学上可接受的载体制成可注射流体、气雾剂、乳膏、凝胶剂、丸剂、糖浆剂、透皮贴剂、胶囊剂、微囊、片剂、颗粒剂或口服液剂型的药物。
本发明提供的具有抗前列腺癌作用的中药复方组合物制成片剂时把具有抗前列腺癌作用的中药复方组合物和载体乳糖或玉米淀粉，需要时加入润滑剂硬脂酸镁，混合均匀，然后压制成片剂。本发明提供的具有抗前列腺癌作用的中药复方组合物制成胶囊剂时把具有抗前列腺癌作用的中药复方组合物和载体乳糖或玉米淀粉混合均匀，整粒，然后装胶囊制成胶囊剂。本发明提供的具有抗前列腺癌作用的复方制成颗粒剂时，把具有抗前列腺癌作用的中药复方组合物和稀释剂乳糖或玉米淀粉混合均匀，整粒，干燥，制成颗粒剂。
本发明还公开了所提供的具有抗前列腺癌作用的中药复方组合物或其药学上可接受的药物制剂在制备治疗与雄激素受体突变和异常扩增相关的恶性肿瘤疾病的药物中的应用。
为了实现以上目的，本发明提供的具有抗前列腺癌作用的中药复方组合物，它是由下列重量份数的原料制成：臭椿皮4～12份，白花蛇舌草12～30份，莪术3～15份，丹参15～30份，夏枯草5～15份，甘草4～12份。
作为优选方案，以上所述的一种具有抗前列腺癌作用的中药复方组合物，它是由下列重量份数的原料制成：臭椿皮6～9份，白花蛇舌草15～24份，莪术4～12份，丹参18～21份，夏枯草6～10份，甘草6～9份。
本发明在中医理论的指导下，辩证施治，结合前列腺癌的发病机理和发病症状，以中医治疗前列腺癌的益气养血、健肾排毒、散瘀止痛、活血止血、调节内分泌、利尿通淋、消瘀散结的扶正抗癌整体治疗理念为治则进行配伍组方。基于以上治则，经过大量文献检索和实验验证，筛选出本发明所述的一种具有抗前列腺癌作用的中药复方组合物，精选臭椿皮，白花蛇舌草，莪术，丹参，夏枯草，甘草6味中药进行组方：方中臭椿皮，味苦、涩、性寒，具清热燥湿、收涩止血之功，含有抗肿瘤成分。白花蛇舌草，味苦、甘，性寒，清热解毒，利湿通淋，具有较强的清热解毒作用，常用于热毒等证，其含有的齐墩果酸、三萜酸、香豆素类及多糖类等有效成分和有效部位均具有抗肿瘤作用，其中三萜类抗肿瘤效果最强。与臭椿皮合用相得益彰，效高力宏，二者共为方中君药。莪术，味苦性温，活血化瘀、消肿止痛之功卓著，行气活血，止痛祛瘀，有效成分莪术油是公认的抗癌良药。丹参，味苦，性微寒，活血化瘀，养血安神，凉血消痈，主要含有脂溶性的二萜类成分和水溶性的酚酸成分。二萜类成分丹参酮ⅡA是目前丹参中报道最多的抗肿瘤活性成分。莪术与丹参两者均具有散瘀止痛、活血养血的功效，共为臣药。夏枯草，味苦、辛，性寒，为常用清热药，具有清肝明目、散结解毒的功效；其在此方中软坚散结之功明显，奏辅佐君药进一步软化肿瘤部位并排除痈毒之效，为佐药。甘草，味甘，性平，补脾益气、清热解毒、调和诸药，在该方中主要起到促进机体免疫功能调整，恢复正气的作用，为使药。6味药按君臣佐使组方原则严格配伍，共奏益气养血、健肾排毒、散瘀止痛、活血止血、调节内分泌、利尿通淋、消瘀散结之功效。
本发明中药复方组合物制备方法：取上述重量份数的中药复方组合物每剂方药六味中药材生药干品若干克，加入中药复方组合物药材量的10～15倍体积蒸馏水浸泡1～3小时；于中药自动煎药器煎煮，煎煮次数为1至3次，每次煎煮0.5～1小时；制备得到的中药粗提物于中药自动煎药器上开盖蒸发浓缩至终体积为初提物体积的5～10％；将所得到的中药浓缩液通过0.22微米微孔滤膜过滤除菌后得到中药水煎剂母液，于4摄氏度冰箱保存备用。
本发明提供的具有抗前列腺癌作用的中药复方组合物与现有技术相比具有以下优势：
(1)本发明提供的具有抗前列腺癌作用的中药复方组合物，根据中医药理论和前列腺癌发病机制选取原料配伍制成，各组分配比严谨，邪正兼顾，君臣佐使，升降沉浮，共奏清热解毒、行气活血、利尿通淋、软坚散结、消淤止痛、益气养血之功，是可治疗湿热蕴积、瘀血内结型前列腺癌的良方，不仅能明显抑制荷瘤小鼠的前列腺癌生长和转移，提高生存质量和免疫机能，延长生存期，改善生化指标，缩小病灶范围，与化疗药物环磷酰胺联合用药具有增效减毒的作用，且无不良反应，显示了很好的抗癌扶正的功效。
(2)本发明提供的具有抗前列腺癌作用的中药复方组合物，通过下调雄激素受体的蛋白水平，抑制其下游基因的表达，阻断雄激素受体信号通路，从而实现抑制前列腺癌的目的，药物疗效确切，作用机制清楚，在前列腺癌治疗方面取得新突破，可在一定程度上填补我国目前前列腺癌治疗用药基本依赖进口而自主知识产权药物匮乏的领域空白。
(3)本发明提供的具有抗前列腺癌作用的中药复方组合物，制备方法成本低、环保，可实现工业化大生产。
(4)本发明提供的具有抗前列腺癌作用的中药复方组合物，可以和药学上可接受的载体制备成各种剂型，方便临床服用，有望开发成新的抗前列腺癌药物。
术语和定义
去势抵抗性前列腺癌(CRPC)：前列腺癌病人经去势治疗(切除睾丸或化学去势降低雄激素)后复发，去势治疗无效的前列腺癌称为去势抵抗性前列腺癌，这种前列腺癌是威胁前列腺癌患者的最大杀手。
前列腺癌细胞PC-3：从人前列腺癌骨转移肿瘤中分离出来的分化程度低、雄激素非依赖性的前列腺癌细胞，不表达内源性的雄激素受体，具有中等程度的转移潜能，表现出显著的上皮细胞癌特征，被广泛用于雄激素抵抗性的前列腺癌的研究。
前列腺癌细胞LNCaP：由Horoszewicz等在1980年从前列腺癌患者的淋巴结转移灶标本中建立起来的，保留了前列腺癌肿瘤细胞学及其早期分化功能的特征，具有显著的上皮细胞特性，表达雄激素受体，是典型的具有雄激素依赖性的前列腺癌细胞。
前列腺癌细胞22RV1：是由雄激素依赖性前列腺癌CWR22细胞异种移植到小鼠中进行 连续传代后而分离的人前列腺癌上皮细胞系。此细胞系表达前列腺特异抗原，依赖于雄激素的刺激而生长，在裸鼠中具有很强的成瘤能力。
细胞增殖：细胞以分裂的方式产生新的个体和后代的过程。单细胞生物，以细胞分裂的方式产生新的个体。多细胞生物，以细胞分裂的方式产生新的细胞，用来补充体内衰老和死亡的细胞；同时，多细胞生物可以由一个受精卵，经过细胞的分裂和分化，最终发育成一个新的多细胞个体。通过细胞分裂，可以将复制的遗传物质，平均地分配到两个子细胞中去。细胞增殖是生物体生长、发育、繁殖和遗传的基础，是生物体的重要生命特征。
细胞凋亡：为维持内环境稳定，由基因控制的细胞自主的、有序的、细胞程序化死亡。细胞凋亡涉及一系列基因的激活、表达以及调控等的作用，是为适应生存环境而主动争取的一种细胞死亡过程。
细胞迁移：也称为细胞爬行、细胞移动或细胞运动，是指细胞在接收到迁移信号或感受到某些物质的梯度后而产生的移动。细胞迁移为细胞头部伪足的延伸、新的黏附建立、细胞体尾部收缩在时空上的交替过程。细胞迁移是正常细胞的基本功能之一，是机体正常生长发育的生理过程，也是活细胞普遍存在的一种运动形式。胚胎发育、血管生成、伤口愈合、免疫反应、炎症反应、动脉粥样硬化、癌症转移等过程中都涉及细胞迁移。
细胞侵袭：肿瘤细胞穿越基底膜游走至间质或其他组织进行远端转移的能力。基底膜是一种特化的细胞外基质，是肿瘤细胞转移过程中必须穿越的物理屏障。基底膜的组成成分通过和细胞表面受体整合素相互作用。肿瘤细胞通过分泌基质降解酶类破坏基底膜的组织结构，同时调节细胞外基质受体整合素的表达，为穿过基底膜和在靶器官粘附、增殖创造有利条件。
肿瘤生长：肿瘤细胞因分裂增生而造成原位肿瘤体积增大并侵入周围组织间隙、淋巴管或血管内，与邻近的正常组织紧密连接在一起而无明显界限。
肿瘤转移：肿瘤细胞由其原发部位侵入淋巴管、血管或体腔部位，随血流、淋巴流带到另一部位或器官继续生长形成与原发肿瘤同样类型肿瘤的过程。
前列腺癌生长与转移：前列腺癌恶变的过程中，由于肿瘤细胞增殖及迁移、侵袭的能力增强而引起肿瘤细胞群体动力学、肿瘤细胞的凋亡率、肿瘤血管生成、肿瘤演进和异质性恶化的过程。前列腺癌在生长与转移的过程中变得越来越富有侵袭性，最终造成肿瘤生长加快、产生周围组织的浸润和远端转移。
雄激素受体(AR)：雄激素受体属于核受体超家族中的类固醇受体。人类雄激素受体是一个单拷贝基因，分子量约为110千道尔顿，一般由四个结构域组成：N端转录激活区(N-terminal domain，NTD)、DNA结合区(DNA binding domain，DBD)、铰链区和配体结合区(Ligand binding domain，LBD)。雄激素受体与前列腺癌的发生发展密切相关。雄激素 如双氢睾酮(Dihydrotestosterone，DHT)与雄激素受体结合后，雄激素受体可以进入细胞核与转录激活域相结合从而启动下游基因表达。目前临床使用的雄激素受体拮抗剂如比卡鲁胺(Bicalutamide)及MDV3100(Enzalutamide)都是与雄激素受体的配体结合区(LBD)结合而抑制了其转录活性。
雄激素受体的转录活性：雄激素受体是典型的核受体转录因子，通过介导雄激素的作用调控多种真核基因的表达。其具体功能为通过转录激活的方式调控其下游靶基因的转录与表达，在配体依赖性途径与配体非依赖性途径下均能发挥转录活性。
缺失配体结构域的雄激素受体：雄激素受体LBD区由12段不连续的α螺旋组成，形成一个配体结合口袋，具有结合配体的功能。雄激素受体在突变的情况下可能缺失LBD区，缺失了LBD区的雄激素受体具有与完整雄激素受体相同的组成性转录激活功能。
荧光素酶报告基因检测(Luciferase assay)：荧光素酶(Luciferase,Luc)报告基因检测系统是以荧光素(Luciferin)为底物来检测萤火虫荧光素酶(Firefly luciferase)活性的一种报告系统。荧光素酶可以催化Luciferin氧化成Oxy-luciferin，在Luciferin氧化的过程中，会发出生物荧光(Bioluminescence)。然后可以通过荧光测定仪也称化学发光仪(Luminometer)或液闪测定仪测定Luciferin氧化过程中释放的生物荧光。荧光素和荧光素酶这一生物发光体系，可以极其灵敏、高效地检测基因的表达，是检测转录因子与目的基因启动子区DNA相互作用的一种检测方法。
附图说明
图1所示为本发明中药复方组合物对各株前列腺癌细胞增殖活性的抑制效果。
图1(A)表示本发明中药复方组合物对各株前列腺癌细胞增殖的抑制及在正常前列腺上皮细胞上的选择性效果。
图1(B)表示本发明中药复方组合物对LNCaP细胞的增殖抑制效果呈时间梯度及浓度梯度依赖性。
图1(C)表示本发明中药复方组合物对22RV1细胞的增殖抑制效果呈时间梯度及浓度梯度依赖性。
图1(D)表示本发明中药复方组合物对PC-3细胞的增殖抑制效果呈时间梯度及浓度梯度依赖性。
图1(E)表示本发明中药复方组合物对病人肿瘤组织源性原代分离的前列腺癌细胞的增殖抑制效果呈时间梯度及浓度梯度依赖性。
图2所示为本发明中药复方组合物对各株前列腺癌细胞的克隆形成的抑制效果。
图2左边表示本发明中药复方组合物分别对LNCaP，22RV1的克隆形成抑制效果的白光 照片。
图2右边表示本发明中药复方组合物分别对LNCaP，22RV1的克隆形成抑制效果的统计结果。
图3所示为本发明中药复方组合物对前列腺癌细胞的诱导凋亡作用。
图3(A)表示本发明中药复方组合物对LNCaP细胞的诱导凋亡作用。
图3(B)表示本发明中药复方组合物对22RV1细胞的诱导凋亡作用。
图3(C)表示本发明中药复方组合物对LNCaP，22RV1的诱导凋亡作用的统计结果。
图4所示为本发明中药复方组合物对前列腺癌细胞迁移、侵袭的抑制效果。
图4上表示本发明中药复方组合物对PC-3细胞横向迁移过程的抑制作用。
图4中表示本发明中药复方组合物对LNCaP细胞纵向迁移过程的抑制作用。
图4下表示本发明中药复方组合物对22RV1细胞侵袭过程的抑制作用。
图5所示为本发明中药复方组合物通过影响雄激素受体信号通路从而抑制前列腺癌的分子机理。
图5(A)表示本发明中药复方组合物对LNCaP细胞中在双氢睾酮(DHT)诱导下检测雄激素受体转录活性的荧光素酶报告基因表达的抑制效果。
图5(B)表示本发明中药复方组合物对22RV1细胞中在双氢睾酮(DHT)诱导下检测雄激素受体转录活性的荧光素酶报告基因表达的抑制效果。
图5(C)表示本发明中药复方组合物对PC-3细胞中在双氢睾酮(DHT)诱导下外源转染野生型雄激素受体(AR)后检测雄激素受体转录活性的荧光素酶报告基因表达的抑制效果。
图5(D)表示本发明中药复方组合物不影响LNCaP细胞中雄激素受体(AR)基因自身的转录水平。
图5(E)表示本发明中药复方组合物下调LNCaP细胞中雄激素受体(AR)蛋白的表达水平。
图5(F)表示本发明中药复方组合物抑制雄激素受体(AR)对其三个经典下游靶基因TMPRSS2，FKBP5，PSA的转录激活。
图6所示为本发明中药复方组合物在裸鼠皮下荷瘤体内动物模型中抑制22RV1异种移植肿瘤的生长。
图6(A)表示本发明中药复方组合物对22RV1异种移植肿瘤生长模型中剥离的肿瘤体积抑制效果的白光照片。
图6(B)表示本发明中药复方组合物对22RV1异种移植肿瘤生长模型中肿瘤体积随天数增长的抑制效果的统计结果。
图6(C)表示本发明中药复方组合物对22RV1异种移植肿瘤生长模型中剥离的肿瘤重量抑制效果的白光照片。
图7所示为本发明中药复方组合物在裸鼠原位异种移植肿瘤生长转移体内动物模型中抑制22RV1-luc异种移植肿瘤的生长与转移。
图7(A)表示本发明中药复方组合物对22RV1-luc异种移植肿瘤生长转移模型中肿瘤荧光强度抑制效果的活体成像结果。
图7(B)表示本发明中药复方组合物对22RV1-luc异种移植肿瘤生长转移模型中原位肿瘤转移到全身脏器形成转移结节的抑制效果的统计结果。
图7(C)表示本发明中药复方组合物对22RV1-luc异种移植肿瘤生长转移模型中全身肿瘤荧光强度随天数增长的抑制效果的统计结果。
图7(D)表示本发明中药复方组合物在灌胃给药方式下对裸鼠体重的影响。
具体实施方式
结合以下具体实施例和附图，对本发明的技术作进一步的详细说明。本发明的保护内容不局限于以下实施例。在不背离发明构思的精神和范围下，本领域技术人员能够想到的变化和优点都被包括在本发明中，并且以所附的权利要求书为保护范围。实施例所描述的具体的物料配比、工艺条件及其结果仅用于说明本发明，而不应当也不会限制权利要求书中所详细描述的本发明。实施本发明的过程、条件、试剂、实验方法等，除以下专门提及的内容之外，均为本领域的普遍知识和公知常识，本发明没有特别限制内容。
实施例1：本发明中药复方组合物的制备
本实施例中具有抗前列腺癌作用的中药复方组合物，由以下原料制成：取臭椿皮9份，白花蛇舌草30份，莪术15份，丹参30份，夏枯草15份，甘草9份，加入中药复方组合物药材量的15倍体积蒸馏水浸泡3小时；于中药自动煎药器内煎煮，煎煮次数为2次，每次煎煮1小时；制备得到的中药粗提物于中药自动煎药器上开盖蒸发浓缩至终体积浓缩为70毫升；浓缩得到的中药水煎剂母液终浓度约为1.5克生药/毫升。将所得到的中药浓缩液通过0.22微米微孔滤膜过滤除菌后得到中药水煎剂母液，于4摄氏度冰箱保存备用。
以下实施例2-7中所采用的中药复方组合物为实施例1所制备
实施例2：本发明中药复方组合物对不同前列腺癌细胞的增殖活性、克隆形成的抑制作用及对不同前列腺癌细胞的诱导凋亡作用
技术方法：
1.细胞的培养
本实验中所用的细胞(LNCaP，22RV1，PC-3，PNT1A)均购于中科院上海细胞库；另 一株细胞来自于上海市长海医院前列腺癌病人肿瘤组织源性原代分离的前列腺癌细胞(PCa原代细胞)，细胞培养于37摄氏度恒温培养箱(湿度95％，CO2浓度5％)中，培养基为含10％胎牛血清(Front)的RPMI-1640(Gibco)。
2.SRB(磺酰罗丹明)法测定细胞增殖
不同的前列腺癌细胞株以5×103个/孔密度接种至96孔板(Corning)，24小时后，加入不同浓度本发明中药复方组合物，对照组加入等量的PBS，各组设6个复孔。药物处理48小时后，加入预冷却的TCA(三氯乙酸，50％，w/v)于4摄氏度孵育60分钟固定细胞。固定后，流水冲洗5遍，风干。每孔加入50微升SRB染液(4％，w/v)，室温孵育10分钟染色。将染液吸出，每孔加入1％醋酸100微升洗5遍，除去未结合染料。风干后，每孔加入浓度为10毫摩尔Tris溶液100微升，震荡溶解结合的SRB染料。将96孔板置于酶标仪(SPECTRA MAX 190)中，在515纳米波长下测定OD值。统计分析中药复方组合物对于细胞增殖水平的影响。为检测本发明中药复方组合物对前列腺癌细胞株的抑制作用是否呈浓度梯度依赖性及时间浓度梯度依赖性，接种不同的前列腺癌细胞株以5×103个/孔密度至96孔板，24小时后，加入不同浓度本发明中药复方组合物，对照组加入等量的PBS，各组设6个复孔。分别继续培养24小时,48小时和72小时后，用上述同样的方法检测中药复方组合物对于细胞增殖水平的影响。
Figure PCTCN2016101970-appb-000001
3.克隆形成实验
克隆形成法(Colony formation)可用来检测肿瘤细胞体外形成克隆的增殖和成瘤能力。肿瘤细胞在体内可以通过自身分裂增殖最终形成肉眼可见的实体瘤，在体外实验时，可以根据肿瘤细胞的克隆形成能力来反映其在体内生成实体瘤能力的强弱。将1000个前列腺癌细胞接入到6孔板中，待细胞贴壁后加入不同浓度本发明中药复方组合物，一周后用多聚甲醛将细胞固定，然后用结晶紫染色，拍照并统计克隆数，每组实验重复三次。
4.流式细胞术检测细胞凋亡
在正常细胞中，磷脂酰丝氨酸(Phosphatidylserine，PS)只分布在细胞膜脂质双层的内侧，而在细胞凋亡早期，细胞膜中的磷脂酰丝氨酸(PS)由脂膜内侧翻向外侧。Annexin V是一种分子量为35～36kD的Ca2+依赖性磷脂结合蛋白，与磷脂酰丝氨酸有高度亲和力，故可通过细胞外侧暴露的磷脂酰丝氨酸与凋亡早期细胞的胞膜结合。因此Annexin V被当作为检测细胞早期凋亡的灵敏指标之一。将AnnexinV进行荧光素(EGFP、FITC等)标记，以标记了的AnnexinV作为荧光探针，利用荧光显微镜或流式细胞仪可检测细胞凋亡的发生。 碘化丙啶(Propidium iodide,PI)是一种核酸染料，它不能透过完整的细胞膜，但对凋亡中晚期的细胞和死细胞，PI能够透过细胞膜而使细胞核染红。因此将AnnexinV与PI匹配使用，就可以将处于不同凋亡时期的细胞区分开来。具体操作步骤如下：不同前列腺癌细胞经过本发明中药复方组合物不同浓度的药液处理36小时后，收集悬浮细胞离心(2000转/分钟，离心5分钟)；贴壁细胞用不含EDTA的胰酶消化收集；用PBS洗涤细胞二次(2000转/分钟，离心5分钟)收集1～5×105细胞；加入500微升的Bindingbuffer悬浮细胞；加入5微升Annexin V-FTIC混匀后，加入5微升PI，混匀；室温、避光、反应5～15分钟；在1小时内，进行流式细胞仪的上机检测。
实验结果：
如图1所示，本发明中药复方组合物对各株前列癌细胞，包括病人组织来源的原代前列腺癌细胞的增殖都有很显著的抑制效果，其48小时半数抑制浓度约为5微克/毫升。本发明中药复方组合物除了对常用前列腺癌细胞株及病人组织来源的原代前列腺癌细胞的增殖抑制效果显著之外，对正常前列腺上皮细胞株PNT1A虽然在高浓度下(100微克/毫升)也有轻微的抑制，但在低浓度下抑制效果很小，显示出其对正常细胞和前列腺癌细胞之间具有很好的选择性。同时，本发明中药复方组合物对各株前列腺癌细胞增殖的抑制效果呈现浓度梯度依赖性及时间梯度依赖性。另外，如图2所示，本发明中药复方组合物也强烈抑制各株前列腺癌细胞的克隆形成。图3显示本发明中药复方组合物显著促进前列腺癌细胞LNCaP，22RV1的凋亡。实验结果提示本发明中药复方组合物可以作为抗前列腺癌生长的潜在药物。
实施例3：本发明中药复方组合物对前列腺癌细胞迁移、侵袭能力的抑制作用
技术方法：
1.划线迁移实验
为研究本发明中药复方组合物是否抑制前列腺癌细胞的横向迁移，将PC-3细胞接种至6孔板，在37摄氏度5％CO2培养箱中常规培养24小时，至细胞长至100％满。更换无血清培养基，继续培养12小时。在长满细胞的培养孔中，用200微升的灭菌枪头进行划痕，划痕后用PBS洗细胞两次，将浮起的细胞吸去，每孔中加入1毫升完全培养基。分别向细胞培养孔中加入不同浓度中药复方组合物，将培养板放入CO2培养箱，37摄氏度继续常规培养24小时。显微镜下观察细胞向划线部分运动的情况，拍照。统计分析不同剂量药物组迁移进入划线区域的细胞数量，确定药物对细胞迁移能力的影响。
2.Transwell小室迁移实验
Transwell小室底部所铺的透水聚碳酸酯薄膜上有大量直径8微米的微孔，实验时，将Transwell小室放入24孔板中，聚碳酸酯薄膜将24孔板的每个孔分别分为上、下两室，Transwell 小室内称上室，培养板内称下室，上室内盛装上层培养液，下室内盛装下层培养液，上下层培养液以聚碳酸酯膜相隔。将细胞接种在上室内，由于聚碳酸酯膜有通透性，下层培养液中的成分可以诱导到上室内的细胞向下室迁移，使得细胞从薄膜的上室面移动到下室面，因此本实验是研究细胞运动的经典实验。为研究本发明中药复方组合物是否抑制前列腺癌细胞的纵向迁移，利用Transwell小室，在上室接入过量的LNCaP细胞，这样细胞就会向下室迁移；同时加入不同浓度的中药复方组合物，12小时后拍照统计细胞迁移率。
Figure PCTCN2016101970-appb-000002
3.Transwell法检测细胞侵袭
将Transwell小室放入24孔板中，上室内盛装上层培养液，下室内盛装下层培养液，上下层培养液以聚碳酸酯膜相隔，Transwell小室上室预先铺一层胶原基质Matrigel，再将细胞接种其中。由于聚碳酸酯膜有通透性，下层培养液中的成分可以诱导上室内的细胞向下迁移，使得细胞从薄膜的上室面移动到下室面。但是，肿瘤细胞若要运动至下室，即必须先利用自身释放的各种化学物质溶解胶原基质，再通过溶解出的空洞运动，这一过程，与肿瘤细胞在体内的浸润过程相似，可以用来反映肿瘤细胞的侵袭浸润能力。因此本实验是研究细胞侵袭的经典实验。取对数生长期的肿瘤细胞以5×104个/孔接种到Transwell小室的上室中，加药组分别加入相应浓度的中药复方组合物，对照组加入等量的PBS。下室中分别各加入完全培养基。37摄氏度5％CO2培养箱中培养12小时。取出Transwell小室，用棉签蘸水擦拭Transwell小室的上室一面，将未穿膜的细胞擦掉。将Transwell小室在4％多聚甲醛中室温固定10分钟，经1％结晶紫染色10分钟后用自来水冲洗。显微镜下拍照，计数每孔内上下左右中5个视野的细胞数目。每组平均设3个复孔。统计比较不同剂量中药复方组合物加药组的穿膜细胞数量，确定中药复方组合物对细胞侵袭能力的影响。
Figure PCTCN2016101970-appb-000003
实验结果：
结果如图4所示，本发明中药复方组合物在较低剂量下(2.5微克/毫升)就可以有效抑制PC-3的横向迁移、LNCaP的纵向迁移及22RV1的侵袭能力。图4左边分别是三种细胞的实验白光照片结果，图4右边分别是三种细胞的统计结果。实验结果提示本发明中药复方组合物可以作为抗前列腺癌转移的潜在药物。
实施例4：本发明中药复方组合物体外抑制雄激素受体的转录活性
技术方法：双荧光素酶报告基因检测
本实验所用质粒为MMTV-LUC和Renila。MMTV-LUC是带有雄激素受体转录激活元件(AR response elements)和荧光素酶的报告基因质粒，雄激素受体与雄激素结合后可以启动该质粒表达出荧光素酶，荧光素酶可以催化其底物荧光素发出荧光，通过检测荧光的强度便能检测雄激素受体激活的程度；Renila内参报告基因带有海肾荧光素酶的启动子区域，能表达海肾荧光素酶，通过该质粒可以确定每组细胞的转染效率是否一致，雄激素对Renila内参报告基因质粒没有影响。为初步探究本发明中药复方组合物能否抑制雄激素受体(Androgen receptor,AR)的转录活性，我们分别在前列腺癌细胞株22RV1和LNCaP中用脂质体2000(Lipofectamine 2000 Reagent)转染MMTV-LUC和Renila两种质粒，此外还在不表达AR的PC-3细胞中转入外源野生型AR；细胞转染24小时后加入雄激素双氢睾酮(Dihydrotestosterone，DHT)激活MMTV-LUC报告基因表达萤火虫荧光素酶；在加入DHT的同时再加入不同浓度中药复方组合物，处理12小时后裂解细胞并测定两种报告基因的荧光表达强度，最终结果用MMTV-LUC的活性与内参质粒Renila的活性相比表示，每组实验重复三次。
实验结果：
如图5A、5B、5C所示，本发明中药复方组合物在不同前列腺癌细胞株中都能够剂量依赖性地降低雄激素(DHT)诱导的MMTV-LUC报告基因的活性，并且在低浓度(约2.5微克/毫升)就能够达到半数抑制率(IC50)，证明本发明中药复方组合物可以体外抑制雄激素受体的转录活性。
实施例5：本发明中药复方组合物显著下调雄激素受体蛋白水平及其靶基因的转录水平
技术方法：
1.聚合酶链式反应(Polymerase chain reaction，PCR)实验
LNCaP细胞经不同浓度中药复方组合物处理后，用TRIzol分离提取RNA，RNA经反转录后得到cDNA，用AR特异性引物通过反转录PCR(RT-PCR)定性检测AR基因的mRNA表达水平；用不同的AR下游靶基因特异性引物(TMPRSS2，FKBP5和PSA)通过荧光实时定量PCR(Q-PCR)检测三者的mRNA的表达水平。
2.免疫印迹(Westernblot)实验
LNCaP细胞经加不同浓度中药复方组合物处理后，细胞经裂解提取蛋白，蛋白经煮沸变性后用聚丙烯酰胺凝胶电泳分离蛋白质样品，然后转移到硝酸纤维素薄膜上，首先用雄激素受体AR的抗体孵育两小时，再用带有荧光标记的二抗孵育一小时，最后用Licor Odyssey双色红外激光成像系统检测本发明中药复方组合物对AR蛋白表达水平的影响。
实验结果：
如图5E所示，免疫印迹实验(Western blot)证明本发明中药复方组合物能够显著抑制雄激素受体的蛋白表达水平；随着加药浓度的增大，AR的蛋白表达量随之明显降低，提示本发明中药复方组合物对前列腺癌肿瘤细胞增殖和迁移的抑制作用可能是通过抑制雄激素受体信号通路实现的。RT-PCR实验发现在一定浓度下本发明中药复方组合物并没有影响AR的mRNA水平(图5D)，但显著抑制了其下游靶基因的转录激活(图5F)。Q-PCR实验显示AR下游的三个经典代表性靶基因TMPRSS2，FKBP5和PSA的转录水平均受到本发明中药复方组合物的显著抑制。以上实验结果从多个角度证明了本发明中药复方组合物对前列腺癌生长与转移的抑制是通过抑制雄激素受体信号通路活性而实现的。
实施例6：本发明中药复方组合物在裸鼠前列腺癌皮下荷瘤生长模型及前列腺癌原位生长转移模型中的抑制效果
技术方法：
1.本发明中药复方组合物对裸鼠前列腺癌皮下荷瘤生长模型的抑制作用
利用人前列腺癌细胞22RV1建立裸鼠前列腺癌皮下荷瘤生长模型。将500万个22RV1细胞皮下注射到7周龄雄性免疫缺陷小鼠(BLAB/c-nude，裸鼠)右侧背部皮下，待皮下肿瘤长到100立方毫米左右时，将小鼠分为8组(平均肿瘤体积相同)，依次为模型组，对照组，中药复方(不含臭椿皮)组，中药复方低剂量组，中药复方高剂量组，复方苦参注射液组，环磷酰胺(Cyclophosphamide，CTX)组，CTX+中药复方高剂量组。给药途径及给药剂量依次如下所述：模型组小鼠不做任何处理，对照组小鼠每天给予等量的PBS灌胃(0.1毫升)，中药复方(不含臭椿皮)组小鼠每天给予等量的不含臭椿皮的中药复方(0.1毫升，具体煎制方法与本发明中药复方一致，不加臭椿皮生药即可，相应减少浓缩终体积,实际剂量15克/千克/天)，中药复方低剂量组小鼠每天给予1/2量的中药复方母液灌胃(0.05毫升，实际剂量7.5克/千克/天)，中药复方高剂量组小鼠每天给予中药复方母液灌胃(0.1毫升，母液浓度1.5克生药/毫升，实际剂量15克/千克/天)，复方苦参注射液组小鼠每天给予0.1毫升复方苦参注射液腹腔注射(实际剂量0.6克/千克/天)，CTX组小鼠每天给予0.05毫升CTX腹腔注射(实际剂量0.05克/千克/天)，CTX+中药复方高剂量组小鼠每天给予0.05毫升CTX腹腔注射及0.1毫升中药复方母液灌胃(实际剂量0.05克/千克/天+15克/千克/天)。每隔2天量取一次小鼠背部皮下荷瘤的肿瘤体积，按照公式体积＝长×宽2×0.52计算，统计肿瘤体积。每隔2天量取一次小鼠体重。施药14天后处死小鼠，取皮下肿瘤，拍照。
2.本发明中药复方组合物对裸鼠前列腺癌原位荷瘤生长转移模型的抑制作用
将转移性前列腺癌细胞22RV1转入萤火虫荧光素酶(Luciferase)表达质粒(该质粒带有 G418抗性筛选标记)，并用G418筛选稳转细胞系，稳转细胞可以表达荧光素酶，荧光素酶与其底物荧光素接触后会发出荧光，荧光的范围和强度与肿瘤的位置和体积呈正相关性，这样即可以通过动物活体成像系统检测到肿瘤细胞的数量和位置。构建好的稳转细胞系命名为22RV1-luc，将22RV1-luc细胞用基质胶混匀注射到小鼠前列腺背侧叶，待肿瘤异种移植成功后通过活体成像技术可以检测到小鼠体内的22RV1-luc细胞的生长和转移情况。具体操作过程为：7周龄雄性免疫缺陷型小鼠麻醉后，行腹腔开口解剖，暴露前列腺，将50万个22RV1-luc细胞用基质胶混匀后注射到小鼠前列腺背侧叶，从第0天开始通过活体成像系统检测荧光值并将小鼠随机分组及给药，给药方式与前一个动物实验相同，每隔3天检测一次并统计荧光强度随天数增大的生长曲线图，每隔2天量取一次小鼠体重。在实验进行到第15天时结束实验，处死小鼠，剥离小鼠脏器统计转移结节数。
实验结果：
结果如图6所示，在裸鼠前列腺癌皮下荷瘤生长模型中，8个组别的小鼠皮下肿瘤大小明显有差异(图6A)。图6B显示通过肿瘤体积随着实验天数增长的测定表明，模型组与对照组肿瘤体积最大，且这两组之间没有显著差异，说明造模成功。中药复方(不含臭椿皮)组的肿瘤终体积相对于对照组来说有轻微的减少，说明有一定的效果，但是效果不太明显；中药复方低剂量组的效果较不含臭椿皮的实验组效果好，但仍然不太显著；中药复方高剂量组效果较显著，说明本发明的中药复方能有效抑制前列腺癌肿瘤的生长；复方苦参注射液组作为一个已经成药的中药复方阳性对照，在本实验中也显示出了很好的抑制肿瘤的效果，但从实验结果来看，抑制效果不及本发明的中药复方的抑制效果强；CTX组作为传统化疗药物的阳性对照，对肿瘤有明显的抑制效果；CTX+中药复方高剂量组对肿瘤抑制的效果最明显，是所有实验组中肿瘤体积最小的组，说明本发明的中药复方组合物能增强传统化疗药物的抑瘤效果。对剥离肿瘤的重量统计也说明了类似的实验结果(图6C)。
此外，如图7所示，在裸鼠前列腺癌原位荷瘤生长转移模型中，图7A为用药14天后对小鼠前列腺原位肿瘤的治疗效果图(n≥5)，通过动物活体成像系统拍照图片，图中彩色区域代表荧光信号，表明该区域有肿瘤细胞聚集，荧光强度越深表明肿瘤越大。实验结果与前一个动物实验的结果类似，中药复方高剂量组效果较显著，说明本发明的中药复方组合物能有效抑制前列腺癌肿瘤的生长(图7C)和转移(图7B)，且这种抑制效果较阳性中药复方——复方苦参注射液要强，与传统化疗药物CTX的抑制效果相当；以上各组的中药复方组合物处理对小鼠体重均没有明显影响(图7D)，初步说明本发明的中药复方组合物在有效剂量下对机体的毒副作用较小；CTX处理下小鼠体重有所下降。结合小鼠体重的实验结果来看，CTX作为一个化疗药物对机体存在一定的毒性，造成了小鼠体重的轻微下降，且与CTX组的小鼠 体重相比，本发明的中药复方组合物与CTX联合用药时，对肿瘤生长与转移的抑制效果是所有组别中最显著的，且小鼠体重得以恢复(图7D)，说明本发明的中药复方组合物不仅能增强传统化疗药物的抑瘤效果，还能改善因传统化疗药物的毒性而造成的对机体的损伤，从而证明本发明的中药复方组合物具有对传统化疗药物增效减毒的效果。
实施例7：本发明中药复方组合物对荷瘤裸鼠肿瘤抑制的增效减毒作用及免疫功能指标的影响
技术方法：
1.本发明中药复方组合物对裸鼠脾脏指数的影响
在观察到模型组、对照组与给药组的裸鼠生存质量及生活状态的不同后，尤其是化疗药物CTX组荷瘤小鼠出现不同程度皮肤粗糙，精神萎靡，懒散怠动，反应迟钝体重减轻等状态，而本发明的中药复方联合用药组小鼠则皮肤顺滑，精神良好，活动灵敏，体重正常，原位肿瘤块较小，因此推测本发明的中药复方组合物对化疗药物的增效减毒效果有可能是通过改善小鼠免疫功能、提高各项免疫指标而实现的。因此，将实施例6中原位肿瘤生长转移模型的裸鼠在实验结束处死后，称量脾脏重量及体重，计算脾脏指数。
Figure PCTCN2016101970-appb-000004
2.本发明中药复方组合物对裸鼠血清白细胞数目的影响
将实施例6中原位肿瘤生长转移模型的裸鼠在实验结束处死后，眼眶取血，用枪吸管准确吸血20微升，枪头外壁无血，迅速将血轻轻放入盛有380微升白细胞稀释液中，摇匀。按红细胞计数的方法，将白细胞混悬液滴入计数池内，静置2至3分钟，待白细胞下沉后计数。在低倍镜下，白细胞呈圆形，浆透亮，核呈紫黑色，稍有折光，借此特点可与杂质相区别。白细胞数目＝5个大格的总数×500，计数后统计各组间差异。
3.本发明中药复方组合物对刀豆素A(Concanamycin A，Con A)诱导的T细胞增生反应的影响
将实施例6中原位肿瘤生长转移模型的裸鼠在实验结束处死后，取脾脏称重后放入预先准备的装有预冷PBS的试管中，将试管中的脾脏和洗液倒入平皿中，把脾脏放入细胞滤器中用镊子挤压脾脏，得到细胞悬液。之后将细胞悬液收集到试管中，1500转/分钟，离心6分钟,倒掉洗液，加红细胞裂解液裂解3分钟，待观察到液体颜色变浅后再加洗液PBS，用滤膜过滤到另一个试管中，再次1500转/分钟，离心6分钟，倒掉洗液与细胞培养液，加Con A(工作浓度5微克/毫升)刺激T淋巴细胞增生，置于37摄氏度恒温培养箱(湿度95％，CO2 浓度5％)培养72小时。SRB法在酶标仪上测定T细胞的吸光度值(A值)从而检测其增殖情况。
实验结果：
结果如表1所示，本发明中药复方组合物对荷瘤裸鼠肿瘤抑制的增效减毒作用及对荷瘤裸鼠免疫功能的三个代表性指标——脾脏指数、白细胞数目及刀豆素A(Concanamycin A，Con A)诱导的T细胞增生反应的影响均具有显著性差异。
表1显示小鼠免疫功能的三个典型指标的统计结果。中药复方组合物的各个给药组与模型组对照组相比，三个指标均无显著性差异。CTX组的三个指标较模型组对照组相比，均出现显著差异，再次验证了化疗药物CTX对机体的毒性，对免疫系统有一定的损伤，而本发明的中药复方组合物与之联合用药后，能明显恢复其对小鼠造成的毒性作用，显著改善荷瘤小鼠的生存质量。实验结果证明，与CTX相比，本发明的中药复方组合物毒副作用小，不会造成免疫器官萎缩，且与CTX联用具有明显的增效减毒效果，提示本发明中药复方组合物的抗肿瘤活性与免疫调节作用具有直接的相关性。综上所述，本发明的中药复方组合物不仅具有抑制前列腺癌生长与转移的功能，还能增强传统化疗药物的抑瘤效果，改善因传统化疗药物的毒性而造成的对机体的损伤，从而证明本发明的中药复方组合物具有对传统化疗药物增效减毒的效果。
表1本发明中药复方组合物对荷瘤裸鼠肿瘤抑制的增效减毒作用及免疫功能指标的影响
Figure PCTCN2016101970-appb-000005
注：与模型组对照组比较，*P＜0.05，**P＜0.01
与CTX组比较，^^P＜0.01
实施例8：本发明中药复方组合物优选方案的验证
为了验证本发明中药复方组合物各组分配比的优选效果，称取如表2所示不同配比组合的六味中药材干品，按照实施例1中的方法制成中药水煎剂并与实施例1中的中药复方组合物进行实施例2-7的平行实验，发现本实施例中不同的中药复方组合物8a-8g药效与实施例1中的中药复方组合物一样，均能达到实施例2-7同样的技术效果，因此验证了本发明中药复方组合物中各组分配比的优选方案。
表2不同配比的中药复方组合物
Figure PCTCN2016101970-appb-000006
上述实施例仅为了说明本发明的技术构思及特点，其目的在于让本领域技术人员能够了解本发明的内容并据以实施，并不能以此限制本发明的保护范围。凡是根据本发明内容的实质所作出的等效变化或修饰，都应涵盖在本发明保护范围内。

Claims (15)

  1. 一种中药复方组合物，其特征在于，包括下列重量份数的中药材：臭椿皮4～12份，白花蛇舌草12～30份，莪术3～15份，丹参15～30份，夏枯草5～15份，甘草4～12份。
  2. 根据权利要求1所述的中药复方组合物，其特征在于，包括下列重量份数的中药材：臭椿皮6～9份，白花蛇舌草15～24份，莪术4～12份，丹参18～21份，夏枯草6～10份，甘草6～9份。
  3. 如权利要求1或2的中药复方组合物的制备方法，其特征在于，包括以下步骤：
    (1)浸泡：取权利要求1或2所述六味中药材，加入蒸馏水浸泡，得到生药蒸馏水混合物；
    (2)煎煮：将步骤(1)所述的生药蒸馏水混合物进行煎煮，得到中药粗提物；
    (3)浓缩：将步骤(2)制备得到的中药粗提物浓缩，得到中药浓缩液；
    (4)除菌：取步骤(3)制备得到的中药浓缩液通过微孔滤膜过滤除菌，即得到所述中药复方组合物。
  4. 将权利要求1所述的中药复方组合物应用于制备治疗恶性肿瘤药物的应用。
  5. 根据权利要求4所述的应用，其特征在于，所述恶性肿瘤包括前列腺癌、乳腺癌、肺癌、结肠癌、脑癌、皮肤癌、膀胱癌、肾癌。
  6. 将权利要求1所述的中药复方组合物应用于制备治疗由雄激素受体异常扩增和突变导致的恶性肿瘤细胞的增殖、生长、迁移和浸润的药物的应用。
  7. 如权利要求1所述的中药复方组合物应用于制备治疗前列腺癌药物的用途。
  8. 将权利要求1所述的中药复方组合物应用于抑制前列腺癌细胞的生长、增殖、转移、迁移、侵袭和促进前列腺癌细胞的凋亡的用途。
  9. 如权利要求7所述的用途，其特征在于，所述前列腺癌为去势抵抗性前列腺癌或雄激素依赖性前列腺癌。
  10. 将权利要求1所述的中药复方组合物应用于体外抑制双氢睾酮诱导的雄激素受体的转录活性以及缺失配体结构域的雄激素受体的转录活性的用途，或用于体外抑制雄激素受体活性。
  11. 将权利要求1所述的中药复方组合物应用于体外抑制雄激素受体突变的前列腺癌细胞株22RV1的增殖、抑制雄激素依赖性前列腺癌细胞株LNCaP的增殖的用途。
  12. 将权利要求1所述的中药复方组合物应用于体外抑制前列腺癌细胞PC-3和LNCaP以及22RV1的迁移的用途，或体外抑制比卡鲁胺或MDV3100治疗无效的雄激素受体突变细胞株22RV1细胞增殖的用途。
  13. 将权利要求1所述的中药复方组合物应用于体内抑制前列腺癌细胞的转移，或体内抑制去势抵抗性前列腺癌的生长和转移，或在小鼠体内抑制前列腺癌细胞22RV1的生长或转移。
  14. 如权利要求4-13所述的用途，其特征在于，将所述中药复方组合物和药学上可接受的载体制成可注射流体、气雾剂、乳膏、凝胶剂、丸剂、糖浆剂、透皮贴剂、胶囊剂、微囊、片剂、颗粒剂或口服液剂型的药物。
  15. 根据权利要求4-13所述的应用，其特征在于，将所述中药复方组合物单独使用或与其他药物联合使用。
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