CN115212246A - Pharmaceutical composition for inhibiting prostate cancer metastasis and preparation method thereof - Google Patents
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- CN115212246A CN115212246A CN202210758425.3A CN202210758425A CN115212246A CN 115212246 A CN115212246 A CN 115212246A CN 202210758425 A CN202210758425 A CN 202210758425A CN 115212246 A CN115212246 A CN 115212246A
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Abstract
The invention provides a pharmaceutical composition for inhibiting prostate cancer metastasis and a preparation method and application thereof, wherein the pharmaceutical composition comprises, by weight, 50-75 parts of ginseng, 50-75 parts of glabrous sarcandra herb, 25-40 parts of parasitic loranthus and 25-40 parts of astragalus membranaceus. The composition can remarkably inhibit the expression and mRNA level of key proteins including ITGB3, ras and the like in an ITGB3-Ras signal pathway, and can remarkably inhibit the metastasis of prostate cancer; the invention can obviously inhibit the growth of prostate cancer; the invention hits the prostate cancer pathogenesis, has the efficacies of tonifying qi and strengthening body resistance, removing blood stasis and detoxifying, and eliminating mass and dissipating stagnation, and has the characteristics of balancing yin and yang, performing both purgation and tonification, tonifying without stagnation, tonifying without dryness, and eliminating without damaging the body resistance.
Description
Technical Field
The invention belongs to the technical field of anticancer drug research, relates to a tumor metastasis inhibition drug technology, and particularly relates to a pharmaceutical composition for inhibiting prostate cancer metastasis and a preparation method thereof.
Background
Prostate cancer is one of the more common malignant tumors of the male genitourinary system, and has the characteristics of high morbidity, high mortality and difficult prevention and treatment. Aggressive growth and distant metastasis are one of the main features of their malignant biological behavior and are also the leading factors that contribute to poor prognosis in prostate cancer patients. Therefore, research for treating metastatic prostate cancer is one of the major research points at present.
Currently, in the research of related western medicines, common western medicines are mainly used for treating prostate cancer before metastasis, such as docetaxel [1] . However, the western medicines not only have poor effect on treating the metastatic prostate cancer, but also have certain side effects [2] . In order to overcome the defects of common western medicines in the aspect of treating metastatic prostate cancer, the existing method mainly focuses on combined medicine [3]-[4] Or drug-assisted chemotherapy [5] On the other hand, although these regimens have some effect in treating metastatic prostate cancer, they still do not overcome the disadvantages of greater side effects and there are still few effective treatment regimens available.
Classic works of traditional Chinese medicine have been well documented in the treatment of tumors, such as "ovarian cyst" in the Nei Jing, "abdominal mass" in the pathogenesis treatises, "accumulation" in the difficultly Jing and "renal rock" and "mammary rock" in the later generations, and these names are basically tumors of organs and tissues such as the stomach and intestine, the liver, the pancreas and the breast. In addition, the book of Wei Ji treasure book cloud: the six excesses, seven emotions, internal injuries, overeating, etc. cause imbalance of yin and yang and disorder of internal organs, produce qi stagnation, phlegm and fluid retention, blood stasis, etc. which are retained in the human body to form stagnation, retention and rock. Therefore, the treatment of tumors by using traditional Chinese medicines is an effective choice. Meanwhile, there are some active reports of Chinese herbs in treating cancer metastasis, for example, li Feifei [6] The clinical research of the traditional Chinese medicine for preventing and treating colorectal cancer recurrence and metastasis is summarized by Chen Xiaochao [7] Et al summarize the study of traditional Chinese medicine for treating colorectal cancer metastasis. In addition, the combination of Chinese and Western medicine has been studied in treating cancer metastasis, such as Yang Lu [8] The effect of the traditional Chinese medicine stasis-dissipating and vein-relaxing granules in combination with western medicines on treating malignant tumor bone metastasis is researched by Li Haoze [9] The advantages of the combination of Chinese and Western medicine therapy in preventing and treating tumor recurrence and metastasis are summarized by the people.
At present, the treatment aspect of the prostate cancer in the traditional Chinese medicine is basically limited in the aspects of inhibiting tumor cell proliferation, inducing apoptosis, regulating an immune system, resisting tumor angiogenesis, regulating cell autophagy and the like, the research on how to inhibit prostate cancer metastasis is not enough, and the research on related formulas is rare, so that the treatment effect of the existing traditional Chinese medicine technology on inhibiting the prostate cancer is still unsatisfactory.
In summary, no matter in western medicine or traditional Chinese medicine, no effective scheme and corresponding knowledge are available for how to inhibit prostate cancer metastasis. In particular, to the best of the inventors' knowledge, the existing related traditional Chinese medicine research rarely relates to how to inhibit prostate cancer metastasis. Therefore, there is a need in the art for a Chinese medicinal formulation that is effective in inhibiting prostate cancer metastasis.
Reference in this section:
[1] deng Jiao, he Zhongming, liu Fei Nego, oudensday, xia Chengxing, yang Delin research on docetaxel treatment of prostate cancer has progressed [ J ] modern oncology, 2021,29 (21): 3855-3858.
[2]Sweeney CJ,Chen YH,Carducci M,Liu G,Jarrard DF,Eisenberger M,Wong YN,Hahn N,Kohli M,Cooney MM,Dreicer R,Vogelzang NJ,Picus J,Shevrin D,Hussain M,Garcia JA,DiPaola RS.Chemohormonal Therapy in Metastatic Hormone-Sensitive Prostate Cancer.N Engl J Med.2015；373(8):737-46.
[3] Zhang Xinjun. Effect of goserelin in combination with bicalutamide applied post-operatively to patients with metastatic prostate cancer [ J ]. Henan medical research, 2021,30 (30): 5686-5688.
[4] Zhang Dong, cheng Yue, yan Zejun therapeutic progress for oligometastatic prostate cancer [ J ] modern J of urology 2019,24 (11): 963-965.
[5] Qi Feng, louxexin, li Xiao, cai Hongzhou, lengxus, yu Bin, zou Qing, xu Zicheng. Analysis of the efficacy of docetaxel neoadjuvant chemotherapy in the local progression and treatment of oligometastatic prostate cancer [ J ]. J.Clin urology J. 2022,37 (06): 447-451.
[6] Li Feifei, zhang Zhiwei, yu Hongjie and Zhang Xiaoxiao clinical research progress of traditional Chinese medicine for preventing and treating colorectal cancer recurrence and metastasis [ J ] modern J.Med. Med. Med and Western medicine, 2021,30 (36) 4078-4083.
[7] Chen Xiaochao, yang Xiangdong, tan Tianying, study overview of TCM for colorectal cancer metastasis [ J ] modern clinical medicine, 2021,47 (06): 465-467.
[8] Yang Lu, wang Zhongji, qijingj, zhang Chengcheng, qian Xinle clinical study of the combination of Chinese medicine stagnation-dissipating and collaterals-dredging particles and western medicines for treating malignant tumor bone metastasis [ J ]. Shanghai J.Med.J., 2022,56 (02): 49-53.
[9] Li Haoze, zhou Jing, li Qi, quqing, advantages of the combination therapy of Chinese and Western medicine for preventing and treating tumor recurrence and metastasis and prospect [ J ]. Shanghai J.J. Med.Med.Med. 2022,56 (02): 91-95+100.
Disclosure of Invention
In view of the disadvantages of the prior art and the need in the art, it is an object of the present invention to provide a pharmaceutical composition that can inhibit prostate cancer metastasis.
In order to realize the purpose, the invention provides the following technical scheme:
the pharmaceutical composition for inhibiting prostate cancer metastasis comprises, by weight, 50-75 parts of ginseng, 50-75 parts of glabrous sarcandra herb, 25-40 parts of parasitic loranthus and 25-40 parts of astragalus membranaceus.
The present inventors have conducted extensive studies on the treatment of prostate cancer in the previous research works. For example, in the influence of the Chinese medicinal prescription for strengthening body resistance and eliminating diseases on the proliferation and apoptosis of human prostatic cancer DU-145 cells [1] Study on the effects and mechanism of human prostate cancer DU-145 apoptosis of Chinese herbal compound for strengthening body resistance and inhibiting tumor [2] The effect of traditional Chinese medicine (trade name: delishen injection) consisting of the whole-component extract of ginseng, astragalus, toad shortbread and cantharis on the aspect of treating the prostatic cancer is researched; inhibition of PC-3 cell proliferation in Ginseng radix Humuli Shang Duiren prostate cancer [4] The effect of the ginseng-walnut decoction on the treatment of the prostate cancer is researched.
In addition, the inventor also researches the traditional Chinese medicine for regulating and controlling the PC-3 cell metastasis effect of human prostate cancer, and configures the traditional Chinese medicine compound consisting of ginseng, turmeric and glabrous sarcandra herb in contrast research on the PC-3 cell metastasis inhibition effect of the dutasteride and the traditional Chinese medicine compound for strengthening body resistance and inhibiting tumors on human prostate cancer. The research finds that the traditional Chinese medicine compound can inhibit the proliferation of the PC-3 cells of the human prostate cancer under the condition of losing nests.
However, the methods in these studies are either effective only in inhibiting tumor cell proliferation or have poor inhibition ability against metastasis of prostate cancer cells, and it is difficult to achieve the metastasis inhibition effect at low drug concentrations.
In addition to the above studies by the inventors, there are some Chinese herbs in the art for inhibiting prostate cancer metastasis, for example, teachers have advanced [5] The inventor finds that the Zilongjin (namely compound Bailong tablet, the main components of which are liquorice, black nightshade, salvia miltiorrhiza and the like) can reduce the invasiveness of cells; li Xiaojiang [6] The research progress of the traditional Chinese medicine for treating the bone metastasis of the prostatic cancer is summarized; hu Meichun [7-8] Based on the active component ailanthone, the Chinese medicinal composition consisting of 6 medicaments of ailanthus bark, oldenlandia diffusa, zedoary and the like is prepared, and the composition can be used for treating the growth and the metastasis of the prostatic cancer.
However, the existing traditional Chinese medicines for inhibiting prostate cancer metastasis are still very few, and the compatibility of various traditional Chinese medicines is poor, so that the development of a new traditional Chinese medicine is difficult; meanwhile, in the aspect of western medicine, people have the same deficiency of theoretical understanding of prostate cancer metastasis, so that theoretical guidance for developing new drugs is lacked in both traditional Chinese medicine and western medicine.
The inventor finds that the composition can effectively inhibit the prostate cancer metastasis through a large amount of grope. From the theory of traditional Chinese medicine, ginseng is the monarch drug, and the compendium of materia medica says "ginseng, … … for treating all deficiency syndromes of men and women, frequent urination and dripping"; ginseng is mainly used for tonifying primordial qi, restoring pulse, strengthening spleen and tonifying lung, while the spleen qi is healthy to promote digestion of food essence, while the lung qi is healthy to regulate water passage, so that the qi transformation function is normal to discharge water metabolized in vivo out of the body through urine. Herba Pileae Scriptae has effects in promoting blood circulation, removing toxic substances, clearing away heat, cooling blood, removing speckle, dispelling blood stasis, dispelling pathogenic wind, removing dampness, and dredging collaterals; herba Taxilli has effects in nourishing liver and kidney, strengthening tendons and bones, removing rheumatism, dredging channels and collaterals, nourishing blood, and preventing miscarriage; radix astragali has effects of invigorating spleen, invigorating middle warmer, invigorating yang, lifting sink, invigorating defensive qi, consolidating superficial resistance, promoting urination, removing toxic substance, and promoting granulation. The whole formula hits the prostate cancer pathogenesis, has the efficacies of tonifying qi and strengthening body resistance, removing blood stasis and detoxifying, and eliminating mass and dissipating stagnation, and has the characteristics of balancing yin and yang, performing both purgation and tonification, tonifying without stagnation, tonifying without dryness, and eliminating without damaging the body resistance.
As can be seen from the examples of the present invention, the composition of the present invention can inhibit the migration and invasion of PC-3 and DU-145 at low concentrations, and shows a high efficacy in inhibiting prostate cancer metastasis.
Integrins (Integrins) are composed of alpha and beta subunits, are transmembrane proteins on cell membranes which mediate the interaction between intercellular cells or between cells and extracellular matrix (ECM), participate in a series of physiological processes such as cell proliferation, differentiation, senescence and apoptosis, and are closely related to pathological processes such as tumorigenesis and metastasis. There are 24 members of the integrin family that have been identified, at least 8 of which are highly expressed in prostate cancer tissue [9] . It is reported that Integrin beta 6 is obviously up-regulated in the development process of prostate cancer, and phosphorylation activation of Integrin beta 1 can promote lymphatic metastasis and bone metastasis of prostate cancer. Activated integrins activate FAK (Focal adhesion kinase), which in turn activates the Src gene family, causing cells to escape from nestling. After the metastasis, it mediates the adhesion of tumor cells and normal organs by activating an adhesion molecule E-cadherin, and promotes the prostate cancer to generate bone metastasis and lymphatic metastasis.
As can be seen in the examples of the present invention, the composition of the present invention can inhibit the expression and mRNA level of key proteins including ITGB3, ras and the like in the ITGB3-Ras signaling pathway, thereby inhibiting the migration and invasion ability of prostate cancer cells.
As can be seen from the comparative examples of the invention, the existing other traditional Chinese medicines can not inhibit the expression of key proteins of ITGB3 and Ras in the aspects of migration and invasion experiments of PC-3 and DU-145.
As can be seen from the examples of the present invention, the composition of the present invention can also significantly inhibit the growth of prostate cancer tumors.
The results show that the invention provides a traditional Chinese medicine composition capable of obviously inhibiting the growth and metastasis of prostate cancer.
Preferably, the pharmaceutical composition further comprises 1-2 parts of docetaxel. According to an embodiment of the invention, on the basis of the traditional Chinese medicine formula provided by the invention, the traditional Chinese medicine formula is combined with docetaxel, so that better effects of inhibiting tumor growth and metastasis can be obtained. This demonstrates that the traditional Chinese medicine formulation provided by the present invention can be used in combination with docetaxel and achieve a synergistic effect in inhibiting tumor growth and metastasis.
Preferably, the pharmaceutical composition consists of the following components in parts by weight: 75 parts of ginseng, 75 parts of glabrous sarcandra herb, 25 parts of Chinese taxillus twig, 35 parts of astragalus and 1 part of docetaxel.
As an alternative embodiment of the present invention, the pharmaceutical composition is in the form of any one of a water decoction, a tablet, a powder, an oral liquid, a pill, a tincture, and a syrup.
The invention provides a preparation method for preparing the composition, which comprises the following steps: decocting the ginseng, the glabrous sarcandra herb, the parasitic loranthus and the astragalus root in parts by weight with water, and concentrating liquid medicine to prepare liquid medicine containing 4.5-20 g/mL of crude drugs. When said composition further comprises docetaxel, said parts by weight of docetaxel is combined with the aforementioned liquid medicine.
As an optional embodiment of the invention, the decoction is carried out for 2-3 times, each time of the decoction is 2-3 hours, and the decoction liquids are combined after the decoction is finished.
The invention provides application of the composition in preparing a medicament for treating prostatic cancer. In particular, the invention provides application of the composition in preparing a medicament for inhibiting prostate cancer metastasis, and particularly provides application in preparing a medicament for inhibiting prostate cancer metastasis in the aspect of inhibiting ITGB3 and Ras expression.
The invention has the beneficial effects that:
the composition can remarkably inhibit the expression and mRNA level of key proteins including ITGB3, ras and the like in an ITGB3-Ras signal pathway, and can remarkably inhibit the metastasis of prostate cancer; the invention can obviously inhibit the growth of prostate cancer; the invention hits the prostate cancer pathogenesis, has the efficacies of tonifying qi and strengthening body resistance, removing blood stasis and detoxifying, and eliminating mass and dissipating stagnation, and has the characteristics of balancing yin and yang, performing both purgation and tonification, tonifying without stagnation, tonifying without dryness, and eliminating without damaging the body resistance.
References in this section:
[1] zhou Shiyi, zhang Shuwu, shao Ji Chun Fuzheng Xiaoxiao Chinese medicinal formula effects on proliferation and apoptosis of human prostate cancer DU-145 [ J ] Chengdu TCM university report, 2011,34 (04): 51-53.
[2] Shao Jichun, zhou Shiyi, zhang Shuwu. Fuzheng tumor-inhibiting Chinese herbal compound has effects on human prostatic cancer DU-145 apoptosis and mechanism research [ J ]. Chinese journal of Male science, 2016,30 (04): 13-18+29.
[3] Zhang Shuwu, zhang Peihai, chang Degui, wang Jiuyuan, qu Xiaowei, shao Ji spring ginseng huhu Shang Duiren prostate cancer PC-3 cell proliferation inhibitory effect [ J ]. Chinese journal of andrology, 2007 (06): 7-9.
[4] Zhang Shuwu, zhang Peihai, chang Degui, wang Jiuyuan, qu Xiaowei, shao Ji spring ginseng huhu Shang Duiren prostate cancer PC-3 cell proliferation inhibitory effect [ J ]. Chinese journal of andrology, 2007 (06): 7-9.
[5] The preparation is named as Zhichang, yu Lizhang, yanqu, ding Yi, li Hongwei, yang Fengguang, liu Wujiang, youjin, li Xuesong, liang Yunyan, wang Daishu, the influence of Zilongjin on the in vitro proliferation and invasion capacity of metastatic prostate cancer [ J ]. China journal of surgery 2003 (06): 76.
[6] Li Xiaojiang, li Yangyang, mou Ruiyu, zhaoyang, wu Mingxin, gu Yingjie research on the treatment of bone metastasis of prostate cancer by traditional Chinese medicine [ J ] Chinese herbal medicine, 2018,49 (04): 965-969.
[7] Hu Meichun research on the function and mechanism of Chinese herbal medicine monomer and Chinese herbal medicine compound for inhibiting the growth and metastasis of prostate cancer [ D ] university of east China, 2015.
[8] Yi Zhengfang, hu Meichun, liu Mingyao A Chinese medicinal compound composition and its application in resisting prostatic cancer, 2019.
[9]Seguin L,Desgrosellier JS,Weis SM,Cheresh DA.Integrins and cancer:regulators of cancer stemness,metastasis,and drug resistance.Trends Cell Biol.2015；25(4):234-40.
Description of the drawings:
FIG. 1 is a graph showing the results of the inhibition of metastasis of prostate cancer cells in vitro by the composition (Fz for short) of example 1 of the present invention; wherein, part (A) is a Transwell invasion cell experiment, and part (B) is a scratch experiment;
FIG. 2 is the results of proteomics analysis of the present invention; wherein, part A investigates and utilizes iTRAQ quantitative proteomics technology to screen protein expression change in PC-3 cells before and after the composition treatment of the invention example 1, and part B bioinformatics analysis shows that ITGB3-Ras signal path is regulated by the composition of the invention example 1;
FIG. 3 shows the results of the composition (Fz) of example 1 of the present invention inhibiting the proliferation of prostate cancer cells in vitro; wherein, part (A) is the detection result of MTT method, and part (B) is the detection result of clone formation method;
FIG. 4 shows the results of the composition of example 1 (Fz) of the present invention for inhibiting the growth and metastasis of prostate cancer cells in vivo; wherein, the measurement of the subcutaneous tumor volume of the mouse in part (A) shows that the composition in part (B) inhibits the growth of tumor tissue, the HE staining of the tumor tissue section of part (B) shows that the anisotropic cells of the control group are arranged like adenoid organ, the composition in part (B) shows that the tumor cells are largely necrotic, nuclearly disintegrated and nuclearly decomposed, and the HE staining of part (C) shows that the naked mouse is respectively injected with DU-145WT + Fz intragastrically (left), DU-145WT + normal saline intragastrically (middle) and DU-145shITGB3+ normal saline intragastrically (right) through femoral artery, and the bone metastasis condition is detected by in vivo imaging after 3 weeks;
FIG. 5 is a graph showing the results of an experiment for detecting the levels of ITGB3 protein and mRNA in PC-3 (higher metastatic potential) and LNCap (low metastasis) cells by immunoblotting and real-time quantitative PCR, indicating that the metastatic potential of prostate cancer cells is positively correlated with the expression of ITGB 3;
FIG. 6 shows the results of the experiment of the composition of example 1 (abbreviated as Fz) of the present invention in inhibiting the ITGB3-Ras signaling pathway; wherein part (A) shows that high expression of ITGB3 in PC-3 cells improves the metastatic potential of the cells, and parts (B) and (C) show that ITGB3 is knocked down in DU-145, so that the metastatic potential of the cells is reduced.
Detailed Description
The present invention is described in detail below by way of examples, and it should be noted that the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention.
Example 1
1. Preparation of the composition
(1) According to the weight portion, 75 portions of ginseng, 75 portions of glabrous sarcandra herb, 25 portions of Chinese taxillus twig and 35 portions of astragalus root are taken.
(2) Pulverizing, adding water to 2-3 cm above the surface of the raw materials, decocting for 3 times, mixing water decoctions, and concentrating to obtain medicinal liquid containing crude drugs of 3.2, 4.5, 6.4, 9.0, 18.0 and 20.0 g/mL.
2. Prostate cancer cell proliferation and metastasis
i) Cell culture and passage: culturing immortalized prostate cell line RWPE-1 and prostate cancer cell lines PC-3 and DU-145 in RPMI-1640 medium containing 10% fetal calf serum by ATCC standard cell culture method at 37 deg.C and 5% CO 2 Culturing and subculturing in an incubator.
ii) preparation of drug-containing serum: 30 SD male rats with the body weight of 150-180g are randomly divided into 15 drug-containing serogroups and 15 drug-free serogroups. According to the previous experimental result, the designed dosage is 3.2g crude drug/kg by adopting the conversion of the body surface area dosage, and the intragastric volume is 20ml/kg d -1 . The drug-free serogroup was administered with equal volume of physiological saline for intragastric administration. After 5 days of continuous intragastric administration, the animals were anesthetized, the abdominal aorta was bled, serum was separated, inactivated at 56 ℃ for 40 minutes, and split-packed at-80 ℃ for subsequent in vitro experiments.
iii) Soft agar colony formation assay: taking each prostate cancer cell line in logarithmic growth phase, preparing the medium containing different concentrations (0,5, 10, 15, 20, 30 μ g/ml, the same below) of the composition into 2 × 10 density 3 The single cell suspension is prepared by spreading 1ml on soft agar (12-well plate), and transferring to culture dish in CO after the cell agar suspension is solidified 2 The cells were cultured at 37 ℃ for 7 to l0 days in an incubator, and the cell colonies were observed and counted. After repeated experiments, the colony forming conditions of various cells are analyzed statistically, and the prostate cancer cells of the composition are determinedThe effect of proliferative capacity.
iv) immunoblot experiments: after treating cells for 24h by using compositions with different concentrations, extracting intracellular proteins to carry out an immunoblotting experiment, and detecting the changes of expression and phosphorylation levels of cyclins (comprising CDK2, p27, cyclin D1, CDK4, p53 and the like); detecting the expression change of apoptosis-related proteins (including caspase3, cleared-caspase 3, PARP, cleared-PARP and the like) to determine whether the composition can induce cycle arrest and apoptosis of prostate cancer cells.
v) scratch test: the prostate cancer cell lines are resuspended in culture media containing different concentrations of compositions, inoculated in six-well plates respectively, vertically scratched on the surface of a monolayer of cells, and observed under an inverted microscope after being cultured for 0,24 and 48 hours, so that the effect of the composition on inhibiting the migration capability of the prostate cancer cells is clarified.
vi) Transwell cell experiments: placing low-nutrient culture solution in upper chamber of the chamber, placing high-nutrient culture solution in lower chamber, spreading Matrigel (simulated extracellular matrix) on bottom filter membrane of upper chamber of Transwell in advance, and adjusting concentration of cell suspension of each group to 1 × 10 with culture medium containing different concentration compositions 5 And/ml, adding sample in the upper chamber, continuously culturing for 48h, sucking culture solution in the upper chamber and the lower chamber, observing the number of cells penetrating through Matrigel in each concentration group by an inverted microscope after crystal violet staining, and determining the inhibition effect of the composition on the invasion capacity of the prostate cancer cells. As with the above method, the chamber was not plated with Matrigel and the effect of the composition on the ability of prostate cancer cells to migrate was investigated.
3. Molecular mechanism research for inhibiting prostate cancer metastasis
i) Effect of composition on ITGB3-Ras signaling pathway: detecting the expression quantity and phosphorylation level changes of ITGB3-Ras signal path key genes (including ITGB3, ras, FAK, ERK, raf, PAK and the like) and key markers and transcription factors (including E-cadherin, vimentin, snail, ZEB and the like) in the EMT process before and after the treatment of the composition by using an immunoblotting experiment; extracting nucleic acid in cells, designing specific primers of key genes in the ITGB3-Ras passage, and detecting mRNA level changes of the proteins in the cells before and after treatment of the composition by a real-time quantitative PCR method; by means of immunofluorescence, nuclear-cytoplasmic separation and the like, subcellular localization change of the protein is researched, and the regulation and control effect of the 'strengthening body resistance and tumor inhibition' prescription on an ITGB3-Ras signal channel in prostate cancer cells and the influence of an EMT process are determined.
ii) construction of a cell model for stable overexpression/knockdown of ITGB 3: PC-3 and DU-145 cells stably expressed and knocked down by ITGB3 are constructed by using a lentivirus technology. The cell model is used for repeating the cytotoxicity experiment and the metastasis invasion experiment of the composition, detecting the expression and phosphorylation level of key proteins in the ITGB3-Ras signal channel, and compared with the result of a prior wild type parent cell, the regulation and control mechanism of the ITGB3-Ras signal channel in inhibiting the prostate cancer cell EMT and metastasis in the composition is elucidated from the in vitro level.
4. The effect of the composition on inhibiting prostate cancer was verified at the in vivo level and preclinical evaluation was performed
i) Nude mouse model of prostate cancer bone metastasis: and subcutaneous tumor model in bone metastasis model, 150 nude mice of 4 weeks of age were randomly divided into blank group, model group, composition-tolerant dose group, high dose group, medium dose group, low dose group, positive control group (docetaxel). After the model group and the mice of each treatment group are anesthetized with pentobarbital, the injection density is 10 slowly in the femoral artery 7 20-40 mul of cell suspension per ml, and 4-5 weeks after injection can form a metastasis focus. Administration was started 24h after injection of the parent aneurysm. According to the body surface area dose reduction algorithm and the results of preliminary experiments, the administration dose is designed to be 4.5 times of the low dose (4.5 g crude drug/kg), 9 times of the medium dose (9 g crude drug/kg), 18 times of the high dose (18 g crude drug/kg) and 30 times of the tolerance dose group (30 g crude drug/kg). The intragastric volume is 20ml/kg d -1 . Once a day for 30 days; the blank group and the model group are administered with 20 ml/kg. D of normal saline by intragastric gavage -1 . Docetaxel 10 mg/kg. D is injected into abdominal cavity of positive control group -1 . The tumor bone metastasis condition in the body of the mouse is observed, the pathological form is observed by HE staining, and comparative analysis is carried out, so that the function of the composition for inhibiting the proliferation and the metastasis of the prostatic cancer is determined from the in vivo level. In the subcutaneous tumor model, C57BL/6J mice were grouped according to the bone metastasis grouping protocol described above and injected subcutaneously with murine prostate cancer cell linesRM-1, the dosing regimen was the same as for the bone metastasis model. The size and the number of tumors in the mice are detected, and the inhibition effect of the composition on the growth of the prostate cancer is determined.
ii) cell models in combination with animal models: inoculating the stable cell line into a femoral artery injection mouse model of prostate cancer bone metastasis, repeating the experiment, and determining the influence of an ITGB3-Ras signal channel on the tumor inhibition effect of the stable cell line by detecting the differential inhibition rate of a composition on an ITGB3 knockdown and over-expression model; taking a bone marrow cavity to carry out experiments such as immunohistochemical staining, real-time quantitative PCR and the like, detecting the expression and phosphorylation levels of key proteins, proliferation related markers (including Ki67, PCNA and the like) and metastasis related markers in the ITGB3-Ras signal path, and further determining the molecular mechanism of the ITGB3-Ras signal path in the process of inhibiting the prostate cancer metastasis by the composition.
5. Results of the experiment
i) As shown in figure 3, the drug-containing serum of the composition (in vitro cell experiments, the compositions refer to the drug-containing serum) induces the death of prostate cancer cells under higher concentration, and preliminarily verifies the anti-tumor activity.
In addition, as shown in figure 1, at lower concentrations (5 μ g/ml, not significantly causing cell death), the composition was able to inhibit the in vitro invasion and migration of prostate cancer cells PC-3 and DU-145 with a certain metastatic potential.
ii) as shown in part a and part B of fig. 4, in the subcutaneous tumor model, the composition was found to inhibit tumor growth with substantial necrosis of tumor cells. As shown in part C of fig. 4, in the femoral artery injection model, the composition was found to significantly inhibit bone metastasis of prostate cancer, and knocking out the expression of ITGB3 produced a tumor-suppressing effect similar to that of the composition.
iii) As shown in figure 2, the protein expression change of PC-3 cells before the composition treatment is detected by using a proteomics technology, and the ITGB3-Ras signal channel is in a key position. We subsequently examined the levels of the background protein and mRNA of ITGB3 and the expression level of the key protein Ras downstream of ITGB3 within PC-3 cells and LNCap cells with low metastatic potential, showing that ITGB3 and Ras are more highly expressed in PC-3 cells with higher metastatic potential, suggesting that the ITGB3-Ras signaling pathway may promote the metastasis of prostate cancer, and that the composition may suppress the metastasis of prostate cancer by inhibiting this pathway (as shown in fig. 5).
iv) on the basis, through expression intervention and functional antagonism, the over-expression of ITGB3 is found to improve the transfer potential of PC-3 cells, and meanwhile, the transfer capacity of the PC-3 cells can be recovered to a lower state through treatment by using the composition; in contrast, the knock-down of ITGB3 can weaken the metastatic ability of DU-145, and the inhibition effect of the composition on the metastasis is simulated, which indicates that the inhibition effect of the composition on the ITGB3-Ras signal pathway is an important reason for inhibiting the prostate cancer metastasis. Meanwhile, after the ITGB3 is knocked down, the invasion and migration capacity of prostate cancer cells can be further inhibited by treating the composition, and the result shows that an ITGB3-Ras channel is probably not the only channel for the formula to play a role, so that the advantage of multiple targets of the traditional Chinese medicine compound is reflected. To this end we have preliminarily demonstrated the important role played by the ITGB3-Ras signaling pathway in this approach to inhibit prostate cancer metastasis (as shown in figure 6).
Example 2
The same as example 1 was repeated except that 50 parts of ginseng, 50 parts of glabrous sarcandra herb, 40 parts of loranthus parasiticus and 40 parts of astragalus root were taken at the time of preparing the composition.
Referring to the method of example 1, the inhibitory effect on prostate cancer metastasis was examined, and it was found that the in vitro invasion and migration of prostate cancer cells PC-3 and DU-145 having a certain metastatic potential can be inhibited at a drug-containing serum concentration of 5. Mu.g/ml.
Example 3
The procedure of example 1 was repeated, except that 60 parts of ginseng, 60 parts of glabrous sarcandra herb, 30 parts of loranthus parasiticus and 30 parts of astragalus root were used to prepare the composition.
Referring to the method of example 1, the inhibitory effect on prostate cancer metastasis was examined, and it was found that the in vitro invasion and migration of prostate cancer cells PC-3 and DU-145 having a certain metastatic potential can be inhibited at a drug-containing serum concentration of 5. Mu.g/ml.
Example 4
In this example, the composition of this example was comprised of the composition of example 1 and docetaxel, wherein the weight ratio of the composition of example 1: docetaxel = 210. The composition of example 1 was used at 210 times the weight of docetaxel as used.
Considering the inhibitory effect on prostate cancer metastasis by the method of example 1, the composition of example 1 required only a drug-containing serum concentration of 2 μ g/ml to achieve the same inhibitory effect on the invasion and migration of PC-3 and DU-145 in vitro as in example 1; the composition of example 1 required only a drug-containing serum concentration of 5. Mu.g/ml in this example, and the same inhibitory effect on the proliferation activity of PC-3 and DU-145 as in example 1 was achieved. This suggests that the composition of example 1 has a synergistic effect with docetaxel and that docetaxel can be administered at very low doses, which is very beneficial in avoiding the side effects of docetaxel.
Comparative example 1
Using ginseng Hu Heshang from "inhibitory action on PC-3 cell proliferation of Ginseng radix Hu He Shang Duiren for prostate cancer", the inhibition of migration and invasion of PC-3 and DU-145 was examined with reference to example 1, and it was found that this formulation could not inhibit the expression of ITGB3 and Ras proteins.
Claims (10)
1. The pharmaceutical composition for inhibiting prostate cancer metastasis is characterized by comprising, by weight, 50-75 parts of ginseng, 50-75 parts of glabrous sarcandra herb, 25-40 parts of parasitic loranthus and 25-40 parts of astragalus membranaceus.
2. The pharmaceutical composition of claim 1, further comprising 1 to 2 parts of docetaxel.
3. The pharmaceutical composition according to claim 2, wherein the pharmaceutical composition comprises the following components in parts by weight: 75 parts of ginseng, 75 parts of glabrous sarcandra herb, 25 parts of Chinese taxillus twig, 35 parts of astragalus and 1 part of docetaxel.
4. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition is in the form of any one of a decoction, a tablet, a powder, an oral liquid, a pill, a tincture, and a syrup.
5. The pharmaceutical composition of claim 2, wherein the formulation of the ginseng, the glabrous sarcandra herb, the parasitic loranthus and the astragalus root in the pharmaceutical composition is any one of decoction, tablet, powder, oral liquid, pill, tincture and syrup.
6. A method for preparing a pharmaceutical composition for inhibiting prostate cancer metastasis, the pharmaceutical composition being the pharmaceutical composition according to any one of claims 1 to 3, the pharmaceutical composition being in the form of a decoction, the method comprising:
decocting the ginseng, the glabrous sarcandra herb, the parasitic loranthus and the astragalus root in parts by weight with water, and concentrating liquid medicine to prepare liquid medicine containing 3.5-20 g/mL of crude drugs.
7. The preparation method according to claim 6, wherein the decoction is carried out for 2 to 3 times, each time for 2 to 3 hours, and the decoction is combined after the decoction.
8. Use of a pharmaceutical composition according to any one of claims 1 to 5 or prepared according to the process of claim 6 or 7 for the manufacture of a medicament for the treatment of prostate cancer.
9. Use of a pharmaceutical composition according to any one of claims 1 to 5 or a pharmaceutical composition prepared by a process according to claim 6 or 7 for the manufacture of a medicament for inhibiting prostate cancer metastasis.
10. The use according to claim 9, for the preparation of a medicament for inhibiting prostate cancer metastasis, for inhibiting ITGB3 and Ras expression.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1491694A (en) * | 2003-08-27 | 2004-04-28 | 山东中医药大学 | Anti-cancer medicine and its producing method |
| CN1954838A (en) * | 2005-10-26 | 2007-05-02 | 黄振华 | Medical composite of antineoplastic |
| CN105194550A (en) * | 2015-10-15 | 2015-12-30 | 华东师范大学 | Compound traditional Chinese medicine composition and application thereof to resistance to prostate cancer |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1491694A (en) * | 2003-08-27 | 2004-04-28 | 山东中医药大学 | Anti-cancer medicine and its producing method |
| CN1954838A (en) * | 2005-10-26 | 2007-05-02 | 黄振华 | Medical composite of antineoplastic |
| CN105194550A (en) * | 2015-10-15 | 2015-12-30 | 华东师范大学 | Compound traditional Chinese medicine composition and application thereof to resistance to prostate cancer |
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| 朱开昕等: "桑寄生药理作用及临床应用研究进展" * |
| 朱开昕等: "桑寄生药理作用及临床应用研究进展", 现代医学与健康研究电子杂志, vol. 2, no. 12, pages 189 - 190 * |
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Application publication date: 20221021 |