CN1810247A - Pharmaceutical effect of berbamine derivative EBB in inhibiting invasive metastasis of human body tumor cell - Google Patents

Abstract

The present invention relates to the pharmaceutical application of berbamine derivative EBB in inhibiting invasive metastasis of human body tumor cell. The medicines can inhibit the metastasis of tumor cell through protecting basement membrane against destruction, and has obvious effects of lowering the expression of MMP-2 and MMP-9, strengthening the expression of TIMP and thus raising TIMP/MMP ratio. The berbamine derivative EBB is compound soluble in dilute hydrochloric acid solution, and may be prepared into corresponding water solution or powder in conventional process, into capsule or tablet via adding pharmaceutically acceptable carrier and supplementary material, or into capsule or tablet via adding supplementary medicine. The clinic dosage of berbamine derivative EBB is 10-120 mg/day, preferably 10-80 mg/day.

Description

Effect of berbamine derivative EBB is in the medicinal application that suppresses the human body tumour cell Invasion and Metastasis

Technical field

The present invention relates to effect of berbamine derivative EBB in the medicinal application that suppresses the human body tumour cell Invasion and Metastasis, this class material is not damaged by the protecting group counterdie and is suppressed the transfer of tumor cell.

Background technology

Neoplasm metastasis is the main cause of anticancer therapy failure and death.According to clinical statistics, there is tumor patient more than 80% to die from the invasion and attack and the transfer of tumor approximately, so the research of aspect receives the special concern of people always.Tumor invasion and transfer are active processes, and Litta once was summarised as three steps to this process: (1) tumor cell and extracellular matrix components adhere to; (2) tumor cell discharges or induces release proteolytic enzyme, degradation of cell epimatrix; (3) degraded areas tumor cell migration under the chemotactic factor guiding.Perfect extracellular matrix and basement membrane can limit invasion by tumor cells and transfer (Liotta LA.Tumor invasion andmetastases-role of the extracellular-matrix:Rhoads Memorial Awardlecture.Cancer Res, 1986,46 (1): 1-7.).

On the histology, the destruction of basement membrane integrity is considered to a sign of malignant tumor invasion and attack beginning.The biological character of tumor cell is different from normal cell, mutual relation in the extracellular environment of studying these cells and being relied between various dissimilar molecules and the mesenchyma stroma of tumors has been found many new cell adhesion molecules and cell transportable molecule and various types of digestive enzyme.

It is key link in the cancerous cell Invasion and Metastasis process that extracellular matrix and basement membrane are reinvented, and needs expression and activation by means of the protein degradation enzyme.Synthetic and a large amount of extracellular matrix degrading enzymes of secretion of tumor cell, the degradation of cell epimatrix is the important step of tumor cell invasion, transfer.The success of tumor cell invasion and transfer to a great extent, the a series of barrier (main component of barrier: each Collagen Type VI, laminin, fibronectin, elastin and proteoglycan etc.) of organizing that depended on these enzymatic degradation just can be categorized as metalloprotein enzyme, serine stretch protein class, thiol protease class, acidic protein enzyme etc. with digestive enzyme according to the physics and chemistry of these zymolyte compositions, biology characteristics.Also can be divided into protease and glycosidase enzyme.Protein ingredient in the former the main degradation of cell epimatrix, as the IV Collagen Type VI, the core protein part in laminin and the Dan Baijutang, the latter mainly degrade wherein glycoprotein and the polysaccharide chain in the Dan Baijutang.The protease of now having discerned has four kinds: and serine protease, cysteine proteinase, aspartic protease, matrix metalloproteinase (matrix metalloproteinase, MMPs).They can the matrix degradation composition, and has specificity in various degree.Matrix metalloproteinase is one group of important extracellular matrix degradation enzyme, and it plays key effect by the degraded of heterogeneity in the pair cell epimatrix in tumor invasion shifts.

It is crucial to it is found that MMPs plays a part in the process to the degraded of extracellular matrix effective ingredient, to metalloprotein enzyme (matrix metalloproteinases, MMPs) and tissue inhibiting (tissue inhibitors of metalloproteinases, TIMPs) research is maximum, the MMPs that has found at present has 25 kinds, is divided into membranous type and secreting type.The function of TIMPs mainly is to suppress MMPs natively, plays an important role in the metabolism of regulating extracellular matrix.The dynamic equilibrium relation of both expressions has determined the degradation of extracellular matrix degree, has also determined the invasion and attack and the transfer of tumor then.Its model of action is comparatively complicated, the two was combined into complex with 1: 1 ratio under most of situations, make MMPs loss of activity (Nelson AR, FingletonB, Rothenberg ML, et al.Matrix metalloproteinase:biologic activity andclinical implication[J] .J Clin Oncol, 2000,18 (5): 1135～11491).More and more studies show that the equal high expressed MMPs of the cell level of malignant phenotype and Invasion and Metastasis phenotype.The two large divisions who forms extracellular matrix, be in a basement membrane and the matter, the basement membrane formation blocks barrier together, this barrier is that the IV Collagen Type VI is a main component with basement membrane collagen, so to its degraded is the committed step of cell invasion and transfer, therefore (MMP-2 MMP-9) is studied maximum to IV Collagen Type VI enzyme.Its grade malignancy of different types of tumor such as breast carcinoma and MMP-2, MMP-9 too much expresses (the Hanemaaier R that is proportionate, Verheijen J H, Mayuire TM, et al.Increasedgelatinase-A and gelatinase-B activities in mallignant vs benignbreast tumors[J] .Int J Cancer, 2000,86:204～207).Many discovering, MMP-2, MMP-9 and TIMPs particularly TIMP-1 ,-2 results of interaction and tumor cell invasion and transfer have dependency, a MMP-2/TIMP-2 ratio even an index of judging as some malignancy of tumor degree and prognosis.Find in the research of and malignant tumor optimum such as Still at prostate relatively; normal or benign prostate disease MMP-2/TIMP-2 ratio is about about 1; malignant tumor or prognosis mala disease MMP-2/TIMP-2 ratio are much in 1 (StillK; Robson CN; Autzen P; et al.Localization and quantification of mRNAfor matrix metalloproteinase-2 (MMP-2) and tisue inhibitor of matrixmetalloproteinase22 (TIMP-2) in human benign and malignant protatictissue[J] .Prostate; 2000,42 (1): 18～25).Gong etc. have studied MMP-2, MMP-9 and TIMP-1 are expressed in the relation in neoplastic process and the pernicious prognosis, find MMP-2, MMP-9/TIMP-1 may become new index (the Gong YI that clinical tumor is studied and judged, Xu GM, Huang WD, et al.Expression of natrix metalloproteinases and thetissue inhibitors of metalloproteinases and their local invasiveness andmetastasis in Chinese human pancreatic cancer[J] .J SurgOncol, 2000,73 (2): 95～99).At present, at IV Collagen Type VI enzyme particularly in the research of MMP-2, except that find with TIMP-2 in close relations, also find membranous type matrix metalloproteinase (MT1-MMP) participating in activation MMP-2, the basement membrane components degraded is increased, then trigger cell invasion and attack and transfer (Dalberg K, Eriksson E, Enberg U, et al.Gelatinase A, membranetype 1 matrix metalloproteinase, and extracellular matrixmetalloproteinase inducer mRNA expression:correlationwith invasivegrowth of breast cancer[J] .World J Surg, 2000,24 (3): 334～340).The inhibition of MMPs may be on two levels, the one, in substrate, can not be converted into activated state, the 2nd, its activity is by natural metalloprotein enzyme inhibition factor (the tissueinhibitor of metalloproteinase that organizes in the body, TIMPs) by suppressing (Yoshimoto M with the bonded mode of activity form, Itoh F, Yamamoto H, et al.Expression ofMMP-7 (PUMP-1) mRNA inhuman colorectal cancers.Int JCancer, 1993,19; 54 (4): 614-618.).Koshiba etc. utilize gelatinase figure (Gelatinzymograph) to detect hiding and activated form of MMP-2 in 13 routine normal pancreatic tissues, 14 routine chronic pancreatitis tissues and the 33 routine cancer of pancreas tissues, and analyze with Western blot and further to have confirmed this result.The result is presented at the form of hiding that has detected MMP-2 in all cancer of pancreas, chronic pancreatitis and the normal structure, the MMP-2 activity form is all expressed (33/33) in institute in a organized way in the cancer of pancreas, the activity ratio of its activity ratio in chronic pancreatitis and the normal structure.Activity ratio in the pT3 phase tumor is significantly higher than pT1 phase tumor.Those have the activity ratio of MMP-2 in the tissue of metastases in local lymph node and metastasis also to be significantly higher than not have the tissue that shifts in the cancer of pancreas tissue.The activity ratio of the patient MMP-2 of recurrence also is significantly higher than the tissue that does not have transfer in 6 months.The activation rate of the patient MMP-2 of recurrence is significantly higher than the patient who does not have recurrence in 6 months in 6 months.The activation of results suggest MMP-2 has played important function (Koshiba T in the cancer of pancreas invasion and attack with in shifting, Hosotani R, Wada M, et al.Involvement of matrix metallo-proteinase-2activity in vasion andmetastasis of pancreatic carcino-ma.Cancer, 1998,15; 82 (4): 642-650.).

The TIMP-2 overexpression, can suppress the formation of lung metastasis after the intravenous injection of transitivity ras transformed mouse embryo fibroblast nude mice, and the imbibition characteristic of the speed of growth of in-vivo tumour and cancerous cell (Delercd YA, Perez N, Shimada H, et al.Inhibition of invasionand metastasis in cells tansfected with a inhibitor ofmetalloproteinases.Can-cer Res, 1992,52:701-708.).

The experiment in vitro result shows, the MMP-2 of the malignant cell strain that ras is gene induced expresses to be increased, the monoclonal anti physical ability that activates MMP-2 promotes the invasive ability of A2085 cell strain, and the ability that the monoclonal antibody of inhibition MMP-2 then makes the A2085 cell strain penetrate reconstituted basement membrane obviously weakens.

At above Invasion and Metastasis mechanism, the medicine that people just are being devoted to study target spot novelty, the invasion and attack of effect effective antitumour and are shifting, as suppress the antineoplastic invasion of the antineoplastic invasion of the antineoplastic invasion of adherent antineoplastic invasion and diversion medicaments, inhibition matrix metalloproteinase and diversion medicaments, inhibition angiogenesis and diversion medicaments, intervention signal conduction and diversion medicaments etc.

Calmodulin antagonist is that (calmodulin CaM) is the medicine of target spot to a class, and their pharmacological action is very extensive with calmodulin.Common have a psychosis class medicine, as trifluoperazine (TFP), chlorpromazine; Class medicine of flaccid muscles is as w-7 and analog thereof; Calcium antagonist is as verapamil.The target spot calmodulin of calmodulin antagonist is Ca in the cell ²⁺Main signal transduction molecule in the signal transduction path, analytic metabolism processes such as the propagation of participation and adjusting cell, differentiation, motion, become a kind of potential chemotherapy of tumors target spot (Hait WN, Lazo Js, et al.Calmodulin:a potential target for cancer chemotherapeutic agents.[J] .JClin Oncol, 1986 Jun; 4 (6): 994-1012).

EBB is domestic at first synthetic effect of berbamine derivative, for a kind of be the calmodulin antagonist of target spot with the calmodulin.The basic structure skeleton of calmodulin antagonist is:

The structural formula of berbamine and its derivant EBB is:

R＝-CH ₂CH ₂CH ₂CH ₂-OCH ₂CH ₃

Berbamine EBB

Before studies show that, EBB all demonstrates in vivo and in vitro to tumor cell proliferation inhibition effect (LIU Jiewen, QI Shuling, ZHU Huifang, et al.The effect of calmodulin antagonistberbamine derivative-EBB on hepatoma in vitro and in vivo.Chinese Medical Journal2002,115 (5): 759-762.) and drug resistance inversion effect (Qi Shuling, Liu Jiewen, Fu Jin etc., calmodulin antagonist EBB reverses the research of K562/VCR cell line multidrug resistance, Chinese experimental hematology's magazine, 1996 03 phases) chemotherapy anticarcinogen commonly used there is cooperative effect.Do not see report as yet about the research of calmodulin antagonist effect of berbamine derivative EBB inhibition tumor cell invasion and transferance at present.

Summary of the invention

The objective of the invention is to open berbamine and derivant EBB thereof to suppressing the medicinal application of human body tumour cell Invasion and Metastasis.This medicine is not damaged by the protecting group counterdie and is suppressed the invasion by tumor cells transfer, has the MMP-2 of reduction and MMP-9 and expresses, and strengthen TIMP simultaneously and express, thus the remarkable result of raising TIMP/MMP ratio.

The present invention is that to utilize people's fibroma cell line HT1080, Proliferation of Human Ovarian Cell be that ES-2 and SKOV3, human melanoma cell are that human tumor high-transfer cells such as M21, human lung cancer cell line BE-1 system carries out the conclusion that effect of berbamine derivative EBB antineoplastic invasion and transfer research are drawn, MMP-2 that described human tumor high-transfer cell is and MMP-9 express and are higher than normal cell (Kanamori Y, Matsushima M, Minaguchi T.Cancer Res.1999,59 (17): 4225-4227).

The known cancer cell surface can be expressed a lot of adhesion molecules at present, has integration plain with neoplasm metastasis is closely-related, as CD44 or the like, wherein integrates element and divides a lot of subtribes again, as α ₂β ₁Be a kind of receptor of collagen, relevant with neoplasm metastasis.Eguchi etc. are with a kind of agonist TPA of strong Protein kinase C α on the stimulation melanin oncocyte in advance ₂β ₁Integrate plain expression, found that with the adherent cell number of type i collagen increases, and this phenomenon can be by calmodulin antagonist W-7 and anti-α ₂Antibody suppresses, but can not be by inhibitors of protein kinase C such as H-7, and the calphostin blocking-up.This obviously melanoma cell and type i collagen stick ability to be strengthened and α ₂β ₁It is relevant to integrate plain expression increase and the kinase whose activation of CaM, and the activation of PKC no thanks to.Calmodulin antagonist then can suppress the kinase whose activation of CaM just, thereby weakens α ₂β ₁Integrate the plain ability of sticking, reach purpose (the Eguchi H that suppresses transfer, Horikoshi T et al.The expression of intergin alpha 2 beta 1 and attachment to type I collagenof melanoma cells are preferentially induced by tumor promoter, TPA (12-0-tetradecanoyl phorbol-13-acetate) .[J] .Br J Dermatol.1996Jan; 134 (1): 33-9).In addition, the former agonist of urokinase type plasmin (urokinase-type plasminogen activator, uPA), matrix metalloproteinase (matrixmetalloproteinase, MMPs) be extracellular matrix (extracellular matrix, two kinds of enzymes that ECM) relate generally in the degradation process.Ca ²⁺But ion is to regulate main cofactor Farias in cell growth and the protease-producing strain signal pathway etc. at the external calcium antagonist verapamil dose dependent ground stimulation uPA that confirmed, MMP-9 produces, and in the BALB/c mouse spontaneous metastasis model, obtained 51.3% metastasis inhibition rate (Farias EF, Aguirre GhisoJA, et al.Verapamil inhibits tumor protease production, local invasion andmetastasis development in murine caricinoma cells.[J] .Int J Cancer.1998Dec 9; 78 (6): 727-34).Zhao Huanyus etc. use the Lewis lung cancer cell seeding in C ₅₇BL/6J mice groin is subcutaneous, observes the spontaneous lung transfer ability, has confirmed that equally also the verapamil of various dose all can significantly suppress the transfer of Lewis lung cancer cell spontaneous lung, and along with the increase of dosage, lung metastasis inhibition rate rises to 85.9% by 43.9%.Known Lewis lung cancer is an indifferent epithelium sample cancer in the spontaneous lung, often assemble the transfer of blood road takes place by induced platelet, research has confirmed that also various concentrations of verapamil all can suppress the inductive platelet aggregation of Lewis lung cancer, along with drug level increases, platelet aggregation inhibition rate by 19% rise to 76.2% (ZhaoHY et al.An experimental study on the inhibition of spontaneous tumormetastasis of Lewis lung carcinoma by the calcium antagonistverapamil.[J] .Chinese Clinical Oncology, 2002 Feb.7 (1): 5-7).

In a word, in tumor cell invasion and the transfer process, the degraded of ECM and the destruction of BM are that cancerous cell is broken through the committed step that the normal cell basement membrane forms metastasis elsewhere.MMPs and TIMPs family are one of The key factor wherein; The former is relevant with degraded with the destruction of ECM and BM composition, and the latter is the specificity suppressioning factor of MMPs, and the integrity of ECM and BM has been kept in both dynamic equilibrium.Therefore, making the active and active maintenance of the TIMPs dynamic equilibrium of cell MMPs, keep ECM and BM integrity, is one of fundamental mechanism of anticancer Invasion and Metastasis treatment.

After the applicant handled HT1080, ES-2, SKOV3, M21, EB-1 cell line 24h with the EBB of 2,5 and 10 μ g/ml concentration, extracellular MMP-2, MMP-9 enzymatic activity were attenuating trend; The RT-PCR testing result shows that MMP-2, MMP-9 and the expression of TIMP-1 on the mRNA level all change to some extent, and the mRNA expression of MMP-2 and MMP-9 is tangible downward trend all along with the rising of drug level; The mRNA of TIMP-1 expresses then opposite, along with the rising of drug level, is tangible ascendant trend.The protein active of extracellular MMP-2, MMP-9 is expressed all consistent attenuatings with the interior mRNA of cell, has reduced the degraded damage capability of HT1080 cell to ECM and BM.This variation is confirmed in the invasion and attack experiment, along with rising HT1080, ES-2, the SKOV3 cell of drug level are crossed the decline of theca cell number.Thereby certain density EBB can suppress the invasive ability of HT1080, ES-2, SKOV3 cell.Experiment shows that the anti-invasion ability of EBB is to express by combine the mRNA that influences MMP-2, MMP-9 and TIMP-1 with calmodulin, i.e. inhibition to MMP-2, MMP-9 reaches relevant to the activation of TIMP-1, and keep the integrity of basement membrane thus, suppress invasion by tumor cells and transfer.In this experiment, also studied the influence of EBB to HT1080, ES-2, SKOV3, M21, EB-1 cell adhesion ability, the result shows that EBB can not change the adhesive capacity of HT1080, ES-2, SKOV3, M21, EB-1 cell.

The experiment detailed content is as follows:

Experiment one: the MMT method is measured drug susceptibility

Method: choose exponential phase human tumour cell line HT1080, ES-2, SKOV3, M21, EB-1, adjust cell concentration 1 * 10 ⁵/ ml is inoculated in 96 orifice plates, and every porocyte number is 2 * 10 ⁴, overnight incubation.Add variable concentrations medicine (EBB), final volume is 200 μ l, continues to cultivate 72 hours.Experiment is divided into blank group and 7 Concentraton gradient medicine groups, does 3 parallel holes for every group.Cultivate and finish preceding 4h, it is the MTT of 5mg/ml that every hole adds 20 μ l concentration, continues to cultivate 4 hours.Abandon supernatant, every hole adds the DMSO of 100 μ l, shakes 10 minutes.546nm measures absorbance, calculates IC ₅₀Value.

Result: EBB acts on 72h to human tumour cell lines such as HT1080, ES-2, SKOV3, M21, EB-1 in 0.469-30 μ g/ml concentration range, all produce cytotoxicity; There is factor graded effect in its effect, and along with factor amount increases, the inhibition of cell growth is obvious more; IC ₅₀Scope at 2-10 μ g/ml.

Experiment two: cell invasion analysis

Method: use the transwell cell and carry out the experiment of cell invasion reconstruction basement membrane.Spread ECM glue and 5 μ g fibronectin with 10 μ g respectively on the upper and lower surface of transwell cell filter membrane (8 μ m aperture), ECM glue is rebuild behind the 1h standby with 50 μ l culture medium.Human tumour cell lines such as HT1080, ES-2, SKOV3 handle 48h respectively through 2,5 and 10 μ g/ml concentration EBB, are resuspended in the RPMI1640 culture medium that contains 0.1%BSA, add 2.5 * 10 in every cell ⁵Cell is cultivated 10h for 37 ℃.HE dyeing, statistics invasion and attack cell number.Experiment repeats 3 times, is calculated as follows medicine to the tumor cell invasion suppression ratio.

The result: handle human tumour cell line 48h such as HT1080, ES-2, SKOV3, M21, EB-1 with 2,5 and 10 μ g/ml concentration EBB after, invasion and attack cell number and drug level are inverse ratio, invasion and attack suppression ratio and drug level proportional (seeing Table 1).Each experimental group and matched group are done the t check, all have extremely significantly statistical significance (p＜0.001), show that EBB can obviously reduce HT1080, ES-2, SKOV3 human tumour cell line's invasive ability.

EBB sees Fig. 1, shown in Figure 2 to the inhibitory action of HT1080, ES-2 cell invasion ability.

Variable concentrations EBB is as shown in table 1 to HT1080, SKOV3, ES-2 cell inhibiting rate.

Table 1

Tab.1 Inhibitic effection of EBB on the invasive ability of

HT1080，SKOV3，ES-2cells in vitro

Cell line	Group	No.of invading cells( X±S)	Ratio of inhibitory(％)	P value
					HT1080	control 2μg/ml 5μg/ml 10μg/ml	89.47±1.856 61.53±0.8743 36.67±1.568 25.13±0.267	31.13±2.265 59.91±2.566 71.58±0.5960	p＝0.0002 p＜0.0001 p＜0.0001
SKOV3	control 2μg/ml 5μg/ml 10μg/ml	109.3±3.578 78.67±6.067 64.93±5.060 46.53±7.856	27.87±5.967 40.16±6.801 60.95±8.411	p＝0.0121 p＜0.0020 p＜0.0019
					ES-2	control 2μg/ml 5μg/ml 10μg/ml	137.3±3.238 96.07±1.333 47.87±2.888 22.80±2.835	30.00±1.372 65.10±2.324 83.29±2.384	p＝0.003 p＜0.0001 p＜0.0001

Experiment three: gelatinase determination of activity

Method: with 2 * 10 ⁵Human tumour cell lines such as/ml HT1080, SKOV3, EB-1 are inoculated in 6 orifice plates, contain among the serum RPMI1640 cultivate 24h after, cultivate with the serum-free RPMI1640 that contains 2,5 and 10 μ g/ml concentration EBB respectively again, establish blank and medium controls simultaneously, continue to cultivate 24h.Collect matched group and medication group supernatant, electrophoresis under 4 ℃ of low temperature.Electrophoresis finishes with rinsed with deionized water twice, moves into the 2.5%Triton-100 eluting, hatches 16h for 37 ℃.Wash glue twice with deionized water, dyeing 1h, decolouring 2h, visible white gelatinase band.

Result: add gelatinase substrate gelatin in the SDS-polyacrylamide gel, can be used to detect the activity of gelatinase MMP-2 and MMP-9 in the cell conditioned medium.HT1080 (see figure 3), ES-2 (see figure 4), EB-1 (see figure 5) human fibrosarcoma cell express MMP-2 and MMP-9 simultaneously, and after the EBB effect, the activity of MMP-2 and MMP-9 all reduces.Wherein do not see notable difference between the activity of MMP-2 and the matched group during medication 2 μ g/ml; Increase to the visible notable difference of 5 or 10 μ g/ml.Do not see notable difference between the activity of MMP-9 and the matched group when medication 2 or 5 μ g/ml; Increase to the visible difference of 10 μ g/ml.SKOV3 only expresses MMP-2, and its activity is also visible difference (see figure 6) between each medication group and matched group.

Experiment four: the RT-PCR method detects MMP-2, MMP-9 and TIMP-1mRNA expresses

Method: handle human tumour cell line 24h such as HT1080, ES-2 respectively with 2,5 and 10 μ g/ml concentration EBB, respectively get 0.5-1 * 10 ⁷Cell is pressed the explanation of Trizol examination factor box and is extracted total RNA, carries out reverse transcription reaction by the explanation of M-MuLV examination factor box, and the PCR reaction condition of MMP-2, MMP-9 and TIMP-1 is 94 ℃ of 10min, 94 ℃ of 1min, 55 ℃ of 30s, 72 ℃ of 1min, and 72 ℃ are extended 10min.Sepharose electrophoresis measures MMP-2, MMP-9 and TIMP-1mRNA expresses, and takes pictures.

The result: experiment shows that can tumor cell effectively degrade and attack basement membrane and shift, and depends on expression of MMPs mRNA and activity.Handle cell 24h such as HT1080, ES-2 with 2,5 and 10 μ g/ml concentration EBB after, the mRNA of MMP-2 and MMP-9 expresses all downward trend.Wherein MMP-9 is more obvious.For the HT1080 cell, the mRNA of MMP-2 expresses with the difference of contrast less when medication 2 or 5 μ g/ml; (see figure 7) differs greatly during 10 μ g/ml.It is visible notable difference that the mRNA of MMP-9 is expressed between 2 μ g/ml and the matched group; As seen notable difference (see figure 8) extremely between 5 μ g/ml group, 10 μ g/ml group and the matched group.For the ES-2 cell, the mRNA of MMP-2 expresses with the difference of contrast less when medication 2 or 5 μ g/ml; Visible difference (see figure 10) during 10 μ g/ml.As seen the mRNA of MMP-9 is expressed between each dosing group and the matched group extremely notable difference (see figure 10).This shows that EBB handles cellular matrix metalloprotein enzyme secretions such as HT1080, ES-2 are suppressed.

EBB has rising effect (HT1080 sees Fig. 9, and ES-2 sees Figure 10) to the expression of matrix metalloprotease tissue inhibitive factor TIMP-1mRNA.

Experiment five: electron microscopic observation

Method: handle human tumour cell line 24h such as HT1080, ES-2, M21 respectively with 2,5 and 10 μ g/ml concentration EBB, respectively get 1-9 * 10 ⁷Cell is done ultrathin section and is observed.Swelling all takes place in result: electron microscopic observation HT1080 (seeing Figure 11), ES-2 (seeing Figure 12), M21 intracellular plastochondrias such as (seeing Figure 13), even the cavity change, and ridge form irregular arrangement, quantity tail off, and there have fat to ooze to be existing.

Description of drawings

Accompanying drawing 1 variable concentrations EBB is to the inhibitory action picture group of HT1080 cell invasion ability.

Wherein: the EBB concentration of Fig. 1-A, Fig. 1-B, Fig. 1-C, Fig. 1-D is respectively control, 2 μ g/ml, 5 μ g/ml, 10 μ g/ml.

Accompanying drawing 2 variable concentrations EBB are to the inhibitory action picture group of ES-2 cell invasion ability.

Wherein: the EBB concentration of Fig. 2-E, Fig. 2-F, Fig. 2-G, Fig. 2-H is respectively control, 2 μ g/ml, 5 μ g/ml, 10 μ g/ml.

Accompanying drawing 3 variable concentrations EBB are to HT-1080 cell MMP-2 and the active influence of MMP-9.

Among the figure: 1, marker 2, control 3,2 μ g/ ml 4,5 μ g/ ml 5,10 μ g/ml.

Accompanying drawing 4 variable concentrations EBB are to ES-2 cell MMP-2 and the active influence of MMP-9.

Among the figure: 1, marker 2, control 3,2 μ g/ ml 4,5 μ g/ ml 5,10 μ g/ml.

Accompanying drawing 5 variable concentrations EBB are to BE-1 cell MMP-2 and the active influence of MMP-9.

Among the figure: 1, marker 2, control 3,2 μ g/ ml 4,5 μ g/ ml 5,10 μ g/ml.

Accompanying drawing 6 variable concentrations EBB are to SKOV3 cell MMP-2 and the active influence of MMP-9.

Among the figure: 1, marker 2, control 3,2 μ g/ ml 4,5 μ g/ ml 5,10 μ g/ml.

Accompanying drawing 7 RT-PCR methods detect variable concentrations EBB to HT1080 cell MMP-2 reaction result.

Among the figure: 1, marker 2, control 3,2 μ g/ ml 4,5 μ g/ ml 5,10 μ g/ml.

Accompanying drawing 8 RT-PCR methods detect variable concentrations EBB to HT1080 cell MMP-9 reaction result.

Among the figure: 1, marker 2, control 3,2 μ g/ ml 4,5 μ g/ml, 5,10 μ g/ml.

Accompanying drawing 9 RT-PCR methods detect variable concentrations EBB to HT1080 cell TIMP-1 gene reaction result.

Among the figure: 1, marker 2, control 3,2 μ g/ ml 4,5 μ g/ ml 5,10 μ g/ml.

Accompanying drawing 10 RT-PCR methods detect variable concentrations EBB to ES-2 cell MMP-2, MMP-9, TIMP-1 gene reaction result.

Among the figure: MMP-9:1, control 2,2 μ g/ ml 3,5 μ g/ ml 4,10 μ g/ml.

MMP-2：6、control 7、2μg/ml 8、5μg/ml 9、10μg/ml。

TIMP-1：10、control 11、2μg/ml 12、5μg/ml 13、10μg/ml。

Accompanying drawing 11 variable concentrations EBB influence the electron microscopic observation picture group to HT1080, ES-2, M21 cellular morphology structure.

Wherein: the EBB concentration of Figure 11-A, Figure 11-B, Figure 11-C is respectively control, 5 μ g/ml, 10 μ g/ml.

Accompanying drawing 12 variable concentrations EBB influence the electron microscopic observation picture group to ES-2 cellular morphology structure.

Wherein: the EBB concentration of Figure 12-D, Figure 12-E, Figure 12-F is respectively control, 5 μ g/ml, 10 μ g/ml.

Accompanying drawing 13 variable concentrations EBB influence the electron microscopic observation picture group to M21 cellular morphology structure.

Wherein: the EBB concentration of Figure 13-G, Figure 13-H, Figure 13-I is respectively control, 5 μ g/ml, 10 μ g/ml.

The specific embodiment

Effect of berbamine derivative EBB is the chemical compound that is dissolved in dilute hydrochloric acid, can make corresponding water preparation or powder according to a conventional method.Also can use physiology acceptable carrier and adjuvant and make capsule and tablet.Can also with other mutually the accessory drugs thing be mixed and made into capsule and tablet.Effect of berbamine derivative EBB clinical dosage is 10～120mg/d, is preferably 10～80mg/d.

Claims (1)

1, the effect of berbamine derivative EBB of general formula (I) is in the medicinal application that suppresses the human body tumour cell Invasion and Metastasis, general formula:

Wherein: R=-CH ₂CH ₂CH ₂CH ₂-OCH ₂CH ₃

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