CN1092321A

CN1092321A - Avoid the method for multi-drug resistance in the cancerous cell

Info

Publication number: CN1092321A
Application number: CN93119621.3A
Authority: CN
Inventors: P·M·乔哈利; I·罗宁森
Original assignee: University of Illinois
Current assignee: University of Illinois
Priority date: 1992-09-17
Filing date: 1993-09-18
Publication date: 1994-09-21
Also published as: MX9305737A; AU5162293A; IL107032A0; WO1994006938A1

Abstract

The present invention is directed to the method that multi-drug resistance occurs in the tumor cells during cancer chemotherapy of avoiding.Particularly, it is related to the use kinases inhibitor, suppresses the inducing action of multi-drug resistance (MDR1) gene that caused by chemotherapeutics.Prove that herein MDR1 gene expression (it cause to after with the tumor cell resistance of some chemotherapeutics treatment) will derive when producing reaction with various cytotoxic drugs treatments.Prove also that herein kinases inhibitor can suppress this cell effect.Therefore, when kinases inhibitor before with cytotoxic drug treatment cancer patient administration or/and during administration simultaneously, can be used for avoiding in the various tumor cells, the MDR1 inducing action that causes by chemotherapeutics.

Description

Avoid the method for multi-drug resistance in the cancerous cell

The present invention is directed to the method for avoiding occurring in the tumor cell multi-drug resistance during the cancer chemotherapy.Particularly, it relates to and uses kinases inhibitor to avoid by the inducing action of chemotherapeutics to multi-drug resistance (MDR1) gene.This paper proves, will induce MDR1 gene expression (it causes the resistance of subsequently some chemotherapeutics being treated in tumor cell) when reacting to producing with various cytotoxic drug treatments.This paper proves that also kinases inhibitor can suppress this cell effect.Therefore imposing before the cytotoxic drug to the cancer patient and/or the while, kinases inhibitor can be used for avoiding at various tumor cells the MDR1 inducing action that causes by chemotherapeutics.

Chemotherapy is the principal mode of conventional treatment of cancer now.Yet follow a subject matter of cancer chemotherapy to be, in therapeutic process, tumor cell just produces the resistance at the cancer therapy drug cytotoxic effect.Observed tumor cell even can produce resistance simultaneously already to the different several chemotherapeutics of structure difference and the mechanism of action.This phenomenon is referred to as multi-drug resistance.Most of documents of multi-drug resistance and clinical Related Mechanism all are related to the representation of P-glycoprotein, i.e. MDR1 gene outcome in the relevant tumor cell.

The P-glycoprotein is that a kind of extensive specificity that is positioned at cell membrane flows to pump, it (comprises for example anthracycline antibiotics of some widely used anticarcinogen by reducing many lipophilic cytotoxic drugs, Changchun alkaloid, epipodophyllotoxin, actinomycin D and TAXOL) in cell, accumulate and the performance function, just produce cell resistance (Pastan andGottesman thus to these medicines, 1991, Annu.Rev.Med.42:277-286; Roninson(Ed.), 1991, Molecular and Cellular Biology of Multidrug Resistance in Tumor cells, plenum Press, New York; Schinkel and Borst 1991, Seminars in Cancer Biology 2:213-226).

Normal epidermis and endothelial tissue (people such as Cordon-Cardo, 1990 J.Histochem.Cytochem.38:1277-1287 at several types; People such as Thiebaut, Proc.Natl.Acad.Sci.USA84:7735-7738), and in raw blood stem cell (Chaudhary and Roninson, 1991, Cell 66:85-94) (people such as Neyfakh and in the subpopulation of mature lymphocyte, 1989, Exp.Cell Res.185:496-505) expressing human P-glycoprotein.The more important thing is, before chemotherapy or after the chemotherapy, in most of type human tumors, all detect MDR1 mRNA or P-glycoprotein (people such as Goldstein, 1989, J.Natl.Cancer Inst.81:116-124; People such as Noonan, 1990, Proc.Natl.Acad.Sci.USA87:7160-7164).The top level of MDR1 is expressed usually and is found in the deutero-tumor of normal structure of expressing MDR1, for example gastric cancer, adrenocortical carcinoma or colorectal carcinoma.In the entity tumor and leukemia of other type, MDR1 expresses quite to hang down usually and maybe can not detect before the treatment, but after chemotherapy, then the substantial portion of these pernicious focuses is expressed the people such as MDR1(Goldstein of high level, 1989, J.Natl.Cancer Inst.81:116-124).Before the present invention, people believe usually that MDR1 expresses after the chemotherapy to be increased, and is owing to selected in the body to have produced due to the rare tumor cell that is pre-stored in of chemotherapeutics resistance because of MDR1 expresses.

Even low-level MDR1 expresses, can cause that also several dissimilar cancers lose reaction (people such as Chan, 1990, J.Clin.Oncol.8:689-704 to chemotherapy; People such as Chan, 1991, N.Engl.J.Med.325:1608-1614; People such as Musto, 1991, Brit.J.Haematol.77:50-53), this fact shows that the multi-drug resistance of P-glycoprotein mediation represents a kind of important composition of clinical medicine resistance.In view of many clinical and clinical researches are emphasized to adopt materia medica strategy (Ford and Hiat for suppressing P-glycoprotein function, 1990, Pharmacol.Rev.42:155-199), and before the present invention, seldom understand relevant under the corresponding conditions of cancer chemotherapy, to inducing or raise all factors that the P-P-glycoprotein expression works in the tumor.Understand these factors, improving one's methods that research avoids that P-glycoprotein in the tumor occurs can be provided, the multi-drug resistance that reduces cancer thus takes place, and causes cancer chemotherapy more effective.

Many gene transfer studies have shown that, the MDR1 gene expression that improves is enough to cause multi-drug resistance phenotype (Roninson(Ed.), 1991, Molecular and Cellular Biology of Multidrug Resistance in Tumor Cell, Plenum Press, New York).For example be transformed into multi-drug resistance pro rata with the recombinant retrovirus mice infected NIH 3T3 cell of carrier MDR1 cDNA, the density of people P-glycoprotein surperficial with it.Whether this kind dependency is not subjected to exist cytotoxicity to select institute to influence (people such as Choi, 1991, Proc.Natl.Acad.Sci.USA 88:7386-7390).

In addition, some other biochemistry changes relevant all the time this fact with the multi-drug resistance cell, shows that such variation also can work to multi-drug resistance, may work by expression or the function that influences the P-glycoprotein.What these variations were the most outstanding is the activity that increases Protein kinase C (PKC), (the people such as Aguino of discovery in the multi-drug resistance cell line that said protein kinase obtains after the rapid cytotoxin of many (although not being whole) multistep is selected, 1990, Caner Commun.2:243-247; People such as Fine, 1988, Proc.Natl.Acad.Sci.USA 85:582-586; People such as O ' Brian, 1989, FEBS Lett.246:78-82; People such as Posada, 1989, Cancer Commun.1:285-292).PKC activates and shows drug resistance level (Ferguson and Cheng, 1987, the Cancer Res.47:433-441 that will increase in some drugs sensitivity and the multi-drug resistance cell line; People such as Fine, 1988, Proc.Natl.Acad.Sci.USA85:582-586; People such as Yu, 1991, Cancer Commun.3:181-189).But though PCK clearly phosphoric acid turn to P-glycoprotein (people such as Chambers, 1990, Biochem.Biophys.Res.Commun.169:253-259; People such as Chambers, 1990, J.Biol.Chem.265:7679-7686; People such as Hamada, 1987, Cancer Res.47:2860-2865), but do not know whether such phosphorylation will be born all responsibilities to viewed drug resistance variation.Can reverse multi-drug resistance (people such as O ' Brian, 1989, the FEBS Lett.246:78-82 in some P-P-glycoprotein expression cell line though shown some pkc inhibitor already; People such as Posada, 1989, Cancer Commun.1:285-292; People such as Palayoor, 1987, Biochem.Biophys.Res.Commun.148:718-725), but show on evidence, at least some observation on to influence be since test compound to the direct inhibition of P-glycoprotein function, but not to the inhibition of the phosphorylation of PKC mediation (people such as Ford, 1990, Cancer Res.50:1748-1756; People such as Sato, 1990, Biochem.Biophys.Res.Commun.173:1252-1257).Top research does not prove that the PKC interaction factor may be to expression rather than influential to the phosphorylation or the function of P-glycoprotein.

There are several laboratorys the factor of regulating MDR1 gene expression in normal cell and the malignant cell to be carried out research.The obvious example that the normal physiological of a MDR1 homologue is regulated finds in the Mus endometrium, and wherein mice mdr expression of gene is induced (people such as Arceci, 1990, Mol.Repro.Dev.25:101-109 by steroid hormone when pregnancy begins; People such as Bates, 1989, Mol.Cell.Biol.9:4337-4344).In rats'liver, find that the mdr expression of gene can be induced by several carcinogenic or Cytotoxic heterobiotins, similar inducing action is also observed (people such as Fairchild, 1987, Proc.Natl.Acad.Sci.USA84:7701-7705 during liver regeneration; People such as Thorgeirsson, 1987, Science, 236:1120-1122).The homologue of a kind of Rodents of MDR1 in addition also can derive (people such as Chin, 1990, Cell Growth Diff.1:361-365) to using some cytotoxic drug to treat in aitiogenic several cell line.On the contrary, in same research, all do not detect the inducing action of cytotoxic drug among the human cell line of any test to people MDR1 gene.Other research worker does not detect the MDR1 inducing action (Schinkel and Borst, 1991, Sem.Cancer Biol.2:213-226) that is produced owing to the cytotoxic drug treatment yet.

Yet several researchs show that people's MDR1 gene may be responsive to the inducing action of being emphasized also under certain condition.Like this in some human cell line the expression meeting of MDR1 because with heat shock, arsenite (people such as Chin, 1990 J.Biol.Chem.265:221-226) or the treatment of some differentiation agents (people such as Mickley, 1989, J.Biol.Chem.264:18031-18040; People such as Bates, 1989, Mol.Cell Biol.9:4337-4344) and improve.Some cytotoxicity P-glycoprotein substrate, it is reported can stimulate from people MDR1 promoter transcription reporter gene (people such as Kohno, 1989, Biochem.Biophys.Res.Commun.165:1415-1421; People such as Tanimura, 1992, Biochem.Biophys.Res.Commun.183:917-924), and the expression (people such as Licht that after prolong exposing, can improve P-glycoprotein in the mesothelioma cell line, 1991, Int.J.Cancer 49:630-637).Although this class report of MDR1 inducing action is arranged, but also never certainly, when short burst is exposed to the employed any medicament of cancer chemotherapy, whether can induce MDR1 expression of gene in people's cell, and whether such inducing action can be avoided by medicament.

Recently, people such as Kioka have reported, add flavonoid, Tricetin can be avoided by arsenite (a kind of chemical compound that is not used in the treatment cancer, but known it can activate the approach of transcribing of the heat shock response composition mediation of promoter) the expression (Kioka etc., 1992 FEBS Lett 301:307-309s) of inductive MDR1 in hepatoma carcinoma cell.Though do not mention in people's such as Kioka the document, but the active inhibitory action of PKC is one of biological action of Tricetin (people such as Gschwendt, 1984, Biochem.Biophys.Res.Commun.124:63), therefore the PCK inhibitory action that is produced by Tricetin is possibly to observed inhibitory action negative part responsibility by the MDR1 inducing action that arsenite produced.But it should be noted that, those skilled in the art believe Tricetin suppress ability by the responsive transcription of heat shock response composition mediation have nothing to do with the PKC inhibitory action (Kantengwa and Polla 1991, Biochem.Biophys.Res.Commun.180:308-314).And people's such as Kioka document does not propose the MDR1 inducing action that non-flavonoid pkc inhibitor can suppress arsenite yet, or but when being used in combination with chemotherapeutic agents or any other medicament of not knowing activation heat shock reaction composition mediated pathways, Tricetin can suppress the inducing action that MDR1 expresses.

The present invention relates to use kinases inhibitor to avoid multi-drug resistance appearance in the cancerous cell, and the external method that is accredited as the kinases inhibitor of realizing that this purpose is useful.

The present invention's part (no matter whether being transported by the P-glycoprotein) cancer therapy drug can induce the MDR1 gene to express this discovery in various tissue-derived human tumor cells.MDR1 gene expression be increased in RNA and protein level is all observed.The MDR1 inducing action is also observed when handling cell with the PKC agonist.And, this inducing action that is produced by cytotoxic drug or PKC agonist, can avoid by handling cell with kinases inhibitor, this shows in that MDR1 is gene induced and has been involved in protein kinase mediated approach on, and kinases inhibitor can be used for stoping the MDR1 gene to express in being exposed to the cancerous cell of chemotherapeutics.Particularly, because of the kinases inhibitor to the PKC non-activity can not suppress the MDR1 inducing action, this inhibitory action is just relevant with the PKC inhibitory action, thereby the kinases inhibitor that PKC has an effect effect is then suppressed this reaction effectively.

When exposing in external short-term, chemotherapeutics can induce this fact of the expression of MDR1 in people's cell to show that cancer chemotherapy can directly be induced the anti-agent of multiple medicines thing, rather than is undertaken by the selection of the rare mutation that is pre-existing in.This directly inducing many occurs in patient's Drug therapy process, this to small part be malignant change through treatment with respect to untreated malignant change, the reason that MDR1 expresses increases this phenomenon appears.Therefore before relating to or use kinases inhibitor simultaneously, be useful for avoiding MDR1 to induce, and avoided the appearance of multi-drug resistance cancerous cell thus, thereby receive better therapeutic effect with the chemotherapy of cytotoxic drug.

The present invention is commented with the method for embodiment, show that in these embodiments the PKC agonist can induce the expression of MDR1 in normal circumference blood lymphocyte (PBL) and tumor cell, in addition, various cytotoxicity cancer therapy drugs also show and can activate the MDR1 gene.The more important thing is that it is all the more so under the situation of having only seldom or do not have detectable P-glycoprotein in the former tumor cell of this MDR1 inducing action, particularly treatment that can stop by PKC agonist or cytotoxic drug mediation that kinases inhibitor is proved to be.Various uses is included among the present invention described herein, comprises that (being limited to but have more than) prevents the appearance of multi-drug resistance tumor cell during cancer chemotherapy.

Briefly introducing of relevant accompanying drawing

Fig. 1. phorbol ester (TPA), DG (DOG) and Staurospcrine(staur) to P-glycoprotein function and in the influence of H9 expression of cell lines.

A. the Rh123 accumulation of the cell 3hr that is untreated and handles through TPA or DOG.

B. untreated cell reaches with Staurosporine and gives the cell 3hr Rh123 accumulation that processing is handled with TPA or DOG then.

C. the cell that is untreated with IgG2a homotype tester dyeing and handles with TFA or DOG.

D. same C is with the UIC2 antibody staining.

E. untreated cell reaches and only handles with Staurosporine or give the cell of handling with TPA or DOG again after the processing with Staurosporine and be infected with through UIC2.

The influence that Fig. 2 .cDNA-PCR analysis TPA, DOG and Staurosporine express in different cell lines MDR1 mRNA.In each swimming lane, last bands of a spectrum (167bp) are corresponding to MDR1, and following bands of a spectrum (120bp) are corresponding to β ₂The specific PCR product of-microglobulin.

A.TPA or DOF(with or give processing without Staurosporine) influence that MDR1 mRNA is expressed in the H9 cell.

B.TPA in the H9 cell to the induction time process of MDR1 mRNA.The PCR that two negative controls (neg.con.) replace cDNA to finish corresponding to water or reverse transcription mixture with no RNA.

C.TPA or DOG in the K562 cell and TPA in the MCF-7 cell to the inducing action of MDR1 mRNA.

Fig. 3. the flow cytometry analysis that drug-induced MDR1 expresses.

A. there are not 30 μ M isoptins (VER) (left side) or having under 30 μ M isoptin (right side) situations effluent of P-glycoprotein-conveying fluorescent dye from the K562 cell.Last figure: from the effusive Rh123 of untreated cell (-) and from 50 μ M Ara-C(ARA) handles cell 12 hours or handle 2 days or 3 days effusive Rh123 of cell with 10 μ M Ara-C.Figure below: reach from the effusive DiOC of cell that handles 36 hours with 1 μ g/ml vinblastine (VBL) from untreated cell ₂(3).

B. the P-P-glycoprotein expression that in the KGI leukaemia that Ara-C handled, increases.Left figure: handle the accumulation of Rh123 in 1.5 days the cell in the untreated cell or with 10 μ M Ara-C.Right figure: the indirect immumofluorescent method labelling that same cell carries out with anti-P-glycoprotein UIC2 antibody or IgG2a homotype tester.

C. be exposed to different medicaments and remained in the no drug media, and by using DiOC ₂(3) count density (left figure) of (transverse axis) and UIC2 antibody double labelling or the K562 cell that draws with IgG2a homotype contrast (right figure) analysis of algae erythrin (PE) (hang down to axle) indirect labelling.From top to bottom: untreated cell, with the 60ng/ml amycin handle there is no in 3 days medicine grow the cell in 5 weeks, handle with 30 μ M chlorambucils (CHL) and there is no the grow cell in 2 weeks of medicine in 5 days, with 10 μ M Ara-C handle there is no in 3 days medicine grew for 5 weeks (this experiment use other detect in half amount of used antibody) cell, with Ara-C by top described processing and from medicine, shifting out the Later Zhou Dynasty, one of the Five Dynasties with the fluorescent active cell isolating gloomy Rh123 cytoclasis of art of classifying.

Fig. 4. the cDNA-PCR that MDR1 mRNA expresses in the cell that drug treating is crossed analyzes.Go up bands of a spectrum (167bp) in each swimming lane corresponding to MDR1, following bands of a spectrum (120bp) are corresponding to β ₂The specific PCR product of-microglobulin increases in the pipe that separates.

A. in the K562 cell Ara-C to the inducing action of MDR1.Cellular exposure in shown in Ara-C concentration 4.5 days.The cell growth relevant with untreated cell is by extracting MTT test determination simultaneously with RNA.

B. the inducing action of MDR1 in the K562 cell of handling with different pharmaceutical.The time that is exposed to medicine is marked.Medicine and concentration thereof are as follows: a untreated cell; DAU, 250ng.The ml daunorubicin; ADR, the 500ng/ml amycin; VBL, the 20ng/ml vinblastine; VP, 1 μ g/ml etoposide; MTX, 200ng/ml methotrexate, CDDP, 3 μ g/ml cisplatins; CHL, 50 μ M chlorambucils; 5FU, 2 μ g/ml 5-fluorouracil, HU30 μ M hydroxyurea.

The inducing action of MDR1 in the C.KB-3-1 cancerous cell, untreated or handle 2 days situation with 200ng/ml amycin or 10 μ M Ara-C.

D. the inducing action of MDR1 in the EJ cancerous cell, untreated (-) or handle 4 days situation with 10 μ M Ara-C.

What E. drug-induced MDR1 expressed in the K562 cell keeps.Cell was handled 3 days with 60ng/ml amycin, 10 μ M Ara-C or 200ng/ml methotrexate, and cultivated the time of being indicated in no drug media.

Fig. 5. kinases inhibitor to cytotoxic drug in the H9 cell to the influence of MDR1 mRNA inducing action.In each test, inhibitor Staurosporine(ST), H7, ISO-H7(IH7) or HA1004(HA) added twice, for the first time being right after before relative medicine adds, for the second time after specific a period of time.

The A.H9 cell, untreated or handled 22 hours with 50 μ M Ara-C.Inhibitor begins and adding after 16 hours in experiment by the concentration of being indicated.

The B.H9 cell, undressed or handled 22 hours with the 200ng/ml amycin.The inhibitor of equal quantities (0.03 μ M Staurosporine, 10 μ M H7, HA-1004 and ISO-H7) begins and adding after 16 hours in experiment.

The C.H9 cell, unprocessed mistake or handled 36 hours with 40ng/ml vinblastine or 200ng/ml ammonia first psychopsid.Equal quantities inhibitor (0.1 μ M Staurosporine; 50 μ M H7) begin and adding after 24 hours in experiment.

Fig. 6. the vinblastine resistance in the K562 cell that Ara-C or amycin were handled.

A. unprocessed mistake and the cell handled with Ara-C or amycin (ADR) in, the growth inhibited effect of vinblastine.Cell is pressed Fig. 3 C and is handled, and grows for 6 weeks down in no medicated strip spare.The vinblastine inhibitory action detects carried out 10 days.

B. in the cell of unprocessed mistake and the gloomy colony of Rh123 of the cell handled of Ara-C and Rh123 become clear in the colony growth inhibited effect of vinblastine.The cell that Ara-C handled shifts out from medicine and back six week is divided into gloomy colony of Rh123 and the bright colony of Rh123 by the dyeing of Rh123 outflow method and by fluorescent active cell classification method.The purity of this gloomy colony of Rh123〉60%(FIG.3C), and the bright colony of Rh123 purity is 90-95%.In classification one week of back, the vinblastine inhibitory action detects carried out 7 days.

The present invention relates to use kinases inhibitor to avoid in cancerous cell, occurring the multi-drug resistance Phenotype.Cytotoxic drug to MDR1 induce and kinases inhibitor avoids the discovery of the ability of this inducing action will give below fully to narrate and illustrate.For ease of discussing, the present invention is introduced with regard to the active two kinds of albumen enzyme inhibitors of anti-PKC and one group of special human tumor cell line.But its principle is applicable to any kinases inhibitor of use and is used for various body outer cell lines and the in-vivo tumour with the wide range of any chemotherapeutics treatment.

The inducing action of MDR1 gene expression

TPA(12-O-myristyl phorbol-13-acetas); a kind of effective PKC activator; and DG; the agent of a kind of PKC physiological stimulation shows among the embodiment 1 can be increased among the normal person PBL and MDR1 expression of gene from dissimilar leukemia or the deutero-cell line of entity tumor below.All observe in all experimental cells systems that act on the preceding express P-glycoprotein of treatment of TPA, and also in not having some other cell line of detectable P-glycoprotein (but not all this other cell line), observe.But be to use the TPA higher than experimental concentration described herein, MDR1 expresses and may be induced in unresponsive cell line.The effect of viewed TPA and DG shows, the expression of MDR1 can mediate signal transduction pathway by PKC and regulates in people's cell.

In the cell of handling with the PKC agonist, all observe the increase that MDR1 expresses at P-glycoprotein and MDR1 mRNA level, raise Tong S Φ fraud (14) badger dumpling of wasting time of the MDR1 mRNA degraded of the steady state levels of increase is played and is stopped the distant quilt of mould

The horsefly meal O that makes a noise is plunderred stricture of vagina stern ˇ cherry (people such as Ueda, 1987, J.Biol Chem.262:505-508) and is comprised and be responsible for stimulating the AP-1 site of transcribing (people such as Angel, 1987, Cell, 49:729-739 by TPA; People such as Lee, 1987, Cell, 49:741-752).This AP-1 site and on every side sequence in people MDR1 gene and its Rodents homologue, guard.(people such as Hsu, 1990, Mol.Cell.Biol.10:3596-3606; People such as Teeter, 1991, Cell Groth Diff.2:429-437).The AP-1 sequence table of hamster pgp1 gene is bright to be the positivity regulon (people such as Teeter of its promoter basically, 1991, Cell Growth Diff.2:429-437), but this homologue mice mdrla(mdr3) the corresponding composition of gene is then had a negativity regulating action (people such as Ikeguchi, 1991, DNA Cell Biol.10:639-649).Therefore, the AP-1 composition of this paper sponsor MDR1 promoter may have been expressed directly effect to the MDR1 that is stimulated by the PKC agonist.

Can explain former discovery by the PKC-mediated pathways to the inducing action of MDR1 gene expression, the multi-drug resistance cell line that promptly optionally increases the P-P-glycoprotein expression usually has people such as high-level PKC(Aquino, 1990, Cancer Commun.2:243-247; People such as Fine, 1988, Proc.Natl.Acad.Sci.USA85:582-586; People such as O ' Brian, 1989, FEBS Lett.246:78-82; People such as Posada, 1989, Cancer Commun.1:285-292).By inference, PKC is active to be increased, and can represent during the selection of this cell line, MDR1 gene expression is increased the earlier results of being responsible for.Yet this explanation can not be got rid of the activity that can further be improved the P-glycoprotein by the inductive phosphorylation of PKC.Find evidence in this back one hypothesis research with people such as Yu (Cancer Commun.3:181-189), they find, in the multi-drug resistance subbreed of MCF-7 cell (obtaining after the MDR1 cDNA transfection of transcribing with allogeneic promoter), the level of drug resistance can be expressed high-level PKC2 carrier and increase because of introducing.The resistance increase is accompanied by the phosphorylation of P-glycoprotein in this PKC2 transfection body increases, and its expression of not obvious change.

PKC plays central role in the various signal transduction pathways relevant with different adaptations, propagation and atomization.Induce the expression of MDR1 in normal and tumor hematopoietic cell even found the PKC kinetins, but also can use the hemopoietic growth factor that works by the PKC mediated pathways, but do not obtain equifinality.And the PKC agonist is not only induced the expression of MDR1 in hemopoietic source cell system, also induces the expression in epithelium source cell system, and this PKC mediation regulating action that shows that MDR1 expresses can play general physiological action.

PKC mediation mechanism has been tied to the transcription response (people such as Kaina of the DNA that is subjected to UV ray or alkylating reagent infringement, 1989, In M.W.Lambert and J.Laval(Ed.), DNA Repair Mechanism and Their Bialogical Implication in Mammalian Cells, Plenum Press, New York; Papathanasiou and Fornace, 1991, PP.13-36 In R.F.Ozols(Ed.), Molecular and Clinical Advances in Anticancer Drug Resistance, Kluwer Academic Pullishers, Boston, MA).PKC activates and also to be associated with other cytotoxic drug CyIocide (people such as Kharbanda for example, 1991, Biochemistry 30:7947-7952) or the cell effect of amycin (people such as Posada, 1989, Cuncer Res.49:6634-6639).Therefore, the inducing action that the MDR1 of PKC mediation expresses may be the part to the general stress of dissimilar primary cellular defect (comprising the infringement that is caused by the cytotoxicity chemotherapeutics) generation.

In fact, the invention discloses the expression of MDR1 in human leukemia and entity tumor derived cell system, can be exposed to the various cytotoxic drugs of cancer chemotherapy use and induce (face embodiment 2 as follows) by short burst.MDR1 induces rna level and protein level, can in cell subsets body, observe with chemotherapeutics (Ara-C, methotrexate, 5-fluorouracil, chlorambucil, cisplatin, hydroxyurea) treatment P-glycoprotein agent delivery (amycin, daunorubicin, vinblastine, etoposide) treatment or that can't help the conveying of P-glycoprotein.Because MDR1 is provided by the resistance that does not provide second group of medicine, and because MDR1 induces and can produce (being shorter than generation cell generation time in many cases) after the short time is exposed to medicine, these discoveries show that the cytotoxicity selection of expressing the MDR1 cell may not cause the increase of observed MDR1 expression.In visible cell injury simultaneously, MDR1 induces and promptly becomes and can check, this shows that it is likely the indirect consequence of this damage, rather than to the direct reaction of specific reagent.

What is more important is treated inductive MDR1 with cytotoxic drug and is expressed in to remove and does not disappear after the medicine, and can keep in the cultured cells several at least weeks in no pharmaceutical culture medium.The P-glycoprotein positive cell of growing under no medicine condition shows aspect its differentiation not have significant change.Therefore, it is a kind of stabilization that drug-induced MDR1 expresses, and it is not limited to death or stops the cell of differentiation.Except increasing the MDR1 expression, the resistance that the cell of drug treating also shows vinblastine (a kind of P-glycoprotein is carried medicine) improves 2-3 doubly, and this resistance cell with expression MDR1 especially is relevant.In addition, the drug treating cell resistance that also shows chlorambucil (a kind of the P-of can't help glycoprotein carry chemotherapeutics) increases to some extent.Back one finds to show that after with the cytotoxic drug treatment, some other clinical relevant drug resistance mechanism can be expressed with MDR1 and be derived jointly.

To sum up state, these discoveries show the various Drug therapy human tumor cells that use with cancer chemotherapy, can directly induce MDR1 to express, and do not realize (missed as former people and believe) by selecting to give the rex that pre-exists.The increase that produces aspect multi-drug resistance is stable, and is enough to reduce external and the interior reaction to chemotherapeutics of body.The inducing action that the MDR1 of medicine mediation expresses seemingly appears at during the cancer chemotherapy probably, and can explain that to small part viewed MDR1 expresses the incidence rate that increases in Drug therapy people tumor.Therefore the invention provides first part of material of MDR1 inducing action under clinical correlated condition, and point out that PKC plays central role in this inducing action.This back one supposition provides the basis of avoiding the inductive amic therapy method of MDR1 during the cancer chemotherapy by the PKC inhibitory action.

Use kinases inhibitor to avoid MDR1 to induce

The present invention proves kinases inhibitor, and the active inhibitor of anti-PKC is particularly arranged, and can stop and induce the MDR1 gene to express in tumor cell.For example; Staurosporine; a kind of effective but nonselective pkc inhibitor (R ü egg and Burgess; 1989, Trends Pharmac.Sci.10:218-220), can stop with TPA; DG and many chemotherapy cytotoxic drugs; comprise Ara-C, vinblastine, the MDR1 in the P-glycoprotein negative cells of methotrexate and amycin treatment induces.Another kind of kinases inhibitor, H7 also can stop the MDR1 that is produced by the chemotherapy medicine to induce.These discoveries provide PKC can with the evidence that participates in this inducing action, and provide the probability of using kinases inhibitor to suppress MDR1 gene expression.And, observe in the chemotherapy medicine inducing tumor cell can't help the resistance of at least a medicine that the P-glycoprotein carries, hint is treated by chemotherapeutics, other approach that produces drug resistance can be expressed by coinduction with MDR1, and be created in all mechanism of using kinases inhibitor to suppress to occur drug resistance in the tumor cell, and be not inducing of MDR1.

Yet in some P-glycoprotein positive cell line, when independent use Staurosporine, increase the expression of P-glycoprotein significantly, it should be noted that, Staurosporine is the P-glycoprotein inhibitors, can directly combine with the P-glycoprotein (people such as Sato, 1990, Biochem.Biophys.Res.Commun.173:1252-1257).In addition, two kinds of other P-glycoprotein binding compounds, loop chain rhzomorph A and isoptins (being also referred to as pkc inhibitor) also increase the expression of P-glycoprotein in some P-glycoprotein positive cell line.Therefore can believe, these medicaments and Staurosporine be by one common, also unknown now mechanism works.These presentation of results, it is negative or avoid increasing MDR1 near negative tumor that kinases inhibitor is used for the P-glycoprotein, may be more effective in the tumor of express P-glycoprotein than being used for most of tumor cell.Yet, it should be noted that, Staurosporine only increases this discovery of P-P-glycoprotein expression in minority hematopoietic cell system, do not represent that the P-P-glycoprotein expression increase that is produced by pkc inhibitor is the general aspects of P-glycoprotein positive tumor cell, and do not show the patient who suffers from P-glycoprotein positive tumor yet, can not avoid further being benefited the tumor cell from using kinases inhibitor by drug-induced multi-drug resistance.

Negative entity tumor of P-glycoprotein or leukemia can be passed through the biopsy material of patient's tumor, operation or hematology's sample, use technology known in the art to analyze, and identified (Roninson(Ed.), 1991, Molewlar and Cellular Biology of Multidrug Resistance in Tumor Cells, Plenum Press, New York).These technology include, but is not limited to carry out immunocytochemistry, immunohistochemistry or immunofluorescence with P-glycoprotein specific antibody and detect, and carry fluorescent dye to carry out vigor dyeing with the P-glycoprotein; Northern dot blotting or carry out slot blot hybridization with the MDR1-specific dna probe; Or the cDNA-PCR of MDR1 mRNA analysis etc.Some operational example that detects above is as being introduced among the

embodiment

1 and 2 below.Should be understood that some is the cell line that the basis seems P-glycoprotein feminine gender with albumen or Function detection, still can demonstrate by the detected MDR1 mRNA(of cDNA-PCR and see Table 1).This shows the detection based on albumen or function, may be preferably from the main standard of using the benefited tumor of protease inhibitor as identifying.In addition, cDNA-PCR or other method of measuring MDR1 mRNA can be used, but will be clear that simultaneously MDR1 still may be the sign of the negative tumor of P-glycoprotein at the level of K562 cell or high slightly (for example 2 times) horizontal expression.

Because Shi Yan protease inhibitor herein, it is nonselective suppressing on the activity at it, the effect that is them is not specific to PKC, and the research that this paper introduced provides evidence: they suppress the active ability of PKC may be to stop the inductive key factor of MDR1.For example, two kinds of effective pkc inhibitors, Staurosporine and H9 can suppress the MDR1 inducing action by the cytotoxic drug generation.Opposite, HA1004, the active kinases inhibitor of a kind of nonreactive PKC, it is invalid fully to show stoping MDR1 to induce.Therefore, any kinases inhibitor that can suppress PKC, no matter whether it is to PKC tool specificity, all inducing of MDR1 very likely is useful in the tumor cell to avoiding.

Therefore, any can stop chemotherapeutics to MDR1 produce inductive protein kinase catalyst (measure as any method of being introduced by following embodiment 1, for example the cDNA-PCR of fluorescent dye deposition, MDR1 mRNA or with the dyeing of P-glycoprotein specific antibody) all can be used in the practice of the inventive method.This inhibitor can suffer from entity tumor or leukemic cancer patient carry out the chemotherapeutics treatment before or administration simultaneously.Any common cancer therapy drug that is used for cancer chemotherapy all is included in the scope of the present invention, and includes, but is not limited to Ara-C, amycin, daunorubicin, vinblastine, etoposide, methotrexate, 5-fluorouracil chlorambucil, cisplatin and hydroxyurea.

A lot of chemical compounds that can suppress PKC were carried out applied research possible in the cancer chemotherapy of vivo and vitro.Yet, should be noted that (Powis and Kozikowski, 1991, Clin Biochem.24:385-397 when finding that such chemical compound shows the selective growth inhibitory action for the tumor relevant with normal cell; Grunicke etc., 1989, Adv.Enzyme Regul.28:201-216), they and may not be certain to show or mean and can avoid the expression of MDR1 in cancerous cell.In vitro study shows, suppresses the anti-proliferative effect that pkc inhibitor can appear in the approximately identical dosage of active dose (people such as Grunicke, 1989, Adv.Enzyme Regul.28:201-216) with its PKC.The chemical compound of experiment comprises Staurosporine and benzoyl derivant CGP41 251 thereof in the body; (MTD Staurosporine is 1mg/Kg to find they to show antitumous effect with 1/10 place of its maximum tolerated dose (MTD) in nude mice; and CGP41 251 is 250mg/Kg) (people such as Meyer; 1989; Int.J.Cancer 43:851-856); other Staurosporine analog that shows anti-tumor activity in vivo comprises people such as UCN-01(Takahashi; 1987; J.Antibiot.40:1782-1784) and 8-N-(diethylamino ethyl) rebeccamycin.(BMY 27557) (people such as Schurig; 1990, Proc.Amer.Assoc.Cancer Res.31:Abs 2469).The preferred dose of back one chemical compound intraperitoneal administration is injected every day * 9 to 64mg/Kg single dose scope at 12mg/Kg/.Another group effective inhibitor of PKC after deliberation with comprising ether fat analog, comprises the cetyl phosphocholine, ET-18-OCH as anticarcinogen ₃, ilmfosine, SRI62-834 and BM41440(Powis and Kozikowski, 1991, Clin Biochem.24:385-397; People such as Grunicke, 1989, Adv.Enzyme Regul.28:201-216).In these medicaments some has been used for clinical trial.Oral in these trials MTD value ilmofosine is 200mg/ (people such as Berdel every day, 1988, Proc.Amer.Cancer Res 29:Abs.2050), and BM41440 is 5mg/Kg body weight (people such as Herrmann, 1987, Lipids 22:962-966).

The also local cutaneous metastatic treatment that is used for mastocarcinoma of cetyl phosphocholine, each patient of dosage range 0.2-38.5g/ uses 3-128 week (people such as Unger, 1990, Cancer Treat Rev.17:243-246).This group chemical compound also by test as the cathartic of autologous bone marrow transplantation (people such as Vogler, 1991, Exp.Hematol, 99:557Abs.).Another kind of pkc inhibitor, suramin have been used for parasitic disease, and make assessment over against its clinical trial as antitumor agent.With the suramin continuous infusion, its speed is 14 days, reaches peak value 300 μ g/ml during end, show the carcinoma of prostate that hormone is not answered have activity (people such as Meyer, 1992, J.Clin.Oncol.10:875-877).One of another kind of pkc inhibitor, the flavonoid quercetin, when by 20mg/Kg dosage when intraperitoneal is used to nude mice, show the antitumor effectiveness that can improve cisplatin, it is the medicine that a kind of P-of can't help glycoprotein is carried, (people such as Grunicke, 1989, Adv.Enzyme Regul.28:201-216).

Though except following example 1 and 2 described Staurosporine, above chemical compound also do not have a kind of inductive ability of MDR1 of causing by cytotoxic drug avoided of being tested, but show very effectively that from result disclosed by the invention they have such effect probably, because they all can suppress PKC.About in the animal body of these and other pkc inhibitor and the availability of clinical testing data this area professional can be used in combination the cancer therapy drug of these chemical compounds with routine, thereby avoid chemotherapeutic period multi-drug resistance to occur.These chemical compounds can and/or be granted the cancer patient before the chemotherapeutics treatment simultaneously, and its dosage range is about 1-250mg/Kg body weight, adopts injection/continuous infusion or topical therapeutic repeatedly.

In addition, the vitro detection technology can be developed, any chemical compound of inducing MDR1 gene expression that causes by chemotherapeutics can be stoped so that differentiate fast.For example, can be with H-9 or K562 leukaemia system, be exposed to the 10 μ M Ara-C that regulate under the conditions of tissue culture, the 200ng/ml amycin, 200ng/ml methotrexate or 40ng/ml vinblastine 10-36 hour (though surpass 1 hour any incubation time all can, see Fig. 2 B) before, handled about 30 minutes with test compound, then with to compare in the same old way, estimate this chemical compound and stop the inductive ability of MDR1 that produces by medicine.Plant detection method thus and identify the chemical compound that can stop induced by chemotherapeutic agents MDR1, kinases inhibitor not necessarily, but can be used for patient treatment by the mode identical with kinases inhibitor.

Embodiment 1

Kinases inhibitor stops Protein kinase C in normal cell and the tumor cell

The MDR1 of agonist mediation induces

1.1 materials and methods

1.1.1 cell line and drug treating

Normal person PBL be behind informed consent by venipuncture from healthy volunteer obtain, then at Histopaque-1077(Sigma, St.Louis MO) goes up and through the density gradient centrifugation low-density mononuclear cell to be separated.KG1 cell line remains on and is added with 20% hyclone (FCS) and 2mM L-glutaminate, (GIBCO Lab. in the Iscove ' s modifaed Dulbecco culture medium of 100 units/ml penicillin and 100 μ g/ml streptomycins, Grand Island, NY).MCF-7, EJ, KB-3-1, Hela and HT-1080 cell line remain on and are added with 10%FCS and 2mM L-glutaminate, among the DMTM of 100 units/ml penicillin and 100 μ g/ml streptomycins.All other cell lines remain on and are added with 10%FCS and 2mM L-glutaminate, in the RPMI culture medium of 100 units/ml penicillin and 100 μ g/ml streptomycins.

Preparation contains 100 μ g/ml TPA(Sigma in dimethyl sulfoxide (DMSO), and St.Louis is MO) with 30mM 1; 2-dioctyl acylglycerol (DOG or DiCg) (Molecular Probes; Eugene, stock solution OR) is stored in-30 ℃.Compare with DMSO solution and experimental results show that DMSO is to the function of P-glycoprotein and express and all do not have influence.TPA with variable concentrations handles different cell lines, depends on viewed cytotoxicity.PBL handles with 1ng/ml TPA, and H9 and K562 cell are handled with 10ng/ml, and other cell is handled with 10 μ g/ml with the 100ng/ml processing for KG1a and KG1 cell.The DOG of same variable concentrations is used to handle different cell line.PBL handles with 75 μ M DOG like this, and H9 and K562 cell were with two part of 75 μ M dosage DOG(2 hours at interval) handle.Flow cytometry is analyzed or RNA extract before with cellular exposure in TPA or DOG8-12 hour.Staurosporine(Sigma, St.Louis MO) is used for the KG1a cell with 100mM concentration, and handles other cell with 30mM concentration; Adding before TPA or the DOG 30 minutes with its adding.

1.1.2 flow cytometry detects

Detect P-glycoprotein activity with rhodamine (Rh123) accumulation test.In this detects, will contain 100ng/ml Rh123(Sigma through drug treating or undressed cell washing three times, St.Louis, in culture medium MO) with it in 37 ℃ of insulations 1.5-2 hour.Washed cell then is with Propidium iodide(PI) dye and place ice until analyzing.The cell of monolayer growth is suspended with phosphate buffered saline (PBS) (PBS) 20mM ethylidene-dioxy base tetraacethyl (Sigma) in (PH7.4), and before Rh123 dyeing, wash 3 times.Use the Rh123 effluent to detect (Chaudhary and Roninson, 1991, Cell 66:85-94) rather than Rh123 accumulation in some experiments.

Utilize the specific mice IgG2a of P-glycoprotein monoclonal antibody (mAB) UIC2 to analyze expression (the Mechetner and Roninson of P-glycoprotein at cell surface, 1992, Proc.Natl.Acad.Sci.USA89:5824-5828) obtain from Sigma company with Mus IgG2a homotype control antibodies.With UIC2mAB or homotype tester dyeing PBL the time, in 4 ℃, with 10 μ g antibody stainings 10 ⁶ Individual cell 30 minutes, and (NJ) dyeing is 30 minutes, adds 2%FCS with PBS and is diluted by 1: 2 for Fisher Scientific, Fairlann with the bonded goat anti-mouse IgG 2a antibody of 10 μ g FITC after the washing secondary.Add 2%FCS with cell washing 2 times with ice-cooled PBS, use with PI dyeing and in insulation on ice to straight the analysis.The cell of other type that dyes uses same quadrat method basically, except per 10 ⁶Individual cell uses this point of second kind of antibody of 2 μ g need not.In some experiments, use algae erythrin (PE) bonded mountain sheep anti mouse IgG2a, do not add PI in this case as second kind of antibody.The flow cytometry analysis is carried out on Coulter Epics 753 flow-cytometers.

1.1.3 RNA extracts and cDNA-PCR analyzes

Adopt small-scale dodecyl sodium sulfate extraction method (Peppel and Baglioni, 1990, Bio Techniques 9:711-713) from about 10 ⁶Extract RNA in the individual cell.MDR1 and β ₂The amplification of the cDNA synthesized polymer enzyme chain reaction (PCR) of-microglobulin cDNA sequence is carried out (people such as Noonan, 1990, Proc.Natl.Acad.Sci.USA87:7160-7164 by the document record basically; Noonan and Roninson, 1991, PP.319-333 In Roninson(Ed.), Melecular and Cellular Biology of Multidrug Resistance in Tumor Cells, Plenum Press, New York), carry out following improvement simultaneously: (ⅰ) after initial heated sample to 94 ℃, the Taq archaeal dna polymerase is joined in the PCR mixture.(ⅱ) for calculating in the process cell of dissimilar processing, the degeneration of different RNA is with the β that obtains after 28 PCR circulations ₂-microglobulin band productive rate is as making the isostatic main standard of cDNA template initial amount in the different preparations.Detect with the radioautogram method ³²The PCR product of P labelling.

1.2 embodiment describes in detail

The function of use detection method detects when can induce lymph sample differentiation or activated different chemicals treatment people PBL, the active change of P-glycoprotein, this method is analyzed the fluorescent mitochondrion dyestuff that Rh123(P-glycoprotein is carried based on flow cytometry) the cell accumulation.In this detects, express seldom or the cell that do not have a P-P-glycoprotein expression is dyed very vivid color by Rh123, and the active cell of tool higher level P-glycoprotein presents the Rh123 faintly colored.The cell of handling with calcium ion carrier A 23187, IL-12 or IL-2 is not observed the P-glycoprotein is had a significant effect.On the contrary, handle PBL with phorbol ester TPA and cause that then the dim cell number of Rh123 significantly increases.Add 30 μ M isoptins (a kind of P-glycoprotein inhibitors) and can stop the increase of Rh123 faintly colored colony.Because the cytosis of the clear TPA of understanding is to stimulate PKC, also test the whether physiological stimulation agent of DOG(cell permeability DG and PKC thus) also influential to Rh123 deposition among the PBL.Handle PBL with DOG and also can reduce the Rh123 accumulation that produces by PBL.

For determining whether that active influence is because the result that the P-P-glycoprotein expression increases to viewed PKC stimulant to the P-glycoprotein, with the P-glycoprotein extracellular antigenic site of monoclonal antibody UIC2(identification by people MDR1 gene code) the indirect labelling immumofluorescent method PBL that dyes undressed PBL and handle with TPA.The P-glycoprotein level of handling with TPA that increases cell surface significantly.As coming with polymerase chain reaction (PCR) amplification MDR1 cDNA sequence detecting like that, the increase of this P-glycoprotein is accompanied by the corresponding increase of MDR1 mRNA level in the PBL total group of TPA processing.Therefore the active increase of the inductive P-glycoprotein of TPA, to small part be because the cause that is activated in rna level and protein level MDR1 gene expression.

Because PBL comprises the allos conductor of many different subtypes, the cloned cell line of therefore a series of leukemia being derived is tested, the variation of the P-P-glycoprotein expression after measuring it and handling with TPA.As concluding in the table 1, all P-glycoprotein positive cells systems show in the P-P-glycoprotein expression of letting alone that is exposed to TPA and increase greatly before handling with TPA.Such cell comprises people KG1 and KG1a stem cell, picture leukaemia system, its high-caliber relatively P-glycoprotein reflects expression (the Chaudhary and Roninson of this albumen in the normal hematopoiesis stem cell probably, 1991, Cell, 66:85-94), this class cell also comprises Mus EL4 thymoma and LBRM33 thymoma cell line.

The influence that table 1.TPA expresses MDR1

The untreated TPA of cell line detects

Handle

Normal cell

PBL + ++ F,A,R

Human hematopoietic cell system

KG1 (acute myelogenous leukemia) ++ +++F, A

KG1a (acute myelogenous leukemia) ++ +++F, A

K562 (chronic lymphocytic leukemia)-++ F, A, R

H9 (T chronic myeloid leukemia)-++ F, A, R

HL-60 (promyelocytic leukemia)--F

THP-1 (promyelocytic leukemia)--F

Jurkat, clone E6-1 (T chronic myeloid leukemia)--F

Molt-4 (T chronic myeloid leukemia)--F

U937 (histocytic lymphoma)--F

The influence that table 1 (continuing) .TPA expresses MDR1

The untreated TPA of cell line detects

Handle

Mus hematopoietic cell system

KL4 (thymoma) ++ +++F

LBRM 33, clone 4A2 (lymphoma)+++ F

People's solid tumor cell system

EJ (bladder cancer)+++ F

MCF-7 (breast carcinoma)--R

HeLa (cervical cancer)--F, R

KB-3-1 (Hela subbreed)--F, R

HT 1080 (fibrosarcoma)--F, R

In table 1, MDR1 expresses the Function detection (F) by the Rh123 accumulation, and UIC2 antibody staining (A) or cDNA-PCR detect MDR1 mRNA(R) estimate, and be expressed as relative value.If being detected by Rh123 or UIC2 dyeing does not have detectable P-P-glycoprotein expression, and its MDR1 mRNA level is not higher than this value of KB-3-1 cell, thinks that so this cell is negative.

In the cell line of no detectable P-P-glycoprotein expression, H9 and K562 leukaemia system obviously show by TPA or DOG and induce MDR1 mRNA and P-glycoprotein.Flow cytometry the analysis showed that with TPA or DOG handles the main cell colony (Fig. 1) that express P-glycoprotein just appears in these cell lines.These variations are accompanied by in the cell of TPA or DOG processing, and the raising of the MDR1 mRNA level of steady statue (Fig. 2 A, B).Show as Fig. 2 B, MDR1 mRNA became in the H9 cell and can detect after adding TPA in 2 hours, and continuing increases till at least 5 little time points, this shows TPA reaction very fast, and causes that by TPA the probability of MDR1 transcriptional activation is consistent in these cells.

MDR1 expressed to increase to have more than and is limited to hematopoietic cell after TPA handled, in the deutero-cell line of some solid carcinoma, also observe, comprise the EJ transitional cell bladder carcinoma cell line of expressing low-level P-glycoprotein, and the MCF-7 mastocarcinoma cell (Fig. 2 C) that the MDR1 detection of expression does not go out when not having TPA and handling.As concluding in the table 1, the P-glycoprotein negative cells of most of test ties up to show after TPA handles does not induce MDR1 to express.Yet should indicate, these cell lines are for the respond with TPA concentration.

For attempting to offset the inducing action of the MDR1 gene expression that causes by the PKC agonist, handle each cell line with a kind of effective protein proteins inhibitors of kinases Staurosporine.Unexpectedly, Staurosporine causes that separately the middle P-P-glycoprotein expression of cell line (KG1, KG1a, Mus EL4 and LBRM33 cell line) that the P-glycoprotein has been positive obviously increases.Two kinds of other chemical compound cyclosporin A and isoptin (being known P-glycoprotein inhibitors and pkc inhibitor) also find to increase dyestuff effluent in P-P-glycoprotein expression and/or KG1 and the EL4 cell.Pkc inhibitor to this effect of P-P-glycoprotein expression, makes and to be good at analyzing the interaction between the Staurosporine and PKC agonist in some cell lines like this in P-glycoprotein positive cell line.But Staurosporine does not induce MDR1 to express in the H9 cell of P-glycoprotein feminine gender.Handled preceding 30 minutes with TPA or DOG, add Staurosporine in the H9 cell, then eliminated the MDR1 that causes by these medicaments fully and induced, proved as flow cytometry (Fig. 1) and cDNA-PCR detection (Fig. 2 A).Staurosporine also suppresses the effect of TPA and DOG in the normal cell.

Embodiment 2

Kinases inhibitor stops cytotoxicity in the tumor cell

The MDR1 inducing action of medicine mediation

2.1 material and method

2.1.1 flow cytometry analysis

With 100ng/ml Rh123 among the 5ml DMEM that is added with 10% hyclone or 10ng/ml DiOC ₂(3) in 37 ℃ with K562 cell dyeing 10 minutes.After washing 2 times,, do not have at 5ml and to make cell flow out dyestuff 3 hours (concerning Rh123) or 2 hours in the dye media (to DiOC in 37 ℃ ₂(3)), as existing document put down in writing (Chaudhary and Roninson, 1991, Cell, 66:85-94).In double-label experiment, with the 3ng/ml DiOC in the 5ml medium ₂(3) dyeing.Every kind of outflow test reaches under the situation that does not have isoptin at existence 30 μ M isoptins respectively to be carried out.The KG1 cell dyeed 3 hours with the 100ng/ml Rh123 in the 5ml medium in 37 ℃, and analyzed under no effluent situation.Indirect immunofluorescence labelling (Chaudhary and Roninson, 1991, Cell 66:85-94), is per 2 * 10 ⁶Individual cell uses 2 μ g one-level antibody (UIC2 or Mus IgG2a homotype tester are available from Sigma company) and 10 μ g secondary antibody (the bonded F[ab ' of goat anti-mouse igg PE-] z segment (Sigma)) to finish.Concerning KG1 cell line per 2 * 10 ⁶Individual cell uses 1 μ g secondary antibody.Flow cytometry analysis and streaming classification art by the document record carry out (Chaudhary and Roninson, 1991, Cell, 66:85-94); Non-vigor cell perhaps discharges from analyze by propidium iodide accumulation in the experiment of not using the algae Erythrin according to its improper volume or granularity.

2.1.2 the growth inhibited effect detects

By every hole 3000 cells cell shop is spread on the 96 hole microdroplet flat boards and (to make two parts of same sample simultaneously), and it is grown in each medicine that improves concentration.Cell growth after 7-10 days by the MTT detection method analyzed (people such as PauWels, 1988, J.Virol.Meth.20:309-321).

2.2 embodiment describes in detail

Studies have shown that in the previous embodiment 1 that the PKC agonist can induce MDR1 to express, show that PKC plays an important role in the multi-drug resistance reaction in activating tumor cell.PKC also influences cell effect (the Papathanasiou and Fornace to dissimilar cellular stress, 1991, PP.13-36 In R.F.Ozols(Ed.), Molecular and Clinical Advances in Anticancer Drug Resistance, Kluwer Academic Publishers, Boston, MA).Especially PKC is by with 1-β-D-cytosine arabinoside (Ara-C), a kind of effective anti-leukemia medicine treatment and activate (people such as Kharbanda, 1991, Biochemistry, 30:7947-7952).Therefore, experimentize that it be can't help the P-glycoprotein and carries whether to test Ara-C() in K562 leukaemia to any effect of P-glycoprotein function tool.Shown in Fig. 3 a, the K562 cellular exposure caused 3-17% living cells subpopulation (by rhodamine-123(Rh123) outflow shows) to occur in Ara-C12-72 hour.It is responsive that Rh123 flows out P-glycoprotein preparation isoptin.The appearance of Rh123 faintly colored cell follows Ara-C to handle in the K562 cell, is related to β ₂The dose dependent that the MDR1 mRNA of-microglobulin expresses increases, as (Fig. 4 a) shown in the result that PCR amplification detected of cDNA sequence.

Also test other many chemotherapeutics to inducing the ability of the expression of MDR1 in the K562 cell.All all can induce MDR1 mRNA to express (Fig. 4 b) to find amycin, daunorubicin, vinblastine, etoposide, methotrexate, 5-fluorouracil, chlorambucil, cisplatin and hydroxyurea, and Rh123 or DiOC ₂(3) (another kind of P-glycoprotein is carried dyestuff, Chaudhary and Roninson, and 1991, Cell, 66:85-94) (Fig. 3 is a) with the treated cell concentration outflow of 3-10%.And these medicines only have preceding 4 kinds to carry (Roninson(Ed.) by the P-glycoprotein, 1991, and Molecular and Cellular Biology of Multidrug Resistance in Tumor Cells, Plenum Press, New York).Above result and MDR1 induced only needs this fact of short time drug exposure to combine, and shows that the cytotoxicity selection of expressing the MDR1 cell do not take the main responsibility to the appearance of the positive subpopulation of P-glycoprotein.

The ability that cytotoxic drug induces MDR1 to express has more than and is limited to the K562 cell.Ara-C improves the expression of P-glycoprotein in KG1 leukaemia's (it contained quite a lot of this albumen in the past in drug treating), as (Fig. 3 a) shown in Rh123 accumulation or monoclonal antibody UIC2 immunocompetence.Ara-C also activates MDR1 mRNA in H9 T chronic myeloid leukemia (Fig. 5) KB-3-1 epidermoid carcinoma (Fig. 4 C), and the expression in the EJ transitional cell bladder carcinoma cell line (Fig. 4 d), although inductive amplitude is low in these cancerous cell lines.In addition, MDR1 is induced (Fig. 5) in the H9 cell line of handling with amycin, vinblastine and methotrexate, and is induced (Fig. 4 C) in the KB-3-1 cell of handling with amycin.But in HL60 leukaemia, do not detect P-glycoprotein inducing action with above-mentioned same chemicals treatment.In all cases, inducing action just becomes and can detect in the observability cell injury, and so-called observability cell injury shows as the cell enlargement, and graininess increases, and cell shape changes and growth is suppressed (Fig. 4 A).In addition, when not having medicine, some cell line continuous passage some months also can cause MDR1 to express a small amount of increasing, and this is because in the untreated cell due to the transmutability of basic MDR1 mRNA level.

Then, whether the test cytotoxicity is handled afterwards drug-induced MDR1 expression and is kept getting off, for this reason, with the Ara-C of the dense property of cytotoxicity, amycin, chlorambucil or methotrexate were handled the K562 cell 3-5 days, and it is grown under no medicine condition.When analyzing different time points by the dyestuff outflow and with the immunofluorescence method of testing (Fig. 3 C) of UIC2 labelling or by cDNA-PCR amplification (Fig. 4 e) method, the expression of MDR1 in the survivaling cell.In treated cell subsets body, keep several weeks (handling in the colony up to 11 weeks) at least after being expressed in medicine and removing of MDR1 at Ara-C.The male K562 cell of P-glycoprotein is in its size, and graininess and the relevant antigenic mark expression aspect of differentiation have no significant change.The growth inhibited of carrying out with vinblastine, P-glycoprotein substrate detects, and also six weeks still there was the multi-drug resistance cell in proof after removing Ara-C or amycin.With ID ₁₀It doubly is the vinblastine resistance of feature that value increases about 2-3, is associated with the Rh123 faintly colored subpopulation (Fig. 6) of cell especially.Therefore, drug treating causes the successive induction of MDR1 expression and cause the drug resistance that it is relevant in the subpopulation of treated cell.And also find, the resistance of the cytotoxic effect of chlorambucil (a kind of can't help the chemotherapy alkylation medicament that the P-glycoprotein carries), the K562 cell of handling through Ara-C and amycin is higher than untreated cell.This result shows that after the chemotherapeutics treatment, other approach or the mechanism of clinical related drugs resistance can be expressed to be gone out by coinduction with MDR1.

Whether participate in the inducing action that caused by cytotoxic drug for measuring PKC, with two kinds of KPC inhibitor, the MDR1 mRNA that Staurosporine and H7 are used for blocking in the H9 cell induces.The adding of this two chemical compound has been blocked by Ara-C, and the MDR1 that amycin, methotrexate and vinblastine cause induces, as by cDNA-PCR(Fig. 5) and handling dyestuff that cell carries out with Ara-C, to flow out test detected.But use ISO-H7, the analog of a kind of H7 is wanted weak 10 times to the effect of protein kinase, do not observe the obvious suppression effect (people such as Pelosin, 1990, Biochem.Biophys.Res.Commun.169:1040-1048).Handle the K562 cell with Ara-C and also observe analog result.By research is played inhibiting specificity to PKC, will improve the H7(of dosage to PKC IC ₅₀=40 μ M are 2.3 μ M to protein kinase A) and HA1004, a kind of non-PKC specificity protein kinase inhibitor is (to PKC IC ₅₀=40 μ M, to protein kinase A is 2.3 μ M) (people such as Hidaka, 1984, Biochemistry, 23:5036-5041) effect of the two compares, shown in Fig. 5 a, it is 10 μ M or higher that H7 suppresses to induce used concentration by the MDR1 that Ara-C causes, but HA1004 promptly uses 60 μ M also to show no obvious suppression effect.The PKC role was consistent when these results caused that with cytotoxic drug MDR1 induces.

The digital proof that this paper provided, various chemotherapeutics comprise and can't help those medicines that the P-glycoprotein carries, and all can directly induce MDR1 to express, and be not to realize by the gene mutation of selecting to be pre-stored in.Drug-induced MDR1 expresses and is confined to treated cell subsets body, and follows the P-glycoprotein to carry the resistance appropriateness of medicine to increase (about 2-3 doubly under the situation of K562).This increase is enough to reduce the interior reaction to chemotherapy of body, and the selection that improves the gene mutation body of tool higher level drug resistance.The MDR1 induced expression effect of medicine mediation can come across during the cancer chemotherapy, and calculates the occurrence rate that increases the MDR1 expression in the tumor of treatment probably.Therefore, pkc inhibitor can stop this fact of MDR1 inducing action to show, in cancer chemotherapy this type of medicament is used in combination with cytotoxic drug, cures the probability of cancerous cell to reach high level.

The embodiment scope that the present invention is not exemplified is limit, and these schemes only attempt to use as the detailed description of each side of the present invention.In fact, from description and appended each figure of front, the of the present invention various modifications except that the indicated and new introduction of this paper all are conspicuous for a person skilled in the art.This class is revised and all certainly will be fallen among the claims scope.

All documents that this paper quoted all are incorporated herein by reference in full.

Claims

1, a kind of method of avoiding inducing the MDR1 gene to express in the cancerous cell with the chemotherapeutic agents treatment comprises the individuality that carries out cancer chemotherapy is used kinases inhibitor.

2, the process of claim 1 wherein this kinases inhibitor Profilin kinase c activity.

3, the method for claim 1, wherein carry out immunocompetence mensuration when using anti-P-glycoprotein antibody, the P-glycoprotein carries the accumulation of dyestuff or effluent to measure, or when MDR1 mRNA expressed test determination, the MDR1 P-glycoprotein that this cancerous cell contains seldom or can not detect.

4, the method for claim 3, cancerous cell wherein is from the hemopoietic tumor.

5, the method for claim 3, cancerous cell wherein is from entity tumor.

6, claim 1,2,3,4 or 5 method, wherein inhibitor before chemotherapeutics administration and with its administration simultaneously.

7, claim 1,2,3,4 or 5 method, wherein inhibitor and chemotherapeutics administration simultaneously.

8, identify the method that causes the inhibitor of the gene induced effect of MDR1 by chemotherapeutics, comprising:

(a). tumor cell line is exposed to the test inhibitor;

(b). cytotoxic drug is added in this cell;

(c). in the presence of test inhibitor and cytotoxic drug with this cell culture at least one hour; And

(d). by accumulation or the outflow that its mRNA level, P-glycoprotein level or P-glycoprotein are carried dyestuff, measure the expression of MDR1 gene in this cultured cell.

9, the method for claim 8, its Chinese medicine is Ara-C.

10, the method for claim 8, its Chinese medicine is a vinblastine.

11, the method for claim 8, its Chinese medicine is an amycin.

12, the method for claim 8, its Chinese medicine is a methotrexate.

13, the method for claim 8, wherein tumor cell line is the H9 leukemia.

14, the method for claim 8, wherein tumor cell line is the K562 leukemia.

15, in the cancer cell of avoiding treating with chemotherapeutics, the method for the inducing action of MDR1 gene expression comprises to the individuality that carries out cancer chemotherapy and uses inhibitor, and wherein inhibitor is determined by claim 8,9,10,11,12,13 or 14.