CN101560546B - Application of Phosphatidylinositol4-kinase type II alpha PI4KIIalpha - Google Patents
Application of Phosphatidylinositol4-kinase type II alpha PI4KIIalpha Download PDFInfo
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- CN101560546B CN101560546B CN 200810104272 CN200810104272A CN101560546B CN 101560546 B CN101560546 B CN 101560546B CN 200810104272 CN200810104272 CN 200810104272 CN 200810104272 A CN200810104272 A CN 200810104272A CN 101560546 B CN101560546 B CN 101560546B
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Abstract
The invention discloses a new biological function of Phosphatidylinositol4-kinase type II alpha PI4KIIalpha and an application based on the new function. PI4KIIalpha plays a key role in regulating andcontrolling a hypoxia-inducible factor HIF-1alpha and a downstream vascular endothecial growth factor VEGF. The selective reduction of PI4KIIalpha expression can obviously inhibit tumor growth and tumor angiogenesis. The PI4KIIalpha can be used as a target molecule for intervening in diseases which are relevant to a gene regulated and controlled by the HIF-1alpha and the diseases which are relevant to angiogenesis. In addition, the target molecule can be used for screening medicines for the relevant diseases.
Description
Background technology
Lipositol (PI) signal path plays the role of a nucleus in the signal path of transmembrane signal transduction and multiple somatomedin and hormone regulation Growth of Cells.Phosphatidylinositol4 4-kinase PI4K is first kinases that plays an important role in the PI signal path, PI4-K D4 position phosphorylation on catalysis lipositol (PI) ring at first in the lipositol working cycle, produce 4-phosphoric acid phosphatidylinositols (4-phosphatidyl-inositide, PIP), then further catalyze and synthesize (4 by the PIP5-K kinases, 5-phosphatidyl-inosididediphosphate, PIP2); PIP2 is at Phospholipase C (Phospholipase C, PLC) be hydrolyzed to InsP3 (1 under effect, 4,5-trisphosphoinositol, IP3) and triglyceride (Diacylglycerol, DAG), as the second messenger, cause Intracellular calcium release and activated protein kinase C (Protein kinase C, PKC), the double messenger system in active cell.In addition, PIP2 is also the substrate of PI3-K, thereby PI4K affects the PI3-K signal path.Research in recent years is found PIP, and PIP2 itself also has important biomolecule and learns function.Therefore, PI4K is the key molecule in the PI signal path, has caused that about the functional study of PI4K people more and more pay close attention to.
The relevant signal path of PI4K in born of the same parents:
PI4K has two types, two types and three types, and wherein two types comprise α and two kinds of hypotypes of β (PI4KII α and PI4KII β).The genes encoding of PI4KII α and protein sequence are seen respectively GenBank accessionnumber:NM_018425 (SEQ ID NO:10) and GenBank accession number:NP_060895 (SEQ ID NO:9), and its major function and film transhipment are closely related.In addition, PI4KII α is playing an important role aspect neurotransmitter release, host cell activation, regulation and control endothelial cell growth factor (ECGF) EGF.But its biological function also is elucidated far away.Its effect and mechanism in tumour at home and abroad is not reported.
Have the PI kinases vigor of bibliographical information cancer patient to increase, the amount of PKC obviously increases in cell, illustrate the lipositol signal path may with Tumor-assaciated.Also there are some circumstantial evidences to show that it may work in tumour about PI4KII α.As: the albumen valosincontaining protein (VCP) of PI4KII α and regulating cell migration is the location altogether, and the VCP rise is a mark of cancer diagnosis.PI4KII α can regulate and control the expression with the closely-related Urogastron of tumor growth (epidermalgrowth factor receptor, EGFR).Most of lipositol kinases enzyme in cancer cells is lived and is raised, comprising PI4K.But so far not about PI4K direct evidence and the Study on Molecular Mechanism relevant with tumor growth.
Seek the antitumor target spot of special signal path and the focus that anti-angiogenic rebirth is present antitumor strategy.Signaling molecule PKC, PLC and PI3-K take the PI4K downstream seeks the existing report of work of new drug as target molecule in the world.We study and find that PI4KII α plays a crucial role in hypoxia inducible factor HIF-1 α are synthetic.Due to the downstream gene that is subjected to the regulation and control of HIF-1 alpha transcriptional---vascular endothelial growth factor VEGF controls vasculogenesis by propagation, survival, the transfer of regulation and control endotheliocyte, so HIF-1 α is directly related with tumor growth and angiogenesis, regulation and control HIF-1 α is important antineoplastic available strategy.PI4KII α may play a significant role it to the crucial regulating and controlling effect of HIF-1 α in tumor growth and angiogenic process.
Summary of the invention
We find to cross expression PI4KII α in HeLa Cells and people's kidney epithelium HEK-293 cell first, the protein expression that can significantly raise hypoxia inducible factor HIF-1 α with and the expression of downstream angiogenesis promoting gene VEGF and nitricoxide synthase NOS.Going deep into Study on Molecular Mechanism shows: PI4KII α is PI3K to the regulating and controlling effect of HIF-1 α, and the MAPK signal path relies on, and synthesizes realization by regulating and controlling it, and on its stability and not impact of transcriptional level.By the special inhibition of RNA perturbation technique PI4KII alpha expression, the expression of HIF-1 α is also significantly suppressed, and can not make its rising even add hypoxia inducible.This is an important discovery, and be also the most important theories basis that we should invent: namely: PI4KII α is that HIF-1 α is synthetic necessary, is the key of regulation and control HIF-1 α, and indication PI4KII α has regulating and controlling effect to the relevant function of HIF-1 α.Based on the vital role of HIF-1 α in tumor growth and angiogenesis, we have further studied the effect of PI4KII α in tumour.The research discovery, along with the growth of tumour, the protein expression of PI4KII α also significantly raises.By PI4KII alpha expression in the special reduction tumour cell of RNA perturbation technique, can significantly suppress tumor growth and neonate tumour blood vessel.And all play remarkable effect in the broad variety tumours such as mammary cancer, cervical cancer.So far, we have disclosed the molecular mechanism of the new function and efficacy of PI4KII α aspect regulation and control hypoxia inducible factor HIF-1 α and neonate tumour blood vessel first.Result of study indication Phosphatidylinositol4 4-kinase PI4KII α may be the crucial target spot that suppresses tumor growth and angiogenesis.
In one aspect, the invention provides a kind of method for the treatment of disease, the method comprises take PI4K as target spot, suppresses the expression of PI4K or the activity of PI4K, and wherein said inhibition can be directly or indirectly.
In one embodiment, wherein disturb to suppress the expression of PI4K by RNA.
In one embodiment, wherein implementing the little RNA sequence that described RNA disturbs is:
Positive-sense strand: UGAAGCAGAACCUCUUCCUCATT (SEQ ID NO:1)
Antisense strand: UCAGGAAGAGGUUCUGCUUCATT (SEQ ID NO:2).
In one embodiment, wherein by the PI4K gene is carried out the expression that gene knockout suppresses PI4K.
In one embodiment, the activity that wherein suppresses PI4KI by PI4K enzyme antagonist.
In one embodiment, wherein said antagonist comprises No. 12, medicine-ocean, No. 1, ocean of marine active substance medicine.
In one embodiment, No. 12, medicine-ocean, described No. 1, ocean of marine active substance medicine is from Syngnathidae and sour jujube skin marine animal.
In one embodiment, wherein said disease is tumour or cancer.
In one embodiment, wherein said tumour or cancer comprise cervical cancer and mammary cancer.
In one embodiment, wherein said disease is the disease relevant to angiogenesis.
In one embodiment, the wherein said disease relevant to angiogenesis comprises tumour, sacroiliitis, diabetes or retinal abnormalities.
In the above-described embodiment, described PI4K enzyme is the PI4KII alpha hypotype.
In yet another aspect, the invention provides the method for a kind for the treatment of disease relevant to angiogenesis, the method comprises take PI4K as target spot, the expression of enhancing PI4K or the activity of PI4K, and wherein said enhancing can be directly or indirectly.
In one embodiment, wherein express to strengthen the expression of PI4K by mistake.
In one embodiment, wherein strengthen the activity of PI4K by agonist.
In one embodiment, described agonist is No. 22, medicine-ocean, No. 13, ocean medicine.
In one embodiment, No. 22, No. 13 medicine-oceans of described marine active substance medicine is from Syngnathidae and sour jujube skin marine animal.
In one embodiment, the wherein said disease relevant to angiogenesis comprises wound healing or tissue regeneration.
In foregoing embodiments, described PI4K enzyme is the PI4KII alpha hypotype.
In one aspect of the method, the invention provides the method for a kind for the treatment of disease relevant to HIF-1 α institute regulatory gene, the method comprises take PI4K as target spot, correspondingly suppresses or strengthen expression or its activity of PI4K, wherein said inhibition or to strengthen can be directly or indirectly.
In one embodiment, described PI4K is the PI4KII alpha hypotype.
In one embodiment, the wherein said disease relevant to HIF-1 α institute regulatory gene comprises diabetes, tumor drug resistance or cardiovascular and cerebrovascular diseases.
In yet another aspect, the invention provides a kind of method of screening of medicaments, wherein, the method comprises take PI4K as target spot, the material that screening inhibition PI4K genetic expression or inhibition PI4K enzyme are lived.
In one embodiment, the activeconstituents of wherein said medicine is the sense-rna fragment of PI4K gene or the antagonist of PI4K.
In foregoing embodiments, described PI4K is the PI4KII alpha hypotype.
In one embodiment, wherein said medicine can be used for treating tumour, cancer, sacroiliitis, diabetes, or retinal abnormalities.
In yet another aspect, the invention provides a kind of method of screening of medicaments, wherein, the method comprises take PI4K as target spot, the material that screening enhancing PI4K genetic expression or enhancing PI4K enzyme are lived.
In one embodiment, the activeconstituents of wherein said medicine is the agonist of PI4K.
In one embodiment, wherein said medicine can be used for promoting wound healing or tissue regeneration.
In foregoing embodiments, described PI4K is the PI4KII alpha hypotype.
In yet another aspect, the invention provides a kind of medicine, it comprises and can suppress PI4K genetic expression or suppress the activeconstituents that the PI4K enzyme is lived.
In one embodiment, wherein said activeconstituents is the sense-rna fragment of PI4K gene or the antagonist of PI4K.
In foregoing embodiments, described PI4K is the PI4KII alpha hypotype.
In one embodiment, wherein said activeconstituents is little RNA sequence:
Positive-sense strand: UGAAGCAGAACCUCUUCCUCATT (SEQ ID NO:1)
Antisense strand: UCAGGAAGAGGUUCUGCUUCATT (SEQ ID NO:2).
In one embodiment, wherein said medicine can be used for treating tumour, cancer, sacroiliitis, diabetes, or retinal abnormalities.
In yet another aspect, the invention provides a kind of medicine, it comprises can strengthen PI4K genetic expression or enhancing PI4K enzyme activeconstituents alive.
In one embodiment, wherein said activeconstituents is the agonist of PI4K.
In foregoing embodiments, described PI4K is the PI4KII alpha hypotype.
In one embodiment, wherein said medicine can be used for promoting wound healing or tissue regeneration.
In one embodiment, wherein said activeconstituents also makes up with routine and suppresses cancer drug.
The compound method of medicine of the present invention, formulation, route of administration, the determining of the auxiliary material that wherein comprises and carrier etc. and effective dose well known to a person skilled in the art.For example, medicine of the present invention can also comprise pharmaceutical carrier except above-mentioned activeconstituents.In this article, medicinal carrier refers to nontoxic weighting agent, thinner, adjuvant or other pharmaceutical adjuncts.According to the known technology of this area, can described medicine be made various formulations according to the needs of therapeutic purpose, route of administration.
In this article, administration medicine also needs according to the characteristic of corresponding preparations and whether the comfort level of described activeconstituents and administration can be provided in vivo and choose suitable mode from the known administering mode of one of ordinary skill in the art.The significant quantity of administration changes to some extent according to the situation of action time of dosage form and expectation and treatment target, the required amount of actual therapeutic can by the doctor according to practical situation (as, patient's weight, age, sex, healthy state, diet, administration frequency, medication, secretion excretion and disease seriousness etc.) and determine easily.
Take PI4KII α as target spot has following advantage:
1.PI4KII α is the stream signal molecule of PI signal path, suppresses tumour efficient high;
With the special hypotype of PI4K as target molecule, specificity is good, few side effects;
3. take the molecule of the special signal path of PI as target molecule, toxicity is little.
In addition, this invention is not only significant at anti-tumor aspect and use, and is intervening significant and application too aspect the disease relevant with HIF-1 α institute regulatory gene, for example: HIF-1 α and diabetes, drug resistance of tumor, cardiovascular and cerebrovascular diseases are all closely related.Equally, the present invention is significant aspect intervention and relevant diseases of angiogenesis and application also, for example: sacroiliitis, diabetic retina is abnormal, and wound healing, tissue regeneration etc. are all closely related with angiogenesis.So, the discovery of the neontology function of our patent protection Phosphatidylinositol4 4-kinase two type alpha hypotypes (PI4KII α) and based on the application of this new function.The PI4KII α disease that to be a very potential intervention relevant to HIF-1 α institute regulatory gene and the target molecule of relevant diseases of angiogenesis can carry out drug screening and the research and development of above-mentioned relative disease with PI4KII α as target molecule.
Description of drawings
Fig. 1 represented to express the protein content that PI4KII α can significantly improve HIF-1 α in people's kidney epithelium HEK-293 cell.A means the figure that crosses expression efficiency that detects PI4KII α, wherein the method by immunoblotting with the antibody test of PI4KII α in people's kidney epithelium HEK-293 cell GFP-PI4KII α cross expression efficiency; B detected to express PI4KII α for the figure of the impact of HIF-1 α expressing quantity, wherein used the protein content of HIF-1 α in the HEK-293 cell of antibody test Different treatments of HIF-1 α, with Actin as internal reference.H:1% anoxic 24 hours, the Co:0.2mM cobalt chloride was processed 6 hours, N: normally cultivate control cells, GFP: cross and express the PEGFP-C1 plasmid cell of 48 hours, GFP-PI4KII α: cross and express the PEGFP-PI4KII α plasmid cell of 48 hours.
Fig. 2 represents that RNA disturbs PI4KII α can significantly reduce under normal oxygen or anoxia condition the protein content of HIF-1 α in various kinds of cell.A is the figure to the evaluation of the jamming effectiveness of PI4KII α, wherein detects the gene jamming effectiveness of PI4KII α in three kinds of different cells (people's kidney epithelium HEK-293 cell, HeLa Cells, MCF-7 Human Breast Cancer Cells) with the method for immunoblotting; B detects to disturb PI4KII α for the figure of the impact of HIF-1 α protein content in HeLa Cells and MCF-7 Human Breast Cancer Cells under normal oxygen condition, wherein use and disturb PI4KII α for the impact of HIF-1 α protein content in cell under the normal oxygen condition of antibody test of HIF-1 α in two kinds of different carcinoma cells (HeLa Cells, MCF-7 Human Breast Cancer Cells).The contrast: transfection the cell of non-specific little RNA, PI4KII α: transfection the cell of little RNA of PI4KII alpha specific, C detects to disturb PI4KII α for the impact of HIF-1 α protein content in people's kidney epithelium HEK-293 cell under normal oxygen and anoxic incentive condition, and wherein the method with immunoblotting detects the anoxic stimulation or often disturbs PI4KII α for the impact of HIF-1 α protein content in people's kidney epithelium HEK-293 cell under oxygen condition.
Fig. 3 shows that PI4KII α can regulate and control the gene of hypoxia inducible factor HIF-1 α downstream promotion angiogenesis: VEGF and iNOS.A showed to express PI4KII α to the figure of the impact of the mrna expression of VEGF in cell, wherein adopt the method for RT-PCR to detect the mrna expression level of VEGF in Different treatments descendant kidney epithelium HEK-293 cell, with the β-actin in cell as internal reference, H:1% anoxic 24 hours, the Co:0.2mM cobalt chloride was processed 6 hours, N: normally cultivate control cells, GFP: cross and express the PEGFP-C1 plasmid cell of 48 hours, GFP-PI4KII α: cross and express the PEGFP-PI4KII α plasmid cell of 48 hours; B showed to express PI4KII α to the figure of the impact of the protein expression of iNOS in cell, and wherein the method by immunoblotting detects after Different treatments the protein content of iNOS in cell, the same A of the meaning of each symbol with the antibody of iNOS.
Fig. 4 shows that the protein content of HIF-1 α in tumour and PI4KII α is all along with tumor growth increases.A means the figure of the weight of tumor growth different number of days, wherein mouse is become fibrous tumours MCA205 injection cell in the C57/BL6 Mice Body, injected rear 7 days, and 10 days, 14 days, respectively get a mouse after 17 days, put to death the taking-up tumour and take weight; When B represents the tumor growth different time, PI4KII α and HIF-1 α protein content detect, wherein with the tumour cracking in A, with the HIF-1 α in the method detection tumour of immunoblotting and the protein content of PI4KII α.
Fig. 5 shows that RNA disturbs PI4KII α to reach the purpose that suppresses the tumor growth that HeLa Cells induces by angiogenesis inhibiting.A means the figure of the affects on the growth that disturbs the tumour that PI4KII α induces HeLa Cells in Mice Body, wherein with transfection contrast little RNA and the little RNA of PI4KII alpha specific the Hela cell be expelled to mouse web portion according to above-described method, measured the size of a tumour in every two days, until the 18th day of tumor injection; B disturbs PI4KII α on the impact of human cervical carcinoma HeLa induced tumor content of hemachrome, wherein the tumour that grows into 18 days in A is taken out, and adopts Drabkin ' s methemoglobin method to measure the content of hemachrome of tumour, compares; C shows that the antibody with anti-CD-31 disturbs PI4KII α to the figure of human cervical carcinoma HeLa induced tumor medium vessels growing state by the means detection of immunohistochemical methods, wherein the tumour of cultivating in A 18 days is taken out section, adopt the antibody of anti-CD31 by the method for immunohistochemical methods, relatively the blood vessel situation in the tumour of Different treatments.
Fig. 6 shows that RNA disturbs PI4KII α can obviously suppress the tumor growth of MCF-7 Human Breast Cancer Cells and people's kidney epithelium HEK-293 cell induction.A shows to disturb PI4KII α to the figure of MCF-7 Human Breast Cancer Cells induced tumor affects on the growth, wherein with transfection contrast little RNA and the little RNA of PI4KII alpha specific the MCF-7 cell be expelled to mouse web portion according to above-described method, measured the size of a tumour in every two days, until the 18th day of tumor injection; B shows to disturb PI4KII α on the figure of the impact of people's kidney epithelium HEK-293 cell induction tumor growth, wherein with transfection contrast little RNA and the little RNA of PI4KII alpha specific the HEK-293 cell be expelled to mouse web portion according to above-described method, measured the size of a tumour in every two days, until the 18th day of tumor injection, select 4 days, 8 days, the tumor growth situation compared in 12 days.
Fig. 7 shows that classical cancer drug vincristine(VCR) (VCR) and the Vinorelbine (VNR) of pressing down has restraining effect for the enzyme work of PI4K.A is the figure of the impact that shows that Vinorelbine and vincristine(VCR) are lived on the PI4K enzyme, and wherein VCR and the VNR with different concns processed the HeLa cell after 1-3 hour, detected the enzyme of PI4K according to method mentioned above and lived, and take the solvent treatment group as contrast, compared processing; B shows that Vinorelbine and vincristine(VCR) on the figure of the impact of HIF-1 α in cell, wherein adopt the method for immunoblotting to detect the VCR of different concns and the protein content that VNR processes HeLa cell HIF-1 α in cell after 1-3 hour.
Fig. 8 is presented at and screens the molecule that PI4KII α is had regulating and controlling effect in the marine active extract.A shows that the work to the PI4K enzyme that screening obtains has inhibiting marine active extract, wherein the different marine extracts of the HeLa Cells of logarithmic phase process are processed after 1-3 hour with corresponding lysate cracking, adopt the method for above-mentioned PI4K enzyme activity determination to carry out its enzyme biopsy survey, will have inhibiting drug treating the data obtained to compare analysis to the work of PI4K enzyme; The work to the PI4K enzyme that B demonstration screening obtains has the marine active extract of activation, wherein processes equally with A kind sample, will have the drug treating the data obtained of activation to compare analysis to the work of PI4K enzyme.
Our invention better is described and understands below by embodiment, but our invention and application are not limited to the scope of embodiment.As do not specialize part, can be according to " Biochemistry and Molecular Biology experimental technique " (Higher Education Publishing House, Yang Angang, Mao Jifang, medicine vertical wave chief editor) that those skilled in the art were familiar with; " protein electrophorese technology " (Science Press, Guo Yaojun writes); " protein interaction molecular cloning handbook (press of Tsing-Hua University, Erica Golemis writes); " cell experiment guide " (Huang Peitang etc. translate for Science Press, the work such as D.L. Spector); In the reference that the laboratory manual such as " fine works molecular biology experiment guide " (Yan Ziying etc. translate for Science Press, F. this uncle R. Boulogne top grade work difficult to understand) and this paper quote, listed method is implemented.
Embodiment
Embodiment 1: materials and methods
Cell cultures
HeLa Cells (ATCC:CCL 2), MCF-7 Human Breast Cancer Cells (ATCC:HTB-22) and people's kidney epithelium HEK-293 (ATCC:CRL-1573) cell are available from U.S.'s typical cells Culture Center (ATCC).Cell is cultivation in DMEM nutritional blend (Kaighn ' s improve), this nutritional blend contains 10% tire calf serum (Hycloe), Vetstrep (100 μ g/ml), and penbritin (100 units/ml), cell is put in 37 ℃ of cell culture incubators, 5%CO
2Condition under cultivate.Operation instruction according to manufacturers, by using cationic lipid: Lipofectamine 2000 (invitrogen), in 6 holes or 6-cm plate, described cell is carried out transfection (30%-60% converges) (concrete grammar is referring to invitrogen Lipofectamine 2000 product descriptions).Transfection is cultivated 6 hour cells afterwards and is changed liquid to containing continuation cultivation in 10% foetal calf serum and two anti-DMEM substratum.
Sxemiquantitative RT-PCR
Operation instruction according to manufacturers, with the total RNA in TRIzol (invitrogen) extraction cell, and (German Eppendorf produces through ultraviolet spectrophotometer, model is 603105430) get 2 μ g after quantitatively and carry out reverse transcription, the concrete experimental technique of reverse transcription is as follows: the RNA of 2 μ g and appropriate reversed transcriptive enzyme (MMLTV-RT, Promega), RNA enzyme inhibitors (Promega) hatched 1 hour jointly at 42 ℃, then hatch and obtained the total cDNA of cell in 10 minutes for 72 ℃.Subsequently with the upstream primer (CCATGAACTTTCTGCTGTCTT) (SEQ ID NO:5) of 20nMVEGF, the downstream primer of 20nM VEGF (ATCGCATCAGGGGCACACAG) (SEQ ID NO:6), 4 μ l cDNA, 12.5 μ l premix polysaccharase mixture (takara) and sterilized water are mixed to cumulative volume 25 μ l, carry out the PCR experiment of 25 circulations: 94 ℃ 30 seconds, 60 ℃ 30 seconds, 72 ℃ 30 seconds.The PCR product carries out agarose electrophoresis and carries out semi-quantitative analysis by gel imaging system (BIO-PRINT gel imaging system, France).With intracellular Actin muscle (β-actin) as internal reference.
Immunoblotting
After cell process respective handling (as transient transfection, anaerobic treatment etc.), use 0.25% trypsin digestion cell, 800g discards the supernatant collecting cell after centrifugal 5 minutes, then cleans cell 2 times with PBS, and with RIPA lysate Tris.HCl, 50mmol/L, pH 7.5; NaCl, 150mmol/L; NP-40,1%; Sodium desoxycholate, 0.5%; SDS, 0.1%; EDTA, 1mmol/L; 1.2mM phenylmethylsulfonyl fluoride (PMSF), 1 μ g/ml leupeptin (leupeptin), 1 μ g/ml Trypsin inhibitor,Trasylol (aprotinin)) cracking 30 minutes, centrifugal 15 minutes of split product 12000g.Get supernatant and 2 * SDS sample loading buffer (0.1MTris-cl; 4%SDS; 0.2% bromine Finland, 20% glycerine, 0.1MDTT) mix, 100 ℃ were boiled 10 minutes, and the centrifuging and taking supernatant carries out the SDS-PAGE electrophoresis, transferring film (nitrocellulose membrane after electrophoresis, PALL), after 1.5 hours, use respectively antibody (concrete weaker concn is referring to the specification sheets of antibody used) (mouse-anti HIF-1 Alpha antibodies as anti-in rabbit, the Santa curz of corresponding protein with the confining liquid sealing that contains 5% skim-milk; The anti-PI4KII Alpha antibodies of goat-anti rabbit, Abgent; The anti-goat-anti Actin of rabbit antibody, Santacurz; The anti-iNOS antibody of goat-anti rabbit, Santa curz) hatched 1.5 hours, use TTBS (50mMTris/HCl, 140mM NaCl, 0.05%Tween-20, PH 7.2) wash described film 3 times, each 15 minutes.Corresponding two anti-(determine that according to the situation of primary antibodie two is anti-, BIOS) hatch 1 hour, again wash film 3 times, after each 15 minutes, luminous substrate on the NC film is carried out exposure tests (concrete grammar is with reference to fine works molecular biology experiment guide).
The biopsy of PI4K enzyme is surveyed
With lysate (100mM NaCl, 300mM sucrose, 3mM MgCl
2, 1%TritonX-100,5mM CaCl
2, 10mM Pipes, 1.2mM phenylmethylsulfonyl fluoride (PMSF), 1 μ g/ml leupeptin (leupeptin), 1 μ g/ml Trypsin inhibitor,Trasylol (aprotinin), 10mM NaF and 5mM EDTA (PH6.8)) and lysing cell (10
6Cell/ml lysate), cracking on ice 20 minutes, the centrifugal 10min of 650g removes nucleus and larger cell debris, keeps the supernatant that contains PI4K albumen.The detection damping fluid (50mM Tris-HCl, 2mMEGTA (ethylene glycol diethyl ether ethylenediamine tetraacetic acid (EDTA)), 2mM EDTA, the 10mM Mg that the PI-4K protein solution 10 μ l that extract are added 20 μ l
2+, 0.4%TritonX-100,1.2mM phenylmethylsulfonyl fluoride (PMSF), 1 μ g/ml leupeptin (leupeptin), 1 μ g/ml Trypsin inhibitor,Trasylol (aprotinin)).Add the 15 good PI4K mixed solutions of μ l aforementioned processing in each Eppendorf pipe, 10 μ l contain the PI liposome solutions of 500 μ g/ml, 2 μ Ci[γ-
32P] ATP solution.Vibration evenly is placed in 37 ℃ of water-baths, reaction 5-10min.Take out the Eppendorf pipe and place on ice, add the 1mol hydrochloric acid soln of 100 μ l, then add the chloroform of 300 μ l: methyl alcohol (2: 1, v/v) mixed solution, termination reaction.Vibrated two minutes, centrifugal 1min.Solution is divided into two-phase, and upper is water layer mutually, and lower is organic layer mutually.Water layer is siphoned away, keep organic phase.Use again 1N HCl: CH
3OH (1: 1, v/v), wash twice.Collect organic phase, vacuum-evaporation.With chloroform dissolved phosphorus acidifying product P I (4) P, do the experiment of exhibition layer with the TLC thin plate.The exhibition layer is 25min approximately, and the TLC thin plate is placed in airing in ventilating kitchen, and thin plate is put in iodine steam dyeing 1min in the iodine cylinder.The location of PI4K phosphorylation product P I (4) P on the TLC thin plate is to determine according to the position of standard P I (4) P on thin plate.Use is shielded memory technology (Wang, Y.J., Wang with phosphorus, J., Sun, H.Q., Martinez, M., Sun, Y.X., Macia, E., Kirchhausen, T., Albanesi, J.P., Roth, M.G., and Yin, H.L. (2003) .Phosphatidylinositol 4 phosphate regulates targeting of clathrin adaptor AP-1complexes to the Golgi.Cell 114,299-310.) be Storm 820 ImagingSystem (the Amersham Pharmacia Biotech) measurement of principle
32The radioactivity of P-PI (4) P band.According to what obtain
32P-PI (4) P radioactivity is done a relative quantitative analysis to the enzyme work of PI4K in lysate, and is detected
32P-PI (4) P radioactivity is directly proportional to the enzyme work of PI4K.
Zooscopy
Carry out testing in body according to the good laboratory standard (GLP regulations) of the non-clinical labororatory research of Food and Drug Administration with according to the German Animal Protection Law as the legislation basis.
C57BL/6 mouse and BALB/C nude mice (dimension tonneau China) maintenance (laminar air flow equipment under the SPF condition, Scantainer, Scanbur), (the MCA205 cell separates from the C57BL/6 mouse to be used separately as the MCA205 mouse tumor cell, separation method reference Shu, S.Y., S.A.Rosenberg.1985.Adoptive immunotherapy of newly induced murine sarcomas.CancerRes.45:1657-1662) and HeLa Cells, the acceptor of MCF-7 Human Breast Cancer Cells.2 * 10
5/ 0.2ml MCA205 cell is inoculated into the C57/BL6 mouse web portion both sides in 6-8 week, inoculates rear 3 days, and 7 days, 10 days, after 17 days, mouse is put to death, take out tumour and adopt the method for above-mentioned immunoblotting to be used for the protein content detection of HIF-1 α and PI4KII α.5 * 10
6After/0.2ml HeLa cell or the MCF-7 cell corresponding genetic manipulation of process (the little RNA of transient transfection PI4KII alpha specific or the little RNA of non-specific contrast), through incubated overnight, with the belly both sides that are injected into mouse after 0.25% trysinization, the both sides of a same mouse are respectively the cells of experimental group and control group.measure observation through the sizes of 18 days, put to death mouse and adopt Drabkin ' s methemoglobin method (Drabkin, D. (1949) .The standardardization of hemoglobin measurement.Am.J.Med.Sci.217, 710-711.) content of hemachrome of measurement tumour and method detection tumour medium vessels growing state (A Giatromanolaki. (1997) the Comparativeevaluation of angiogenesis assessment with anti-factor-VIII and anti-CD31immunostaining in non-small cell lung cancer.Clinical Cancer Research that makes immunohistochemical methods of the antibody of anti-CD-31, Vol 3, Issue 12 2485-2492).
Embodiment 2: cross and express PI4KII α to studying with the adjusting function of the closely-related hypoxia inducible factor HIF-1 of tumor growth α in tumour cell
People's kidney epithelium HEK293 cell is transient transfection plasmid PEGFP-PI4KII α or PEGFP-C1 (PEGFP-C1 buys from Promage, and PEGFP-PI4KII α builds according to " fine works molecular biology experiment guide ") according to the method described above.After 48 hours, lysing cell detects the PI4KII α in cell with the method for above-mentioned immunoblotting, to determine transfection efficiency.On the basis of the GFP-PI4KII alpha expression that transfection detected, detect the protein content of HIF-1 α in overexpressing cell, and with albumen Actin muscle (Actin) internal reference as a comparison of running one's home in cell.For the reliability that guarantees to test, we have designed the experiment of positive control, we adopt the 0.2mM cobalt chloride to process cell 6 hours (Co), and in the cell under the HIF-1 α protein content in the 1% oxygen treatments applied cell HEK-293 of 24 hours (H) is cultivated with normal oxygen, this protein content compares.
This result proved that expressing PI4KII α can cause HIF-1 α albumen in the increase (Fig. 1) of cell paste intensive amount as cobalt chloride or anoxic.
Synthetic one section special little RNA sequence (positive-sense strand: UGAAGCAGAACCUCUUCCUCATT (SEQ ID NO:1) that disturbs PI4KII α; Antisense strand: UCAGGAAGAGGUUCUGCUUCATT (SEQ ID NO:2)) and the little RNA sequence of nonspecific contrast (positive-sense strand: UUCUCCGAACGUGUCACGUTT (SEQ ID NO:3); Antisense strand: ACGUGACACGUUCGGAGAATT (SEQ ID NO:4)).Explanation is according to the method described above carried out cultivating 72-96 hour after transfection to HeLa cell and MCF-7 cell, cell is carried out cracking and carry out above-mentioned immunoblotting and detect the protein content of PI4KII α to determine the interference effect of described interference sequence, the protein content of HIF-1 α detect interference cell and control cells on proof is disturbed the jamming effectiveness of little RNA very high basis in, compare, with the albumen Actin muscle (Actin) of running one's home in cell as internal reference.In order to determine under anoxia condition, whether PI4KIIa also has corresponding regulating and controlling effect to HIF-1 α, the HEK-293 cell at transient transfection added cobalt chloride to process 6 hours in 66 hours after the little RNA of interference PI4KIIa specificity or the little RNA of nonspecific contrast, then jamming effectiveness and corresponding HIF-1 α are detected comparison.
This result proves, no matter under normal oxygen condition or anoxia condition, disturbs PI4KII α can suppress significantly the protein content (Fig. 2) of HIF-1 α in cell.
The adjusting function research of embodiment 4.PI4KII α to HIF-1 α downstream Angiogensis gene
Cross expression GFP-PI4KII α and GFP in people's kidney epithelium HEK293 cell, after transfection 48 hours, carry out the RT-PCR experiment according to this paper aforesaid method and detect the mRNA level of VEGF in overexpressing cell and control cells and compare, and the housekeeping gene Actin muscle (β-actin) as internal reference relatively.Use VEGF (primer sequence is seen embodiment 1) and Actin muscle (corresponding upstream and downstream primer (β-actin upstream primer: TCCTGTGGCATCCACCAAAC (SEQID NO:7) of β-actin); β-actin downstream primer: GAAGCATTTGCGGTGGACCA (SEQ IDNO:8)).Exactness for the confirmatory experiment method, we have set up positive control, be that the 0.2mM cobalt chloride is processed cell 6 hours (Co), in the cell under the VEGFmRNA content in the 1% oxygen treatments applied cell HEK-293 of 24 hours (H) is cultivated with normal oxygen, the mRNA content of this gene compares.For the further checking PI4KII α impact for HIF-1 α downstream regulatory gene, we have selected to detect another one and have been subjected to HIF-1 α regulation and control and with angiogenesis, the protein induced type nitricoxide synthase (iNOS) of close ties are arranged in human cervical carcinoma cell HeLa.Experimental design is identical with the detection of scheme and VEGF.
This result proves: excessively express PI4KII α and can significantly raise and the downstream gene VEGF of the closely-related hypoxia inducible factor HIF-1 of angiogenesis α and the transcript and expression (Fig. 3) of iNOS.
The mouse of growth logarithmic phase becomes fibroma MCA205 cell, and (the MCA205 cell separates from the C57BL/6 mouse, separation method reference Shu, S.Y., S.A.Rosenberg.1985.Adoptiveimmunotherapy of newly induced murine sarcomas.Cancer Res.45:1657-1662) with washing 3 times with PBS after 0.25% trysinization collection.Regulating final cell concn by cell counting is 1 * 10
6/ ml is then injected into C57/BL6 mouse web portion both sides, every side injection 0.2ml cell suspension.Tumor planting 3 days afterwards, 7 days, 10 days, get respectively a mouse in the time of 17 days out, take out tumour after putting to death, use RIPA lysate homogenate cracking 15 minutes after measuring weight, get 50 μ l out from cleavage mixture, 12000g adopts BCA method (with reference to the cell experiment guide) to survey the split product total protein concentration after centrifugal 15 minutes, remaining cleavage mixture adds the SDS sample loading buffer and boiled 15 minutes, be used for detecting PI4KII α, the protein content of Actin and HIF-1 α according to the western blotting method of embodiment 1.
The results show, along with the growth of tumour, the PI4KII α in tumor tissues and the protein content of HIF-1 α all obviously increase (Fig. 4).
The neonate tumour blood vessel that embodiment 6.PI4KII α induces HeLa Cells and the regulation and control of tumor growth
The PI4KII alpha specific of the aforementioned sequence of HeLa Cells transient transfection (sequence described in embodiment 3) disturbs after little RNA or the little RNA of non-specific contrast 20 hours, collects afterwards with 0.25% trysinization and washes 3 times with PBS.Regulating final cell concn by cell counting is 2.5 * 10
7Then/ml is expelled to respectively BALB/C nude mice mouse web portion both sides, every side injection 0.2ml cell suspension.Often use every other day the vernier caliper measurement size after injection cell, when tumor growth to 18 day, put to death mouse, take out tumour and adopt Drabkin ' s methemoglobin method to measure the content of hemachrome of tumour and method (concrete grammar is with reference to the cell experiment guide) the detection tumour medium vessels growing state of making immunohistochemical methods of the antibody (available from BD company) of anti-CD-31.
This result shows: disturb significantly to suppress after PI4KII α the new life (Fig. 5 B, C) of the tumour medium vessels that HeLa Cells induces, and suppress the growth (Fig. 5 A) of tumour.
The regulation and control of embodiment 7.PI4KII α to the tumor growth of MCF-7 Breast Cancer Cell and people's kidney epithelium HEK-293 cell induction
This experimental result shows, disturbs the tumor growth (Fig. 6) that can obviously suppress MCF-7 Breast Cancer Cell and people's kidney epithelium HEK-293 cell induction after PI4KII α.
HeLa Cells is cultured to logarithmic phase, low serum (1% serum) pre-treatment is after 12 hours, add different concns (25nM in original substratum, 50nM) VCR or different concns (15 μ M, 60 μ M) VNR and cell were hatched 1-3 hour jointly, after 0.25% trysinization, cell is according to the vigor of measuring the PI4K in PI4K enzyme method detection cell pyrolysis liquid alive in embodiment 1, cell for detection of HIF-1 α protein content is used the cracking of RIPA lysate after relative medicine is processed, do immunoblot experiment according to the method for describing in embodiment 1.
This result shows: the cancer drug that presses down of these two kinds of classics of vincristine(VCR) and Vinorelbine has obvious restraining effect for the enzyme work of PI4K, and certain dose-dependently (Fig. 7 A) is arranged, these two kinds of medicines of while also have restraining effect (Fig. 7 B) for the protein content of the transcription factor HIF-1 α that PI4KII α regulates and controls.
Work has the medicine of adjusting function to the PI4K enzyme in embodiment 9. screening
Choose more than 20 and plant Syngnathidae and sour jujube skin marine animal, grind through freeze-drying and obtain meal, again through keeping supernatant liquor after 95% and 50% ethanol extracted twice, pass through again sherwood oil, ethyl acetate, the fractionation of propyl carbinol and water obtains different deliquescent each compositions, and then the technology by column chromatography obtains numerous monomeric compounds, for detection of the impact of in HeLa Cells, the PI4K enzyme being lived.Concrete detection method is as follows: HeLa Cells is cultured to logarithmic phase, low serum (1% serum) pre-treatment added different marine animal effective extracts and cell jointly to hatch the method that adopts above-mentioned PI4K enzyme biopsy to survey after 1-3 hour in original substratum in cell culture incubator and detects various different pharmaceuticals for the impact alive of the enzyme of PI4K after 12 hours.
Screen altogether in this experiment screening process and obtain the marine active substances that 10 kinds of rise PI4K enzymes are lived, lower the marine active substances (Fig. 8) that the PI4K enzyme is lived for 12 kinds.
Reference:
Carter?CA，Protein?kinase?C?as?a?drug?target：implications?for?drug?or?dietprevention?and?treatment?of?cancer.Curr?Drug?Targets，2000，1(2)：163-183.
Dai，R.M.，Chen，E.，Longo，D.L.，Gorbea，C.M.，and?Li，C.C.(1998).Involvement?of?valosin-containing?protein，an?ATPase?Co-purified?withIkappaBalpha?and?26?S?proteasome，in?ubiquitin-proteasome-mediateddegradation?of?IkappaBalpha.J.Biol.Chem.273，3562-3573.
Minogue，S.，Waugh，M.G.，De?Matteis，M.A.，Stephens，D.J.，Berditchevski，F.，and?Hsuan，J.J.(2006).Phosphatidylinositol?4-kinase?isrequired?for?endosomal?trafficking?and?degradation?of?the?EGF?receptor.J.CellSci.119.571-581.
Weber，G.，Shen，F.，Prajda，N.，Yeh，Y.A.，Yang，H.，Herenyiova，M.，andLook，K.Y.(1996).Increased?signal?transduction?activity?and?down-regulationin?human?cancer?cells.Anticancer?Res.16，3271-3282.
Yamamoto，S.，Tomita，Y.，Hoshida，Y.，Toyosawa，S.，Inohara，H.，Kishino，M.，Kogo，M.，Nakazawa，M.，Murakami，S.，Iizuka，N.，Kidogami，S.，Monden，M.，Kubo，T.，Ijuhin，N.，and?Aozasa，K.(2004b).Expression?level?ofvalosin-containing?protein(VCP)as?a?prognostic?marker?for?gingivalsquamous?cell?carcinoma.Ann.Oncol.15，1432-1438.
Sequence table
Claims (1)
1. medicine that can be used for treating cancer, it comprises and can suppress PI4KII α genetic expression or suppress the activeconstituents that PI4KII α enzyme is lived, and wherein said activeconstituents is the sense-rna fragment of PI4KII α gene, and its sequence is:
Positive-sense strand: UGAAGCAGAACCUCUUCCUCATT (SEQ ID NO:1)
Antisense strand: UCAGGAAGAGGUUCUGCUUCATT (SEQ ID NO:2).
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| Jun Guo等.Phosphatidylinositol 4-kinase type IIα is responsible for the phosphatidylinositol 4-kinase activity associated with synaptic vesicles.《PNAS》.2003,第100卷(第7期),3995－4000. * |
| Mark G. Waugh等.Lipid and Peptide Control of Phosphatidylinositol 4-Kinase IIα Activity on Golgi-endosomal Rafts.《THE JOURNAL OF BIOLOGICAL CHEMISTRY》.2006,第281卷(第7期),3757-3763. * |
| 李春香等.海洋药用动物海龙的研究.《氨基酸和生物资源》.2001,第23卷(第2期),6－9. * |
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