CN106727461A - A kind of application of KDM1A micromolecular inhibitors in growth of tumour cell and transfer is suppressed - Google Patents
A kind of application of KDM1A micromolecular inhibitors in growth of tumour cell and transfer is suppressed Download PDFInfo
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Abstract
本发明涉及一种KDM1A小分子抑制剂在制备抑制肿瘤细胞生长和转移药物中的应用，所述KDM1A小分子抑制剂可抑制细胞内组蛋白去甲基酶KDM1A的活性，并抑制组蛋白H3K4me2的去甲基化，从而导致TIMP3表达上调，促使细胞外MMP2表达的下调，同时抑制细胞内JNK激酶活性，有效地抑制了肿瘤细胞的侵袭和转移；所述KDM1A小分子抑制剂为2‑PCPA。与现有技术相比，本发明阐明了KDM1A小分子抑制剂2‑PCPA抑制肺癌细胞增殖、迁移和侵袭的分子机制，并在此基础上研究其治疗肺癌的效果，填补了用表观遗传药物治疗肺癌领域的空白，2‑PCPA可用于制备抑制肺癌细胞增殖和转移的小分子药物，从而提高肺癌的治疗效果，延长肺癌患者的生命。
The present invention relates to the application of a KDM1A small molecule inhibitor in the preparation of drugs for inhibiting tumor cell growth and metastasis. The KDM1A small molecule inhibitor can inhibit the activity of intracellular histone demethylase KDM1A and inhibit the expression of histone H3K4me2 Demethylation, thereby leading to the upregulation of TIMP3 expression, the downregulation of extracellular MMP2 expression, and the inhibition of intracellular JNK kinase activity, effectively inhibiting the invasion and metastasis of tumor cells; the KDM1A small molecule inhibitor is 2‑PCPA. Compared with the prior art, the present invention clarifies the molecular mechanism of the KDM1A small molecule inhibitor 2-PCPA inhibiting the proliferation, migration and invasion of lung cancer cells, and studies its effect on the treatment of lung cancer on this basis, filling the gap of using epigenetic drugs As a blank in the field of lung cancer treatment, 2‑PCPA can be used to prepare small molecule drugs that inhibit the proliferation and metastasis of lung cancer cells, thereby improving the therapeutic effect of lung cancer and prolonging the life of lung cancer patients.
Description
Technical field
Suppressing tumour cell life the present invention relates to therapeutic field of tumor, more particularly to a kind of KDM1A micromolecular inhibitors
Application in long and transfer, is particularly preparing the application in suppressing growth of tumour cell and diversion medicaments.
Background technology
Lung cancer is that in world wide, rate of rise is most fast, and patient numbers are also one of death rate highest cancer at most.
At present, the means of conventional lung cancer therapy mainly include：Surgery excision, radiation and chemotherapy, targeted drug treatment, and it is just emerging
Immune cell therapy for rising etc..Even so, because the early stage of lung cancer is difficult to be found, when the lung cancer of Most patients is found
Through being middle and advanced stage, cause the effect of lung cancer therapy extremely limited, patient is dead because of the recurrence and transfer of uncontrollable lung cancer.
Therefore, the molecular mechanism of proliferation of lung cancer cells, migration, and transfer is furtherd investigate, it will help find the medicine of new treatment lung cancer
Thing target spot.
Epigenetic is that modulate tumor cell is produced, one of important mechanisms of propagation and transfer.Epigenetic regulation is main
Including the following aspects：Histone modification, DNA methylation and demethylation, non-coding RNA etc..Wherein, many participation group eggs
The enzyme of white modification, including histone H 3 K4me2 demethyl enzyme KDM1A, histone H 3 K27me3 transmethylases EZH2 etc., swollen
Abnormal expression is raised in oncocyte.But its mechanism of action is not also fully aware of, and KDM1A micromolecular inhibitors are there is no at present in suppression
Application report in growth of tumour cell processed and transfer.
The content of the invention
It is an object of the invention to overcome defect of the prior art, there is provided prepared by a kind of KDM1A micromolecular inhibitors
Suppress the application in growth of tumour cell and diversion medicaments.
The present invention is adopted the following technical scheme that：
A kind of KDM1A micromolecular inhibitors are preparing the application in suppressing growth of tumour cell and diversion medicaments.
Preferably, the KDM1A micromolecular inhibitors are 2-PCPA.
Preferably, the tumour cell is cancer cell.
Preferably, the tumour cell is lung carcinoma cell.
Preferably, the PC9 and A549 in the tumour cell behaviour NSCLC cell lines.
Preferably, the cell administration amount of the micromolecular inhibitor 2-PCPA is 2 μm~20 μm.
2-PCPA, full name is Tranylcypromine hydrochloride, is irreversible monoamine oxidase
(monoamine oxidase, MAO) inhibitor.It can increase the activity of norepinephrine in serum
The transmission of (noradrenergic activity) and enhancing dopamine, for calm and antidepressants.Additionally, 2-PCPA may be used also
(it is also called with inhibition of histone demethyl enzyme KDM1A：LSD1, BHC110) activity, and inhibition of histone H3K4me2 goes first
Base (IC50=>50μM)
The present invention have studied effects of the histone H 3 K4me2 demethyl enzyme KDM1A in the growth of tumour cell and transfer
Mechanism.KDM1A is mainly and promotes tumour thin by suppressing a kind of expression of TIMP3 (TIMP)
The migration and invasion and attack of born of the same parents.Matrix metalloproteinase MMP2 in extracellular matrix sends out in the migration of tumour cell and invasive procedure
Important function has been waved, and TIMP3 can suppress the activity or protein level of MMP2, so as to suppress the migration and invasion and attack of tumour cell
Ability.
The present invention is investigated and uses KDM1A micromolecular inhibitors 2-PCPA, the activity of interior KDM1A capable of inhibiting cell, and
The demethylation of inhibition of histone H3K4me2, so as to cause TIMP3 up-regulateds, promotes the downward of extracellular MMP2 expression, together
When suppress intracellular JNK kinase activities, restrained effectively the invasion and attack and transfer of tumour cell.
Compared with prior art, the invention has the advantages that：
Clear illustration of the present invention micromolecular inhibitor 2-PCPA suppresses the molecule machine of tumor cell proliferation, migration and invasion and attack
System, especially suppresses the molecular mechanism of proliferation of lung cancer cells, migration and invasion and attack, and studies the effect of its treatment lung cancer on this basis
Really, the blank with epigenetic drug therapy lung cancer field has been filled up, micromolecular inhibitor 2-PCPA can be used to prepare suppression lung
Cancer cell multiplication and the small-molecule drug of transfer, so as to improve the therapeutic effect of lung cancer, extend the life of patients with lung cancer；
Brief description of the drawings
Fig. 1 is influence result figure of the KDM1A micromolecular inhibitors (2-PCPA) to proliferation of lung cancer cells ability；
Fig. 2 is influence result figure of the KDM1A micromolecular inhibitors (2-PCPA) to lung carcinoma cell invasion ability；
Fig. 3 is influence result figure of the KDM1A micromolecular inhibitors (2-PCPA) to lung carcinoma cell transfer ability；
Fig. 4 is influence result figure of the KDM1A micromolecular inhibitors (2-PCPA) to Increase Apoptosis of Lung Cancer Cells；
Fig. 5 is influence result figure of the KDM1A micromolecular inhibitors (2-PCPA) to internal proliferation of lung cancer cells ability；
Fig. 6 is influence result figure of the KDM1A micromolecular inhibitors (2-PCPA) to JNK signal paths in lung carcinoma cell；
Fig. 7 is shadow of the KDM1A micromolecular inhibitors (2-PCPA) to histone modification in lung carcinoma cell (H3K4me2) level
Ring result figure；
Fig. 8 is the influence result figure that overexpression TIMP3 is expressed JNK tyrosine phosphorylations and MMP2；
Fig. 9 is to cut down the influence result figure that TIMP3 is expressed JNK tyrosine phosphorylations and MMP2.
Specific embodiment
With reference to the accompanying drawings and examples, specific embodiment of the invention is further described.Following examples are only
For clearly illustrating technical scheme, and can not be limited the scope of the invention with this.Hereinafter implement
Method therefor in example, unless otherwise specified, is conventional method.
KDM1A micromolecular inhibitors of the present invention, can suppress propagation and the transfer of all kinds of tumour cells, including blood
Cancer, osteocarcinoma, lymph cancer, intestinal cancer, liver cancer, stomach cancer, lung cancer, kidney, cancer of the esophagus etc.；KDM1A micromolecular inhibitors include 2-PCPA,
Cyclopropyl amine derivatives etc..
Hereinafter KDM1A micromolecular inhibitors 2-PCPA is only illustrated on the propagation of lung carcinoma cell and transfer influence.
1. the culture of lung carcinoma cell, amplification and passage；
From PC9 and A549 in people's NSCLC cell lines, the growth in DMEM culture mediums (HyClone companies).Including
The people NSCLC cell lines of H1650, H292, H1975,95D and HCC827 are raw in RPMI-1640 culture mediums (Hyclone companies)
It is long.Contain penicillin (100U/ml) and streptomysin (100mg/ in the DMEM culture mediums and the RPMI-1640 culture mediums
Ml) (Life Technologies companies) and 10%FBS (Gibco companies).By above-mentioned cell at 37 DEG C, in 5%CO2Tide
Incubated in wet atmosphere.
2. the use of micromolecular inhibitor 2-PCPA；
Gefitinib (#S1025) is ordered from Selleck companies.Ordered from Enzo Life Science companies
Tranylcypromine (Parnate or 2-PCPA) (#EI-217).
PC9 and A549 cells are cultivated in 6 orifice plates, until cell density reaches 30-40%.Experiment test group is by cell
Density is processed 48 hours for the 2-PCPA of the cell various concentrations of 30-40%, and concentration includes 0 μm, 2 μm, 4 μm, 10 μm, 15 μ
m、20μm.Cell lysate is prepared using the cell after 2-PCPA treatment and for the analysis of Western blotting.Separately
Outward, the cell after being processed using 2-PCPA will also be used in cell propagation, invasion and attack and migration experiment.
3. influence of detection KDM1A micromolecular inhibitors (2-PCPA) to proliferation of lung cancer cells ability；
Cell after using 2-PCPA to process is inoculated into 96 orifice plates with the density of 1000 cells in every hole.Using different
Condition is processed, and 6 times are replicated after the treatment of each condition.In different time points, the explanation according to manufacturer is used
The mono- Solution Cell Proliferations of CellTiter96AQueous determine (MTS) (Promega company G3580) and carry out the propagation survey of MTS cells
It is fixed.Alternately, counted after cell number suitably dilution.
Influence results of the 2-PCPA to proliferation of lung cancer cells ability is as shown in figure 1,2-PCPA can effectively press down as shown in Figure 1
Proliferation of lung cancer cells processed.
4. influence of detection KDM1A micromolecular inhibitors (2-PCPA) to lung carcinoma cell invasion ability；
In 24 orifice plates (BD Biosciences companies, article No.：353504) each bottom in has the film of 8 micron pore sizes
(BD353097) in Transwell cells, 100 microlitres of dilutions are added (to carry out 1 with serum free medium：8 dilution)
Matrigel (BD Biosciences companies, article No.：356234).Using the cell that Trypsin Induced is adherent, and by cell
It is resuspended in the culture medium containing 1%FBS.By 5 × 104Individual cell is suspended in 100 microlitres of culture medium, and thin
Born of the same parents' suspension is layered on the cell on film.Cell portion under film adds 600 microlitres containing 20%FBS of culture medium.Cell is existed
37 DEG C, 5%CO2Under conditions of cultivate 48 hours, then, do not have in cell on the film with cotton swab each Transwell cell
The cell of intrusion is removed.And for being attached to the cell of the intrusion of film lower surface, in the methyl alcohol containing 0.1% crystal violet,
At 37 DEG C, dye 30 minutes, then washed twice with 1xPBS solution.The cell of dyeing is in microscope (multiplication factor：Under 100x)
Observed, and recorded the number of all migrating cells.Each experiment condition is in triplicate.
Influence results of the 2-PCPA to lung carcinoma cell invasion ability as shown in Fig. 2 as shown in Figure 2 compared with control group,
The invasion cell number of PC9 and A549 after use 2-PCPA treatment of the present invention is significantly less than the invasion cell of control group
Number, i.e. 2-PCPA can effectively suppress the invasion and attack of lung carcinoma cell.
5. influence of detection KDM1A micromolecular inhibitors (2-PCPA) to lung carcinoma cell transfer ability；
The cultured cells in 6 orifice plates, until the density of cell reaches 80%.Then, with a rifle for aseptic 10 microlitre
Head carries out the cut of horizontal direction.With phosphate buffered saline (PBS) (1xPBS) slightly washed cell removing cell fragment.Selection
Three different regions of each culture dish carry out similar cut, and compare the degree and distance of cell boundary migration.Respectively
0, cut was shot in 24,48, and 72 hours, to assess and calculate the degree of cell migration.Each experiment condition weight
It is multiple three times.
Influence results of the 2-PCPA to lung carcinoma cell transfer ability as shown in figure 3, as shown in Figure 3 compared with control group,
The cell migration degree of PC9 and A549 after use 2-PCPA treatment of the present invention is respectively less than the cell migration journey of control group
Degree, i.e. 2-PCPA can effectively suppress the migration of lung carcinoma cell.
6. influence of detection KDM1A micromolecular inhibitors (2-PCPA) to lung carcinoma cell clonality；
Cell is inoculated into 10 centimetres of culture dish with the density of each 1000 cell of culture dish, 12 are continuously cultivated
My god.Then, cell is fixed in ice-cold methanol solvate, and is dyeed with crystal violet solution.The bottom of culture dish is placed on
Face is taken pictures, and bacterium colony to can visually see is counted.Each experiment condition is in triplicate.
7. influence of detection KDM1A micromolecular inhibitors (2-PCPA) to Increase Apoptosis of Lung Cancer Cells；
Cell is washed twice in cold phosphate buffered saline (PBS) (1xPBS).Then, it is resuspended in 1 × combination buffer,
Every milliliter contains 1 × 106Cell, FITC by 100 microlitres of cell suspending liquid with 5 microlitres and is withered using FITC annexin Vs
Mixing in the PI (BD556547) that 5 microlitres of detection kit of dying, according to the explanation of manufacturer.Use Accuri C6 fluidic cells
The cell of instrument (BD Biosciences companies, the U.S.) dyeing is analyzed.
Influence results of the 2-PCPA to Increase Apoptosis of Lung Cancer Cells is as shown in figure 4,2-PCPA withers to lung carcinoma cell as shown in Figure 4
Die substantially without influence.
8. influence of detection KDM1A micromolecular inhibitors (2-PCPA) to the lung carcinoma cell cycle；
Cell is centrifuged, is washed twice with PBS, and be incubated overnight with 70% cold ethanol at 4 DEG C.Cell is according to manufacture
The explanation of business is mixed with PI-RNA enzyme dyeings buffer solution (BD Pharmingen companies, #550825).Use BD Accuri C6
The cell of fluidic cell dyeing is analyzed.The FlowJo7.6.1 Software on Drawing that result is used.
9. influence of detection KDM1A micromolecular inhibitors (2-PCPA) to internal proliferation of lung cancer cells ability；
In 100 microlitres of 1xPBS solution 2 × 10 will be suspended in6Individual lung carcinoma cell is subcutaneously injected into 5 weeks right sides of big nude mice
Side arm-pit areas.Then, tumor size was determined every 5 to 7 days, is measured using digital instrument, and tumor group is calculated based on this formula
The volume (V) knitted：V=0.5*A*B2, wherein A=major diameters and B=minor axis.After 5 weeks, the tumor tissues of heteroplastic transplantation are divided
From, take pictures, and storage is in liquid nitrogen.
2-PCPA is to the influence result of internal proliferation of lung cancer cells ability as shown in figure 5, as shown in Figure 5, control group swells
The Volume Changes and weight change amplitude of knurl are all higher than experimental group, from Fig. 5 it can also be seen that the volume of control group tumour is significantly greater than
Experimental group, i.e. 2-PCPA can preferably suppress the propagation of internal lung carcinoma cell.
10. influence of detection KDM1A micromolecular inhibitors (2-PCPA) to JNK signal paths in lung carcinoma cell；
Carry out expressions of the Western blotting detection 2-PCPA to phosphorylation JNK kinases in lung carcinoma cell.2-
PCPA is to the influence result of JNK signal paths in lung carcinoma cell as shown in fig. 6, it will be appreciated from fig. 6 that in 2-PCPA is capable of inhibiting cell
JNK kinase activities.
Influence of 11. detections KDM1A micromolecular inhibitors (2-PCPA) to MMP2 expression in lung carcinoma cell；
Carry out expressions of the Western blotting detection 2-PCPA to MMP2 in lung carcinoma cell.Can by testing result
Obtaining 2-PCPA can make the MMP2 of lung carcinoma cell China and foreign countries express downward.
Influence of 12. detections KDM1A micromolecular inhibitors (2-PCPA) to H3K4me2 levels in lung carcinoma cell；
Carry out influences of the Western blotting detections 2-PCPA to the expression of H3K4me2 in lung carcinoma cell.2-
PCPA is to the influence result of H3K4me2 levels in lung carcinoma cell as shown in fig. 7, during 2-PCPA can make lung carcinoma cell as shown in Figure 7
The expression reduction of KDM1A, the expression of TIMP3 is raised, and the level of histone modification H3K4me2 is raised.
The influence that 13. detection overexpression TIMP3 are expressed JNK tyrosine phosphorylations and MMP2；
Carry out Western blotting detection overexpression TIMP3 to JNK tyrosine phosphorylations in lung carcinoma cell and MMP2
The influence of expression.Its result is as shown in figure 8, overexpression TIMP3 can make JNK kinase phosphorylations in lung carcinoma cell as shown in Figure 8
Change and weaken, extracellular MMP2 expression is lowered.
The influence that 14. detection abatement TIMP3 are expressed JNK tyrosine phosphorylations and MMP2
Carry out tables of the Western blotting detections abatement TIMP3 to JNK tyrosine phosphorylations and MMP2 in lung carcinoma cell
Up to horizontal influence.Its result is as shown in figure 9, abatement TIMP3 can make JNK tyrosine phosphorylation liters in lung carcinoma cell as shown in Figure 9
Height, extracellular MMP2 expression enhancing.
From above-described embodiment, the micromolecular inhibitor 2-PCPA of KDM1A, the activity of interior KDM1A capable of inhibiting cell, and
The demethylation of inhibition of histone H3K4me2, so as to cause TIMP3 up-regulateds, promotes the downward of extracellular MMP2 expression, together
When suppress intracellular JNK kinase activities, restrained effectively the invasion and attack and transfer of lung carcinoma cell, filled up and used epigenetic medicine
The blank in treatment lung cancer field, while also for the treatment of other tumour cells provides foundation.
Specific embodiment of the invention has been described in detail above, but it is only used as example, and the present invention is not intended to limit
In particular embodiments described above.To those skilled in the art, any equivalent modifications carried out to the practicality and replace
In generation, is also all among scope of the invention.Therefore, the equalization made without departing from the spirit and scope of the invention is converted and repaiied
Change, all should be contained within the scope of the invention.
Claims (5)
1. a kind of KDM1A micromolecular inhibitors are preparing the application in suppressing growth of tumour cell and diversion medicaments.
2. application according to claim 1, it is characterised in that the KDM1A micromolecular inhibitors are 2-PCPA.
3. application according to claim 1, it is characterised in that the tumour cell is cancer cell.
4. application according to claim 1, it is characterised in that the tumour cell is lung carcinoma cell.
5. application according to claim 1, it is characterised in that PC9 in the tumour cell behaviour NSCLC cell lines and
A549。
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| WO2014194280A2 (en) * | 2013-05-30 | 2014-12-04 | The Board of Regents of the Nevada System of Higher Education on behalf of the University of | Novel suicidal lsd1 inhibitors targeting sox2-expressing cancer cells |
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2016
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| WO2014194280A2 (en) * | 2013-05-30 | 2014-12-04 | The Board of Regents of the Nevada System of Higher Education on behalf of the University of | Novel suicidal lsd1 inhibitors targeting sox2-expressing cancer cells |
Non-Patent Citations (2)
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| DANTE ROTILI等: "Pan-Histone Demethylase Inhibitors Simultaneously Targeting Jumonji C and Lysine-Specific Demethylases Display High Anticancer Activities", 《JOURNAL OF MEDICINAL CHEMISTRY》 * |
| POLLOCK等: "Lysine-specific histone demethylase 1 inhibitors control breast cancer proliferation in ERa-dependent and independent manners", 《ACS CHEM BIOL.》 * |
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| CN110218791A (en) * | 2019-05-15 | 2019-09-10 | 张鹏 | The purposes of SPZ1 and its purposes, pharmaceutical composition and the drug screening method of inhibitor |
| CN110218791B (en) * | 2019-05-15 | 2023-09-01 | 深圳市龙岗区耳鼻咽喉医院 | Use of SPZ1 and use of inhibitor thereof, pharmaceutical composition and pharmaceutical screening method |
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