CN108379268A - Nifeviroc application in preparation of anti-tumor drugs - Google Patents
Nifeviroc application in preparation of anti-tumor drugs Download PDFInfo
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- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
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Abstract
The present invention provides nifeviroc application in preparation of anti-tumor drugs, and provides a kind of antitumor drug including nifeviroc.It is proved by cell experiment, after nifeviroc acts on tumour cell, may interfere with cell cycle progression, inhibit cell growth, make cell cycle arrest in the G0/G1 phases；Can also be by promoting intracellular reactive oxygen content to increase, and cause the reduction of mitochondrial membrane potential, inducing cell apoptosis；At the same time, effect of the nifeviroc in inhibiting tumor cell invasion transfer is notable, by the adhesion strength for inhibiting tumour cell extracellular matrix, the directional migration ability and non-directional transfer ability and vitro invasion ability of reduction cell, in-vivo tumour volume can also be inhibited to increase, it is seen that nifeviroc (Nifeviroc) has good antitumor activity, can be used for preparing antitumor drug, and it without apparent side effect, has a good application prospect.
Description
Technical field
The present invention relates to the applications of nifeviroc, and in particular to nifeviroc application in preparation of anti-tumor drugs belongs to
In pharmaceutical technology field.
Background technology
Malignant tumour is to seriously threaten the great chronic disease of human health, is the public health of China or even global most serious
One of problem, tumour control the health strategy emphasis for having become countries in the world government.According to the World Health Organization (WHO) international cancer
The Global Raport of research administration (IARC) publication shows that China's tumor incidence and the death rate are in medium level, in the whole world 184
In a countries and regions, the 72nd and the 30th are occupied respectively, and lung cancer, liver cancer, breast cancer, gastric cancer and colon cancer are main normal
See malignant tumour.For morning, mid-term patient, operation is the therapeutic scheme of first choice.But it since tumour onset is hidden, examines in early days
Disconnected difficulty, lesion has belonged to Locally Advanced or DISTANT METASTASES IN has occurred when most of patients head is examined, for this kind of patient, clinically usually
Use the general treatment measures based on radiotherapy, chemotherapy.
Although chemotherapy drugs in combination physiotherapy has been made significant headway in the treatment of tumour, traditional chemicotherapy
It is a kind for the treatment of means for the purpose of killing, toxic side effect is big, and to the poor selectivity of tumour cell, multiple medicine easily occurs in patient
Drug resistance phenomenon (multidrug resistance, MDR), causes some patientss relatively low to the sensibility of chemicotherapy, final long-term
Survival rate is not improved significantly, and survival rate is only 15% within 5 years.At the same time, the recurrence and transfer of tumour are clinical treatments
In maximum problem, directly affect the prognosis of patient.How improving curative effect of medication, mitigating the adverse reaction of chemotherapy is tumour medicine
Research Emphasis, and improve patient clinical symptom, the important channel improved the quality of living.
Molecular targeted agents, which enter specifically to be combined with carcinogenic target spot in vivo, has an effect, and passes through the table of down-regulation protein
The activation of downstream gene is reached or inhibits, programmatically reversing tumor cell differentiation；Or indirectly by the new green blood of target tumor
Pipe, makes tumour cell ischemic and generates apoptosis, necrosis.Compared with classic chemotherapy drug, molecular targeted therapy is mainly for lesion
Cell has specificity antineoplastic effect, can reduce the damage to body normal structure, reduces toxic side effect.From FDA approvals
First monoclonal antibody (Rituximab) in city is used successfully to the targeted therapy of tumor patient, molecular targeted in the past 20 years to control
Treatment experienced fast development, at present there are many drug be used for clinical treatment, respectively tumor angiogenesis factor (VEGF) by
Body inhibitor, tyrosine kinase inhibitor, proteasome inhibitor, EGF-R ELISA (EGFR) inhibitor, cell week
Stage dependent kinases inhibitor, platelet derived growth factor (PDGF) acceptor inhibitor, Cycloxygenase (COX-2) inhibit
Agent etc..It is wherein the most commonly used with Tarceva (Erlotinib) and Gefitinib (Iressa).
Clinical studies show, above-mentioned targeted drug are better than traditional change in terms of alleviating symptom, improving patients ' life quality
Treatment scheme.But still there is the problems such as efficient low, patient clinical cardinal symptom improves unobvious, toxic reaction is more.Therefore,
Novel, efficient, hypotoxicity molecular targeted agents are developed to controlling tumor progression, the quality of life for improving patient has important meaning
Justice.
As g protein coupled receptor superfamily (GPCR) member, CCR5 early stages are considered as the source of autoimmune disease
Head, such as rheumatoid arthritis, multiple sclerosis.With deepening continuously to chemotactic factor (CF) and its receptor research, Ren Menfa
Disease problem --- the Immune Deficiency Syndrome (acquired that existing CCR5 is not captured with the mankind
Immunodeficiency syndrome, AIDS) it is closely related.Chemokine receptor CCR 5 is accredited as I types for the first time within 1996
The accessory receptor of human immunodeficiency virus (HIV-1), is mainly expressed in immature dendritic cell, natural killer cells, list
Nucleus and resting stage memory T-lymphocyte surface can assist inhibition of HIV to invade host cell, the weight as drug screening
Target spot is wanted to be widely used in the design and research and development of anti-Chinese mugwort drug.In recent years, more and more studies have shown that in addition to entering as HIV
Invade CD4+Outside the chemotaxis of the cooperative expert systems mediated immunity cell of T cell, CCR5 also participates in the occurrence and development process of tumour,
And it plays a significant role.Clinical studies show, CCR5 overexpressions in tumor patient, and its poor prognosis can be predicted.Cause
This, CCR5 is expected to become new cancer target and diagnosis, prognostic marker, and targeting blocking drugs are developed for CCR5, will
New approach is opened up for tumor patient treatment.
Invention content
For deficiencies of the prior art, the purpose of the present invention is to provide nifevirocs to prepare antineoplastic
Application in object solves the problems such as existing drug is ineffective to oncotherapy and toxic reaction is more.
To achieve the above object, the present invention adopts the following technical scheme that：Nifeviroc answering in the preparation of antitumor drugs
With the molecular formula of the nifeviroc is C33H42N4O6, structural formula is as follows,
Further, the targeted tumour of the antitumor drug is lung cancer, liver cancer, gastric cancer, colon cancer, cancer of the esophagus and mammary gland
It is one or more in cancer.
Further, the nifeviroc is the main active of antitumor drug.
Further, the dosage form of the antitumor drug include tablet, capsule, granule, injection, injectable powder,
Oral administration solution, oral administration mixed suspension, emulsion for injection, Orally taken emulsion, sustained-release tablet or Dospan.
Compared with prior art, the present invention has the advantages that：
1, nifeviroc (Nifeviroc) of the present invention has very strong affinity to CCR5, for high activity, Gao Xuan
The CCR5 of selecting property targets blocking drugs.Nifeviroc can block cell surface CCR5, inhibit signal path in tumour cell
Activation or conduction, restrained effectively growth and the proliferation of tumour cell, while also can inhibit the invasion and transfer of tumour cell.
2, after nifeviroc (Nifeviroc) of the invention effect tumour cell, cell cycle progression is may interfere with, cell is made
Cycle arrest is in the G0/G1 phases；Can also be by promoting intracellular reactive oxygen content to increase, and cause the drop of mitochondrial membrane potential
It is low, inducing cell apoptosis；At the same time, effect of the nifeviroc in inhibiting tumor cell invasion transfer is notable, passes through inhibition
The adhesion strength of tumour cell extracellular matrix reduces the directional migration ability and non-directional transfer ability and vitro invasion of cell
Ability, it is seen that nifeviroc has good antitumor activity, can be used for preparing antitumor drug, and without apparent secondary work
With having a good application prospect.
Description of the drawings
Fig. 1 is inhibited proliferation of the nifeviroc to different tumour cells of various concentration；
Fig. 2 is nifeviroc under different time to the inhibited proliferation of non-small cell lung cancer cell；
Fig. 3 is influence of the nifeviroc to different apoptosis of tumor cells rates；
Fig. 4 is influence of the nifeviroc of various concentration to non-small cell lung cancer cell apoptosis rate；
Fig. 5 is influence of the nifeviroc of various concentration to cell proliferation of NSCLC；
Fig. 6 is influence of the nifeviroc of various concentration to non-small cell lung cancer cell period profile；
Fig. 7 is that the nifeviroc of various concentration induces the situation of change of ROS in non-small cell lung cancer cell；
Fig. 8 is influence of the nifeviroc of various concentration to non-small cell lung cancer cell mitochondrial membrane potential；
Fig. 9 is influence of the nifeviroc of various concentration to non-small cell lung cancer cell adhesion strength；
Figure 10 is the influence that the nifeviroc of various concentration migrates non-small cell lung cancer cell；
Figure 11 is the influence that scarification detects that nifeviroc migrates non-small cell lung cancer cell；
Figure 12 is the influence that the nifeviroc of various concentration invades non-small cell lung cancer cell；
Figure 13 is the influence that nifeviroc changes knurl.
Specific implementation mode
With reference to specific embodiments and the drawings, invention is further described in detail.To experiment side in the following example
What method was not particularly illustrated, be routine operation, or operate according to the normal condition proposed by manufacturer.
Embodiment 1CCK-8 methods detect cell Proliferation
Taking the good cell of growth conditions respectively, (Non-small Cell Lung Cancer A 549, non-small cell lung cancer NCI-H520 are thin
Born of the same parents, human bronchial epithelial HBE cells, hepatoma Hep G 2 cells and MCF-7 Breast Cancer Cell), with 0.25% trypsin digestion,
Termination digests after microscopic observation cell retraction, 1000rpm centrifugation 5min, collection cell, after Trypan Blue, cell counter meter
Number cell, cell density is adjusted to 4 × 104A/ml is inoculated in 96 orifice plates, per 100 μ l (3 × 10 of hole3A cell), it sets
CO2It is cultivated in constant incubator.Wherein set control group, administration group (concentration of Nifeviroc is respectively 1.875,3.75,
7.5,15,30 μM), 5 multiple holes of every group of setting discard original fluid after cell is adherent, and administration group is separately added into same volume
The Nifeviroc of various concentration, is grouped intervention, and isometric culture medium is added in control group.Administration group and control group difference
It is returned to zero with the blank group of the CCK-8 solution of not inoculating cell.The pending time terminate after into hole be added CCK-8 solution, 37 DEG C
It is incubated 4h, measures the absorbance (OD values) in each hole at 450nm with all-wave length microplate reader, cell survival rate is calculated according to OD values,
Calculation formula is：Cell survival rate (%)=administration group OD values/control group OD value × 100%, observation different time, various concentration
Influences of the Nifeviroc to different cell Proliferations.
Make as shown in Figure 1, Nifeviroc all has inhibition to the proliferation of tumour cell A549, H520, HepG2 and MCF-7
With, and dose dependent is presented, i.e., in certain concentration range, with the increase of drug dose, tumour cell is killed
Wound effect gradually increases.After various concentration (0-30 μM) Nifeviroc acts on HBE cells (normal cell), except 30 μM of dosage have
Certain growth inhibition effect, inhibiting rate are 10.96 ± 1.54%, the cell viability of remaining concentration group equal nothing compared with the control group
Notable difference (p>0.05).
As shown in Fig. 2, various concentration (0-30 μM) Nifeviroc act on respectively A549 cells for 24 hours, after 48h, 72h, with
The increase of drug dose and the extension of action time, Nifeviroc constantly increase the inhibiting effect of A549 cell growths, in bright
Aobvious dose dependent and time dependence.When drug concentration increases to 30 μM from 1.875 μM, administration group cell proliferation rate for 24 hours
By under 95.63 ± 0.97% near 64.21 ± 1.50%, half-inhibition concentration (IC50) is 18 μM, 48h administration group cell Proliferations
Rate drops to 24.00 ± 2.65% by 96.36 ± 0.81%, and half-inhibition concentration (IC50) is 13.22 μM, and 72h administration groups are thin
Born of the same parents' proliferation rate drops to 4.0 ± 0.78% by 93.23 ± 0.9741%, and half-inhibition concentration (IC50) is 9.38 μM.
Embodiment 2Annexin V-FITC/PI double-stainings detect Apoptosis
By the good cell of growth conditions, (Non-small Cell Lung Cancer A 549, non-small cell lung cancer NCI-H520 are thin respectively
Born of the same parents, human bronchial epithelial HBE cells, hepatoma Hep G 2 cells and MCF-7 Breast Cancer Cell), with 0.25% trypsin digestion,
Single cell suspension is prepared, with 1 × 105The density in a/hole is seeded in 6 well culture plates, sets 37 DEG C, 5%CO2Incubator culture.It waits for
After cell is adherent, discard original fluid, be added same volume various concentration Nifeviroc (0,1.875,3.75,7.5,15,
30 μM), it is grouped intervention, every group sets 3 multiple holes.After the Nifeviroc effects 48h of various concentration, with the pancreas without EDTA
Enzyme distinguishes vitellophag, and cell is collected by centrifugation；The PBS buffer solution for being separately added into 4 DEG C of precoolings washs cell 3 times, 1000rpm centrifugations
5min abandons supernatant.Cell is resuspended with Binding Buffer (1 ×), 5 μ l Annexin V- are added to each group cell respectively
FITC and 5 μ l PI propidium iodide stain liquid, gently mixing, room temperature are protected from light incubation 15min.It is withered with flow cytomery cell
Rate is died, data are analyzed using FlowJo softwares.
As shown in figure 3, Nifeviroc all has apparent rush apoptosis to tumour cell A549, H520, HepG2 and MCF-7
Effect.Comparing the apoptosis rate of each group cell can find, Nifeviroc is in dose dependent to the apoptosis-promoting effect of tumour cell,
I.e. with the increase of Nifeviroc concentration, apoptosis rate gradually increases.A concentration of 15 μM of Nifeviroc and tumour cell
After acting on 48h, live cell fraction drops to 40.10 ± 1.44% by 99.51 ± 0.41%, early apoptosis of cells rate (early
Apoptotic cells) by 0.14 ± 0.19% 18.77 ± 3.28% are increased to, cell late apoptic/necrosis rate (late
Apoptotic cells/necrotic cells) by 0.22 ± 0.24% increase to 34.32 ± 3.02%.
As shown in figure 4, after Nifeviroc and A549 the cytosis 48h of various concentration, each concentration group (1.875,3.75,
7.5,15,30 μM) the total apoptosis rate of cell be respectively 5.36 ± 0.39%, 6.16 ± 0.19%, 13.41 ± 0.73%, 39.60
± 2.91% and 66.71 ± 3.48%, wherein the apoptosis rate of a concentration of 15 μM and 30 μM dosage groups is apparently higher than blank pair
According to group (* p<0.05), compared with remaining dosage group, there is significant difference (* p<0.05), and with the raising of drug concentration,
Apoptosis is gradually changed into late apoptic by early apoptosis.
Nifeviroc is can be seen that Non-small Cell Lung Cancer A 549, non-small cell lung cancer NCI- from embodiment 1-2
The kinds of tumor cells such as H520 cells, hepatoma Hep G 2 cells, MCF-7 Breast Cancer Cell all have inhibiting effect, following embodiments
Verification experimental verification is carried out by taking representative Non-small Cell Lung Cancer A 549 as an example.
3 cell clonal formation of embodiment is tested
By 0.25% trypsin digestion of the good Non-small Cell Lung Cancer A 549 of growth conditions, prepare unicellular outstanding
Liquid is seeded in the density in 100/hole in 24 well culture plates, sets 37 DEG C, 5%CO2Incubator culture.After cell is adherent, abandon
Original fluid is removed, the Nifeviroc (0,1.875,3.75,7.5,15,30 μM) of same volume various concentration is added, is grouped
Intervene, every group sets 3 multiple holes.After the Nifeviroc effects 48h of various concentration, changes fresh culture and continue culture 2-3 weeks, often
72h is changed the liquid once；When occurring macroscopic clone in culture plate, culture is terminated, and discard cell culture fluid, it is small with PBS
The heart embathes 2 times, fixes 15min at room temperature；Fixer is removed, violet staining 30min is added；Dye liquor is slowly washed away with flowing water, in
It is dry in air at room temperature, it takes pictures under mirror and counts cell colony, calculate cloning efficiency.As shown in Figure 5.
Compared with CCK-8 is tested, cell clonal formation experiment can more embody drug to the long term of tumour growth, help
In the influence for understanding pharmaceutical intervention and restoring to cell proliferative capacity, often this recovery capability may be multiple after treating malignant tumor
The power of hair.From fig. 5, it can be seen that after Nifeviroc is acted on, A549 Cell colonies assays are declined, and with
Drug dose increases, and Cell colonies assay continuously decreases, and the difference between each group has conspicuousness.
4 cell cycle of embodiment is detected
By 0.25% trypsin digestion of the good Non-small Cell Lung Cancer A 549 of growth conditions, prepare unicellular outstanding
Liquid, with 1 × 105The density in a/hole is seeded in 6 well culture plates, sets 37 DEG C, 5%CO2Incubator culture.After cell is adherent,
Original fluid is discarded, the Nifeviroc (0,1.875,3.75,7.5,15,30 μM) of same volume various concentration is added, is divided
Group is intervened, and every group sets 3 multiple holes.After the Nifeviroc effects 48h of various concentration, trypsin digestion is used respectively, is collected by centrifugation
The PBS washing cells of 4 DEG C of precoolings are used in combination in cell.Take 300 μ l PBS that each group cells are resuspended, be respectively added slowly to 700 μ l, -20 DEG C
The absolute ethyl alcohol of precooling is set 4 DEG C of refrigerators and is fixed overnight.3000rpm centrifuges 5min, discards ethyl alcohol fixer, clear with the PBS of precooling
It washes primary.100 μ l RNase A are added in 37 DEG C of water-bath 30min, are eventually adding 400 μ l PI dyeing liquors, 4 DEG C or are protected from light on ice
It is incubated 30min.With flow cytomery, red fluorescence at excitation wavelength 488nm is recorded, Flowjo softwares is used in combination to analyze cell
Period.
The results are shown in Figure 6, after A549 cells act on 48h with various concentration Nifeviroc, G0/G1 phases cell institute accounting
Example increases in concentration dependent, respectively 52.15 ± 2.32%, 55.47 ± 1.49%, 60.69 ± 2.00%, 72.90 ±
1.05%, 77.73 ± 1.32%, each group corresponding S phases and G2/M phase cell proportions significantly reduce.Compared with the control group, as a result
With significant difference (* p<0.05).This result shows that, Nifeviroc by by A549 cell-cycle arrests in the G0/G1 phases,
And then effectively inhibit cell Proliferation.
The measurement of 5 intracellular ROS level of embodiment
By 0.25% trypsin digestion of the good Non-small Cell Lung Cancer A 549 of growth conditions, prepare unicellular outstanding
Liquid, with 1 × 105The density in a/hole is seeded in 6 well culture plates, is placed in 37 DEG C, 5%CO2Incubator culture.Wait for that cell is adherent
Afterwards, original fluid is discarded, the Nifeviroc (0,1.875,3.75,7.5,15,30 μM) of same volume various concentration is added, into
Row grouping is intervened, and every group sets 3 multiple holes.After the Nifeviroc effects 48h of various concentration, is digested and collected thin respectively with pancreatin
Born of the same parents, the PBS being pre-chilled with 4 DEG C wash cell, and 1000rpm centrifuges 5min, abandons supernatant.It is (living that each group cell is suspended in DCFH-DA
Property oxygen ROS fluorescence probes, final concentration：10 μM) in working solution, set 37 DEG C of incubation 20min in cell incubator.Per 5min by cell
Mixing is primary, to ensure that probe comes into full contact with cell.Cell is washed with serum free medium 3 times, and stream is used after collecting cell
Formula cell instrument detects the variation of each group intracellular ROS level, and data are analyzed using FlowJo softwares.
As shown in fig. 7, after (1.875,3.75,7.5,15,30 μM) interventions of various concentration Nifeviroc, A549 cells
Interior ROS levels increase 15%, 26.7%, 65.9%, 171.1% and 211.9% respectively, have certain dose-response relationship.
Wherein Nifeviroc is 15 μM a concentration of, 30 μM of processing group ROS contents dramatically increase, intracellular DCF fluorescence intensity levels difference
It is 2.71,3.12 times of control group.Thus illustrate, Nifeviroc can damage cells interior hydrogen reduction system, and oxygen occurs for inducing cell
Change stress, this acts on during inducing apoptosis of tumour cell and plays a significant role.
6 mitochondrial membrane potential of embodiment detects
By 0.25% trypsin digestion of the good Non-small Cell Lung Cancer A 549 of growth conditions, prepare unicellular outstanding
Liquid, with 1 × 105The density in a/hole is seeded in 6 well culture plates, sets 37 DEG C, 5%CO2Incubator culture.After cell is adherent,
Original fluid is discarded, the Nifeviroc (0,1.875,3.75,7.5,15,30 μM) of same volume various concentration is added, is divided
Group is intervened, and every group sets 3 multiple holes.After the Nifeviroc effects 48h of various concentration, collected by trypsinisation cell is used respectively, is used in combination
PBS washs cell.Each group cell is resuspended in 0.5ml culture solutions (containing serum and phenol red), 0.5ml JC-1 dyes are separately added into
Color working solution, mixes well, and is placed in 37 DEG C of incubation 20min in cell incubator.It is washed with the JC-1 dye solutions (1 ×) of precooling
Wash cell 3 times, after cell is resuspended, with the variation of flow cytomery each group mitochondrial membrane potential in anoxic, data use
FlowJo softwares are analyzed.
It is the mark of mitochondrial damages that mitochondrial membrane permeability, which increases, and mitochondrial membrane potential (Δ Ψ m) reduces.Just
Under normal physiological conditions, there is higher potential difference inside and outside mitochondrial membrane.As ROS and Mitochondria permeability transition complexity albumen phase
In conjunction with, make mitochondrial membrane hole path open.Mitochondrial membrane is impaired so that the potential difference inside and outside film reduces, i.e. Δ Ψ m are reduced, to
Start the cascade reaction of apoptosis, and eventually leads to cell death.
As shown in figure 8, after various concentration Nifeviroc effect A549 cells 48h, with the raising of Nifeviroc concentration,
It observes in cell that red fluorescence gradually weakens and green fluorescence increases with drug concentration and gradually increased, shows cell mitochondrial
The JC-1 of middle polymer form is constantly being reduced, and the JC-1 of monomeric form is gradually increasing, and the Δ Ψ m of cell are with Nifeviroc
Concentration increases and is remarkably decreased.When drug concentration is 7.5 μM, the cell proportion that Δ Ψ m are reduced is (18.47 ± 1.95) %,
With the increase of drug concentration, the Δ Ψ m that A549 cells lose increase, when drug concentration is 30 μM, the cell of Δ Ψ m reductions
Ratio reaches (51.17 ± 3.26) %, illustrates that Nifeviroc can be such that A549 mitochondrial membrane potential in anoxic degree of depolarization increases,
With certain dose-dependent effect.
7 Cell-matrix adhesion of embodiment is tested
With serum free medium by FN (1g/L) diluting stock solutions to required concentration, the culture of 24 holes is added with 200 holes μ l/
Plate, 4 DEG C are coated with basilar memebrane overnight, and BSA is control substrate.Next day, which inhales, abandons liquid in culture plate, PBS rinses 3 times, ultraviolet light irradiation
Sterilizing.In order to reduce non-specific binding, the serum free medium that 200 μ l contain BSA (10g/L) is added per hole, 37 DEG C are incubated 1h,
To prepare aquation basilar memebrane.By various concentration Nifeviroc (0,25,50,100,200nM) effect non-small cell lung cancer for 24 hours
A549 cells, with trypsin digestion, 1000rpm centrifuges 5min and collects cell.With 1 × 105A/hole be inoculated in coating FN or
In 24 porocyte culture plates of BSA, every group sets 3 parallel holes.37 DEG C, saturated humidity, 5%CO2It is cultivated in incubator.It is taken after 1h
Go out tissue culture plate, cleaned 3 times with PBS, to remove nonadherent cell.Add MTT solution (5mg/ml) 200 μ l per hole, 37 DEG C
It is incubated 4h.Careful inhale abandons culture supernatant, and 200 μ lDMSO are added per hole, and shaken at room temperature 10min makes crystal fully dissolve.Make
Absorbance of each hole at 490nm is measured with microplate reader, is with reference to calculating adhesion rate with control substrate group attached cell.
During invasion and metastasis of tumor, adherency is considered as initiating step.Tumour cell is by reducing homogenous cell
Between adhesion strength, and increase with the adhesion strength of extracellular matrix, obtain migration, invasive ability.Therefore, it reduces between cell and matrix
Adhesion strength be bound to that the invasion transfer ability of tumour cell can be inhibited.As shown in figure 9, be respectively 25 through concentration, 50,100,
After the Nifeviroc effects for 24 hours of 200nM, the adhesion rate of A549 cells on extracellular matrix FN be respectively (94.56 ±
2.96) %, (75.25 ± 2.37) %, (51.29 ± 1.46) %, (43.33 ± 2.64) % are significantly lower than control group
(100%).
Embodiment 8Transwell migration experiments
The Non-small Cell Lung Cancer A 549 of logarithmic growth phase is based on 37 DEG C, 5%CO with free serum culture2Incubator
Middle hungry culture 12h.With 0.25% trypsin digestion and cell, 1000rpm centrifuges 5min and collects cell.It is thin that PBS washings are pre-chilled
Born of the same parents 2 times are resuspended cell with serum free medium, prepare single cell suspension.Trypan Blue simultaneously counts cell, adjusts cell density
To 1 × 105A/ml.The cell compartments that aperture is 8 μm are positioned in 24 orifice plates.200 μ l cell suspensions (2 × 10 of accurate measuring4
A cell) it is inoculated in interior on transwell.Be separately added into lower room 800 μ l containing various concentration Nifeviroc (0,25,
50,100,200nM) culture medium (containing 10%FBS), 3 parallel control holes of every group of setting.37 DEG C, saturated humidity, 5%CO2Training
It supports and is cultivated in case.It takes out cell afterwards for 24 hours, wipes the cell in upper chamber away with swab stick.1% paraformaldehyde fixes 30min.PBS is buffered
Liquid rinse 3 times, each 5min.At room temperature, 1% violet staining 10min.Distilled water cleans 3 times, each 5min.It is impregnated in PBS
5min returns indigo plant, takes pictures under the microscope and calculate relative mobility.
As shown in Figure 10, through concentration be respectively 25,50,100, after the Nifeviroc effects for 24 hours of 200nM, A549 cells
Transfer ability is substantially reduced, and the relative mobility of cell is respectively：(66.51 ± 2.32) %, (53.05 ± 4.15) %,
(38.46 ± 3.60) %, (39.24 ± 3.01) %.Except 25nM dosage groups, remaining 3 drug-treated group (50nM, 100nM,
200nM) compared with blank control group, significant difference (* p<0.05).Wherein 100nM, 200nM dosage group inhibit migration
Effect is the most apparent.The trend that gradient reduces is presented with the increase of drug dose for the transfer ability of administration group cell.
9 cell scratch experiment of embodiment
The good Non-small Cell Lung Cancer A 549 of growth conditions is taken, with trypsin digestion cell, 1000rpm centrifuges 5min and receives
Collect cell, using Trypan Blue and cell is counted, with 1 × 106A/hole is inoculated in 6 porocyte culture plates, routine culture.
Next day waits for that cell confluency reaches 100%, and cut is quickly gently done in culture plate bottom with 10 μ l pipette tips.It discards in hole and cultivates
Liquid is cleaned 3 times with sterile PBS, with removal floating cell fragment, be added Nifeviroc containing various concentration (0,25,50,100,
Serum free medium 200nM).Each concentration is all provided with 3 multiple holes, 37 DEG C, 5%CO2It is cultivated in incubator, respectively at 0h, for 24 hours
Observe and take pictures under inverted microscope, using Image J softwares calculate iuntercellular away from.
As shown in figure 11, healing rate of the cellular control unit in cut both sides is significantly stronger than drug-treated group.Experimental group is thin
The trend that gradient reduces is presented with the increase of drug concentration for the transfer ability of born of the same parents.It can be seen that Nifeviroc is to A549 cells
Directional migration ability and non-directional transfer ability all have significant inhibiting effect.
Embodiment 10Transwell matrigel invasions are tested
The Matrigel matrigels dispensed in advance is ice bath melted under aseptic condition, dilute 2 times with serum free medium.
The cells transwell that aperture is 8 μm are positioned in 24 orifice plates, 50 μ l Matrigel glue is gently drawn respectively and is added often
A small interior makes it be laid in the upper chamber face of cell basilar memebrane.24 orifice plates are placed in 37 DEG C of constant incubators, 2h is stood, makes
Matrigel solidifies plastic.After matrigel solidification, small indoor residual liquid is sucked.It is added 50 μ l serum free mediums, 37
DEG C be incubated 30min.It will be by the Non-small Cell Lung Cancer A 549 of Nature enemy, with 0.25% trypsin digestion, with no blood
Clear culture medium prepares single cell suspension, counts cell and adjusts cell density to 2 × 105A/ml, 200 μ l cells of accurate measuring
Suspension (4 × 104A cell) it is inoculated in coated small interior.800 μ l are separately added into lower room containing various concentration
Nifeviroc (0,25,50,100,200nM) RPMI-1640 culture mediums (contain 10%FBS), 3 parallel controls of every group of setting
Hole, 37 DEG C, saturated humidity, 5%CO2It is cultivated in incubator.The cells traswell are taken out after for 24 hours from culture plate, it is small with swab stick
The heart wipes the cell in upper chamber away.1% paraformaldehyde fixes 30min.PBS buffer solution rinse 3 times, each 5min.At room temperature, 1%
Violet staining 10min.Distilled water cleans 3 times, each 5min.5min is impregnated in PBS, returns indigo plant.It under the microscope and takes pictures, calculates
Opposite invasion rate.
As shown in figure 12, after various concentration Nifeviroc effects for 24 hours, the invasive ability of A549 cells is declined,
With the raising of drug dose, inhibiting effect is further apparent.
The influence that 11 nifeviroc of embodiment changes knurl
By 0.25% trypsin digestion of the good Non-small Cell Lung Cancer A 549 of growth conditions, prepare unicellular outstanding
Liquid.The little NOD/SCID mouse of 5-7 week old weight differences are chosen, every mouse counts 1.5 × 10 respectively7A A549 cells armpit
Lower inoculation tumor formation.Mouse tumor formation situation is observed, and measures its longest diameter (L) and shortest diameter (S), waits for gross tumor volume (V=L × S2/
2) reach 100mm3Afterwards, it is randomly divided into control group and administration group, every group parallel equipped with 10, according to dosage (gives by daily single
Medicine group dose is the physiological saline of 10mg/kg Nifeviroc, and control group is the physiological saline of same volume).Every 3 days
Tumor volume situation of change is measured and compared, after 3 weeks, puts to death mouse, tumor resection tissue calculates tumour inhibiting rate.Such as Figure 13 institutes
Show.
As can be seen from Figure 13, administration group gross tumor volume is obviously reduced than control group, shows that nifeviroc can significantly press down
The growth of tumour processed and volume increase, and have notable curative effect to tumour.It is administered simultaneously the NOD/SCID mouse of group and control group
Weight has no notable difference, shows nifeviroc non-evident effect.
Finally illustrate, the above examples are only used to illustrate the technical scheme of the present invention and are not limiting, although with reference to compared with
Good embodiment describes the invention in detail, it will be understood by those of ordinary skill in the art that, it can be to the skill of the present invention
Art scheme is modified or replaced equivalently, and without departing from the objective and range of technical solution of the present invention, should all be covered at this
In the right of invention.
Claims (4)
1. the molecular formula of nifeviroc application in preparation of anti-tumor drugs, the nifeviroc is C33H42N4O6, structural formula
As follows：
2. nifeviroc application in preparation of anti-tumor drugs according to claim 1, which is characterized in that described antitumor
The targeted tumour of drug is one or more in lung cancer, liver cancer, gastric cancer, cancer of the esophagus, colon cancer and breast cancer.
3. nifeviroc application in preparation of anti-tumor drugs according to claim 1, which is characterized in that the Ni Feiwei
Sieve is the main active of antitumor drug.
4. nifeviroc application in preparation of anti-tumor drugs according to claim 1, which is characterized in that described antitumor
The dosage form of drug includes tablet, capsule, granule, injection, injectable powder, oral administration solution, oral administration mixed suspension, injection
Emulsion, Orally taken emulsion, sustained-release tablet or Dospan.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110882236A (en) * | 2019-11-26 | 2020-03-17 | 上海市同仁医院 | Anti-tumor composition, pharmaceutical preparation for treating cancer and application of pharmaceutical preparation |
| CN113122497A (en) * | 2021-04-26 | 2021-07-16 | 重庆理工大学 | Engineered mitochondria and methods of making the same |
-
2018
- 2018-05-24 CN CN201810509561.2A patent/CN108379268A/en active Pending
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| 周玉丽等: "趋化因子CCL5及其受体CCR5在乳腺癌中作用的研究进展", 《肿瘤》 * |
| 欧周罗: "趋化因子结合蛋白在肿瘤中的作用及临床意义", 《延安大学学报》 * |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110882236A (en) * | 2019-11-26 | 2020-03-17 | 上海市同仁医院 | Anti-tumor composition, pharmaceutical preparation for treating cancer and application of pharmaceutical preparation |
| CN110882236B (en) * | 2019-11-26 | 2022-12-02 | 上海市同仁医院 | Anti-tumor composition, pharmaceutical preparation for treating cancer and application of pharmaceutical preparation |
| CN113122497A (en) * | 2021-04-26 | 2021-07-16 | 重庆理工大学 | Engineered mitochondria and methods of making the same |
| CN113122497B (en) * | 2021-04-26 | 2023-08-11 | 重庆理工大学 | Engineered mitochondria and method of making same |
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Application publication date: 20180810 |