CN107737124A - A kind of application of curcumin analogue in antineoplastic is prepared - Google Patents
A kind of application of curcumin analogue in antineoplastic is prepared Download PDFInfo
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- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
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- C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
- C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
- C07D213/60—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
- C07D213/62—Oxygen or sulfur atoms
- C07D213/63—One oxygen atom
- C07D213/68—One oxygen atom attached in position 4
Abstract
A kind of application of curcumin analogue in antineoplastic is prepared, it is characterized in that, the structure of described curcumin analogue is shown below, and described antineoplastic is used for selective killing tumour cell to normal cytotoxic, can be used as a kind of potential antineoplastic.
Description
Technical field
The invention belongs to pharmaceutical technology field, and in particular to two kinds of compounds CAA and CAI answering in antineoplastic is prepared
With.
Background technology
Tumour is one of the main reason for causing human death, and its incidence of disease and fatal rate totally become in what is risen every year
Gesture.Statistics display, from 2010, malignant tumour oneself turn into the dead primary cause of the death of urban and rural residents.The prevention of visual tumors and control
Treat very urgent.Drug therapy is one of essential therapeutic arsenals of tumour.At present, although have developed numerous antineoplastics,
Effectively extend the life of patient or improve the life quality of patient.But the drug research and development of tumour is also faced with huge
Big challenge, if antineoplastic is mostly cell toxicity medicament, its side effect is obvious, limits the clinical practice of these medicines.Nearly two
Over 10 years, the targeted therapies of tumour have obtained the target tumor signal egg such as development at full speed, Imatinib, Herceptin
White medicine shows the therapeutic effect of great prospect and relatively low toxic side effect in clinical studies.It is however, acquired resistance to
The appearance of medicine and the polytropy of Oncogenome so that targeted therapies are equally faced with huge challenge.
Increasing result of study proves the exclusive biochemical characteristic change of target tumor, is expected to overcome and solves acquired
This intractable problem of resistance.There are some researches show reactive oxygen species (the Reactive oxygen in malignant cell
Species, ROS) it is horizontal considerably beyond normal cell, so as to cause tumour cell over oxidation stress phenomenon.Tumour
ROS increase may be to tumour in cell generation, development important role, appropriate ROS contribute to the increasing of tumour cell
Grow and break up.However, excessive ROS can killing tumor cell.Tumour cell be chronically at abnormal oxidation stress under conditions of, production
The tolerance to endogenous ROS is given birth to, but exogenous ROS attack becomes also more sensitive than normal cell on the contrary.Thus,
The change of environment, causes the difference of tumour cell and normal cell in the reduction of tumour cell internal oxidition, to promote the swollen of ROS generations
The exploitation of oncocyte selective killing medicine provides foundation.
The present inventor is devoted for years to study and its resist in the structure of modification of curcumin analogue (Curcumin Analogs)
Tumor promotion is screened, and two kinds of specific molecule curcumin analogue A (Curcumin have been filtered out from hundreds of compounds
Analogs A, CAA) and curcumin analogue I (Curcumin Analogs I, CAI), it is for some specific cancer tools
There is very excellent drug effect.In addition, we expand research for its pharmacological mechanism, its suppression for tumour cell is found
Make to use and be to rely on the caused of ROS.Also, in order to improve the antitumor action of compound, we further study
A kind of influence of CAA, CAI the combination piperlongumine (Piperlongumine, PL, ROS derivants) to its antitumor activity.As a result
Demonstrate CAA and CAI has preferable selecting cell toxic action for tumour cell, and to normal cell without the secondary work of notable poison
With.And being combined PL has antitumor sensitization to compound.
The content of the invention
Present invention aims at provide a kind of synthesis and application of the antitumor curcumin analogue of selectivity, the antitumor ginger
Flavine analog has good inhibiting effect to tumour cell, meanwhile, to normal cell without notable toxic side effect.
A kind of application of curcumin analogue in antineoplastic is prepared, the structure of described curcumin analogue are as follows
Shown in formula：
Described antineoplastic is for selective killing tumour cell and to normal cytotoxic, specific synthetic method
See embodiment 1 and embodiment 2.
Preferably, described curcumin analogue activates oxidative stress and apoptosis to select by improving cell ROS levels
Selecting property killing tumor cell.
Preferably, described curcumin analogue is selected by activating caspase-3, caspase-8 and caspase-9
Selecting property killing tumor cell.
Preferably, described tumour is lymthoma, breast cancer or/and colon cancer.
It is preferred that the not all tumour of tumour of the present invention, but specific tumour, it can such as be induced by specific mechanism
The tumour of apoptosis.As described in Example 3, preferably described tumour cell is the cell of compound CAA and CAI energy Inhibit proliferaton, is wrapped
Include human tissue cell's lymthoma U937 cells, (T lymphocytic leukemia cells E6-1, human breast carcinoma are thin for human blood tumour cell
Born of the same parents (triple negative breast cancer cell MDA-MB-231, MDA-MB-468), people's inflammatory breast cancer SUM149 cells and human colon carcinoma are thin
Born of the same parents (HCT-116).
Mechanism Study result shows (embodiment 4-6) that CAA and CAI external evoked apoptosis of tumor cells effect are to pass through
Adjusting mitochondrial oxidation stress be caused by signal path.CAA and CAI can produce oxidative stress with inducing mitochondrial, so as to swash
Caspase-3/8/9 living, and notable inducing apoptosis of tumour cell.It is important that CAA and CAI does not have to normal non-tumor cell
Significant toxicity, the i.e. compound have the function that selective killing tumour.Preferably, described normal cell is being selected from people just
Normal fibroblast NHF cells, human peripheral blood monocyte PBMCs cells and people's normal colon mucosa cell NCM 460 are thin
Born of the same parents.
In addition, being in vitro combined compound CAA, CAI with various clinical antineoplastic respectively, synergistic antitumor can be played
Drug effect.According to embodiment 7, combination CAA and piperlongumine (Piperlongumine, PL), can be remarkably reinforced in low concentration
The apoptosis such as U-937, E6-1, SUM149 and MDA-MB-231；And in the case of drug combination, to normal cell
The cell of NHF, PBMCs, NCM 460 will not cause toxicity.Preferably, described antineoplastic include curcumin analogue and
The combination of piperlongumine.
In addition, after combination CAA, CAI, it is effectively improved FOLFOX therapies and colon carcinoma cell line HCT116 maximum is treated
Imitate (embodiment 7).Preferably, described antineoplastic is when treating colon cancer, described curcumin analogue conduct
FOLFOX therapies sensitizer uses.
Preferably, described curcumin analogue is CAA, for suppressing the growth of nude mouse tumor cell transplantation knurl.
Preferably, the tumour cell is selected from chronic granulocytic leukemia MV-4-11 cells, people's triple negative breast cancer
MDA-MB-231 cells.
Preferably, described antineoplastic is used to suppress tumor cell proliferation.
Preferably, the tumour cell is selected from human tissue cell's lymthoma U937 cells, people's triple negative breast cancer MDA-
MB-231 cells and human colon carcinoma HCT116 cells.
The compound CAA being related in the present invention can exist in the form of antineoplastic pharmaceutical compositions, and the composition contains
There are the compound CAA and PL of therapeutically effective amount, and pharmaceutically acceptable carrier.
Pharmaceutically acceptable carrier described above refers to the conventional pharmaceutical carrier of pharmaceutical field, such as：Diluent, figuration
Agent such as water etc., filler such as starch, sucrose etc.；Adhesive such as cellulose derivative, alginates, gelatin and polyvinylpyrrolidine
Ketone；Wetting agent such as glycerine；Disintegrant such as agar, calcium carbonate and sodium acid carbonate；Sorbefacient such as quaternary ammonium compound；Surface-active
Agent such as hexadecanol；Absorption carrier such as kaolin and soap clay；Lubricant such as talcum powder, calcium stearate/magnesium, polyethylene glycol etc..
It can in addition contain add other assistant agents such as flavouring agent, sweetener etc. in the composition.
The present invention can be in the form of compositions by oral, and the mode of nasal inhalation, rectum or parenteral is applied to
Need the patient of this treatment.For it is oral when, conventional solid pharmaceutical preparation such as tablet, pulvis, granula, capsule can be made into
Deng liquid preparation such as water or oil-suspending agent or other liquid preparations such as syrup, elixir etc. is made；, can during for parenteral
It is made into solution, water or oleaginous suspension of injection etc..
The various formulations of pharmaceutical composition of the present invention can be prepared according to the conventional production process of pharmaceutical field.Such as make work
Property composition mixes with one or more carriers, is then made into required formulation.
In vivo in activity, CAA has extremely strong inhibitory action to specific tumors.In the specific embodiment party of the present invention
In formula, the cell in the tumour is to be produced by activating the Caspase signal paths in tumor tissues and induced tumor cell
(embodiment 8) of raw apoptosis.
Brief description of the drawings
Fig. 1 is CAA and CAI chemical synthesis schematic diagram.
Fig. 2 is the propagation of CAA and CAI dose-dependent inhibition Several Kinds of Malignancy.WST-1 methods detect medicine to a variety of swollen
The influence of tumor cell growth.The tumour cell of detection includes：It is M4 myelomonocytic leukemia cell MV-4-11 (A), non-small thin
It is born of the same parents' lung carcinoma cell NCI-H23 (B), human osteosarcoma cell Saos-2 (C), T lymphocytic leukemia cells E6-1 (D), non-small thin
Born of the same parents' lung cell A549 (E), human osteosarcoma cell MG-63 (F), human tissue cell lymphoma cell U-937 (G), human colon carcinoma
Cell HCT-116 (H), triple negative breast cancer cell MDA-MB-231 (J), triple negative breast cancer cell (MDA-MB-468).
Fig. 3 is that apoptosis occurs for CAA and CAI selective inductions kinds of tumor cells, does not have notable toxicity to normal cell.Make
Included with Tali imaging-type polychrome cell counters, influence of the detection compound to apoptosis of many kinds, the cell of detection：T drenches
Bar cell leukemia cell E6-1 (A), people triple negative breast cancer cell MDA-MB-231 (B), property breast cancer cell SUM149
(C), human colon cancer cell HCT-116 (D), human fibroblast cell line NHF (F), the non-tumour source galactophore epithelial cell of people
MCF10A (G), human peripheral blood mononuclear cell PBMCs cells (H), people's colonic crypt cells NCM460 (I).Staining cell is in fluorescence
Apoptosis situation (E-J) is detected under microscope.
Fig. 4 be CAA and CAI by adjust mitochondrial oxidation stress signal path and induced tumor apoptosis.(A-
C) TMRM methods detection compound CAA and CAI is to T lymphocytic leukemia cells E6-1 (A), people's triple negative breast cancer cell MDA-
MB-231 (B) and human fibroblast cell line NHF (C) mitochondrial membrane potential in anoxic stable state influence.(D) E6-1 cells TMRM and
Hoechst coloration results.(E) NHF cells TMRM and Hoechst coloration result.(F) after the processing of E6-1 cell drugs, Western
The situation that blot methods detection cytochrome c discharges from endochylema.(G-H) Western blot methods detect apoptosis-related protein in E6-1
Expression in cell.
Fig. 5 is CAA and CAI to Caspase-3 in human T lymphocyte's leukaemia E6-1 cells, 8,9, λ-H2AX activation.
1.2×106Individual tumour cell is incubated at 37 DEG C with 1640 culture medium, updates nutrient solution after 24 hours.Add the CAA of various concentrations
With CAI and curcumin, using Doxorubicin as positive control, and set and add NAC to judge the apoptosis-induced mechanism of compound, locate
Reason cell collects cell extraction total protein after 24 hours, procaspase-3, caspase- are detected respectively with Western Blot
3rd, procaspase-8, caspase-8, procaspase-9, caspase-9 and λ-H2AX content, β-actin are as calibration
Albumen.
Fig. 6 combinations CAA can effectively improve piperlongumine (PL) anti-leukocythemia (A, B) and the effect of breast cancer (C, D), this
For kind enhancing effect because NAC addition is totally constrained, this compound combination is remarkably reinforced antitumor activity, but to normal cell
Influence smaller (E-G).
Fig. 7 FOLFOX add low concentration (0.5 μM of CAA, 1 μM of CAI) chemical combination as a kind of important colon cancer therapy
Thing, you can good sensitization is played to FOLFOX therapies.
Antitumous effect in Fig. 8 CAA and CAI bodies.(A) CAA effectively suppresses chronic granulocytic leukemia MV-4-11 cells
The growth of xenograft tumor, and on mouse weight without influence.(B) CAA significantly inhibits triple negative breast cancer MDA-MB-231 cells
The growth of xenograft tumor.
Embodiment
The present invention further illustrates below in an example.These embodiments simply to illustrate that mesh of the invention
, rather than for limiting the scope of the present invention.
The CAA of embodiment 1 synthesis
10mmol TMBs, 5mmol N- methyl -4- piperidones are dissolved in 10mL absolute ethyl alcohols
In, (5-8 DEG C) stirring 10min, solution are unchanged under the conditions of ice-water bath.Weigh sodium hydrate solid 40g and be dissolved in 60ml distillations
Water, it is configured to 40% (w/w) sodium hydroxide solution.40% sodium hydroxide solution 1.0mL is slowly added dropwise while stirring in reaction
In solution, after stirring reaction 2h, there are a large amount of insoluble pale yellow precipitates, reaction solution is detected with TLC, to 254nm uviol lamps,
The black splotch of raw material 3,4,5-Trimethoxybenzaldehyde no longer changes, and product spot is obvious under 365nm.Stop reaction, will be anti-
Liquid is answered to filter, product is first washed with water, and is then washed twice with 10% ethanol.Solid powder is mixed into sample, silica gel column chromatography purifying, stream
Dynamic is mutually petroleum ether：Ethyl acetate=4:1 to 2:1, gradient elution.After solvent is removed in rotation, after being dried in vacuum overnight at 30 DEG C
(3E, 5E) -1- methyl -3,5- bis- (3,4,5- trimethoxy benzylidene) piperidin-4-one, yellow powder, yield 16.9%, melt
139.55~142.4 DEG C of point.1H-NMR(CDCl3),δ:7.786 (s, 2H, Ar-CH=C × 2), 6.649 (s, 4H, Ar-H2×2,
Ar-H6×2),3.959(s,6H,4-OCH3×2),3.916(s,12H,3-OCH3×2,5-OCH3×2),3.849(s,4H,
CH2-N-CH2),2.509(s,3H,N-CH3).ESI-MS m/z:470.0(M+1)+,calcd for C26H31NO7:469.53。
Specific synthetic route is shown in Fig. 1.
The CAI of embodiment 2 synthesis
First 5mmol piperidone hydrochloride monohydrates are dissolved in 2ml distilled water, add 10mmol2,3- dimethoxy benzenes
After formaldehyde, the dissolving of 10mL absolute ethyl alcohols is poured into, (5-8 DEG C) stirring 10min, solution are unchanged under the conditions of ice-water bath.Weigh hydrogen
Sodium oxide molybdena solid 40g is dissolved in 60ml distilled water, is configured to 40% (w/w) sodium hydroxide solution.It is slowly added dropwise while stirring
40% sodium hydroxide solution 1.5mL is in reaction solution, after stirring reaction 2h, a large amount of insoluble yellow mercury oxides occurs, is examined with TLC
Reaction solution is surveyed, to 254nm uviol lamps, raw material 2, the black splotch of 3- dimethoxy benzaldehydes no longer changes, product under 365nm
Spot is obvious.Stop reaction, by reacting liquid filtering, product is first washed with water, and is then washed twice with 10% ethanol.Solid powder is mixed
Sample, silica gel column chromatography purifying, mobile phase is petroleum ether：Ethyl acetate=4:1 to 2:1, gradient elution.After solvent is removed in rotation, 30
Product as yellow powder (3E, 5E) -3,5- two (2,3- dimethoxybenzylidens) piperidines -4- is obtained after being dried in vacuum overnight at DEG C
Ketone, orange-yellow powder, yield 27.36%, 70.2~76.0 DEG C of fusing point.1H-NMR(CDCl3)δ:7.978(2H,s,Ar-CH×
2), 7.068 (2H, t, J=8.0Hz, Ar-H2× 2), 6.947 (2H, d, J=8.0Hz, Ar-H3× 2), 6.815 (2H, d, J=
7.5Hz,Ar-H4×2),4.025(4H,s,N-CH2×2),3.887(6H,s,2-O-CH3×2),3.834(6H,s,3-O-CH3
×2),1.744(1H,s,-NH).ESI-MS m/z:396.3(M+1)+,calcd for C23H25NO5:395.45。
Specific synthetic route is shown in Fig. 1.
The propagation of a variety of people source malignant tumours of the CAA and CAI dose dependents of embodiment 3
Human blood tumor cell line (T lymphocytic leukemia cell E6-1, M4 myelomonocytic leukemia cells MV-4-
11st, human tissue cell lymphoma cell U-937), Breast cancer lines (triple negative breast cancer cell MDA-MB-231, MDA-
MB-468), cell line of human osteosarcoma (MG-63, Saos-2), non-small cell lung cancer cell strain (NCI-H23, A549) and people's colon
JEG-3 (HCT-116), it is derived from American Cell, strain library (ATCC).Cell is inoculated in 96 well culture plates, and cell hangs
Liquid is containing 10% heat-inactivated fetal bovine serum, the 1640 or DMEM culture mediums of 10mg/mL gentamicins, and cell density is added per hole is
5000 cell.Contain 5%CO in 37 DEG C2Cultivated in the incubator of saturated humidity.The various concentrations chemical combination for being dissolved in DMSO after 24h
Thing is added in culture plate, is incubated 48h, and 4h adds 5mg/mL water-soluble tetrazolium salts (WST-1) 20 μ L per hole before terminating culture.It is incubated
Finish, add water fully to dissolve, each hole absorbance value (Abs) is determined under enzyme-linked immunosorbent assay instrument 450nm wavelength, blank control is
DMSO groups.Growth of tumour cell rate is mapped with the various concentrations of same medicine, dose-effect curve is can obtain, as a result sees accompanying drawing
2.It can be seen that CAA, CAI can all play good inhibited proliferation for more plants of different types of tumour cells.
Embodiment 4 CAA, CAI selective induction kinds of tumor cells apoptosis, and there is no notable toxicity to make to normal cell
With
Human blood tumor cell line (T lymphocytic leukemia cell E6-1), people's triple negative breast cancer cell line (MDA-
MB-231), cell line of human osteosarcoma (MG-63, Saos-2), non-small cell lung cancer cell strain (NCI-H23, A549) and people's colon
JEG-3 (HCT-116) is derived from American Cell, strain library (ATCC), inflammatory breast cancer cell SUM149 by the U.S.
Stephen doctors Ethier present (Wayne State University, Detroit, MI, USA)；Normal cell strain includes
Human fibroblast cell line's (NHF, being purchased from Coriell institute for Medical Research, the U.S.), people's colon glue
Theca cell system (NCM460, being purchased from INCELL companies, the U.S.), people are non-, and tumour source galactophore epithelial cell (MCF10A, is purchased from the U.S.
ATCC companies).Human peripheral blood mononuclear cell's PBMCs cells, instructed according to school's ethics, from the voluntarily strong of Canadian University of Windsor
Extract and separate in health volunteer body.
Cell is inoculated in 6 well culture plates, and density is 2.75 × 105Individual/hole, after overnight incubation, collect cell, centrifugation,
Be dissolved in DMSO CAA, CAI, curcumin or taxol (TAX) or Doxorubicin (DOX) add culture plate in, be incubated 48 or
72h.Flushed three times with 1 × PBS, then make cell be suspended in Annexin V binding buffer (10mM HEPES,
140mM NaCl,2.5mM CaCl2,pH 7.4).Then at 37 DEG C with Annexin V (Life Technologies Inc, business
Article Number：A13201, Burlington, Canada) and propidium iodide (Life Technologies Inc, commodity
Number：P3566, Burlington, Canada) dyeing 15 minutes after, useImaging-type polychrome cell counter
(LifeTechnologies Inc, article number：T10796, Burlington, Canada) quantitative analysis results.
Use green fluorescence (excitation 458nm；Emission 525/20nm) and red fluorescence (excitation
530nm；Emission 585nm) passage quantitative analysis results, cell mass be respectively expressed as viable apoptotic cell (green), evening
Phase apoptotic cell (green and red combination, i.e. yellow) and non-viable non-apoptotic cell (red).As a result (accompanying drawing 3A-E) show CAA, CAI and
Curcumin induces E6-1, MDA-MB-231, SUM149 and HCT119 Apoptosis, and CAA, CAI with dosage-dependent manner
Compared with curcumin, the effect of induced tumor apoptosis can be played in lower dosage, shows that CAA, CAI effect are better than
Guide's curcumin, and slightly it is better than the TAX and DOX of same dose.Importantly, compound CAA, CAI is to normal human cell's strain
NHF, MCF10A, PBMCs and NCM-460 do not have significant apoptosis-induced effect, with reference to the accompanying drawings 3F-J, and CAA, CAII are to NCM460
The toxic action of cell is less than DOX, and is less than TAX to the toxic action of NHF cells.
Embodiment 5 CAA, CAI be by adjust mitochondrial oxidation stress signal path cause tumor cell induction apoptosis
Mitochondria during breathing aoxidizes, cause the mal-distribution of proton and other ion concentrations in inner membrance both sides and
Form mitochondrial membrane potential (Mitochondrial membrane potential, MMP).The oxidation of mitochondria glutathione and
The increase of reactive oxygen species, MMP depolarising will be caused so that the opening in membrane channels hole, cromoci (Cytochrome C,
Cyto C) etc. content be discharged into kytoplasm, MMP is destroyed.Recent study shows that MMP becomes in Apoptosis early stage pathology
Begin to decline before changing, the process is earlier than DNA fragmentation.Therefore, we use two kinds of different dyestuffs
Tetramethylrhodamine methyl ester (TMRM) and Hoechst and cytosis, after TMRM enters mitochondria,
Intense red fluorescence is presented, after MMP is destroyed, membrane channels hole opens, and fluorescence disappears, and Hoechst then can be thin with apoptosis
The DNA of born of the same parents is directly in conjunction with whether in the fine and close dense dye blue-fluorescence of chunky shape, analyzing CAA, CAI with this can cause that MMP's is broken
It is bad, so as to cause apoptosis of tumor cells.
T lymphocytic leukemia cells E6-1, people's triple negative breast cancer cell line (MDA-MB-231), human fibroblasts
It is the medicine (E6-1 such as (NHF) and CAA, CAI, curcumin：0.5 μM of taxol, 1 μM of CAA, 2 μM of CAI, 10 μM of curcumins and
Blank control DMSO；NHF：1 μM of Doxorubicin, 1 μM of CAA, 2 μM of CAI, 10 μM of curcumins and blank control DMSO) it is incubated
After 48h, the mitochondrial membrane potential variation of cell is detected using TMRM methods.As a result quantitatively divided with the ratio of TMRM positive cells
Analysis.The cell sample detected, further with 10 μM of Hoechst dyestuffs redye 45 minutes, under inverted fluorescence microscope
Observe result (Leica Microsystems, Wetzlar, Germany).
Fig. 4 shows, in the result of E6-1 and MDA-MB-231 cells, more obvious TMRM occur in DMSO control groups
Positive findings (red fluorescence is more strong), when illustrating not dosing, cell MMP is normal；After 1 μM of CAA is added, basic observation
It is completely destroyed less than red fluorescence, MMP；In addition, preferable MMP executions can also be played by adding 2 μM of CAI.
In terms of Hoechst coloration results, in addition to curcumin group is added, there is the apoptosis situation of obvious karyon fragmentation in remaining each group.
Above phenomenon illustrates the destruction of CAA, CAI to MMP, then causes apoptosis of tumor cells.And for NHF cells, TMRM
Dyeing and Hoechst coloration results show that CAA, CAI change on the MMP of normal cell and apoptosis situation aspect is substantially without influence
(Fig. 4 A-E).
The increase of reactive oxygen species in mitochondria, is to cause MMP to disappear, the major reason of Apoptosis.By detect CAA,
Influences of the CAI to E6-1 cells, MV-4-11 cell TMRM positive findingses, and add antioxidant NAC, thus CAA, CAI for
Whether MMP destruction is caused due to its sedimentation to intracellular ROS.(accompanying drawing 4A-E) is understood by result, CAA, CAI couple
The reduction effect of tumour cell TMRM positive rates, substantially can be reversed by NAC addition, illustrate compound for the broken of MMP
Bad effect is exactly to be played by ROS accumulation.
After MMP is destroyed, Intramitochondrial Cyto C are released into kytoplasm；Succinate dehydrogenase A (Succinate
Dehydrogenase-A, SDHA) it is the marker enzyme for reflecting mitochondrial function, after MMP loses stable state, apoptosis, cause
SDHA vigor declines.Therefore, in order to further confirm that the effect of CAA, CAI for tumour cell MMP, we further have detected
Influence of the two compounds to Cyto C in E6-1 cell cytoplasms and mitochondria and SDHA expressions.By E6-1 respectively with 1 μ
M Doxorubicins, 1 μM of CAA, 2 μM of CAI, 10 μM of curcumins and blank control DMSO are incubated 24 hours altogether, according to manipulator
Volume, uses mitochondria separating kit (Abcam Canada, article number：Ab110170, Toronto, Canada) cell is trained
Nutrient solution carries out the separation of kytoplasm and mitochondria, after Coomassie Brilliant Blue determines protein concentration, carries out Westren Blot analyses.Knot
Fruit contrasts as shown in accompanying drawing 4F with blank DMSO groups, and Cyto C expression quantity substantially increases in CAA, CAI group kytoplasm, and SDHA is then bright
It is aobvious to reduce.Correspond to therewith, Intramitochondrial Cyto C and SDHA are in then inverse variation with situation in kytoplasm.Thus, further really
The interference effect of CAA, CAI to MMP stable states is demonstrate,proved, so as to cause the apoptosis of tumour cell (Fig. 4 G-H).
Activation of embodiment 6 CAA, the CAI to Caspase-3/8/9 and λ-H2AX
Caspase passages are important rush antiapoptotic signals albumen.It is intracellular to E6-1 that we have detected CAA, CAI
Caspase-3, caspase-8 and caspase-9 influence.Such as accompanying drawing 5, caspase-3, caspase-8 and caspase-9's
Activity form is substantially activated after 1 μM of CAA, 2 μM of CAI processing 24h respectively, and its precursor procaspase-3,
Procaspase-8 and procaspase-9 expression quantity significantly reduces, and it is probably that CAA, CAI inducing cell wither to illustrate Caspase
The signal of interest transduction pathway died.After adding NAC, the caspase activation effects of compound disappear.
λ-H2AX, are a member of histone family, and its major function is gene transcription regulation, DNA damage reparation, cell week
Period regulation etc..As a kind of important cancer suppressor protein, the ability of its DNA damage reparation ensure that the normal apoptotic of cell, unlikely
In generation canceration.Therefore, we also further have detected the activation situation of CAA, CAI to λ-H2AX.From 5,1 μM of accompanying drawing
CAA, 2 μM of CAI can play obvious activation to λ-H2AX, and its action intensity is identical with 10 μM of curcumins.Likewise,
This activation also can be because of NAC addition and by complete antagonism, and the antitumor activity for demonstrating compound again is to rely on
In ROS generation.
Embodiment 7 is combined the antitumor activity that CAA significantly increases PL, FOLFOX pharmaceutical composition, is aligned without influenceing it
The hypotoxicity of normal cell
Piperlongumine (Piperlongumine, PL) is the alkaloids antitumoral compounds for being isolated from the Bi roots of grass, can be passed through
Promote ROS generation, cause the cells such as PI3K/Akt/mTOR, JAK/STAT propagation associated signal paths suppression, for including
The selective lethal effect of the Several Kinds of Malignancy such as breast cancer, prostate cancer, stomach cancer.Therefore, we further join CAA and PL
With whether the compound of detection two identical mechanism of action of combination can play the synergistic effect of pharmacological activity, method：Same embodiment
4.As shown in accompanying drawing 6A, 6B, in the case where being simply added into 2 μM of PL or 0.5 μM of CAA, E6-1, U937 cell occur certain
Degree apoptosis, but it is more faint.After adding PL and CAA at the same time, the enhancing of Apoptosis degree, and it is significantly greater than list plus two
The summation of effect during individual compound.Identical, this synergistic effect is in CAA connection and PL treatment breast cancer SUM149, MDA-MB-
Equally existed (accompanying drawing 6C-D) in the experiment of 231 cells.It is and thin for normal peripheral blood mononuclear cells system PBMCs, human desmocyte
Born of the same parents system NHF and colonic crypt cells NCM460 cells, combination CAA and PL and single plus PL apoptosis-induced effect are essentially identical
(accompanying drawing 6E-G).Thus, while illustrating that being combined CAA can maintain to normal cell hypotoxicity, PL is significantly greatly strengthened
To the inhibitory action of tumour cell.
In addition, we further have detected sensitizations of the CAA for clinically conventional chemotherapeutics.FOLFOX medicines
Composition, it is a kind of clinical commonly used drug 5 FU 5 fluorouracil (5-FU), Calciumlevofolinate (FA) and the medicine of oxaliplatin (OX) three connection
Cancer immunotherapies are more conventional in the aftertreatment of colon cancer at present.Its toxicity is neurotoxicity, phlebitis, bone
Marrow suppresses, and clinical application need to strictly control dosage, it is significant in FOLFOX Clinical practice to develop its sensitizer.Cause
This, in our current research, we have detected the maximum effective dose of FOLFOX therapies first.From 7,12.5 μM of 5-FU of accompanying drawing,
5 μM of FA and 1.25 μM of OX drug regimen has arrived at maximum suppression activity to HCT-116 cells, even if by 5-FU, FA
Increasing concentrations, it is acted on also, and without any change, (height of green cylindricality represents viable apoptotic cell quantity, and it is dense to increase medicine
Degree, its height are also unchanged).Now, the CAA of 0.5 μM of low concentration is added, its antitumor activity is remarkably reinforced, and has exceeded 50 μM
The therapeutic effect that 5-FU, 10 μM of FA and 1.25 μM of OX are combined.Identical, the effect of 1 μM of CAI also can substantially increase FOLFOX.
Thus, it was demonstrated that CAA, CAI for the chemotherapeutics of Clinical practice also have good sensitization.
The CAA of embodiment 8 substantially suppresses mouse MV-4-11 chronic myelomonocytic leukemias and MDA-MB-231 tri- is negative
The growth of breast cancer transplantable tumor
People's chronic myelomonocytic leukemia MV-4-11 cells, to be derived from American Cell, strain library (ATCC).Using
Iscove's Modified Dulbecco's Medium cultivate (ATCC, article number：30-2005, Manassas, USA).Move
Plant in CD-1 nude mices, male, from Charles River Laboratories, QC, Canada；
People triple negative breast cancer MDA-MB-231 cell (ATCC, article number：HTB-26&HTB-132,Manassas,VA,
USA) using Dulbecco ' s Modified Eagles cultures (Sigma-Aldrich, Mississauga, Canada), transplanting
In female CD-1 nude mices (Charles River Laboratories, QC, Canada).
Mouse cultivates specification (University of Windsor according to animal feeding administration committee of University of Windsor
Animal Care Committee, AUPP#14-15) raised, cultivated under the illumination/dark condition of daily 12 hours.Carefully
Cell lysis liquid withBasement Membrane Matrix (VWR International, commodity
Number：47743-715, Mississauga, Canada) mixing after formed 0.2mL volumes parenteral solution, sterile stripping subcutaneous vaccination,
MV-4-11 cells：Per mouse bilateral oxter subcutaneous vaccination 5 × 106Cell/0.2mL；MDA-MB-231 cells：Per mouse one side oxter
Subcutaneous vaccination 2 × 106Cell/0.2mL.Administration group and DMSO blank control groups are set respectively, wherein injection curcumin group 4, gives
Give CAA groups and solvent control group every group 5.To be subcutaneously injected, dosage is administering mode：Per 300mLPBS 7mg/kgization
Compound (MV-4-11 transplantable tumors), per 300mLPBS 3mg/kg compounds (MDA-MB-231 transplantable tumors) or same volume concentration
DMSO, weekly administration three times, are administered five weeks.
After inoculated tumour cell, the size of start recording gross tumor volume and the change of mouse weight.Gross tumor volume calculates public
Formula is as follows：Volume=1/2 (Length × Width2).By experimental result (accompanying drawing 8A) as can be seen that giving same dose
Treat MV-4-11 cell transplantation knurls, CAA therapeutic effect will be substantially better than curcumin, and during being administered, two groups of administration groups with
The body weight of control group mice is in the faint increase trend of identical, illustrates curcumin and CAA to mouse and has no toxic side effect.Peel off swollen
The photo of knurl more intuitively presents CAA and the strong inhibition of MV-4-11 transplantable tumors is acted on.And in MDA-MB-231 cell transplantation knurls
In model (accompanying drawing 8B), CAA equally suppresses tumour growth potently, and since administration, CAA almost completely inhibit the life of tumour
It is long.CAA is on the body weight increase of mouse without influence.The photo of tumour is intuitively presented in the outstanding anti-breast cancer bodies of CAA after stripping
Activity.
Claims (12)
- A kind of 1. application of curcumin analogue in antineoplastic is prepared, it is characterised in that described curcumin analogue Structure be shown below：Described antineoplastic is for selective killing tumour cell and to normal cytotoxic.
- 2. application of the curcumin analogue according to claim 1 in antineoplastic is prepared, it is characterised in that described Curcumin analogue by improve cell ROS level, activate oxidative stress and apoptosis carrys out selective killing tumour cell.
- 3. application of the curcumin analogue according to claim 1 in antineoplastic is prepared, it is characterised in that described Curcumin analogue by activating caspase-3, caspase-8 and caspase-9 come selective killing tumour cell.
- 4. application of the curcumin analogue according to any one of claims 1 to 3 in antineoplastic is prepared, its feature It is, described tumour is lymthoma, breast cancer, colon cancer.
- 5. application of the curcumin analogue according to claim 4 in antineoplastic is prepared, it is characterised in that described Tumour cell is selected from human tissue cell's lymthoma U937 cells, human T lymphocyte leukaemia E6-1 cells, people's triple negative breast cancer MDA-MB-231 cells, people's inflammatory breast cancer SUM149 cells and human colon carcinoma HCT116 cells.
- 6. application of the curcumin analogue according to claim 1 in antineoplastic is prepared, it is characterised in that described Normal cell be selected from people normal fibroblast NHF cells, human peripheral blood monocyte PBMCs cells and people's Normal Colon The cells of mucomembranous cell NCM 460.
- 7. application of the curcumin analogue according to claim 1 in antineoplastic is prepared, it is characterised in that described Antineoplastic include the combination of curcumin analogue and piperlongumine.
- 8. application of the curcumin analogue according to claim 7 in antineoplastic is prepared, it is characterised in that described Antineoplastic when treating colon cancer, described curcumin analogue uses as FOLFOX therapy sensitizers.
- 9. application of the curcumin analogue according to claim 1 in antineoplastic is prepared, it is characterised in that described Curcumin analogue be CAA, for suppressing the growth of nude mouse tumor cell transplantation knurl.
- 10. application of the curcumin analogue according to claim 9 in antineoplastic is prepared, it is characterised in that institute State tumour cell and be selected from chronic granulocytic leukemia MV-4-11 cells, people's triple negative breast cancer MDA-MB-231 cells.
- 11. application of the curcumin analogue according to claim 1 in antineoplastic is prepared, it is characterised in that institute The antineoplastic stated is used to suppress tumor cell proliferation.
- 12. application of the curcumin analogue according to claim 11 in antineoplastic is prepared, it is characterised in that institute State tumour cell and be selected from human tissue cell's lymthoma U937 cells, people's triple negative breast cancer MDA-MB-231 cells and people's colon Cancer HCT116 cells.
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| CN108619145A (en) * | 2018-04-23 | 2018-10-09 | 温州医科大学附属第医院 | Application of the compound in treating tumour |
| CN108771670A (en) * | 2018-05-16 | 2018-11-09 | 广东食品药品职业学院 | A kind of pharmaceutical composition and its application in preparing antiprostate cancer |
| CN109078014A (en) * | 2018-03-26 | 2018-12-25 | 温州医科大学 | 3,4- dimethoxybenzyliden piperidines keto analog is preparing the application in anti-lung-cancer medicament |
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| CN109078014A (en) * | 2018-03-26 | 2018-12-25 | 温州医科大学 | 3,4- dimethoxybenzyliden piperidines keto analog is preparing the application in anti-lung-cancer medicament |
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| CN108771670A (en) * | 2018-05-16 | 2018-11-09 | 广东食品药品职业学院 | A kind of pharmaceutical composition and its application in preparing antiprostate cancer |
| CN110452163A (en) * | 2019-08-13 | 2019-11-15 | 温州医科大学附属第二医院、温州医科大学附属育英儿童医院 | A kind of application of the preparation method of curcumin derivate and its anti-prostate cancer |
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| CN116082251B (en) * | 2023-04-11 | 2023-06-30 | 齐泽(云南)生物科技有限公司 | A pharmaceutical compound |
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