CN107595859A - Applications of the cucurbitacin C in broad-spectrum anti-cancer drug is prepared - Google Patents
Applications of the cucurbitacin C in broad-spectrum anti-cancer drug is prepared Download PDFInfo
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- DGIGXLXLGBAJJN-BTCRBLTISA-N Cucurbitacin C Natural products CC(=O)OC(C)(C)C=CC(=O)[C@@](C)(O)[C@H]1[C@H](O)C[C@@]2(C)[C@@H]3CC=C4[C@@H](CC[C@H](O)C4(C)C)[C@@]3(CO)C(=O)C[C@]12C DGIGXLXLGBAJJN-BTCRBLTISA-N 0.000 title claims abstract description 69
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Abstract
Application the present invention relates to cucurbitacin C antitumor efficacy and in antineoplastic, cucurbitacin C has the effect of suppressing kinds of tumor cells growth, causes the growth inhibition and apoptosis of tumour cell by influenceing Cellular cycle and apoptosis GAP-associated protein GAP and suppression Akt signal paths.Present invention firstly discloses cucurbitacin C antitumor efficacy, and it is from the frequently seen plants such as cucumber, abundant raw material, it is cheap, toxic side effect is small, available for the antineoplastics such as treatment prostate cancer, carcinoma of urinary bladder, liver cancer, breast cancer, colorectal cancer, lung cancer are prepared, has broad application prospects.
Description
Technical field
The invention belongs to biomedicine technical field, specifically, is related to applications of the cucurbitacin C in antineoplastic.
Background technology
Cancer be a kind of cancer cell abnormality proliferation and differentiation disease, by inducing cell apoptosis, cell growth arrest and
Suppress angiogenesis, cell metabolism and invasion and attack come target cancer cell chemotherapy be still main strategy of cancer treatment it
One.Natural products is most important cancer therapy drug, and 75% antitumoral compounds for treating cancer are natural products or therewith
Related compound.Natural products has the characteristics that effect is steady, toxic side effect is small and tolerance is high, is played in clinical treatment
More and more important effect.At present, the research and development of natural products turn into a kind of worldwide trend, and scientific research personnel is more and more
Notice chemically synthetic drug turn to natural drug, it is desirable to safer effective new drug is developed from natural products.Cause
This, natural products has huge potentiality in anticancer.
Cucurbitacin C is the principal element that cucumber bitter taste is formed, and the material base of cucumber medical value.《Compendium of Materia Medica》
Record cucumber：Main heat-clearing is quenched one's thirst, dredging water passages；Children's hot dysentery；Edema tripe swollen, four limbs edema；Abscess of throat；Fox thorn poison is swollen etc..《This
Grass is picked up any lost article from the road》Record cucumber leaf：Main children dodge tinea, one-year-old to take a leaf, and raw Norway twists juice clothes, must told down good.At present, cucumber bitter taste synthesizes
Successfully being cracked with regulatory molecule mechanism, be related to 11 genes altogether, its bitter substance cucurbitacin C is responsible for synthesizing by 9 genes, and
And to prepare cucurbitacin C techniques ripe for extraction from cucumber fruits and cucumber leaf.However, cucurbitacin C medical value is not yet
Obtain effectively exploring and excavating.
The content of the invention
It is an object of the invention to provide the new application of cucurbitacin C cancer suppressing functions and its action target spot in cancer cell.
It is a further object of the present invention to provide applications of the cucurbitacin C in antineoplastic.
In order to realize the object of the invention, the present invention provides applications of the cucurbitacin C in broad-spectrum anti-cancer drug is prepared.Specifically
Be related to cucurbitacin C by suppress cell growth, transfer and inducing cell apoptosis antitumor efficacy and cucurbitacin C in tumour cell
In action target spot.
Tumour of the present invention includes but is not limited to by prostate cancer, carcinoma of urinary bladder, liver cancer, breast cancer, colorectal cancer or lung cancer
Caused tumour.It is preferred that the tumour caused by prostate cancer, carcinoma of urinary bladder or liver cancer.
The cucurbitacin C of the present invention is a kind of tetracyclic triterpenes chemical combination of the middle extraction from cucumber leaves, cucumber fruits, seed
Thing, molecular formula C32H48O8, No. CAS：5988-76-1, chemical constitution are as follows：
The cucumber cucurbitacine C of the present invention can refer to CN104892713 (denominations of invention：Cucurbitacin C and the like preparation
Method and application) described in method be prepared.
Cucumber cucurbitacine C can induce growth of tumour cell Cycle Arrest and apoptosis, and cause tumour cell cycle albumen and
The change of apoptosis regulation GAP-associated protein GAP.
Cucumber cucurbitacine C can suppress the invasion and attack and transfer of tumour cell.
The present invention also provides a kind of broad-spectrum anti-cancer drug or pharmaceutical composition, and its active component is cucurbitacin C.
Preferably, the medicine or pharmaceutical composition include at least one pharmaceutical excipient.The medicine or drug regimen
The formulation of thing can be tablet, powder, capsule, granule, supensoid agent, syrup, oral liquid, ointment, patch or injection
Agent etc..Method of administration includes external application, oral or injection.
The present invention also provides the anti-tumor agent using Akt signal paths as target spot, and its active component is cucurbitacin C.It is described
Cucurbitacin C causes the growth inhibition and apoptosis of tumour cell by suppressing phosphorylation Akt levels.
The present invention also provides the anti-tumor agent using tumour cell cycle albumen as target spot, and its active component is cucurbitacin C.
Wherein, the tumour cell cycle albumen includes but is not limited to cyclin A, cyclin D1.
Result of study shows that the cucurbitacin C being separated to from cucumber leaves and fruit can effectively suppress to include prostate
The growth of the kinds of tumor cells such as cancer, carcinoma of urinary bladder and liver cancer, its half effective inhibition concentration (IC50) some tumour cells as before
As little as 19.6nM in row adenocarcinoma cell PC-3.Low concentration cucurbitacin C (≤100nM) can be hindered with induced tumor cell growth cycle
It is stagnant, raise cucurbitacin C concentration (1 μM) then inducing apoptosis of tumour cell.Meanwhile cucurbitacin C can also prevent the invasion and attack of tumour cell
And migration.Cucurbitacin C causes the life of tumour cell by influenceing Cellular cycle and apoptosis GAP-associated protein GAP and suppression Akt signal paths
Long suppression and apoptosis.
The present invention has advantages below：
Present invention firstly discloses cucurbitacin C antitumor efficacy, it is from frequently seen plants such as cucumber, abundant raw material,
Cheap, toxic side effect is small, and available for preparing, smelting treatment prostate cancer, carcinoma of urinary bladder, liver cancer, breast cancer, colorectal cancer, lung cancer etc. are anti-
Tumour medicine, have broad application prospects.
Brief description of the drawings
ICs of the Fig. 1 for cucurbitacin C in the embodiment of the present invention 1 to various tumour cells50It is worth (B) and inhibiting rate curve (A)；Its
In, cucurbitacin C (including prostate gland cancer cell LNCaP, DU145 and PC-3 in kinds of tumor cells；Transitional cell bladder carcinoma cell line T24；Liver
Cancer cell HepG2) half effective inhibition concentration (IC50) it is respectively 19.6nM (PC-3) to 158.7nM.
Fig. 2 is the tumor cell cloning efficiency after cucurbitacin C processing in the embodiment of the present invention 1；Wherein, A represents clone
Experiment is formed, B represents cloning efficiency result of calculation.
Fig. 3 is cucurbitacin C induced tumors cell-cycle arrest (A) and Apoptosis (B) in the embodiment of the present invention 1.
Fig. 4 is that cucurbitacin C significantly inhibits tumor cell migration in the embodiment of the present invention 1；Wherein, A is cell scratch experiment；
B is cut healing rate result of calculation.
Fig. 5 is influences of the cucurbitacin C to tumour cell cycle albumen and apoptosis-related protein in the embodiment of the present invention 2.Its
In, A：100nM cucurbitacins C handled tumour cell after 24 hours, and western blot detect its cell cycle GAP-associated protein GAP
Cyclin A and cyclin D1, apoptosis-related protein bcl-2, cleaved caspase-3's and cleaved caspase-9
Influence.B：Various concentrations cucurbitacin C handled tumour cell after 24 hours, cell cycle albumen and cell growth GAP-associated protein GAP
P21 and p53 influence.β-actin are used as internal reference albumen.
Fig. 6 is that cucurbitacin C suppresses tumour cell phosphorylation akt in the embodiment of the present invention 2.Wherein, A：100nM cucurbitacins C
Tumour cell PC-3, T24 and HepG2 are handled after 24 hours, western blot detect it to akt and phosphorylation akt (p-
aktSer473) influence.B：Cucurbitacin C handles tumour cell different time points to akt and p-aktSer473Influence.β-actin
As internal reference albumen.
Embodiment
Following examples are used to illustrate the present invention, but are not limited to the scope of the present invention.Unless otherwise specified, embodiment
In the conventional meanses that are well known to those skilled in the art of used technological means, raw materials used is commercial goods.
Antitumor activities of the cucurbitacin C of embodiment 1 to kinds of tumor cells
1st, influences of the mtt assay detection cucurbitacin C to tumour cell term growth
1.1 experimental cell
Human bladder cancer cell strain T24, human prostate cancer cell line LNCaP, DU145 and PC-3 and human hepatoma cell strain
HepG2.Above-mentioned cell line is incubated in the complete culture solution of 10% hyclone of addition respectively.100IU/ is added in nutrient solution
ML penicillin and 100 μ g/mL streptomysins and 10mM HEPES, are placed in 37 DEG C, 5%CO2Cultivated in incubator.Logarithm will be in
Growth period cell is inoculated in 96 well culture plates with about 5000, every hole cell, and per the μ L culture mediums of pore volume 100, culture 24 is small
When.
1.2 Experimental agents
Cucurbitacin C is dissolved in DMSO, stock concentration 2mM.
1.3 inhibiting tumor cell determinations of activity
It is measured using mtt assay：
Cucurbitacin C with culture medium be diluted to 5nM, 10nM, 50nM, 100nM, 0.5 μM, 1 μM, 10 μM, it is 48 small to continue culture
When.Then, culture medium is discarded, 100 μ L MTT (2.5ug/ul) are added per hole, after 37 DEG C are incubated 4 hours, adds 200 μ L immediately per hole
Lysate, 37 DEG C of overnight incubations, optical density (OD) value, Detection wavelength 570nm, finally, according to following public affairs are determined with ELIASA
Formula calculates inhibiting rate of the medicine to tumour cell, and calculates IC according to middle efficacious prescriptions journey50Value：
Inhibiting rate (%)=(ODControl-ODExperiment)/ODControl× 100%
Control group mean OD value-administration group mean OD value)/control group mean OD value × 100%
Wherein, ODControlFor control group mean OD value, ODExperimentFor administration group mean OD value.ODControl、ODExperimentTo have removed ODBlank
Experiment value.
1.4 experimental result
The numerical value measured is tested after analysis, draws the IC to various tumour cells50Value and inhibiting rate curve.Fig. 1 data
Show, cucurbitacin C can effectively suppress a variety of growth of cancer cells, and inhibition is obvious at low concentrations.
2nd, influences of the Clone formation method detection cucurbitacin C to tumour cell long term growth
500 HepG2 and DU145 cells kinds added in six orifice plates, after 24 hours 20ml containing various concentrations (0nM,
10nM, 50nM, 100nM) cucurbitacin C complete medium, change a subculture per 3-4, cultivate 14 days.After culture terminates, use
4% paraformaldehyde is fixed, 0.1% violet staining.>=100 μm of clone's numbers are counted under 40 times of mirrors, and according to following
Formula calculates cloning efficiency：
Cloning efficiency (%)=administration group clone's number/control group mean OD value × 100%
Tumor cell cloning efficiency after cucurbitacin C processing is shown in Fig. 2.Fig. 2 as shown by data, in DU145 cells, 10-
50nM cucurbitacins C can significantly inhibit Clone formation quantity and size, and in HepG2 cells, 50-100nM, which has, to be significantly inhibited
Effect.
3rd, cucurbitacin C inducing cell cycle arrests and apoptosis
6 orifice plates are inoculated in after exponential phase cell dissociation, change the culture of the cucurbitacin C containing various concentrations after 24 into
Liquid, handle 48 hours, pancreatin digestion, and with 70% ice-cold in advance alcohol in -20 DEG C of fixations.Using 50 μ g/ml propidium iodides
(propidium iodide, PI), 10 μ g/ml DNase-free RNase are handled 15 minutes, are filtered and enter after flow cytometer
Row cell cycle analysis (FACS Calibur BD), each sample record 105Individual cell data.
For Apoptosis assay, the cell after above-mentioned cucurbitacin C processing, Annexin V-FITC and PI dyeing are resuspended in
15 minutes in liquid, then Apoptosis assay, each 10,000 cell data of sample record are carried out in flow cytometer.
Fig. 3 as shown by data, is compared and cellular control unit, low concentration cucurbitacin C significantly induce DU145 and LNCaP after handling
The cell G1 phases block, T24 the and HepG2 cell G2/M phases block.Also, high concentration (1 μM) cucurbitacin C processing causes G1 early stages
(sub-G1) block, show that high concentration cucurbitacin C can inducing cell apoptosis.Further cell apoptosis assay is shown, 24 hours calabashes
Lu Su C (100nM) processing can significantly induce T-24 and HepG2 early apoptosis.10nM Doxorubicins (Doxorubicin) are as sun
Property control, to induction cancer cell late apoptic and death.
4th, cucurbitacin C significantly inhibits tumor cell migration
6 orifice plates are inoculated in after exponential phase cell (DU145, T24) digestion, are grown after cell to after converging plus without blood
Clear culture medium Nature enemy is stayed overnight, and with 200 μ l pipette tips scratchings, after culture medium cleaning and removing residual cell, adds 20nM cucurbitacins C
Processing, control group and treatment group were taken pictures in 0 hour, 12 hours and 24 hours.
Fig. 4 results show that 20nM cucurbitacins C can significantly inhibit prostate gland cancer cell DU145 and transitional cell bladder carcinoma cell line T24 cells
Migration.
Anti-tumor target researchs of the cucurbitacin C of embodiment 2 to tumour cell
1st, influences of the cucurbitacin C to tumour cell cycle albumen and apoptosis-related protein
The medicine of induced tumor cell-cycle arrest and apoptosis is potential antineoplastic.Swollen to find cucurbitacin C
The action target spot of oncocyte, the present invention have detected after cucurbitacin C processing to tumour cell cycle albumen and apoptosis-related protein
Influence.Prostate cancer, carcinoma of urinary bladder and liver cancer cells are grown to after converging, and after adding cucurbitacin C processing 24 hours, collect cell cracking
Immunoblot experiment (western blot) is carried out afterwards.Result of study finds that cyclin A and cyclin D1 are at cucurbitacin C
Significantly reduced in three kinds of tumour cells after reason, and the dosage Cheng Yi of p21 albumen expression quantity and cucurbitacin C in tumour cell
The relation of relying, show that cucurbitacin C suppresses growth of tumour cell by acting on cyclin.Also, cucurbitacin C can be lured significantly
The expression of apoptosis-related protein cleaved caspase-3 in prostate gland cancer cell LNCaP is led, and hepatocellular carcinoma H22 shows
For cleaved caspase-9 notable aggregation, bcl-2 is unchanged in three kinds of cells, shows cucurbitacin C in different cells
In pass through the apoptosis (Fig. 5) of different mechanism of action induced tumor cells.
2nd, serine/threonine protein kitase B (protein kinase B, PKB/Akt) signal pathway is cucurbitacin C suppressions
The action target spot of growth of tumour cell processed
Akt is responsible for a maincenter molecule of many growth factor and cell factor downstream signal transmission, in regulating cell
Propagation in survival with playing vital effect.Akt overactivity and the generation tight association of kinds cancer.The present invention
It was found that 100nM cucurbitacins C significantly suppress phosphorylation Akt Ser473 (p- in prostate cancer, carcinoma of urinary bladder and liver cancer
aktSer473).In Human Prostate Cancer PC-3 Cell Line, suppression of the cucurbitacin C to p-akt (Ser473) starts from 3 small after processing
When, the peak value that reached suppression by the 10th hour；For transitional cell bladder carcinoma cell line T24, cucurbitacin C goes out earlier to p-akt depression effect
It is existing, the peak value (Fig. 6) of suppression is reached after about handling after 1 hour or so, 24 hours.
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be modified or improved, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Claims (9)
1. applications of the cucurbitacin C in broad-spectrum anti-cancer drug is prepared.
2. application according to claim 1, it is characterised in that the tumour include by prostate cancer, carcinoma of urinary bladder, liver cancer,
Tumour caused by breast cancer, colorectal cancer or lung cancer.
3. application according to claim 2, it is characterised in that the tumour is included by prostate cancer, carcinoma of urinary bladder or liver cancer
Caused tumour.
4. according to any described applications of claim 1-3, it is characterised in that the cucurbitacin C comes from cucumber.
5. a kind of broad-spectrum anti-cancer drug or pharmaceutical composition, it is characterised in that active component is cucurbitacin C.
6. medicine according to claim 5 or pharmaceutical composition, it is characterised in that including at least one pharmaceutical excipient；
The formulation of the medicine or pharmaceutical composition is tablet, powder, capsule, granule, supensoid agent, syrup, oral liquid, ointment
Agent, patch or injection.
7. medicine according to claim 5 or pharmaceutical composition, it is characterised in that method of administration include external application, it is oral or
Injection.
8. using Akt signal paths as the anti-tumor agent of target spot, it is characterised in that active component is cucurbitacin C.
9. using tumour cell cycle albumen as the anti-tumor agent of target spot, it is characterised in that active component is cucurbitacin C；Wherein
The tumour cell cycle albumen includes cyclin A, cyclin D1.
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| CN115671115A (en) * | 2021-07-23 | 2023-02-03 | 中国医学科学院药物研究所 | Application of cucurbitacin E in preparation of medicine for preventing or treating adrenocortical carcinoma |
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