CN109498618A - A kind of pharmaceutical composition inhibiting ovarian cancer cell - Google Patents
A kind of pharmaceutical composition inhibiting ovarian cancer cell Download PDFInfo
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- CN109498618A CN109498618A CN201910003046.1A CN201910003046A CN109498618A CN 109498618 A CN109498618 A CN 109498618A CN 201910003046 A CN201910003046 A CN 201910003046A CN 109498618 A CN109498618 A CN 109498618A
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/365—Lactones
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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Abstract
A kind of pharmaceutical composition inhibiting ovarian cancer cell, belongs to biomedicine technical field.The composition is composed of the brefeldin A (BFA) of 200ng/mL and the cis-platinum (CDDP) of 4.0~4.5 μ g/mL.The composition of medicine can significantly inhibit the growing multiplication of ovarian cancer cell and promote the apoptosis of ovarian cancer cell, have the function of adjusting and ovarian cellular apoptosis related gene and protein, be significantly better than independent medication, have the function of cooperateing with ovarian cancer resistance.A kind of its targeted drug combination that can be used as regulation gonad cell apoptosis pathway provides a kind of new complex treatment strategy for clinically oophoroma.
Description
Technical field
The present invention relates to biomedicine technical field, specially a kind of pharmaceutical composition for inhibiting ovarian cancer cell.
Background technique
Cancer is to seriously threaten the major disease of human survival and social development.Wherein, oophoroma (ovarian
It carcinoma is) one of most common three big malignant tumour in women, disease incidence occupies third, and only this is in breast cancer, cervical carcinoma
Later.But since ovary is located at pelvic cavity deep, early lesion is not easy to be found, once symptom occur belongs to advanced stage more, therefore it is dead
The rate of dying occupies the first place of gynecological tumor.
The treatment mode of current clinically oophoroma is the composite treatment based on operation plus chemotherapy.Chemotherapy both can be independent
Applied to the palliative treatment of advanced cancer, but also as a part of complex treatment, combination therapy is carried out with operation, radiotherapy etc.,
And chemotherapy can prevent the transfer of oophoroma, recurrence, improve the long-term survival rate of patient.Cis-platinum (cis-dichlorodiamine
Platinum, CDDP) it is the platinum complex for being initially applied to clinical antineoplastic, with curative for effect, anticancer spectrum is wide, anticancer activity
High feature is mainly used for the treatment of a variety of solid tumors such as oophoroma, lung cancer.But its toxic side effect is also larger, such as kidney poison
Property, bone marrow suppression, neurotoxicity etc..Since cis-platinum and a variety of anticancer drugs have good synergistic effect, and without crossing drug resistant
Property, clinically mainly solves the problems, such as this by combined chemotherapy at present, therefore finds that a kind of toxic side effect is lower, anticancer effect
More preferably, more reasonably comprehensive therapeutic plan has important clinical meaning.
Natural products has the diversity of structure and function, is the important materials of R and D disease treatment novel drugs,
And mycetogenetic secondary metabolite is considered as the important sources of new drug primary structure.Wherein, brefeldin A
(brefeldin A, BFA) is exactly a kind of important secondary metabolite in fungi, it is a kind of macrolide antibiotics, tool
There are many bioactivity, including antitumor, antibacterial, antiviral activity etc..Studies have shown that BFA can promote kinds of tumor cells
Apoptosis, such as lung cancer, prostate cancer, liver cancer, breast cancer cell.Therefore, BFA is one of potential anticancer drug candidate.
Summary of the invention
It is an object of the invention to overcome above-mentioned background technique difficult, a kind of pharmaceutical composition for inhibiting ovarian cancer cell is provided
Object.
In order to achieve the above objectives, the technical solution of use are as follows: a kind of pharmaceutical composition for inhibiting ovarian cancer cell, it is described to be somebody's turn to do
Composition is composed of the brefeldin A (BFA) of 200ng/mL and the cis-platinum (CDDP) of 4.0-4.5 μ g/mL.
Preferably, the brefeldin A (BFA) and cis-platinum (CDDP) drug combination can inhibit the life of ovarian cancer cell
Long proliferation.
Preferably, the brefeldin A (BFA) and cis-platinum (CDDP) drug combination promote ovarian cellular apoptosis.
Preferably, the brefeldin A (BFA) and cis-platinum (CDDP) drug combination influence ovarian cellular apoptosis phase
Close the expression of albumen.
Preferably, the brefeldin A (BFA) and cis-platinum (CDDP) drug combination influence ovarian cellular apoptosis phase
The gene expression of pass.
Having the beneficial effect that using the above scheme
1. BFA (effective inhibition concentration IC is being used alone in this pharmaceutical composition for inhibiting ovarian cancer cell50For
400ng/mL) and CDDP (effective inhibition concentration IC50When for 9 μ g/mL), the survival rate of cell is in decreasing trend, but when using
IC50Concentration (i.e. 200ng/mL BFA+4.5 μ g/mL CDDP) Combined Treatment cell of each half, cell survival rate further drop
It is low, so significantly suppressing the growing multiplication of ovarian cancer cell when using BFA and CDDP drug combination.
2. this pharmaceutical composition for inhibiting ovarian cancer cell has BFA group, CDDP group and BFA+ compared with the control group
Total apoptosis rate of CDDP group cell is respectively 4.4%, 6.3%, 17.8%, is all remarkably higher than control group 2.9%；And BFA+CDDP
Total apoptosis rate of group cell is also significantly higher than independent medication group BFA group and CDDP group, and it is significant to show BFA and CDDP drug combination
Increase the apoptosis rate of ovarian cancer cell.
3. this pharmaceutical composition for inhibiting ovarian cancer cell, in BFA+CDDP drug combination group cell Bax mRNA and
The expression of Caspase3 mRNA is significantly higher than independent medication group, significantly shows BFA and CDDP drug combination and promotes
The up-regulation of gene participating in apoptosis expression.
4. this pharmaceutical composition for inhibiting ovarian cancer cell, when BFA+CDDP drug combination group cell Bax protein expression
Level is also significantly higher than each independent medication group, and Pro-caspase3 protein expression level is also substantially less than each independent medication group,
Cleaved-caspase3 protein expression level is also significantly higher than each independent medication group, and the prominent BFA and CDDP joint that shows is used
Medicine promotes the up-regulation of cell death related protein expression, promotes the generation of Apoptosis.
Detailed description of the invention
Fig. 1 is the BFA of the present invention and CDDP inhibiting effect that medication grows oophoroma SKOV-3 cell alone or in combination
Schematic diagram；
Fig. 2 is BFA and CDDP facilitation of the medication to oophoroma SKOV-3 Apoptosis alone or in combination；
Fig. 3 BFA and the CDDP influence that oophoroma SKOV-3 gene participating in apoptosis is expressed in medication alone or in combination；
Fig. 4 BFA and the CDDP influence that oophoroma SKOV-3 cell death related protein is expressed in medication alone or in combination.
Specific embodiment
It is further described the present invention below with reference to embodiment, but the present invention is not limited only to following embodiments, it is anticipated that this
For field technical staff in the case where combining the prior art, there may be many variations for performance.
It is a kind of inhibit ovarian cancer cell pharmaceutical composition, described the composition by 200ng/mL brefeldin A
(BFA) it is composed with the cis-platinum (CDDP) of 4.0~4.5 μ g/mL.
The brefeldin A (BFA) and cis-platinum (CDDP) drug combination can inhibit the growing multiplication of ovarian cancer cell.
The brefeldin A (BFA) and cis-platinum (CDDP) drug combination promote ovarian cellular apoptosis.
The brefeldin A (BFA) and cis-platinum (CDDP) drug combination influence ovarian cellular apoptosis GAP-associated protein GAP
Expression.
The brefeldin A (BFA) and cis-platinum (CDDP) drug combination influence the relevant base of ovarian cellular apoptosis
Because of expression.
Brefeldin A and cisplatin combined medication can significantly inhibit the growing multiplication of ovarian cancer cell, and can significantly promote
Into the apoptosis of ovarian cancer cell, has the function of adjusting and ovarian cellular apoptosis related gene and protein, be significantly better than list
Private medicine has the function of cooperateing with ovarian cancer resistance.A kind of its targeted drug combination that can be used as regulation gonad cell apoptosis pathway,
A kind of new complex treatment strategy is provided for clinically oophoroma.One, experimental method
(1) drug
Brefeldin A (is purchased from Cell Signaling Technology company), and cisplatin injections (are purchased from Shandong
Shandong drugmaker).
(2) cell line
Abortion syndrome SKOV-3.Cultivated using 1640 culture mediums, in culture medium containing 10% fetal calf serum,
100 μ g/mL streptomysins, 100U/mL penicillin, are placed in 37 DEG C, 95% air, 5%CO2, saturated humidity carbon dioxide culture
It is cultivated in case.Cell growth pattern is adherent growth, and passage in every 2-3 days is primary.
(3) reagent and instrument
Fetal calf serum is purchased from Zhejiang Tian Hang Biotechnology Co., Ltd, and RPMI-1640 culture medium is purchased from Gibco company, PCR
Primer is synthesized by Shanghai Sheng Gong biotech firm, and M-MLV reverse transcriptase and RNase inhibitor are purchased from Promega company, dNTP Mix
(10mM) is purchased from Shanghai Sheng Gong biotech firm, and RNAiso TM Plus is purchased from TaKaRa Biotechnology company, q-RT-
PCR kit is purchased from Bio-Rad company, rabbit-anti people β-actin monoclonal antibody, rabbit-anti people Bax are mostly anti-, rabbit-anti people Caspase3 is mostly anti-and
The secondary antibody of horseradish peroxidase-labeled is purchased from Protein-Tech Group company.
Biological clean bench (BCM-1300A) is purchased from safe and sound air technique company, carbon dioxide incubator (Gold-
Sim it) is purchased from Xi Meng international corporation, inverted fluorescence microscope (IX73) is purchased from Olympus company, program temperature reduction box (5100-
0001) it is purchased from Nalgene company, flow cytometer (Gallios) is purchased from Beckman Coulter company, PCR instrument (CFX
Connect TM Optics Module) and electrophoresis apparatus (PowerPac Basic) be purchased from Bio-Rad company, gel imaging system
(Gel Doc XR) the Bio-Rad company of system.
(4) mtt assay detects cell survival rate
It takes logarithmic phase oophoroma SKOV-3 cell culture for 24 hours, 0,100,200,300,400,450,500ng/ is respectively set
The μ g/mL CDDP of mL BFA and 0,2,4,6,8,9,10 is experimental group.Drug-treated for 24 hours after, be added MTT reagent, be incubated for 4h after abandon
Supernatant adds DMSO reagent, and the light absorption value (A) in each hole is measured under 570nm wavelength.Cell survival rate calculation formula is as follows:
Survival rate (%)=(experimental group A value-zeroing group A value)/(control group A value-zeroing group A value).
(5) AnnexinV/PI Apoptosis by Flow Cytometry rate
Cell is collected, PBS solution rinses 2 times, and centrifugation is dyed, specific steps are detailed in explanation with Annexin V/PI
After being protected from light 15min at room temperature, cell is resuspended with appropriate amount of buffer solution in book (BBI, BS7127), living with flow cytomery
Cell percentage, viable apoptotic cell percentage, non-viable apoptotic cell percentage and non-viable non-apoptotic cell percentage.Wherein, cell
Total apoptosis rate=viable apoptotic cell percentage+non-viable apoptotic cell percentage+non-viable non-apoptotic cell percentage.
(6) real-time fluorescence quantitative PCR (q-RT-PCR) detects gene expression dose
Drug-treated for 24 hours after, lytic cell extracts its total serum IgE, and ultramicrospectrophotometer detection total serum IgE purity is simultaneously surveyed
Its fixed concentration, agarose gel electrophoresis detect the integrality of total serum IgE, synthesize cDNA according to q-RT-PCR RNA isolation kit reverse transcription.
Primer:
β-Actin, F:5 '-CGGGAAATCGTGCGTGAC-3 ',
R:5′-CAGGAAGGAAGGC TGGAAG-3′；Bax,
F:5 '-CCCGAGAGGTCTTTTTCCGAG-3 ', R:5 '-CCAGCCCATGATGGTTCTGAT-3 '；Caspase3,
F:5 '-GAAATTGTGGAATTGATGCGTGA-3 ', R:5 '-CTACAACGATCCCCTCTGAAAAA-3 '.CDNA is pressed 1: 10
It is used as template after dilution, establishes following reaction system: 10 μ L of total volume, including 1 μ L cDNA, 5 μ L Sso Fast Eva
Green 3 ' primer of supermix, 0.5 μ L, 0.5 μ L, 5 ' primer, 3 μ L sterile waters.PCR response procedures are as follows: 94 DEG C of 60s, 95 DEG C
20s, 56 DEG C (β-Ac tin and Bax) or 60.4 DEG C of (Caspase3) 30s, 40 cycle periods.Each sample sets the repetition of 3 pipes.
Using β-ac tin as internal reference, using 2-△△CTMethod, with the relative expression quantity of the 3 duplicate mean value calculation genes of pipe.
(7) protein immunoblot (Western blot) detects protein expression level
Drug-treated for 24 hours after, lytic cell extracts its gross protein, using BCA method measurement protein concentration, take one
Quantitative Western quality sample, progress protein denaturation, loading, electrophoresis, transferring film, skim milk are closed, primary antibody is incubated for, secondary antibody is incubated for,
It exposure development and takes pictures, the analysis of band gray value is carried out using IPP software.
(8) statistical analysis
It is analyzed using 19.0 statistical software of SPSS, experimental data is usedIt indicates, IPP software analyzes western blot
The gray value of band, two comparison among groups are analyzed using independent samples t test, and more comparison among groups use one-way analysis of variance
(one-way ANOVA) indicates that difference is statistically significant with P < 0.05.
Two, experimental result
As Fig. 1 (* and * * indicate compared with the control group, P < 0.05 and P < 0.01；# and ## indicate compared with drug combination group,
P < 0.05 and P < 0.01) Figure 1A the results show that being respectively adopted 0,100,200,300,400,450,500ng/mL BFA processing
Cell, cell survival rate are in decreasing trend, and drug half effective inhibition concentration (IC50) it is 400ng/mL；Figure 1B the results show that
0,2,4,6,8,9,10 μ g/mL CDDP processing cell is respectively adopted, cell survival rate is in decreasing trend, and IC50For 9 μ g/mL；
Fig. 1 C is the results show that using the two IC50Concentration (i.e. 200ng/mL BFA+4.5 μ g/mL CDDP) Combined Treatment of each half is thin
Born of the same parents, cell survival rate further decrease, and show that BFA and CDDP drug combination significantly suppresses the growing multiplication of ovarian cancer cell.
As (D1, D2, D3 and D4 quadrant respectively represent living cells, viable apoptotic cell, non-viable apoptotic cell and necrosis to Fig. 2
Cell percentage) the results show that compared with the control group, total apoptosis rate of BFA group, CDDP group and BFA+CDDP group cell is respectively
4.4%, 6.3%, 17.8%, it is all remarkably higher than control group 2.9%；And total apoptosis rate of BFA+CDDP group cell is also significantly higher than
Independent medication group BFA group and CDDP group show that BFA and CDDP drug combination significantly increases the apoptosis rate of ovarian cancer cell.
As Fig. 3 (* and * * indicate compared with the control group, P < 0.05 and P < 0.01；# and ## indicate compared with drug combination group,
P < 0.05 and P < 0.01) Bax be in Bcl2 protein family promote Apoptosis the factor.Caspase3 is in apoptosis process
Most important terminal shears enzyme, and shearing occurs and is activated, the generation finally induced cell apoptosis.Fig. 3 is the results show that BFA+
The expression of CDDP drug combination group cell Bax mRNA and Caspase3mRNA are significantly higher than independent medication group, show BFA
The up-regulation of gene participating in apoptosis expression is significantly promoted with CDDP drug combination.As (* and * * is indicated and compareed Fig. 4
Group is compared, P < 0.05 and P < 0.01；# and ## indicates P < 0.05 and P < 0.01 compared with drug combination group) Pro-caspase3 egg
White is the precursor of Caspase3 albumen, when Apoptosis occurs, shearing occurs and is activated to form splitting for Caspase3 albumen
Solve albumen, i.e. Cleaved-caspase3 albumen.Fig. 4 is the results show that compared with the control group, BFA+CDDP drug combination group cell
Bax protein expression level is also significantly higher than each independent medication group, and Pro-caspase3 protein expression level is also substantially less than each list
Private medicine group, Cleaved-caspase3 protein expression level are also significantly higher than each independent medication group, show BFA and CDDP joint
Medication significantly promotes the up-regulation of cell death related protein expression, promotes the generation of Apoptosis.
Claims (5)
1. a kind of pharmaceutical composition for inhibiting ovarian cancer cell, it is characterised in that: the composition is luxuriant and rich with fragrance by the mine-laying of 200ng/mL
The cisplatin combination of moral rhzomorph A and 4.0~4.5 μ g/mL form.
2. the pharmaceutical composition according to claim 1 for inhibiting ovarian cancer cell, it is characterised in that: described pharmaceutical composition
Application in terms of inhibiting ovarian cancer cell growth proliferation.
3. the pharmaceutical composition according to claim 1 for inhibiting ovarian cancer cell, it is characterised in that: described pharmaceutical composition
Application in terms of promoting ovarian cellular apoptosis.
4. the pharmaceutical composition according to claim 1 for inhibiting ovarian cancer cell, it is characterised in that: described pharmaceutical composition
In the application for promoting ovarian cellular apoptosis protein expression.
5. the pharmaceutical composition according to claim 1 for inhibiting ovarian cancer cell, it is characterised in that: described pharmaceutical composition
In the application for promoting ovarian cellular apoptosis related gene expression.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111296356A (en) * | 2019-12-03 | 2020-06-19 | 北京银丰鼎诚生物工程技术有限公司 | Method for establishing rat ovarian premature senility model by using cisplatin |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000015766A1 (en) * | 1998-09-16 | 2000-03-23 | Oncopharmaceutical, Inc. | Treatment of oncologic tumors with an injectable formulation of a golgi apparatus disturbing agent |
| CN103739644A (en) * | 2013-12-31 | 2014-04-23 | 浙江工业大学 | Brefeldin A glycosylated derivative and preparation and application thereof |
-
2019
- 2019-01-02 CN CN201910003046.1A patent/CN109498618A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000015766A1 (en) * | 1998-09-16 | 2000-03-23 | Oncopharmaceutical, Inc. | Treatment of oncologic tumors with an injectable formulation of a golgi apparatus disturbing agent |
| CN103739644A (en) * | 2013-12-31 | 2014-04-23 | 浙江工业大学 | Brefeldin A glycosylated derivative and preparation and application thereof |
Non-Patent Citations (4)
| Title |
|---|
| TSENG CN 等: "Brefeldin A Induces Apoptosis by Activating the Mitochondrial and Death Receptor Pathways and Inhibits Focal Adhesion Kinase-Mediated Cell Invasion", 《BASIC & CLINICAL PHARMACOLOGY & TOXICOLOGY》 * |
| 徐路 等: "线粒体相关内质网膜在顺铂诱导卵巢癌细胞凋亡中的作用", 《中国病理生理杂志》 * |
| 郑翔: "布雷菲德菌素A通过线粒体凋亡途径增强顺铂抗肺癌细胞的作用", 《中国生物化学与分子生物学报》 * |
| 陈慧 等: "《妇产科基础与实践》", 30 June 2017 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111296356A (en) * | 2019-12-03 | 2020-06-19 | 北京银丰鼎诚生物工程技术有限公司 | Method for establishing rat ovarian premature senility model by using cisplatin |
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