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Cantharidin induces apoptosis and suppresses cell migration and invasion of MDA-MB-231 human breast cancer cells through inhibition of EGFR-STAT3 signaling

DC Field Value Language
dc.contributor.advisor김영식-
dc.contributor.author박민경-
dc.date.accessioned2017-10-31T08:19:06Z-
dc.date.available2020-10-06T10:26:58Z-
dc.date.issued2017-08-
dc.identifier.other000000145531-
dc.identifier.urihttps://hdl.handle.net/10371/137927-
dc.description학위논문 (석사)-- 서울대학교 대학원 약학대학 약학과, 2017. 8. 김영식.-
dc.description.abstractBreast cancer is the most frequently diagnosed life-threatening cancer in women. Common chemotherapeutic agents target three receptors: estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2). Breast cancer cells that lack ER, PR and HER2 are referred to as triple negative breast cancer (TNBC). TNBC occurs in about 15~20 % of diagnosed breast cancer. TNBC is known to increase recurrence and mortality rate within 5 years of cancer detection and is thus considered to have poorer prognosis. Recently, the signal transducer and activator of transcription 3 (STAT3) is reported as a key factor in TNBC treatment, due to high levels of STAT3 expression in TNBC.
Bioassay-guided fractionation and purification were performed to isolate the cytotoxic compound from cantharides. The dried cantharides were crushed and extracted with acetonitrile and then separated into methylene chloride, acetonitrile, n-hexane and water layers. Methylene chloride and acetonitrile layerwhich had strong activity were further separated and purificated using various chromatographic techniques. The extract was fractionated into 4 fractions by Prep-LC, and the third fraction showing cytotoxic activity was separated using CCC (Counter-Current Chromatography). Separated fraction was structurally determined and identified as cantharidin by comparing NMR and HRMSdata with literature value.
Cantharidin is an active constituent of the blister beetles, belonging to Meloidae family, which is traditionally used to treat wart and relieve blood stasis. Moreover, numerous studies have revealed that cantharidin has a cytotoxic effect on cancer cells.
However, there have been no reports on cantharidins effect on inhibiting cell growth of TNBC. Herein, we demonstrated that cantharidin induced cell death in one of TNBC cells, MDA-MB-231, by suppressing STAT3 activation. The result showed that cantharidin reduced STAT3 tyrosine-705 in MDA-MB-231 cells.Cantharidinsignificantly inhibited activation of EGFR, Src, and STAT3, not affecting JAK-STAT3 signaling.Moreover, cantharidin inhibits cell proliferation and induces apoptosis by regulating a transcription of STAT3 target genes such as cox-2, cyclin D1, bcl-2, caspase-3 and parp1.
Taken together, this study provides that cantharidin may be used as a potential therapeutic agent against TNBC, especially MDA-MB-231 cells, by reducing EGFR-STAT3 signaling.
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dc.description.tableofcontentsI. INTRODUCTION 1
1. Triple negative breast cancer (TNBC). 1
2. Signal transducer and activator of transcription 3 (STAT3) 2
3. EGFR (Epidermal growth factor receptor) 5
4. EGFR-STAT3 pathway. 6
5. Cantharides 7
II. MATERIALS AND METHODS 8
1. MATERIALS 8
1.1. Insect materials. 8
1.2. Chemicals and reagents. 8
1.3. Instruments.. 9
1.4. Antibodies 10
2. METHODS 11
2.1. Cell culture. 11
2.2. Cell viability assay 11
2.3. Preparation of nuclear and cytosolic fractions. 12
2.4. Luciferase reporter assay 12
2.5. Western blot analysis 13
2.6. Wound-healing assay 14
2.7. Annexin V & PI analysis 15
2.8. Invasion assay. 15
2.9. Extraction 16
2.10. Preparative HPLC separation 17
2.11. Measurement of the partition coeffient (K) 17
2.12. HPCCC separation. 18
2.13. Identification of isolated compound. 19
2.14. Statistical analysis. 19
III. RESULTS 20
1. Extraction and solvent partition 20
2. Separation of methylene chloride and acetonitrile fraction by preparative HPLC and the cell viability of obtained four fractions. 22
3. Separation of MA3 fraction by HPCCC and the cell viability of separated fractions 25
4. Identification of compound 28
5. Effect of cantharidin on the cell viability in MDA-MB-231 cells. 33
6. Inhibitory effect of cantharidin on STAT3 activation 35
7. Effect of cantharidin on STAT3 upstream signaling pathways. 38
8. Effect of cantharidin on STAT3 associated proteins. 41
9. Effect of cantharidin on expression of STAT3 downstream genes. 43
10. Induction of caspase-mediated apoptosis by cantharidin. 45
11. Effect of cantharidin on changes in cell morphology and apoptosis. 47
12. Effect of cantharidin on cell migration. 51
13. Effect of cantharidin on cell invasion. 53
IV. DISCUSSION. 55
V. CONCLUSION. 60
REFERENCES. 61
ABSTARCT IN KOREAN. 68
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dc.formatapplication/pdf-
dc.format.extent1495717 bytes-
dc.format.mediumapplication/pdf-
dc.language.isoen-
dc.publisher서울대학교 대학원-
dc.subjectTriple negative breast cancer-
dc.subjectMDA-MB-231-
dc.subjectEGFR-
dc.subjectSTAT3-
dc.subjectCantharidin-
dc.subjectApoptosis-
dc.subjectBioassay-guided-
dc.subjectHPCCC-
dc.subject.ddc615-
dc.titleCantharidin induces apoptosis and suppresses cell migration and invasion of MDA-MB-231 human breast cancer cells through inhibition of EGFR-STAT3 signaling-
dc.typeThesis-
dc.description.degreeMaster-
dc.contributor.affiliation약학대학 약학과-
dc.date.awarded2017-08-
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College of Pharmacy (약학대학)Dept. of Pharmacy (약학과)Theses (Master's Degree_약학과)
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