16P - Carvacrol induces growth inhibition and circumvents chemoresistance via inhibition of STAT3/Skp2/p27 pathway in non-small lung cancer cells

Date 07 May 2017
Event ELCC 2017
Session Poster Display Session
Topics Non-Small-Cell Lung Cancer, Metastatic
Lung and other Thoracic Tumours
Pathology/Molecular Biology
Presenter Kyung-Chan Kim
Citation Annals of Oncology (2017) 28 (suppl_2): ii1-ii5. 10.1093/annonc/mdx090
Authors K. Kim1, C. Lee2
  • 1Internal Medicine, Daegu Catholic University Hospital, 42472 - Daegu/KR
  • 2Biochemistry & Molecular Biology, Yeungnam University School of Medicine, 42415 - Daegu/KR

Abstract

Background

S-phase kinase-associated protein 2 (Skp2) which constitues SCF complex and plays a role to recognize the substrates has been known to act as a proto-oncogene. In this study, we examined the effect of carvacrol, an active compound of oregano, on Skp2 inactivation and the underlying mechanisms in non-small lung cancer cells.

Methods

Reagents and antibodies. Carvacrol was obtained from Sigma-Aldrich. A549 and H460 cells were purchased from the ATCC. For Western blot analysis, specific antibodies against Skp2, phospho-STAT3, STAT3, p21, p27, and GAPDH, as well as secondary antibodies were obtained from Santa Cruz Biotechnology.

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Cell viability measurement. Cells were treated with carvacrol for 24 h and stained wtih 0.4% Trypan blue solution. Dye-excluding viable cells were counted under the microscope.

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Clonogenic assay. Cells were seeded into 24 well plates and treated with carvacrol and then cultured for the next 7 to 10 days to form colonies. Colonies of > 50 cells were stained with crystal violet.

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Ectopic expression of Skp2. To ectopically express Skp2, the recombinant plasmid, pcDNA3-Skp2-myc, was constructed and transfected into cells using Lipofectamine 2000. To establish stable cell lines, the transfected cells were cultured in the presence of 400 μg/ml of G418.

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siRNA transfection. To reduce Skp2 expression, cells were transfected with siRNA targeting Skp2 or control siRNA.

Results

Carvacrol treatment of A549 as well as H460 cells caused to reduce Skp2 protein level in dose-dependent manner. RT-PCR assay was also found that Skp2 mRNA level was reduced by carvacrol, suggesting the transcriptional down-regulation of Skp2 expression by carvacrol. We next found that the cytotoxic effect of carvacrol was attenuated in Skp2 overexpression in A459 cells and further observed its synergistic anti-proliferative effect in cells transfected with Skp2 specific siRNA. In addition, carvacrol was found to result in apoptotic cell death.

Conclusions

Taken together, these data indicate that carvacrol targets Skp2 to inhibit cell proliferation and to cause apoptotic cell death in non-small lung cancer cells.

Clinical trial identification

Legal entity responsible for the study

Yeungnam University College of Medicine, Republic of Korea

Funding

Yeungnam University College of Medicine, Republic of Korea

Disclosure

All authors have declared no conflicts of interest.