Acta Pharmacologica Sinica (2009) 30: 1297–1306; doi: 10.1038/aps.2009.124

Acta Pharmacologica Sinica (2009) 30: 1297–1306; doi: 10.1038/aps.2009.124; published online 24 August 2009

Original Article

n-Butylidenephthalide induced apoptosis in the A549 human lung adenocarcinoma cell line by coupled down-regulation of AP-2α and telomerase activity

Chyou-wei WEI^1,^#, Chai-ching LIN^2,^#, Yung-luen YU^{3, #}, Chai-yi LIN⁴, Po-cheng LIN⁵, Min-tze WU⁶, Cheng-jueng CHEN⁷, Wen-liang CHANG⁸, Shinn-zong LIN⁹, Yi-lin Sophia CHEN^10,*, Horng-jyh HARN^11,*

¹Institute of Biomedical Nutrition, College of Medicines and Nursings, Hungkuang University, Taichung, Taiwan, China; ²Graduate Institute of Biotechnology, National Ilan University Ilan, Taiwan, China; ³Graduate Institute of Cancer Biology and Center for Molecular Medicine, China Medical University and Hospital, Taichung, Taiwan, China; Department of Biotechnology, Asia University, Taichung, Taiwan, China; ⁴Graduate Institute of Biotechnology, National Ilan University, Ilan, Taiwan, China; ⁵Graduate Institute of Biotechnology, National Dong Hwa University, Hualien, Taiwan, China; ⁶Biotechnology Division, Taiwan Agricultural Research Institute Council of Agriculture, Taichung, Taiwan, China; ⁷Division of Traumatology, Department of Surgery; Tri-Service General Hospital, Taipei, Taiwan, China; ⁸School of Pharmacy, National Defense Medical Center, Taipei, Taiwan, China; ⁹Center for Neuropsychiatry, China Medical University and Hospital, Taichung, Taiwan, China; ¹⁰Graduate Institute of Biotechnology, National Ilan University Ilan, Taiwan, China; ¹¹Department of Pathology, China Medical University and Hospital, Taichung, Taiwan, China

Aim: To investigate the role of hTERT gene expression and AP-2α in n-butylidenephthalide (n-BP)-induced apoptosis in A549 lung cancer cells.

Methods: Viability of A549 cells was measured by MTT assay. Protein expression was determined by Western blot. Telomerase activity was measured using the modified telomere repeat amplification protocol (TRAP) assay. Xenograft mice were used as a model system to study the cytotoxic effect of n-BP in vivo. The morphology of tumor was examined by immunohistochemical staining.

Results: The growth of A549 lung cancer cells treated with n-BP was significantly inhibited. Telomerase activity and hTERT mRNA expression were determined by telomeric repeat amplification protocol and reverse transcription-polymerase chain reaction, respectively. n-BP inhibited telomerase activity and hTERT mRNA expression in A549 cells while overexpression of hTERT could abolish BP-induced growth inhibition in the A549 cells. We also showed that hTERT promoter activity in the presence of n-BP was mediated via AP-2α. We saw an inhibition of tumor growth when nude mice carrying A549 subcutaneous xenograft tumors were treated with n-BP. Immunohistochemistry of this tumor tissue also showed a decrease in the expression of hTERT.

Conclusion: The antiproliferative effects of n-BP on A549 cells in vitro and in vivo suggest a novel clinical application of this compound in the treatment of lung cancers.

Keywords: human telomerase reverse transcriptase; n-butylidenephthalide; AP-2 xenograft; immunohistochemistry

This work was supported by Grants 96-2320-B-303-001-MY3 and 96-2320-B-303-002-MY2 from National Science Council of the Republic of China and was partially supported by the National Science Council Grants, NSC96 2320-B-039-032-MY3 and NSC96 3111-B-039-003 to Yung-luen YU.

Chyou-wei WEI, Chai-ching LIN, and Yung-luen YU contributed equally as first authors, and Yi-lin Sophia CHEN and Horng-jyh HARN shared equal corresponding authorship.

^#These authors contributed equally to the work.
* To whom correspondence should be addressed.
Emal a221865880@yahoo.com.tw (Yi-lin Sophia CHEN) and d15977@mail.cmuh.org.tw (Horng-jyh HARN)
Received 2009-05-31 Accepted 2009-07-14

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