大部分的口腔癌病患死於原位癌的轉移和擴散，尋找有效抗癌和抗轉移的藥物療法來增進病患的生活品質和存活率，對治療口腔癌而言非常重要。爵床草是台灣民間常用草藥，常被用來消炎止熱和治療口瘡、乳瘤。我們利用MTS分析，發現處以0.3 μM的JA 48小時，即可有效抑制口腔癌細胞OC-2和HSC3的增生，但不影響正常口腔上皮細胞生長。OC-2經JA處理後，其sub-G1的DNA含量增加，並可觀察到DNA固縮和梯度斷裂的現象，說明JA可使OC-2走向細胞凋亡。除此之外，0.3 μM的JA亦可有效降低OC-2的遷移能力。利用流式細胞儀觀察細胞膜表面，發現JA使得細胞表面的α3、β1和α2 integrin的表現增加。以抗體阻斷integrin α3可以有效使OC2恢復被JA所抑制的遷移能力。細胞貼附實驗指出JA能提高OC-2在laminin環境下的貼附能力。當我們進一步以免疫螢光染色觀察細胞，發現在JA處理下的OC-2，其外形、細胞骨架和細胞邊緣的filopodia的密度和長度明顯減少，利用GST-pull down assay偵測到Cdc42-GTP和Rac-GTP的表現量降低。推論爵床素A可能透過刺激口腔癌細胞大量表現integrin α3，使細胞貼附能力增加或是透過改變細胞骨架來降低細胞遷移能力。希望本研究對於JA所做的抗癌及抗轉移效應評估能提供口腔癌治療在研發上一些幫助。

Most patients’ death arise from the metastatic spread of primary tumors. There is an urgent need for the finding of novel anti-tumor and anti–metastatic drugs to improve the quality of life and survival rate of patents. Justicia procumbens is an herb and used to reduce fever, inflammation, and canker in Taiwan. We’ve detected that 0.3 μM JA could effectively inhibit the proliferation of oral cancer cells, OC-2 and HSC3, but not normal oral keratinocytes in MTS assay. JA-treated OC2 underwent apoptosis as suggested by increased sub-G1 DNA contents, condensed nuclear morphologh and DNA fragmentiation. The migration activity of OC-2 was also significantly reduced by treatment with low concentration of JA. The expression of α3-, β1- and α2-integrin on the surface of OC-2 was assessed by flow cytometry and showed to be increased by a JA treatment. Blocked integrin α3 can reverse JA-induced migration inhibition. JA-treated cells also showed significantly higher adhesion to laminin. In conclusion, these results indicate a novel anti-tumor role of JA via reducing reorganization of cytoskeleton, increasing integrin 3 expression and adhesion ability, to inhibit tumor cells migration. Furthermore, immunofluorescence staining of phalloidin/actin indicated that JA-treated cells exhibited cytoskeleton rearrangement and significant decreases in the number and length of filopodia. Activity of Rho-family small GTPases was detected by a GST-pull down assay and showed that GTP-loaded Cdc42 and Rac were decreased in JA-treated cells. In the future, we hope these findings will be helpful to further understand the mechanisms underlying tumor progression in oral cancer, and subsequently improve the cancer prevention and treatment.

1. Ziober, A.F., Falls, E.M. & Ziober, B.L. The extracellular matrix in oral squamous cell carcinoma: friend or foe? Head Neck 28, 740-9 (2006).
2. Renner, G. Small cell carcinoma of the head and neck: a review. Semin Oncol 34, 3-14 (2007).
3. Bsoul, S.A., Huber, M.A. & Terezhalmy, G.T. Squamous cell carcinoma of the oral tissues: a comprehensive review for oral healthcare providers. J Contemp Dent Pract 6, 1-16 (2005).
4. Schwentner, I., Obrist, P., Thumfart, W. & Sprinzl, G. Distant metastasis of parotid gland tumors. Acta Otolaryngol 126, 340-5 (2006).
5. Lippman, S.M. & Lee, J.J. Reducing the "risk" of chemoprevention: defining and targeting high risk--2005 AACR Cancer Research and Prevention Foundation Award Lecture. Cancer Res 66, 2893-903 (2006).
6. Eccles, S.A. & Welch, D.R. Metastasis: recent discoveries and novel treatment strategies. Lancet 369, 1742-57 (2007).
7. Gilcrease, M.Z. Integrin signaling in epithelial cells. Cancer Lett 247, 1-25 (2007).
8. Ridley, A.J. & Hall, A. The small GTP-binding protein rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors. Cell 70, 389-99 (1992).
9. Etienne-Manneville, S. & Hall, A. Rho GTPases in cell biology. Nature 420, 629-35 (2002).
10. Tsao, A.S., Kim, E.S. & Hong, W.K. Chemoprevention of cancer. CA Cancer J Clin 54, 150-80 (2004).
11. Mohagheghzadeh, A., Schmidt, T.J. & Alfermann, A.W. Arylnaphthalene lignans from in vitro cultures of Linum austriacum. J Nat Prod 65, 69-71 (2002).
12. Asano, J., Chiba, K., Tada, M. & Yoshii, T. Antiviral activity of lignans and their glycosides from Justicia procumbens. Phytochemistry 42, 713-7 (1996).
13. Day, S.H., Chiu, N.Y., Won, S.J. & Lin, C.N. Cytotoxic lignans of Justicia ciliata. J Nat Prod 62, 1056-8 (1999).
14. Su, C.L. et al. Caspase-8 acts as a key upstream executor of mitochondria during justicidin A-induced apoptosis in human hepatoma cells. FEBS Lett 580, 3185-91 (2006).
15. Lee, J.C. et al. Justicidin A decreases the level of cytosolic Ku70 leading to apoptosis in human colorectal cancer cells. Carcinogenesis 26, 1716-30 (2005).
16. Giannelli, G. et al. Role of the alpha3beta1 and alpha6beta4 integrins in tumor invasion. Clin Exp Metastasis 19, 217-23 (2002).
17. Hood, J.D. & Cheresh, D.A. Role of integrins in cell invasion and migration. Nat Rev Cancer 2, 91-100 (2002).
18. Chambers, A.F., MacDonald, I.C., Schmidt, E.E., Morris, V.L. & Groom, A.C. Clinical targets for anti-metastasis therapy. Adv Cancer Res 79, 91-121 (2000).
19. Fulda, S. & Debatin, K.M. Extrinsic versus intrinsic apoptosis pathways in anticancer chemotherapy. Oncogene 25, 4798-811 (2006).
20. Leist, M. & Nicotera, P. The shape of cell death. Biochem Biophys Res Commun 236, 1-9 (1997).
21. Kerr, J.F., Winterford, C.M. & Harmon, B.V. Apoptosis. Its significance in cancer and cancer therapy. Cancer 73, 2013-26 (1994).
22. Rupinder, S.K., Gurpreet, A.K. & Manjeet, S. Cell suicide and caspases. Vascul Pharmacol 46, 383-93 (2007).
23. Riedl, S.J. & Salvesen, G.S. The apoptosome: signalling platform of cell death. Nat Rev Mol Cell Biol 8, 405-13 (2007).
24. Hansen, L.A. et al. Retinoids in chemoprevention and differentiation therapy. Carcinogenesis 21, 1271-9 (2000).
25. Massimi, M. & Devirgiliis, L.C. Adhesion to the extracellular matrix is positively regulated by retinoic acid in HepG2 cells. Liver Int 27, 128-36 (2007).
26. Janes, S.M. & Watt, F.M. New roles for integrins in squamous-cell carcinoma. Nat Rev Cancer 6, 175-83 (2006).
27. Levy, L., Broad, S., Diekmann, D., Evans, R.D. & Watt, F.M. beta1 integrins regulate keratinocyte adhesion and differentiation by distinct mechanisms. Mol Biol Cell 11, 453-66 (2000).
28. Watt, F.M. Role of integrins in regulating epidermal adhesion, growth and differentiation. Embo J 21, 3919-26 (2002).
29. Adachi, M. et al. Reduced integrin alpha3 expression as a factor of poor prognosis of patients with adenocarcinoma of the lung. J Clin Oncol 16, 1060-7 (1998).
30. Ura, H., Denno, R., Hirata, K., Yamaguchi, K. & Yasoshima, T. Separate functions of alpha2beta1 and alpha3beta1 integrins in the metastatic process of human gastric carcinoma. Surg Today 28, 1001-6 (1998).
31. Owens, D.M. & Watt, F.M. Influence of beta1 integrins on epidermal squamous cell carcinoma formation in a transgenic mouse model: alpha3beta1, but not alpha2beta1, suppresses malignant conversion. Cancer Res 61, 5248-54 (2001).
32. Dedhar, S., Saulnier, R., Nagle, R. & Overall, C.M. Specific alterations in the expression of alpha 3 beta 1 and alpha 6 beta 4 integrins in highly invasive and metastatic variants of human prostate carcinoma cells selected by in vitro invasion through reconstituted basement membrane. Clin Exp Metastasis 11, 391-400 (1993).
33. Mehlen, P. & Puisieux, A. Metastasis: a question of life or death. Nat Rev Cancer 6, 449-58 (2006).
34. Boutahar, N., Guignandon, A., Vico, L. & Lafage-Proust, M.H. Mechanical strain on osteoblasts activates autophosphorylation of focal adhesion kinase and proline-rich tyrosine kinase 2 tyrosine sites involved in ERK activation. J Biol Chem 279, 30588-99 (2004).
35. Nobes, C.D. & Hall, A. Rho, rac, and cdc42 GTPases regulate the assembly of multimolecular focal complexes associated with actin stress fibers, lamellipodia, and filopodia. Cell 81, 53-62 (1995).
36. Wiesner, S., Legate, K.R. & Fassler, R. Integrin-actin interactions. Cell Mol Life Sci 62, 1081-99 (2005).
37. Scott, G.K. et al. Coordinate suppression of ERBB2 and ERBB3 by enforced expression of micro-RNA miR-125a or miR-125b. J Biol Chem 282, 1479-86 (2007).
38. Tran, N. et al. MicroRNA expression profiles in head and neck cancer cell lines. Biochem Biophys Res Commun 358, 12-7 (2007).