1. Volume 55, Issue 2, Pages 441-451 (February 2009)
Metastin Inhibits Migration and Invasion of Renal Cell Carcinoma with Overexpression of Metastin Receptor 
Sunao Shoji, Xian Yan Tang, Shinobu Umemura, Johbu Itoh, Susumu Takekoshi, Masanori Shima, Yukio Usui, Yoshihiro Nagata, Toyoaki Uchida, Robert Yoshiyuki Osamura, Toshiro Terachi 
European Urology 
Volume 55, Issue 2, Pages (February 2009)
DOI: /j.eururo
Copyright © 2008 European Association of Urology Terms and Conditions

2. Fig. 1
Expression level of metastin and metastin receptor in renal cell carcinoma (RCC). (A) Result of reverse transcription-polymerase chain reaction (RT-PCR) for detection of metastin receptor messenger RNA (mRNA) in RCC and paired kidney cortex samples. The metastin receptor mRNA ratio (mRNA copy of metastin receptor ratio to β-actin) was prominently higher in the RCC samples (mean±SD: 118±948×10−4%) than in the control normal tissue samples (mean±SD: 3.00±29.0×10−4%) (p=0.011). (B) Result of RT-PCR for detection of metastin. Metastin mRNA ratio (mRNA copy of metastin ratio to β-actin) in RCC samples was not statistically different from control normal tissue samples. (C) Ratio of metastin/metastin receptor mRNA (metastin mRNA ratio/metastin receptor mRNA ratio) was also low in most of the RCC samples (mean±SD: ±0.960) compared with the control samples (mean±SD: 3.70±5.57) (p<0.001).
European Urology  , DOI: ( /j.eururo )
Copyright © 2008 European Association of Urology Terms and Conditions

3. Fig. 2
Immunohistochemical study of metastin and metastin receptor. Both renal cell carcinoma (RCC) cells (A) and renal tubule cells (B) are positively stained for metastin. But, metastin receptor is strongly stained in RCC cells (C) compared with renal tubule cells (D).
European Urology  , DOI: ( /j.eururo )
Copyright © 2008 European Association of Urology Terms and Conditions

4. Fig. 3
Migration and invasive assays. (A) Migration assay of Caki-1 and ACHN cells: 14h (Caki-1) and 16h (ACHN) after the experimental wounds were made, the average numbers of the re-migrated cells in the spaces between scraped edges on both sides of the wound were evaluated. The photograph and the graph show the numbers of re-migrated cells are in a metastin dose-dependent pattern. (B) Matrigel invasive assay. The numbers of invaded Caki-1 and ACHN cells at the reverse side of the membrane were counted. Fewer cells invaded the membrane in 1000nmol/l metastin chambers than in metastin-negative (metastin 0nmol/l) chambers.
European Urology  , DOI: ( /j.eururo )
Copyright © 2008 European Association of Urology Terms and Conditions

5. Fig. 4
Visualization of focal adhesions and stress (actin) fibers. (A and D) Expression of actin fibers (rhodamine phalloidin–positive red fibers), focal adhesions (vinculin-positive cyan patches), and microtubules (α-tubulin–positive, green) in Caki-1 and ACHN cells were observed. (B and E) After treatment with metastin, excessive formation of actin fibers (rhodamine phalloidin–positive red fibers) and focal adhesions (vinculin-positive cyan patches) were identified in Caki-1 and ACHN cells. (C and F) When supplied with metastin and Rho-kinase inhibitor (Y-27623), the actin fibers and focal adhesions were prominently decreased in number. In contrast, microtubules (α-tubulin–positive, green) show no significant changes after being supplied with metastin or metastin+Rho-kinase inhibitor Y (A–F, third column).
European Urology  , DOI: ( /j.eururo )
Copyright © 2008 European Association of Urology Terms and Conditions

6. Fig. 5
Schematic explanation of the mechanism of the effect of metastin on RCC cells. (A) Rho-family proteins regulate cell adhesion and motility through sequential activation of its family members, resulting in the formation of stress fiber and focal adhesion patches. (B) Metastin stimulates RhoA and Rho kinase, resulting in the induction of excessive formations of stress fiber and focal adhesion. At the same time, metastin inhibits Rac1, and the formation of filopodia and lamellipodia are inhibited. (C) The Rho-kinase inhibitor (Y-27623) inhibits downstream of RhoA, and stress fiber formation and focal adhesion patches are decreased.
European Urology  , DOI: ( /j.eururo )
Copyright © 2008 European Association of Urology Terms and Conditions