Figure 1
MTX-induced apoptotic cell death positively correlates with WWOX expression in SCC cells. (a) SCC-4, SCC-9 and SCC-15 cells were seeded on 96-well cell culture plates. One day later, cells were treated with the indicated doses of MTX and cultured for 48 and 72 h, respectively. Cell viability was determined by crystal violet staining. The percentages of viable cells after MTX treatment were measured versus untreated control cells as described in the Methods. Representative results of six independent experiments are shown. (b) SCC-4, SCC-9 and SCC-15 cells were treated with 10 μg/ml MTX for the indicated times, followed by examining cell morphology under a light microscope ( × 200). Enlarged are representative dead cells (see the insert). Dead cells are visualized mainly as ‘whitish punctates’ in the pictures. Scale bar, 60 μm. (c) MTX-induced phosphatidylserine exposure on the surface of SCC-15, but not SCC-9 cells. SCC-9 and SCC-15 cells were treated with or without MTX (10 μg/ml) for 72 h. The binding of annexin V–FITC to phosphatidylserine on the cell surface was examined using a fluorescence microscope. (d) Western blot analysis of lysates from SCC-4, SCC-9 and SCC-15 cells was performed to determine the expression levels of endogenous WWOX protein. The specific bands in the western blots were quantified using a densitometer. The bar graph depicts the ratio of WWOX to β-actin protein levels in SCC-4, SCC-9 or SCC-15 cells, respectively. (e) SCC-4, SCC-9 and SCC-15 cells were treated with the indicated doses of MTX for 72 h. WWOX protein levels were determined using western blotting. β-Actin was used as an internal control. (f) SCC-15 and SCC-9 cells were treated with 10 μg/ml MTX and cultured for various time intervals. Total RNA was extracted from the SCC cells using TRIZOL reagent. The mRNA levels of WWOX and β-actin were determined by reverse transcription PCR. The PCR products were visualized on 1.2% agarose gels stained by ethidium bromide and quantified using a densitometer. The bar graphs depict the ratio of WWOX to β-actin mRNA levels in SCC-15 (upper panel) and SCC-9 cells (lower panel). (g) SCC-15 and SCC-9 cells were treated with 10 μg/ml MTX for 48 or 72 h. Quantification of WWOX mRNA expression in MTX-treated cells was performed by real-time PCR analysis, and data were analyzed using the comparative Ct method. (h) SCC-15 cells were treated with 10 μg/ml MTX and cultured for 24, 48 and 72 h. WWOX protein expression and the cleavage of caspase-9 and -3 were determined using western blotting. β-Actin was used as a loading control. (i) MTX-induced upregulation of WWOX protein is associated with tumor regression in patients with SCC. Continuous intra-arterial infusion of MTX (50 mg/day, 4–11 days) caused complete tumor regression in SCC patients.21, 22, 23 Cancer cells in the SCC tumor sections showed condensed chromatin within the shrinking cancer areas after intra-arterial MTX infusion (arrows in the upper right panel; H&E stain). WWOX protein expression was markedly upregulated in the cancer cells after MTX treatment, as determined by immunohistochemistry (lower panels). Scale bar, 100 μm. (j) SCC cancer cells with condensed chromatin in the tumor sections (arrows in i) showed active caspase-3 staining (upper panel) and positive TUNEL (lower panel) after intra-arterial MTX treatment. Scale bar, 20 μm. (a, f and g) Data represent means±S.D. (n=4 per group). Comparisons with untreated control were made by Student’s t-test. *P<0.05, **P<0.01 and #P<0.001
