Bottom, data represent the quantitative results for the top panel. FL prazosin accumulation assays. Moreover, the cisplatin-resistant cells underwent morphological changes, from round to spindle-shaped, increased expression of epithelial-to-mesenchymal transition (EMT)-related molecules such as N-cadherin, and showed increased cell migration when compared with the parental cell lines. These results suggest that these newly established cell lines have acquired drug resistance and EMT induction. = 5). Cisplatin-resistance was observed GV-196771A in YD-8/CIS, YD-9/CIS, and YD-38/CIS cells. Resistance to 5-fluorouracil, paclitaxel, and CKD-602 was not obtained in YD-8 and YD-38 cells. YD-9/CIS cells only showed cross-resistance to paclitaxel and CKD-602. * < 0.01 versus parental cells. 2.2. MDR-Related Gene Expression Was Altered in the Cisplatin-Resistant Cell Lines. MDR1 Expression Increased in YD-8/CIS and YD-9/CIS Cells, and BCRP Levels Increased in YD-8/CIS and YD-38/CIS Cells Next, we investigated the expression of MDR-related genes in both the parental and cisplatin-resistant cell lines. The mRNA expression of MDR-related genes was investigated by qPCR (Physique 2a). In YD-8/CIS cells, (>2.4-fold change), (>7.2-fold change) and (>2.5-fold change) were upregulated. In YD-9/CIS cells, (>7.5-fold change), (>2.7-fold change), and (>1.6-fold change) were upregulated. In YD-38/CIS cells, (>3.7-fold change) was upregulated. We confirmed the GV-196771A expression changes in and by Western blot (Physique 2b). BCRP was upregulated in YD-8/CIS and YD-38/CIS cells, while MDR1 was downregulated only in YD-38/CIS cells. Conversely, MDR1 was overexpressed in YD-8/CIS and YD-9/CIS cells. Our results show that compared with their parental cells, BCRP was upregulated in YD-8/CIS and YD-38/CIS cells, while MDR1 was upregulated in YD-9/CIS cells. Open in a separate window Physique 2 Differences in expression of multi-drug resistant (MDR)-related genes and proteins in parental and cisplatin-resistant OSCC cell lines. (a) The expression of MDR-related genes was measured by qPCR (imply SD; = 3). * < 0.01 versus parental cells. (b) Left, the expression of MDR1 and BCRP proteins was determined by a Western blot assay. Right, data represent quantitative results for left panel. Exposure to cisplatin induces upregulation of BCRP in YD-8 and YD-38 cells, and upregulation of MDR1 in YD-8 and YD-9 cells. 2.3. Acquisition of Resistance to Cisplatin Increases MDR1 Activity in YD-9/CIS Cells and BCRP Activity in YD-8/CIS, YD-9/CIS, and YD-38/CIS Cells We also examined MDR1 protein activity using rhodamine 123, and BCRP protein activity using bodipy FL prazosin. Physique 3a shows that the accumulation of rhodamine 123 was reduced in YD-9/CIS cells (34.4%) compared to parental cells (54.2%). In the GV-196771A three cisplatin-resistant cell lines, the intracellular accumulation of bodipy FL prazosin was less than in their parental cell lines (Physique 3b; YD-8/CIS, 17.5%; YD-8, 65.0%; YD-9/CIS, 25.8%; YD-9, 66.0%; YD-38/CIS, 52.0%; YD-38, 78.2%). These results show that YD-8, YD-9, and YD-38 cells developed cisplatin-resistance by increasing the activity of MDR1 or BCRP. Open in a separate window Physique 3 Functional assay for multidrug resistance protein1 or P glycoprotein (MDR1) and breast cancer resistance protein (BCRP) expression in parental and cisplatin-resistant OSCC cell lines. (a) Top, a rhodamine123 assay was used to monitor MDR1 activity. Bottom, data represent the quantitative results for the top panel. (b) HDAC5 Top, a prazosin assay was used to monitor BCRP activity. Bottom, data represent the quantitative results for the top panel. Cells were incubated for 30 min with growth medium alone (blank, thin solid collection), or 1 g/mL rhodamine123, or 250 nM prazosin (heavy solid collection). Cisplatin treatment is sufficient to induce MDR1 activity GV-196771A in YD-9 cells and BCRP activity in YD-8, YD-9, and YD-38 cells (imply SD; = 3). * < 0.01 versus parental cells. 2.4. Cell Lines Which Acquired Cisplatin-Resistance Displayed Increased EMT-Related Markers, Including Cell Mobility, Increased N-Cadherin Expression, and Decreased E-Cadherin Expression It has been reported that this acquisition of chemoresistance is usually associated with an EMT phenotype. We assessed this in our cell lines by immunofluorescence, Western blot, and wound healing assays. Physique 4a,b shows morphological changes between.