and C.O. outcome of GC patients treated with chemotherapy. 0.05), whereas no differential response was observed in response to ML-109 5-FU treatment (Figure 3B). Moreover, MKN74_CD44v6 cells also exhibited decreased apoptosis levels in response to cisplatin in comparison to both isogenic counterparts, 0.001 (Figure 3C). These results indicate that de novo expression of CD44v6 in GC cells allows tolerating cisplatin treatment. To confirm these ML-109 results in a model that better mimics the natural context, we modeled the expression of CD44v6 in two non-edited GC cells that endogenously overexpress CD44v6 in all their cells (GP202 and MKN45; Figure S1) and treated them with cisplatin. Specific siRNAs were used to inhibit CD44v6 expression, with 50% and 90% inhibition levels being obtained for MKN45 and GP202, respectively (Figure 4A). CD44v6 depleted cells displayed higher cisplatin-induced apoptosis levels when compared to siRNA scramble controls, 0.001 (Figure 4B), indicating that CD44v6 expression inhibition renders cancer cells more sensitive to the effects of this drug. Although the siRNAs we used in this experiment can efficiently inhibit the expression of CD44v6, our results could have been reinforced if an additional set of CD44v6 siRNAs had also been used. Nevertheless, taken together, these results consistently indicate that, in these GC controlled models, CD44v6 modulates response ML-109 to cisplatin treatment. Open in a separate window Figure 3 Assessing the response of the isogenic MKN74 cells to conventionally used chemotherapeutic agents: (A) Percentage cell survival upon incubation with cisplatin or (B) 5-FU for 48 h (compared to vehicle control) in MKN74 cells; (C) Percentage of apoptotic cells in MKN74 cells incubated with 10 M cisplatin or vehicle (0.9% NaCl) for 48 h. Results are expressed as the average + SD of at least three independent experiments. Statistically significant results were determined by Two-way ANOVA with Tukeys multiple comparisons test (* 0.05; ** 0.001; **** 0.0001). Open in a separate window Figure 4 CD44v6-induced modulation to cisplatin response in GC cells is possibly mediated by pSTAT3 or pP38. (A) Immunofluorescence and Western blotting of CD44v6 in GP202 (left) and MKN45 (right) upon incubation with CD44v6 specific siRNAs, compared with incubation with scramble siRNA. Expression inhibition ML-109 levels were, on average, ~90% and ~50% for GP202 and MKN45, respectively, in two independent experiments for each cell line. CD44v6 is represented in green; (B) Percentage of apoptotic cells in GP202 and MKN45 cell lines in response to 48 h treatment with cisplatin (concentrations ML-109 selected according to Figure S3) or vehicle, following a 24 h incubation with scramble or CD44v6 siRNAs. Results are expressed as the average + SD of at least three independent experiments. Statistically significant results were determined by Two-way ANOVA with Tukeys multiple comparisons test (* 0.05; ** 0.001; **** 0.0001); (C) Immunofluorescence of pP38 (seen in red) in GC cell lines treated with vehicle control or with cisplatin for 48 h (following a 24 h pre-incubation with scramble or CD44v6 siRNAs). Percentage of cells with nuclear pP38 expression is shown underneath the corresponding experimental conditions; (D) Western blotting of pSTAT3 in MKN74 isogenic cell lines in vehicle control and following 24 and 48 h with 10 M cisplatin treatment. CD44v6 and pSTAT3 were run in the same gel against the same tubulin; (E) Immunofluorescence of pSTAT3 (seen in green) in vehicle and cisplatin treated cell lines. In all immunofluorescence images, nuclei are stained with DAPI (seen in blue) and white scale bars represent a distance of 50 m. 2.3. CD44v6-Induced Modulation to Cisplatin Response in GC Cells is Possibly Mediated by pSTAT3 or pP38 Since activation of STAT3 or P38 signaling pathways have been described as mediators of survival in response to cisplatin, we evaluated the levels of pSTAT3 and/or pP38 in the MKN74 isogenic model and in the GC cells that endogenously overexpress CD44v6, GP202 and MKN45 (Figure 4CCE). Regarding the isogenic model, none of the three cell lines present the phosphorylated form of STAT3, pSTAT3, at basal Pdgfra level (Figure 4D,E; and previously observed in Figure 2E). However, following 24 h of cisplatin treatment, pSTAT3 levels increased in MKN74_CD44v6 overexpressing.