Depletion of HAS2 in HCT116 and DLD1 cells, which express high levels of HAS2, critically increased sensitivity of radiation/oxaliplatin\mediated apoptotic cell death. the novel mechanisms behind the constitutive activation of HAS2 signaling in CRC, thereby highlighting its potential as a therapeutic target. values?0.05 were considered significant. 3.?RESULTS 3.1. HAS2 expression correlates with the malignant phenotype of colorectal cancer tissues and cell lines To investigate the role of HAS2 in CRC, we first compared the expression levels of HAS2 between neoplastic and nonCneoplastic tissues of human CRC patients, by immunohistochemistry (IHC). Importantly IHC analysis revealed that various CRC tissues show higher expression of HAS2 in neoplastic tissues compared to nonCneoplastic tissues (Figure?1A). A similar observation was made in the CRC patient tissue samples using western blot analysis (Figure?1B). Further, we analyzed the levels of HAS2 in six different CRC cell lines. Notably, western blot analysis and immunostaining experiments revealed that four cell lines (HD29, WiDr, DLD1 and HCT116) expressed higher levels of HAS2 compared with SW480 and RKO CRC cells (Figure?1C,D). Interestingly, when these CRC cell lines were exposed individually to ionizing radiation (10?Gy) or oxaliplatin (40?mol/L), an increased apoptotic cell death was observed in SW480 and RKO Rabbit Polyclonal to ACSA CRC cells that express comparatively low levels of HAS2 (Figure?1E). Taken together, these results demonstrate that HAS2 levels correlate with the malignant phenotype of CRC. Open in a separate window Figure 1 Correlation of hyaluronic acid synthase 2 (HAS2) with the colorectal cancer (CRC) tissues and cell lines. Zaldaride maleate A, Immunohistochemistry of HAS2 expression in nonCneoplastic and CRC tissue samples (n?=?32) and scores for HAS2 staining. Scale bars: 50?m. B, Western blot analysis of HAS2 expression levels in 10 paired CRC and tumor\free tissue samples. (C) Western blot analysis and (D) immunofluorescence staining for Zaldaride maleate comparison Zaldaride maleate of HAS2 levels in CRC cell lines. E, Flow cytometric analysis of the apoptosis rate in CRC cell lines after therapeutic treatment (irradiation?=?10?Gy, Oxaliplatin?=?40?mol/L). Data are presented as mean??SD from one of three independent experiments with similar results. *P?0.05 vs control; **P?0.01 vs control; ***P?0.001 vs control 3.2. Knockdown of HAS2 sensitizes colorectal cancer cells to anticancer treatment Because high levels of HAS2 were related to apoptosis of CRC in response to anticancer treatment, we postulated that HAS2 might have a role in CRC malignancy. Hence, we depleted endogenous HAS2 molecule by siRNA transfection system in the HCT116 and DLD1 cell lines, which express high levels of HAS2 (Figure?2A). Importantly, knockdown of HAS2 reduced the growing ability (Figure?2B) and colony formation efficiency of the malignant CRC cell lines (Figure?2C). Colony forming ability could be suggestive of cell responses such as proliferation, differentiation and cell death.22 Therefore, we tested whether HAS2 depletion could sensitize CRC cells to radiation and oxaliplatin. By FACS analysis using Annexin V and propidium iodide (PI) double staining, measurement of apoptosis of CRC cells after transfection with Zaldaride maleate HAS2 siRNA followed by treatment with radiation or oxaliplatin indicated an increase in apoptotic cell death (Figure?2D). In agreement with these results, the cleaved forms of caspase\3 and PARP, the hallmarks of apoptosis,23 were increased in HAS2\depleted CRC cells compared with that in the control cells (Figure?2E). Collectively, these data suggest that HAS2 depletion sensitizes CRC cells to anticancer treatment. Open in a separate window Figure 2 Hyaluronic acid synthase 2 (HAS2) is required for therapeutic resistance of colorectal cancer (CRC) cell lines. A, Western blot analysis for validation of HAS2 siRNA efficacy. B, Growth of HCT116 and DLD1 CRC cell lines transfected with siRNA against HAS2 (for 4?d). C, Quantification of colony formation in HAS2 knockdown HCT116 and DLD1 cells. D, Apoptosis assay after therapeutic treatment of HCT116 and DLD1 CRC cells transfected with siHAS2. E, Western blot analysis for the expression of cleaved caspase3 and cleaved PARP in HAS2\depleted HCT116 and DLD1 cells treated with irradiation and oxaliplatin. Data are presented as mean??SD from one of three independent experiments with similar results. *P?0.05 vs control; **P?0.01 vs control; ***P?0.001 vs control 3.3. Knockdown of HAS2 suppresses metastatic ability of colorectal cancer cells through epithelial\mesenchymal transition To further investigate the role of HAS2 in CRC malignancy regulation, we next examined the effect of HAS2 on the metastatic ability of CRC. To this end, we first investigated migration and invasion of HCT116 and DLD1 CRC cells after transfection with HAS2 siRNA. By Boyden chamber assay, we observed that siRNA\mediated HAS2 depletion effectively suppresses migration and invasion.