Hypoxia is common in solid tumors and results in the activation of hypoxia-response genes. hypoxia-induced EMT and abolished the unique feature of GC7. GC7 enhanced sensitivity to doxorubicin in HCC by reversing hypoxia-induced EMT via the HIF-1-mediated signaling pathway. We suggest a new method of enhancing cytotoxicity of chemotherapy and improving the long-term survival rate in HCC. test were applied to assess the effects of doxorubicin and the combined treatment. Other analysis for comparing two groups was performed using Students t-tests and a P-value less than 0.05 was considered to be statistically significant. Results Hypoxia induces chemoresistance to doxorubicin and low concentrations of GC7 sensitizes HCC cells to doxorubicin To assess the role of hypoxia in chemotherapy sensitivity, we used the CCK8 assay to detect cell viability of HCC cells in different conditions. Huh7, Hep3W, SNU387 and SNU449 cells were more sensitive to doxorubicin under conditions of hypoxia compared with normoxia (Physique 1E-H). Hence, hypoxia can induce chemoresistance to doxorubicin in these four types of HCC SRT1720 HCl cell line. Physique 1 Hypoxia induces chemoresistance to doxorubicin. A-D: The cytotoxicity of GC7 SRT1720 HCl in HCC cells. HCC cells were incubated with different concentrations of GC7 for 48 h. The value of CCK8 was the treated HCC cells that were normalized to the control group treated … In a recent study, Tariq et al. indicated that hypusinated eIF5A was indispensible for the expression of HIF-1 in hypoxia [23]. As an inhibitor of active eIF5A2, GC7 has been widely used in recent studies and can reverse doxorubicin-induced EMT by inhibiting activation of eIF5A2 [22]. Thus, GC7 was applied throughout our study. As GC7 is usually cytotoxic to HCC cells, we used the CCK8 assay to initially detect an appropriate concentration of GC7. The cytotoxicity of GC7 in all four HCC cell lines was rare when a dose of 0 to 20 M was employed. However, the viability of the HCC cell lines was significantly inhibited at higher concentrations of GC7 (40-100 M) (Physique 1A-Deb). Finally, the preferred concentration (20 M) was used in our study. Interestingly, co-treatment with GC7 significantly enhanced the sensitivity of Huh7 and Hep3W cell lines to doxorubicin in hypoxic conditions (Physique 1E-H). Therefore, low concentrations of GC7 can enhance the sensitivity to doxorubicin in both Huh7 and Hep3W cell lines. GC7 enhanced sensitivity to doxorubicin and regulated doxorubicin-induced EMT in epithelial phenotype HCC cells To evaluated whether hypoxia induced doxorubicin resistance in HCC epithelial phenotype cells, we speculated whether EMT contributed to drug resistance [24]. Western blot analysis showed that doxorubicin treatment led to significant down-regulation of E-cadherin and up-regulation of Vimentin in HCC cells (Physique 2A). These results suggested that doxorubicin could induce Rabbit Polyclonal to HARS EMT in HCC cells. Interestingly, doxorubicin treatment with GC7 reversed doxorubicin-induced EMT in epithelial phenotype HCC cells, but not in HCC mesenchymal phenotype cells (Physique 2A). Immunofluorescent staining also showed comparable results that were consistent with the Western blot analysis (Physique 2B). These data suggested that GC7 could reverse SRT1720 HCl doxorubicin-induced EMT in HCC cells common of an epithelial phenotype. Furthermore, the CCK-8 assay was used to measure cell viability in cells treated with doxorubicin alone or doxorubicin plus GC7, and the results showed that the sensitivity of doxorubicin was reduced in HCC epithelial cells after co-treatment with GC7 (Physique 2C, ?,2D).2D). In contrast, there was no significant difference between the two groups (Physique 2E, ?,2F2F). Physique 2 GC7 enhanced sensitivity to doxorubicin and regulated doxorubicin-induced EMT in epithelial phenotype HCC cells. A: GC7 reversed the manifestation of doxorubicin-induced EMT-markers in HCC cells. Western blotting was used to examine the manifestation of E-cadherin … EMT is usually induced during hypoxia and GC7 prevents EMT in HCC cells with an epithelial phenotype From previous experience, we observed that hypoxia could induce chemoresistance to doxorubicin and GC7 significantly sensitized two of the four HCC cell lines to doxorubicin. However, the exact mechanism.