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  • br aPKC mediated ROS inhibition enhances gemcitabine resista

    2020-08-03


    3.5. aPKCι-mediated ROS inhibition enhances gemcitabine resistance in GBC
    Next, we further investigated whether aPKCι would influence the sensitivity of GBC MPP+ Iodide to commonly used chemotherapeutic drugs gemcitabine. Given that ROS has been reported to participate in che-moresistance, we speculated that aPKCι may involve in this process. We treated aPKCι or Nrf2 silencing GBC cells with various concentrations of gemcitabine for 72 h to analyze the cell viability by CCK-8 assays. Indeed, when aPKCι was knocked down, the cells exhibited more sen-sitivity to gemcitabine than the negative control group, with a drama-tically decreased of IC50 (Fig. 5A and S4A). Then, we examined the viability of the two GBC cell lines with the indicated dose of 
    gemcitabine for 24, 48 and 72 h. The results showed that the sensitivity of the cells to gemcitabine increased with time in the aPKCι knockdown group (Fig. 5B and S4B). Notably, we also observed similar results that the loss of Nrf2 could help GBC cells to overcome the obstacle of che-moresistance. Moreover, we found that aPKCι or Nrf2 depletion led to ROS accumulation in gemcitabine-treated cells (Fig. 5C and S4C). Consistently, aPKCι deficiency decreased the Nrf2 protein and its target genes expression levels in cells treated with gemcitabine (Fig. 5D–E and S4D and E). Furthermore, B-cell lymphoma 2 (Bcl2) protein was de-creased, while Bcl-2-associated X (Bax) protein was increased after aPKCι knockdown in GBC cells treated with gemcitabine (Fig. 5D and S4D). In vivo, gemcitabine had slight suppression effect on the growth of xenograft tumors in the negative control group. However, when
    Fig. 6. The DLL motif is required for aPKCι-mediated ROS inhibition, cell growth and gemcitabine resistance. (A) The expression levels of aPKCι, Nrf2 and Keap1 proteins were determined in GBC cells after aPKCι knockdown, re-expression, DLL deletion or missense mutant, as indicated. N.S. no significance. (B) The mRNA levels of Nrf2 target genes were measured by qPCR in GBC cells with the indicated treatments. (C) Relative ROS levels were detected in GBC cells with the indicated treatments. (D) GBC cells with indicated treatments were subjected to soft agar growth assay. Scale bar, 50 μm. (E) Cell proliferation was assessed by CCK-8 in GBC cells with gemcitabine treatment. **P < 0.01. Data are derived from three independent experiments and presented as means ± SDs.
    aPKCι was depleted, the tumors were significantly repressed in the presence of gemcitabine (Fig. 5F and G). Therefore, aPKCι improves the resistance of GBC cells to gemcitabine through ROS inhibition.
    3.6. The DLL motif is required for aPKCι-mediated ROS inhibition, cell growth and gemcitabine resistance
    To further validate whether the aPKCι-Keap1 interaction through 
    the DLL motif is associated with these biological functions, aPKCι-WT (wild-type), aPKCι-DLL257 deletion mutant (M1) and aPKCι-DLL257 mutant (M2) vectors were transfected into aPKCι-deficient cells. We found that re-expression of aPKCι-WT, but not mutant M1 or M2, re-versed the reduction of the level of Nrf2 protein and its target genes induced by aPKCι knockdown (Fig. 6A and B). In addition, we examined the intracellular ROS levels in aPKCι-deficient cells with the above vectors. As shown in Fig. 6C, mutant M1 or M2 did not reduce the
    (caption on next page)
    Fig. 7. aPKCι is frequently upregulated and correlated with poor prognosis in patients with GBC. (A) Representative images of IHC staining of aPKCι, Nrf2 and Keap1 expression in 72 GBC samples and pair-matched normal tissues. (B) Quantification of aPKCι, Nrf2 and Keap1 expression levels in 72 GBC samples and pair-matched normal tissues. (C) The histogram showed the correlation of low or high aPKCι expression with the expression levels of Nrf2 and Keap1. (D) The protein levels of aPKCι, Nrf2 and Keap1 in 8 representative GBC samples (T) and pair-matched normal tissues (N) were evaluated by western blotting. (E) Correlation between aPKCι and clinicopathologic characteristics in patients with GBC. The clinical stage was according to 7th AJCC staging criteria. (F) Kaplan-Meier analysis showed the correlation between aPKCι expression and the overall survival of patients with GBC. *P < 0.05, **P < 0.01.
    intracellular ROS levels in both NOZ and GBC-SD cells. Likewise, mu-tant M1 or M2 had no obvious effect on cell growth when aPKCι was silenced (Fig. 6D). Consistently, aPKCι-WT rescued the defects of cell proliferation in aPKCι-deficient cells treated with gemcitabine; how-ever, transfection with mutant M1 or M2 failed to produce this effect (Fig. 6E). These results suggest that the aPKCι-Keap1 interaction through the DLL motif is required for cell growth, ROS inhibition, and chemoresistance in GBC cells.
    3.7. aPKCι is frequently upregulated and correlated with poor prognosis in patients with GBC