The selective inhibition of nuclear PKCζ restores the effectiveness of chemotherapeutic agents in chemoresistant cells

Figures & data

Figure 1 Redox stress induces PKCζ nuclear translocation protecting PKCζ-overexpressing HeLa cells by its own apoptotic effects. (A) PKCζGFP-overexpressing HeLa cells in resting condition (left) and after a 30 min treatment with H₂O₂ 1 mM (right); histograms represent the ratio between the nuclear fluorescence and the cytosolic fluorescence, (184 ± 12.1). (B) Endogenous PKCζ in HeLa cells in resting conditions and after 30 min of treatment with H₂O₂. (C)(i) Increase dose-dependent in cell viability in PKCζGFP overexpressing HeLa cells: the histograms represents apoptotic counts expressed as difference of % fluorescent cells treated (30 min with different H₂O₂ concentrations) compared with resting condition (50 µM: 1.7 ± 1.3, 100 µM: 4.9 ± 1.1, 1 mM: 15.7 ± 0.8, 2 mM: 18.5 ± 1.3). (C)(ii) Caspase 3 activity assay: gray line, untreated cells; black line, PKCζGFP-overexpressing cells; dotted-gray line, mock cells treated with H₂O₂; dotted-black line, PKCζGFP-overexpressing cells treated with H₂O₂. (D) Apoptotic counts in HeLa cells overexpressing different PKC isoforms (PKCα, PKCε, PKCζ) treated with oxidant agent (H₂O₂ 1 mM) and compared with mock cells (Mock: 1.0 ± 2.1; PKCα −4.7 ± 0.3; PKCε 7.3 ± 0.8; PKCζ 15.7 ± 0.8).

Figure 2 Nuclear PKCζ protects HeLa cells by different apoptotic stimuli. (A) H₂O₂ (100 µM 30 min) pretreatment induces PKCζ nuclear translocation in PKCζGFP-overexpressing cells, protecting from apoptotic effects of (i) ceramide (30 µM 2 h) and (ii) okadaic acid (0.5 µM 2 h) and (iii) but not from of anti-Fas (4 µg/ml). The upper parts show PKCζGFP-overexpressing HeLa cells following the apoptotic stimulus (left) and following the apoptotic stimulus but previously accompanied by H₂O₂ 100 µM pretreatment (right). The lower parts show the antiapoptotic effects promoted by nuclear translocation of PKCζGFP, mediating H₂O₂ pre-treatment (cell viability in ceramide-treated cells: Δ% −5.3 ± 1.11 vs. 12.1 ± 0.95 in H₂O₂ pre-treated HeLa cells; Okadaic acid-treated cells Δ% 2.0 ± 1.21 vs. 14.5 ± 1.23 in H₂O₂ pre-treated cells; antiFAS-treated cells: Δ% −1.4 ± 1.73 vs. 1.6 ± 1.57 in H₂O₂ pre-treated HeLa cells). (B) Anti-FAS induces nuclear perturbation as confirmed by nuclear-GFP distribution, where it appears cytosolic-redistributed after 2 h of anti-FAS treatment. (C)(i) LMB (4 µg/ml 2 h) pretreatment induces PKCζ nuclear accumulation in PKCζGFP overexpressing cells, protecting from the apoptotic effects of ceramide (after 2 h of ceramide 30 µM; Δ% cells viability in pre-treated LMB cells: −13.4 ± 7.03 vs. −47.2 ± 8.33 in mock cells. Δ% cells viability in pre-treated LMB cells: −11.0 ± 3.38). (iii) Immunofluorescence of endogenous PKCζ: effect of LMB 4 µg/ml 2 h.

Figure 3 Effects of constitutively nuclear PKCζ (nuPKCζ), constitutively active and constitutively nuclear PKCζ (nuPKCζ-act) and selectively nuclear PKCζ inhibitor (nu-inhPKCζ) on cell viability. (A) Cellular localization and schematic structure of the chimera PKCζ: (i) nuPKCζ and (ii) nuPKCζ-act. (B) Nuclear localization of the different PKCζ chimeras: the histograms represent the ratio between the nuclear and cytosolic average fluorescence intensity, expressed as a percentage, for the three chimera proteins in resting conditions (nuclear distribution of: nuPKCζ-act 4.71 ± 0.80 or nuPKCζ 2.76 ± 0.41 vs. 0.41 ± 0.03 for PKCζ-GFP). (C) Apoptotic counts of HeLa cells expressing the different PKCζ mutants following ceramide treatment (cell viability in nuPKCζ-act and nuPKCζ cells: Δ% 12.6 ± 0.40 or 6.3 ± 1.08 vs. −5.3 ± 1.11 in PKCζGFP expressing HeLa cells, respectively). (D) Cellular localization and schematic structure of the selectively nuclear PKCζ inhibitor (nu-inhPKCζ). (E) Effects of nu-inhPKCζ overexpression on cell viability following LMB pretreatment and/or C2-ceramide treatment (Δ% cells viability in pre-treated LMB cells ceramide untreated: −4.6 ± 2.31 vs. −60.0 ± 8.60 after ceramide treatment or in not LMB pre-treated −48.6 ± 8.71).

Figure 4 Role of PKCζ nuclear translocation in chemoresistance cells. (A) Doxorubicin resistance of two different clones selected by culturing HeLa cells in presence of Doxorubicin 20 µM. The graph represents a time course of cell viability of three different clones of HeLa cells: a mock clone (black line) and two selected doxorubicin-resistant clones of HeLa cells (gray and dark gray) (Mock: 43.3 ± 4.63, 48.56 ± 4.24, 35.33 ± 4.57, 21.56 ± 3.06, 13.11 ± 2.26, 3.67 ± 0.97; DrHeLa1: 44.25 ± 5.82, 42.75 ± 5.45, 37.50 ± 7.09, 50.75 ± 3.53, 48.88 ± 4.53, 48.88 ± 5.54; DrHeLa2: 56.50 ± 4.42, 48.25 ± 5.58, 55.50 ± 6.61, 63.63 ± 5.48, 61.50 ± 6.31, 56.88 ± 5.03). (B) Time course of ROS production, expressed as normalized values of fluorescence intensity (mock and two doxorubicin-resistant HeLa cells). The histograms represent the increase in ROS production expressed as a percentage respect to mock cells (DrHeLa1 45.1% ± 20.3; DrHeLa2 49.2% ± 25.5). (C) Immunofluorescence of endogenous PKCζ in mock HeLa cells and in two different clones of doxorubicin-resistant HeLa cells. The histograms on the right represent the nuclear distribution of endogenous PKCζ, expressed as ratio between nuclear and cytosolic average fluorescence intensity (mock cells: 0.43 ± 0.04 vs. 1.31 ± 0.16 or 1.11 ± 0.10 in clone 1 or 2, respectively). (D) Apoptotic counts of doxorubicin-resistant HeLa cells transiently expressing nu-inhPKCζ. The histograms represents the difference of percentage of fluorescence following different doxorubicin treatment (20 µM 3 h or 24 h) compared with resting conditions (nu-inhPKCζ expressing DrHeLa1: 46.8 ± 5.23 vs. 28.3 ± 2.75 or 26.1 ± 1.33 after 3 h or 24 h of doxorubicin exposure, respectively; clone2: 41.9 ± 5.66 vs. 25.7 ± 1.45 or 16.6 ± 1.74).