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. 2020 Jun 12:10:949.
doi: 10.3389/fonc.2020.00949. eCollection 2020.

"VSports在线直播" Broad Spectrum Deubiquitinase Inhibition Induces Both Apoptosis and Ferroptosis in Cancer Cells

Li Yang  1   2 , Xin Chen  2 , Qianqian Yang  2 , Jinghong Chen  2   3 , Qingtian Huang  2 , Leyi Yao  2 , Ding Yan  2 , Jiawen Wu  2 , Peiquan Zhang  2 , Daolin Tang  4 , Nanshan Zhong  2 , Jinbao Liu  2
Affiliations

Broad Spectrum Deubiquitinase Inhibition Induces Both Apoptosis and Ferroptosis in Cancer Cells

"V体育2025版" Li Yang et al. Front Oncol. .

Abstract

Proteasomal deubiquitinase (DUB) inhibition has been found to be effective in experimental cancer therapy by inducing proteasome inhibition and apoptosis. Ferroptosis is a form of regulated cell death characterized by an iron-dependent lipid peroxidation. Antioxidant enzyme glutathione peroxidase 4 (GPX4) plays a key role in blocking ferroptosis through directly reducing phospholipid hydroperoxides production. Since cytoplasmic DUB inhibition can promote protein degradation in the cell, we hypothesize that DUB inhibition induces GPX4 degradation. Here we used palladium pyrithione complex (PdPT), a broad spectrum deubiquitinase inhibitor, to explore its cell death induction and anti-cancer effect in vitro, ex vivo, and in vivo. Mechanically, caspase activation and GPX4 protein degradation are required for PdPT-induced apoptosis and ferroptosis, respectively VSports手机版. Notably, PdPT-induced multiple deubiquitinase inhibition is essential for proteasomal degradation of GPX4. These findings not only identify a novel mechanism of post-translational modification of GPX4 in ferroptosis, but also suggest a potential anti-caner therapeutic strategy using Pan-DUB inhibition. .

Keywords: GPX4; apoptosis; cancer; deubiquitinase; ferroptosis. V体育安卓版.

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"V体育平台登录" Figures

Figure 1
Figure 1
PdPT is an inhibitor of multiple DUBs. a and b The in situ and in vitro effects of PdPT on proteasome peptidase activity. Lysates were from PdPT or BTZ-treated A549 (A) and NCI-H1299 (B). Cells were treated with the indicated doses of PdPT and then the proteasome chymotrypin-like activity were analyzed. (C) Purified 20S proteasomes were treated with the indicated doses of PdPT and then the chymotrypin-like activity was measured using specific synthetic fluorogenic substrates. BTZ was used as a positive control. Values are expressed as mean ± SD (n = 3). **P < 0.01, compared with each control. d and e The effect of PdPT on DUB activities. Purified 26S proteasomes (D) or A549 cell lysates (E) were exposed to PdPT (1.25, 2.5, and 5.0 μM) and the dynamic DUB activity was measured. NEM was used as a positive control. (F) Abolishment of Ub-VS labeling of proteasomal DUBs by PdPT. Lysates from PdPT (2.5, 25 μM) or b-AP15 (25 μM)-treated A549 and NCI-H1299 cells were incubated with labeled HA-UbVS at 37°C for 30 min. The levels of HA and DUBs were assayed using western blot. (G) A549, NCI-H1299, and A549/DDP cells were treated with indicated PdPT for 6 h. Total ubiquitinated proteins (Ubs.), K48-linked ubiquitinated proteins (K48), and PARP proteins were detected using western blot. Bortezomib(BTZ, 100 nM) and b-AP15 (1 μM) were used as a positive control and CDDP (10 μM) was used as a negative control.
Figure 2
Figure 2
Anti-cancer activity of PdPT in vitro. (A) A549, NCI-H1299, A549/DDP and BEAS-2B cells were exposed to indicated concentration of PdPT for 24 or 48 h. Cell viability was detected by MTS assay (n = 3; *P < 0.05). (B) A549, NCI-H1299, A549/DDP and BEAS-2B cells were exposed to indicated CDDP for 24 or 48 h. Cell viability was detected by MTS assay (n = 3; *P < 0.05). (C,D) A549 and NCI-H1299 cells were treated with indicated PdPT for 24 h. Apoptotic cells were detected with Annexin V-FITC and PI double stain followed by flow cytometry. (E) In parallel, fluorescence microscope were used to monitor the levels of Annexin V- or PI-positive cells.
Figure 3
Figure 3
PdPT induces apoptosis and ferroptosis in non-small cell lung cancer cells. (A) A549 and NCI-H11299 cells were treated with PdPT (5 μM) in the absence or presence of indicated cell death inhibitors for 24 h. BAF, bafilomycin A1 (100 nM); E64d (10 μM); necrostatin-1 (50 μM); zVAD, z-VAD-FMK (50 μM); DFO, deferoxamine (100 μM); Cell viabilities were analyzed by MTS. Mean ± SD (n = 3). *P < 0.05, vs. control. (B) A549 cells were treated with PdPT (5 μM), b-AP15 (1 μM) and RSL3 (10 μM) in the presence or absence of z-VAD-FMK (50 μM), ferrostatin-1 (2.5 μM), or deferoxamine (100 μM) for 24 h. The levels of Annexin V- or PI-positive cells were observed using a fluorescence microscope. (C,D) A549 and NCI-H1299 cells were treated with PdPT (5 μM) in the presence or absence of z-VAD-FMK (50 μM) or DFO (100 μM) for 24 h. The levels of Annexin V- or PI-positive cells were assayed by flow cytometry. Mean ± SD (n = 3). *P < 0.05, vs. control. (E) Western blot analysis of cleaved-PARP and cleaved-caspases in A549, NCI-H1299 and A549/DDP cells following treatment with PdPT for 24 h. (F) Western blot analysis of indicated proteins in A549, NCI-H1299, and A549/DDP cells following treatment with PdPT for indicated times.
Figure 4
Figure 4
PdPT induces GPX4 protein degradation. (A,B) A549 and NCI-H1299 cells were treated with PdPT for indicated does or time and then PARP and GPX4 protein levels were assayed using western blot. (C) A549 and NCI-H1299 cells were treated with PdPT (5 μM) in the absence or presence of z-VAD-FMK (50 μM), ferrostatin-1 (2.5 μM), deferoxamine (100 μM) for 24 h. The protein levels of PARP and GPX4 were assayed using western blot. (D) A549 and NCI-H1299 cells were treated with PdPT (5 μM) in the absence or presence of bortezomib (BTZ, 100 nM) for 24 h. The protein level of GPX4 was assayed using western blot. (E) A549 and NCI-H1299 cells were treated with PdPT (5 μM), CDDP (10 μM), BTZ (100 nM), and b-AP15 (1 μM) for 24 h, and then indicated proteins were assayed using western blot. (F,G) Immunoprecipitation analysis of GPX4-Ub complex formation in A549 and NCI-H1299 cells following PdPT treatment for 24 h.
Figure 5
Figure 5
PdPT induces cytotoxity by apoptosis and ferroptosis in cancer cells from AML patients. (A) Cancer cells from six AML patients (Pt) and peripheral blood mononuclear cells from six healthy volunteers (Nm) were treated with PdPT at the indicated doses for 24 h, and the cell viability was detected with the MTS assay. The scatter plot of the IC50 values in each group is shown. (B,C) Cancer cells from AML patients were incubated with PdPT at the indicated doses for 24 h. Cell death was analyzed with annexin V/PI staining followed by flow cytometry (B), and the results are summarized (C). Mean ± SD (n = 3). *P < 0.05, vs. control. (D) AML cancer cells were treated with the indicated doses of PdPT, CDDP (5 μM) or BTZ (50 nM) for 24 h, and then cells were stained with Annexin V/PI (A/P) and imaged under a fluorescent microscope. The phase contrast and fluorescent images were taken and merged. Scale bar = 50 μm. Peripheral mononuclear cells of AML cancer cells were treated with PdPT, BTZ (50 nM) for 24 h, followed by detecting total ubiquitinated proteins (Ubs.) (E), caspase-3, cleaved-caspase3, caspase 8 (F), and GPX4 (G) with western blotting analyses. GAPDH was used as a loading control. C, control.
Figure 6
Figure 6
Anti-cancer activity of PdPT in vivo. Nude BALB/c mice bearing A549 and NCI-H1299 xenografts tumors were treated with either vehicle (Veh) or PdPT (7.5 mg/kg/day, intraperitoneally) for 15 days when the average tumor size reached at 50 mm3. Tumor size was recorded every other day. Tumor size (A), tumor images (B), tumor weight (C), and body weight (D) were shown (n = 6 mice/group, ***P < 0.001, compared with each control). (E,F) Representative image of immunohistochemistry staining and quantification for total ubiquitinated proteins (Ubs), cleaved-caspase 3, and GPX4 in nude mouse tumor tissues (200 ×). Mean±SD. (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, vs. Vehicle.
Figure 7
Figure 7
Chemical structure of PdPT (A) and a schematic illustration of the proposed model depicting that PdPT induces caspase-dependent apoptosis and GPX4-dependent ferroptosis by inhibition of DUBs (B).
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