LCS-1 inhibition of superoxide dismutase 1 induces ROS-dependent death of glioma cells and degradates PARP and BRCA1

Abstract

Gliomas are characterized by high morbidity and mortality, and have only slightly increased survival with recent considerable improvements for treatment. An innovative therapeutic strategy had been developed via inducing ROS-dependent cell death by targeting antioxidant proteins. In this study, we found that glioma tissues expressed high levels of superoxide dismutase 1 (SOD1). The expression of SOD1 was upregulated in glioma grade III and V tissues compared with that in normal brain tissues or glioma grade I tissues. U251 and U87 glioma cells expressed high levels of SOD1, low levels of SOD2 and very low levels of SOD3. LCS-1, an inhibitor of SOD1, increased the expression SOD1 at both mRNA and protein levels slightly but significantly. As expected, LCS-1 caused ROS production in a dose- and time-dependent manner. SOD1 inhibition also induced the gene expression of HO-1, GCLC, GCLM and NQO1 which are targeting genes of nuclear factor erythroid 2-related factor 2, suggesting the activation of ROS signal pathway. Importantly, LCS-1 induced death of U251 and U87 cells dose- and time-dependently. The cell death was reversed by the pretreatment of cells with ROS scavenges NAC or GSH. Furthermore, LCS-1 decreased the growth of xenograft tumors formed by U87 glioma cells in nude mice. Mechanistically, the inhibition of P53, caspases did not reverse LCS-1-induced cell death, indicating the failure of these molecules involving in cell death. Moreover, we found that LCS-1 treatment induced the degradation of both PARP and BRCA1 simultaneously, suggesting that LCS-1-induced cell death may be associated with the failure of DNA damage repair. Taking together, these results suggest that the degradation of both PARP and BRCA1 may contribute to cell death induced by SOD1 inhibition, and SOD1 may be a target for glioma therapy.

Keywords: BRCA1; LCS-1; PARP; ROS; SOD1; cell death; glioma.

Figure 1

The expression of SOD1 in glioma tissues. (A) SOD1 IHC scores of normal brain tissues, tumor adjacent tissues and glioma grade I, II, III and IV tissues. * P < 0.05 compared with the normal brain tissue (Nor) or glioma grade I tissues. (B) Staining intensity of glioma with different histopathological types. Staining intensity was scored using a four-tier scale and defined as follows: negative staining (0-80); weak staining (80-200); moderate staining (200-300); strong staining (300-400). Nor, normal brain tissue; Adj, glioma adjacent tissues; I, well differentiated glioma; II, moderately differentiated glioma; III, poorly differentiated glioma; and IV, non-differentiated glioma. (C) SOD1 IHC staining for full glioma microarray. (D-E) Representative SOD1 weak (D), moderate staining (E) in normal brain tissues. (F-H) Representative SOD1 weak (F), moderate (G), strong staining (H) in glioma adjacent tissues. (I-K) Representative SOD1 weak (I), moderate (J), strong staining (K) in glioma grade I tissues. (L, M) Representative SOD1 weak (L), moderate (M), strong staining (N) in glioma grade II tissues. (O-Q) Representative SOD1 weak (O), moderate (P), strong staining (Q) in glioma grade III tissues. (R) Representative SOD1 strong staining in glioma grade IV tissues.

Figure 2

The expression of SOD1, SOD2 and SOD3 in glioma cell lines. (A) qRT-PCR analysis of SOD1, SOD2 and SOD3 mRNA levels in U251 cells. (B) qRT-PCR analysis of SOD1, SOD2 and SOD3 mRNA levels in U87 cells. (C) FACS analysis of SOD1 and SOD2 protein levels in U251 and U87 cells. (D) qRT-PCR analysis of SOD1 and 2 mRNA levels in U251 cells treated with 10 µM LCS-1 for the indicated time periods. * P < 0.05 compared with the medium groups. (E) qRT-PCR analysis of SOD1, SOD2 and SOD3 mRNA levels in U87 cells treated with 10 µM LCS-1 for the indicated time periods. * P < 0.05 compared with the medium groups. (F, G) Western blot analysis of the protein levels of SOD1 in U251 (F) and U87 (G) cells. (H, I) The quantitative data from F (H) and G (I) respectively. * P < 0.05 compared with the medium groups.

Figure 3

LCS-1 induces the production of ROS. (A) DCF staining analysis of ROS levels in U251 and U87 cells treated with the indicated doses of LCS-1 for the indicated time periods. (B) qRT-PCR analysis of the mRNA levels of NRF2-targeted genes in U251 cells treated with 10 µM LCS-1 for the indicated time periods. * P < 0.05 compared with the control groups. (C) qRT-PCR analysis of the mRNA levels of NRF2-targeted genes in U87 cells treated with 10 µM LCS-1 for the indicated time periods. * P < 0.05 compared with the control groups. (D) Western blot analysis of the protein levels of HO-1 in U87 cells treated with 10 μM LCS-1 for the indicated time periods. (E) Quantitative data from (D) * P < 0.05 compared with the medium groups.

Figure 4

LCS-1 induces cell death in glioma cells. (A-B) PI/FITC-Annexin V staining of death of U251 (A) and U87 (B) cells treated with the indicated doses of LCS-1 for 24 h (C, D) PI/FITC-Annexin V staining of death of U251 (C) and U87 (D) cells treated with 20 µM LCS-1 for the indicated time periods. (E-H) Quantitative data from A (E), B (F), C (G) and D (H) respectively. * P < 0.05 compared with the control groups.

Figure 5

ROS scavenges reverse LCS-1-induces cell death. (A-D) PI/FITC-Annexin V staining of death of U251glioma cells pretreated with the indicated doses of NAC (A), or GSH (B) for 1 h and re-treated with 20 µM LCS-1 for 24 h, and U87 cells pretreated with indicated doses of NAC (C), or GSH (D) for 1h and retreated with 20 µM LCS-1 for 24 h (E-H) Quantitative data from A (E), B (F), C (G) and D (H) respectively. * P < 0.05 compared with LCS-1-treated alone groups.

Figure 6

LCS-1 decreases tumor growth in nude mice. (A) Tumor growth curve from nude mice implanted with U87 cells and treated with or without LCS-1. * P < 0.05 compared with the vehicle groups. (B) Tumors from nude mice implanted with U87 cells and treated with or without LCS-1. (C) The weights of tumors from nude mice implanted with U87 cells and treated with or without LCS-1. * P < 0.05 compared with the vehicle group. (D) The weights of mice implanted with U87 cells and treated with or without LCS-1.

Figure 7

LCS-1-induced cell death is P53 in-dependent. (A) qRT-PCR analysis of the mRNA levels of P53-targeted genes in U251 cells treated with the indicated doses of LCS-1 for 12 h. * P < 0.05 compared with the medium groups. (B) qRT-PCR analysis of the mRNA levels of P53-targeted genes in U87 cells treated with 10 μM LCS-1 for the indicated time periods. * P < 0.05 compared with the medium groups. (C, D) PI/FITC-Annexin V staining of death of U251 (C) and U87 (D) cells pretreated with the indicated doses of P53 inhibitor Pifithrin-α (PFT-α) for 1 h and retreated with 20 μM LCS-1 for 24 h. (E, F) Quantitative data from C (E) and D (F). * P < 0.05 compared with LCS-1-treated alone groups.

Figure 8

LCS-1-induced cell death is Caspase in-dependent. (A) Western blot analysis of caspase 3 activation in U251 and U87 cells treated with the indicated doses of LCS-1 for 24 h. U87 cells treated with 1 μM Staurosporine (Stsp) for 24 h as positive controls. GAPDH protein levels were measured as loading controls. (B) PI/FITC-Annexin V staining of death of U251 glioma cells pretreated with the indicated doses of pan-caspase inhibitor Z-vad-FMK (Z-vad) for 1 h, and retreated with 20 μM LCS-1 for 24 h (C) PI/FITC-Annexin V staining of death of U87 glioma cells pretreated with the indicated doses of pan-caspase inhibitor Z-vad-FMK (Z-vad) for 1 h. and retreated with 20 μM LCS-1 for 24 h (D) Quantitative data from B and C respectively.

Figure 9

LCS-1 induces degradation of both PARP and BRCA1. (A) PI/FITC-Annexin V staining of death of U251 glioma cells pretreated with the indicated doses of PARP inhibitor PJ34 for 1 h. and retreated with 20 μM LCS-1 for 24 h (B) PI/FITC-Annexin V staining of death of U87 glioma cells pretreated with the indicated doses of PARP inhibitor PJ34 for 1 h. and retreated with 20 μM LCS-1 for 24 h. (C, D) Quantitative data of A (C) and B (D) respectively. * P < 0.05 compared with LCS-1-treated alone groups. (E, F) Western blot analysis of the protein levels of PARP, BRCA1 and BRCA2 in U251 (E) and U87 (F) cells treated with the indicated doses of LCS-1 for 24 h. GAPDH protein levels were measured as loading controls. (G, H) Western blot analysis of the protein levels of PARP, BRCA1 and BRCA2 in U251 (G) and U87 (H) cells treated with 20 μM LCS-1 for the indicated time periods. GAPDH protein levels were measured as loading controls. (I) qRT-PCR analysis of the mRNA levels of PARP1, PARP2, BRCA1 and BRCA2 in U251 cells treated with the indicated doses of LCS-1 for 12 h. * P < 0.05 compared with the control groups. (J) Western blot analysis of the phosphorylated levels of H2AX in U251 and U87 cells treated with 20 μM LCS-1 for the indicated time periods. GAPDH protein levels were measured as loading controls. (K) Western blot analysis of the protein levels of PARP, BRCA1 in U87 cells treated with the indicated doses of EGF, or IL-6 for 24 h. GAPDH protein levels were measured as loading controls. (L) PI/FITC-Annexin V staining of death of U87 glioma cells pretreated with 20 ng/mL EGF, or 20 ng/mL IL-6 for 24 h. and retreated with 20 μM LCS-1 for 24 h. (M) Quantitative data from L. * P < 0.05 compared with LCS-1-treated alone group.

Figure 10

Model of the mechanism by which LCS-1 induces cell death. LCS-1 inhibited the enzyme activity of SOD1, resulting in the accumulation of ROS, leading to the induction of DNA damage. Meanwhile, LCS-1 induces the degradation of PARP. The dysfunction of PARP inhibits DNA damage repair via blocking both PARylation-mediated and EJ-mediated pathways. Furthermore, LCS-1 induces the degradation of BRCA1, eliciting the block of HR-mediated pathway. The inhibition of these three repair pathways results in death of glioma cells.