A small molecule targeting glutathione activates Nrf2 and inhibits cancer cell growth through promoting Keap-1 S-glutathionylation and inducing apoptosis

Abstract

The level of glutathione (GSH) is increased in many cancer cells. Consuming intracellular GSH by chemical small molecules that specifically target GSH is a new strategy to treat cancer. Recently, we synthesized and proved that a new compound 2-(7-(diethylamino)-2-oxo-2H-chromen-3-yl)cyclohexa-2,5-diene-1,4-dione (PBQC) could target to and consume intracellular GSH specifically, but, it is not clear if PBQC can affect cancer cell growth and the activity of the nuclear factor-erythroid 2-related factor 2 (Nrf2) which is a key factor involved in regulation of cancer cell growth. In this study, we addressed these questions. We found that PBQC suppressed cancer cell growth through increasing the activity of Nrf2, while it did not inhibit normal vascular endothelial cell growth. Furthermore, we demonstrated that PBQC can cause Keap-1 protein S-glutathionylation and promote Nrf2 nuclear translocation as well as the expression of pro-apoptosis genes. As a result, the cancer cells underwent apoptosis. Here, we provide a new Nrf2 activator, PBQC that can promote the expressions of pro-apoptosis genes downstream Nrf2. The data suggest that PBQC is a potential lead-compound for development of new anti-cancer drugs.

Fig. 1. PBQC up regulated Nrf2 activity. (A) PBQC molecular structure is shown. (B) HeLa cells were stably transfected with ARE–luciferase reporter gene, and incubated Luci–HeLa cells with 0.1% DMSO (control) or PBQC at 1, 5, 10 μM for 6, 12, 24 and 48 h. The cell viability was analyzed by SRB assay and Luci–HeLa cells were lysed and luciferase activities were measured, setting the control group activity to 1. (*p < 0.05, **p < 0.01, ***p < 0.001, vs. control, n = 3).

Fig. 2. PBQC inhibited the growth of tumor cells but did not affect the normal vascular endothelial cell viability. (A) HeLa cells were exposed to compound PBQC at 1, 5 and 10 μM for 6, 12, 24 and 48 h, respectively. The cells obviously underwent morphological changes with the extension of time and the increase of PBQC concentration. The cell viability was measured by SRB assay. Bar as present 22 μM. (B) Effects of compound PBQC on human umbilical vein endothelial cell viability, the cells were treated with PBQC at the concentrations of 1, 5 and 10 μM or treated with DMSO 0.1% (v/v) (control) for 48 h. Cell viability was analyzed by SRB assay and illustrated in the labelled column. Set the control group activity to 1. Bar as present 22 μM. (*p < 0.05, **p < 0.01, ***p < 0.001, vs. control, n = 3).

Fig. 3. PBQC up regulated Nrf2 in HeLa cells. (A) Western blot analysis showed that Nrf2 level was up-regulated by PBQC prominently at 12 h and 24 h. (B) 100 nM si-Nrf2 dramatically decreased Nrf2 protein. And 10 μM PBQC prevented the decline of Nrf2. Set the control group activity to 1. (*p < 0.05, vs. control, n = 3).

Fig. 4. PBQC decreased the level of glutathione in Hela cells. (A) We treated the cells with 10 μM PBQC for 1, 3 and 6 h, then, detected the changes of the blue fluorescence by confocal microscopy. Bar as present 50 μM. (B) The blue fluorescence intensity was quantified by Image J. (C) We treated the cells with 10 μM PBQC for 1, 3 and 6 h, then, detected the level of glutathione by the total glutathione assay kit (Beyotime Institute of Biotech, Jiangsu, China), following the manufacture's instruction. (*p < 0.05, **p < 0.01, vs. control, n = 3).

Fig. 5. PBQC promoted Keap-1 glutathionylation and Nrf2 nuclear translocation. (A) WB analysis of co-IP of Keap-1 with glutathione antibody in HeLa cells treated with PBQC (0.1 and 10 μM) with 1% CS for 1, 3 and 6 h, quantification of co-immunoprecipitated Keap-1 levels. (B) Immunofluorescence assay of Nrf2 in HeLa cells incubated with PBQC (10 μM) for 3, 6, 12 and 24 h, nuclei were labeled with PI. Bar as present 50 μM. (*p < 0.05, **p < 0.01, vs. control, n = 3).

Fig. 6. PBQC up regulated the expressions of anti-oxidant genes and decreased the intracellular ROS level. (A and B) RT-PCR analysis of mRNA levels of Nrf2, HO-1 and GCLC treated with PBQC (1, 5 and 10 μM) PBQC for indicated times. (C) Incubated HeLa cells with 0.1% DMSO (control) or PBQC at 1, 5 and 10 μM for 1, 6, 12, 18 and 24 h, then used 5 μM DCHF probe treated all cells for 30 min. The fluorescence intensity was observed by inverted fluorescence microscope (200×) and the exciting light of DCHF probe was blue light. ROS expression level was quantified by GraphPad Prism 5 software. Bar as present 22 μM. (*p < 0.05, **p < 0.01, vs. control, n = 3).

Fig. 7. The varying degree of Nrf2 activation had different effects on the downstream Bcl-2 and Bax gene expressions. (A) RT-PCR analysis of mRNA levels of Nrf2, HO-1, GCLC, Bcl-2 and Bax in HeLa cells treated with PBQC (0.1 μM) for indicated times. (B) RT-PCR analysis of mRNA levels of Bcl-2 and Bax in HeLa cells treated with PBQC at 1, 5 and 10 μM for 1, 3, 6, 12 and 24 h, set the control group activity to 1. (#p > 0.05, *p < 0.05, **p < 0.01, vs. control, n = 3).

Fig. 8. Significant activation of Nrf2 by PBQC promoted NQO1 and p53 expressions. (A) RT-PCR analysis of mRNA levels of NQO1 in HeLa cells treated with PBQC at 1, 5 and 10 μM for 1, 3, 6, 12 and 24 h. (B) Western blot analysis of the protein level of p53, Bcl-2, Bax and β-actin as a normalization control and quantitative statistics (C and D). HeLa cells were treated with 0.1% DMSO (control) or PBQC at 1, 5 and 10 μM for 3, 6, 12 and 24 h. We set the control group activity to 1. (*p < 0.05, **p < 0.01, vs. control, n = 3).

Fig. 9. PBQC induced cancer cell apoptosis. (A) Effects of compound PBQC on LDH activity of HeLa cells. The culture medium was collected as samples for LDH assay after 48 h treatment at the concentration of 10 μM. Apoptosis-associated markers illustrated PBQC-accelerated cell dying in HeLa cells. (B) Hoechst 33258 staining testified that extra added PBQC (0, 1, 5 and 10 μM for 24 h) can increase the rate of HeLa cell-apoptosis. Microscope images (200×) were taken under a fluorescent microscope (Nikon). Apoptotic cells were quantitative. Bar as present 22 μM. (C) The changes of apoptosis of HeLa cells were observed by TUNEL assay. HeLa cells treated with 0.1% DMSO (control) or 1, 5 and 10 μM PBQC for 24 h were stained with TUNEL kit and confocal microscopic imaging was performed. Blue channel (405–640 nm), Ex = 405 nm; green channel (500–578 nm), Ex = 488 nm; statistics of the number of apoptosis cells. Bar as present 50 μM. (D) Western blot analysis of the protein levels of PARP and β-actin as a normalization control and quantitative statistics. HeLa cells were treated with 0.1% DMSO (control) or PBQC at 1, 5 and 10 μM for 6, 12, 24 and 48 h. (*p < 0.05, **p < 0.01, ***p < 0.001, vs. control, n = 3).

Fig. 10. Schematic presentation of PBQC activating Nrf2 and inducing apoptosis. (A) PBQC treatment increases Keap-1 S-glutathionylation and promotes Nrf2 activity. Subsequently, the activated Nrf2 gets into the nucleus, anti-oxidative signaling pathway is activated. The significant activation of Nrf2 by PBQC up-regulates NQO1 and p53 and specifically inhibits the increase of Bcl-2 expression. The pro-apoptotic protein Bax is significantly increased. As a result, the cancer cells undergo apoptosis.