p62 Suppressed VK3-induced Oxidative Damage Through Keap1/Nrf2 Pathway In Human Ovarian Cancer Cells

Abstract

Imbalance of redox homeostasis may be responsible for the resistance of cancer to chemotherapy. Currently, increasing studies demonstrated that vitamin K3 (VK3), which promoted the production of ROS, had potential to be developed as an anti-tumor agent. We found SKOV3/DDP cells with high levels of p62 were insensitive to VK3 compared with SKOV3 cells. Furthermore, Nrf2 downstream antioxidant genes such as HO-1(heme oxygenase 1) and NQO1 (NAD (P) H: quinone oxidoreductase 1) were upregulated in SKOV3/DDP cells with VK3 treatment, which indicated VK3 activated Nrf2 signaling in SKOV3/DDP cells. Moreover, co-localization of p62 and Keap1 was also observed. Suppression of p62 expression increased the apoptosis induced by VK3, and the expression of Nrf2, HO-1 and NQO1 were all downregulated in SKOV3/DDP cells. Our results suggested that overexpressed p62 may protect cells from oxidative damage caused by VK3 through activating Keap1/Nrf2 signaling in ovarian cancer.

Keywords: Drug resistance; Keap1; Nrf2; ROS; VK3; p62.

Figure 1

VK3 promoted the apoptosis of SKOV3 ovarian cancer cells. (A) SKOV3 and SKOV3/DDP cells were treated with VK3 for 8 and 16 h. The MTT assay was used to examine the cell viability. Data are presented as mean ± SD, n = 3. (B) Both cells were treated with 15 µM VK3 and stained with Hoechst 33342. Cell morphology was observed by fluorescence microscopy. Arrows indicate apoptotic cells. Scale bar = 20 µm. (C) Cells were stained with Annexin V-FITC/PI, and the ratio of apoptosis was detected using a FACScan flow cytometer. Data are presented as mean ± SD, n = 3. (D) Apoptotic rate in (C) was quantified in both cells. Data are presented as mean ± SD, n = 3. *P < 0.05 and **P < 0.01 compared with untreated cells. (E) Western blotting was used to analyze the expression of cleaved caspase-3. (F) The expression of cleaved caspase-3 was quantified. Data are presented as mean ± SD, n = 3. **P < 0.01 compared with untreated cells.

Figure 2

Inhibition of ROS reduces VK3-induced cell death in ovarian cancer cells. (A) Both cells were treated with VK3 (15 µM) for 8 or 16 h and ROS generation was determined using 50 µM DCFH-DA. DCF fluorescence intensity was detected by fluorescence microscopy (100×). (B) Quantification of DCF fluorescence intensity in (A). Data are presented as mean ± SD, n = 3. **P < 0.01 compared with control. (C) SKOV3 cells pretreated with 40 μM NAC for 1h were stained with Annexin V-FITC/PI. FACScan was used to count positively stained cells. (D) Quantitation of apoptotic rate in SKOV3 cells in (C). Data are presented as mean ± SD, n = 3. *P < 0.05 compared with 8 h VK3 treatment; ^#P < 0.05 compared with 16 h VK3 treatment. (E) The MTT assay was used to examine the cell viability with 40 µM NAC pretreatment followed by 15 µM VK3 culture. Data are presented as mean ± SD, n = 3. *P < 0.05 compared with VK3 treatment alone.

Figure 3

VK3 activates the Nrf2 pathway in SKOV3/DDP cells. (A) Both cells were treated as before. Nucleus extracts were subjected to immunoblot analysis with anti-Nrf2 and anti-LaminA/C. (B) Quantitation of nucleus Nrf2 protein level in (A). Data are presented as mean ± SD, n = 3. *P < 0.05 compared with untreated cells. (C) Total RNAs were prepared and NQO-1 and HO-1 mRNA levels were analyzed by RT-PCR. (D, E) Quantitation of HO-1 and NQO1 levels in (C). Data are presented as mean ± SD, n = 3. *P < 0.05 compared with SKOV3 cells. (F) The expression of HO-1 and NQO1 were examined by western blotting. (G, H) Quantitation of HO-1 and NQO1 levels in (E).Data are presented as mean ± SD, n = 3. *P < 0.05 compared with SKOV3 cells.

Figure 4

Knockdown of p62 inhibited the translocation of Nrf2 in SKOV3/DDP cells with VK3 treatment. (A) si-p62 or si-scrambled were transfected with SKOV3/DDP cells. After treated with 15 µM VK3 for 16h, cell lysates were subjected to immunoblot analysis (B) Cells were treated as (A), Immunofluorescence was performed with anti-Nrf2 antibodies and detected by fluorescence microscopy (scale bar, 25 µm).

Figure 5

p62 inhibition increased VK3-induced apoptosis in SKOV3/DDP cells. (A) SKOV3/DDP cells were transfected with p62 or control siRNA. After treatment with 15 µM VK3 for 16h, MTT assays was used to evaluate cell viability. Data are presented as mean ± SD, n = 3. *P < 0.05 compared with si-scrambled + VK3 treatment group. (B) SKOV3/DDP cells were treated as (A). And the expression of cleaved caspase-3 was analyzed by Western blotting. (C) The expression of cleaved caspase-3 in (B) was quantified. Data are presented as mean ± SD, n = 3. *P < 0.05 compared with si-scrambled + VK3 treatment group.

Figure 6

The interaction between p62 and Keap1 increased with VK3 treatment in SKOV3/DDP cells. (A) Both cells were treated with 15 µM VK3 for 8 or 16 h. Cell lysates were subjected to immunoblot analysis with anti-p62. (B) The expression of p62 in (A) was quantified. Data are presented as mean ± SD, n = 3. *P < 0.01 compared with untreated cells. (C) SKOV3/DDP cells were treated as before, and total p62 and Keap1 were detected by western blotting. (D) Cell lysates were immunoprecipitated with anti-Keap1 antibody and immunoblotting was performed with anti-p62 and anti-Keap1 antibodies. (E) Cells were treated with 15 µM VK3 for 8 h. Immunofluorescence of p62 and Keap1 was detected by fluorescence microscopy (scale bar, 25 µm).

Figure 7

Schematic representation of the p62- Keap1- Nrf2 pathway in ovarian cancer cells with the treatment of VK3. Our study provides evidence that p62 promotes Nrf2 signaling through interacting with Keap1, which blocks VK3-induced apoptosis by inhibiting ROS production in SKOV3/DDP cells.