Targeting the mitochondria activates two independent cell death pathways in ovarian cancer stem cells

Abstract

Cancer stem cells are responsible for tumor initiation and chemoresistance. In ovarian cancer, the CD44+/MyD88+ ovarian cancer stem cells are also able to repair the tumor and serve as tumor vascular progenitors. Targeting these cells is therefore necessary to improve treatment outcome and patient survival. The previous demonstration that the ovarian cancer stem cells are resistant to apoptotic cell death induced by conventional chemotherapy agents suggests that other forms of targeted therapy should be explored. We show in this study that targeting mitochondrial bioenergetics is a potent stimulus to induce caspase-independent cell death in a panel of ovarian cancer stem cells. Treatment of these cells with the novel isoflavone derivative, NV-128, significantly depressed mitochondrial function exhibited by decrease in ATP, Cox-I, and Cox-IV levels, and by increase in mitochondrial superoxide and hydrogen peroxide. This promotes a state of cellular starvation that activates two independent pathways: (i) AMPKα1 pathway leading to mTOR inhibition; and (ii) mitochondrial MAP/ERK kinase/extracellular signal-regulated kinase pathway leading to loss of mitochondrial membrane potential. The demonstration that a compound can specifically target the mitochondria to induce cell death in this otherwise chemoresistant cell population opens a new venue for treating ovarian cancer patients.

Figure 1

NV-128 depresses mitochondrial function in rapamycin-resistant ovarian cancer stem cells: (a) ovarian cancer cells were treated with increasing concentrations of Rapamycin or NV-128 for 24h and percentage of viable cells quantified using Celltiter⁹⁶ assay; OCSCs were treated with NV-128 (10 μg/ml) at time points shown and (b) status of Cox-I, III, and IV determined by Western blot analysis; (c) effect on mitochondrial membrane potential and mitochondrial mass quantified using MitoTracker red and Mitotracker green, respectively; (d) levels of ATP and ADP quantified as described in the Materials and Methods section; (e) levels of mitochondrial superoxide and cellular hydrogen peroxide quantified using MitoSox and CM-H2DCFDA dyes, respectively; b-e shows results for OCSC1, similar results were observed with other cells tested. C, control. *^{, #} p < 0.001 compared to control. n = 3.

Figure 2

NV-128 activates the ERK pathway through ROS. (a) OCSCs were treated with NV-128 (10 μg/ml) at time points shown and mitochondrial fractions analyzed for levels of phosphorylated and total ERK1/2; (b) OCSCs were pre-teated with the ROS scavenger MnTBAP (500μM) for 1h prior to NV-128 treatment and effect on mitochondrial superoxide determined by flow cytometry; (c) effect of MnTBAP pre-pretreatment on mitochondrial ERK and AMPKα1 activation analyzed by Western blot analysis and a densitometer graph depicting levels of pERK1/2. * p = 0.0001 compared to NV-128 alone, ** p = 0.01 compared to control. Results shown are for OCSC1, similar results were observed with other cells tested. C, control. n = 3.

Figure 3

NV-128-induced inhibition of mTOR is independent of the MEK/ERK pathway. (a) OCSCs were pre-treated with U0126 (10 μM) for 1h prior to NV-128 treatment and effect on ERK and mTOR activity determined using Western blot analysis; (b) effect on cell morphology analyzed using a real-time video imaging system. Results shown are for OCSC1, similar results were observed with other cells tested. C, control. n = 3.

Figure 4

NV-128-induced loss of MMP was secondary to ERK-induced mitochondrial translocation of Bax. OCSCs were pre-treated with U0126 (10 μM) for 1h prior to NV-128 treatment: (a) effect on mitochondrial membrane potential determined using JC1 dye; (b) effect on mitochondrial Bax determined by Western blot analysis using mitochondrial fractions. Results shown are for OCSC1, similar results were observed with other cells tested. C, control. n = 3

Figure 5

NV-128-induced loss of ATP inhibits mTOR. OCSCs were treated with NV-128 in the presence of 20% FBS: (a) effect on ATP levels determined as described in the Methods section; (b) effect on pERK and mTOR target, pS6k determined by Western blot; (c) NV-128 activates the AMPK pathway. OCSCs were treated with NV-128 (10 μg/ml) at time points shown and phosphorylation status of AMPKα1 determined by Western blot analysis. Results shown are for OCSC1, similar results were observed with other cells tested. C, control (without FBS). ^# p < 0.001 compared to NV-128 alone. n = 3.

Figure 6

Proposed model for NV-128-induced casapase-independent cell death in OCSCs. By targeting the mitochondria, NV-128 activates two independent cell death pathways. Degradation of Cox-IV leads to ATP loss and increase mitochondrial ROS. ATP loss leads to inhibition of mTOR pathway and autophagic cell death. ROS activates the ERK/Bax axis leading to loss of mitochondrial membrane potential and EndoG-dependent DNA fragmentation.