Phenethyl Isothiocyanate Exposure Promotes Oxidative Stress and Suppresses Sp1 Transcription Factor in Cancer Stem Cells

Abstract

Aldehyde dehydrogenase 1 (ALDH1) is a cytosolic marker of cancer stem cells (CSCs), which are a sub-population within heterogeneous tumor cells. CSCs associate with therapy-resistance, self-renewal, malignancy, tumor-relapse, and reduced patient-survival window. ALDH1-mediated aldehyde scavenging helps CSCs to survive a higher level of oxidative stress than regular cancer cells. Cruciferous vegetable-derived phenethyl isothiocyanate (PEITC) selectively induces reactive oxygen species (ROS), leading to apoptosis of cancer cells, but not healthy cells. However, this pro-oxidant role of PEITC in CSCs is poorly understood and is investigated here. In a HeLa CSCs model (hCSCs), the sphere-culture and tumorsphere assay showed significantly enriched ALDH^hi CSCs from HeLa parental cells (p < 0.05). Aldefluor assay and cell proliferation assay revealed that PEITC treatments resulted in a reduced number of ALDH^hi hCSCs in a concentration-dependent manner (p < 0.05). In the ROS assay, PEITC promoted oxidative stress in hCSCs (p ≤ 0.001). Using immunoblotting and flow cytometry techniques, we reported that PEITC suppressed the cancer-associated transcription factor (Sp1) and a downstream multidrug resistance protein (P-glycoprotein) (both, p < 0.05). Furthermore, PEITC-treatment of hCSCs, prior to xenotransplantation in mice, lowered the in vivo tumor-initiating potential of hCSCs. In summary, PEITC treatment suppressed the proliferation of ALDH1 expressing cancer stem cells as well as key factors that are involved with drug-resistance, while promoting oxidative stress and apoptosis in hCSCs.

Keywords: ALDH1; apoptosis; cancer stem cells; phenethyl isothiocyanate; reactive oxygen species.

Figure 1

Sphere culture method enriches aldehyde dehydrogenase^hiCD44^hi HeLa cervical cancer stem-like sphere culture model (ALDH^hiCD44^hi hCSCs) population from parental HeLa cells. (a) Representative fluorescence-activated cell sorting (FACS) dot plots showing enrichment of ALDH^hi cancer stem-like cells from day 0 to day 10 post sphere formation in low anchorage dishes. The ALDH^hi gated cells were mostly CD44 positive (b). Bar graphs representing aldehyde dehydrogenase enrichment in HeLa cultures showing cancer stem cells enrichment level was significantly higher by end of the tenth day. All data represent means ± SEM, *** p ≤ 0.001. DEAB: diethylaminobenzaldehyde.

Figure 2

Phenethyl isothiocyanate (PEITC) attenuates aldehyde dehydrogenase 1(ALDH) expressing HeLa cancer stem cells (hCSCs) in a concentration dependent manner. Representative fluorescence-activated cell sorting (FACS) dot plots showing PEITC reduced ALDH1 expressing HeLa cells (a) and hCSCs (c). Bar graphs showing the reduction of ALDH high cells in HeLa (b) and in hCSCs, hCSCs + PEITC, and hCSCs + Disulfiram treatments, using Disulfiram as a known ALDH-inhibiting agent (positive control) (d). Bar diagrams showing attenuation of ALDH high hCSCs by PEITC in a concentration dependent manner (e). All data represent means ± SEM, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. DEAB: diethylaminobenzaldehyde. DMSO: dimethyl sulfoxide control.

Figure 3

Phenethyl isothiocyanate (PEITC) induces reactive oxygen species (ROS) in HeLa cancer stem cells (hCSCs). (a) Representative fluorescence-activated cell sorting (FACS) histograms showing PEITC induces ROS in hCSCs in 3 hr, which can be replenished by exogenous glutathione (GSH). H₂O₂ was used as a positive control (b). Bar diagram showing the ROS induction by PEITC in hCSCs. All data represent means ± SEM. One-way ANOVA, followed by a Dunnett’s post hoc test, was used to compare multiple means. The pairs with significant mean differences are shown, *** p ≤ 0.001. DCF-DA: dichlorofluorescin diacetate.

Figure 4

Phenethyl isothiocyanate (PEITC) suppresses HeLa cancer stem cells (hCSCs) proliferation. (a) Representative photomicrographs showing attenuation of hCSCs-sphere formation (day 7) by PEITC in a concentration dependent manner (400-μm scale). (b) Bar graphs showing concentration-dependent effects of PEITC on the viability of hCSCs after 24 h treatment. The dotted line represents the baseline cell viability for dimethyl sulfoxide (DMSO), which served as a vehicle control to determine statistical significance. (c) Bar graphs showing the percentage of early apoptotic cells obtained from Propidium Iodide and Annexin V assay. All data represent means ± SEM, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

Figure 5

Phenethyl isothiocyanate (PEITC) suppresses transcription factor Sp1 and multidrug resistance protein P-Gp/ABCB1 in HeLa cancer stem cells. (a) Western blots showing PEITC attenuated Sp1 and the corresponding bar graphs with relative Sp1 expression (b). (c) Bar graphs showing 10 µM PEITC suppresses multi-drug resistance protein P-Gp (ABCB1) as compared to vehicle control. All data represent means ± SEM, * p ≤ 0.05.