qPCR analysis to determine fold change in mRNA expression of catalase in MDA-MB-468 cells at 4 h following treatment with 1 mM DETA-NONOate [a]. Immnoblot analysis of the expression of catalase in lysates of MDA-MB-468 cells following treatment with 1 mM DETA-NONOate [b, left panel].

<p>Densitometric analysis of catalase expression utilizing the Image Quant software [b, right panel]. Enzyme activity of catalase in 100 µg of cell lysate of MDA-MB-468 cells with or without the addition of 1 mM DETA-NONOate [c, left panel] Enzyme activity of catalase in 100 µg of cell lysate of MCF-7 cells with or without the addition of 1 mM DETA-NONOate [c, right panel]. Silencing of catalase in MDA-MB-468 cells utilizing siRNA against human catalase depicted by immunobloting and qPCR [d, left and right panel]. H₂O₂ production in MDA-MB-468 cells either transfected with scramble controls or siRNA against catalase, at 24 h following treatment with 1 mM DETA-NONOate [e]. Annexin V binding assay at 24 h, in MDA-MB-468 cells either transfected with scrambled controls [siScramble] or siRNA against the human catalase gene, following treatment with 1 mM DETA-NONOate [1 mM DETA] [f]. Graphical representation of the quantification of cells undergoing apoptosis in either scramble controls or in catalase silenced MDA-MB-468, following treatment with 1 mM DETA-NONOate [g]. Caspase-3 activity in MDA-MB-468 at 24 h following treatment with 1 mM DETA-NONOate in either scrambled controls [siScramble] or silenced for human catalase gene [siCAT] [h]. Results are expressed as the means from three independent experiments performed in duplicate; <i>bars</i>, SE wherever applicable. Western blot images are representative of three different experiments. GAPDH is probed as an equal loading control wherever applicable.</p

Immunoblot analysis of the dephosphorylation of pFOXO1 at serine 256, in MDA-MB-468 cell lysates, over time following treatment with 1 mM DETA-NONOate [a, left panel]. Immunoblot analysis of the dephosphorylation of pFOXO1 at serine 256 in MCF-7 cell lysates at 24 h post-treatment with 1 mM DETA-NONOate [a right panel].

<p>Immunoblot analysis of dephosphorylation of pFOXO1 upon DETA-NONOate treatment in either scrambled controls or MDA-MB-468 cells silenced for the catalytic subunit of PP2A [PP2A siRNA] [b]. Immunoblot analysis of FOXO1 in MDA-MB-468 nuclear fractions at 24 h post-treatment with 1 mM DETA-NONOate [c ]. Silencing of FOXO1 in MDA-MB-468 cells utilizing siRNA against human FOXO1 [d left panel] qPCR analysis to determine the fold change in mRNA expression of FOXO1 in MDA-MB-468 transfected with either scrambled controls [SiScramble] or siRNA against the human FOXO1 gene [siFOXO] [d, right panel]. Annexin V binding assay at 24 h, in MDA-MB-468 cells either transfected with scrambled controls [siScramble] or siRNA against the human FOXO1 gene, following treatment with 1 mM DETA-NONOate [1 mM DETA] [e]. Caspase-3 activity in MDA-MB-468 at 24 h following treatment with 1 mM DETA-NONOate in either scrambled controls [siScramble] or silenced for human FOXO1 gene [siFOXO] [f]. Results are expressed as the means from three independent experiments performed in duplicate; <i>bars</i>, SE wherever applicable. Western blot images are representative of three different experiments. GAPDH and Lamin A are probed as equal loading controls for cytosolic and nuclear nuclear fractions respectively.</p

qPCR analysis to compare the expression of catalase mRNA in MDA-MB-468 cells transfected with scrambled controls, siRNA against FOXO1 and SiRNA against PP2A following treatment with 1 mM DETA-NONOate for 4 h [a]. Immunoblot analysis of catalase protein expression in the lysates of MDA-MB-468 cells transfected with scrambled controls, siRNA against FOXO1 and siRNA against PP2A at 24 h following treatment with 1 mM DETA-NONOate [b].

<p>Immunoblot analysis of total and pFOXO1 in lysates of MDA–MB-468 cells at 24 h, following overexpression of either the human catalase gene [MDAOXCAT] or empty vectors in scrambled controls or in cells silenced for the catalytic subunit of PP2A [c–d]. Results are expressed as the means from three independent experiments performed in duplicate; <i>bars</i>, SE wherever applicable. Western blot images are representative of three different experiments. GAPDH is probed as an equal loading control wherever applicable.</p

Attenuation of Antioxidant Capacity in Human Breast Cancer Cells by Carbon Monoxide through Inhibition of Cystathionine β‑Synthase Activity: Implications in Chemotherapeutic Drug Sensitivity

Drug resistance is a major impediment to effective treatment of breast cancer. Compared to normal cells, cancer cells have an increased antioxidant potential due to an increased ratio of reduced to oxidized glutathione (GSH/GSSG). This is known to confer therapeutic resistance. Here, we have identified a mechanism, unique to breast cancer cells, whereby cystathionine β-synthase (CBS) promotes elevated GSH/GSSG. Lentiviral silencing of CBS in human breast cancer cells attenuated GSH/GSSG, total GSH, nuclear factor erythroid 2-related factor 2 (Nrf2), and processes downstream of Nrf2 that promote GSH synthesis and regeneration of GSH from GSSG. Carbon monoxide (CO) reduced GSH/GSSG in three breast cancer cell lines by inhibiting CBS. Furthermore, CO sensitized breast cancer cells to doxorubicin. These results provide insight into mechanism(s) by which CBS increases the antioxidant potential and the ability for CO to inhibit CBS activity to alter redox homeostasis in breast cancer, increasing sensitivity to a chemotherapeutic