L-Buthionine sulfoximine is a specific γ-glutamylcysteine synthetase inhibitor that blocks the rate-limiting step of glutathionine biosynthesis and in doing so depletes the intracellular glutathionine pool in both cultured cells and in whole animals. Glutathionine (GSH) is a water-soluble tripeptide composed of glutamine, cysteine, and glycine. Reduced glutathione is the most abundant intracellular small molecule thiol present in mammalian cells and it serves as a potent intracellular antioxidant protecting cells from toxins such free radicals. Changes in glutathionine homeostasis have been implicated in the etiology and progression of a
L-Buthionine sulfoximine is a specific γ-glutamylcysteine synthetase inhibitor that blocks the rate-limiting step of glutathionine biosynthesis and in doing so depletes the intracellular glutathionine pool in both cultured cells and in whole animals.
Glutathionine (GSH) is a water-soluble tripeptide composed of glutamine, cysteine, and glycine.
Reduced glutathione is the most abundant intracellular small molecule thiol present in mammalian cells and it serves as a potent intracellular antioxidant protecting cells from toxins such free radicals.
Changes in glutathionine homeostasis have been implicated in the etiology and progression of a variety of human diseases, including breast cancer.
In particular, studies have shown that elevated levels of glutathionine prevent apoptotic cell death whereas depletion ofglutathionine facilitates apoptosis.
Buthionine sulfoximin depletes cellular glutathionine and sensitizes tumor cells to apoptosis induced by standard chemotherapeutic agents.
Buthionine sulfoximine (BSO) plus estradiol induce apoptosis in MCF-7:2A cells.
(a) Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) staining for apoptosis in MCF-7:2A cells following BSO plus 17β-estradiol (E2) treatment for 96 h were performed as described in Materials and methods. Slides were photographed through a brightfield microscope under 100 × magnification. TUNEL-positive cells were stained black (white arrows). Columns (right), mean percentage of apoptotic cells (annexin V-positive cells) from three independent experiments performed in triplicate; bars, standard error of the mean (SEM). *, p < 0.001 compared with control cells; #, p < 0.001 compared with estradiol-treated cells.
(b) Annexin V staining for apoptosis. Cells were seeded in 100 mm plates at a density of 1 × 106 per plate and after 24 h were treated with ethanol vehicle (control), 1 nM E2, or BSO plus E2 for 72 h and then stained with fluorescein isothiocyanate (FITC)-annexin V and propidium iodide (PI) and analyzed by flow cytometry. PI was used as a cell viability marker. Representative cytograms are shown for each group. Quantitation of apoptosis (percentage of control) in the different treatment groups is shown on the right. bars, ± SEM. *, p < 0.05 compared with control cells; #, p < 0.01 compared with estradiol-treated cells.
Apoptosis (programmed cell death) is required for normal development and tissue homeostasis in multicellular organisms.
Deregulation of apoptosis is fundamental to many diseases, such as cancer, stroke, heart disease, neurodegenerative disorders, and autoimmune disorders. There are two main pathways for apoptosis, namely the extrinsic receptor mediated pathway and the intrinsic mitochondria-mediated pathway.
Components of the extrinsic pathway include the death receptors FasR/FasL, DR4/DR5, and tumor necrosis factor (TNF), whereas the intrinsic pathway centers on the Bcl-2 family of proteins which comprises both proapoptotic proteins, such as Bax, Bak, and Bid and antiapoptotic proteins, such as Bcl-2 and Bcl-xL.
The Bcl-2 family proteins regulate apoptosis by altering mitochondrial membrane permeabilization which leads to the release of apoptogenic factors such as cytochrome c, procaspases, and apoptosis inducing factor (AIF).
In particular, Bcl-2 and Bcl-xL inhibit apoptosis by maintaining mitochondrial membrane integrity whereas Bax and Bak facilitate apoptosis by initiating the loss of outer mitochondrial integrity.
Apart from its action on the mitochondria, there is also evidence that Bcl-2 possesses antioxidant property.
Bcl-2 overexpression increases cellular glutathionine level which is associated with increased resistance to chemotherapy-induced apoptosis whereas glutathionine depletion restores apoptosis in Bcl-2 expressing cells.
Currently, estrogen deprivation using aromatase inhibitors is one of the standard treatments for postmenopausal women with estrogen receptor (ER)-positive breast cancer.
Unfortunately, a major clinical problem with the use of prolonged estrogen deprivation is the development of drug resistance (that is, hormone-independent growth).
Our laboratory as well as other investigators, have instigated a major effort in studying antihormone resistance in breast cancer and have developed model systems of estrogen deprivation that are sensitive or resistant to the apoptotic actions of estrogen.
In particular, we have previously reported the development of an estrogen deprived breast cancer cell line, MCF-7:5C, which undergoes estradiol-induced apoptosis after 2 days of treatment via the mitochondrial pathway.
In contrast, we have another estrogen deprived breast cancer cell line, MCF-7:2A, which appears to be resistant to estradiol-induced apoptosis.
L-Buthionine sulfoximine (BSO) is a specific γ-glutamylcysteine synthetase inhibitor that blocks the rate-limiting step of glutathionine (GSH) biosynthesis and in doing so depletes the intracellular GSH pool in both cultured cells and in whole animals.
GSH is a water-soluble tripeptide composed of glutamine, cysteine, and glycine. Reduced glutathione is the most abundant intracellular small molecule thiol present in mammalian cells and it serves as a potent intracellular antioxidant protecting cells from toxins such free radicals. Changes in GSH homeostasis have been implicated in the etiology and progression of a variety of human diseases, including breast cancer. In particular, studies have shown that elevated levels of GSH prevent apoptotic cell death whereas depletion of GSH facilitates apoptosis. BSO depletes cellular GSH and sensitizes tumor cells to apoptosis induced by standard chemotherapeutic agents.
Source: Joan S Lewis-Wambi, Helen R Kim, Chris Wambi, Roshani Patel, Jennifer R Pyle,Andres J Klein-Szanto and V Craig Jordan