Glutathione and Glutathione Utilizing Enzymes Are Key Determinants in the Sensitivity of Myeloma Cells to Arsenic Trioxide as Well as Its Mechanism of Action.
Journal - ASH Annual Meeting Abstracts
Arsenic trioxide (ATO, Trisenox) is currently being tested inclinical trials as a single agent or in combination with otheragents that have activity in multiple myeloma (MM). We and othershave demonstrated that glutathione (GSH) levels can influencethe ability of ATO to induce cell death in MM cell lines/patientsamples and based on these data have initiated a trial to testthe safety and efficacy of the combination of ATO and ascorbicacid for the treatment of refractory/relapsed MM. Thereforewe performed expression profiling on 4 MM cell lines treatedwith ATO over a 48 timecourse. Affymetrix Hu133 2.0 plus arrayswere hybridized and up to 7035 out of 54,675 probe sets displayeda change and up to 1546 probes sets displayed 2 fold or greaterchanges compared to untreated cells at 6 hrs. By only lookingat genes that increased in all four cell lines we restrictedour search to less than 365 probes sets at any given time point.Interestingly the cells appear to have initiated several pathwaysthat are consistent with an attempt to enhance GSH synthesis.Upregulation of transporters of cysteine (xCT) and glycine (Glyt1)as well as enzymes that convert methionine to cysteine (cystathionase)and serine to glycine (serine hydroxymethyl transferase-1) wasobserved. Moreover the rate-limiting step of the glutathionesalvage pathway gamma-glutamyltransferase is also upregulated.Together this suggests an increase in the building blocks forGSH that can be used for de novo synthesis. The rate limitingstep for this reaction is performed by gamma-glutamate cysteineligase which both the catalytic and modifier subunits are upregulated.GSH can also be regenerated from GSSG by glutathione reductase(GR) in an NADPH-dependent fashion. Both GR and the NADPH generatingmalic enzyme are also upregulated following treatment with ATO.While consistent with our previous findings the data do notprovide much insight as to how the GSH is utilized. The onlyGSH utilizing enzyme that was observed to be upregulated werethe cytosolic and mitochondrial forms of glutaredoxin. Glutathioneperoxidase (GPx) activity is not altered by ATO treatment. HoweverGPx baseline expression and activity do correlate with sensitivityof MM cell lines to ATO. We also determined GSTP1 activity inthe cells and found that it was expressed in 4/5 MM cell linestested. In contrast to GPx, GSTP1 baseline expression did notcorrelate with sensitivity to ATO. However this pattern of expressioncorrelated with our previous findings regarding these cellsdemonstrating differences in caspase dependence of ATO-inducedcell death. The one line that did not express GSTP1, RPMI 8226,also utilizes caspase-independent mechanisms of cell death.Transfection of the GSTP1A allele into these cells could rendercells more resistant to ATO-induced apoptosis at concentrationsof ATO that are not likely to be achieved in patients. Interestinglytransfection of the GSTP1B allele could not render cells moreresistant, however like GSTP1A it resulted in inhibition ofthe caspase-independent pathway. Taken together these data confirmthat GSH is an important modulator of ATO therapy and that GPxexpression may determine the sensitivity of cells to ATO whileGSTP1 can affect the mechanism of action by which ATO-inducesapoptosis.