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Improving T-ALL sensitivity to parthenolide
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Figure 5. Effects of parthenolide (PTL) and xCT modifying agents on mesenchymal stem cell (MSC) viability. (A) Average live MSC counts from 3 fields (x10 magnification) following treatment with PTL (10 μM), sulfasalazine (SSZ) (300 μM), or a combination of both agents measured by fluorescence microscopy. (B) Average live MSC counts from 3 fields (x10 mag- nification) following treatment with PTL (10 μM), pre-treated with xCT, or scramble control siRNA. (C) Viability of MSC following treatment with PTL (10 μM) and SSZ (300 μM), or PTL after pre-treatment with xCT siRNA, measured by flow cytometry. Treatment with 70% ethanol served as a positive control for inducing toxicity. Data represent mean±Standard Deviation (n=3). Live cell counts by fluorescence microscopy were calculated using the ImageJ particle analysis software. Results were analyzed by one-way ANOVA (A-C). ***P≤0.001.
quency than standard PTL.16 Some of these delivery sys- tems are inexpensive and formulations are readily scalable.18 Consequently, they should facilitate the use of PTL at clin- ically relevant doses. However, the efficacy of many thera- peutic drugs may be compromised by the host BM microenvironment. Therapy-induced niches, which pro- tect leukemia cells against standard first-line induction agents, have been described in ALL.12,20 In the present study, we investigated whether resistance to PTL, reported in a minority of T-ALL cases,8 may be conferred by BM-derived MSC.
In vitro PTL treatment reduced viability of T-ALL cells to less than 30%, confirming previous results in a separate cohort of pediatric cases.8 While there were differences in the responses of individual patient samples, there was no correlation between PTL cytotoxicity with karyotype or MRD risk status, which may be a result of the heteroge- neous nature of this disease. PTL was shown to increase ROS stress and lower rGSH levels in T-ALL. The anti- oxidative compound NAC blocked ROS upregulation and diminished PTL cytotoxicity, suggesting that PTL toxicity is, at least in part, related to ROS stress in T-ALL. These findings concur with reports of increased ROS stress in pri- mary AML and CLL cell lines following PTL treatment.6,21,22
The protective effect provided by NAC may be attributed to the role of cysteine as the rate limiting amino acid in rGSH production.23 Reduction of cystine to cysteine and subsequent supply to leukemia cells is crucial for GSH syn- thesis in these cells. GSH levels elevate the anti-oxidative capacity of cells, which may provide protection against PTL. In AML and breast cancer, populations of cells endure lower levels of ROS stress and these cells are more resistant to therapy.24,25 It is unclear whether ROS is actively elimi- nated by rGSH or if PTL directly interacts with rGSH, lead- ing to a reduction in anti-oxidative capacity and increased ROS accumulation. One function of rGSH is to detoxify cells from reactive electrophiles,26 which PTL contains in the
form of an a,β-unsaturated carbonyl group. PTL can direct- ly interfere with rGSH synthesis in AML, causing depletion of rGSH, allowing ROS levels to increase.21 Whether PTL directly blocked rGSH synthesis was beyond the scope of this study. However, it is evident that ROS levels increased and rGSH levels dropped, putting the cells under higher lev- els of stress, which is likely to drive apoptosis.
As these data indicate that ROS levels may be linked to PTL cytotoxicity, it is possible that the BM microenviron- ment provides resistance to drug activity by protecting leukemia cells from ROS stress. CLL cells had decreased sensitivity to the ROS inducing agents fludarabine and oxi- platin when co-cultured with BM stromal cells.9 Chemo- protection was derived from the generation and release of cysteine by stromal cells for uptake by CLL cells. The BM microenvironment can also play a role in leukemia drug resistance, mediated by a diverse set of mechanisms, both by direct cell adhesion11,13,27 and/or soluble factor release.12,20,28,29 In this study, the protective effects of MSC against PTL toxicity were related to decreases in ROS stress and preserved rGSH levels, suggesting that MSC increased the anti-oxidative stress capacity of T-ALL cells and thereby conferred resistance to PTL. To eliminate variability, MSC were generated from a single normal BM donor, allowing direct comparison of the effects on the patients' samples. This may have introduced bias but our results are consis- tent with those reported in CLL.9 These data also suggest that protection is provided by one or more secretable fac- tors, as preventing direct contact with MSC still provided T- ALL cells with protection against PTL. However, protection was not to the same extent as observed in direct contact, suggesting involvement of other factors.
Mesenchymal stem cell conditioned media contained sig- nificantly higher levels of thiols, an important feature of the anti-oxidative compound cysteine, compared to uncondi- tioned media. A key mechanism by which normal cells synthesize cysteine is via intake of extracellular cystine
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