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Improving T-ALL sensitivity to parthenolide
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Figure 3. Combination of parthenolide (PTL) with sulfasalazine (SSZ) overcomes protective effects of mesenchymal stem cells (MSC). (A) Viability of acute lym- phoblastic leukemia (ALL) samples (patients 2, 5, 6, 9, and 10) treated with PTL (10 μM), SSZ (300 μM) or a combination of both agents for 24 hours (h) with or without MSC. (B) Viability of samples (patients 1, 2, 5, 6, and 10) following 24-h exposure to PTL (10 μM) or a combination of PTL with SSZ (300 μM) separated from MSC by tran- swell inserts. Symbols represent the average viability in replicate samples. Each symbol represents an individual patient. Lines represent median and interquartile range. (C) Thiol concentration in media following incubation with MSC with or without SSZ treatment (n=4). Data represent mean±Standard Deviation. Results were ana- lyzed by one-way ANOVA (A) or paired t-test (B and C). *P≤0.05, **P≤0.01.
were significantly lower in MSC conditioned cells (353, range 200-617) compared to those cultured without MSC (573, range 321-796; P=0.05) (Figure 2B).
To examine whether these observed protective effects were specific to MSC, T-ALL cells were co-cultured on BM stromal cells and fibroblasts. PTL toxicity was reduced in co-cultures containing stromal cells and fibrob- lasts but not to the same extent as cultures containing MSC (Online Supplementary Figure S4A). T-ALL cells con- ditioned with stroma had similar ROS levels to those con- ditioned with MSC. ROS levels in cells cultured on fibroblasts were similar to those observed in unsupported cultures (Online Supplementary Figure S4B).
Parthenolide is known to lower rGSH levels in leukemia cells, therefore the ability of MSC to modulate this effect was examined. T-ALL cells conditioned with MSC had significantly higher levels of rGSH (median 131nM, range 108-135nM) compared to those without conditioning (82nM, range 60-99nM; P=0.03). Following PTL treatment, rGSH levels were significantly decreased in both MSC conditioned (P=0.04) and unconditioned (P=0.0003) cells, indicating reduced anti–oxidant activity. However, rGSH levels were still higher in MSC condi- tioned cells (median 87nM, range 66-117nM) compared to those without MSC conditioning (26nM, range 7-35nM; P=0.01) (Figure 2C).
As cysteine is crucial for GSH synthesis in leukemia, it is possible that MSC release cysteine into the surrounding media, thereby moderating the anti-oxidant effects of PTL. A thiol detection assay was used, since cysteine con- tains a free thiol group, to investigate cysteine release. Thiol concentrations were significantly higher in media removed from MSC cultures (91±17 μM) compared to
background media levels (62±17 μM; P=0.0002), repre- senting a 1.5-fold increase over background (Figure 2D).
Blocking thiol release overcomes protective effect
The thiol functional group plays a major role in intracel- lular anti-oxidant defenses. Cysteine residues eliminate ROS, usually by converting them to 2H2O or H2O and O2, and reduce oxidized protein thiols.14 SSZ, an inhibitor of the cystine uptake antiporter xc-, was used to inhibit cys- tine uptake and block subsequent cysteine generation and release in MSC, to determine what effect this would have on the protection conferred by MSC. The combination of PTL and SSZ significantly reduced the median viability of T-ALL cells with MSC conditioning to 38% (range 11- 42%) compared to PTL (71%, range 59-95%; P=0.002) or SSZ alone (88%, range 69-109%; P=0.04) (Figure 3A). The treatment combination reduced viabilities to levels comparable to those observed in cells treated with both agents without MSC conditioning (16%, range 5-24%). In the absence of MSC, SSZ had low cytotoxicity (92% median survival, range 77-102%) and when combined with PTL there was no significant difference in toxicity compared to PTL alone (P=0.9). Addition of PTL+ SSZ to T-ALL cells conditioned with BM stroma or fibroblasts also reduced leukemia cell survival, although to a lesser extent (Online Supplementary Figure S4C). Interestingly, the effects of combining these agents were more remark- able in samples that were relatively resistant to PTL. Fifty- six percent of cells from patient 9 survived treatment with PTL, but viability was reduced to only 6% when PTL was combined with SSZ. Leukemia burden in NSG mice engrafted with this sample was significantly reduced when treated with PTL and SSZ compared to placebos
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