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B.C. Ede et al.
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Figure 4. xCT knockdown in mesenchymal stem cells (MSC) overcomes MSC mediated resistance to parthenolide (PTL). Viability of T-cell acute lymphoblastic leukemia (T-ALL) samples (patients 5, 6, 8-10) treated with 10 μM PTL for 24 hours (h) in the presence of MSC pre-treated with xCT or scramble control siRNA, in direct contact (A) or samples (1, 2, 6, 8, and 10) in transwell inserts (B). Symbols represent the average viability in duplicate samples. Each symbol represents an individual patient. Lines represent median and interquartile range. (C) Thiol concentration in media following 24-h incubation with MSC pre-treated with xCT or scram- ble control siRNA compared to background media. Data represent mean±Standard Deviation. (n=4). Results were analyzed by one-way ANOVA (A) or paired t-tests (B and C). *P≤0.05, **P≤0.01.
(P<0.0001) (Online Supplementary Figure S5). Using SSZ alone and in combination with PTL had minimal effects on normal hemopoietic cells (Online Supplementary Figure S6).
In transwell experiments, using SSZ with PTL abrogated the protective effects of MSC support, reducing T-ALL survival from 58% (range 20-71%) to 14% (range 9-43%; P=0.01) (Figure 3B). Media harvested from cultures of SSZ treated MSC contained thiol levels similar to back- ground media (53±19 μM vs. 61±10 μM, respectively). In contrast, media from untreated MSC contained signifi- cantly higher thiol levels (90±11μM; P=0.005) compared to media from SSZ treated cells, representing a 1.5-fold increase over background media (Figure 3C).
As a second approach to block the cystine uptake antiporter xc-, and thereby prevent cysteine release from MSC, xCT was targeted with siRNAs. Relative gene expression levels of xCT were significantly lower in MSC 48 h after transfection with either siRNA 1 (17±9%) or siRNA 2 (9±4%) compared to scramble control siRNA (P≤0.0001) (Online Supplementary Figure S7A). Protein available from xCT siRNA-1 showed a significant reduc- tion in xCT expression (3±0.1%) relative to scramble con- trol levels quantified by western blotting densitometry (P=0.002) (Online Supplementary Figure S7B).
The viability of PTL treated T-ALL cells, co-cultured with MSC treated with xCT siRNA-1 (27%, range 3-28%) or -2 (35%, range 24-42%), was significantly lower than cells co-cultured with scramble control MSC (49%, range 46-72%; P<0.05) (Figure 4A). As siRNA-1 provided the best reduction in MSC protection, it was selected for fur- ther experiments. When transwell inserts were used to separate xCT knockdown MSC from T-ALL cells, the median viability after PTL treatment was significantly
lower (13%, range 7-30%) compared to scramble control MSC cells (24%, range 15-74%; P=0.05) (Figure 4B). Scramble control MSC media had a significantly higher thiol concentration (86±2 μM) compared to media har- vested from xCT knockdown MSC (46±1 μM; P=0.002) (Figure 4C). Media from xCT knockdown MSC did not show any increase in thiol levels above background media (53±1 μM).
To confirm that the observed effects of interfering with xCT were a result of blocking cystine uptake, and not a result of toxicity, the viability and morphology of treated MSC were assessed. Treatment with PTL, SSZ or both agents in combination had no effect on the viability and confluency of MSC (P≥0.37) (Figure 5A) nor did siRNA treatment (P≥0.82) (Figure 5B and Online Supplementary Figure S8). This was also confirmed by flow cytometry (Figure 5C).
Discussion
To date, PTL is the only drug that has been shown to be capable of completely eradicating childhood ALL in NSG xenografts, as a single agent.8 Most studies, using such models, report reduction in leukemia burden but levels often increase on cessation of treatment. Consequently, there is much interest in the application of PTL for cancer therapy. Several groups are developing strategies to improve the bioavailability of PTL, without having detri- mental effects on its pharmacokinetic properties.15-19 PTL can be successfully sequestered into nanoscopic vectors which can achieve equivalent toxicity to unmodified PTL.16- 18 Nanoparticle PTL formulations can be used in vivo at 40- fold lower doses with 20-fold lower administration fre-
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haematologica | 2018; 103(9)