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. 2016 Nov 8;7(45):73147-73159.
doi: 10.18632/oncotarget.12246.

Adipocytes cause leukemia cell resistance to daunorubicin via oxidative stress response

Xia Sheng  1 , Jonathan Tucci  1 , Jean-Hugues Parmentier  1 , Lingyun Ji  2 , James W Behan  1 , Nora Heisterkamp  3   4   5 , Steven D Mittelman  1   4   6
Affiliations

Adipocytes cause leukemia cell resistance to daunorubicin via oxidative stress response

Xia Sheng et al. Oncotarget. .

Abstract

Adipocytes promote cancer progression and impair treatment, and have been shown to protect acute lymphoblastic leukemia (ALL) cells from chemotherapies. Here we investigate whether this protection is mediated by changes in oxidative stress. Co-culture experiments showed that adipocytes protect ALL cells from oxidative stress induced by drugs or irradiation. We demonstrated that ALL cells induce intracellular ROS and an oxidative stress response in adipocytes. This adipocyte oxidative stress response leads to the secretion of soluble factors which protect ALL cells from daunorubicin (DNR). Collectively, our investigation shows that ALL cells elicit an oxidative stress response in adipocytes, leading to adipocyte protection of ALL cells against DNR.

Keywords: ALL; adipocyte; drug resistance; glutathione; oxidative stress.

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Conflict of interest statement

CONFLICTS OF INTEREST

The authors disclose no potential conflicts of interest.

Figures

Figure 1
Figure 1. Adipocytes protect ALL from oxidative stress induced cell death
(A) ALL cells co-cultured in TransWells over 3T3-L1 and ChubS7 pre-adipocytes (hatched bars) and adipocytes (black bars) with 72 hour DNR treatment (8093 35 nM, BV173 100 nM, RS4;11 100 nM, and Nalm6 200 nM). No feeder condition is where ALL cells were cultured alone (n = 3–5). (B) One representative image of western blot of Caspase 3 (37 kDa) and cleaved caspase 3 (19 and 17 kDa) in BV173 cells treated with DNR for 24 hours in the presence or absence of adipocytes. Images were histogram stretched in a consistent manner to increase brightness for publication. Quantification of the ratio of cleaved (both bands) over total caspase 3 (band at 37 kDa) is shown on the right (n = 3). (C) BV173 ALL cells cultured over 3T3-L1 cells and treated with various doses of doxorubicin (Dox). (D) 8093 cells treated with 35 nM DNR in 3T3-L1 ACM and ALCM (left, n = 6); BV173 cells treated with 100 nM DNR in ChubS7 ACM and ALCM (right, n = 5). (E) Quantification of cleaved over total caspase 3 of 8093 ALL cells after 24 hours treatment with 25 nM DNR with and without ALCM. (F) Annexin V staining of human ALL cells after 24 hour exposure to DNR with or without ALCM. *P < 0.05, **P < 0.01, ***P < 0.001 All asterisks indicate comparison to control no feeder (NF gray bar) or no DNR no adipocyte conditions (white bar) unless otherwise indicated.
Figure 2
Figure 2. Adipocytes protect ALL cells from oxidative stress
(A) Measurement of % ROShigh population in Nalm6 cells treated with DNR for 6 hours. % ROShigh based on gates defined by cells without DNR treatment. Adipo condition is significantly different than both Fibro and No Feeder by repeated measure ANOVA (p < 0.05). (B) CellRox® intensity by flow cytometry measures intracellular ROS after 24 hours exposure to DNR or DNR plus ALCM. (C) GCLC and GCLM gene expressions by qPCR in 8093 and BV173 cells treated with DNR, with or without adipocytes present (n = 3). Asterisks and p-values indicate comparisons to the no-daunorubicin, no 3T3-L1 condition (white bar), unless otherwise noted. (D) ALL cells co-cultured with 3T3-L1 pre-adipocytes and adipocytes with chaetocin treatment (BV173 and Nalm6 100 nM, RS4;11 50 nM, n = 3–4). (E) Irradiated BV173 ALL cells co-cultured with 3T3-L1 pre-adipocytes and adipocytes (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001.
Figure 3
Figure 3. ALL cells induce oxidative stress in adipocytes
(A) Representative fluorescent confocal microscopy images of 3T3-L1 adipocytes alone, with ALL in TransWells, or treated with 20 mM BSO for 48 hours. Top: DCF (green) only, bottom: DIC images. (B) Quantification of fluorescence (FITC) from 3 images similar to A. *indicate t-tests on log-transformed pixel counts. (C) 3T3-L1 gene expression by qPCR with (gray bar) and without (white bar) 8093 cells; n = 3. (D) Western blot of HO-1 expression in 3T3-L1 adipocytes exposed to DNR, ALL, or both. GAPDH was used as loading control. E. GSH levels measured in media after conditioning with BV173 ALL cells, 3T3-L1 adipocytes, or both, for 48 hours. *P < 0.05, **P < 0.01, ***P < 0.001.
Figure 4
Figure 4. Oxidative stress in adipocytes leads to secretion of survival factors that protect ALL cells from DNR
(A) Representative western blot of HIF-1α in ChubS7 adipocytes treated with CoCl2. GAPDH was used as loading control. (B) 3T3-L1 gene expression by qPCR with CoCl2 treatment for 24 hours (n = 4). (C) Viable cell number of DNR-treated 8093 ALL cells (left) or BV173 cells (right) in the presence of ACM from 3T3-L1 or ChubS7. ACM spiked with CoCl2 were denoted as (+ CoCl2) and ACM conditioned in the presence of 30 μM CoCl2 as (w/ CoCl2, n = 4–8). *P < 0.05 All asterisks indicate comparison to control conditions unless otherwise indicated.
Figure 5
Figure 5. Glutathione synthesis is partially involved in adipocyte protection of ALL cells
(A) Intracellular GSH quantification in 3T3-L1 adipocytes (n = 3). Viable cell number of BV173 and Nalm6 cells treated with DNR (100 and 200 nM, respectively) while co-cultured with 3T3-L1 (B) or ChubS7 (C) adipocytes that were pre-treated with 20 mM BSO for 24 hours (n = 8–10). (D) Viable cell number of BV173 and Nalm6 cells treated with DNR while co-cultured with 3T3-L1 adipocytes that were pre-treated with 250 nM AUR for 24 hours (n = 3).*p < 0.05, **p < 0.01, ***p < 0.001 All asterisks indicate comparison to no feeder or vehicle (Veh) conditions unless otherwise indicated.
Figure 6
Figure 6. Exogenous antioxidants protect ALL cells from DNR
(A) BV173 and Nalm6 treated with DNR or DNR + GSH (20 mM, n = 4). (B) BV173 and Nalm6 treated with DNR or DNR + NAC (20 mM, n = 3–4). (C) Cell viability and intracellular ROS evaluation using flow cytometry with DAPI and CellROX® staining of BV173 (top two panels) and Nalm6 (bottom two panels) treated with DNR alone or DNR and NAC n = 5. *P < 0.05, **P < 0.01, ***P < 0.001 All asterisks indicate comparison to control conditions unless otherwise indicated.
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