Vincristine could partly suppress stromal support to T-ALL blasts during pegylated arginase I treatment
© Kwong-Lam and Chi-Fung; licensee BioMed Central Ltd. 2013
Received: 6 February 2013
Accepted: 28 March 2013
Published: 10 April 2013
Relapsed T-lineage acute lymphoblastic leukemia (T-ALL) has been an incurable disease. Recent reports showed that an L-arginine depleting enzyme, pegylated arginase (BCT-100) may be effective against T-ALL cells. On the other hand, studies including ours had shown the symbiosis of ALL blasts and human mesenchymal stromal cells (hMSCs) in bone marrow microenvironment during L-asparaginase treatment. As L-asparaginase and BCT-100 both act by depleting lymphoid cells of specific amino acid, we hypothesized that hMSCs may also protect T-ALL blasts from BCT-100 treatment in co-culture and such protection may be abrogated by pre-treating hMSCs with vincristine (VCR).
XTT assay was used to test sensitivities of T-ALL cell lines and hMSCs to BCT-100. Apoptosis of T-ALL cell lines with or without BCT-100 treatment were tested by annexin V / propidium iodide (AV/PI) assay using flow cytometer. Western blotting was performed to analyze the expression of ornithine transcarbamylase (OTC), an enzyme involved in L-arginine metabolism which may account for BCT-100 resistance.
hMSCs were resistant to BCT-100 while CCRF-CEM, Jurkat and MOLT-4 were very sensitive to it. hMSCs could protect all the three cell lines from BCT-100 treatment in transwell co-culture. All the 3 T-ALL cell lines were also found to be rescued by an L-arginine precursor citrulline, while the breakdown product of BCT-100, ornithine only had limited salvaging effect on CCRF-CEM but not Jurkat and MOLT-4. Both hMSCs and 3 T-ALL cell lines express citrulline synthesis enzyme, ornithine transcarbamylase (OTC) at basal level while only hMSCs could express OTC at relatively higher level under BCT-100 treatment. Treating hMSCs with vincristine before co-culturing with T-ALL could resume the cytotoxicity of BCT-100 to CCRF-CEM and MOLT-4 cells.
Our results suggest a possible strategy to overcome resistance to BCT-100 from cancer microenvironments by suppressing hMSCs either in marrow or in the perivascular niche using vincristine.
KeywordsArginase Bone marrow Lymphoid leukemic cells Mesenchymal stromal cell Ornithine transcarbamylase Stromal suppression
Acute lymphoblastic leukemia (ALL) is the commonest form of pediatric malignancies which originates from lymphoid precursors . ALL can be divided into 2 sub-types, B-lineage ALL (B-ALL) accounts for 85% of all cases and the remaining 15% are T-lineage ALL (T-ALL) . With the advances in anti-cancer drugs development and treatment regimen design, the long term event free survival of childhood ALL can be up to 85% these days . However, T-ALL is known to have less favorable prognosis and relapse is more common probably due to emergence of chemo-resistance . For patients with relapsed T-ALL, there is still no effective curative strategy up to the moment.
L-asparaginase is a commonly used chemotherapeutic agent in both B-ALL and T-ALL. Its main mechanism is by depletion of L-asparagine, a non-essential amino acid. Leukemic blasts lack L-asparaginase resulting in chemoresistance. It has been shown that mesenchymal stromal cells (MSCs) may contribute to L-asparaginase resistance of B-ALL blasts in bone marrow microenvironment by secreting asparagine . On the other hand, MSCs have a peculiar chemo-sensitivity pattern. MSCs are resistant to most of the commonly used chemotherapeutic agents for leukemia, but remain sensitive to anti-microtubule agents such as paclitaxel and vincristine (VCR) . In almost all currently adopted protocols for childhood ALL, it is recommended to administer VCR 1 or 2 days prior to L-asparaginase administration . Traditional view was that such practice can suppress the allergic reaction to asparaginase. However, our previous work provided a new rationale on this treatment design. We showed that vincristine can temporarily disrupt the stromal support to leukemic blasts and retain the L-asparagine depleted microenvironment . However, the high intrinsic asparaginase resistance in T-ALL blasts implies B-ALL chemotherapy regimens may not be as effective as in T-ALL patients [5, 9]. Drugs with higher efficacy against T-ALL are urgently needed.
Recent reports showed that another amino acid depleting agent, pegylated arginase I (BCT-100), is effective against T-ALL both in vitro and in vivo [10, 11]. BCT-100 is purified from B. subtilis followed by pegylation for prolonging its bio-activity . Arginase breaks down L-arginine into ornithine and urea. This has been proposed as a novel anti-cancer agent because many types of cancer cells cannot synthesize L-arginine [12, 13]. However, cells may potentially be resistant to BCT-100 if they express ornithine transcarbamylase (OTC) or they are able to utilize citrulline under an L-arginine starvation setting . As the nutrient-depleting mechanism of BCT-100 is similar to L-asparaginase, we suspect that T-ALL blasts may acquire chemo-resistance to BCT-100 in a manner similar to that of L-asparaginase resistance induced by hMSCs to B-ALL. Therefore we hypothesized that: 1) hMSCs may protect T-ALL blasts from BCT-100 induced cytotoxicity by providing soluble factors involved in L-arginine metabolism; and 2) BCT-100 resistance induced by hMSCs may be overcome by pre-treating MSCs with vincristine.
Results and discussion
T-ALL cell lines were BCT-100 sensitive while hMSCs were BCT-100 resistant
hMSCs partly protected T-ALL cells from BCT-100 induced apoptosis
We first hypothesized the involvement of cytokines in the hMSCs/T-ALL blasts interaction during BCT-100 treatment. A previous report showed that IL-8 was up-regulated in T-ALL cells refractory to chemotherapy . IL-8 is known to be secreted by both hMSCs and T-ALL cells [19, 20]. However, other reports reported that CCRF-CEM, Jurkat, MOLT-4 and primary T-ALL cells do not express IL-8 receptors CXCR1 and CXCR2 [19, 21]. It implies that IL-8 works on the cancer micro-environment mainly by enhancing angiogenesis and immune cell migration . Therefore IL-8 may not be an important factor in stromal protection to T-ALL blasts against BCT-100. Therefore, we then investigated whether the possible soluble factors involved are chemicals mainly involve in arginine metabolism.
Ornithine and citrulline could partly rescue T-ALL cells
hMSCs protected T-ALL cell lines against BCT-100 induced cytotoxicity possibly in part by their sustained OTC expression
Vincristine pre-treatment on hMSCs could potentially override the protective effect of hMSCs on T-ALL cells during BCT-100 treatment
Differential toxicity of BCT-100 to T-ALL blasts and hMSCs was observed. BCT-100 induced significant cytotoxicity to 3 T-ALL cell lines including CCRF-CEM, Jurkat and MOLT-4 but not hMSCs. With such differential response between T-ALL cells and hMSCs as basis, the interaction of hMSCs and T-ALL blasts during BCT-100 treatment was further investigated. hMSCs could partly rescue all 3 T-ALL cell lines from BCT-100 induced toxicity. While testing for the involvement of L-arginine metabolic pathway substrates in the rescue mechanism, all the 3 T-ALL cell lines tested could utilize citrulline for enhancing survival during BCT-100-induced L-arginine deprivation. On the other hand, only CCRF-CEM could marginally utilize ornithine for survival during BCT-100 treatment. hMSCs and all 3 T-ALL cell lines expressed OTC, which means both hMSCs and T-ALL blasts should be capable of converting ornithine into citrulline and eventually recycling L-arginine even under BCT-100 treatment. However, the expression of OTC could also be suppressed in both hMSCs and T-ALL cell lines during BCT-100 treatment. Despite the decrease in OTC expression, expression in hMSCs remained relatively higher than that of the three T-ALL cell lines. The differential OTC expression between hMSCs and T-ALL cells during BCT-100 treatment suggested that hMSCs could potentially protect T-ALL cells from BCT-100 treatment by their sustained OTC expression. In order to suppress the stromal protection to T-ALL blasts, hMSCs could be pre-treated with vincristine (VCR). The protective effect of hMSCs to 2 of the 3 T-ALL cell lines against BCT-100 were abolished with this approach. This suggests a possible synergy between VCR and BCT-100 in treating T-ALL in the cancer microenvironment, similar to the synergy of VCR and L-asparaginase in ALL chemotherapy protocol. Further investigation using animal studies may help to improve the efficacy of BCT-100, especially when in combination with VCR.
Material & methods
Bone marrow mononuclear cells from healthy young adults’ bone marrow transplantation donor were obtained with written informed consent under approval of Institutional Review Board (The Hong Kong University and Hong Kong West Cluster Hospitals). The bone marrow-derived human MSCs were expanded and characterized in vitro according to our previous published method . The human telomerase reverse transcriptase immortalized MSCs (hTertMSCs) was a gift from Prof. D. Campana (St Jude Children's Research Hospital, Memphis, Tennessee) . hTertMSCs or hMSCs were cultured in low-glucose Dulbeco's Minimal Essential Medium (DMEM-LG) supplemented with 10% fetal bovine serum (FBS), 1% penicillin/ streptomycin (P/S), and 1% L-glutamine. CCRF-CEM, Jurkat and MOLT-4 are T cell lineage ALL cell lines purchased from American Type Culture Collection (ATCC). The 3 T-ALL cell lines were cultured in RPMI 1640 supplemented with 10% FBS, 1% HEPES, 1% Penicillin / Streptomycin.
Antibodies and chemicals
BCT-100 was provided by Dr Cheng NM, Paul of Bio-Cancer Treatment Ltd. Vincristine was purchased from Eli Lily (Eli Lily and Company Incorporated, UK). Mouse Anti-human OTC antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit anti-human β-actin antibody was purchased from Cell Signaling Technology (Danvers, MA). L-citrulline and L-ornithine were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA).
Transwell co-culture of MSCs and T-ALL cell lines
hTertMSCs or hMSCs were seeded at the density of 1 × 104 cells/well onto 12-well plates. They were then treated with or without vincristine sulfate (VCR, Eli Lily and Company Incorporated, UK) as previously described . Both MSCs were washed with phosphate buffered saline and then incubated with RPMI 1640 medium supplemented with 10% FBS, 1% HEPES, 1% penicillin/streptomycin and 1% L-glutamine. ALL blasts (1 × 105) (CCRF-CEM, Jurkat or MOL-4) were seeded onto the transwell insert of the 12-well plates seeded with the previously seeded MSCs. MSCs/T-ALL blasts co-culture was treated with or without 1 U/mL BCT-100 for 36 hours.
Cells were plated at 104 per well in 96 well plate with increasing concentration of BCT-100 (0.3125 U/ml, 0.625 U/ml, 1.25 U/ml, 2.5 U/ml, 5 U/ml and 10 U/ml) for 48 hours at 37°C. Cell viability was determined by a colorimetric method using XTT cell proliferation assay kit according to manufacturer’s instructions (Roche Diagnostics, Switzerland).
Protein extraction and Western blot analysis
Protein extraction and Western blot analysis were carried out as follow. After treatment, cells were washed with phosphate buffered saline, and then resuspended in lysis buffer (phosphate-buffered saline containing 1% Nonidet P-40, 0.5% sodium deoxycholate and 0.1% SDS) containing the protease inhibitors (100 μg/ml phenylmethylsulfonyl fluoride and 1 mM sodium fluoride). The lysate was incubated on ice for 20 min, and then centrifuged at 13,200 rpm for 20 min at 4°C. Protein concentration was determined using the Bio-Rad protein assay. Whole cell lysate containing 15 μg of protein from each sample were used in immunoblotting, and subsequently the gels were electro-blotted onto PVDF membranes (Immobilon-P, Millipore). Antibodies purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and Cell Signaling Co. (Danvers, MA) were used to detect the proteins of interest. The horseradish peroxidase conjugated antibodies against mouse and rabbit IgG were used as secondary antibodies (Sigma-Aldrich, St. Louis, MO). The secondary antibody binding was detected by ECL Western detection blotting reagents (GE Healthcare, USA) and detected by Fuji Medical X-Ray Film (Fuji, Japan).
AV/PI (Annexin V-FITC Kit, Becton, Dickinson and Company, NJ) was used to assess the extent of apoptosis of T-ALL cells after exposure to arginase treatment. T-ALL blasts in transwell co-culture were harvested, washed and then re-suspended in 1X AV binding buffer provided by the commercial kit. The cells were labeled with AV-FITC and propidium iodide (PI) following the manufacturer's instruction and analyzed by flow cytometry. Flow cytometric analysis was performed with a BD LSR II (BD Biosciences, San Jose, CA). Ten thousand events per sample were collected into list mode files and analyzed by FlowJo.
Experiments were performed for 3 times with consistent results. Comparisons between mean values were made using two way's analysis of variance (ANOVA) or paired Student t test (one-tailed). The differences were considered statistically significant when p < 0.05.
T-lineage acute lymphoblastic leukemia
- BCT-100 (brand name):
Human mesenchymal stromal cells
- XTT assay:
2,3-bis-(2-methoxy-4-nitro- 5-sulfophenyl)-2H-tetrazolium- 5-carboxanilide assay, vincristine (VCR)
- AV/PI assay:
Annexin V / propidium iodide assay
Human telomerase reverse transcriptase immortalized mesenchymal stromal cells.
Dr Dee MF was responsible for proof-reading of the manuscript. Dr. Cheng NM (Bio-Cancer Treatment Ltd., Hong Kong ) provided BCT-100.
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