The online version of this article (doi:10.1186/s13045-015-0176-7) contains supplementary material, which is available to authorized users.
Shiow-Lin Pan and Che-Ming Teng contributed equally to this work.
The authors declare that they have no competing interests.
MWC performed almost all the experiments. MJL and JPL provided the HDAC6 inhibitor tubastatin A. YLC was responsible for preparing the materials. JCW and SLP designed the animal study. SLP and CMT organized the manuscript. All authors approved the final manuscript.
Combination therapy is a key strategy for minimizing drug resistance, a common problem in cancer therapy. The microtubule-depolymerizing agent vincristine is widely used in the treatment of acute leukemia. In order to decrease toxicity and chemoresistance of vincristine, this study will investigate the effects of combination vincristine and vorinostat (suberoylanilide hydroxamic acid (SAHA)), a pan-histone deacetylase inhibitor, on human acute T cell lymphoblastic leukemia cells.
Cell viability experiments were determined by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay, and cell cycle distributions as well as mitochondria membrane potential were analyzed by flow cytometry. In vitro tubulin polymerization assay was used to test tubulin assembly, and immunofluorescence analysis was performed to detect microtubule distribution and morphology. In vivo effect of the combination was evaluated by a MOLT-4 xenograft model. Statistical analysis was assessed by Bonferroni’s t test.
Cell viability showed that the combination of vincristine and SAHA exhibited greater cytotoxicity with an IC50 value of 0.88 nM, compared to each drug alone, 3.3 and 840 nM. This combination synergically induced G2/M arrest, followed by an increase in cell number at the sub-G1 phase and caspase activation. Moreover, the results of vincristine combined with an HDAC6 inhibitor (tubastatin A), which acetylated α-tubulin, were consistent with the effects of vincristine/SAHA co-treatment, thus suggesting that SAHA may alter microtubule dynamics through HDAC6 inhibition.
These findings indicate that the combination of vincristine and SAHA on T cell leukemic cells resulted in a change in microtubule dynamics contributing to M phase arrest followed by induction of the apoptotic pathway. These data suggest that the combination effect of vincristine/SAHA could have an important preclinical basis for future clinical trial testing.
Additional file 1: Figure S2. The effect of vincristine and SAHA co-treatment in acute myeloid leukemic cell lines. (A) HL60 (acute promyelocytic leukemia) cells were co-treated with the indicated vincristine and SAHA, and the CI values of the sub-G1 phase were analyzed. (B) The cell viability of U937 (acute monocytic leukemia) after the indicated treatment. V, vincristine; S, SAHA; Tuba, tubastatin A.13045_2015_176_MOESM1_ESM.jpg
Additional file 2: Figure S1. The effect of vincristine combined with various doses of SAHA. (A) MOLT-4 cells were treated with various SAHA alone or combined with 1 and 3 nM vincristine for 48 h. The cell viability was evaluated by MTT assay. (B) The distribution of cell cycle after MOLT-4 cells were treated with various concentrations of SAHA alone or in combination with vincristine 1 or 3 nM for 24 and 48 h. The quantitative data are shown in the time course. (C) The combination effect on G2/M arrest (left figure) and apoptosis (right figure) were used by the combination index (CI).13045_2015_176_MOESM2_ESM.jpg
Additional file 3: Figure S3. The results of vincristine- (0.3 and 3 μM), or SAHA (5 and 50 μM) alone, or combination of both in vitro tubulin polymerization assay. (A) The figure shows more comprehensive results on in vitro tubulin polymerization, including paclitaxel (10 μM), vincristine (0.3, 3, and 10 μM), SAHA (S, 5 and 10 μM) alone, and SAHA combined with vincristine (S 50 + V 0.3 and S 5 + V3). (B) The absorbance of different drugs at the endpoint (30 min).13045_2015_176_MOESM3_ESM.jpg
Additional file 4: Figure S4. The quantity of abnormal spindles in vincristine alone compared to combination with SAHA. MOLT-4 cells were treated with SAHA (0.5 μM) and vincristine alone (3 nM) or co-treatment for 24 h and then stained with β-tubulin and DAPI. The figure shows the quantity of abnormal spindles by the immunofluorescence images.13045_2015_176_MOESM4_ESM.jpg
Additional file 5: Figure S5. The cell toxicity of normal cells after combination treatment. The cell viability (MTT assay) of (A) HUVEC (human umbilical vein endothelial cell) and (B) BEAS-2B (human bronchial epithelial cell) after co-treating with the indicated drugs for 48 h.13045_2015_176_MOESM5_ESM.jpg
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- The synergic effect of vincristine and vorinostat in leukemia in vitro and in vivo
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