br All animal experiments were performed in

All animal experiments were performed in accordance with the NIH guidelines on laboratory animal welfare and approved by the Institutional Animal Care and Use Committee of the National Taiwan University College of Medicine (IACUC number: 20110303).
Data are expressed as mean values ± standard errors of the means (SEM) and were analyzed using a one-way analysis of variance (ANOVA). If the ANOVA indicated significant intergroup differences, Tukey's post hoc test was used to determine the pairs of groups ex-hibiting these differences. A p value < 0.05 was considered statistically significant.
Fig. 4. MPT0G211 treatment markedly inhibited SSH1 expression and cofilin phosphorylation in MDA-MB-231 cells.
Fig. 5. Actin distribution in MDA-MB-231 cells in response to MPT0G211 treatment.
(A) MDA-MB-231 cells were treated with MPT0G211 or tubastatin A (10 μM) for 24 h, after which whole-cell extracts were subjected to western blotting. (B) Cells were treated as described in (A), incubated with a stain™ 488 phalloidin antibody and DAPI, and photographed using a ZEISS LS 510 META confocal microscope under 200× magnification. Scale bar = 50 μm. (C, D) MDA-MB-231 cells were incubated with MPT0G211 or tubastatin A (1, 10 μM) for 24 h, after which total cell lysates were immunoprecipitated with an anti-F-actin antibody and immunoblotted for cortactin (C) or subjected to western blotting with the indicated antibodies
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parenchyma that caused by cancer cells. The scale bar = 5 mm. (C) Body weights of the animals. (D) Paraffin sections of lungs stained with hematoxylin and eosin or Cl-Amidine as indicated. Sections were examined using light microscopy (100 × for hematoxylin and eosin, phospho-cofilin and acetyl-H3 stains; 200 × for acetyl-α-tubulin). Arrows indicate alveolar damage. Scale bars are labeled as appropriate. The results in (B, C) are shown as means ± standard errors of the means. *p < 0.05, **p < 0.01 and ***p < 0.001 versus the control group.
4. Results
4.1. The effects of MPT0G211 on the inhibition of HDAC activity and suppression of tumor cell migration
The structure of MPT0G211 is shown in Fig. 1A. Compared with the HDAC6 inhibitors tubastatin A and ACY-1215 (IC50 = 15 and 4.7 nM, respectively) [17,23], our previous study has demonstrated that MPT0G211 not only more potently inhibited HDAC6 activity (IC50 = 0.291 nM), but also exhibited much higher selectivity for HDAC6 versus other HDAC family of enzymes [20]. We confirmed that the HDAC6 activity-specific inhibition mediated by MPT0G211 was not due to a decrease in HDAC6 levels, as treatment for 6–24 h did not downregulate HDAC6 protein expression in MDA-MB-231 and MCF-7 cells (Fig. 1B).
α-Tubulin is the cytosolic target of HDAC6, whereas histone H3/H4 is known as a downstream target of class I HDACs in the nucleus. Therefore, we used immunoblotting to examine whether MPT0G211 more strongly induced the hyperacetylation of α-tubulin vs. histone H3. As shown in Fig. 1C, α-tubulin acetylation increased significantly in response to treatment with a low concentration (0.1 μM) of MPT0G211 in MDA-MB-231 and MCF-7 cells. The corresponding lack of effect on histone acetylation confirmed the selective inhibitory effect of MPT0G211 on HDAC6 activity. Similar effect was observed on α-tu-bulin acetylation after cells treated with tubastatin A; however, tubas-tatin A also cause acetylation of histone H3 at low concentration, in-dicating that MPT0G211 is a more selective HDAC6 inhibitor.
As TNBC cells are highly metastatic [24], we next examined the capacity of MPT0G211 to inhibit the migration of breast cancer cells. As shown in Fig. 2A, neither MPT0G211 (1 μM) nor paclitaxel (0.01 μM) treatment at low concentrations significantly inhibited the migration of MDA-MB-231 cells, and a high concentration (10 μM) of MPT0G211 had only a slight inhibitory effect. However, the combination MPT0G211 with paclitaxel inhibited breast cancer migration in a con-centration-dependent manner. By contrast, tubastatin A did not mark-edly inhibit breast cancer cell migration either alone or in combination with paclitaxel (Fig. 2A). These results suggest that relative to tubas-tatin A, MPT0G211 is a more potent inhibitor of breast cancer cell migration. We further evaluated the cell viability and cell growth in-hibition of MPT0G211 by MTT and SRB assays. There was no significant cytotoxicity observed by MPT0G211 (10 μM) treatment for 12 and 24 h (Fig. 2B), and the growth inhibition GI50 of MPT0G211 were 16.19, 5.9 μM in TNBC MDA-MB-231 and MCF-7 cells, respectively (Fig. 2C). These results suggested that the anti-metastasis effect of MPT0G211 was not major due to cell death. We also evaluated the inhibitory effect of migration for MPT0G211 in another TNBC MDA-MB-468 cells. As shown in supplemental Fig. 1, the result showed that combination of MPT0G211 and paclitaxel further inhibit MDA-MB-468 cell migration in a dose-dependent manner, while comparing with MPT0G211 or pa-clitaxel alone group. There was no significant cytotoxicity observed after MPT0G211 treatment at 48 h in MDA-MB-468 cells (Supplemental Fig. 1 B, C).