Fig 1.
MT-4 induces apoptosis in different cancer cell lines.
(A) Chemical structure of MT-4. (B) Two cancer cell lines and HUVECs were treated with MT-4 at different concentrations for cell viability. The IC50 of MT-4 was evaluated and compared to control by MTT assay after 48 h. The cells were incubated with MT-4 (0.025 to 3 μM) or DMSO (Control, CTL), and the absorbance was read at 570 nm. Data are presented as the mean of three replicate experiments. (C) NCI-ADR/res cells were treated with or without indicated agent (blank) and co-treated with 10 μM rhodamine 123 (RHO). After incubation for 60 min at 37°C, cells were washed with cold PBS and collected by trypsinization. Bound drugs were analyzed by flow cytometry. VPL, verapamil (30 μM); CYC, cyclosporine A (10 μM); paclitaxel (10 μM) and MT-4 (0.3, 1 and 3 μM). The sensitive cells (A2780) were as a surrogate standard to define the accumulation of Rho123 within the cells (Phase 2).
Table 1.
IC50 from MTT assay (μM).
Fig 2.
MT-4 induces G2/M arrest and regulatory proteins.
(A) A2780 and NCI-ADR/res cells were treated with various concentrations of MT-4 (0.05 to 3 μM) or DMSO (Control, CTL). After incubation for 24 h and 48 h, cells were stained with propidium iodide, and cell cycles were analyzed by flow cytometry. Quantitative data are presented from three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001. (B) Both cell lines were treated with 0.3 μM MT-4 for different time intervals. Whole cell lysates were analyzed by western blot for G2/M protein. Similar results were obtained in three independent experiments. (C) Inhibition of tubulin polymerization. Tubulin in the reaction buffer was incubated at 37°C in the presence of DMSO (Control, CTL), the indicated concentration of MT-4 (3 and 10 μM), or 10 μM vincristine and 40 μM paclitaxel. The microtubule assembly was evaluated by measuring absorbance at 340 nm using a spectrophotometer.
Fig 3.
MT-4 induces apoptosis and activates caspases in an ovarian cell line.
(A) The cells were treated with MT-4 at various concentrations and then analyzed using a cell death detection assay after 48 h. Quantitative data are presented from three independent experiments. *p < 0.05, **p < 0.01. (B) Both cell lines were treated with various concentrations (0.05 to 3 μM) of MT-4. After incubation for 48 h, whole cell lysates were subjected to western blot protein analysis. (C) NCI-ADR/res cells were treated with different concentrations of paclitaxel (0.1 to 10 μM) and vincristine (0.1 to 10 μM). After incubation for 48 h, the cell lysates were subjected to western blot analysis. Similar results were obtained in three independent experiments.
Fig 4.
Activation of p38 MAPK contributes to MT-4-induced apoptosis.
(A) A2780 and NCI-ADR/res cells were treated with MT-4 (0.3 and 1 μM) or DMSO (Control, CTL) or in the presence of SB203580 (10, 20, and 50 μM). After incubation for 48 h, viable cells were analyzed by MTT assay. *p < 0.05, **p < 0.01, ***p < 0.001. Data represent the mean ± S.E.M. of at least three independent experiments. (B) The cells were treated with DMSO (Control, CTL) and 0.3 μM MT-4 alone, SB203580 alone (20 μM or 50 μM), or a combination of both reagents for 48 h. Cell apoptosis via PARP and p38 protein was evaluated by western blot. (C) Both cells were transfected with or without 2 μg of dominant-negative (DN)-p38 plasmid and then incubated with MT-4 (0.3 μM) for 48h. Whole cell lysates were subjected to western blot analysis. (D) NCI-ADR/res cells were treated with 0.3 μM MT-4 for different time intervals. Whole cell lysates were subjected to western blot analysis for MKK3/6 and p38 protein. Similar results were obtained in three independent experiments.
Fig 5.
MT-4 regulated HSP27 protein expression.
(A) HSP27, HSP70, and HSP90 mRNAs were analyzed from the Bonome dataset. (B) A2780 and NCI-ADR/res cells were lysed and analyzed by western blot for HSP27 protein. Lane 1, A2780 and Lane 2, NCI-ADR/res. (C) NCI-ADR-res cells were treated with the indicated concentrations of MT-4 (0.05 to 3 μM) and DMSO (Control, C) for 48 h. (D) NCI-ADR/res cells were treated with 0.3 μM MT-4, 10 μM paclitaxel (T), or 10 μM vincristine (Vin) or DMSO (Control, C) for 48 h. (E) NCI-ADR/res cells were treated with 0.3 μM MT-4, SB203580 alone (20 μM or 50 μM) or a combination of both reagents for 48 h. Whole cell lysates were evaluated using western blot analysis for the indicated proteins. (F) NCI-ADR/res cells were transfected with or without 2 μg of domain-negative p38 plasmid and then incubated with MT-4 (0.3 μM) for 48 h. Whole cell lysates were subjected to western blot analysis. Similar results were obtained in at least three other independent experiments. (G) Co-immunoprecipitation (Co-IP) assay and western blot assay. NCI-ADR/res cells were treated as indicated, and lysates were subjected to a Co-IP assay with anti-caspase-3 and were blotted with anti-HSP27 and anti-caspase-3. (H) The cells were transfected with or without 5 μg of wild-type HSP27 plasmid and then incubated with MT-4 (0.3 μM) for 48 h. Whole cell lysates were subjected to western blot analysis and (I) cell death detection kit assays. *p < 0.05. The data represent the mean ± S.E.M. of at least three independent experiments. Densitometric analysis shows fold change in protein levels compared with control group by image J.
Fig 6.
MT-4 triggers apoptosis in an ovarian xenograft animal model.
Xenograft nude mice were intravenously (i.v.) and intraperitoneally (i.p.) administered MT-4 (5 mg/kg and/or 20 mg/kg) daily, paclitaxel (20 mg/kg, i.v.) every 4 days for a total of 5 days, or a vehicle after tumor cell (A2780 and NCI-ADR/res) implantation. (A-B) Tumor size and body weight of the A2780 xenograft mice. (C) Tumor size of the NCI-ADR/res xenografts. * p < 0.05, **p < 0.01 compared to controls.