Fig. 11-Ethyl-2-[[3-ethyl-5-(3-methylbenzothiazolin-2-yliden)]-4-oxothiazolidin-2-ylidenemethyl] pyridinium chloride (MKT-077) induces growth inhibitory effects in medullary thyroid carcinoma (MTC) cell culture. (A) TT and MZ-CRC-1 cells in 24-well plates were treated with serially increasing doses of MKT-077 and vandetanib for 48 hours. Cells were then allowed to recover in drug-free fresh medium for 48 hours, prior to measurement of cell viability by MTT assay. Data (mean±SD, n=6) are expressed as the percentage of vehicle-treated control. (B, C) TT and MZ-CRC-1 cells were treated with MKT-077 at indicated doses for 48 hours prior to cell cycle analysis using Hoechst 33342. Control cells were treated with equal volume of dimethyl sulfoxide. (B) Data are expressed as the percentage of living cells in each cell cycle phase. (C) Number of apoptotic cells was estimated by counting cells in the subG0/G1 phase. (D) Total lysates of TT and MZ-CRC-1 cells treated with different doses of MKT-077 for 48 hours were analyzed by Western blotting for indicated proteins. β-Actin is the control for equal loading. Ki67, MKI67-encoded protein; E2F-1, E2 promoter binding factor 1; PARP, poly(ADP-ribose) polymerase; COX IV, cytochrome c oxidase; PGC-1α, peroxisome proliferator-activated receptor gamma coactivator 1α; RET, rearranged during transfection.
Fig. 2Differential mitochondrial accumulation of 1-ethyl-2-[[3-ethyl-5-(3-methylbenzothiazolin-2-yliden)]-4-oxothiazolidin-2-ylidenemethyl] pyridinium chloride (MKT-077) in medullary thyroid carcinoma (MTC) cells. (A) TT and MZ-CRC-1 cells were treated with 1 µM MKT-077 and 100 nM MitoTracker Green FM (Thermo Fisher Scientific) for 30 minutes. Pictures were then taken under a fluorescent microscope (×400). (B, C) TT and MZ-CRC-1 cells, treated with increasing doses of MKT-077 for 1 hour, were analyzed by flow cytometry to measure red fluorescence (PE channel, 575 nm) (B). (C) Data of 20,000 cells per dose were normalized to dimethyl sulfoxide-treated control and presented as mean±SD. (D) TT and MZ-CRC-1 cells were stained with 5 nM TMRE for 15 minutes and analyzed by flow cytometry to measure red fluorescence (PE channel, 575 nm). Unstained cells were used as negative controls. (E) Total lysates of TT and MZ-CRC-1 cells were analyzed by Western blotting for indicated proteins. GAPDH is the control for equal loading. TMRE, tetra-methyl-rhodamine ethyl ester perchlorate; RET, rearranged during transfection; PGC-1α, peroxisome proliferator-activated receptor gamma coactivator 1α; COX IV, cytochrome c oxidase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
Fig. 31-Ethyl-2-[[3-ethyl-5-(3-methylbenzothiazolin-2-yliden)]-4-oxothiazolidin-2-ylidenemethyl] pyridinium chloride (MKT-077) effects are mediated independently of TP53. (A) Western blot analysis of total lysates. TT cells were infected for 5 days with lentiviral pLKO.1 expressing TP53-targeting shRNA (shp53) or the control virus pLKO.1 prior to the treatment with indicated doses of MKT-077 for 48 hours. (B) TT cells infected with lentiviral shp53 or the control pLKO.1 in 24-well plates were treated with increasing doses of MKT-077 for 48 hours. Cells were then allowed to recover in drug-free fresh medium for 48 hours prior to the measurement of cell viability by MTT assay. Data (mean±SD, n=4) are expressed as the percentage of vehicle-treated control. Ki67, MKI67-encoded protein; E2F-1, E2 promoter binding factor 1; RET, rearranged during transfection; PARP, poly(ADP-ribose) polymerase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; NS, not significant.
Fig. 4The reactive oxygen species (ROS) scavenger, N-acetyl-cysteine (NAC), can attenuate 1-ethyl-2-[[3-ethyl-5-(3-methylbenzothiazolin-2-yliden)]-4-oxothiazolidin-2-ylidenemethyl] pyridinium chloride (MKT-077)-induced ROS production and cell death, but not RET down-regulation. (A) TT cells treated with MKT-077 for 24 hours were treated with 1 µM 5-(and-6)-carboxy-2',7'-dichlorodihydrofluorescein diacetate (carboxy-H2DCFDA) for 1 hour and then incubated for 2 hours in a dye-free culture medium. Cells were harvested and analyzed by flow cytometry to measure green fluorescence (fluorescein isothiocyanate [FITC] channel, 525 nm). (B) Cells were pretreated with 5 mM NAC for 2 hours, and then incubated with indicated doses of MKT-077 for 24 hours before measurement of carboxy-H2DCFDA fluorescence by flow cytometry. (C) Cells were treated with indicated doses of MKT-077 in the presence of 5 mM NAC in the medium containing 2% fetal bovine serum (FBS) for 24 hours. Total cell lysates were analyzed by Western blotting for indicated proteins. GAPDH is the control for equal loading. PARP, poly(ADP-ribose) polymerase; RET, rearranged during transfection; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
Fig. 51-Ethyl-2-[[3-ethyl-5-(3-methylbenzothiazolin-2-yliden)]-4-oxothiazolidin-2-ylidenemethyl] pyridinium chloride (MKT-077) suppresses TT xenografts in athymic mice. (A) Athymic mice bearing TT xenografts were treated with MKT-077 (10 mg/kg/dose). Drug was administered intraperitoneal every two days beginning from day 22 after tumor implantation. The control group was treated with the vehicle only (METHODS). Changes in tumor sizes were determined at indicated time-points. (B) Weights of tumors collected at the end of the treatment. Data are mean±SE (n=8). aP<0.05; bP<0.001.