Fig 1.
Chaetocin inhibits cell viability in melanoma cells.
Cells (A375, Sk-Mel-28, IGR37, LU-1205, MV3, and normal melanocytes) were treated with different doses of chaetocin for 24 h, and cell viability was determined by MTT assay. (A) Chaetocin suppressed cell proliferation in a dose-dependent manner. (B) The IC50 was determined when various cells were treated by chaetocin for 24 h. (C-D) Sk-Mel-28 and A375 cells were treated with 10 μM chaetocin for 24, 48 and 72 h, and displayed a time-dependent inhibitory effect on cell viability. Data represented as means ± SEM (n = 3). *P< 0.05 and **P< 0.01, with the untreated controls. #P< 0.05, compared with normal melanocytes.
Fig 2.
Chaetocin induces apoptosis in human melanoma cells.
Apoptosis was detected by Annexin V-FITC/PI using flow cytometry. (A) The representative plots of flow cytometry for chaetocin-induced apoptosis in Sk-Mel-28 and A375 cells which were treated with 0, 5 and 10 μM chaetocin for 24 h. (B) The cells were treated with indicated concentrations of chaetocin for 24 h, and demonstrated an increasing apoptotic rate in a dose-dependent manner. (C-D) Sk-Mel-28 (C) and A375 (D) cells were treated without or with 10 μM chaetocin for 24, 48 and 72 h, and apoptotic cells were determined with flow cytometry. Data represented as means ± SEM (n = 3). *P< 0.05 and **P< 0.01, compared with the controls.
Fig 3.
Chaetocin reduces mitochondrial membrane potential (Δψm).
SK-Mel-28 and A375 cells were incubated in the presence or absence of chaetocin for 12 h, and mitochondrial membrane potential was analyzed as described in Materials and methods. Chaetocin significantly decreased mitochondrial membrane potential of Sk-Mel-28 and A375 cells. Data represented as means ± SEM (n = 3). **P< 0.01, compared with the controls.
Fig 4.
Chaetocin treatment produces ROS and N-Acetyl Cysteine (NAC) reduces chaetocin-induced apoptotic effects in human melanoma cells.
(A-B): Chaetocin treatment resulted in ROS generation in Sk-Mel-28 (A) and A375 (B) cells, as detected by a fluorescence plate reader. Data represented as means ± SEM (n = 3). **P< 0.01, compared with controls. (C-D): NAC pre-treatment reduced chaetocin-induced ROS generation in Sk-Mel-28 (C) and A375 (D) cells. (E-F) NAC pre-treatment attenuated chaetocin-induced apoptosis in Sk-Mel-28 (E) and A375 (F) cells. Data represented as means ± SEM (n = 3). **P< 0.01.
Fig 5.
The effects of chaetocin treatment on the expression of Nrf2, SOD2 and catalase.
(A-B) The cells were treated with 0, 5 and 10 μM chaetocin for 4 and 12 h, and the protein levels of Nrf2, SOD2, and catalase from Sk-Mel-28 (A) and A375 cells (B) were evaluated Western blotting. The results of Western blotting were scanned and analyzed by NIH image J 3.0. The protein levels of Nrf2, SOD2 and catalase were quantified by densitometric ratio of targeted protein/β-actin. Data represented as means ± SEM (n = 3). *P< 0.05 and **P< 0.01, compared with the controls. SOD2: Superoxide dismutase 2.
Fig 6.
The effects of chaetocin treatment on cytochrome c release and Bax and Bcl-2 expression in melanoma cells.
(A) The cells were treated with 0, 5 and 10 μM chaetocin for 48 h, and the protein expression of cytochrome c, Bax and Bcl-2 in Sk-Mel-28 and A375 cells was analyzed with Western blotting. (B) Pre-treatment with N-acetyl cysteine (NAC) counteracted the chaetocin-mediated effects on the protein expression of cytochrome c, Bax and Bcl-2 in the cells. (C-D) The protein levels of cytochrome c, Bax and Bcl-2 were quantified by densitometric ratio of targeted protein/β-actin. Data represented as means ± SEM (n = 3). *P< 0.05 and **P< 0.01, compared with the controls; ##P< 0.01, compared with 10μM chaetocin.
Fig 7.
The effects of chaetocin treatment on the expression level of caspase-9/-3.
Sk-Mel-28 and A375 cells were treated with 0, 5 and 10 μM chaetocin for 48 h, and cellular lysates were then prepared and subjected to SDS-PAGE analysis. (A): Representative western blot images for procaspase-9, cleaved caspase-9, procaspase-3, and cleaved caspase-3. (B-C): The relative protein levels of procaspase-9, cleaved caspase-9, procaspase-3, and cleaved caspase-3 in Sk-Mel-28 (B) and A375 (C) cells were quantified by densitometry. The data are represented as means ± SEM (n = 3). *P< 0.05 and **P< 0.01, compared with the controls.
Fig 8.
Chaetocin treatment elevates the activities of caspase-9 and caspase-3.
(A-B): Sk-Mel-28 and A375 cells were treated with 0, 5 and 10 μM chaetocin for 48 h, and their caspase-9 and caspase-3 activities were measured as described in Materials and methods. Data represented as means ± SEM (n = 3). *P< 0.05 and **P< 0.01, compared with the controls.
Fig 9.
Chaetocin inhibits tumor growth in xenografts.
Mice xenografted with Sk-Mel-28 and A375 cells were intraperitoneally injected with chaetocin (2 mg/kg/day) for 20 days when the tumor volume reached 100 ± 10 mm3 (on 12th days of cell inoculations). (A-B): Tumor volume was assessed every 4 days, and average tumor weight was determined after the mice were sacrificed at the end of treatment. *P< 0.05 and **P< 0.01 vs controls, n = 6/group. (C): Immunohistochemical staining was used to detect PCNA expression (dark brown) in the xenograft sections. Original magnification: 200 ×. (D): TUNEL assay was used to evaluate cellular apoptosis (green) in Sk-Mel-28 and A375 xenografts from control (saline) and chaetocin treated mice. Original magnification: 200 ×. (E): Western blot analysis of the protein expression levels of active caspase-9/-3 (cleaved caspase-9/-3), Bax and Bcl-2 in the tumor lysates of control and chaetocin-treated mice. (F): Quantification of cleaved caspase-9/-3, Bax and Bcl-2 protein levels in tumor the lysates of control and chaetocin-treated mice. *P< 0.05 and **P< 0.01 vs controls, n = 6/group.