FIGURE 1

FIGURE 1. CUMA induces morphological change in A375-R and inhibition of A375-R colony formation. (A) Chemical structure of cumingianoside A. (B) Long-term proliferation assay of A375-R treated with vehicle and indicated concentrations of CUMA for six days; top, quantification of crystal violet absorbance at 595 nm; bottom, colony stained with crystal violet (C) A375-R melanoma cells were treated with 20 µM of CUMA for 24 h and 48 h and the morphological changes were recorded by light microscopy (200 ?, 400 ? magnification). Data are mean ± SD, n = 3. Different letters indicate significant difference with P ? 0.05.
FIGURE 2. CUMA treatment induces G2/M cell cycle arrest in A375-R melanoma cells. (A) Cells were treated with vehicle and indicated concentrations of CUMA for 12 h, 24 h, and 48 h, respectively. Top, the cell cycle distribution was measured by PI staining and flow cytometry. Bottom, the percentage of cells in G0/G1, S and G2 phase are presented as mean ± SD of three independent experiments. (B) A375-R cell were treated with 20 µM CUMA for indicated time period and cell cycle-related proteins were detected by western blotting.
FIGURE 3. CUMA treatment induces apoptosis in A375-R melanoma cells. (A) Cells were treated with vehicle and indicated concentrations of CUMA for 72 h. Cisplatin (CP) was used as a positive control. Induction of apoptosis was measured by PI/Annexin double staining and flow cytometry. Q1: dead cells, Q2: late apoptotic cells, Q3: live cells, Q4: early apoptotic cells. (B) Apoptotic fraction represents the sum of Q2 and Q4. Data are mean ± SD of three independent experiments. Different letters indicate significant difference with P ? 0.05. (C) A375-R cell were treated with 20 µM CUMA for indicated time period and apoptosis-related proteins were detected by western blotting.
FIGURE 4. CUMA inhibits BRAFV600E mutant melanoma with acquired resistance to PLX4032 in xenograft model. NOD/SCID mice were inoculated with A375-R melanoma cells and when the tumor reached around 100 mm3 were orally treated with vehicle (tumor control), CUMA (50 mg/kg/day and 75 mg/kg/day; CUMA50 and CUMA75), PLX4032 (50 mg/kg/day), CUMA50 and PLX4032 in combination (50 mg/kg/every other day and 50 mg/kg/day; CUMA50+PLX4032). (A) Tumor volumes were measured every three days and presented as mean ± SD, n ? 6 in each treatment group. (B) At the end of the study tumors were excised and the weight was presented as mean ± SD, n ? 6 for each treatment group. (C) Top, the expression of Ki67, cleaved caspase 3, cleaved PARP and VEGF (brown staining) were examined by immunohistochemistry. Nucleus was stained blue with hematoxylin. Bottom, quantitative data of the detected proteins. Data are mean ± SD, n ? 3. Different letters indicate significant difference with P ? 0.05. (D) Mice body weights were measured every three days and presented as mean ± SD, n ? 6 for each treatment group. (E) Histopathological analysis of the liver and spleen was examined by H&E staining. The integrity of the portal vein and renal glomeruli were examined among groups. (scale bar ? 20 µm)
FIGURE 5. CUMA treatment induces ER stress and autophagy in A375-R melanoma cells. (A) A375-R cell were treated with 20 µM CUMA for indicated time period and ER stress related proteins were detected by western blotting. (B) A375-R cell were treated with 20 µM CUMA for 24 h and autophagy related genes expression were examined by qPCR. (C) A375-R cells were treated with vehicle or 20 µM CUMA for 2 h and LC3B puncta were detected by immunofluoresent staining. (D) A375-R cells were treated with 20 µM CUMA, Baf A1 or CUMA and Baf A1 in combination for 2 h and the autophagic flux was analyzed by measuring the conversion of LC3B-I to LC3B-II. (E) The expression of LC3B (brown staining) in tumor tissues was examined by immunohistochemistry. Nucleus was stained blue with hematoxylin. (F) A375-R cell were treated with CUMA (20 µM), 4-PBA (1 mM), CUMA and 4-PBA in combination for 24 h and the protein expression were analyzed by western blotting. Tg (100 nM), an ER stress inducer was used as a reference drug in this study.

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