Journal: Journal of Pharmaceutical Analysis

Article Title: Tetrandrine targeting SIRT5 exerts anti-melanoma properties via inducing ROS, ER stress, and blocked autophagy

doi: 10.1016/j.jpha.2024.101036

Figure Lengend Snippet: Tetrandrine (TET) inhibited cell proliferation and viability in melanoma cells. (A) Melanoma cells (A375 and MV3) were incubated with a series of different TET concentrations for 48 h. Cell viability was measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. The half-inhibitory concentrations (IC 50 ) values of TET for 48 h in the both cell lines were marked in the upper right corner. (B) Viability of A375 and MV3 cells was tested after treated with TET. (C, D) Morphology of A375 and MV3 cells under different TET concentrations of treatment (5, 10 and 15 μM) for 48 h (C), and Histograms show the effect of TET on the viability of the cells (D). (E) 5-ethynyl-2’-deoxyuridine (EdU) assay evaluated the proliferation capacity of cells treated with dimethyl sulfoxide (DMSO) or TET (10 μM) for 48 h. Quantity of EdU-positive A375 and MV3 cells were calculated. (F) Clonogenicity of A375 and MV3 cells was assessed after treated with DMSO or TET (10 μM). The colonies were cultured for 7 days. Crystal violet was solved by 75% ethyl alcohol and the absorbance of each well was assessed by microplate reader (560 OD). (G) Western blot assay detected the expression of cell cycle-related proteins after treated with different concentrations of TET. (H) The cell cycle of A375 and MV3 cells after treated with TET (10 μM) were detected by flow cytometry. Relative proportions of cells in different phases were presented in bar charts. (I) Apoptosis of A375 and MV3 cells after treated with TET (10 μM) for 48 h. Apoptosis rate was presented in bar charts. (J) Western blot assay detected the expressions of apoptosis-associated proteins in melanoma cells after treating with DMSO and TET (5, 10, and 15 μM). (K) The self-renewal capability of melanoma cells was investigated by soft agar assay after incubating with DMSO or TET (10 μM) for 14 days. All data were analyzed by unpaired Student's t -tests and shown as the means ± standard deviation (SD). ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, ns: not statistically significant. DAPI: 4',6-diamidino-2-phenylindole; FITC: fluorescein isothiocyanate; PI: propidium iodide; c-PARP: cleaved poly (ADP-ribose) polymerase; PUMA: P53 up-regulated modulator of apoptosis; DAPDH: glyceraldehyde-3-phosphate dehydrogenase; OD: optical density.

Article Snippet: Anti-LC3B (Cat. No.: 314600-1-AP), UNC-52-like kinase 1 (ULK1; Cat. No.: 20986-1-AP), p62(18420-1-AP), Syntax 17 (Cat. No.: 17815-1-AP), glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Cat. No.: 60004-1-Ig), Tubulin (Cat. No.: 66031-1-lg), activating transcription factor 6 (ATF6; Cat. No.: 24169-1-ap), inositol requiring enzyme 1 alpha (IRE1α; Cat. No.: 27528-1-AP), double-stranded RNA-dependent protein kinase-like ER kinase (PERK; Cat. No.: 24390-1-AP), CCAAT/enhancer-binding protein homologous protein (CHOP; Cat. No.: 15204-1-AP), phosphatase and tensin homolog (PTEN)-induced kinase 1 (PINK1; Cat. No.: 23274-1-AP), nuclear dot protein 52 kDa (NDP52; Cat. No.: 12229-1-AP), optineurin (OPTN; Cat. No.: 10837-1-AP), P53 up-regulated modulator of apoptosis (PUMA; Cat. No.: 55120-1-AP) and sirtuin 5 (SIRT5; Cat. No.: 15122-1-AP) were purchased from Proteintech (Wuhan, China).

Techniques: Incubation, MTT Assay, EdU Assay, Cell Culture, Western Blot, Expressing, Flow Cytometry, Soft Agar Assay, Standard Deviation

Journal: Journal of Pharmaceutical Analysis

Article Title: Tetrandrine targeting SIRT5 exerts anti-melanoma properties via inducing ROS, ER stress, and blocked autophagy

doi: 10.1016/j.jpha.2024.101036

Figure Lengend Snippet: Tetrandrine (TET) brought about mitophagy and massive reactive oxygen species (ROS) accumulation. (A) Mitochondria (Mito) morphology in both A375 and MV3 cells was observed after treated with dimethyl sulfoxide (DMSO) and TET (10 μM) for 48 h. (B) Left: The co-localizations between mitochondria (red) and LC3B (green) in A375 and MV3 cells were detected by using immunofluorescence (IF) assay after treated with DMSO or TET (10 μM) for 48 h. Right: The intensity of green and red signals in melanoma cells per slide was calculated by ImageJ software. (C) Western blot assay detected the expressions of mitophagy-related proteins after treated with TET (5, 10 and 15 μM) for 48 h and DMSO. (D) Intracellular ROS levels was evaluated by using dichlorodihydrofluorescein (DCFH)-diacetate (DA)-associated fluorescent imaging (left) and flow cytometry (right) after treated with TET (10 μM). (E–H) The levels of intracellular ROS (E), intracellular superoxide anion (F), mitochondrial superoxide anion (G), and mitochondrial hydroxyl radicals (H) in melanoma cells after treated with TET (10 μM) were respectively evaluated by O13 Red, dihydroethidium (DHE), MitoROS 580, and MitoROS OH580 probes. (I, J) Left: Cell morphology of A375 and MV3 cells was observed after respectively treated with DMSO, TET (10 μM), N-acetylcysteine (NAC) (20 μM), or TET + NAC for 48 h. Right: Cell numbers of MV3 and A375 cells were counted (I), and the viability of A375 and MV3 cells was also assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) method (J). (K, L) The morphological structures of microtubule (tubulin antibody) (K) and microfilaments (phalloidin dyes) (L) was detected by IF assay in A375 and MV3 cells after treated with DMSO, TET (10 μM), NAC (20 μM) or TET + NAC for 48 h. (M) After transfected with green fluorescent protein (GFP)-red fluorescent protein (RFP)-LC3 plasmids, the level of autophagy was detected by IF assay in A375 and MV3 cells after treated with DMSO, TET (10 μM), NAC (20 μM) or TET + NAC for 48 h. (N) Western blot detected the expressions of ER stress, autophagy, and apoptosis markers in A375 and MV3 cells after treated with DMSO, TET (10 μM), NAC (20 μM) or TET + NAC for 48 h. All data were analyzed by unpaired Student's t -tests and are shown as the means ± standard deviation (SD). ∗∗ P < 0.01, ∗∗∗ P < 0.001. PERK: double-stranded RNA-dependent protein kinase-like ER kinase; IRE1α: inositol requiring enzyme 1 alpha; PINK1: phosphatase and tensin homolog (PTEN)-induced kinase 1; NDP52: nuclear dot protein 52 kDa; OPTN: optineurin; c-PARP: cleaved poly (ADP-ribose) polymerase; ATF6: activating transcription factor 6; GAPDH: glyceraldehyde-3-phosphate dehydrogenase.

Article Snippet: Anti-LC3B (Cat. No.: 314600-1-AP), UNC-52-like kinase 1 (ULK1; Cat. No.: 20986-1-AP), p62(18420-1-AP), Syntax 17 (Cat. No.: 17815-1-AP), glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Cat. No.: 60004-1-Ig), Tubulin (Cat. No.: 66031-1-lg), activating transcription factor 6 (ATF6; Cat. No.: 24169-1-ap), inositol requiring enzyme 1 alpha (IRE1α; Cat. No.: 27528-1-AP), double-stranded RNA-dependent protein kinase-like ER kinase (PERK; Cat. No.: 24390-1-AP), CCAAT/enhancer-binding protein homologous protein (CHOP; Cat. No.: 15204-1-AP), phosphatase and tensin homolog (PTEN)-induced kinase 1 (PINK1; Cat. No.: 23274-1-AP), nuclear dot protein 52 kDa (NDP52; Cat. No.: 12229-1-AP), optineurin (OPTN; Cat. No.: 10837-1-AP), P53 up-regulated modulator of apoptosis (PUMA; Cat. No.: 55120-1-AP) and sirtuin 5 (SIRT5; Cat. No.: 15122-1-AP) were purchased from Proteintech (Wuhan, China).

Techniques: Immunofluorescence, Software, Western Blot, Imaging, Flow Cytometry, Transfection, Standard Deviation

Journal: Journal of Pharmaceutical Analysis

Article Title: Tetrandrine targeting SIRT5 exerts anti-melanoma properties via inducing ROS, ER stress, and blocked autophagy

doi: 10.1016/j.jpha.2024.101036

Figure Lengend Snippet: Sirtuin 5 (SIRT5) depletion cooperating with tetrandrine (TET) treatment accelerated melanoma cell death. (A) The knockdown efficiency of two SIRT5 shRNA fragments was evaluated in A375 and MV3 cells by Western blot assay. (B) Reactive oxygen species (ROS) level was detected by utilizing immunofluorescence (IF) and flow cytometry. (C–F) The levels of intracellular ROS (C), intracellular superoxide anion (D), mitochondrial superoxide anion (E), and mitochondrial hydroxyl radicals (F) in shGFP and SIRT5 depletion melanoma cells were respectively evaluated by O13 Red, dihydroethidium (DHE), MitoROS 580, and MitoROS OH580 probes. (G, H) Western blot assay detected the expressions of endoplasmic reticulum (ER) stress, autophagy, mitophagy and apoptosis associated proteins between shGFP vs. shSIRT5 and OE-GFP vs. OE-SIRT5 groups after treated with dimethyl sulfoxide (DMSO) or tetrandrine (TET) (10 μM) for 48 h. (I, J) After subcutaneously injecting the cells with stably overexpressing SIRT5 or green fluorescent protein (GFP) into mice, they were treated by TET (120 mg/kg/day), qod, 30 days in total. Tumor growth was daily monitored and measured every 5 days. The xenograft tumors ( n = 6) were dissected, weighed and measured on the 30th day. (K) The expression of SIRT5 and Ki67 in xenograft tumor tissues was detected by immunocytochemistry (IHC) stain. The value of IHC positive signal in panels were quantified. All data were analyzed by unpaired Student's t -tests and are shown as the means ± standard deviation (SD). ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, ns: not statistically significant. DCFH-DA: dichlorodihydrofluorescein diacetate; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFPi: GFP interfering; SIRT5i: SIRT5 interfering; ATF6: activating transcription factor 6; PERK: double-stranded RNA-dependent protein kinase-like ER kinase; IRE1α: inositol requiring enzyme 1 alpha; PINK1: phosphatase and tensin homolog (PTEN)-induced kinase 1; NDP52: nuclear dot protein 52 kDa; c-PARP: cleaved poly (ADP-ribose) polymerase; V: vector.

Article Snippet: Anti-LC3B (Cat. No.: 314600-1-AP), UNC-52-like kinase 1 (ULK1; Cat. No.: 20986-1-AP), p62(18420-1-AP), Syntax 17 (Cat. No.: 17815-1-AP), glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Cat. No.: 60004-1-Ig), Tubulin (Cat. No.: 66031-1-lg), activating transcription factor 6 (ATF6; Cat. No.: 24169-1-ap), inositol requiring enzyme 1 alpha (IRE1α; Cat. No.: 27528-1-AP), double-stranded RNA-dependent protein kinase-like ER kinase (PERK; Cat. No.: 24390-1-AP), CCAAT/enhancer-binding protein homologous protein (CHOP; Cat. No.: 15204-1-AP), phosphatase and tensin homolog (PTEN)-induced kinase 1 (PINK1; Cat. No.: 23274-1-AP), nuclear dot protein 52 kDa (NDP52; Cat. No.: 12229-1-AP), optineurin (OPTN; Cat. No.: 10837-1-AP), P53 up-regulated modulator of apoptosis (PUMA; Cat. No.: 55120-1-AP) and sirtuin 5 (SIRT5; Cat. No.: 15122-1-AP) were purchased from Proteintech (Wuhan, China).

Techniques: Knockdown, shRNA, Western Blot, Immunofluorescence, Flow Cytometry, Stable Transfection, Expressing, Immunocytochemistry, Staining, Standard Deviation, Plasmid Preparation