Journal: Cancers

Article Title: TOM40 Inhibits Ovarian Cancer Cell Growth by Modulating Mitochondrial Function Including Intracellular ATP and ROS Levels

doi: 10.3390/cancers12051329

Figure Lengend Snippet: TOM40 is highly expressed by human epithelial ovarian cancer (EOC) cells. ( A ) TOM40 mRNA levels were measured by real-time polymerase chain reaction (PCR) in human ovarian surface epithelial (HOSE) cells and EOC cell lines. Fold expression is expressed as the ratio of TOM40 mRNA/actin mRNA. Results represent the mean ± Standard Error., n = 3. Box plots were used to compare the expression levels of TOM40 in HOSE cells and EOC cell lines; * p < 0.05. ( B ) TOM40 protein expression levels were examined by western blot analysis in HOSE cells and EOC cells. TOM40 band intensities were quantified relative to α-actinin using Image J 1.48v software (Right, box plot); * p < 0.05. ( C ) mRNA expression levels of TOM40 in the tumors of patients with ovarian cancer were analyzed using data from the Gene Expression Omnibus (GEO) database (GEO accession numbers GSE18520, GSE26712, and GSE9899). p values compare normal tissue or tissue with low malignant potential (LMP) to cancerous tumor tissue (** p < 0.01 and *** p < 0.001). ( D ) Representative images of immunohistochemical staining of TOM40 expression in normal epithelial ovarian tissue and in benign, borderline, and cancerous ovarian tumors: The boxed regions are displayed at high magnification in the inset (scale bar: 100 μm). ( E ) Box plot depiction of immunohistochemical staining data. The histoscores were computed based on the intensity and tissue area of positive staining. N, normal; Be, benign, Bo, borderline; Ca, epithelial ovarian cancer. ( F , G ) Kaplan–Meier plots of disease-free survival and overall survival for ovarian cancer patients as categorized by TOM40 expression. Survival data for only 181 of 201 patients with EOC were available to construct Kaplan–Meier survival curves. The detailed information regarding statistical tests is available in the Materials and Methods section.

Article Snippet: Briefly, sections were incubated with rabbit polyclonal anti-TOM40 antibody (Cat. 18409-1-AP; Proteintech Group, Inc, Rosemont, IL, USA) at a dilution of 1:150, overnight at 4 °C.

Techniques: Real-time Polymerase Chain Reaction, Expressing, Western Blot, Software, Immunohistochemical staining, Staining, Construct

Journal: Cancers

Article Title: TOM40 Inhibits Ovarian Cancer Cell Growth by Modulating Mitochondrial Function Including Intracellular ATP and ROS Levels

doi: 10.3390/cancers12051329

Figure Lengend Snippet: Clinicopathologic significance of TOM40 protein expression in epithelial ovarian cancers.

Article Snippet: Briefly, sections were incubated with rabbit polyclonal anti-TOM40 antibody (Cat. 18409-1-AP; Proteintech Group, Inc, Rosemont, IL, USA) at a dilution of 1:150, overnight at 4 °C.

Techniques: Expressing, Diagnostic Assay

Journal: Cancers

Article Title: TOM40 Inhibits Ovarian Cancer Cell Growth by Modulating Mitochondrial Function Including Intracellular ATP and ROS Levels

doi: 10.3390/cancers12051329

Figure Lengend Snippet: Univariate and multivariate analyses of disease-free survival and overall survival in epithelial ovarian cancer patients.

Article Snippet: Briefly, sections were incubated with rabbit polyclonal anti-TOM40 antibody (Cat. 18409-1-AP; Proteintech Group, Inc, Rosemont, IL, USA) at a dilution of 1:150, overnight at 4 °C.

Techniques:

Journal: Cancers

Article Title: TOM40 Inhibits Ovarian Cancer Cell Growth by Modulating Mitochondrial Function Including Intracellular ATP and ROS Levels

doi: 10.3390/cancers12051329

Figure Lengend Snippet: TOM40 promotes the proliferation of epithelial ovarian cancer (EOC) cell lines and EOC xenografts. EOC cell lines (RMUS-S, TOV-112D, SK-OV-3, OVCA-429, and OVCA-433) that stably express sh-control and sh-TOM40 were established by lentivirus and selected with 3 μg/ml puromycin for 15 days. ( A ) Numbers of EOC cells that stably express sh-TOM40 were counted 48 hours after seeding with a LUNAII TM automated cell counter. Data are expressed as the mean ± minimum to maximum, n = 4; ** p < 0.01 and *** p < 0.001. TOM40 knockdown was examined by western blot analysis using an anti-TOM40 antibody (images to the right of each box plot). ( B ) The comparative growth rate of sh-control and sh-TOM40 ovarian cancer cell lines was measured daily with a crystal violet assay on days 1 through 4 post-seeding. Data are expressed as the mean ± Standard Deviation (S.D.)., n = 4; *** p < 0.001. ( C ) The long-term growth rates of EOC cells that stably express sh-TOM40 were evaluated via a colonization assay (RMUG-S, TOV-112D, and SK-OV-3 were evaluated 10 days post-seeding; OVCA-429 and OVCAR-433 were evaluated 11 days post-seeding). (Images, top half of panel) Representative images of crystal violet staining of EOC cells that stably express sh-control or sh-TOM40. (Bar graph, lower half of panel) Crystal violet intensities were measured at 595 nm using an ELISA reader. Data are expressed as the mean ± S.D., n = 4. ( D ) BALB/c nude mice were inoculated subcutaneously into both flanks with 3 × 10 6 RMUG-S cells that stably express sh-control or sh-TOM40. Six tumors were used in each group. Shown is an image of the aforementioned xenograft tumors, which were dissected from the mice 68 days after inoculation. The scale bar is 5 mm. ( E ) Tumor volume was monitored at 14, 26, 40, 54, and 68 days after inoculation with RMUG-S cells that stably express sh-control or sh-TOM40. Tumor growth is expressed as the mean tumor volume ± Standard Error (S.E.), n = 6; ** p < 0.01 and *** p < 0.001. ( F ) Final volume (left box plot) and final weight (right box plot) of tumors were measured after sacrifice at 68 days post-inoculation. Data are expressed as the mean ± minimum to maximum, n = 6; *** p < 0.001.

Article Snippet: Briefly, sections were incubated with rabbit polyclonal anti-TOM40 antibody (Cat. 18409-1-AP; Proteintech Group, Inc, Rosemont, IL, USA) at a dilution of 1:150, overnight at 4 °C.

Techniques: Stable Transfection, Western Blot, Crystal Violet Assay, Standard Deviation, Staining, Enzyme-linked Immunosorbent Assay

Journal: Cancers

Article Title: TOM40 Inhibits Ovarian Cancer Cell Growth by Modulating Mitochondrial Function Including Intracellular ATP and ROS Levels

doi: 10.3390/cancers12051329

Figure Lengend Snippet: TOM40 expression is positively correlated with intracellular ATP levels. ( A ) Intracellular ATP levels were measured with a luciferase activity assay in whole cell lysates from epithelial ovarian cancer (EOC) cell lines and immortalized HOSEs (iHOSE). Luciferase activity (ATP levels) is expressed as a ratio of luciferase levels to protein quantity (bar graph, left). Results represent the means ± Standard Error (S.E.)., n = 4. Box plot (right) represents the comparative intracellular ATP levels of EOC cells compared to iHOSE cells. (Cancer, n = 14; iHOSE, n = 2). ( B , D ) Intracellular ATP levels were measured with an ATP assay kit based on luciferase activity. The relative intracellular ATP levels were quantified as a ratio of luciferase levels to protein quantity. Results are the mean ± S.E. (B, n = 12; D , cancer cell, n = 20; iHOSE, n = 8; ** p < 0.01 and *** p < 0001). ( C , E ) Cellular mitochondrial levels were measured by real-time PCR using total cellular DNA samples. The relative mitochondrial number was quantified as a ratio of mitochondrial 16S rRNA/ß2-microglobulin levels. Fold induction was calculated as a relative expression by iHOSE-empty vector cells. Results represent the mean ± S.E. (C, n = 9; E, n = 5; # p > 0.05, * p < 0.05, ** p < 0.01, and *** p < 0.001). Box plot in C represents the relative mitochondrial number of sh-control EOC cells compared to sh-control iHOSE cells (cancer, n = 5; iHOSE, n = 2).

Article Snippet: Briefly, sections were incubated with rabbit polyclonal anti-TOM40 antibody (Cat. 18409-1-AP; Proteintech Group, Inc, Rosemont, IL, USA) at a dilution of 1:150, overnight at 4 °C.

Techniques: Expressing, Luciferase, Activity Assay, ATP Assay, Real-time Polymerase Chain Reaction, Plasmid Preparation

Journal: Cancers

Article Title: TOM40 Inhibits Ovarian Cancer Cell Growth by Modulating Mitochondrial Function Including Intracellular ATP and ROS Levels

doi: 10.3390/cancers12051329

Figure Lengend Snippet: The knockdown of TOM40 expression increases the mitochondrial membrane potential. Epithelial ovarian cancer (EOC) cells that stably express sh-control and sh-TOM40 were seeded at 2.5 × 10 6 cells per wells and stained with 2 μM tetraehylbenzimidazolylcarbocyanine iodide (JC-1) for 15 min. The JC-1 staining was measured by fluorescence-activated cell sorting (FACS) analysis and fluorescence microscopy imaging. ( A ) Representative images of FACS analysis of 5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) staining: Quantification of the percentage of cells is shown in the top-left and top-right regions of dot plots to count JC-1 aggregation (red fluorescent). The overlay histograms are representative images of three separate experiments (The white area is sh-control cells, and the red area is sh-TOM40 cells). The results are expressed as the mean ± Standard Deviation (S.D.)., n = 3. ( B ) Representative fluorescent images of JC-1 staining images: The scale bar is 100 µm. EOC cells that stably express sh-control and sh-TOM40 were trypsinized, and 1 × 10 6 cells were confined into a round-bottom tube. The cells were stained with a mixture of 100 nM MitoTracker Green FM and 25 nM tetramethylrhodamine ethyl ester (TMRE) for 15 min at 37 °C CO 2 incubator (5% CO 2 and 37 °C). The levels of MitoTracker Green FM and TMRE staining were measured using FACS analysis. ( C ) Representative images of histograms of MitoTracker and TMRE staining: Quantification of the percentage of cells is shown in the blue line region of histogram. The white area is sh-control cells, while the green or red area is sh-TOM40 cells in histogram. The bar graph displays the relative stained cells. The results are expressed as the mean ± S.D., n = 4 (TOV-112D and OVCAR-3); n = 6 (OVCA-429 and RMUG-S). # p > 0.05; * p < 0.05; *** p < 0.001; and **** p < 0.0001. ( D ) Representative fluorescent images of TMRE staining of EOC cell lines that stably express sh-control or sh-TOM40: The Scale bar is 200 μm.

Article Snippet: Briefly, sections were incubated with rabbit polyclonal anti-TOM40 antibody (Cat. 18409-1-AP; Proteintech Group, Inc, Rosemont, IL, USA) at a dilution of 1:150, overnight at 4 °C.

Techniques: Expressing, Stable Transfection, Staining, Fluorescence, FACS, Microscopy, Imaging, Standard Deviation

Journal: Cancers

Article Title: TOM40 Inhibits Ovarian Cancer Cell Growth by Modulating Mitochondrial Function Including Intracellular ATP and ROS Levels

doi: 10.3390/cancers12051329

Figure Lengend Snippet: The knockdown of TOM40 expression generates intracellular reactive species which reduce cell growth. ( A ) Epithelial ovarian cancer (EOC) cells that stably express sh-control and sh-TOM40 were seeded at 1 × 10 6 cells in a 60-cm culture dish. The cells were stained with 20 μM 2’,7’-dichlorofluorescin diacetate (DCFDA) for 30 min at CO 2 incubator (5% CO 2 and 37 °C). The levels of DCFDA staining were measured by FACS analysis. Representative images of histograms: quantification of the percentage of cells in shown the blue line region of histogram. The white area is sh-control cells, while the green is sh-TOM40 cells in histogram. The results are expressed as the mean ± S.D., n = 4 (TOV-112D and OVCAR-3); n = 6 (OVCA-429 and RMUG-S). *** p < 0.001 and **** p < 0.0001. ( B ) EOC cells that stably express sh-control and sh-TOM40 were seeded at 3 × 10 5 RMUG-S and 2 × 10 5 TOV-112D cells in a 24-well plate. The sh-TOM40-expressed RMUG-S cells were treated with 0.25 mM N-acetyl-L-cysteine (NAC), 0.5 mM NAC, 0.5 mM glutathione (GSH), or 1 mM GSH for 72 h. The sh-TOM40 expressed TOV-112D cells were treated with 0.25 mM NAC, 1 mM NAC, 0.5 mM GSH, or 1 mM GSH for 48 h. The growth rate was measured by crystal violet assay. Representative images of the crystal violet stained cells (bottom panel): Data in the bar graph are expressed as the mean of fold change ± S.D., n = 4. * p < 0.05; ** p < 0.01; and **** p < 0.0001.

Article Snippet: Briefly, sections were incubated with rabbit polyclonal anti-TOM40 antibody (Cat. 18409-1-AP; Proteintech Group, Inc, Rosemont, IL, USA) at a dilution of 1:150, overnight at 4 °C.

Techniques: Expressing, Stable Transfection, Staining, Crystal Violet Assay

Journal: Cancers

Article Title: TOM40 Inhibits Ovarian Cancer Cell Growth by Modulating Mitochondrial Function Including Intracellular ATP and ROS Levels

doi: 10.3390/cancers12051329

Figure Lengend Snippet: Metformin reduces EOC cell growth in an AMP-activated protein kinase (AMPK)-independent manner. ( A ) Epithelial ovarian cancer (EOC) cell lines that stably express sh-control or sh-TOM40 were harvested at 70% confluency. The expression levels of indicated proteins were measured by western blot analysis. ( B ) sh-TOM40 stably expressed EOC cells were introduced to si-Luciferase or si-AMPKα1 and then incubated in a CO 2 incubator (5% CO 2 and 37 ℃) for 72 h. Cell viability was assessed by crystal violet assay (Left panel). Data in the bar graph are expressed as the mean of absorbance ± Standard Deviation (S.D.)., n = 16. # p > 0.05. The expression levels of indicated proteins including anti-AMPKα1 were measured by western blot analysis (Right panel). ( C ) Cell viability (relative absorbance at 550 nm) as measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolum bromide (MTT) assay. OVCAR-3 that stably express an empty vector or TOM40 was cultured with or without 25 mM glucose and with or without 1 mM AICAR, 5 mM metformin, or 1 mM aspirin for 48 h. AICAR, 5-aminoimidazole-4-carboxamid ribonucleotide. ( D ) SK-OV-3 that stably express an empty vector or TOM40 was cultured with or without 25 mM glucose and with or without 5 mM metformin for 48 h. Results are shown as the relative means ± S.D., n = 6; # p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. ( E ) Western blot analysis of proteins expressed by EOC cell lines, OVCAR-3 and SK-OV-3, that stably express empty vectors or TOM40 and were cultured in dulbecco modified eagle medium (DMEM) with or without 25mM glucose and with or without 1 mM AICAR or 5 mM metformin for 24 h.

Article Snippet: Briefly, sections were incubated with rabbit polyclonal anti-TOM40 antibody (Cat. 18409-1-AP; Proteintech Group, Inc, Rosemont, IL, USA) at a dilution of 1:150, overnight at 4 °C.

Techniques: Stable Transfection, Expressing, Western Blot, Luciferase, Incubation, Crystal Violet Assay, Standard Deviation, MTT Assay, Plasmid Preparation, Cell Culture, Modification