Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: SNCG promotes the progression and metastasis of high-grade serous ovarian cancer via targeting the PI3K/AKT signaling pathway

doi: 10.1186/s13046-020-01589-9

Figure Lengend Snippet: SNCG accelerates ovarian cancer cell proliferation, facilitates cell migration and invasion in vitro. a Western blot analysis of SNCG expression in different ovarian cancer cell lines. b The transfection efficiency was confirmed by Western blotting and qRT-PCR in SKOV3, HO-8901 PM, and OVCAR3 cells. c The MTT assay was used to detect ovarian cancer cell viability. d A soft agar assay was used to examine the proliferation of ovarian cancer cells. e Cell migration and invasion capabilities were determined using transwell assays (original magnification, × 200). f and g SKOV3 and HO-8910 PM cell transfectants were plated on FN and stained for SNCG, phalloidin, and nuclear. Moreover, cells were stained for Vimentin, MMP9, and F-actin. The individual or merged images visualized by a laser scanning confocal microscope (original magnification, × 1000). ▲ , P < 0.05. *, P < 0.001. Ctrl: control; Ove: overexpression; Scr: scramble; sh1: small hairpin RNA 1; sh2: small hairpin RNA 2; sh3: small hairpin RNA 3

Article Snippet: Human ovarian tumor cell lines SKOV3, OVCAR3, OVCAR5, ES-2 were purchased from Keygen Biotech (Jiang Su, China).

Techniques: Migration, In Vitro, Western Blot, Expressing, Transfection, Quantitative RT-PCR, MTT Assay, Soft Agar Assay, Staining, Microscopy, Control, Over Expression

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: SNCG promotes the progression and metastasis of high-grade serous ovarian cancer via targeting the PI3K/AKT signaling pathway

doi: 10.1186/s13046-020-01589-9

Figure Lengend Snippet: SNCG induces ovarian cancer progression via activating the PI3K/AKT signaling pathway. a-b A phospho-kinase array kit was performed on protein lysates of SKOV3-sh1 and control cells. Thirteen proteins were obvious changes in their phosphorylation status and highlighted by boxes. c Western blot analysis of the levels of Akt, p-Akt (Sec473), p70S6 kinase, p-p70S6 kinase (Thr389), mTOR, and p-mTOR (Sec2448) in SKOV3 and HO-8901 PM cells transfected with SNCG-shRNA or Scr, and OVCAR3 cells transfected with SNCG-Ove or Ctrl. d Expression levels of Akt, p-Akt (Sec473), p70S6 kinase, p-p70S6 kinase (Thr389), mTOR, and p-mTOR (Sec2448) in cells transfected with SNCG shRNA, Scr, SNCG Ove, Ctrl, IGF-1, and DMSO were determined by Western blot. e Expression levels of Akt, p-Akt (Sec473), p70S6 kinase, p-p70S6 kinase (Thr389), mTOR, and p-mTOR (Sec2448) in cells transfected with SNCG shRNA, Scr, SNCG Ove, Ctrl, LY294002, and DMSO were determined by Western blot. f Wound healing assay (upper, original magnification × 40) and cell colony formation (lower) of cells transfected with SNCG shRNA, Scr, SNCG Ove, Ctrl, IGF-1, LY294002, and DMSO confirmed the effect of SNCG on the PI3K/AKT signaling pathway. ▲ , P < 0.05. *, P < 0.001. IGF-1: insulin-like growth factor 1; sh-SNCG: small hairpin RNAs of SNCG

Article Snippet: Human ovarian tumor cell lines SKOV3, OVCAR3, OVCAR5, ES-2 were purchased from Keygen Biotech (Jiang Su, China).

Techniques: Control, Phospho-proteomics, Western Blot, Transfection, shRNA, Expressing, Wound Healing Assay

Journal: Nanoscale Research Letters

Article Title: Functionalized Folate-Modified Graphene Oxide/PEI siRNA Nanocomplexes for Targeted Ovarian Cancer Gene Therapy

doi: 10.1186/s11671-020-3281-7

Figure Lengend Snippet: Cellular uptake of different nanocomplexes. Confocal laser scanning microscopy (CLSM) images of the cellular uptake of FITC-labeled different nanocomplexes in human ovarian cancer SKOV3 cells after incubation for 4 h. Blue, nuclei (DAPI-labeled); red, cytomembrane (Dil-labeled); green, nanocomplexes (FITC-labeled). Scale bars represent 100 μm, but the scale bar is 10 μm in single cell

Article Snippet: The SKOV3 (human ovarian cancer cells) was purchased from Keygen (China).

Techniques: Confocal Laser Scanning Microscopy, Labeling, Incubation

Journal: Nanoscale Research Letters

Article Title: Functionalized Folate-Modified Graphene Oxide/PEI siRNA Nanocomplexes for Targeted Ovarian Cancer Gene Therapy

doi: 10.1186/s11671-020-3281-7

Figure Lengend Snippet: Cellular internalization and lysosomal escape of PEG-GO-PEI-FA observed by CLSM. SKOV3 ovarian cancer cells incubated with a PEI, b PEG-GO-PEI-FA, and c PEG-GO-PEI-FA for 0.5, 1, 2, 4, and 8 h. Those in blue were nuclei stained with DAPI, green were nanocomplexes labeled with FITC, and those in red represented endosomes and lysosome fluorescence after staining with LysoTracker red. Scale bars represent 100 μm, but the scale bar is 10 μm in single cell

Article Snippet: The SKOV3 (human ovarian cancer cells) was purchased from Keygen (China).

Techniques: Incubation, Staining, Labeling, Fluorescence

Journal: Nanoscale Research Letters

Article Title: Functionalized Folate-Modified Graphene Oxide/PEI siRNA Nanocomplexes for Targeted Ovarian Cancer Gene Therapy

doi: 10.1186/s11671-020-3281-7

Figure Lengend Snippet: In vitro cytotoxicity of PEG-GO-PEI-FA/siRNA in ovarian cancer SKOV3 cells via CCK-8 assay. a SKOV3 cells were treated with PEG-GO-PEI and PEG-GO-PEI-FA at different concentrations (10–500 μg/mL) at 12 and 24 h to get the optimal dose of nanocarrier. b The cytotoxicity of PEG-GO-PEI-FA/siRNA, PEG-GO-PEI/siRNA, and Lipo2000/siRNA were measured in different time points (4–48 h) at 100 μg/mL. Error bars represent ± SD; * p < 0.05 (Student’s t test)

Article Snippet: The SKOV3 (human ovarian cancer cells) was purchased from Keygen (China).

Techniques: In Vitro, CCK-8 Assay

Journal: Cancer Medicine

Article Title: ONC201 induces the unfolded protein response (UPR) in high‐ and low‐grade ovarian carcinoma cell lines and leads to cell death regardless of platinum sensitivity

doi: 10.1002/cam4.3858

Figure Lengend Snippet: ONC201 inhibits viability of OVCA cells. (A and B) ONC201 reduces cell viability of SKOV3, OV433, CaOV3, TOV112D, VOA4627, and VOA1312 cells in a dose and time‐dependent manner. Cells were treated with either vehicle or drug at the indicated concentration (0/1/10/25/50/100/200 µM) for (A) 48 h and (B) 72 h MTT assay results are shown. The absorbance was read at 540 nm using an automated microplate reader. The percentage of cell viability was calculated to compare the vehicle group. Data were expressed as the percent cell proliferation relative to the control as mean ± SD from triplicate wells. (C) ONC201 inhibits OVCA migration. Cells were treated by ONC201 (0/1/10/100 µM) and migration of cells was evaluated via wound healing assay. Cells were seeded and left overnight. The following day a “wound” was created using 200 µl pipet tips and treated with ONC201. After incubation with ONC201 for 8–24 h depending on the cell line, cells were washed by DPBS and were stained with crystal violet. Dose‐dependent decreased cell migration was noted in all cell lines. A representative image from three independent experiments is shown (image magnification ×200). A p ‐value ≤0.05 is presented as * and p ≤ 0.01 as **

Article Snippet: High‐grade human ovarian cancer cells (TOV112D, OV433, SKOV3, and CaOV3) were bought from American Type Culture Collection (ATCC).

Techniques: Concentration Assay, MTT Assay, Control, Migration, Wound Healing Assay, Incubation, Staining

Journal: Cancer Medicine

Article Title: ONC201 induces the unfolded protein response (UPR) in high‐ and low‐grade ovarian carcinoma cell lines and leads to cell death regardless of platinum sensitivity

doi: 10.1002/cam4.3858

Figure Lengend Snippet: ONC201 downregulates PI3K/AKT and ERK/MEK signaling pathway. ONC201 (20 µM) inhibits AKT and ERK in high‐ and low‐grade OVCA cells. Lysates were collected from SKOV3 and VOA4627 cell lines at 48, 72, and 96 h and western blot was completed with indicated antibodies. A p ‐value ≤0.05 is presented as * and p ≤ 0.01 as **

Article Snippet: High‐grade human ovarian cancer cells (TOV112D, OV433, SKOV3, and CaOV3) were bought from American Type Culture Collection (ATCC).

Techniques: Western Blot

Journal: Cancer Medicine

Article Title: ONC201 induces the unfolded protein response (UPR) in high‐ and low‐grade ovarian carcinoma cell lines and leads to cell death regardless of platinum sensitivity

doi: 10.1002/cam4.3858

Figure Lengend Snippet: ONC201 activates ER stress. The expression of UPR‐related genes was evaluated by qPCR in SKOV3 (A), VOA1312 (B), OV433 (C), and CaOV3 (D). CHOP expression was induced 5–15 fold in all cell lines, suggesting that ONC201 prompts ER stress in human OVCA cells. (E) ONC201 (20 µM) upregulated protein expression of CHOP and ATF4. A p ‐value ≤0.05 is presented as * and p ≤ 0.01 as **

Article Snippet: High‐grade human ovarian cancer cells (TOV112D, OV433, SKOV3, and CaOV3) were bought from American Type Culture Collection (ATCC).

Techniques: Expressing

Journal: Cancer Medicine

Article Title: ONC201 induces the unfolded protein response (UPR) in high‐ and low‐grade ovarian carcinoma cell lines and leads to cell death regardless of platinum sensitivity

doi: 10.1002/cam4.3858

Figure Lengend Snippet: ONC201 promotes apoptosis both in high‐ and low‐grade OVCA cells. (A) SKOV3 and VOA4627 cells treated with or without ONC201 (20 µM) were double‐stained with Annexin V and PI, and then analyzed by flow cytometry. The percentages of viable cells (compared to vehicle) are shown. (B) Representative flow diagram of Annexin V‐PI staining. (C) Caspase3/7 activity was measured by Caspase‐Glo 3/7 assay. SKOV3 and VOA4627 cells were treated with ONC201 (20 µM) for 48, 72 h. Caspase3/7 activity was increased from 48 to 72 h. (D) ONC201‐treated lysates were collected from SKOV3 and VOA4627 cell lines and blotted with Wee1 antibodies. A p ‐value ≤0.05 is presented as * and p ≤ 0.01 as **

Article Snippet: High‐grade human ovarian cancer cells (TOV112D, OV433, SKOV3, and CaOV3) were bought from American Type Culture Collection (ATCC).

Techniques: Staining, Flow Cytometry, Activity Assay, Caspase-Glo Assay

Journal: Cancer Medicine

Article Title: ONC201 induces the unfolded protein response (UPR) in high‐ and low‐grade ovarian carcinoma cell lines and leads to cell death regardless of platinum sensitivity

doi: 10.1002/cam4.3858

Figure Lengend Snippet: ONC201 leads cells to intrinsic rather than TRAIL‐induced cell death. (A) ONC201 leads to a loss of mitochondrial membrane potential (MMP). MMP was measured by JC‐1 assay. JC‐1 monomers (green) were increased by ONC201 (20 µM) treatment over time both in SKOV3 and VOA4627 cells. (B) After treating with ONC201 (20 µM) for 48 and 72 h, cells are harvested and cell extracts were analyzed by western blotting to detect the expression of cleaved PARP, BIM, and Mcl‐1. (C and D) ONC201 (20 µM) led OVCA cells to death via TRAIL‐independent pathway. Lysates were collected from SKOV3 and VOA4627 cell lines and blotted with TRAIL antibodies. Whereas, the expression of DR5 gene was confirmed by qPCR in SKOV3, OV433, CaOV3, and VOA1312. (E) ONC201 does not activate Caspase‐8. Caspase‐8 (p18/p10) was found intact both in ONC201 treated and control SKOV3 cells. A p ‐value ≤0.05 is presented as * and p ≤ 0.01 as **

Article Snippet: High‐grade human ovarian cancer cells (TOV112D, OV433, SKOV3, and CaOV3) were bought from American Type Culture Collection (ATCC).

Techniques: Membrane, Western Blot, Expressing, Control

Journal: Cancer Medicine

Article Title: ONC201 induces the unfolded protein response (UPR) in high‐ and low‐grade ovarian carcinoma cell lines and leads to cell death regardless of platinum sensitivity

doi: 10.1002/cam4.3858

Figure Lengend Snippet: ONC201 increases endoplasmic reticulum stress in both high‐grade and low‐grade ovarian cancer cells; overwhelming pro‐survival signals and activating the pro‐death arm of the unfolded protein response pathway. In addition, a weekly oral dose of ONC201 lowers the tumor burden in mice. This is a promising therapeutic agent in OVCA treatment and should be considered for clinical translation

Article Snippet: High‐grade human ovarian cancer cells (TOV112D, OV433, SKOV3, and CaOV3) were bought from American Type Culture Collection (ATCC).

Techniques:

Journal: Translational Cancer Research

Article Title: Fluid shear stress-induced IL-8/CXCR signaling in human ovarian cancer cells

doi: 10.21037/tcr.2019.08.22

Figure Lengend Snippet: The effects of fluid shear stress on IL-8 mRNA expression in SKOV3 cells. (A) Quantitative reversal transcription-polymerase chain reaction detection of IL-8 mRNA expression. The increase of IL-8 mRNA expression by shear stress was time-dependent. The biphasic response of IL-8 mRNA expression was found in experiments in which the applied low shear stress was 0.5, 1.5, or 2.0 dyne/cm 2 , respectively (**P<0.01 vs. control group). (B) SKOV3 cells were exposed to different shear stresses (0.5, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, or 5.0 dyne/cm 2 ) for 1 or 2 h. IL-8 mRNA expression was negatively associated with the intensity of shear stress (**P<0.01 vs. control group).

Article Snippet: Human ovarian cancer SKOV3 cells were obtained from Nanjing KeyGen Biotech.

Techniques: Shear, Expressing, Polymerase Chain Reaction, Control

Journal: Translational Cancer Research

Article Title: Fluid shear stress-induced IL-8/CXCR signaling in human ovarian cancer cells

doi: 10.21037/tcr.2019.08.22

Figure Lengend Snippet: The effects of fluid shear stress on IL-8 protein production in SKOV3 cells. (A) IL-8 protein levels in media were quantified using quantitative sandwich ELISA. IL-8 secretion increased obviously when SKOV3 cells subjected to fluid shear stress (0.5, 1.5, 2.0 dyne/cm 2 ) for 5 h (**P<0.01 vs. control group). Then IL-8 secretion gradually decreased at 8 h of stimulation by shear stress compared with that of 5 h group. The increase of IL-8 secretion by shear stress was time-dependent. (B) SKOV3 cells untreated with fluid shear stress secreted extraordinarily little IL-8 in culture media. IL-8 secretion increased obviously when SKOV3 cells subjected to fluid shear stress (0.5, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0 dyne/cm 2 ) for 5 and 6 h (**P<0.01 vs. control group). The secretion was most when SKOV3 cells subjected to fluid shear stress 1.5 dyne/cm 2 . Force-dependent relation of the production of IL-8 protein in SKOV3 cells subjected to shear stress was same as the expression of IL-8 mRNA.

Article Snippet: Human ovarian cancer SKOV3 cells were obtained from Nanjing KeyGen Biotech.

Techniques: Shear, Sandwich ELISA, Control, Expressing

Journal: Translational Cancer Research

Article Title: Fluid shear stress-induced IL-8/CXCR signaling in human ovarian cancer cells

doi: 10.21037/tcr.2019.08.22

Figure Lengend Snippet: Fluid shear stress-induced expression of IL-8-green fluorescence protein reporter gene in SKOV3 cells. -102–+61bp in 5’-flanking regions of IL-8 gene (IL-8 USCS) was isolated from genomic DNA of SKOV3 cells and pEGFP1-IL8USCS. The SKOV3 cells were transfected by Dosper liposomal transfection regent and selected by G418 and stimulated with 1.5 dyne/cm 2 shear stress for 3 hours. [1] SKOV3 cells alone; [2] empty pEGFP1 transfected cells without stimulation; [3] pEGFP1-IL8USCS transfected cells with no stimulation; [4] pEGFP1-IL8USCS transfected cells stimulated with shear stress for 3 hours (**P<0.01 vs. the 3rd group; n=3).

Article Snippet: Human ovarian cancer SKOV3 cells were obtained from Nanjing KeyGen Biotech.

Techniques: Shear, Expressing, Fluorescence, Isolation, Transfection

Journal: Translational Cancer Research

Article Title: Fluid shear stress-induced IL-8/CXCR signaling in human ovarian cancer cells

doi: 10.21037/tcr.2019.08.22

Figure Lengend Snippet: CXCR2 took part in the fluid shear stress-induced IL-8 expression. (A) Immunofluorescence of SKOV3 cells showing CXCR2 was present in the surface of SKOV3 cells (×1,000 magnification). (B) Determination of CXCR2 mRNA expression in SKOV3 cells by RT-PCR and Northern blot. Total RNAs were isolated from SKOV3 cells that were untreated or treated for 1 hour with 1.5 dyne/cm 2 shear stress. (C) Inhibitory effect of CXCR2 mutant on shear stress-induced expression of IL-8 reporter gene in SKOV3 cells. The SKOV3 cells were co-transfected with pcDNA3-mt CXCR2 and pEGFP1-IL8USCS and selected by G418, then stimulation by 1.5 dyne/cm 2 shear stress for 3 hours. The green fluorescent protein expression was analyzed by flow cytometry. A relative fluorescence intensity was calculated referred to the mean fluorescence intensity of the SKOV3 cells alone. [1] SKOV3 cells alone; [2] empty pcDNA3 and pEGFP1-IL8 USCS co-transfected cells without stimulation; [3] pEGFP1-IL8USCS transfected cells stimulated with 1.5 dyne/cm 2 shear stress for 3 hours; [4] pEGFP1-IL8USCS and pcDNA-mtCXCR2 co-transfected cells without stimulation; [5] pEGFP1-IL8USCS and pcDNA-mtCXCR2 co-transfected cells with stimulation (**P<0.01 vs. the 3rd group; n=3).

Article Snippet: Human ovarian cancer SKOV3 cells were obtained from Nanjing KeyGen Biotech.

Techniques: Shear, Expressing, Immunofluorescence, Reverse Transcription Polymerase Chain Reaction, Northern Blot, Isolation, Mutagenesis, Transfection, Flow Cytometry, Fluorescence

Journal: Translational Cancer Research

Article Title: Fluid shear stress-induced IL-8/CXCR signaling in human ovarian cancer cells

doi: 10.21037/tcr.2019.08.22

Figure Lengend Snippet: Fluid shear stress-induced NF-kB activation in SKOV3 cells. (A) Phosphorylation, and degradation of IkBa in SKOV3 cells exposed to 1.5 dyne/cm 2 fluid shear stress. (B) NF-kB nuclear translocation in SKOV3 cells exposed to 1.5 dyne/cm 2 fluid shear stress. Photographs were taken by using laser confocal microscope: [A] control; [B] 0.5-hour exposure; [C] 1-hour exposure; [D] 1.5 hours exposure; [E] 2 hours exposure.

Article Snippet: Human ovarian cancer SKOV3 cells were obtained from Nanjing KeyGen Biotech.

Techniques: Shear, Activation Assay, Phospho-proteomics, Translocation Assay, Microscopy, Control

Journal: Biochemical pharmacology

Article Title: Growth differentiation factor 15 stimulates rapamycin-sensitive ovarian cancer cell growth and invasion

doi: 10.1016/j.bcp.2012.10.007

Figure Lengend Snippet: GDF15 promotes anchorage-independent growth of ovarian cancer cells. (A) CaOv3, OvCar3, and SKOv3 cells were incubated in serum-free media for 48 h. GDF15 concentration was then determined by ELISA in media from cell lines. (B) SKOv3, OvCar3, and CaOv3 ovarian cancer cells were plated in matrigel, and maintained in media containing 20 ng/mL rhGDF15 or vehicle control. Media was changed twice a week for 3–4 weeks. Representative photographs taken with 4× objective lens are shown. Matrigel was dissolved using dispase and cells were counted by trypan blue exclusion. Average fold change in anchorage-independent (AI) cell growth is shown for rhGDF15 group (GDF) versus control vehicle group (C) per line. (C) SKOv3 cells were stably transfected with empty vector control or GDF15 expression plasmid. Three GDF15 stable clones (G1, G3, G5) were selected and analyzed by real-time PCR for GDF15 transcript level versus control clone (C). Levels of GDF15 transcript were normalized to RPLPO internal control transcript. Values reflect the average fold change in normalized GDF15 transcript expression per stable clone relative to control clone. (D) Control clone (C) and two GDF15 stable clones (G3 and G5) were plated in matrigel. Media was changed twice a week for 3–4 weeks. Matrigel was then dissolved using dispase and cells were counted by trypan blue exclusion. Average fold change in anchorage-independent (AI) growth is shown for the GDF15 clones relative to control clone. (E) Growth of SKOv3 parental cells treated with vehicle control or 20 ng/mL rhGDF15, or stable control clone or GDF15 clones G1 or G3 was assessed by Real-Time Cell Analysis. Cell index reflects growth over 24 h, and represents the average of triplicate cultures per group.

Article Snippet: Stable transfectants were developed by transfecting empty pCMV vector or GDF15 expression plasmid (Origene) into SKOv3 using Lipofectamine transfection reagent.

Techniques: Incubation, Concentration Assay, Enzyme-linked Immunosorbent Assay, Stable Transfection, Transfection, Plasmid Preparation, Expressing, Clone Assay, Real-time Polymerase Chain Reaction

Journal: Biochemical pharmacology

Article Title: Growth differentiation factor 15 stimulates rapamycin-sensitive ovarian cancer cell growth and invasion

doi: 10.1016/j.bcp.2012.10.007

Figure Lengend Snippet: GDF15 promotes invasiveness of ovarian cancer cells. (A) SKOv3 cells were plated in Boyden chambers in the presence of 10% FBS plus vehicle control (C) or 20 ng/mL rhGDF15. (B) SKOv3 stable control clone, GDF15 stable clone 1, and GDF15 stable clone 3 cells were plated in Boyden invasion chambers in the presence of 10% FBS. For both (A) and (B), photos were taken after 24 h of invasion, and the number of invaded cells was counted in ten different fields per sample. Representative photos are shown. Values reflect the total number of invaded cells in triplicate cultures per group. (C) Representative photos of stable control and GDF15 clones are shown at 10× magnification. Western blotting for mesenchymal marker N-cadherin was performed twice; a representative blot is shown for total cell lysates from control and GDF15 stable clones. Quantification of N-cadherin was normalized to actin, and is shown relative to control clone. (D) SKOv3, SKOv3 stable control clone, GDF15 stable clone 1, and GDF15 stable clone 3 cells were plated in the presence of 5% FBS in the upper chamber of XCelligence CIM-plates. Medium containing 10% FBS, and 20 ng/mL rhGDF15 where indicated, was placed into the plate’s corresponding bottom chambers. Cell index reflects cell migration measured every 15 min for 10 h, and represents the average of triplicate cultures per group. (E) Real-time PCR was performed for invasion markers MMP-2, MMP-9, and VEGF in SKOv3 control and GDF15 stable clones 1, 3, and 5. Values reflect the fold change in transcript normalized to RPLPO housekeeping gene. (F) A tumor tissue array consisting of 122 tumor samples and 10 normal tissue samples was stained for MMP2 and MMP9. Photos were taken at 5× magnification under the microscope, and representative photos are shown. (G) SKOv3 control and GDF15 clones were plated in Boyden chambers, and treated for 24 h with DMSO (C), 1 µM (labeled “1”) or 10 µM (labeled “10”) of the pan-MMP inhibitor GM6001. The number of invaded cells was counted in ten different fields per sample. Values reflect the average number of invaded cells in triplicate cultures per group.

Article Snippet: Stable transfectants were developed by transfecting empty pCMV vector or GDF15 expression plasmid (Origene) into SKOv3 using Lipofectamine transfection reagent.

Techniques: Stable Transfection, Clone Assay, Western Blot, Marker, Migration, Real-time Polymerase Chain Reaction, Staining, Microscopy, Labeling

Journal: Biochemical pharmacology

Article Title: Growth differentiation factor 15 stimulates rapamycin-sensitive ovarian cancer cell growth and invasion

doi: 10.1016/j.bcp.2012.10.007

Figure Lengend Snippet: PI3K/mTORc1 and MAPK signaling are activated by GDF15 in ovarian cancer cells. (A) SKOv3 cells were serum starved overnight, and then stimulated with 20 ng/mL rhGDF15 for 2 or 5 min, or with the corresponding volume of vehicle control for 5 min. Western blots of total protein lysates were performed at least 3 times for p-Thr180/ Tyr182 p38MAPK, total p38, p-Thr202/Tyr204 p42/p44 Erk1/2, total Erk1/2, p-S473 Akt, and total Akt; representative blots are shown. Quantification is shown as a ratio of phospho-protein to total protein, and is shown relative to vehicle control. (B) SKOv3 cells were plated in matrigel. Cells were maintained in media containing vehicle control, 20 ng/mL rhGDF15, or GDF15 plus 1 µM PI3K inhibitor LY294002, 100 nM MEK inhibitor PD0325901, or 10 µM p38MAPK inhibitor SB203580. Media and drugs were changed twice a week for 3–4 weeks. Matrigel was dissolved using dispase and cells were counted by trypan blue exclusion. Fold change in anchorage-independent (AI) growth is shown relative to the vehicle control group; ** p < 0.005 for GDF15-stimulated versus control vehicle, and for inhibitor + GDF15 groups versus GDF15 alone. (C) Total protein lysates from SKOv3 stable control and GDF15 clones 1, 3, and 5 were Western blotted at least twice for phosphorylated and total p38MAPK, Akt, and 4EBP1; representative blots are shown. Quantification is shown as a ratio of phosphorylated to total protein, and is shown relative to control clone. (D) (Left) GDF15 stable clone 1 (G1) and clone 3 (G3) and control empty vector clone cells were treated with 10 nM or 100 nM of rapamycin, or with (right) 1, 5, or 10 µM TGF beta receptor type II inhibitor SB431542. Control groups (C) were treated with DMSO alone. After 72 h, MTS proliferation assays were performed. Proliferation is shown as a percentage of the control vehicle group per cell line, and reflects the average of six replicates. (E) SKOv3 cells were plated in matrigel. Cells were maintained in media containing vehicle control, 20 ng/mL GDF15, or GDF15 plus 100 nM rapamycin or 5 µM TGF beta receptor type II inhibitor SB431542. Media and drugs were changed twice a week for 3–4 weeks. Matrigel was dissolved using dispase and cells were counted by trypan blue exclusion. Fold change in anchorage-independent (AI) growth is shown relative to the control group. (F) GDF15 stable clone 1 (G1) and clone 3 (G3) and control empty vector clone cells were treated with DMSO control (C) and the concentrations shown for LY294002, PD0325901, or SB203580. After 72 h, MTS proliferation assays were performed. Proliferation is shown as a percentage of the control vehicle group per cell line, and reflects the average of six replicates.

Article Snippet: Stable transfectants were developed by transfecting empty pCMV vector or GDF15 expression plasmid (Origene) into SKOv3 using Lipofectamine transfection reagent.

Techniques: Western Blot, Clone Assay, Stable Transfection, Plasmid Preparation

Journal: Biochemical pharmacology

Article Title: Growth differentiation factor 15 stimulates rapamycin-sensitive ovarian cancer cell growth and invasion

doi: 10.1016/j.bcp.2012.10.007

Figure Lengend Snippet: Rapamycin inhibits GDF15-mediated ovarian cancer cell invasion. (A) SKOv3 cells were plated in Boyden invasion chambers in the presence of vehicle control, 20 ng/ mL GDF15, or GDF15 plus 100 nM rapamycin (Rp). Photos were taken after 24 h of invasion (representatives shown), and the number of invaded cells was counted in ten different fields per sample. Values reflect the total number of invaded cells in triplicate cultures per group. (B) SKOv3 cells were treated with vehicle control, 20 ng/mL GDF15, or 20 ng/mL GDF15 plus 100 nM rapamycin (Rp). Real-time PCR was performed for MMP-9 and VEGF. Values reflect the fold change in transcript normalized to RPLPO housekeeping gene. (C) SKOv3 GDF stable clones 1 and 3 were plated in Boyden invasion chambers in the presence of vehicle control (C) or 100 nM rapamycin (Rp). After 24 h of invasion, the number of invaded cells was counted in ten different fields per sample. Values reflect the total number of invaded cells in triplicate cultures per group. (D) SKOv3, SKOv3 stable control clone, GDF15 stable clone 1, and GDF15 stable clone 3 were plated in the presence of 5% FBS, and 100 nM rapamycin (Rp) where indicated, in the upper chamber of XCelligence CIM-plates. Medium containing 10% FBS, and 20 ng/mL rhGDF15 (G) where indicated, was placed into the plate’s corresponding bottom chambers. CIM-plates were monitored every 15 min for 10 h. Standardized cell index (CI) values were plotted linearly against time. The CI slope per hour was calculated and is shown here as a measure of migration. Values reflect the average of triplicates at each time point.

Article Snippet: Stable transfectants were developed by transfecting empty pCMV vector or GDF15 expression plasmid (Origene) into SKOv3 using Lipofectamine transfection reagent.

Techniques: Real-time Polymerase Chain Reaction, Clone Assay, Stable Transfection, Migration

Journal: Biochemical pharmacology

Article Title: Growth differentiation factor 15 stimulates rapamycin-sensitive ovarian cancer cell growth and invasion

doi: 10.1016/j.bcp.2012.10.007

Figure Lengend Snippet: Neutralization of secreted GDF15 reduces invasion and growth of GDF15-overexpressing ovarian cancer cells. (A) Concentrations of secreted GDF15 were determined by ELISA in media from SKOv3 and Tov21 cell lines. Cells were incubated in serum-free media for 48 h prior to ELISA. Values reflect the average fold expression in three samples per group. (B) Tov21 cells were either untreated, or treated with 1 µg/mL control IgG or GDF15 mAb (147627; R&D Systems) for 24 h. The concentration of GDF15 in media was measured by ELISA in triplicates per group. Values reflect the average fold expression per group. (C) Tov21 cells were treated with 1 µg/mL control IgG or GDF15 mAb for 24 h. Real-time PCR was then performed for MMP-2, MMP-9, and VEGF, and normalized to RPLPO. Values reflect the average fold change in normalized transcript. (D) Tov21 cells were plated in matrigel and treated with 1 µg/mL control IgG or GDF15 mAb (147627); media was changed twice a week for 3–4 weeks. Matrigel was dissolved using dispase and cells were counted by trypan blue exclusion. The percentage of anchorage-independent (AI) growth is shown relative to the control group. (E) Tov21 cells were plated in Boyden chambers and treated with 1 µg/mL control IgG or GDF15 mAb (147627). After 24 h, the number of invaded cells was counted in ten different fields per sample. Values reflect the total number of invaded cells in triplicate cultures per group.

Article Snippet: Stable transfectants were developed by transfecting empty pCMV vector or GDF15 expression plasmid (Origene) into SKOv3 using Lipofectamine transfection reagent.

Techniques: Neutralization, Enzyme-linked Immunosorbent Assay, Incubation, Expressing, Concentration Assay, Real-time Polymerase Chain Reaction

Journal: Biochemical pharmacology

Article Title: Growth differentiation factor 15 stimulates rapamycin-sensitive ovarian cancer cell growth and invasion

doi: 10.1016/j.bcp.2012.10.007

Figure Lengend Snippet: GDF15 knockdown reduces invasion and growth in association with reduced p-4EBP1 in GDF15-overexpressing ovarian cancer cells. (A) Real-time PCR was performed for GDF15 in SKOv3 and Tov21 cells, and normalized to the level of internal control transcript RPLPO. Values reflect the average fold change in normalized GDF15 transcript. (B) Tov21 cells were infected with lentiviral GDF15 shRNA or control shRNA. Real-time PCR was then performed for GDF15 and RPLPO. Values reflect the average fold change in normalized GDF15 transcript. (C) Tov21 cells were infected with lentiviral GDF15 shRNA or control shRNA. Real-time PCR was performed for MMP-2, MMP-9, and VEGF, and normalized to RPLPO. Values reflect the average fold change in normalized transcript. (D) Tov21 cells were plated in matrigel and infected with lentiviral control or GDF15 shRNA; media and virus were changed twice a week for 3–4 weeks. Matrigel was dissolved using dispase and cells were counted by trypan blue exclusion. The percentage of anchorage-independent (AI) growth is shown. (E) Tov21 cells were plated in Boyden chambers and infected with control or GDF15 shRNA. After 24 h, the number of invaded cells was counted in ten different fields per sample. Values reflect the total number of invaded cells in triplicate cultures per group. (F) Tov21 cells were infected with lentiviral control shRNA or GDF15 shRNA for 48 h. Western blots were performed at least twice for phosphorylated and total 4EBP1, Erk1/2, p38, and Akt; representative blots are shown. Quantification is shown as a ratio of phosphorylated to total protein above each blot.

Article Snippet: Stable transfectants were developed by transfecting empty pCMV vector or GDF15 expression plasmid (Origene) into SKOv3 using Lipofectamine transfection reagent.

Techniques: Real-time Polymerase Chain Reaction, Infection, shRNA, Western Blot

Journal: The Journal of Pathology

Article Title: Mesothelial‐to‐mesenchymal transition as a possible therapeutic target in peritoneal metastasis of ovarian cancer

doi: 10.1002/path.4889

Figure Lengend Snippet: AFMCs undergo MMT ex vivo and favour tumour progression in a subcutaneous xenograft mouse model. (A) Representative microscopy images of HPMCs, HPMCs treated with TGF‐β1 plus IL‐1β (T + I), and AFMCs in culture. Under phase contrast, the altered morphology of AFMCs is similar to that observed in HPMCs T + I (scale bar: 100 µm). Immunofluorescence staining for calretinin (green) confirms the MC nature, and positive α‐SMA (red) expression indicates AFMC conversion into myofibroblasts. [4′,6‐Diamidino‐2‐phenylindole: blue. Scale bars: 25 µm.] (B) Transcript levels of MMT markers, analysed by RT‐qPCR in HPMCs ( n = 8), HPMCs T + I ( n = 8), and AFMCs ( n = 5). E‐cadherin expression is repressed and, conversely, the expression of Snail and VEGF is induced in AFMCs and HPMCs T + I as compared with control HPMCs. Bar graphics represent mean ± standard error of the mean (SEM). Symbols represent the statistical differences between groups (* p ≤ 0.05; ** p ≤ 0.005). A.U., absolute units. (C) SKOV3‐luc‐D3 cells were co‐inoculated with control HPMCs into the left flank of mice, and with either HPMCs T + I ( n = 7) (a) or AFMC‐derived myofibroblasts ( n = 5) (b) in the right flank. Mice of both groups were monitored for 36 days. Representative bioluminescence images show the subcutaneous growth of SKOV3‐luc‐D3 cells plus HPMCs (orange circle) (a, b) as compared with SKOV3‐luc‐D3 cells plus HPMCs T + I (a, blue circle) or SKOV3‐luc‐D3 cells plus trans‐differentiated AFMCs (b, blue circle), over the duration of the experiment. Quantification of bioluminescence showed that tumour growth was increased in the right flank, where MCs that had undergone MMT had been used (a, b). Graphs represent mean average radiance (expressed as photons/s/cm 2 /sr) of SKOV3‐luc‐D3 cells ± SEM. Symbols represent the statistical differences between groups (** p ≤ 0.01; **** p ≤ 0.0001). dpi, days post‐inoculation.

Article Snippet: To induce MMT in vitro , HPMCs were treated with 0.5 ng/ml TGF‐β1 (R&D Systems, Minneapolis, MN, USA) plus 2.5 ng/ml interleukin (IL)‐1β (R&D Systems) (T + I) for 72 h . The human ovarian carcinoma cell line SKOV3 expressing luciferase (SKOV3‐luc‐D3) (Caliper Life Sciences, Hopkinton, MA, USA) was cultured in McCoy's 5A medium supplemented with 10% FBS, with geneticin used as a selection agent.

Techniques: Ex Vivo, Microscopy, Immunofluorescence, Staining, Expressing, Quantitative RT-PCR, Control, Derivative Assay

Journal: The Journal of Pathology

Article Title: Mesothelial‐to‐mesenchymal transition as a possible therapeutic target in peritoneal metastasis of ovarian cancer

doi: 10.1002/path.4889

Figure Lengend Snippet: OvCa‐secreted TGF‐β transforms the pre‐metastatic peritoneum, favouring tumour progression. (A) Representative images of in vivo monitoring of SKOV3‐luc‐D3 cells in mice pre‐conditioned with TGF‐β1‐encoding adenovirus or control. Quantification of bioluminescence showed that tumour growth was increased in mice pre‐conditioned with TGF‐β1 adenovirus ( n = 6) as compared with control adenoviral pretreatment ( n = 6). (B) (a) Representative images of E‐cadherin immunostaining in the mesothelial monolayer of a mouse killed 2 days after being pre‐conditioned with conditioned medium (CM) from OvCa cells or control medium. Scale bars: 25 µm. (b) Diagram of experimental design. (c) Representative images of in vivo monitoring of SKOV3‐luc‐D3 cells and quantification of bioluminescence showed that intraperitoneal tumour growth was higher in mice pretreated with SKOV3 medium (CM). The TGF‐β receptor I inhibitor (GW) reduced tumour growth to levels comparable to those of mice whose peritoneums had not been pre‐conditioned (control medium). n = 6 per group. All mice were monitored for 41 days. Graphs represent mean average radiance (expressed as photons/s/cm 2 /sr) of SKOV3‐luc‐D3 cells ± standard error of the mean. Symbols represent the statistical differences over time between groups (** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001). dpi, days post‐inoculation; i.p., intraperitoneal.

Article Snippet: To induce MMT in vitro , HPMCs were treated with 0.5 ng/ml TGF‐β1 (R&D Systems, Minneapolis, MN, USA) plus 2.5 ng/ml interleukin (IL)‐1β (R&D Systems) (T + I) for 72 h . The human ovarian carcinoma cell line SKOV3 expressing luciferase (SKOV3‐luc‐D3) (Caliper Life Sciences, Hopkinton, MA, USA) was cultured in McCoy's 5A medium supplemented with 10% FBS, with geneticin used as a selection agent.

Techniques: In Vivo, Control, Immunostaining

Journal: The Journal of Pathology

Article Title: Mesothelial‐to‐mesenchymal transition as a possible therapeutic target in peritoneal metastasis of ovarian cancer

doi: 10.1002/path.4889

Figure Lengend Snippet: The TGF‐β1–Smad3 pathway is activated in AFMCs, and truncated in OvCa cells. (A) Double immunofluorescence staining for α‐SMA (red) and pSmad3 (green) in HPMCs T + I and AFMCs indicates Smad3‐dependent TGF‐β1 pathway activation in both in vitro and ex vivo trans‐differentiated MCs as compared with double‐negative control cells. Scale bars: 25 µm. pSmad3‐positive nuclei were quantified; the box plot represents mean ± standard error of the mean. Symbols represent the statistical differences between groups. (B) Treatment of HPMCs and SKOV3 cells with TGF‐β1 for 1 and 6 h. Immunofluorescence images show that, upon TGF‐β1 treatment, pSmad3 translocates to the nucleus in HPMCs (a, c) and remains cytoplasmic in OvCa cells (b, d). 4′,6‐Diamidino‐2‐phenylindole: blue. Scale bars: 25 µm.

Article Snippet: To induce MMT in vitro , HPMCs were treated with 0.5 ng/ml TGF‐β1 (R&D Systems, Minneapolis, MN, USA) plus 2.5 ng/ml interleukin (IL)‐1β (R&D Systems) (T + I) for 72 h . The human ovarian carcinoma cell line SKOV3 expressing luciferase (SKOV3‐luc‐D3) (Caliper Life Sciences, Hopkinton, MA, USA) was cultured in McCoy's 5A medium supplemented with 10% FBS, with geneticin used as a selection agent.

Techniques: Double Immunofluorescence Staining, Activation Assay, In Vitro, Ex Vivo, Negative Control, Immunofluorescence

Journal: The Journal of Pathology

Article Title: Mesothelial‐to‐mesenchymal transition as a possible therapeutic target in peritoneal metastasis of ovarian cancer

doi: 10.1002/path.4889

Figure Lengend Snippet: Lentiviral knockdown of Smad3 reduces tumour progression in the peritoneum. (A) Western blot shows the expression level of Smad3 in parietal peritoneum lysates of mice injected in a preliminary assay with PBS ( n = 2), control lentiviral particles ( n = 3), or Smad3 shRNA‐producing lentiviral particles ( n = 3). Expression of β‐actin was employed as a loading control. (B) (a) Representative images of SKOV3‐luc‐D3 cell bioluminescence in mice pre‐conditioned with lentiviral particles producing Smad3 shRNA or control. Quantification of bioluminescence showed that tumour growth was significantly reduced in mice pre‐conditioned with lentiviral particles producing Smad3 shRNA ( n = 9) as compared with controls ( n = 9). Mice of both groups were monitored for 41 days. The graph represents mean average radiance (expressed as photons/s/cm 2 /sr) of SKOV3‐luc‐D3 cells ± standard error of the mean. Symbols represent the statistical differences over time between both groups (** p ≤ 0.01; *** p ≤ 0.001; p ≤ 0.0001). dpi, days post‐inoculation. (b) Representative images show a decrease in the number of metastases in Smad3 knockdown mice as compared with the control group. Tumours are outlined in white.

Article Snippet: To induce MMT in vitro , HPMCs were treated with 0.5 ng/ml TGF‐β1 (R&D Systems, Minneapolis, MN, USA) plus 2.5 ng/ml interleukin (IL)‐1β (R&D Systems) (T + I) for 72 h . The human ovarian carcinoma cell line SKOV3 expressing luciferase (SKOV3‐luc‐D3) (Caliper Life Sciences, Hopkinton, MA, USA) was cultured in McCoy's 5A medium supplemented with 10% FBS, with geneticin used as a selection agent.

Techniques: Knockdown, Western Blot, Expressing, Injection, Control, shRNA