Journal: Pharmaceuticals

Article Title: Hydrophilic and Lipophilic Carbon Dots Impart Thermosensitivity to Doxorubicin Loaded Phospholipid Liposomes

doi: 10.3390/ph19050668

Figure Lengend Snippet: Comparative cytotoxicity of either empty or DOX-loaded PEGylated DPPC liposomes (Lip), or CD (Lip-CD) and CD-C16 (Lip-CD16) containing liposomes against PC3 cells following an 1 h incubation at ( A ) 37 or ( B ) 40°C. DOX concentration was 5 or 10 μM, while the concentration of empty liposomes corresponded to the liposomal concentration in experiments at the highest DOX concentration (10 μΜ). The cytotoxicity was assessed after a 24 h post-incubation period employing the standard MTT assay. Cell survival data are the mean of at least six independent values while error bars represent one standard deviation. The statistical significance was derived by Student’s t -test following the assignment: * p < 0.05, ** p < 0.01, *** p < 0.001; ns denotes no statistical significance, p > 0.05.

Article Snippet: The human prostate cancer PC3 cell line as well as the non-cancerous human embryonic kidney HEK293 of the cell bank from the Institute of Nanoscience and Nanotechnology, NCSR Demokritos, were purchased from the American Type Culture Collection (ATCC CRL-1435TM and CRL-1573TM, respectively; Manassas, VA, USA).

Techniques: Liposomes, Incubation, Concentration Assay, MTT Assay, Standard Deviation, Derivative Assay

Journal: Cells

Article Title: Senescent Stroma-Derived Glutamine: A Driver of Aggressiveness in Prostate and Ovarian Cancer Cells

doi: 10.3390/cells15090770

Figure Lengend Snippet: The metabolic component secreted by senescent HPFs and HOFs is involved in the acquisition of a pro-invasive phenotype in prostate and ovarian cancer cells. ( A ) Quantification of SA-β-Gal staining of human prostate fibroblasts (HPFs) and human ovarian fibroblasts (HOFs) treated for 24 h with 5 nM Docetaxel (DTX) and 20 μM cisplatin (CPT), respectively, and then cultured in drug-free medium for an additional 6 days. For each condition, images were taken from five randomly selected fields, and both the total number of cells and the number of blue (SA-β-Gal positive) cells were counted. Bar graphs represent the average ratio of positive cells to the total cell count. Representative images of the stained cells are shown below the bar graphs (magnification 20×, scale bar 100 μm). ( B ) Representative immunoblots of p16 and p21 protein levels in DTX-treated HPFs and CPT-treated HOFs. β-actin was used as loading control. ( C ) Invasion assay: PC3 and SKOV3 cells were incubated with CM CTRL and CM SEN conditioned media (CM) from senescent and non-senescent (CTRL) fibroblasts for 72 h and then seeded in Boyden chambers. Cells were allowed to invade for 16 h. Representative images of the filters are shown below the bar graphs (magnification 20×, scale bar 100 μm). ( D ) mRNA expression level of EMT key genes in PC3 and SKOV3 cells incubated with CM CTRL or CM SEN for 72 h. ( E ) Invasion assay: PC3 and SKOV3 cells were incubated for 72 h with boiled and non-boiled CM CTRL or CM SEN and then allowed to invade for 16 h. Representative images of the filters are shown below the bar graphs (magnification 20×, scale bar 100 μm). ( F ) GC–MS analysis of metabolites in CM-CTRL or CM-SEN. Data reported are normalized to CM CTRL. * symbol indicates metabolite levels significantly increased both in senescent CM from HPFs and HOFs. Data are means ± SEM of three independent experiments. Statistical significance was assessed by unpaired Student t -test ( A , C , D , F ) or one-way Anova followed by Tukey’s multiple comparisons ( E ). * p < 0.05; ** p < 0.01; *** p < 0.001; ( ϕϕϕ p < 0.001; ϕϕϕϕ p < 0.0001; $$ p < 0.01; $$$ p < 0.001). In panel ( E ), * indicates CM SEN boiled vs. SEN non-boiled; ϕ indicates CM CTRL boiled vs. CM CTRL non-boiled; $ indicates CM SEN vs. CM CTRL.

Article Snippet: Human prostate (PC3) and ovarian (SKOV3) cancer cell lines were obtained from ATCC (PC3: CVCL_E2RM; SKOV3: CVCL_0532).

Techniques: Staining, Cell Culture, Cell Characterization, Western Blot, Control, Invasion Assay, Incubation, Expressing, Gas Chromatography-Mass Spectrometry

Journal: Cells

Article Title: Senescent Stroma-Derived Glutamine: A Driver of Aggressiveness in Prostate and Ovarian Cancer Cells

doi: 10.3390/cells15090770

Figure Lengend Snippet: Gln availability drives invasive ability in PC3 and SKOV3 cells. ( A ) Invasion assay: PC3 and SKOV3 cells were incubated for 72 h with DMEM in presence or absence of 2 mM Glutamine (Gln) and then seeded in Boyden chambers. Representative images of the filters are shown below the bar graphs (magnification 20×, scale bar 100 μm). ( B ) Invasion assay: PC3 and SKOV3 were incubated with CM CTRL and CM SEN for 72 h and with ASNase 1 U/mL during the last 48 h. Then, cells were seeded in Boyden chambers; ( C ) mRNA expression level of Gln transporter SLC1A5 in PC3 and SKOV3 cells incubated with CM CTRL or CM SEN for 72 h. ( D ) PC3 and SKOV3 cells were incubated for 72 h with CM CTRL or CM SEN. Then, GC–MS analysis was performed to measure Glu and Gln intracellular content. ( E ) Invasion assay: PC3 and SKOV3 cells were incubated for 72 h with DMEM and then seeded in a Boyden chamber in presence or absence of BPTES 1 μM for 16 h. Representative images of the filters are shown below the bar graphs (magnification 20×, scale bar 100 μm). ( F ) Invasion assay: PC3 and SKOV3 cells were incubated for 72 h with boiled and non-boiled CM; BPTES 1 μM was added during the final 16 h of incubation. Data are means ± SEM of three independent experiments. Statistical significance was assessed by Student t -test ( A , C – E ) or one-way Anova followed by Tukey’s multiple comparisons ( B , F ). * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.001; ϕ p < 0.05; ϕϕ p < 0.01; ϕϕϕ p < 0.001; $ p < 0.05; $$ p < 0.01; $$$ p < 0.001. In panel ( E ), * indicates CM SEN + ASNase vs. CM SEN; ϕ indicates CM CTRL + ASNase vs. CM CTRL; $ indicates CM SEN vs. CM CTRL. In panel ( F ), * indicates treated CM SEN vs. CM SEN, ϕ indicates treated CM CTRL vs. CM CTRL, $ indicates CM SEN vs. CM CTRL.

Article Snippet: Human prostate (PC3) and ovarian (SKOV3) cancer cell lines were obtained from ATCC (PC3: CVCL_E2RM; SKOV3: CVCL_0532).

Techniques: Invasion Assay, Incubation, Expressing, Gas Chromatography-Mass Spectrometry

Journal: Cells

Article Title: Senescent Stroma-Derived Glutamine: A Driver of Aggressiveness in Prostate and Ovarian Cancer Cells

doi: 10.3390/cells15090770

Figure Lengend Snippet: Gln confers aggressiveness to prostate and ovarian cancer cells. ( A , B ) PC3 and SKOV3 cells were conditioned for 72 h with CM from HPFs and HOFs; BPTES 1 μM was added during the final 16 h of incubation. Then, cells were detached and grown as spheroids for 7 days. Volumes of prostatic and ovarian spheroids were calculated as described in M and M. Representative images of spheroids are shown below the bar graphs (magnification 20×, scale bar 100 μm). ( C ) Prostate and ovarian cancer cells were incubated for 72 h with or without 2 mM Gln. Then, cells were detached and grown as spheroids for 7 days. Volumes of prostatic and ovarian spheroids were calculated as described in M and M. Representative images of spheroids are shown below the bar graphs (magnification 20×, scale bar 100 μm). Data are represented as mean ± SEM of three independent experiments. Statistical significance was assessed by t -test. **** p < 0.0001.

Article Snippet: Human prostate (PC3) and ovarian (SKOV3) cancer cell lines were obtained from ATCC (PC3: CVCL_E2RM; SKOV3: CVCL_0532).

Techniques: Incubation

Journal: Cells

Article Title: Senescent Stroma-Derived Glutamine: A Driver of Aggressiveness in Prostate and Ovarian Cancer Cells

doi: 10.3390/cells15090770

Figure Lengend Snippet: GS silencing in senescent fibroblasts affects EMT program in prostate and ovarian cancer cells. ( A ) Immunoblot of GS protein levels in senescent and CTRL fibroblasts. Vinculin was used as loading control. ( B ) GS protein levels in senescent HPFs and HOFs following 48 h gene silencing. HSP90 immunoblot was performed to ensure equal loading. ( C ) Invasion assay: PC3 and SKOV3 cells were incubated for 72 h with CM from GS silenced HPFs and HOFs and then let to invade for 16 h. Representative images of filters are shown below the bar graphs (magnification 20×, scale bar 100 μm). ( D ) mRNA expression level of EMT markers in PC3 and SKOV3 cells after 72 h incubation with CM from GS-silenced fibroblasts. Data are mean ± SEM of three independent experiments. Statistical significance was assessed by Student t -test. t -test * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: Human prostate (PC3) and ovarian (SKOV3) cancer cell lines were obtained from ATCC (PC3: CVCL_E2RM; SKOV3: CVCL_0532).

Techniques: Western Blot, Control, Invasion Assay, Incubation, Expressing

Journal: Cells

Article Title: Senescent Stroma-Derived Glutamine: A Driver of Aggressiveness in Prostate and Ovarian Cancer Cells

doi: 10.3390/cells15090770

Figure Lengend Snippet: Senescent stroma-derived Gln is uploaded by PC3. CTR and senescent HPFs were incubated for 24 h with 13 C glucose, then cell lysates and CM were collected for GC–MS analysis. PC3 cells were incubated with CM for further 24 h and the amount of 13 C Gln inside cells was measured. Data are represented as mean ± SEM of three independent experiments. Statistical significance was assessed by Student t -test. * p < 0.05.

Article Snippet: Human prostate (PC3) and ovarian (SKOV3) cancer cell lines were obtained from ATCC (PC3: CVCL_E2RM; SKOV3: CVCL_0532).

Techniques: Derivative Assay, Incubation, Gas Chromatography-Mass Spectrometry

Journal: Cells

Article Title: Senescent Stroma-Derived Glutamine: A Driver of Aggressiveness in Prostate and Ovarian Cancer Cells

doi: 10.3390/cells15090770

Figure Lengend Snippet: Gln-dependent NRF2/ETS1 pathway drives the invasive abilities of cancer cells. ( A ) Total ROS production was measured with a DCFDA probe in PC3 and SKOV3 cells after 72 h incubation with CM CTRL and CM SEN. ( B ) Total ROS production measured with DCFDA probe in PC3 and SKOV3 cells after incubation with boiled and non-boiled CM CTRL for 72 h in the presence or absence of ASNase 1 U/mL during the last 48 h. ( C ) Total ROS measured as described above. Cells were conditioned for 72 h, BPTES 1 μM was added during the final 16 h of incubation. ( D ) NRF2 protein level in PC3 and SKOV3 cells incubated either with DMEM in presence or absence of 2 mM Gln with CM CTRL and CM SEN. HSP90 immunoblot was performed to ensure equal loading. ( E ) ETS1 protein level in PC3 and SKOV3 cells incubated either with DMEM in presence or absence of 2 mM Gln or with CM CTRL or CM SEN. Actin was used as loading control. ( F ) GSH/GSSG ratio in prostate and ovarian cancer cells incubated for 72 h with CM CTRL or CM SEN. Data are normalized to protein content. ( G ) PC3 and SKOV3 cells were incubated for 72 h with CM CTRL or CM SEN; BPTES 1 μM was added during the final 16 h of incubation. Representative confocal microscopy images show ETS1 nuclear translocation. Bar graphs show the nuclear fluorescence intensity of ETS1 signal. Fluorescence intensity was quantified with ImageJ software (red: ETS1, blue: DAPI; scale bar 44 μm). Data are mean ± SEM of three independent experiments. Statistical significance was assessed by Student t -test ( A , C , F ) or one-way Anova followed by Tukey’s multiple comparisons ( B , G ). * p < 0.05; ** p < 0.01; φ p < 0.05; $$ p < 0.01. In panel ( G ), * indicates CM CTRL + BPTES vs. CM CTRL, $ indicates CM SEN vs. CM CTRL, φ indicates CM SEN + BPTES vs. CM SEN.

Article Snippet: Human prostate (PC3) and ovarian (SKOV3) cancer cell lines were obtained from ATCC (PC3: CVCL_E2RM; SKOV3: CVCL_0532).

Techniques: Incubation, Western Blot, Control, Confocal Microscopy, Translocation Assay, Fluorescence, Software

Journal: Cell Reports Methods

Article Title: Biobank of genetically defined murine prostate cancer tumoroids uncovers oncogenic pathways and drug vulnerabilities driven by PTEN-loss

doi: 10.1016/j.crmeth.2026.101370

Figure Lengend Snippet: The PDPK1/AKT/FLT DPI and tenovin-6 (T6) show high anti-cancer efficacy in murine tumoroids and human PCa cell lines (A) Dose-response curves for DPI (top) and T6 (bottom) for in vivo and in vitro Pten KO (left), Pten/Stat3 KO (middle), and Pten/Tp53 KO (right) tumoroids. Points represent means of technical duplicates per tumoroid line ( N = 3). Curve fitting was performed using GraphPad Prism 8.0.2. (B) Bar graphs showing means and ±SD of half-maximal inhibitory concentration (IC50) for DPI (top) and T6 (bottom) for in vivo and in vitro tumoroid lines of all genotypes ( N = 3). Statistical analysis was performed using GraphPad Prism 8.0.2 (one-way ANOVA, Tukey’s test). p > 0.05 if not specified otherwise, ∗ p ≤ 0.05. (C) Bar graphs depicting means and ±SD of IC50 values of DPI (left) and T6 (right) on human PCa cell lines. 22RV1: primary PCa; LNCaP: metastatic PCa; DU145, PC3: metastatic castration-resistant PCa ( N = 3). Statistical analysis was performed using GraphPad Prism 8.0.2 (one-way ANOVA). p > 0.05 if not specified otherwise, ∗ p ≤ 0.05; ∗∗ p ≤ 0.01. (D) Heatmaps of synergy scores calculated with the highest single agent (HSA) model for DPI and enzalutamide (left), and T6 and enzalutamide (right) on the human LNCaP cell line. Values > 0 represent synergistic effects, and values < 0 represent antagonistic effects. IC50 concentrations of respective compounds are underlined ( N = 3). See also .

Article Snippet: Human PC3 metastatic PCa cell line , ATCC , CRL-1435; RRID:CVCL_0035.

Techniques: In Vivo, In Vitro, Concentration Assay