Journal: Antioxidants

Article Title: THC and CBD Induce Heme Oxygenase-1-Dependent Cell Death and Trigger Mitochondrial Dysfunction in Human Melanoma and Cutaneous Squamous Cell Carcinoma Cells

doi: 10.3390/antiox15030286

Figure Lengend Snippet: Effect of THC ( A ), CBD ( B ), mAEA ( C ), and JWH-133 ( D ) on metabolic activity of A375 and A431 cells. Cells were incubated with the respective cannabinoid at the indicated concentrations for the indicated times. The values given are based on WST-1 assays. All percentage values shown refer to the respective time-matched vehicle control, which was set to 100%. The data are mean values ± SEM of n = 8–9 per group from 3 independent experiments. * p ≤ 0.05 vs. corresponding vehicle control; one-way ANOVA with Dunnett’s post hoc test.

Article Snippet: A375 (human melanoma cell line; #300110; RRID:CVCL_0132) and A431 cells (human skin epidermoid carcinoma cell line; #300112; RRID:CVCL_0037) were obtained from CLS Cell Lines Service (Eppelheim, Germany).

Techniques: Activity Assay, Incubation, Control

Journal: Antioxidants

Article Title: THC and CBD Induce Heme Oxygenase-1-Dependent Cell Death and Trigger Mitochondrial Dysfunction in Human Melanoma and Cutaneous Squamous Cell Carcinoma Cells

doi: 10.3390/antiox15030286

Figure Lengend Snippet: Effect of THC ( A ), CBD ( B ), and mAEA ( C ) on activation of caspases-3/7 in A375 and A431 cells. Cells were incubated with the respective cannabinoid at the indicated concentrations for the indicated times. The values given are based on caspase-3/7 activity assays. All percentage values shown refer to the respective time-matched vehicle control, which was set to 100%. The data are mean values ± SEM of n = 9 per group from 3 independent experiments. * p ≤ 0.05 vs. corresponding vehicle control; one-way ANOVA with Dunnett’s post hoc test.

Article Snippet: A375 (human melanoma cell line; #300110; RRID:CVCL_0132) and A431 cells (human skin epidermoid carcinoma cell line; #300112; RRID:CVCL_0037) were obtained from CLS Cell Lines Service (Eppelheim, Germany).

Techniques: Activation Assay, Incubation, Activity Assay, Control

Journal: Antioxidants

Article Title: THC and CBD Induce Heme Oxygenase-1-Dependent Cell Death and Trigger Mitochondrial Dysfunction in Human Melanoma and Cutaneous Squamous Cell Carcinoma Cells

doi: 10.3390/antiox15030286

Figure Lengend Snippet: Effect of THC and CBD on activation of caspase-8 ( A – D ) and caspase-9 ( E – H ) in A375 and A431 cells. Cells were incubated with THC or CBD at the indicated concentrations for 6 h. The values given are based on caspase activity assays. All percentage values shown refer to the respective time-matched vehicle control, which was set to 100%. The data are mean values ± SEM of n = 9 per group from 3 independent experiments. * p ≤ 0.05 vs. corresponding vehicle control; one-way ANOVA with Dunnett’s post hoc test.

Article Snippet: A375 (human melanoma cell line; #300110; RRID:CVCL_0132) and A431 cells (human skin epidermoid carcinoma cell line; #300112; RRID:CVCL_0037) were obtained from CLS Cell Lines Service (Eppelheim, Germany).

Techniques: Activation Assay, Incubation, Activity Assay, Control

Journal: Antioxidants

Article Title: THC and CBD Induce Heme Oxygenase-1-Dependent Cell Death and Trigger Mitochondrial Dysfunction in Human Melanoma and Cutaneous Squamous Cell Carcinoma Cells

doi: 10.3390/antiox15030286

Figure Lengend Snippet: Effect of THC and CBD on PARP cleavage ( A – D ), LC3-I to LC3-II conversion ( E – H ), and GPX4 expression ( I – L ) in A375 and A431 cells. The cells were incubated with THC or CBD at the indicated concentrations for 24 h. The values given in the bar charts are based on densitometric analyses of the blots. PARP, cleaved PARP (cl. PARP), LC3A/B-I, LC3A/B-II and GPX4 levels were normalized to β-actin. All percentage values shown refer to the respective vehicle control, which was set to 100%. The blots shown are representative. The data are mean values ± SEM of n = 3 ( I – L ), n = 4 ( D , F , G ), n = 5 ( A , B , E , H ) or n = 6 ( C ) independent experiments. * p ≤ 0.05 vs. corresponding vehicle control; one-way ANOVA with Dunnett’s post hoc test.

Article Snippet: A375 (human melanoma cell line; #300110; RRID:CVCL_0132) and A431 cells (human skin epidermoid carcinoma cell line; #300112; RRID:CVCL_0037) were obtained from CLS Cell Lines Service (Eppelheim, Germany).

Techniques: Expressing, Incubation, Control

Journal: Antioxidants

Article Title: THC and CBD Induce Heme Oxygenase-1-Dependent Cell Death and Trigger Mitochondrial Dysfunction in Human Melanoma and Cutaneous Squamous Cell Carcinoma Cells

doi: 10.3390/antiox15030286

Figure Lengend Snippet: Effect of THC and CBD on the expression of HO-1 and HO-2 at the mRNA ( A – D ) and protein ( E – H ) level in A375 and A431 cells. Cells were incubated with THC or CBD at the indicated concentrations for 6 h ( A – D ) or 24 h ( E – H ). The values given in the bar charts are based on quantitative RT-PCR ( A – D ) or densitometric analyses of the blots ( E – H ). HO mRNA levels were normalized to PPIA mRNA levels and HO protein levels to β-actin. All percentage values shown refer to the respective vehicle control, which was set to 100%. The blots shown are representative. The mRNA and protein data are mean values ± SEM of n = 3 ( A – D ), n = 7 ( E ), n = 5 ( F ) or n = 6 ( G , H ) independent experiments. * p ≤ 0.05 vs. corresponding vehicle control; one-way ANOVA with Dunnett’s post hoc test.

Article Snippet: A375 (human melanoma cell line; #300110; RRID:CVCL_0132) and A431 cells (human skin epidermoid carcinoma cell line; #300112; RRID:CVCL_0037) were obtained from CLS Cell Lines Service (Eppelheim, Germany).

Techniques: Expressing, Incubation, Quantitative RT-PCR, Control

Journal: Antioxidants

Article Title: THC and CBD Induce Heme Oxygenase-1-Dependent Cell Death and Trigger Mitochondrial Dysfunction in Human Melanoma and Cutaneous Squamous Cell Carcinoma Cells

doi: 10.3390/antiox15030286

Figure Lengend Snippet: Influence of the HO-1 inhibitor SnPPIX on the decrease in metabolic activity ( A , D , G , J ) and cell number ( B , E , H , K ) as well as on the increase in caspase-3/7 activity ( C , F , I , L ) mediated by THC or CBD in A375 and A431 cells. Cells were pre-treated with SnPPIX (25 µM) or its vehicle for 1 h, followed by a 48-h (A375 cells) or 24-h (A431 cells) co-incubation with the indicated concentrations of THC or CBD or its vehicle. The data are mean values ± SEM of n = 9 from 3 independent experiments ( A – J ) or n = 12 from 4 independent experiments ( K , L ). * p ≤ 0.05 vs. corresponding vehicle control; # p ≤ 0.05 vs. corresponding THC- or CBD-treated group; one-way ANOVA with Bonferroni’s post hoc test.

Article Snippet: A375 (human melanoma cell line; #300110; RRID:CVCL_0132) and A431 cells (human skin epidermoid carcinoma cell line; #300112; RRID:CVCL_0037) were obtained from CLS Cell Lines Service (Eppelheim, Germany).

Techniques: Activity Assay, Incubation, Control

Journal: Antioxidants

Article Title: THC and CBD Induce Heme Oxygenase-1-Dependent Cell Death and Trigger Mitochondrial Dysfunction in Human Melanoma and Cutaneous Squamous Cell Carcinoma Cells

doi: 10.3390/antiox15030286

Figure Lengend Snippet: Effect of THC and CBD on mitochondrial membrane potential (ΔΨm) of A375 ( A ) and A431 cells ( B ). The cells were incubated with THC or CBD at the indicated concentrations for 24 h. ΔΨm was assessed using the JC-10 fluorescence ratio (orange to green) as described in and expressed relative to the vehicle control (set to 100%). Data are mean values ± SEM of n = 9 from 3 independent experiments. All percentage values shown refer to the respective vehicle control, which was set to 100%. * p ≤ 0.05 vs. corresponding vehicle control; one-way ANOVA with Dunnett’s post hoc test.

Article Snippet: A375 (human melanoma cell line; #300110; RRID:CVCL_0132) and A431 cells (human skin epidermoid carcinoma cell line; #300112; RRID:CVCL_0037) were obtained from CLS Cell Lines Service (Eppelheim, Germany).

Techniques: Membrane, Incubation, Fluorescence, Control

Journal: Antioxidants

Article Title: THC and CBD Induce Heme Oxygenase-1-Dependent Cell Death and Trigger Mitochondrial Dysfunction in Human Melanoma and Cutaneous Squamous Cell Carcinoma Cells

doi: 10.3390/antiox15030286

Figure Lengend Snippet: Effect of THC and CBD on mitochondrial HO-1 protein levels in A375 ( A , B ) and A431 cells ( C , D ). The cells were treated with the indicated concentrations of THC or CBD for 24 h. Thereafter, the corresponding proteins in the mitochondrial fractions were determined using Western blot analysis. The values given in the bar charts are based on densitometric analyses of the blots. Mitochondrial HO-1 was normalized to VDAC. All percentage values shown refer to the respective vehicle control, which was set to 100%. The blots shown are representative. In ( B ) the same VDAC blot is shown as in D, and in ( C ) as in C, as the same membranes were stripped and reprobed for different target proteins. The data are mean values ± SEM of n = 4 ( A – C ) or n = 3 ( D ) independent experiments. * p ≤ 0.05 vs. corresponding vehicle control; one-way ANOVA with Dunnett’s post hoc test.

Article Snippet: A375 (human melanoma cell line; #300110; RRID:CVCL_0132) and A431 cells (human skin epidermoid carcinoma cell line; #300112; RRID:CVCL_0037) were obtained from CLS Cell Lines Service (Eppelheim, Germany).

Techniques: Western Blot, Control

Journal: Antioxidants

Article Title: THC and CBD Induce Heme Oxygenase-1-Dependent Cell Death and Trigger Mitochondrial Dysfunction in Human Melanoma and Cutaneous Squamous Cell Carcinoma Cells

doi: 10.3390/antiox15030286

Figure Lengend Snippet: Effect of THC and CBD on the concentrations of subunits of mitochondrial respiratory chain complexes of A375 ( A – D ) and A431 cells ( E – H ). The cells were treated with the indicated concentrations of THC or CBD for 24 h. Thereafter, the corresponding proteins in the mitochondrial fractions were determined using Western blot analysis. The values given in the bar charts are based on densitometric analyses of the blots. Mitochondrial proteins were normalized to VDAC. All percentage values shown refer to the respective vehicle control, which was set to 100%. The blots shown are representative. In ( B ) the same VDAC blot is shown as in A, in ( D ) as in B, and in ( H ) as in D, as the same membranes were stripped and reprobed for different target proteines. The data are mean values ± SEM of n = 4 independent experiments. * p ≤ 0.05 vs. corresponding vehicle control; one-way ANOVA with Dunnett’s post hoc test.

Article Snippet: A375 (human melanoma cell line; #300110; RRID:CVCL_0132) and A431 cells (human skin epidermoid carcinoma cell line; #300112; RRID:CVCL_0037) were obtained from CLS Cell Lines Service (Eppelheim, Germany).

Techniques: Western Blot, Control

Journal: Antioxidants

Article Title: THC and CBD Induce Heme Oxygenase-1-Dependent Cell Death and Trigger Mitochondrial Dysfunction in Human Melanoma and Cutaneous Squamous Cell Carcinoma Cells

doi: 10.3390/antiox15030286

Figure Lengend Snippet: Effect of THC and CBD on the release of mitochondrial cytochrome c (Cyt c) into the cytosol of A375 ( A , B ) and A431 cells ( C , D ). The cells were incubated with THC or CBD at the indicated concentrations for 24 h. The values given in the bar charts are based on densitometric analyses of the blots. Cyt c levels were normalized on VDAC in mitochondrial fractions and on GAPDH in cytosolic fractions. The blots shown are representative. In ( A ) the same VDAC blot is shown as in B, in ( C ) as in C, and in ( D ) as in H, as the same membranes were stripped and reprobed for different target proteines. The data are mean values ± SEM of n = 4 (( A , B , D ) left graph each; ( C )) or n = 3 (( A , B , D ) right graph each) independent experiments. All percentage values shown refer to the respective vehicle control, which was set to 100%. * p ≤ 0.05 vs. corresponding vehicle control with Dunnett’s post hoc test.

Article Snippet: A375 (human melanoma cell line; #300110; RRID:CVCL_0132) and A431 cells (human skin epidermoid carcinoma cell line; #300112; RRID:CVCL_0037) were obtained from CLS Cell Lines Service (Eppelheim, Germany).

Techniques: Incubation, Control

Journal: Antioxidants

Article Title: THC and CBD Induce Heme Oxygenase-1-Dependent Cell Death and Trigger Mitochondrial Dysfunction in Human Melanoma and Cutaneous Squamous Cell Carcinoma Cells

doi: 10.3390/antiox15030286

Figure Lengend Snippet: Influence of THC and CBD on the mitochondrial structure of A375 and A431 cells. Representative images from the transmission electron microscopy of A375 ( A – C ) and A431 cells ( D – F ) treated for 24 h with vehicle ( A , D ), 6 µM THC ( B , E ) or 6 µM CBD ( C , F ). The images on the right of a treatment group (scale bar: 500 nm) are enlarged views of the areas outlined by white boxes in the corresponding left image (scale bar: 2 µm). The structures highlighted with white arrows in the enlarged views represent intact mitochondria (vehicle) vs. damaged mitochondria (THC, CBD), while white arrowheads mark altered mitochondria-associated ER membranes (CBD).

Article Snippet: A375 (human melanoma cell line; #300110; RRID:CVCL_0132) and A431 cells (human skin epidermoid carcinoma cell line; #300112; RRID:CVCL_0037) were obtained from CLS Cell Lines Service (Eppelheim, Germany).

Techniques: Transmission Assay, Electron Microscopy

Journal: Antioxidants

Article Title: THC and CBD Induce Heme Oxygenase-1-Dependent Cell Death and Trigger Mitochondrial Dysfunction in Human Melanoma and Cutaneous Squamous Cell Carcinoma Cells

doi: 10.3390/antiox15030286

Figure Lengend Snippet: Effect of THC and CBD on oxygen consumption rate (OCR) of A375 ( A – D ) and A431 cells ( E – H ). The cells were incubated with THC or CBD at the indicated concentrations for 24 h. A mitochondrial stress test was then carried out and OCR values were determined using the Seahorse XFe24 Analyzer. Therefore, oligomycin (port A), FCCP (port B) and antimycin A/rotenone (port C) were loaded into the respective ports of the sensor cartridges and released into the wells at the specified times, as marked by enclosed uppercase letters with arrows in the time-course panels. From this assay, time courses of OCR in both cell lines treated with THC ( A , E ) or CBD ( C , G ) and calculations of basal respiration (basal), ATP-linked respiration (ATP), spare respiratory capacity and proton leak ( B , D , F , H ) are shown. The data represent mean values ± SEM of n = 3 per group from 3 independent experiments. * p ≤ 0.05 vs. corresponding vehicle control; one-way ANOVA with Dunnett’s post hoc test.

Article Snippet: A375 (human melanoma cell line; #300110; RRID:CVCL_0132) and A431 cells (human skin epidermoid carcinoma cell line; #300112; RRID:CVCL_0037) were obtained from CLS Cell Lines Service (Eppelheim, Germany).

Techniques: Incubation, Control

Journal: Bioconjugate chemistry

Article Title: Simple Method To Prepare Oligonucleotide-Conjugated Antibodies and Its Application in Multiplex Protein Detection in Single Cells.

doi: 10.1021/acs.bioconjchem.5b00613

Figure Lengend Snippet: Fig. 6. Detection of proteins in cell lysates. A panel of 24-plex antibody binders was used to detect their respective target proteins in MCF7, K562, A549, and A431 cell lysates. The cell lysates were diluted to different concentrations and reacted with a mixture of antibody binders. Only selected examples of protein targets CSTB (A), CASP3 (B), Ki-67(C), and GATA3 (D) are shown. Ct values were shown as the means ± SD. Ct: cycle threshold.

Article Snippet: MCF7, K562, A549, and A431 cell lysates were obtained from Novus (Littleton, CO).

Techniques:

Journal: BMC cancer

Article Title: Identification of the angiogenic gene signature induced by EGF and hypoxia in colorectal cancer.

doi: 10.1186/1471-2407-13-518

Figure Lengend Snippet: Figure 5 EGF receptor is autophosphorylated in Caco-2 and activates downstream signalling pathways. Caco-2 cells were stimulated with 20 ng/mL EGF for the time periods indicated. Western blotting for (a) phosphorylated EGFR or total EGFR and (b) antibodies recognising signal- ling enzymes is shown. Cell lysate of EGF-treated A431 cells was used as positive control. α-tubulin is shown as a loading control. Densitometry was performed using Phoretix 1D analysis software against α-tubulin (for ERK, data for p42 and p44 are shown).

Article Snippet: Whole cell lysate of EGF-treated A431 epithelial carcinoma cells used as positive control was from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Western Blot, Positive Control, Control, Software

Journal: Cancers

Article Title: Implication of COPB2 Expression on Cutaneous Squamous Cell Carcinoma Pathogenesis

doi: 10.3390/cancers14082038

Figure Lengend Snippet: Effect of COPB2 knockdown on biologic behavior of cutaneous squamous cell carcinoma (cSCC) cells: ( A ) (i) COPB2 protein expression decreased predominantly after COPB2 knockdown in both HSC-1 and A431 cells (Magnification, 400×; Scale bar, 50 μm); (ii) Proliferation ability of both HSC-1 and A431 cells decreased significantly after COPB2 knockdown at the indicated time points (Both p < 0.001, Mann-Whitney U-test). ( B ) (i) Representative patterns of cell invasion in each group of cSCC cells (Magnification, 100×; Scale bar, 200 μm). (ii) Invasion ability of both HSC-1 and A431 cells decreased significantly after COPB2 knockdown (Both p = 0.002, Mann-Whitney U-test). ( C ) Apoptotic cells increased predominantly after COPB2 knockdown in both HSC-1 and A431 cells. ( D ) (i) Tumor nodules of xenograft mouse models obtained from control and shCOPB2 groups of A431 cells (Scale bar, 1 cm). (ii) Volume of the tumor nodules increased significantly in control group compared to that in the COPB2 knockdown group at the indicated days (* p = 0.008, Mann-WhitneyU-test). (iii) Representative patterns of apoptotic cells in tumor nodules from each group of A431 cells-xenograft models (Magnification, 400×; Scale bar, 50 μm). Apoptotic cells were predominantly higher in the COPB2 knockdown group than in the control group in A431 cells ( p < 0.001, Mann-Whitney U-test).

Article Snippet: COPB2 knockdown stable HSC-1 and A431, as well as related control cells, were established using pGFP-C-shCOPB2 lentiviral particles (OriGene, Rockville, MD, USA) and maintained in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% fetal bovine serum (FBS; Invitrogen, Waltham, MA, USA) and 1% streptomycin/penicillin (Gibco, Waltham, MA, USA).

Techniques: Expressing, MANN-WHITNEY