Journal: Heliyon

Article Title: Exercise potentially prevents colorectal cancer liver metastases by suppressing tumor epithelial cell stemness via RPS4X downregulation

doi: 10.1016/j.heliyon.2024.e26604

Figure Lengend Snippet: Knockdown of RPS4X expression reduced tumor stemness. (A) The RPS4X gene expression level was assessed by qPCR. GAPDH served as an internal parameter (n = 3). (B) The cell proliferation ability of SW48 was determined using the CCK-8 assay (n = 3). (C–D) Cell migration and invasion ability of SW48 were evaluated (n = 3). Data are presented as mean ± SD. *P < 0.05 (E–F) Cell apoptotic rate of SW48 was evaluated (n = 3). Data are presented as mean ± SD. **P < 0.01. (G) Tumor growth curve and RPS4X down-regulation in tumors from mice (n = 5 per group). Data are presented as mean ± SD. *P < 0.05 **P < 0.01 ***P < 0.001. (H) Representative images showing the tumors harvested from SW48-bearing mice (n = 5 per group). (I)Weight of the harvested tumors from tumor-bearing mice (n = 5 per group). Data are presented as mean ± SD. **P < 0.01.

Article Snippet: SW48 (catalog KG536) cancer cell lines were purchased from KeyGEN.

Techniques: Knockdown, Expressing, Gene Expression, CCK-8 Assay, Migration

Journal: Oncotarget

Article Title: Therapeutic efficacy of SYM004, a mixture of two anti-EGFR antibodies in human colorectal cancer with acquired resistance to cetuximab and MET activation

doi: 10.18632/oncotarget.18749

Figure Lengend Snippet: (A) Western blot analysis of protein expression in GEO, SW48, GEO-CR and SW48-CR cells treated with cetuximab (5 μg/ml) and SYM004 (5 μg/ml) was performed. Total cell protein extracts were subjected to immuneblotting with the indicated antibodies, as described in Materials and Methods. (B) Two mg of GEO cell or of GEO-CR cell protein extracts were immune-precipitated with a specific anti-MET antibody and then were immune-blotted with a specific anti-EGFR antibody, as described in Materials and Methods. (C) Western blot analysis of protein expression in SW48, SW48-CR, SW48H2 and SKBR3 was performed. Total cell protein extracts were subjected to immuneblotting with the indicated antibodies, as described in Materials and Methods. (D) HER2 gene amplified SW48 cells line (SW48H2) are exposed to different concentration of cetuximab (range, 0.001 to 10 μg/ml) and SYM004 (range, 0.001 to 10 μg/ml) for 96 hours and evaluated for proliferation by MTT staining, as described in Materials and Methods.

Article Snippet: The human SW48 (catalogue number: HTL99020) ( KRAS, NRAS, BRAF and PIK3CA WT), colon cancer cell line was obtained from IRCCS “Azienda Ospedaliera Universitaria San Martino-IST Istituto Nazionale per la Ricerca sul Cancro, Genova” Italy.

Techniques: Western Blot, Expressing, Amplification, Concentration Assay, Staining

Journal: Oncotarget

Article Title: Therapeutic efficacy of SYM004, a mixture of two anti-EGFR antibodies in human colorectal cancer with acquired resistance to cetuximab and MET activation

doi: 10.18632/oncotarget.18749

Figure Lengend Snippet: (A-B) Mice were injected subcutaneously in the right flank with SW48 cells as described in the Materials and Methods. After two weeks (average tumor size 200-300 mm 3 ) mice were treated intraperitoneally with: PBS control, cetuximab (1 mg twice a week), SYM004 (50 mg/kg twice a week). The treatment was continued for 30 weeks. Each group consisted of 10 mice. Tumor volumes were measured three times a week. Animals were sacrificed when tumors achieved 2.000 mm 3 in size. Abbreviations: CTR, control; A, median tumor volume (mm 3 ); B, alive mice/total mice; C, number of mice without clinical evidence of progression.

Article Snippet: The human SW48 (catalogue number: HTL99020) ( KRAS, NRAS, BRAF and PIK3CA WT), colon cancer cell line was obtained from IRCCS “Azienda Ospedaliera Universitaria San Martino-IST Istituto Nazionale per la Ricerca sul Cancro, Genova” Italy.

Techniques: Injection, Control

Journal: Oncotarget

Article Title: Therapeutic efficacy of SYM004, a mixture of two anti-EGFR antibodies in human colorectal cancer with acquired resistance to cetuximab and MET activation

doi: 10.18632/oncotarget.18749

Figure Lengend Snippet: (A-B) SW48 cells were injected s.c. into the right flank of seven nude mice. After two weeks mice were treated with Cetuximab (1 mg twice a week) by i.p. injection. Treatment was continued until disease progression. The black arrows indicate the time of progression to cetuximab. At progression phase mice were assigned to SYM004 treatment (50 mg/Kg twice a week) by i.p. injection. The treatment was continued until 30 weeks. At week 30 four out of seven mice were still responding to SYM004 (as indicated by double asterisk). Abbreviations: PD, progression disease; PR, partial response; SD, stable disease.

Article Snippet: The human SW48 (catalogue number: HTL99020) ( KRAS, NRAS, BRAF and PIK3CA WT), colon cancer cell line was obtained from IRCCS “Azienda Ospedaliera Universitaria San Martino-IST Istituto Nazionale per la Ricerca sul Cancro, Genova” Italy.

Techniques: Injection, Biomarker Discovery

Journal: JNCI Journal of the National Cancer Institute

Article Title: Role of Reactive Oxygen Species in the Abrogation of Oxaliplatin Activity by Cetuximab in Colorectal Cancer

doi: 10.1093/jnci/djv394

Figure Lengend Snippet: Combination of cetuximab and oxaliplatin in vitro. A and B ) Thiazolyl blue tetrazolium bromide (MTT) assay assessed the effect of a fixed concentration of cetuximab (IC 20 1.4 μg/mL) with oxaliplatin (1-2-3-5 μM) or SN-38 (0.01–1 μM) on growth inhibition at 72 hours in SW48 cells. Results are presented as mean ± SD of three independent experiments. Statistical significance was calculated by two-tailed Student’s t test (* P < .05, † P < .01, and ‡ P < .001). Combination index calculations were performed by Calcusyn software. Values of 1 indicate additivity; values greater than 1 indicate antagonism and of less than 1 indicate synergism. C ) H2AX phosphorylation was detected following continuous treatment of 50 μM oxaliplatin (3 hours) or 1 μM SN-38 (1 hour) in combination with 100 μg/mL cetuximab in SW48 cells. Protein samples were collected post-treatment at the indicated times, and calnexin was used as a loading control. The result of three independent experiments is presented. D ) SW48 and DLD-1 cells were treated with oxaliplatin (50 μM) or SN-38 (1 μM) and cetuximab (100 μg/mL) and with oxaliplatin (100 μM) or SN-38 (1 μM) and cetuximab (100 μg/mL) for 18 hours, respectively. β-Actin was used as loading control. The result of three independent experiments is presented. E and F ) Apoptosis was measured by caspase 3/7 activity. SW48 cells were treated with oxaliplatin (50 μM), SN-38 (1 μM), or their combination with cetuximab (100 μg/mL) for 18 hours. Isotoxic concentrations (IC 75 ) of oxaliplatin (100 μM) and SN-38 (1 μM) were used for the DLD-1 cells. Results are presented as fold-increase to untreated samples and as mean ± SD of three independent experiments. Statistical significance was calculated by two-tailed Student’s t test (* P < .05, † P < .01, and ‡ P < .001).

Article Snippet: Merck Serono (Darmstadt) provided the SW48 cell line, and Bert Vogelstein (Johns Hopkins University) the DLD-1 isogenic KRAS wild-type cell line.

Techniques: In Vitro, MTT Assay, Concentration Assay, Inhibition, Two Tailed Test, Software, Phospho-proteomics, Control, Activity Assay

Journal: JNCI Journal of the National Cancer Institute

Article Title: Role of Reactive Oxygen Species in the Abrogation of Oxaliplatin Activity by Cetuximab in Colorectal Cancer

doi: 10.1093/jnci/djv394

Figure Lengend Snippet: Cetuximab and oxaliplatin treatment effects on cellular reactive oxygen species (ROS) levels. A ) SW48 cells were treated with oxaliplatin (10–100 μM) or SN-38 (0.01–1 μM) for one hour. ROS were detected with H 2 DCFDA reagent and flow cytometry analysis. Results are presented as fold-increase in mean fluorescence intensity normalized to untreated and mean ± SD (n = 3). Symbols indicate statistical significance (two-tailed Student’s t test; * P < .05, † P < .01, and ‡ P < .001). B ) Caspase 3/7 activity detected apoptosis following 24 hours’ continuous treatment of oxaliplatin (50 μM), SN38 (1 μM), or their combination with the antioxidants NAC (1mM) or ascorbic acid (100 μM) in the SW48 cell line and oxaliplatin (100 μM) or SN-38 (1 µM) with ascorbic acid (100 µM) in DLD-1 cells. Results are presented as fold-increase to untreated sample and are shown as mean ± SD (n = 3). Statistical analysis conducted as above. C and D ) Levels of ROS were measured as described in (A) . SW48 and DLD-1 cells were treated with 50 μM or 100 μM oxaliplatin, respectively, SN-38 (1 µM), cetuximab (100 μg/mL), or gefitinib (1 µM) for one hour. Results are presented as fold-increase in mean fluorescence intensity normalized to untreated control. Each experiment was repeated three times, and results are shown as mean ± SD. Symbols indicate statistical significance (* P < .05, † P < .01, and ‡ P < .001), and two-tailed Student’s t test was used for analysis. E ) SW48 and DLD-1 cells were treated as in (C and D) , and samples processed by immunoblotting calnexin were used as a loading control. The result of three independent experiments is presented.

Article Snippet: Merck Serono (Darmstadt) provided the SW48 cell line, and Bert Vogelstein (Johns Hopkins University) the DLD-1 isogenic KRAS wild-type cell line.

Techniques: Flow Cytometry, Fluorescence, Two Tailed Test, Activity Assay, Control, Western Blot

Journal: JNCI Journal of the National Cancer Institute

Article Title: Role of Reactive Oxygen Species in the Abrogation of Oxaliplatin Activity by Cetuximab in Colorectal Cancer

doi: 10.1093/jnci/djv394

Figure Lengend Snippet: In vitro and in vivo expression of DUOX2 following oxaliplatin and cetuximab treatment. A ) SW48 cells were treated with cetuximab (100 μg/mL), gefitinib (1 μM), oxaliplatin (50 μM), or their combination for six hours. DLD-1 cells were treated with cetuximab (100 μg/mL), gefitinib (1 μM), and oxaliplatin (100 μM). mRNA levels of DUOX2 were measured by real-time polymerase chain reaction (RT-PCR). Results (2 -ΔΔCT ) are presented as mean ± SD of three independent experiments. GADPH was used as endogenous control and untreated sample as calibrator. Statistical significance was calculated by two-tailed Student’s t test (* P < .05, † P < .01, and ‡ P < .001). B ) SW48 and DLD-1 cells were treated in the same conditions described in (A) and sample processed by immunoblotting. Calnexin was used as a loading control. The result of three independent experiments is presented. C ) 5 x 10 6 DLD-1 cells were injected into a single site on the flank of nude mice (nu/nu). Mice were assigned randomly to one of four experimental groups: control and n = 3 for each treatment group and drug treated for 24 hours by intraperitoneal injection with control (saline), cetuximab (30mg/kg in saline), oxaliplatin (8mg/kg in saline), oxaliplatin + cetuximab (as before). mRNA for DUOX2 (C, left panel) and DUOXA2 (C, right panel) was determined using real-time polymerase chain reaction and results expressed as a fold-change (mean ± SD) normalized to untreated control. Xenograft serial sections (5 μM) were prepared and DUOX2 in situ hybridization (ISH) (D) , and DUOX2 immunohistochemistry (IHC) (E) was conducted. F ) Representative images at 40x magnification resulting from ISH and IHC analysis ( scale bars = 50 µM). Results are expressed as mean ± SD. Xenograft data was normally distributed according to Shapiro-Wilks testing, except IHC, where data was log 10 -transformed and then determined to be normally distributed. Statistics was performed using two-tailed Student’s t test with Welch’s correction.

Article Snippet: Merck Serono (Darmstadt) provided the SW48 cell line, and Bert Vogelstein (Johns Hopkins University) the DLD-1 isogenic KRAS wild-type cell line.

Techniques: In Vitro, In Vivo, Expressing, Real-time Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Control, Two Tailed Test, Western Blot, Injection, Saline, In Situ Hybridization, Immunohistochemistry, Transformation Assay

Journal: JNCI Journal of the National Cancer Institute

Article Title: Role of Reactive Oxygen Species in the Abrogation of Oxaliplatin Activity by Cetuximab in Colorectal Cancer

doi: 10.1093/jnci/djv394

Figure Lengend Snippet: Regulation of DUOX2 transcription by oxaliplatin and cetuximab. A ) SW48 and DLD-1 cells were treated with 50 μM or 100 μM oxaliplatin, respectively, in combination with cetuximab (100 μg/mL) for one hour. β-actin and calnexin were used as loading controls. The result of three independent experiments is presented. B ) Following 72 hours’ transfection with scrambled siRNA or STAT1 siRNA (50nM), the SW48 cells were treated with oxaliplatin (50 μM) for six hours. β-actin was used as a loading control. The result of three independent experiments is presented. C and D ) Following three hours’ oxaliplatin treatment (50 μM and 100 μM in SW48 and DLD-1 cells, respectively), binding of STAT1 to the DUOX2 promoter was measured by chromatin immunoprecipitation. Rabbit IgG was used as negative control. Values are normalized to INPUT samples and presented as % input. Each experiment was repeated in triplicates, and results are presented as mean ± SD. Statistical analysis was performed with two-way analysis of variance (ANOVA) and Bonferroni post-test. Correction for multiple comparisons was applied for STAT1 IP-oxaliplatin vs STAT1 IP-untreated and rabbit IgG-oxaliplatin vs rabbit Ig-untreated for both cell lines (* P < .05, † P < .01, and ‡ P < .001). E ) Following 72 hours’ transfection (50nM) with STAT1 siRNA, the SW48 and DLD-1 cells were treated with 50 μM or 100 μM oxaliplatin, respectively, for 18 hours. Levels of PARP cleavage measured apoptosis and β-Actin was used as a loading control. The experiment presented is representative of three independent experiments. F ) Caspase 3/7 activity was measured in the same experimental conditions in (E) . Results are presented as mean ± SD of three independent experiments. Statistical analysis was performed with two-way ANOVA and Bonferroni post-test. Correction for multiple comparisons was applied for scrambled vs scrambled-oxaliplatin, scrambled vs STAT1 siRNA, scrambled vs scrambled-oxaliplatin, scrambled-oxaliplatin vs STAT1 siRNA, and scrambled-oxaliplatin vs STAT1 siRNA-oxaliplatin.

Article Snippet: Merck Serono (Darmstadt) provided the SW48 cell line, and Bert Vogelstein (Johns Hopkins University) the DLD-1 isogenic KRAS wild-type cell line.

Techniques: Transfection, Control, Binding Assay, Chromatin Immunoprecipitation, Negative Control, Activity Assay

Journal: JNCI Journal of the National Cancer Institute

Article Title: Role of Reactive Oxygen Species in the Abrogation of Oxaliplatin Activity by Cetuximab in Colorectal Cancer

doi: 10.1093/jnci/djv394

Figure Lengend Snippet: Role of p38 and ERK kinases in oxaliplatin-induced apoptosis. A ) Activation of p38 and ERK1/2 was assessed by immunoblotting in the SW48 cells treated with oxaliplatin (50 μM), cetuximab (100 μg/mL), and the antioxidants NAC (1mM) or ascorbic acid (AA) (100 μM) for 24 hours. Calnexin was used as a loading control. The result of three independent experiments is presented. B ) SW48 cells were treated with oxaliplatin (50 μM), SB202190 (0.1-1-10 μM), or pimasertib (0.1-1-10 μM) for 24 hours. Viable cells (%) were measured by the Cell Titre Glo assay (Promega), and treated samples were normalized to untreated control (mean ± SD; n = 3). Statistical significance was measured by two-tailed Student’s t test (* P < .05, † P < .01, and ‡ P < .001). C ) Treatment conditions of (B) were used. Caspase 3/7 activity in treated samples is normalized to untreated control and presented as fold-increase. Results are presented as mean ± SD (n = 3), and statistical analysis was performed as in (B) . D ) Apoptosis was assessed by immunoblotting of PARP cleavage. The SW48 and DLD-1 cell lines were treated with 50 μM or 100 μM oxaliplatin, respectively, in combination with SB202190 (10 μM) or pimasertib (10 μM). Calnexin was used as a loading control. The result presented is representative of three independent experiments. E ) SW48 and DLD-1 cells were transfected with scrambled or p38 siRNA (50nM and 100nM, respectively). After 48 hours’ siRNA transfection, cells were treated for 24 hours with 50 μM (SW48) or 100 μM (DLD-1) oxaliplatin. Calnexin was used as loading control. The results presented are representative of three independent experiments.

Article Snippet: Merck Serono (Darmstadt) provided the SW48 cell line, and Bert Vogelstein (Johns Hopkins University) the DLD-1 isogenic KRAS wild-type cell line.

Techniques: Activation Assay, Western Blot, Control, Glo Assay, Two Tailed Test, Activity Assay, Transfection