kras g12c mutant human lung cancer cell line nci h358 cells  (ATCC)

Journal: ACS Chemical Biology

Article Title: Distal Covalent Targeting Suppresses Signaling of Oncogenic K‑Ras(G13C) in Cancer Cells

doi: 10.1021/acschembio.5c00249

Figure Lengend Snippet: (A) Alignment of K-Ras­(G13C) to adagrasib-bound K-Ras­(G12C). (B) Modular design of shift-register electrophiles to repurpose K-Ras­(G12C) covalent inhibitors for the G13C mutation. (C) Proximity scanning of Cys13 accessibility using unbranched alkyl linkers. (D) Labeling efficiency of G13C-targeting covalent inhibitors with various linker lengths. Conditions: K-Ras­(G13C)•GDP or •GppNHp (1 μM), compound (100 μM), RT incubation. Each box represents the mean of two independent runs. (E) Schematic summary of an expanded linker screening campaign. (F) Lead 1,3-disubstituted aromatic linked compounds and their covalent modification kinetics. Conditions: K-Ras­(G13C)•GDP or •GppNHp (200 nM), compound (10 μM), RT incubation.

Article Snippet: HCT116/KRAS G13C heterozygous and homozygous (SL753) cancer biomarker mutant cell lines were obtained from GeneCopoeia, Inc. (Rockville, MD).

Techniques: Mutagenesis, Labeling, Incubation, Modification

Journal: ACS Chemical Biology

Article Title: Distal Covalent Targeting Suppresses Signaling of Oncogenic K‑Ras(G13C) in Cancer Cells

doi: 10.1021/acschembio.5c00249

Figure Lengend Snippet: (A) Chemical structure of lead compound G13Ci-22 . (B) Aligned ligand poses of G13Ci-22 (CovDock) and MRTX1133 (PDB Entry 7RPZ ) in the corresponding Switch-II Pocket. (C) Covalent modification kinetics of K-Ras­(G13C) by inhibitors with the optimal 3-acrylamidobenzene carbonyl linker and alternative Switch-II Pocket binding moieties. Conditions: K-Ras­(G13C)•GDP or •GppNHp (200 nM), Compound (10 μL), RT incubation. (D) Chemical structures of the alternative Switch-II Pocket binding moieties. The optimal linker and Cys-warhead, same with those in G13Ci-22 , were omitted in the chemical structures.

Article Snippet: HCT116/KRAS G13C heterozygous and homozygous (SL753) cancer biomarker mutant cell lines were obtained from GeneCopoeia, Inc. (Rockville, MD).

Techniques: Modification, Binding Assay, Incubation

Journal: ACS Chemical Biology

Article Title: Distal Covalent Targeting Suppresses Signaling of Oncogenic K‑Ras(G13C) in Cancer Cells

doi: 10.1021/acschembio.5c00249

Figure Lengend Snippet: (A) Mutant selectivity of G13Ci-22 against relevant cyslight K-Ras proteins. (B) Thermal stabilization of K-Ras­(G13C) proteins due to covalent modification by G13Ci-22 . (C) Schematic description of guanine nucleotide cycles of K-Ras, K-Ras­(G13C), and covalently inhibited K-Ras­(G13C) GTPases. G13Ci-22 inhibited Ras-RafRBD binding (D), GTPase activity (E), and nucleotide exchange (F) of K-Ras­(G13C) oncoprotein.

Article Snippet: HCT116/KRAS G13C heterozygous and homozygous (SL753) cancer biomarker mutant cell lines were obtained from GeneCopoeia, Inc. (Rockville, MD).

Techniques: Mutagenesis, Modification, Binding Assay, Activity Assay

Journal: ACS Chemical Biology

Article Title: Distal Covalent Targeting Suppresses Signaling of Oncogenic K‑Ras(G13C) in Cancer Cells

doi: 10.1021/acschembio.5c00249

Figure Lengend Snippet: (A) Immunoblot of HCT116 cell and its genetically engineered derivative cells treated with G13Ci-22 at various concentrations. (B) Time-course immunoblot of the homozygous HCT116 (K-Ras­(G13C/G13C)) cell treated with 10 nM of G13Ci-22 . (C) Immunoblot of human cancer cell lines NCI-H1355 and NCI-H1734, which harbor K-Ras­(G13C) mutation, treated with G13Ci-22 .

Article Snippet: HCT116/KRAS G13C heterozygous and homozygous (SL753) cancer biomarker mutant cell lines were obtained from GeneCopoeia, Inc. (Rockville, MD).

Techniques: Western Blot, Mutagenesis

Journal: Journal of Clinical Oncology

Article Title: Onvansertib in Combination With Chemotherapy and Bevacizumab in Second-Line Treatment of KRAS -Mutant Metastatic Colorectal Cancer: A Single-Arm, Phase II Trial

doi: 10.1200/JCO-24-01266

Figure Lengend Snippet: PLK1 regulates the hypoxia pathway, related to . (A, B) KRAS -mutant CRC cells were treated with DMSO or Onv at the indicated doses for 20 hours and then exposed to hypoxia or kept in normoxia for 4 hours. (A) Heatmap of hypoxia-related genes significantly regulated by Onv in HCT116 and SW620 cells based on the RNA-seq analysis. (B) Expression of hypoxia-related genes in LoVo and DLD-1 cells, assessed by RT-qPCR and normalized to the housekeeping gene RPLP0 . Bar graphs represent expression relative to Normoxia_DMSO sample. (C, D) SW620 and HCT116 cells were transfected with nontargeting control siRNA (siNTC) or PLK1 targeting siRNA (siPLK1) for 20 hours and then exposed to hypoxia for 4 hours. (C) Left: Simple Western images of PLK1, HIF1α, and β-actin. Right: HIF1α and PLK1 protein expression normalized to β-actin. (D) Expression of hypoxia-related genes assessed by RT-qPCR and normalized to the housekeeping gene RPLP0 . Bar graphs represent expression relative to Normoxia_siNTC. (B-D) Data are shown as mean ± SEM of at least three independent biological replicates. HIF1α, hypoxia-inducible factor 1α; Hx, hypoxia; Nx, normoxia; Onv, onvansertib; PLK1, polo-like kinase 1; RT-qPCR, real-time quantitative polymerase chain reaction.

Article Snippet: Human KRAS -mutant colorectal cancer cell lines HCT116, SW620, LoVo, and DLD-1 (ATCC, Manassas, VA) were cultured in RPMI (Cat# 30-2001, ATCC) supplemented with 10% FBS (Cat#16000069, ThermoFisher Scientific, Waltham, MA) and 1× penicillin-streptomycin solution (Cat#30-2300, ATCC).

Techniques: Mutagenesis, RNA Sequencing, Expressing, Quantitative RT-PCR, Transfection, Control, Simple Western, Real-time Polymerase Chain Reaction

Journal: Cancer Science

Article Title: A Mutual Interaction Between GSTP1 and p53 Improves the Drug Resistance and Malignant Biology of Pancreatic Cancer

doi: 10.1111/cas.70019

Figure Lengend Snippet: The expression of GSTP1 and p53 in PC specimens. (A) Low and high GSTP1 expression in two normal pancreas samples. (B) Low and high GSTP1 expression in two PC samples. (C) Low and high p53 expression in two PC samples. (D) High GSTP1 and mtp53 expression in one PC sample. (E) Low GSTP1 and mtp53 expression in one PC sample. (F) High GSTP1 and wtp53 expression in one serial section of PC tissue. (G) Kaplan–Meier analysis of high and low expression of GSTP1 in PC patients. (H) Kaplan–Meier analysis of Mtp53 and wtp53 expression in PC patients. (I) Kaplan–Meier analysis of high and low expression of GSTP1 in wtp53 PC patients.

Article Snippet: The wtp53 Capan‐2 (Kras mutant) cell line was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) [ ].

Techniques: Expressing

Journal: Cancer Science

Article Title: A Mutual Interaction Between GSTP1 and p53 Improves the Drug Resistance and Malignant Biology of Pancreatic Cancer

doi: 10.1111/cas.70019

Figure Lengend Snippet: The clinical significance of GSTP1 expression in 40 cases of human PC samples containing wildtype p53.

Article Snippet: The wtp53 Capan‐2 (Kras mutant) cell line was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) [ ].

Techniques: Expressing

Journal: Cancer Science

Article Title: A Mutual Interaction Between GSTP1 and p53 Improves the Drug Resistance and Malignant Biology of Pancreatic Cancer

doi: 10.1111/cas.70019

Figure Lengend Snippet: The relationship between GSTP1 and p53 in vitro. (A) GSTP1 and p53 protein expression in six PC cell lines. (B) GSTP1 mRNA expression in six PC cell lines. (C) GSTP1 and p53 expression in GSTP1‐ or p53‐silenced mtp53 BxPC‐3 cells. (D) GSTP1 and p53 expression in GSTP1‐ or p53‐silenced mtp53 PANC‐1 cells. (E) GSTP1 and p53 expression in GSTP1‐ or p53‐silenced wtp53 Capan‐2 cells. (F) GSTP1 and p53 expression in the GFP, GSTP1‐GFP, GSTP1‐GFP plus si‐p53, and si‐p53 groups of SW1990 cells with wtp53. 1: sictrl group; 2: si1‐GSTP1 group; 3: si2‐GSTP1 group; 4: si‐p53 group; 5: GFP group; 6: GSTP1‐GFP group; 7: GSTP1‐GFP + si‐p53 group; 8: Si‐p53 group. The experiments were performed in triplicate. Error bars ± SDs. * p < 0.05 and ** p < 0.01 compared with the control.

Article Snippet: The wtp53 Capan‐2 (Kras mutant) cell line was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) [ ].

Techniques: In Vitro, Expressing, Control

Journal: Cancer Science

Article Title: A Mutual Interaction Between GSTP1 and p53 Improves the Drug Resistance and Malignant Biology of Pancreatic Cancer

doi: 10.1111/cas.70019

Figure Lengend Snippet: GSTP1 inhibited cell proliferation and drug resistance in a wtp53‐dependent manner in vitro. (A) MTT assays in si1‐GSTP1 and sictrl transfected wtp53 Capan‐2 cells cultured within 4 days. (B) MTT assays of GFP, GSTP1‐GFP and GSTP1‐GFP plus si‐p53 transfected SW1990 cells with wtp53 cultured within 4 days. (C, D) MTT assays in si1‐GSTP1 and sictrl transfected wtp53 Capan‐2 cells under OXA (C) or GEM (D) treatment for 2 days. (E, F) MTT assays in GFP, GSTP1‐GFP and GSTP1‐GFP plus si‐p53 transfected SW1990 cells with wtp53 under OXA (E) or GEM (F) treatment for 2 days. The experiments were performed in triplicate. Error bars ± SDs. * p < 0.05 and ** p < 0.01 compared with the control.

Article Snippet: The wtp53 Capan‐2 (Kras mutant) cell line was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) [ ].

Techniques: In Vitro, Transfection, Cell Culture, Control

Journal: Cancer Science

Article Title: A Mutual Interaction Between GSTP1 and p53 Improves the Drug Resistance and Malignant Biology of Pancreatic Cancer

doi: 10.1111/cas.70019

Figure Lengend Snippet: GSTP1 regulated cell proliferation and drug resistance via p53/p21‐ and Bax/Bcl2‐mediated signaling pathways. (A, B) p53/p21 and Bax/Bcl2 protein expression in si1‐GSTP1 and sictrl transfected wtp53 Capan‐2 cells with or without OXA (A) or GEM (B) treatment for 2 days. (C, D) p53/p21 and Bax/Bcl2 protein expression in GFP and GSTP1‐GFP transfected SW1990 cells with wtp53 with or without OXA (C) or GEM (D) treatment for 2 days. 1: Sictrl group; 2: si1‐GSTP1 group; 3: Sictrl with OXA (A) or GEM (B) treatment group; 4: si1‐GSTP1 with OXA (A) or GEM (B) treatment group; 5: GFP group; 6: GSTP1‐GFP group; 7: GFP with OXA (C) or GEM (D) treatment group; 8: GSTP1‐GFP with OXA (C) or GEM (D) treatment group. The experiments were performed in triplicate. Error bars ± SDs. * p < 0.05 and ** p < 0.01 compared with the control.

Article Snippet: The wtp53 Capan‐2 (Kras mutant) cell line was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) [ ].

Techniques: Protein-Protein interactions, Expressing, Transfection, Control

Journal: Cancer Science

Article Title: A Mutual Interaction Between GSTP1 and p53 Improves the Drug Resistance and Malignant Biology of Pancreatic Cancer

doi: 10.1111/cas.70019

Figure Lengend Snippet: GSTP1 mediated cell invasion and migration via partially regulating wtp53 mediated EMT signaling. (A) Cell invasion and migration in sictrl, si1‐GSTP1 and si2‐GSTP1 transfected wtp53 Capan‐2 cells. (B) Cell invasion and migration of GFP, GSTP1‐GFP and GSTP1‐GFP plus si‐p53 transfected SW1990 cells with wtp53. (C) Protein expression of EMT epithelial and mesenchymal biomarkers in sictrl, si1‐GSTP1 and si2‐GSTP1 transfected wtp53 Capan‐2 cells. (D) Protein expression of EMT epithelial and mesenchymal biomarkers in GFP and GSTP1‐GFP transfected SW1990 cells with wtp53. 1: Sictrl group; 2: si1‐GSTP1 group; 3: si2‐GSTP1 group; 4: GFP group; 5: GSTP1‐GFP group. The experiments were performed in triplicate. Error bars ± SDs. * p < 0.05 and ** p < 0.01 compared with the control.

Article Snippet: The wtp53 Capan‐2 (Kras mutant) cell line was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) [ ].

Techniques: Migration, Transfection, Expressing, Control

Journal: Cancer Science

Article Title: A Mutual Interaction Between GSTP1 and p53 Improves the Drug Resistance and Malignant Biology of Pancreatic Cancer

doi: 10.1111/cas.70019

Figure Lengend Snippet: The feedback regulation of GSTP1 and wtp53 mediated the malignant progression of PC in vitro. (A) GSTP1 was not coimmunoprecipitated with p53 in mtp53 PANC‐1 or wtp53 Capan‐2 cells. (B) P53 protein could bind to the GSTP1 DNA promoter in wtp53 Capan‐2 or SW1990 cells. (C) P53 protein could not bind to the GSTP1 DNA promoter in mtp53 BxPC‐3 or PANC‐1 cells. (D) IF staining of p53 (TRITC, red) and nuclei (Hoechst, blue) in si1‐GSTP1 and sictrl transfected wtp53 Capan‐2 cells. (E) IF staining of p53 (TRITC, red) and nuclei (Hoechst, blue) in GFP and GSTP1‐GFP transfected SW1990 cells with wtp53. (F) IF staining of p53 (TRITC, red) and nuclei (Hoechst, blue) in si1‐GSTP1 and sictrl transfected mtp53 BxPC‐3 cells. (G) IF staining of p53 (TRITC, red) and nuclei (Hoechst, blue) in si1‐GSTP1 and sictrl transfected mtp53 PANC‐1 cells.

Article Snippet: The wtp53 Capan‐2 (Kras mutant) cell line was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) [ ].

Techniques: In Vitro, Staining, Transfection

Journal: Cancer Science

Article Title: A Mutual Interaction Between GSTP1 and p53 Improves the Drug Resistance and Malignant Biology of Pancreatic Cancer

doi: 10.1111/cas.70019

Figure Lengend Snippet: GSTP1 inhibited tumor growth in coordination with wtp53 in vivo. (A) Subcutaneous tumor volumes in GFP and GSTP1‐GFP transfected SW1990 cells with wtp53 in 5 nude mice. (B) HE staining of the subcutaneous tumor. (C) GSTP1 and p53 expression in subcutaneous tumors in the GFP and GSTP1‐GFP groups, as determined by WB. (D) ki67 expression in the subcutaneous tumors of the GFP and GSTP1‐GFP groups was compared via IHC. (E) Liver metastases in GFP and GSTP1‐GFP transfected SW1990 cells with wtp53 in 5 nude mice. (F) HE staining of liver metastases. (G) Representative images from the IHC assays of GSTP1 and p53 protein expression in the GFP and GSTP1‐GFP groups in vivo. (H) Statistical data from the IHC assays of GSTP1 and p53 protein expression in the GFP and GSTP1‐GFP groups in vivo. Error bars ± SDs. * p < 0.05 and ** p < 0.01 compared with the control.

Article Snippet: The wtp53 Capan‐2 (Kras mutant) cell line was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) [ ].

Techniques: In Vivo, Transfection, Staining, Expressing, Control