Journal: Cancers

Article Title: Polo-like Kinase 1 Inhibitors Demonstrate In Vitro and In Vivo Efficacy in Preclinical Models of Small Cell Lung Cancer

doi: 10.3390/cancers17030446

Figure Lengend Snippet: Assessment of in vitro antiproliferative activity of targeted agents in a panel of SCLC cell lines ( a ); effect of volasertib ( b ) and onvansertib ( c ) on proliferation of SCLC cell lines. Cells were treated for 72 h with indicated agents. Cell proliferation was determined using colorimetric or luminescent assays depending on the degree of clustering of SCLC cell lines in culture. Values represent the mean ± S.D. from a minimum of 3 independent experiments. Blue and orange curves define cell lines with non-disruptive and disruptive p53 mutations, respectively. Basal protein expression in SCLC cell lines ( d ). SCLC subtype based on expression are indicated after each cell line: ASCL1 (A), POU2F3 (P), YAP1 (Y).

Article Snippet: We accessed the gene expression data of human SCLC cell lines previously generated on the Illumina HT-12 platform and deposited in the NCBI Gene Expression Omnibus (GEO accession GSE55830) [ ].

Techniques: In Vitro, Activity Assay, Expressing

Journal: Cancers

Article Title: Polo-like Kinase 1 Inhibitors Demonstrate In Vitro and In Vivo Efficacy in Preclinical Models of Small Cell Lung Cancer

doi: 10.3390/cancers17030446

Figure Lengend Snippet: SCLC cell line sensitivity to PLK1 inhibitors measured by IC 50 concentration.

Article Snippet: We accessed the gene expression data of human SCLC cell lines previously generated on the Illumina HT-12 platform and deposited in the NCBI Gene Expression Omnibus (GEO accession GSE55830) [ ].

Techniques: Concentration Assay, Mutagenesis

Journal: Cancers

Article Title: Polo-like Kinase 1 Inhibitors Demonstrate In Vitro and In Vivo Efficacy in Preclinical Models of Small Cell Lung Cancer

doi: 10.3390/cancers17030446

Figure Lengend Snippet: Effect of TP53 mutational status on PLK1 inhibitor sensitivity. Comparison of mean IC 50 to TP53 gene mutation status ( a ). TP53 gene status in 166 tumor samples in cbioportal.org ( b ) and 50 SCLC cell lines from publicly available CCLE data ( c ). Activity of PLK1 inhibitor onvansertib in parental and resistant H526 cells (IC 50 concentration in the resistant vs. parent: 447 nM vs. 51 nM) ( d ). Gene expression profiles of matched parental and PLK1 inhibitor resistant H526 cells from 3 separate samples ( e ). Heatmap shows the top differential gene expression ( p -adj < 0.5; logFC > 4 cut-off) with red indicating high and blue indicating low natural log-transformed expression.

Article Snippet: We accessed the gene expression data of human SCLC cell lines previously generated on the Illumina HT-12 platform and deposited in the NCBI Gene Expression Omnibus (GEO accession GSE55830) [ ].

Techniques: Comparison, Mutagenesis, Activity Assay, Concentration Assay, Gene Expression, Transformation Assay, Expressing

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: De novo and histologically transformed small cell lung cancer is sensitive to lurbinectedin treatment through the modulation of EMT and NOTCH signaling pathways

doi: 10.1158/1078-0432.CCR-23-0471

Figure Lengend Snippet: A. Cell viability IC50 values in 14 human and 3 murine SCLC cell lines in response to 24 hrs treatment indicates high susceptibility of SCLC cells to lurbinectedin. B. Flow cytometry analysis of apoptotic induction showing percentage of apoptotic cell in different SCLC cell lines in response to respective IC50 concentration of lurbinectedin for 24hr and 48 hrs treatment. Grey color: Control, Blue: 24 hrs lurbinectedin treatment, Orange: 48 hrs lurbinectedin treatment. Results shown as mean ± SD. P values were calculated by Student’s t test (*p < 0.05, **p < 0.01, ***p < 0.001) C. Spearman correlation showing genes associated with sensitivity and resistance in response to lurbinectedin treatment comparing the gene signature with previously published gene signature of SCLC cell lines.

Article Snippet: Cell lines and cell cultures Human SCLC cell lines were obtained from the American Type Culture Collection (ATCC) and the European Collection of Authenticated Cell Cultures (ECACC).

Techniques: Flow Cytometry, Concentration Assay, Control

Journal: Scientific Reports

Article Title: Dynamic phenotypic reprogramming and chemoresistance induced by lung fibroblasts in small cell lung cancer

doi: 10.1038/s41598-024-52687-z

Figure Lengend Snippet: Fibroblast-derived IL-6 promoted phenotypic reprogramming of SCLC cells. ( a , b ) H69 and H2227 cells co-cultured with MRC-5 cells ( a ) or HFL1 ( b ) respectively and the expression of NE genes in H69 and H2227 cells was measured by qRT-PCR (n = 3); ( c ) The protein expression of NE markers was determined by Western blot analysis and the blots were cropped prior to hybridisation with antibodies, original images were displayed in . ( d , e ) Antibody arrays were performed in serum-free culture medium from separately cultured or co-cultured H2227 and MRC-5 cells, membranes were scanned by chemiluminescence instrument ( d ) and quantified by image J software, the levels of cytokines were normalized to positive controls ( e ); ( f ) The expression of NE genes in H2227 cells cultured separately or co-cultured with MRC-5 cells which transfected with siRNA targeted to IL6, CCL20 or IGFBP1 or negative control were determined by qRT-PCR (n = 3); ( g ) Western blot was used to analyze the expression of NE markers in H69 and H2227 cells which cultured separately or co-cultured with MRC-5 cells which transfected with siRNA targeted to IL6 or negative control, the blots were cropped prior to hybridisation with antibodies, original images were displayed in . Quantification of blots were performed using Image J software (n = 2). * P < 0.05, ** P < 0.01.

Article Snippet: Human SCLC cell lines (H69, H2227, H1092, H446, 21H, H196 and SBC-5) were obtained from National collection of Authenticated cell cultures (Shanghai, China) or Procell Life Science and Technology company (Wuhan, Hubei, China) in 2021.

Techniques: Derivative Assay, Cell Culture, Expressing, Quantitative RT-PCR, Western Blot, Hybridization, Software, Transfection, Negative Control

Journal: Scientific Reports

Article Title: Dynamic phenotypic reprogramming and chemoresistance induced by lung fibroblasts in small cell lung cancer

doi: 10.1038/s41598-024-52687-z

Figure Lengend Snippet: Evaluation and validation of drug sensitivity upon fibroblasts stimulation in SCLC. ( a , b ) The log fold change of predicted sensitivity scores of drugs based on GDSC2 database between high- and low-infiltration group in George’s cohort, Log FC > 0 means high-infiltration group is more resistant to these drugs (blue bars), Log FC < 0 represents high-infiltration group is more sensitive to these drugs (orange bars); ( c , d ) NE type SCLC cells were separately cultured or co-cultured with MRC-5 for 7 days, then reseeded and treated with cisplatin or etoposide for another 48 h, cell viability was detected by CCK-8 reagent (n = 3). ( e , f ) CCK-8 reagent was used to test the viability of separately cultured or co-cultured H69 and H2227 cells which pretreated with or without AZD-1480 and exposed to Cisplatin ( e ) or Etoposide ( f ). * or #, P < 0.05, ** or ##, P < 0.01. *, Co-Veh vs. Mo-Veh. #, Co-AZD vs. Mo-AZD.

Article Snippet: Human SCLC cell lines (H69, H2227, H1092, H446, 21H, H196 and SBC-5) were obtained from National collection of Authenticated cell cultures (Shanghai, China) or Procell Life Science and Technology company (Wuhan, Hubei, China) in 2021.

Techniques: Biomarker Discovery, Cell Culture, CCK-8 Assay

Journal: Translational Cancer Research

Article Title: PTPMT1 inhibition induces apoptosis and growth arrest of human SCLC cells by disrupting mitochondrial metabolism

doi: 10.21037/tcr-2024-2379

Figure Lengend Snippet: PTPMT1 knockdown reduces cell growth in A549 cells. PTPMT1 mRNA expression (A) and protein level (B) were significantly decreased in PTPMT1-shRNA-transfected cells. (C) CCK-8 assay for cell proliferation of PTPMT1-shRNA-transduced A549. (D) CCK-8 assay for cell proliferation of PTPMT1-shRNA-transduced H69 cells. ***, P<0.001, vs . scramble (control). PTPMT1, protein tyrosine phosphatase mitochondrial 1; mRNA, messenger RNA; CCK-8, cell counting kit 8; shRNA, short-hairpin RNA.

Article Snippet: NCI-H69 (a human SCLC cell line) was purchased from Procell (Wuhan, China) and cultured in 1640 medium (Gibco, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco).

Techniques: Knockdown, Expressing, shRNA, Transfection, CCK-8 Assay, Control, Cell Counting

Journal: Translational Cancer Research

Article Title: PTPMT1 inhibition induces apoptosis and growth arrest of human SCLC cells by disrupting mitochondrial metabolism

doi: 10.21037/tcr-2024-2379

Figure Lengend Snippet: PTPMT1 inhibition induces cell death in A549 cells. (A,B) The percentage of apoptosis in the A549 and H69 cells transduced with PTPMT1-shRNA or control shRNA assessed by annexin-V assay and flow cytometry. ***, P<0.0001, sh-PTPMT1 vs . scramble (control). (C) The cell viability of A549 cells treated with alexidine dihydrochloride in the presence of Fer-1. (D) The cell viability of H69 cells treated with alexidine dihydrochloride in the presence of Fer-1. ns, no significance; **, P<0.01, alexidine dihydrochloride 2.5 µM vs . control (DMSO); ***, P<0.001, alexidine dihydrochloride 5 µM/10 µM vs . control (DMSO). PTPMT1, protein tyrosine phosphatase mitochondrial 1; shRNA, short-hairpin RNA; DMSO, dimethylsulfoxide.

Article Snippet: NCI-H69 (a human SCLC cell line) was purchased from Procell (Wuhan, China) and cultured in 1640 medium (Gibco, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco).

Techniques: Inhibition, Transduction, shRNA, Control, Annexin V Assay, Flow Cytometry

Journal: Translational Cancer Research

Article Title: PTPMT1 inhibition induces apoptosis and growth arrest of human SCLC cells by disrupting mitochondrial metabolism

doi: 10.21037/tcr-2024-2379

Figure Lengend Snippet: PTPMT1 inhibition reduces Glut expression. (A-E) The qRT-PCR analysis of the transcriptome differentially sequenced genes in H69 cells. *, P<0.05; **, P<0.01; ***, P<0.001, AD 5 µM vs . control (DMSO). (F,G) The H69 cells were transduced with PTPMT1 shRNA. The expression of Glut1 and Glut3 was determined by qRT-PCR. ***, P<0.001, sh-PTPMT1 vs . scramble (control). (H) The H69 cells were transduced with PTPMT1-shRNA or control vectors, and stained with JC-1 dye, and the ratio of the average fluorescence intensity of red/green was determined by flow cytometry. ***, P<0.001, sh-PTPMT1 vs . scramble (control). PTPMT1, protein tyrosine phosphatase mitochondrial 1; qRT-PCR, quantitative real-time polymerase chain reaction; shRNA, short-hairpin RNA; DMSO, dimethylsulfoxide; AD, alexidine dihydrochloride; mRNA, messenger RNA.

Article Snippet: NCI-H69 (a human SCLC cell line) was purchased from Procell (Wuhan, China) and cultured in 1640 medium (Gibco, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco).

Techniques: Inhibition, Expressing, Quantitative RT-PCR, Control, Transduction, shRNA, Staining, Fluorescence, Flow Cytometry, Real-time Polymerase Chain Reaction

Journal: Journal of Radiation Research

Article Title: hTERT promoter methylation promotes small cell lung cancer progression and radiotherapy resistance

doi: 10.1093/jrr/rraa052

Figure Lengend Snippet: The promoter of hTERT is methylated and correlates with radiotherapy in SCLC. ( A ) mRNA levels of hTERT in normal and SCLC tissues. ( B ) mRNA levels of hTERT in normal tissues and SCLC patients with or without radiotherapy. ( C ) Methylation status of the hTERT promoter in normal and SCLC tissues. ( D ) Methylation status of the hTERT promoter in normal tissues and SCLC patients with or without radiotherapy. ( E ) The correlation of hTERT and DNMT3B was analyzed in the TCGA lung cancer dataset. ( F ) mRNA expression of DNMT3B in H20 cells transfected with DNMT3B shRNA was determined by qPCR. ( G ) Methylation status of the hTERT promoter in H20 cells transfected with DNMT3B shRNA. Data are shown as mean ± S.D. * P < 0.05; * * P < 0.01; * * * P < 0.001; ns, not significant (Student’s t-test).

Article Snippet: H20 and H446 human SCLC cell lines were purchased from the China Center for Type Culture Collection.

Techniques: Methylation, Expressing, Transfection, shRNA

Journal: Journal of Radiation Research

Article Title: hTERT promoter methylation promotes small cell lung cancer progression and radiotherapy resistance

doi: 10.1093/jrr/rraa052

Figure Lengend Snippet: The methylation of hTERT promotes SCLC proliferation. ( A ) mRNA expression of hTERT in H20 cells transfected with hTERT expression plasmid was determined by qPCR. ( B ) Protein expression of hTERT in H20 cells transfected with hTERT expression plasmid was determined by western blot. Cell viability of H20 cells ( C ) or H446 cells ( D ) transfected with hTERT expression plasmid was determined by cell count assay. Cell viability of H20 cells ( E ) or H446 cells ( F ) transfected with hTERT expression plasmid was determined by MTT assay. ( G ) Methylation status of the hTERT promoter in the normal pulmonary epithelium BEAS-2B cells or SCLC cells H20 and H446. ( H ) Methylation status of the hTERT promoter in H20 and H446 cells treated with 5-Aza. ( I ) mRNA expression of hTERT in H20 and H446 cells treated with 5-Aza. Cell viability of H20 cells ( J ) or H446 cells ( K ) treated with 5-Aza and transfected with or without hTERT expression plasmid was determined by cell count assay. ( L ) mRNA expression of hTERT in H20 cells transfected with hTERT shRNA was determined by qPCR. ( M ) Cell viability of H20 and H446 cells transfected with hTERT shRNA was determined by cell count assay. Data are shown as mean ± S.D. * P < 0.05; * * P < 0.01; * * * P < 0.001; ns, not significant (ANOVA test in E and G, others Student’s t-test).

Article Snippet: H20 and H446 human SCLC cell lines were purchased from the China Center for Type Culture Collection.

Techniques: Methylation, Expressing, Transfection, Plasmid Preparation, Western Blot, Cell Counting, MTT Assay, shRNA

Journal: Journal of Radiation Research

Article Title: hTERT promoter methylation promotes small cell lung cancer progression and radiotherapy resistance

doi: 10.1093/jrr/rraa052

Figure Lengend Snippet: The methylation of hTERT promotes SCLC migration and invasion. ( A ) Transwell assay of migration and invasion of H20 cells transfected with hTERT expression plasmid or vector. ( B ) Statistical results of the Transwell assay in (A). ( C ) qPCR analysis of the expression of epithelioid markers OCLN and JUP or the mesenchymal markers ZEB1, ZEB2, TWIST1 and FN1 in H20 cells transfected with hTERT expression plasmid or vector. ( D ) Transwell assay of migration and invasion of H20 cells treated with 5-Aza. ( E ) Statistical results of the Transwell assay in (D). ( F ) qPCR analysis of the expression of epithelioid markers OCLN and JUP or the mesenchymal markers ZEB1, ZEB2, TWIST1 and FN1 in H20 cells treated with 5-Aza. Data are shown as mean ± S.D. * P < 0.05; * * P < 0.01; * * * P < 0.001; ns, not significant (Student’s t-test).

Article Snippet: H20 and H446 human SCLC cell lines were purchased from the China Center for Type Culture Collection.

Techniques: Methylation, Migration, Transwell Assay, Transfection, Expressing, Plasmid Preparation

Journal: Journal of Radiation Research

Article Title: hTERT promoter methylation promotes small cell lung cancer progression and radiotherapy resistance

doi: 10.1093/jrr/rraa052

Figure Lengend Snippet: The methylation of hTERT promotes SCLC radiotherapy resistance. Methylation status of the hTERT promoter in H20 ( A ) and H446 ( B ) cells treated with a single dose of 5 Gy irradiation. mRNA level of hTERT in H20 ( C ) and H446 ( D ) cells treated with a single dose of 5 Gy irradiation. Cell viability of H20 cells ( E ) or H446 cells ( F ) transfected with hTERT expression plasmid and treated with a gradient dose of irradiation. Cell viability of H20 cells ( G ) or H446 cells ( H ) treated with 5-Aza and treated with a gradient dose of irradiation. Data are shown as mean ± S.D. * * * P < 0.001; ns, not significant (ANOVA test in (E) and (H), others Student’s t-test).

Article Snippet: H20 and H446 human SCLC cell lines were purchased from the China Center for Type Culture Collection.

Techniques: Methylation, Irradiation, Transfection, Expressing, Plasmid Preparation

Journal: Journal of Radiation Research

Article Title: hTERT promoter methylation promotes small cell lung cancer progression and radiotherapy resistance

doi: 10.1093/jrr/rraa052

Figure Lengend Snippet: EZH2 acted as the downstream effector of hTERT. ( A ) Correlation of hTERT and EZH2 analysed in the TCGA lung cancer dataset. ( B ) mRNA levels of EZH2 in H20 or H446 cells transfected with hTERT expression plasmid were determined by qPCR. ( C ) mRNA levels of EZH2 in H20 or H446 cells treated with 5-Aza and transfected with or without hTERT expression plasmid were determined by qPCR. ( D ) mRNA levels of EZH2 in H20 cells transfected with EZH2 expression plasmid were determined by qPCR. ( E ) Cell viability of H20 cells transfected with EZH2 expression plasmid was determined by cell count assay. ( F ) Cell viability of H20 cells transfected with EZH2 expression plasmid was determined by MTT assay. ( G ) Transwell assay of migration and invasion of H20 cells transfected with EZH2 expression plasmid or vector. ( H ) Cell viability of H20 cells transfected with EZH2 expression plasmid and treated with a gradient dose of irradiation. Data are shown as mean ± S.D. * * P < 0.01; * * * P < 0.001; ns, not significant (ANOVA test in F and H, others Student’s t-test).

Article Snippet: H20 and H446 human SCLC cell lines were purchased from the China Center for Type Culture Collection.

Techniques: Transfection, Expressing, Plasmid Preparation, Cell Counting, MTT Assay, Transwell Assay, Migration, Irradiation

Journal: Cell reports

Article Title: WEE1 inhibition enhances the antitumor immune response to PD-L1 blockade by the concomitant activation of STING and STAT1 pathways in SCLC

doi: 10.1016/j.celrep.2022.110814

Figure Lengend Snippet: (A) Cell viability IC 50 values were determined in response to treatment with AZD1775 (0–10 μM) for 5 days in 16 human and three murine SCLC cell lines. The data shown represent the means ± SD of three individual experiments. (B) Cells were treated with 1 μM AZD1775 for 24 or 48 h. Then, the proportion of apoptotic cells was detected using annexin V-propidium iodide-based flow cytometry. Bars represent mean ± SD of triplicate. Statistical significance was determined using Student’s t test (**p < 0.01, ***p < 0.001). (C) Cells were treated with 1 μM AZD1775 for 16 h. Cell-cycle states were detected with EdU-DAPI-based flow cytometry. (D) Cells were treated with 1 μM AZD1775 for 8, 24, or 48 h. Western blots show protein expression of phospho-WEE1, phospho- and total CDK1, γH2AX, cleaved PARP, and actin (loading control) at each time indicated. (E) Tumor-growth curves of subcutaneous tumors in nude mice with conditional loss of Trp53 , p130 , and Rb1 (RPP; top) and Trp53 , Rb1 , and MYC T58A (RPM; bottom) treated with vehicle or 60 mg/kg of AZD1775 (n = 5 per group). Bars represent mean ± SE. Statistical significance was determined using Student’s t test (**p < 0.01). (F) Western blots showing phosho-WEE1, γH2AX, and actin (loading control) of RPP or RPM tumors from (E). See also .

Article Snippet: SCLC human-derived cell lines were obtained from the American Type Culture Collection (ATCC) and European Collection of Authenticated Cell Cultures (ECACC) ( ).

Techniques: Flow Cytometry, Western Blot, Expressing, Control

Journal: Cell reports

Article Title: WEE1 inhibition enhances the antitumor immune response to PD-L1 blockade by the concomitant activation of STING and STAT1 pathways in SCLC

doi: 10.1016/j.celrep.2022.110814

Figure Lengend Snippet: (A) Quantification of cells containing micronuclei (MN) after 1 μM AZD1775 treatment for 24 h. Bars represent mean ± SD of eight areas. Statistical significance was determined using Student’s t test (***p < 0.001). (B) Western blots showing the protein expression of STING pathway, phospho (p)- and total (t)-STING, p- and t-TBK1, p- and t-IRF3, cGAS, and actin (loading control) in SCLC cells treated with 1 μM AZD1775 for 8, 24, and 48 h. (C) Quantitative mRNA expression of IFN-α , IFN-β after treatment with 1 μM AZD1775 for 48 h in SCLC cells (H526, H82, H446, RPP, and RPM). Bars represent mean ± SD of triplicate. Statistical significance was determined using Student’s t test (**p < 0.01, ***p < 0.001). (D and E) Quantitative mRNA expression of (D) CXCL10 and (E) CCL5 after treatment with 1 μM AZD1775 for 48 h in SCLC cells (H526, H82, H446, RPP, and RPM). Bars represent mean ± SD of triplicate. Statistical significance was determined using Student’s t test (**p < 0.01, ***p < 0.001). See also – .

Article Snippet: SCLC human-derived cell lines were obtained from the American Type Culture Collection (ATCC) and European Collection of Authenticated Cell Cultures (ECACC) ( ).

Techniques: Western Blot, Expressing, Control

Journal: Cell reports

Article Title: WEE1 inhibition enhances the antitumor immune response to PD-L1 blockade by the concomitant activation of STING and STAT1 pathways in SCLC

doi: 10.1016/j.celrep.2022.110814

Figure Lengend Snippet: (A and B) HALLMARK pathway enrichment analyses of differentially expressed genes (DEGs) from RPP tumors on the mice treated with vehicle, AZD1775, or AZD1775 plus anti-PD-L1 antibody for 21 days. (A) AZD1775 versus vehicle and (B) AZD1775 plus anti-PD-L1 antibody versus vehicle (n = 5 per group). (C and D) HALLMARK pathway enrichment analyses of DEGs from RPM tumors on the mice treated with vehicle, AZD1775, or AZD1775 plus anti-PD-L1 antibody for 18 days. (C) AZD1775 versus vehicle and (D) AZD1775 plus anti-PD-L1 antibody versus vehicle (n = 4 per group). (E) Quantitative mRNA expression of IFN-γ after treatment with 1 μM of AZD1775 for 48 h in SCLC cells (H526, H82, H446, RPP, and RPM cells). The data shown represent the means ± SD of triplicate. p values were calculated by Student’s t test (**p < 0.01, ***p < 0.001). (F) Western blots show expression of p-WEE1, p- and total STAT1, and actin (loading control) in SCLC cells (H526, H82, H446). Cells were treated with 1 μM AZD1775 for 24 h. (G) Quantitative mRNA expression of IRF1 after treatment with 1 μM AZD1775 for 48 h in SCLC cells (H82, H446). The data shown represent the means ± SD of triplicate. p values were calculated by Student’s t test (*p < 0.05). See also .

Article Snippet: SCLC human-derived cell lines were obtained from the American Type Culture Collection (ATCC) and European Collection of Authenticated Cell Cultures (ECACC) ( ).

Techniques: Expressing, Western Blot, Control