Journal: Frontiers in Genetics

Article Title: Whole Genome Assembly of Human Papillomavirus by Nanopore Long-Read Sequencing

doi: 10.3389/fgene.2021.798608

Figure Lengend Snippet: Nanopore sequencing found tandem HPV genomes in cervical cancer tissues and CaSki cells, respectively. (A) An 11.54 kb-long HPV 35 tandem genomic sequence was obtained by nanopore sequencing. The 11.54 kb-long sequence was aligned to HPV35 16B (NCBI: KX514416.1), which consists of three genomic segments, E1-E7-E6 (1567–10 nt), a whole genome frame (7894–10 nt), and URR-L1 (1561–10 nt. (B) A 21.18 kb-long HPV tandem sequence flanked with human genomic sequence at one end was obtained by nanopore sequencing from CaSki cells. The sequence was aligned to HPV16 (NCBI: U89348), in which a truncated genome (470–7905 nt) was connected to another truncated one (470–6905 nt) in a head-to-tail manner. (C) Another 15.07 kb-long HPV tandem sequence was obtained by nanopore sequencing from CaSki cells and was flanked at one end by the human gene; concatemers are formed by joining of incomplete HPV genomes with two spliced sites at 2032 nt and 4,586 nt.

Article Snippet: As a proof of concept, in this study, we completed whole genome sequencing from a cervical cancer tissue and a CaSki cell line with Oxford Nanopore Technologies.

Techniques: Nanopore Sequencing, Sequencing

Journal: Frontiers in Genetics

Article Title: Whole Genome Assembly of Human Papillomavirus by Nanopore Long-Read Sequencing

doi: 10.3389/fgene.2021.798608

Figure Lengend Snippet: Physical state determination of the HPV genome by exonuclease V (ExoV)-qPCR–based assay.

Article Snippet: As a proof of concept, in this study, we completed whole genome sequencing from a cervical cancer tissue and a CaSki cell line with Oxford Nanopore Technologies.

Techniques:

Journal: Frontiers in Genetics

Article Title: Whole Genome Assembly of Human Papillomavirus by Nanopore Long-Read Sequencing

doi: 10.3389/fgene.2021.798608

Figure Lengend Snippet: HPV integration sites identified by nanopore sequencing in CaSki cells and a cervical cancer tissue. (A) Distribution of HPV integration sites in different function regions of human genes identified from CaSki cells (HPV16) and a cervical cancer tissue (HPV35) by nanopore sequencing. (B) The HPV integration site was amplified by PCR from the CaSki cells, followed by agarose gel electrophoresis. C-33A cells (HPV-negative cervical cancer cells) was used as a negative control. (C) Sanger sequencing of the HPV integration site. PCR products were subjected to Sanger sequencing. Peaks of nucleotides at integration sites were shown. The cellular sequence from PRR30 was boxed with green color, and viral sequence was boxed with red color. (D) The sequence of the integration site located in PRR30 gene. The exon region of PRR30 gene was labeled with green color, and the URR region of HPV genome was labeled with red color. Three random nucleotides at the breakpoint (BP) were labeled with blue color.

Article Snippet: As a proof of concept, in this study, we completed whole genome sequencing from a cervical cancer tissue and a CaSki cell line with Oxford Nanopore Technologies.

Techniques: Nanopore Sequencing, Amplification, Agarose Gel Electrophoresis, Negative Control, Sequencing, Labeling

Journal: Molecular cancer therapeutics

Article Title: Glutaminase inhibitors induce thiol-mediated oxidative stress and radio-sensitization in treatment resistant cervical cancers

doi: 10.1158/1535-7163.MCT-20-0271

Figure Lengend Snippet: (A). Clonogenic cell survival assays for CaSki, C33A, SiHa, and SiHa PTEN−/− in buffered media with and without glutamine (Gln+ and Gln−). Cells were grown in glutamine and pyruvate-free media (CaSki-5 days and C33A, SiHa and SiHa PTEN−/− - 10 days) and plated for clonogenic cell survival assay in media supplemented with 2 mM glutamine. Cell lines with PI3K pathway mutation are marked by #. (B). TCGA primary cervical cancer (TCGA-CESC) samples with PTEN-inactivating alterations, including 4.8% with deep copy number loss (−2), 22.8% with shallow copy number loss (−1), and ~7.2% with single-nucleotide variants (mut). “−2” (or deep deletion) indicates a deep loss, possibly a homozygous deletion, and “−1” (or shallow deletion) indicates a shallow loss, possibly a heterozygous deletion. (C). PTEN alterations significantly down-regulate PTEN gene expression (FPKM) in cervix tumor samples from the TCGA-CESC cohort. Samples with mutations and shallow deletions show lower PTEN expression compared to samples with wild type (WT) PTEN. Samples with deep deletions show the lowest PTEN expression levels. (D). Changes in glutamine and glutamate metabolism related gene expression after PTEN loss in SiHa cell lines. SiHa_1, SiHa_2, and SiHa_3 (biological replicates of SiHa) cells have wild-type PTEN, while PTEN−/−_1, PTEN−/−_2, and PTEN−/−_3 (biological replicates of SiHa PTEN−/−) cells are derived through knocking out PTEN in SiHa cells (See Methods and Supplementary Figure 1). (E). Changes in glycolysis and gluconeogenesis related gene expression after PTEN loss in SiHa cell lines.

Article Snippet: For tumor generation, 3.5×10 6 SiHa and or CaSki cells were injected subcutaneously into the left flank of 6–8 week old, female nude mice (Crl:NU(NCr)-Foxn1 nu, Charles River Laboratories, Wilmington, MA) in a half matrigel, half serum-free IMDM mixture.

Techniques: Clonogenic Cell Survival Assay, Mutagenesis, Gene Expression, Expressing, Derivative Assay

Journal: Molecular cancer therapeutics

Article Title: Glutaminase inhibitors induce thiol-mediated oxidative stress and radio-sensitization in treatment resistant cervical cancers

doi: 10.1158/1535-7163.MCT-20-0271

Figure Lengend Snippet: (A-C) For clonogenic cell survival assays in CaSki, SiHa, and SiHa PTEN−/− cells were treated with 200 nM telaglenastat monotherapy (CB-839), 25 nM auranafin and 125 μM L-buthionine sulfoximine for 96h. Plating efficiencies for CaSki cells were Ctrl - 109±2, CB-839 - 76±6 , CB-839+AUR - 44±3, CB-839+BSO - 0±0.56, CB-839+BA - 0±0, BA- 39±3), SiHa cells Ctrl - 130±4, CB-839 - 131±12, CB-839+AUR - 124±5, CB-839+BSO - 10±4, CB-839+BA - 6±1.5, BA - 114±4 and, SiHa PTEN−/− cells Ctrl - 65±3., CB-839 - 48±1.5, CB-839+AUR - 35±4, CB-839+BSO - 2±1, CB-839+BA - 2±0.5, BA- 56±3. Statistical analysis: Error bars represent ± SD of N=3 experiments performed on different days. Two tailed paired student’s test was performed and asterisks represents * p <0.05. (D-F) Clonogenic cell survival assays in CaSki, SiHa, and SiHa PTEN−/− with CB839 (200nM) plus increasing single fraction doses of radiation. (G). Cell viability study of normal cervix epithelial cells HCvEpC with and without 200 nM telaglenastat (CB-839) and increasing doses of radiation. (H) Tumor growth delay of SiHa xenografts treated with 75mg/kg of telaglenastat (CB-839) and 4 Gy single fraction dose of tumor directed radiation delivered via SARRP. Statistical Analysis for xenograft studies was performed using Linear mixed model fit by REML (Restricted Maximum Likelihood) as described in the Materials and Methods. Ctrl vs 4 Gy, Ctrl vs CB-839, Ctrl vs CB-839+ 4 Gy, p<0.05; CB-839+ 4 Gy vs CB-839, p<0.05; 4 Gy vs CB-839+ 4 Gy p<0.05. (I) Schematic diagram of proposed primary mechanism of action of CB-839 in cervical cancer.

Article Snippet: For tumor generation, 3.5×10 6 SiHa and or CaSki cells were injected subcutaneously into the left flank of 6–8 week old, female nude mice (Crl:NU(NCr)-Foxn1 nu, Charles River Laboratories, Wilmington, MA) in a half matrigel, half serum-free IMDM mixture.

Techniques: Two Tailed Test

Journal: Molecular cancer therapeutics

Article Title: Glutaminase inhibitors induce thiol-mediated oxidative stress and radio-sensitization in treatment resistant cervical cancers

doi: 10.1158/1535-7163.MCT-20-0271

Figure Lengend Snippet: (A) Consumption rate of Glutamine (Gln) (nmol/cell/h) for CaSki, SiHa and SiHa PTEN−/− cells in media with 13C5 labeled Gln. Results are normalized to glucose uptake. (B) Glucose uptake quantified by 18F-Fluoro-deoxy-glucose (FDG) uptake for CaSki, SiHa and SiHa PTEN−/− cells. (C) Glucose consumption from media for cells cultured in the presence and absence of glutamine starvation. (D-G) Cell proliferation assays for CaSki, C33A, SiHa and SiHa PTEN−/− cells in media with and without (Gln+ and Gln−) glutamine. Cells were seeded in 12 well plate at the density of 4000 cells per well. Relative cell density is plotted in Y axis with time as a function in X-axis. (H-K) Cell viability assays for CaSki, C33A, SiHa and SiHa PTEN−/− cells in media with (Gluc+) and without glucose (Gluc−) and or glutamine (Gln+ and Gln−). Statistical analysis: Error bars represent ± SD of N=3 experiments performed on different days. Two tailed paired student’s test was performed and asterisks represents * p <0.05.

Article Snippet: For tumor generation, 3.5×10 6 SiHa and or CaSki cells were injected subcutaneously into the left flank of 6–8 week old, female nude mice (Crl:NU(NCr)-Foxn1 nu, Charles River Laboratories, Wilmington, MA) in a half matrigel, half serum-free IMDM mixture.

Techniques: Labeling, Cell Culture, Two Tailed Test

Journal: Molecular cancer therapeutics

Article Title: Glutaminase inhibitors induce thiol-mediated oxidative stress and radio-sensitization in treatment resistant cervical cancers

doi: 10.1158/1535-7163.MCT-20-0271

Figure Lengend Snippet: A-D Glutamine carbon is used to generate reduced glutathione (GSH) and tricarboxylic acid (TCA) cycle intermediates in PI3K activated cervical cancer cells. CaSki SiHa, and SiHa PTEN−/− cells were grown with 13C5 labeled glutamine (Gln) for 18 hrs, and isotope incorporation into (A) glutamate (M+5 fraction), (B) α-ketoglutarate (α-KG M+5 fraction) and reduced glutathione (GSH represented as relative intensity of M+5 fraction (C) and peak areas of 13C5Gln labeled GSH (D) were quantified by liquid chromatography mass spectroscopy as described in the materials and methods. (E-F) SiHa cells divert glucose carbon into the tricarboxylic acid cycle (TCA) intermediates under conditions of glutamine deprivation (Gln−). SiHa, SiHa PTEN−/− and CaSki cells were grown with 13C6 glucose for 18hrs, followed by 48hrs of media with glutamine (Gln+) or without glutamine (Gln−), and 13C6 glucose isotope incorporation in m+3 tricarboxylic acid (TCA) cycle intermediates (E) Malate and (F) Citrate were quantified by liquid chromatography mass spectrometry as described in the materials and methods. (G-H) Glutamine deprivation results in decreased generation of nucleotide precursors (G) adenosine and (H) dGMP in cervical cancer cells. Statistical analysis: Error bars represent ± SD of N=3 experiments performed on different days. Two tailed paired student’s test was performed and asterisks represents * p <0.05.

Article Snippet: For tumor generation, 3.5×10 6 SiHa and or CaSki cells were injected subcutaneously into the left flank of 6–8 week old, female nude mice (Crl:NU(NCr)-Foxn1 nu, Charles River Laboratories, Wilmington, MA) in a half matrigel, half serum-free IMDM mixture.

Techniques: Labeling, Liquid Chromatography, Mass Spectrometry, Two Tailed Test

Journal: Molecular cancer therapeutics

Article Title: Glutaminase inhibitors induce thiol-mediated oxidative stress and radio-sensitization in treatment resistant cervical cancers

doi: 10.1158/1535-7163.MCT-20-0271

Figure Lengend Snippet: (A-B). Total Glutathione (GSH) level (moles of GSH/mg of protein) and percent GSSG (% of total cellular GSH present in the form of GSSG) for CaSki, SiHa and SiHa PTEN−/− cells grown with (Gln+) and without (Gln−) glutamine for 48 hrs. (C). Reactive oxygen species (ROS) quantified by 2’,7’-dichlorodihydrofluorescein diacetate (H2DCFDA) oxidation in CaSki, SiHa and SiHa PTEN−/− cells with (Gln+) and without (Gln−) glutamine. (D). NADPH/NADP ratio for CaSki, SiHa and SiHa PTEN−/− cells grown with (Gln+) and without (Gln−) glutamine. (E). Thioredoxin reductase (TR) activity (mU/mg of protein) for CaSki, SiHa and SiHa PTEN−/− cells grown with (Gln+) and without (Gln−) glutamine for 48 hrs. Statistical analysis: Error bars represent ± SD of N=3 experiments performed on different days. Two tailed paired student’s test was performed and asterisks represents * p <0.05.

Article Snippet: For tumor generation, 3.5×10 6 SiHa and or CaSki cells were injected subcutaneously into the left flank of 6–8 week old, female nude mice (Crl:NU(NCr)-Foxn1 nu, Charles River Laboratories, Wilmington, MA) in a half matrigel, half serum-free IMDM mixture.

Techniques: Activity Assay, Two Tailed Test

Journal: Molecular cancer therapeutics

Article Title: Glutaminase inhibitors induce thiol-mediated oxidative stress and radio-sensitization in treatment resistant cervical cancers

doi: 10.1158/1535-7163.MCT-20-0271

Figure Lengend Snippet: (A) Dose response curve for telaglenastat monotherapy in CaSki, SiHa and SiHa PTEN−/− cells. (B-D). Telaglenastat alteration of intracellular redox pools were measured by quantifying levels of reduced glutathione (GSH), percent oxidized glutathione (%GSSG) and thioredoxin reductase (TR) activity after treating cells with 50 nM of telaglenastat for 48hr. (E-F). Cytotoxic effects of telaglenastat (CB-839) can be rescued by the additional of the thiol antioxidant, N-acetylcysteine (NAC). Clonogenic cell survival assays were performed in CaSki (E) and SiHa PTEN−/− (F) cells 96 h after incubation with 200 nM telaglenastat (CB-839) with (CB + NAC) and without NAC rescue. Statistical analysis: Error bars represent ± SD of N=3 experiments performed on different days. Two tailed paired student’s test was performed and asterisks represents * p <0.05. (G-H) In vivo efficacy of telaglenastat (CB-839) monotherapy in mice with (G) CaSki and SiHa (H) xenografts treated with 50 and 100 mg/Kg for 37 days. Statistical analysis was performed by Linear mixed model fit by REML (Restricted Maximum Likelihood). For CaSki xenograft Ctrl vs CB-839 50 mg/KG, Ctrl vs CB-839 100 mg/KG, the tumor volumes of both treated groups were smaller than Ctrl group with p<0.05. There was no significant difference between CaSki CB-839 50 mg/KG vs CaSki CB-839 100 mg/KG. For SiHa xenograft model Ctrl vs CB-839 50 mg/KG, p<0.05; Ctrl vs CB-839 100 mg/KG, p>0.05; CB-839 50 mg/KG vs CB-839 100 mg/KG, p>0.05.

Article Snippet: For tumor generation, 3.5×10 6 SiHa and or CaSki cells were injected subcutaneously into the left flank of 6–8 week old, female nude mice (Crl:NU(NCr)-Foxn1 nu, Charles River Laboratories, Wilmington, MA) in a half matrigel, half serum-free IMDM mixture.

Techniques: Activity Assay, Incubation, Two Tailed Test, In Vivo

Journal: Frontiers in Endocrinology

Article Title: Natural Compounds in Sex Hormone-Dependent Cancers: The Role of Triterpenes as Therapeutic Agents

doi: 10.3389/fendo.2020.612396

Figure Lengend Snippet: Triterpenoid compounds with reported in vivo antiproliferative activity.

Article Snippet: Cucurbitane , Cucurbitane B Cucurbitane B Cucurbitane B Cucurbitane B Cucurbitane D , MDA-MB-468 cells implanted into the breasts of nude mice MDA-MB-231 human breast cancer orthotopic xenografts in immunodeficient Sprague-Dawley mice PC-3 and LNCaP xenograft male athymic mouse model MDA-MB-231 cells and 4T-1 cells orthotopically implanted in the mammary fat pad of female athymic BALB/c mice and nude mice Athymic nude mice with cervical cancer (CaSki) cell-derived orthotopic xenograft tumors , Tumor volume was reduced by 55% Significantly reduced tumor volume Significantly reduced the rate of in vivo tumor-formation Tumor size reduction by 79% (vs. control) at day 31 Tumor volume was reduced by 55% (MDA-MB-231 cells) and 39% (4T-1) vs. control Significant reduction of tumor volume and weight; decreased expression of pAKT and pSTAT3 proteins , 1.0 mg/kg; 6 weeks; i.p 0.5 mg/kg and 1.0 mg/kg; 3 times/week; 36 days 0.1 μmol, 5 days, oral gavage; 2 weeks pre-treatment before cancer cell implantation; 2 mg/kg daily; 60 days; oral gavage. 1 mg/kg body weight; intra-tumoral , ( – ) , .

Techniques: In Vivo, Activity Assay, Inhibition, Control, Expressing, Phospho-proteomics, Transgenic Assay

Journal: Cancer Cell International

Article Title: Overexpression of trefoil factor 3 (TFF3) contributes to the malignant progression in cervical cancer cells

doi: 10.1186/s12935-016-0379-1

Figure Lengend Snippet: Forced expression of TFF3 in cervical cancer cells modulates the expression of malignant progression-related gene markers. a Determination of endogenous expression of TFF3 protein by Western blot in the cervical cancer cells lines CaSki, SiHa, Me180 and Hela and human non-tumor keratinocyte line HaCaT. b Semi-quantitative RT-PCR analysis was used to assess the mRNA levels of TFF3 in SiHa and Hela cells with either forced or depleted expression of TFF3 as described in “ ” section. c Western blot analysis was used to assess the protein levels of TFF3 in SiHa and Hela cells with either forced or depleted expression of TFF3. d , e Quantitative PCR analysis quantifying the change in expression of various genes associated with malignant progression in SiHa-TFF3/Hela cells. Change in gene expression is expressed as fold difference, respectively. Fold change values are representative of three independent experiments

Article Snippet: Human cervical cancer cell lines SiHa, CaSki, Hela, Me180 and human non-tumor keratinocyte line HaCaT were obtained from Nanjing KeyGen Biotech Co, Ltd (Nanjing,China).

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Gene Expression