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Cisplatin enhances cell stiffness and decreases invasiveness rate in prostate cancer cells by actin accumulation: Confocal and atomic force microscopy
<p><strong>Summary</strong></p> <p>Dataset of imaging data related to the publication Raudenska, M., Kratochvilova, M., Vicar, T., Gumulec, J., Balvan, J., Polanska, H. Pribyl, J. & Masarik, M.:Cisplatin enhances cell stiffness and decreases invasiveness rate in prostate cancer cells by actin accumulation. <em>Scientific Reports </em><strong>2019, </strong>9, 1660</p> <p>This dataset includes image data of <em>atomic force microcopy</em> (Young modulus) and <em>confocal microscopy</em>(staining of F-actin and β-tubulin) of prostate cell lines PNT1A, 22Rv1, and PC-3. </p> <p><strong>Materials and Methods</strong></p> <p><em>Cells, cell culture conditions</em></p> <p>Cells confluent up to 50–60% were washed with a FBS-free medium and treated with a fresh medium with FBS and required antineoplastic drug concentration (IC50 concentration for the particular cell line). The cells were treated with 93 µM (PC-3), 38 µM (PNT1A), and 24 µM (22Rv1) of cisplatin (Sigma-Aldrich, St. Louis, Missouri), respectively. IC50 concentrations used for treatment with docetaxel (Sigma-Aldrich, St. Louis, Missouri) were 200nM for PC-3, 70nM for PNT1A, and 150nM for 22Rv1. </p> <p><em>Long-term zinc (II) treatment of cell cultures</em></p> <p>Cells were cultivated in the constant presence of zinc(II) ions. Concentrations of zinc(II) sulphate in the medium were increased gradually by small changes of 25 or 50 µM. The cells were cultivated at each concentration no less than one week before harvesting and their viability was checked before adding more zinc. This process was used to select zinc resistant cells naturally and to ensure better accumulation of zinc within the cells (accumulation of zinc is usually poor during the short-term treatment of prostate cancer cells). Total time of the cultivation of cell lines in the zinc(II)-containing media exceeded one year. Resulting concentrations of zinc(II) in the media (IC50 for the particular cell line) were 50 µM for the PC-3 cell line, 150 µM for the PNT1A cell line, and 400 µM for the 22Rv1 cell line. The concentrations of zinc(II) in the media and FBS were taken into account. </p> <p><em>Actin and tubulin staining</em></p> <p>β-tubulin was labeled with anti- β tubulin antibody [EPR1330] (ab108342) at a working dilution of 1/300. The secondary antibody used was Alexa Fluor® 555 donkey anti-rabbit (ab150074) at a dilution of 1/1000. Actin was labeled with Alexa Fluor™ 488 Phalloidin (A12379, Invitrogen); 1 unit per slide. For mounting Duolink® In Situ Mounting Medium with DAPI (DUO82040) was used. The cells were fixed in 3.7% paraformaldehyde and permeabilized using 0.1% Triton X-100. </p> <p><em>Confocal microscopy</em></p> <p>The microscopy of samples was performed at the Institute of Biophysics, Czech Academy of Sciences, Brno, Czech Republic. Leica DM RXA microscope (equipped with DMSTC motorized stage, Piezzo z-movement, MicroMax CCD camera, CSU-10 confocal unit and 488, 562, and 714 nm laser diodes with AOTF) was used for acquiring detailed cell images (100× oil immersion Plan Fluotar lens, NA 1.3). Total 50 Z slices was captured with Z step size 0.3 μm.</p> <p><em>Atomic force microscopy</em></p> <p>We used the bioAFM microscope JPK NanoWizard 3 (JPK, Berlin, Germany) placed on the inverted optical microscope Olympus IX‑81 (Olympus, Tokyo, Japan) equipped with the fluorescence and confocal module, thus allowing a combined experiment (AFM‑optical combined images). The maximal scanning range of the AFM microscope in X‑Y‑Z range was 100‑100‑15 µm. The typical approach/retract settings were identical with a 15 μm extend/retract length, Setpoint value of 1 nN, a pixel rate of 2048 Hz and a speed of 30 µm/s. The system operated under closed-loop control. After reaching the selected contact force, the cantilever was retracted. The retraction length of 15 μm was sufficient to overcome any adhesion between the tip and the sample and to make sure that the cantilever had been completely retracted from the sample surface. Force‑distance (FD) curve was recorded at each point of the cantilever approach/retract movement. AFM measurements were obtained at 37°C (Petri dish heater, JPK) with force measurements recorded at a pulling speed of 30 µm/s (extension time 0.5 sec).</p> <p>The Young's modulus (E) was calculated by fitting the Hertzian‑Sneddon model on the FD curves measured as force maps (64x64 points) of the region containing either a single cell or multiple cells. JPK data evaluation software was used for the batch processing of measured data. The adjustment of the cantilever position above the sample was carried out under the microscope by controlling the position of the AFM‑head by motorized stage equipped with Petri dish heater (JPK) allowing precise positioning of the sample together with a constant elevated temperature of the sample for the whole period of the experiment. Soft uncoated AFM probes HYDRA-2R-100N (Applied NanoStructures, Mountain View, CA, USA), i.e. silicon nitride cantilevers with silicon tips are used for stiffness studies because they are maximally gentle to living cells (not causing mechanical stimulation). Moreover, as compared with coated cantilevers, these probes are very stable under elevated temperatures in liquids – thus allowing long-time measurements without nonspecific changes in the measured signal.</p> <p><em>Image analysis</em></p> <p>Fluorescence microscopy data were analyzed in ImageJ 1.52h and Python 3.7.1 as follows: cells were manually segmented using actin fluorescence channel, two regions were created for analysis: whole cell and cell periphery, lining a 4 μm thick region around cell border and including most of periphery actin cytoskeleton. In these two regions following parameters were measured for both actin and tubulin fluorescence: Integrated intensity, median intensity, and following regions were measured to describe cell morphology: Cell area, Maximum caliper (max feret diameter), roundness, and aspect ratio. Moreover, stress fibers were manually segmented in every cell and following parameters were measured: number of fibers per cell, feret angle of fiber, integrated intensity, fiber length, mean intensity. Next, a standard deviation of feret angles of individual fibers was calculated relatively to mean of feret angle using a circstd function from scipy package for Python.</p> <p><strong>Identification of files</strong></p> <p><em>Microscopy data</em></p> <p>Files are separated into individual zip files. The dataset of <em>confocal microscopy </em>is separated based on treatments: untreated control, docetaxel-treated cells, cisplatin-treated cells, zinc-treated cells. Filenames actin_tubulin_Zstack_cisplatin.zip, actin_tubulin_Zstack_untreated_control.zip, actin_tubulin_Zstack_zinc.zip, actin_tubulin_Zstack_docetaxel.zip. Files included in these ZIP archives are named as follows: "cellline_treatment_FOV". Files are 3-layer 16bit tiff files with layer sequence as follows: F-Actin (Phalloidin)/b-tubulin/Hoechst 33342. The dataset contains 242 FOVs of three cell line types/three treatments + one control, files are Z-stacks made of 50 slices.</p> <p>The dataset of <em>atomic force microscopy </em>(AFM) is included in one ZIP archive "AFM_YoungModulus_SetpointHeight.zip", which includes data on Young modulus and Setpoint Height of cell lines 22Rv1, PNT1A and PC-3 and treatments zinc, docetaxel, cisplatin (+control), i.e. identical like for confocal microscopy. The file naming is as follows: "AFM_cellline_treatment_FOV_Youngmodulus.tif" for Young modulus and "AFM_cellline_treatment_FOV_setpointheight.tif" for setpoint height. The data are filtered 32-bit tiff images, where the pixel value correspond to cell stiffness (young modulus) in Pa or setpoint height in m.</p> <p><em>Confocal microscopy analysis files</em></p> <p>Following files are csv tables including image analysis of actin/tubulin staining captured by confocal microscope:</p> <p>Cytoskeleton_fluo_analysis_Cell_Cell_periphery_morphology.csv: table includes analyzed data for actin and tubulin staining in following cellular regions: cell, cell periphery. Standard ImageJ parameters regarding intensity and morphology included.</p> <p>Cytoskeleton_fluo_analysis_Fibers.csv: table includes results of manual segmentation and consequent analysis of actin stres fibers in the cells. Apart from standard ImageJ parameters, also number of stress fibers per cell and standard deviation of fiber angle relative to the cell mean angle (for details see methods) are included.</p>
Genome editing of LKB1 gene by CRISPR/Cas9 in lung cancer cells and evaluation of its role in metformin and cisplatin response.
<pre>LKB1 is an important upstream inhibitor of the mTOR/S6Ks pathway. Loss of LKB1 is often associated with cancer, including in A549 lung cancer cell line, boosting the transformation of pre-malignant neoplasic cells. Metformin, a natural compound derived from Galega officinalis, is mainly used as a treatment for diabetes mellitus type 2 (DM2), but recently, it has been associated to lower incidence of cancer. One of the main mechanisms is by activation of AMPK through different pathways, including LKB1 activation. Activation of AMPK inhibits the mTOR pathway, protein synthesis and thus cell growth. The combination of metformin and cisplatin, a main chemothepeutic drug for lung cancer, has shown to improve treatment of cancer cells to cisplatin and also to hinder the cisplatin associated resistance common in lung cancer. Here we aim to use the CRISPR/Cas9 technology to correct the mutation presented in A549 lung cancer cells and improve their response to metformin or to the combination between metformin and cisplatin. Besides, we will assess the mTOR signaling pathway status, as well as cell growth, proliferation, cell cycle and sensitivity to apoptosis induction by cisplatin. This project may be useful as a proof of principle that in the future therapies must consider the genomic background of cancer cells before administration of a specific anticancer drug.<br><br></pre>
Valproic Acid Synergizes With Cisplatin and Cetuximab in vitro and in vivo in Head and Neck Cancer by Targeting the Mechanisms of Resistance - Unpublished data
<p>Antitumor effects of valproic acid (VPA) in combination with Cisplatin/Cetuximab doublet in head and neck squamous cell carcinoma (HNSCC) models. We reported unpublished data of the effects of this combination on cell cycle and 3D cell cultures</p>
Transcriptome Analysis of Cisplatin, Cannabidiol, and Intermittent Serum Starvation Alone and in Various Combinations on Colorectal Cancer Cells
<p>* See README file for the description of data files available in this repository</p> <p>1. Study Description:</p> <p>Platinum-derived chemotherapy medications are often combined with other conventional therapies for treating different tumours, including colorectal cancer. However, the development of drug resistance and multiple adverse effects remain common in clinical settings. Thus, there is a necessity to find novel treatments and drug combinations that could effectively target colorectal cancer cells and lower the probability of disease relapse. To find potential synergistic interaction, we designed multiple different combinations between cisplatin, cannabidiol, and intermittent serum starvation on colorectal cancer cell lines. Based on the cell viability assay, we found that combinations between cannabidiol and intermittent serum starvation, cisplatin, and intermittent serum starvation, as well as cisplatin, cannabidiol and intermittent serum starvation can work in a synergistic fashion on different colorectal cancer cell lines. Furthermore, we analyzed differentially expressed genes and affected pathways in colorectal cancer cell lines to understand further the potential molecular mechanisms behind the treatments and their interactions. We found that synergistic interaction between cannabidiol and intermittent serum starvation can be related to changes in the transcription of genes responsible for cell metabolism and cancer’s stress pathways. Moreover, when we added cisplatin to the treatments, there was a strong enrichment of genes taking part in G2/M cell cycle arrest and apoptosis.</p> <p> </p> <p>2. Bioinformatics workflow:</p> <p>Initial quality control was conducted using FastQC v0.11.9 https://www.bioinformatics.babraham.ac.uk/projects/fastqc/. Sequencing reads were trimmed of adapter sequences and low-quality bases using Trimmomatic. Trimmed sequence files were examined with FastQC to verify the trimming results. Trimmed sequencing reads were mapped to Human genome (GRCh37, Ensembl) downloaded from Illumina iGenome website (<a href="https://support.illumina.com/sequencing/sequencing_software/igenome.html">https://support.illumina.com/sequencing/sequencing_software/igenome.html</a>). Mapping was done using splice aware aligner HISAT2 2.1.0. Alignment files in SAM format were converted to BAM, sorted and indexed with samtools v.1.3.1. Mapping quality and statistics were collected with QualiMap software package v.2.2.2 <a href="http://qualimap.conesalab.org/">http://qualimap.conesalab.org/</a> The counts if reads mapping to features (genes) were counted using FeatureCounts v.2.0.1 software.</p> <p>Data exploration, visualization and statistical comparisons were conducted using R language version 4.2.2. Pair-wise comparisons between experimental groups were done with DESeq2 v.2.1.36 as described in the package manual. To decrease computational time, only the genes with at least 5 reads across 3 samples were kept in the analysis. In addition to hard threshold filtering mentioned above, DESeq2 implements independent filtering based on mean of normalized count as a filter statistic.</p> <p>We used hierarchical clustering (HC) and principal components analysis (PCA) to investigate the relationship between samples and detect potential outliers. Prior to HC and PCA analysis, DESeq2 normalized values underwent variance stabilizing transformation with using vst() function from DESeq2. HC was done using hclust() function implemented in R, with the clustering method set as “complete” for the matrices of sample-to-sample distances, and “Ward.D2” in case of the sample and gene clustering based on top 500 most variable genes. The distance measure in HC analysis was set to “euclidean”. Principal components analysis (PCA), applied to top 500 highly variable genes, was conducted using prcomp() function implemented in R with default options.</p> <p>Differentially expressed genes (DEGs) were detected with DESeq2 function results() with default options. DESeq2 uses Wald test to determine significantly changed genes between groups. The independent filtering option was set to TRUE with alpha threshold (adjusted p-value) kept at 0.1. Multiple comparison adjustment was done using Bejamini-Hochberg procedure.</p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p>
The essential oil of Polygonum minus modulating cisplatin-induced hepatotoxicity through inflammatory and apoptotic pathway
<p>Oxidative stress, inflammation and apoptosis are thought as primary mediators of cisplatin-induced hepatotoxicity. The objective of this study was to determine the protective effect of <em>Polygonum minus</em> essential oil in cisplatin-induced hepatotoxicity. A total of forty-two male rats were randomly divided into seven groups: control, cisplatin, positive control β-caryophyllene 150 mg/kg (BCP), PmEO 100 mg/kg + cisplatin (PmEO100CP), PmEO 200 mg/kg + cisplatin (PmEO200CP), PmEO 400 mg/kg + cisplatin (PmEO400CP) and PmEO 400 mg/kg (PmEO400). Rats in the BCP, PmEO100CP, PmEO200CP, PmEO400CP and PmEO400 group received respective treatment orally for 14 consecutive days prior to cisplatin injection. All animals except for those in the control group and PmEO400 were administered with a single dose of cisplatin (10 mg/kg) intraperitoneally on day 15 and were sacrificed on day 18. PmEO100CP pretreatment protected against cisplatin-induced hepatotoxicity by decreasing CYP2E1, malondialdehyde, 8-OHdG and protein carbonyl which was accompanied by increased antioxidant status as compared to cisplatin alone group. PmEO100CP pretreatment also attenuated changes in liver inflammatory markers. PmEO100CP administration also reduced cisplatin-induced apoptosis. In conclusion, our results suggested that <em>P. minus</em> essential oil at a dose of 100 mg/kg may protect against cisplatin-induced hepatotoxicity possibly via inhibition of oxidative stress, inflammation and apoptosis.</p>
Atomic resolution X-ray crystal structure of cisplatin bound to hen egg white lysozyme stored for 5 years ‘on the shelf’
<p>These are the raw diffraction images for crystals 1 and 2 underpinning PDBe code 5LXW.</p>
Acidic Microenvironment Enhances Cisplatin Resistance in Bladder Cancer via Bcl-2 and XIAP
Open the record for dataset details and reuse information.
Original dataset for the species sensitivity distribution (SSD) curves of the antineoplastics Imatinib, Cisplatin and 5-Fluorouracil
<p>Original dataset (LC<sub>50</sub> and EC<sub>50</sub> for freshwater species) for the species sensitivity distribution (SSD) curves of the antineoplastics Imatinib (IM), Cisplatin (CisPt) and 5-Fluorouracil (5-FU).</p>
Trial of Postoperative Radiation, Cisplatin, and Panitumumab in Locally Advanced Head and Neck Cancer
ClinicalTrials.gov study NCT00798655. IPD Sharing: Not stated. Countries: 1. Publications: 1.
Radiation Therapy, Paclitaxel, and Cisplatin in Treating Patients With Cancer of the Cervix
ClinicalTrials.gov study NCT00003377. IPD Sharing: Not stated. Countries: 1. Publications: 1.
Triapine, Cisplatin, and Radiation Therapy in Treating Patients With Cervical Cancer or Vaginal Cancer
ClinicalTrials.gov study NCT00941070. IPD Sharing: Not stated. Countries: 1. Publications: 2.
Trial of 2 Cycles of Induction Chemo With Abraxane, Cetuximab, Cisplatin, & 5-FU for Advanced Head and Neck Cancer
ClinicalTrials.gov study NCT00736944. IPD Sharing: NO. Countries: 1. Publications: 10.
Gemcitabine Hydrochloride and Cisplatin With or Without Veliparib or Veliparib Alone in Treating Patients With Locally Advanced or Metastatic Pancreatic Cancer
ClinicalTrials.gov study NCT01585805. IPD Sharing: Not stated. Countries: 3. Publications: 1.
Cetuximab and Cisplatin in Treating Patients With Advanced, Persistent, or Recurrent Cervical Cancer
ClinicalTrials.gov study NCT00101192. IPD Sharing: Not stated. Countries: 1. Publications: 1.
Nab-Paclitaxel, Cetuximab, Cisplatin, and Radiation Therapy in Treating Patients With Recurrent Head and Neck Cancer
ClinicalTrials.gov study NCT00833261. IPD Sharing: Not stated. Countries: 1. Publications: 1.
Testing the Addition of a New Anti-Cancer Drug, Triapine, to the Usual Chemotherapy Treatment (Cisplatin) During Radiation Therapy for Advanced-stage Cervical and Vaginal Cancers
ClinicalTrials.gov study NCT02466971. IPD Sharing: Not stated. Countries: 3. Publications: 2.
Reduced-Dose Intensity-Modulated Radiation Therapy With or Without Cisplatin in Treating Patients With Advanced Oropharyngeal Cancer
ClinicalTrials.gov study NCT02254278. IPD Sharing: Not stated. Countries: 4. Publications: 1.
A Study of Aflibercept Administered in Combination With Pemetrexed and Cisplatin in Participants With Advanced Carcinoma
ClinicalTrials.gov study NCT00794417. IPD Sharing: Not stated. Countries: 2. Publications: 2.
Cisplatin-Based Chemotherapy and/or Surgery in Treating Young Patients With Adrenocortical Tumor
ClinicalTrials.gov study NCT00304070. IPD Sharing: YES. Countries: 4. Publications: 1.
Radiation + Cisplatin or Panitumumab in Locally Advanced Stage III or Stage IV Head and Neck Cancer
ClinicalTrials.gov study NCT00820248. IPD Sharing: NO. Countries: 1. Publications: 3.
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