Diverse Cell Lines Research Articles

A verified genomic reference sample for assessing performance of cancer panels detecting small variants of low allele frequency

BackgroundOncopanel genomic testing, which identifies important somatic variants, is increasingly common in medical practice and especially in clinical trials. Currently, there is a paucity of reliable genomic reference samples having a suitably large number of pre-identified variants for properly assessing oncopanel assay analytical quality and performance. The FDA-led Sequencing and Quality Control Phase 2 (SEQC2) consortium analyze ten diverse cancer cell lines individually and their pool, termed Sample A, to develop a reference sample with suitably large numbers of coding positions with known (variant) positives and negatives for properly evaluating oncopanel analytical performance.ResultsIn reference Sample A, we identify more than 40,000 variants down to 1% allele frequency with more than 25,000 variants having less than 20% allele frequency with 1653 variants in COSMIC-related genes. This is 5–100× more than existing commercially available samples. We also identify an unprecedented number of negative positions in coding regions, allowing statistical rigor in assessing limit-of-detection, sensitivity, and precision. Over 300 loci are randomly selected and independently verified via droplet digital PCR with 100% concordance. Agilent normal reference Sample B can be admixed with Sample A to create new samples with a similar number of known variants at much lower allele frequency than what exists in Sample A natively, including known variants having allele frequency of 0.02%, a range suitable for assessing liquid biopsy panels.ConclusionThese new reference samples and their admixtures provide superior capability for performing oncopanel quality control, analytical accuracy, and validation for small to large oncopanels and liquid biopsy assays.

Sunitinib Combined with Th1 Cytokines Potentiates Apoptosis in Human Breast Cancer Cells and Suppresses Tumor Growth in a Murine Model of HER-2pos Breast Cancer.

Although immune-based therapies have made remarkable inroads in cancer treatment, they usually must be combined with standard treatment modalities, including cytotoxic drugs, to achieve maximal clinical benefits. As immunotherapies are further advanced and refined, considerable efforts will be required to identify combination therapies that will maximize clinical responses while simultaneously decreasing the unpleasant and sometimes life-threatening side effects of standard therapy. Over the last two decades, evidence has emerged that Th1 cytokines can play a central role in protective antitumor immunity and that combinations of Th1 cytokines can induce senescence and apoptosis in cancer cells. To explore the possibility of combining targeted drugs with Th1-polarizing vaccines, we undertook a study to examine the impact of combining Th1 cytokines with the relatively broad-spectrum receptor tyrosine kinase antagonist, sunitinib. We found that when a panel of five phenotypically diverse human breast cancer cell lines was subjected to treatment with sunitinib plus recombinant Th1 cytokines IFN-γ and TNF-α, synergistic effects were observed across a number of parameters including different aspects of apoptotic cell death. Interestingly, sunitinib was found to have a profoundly suppressive effect of T cell's capacity to secrete IFN-γ, indicating that in vivo use of this drug may hinder robust Th1 responses. Nonetheless, this suppression was circumvented in a mouse model of HER-2pos breast disease by supplying recombinant interferon-gamma to achieve a combination therapy significantly more potent than either agent.

Novel Human-Derived RET Fusion NSCLC Cell Lines Have Heterogeneous Responses to RET Inhibitors and Differential Regulation of Downstream Signaling.

Rearranged during transfection (RET) rearrangements occur in 1% to 2% of lung adenocarcinomas as well as other malignancies and are now established targets for tyrosine kinase inhibitors. We developed three novel RET fusion-positive (RET+) patient-derived cancer cell lines, CUTO22 [kinesin 5B (KIF5B)-RET fusion], CUTO32 (KIF5B-RET fusion), and CUTO42 (echinoderm microtubule-associated protein-like 4-RET fusion), to study RET signaling and response to therapy. We confirmed each of our cell lines expresses the RET fusion protein and assessed their sensitivity to RET inhibitors. We found that the CUTO22 and CUTO42 cell lines were sensitive to multiple RET inhibitors, whereas the CUTO32 cell line was >10-fold more resistant to three RET inhibitors. We discovered that our RET+ cell lines had differential regulation of the mitogen-activated protein kinase and phosphoinositide 3-kinase/protein kinase B (AKT) pathways. After inhibition of RET, the CUTO42 cells had robust inhibition of phosphorylated AKT (pAKT), whereas CUTO22 and CUTO32 cells had sustained AKT activation. Next, we performed a drug screen, which revealed that the CUTO32 cells were sensitive (<1 nM IC50) to inhibition of two cell cycle-regulating proteins, polo-like kinase 1 and Aurora kinase A. Finally, we show that two of these cell lines, CUTO32 and CUTO42, successfully establish xenografted tumors in nude mice. We demonstrated that the RET inhibitor BLU-667 was effective at inhibiting tumor growth in CUTO42 tumors but had a much less profound effect in CUTO32 tumors, consistent with our in vitro experiments. These data highlight the utility of new RET+ models to elucidate differences in response to tyrosine kinase inhibitors and downstream signaling regulation. Our RET+ cell lines effectively recapitulate the interpatient heterogeneity observed in response to RET inhibitors and reveal opportunities for alternative or combination therapies. SIGNIFICANCE STATEMENT: We have derived and characterized three novel rearranged during transfection (RET) fusion non-small cell lung cancer cell lines and demonstrated that they have differential responses to RET inhibition as well as regulation of downstream signaling, an area that has previously been limited by a lack of diverse cell line modes with endogenous RET fusions. These data offer important insight into regulation of response to RET tyrosine kinase inhibitors and other potential therapeutic targets.

Analysis of the PD-1 Ligands Among Gastrointestinal Cancer Patients: Focus on Cancer Immunity.

Many types of gastrointestinal cancer have shown promising outcomes after checkpoint blockade immunotherapy; however, it remains largely unclear about the expression profiles of programmed death 1 (PD-1) ligands (CD274 and PDCD1LG2) in the context of human pan-cancer. This work comprehensively analyzed the expression pattern of the PD-1 ligands and the clinical significance in the prognosis prediction among the seven types of gastrointestinal malignancies collected from The Cancer Genome Atlas (TCGA) and the Cancer Cell Line Encyclopedia (CCLE) database. Furthermore, the correlation of CD274/PDCD1LG2 with cancer immunity was also explored. The patients with liver hepatocellular carcinoma (LIHC) receiving cytokine-induced killer (CIK) cell immunotherapy at our cancer center were enrolled. CD274 and PDCD1LG2 displayed inconsistent gene expression levels among the diverse cancer cell lines. Typically, the abnormal expression level of CD274 and PDCD1LG2 was detected in both esophageal carcinoma (ESCA) and stomach adenocarcinoma (STAD), where PDCD1LG2 was related to the overall survival (OS) of the patients in ESCA (p = 0.015) and STAD (p = 0.025). High-serum CD274 and PDCD1LG2 levels predicted a worse survival in the patients with LIHC receiving CIK therapy. More importantly, the expression level of CD274 and PDCD1LG2 was significantly correlated with the degree of Estimation of STromal and Immune cells in MAlignant Tumor tissues using the Expression data (ESTIMATE). In addition, we found that CD274 and PDCD1LG2 were correlated with gene markers in tumor-infiltrating immune cells. Furthermore, the expression of CD274 and PDCD1LG2 was correlated with tumor mutation burden (TMB), microsatellite instability (MSI), mismatch repair (MMR), and DNA methyltransferase (DNMT) of different types of cancers. The present work comprehensively analyzed a RNA sequencing of the PD-1 ligands across the seven distinct types of gastrointestinal cancers, which provided clues for further studies in cancer immunity and development.

Fangchinoline diminishes STAT3 activation by stimulating oxidative stress and targeting SHP-1 protein in multiple myeloma model

IntroductionThe development of cancer generally occurs as a result of various deregulated molecular mechanisms affecting the genes that can control normal cellular growth. Signal transducer and activator of transcription 3 (STAT3) pathway, once aberrantly activated can promote carcinogenesis by regulating the transcription of a number of oncogenic genes. ObjectivesHere, we evaluated the impact of fangchinoline (FCN) to attenuate tumor growth and survival through modulation of oncogenic STAT3 signaling pathway using diverse tumor cell lines and a xenograft mouse model. MethodsTo evaluate the action of FCN on STAT3 cascade, protein levels were analyzed by Western blot analysis and electrophoretic mobility shift assay (EMSA). Translocation of STAT3 was detected by immunocytochemistry. Thereafter, FCN-induced ROS was measured by GSH/GSSG assay and H2DCF-DA. FCN-induced apoptosis was analyzed using Western blot analysis and flow cytometry for various assays. Finally, anti-cancer effects of FCN in vivo was evaluated in a myeloma model. ResultsWe noted that FCN abrogated protein expression levels of STAT3 and upstream signals (JAK1/2 and Src). In addition, FCN also attenuated DNA binding ability of STAT3 and its translocation into the nucleus. It altered the levels of upstream signaling proteins, increased SHP-1 levels, and induced substantial apoptosis in U266 cells. FCN also promoted an increased production of reactive oxygen species (ROS) and altered GSSG/GSH ratio in tumor cells. Moreover, FCN effectively abrogated tumor progression and STAT3 activation in a preclinical myeloma model. ConclusionOverall, this study suggests that FCN may have a tremendous potential to alter abnormal STAT3 activation and induce cell death in malignant cells along with causing the suppression of pathogenesis and growth of cancer through a pro-oxidant dependent molecular mechanism.

Severe acute respiratory syndrome coronavirus 2 ORF3a induces the expression of ACE2 in oral and pulmonary epithelial cells and the food supplement Vita Deyun® diminishes this effect.

The coronavirus disease 2019 (COVID-19) pandemic has become a serious global health problem and numerous studies are currently being conducted to improve understanding of the components of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus, as well as to identify solutions that mitigate the effects of COVID-19 symptoms. The nutritional supplement Vita Deyun® is composed of silymarin, glutathione, vitamin C and selenium. Studies of its individual components have demonstrated their benefits as anti-inflammatory agents, antioxidants and enhancers of the immune response. Therefore, the present study aimed to evaluate the in vitro effects of Vita Deyun on the expression of angiotensin-converting enzyme 2 (ACE2) in diverse cell lines, as well as in the presence or absence of the SARS-CoV-2 open reading frame (ORF)3a protein. Through reverse transcription-quantitative PCR, the use of viral particles containing SARS-CoV-2 ORF3a and bioinformatics analysis via the National Center for Biotechnology Information databases, ACE2 was determined to be highly expressed in oral and skin epithelial cells, with a lower expression observed in lung cells. Notably, the expression of SARS-CoV-2 ORF3a increased the level of ACE2 expression and Vita Deyun treatment diminished this effect. In addition, Vita Deyun treatment markedly decreased interleukin-18 mRNA levels. The combination of phytonutrients in Vita Deyun may help to boost the immune system and could reduce the effects of COVID-19. Ongoing clinical studies are required to provide evidence of the efficacy of Vita Deyun.

The sphingosine kinase 1 activator, K6PC-5, attenuates Ebola virus infection

SummaryEbola virus (EBOV) is responsible for outbreaks with case fatality rates of up to 90% and for an epidemic in West Africa with more than ten thousand deaths. EBOV glycoprotein (EBOV-GP) is the only viral surface protein and is responsible for viral entry into cells. Here, by employing pseudotyped EBOV-GP viral particles, we uncover a critical role for sphingolipids in inhibiting viral entry. Sphingosine kinase 1 (SphK1) catalyzes the phosphorylation of sphingosine to sphingosine 1-phosphate (S1P). The administration of the SphK1 activator, K6PC-5, or S1P, or the overexpression of SphK1 consistently exhibited striking inhibitory effects in EBOV-GP-driven entry in diverse cell lines. Finally, K6PC-5 markedly reduced the EBOV titer in infected cells and the de novo production of viral proteins. These data present K6PC-5 as an efficient tool to inhibit EBOV infection in endothelial cells and suggest further studies to evaluate its systemic effects.

High-resolution quantitative profiling of tRNA abundance and modification status in eukaryotes by mim-tRNAseq

SummaryMeasurements of cellular tRNA abundance are hampered by pervasive blocks to cDNA synthesis at modified nucleosides and the extensive similarity among tRNA genes. We overcome these limitations with modification-induced misincorporation tRNA sequencing (mim-tRNAseq), which combines a workflow for full-length cDNA library construction from endogenously modified tRNA with a comprehensive and user-friendly computational analysis toolkit. Our method accurately captures tRNA abundance and modification status in yeast, fly, and human cells and is applicable to any organism with a known genome. We applied mim-tRNAseq to discover a dramatic heterogeneity of tRNA isodecoder pools among diverse human cell lines and a surprising interdependence of modifications at distinct sites within the same tRNA transcript.

Enhancing CRISPR deletion via pharmacological delay of DNA-PKcs.

CRISPR-Cas9 deletion (CRISPR-del) is the leading approach for eliminating DNA from mammalian cells and underpins a variety of genome-editing applications. Target DNA, defined by a pair of double-strand breaks (DSBs), is removed during nonhomologous end-joining (NHEJ). However, the low efficiency of CRISPR-del results in laborious experiments and false-negative results. By using an endogenous reporter system, we show that repression of the DNA-dependent protein kinase catalytic subunit (DNA-PKcs)—an early step in NHEJ—yields substantial increases in DNA deletion. This is observed across diverse cell lines, gene delivery methods, commercial inhibitors, and guide RNAs, including those that otherwise display negligible activity. We further show that DNA-PKcs inhibition can be used to boost the sensitivity of pooled functional screens and detect true-positive hits that would otherwise be overlooked. Thus, delaying the kinetics of NHEJ relative to DSB formation is a simple and effective means of enhancing CRISPR-deletion.

Discovery of 4-alkoxy-2-aryl-6,7-dimethoxyquinolines as a new class of topoisomerase I inhibitors endowed with potent invitro anticancer activity.

In our attempt to develop potential anticancer agents targeting Topoisomerase I (TOP1), two novel series of 4-alkoxy-2-arylquinolines 14a-p and 19a-c were designed and synthesized based on structure activity relationships of the reported TOP1 inhibitors and structural features required for stabilization of TOP1-DNA cleavage complexes (TOP1ccs). The invitro anticancer activity of these two series of compounds was evaluated at one dose level using NCI-60 cancer cell lines panel. Compounds 14e-h and 14m-p, with p-substituted phenyl at C2 and propyl linker at C4, were the most potent and were selected for assay at five doses level in which they exhibited potent anticancer activity at sub-micromolar level against diverse cancer cell lines. Compound 14m was the most potent with full panel GI50 MG-MID 1.26μM and the most sensitive cancers were colon cancer, leukemia and melanoma with GI50 MG-MID 0.875, 0.904 and 0.926μM, respectively. Melanoma (LOX IMVI) was the most sensitive cell line to all tested compounds displaying GI50 from 0.116 to 0.227μM, TGI from 0.275 to 0.592μM and LC50 at sub-micromolar concentration against almost of the tested compounds. Compounds 14e-h and 14m-p were assayed using TOP1-mediated DNA cleavage assay to evaluate their ability to stabilize TOP1ccs resulting in cancer cell death. The morpholino analogs 14h and 14p exhibited moderate TOP1 inhibitory activity compared to 1μM camptothecin suggesting their use as lead compounds that can be optimized for the development of more potent anticancer agents with potential TOP1 inhibitory activity. Finally, Swiss ADME online web tool predicted that compounds 14h and 14p possessed good oral bioavailability and druglikeness characteristics.

Bioactive peptide of Cicer arietinum L. induces apoptosis in human endometrial cancer via DNA fragmentation and cell cycle arrest.

Chickpea seed proteins are alleged source of nutraceuticals. These seed proteins were subjected to different proteases to produce peptides. The efficacy of these peptides was confirmed using six diverse human cancer cell lines (PA-1, Ishikawa cells, A549, MCF-7, HepG2, MDA-MB-231). Alcalase generated peptides exhibited the highest antagonistic inhibition of Ishikawa cells. Flow cytometric analysis revealed that chickpea peptide induced S and G2 phase arrest of cell cycle in a dose dependent manner. DNA fragmentation and apoptosis occurred by down regulation of Bcl-2 expression, upregulation of Bax expression and promotion of caspase-3 initiation. Chickpea peptidesascertain potential antiproliferative molecule that can be deployed in cancer treatment regimes.

Homobivalent Lamellarin-Like Schiff Bases: In Vitro Evaluation of Their Cancer Cell Cytotoxicity and Multitargeting Anti-Alzheimer's Disease Potential.

Marine alkaloids belonging to the lamellarins family, which incorporate a 5,6-dihydro-1-phenylpyrrolo[2,1-a]isoquinoline (DHPPIQ) moiety, possess various biological activities, spanning from antiviral and antibiotic activities to cytotoxicity against tumor cells and the reversal of multidrug resistance. Expanding a series of previously reported imino adducts of DHPPIQ 2-carbaldehyde, novel aliphatic and aromatic Schiff bases were synthesized and evaluated herein for their cytotoxicity in five diverse tumor cell lines. Most of the newly synthesized compounds were found noncytotoxic in the low micromolar range (<30 μM). Based on a Multi-fingerprint Similarity Search aLgorithm (MuSSeL), mainly conceived for making protein drug target prediction, some DHPPIQ derivatives, especially bis-DHPPIQ Schiff bases linked by a phenylene bridge, were prioritized as potential hits addressing Alzheimer’s disease-related target proteins, such as cholinesterases (ChEs) and monoamine oxidases (MAOs). In agreement with MuSSeL predictions, homobivalent para-phenylene DHPPIQ Schiff base 14 exhibited a noncompetitive/mixed inhibition of human acetylcholinesterase (AChE) with Ki in the low micromolar range (4.69 μM). Interestingly, besides a certain inhibition of MAO A (50% inhibition of the cell population growth (IC50) = 12 μM), the bis-DHPPIQ 14 showed a good inhibitory activity on self-induced β-amyloid (Aβ)1–40 aggregation (IC50 = 13 μM), which resulted 3.5-fold stronger than the respective mono-DHPPIQ Schiff base 9.

One-Pot Simultaneous Detection of Multiple DNA and MicroRNA by Integrating the Cationic-Conjugated Polymer and Nuclease-Assisted Cyclic Amplification

A simple biosensor based on the cationic polymers and nuclease-assisted cyclic amplification has been constructed for the one-pot multiple detections of DNA and microRNA. In this work, we designed and synthesized a broad-spectrum polymer quencher, diazobenzene-containing poly(p-phenylethynylene) (azo-PPE(+)), to efficiently quench multiple fluorophores. Based on this concept, we prepared three DNA probes modified with three fluorophores (FAM, TAMRA, and Cy5), which entirely complement the target nucleic acids. In the presence of the target nucleic acid, the DNA probe hybridizes with the target and forms a duplex chain that is subsequently cleaved by nuclease into oligonucleotide fragments. Meanwhile, the target nucleic acid is released and rehybridized with other DNA probes, resulting in the cyclic degradation of the DNA probe. Due to the weak electrostatic interaction between the oligonucleotide fragment and azo-PPE(+), the fluorescence signal is restored and the target could be specifically detected. The combination of nuclease-assisted cyclic amplification and azo-PPE(+) enables a one-pot multiple nucleic acid detection with high sensitivity and specificity, even for the single-base-mismatched target. Moreover, the biosensor performs well in real biological samples and can distinguish miRNA expression in diverse cell lines, demonstrating its potential clinical diagnostic prospects.

Automated Quantitative Analysis of Airway Epithelial Cell Detachment Upon Fungal Challenge.

Host-pathogen interactions involve a complex interplay between host and pathogen factors, resulting in either host protective immunity or establishment of disease. One of the hallmarks for disease progression is host tissue destruction. The first host surface to interact with the opportunistic respiratory fungal pathogen, Aspergillus fumigatus, is the airway epithelium. Unravelling the mechanisms involved in airway epithelial cell damage by A. fumigatus is essential to understanding the establishment and progression of infection in the host. Although host cell damage can be measured in vitro by indirect cell lysis assays, here, we describe an automated, simple, and low-cost assay to directly visualize and quantify epithelial cell line damage after challenge with A. fumigatus. We employ the previously characterized tissue noninvasive A. fumigatus ΔpacC mutant to demonstrate the quantitative difference in cell damage relative to its parental tissue invasive strain. This assay is easily scaled up for high-throughput screening of multiple Aspergillus mutants and can be adapted to suit diverse host cell lines, different time points of infection, challenge with other microbes, and drugs or novel compounds.

Mass Cytometry – Based Cytotoxicity Assay for Profiling Natural Killer Cells

Phenotypic and functional assessment of Natural Killer (NK) cell activity against cancer cells in a clinical setting requires the integration of labor-intensive multicolor flow cytometry, chromium (Cr) release assays, and CD107a degranulation assays. To merge these into a single high-throughput assay, we developed a method for profiling NK-cancer cell interactions and simultaneous target killing using mass cytometry. In this study, a multiparameter 43-marker mass cytometry panel was applied to a co-culture of immune cells from healthy donors with diverse target cancer cell lines. DNA content, combined with classical CD45 surface staining, were used as gating parameters for co-cultures of immune cells (CD45high/DNAlow) with hematological (CD45low/DNAhigh) and solid cancer cell lines (CD45neg /DNAhigh), recapitulating flow cytometry forward and side scatter. This allows for discrimination of cancer cells from immune populations based on relative “size” and simultaneous assessment of phenotypical changes in both populations. In addition, the sensitivity of mass cytometry allows for the simultaneous detection of changes in NK cell phenotype and their degranulation upon target recognition, and for the analysis of target cells for cytotoxic protein granzyme B content, and cell death. These findings have broad applicability in clinical settings with the aim to phenotype and assess functional changes following NK-cancer cell interactions.

TACIMA-218: A Novel Pro-Oxidant Agent Exhibiting Selective Antitumoral Activity.

We report the discovery, via a unique high-throughput screening strategy, of a novel bioactive anticancer compound: Thiol Alkylating Compound Inducing Massive Apoptosis (TACIMA)-218. We demonstrate that this molecule engenders apoptotic cell death in genetically diverse murine and human cancer cell lines, irrespective of their p53 status, while sparing normal cells. TACIMA-218 causes oxidative stress in the absence of protective antioxidants normally induced by Nuclear factor erythroid 2-related factor 2 activation. As such, TACIMA-218 represses RNA translation and triggers cell signaling cascade alterations in AKT, p38, and JNK pathways. In addition, TACIMA-218 manifests thiol-alkylating properties resulting in the disruption of redox homeostasis along with key metabolic pathways. When administered to immunocompetent animals as a monotherapy, TACIMA-218 has no apparent toxicity and induces complete regression of pre-established lymphoma and melanoma tumors. In sum, TACIMA-218 is a potent oxidative stress inducer capable of selective cancer cell targeting.

An integrated pan-cancer analysis of TFAP4 aberrations and the potential clinical implications for cancer immunity.

Studies have shown that transcription factor activating enhancer binding protein 4 (TFAP4) plays a vital role in multiple types of cancer; however, the TFAP4 expression profile is still unknown, as is its value within the human pan‐cancer analysis. The present study comprehensively analysed TFAP4 expression patterns from 33 types of malignancies, along with the significance of TFAP4 for prognosis prediction and cancer immunity. TFAP4 displayed inconsistent levels of gene expression across the diverse cancer cell lines, and displayed abnormal expression within most malignant tumours, which closely corresponded to overall survival. More importantly, the TFAP4 level was also significantly related to the degree of tumour infiltration. TFAP4 was correlated using gene markers in tumour‐infiltrating immune cells and immune scores. TFAP4 expression was correlated with tumour mutation burden and microsatellite instability in different cancer types, and enrichment analyses identified TFAP4‐associated terms and pathways. The present study comprehensively analysed the expression of TFAP4 across 33 distinct types of cancers, which revealed that TFAP4 may possibly play a vital role during cancer formation and development. TFAP4 is related to differing degrees of immune infiltration within cancers, which suggests the potential of TFAP4 as an immunotherapy target in cancers. Our study demonstrated that TFAP4 plays an important role in tumorigenesis as a prognostic biomarker, which highlights the possibility of developing new targeted treatments.

Stimuli responsive nanogels with intrinsic fluorescence: Promising nanovehicles for controlled drug delivery and cell internalization detection in diverse cancer cell lines

Nanogels are an important class of nanomaterials with a wide field of potential applications in nanomedicine. Fluorescent nanogels have the great advantage of the application in theranostics. In this study, intrinsic fluorescent nanogels, crosslinked with fluorescein O,O′-diacrylate (FDA), were synthetized and studied for first time, via a robust methodology of one–pot, resulting in yields in scale of grams. This new type of nanovehicles avoid the susceptibility to leaching, inhomogeneity, or degradation of the fluorescent moieties, observed in others approaches using encapsulation or conjugation of fluorescent molecules, additionally they show multi-stimuli response to temperature and pH. Both anionic (AFN) and cationic fluorescent nanogels (CFN) were successfully produced, which showed strong fluorescence intensity at neutral pH, the fluorescence was quenched at pH 5. Cisplatin (CDDP) and 5-fluorouracil (5-FU) were loaded into the nanogels, leading to partially quenching of the fluorescence. The kinetics of drug release and cell-viability studies are also reported. Cell-internalization studies were performed by fluorescence microscopy in non-small lung adenocarcinoma cell line NCI-H1437 and human colorectal cancer cell line HCT-116; demonstrating that the AFN were not internalized and CFN were easily internalized at concentrations of 100 µg/mL in 5 min, irrespective of the molecular weight of the Poly(ethylenglycol)-chains. The developed method can be expanded to the preparation of other fluorescent nanogel types; whereas the prepared AFN and CFN could be studied as nanocarriers to be internalized in diverse cell-lines.

Development of a UPLC-MS/MS Method for the Quantification of VS-5584 and Its Application in Pharmacokinetic Studies in Rats

VS-5584 is a small-molecular compound that showed equivalent activity against mTOR and all class I PI3K isoforms and demonstrated preclinical activity in diverse cancer cell lines and xenograft tumor model, and rational combination of VS-5584 and other target therapies achieved promising outcomes in oncology. In the present study, we established and validated a simple and sensitive UPLC-MS/MS method for the determination of VS-5584 in plasma samples. VS-5584 was separated via an Acquity UPLC BEH C18 column, with a mobile phase composed of acetonitrile and 0.2% formic acid in water (40 : 60). The calibration curve displayed a good linearity in the range of 1.0–1000 ng/mL, with satisfactory accuracy (−13.6% < RE% < 8.8%) and precision (CV%, less than 9.2%). The validated method was then applied to a pharmacokinetic study in rats. After administration of 10 mg/kg, VS-5584 was absorbed quickly and reached a peak concentration of 473.2 ± 72.0 ng/mL after 20 min. The established method allows for the quantification of VS-5584 in rat plasma in detail and can be utilized to successfully describe the pharmacokinetic profile of VS-5584.

Synthesis, Characterization, and Biological Activity of Anthraquinone-Substituted Imidazolium Salts for the Treatment of Bladder Cancer

BACKGROUND: Bladder cancer is one of the most common types of cancer diagnosed each year, and more than half of patients have non-muscle invasive bladder cancer (NMIBC). The standard of care for patients with high-grade NMIBC is Bacillus Calmette-Guerin (BCG). Unfortunately, multiple BCG shortages have limited access to this treatment. Available alternatives using intravesical administration of chemotherapy have some efficacy, but lack prospective validation and long-term outcomes. Development of novel intravesical therapies may provide more active alternatives to BCG for patients with high-grade NMIBC. OBJECTIVE: To develop an optimal imidazolium salt for the intravesical treatment of NMIBC and determine preliminary in vitro activity of anthraquinone-substituted imidazolium salts. METHODS: The development of the anthraquinone-substituted imidazolium salts was undertaken in an attempt to increase the potency of this class of compounds by incorporating the quinone functional group observed in the chemotherapeutics doxorubicin, valrubicin, and mitomycin. All compounds were characterized by 1H and 13C NMR spectroscopy and infrared spectroscopy. Furthermore, these imidazolium salts were tested for in vitro cytotoxicity by the Developmental Therapeutics Program (DTP) on the NCI-60 human tumor cell line screening. Additional in vitro testing was performed against diverse bladder cancer cell lines (RT112, TCCSUP, J82, and UMUC13) using CellTiter-Glo® assays and colony-forming assays. RESULTS: The NCI-60 cell line screening indicated that compound 7 had the highest activity and was concluded to be the optimal compound for further study. Using CellTiter-Glo® assays on bladder cancer cell lines, 50% growth inhibitory concentration (IC50) values were determined to range from 32–50μM after an exposure of 1 h, for compound 7. Further evaluation of the compound by colony-forming assays showed the complete inhibition of growth at 10 days post a 100μM dose of compound 7 for 1 h. CONCLUSIONS: The most active lipophilic anthraquinone imidazolium salt, compound 7, could be a viable treatment for non-muscle invasive bladder cancer as it exhibits a cell-killing effect at a 1 h time period and completely inhibits cancer regrowth in colony-forming assays.