monitoring of reactive oxygen species (ROS) in tumors during treatment with anticancer therapy is important for understanding the mechanism of action and in the design of new anticancer drugs. In ...this work, a platinized nanoelectrode is placed into a single cell for detection of the ROS signal, and drug-induced ROS production is then recorded. The main advantages of this method are the short incubation time with the drug and its high sensitivity which allows the detection of low intracellular ROS concentrations. We have shown that our new method can measure the ROS response to chemotherapy in tumor-bearing mice in real-time. ROS levels were measured
inside the tumor at different depths in response to doxorubicin. This work provides an effective new approach for the measurement of intracellular ROS by platinized nanoelectrodes.
Patients with metastatic melanoma are difficult to treat and have a very poor prognosis because of high resistance to therapy. Recent evidence indicates that tumors could overcome death through ...autophagy, a survival mechanism, which cancer cells use under lack of energy and nutrient deprivation. Melanoma cells have different sensitivity to temozolomide (TMZ) treatment. In this study, we showed that the combination of autophagy inhibitors chloroquine or LY294002 and TMZ induced enhanced cytotoxicity of alkylating agents on human melanoma cell lines. All assays were performed on patient-derived melanoma cell lines. The effectiveness of the combined treatment of TMZ and autophagy inhibitors was determined using an MTT assay. Next, we analyzed the expression mRNA level of Beclin 1, LC3B, and p62/STSQM1 and the relative expression of LC3B protein under combined treatment. Autophagic flux was determined by analysis of colocalization of Lysotracker Red and LC3B puncta. Apoptosis was measured by Annexin V/PI staining. Cell cycle analyses were carried out by flow cytometry. We showed that autophagy inhibition could enhance melanoma cell death combined with TMZ therapy. Chloroquine synergistically enhanced the TMZ-induced growth arrest and increased the G0/G1 population in Mel Z and Mel IL cell lines, but not Mel MTP. The expression analysis showed that autophagy involvement in TMZ enhanced cytotoxicity. Furthermore, LY294002, an early-stage autophagy, and PI3K inhibitor were found to exert similar effects. Both chloroquine and LY294002 improved the cytotoxic effect of TMZ treatment, making this combination applicable as a potent antitumor treatment for metastatic melanoma.
Summary
Objective
The problem of drug resistance to BRAF-targeted therapy often occurs in melanoma treatment. Activation of PI3K/AKT/mTOR signaling pathway is one of the mechanisms of acquired ...resistance and a potential target for treatment. In the current research, we investigated that dual inhibition of mTOR and MEK synergistically reduced the viability of melanoma cells in vitro.
Methods
A combination of rapamycin (a macrolide immunosuppressant, mTOR inhibitor) and binimetinib (an anti-cancer small molecule, selective inhibitor of MEK) was studied using a panel of melanoma cell lines, including patient-derived cells.
Results
It was found, that combinatorial therapy of rapamycin (250 nM) and binimetinib (2 μM) resulted in 25% of cell viability compared to either rapamycin (85%) or binimetinib alone (50%) for A375 and vemurafenib-resistant Mel IL/R cells. The suppressed activation of mTOR and MEK by combined rapamycin and binimetinib treatment was confirmed using Western blot assay. Cell death occured via the apoptosis pathway; however, the combination treatment significantly increased the apoptosis only for Mel IL/R cells. The enhanced cytotoxic effect was also associated with enhanced cell cycle arrest in the G0/G1 phase.
Conclusion
In general, we provide the evidence that dual inhibition of mTOR and MEK could be promising for further preclinical investigations.
•A microarray-based method was evaluated to identify ctDNA BRAF mutations in melanoma patients.•The presence of BRAF mutations in cfDNA correlates with tumor progression (P=0.005).•Increased levels ...of cfDNA correlate with tumor progression (P=0.02).•Coincidence of genotypes between the tumor DNA and ctDNA was 65%.
Background: Circulating tumor DNA (ctDNA) holds great potential for cancer therapy and can provide diagnostic and prognostic information before and during treatment.
Methods: Plasma DNA samples from 97 melanoma patients, 20 healthy donors and 3 patients with benign skin tumors were analyzed by microarray analysis and droplet digital PCR (ddPCR).
Results: A microarray for simultaneous detection of six BRAF V600 mutations in ctDNA has been developed. The method allows the detection of 0.05% mutated DNA from WT DNA background. For paired samples (pre-surgery plasma and tumor tissue) isolated from 74 patients, the concordance of genotypes between tumor DNA and ctDNA was 65% (48/74). BRAF mutations in ctDNA were detected in 27/50 patients with BRAF-positive tumors and in 3/24 patients with BRAF wild-type tumors. The presence of ctDNA BRAF mutations in 23 plasma samples from melanoma patients undergoing therapy correlated significantly with tumor progression (P=0.005). The increase in cell-free DNA levels measured by ddPCR also correlated with disease progression (P=0.02). The concordance of results obtained by microarray identification of BRAF mutations and those obtained by ddPCR was 91%.
Conclusion: The novel microarray-based approach can be a useful non-invasive tool for accurate identification of ctDNA BRAF mutations to monitor disease progression.
Herein, we describe the design, synthesis, and biological evaluation of novel betulin and N-acetyl-d-galactosamine (GalNAc) glycoconjugates and suggest them as targeted agents against hepatocellular ...carcinoma. We prepared six conjugates derived via the C-3 and C-28 positions of betulin with one or two saccharide ligands. These molecules demonstrate high affinity to the asialoglycoprotein receptor (ASGPR) of hepatocytes assessed by in silico modeling and surface plasmon resonance tests. Cytotoxicity studies in vitro revealed a bivalent conjugate with moderate activity, selectivity of action, and cytostatic properties against hepatocellular carcinoma cells HepG2. An additional investigation confirmed the specific engagement with HepG2 cells by the enhanced generation of reactive oxygen species. Stability tests demonstrated its lability to acidic media and to intracellular enzymes. Therefore, the selected bivalent conjugate represents a new potential agent targeted against hepatocellular carcinoma. Further extensive studies of the cellular uptake in vitro and the real-time microdistribution in the murine liver in vivo for fluorescent dye-labeled analogue showed its selective internalization into hepatocytes due to the presence of GalNAc ligand in comparison with reference compounds. The betulin and GalNAc glycoconjugates can therefore be considered as a new strategy for developing therapeutic agents based on natural triterpenoids.
Background: More than 500 thousand new cases of malignant neoplasms are registered annually in the Russian Federation, of which more than 50 thousand new cases are due to hereditary forms. Improving ...the diagnosis of these diseases will make it possible to detect tumors at the early stages and take timely preventive and therapeutic measures.
Aims: Creation of a database and development of a software for the NGS data analysis for the prevention and early diagnosis of hereditary forms of oncological diseases.
Methods: The present study used 636 DNA samples obtained from cancer patients with a high hereditary risk or a burdened family history. DNA was isolated from blood lymphocytes. DNA libraries were prepared with a KAPA Target Enrichment Panel (Roche). The panel included probes for targeted enrichment of the coding region of 44 genes. NGS was performed on the MiSeq platform (Illumina).
Results: We identified 65 pathogenic/ probably pathogenic nucleotide sequence variants in 96 patients in the ATM, BLM, BRCA1, BRCA2, CHEK2, EPCAM, MEN1, MLH1, MSH2, MSH3, MSH6, MUTYH, PALB2, TP53 genes. We also identified 2858 nucleotide sequence variants of unknown clinical significance. Conclusions: We have created a local database that contains both genetic variants and clinical and anamnestic data. The database contains 4763 nucleotide sequence variants at the moment, among which 2522 are unique variants identified in a single patient.
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•Cationized polysaccharide microgels serve as a core for anionic liposome satellites.•Polylysine links alginate and liposomes functionalities in a multifunctional ...carrier.•Electrostatic bonding of polylysine to anionic species restricts its cytotoxicity.
Multi-liposomal assemblies have been prepared via electrostatic adsorption of cationic polylysine over anionic alginate microgels followed by adsorption of anionic liposomes over the polylysine layer. The adsorbed liposomes (up to 150 per one assembly) retained their integrity; the assemblies are stable (do not dissociate) in physiological solution with NaCl = 0.15 M. The assemblies showed a low cytotoxicity; incorporation of Dox into liposomes did not alter the drug efficacy towards melanoma cells. The results can be used to obtain biocompatible and biodegradable multi-liposomal containers for encapsulation and delivery of drugs and increase their therapeutic effect.
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