Tumor alkalization therapy: misconception or good therapeutics perspective? - the case of malignant ascites.

Tumor acidity has been identified as a key factor in promoting cancer progression, metastasis, and resistance. Tumor alkalization therapy has emerged as a potential strategy for cancer treatment. This article provides preclinical and clinical evidence for tumor alkalization therapy as a promising cancer treatment strategy. The potential of tumor alkalization therapy using sodium bicarbonate in the treatment of malignant ascites was studied. The concept of intraperitoneal perfusion with an alkalizing solution to increase the extracellular pH and its antitumor effect were explored. The significant extension in the overall survival of the Ehrlich ascites carcinoma mice treated with sodium bicarbonate solution compared to those treated with a sodium chloride solution was observed. In the sodium bicarbonate group, mice had a median survival of 30 days after tumor cell injection, which was significantly (p<0.05) different from the median survival of 18 days in the sodium chloride group and 14 days in the intact group. We also performed a case study of a patient with ovarian cancer malignant ascites resistant to previous lines of chemotherapy who underwent intraperitoneal perfusions with a sodium bicarbonate solution, resulting in a significant drop of CA-125 levels from 5600 U/mL to 2200 U/mL in and disappearance of ascites, indicating the potential effectiveness of the treatment. The preclinical and clinical results obtained using sodium bicarbonate perfusion in the treatment of malignant ascites represent a small yet significant contribution to the evolving field of tumor alkalization as a cancer therapy. They unequivocally affirm the good prospects of this concept.

Cell-penetrating peptide and cationic liposomes mediated siRNA delivery to arrest growth of chronic myeloid leukemia cells in vitro.

Gene silencing through RNA interference (RNAi) is a promising therapeutic approach for a wide range of disorders, including cancer. Non-viral gene therapy, using specific siRNAs against BCR-ABL1, can be a supportive or alternative measure to traditional chronic myeloid leukemia (CML) tyrosine kinase inhibitor (TKIs) therapies, given the prevalence of clinical TKI resistance. The main challenge for such approaches remains the development of the effective delivery system for siRNA tailored to the specific disease model. The purpose of this study was to examine and compare the efficiency of endosomolytic cell penetrating peptide (CPP) EB1 and PEG2000-decorated cationic liposomes composed of polycationic lipid 1,26-bis(cholest-5-en-3-yloxycarbonylamino)-7,11,16,20-tetraazahexacosane tetrahydrochloride (2Ð¥3) and helper lipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) for anti-bcr-abl siRNA delivery into the K562 human CML cell line. We show that both EB1 and 2Ð¥3-DOPE-DSPE-PEG2000 (0.62 % mol.) liposomes effectively deliver siRNA into K562 cells by endocytic mechanisms, and the use of liposomes leads to more effective inhibition of expression of the targeted gene (BCR-ABL1) and cancer cell proliferation. Taken together, these findings suggest that PEG-decorated cationic liposomes mediated siRNA delivery allows an effective antisense suppression of certain oncogenes, and represents a promising new class of therapies for CML.

Management and Treatment of Non-small Cell Lung Cancer with MET Alteration and Mechanisms of Resistance.

OPINION STATEMENT: MET-driven tumors are a heterogenous group of non-small cell lung cancers (NSCLC) with activating mutations. Pathologic activation of MET can be achieved with increased number of gene copies overexpression, or decreased protein degradation through several mechanisms, including mutations, amplifications, or fusions. Besides its role as primary driver, MET activation might also mediate resistance to kinase inhibitors in NSCLC with various other actionable alterations. While checkpoint inhibitors have modest efficacy in MET-driven tumors, several approaches of targeted blockade are available. Among them the most promising are small tyrosine kinase inhibitors, antibody-drug conjugates, and bispecific antibodies. Unfortunately, resistance is virtually inevitable. Resistance to small kinase inhibitors might be mediated by kinase domain mutations or activation of shunting cascades. Various resistance mechanisms might be present in one patient, making it overcoming an unresolved problem.

Tumor acidity: From hallmark of cancer to target of treatment.

Tumor acidity is one of the cancer hallmarks and is associated with metabolic reprogramming and the use of glycolysis, which results in a high intracellular lactic acid concentration. Cancer cells avoid acid stress major by the activation and expression of proton and lactate transporters and exchangers and have an inverted pH gradient (extracellular and intracellular pHs are acid and alkaline, respectively). The shift in the tumor acid-base balance promotes proliferation, apoptosis avoidance, invasiveness, metastatic potential, aggressiveness, immune evasion, and treatment resistance. For example, weak-base chemotherapeutic agents may have a substantially reduced cellular uptake capacity due to "ion trapping". Lactic acid negatively affects the functions of activated effector T cells, stimulates regulatory T cells, and promotes them to express programmed cell death receptor 1. On the other hand, the inversion of pH gradient could be a cancer weakness that will allow the development of new promising therapies, such as tumor-targeted pH-sensitive antibodies and pH-responsible nanoparticle conjugates with anticancer drugs. The regulation of tumor pH levels by pharmacological inhibition of pH-responsible proteins (monocarboxylate transporters, H+-ATPase, etc.) and lactate dehydrogenase A is also a promising anticancer strategy. Another idea is the oral or parenteral use of buffer systems, such as sodium bicarbonate, to neutralize tumor acidity. Buffering therapy does not counteract standard treatment methods and can be used in combination to increase effectiveness. However, the mechanisms of the anticancer effect of buffering therapy are still unclear, and more research is needed. We have attempted to summarize the basic knowledge about tumor acidity.

Minimal PK/PD model for simultaneous description of the maximal tolerated dose and metronomic treatment outcomes in mouse tumor models.

PURPOSE: Metronomic chemotherapy (MC) is a promising approach where, in contrast to the conventional maximal tolerated dose (MTD) strategy, regular fractionated doses of the drug are used. This approach has proven its efficacy, although drug dosing and scheduling are often chosen empirically. Pharmacokinetic/pharmacodynamic (PK/PD) models provide a way to choose optimal protocols with computational methods. Existing models are usually too complicated and are valid for only a subset of drug schedules. To address this issue, we propose herein a simple model that can describe MC and MTD regimens simultaneously. METHODS: The minimal model comprises tumor suppression due to antiangiogenic drug effect together with a cell-kill term, responsible for its cytotoxicity. The model was tested on data obtained on tumor-bearing mice treated with gemcitabine in ether MTD, MC, or combined (MTD + MC) regimens. RESULTS: We conducted a number of tests in which data were divided in various ways into training and validation sets. The model successfully described different trends in the MTD and MC regimens. With parameters obtained by fitting the model to MTD data, the simulations correctly predicted trends in both the MC and combined therapy groups. CONCLUSION: Our results demonstrate that the proposed model presents a minimal yet efficient tool for modeling outcomes in different treatment regimens in mice. We hope that this model has the potential for use in clinical practice in the development of patient-specific chemotherapy scheduling protocols based on observed treatment response.

New Data on Organization and Spatial Localization of B-Chromosomes in Cell Nuclei of the Yellow-Necked Mouse Apodemus flavicollis.

The gene composition, function and evolution of B-chromosomes (Bs) have been actively discussed in recent years. However, the additional genomic elements are still enigmatic. One of Bs mysteries is their spatial organization in the interphase nucleus. It is known that heterochromatic compartments are not randomly localized in a nucleus. The purpose of this work was to study the organization and three-dimensional spatial arrangement of Bs in the interphase nucleus. Using microdissection of Bs and autosome centromeric heterochromatic regions of the yellow-necked mouse (Apodemus flavicollis) we obtained DNA probes for further two-dimensional (2D)- and three-dimensional (3D)- fluorescence in situ hybridization (FISH) studies. Simultaneous in situ hybridization of obtained here B-specific DNA probes and autosomal C-positive pericentromeric region-specific probes further corroborated the previously stated hypothesis about the pseudoautosomal origin of the additional chromosomes of this species. Analysis of the spatial organization of the Bs demonstrated the peripheral location of B-specific chromatin within the interphase nucleus and feasible contact with the nuclear envelope (similarly to pericentromeric regions of autosomes and sex chromosomes). It is assumed that such interaction is essential for the regulation of nuclear architecture. It also points out that Bs may follow the same mechanism as sex chromosomes to avoid a meiotic checkpoint.

Amino acid residue at the 165th position tunes EYFP chromophore maturation. A structure-based design.

For the whole GFP family, a few cases, when a single mutation in the chromophore environment strongly inhibits maturation, were described. Here we study EYFP-F165G - a variant of the enhanced yellow fluorescent protein - obtained by a single F165G replacement, and demonstrated multiple fluorescent states represented by the minor emission peaks in blue and yellow ranges (~470 and ~530 nm), and the major peak at ~330 nm. The latter has been assigned to tryptophan fluorescence, quenched due to excitation energy transfer to the mature chromophore in the parental EYFP protein. EYFP-F165G crystal structure revealed two general independent routes of post-translational chemistry, resulting in two main states of the polypeptide chain with the intact chromophore forming triad (~85%) and mature chromophore (~15%). Our experiments thus highlighted important stereochemical role of the 165th position strongly affecting spectral characteristics of the protein. On the basis of the determined EYFP-F165G three-dimensional structure, new variants with ~ 2-fold improved brightness were engineered.

Impacts of OrX and cAMP-insensitive Orco to the insect olfactory heteromer activity.

Insect odorant receptors (ORs) have been suggested to function as ligand-gated cation channels, with OrX/Orco heteromers combining ionotropic and metabotropic activity. The latter is mediated by different G proteins and results in Orco self-activation by cyclic nucleotide binding. In this contribution, we co-express the odor-specific subunits DmOr49b and DmOr59b with either wild-type Orco or an Orco-PKC mutant lacking cAMP activation heterologously in mammalian cells. We show that the characteristics of heteromers strongly depend on both the OrX type and the coreceptor variant. Thus, methyl acetate-sensitive Or59b/Orco demonstrated 25-fold faster response kinetics over o-cresol-specific Or49b/Orco, while the latter required a 10-100 times lower ligand concentration to evoke a similar electrical response. Compared to wild-type Orco, Orco-PKC decreased odorant sensitivity in both heteromers, and blocked an outward current rectification intrinsic to the Or49b/Orco pair. Our observations thus provide an insight into insect OrX/Orco functioning, highlighting their natural and artificial tuning features and laying the groundwork for their application in chemogenetics, drug screening, and repellent design.

Triphenilphosphonium Analogs of Chloramphenicol as Dual-Acting Antimicrobial and Antiproliferating Agents.

In the current work, in continuation of our recent research, we synthesized and studied new chimeric compounds, including the ribosome-targeting antibiotic chloramphenicol (CHL) and the membrane-penetrating cation triphenylphosphonium (TPP), which are linked by alkyl groups of different lengths. Using various biochemical assays, we showed that these CAM-Cn-TPP compounds bind to the bacterial ribosome, inhibit protein synthesis in vitro and in vivo in a way similar to that of the parent CHL, and significantly reduce membrane potential. Similar to CAM-C4-TPP, the mode of action of CAM-C10-TPP and CAM-C14-TPP in bacterial ribosomes differs from that of CHL. By simulating the dynamics of CAM-Cn-TPP complexes with bacterial ribosomes, we proposed a possible explanation for the specificity of the action of these analogs in the translation process. CAM-C10-TPP and CAM-C14-TPP more strongly inhibit the growth of the Gram-positive bacteria, as compared to CHL, and suppress some CHL-resistant bacterial strains. Thus, we have shown that TPP derivatives of CHL are dual-acting compounds targeting both the ribosomes and cellular membranes of bacteria. The TPP fragment of CAM-Cn-TPP compounds has an inhibitory effect on bacteria. Moreover, since the mitochondria of eukaryotic cells possess qualities similar to those of their prokaryotic ancestors, we demonstrate the possibility of targeting chemoresistant cancer cells with these compounds.

Tetracenomycin X inhibits translation by binding within the ribosomal exit tunnel.

The increase in multi-drug resistant pathogenic bacteria is making our current arsenal of clinically used antibiotics obsolete, highlighting the urgent need for new lead compounds with distinct target binding sites to avoid cross-resistance. Here we report that the aromatic polyketide antibiotic tetracenomycin (TcmX) is a potent inhibitor of protein synthesis, and does not induce DNA damage as previously thought. Despite the structural similarity to the well-known translation inhibitor tetracycline, we show that TcmX does not interact with the small ribosomal subunit, but rather binds to the large subunit, within the polypeptide exit tunnel. This previously unappreciated binding site is located adjacent to the macrolide-binding site, where TcmX stacks on the noncanonical basepair formed by U1782 and U2586 of the 23S ribosomal RNA. Although the binding site is distinct from the macrolide antibiotics, our results indicate that like macrolides, TcmX allows translation of short oligopeptides before further translation is blocked.

Two independent routes of post-translational chemistry in fluorescent protein FusionRed.

The crystal structure of monomeric red fluorescent protein FusionRed (λex/λem 580/608 mn) has been determined at 1.09 Å resolution and revealed two alternative routes of post-translational chemistry, resulting in distinctly different products. The refinement occupancies suggest the 60:40 ratio of the mature Met63-Tyr64-Gly65 chromophore and uncyclized chromophore-forming tripeptide with the protein backbone cleaved between Met63 and the preceding Phe62 and oxidized Cα-Cß bond of Tyr64. We analyzed the structures of FusionRed and several related red fluorescent proteins, identified structural elements causing hydrolysis of the peptide bond, and verified their impact by single point mutagenesis. These findings advance the understanding of the post-translational chemistry of GFP-like fluorescent proteins beyond the canonical cyclization-dehydration-oxidation mechanism. They also show that impaired cyclization does not prevent chromophore-forming tripeptide from further transformations enabled by the same set of catalytic residues. Our mutagenesis efforts resulted in inhibition of the peptide backbone cleavage, and a FusionRed variant with ~30% improved effective brightness.

Insights into the improved macrolide inhibitory activity from the high-resolution cryo-EM structure of dirithromycin bound to the E. coli 70S ribosome.

Macrolides are one of the most successful and widely used classes of antibacterials, which kill or stop the growth of pathogenic bacteria by binding near the active site of the ribosome and interfering with protein synthesis. Dirithromycin is a derivative of the prototype macrolide erythromycin with additional hydrophobic side chain. In our recent study, we have discovered that the side chain of dirithromycin forms lone pair-π stacking interaction with the aromatic imidazole ring of the His69 residue in ribosomal protein uL4 of the Thermus thermophilus 70S ribosome. In the current work, we found that neither the presence of the side chain, nor the additional contact with the ribosome, improve the binding affinity of dirithromycin to the ribosome. Nevertheless, we found that dirithromycin is a more potent inhibitor of in vitro protein synthesis in comparison with its parent compound, erythromycin. Using high-resolution cryo-electron microscopy, we determined the structure of the dirithromycin bound to the translating Escherichia coli 70S ribosome, which suggests that the better inhibitory properties of the drug could be rationalized by the side chain of dirithromycin pointing into the lumen of the nascent peptide exit tunnel, where it can interfere with the normal passage of the growing polypeptide chain.

Structure of Dirithromycin Bound to the Bacterial Ribosome Suggests New Ways for Rational Improvement of Macrolides.

Although macrolides are known as excellent antibacterials, their medical use has been significantly limited due to the spread of bacterial drug resistance. Therefore, it is necessary to develop new potent macrolides to combat the emergence of drug-resistant pathogens. One of the key steps in rational drug design is the identification of chemical groups that mediate binding of the drug to its target and their subsequent derivatization to strengthen drug-target interactions. In the case of macrolides, a few groups are known to be important for drug binding to the ribosome, such as desosamine. Search for new chemical moieties that improve the interactions of a macrolide with the 70S ribosome might be of crucial importance for the invention of new macrolides. For this purpose, here we studied a classic macrolide, dirithromycin, which has an extended (2-methoxyethoxy)-methyl side chain attached to the C-9/C-11 atoms of the macrolactone ring that can account for strong binding of dirithromycin to the 70S ribosome. By solving the crystal structure of the 70S ribosome in complex with dirithromycin, we found that its side chain interacts with the wall of the nascent peptide exit tunnel in an idiosyncratic fashion: its side chain forms a lone pair-π stacking interaction with the aromatic imidazole ring of the His69 residue in ribosomal protein uL4. To our knowledge, the ability of this side chain to form a contact in the macrolide binding pocket has not been reported previously and potentially can open new avenues for further exploration by medicinal chemists developing next-generation macrolide antibiotics active against resistant pathogens.

Size-tunable MRI-visible nitroxide-based magnetic mixed micelles: preparation, stability, and theranostic application.

Metal-free magnetic mixed micelles (mean diameter: 16 nm) composed of biocompatible surfactant Tween 80 and hydrophobic pyrrolidine-N-oxyl radical were prepared by mixing them in phosphate-buffered saline. The magnetic mixed micelles were characterized by dynamic light scattering and small angle neutron scattering measurements. The stability of the micelles is found to depend on the length of alkyl side chain in the nitroxide compounds and degree of unsaturation in the hydrophobic chain in the surfactant. The size of the mixed micelle can be tuned by changing the molar ratio of Tween 80 and nitroxyl radical. In view of theranostic application of the micelle, the cytotoxicity and stability in a physiological environment was investigated; the mixed micelle exhibited no cytotoxicity, high colloidal stability and high resistance towards reduction by large excess ascorbic acid. The in vitro and in vivo magnetic resonance imaging (MRI) revealed sufficient contrast enhancement in the proton longitudinal relaxation time (T 1) weighted images. In addition, hydrophobic fluorophores and an anticancer drug are stably encapsulated in the mixed micelles and showed fluorescence (FL) upon reduction by ascorbic acid and cytotoxicity to cancer cells, respectively. For example, the paclitaxel-loaded mixed micelles efficiently suppressed cancer cell growth. Furthermore, they were found to give higher MRI contrast (higher r 1 value) in vitro than the micelles without paclitaxel. The magnetic mixed micelles presented here are promising theranostic agents in nanomedicine due to their high biocompatibility and high resistivity towards reduction as well as functioning as a drug carrier in therapy and MR or FL imaging probe in diagnosis.

Magnetic Mixed Micelles Composed of a Non-Ionic Surfactant and Nitroxide Radicals Containing a D-Glucosamine Unit: Preparation, Stability, and Biomedical Application.

Metal-free magnetic mixed micelles (mean diameter: < 20 nm) were prepared by mixing the biocompatible non-ionic surfactant Tween 80 and the non-toxic, hydrophobic pyrrolidine-N-oxyl radicals bearing a D-glucosamine unit in pH 7.4 phosphate-buffered saline (PBS). The time-course stability and in vitro magnetic resonance imaging (MRI) contrast ability of the mixed micelles was found to depend on the length of the alkyl chain in the nitroxide radicals. It was also confirmed that the mixed micelles exhibited no toxicity in vivo and in vitro and high stability in the presence of a large excess of ascorbic acid. The in vivo MRI experiment revealed that one of these mixed micelles showed much higher contrast enhancement in the proton longitudinal relaxation time (T1) weighted images than other magnetic mixed micelles that we have reported previously. Thus, the magnetic mixed micelles presented here are expected to serve as a promising contrast agent for theranostic nanomedicines, such as MRI-visible targeted drug delivery carriers.

Insertion of the voltage-sensitive domain into circularly permuted red fluorescent protein as a design for genetically encoded voltage sensor.

Visualization of electrical activity in living cells represents an important challenge in context of basic neurophysiological studies. Here we report a new voltage sensitive fluorescent indicator which response could be detected by fluorescence monitoring in a single red channel. To the best of our knowledge, this is the first fluorescent protein-based voltage sensor which uses insertion-into-circular permutant topology to provide an efficient interaction between sensitive and reporter domains. Its fluorescent core originates from red fluorescent protein (FP) FusionRed, which has optimal spectral characteristics to be used in whole body imaging techniques. Indicators using the same domain topology could become a new perspective for the FP-based voltage sensors that are traditionally based on Förster resonance energy transfer (FRET).

Resistance mechanisms to drug therapy in breast cancer and other solid tumors: An opinion.

Cancer is an important contributor to mortality worldwide. Breast cancer is the most common solid tumor in women. Despite numerous drug combinations and regimens, all patients with advanced breast cancer, similarly to other solid tumors, inevitably develop resistance to treatment. Identified mechanisms of resistance could be classified into intra- and extracellular mechanisms. Intracellular mechanisms include drug metabolism and efflux, target modulations and damage restoration. Extracellular mechanisms might be attributed to the crosstalk between tumor cells and environmental factors. However, current knowledge concerning resistance mechanisms cannot completely explain the phenomenon of multi-drug resistance, which occurs in the vast majority of patients treated with chemotherapy. In this opinion article, we investigate the role of these factors in the development of drug-resistance.

Functioning of Fluorescent Proteins in Aggregates in Anthozoa Species and in Recombinant Artificial Models.

Despite great advances in practical applications of fluorescent proteins (FPs), their natural function is poorly understood. FPs display complex spatio-temporal expression patterns in living Anthozoa coral polyps. Here we applied confocal microscopy, specifically, the fluorescence recovery after photobleaching (FRAP) technique to analyze intracellular localization and mobility of endogenous FPs in live tissues. We observed three distinct types of protein distributions in living tissues. One type of distribution, characteristic for Anemonia, Discosoma and Zoanthus, is free, highly mobile cytoplasmic localization. Another pattern is seen in FPs localized to numerous intracellular vesicles, observed in Clavularia. The third most intriguing type of intracellular localization is with respect to the spindle-shaped aggregates and lozenge crystals several micrometers in size observed in Zoanthus samples. No protein mobility within those structures was detected by FRAP. This finding encouraged us to develop artificial aggregating FPs. We constructed "trio-FPs" consisting of three tandem copies of tetrameric FPs and demonstrated that they form multiple bright foci upon expression in mammalian cells. High brightness of the aggregates is advantageous for early detection of weak promoter activities. Simultaneously, larger aggregates can induce significant cytostatic and cytotoxic effects and thus such tags are not suitable for long-term and high-level expression.

Abnormal expression of ATP1A1 and ATP1A2 in breast cancer.

Breast cancer is the first in incidence and the second in death among all solid tumors occurring in women. The identification of molecular genetic abnormalities in breast cancer is important to improve the results of treatment. In the present study, we analyzed microarray data of breast cancer expression profiling (NCBI GEO database, accession GSE65194), focusing on Na +/K +-ATPase coding genes. We found overexpression of the ATP1A1 and down-regulation of the ATP1A2. We expect that our research could help to improve the understanding of predictive and prognostic features of breast cancer.

Pulse mode of laser photodynamic treatment induced cell apoptosis.

One of the factors limiting photodynamic therapy (PDT) is hypoxia in tumor cells during photodynamic action. PDT with pulse mode irradiation and appropriate irradiation parameters could be more effective in the singlet oxygen generation and tissue re-oxygenation than continuous wave (CW) mode. We theoretically demonstrate differences between the cumulative singlet oxygen concentration in PDT using pulse mode and CW mode of laser irradiation. In vitro experimental results show that photodynamic treatment with pulse mode irradiation has similar cytotoxicity to CW mode and induces mainly cell apoptosis, whereas CW mode induces necrotic cell death. We assume that the cumulative singlet oxygen concentration and the temporal distribution of singlet oxygen are important in photodynamic cytotoxicity and apoptosis initiation. We expect our research may improve irradiation protocols and photodynamic therapy efficiency.