Tracking the cellular immune response from early premalignancy to active multiple myeloma in a new model.

To calibrate a murine model to study premalignant to malignant multiple myeloma, mice were inoculated with different amounts of myeloma cells, and changes in the immune profile were tracked for over 200 days. The model highlights the development of T-cell exhaustion and suppressor before the appearance of clinical symptoms.

Synthesis and anticancer properties of novel dolastatin 10 analogs featuring five-membered heterocyclic rings with a linkable group at the C-terminus.

Dolastatin 10 (Dol-10), a natural marine-source pentapeptide, is a powerful antimitotic agent regarded as one of the most potent anticancer compounds found to date. Dol-10 however, lacks chemical conjugation capabilities, which restricts the feasibility of its application in targeted drug therapy. This limitation has spurred the prospect that chemical structure of the parent molecule might allow conjugation of the derivatives to drug carriers such as antibodies. By first employing docking studies, we designed and prepared a series of novel Dol-10 analogs with a modified C-terminus, preserving high potency of the parent compound while enhancing conjugation capability. The modifications involved the introduction of a methyleneamine functionality at position 4 of the 1,3-thiazole ring, along with the substitution of the thiazole ring with a 1,2,3-triazole moiety, furnished with methylenehydroxy, carboxy, methyleneamine, and N(Me)-methyleneamine tethering functionalities at position 4. Among the synthesized pentapeptides, DA-1 exhibited the highest potency in prostate cancer (PC-3) cells, eliciting apoptosis (IC50 0.2 ± 0.1 nm) and cell cycle arrest at the mitotic stage after at least 6 days of culture. This delayed response suggests the accumulation of cellular stress or significant physiological alterations that profoundly impact the cell cycle. We believe that these novel Dol-10 derivates represent a new and straightforward route for the development of C-terminus modified Dol-10-based microtubule inhibitors, thereby advancing targeted anticancer therapy.

Turn-on Coumarin Precursor: From Hydrazine Sensor to Covalent Inhibition and Fluorescence Detection of Rabbit Muscle Aldolase.

Hydrazine, a highly toxic compound, demands sensitive and selective detection methods. Building upon our previous studies with pre-coumarin OFF-ON sensors for fluoride anions, we extended our strategy to hydrazine sensing by adapting phenol protecting groups (propionate, levulinate, and γ-bromobutanoate) to our pre-coumarin scaffold. These probes reacted with hydrazine, yielding a fluorescent signal with low micromolar limits of detection. Mechanistic studies revealed that hydrazine deprotection may be outperformed by a retro-Knoevenagel reaction, where hydrazine acts as a nucleophile and a base yielding a fluorescent diimide compound (6,6'-((1E,1'E)-hydrazine-1,2diylidenebis(methaneylylidene))bis(3(diethylamino)phenol, 7). Additionally, our pre-coumarins unexpectedly reacted with primary amines, generating a fluorescent signal corresponding to phenol deprotection followed by cyclization and coumarin formation. The potential of compound 3 as a theranostic Turn-On coumarin precursor was also explored. We propose that its reaction with ALDOA produced a γ-lactam, blocking the catalytic nucleophilic amine in the enzyme's binding site. The cleavage of the ester group in compound 3 induced the formation of fluorescent coumarin 4. This fluorescent signal was proportional to ALDOA concentration, demonstrating the potential of compound 3 for future theranostic studies in vivo.

Thioxobimanes.

Dioxobimanes, colloquially known as bimanes, are a well-established family of N-heterobicyclic compounds that share a characteristic core structure, 1,5-diazabicyclo[3.3.0]octadienedione, bearing two endocyclic carbonyl groups. By sequentially thionating these carbonyls in the syn and anti isomers of the known (Me,Me)dioxobimane, we were able to synthesize a series of thioxobimanes, representing the first heavy-chalcogenide bimane variants. These new compounds were extensively characterized spectroscopically and crystallographically, and their aromaticity was probed computationally. Their potential role as ligands for transition metals was demonstrated by synthesizing a representative gold(I)-thioxobimane complex.

The emerging role of selenium metabolic pathways in cancer: New therapeutic targets for cancer.

Selenium (Se) is incorporated into the body via the selenocysteine (Sec) biosynthesis pathway, which is critical in the synthesis of selenoproteins, such as glutathione peroxidases and thioredoxin reductases. Selenoproteins, which play a key role in several biological processes, including ferroptosis, drug resistance, endoplasmic reticulum stress, and epigenetic processes, are guided by Se uptake. In this review, we critically analyze the molecular mechanisms of Se metabolism and its potential as a therapeutic target for cancer. Sec insertion sequence binding protein 2 (SECISBP2), which is a positive regulator for the expression of selenoproteins, would be a novel prognostic predictor and an alternate target for cancer. We highlight strategies that attempt to develop a novel Se metabolism-based approach to uncover a new metabolic drug target for cancer therapy. Moreover, we expect extensive clinical use of SECISBP2 as a specific biomarker in cancer therapy in the near future. Of note, scientists face additional challenges in conducting successful research, including investigations on anticancer peptides to target SECISBP2 intracellular protein.

Gold nanoparticles stabilize peptide-drug-conjugates for sustained targeted drug delivery to cancer cells.

BACKGROUND: Peptide-drug-conjugates (PDCs) are being developed as an effective strategy to specifically deliver cytotoxic drugs to cancer cells. However one of the challenges to their successful application is the relatively low stability of peptides in the blood, liver and kidneys. Since AuNPs seem to have a longer plasma half-life than PDCs, one approach to overcoming this problem would be to conjugate the PDCs to gold nanoparticles (AuNPs), as these have demonstrated favorable physico-chemical and safety properties for drug delivery systems. We set out to test whether PEG coated-AuNPs could provide a suitable platform for the non-covalent loading of pre-formed PDCs and whether this modification would affect the bioavailability of the PDCs and their cytotoxicity toward target cancer cells. METHODS: Peptides specifically internalized by A20 murine lymphoma cells were isolated from a phage library displaying 7mer linear peptides. Peptide specificity was validated by flow cytometry and confocal microscopy. PDCs were synthesized containing a selected peptide (P4) and either chlorambucil (Chlor), melphalan (Melph) or bendamustine (Bend). Gold nanoparticles were sequentially coated with citrate, PEG-6000 and then PDC (PDC-PEG-AuNP). The physico-chemical properties of the coated particles were analyzed by electrophoresis, TEM, UV-VIS and FTIR. Stability of free and PDC-coated AuNP was determined. RESULTS: Biopanning of the phage library resulted in discovery of several novel peptides that internalized into A20 cells. One of these (P4) was used to synthesize PDCs containing either Chlor, Melph or Bend. All three PDCs specifically killed A20 target cells, however they had short half-lives ranging from 10.6 to 15.4 min. When coated to PEG-AuNPs, the half-lives were extended to 21.0-22.3 h. The PDC-PEG-AuNPs retained cytotoxicity towards the target cells. Moreover, whereas pre-incubation for 24 h of free PDCs almost completely abolished their cytotoxic activity, the PDC-PEG-AuNPs were still active even after 72 h pre-incubation. CONCLUSIONS: Peptide-drug-conjugates hold potential for improving the target efficacy of chemotherapeutic drugs, however their short half-lives may limit their application. This hurdle can be overcome by easily conjugating them to gold nanoparticles. This conjugation also opens up the possibility of developing slow release formulations of targeted drug delivery systems containing PDCs.

The non-ionizing electromagnetic stimulation enhanced antibody production (NESEAP) effect - Discovery and technological applications.

The rise of biological therapeutics in the global pharmaceuticals market has escalated the demand for quality monoclonal antibodies for healthcare and scientific applications. Reducing costs while enhancing production yields without compromising quality are the main challenges to the growth of this industry today. Over the last two decades non-ionizing radiation has been demonstrated to elicit targeted biological responses in a frequency and dose dependent manner. We hypothesize and design a millimeter wave radiation procedure to enhance the yields of antibody-producing hybridoma cell lines. We demonstrate this method enhances the production of IgA and IgG antibodies from MOPC315.BM and U13.6 cells by a factor of 24.05 ± 3.32 and 1.41 ± 0.03 respectively relative to untreated cells. No treatment associated cytotoxicity was observed in either cell line corroborating physiological viability of irradiated cells. Our results demonstrate proof-of-concept of a novel technique to significantly enhance antibody yields from hybridoma cells which could lead to a reduction in antibody production costs. Further studies will focus on scaling up of this technology and employment of non-contact, tuned electromagnetic stimulation of biological systems for targeted responses.

Au nanodyes as enhanced contrast agents in wide field near infrared fluorescence lifetime imaging.

The near-infrared (NIR) range of the electromagnetic (EM) spectrum offers a nearly transparent window for imaging tissue. Despite the significant potential of NIR fluorescence-based imaging, its establishment in basic research and clinical applications remains limited due to the scarcity of fluorescent molecules with absorption and emission properties in the NIR region, especially those suitable for biological applications. In this study, we present a novel approach by combining the widely used IRdye 800NHS fluorophore with gold nanospheres (GNSs) and gold nanorods (GNRs) to create Au nanodyes, with improved quantum yield (QY) and distinct lifetimes. These nanodyes exhibit varying photophysical properties due to the differences in the separation distance between the dye and the gold nanoparticles (GNP). Leveraging a rapid and highly sensitive wide-field fluorescence lifetime imaging (FLI) macroscopic set up, along with phasor based analysis, we introduce multiplexing capabilities for the Au nanodyes. Our approach showcases the ability to differentiate between NIR dyes with very similar, short lifetimes within a single image, using the combination of Au nanodyes and wide-field FLI. Furthermore, we demonstrate the uptake of Au nanodyes by mineral-oil induced plasmacytomas (MOPC315.bm) cells, indicating their potential for in vitro and in vivo applications.

Phyto-Photodynamic Therapy of Prostate Cancer Cells Mediated by Yemenite 'Etrog' Leave Extracts.

Cancer therapy, from malignant tumor inhibition to cellular eradication treatment, remains a challenge, especially regarding reduced side effects and low energy consumption during treatment. Hence, phytochemicals as cytotoxic sensitizers or photosensitizers deserve special attention. The dark and photo-response of Yemenite 'Etrog' leaf extracts applied to prostate PC3 cancer cells is reported here. An XTT cell viability assay along with light microscope observations revealed pronounced cytotoxic activity of the extract for long exposure times of 72 h upon concentrations of 175 µg/mL and 87.5 µg/mL, while phototoxic effect was obtained even at low concentration of 10.93 µg/mL and a short introduction period of 1.5 h. For the longest time incubation of 72 h and for the highest extract concentration of 175 µg/mL, relative cell survival decreased by up to 60% (below the IC50). In combined phyto-photodynamic therapy, a reduction of 63% compared to unirradiated controls was obtained. The concentration of extract in cells versus the accumulation time was inversely related to fluorescence emission intensity readings. Extracellular ROS production was also shown. Based on an ATR-FTIR analysis of the powdered leaves and their liquid ethanolic extract, biochemical fingerprints of both polar and non-polar phyto-constituents were identified, thereby suggesting their implementation as phyto-medicine and phyto-photomedicine.

A lone spike in blood glucose can enhance the thrombo-inflammatory response in cortical venules.

How transient hyperglycemia contributes to cerebro-vascular disease has been a challenge to study under controlled physiological conditions. We use amplified, ultrashort laser-pulses to physically disrupt brain-venule endothelium at targeted locations. This vessel disruption is performed in conjunction with transient hyperglycemia from a single injection of metabolically active D-glucose into healthy mice. The observed real-time responses to laser-induced disruption include rapid serum extravasation, platelet aggregation, and neutrophil recruitment. Thrombo-inflammation is pharmacologically ameliorated by a platelet inhibitor, by a scavenger of reactive oxygen species, and by a nitric oxide donor. As a control, vessel thrombo-inflammation is significantly reduced in mice injected with metabolically inert L-glucose. Venules in mice with diabetes show a similar response to laser-induced disruption and damage is reduced by restoration of normo-glycemia. Our approach provides a controlled method to probe synergies between transient metabolic and physical vascular perturbations and can reveal new aspects of brain pathophysiology.

Drug resistance to chlorambucil in murine B-cell leukemic cells is overcome by its conjugation to a targeting peptide.

Targeting drugs through small-molecule carriers with a high affinity to receptors on cancer cells can overcome the lack of target cell specificity of most anticancer drugs. These targeted carrier-drug conjugates are also capable of reversing drug resistance in cancer cells. Although many targeted drug delivery approaches are being tested, the linkage of several and different drugs to a single carrier molecule might further enhance their therapeutic efficacy, particularly if the drugs are engineered for variable time release. This report shows that murine B-cell leukemic cells previously resistant to a chemotherapeutic drug can be made sensitive to that drug as long as it is conjugated to a targeting peptide and, in particular, when the conjugate contains multiple copies of the drug. Using a 13mer peptide (VHFFKNIVTPRTP) derived from the myelin basic protein (p-MBP), dendrimer-based peptide conjugates containing one, two, or four molecules of chlorambucil were synthesized. Although murine hybridomas expressing antibodies to either p-MBP (MBP cells) or a nonrelevant antigen (BCL-1 cells) were both resistant to free chlorambucil, exposure of the cells to the p-MBP-chlorambucil conjugate completely reversed the drug resistance in MBP, but not BCL-1 cells or normal spleen cells. Moreover, at equivalent drug doses, there was significant enhancement in the cytotoxic activity of multidrug versus single-drug copy conjugates. On the basis of these results, the use of multifunctional dendrone linkers bearing several covalently bound cytotoxic agents allows the development of more effective targeted drug systems and enhances the efficacy of currently approved drugs for B-cell leukemia.

Multifractal analysis of cellular ATR-FTIR spectrum as a method for identifying and quantifying cancer cell metastatic levels.

Cancer is a leading cause of mortality today. Sooner a cancer is detected, the more effective is the treatment. Histopathological diagnosis continues to be the gold standard worldwide for cancer diagnosis, but the methods used are invasive, time-consuming, insensitive, and still rely to some degree on the subjective judgment of pathologists. Recent research demonstrated that Attenuated Total Reflection-Fourier Transform Infrared (ATR-FTIR) spectroscopy can be used to determine the metastatic potential of cancer cells by evaluating their membrane hydration. In the current study, we demonstrate that the conversion of ATR-FTIR spectra using multifractal transformation generates a unique number for each cell line's metastatic potential. Applying this technique to murine and human cancer cells revealed a correlation between the metastatic capacity of cancer cells within the same lineage and higher multifractal value. The multifractal spectrum value was found to be independent of the cell concentration used in the assay and unique to the tested lineage. Healthy cells exhibited a smaller multifractal spectrum value than cancer cells. Further, the technique demonstrated the ability to detect cancer progression by being sensitive to the proportional change between healthy and cancerous cells in the sample. This enables precise determination of cancer metastasis and disease progression independent of cell concentration by comparing the measured spectroscopy derived multifractal spectrum value. This quick and simple technique devoid of observer bias can transform cancer diagnosis to a great extent improving public health prognosis worldwide.

Fractal and textural imaging identify new subgroups of patients with colorectal cancer based on biophysical properties of the cancer cells.

Colorectal cancer (CRC) can been sub-divided, based on the generation of tertiary lymphoid structures (TLS), into CRC with a Crohn's like lymphoid reaction (CLR) representing de novo formation of TLSs or CRC lacking TLSs that show Diffuse Inflammatory infiltration (DII). The association between TLS, early treatment initiation and longer survival highlights the need for deeper patient stratification that could lead to more targeted therapies. We hypothesized that such stratification might be achieved by using digital image analyses. Here we retrospectively analyzed 35 CRC patient samples classified as CLR or DII by digital analysis, focusing on the parameters Fractal dimension, Lacunarity and the textural features Angular second momentum, Correlation, Inverse difference momentum and Entropy. Significant differences in the grades of these parameters between the two patient groups provided preliminary data that additional biophysical information can divide CRC into at least 3 subgroups which encompass CLR and DII. Additional studies are needed to test if this sub-classification aids in the selection of targeted therapy for patients with CRC.

Novel Cyclic Peptides for Targeting EGFR and EGRvIII Mutation for Drug Delivery.

The epidermal growth factor-epidermal growth factor receptor (EGF-EGFR) pathway has become the main focus of selective chemotherapeutic intervention. As a result, two classes of EGFR inhibitors have been clinically approved, namely monoclonal antibodies and small molecule kinase inhibitors. Despite an initial good response rate to these drugs, most patients develop drug resistance. Therefore, new treatment approaches are needed. In this work, we aimed to find a new EGFR-specific, short cyclic peptide, which could be used for targeted drug delivery. Phage display peptide technology and biopanning were applied to three EGFR expressing cells, including cells expressing the EGFRvIII mutation. DNA from the internalized phage was extracted and the peptide inserts were sequenced using next-generation sequencing (NGS). Eleven peptides were selected for further investigation using binding, internalization, and competition assays, and the results were confirmed by confocal microscopy and peptide docking. Among these eleven peptides, seven showed specific and selective binding and internalization into EGFR positive (EGFR+ve) cells, with two of them-P6 and P9-also demonstrating high specificity for non-small cell lung cancer (NSCLC) and glioblastoma cells, respectively. These peptides were chemically conjugated to camptothecin (CPT). The conjugates were more cytotoxic to EGFR+ve cells than free CPT. Our results describe a novel cyclic peptide, which can be used for targeted drug delivery to cells overexpressing the EGFR and EGFRvIII mutation.

Validation of a Mathematical Model Describing the Dynamics of Chemotherapy for Chronic Lymphocytic Leukemia In Vivo.

In recent years, mathematical models have developed into an important tool for cancer research, combining quantitative analysis and natural processes. We have focused on Chronic Lymphocytic Leukemia (CLL), since it is one of the most common adult leukemias, which remains incurable. As the first step toward the mathematical prediction of in vivo drug efficacy, we first found that logistic growth best described the proliferation of fluorescently labeled murine A20 leukemic cells injected in immunocompetent Balb/c mice. Then, we tested the cytotoxic efficacy of Ibrutinib (Ibr) and Cytarabine (Cyt) in A20-bearing mice. The results afforded calculation of the killing rate of the A20 cells as a function of therapy. The experimental data were compared with the simulation model to validate the latter's applicability. On the basis of these results, we developed a new ordinary differential equations (ODEs) model and provided its sensitivity and stability analysis. There was excellent accordance between numerical simulations of the model and results from in vivo experiments. We found that simulations of our model could predict that the combination of Cyt and Ibr would lead to approximately 95% killing of A20 cells. In its current format, the model can be used as a tool for mathematical prediction of in vivo drug efficacy, and could form the basis of software for prediction of personalized chemotherapy.

Search for Synergistic Drug Combinations to Treat Chronic Lymphocytic Leukemia.

Finding synergistic drug combinations is an important area of cancer research. Here, we sought to rationally design synergistic drug combinations with an inhibitor of BTK kinase, ibrutinib, which is used for the treatment of several types of leukemia. We (a) used a pooled shRNA screen to identify genes that protect cells from the drug, (b) identified protective pathways via bioinformatics analysis of these gene sets, and (c) identified drugs that inhibit these pathways. Based on this analysis, we established that inhibitors of proteasome and mTORC1 could synergize with ibrutinib both in vitro and in vivo. We suggest that FDA-approved inhibitors of these pathways could be effectively combined with ibrutinib for the treatment of chronic lymphocytic leukemia (CLL).

The Impact of Induction Regimes on Immune Responses in Patients with Multiple Myeloma.

Current standard frontline therapy for newly diagnosed patients with multiple myeloma (NDMM) involves induction therapy, autologous stem cell transplantation (ASCT), and maintenance therapy. Major efforts are underway to understand the biological and the clinical impacts of each stage of the treatment protocols on overall survival statistics. The most routinely used drugs in the pre-ASCT "induction" regime have different mechanisms of action and are employed either as monotherapies or in various combinations. Aside from their direct effects on cancer cell mortality, these drugs are also known to have varying effects on immune cell functionality. The question remains as to how induction therapy impacts post-ASCT immune reconstitution and anti-tumor immune responses. This review provides an update on the known immune effects of melphalan, dexamethasone, lenalidomide, and bortezomib commonly used in the induction phase of MM therapy. By analyzing the actions of each individual drug on the immune system, we suggest it might be possible to leverage their effects to rationally devise more effective induction regimes. Given the genetic heterogeneity between myeloma patients, it may also be possible to identify subgroups of patients for whom particular induction drug combinations would be more appropriate.

Broadband Infrared Spectroscopy for Non-Contact Measurement of Neurological Disease Biomarkers in Cerebrospinal Fluid.

Cerebrospinal fluid (CSF) is a clear and colorless biological fluid which circulates within brain ventricles (cavities), the spinal cord's central canal, the space between the brain and the spinal cord, as well as their protective coverings, the meninges. Cerebrospinal fluid contains different constituents, such as albumin and lactate, whose levels are used clinically as biomarkers of neurodegenerative disorders. In current clinical practice, analysis of CSF content for the diagnosis of central nervous system disorders requires an invasive procedure known as lumbar puncture or spinal tap. With the aim of developing a noninvasive alternative, we report here the spectral behavior of albumin and lactate over a broad wavelength range of 600-2000 nm, after each was added separately at varying normal and abnormal concentration levels to artificial CSF ( aCSF). Spectral measurements were conducted simultaneously by two different spectrometers working at different spectral ranges in transmittance mode. Spectral analysis revealed that albumin and lactate each possesses its own first and second derivative absorbance spectra fingerprint between 1660 and 1810 nm. Distinguishing albumin from lactate by their spectral data enabled the differentiation between aCSF conditions modeling different neurological disorders. Spectral changes of each compound strongly correlated ( R2 > 0.9) with absorbance derivative spectra peaks at specific wavelengths, when analyzed by linear regression with variations in their concentration. These findings suggest the feasibility of CSF biomarker assessment by broadband infrared spectroscopy.

Recent Innovations in Peptide Based Targeted Drug Delivery to Cancer Cells.

Targeted delivery of chemotherapeutics and diagnostic agents conjugated to carrier ligands has made significant progress in recent years, both in regards to the structural design of the conjugates and their biological effectiveness. The goal of targeting specific cell surface receptors through structural compatibility has encouraged the use of peptides as highly specific carriers as short peptides are usually non-antigenic, are structurally simple and synthetically diverse. Recent years have seen many developments in the field of peptide based drug conjugates (PDCs), particularly for cancer therapy, as their use aims to bypass off-target side-effects, reducing the morbidity common to conventional chemotherapy. However, no PDCs have as yet obtained regulatory approval. In this review, we describe the evolution of the peptide-based strategy for targeted delivery of chemotherapeutics and discuss recent innovations in the arena that should lead in the near future to their clinical application.

syn-Bimane as a chelating O-donor ligand for palladium(ii).

A cationic Pd(ii) complex containing syn-(Me,Me)bimane as a ligand was prepared and fully characterized. This complex represents the first well-defined case of a bimane scaffold coordinated to a metal center. The strongly-fluorescent syn-bimane chelates the Pd(ii) center via its carbonyl oxygen atoms, affording a non-fluorescent complex. The crystal structure of this complex shows that the coordinated bimane departs from planarity, with its bicyclic framework bent about the N-N bond. Spectroscopic evidence demonstrates that bimane coordination is reversible in solution.