Design, Synthesis and Antimicrobial Properties of New Tetracyclic Quinobenzothiazine Derivatives.

A new method for modifying the structure of tetracyclic quinobenzothiazinium derivatives has been developed, allowing introduction of various substituents at different positions of the benzene ring. The method consists of reacting appropriate aniline derivatives with 5,12-(dimethyl)thioquinantrenediinium bis-chloride. A series of new quinobenzothiazine derivatives was obtained with propyl, allyl, propargyl and benzyl substituents in 9, 10 and 11 positions, respectively. The structure of the obtained compounds was analyzed by 1H and 13C NMR (HSQC, HMBC) and X-ray analysis. All the compounds were tested against reference strains Staphylococcus aureus ATCC 29213 and Enterococcus faecalis ATCC 29212, and representatives of multidrug-resistant clinical isolates of methicillin-resistant S. aureus (MRSA) and vancomycin-resistant E. faecalis (VRE). In addition, all the compounds were evaluated in vitro against Mycobacterium smegmatis ATCC 700084 and M. marinum CAMP 5644. 9-Benzyloxy-5-methyl-12H-quino [3,4-b][1,4]benzothiazinium chloride (6j), 9-propoxy-5-methyl-12H-quino[3,4-b][1,4]benzothiazinium chloride (6a) and 9-allyloxy-5-methyl-12H-quino[3,4-b][1,4]benzothiazinium chloride (6d) demonstrated high activity against the entire tested microbial spectrum. The activities of the compounds were comparable with oxacillin, tetracycline and ciprofloxacinagainst staphylococcal strains and with rifampicin against both mycobacterial strains. Compound 6j had a significant effect on the inhibition of bacterial respiration as demonstrated by the MTT assay. The compounds showed not only bacteriostatic activity, but also bactericidal activity. Preliminary in vitro cytotoxicity screening of the compounds performed using normal human dermal fibroblasts (NHDF) proved that the tested compounds showed an insignificant cytotoxic effect on human cells (IC50 > 37 µM), making these compounds interesting for further investigation. Moreover, the intermolecular similarity of novel compounds was analyzed in the multidimensional space (mDS) of the structure/property-related in silico descriptors by means of principal component analysis (PCA) and hierarchical clustering analysis (HCA), respectively. The distance-oriented structure/property distribution was related with the experimental lipophilic data.

Combination of LIGHT (TNFSF14)-Armed Myxoma Virus Pre-Loaded into ADSCs and Gemcitabine in the Treatment of Experimental Orthotopic Murine Pancreatic Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is a deadly neoplasm. Oncolytic viruses have tumorolytic and immune response-boosting effects and present great potential for PDAC management. We used LIGHT-armed myxoma virus (vMyx-LIGHT) loaded ex vivo into human adipose-derived mesenchymal stem cells (ADSCs) to evaluate murine PDAC treatment in conjunction with gemcitabine (GEM). The cytotoxicity of this treatment was confirmed in vitro using human and murine pancreatic cancer cell cultures, which were more sensitive to the combined approach and largely destroyed. Unlike cancer cells, ADSCs sustain significant viability after infection. The in vivo administration of vMyx-LIGHT-loaded ADSCs and gemcitabine was evaluated using immunocompetent mice with induced orthotopic PDAC lesions. The expression of virus-encoded LIGHT increased the influx of T cells to the tumor site. Shielded virus followed by gemcitabine improved tumor regression and survival. The addition of gemcitabine slightly compromised the adaptive immune response boost obtained with the shielded virus alone, conferring no survival benefit. ADSCs pre-loaded with vMyx-LIGHT allowed the effective transport of the oncolytic construct to PDAC lesions and yielded significant immune response; additional GEM administration failed to improve survival. In view of our results, the delivery of targeted/shielded virus in combination with TGF-ß ablation and/or checkpoint inhibitors is a promising option to improve the therapeutic effects of vMyx-LIGHT/ADSCs against PDAC in vivo.

Towards Property Profiling: SYNTHESIS and SAR Probing of New Tetracyclic Diazaphenothiazine Analogues.

A series of new tertiary phenothiazine derivatives containing a quinoline and a pyridine fragment was synthesized by the reaction of 1-methyl-3-benzoylthio-4-butylthioquinolinium chloride with 3-aminopyridine derivatives bearing various substituents on the pyridine ring. The direction and mechanism of the cyclization reaction of intermediates with the structure of 1-methyl-4-(3-pyridyl)aminoquinolinium-3-thiolate was related to the substituents in the 2- and 4-pyridine position. The structures of the compounds were analyzed using 1H, 13C NMR (COSY, HSQC, HMBC) and X-ray analysis, respectively. Moreover, the antiproliferative activity against tumor cells (A549, T47D, SNB-19) and a normal cell line (NHDF) was tested. The antibacterial screening of all the compounds was conducted against the reference and quality control strain Staphylococcus aureus ATCC 29213, three clinical isolates of methicillin-resistant S. aureus (MRSA). In silico computation of the intermolecular similarity was performed using principal component analysis (PCA) and hierarchical clustering analysis (HCA) on the pool of structure/property-related descriptors calculated for the novel tetracyclic diazaphenothiazine derivatives. The distance-oriented property evaluation was correlated with the experimental anticancer activities and empirical lipophilicity as well. The quantitative shape-based comparison was conducted using the CoMSA method in order to indicate the potentially valid steric, electronic and lipophilic properties. Finally, the numerical sampling of similarity-related activity landscape (SALI) provided a subtle picture of the SAR trends.

Myxoma Virus Expressing LIGHT (TNFSF14) Pre-Loaded into Adipose-Derived Mesenchymal Stem Cells Is Effective Treatment for Murine Pancreatic Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is a weakly immunogenic fatal neoplasm. Oncolytic viruses with dual anti-cancer properties-oncolytic and immune response-boosting effects-have great potential for PDAC management. Adipose-derived stem cells (ADSCs) of mesenchymal origin were infected ex vivo with recombinant myxoma virus (MYXV), which encodes murine LIGHT, also called tumor necrosis factor ligand superfamily member 14 (TNFSF14). The viability and proliferation of ADSCs were not remarkably decreased (1-2 days) following MYXV infection, in sharp contrast to cells of pancreatic carcinoma lines studied, which were rapidly killed by the infection. Comparison of the intraperitoneal (IP) vs. the intravenous (IV) route of ADSC/MYXV administration revealed more pancreas-targeted distribution of the virus when ADSCs were delivered IP to mice bearing orthotopically injected PDAC. The biodistribution, tumor burden reduction and anti-tumor adaptive immune response were examined. Bioluminescence data, used to assess the presence of the luciferase-tagged virus after IP injection, indicated enhanced trafficking into the pancreata of mice bearing orthotopically-induced PDAC, as compared to tumor-free animals, resulting in extended survival of the treated PDAC-seeded animals and in the boosted expression of key adaptive immune response markers. We conclude that ADSCs pre-loaded with transgene-armed MYXV and administered IP allow for the effective ferrying of the oncolytic virus to sites of PDAC and mediate improved tumor regression.

COVID-19 Autopsies: A Case Series from Poland.

This paper presents autopsy findings of 3 COVID-19 patients randomly selected for post-mortem from two tertiary referral Polish hospitals. Analysis of macroscopic, histopathological findings with clinical features was performed. All 3 deceased patients were Caucasian males (average age 61 years, range from 56 to 68 years). Using real-time polymerase chain reaction assay, the patients were confirmed (antemortem) to have severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Two patients were obese, and 1 patient had type 2 diabetes mellitus. The medical history of 1 patient included hemorrhagic pancreatitis, gangrenous cholecystitis, Acinetobacter baumanii sepsis, and cholecystectomy. Pulmonary embolism was diagnosed in 2 patients. At autopsy, in 1 case, the lungs showed bilateral interstitial pneumonia with diffuse alveolar damage (DAD), while in another case, interstitial pulmonary lymphoid infiltrates and enlarged atypical pneumocytes were present but without DAD. Microthrombi in lung vessels and capillaries were observed in 2 cases. This study revealed thrombotic complications of COVID-19 and interstitial pneumonia with DAD presence as the main autopsy findings in patients with SARS-CoV-2 infection that was confirmed antemortem with molecular tests. Autopsy studies using tissue sections handled in accordance with SARS-CoV-2 biosafety guidelines are urgently needed, especially in the case of subjects who were below the age of 60.

Potassium Ferrate (VI) as the Multifunctional Agent in the Treatment of Landfill Leachate.

Possible use of potassium ferrate (VI) (K2FeO4) for the treatment of landfill leachate (pH = 8.9, Chemical Oxygen Demand (COD) 770 mg O2/L, Total Organic Carbon (TOC) 230 mg/L, Total Nitrogen (Total N) 120 mg/L, Total Phosphorus (Total P) 12 mg/L, Total Coli Count (TCC) 6.8 log CFU/mL (Colony-Forming Unit/mL), Most Probable Number (MPN) of fecal enterococci 4.0 log/100 mL, Total Proteolytic Count (TPC) 4.4 log CFU/mL) to remove COD was investigated. Central Composite Design (CCD) and Response Surface Methodology (RSM) were applied for modelling and optimizing the purification process. Conformity of experimental and predicted data (R2 = 0.8477, Radj2 = 0.7462) were verified using Analysis of Variance (ANOVA). Application of K2FeO4 using CCD/RSM allowed to decrease COD, TOC, Total N, Total P, TCC, MPN of fecal enterococci and TPC by 76.2%, 82.6%, 68.3%, 91.6%, 99.0%, 95.8% and 99.3%, respectively, by using K2FeO4 0.390 g/L, at pH = 2.3 within 25 min. Application of equivalent amount of iron (as FeSO4 × 7H2O and FeCl3 × 6H2O) under the same conditions allowed to diminish COD, TOC, Total N, Total P, TCC, MPN of fecal enterococci and TPC only by 38.1%, 37.0%, 20.8%, 95.8%, 94.4%, 58.2%, 90.8% and 41.6%, 45.7%, 29.2%, 95.8%, 92.1%, 58.2%, 90.0%, respectively. Thus, K2FeO4 could be applied as an environmentally friendly reagent for landfill leachate treatment.

Myxoma Virus-Loaded Mesenchymal Stem Cells in Experimental Oncolytic Therapy of Murine Pulmonary Melanoma.

Oncolytic viruses can target neoplasms, triggering oncolytic and immune effects. Their delivery to melanoma lesions remains challenging. Bone-marrow-derived mesenchymal stem cells (MSCs) were shown to be permissive for oncolytic myxoma virus (MYXV), allowing its transfer to melanoma cells, leading to their killing. Involvement of progeny virus was demonstrated in the transfer from MSCs to co-cultured melanoma cells. The inhibitory effect of virus on melanoma foci formation in murine lungs was revealed using melanoma cells previously co-cultured with MYXV-infected MSCs. Virus accumulation and persistence in lungs of lesion-bearing mice were shown following intravenous administration of MSC-shielded MYXV construct encoding luciferase. Therapy of experimentally induced lung melanoma in mice with interleukin (IL)-15-carrying MYXV construct delivered by MSCs led to marked regression of lesions and could increase survival. Elevated natural killer (NK) cell percentages in blood indicated robust innate responses against unshielded virus only. Lung infiltration by NK cells was followed by inflow of CD8+ T lymphocytes into melanoma lesions. Elevated expression of genes involved in adaptive immune response following oncolytic treatment was confirmed using RT-qPCR. No adverse pathological effects related to MSC-mediated oncolytic therapy with MYXV were observed. MSCs allow for safe and efficient ferrying of therapeutic MYXV to pulmonary melanoma foci triggering immune effects.

Mesenchymal stem cells as carriers for systemic delivery of oncolytic viruses.

Progress in genetic engineering led to the emergence of some viruses as potent anticancer therapeutics. These oncolytic viruses combine self-amplification with dual antitumor action: oncolytic (destruction of cancer cells) and immunostimulatory (eliciting acquired antitumor response against cancer epitopes). As any other viruses, they trigger antiviral response upon systemic administration. Mesenchymal stem cells are immature cells capable of self-renewing and differentiating into many cell types that belong to three germinal layers. Due to their inherent tumor tropism mesenchymal stem cells loaded with oncolytic virus can improve delivery of the therapeutic cargo to cancer sites. Shielding of oncolytic viral construct from antiviral host immune response makes these cells prospective delivery vehicles to even hard-to-reach metastatic neoplastic foci. Use of mesenchymal stem cells has been criticized by some investigators as limiting proliferative abilities of primary cells and increasing the risk of malignant transformation, as well as attenuating therapeutic responses. However, majority of preclinical studies indicate safety and efficacy of mesenchymal stem cells used as carriers of oncolytic viruses. In view of contradictory postulates, the debate continues. The review discusses mesenchymal stem cells as carriers for delivery of genetically engineered oncolytic constructs and focuses on systemic approach to oncoviral treatment of some deadly neoplasms.

Taguchi Method and Response Surface Methodology in the Treatment of Highly Contaminated Tannery Wastewater Using Commercial Potassium Ferrate.

The potential implementation of Envifer®, a commercial product containing potassium ferrate (40.1% K2FeO4), for the purification of highly contaminated tannery wastewater from leather dyeing processes was proposed. The employment of the Taguchi method for optimization of experiments allowed the discoloration (98.4%), chemical oxygen demand (77.2%), total organic carbon (75.7%), and suspended solids (96.9%) values to be lowered using 1.200 g/L K2FeO4 at pH 3 within 9 min. The application of the central composite design (CCD) and the response surface methodology (RSM) with the use of 1.400 g/L K2FeO4 at pH 4.5 diminished the discoloration, the chemical oxygen demand, the total organic carbon, and suspended solids within 9 min. The Taguchi method is suitable for the initial implementation, while the RSM is superior for the extended optimization of wastewater treatment processes.

Crystal structures and other properties of ephedrone (methcathinone) hydrochloride, N-acetylephedrine and N-acetylephedrone.

PURPOSE: Three compounds obtained from ephedrine were identified and characterized by various instrumental analytical methods. Ephedrone (methcathinone) hydrochloride and its fundamental derivatives N-acetylephedrine and N-acetylephedrone were analyzed as precursors of a cathinone derivative. METHODS: The obtained samples were analyzed by gas chromatography coupled with mass spectrometry, nuclear magnetic resonance spectroscopy, infrared and Raman spectroscopy, and X-ray crystallography. RESULTS: The three compounds were confirmed as: N-methyl-2-amino-1-phenylpropan-1-one (methcathinone) hydrochloride, N-acetyl-N-methyl-2-amino-1-phenylpropan-1-one (cathinone derivative), and N-acetyl-N-methyl-2-amino-1-phenylpropan-1-ol (acetyl derivative of ephedrine). CONCLUSIONS: X-ray crystallography is especially useful for identifying the new designer drugs and their different precursor forms.

Derivatives of Graphene Oxide as Potential Drug Carriers.

Chemically functionalized graphene oxides could be used as novel drug carriers. Covalent alterations of graphene oxides lead to surface changes via formation of chemical bonding while non-covalent ones involve van der Waals forces, hydrogen bonding, and π-π stacking interactions. Covalent modifications appear to be superior as they can yield compounds with desired properties and carriers prepared by other methods are less stable. Synthesis of graphene oxide-iminodiacetic acid and graphene oxide-glycine involves nucleophilic substitution of graphene oxide nanoparticles with iminodiacetic acid or glycine. As the first step, iminodiacetic acid or glycine were transformed into iminodiacetic acid or glycine methyl ester hydrochlorides, respectively, for C-terminus protection. The obtained product, activated in situ, was then used to form amide bonds between graphene oxide and iminodiacetic acid or glycine.

Synthesis and Antimicrobial Activity of Sulfur Derivatives of Quinolinium Salts.

A novel method for cleavage of the dithiine ring in 5,12-(dimethyl)-thioqinantrenium bis-chloride 1 "via" reaction with sodium hydrosulfide leads to 1-methyl-3-mercaptoquinoline-4(1H)-thione 2. Further transformation of thiol and thione functions of compound 2 leads to a series of sulfide and disulfide derivatives of quinolinium salts 4 and 6. 1-Methyl-4-chloro-3-benzylthioquinoline chloride 8 was obtained by N-alkylating 4-chloro-3-benzylthioquinoline using dimethyl sulfate. Antimicrobial activity of the obtained compounds was investigated using six Gram-positive and six Gram-negative bacterial strains, as well as Candida albicans yeast. Greater activity was demonstrated towards Gram-positive strains. MIC values for compounds and with benzylthio 4d and benzoylthio 4f substituents in 3-quinoline position were found to be in the 0.5-1 µg/mL range, at a level similar to that of ciprofloxacin (reference). Compounds 4d and 4f also demonstrated interesting antifungal properties (MIC = 1).

Bis-anthracycline WP760 abrogates melanoma cell growth by transcription inhibition, p53 activation and IGF1R downregulation.

Anthracycline chemotherapeutics, e.g. doxorubicin and daunorubicin, are active against a broad spectrum of cancers. Their cytotoxicity is mainly attributed to DNA intercalation, interference with topoisomerase activity, and induction of double-stranded DNA breaks. Since modification of anthracyclines can profoundly affect their pharmacological properties we attempted to elucidate the mechanism of action, and identify possible molecular targets, of bis-anthracycline WP760 which previously demonstrated anti-melanoma activity at low nanomolar concentrations. We studied the effect of WP760 on several human melanoma cell lines derived from tumors in various development stages and having different genetic backgrounds. WP760 inhibited cell proliferation (IC50 = 1-99 nM), impaired clonogenic cell survival (100 nM), and inhibited spheroid growth (≥300 nM). WP760 did not induce double-stranded DNA breaks but strongly inhibited global transcription. Moreover, WP760 caused nucleolar stress and led to activation of the p53 pathway. PCR array analysis showed that WP760 suppressed transcription of ten genes (ABCC1, MTOR, IGF1R, EGFR, GRB2, PRKCA, PRKCE, HDAC4, TXNRD1, AKT1) associated with, inter alia, cytoprotective mechanisms initiated in cancer cells during chemotherapy. Furthermore, WP760 downregulated IGF1R and upregulated PLK2 expression in most of the tested melanoma cell lines. These results suggest that WP760 exerts anti-melanoma activity by targeting global transcription and activation of the p53 pathway and could become suitable as an effective therapeutic agent.

Methotrexate and Cytarabine-Loaded Nanocarriers for Multidrug Cancer Therapy. Spectroscopic Study.

Determining the properties of nanoparticles obtained by novel methods and defining the scope of their application as drug carriers has important practical significance. This article presents the pioneering studies concerning high degree incorporation of cytarabine (AraC) and methotrexate (MTX) into liposome vesicles. The main focus of this study were cytarabine-methotrexate-dipalmitoylphosphatidylcholine (DPPC) interactions observed in the gel and fluid phases of DPPC bilayers. The proposed new method of use the Transmittance2919/2850 ratio presented in our research is sensitive to subtle changes in conformational order resulting from rotations, kinks and bends of the lipid chains. The transition temperatures characterized by Fourier Transform Infrared Spectroscopy (FT-IR) were consistent with the results obtained by Differential Scanning Calorimetry (DSC). Transmission Electron Microscopy (TEM) was used in order to determine the size and shape of the liposomes obtained. The mutual interactions occurring between the drugs studied and the phospholipids were analyzed using the Nuclear Magnetic Resonance (NMR).

Synthesis and In Vitro Antiproliferative Activity of Novel Phenyl Ring-Substituted 5-Alkyl-12(H)-quino[3,4-b][1,4]benzothiazine Derivatives.

A novel series of tetracyclic quinobenzothiazine derivatives was synthetized. Compounds containing a substituent (hydroxyl, methyl, phenyl, piperidyl, or piperazinyl) in positions 9 and 11 were obtained by cyclization of suitable 4-aminoquinolinium-3-thiolates. Quinobenzothiazine 10-O-substituted derivatives were obtained by alkylating the hydroxyl group in position 10 of the parent (quinobenzothiazine) system. Antiproliferative activity of the synthesized compounds was studied using cultured neoplastic cells (MDA-MB-231, SNB-19, and C-32 cell lines). Four selected compounds were investigated in more detail for cytotoxicity and antiproliferative effect. Transcriptional activity of genes regulating cell cycle (TP53), apoptosis (BAX, BCL-2), as well as proliferation (H3) were assessed. Finally, the ability of the selected compounds to bind DNA was checked in the presence of ethidium bromide.

Can high dose rates used in cancer radiotherapy change therapeutic effectiveness?

Current cancer radiotherapy relies on increasingly high dose rates of ionising radiation (100-2400 cGy/min). It is possible that changing dose rates is not paralleled by treatment effectiveness. Irradiating cancer cells is assumed to induce molecular alterations that ultimately lead to apoptotic death. Studies comparing the efficacy of radiation-induced DNA damage and apoptotic death in relation to varying dose rates do not provide unequivocal data. Whereas some have demonstrated higher dose rates (single dose) to effectively kill cancer cells, others claim the opposite. Recent gene expression studies in cells subject to variable dose rates stress alterations in molecular signalling, especially in the expression of genes linked to cell survival, immune response, and tumour progression. Novel irradiation techniques of modern cancer treatment do not rely anymore on maintaining absolute constancy of dose rates during radiation emission: instead, timing and exposure areas are regulated temporally and spatially by modulating the dose rate and beam shape. Such conditions may be reflected in tumour cells' response to irradiation, and this is supported by the references provided.

Antitumor effects of recombinant antivascular protein ABRaA-VEGF121 combined with IL-12 gene therapy.

Development and neoplastic progression strongly rely on tumor microenvironment cells. Various kinds of cells that form such tumor milieu play substantial roles in angiogenesis and immunosuppression. Attempts to inhibit tumor vascularization alter tumor milieu and enhance immune response against the tumor. Anticancer therapeutic strategy bringing together antiangiogenic and immunostimulating agents has emerged as a promising approach. We here investigated whether therapy directed against preexisting vessels, combined with an immunomodulatory factor would be equally effective in arresting tumor growth. To this goal, we investigated the effectiveness of ABRaA-vascular endothelial growth factor isoform 121 (VEGF121), an antivascular drug constructed by us. It is a fusion protein composed of VEGF121, and abrin A chain (translation-inhibiting toxin). We used it in combination with interleukin (IL-12) gene therapy and tried to inhibit B16-F10 melanoma tumor growth. ABRaA-VEGF121 is a chimeric recombinant protein capable of destroying tumor vasculature and triggering necrosis in the vicinity of damaged vessels. IL-12 cytokine, in turn, activates both specific and non-specific immune responses. Our results demonstrate that combination of ABRaA-VEGF121 antivascular agent with immunostimulatory cytokine IL-12 indeed inhibits tumor growth more effectively than either agent alone, leading to complete cure of ca. 20 % mice. Post-therapeutic analysis of tumors excised from mice treated with combination therapy showed decreased numbers of blood microvessels in the tumor microenvironment, lowered numbers of regulatory T lymphocytes, as well as showed higher levels of CD4(+) and CD8(+) as compared to control mice. It seems that bringing together antivascular strategy and the action of immunostimulating agents indeed inhibits growth of tumors.

Synthesis and in vitro antiproliferative activity of novel 12(H)-quino[3,4-b][1,4]benzothiazine derivatives.

Novel method of N-dealkylating quinobenzothiazinium salts 2, promoted by reaction with benzimidazole, led to a series of new azaphenothiazine derivatives having 12(H)-quino[3,4-b][1,4] benzothiazine 4 structure. Reaction of compounds 4 in an alkaline milieu with alkylating agents occur as N-alkylation of the thiazine nitrogen and yields quinobenzothiazine derivatives 7. In vitro antiproliferative activity of compounds 4 and 7 was tested using two cancer cell lines (SNB-19 and C-32) and cisplatin as a reference. Most of the studied azaphenothiazine derivatives showed activity against both cell lines investigated (5.6-12.4 µg/ml concentration range tested). Compounds 4(b-e) containing a halogen atom or methyl group at the 9-position of the quinobenzothiazine ring show activity in the tested concentration range only against C-32 cell line. Compound 4f with methyl group in 11-position of quinobenzothiazine ring lacked activity against either cell line. The presence of additional aminoalkyl substituents at the thiazine nitrogen atom in compounds 7 increases their activity against both examined cell lines, when compared to compounds 4.

Vasostatin increases oxygenation of B16-F10 melanoma tumors and raises therapeutic efficacy of cyclophosphamide.

One of the preconditions of effective anticancer therapy is efficient transfer of the therapeutic agent (chemotherapeutic) to tumor cells. Fundamental barriers making drug delivery and action difficult include underoxygenation, elevated interstitial pressure, poor and abnormal tumor blood vascular network and acidic tumor milieu. In this study we aimed at developing an optimized scheme of administering a combination of an angiogenesis-inhibiting drug (vasostatin) and a chemotherapeutic (cyclophosphamide) in the therapeutic treatment of mice bearing experimental B16-F10 melanoma tumors. We report that the strongest tumor growth inhibition was observed in mice that received two, three or four vasostatin doses in combination with one injection of cyclophosphamide (i.e., V2 + CTX, V3 + CTX or V4 + CTX schemes). Double administration of vasostatin increases oxygenation of B16-F10 tumors. On the other hand, its five-fold administration lowers tumor oxygenation, breaks down tumor vascular network (increasing hypoxia) and leads in consequence to death of cancer cells and appearance of necrotic areas in the tumor. A decreased cyclophosphamide dose in combination with two doses of vasostatin (V2 + CTX scheme) inhibits tumor growth similarly to a larger dose of cyclophosphamide alone.

Properties of B16-F10 murine melanoma cells subjected to metabolic stress conditions.

Neoplastic cells which co-form tumors are usually subjected to various stress factors, mainly hypoxia and shortage of nutrient factors. Such cells employ different strategies that permit their survival under such conditions. Experiments in vitro are usually carried out in the presence of 21% oxygen and medium supplemented with 10% FBS. Altering these parameters can approximate the in vivo conditions found within tumor mass. The present paper reports certain properties (especially ability to metastasize) of B16-F10 cells able to grow upon exposure to altered growth conditions (medium supplemented with 0.06% FBS or presence of 1% oxygen for 24 or 72 hours). These properties were compared with those of control cells cultured in the presence of 21% oxygen and in medium supplemented with 10% FBS. Some properties of the cells exposed to medium supplemented with 0.06% FBS differ from those of cells cultured under low oxygenation conditions (ability to form metastases, to migrate, or to express various proteins). Only the partial deprivation of oxygen did increase both the number of migrating cells and the number of metastases formed. Serum deficiency enhanced only the cell ability to metastasize, but not to migrate. It appears that cultured B16-F10 cells employ different adaptation strategies under conditions of oxygen shortage and those of serum deficiency. Under oxygen deprivation, such cells most likely undergo an epithelial-mesenchymal transition, whereas serum deficiency ("starvation"), while increasing the tumorigenicity of B16-F10 cells, does not induce the epithelial-mesenchymal transition.