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Purpose: to study the expression of vascular endothelial growth factor (vegf-a) and its receptors (vegfr-1 and vegfr-2) in renal cell carcinoma (rcc) cells and assess the effect of the expression levels of these markers on the tumor characteristics and prognosis of patients with rcc.Material and methods. The study included 65 patients with rcc (pt1a-t4n0/+m0/+). All patients underwent radical surgery. Histological tumor tissue samples obtained during surgery were used for the study. Expression of vegfa, vegfr-1, -2 was studied by immunohistochemical staining using appropriate antibodies to receptors and growth factors.Results. Expression of vegf and vegfr-1 and vegfr-2 receptors was ound in the cytoplasm and on the membrane of primary tumor cells of patients with rcc. There was a significant direct correlation of overexpression of the markers with g 3-4 anaplasia (vegfr-1, -2) and signs of significant tumor extension, including high pt category (vegfr-1, -2), larger size of the primary tumor (vegfr-1 , -2), tumor invasion of paranephria (vegf, vegfr-1), tumor venous thrombosis (vegfr-1, -2), multiple metastases (vegf-2), metastases in the adrenal gland (vegf, vegfr-2) and liver (vegfr-1) (p0.05).Conclusion. Expression of vegfa, as well as vegfr-1 and vegfr-2 receptors, was found on the surface and in the cytoplasm of cells of the primary tumor of patients with rcc (pt1a-t4n0/+m0/+). There was a significant correlation between vegf/vegfr overexpression with a high grade (g3-4) tumor anaplasia and significant tumor extension. In univariate analysis, a significant adverse effect on specific survival of vegfr-2 overexpression was observed. In regression analysis, vegfr-2 overexpression tended to independently affect specific survival. These results show the importance of vegf/vegfr expression as biomarkers in renal cell carcinoma.

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.

Novel 4-amino-5-cyano-2-sulfonylpyrimidines were prepared based on three-component cyclization between isothiouronium salts, benzaldehydes and malononitrile, followed by oxidation of the sulfide moiety with Oxone. The cytotoxic activity of the synthesized compounds, as well as the induction of apoptosis, inhibition of the cell cycle and proliferation tests were performed on selected cancer cell lines A431, A549, A375, HCT 116, MCF7, LNCap and SH-SY5Y.

Introduction. Cutaneous melanoma is a challenge to treat due to rapid progression of disease and acquired resistance to therapy. Autophagy and the epithelial-to-mesenchymal transition (EMT) are closely interrelated and play a key role in tumor progression. Targeted co-inhibition of MEK and mTOR kinases is a potential target for melanoma therapy by downregulatoin of the EMT.Objective: to study the effect of MEK and mTOR co-inhibition on cell viability, ability to form 3D-spheroids and migratory capacity of melanoma cell lines, and correlation of these changes with EMTand autophagy-related markers.Material and Methods. Melanoma cell lines Mel Z and Mel MTP were derived from patients, who were treated at the N.N. Blokhin National Medical Research Center of Oncology. The antiproliferative effect of binimetinib and/or rapamycin was studied by the MTT -test. 3D spheroids were formed using RGD peptides. Cell migration and invasion were assessed by a Boyden chamber migration assay. The expression levels of autophagy and EMT markers were investigated by immunocytochemistry or immunoblotting.Results. Rapamycin increased cytotoxicity of binimetinib in both 2D and 3D melanoma cell line cultures. At the same time, binimetinib and rapamycin reduced invasion, but not migration capacity of melanoma cells in vitro. The effectiveness of the combination was associated with a decrease in the EMT markers (N-cadherin and β-catenin) and autophagy markers (Beclin 1, p62/SQST M1 and LC3BII ) in melanoma cells.Conclusion. Inactivation of autophagy and EMT leads to overcoming the resistance to current anti-melanoma therapy and can be considered as a promising target for the treatment of melanoma.

The possibilities of using fluorescence-based analytical methods and their advantages for visualization and identification of the type of programmed cell death in human tumor cells exposed to flavonoids have been analyzed in experiments in vitro. HeLa cervical cancer cells and A498 kidney carcinoma cells were used as the objects of the study, and exposure to the flavonoid-containing extract of common hedgehyssop (Gratiola officinalis L.) was tested as the experimental treatment. The following fluorescence-based techniques were used: the “live and dead” test with double staining by propidium iodide and acridine orange and double staining with annexin and propidium iodide. Autophagy induction was confirmed by fluorescence-based tests implemented in a Muse cell analyzer with a Muse Autophagy LC3-Antibody Based Kit. The use of double staining with acridine orange and propidium iodide fluorescent dyes in the “live and dead” test and comparison with phase contrast microscopy enables the visualization of apoptotic body and autophagosome formation processes in the cells and can, therefore, be used as a method of screening assessment of the efficiency of various chemotherapy drugs.

1-Imidoylbenzotriazoles [N-aryl-1-(1H-benzotriazol-1-yl)-2,2,2-trifluoroethan-1-imines] reacted with tosylmethyl isocyanide according to van Leusen’s procedure to give difficultly accessible 1-aryl-4-(4-methylbenzenesulfonyl)-5-(trifluoromethyl)-1H-imidazoles in good yields (81–94%). The initial imidoylbenzotriazoles were conveniently synthesized by reaction of sodium benzotriazolide with the corresponding imidoyl chlorides in THF. The reactions were carried out with a wide series of imidoylbenzotriazoles containing various electron-donating and electron-withdrawing substituents in the N-aryl fragment.

Lanthanide-doped upconversion nanoparticles (UCNPs) are promising bioimaging agents that emit light under near infra-red excitation, capable of penetrating deep in biotissues with a high signal-to-noise ratio. Their successful implementation is principally associated with surface functionalization. Here, we report on UCNP surface modification with highly hydrophilic, endogenous, non-toxic, non-immunogenic colominic acid, conferring "stealth" properties. We proposed surface functionalization of UCNPs based on a two-step strategy, which consists of hydrophilization with polyethyleneimine and attachment of colominic acid by electrostatic or covalent bond formation. Analysis revealed that regardless of the nature of the bond, colominic acid acted as a non-cytotoxic UCNP surface coating with low nonspecific blood protein adsorption. UCNP-colominic acid nanocomplexes exhibited low uptake by macrophages in vitro, which plays an active role in inflammatory reactions. We demonstrated the superiority of colominic acid compared to polyethylene glycol coating in terms of the prolonged circulation time in the bloodstream of small animals when injected intravenously. The colominic acid coating made it possible to prolong the UCNP circulation time up to 3 h. This led to the efficient UCNP accumulation in the inflammation site due to microvascular remodeling, accompanied by an enhanced uptake and retention effect. UCNP-assisted imaging of inflammation in the whole-body mode as well as local visualization of blood vessels were acquired in vivo. These collective findings validate the functional significance of UCNP decoration with colominic acid for their application in bioimaging.

Theranostics is the direction in modern biomedicine aimed at developing drugs that combine the capabilities of diagnosis and therapy of tumors in one agent. Upconversion nanophosphors (UCNPs) are inorganic crystalline materials that can be used to create a nanoplatform providing diagnostic and therapeutic modalities. They have been proposed as luminescent markers for optical imaging of biological tissue due to their anti-Stokes luminescence, lack of photodegradation and low toxicity. In this article, UCNPs as a theranostic agent for both optical imaging and delivery of anticancer drugs have been offered. To obtain biocompatible nanocomplexes, UCNP surface with a core/shell structure of NaYF4:Yb3+Tm3+/NaYF4 was modified with polylactic acid in the presence of various stabilizers (dextran, polyvinyl alcohol, and poly-N-vinylpyrrolidone). To give the therapeutic modality to the nanocomplex, the antitumor antibiotic doxorubicin was loaded into the polymer shell. The loading efficiency was up to 0.1 mg per 1 mg UCNPs. The toxicity and the intracellular accumulation of nanocomplexes were evaluated in vitro. It was concluded that the modification of UCNPs with polylactic acid provides the transport of doxorubicin, allowing the combination of diagnostic and therapeutic modalities in one agent.

Formulation of promising anticancer herbal drug curcumin as a nanoscale-sized curcumin (nanocurcumin) improved its delivery to cells and organisms both in vitro and in vivo. We report on coupling nanocurcumin with upconversion nanoparticles (UCNPs) using Poly (lactic-co-glycolic Acid) (PLGA) to endow visualisation in the near-infrared transparency window. Nanocurcumin was prepared by solvent-antisolvent method. NaYF4:Yb,Er (UCNP1) and NaYF4:Yb,Tm (UCNP2) nanoparticles were synthesised by reverse microemulsion method and then functionalized it with PLGA to form UCNP-PLGA nanocarrier followed up by loading with the solvent-antisolvent process synthesized herbal nanocurcumin. The UCNP samples were extensively characterised with XRD, Raman, FTIR, DSC, TGA, UV-VIS-NIR spectrophotometer, Upconversion spectrofluorometer, HRSEM, EDAX and Zeta Potential analyses. UCNP1-PLGA-nanocurcumin exhibited emission at 520, 540, 660 nm and UCNP2-PLGA-nanocurmin showed emission at 480 and 800 nm spectral bands. UCNP-PLGA-nanocurcumin incubated with rat glioblastoma cells demonstrated moderate cytotoxicity, 60–80% cell viability at 0.12–0.02 mg/mL marginally suitable for therapeutic applications. The cytotoxicity of UCNPs evaluated in tumour spheroids models confirmed UCNP-PLGA-nanocurcumin therapeutic potential. As-synthesised curcumin-loaded nanocomplexes were administered in tumour-bearing laboratory animals (Lewis lung cancer model) and showed adequate contrast to enable in vivo and ex vivo study of UCNP-PLGA-nanocurcumin bio distribution in organs, with dominant distribution in the liver and lungs. Our studies demonstrate promise of nanocurcumin-loaded upconversion nanoparticles for theranostics applications.

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.

Melanoma is one of the most aggressive and lethal form of cancer. Photodynamic therapy (PDT) is a clinically approved technique for cancer treatment, including non-melanoma skin cancer. However, the most of conventional photosensitizers are of low efficacy against melanoma due to the possible dark toxicity at high drug concentrations, melanin pigmentation, and induction of anti-oxidant defense mechanisms. In the current research we propose non-toxic flavin mononucleotide (FMN), which is a water-soluble form of riboflavin (vitamin B2) as a promising agent for photodynamic therapy of melanoma. We demonstrated selective accumulation of FMN in melanoma cells in vivo and in vitro in comparison with keratinocytes and fibroblasts. Blue light irradiation with dose 5 J/cm2 of melanoma cells pre-incubated with FMN led to cell death through apoptosis. Thus, the IC50 values of human melanoma A375, Mel IL, and Mel Z cells were in a range of FMN concentration 10–30 µM that can be achieved in tumor tissue under systemic administration. The efficiency of reactive oxygen species (ROS) generation under FMN blue light irradiation was measured in single melanoma cells by a label-free technique using an electrochemical nanoprobe in a real-time control manner. Melanoma xenograft regression in mice was observed as a result of intravenous injection of FMN followed by blue-light irradiation of tumor site. The inhibition of tumor growth was 85–90% within 50 days after PDT treatment.

Background: To improve quality of surgery in oncology, it is necessary to completely remove the tumor, including its metastases, to minimize injury to normal tissues and to reduce duration of an intervention. Modern methods of detection based on radiological computerized tomography and magnetic resonance imaging can identify a tumor after its volume has become big enough, i.e. it contains more than 10 billion cells. Therefore, an improvement of sensitivity and resolution ability of diagnostic tools to identify early stages of malignant neoplasms seems of utmost importance. Aim: To demonstrate the potential of a new class of anti-Stokes luminescence nanoparticles for deep optical imaging with high contrast of malignant tumors. Materials and methods: Upconversion nanoparticles with narrow dispersion and a size of 70 to 80 nm, with a core/shell structure of NaYF4:Yb3+:Tm3+/NaYF4 were used in the study. The nanoparticles have an intensive band of anti-Stokes photoluminescence at a wavelength of 800 nm under irradiation with a wavelength of 975 nm (both wavelengths are within the transparency window for biological tissues). The conversion coefficient of the excitation radiation into the anti-Stokes luminescence was 9%. To increase the time during which nanoparticles can circulate in blood flow of small animals, the nanoparticles were covered by a biocompatible amphiphilic polymer shell. As a tumor model we used Lewis epidermoid carcinoma transfected to mice. Results: We were able to obtain stable water colloids of nanoparticles covered with amphiphilic polymer that could preserve their initial size at least for one month. The use of upconversion nanoparticles with a hydrophilic shell made of intermittent maleic anhydride and octadecene co-polymer with subsequent coating with diglycidyl polyethylene glycol ether allowed for reduction of non-specific reaction of nanoparticles with plasma proteins. In its turn, it resulted in an increased time of their circulation in blood flow of small animals for up to 1 hour. With the Lewis lung carcinoma transfected to mice model we demonstrated аn in-life transportation of upconversion nanoparticles into the tumor with a high degree of localization due to a passive EPR effect. The contrast of luminescent signal in the tumor compared to adjacent tissues was at least 70%. The possibility of visualization of upconverted nanoparticles up to 15 mm of biological tissue was shown. Conclusion: The optical imaging techniques with anti-Stokes photoluminescent markers ensure a high contract real-time detection of tumor tissues that allows for their use for intra-operative diagnostics.

The dynamics of the production of nitric oxide (NO) metabolites: nitrites, nitrates, volatile nitrosamines and iNOS expression was studied in mice with subcutaneous transplanted, spontaneous and chemical- induced tumors. Tumor growth was accompanied by increased production of nitrites + nitrates in tumors or their release with urine that not dependent on tumor histotype. The total concentration of nitrites and nitrates in tumors reached micromolar levels characteristic of nitrosative stress. The ability of peritoneal macrophages + monocytes to generates nitrites was suppressed at the stage of intensive growth of the Lewis lung carcinoma, which may indicate a decrease in the cytotoxic properties of immune cells. The possibility of formation in the Erlich carcinoma of volative N-nitrosodimethylamine and N-nitrosodiethylamine compounds with pronounced carcinogenic properties was demonstrated. A positive expression of iNOS was revealed in some areas of lung carcinoma at all investigated time points using the immunohistochemical method. The lungs metastases were not stain or weakly stained. This may indicate selection of the cells with a low activity of iNOS migrating in the lungs.

A novel surface modification approach of brightly luminescent upconversion nanoparticles (UCNPs) is reported. Inorganic core@shell UCNPs (core – NaYF4 co-doped with Yb3+ and Tm3+ ions, shell – NaYF4) were modified by intercalation with amphiphilic copolymer poly(maleic anhydride-alt-1-octadecene) followed by cross-linking with poly(ethylene glycol) diglycidyl ether (PEG-DGE). The proposed approach enables preparation of UCNPs with an outmost PEG-containing layer, which provides steric stabilization and low non-specific protein adsorption. Intravenous injection of PEG-functionalized UCNPs into the mice results in extension of the UCNP blood circulation time up to 1 hour. In vivo epi-luminescence imaging of the mouse model with Lewis lung carcinoma is ensured by the high quantum yield of the modified UCNPs and passive targeting associated with efficient UCNP accumulation in solid tumors.

Melanoma is one of the most aggressive and treatment-resistant tumors that responsible for majority of skin-cancer related deaths. Here we propose a combination of MEK inhibitor binimetinib with metformin as a promising therapy against human melanoma cells in vitro, including BRAF -mutated A375, Mel Z, and Mel IL cells, and NRAS-mutated Mel MTP and Mel Me cells. Additionally, we obtained two close to clinical practice models of melanoma progression. The first one was vemurafenib-resistant Mel IL/R melanoma cells with acquired resistance to BRAF inhibition-targeted therapy, and the second one was tumor spheroids, which are 3D in vitro model of small-size solid tumors in vivo. The cytotoxicity of binimetinib and metformin was synergistic in both 2D and 3D melanoma culture and mediated through apoptotic pathway. The combination reduced the number of melanoma-formed colonies, inhibited cell invasion and migration, and led to G0/G1 cell cycle arrest through cyclin D/CDK4/CDK6 pathway. The mechanism of metformin and binimetinib synergy in melanoma cells was associated with increased activation of p-AMPKα and decreased p-ERK, but not with alterations in p-mTOR. In summary, the combination of metformin and binimetinib resulted in stronger anti-proliferative effects on melanoma cells compared to binimetinib alone, and therefore could be promising for clinical applications.

Alofanib (formerly known as RPT835) is a novel allosteric FGFR2 inhibitor with activity in FGFR2-expressing triple-negative breast cancer [Tjulandin S, et al. San Antonio Breast Cancer Symposium 2014]. Early data suggest that combining FGFR2 inhibitors with platinum-containing cytotoxic agents for the treatment of epithelial ovarian cancer may yield increased antitumor activity [Cole C, et al. Cancer Biol Ther. 2010]. We investigated antitumor activity of alofanib in ovarian cancer in vitro and in vivo. To assess the efficacy of alofanib on FGF-mediated cell proliferation, ovarian cancer (SKOV-3) FGFR2-expressing cells were incubated in a 96-well microculture plate and were treated with serially diluted RPT835. Basic FGF was added at a concentration of 25 ng/ml. Control wells were left untreated. Cell growth inhibition was determined using Promega's Cell Titer-Glo® assay. Immunocompromised mice were used for xenotransplantation of FGFR2 high-expressing ovarian cancer cells (SCOV-3). Seventy animals with measurable tumors were selected on day 10 and randomized into control groups (no treatment or chemotherapy alone (paclitaxel + carboplatin) and treatment groups (alofanib orally or intravenously (different dose levels) in combination with chemotherapy). Measurements of tumor volume (mm3) were performed by digital calipers every 3 days during 30 days after tumor inoculation. Basic FGF significantly increased proliferation of the ovarian cancer cells in untreated control group (P = 0.001). Alofanib treatment resulted in growth inhibition of SKOV-3 cell line in vitro. Treatment of alofanib in combination with paclitaxel/carboplatin demonstrated significant tumor growth delay phenotype in all treatment groups compared to control non-treatment groups. Alofanib exhibited a dose-dependent effect on tumor growth. Daily intravenous regimen of alofanib (total maximum dose per week was 350 mg/kg) demonstrated dramatic effect (inhibiting growth by 80% and by 53% in comparison with vehicle and chemotherapy group alone, respectively (P These results provide strong rationale for evaluation of alofanib in combination with paclitaxel and carboplatin in patients with ovarian cancer. Citation Format: Sergei Tjulandin, Mikhail Byakhov, Evgenia Stepanova, Dmitry Khochenkov, Daniel Harrison, Ilya Tsimafeyeu. Alofanib, a novel allosteric FGFR2 inhibitor, shows potent antitumor activity in ovarian cancer with FGFR2 expression. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 796. doi:10.1158/1538-7445.AM2015-796