Metal-(Acyclic Diaminocarbene) Complexes Demonstrate Nanomolar Antiproliferative Activity against Triple-Negative Breast Cancer.

Invited for the cover of this issue are Tatiyana Serebryanskaya, Mikhail Kinzhalov and co-workers at St. Petersburg State University, the Research Institute for Physical Chemical Problems, Belarusian State University, Togliatti State University and Blokhin National Medical Research Center of Oncology. The image depicts the shield of Pallas Athena with the structure of a palladium carbene complex that protects against triple-negative breast cancer. Read the full text of the article at 10.1002/chem.202400101.

Metal-(Acyclic Diaminocarbene) Complexes Demonstrate Nanomolar Antiproliferative Activity against Triple-Negative Breast Cancer.

Hydrolytically stable PdII and PtII complexes supported by acyclic diaminocarbene ligands represent a novel class of structural organometallic anticancer agents exhibiting nanomolar antiproliferative activity in a panel of cancer cell lines (IC50 0.07-0.81 µM) and up to 300-fold selectivity for cancer cells over normal primary fibroblasts. The lead drug candidate was 300 times more potent than cisplatin in vitro and showed higher efficacy in reducing the growth of aggressive MDA-MB-231 xenograft tumors in mice.

Electrochemical Nanopipette Sensor for In Vitro/In Vivo Detection of Cu2+ Ions.

In vitro/in vivo detection of copper ions is a challenging task but one which is important in the development of new approaches to the diagnosis and treatment of cancer and hereditary diseases such as Alzheimer's, Wilson's, etc. In this paper, we present a nanopipette sensor capable of measuring Cu2+ ions with a linear range from 0.1 to 10 µM in vitro and in vivo. Using the gold-modified nanopipette sensor with a copper chelating ligand, we evaluated the accumulation ability of the liposomal form of an anticancer Cu-containing complex at three levels of biological organization. First, we detected Cu2+ ions in a single cell model of human breast adenocarcinoma MCF-7 and in murine melanoma B16 cells. The insertion of the nanoelectrode did not result in leakage of the cell membrane. We then evaluated the distribution of the Cu-complex in MCF-7 tumor spheroids and found that the diffusion-limited accumulation was a function of the depth, typical for 3D culture. Finally, we demonstrated the use of the sensor for Cu2+ ion detection in the brain of an APP/PS1 transgenic mouse model of Alzheimer's disease and tumor-bearing mice in response to injection (2 mg kg-1) of the liposomal form of the anticancer Cu-containing complex. Enhanced stability and selectivity, as well as distinct copper oxidation peaks, confirmed that the developed sensor is a promising tool for testing various types of biological systems. In summary, this research has demonstrated a minimally invasive electrochemical technique with high temporal resolution that can be used for the study of metabolism of copper or copper-based drugs in vitro and in vivo.

Selective and Reversible 1,3-Dipolar Cycloaddition of 2-(2-Oxoindoline-3-ylidene)acetates with Nitrones in the Synthesis of Functionalized Spiroisoxazolidines.

The 1,3-dipolar cycloaddition of 2-(2-oxoindoline-3-ylidene)acetates with functionalized aldo- and ketonitrones proceeds with good selectivity to provide new highly functionalized 5-spiroisoxazolidines. A characteristic feature of these reactions is reversibility that allows for the control of the diastereoselectivity of cycloaddition. The reduction of obtained adducts using zinc powder in acetic acid leads to 1,3-aminoalcohols or spirolactones. For a number of the spiro compounds obtained, anticancer activity was found.

Nanocurcumin-Loaded UCNPs for Cancer Theranostics: Physicochemical Properties, In Vitro Toxicity, and In Vivo Imaging Studies.

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.

Immunochemical expression of fibroblast growth factor and its receptors in primary tumor cells of renal cell carcinoma.

BACKGROUND: There is the evidence of the role of the fibroblast growth factor and its receptors (FGF/FGFR) signaling pathway in tumorogenesis of renal cell carcinoma (RCC). We conducted a study aimed at evaluating of FGF2, FGFR1, and FGFR2 expression in primary tumor cells and assessing of these molecules expression levels effect on the characteristics of the tumor process and prognosis of patients with RCC. METHODS: Expression of FGF2, FGFR1, and FGFR2 was investigated in 65 primary RCC specimens by immuhistochemical staining using the appropriate antibodies. Expression levels were evaluated by the semi-quantitative method. A search for correlations of expression levels of investigated growth factors and receptors with RCC features and patients outcomes was performed. RESULTS: Cytoplasm and membrane expression of FGF2, FGFR1, and FGFR2 was found in the primary tumor cells of RCC patients. FGF2 staining was detected in 60.0% of cases (44.2 ± 5.4 HS). It was noted higher frequency and intensity of FGFR2 expression (66.2%; 46.6 ± 6.3 HS) comparing with FGFR1 (32.3%; 7.5 ± 2.2 HS). The correlation was revealed between the investigated markers overexpression and Fuhrman grade G3-4, as well as features of advanced RCC (paranephric fat tumor invasion, venous wall tumor invasion, adrenal and liver metastases). FGFR2 overexpression showed negative influence on cancer-specific survival in univariate analysis, however, lost its predictive value in multivariable analysis. CONCLUSION: Expression of FGF2 and its receptors was found on the surface and in the cytoplasm of RCC primary tumor cells and needs following investigations.

Rapamycin synergizes the cytotoxic effects of MEK inhibitor binimetinib and overcomes acquired resistance to therapy in melanoma cell lines in vitro.

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.

Local Overheating of Biotissue Labeled With Upconversion Nanoparticles Under Yb3+ Resonance Excitation.

Local overheating of biotissue is a critical step for biomedical applications, such as photothermal therapy, enhancement of vascular permeability, remote control of drug release, and so on. Overheating of biological tissue when exposed to light is usually realized by utilizing the materials with a high-absorption cross section (gold, silica, carbon nanoparticles, etc.). Here, we demonstrate core/shell NaYF4:Yb3+, Tm3+/NaYF4 upconversion nanoparticles (UCNPs) commonly used for bioimaging as promising near-infrared (NIR) absorbers for local overheating of biotissue. We assume that achievable temperature of tissue labeled with nanoparticles is high enough because of Yb3+ resonance absorption of NIR radiation, whereas the use of auxiliary light-absorbing materials or shells is optional for photothermal therapy. For this purpose, a computational model of tissue heating based on the energy balance equations was developed and verified with the experimentally obtained thermal-graphic maps of a mouse in response to the 975-nm laser irradiation. Labeling of biotissue with UCNPs was found to increase the local temperature up to 2°C compared to that of the non-labeled area under the laser intensity lower than 1 W/cm2. The cellular response to the UCNP-initiated hyperthermia at subcritical ablation temperatures (lower than 42°C) was demonstrated by measuring the heat shock protein overexpression. This indicates that the absorption cross section of Yb3+ in UCNPs is relatively large, and microscopic temperature of nanoparticles exceeds the integral tissue temperature. In summary, a new approach based on the use of UCNP without any additional NIR absorbers was used to demonstrate a simple approach in the development of photoluminescent probes for simultaneous bioimaging and local hyperthermia.

The Susceptibility of Human Melanoma Cells to Infection with the Leningrad-16 Vaccine Strain of Measles Virus.

Oncolytic viruses, including live attenuated measles virus (MV) vaccine strains, have recently been shown as promising therapeutic agents against human malignancies. In this study, the oncolytic potential of the attenuated vaccine strain Leningrad-16 (L-16) of MV was evaluated in a panel of human metastatic melanoma cell lines. The L-16 measles virus was shown to replicate within melanoma cells mediating direct cell killing of tumor cells, although all melanoma cell lines varied in regard to their ability to respond to L-16 MV infection, as revealed by the different pattern of the Interferon Stimulated Gene expression, cytokine release and mechanisms of cell death. Furthermore, the statistically significant L-16 measles virus related tumor growth inhibition was demonstrated in a melanoma xenograft model. Therefore, L-16 MV represents an appealing oncolytic platform for target delivery of therapeutic genes along with other attenuated measles virus strains.

Expression of Receptor Tyrosine Kinases on Peripheral Blood Mononuclear Cells and Tumor-Infiltrating Lymphocytes in Patients with Renal Cell Carcinoma and Healthy Donors.

BACKGROUND: Very little is known about receptor tyrosine kinase (RTK) expression on peripheral blood mononuclear cells (PBMC) in humans including renal cell carcinoma (RCC) patients. OBJECTIVES: The primary objective of this study was to evaluate expression levels of major RTKs on PBMC and tumor-infiltrating lymphocytes (TIL) isolated from RCC patients. The secondary aim was to compare levels of RTK expression in RCC patients before surgery and on the 180th day after surgery (lymphocyte lifetime) and to compare them with the expression in healthy donors. In addition, we compared RTK and PD-L1 expression in TIL. METHODS: Tumor and blood samples were obtained from 20 patients with primary RCC immediately after surgical resection. Blood samples were collected from 20 healthy donors. Tumors were harvested into RPMI 1640 medium (Gibco) and processed within 4 h. TIL isolation was performed using a modified protocol [Baldan et al. Br J Cancer. 2015;112:1510-18]. Expression of RTKs was evaluated with NovoExpress Software. Twenty tumors from the same patients were stained with PD-L1 IHC assay (clone SP142; Ventana). RESULTS: PBMC and TIL express RTKs in humans. The RTK expression level was significantly lower on peripheral blood cells in patients with RCC (mean 41%, range 27.1-62.6%) as compared with healthy donor PBMC (mean 77.1%, range 72.1-80.1%, all p < 0.05). Furthermore, RTK expression was significantly downregulated on intratumoral cells (mean 40%, range 23.2-52.3%) in comparison with healthy donor PBMC. There was no significant recovery of RTK expression on the 180th day except for VEGFR2. Five of 20 (25%) patients were PD-L1 positive. PD-L1 expression on TIL was strongly associated with downregulated expression of PDGFRα (p = 0.017) and PDGFRß (p = 0.024). CONCLUSIONS: PBMC and TIL had similar low RTK expression levels in RCC patients. PBMC of healthy humans had a significantly higher expression of RTK. PD-L1 and PDGFRα-ß expression could correlate. Comprehensive basic and clinical studies will be needed to define a biological role of RTKs on different lymphocyte subsets and correlations between clinical outcomes and expression levels.

Expression of growth factors and their receptors in the primary renal cell carcinoma: new data and review.

INTRODUCTION: The aim of our study was to investigate expression levels and the prognostic value of multiple growth factors and their receptors in the primary tumor cells of renal cell carcinoma (RCC). MATERIAL AND METHODS: Expression of vascular endothelial growth factor (VEGF)A, fibroblast growth factor (FGF)2, vascular endothelial growth factor receptor (VEGFR)1, VEGFR2, FGFR1, FGFR2, platelet-derived growth factor receptor (PDGFR)α, and PDGFRß was investigated in 65 primary RCC specimens by immuhistochemical staining using the appropriate antibodies. Expression levels were evaluated by the semi-quantitative method. A search for correlations of expression levels of investigated growth factors and receptors with RCC features and patients outcomes was performed. RESULTS: Expression of all growth factors and their receptors was detected both on the surface and in the cytoplasm of the primary tumor cells in RCC patients. The expression of all analyzed factors was interconnected. FGFR2 expression correlated with the largest number of other growth factors and receptors. A strong correlation was revealed between high expression of the studied markers, high Fuhrman grade, and advanced RCC stages. In a univariate analysis overexpression of VEGFR2 (p <0.0001) and FGFR2 (p = 0.014) had negative influence on cancer-specific survival. CONCLUSIONS: Expression of growth factors and tyrosine kinase receptors in the primary tumor cells is strongly interconnected and associated with unfavorable features of RCC.

Metformin increases antitumor activity of MEK inhibitor binimetinib in 2D and 3D models of human metastatic melanoma cells.

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.

Inhibition of endoplasmic reticulum stress-induced autophagy sensitizes melanoma cells to temozolomide treatment.

The incidence of malignant melanoma is increasing. The discovery of agents specifically targeting the mutated cascades has provided a good response for patients with oncogenic B-Raf proto-оncogene, serine/threonine kinase (BRAF). However, numerous studies continue to focus on novel methods of treatment to overcome acquired resistance to novel drugs. Recently, it has been revealed that inhibition of endoplasmic reticulum (ER) stress chaperon 78 kDa glucose-regulated protein 78 (GRP78) leads to down-regulation of autophagy and increased sensitivity to temozolomide (TMZ) treatment. Melanoma cells have a different sensitivity to TMZ treatment, which corresponds to the basal autophagy level. In the present study, we demonstrated that downregulation of GRP78 mitigated chemoresistance to TMZ in three melanoma cell lines. We found that downregulation of GRP78 led to inhibition of autophagy, cell cycle arrest in the G0/G1 phase, and activation of caspase-7-induced apoptosis, and this was affected by the initial autophagy level. Moreover, inhibition of GRP78 mitigated the combined TMZ and chloroquine effect. Our data revealed that autophagy inhibition through downregulation of ER stress response could overcome resistance to TMZ treatment in melanoma cells with a high basal level of autophagy treatment, which makes this combination a potential potent antitumor treatment for metastatic melanoma.

Autophagy inhibitors chloroquine and LY294002 enhance temozolomide cytotoxicity on cutaneous melanoma cell lines in vitro.

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.

Targeting FGFR2 with alofanib (RPT835) shows potent activity in tumour models.

Alofanib (RPT835) is a novel selective allosteric inhibitor of fibroblast growth factor receptor 2 (FGFR2). We showed previously that alofanib could bind to the extracellular domain of FGFR2 and has an inhibitory effect on FGF2-induced phoshphorylation of FRS2α. In the present study, we further showed that alofanib inhibited phosphorylation of FRS2α with the half maximal inhibitory concentration (IC50) values of 7 and 9 nmol/l in cancer cells expressing different FGFR2 isoforms. In a panel of four cell lines representing several tumour types (triple-negative breast cancer, melanoma, and ovarian cancer), alofanib inhibited FGF-mediated proliferation with 50% growth inhibition (GI50) values of 16-370 nmol/l. Alofanib dose dependently inhibited the proliferation and migration of human and mouse endothelial cells (GI50 11-58 nmol/l) compared with brivanib and bevacizumab. Treatment with alofanib ablated experimental FGF-induced angiogenesis in vivo. In a FGFR-driven human tumour xenograft model, oral administration of alofanib was well tolerated and resulted in potent antitumour activity. Importantly, alofanib was effective in FGFR2-expressing models. These results show that alofanib is a potent FGFR2 inhibitor and provide strong rationale for its evaluation in patients with FGFR2-driven cancers.

Targeting liposomes loaded with melphalan prodrug to tumour vasculature via the Sialyl Lewis X selectin ligand.

Earlier we showed that liposome formulation of DL-melphalan lipophilic prodrug bearing tetrasaccharide Sialyl Lewis X (SiaLeX) caused prolonged therapeutic effect on mammary cancer in mice. Here, we compare antivascular effect of SiaLeX-liposomes loaded with diglyceride ester of melphalan (Mlph) against SiaLeX-free formulation in Lewis lung carcinoma model. METHODS: Liposomes of egg phosphatidylcholine/yeast phosphatidylinositol/1,2-dioleoyl glycerol (DOG) conjugate of Mlph/±SiaLeX-PEG8-15-DOG, 8:1:1:0.2 by mol, were prepared by standard extrusion. After two intravenous injections with Mlph or liposomes under either standard or delayed treatment protocols, vascular-disrupting effects of the preparations were evaluated basing on tumour section histomorphology, lectin perfusion assay and immunohistochemistry (anti-CD31 staining) data. Also, untreated mice were administered with fluorescently-labelled liposomes to assess their distribution in tumour sections with confocal laser scanning microscopy. RESULTS: Two injections of SiaLeX-liposomes reproducibly caused severe injuries of tumour vessels. SiaLeX-liposomes co-localized with CD31 marker on vascular endothelium while the non-targeted formulation extravasated into tumour. DISCUSSION: Cytotoxic SiaLeX-liposomes exhibit superior vascular-disrupting properties compared to non-targeted liposomes, yet the effect starts to transform into gain in tumour growth inhibition only under delayed treatment regimen. CONCLUSION: SiaLeX-ligand provides targeting of cytotoxic liposomes to tumour endothelium and subsequent antivascular effect.