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Molecular targeted cancer therapy is a promising treatment strategy. Considering the central role of the epidermal growth factor receptor in cell proliferation and survival, there are indications that targeted agents like tyrosine kinase inhibitors, i. e., erlotinib, may enhance the antitumor treatment by radiation. The aim of this study is to analyze the inactivation effects of g-rays and to test the radiosensitizing potential of erlotinib on human lung adenocarcinoma cells in vitro. Irradiations were performed with doses ranging from 1 Gy to 8 Gy. In order to increase the radiosensitivity of CRL-5876 lung adenocarcinoma cells, the cells were treated with a clinically relevant concentration of 2 µM erlotinib. The effects of single and combined treatments were monitored using clonogenic survival, cell viability and proliferation assays at different time points. For the detection and visualization of the phosphorylated histone H2AX (γ-H2AX), an important biological marker of DNA double-strand break formation, fluorescence immunocytochemistry, was performed. The response to the treatment was monitored at four time points: 30 min, 2, 6, and 24 h. Irradiations with g-rays resulted in significant cell inactivation regarding all analyzed biological endpoints. Combined treatments revealed consistent cell inactivation. Moreover, compared to g-rays alone, elevated levels of g-H2AX foci were observed after pretreatment with erlotinib, indicating radiosensitization through impaired DNA repair. [Projekat Ministarstva nauke Republike Srbije, br. 173046 i br. 171019]

This study was conducted in order to evaluate the ability of carbon ions to induce DNA double-strand breaks and apoptosis in the radio-resistant human HTB140 melanoma cells. The cells were irradiated with 12C ions having the linear energy transfer of 258 keV/mm. Irradiations were performed in the dose range from 2 to 16 Gy. Induction of DNA double-strand breaks was evaluated 2 hour after irradiation through expression of gH2AX protein. Increased level of gH2AX detected in irradiated samples was especially high after irradiation with 12 and 16 Gy. Dose dependent increase of apoptosis was detected 48 hour after irradiation by flow-cytometry, with the maximum value of 20.4% after irradiation with 16 Gy, and the apoptotic index of 9.3. Pro-apoptotic effects of carbon ion beams were confirmed by changes of key molecules of the mitochondrial apoptotic pathway, p53 protein expression, Bax/Bcl-2 ratio and caspase-3 activation.

Abstract Background Considering that HTB140 melanoma cells have shown a poor response to either protons or alkylating agents, the effects of a combined use of these agents have been analysed. Methods Cells were irradiated in the middle of the therapeutic 62 MeV proton spread out Bragg peak (SOBP). Irradiation doses were 12 or 16 Gy and are those frequently used in proton therapy. Four days after irradiation cells were treated with fotemustine (FM) or dacarbazine (DTIC). Drug concentrations were 100 and 250 μM, values close to those that produce 50% of growth inhibition. Cell viability, proliferation, survival and cell cycle distribution were assessed 7 days after irradiation that corresponds to more than six doubling times of HTB140 cells. In this way incubation periods providing the best single effects of drugs (3 days) and protons (7 days) coincided at the same time. Results Single proton irradiations have reduced the number of cells to ~50%. FM caused stronger cell inactivation due to its high toxicity, while the effectiveness of DTIC, that was important at short term, almost vanished with the incubation of 7 days. Cellular mechanisms triggered by proton irradiation differently influenced the final effects of combined treatments. Combination of protons and FM did not improve cell inactivation level achieved by single treatments. A low efficiency of the single DTIC treatment was overcome when DTIC was introduced following proton irradiation, giving better inhibitory effects with respect to the single treatments. Most of the analysed cells were in G1/S phase, viable, active and able to replicate DNA. Conclusion The obtained results are the consequence of a high resistance of HTB140 melanoma cells to protons and/or drugs. The inactivation level of the HTB140 human melanoma cells after protons, FM or DTIC treatments was not enhanced by their combined application.

Quercetin, a well-known flavonoid with significant medicinal potential, was derivatized at the C8 position with a tetrahydroisoquinoline (THIQ) moiety, and physicochemical and pharmacological properties, inhibition potential, antioxidant activity, and cytotoxicity of new compounds were evaluated. Physicochemical and pharmacological properties, including lipophilicity, membrane permeability, and P-glycoprotein substrate affinity, were assessed theoretically using the SwissADME software. The metal-chelating ability of the new compounds was evaluated on metal ions Fe 2+ , Zn 2+ , and Cu 2+ , whose homeostasis disruption is linked to the development of Alzheimer's disease. Inhibition potential was tested on the cholinergic enzymes acetylcholinesterase and butyrylcholinesterase, as well as Na + , K + -ATPase, an enzyme commonly overexpressed in tumours. Antioxidant potential was assessed using the DPPH assay. Cytotoxicity studies were conducted on healthy MRC-5 cells and three cancer cell lines: HeLa, MDA-231, and MDA-468. The results indicated that derivatization of quercetin with THIQ yielded compounds with lower toxicity, preserved chelating ability, improved antioxidant potential, increased selectivity toward the cholinergic enzyme butyrylcholinesterase, and enhanced inhibition potential toward Na + , K + -ATPase and butyrylcholinesterase compared to quercetin alone. Therefore, the synthesized derivatives represent compounds with an improved profile and could be promising candidates for further optimization in developing drugs for neurodegenerative and cancer diseases.

Apart from cytotoxicity, inhibitors of the COX-2 enzyme have demonstrated additional effects important for cancer treatment (such as radiosensitization of tumor cells and cell antimigratory effects); however, the relationship between the inhibition of other inflammation-related enzyme 5-LOX inhibitors and anticancer activity is still not well understood. In our study, the cytotoxicity of thirteen COX-2 and 5-LOX inhibitors previously presented by our group ( 1 - 13 ) was tested on three cancer cell lines (HCT 116, HT-29 and BxPC-3) and one healthy cell line (MRC-5). Compounds 3 , 5 , 6 and 7 showed moderate cytotoxicity, but good selectivity towards cancer cell lines. IC 50 values were in the range of 22.99-51.66 µM (HCT 116 cell line), 8.63-41.20 µM (BxPC-3 cell line) and 24.78-81.60 µM (HT-29 cell line; compound 7 > 100 µM). In comparison to tested, commercially available COX-2 and 5-LOX inhibitors, both cytotoxicity and selectivity were increased. The addition of compounds 6 and 7 to irradiation treatment showed the most significant decrease in cell proliferation of the HT-29 cell line ( p < 0.001). The antimigratory potential of the best dual COX-2 and 5-LOX inhibitors (compounds 1 , 2 , 3 and 5 ) was tested by a wound-healing assay using the SW620 cell line. Compounds 1 and 3 were singled out as compounds with the most potent effect (relative wound closure was 3.20% (24 h), 5,08% (48 h) for compound 1 and 3.86% (24 h), 7.68% (48 h) for compound 3 ). Considering all these results, compound 3 stood out as the compound with the most optimal biological activity, with the best dual COX-2 and 5-LOX inhibitory activity, good selectivity towards tested cancer cell lines, significant cell antimigratory potential and a lack of toxic effects at therapeutic doses.

The literature data emphasize that nanoparticles might improve the beneficial effects of near-infrared light (NIR) on wound healing. This study investigates the mechanisms of the synergistic wound healing potential of NIR light and silver metal-organic frameworks combined with nitrogen- and sulfur-doped carbon dots (AgMOFsN-CDs and AgMOFsS-CDs, respectively), which was conducted by testing the fibroblasts viability, scratch assays, biochemical analysis, and synchrotron-based Fourier transform infrared (SR-FTIR) cell spectroscopy and imaging. Our findings reveal that the combined treatment of AgMOFsN-CDs and NIR light significantly increases cell viability to nearly 150% and promotes cell proliferation, with reduced interleukin-1 levels, suggesting an anti-inflammatory response. SR-FTIR spectroscopy shows this combined treatment results in unique protein alterations, including increased α-helix structures and reduced cross-β. Additionally, protein synthesis was enhanced upon the combined treatment. The likely mechanism behind the observed changes is the charge-specific interaction of N-CDs from the AgMOFsN-CDs with proteins, enhanced by NIR light due to the nanocomposite's optical characteristics. Remarkably, the complete wound closure in the in vitro scratch assay was achieved exclusively with the combined NIR and AgMOFsN-CDs treatment, demonstrating the promising application of combined AgMOFsN-CDs with NIR light photodynamic therapy in regenerative nanomedicine and tissue engineering.

Selective cytotoxicity of ZnO nanoparticles among different cell types and cancer and non-cancerous cells has been demonstrated earlier. In the view of anticancer potential of ZnO nanoparticles and their presence in numerous industrial products, it is of great importance to carefully evaluate their effects and mechanisms of action in both cancerous and healthy cells. In this paper, the effects of ZnO nanoparticles on cancerous HeLa and non-cancerous MRC-5 cells are investigated by studying the changes in the vibrational properties of the cells using Raman spectroscopy. Both types of cells were incubated with ZnO nanoparticles of average size 40 nm in the doses from the range 10-40 µg/ml for the period of 48 h, after which Raman spectra were collected. Raman modes' intensity ratios I 1659 /I 1444 , I 2855 /I 2933 and I 1337 /I 1305 were determined as spectral markers of the cytotoxic effect of ZnO in both cell types. Non-negative principal component analysis was used instead of standard one for analysis and detection of spectral features characteristic for nanoparticle-treated cells. The first several non-negative loading vectors obtained in this analysis coincided remarkably well with the Raman spectra of particular biomolecules, showing increase of lipid and decrease of nucleic acids and protein content. Our study pointed out that Raman spectral markers of lipid unsaturation, especially I 1270 /I 1300 , are relevant for tracing the cytotoxic effect of ZnO nanoparticles on both cancerous and non-cancerous cells. The change of these spectral markers is correlated to the dose of applied nanoparticles and to the degree of cellular damage. Furthermore, great similarity of spectral features of increasing lipids to spectral features of phosphatidylserine, one of the main apoptotic markers, was recognized in treated cells. Finally, the results strongly indicated that the degree of lipid saturation, presented in the cells, plays an important role in the interaction of cells with nanoparticles., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

In this study, C-doped TiO 2 nanoparticles (C-TiO 2 ) were prepared and tested as a photosensitizer for visible-light-driven photodynamic therapy against cervical cancer cells (HeLa). X-ray diffraction and Transmission Electron Microscopy confirmed the anatase form of nanoparticles, spherical shape, and size distribution from 5 to 15 nm. Ultraviolet-visible light spectroscopy showed that C doping of TiO 2 enhances the optical absorption in the visible light range caused by a bandgap narrowing. The photo-cytotoxic activity of C-TiO 2 was investigated in vitro against HeLa cells. The lack of dark cytotoxicity indicates good biocompatibility of C-TiO 2 . In contrast, a combination with blue light significantly reduced the survival of HeLa cells: illumination only decreased cell viability by 30% (15 min of illumination, 120 µW power), and 60% when HeLa cells were preincubated with C-TiO 2 . We have also confirmed blue light-induced C-TiO 2 -catalyzed generation of reactive oxygen species in vitro and intracellularly. Oxidative stress triggered by C-TiO 2 /blue light was the leading cause of HeLa cell death. Fluorescent labeling of treated HeLa cells showed distinct morphological changes after the C-TiO 2 /blue light treatment. Unlike blue light illumination, which caused the appearance of large necrotic cells with deformed nuclei, cytoplasm swelling, and membrane blebbing, a combination of C-TiO 2 /blue light leads to controlled cell death, thus providing a better outcome of local anticancer therapy., (© 2021. The Author(s), under exclusive licence to European Photochemistry Association, European Society for Photobiology.)

Objects of the present study are improved fullerene C 60 drug carrier properties trough encapsulation by microbial polysaccharides, levan (LEV), pullulan (PUL), and their hydrophobized cholesterol-derivatives (CHL and CHP), that show better interaction with cancer cells. The zeta potential, polydispersity index, and the diameter of particles were determined, and their cytotoxicity against three cancer cell lines were tested. Biochemical changes in HeLa cells are analyzed by synchrotron radiation (SR) FTIR spectro-microscopy combined with the principal component analysis (PCA). The most significant changes occur in HeLa cells treated with LEV-C 60 and correspond to the changes in the protein region, i.e. Amide I band, and the changes in the structure of lipid bodies and membrane fluidity are evident. The highest cytotoxicity was also induced by LEV-C 60 . In HeLa cells, cytotoxicity could not be strictly associated with biochemical changes in lipids, proteins and nucleic acids, but these findings are significant contribution to the study of the mechanism of interaction of C 60 -based nanoparticles with cellular biomolecules. In conclusion, LEV, PUL, CHL, and CHP enhanced fullerene C 60 potential to be used as target drug delivery system with the ability to induce specific intracellular changes in HeLa cancer cells., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Two new dinuclear bimetallic complexes, [{PdCl(bipy)}{μ-(pyrazine)}{PtCl(bipy)}]Cl(ClO4) (1) (bipy is 2,2'-bipyridine) and [{PdCl(en)}{μ-(pyrazine)}{PtCl(en)}]Cl(ClO4) (2) (en is ethylenediamine), have been synthesized and characterized by elemental microanalysis, IR, (1)H NMR spectroscopy and MALDI-TOF mass spectrometry. The pKa values of the coordinated water molecules of the diaqua species were determined as well. Substitution reactions of complexes (1) and (2) with thiourea (Tu), l-methionine (l-Met), l-cysteine (l-Cys), l-histidine (l-His) and guanosine-5'-monophosphate (5'-GMP) were studied under the pseudo-first order conditions as a function of nucleophile concentration and temperature. The order of reactivity of nucleophiles was: Tu > l-Met > l-Cys > l-His > 5'-GMP. Substitution reactions with Tu, l-Cys and l-His were followed by decomposition of bimetallic complexes to the corresponding substituted mononuclear complexes [Pd(N-N)(Nu)2] and [Pt(N-N)(Nu)2] (N-N = bipy, en), releasing the bridging ligand. However, the structures of starting bimetallic complexes were preserved during the reactions with l-Met and 5'-GMP. The absorption spectroscopic study of interactions of calf-thymus DNA (CT-DNA) with complexes (1), (2) and [{PdCl(bipy)}{μ-(NH2(CH2)6H2N)} {PtCl(bipy)}]Cl(ClO4) (3), has shown that all the complexes exhibit high intrinsic binding constants (Kb = 10(4)-10(5) M(-1)). DNA-ethidium bromide (DNA-EB) fluorescence was quenched after addition of complexes (1), (2) or (3), indicating displacement of intercalating EB by complexes. All complexes have shown good binding affinity to bovine serum albumin protein (BSA). Chemosensitivity of A375 (human melanoma) and HeLa (human cervical cancer) cell lines toward complexes (1), (2) and (3) was analyzed by SRB assay. Complex (1) displayed significant inhibitory effect on the growth of both cell lines.

Ionizing radiation induces DNA double strand breaks (DSBs) that trigger phosphorylation of the histone protein H2AX (γH2AX). Immunofluorescent staining visualizes formation of γH2AX foci, allowing their quantification. This method, as opposed to Western blot assay and Flow cytometry, provides more accurate analysis, by showing exact position and intensity of fluorescent signal in each single cell. In practice there are problems in quantification of γH2AX. This paper is based on two issues: the determination of which technique should be applied concerning the radiation dose, and how to analyze fluorescent microscopy images obtained by different microscopes. HTB140 melanoma cells were exposed to γ-rays, in the dose range from 1 to 16 Gy. Radiation effects on the DNA level were analyzed at different time intervals after irradiation by Western blot analysis and immunofluorescence microscopy. Immunochemically stained cells were visualized with two types of microscopes: AxioVision (Zeiss, Germany) microscope, comprising an ApoTome software, and AxioImagerA1 microscope (Zeiss, Germany). Obtained results show that the level of γH2AX is time and dose dependent. Immunofluorescence microscopy provided better detection of DSBs for lower irradiation doses, while Western blot analysis was more reliable for higher irradiation doses. AxioVision microscope containing ApoTome software was more suitable for the detection of γH2AX foci.

Response of human HTB140 melanoma cells to proton irradiation in combination with fotemustine (FM) was investigated. Effects of these agents were analysed on cell proliferation and induction of apoptosis. Cells pretreated with 100- or 250-µM of FM were irradiated in the middle of the therapeutic 62-MeV proton spread-out Bragg peak, with a dose of 16 Gy. All treatments reduced proliferation and survival of melanoma cells. The most pronounced effects of the combined treatment were obtained for cell survivals. The level of apoptosis increased after all applied treatments. Particularly good pro-apoptotic effect was achieved when proton irradiation was combined with 250 μM of FM. This was followed by the increased expression of p53 gene. The obtained results have shown that combined application of FM and protons significantly reduced growth of this resistant melanoma cell line.