Synergistic Effects of Melatonin on Radiosensitization in Carbon-ion Radiotherapy.

BACKGROUND/AIM: Despite the established antitumor effectiveness and synergistic interactions of melatonin with photon irradiation, its role in carbon-ion radiotherapy remains uncertain. This study aimed to elucidate the mechanisms and potential clinical advantages of combining exogenous melatonin therapy with carbon-ion radiotherapy. MATERIALS AND METHODS: The investigation assessed the impact of combining exogenous melatonin with photon or carbon-ion irradiation on cell-cycle modulation and DNA-repair capability using the melanoma cell line B16F10. RNA sequencing and bioinformatics analysis were conducted to explore mechanisms and evaluate potential clinical benefits, with validation performed on the osteosarcoma cell line LM8. RESULTS: Pre-treatment with melatonin reduced the survival fraction of B16F10 and LM8 cells upon exposure to photon and carbon-ion radiation. Mechanistically, melatonin was found to inhibit G2/M arrest, preserve DNA damage, and suppress key genes involved in DNA double-strand break repair after 8 Gy carbon-ion radiation. Furthermore, RNA sequencing and bioinformatics analysis revealed favorable changes in genes associated with survival and metastasis, highlighting potential clinical significance. LM8 cells treated with melatonin exhibited increased radiosensitivity and suppression of DNA-repair proteins. CONCLUSION: The combination of exogenous melatonin not only heightened radiosensitivity and modulated hallmark tumor gene sets in vitro but also markedly suppressed the efficiency of DNA double-strand break-repair pathway, thus enhancing the cytotoxicity of carbon-ion radiotherapy.

Wavelength-dependent photobiomodulation (PBM) for proliferation and angiogenesis of melanoma tumor in vitro and in vivo.

Photobiomodulation (PBM) has widely been used to effectively treat complications associated with cancer treatment, including oral mucositis, radiation dermatitis, and surgical wounds. However, the safety of PBM against cancer still needs to be validated as the effects of PBM on cancer cells and their mechanisms are unclear. The current study investigated the wavelength-dependent PBM effects by examining four different laser wavelengths (405, 532, 635, and 808 nm) on B16F10 melanoma tumor cells. In vitro tests showed that PBM with 808 nm promoted both proliferation and migration of B16F10 cells. In vivo results demonstrated that PBM with 808 nm significantly increased the relative tumor volume and promoted angiogenesis with overexpression of VEGF and HIF-1α. In addition, PBM induced the phosphorylation of factors closely related to cancer cell proliferation and tumor growth and upregulated the related gene expression. The current result showed that compared to the other wavelengths, 808 nm yielded a significant tumor-stimulating effect the malignant melanoma cancer. Further studies will investigate the in-depth molecular mechanism of PBM on tumor stimulation in order to warrant the safety of PBM for clinical cancer treatment.

Experimental evidence that carbon-ion radiotherapy utilizes cytotoxic T lymphocyte-mediated anti-tumor immunity for shrinking tumors compared to X-ray therapy.

The therapeutic efficacy of radiotherapy (RT) is primarily driven by two factors: biophysical DNA damage in cancer cells and radiation-induced anti-tumor immunity. However, Anti-tumor immune responses between X-ray RT (XRT) and carbon-ion RT (CIRT) remain unclear. In this study, we, employed mouse models to assess the immunological contribution, especially cytotoxic T-lymphocyte (CTL)-mediated immunity, to the therapeutic effectiveness of XRT and CIRT in shrinking tumors. We irradiated mouse intradermal tumors of B16F10-ovalbumin (OVA) mouse melanoma cells and 3LL-OVA mouse lung cancer cells with carbon-ion beams or X-rays in the presence or absence of CTLs. CTL removal was performed by administration of anti-CD8 monoclonal antibody (mAb) in mice. Based on tumor growth delay, we determined the tumor growth and regression curves. The enhancement ratio (ER) of the slope of regression lines in the presence of CTLs, relative to the absence of CTLs, indicates the dependency of RT on CTLs for shrinking mouse tumors, and the biological effectiveness (RBE) of CIRT relative to XRT were calculated. Tumor growth curves revealed that the elimination of CD8+ CTLs by administrating anti-CD8 mAb accelerated tumor growth compared to the presence of CTLs in both RTs. The ERs were larger in CIRT compared to XRT in the B16F10-OVA tumor models, but not in the 3LL-OVA models, suggesting a greater contribution of CTL-mediated anti-tumor immunity to tumor reduction in CIRT compared to XRT in the B16F10-OVA tumor model. In addition, the RBE values for both models were larger in the presence of CTLs compared to models without CTLs, suggesting that CIRT may utilize CTL-mediated anti-tumor immunity more than X-ray. The findings from this study suggest that although immunological contribution to therapeutic efficacy may vary depending on the type of tumor cell, CIRT utilizes CTL-mediated immunity to a greater extent compared to XRT.

Combining large-spot low-fluence 1064-nm and fractional 1064-nm picosecond lasers for promoting protective melanosome autophagy via the PI3K/Akt/mTOR signalling pathway for the treatment of melasma.

Melasma is a common condition of hyperpigmented facial skin. Picosecond lasers are reported to be effective for the treatment of melasma. We aimed to identify the most effective therapeutic mode and elucidate the potential molecular mechanisms of picosecond lasers for the treatment of melasma. Female Kunming mice with melasma-like conditions were treated using four different picosecond laser modes. Concurrently, in vitro experiments were conducted to assess changes in melanin and autophagy in mouse melanoma B16-F10 cells treated with these laser modes. Changes in melanin in mouse skin were detected via Fontana-Masson staining, and melanin particles were evaluated in B16-F10 cells. Real-time polymerase chain reaction and western blotting were used to analyse the expression levels of melanosome and autophagy-related messenger ribonucleic acid (mRNA) and proteins. A combination of large-spot low-fluence 1064-nm and fractional 1064-nm picosecond lasers resulted insignificant decreases in melanin as well as in mRNA and protein expression of melanin-synthesizing enzymes (TYR, TRP-1 and MITF). This combination also led to increased expression of the autophagy-related proteins, Beclin1 and ATG5, with a marked decrease in p62 expression. Intervention with the PI3K activator, 740 Y-P, increased TYR, TRP-1, MITF, p-PI3K, p-AKT, p-mTOR and p62 expression but decreased the expression of LC3, ATG5 and Beclin1. A combination of large-spot low-fluence 1064-nm and fractional 1064-nm picosecond lasers proved more effective and safer. It inhibits melanin production, downregulates the PI3K/AKT/mTOR pathway, enhances melanocyte autophagy and accelerates melanin metabolism, thereby reducing melanin content.

Hypofractionated radiotherapy combined with lenalidomide improves systemic antitumor activity in mouse solid tumor models.

Background: Hypofractionated radiotherapy (hRT) can induce a T cell-mediated abscopal effect on non-irradiated tumor lesions, especially in combination with immune checkpoint blockade (ICB). However, clinically, this effect is still rare, and ICB-mediated adverse events are common. Lenalidomide (lena) is an anti-angiogenic and immunomodulatory drug used in the treatment of hematologic malignancies. We here investigated in solid tumor models whether lena can enhance the abscopal effect in double combination with hRT. Methods: In two syngeneic bilateral tumor models (B16-CD133 melanoma and MC38 colon carcinoma), the primary tumor was treated with hRT. Lena was given daily for 3 weeks. Besides tumor size and survival, the dependence of the antitumor effects on CD8+ cells, type-I IFN signaling, and T cell costimulation was determined with depleting or blocking antibodies. Tumor-specific CD8+ T cells were quantified, and their differentiation and effector status were characterized by multicolor flow cytometry using MHC-I tetramers and various antibodies. In addition, dendritic cell (DC)-mediated tumor antigen cross-presentation in vitro and directly ex vivo and the composition of tumor-associated vascular endothelial cells were investigated. Results: In both tumor models, the hRT/lena double combination induced a significant abscopal effect. Control of the non-irradiated secondary tumor and survival were considerably better than with the respective monotherapies. The abscopal effect was strongly dependent on CD8+ cells and associated with an increase in tumor-specific CD8+ T cells in the non-irradiated tumor and its draining lymph nodes. Additionally, we found more tumor-specific T cells with a stem-like (TCF1+ TIM3- PD1+) and a transitory (TCF1- TIM3+ CD101- PD1+) exhausted phenotype and more expressing effector molecules such as GzmB, IFNγ, and TNFα. Moreover, in the non-irradiated tumor, hRT/lena treatment also increased DCs cross-presenting a tumor model antigen. Blocking type-I IFN signaling, which is essential for cross-presentation, completely abrogated the abscopal effect. A gene expression analysis of bone marrow-derived DCs revealed that lena augmented the expression of IFN response genes and genes associated with differentiation, maturation (including CD70, CD83, and CD86), migration to lymph nodes, and T cell activation. Flow cytometry confirmed an increase in CD70+ CD83+ CD86+ DCs in both irradiated and abscopal tumors. Moreover, the hRT/lena-induced abscopal effect was diminished when these costimulatory molecules were blocked simultaneously using antibodies. In line with the enhanced infiltration by DCs and tumor-specific CD8+ T cells, including more stem-like cells, hRT/lena also increased tumor-associated high endothelial cells (TA-HECs) in the non-irradiated tumor. Conclusions: We demonstrate that lena can augment the hRT-induced abscopal effect in mouse solid tumor models in a CD8 T cell- and IFN-I-dependent manner, correlating with enhanced anti-tumor CD8 T cell immunity, DC cross-presentation, and TA-HEC numbers. Our findings may be helpful for the planning of clinical trials in (oligo)metastatic patients.

Combination low-dose cyclophosphamide with check-point blockade and ionizing radiation promote an abscopal effect in mouse models of melanoma.

PURPOSE: The complex strategy of hypo-fractionated radiotherapy (HFRT) in combination with an immune checkpoint inhibitor (ICI) can stimulate a potential systemic antitumor response; however, the abscopal effect is always precluded by the tumor microenvironment, which may limit sufficient T-cell infiltration of distant nonirradiated tumors for certain kinds of inhibitory factors, such as regulatory T-cells (Tregs). Additionally, low-dose cyclophosphamide (LD-CYC) can specifically kill regulatory Tregs and strongly synergize antigen-specific immune responses, which could promote an abscopal effect. MATERIALS AND METHODS: We explored whether a triple regimen consisting of HFRT, ICI, and LD-CYC could achieve a better systemic antitumor response in bilateral mouse tumor models. RESULT: Our data demonstrate that LD-CYC combined with HFRT and antiprogrammed cell death ligand 1 (PDL-1) therapy could enhance the abscopal effect than only HFRT/antiPDL-1 or HFRT alone. Surprisingly, repeat CYC doses cannot further restrain tumor proliferation but can prolong murine overall survival, as revealed by the major pathologic responses. These results are associated with increased CD8 + effector T-cell infiltration, although LD-CYC did not upregulate PDL-1 expression in the tumor. CONCLUSIONS: Compared with traditional strategies, for the first time, we demonstrated that a triple treatment strategy remarkably increased the number of radiation-induced tumor-infiltrating CD8 + T-cells, effectively decreasing infiltrating Tregs, and promoting an abscopal effect. Thus, we describe a novel and effective therapeutic approach by combining multiple strategies to target several tumor-mediated immune inhibitory mechanisms.

The Impact of Dose Rate on the Tumor Microenvironment Using Flattening-filter-free Beams.

AIMS: Recently, dose delivery technology has rapidly evolved with flattening filter-free beams (FFF), and the biological effects of high dose rates are a matter of interest. We hypothesized that FFF beams at different dose rates obtained with modern linear accelerators have different effects on the TME. MATERIALS AND METHODS: The B16-F10 melanoma syngeneic tumor model was established, and mice were randomized to 2 different doses (2 Gy and 10 Gy) and 3 different dose rates (1 Gy/min, 6 Gy/min, and 14 Gy/min) along with the control group. Euthanasia was performed on the seventh day after RT, and intracardiac blood was collected for a comet assay. Tumors were harvested and examined histomorphologically and immunohistochemically. Statistical analyses were performed using SPSS software version 23 (SPSS Inc., Chicago, IL, USA). RESULTS: The daily growth rate was uniform, and no difference was observed between tumor volumes across all three dose rates for each dose. Deoxyribonucleic acid (DNA) damage in blood mononuclear cells was not affected by dose or dose rate. In the TME histomorphological examination, the number of mitosis is less in the 10 Gy arm, whereas the pleomorphism score was greater. Nevertheless, varying dose rates had no effect on the number of mitosis or the pleomorphism score. The severity of the inflammation, cell densities in the TME, and expression of immunohistochemical markers were comparable across all doses and dose rates. CONCLUSION: In our study involving the B16-F10 syngeneic tumor model, varying dose rates obtained with FFF beams had no effect on tumor volume, blood mononuclear cell DNA damage, or TME parameters. However, in order to fully understand the biological impacts of novel techniques, our study should be validated with alternative preclinical setups.

Synthesis and biological evaluation of 125I-erythropoietin as a potential radiopharmaceutical agent for tumours

Erythropoietin (EPO) is a glycoprotein hormone responsible for regulating erythropoiesis. Expression of EPO and EPO receptors (EPOr) has recently been demonstrated in some neoplastic cell lines and tumours, suggesting a potential new target for therapy. In this work, EPO was labeled with iodine-125 using the lactoperoxidase method, known to prevent damage to protein during radioiodination, and labeling conditions were optimized. In vitro stability studies have shown that 125I-EPO is radiochemically stable for 20 days after radiolabeling. In vitro cell binding studies have demonstrated very low binding (<2 percent) of EPO to normal and neoplastic cell lines tested. As expected, the biodistribution in healthy mice exhibited comparatively high rates of fixation in the organs of the excretory system. Thyroid also proved to be a critical organ which may indicate in vivo dissociation of 125I-EPO. In mice with induced melanoma, only a residual fixation in the tumour was evident. Further studies are warranted on other tumoral cell lines to better understand the binding process and internalization into cells. Studies on EPO labeled with carbon-11 could be valuable, since there is a greater chance of preserving the biological activity of the protein using this method.

A eritropoetina (EPO) é um hormônio glicoprotéico responsável pela regulação da eritropoese. Recentemente foi demonstrado que os receptores de EPO (EPOr) estão expressos em algumas linhas celulares neoplásicas, o que sugere a sua potencialidade como um novo alvo terapêutico. Neste trabalho a EPO foi radiomarcada com iodo-125 através do método da lactoperoxidase, menos agressivo para a viabilidade biológica das proteínas. A 125I-EPO revelou ser radioquimicamente estável durante 20 dias após a síntese. Um estudo biológico in vitro em linhas celulares tumorais demonstrou que a 125I-EPO apresenta uma ligação muito fraca (<2 por cento), tanto em células normais como nas linhagens tumorais testadas. A biodistribuição em camundongos saudáveis apresentou taxas de fixação relativamente maiores nos órgãos excretores e a tireóide revelou ser o órgão crítico, o que pode indicar a dissociação in vivo da 125I-EPO. No estudo em camundongos com melanoma induzido a fixação no tumor foi residual. Serão, no entanto, necessários novos estudos em outras linhagens tumorais para entender o seu processo de internalização e ligação nas células. Estudos da EPO radiomarcada com carbono-11 poderão também revelar-se interessantes, já que neste método há maior probabilidade da atividade biológica ser preservada.

Expresión del gen PRL-1, en células de melanoma murino B-16, inducidas a supresión del crecimiento con el sensibilizador a la radiación ultravioleta: bromodeoxiuridina / Expression of the PRL-1 gene in B-16 murine melanoma cells induce to growth suppression in the ultraviolet radiation sensitizer bromodeoxiuridin

El cáncer es una enfermedad del genoma, en el cual múltiples aberraciones genéticas conducen a la activación de oncogenes y a la inactivación de genes supresores tumorales, promoviendo un desequilibrio en el control de la proliferación, diferenciación y muertecelular. Las células tumorales pueden ser inducidas a un crecimiento controlado y a diferenciación terminal in vitro por tratamiento farmacológico que conduce a la restauración parcial del fenotipo celular normal. Sin embargo, el uso de inductores farmacológicos de diferenciarón, supresión del crecimiento y muerte celular como el ácido retinoíco, vitamina D, L-tirosina y bromodeoxiuridina, es limitado debido a que no se conocen los mecanismos moleculares y sus blancos de acción. La Bromodeoxiuridina, es un análogo de la timina, sensibilizador a la radiación ultravioleta e inductor de la supresión del crecimiento. Sin embargo, las bases moleculares de estas acciones no se conocen muy bien. La identificación de genes blancos moleculares (como cinasas y fosfatasas) de la Bromodeoxiuridina (BrdU) y su papel en el control de la proliferación celular y la radiosensibilización, conduciría al entendimiento y desarrollo de potenciales agentes terapéuticos para el control de la proliferación en células tumorales, como tratamiento alternativo a la quimioterapia y radioterapia convencionales, las cuales son altamente agresivas no sólo para las células tumorales, sino también para las células normales en proliferación. Los genes que codifican para proteínas fosfatasas se han implicado en la tumorigénesis. Recientemente en el laboratorio de fisiología molecular del INS se clonó y se secuenció el cADN que codifica para la tirosina fosfatasa PRL-1. Sin embargo, no se sabe si sus niveles de expresión cambian en células de melanoma proliferantes e inducidas a supresión del crecimiento y diferenciación celular. Por lo tanto, el objetivo de este trabajo fue determinar el nivel de expresión de ARN del gen que codifica para la tirosina-fosfatasa PRL-1, en células de melanoma murino B-16 inducidas a supresión del crecimiento y diferenciación celular. Por lo tanto, el objetivo de este trabajo fue determinar el nivel de expresión del ARN del gen que codifica para la tirosina-fosfatasa PRL-1, en células de melanoma murino B-16 inducidas a supresión del crecimiento por el tratamiento con Bromodeoxiuridina, radiación ultravioleta tipo C y sensibilización...