Effetti biologici e meccanismi molecolari indotti dagli ultrasuoni a bassa intensità su cheratinociti e melanoma umano

Negli ultimi anni gli Ultrasuoni (US) a bassa intensità sono stati largamente utilizzati come strumento terapeutico. Il loro vasto utilizzo in diversi campi ha così guidato i ricercatori a studiare i relativi bioeffetti da essi indotti, tra i più significativi abbiamo la porazione (sonoporazione) della membrana, l’attivazione di processi citotossici e infiammatori. Attualmente una metodologia sperimentale univoca accetta per studiare e descrivere quantitativamente il rischio biologico è ancora mancante. Come noto, i cheratinociti sono il principale tipo cellulare della pelle e i più esposti a fattori ambientali, fornendo la prima linea di barriera difensiva nell’ospite e partecipando inoltre attivamente alla risposta immunitaria. Abbiamo così studiato se l’energia meccanica trasportata dagli US a bassa intensità alla frequenza di 1 MHz potesse generare uno stress meccanico in grado di indurre il rilascio della citochina infiammatoria IL-6 nella linea cellulare di cheratinociti umani HaCaT. I nostri risultati evidenziano come gli US siano in grado di indurre overespressione e secrezione di IL-6, associata all’attivazione del fattore nucleare - κB (NF-κB). Inoltre, sono state osservate alterazione nella morfologia e permeabilità di membrana insieme a una ridotta vitalità cellulare in funzione dei parametri di esposizione. In tale contesto, sono stati indagati i bioeffetti degli US sulle linee di melanoma umano SK-MEL-5 e SK-MEL-28, le quali presentano una diversa espressione di p53. Preliminarmente, tali dati hanno mostrato un aumento dei livelli di mRNA di IL-6 esclusivamente nelle cellule SK-MEL-28 accompagnato da un arresto del ciclo cellulare in G2/M più evidente nelle SK-MEL-5, suggerendo un potenziale ruolo di p53 nel modulare i differenti effetti indotti dagli US. Questi risultati possono pertanto aprire la strada per studiare ulteriormente i meccanismi molecolari legati all’esposizione agli US

The kinesin Eg5 inhibitor K858 induces apoptosis but also survivin-related chemoresistance in breast cancer cells

Inhibitors of kinesin spindle protein Eg5 are characterized by pronounced antitumor activity. Our group has recently synthesized and screened a library of 1,3,4-thiadiazoline analogues with the pharmacophoric structure of K858, an Eg5 inhibitor. We herein report the effects of K858 on four different breast cancer cell lines: MCF7 (luminal A), BT474 (luminal B), SKBR3 (HER2 like) and MDA-MB231 (basal like). We demonstrated that K858 displayed anti-proliferative activity on every analyzed breast cancer cell line by inducing apoptosis. However, at the same time, we showed that K858 up-regulated survivin, an anti-apoptotic molecule. We then performed a negative regulation of survivin expression, with the utilization of wortmannin, an AKT inhibitor, and obtained a significant increase of K858-dependent apoptosis. These data demonstrate that K858 is a potent inhibitor of replication and induces apoptosis in breast tumor cells, independently from the tumor phenotype. This anti-proliferative response of tumor cells to K858 can be limited by the contemporaneous over-expression of survivin; consequently, the reduction of survivin levels, obtained with AKT inhibitors, can sensitize tumor cells to K858-induced apoptosis

Differential effects on membrane permeability and viability of human keratinocyte cells undergoing very low intensity megasonic fields

Among different therapeutic applications of Ultrasound (US), transient membrane sonoporation (SP) - a temporary, non-lethal porosity, mechanically induced in cell membranes through US exposure - represents a compelling opportunity towards an efficient and safe drug delivery. Nevertheless, progresses in this field have been limited by an insufficient understanding of the potential cytotoxic effects of US related to the failure of the cellular repair and to the possible activation of inflammatory pathway. In this framework we studied the in vitro effects of very low-intensity US on a human keratinocyte cell line, which represents an ideal model system of skin protective barrier cells which are the first to be involved during medical US treatments. Bioeffects linked to US application at 1 MHz varying the exposure parameters were investigated by fluorescence microscopy and fluorescence activated cell sorting. Our results indicate that keratinocytes undergoing low US doses can uptake drug model molecules with size and efficiency which depend on exposure parameters. According to sub-cavitation SP models, we have identified the range of doses triggering transient membrane SP, actually with negligible biological damage. By increasing US doses we observed a reduced cells viability and an inflammatory gene overexpression enlightening novel healthy relevant strategies

Folate-based single cell screening using surface enhanced Raman microimaging

Recent progress in nanotechnology and its application to biomedical settings have generated great advantages in dealing with early cancer diagnosis. The identification of the specific properties of cancer cells, such as the expression of particular plasma membrane molecular receptors, has become crucial in revealing the presence and in assessing the stage of development of the disease. Here we report a single cell screening approach based on Surface Enhanced Raman Scattering (SERS) microimaging. We fabricated a SERS-labelled nanovector based on the biofunctionalization of gold nanoparticles with folic acid. After treating the cells with the nanovector, we were able to distinguish three different cell populations from different cell lines (cancer HeLa and PC-3, and normal HaCaT lines), suitably chosen for their different expressions of folate binding proteins. The nanovector, indeed, binds much more efficiently on cancer cell lines than on normal ones, resulting in a higher SERS signal measured on cancer cells. These results pave the way for applications in single cell diagnostics and, potentially, in theranostic

AR and PI3K/AKT in Prostate Cancer: A Tale of Two Interconnected Pathways

Prostate cancer (PCa) is the most common cancer in men. The androgen receptor (AR) has a pivotal role in the pathogenesis and progression of PCa. Many therapies targeting AR signaling have been developed over the years. AR signaling inhibitors (ARSIs), including androgen synthesis inhibitors and AR antagonists, have proven to be effective in castration-sensitive PCa (CSPC) and improve survival, but men with castration-resistant PCa (CRPC) continue to have a poor prognosis. Despite a good initial response, drug resistance develops in almost all patients with metastatic CRPC, and ARSIs are no longer effective. Several mechanisms confer resistance to ARSI and include AR mutations but also hyperactivation of other pathways, such as PI3K/AKT/mTOR. This pathway controls key cellular processes, including proliferation and tumor progression, and it is the most frequently deregulated pathway in human cancers. A significant interaction between AR and the PI3K/AKT/mTOR signaling pathway has been shown in PCa. This review centers on the current scene of different AR and PI3K signaling pathway inhibitors, either as monotherapy or in combination treatments in PCa, and the treatment outcomes involved in both preclinical and clinical trials. A PubMed-based literature search was conducted up to November 2022. The most relevant and recent articles were selected to provide essential information and current evidence on the crosstalk between AR and the PI3K signaling pathways. The ClinicalTrials.gov registry was used to report information about clinical studies and their results using the Advanced research tool, filtering for disease and target

Immunotherapy in prostate cancer. Recent advances and future directions

In recent years, immunotherapy has been proposed for the treatment of asymptomatic or minimally symptomatic metastatic castrate-resistant prostate cancer (PCa). Clinical trials using Sipuleucel-T have demonstrated a survival benefit in PCa patients, suggesting that this cancer is linked to a limited immune response. However, the outcome of PCa treated with immune therapeutics has limited benefits in monotherapy: novel vaccination approaches and immune checkpoint blockade gave disappointing results. Several combinations of therapies, such as novel cancer vaccines or checkpoint inhibitors with different immunotherapeutic agents, combined with hormone therapy (enzalutamide, abiraterone acetate), radiotherapy or radium-223, DNA-damaging agents (olaparib), or chemotherapy (docetaxel) hold great promise for eliciting an immune response and improving clinical outcomes in PCa. The goal of immunotherapy is to overcome immunosuppression and destroy cancer cells, or at least to induce those pathways that go back from ‘the escape phase to equilibrium phase’according to the definition of cancer immunoediting. The aim of this review is to analyse the immune responses during PCa progression and to present the current data regarding immune therapies for PCa

Resistance to the mTOR inhibitor everolimus is reversed by the downregulation of survivin in breast cancer cells

Everolimus (RAD001) is an inhibitor of mammalian target of rapamycin used in combination with exemestane to treat hormone receptor-positive advanced breast cancer. However, not all patients are equally sensitive to RAD001 and certain patients develop resistance. Therefore, the present study analyzed the mechanisms involved in the resistance of breast cancer cells to RAD001 in order to identify a potential tool to overcome it. The effects of RAD001 on the inhibition of cell viability, on the induction of apoptosis and autophagy and on the regulation of survivin, an anti-apoptotic protein, were evaluated in two breast cancer cell lines: BT474 (luminal B) and MCF7 (luminal A). RAD001 was demonstrated to induce autophagy in the two cell lines at following a short period of treatment (4 h) and to induce apoptosis exclusively in BT474 cells following longer periods of treatment (48 h). RAD001 induced the downregulation of survivin in BT474 cells and its upregulation in MCF7 cells. Consequently, inhibiting survivin with YM155 resulted in the acquired resistance of MCF7 cells to RAD001 being reverted, restoring RAD001-induced apoptosis. These data demonstrated that RAD001 exerted anti-proliferative and pro-apoptotic effects on breast cancer cells, but that these effects were repressed by the simultaneous up-regulation of survivin. Finally, the results demonstrated that inhibiting the expression of survivin resulted in the restoration of the anti-neoplastic activity of RAD001

Growth arrest and apoptosis induced by kinesin Eg5 inhibitor K858 and by its 1,3,4-thiadiazoline analogue in tumor cells

Tumors are complex and heterogeneous but, despite this, they share the ability to proliferate continuously, irrespective of the presence of growth signals, leading to a higher fraction of actively growing and dividing cells compared with normal tissues. For this reason, the cytotoxic antimitotic treatments remain an important clinical tool for tumors. Among these drugs, antitubulin compounds constitute one of the most effective anticancer chemotherapies; however, they cause dose-limiting side effects. Therefore, it is still necessary to develop compounds with new targets and new mechanisms of action to reduce side effects or chemoresistance. Mitosis-specific kinesin Eg5 can represent an attractive target for discovering such new anticancer agents because its role is fundamental in mitotic progression. Therefore, we analyzed the effects induced by an inhibitor of kinesin Eg5, K858, and by its 1,3,4-thiadiazoline analogue on human melanoma and prostate cancer cell lines. We found that both compounds have an antiproliferative effect, induce apoptosis, and can determine a downmodulation of survivin

SERS-based diagnostics: selective targeting of different human cancer cells using functionalized gold nanoparticles

In the last few decades, the development of novel spectroscopic techniques, often combining a very high sensitivity with huge spatial resolution, allowed the possibility of investigating down-scaled phenomena, as for example biochemical and biophysical processes in living systems. Surface Enhanced Raman Spectroscopy (SERS) is one of the most established techniques in this framework [1]. SERS is based on the plasmonic resonance of metal nanostructures: the collective electronic excitation at the metal surface can indeed be excited by light and give rise to the localization of strong electromagnetic fields close to the nanoparticle surface, which can be used for spectroscopy. In the past years, SERS has allowed to reach not only the threshold of single molecule vibrational spectroscopy [2], but also the implementation of devices in the field of biosensing, capable of detecting specific biomolecules at very low concentration by means of Raman spectroscopy [3]. Moreover, the implementation of SERS-labelled nanomaterials, such as functionalized metallic nanoparticles, paved the way for the application of these systems in the emerging field of nanomedicine [4]. Much interest has lately risen, indeed, around the concept of “theranostics”, i.e. combining diagnostics with therapy, the latter to perform selectively on cancer cells without damaging the healthy tissue [4]. Plasmonics-based theranostics is often designed combining SERS and photothermal bleaching. One of the open problems in biomedicine is the early detection of cancer, i.e. the capability of reveal the presence of the disease when it is not yet advanced. Addressing this problem, we designed a biocompatible system based on gold nanoparticles functionalized with the Raman active bifunctional linker 4-aminothiophenol and further conjugated with folic acid, a biomolecule with an essential role in cell reproduction. Our system can be considered a nanobiovector, as it is capable of targeting a specific kind of cell and locate on the folate receptors, on the cell membrane [5]. Folic acid receptors are generally overexpressed in many types of cancer cells, as these reproduce more frequently then ordinary ones [6]. The presence of folate receptors on the membrane strongly depends on the physiology of the cell line considered. In this presentation, we will show that the high specificity of our system allowed us not only to target cancer cells, but also to be able to distinguish different cell lines based on their level of expression of folate receptors [5]