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

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

Pro-Inflammatory effects of 1-MHz Pulsed Ultrasound in Human Keratinocytes

Low intensity pulsed ultrasound (LIPUS) of ~1-MHz frequency has been widely applied since the 1950s for both therapeutic and diagnostic tool [1]. The extensive clinical application of LIPUS suggests the need to better understand the related biological effects which are mostly linked to mechanical stress [2]. In this framework, we investigated whether mechanical energy transported by 1 MHz LIPUS could generate a mechanical stress able to modulate and induce the release of inflammatory cytokines such IL-6 in a human keratinocyte cell line (HaCaT). Human keratinocytes are often used to study epidermis homeostasis and its pathophysiology. In particular, these cells respond to both chemicals and physical agents, reporting a different production of cytokines, such as IL-1 and IL-6, in relation to the stimulus and the dose of the stimulus [3]. At first, we analysed the gene expression and protein secretion of the pro-inflammatory cytokine IL-6 by Real Time-qPCR and ELISA, respectively, at varying exposure parameters. Then, we focused on the activation of Nuclear Factor-κB (NF-κB), a pleiotropic regulator of many cellular pathways involved in cytokine production, cell-cycle regulation and apoptosis. Related effects on the cell cycle distribution and apoptosis have been also evaluated through flow cytometry analysis and by following the gene expression of Bax/Bcl-2. Our results pointed out that 1 hour exposure of 1 MHz LIPUS at spatial-peak temporal-average Intensity (Ispta) of 65 mW/cm2 (Figure 1a) promotes both significant IL-6 gene overexpression and protein secretion (Figure 1b), associated with the activation of Nuclear Factor-κB (Figure 1c). Furthermore, we observed a reduced cell viability dependent on exposure parameters together with alterations in membrane permeability and cell proliferation, paving the way for further investigating the molecular mechanisms related to ultrasound exposure

In vitro biological effects induced by ultrasounds-cell interaction

Over the last few years, low intensity ultrasounds (US) have been widely used as therapeutic tool The widespread use of US in different application fields drove researchers to study the related bioeffects the more significant range from membrane poration (sonoporation) to the triggering of cytotoxic and inflammatory processes. Currently, an univocal experimental methodology accepted to investigate and quantitatively describe the biological risk with proper indicators is still lacking. As well known, the primary function of skin is to protect the body to potentially harmful external substances an microorganisms 3 Keratinocytes comprise primarily the epidermis and with their tight junctions form a barrier between the host and the environment 3 4 For this reason, keratinocytes directly interact with non invasive medical treatments US In this regard, firstly we developed experimental setup to define the in vitro effects of very low intensity 0 5 and 1 MHz US in immortalized human keratinocytes HaCaT and human melanoma SK MEL 28 cells By confocal fluorescence microscopy we pointed out, for the first time even in the subcavitation regime, relevant modifications in the membrane morphology and functionality. In this framework, we investigated the possible activation of inflammatory pathways, cytokine interleukine 6 (IL 6 inducible nitric oxide synthase iNOS and Nuclear Factor κ B NF kB) activation In addition, cell proliferation was evaluated by means of the MTT proliferation assay and by flow cytometry to detect apoptotic cells (sub G 1 DNA content

Effect of 1‑MHz ultrasound on the proinflammatory interleukin‑6 secretion in human keratinocytes

Keratinocytes, the main cell type of the skin, are one of the most exposed cells to environmental factors, providing a first defence barrier for the host and actively participating in immune response. In fact, keratinocytes express pattern recognition receptors that interact with pathogen associated molecular patterns and damage associated molecular patterns, leading to the production of cytokines and chemokines, including interleukin (IL)-6. Herein, we investigated whether mechanical energy transported by low intensity ultrasound (US) could generate a mechanical stress able to induce the release of inflammatory cytokine such IL-6 in the human keratinocyte cell line, HaCaT. The extensive clinical application of US in both diagnosis and therapy suggests the need to better understand the related biological effects. Our results point out that US promotes the overexpression and secretion of IL-6, associated with the activation of nuclear factor-κB (NF-κB). Furthermore, we observed a reduced cell viability dependent on exposure parameters together with alterations in membrane permeability, paving the way for further investigating the molecular mechanisms related to US exposure