Ultrasound-mediated activity by various metal-porphyrin complexes in human colon cancer and fibroblast cells

Porphyrins are known photosensitisers as their tetrapyrrole ring structure can be activated by light to generate ROS which causes damage to cell structures and leads to cell death.Evidences suggest their ability to also behave as sonosensitisers in SDT of cancer.This is an innovative approach developed from photodynamic therapy in which a physical stimulus,e.g.US instead of light,is used to trigger the cytotoxic nature of sonosensitisers,which are ineffective on their own.The ability of porphyrin to generate ROS following electronic excitation, renders porphyrins an excellent platform to explore the sonodynamic process.Moreover,some studies suggest that the cytotoxic effect of porphyrins and US might be selective for cancer cells.The aim of this work is to investigate the effects induced by US exposure of metal complexes of TMPyP,i.e.TMPyP-Zn(II) andTMPyP-Pd(II),on cancer and non-cancerous cells,in order to evaluate possible differences in responsiveness to the treatment.SDT was performed with low intensity US on HT-29 and HDF 106-05 cells,previously incubated with P-Zn or P-Pd. Effects of light exposure of the same P-Zn or P-Pd concentrations were evaluated on both cell lines,as a reference.To highlight possible differences between HT-29 and HDF 106-05 cells,basal content of GSH and porphyrin cellular uptake were determined.Effects of the SDT with P-Zn or P-Pd were then evaluated on cell growth,ROS production and MMP.Due to the interaction of US with cell membrane for the energy transfer to intracellular sonosensitisers, differences on cell membrane response were also investigated during US exposure. Therefore,the effects of US on cell membrane poration and calcium flux and cell membrane fluidity were assessed.HT-29 and HDF 106-05 cells showed similar intracellular GSH levels suggesting a similar role of GSH system in defence against ROS.The maximum P-Zn and P-Pd uptake was reached on both cell lines after 24 hours.Effects of SDT with US and P-Zn or P-Pd used at non-cytotoxic concentration per se, resulted in significant ROS production and cytotoxicity only on HT-29 cells.The results also showed a porphyrin dose-dependent sonodynamic effect,ROS-mediated cytotoxicity and mitochondrial function impairment only on HT-29 cells.Conversely, P-Zn or P-Pd under light exposure showed similar cytotoxicity on both cell lines.It is worth mentioning that US was not able to induce membrane poration on both cell lines.However,HT-29 and HDF 106-05 cells showed different behaviour during US exposure in terms of membrane fluidity and calcium flux.HT-29 cells showed a significant increase in membrane fluidity along with an increase of calcium flux,whereas HDF 106-05 cells showed a significant decrease in cell membrane fluidity and an increase of calcium flux followed by a rapid decrease.Finally,the data show that the efficacy of SDT with US and metal-porphyrin complexes in killing cancer cells is dependent on ROS generation triggered by US-mediated sensitiser activation.The US-mediated sensitiser activation seems to be selective according to cell type, being effective against cancer cells but not to non-cancerous cells,despite what has been observed for photodynamic treatment.Experimental evidence suggests that outcome and selectivity of the sonodynamic treatment,supported by intramembrane cavitation hypothesis of sonosensitiser activation,could be strongly regulated by different ROS production and cell membrane properties in response to US exposure.