Abstract
Hepatocellular carcinoma (HCC) presents a significant therapeutic challenge, necessitating novel approaches beyond conventional treatments. This study investigates the combined cytotoxic effects of nano-titanium dioxide (nano-TiO₂), copper (II) phthalocyanine (CuPc), and copper (II) phthalocyanine-modified nano-TiO₂-(nano-TiO₂/Pc) on HepG2 hepatocellular carcinoma cells using sonodynamic therapy (SDT), photodynamic therapy (PDT), and sonophotodynamic therapy (SPDT). The results show that individual treatments with nano-TiO₂ or CuPc alone did not induce significant cytotoxicity. However, when combined with SDT or PDT, a noticeable decrease in cell viability was observed. Strikingly, SPDT combined with nano-TiO₂/Pc demonstrated the most significant cytotoxic effect, achieving up to 83.80% apoptosis in HepG2 cells. This was associated with a marked reduction in Bcl-2 protein levels and an increase in cleaved caspase-3, cleaved caspase-9, cytochrome-c (cyt-c), and Bax indicating the activation of both intrinsic and extrinsic apoptotic pathways. Furthermore, SPDT-nano-TiO₂/Pc significantly increased oxidative stress, as evidenced by decreased levels of superoxide dismutase (SOD), catalase (CAT), and glutathione (GSH), along with elevated levels of malondialdehyde (MDA). These findings suggest that phthalocyanine-mediated SPDT effectively enhances mitochondrial apoptosis and disrupts the tumor cytoplasmic membrane, highlighting the potential of combining SDT and PDT with nano-TiO₂/Pc as a promising strategy for cancer treatment.