The Antitumor Effect Andlts Mechanism By Photoexcited TiO₂Combined With Bufalin On Human Melanoma A375Cells

ObjectiveBased on the Photodynamic therapy（PDT）, we applied TiO₂nanoparticles withhigh percentage of reactive facets, and combined them with Bufalin, to evaluate theantitumor effect of photoexcited TiO₂combined with Bufalin on human A375melanoma cells, and the mechanisms were also investigated, providing experimentaldata for new therapy of melanoma.MethodsThe antitumor effect on A375cells induced by photoexcited TiO₂combined with BufalinA375cells were treated with TiO₂（TiO₂12.5μg/mL,25μg/mL,50μg/mL,100μg/mL,200μg/mL）,UVA（UVA800μW/cm2,75min）, photoexcited TiO₂（TiO₂12.5μg/mL,25μg/mL,50μg/mL,100μg/mL,200μg/mL, UVA,800μW/cm2,75min）,Bufalin （Bu5nM,10nM,20nM,40nM,80nM）, respectively. The inhibitory rate wascalculated through MTT assay12h,24h,48h and72h later, in order to investigate theeffect of photoexcited TiO₂and Bufalin on A375cells proliferation.A375cells were treated with photoexcited TiO₂（TiO₂25μg/mL, UVA800μW/cm2,75min）,Bufalin （Bu10nM）, photoexcited TiO₂combined withBufalin（TiO₂25μg/mL, UVA800μW/cm²,75min, Bu10nM） respectively. Theinhibitory rate was calculated through MTT assay12h,24h,48h and72h later in orderto investigate the effect of photoexcited TiO₂combined with Bufalin on A375cellsproliferation. Morphological changes of A375cells were observed by lightmicroscope.The mechanism of antitumor effect on A375cells induced byphotoexcited TiO₂combined with BufalinA375cells were treated with photoexcited TiO₂combined with （TiO₂25μg/mL,UVA800μW/cm2,75min, Bu10nM）,photoexcited TiO₂（TiO₂25μg/mL, UVA800μW/cm2,75min）, and Bufalin（Bu10nM） respectively. With the application offlow cytometry, cell cycle, apoptosis, and Reactive oxygen species （ROS） of A375 cells were determinated by PI staining, Annexin-V/PI double staining and DCFH-DAstaining respectively. The expression of Bax and Bcl-2was investigated by WesternBlot method.ResultsThe antitumor effect on A375cells induced by photoexcited TiO₂combined with BufalinThe cell growth of A375was both inhibited when treated with eitherphotoexcited TiO₂or Bufalin. However, the inhibitory rate was significantly increasedwhen A375cells were induced by photoexcited TiO₂combined with Bufalin（P<0.05）.In addition, A375cells shrinked, becoming smaller and rounder compared withnormal cells.The mechanism of antitumor effect on A375cells induced byphotoexcited TiO₂combined with BufalinWhen A375cells were treated with either photoexcited TiO₂or Bufalin, the ROScontent was increased, the proportion of Bax/Bcl-2increased as well, the apoptosis orcell cycle arrest were also observed. Flow cytometry analysis showed A375cells wereinduced to apoptosis when treated with photoexcited TiO₂, while A375cells werearrested in S phase when treated with Bufalin. However, the apoptosis or cell cyclearrest were also observed when A375cells were treated by photoexcited TiO₂combined with Bufalin, with significant high ROS accumulation （P<0.05）.ConclusionWhen A375cells were treated with photoexcited TiO₂combined with Bufalin,the ROS content was increased, the apoptosis and cell cycle arrest were also observed.More importantly, the inhibitory rate was significantly increased when A375cellswere induced by photoexcited TiO₂combined with Bufalin, compared with A375treated with either photoexcited TiO₂or Bufalin. Photoexcited TiO₂combined withBufalin may be regarded as a new promising treatment for melanoma.