Photodynamic action of palmatine hydrochloride on colon adenocarcinoma HT-29 cells

Palmatine hydrochloride (PaH) is a natural active compound from a traditional Chinese medicine (TCM). The present study aims to evaluate the effect of PaH as a new photosensitizer on colon adenocarcinoma HT-29 cells upon light irradiation. Firstly, the absorption and fluorescence spectra of PaH were measured using a UV-vis spectrophotometer and RF-1500PC spectrophotometer, respectively. Singlet oxygen (1O2) production of PaH was determined using 1, 3-diphenylisobenzofuran (DPBF). Dark toxicity of PaH was estimated using the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Cellular uptake of PaH in HT-29 cells was detected at different time intervals. Subellular localization of PaH in HT-29 cells was observed using confocal laser fluorescence microscopy. For photodynamic treatment, HT-29 cells were incubated with PaH and then irradiated by visible light (470 nm) from a LED light source. Photocytotoxicity was investigated 24 h after photodynamic treatment using MTT assay. Cell apoptosis was observed 18 h after photodynamic treatment using a flow cytometry with Annexin V/PI staining. Results showed that PaH has an absorption peak in the visible region from 400 nm to 500 nm and a fluorescence emission peak at 406 nm with an excitation wavelength of 365 nm. PaH was activated by the 470 nm visible light from a LED light source to produce 1O2. Dark toxicity showed that PaH alone treatment had no cytotoxicity to HT-29 cancer cells and NIH-3T3 normal cells after incubation for 24 h. After incubation for 40 min, the cellular uptake of PaH reached to the maximum and PaH was located in mitochondria. Photodynamic treatment of PaH demonstrated a significant photocytotoxicity on HT-29 cells. The rate of cell death increased significantly in a PaH concentration-dependent and light dose-dependent manner. Further evaluation revealed that the early and late apoptotic rate of HT-29 cells increased remarkably up to 21.54% and 5.39% after photodynamic treatment of PaH at the concentration of 5 μM and energy density of 10.8 J/cm2. Our findings demonstrated that PaH as a naturally occurring photosensitizer has potential in photodynamic therapy on colon adenocarcinoma.