Photodynamic therapy with the novel photosensitizer chlorophyllin f induces apoptosis and autophagy in human bladder cancer cells.

BACKGROUND AND OBJECTIVES: Our group recently synthesized a new, low-cost photosensitizer, chlorophyllin f. In this study, the effects of chlorophyllin f-mediated photodynamic therapy (PDT) and its potential mechanisms were examined in human bladder cancer cells. MATERIALS AND METHODS: MitoTracker® Green probe and LysoTracker® Green probe were used to label mitochondria and lysosomes, revealing the intracellular localization of chlorophyllin f in 5637 and T24 cells by confocal laser scanning microscopy (CLSM). The cells were treated with chlorophyllin f-mediated PDT; the photo-cytotoxicity of chlorophyllin f was monitored using the Cell Counting Kit-8 assay, and apoptosis was measured by Annexin V-FITC/PI dual staining. Western blotting, transmission electron microscopy (TEM), and staining with Cyto-ID® Autophagy Detection dye, monodansylcadaverine (MDC) and acridine orange were performed to assess autophagy. The role of autophagy was examined by measuring cell viability and apoptosis in both cell lines pretreated with the autophagy inhibitor 3-methyladenine (3-MA). RESULTS: Chlorophyllin f showed affinity for mitochondria and lysosomes. It exhibited significant photocytotoxicity, resulting in a maximum of 86.51% and 84.88% cell death in 5637 and T24 cells, respectively. Additionally, chlorophyllin f-mediated PDT (f-PDT) also induced a significantly higher percentage of apoptosis in treated cells compared to the control groups (P < 0.05). Moreover, the expression of Beclin1 protein and the proportion of LC3-II:LC3-I in both cell lines significantly increased after f-PDT. Autophagy, characterized by an increase in the formation of Cyto-ID® Autophagy Detection dye-labeled autophagosomes, MDC fluorescent dye-labeled autophagic vacuoles and acridine orange-labeled acidic vesicular organelles (AVOs), was observed in f-PDT-treated cells. TEM also revealed double-membrane autophagosome structures 1 hour after f-PDT. Most importantly, when pretreated with 3-MA, the two cell lines showed more significant photo-cytotoxicity and apoptotic cell death compared to those exposed to f-PDT alone (P < 0.05). CONCLUSION: Chlorophyllin f-mediated PDT exerts anti-tumor activity by inducing apoptosis and autophagy, and most importantly, autophagy inhibition enhances f-PDT-mediated apoptotic cell death. These results suggest that chlorophyllin f is a new, effective photosensitizer and that the combination of f-PDT with autophagy inhibitors may be an attractive therapeutic strategy against human non-muscle invasive bladder cancer.

Analysis of urinary calculi composition by infrared spectroscopy: a prospective study of 625 patients in eastern China.

Urolithiasis is a common urologic disease whose prevalence is about 1-20% and increasing throughout the world. The recurrence rate after treatment is more than 50%. Urinary stone analysis is important in determining the possible etiology and the pathophysiology of stone formation. A better understanding of the stone composition may help prevent urinary stone formation. From March 2007 to December 2008, physical analysis of urolithiasis in patients who lived in eastern China for more than 5 years and underwent surgery or shock wave lithotripsy in our hospital or passed their stones spontaneously was carried out using the Fourier transform infrared spectroscopy (FT-IR). Clinical and demographic findings were evaluated and compared with the stone components. Stone analysis was performed in 625 patients. The FT-IR evaluation showed that 234 (37.4%) were pure, and the most frequent was calcium oxalate (33.9%), followed by calcium phosphate (2.7%), and uric acid (0.8%). 391 (62.6%) were mixed stone, calcium oxalate (43.2%) was the most commonly major component, followed by calcium phosphate (16.3%), cystine (1.3%), uric acid (1.1%), and struvite (0.6%). Uric acid (p = 0.029) was the major component found more frequently in men, while struvite (p = 0.037) was more frequent in women. Uric acid (p = 0.031) was more common in lower urinary tract stones, and its formers with the mean age of 55 years were older than those with other components (p = 0.039). In eastern China, the most commonly found pure stone was calcium oxalate, while the most frequent mixed stone was calcium oxalate and calcium phosphate mixture. Stone location, gender, and age may influence stone component.