蔡德甫^1#、林致凡^2#、楊尚哲²、鄧雅旻²、陳宏恩¹、林宜佳¹、仇光宇¹、黃一勝^1,2,3,4

¹新光醫院外科部泌尿科、²新光醫院中央研究室、³台北醫學大學醫學院、⁴輔仁大學醫學院

Te-Fu Tsai^1#, Ji-Fan Lin^2#, Shan-Che Yang², Ya-Ming Teng²,Hung-En Chen¹, Yi-Chia Lin¹,

Kuang-Yu Chou¹, and Thomas I-Sheng Hwang^1,3,4,5

Department of Urology, Shin Kong Wu Ho-Su Memorial Hospital¹, Central Laboratory, Shin Kong Wu Ho-Su Memorial Hospital², Department of Urology, Taipei Medical University³, Division of Urology, School of Medicine, Fu-Jen Catholic University⁴, Taipei, Taiwan.

# These authors contributed equally to this work.

Background: Quinazolines which processing a wide spectrum of biological properties such as antibacterial, antifungal, antivirus, and anticancer activities, are considered as one of the most important heterocycles in medicinal chemistry. The derivatives of quinazolinone are known to inhibit cell metastasis and promote cell death in various types of cancer. Here, we described for the first time that the novel quinazoline derivative MJ-56 (6-pyrrolidinyl-2-(3-bromostyryl)quinazoline-4-one) emitted green fluorescent in the cytosol and exhibits phototoxicity toward human bladder cancer cells (5637 and T24) under blue-light exposure.

Materials and Methods: Human BC cell lines (5637 and T24) and immortalized uroepithelial cell line (SV-Huc-1) were used in this study. Observation of the fluorescent in MJ-56 treated cells with or without MitoTracker or LysoTracker was conducted by using a fluorescent microscopy with proper filter sets. Cells were treated with indicated concentrations of MJ-56 for 1 hours with or without the blue light irradiation. The cell viability was detected by (1) WST-1 reagent immediately one hour post-treatment, (2) Cytosmart System that records the cell morphology and calculates cell viability automatically for 36 hours, and (3) colony formation assays. The ADT/ATP levels in MJ-56 treated cells were detected using a commercial kit.

Results: We found that green fluorescent was readily detectable in the cytosol of cells treated with 0.125 μM MJ-56 for one hour. Vital staining of mitochondria or lysosomes using MitoTracker or LysoTracker, respectively, demonstrated that the fluorescent caused by MJ-56 was not located in either organelles. Administration of MJ-56 for 24 hours did not cause significant loss of cell viability in both 5637 and T24 cells. However, treatment of 0.125 μM MJ-56 for one hour and exposed to blue light for 15 minutes significantly reduced cell viability in 5637 and T24 cells. To understand the impact of MJ-56 on normal urothelial cells, we performed the same treatment using SV-Huc-1, an immortalized urothelial cell line. The results showed that MJ-56 has small effect on SV-Huc-1 cells compared to BC cells, even with the blue light exposure. The caspase 3/7 activities in cells treated with MJ-56 and exposed to blue light were significantly increased one hour post treatment. However, the DNA fragmentation cannot be detected at 1, 6, or 24 hours-post-treatment due to the significantly loss of viable cells. The increased ADP/ATP ratio in treated cells suggested that MJ-56 may induce necrosis rather than apoptosis in BC cells.

Conclusions: Our results demonstrated that MJ-56 exhibits phototoxicity to bladder cancer cells with minimal impact on urothelial cells, indicating a potential novel therapeutic agent against bladder cancer. However, the mechanism underlying MJ-56 induced cell death as well as the translational studies warrant further investigation.