Purpose: Previous studies have shown that the concentration of cell-free DNA was higher and its strand length longer in body fluids obtained from patients with cancer as compared to patients with benign diseases.We hypothesized that analysis of both DNA copy number and strand length of cell-free DNA from an amplified chromosomal region could improve the diagnosis of malignant diseases in body fluids. Experimental Design: To test this hypothesis, we used ovarian cancer effusion as an example and applied a quantitative real-time PCR to measure the relative copy number and strand length of DNA fragments from one of the most frequently amplified genes, cyclin E, in ovarian serous carcinomas. Results: As compared with nonamplified chromosomal loci, including h-actin, p53, 2p24.1, and 4p15.31, measurement of cyclin E DNA copy number (100 bp) had the best performance in distinguishing malignant (n = 88) from benign (n = 70) effusions after normalization to effusion volume or Line-1 DNA with areas under the receiver operating characteristics curve (AUC) of 0.832 and 0.847, respectively. Different DNA lengths of the cyclin E locus were further analyzed and we found that the AUC was highest by measuring the 400-bp cyclin E locus (AUC = 0.896). The AUC was improved to 0.936 when it was combined with the length integrity index as defined by the relative abundance of 400 bp cyclin E to 100 bp p53 loci. Cyclin E real-time PCR assay had a higher sensitivity (95.6%) than routine cytology examination (73.9%) and was able to diagnose false-negative cytology cases in this study. Conclusions: The above findings indicate that measurement of the DNA copy number and strand length of the cyclin E locus is a useful cancer diagnostic tool.

Introduction: Oxalate in urine can cause tubular cellular damage by the production of free radicals. Then, cell death and cellular debris may promote the retention of calcium oxalate crystals and finally the formation of stones. The two most abundant urinary proteins, Tamm-Horsfall protein (THP) and albumin, were tested for the effects of antioxidants. Materials and Methods: By using xanthine-xanthine oxidase reaction, purified THP and albumin were tested for the inhibitory effect. OD₂₉₅ was used as a spectrophotometric method to measure the production of uric acid during the reaction. Results and Conclusions: Both proteins can inhibit the reaction of xanthine oxidase on xanthine, although the effect was decreased after enzymatic deglycosylation of sialic acid. Albumin has an IC₅₀ of 10.7 nM in native condition and 11.9 nM after deglycosylation, whereas THP has 69.6 nM in native condition and 102.0 nM in deglycosylated condition. The data indicates that THP and albumin have an antioxidant effect. Sialic acid in THP has partly an inhibitory effect and is associated with calcium oxalate formation. Studies have indicated that further investigation of the role of free radicals in the formation of urolithiasis and of sialic acid in protein function is needed.

The Hedgehog (Hh) signaling pathway is a critical regulator of embryonic patterning, and aberrant Hh pathway activation has been implicated in a diverse spectrum of cancers. Therefore, components of the Shh pathway (such as Shh, SMO, and GLI1/2) are viable therapeutic targets for anti-tumor strategy. Smo antagonists such as GDC-0449 and NVP-LDE225 have received FDA approval for treating basal cell carcinoma. However, acquired resistance has emerged as a challenge to targeted therapeutics and may limit their anti-cancer efficacy. Studies have linked the hyperactive phosphatidylinositol-3-kinase (PI3K)/AKT/mTOR signaling pathway in a variety of human cancers and drug resistance condition. The synergistic role of PI3K/AKT/mTOR in Hh signaling in embryonic development and Hh-dependent tumors has been reported. Both the PI3K/Akt/mTOR and Hh pathway also play important role in maintaining the stem cell-like properties of cancer stem cell (CSCs). We report here the development of a potent dual mTOR-PI3K inhibitor designated DCB-HDG2-57 with Hh signaling pathway antagonist activity. In vitro biochemical assays showed that DCB-HDG2-57 inhibited recombinant pI3Kα and mTOR kinase with IC50 of 27.2 and 133 nM, respectively. DCB-HDG2-57 is also highly active in cellular assays, as evidenced by inhibition of phosphorylation of cellular downstream targets of PI3K-mTOR kinases ribosomal protein S6 kinase (S6K1, Ser240/24) in the colon (LS-180) and pancreatic cancer (MiaPaCa2 and PANC1) cell lines. Interestingly, DCB-HDG2-57 demonstrated Hh signaling pathway antagonist activity in a 293 cell-based Gli-luciferase inhibition assay upon agonist treatment, and retains inhibition activity against the Smo wild-type and D473H mutant co-transfection with IC50 of 261.8 and 327.7 nM, respectively. In addition, DCB-HDG2-57 can significantly decrease the cancer stem cell liked side population of PANC1 pancreatic cell, and inhibited the migration of E3LZ10.7 pancreatic cancer cell. DCB-HDG2-57 demonstrated in vivo tumor growth inhibition activity in LS-180 colon cancer xenograft model and reduced the Gli-1 RNA abundance in the tumor. Taken together, our data demonstrate that combined pharmacological blockade of mTOR-PI3K and Hh pathway of DCB-HDG2-57 can provide a therapeutic strategy for targeting ligand-dependent Hh cancer. Citation Format: Ying-Shuan Lee, Mann-Yan Kuo, Chia Wei Liu, Yu-Wen Tseng, Win Yin Wei, Shian-Yi Chiou, Her Sheng Lin, Y.-Y. Lu, Chu-Bin Liao, Shao-Zheng Pen. A dual mTOR-PI3K inhibitor DCB-HDG2-57 with Hedgehog signaling pathway antagonist activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 134. doi:10.1158/1538-7445.AM2017-134