A BODIPY-Naphtholimine-BF2 Dyad for Precision Photodynamic Therapy, Targeting, and Dual Imaging of Endoplasmic Reticulum and Lipid Droplets in Cancer.

Currently, effective therapeutic modalities for pancreatic ductal adenocarcinoma (PDAC) are quite limited, leading to gloomy prognosis and ∼6-months median patient survival. Recent advances showed the promise of photodynamic therapy (PDT) for PDAC patients. Next generation photosensitizers (PS) are based on "organelle-targeted-PDT" and provide new paradigm in the field of precision medicines to address the current challenge for treating PDAC. In this investigation, we have constructed a novel PS, named as N b B, for precise and simultaneous targeting of endoplasmic reticulum (ER) and lipid droplets (LDs) in PDAC, based on the fact that malignant PDAC cells are heavily relying on ER for hormone synthesis. Our live cell imaging and fluorescence recovery after photobleaching (FRAP) experiments revealed that N b B is quickly targeted to ER and subsequently to LDs and shows simultaneous dual fluorescence color due to polar and nonpolar milieu of ER and LDs. Interestingly, the same molecule generates triplet state and singlet oxygen efficiently and causes robust ER stress and cellular lipid peroxidation, leading to apoptosis in two different PDAC cells in the presence of light. Together, we present, for the first time, a potential next generation precision medicine for ER-LD organelle specific imaging and PDT of pancreatic cancer.

Non-enzymatic function of WRN RECQL helicase regulates removal of topoisomerase-I-DNA covalent complexes and triggers NF-κB signaling in cancer.

Mutation in Werner (WRN) RECQL helicase is associated with premature aging syndrome (Werner syndrome, WS) and predisposition to multiple cancers. In patients with solid cancers, deficiency of the WRN RECQL helicase is paradoxically associated with enhanced overall survival in response to treatment with TOP1 inhibitors, which stabilize pathological TOP1-DNA-covalent-complexes (TOP1cc) on the genome. However, the underlying mechanism of WRN in development of chemoresistance to TOP1 inhibitors is not yet explored. Our whole-genome transcriptomic analysis for ~25,000 genes showed robust activation of NF-κB-dependent prosurvival genes in response to TOP1cc. CRISPR-Cas9 knockout, shRNA silencing, and under-expression of WRN confer high-sensitivity of multiple cancers to TOP1 inhibitor. We demonstrated that WRN orchestrates TOP1cc repair through proteasome-dependent and proteasome-independent process, unleashing robust ssDNA generation. This in turn ensues signal transduction for CHK1 mediated NF-κB-activation through IκBα-degradation and nuclear localization of p65 protein. Intriguingly, our site-directed mutagenesis and rescue experiments revealed that neither RECQL-helicase nor DNA-exonuclease enzyme activity of WRN (WRNE84A , WRNK577M , and WRNE84A-K577M ) were required for TOP1cc removal, ssDNA generation and signaling for NF-κB activation. In correlation with patient data and above results, the TOP1 inhibitor-based targeted therapy showed that WRN-deficient melanoma tumors were highly sensitive to TOP1 inhibition in preclinical in vivo mouse model. Collectively, our findings identify hitherto unknown non-enzymatic role of WRN RECQL helicase in pathological mechanisms underlying TOP1cc processing and subsequent NF-κB-activation, offering a potential targeted therapy for WRN-deficient cancer patients.

Membrane damage precedes DNA damage in hydroxychavicol treated E. coli cells and facilitates cooperativity with hydrophobic antibiotics.

Hydroxychavicol (HC), found abundantly in Piper betle leaves is credited with antimicrobial property. Previously we had shown HC induces reactive oxygen species mediated DNA damage in bacterial cells. HC also resulted in membrane compromise revealing its pleiotropic effects on cellular targets. The kinetics and exact sequence of events leading to inhibition of growth and cell death in E. coli after HC treatment remains poorly understood. We show that sub-lethal concentration (125 µg/mL) of HC causes cellular filamentation within 1 h of treatment, while a higher concentration (750 µg/mL) induces cell breakage. HC-treated cells were found to experience oxidative stress as early as 10 min, while evidence of membrane damage was apparent at 30 min. DNA damage repair genes were found to be activated at 60 min. Interestingly, HC-induced cell permeabilization was inhibited and enhanced by external Mg2+ and EDTA, respectively, suggesting that HC damages the outer membrane. Kinetic experiments revealed that HC-treated cells underwent oxidative stress, membrane damage and DNA damage in that order. Because gram negative bacteria such as E. coli are refractory to several antibiotics due to the presence of the outer membrane, we hypothesized that HC pretreatment would sensitize E. coli to hydrophobic antibiotics. Our study reveals for the first time that HC could sensitize bacteria to clinically used antibiotics due to its outer membrane damaging property.

Hydroxychavicol, a key ingredient of Piper betle induces bacterial cell death by DNA damage and inhibition of cell division.

Antibiotic resistance is a global problem and there is an urgent need to augment the arsenal against pathogenic bacteria. The emergence of different drug resistant bacteria is threatening human lives to be pushed towards the pre-antibiotic era. Botanical sources remain a vital source of diverse organic molecules that possess antibacterial property as well as augment existing antibacterial molecules. Piper betle, a climber, is widely used in south and south-east Asia whose leaves and nuts are consumed regularly. Hydroxychavicol (HC) isolated from Piper betle has been reported to possess antibacterial activity. It is currently not clear how the antibacterial activity of HC is manifested. In this investigation we show HC generates superoxide in E. coli cells. Antioxidants protected E. coli against HC induced cell death while gshA mutant was more sensitive to HC than wild type. DNA damage repair deficient mutants are hypersensitive to HC and HC induces the expression of DNA damage repair genes that repair oxidative DNA damage. HC treated E. coli cells are inhibited from growth and undergo DNA condensation. In vitro HC binds to DNA and cleaves it in presence of copper. Our data strongly indicates HC mediates bacterial cell death by ROS generation and DNA damage. Damage to iron sulfur proteins in the cells contribute to amplification of oxidative stress initiated by HC. Further HC is active against a number of Gram negative bacteria isolated from patients with a wide range of clinical symptoms and varied antibiotic resistance profiles.