Reserpine Induces Apoptosis and Cell Cycle Arrest in Hormone Independent Prostate Cancer Cells through Mitochondrial Membrane Potential Failure.

BACKGROUND: Reserpine, an indole alkaloid commonly used for hypertension, is found in the roots of Rauwolfia serpentina. Although the root extract has been used for the treatment of cancer, the molecular mechanism of its anti-cancer activity on hormonal independent prostate cancer remains elusive. METHODS: we evaluated the cytotoxicity of reserpine and other indole alkaloids, yohimbine and ajmaline on Prostate Cancer cells (PC3) using MTT assay. We investigated the mechanism of apoptosis using a combination of techniques including acridine orange/ethidium bromide staining, high content imaging of Annexin V-FITC staining, flow cytometric quantification of the mitochondrial membrane potential and Reactive Oxygen Species (ROS) and cell cycle analysis. RESULTS: Our results indicate that reserpine inhibits DNA synthesis by arresting the cells at the G2 phase and showed all standard sequential features of apoptosis including, destabilization of mitochondrial membrane potential, reduced production of reactive oxygen species and DNA ladder formation. Our in silico analysis further confirmed that indeed reserpine docks to the catalytic cleft of anti-apoptotic proteins substantiating our results. CONCLUSION: Collectively, our findings suggest that reserpine can be a novel therapeutic agent for the treatment of androgen-independent prostate cancer.

Synthesis and characterization of cystamine conjugated chitosan-SS-mPEG based 5-Fluorouracil loaded polymeric nanoparticles for redox responsive drug release.

The principle objective of this study was to develop and characterize redox responsive polymeric nanoparticles (PNPs) as a stimuli responsive drug delivery system. The chitosan-cystamine-methoxy poly(ethylene glycol) (CH-SS-mPEG) copolymer was synthesized by conjugation of cystamine appended chitosan with carboxylic acid-terminated mPEG and characterized by FTIR, 1H NMR, XRD analysis and colorimetric assay. This copolymer could be formulated as 5-Fluorouracil (5-FU) loaded PNPs and the characteristics of PNPs were evaluated. Moreover, folic acid functionalized PNPs were prepared for folate receptor targeted drug delivery. Drug release studies indicated that the redox sensitive PNPs were stable in physiological condition while quickly releasing 5-FU in the trigger of redox potential due to the cleavage of the disulfide linkages. In contrast, less quantity of drug was released from the reduction insensitive chitosan-g-methoxy poly(ethylene glycol) (CH-g-mPEG) based PNPs under both reduction sensitive and non-reductive conditions. From the cytotoxicity studies, it was evident that 5-FU loaded PNPs had higher toxicity against MCF7 cells when compared to 5-FU free PNPs. Subsequently, cellular uptake studies showed significantly increased internalization of folic acid attached PNPs. In conclusion, the developed PNPs appeared to be of great promise in redox responsive drug release for targeted drug delivery.

Cytocompatible chitosan-graft-mPEG-based 5-fluorouracil-loaded polymeric nanoparticles for tumor-targeted drug delivery.

Biodegradable materials like chitosan (CH) and methoxy polyethylene glycol (mPEG) are widely being used as drug delivery carriers for various therapeutic applications. In this study, copolymer (CH-g-mPEG) of CH and carboxylic acid terminated mPEG was synthesized by carbodiimide-mediated acid amine reaction. The resultant hydrophilic copolymer was characterized by Fourier transform infrared spectroscopy and 1H NMR studies, revealing its relevant functional bands and proton peaks, respectively. Blank polymeric nanoparticles (B-PNPs) and 5-fluorouracil loaded polymeric nanoparticles (5-FU-PNPs) were formulated by ionic gelation method. Furthermore, folic acid functionalized FA-PNPs and FA-5-FU-PNPs were prepared for folate receptor-targeted drug delivery. FA-5-FU-PNPs were characterized by particle size, zeta potential, and in vitro drug release studies, resulting in 197.7 nm, +29.9 mv, and sustained drug release of 88% in 24 h, respectively. Cytotoxicity studies were performed for FA-PNPs and FA-5-FU-PNPs in MCF-7 cell line, which exhibited a cell viability of 80 and 41%, respectively. In vitro internalization studies were carried out for 5-FU-PNPs and FA-5-FU-PNPs which demonstrated increased cellular uptake of FA-5-FU-PNPs by receptor-mediated transport. Significant (p < .01) reduction (1.5-fold) of reactive oxygen species (ROS) accumulation was observed in lipopolysaccharides-stimulated RAW264.7 macrophages, revealing its potent antioxidant property. From the obtained results, it is concluded that folic acid functionalization of 5-FU-PNPs is an ideal approach for sustained and targeted drug delivery, thereby influencing better therapeutic effect.

Expression of heterologous oxalate decarboxylase in HEK293 cells confers protection against oxalate induced oxidative stress as a therapeutic approach for calcium oxalate stone disease.

Oxalates stimulate alterations in renal epithelial cells and thereby induce calcium oxalate (CaOx) stone formation. Bacillus subtilis YvrK gene encodes for oxalate decarboxylase (OxdC) which degrades oxalate to formate and CO2. The present work is aimed to clone the oxdC gene in a mammalian expression vector pcDNA and transfect into Human Embryonic Kidney 293 (HEK293) cells and evaluate the oxdC expression, cell survival rate and oxalate degrading efficiency. The results indicate cell survival rate of HEK293/pcDNAOXDC cells pre-incubated with oxalate was enhanced by 28%. HEK293/pcDNAOXDC cells expressing OxdC treated with oxalate, significantly restored antioxidant activity, mitochondrial membrane potential and intracellular reactive oxygen species (ROS) generation compared with HEK293/pcDNA. Apoptotic marker caspase 3 downregulation illustrates HEK293/pcDNAOXDC cells were able to survive under oxalate-mediated oxidative stress. The findings suggest HEK293 cells expressing oxdC capable of degrading oxalate protect cells from oxidative damage and thus serve as a therapeutic option for prevention of CaOx stone disease. [Formula: see text].