Development and validation of a sensitive LC/MS/MS method for the determination of γ-tocotrienol in rat plasma: application to pharmacokinetic studies.

γ-Tocotrienol has attracted much attention owing to its multiple health benefits. This study developed and validated a simple, specific, sensitive and reliable LC/MS/MS method to analyze γ-tocotrienol in rat plasma. Plasma samples (50 µL) were extracted with internal standard solution (25 ng/mL of itraconazole) in acetonitrile (200 µL) with an average recovery of 44.7% and an average matrix effect of -2.9%. The separation of γ-tocotrienol and internal standard from the plasma components was achieved with a Waters XTerra® MS C(18) column with acetonitrile-water as mobile phase. Analysis was performed under positive ionization electrospray mass spectrometer via the multiple reaction monitoring. The standard curve was linear over a concentration range of 10-1000 ng/mL with correlation coefficient values >0.997. The method was validated with intra- and inter-day accuracy (relative error) ranging from 1.79 to 9.17% and from 2.16 to 9.66%, respectively, and precision (coefficient of variation) ranged from 1.94 to 9.25% and from 2.37 to 10.08%, respectively. Short-term stability, freeze-thaw stability and the processed sample stability tests were performed. This method was further applied to analyze γ-tocotrienol plasma concentrations in rats at various time points after administration of a 2 mg/kg single intravenous dose, and a pharmacokinetic profile was successfully obtained.

Exploiting Arginine Auxotrophy with Pegylated Arginine Deiminase (ADI-PEG20) to Sensitize Pancreatic Cancer to Radiotherapy via Metabolic Dysregulation.

Distinct metabolic vulnerabilities of cancer cells compared with normal cells can potentially be exploited for therapeutic targeting. Deficiency of argininosuccinate synthetase-1 (ASS1) in pancreatic cancers creates auxotrophy for the semiessential amino acid arginine. We explored the therapeutic potential of depleting exogenous arginine via pegylated arginine deiminase (ADI-PEG20) treatment as an adjunct to radiotherapy. We evaluated the efficacy of treatment of human pancreatic cancer cell lines and xenografts with ADI-PEG20 and radiation via clonogenic assays and tumor growth delay experiments. We also investigated potential mechanisms of action using reverse-phase protein array, Western blotting, and IHC and immunofluorescence staining. ADI-PEG20 potently radiosensitized ASS1-deficient pancreatic cancer cells (MiaPaCa-2, Panc-1, AsPc-1, HPAC, and CaPan-1), but not ASS1-expressing cell lines (Bxpc3, L3.6pl, and SW1990). Reverse phase protein array studies confirmed increased expression of proteins related to endoplasmic reticulum (ER) stress and apoptosis, which were confirmed by Western blot analysis. Inhibition of ER stress signaling with 4-phenylbutyrate abrogated the expression of ER stress proteins and reversed radiosensitization by ADI-PEG20. Independent in vivo studies in two xenograft models confirmed significant tumor growth delays, which were associated with enhanced expression of ER stress proteins and apoptosis markers and reduced expression of proliferation and angiogenesis markers. ADI-PEG20 augmented the effects of radiation by triggering the ER stress pathway, leading to apoptosis in pancreatic tumor cells.

A novel N-alkylated prodigiosin analogue induced death in tumour cell through apoptosis or necrosis depending upon the cell type.

PURPOSE: To investigate the mechanism of cell death induced by the N-alkylated prodigiosin analogue, 2,2'-[3-methoxy-1'amyl-5'-methyl-4-(1''-pyrryl)] dipyrryl-methene (MAMPDM) in S-180 and EL-4 tumour cell lines. METHODS: Effect of MAMPDM on cell viability was assessed by MTT dye conversion. Induction of apoptosis was assessed by monitoring caspase 3 activity using a fluorogenic substrate, fragmentation of DNA by gel electrophoresis and sub-diploid DNA containing cells by flowcytometry. Necrosis was estimated by flowcytometric analysis of the uptake of propidium iodide. RESULTS: MAMPDM inhibited the proliferation of murine fibrosarcoma, S-180 cells and induced cell death. Investigations into the mechanism of cell death by MAMPDM in S-180 cells showed absence of hallmarks of apoptotic cell death such as activation of caspase 3, DNA fragmentation and presence of cells with sub-diploid DNA content. However, there was a rapid loss of membrane integrity as assessed by uptake of propidium iodide, which is characteristic of necrosis. In contrast to induction of necrosis in S-180 cells, MAMPDM induced apoptotic cell death in EL-4 cells as evident by activation of caspase 3, fragmentation of DNA and sub-diploid DNA containing cells. CONCLUSIONS: MAMPDM could induce cell death by either apoptosis or necrosis depending upon the cell type. This would be of advantage in elimination of tumor cells defective in apoptotic pathway and therefore, refractory to the conventional therapies.

Hypoxia-targeted gold nanorods for cancer photothermal therapy.

Tumor hypoxia is a well-recognized driver of resistance to traditional cancer therapies such as chemotherapy and radiation therapy. We describe development of a new nanoconstruct composed of gold nanorods (GNRs) conjugated to carbonic anhydrase IX (CAIX) antibody that specifically binds to CAIX, a biomarker of hypoxia, to facilitate targeting tumor hypoxic areas for focused photothermal ablation. Physicochemical characterization studies confirmed the size, shape, monodispersity, surface charge, and serum stability of the GNRs. Enzyme-linked immunosorbent assays and cellular binding and uptake studies confirmed successful conjugation of antibody to the GNRs and specificity for CAIX. Near-infrared irradiation of CAIX-overexpressing cells treated with GNR/anti-CAIX resulted in significantly higher cell death than cells treated with control GNRs. In vivo biodistribution studies using hyperspectral imaging and inductively coupled plasma mass spectrometry confirmed intravenous administration results not only in greater accumulation of GNR/anti-CAIX in tumors than control GNRs but also greater penetration into hypoxic areas of tumors. Near-infrared ablation of these tumors showed no tumor regression in the sham-treated group, regression but recurrence in the non-targeted-GNR group, and complete tumor regression in the targeted-GNR group. GNR/anti-CAIX nanoconstructs show promise as hypoxia targeting and photothermal ablation agents for cancer treatment.

Identification of a red-pigmented bacterium producing a potent anti-tumor N-alkylated prodigiosin as Serratia marcescens.

A bacterial strain producing a novel prodigiosin analogue 2,2'-[3-methoxy-1'amyl-5'-methyl-4-(1''-pyrryl)] dipyrrylmethene (MAMPDM) possessing potent cytotoxic activity towards cancer cells was isolated and identified. The bacterial cells were spherical and occurred singly, and some of the biochemical tests matched with Micrococcus. Therefore, the isolate was earlier tentatively reported to be Micrococcus sp. In the present studies, analytical profile index (API) suggested this organism to be Klebsiella. However, Klebsiella is not known to produce the red pigment prodigiosin, which is produced by Serratia species and some other bacteria. Based on other biochemical characteristics, particularly DNase, gelatinase, lipase, ornithine decarboxylase, presence of a cell-associated N-alkylated prodigiosin (MAMPDM) and organic solvent tolerance, the strain has now been identified as a variant of Serratia marcescens. 16S rRNA gene analysis conclusively established this organism as S. marcescens ost3. The red pigment (MAMPDM) of this organism showed selective cytotoxic activity in cancer cell lines of different origin (LS-A and U937) and reduced toxicity to non-malignant cells. The LC50 of MAMPDM was 1.59 microM and 0.176 microM for U937 and LS-A cells, respectively, while there was no effect on the viability of L929, a non-malignant cell line, at these concentrations. Thus, S. marcescens ost3 may serve as a source of a new anti-cancer compound.

Integrin αvß3-targeted gold nanoshells augment tumor vasculature-specific imaging and therapy.

PURPOSE: Gold nanoshells (NSs) have already shown great promise as photothermal actuators for cancer therapy. Integrin αvß3 is a marker that is specifically and preferentially overexpressed on multiple tumor types and on angiogenic tumor neovasculature. Active targeting of NSs to integrin αvß3 offers the potential to increase accumulation preferentially in tumors and thereby enhance therapy efficacy. METHODS: Enzyme-linked immunosorbent assay (ELISA) and cell binding assay were used to study the in vitro binding affinities of the targeted nanoconjugate NS-RGDfK. In vivo biodistribution and tumor specificity were analyzed using 64Cu-radiolabeled untargeted and targeted NSs in live nude rats bearing head and neck squamous cell carcinoma (HNSCC) xenografts. The potential thermal therapy applications of NS-RGDfK were evaluated by subablative thermal therapy of tumor xenografts using untargeted and targeted NSs. RESULTS: ELISA and cell binding assay confirmed the binding affinity of NS-RGDfK to integrin αvß3. Positron emission tomography/computed tomography imaging suggested that tumor targeting is improved by conjugation of NSs to cyclo(RGDfK) and peaks at ~20 hours postinjection. In the subablative thermal therapy study, greater biological effectiveness of targeted NSs was implied by the greater degree of tumor necrosis. CONCLUSION: The results presented in this paper set the stage for the advancement of integrin αvß3-targeted NSs as therapeutic nanoconstructs for effective cancer therapy.