Victoria Palau

Mechanism of Action of Two Flavone Isomers Targeting Cancer Cells with Varying Cell Differentiation Status.

Apoptosis can be triggered in two different ways, through the intrinsic or the extrinsic pathway. The intrinsic pathway is mediated by the mitochondria via the release of cytochrome C while the extrinsic pathway is prompted by death receptor signals and bypasses the mitochondria. These two pathways are closely related to cell proliferation and survival signaling cascades, which thereby constitute possible targets for cancer therapy. In previous studies we introduced two plant derived isomeric flavonoids, flavone A and flavone B which induce apoptosis in highly tumorigenic cancer cells of the breast, colon, pancreas, and the prostate. Flavone A displayed potent cytotoxic activity against more differentiated carcinomas of the colon (CaCo-2) and the pancreas (Panc28), whereas flavone B cytotoxic action is observed on poorly differentiated carcinomas of the colon (HCT 116) and pancreas (MIA PaCa). Apoptosis is induced by flavone A in better differentiated colon cancer CaCo-2 and pancreatic cancer Panc 28 cells via the intrinsic pathway by the inhibition of the activated forms of extracellular signal-regulated kinase (ERK) and pS6, and subsequent loss of phosphorylation of Bcl-2 associated death promoter (BAD) protein, while apoptosis is triggered by flavone B in poorly differentiated colon cancer HCT 116 and MIA PaCa pancreatic cancer cells through the extrinsic pathway with the concomitant upregulation of the phosphorylated forms of ERK and c-JUN at serine 73. These changes in protein levels ultimately lead to activation of apoptosis, without the involvement of AKT.

Exacerbation of Celecoxib-Induced Renal Injury by Concomitant Administration of Misoprostol in Rats

Nonsteroidal anti-inflammatory drugs (NSAIDs) can produce adverse effects by inhibiting prostaglandin (PG) synthesis. A PGE1 analogue, misoprostol, is often utilized to alleviate NSAID-related gastrointestinal side effects. This study examined the effect of misoprostol on celecoxib renal toxicity. Additionally, the effects of these drugs on cardiovascular parameters were evaluated. Four randomized rat groups were orally gavaged for 9 days, two groups receiving vehicle and two groups receiving misoprostol (100 µg/kg) twice daily. Celecoxib (40 mg/kg) was co-administered once daily to one vehicle and one misoprostol group from days 3 to 9. Urine and blood samples were collected and blood pressure parameters were measured during the study period. Hearts and kidneys were harvested on final day. Day 2 urinary electrolyte samples revealed significant reductions in sodium excretion in misoprostol (0.12±0.05 µmol/min/100 g) and misoprostol+celecoxib groups (0.07±0.02 µmol/min/100 g). At day 3, all treatment groups showed significantly reduced sodium excretion. Potassium excretion diminished significantly in vehicle+celecoxib and misoprostol+celecoxib groups from day 3 onward. Urinary kidney injury molecule-1 levels were significantly increased in vehicle+celecoxib (0.65±0.02 vs. 0.35±0.07 ng/mL, p = 0.0002) and misoprostol+celecoxib (0.61±0.06 vs. 0.37±0.06 ng/mL, p = 0.0015) groups when compared to baseline; while plasma levels of cardiac troponin I increased significantly in vehicle+celecoxib (p = 0.0040) and misoprostol+misoprostol (p = 0.0078) groups when compared to vehicle+vehicle. Blood pressure parameters increased significantly in all misoprostol treated groups. Significant elevation in diastolic (p = 0.0071) and mean blood pressure (p = 0.0153) was noted in misoprostol+celecoxib compared to vehicle+celecoxib. All treatments produced significant tubular dilatation/necrosis compared to control. No significant myocardial changes were noticed; however, three animals presented with pericarditis. Kidney, heart, and plasma celecoxib levels revealed no significant change between vehicle+celecoxib and misoprostol+celecoxib. Concomitant misoprostol administration did not prevent celecoxib renal toxicity, and instead exacerbated renal side effects. Misoprostol did not alter plasma or tissue celecoxib concentrations suggesting no pharmacokinetic interaction between celecoxib and misoprostol.

Synthesis and biological activity of fused tetracyclic Pyrrolo[2,1-c][1,4]benzodiazepines

Cancer remains the second major cause of death in the world. Thus, there is a pressing need to identify potential synthetic route for the development of novel anticancer agents which will serve as lead compounds to effectively combat this life-threatening epidemic. Pyrrolo[2,1-c][1,4]benzodiazepines (PBDs) have sparked a great interest as lead compounds because of their cancerostatic and anti-infective properties. The twisted molecular structure of PBD analogs provides both helical and chiral elements. In an effort to expand novel PBDs that interact with the key exocyclic amino group of the DNA-guanine base, we hypothesized that construction of a fused cyclic active system, would likely serve as an electrophilic site when compared to traditional electrophilic C11-N10 imine group. To examine our theory, we report herein the synthesis and cell viability/cytotoxicity of a series of PBD analogs using NCI-60 cell lines screening. Thus, compounds 1–13 were synthesized and fully characterized. The selected PBDs were found to have marginal inhibition of growth, up to 30%, for certain cell lines.

Development of reversed-phase high performance liquid chromatography methods for quantification of two isomeric flavones and the application of the methods to pharmacokinetic studies in rats

Isomers 5,7-dihydroxy-3,6,8-trimethoxy-2-phenyl-4H-chromen-4-one (5,7-dihydroxy-3,6,8 trimethoxy flavone) (flavone A) and 3,5-dihydroxy-6,7,8-trimethoxy-2-phenyl-4H-chromen-4-one (3,5-dihydroxy-6,7,8-trimethoxy flavone) (flavone B) have recently demonstrated differential antineoplastic activities against pancreatic cancer in vitro. These studies also indicated that these compounds target highly tumorigenic cells while sparing normal cells. The in vivo antitumor activities of these flavones have not been determined, and detection protocols for these compounds are needed to conduct pre-clinical assays following intravenous dosing. Here, we report methods developed using acetonitrile to extract two flavone isomers and corresponding internal standards, celecoxib and diclofenac, from rat plasma. Separation was achieved using a Shimadzu liquid chromatography system with a C18 column and mobile phase acetonitrile/water (60:40 and 70:30 for flavones A and B, respectively) containing 0.2% acetic acid and 0.05% triethylamine at a flow rate of 0.4mL/min and detection at 245nm. Calibration curves ranging from 250 to 2500ng/mL and 2500 to 100,000ng/mL for both flavones were linear (r(2)≥0.99) with the lower limits of quantification being 250ng/mL. Recovery of concentrations 250, 1000, 2500, 5000, and 100,000ng/mL ranged from 87 to 116% and 84 to 103% (n=3) for flavone A and B, respectively. Stability of both flavones after a freezing/thawing cycle yielded a mean peak ratio ≥0.92 when compared to freshly extracted samples. Intravenous administration of a 20mg/kg dose in rats yielded half-lives of 83.68±56.61 and 107.45±53.31min with clearance values of 12.99±13.78 and 80.79±35.06mL/min/kg for flavones A and B, respectively.

γ-Tocotrienol induces apoptosis in pancreatic cancer cells by upregulation of ceramide synthesis and modulation of sphingolipid transport

BackgroundCeramide synthesis and metabolism is a promising target in cancer drug development. γ-tocotrienol (GT3), a member of the vitamin E family, orchestrates multiple effects that ensure the induction of apoptosis in both, wild-type and RAS-mutated pancreatic cancer cells. Here, we investigated whether these effects involve changes in ceramide synthesis and transport.MethodsThe effects of GT3 on the synthesis of ceramide via the de novo pathway, and the hydrolysis of sphingomyelin were analyzed by the expression levels of the enzymes serine palmitoyl transferase, ceramide synthase-6, and dihydroceramide desaturase, and acid sphingomyelinase in wild-type RAS BxPC3, and RAS-mutated MIA PaCa-2 and Panc 1 pancreatic cancer cells. Quantitative changes in ceramides, dihydroceramides, and sphingomyelin at the cell membrane were detected by LCMS. Modulation of ceramide transport by GT3 was studied by immunochemistry of CERT and ARV-1, and the subsequent effects at the cell membrane was analyzed via immunofluorescence of ceramide, caveolin, and DR5.ResultsGT3 favors the upregulation of ceramide by stimulating synthesis at the ER and the plasma membrane. Additionally, the conversion of newly synthesized ceramide to sphingomyelin and glucosylceramide at the Golgi is prevented by the inhibition of CERT. Modulation ARV1 and previously observed inhibition of the HMG-CoA pathway, contribute to changes in membrane structure and signaling functions, allows the clustering of DR5, effectively initiating apoptosis.ConclusionsOur results suggest that GT3 targets ceramide synthesis and transport, and that the upregulation of ceramide and modulation of transporters CERT and ARV1 are important contributors to the apoptotic properties demonstrated by GT3 in pancreatic cancer cells.

A Systems Medicine Approach to the Role of Vitamins in Protecting the Gastrointestinal Tract From Oxidative Stress

This chapter focuses on the role of antioxidant vitamins in protecting the gastrointestinal (GI) tract from oxidative stress. A systems medicine approach is used since it alone is sufficiently comprehensive to capture the broad range of relevant complexities and interrelationships relevant to GI protection. Systems medicine utilizes and integrates the vast amount of information gained from genomics and metagenomics, as well as environmental factors, and applies this information to better patient care. A major goal of system medicine is to develop paradigms to treat or slow the progression of chronic diseases, that is, disease prevention. GI disorders are quintessential examples of chronic inflammation with its attendant oxidative stress. Genomics and metagenomics have provided great insight into the underlying molecular mechanisms for GI disorders and will eventually help define individualized diets to minimize chronic inflammation. While the role of antioxidant nutrients and micronutrients is promising, there is a need for large-scale well-designed clinical trials supported by studies using animal models.

Abstract 3568: Delta-tocotrienol and simvastatin induce cytotoxicity and synergy in BRAF mutant SK-MEL-28 but not in wild type BRAF SK-MEL-2 melanoma cancer cells

Targeting the mutant BRAF protein is an accepted approach to the treatment of metastatic melanoma. Potent and specific BRAF inhibitors like vemurafenib and dabrafenib are superior to chemotherapy in treatment of BRAF mutant melanomas which represent nearly 50% of all melanomas. Previous studies have shown that certain isoforms of vitamin E and statins can have synergistic anti-cancer activity. We determined whether a combination of delta-tocotrienol (DT3), an unsaturated vitamin E isoform, and simvastatin, an HMG-CoA reductase inhibitor, can exert an anti-neoplastic activity on BRAF-mutated SK-MEL-28 and BRAF-wild type SK-MEL-2 melanoma cell lines and whether a differential effect would be evident. MTS assays were used to analyze cytotoxicity. SK-MEL-28 and SK-MEL-2 cells were cultured in MEM media containing 10% serum and plated in 96-well culture plates for 24 hours then treated with DT3 (0-40 μM), simvastatin (0-5 μM), or a combination and dosed again at 48 hours. SK-MEL-28 and SK-MEL-2 cells grown in 60 mm plates and were treated with DT3 at concentrations of 40, 30, 20 μM, simvastatin at a concentrations of 20, 10, 5 μM or dissolution vehicle as a control for 6 h. Protein concentration of cell lysates was measured spectrophotometrically (GLO Max Multi+, Promega), using a BCA protein assay kit. The samples were run in SDS PAGE and blotted onto nitrocellulose membranes. Membranes were incubated with antibodies against Hsp 70 (Enzo Life Sciences, Farmingdale, NY), Hsp 90 (Santa Cruz, Dallas, TX), pS6 and pBAD (Cell Signaling, Danvers, MA). Using MTS assay, we found that DT3 (IC50 38.8 μM) and simvastatin (IC50 22.7μM) have cytotoxic effects on melanoma cell line SK-MEL-28, but on the SK-MEL-2 cells DT3 does not have an effect at the concentrations studied (10-40 μM DT3) yet simvastatin (IC50 16.9 μM) does have cytotoxicity. Further studies determined that combinations of these drugs display a synergistic effect on SK-MEL-28 by inhibition of pS6 and pBAD and subsequent apoptosis. However, these effects are not observed in SK-MEL-2 cells; treated SK-MEL-2 cells show over-expression of Hsp70 and Hsp90 suggestive of a rescue effect leading to lesser cytotoxic activity. The selective cytotoxicity observed in BRAF-mutated cells and not in wild type BRAF melanoma cell lines by both DT3 and simvastatin warrants further research into the potential therapeutic use of these combinations. This observation has added importance in the light of recent findings that show the acquisition of BRAF mutation is an early event in melanogenesis and hence these compounds may have a key role in chemoprevention approaches to melanoma. Citation Format: Kelley Cross, Victoria Palau, Marianne Brannon, Janet Lightner, Megan Dycus, William Stone, Koyamangalath Krishnan. Delta-tocotrienol and simvastatin induce cytotoxicity and synergy in BRAF mutant SK-MEL-28 but not in wild type BRAF SK-MEL-2 melanoma cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3568.