Growth inhibitory and anti-metastatic activity of epithelial cell adhesion molecule targeted three-way junctional delta-5-desaturase siRNA nanoparticle for breast cancer therapy.

8-Hydroxyoctanoic acid (8-HOA) produced through cyclooxygenase-2 (COX-2) catalyzed dihomo-γ-linolenic acid (DGLA) peroxidation in delta-5-desaturase inhibitory (D5D siRNA) condition showed an inhibitory effect on breast cancer cell proliferation and migration. However, in vivo use of naked D5D siRNA was limited by off-target silencing and degradation by endonucleases. To overcome the limitation and deliver the D5D siRNA in vivo, we designed an epithelia cell adhesion molecule targeted three-way junctional nanoparticle having D5D siRNA. In this study, we have hypothesized that 3WJ-EpCAM-D5D siRNA will target and inhibit the D5D enzyme in cancer cells leading to peroxidation of supplemented DGLA to 8-HOA resulting in growth inhibitory effect in the orthotopic breast cancer model developed by injecting 4T1 cells. On analysis, we observed a significant reduction in tumor size and metastatic lung nodules in animals treated with a combination of 3WJ-EpCAM-D5D siRNA and DGLA through activating intrinsic apoptotic signaling pathway and by reducing endothelial-mesenchymal damage.

Nitric oxide synthase inhibitors 1400W and L-NIO inhibit angiogenesis pathway of colorectal cancer.

BACKGROUND: It has been widely accepted that angiogenesis plays fundamental roles in colorectal cancer development, and therapeutic targeting of this pathway has achieved promising outcome. Recent reports have highlighted the involvement of nitric oxide synthases (NOS) in the development of angiogenesis in cancer; however, the mechanism and therapeutic value of NOS inhibitors in colon cancer are largely unknown. OBJECTIVE: In this study, we investigated the effects and mechanism of the NOS inhibitors 1400W and L-NIO on the angiogenesis pathway in colorectal cancer cells. METHODS: Two colorectal cancer cell lines, HT 29 and HCT 116, were used for in vitro study. The expression of iNOS and eNOS in cells was knocked down via shRNA transfection. MTS assays and wound healing assays were performed to assess cell proliferation and migration after shRNA transfection or treatment with 1400W, L-NIO, and 5-fluorouracil. Human angiogenesis PCR arrays and proteome profiler human angiogenesis arrays were used to detect changes in key genes/proteins involved in modulating angiogenesis after 1400W and L-NIO treatment. RESULTS: Knockdown of iNOS and eNOS significantly inhibited colorectal cancer cell growth. Treatment with NOS inhibitors inhibited colorectal cancer cell growth and migration, and was associated with suppression of the expression of key genes/proteins involved in the angiogenesis pathway. In addition, the combined use of NOS inhibitors with 5-fluorouracil showed enhanced inhibition of cell proliferation and migration. CONCLUSION: NOS inhibitors could suppress colorectal cancer cell growth and migration, likely via suppressing the angiogenesis pathway.

Dihomo-γ-linolenic acid inhibits xenograft tumor growth in mice bearing shRNA-transfected HCA-7 cells targeting delta-5-desaturase.

BACKGROUND: We previously demonstrated that knockdown of delta-5-desaturase via siRNA transfection together with dihomo-γ-linolenic acid supplementation inhibited colon cancer cell growth and migration, by promoting the production of the anti-cancer byproduct 8-hydroxyoctanoic acid from Cyclooxygenase-2-catalyzed dihomo-γ-linolenic acid peroxidation. Here, we extend our study to investigate the effects of delta-5-desaturase-knockdown and the resulting intensified dihomo-γ-linolenic acid peroxidation in xenograft tumor mice model. METHODS: Four-week old nude mice bearing the human colon cancer cell HCA-7/C29 vs. its delta-5-desaturase knockdown analog (via shRNA transfection) were subject to 4-week treatments of: vehicle control, dihomo-γ-linolenic acid supplementation, 5-Fluorouracil, and combination of dihomo-γ-linolenic acid and 5-Fluorouracil. Tumor growth was monitored during the treatment. At the endpoint, the mice were euthanized and the tumor tissues were collected for further mechanism analysis. RESULTS: Delta-5-desaturase knockdown (shRNA) together with dihomo-γ-linolenic acid supplementation increased 8-hydroxyoctanoic acid production to a threshold level in xenograft tumors, which consequently induced p53-dependent apoptosis and reduced tumors significantly. The promoted 8-hydroxyoctanoic acid formation was also found to suppress the tumors' metastatic potential via regulating MMP-2 and E-cadherin expressions. In addition, our in vivo data showed that delta-5-desaturase knockdown along with dihomo-γ-linolenic acid supplementation resulted in anti-tumor effects comparable to those of 5-Fluorouracil. CONCLUSIONS: We have demonstrated that our paradigm-shifting strategy of knocking down delta-5-desaturase and taking advantage of overexpressed Cyclooxygenase-2 in tumor cells can be used for colon cancer suppression. Our research outcome will lead us to develop a better and safer anti-cancer therapy for patients.

Knockdown delta-5-desaturase in breast cancer cells that overexpress COX-2 results in inhibition of growth, migration and invasion via a dihomo-γ-linolenic acid peroxidation dependent mechanism.

BACKGROUND: Cyclooxygenase-2 (COX-2), the inducible COX form, is a bi-functional membrane-bound enzyme that typically metabolizes arachidonic acid (downstream ω-6 fatty acid) to form 2-series of prostaglandins known to be involved in cancer development. Overexpression of COX-2 has been found in a majority of breast carcinomas, and has also been associated with increased severity and the development of the metastasis. Our lab recently demonstrated that COX-2 can also metabolize dihomo-γ-linolenic acid (DGLA, a precursor of ω-6 arachidonic acid) to produce an anti-cancer byproduct, 8-hydroxyoctanoic acid (8-HOA) that can inhibit growth and migration of colon and pancreatic cancer cells. We thus tested whether our strategy of knocking down delta-5-desaturase (D5D, the key enzyme that converts DGLA to arachidonic acid) in breast cancer cells overexpressing COX-2 can also be used to promote 8-HOA formation, thereby suppressing cancer growth, migration, and invasion. METHODS: SiRNA and shRNA transfection were used to knock down D5D expression in MDA-MB 231 and 4 T1 cells (human and mouse breast cancer cell lines expressing high COX-2, respectively). Colony formation assay, FITC Annexin V/PI double staining, wound healing and transwell assay were used to assess the effect of our strategy on inhibition of cancer growth, migration, and invasion. GC/MS was used to measure endogenous 8-HOA, and western blotting was performed to evaluate the altered key protein expressions upon the treatments. RESULTS: We demonstrated that D5D knockdown licenses DGLA to inhibit growth of breast cancer cells via promoting formation of 8-HOA that can inhibit histone deacetylase and activate cell apoptotic proteins, such as procaspase 9 and PARP. Our strategy can also significantly inhibit cancer migration and invasion, associated with altered expression of MMP-2/- 9, E-cadherin, vimentin and snail. In addition, D5D knockdown and DGLA supplementation greatly enhanced the efficacy of 5-fluorouracil on breast cancer growth and migration. CONCLUSIONS: Consistent to our previous studies on colon and pancreatic cancer, here we demonstrate again that the high level of COX-2 in breast cancer cells can be capitalized on inhibiting cancer growth and migration. The outcome of this translational research could guide us to develop new anti-cancer strategy and/or to improve current chemotherapy for breast cancer treatment.

Acridine Orange Conjugated Polymersomes for Simultaneous Nuclear Delivery of Gemcitabine and Doxorubicin to Pancreatic Cancer Cells.

Considering the systemic toxicity of chemotherapeutic agents, there is an urgent need to develop new targeted drug delivery systems. Herein, we have developed a new nuclear targeted, redox sensitive, drug delivery vehicle to simultaneously deliver the anticancer drugs gemcitabine and doxorubicin to the nuclei of pancreatic cancer cells. We prepared polymeric bilayer vesicles (polymersomes), and actively encapsulated the drug combination by the pH gradient method. A redox-sensitive polymer (PEG-S-S-PLA) was incorporated to sensitize the formulation to reducing agent concentration. Acridine orange (AO) was conjugated to the surface of the polymersomes imparting nuclear localizing property. The polymersomes' toxicity and efficacy were compared with those of a free drug combination using monolayer and three-dimensional spheroid cultures of pancreatic cancer cells. We observed that the redox sensitive, nuclear-targeted polymersomes released more than 60% of their encapsulated contents in response to 50 mM glutathione. The nanoparticles are nontoxic; however, the drug encapsulated vesicles have significant toxicity. The prepared formulation can increase the drug's therapeutic index by delivering the drugs directly to the cells' nuclei, one of the key organelles in the cells. This study is likely to initiate research in targeted nuclear delivery using other drug formulations in other types of cancers.

Radical [1,3] Rearrangements of Breslow Intermediates.

Breslow intermediates that bear radical-stabilizing N substituents, such as benzyl, cinnamyl, and diarylmethyl, undergo facile homolytic C-N bond scission under mild conditions to give products of formal [1,3] rearrangement rather than benzoin condensation. EPR experiments and computational analysis support a radical-based mechanism. Implications for thiamine-based enzymes are discussed.

20-Hydroxyeicosatetraenoic Acid Is a Key Mediator of Angiotensin II-induced Apoptosis in Cardiac Myocytes.

Cardiomyocyte apoptosis is involved in a variety of cardiac stresses, including ischemia-reperfusion injury, heart failure, and cardiomyopathy. Both Angiotensin II (Ang II) and 20-hydroxyeicosatetraenoic acid (20-HETE) induce apoptosis in cardiomyocytes. Here, we examined the relationship between 20-HETE and Ang II in cardiomyocyte apoptosis. Apoptosis was examined using flow cytometry in primary cultured rat cardiomyocytes treated with control, Ang II, and Ang II plus HET0016 (a 20-HETE formation inhibitor). The results demonstrated that the treatment of cardiomyocytes with Ang II or 20-HETE significantly increased the percentage of apoptotic cells and that Ang II-induced apoptosis was markedly attenuated by HET0016 or losartan (an AT1 receptor antagonist). In apoptotic mechanism experiments, Ang II or 20-HETE treatment significantly reduced mitochondrial membrane potential, indicating that a mitochondria-dependent mechanism is involved. Ang II-induced alteration in mitochondrial membrane potential was significantly attenuated by HET0016. Treatment of cardiomyocytes with Ang II also increased superoxide production, and this effect of Ang II was attenuated by HET0016. Treatment of cardiomyocytes with Ang II significantly increased CYP4A1 expression and 20-HETE production, as measured by Western blot, real-time RT-PCR, and mass spectrometric analysis. All results suggest that 20-HETE may play a key role in Ang II-induced apoptosis in cardiomyocytes by a mitochondrial superoxide-dependent pathway.

Autistic children exhibit decreased levels of essential Fatty acids in red blood cells.

Omega-6 (n-6) and omega-3 (n-3) polyunsaturated fatty acids (PUFA) are essential nutrients for brain development and function. However, whether or not the levels of these fatty acids are altered in individuals with autism remains debatable. In this study, we compared the fatty acid contents between 121 autistic patients and 110 non-autistic, non-developmentally delayed controls, aged 3-17. Analysis of the fatty acid composition of red blood cell (RBC) membrane phospholipids showed that the percentage of total PUFA was lower in autistic patients than in controls; levels of n-6 arachidonic acid (AA) and n-3 docosahexaenoic acid (DHA) were particularly decreased (p<0.001). In addition, plasma levels of the pro-inflammatory AA metabolite prostaglandin E2 (PGE2) were higher in a subset of the autistic participants (n=20) compared to controls. Our study demonstrates an alteration in the PUFA profile and increased production of a PUFA-derived metabolite in autistic patients, supporting the hypothesis that abnormal lipid metabolism is implicated in autism.

Delta-6-desaturase activity and arachidonic acid synthesis are increased in human breast cancer tissue.

Omega-6 (n-6) arachidonic acid (AA) and its pro-inflammatory metabolites, including prostaglandin E2 (PGE(2)), are known to promote tumorigenesis. Delta-6 desaturase (D6D) is the rate-limiting enzyme for converting n-6 linoleic acid (LA) to AA. Our objective was to determine if AA synthesis, specifically D6D activity, and PGE(2) levels are increased in cancerous breast tissue, and whether these variables differ between estrogen receptor positive (ER+) and negative (ER-) breast cancers. Gas chromatography was performed on surgical breast tissue samples collected from 69 women with breast cancer. Fifty-four had ER+ breast cancer, and 15 had ER- breast cancer. Liquid chromatography-mass spectrometry was used to determine PGE(2) levels. Lipid analysis revealed higher levels of LA metabolites (C18:3 n-6, C20:3 n-6, and AA) in cancerous tissue than in adjacent noncancerous tissue (P < 0.01). The ratio of LA metabolites to LA, a measure of D6D activity, was increased in cancerous tissue, suggesting greater conversion of LA to AA (P < 0.001), and was higher in ER- than in ER+ patients, indicating genotype-related trends. Similarly, PGE(2) levels were increased in cancerous tissue, particularly in ER- patients. The results showed that the endogenous AA synthetic pathway, D6D activity, and PGE(2) levels are increased in breast tumors, particularly those of the ER- genotype. These findings suggest that the AA synthetic pathway and the D6D enzyme in particular may be involved in the pathogenesis of breast cancer. The development of drugs and nutritional interventions to alter this pathway may provide new strategies for breast cancer prevention and treatment.

Development of biocompatible polymeric nanoparticles for in vivo NIR and FRET imaging.

The majority of near-infrared (NIR) fluorophores are organic molecules that show significant overlap between the excitation and emission spectra and therefore exhibit high fluorescence backgrounds during in vivo imaging. Recently, cyanine dyes with a large Stokes shift have shown great promise for NIR imaging but often undergo rapid photodegradation and nonspecific protein adsorption. Alternatively, fluorescence resonance energy transfer (FRET) is a promising technique to generate a larger gap between the excitation and emission maxima and thus can reduce the background signal. Here, we report the rational design of FRET-based polymeric nanoparticles for NIR and FRET imaging. The particles were assembled from diblock copolymers of poly(d,l-lactic-co-glycolic acid) and maleimide-activated poly(ethylene glycol), which were also encapsulated with both the donor (1,1'-dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine) and acceptor (1,1'-dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanine) fluorophores. Because of their extreme hydrophobicity, thousands of fluorophores could be encapsulated inside a single particle without causing leakage. FRET resulted in a large Stokes shift (>100 nm) of the emission maxima, and the transfer efficiency could be fine-tuned by further adjusting the doping ratio of the donor and acceptor fluorophores. The optimized formulation was less than 100 nm in size, brighter than quantum dots, stable in biological media, and demonstrated similar biodistribution to most nanomaterials. Additional animal phantom studies demonstrated that the FRET imaging platform developed could have far-reaching applications in optical imaging.

An advanced Electron Spin Resonance (ESR) spin-trapping and LC/(ESR)/MS technique for the study of lipid peroxidation.

There are two types of nutritionally important polyunsaturated fatty acids (PUFAs), namely ω-6s and ω-3s. PUFAs and their metabolites generated from lipid peroxidation via cyclooxygenase (COX) and lipoxygenase (LOX) are believed to be involved in a variety of physiological and pathological processes in the human body. Both COX- and LOX-catalyzed PUFA peroxidation are complex events that generate a series of radicals, which may then bind proteins, target DNA/RNA, and lead to a number of biological changes. However, due to the lack of an appropriate method, it was not possible until recently to identify the short-lived PUFA-derived radicals in COX-/LOX-catalyzed peroxidation. Failure to characterize free radicals during peroxidation has greatly restricted our knowledge about COX/LOX biology in human health. Here we review the development and refinement of combined ESR spin trapping and LC/ESR/MS to characterize PUFA-derived radicals formed from in vitro (cell-free) peroxidation. We also present the most recent approach for studying peroxidation in cells which allows us to directly assess the potential bioactivity of PUFA-derived free radicals. This advanced technique has resulted in a major breakthrough in radical structural characterization, as well as assessment of free radical-associated cell growth response, thereby greatly improving our knowledge of PUFAs, COX-/LOX-catalyzed lipid peroxidation, and their related biological consequences.

Solvent-assisted slow conversion of a dithiazole derivative produces a competitive inhibitor of peptide deformylase.

Due to its potential as an antibiotic target, E. coli peptide deformylase (PDF(Ec)) serves as a model enzyme system for inhibitor design. While investigating the structural-functional and inhibitory features of this enzyme, we unexpectedly discovered that 2-amino-5-mercapto-1,3,4-thiadiazole (AMT) served as a slow-binding inhibitor of PDF(Ec) when the above compound was dissolved only in dimethylformamide (DMF), but not in any other solvent, and allowed to age. The time dependent inhibitory potency of the DMF-dissolved AMT was correlated with the broadening of the inhibitor's 295 nm spectral band toward the visible region, concomitant with the increase in the mass of the parent compound by about 2-fold. These data led to the suggestion that DMF facilitated the slow dimerization of AMT (via the formation of a disulfide bond), and that the dimeric form of AMT served as an inhibitor for PDF(Ec). The latter is not caused by the simple oxidation of sulfhydryl groups by oxidizing agents such as H(2)O(2). Newly synthesized dimeric/dithiolated form of AMT ("bis-AMT") exhibited similar spectral and inhibitory features as given by the parent compound when incubated with DMF. The computer graphic modeling data revealed that bis-AMT could be reliably accommodated within the active site pocket of PDF(Ec), and the above enzyme-ligand interaction involves coordination with the enzyme resident Ni(2+) cofactor. The mechanism of the DMF-assisted activation of AMT (generating bis-AMT), the overall microscopic pathway for the slow-binding inhibition of PDF(Ec) by bis-AMT, and the potential of bis-AMT to serve as a new class of antibiotic agent are presented.

Fluorescent liposomes for differential interactions with glycosaminoglycans.

We have successfully synthesized a lipid containing the pyranine dye as the hydrophilic headgroup. This lipid was incorporated into liposomes with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine as the major component. The resultant liposomes displayed differential modulations in fluorescence emission intensity in the presence of nanomolar concentrations of different glycosaminoglycans. Linear discriminant analysis of the fluorescence response data demonstrate that the liposomes are able to distinguish between different GAGs. In addition, we also demonstrate that the liposomes incorporating the pyranine lipid are able to distinguish between dilute serum from healthy individuals and serum containing elevated chondroitin sulfate (simulated serum from an Alzheimer's disease patient).

EpCAM-Targeted 3WJ RNA Nanoparticle Harboring Delta-5-Desaturase siRNA Inhibited Lung Tumor Formation via DGLA Peroxidation.

Knocking down delta-5-desaturase (D5D) expression by D5D small interfering RNA (siRNA) has been reported that could redirect the cyclooxygenase-2 (COX-2)-catalyzed dihomo-γ-linolenic acid (DGLA) peroxidation from producing prostaglandin E2 to 8-hydroxyoctanoic acid (8-HOA), resulting in the inhibition of colon and pancreatic cancers. However, the effect of D5D siRNA on lung cancer is still unknown. In this study, by incorporating epithelial cell adhesion molecule (EpCAM) aptamer and validated D5D siRNA into the innovative three-way junction (3WJ) RNA nanoparticle, target-specific accumulation and D5D knockdown were achieved in the lung cancer cell and mouse models. By promoting the 8-HOA formation from the COX-2-catalyzed DGLA peroxidation, the 3WJ-EpCAM-D5D siRNA nanoparticle inhibited lung cancer growth in vivo and in vitro. As a potential histone deacetylases inhibitor, 8-HOA subsequently inhibited cancer proliferation and induced apoptosis via suppressing YAP1/TAZ nuclear translocation and expression. Therefore, this 3WJ-RNA nanoparticle could improve the targeting and effectiveness of D5D siRNA in lung cancer therapy.

In Vitro Photodynamic Therapy of Mononuclear and Dinuclear Iridium(III) Bis(terpyridine) Complexes.

Three mononuclear or dinuclear bis(terpyridine) (tpy) iridium(III) complexes bearing pyren-1-yl (pyr) group(s) were synthesized. Their photophysical properties in water and in vitro photodynamic therapy (PDT) effects toward the human lung epithelial cancer cell line A549 and the human epidermal skin cancer cell line A431 were investigated to evaluate the effects of dinuclear versus mononuclear complexes and the impact of the oligoether substituent at the ligand. All complexes possessed pyr-tpy ligand-associated charge transfer (1CT)/1π,π* absorption bands at 350-550 nm, with the dinuclear complex Ir3 showing the much enhanced absorptivity of this band. These complexes exhibited dual emission upon excitation at >430 nm in most cases, with the emitting states being ascribed to 1ILCT (intraligand charge transfer) and 3π,π*/3CT states, respectively. All complexes exhibited relatively weak to moderate cytotoxicity in the dark but high photocytotoxicity upon broadband visible light irradiation. Among them, the dinuclear complex Ir3 showed the highest intracellular reactive oxygen species (ROS) generation and PDT efficiency compared to its mononuclear counterpart Ir1. Introducing an oligoether substituent on one of the tpy ligands in Ir2 also improved its intracellular ROS generation and PDT efficacy compared to those induced by Ir1. Ir3 induced both mitochondrial dysfunction and lysosomal damage upon light activation toward both cell lines, whereas Ir1 and Ir2 caused both mitochondrial dysfunction and lysosomal damage in A431 cells but only lysosomal damage in A549 cells. The dominant cell death pathway induced by Ir1-Ir3 PDT is apoptosis.

Angiotensin II enhances GABA(B) receptor-mediated responses and expression in nucleus tractus solitarii of rats.

Angiotensin II (ANG II) increases GABA(B) receptor expression in neuronal cultures from the nucleus tractus solitarii (NTS). In the present study, the chronic effects of ANG II on GABA(B) receptor expression and activity were examined in the NTS of Sprague-Dawley rats. Intracerebroventricular infusion of ANG II caused a significant elevation in blood pressure (BP) and an increase in GABA(B) receptor expression in the NTS. Conversely, chronic N(G)-nitro-l-arginine methyl ester (l-NAME) treatment also increased BP, but had no effect on GABA(B) receptor expression in the NTS. Next, we examined the BP response to the GABA(B) receptor agonist baclofen microinjected into the NTS of ANG II- or artificial cerebrospinal fluid (aCSF)-infused rats. NTS microinjection of baclofen increased BP in both groups of rats. However, the pressor response to baclofen was enhanced in ANG II-infused rats compared with aCSF-infused rats. In addition, bilateral microinjection of the GABA(B) receptor antagonist CGP-35348 into the NTS evoked a decrease in BP in both group of rats, and the depressor responses to CGP-35348 were enhanced in the ANG II-infused rats. In contrast, the pressor responses to the GABA(A) receptor agonist muscimol and the depressor responses to the GABA(A) receptor antagonist bicuculline were comparable between aCSF- and ANG II-infused rats. These results indicate that chronic ANG II infusion stimulates GABA(B) receptor expression and augments GABA(B) receptor-mediated responses in the NTS. This effect could contribute to the central nervous system actions of ANG II that result in dampening of baroreflexes and elevation in arterial BP.

Dihomo-γ-linolenic acid inhibits growth of xenograft tumors in mice bearing human pancreatic cancer cells (BxPC-3) transfected with delta-5-desaturase shRNA.

We recently reported that siRNA-knockdown of delta-5-desaturase (D5D), the rate-limiting enzyme converting upstream ω - 6 dihomo-γ-linolenic acid (DGLA) to arachidonic acid, promoted formation of the anti-cancer byproduct 8-hydroxyoctanoic acid (8-HOA) from COX-2-catalyzed DGLA peroxidation, consequently suppressing pancreatic cancer cell growth, migration and invasion. In this study, we have further investigated the anti-tumor effects of D5D-knockdown and the resulting intensified COX-2-catalyzed DGLA peroxidation in subcutaneous xenograft tumors. Four-week old female nude mice (Jackson Laboratory, J:Nu-007850) were injected with human pancreatic cancer cell line BxPC-3 or its D5D knockdown counterpart (via shRNA), followed by 4-week treatments of: vehicle control, DGLA supplementation (8 mg/mouse, twice a week), gemcitabine (30 mg/kg, twice a week), and a combination of DGLA and gemcitabine. In D5D-knockdown tumors, DGLA supplementation promoted 8-HOA formation to a threshold level (> 0.3 µg/g) and resulted in significant tumor reduction (30% vs. control). The promoted 8-HOA not only induced apoptosis associated with altered expression of Bcl-2, cleaved PARP, procaspase 3 and procaspase 9, but also suppressed the tumor metastatic potential via altering MMP-2 and E-cadherin expression. DGLA supplementation resulted in similar anti-tumor effects to those of gemcitabine in our experiments, while the combined treatment led to most significant inhibitory effect on D5D-knockdown tumor growth (70% reduction vs. control). Compared to conventional COX-2 inhibition in cancer treatment, our new strategy that takes advantage of overexpressed COX-2 in cancer cells and tumors, and of abundant ω - 6 fatty acids in the daily diet, should lead us to develop a better and safer anti-pancreatic cancer therapy for patients.

Celastrol suppresses nitric oxide synthases and the angiogenesis pathway in colorectal cancer.

The thunder god vine (Tripterygium wilfordii Hook. F) is traditionally used for inflammation-related diseases in traditional Chinese medicine. In recent years, celastrol (a natural compound from the root of the thunder god vine) has attracted great interest for its potential anticancer activities. The free radical nitric oxide (NO) is known to play a critical role in colorectal cancer growth by promoting tumour angiogenesis. However, how celastrol influences the NO pathway and its mechanism against colorectal cancer is largely unknown. In this study, we investigated the effects and mechanism of celastrol on nitric oxide synthase (NOS) and the angiogenesis pathway in colorectal cancer. Our data show that celastrol inhibited HT-29 and HCT116 cell proliferation, migration, and NOS activity in the cytoplasm. The antiproliferation activity of celastrol was associated with the inhibition of iNOS and eNOS in colorectal cancer cells. Treatment with celastrol inhibited colorectal cancer cell growth and migration, and was associated with suppression of the expression of key genes (TYMP, CDH5, THBS2, LEP, MMP9, and TNF) and proteins (IL-1b, MMP-9, PDGF, Serpin E1, and TIMP-4) involved in the angiogenesis pathway. In addition, combinational use of celastrol with 5-fluorouracil, salinomycin, 1400 W, and L-NIO showed enhanced inhibition of colorectal cancer cell proliferation and migration. In sum, our study suggests that celastrol could suppress colorectal cancer cell growth and migration, likely through suppressing NOS activity and inhibiting the angiogenesis pathway.

Specific delivery of delta-5-desaturase siRNA via RNA nanoparticles supplemented with dihomo-γ-linolenic acid for colon cancer suppression.

We have previously demonstrated that DGLA treatment along with Delta-5-Desaturase (D5D) siRNA in various types of cancer cells enhances the formation of 8-HOA from COX-2-catalyzed DGLA peroxidation, which in turn inhibits cancer cell growth and migration. However, delivery of naked siRNA remains a formidable challenge due to its "off-target" effect. In this study, we employed RNA nanotechnology for specific delivery of D5D-siRNA to xenograft colon tumors using 3WJ RNA nanoparticles. When a targeting module, i.e., the EpCAM aptamer, was incorporated, the 3WJ pRNA nanoparticles were able specifically deliver D5D siRNA to human colon cancer HCA-7 cells both in vitro and in vivo, resulting in significant downregulation of D5D expression. Co-treatment with DGLA in combination with 3WJ-EpCAM-siRNA induced a higher DGLA/AA ratio and enhanced formation of 8-HOA at a threshold level, and in HCA-7 tumor-bearing mice, induced significant tumor suppression. We further confirmed that 8-HOA formation, promoted by COX-2-catalyzed DGLA peroxidation, inhibited HDAC and consequently induced apoptosis in tumor cells. Therefore, the 3WJ RNA nanoparticle system holds great promise as a suitable therapeutic delivery platform for colon cancer therapy.

Cadmium generates reactive oxygen- and carbon-centered radical species in rats: insights from in vivo spin-trapping studies.

Cadmium (Cd) is a known industrial and environmental pollutant. In the present work, an in vivo spin-trapping technique was used in conjunction with electron spin resonance (ESR) spectroscopy to investigate free radical generation in rats following administration of cadmium chloride (CdCl2, 40 micromol/kg) and the spin trapping agent alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (POBN, 1 g/kg). In Cd-treated rats, POBN radical adducts were formed in the liver, were excreted into the bile, and exhibited an ESR spectrum consistent with a carbon-centered radical species probably derived from endogenous lipids. Isotope substitution of dimethyl sulfoxide [(CH3)2SO] with 13C demonstrated methyl radical formation (POBN/*13CH3). This adduct indicated the production of hydroxyl radical, which reacted with [(13CH3)2SO] to form *13CH3, which then reacted with POBN to form POBN/*13CH3. Depletion of hepatic glutathione by diethyl maleate significantly increased free radical production, whereas inactivation of Kupffer cells by gadolinium chloride and chelation of iron by desferal inhibited it. Treatment with the xanthine oxidase inhibitor allopurinol, the catalase inhibitor aminobenzotriazole, or the cytochrome P450 inhibitor 3-amino-1,2,4-triazole had no effect. This is the first study to show Cd generation of reactive oxygen- and carbon-centered radical species by involvement of both iron mediation through iron-catalyzed reactions and activation of Kupffer cells, the resident liver macrophages.