Iminodibenzyl induced redirected COX-2 activity inhibits breast cancer progression.

Knocking down delta-5-desaturase (D5D) by siRNA or shRNA is a promising strategy to achieve 8-hydroxyoctanoic acid (8-HOA) production for cancer inhibition. However, the RNAi-based strategy to stimulate 8-HOA is restricted due to endonucleases mediated physiological degradation and off-target effects. Thus, to get persistent 8-HOA in the cancer cell, we recognized a D5D inhibitor Iminodibenzyl. Here, we have postulated that Iminodibenzyl, by inhibiting D5D activity, could shift the di-homo-gamma-linolenic acid (DGLA) peroxidation from arachidonic acid to 8-HOA in high COX-2 microenvironment of 4T1 and MDA-MB-231 breast cancer cells. We observed that Iminodibenzyl stimulated 8-HOA caused HDAC activity reduction resulting in intrinsic apoptosis pathway activation. Additionally, reduced filopodia and lamellipodia, and epithelial-mesenchymal transition markers give rise to decreased cancer cell migration. In the orthotopic breast cancer model, the combination of Iminodibenzyl and DGLA reduced tumor size. From in vitro and in vivo studies, we concluded that Iminodibenzyl could reprogram COX-2 induced DGLA peroxidation to produce anti-cancer activity.

Delta-5-desaturase: A novel therapeutic target for cancer management.

Delta-5 desaturase (D5D) is a rate-limiting enzyme that introduces double-bonds to the delta-5 position of the n-3 and n-6 polyunsaturated fatty acid chain. Since fatty acid metabolism is a vital factor in cancer development, several recent studies have revealed that D5D activity and expression could be an independent prognostic factor in cancers. However, the mechanistic basis of D5D in cancer progression is still controversial. The classical concept believes that D5D could aggravate cancer progression via mediating arachidonic acid (AA)/prostaglandin E2 production from dihomo-γ-linolenic acid (DGLA), resulting in activation of EP receptors, inflammatory pathways, and immunosuppression. On the contrary, D5D may prevent cancer progression through activating ferroptosis, which is iron-dependent cell death. Suppression of D5D by RNA interference and small-molecule inhibitor has been identified as a promising anti-cancer strategy. Inhibition of D5D could shift DGLA peroxidation pattern from generating AA to a distinct anti-cancer free radical byproduct, 8-hydroxyoctanoic acid, resulting in activation of apoptosis pathway and simultaneously suppression of cancer cell survival, proliferation, migration, and invasion. Hence, understanding the molecular mechanisms of D5D on cancer may therefore facilitate the development of novel therapeutical applications. Given that D5D may serve as a promising target in cancer, in this review, we provide an updated summary of current knowledge on the role of D5D in cancer development and potentially useful therapeutic strategies.

Iminodibenzyl redirected cyclooxygenase-2 catalyzed dihomo-γ-linolenic acid peroxidation pattern in lung cancer.

Cyclooxygenase-2 (COX-2) is up-regulated by redox imbalance and is considered a target for cancer therapy. The rationale of the COX-2 inhibitor lies in suppressing COX-2 catalyzed peroxidation of omega-6 polyunsaturated fatty acids (PUFAs), which are essential and pervasive in our daily diet. However, COX-2 inhibitors fail to improve cancer patients' survival and may lead to severe side effects. Here, instead of directly inhibiting COX-2, we utilize a small molecule, iminodibenzyl, which could reprogram the COX-2 catalyzed omega-6 PUFAs peroxidation in lung cancer by inhibiting delta-5-desaturase (D5D) activity. Iminodibenzyl breaks the conversion from dihomo-γ-linolenic acid (DGLA) to arachidonic acid, resulting in the formation of a distinct byproduct, 8-hydroxyoctanoic acid, in lung cancer cells and solid tumors. By utilizing COX-2 overexpression in cancer, the combination of DGLA supplementation and iminodibenzyl suppressed YAP1/TAZ pathway, decreasing the tumor size and lung metastasis in nude mice and C57BL/6 mice. This D5D inhibition-based strategy selectively damaged lung cancer cells with a high COX-2 level, whereas it could avoid harassing normal lung epithelial cells. This finding challenged the COX-2 redox basis in cancer, providing a new direction for developing omega-6 (DGLA)-based diet/regimen in lung cancer therapy.

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.

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.

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.

Free radical-dependent inhibition of prostaglandin endoperoxide H Synthase-2 by nitro-arachidonic acid.

Prostaglandin endoperoxide H synthase (PGHS) is a heme-enzyme responsible for the conversion of arachidonic acid (AA) to prostaglandin H2 (PGH2). PGHS have both oxygenase (COX) and peroxidase (POX) activities and is present in two isoforms (PGHS-1 and -2) expressed in different tissues and cell conditions. It has been reported that PGHS activity is inhibited by the nitrated form of AA, nitro-arachidonic acid (NO2AA), which in turn could be synthesized by PGHS under nitro-oxidative conditions. Specifically, NO2AA inhibits COX in PGHS-1 as well as POX in both PGHS-1 and -2, in a dose and time-dependent manner. NO2AA inhibition involves lowering the binding stability and displacing the heme group from the active site. However, the complete mechanism remains to be understood. This review describes the interactions of PGHS with NO2AA, focusing on mechanisms of inhibition and nitration. In addition, using a novel approach combining EPR-spin trapping and mass spectrometry, we described possible intermediates formed during PGHS-2 catalysis and inhibition. This literature revision as well as the results presented here strongly suggest a free radical-dependent inhibitory mechanism of PGHS-2 by NO2AA. This is of relevance towards understanding the underlying mechanism of inhibition of PGHS by NO2AA and its anti-inflammatory potential.

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.

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.

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.

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.

Evaluation of Serum CEA, CA19-9, CA72-4, CA125 and Ferritin as Diagnostic Markers and Factors of Clinical Parameters for Colorectal Cancer.

Blood-based protein biomarkers have recently shown as simpler diagnostic modalities for colorectal cancer, while their association with clinical pathological characteristics is largely unknown. In this study, we not only examined the sensitivity and reliability of single/multiple serum markers for diagnosis, but also assessed their connection with pathological parameters from a total of 279 colorectal cancer patients. Our study shown that glycoprotein carcinoembryonic antigen (CEA) owns the highest sensitivity among single marker in the order of CEA > cancer antigen 72-4 (CA72-4) > cancer antigen 19-9 9 (CA19-9) > ferritin > cancer antigen 125 (CA125), while the most sensitive combined-markers for two to five were: CEA + CA72-4; CEA + CA72-4 + CA125; CEA + CA19-9 + CA72-4 + CA125; and CEA + CA19-9 + CA72-4 + CA125 + ferritin, respectively. We also demonstrated that patients who had positive preoperative serum CEA, CA19-9, or CA72-4 were more likely with lymph node invasion, positive CA125 were prone to have vascular invasion, and positive CEA or CA125 were correlated with perineural invasion. In addition, positive CA19-9, CA72-4, or CA125 was associated with poorly differentiated tumor, while CEA, CA19-9, CA72-4, CA125 levels were positively correlated with pathological tumor-node-metastasis stages. We here conclude that combined serum markers can be used to not only diagnose colorectal cancer, but also appraise the tumor status for guiding treatment, evaluation of curative effect, and prognosis of patients.

The Autophagy Receptor Adaptor p62 is Up-regulated by UVA Radiation in Melanocytes and in Melanoma Cells.

UVA (315-400 nm) is the most abundant form of UV radiation in sunlight and indoor tanning beds. However, much remains to be understood about the regulation of the UVA damage response in melanocytes and melanoma. Here, we show that UVA, but not the shorter waveband UVB (280-315 nm), up-regulates adaptor protein p62 in an Nrf2- and reactive oxygen species (ROS)-dependent manner, suggesting a UVA-specific effect on p62 regulation. UVA-induced p62 up-regulation was inhibited by a mitochondria-targeted antioxidant or Nrf2 knockdown. In addition, p62 knockdown inhibited UVA-induced ROS production and Nrf2 up-regulation. We also report here a novel regulatory feedback loop between p62 and PTEN in melanoma cells. PTEN overexpression reduced p62 protein levels, and p62 knockdown increased PTEN protein levels. As compared with normal human skin, p62 was up-regulated in human nevus, malignant melanoma and metastatic melanoma. Furthermore, p62 was up-regulated in melanoma cells relative to normal human epidermal melanocytes, independent of their BRAF or NRAS mutation status. Our results demonstrated that UVA up-regulates p62 and induces a p62-Nrf2 positive feedback loop to counteract oxidative stress. Additionally, p62 forms a feedback loop with PTEN in melanoma cells, suggesting p62 functions as an oncogene in UVA-associated melanoma development and progression.

Techniques for Detecting Reactive Oxygen Species in Pulmonary Vasculature Redox Signaling.

Redox signaling plays important roles in regulating pulmonary vasculature function. Aberrant redox signaling, e.g., overproduction of reactive oxygen species (ROS) that exceeds the capability of cellular antioxidant mechanisms, has been found to alter vasculature function and remodel blood vessel structure, thus contributes to pathological processes of pulmonary vasculature. The regulation of pulmonary vasculature via ROS is a very complicated process with various biological events involved, however, the specific effect of individual ROS and the underlying mechanism still remain unclear. Most of ROS are present as free radical forms with extremely short lifetime, which makes it very difficult to detect the ROS and investigate their bioactivities. Therefore, developing specific and sensitive methods to detect ROS in complex biological system is essential for us to advance our knowledge in pulmonary vasculature regulation. In this chapter, we introduce several commonly used techniques for the detection of ROS in vitro and in vivo, including chemiluminescence-based assay, fluorescence-based assay, cytochrome c reduction method, genetically encoded fluorescent probes, as well as ESR spin trapping technique. We also discuss the advantages, limitations, and recent technical advances of each individual technique as well as their applications in pulmonary vasculature studies. We believe that technical advance in the detection of ROS will provide us with a better understanding on how to maintain normal pulmonary vasculature functions under oxidative stress.

A novel multi-hyphenated analytical method to simultaneously determine xanthine oxidase inhibitors and superoxide anion scavengers in natural products.

Natural products, such as rosmarinic acid and apigenin, can act as xanthine oxidase inhibitors (XOIs) as well as superoxide anion scavengers, and have potential for treatment of diseases associated with high uric acid levels and oxidative stress. However, efficient simultaneous screening of these two bioactivities in natural products has been challenging. We have developed a novel method by assembling a multi-hyphenated high performance liquid chromatography (HPLC) system that combines a photo-diode array, chemiluminescence detector and a HPLC system with a variable wavelength detector, to simultaneously detect components that act as both XOIs and superoxide anion scavengers in natural products. Superoxide anion scavenging activity in the analyte was measured by on-line chemiluminescence chromatography based on pyrogallol-luminol oxidation, while xanthine oxidase inhibitory activity was determined by semi-on-line HPLC analysis. After optimizing multiple elements, including chromatographic conditions (e.g., organic solvent concentration and mobile phase pH), concentrations of xanthine/xanthine oxidase and reaction temperature, our validated analytical method was capable of mixed sample analysis. The final results from our method are presented in an easily understood visual format including comprehensive bioactivity data of natural products.

Probing the structural basis and adsorption mechanism of an enzyme on nano-sized protein carriers.

Silica nanoparticles (SiNPs) are important nano-sized, solid-state carriers/hosts to load, store, and deliver biological or pharmaceutical cargoes. They are also good potential solid supports to immobilize proteins for fundamental protein structure and dynamics studies. However, precaution is necessary when using SiNPs in these areas because adsorption might alter the activity of the cargoes, especially when enzymes are loaded. Therefore, it becomes important to understand the structural basis of the cargo enzyme activity changes, if there is any. The high complexity and dynamics of the nano-bio interface present many challenges. Reported here is a comprehensive study of the structure, dynamics, and activity of a model enzyme, T4 lysozyme, upon adsorption to a few surface-modified SiNPs using several experimental techniques. Not surprisingly, a significant activity loss on each studied SiNP was found. The structural basis of the activity loss was identified based on results from a unique technique, the Electron Paramagnetic Resonance (EPR) spectroscopy, which probes structural information regardless of the complexity. Several docking models of the enzyme on SiNPs with different surfaces, at different enzyme-to-SiNP ratios are proposed. Interestingly, we found that the adsorbed enzyme can be desorbed via pH adjustment, which highlighted the potential to use SiNPs for enzyme/protein delivery or storage due to the high capacity. In order to use SiNPs as enzyme hosts, minimizing the enzymatic activity loss upon adsorption is needed. Lastly, the work outlined here demonstrate the use of EPR in probing structural information on the complex (inorganic)nano-bio interface.

Inhibition of cancer migration and invasion by knocking down delta-5-desaturase in COX-2 overexpressed cancer cells.

We recently reported that knockdown of delta-5-desaturase (a key enzyme that converts dihomo-γ-linolenic acid, DGLA, to the downstream ω-6 arachidonic acid) promotes formation of an anti-cancer byproduct 8-hydroxyoctanoic acid from cyclooxygenase (COX)-catalyzed DGLA peroxidation. 8-hydroxyoctanoic acid can exert its growth inhibitory effect on cancer cells (e.g. colon and pancreatic cancer) by serving as a histone deacetylase inhibitor. Since histone deacetylase inhibitors have been well-known to suppress cancer cell migration and invasion, we thus tested whether knockdown of delta-5-desaturase and DGLA treatment could also be used to inhibit cancer migration and invasion of colon cancer and pancreatic cancer cells. Wound healing assay, transwell assay and western blot were used to assess cell migration and invasion as well as the associated molecular mechanisms. Formation of threshold level of 8-hydroxyoctanoic acid was quantified from COX-catalyzed DGLA peroxidation in the cancer cells that overexpress COX-2 and their delta-5-desaturases were knocked down by shRNA transfection. Our results showed that knockdown of delta-5-desaturase along with DGLA supplement not only significantly inhibited cell migration, but also improved the efficacies of 5-flurouracil and gemcitabine, two frontline chemotherapy drugs currently used in the treatment of colon and pancreatic cancer, respectively. The molecular mechanism behind these observations is that 8-hydroxyoctanoic acid inhibits histone deacetylase, resulting in downregulation of cancer metastasis promotors, e.g., MMP-2 and MMP-9 as well as upregulation of cancer metastasis suppressor, e.g. E-cadherin. For the first time, we demonstrated that we could take the advantage of the common phenomenon of COX-2 overexpression in cancers to inhibit cancer cell migration and invasion. With the shifting paradigm of COX-2 cancer biology, our research outcome may provide us a novel cancer treatment strategy.