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Find video protocols related to scientific articles indexed in Pubmed.
PARP inhibitor attenuated colony formation can be restored by MAP kinase inhibitors in different irradiated cancer cell lines.
Int. J. Radiat. Biol.
PUBLISHED: 06-18-2014
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Purpose: Sensitizing cancer cells to irradiation is a major challenge in clinical oncology. We aimed to define the signal transduction pathways involved in poly(ADP-ribose) polymerase (PARP) inhibitor-induced radiosensitization in various mammalian cancer lines. Materials and methods: Clonogenic survival assays and Western blot examinations were performed following telecobalt irradiation of cancer cells in the presence or absence of various combinations of PARP- and selective mitogen-activated protein kinase (MAPK) inhibitors. Results: HO3089 resulted in significant cytotoxicity when combined with irradiation. In human U251 glioblastoma and A549 lung cancer cell lines, Erk1/2 and JNK/SAPK were found to mediate this effect of HO3089 since inhibitors of these kinases ameliorated it. In murine 4T1 breast cancer cell line, p38 MAPK rather than Erk1/2 or JNK/SAPK was identified as the main mediator of HO3089's radiosensitizing effect. Besides the aforementioned changes in kinase signaling, we detected increased p53, unchanged Bax and decreased Bcl-2 expression in the A549 cell line. Conclusions: HO3089 sensitizes cancer cells to photon irradiation via proapoptotic processes where p53 plays a crucial role. Activation of MAPK pathways is regarded the consequence of irradiation-induced DNA damage, thus their inhibition can counteract the radiosenzitizing effect of the PARP inhibitor.
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Synthesis and functional survey of new Tacrine analogs modified with nitroxides or their precursors.
Eur J Med Chem
PUBLISHED: 03-03-2014
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A series of new Tacrine analogs modified with nitroxides or pre-nitroxides on 9-amino group via methylene or piperazine spacers were synthesized; the nitroxide or its precursors were incorporated into the Tacrine scaffold. The new compounds were tested for their hydroxyl radical and peroxyl radical scavenging ability, acetylcholinesterase inhibitor activity and protection against A?-induced cytotoxicity. Based on these assays, we conclude that Tacrine analogs connected to five and six-membered nitroxides via piperazine spacers (9b, 9b/HCl and 12) exhibited the best activity, providing direction for further development of additional candidates with dual functionality (anti Alzheimer's and antioxidant).
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HO-3867, a safe STAT3 inhibitor, is selectively cytotoxic to ovarian cancer.
Cancer Res.
PUBLISHED: 03-03-2014
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STAT3 is well corroborated preclinically as a cancer therapeutic target, but tractable translational strategies for its blockade by small molecule inhibitors have remained elusive. In this study, we report the development of a novel class of bifunctional STAT3 inhibitors, based on conjugation of a diarylidenyl-piperidone (DAP) backbone to an N-hydroxypyrroline (-NOH) group, which exhibits minimal toxicity against normal cells and good oral bioavailability. Molecular modeling studies of this class suggested direct interaction with the STAT3 DNA binding domain. In particular, the DAP compound HO-3867 selectively inhibited STAT3 phosphorylation, transcription, and DNA binding without affecting the expression of other active STATs. HO-3867 exhibited minimal toxicity toward noncancerous cells and tissues but induced apoptosis in ovarian cancer cells. Pharmacologic analysis revealed greater bioabsorption and bioavailability of the active (cytotoxic) metabolites in cancer cells compared with normal cells. The selective cytotoxicity of HO-3867 seemed to be multifaceted, eliciting differential activation of the Akt pathway in normal versus cancer cells. RNAi attenuation experiments confirmed the requirement of STAT3 for HO-3867-mediated apoptosis in ovarian cancer cells. In vivo testing showed that HO-3867 could block xenograft tumor growth without toxic side effects. Furthermore, in primary human ovarian cancer cells isolated from patient ascites, HO-3867 inhibited cell migration/invasion and survival. Our results offer preclinical proof-of-concept for HO-3867 as a selective STAT3 inhibitor to treat ovarian cancer and other solid tumors where STAT3 is widely upregulated.
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A quinazoline-derivative compound with PARP inhibitory effect suppresses hypertension-induced vascular alterations in spontaneously hypertensive rats.
Biochim. Biophys. Acta
PUBLISHED: 02-25-2014
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Oxidative stress and neurohumoral factors play important role in the development of hypertension-induced vascular remodeling, likely by disregulating kinase cascades and transcription factors. Oxidative stress activates poly(ADP-ribose)-polymerase (PARP-1), which promotes inflammation and cell death. We assumed that inhibition of PARP-1 reduces the hypertension-induced adverse vascular changes. This hypothesis was tested in spontaneously hypertensive rats (SHR).
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PARP-inhibitor treatment prevents hypertension induced cardiac remodeling by favorable modulation of heat shock proteins, Akt-1/GSK-3? and several PKC isoforms.
PLoS ONE
PUBLISHED: 01-01-2014
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Spontaneously hypertensive rat (SHR) is a suitable model for studies of the complications of hypertension. It is known that activation of poly(ADP-ribose) polymerase enzyme (PARP) plays an important role in the development of postinfarction as well as long-term hypertension induced heart failure. In this study, we examined whether PARP-inhibitor (L-2286) treatment could prevent the development of hypertensive cardiopathy in SHRs. 6-week-old SHR animals were treated with L-2286 (SHR-L group) or placebo (SHR-C group) for 24 weeks. Wistar-Kyoto rats were used as aged-matched, normotensive controls (WKY group). Echocardiography was performed, brain-derived natriuretic peptide (BNP) activity and blood pressure were determined at the end of the study. We detected the extent of fibrotic areas. The amount of heat-shock proteins (Hsps) and the phosphorylation state of Akt-1(Ser473), glycogen synthase kinase (GSK)-3?(Ser9), forkhead transcription factor (FKHR)(Ser256), mitogen activated protein kinases (MAPKs), and protein kinase C (PKC) isoenzymes were monitored. The elevated blood pressure in SHRs was not influenced by PARP-inhibitor treatment. Systolic left ventricular function and BNP activity did not differ among the three groups. L-2286 treatment decreased the marked left ventricular (LV) hypertrophy which was developed in SHRs. Interstitial collagen deposition was also decreased by L-2286 treatment. The phosphorylation of extracellular signal-regulated kinase (ERK)1/2(Thr183-Tyr185), Akt-1(Ser473), GSK-3?(Ser9), FKHR(Ser256), and PKC ?(Ser729) and the level of Hsp90 were increased, while the activity of PKC ?/?II(Thr638/641), ?/?(410/403) were mitigated by L-2286 administration. We could detect signs of LV hypertrophy without congestive heart failure in SHR groups. This alteration was prevented by PARP inhibition. Our results suggest that PARP-inhibitor treatment has protective effect already in the early stage of hypertensive myocardial remodeling.
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A novel curcumin analog (H-4073) enhances the therapeutic efficacy of cisplatin treatment in head and neck cancer.
PLoS ONE
PUBLISHED: 01-01-2014
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Chemotherapy constitutes the standard modality of treatment for localized head and neck squamous cell carcinomas (HNSCC). However, many patients fail to respond and relapse after this treatments due to the acquisition of chemo-resistance. Therefore, there is an urgent need to develop novel drugs that could reverse the resistant phenotype. Curcumin, the constituent of the spice turmeric has been shown to have anti-inflammatory, anti-oxidant and anti-proliferative properties in several tumor types. However, use of curcumin has been limited due to its poor bio-absorption. Recently, a novel class of curcumin analogs, based on diarylidenylpiperidones (DAP), has been developed by incorporating a piperidone link to the beta-diketone structure and fluoro substitutions on the phenyl groups. In this study, we evaluated the effectiveness of H-4073, a parafluorinated variant of DAP, using both in vitro and in vivo head and neck cancer models. Our results demonstrate that H-4073 is a potent anti-tumor agent and it significantly inhibited cell proliferation in all the HNSCC cell lines tested in a dose-dependent manner. In addition, pretreatment of cisplatin-resistant HNSCC cell lines with H-4073 significantly reversed the chemo-resistance as observed by cell viability assay (MTT), apoptosis assay (Annexin V binding) and cleaved caspase-3 (Western blot). H-4073 mediated its anti-tumor effects by inhibiting JAK/STAT3, FAK, Akt and VEGF signaling pathways that play important roles in cell proliferation, migration, survival and angiogenesis. In the SCID mouse xenograft model, H-4073 significantly enhanced the anti-tumor and anti-angiogenesis effects of cisplatin, with no added systemic toxicity. Interestingly, H-4073 inhibited tumor angiogenesis by blocking VEGF production by tumor cells as well as directly inhibiting endothelial cell function. Taken together, our results suggest that H-4073 is a potent anti-tumor agent and it can be used to overcome chemotherapy resistance in HNSCC.
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Synthesis and potential use of 1,8-naphthalimide type (1)O2 sensor molecules.
Photochem. Photobiol. Sci.
PUBLISHED: 10-23-2013
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New double (fluorescent and spin) sensor molecules containing 4-amino substituted 1,8-naphthalimide as a fluorophore and a sterically hindered amine (pre-nitroxide) or pyrroline nitroxide as a quencher and radical capturing moiety were synthesized. All sensors were substituted with a diethylaminoethyl side-chain to increase the water solubility. Steady state fluorescence properties of these compounds and their responses to ROS in vitro are reported with perspectives of plant physiology use in vivo.
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Pulmonary hypertension secondary to left-heart failure involves peroxynitrite-induced downregulation of PTEN in the lung.
Hypertension
PUBLISHED: 01-21-2013
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Pulmonary hypertension (PH) that occurs after left-heart failure (LHF), classified as Group 2 PH, involves progressive pulmonary vascular remodeling induced by smooth muscle cell (SMC) proliferation. However, mechanisms involved in the activation of SMCs remain unknown. The objective of this study was to determine the involvement of peroxynitrite and phosphatase-and-tensin homolog on chromosome 10 (PTEN) in vascular SMC proliferation and remodeling in the LHF-induced PH (LHF-PH). LHF was induced by permanent ligation of left anterior descending coronary artery in rats for 4 weeks. MRI, ultrasound, and hemodynamic measurements were performed to confirm LHF and PH. Histopathology, Western blot, and real-time polymerase chain reaction analyses were used to identify key molecular signatures. Therapeutic intervention was demonstrated using an antiproliferative compound, HO-3867. LHF-PH was confirmed by significant elevation of pulmonary artery pressure (mean pulmonary artery pressure/mm Hg: 35.9±1.8 versus 14.8±2.0, control; P<0.001) and vascular remodeling. HO-3867 treatment decreased mean pulmonary artery pressure to 22.6±0.8 mm Hg (P<0.001). Substantially higher levels of peroxynitrite and significant loss of PTEN expression were observed in the lungs of LHF rats when compared with control. In vitro studies using human pulmonary artery SMCs implicated peroxynitrite-mediated downregulation of PTEN expression as a key mechanism of SMC proliferation. The results further established that HO-3867 attenuated LHF-PH by decreasing oxidative stress and increasing PTEN expression in the lung. In conclusion, peroxynitrite and peroxynitrite-mediated PTEN inactivation seem to be key mediators of lung microvascular remodeling associated with PH secondary to LHF.
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HO-3867, a curcumin analog, sensitizes cisplatin-resistant ovarian carcinoma, leading to therapeutic synergy through STAT3 inhibition.
Cancer Biol. Ther.
PUBLISHED: 11-01-2011
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Cisplatin resistance is a major obstacle in the treatment of ovarian cancer. Drug combinations with synergistic or complementary functions are a promising strategy to overcome this issue. We studied the anticancer efficacy of a novel compound, HO-3867, used in combination with cisplatin against chemotherapy-resistant ovarian cancer. A2780R cells, a cisplatin-resistant human ovarian cancer cell line, were exposed to 1, 5, or 10 uM of HO-3867 alone or in combination with cisplatin (10 ug/ml) for 24 hours. Cell viability (MTT), proliferation (BrdU), cell-cycle analysis (FACS), and protein expression (western blot) were used for in vitro studies. STAT3 overexpression was performed using transfected STAT3 cDNA. In vivo studies used cisplatin-resistant xenograft tumors grown in nude mice and treated with 100-ppm HO-3867 and weekly injections of 4-mg/kg cisplatin. HO-3867/cisplatin combination treatment significantly inhibited cisplatin-resistant cell proliferation in a concentration-dependent manner. The inhibition was associated with increased expression of p53 and p21, and decreased expression of cdk5 and cyclin D1. Apoptosis was induced by activation of Bax, cytochrome c release, and stimulated cleavage of caspase-9, caspase-3, and PARP. Overexpression of STAT3 decreased the HO-3867-induced apoptosis. The combination treatment significantly inhibited the growth of cisplatin-resistant xenograft tumors with significant downregulation of pSTAT3, and without apparent toxicity to healthy tissues. The combination treatment exhibited synergistic anticancer efficacy, which appears largely due to HO-3867-induced downregulation of pSTAT3. The results, combined with the previously-reported safety features of HO-3867, suggest the potential use of this compound as a safe and effective adjuvant for the treatment of ovarian cancer.
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Structure and dynamics of a conformationally constrained nitroxide side chain and applications in EPR spectroscopy.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 09-12-2011
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A disulfide-linked nitroxide side chain (R1) is the most widely used spin label for determining protein topology, mapping structural changes, and characterizing nanosecond backbone motions by site-directed spin labeling. Although the internal motion of R1 and the number of preferred rotamers are limited, translating interspin distance measurements and spatial orientation information into structural constraints is challenging. Here, we introduce a highly constrained nitroxide side chain designated RX as an alternative to R1 for these applications. RX is formed by a facile cross-linking reaction of a bifunctional methanethiosulfonate reagent with pairs of cysteine residues at i and i + 3 or i and i + 4 in an ?-helix, at i and i + 2 in a ?-strand, or with cysteine residues in adjacent strands in a ?-sheet. Analysis of EPR spectra, a crystal structure of RX in T4 lysozyme, and pulsed electron-electron double resonance (ELDOR) spectroscopy on an immobilized protein containing RX all reveal a highly constrained internal motion of the side chain. Consistent with the constrained geometry, interspin distance distributions between pairs of RX side chains are narrower than those from analogous R1 pairs. As an important consequence of the constrained internal motion of RX, spectral diffusion detected with ELDOR reveals microsecond internal motions of the protein. Collectively, the data suggest that the RX side chain will be useful for distance mapping by EPR spectroscopy, determining spatial orientation of helical segments in oriented specimens, and measuring structural fluctuations on the microsecond time scale.
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Synthesis and study of new paramagnetic resveratrol analogues.
Bioorg. Med. Chem.
PUBLISHED: 08-16-2011
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New resveratrol analogues containing five- and six-membered nitroxides and isoindoline nitroxides were synthesized. These new compounds were compared to resveratrol based on their ABTS radical scavenging ability as well on their capacity to suppress inflammatory process in macrophages induced by lipopolysaccharides. The ABTS and ROS scavenging activities of new molecules were the same or weaker than that of resveratrol, but some of paramagnetic resveratrol derivatives suppressed nitrite and TNF? production more efficiently than resveratrol. Based on these results the new nitroxide and phenol containing hybrid molecules can be considered as new antioxidant and anti-inflammatory agents.
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Amelioration of doxorubicin-induced cardiotoxicity by an anticancer-antioxidant dual-function compound, HO-3867.
J. Pharmacol. Exp. Ther.
PUBLISHED: 07-28-2011
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Doxorubicin (DOX) is a drug commonly used for the treatment of cancer. The development of resistance to DOX is common, and high cumulative doses cause potentially lethal cardiac side effects. HO-3867 (3,5-bis(4-fluorobenzylidene)-1-[(2,2,5,5-tetramethyl-2,5-dihydro-1-hydroxy-pyrrol-3-yl)methyl]piperidin-4-one), a synthetic curcumin analog, has been shown to exhibit both anticancer and cardioprotective effects. However, its cardioprotection in the setting of a conventional cancer therapy has not been established. This work investigated the use of HO-3867 and DOX to achieve a complementary outcome, i.e., increased toxicity toward cancer cells, and reduced cardiac toxicity. Combination treatment was investigated using DOX-resistant MCF-7 breast cancer cells [MCF-7 multidrug-resistant (MDR)] and BALB/c mice. Lower doses of HO-3867 and DOX (5 and 2.5 ?M, respectively) reduced viability of MCF-7 MDR cells to an extent significantly greater than that when either drug was used alone, an effect equivalent to that induced by exposure to 50 ?M DOX. In normal cardiac cells, the loss of viability from combination treatment was significantly lower than that induced by 50 ?M DOX. Increases in apoptotic markers, e.g., cleaved caspase-3, and decreases in fatty acid synthase and pAkt expressions were observed by Western blotting. Mice treated with both HO-3867 and DOX showed significant improvement in cardiac functional parameters compared with mice treated with DOX alone. Reduced expression of Bcl-2 and pAkt was observed in mice treated with DOX alone, whereas mice given combination treatment showed levels similar to control. The study indicates that combination treatment of HO-3867 and DOX is a viable option for treatment of cancer with reduced cardiotoxic side effects.
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Synthesis of N-substituted 3,5-bis(arylidene)-4-piperidones with high antitumor and antioxidant activity.
J. Med. Chem.
PUBLISHED: 07-06-2011
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A series of 3,5-bis(arylidene)-4-piperidone (DAP) compounds are considered as synthetic analogues of curcumin for anticancer properties. We performed structure-activity relationship studies by synthesizing a number of DAPs N-alkylated or acylated with nitroxides or their amine precursors as potent antioxidant moieties. Both subtituents on arylidene rings and on piperidone nitrogen (five- or six-membered, 2- or 3-substituted or 3,4-disubstituted isoindoline nitroxides) were varied. The anticancer efficacy of the new DAP compounds was tested by measuring their cytotoxicity to cancer cell lines A2780 and MCF-7 and to the H9c2 cell line. The results showed that all DAP compounds induced a significant loss of cell viability in the human cancer cell lines tested; however, only pyrroline appended nitroxides (5c (Selvendiran, K.; Tong, L.; Bratasz, A.; Kuppusamy, L. M.; Ahmed, S.; Ravi, Y.; Trigg, N. J.; Rivera, B. K.; Ka?lai, T.; Hideg, K.; Kuppusamy, P. Mol. Cancer Ther. 2010, 9, 1169-1179), 5e, 7, 9) showed limited toxicity toward noncancerous cell lines. Computer docking simulations support the biological activity tested. These results suggest that antioxidant-conjugated DAPs will be useful as a safe and effective anticancer agent for cancer therapy.
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Regulation of kinase cascade activation and heat shock protein expression by poly(ADP-ribose) polymerase inhibition in doxorubicin-induced heart failure.
J. Cardiovasc. Pharmacol.
PUBLISHED: 06-24-2011
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Cardiomyopathy is one of the most severe side effects of the chemotherapeutic agent doxorubicin (DOX). The formation of reactive oxygen species plays a critical role in the development of cardiomyopathies, and the pathophysiological cascade activates nuclear enzyme poly(ADP-ribose) polymerase (PARP), and kinase pathways. We characterized the effects of the PARP-inhibitor and kinase-modulator compound L-2286 in DOX-induced cardiac injury models. We studied the effect of the established superoxide dismutase-mimic Tempol and compared the effects of this agent with those of the PARP inhibitor. In the rat H9C2 cardiomyocytes, in which DOX-induced poly(ADP-ribosyl)ation, L-2286 protected them from the DOX-induced injury in a concentration-dependent manner. In the in vivo studies, mice were pretreated (for 1 week) with L-2286 or Tempol before the DOX treatment. Both the agents improved the activation of cytoprotective kinases, Akt, phospho-specific protein kinase C ϵ, ?/? and suppressed the activity of cell death promoting kinases glycogen synthase kinase-3?, JNK, and p38 mitogen-activated protein kinase, but the effect of PARP inhibitor was more pronounced and improved the survival as well. L-2286 activated the phosphorylation of proapoptotic transcription factor FKHR1 and promoted the expression of Hsp72 and Hsp90. These data suggest that the mode of the cytoprotective action of the PARP inhibitor may include the modulation of kinase pathways and heat shock protein expression.
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A rigid disulfide-linked nitroxide side chain simplifies the quantitative analysis of PRE data.
J. Biomol. NMR
PUBLISHED: 05-06-2011
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The measurement of (1)H transverse paramagnetic relaxation enhancement (PRE) has been used in biomolecular systems to determine long-range distance restraints and to visualize sparsely-populated transient states. The intrinsic flexibility of most nitroxide and metal-chelating paramagnetic spin-labels, however, complicates the quantitative interpretation of PREs due to delocalization of the paramagnetic center. Here, we present a novel, disulfide-linked nitroxide spin label, R1p, as an alternative to these flexible labels for PRE studies. When introduced at solvent-exposed ?-helical positions in two model proteins, calmodulin (CaM) and T4 lysozyme (T4L), EPR measurements show that the R1p side chain exhibits dramatically reduced internal motion compared to the commonly used R1 spin label (generated by reacting cysteine with the spin labeling compound often referred to as MTSL). Further, only a single nitroxide position is necessary to account for the PREs arising from CaM S17R1p, while an ensemble comprising multiple conformations is necessary for those observed for CaM S17R1. Together, these observations suggest that the nitroxide adopts a single, fixed position when R1p is placed at solvent-exposed ?-helical positions, greatly simplifying the interpretation of PRE data by removing the need to account for the intrinsic flexibility of the spin label.
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Dynamics of tropomyosin in muscle fibers as monitored by saturation transfer EPR of bi-functional probe.
PLoS ONE
PUBLISHED: 03-17-2011
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The dynamics of four regions of tropomyosin was assessed using saturation transfer electron paramagnetic resonance in the muscle fiber. In order to fully immobilize the spin probe on the surface of tropomyosin, a bi-functional spin label was attached to i,i+4 positions via cysteine mutagenesis. The dynamics of bi-functionally labeled tropomyosin mutants decreased by three orders of magnitude when reconstituted into "ghost muscle fibers". The rates of motion varied along the length of tropomyosin with the C-terminus position 268/272 being one order of magnitude slower then N-terminal domain or the center of the molecule. Introduction of troponin decreases the dynamics of all four sites in the muscle fiber, but there was no significant effect upon addition of calcium or myosin subfragment-1.
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Direct detection of free radicals and reactive oxygen species in thylakoids.
Methods Mol. Biol.
PUBLISHED: 02-01-2011
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In plants, reactive oxygen species (ROS), also known as active oxygen species (AOS), are associated with normal, physiologic processes as well as with responses to adverse conditions. ROS are connected to stress in many ways: as primary elicitors, as products and propagators of oxidative damage, or as signal molecules initiating defense or adaptation. The photosynthetic electron transport is a major site of oxidative stress by visible or ultraviolet light, high or low temperature, pollutants or herbicides. ROS production can be presumed from detecting oxidatively damaged lipids, proteins, or pigments as well as from the alleviating effects of added antioxidants. On the contrary, measuring ROS by special sensor molecules provides more direct information. This chapter focuses on the application of spin trapping electron paramagnetic resonance (EPR) spectroscopy for detecting ROS: singlet oxygen and oxygen free radicals in thylakoid membrane preparations.
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Synthesis and study of 2-amino-7-bromofluorenes modified with nitroxides and their precursors as dual anti-amyloid and antioxidant active compounds.
Eur J Med Chem
PUBLISHED: 01-14-2011
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A series of 2-aminofluorenes N-alkylated with nitroxides or their precursors were synthesized. The new compounds were tested on hydroxyl radical and peroxyl radical scavenging ability and inflammatory assay on the endothelial brain cells. In agreement with ROS scavenging ability the same compound 7-bromo-N -[(1-Oxyl-2,2,6,6-tetramethyl-1,2,3,6-tetrahydropyridine-4yl)methyl]-9H-fluoren-2-amine (3b) and its hydroxylamine salt (3b/OH/HCl) showed the anti-inflammatory property on the endothelial brain cells.
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Induction of mitochondrial destabilization and necrotic cell death by apolar mitochondria-directed SOD mimetics.
Mitochondrion
PUBLISHED: 01-06-2011
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In this paper, we present evidence, for the first time, that increasing the lipophilicity of mitochondria targeting SOD mimetics reverses their cytoprotective properties, destabilizing the mitochondrial membrane system and promoting cell death. A new mitochondria-directed apolar SOD mimetic, HO-3814, was found to provoke mitochondrial swelling and loss of mitochondrial membrane potential, and these effects were not inhibited by cyclosporine A. HO-3814-induced cell death was predominantly necrotic, caspase-independent, and not affected by mitochondrial permeability transition inhibitors or cyclophilin D-suppression, inhibitors of mitogen-activated protein kinases or Akt, or various antioxidants. In contrast, Bcl-2 overexpression diminished the effects of HO-3814.
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Cellular uptake, retention and bioabsorption of HO-3867, a fluorinated curcumin analog with potential antitumor properties.
Cancer Biol. Ther.
PUBLISHED: 11-15-2010
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Curcumin, a naturally-occurring compound found in the rhizome of Curcuma longa plant, is known for its antitumor activities. However, its clinical efficacy is limited due to poor bioabsorption. A new class of synthetic analogs of curcumin, namely diarylidenylpiperidone (DAP), has been developed with substantially higher anticancer activity than curcumin. However, its cellular uptake and bioabsorption have not been evaluated. In this study we have determined the absorption of a representative DAP compound, HO-3867, using optical and electron paramagnetic resonance spectrometry. The cellular uptake of HO-3867 was measured in a variety of cancer cell lines. HO-3867 was taken in cells within 15 minutes of exposure and its uptake was more than 100-fold higher than curcumin. HO-3867 was also retained in cells in an active form for 72 hours and possibly longer. HO-3867 was substantially cytotoxic to all the cancer cells tested. However, there was no direct correlation between cellular uptake and cytotoxicity suggesting that the cytotoxic mechanisms could be cell-type specific. When administered to rats by intraperitoneal injection, significantly high levels of HO-3867 were found in the liver, kidney, stomach, and blood after 3 hours. Also, significant accumulation of HO-3867 was found in murine tumor xenografts with a dose-dependent inhibition of tumor growth. The results suggest that the curcumin analog has substantially higher bioabsorption when compared to curcumin.
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HO-3867, a synthetic compound, inhibits the migration and invasion of ovarian carcinoma cells through downregulation of fatty acid synthase and focal adhesion kinase.
Mol. Cancer Res.
PUBLISHED: 08-16-2010
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Fatty acid synthase (FAS) and focal adhesion kinase (FAK), which are overexpressed in a variety of human epithelial tumors, play a key role in the migration and invasion of cancer cells. Hence, strategies targeted at inhibiting the FAS/FAK proteins may have therapeutic potential for cancer treatment. The goal of the present study was to determine the effect of HO-3867, a synthetic compound, on the migratory ability of ovarian cancer cells and to understand the mechanistic pathways including the involvement of FAS, FAK, and associated signaling proteins. The study was done using two established human ovarian cancer cell lines, A2780 and SKOV3. Incubation with 10 ?mol/L HO-3867 for 24 hours significantly inhibited the native as well as the vascular endothelial growth factor (VEGF)-mediated migration and invasion of the cells. HO-3867 significantly attenuated FAS and FAK protein levels apparently through accelerated ubiquitin-dependent degradation, as shown by a clear downregulation of isopeptidase USP2a. Exposure of cells to HO-3867 also significantly inhibited FAS activity and mRNA levels and a number of downstream proteins, including phospho-extracellular signal-regulated kinase 1/2, phospho-human epidermal growth factor receptor 1, sterol regulatory element binding protein 1, VEGF, and matrix metalloproteinase 2. Western blot and immunohistochemical analyses of A2780 xenograft tumors in mice treated with HO-3867 showed significant reduction in FAS, FAK, VEGF, and downstream protein levels when compared with the untreated control. Collectively, the results showed that HO-3867 suppressed the migration and invasion of ovarian cancer cells by inhibiting the expression or activity of FAS and FAK proteins. The study suggests that molecular targeting of FAS and FAK by HO-3867 may be a potential strategy for ovarian cancer therapy.
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Anticancer efficacy of a difluorodiarylidenyl piperidone (HO-3867) in human ovarian cancer cells and tumor xenografts.
Mol. Cancer Ther.
PUBLISHED: 05-04-2010
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The purpose of this study was to evaluate the anticancer potency and mechanism of a novel difluorodiarylidenyl piperidone (H-4073) and its N-hydroxypyrroline modification (HO-3867) in human ovarian cancer. Studies were done using established human ovarian cancer cell lines (A2870, A2780cDDP, OV-4, SKOV3, PA-1, and OVCAR3) as well as in a murine xenograft tumor (A2780) model. Both compounds were comparably and significantly cytotoxic to A2780 cells. However, HO-3867 showed a preferential toxicity toward ovarian cancer cells while sparing healthy cells. HO-3867 induced G(2)-M cell cycle arrest in A2780 cells by modulating cell cycle regulatory molecules p53, p21, p27, cyclin-dependent kinase 2, and cyclin, and promoted apoptosis by caspase-8 and caspase-3 activation. It also caused an increase in the expression of functional Fas/CD95 and decreases in signal transducers and activators of transcription 3 (STAT3; Tyr705) and JAK1 phosphorylation. There was a significant reduction in STAT3 downstream target protein levels including Bcl-xL, Bcl-2, survivin, and vascular endothelial growth factor, suggesting that HO-3867 exposure disrupted the JAK/STAT3 signaling pathway. In addition, HO-3867 significantly inhibited the growth of the ovarian xenografted tumors in a dosage-dependent manner without any apparent toxicity. Western blot analysis of the xenograft tumor tissues showed that HO-3867 inhibited pSTAT3 (Tyr705 and Ser727) and JAK1 and increased apoptotic markers cleaved caspase-3 and poly ADP ribose polymerase. HO-3867 exhibited significant cytotoxicity toward ovarian cancer cells by inhibition of the JAK/STAT3 signaling pathway. The study suggested that HO-3867 may be useful as a safe and effective anticancer agent for ovarian cancer therapy.
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Trimetazidine, administered at the onset of reperfusion, ameliorates myocardial dysfunction and injury by activation of p38 mitogen-activated protein kinase and Akt signaling.
J. Pharmacol. Exp. Ther.
PUBLISHED: 02-18-2010
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Trimetazidine [1-(2,3,4-trimethoxybenzyl)piperazine; TMZ] is an anti-ischemic cardiac drug; however, its efficacy and mechanism of cardioprotection upon reperfusion are largely unknown. The objective of this study was to determine whether TMZ, given before reperfusion, could attenuate myocardial reperfusion injury. Ischemia/reperfusion (I/R) was induced in rat hearts by ligating the left anterior descending (LAD) coronary artery for 30 min followed by 48 h of reperfusion. TMZ (5 mg/kg b.wt.) was administered 5 min before reperfusion. The study used three experimental groups: control (-I/R; -TMZ), I/R (+I/R; -TMZ), and TMZ (+I/R; +TMZ). Echocardiography and EPR oximetry were used to assess cardiac function and oxygenation, respectively. The ejection fraction, which was significantly depressed in the I/R group (62 +/- 5 versus 84 +/- 3% in control), was restored to 72 +/- 3% in the TMZ group. Myocardial pO2 in the TMZ group returned to baseline levels (approximately 20 mm Hg) within 1 h of reperfusion, whereas the I/R group showed a significant hyperoxygenation even after 48 h of reperfusion. The infarct size was significantly reduced in the TMZ group (26 +/- 3 versus 47 +/- 5% in I/R). TMZ treatment significantly attenuated superoxide levels in the tissue. Tissue homogenates showed a significant increase in p38 and p-Akt and decrease in caspase-3 levels in the TMZ group. In summary, the results demonstrated that TMZ is cardioprotective when administered before reperfusion and that this protection appears to be mediated by activation of p38 mitogen-activated protein kinase and Akt signaling. The study emphasizes the importance of administering TMZ before reflow to prevent reperfusion-mediated cardiac injury and dysfunction.
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Resolving Conformational and Rotameric Exchange in Spin-Labeled Proteins Using Saturation Recovery EPR.
Appl Magn Reson
PUBLISHED: 02-17-2010
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The function of many proteins involves equilibria between conformational substates, and to elucidate mechanisms of function it is essential to have experimental tools to detect the presence of conformational substates and to determine the time scale of exchange between them. Site-directed spin labeling (SDSL) has the potential to serve this purpose. In proteins containing a nitroxide side chain (R1), multicomponent electron paramagnetic resonance (EPR) spectra can arise either from equilibria involving different conformational substates or rotamers of R1. To employ SDSL to uniquely identify conformational equilibria, it is thus essential to distinguish between these origins of multicomponent spectra. Here we show that this is possible based on the time scale for exchange of the nitroxide between distinct environments that give rise to multicomponent EPR spectra; rotamer exchange for R1 lies in the ?0.1-1 ?s range, while conformational exchange is at least an order of magnitude slower. The time scales of exchange events are determined by saturation recovery EPR, and in favorable cases, the exchange rate constants between substates with lifetimes of approximately 1-70 ?s can be estimated by the approach.
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Synthesis and study of new paramagnetic and diamagnetic verapamil derivatives.
Bioorg. Med. Chem.
PUBLISHED: 02-16-2010
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New derivatives of verapamil (1) modified with nitroxides and their precursors were synthesized and screened for reactive oxygen species (ROS)-scavenging activities. The basic structure was modified by changing the nitrile group to an amide or the methyl substituent on tertiary nitrogen with nitroxides and their reduced forms (hydroxylamine and secondary amines). Among the new verapamil derivatives compound 16B [Mohan, I. K.; Kahn, M.; Wisel, S.; Selvendiran, K.; Sridhar, A.; Carnes, C.A.; Bognár, B.; Kálai, T.; Hideg, K.; Kuppusamy, P. Am. J. Physiol. Heart Circ. Physiol.2009, 296, 140], modified with hydroxylamine salt of 2,2,6,6-tetramethyl-1,2,3,6-tetrahydropyridine-1-yloxyl proved to be the best ROS scavenger in vitro and protected HSMC and CHO cells against H(2)O(2) induced damage.
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Safe and targeted anticancer efficacy of a novel class of antioxidant-conjugated difluorodiarylidenyl piperidones: differential cytotoxicity in healthy and cancer cells.
Free Radic. Biol. Med.
PUBLISHED: 01-23-2010
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The development of smart anticancer drugs that can selectively kill cancer cells while sparing the surrounding healthy tissues/cells is of paramount importance for safe and effective cancer therapy. We report a novel class of bifunctional compounds based on diarylidenyl piperidone (DAP) conjugated to an N-hydroxypyrroline (NOH; a nitroxide precursor) group. We hypothesized that the DAP would have cytotoxic (anticancer) activity, whereas the NOH moiety would function as a tissue-specific modulator (antioxidant) of cytotoxicity. The study used four DAPs, namely H-4073 and H-4318 without NOH and HO-3867 and HO-4200 with NOH substitution. The goal of the study was to evaluate the proof-of-concept anticancer-versus-antioxidant efficacy of the DAPs using a number of cancerous (breast, colon, head and neck, liver, lung, ovarian, and prostate cancer) and noncancerous (smooth muscle, aortic endothelial, and ovarian surface epithelial) human cell lines. Cytotoxicity was determined using an MTT-based cell viability assay. All four compounds induced significant loss of cell viability in cancer cells, whereas HO-3867 and HO-4200 showed significantly less cytotoxicity in noncancerous cells. EPR measurements showed a metabolic conversion of the N-hydroxylamine function to nitroxide with significantly higher levels of the metabolite and superoxide radical-scavenging (antioxidant) activity in noncancerous cells compared to cancer cells. Western blot analysis showed that the DAP-induced growth arrest and apoptosis in cancer cells were mediated by inhibition of STAT3 phosphorylation at the Tyr705 and Ser727 residues and induction of apoptotic markers of cleaved caspase-3 and PARP. The results suggest that the antioxidant-conjugated DAPs will be useful as safe and effective anticancer agents for cancer therapy.
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Site-directed spin labeling of a genetically encoded unnatural amino acid.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 12-07-2009
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The traditional site-directed spin labeling (SDSL) method, which utilizes cysteine residues and sulfhydryl-reactive nitroxide reagents, can be challenging for proteins that contain functionally important native cysteine residues or disulfide bonds. To make SDSL amenable to any protein, we introduce an orthogonal labeling strategy, i.e., one that does not rely on any of the functional groups found in the common 20 amino acids. In this method, the genetically encoded unnatural amino acid p-acetyl-L-phenylalanine (p-AcPhe) is reacted with a hydroxylamine reagent to generate a nitroxide side chain (K1). The utility of this scheme was demonstrated with seven mutants of T4 lysozyme, each containing a single p-AcPhe at a solvent-exposed helix site; the mutants were expressed in amounts qualitatively similar to the wild-type protein. In general, the EPR spectra of the resulting K1 mutants reflect higher nitroxide mobilities than the spectra of analogous mutants containing the more constrained disulfide-linked side chain (R1) commonly used in SDSL. Despite this increased flexibility, site dependence of the EPR spectra suggests that K1 will be a useful sensor of local structure and of conformational changes in solution. Distance measurements between pairs of K1 residues using double electron electron resonance (DEER) spectroscopy indicate that K1 will also be useful for distance mapping.
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VLDL lipolysis products increase VLDL fluidity and convert apolipoprotein E4 into a more expanded conformation.
J. Lipid Res.
PUBLISHED: 12-03-2009
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Our previous work indicated that apolipoprotein (apo) E4 assumes a more expanded conformation in the postprandial period. The postprandial state is characterized by increased VLDL lipolysis. In this article, we tested the hypothesis that VLDL lipolysis products increase VLDL particle fluidity, which mediates expansion of apoE4 on the VLDL particle. Plasma from healthy subjects was collected before and after a moderately high-fat meal and incubated with nitroxyl-spin labeled apoE. ApoE conformation was examined by electron paramagnetic resonance spectroscopy using targeted spin probes on cysteines introduced in the N-terminal (S76C) and C-terminal (A241C) domains. Further, we synthesized a novel nitroxyl spin-labeled cholesterol analog, which gave insight into lipoprotein particle fluidity. Our data revealed that the order of lipoprotein fluidity was HDL approximately LDL
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Site-specific DNA structural and dynamic features revealed by nucleotide-independent nitroxide probes.
Biochemistry
PUBLISHED: 08-05-2009
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In site-directed spin labeling, a covalently attached nitroxide probe containing a chemically inert unpaired electron is utilized to obtain information on the local environment of the parent macromolecule. Studies presented here examine the feasibility of probing local DNA structural and dynamic features using a class of nitroxide probes that are linked to chemically substituted phosphorothioate positions at the DNA backbone. Two members of this family, designated as R5 and R5a, were attached to eight different sites of a dodecameric DNA duplex without severely perturbing the native B-form conformation. Measured X-band electron paramagnetic resonance (EPR) spectra, which report on nitroxide rotational motions, were found to vary depending on the location of the label (e.g., duplex center vs termini) and the surrounding DNA sequence. This indicates that R5 and R5a can provide information on the DNA local environment at the level of an individual nucleotide. As these probes can be attached to arbitrary nucleotides within a nucleic acid sequence, they may provide a means to "scan" a given DNA molecule in order to interrogate its local structural and dynamic features.
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PARP inhibition delays transition of hypertensive cardiopathy to heart failure in spontaneously hypertensive rats.
Cardiovasc. Res.
PUBLISHED: 05-14-2009
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Oxidative stress followed by abnormal signalling can play a critical role in the development of long-term, high blood pressure-induced cardiac remodelling in heart failure (HF). Since oxidative stress-induced poly(ADP-ribose)polymerase (PARP) activation and cell death have been observed in several experimental models, we investigated the possibility that inhibition of nuclear PARP improves cardiac performance and delays transition from hypertensive cardiopathy to HF in a spontaneously hypertensive rat (SHR) model of HF.
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Cardioprotection by HO-4038, a novel verapamil derivative, targeted against ischemia and reperfusion-mediated acute myocardial infarction.
Am. J. Physiol. Heart Circ. Physiol.
PUBLISHED: 03-14-2009
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Many cardiac interventional procedures, such as coronary angioplasty, stenting, and thrombolysis, attempt to reintroduce blood flow (reperfusion) to an ischemic region of myocardium. However, the reperfusion is accompanied by a complex cascade of cellular and molecular events resulting in oxidative damage, termed myocardial ischemia-reperfusion (I/R) injury. In this study, we evaluated the ability of HO-4038, an N-hydroxypiperidine derivative of verapamil, on the modulation of myocardial tissue oxygenation (Po(2)), I/R injury, and key signaling molecules involved in cardioprotection in an in vivo rat model of acute myocardial infarction (MI). MI was created in rats by ligating the left anterior descending coronary artery (LAD) for 30 min followed by 24 h of reperfusion. Verapamil or HO-4038 was infused through the jugular vein 10 min before the induction of ischemia. Myocardial Po(2) and the free-radical scavenging ability of HO-4038 were measured using electron paramagnetic resonance spectroscopy. HO-4038 showed a significantly better scavenging ability of reactive oxygen radicals compared with verapamil. The cardiac contractile functions in the I/R hearts were significantly higher recovery in HO-4038 compared with the verapamil group. A significant decrease in the plasma levels of creatine kinase and lactate dehydrogenase was observed in the HO-4038 group compared with the verapamil or untreated I/R groups. The left ventricular infarct size was significantly less in the HO-4038 (23 +/- 2%) compared with the untreated I/R (36 +/- 4%) group. HO-4038 significantly attenuated the hyperoxygenation (36 +/- 1 mmHg) during reperfusion compared with the untreated I/R group (44 +/- 2 mmHg). The HO-4038-treated group also markedly attenuated superoxide production, increased nitric oxide generation, and enhanced Akt and Bcl-2 levels in the reperfused myocardium. Overall, the results demonstrated that HO-4038 significantly protected hearts against I/R-induced cardiac dysfunction and damage through the combined beneficial actions of calcium-channel blocking, antioxidant, and prosurvival signaling activities.
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Inhibition of vascular smooth-muscle cell proliferation and arterial restenosis by HO-3867, a novel synthetic curcuminoid, through up-regulation of PTEN expression.
J. Pharmacol. Exp. Ther.
PUBLISHED: 03-10-2009
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Phosphatase and tensin homolog (PTEN), a tumor suppressor gene, has been shown to play a vital role in vascular smooth muscle cell (SMC) proliferation and hence is a potential therapeutic target to inhibit vascular remodeling. The goal of this study was to evaluate the efficacy and mechanism of HO-3867 [((3E,5E)-3,5-bis[(4-fluorophenyl)methylidene]-1-[(1-hydroxy-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrol-3-yl)methyl]piperidin-4-one)], a new synthetic curcuminoid, in the inhibition of vascular SMC proliferation and restenosis. Experiments were performed using human aortic SMCs and a rat carotid artery balloon injury model. HO-3867 (10 microM) significantly inhibited the proliferation of serum-stimulated SMCs by inducing cell cycle arrest at the G(1) phase (72% at 24 h) and apoptosis (at 48 h). HO-3867 significantly increased the phosphorylated and total levels of PTEN in SMCs. Suppression of PTEN expression by PTEN-small interfering RNA transfection reduced p53 and p21 levels and increased extracellular signal-regulated kinase 1/2 phosphorylation, resulting in decreased apoptosis. Conversely, overexpression of PTEN by cDNA transfection activated caspase-3 and increased apoptosis. Furthermore, HO-3867 significantly down-regulated matrix metalloproteinase (MMP)-2, MMP-9, and nuclear factor (NF)-kappaB expressions in SMCs. Finally, HO-3867 inhibited arterial neointimal hyperplasia through overexpression of PTEN and down-regulation of MMPs and NF-kappaB proteins. HO-3867 is a potent drug, capable of overexpressing PTEN, which is a key target in the prevention of vascular remodeling, including restenosis.
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New poly(ADP-ribose) polymerase-1 inhibitors with antioxidant activity based on 4-carboxamidobenzimidazole-2-ylpyrroline and -tetrahydropyridine nitroxides and their precursors.
J. Med. Chem.
PUBLISHED: 02-28-2009
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4-Carboxamidobenzimidazoles were previously described as PARP inhibitor compounds. Here we report upon 4-carboxamido-1H-benzimidazoles substituted in the 2-position with nitroxides or their amine or hydroxylamine precursors. Among the new molecules, a highly active PARP inhibitor 4h (IC(50) = 14 nM) was identified with antioxidant/radical scavenger activity. We concluded that in most cases sterically hindered amines are better PARP inhibitors than their oxidized form and structural changes in the 2-substituted 4-carboxamido-1H-benzimidazoles (such as N-substitution or changing the position of the carboxamide group) were detrimental to PARP inhibition activity but not to antioxidant activity. These results indicate the advantages of combining an antioxidant nitroxide or nitroxide precursor with a PARP inhibitor molecule to decrease or eliminate the deleterious processes initiated by reactive oxygen and reactive nitrogen species (ROS and RNS). The radical scavenging capability of 4h was demonstrated by EPR study of urine collected after drug administration.
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Pharmacological preconditioning of mesenchymal stem cells with trimetazidine (1-[2,3,4-trimethoxybenzyl]piperazine) protects hypoxic cells against oxidative stress and enhances recovery of myocardial function in infarcted heart through Bcl-2 expression.
J. Pharmacol. Exp. Ther.
PUBLISHED: 02-13-2009
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Stem cell transplantation is a possible therapeutic option to repair ischemic damage to the heart. However, it is faced with a number of challenges including the survival of the transplanted cells in the ischemic region. The present study was designed to use stem cells preconditioned with trimetazidine (1-[2,3,4-trimethoxybenzyl]piperazine; TMZ), a widely used anti-ischemic drug for treating angina in cardiac patients, to increase the rate of their survival after transplantation. Bone marrow-derived rat mesenchymal stem cells (MSCs) were subjected to a simulated host tissue environment by culturing them under hypoxia (2% O(2)) and using hydrogen peroxide (H(2)O(2)) to induce oxidative stress. MSCs were preconditioned with 10 microM TMZ for 6 h followed by treatment with 100 microM H(2)O(2) for 1 h and characterized for their cellular viability and metabolic activity. The preconditioned cells showed a significant protection against H(2)O(2)-induced loss of cellular viability, membrane damage, and oxygen metabolism accompanied by a significant increase in HIF-1alpha, survivin, phosphorylated Akt (pAkt), and Bcl-2 protein levels and Bcl-2 gene expression. The therapeutic efficacy of the TMZ-preconditioned MSCs was evaluated in an in vivo rat model of myocardial infarction induced by permanent ligation of left anterior descending coronary artery. A significant increase in the recovery of myocardial function and up-regulation of pAkt and Bcl-2 levels were observed in hearts transplanted with TMZ-preconditioned cells. This study clearly demonstrated the potential benefits of pharmacological preconditioning of MSCs with TMZ for stem cell therapy for repairing myocardial ischemic damage.
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HO-3867, a STAT3 inhibitor induces apoptosis by inactivation of STAT3 activity in BRCA1-mutated ovarian cancer cells.
Cancer Biol. Ther.
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BRCA1 plays an important role in DNA damage and repair, homologous recombination, cell-cycle regulation and apoptosis. BRCA-mutated ovarian cancer often presents at an advanced stage, however, tend to have better response to platinum-based chemotherapy as compared with sporadic cases of epithelial ovarian cancer (EOC). In spite of this, most patients will develop a recurrence and eventually succumb to the disease. Preclinical studies are currently investigating natural compounds and their analogs for tumor-directed targets in ovarian cancer. The aim of this study is to investigate whether the STAT3 inhibitor HO-3867, a novel curcumin analog, has a therapeutic effect on BRCA1-mutated ovarian cancer. Our novel agent, HO-3867 and a commercial STAT3 inhibitor, STATTIC, significantly inhibited BRCA-mutated ovarian cancer cells in vitro in a dose- and time-dependent manner. BRCA-mutated ovarian cancer cells treated with HO-3867 exhibited a significant degree of apoptosis with elevated levels of cleaved caspase-3, caspase-7 and PARP. HO-3867 treatment induced more reactive oxygen species (ROS) in BRCA-mutated cells compared with wild-type cells, however, there was no increased ROS when benign ovarian surface epithelial cells were treated with HO-3867. BRCA1-mutated cancer cells had higher expression of Tyrosine-phosphorylated STAT3 (pTyr705) as compared with other STAT proteins. Furthermore, treatment of these cells with HO-3867 resulted in decreased expression of pTyr705 and its downstream targets cyclin D1, Bcl-2 and survivin. In addition, overexpression of STAT3 cDNA provided resistance to HO-3867-induced apoptosis. Our results show that HO-3867, a potent STAT3 inhibitor, may have a role as a biologically targeted agent for BRCA1-mutated cancers either as an adjunct to cytotoxic chemotherapy or as a single agent.
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The influence of spin-labeled fluorene compounds on the assembly and toxicity of the a? peptide.
PLoS ONE
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The deposition and oligomerization of amyloid ? (A?) peptide plays a key role in the pathogenesis of Alzheimers disease (AD). A? peptide arises from cleavage of the membrane-associated domain of the amyloid precursor protein (APP) by ? and ? secretases. Several lines of evidence point to the soluble A? oligomer (A?O) as the primary neurotoxic species in the etiology of AD. Recently, we have demonstrated that a class of fluorene molecules specifically disrupts the A?O species.
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