Brian K. Rivera

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

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% O2) and using hydrogen peroxide (H2O2) to induce oxidative stress. MSCs were preconditioned with 10 μM TMZ for 6 h followed by treatment with 100 μM H2O2 for 1 h and characterized for their cellular viability and metabolic activity. The preconditioned cells showed a significant protection against H2O2-induced loss of cellular viability, membrane damage, and oxygen metabolism accompanied by a significant increase in HIF-1α, 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.

Hypoxia induces chemoresistance in ovarian cancer cells by activation of signal transducer and activator of transcription 3

Signal transducer and activator of transcription 3 (STAT3) is activated in a variety of human cancers, including ovarian cancer. The molecular mechanism by which the STAT3 is activated in cancer cells is poorly understood. We observed that human ovarian xenograft tumors (A2780) in mice were severely hypoxic (pO2 ∼ 2 mmHg). We further observed that hypoxic exposure significantly increased the phosphorylation of STAT3 (pSTAT3) at the Tyr705 residue in A2780 cell line. The pSTAT3 (Tyr705) level was highly dependent on cellular oxygenation levels, with a significant increase at <2% O2, and without any change in the pSTAT3 (Ser727) or total STAT3 levels. The pSTAT3 (Tyr705) elevation following hypoxic exposure could be reversed within 12 hr after returning the cells to normoxia. The increased level of pSTAT3 was partly mediated by increased levels of reactive oxygen species generation in the hypoxic cancer cells. Conventional chemotherapeutic drugs cisplatin and taxol were far less effective in eliminating the hypoxic ovarian cancer cells suggesting a role for pSTAT3 in cellular resistance to chemotherapy. Inhibition of STAT3 by AG490 followed by treatment with cisplatin or taxol resulted in a significant increase in apoptosis suggesting that hypoxia‐induced STAT3 activation is responsible for chemoresistance. The results have important clinical implications for the treatment of hypoxic ovarian tumors using STAT3‐specific inhibitors.

Synthesis of N-substituted 3,5-bis(arylidene)-4-piperidones with high antitumor and antioxidant activity.

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.; Kálai, 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.

Anticancer Efficacy of a Difluorodiarylidenyl Piperidone (HO-3867) in Human Ovarian Cancer Cells and Tumor Xenografts

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 G2-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. Mol Cancer Ther; 9(5); 1169–79. ©2010 AACR.

Hyperbaric oxygenation enhances transplanted cell graft and functional recovery in the infarct heart

A major limitation to the application of stem-cell therapy to repair ischemic heart damage is the low survival of transplanted cells in the heart, possibly due to poor oxygenation. We hypothesized that hyperbaric oxygenation (HBO) can be used as an adjuvant treatment to augment stem-cell therapy. Therefore, the goal of this study was to evaluate the effect of HBO on the engraftment of rat bone marrow-derived mesenchymal stem cells (MSCs) transplanted in infarct rat hearts. Myocardial infarction (MI) was induced in Fisher-344 rats by permanently ligating the left-anterior-descending coronary artery. MSCs, labeled with fluorescent superparamagnetic iron oxide (SPIO) particles, were transplanted in the infarct and peri-infarct regions of the MI hearts. HBO (100% oxygen at 2 ATA for 90 min) was administered daily for 2 weeks. Four MI groups were used: untreated (MI); HBO; MSC; MSC+HBO. Echocardiography, electro-vectorcardiography, and magnetic resonance imaging were used for functional evaluations. The engraftment of transplanted MSCs in the heart was confirmed by SPIO fluorescence and Prussian-blue staining. Immunohistochemical staining was used to identify key cellular and molecular markers including CD29, troponin-T, connexin-43, VEGF, alpha-smooth-muscle actin, and von Willebrand factor in the tissue. Compared to MI and MSC groups, the MSC+HBO group showed a significantly increased recovery of cardiac function including left-ventricular (LV) ejection fraction, fraction shortening, LV wall thickness, and QRS vector. Further, HBO treatment significantly increased the engraftment of CD29-positive cells, expression of connexin-43, troponin-T and VEGF, and angiogenesis in the infarct tissue. Thus, HBO appears to be a potential and clinically-viable adjuvant treatment for myocardial stem-cell therapy.

HO-3867, a synthetic compound, inhibits the migration and invasion of ovarian carcinoma cells through downregulation of fatty acid synthase and focal adhesion kinase.

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 μmol/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. Mol Cancer Res; 8(9); 1188–97. ©2010 AACR.

HO-3867, a curcumin analog, sensitizes cisplatin-resistant ovarian carcinoma, leading to therapeutic synergy through STAT3 inhibition

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.

Myocardial oxygenation and functional recovery in infarct rat hearts transplanted with mesenchymal stem cells

Stem cell therapy for myocardial tissue repair is limited by the poor survival of transplanted cells, possibly because of inadequate supply of oxygen and nutrients. The purpose of this study was to assess the oxygenation level and functional recovery after allogenic transplantation of mesenchymal stem cells (MSC) in a rat model of myocardial infarction (MI). Myocardial oxygen tension (Po(2)) was measured by electron paramagnetic resonance oximetry using an implantable oxygen-sensing spin probe (OxySpin). MSCs incubated with OxySpins showed substantial uptake of the probe without affecting its oxygen sensitivity or calibration. The cells internalized with OxySpins were able to differentiate into osteogenic, adipogenic, cardiomyocyte, and endothelial cell lineages. The labeled cells tested positive for CD44 and CD29 markers and negative for the hematopoietic markers CD14 and CD45. For the in vivo studies, MI was induced in rats by permanently ligating the left anterior descending coronary artery. MSCs with OxySpins were transplanted in the infarct region of hearts. A significant increase in Po(2) was observed in the MSC group compared with the untreated MI group (18.1 +/- 2.6 vs. 13.0 +/- 1.8 mmHg, n = 4, P < 0.05) at 4 wk after transplantation. Echocardiography showed a significant improvement in ejection fraction and fraction shortening, which inversely correlated with the magnitude of fibrosis in the treated hearts. The cell-transplanted hearts also showed an increase in vascular endothelial growth factor level and capillary density in the infarct region. The study established our ability to measure and correlate changes in myocardial tissue oxygenation with cardiac function in infarcted rat hearts treated with MSCs.

Safe and targeted anticancer efficacy of a novel class of antioxidant-conjugated difluorodiarylidenyl piperidones: Differential cytotoxicity in healthy and cancer cells

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.

Inhibition of vascular smooth-muscle cell proliferation and arterial restenosis by HO-3867, a novel synthetic curcuminoid, through up-regulation of PTEN expression

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 μM) significantly inhibited the proliferation of serum-stimulated SMCs by inducing cell cycle arrest at the G1 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)-κB expressions in SMCs. Finally, HO-3867 inhibited arterial neointimal hyperplasia through overexpression of PTEN and down-regulation of MMPs and NF-κB proteins. HO-3867 is a potent drug, capable of overexpressing PTEN, which is a key target in the prevention of vascular remodeling, including restenosis.