Decreased NMIIA heavy chain phosphorylation at S1943 promotes mitoxantrone resistance by upregulating BCRP and N-cadherin expression in breast cancer cells.

Mitoxantrone (MX) is an effective treatment for breast cancer; however, high efflux of MX that is accomplished by breast cancer resistance protein (BCRP) leads to acquired multidrug resistance (MDR), reducing MX's therapeutic efficacy in breast cancer. Non-muscle myosin IIA (NMIIA) and its heavy phosphorylation at S1943 have been revealed to play key roles in tumor metastasis and progression, including in breast cancer; however, their molecular function in BCRP-mediated MDR in breast cancer remains unknown. In this study, we revealed that the expression of NMIIA heavy chain phosphorylation at S1943 was downregulated in BCRP-overexpressing breast cancer MCF-7/MX cells, and stable expression of NMIIA-S1943A mutant increased BCRP expression and promoted the resistance of MCF-7/MX cells to MX. Meanwhile, NMIIA S1943 phosphorylation induced by epidermal growth factor (EGF) was accompanied by the downregulation of BCRP in MCF-7/MX cells. Furthermore, stable expression of NMIIA-S1943A in MCF-7/MX cells resulted in upregulation of N-cadherin and the accumulation of ß-catenin on the cell surface, which inhibited the nucleus translocation of ß-catenin and Wnt/ß-catenin-based proliferative signaling. EGF stimulation of MCF-7/MX cells showed the downregulation of N-cadherin and ß-catenin. Our results suggest that decreased NMIIA heavy phosphorylation at S1943 increases BCRP expression and promotes MX resistance in breast cancer cells via upregulating N-cadherin expression.

Metformin Alleviates Sepsis-Associated Myocardial Injury by Enhancing AMP-Activated Protein Kinase/Mammalian Target of Rapamycin Signaling Pathway-Mediated Autophagy.

ABSTRACT: Sepsis-associated myocardial injury is one of the main causes of death in intensive care units, and current clinical treatments have not been satisfactory. Therefore, finding an effective intervention is an urgent requirement. Metformin, an anti-type 2 diabetes drug, has been reported to be an autophagic activator agent that confers protection in some diseases. However, it is unclear whether it can provide defense against sepsis-associated myocardial injury. In this study, we investigated the cardioprotective effects of metformin pretreatment against lipopolysaccharide (LPS)-induced myocardial injury in C57BL/6J mice or H9c2 cells and the possible underlying mechanisms. Metformin was administered at a dose of 100 mg/kg for a week before LPS intraperitoneal injection. Twenty-four hours after LPS intervention, echocardiographic evaluation, reactive oxygen species measurement, Hoechst staining, western blotting, hematoxylin and eosin staining, and enzyme-linked immunosorbent assay were performed. Inhibitors of autophagy and AMP-activated protein kinase (AMPK) were used to further clarify the mechanisms involved. Metformin pretreatment effectively attenuated cardiac dysfunction, reduced the levels of myocardial enzymes, and alleviated cardiac hydroncus in LPS-treated mice. In addition, metformin restored the LPS-disrupted antioxidant defense and activated LPS-reduced autophagy by modulating the AMPK/mammalian target of rapamycin (AMPK/mTOR) pathway both in vivo and in vitro. The antioxidant effects of metformin on cardiomyocytes were abolished by the autophagy inhibitor 3-methyladenine (3-MA). Treatment with compound C, an AMPK inhibitor, reversed the metformin-induced autophagy in LPS-treated H9c2 cells. In conclusion, metformin pretreatment alleviates LPS-induced myocardial injury by activating AMPK/mTOR pathway-mediated autophagy.

Chrysin prevents lipopolysaccharide-induced acute lung injury in mice by suppressing the IRE1α/TXNIP/NLRP3 pathway.

Acute lung injury (ALI) remains a serious challenge in the intensive care unit. Inflammation plays a key role in the progression of ALI. Chrysin (CHR) is a natural flavonoid with anti-inflammatory functions. We investigated the anti-inflammatory effects in a mouse model of ALI induced by lipopolysaccharide (LPS), and identified the underlying mechanisms of its action. Following CHR administration, mice were challenged with LPS intratracheally for 6 h to induce ALI. Compared to mice challenged with LPS alone, the presence of CHR showed a reduction in the development of lung injuries, as confirmed by histopathological observation. Pre-treatment with CHR attenuated inflammation by reducing the production of myeloperosidase (MPO), and pro-inflammatory cytokine levels in the lung and bronchoalveolar lavage fluid (BALF). Furthermore, CHR improved lung edema by reducing the vascular permeability, as demonstrated by less evans blue staining in the lung tissue and low levels of protein in BALF. In addition, our results proved that CHR improved the antioxidant capacity by increasing the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in the lung tissue. Results of western blot assays suggested that CHR suppressed the LPS-induced expression of glucose-regulated protein 78 (GRP78) and phosphorylated inositol-requiring enzyme 1α (p-IRE1α). We also found that CHR suppressed the expression of thioredoxin interaction protein (TXNIP), nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) and cleaved caspase-1. In conclusion, CHR improved vascular permeability and mitigated the inflammatory response of lung tissue by suppressing the IRE1α/TXNIP/NLRP3 pathway, thereby alleviating LPS-induced ALI in the lungs of mice.

Epithelial cell adhesion molecule promotes breast cancer resistance protein-mediated multidrug resistance in breast cancer by inducing partial epithelial-mesenchymal transition.

Overexpression of breast cancer resistance protein (BCRP) plays a crucial role in the acquired multidrug resistance (MDR) in breast cancer. The elucidation of molecular events that confer BCRP-mediated MDR is of major therapeutic importance in breast cancer. Epithelial cell adhesion molecule (EpCAM) has been implicated in tumor progression and drug resistance in various types of cancers, including breast cancer. However, the role of EpCAM in BCRP-mediated MDR in breast cancer remains unknown. In the present study, we revealed that EpCAM expression was upregulated in BCRP-overexpressing breast cancer MCF-7/MX cells, and EpCAM knockdown using siRNA reduced BCRP expression and increased the sensitivity of MCF-7/MX cells to mitoxantrone (MX). The epithelial-mesenchymal transition (EMT) promoted BCRP-mediated MDR in breast cancer cells, and EpCAM knockdown partially suppressed EMT progression in MCF-7/MX cells. In addition, Wnt/ß-catenin signaling was activated in MCF-7/MX cells, and the inhibition of this signaling attenuated EpCAM and BCRP expression and partially reversed EMT. Together, this study illustrates that EpCAM upregulation by Wnt/ß-catenin signaling induces partial EMT to promote BCRP-mediated MDR resistance in breast cancer cells. EpCAM may be a potential therapeutic target for overcoming BCRP-mediated resistance in human breast cancer.

Baicalein attenuates monocrotaline-induced pulmonary arterial hypertension by inhibiting vascular remodeling in rats.

BACKGROUND: Pulmonary arterial hypertension (PAH) is a devastating cardiopulmonary disorder characterized by elevated pulmonary arterial pressure (PAP) and right ventricular hypertrophy (RVH) driven by progressive vascular remodeling. Reversing adverse vascular remodeling is an important concept in the treatment of PAH. Endothelial injury, inflammation, and oxidative stress are three main contributors to pulmonary vascular remodeling. Baicalein is a natural flavonoid that has been shown to possess anti-proliferative, anti-inflammatory, anti-oxidative, and cardioprotective properties. We hypothesized that baicalein may prevent the progression of PAH and preserve the right heart function by inhibiting pulmonary arterial remodeling. METHODS: Male Sprague-Dawley rats were distributed randomly into 4 groups: control, monocrotaline (MCT)-exposed, and MCT-exposed plus baicalein treated rats (50 and 100 mg/kg/day for 2 weeks). Hemodynamic changes, RVH, and lung morphological features were examined on day 28. Apoptosis was determined by TUNEL staining, and the mRNA levels of tumor necrosis factor alpha (TNF-α), interleukin-1ß (IL-1ß), and IL-6 were detected by qRT-PCR. The changes in oxidative indicators, including malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) were measured using corresponding commercial kits. The levels of Bax, Bcl-2, and cleaved caspase-3, and the activation of mitogen-activated protein kinase (MAPK) and NF-κB were assessed by western blotting. RESULTS: MCT induced an increase in hemodynamic parameters and RVH, which were attenuated by baicalein treatment. Baicalein also blocked MCT-induced pulmonary arterial remodeling. The levels of apoptotic (Bax/Bcl-2 ratio and cleaved caspase-3) and inflammatory (IL-6, TNF-α, and IL-1ß) biomarkers in lung tissue were lower in baicalein-treated groups. Baicalein also decreased MDA level, and increased SOD and GSH-Px activity in rat pulmonary tissue. Furthermore, baicalein inhibited MCT-induced activation of the MAPK and NF-κB pathways. CONCLUSION: Baicalein ameliorates MCT-induced PAH by inhibiting pulmonary arterial remodeling at least partially via the MAPK and NF-κB pathways in rats.

Nebivolol ameliorated kidney damage in Zucker diabetic fatty rats by regulation of oxidative stress/NO pathway: Comparison with captopril.

The aim was to evaluate the effects and mechanisms of nebivolol on renal damage in Zucker diabetic fatty (ZDF) rats, in comparison with those of atenolol and captopril. Animals were divided into: control lean Zucker rats, ZDF rats, ZDF rats orally treated with nebivolol (10 mg/kg), atenolol (100 mg/kg) or captopril (40 mg/kg) for 6 months. Systolic blood pressure (SBP), blood glucose, kidney structure and function, plasma and kidney levels of nitric oxide (NO) and asymmetric dimethylarginine (ADMA), and oxidant status were evaluated. Kidney expressions of adenosine monophosphate-activated protein kinase (AMPK), NADPH oxidase (NOX) isoforms 2 and 4 and subunit p22phox , nitric oxide synthase (NOS) isoforms, endothelial NOS (eNOS) uncoupling, protein arginine N-methyltransferase (PRMT) 1, and dimethylarginine dimethylaminohydrolase (DDAH) 1 and 2 were tested. All drugs induced a similar control of SBP. Nebivolol did not affect the increased plasma glucose. Unlike atenolol, nebivolol prevented the decrease in plasma insulin, and, like captopril, it reduced plasma lipid contents. Nebivolol ameliorated, to a greater extent than captopril, damages to renal structure and function, which were associated with an improvement in interlobular artery dysfunction. Nebivolol elevated kidney phosphorylation of AMPK, attenuated NOX4 and p22phox expression and oxidative stress marker levels. Nebivolol increased plasma and renal NO, enhanced expressions of eNOS, p-eNOS and neuronal NOS, and suppressed eNOS uncoupling and inducible NOS expression. High ADMA in plasma and kidney were decreased by nebivolol through increasing DDAH2 and decreasing protein arginine N-methyltransferase 1. Long-term treatment of nebivolol ameliorated diabetic nephropathy, at least in part, via regulation of renal oxidative stress/NO pathway.

Overproduction of reactive oxygen species and activation of MAPKs are involved in apoptosis induced by PM2.5 in rat cardiac H9c2 cells.

Epidemiological studies show a positive correlation between the air levels of fine particulate matter (PM2.5) and cardiovascular disorders, but how PM2.5 affects cardiomyocytes has not been studied in great deal. The aim of the present study was to obtain an insight into the links among intracellular levels of reactive oxygen species (ROS), apoptosis and mitogen-activated protein kinases (MAPKs) in rat cardiac H9c2 cells exposed to PM2.5. H9c2 cells were incubated with PM2.5 at 100-800 µg ml(-1) to evaluate the effects of PM2.5 on cell viability, cell apoptosis, intracellular levels of ROS and expression of apoptosis-related proteins as well as activation of MAPKs. PM2.5 decreased cell viability, increased the cell apoptosis rate and intracellular ROS production in a concentration-dependent manner. PM2.5 decreased the Bcl-2/Bax ratio and increased cleaved caspase-3 levels. A Western blots study showed up-regulation of phosphorylated MAPKs including extracellular signal-regulated protein kinases (ERKs), c-Jun NH2-terminal kinases (JNKs) and p38 MAPK in the PM2.5-treated cells. The p38 MAPK inhibitor SB239063 attenuated whereas the ERKs inhibitor PD98059 augmented the effects of PM2.5 on apoptosis and the expression of related proteins. In conclusion, PM2.5 decreases cell viability and increases apoptosis by enhancing intracellular ROS production and activating the MAPKs signaling pathway in H9c2 cells. The MAPKs signaling pathway could be a new promising target for clinical therapeutic strategies against PM2.5-induced cardiac injury.

Down-regulation of c-fos by shRNA sensitizes adriamycin-resistant MCF-7/ADR cells to chemotherapeutic agents via P-glycoprotein inhibition and apoptosis augmentation.

Multidrug resistance (MDR) is a major hurdle in the treatment of cancer. Research indicated that the main mechanisms of most cancers included so-called "pump" (P-glycoprotein, P-gp) and "non-pump" (apoptosis) resistance. Identification of novel signaling molecules associated with both P-gp and apoptosis will facilitate the development of more effective strategies to overcome MDR in tumor cells. Since the proto-oncogene c-fos has been implicated in cell adaptation to environmental changes, we analyzed its role in mediating "pump" and "non-pump" resistance in MCF-7/ADR, an adriamycin (ADR)-selected human breast cancer cell line with the MDR phenotype. Elevated expression of c-fos in MCF-7/ADR cells and induction of c-fos by ADR in the parental drug-sensitive MCF-7 cells suggested a link between c-fos and MDR phenotype. Down-regulation of c-fos expression via shRNA resulted in sensitization of MCF-7/ADR cells to chemotherapeutic agents, including both P-gp and non-P-gp substrates. Further results proved that c-fos down-regulation in MCF-7/ADR cells resulted in decreased P-gp expression and activity, enhanced apoptosis, and altered expression of apoptosis-associated proteins (i.e., Bax, Bcl-2, p53, and PUMA). All above facts indicate that c-fos is involved in both P-gp- and anti-apoptosis-mediated MDR of MCF-7/ADR cells. Based on these results, we propose that c-fos may represent a potential molecular target for resistant cancer therapy, and suppressing c-fos gene expression may therefore be an effective means to temper breast cancer cell's MDR to cytotoxic chemotherapy.

[The cytotoxicity of indirubin derivative PHII-7 against human breast cancer MCF-7 cells and its mechanisms].

OBJECTIVE: To observe the cytotoxicity of indirubin derivative PHII-7 against human breast cancer MCF-7 cells and to study its primary mechanisms. METHODS: The proliferation of MCF-7 cells was detected using MTT colorimetry. Annexin V/PI double staining was applied to detect the apoptosis rate of MCF-7 cells. The distribution of cell cycles was detected using PI staining and flow cytometry (FCM). The levels of reactive oxygen species (ROS) in MCF-7 cells were detected by DCFH-DA staining. The mRNA and protein levels of c-fos were detected using RT-PCR and Westem blot analysis. RESULTS: PHII-7 at different concentrations inhibited the proliferation of MCF-7 cells in a concentration-dependent manner, with the inhibitory rate ranging from 43.13% to 90.90% (P < 0.05). The inhibition was strengthened along with increased concentrations. PHII-7 at different concentrations could induce the apoptosis of MCF-7 cells. The early apoptosis rate was 1.43% +/- 0.02%, 9.14% +/- 0.36%, and 45.79% +/- 8.46%, respectively with the action of 1.25, 2.50, and 5.00 micromol/L PHII-7, respectively, showing dose-dependent manner. FCM analysis found that the proportion of MCF-7 cells in the G0/G1 phase and the S phase decreased after treatment with PHII-7, and the ratio of MCF-7 cells in the G2/M phase obviously increased (P < 0.01). The intra-cellular ROS level was significantly elevated 2 h after pretreatment with PHII-7. The levels of the protooncogene c-fos mRNA and protein were down-regulated in a dose-dependent manner after action of PHII-7. CONCLUSIONS: PHII-7 exerted obvious in vitro cytotoxic effects on MCF-7 cells. Its mechanisms might be associated with arresting the cell cycle, regulating the redox equilibrium, and down-regulating the expression of the protooncogene.

Downregulation of cytokeratin 18 induces cellular partial EMT and stemness through increasing EpCAM expression in breast cancer.

Induction of epithelial-mesenchymal transition (EMT) and cancer stem cell (CSC) characteristics underlie the development of metastasis, chemoresistance, and tumor recurrence in breast cancer. Downregulation of cytokeratin 18 (CK18) is a critical molecular event of EMT; however, its importance in the induction of EMT and CSC features has not been defined to date. This study aimed to investigate the biological significance and underlying molecular mechanisms of CK18 in inducing EMT phenotype and stemness properties of breast cancer cells. Three breast cancer cell lines (i.e., non-metastatic MCF-7, highly metastatic MDA-MB-231, and mitoxantrone (MX)-selected resistant MCF-7/MX cells) and two CK18-knockdown stable cell clones (MCF-7-shCK18-7D and 3C) were used to determine the association between CK18 and EMT and stemness. CK18 expression was extremely low in highly metastatic, resistant, and transforming growth factor (TGF)-ß1/tumor necrosis factor (TNF)-α-treated breast cancer cells with mesenchymal phenotype and increased expression of CSC markers. Depletion of CK18 promoted partial EMT and the acquisition of stemness properties in breast cancer MCF-7 cells. Mechanistically, CK18 interference in MCF-7 cells activated the Wnt/ß-catenin signaling, resulting in the up-regulation of epithelial cell adhesion molecule (EpCAM). Consistently, the stemness properties and metastasis can be attenuated by further knockdown of EpCAM in CK18-depleted cells. In conclusion, downregulation of CK18 promotes partial EMT and enhances breast cancer stemness by increasing EpCAM expression partly via the Wnt/ß-catenin pathway. These findings indicate that CK18 may serve as a potential treatment target for advanced breast cancer.

Downregulation of cytokeratin 18 enhances BCRP-mediated multidrug resistance through induction of epithelial-mesenchymal transition and predicts poor prognosis in breast cancer.

Multiple drug resistance (MDR) and metastasis have been identified as the two major causes of the poor prognosis of patients with breast cancer. However, the relationship between MDR and metastasis has not been characterized. Epithelial­mesenchymal transition (EMT), a process known to promote metastasis in cancer, has been shown to be associated with the MDR phenotype of many tumor types. Reduced cytokeratin 18 (CK18) expression is thought to be one of the hallmarks of EMT, and the role of CK18 in MDR of metastatic breast cancer remains unknown. In the present study, we revealed that the expression of CK18 was significantly downregulated in breast cancer tissues and in an MDR cell line overexpressing breast cancer resistant protein (BCRP), and the presence of low levels of CK18 was associated with TNM stage, lymph node metastasis, and unfavorable survival in breast cancer patients. Further results demonstrated that CK18 stable knockdown using shRNA increased BCRP expression and induced the EMT process in human breast cancer MCF­7 cells. Moreover, CK18 knockdown was associated with the activation of the NF­κB/Snail signaling pathway, which has been revealed to regulate EMT and BCRP. Based on these findings, we concluded that CK18 knockdown enhanced BCRP­mediated MDR in MCF­7 cells through EMT induction partly via the NF­κB/Snail pathway. These findings provide a valuable insight into the potential role of CK18 in MDR, migration and invasion of breast cancer cells. Reduced expression of CK18 may be a novel biomarker for predicting the poor prognosis of breast cancer patients.

Nephroprotective effects of nebivolol in 2K1C rats through regulation of the kidney ROS-ADMA-NO pathway.

BACKGROUND: To evaluate the protective effect of nebivolol against kidney damage and elucidate the underlying mechanism in a two-kidney, one-clip (2K1C) rat model. METHODS: 2K1C rats were obtained by clipping left renal artery of male Wistar rats and were considered hypertensive when systolic blood pressure (SBP) was ≥160mmHg 4 weeks after surgery. The 2K1C hypertensive rats were divided into untreated, nebivolol (10mg/kg, ig), and atenolol (80mg/kg, ig) treatment groups. The treatments lasted for 8 weeks. SBP, kidney structure and function, plasma and kidney angiotensin (Ang) II, nitric oxide (NO), asymmetric dimethylarginine (ADMA), and the oxidant status were examined. Kidney protein expression of NADPH oxidase (Nox) isoforms and its subunit p22phox, nitric oxide synthase (NOS) isoforms, protein arginine N-methyltransferase (PRMT) 1, and dimethylarginine dimethylaminohydrolase (DDAH) 1 and 2 was tested by western blotting. RESULTS: Nebivolol and atenolol exerted similar hypotensive effects. However, atenolol had little effect while nebivolol significantly ameliorated the functional decline and structural damage in the kidney, especially in non-clipped kidney (NCK), which was associated with the reduction of Ang II in NCK. Moreover, nebivolol inhibited the NCK production of reactive oxygen species (ROS) by decreasing Nox2, Nox4, and p22phox expression. Further, nebivolol reduced the plasma and kidney ADMA levels by increasing DDAH2 expression and decreasing PRMT1 expression. Nebivolol also increased the NCK NO level by ameliorating the expression of kidney NOS isoforms. CONCLUSIONS: Our results demonstrated that long-term treatment with nebivolol had renoprotective effect in 2K1C rats partly via regulation of kidney ROS-ADMA-NO pathway.

Baicalein attenuates monocrotaline-induced pulmonary arterial hypertension by inhibiting endothelial-to-mesenchymal transition.

AIMS: Endothelial-to-mesenchymal transition (EndoMT) was shown to lead to endothelial cell (EC) dysfunction in pulmonary arterial hypertension (PAH). Baicalein was reported to inhibit epithelial-to-mesenchymal transition (EMT), a biological process that has many regulatory pathways in common with EndoMT. Whether it can attenuate PAH by inhibiting EndoMT remains obscure. MAIN METHODS: PAH was induced by a single subcutaneous injection of MCT (60 mg/kg) in male Sprague Dawley rats. Two weeks after MCT administration, the rats in the treatment groups received baicalein orally (50 or 100 mg/kg/day) for an additional 2 weeks. Hemodynamic changes and right ventricular hypertrophy (RVH) were evaluated on day 28. Cardiopulmonary interstitial fibrosis was detected using Masson's trichrome, Picrosirius-red, and immunohistochemical staining. The reactivity of pulmonary arteries (PAs) was examined ex vivo. The protein expresson of EndoMT molecules, bone morphogenetic protein receptor 2 (BMPR2), and nuclear factor-κB (NF-κB) was examined to explore the mechanism of protective action of baicalein. KEY FINDINGS: Baicalein (50 and 100 mg/kg) significantly alleviated MCT-induced PAH and cardiopulmonary interstitial fibrosis. Furthermore, baicalein treatment enhanced PA responsiveness to acetylcholine (ACh) in PAH rats. The upregulation of EndoMT molecules (N-cadherin, vimentin, Snail, and Slug) strongly suggest that EndoMT participates in MCT-induced PAH, which was reversed by baicalein (50 and 100 mg/kg) treatment. Moreover, baicalein partially reversed MCT-induced reductions in BMPR2 and NF-κB activation in the PAs. SIGNIFICANCE: Baicalein attenuated MCT-induced PAH in rats by inhibiting EndoMT partially via the NF-κB-BMPR2 pathway. Thus, baicalein might be considered as a promising treatment option for PAH.

[Effects of PKCα/c-fos, Bax/Bcl-2 expression on the proliferation and apoptosis of rat pulmonary arterial smooth muscle cells in hypoxia].

OBJECTIVE: To explore the effects of PKCα/c-fos, Bax/Bcl-2 on the proliferation and apoptosis of rat pulmonary arterial smooth musclecells(PASMCs) in hypoxia. METHODS: The PASMCs of rats had been isolated and cultured, and then were cultured under normoxia (5%CO2、21%O2、74%N2)and hypoxia(5%CO2,2% O2,93%N2)condition for 24 h and 48 h, respectively. The proliferation of PASMCs was tested by methylthiazolyltetrazolium (MTT). The changes of PASMCs apoptosis were detected by Annexin V-FITC/PI staining combined flow cytometry. RT-PCR and Western blot analysis were performed to detect the gene and protein levels of PKCα/c-fos, Bax/Bcl-2, respectively. All experiments were repeated three times with at least triplicate samples. RESULTS: The proliferation of PASMCs in hypoxia group were stronger than that of normoxia group after 24 h and 48 h (P < 0.01), while the apoptosis rate did not change significantly. Meanwhile, the higher levels of PKCα, c-fos, Bcl-2 mRNA and proteins after hypoxia for 24 h and 48 h were detected by RT-PCR and Western blot analysis, while the ex-pression levels of Bax had no significant difference under normoxic and hypoxic conditions. CONCLUSIONS: Elevated proliferation and expression of PKCα, c-fos, Bcl-2 was observed in rat PASMCs in hypoxia, and while the apoptosis rate had no significant change.

Limb ischemic preconditioning protects endothelium from oxidative stress by enhancing nrf2 translocation and upregulating expression of antioxidases.

Remote ischemic preconditioning is often performed by limb ischemic preconditioning (LIPC), which has been demonstrated to be beneficial to various cells, including endothelial cells. The mechanisms underlying the protection have not been well clarified. The present study was designed to observe the effects of sera derived from rats after LIPC on human umbilical vein endothelial cells (HUVECs) injured by hydrogen peroxide (H2O2) -induced oxidative stress and explore the involvement of redox state in the protection. Incubation with 1 mM H2O2 for 2 h induced a significant reduction in HUVECs' viability with increased production of malondialdehyde (MDA) and reactive oxygen species (ROS). Preincubation with early preconditioning serum (EPS) or delayed preconditioning serum (DPS) derived from rats subjected to LIPC alleviated these changes. Both EPS and DPS increased the nuclear translocation of transcription factor nuclear factor E2-related factor 2 (Nrf2) and the expression of antioxidases. The protective effects of EPS and DPS were blocked neither by MEK/ERK inhibitors U0126 nor by PI3K/Akt inhibitors LY294002. In conclusion, the present study provides the evidence that LIPC protects the HUVECs from H2O2-induced injury by, at least partially, enhancement of Nrf2 translocation and upregulation of antioxidases via signaling pathways independent of MEK/ERK and PI3K/Akt.

Ginsenoside Rg1 reduces toxicity of PM(2.5) on human umbilical vein endothelial cells by upregulating intracellular antioxidative state.

Ambient airborne particulate matter (PM) is an important environmental pollutant responsible for many human diseases. Oxidative stress is suggested to be involved in PM-induced cell injury. The present study is designed to study unsalutary effects of the organic extracts of PM with an aerodynamic diameter of less than 2.5µm (PM(2.5)) and protective effect of Ginsenoside Rg1 (Rg1) against PM(2.5) on human umbilical vein endothelial cells (HUVECs) in vitro. Cytotoxic effects of the organic extract PM(2.5) on HUVECs were measured by means of HUVEC cell viability and the generation of intracellular reactive oxygen species (ROS). Expression of heme oxygenase-1(HO-1) and Nuclear factor-erythroid 2-related factor 2 (Nrf2) and Nrf2 cytoplasm-nucleus location were assayed. The present results showed that PM(2.5) (50-800µg/ml) decreased HUVEC viability and increased intracellular generation of ROS and malondialdehyde (MDA) in a concentration dependent manner, but increased HO-1 expression without concentration dependence. Rg1 (10 and 40µg/ml) diminished PM(2.5)-induced HUVEC viability, decrease ROS and MDA generation, increased HO-1 and Nrf2 expression and promoted Nrf2 translocation to nucleus in a concentration dependent manner. These results suggested that organic extracts of PM(2.5) increase oxidative stress and decrease cell viability; Rg1 antagonize PM(2.5)-induced excess oxidative stress; HO-1 expression increase and Nrf2 translocation to nucleus may be involved in the effects of both PM(2.5) and Rg1 on HUVECs.

Cytosine arabinoside promotes cytotoxic effect of T cells on leukemia cells mediated by bispecific antibody.

Chemotherapeutic drugs can enhance an immune response of the host against the tumor in addition to killing cancer cells by direct cytotoxicity. Therefore, the combination of chemotherapy and immunotherapy is a promising approach for eliminating tumors, particularly in advanced stages. A strategic medication is to use a bispecific antibody format that is capable of recruiting polyclonal T cells around antibody-target-expressing tumor cells. Recently, we have constructed a bispecific antibody, anti-CD3×anti-CD19, in a diabody configuration. In this study, we measured B7 family members B7.1 (CD80) and B7.2 (CD86) expressed on a CD19(+) human leukemia cell line, Nalm-6, stimulated by cytosine arabinoside (Ara-C). We found that a low concentration of Ara-C could upregulate CD80 expressed on CD19(+) Nalm-6 cells. The cytotoxicity of T lymphocytes against Nalm-6 cells in vitro and in vivo mediated by the anti-CD3×anti-CD19 diabody with or without a low dose of Ara-C was compared. The combination of the anti-CD3×anti-CD19 diabody and Ara-C showed the greatest effectiveness in enhancing the cytotoxicity of T cells against the tumor cells in vitro and in vivo. Activated T cells expressed higher levels of CD25 and CD69 and released more interleukin 2. Both perforin/granzyme B system and Fas/FasL pathway were involved in the diabody-induced T-cell cytotoxicity. Moreover, the activated T cells could upregulate ICAM-3 expression on Nalm-6 cells, and inhibition of LFA-1-ICAM-3 interaction impaired cytotoxicity of T cells. It was noted that Ara-C could upregulate CD80 expressed on two of five specimens of acute B lymphoblastic leukemia patient-derived cells. Cytotoxicity of T cells against these two patient-derived cells was enhanced in the presence of the anti-CD3×anti-CD19 diabody. These findings indicate that treatment strategy using both cytotoxic lymphocyte-based immunotherapy and chemotherapy may have synergistic effects.

PHII-7 inhibits cell growth and induces apoptosis in leukemia cell line K562 as well as its MDR- counterpart K562/A02 through producing reactive oxygen species.

Multidrug resistance (MDR) is a major obstacle that hinders the efficacy of chemotherapy in many human malignancies. PHII-7 is a derivative of indirubin, which was designed and synthesized by our laboratory. Our preliminary work indicated its potent antitumor activities in vitro and in vivo. Furthermore, based on the model of MDR cell line, we found its powerful effects in inhibiting the expression of P-glycoprotein (P-gp) and killing multidrug-resistant (MDR) cells with the detailed mechanism remained to be explored. Reactive oxygen species are known for high reactive activity as they possess unmatched electrons. In this study, we showed that PHII-7 generated equal reactive oxygen species in parental K562 and its counterpart MDR K562/A02 cells. Pre-incubation with thiol antioxidants glutathione or N-acetyl-cysteine(NAC) almost abolished the cytotoxicity of PHII-7. Moreover, NAC abrogated DNA damage, cell cycle arrests and apoptosis induced by PHII-7. Our results collectively indicated that reactive oxygen species production induced by PHII-7 contributed to both apoptosis and cell cycle arrets in MDR K562/A02 cells, thus extending our prior related findings. Notably, JNK phosphorylation was also induced by PHII-7 and pre-incubated of K562/A02 cells with NAC or inhibitor of JNK(SP006125) eliminated P-gp downregulation. Taken together, our results may provide a detailed biochemical basis for further clinical application of PHII-7.

A novel indirubin derivative PHII-7 potentiates adriamycin cytotoxicity via inhibiting P-glycoprotein expression in human breast cancer MCF-7/ADR cells.

Multidrug resistance (MDR) is a major impediment to the effective chemotherapy of many human malignancies, and novel MDR reversal agents are desirable for combination therapy to reduce MDR, enhance anti-tumor activity and reduce side effects. Overexpression of P-glycoprotein (P-gp) is the most prevalent cause of MDR in cancer tissues, and resistance to apoptosis is a common characteristic for the multidrug resistant cancer cells. Our group has synthesized a novel potent anti-tumor indirubin derivative, PHII-7. In this study, MCF-7/ADR cells, an adriamycin (ADR)-selected human breast tumor cell line with the MDR phenotype, were used to investigate the anticancer properties of this novel indirubin derivative. Cytotoxicity and apoptosis assays showed that PHII-7 significantly inhibited cell growth, induced apoptosis, potentiated ADR cytotoxicity and restored chemotherapy sensitivity in the MDR cancer cells. Further studies indicated that by down-regulation of P-gp expression, PHII-7 partially inhibited P-gp efflux pump function and increased intracellular accumulation of Rhodamine 123, a P-gp substrate. These results provide a biochemical basis for possible clinical application of PHII-7 alone or in combination with conventional antineoplastic agents in the treatment MDR tumors.

Efficient inhibition of human B-cell lymphoma in SCID mice by synergistic antitumor effect of human 4-1BB ligand/anti-CD20 fusion proteins and anti-CD3/anti-CD20 diabodies.

Here we constructed and produced a recombinant human 4-1BB ligand (4-1BBL)/anti-CD20 fusion protein and examined its antitumor activity, alone and in combination with an anti-CD3/anti-CD20 bispecific diabody. The 4-1BBL/anti-CD20 fusion protein retained both the costimulatory activity of 4-1BBL on T cells and the tumor targeting ability of CD20 antibody on B cells. The fusion protein bound as efficiently to 4-1BB- and CD20-positive cells as its respective parental antibodies, and was capable of cross-linking human T lymphocytes and CD20-positive tumor cells. Combination treatment with 4-1BBL/anti-CD20 fusion protein and anti-CD3/anti-CD20 diabody led to significantly increased T-cell cytotoxicity to human B-lymphoma cells in vitro and drastically more potent tumor inhibitory activity in vivo in xenografted B-cell lymphoma in severe combined immunodeficiency disease mice. Mechanistic studies revealed that the combination treatment remarkably inhibited apoptosis of human peripheral blood lymphocytes, accompanied by upregulation of Bcl-XL and Bf1-1, perforin and granzyme B mRNA, and increased interleukin-2 production. Taken together, these results suggest that targeted delivery of 4-1BBL to the tumor site, when combined with anti-CD3/anti-CD20 diabody, could strongly potentiate the antitumor activity of the diabody, thus may have significant clinical application in the treatment of human CD20-positive B-cell malignancies.