Differential effect of ascorbic acid and n-acetyl-L-cysteine on arsenic trioxide-mediated oxidative stress in human leukemia (HL-60) cells

Arsenic trioxide (ATO) has been recommended for the treatment of refractory cases of acute promyelocytic leukemia (APL). Recent studies in our laboratory indicated that oxidative stress plays a key role in ATO-induced cytotoxicity in human leukemia (HL-60) cells. In the present investigation, we performed the MTT assay and trypan blue exclusion test for cell viability. We also performed the thiobarbituric acid test to determine the levels of malondialdehyde (MDA) production in HL-60 cells coexposed to either ascorbic acid (AA) and ATO or to n-acetyl-L-cysteine (NAC) and ATO. The results of MTT assay indicated that AA exposure potentiates the cytotoxicity of ATO in HL-60 cells, as evidenced by a gradual increase in MDA levels with increasing doses of AA. In contrary, the addition of NAC to ATO-treated HL-60 cells resulted in a dose-dependent decrease of MDA production. From these results, we conclude that the addition of the AA to ATO-treated HL-60 cells enhances the formation of reactive oxygen species (ROS), whereas the addition of NAC under the same experimental condition significantly (p < .05) decreases the level of ROS formation. On the basis of these direct in vitro findings, our studies provide evidence that AA may extend the therapeutic spectrum of ATO. The coadministration of NAC with ATO shows a potential specificity for tumor cells, indicating that it may not enhance the clinical outcome associated with ATO monotherapy in vivo.     

Investigation of Gallic Acid Induced Anticancer Effect in Human Breast Carcinoma MCF‐7 Cells

Gallic acid (GA), a polyhydroxylphenolic compound abundantly distributed in plants, fruits, and foods, has been reported to have various biological activities including an anticancer effect. In this study, we extensively investigated the anticancer effect of GA in human breast carcinoma MCF‐7 cells. Our study indicated that treatment with GA resulted in inhibition of proliferation and induction of apoptosis in MCF‐7 cells. Then, the molecular mechanism of GA's apoptotic action in MCF‐7 cells was further investigated. The results revealed that GA induced apoptosis by triggering the extrinsic or Fas/FasL pathway as well as the intrinsic or mitochondrial pathway. Furthermore, the apoptotic signaling induced by GA was amplified by cross‐link between the two pathways. Taken together, our findings may be useful for understanding the mechanism of action of GA on breast cancer cells and provide new insights into the possible application of such compound and its derivatives in breast cancer therapy.     

Ca2+‐Inactivated K+ Current is Modulated by Endothelin‐1 in B‐16 Murine Melanoma Cells

It is well established that endothelin‐1 (ET‐1) plays a role in differentiation and proliferation in a variety of cells such as fibroblasts and human melanoma cells via a receptor‐mediated mechanism. However, whether ET‐1 modulates ion channel activity in these cell types is still unknown. In this report, we recorded the voltage‐dependent outward K⁺ current in cultured B16 melanoma cells using the patch‐clamp technique. Biophysical and pharmacological properties of the K⁺ current, and the effect of ET‐1 on the K⁺ current were investigated. When cells were loaded with a Ca²⁺‐chelating agent (EGTA or BAPTA), the K⁺ current amplitude gradually increased with time after establishment of the whole cell configuration. Replacement of Ca²⁺ with Co²⁺ in the extracellular medium caused no significant modulation of the K⁺ current amplitude. Addition of BaCl₂ or quinidine to the extracellular solution reduced the K⁺ current amplitude, whereas the K⁺ current was insensitive to tetraethylammonium. ET‐1 (10 nM) reversibly decreased the K⁺ current amplitude and accelerated the decay of the K⁺ current. The ET‐1‐induced inhibitory effect displayed no desensitization following repeated ET‐1 application. Pretreatment with pertussis toxin (PTX) or perfusion of cells with the protein kinase C (PKC) inhibitor H‐7 abolished the inhibitory effect of ET‐1 on the K⁺ current. We conclude that the outward K⁺ current recorded in murine B‐16 melanoma cells represents a Ca²⁺‐inactivated K⁺ current, and that the inhibitory effect of ET‐1 on the K⁺ current may reveal a novel mechanism to control the differentiation and proliferation of melanoma cells.     

2‐Deoxy‐d‐Glucose Sensitizes Human Ovarian Cancer Cells to Cisplatin by Increasing ER Stress and Decreasing ATP Stores in Acidic Vesicles

Cisplatin is a commonly used chemotherapeutic agent; however, the development of acquired resistance limits its application. Here, we demonstrate that 2‐deoxy‐d ‐glucose (2‐DG) enhanced the antitumor effects of cisplatin in SKOV3 cells, which include inhibition of proliferation and promotion of apoptosis. Additionally, either cisplatin or 2‐DG alone could upregulate the endoplasmic reticulum (ER) stress‐associated protein glucose‐regulated protein‐78 (GRP78). Moreover, exposure to 2‐DG increased the expression of GRP78 induced by cisplatin. Cisplatin also upregulated ER stress‐associated apoptotic protein 153/C/EBP homology protein (CHOP) in SKOV3 cells. While treatment with 2‐DG alone could not upregulate the CHOP expression, a combination of both 2‐DG and cisplatin increased the protein levels of CHOP above those induced by Cisplatin alone. Finally, cisplatin mediated an increase in ATP stores within acidic vesicles, whereas 2‐DG decreased this effect. These data demonstrate that 2‐DG sensitizes SKOV3 cells to cisplatin by increasing ER stress and decreasing ATP stores in acidic vesicles.     

Interaction of COP1 and UVR8 regulates UV‐B‐induced photomorphogenesis and stress acclimation in Arabidopsis

The ultraviolet‐B (UV‐B) portion of the solar radiation functions as an environmental signal for which plants have evolved specific and sensitive UV‐B perception systems. The UV‐B‐specific UV RESPONSE LOCUS 8 (UVR8) and the multifunctional E3 ubiquitin ligase CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1) are key regulators of the UV‐B response. We show here that uvr8‐null mutants are deficient in UV‐B‐induced photomorphogenesis and hypersensitive to UV‐B stress, whereas overexpression of UVR8 results in enhanced UV‐B photomorphogenesis, acclimation and tolerance to UV‐B stress. By using sun simulators, we provide evidence at the physiological level that UV‐B acclimation mediated by the UV‐B‐specific photoregulatory pathway is indeed required for survival in sunlight. At the molecular level, we demonstrate that the wild type but not the mutant UVR8 and COP1 proteins directly interact in a UV‐B‐dependent, rapid manner in planta. These data collectively suggest that UV‐B‐specific interaction of COP1 and UVR8 in the nucleus is a very early step in signalling and responsible for the plant's coordinated response to UV‐B ensuring UV‐B acclimation and protection in the natural environment.     

Epac1‐induced cellular proliferation in prostate cancer cells is mediated by B‐Raf/ERK and mTOR signaling cascades

cAMP‐dependent, PKA‐independent effects on cell proliferation are mediated by cAMP binding to EPAC and activation of Rap signaling. In this report, we employed the analogue 8‐CPT‐2‐O‐Me‐cAMP to study binding to EPAC and subsequent activation of B‐Raf/ERK and mTOR signaling in human cancer cells. This compound significantly stimulated DNA synthesis, protein synthesis, and cellular proliferation of human 1‐LN prostate cancer cells. By study of phosphorylation‐dependent activation, we demonstrate that EPAC‐mediated cellular effects require activation of the B‐Raf/ERK and mTOR signaling cascades. RNAi directed against EPAC gene expression as well as inhibitors of ERK, PI 3‐kinase, and mTOR were employed to further demonstrate the role of these pathways in regulating prostate cancer cell proliferation. These studies were then extended to several other human prostate cancer cell lines and melanoma cells with comparable results. We conclude that B‐Raf/ERK and mTOR signaling play an essential role in cAMP‐dependent, but PKA‐independent, proliferation of cancer cells. J. Cell. Biochem. 108: 998–1011, 2009. © 2009 Wiley‐Liss, Inc.     

Inhibition of Hypoxia Inducible Factor 1α by siRNA‐Induced Apoptosis in Human Retinoblastoma Cells

Hypoxia, which activates the hypoxia inducible factor 1α (HIF‐1α), is an essential feature of retinoblastoma (RB) and contributes to poor prognosis and resistance to conventional therapy. In this study, the effect of HIF‐1α knockdown by small interfering RNA (siRNA) on cell proliferation, apoptosis, and apoptotic pathways of human Y‐79 RB cells was first investigated. Exposure to hypoxia induced the increased expression of HIF‐1α both in mRNA and protein levels. Then, knockdown of HIF‐1α by siRNA_HIF‐1α resulted in inhibition of cell proliferation and induced cell apoptosis in human Y‐79 RB cells under both normoxic and hypoxic conditions, with hypoxic conditions being more sensitive. Furthermore, knockdown of HIF‐1α could enhance hypoxia‐induced slight increase of Bax/Bcl‐2 ratio and activate caspase‐9 and caspase‐3. These results together indicated that suppression of HIF‐1α expression may be a promising strategy for the treatment of human RB in the future.     

Pinitol attenuates LPS‐induced pneumonia in experimental animals: Possible role via inhibition of the TLR‐4 and NF‐κB/IκBα signaling cascade pathway

Pneumonia is a chronic disorder of the respiratory system associated with worsening quality of life and a significant economic burden. Pinitol, a plant cyclic polyol, has been documented for immune‐inflammatory potential. The aim of present investigation was to evaluate the potential and possible mechanism of action of pinitol against lipopolysaccharide (LPS)‐induced pneumonia in the experimental animal model. Pneumonia was induced in Sprague‐Dawley rats by intratracheal administration of LPS (2 mg/kg). Animals were treated with either vehicle or dexamethasone or pinitol (5 or 10 or 20 mg/kg). Potential of pinitol against LPS‐induced pulmonary insult was assessed based on behavioral, biochemical, molecular, and ultrastructural studies. Intratracheal instillation of LPS induced significant (P < .05) inflammatory infiltration in bronchoalveolar lavage fluid (BALF) and lung tissue reflected by elevated pleural effusion volume, lung edema, BALF polymorphonuclear leukocytes count and lung myeloperoxidase levels, which was attenuated by pinitol (10 and 20 mg/kg) administration. Pinitol also markedly (P < .05) inhibited LPS‐induced alterations in electrocardiographic, hemodynamic changes, right ventricular, and lung function tests. The LPS‐induced downregulated nuclear factor erythroid 2–related factor 2 (Nrf‐2) and heme oxygenase‐1 (HO‐1), whereas upregulated transforming growth factor‐β (TGF‐β), tumor necrosis factor‐α (TNF‐α), interleukin‐1β (IL‐1β), IL‐6, NOD‐, LRR‐, and pyrin domain‐containing protein 3 (NLRP3), and inducible nitric oxide synthase (iNOs) lung messenger RNA expressions were significantly (P < .05) inhibited by pinitol. Western blot analysis suggested pinitol markedly (P < .05) decreased nuclear factor‐κB (NF‐κB), inhibitor of nuclear factor κB (IkBα), toll‐like receptor 4 (TLR‐4), and cyclooxygenase‐II (COX‐II) protein expressions in the lung. These findings were further supported by histological and ultrastructural analyses of lung tissue that show pinitol significantly (P < .05) ameliorates LPS‐induced aberrations in lung tissue. In conclusion, pinitol attenuated LPS‐induced pneumonia via inhibition of TLR‐4 to downregulate the NF‐κB/IκBα signaling cascade and thus ameliorated the production of proinflammatory cytokines (TNF‐α, ILs, NLRP3, and TGF‐β), inflammatory mediators (COX‐II and iNOs) and elevated oxidative stress (Nrf‐2 and HO‐1).     

Regulation of polyamine metabolism in Pyropia cinnamomea (W.A. Nelson), an important mechanism for reducing UV‐B‐induced oxidative damage

It is generally accepted that ultraviolet (UV) radiation can have adverse affects on phototrophic organisms, independent of ozone depletion. The red intertidal seaweed Pyropia cinnamomea W.A. Nelson (previously Porphyra cinnamomea Sutherland et al. 2011), similar to many other intertidal macrophytes, is exposed to high levels of UV radiation on a daily basis due to emersion in the upper littoral zone. It has been shown that seaweeds, like higher plants, respond to an increased activity of antioxidative enzymes when exposed to stress. However, earlier investigations have shown that P. cinnamomea also compensates for stress due to UV radiation by increasing polyamine (PA) levels, especially bound‐soluble and bound‐insoluble PAs. The PA precursor putrescine (PUT) can be synthesized via two enzymatic pathways: arginine decarboxylase (ADC) and ornithine decarboxylase (ODC). Both of these enzymes showed increased activity in P. cinnamomea under UV stress. In higher plants, ADC is the enzyme responsible for increased PA levels during stress exposure, while ODC is correlated with cell division and reproduction. However, there are contrary findings in the literature. Using two irreversible inhibitors, we identified the enzyme most likely responsible for increased PUT synthesis and therefore increased stress tolerance in P. cinnamomea. Our results show that changes in the PA synthesis pathway in P. cinnamomea under UV stress are based on an increased activity of ADC. When either inhibitor was added, lipid hydroperoxide levels increased even under photosynthetically active radiation, suggesting that PAs are involved in protection mechanisms under normal light conditions as well. We also show that under optimum or low‐stress conditions, ODC activity is correlated with PUT synthesis.