Oxidative Stress In Cancer Cells Research Articles

Abstract 1739: A novel HSP90 inhibitor induces metabolic oxidative stress in head and neck squamous cancers and prevents migration and stem cell EMT even under hypoxic conditions

Abstract INTRODUCTION: Advanced head and neck squamous cell carcinoma (HNSCC) remains a challenge to treat. In HNSCC, hypoxia is a negative prognostic marker with altered metabolism, increased invasiveness, and resistance to therapy. Natural withanolides exhibit potent, highly selective anticancer effects in HNSCCs in vitro and in vivo through a novel anticancer mechanism. These drugs [withaferin A and the novel compound, withalongolide A-triacetate(WGA-TA)] selectively induce metabolic oxidative stress in cancer cells while inhibiting HSP90 chaperone function via disruption of CDC37 (co-chaperone) binding. We hypothesize that WGA-TA is a potent, novel therapeutic drug for HNSCC that will decrease tumor migration and cancer stem cell endothelial-mesenchymal transition (EMT) even in the setting of hypoxia. METHODS: Human HNSCC cell lines MDA-1986, UMSCC11A, 11B and 12 were grown either under normoxic or hypoxic conditions. CM-H2DCFDA level defined accumulation of intracellular reactive oxygen species. Effects on proliferation and apoptosis were evaluated by flow cytometry (FC), Western blotting (WB), and RT PCR. Viability was evaluated by MTS and clonigenic assay. Scratch wound-healing and Boyden chamber assays were used to follow cell migration. RESULTS: WGA-TA treatment of both hypoxic and normoxic cells showed dose dependent induction of oxidation (p<0.01 vs. controls). This effect was completely blocked by N-acetyl cysteine(NAC) under both conditions (p<0.001). Dose dependent changes in biomarkers of oxidative stress (200-400% increase in HSP32 and HSP70; 80-90% decrease in HSF-1) with functional inhibition of Akt, mTOR, the EMT marker vimentin (80-90%), and cancer stem cell marker (Bmi-1, CD44 & ALDH each 50-90% inhibition (p<0.01 vs. control) and modulation of glycolytic proteins (20-80%; p<0.01) was observed under both conditions after WGA-TA treatment. Oxidative stress effect was further enhanced by hypoxia or pretreatment with L-Buthione-[S,R]-sulfoxime (BSO), whereas NAC treatment completely abrogated this effect even at 500nM WGA-TA. Treatment of hypoxic cells with WGA-TA did not alter the IC50 values (50-100 nM) and NAC pretreatment did not inhibit proliferation, whereas BSO decreased IC50 values by 10-40% indicating the role of ROS in reducing cell proliferation and viability. Dose-dependent induction of apoptosis (starting at 500nM WGA-TA) on FC was confirmed by caspases 3 and PARP cleavage on WB and accelerated by BSO but blocked by NAC treatment. Finally 500-1000 nM WGA-TA concentrations prevented HNSCC cell migration by 80-90% (p<0.01 vs. control). CONCLUSION: WGA-TA potently induces glycolytic oxidative stress in HNSCC leading to decrease in CSC EMT as well as,induction of apoptosis and loss of tumor cell migration even in hypoxic conditions. These exciting findings warrant further evaluation in translational in vivo models. Citation Format: Chitra Subramanian, Qing Zhu, Eileen Brandes, Peter T. White, Barbara N. Timmermann, Mark S. Cohen. A novel HSP90 inhibitor induces metabolic oxidative stress in head and neck squamous cancers and prevents migration and stem cell EMT even under hypoxic conditions. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1739. doi:10.1158/1538-7445.AM2015-1739

Abstract 2912: Nitric oxide mediates metabolic coupling of omentum-derived adipose to ovarian and endometrial cancer cells

Abstract Omental adipose stromal cells (O-ASCs) are multipotent population of mesenchymal stem cells contained in the omentum tissue which promote endometrial and ovarian tumor proliferation, migration and drug resistance. The mechanistic underpinnings of O-ASCs role in tumor progression and growth are unclear. Here, we report a novel nitric oxide (NO) mediated metabolic coupling between O-ASCs and gynecological cancer cells in which O-ASCs support NO homeostasis in malignant cells. NO is synthesized endogenously by the conversion of L-arginine into citrulline through nitric oxide synthase (NOS). Ovarian cancers have high affinity for NO which has solubility in lipids. This explains the proximity of many carcinomas to fatty tissue, and thus the high positive correlation between obesity and cancer. Through arginine depletion in the media using L-arginase and NOS inhibition in cancer cells using L-NAME, we demonstrate that patient derived O-ASCs increase NO levels in ovarian and endometrial cancer cells and promote proliferation in these cells. O-ASCs and cancer cell cocultures revealed that cancer cells utilize O-ASCs-secreted arginine and in turn secrete citrulline in the microenvironment. Interestingly, citrulline increased adipogenesis potential of the O-ASCs. Furthermore, we found that O-ASCs increased NO synthesis in cancer cells which led to decrease in mitochondrial respiration in these cells. Our findings suggest that O-ASCs upregulate glycolysis and reduce oxidative stress in cancer cells by increasing NO levels through paracrine metabolite secretion. Significantly, we found that O-ASC mediated chemoresistance in cancer cells can be deregulated by altering NO homeostasis. A combined approach of targeting secreted arginine through L-arginase, along with targeting microenvironment secreted factors induced increased NO synthesis in cancer cells using L-NAME, may be a viable therapeutic approach for targeting ovarian and endometrial cancers. Interestingly, we found that NO modulates s-nitrosation of cellular proteins mediating energy metabolism and cellular differentiation. O-ASCs were found to synthesize higher levels of NO during their differentiation to fat cells which leads to higher protein s-nitrosation. Inhibition of s-nitrosation of cellular proteins showed that NO-mediated protein modifications are strongly associated with O-ASCs adipogenesis. In summary, our data indicate that modulation of intrinsic NO substantially affects tumor growth and drug toxicity in ovarian and endometrial cancer cells. Our study will lead to significant advances in the understanding of the omentum in altering cancer metabolism and lead to novel therapeutics. Citation Format: Bahar Salimian Rizi, Kevin Chen, Inka Cajo Didelija, Juan Marini, Ann H. Klopp, Deepak Nagrath. Nitric oxide mediates metabolic coupling of omentum-derived adipose to ovarian and endometrial cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2912. doi:10.1158/1538-7445.AM2015-2912

Zinc supplementation induces apoptosis and enhances antitumor efficacy of docetaxel in non-small-cell lung cancer.

BackgroundExposure to exogenous zinc results in increased apoptosis, growth inhibition, and altered oxidative stress in cancer cells. Previous studies also suggested that zinc sensitizes some cancer cells to cytotoxic agents depending on the p53 status. Therefore, zinc supplementation may show anticancer efficacy solely and may increase docetaxel-induced cytotoxicity in non-small-cell lung cancer cells.MethodsHere, we report the effects of several concentrations of zinc combined with docetaxel on p53-wild-type (A549) and p53-null (H1299) cells. We evaluated cellular viability, apoptosis, and cell cycle progression as well as oxidative stress parameters, including superoxide dismutase, glutathione peroxidase, and malondialdehyde levels.ResultsZinc reduced the viability of A549 cells and increased the apoptotic response in both cell lines in a dose-dependent manner. Zinc also amplified the docetaxel effects and reduced its inhibitory concentration 50 (IC50) values. The superoxide dismutase levels increased in all treatment groups; however, glutathione peroxidase was slightly increased in the combination treatments. Zinc also caused malondialdehyde elevations at 50 μM and 100 μM.ConclusionZinc has anticancer efficacy against non-small-cell lung cancer cells in the presence of functionally active p53 and enhances docetaxel efficacy in both p53-wild-type and p53-deficient cancer cells.

Antioxidants and Lipid Peroxidation Status In Women with Breast Cancer

Introduction: Breast cancer is one of the most common malignancy in women and is the leading cause of cancer related death worldwide. Reactive oxygen species (ROS) play an important role in the development of tumours. Several mechanisms leading to oxidative stress have been proposed in cancer patients. In this study we aimed to measure and compare the levels of zinc, superoxide dismutase, catalase, vitamin C, vitamin E, and malondialdehyde in breast cancer patients and age matched healthy controls. Methods: In this comparative cross sectional study, 30 confirmed breast cancer cases and 30 age and sex matched controls were enrolled after taking their consent. Blood sample was collected in EDTA vial, and the parameters of oxidative stress were evaluated by spectrophotometric and atomic absorption spectrophotometric methods. Results: Zinc and MDA level were significantly higher (p<0.001) in cases (106.4 µg/dl and 7.12 nmol/ml) compared to controls (89.8µg/ dl and 3.99nmol/ml). In contrast, there were lower catalase, vitamin C and E levels in cases (28.7±14.56, 0.92±0.35 and 0.68±0.13 mg/dl) compared to controls (37.1±14.7,1.62±0.59 and 1.02±0.22 mg/dl) and were statistically significant (p<0.05 and p<0.001). Whereas, superoxide dismutase was lower in cases compared to controls, but was not statistically significant. Conclusion: Zinc was found to be higher in our study as it is a pivotal element in all rapidly growing tissues. Enhanced lipid peroxidation observed in the circulation of breast cancer patients in our study can be attributed to over production of ROS and deficiency of antioxidant defences. Oxidative stress in cancer cells may trigger excessive consumption of antioxidants as a compensatory mechanism which lead to its lower concentration.

Selective induction of oxidative stress in cancer cells via synergistic combinations of agents targeting redox homeostasis

Cancer cell resistance to chemotherapy is still a heavy burden that impairs the response of many cancer patients to conventional chemotherapy. Using drug combinations is one therapeutic approach to overcome the developing resistance to any one drug. Oxidative stress is now a generally regarded hallmark of cancer that can be one approach to selectively target cancer cells while sparing normal cells. With the aim of increasing oxidative stress in cancer cells to a lethal set point, we have generated and combined several series of redox active compounds that act at different points of the cellular oxidative cascade. The premise of such combinations is to deplete of endogenous antioxidant defence proteins (e.g., Glutathione) while concomitantly increasing the generation of ROS via metal redox recycling and Fenton chemistry which eventually leads to the disruption of cellular redox homeostasis and induction of cell death. Through this approach, we have identified highly synergistic combinations of two distinctive classes of compounds (Azines and Copper(II) complexes of 2-pyridyl ketone thiosemicarbazones) which are capable of eliminating cancer cells without concomitant increase in toxicity toward normal cells. In one of our most potent combinations, a combination index (CI) value of 0.056 was observed, representing a 17 fold enhancement in activity beyond additive effects. Such new combination regimen of redox active compounds can be one step closer to potentially safer low dose chemotherapy.

UCP2 inhibition sensitizes breast cancer cells to therapeutic agents by increasing oxidative stress

Modulation of oxidative stress in cancer cells plays an important role in the study of the resistance to anticancer therapies. Uncoupling protein 2 (UCP2) may play a dual role in cancer, acting as a protective mechanism in normal cells, while its overexpression in cancer cells could confer resistance to chemotherapy and a higher survival through downregulation of ROS production. Thus, our aim was to check whether the inhibition of UCP2 expression and function increases oxidative stress and could render breast cancer cells more sensitive to cisplatin (CDDP) or tamoxifen (TAM). For this purpose, we studied clonogenicity, mitochondrial membrane potential (ΔΨm), cell viability, ROS production, apoptosis, and autophagy in MCF-7 and T47D (only the last four determinations) breast cancer cells treated with CDDP or TAM, in combination or without a UCP2 knockdown (siRNA or genipin). Furthermore, survival curves were performed in order to check the impact of UCP2 expression in breast cancer patients. UCP2 inhibition and cytotoxic treatments produced a decrease in cell viability and clonogenicity, in addition to an increase in ΔΨm, ROS production, apoptosis, and autophagy. It is important to note that CDDP decreased UCP2 protein levels, so that the greatest effects produced by the UCP2 inhibition in combination with a cytotoxic treatment, with regard to treatment alone, were observed in TAM+UCP2siRNA-treated cells. Moreover, this UCP2 inhibition caused autophagic cell death, since apoptosis parameters barely increased after UCP2 knockdown. Finally, survival curves revealed that higher UCP2 expression corresponded with a poorer prognosis. In conclusion, UCP2 could be a therapeutic target in breast cancer, especially in those patients treated with tamoxifen.

Reduction of graphene oxide by resveratrol: a novel and simple biological method for the synthesis of an effective anticancer nanotherapeutic molecule.

ObjectiveGraphene represents a monolayer or a few layers of sp2-bonded carbon atoms with a honeycomb lattice structure. Unique physical, chemical, and biological properties of graphene have attracted great interest in various fields including electronics, energy, material industry, and medicine, where it is used for tissue engineering and scaffolding, drug delivery, and as an antibacterial and anticancer agent. However, graphene cytotoxicity for ovarian cancer cells is still not fully investigated. The objective of this study was to synthesize graphene using a natural polyphenol compound resveratrol and to investigate its toxicity for ovarian cancer cells.MethodsThe successful reduction of graphene oxide (GO) to graphene was confirmed by UV-vis and Fourier transform infrared spectroscopy. Dynamic light scattering and scanning electron microscopy were employed to evaluate particle size and surface morphology of GO and resveratrol-reduced GO (RES-rGO). Raman spectroscopy was used to determine the removal of oxygen-containing functional groups from GO surface and to ensure the formation of graphene. We also performed a comprehensive analysis of GO and RES-rGO cytotoxicity by examining the morphology, viability, membrane integrity, activation of caspase-3, apoptosis, and alkaline phosphatase activity of ovarian cancer cells.ResultsThe results also show that resveratrol effectively reduced GO to graphene and the properties of RES-rGO nanosheets were comparable to those of chemically reduced graphene. Biological experiments showed that GO and RES-rGO caused a dose-dependent membrane leakage and oxidative stress in cancer cells, and reduced their viability via apoptosis confirmed by the upregulation of apoptosis executioner caspase-3.ConclusionOur data demonstrate a single, simple green approach for the synthesis of highly water-dispersible functionalized graphene nanosheets, suggesting a possibility of replacing toxic hydrazine by a natural and safe phenolic compound resveratrol, which has similar efficacy in the reduction of GO to rGO. Resveratrol-based GO reduction would facilitate large-scale production of graphene-based materials for the emerging graphene-based technologies and biomedical applications.

Combinations of Ashwagandha leaf extracts protect brain-derived cells against oxidative stress and induce differentiation.

BackgroundAshwagandha, a traditional Indian herb, has been known for its variety of therapeutic activities. We earlier demonstrated anticancer activities in the alcoholic and water extracts of the leaves that were mediated by activation of tumor suppressor functions and oxidative stress in cancer cells. Low doses of these extracts were shown to possess neuroprotective activities in vitro and in vivo assays.Methodology/Principal FindingsWe used cultured glioblastoma and neuroblastoma cells to examine the effect of extracts (alcoholic and water) as well as their bioactive components for neuroprotective activities against oxidative stress. Various biochemical and imaging assays on the marker proteins of glial and neuronal cells were performed along with their survival profiles in control, stressed and recovered conditions. We found that the extracts and one of the purified components, withanone, when used at a low dose, protected the glial and neuronal cells from oxidative as well as glutamate insult, and induced their differentiation per se. Furthermore, the combinations of extracts and active component were highly potent endorsing the therapeutic merit of the combinational approach.ConclusionAshwagandha leaf derived bioactive compounds have neuroprotective potential and may serve as supplement for brain health.

DAPK2 regulates oxidative stress in cancer cells by preserving mitochondrial function.

Death-associated protein kinase (DAPK) 2 is a serine/threonine kinase that belongs to the DAPK family. Although it shows significant structural differences from DAPK1, the founding member of this protein family, DAPK2 is also thought to be a putative tumour suppressor. Like DAPK1, it has been implicated in programmed cell death, the regulation of autophagy and diverse developmental processes. In contrast to DAPK1, however, few mechanistic studies have been carried out on DAPK2 and the majority of these have made use of tagged DAPK2, which almost invariably leads to overexpression of the protein. As a consequence, physiological roles of this kinase are still poorly understood. Using two genetically distinct cancer cell lines as models, we have identified a new role for DAPK2 in the regulation of mitochondrial integrity. RNA interference-mediated depletion of DAPK2 leads to fundamental metabolic changes, including significantly decreased rate of oxidative phosphorylation in combination with overall destabilised mitochondrial membrane potential. This phenotype is further corroborated by an increase in the production of mitochondrial superoxide anions and increased oxidative stress. This then leads to the activation of classical stress-activated kinases such as ERK, JNK and p38, which is observed on DAPK2 genetic ablation. Interestingly, the generation of oxidative stress is further enhanced on overexpression of a kinase-dead DAPK2 mutant indicating that it is the kinase domain of DAPK2 that is important to maintain mitochondrial integrity and, by inference, for cellular metabolism.

The role of oxidative stress in anticancer activity of sesquiterpene lactones.

Sesquiterpene lactones (SLs) are plant-derived compounds that are abundant in plants of the Asteraceae family and posses a broad spectrum of biological activities, ranging from anti-inflammatory, phytotoxic, antibacterial, and antifungal to cytotoxic/anticancer. In recent years, anticancer properties of these compounds and molecular mechanisms of their action have been studied extensively on numerous cell lines and also on experimental animals. SLs have been shown to disrupt cellular redox balance and induce oxidative stress in cancer cells. Oxidative stress is associated with increased production of reactive oxygen species (ROS) which in turn can promote many aspects of cancer development and progression. On the other hand, ROS, which initiate apoptosis via the mitochondrial-dependent pathway, can also be used to kill cancer cells, if they can be generated in cancer. One of the most important regulators of the redox equilibrium in the cells is reduced glutathione (GSH). In cancer cells, GSH levels are higher than in normal cells. Therefore, SL can induce apoptosis of cancer cells by decreasing intracellular GSH levels. The use of SL which can affect intracellular redox signaling pathways can be considered an interesting approach for cancer treatment. In this review, we give a brief description of the mechanisms and pathways involved in oxidative stress-induced anticancer activity of SL.

Nitric oxide mediates metabolic coupling of omentum-derived adipose stroma to ovarian and endometrial cancer cells.

Omental adipose stromal cells (O-ASC) are a multipotent population of mesenchymal stem cells contained in the omentum tissue that promote endometrial and ovarian tumor proliferation, migration, and drug resistance. The mechanistic underpinnings of O-ASCs' role in tumor progression and growth are unclear. Here, we propose a novel nitric oxide (NO)-mediated metabolic coupling between O-ASCs and gynecologic cancer cells in which O-ASCs support NO homeostasis in malignant cells. NO is synthesized endogenously by the conversion of l-arginine into citrulline through nitric oxide synthase (NOS). Through arginine depletion in the media using l-arginase and NOS inhibition in cancer cells using N(G)-nitro-l-arginine methyl ester (l-NAME), we demonstrate that patient-derived O-ASCs increase NO levels in ovarian and endometrial cancer cells and promote proliferation in these cells. O-ASCs and cancer cell cocultures revealed that cancer cells use O-ASC-secreted arginine and in turn secrete citrulline in the microenvironment. Interestingly, citrulline increased adipogenesis potential of the O-ASCs. Furthermore, we found that O-ASCs increased NO synthesis in cancer cells, leading to decrease in mitochondrial respiration in these cells. Our findings suggest that O-ASCs upregulate glycolysis and reduce oxidative stress in cancer cells by increasing NO levels through paracrine metabolite secretion. Significantly, we found that O-ASC-mediated chemoresistance in cancer cells can be deregulated by altering NO homeostasis. A combined approach of targeting secreted arginine through l-arginase, along with targeting microenvironment-secreted factors using l-NAME, may be a viable therapeutic approach for targeting ovarian and endometrial cancers.

Redox-active and redox-silent compounds: synergistic therapeutics in cancer.

Tumours exhibit higher basal levels of reactive oxygen species (ROS) and altered redox environment compared to normal cells. Excessive level of ROS can be toxic to these cells, thus they become more vulnerable to damage by further ROS insults induced by pharmacological agents. However, the upregulation of antioxidant capacity in adaptation to intrinsic oxidative stress in cancer cells can confer drug resistance. Therefore, abrogation of such drug-resistant mechanisms by redox modulation could have significant therapeutic implications. Many redox-modulating agents have been developed. The redox-active system epitomised by ascorbate-driven quinone redox cycling, and the group of redox-silent vitamin E analogues represented by α-tocopheryl succinate have been shown to induce selective cancer cell death in different types of cancer. These compounds synergistically act by destabilising organelles like mitochondria, unleashing their apoptogenic potential, which results in efficient death of malignant cells and suppression of tumour growth. Consistent with this notion, clinical trials that aim to examine the therapeutic performance of novel redox-modulating drugs in cancer patients are currently under way.

Interleukin 6 Alters Localization of hMSH3, Leading to DNA Mismatch Repair Defects in Colorectal Cancer Cells

Elevated microsatellite alterations at selected tetranucleotide repeats (EMAST) is the most common DNA mismatch repair defect in colorectal cancers, observed in approximately 60% of specimens. This acquired genotype correlates with metastasis and poor outcomes for patients, and is associated with intra-epithelial inflammation and heterogeneous nuclear levels of the mismatch repair protein hMSH3. Inflammation and accompanying oxidative stress can cause hMSH3 to change its intracellular location, but little is known about the source of oxidative stress in cancer cells. We investigated whether cytokines mediate this process. We analyzed levels of interleukin 6 (IL6) and its receptor (IL6R) in human colon and lung cancer cell lines by flow cytometry and enzyme-linked immunosorbent assay; proteins were localized by immunofluorescence and immunoblot analyses. IL6 signaling was blocked with antibodies against IL6, soluble glycoprotein 130 Fc fragments, and the signal transducers and activators of transcription 3 inhibitor NSC74859; a constitutively active form of STAT3 was expressed in colon and lung cancer cell lines to replicate IL6R signaling. EMAST was detected by DNA fragment analysis. Immunohistochemistry was used to examine levels of IL6 in 20 colorectal tumor and adjacent nontumor tissues. Incubation of colon and lung cancer cell lines with IL6, but not other cytokines, caused hMSH3, but no other mismatch repair proteins, to move from the nucleus to the cytosol after generation of oxidative stress; inhibition of IL6 signaling prevented this shift. Expression of constitutively active STAT3 also caused hMSH3 to translocate from the nucleus to the cytoplasm in cancer cell lines. Incubation of cells with IL6 led to tetranucleotide frameshifts, the signature for EMAST. EMAST-positive colorectal tumors had significantly higher levels of IL6 than EMAST-negative tumors. IL6 signaling disrupts the nuclear localization of hMSH3 and DNA repair, leading to EMAST in cancer cell lines. Inflammatory cytokines might therefore promote genetic alterations in human cancer cells.

Different effects of LDH-A inhibition by oxamate in non-small cell lung cancer cells.

Higher rate of glycolysis has been long observed in cancer cells, as a vital enzyme in glycolysis, lactate dehydrogenase A (LDH-A) has been shown with great potential as an anti-cancer target. Accumulating evidence indicates that inhibition of LDH-A induces apoptosis mediated by oxidative stress in cancer cells. To date, it's still unclear that whether autophagy can be induced by LDH-A inhibition. Here, we investigated the effects of oxamate, one classic inhibitor of LDH-A in non-small cell lung cancer (NSCLC) cells as well as normal lung epithelial cells. The results showed that oxamate significantly suppressed the proliferation of NSCLC cells, while it exerted a much lower toxicity in normal cells. As previous studies reported, LDH-A inhibition resulted in ATP reduction and ROS (reactive oxygen species) burst in cancer cells, which lead to apoptosis and G2/M arrest in H1395 cells. However, when being exposed to oxamate, A549 cells underwent autophagy as a protective mechanism against apoptosis. Furthermore, we found evidence that LDH-A inhibition induced G0/G1 arrest dependent on the activation of GSK-3β in A549 cells. Taken together, our results provide useful clues for targeting LDH-A in NSCLC treatment and shed light on the discovery of molecular predictors for the sensitivity of LDH-A inhibitors.

Abstract 1232: Antiproliferative, antioxidant and antiapoptotic effect of rhamnetin in human prostate cancer cells

Abstract Persistent oxidative stress due to generation of reactive oxygen species (ROS) has been associated with malignancy and cancer progression. Using a panel of human prostate cancer cells, we observed increased production of intracellular ROS, compared to normal human prostate epithelial cells, accompanied by elevated expression of antioxidant enzymes viz. superoxide dismutase, catalase, peroxiredoxin-1 and thioredoxin that may be a likely mechanism to endure increased oxidative stress. There is a need for novel antioxidant agent that can effectively suppress the generation of increased ROS in cancer cells. Rhamnetin (3,5,3,4-tetrahydroxy-7-methoxyflavone), remains highest on oxygen radical absorbance capacity scale, however its anti-proliferative activities has not been assessed. Rhamnetin is an O-methylated flavonol, abundantly present in cloves, sweet annie, annual wormwood, green vegetables and orchard grass. Previous studies (Shukla et al. Proc. Amer. Assoc. Cancer Res. 102: 5576, 2011) using androgen-responsive human prostate cancer LNCaP and androgen-refractory PC-3 cells, we have demonstrated that rhamnetin possess anticancer activity, induces apoptosis, and inhibits cancer cell growth. In the present study we assessed the antioxidant and anti-proliferative ability of rhmanetin in prostate cancer. Treatment of LNCaP and PC-3 cells with 100μM paraquat, an oxidative stress-inducing agent, induced cellular proliferation in both these cells. Treatment of cells with 5-80μM rhamnetin significantly decreased paraquat-induced ROS production and inhibited cell proliferation in dose-dependent manner. Similar effects were noted with the addition of recombinant Klotho protein to these cells, which is known to offer resistance to oxidative stress at the cellular level. Rhamnetin treatment also increased Klotho protein levels in both cell lines, along with increase in the expression of p53 and p21/waf1. Furthermore, rhamnetin treatment resulted in the increased expression of caspase-3, caspase-9 causing induction of apoptosis, which may be one of the protective mechanism(s) to decrease cell proliferation and enhance resistance to oxidative stress in cancer cells. Taken together, these results demonstrate that rhamnetin acts as a potent antioxidant agent, reduces intracellular ROS levels, increases Klotho protein expression, induces apoptosis in prostate cancer cells, which may have a significant role in the inhibition of prostate cancer progression. Citation Format: Christine Oak, Natarajan Bhaskaran, Sanjay Gupta, Sanjeev Shukla. Antiproliferative, antioxidant and antiapoptotic effect of rhamnetin in human prostate cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1232. doi:10.1158/1538-7445.AM2014-1232

A selenosemicarbazone complex with copper efficiently down-regulates the 90-kDa heat shock protein HSP90AA1 and its client proteins in cancer cells.

BackgroundThe 90-kDa heat shock protein HSP90AA1 is critical for the stability of several proteins that are important for tumor progression and thus, is a promising target for cancer therapy. Selenosemicarbazone metal complexes have been shown to possess anticancer activity through an unknown molecular mechanism.MethodsThe MTT assay, fluorescence-activated cell sorting, and fluorescent microscopy were used to analyze the mechanism of the anti-cancer activity of the selenosemicarbazone metal complexes. Additionally, RNA-seq was applied to identify transcriptional gene changes, and in turn, the signaling pathways involved in the process of 2-24a/Cu-induced cell death. Last, the expression of HSP90AA1, HSPA1A, PIM1, and AKT proteins in 2-24a/Cu-treated cells were investigated by western blot analysis.ResultsA novel selenosemicarbazone copper complex (2-24a/Cu) efficiently induced G2/M arrest and was cytotoxic in cancer cells. 2-24a/Cu significantly induced oxidative stress in cancer cells. Interestingly, although RNA-seq revealed that the transcription of HSP90AA1 was increased in 2-24a/Cu-treated cells, western blotting showed that the expression of HSP90AA1 protein was significantly decreased in these cells. Furthermore, down-regulation of HSP90AA1 led to the degradation of its client proteins (PIM1 and AKT1), which are also cancer therapy targets.ConclusionOur results showed that 2-24a/Cu efficiently generates oxidative stress and down-regulates HSP90AA1 and its client proteins (PIM1, AKT1) in U2os and HeLa cells. These results demonstrate the potential application of this novel copper complex in cancer therapy.

Serum metabolomic signatures of lymph node metastasis of esophageal squamous cell carcinoma.

Lymph node metastasis was recently proven to be the single most important prognostic factor for esophageal cancer, an important malignant tumor with poor prognosis. A global metabolomics approach was applied to study lymph node metastasis of esophageal squamous cell carcinoma (ESCC). Metabolomics analyses were performed using gas chromatography/mass spectrometry together with univariate and multivariate statistical analyses. There were clear metabolic distinctions between ESCC patients and healthy subjects. ESCC patients could be well-classified according to lymph node metastasis. We further identified a series of differential serum metabolites for ESCC and lymph node metastatic ESCC patients, suggesting metabolic dysfunction in proliferation (aerobic glycolysis, glutaminolysis, fatty acid metabolism, and branched-chain amino acid consumption), apoptosis, migration, immune escape, and oxidative stress of cancer cells in metastatic ESCC patients. In total, three serum metabolites (valine, γ-aminobutyric acid, and pyrrole-2-carboxylic acid) were selected by binary logistic regression analysis, and their combined use resulted in high diagnostic capacity for ESCC metastasis by receiver operating characteristic analysis. The present metabolomics study staged ESCC patients by lymph node metastasis, and the results suggest promising applications of this approach in prognostic prediction, tailored therapeutics, and understanding the pathological mechanisms of poor prognosis of ESCC patients.

Nanoparticle induced oxidative stress in cancer cells: adding new pieces to an incomplete jigsaw puzzle.

Nanotechnology is an emerging field with many promising applications in drug delivery systems. Because of outstanding developments in this field, rapidly increasing research is directed to the development of nanocarriers that may enhance the availability of drugs to the target sites. Substantial fraction of information has been added into the existing scientific literature focusing on the fact that nanoparticles usually generate reactive oxygen species to a greater extent than micro-sized particles. It is worth mentioning that oxidative stress regulates an array of cell signaling cascades that resulted in cancer cell damage. Accumulating experimental evidence over the years has shown that wide-ranging biological mechanisms are triggered by these NPs in cultured cells due to the unique properties of engineered nanoparticles. In this review, we have attempted to provide an overview of the signaling cascades that are activated by oxidative stress in cancer cells in response to different kinds of nanomaterials, including quantum dots, metallic and polymeric nanoparticles.

Microwave-assisted synthesis and evaluation of acylhydrazones as potential inhibitors of bovine glutathione peroxidase

Glutathione peroxidases (GPx) were the first selenocysteine enzymes identified. They play critical roles in cellular defense to excessH(2)O(2) and lipid peroxides, with GPx1 contributing the most cellular activity. In the treatment of cancer, for example, in lymphomas and leukaemias, evidence has been accumulating that up-regulation of the GPx system may serve to protect cancer cells from oxidative stress caused by anticancer drugs. We hypothesize that small molecules which block GPx1 could help overcome acquired resistance to anticancer drugs by raising the level of oxidative stress in cancer cells. Our previous efforts identified an acylhydrazone as a lead structure for the inhibition of GPx1. Now we report the microwave-supported synthesis and inhibitory screening of a series of 78 analogs. The special conformational isomerism resulting of the acylhydrazone functionality was investigated by the analysis of distinct NMR data and crystal structures, indicating that conformers at the C(O)-N hydrazide bond are responsible for this phenomena. Though some of the analogs showed poor aqueous solubility and could not be tested in the enzyme assay, the combinatorial approach led to the identification of a closely related isomer of the lead compound with increased inhibitory activity: N'-[1-(4-hydroxyphenyl)ethylidene]-2-(1H-imidazol-1-yl)acetohydrazide. This success supports the idea that novel GPx1 inhibitors can be developed by drug-design methods and paves the way for a new class of GPx1 inhibitors.

The Proto-Oncoprotein c-Cbl Protects Cells against Oxidative Stress by Down- Regulating Apoptosis and is Highly Expressed in Several Cancers

Objective: To determine the role of c-Cbl in the apoptotic process, using several means, stressing the role of oxidative stress in cancer cells. Methods: Prostatic epithelial cell apopotosis of c-Cbl-/-mice were compared to wildtype mice (n: 6 per condition), upon testosterone-antagonist flutamide. c-Cbl-deficient mouse embryonic fibroblasts (MEFs) were compared to wild type MEFs under etoposide or hydrogen peroxide treatments. The use of c-Cbl RNA silencing in the human prostate adenocarcinoma cell line LNCaP allowed to reveal c-Cbl’s role in LNCaPs’ apoptosis. The role of the p38-MAPK stress pathway in the LNCAP c-Cbl anti-apoptotic effect as well as its relationship with the well-documented Grb2-associated Tyrosine-Kinase-Receptor (TKR) down-regulation were investigated, using c-Cbl and/or Grb2 RNA silencing. Human c-Cbl protein expression was analysed by Western blotting and immunostaining, comparing prostatic adenocarcinoma (x22) to benign prostatic hypertrophia (x6). In situ tissue microarrays were used to assess several human malignancies (x17 and x6 spots/tissue) and to compare the magnitude of c-Cbl and oxidative stress expression. Results: The cellular apoptotic threshold decreased in Mouse c-Cbl-/-prostatic cells and c-Cbl-/- MEFs. Only hydrogen peroxide in c-Cbl-/-MEFs induced apoptosis up to six times more than controls. Similar results were found in LNCaPs. c-Cbl down-regulates the activation of the apoptotic ASK1-p38MAPK stress pathway. c-Cbl is overexpressed in prostate, ovary, uterus, brain, lung, colon, rectum adenocarcinoma and in rhabdomyosarcoma. We found a correlation between malignant oxidative stress and c-Cbl over-expression. Conclusions: c-Cbl increases the cellular apoptotic threshold of wild type MEFs and mouse prostate cells. c-Cbl behaves as a strong cellular protector against oxidative stress in MEFs and LNCaPs. The p38-MAPK pathway is down-regulated by c-Cbl, possibly independently of the Grb2-associated TKR down-regulation. A high c-Cbl expression in several cancers often associated with high oxidative stress expression has been found, suggesting that c-Cbl could thereby promote their survival.