Arsenic induced apoptosis in malignant melanoma cells is enhanced by menadione through ROS generation, p38 signaling and p53 activation

Introduction  Resistance to apoptosis is a prominent feature of melanoma. Pharmacological concentration of arsenic in combination with a widely known oxidant, menadione was explored in this study to synergistically sensitize malignant melanoma cells to apoptosis. The molecular mechanism of apoptosis and the signaling-pathways involved were thoroughly investigated. Materials methods and results  Menadione synergized NaAsO₂ to significantly increase ROS generation and facilitate the major apoptotic signaling events: alteration of mitochondrial membrane potential, cytochrome c release and anti-apoptotic protein Bcl-2 down-regulation and subsequent activation of caspase-9 and caspase-3 followed by poly-ADP-ribose polymerase-1 cleavage. Antioxidant N-acetyl-l-cysteine antagonized these events. Investigation of the signaling-pathway revealed significant suppression of AP-1 activity but not NF-κB upon NaAsO₂ and menadione application. An increase in p38 phosphorylation and p53 protein expression did also dictate the apoptotic response. Suppression of p38 activation with SB203580 and inhibition of p53 expression by siRNA attenuated apoptosis. Transfection of p53, in p53 null HCT cells augmented the apoptotic events. Moreover, the treatment also led to tumor size reduction in BALB/c mice developed by intra-dermal B16 mouse melanoma cell injection; however, it had no detectable pro-proliferative or pro-apoptotic effect on non-tumor keratinocytes, normal fibroblasts or PBMC. Conclusion  This study thus provides an insight into innovative mechanisms of melanoma sensitization, a proper cure against which is still elusive. Taken together, our data also provides the first evidence of arsenic activity accentuation by menadione through modulation of specific signaling-pathways.     

Regulation of cell survival by sphingosine-1-phosphate receptor S1P1 via reciprocal ERK-dependent suppression of Bim and PI-3-kinase/protein kinase C-mediated upregulation of Mcl-1

Although the ability of bioactive lipid sphingosine-1-phosphate (S1P) to positively regulate anti-apoptotic/pro-survival responses by binding to S1P₁ is well known, the molecular mechanisms remain unclear. Here we demonstrate that expression of S1P₁ renders CCL39 lung fibroblasts resistant to apoptosis following growth factor withdrawal. Resistance to apoptosis was associated with attenuated accumulation of pro-apoptotic BH3-only protein Bim. However, although blockade of extracellular signal-regulated kinase (ERK) activation could reverse S1P₁-mediated suppression of Bim accumulation, inhibition of caspase-3 cleavage was unaffected. Instead S1P₁-mediated inhibition of caspase-3 cleavage was reversed by inhibition of phosphatidylinositol-3-kinase (PI3K) and protein kinase C (PKC), which had no effect on S1P₁ regulation of Bim. However, S1P₁ suppression of caspase-3 was associated with increased expression of anti-apoptotic protein Mcl-1, the expression of which was also reduced by inhibition of PI3K and PKC. A role for the induction of Mcl-1 in regulating endogenous S1P receptor-dependent pro-survival responses in human umbilical vein endothelial cells was confirmed using S1P receptor agonist FTY720-phosphate (FTY720P). FTY720P induced a transient accumulation of Mcl-1 that was associated with a delayed onset of caspase-3 cleavage following growth factor withdrawal, whereas Mcl-1 knockdown was sufficient to enhance caspase-3 cleavage even in the presence of FTY720P. Consistent with a pro-survival role of S1P₁ in disease, analysis of tissue microarrays from ER⁺ breast cancer patients revealed a significant correlation between S1P₁ expression and tumour cell survival. In these tumours, S1P₁ expression and cancer cell survival were correlated with increased activation of ERK, but not the PI3K/PKB pathway. In summary, pro-survival/anti-apoptotic signalling from S1P₁ is intimately linked to its ability to promote the accumulation of pro-survival protein Mcl-1 and downregulation of pro-apoptotic BH3-only protein Bim via distinct signalling pathways. However, the functional importance of each pathway is dependent on the specific cellular context.     

Asparanin A induces G2/M cell cycle arrest and apoptosis in human hepatocellular carcinoma HepG2 cells

We recently established that asparanin A, a steroidal saponin extracted from Asparagus officinalis L., is an active cytotoxic component. The molecular mechanisms by which asparanin A exerts its cytotoxic activity are currently unknown. In this study, we show that asparanin A induces G₂/M phase arrest and apoptosis in human hepatocellular carcinoma HepG2 cells. Following treatment of HepG2 cells with asparanin A, cell cycle-related proteins such as cyclin A, Cdk1 and Cdk4 were down-regulated, while p21^WAF1/Cip1 and p-Cdk1 (Thr14/Tyr15) were up-regulated. Additionally, we observed poly (ADP-ribose) polymerase (PARP) cleavage and activation of caspase-3, caspase-8 and caspase-9. The expression ratio of Bax/Bcl-2 was increased in the treated cells, where Bax was also up-regulated. We also found that the expression of p53, a modulator of p21^WAF1/Cip1 and Bax, was not affected in asparanin A-treated cells. Collectively, our findings demonstrate that asparanin A induces cell cycle arrest and triggers apoptosis via a p53-independent manner in HepG2 cells. These data indicate that asparanin A shows promise as a preventive and/or therapeutic agent against human hepatoma.     

Methylated chrysin induces co-ordinated attenuation of the canonical Wnt and NF-kB signaling pathway and upregulates apoptotic gene expression in the early hepatocarcinogenesis rat model

Hepatocellular carcinoma (HCC), a highly aggressive form of solid tumor, has been increasing in South East Asia. The lack of effective therapy necessitates the introduction of novel chemopreventive strategies to counter the substantial morbidity and mortality associated with the disease. Recently, we reported that dimethoxy flavone (DMF), a methylated flavone derived from chrysin, significantly suppressed the development of preneoplastic lesions induced by N-nitrosodiethylamine (DEN) in rats, although the mechanism of action was not known. In the present study, we have investigated the effects of DMF administration on gene expression changes related to the inflammation-mediated NF-kB pathway, Wnt pathway and apoptotic mediators in DEN-induced preneoplastic nodules. There was a significant increase in inflammatory markers like cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) and a decrease in apoptotic mediators like p53, caspase-3 and bax in DEN-treated rats when compared to the control group. Activation of NF-kB was noticed by an elevated expression of nuclear protein expression of NF-kB and cytoplasmic phospho-IkBαSer^32/36 in the same animals. Likewise, upregulation of canonical Wnt pathway was noticed by elevated expression of nuclear protein levels of phospho-β-cateninThr³⁹³ and cytoplasmic casein kinase-2 (CK2), Dvl2 and cyclin D1 levels, along with a simultaneous decrease in expression of phospho-GSK3β^Ser9. Dietary DMF (100 mg/kg) administration inhibited liver nodule incidence and multiplicity by 82% and 78%, respectively. DMF also reversed the activation of NF-kB and Wnt pathway as shown by the decrease in protein expression of several proteins. Results of the present investigation provide evidence that attenuation of Wnt pathway and suppression of inflammatory response mediated by NF-kB could be implicated, in part, in the chemopreventive effects of methylated flavone. Therefore, the present findings hold great promise for the utilization of DMF as an effective chemotherapeutic agent in treating early stages of liver cancer.     

Measurement of menadione in urine by HPLC

Menadione is a metabolite of vitamin K that is excreted in urine. A high performance liquid chromatography (HPLC) method using a C₃₀ column, post-column zinc reduction and fluorescence detection was developed to measure urinary menadione. The mobile phase was composed of 95% methanol with 0.55% aqueous solution and 5% DI H₂O. Menaquinone-2 (MK-2) was used as an internal standard. The standard calibration curve was linear with a correlation coefficient (R²) of 0.999 for both menadione and MK-2. The lower limit of quantification (LLOQ) was 0.3 pmole menadione/mL urine. Sample preparation involved hydrolysis of menadiol conjugates and oxidizing the released menadiol to menadione. Using this method, urinary menadione was shown to increase in response to 3 years of phylloquinone supplementation. This HPLC method is a sensitive and reproducible way to detect menadione in urine.     

Multiple signals modulate the activity of the complex sensor kinase TodS

The reason for the existence of complex sensor kinases is little understood but thought to lie in the capacity to respond to multiple signals. The complex, seven‐domain sensor kinase TodS controls in concert with the TodT response regulator the expression of the toluene dioxygenase pathway in Pseudomonas putida F1 and DOT‐T1E. We have previously shown that some aromatic hydrocarbons stimulate TodS activity whereas others behave as antagonists. We show here that TodS responds in addition to the oxidative agent menadione. Menadione but no other oxidative agent tested inhibited TodS activity in vitro and reduced P_todX expression in vivo. The menadione signal is incorporated by a cysteine‐dependent mechanism. The mutation of the sole conserved cysteine of TodS (C320) rendered the protein insensitive to menadione. We evaluated the mutual opposing effects of toluene and menadione on TodS autophosphorylation. In the presence of toluene, menadione reduced TodS activity whereas toluene did not stimulate activity in the presence of menadione. It was shown by others that menadione increases expression of glucose metabolism genes. The opposing effects of menadione on glucose and toluene metabolism may be partially responsible for the interwoven regulation of both catabolic pathways. This work provides mechanistic detail on how complex sensor kinases integrate different types of signal molecules.     

Sorting out the role of nitric oxide in cadmium-induced Arabidopsis thaliana programmed cell death

As a vital cell-signaling molecule, nitric oxide (NO) has been reported to regulate toxic metal responses in plants. Our recent report has suggested that caspase-3-like protease activation was detected in Arabidopsis (Arabidopsis thaliana) after Cd²⁺ treatment. NO contributed caspase-3-like protease activation in Cd²⁺ induced Arabidopsis thaliana programmed cell death (PCD), which was mediated by MPK6. It was first shown that NO promotes Cd²⁺-induced Arabidopsis PCD by promoting MPK6-mediated caspase-3-like activation. Our study contributed to the understanding of NO signaling pathway in Cd²⁺-induced Arabidopsis thaliana PCD. Although several studies have revealed that NO regulates plant PCD, compared with the study of signaling pathways involved in animal cell apoptosis, the mechanism of NO function still remains elusive and the molecular mechanisms of MAPK are far from clear in Cd²⁺-induced PCD. By using the fluorescence techniques and the Arabidopsis seedlings as the reference model, the subsequent researches have been performed to obtain comprehensive understanding of Cd²⁺-induced plant PCD.     

Menadione (vitamin K) enhances the antibiotic activity of drugs by cell membrane permeabilization mechanism

Menadione, vitamin K3, belongs to the class of lipid-soluble vitamins and lipophilic substances as menadione cause disturbances in the bacterial membrane, resulting in damage to the fundamental elements for the integrity of the membrane, thus allowing increased permeability. Accordingly, the aim of this study was to evaluate in vitro the antibiotic-modifying activity of menadione in multiresistant strains of Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli, with a gradual increase in its subinhibitory concentration. In addition, menadione was compared with cholesterol and ergosterol for similarity in mechanism of drug modulatory action. Antibiotic-modifying activity and antibacterial effect were determined by the broth microdilution assay. Menadione, cholesterol and ergosterol showed modulatory activity at clinically relevant concentrations, characterizing them as modifiers of bacterial drug resistance, since they lowered the MIC of the antibiotics tested. This is the first report of the antibacterial activity of menadione and its potentiation of aminoglycosides against multiresistant bacteria.     

Stigmasterol isolated from marine microalgae Navicula incerta induces apoptosis in human hepatoma HepG2 cells

Plant sterols have shown potent anti-proliferative effects and apoptosis induction against breast and prostate cancers. However, the effect of sterols against hepatic cancer has not been investigated. In the present study, we assessed whether the stigmasterol isolated from Navicula incerta possesses apoptosis inductive effect in hepatocarcimona (HepG2) cells. According to the results, Stigmasterol has up-regulated the expression of pro-apoptotic gene expressions (Bax, p53) while down-regulating the anti-apoptotic genes (Bcl-2). Probably via mitochondrial apoptosis signaling pathway. With the induction of apoptosis caspase-8, 9 were activated. The DNA damage and increase in apoptotic cell numbers were observed through Hoechst staining, annexin V staining and cell cycle analysis. According to these results, we can suggest that the stigmasterol shows potent apoptosis inductive effects and has the potential to be tested as an anti-cancer therapeutic against liver cancer. [BMB Reports 2014; 47(8): 433-438]     

PDCD6 additively cooperates with anti-cancer drugs through activation of NF-κB pathways

The expression of programmed cell death 6 (PDCD6) is known to be down-regulated in cancer cell lines and ovarian cancer tissues compared to normal cells and tissues. In the current study, we characterized the specific function of PDCD6 as a novel pro-apoptotic protein. To define the roles of PDCD6 and cisplatin in tumorigenesis, we either over-expressed PDCD6 or treated it with cisplatin in SKOV-3 ovarian cancer cells. Both PDCD6 and cisplatin respectively inhibited cancer cell proliferation in a dose-dependent manner. The combined treatment of PDCD6 and cisplatin was more effective at suppressing cell growth than with either drug treatment alone, but had no effect with the treatment of caspase-3 and caspase-9 inhibitors. Cleavages of caspase-3, -8, -9, and poly (ADP-ribose) polymerase (PARP) in PDCD6-overexpressing cells were significantly increased after cisplatin treatment. Cell cycle analysis highly correlated with down-regulation of cyclin D1 and CDK4, and the induction of p16 and p27 as a cyclin-dependent kinase inhibitor. Additionally, PDCD6 also suppressed the phosphorylation of signaling regulators downstream of PI3K, including PDK1 and Akt. PDCD6 promotes TNFα-dependent apoptosis through the activation of NF-κB signaling pathways, increasing Bax, p53, and p21 expression, while also down-regulating Bcl-2 and Bcl-xL expression. The p21 and p53 promoter luciferase activities were enhanced by PDCD6, while there was no affect in p53^−/− and p21^−/−. At the same time, p53 activity was confirmed by UV irradiation and siPDCD6. Taken together, these results provide evidence that PDCD6 can mediate the pro-apoptotic activity of cisplatin or TNFα through the down-regulation of NF-κB expression.     

Increase in the nuclear localization of PTEN by the Toxoplasma GRA16 protein and subsequent induction of p53‐dependent apoptosis and anticancer effect

This study investigated the efficacy of Toxoplasma GRA16, which binds to herpes virus‐associated ubiquitin‐specific protease (HAUSP), in anticancer treatment, and whether the expression of GRA16 in genetically modified hepatocellular carcinoma (HCC) cells (GRA16‐p53‐wild HepG2 and GRA16‐p53‐null Hep3B) regulates PTEN because alterations in phosphatase and tensin homologue (PTEN) and p53 are vital in liver carcinogenesis and the abnormal p53 gene appears in HCC. For this purpose, we established the GRA16 cell lines using the pBABE retrovirus system, assessed the detailed mechanism of PTEN regulation in vitro and established the anticancer effect in xenograft mice. Our study showed that cell proliferation, antiapoptotic factors, p‐AKT/AKT ratio, cell migration and invasive activity were decreased in GRA16‐stable HepG2 cells. Conversely, the apoptotic factors PTEN and p53 and apoptotic cells were elevated in GRA16‐stable HepG2 cells but not in Hep3B cells. The change in MDM2 was inconspicuous in both HepG2 and Hep3B; however, the PTEN level was remarkably elevated in HepG2 but not in Hep3B. HAUSP‐bound GRA16 preferentially increased p53 stabilization by the nuclear localization of PTEN rather than MDM2‐dependent mechanisms. These molecular changes appeared to correlate with the decreased tumour mass in GRA16‐stable‐HepG2 cell‐xenograft nude mice. This study establishes that GRA16 is a HAUSP inhibitor that targets the nuclear localization of PTEN and induces the anticancer effect in a p53‐dependent manner. The efficacy of GRA16 could be newly highlighted in HCC treatment in a p53‐dependent manner.     

Livin abrogates apoptosis of SPC-A1 cell by regulating JNKI signaling pathway

Livin, a novel member of inhibitors of apoptosis protein, is highly expressed in tumor tissues. It is a potential target in tumor therapy. Silencing its gene expression has been found to promote tumor cell apoptosis or increase tumor sensitivity to therapies. This paper studied the effect of livin anti-apoptotic activity and examined its molecular mechanisms. In the study, higher levels of cell apoptosis were measured by FACS in the experiment group with livin expression silenced than that in controls (P < 0.05). After livin gene expression was knocked down, cleaved caspase-3 protein was up-regulated but caspase-3 mRNA expression was almost the same, the phosphorylated JNK1 protein was down-regulated but JNK1 mRNA and total JNK1 protein expression was approximately the same too. The results suggest that livin may exert anti-apoptotic action on SPC-A1 by activating JNK1 signaling pathway and inhibiting caspase-3 activation.     

Biphasic behavior of changes in elemental composition during staurosporine-induced apoptosis

Although the identification of events that occur during apoptosis is a fundamental goal of apoptotic cell death research, little is know about the precise sequence of changes in total elemental composition during apoptosis. We evaluated total elemental composition (Na, Mg, P, Cl, S, and K) in relation to molecular and morphological features in human U937 cells induced to undergo apoptosis with staurosporine, an intrinsic pathway activator. To evaluate total elemental content we used electron probe X-ray microanalysis to measure simultaneously all elements from single, individual cells. We observed two phases in the changes in elemental composition (mainly Na, Cl and K). The early phase was characterized by a decrease in intracellular K (P < 0.001) and Cl (P < 0.001) content concomitant with cell shrinkage, and preceded the increase in proteolytic activity associated with the activation of caspase-3. The later phase started with caspase-3 activation, and was characterized by a decrease in the K/Na ratio (P < 0.001) as a consequence of a significant decrease in K and increase in Na content. The inversion of intracellular K and Na content was related with the inhibition of Na⁺/K⁺ ATPase. This later phase was also characterized by a significant increase (P < 0.001) in intracellular Cl with respect to the early phase. In addition, we found a decrease in S content and an increase in the P/S ratio. These distinctive changes coincided with chromatin condensation and DNA fragmentation. Together, these findings support the concept that changes in total elemental composition take place in two phases related with molecular and morphological features during staurosporine-induced apoptosis.     

Nutlin-3 downregulates p53 phosphorylation on serine392 and induces apoptosis in hepatocellular carcinoma cells

Drug-resistance and imbalance of apoptotic regulation limit chemotherapy clinical application for the human hepatocellular carcinoma (HCC) treatment. The reactivation of p53 is an attractive therapeutic strategy in cancer with disrupted-p53 function. Nutlin-3, a MDM2 antagonist, has antitumor activity in various cancers. The post-translational modifications of p53 are a hot topic, but there are some controversy ideas about the function of phospho-Ser³⁹²-p53 protein in cancer cell lines in response to Nutlin-3. Therefore, we investigated the relationship between Nutlin-3 and phospho-Ser³⁹²-p53 protein expression levels in SMMC-7721 (wild-type TP53) and HuH-7 cells (mutant TP53). We demonstrated that Nutlin-3 induced apoptosis through down-regulation phospho-Ser³⁹²-p53 in two HCC cells. The result suggests that inhibition of p53 phosphorylation on Ser³⁹² presents an alternative for HCC chemotherapy. [BMB Reports 2014; 47(4): 221-226]     

Hormesis in Cholestatic Liver Disease; Preconditioning with Low Bile Acid Concentrations Protects against Bile Acid-Induced Toxicity

Introduction

Cholestasis is characterized by accumulation of bile acids and inflammation, causing hepatocellular damage. Still, liver damage markers are highest in acute cholestasis and drop when this condition becomes chronic, indicating that hepatocytes adapt towards the hostile environment. This may be explained by a hormetic response in hepatocytes that limits cell death during cholestasis.

Aim

To investigate the mechanisms that underlie the hormetic response that protect hepatocytes against experimental cholestatic conditions.

Methods

HepG2.rNtcp cells were preconditioned (24 h) with sub-apoptotic concentrations (0.1–50 μM) of various bile acids, the superoxide donor menadione, TNF-α or the Farsenoid X Receptor agonist GW4064, followed by a challenge with the apoptosis-inducing bile acid glycochenodeoxycholic acid (GCDCA; 200 μM for 4 h), menadione (50 μM, 6 h) or cytokine mixture (CM; 6 h). Levels of apoptotic and necrotic cell death, mRNA expression of the bile salt export pump (ABCB11) and bile acid sensors, as well as intracellular GCDCA levels were analyzed.

Results

Preconditioning with the pro-apoptotic bile acids GCDCA, taurocholic acid, or the protective bile acids (tauro)ursodeoxycholic acid reduced GCDCA-induced caspase-3/7 activity in HepG2.rNtcp cells. Bile acid preconditioning did not induce significant levels of necrosis in GCDCA-challenged HepG2.rNtcp cells. In contrast, preconditioning with cholic acid, menadione or TNF-α potentiated GCDCA-induced apoptosis. GCDCA preconditioning specifically reduced GCDCA-induced cell death and not CM- or menadione-induced apoptosis. The hormetic effect of GCDCA preconditioning was concentration- and time-dependent. GCDCA-, CDCA- and GW4064- preconditioning enhanced ABCB11 mRNA levels, but in contrast to the bile acids, GW4064 did not significantly reduce GCDCA-induced caspase-3/7 activity. The GCDCA challenge strongly increased intracellular levels of this bile acid, which was not lowered by GCDCA-preconditioning.

Conclusions

Sub-toxic concentrations of bile acids in the range that occur under normal physiological conditions protect HepG2.rNtcp cells against GCDCA-induced apoptosis, which is independent of FXR-controlled changes in bile acid transport.     

Production of superoxide anion during the oxidation of hemoglobin by menadione.

Menadione in the presence of oxyhemoglobin will accelerate the formation of methemoglobin and result in the generation of superoxide anion. Menadione appears to oxidize slowly ferrohemoglobin to ferrihemoglobin, while forming menadione semiquinone in the process. Menadione semiquinone is known to react with molecular oxygen to yield superoxide anion. The superoxide anion appears to be the source of hydrogen peroxide which accounts for most of the observed methemoglobin formation when hemoglobin is reacted with menadione.     

Hsp70 regulates the doxorubicin-mediated heart failure in Hsp70-transgenic mice

The aim of this study was to investigate the potential protective effect of the Hsp70 protein in the cardiac dysfunction induced by doxorubicin (DOX) and the mechanisms of its action. For this purpose, we used both wild-type mice (F1/F1) and Hsp70-transgenic mice (Tg/Tg) overexpressing human HSP70. Both types were subjected to chronic DOX administration (3 mg/kg intraperitoneally every week for 10 weeks, with an interval from weeks 4 to 6). Primary cell cultures isolated from embryos of these mice were also studied. During DOX administration, the mortality rate as well as weight reduction were lower in Tg/Tg compared to F1/F1 mice (P < 0.05). In vivo cardiac function assessment by transthoracic echocardiography showed that the reduction in left ventricular systolic function observed after DOX administration was lower in Tg/Tg mice (P < 0.05). The study in primary embryonic cell lines showed that the apoptosis after incubation with DOX was reduced in cells overexpressing Hsp70 (Tg/Tg), while the apoptotic pathway that was activated by DOX administration involved activated protein factors such as p53, Bax, caspase-9, caspase-3, and PARP-1. In myocardial protein extracts from identical mice with DOX-induced heart failure, the particular activated apoptotic pathway was confirmed, while the presence of Hsp70 appeared to inhibit the apoptotic pathway upstream of the p53 activation. Our results, in this DOX-induced heart failure model, indicate that Hsp70 overexpression in Tg/Tg transgenic mice provides protection from myocardial damage via an Hsp70-block in p53 activation, thus reducing the subsequent apoptotic mechanism.