DNA-PKcs is important for Akt activation and gemcitabine resistance in PANC-1 pancreatic cancer cells

Pancreatic cancer is one of the most aggressive human malignancies with extremely poor prognosis. The moderate activity of the current standard gemcitabine and gemcitabine-based regimens was due to pre-existing or acquired chemo-resistance of pancreatic cancer cells. In this study, we explored the potential role of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) in gemcitabine resistance, and studied the underlying mechanisms. We found that NU-7026 and NU-7441, two DNA-PKcs inhibitors, enhanced gemcitabine-induced cytotoxicity and apoptosis in PANC-1 pancreatic cancer cells. Meanwhile, PANC-1 cells with siRNA-knockdown of DNA-PKcs were more sensitive to gemcitabine than control PANC-1 cells. Through the co-immunoprecipitation (Co-IP) assay, we found that DNA-PKcs formed a complex with SIN1, the latter is an indispensable component of mammalian target of rapamycin (mTOR) complex 2 (mTORC2). DNA-PKcs–SIN1 complexation was required for Akt activation in PANC-1 cells, while inhibition of this complex by siRNA knockdown of DNA-PKcs/SIN1, or by DNA-PKcs inhibitors, prevented Akt phosphorylation in PANC-1 cells. Further, SIN1 siRNA-knockdown also facilitated gemcitabine-induced apoptosis in PANC-1 cells. Finally, DNA-PKcs and p-Akt expression was significantly higher in human pancreatic cancer tissues than surrounding normal tissues. Together, these results show that DNA-PKcs is important for Akt activation and gemcitabine resistance in PANC-1 pancreatic cancer cells.     

Cepharanthine triggers apoptosis in a human hepatocellular carcinoma cell line (HuH-7) through the activation of JNK1/2 and the downregulation of Akt

Cepharanthine (CEP), a biscoclaurine alkaloid, has been reported to induce cell death, however, the molecular mechanism of this phenomenon remains unclear. We herein report that CEP induced apoptosis in HuH-7 cells through nuclear fragmentation, DNA ladder formation, cytochrome c release, caspase-3 activation and poly-(ADP-ribose)-polymerase cleavage. CEP triggered the generation of reactive oxygen intermediates, the activation of mitogen activated protein kinase (MAPK) p38, JNK1/2 and p44/42, and the downregulation of protein kinase B/Akt. Antioxidants and SP600125, an inhibitor of JNK1/2, but not inhibitors of p38 MAPK and MEK1/2, significantly prevented cell death, thus implying that reactive oxygen species and JNK1/2 play crucial roles in the CEP-induced apoptosis of HuH-7 cells.     

PKCη confers protection against apoptosis by inhibiting the pro-apoptotic JNK activity in MCF-7 cells

Apoptosis is frequently regulated by different protein kinases including protein kinase C family enzymes. Both inhibitory and stimulatory effects were demonstrated for several of the different PKC isoforms. Here we show that the novel PKC isoform, PKCη, confers protection against apoptosis induced by the DNA damaging agents, UVC irradiation and the anti-cancer drug — Camptothecin, of the breast epithelial adenocarcinoma MCF-7 cells. The induced expression of PKCη in MCF-7 cells, under the control of the tetracycline-responsive promoter, resulted in increased cell survival and inhibition of cleavage of the apoptotic marker PARP-1. Activation of caspase-7 and 9 and the release of cytochrome c were also inhibited by the inducible expression of PKCη. Furthermore, JNK activity, required for apoptosis in MCF-7, as indicated by the inhibition of both caspase-7 cleavage and cytochrome c release from the mitochondria in the presence of the JNK inhibitor SP600125, was also suppressed by PKCη expression. Hence, in contrast to most PKC isoforms enhancing JNK activation, our studies show that PKCη is an anti-apoptotic protein, acting as a negative regulator of JNK activity. Thus, PKCη could represent a target for intervention aimed to reduce resistance to anti-cancer treatments.     

Celecoxib induces p53-PUMA pathway for apoptosis in human colorectal cancer cells

Celecoxib, a clinical non-steroidal anti-inflammatory drug, displays anticarcinogenic and chemopreventive activities in human colorectal cancers, although the mechanisms of apoptosis by celecoxib are poorly understood. The existence of functional p53 but not securin in colorectal cancer cells was higher on the induction of cytotoxicity than the p53-mutational colorectal cancer cells following celecoxib treatment. The p53-wild type HCT116 cells were more susceptible to increase ∼25% cell death than the p53-null HCT116 cells after treatment with 100 μM celecoxib for 24 h. Transfection with a small interfering RNA of p53 reduced the celecoxib-induced cytotoxicity in the RKO (p53-wild type) colorectal cancer cells. Celecoxib (80-100 μM for 24 h) significantly increased total p53 proteins and the phosphorylated p53 proteins at serine-15, -20, -46, and -392 in RKO cells. However, the phospho-p53 (serine-15, -20, and -392) proteins were presented on the nuclei of cells but the phospho-p53 (serine-46) protein was located on the cytoplasma of apoptotic cells following treatment with celecoxib. Interestingly, the p53 up-regulated modulator of apoptosis (PUMA) protein, which located on the mitochondria, was induced by celecoxib in the p53-functional colorectal cancer cells but not in the p53-mutational cells. Together, this study provides the first time that celecoxib induces the various phosphorylated sites of p53 and activates p53-PUMA pathway, which potentiates the apoptosis induction in human colorectal cancer cells.     

Hydrogen peroxide is a mediator of indole-3-acetic acid/horseradish peroxidase-induced apoptosis

Recently, we reported that a combination of indole-3-acetic acid (IAA) and horseradish peroxidase (HRP) induces apoptosis in G361 human melanoma cells. However, the apoptotic mechanism involved has been poorly studied. It is known that when IAA is oxidized by HRP, free radicals are produced, and since oxidative stress can induce apoptosis, we investigated whether reactive oxygen species (ROS) are involved in IAA/HRP-induced apoptosis. Our results show that IAA/HRP-induced free radical production is inhibited by catalase, but not by superoxide dismutase or sodium formate. Furthermore, catalase was found to prevent IAA/HRP-induced apoptotic cell death, indicating that IAA/HRP-produced hydrogen peroxide (H2O2) may be involved in the apoptotic process. Moreover, the antiapoptotic effect of catalase is potentiated by NADPH, which is known to protect catalase. On further investigating the IAA/HRP-mediated apoptotic pathway, we found that the IAA/HRP reaction leads to caspase-3 activation and poly(ADP-ribose) polymerase (PARP) cleavage, which was also blocked by catalase. Additionally, we found that IAA/HRP produces H2O2 and induces peroxiredoxin (Prx) sulfonylation. Consequently, our results suggest that H2O2 plays a major role in IAA/HRP-induced apoptosis.     

Chemopreventive properties of Hibiscus sabdariffa L. on human gastric carcinoma cells through apoptosis induction and JNK/p38 MAPK signaling activation

Gastric cancer is a common malignancy in many countries of the world, especially in Asia. Prevention is likely to be the most effective means of not only reducing the incidence but also mortality from this disease. The term 'chemoprevention' has been referred to the prevention of cancer using specific agents to suppress or reverse the carcinogenic process. In recent years, attention has been focused on the anticancer properties of edible plants, an important role in the prevention of disease. Hibiscus sabdariffa Linne (Malvaceae), an attractive plant believed to be native to Africa, is cultivated in the Sudan and Eastern Taiwan. The purpose of this study was to examine whether the plant, H. sabdariffa extracts (HSE), affects the apoptosis of AGS cells. Using a set of apoptotic detection assays, they showed that HSE induced cytotoxicity and apoptosis of AGS cells in a concentration-dependent manner but is ineffective in Chang liver cells. The result also revealed increased phosphorylation in p38, JNK and c-Jun, cytochrome c release, and expression of Fas, FasL, Bax, and t-Bid in the HSE-treated AGS cells. We further used MAPK inhibitors to evaluate their effect on the HSE-induced AGS death. The data showed that SB203580 (p38 inhibitor), JNK inhibitor I and II or transfection with the mutant JNK expression vector had strong potential in inhibiting AGS cells apoptosis and related proteins expression. Finally, we suggested that HSE mediated AGS apoptosis via the JNK/p38 signaling cascade. According to these results, HSE could be developed as a chemopreventive agent.     

Vitamin K3-2,3-epoxide induction of apoptosis with activation of ROS-dependent ERK and JNK protein phosphorylation in human glioma cells

2-Methyl-1,4-naphthoquinone (menadione or vitamin K3; EPO) and K3-2,3-epoxide (EPO1), but not vitamin K3-3-OH (EPO2), exhibited cytotoxicity that caused DNA fragmentation and chromatin condensation in U87 and C6 cells. EPO1 showed more-potent cytotoxicity than EPO, and the IC₅₀ values of EPO and EPO1 in U87 cells were 37.5 and 15.7 μM, respectively. Activation of caspase 3 enzyme activity with cleavage of caspase 3 protein was detected in EPO1-treated U87 and C6 cells, and the addition of the caspase 3 peptidyl inhibitor, DEVD-FMK, reduced the cytotoxic effect of EPO1. An increase in the intracellular ROS level by EPO1 was observed in the DCHF-DA analysis, and EPO1-induced apoptosis and caspase 3 protein cleavage were prevented by adding the antioxidant, N-acetyl-cysteine (NAC), with decreased ROS production elicited by EPO1. Activation of ERK and JNK, but not p38, via phosphorylation induction was identified in EPO1- but not EPO- or EPO2-treated U87 and C6 cells, and this was blocked by adding NAC. However, the ERK inhibitor, PD98059, and the JNK inhibitor, SP600125, showed no effect on EPO1-induced cytotoxicity in either cell type. Our findings demonstrate that 2,3-epoxide substitution significantly potentiates the apoptotic effect of vitamin K3 via stimulating ROS production, which may be useful in the chemotherapy of glioblastoma cells.     

42- and 63-bp anti-MDR1-siRNAs bearing 2′-OMe modifications in nuclease-sensitive sites induce specific and potent gene silencing

DsRNAs longer than 30 bp induce interferon response and global changes in gene expression profile in mammalians. 21 bp siRNA and 25/27 bp dsiRNA acting via RNA interference mechanism are used for specific gene silencing in this class of organisms. We designed selectively 2′-O-methyl-modified 42 and 63 bp anti-MDR1-siRNAs that silence the expression of P-glycoprotein and restore the sensitivity of drug-resistant cancer cells to cytostatic more efficiently than canonical 21 bp siRNAs. We also show that they act in a Dicer-independent mode and are devoid of immunostimulating properties. Our findings suggest that 42 and 63 bp siRNAs could be used as potential therapeutics.     

Bufalin induces autophagy-mediated cell death in human colon cancer cells through reactive oxygen species generation and JNK activation

Colorectal cancer is the second most common cause of cancer death in the world and about half of the patients with colorectal cancer require adjuvant therapy after surgical resection. Therefore, the eradication of cancer cells via chemotherapy constitutes a viable approach to treating patients with colorectal cancer. In this study, the effects of bufalin isolated from a traditional Chinese medicine were evaluated and characterized in HT-29 and Caco-2 human colon cancer cells. Contrary to its well-documented apoptosis-promoting activity in other cancer cells, bufalin did not cause caspase-dependent cell death in colon cancer cells, as indicated by the absence of significant early apoptosis as well as poly(ADP-ribose) polymerase and caspase-3 cleavage. Instead, bufalin activated an autophagy pathway, as characterized by the accumulation of LC3-II and the stimulation of autophagic flux. The induction of autophagy by bufalin was linked to the generation of reactive oxygen species (ROS). ROS activated autophagy via the c-Jun NH₂-terminal kinase (JNK). JNK activation increased expression of ATG5 and Beclin-1. ROS antioxidants (N-acetylcysteine and vitamin C), the JNK-specific inhibitor SP600125, and JNK2 siRNA attenuated bufalin-induced autophagy. Our findings unveil a novel mechanism of drug action by bufalin in colon cancer cells and open up the possibility of treating colorectal cancer through a ROS-dependent autophagy pathway.     

Differential requirement of MKK4 and MKK7 in JNK activation by distinct scaffold proteins

Different scaffold proteins play distinct roles in various signaling pathways by recruiting different downstream molecules. Here, using MKK4(-/-) and MKK4(-/-)/7(-/-) murine embryonic fibroblast cells, we examined differential employment of MKK4 and MKK7 by scaffold proteins Axin, Dvl, and Epstein-Barr virus latent membrane protein-1 (LMP-1) in mediating JNK activation. We present evidence that Axin depends mainly on MKK7 for activation of JNK, while Dvl depends almost equally on MKK4 and MKK7 for JNK activation, In contrast, LMP-1-induced JNK activation is primarily dependent on MKK4. Our results demonstrate that Axin, Dvl, and LMP-1 differentially utilize MKK4 and MKK7 for JNK activation.     

Cadmium activates CaMK-II and initiates CaMK-II-dependent apoptosis in mesangial cells

Cadmium is a toxic metal that initiates both mitogenic responses and cell death. We show that Cd(2+) increases phosphorylation and activity of Ca(2+)/calmodulin-dependent protein kinase II (CaMK-II) in mesangial cells, in a concentration-dependent manner. Activation is biphasic with peaks at 1-5 min and 4-6 h. Cadmium also activates Erk, but this appears to be independent of CaMK-II. At 10-20 microM, Cd(2+) initiates apoptosis in 25-55% of mesangial cells by 6h. Inhibition of CaMK-II, but not of Erk, suppresses Cd(2+)-induced apoptosis. We conclude that activation of CaMK-II by Cd(2+) contributes to apoptotic cell death, independent of Erk activation.     

Activation of Bak in ultrasound-induced, JNK- and p38-independent apoptosis and its inhibition by Bcl-2

The molecular mechanisms underlying ultrasound-induced apoptosis remain poorly understood. We have demonstrated that in Jurkat cells, the over-expression of the anti-apoptotic protein Bcl-2 inhibited ultrasound-induced apoptosis, but not necrosis. Inhibition of caspase activity also protected the cells from apoptosis, but not from necrosis, showing the involvement of different mechanisms in ultrasound-induced apoptosis and necrosis. Bak, a pro-apoptotic member of the Bcl-2 family proteins, was activated by ultrasound and its activation was completely inhibited by Bcl-2 over-expression, but not by caspase inhibition. Antioxidant N-acetyl cysteine did not protect the cells from ultrasound-induced apoptosis or necrosis, nor did the inhibition of either c-Jun N-terminal kinase or p38, key factors in the radical oxygen species (ROS)-mediated cell stress response, suggesting that ROS do not play a crucial role in ultrasound-induced apoptosis. Our results confirm that ultrasound induces apoptosis via a pathway that involves Bak, Bcl-2, and caspases, but not ROS.     

Differential regulation of CIDEA and CIDEC expression by insulin via Akt1/2- and JNK2-dependent pathways in human adipocytes

Both insulin and the cell death-inducing DNA fragmentation factor-α-like effector (CIDE) family play important roles in apoptosis and lipid droplet formation. Previously, we reported that CIDEA and CIDEC are differentially regulated by insulin and contribute separately to insulin-induced anti-apoptosis and lipid droplet formation in human adipocytes. However, the upstream signals of CIDE proteins remain unclear. Here, we investigated the signaling molecules involved in insulin regulation of CIDEA and CIDEC expression. The phosphatidylinositol 3-kinase (PI3K) inhibitors wortmannin and PI-103 blocked both insulin-induced downregulation of CIDEA and upregulation of CIDEC. The Akt inhibitor API-2 and the c-Jun N-terminal kinase (JNK) inhibitor SP600125 selectively inhibited insulin regulation of CIDEA and CIDEC expression, respectively, whereas the MAPK/ERK kinase inhibitor U0126 and the p38 inhibitor SB203580 did not. Small interfering RNA-mediated depletion of Akt1/2 prevented insulin-induced downregulation of CIDEA and inhibition of apoptosis. Depletion of JNK2, but not JNK1, inhibited insulin-induced upregulation of CIDEC and lipid droplet enlargement. Furthermore, insulin increased both Akt and JNK phosphorylation, which was abrogated by the PI3K inhibitors. These results suggest that insulin regulates CIDEA and CIDEC expression via PI3K, and it regulates expression of each protein via Akt1/2- and JNK2-dependent pathways, respectively, in human adipocytes.     

(2Alpha,3beta)-2,3-dihydroxyolean-12-en-28-oic acid, a new natural triterpene from Olea europea, induces caspase dependent apoptosis selectively in colon adenocarcinoma cells

Triterpenoids are known to induce apoptosis and to be anti-tumoural. Maslinic acid, a pentacyclic triterpene, is present in high concentrations in olive pomace. This study examines the response of HT29 and Caco-2 colon-cancer cell lines to maslinic-acid treatment. At concentrations inhibiting cell growth by 50-80% (IC50HT29=61+/-1 microM, IC80HT29=76+/-1 microM and IC50Caco-2=85+/-5 microM, IC80Caco-2=116+/-5 microM), maslinic acid induced strong G0/G1 cell-cycle arrest and DNA fragmentation, and increased caspase-3 activity. However, maslinic acid did not alter the cell cycle or induce apoptosis in the non-tumoural intestine cell lines IEC-6 and IEC-18. Moreover, maslinic acid induced cell differentiation in colon adenocarcinoma cells. These findings support a role for maslinic acid as a tumour suppressant and as a possible new therapeutic tool for aberrant cell proliferation in the colon. In this report, we demonstrate for the first time that, in tumoural cancer cells, maslinic acid exerts a significant anti-proliferation effect by inducing an apoptotic process characterized by caspase-3 activation by a p53-independent mechanism, which occurs via mitochondrial disturbances and cytochrome c release.     

The JNK inhibitor SP600125 enhances dihydroartemisinin-induced apoptosis by accelerating Bax translocation into mitochondria in human lung adenocarcinoma cells

The C-Jun N-terminal Kinase (JNK) inhibitor SP600125 is widely used to inhibit the JNK-mediated Bax activation and cell apoptosis. However, this report demonstrates that SP600125 synergistically enhances the dihydroartemisinin (DHA)-induced human lung adenocarcinoma cell apoptosis by accelerating Bax translocation and subsequent intrinsic apoptotic pathway involving mitochondrial membrane depolarization, cytochrome c release, caspase-9 and caspase-3 activation. The dynamical analysis of GFP-Bax mobility inside single living cells using fluorescence recovery after photobleaching revealed that SP600125 aggravated the DHA-induced decrease of Bax mobility and Bax translocation. These results for the first time present a novel pro-apoptotic action of SP600125 in DHA-induced apoptosis.     

Src mediates ERK reactivation in gefitinib resistance in non-small cell lung cancer

To study epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) resistance mechanisms, we established a novel gefitinib-resistant lung cancer cell line derived from an EGFR-mutant non-small cell lung cancer cell line (PC-9) pretreated with 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (designated PC9-GR). We found that gefitinib substantially suppressed the EGFR signaling pathway, whereas ERK was reactivated after several hours in PC9-GR but not in PC-9. The combination of gefitinib with ERK inhibition (by U0126) restored gefitinib susceptibility in PC9-GR, but PI3K-Akt inhibition with LY294002 did not. Although the levels of phosphorylated Src were up-regulated simultaneously with ERK reactivation, neither ERK suppression using U0126 nor an ERK-specific siRNA induced Src phosphorylation. Furthermore, dual inhibition of EGFR and Src restored gefitinib sensitivity in PC9-GR in vitro and in vivo. In conclusion, our results indicate that Src-mediated ERK reactivation may play a role in a novel gefitinib resistance mechanism, and that the combined use of gefitinib with a Src inhibitor may be a potent strategy to overcome this resistance.     

Synthesis and in vitro anti-HSV-1 activity of a novel Hsp90 inhibitor BJ-B11

In this study, a novel Hsp90 inhibitor BJ-B11, was synthesized and evaluated for in vitro antiviral activity against several viruses. Possible anti-HSV-1 mechanisms were also investigated. BJ-B11 displayed no antiviral activity against coxsackievirus B₃ (CVB₃), human respiratory syncytial virus (RSV) and influenza virus (H1N1), but exhibited potent anti-HSV-1 and HSV-2 activity with EC₅₀ values of 0.42 ± 0.18 μM and 0.60 ± 0.21 μM, respectively. Additionally, the inhibitory effects of BJ-B11 against HSV-1 were likely to be introduced at early stage of infection. Our results indicate that BJ-B11 with alternative mechanisms of action is potent as an anti-HSV clinical trial candidate.