Yong-Jin Lee

Reactive oxygen species and PI3K/Akt signaling play key roles in the induction of Nrf2-driven heme oxygenase-1 expression in sulforaphane-treated human mesothelioma MSTO-211H cells

The nuclear factor erythroid-derived 2 related factor 2 (Nrf2)/heme oxygenase (HO)-1 induction plays cytoprotective roles against oxidative injury, apoptosis, and anticancer therapy; however, little is known about its regulation in human mesothelioma MSTO-211H cells. In this study, we investigated Nrf2/HO-1 induction in response to sulforaphane and determined the signaling pathways involved in this process. Sulforaphane treatment decreased cell viability and triggered a rapid and transient increase in the intracellular ROS levels. Pretreatment with N-acetylcysteine (NAC) prevented sulforaphane-induced cytotoxicity. Erk1/2 was activated within 1h of sulforaphane addition, whereas Akt phosphorylation was suppressed until the first 8h, and was then maintained at an elevated level until 72h, displaying a biphasic regulatory feature. Nrf2 protein levels in both nuclear and whole cell lysates were increased after sulforaphane treatment and were decreased by pretreatment with NAC, actinomycin D and cycloheximide. Activation of the Nrf2/HO-1 system after sulforaphane treatment was suppressed by pretreatment with NAC or Ly294002, a PI3K inhibitor. Knockdown of Nrf2 with siRNA decreased cell viability and attenuated sulforaphane-induced HO-1 up-regulation. Overall, our results indicate that ROS generation and/or activation of PI3K/Akt signaling regulate cell survival and Nrf2-driven HO-1 expression in sulforaphane-treated MSTO-211H cells.

Synergistic anti-cancer effects of resveratrol and chemotherapeutic agent clofarabine against human malignant mesothelioma MSTO-211H cells.

Dietary phytochemicals as adjuvants have been suggested to play important roles in enhancing chemotherapeutic potential owing to multitargeted chemopreventive properties and lack of substantial toxicity. Here, we investigated the efficacy of the combined treatment of various phytochemicals with the anticancer drug clofarabine in malignant mesothelioma MSTO-211H cells and normal mesothelial MeT-5A cells. The combined treatment of resveratrol and clofarabine produced a synergistic antiproliferative effect in MSTO-211H cells, but not in MeT-5A cells. In MSTO-211H cells, the nuclear accumulation of Sp1 and the levels of p-Akt, Sp1, c-Met, cyclin D1, and p21 were effectively decreased by the combined treatment of them. In combination with clofarabine, the ability of resveratrol to reduce the contents of Sp1 and its target gene products was also evident in a time- and dose-dependent experiment. The inhibition of phosphoinositide 3-kinase using Ly294002 augmented a decrease in the p21 level induced by their combination, but it showed no significant effects on expression of Sp1 and cyclin D1. Taken together, the data provide evidence that the synergistic antiproliferative effect of resveratrol and clofarabine is linked to the inhibition of Akt and Sp1 activities, and suggest that this combination may have therapeutic value in treatment of malignant mesothelioma.

The flavonoid resveratrol suppresses growth of human malignant pleural mesothelioma cells through direct inhibition of specificity protein 1

Resveratrol (Res), from the skin of red grapes, induces apoptosis in some malignant cells, but there are no reports on the apoptotic effect of Res on human malignant pleural mesothelioma. We found that Res interacts with specificity protein 1 (Sp1). The IC50 for Res was 17 µM in MSTO-211H cells. Cell viability was decreased and apoptotic cell death was increased by Res (0-60 µM). Res increased the Sub-G1 population in MSTO-211H cells and significantly suppressed Sp1 protein levels, but not Sp1 mRNA levels. Res modulated the expression of Sp1 regulatory proteins including p21, p27, cyclin D1, Mcl-1 and survivin in mesothelioma cells. After treatment with Res, apoptosis signaling cascades were activated by the activation of Bid, Bim, caspase-3 and PARP, upregulation of Bax and downregulation of Bcl-xL. Res (20 mg/kg daily for 4 weeks) effectively suppressed tumor growth in vivo in BALB/c athymic (nu+/nu+) mice injected with MSTO-211H cells, an effect that was mediated by inhibition of Sp1 expression and induction of apoptotic cell death. Our results strongly suggest that Sp1 is a novel molecular target of Res in human malignant pleural mesothelioma.

Synergistic inhibition of mesothelioma cell growth by the combination of clofarabine and resveratrol involves Nrf2 downregulation

We previously reported that MSTO-211H cells have a higher capacity to regulate Nrf2 activation in response to changes in the cellular redox environment. To further characterize its biological significance, the response of Nrf2, a transcription factor that regulates ARE-containing genes, on the synergistic cytotoxic effect of clofarabine and resveratrol was investigated in mesothelioma cells. The combination treatment showed a marked growth-inhibitory effect, which was accompanied by suppression of Nrf2 activation and decreased expression of heme oxygenase-1 (HO-1). While transient overexpression of Nrf2 conferred protection against the cytotoxicity caused by their combination, knockdown of Nrf2 expression using siRNA enhanced their cytotoxic effect. Pretreatment with Ly294002, a PI3K inhibitor, augmented the decrease in HO-1 level by their combination, whereas no obvious changes were observed in Nrf2 levels. Altogether, these results suggest that the synergistic cytotoxic effect of clofarabine and resveratrol was mediated, at least in part, through suppression of Nrf2 signaling. [BMB Reports 2012; 45(11): 647-652]

Hesperidin Induces Apoptosis by Inhibiting Sp1 and Its Regulatory Protein in MSTO-211H Cells

Hesperidin, a flavanone present in citrus fruits, has been studied as potential therapeutic agents that have anti-tumor activity and apoptotic effects in several cancers, but there is no report about the apoptotic effect of hesperidin in human malignant pleural mesothelioma through the specificity protein 1 (Sp1) protein. We investigated whether hesperidin inhibited cell growth and regulated Sp1 target proteins by suppressing the levels of Sp1 protein in MSTO-211H cells. The IC50 value of hesperidin was determined to be 152.3 μM in MSTO-211H cells for 48 h. Our results suggested that hesperidin (0-160 μM) decreased cell viability, and induced apoptotic cell death. Hesperidin increased Sub-G1 population in MSTO-211H cells. Hesperidin significantly suppressed mRNA/protein level of Sp1 and modulated the expression level of the Sp1 regulatory protein such as p27, p21, cyclin D1, Mcl-1, and survivin in mesothelioma cells. Also, hesperidin induced apoptotic signaling including: cleavages of Bid, caspase-3, and PARP, upregulation of Bax, and down-regulation of Bcl-xl in mesothelioma cells. These results show that hesperidin suppressed mesothelioma cell growth through inhibition of Sp1. In this study, we demonstrated that Sp1 acts as a novel molecular target of hesperidin in human malignant pleural mesothelioma.

Resveratrol contributes to chemosensitivity of malignant mesothelioma cells with activation of p53.

Resveratrol is a naturally occurring polyphenolic phytoalexin with chemopreventive properties. We previously reported a synergistic anti-proliferative effect of resveratrol and clofarabine against malignant mesothelioma (MM) cells. Here, we further investigated molecular mechanisms involved in the synergistic interaction of these compounds in MM MSTO-211H cells. Resveratrol, in combination with clofarabine, time-dependently induced a strong cytotoxic effect with the nuclear accumulation of phospho-p53 (p-p53) in MSTO-211H cells, but not in normal mesothelial MeT-5A cells. Combination treatment up-regulated the levels of p-p53, cleaved caspase-3, and cleaved PARP proteins. Gene silencing with p53-targeting siRNA attenuated the sensitivity of cells to the combined treatment of two compounds. Analyses of p53 DNA binding assay, p53 reporter gene assay, and RTP-CR toward p53-regulated genes, including Bax, PUMA, Noxa and p21, demonstrated that induced p-p53 is transcriptionally active. These results were further confirmed by the siRNA-mediated knockdown of p53 gene. Combination treatment significantly caused the accumulation of cells at G1 phase with the increases in the sub-G0/G1 peak, DNA ladder, nuclear fragmentation, and caspase-3/7 activity. Taken together, these results demonstrate that resveratrol and clofarabine synergistically elicit apoptotic signal via a p53-dependent pathway, and provide a scientific rationale for clinical evaluation of resveratrol as a promising chemopotentiator in MM.

Knockdown of Bcl-xL Enhances Growth-Inhibiting and Apoptosis-Inducing Effects of Resveratrol and Clofarabine in Malignant Mesothelioma H-2452 Cells

Mcl-1 and Bcl-xL, key anti-apoptotic proteins of the Bcl-2 family, have attracted attention as important molecules in the cell survival and drug resistance. In this study, we investigated whether inhibition of Bcl-xL influences cell growth and apoptosis against simultaneous treatment of resveratrol and clofarabine in the human malignant mesothelioma H-2452 cells. Resveratrol and clofarabine decreased Mcl-1 protein levels but had little effect on Bcl-xL levels. In the presence of two compounds, any detectable change in the Mcl-1 mRNA levels was not observed in RT-PCR analysis, whereas pretreatment with the proteasome inhibitor MG132 led to its accumulation to levels far above basal levels. The knockdown of Bcl-xL inhibited cell proliferation with cell accumulation at G2/M phase and the appearance of sub-G0/G1 peak in DNA flow cytometric assay. The suppression of cell growth was accompanied by an increase in the caspase-3/7 activity with the resultant cleavages of procaspase-3 and its substrate poly (ADP-ribose) polymerase, and increased percentage of apoptotic propensities in annexin V binding assay. Collectively, our data represent that the efficacy of resveratrol and clofarabine for apoptosis induction was substantially enhanced by Bcl-xL-lowering strategy in which the simultaneous targeting of Mcl-1 and Bcl-xL could be a more effective strategy for treating malignant mesothelioma. Graphical Abstract

Resveratrol and clofarabine induces a preferential apoptosis-activating effect on malignant mesothelioma cells by Mcl-1 down-regulation and caspase-3 activation.

We previously demonstrated that resveratrol and clofarabine elicited a marked cytotoxicity on malignant mesothelioma (MM) MSTO-211H cells but not on the corresponding normal mesothelial MeT-5A cells. Little is known of the possible molecules that could be used to predict preferential chemosensitivity on MSTO-211H cells. Resveratrol and clofarabine induced down-regulation of Mcl-1 protein level in MSTO-211H cells. Treatment of cells with cycloheximide in the presence of proteasome inhibitor MG132 suggested that Mcl-1 protein levels were regulated at the post-translational step. The siRNA-based knockdown of Mcl-1 in MSTO-211H cells triggered more growth-inhibiting and apoptosis-inducing effects with the resultant cleavages of procaspase-3 and its substrate PARP, increased caspase-3/7 activity, and increased percentage of apoptotic propensities. However, the majority of the observed changes were not shown in MeT-5A cells. Collectively, these studies indicate that the preferential activation of caspase cascade in malignant cells might have important applications as a therapeutic target for MM. [BMB Reports 2015; 48(3): 166-171]

ERK1/2 activation in quercetin-treated BEAS-2B cell plays a role in Nrf2-driven HO-1 expression

Quercetin, a member of the flavonoids, has been heralded as chemopreventive agent, generally because of its free radical-scavenging and antiproliferative activities. In this process, NF-E2-related factor-2 (Nrf2) plays a role in protecting cells from oxidative damage; however, the exact mechanisms in which bronchial epithelial cells respond to quercetin by activating Nrf2 are not fully understood. Here, we report the role of extracellular signal-regulated kinase (ERK) activation in response to quercetin. Quercetin treatment decreased the viability of human bronchial epithelial BEAS-2B cells in a dose- and time-dependent manner. ERK1/2 was activated within 10 min of quercetin addition and then remained increased above control level throughout 48 h, whereas AKT phosphorylation was suppressed from the first 1 h of quercetin treatment. Inhibition of Erk1/2 phosphorylation attenuated quercetin-induced loss of cell viability. Nrf2 levels in both nuclear and whole cell lysates increased after quercetin treatment and was accompanied by the increase of heme oxygenase-1 (HO-1) protein level, indicating that increased HO-1 expression is Nrf2-mediated. Activation of the Nrf2/HO-1 system after quercetin treatment was suppressed by the pretreatment with MEK inhibitor, PD98059. Also, silencing Nrf2 with siRNA decreased cell viability and augmented cytotoxic effect of anticancer drug LBH589. Overall, our results indicate that quercetin induces the up-regulation of Nrf2-mediated HO-1 expression, at least in part, via MEK/ERK1/2 signaling, as an adaptive mechanism which may provide advantages for cell proliferation and enhance survival despite endogenous and/or exogenous oxidative insults.

Quercetin exerts preferential cytotoxic effects on malignant mesothelioma cells by inducing p53 expression, caspase-3 activation, and apoptosis

Quercetin, a naturally occurring flavonoid, has been heralded as a promising chemopreventive agent. This study was undertaken to probe the molecular mechanisms underlying the anti-cancer activity of quercetin in malignant mesothelioma (MM) cells. Quercetin at low doses elicited apoptotic cell death on MM MSTO-211H cells, as signified by pyknotic and fragmented nuclei, increased annexin V binding, and increased proportion of cells with hypodiploid DNA. Preceding these changes, quercetin induced up-regulation of p53 at both mRNA and protein levels without altering its ubiquitination, and increased caspase-3/7 activity with the resultant cleavages of procaspase-3 and PARP. Analyses of nuclear p53 level, p53 reporter gene, and RT-PCR toward p53-regulated genes demonstrated that induced p53 is transcriptionally active. The proportion of cells at sub-G0/G1 peak and G2/M phase increased in a quercetin concentration-dependent fashion, which were blocked by the pan-caspase inhibitor Z-VAD. Additionally, quercetin and gemcitabine produced a significant synergistic effect on inhibiting MS-TO-211H cell growth. Given that quercetin induced preferential p53-upregulating, growth-inhibiting, and apoptosis-activating effects on MM cells, the use of quercetin may be a potential therapeutic strategy for enhancing anti-cancer efficacy of existing chemotherapy in MM.