Ji Hoon Jung

Suppression of STAT3 and HIF-1 alpha mediates anti-angiogenic activity of betulinic acid in hypoxic PC-3 prostate cancer cells.

Background Signal transducer and activator of transcription 3 (STAT3) is a transcription factor that regulates various cellular processes such as cell survival, angiogenesis and proliferation. In the present study, we examined that betulinic acid (BA), a triterpene from the bark of white birch, had the inhibitory effects on hypoxia-mediated activation of STAT3 in androgen independent human prostate cancer PC-3 cells. Methodology/Principal Findings BA inhibited the protein expression and the transcriptional activities of hypoxia-inducible factor-1α (HIF-1α) under hypoxic condition. Consistently, BA blocked hypoxia-induced phosphorylation, DNA binding activity and nuclear accumulation of STAT3. In addition, BA significantly reduced cellular and secreted levels of vascular endothelial growth factor (VEGF), a critical angiogenic factor and a target gene of STAT3 induced under hypoxia. Furthermore, BA prevented in vitro capillary tube formation in human umbilical vein endothelial cells (HUVECs) maintained in conditioned medium of hypoxic PC-3 cells, implying anti-angiogenic activity of BA under hypoxic condition. Of note, chromatin immunoprecipitation (ChiP) assay revealed that BA inhibited binding of HIF-1α and STAT3 to VEGF promoter. Furthermore, silencing STAT3 using siRNA transfection effectively enhanced the reduced VEGF production induced by BA treatment under hypoxia. Conclusions/Significance Taken together, our results suggest that BA has anti-angiogenic activity by disturbing the binding of HIF-1α and STAT3 to the VEGF promoter in hypoxic PC-3 cells.

Activation of reactive oxygen species/AMP activated protein kinase signaling mediates fisetin-induced apoptosis in multiple myeloma U266 cells

We investigated the molecular mechanisms responsible for fisetin-induced apoptosis in U266 cells. Fisetin elicited the cytotoxicity in U266 cells, manifested as an increased fraction of the cells with sub-G1 content or stained positively with TUNEL labeling. Fisetin enhanced caspase-3 activation, downregulation of Bcl-2 and Mcl-1(L), and upregulation of Bax, Bim and Bad. Fisetin activated AMPK as well as its substrate acetyl-CoA carboxylase (ACC), along with a decreased phosphorylation of AKT and mTOR. Fisetin also stimulated generation of ROS in U266 cells. Conversely, compound C or N-acetyl-L-cystein blocked fisetin-induced apoptosis. Our data suggest that fisetin-induced apoptosis in U266 cells is through ROS and AMPK pathways.

Brazilin Induces Apoptosis and G2/M Arrest via Inactivation of Histone Deacetylase in Multiple Myeloma U266 Cells

Although brazilin [7,11b-dihydrobenz(b)indeno[1,2-d]pyran-3,6a,9,10(6H)-tetrol] isolated from Caesalpinia sappan was known to have various biological activities, including anti-inflammation, antibacteria, and antiplatelet aggregation, there is no report yet on its anticancer activity. In the present study, the anticancer mechanism of brazilin was elucidated in human multiple myeloma U266 cells. We found that brazilin significantly inhibited the activity of histone deacetylases (HDACs), transcription factors involved in the regulation of apoptosis and cell cycle arrest in U266 cells. Consistently, brazilin enhanced acetylation of histone H3 at Lys 23, indicating activation of histone acetyltransferase (HAT), and also suppressed the expressions of HDAC1 and HDAC2 at both protein and mRNA levels. Additionally, brazilin significantly increased the number of sub-G1 cell population and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells undergoing apoptosis and also activated caspase-3 and regulated the expression of Bcl-2 family proteins, including Bax, Bcl-x(L), and Bcl-2 in U266 cells, indicating that brazilin induces apoptosis through the mitochondria-dependent pathway. Interestingly, cell cycle analysis revealed that brazilin induced G2/M phase arrest along with apoptosis induction. Consistently, brazilin attenuated the expression of cyclin-dependent kinases (CDKs), such as cyclin D1, cyclin B1, and cyclin E, and also activated p21 and p27 in U266 cells. Furthermore, HAT inhibitor anacardic acid reversed activation of acetyl-histone H3 and cleavage of PARP induced by brazilin, while pan-caspase inhibitor Z-VAD-FMK001 did not affect the expression of HDAC induced by brazilin that brazilin mediates apoptosis via inactivation of HDAC in U266 cells. Notably, brazilin significantly potentiated the cytotoxic effect of standard chemotherapeutic agents, such as bortezomib or doxorubicin, in U266 cells. When our findings are taken together, they suggest that brazilin has potential as a chemotherapeutic agent alone or in combination with an anticancer agent for multiple myeloma treatment.

Icariside II Induces Apoptosis in U937 Acute Myeloid Leukemia Cells: Role of Inactivation of STAT3-Related Signaling

Background The aim of this study is to determine anti-cancer effect of Icariside II purified from the root of Epimedium koreanum Nakai on human acute myeloid leukemia (AML) cell line U937. Methodology/Principal Findings Icariside II blocked the growth U937 cells in a dose- and time-dependent manner. In this anti-proliferation process, this herb compound rendered the cells susceptible to apoptosis, manifested by enhanced accumulation of sub-G1 cell population and increased the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells. Icariside II was able to activate caspase-3 and cleaved poly (ADP-ribose) polymerase (PARP) in a time-dependent manner. Concurrently, the anti-apoptotic proteins, such as bcl-xL and survivin in U937 cells, were downregulated by Icariside II. In addition, Icariside II could inhibit STAT3 phosphorylation and function and subsequently suppress the activation of Janus activated kinase 2 (JAK2), the upstream activators of STAT3, in a dose- and time-dependent manner. Icariside II also enhanced the expression of protein tyrosine phosphatase (PTP) SH2 domain-containing phosphatase (SHP)-1, and the addition of sodium pervanadate (a PTP inhibitor) prevented Icariside II-induced apoptosis as well as STAT3 inactivation in STAT3 positive U937 cells. Furthermore, silencing SHP-1 using its specific siRNA significantly blocked STAT3 inactivation and apoptosis induced by Icariside II in U937 cells. Conclusions/Significance Our results demonstrated that via targeting STAT3-related signaling, Icariside II sensitizes U937 cells to apoptosis and perhaps serves as a potent chemotherapeutic agent for AML.

Translationally controlled tumor protein induces human breast epithelial cell transformation through the activation of Src.

Translationally controlled tumor protein (TCTP) is implicated in cell growth and malignant transformation. TCTP has been found to interact directly with the third cytoplasmic domain of the α subunit of Na,K-ATPase, but whether this interaction has a role in tumorigenesis is unclear. In this study, we examined TCTP-induced tumor progression signaling networks in human breast epithelial cells, using adenoviral infection. We found that TCTP (a) induces Src release from Na,K-ATPase α subunit and Src activation; (b) phosphorylates tyrosine residues 845, 992, 1086, 1148 and 1173 on anti-epidermal growth factor receptor (EGFR); (c) activates PI3K (phosphatidylinositol 3-kinase )–AKT, Ras–Raf–MEK–ERK1/2, Rac–PAK1/2, MKK3/6–p38 and phospholipase C (PLC)-γ pathways; (d) enhances NADPH oxidase-dependent reactive oxygen species (ROS) generation; (e) stimulates cytoskeletal remodeling and cell motility and (f) upregulates matrix metalloproteinase (MMP) 3 and 13. These findings suggest that TCTP induces tumorigenesis through distinct multicellular signaling pathways involving Src-dependent EGFR transactivation, ROS generation and MMP expression.

Janus activated kinase 2/signal transducer and activator of transcription 3 pathway mediates icariside II-induced apoptosis in U266 multiple myeloma cells

Although the flavonoid icariside II exhibits anti-inflammatory and anti-cancer activities, its molecular targets/pathways in human multiple myeloma cells are poorly understood. To analyze the effects on signal transducer and activator of transcription 3 (STAT3) signaling and apoptosis, U266 multiple myeloma cells were treated with icariside II and performed Western blotting, electrophoretic mobility gel shift assay (EMSA), RT-PCR, proliferation assay, cell cycle analysis and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Icariside II inhibited STAT3 activation and enhanced the expression of SHP-1 and PTEN through inhibiting Janus activated kinase 2 (JAK2) and c-Src. Icariside II down-regulated the expression of STAT3 target genes Bcl-2, Bcl-x(L), survivin, cyclin D(1), COX-2 and vascular endothelial growth factor (VEGF). Also, icariside II enhanced poly (ADP-ribose) polymerase (PARP) cleavage and caspase-3 activation. Pervanadate reversed the icariside II-mediated STAT3 inactivation and also blocked the cleavages of caspase-3 and PARP, suggesting involvement of STAT3 pathway in icariside II-induced apoptosis. Furthermore, icariside II enhanced the apoptotic effects of clinically used drugs thalidomide and bortezomib in U266 cells. Icariside II could be a potential therapeutic intervention agent alone or in combination with current drugs for multiple myeloma as a novel blocker of STAT3 signaling cascades at multiple levels, contributing to its anti-proliferative and anti-apoptosis.

Tanshinone IIA Induces Autophagic Cell Death via Activation of AMPK and ERK and Inhibition of mTOR and p70 S6K in KBM-5 Leukemia Cells

Although tanshinone IIA (Tan IIA) from Salviae miltiorrhizae was known to induce apoptosis in various cancers, its underlying mechanism of autophagic cell death was not reported yet. Thus, in the present study, the molecular mechanism of autophagic cell death by Tan IIA was investigated in KBM‐5 leukemia cells. Tan IIA significantly increased the expression of microtubule‐associated protein light chain 3 (LC3) II as a hallmark of autophagy in western blotting and immunofluorescence staining. Tan IIA augmented the phosphorylation of adenosine monophosphate‐activated protein kinase (AMPK) and attenuated the phosphorylation of mammalian target of rapamycin (mTOR) and p70 S6K in a dose‐dependent manner. Conversely, autophagy inhibitor 3‐methyladenine partly reversed the cytotoxicity and the phosphorylation of AMPK, mTOR and p70 S6K induced by Tan IIA in KBM‐5 leukemia cells. In addition, Tan IIA dramatically activated the extracellular signal regulated kinase (ERK) signaling pathway including Raf, ERK and p90 RSK in a dose‐dependent and time‐dependent manner. Consistently, ERK inhibitor PD184352 suppressed LC3‐II activation induced by Tan IIA, whereas PD184352 and PD98059 did not affect poly (ADP‐ribose) polymerase cleavage and sub‐G1 accumulation induced by Tan IIA in KBM‐5 leukemia cells. Furthermore, Tan IIA could induce autophagy via LC3‐II activation in various cancer cells such as prostate (PC‐3), multiple myeloma (U266), lung (NCI‐H460), and breast (MDA‐MB‐231) cells. Overall, these findings suggest that Tan IIA induces autophagic cell death via activation of AMPK and ERK and inhibition of mTOR and p70 S6K in KBM‐5 cells as a potent natural compound for leukemia treatment. Copyright

Inhibition of STAT3 signaling and induction of SHP1 mediate antiangiogenic and antitumor activities of ergosterol peroxide in U266 multiple myeloma cells

BackgroundErgosterol peroxide (EP) derived from edible mushroom has been shown to exert anti-tumor activity in several cancer cells. In the present study, anti-angiogenic activity of EP was investigated with the underlying molecular mechanisms in human multiple myeloma U266 cells.ResultsDespite weak cytotoxicity against U266 cells, EP suppressed phosphorylation, DNA binding activity and nuclear translocalization of signal transducer and activator of transcription 3 (STAT3) in U266 cells at nontoxic concentrations. Also, EP inhibited phosphorylation of the upstream kinases Janus kinase 2 (JAK2) and Src in a time-dependent manner. Furthermore, EP increased the expression of protein tyrosine phosphatase SHP-1 at protein and mRNA levels, and conversely silencing of the SHP-1 gene clearly blocked EP-mediated STAT3 inactivation. In addition, EP significantly decreased vascular endothelial growth factor (VEGF), one of STAT3 target genes at cellular and protein levels as well as disrupted in vitro tube formation assay. Moreover, EP significantly suppressed the growth of U266 cells inoculated in female BALB/c athymic nude mice and immunohistochemistry revealed that EP effectively reduced the expression of STAT3 and CD34 in tumor sections compared to untreated control.ConclusionThese findings suggest that EP can exert antitumor activity in multiple myeloma U266 cells partly with antiangiogenic activity targeting JAK2/STAT3 signaling pathway as a potent cancer preventive agent for treatment of multiple myeloma cells.

Apoptosis Induced by Tanshinone IIA and Cryptotanshinone Is Mediated by Distinct JAK/STAT3/5 and SHP1/2 Signaling in Chronic Myeloid Leukemia K562 Cells

Though tanshinone IIA and cryptotanshinone possess a variety of biological effects such as anti-inflammatory, antioxidative, antimetabolic, and anticancer effects, the precise molecular targets or pathways responsible for anticancer activities of tanshinone IIA and cryptotanshinone in chronic myeloid leukemia (CML) still remain unclear. In the present study, we investigated the effect of tanshinone IIA and cryptotanshinone on the Janus activated kinase (JAK)/signal transducer and activator of transcription (STAT) signaling during apoptotic process. We found that both tanshinone IIA and cryptotanshinone induced apoptosis by activation of caspase-9/3 and Sub-G1 accumulation in K562 cells. However, they have the distinct JAK/STAT pathway, in which tanshinone IIA inhibits JAK2/STAT5 signaling, whereas cryptotanshinone targets the JAK2/STAT3. In addition, tanshinone IIA enhanced the expression of both SHP-1 and -2, while cryptotanshinone regulated the expression of only SHP-1. Both tanshinone IIA and cryptotanshinone attenuated the expression of bcl-xL, survivin, and cyclin D1. Furthermore, tanshinone IIA augmented synergy with imatinib, a CML chemotherapeutic drug, better than cryptotanshinone in K562 cells. Overall, our findings suggest that the anticancer activity of tanshinone IIA and cryptotanshinone is mediated by the distinct the JAK/STAT3/5 and SHP1/2 signaling, and tanshinone IIA has the potential for combination therapy with imatinib in K562 CML cells.

Melatonin Suppresses the Expression of 45S Preribosomal RNA and Upstream Binding Factor and Enhances the Antitumor Activity of Puromycin in MDA-MB-231 Breast Cancer Cells

Since the dysregulation of ribosome biogenesis is closely associated with tumor progression, in the current study, the critical role of ribosome biogenesis related signaling was investigated in melatonin and/or puromycin induced apoptosis in MDA-MB-231 breast cancer cells. Despite its weak cytotoxicity, melatonin from 3 mM attenuated the expression of 45S pre-ribosomal RNA (pre-rRNA), UBF as a nucleolar transcription factor, and fibrillarin at mRNA level and consistently downregulated nucleolar proteins such as UBF and fibrillarin at protein level in MDA-MB-231 cells. Furthermore, immunofluorescence assay revealed that UBF was also degraded by melatonin in MDA-MB-231 cells. In contrast, melatonin attenuated the expression of survival genes such as Bcl-xL, Mcl-1, cyclinD1, and cyclin E, suppressed the phosphorylation of AKT, mTOR, and STAT3, and cleaved PARP and activated caspase 3 only at a high concentration of 12 mM. However, combined treatment of melatonin (3 mM) and puromycin (1 μM) synergistically inhibited viability, attenuated the expression of 45S pre-rRNA and UBF, and consistently downregulated UBF, XPO1 and IPO7, procaspase 3, and Bcl-xL in MDA-MB 231 cells. Overall, these findings suggest that melatonin can be a cancer preventive agent by combination with puromycin via the inhibition of 45S pre-rRNA and UBF in MDA-MB 231 breast cancer cells.