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Coiled-coil domain containing 3 (CCDC3) was previously shown to regulate liver lipid metabolism as a secretory protein. Here, we report an unexpected intracellular role of CCDC3 as a tumor suppressor in breast cancer (BrC). Bioinformatics datasets analysis showed that CCDC3 is under-expressed in BrCs, while its higher levels are correlated with higher overall survival and lower relapse of cancer patients, and CCDC3 is positively correlated with p53 and its target genes. Ectopic CCDC3 markedly suppressed proliferation, colony formation, and xenograft tumor growth by augmenting p53 activity in BrC cells. Depletion of endogenous CCDC3 by CRISPR-Cas9 increased proliferation and drug resistance of BrC cells by alleviating 5-Fluorouracil (5-FU)-induced p53 level and activity. Mechanistically, CCDC3 bound to the C-termini of p53 and MDM2, consequently stabilizing p53 in the nucleus and impairing MDM2 recruitment of p53 to the 26S proteosome without inhibiting p53 ubiquitination. p53 induced CCDC3 expression by binding to its promoter in BrC cells. Our results unveil a unique mechanism underlying CCDC3 activation of p53 in a positive feedback fashion to suppress BrC growth.

Though limited success through chemotherapy, radiotherapy and surgery has been obtained for efficient cancer therapy for modern decades, cancers are still considered high burden to human health worldwide to date. Recently repurposing drugs are attractive with lower cost and shorter time compared to classical drug discovery, just as Metformin from Galega officinalis, originally approved for treating Type 2 diabetes by FDA, is globally valued at millions of US dollars for cancer therapy. As most previous reviews focused on FDA approved drugs and synthetic agents, current review discussed the anticancer potential of phytochemicals originally approved for treatment of cardiovascular diseases, diabetes, infectious diarrhea, depression and malaria with their molecular mechanisms and efficacies and suggested future research perspectives.

Since heat shock protein (HSP27) is a prognostic marker in cervical cancer, in the present study, the apoptotic mechanism of lambertianic acid (LA) was investigated in human cervical cancers in association with HSP27/STAT3/AKT signaling axis. LA exerted significant cytotoxicity, induced sub-G1 population, and increased the cleavage of Poly (ADP-ribose) polymerase (PARP) and cysteine aspartyl-specific protease 3 (caspase3) in HeLa and Caski cancer cells. Consistently, LA downregulated anti-apopotic genes such as B-cell lymphoma 2 (Bcl-2) and inhibitors of apoptosis proteins (c-IAP) in HeLa and Caski cells. Furthermore, LA-inhibited phosphorylation of HSP27, signal transducer, and activator of transcription 3 (STAT3) and Protein kinase B (AKT) through disturbing the binding of HSP27 with STAT3 or AKT in HeLa cells. Notably, LA upregulated the level of miR216b in HeLa and Caski cells. Consistently, miR216b mimic suppressed phosphorylation of HSP27 and reduced the expression of pro-PARP, while miR216b inhibitor reversed the ability of LA to attenuate phosphorylation of AKT, HSP27, and STAT3 and to reduce the expression of pro-PARP in HeLa cells. Overall, our findings suggest that miRNA216b mediated inhibition of HSP27/STAT3/ AKT signaling axis is critically involved in LA-induced apoptosis in cervical cancers.

Simple Summary Bone scintigraphy is the most widely used radionuclide technique to investigate bone metastasis, primarily due to its reasonable time and cost factor. However, it is important to recognize that bone scintigraphy to assess treatment response sometimes shows a “flare phenomenon”, which can be misinterpreted as disease progression. Distinction between flare phenomenon and disease progression could help in the decision to continue effective treatments in patients with flare phenomenon and to cease ineffective treatments and consider other salvage treatment plans in patients with disease progression. Despite many methods having been tried to answer this question, there was still no reliable way to differentiate between flare phenomenon and progression of bone metastases. Our results suggest that ALP is a useful serologic marker to differentiate flare phenomenon from disease progression on bone scintigraphy in breast or prostate cancer patients with bone metastasis. Abstract The flare phenomenon (FP) on bone scintigraphy after the initiation of systemic treatment seriously complicates evaluations of therapeutic response in patients with bone metastases. The aim of this study was to evaluate whether serum alkaline phosphatase (ALP) can differentiate FP from disease progression on bone scintigraphy in these patients. Breast or prostate cancer patients with bone metastases who newly underwent systemic therapy were reviewed. Pretreatment baseline and follow-up data, including age, pathologic factors, type of systemic therapy, radiologic and bone scintigraphy findings, and ALP levels, were obtained. Univariate and multivariate analyses of these factors were performed to predict FP. An increased extent and/or new lesions were found in 160 patients on follow-up bone scintigraphy after therapy. Among the 160 patients, 80 (50%) had an improvement on subsequent bone scintigraphy (BS), while subsequent scintigraphy also showed an increased uptake in 80 (50%, progression). Multiple regression analysis revealed that stable or decreased ALP was an independent predictor for FP (p < 0.0001). ALP was an independent predictor for FP on subgroup analysis for breast and prostate cancer (p = 0.001 and p = 0.0223, respectively). Results of the study suggest that ALP is a useful serologic marker to differentiate FP from disease progression on bone scintigraphy in patients with bone metastasis. Clinical interpretation for scintigraphic aggravation can be further improved by the ALP data and it may prevent fruitless changes of therapeutic modality by misdiagnosis of disease progression in cases of FP.

Brassinin derived from Chinese cabbage has been reported to act as an anti-cancer agent on prostate, liver, and colon cancer cells. However, its mechanism and impact are largely unknown in colon cancer cells. Here, we first published a report that Brassinin induces apoptosis and inhibits the survival of colon cancer cells by activating p53. We found that Brassinin induces p53 and p21 dose- and time-dependent manner in wild type of p53 colon cancer cells. Interestingly, Brassinin induces apoptosis in wild-type of p53 cancer cells, but not in null-type of p53 cancer cells dose dependently. Additionally, Brassinin induces apoptosis through L5. Furthermore, Brassinin enhanced the apoptotic effect with doxorubicin by activating p53. Altogether, our findings suggest that Brassinin is a new p53 regulator via induce apoptosis and inhibit the proliferation in colon cancer cells.

CCR4-NOT transcription complex subunit 2 (CNOT2), a subunit of the CCR4-NOT complex, has been described in cancer progression. The CNOT complex plays an important role in multiple cellular functions. Recent studies in our laboratory showed that CNOT2 promotes breast cancer cell proliferation and angiogenesis. In addition, CNOT2 signals are critically related to apoptosis induced by atorvastatin in lung cancer cells. Furthermore, depletion of CNOT2 was shown to enhance the antitumor effect of midline 1 interacting protein 1 (MID1IP1) depletion, thus inhibiting c-Myc expression in liver cancer cells. However, the molecular mechanisms related to its oncogenic role remain unclear. Herein, for the first time, we report that CNOT2 inhibition can induce apoptosis in colorectal cancer cells by activating p53. Inhibition of CNOT2 markedly induced apoptosis in various cancer cells like that of the wild-type p53. Furthermore, inhibition of CNOT2 elongated p53 s half-life. Previously, our laboratory demonstrated that MID1IP1 promoted colocalization with c-Myc mediated by CNOT2. Interestingly, inhibition of CNOT2 cannot induce p53 expression without MID1IP1 or apoptosis in cancer cells. In conclusion, our results demonstrate that CNOT2 inhibition induces apoptosis through MID1IP1 by activating p53.

RNA-binding motif protein 10 (RBM10) is an RNA-binding protein frequently deleted or mutated in lung cancer cells. Recent reports showed that the knockdown of RBM10 in human cancer cells enhances the growth of mouse tumor xenografts, suggesting that RBM10 acts as a tumor suppressor. RBM10 also regulates alternative splicing and controls cancer cell proliferation. However, the underlying molecular mechanisms for its tumor suppression role remain largely unclear. Here, we for the first time report that RBM10 can induce apoptosis and inhibit cancer cell proliferation by activating p53. Our analysis of cancer genomic databases showed that patients with wild-type RBM10 and p53 survive longer than do those with mutated p53 or less RBM10. RBM10 overexpression markedly inhibited mitochondrial respiration, cell migration and proliferation of various cancer cells that harbor wild-type p53. Also, RBM10 overexpression elongated p53's half-life by disrupting MDM2-p53 interaction and subsequently repressing p53 ubiquitination, whereas knockdown of RBM10 decreased p53 stability. Altogether, our results demonstrate that RBM10 inhibits cancer cell proliferation and induces apoptosis in part by blocking the MDM2-p53 feedback loop.

Galbanic acid (GBA) is known a sesquiterpene coumarin to have apoptotic, anti-hypoxic, anti-proliferative, anti-hepatitis, anti-angiogenic, anti-bacteria and anti-thrombotic effects. Also, antitumor effect of GBA was reported in prostate, ovary, breast and lung cancers. Nevertheless, the underlying molecular mechanism of GBA was not fully understood to overcome chemoresistance in resistant lung cancer so far. Thus, synergistic antitumor mechanism of GBA and TNF-related apoptosis-inducing ligand (TRAIL) was elucidated in H460 and resistant H460/R non-small cell lung cancer cells (NSCLCs). Combination of GBA and TRAIL significantly exerted cytotoxicity in a dose dependent manner compared to GBA or TRAIL alone in H460/R cells. Also, GBA and TRAIL significantly increased the number of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) positive cells and sub-G1 population in a dose dependent manner in H460/R cells. Consistently, GBA and TRAIL induced cleavages of poly (ADP-ribose) polymerase (PARP), caspase-9 and caspase-8 along with upregulation of death receptor 5 (DR5) and also attenuated the expression of B-cell lymphoma-extra-large (Bcl-xL), B-cell lymphoma 2 (Bcl-2), X-linked inhibitor of apoptosis protein (XIAP) in H460/R cells. Furthermore, combination of GBA and TRAIL remarkably inhibited the expression of decoy receptor 1 (DcR1) and multidrug resistance 1(MDR1) in H460/R cells. Consistently, GBA and TRAIL effectively maintained Rhodamine 123 accumulation in H460/R cells compared to GBA or TRAIL alone by blocking multidrug efflux pump from the cells. Overall, our findings suggest that galbanic acid enhances TRAIL induced apoptosis via inhibition of MDR1 and activation of caspases and DR5 in H460/R cells as a potent TRAIL sensitizer.

Extracted from the root tuber of Ophiopogon japonicas, ophiopogon is well known to have an anti-cancer effect. However, the underlying mechanisms are still largely unknown. Here, we report that Ophiopogon D (OP-D) can inhibit colon cancer cell proliferation and induce apoptosis by inhibiting c-Myc expression through activation of p53 and CNOT2 regulation. Our results showed that OP-D induced p53 expression via ribosomal protein L5 or L11 and inhibited c-Myc expression through CNOT2 in a dose-dependent manner. Additionally, OP-D regulated cyclin D1 and CDK4 which are well known as cell cycle regulatory proteins. Consistently, OP-D inhibited the phosphorylation of AKT expression in a dose-dependent manner. Furthermore, OP-D shortened c-Myc’s half-life in a time-dependent manner. Furthermore, CNOT2 knockdown enhanced the inhibitory effect of OP-D on c-Myc in colon cancer cells. Interestingly, OP-D has increased the apoptotic effect of colon cancer cells when combined with doxorubicin or 5-FU, a treatment already used clinically. Altogether, our results suggested that OP-D regulates colon cancer cell survival and induces apoptosis by inhibiting c-Myc expression via activation of p53 and CNOT2 regulation. Citation Format: Hyun Min Ko, Wona Jee, Do-il Park, Somi Park, Ye-Rin Park, Hyeung-Jin Jang, Ji Hoon Jung. Ophiopogonin D increase apoptosis by activating p53 via ribosomal protein L5 and L11 and inhibiting the expression of c-Myc via CNOT2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 864.

Coiled-coil domain containing 3 (CCDC3) was previously shown to regulate liver lipid metabolism as a secretory protein. Our recent studies revealed an intracellular role of CCDC3 as a tumor suppressor in breast cancer (BrC). Bioinformatics datasets analysis showed that CCDC3 is under-expressed in BrCs, while its higher levels are correlated with higher overall survival and lower relapse of cancer patients. CCDC3 is positively correlated with p53 and its target genes. Ectopic CCDC3 markedly suppressed proliferation, colony formation, and xenograft tumor growth by augmenting p53 activity in BrC cells. In contrast, knockdown of endogenous CCDC3 using CRISPR led to reduction of p53 level and activity, consequently promoting the proliferation of BrC cells. Mechanistically, CCDC3 bound to the C-termini of p53 and MDM2, consequently stabilizing p53 in the nucleus and impairing MDM2 recruitment of p53 to the 26S proteosome without inhibiting p53 ubiquitination. Also, knockdown of CCDC3 caused the resistance of BrC cells to 5-FU to some degrees by alleviating p53 response to this drug. p53 induced CCDC3 expression by binding to its promoter specifically in BrC cells. Our results unveil a unique mechanism underlying CCDC3 activation of p53 in a positive feedback fashion to suppress BrC growth and suggest that CCDC3 deficiency could be a factor for drug resistance of BrCs. Citation Format: Hyemin Lee, Caiyue Li, Ji Hoon Jung, Yiwei Zhang, Jieqiong Wang, Chang Liu, Allyson M. Segall, Roger L. Sheffmaker, Shelya X. Zeng, Hua Lu. Coiled-coil domain containing 3 suppresses breast cancer growth by protecting p53 from proteasome-mediated degradation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 856.

The tumor suppressor p53 acts as a transcription factor that regulates the expression of a number of genes responsible for DNA repair, cell cycle arrest, metabolism, cell migration, angiogenesis, ferroptosis, senescence, and apoptosis. It is the most commonly silenced or mutated gene in cancer, as approximately 50% of all types of human cancers harbor TP53 mutations. Activation of p53 is detrimental to normal cells, thus it is tightly regulated via multiple mechanisms. One of the recently identified regulators of p53 is RNA-binding motif protein 10 (RBM10). RBM10 is an RNA-binding protein frequently deleted or mutated in cancer cells. Its loss of function results in various deformities, such as cleft palate and malformation of the heart, and diseases such as lung adenocarcinoma. In addition, RBM10 mutations are frequently observed in lung adenocarcinomas, colorectal carcinomas, and pancreatic ductal adenocarcinomas. RBM10 plays a regulatory role in alternative splicing. Several recent studies not only linked this splicing regulation of RBM10 to cancer development, but also bridged RBM10′s anticancer function to the p53 pathway. This review will focus on the current progress in our understanding of RBM10 regulation of p53, and its role in p53-dependent cancer prevention.

Though honokiol, derived from the Magnolia tree, was known to suppress renal fibrosis, pulmonary fibrosis, non-alcoholic steatoheptitis, inflammation and cancers, the underlying antifibrotic mechanisms of honokiol are not fully understood in hepatic stellate cells until now. Thus, in the present study, inhibitory mechanism of honokiol on liver fibrosis was elucidated mainly in hepatic stellate cells (HSCs) by 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, cell cycle analysis and western-blotting. Honokiol exerted cytotoxicity in LX-2, HSC-T6 and Hep-G2 cells. Honokiol increased sub G1 population and activated caspase 3 and cleaved poly (ADP-ribose) polymerase (PARP) in HSCs. Moreover, honokiol attenuated the expression of alpha smooth muscle actin (α-SMA), transforming growth factor beta 1 (TGF-β1), phospho-Smad3, phospho-AKT, cyclin D1, c-Myc, Wnt3a, β-catenin, and activated phosphorylation of glycogen synthase kinase 3 beta (GSK3β) in HSCs. Conversely, GSK3β inhibitor SB216763 reversed the effect of honokiol on PARP, α-SMA, phospho-GSK3β, β-catenin and sub G1 population in LX-2 cells. Overall, honokiol exerts apoptotic and antifibrotic effects via activation of GSK3β and inhibition of Wnt3a/β-catenin signalling pathway.

Though epigallocatechin-3-gallate (EGCG), a major compound of green tea, has anti-diabetes, anti-obesity, anti-inflammatory, and antitumor effects, the underlying antitumor molecular mechanism of EGCG was not fully understood so far. Here the sensitizing effect of EGCG to tumor-necrosis-factor-related apoptosis-inducing ligand (TRAIL) was examined in colorectal cancers. Cotreatment of EGCG and TRAIL synergistically enhanced cytotoxicity and sub G1 accumulation, increased the number of terminal deoxynucleotidyl transferase-dT-mediated dUTP nick end labelling (TUNEL)-positive cells in SW480 and HCT116 cells. Furthermore, this cotreatment promoted the cleavages of poly (adenosine diphosphate-ribose) polymerase (PARP) and induced caspase 8 activation compared to TRAIL or EGCG alone in SW480 and HCT116 cells. Of note, cotreatment of EGCG and TRAIL increased the expression of death receptor 5 (DR5) at protein and mRNA levels and also DR5 cell surface level in colon cancer cells. Conversely, depletion of DR5 reduced the apoptotic activity of cotreatment of EGCG and TRAIL to increase cytotoxicity, sub-G1 population and PARP cleavages in colon cancer cells. Overall, our findings provide evidence that EGCG can be a sensitizer of TRAIL via DR5 and caspase 8 mediated apoptosis in colorectal cancer cells.

Though lambertianic acid (LA) was known to exert antitumor effect in liver and prostate cancers, its underlying anticancer mechanism is never reported in breast cancers so far. Thus, in this study, apoptotic mechanism of LA was elucidated in MDA-MB-231 breast cancer cells. Here, LA increased cytotoxicity in MCF-7 and MDA-MB-231 cells; enhanced sub-G1 population, G2/M arrest, and cleaved poly(ADP-ribose) polymerase; activated phosphorylation of AMP-activated protein kinase (AMPK)/acetyl-CoA carboxylase pathway; and also suppressed phosphorylation of AKT and the expression of forkhead box M1 (FOXM1), X-linked inhibitor of apoptosis protein, B-cell lymphoma 2, and CyclinB1 in MDA-MB-231 cells. Furthermore, AMPK inhibitor compound C reversed the effect of LA on FOXM1, Cyclin B1, and cleaved poly(ADP-ribose) polymerase in MDA-MB-231 cells. Notably, immunoprecipitation revealed that LA disturbed the direct binding of AKT and FOXM1 in MDA-MB-231 cells. Overall, these findings suggest that LA-induced apoptosis is mediated via activation of AMPK and inhibition of AKT/FOXM1 signaling pathway.

Simple Summary Herein, the oncogenic role of UBE2M as an E2 NEDD8-conjugating enzyme was explored in hepatocellular carcinoma (HCC) cells, since neddylation plays a critical role in tumorigenesis. To address this issue, human tissue array and TCGA analysis were conducted in HCCs to find overexpression of UBE2M in HCCs. In addition, a differential profile was confirmed in UBE2M-depleted HepG2 cells. Furthermore, UBE2M depletion activated p53 expression and stability, while the ectopic expression of UBE2M disturbed p53 activation and enhanced degradation of exogenous p53 mediated by MDM2 in HepG2 cells via binding to MDM2 and ribosomal protein L11 by immunoprecipitation and immunofluorescence. These findings provide evidence that UBE2M is critically involved in liver cancer progression as a p53 negative regulator by binding to MDM2 and ribosomal protein L11. Abstract Though UBE2M, an E2 NEDD8-conjugating enzyme, is overexpressed in HepG2, Hep3B, Huh7 and PLC/PRF5 HCCs with poor prognosis by human tissue array and TCGA analysis, its underlying oncogenic mechanism remains unclear. Herein, UBE2M depletion suppressed viability and proliferation and induced cell cycle arrest and apoptosis via cleavages of PARP and caspase 3 and upregulation of p53, Bax and PUMA in HepG2, Huh7 and Hep3B cells. Furthermore, UBE2M depletion activated p53 expression and stability, while the ectopic expression of UBE2M disturbed p53 activation and enhanced degradation of exogenous p53 mediated by MDM2 in HepG2 cells. Interestingly, UBE2M binds to MDM2 or ribosomal protein L11, but not p53 in HepG2 cells, despite crosstalk between p53 and UBE2M. Consistently, the colocalization between UBE2M and MDM2 was observed by immunofluorescence. Notably, L11 was required in p53 activation by UBE2M depletion. Furthermore, UBE2M depletion retarded the growth of HepG2 cells in athymic nude mice along with elevated p53. Overall, these findings suggest that UBE2M promotes cancer progression as a p53 negative regulator by binding to MDM2 and ribosomal protein L11 in HCCs.

Though Forkhead box P (FOXP) transcription factors comprising of FOXP1, FOXP2, FOXP3 and FOXP4 are involved in the embryonic development, immune disorders and cancer progression, the underlying function of FOXP3 targeting CD4 + CD25+ regulatory T (Treg) cells and the dual roles of FOXP proteins as an oncogene or a tumor suppressor are unclear and controversial in cancers to date. Thus, the present review highlighted research history, dual roles of FOXP proteins as a tumor suppressor or an oncogene, their molecular networks with other proteins and noncoding RNAs, cellular immunotherapy targeting FOXP3, and clinical implications in cancer progression.

Though ginsenoside metabolite compound K was known to have antitumor effect in several cancers, its underlying apoptotic mechanism still remains unclear so far. Thus, in the present study, the apoptotic mechanism of compound K was explored in colorectal cancer cells (CRCs) in association with leucine rich repeat containing G protein-coupled receptor 5 (LGR5) that was overexpressed in colorectal cancers with poor survival rate. Here compound K significantly reduced viability of HCT116p53+/+ cells better than that of HCT116p53-/- cells. Consistently, compound K increased sub G1 population and attenuated the expression of LGR5, c-Myc, procaspase3, Pin1 in HCT116p53+/+ cells more than in HCT116p53-/- cells. Conversely, caspase 3 inhibitor Z-DEVD-FMK reversed inhibitory effect of compound K on LGR5, c-Myc and procaspase3 in HCT116 cells. Consistently, inhibition of LGR5 using transfection method enhanced suppression of pro-PARP, Bcl-xL c-Myc, Snail and Pin1 in compound K treated HCT116p53+/+ cells. Furthermore, compound K synergistically potentiated antitumor effect of 5-fluorouracil (5-FU) or Doxorubicin to reduce the survival genes and cytotoxicity in HCT116p53+/+ cells. Overall, our findings provide scientific insight that compound K induces apoptosis in colon cancer cells via caspase and p53 dependent LGR5 inhibition with combination therapy potential with 5-FU or doxorubicin.

In the current study, the function of long noncoding RNA (LncRNA) RAB5IF was elucidated in hepatocellular carcinoma (HCCs) in association with LGR5 related signaling. Here TCGA analysis revealed that LncRNA RAB5IF was overexpressed in HCC, and its overexpression level was significantly (p &lt, 0.05) correlated with poor prognosis in patients with HCC. Furthermore, LncRNA RAB5IF depletion suppressed cell proliferation and colony formation, increased sub G1 population, cleavage of poly ADP-ribose polymerase (PARP) and cysteine aspartyl-specific protease (caspase 3) and attenuated the expression of procaspase 3, pro-PARP and B-cell lymphoma 2 (Bcl-2) in HepG2 and Hep3B cells. Furthermore, LncRNA RAB5IF depletion reduced the expression of LGR5 and its downstreams such as &beta, catenin and c-Myc in HepG2 and Hep3B cells. Notably, LGR5 depletion also attenuated the expression of pro-PARP, pro-caspase3, &beta, catenin and c-Myc in HepG2 and Hep3B cells. Conversely, LGR5 overexpression upregulated &beta, catenin and c-Myc in Alpha Mouse Liver 12 (AML-12) normal hepatocytes. Overall, these findings provide novel evidence that LncRNA RAB5IF promotes the growth of hepatocellular carcinoma cells via LGR5 mediated &beta, catenin and c-Myc signaling as a potent oncogenic target.

Herein the underlying apoptotic mechanism of Farnesiferol C (FC) derived from Ferula assafoetida was elucidated in chronic myelogenous leukemia (CML) K562 and KBM5 cells. FC showed significant cytotoxicity in K562 and KBM5 cells, more so than in U937 and UL-60 acute myeloid leukemia (AML) cells. Cleaved PARP and caspase 9/3 attenuated the expression of Bcl2 and induced G1 arrest in K562 and KBM5 cells. Also, FC effectively abrogated the expression of cell cycle related proteins, such as: Cyclin D1, Cyclin E, Cyclin B1 in K562, and KBM5 cells, but caspase 3 inhibitor Z-DEVD-FMK rescued the cleavages of caspase 3 and PARP induced by FC in K562 cells. Of note, FC decreased histone deacetylase 1 (HDAC1) and HDAC2, and enhanced histone H3 acetylation K18 (Ac-H3K18) in K562 and KBM5 cells. Furthermore, combination of FC and Imatinib enhanced the apoptotic effect of Imatinib as a potent Imatinib sensitizer in K562 cells. Overall, our findings provide scientific evidence that inactivation of HDAC and caspase activation mediate FC induced apoptosis in CML cells.

The underlying interaction between melatonin (MLT) and daily fruit intake still remains unclear to date, despite multibiological effects of MLT. Herein, the apoptotic mechanism by co-treatment of MLT and pterostilbene (Ptero) contained mainly in grape and blueberries was elucidated in colorectal cancers (CRCs). MLT and Ptero co-treatment (MLT+Ptero) showed synergistic cytotoxicity compared with MLT or Ptero alone, reduced the number of colonies and Ki67 expression, and also increased terminal deoxynucleotidyl transferase dUTP nick end labeling- (TUNEL) positive cells and reactive oxygen species (ROS) production in CRCs. Consistently, MLT+Ptero cleaved caspase 3 and poly (ADP-ribose) polymerase (PARP), activated sex-determining region Y-Box10 (SOX10), and also attenuated the expression of Bcl-xL, neural precursor cell expressed developmentally downregulated protein 9 (NEDD9), and SOX9 in CRCs. Additionally, MLT+Ptero induced differentially expressed microRNAs (upregulation: miR-25-5p, miR-542-5p, miR-711, miR-4725-3p, and miR-4484, downregulation: miR-4504, miR-668-3p, miR-3121-5p, miR-195-3p, and miR-5194) in HT29 cells. Consistently, MLT +Ptero upregulated miR-25-5p at mRNA level and conversely NEDD9 overexpression or miR-25-5p inhibitor reversed the ability of MLT+Ptero to increase cytotoxicity, suppress colony formation, and cleave PARP in CRCs. Furthermore, immunofluorescence confirmed miR-25-5p inhibitor reversed the reduced fluorescence of NEDD9 and increased SOX10 by MLT+Ptero in HT29 cells. Taken together, our findings provided evidence that MLT+Ptero enhances apoptosis via miR-25-5p mediated NEDD9 inhibition in colon cancer cells as a potent strategy for colorectal cancer therapy.

Though Atorvastatin has been used as a hypolipidemic agent, its anticancer mechanisms for repurposing are not fully understood so far. Thus, in the current study, its apoptotic and autophagic mechanisms were investigated in non-small cell lung cancers (NSCLCs). Atorvastatin increased cytotoxicity, sub G1 population, the number of apoptotic bodies, cleaved poly (ADP-ribose) polymerase (PARP) and caspase 3 and activated p53 in H1299, H596, and H460 cells. Notably, Atorvastatin inhibited the expression of c-Myc and induced ribosomal protein L5 and L11, but depletion of L5 reduced PARP cleavages induced by Atorvastatin rather than L11 in H1299 cells. Also, Atorvastatin increased autophagy microtubule-associated protein 1A/1B-light chain 3II (LC3 II) conversion, p62/sequestosome 1 (SQSTM1) accumulation with increased number of LC3II puncta in H1299 cells. However, late stage autophagy inhibitor chloroquine (CQ) increased cytotoxicity in Atorvastatin treated H1299 cells compared to early stage autophagy inhibitor 3-methyladenine (3-MA). Furthermore, autophagic flux assay using RFP-GFP-LC3 constructs and Lysotracker Red or acridine orange-staining demonstrated that autophagosome-lysosome fusion is blocked by Atorvastatin treatment in H1299 cells. Conversely, overexpression of CCR4-NOT transcription complex subunit 2(CNOT2) weakly reversed the ability of Atorvastatin to increase cytotoxicity, sub G1 population, cleavages of PARP and caspase 3, LC3II conversion and p62/SQSTM1 accumulation in H1299 cells. In contrast, CNOT2 depletion enhanced cleavages of PARP and caspase 3, LC3 conversion and p62/SQSTM1 accumulation in Atorvastatin treated H1299 cells. Overall, these findings suggest that CNOT2 signaling is critically involved in Atorvastatin induced apoptotic and autophagic cell death in NSCLCs.

Though Pyrogallol, one of the natural polyphenols, was known to have anti-inflammatory and antitumor effects in breast and colon cancers, the underlying antitumor mechanisms of Pyrogallol, still remain unclear so far. Here, the antitumor mechanisms of Pyrogallol were elucidated in Hep3B and Huh7 hepatocellular carcinoma cells (HCCs). Pyrogallol showed significant cytotoxicity and reduced the number of colonies in Hep3B and Huh7 cells. Interestingly, Pyrogallol induced S-phase arrest and attenuated the protein expression of CyclinD1, Cyclin E, Cyclin A, c-Myc, S-phase kinase-associated protein 2 (Skp2), p-AKT, PI3K, increased the protein expression of p27, and also reduced the fluorescent expression of Cyclin E in Hep3B and Huh7 cells. Furthermore, Pyrogallol disturbed the interaction between Skp2, p27, and c-Myc in Huh7 cells. Notably, Pyrogallol upregulated miRNA levels of miR-134, and conversely, miR-134 inhibition rescued the decreased expression levels of c-Myc, Cyclin E, and Cyclin D1 and increased the expression of p27 by Pyrogallol in Huh7 cells. Taken together, our findings provide insight that Pyrogallol exerts antitumor effects in HCCs via miR-134 activation-mediated S-phase arrest and inhibition of PI3K/AKT/Skp2/cMyc signaling as a potent anticancer candidate.

Among ribosomal proteins essential for protein synthesis, the functions of ribosomal protein L5 (RPL5) and RPL11 still remain unclear to date. Here, the roles of RPL5 and RPL11 were investigated in association with p53/p21 signaling in the antitumor effect of puromycin mainly in HCT116 and H1299 cancer cells. Cell proliferation assays using 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assays and colony formation assays, cell cycle analysis, Reverse transcription polymerase chain reaction (RT-PCR) and Western blotting were performed in cancer cells. Puromycin exerted cytotoxic and anti-proliferative effects in p53 wild-type HCT116 more than in p53 null H1299 cells. Consistently, puromycin increased sub-G1, cleaved Poly (ADP-ribose) polymerase (PARP), activated p53, p21, and Mouse double minute 2 homolog (MDM2), and attenuated expression of c-Myc in HCT116 cells. Notably, puromycin upregulated the expression of RPL5 and RPL11 to directly bind to MDM2 in HCT116 cells. Conversely, deletion of RPL5 and RPL11 blocked the activation of p53, p21, and MDM2 in HCT116 cells. Also, puromycin enhanced the antitumor effect with reactivating p53 and inducing tumor apoptosis (RITA) or doxorubicin in HCT116 cells. These findings suggest that puromycin induces p53-dependent apoptosis via upregulation of RPL5 or RPL11 for binding with MDM2, and so can be used more effectively in p53 wild-type cancers by combination with RITA or doxorubicin.

Background and purpose The TNF-related apoptosis-inducing ligand (TRAIL) is a promising anticancer agent due to its remarkable ability to selectively kill tumour cells. However, because most tumours exhibit resistance to TRAIL-induced apoptosis, the development of combination therapies to overcome resistance to TRAIL is required for effective cancer therapy. Experimental approach Cell viability and possible synergy between the plant pyranocoumarin decursin and TRAIL was measured by MTT assay and calcusyn software. Reactive oxygen species (ROS) and apoptosis were measured using dichlorodihydrofluorescein and annexin/propidium iodide in cell flow cytometry. Changes in protein levels were assessed with Western blotting. Key results Combining decursin and TRAIL markedly decreased cell viability and increased apoptosis in TRAIL-resistant non-small-cell lung cancer (NSCLC) cell lines. Decursin induced expression of the death receptor 5 (DR5). Inhibition of DR5 attenuated apoptotic cell death in decursin + TRAIL treated NSCLC cell lines. Interestingly, induction of DR5 and CCAAT/enhancer-binding protein homologues protein by decursin was mediated through selective induction of the pancreatic endoplasmic reticulum kinase (PERK)/activating transcription factor 4 (ATF4) branch of the endoplasmic reticulum stress response pathway. Furthermore, enhancement of PERK/ATF4 signalling by decursin was mediated by ROS generation in NSCLC cell lines, but not in normal human lung cells. Decursin also markedly down-regulated expression of survivin and Bcl-xL in TRAIL-resistant NSCLC cells. Conclusions and implications ROS generation by decursin selectively activated the PERK/ATF4 axis of the endoplasmic reticulum stress signalling pathway, leading to enhanced TRAIL sensitivity in TRAIL-resistant NSCLC cell lines, partly via up-regulation of DR5.

Though Moracin D derived from Morus alba was known to have anti-inflammatory and antioxidant activities, the underlying antitumor mechanism of Moracin D was never unveiled so far. Thus, in the recent study, the apoptotic mechanism of Moracin D was elucidated in breast cancer cells. Herein, Moracin D exerted significant cytotoxicity in MDA-MB231 and MCF7 cells. Also, Moracin D increased sub G1 population, cleaved poly (ADP-ribose) polymerase (PARP) and attenuated the expression of pro-cysteine aspartyl-specific protease (procaspase 3), c-Myc, cyclin D1, B-cell lymphoma 2 (Bcl-2),and X-linked inhibitor of apoptosis protein (XIAP) in MDA-MB231 cells. Of note, Moracin D reduced expression of Forkhead box M1 (FOXM1), β-catenin, Wnt3a, and upregulated glycogen synthase kinase 3 beta (GSK 3β) on Tyr216 along with disturbed binding of FOXM1 with β-catenin in MDA-MB-231 cells. Conversely, GSK3β inhibitor SB216763 reversed the apoptotic ability of Moracin D to reduce expression of FOXM1, β-catenin, pro-caspase3 and pro-PARP in MDA-MB-231 cells. Overall, these findings provide novel insight that Moracin D inhibits proliferation and induces apoptosis via suppression of Wnt3a/FOXM1/β-catenin signaling and activation of caspase and GSK3β

Oncogene MYC is deregulated in many human cancers, especially in lymphoma. Previously, we showed that inauhzin (INZ) activates p53 and inhibits tumor growth. However, whether INZ could suppress cancer cell growth independently of p53 activity is still elusive. Here, we report that INZ(c), a second generation of INZ, suppresses c-Myc activity and thus inhibits growth of human lymphoma cells in a p53-independent manner. INZ(c) treatment decreased c-Myc expression at both mRNA and protein level, and suppressed c-Myc transcriptional activity in human Burkitt's lymphoma Raji cells with mutant p53. Also, we showed that overexpressing ectopic c-Myc rescues the inhibition of cell proliferation by INZ(c) in Raji cells, implicating c-Myc activity is targeted by INZ(c). Interestingly, the effect of INZ(c) on c-Myc expression was impaired by disrupting the targeting of c-Myc mRNA by miRNAs via knockdown of ribosomal protein (RP) L5, RPL11, or Ago2, a subunit of RISC complex, indicating that INZ(c) targets c-Myc via miRNA pathways. These results reveal a new mechanism that INZ(c) targets c-Myc activity in human lymphoma cells.

To elucidate the underlying oncogenic mechanism of zinc finger protein 746 (ZNF746), current study was conducted in colorectal cancers (CRCs). Herein, ZNF746 was overexpressed in HCT116, SW620, and SW480 cells, which was supported by CRC tissue microarray and TCGA analysis. Also, DNA microarray revealed the differentially expressed gene profile particularly related to cell cycle genes and c-Myc in ZNF746 depleted HCT116 cells. Furthermore, ZNF746 enhanced the stability of c-Myc via their direct binding through nuclear colocalization by immunoprecipitation and immunofluorescence, while ZNF746 and c-Myc exist mainly in nucleoplasm. Conversely, ZNF746 depletion attenuated phosphorylation of c-Myc (S62) and glycogen synthase kinase 3β (GSK3β) (S9) and also activated p-c-Myc (T58), which was reversed by GSK3 inhibitors such as SB-216763 and Enza. Also, c-Myc degradation by ZNF746 depletion was blocked by knockdown of F-box/WD repeat-containing protein 7 (FBW7) ubiquitin ligase or proteosomal inhibitor MG132. Additionally, the growth of ZNF746 depleted HCT116 cancer cells was retarded with decreased expression of ZNF746 and c-Myc. Overall, these findings suggest that ZNF746 promotes CRC progression via c-Myc stability mediated by GSK3 and FBW7.

The heparan sulfate mimetic PG545 has been shown to exert anti-angiogenic and anti-metastatic activity in vitro and in vivo cancer models. Although much of this activity has been attributed to inhibition of heparanase and heparan sulfate-binding growth factors, it was hypothesized that PG545 may additionally disrupt Wnt signaling, an important pathway underlying the malignancy of pancreatic cancer. We show that PG545, by directly interacting with Wnt3a and Wnt7a, inhibits Wnt/β-catenin signaling leading to inhibition of proliferation in pancreatic tumor cell lines. Additionally, we demonstrate for the first time that the combination of PG545 with gemcitabine has strong synergistic effects on viability, motility and apoptosis induction in several pancreatic cell lines. In an orthotopic xenograft mouse model, combination of PG545 with gemcitabine efficiently inhibited tumor growth and metastasis compared to single treatment alone. Also, PG545 treatment alone decreased the levels of β-catenin and its downstream targets, cyclin D1, MMP-7 and VEGF which is consistent with our in vitro data. Collectively, our findings suggest that PG545 exerts anti-tumor activity by disrupting Wnt/β-catenin signaling and combination with gemcitabine should be considered as a novel therapeutic strategy for pancreatic cancer treatment.

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.

The peptidyl-prolyl cis/trans isomerase Pin1 is overexpressed in a wide variety of cancer cells and thus considered as an important target molecule for cancer therapy. This study demonstrates that decursin, a bioactive compound from Angelica gigas, exert the anti-cancer effect against breast cancer cells via regulation of Pin1 and its related signaling molecules. We observed that decursin induced G1 arrest with decrease in cyclin D1 level in Pin1-expressing breast cancer cells MDA-MB-231, but not Pin1-non-expressing breast cancer cells MDA-MB-157. In addition, decursin significantly reduced protein expression and enzymatic activity of Pin1 in MDA-MB-231 cells. Further, we found that decursin treatment enhanced the p53 expression level and failed to down-regulate Pin1 in the cells transfected with p53 siRNA, indicating the importance of p53 in the decursin-mediated Pin1 inhibition in MDA-MB-231 cells. Decursin stimulated association between Pin1 to p53. Moreover, decursin facilitated p53 transcription in MDA-MB-231 cells. Overall, our current study suggests the potential of decursin as an attractive cancer therapeutic agent for breast cancer by targeting Pin1 protein.

Here, antitumor mechanism of cinnamaldehyde derivative CB-PIC was elucidated in human SW620 colon cancer cells. CB-PIC significantly exerted cytotoxicity, increased sub-G1 accumulation, and cleaved PARP with apoptotic features, while it enhanced the phosphorylation of AMPK alpha and ACC as well as activated the ERK in hypoxic SW620 cells. Furthermore, CB-PIC suppressed the expression of HIF1 alpha, Akt, and mTOR and activated the AMPK phosphorylation in hypoxic SW620 cells. Conversely, silencing of AMPKα blocked PARP cleavage and ERK activation induced by CB-PIC, while ERK inhibitor PD 98059 attenuated the phosphorylation of AMPKα in hypoxic SW620 cells, implying cross-talk between ERK and AMPKα. Furthermore, cotreatment of CB-PIC and metformin enhanced the inhibition of HIF1α and Akt/mTOR and the activation of AMPKα and pACC in hypoxic SW620 cells. In addition, CB-PIC suppressed the growth of SW620 cells inoculated in BALB/c athymic nude mice, and immunohistochemistry revealed that CB-PIC treatment attenuated the expression of Ki-67, CD34, and CAIX and increased the expression of pAMPKα in CB-PIC-treated group. Interestingly, CP-PIC showed better antitumor activity in SW620 colon cancer cells under hypoxia than under normoxia, since it may be applied to chemoresistance. Overall, our findings suggest that activation of AMPKα and ERK mediates CB-PIC-induced apoptosis in hypoxic SW620 colon cancer cells.

This study involves the antitumor potential of 1,2,3,4,6-penta-O-galloyl-beta-D-glucose (PGG) to induce apoptosis and enhance antitumor activity of imatinib in K562 cells. Though PGG was reported to have antitumor activities in breast, prostate, kidney, liver cancers and HL-60 leukemia cells, there is no report on synergistic antitumor effect of PGG with imatinib until now. In the present study, PGG significantly enhanced the cytotoxicty and cleavage of poly (ADP-ribose) polymerase (PARP) in imatinib induced chronic myeloid leukemia K562 cells. Furthermore, oral administration of PGG or imatinib significantly inhibited the growth of K562 cells inoculated in Balb/c athymic nude mice and also immunohistochemistry revealed decreased expression of Ki67 (proliferation), CD34 (blood density) and death domain-associated protein (DAXX) and increased terminal deoxynucleotide transferasemediated dUTPnick end labeling (TUNEL) positive cells as one of the apoptotic feature in tumor sections of K562 mouse xenograft model comparable to imatinib treated group. Overall, our findings suggest the potency of PGG to induce apoptosis and enhance antitumor activity of imatinib inK562 cells. Key words: Chronic myeloid leukemia, apoptosis, 1,2,3,4,6-penta-O-galloyl-beta-D-glucose (PGG), imatinib.

Aim of this study was to identify the molecular mechanisms of tanshinone IIA-induced apoptosis in chronic myelogenous leukemia (CML) cells. Cytotoxicity of tanshinone IIA was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Our data demonstrate that tanshinone IIA induced apoptosis by increasing the sub-G1 DNA contents and DNA fragmentation in KBM-5 CML cell line. In addition, tanshinone IIA significantly reduced mitochondrial membrane potential (MMP), mediated cytochrome c release from mitochondria and activated caspase-3 and 9, indicating mitochondria-dependent apoptosis by tanshinone IIA. Tanshinone IIA attenuated expression of several apoptosis-related proteins such as c-inhibitor of apoptosis protein (IAP) 2, Mcl-1(L) and Bcl-2. Interestingly, although tanshinone IIA notably enhanced the phosphorylation of both c-Jun N-terminal protein kinase (JNK) and p38, JNK inhibitor, but not p38 inhibitor, reversed tanshinone IIA-induced apoptosis. Our findings suggest that tanshinone IIA induces mitochondria-dependent apoptosis via activation of JNK in KBM 5 cells as a potent anti-cancer agent for CML therapy.

// Jihyun Lee 1, * , Eun Jung Sohn 1, * , Sangwook Yoon 1 , Gunho Won 1 , Chang Geun Kim 1 , Ji Hoon Jung 1 , Sung-Hoon Kim 1 1 College of Korean Medicine, Kyung Hee University, Dongdaemun-gu, Seoul, 130-701, Republic of Korea * These authors have contributed equally to this work Correspondence to: Sung-Hoon Kim, email: sungkim7@khu.ac.kr Keywords: tanshinone I, autophagy, p62 ΔUBA , IRE1, mesothelioma cells Received: October 27, 2016 Accepted: January 16, 2017 Published: February 15, 2017 ABSTRACT The aim of present study is to elucidate autophagic mechanism of tanshinone I (Tan I) in H28 and H2452 mesothelioma cells. Herein, Tan I exerted cytotoxicity with autophagic features of autophagy protein 5 (ATG5)/ microtubule-associated protein 1A/1B-light chain 3II (LC3 II) activation, p62/sequestosome 1 (SQSTM1) accumulation and increased number of LC3II punctae, acridine orange-stained cells and autophagic vacuoles. However, 3-methyladenine (3MA) and NH 4 Cl increased cytotoxicity in Tan I treated H28 cells. Furthermore, autophagy flux was enhanced in Tan I-treated H28 cells transfected by RFP-GFP-LC3 constructs, with colocalization of GFP-LC3 punctae with LAMP1 or Lysotracker. Interestingly, C-terminal UBA domain is required for Tan 1 induced aggregation of p62 in H28 cells. Notably, Tan I upregulated CCAAT-enhancer-binding protein homologous protein (CHOP), inositol-requiring protein-1 (IRE1) and p-c-Jun N-terminal kinase (p-JNK), but silencing of IRE1 or p62 and JNK inhibitor SP600125 blocked the LC3II accumulation in Tan I-treated H28 cells. Overall, these findings demonstrate that Tan I exerts antitumor activity through a compromise between apoptosis and p62/SQSTM1-dependent autophagy via activation of JNK and IRE 1 in malignant mesothelioma cells.

Though Farnesiferol c (FC) has been reported to have anti-angiogenic and antitumor activity, the underlying antitumor mechanism of FC still remains unclear. Thus, in the present study, we investigated the apoptotic mechanism of FC in human H1299 and H596 non-small lung cancer cells (NSCLCs). FC significantly showed cytotoxicity, increased sub-G1 accumulation, and attenuated the expression of Bcl-2, Bcl-xL, Survivin and procaspase 3 in H1299 and H596 cells. Furthermore, FC effectively suppressed the mRNA expression of G1 arrest related genes such as Cyclin D1, E2F1 transcription factor and CDC25A by RT-PCR. Interestingly, FC inhibited the expression of c-Myc, ribosomal protein L11 (L11) and nucleolin (NCL) in H1299 and H596 cells. Of note, silencing of L11 by siRNA transfection enhanced the expression of c-Myc through a negative feedback mechanism, while c-Myc knockdown downregulated L11 in H1299 cells. Additionally, combined treatment of FC and puromycin/doxorubicin promoted the activation of caspase 9/3, and attenuated the expression of c-Myc, Cyclin D1 and CDK4 in H1299 cells compared to single treatment. Taken together, our findings suggest that FC induces apoptosis and G1 arrest via regulation of ribosomal protein L11 and c-Myc and also enhances antitumor effect of puromycin or doxorubicin in NSCLCs.

Although 1,2,3,4,6-penta-O-galloyl-beta-d-glucose (PGG) was well known to have antitumor activities in breast, prostate, kidney, liver cancers and HL-60 leukemia via regulation of caspase 3, p53, S-phase kinase-associated protein 2 (Skp2) and insulin receptor signaling, the underlying mechanism of PGG-induced apoptosis linked with reactive oxygen species (ROS) mediated c-Jun N-terminal kinase (JNK) and DAXX was never elucidated in chronic myeloid leukemia (CML) K562 cells until now. Herein PGG significantly decreased the viability of CML cell lines such as K562 and KBM-5 without hurting normal peripheral blood lymphocytes (PBLs). PGG increased the number of TUNEL-positive cells and the sub-G1 cell population as well as activated caspase cascades including caspase-8, -9 and -3 in K562 cells. Interestingly, a significant activation of JNK by PGG was observed by MULTIPLEX assay and Western blotting. Conversely, JNK inhibitor D-JNKi suppressed the cleavages of caspase 3 and PARP induced by PGG in K562 cells. Also, PGG dramatically enhanced generation of ROS and reduced the expression of death-domain-associated protein (DAXX). Of note, ROS inhibitor acetyl-L-cysteine (NAC) reversed JNK-dependent apoptosis and DAXX inhibition induced by PGG. Overall, these findings suggest that ROS-dependent JNK activation and DAXX downregulation are critically involved in PGG-induced apoptosis in K562 cells.

Chronic inflammation of rheumatoid arthritis (RA) is promoted by proinflammatory cytokines and closely linked to angiogenesis. In the present study, we investigated the anti-inflammatory effects of emodin (1,3,8-trihydroxy-6-methyl-anthraquinone) isolated from the root of Rheum palmatum L. in interleukin 1 beta (IL-1β) and lipopolysaccharide (LPS)-stimulated RA synoviocytes under hypoxia. Emodin significantly inhibited IL-1β and LPS-stimulated proliferation of RA synoviocytes in a dose-dependent manner under hypoxic condition. Also, enzyme linked immunosorbent assay (ELISA) revealed that emodin significantly reduced the production of pro-inflammatory cytokines [tumor necrosis factor-alpha (TNF-α), IL-6 and IL-8], mediators [prostagladin E(2) (PGE(2)), matrix metalloproteinase (MMP)-1 and MMP-13] and vascular endothelial growth factor (VEGF) as an angiogenesis biomarker in IL-1β and LPS-treated synoviocytes under hypoxia. Consistently, emodin attenuated the expression of cyclooxygenase 2 (COX-2), VEGF, hypoxia inducible factor 1 alpha (HIF-1α), MMP-1 and MMP-13 at mRNA level in IL-1β and LPS-treated synoviocytes under hypoxia. Furthermore, emodin reduced histone deacetylase (HDAC) activity as well as suppressed the expression of HDAC1, but not HDAC2 in IL-1β and LPS-treated synoviocytes under hypoxia. Overall, these findings suggest that emodin inhibits proinflammatory cytokines and VEGF productions, and HDAC1 activity in hypoxic RA synoviocytes.

Bojungbangdocktang (BJBDT) is a medicinal herbal cocktail that has been used for cancer prevention and treatment in traditional Korean medicine. In the current study, BJBDT was demonstrated to regulate hematopoiesis. BJBDT significantly increased the expression of hematopoietic cytokines interleukin (IL)-3, stem cell factor (SCF), granulocyte-macrophage-colony stimulating factor (GM-CSF), thrombopoietin (TPO) and erythropoietin (EPO) at the level of mRNA and secretion in hematopoietic stem cells (HSCs). Additionally, BJBDT enhanced the phosphorylation of Janus activated kinase 2 (JAK2) and signal transducer and activator of transcription 5 (STAT5) and STAT binding to gamma interferon activated sites (GAS) in HSCs. Furthermore, BJBDT significantly enhanced the growth rate of granulocyte erythrocyte monocyte macrophage colony-forming units (CFU-GEMM) and erythroid burst forming units (BFU-E) in vitro. Moreover, BJBDT increased the level of EPO at mRNA in kidney and plasma, and the numbers of erythroid-specific antigen Ter-119(+) erythroid cells in mice with aplastic anemia induced by 20% benzene. Consistently, histochemical staining revealed BJBDT increased the bone marrow and stromal cells as well as decreased macrophages and adipocytes in bone marrow tissues of mice with aplastic anemia. Taken together, the results suggest that BJBDT can enhance hematopoiesis via hematopoietic cytokine-mediated JAK2/STAT5 pathway as a potent hematopoietic candidate. Copyright © 2010 John Wiley & Sons, Ltd.

Although ZNF746, also known as Parkin-interacting substrate (PARIS), has been reported to suppress peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) and its target gene NRF-1 leading to the neurodegeneration in Parkinson's disease, its function in tumorigenesis has yet to be investigated. Thus, in the present study, the role of ZNF746 in the invasion and epithelial to mesenchymal transition (EMT) in H460 non-small cell lung cancer (NSCLC) cells was investigated. Invasion assay showed that inhibition of ZNF746 using siRNA transfection inhibited the invasion of H460 NSCLC cells using Boyden chamber. Quantitative PCR (qPCR) analysis revealed that the silencing of ZNF746 attenuated the expression of matrix metalloproteinase (MMP)1, MMP2 and MMP9, but not MMP7, in H460 NSCLC cells. Immunoblotting assay revealed that the expression of E-cadherin and β-catenin of epithelial phenotype was upregulated, while Slug was downregulated in ZNF746 siRNA-transfected H460 NSCLC cells. Accordingly, the mRNA expression of E-cadherin was upregulated while vimentin or Slug, Twist, ZEB as EMT key transcriptional factors were suppressed in ZNF746 siRNA-transfected H460 NSCLC cells. Also, mRNA expression of transcriptional marker Nanog and Octamer-binding transcription factor 4 (OCT4), known to enhance malignancy and metastasis in lung adenocarcinoma, was suppressed in ZNF746 siRNA-transfected H460 NSCLC cells. Notably, the endogenous expression of ZNF746 was induced in parallel with Twist at the protein level during hypoxia. Overall, our findings suggest that inhibition of ZNF746 suppresses the invasion and EMT molecules in H460 NSCLC cells and ZNF746 may be an important target molecule in lung tumorigenesis.

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-x(L) 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.

Background Ergosterol 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. Results Despite 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. Conclusion These 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.

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.

Cryptotanshinone is a biologically active compound from the root of Salvia miltiorrhiza. In the present study, we investigated the molecular mechanisms by which cryptotanshinone is in synergy with tumor necrosis factor-alpha (TNF-α) for the induction of apoptosis in human chronic myeloid leukemia (CML) KBM-5 cells. The co-treatment of cryptotanshinone with TNF-α reduced the viability of the cells [combination index (CI)1]. Concomitantly, the co-treatment of cryptotanshinone and TNF-α elicited apoptosis, manifested by enhanced the number of terminal deoxynucleotide transferase-mediated dUTP-nick-end labeling (TUNEL)-positive cells, the sub-G1 cell populations, and the activation of caspase-8 and -3, in comparison with the treatment with either drug alone. The treatment with cryptotanshinone further suppressed TNF-α-mediated expression of c-FLIP(L), Bcl-x(L), but the increased level of tBid (a caspase-8 substrate). Furthermore, cryptotanshinone activated p38 but not NF-κB in TNF-α-treated KBM-5 cells. The addition of a specific p38 MAPK inhibitor SB203580 significantly attenuated cryptotanshinone/TNF-α-induced apoptosis. The combination treatment of cryptotanshinone and TNF-α also stimulated the reactive oxygen species (ROS) generation. N-acetyl-L-cysteine (NAC, a ROS scavenger) was not only able to block cryptotanshinone/TNF-α-induced ROS production but also the activation of caspase-8 and p38 MAPK. Overall, our findings suggest that cryptotanshinone can sensitize TNF-α-induced apoptosis in human myeloid leukemia KBM-5 cells, which appears through ROS-dependent activation of caspase-8 and p38.

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.

The pineal gland hormone melatonin (N-acetyl-5-methoxytryptamine) is well known as an anticancer effector. Recently, a variety of studies have shown that melatonin induces apoptosis in various cancer cell types such as colon cancer, Ewing sarcoma, hepatic cancer, prostate cancer and breast cancer cells. Glioblastoma cancer stem-like cells (GCSLC) are the most common and aggressive primary brain tumor in adults. In the present study, we found that melatonin significantly decreased the sphere formation of GCSLC, and attenuated the expression of c-Myc genes. Furthermore, melatonin suppressed expression of H3K4me3 and H3K79me3 around c-Myc promoter region by Western blotting and Chip assay. Of note, melatonin suppressed the expression of several stemness markers including nestin in GCSLC. Overall, our findings suggest melatonin as a potent anticancer stem cell agent for GCSLC. Citation Format: Hyemin Lee, Ji Hoon Jung, Hyun Joo Lee, Myoung Seok Jeong, Deok-Beom Jung, Hee Young Kwon, Sung-Hoon Kim. Melatonin inhibits stemness of glioblastoma cancer stem-like cells via regulation of histone methylation. [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 94. doi:10.1158/1538-7445.AM2015-94

Though gallotannin was known to have anti-oxidant and antitumor activity, the underlying antitumor mechanism of gallotannin still remains unclear. Thus, in the present study, antitumor mechanism of gallotannin was elucidated in hepatocellular carcinoma cells. Gallotannin significantly exerted cytotoxicity against Hep G2 and Chang hepatocellular carcinoma cells with the accumulation of the sub-G1 population and increase of terminal deoxynucleotidyltransferasedUTP nick end labeling (TUNEL) positive cells as an apoptotic feature. Also, gallotannin attenuated the expression of pro-caspase9, pro-caspase3, Bcl2 and integrin β1 and cleaved poly(ADP)-ribose polymerase (PARP) in Hep G2 and Chang cancer cells. Furthermore, gallotannin suppressed cell repair motility by wound healing assay and also inhibited cell adhesion in Hep G2 cells. Of note, gallotannin attenuated the expression of epithelial cadherin (E-cadherin) to form cell-cell adhesion from the early stage, and also beta-catenin at late phase in Hep G2 cells. Consistently, Immunofluorescence assay showed that E-cadherin or β-catenin expression was suppressed in a time dependent manner by gallotannin. Furthermore, silencing of E-cadherin by siRNA transfection method enhanced PAPR cleavage, caspase 3 activation and sub G1 population and attenuated the cell adhesion induced by gallotannin in Hep G2 cells. Overall, our findings demonstrate that the disruption of cell adhesion junction by suppression of E-cadherin mediates gallotannin enhanced apoptosis in Hep G2 liver cancer cells. Citation Format: Hee Young Kwon, Ji Hoon Jung, Hyun Joo Lee, Myoung Seok Jeong, Deok-Beom Jung, Bonglee Kim, Hyemin Lee, Sung-Hoon Kim. Suppression of E-cadherin mediates gallotannin-induced apoptosis in Hep G2 hepatocelluar carcinoma 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 26. doi:10.1158/1538-7445.AM2015-26

Purpose Laparoscopic gastrectomy in obese patients has been investigated in several studies, but its feasibility has rarely been examined in morbidly obese patients, such as in those with a body mass index (BMI) of ≥30 kg/m2. The present study aimed to evaluate the technical feasibility and safety of laparoscopic gastrectomy in morbidly obese patients with gastric cancer. Materials and Methods A total of 1,512 gastric cancer patients who underwent laparoscopic distal gastrectomy (LDG) were divided into three groups: normal (BMI

Drug resistance causes a serious limitation for the chemotherapy of multiple myeloma. Combination cancer therapy is one of attractive approaches to overcome drug resistance of cancer cells. In the present study, we investigated the synergistic effect of decursin (a major compound in Angelica gigas) and doxorubicin on the induction of apoptosis in three human multiple myeloma cells. Concurrent treatment of decursin and doxorubicin significantly exerted significant cytotoxicity, while doxorubicin or decursin alone had no cytotoxic effect at physiologically relevant concentrations in signal transducer and activator of transcription 3 (STAT3) positive cell lines such as U266 and RPMI8226 cells and also STAT3 negative cell line MM.1S cells. We further demonstrated that the loss of the viability of the cells co-treated with decursin and doxorubicin was due to the induction of apoptosis. Combination treatment of doxorubicin and decursin enhanced the activation of caspapse-9 and 3, the cleavage of poly (ADP-ribose) polymerase (PARP) and sub G1 population compared to either drug alone in three multiple myeloma cells. In addition, the combined treatment of doxorubicin and decursin downregulated the phosphorylation of the mammalian target of rapamycin (mTOR) and its downstream S6K1 and activated the phosphorylation of extracellular signal regulated kinase (ERK) in three multiple myeloma cells. Furthermore, the combined treatment of doxorubicin and decursin reduced mitochondrial membrane potential, suppressed the phosphorylation of JAK2, STAT3 and Src, activated SHP-2 and attenuated the expression of cyclind-D and survivin in STAT3 positive U266 cells. Conversely, the broadly-acting tyrosine phosphatases inhibitor pervanadate reversed STAT3 inactivation and also PARP cleavage and caspase-3 activatio induced by combine treatment of doxorubicin and decursin in U266 cells. Citation Format: Duck-Beom Jung, Soo-Jin Jeong, Hee-Young Kwon, Ji Hoon Jung, Eunjung sohn, Eun Ah Sin, Ji-Hyun Kim, Sun-Mi Cho, Miyong Yun, Hyo-jung Lee, Sung Hoon Kim. Decursin and doxorubicin are in synergy for the induction of apoptosis via STAT3 and/or mTOR pathways in human multiple myeloma cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 836. doi:10.1158/1538-7445.AM2013-836

Though special AT-rich sequence-binding protein 2 (SATB2) is known as a transcriptional regulator of skeletal development and osteogenic differentiation, the underlying role of SATB2 still remains unclear. In the present study, we found that SATB2 localized to the mitotic microtubules, centrosome and midbodies in mitotic cells with α-tubulin. SATB2 knockdown by siRNA transfection reduced the cell viability, and increased the number of ethidium homodimer positive stained cells in SKOV3 ovarian cancer cells. Consistently, cell cycle analysis revealed that SATB2 knockdown accumulated G1 arrest and downregulated the cyclin-dependent kinase 2 (CDK2) expression by western blotting, while immunofluorescence assay revealed that CDK2 and SATB2 were co-localized in SKOV3 cells. Furthermore, proteosomal inhibitor MG132 treatment reversed the downregulation of CDK2 induced by SATB2 knockdown in SKOV3 cells. Overall, our findings suggest that SATB2 regulates the mitosis and G1 phase cell cycle via proteosomal ubiquitination in SKOV3 ovarian cancer cells. Citation Format: Eun Ah Sin, Eun J. Sohn, Ji Hoon Jung, Duckgue Lee, Bonglee Kim, Duck-Beom Jung, Ji-Hyun Kim, Hyo-Jeong Lee, Sung Hoon Kim. SATB2 localizes to mitotic microtubules and centrosome of cell cycles and regulates G1 phase cell cycle via proteosomal-dependent CDK2 in SKOV-3 ovarian cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1740. doi:10.1158/1538-7445.AM2013-1740