Gunho Won

Upregulation of miRNA3195 and miRNA374b Mediates the Anti-Angiogenic Properties of Melatonin in Hypoxic PC-3 Prostate Cancer Cells

Recently microRNAs (miRNAs) have been attractive targets with their key roles in biological regulation through post-transcription to control mRNA stability and protein translation. Though melatonin was known as an anti-angiogenic agent, the underlying mechanism of melatonin in PC-3 prostate cancer cells under hypoxia still remains unclear. Thus, in the current study, we elucidated the important roles of miRNAs in melatonin-induced anti-angiogenic activity in hypoxic PC-3 cells. miRNA array revealed that 33 miRNAs (>2 folds) including miRNA3195 and miRNA 374b were significantly upregulated and 16 miRNAs were downregulated in melatonin-treated PC-3 cells under hypoxia compared to untreated control. Melatonin significantly attenuated the expression of hypoxia-inducible factor (HIF)-1 alpha, HIF-2 alpha and vascular endothelial growth factor (VEGF) at mRNA level in hypoxic PC-3 cells. Consistently, melatonin enhanced the expression of miRNA3195 and miRNA 374b in hypoxic PC-3 cells by qRT-PCR analysis. Of note, overexpression of miRNA3195 and miRNA374b mimics attenuated the mRNA levels of angiogenesis related genes such as HIF-1alpha, HIF-2 alpha and VEGF in PC-3 cells under hypoxia. Furthermore, overexpression of miRNA3195 and miRNA374b suppressed typical angiogenic protein VEGF at the protein level and VEGF production induced by melatonin, while antisense oligonucleotides against miRNA 3195 or miRNA 374b did not affect VEGF production induced by melatonin. Also, overexpression of miR3195 or miR374b reduced HIF-1 alpha immunofluorescent expression in hypoxic PC-3 compared to untreated control. Overall, our findings suggest that upregulation of miRNA3195 and miRNA374b mediates anti-angiogenic property induced by melatonin in hypoxic PC-3 cells.

Generation of ROS by Decursin selectively induces the ER stress pathway components ATF4/PERK leading to the synergistic enhancement of TRAIL‐induced apoptosis

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.

Decursin enhances TRAIL-induced apoptosis through oxidative stress mediated- endoplasmic reticulum stress signalling in non-small cell lung cancers.

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.

Regulation of crosstalk between epithelial to mesenchymal transition molecules and MMP-9 mediates the antimetastatic activity of anethole in DU145 prostate cancer cells.

The underlying antimetastatic mechanism of anethole (1) still remains unclear in association with the molecules of the epithelial to mesenchymal transition (EMT). Herein, the role of the EMT molecules was elucidated in terms of the antimetastatic activity of 1 using DU145 cells. Anethole significantly inhibited the adhesion of DU145 cells to vitronectin-coated plates, as well as migration in a wound-healing assay and invasion using a Boyden chamber. Also, anethole suppressed the expression of MMP-9 in DU145 cells by zymography, ELISA, and RT-PCR. Consistently, the silencing of MMP-9 enhanced the activity of 1 to upregulate the expression of E-cadherin and to attenuate the expression of Vimentin in DU145 cells. Compound 1 enhanced E-cadherin, which is an epithelial marker and attenuated the expression of Vimentin, Twist, and Snail as mesenchymal molecules at the mRNA level. Consistently, anethole upregulated E-cadherin and downregulated the expression of Vimentin, Twist and PI3K, and AKT at the protein level in DU145 cells. Conversely, the antimetastatic effects of 1 to inhibit invasion and the expression of MMP-9 and upregulate E-cadherin were reversed by the EMT inducer TGF-β in DU145 cells. Overall, the present findings suggest that anethole exerts antimetastatic activity via regulation of crosstalk between EMT molecules and MMP-9 on the basis of the in vitro data obtained.

Upregulation of death receptor 5 and activation of caspase 8/3 play a critical role in ergosterol peroxide induced apoptosis in DU 145 prostate cancer cells

BackgroundThough ergosterol peroxide (EP) derived from Neungyi mushrooms (Sarcodon aspratus) was known to have cytotoxic, apoptotic, anti-inflammatory and antimycobacterial effects, the underlying molecular mechanism of EP still remains unclear. Thus, in the present study, the apoptotic mechanism of EP was elucidated in DU 145 prostate cancer cells.MethodsCell viability of prostate cancer cells was measured by MTT assay. To see whether EP induces the apoptosis, FACS, western blot and TUNEL assay were performed. To determine the role of Death receptor (DR) 5 molecules in EP-induced apoptosis in DU 145 prostate cancer cells, the silencing of DR 5 was performed by using siRNAs.ResultsEP showed significant cytotoxicity against DU 145, PC 3, M2182 prostate cancer cells. Also, EP effectively increased the sub G1 population and terminal deoxynucleotidyl transferase DUTP nick end labeling (TUNEL) positive cells in DU 145 prostate cancer cells. Furthermore, western blotting revealed that EP cleaved poly (ADP-ribose) polymerase (PARP) and caspase 8/3, attenuated the expression of fluorescence loss in photobleaching (FLIP), Bcl-XL and Bcl-2 as well as activated Bax, Fas-associated death domain (FADD) and DR 5 in a concentration dependent manner in DU 145 prostate cancer cells. Conversely, caspase 8 inhibitor Z-IETD-FMK blocked the apoptotic ability of EP to cleave PARP and an increase of sub G1 population in DU 145 prostate cancer cells. Likewise, the silencing of DR 5 suppressed the cleavages of PARP induced by EP in DU 145 prostate cancer cells.ConclusionOverall, our findings suggest that ergosterol peroxide induces apoptosis via activation of death receptor 5 and caspase 8/3 in DU 145 prostate cancer cells as a cancer chemopreventive agent or dietary factor.

Blockage of epithelial to mesenchymal transition and upregulation of let 7b are critically involved in ursolic acid induced apoptosis in malignant mesothelioma cell.

Malignant pleural mesothelioma (MPN), which is caused by asbestos exposure, is one of aggressive lung tumors. In the present study, we elucidated the anti-tumor mechanism of ursolic acid in malignant mesotheliomas. Ursolic acid significantly exerted cytotoxicity in a time and dose dependent manner in H28, H2452 and MSTO-211H mesothelioma cells and inhibited cell proliferation by colony formation assay in a dose-dependent fashion. Also, ursolic acid treatment accumulated the sub-G1 population, attenuated the expression of procapase 9, cyclin D1, pAKT, p-glycogen synthase kinase 3-alpha/beta (pGSK3α/β), β-catenin and nuclear factor kappa-light-chain-enhancer of activated B cells (NFkB) and also cleaved caspase 3 and poly (ADP-ribose) polymerase (PARP) in mesothelioma cells. Furthermore, ursolic acid treatment blocked epithelial and mesenchymal transition (EMT) molecules by activating E-cadherin as an epithelial marker and attenuating Vimentin, and Twist as mesenchymal molecules. Interestingly, miRNA array revealed that 23 miRNAs (>2 folds) including let-7b and miRNA3613-5p, miRNA134 and miRNA196b were significantly upregulated while 33 miRNAs were downregulated in ursolic acid treated H2452 cells. Furthermore, overexpression of let 7b using let-7b mimics enhanced the antitumor effect of ursolic acid to attenuate the expression of procaspases 3, pro-PARP, pAKT, β-catenin and Twist and increase sub-G1 accumulation in H2452 mesothelioma cells. Overall, our findings suggest that ursolic acid induces apoptosis via inhibition of EMT and activation of let7b in mesothelioma cells as a potent chemotherapeutic agent for treatment of malignant mesotheliomas.

CCR4-NOT2 Promotes the Differentiation and Lipogenesis of 3T3-L1 Adipocytes via Upregulation of PPARγ, CEBPα and Inhibition of P-GSK3α/β and β-Catenin

Background/Aims: Though CCR4-NOT2 (CNOT2), one of CCR4-NOT complex subunits, was known to be involved in metastasis and apoptosis through transcription and mRNA degradation, its other biological function is poorly understood so far. The aim of this study is to elucidate the molecular role of CNOT2 in the differentiation process of 3T3-L1 preadipocytes. Methods and Results: CNOT2 was overexpressed during the differentiation process of 3T3-L1 preadipocytes. Consistently, mRNA levels of CNOT2, adiponectin, adiponectin 2, PPARγ and CEBPα were enhanced in 3T3-L1 adipocytes. Conversely, CNOT2 depletion by siRNA transfection also reversed the activation of PPARγ and CEBPα and inhibition of GSK3α/β and β-catenin at the protein level in 3T3-L1 preadipocytes. Immunofluorescence assay revealed that CNOT2 was colocalized with PPARγ, but not with CEBPα in 3T3-L1 adipocyte. Consistently, IP western blots revealed that CNOT2 interacted with PPARγ in 3T3-L1 adipocyte. Conclusion: Our findings demonstrate that CNOT2 promotes the differentiation of 3T3-L1 preadipocytes via upregulation of PPARγ, and CEBPα and inhibition of GSK3α/β and β-catenin signaling as a potent molecular target for obesity.

Activation of JNK and IRE1 is critically involved in tanshinone I-induced p62 dependent autophagy in malignant pleural mesothelioma cells: implication of p62 UBA domain

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 NH4Cl 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.

SATB2 is localized to the centrosome and spindle maintenance and its knockdown leads to downregulation of CDK2.

Though special AT-rich sequence-binding protein 2 (SATB2) is reported as a transcriptional regulator of skeletal development and osteogenic differentiation, the underlying mechanism of SATB2 is not fully understood. Herein, we report that SATB2 is localized to the mitotic microtubules, the centrosome, and midbodies in mitotic cells with alpha-tubulin. Moreover, siRNA-mediated disruption of SATB2 in H460 cells caused the defect of nuclear morphology and multinucleate cells. SATB2 siRNA knockdown reduced the viability and downregulated the CDK2 expression in SKOV3 cells. Consistently, cell cycle analysis demonstrated that the silencing of SATB2 induced cell-cycle G1 arrest. Furthermore, proteosomal inhibitor MG132 treatment rescued the downregulation of CDK2 in SATB2-silenced SKOV3 cells. Taken together, our findings suggest that SATB2 regulates the mitosis of cell cycle and affects G1 cell cycle via interaction with CDK2.

Abstract 1015: Misaponin B induces G2-M arrest and autophagy via upregulation of miR1290 in non-small cell lung cancer (NSCLC) A549 cells

Though Mi-saponin B is found to be the potent component among a mixture of saponins, its function still remains unlcear. Thus, in the present study, we investigated the anti-tumor mechanism of Mi-saponin B in non-small cell lung cancer (NSCLC) A549 cells. Mi-saponin B treatment caused cell growth inhibition via G2/M arrest in A549 cells. Moreover, Western blotting and qRT-PCR assays showed that Mi-saponin B induced conversion of LC3I to LC3II, a maker of autophagy. Immunofluorescence assay revealed that Mi-saponin B treatment formed the puncta of GFP-fused LC3II. Conversely, 3MA, an inhibitor of autophagy, attenuated the LC3II expression induced by Mi-saponin B in A549 cells. Also, transmission electron microscopy (TEM) showed that Mi-saponin B induces the autophagic vacuoles. Interestingly, miR1290 was upregulated in a dose dependent manner in Mi-saponin B treated A549 cells by qRT-PCR. Also, combined treatment of miR1290 and Mi-saponin B increased the expression of LC3II at protein and mRNA levels and also enhanced the formation of puncta of LC3II in A549 cells. Overall, our findings for the first time demonstrate that Mi-saponin B induces G2/M arrest and autophagy via upregulation of miR1290 in A549 cells as a potent anti-cancer agent. Citation Format: Gunho Won, Eun Jung Sohn, Myoung Seok Jeong, Sang Wook Yoon, Jihyun Lee, Bonglee Kim, Sung Hoon Kim. Misaponin B induces G2-M arrest and autophagy via upregulation of miR1290 in non-small cell lung cancer (NSCLC) A549 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 1015. doi:10.1158/1538-7445.AM2015-1015