• Asian Pacific Journal of Cancer Prevention
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• v.16 no.14
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• pp.6073-6080
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• 2015

Background: Melanoma differentiation-associated gene-7 (MDA-7)/interleukin-24 (IL-24), a unique tumor suppressor gene, has killing activity in a broad spectrum of cancer cells. Herein, plasmids producing mda-7 proteins fused to different RGD peptides (full RGD4C and shortened RGD, tRGD) were evaluated for apoptosis induction with a hepatocellular carcinoma cell line, Hep-G2. The study aim was to improve the apoptosis potency of mda-7 by tethering to RGD peptides. Materials and Methods: Three plasmids including mda-7, mda-7-RGD and mda-7-tRGD genes beside a control vector were transfected into Hep-G2 cells. After 72 hours incubation, cell viability was evaluated by MTT assay. In addition, the rate of apoptosis was analyzed by flow cytometry using PI/annexin staining. To detect early events in apoptosis, 18 hours after transfection, expression of the BAX gene was quantified by real time PCR. Modeling of proteins was also performed to extrapolate possible consequences of RGD modification on their structures and subsequent attachment to receptors. Results and Conclusions: In MTT assays, while all mda-7 forms showed measurable inhibition of proliferation, unmodified mda-7 protein exhibited most significant effect compared to control plasmid (P<0.001). Again, flow cytometry analysis showed a significant apoptosis induction by simple mda-7 gene but not for those RGD-fused mda-7 proteins. These findings were also supported by expression analysis of BAX gene (P<0.001). Protein modelling analysis revealed that tethering RGD at the end of IL-24/Mda7 disrupt attachment to cognate receptor, IL-20R1/IL-20R2. In conclusion, fusion of RGD4C and shortened RGD peptides to carboxyl terminal of mda7, not only reduce apoptosis property in vitro but also disrupt receptor attachment as demonstrated by protein modelling.

• Molecular & Cellular Toxicology
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• v.5 no.4
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• pp.335-345
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• 2009

A major source of paeoniflorin (PF) which was from the Paeonia lactiflora root, has been used as a herbal medicine in East Asia for its antiallergic, antiinflammatory, and immunoregulatory effects. However, only few details are known about the mechanism of apoptosis induced by this compound. The present study was undertaken to further elucidate the molecular mechanism of apoptosis and the changes of gene expression elicited by PF using DNA microarrays and computational gene-expression analysis tools in human leukemia U937 cells. A comparative global transcription analysis between treatment with PF and anisomycin (AM) that induces apoptosis in U937 cells revealed that c-Jun-$NH_2$-kinase (JNK) pathway related genes were less expressed in PF-treated cells. Elucidation of the mechanisms by which PF conducts its anti-cancer activities through comparative analysis of the gene expression is necessary to provide a solid foundation for its use as a promising agent in prevention and treatment strategies.

• Molecules and Cells
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• v.20 no.1
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• pp.97-104
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• 2005

To investigate apoptosis in HC11 mammary epithelial cells, we compared the gene expression profiles of actively growing and serum-starved apoptotic cells using a mouse apoptosis gene array and $^{33}P$-labeled cDNA prepared from the RNA of the two cultures. Analysis of the arrays showed that expression of several genes such as clusterin, secreted frizzled related protein mRNA (sFRP-1), CREB-binding protein (CBP), and others was higher in the apoptotic cells whereas expression of certain genes including survivin, cell division cycle 2 homolog A (CDC2), and cyclin A was lower. These expression patterns were confirmed by RT-PCR and/or Northern analyses. We compared the expression of some of these genes in the mouse mammary gland under various physiological conditions. The expression levels of genes (clusterin, CBP, and M6P-R) up-regulated in apoptotic conditions were higher at involution than during lactation. On the other hand, genes (Pin, CDC2) downregulated in apoptotic conditions were relatively highly expressed in virgin and pregnant mice. We conclude that certain genes such as clusterin, sFRP-1, GAS1 and CBP are induced in apoptotic mammary epithelial cells, and others are repressed. Moreover, the apoptosis array is an efficient technique for comparing gene expression profiles in different states of the same cell type.

• The Journal of Korean Obstetrics and Gynecology
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• v.19 no.3
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• pp.1-12
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• 2006

Purpose : This study was aimed to investigate the inhibitory effect of Leonurus sibiricus on the proliferation of human uterine leiomyoma cells and the expression of gene related the mechanism of cell apoptosis. Methods : We counted the number of death cells treated with indicated concentration of Leonurus sibiricus and investigated cell death rate by MTS assay. Furthermore, flow cytometry analysis and DNA fragmentation assay were used to dissect between necrosis and apoptosis and then we observed the differential gene expression by western blot analysis. Results : Leonurus sibiricus significantly inhibited the proliferation of uterine leiomyoma cell in a dose-dependent and time dependent manner. Fluorescence activated cell sorter (FACS) analysis indicated that Leonurus sibiricus induced G1 cell cycle arrest. Leonurus sibiricus enhanced the expression of p27 and p53 with cell cycle arrest. Conclusion : These findings suggest that Leonurus sibiricus is a candidate agent for the treatment of uterine leiomyoma. p27, $p53^{1}$ may play an important role in Leonurus sibiricus-induced cell cycle arrest and cell growth inhibition.

• Journal of the East Asian Society of Dietary Life
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• v.25 no.1
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• pp.87-98
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• 2015

We investigated the apoptotic effects of grape skin extracts (GSE) and related gene expressions in human breast cancer MDA-MB-231 cells cultured in the presence of 0, 0.5, 1 and 1.5 mg/mL of GSE for 72 hours. MTT assay, trypan blue and nuclei staining showed lower cellular mitochondrial activities and increased cell deaths with a higher concentration of GSE (p<0.05). Increased cell number with fragmentated DNA of sub-G1 phase was calculated as a measure of apoptotic cell death by FACS analysis (p<0.05). In particular, apoptotic cell death caused markedly increased in the 1 and 1.5 mg/mL of GSE groups, as revealed by flow cytometry (Annexin V-FITC). RT-PCR analysis was performed on apoptotic and preapoptotic genes. Expression of the apoptosis suppressor gene bcl-2 significantly decreased, proapoptotic gene bax was significantly increased and procaspase-3 showing the presence of caspase-3 significantly decreased (p<0.05). Furthermore, bcl-2/bax ratio which is considered to be an important indicator of apoptosis, significantly decreased in a concentration-dependent manner (p<0.05). These results indicated that GSE induces apoptosis in MDA-MB-231 human breast cancer cells.

• Tuberculosis and Respiratory Diseases
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• v.56 no.5
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• pp.450-464
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• 2004

Background : The FHIT (fragile histidine triad) gene is a frequent target of deletions associated with abnormal RNA and protein expression in lung cancer. Previous studies have shown FHIT gene transfer into lung cancer cell line lacking FHIT protein expression resulted in inhibition of tumor cell growth attributable to the induction of apoptosis and reversion of tumorigenecity. However, the mechanism of the tumor suppressor activity of the FHIT gene and the cellular pathways associated with its function are not completely understood. Methods : To gain insight into the biological function of FHIT, we compared the NCI-H358 cell line with its stable FHIT transfectants after treatment with cisplatin or paclitaxel. We investigated the effects of FHIT gene expression on cell proliferation, apoptosis, and activation of caspase system and Bcl-2 family. The induction of apoptosis was evaluated by using DAPI staining and flow cytometry. Activation of caspases and Bcl-2 members was evaluated by Western blot analysis. Results : A significantly increased cell death was observed in FHIT transfectants after cisplatin or paclitaxel treatment and this was attributable to the induction of apoptosis. Remarkable changes in caspases and Bcl-2 family were observed in the transfected cells as compared with the control cells after treatment with paclitaxel. Activation of caspase-3 and caspase-7 was markedly increased in cells expressing FHIT. Expression level of Bcl-2 and Bcl-xL protein was significantly decreased and that of Bax and Bad protein was significantly increased in the transfected cells. Conclusion : FHIT gene delivery into lung cancer cells results in enhanced apoptosis induced by treatment with cisplatin or paclitaxel. The data suggest that apoptosis in FHIT-expressing cells could be related to activation of caspase pathway and Bcl-2 family.

• Asian Pacific Journal of Cancer Prevention
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• v.13 no.3
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• pp.1043-1048
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• 2012

The COPS3 gene has stimulating effect on cell proliferation and progression of osteosarcomas and related cells. However, the features of COPS3 and its potential application as a therapeutic target in other cancers has not yet been studied. In this study, therefore, the effect of COPS3 silencing via COPS3 siRNA on lung cancer cell proliferation was examined. Expression levels of COPS3 gene in COPS3 siRNA infected cells and control siRNA infected cells were compared with real time PCR and Western blot analysis. Cell proliferation levels were comprehensively analyzed by MTT, BrdU incorporationy, and colony formation assays. For mechanistic assessment the effects of COPS3 silencing on cell cycle and apoptosis were analyzed using flow cytometry. Results showed that successful silencing of the COPS3 gene at both translational and transcriptional levels significantly reduced the proliferation and colony formation by lung cancer cells (p<0.01). Flow cytometry showed cell cycle arrest in the G0/G1 phase after COPS3 silencing, and more importantly, apoptosis was induced as a result of COPS3 knockdown, which negatively affected cell survival. Therefore, these results provide another piece of important evidence that the COPS3 gene expressed in lung cancer cells may play a critical role in stimulating proliferation. Down-regulation of COPS3 could significantly inhibit lung cancer cell growth, which was most likely mediated via induction of cell cycle arrest in G0/G1 phase and apoptosis.

• The Journal of Korean Obstetrics and Gynecology
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• v.19 no.2
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• pp.186-198
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• 2006

Purpose : The purpose of this study is to demonstrate the direct inhibitory effect of Hyulbuchukeotang on the proliferation of uterine leiomyoma cells through an experiment treating uterine leiomyoma cells cultivated by explantation with indicated concentrations of Hyulbuchukeotang and to research the gene expression related to cell cycle ill order to discover the connection with apoptosis and its mechanism by analyzing cell cycle. Methods : After primary culture of uterine leiomyoma cells, the cultivated uterine leiomyoma cells were treated with indicated concentrations of Hyulbuchukeotang for 24 hours. The inhibitory effect on the cell proliferation was determined by the cell count assay. The value of a cell count assay represent the percentage of cells in a phase of the cell cycle compared with total cells. In addition, a link between Hyulbuchukeotang and apoptosis was examined through flow cytometric analysis by FACS and DNA fragmentation analysis. Finally, the degree of gene expression related to cell cycle was evaluated by Western blot analysis. Results : The inhibitory effect of Hyulbuchukeotang increase of uterine leiomyoma cells treated with indicated concentrations of Hyulbuchkeotang increases. The result of gene expression related to G1 phase after treating with 100, 250, 500, 1,000 ${\mu}g/ml$ concentrations of Hyulbuchukeotang. on uterine leiomyoma cells is that the gene expression of p27 was increased but that of p53 an p21 remained unchanged and the gene of pRB, pro-caspase 3 was decreased. Conclusion Through the mentioned experiments, it is demonstrated that Hyulbuchkeotang is effective in inhibiting Proliferation of uterine leiomyoma cells by extending cell cycle G1. However it is not considered that the inhibitory effect results from the aptoposis.

• Asian-Australasian Journal of Animal Sciences
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• v.20 no.2
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• pp.153-159
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• 2007

In our previous study, overexpression of extracellular proteinase inhibitor (Expi) gene accelerated apoptosis of mammary epithelial cells, and induced expression of B cell activating factor (BAFF) gene. In this study, we found induction of BAFF-receptor (BAFF-R) gene expression in the Expi-transfected cells. A proliferation-inducing ligand (APRIL) gene is another TNF family member and the closest known relative of BAFF. We found induction of APRIL gene expression in the Expi-overexpressed apoptotic cells. NF-${\kappa}$B gene was also induced in the Expi-overexpressed cells. Expression patterns of BAFF and APRIL pathway-related genes were examined in in vivo mouse mammary gland at various reproductive stages. Expression levels of BAFF gene were very low at early pregnancy, increased from mid-pregnancy, and peaked at lactation, and thereafter decreased at involution stages of mammary gland. Expression of BAFF-R gene was highly induced in involution stages compared to lactation stages. Thus, expression patterns of BAFF-R gene were correlated to apoptotic status of mammary gland: active apoptosis of mammary epithelial cells occurs at involution stage of mammary gland. Expression levels of NF-${\kappa}$B gene were higher in involution stages compared to lactation stages. We analyzed mRNA levels of bcl-2 family genes from different stages of mammary development. Bcl-2 gene expression was relatively constant during lactation and involution stages. There was a slight increase in bcl-xL gene expression in involution stages compared to lactation state. Bax gene expression was highly induced in involution stage. Our results suggest that signaling pathways activated by both BAFF and ARRIL in mammary gland point towards NF-${\kappa}$B activation which causes upregulation of bax.

• Bulletin of the Korean Chemical Society
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• v.28 no.1
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• pp.95-98
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• 2007

Polyethylenimine (PEI) has been widely investigated for delivery of DNA into cells. It was previously reported that there were at least two types of cytotoxicity in PEI-mediated gene delivery, immediate and delayed toxicities. PEI-mediated gene delivery protocols use net cationic complexes with an excess of PEI to maintain equilibrium between the complexed and dissociated forms in solution. In this study, toxicity of free PEI or PEI/ DNA complex was investigated. Human embryonic kidney 293 cells were incubated with free PEI or PEI/DNA complex for 4 hrs. Then, the cells were analyzed at 6, 24, 48, and 96 hrs after the incubation. In MTT assay, the viability of the cells incubated with PEI/DNA complex was continuously decreased with time, while that of the cells incubated with free PEI was not. On the contrary, the expression level of the luciferase gene increased gradually along with time. Release of DNAs from the complexes for transcription produces free PEIs in the cells. This process may proceed slowly due to high charge density of PEI and may be related to delayed toxicity. In addition, apoptotic cells were observed only in the cells incubated with the PEI/DNA complex from 24 hrs after the incubation. The results suggest that PEI/DNA complex contributes to the delayed toxicity by inducing apoptosis and that the delayed toxicity may be related to decomplexation of the complexes in the cells.