• Journal of Physiology & Pathology in Korean Medicine
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• v.25 no.5
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• pp.857-862
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• 2011

The purpose of this study is to investigate the anti-proliferative mechanism by Trichosanthes kirilowii (TCK) in MCF-7 human breast carcinoma cell. In this study, we used human breast cancer cell line, Michigan cancer foundation-7 cells (MCF-7 cells). They were co-incubated with 30~200 ${\mu}g$/ml TCK for 48 hours, and cell viability was measured by Water-soluble tetrazolium salt-1 (WST-1) assay. After MCF-7 cells were exposed to 60 ${\mu}g$/ml of TCK for 0, 3, 6, 12, 24, 48 hours, We performed flow analysis cytometry sorting(FACS) and western blot analysis. We investigated the effect of dose-dependent cell growth inhibition by TCK, which could be proved by WST-1 assay. Also, flow cytometry analysis showed that TCK increased percentage of subG1 phase and G2/M phase cell cycle. In addition, TCK induced apoptosis through the expression of caspase-9, -3 and poly(ADP-ribose) polymerase(PARP) activation. Moreover, we showed that ATM-dependent G2/M phase arrest by DNA damage and phosphorylation of chk2, cdc25C, cdc2(Tyr15). Taken together, these results suggest that by G2/M phase arrest through DNA damage and inducing of apoptosis through intrinsic pathway, TCK may have potential tumor suppressor in breast cancer.

• Journal of Physiology & Pathology in Korean Medicine
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• v.18 no.2
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• pp.427-430
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• 2004

This study was to investigate the cell cycle arrest effect and its mechanism of Radix Aconiti (RA) extract in HepG2 human hepatoma cells. We used the Flow Cytometer to investigate the effects on cell cycle arrest in RA extract-treated HepG2 cells. And protein levels involved in cell cycle progression such as p53, p21, and p21 are detected by Western blotting method. RA extract induced cell cycle arrest as confirmed by increase of G0/G1 cell population, and the mechanisms were related with up-regulation of p53, p21, p27 protein expressin in HepG2 cells. These results suggest that RA may be a valuable agent for the therapeutic intervention of human hepatomas.

• Biomolecules & Therapeutics
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• v.26 no.3
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• pp.328-334
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• 2018

Because of the unsatisfactory treatment options for breast cancer (BC), there is a need to develop novel therapeutic approaches for this malignancy. One such strategy is chemotherapy using non-toxic dietary substances and botanical products. Studies have shown that Panduratin A (PA) possesses many health benefits, including anti-inflammatory, anti-bacterial, anti-oxidant and anticancer activities. In the present study, we provide evidence that PA treatment of MCF-7 BC cells resulted in a time- and dose-dependent inhibition of cell growth with an $IC_{50}$ of $15{\mu}M$ and no to little effect on normal human MCF-10A breast cells. To define the mechanism of these anti-proliferative effects of PA, we determined its effect critical molecular events known to regulate the cell cycle and apoptotic machinery. Immunofluorescence and flow cytometric analysis of Annexin V-FITC staining provided evidence for the induction of apoptosis. PA treatment of BC cells resulted in increased activity/expression of mitochondrial cytochrome C, caspases 7, 8 and 9 with a significant increase in the Bax:Bcl-2 ratio, suggesting the involvement of a mitochondrial-dependent apoptotic pathway. Furthermore, cell cycle analysis using flow cytometry showed that PA treatment of cells resulted in G0/G1 arrest in a dose-dependent manner. Immunoblot analysis data revealed that, in MCF-7 cell lines, PA treatment resulted in the dose-dependent (i) induction of $p21^{WAF1/Cip1}$ and p27Kip1, (ii) downregulation of Cyclin dependent kinase (CDK) 4 and (iii) decrease in cyclin D1. These findings suggest that PA may be an effective therapeutic agent against BC.

• Asian Pacific Journal of Cancer Prevention
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• v.13 no.4
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• pp.1377-1382
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• 2012

Morphine is not only an analgesic treating pain for patients with cancer but also a potential anticancer drug inhibiting tumor growth and proliferation. To gain better insight into the involvement of morphine in the biological characteristics of gastric cancer, we investigated effects on progression of gastric carcinoma cells and the expression of some apoptosis-related genes including caspase-9, caspase-3, survivin and NF-${\kappa}B$ using the MGC-803 human gastric cancer cell line. The viability of cells was assessed by MTT assay, proliferation by colony formation assay, cell cycle progression and apoptosis by flow cytometry and ultrastructural alteration by transmission electron microscopy. The influences of morphine on caspase-9, caspase-3, survivin and NF-${\kappa}B$ were evaluated by semi-quantitative RT-PCR and Western blot. Our data showed that morphine could significantly inhibit cell growth and proliferation and cause cell cycle arrest in the G2/M phase. MGC-803 cells which were incubated with morphine also had a higher apoptotic rate than control cells. Morphine also led to morphological changes of gastric cancer cells. The mechanism of morphine inhibiting gastric cancer progression in vitro might be associated with activation of caspase-9 and caspase-3 and inhibition of survivin and NF-${\kappa}B$.

• Asian Pacific Journal of Cancer Prevention
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• v.13 no.4
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• pp.1425-1430
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• 2012

Cyclin L2 is a novel member of the cyclin family, recently implicated in the regulation of cell cycle progression and/or transcriptional regulation. The present study was undertaken to investigate the effects of overexpression on tumor cell growth and chemosensitivity in human gastric cells in vitro. Cyclin L2 was transfected into human gastric cancer cell line BCG823 and expressed with a mammalian expression vector pcDNA3.1. The effects and mechanisms of cyclin L2 on cell growth, cell cycling and apoptosis were studied. Compared to control vectors, overexpression of cyclin L2 inhibited the growth of BCG823 cells and enhance their chemosensitivity to fluorouracil, docetaxel and cisplatin. The anti-proliferative effects of cyclin L2 could be due to G0/G1 arrest and apoptosis. Cyclin L2 induced G0/G1 arrest and apoptosis involved upregulation of caspase-3 and down regulation Bcl-2 and survivin. The results indicated that overexpression of cyclin L2 protein may promote efficient growth inhibition and enhance chemosensitivity to chemotherapeutic agents in human gastric cancer cells by inducing G0/G1 cell cycle arrest and apoptosis.

• Asian Pacific Journal of Cancer Prevention
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• v.14 no.11
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• pp.6481-6486
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• 2013

Background: Our previous study demonstrated cytotoxicity of a crude extract from Patrinia heterophylla Bunge (PHEB). In the present study, we aimed to investigate the effects of isovaltrate acetoxyhydrin (IA) isolated from PHEB on the gastric cancer cell SGC-7901, in order to explore a potential treatment for gastric cancer. Methods: MTT assays were employed to determine the effects of IA on cell vitality and proliferation, with monitoring of cell morphology changes and examination of apoptosis with Annexin V-PI staining. Flow cytometry was used to assess cell cycle progression and mitochondrial membrane potential. The activity of caspase 3, 9 was evaluated by spectrophotometry, and the protein levels of Bax, Bcl2 and Cyclin B1 were analyzed with Western blotting of total proteins extracted from cultured cells. Results: The results demonstrated direct toxicity of IA towards SGC-7901 cells. Evidence of apoptosis included blebbing and chromatin condensation. Annexin V-PI assays revealed early apoptosis, involving rapid depolarization of mitochondrial membranes and activity of caspase 3, 9 signaling pathways. Western blotting showed that Bcl2 and Bax proteins was down- and up-regulated, respectively, and cyclin B1 was up-regulated. Cell cycle analysis further indicated that IA could induce G2/M phase arrest in SGC-7901 cells. Conclusions: In conclusion, we believe that IA induces apoptosis of SGC-7901 cells, therefore providing a potential therapeutic agent for treatment of gastric cancer.

• BMB Reports
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• v.39 no.6
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• pp.722-729
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• 2006

Recently, we have reported that 3-hydrogenkwadaphnin (3-HK), a diterpene ester isolated from Dendrostellera lessertii (Thymealeaceae), is very effective against leukemia cell lines without any detectable effects on normal cells (Moosavi et al., 2005b). In this study, we report that 3-HK induces $G_1$ cell-cycle arrest, differentiation and apoptosis in APL NB4 cell line. Indeed, the drug between 24 to 96 h induced 7-65% growth inhibition of NB4 cells. Cell viability was also decreased by 2-55% between 24 to 96 h treatments with the drug, respectively. These effects of the drug were also dose-dependent. According to flow cytomtry results, 3-HK (15 nM) induced a significant G1-arrest up to 24 h which was consequently followed with appearance of sub-$G_1$ peak at 72 to 96 h. Hoechst 33258 staining and DNA fragmentation assays confirmed the occurrence of apoptosis among the treated cells. On the other hand, NBT reducing assay, Wright-Giemsa staining, phagocytic activity and expression of cell surface markers (CD11b and CD14) confirmed that the inhibition of proliferation is associated with differentiation especially toward macrophage-like morphology. Interestingly, 3-HK at 5 and 10 nM enhanced the effects of all-trans retinoic acid (ATRA) in NB4 cells. Based on these results, 3-HK might become an ideal candidate for treatment of APL patients pending full exploration of its biological functions.

• Animal cells and systems
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• v.9 no.4
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• pp.191-198
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• 2005

The effects of 6-aminonicotinamide (6-AN) on viability of IMR32 neuroblastoma cells in the presence of ATP or $NAD^+$ have been investigated. 6-AN caused marked reduction in cell viability and similar observations were also made with cells treated with 6-AN+ATP. However, cells treated with $6-AN+NAD^+$ showed cell viability similar to untreated cells. Morphologically, 6-AN and 6-AN+ATP treated cells showed loss of neurites, polyhedric shapes, shrinkage of cell bodies and formation of lysed cells, while $6-AN+NAD^+$ cells did not show any such changes. The flow cytometry analysis demonstrated that 6-AN increased cell population in $G_0/G_1$ phase and decreased cell population in Sand $G_2/M$ phase following a 72 h exposure. Western blot analysis showed that 6-AN stimulated a substantial increase in the level of the cdk inhibitor $p27^{kip1}$, but lowered the levels of cdk2, cyclin E and p-Rb. However, cdc25A and p53R2 were not significantly affected. Immunofluorscence staining of $p27^{kip1}$, cdk2, cyclin E and p-Rb revealed close correlation between the signal observed in the Western blot analysis. 6AN+ATP treated cells showed similar results obtained with 6-AN treated cells in expression of cdk2, cyclin E, p-Rb proteins and $p27^{kip1}$, $6-AN+NAD^+$ cells showed greater expression of cdk2, cyclin E and p-Rb than those in 6-AN and 6-AN+ATP treated cells. The results suggest that 6-AN induced the $G_0/G_1$ phase arrest in IMR32 neuroblastoma cell lines through the increase of $p27^{kip1}$ and the decrease of cdk2, cyclin E and p-Rb.

• Imaging Science in Dentistry
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• v.30 no.4
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• pp.275-279
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• 2000

Purpose: Radiation damage is produced and viable cell number is reduced. We need to know the type of cell death by the ionizing radiation and the amount and duration of cell cycle arrest. In this study, we want to identified the main cause of the cellular damage in the oral cancer cells and normal keratinocytes with clinically useful radiation dosage. Materials and Methods: Human gingival tissue specimens obtained from healthy volunteers were used for primary culture of the normal human oral keratinocytes (NHOK). Primary NHOK were prepared from separated epithelial tissue and maintained in keratinocyte growth medium containing 0.15 mM calcium and a supplementary growth factor bullet kit. Fadu and Hep-2 cell lines were obtained from KCLB. Cells were irradiated in a /sup 137/Cs γ-irradiator at the dose of 10 Gy. The dose rate was 5.38 Gy/min. The necrotic cell death was examined with Lactate Dehydrogenase (LDH) activity in the culture medium. Every 4 day after irradiation, LDH activities were read and compared control group. Cell cycle phase distribution and preG1-incidence after radiation were analyzed by flow cytometry using Propidium Iodine staining. Cell cycle analysis were carried out with a FAC Star plus flowcytometry (FACS, Becton Dickinson, USA) and DNA histograms were processed with CELLFIT software (Becton Dickinson, USA). Results: LDH activity increased in all of the experimental cells by the times. This pattern could be seen in the non-irradiated cells, and there was no difference between the non-irradiated cells and irradiated cells. We detected an induction of apoptosis after irradiation with a single dose of 10 Gy. The maximal rate of apoptosis ranged from 4.0% to 8.0% 4 days after irradiation. In all experimental cells, we detected G2/M arrest after irradiation with a single dose of 10 Gy. Yet there were differences in the number of G2/M arrested cells. The maximal rate of the G2/M ranges from 60.0% to 80.0% 24h after irradiation. There is no significant changes on the rate of the G0/G1 phase. Conclusion: Radiation sensitivity was not related with necrosis but cell cycle arrest and apoptosis. These data suggested that more arrested cell is correlated with more apoptosis.

• BMB Reports
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• v.43 no.4
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• pp.291-296
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• 2010

Cyclin-dependent kinase 2 (CDK2) is a member of serine/threonine protein kinases, which initiates the principal transitions of the eukaryotic cell cycle and is a promising target for cancer therapy. The present study was designed to inhibit cdk2 gene expression to induce cell cycle arrest and cell proliferation suppression. Here, we constructed a series of RNA interference (RNAi) plasmids which can successfully express small interference RNA (siRNA) in the transfected human cells. The results showed that the RNAi plasmids containing the coding sequences for siRNAs down-regulated the cdk2 gene expression in human cancer cells at the mRNA and the protein levels. Furthermore, we found that the cell cycle was arrested at G0G1 phases and the cell proliferation was inhibited by different siRNAs. These results demonstrate that suppression of CDK2 activity by RNAi may be an effective strategy for gene therapy in human cancers.