• Journal of Life Science
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• v.26 no.5
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• pp.555-562
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• 2016

In this study, we investigated the effects of Undaria pinnatifida (UP), Petalonia binghamiae (PB) and Punctaria latifolia (PL) extracts on the inhibition of proliferation and apoptosis in human gastric and breast cancer cells. AGS, MDA-MB-231 and SK-BR-3 cells were treated with 0, 50, 100, and 200 μg/ml concentrations of the extracts to determine their anti-proliferative effects, using the MTT assay. The UP, PB and PL extracts inhibited proliferation of AGS, MDA-MB-231 and SK-BR-3 cells in a dose-dependent manner, and the PL extract was found to be the most effective. DAPI staining was also performed to determine changes in the cell nucleus. Further, the AGS, MDA-MB-231 and SK-BR-3 cells were treated with 0, 50, 100, and 200 μg/ml of only the PL extract. DAPI staining showed increased chromatin condensation, which is indicative of apoptosis, in the 200 μg/ml group. The expression of the Bax, Bcl-2, and PARP proteins in AGS, MDA-MB-231 and SK-BR-3 cells treated with the PL extract was also determined by western blot analysis. The expression of Bax (a pro-apoptotic protein) and cleaved-PARP was increased, whereas the expression of Bcl-2 (an anti-apoptotic protein) was decreased compared with the control. These findings indicate that the PL extract may have potential as an alternative anticancer drug and nutraceutical.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.7
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• pp.3311-3317
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• 2014

Background: The consequence of Rho GDP dissociation inhibitor alpha (RhoGDI${\alpha}$) activity on migration and invasion of estrogen receptor positive ($ER^+$) and negative ($ER^-$) breast cancer cells has not been studied using the proteomic approach. Changes in expression of RhoGDI${\alpha}$ and other proteins interacting directly or indirectly with RhoGDI${\alpha}$ in MCF7 and MDA-MB-231, with different metastatic potentials is of particular interest. Materials and Methods: $ER^+$ MCF7 and ER- MDA-MB-231 cell lines were subjected to two-dimensional electrophoresis (2-DE) and spots of interest were identified by matrix-assisted laser desorption/ionization time of- flight/time-of-flight (MALDI-TOF/TOF) mass spectrometry (MS) analysis after downregulation of RhoGDI${\alpha}$ using short interfering RNA (siRNA) and upregulated using GFP-tagged ORF clone of RhoGDI${\alpha}$. Results: The results showed a total of 35 proteins that were either up- or down-regulated in these cells. Here we identifed 9 and 15 proteins differentially expressed with silencing of RhoGDI${\alpha}$ in MCF-7 and the MDA-MB-231 cells, respectively. In addition, 10 proteins were differentially expressed in the upregulation of RhoGDI${\alpha}$ in MCF7, while only one protein was identified in the upregulation of RhoGDI${\alpha}$ in MDA-MB-231. Based on the biological functions of these proteins, the results revealed that proteins involved in cell migration are more strongly altered with RhoGDI-${\alpha}$ activity. Although several of these proteins have been previously indicated in tumorigenesis and invasiveness of breast cancer cells, some ohave not been previously reported to be involved in breast cancer migration. Hence, these proteins may serve as useful candidate biomarkers for tumorigenesis and invasiveness of breast cancer cells. Conclusions: Future studies are needed to determine the mechanisms by which these proteins regulate cell migration. The combination of RhoGDI${\alpha}$ with other potential biomarkers may be a more promising approach in the inhibition of breast cancer cell migration.

• Journal of Pharmacopuncture
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• v.22 no.1
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• pp.28-34
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• 2019

Background: Breast cancer is a complex, heterogeneous disease and one of the most common malignancies in women worldwide. The efficacy of chemotherapy as an important breast cancer treatment option has been severely limited because of the inherent or acquired resistance of cancer cells. The molecular chaperone heat shock protein 90 (HSP90) upregulated in response to cellular stress is required for functions such as conformational maturation, activation and stability in more than 200 client proteins, mostly of the signaling type. In this study, the expression of HSP90 isoforms including $HSP90{\alpha}$ and $HSP90{\beta}$ in breast cancer cell lines before and after treatment with doxorubicin (DOX) was assessed. Material and Methods: The cell cytotoxicity of DOX in MDA-MB-231 and MCF-7 cell lines was determined using the MTT assay. immunofluorescence and western blotting techniques were used to determine the expression of $HSP90{\beta}$ in the cell lines before and after DOX treatment. Immunofluorescence was also conducted to ascertain the expression of $HSP90{\alpha}$. Results: The MTT assay results showed that the MDA-MB-231 cells ($IC_{50}=14.521{\mu}M$) were more sensitive than the MCF-7 cells ($IC_{50}=16.3315{\mu}M$) to DOX. The immunofluorescence results indicated that the expression of $HSP90{\alpha}$ in both cell lines decreased after exposure to DOX. The western blot and immunofluorescence analyses showed that $HSP90{\beta}$ expression decreased in the MCF-7 cells but increased in the MDA-MB-231 cells after DOX treatment. Conclusion: The obtained results suggested that $HSP90{\alpha}$ and $HSP90{\beta}$ expression levels were reduced in the MCF-7 cells after exposure to DOX. In the MDA-MB-231 cells, $HSP90{\alpha}$ expression was reduced while $HSP90{\beta}$ was found to be overexpressed following DOX treatment.

• Biomolecules & Therapeutics
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• v.17 no.4
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• pp.370-378
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• 2009

N-myc downstream-regulated gene 2 (NDRG2) has recently been found to be a tumor suppressor gene. Although it has been reported that NDRG2 expression in breast cancer cells decreases cell proliferation by inhibiting STAT3 activation via SOCS1 induction, the molecular mechanism of chemotherapeutic agent-induced apoptosis is not well known. To elucidate the effect of NDRG2 on the apoptotic pathway induced by doxorubicin, we established stable cell lines expressing NDRG2 and investigated the effect of NDRG2 expression on the doxorubicin-induced apoptosis. While STAT3 activation was remarkably inhibited by NDRG2 overexpression, the expression level of p21 was increased by NDRG2 expression. We confirmed that NDRG2-expressing cells treated with doxorubicin suppressed STAT3 activation and upregulated p21 expression. NDRG2 expression considerably enhanced TUNEL positive apoptotic cells, poly-ADP ribose polymerase (PARP) cleavage, release of cytochrome c to cytosol, and caspase-3 activity in doxorubicin-induced apoptosis. Bid expression in a resting state and after treatment with doxorubicin increased in MDA-MB-231-NDRG2 cells compared to MDA-MB-231-mock cells. Meanwhile, Bcl-$x_L$ expression decreased in MDA-MB-231-NDRG2 cells compared to MDA-MB-231-mock cells in a resting state and in doxorubicin-treated cells. Collectively, these data suggest that suppression of STAT3 activation by NDRG2 influences the sensitivity to doxorubicin-induced apoptosis of breast cancer cells and this may provide a potential therapeutic benefit to overcome the resistance against doxorubicin in breast cancer.

• Bulletin of the Korean Chemical Society
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• v.35 no.4
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• pp.987-988
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• 2014

• Asian Pacific Journal of Cancer Prevention
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• v.16 no.5
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• pp.1833-1837
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• 2015

An aristolactam-type alkaloid, isolated from Orophea enterocarpa, is enterocarpam-III (10-amino-2,3,4,6-tetramethoxyphenanthrene-1-carboxylic acid lactam). It is cytotoxic to various human and murine cancer cell lines; however, the molecular mechanisms remain unclear. The aims of this study were to investigate cytotoxic effects on and mechanism (s) of human cancer cell death in human hepatocellular carcinoma HepG2 and human invasive breast cancer MDA-MB-231 cells compared to normal murine fibroblast NIH3T3 cells. Cell viability was determined by MTT assay to determine $IC_{10}$, $IC_{20}$ and $IC_{50}$ levels, reactive oxygen species (ROS) production with 2',7'-dichlorohydrofluorescein diacetate and the caspase-3, -8 and -9 activities using specific chromogenic (p-nitroaniline) tetrapeptide substrates, viz., DEVD-NA, IETD-NA and LEHD-NA and employing a microplate reader. Mitochondrial transmembrane potential (MTP) was measured by staining with 3, 3'-dihexyloxacarbocyanine iodide ($DiOC_6$) and using flow cytometry. The compound was cytotoxic to HepG2 and MDA-MB-231 cells with the $IC_{50}$ levels of $26.0{\pm}4.45$ and $51.3{\pm}2.05{\mu}M$, respectively. For murine normal fibroblast NIH3T3 cells, the $IC_{50}$ concentration was $81.3{\pm}10.1{\mu}M$. ROS production was reduced in a dose-response manner in HepG2 cells. The caspase-9 and -3 activities increased in a concentration-dependent manner, whereas caspase-8 activity did not alter, indicating the intrinsic pathway activation. Enterocarpam-III decreased the mitochondrial transmembrane potential (MTP) dose-dependently in HepG2 cells, suggesting that the compound induced HepG2 cell apoptosis via the mitochondrial pathway. In conclusion, enterocarpam-III inhibited HepG2 and MDA-MB-231 cell proliferation and induced human HepG2 cells to undergo apoptosis via the intrinsic (mitochondrial) pathway and induction of caspase-9 activity.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.23
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• pp.10397-10400
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• 2015

Stigmalactam, an aristolactam-type alkaloid extracted from Orophea enterocarpa, exerts cytotoxicity against several human and murine cancer cell lines, but the molecular mechanisms remain elusive. The aims of this study were to identify the mode and mechanisms of human cancer cell death induced by stigmalactam employing human hepatocellular carcinoma HepG2 and human invasive breast cancer MDA-MB-231 cells as models, compared to normal murine fibroblasts. It was found that stigmalactam was toxic to HepG2 and MDA-MB-231 cells with $IC_{50}$ levels of $23.0{\pm}2.67{\mu}M$ and $33.2{\pm}4.54{\mu}M$, respectively, using MTT assays. At the same time the $IC_{50}$ level towards murine normal fibroblast NIH3T3 cells was $24.4{\pm}6.75{\mu}M$. Reactive oxygen species (ROS) production was reduced in stigmalactam-treated cells dose dependently after 4 h of incubation, indicating antioxidant activity, measured by using 2',7',-dichlorohydrofluorescein diacetate and flow cytometry. Caspase-3 and caspase-9 activities were increased in a dose response manner, while stigmalactam decreased the mitochondrial transmembrane potential dose-dependently in HepG2 cells, using 3,3'-dihexyloxacarbocyanine iodide and flow cytometry, indicating mitochondrial pathway-mediated apoptosis. In conclusion, stigmalactam from O. enterocarpa was toxic to both HepG2 and MDA-MB-231 cells and induced human cancer HepG2 cells to undergo apoptosis via the intrinsic (mitochondrial) pathway.

• Nutrition Research and Practice
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• v.3 no.4
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• pp.265-271
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• 2009

The role that antioxidants play in the process of carcinogenesis has recently gained considerable attention. $\alpha$-Lipoic acid, a naturally occurring disulfide molecule, is a powerful antioxidant that reportedly exerts beneficial effects in patients with advanced cancer by reducing the level of reactive oxygen species and increasing glutathione peroxidase activity. In this study, we examined changes in the protein and mRNA expression associated with cell proliferation and apoptosis in MDA-MB-231 breast cancer cultured in the presence of various concentrations (0, 250, 500, and 1000 ${\mu}mol/L$) of $\alpha$-lipoic acid. The results revealed that $\alpha$-lipoic acid inhibited the growth of breast cancer cells in a dose-independent manner (P < 0.05). Additionally, $ErbB_2$ and $ErbB_3$ protein and mRNA expressions were significantly decreased in a dose-dependent manner in response to $\alpha$-lipoic acid (P < 0.05). Furthermore, the protein expression of phosphorylated Akt (p-Akt) levels and total Akt, and the mRNA expression of Akt were decreased dose-dependently in cells that were treated with $\alpha$-lipoic acid (P < 0.05). Bcl-2 protein and mRNA expressions were also decreased in cells that were treated with $\alpha$-lipoic acid (P < 0.05). However, Bax protein and mRNA expressions were increased in cells treated with $\alpha$-lipoic acid (P < 0.05). Finally, caspase-3 activity was significantly increased in a dose-dependent manner in cells treated with $\alpha$-lipoic acid (P < 0.05). In conclusion, we demonstrated that $\alpha$-lipoic acid inhibits cell proliferation and induces apoptosis in MDA-MB-231 breast cancer cell lines.

• Asian Pacific Journal of Cancer Prevention
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• v.13 no.6
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• pp.2979-2984
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• 2012

Recent studies have indicated a link between levels of cyclooxygenase-2 (COX-2) and development of the multidrug resistance (MDR) phenotype. The ATP-binding cassette sub-family G member 2 (ABCG2) is a major MDR-related transporter protein that is frequently overexpressed in cancer patients. In this study, we aimed to evaluate any positive correlation between COX-2 and ABCG2 gene expression using the COX-2 inducer 12-O-tetradecanoylphorbol-13-acetate (TPA) in human breast cancer cell lines. ABCG2 mRNA and protein expression was studied using real-time RT-PCR and flow cytometry, respectively. A significant increase of COX-2 mRNA expression (up to 11-fold by 4 h) was induced by TPA in MDA-MB-231 cells, this induction effect being lower in MCF-7 cells. TPA caused a considerable increase up to 9-fold in ABCG2 mRNA expression in parental MCF-7 cells, while it caused a small enhancement in ABCG2 expression up to 67 % by 4 h followed by a time-dependent decrease in ABCG2 mRNA expression in MDA-MB-231 cells. TPA treatment resulted in a slight increase of ABCG2 protein expression in MCF-7 cells, while a time-dependent decrease in ABCG2 protein expression was occurred in MDA-MB-231 cells. In conclusion, based on the observed effects of TPA in MDA-Mb-231 cells, it is proposed that TPA up-regulates ABCG2 expression in the drug sensitive MCF-7 breast cancer cell line through COX-2 unrelated pathways.

• Journal of Physiology & Pathology in Korean Medicine
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• v.25 no.6
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• pp.968-974
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• 2011

Saussurea lappa Clarke is a well-known transitional medicine in Asia including Korea, China and Japan. It has been reported that Clarke has diverse effects such as anti-viral, anti-inflammatory, anti-cancer in human gastric cells and human prostate cancer cells. However, the anti-cancer effects and the mechanism of actions of Saussurea lappa Clarke are still unknown in breast cancer. In this study, we observed that Saussurea lappa Clarke inhibits the cell growth in a dose- and time-dependent manner in highly-metastatic MDA-MB-231 breast cancer cells. In order to examine whether Saussurea lappa Clarke suppresses cell growth inducing apoptosis cell death or cell cycle arrest, we analyzed DNA contents and cell cycle distribution using a flow cytometer and western blotting in MDA-MB-231 cells. We suggest that Saussurea lappa Clarke dose not induced apoptosis and induced G2/M phase cell cycle arrest. Moreover, Saussurea lappa Clarke also decreased the expression level of metastasis-angiogenesis releated protein such as VEGF. However, dose not changed the expression level of metastasis related protease MMP-1 in highly-metastatic MDA-MB-231 breast cancer cells. Therefore, Saussurea lappa Clarke might be good and useful chemotherapy agent highly-metastatic breast cancer patients.