Attenuated anti‑tumor activity of NK‑92 cells by invasive human breast carcinoma MDA‑MB‑231 cells

Hwan Hee Lee,Hyosun Cho 대한독성 유전단백체 학회 2020 Molecular & cellular toxicology Vol.16 No.2

Background Natural killer (NK) cells are typical innate lymphocytes that directly kill cancer cells. However, the anti-tumor function of NK cells can be modulated by various cancers in a tumor microenvironment. Objective We investigated whether the functional characteristics of NK cells are affected by coculture with human breast carcinoma MCF-7 or MDA-MB231 cells and further examined the underlying molecular mechanisms that are associated with attenuated anti-tumor activity of NK cells. NK-92 cells were cocultured with MCF-7 or MDA-MB-231 cells at a ratio of 1:2 (target:effector) for 6 h. The cytotoxic effect of NK-92 cells was measured using CytoTox96 assay. The frequency of CD56, NKp30+, NKp44+, NKG2D+, and NKG2A+ in NK-92 cells was investigated using individual fluorescent antibodies and flow cytometry. In addition, the production of cytokines was measured using Human Cytokine Array C1 kit. Proteins in cancer cell lysates were quantified and the expressions of PI3K, pAkt, Stat3, and pStat3 were examined by western blot analysis. Results The cytotoxicity of NK-92 cells is greater against MCF-7 than against MDA-MB-231 cells and cocultures with MCF-7 or MDA-MB-231 cells increased the frequency of CD56dim population in NK-92 cells as well as IFN-γ production. The frequency of the NKp30+ or NKG2D+ population in NK-92 cells was significantly decreased in coculture with MCF-7 or MDA-MB-231 cells. However, the frequency of NKG2A+ expression in NK-92 cells was significantly increased in coculture with MCF-7 or MDA-MB-231 cells. The secretions of IL-6, IL-8, monocyte chemoattractant protein-1, and granulocyte– macrophage colony-stimulating factor were significantly higher in supernatant from coculture of NK-92 and MDA-MB-231 cells than in supernatants from NK-92 cell single culture or coculture of NK-92 and MCF-7 cells, which positively correlates with the enhanced expression of pStat3 in MDA-MB-231 cells cocultured with NK-92 cells. Conclusion These findings demonstrate that the anti-tumor function of NK-92 cells is significantly suppressed by an invasive human breast carcinoma, MDA-MB-231, and the impairment of NK-92 cell function is associated with the upregulation of the IL-6/Stat3 signaling pathway.

두릅의 인체 유방암세포 MDA-MB-231 사멸 효과

류민주 ( Min Ju Ryu ),정하숙 ( Ha Sook Chung ) 한국산업식품공학회 2015 산업 식품공학 Vol.19 No.3

본 연구는 인체 유래 유방암세포인 MDA-MB-231 세포증식을 억제하고 세포사멸을 유도하는 천연소재 발굴을 목적으로, 북한산 두릅 추출물을 MDA-MB-231 세포에 처리하여 세포사멸 및 작용기전을 규명하였다. 실험 결과, 두릅추출물 처리 농도가 증가할수록 세포증식이 감소하였고, 세포질의 응축과 핵이 분절되는 등 apoptotic bodies를 형성하였다. 또한 유세포 분석기를 사용하여 MDA-MB-231의 세포주기가 억제되었고, 세포사멸의 특징인 sub-G1 수치가 증가되는 것이 관찰되었고, 두릅 추출물 농도가 증가함에 따라 apoptotic 세포가 유의적으로 증가하였으며, 특히 early apoptosis 보다 late apoptosis가 더 많이 진행됨을 확인할수 있었다. 이를 바탕으로 MDA-MB-231 세포사멸과 관련된 유전자를 확인하고자 RT-PCR과 western blotting을 수행한 결과, 세포사멸의 주요한 조절인자인 anti-apoptotic인 bcl-2 발현이 두릅 추출물 처리 시 농도 의존적으로 감소되었고, 반대로 Bax의 발현은 증가됨을 확인하였다. 이를 통해 불활성화 형태로 존재한 pro-caspase-9과 pro-caspase-3의 발현이 시료 농도가 증가할수록 감소되었고, 활성화된 형태인 cleaved caspase-9과 cleaved caspase-3의 발현은 두릅 추출물 처리 농도에 의존적으로 증가하였다. 뿐만 아니라, 세포사멸 과정의 주요 인자인 PARP 또한 시료 농도가 증가할수록 절단 현상이 비례적으로 증가하였다. 이상의 실험 결과를 근거로, 북한산 두릅 지상부의 70% ethyl alcohol 추출물이 MDA-MB-231 인체 유방암 세포의 증식을 억제하는 효과가 있음을 확인하였고, 세포사멸과 관련된 활성기전을 규명하였다. 추후 암 예방/치료제로서 두릅을 활용하기 위해서는 활성 본체와 안정성 규명 및 임상실험 등 추가 연구가 필요할 것으로 사료된다. The cytotoxic effect and mechanism of Aralia elata were investigated in MDA-MB-231 human breast cancer cells. In this study, Aralia elata inhibited significantly the proliferation of MDA-MB-231 cells, and some typical apoptotic characteristics, such as nuclear fragmentation and chromatin condensation, were observed. In addition, flow cytometry analysis showed that Aralia elata increased the sub-G1 (apoptosis) population and apoptosis further confirmed by Annexin V-FITC and PI double staining. With respect to the mechanism underlying the induction of apoptosis, apoptosis-related mRNA and proteins were measured using a reverse transcription-polymerase chain reaction and western blot analysis. Aralia elata reduced anti-apoptotic Bcl-2 mRNA and protein levels, but pro-apoptotic Bax mRNA and protein expression were increased compared with the controls. Aralia elata also induced the cleavage of caspase-9 followed by the activation of caspase-3, resulting in the activation of poly-ADP-ribose polymerase. These results suggest that apoptotic activity of Aralia elata is probably modulated by a caspase-dependent cascade via the activation of intrinsic pathway. This is the first report to demonstrate the cytotoxic effect of Aralia elata on human breast cancer cells and to provide a possible mechanism for this activity.

Loquat (Eriobotrya japonica) leaf extract inhibits the growth of MDA-MB-231 tumors in nude mouse xenografts and invasion of MDA-MB-231 cells

Mi-Kyoung You,Min-Sook Kim,Kyu-Shik Jeong,Eun Kim,Yong-Jae Kim,Hyeon-A Kim 한국영양학회 2016 Nutrition Research and Practice Vol.10 No.2

BACKGROUND/OBJECFTIVES: The present study was conducted to examine the inhibitory effect of loquat leaves on MDA-MB-231 cell proliferation and invasion. MATERIALS/METHODS: Female athymic nude mice were given a subcutaneous (s.c.) inoculation of MDA-MB-231 cells and randomly grouped to receive a s.c. injection of either 500 ㎎/㎏ ethanol, water extract or vehicle five times a week. Tumor growth, mitotic rate and necrosis were examined. MDA-MB-231 cells were cultured with DMSO or with various concentrations of loquat water or ethanol extract. Proliferation, adhesion, migration, invasion and matrix metalloproteinase (MMP) activity were examined. RESULTS: Tumor growth of xenograft nude mouse was significantly reduced by loquat extracts. The results of mitotic examination revealed that loquat extracts reduced tumor cell division. Both ethanol and water extracts significantly inhibited MDA-MB-231 cell proliferation. The protein expression of ErbB3 was significantly down-regulated by loquat leaf extracts. Loquat leaf extracts increased apoptosis of MDA-MB-231 cells following 24 hour incubation and the ethanol extract was more potent in inducing apoptosis than the water extract. Furthermore, loquat extracts inhibited adhesion, migration and invasion of MDA-MB-231 cells. MMP activity was significantly inhibited by loquat extracts. CONCLUSION: Our results show that extracts of loquat inhibit the growth of tumor in MDA-MB-231 xenograft nude mice and the invasion of human breast cancer cells, indicating the inhibition of tumor cell proliferation and invasion.

MDA-MB-231 유방암 세포에서 석류 유래 나노베지클의 항암효과

김동하(Dong-ha Kim),김지수(Ji-Su Kim),권인숙(In-Sook Kwun),조영은(Young-Eun Cho) 한국영양학회 2024 Journal of Nutrition and Health Vol.57 No.1

Purpose: Cancer is the leading cause of death in Koreans, with breast cancer being the most common among women. Breast cancer readily metastasizes, and the existing treatment processes impose a significant burden on patients. This study examined whether pomegranate-derived exosome-like nanovesicles (PNVs) have anti-cancer effects by inhibiting cell infiltration and metastasis while increasing apoptosis on breast cancer MDA-MB-231 cells. Methods: Initially, exosome-like nanovesicles were isolated from pomegranate using ultracentrifugation. Subsequently, the size range of these nanovesicles was confirmed using nanoparticle tracking analysis. The ability of breast cancer MDA-MB-231 cells to internalize these natural nanovesicles was assessed with flourescence microscope. The anti-cancer effects of the PNVs were confirmed by applying various concentrations of PNVs (10, 50, 100 μg/mL) to MDA-MB-231 cells and systematically assessing their impact on cell viability and migration. Results: The round shape of the lipid bilayer in the PNVs was confirmed, providing crucial insights into their structural properties. We demonstrate that PNVs-associated DiD dye can be efficiently internalized by the MDA-MB-231 cells. The data showed that the PNVs inhibited cell viability, invasion rates, and migration in MDA-MB-231 cells. In addition, PNVs were absorbed into the MDA-MB-231 cells, leading to an increased expression of apoptosis proteins, such as cleaved caspase-3 and phosphorus-JNK, in a concentration-dependent manner. Furthermore, a reduction in cell infiltration and decreased expression of the transition markers MMP-2 and MMP-9 proteins were observed. Conclusion: For the first time, this study suggests that PNVs may be useful in the prevention or treatment of breast cancer by inhibiting the infiltration and metastasis of MDA-MB-231 cells and inducing apoptosis.

Effect of Snake Venom Toxin from Vipera lebetina turanica on Breast Cancer Cells

양가람,송호섭 대한침구의학회 2009 대한침구의학회지 Vol.26 No.3

목적 : 이 연구는 Vipera lebetina turanica의 蛇毒藥鍼液(Snake venom toxin, SVT)이 인간 유방암 세포주인 MCF-7과 MDA-MB-231 세포에서 암세포성장의 억제 및 그 기전에 대하여 살펴보고자 하였다. 방법 : SVT를 처리한 후 MCF-7과 MDA-MB-231의 성장억제를 관찰하기 위해 CCK-8 assay를 시행하였고, apoptosis 평가에는 TUNEL assay를 시행하였다. 세포자멸사 관련 세포기전을 보기 위하여 세포내 활성산소량 및 미토콘드리아의 세포막전위 변화를 측정하였고, 세포자멸사 조절 단백인 Bax, Bcl-2 발현 변화 관찰에는 western blot analysis를 시행하였다. 결과 : MCF-7과 MDA-MB-231 세포에 SVT를 처리한 후, 유방암 세포의 성장, Apoptosis의 유발 및 기전에 미치는 영향을 관찰하여 다음과 같은 결과를 얻었다. 1. MCF-7 세포와 MDA-MB-231 세포에서 SVT를 처리한 후 유방암 세포 성장이 억제되었다. 2. TUNEL assay를 통한 세포자멸사 평가에서 SVT를 처리한 MCF-7세포와 MDA-MB-231 세포 모두 세포자멸사 활성세포의 유의한 증가를 나타내었다. 3. 세포자멸사 관련 세포기전연구에서 SVT를 처리한 MCF-7 세포와 MDA-MB-231 세포에서 세포내 활성산소의 유의한 증가와 미토콘드리아 세포막 전위의 유의한 변동이 관찰되었다. 4. SVT를 처리한 MCF-7세포와 MDA-MB-231세포는 세포자멸사 관련 단백 발현에서 Bax의 유의한 증가와 Bcl-2의 유의한 감소를 나타내었다. 결론 : 이상의 결과는 SVT가 세포내 활성산소를 증가시킴으로써 미토콘드리아의 세포막전위에 변화를 일으켜 유방암 세포주인 MCF-7과 MDA-MB-231 세포에 세포자멸사를 유발하여 증식억제 효과가 있음을 입증한 것이다.

Protein Profiles Associated with Anoikis Resistance of Metastatic MDA-MB-231 Breast Cancer Cells

Akekawatchai, Chareeporn,Roytrakul, Sittiruk,Kittisenachai, Suthathip,Isarankura-Na-Ayudhya, Patcharee,Jitrapakdee, Sarawut Asian Pacific Journal of Cancer Prevention 2016 Asian Pacific journal of cancer prevention Vol.17 No.2

Resistance to anoikis, a cell-detachment induced apoptosis, is one of the malignant phenotypes which support tumor metastasis. Molecular mechanisms underlying the establishment of this phenotype require further investigation. This study aimed at exploring protein expression profiles associated with anoikis resistance of a metastatic breast cancer cell. Cell survival of suspension cultures of non-metastatic MCF-7 and metastatic MDA-MB-231 cells were compared with their adherent cultures. Trypan blue exclusion assays demonstrated a significantly higher percentage of viable cells in MDA-MB-231 than MCF-7 cell cultures, consistent with analysis of annexin V-7-AAD stained cells indicating that MDA-MB-231 possess anti-apoptotic ability 1.7 fold higher than MCF-7 cells. GeLC-MS/MS analysis of protein lysates of MDA-MB-231 and MCF-7 cells grown under both culture conditions identified 925 proteins which are differentially expressed, 54 of which were expressed only in suspended and adherent MDA-MB-231 but not in MCF-7 cells. These proteins have been implicated in various cellular processes, including DNA replication and repair, transcription, translation, protein modification, cytoskeleton, transport and cell signaling. Analysis based on the STITCH database predicted the interaction of phospholipases, PLC and PLD, and 14-3-3 beta/alpha, YWHAB, with the intrinsic and extrinsic apoptotic signaling network, suggesting putative roles in controlling anti-anoikis ability. MDA-MB-231 cells grown in the presence of inhibitors of phospholipase C, U73122, and phospholipase D, FIPI, demonstrated reduced ability to survive in suspension culture, indicating functional roles of PLC and PLD in the process of anti-anoikis. Our study identified intracellular mediators potentially associated with establishment of anoikis resistance of metastatic cells. These proteins require further clarification as prognostic and therapeutic targets for advanced breast cancer.

신선초 추출물이 인체 유방암 세포 MDA-MB-231의 세포 사멸에 미치는 영향

정유진(Yu-Jin Jeong),강금지(Keum Jee Kang) 한국식품영양과학회 2011 한국식품영양과학회지 Vol.40 No.12

AKE의 농도별 처리가 인체 유방암 세포 MDA-MB-231의 세포사멸에 미치는 영향을 확인하기 위하여 세포 화학적인 방법인 MTT 분석, 이중 핵 염색법(Hoechst 33342/EtBr staining), FACS를 통하여 세포사멸을 관찰하였다. MTT 분석 결과, 150 μg/mL 처리 군에서 대조군에 대비하여 약 50%의 세포사멸을 나타내었으며 세포사멸이 농도 의존적으로 증가되었고(p<0.05), 이중 핵 염색법을 이용하여 세포 사의 구분 결과 능동적 세포예정사인 apoptosis가 농도 의존적으로 급격히 증가하였으며(p<0.05), 특히 150 μg/mL 처리 군에서 현저한 증가율을 나타내었다. 보다 더 명확한 세포사멸을 확인하기 위하여 FACS를 이용한 apoptosis 측정 결과, 처리군 간 크게 차이를 보이며 농도 의존적으로 증가되었다. 세포사멸관련 mRNA 유전자 발현을 관찰한 결과, 세포사멸 억제 유전자 Bcl-2는 처리농도가 증가할수록 유의적 증가를 보였으며(p<0.05), 세포사멸 유도 유전자 Bax는 유의적 감소를 나타내었다(p<0.05). 세포사멸의 지표인 Bcl-2/Bax의 비율은 농도 의존적인 감소를 나타내었으며(p<0.05), 세포사멸유도의 마지막 단계의 실행자인 caspase-3의 활성도 첨가 농도 의존적으로 증가하여 세포사멸을 유도하는 것으로 확인되었다(p<0.05). 결론적으로, AKE는 유방암 세포 MDAMB-231의 세포사멸을 유도하는 것으로 나타나 신선초의 항암효과의 가능성을 제시해주었다. 향후 in vivo 실험에서도 신선초의 항암효과에 대한 심층적 연구가 이뤄져야 할 것으로 사료된다. We investigated the effect of Angelica keiskei ethanol (AKE) extract on cell death in MDA-MB-231 human breast cancer cells. MDA-MB-231 cells were cultured in the presence 125, 150 and 175 μg/mL concentrations of AKE for 24 hours. MTT assays demonstrated that mitochondrial dehydrogenase activities decreased in a dose-dependent manner in MDA-MB-231 cells (p<0.05). In contrast, the proportion of dual staining with Hoechst 33342/ethidium bromide(EtBr) for cell death increased in a dose-dependent manner in MDA-MB-231 cells (p<0.05). In particular, the levels of cell death caused by apoptotic program showed marked increases in the 150 and 175 μg/mL AKE groups, as revealed by flow cytometry. An apoptotic suppressor gene, Bcl-2, significantly decreased at the transcript level (p<0.05). The expression levels of proapoptotic genes, both Bax and caspase 3 significantly increased (p<0.05). Furthermore, the ratio of Bcl-2/Bax mRNA which is considered to be an important indicator of apoptosis, significantly decreased in a dose-dependent manner (p<0.05). These results taken together indicate that, the AKE extract used in this study induces cell death in MDA-MB-231 human breast cancer cells.

울금이 MDA-MB-231 세포 및 DMBA로 유발된 흰쥐의 유방암에 미치는 영향

양동선 ( Dong Seon Yang ),양승정 ( Seung Jeong Yang ) 대한한방부인과학회 2013 大韓韓方婦人科學會誌 Vol.26 No.3

Objectives: Breast cancer is the most common cancer among women and has rapidly increasing rate annually. At present, western cancer therapies by surgery, radiation, and anticancer drug have not been fully effective. So many interests are given to herbal medicine on cancer treatment recently. This study was designed to investigate the effects of Curcuma longa L. (CL) on MDA-MB-231 human breast cancer cells and DMBA-induced breast cancer in rats. Methods: In this experiment, MDA-MB-231 cells were cultured in cell culture plates. 0.0625, 0.125, 0.25, 0.5, 1.0 mg/ml of CL extract were tested for their antiproliferative effects on MDA-MB-231 cells by MMT assay. And we induced breast cancer in rats. The changes in tumor`s weight, and the effects on proliferations of splenocyte and thymocyte were investigated. Results: CL showed anti-proliferative effects on MDA-MB-231 cells in proportion to concentration of the CL. DMBA-induced breast cancer in rats, tumor`s weight of the rat was not statistically significant, but showed a tendency to be reduced in the groups treated with CL. Proliferation rate of the rat`s splenocyte and thymocyte increased in proportion to CL. In breast cancer tissue, expression of ER-α was weakened proportionately to the concentration of the CL. Conclusions: These data suggest that CL can prevent the proliferation of breast cancer, then CL is useful to treat patient with breast cancer.

Curcuma zedoaria (christm.) roscoe inhibits proliferation of MDA‑MB231 cells via caspase‑cascade apoptosis

Romen Meitei Lourembam,Amit Singh Yadav,Gopal C. Kundu,Pranab Behari Mazumder 경희대학교 융합한의과학연구소 2019 Oriental Pharmacy and Experimental Medicine Vol.19 No.3

Curcuma zedoaria is a perennial herb that belongs to Zingiberaceae family, found growing lavishly in Manipur; a north eastern part of India. The traditional usages of the plant is not limited to a particular disease but extended over a large number of diseases. Anti-breast cancer activity of ethyl acetate extract of Curcuma zedoaria was conducted on MDA-MB231 breast cancer cell line with the help of MTT assay technique and the mechanistic pathway was established with the help of western blot technique, confocal microscopy, wound healing migration assay and cell cycle analysis. Cell cytotoxicity test of ethyl acetate extracts showed remarkable result on MDA-MB231 in dose dependent manner (p<0.05). Confocal microscopy studies on MDA-MB231 cells revealed the activation of cleaved caspase 9 in treated cells. Western blot analyses confrm the regulation of cytochrome C and suppression of Bcl-2 in treated cells which suggest ethyl acetate extract of Curcuma zedoaria inhibits the cancer cells through cascase-dependent pathways. In cell cycle analysis, after the treatments of the cells; there was an increase in the percentage of SubG0/G1 cells that decreased S and G2 phase indicating the population undergoing cell death (apoptosis or necrosis) thereby, our data suggest that ethyl acetate extracts were able to induce signifcant apoptosis in MDA-MB-231 cells in dose dependent manner. The wound healing migration assay confrms the anti-metastatic nature of the plant extract. With the help one way ANOVA test, all the experiments were executed and it was found that ethyl acetate extract of Curcuma zedoaria inhibits MDA-MB231 cells with the down-regulation of the expression of Bcl-2 through cascase-dependent pathways therefore Curcuma zedoaria is a potent anti-cancer plant especially in breast cancer.

Apoptosis-Mediated Cytotoxicity of Ouabain, Digoxin and Proscillaridin A in the Estrogen Independent MDA-MB-231 Breast Cancer Cells

Katarzyna Winnicka,Krzysztof Bielawski,Anna Bielawska,Wojciech Miltyk 대한약학회 2007 Archives of Pharmacal Research Vol.30 No.10

We examined the effects of three cardiac glycosides, ouabain, digoxin and proscillaridin A, on the proliferation of estrogen independent MDA-MB-231 breast cancer cells. In terms of reduction in cell viability, the compounds rank for both 24 h and 48 h of incubation in MDA-MB-231 cells in the order proscillaridin A > digoxin > ouabain. Digoxin for 24 h and 48 h of incubation in MDA-MB-231 cells proved to be only slightly more potent than ouabain, with IC50 values of 122 ± 2 and 70 ± 2 nM, respectively, compared to 150 ± 2 and 90 ± 2 nM for ouabain. In contrast, proscillaridin A, was much more active and showed a high level of cytotoxic potency, IC50 51 ± 2 and 15 ± 2 nM for 24 h and 48 h of incubation, respectively. The concentrations of digoxin, ouabain and proscillaridin A needed to inhibit [3H]thymidine incorporation into DNA by 50% (IC50) in MDA-MB-231 cells for 24 h of incubation were found to be 124 ± 2 nM, 142 ± 2 nM, and 48 ± 2 nM, respectively. In the present study, we demonstrated that ouabain, digoxin, and proscillaridin A induce apoptosis in MDA-MB-231 cells by increasing free calcium concentration and by activating caspase-3.