• BMB Reports
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• 제48권9호
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• pp.531-536
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• 2015

Gecko proteins have long been used as anti-tumor agents in oriental medicine, without any scientific background. Although anti-tumor effects of Gecko proteins on several cancers were recently reported, their effect on bladder cancer has not been investigated. Thus, we explored the anti-tumor effect of Gecko proteins and its cellular mechanisms in human bladder cancer 5637 cells. Gecko proteins significantly reduced the viability of 5637 cells without any cytotoxic effect on normal cells. These proteins increased the Annexin-V staining and the amount of condensed chromatin, demonstrating that the Gecko proteinsinduced cell death was caused by apoptosis. Gecko proteins suppressed Akt activation, and the overexpression of constitutively active form of myristoylated Akt prevented Gecko proteins-induced death of 5637 cells. Furthermore, Gecko proteins activated caspase 9 and caspase 3/7. Taken together, our data demonstrated that Gecko proteins suppressed the Akt pathway and activated the intrinsic caspase pathway, leading to the apoptosis of bladder cancer cells. [BMB Reports 2015; 48(9): 531-536]

• 동의생리병리학회지
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• 제30권2호
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• pp.131-136
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• 2016

An ethanolic extracts of Scutellaria Baicalensis GEORGI are used to treat cancer, infectious diseases, and inflammation. In the present study, we investigated the effects of an ESBG on the growth and survival of 5637 cells, a human bladder carcinoma cell line. Cells were treated with different concentrations of an ethanolic extract of Scutellaria Baicalensis GEORGI (ESBG), and cell death was assessed using a MTT (3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide) assay. Analyses of the sub G1 peak, caspase-3 and -9 activities, and mitochondrial membrane depolarizations were conducted to confirm cell death by apoptosis. ESBG had a cytotoxic effect on 5637 cells, and increased the sub G1 peak, caspase-3 and -9 activities, and mitochondrial depolarization, indicating ESBG induced apoptosis. Furthermore, MAPK (mitogen-activated protein kinases) inhibitors suppressed this apoptosis. In an in vitro study, a combination of sub-optimal doses of ESBG and paclitaxel, 5-fluorouracil, or docetaxel noticeably suppressed tumor growth by 5637 cells. Our findings provide insight of the mechanisms underlying cellular apoptosis induced by ESBG, and suggest new therapeutic strategies for bladder cancer.

• 대한한방내과학회지
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• 제37권1호
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• pp.16-25
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• 2016

Objectives: Gilgyung-tang (GGT) has been used as one of the main multi-herb formulas to treat “Peo-ong” (lung abscess). In this study, we investigated the inhibitory effects of water extracts of GGT on cell migration and invasion, two critical cellular processes that are often deregulated during metastasis, in human bladder cancer 5637 cells.Methods: Effects on cell viability were quantified using an MTT assay. To analyze the anti-metastatic effects, we conducted a wound healing migration assay, an in vitro invasiveness assay, and a measurement of the transepithelial electrical resistance (TER). The expression of protein and mRNA were measured by Western blotting and real-time polymerase chain reaction (RT-PCR), respectively.Results: GGT markedly inhibited the cell motility and invasiveness of 5637 cells within the concentration range that was not cytotoxic. The inhibitory effects of GGT on cell invasiveness were associated with tightening of the tight junctions (TJs), which was demonstrated by an increase in the TER. The RT-PCR and Western blotting results indicated that GGT decreased the levels of claudin proteins. GGT also inhibited the activity and expression of matrix metalloproteinase (MMP)-2 and -9 and simultaneously increased the levels of tissue inhibitor of metalloproteinase-1 and -2.Conclusions: Our findings suggest that GGT reduces both the migration and the invasion of 5637 cells by modulating the activity of TJs and MMPs.

• 대한한방내과학회지
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• 제37권1호
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• pp.65-76
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• 2016

Objectives: The purpose of this study was to identify the inhibitory effects of Hwangheuk-san (HHS), a Korean multi-herb formula comprising four medicinal herbs, on cell migration and invasion, two critical cellular processes that are often deregulated during metastasis, using the human bladder cancer 5637 cell line.Methods: Cell viability, motility, and invasion were assessed by 3-(4,5-dimethyl-2 thiazolyl)-2,5-diphnyl-2H-tetrazolium bromide (MTT), wound healing migration, and Transwell assays, respectively. Gene expression was detected by Western blot analysis. In addition, the activities of matrix metalloproteinases (MMPs) and the values for transepithelial electrical resistance (TER) were analyzed using a Gelatinase Activity Assay Kit and an Epithelial Tissue Voltohmmeter, respectively.Results: Our data indicated that within the concentration range that was not cytotoxic, HHS effectively inhibited the cell motility and invasiveness of 5637 cells. HHS markedly decreased the expression and activity of MMP-2 and MMP-9, which was associated with unregulation of tissue inhibitors of metalloproteinase (TIMP)-1 and TIMP-2. Further investigation revealed that phosphorylation of phosphatidylinositol 3-kinase (PI3K) and AKT was decreased in HHS-treated 5637 cells, and a PI3K/AKT inhibitor synergistically reduced the inhibition of migration and invasion and also inactivated MMP-2 and MMP-9. Moreover, HHS increased the tightening of tight junctions (TJs), which was demonstrated by an increase in the TER, and reduced the expression the levels of claudin family members (claudin-3 and -4), which are major components involved in the tightening of TJs.Conclusions: The present findings demonstrated that HHS attenuated the migration and invasion of bladder cancer 5637 cells by modulating the activity of the PI3K/Akt signaling pathway and also through TJ tightening.

• 한국식품영양과학회지
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• 제45권10호
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• pp.1422-1429
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• 2016

본 연구에서는 홍차버섯이라고 알려진 콤부차(Kombucha, K)에 플라보노이드 성분 및 각종 기능성 물질이 풍부한 감귤액을 첨가하여 감귤의 생리활성 물질들이 콤부차로 이행되는 효과를 기대하여 감귤 콤부차(citrus Kombucha, CK)를 배양한 후 항산화 능력 및 인체 방광암세포(T-24와 5637)를 이용한 항암 효과를 확인하고 더 나아가 암의 증식을 억제시킬 수 있는 천연소재 탐색을 목적으로 연구를 진행하였다. 항산화 및 총페놀 함량 결과는 K보다 CK의 항산화 능력과 페놀 함량이 높게 확인되었으며 방광암세포 T-24와 5637에 K 혹은 CK를 24시간 처리한 후 MTT assay를 통해 세포독성을 확인한 결과 농도 의존적으로 생존율이 감소하였다. 특히 T-24 세포에서는 CK를 처리하였을 때 현저한 세포의 형태적 변화를 확인하였다. Western immunoblot을 통해 apoptosis 관련 단백질들의 발현을 확인하였는데 T-24에 CK 처리하였을 때 Bcl-2의 발현은 크게 감소하였으며, pro-caspase-9, pro-caspase-8, pro-caspase-3는 농도가 높아질수록 감소하였으며, cleaved caspase-9, cleaved caspase-8, cleaved caspase-3는 농도가 높아질수록 증가하는 경향을 보였다. 또한, cleaved PARP가 증가함을 확인할 수 있었다. 이상의 결과에서 일반 콤부차보다 감귤액을 첨가한 감귤 콤부차가 인체 방광암세포 T-24에 caspase에 의한 apoptosis가 유도됐음을 확인할 수 있었다.

• 생명과학회지
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• 제26권4호
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• pp.431-438
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• 2016

TRAIL은 정상세포에서는 세포독성을 나타내지 않는 반면, 암세포에서는 사멸을 유도하므로 항암제로 각광받고 있지만 많은 암세포에서 TRAIL에 저항성을 가지고 있는 것으로 알려져 있으므로 이를 극복해야하는 큰 어려움이 남아있다. 본 연구에서는 TRAIL에 저항성을 가지는 인간 방광암 세포주인 5637 세포를 이용하여 histone deacetylase 억제제인 sodium butyrate (SB)와 TRAIL을 혼합처리하였을 경우 유발되는 세포사멸 효과와 이와 관련된 분자생물학적 메카니즘을 연구하였다. 세포독성이 없는 조건의 TRAIL과 SB를 혼합처리 하였을 경우 SB 단독처리군 보다 세포사멸이 현저하게 증가하는 것으로 확인되었다. TRAIL과 SB의 혼합처리는 caspases (caspase-3, -8 and -9)의 활성화 및 PARP의 단편화를 유발하였다. 하지만 caspase 억제제에 의하여 TRAIL과 SB의 혼합처리에 의하여 유발되는 apoptosis가 현저하게 억제되는 것으로 나타났다. 또한 TRAIL과 SB의 혼합처리는 세포표면에 존재하는 DR5의 발현 증가 및 c-FLIP의 발현 감소를 유발하였으며, pro-apoptotic protein인 Bax와 세포질 cytochrome c의 발현 증가 및 anti- apoptotic protein인 Bcl-xL의 발현감소와 함께 tBid의 형성을 유발하였다. 이는 SB와 TRAIL의 혼합처리가 안전하고 선택적으로 TRAIL에 저항성을 가지는 방광암 세포에서 치료하는데 효과적인 전략임을 제시하는 결과이다.

• BMB Reports
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• 제53권2호
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• pp.82-87
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• 2020

Circular RNAs (circRNAs), one kind of non-coding RNA, have been reported as critical regulators for modulating gene expression in cancer. In this study, microarray analysis was used to screen circRNA expression profiles of bladder cancer (BC) 5637 cells, T24 cells and normal control SV-HUC-1 cells. The data from the microarray showed that hsa_circ_0075828 (named circCASC15) was most highly expressed in 5637 and T24 cells. circCASC15 was highly expressed in BC tissues and cells. Overexpression of circCASC15 was closely associated with BC tumor stage and promoted cell proliferation significantly in vitro and in vivo. Mechanistically, circCASC15 could act as miR-1224-5p sponge to activate the expression of CREB1 to promote cell proliferation in BC. In short, circCASC15 promotes cell proliferation in BC, which might be a new molecular target for BC diagnosis and therapy.

• Asian Pacific Journal of Cancer Prevention
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• 제14권10호
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• pp.5941-5948
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• 2013

Tumor response to antineoplastic drugs is not always predictable. This is also true for bladder carcinoma, a highly recurrent neoplasia. Currently, the combination of cisplatin and gemcitabine is well accepted as a standard protocol for treating bladder carcinoma. However, in some cases, this treatment protocol causes harmful side effects. Therefore, we investigated the roles of the genes TP53, RASSF1A (a tumor suppressor gene) and hMLH1 (a gene involved in the mismatch repair pathway) in cell susceptibility to cisplatin/gemcitabine treatment. Two bladder transitional carcinoma cell (TCC) lines, RT4 (wild-type TP53) and 5637 (mutated TP53), were used in this study. First, we evaluated whether the genotoxic potential of cisplatin/gemcitabine was dependent on TP53 status. Then, we evaluated whether the two antineoplastic drugs modulated RASSF1A and hMLH1 expression in the two cell lines. Increased DNA damage was observed in both cell lines after treatment with cisplatin or gemcitabine and with the two drugs simultaneously, as depicted by the comet assay. A lack of RASSF1A expression and hypermethylation of its promoter were observed before and after treatment in both cell lines. On the other hand, hMLH1 downregulation, unrelated to methylation status, was observed in RT4 cells after treatment with cisplatin or with cisplatin and gemcitabine simultaneously (wild-type TP53); in 5637 cells, hMLH1 was upregulated only after treatment with gemcitabine. In conclusion, the three treatment protocols were genotoxic, independent of TP53 status. However, cisplatin was the most effective, causing the highest level of DNA damage in both wild-type and mutated TP53 cells. Gemcitabine was the least genotoxic agent in both cell lines. Furthermore, no relationship was observed between the amount of DNA damage and the level of hMLH1 and RASSF1A expression. Therefore, other alternative pathways might be involved in cisplatin and gemcitabine genotoxicity in these two bladder cancer cell lines.

• Asian Pacific Journal of Cancer Prevention
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• 제13권5호
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• pp.1957-1962
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• 2012

Bladder cancer is a common malignancy worldwide. Despite the increased use of cisplatin-based combination therapy, the outcomes for patients with advanced disease remain poor. Recently, altered activation of the PI3K/Akt/mTOR pathway has been associated with reduced patient survival and advanced stage of bladder cancer, making its upstream or downstream components attractive targets for therapeutic intervention. In the present study, we showed that treatment with DTCM-glutaramide, a piperidine that targets PDK1, results in reduced proliferation, diminished cell migration and G1 arrest in 5637 and T24 bladder carcinoma cells. Conversely, no apoptosis, necrosis or autophagy were detected after treatment, suggesting that reduced cell numbers in vitro are a result of diminished proliferation rather than cell death. Furthermore previous exposure to 10 ${\mu}g/ml$ DTCM-glutarimide sensitized both cell lines to ionizing radiation. Although more studies are needed to corroborate our findings, our results indicate that PDK1 may be useful as a therapeutic target to prevent progression and abnormal tissue dissemination of urothelial carcinomas.

• 한국식품영양과학회지
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• 제45권12호
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• pp.1838-1842
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• 2016

콤부차는 미생물의 공생체에 의해 제조되는 신맛이 강한 음료로 최근에는 "장수 식품"의 하나로 주로 러시아를 비롯한 서방 국가들에서 많이 음용되어온 기능성 음료이다. 본 연구에서는 일반적으로 제조되는 콤부차의 기능성을 제주 특산물인 감귤의 과피 추출액과 녹차의 추출액을 첨가한 후 콤부차를 발효시켜 각 성분이 가지는 기능성을 이행시킬 목적으로 감귤/녹차 콤부차를 제조하였고, 제조된 감귤/녹차 콤부차의 기능성을 항암성 중심으로 일반 콤부차와 비교 평가하였다. 또한, 콤부차의 산업화, 표준화 및 안전성 확보를 위하여 일반 세포에 미치는 독성의 정도를 RAW 264.7 마우스 대식세포를 이용하여 조사하였다. 그 결과 일반 콤부차보다 감귤/녹차 콤부차는 항산화력이 약 3배 증가하였으며, 항암성도 현저하게 증가한 것으로 나타났다. 특히 일반 세포에 대한 독성은 현저히 낮아진 것으로 나타났으며, 이는 감귤/녹차 콤부차의 안전한 항암 기능성 음료로의 개발 가능성을 시사하고 있다.