• Asian Pacific Journal of Cancer Prevention
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• 제15권17호
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• pp.7037-7041
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• 2014

Background: 5-Fluorouracil (5-FU) is the most commonly used drug in colon cancer therapy. However, despite impressive clinical responses initially, development of drug resistance to 5-Fu in human tumor cells is the primary cause of failure of chemotherapy. In this study, we established a 5-Fu-resistant human colon cancer cell line for comparative chemosensitivity studies. Materials and Methods: Real time PCR and Western blotting were used to determine gene expression levels. Cell viability was measured by MTT assay. Glucose uptake was assess using an Amplex Red Glucose/Glucose Oxidase assay kit. Results: We found that 5-Fu resistance was associated with the overexpression of Glut1 in colon cancer cells. 5-Fu treatment at low toxic concentration induced Glut1 expression. At the same time, upregulation of Glut1 was detected in 5-Fu resistant cells when compared with their parental cells. Importantly, inhibition of Glut1 by a specific inhibitor, WZB117, significantly increased the sensitivity of 5-Fu resistant cells to the drug. Conclusions: This study provides novel information for the future development of targeted therapies for the treatment of chemo-resistant colon cancer patients. In particular it demonstrated that Glut1 inhibitors such as WZB117 may be considered an additional treatment options for patients with 5-Fu resistant colon cancers.

• Asian Pacific Journal of Cancer Prevention
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• 제15권2호
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• pp.629-635
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• 2014

Background: Acute myeloid leukemia (AML) is a fatal hematological malignancy which is resistant to a variety of chemotherapy drugs. Myeloid cell leukemia-1 (Mcl-1), a death-inhibiting protein that regulates apoptosis, has been shown to be overexpressed in numerous malignancies. In addition, it has been demonstrated that the expression level of the Mcl-1 gene increases at the time of leukemic relapse following chemotherapy. The aim of this study was to target Mcl-1 by small interference RNA (siRNA) and analyze its effects on survival and chemosensitivity of acute myeloid leukemia cell line HL-60. Materials and Methods: siRNA transfection was performed with a liposome approach. The expression levels of mRNA and protein were measured by real-time quantitative PCR and Western blot analysis, respectively. Trypan blue assays were performed to evaluate tumor cell growth after siRNA transfection. The cytotoxic effects of Mcl-1 siRNA (siMcl-1) and etoposide were determined using MTT assay on their own and in combination. Apoptosis was quantified using a DNA-histone ELISA assay. Results: Transfection with siMcl-1 significantly suppressed the expression of Mcl-1 mRNA and protein in a time-dependent manner, resulting in strong growth inhibition and spontaneous apoptosis. Surprisingly, pretreatment with siMcl-1 synergistically enhanced the cytotoxic effect of etoposide. Furthermore, Mcl-1 down-regulation significantly increased apoptosis sensitivity to etoposide. No significant biological effects were observed with negative control siRNA treatment. Conclusions: Our results suggest that specific suppression of Mcl-1 by siRNA can effectively induce apoptosis and overcome chemoresistance of leukemic cells. Therefore, siMcl-1 may be a potent adjuvant in leukemia chemotherapy.

• Tuberculosis and Respiratory Diseases
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• 제62권1호
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• pp.33-42
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• 2007

연구배경: 다양한 고형암에서 HO-1의 높은 발현이 알려져 있고, 그것의 항산화와 항세포고사의 역할로 인해 빠른 암종의 성장에 중요한 역할이 있음이 보고되고 있다. 대표적인 활성산소종 생성 항암제인 Cisplatin은 현재까지 폐암치료에 가장 광범위하게 사용되고 있으나, 여러 내성발생이 임상치료의 주요문제로 대두되고 있다. 저자들은 A549 폐암세포주에서 HO-1의 발현이 증가되었고 HO-1 활성억제제나 siRNA 방법을 통해 생존율의 의미 있는 감소와 세포고사가 유도됨을 보고한 바 있다. 이 연구의 목적은 A549 폐암세포주에 cisplatin 처리시 HO-1의 발현의 증가유무와 기전을 규명하고 실제 HO-1의 억제가 cisplatin에 의한 항암제 감수성을 증가시키는지를 알아보는데 있다. 방 법: 비소세포폐암세포주인 A549, NCI-H23, NCIH157, NCI-H460을 이용하였다. 세포독성은 MTT 방법으로 구하였고, HO-1, Nrf2, MAPK의 발현은 Western blotting으로 확인하였다. 또한 MAPK억제제들을 전처치한 후 cisplatin에 의해 유도된 Nrf2와 HO-1의 발현에 미치는 영향을 역시 Western blotting으로 관찰하였다. A549세포에 활성억제제인 ZnPP나 HO-1 small interfering RNA (siRNA)을 주입하여 cisplatin과의 병합요법시 생존율의 배가효과 유무를 MTT 방법으로 확인하였고, 이러한 효과가 ROS 형성과 HO-1의 발현변화와 관련되는지를 알아보기 위해 $carboxy-H_2DCFDA$ 방법과 Western blotting을 통해 각각 확인하였다. 결 과: Cisplatin 처리시 다른 세포주에 비해 A549 세포가 의의 있게 내성을 보였다. $10{\mu}M$의 농도에서 시간 의존적으로 HO-1, Nrf2, MAPK의 발현이 증가하였고, MAPK 억제제들을 전 처치하였을 때 cisplatin에 의해 유도된 HO-1과 Nrf2의 발현이 억제됨을 확인하였다. HO-1의 활성억제제인 ZnPP와 HO-1 siRNA를 통해 HO-1 mRNA를 직접 억제하는 방법으로 cisplatin과 병합치료시 단독치료에 비해 의의 있는 생존율의 감소를 보였다. 이러한 효과는 활성산소종의 생성 증가와 HO-1의 발현억제에 의한 결과임을 확인하였다. 결 론: Cisplatin 처리시 HO-1의 발현은 MAPKNrf2-HO-1의 경로를 통해 증가하였고, 부분적으로 치료에 대한 내성과 관련이 있었으며, ZnPP 등의 활성억제제나 siRNA를 통한 knock-down 방법으로 HO-1 을 표적으로 억제하는 치료방법을 통해 cisplatin의 항암제 감수성을 증가시켰다.

• Tuberculosis and Respiratory Diseases
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• 제44권3호
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• pp.525-533
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• 1997

연구배경 : 수술적 절제가 불가능한 폐암환자에서 복합화학요법의 역할이 최근 증대되고 있으나 아직 가장 이상적인 복합화학요법은 확립되지 않고 있다. 두 종류 이상의 항암제를 복합투여시 약제간의 상호작용에 의해 항암효과의 상승 혹은 억제를 보일 수 있으나 이를 예측하기가 어려웠다. 본 연구에서는 MTT 검사를 이용하여 두 약제를 여러 농도에서 복합투여후 살해능의 변화를 관찰하였다. 방 법 : 사람의 폐선암세포인 PC-14를 이용하여 cisplatin, mitomycin C, adriamycin 및 etoposide를 여러 농도에서 단독 또는 두 약제를 복합투여하여 항암효과의 변화를 MTT 검사로 측정하고 두 약제 복합투여시의 상호 작용의 결과를 이원배치법을 이용한 Anova분석을 이용하여 측정하였다. 결 과 : 위의 네종류의 약제는 단독투여시 농도에 비례하는 암세포살해능을 보였고 두 약제를 복합투여시 모든 조합에서 암세포살해능의 상승효과를 보였으며 특히 mitomycin C 와 cisplatin 및 adriamycin과 cisplatin을 복합투여시 상승효과가 강하게 나타났다. 결 론 : 위의 결과로 비소세포폐암의 복합화학요법시 mitomycin C와 cisplatin 혹은 adriamycin과 cisplatin을 같이 사용할 경우 항암효과의 극대화를 얻을 수 있으리라 기대된다. 나아가 이번 연구의 디자인은 복합항암화학요법을 필요로 하는 모든 종류의 암에 적용되어 최대항암효과를 얻을 수 있는 약제선정에 도움의 될 수 있으리라 생각된다.

• 치과방사선
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• 제27권2호
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• pp.91-103
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• 1997

The purpose of this study was to aid in the prediction of tumor cell tolerance to radiotherapy and/or chemotherapy. For this study, cell surviving curves were obtained for human epidermoid carcinoma A-253 cell line using semiautomated MTT assay. 2,4,6,8,10 Gy were irradiated at a dose rate of 210 cGy/min using /sup 60/Co Irradiator ALDORADO 8. After irradiation, A-253 cell lines(2×10⁴cells/mil were exposed to bleomycin or cisplatin for 1 hour. The viable cells were determined for each radiation dose with/without 2 /lg/mi of drug at the 3rd day. And they were compared to control values. The results were obtained as follows : 1. The surviving curve with gentle slope was obtained after irradiation of 2, 4, 6, 8, 10 Gy on A-253 cell line. 2. The cytotoxicity of bleomycin or cisplatin at the concentration of 2㎍/ml was great on A-253 cell line. But, there was no significant difference between the cytotoxicity of bleomycin and that of cisplatin. 3. There were significant differences of surviving fractions after irradiation with 2㎍/mi of bleomycin compared with irradiation only on A-253 cell line. 4. There were significant differences of surviving fractions after irradiation with 2㎍/ml of cisplatin compared with irradiation only on A-253 cell line. 5. There were no significant differences of surviving fractions between the groups of irradiation with bleomycin and the groups of irradiation with cisplatin on A-253 cell line.

• Asian Pacific Journal of Cancer Prevention
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• 제13권9호
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• pp.4807-4814
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• 2012

Purpose: The chemoresistance of human hepatocellular carcinoma (HCC) to cytotoxic drugs, especially intrinsic or acquired multidrug resistance (MDR), still remains a major challenge in the management of HCC. In the present study, possible mechanisms involved in MDR of HCC were identified using a 5-fluorouracil (5-FU)-resistant human HCC cell line. Methods: BEL-7402/5-FU cells were established through continuous culturing parental BEL-7402 cells, imitating the pattern of chemotherapy clinically. Growth curves and chemosensitivity to cytotoxic drugs were determined by MTT assay. Doubling times, colony formation and adherence rates were calculated after cell counting. Morphological alteration, karyotype morphology, and untrastructure were assessed under optical and electron microscopes. The distribution in the cell cycle and drug efflux pump activity were measured by flow cytometry. Furthermore, expression of potential genes involved in MDR of BEL-7402/5-FU cells were detected by immunocytochemistry. Results: Compared to its parental cells, BEL-7402/5-FU cells had a prolonged doubling time, a lower mitotic index, colony efficiency and adhesive ability, and a decreased drug efflux pump activity. The resistant cells tended to grow in clusters and apparent changes of ultrastructures occurred. BEL-7402/5-FU cells presented with an increased proportion in S and G2/M phases with a concomitant decrease in G0/G1 phase. The MDR phenotype of BEL-7402/5-FU might be partly attributed to increased drug efflux pump activity via multidrug resistance protein 1 (MRP1), overexpression of thymidylate synthase (TS), resistance to apoptosis by augmentation of the Bcl-xl/Bax ratio, and intracellular adhesion medicated by E-cadherin (E-cad). P-glycoprotein (P-gp) might play a limited role in the MDR of BEL-7402/5-FU. Conclusion: Increased activity or expression of MRP1, Bcl-xl, TS, and E-cad appear to be involved in the MDR mechanism of BEL-7402/5-FU.

• BMB Reports
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• 제51권9호
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• pp.474-479
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• 2018

Cisplatin is one of the most effective chemotherapeutic drugs used in the treatment of HCC, but many patients will ultimately relapse with cisplatin-resistant disease. Used in combination with cisplatin, resveratrol has synergistic effect of increasing chemosensitivity of cisplatin in various cancer cells. However, the mechanisms of resveratrol enhancing cisplatin-induced toxicity have not been well characterized. Our study showed that resveratrol enhances cisplatin toxicity in human hepatoma cells via an apoptosis-dependent mechanism. Further studies reveal that resveratrol decreases the absorption of glutamine and glutathione content by reducing the expression of glutamine transporter ASCT2. Flow cytometric analyses demonstrate that resveratrol and cisplatin combined treatment leads to a significant increase in ROS production compared to resveratrol or cisplatin treated hepatoma cells alone. Phosphorylated H2AX (${\gamma}H2AX$) foci assay demonstrate that both resveratrol and cisplatin treatment result in a significant increase of ${\gamma}H2AX$ foci in hepatoma cells, and the resveratrol and cisplatin combined treatment results in much more ${\gamma}H2AX$ foci formation than either resveratrol or cisplatin treatment alone. Furthermore, our studies show that over-expression of ASCT2 can attenuate cisplatin-induced ROS production, ${\gamma}H2AX$ foci formation and apoptosis in human hepatoma cells. Collectively, our studies suggest resveratrol may sensitize human hepatoma cells to cisplatin chemotherapy via gluta${\gamma}H2AX$mine metabolism inhibition.

• 대한약침학회지
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• 제19권2호
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• pp.129-136
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• 2016

Objectives: The crude extracts of Scutellaria barbata D. Don (SB) have traditionally demonstrated inhibitory effects on numerous human cancers both in vitro and in vivo. Gastric cancer is one of the most common types of cancer on world. The authors investigated the effects of an ethanol extract of Scutellaria barbata D. Don (ESB) on the growth and survival of MKN-45 cells (a human gastric adenocarcinoma cell line). Methods: The MKN-45 cells were treated with different concentrations of ESB, and cell death was examined using an MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay. Analyses of sub-G1 peaks, caspase-3 and -9 activities, and mitochondrial membrane depolarizations were conducted to determine the anti-cancer effects of SB on MKN-45 cells. Also, intracellular reactive oxygen species (ROS) generation was investigated. Results: ESB inhibited the growth of MKN-45 cells, caused cell cycle arrest, and increased the sub-G1 population. In addition, ESB markedly increased mitochondrial membrane depolarization and the activities of caspase-3 and -9. ESB exerted anti-proliferative effects on MKN-45 cells by modulating the mitogen-activated protein kinase (MAPK) signaling pathway and by increasing the generation of ROS. Furthermore, combinations of anti-cancer drugs plus ESB suppressed cell growth more than treatments with an agent or ESB, and this was especially true for cisplatin, etoposide, and doxorubicin. Conclusion: ESB has a dose-dependent cytotoxic effect on MKN-45 cells and this is closely associated with the induction of apoptosis. ESB-induced apoptosis is mediated by mitochondria-, caspase- and MAPK dependent pathways. In addition, ESB enhances ROS generation and increases the chemosensitivity of MKN-45 cells. These results suggest that treatment with ESB can inhibit the proliferation and promote the apoptosis of human gastric adenocarcinoma cells by modulating the caspase-, MAPK- and ROS-dependent pathway.

• 치과방사선
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• 제27권1호
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• pp.43-53
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• 1997

The purpose of this study was to aid in the prediction of tumor cell tolerance to radiotherapy and/or chemotherapy. For this study, cell surviving curves were obtained for mouse lymphoma YAC-1 cell line using semi automated MTT assay. 2, 4,6, 8, 10Gy were irradiated at a dose rate of 210cGy/rnin using /sup 60/Co Irradiator ALDORADO 8. After irradiation, YAC-1 cell lines(3×10⁴cells/ml) were exposed to bleomycin or cisplatin for 1 hour. The viable cells were determined for each radiation dose and/or each concentration of drug at the 4th day. And they were compared to control values. The obtained results were as follows : 1. The surviving curve with gentle slope was obtained after irradiation of 2, 4, 6, 8, 10 Gy on YAC-1 cell line. 2. The cytotoxicity of bleomycin or cisplatin was increased significantly at all concentration of 0.2㎍/ml, 2㎍/ml and 20㎍/ml on YAC-1 cell line (P<0.01). And the cytotoxicity of cisplatin was greater than that of bleomycin at all concentration on YAC-1 cell line (P<0.01). 3. There were no significant differences of surviving fractions among 4Gy, 6Gy and 8Gy after irradiation of each radiation dose with 2㎍/ml of bleomycin compared with irradiation only on YAC-1 cell line. 4. There was significant difference of surviving fraction between 2Gy and 10Gy after irradiation of each radiation dose with 2㎍/ml of cisplatin compared with irradiation only on YAC-1 cell line(P<0.05). 5. There were significant differences of surviving fractions between the groups of irradiation only and the groups of irradiation with 2㎍/ml of bleomycin or cisplatin at all doses of 2, 4, 6, 8 and 10Gy on YAC-1 cell line(P<0.05).

• 대한의생명과학회지
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• 제2권2호
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• pp.153-158
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• 1996

Verapamil은 항암제에 대한 내성을 극복하기 위하여 실험적으로 사용하는 대표적인 약제로 알려져 있다 본 연구에서는 부인과 종양중 자궁경부암 치료시에 발생하는 항암 화학요법제에 대한 내성을 극복하기 위한 기초적인 실험으로 인체 자궁경부암 유래의 HeLa 및 Caski 세포주를 이용, MTT법으로 verapamil과 5-FU 및 DDP를 단독 혹은 병용 처리하여 세포독성 상승효과를 측정하였다. Verapamil 단독투여시 각 세포주는 용량에 비례하여 세포독성이 증가하였으며, 각 항암제와의 병용 투여시에 세포독성의 상승효과가 관찰이 되었으며, HeLa세포주에서 그 효과가 가장 높았다. 이러한 결과를 바탕으로 세포내 내성발현의 기전의 탐구가 필요하며 자궁경부암 화학요법에 verapamil사용의 이론적인 가능성을 제시하고 있다