• Journal of Life Science
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• v.19 no.8
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• pp.1023-1032
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• 2009

Despite the fact that many cancer cells are sensitive to TNF-related apoptosis-inducing ligand (TRAIL)-induced apoptosis, some cancer cells show either partial or complete resistance to TRAIL. Human leukemic K562 and CEM cells also show resistance to TRAIL-induced apoptosis. Novel molecular target and treatment strategies are required to overcome TRAIL resistance of human leukemia cells. Therefore, the purpose of this study was to target key anti-apoptotic molecules deciding TRAIL resistance for sensitization of TRAIL-resistant K562 and CEM cells, and to evaluate the effect of quercetin as a TRAIL sensitizer on these TRAIL-resistant cells. We found that quercetin acted in synergy with TRAIL to enhance TRAIL-induced apoptosis in K562 cells by inhibition of the DNA-PK/Akt signaling pathway, which leads to enhancement of TRAIL-mediated activation of caspases and concurrent cleavage of PARP and up-regulation of Bax. The findings suggest that the DNA-PK/Akt signaling pathway plays an essential role in regulating cells to escape from TRAIL-induced apoptosis, and quercetin could act in synergy with TRAIL to increase apoptosis by inhibition of the DNA-PK/Akt signaling pathway, which overcomes TRAIL-resistance of K562 and CEM cells. This study suggests that DNA-PK might interfere with TRAIL-induced apoptosis in human leukemic cells through activation of the Akt signaling pathway.

• Molecules and Cells
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• v.23 no.2
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• pp.138-144
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• 2007

Previously we showed that the human GM3 synthase gene was expressed during the induction of megakaryocytic differentiation in human leukemia K562 cells by phorbol 12-myristate 13-acetate (PMA). In this study we found that treatment of PMA-induced K562 cells with $G{\ddot{o}}6976$, a specific inhibitor of PKC, and U0126, an inhibitor of the extracellular signal-regulated kinase (ERK) reduced expression of GM3 synthase, whereas wortmannin, an inhibitor of phosphoinositide 3-kinase (PI3K) did not. Moreover, activation of ERK and cAMP response element binding protein (CREB) was prevented by pretreatment with $G{\ddot{o}}6976$ and U0126. PMA stimulated the promoter activity of the 5'-flanking region from -177 to -83 region of the GM3 synthase gene, and mutation or deletion of a CREB site located around -143 of the promoter reduced PMA-stimulated promoter activity, as did the inhibitors $G{\ddot{o}}6976$ and U0126. Our results demonstrate that induction of GM3 synthase during megakaryocytic differentiation in PMA-stimulated human leukemia K562 cells depends upon the PKC/ERK/CREB pathway.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.11
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• pp.4555-4561
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• 2014

Background: Silencing due to methylation of suppressor of cytokine signaling-3 (SOCS-3), a negative regulator gene for the JAK/STAT signaling pathway has been reported to play important roles in leukemogenesis. Imatinib mesylate is a tyrosine kinase inhibitor that specifically targets the BCR-ABL protein and induces hematological remission in patients with chronic myeloid leukemia (CML). Unfortunately, the majority of CML patients treated with imatinib develop resistance under prolonged therapy. We here investigated the methylation profile of SOCS-3 gene and its downstream effects in a BCR-ABL positive CML cells resistant to imatinib. Materials and Methods: BCR-ABL positive CML cells resistant to imatinib (K562-R) were developed by overexposure of K562 cell lines to the drug. Cytotoxicity was determined by MTS assays and $IC_{50}$ values calculated. Apoptosis assays were performed using annexin V-FITC binding assays and analyzed by flow cytometry. Methylation profiles were investigated using methylation specific PCR and sequencing analysis of SOCS-1 and SOCS-3 genes. Gene expression was assessed by quantitative real-time PCR, and protein expression and phosphorylation of STAT1, 2 and 3 were examined by Western blotting. Results: The $IC_{50}$ for imatinib on K562 was 362nM compared to 3,952nM for K562-R (p=0.001). Percentage of apoptotic cells in K562 increased upto 50% by increasing the concentration of imatinib, in contrast to only 20% in K562-R (p<0.001). A change from non-methylation of the SOCS-3 gene in K562 to complete methylation in K562-R was observed. Gene expression revealed down-regulation of both SOCS-1 and SOCS-3 genes in resistant cells. STAT3 was phosphorylated in K562-R but not K562. Conclusions: Development of cells resistant to imatinib is feasible by overexposure of the drug to the cells. Activation of STAT3 protein leads to uncontrolled cell proliferation in imatinib resistant BCR-ABL due to DNA methylation of the SOCS-3 gene. Thus SOCS-3 provides a suitable candidate for mechanisms underlying the development of imatinib resistant in CML patients.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.13
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• pp.5455-5461
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• 2014

Background: Saponins are a major active component for the traditional Chinese medicine, Rubus parvifolius L., which has shown clear antitumor activities. However, the specific effects and mechanisms of saponins of Rubus parvifolius L. (SRP) remain unclear with regard to human chronic myeloid leukemia cells. The aim of this study was to investigate inhibition of proliferation and apoptosis induction effects of SRP in K562 cells and further elucidate its regulatory mechanisms. Materials and Methods: K562 cells were treated with different concentrations of SRP and MTT assays were performed to determine cell viability. Apoptosis induction by SRP was determined with FACS and DAPI staining analysis. Western blotting was used to detect expression of apoptosis and survival related genes. Specific inhibitors were added to confirm roles of STAT3 and AMPK pathways in SRP induction of apoptosis. Results: Our results indicated that SRP exhibited obvious inhibitory effects on the growth of K562 cells, and significantly induced apoptosis. Cleavage of pro-apoptotic proteins was dramatically increased after SRP exposure. SRP treatment also increased the activities of AMPK and JNK pathways, and inhibited the phosphorylation expression level of STAT3 in K562 cells. Inhibition of the AMPK pathway blocked the activation of JNK by SRP, indicating that SRP regulated the expression of JNK dependent oon the AMPK pathway. Furthermore, inhibition of the latter significantly conferred resistance to SRP pro-apoptotic activity, suggesting involvement of the AMPK pathway in induction of apoptosis. Pretreatment with a STAT3 inhibitor also augmented SRP induced growth inhibition and cell apoptosis, further confirming roles of the STAT3 pathway after SRP treatment. Conclusions: Our results demonstrated that SRP induce cell apoptosis through AMPK activation and STAT3 inhibition in K562 cells. This suggests the possibility of further developing SRP as an alternative treatment option, or perhaps using it as adjuvant chemotherapeutic agent for chronic myeloid leukemia therapy.

• Asian Pacific Journal of Cancer Prevention
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• v.16 no.9
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• pp.3715-3721
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• 2015

Leukemia is a clonal disorder with blocked normal differentiation and cell death of hematopoietic progenitor cells. Traditional modalities with most used radiation and chemotherapy are nonspecific and toxic which cause adverse effects on normal cells. Differentiation inducing therapy forcing malignant cells to undergo terminal differentiation has been proven to be a promising strategy. However, there is still scarce of potent differentiation inducing agents. We show here that Angelica sinensis polysaccharide (ASP), a major active component in Dong quai (Chinese Angelica sinensis), has potential differentiation inducing activity in human chronic erythro-megakaryoblastic leukemia K562 cells. MTT assays and flow cytometric analysis demonstrated that ASP inhibited K562 cell proliferation and arrested the cell cycle at the G0/G1 phase. ASP also triggered K562 cells to undergo erythroid differentiaton as revealed by morphological changes, intensive benzidine staining and hemoglobin colorimetric reaction, as well as increased expression of glycophorin A (GPA) protein. ASP induced redistribution of STAT5 protein from the cytoplasm to the nucleus. Western blotting analysis further identified that ASP markedly sensitized K562 cells to exogenous erythropoietin (EPO) by activating EPO-induced JAK2/STAT5 tyrosine phosphorylation, thus augmenting the EPO-mediated JAK2/STAT5 signaling pathway. On the basis of these findings, we propose that ASP might be developed as a potential candidate for chronic myelogenous leukemia inducing differentiation treatment.

• Asian Pacific Journal of Cancer Prevention
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• v.17 no.11
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• pp.4857-4861
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• 2016

Background: Imatinib mesylate, a tyrosine kinase inhibitor specifically targeting the BCR/ABL fusion protein, induces hematological remission in patients with chronic myeloid leukemia (CML). However, the majority of CML patients treated with imatinib develop resistance with prolonged therapy. Dendrophthoe pentandra (L.) Miq. is a Malaysian mistletoe species that has been used as a traditional treatment for several ailments such as smallpox, ulcers, and cancers. Methods: We developed a resistant cell line (designated as K562R) by long-term co-culture of a BCR/ABL positive CML cell line, K562, with imatinib mesylate. We then investigated the anti-proliferative effects of D. pentandra methanol extract on parental K562 and resistant K562R cells. Trypan blue exclusion assays were performed to determine the IC50 concentration; apoptosis and cell cycle analysis were conducted by flow cytometry. Results: D. pentandra extract had greater anti-proliferative effects towards K562R ($IC50=192{\mu}g/mL$) compared to K562 ($500{\mu}g/mL$) cells. Upon treatment with D. pentandra extract at the IC50. concentration: K562 but not K562R demonstrated increase in apoptosis and cell cycle arrest in the G2/M phase. Conclusion: D. pentandra methanol extract exerts potent anti-proliferative effect on BCR/ABL positive K562 cells.

• The Korean Journal of Nuclear Medicine
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• v.38 no.1
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• pp.85-98
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• 2004

Purpose: Cellular uptake of $^{99}mTc$-sestamibi (MIBI) and $^{99}mTc$-tetrofosmin (TF) is low in cancer cells expressing multidrug resistance(MDR) by p-glycoprotein(Pgp) or multidrug related protein(MRP). Verapamil is known to increase cellular uptake of MIBI in MDR cancer cells, but is recently reported to have different effects on tracer uptake in certain cancer cells. This study was prepared to evaluate effects of verapamil on cellular uptake of MIBI and TF in several cancer cells. Materials and Methods: Celluar uptakes of Tc-99m MIBI and TF were measured in erythroleukermia K562 cell, breast cancer MCF7 cell, and human ovarian cancer SK-OV-3 cells, and data were compared with those of doxorubicin-resistant K562(Ad) cells. RT-PCR and Western blot analysis were used for the detection of mdr1 mRNA and Pgp expression, and to observe changes in isotypes of PKC enzyme. Effects of verapamil on MIBI and TF uptake were evaluated at different concentrations upto $200{\mu}M\;at\;1{\times}10^6\;cells/ml\;at\;37^{\circ}C$. Radioactivity in supernatant and pellet was measured with gamma counter to calculate cellular uptake ratio. Toxicity of verapamil was measured with MTT assay. Results: Cellular uptakes of MIBI and TF were increased by time in four cancer cells studied. Co-incubation with verapamil resulted in an increase in uptake of MIBI and TF in K562(Adr) cell at a concentration of $100{\mu}M$ and the maximal increase at $50{\mu}M$ was 10-times to baseline. In contrast, uptakes of MIBI and TF in K562, MCF7, SK-OV3 cells were decreased with verapamil treatment at a concentration over $1{\mu}M$. With a concentration of $200{\mu}M$ verapamil, MIBI and TF uptakes un K562 cells were decreased to 1.5 % and 2.7% of those without verapamil, respectively. Cellular uptakes of MIBI and TF in MCF7 and SK-OV-3 cells were not changed with $10{\mu}M$, but were also decreased with verapamil higher than $10{\mu}M$, resulting 40% and 5% of baseline at $50{\mu}M$. MTT assay of four cells revealed that K562, MCF7, SK-OV3 were not damaged with verapamil at $200{\mu}M$. Conclusion: Although verapamil increases uptake of MIBI and TF in MDR cancer cells, cellular uptakes were further decreased with verapamil in certain cancer cells, which is not related to cytotoxicity of drug. These results suggest that cellular uptakes of both tracers might differ among different cells, and interpretation of changes in tracer uptake with verapamil in vitro should be different when different cell lines are used.

• YAKHAK HOEJI
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• v.40 no.2
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• pp.225-229
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• 1996

The cytotoixic effect of Pylorae Herba (Pyrola japonica Klenze) against L1210 and K-562 cells was studied in vitro. The methanolic extract of Pylorae Radix was add ed to the culture of L1210 cells and K-562 cells for the cytotoxic activity and the ED50 values of hexane, ethylacetate, buthanol and water fractions from methanolic extract were determined using MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazoliumbromide) assay. The active constituent isolated by bioassay guided fractionation followed by purification gave rise to a yellow needle crystal and was clarified to be chimaphilline by the comparison with the published data. The average life spans with it were not prolonged significantly on tumor growth in hybrid female mouse (BDF1-KIST) inoculated subcutaneously with P388 cells.

• Radiation Oncology Journal
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• v.21 no.3
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• pp.227-237
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• 2003

Purpose: The human chronic myelogenous leukemia cell line, K562, expresses the chimeric bcr-abl oncoprotein, whose deregulated protein tyrosine kinase activity antagonizes via DNA damaging agents. Previous experiments have shown that nanomolar concentrations of herbimycin A (HWA) coupled with X-irradiation have a synergistic effect in inducing apoptosis in the Ph-positive K562 leukemia cell line, but genistein, a PTK inhibitor, is non selective for the radiation-induced apoptosils on $p210^{bcr/abl}$ protected K562 cells. In these experiments, the cytoplasmic signal transduction pathways, the Induction on a number of transcription factors and the differential gene expression in this model were investigated. Materials and Methids: K562 cells in the exponential growth phase were used in this study. The cells were irradiated with 0.5-12 Gy, using a 6 Mev Linac (Clinac 1800, Varian, USA). Immediately after irradiation, the cells were treated with $0.25/muM$ of HMA and $25/muM$ of genistein, and the expressions and the activities of abl kinase, MAPK family, NF- kB, c-fos, c-myc, and thymidine kinase1 (TK1) were examined. The differential gene expressions induced by PTK inhibitors were also investigated. Results: The modulating effects of herbimycin A and genistein on the radiosensitivity of K562 cells were not related to the bcr-abl kinase activity. The signaling responses through the MAPK family of proteins, were not involved either in association with the radiation-induced apoptosis, which is accelerated by HMA, the expression of c-myc was increased. The combined treatment of genistein, with irradiation, enhanced NF- kB activity and the TK1 expression and activity. Conclusion: The effects of HMA and genistein on the radiosensitivity on the K562 cells were not related to the bcr-abl kinase activity in this study, another signaling pathway, besides the WAPK family responses to radiation to K562 cells, was found. Further evaluation using this model will provide valuable information for the optional radiosensitization or radioprotection.

• The Korean Journal of Nuclear Medicine
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• v.37 no.3
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• pp.178-189
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• 2003

Purpose: Cellular uptakes of $^{99m}Tc-sestamibi(MIBI)\;and\;\;^{99m}Tc-tetrofosmin$ into cancer cell lines expressing multidrug resistance(MDR) were investigated and compared. The effects of verapamil and cyclosporin A, well-known multidrug resistant reversing agents, on cellular uptakes of both tracers were also compared. Materials and Methods: Doxorubicin-resistant HCT15/CL02 human colorectal cell and doxorubicin-resistant K562(Adr) and vincristine-resistant K562(Vcr) human leukemic cells were studied. RT-PCR analysis was used for the detection of mdr1 mRNA expression. MDR-reversal effects with verapamil and cyclosporine A were evaluated at different drug concentrations after incubation with MIBI and tetrofosmin for 1, 15, 30, 45 and 60 min, using single-cell suspensions at $1{\times}10^6cells/ml$ incubated at $37^{\circ}C$. Radioactivity in supernatants and pellets were measured with gamma well counter. Results: The cellular uptakes of MIBI and tetrofosmin in K562(Adr) and K562(Vcr) were lower than those of parental K562 cell. In HCT15/CL02 cells and K562(Adr) cells, there were no significant difference in cellular uptakes of both tracers, but cellular uptake of MIBI was higher than that of tetrofosmin in K562(Vcr) cells. Coincubation with verapamil resulted in a increase In cellular uptakes of MIBI and tetrofosmin. Verapamil increased cellular uptakes of MIBI and tetrofosmin by HCT15/CL02 cell by 11.9- and 6.8-fold, by K562(Adr) cell by 14.3- and 8-fold and by K562(Vcr) cell by 7- and 5.7-fold in maximum, respectively. Cyciosporin A increased cellular uptakes of MIBI and tetrofosmin by HCT15/CL02 cell by 10- and 2.4-fold, by K562(Adr) cell by 44- and 13-fold and by K562(Vcr) cell by 18.8- and 11.8-fold in maximum, respectively Conclusion: Taking together, MIBI and tetrofosmin are considered as suitable radiopharmaceuticals for defecting multidrug resistance. However, MIBI seems to be a better tracer than tetrofosmin for evaluating MDR reversal effect of the modulators. Since cellular uptakes of both tracers might differ in different cell types, further experiments regarding differences in cellular uptakes between cell types should be explored.