Multiple Drug Resistant Cell Line Research Articles

Downregulation of cytokeratin 18 enhances BCRP-mediated multidrug resistance through induction of epithelial-mesenchymal transition and predicts poor prognosis in breast cancer.

Multiple drug resistance (MDR) and metastasis have been identified as the two major causes of the poor prognosis of patients with breast cancer. However, the relationship between MDR and metastasis has not been characterized. Epithelial‑mesenchymal transition (EMT), a process known to promote metastasis in cancer, has been shown to be associated with the MDR phenotype of many tumor types. Reduced cytokeratin 18 (CK18) expression is thought to be one of the hallmarks of EMT, and the role of CK18 in MDR of metastatic breast cancer remains unknown. In the present study, we revealed that the expression of CK18 was significantly downregulated in breast cancer tissues and in an MDR cell line overexpressing breast cancer resistant protein (BCRP), and the presence of low levels of CK18 was associated with TNM stage, lymph node metastasis, and unfavorable survival in breast cancer patients. Further results demonstrated that CK18 stable knockdown using shRNA increased BCRP expression and induced the EMT process in human breast cancer MCF‑7 cells. Moreover, CK18 knockdown was associated with the activation of the NF‑κB/Snail signaling pathway, which has been revealed to regulate EMT and BCRP. Based on these findings, we concluded that CK18 knockdown enhanced BCRP‑mediated MDR in MCF‑7 cells through EMT induction partly via the NF‑κB/Snail pathway. These findings provide a valuable insight into the potential role of CK18 in MDR, migration and invasion of breast cancer cells. Reduced expression of CK18 may be a novel biomarker for predicting the poor prognosis of breast cancer patients.

A secretomic study on human hepatocellular carcinoma multiple drug-resistant cell lines.

The aim of this study was to identify pivotal dysregulated proteins that are biomarkers for multiple drug resistance (MDR) of human hepatocellular carcinoma (HCC). The secretome profiles of the human HCC MDR cell line BEL7402/5-FU and its parental cell line BEL7402 were comparatively analyzed using isobaric tags for the relative and absolute quantification (iTRAQ)-coupled 2D LC-MS/MS. In total, 279differentially expressed proteins were identified, of which, with a consistent result in the duplex test, 131proteins were overexpressed in BEL7402/5-FU compared to its parental cell line, and 56proteins were underexpressed. Several differentially expressed proteins determined by western blot analysis were also validated. The association of MDR with one of the highly regulated proteins, α-2-HS-glycoprotein (AHSG) was determined. This study detailed the application of iTRAQ technology to MDR biomarkers in the HCC cell secretome. The results showed that differentially expressed proteins that may be associated with MDR of HCC provide valuable additional information with regard to understanding the role of MDR.

Wnt pathway activation and ABCB1 expression account for attenuation of Proteasome inhibitor-mediated apoptosis in multidrug-resistant cancer cells

Multiple drug resistance (MDR) is a major obstacle to attenuating the effectiveness of chemotherapy to many human malignancies. Proteasome inhibition induces apoptosis in a variety of cancer cells and is recognized as a novel anticancer therapy approach. Despite its success, some multiple myeloma patients are resistant or become refractory to ongoing treatment by bortezomib suggesting that chemoresistant cancer cells may have developed a novel mechanism directed against the proteasome inhibitor. The present study aimed to investigate potential mechanism(s) of attenuation in a MDR cell line, MES-SA/Dx5. We found that compared to the parental human uterus sarcoma cell line MES-SA cells, MES-SA/Dx5 cells highly expressed the ABCB1 was more resistant to MG132 and bortezomib, escaping the proteasome inhibitor-induced apoptosis pathway. The resistance was reversed by co-treatment of MG132 and the ABCB1 inhibitor verapamil. The data indicated that ABCB1 might play a role in the efflux of MG132 from the MES-SA/Dx5 cells to reduce MG132-induced apoptosis. Furthermore, the canonical Wnt pathway was found activated only in the MES-SA/Dx5 cells through active β-catenin and related transactivation activities. Western blot analysis demonstrated that Wnt-targeting genes, including c-Myc and cyclin D1, were upregulated and were relevant in inhibiting the expression of p21 in MES-SA/Dx5 cells. On the other hand, MES-SA cells expressed high levels of p21 and downregulated cyclin D1 and caused cell cycle arrest. Together, our study demonstrated the existence and participation of ABCB1 and the Wnt pathway in an MDR cell line that attenuated proteasome inhibitor-induced apoptosis.

JNK signaling maintains the mesenchymal properties of multi-drug resistant human epidermoid carcinoma KB cells through snail and twist1

Background and methodsIn addition to possess cross drug resistance characteristic, emerging evidences have shown that multiple-drug resistance (MDR) cancer cells exhibit aberrant metastatic capacity when compared to parental cells. In this study, we explored the contribution of c-Jun N-terminal kinases (JNK) signaling to the mesenchymal phenotypes and the aberrant motile capacity of MDR cells utilizing a well characterized MDR cell line KB/VCR, which is established from KB human epidermoid carcinoma cells by vincristine (VCR), and its parental cell line KB.ResultsTaking advantage of experimental strategies including pharmacological tool and gene knockdown, we showed here that interference with JNK signaling pathway by targeting JNK1/2 or c-Jun reversed the mesenchymal properties of KB/VCR cells to epithelial phenotypes and suppressed the motile capacity of KB/VCR cells, such as migration and invasion. These observations support a critical role of JNK signaling in maintaining the mesenchymal properties of KB/VCR cells. Furthermore, we observed that JNK signaling may control the expression of both snail and twist1 in KB/VCR cells, indicating that both snail and twist1 are involved in controlling the mesenchymal characteristics of KB/VCR cells by JNK signaling.ConclusionJNK signaling is required for maintaining the mesenchymal phenotype of KB/VCR cells; and JNK signaling may maintain the mesenchymal characteristics of KB/VCR cells potentially through snail and twist1.

The Anticancer Agent Prodigiosin Is Not a Multidrug Resistance Protein Substrate

The brilliant red pigments prodiginines are natural secondary metabolites that are produced by select species of Gram-negative and Gram-positive bacteria. These molecules have received significant attention due to their reported antibacterial, antifungal, immunosuppressive, and anticancer activities. In this study, a Serratia marcescens SER1 strain was isolated and verified using 16s rDNA. The prodigiosin was purified using silica chromatography and was analyzed by (1)H-NMR spectroscopy. The cell cytotoxic effects of the purified prodigiosin on multiple drug resistant cell lines that overexpress MDR1, BCRP, or MRP2 pumps were analyzed. Prodigiosin had nearly identical cytotoxic effects on the resistant cells in comparison to their parental lines. In agreement with the same prodigiosin cytotoxicity, FACS analysis of prodigiosin accumulation and efflux in MDR overexpressing cell lines also indicated that this pro-apoptotic agent operates independently of the presence of the MDR1, BCRP, or MRP transporter and may be a potential treatment for malignant cancer cells that overexpress multidrug resistance transporters.

Multi-Drug–Resistant Cells Enriched From Chronic Myeloid Leukemia Cells by Doxorubicin Possess Tumor-Initiating–Cell Properties

Multiple drug resistance (MDR) occurring during chemotherapy is a major obstacle for treatment of cancers using chemotherapeutic drugs; thus, the mechanisms underlying MDR have attracted intensive attention. Many studies have shown that tumor-initiating cells exhibit a chemotherapeutic tolerance characteristic. However, whether the MDR cells possess tumor-initiating cells properties and its underlying mechanisms remain to be fully elucidated. In this study, we utilized a well-established MDR cell line K562/A02 enriched by doxorubicin from K562 cells to determine if the K562/A02 cells possess tumor-initiating properties and investigated its potential molecular mechanisms. We observed that the expressions of Oct4, Sox2, and Nanog, all of which are well-characterized stem cell markers, in K562/A02 cells were elevated in comparison to parental K562 cells; in addition, we found that K562/A02 cells exhibited more potent in vitro and in vivo tumor-initiating properties, as revealed by sphere assay, self-renewal assay, soft agar assay, and animal studies. Furthermore, our data suggest that snail and twist1, two well known transcriptional factors for the epithelial-mesenchymal transition (EMT) program, may be potentially involved in the acquisition of tumor-initiating properties of K562/A02 cells. Thus, our study demonstrates that MDR K562/A02 cells possess tumor-initiating properties, most likely due to the elevated expressions of snail and twist1.

Abstract 4914: Methylation profiling of CpG islands in multiple drug resistant cell line

Abstract Multiple drug resistance (MDR) is a major issue to attenuate the effectiveness of chemotherapy to many human malignancies. Despite extensively investigation, MDR inhibitors have been discovered serve side effect. Recently, scientists have reported that proteasome inhibitor, Bertezomib (PS-341), induced apoptosis in a variety of cancer cells and recognized as a novel anticancer therapy approach. Previous study revealed that bertezomib inhibits DNA methyltransferase and led to DNA hypomethylation in myeloid leukemia. Despite its success, some patients are resistant to ongoing proteasome inhibitor treatment. These reports suggested that chemoresistant cell line may develop a novel mechanism to against proteasome inhibitor. To verify the hypothesis, we used human CpG island microarray to indentify epigenetic change and genes expression profile in parental human uterus sarcoma cell line (MES-SA) and MES-SA variant drug resistant cell line (Dx5-C5) Our data has shown that the global genes were hypomethylated in both PS-341 treated MES-SA and Dx5-C5 cell line. Furthermore, the Wnt signalling pathway and cell cycle related gene have significant altered in DNA methylation pattern of Dx5-C5. In summary, our study demonstrated that the cell cycle related gene(s) and upstream gene(s) of Wnt pathway altered its DNA methylation pattern and mRNA expression level in MDR cell line. These data may help us to develop a new strategy for treatment of multiple drug resistance cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4914.

Abstract 3560: Evaluation of RNA-stress granules formation as an indicator of response to Darinaparsin in cancer cell lines

Abstract Darinaparsin (ZIO101) is a novel organic arsenic compound demonstrating clinical efficacy and a favorable safety profile in hematological cancers and solid tumors. Its mechanism of action includes disruption of mitochondrial functions and induction of apoptosis. We studied the ability of darinaparsin to induce RNA stress granules, a critical survival adaptation mechanism for cells under stress. Darinaparsin caused a stress granule response similar to sodium arsenite, but at lower concentrations. Treatment of cells with darinaparsin induced the formation of granules containing a number of proteins specifically associated with stress granules, including G3BP (the Ras-GAP SH3 domain binding protein), CCAR1 (Cell Cycle and Apoptosis Regulatory Protein-1), caprin-1 (Cytoplasmic Activation/Proliferation-associated protein1) and AKAP350A (AKAP9). However, darinaparsin led to development of a greater number of smaller granules compared to sodium arsenite-induced stress. We evaluated the effect of 0.5 mM arsenite or 0.05 mM darinaparsin on poorly differentiated uterine sarcoma (MES-SA) and the multiple drug resistant cell line MES-SA/Dx5 that was established from MES-SA cells. MES-SA/Dx5 cells express high levels of mdr-1 mRNA and P-glycoprotein. Both sodium arsenite and darinaparsin led to stress granule formation in both cell lines. However, MES-SA/Dx5 cells were extremely sensitive to darinaparsin treatment and exhibited a prominent loss of cell contact. The amount of cleaved PARP (a marker of apoptosis) determined at 48h and 72h after darinaparsin treatment was consistent with the immunofluorescence findings in that cleaved PARP was significantly elevated in MES-SA/Dx5 cells compared to MES-SA cells. This indicates enhanced cytotoxic effects of darinaparsin on this otherwise chemotherapeutic resistant cell line. We also evaluated the effect of darinaparsin and sodium arsenite on different leukemia cell lines including Jurkat (T-cell lymphoblastic leukemia derived), Oci and THP1 (acute myeloid leukemia derived). THP1 cells express wild type nucleophosmin (NPM), while Oci cells are heterozygous for its mutant form NPMc+. Interestingly, we found that Jurkat cells did not developed stress granules in response to darinaparsin treatment and this cell line showed accelerated toxicity compared with THP1 and Oci cells, which did form stress granules. These studies demonstrate that darinaparsin has a different spectrum of activity than sodium arsenite and can elicit differential toxicity on specific classes of tumor cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3560.

Mitochondrial Expression and Functional Activity of Breast Cancer Resistance Protein in Different Multiple Drug-Resistant Cell Lines

The multidrug resistance (MDR) phenotype is characterized by the overexpression of a few transport proteins at the plasma membrane level, one of which is the breast cancer resistance protein (BCRP). These proteins are expressed in excretory organs, in the placenta and blood-brain barrier, and are involved in the transport of drugs and endogenous compounds. Because some of these proteins are expressed in the mitochondria, this study was designed to determine whether BCRP is expressed at a mitochondrial level and to investigate its function in various MDR and parental drug-sensitive cell lines. By using Western blot analysis, immunofluorescence confocal and electron microscopy, flow cytometry analysis, and the BCRP (ABCG-2) small interfering RNA, these experiments showed that BCRP is expressed in the mitochondrial cristae, in which it is functionally active. Mitoxantrone accumulation was significantly reduced in mitochondria and in cells that overexpress BCRP, in comparison to parental drug-sensitive cells. The specific inhibitor of BCRP, fumitremorgin c, increased the accumulation of mitoxantrone significantly in comparison with basal conditions in both whole cells and in mitochondria of BCRP-overexpressing cell lines. In conclusion, this study shows that BCRP is overexpressed and functionally active in the mitochondria of MDR-positive cancer cell lines. However, its presence in the mitochondria of parental drug-sensitive cells suggests that BCRP can be involved in the physiology of cancer cells.

Relationship between survivin expression and chemosensitivity of human lung cancer cells

To investigate the relationship between survivin expression and the chemosensitivity in multiple drug resistant human lung cancer cell line Human lung cancer cells of the parental cell line H460 and multiple drug resistant cell line H460/cDDP were cultured respectively. RT-PCR and Western blotting were used to detect the mRNA and protein expression of survivin. siRNA targeting survivin was transfected into H460/cDDP cells by Liposomes approach. The expression change of survivin protein after transfection was examined by Western blotting. Cisplatin (DDP) and paclitaxel of different concentrations were added into the culture fluid of the H460/cDDP cells respectively. Methyl thiazolyl tetrazolium (MTT) assay was used to calculate the IC(50) value so as to detect the cytotoxicity of cisplatin and paclitaxel to the cells. The mRNA expression and protein expression of survivin in the H460/cDDP cells were both significantly higher than those in the H460 cells. 24 h, 48 h, and 72 h after transfection of the survivin siRNA the survivin protein expression levels of the H460/cDDP were decreased by 67.0%, 76.2%, and 92.3% respectively. MTT assay showed that the cisplatin IC(50) levels of the H460/cDDP and H460 cells were 6.3 +/- 0.6 mg/L and 0.8 +/- 0.1 mg/L respectively, and the paclitaxel IC(50) levels were 12.7 +/- 1.2 mg/L and 1.5 +/- 0.3 mg/L respectively. 48 h after the transfection of survivin siRNA, the cisplatin and paclitaxel IC(50) levels of the H460/cDDP cells were decreased to 4.0 +/- 0.9 mg/L (P = 0.002) and 8.1 +/- 1.7 mg/L (P = 0.003) respectively. The survivin expression of the resistant cell line H460/cDDP was up-regulated compared with that of the parental cell line H460. Survivin siRNA sensitized the H460/cDDP cells to both cisplatin and paclitaxel. Survivin participates in the multiple drug resistance mechanism of human lung cancer cells.

P-Glycoprotein Mediates Celecoxib-Induced Apoptosis in Multiple Drug-Resistant Cell Lines

In several neoplastic diseases, including hepatocellular carcinoma, the expression of P-glycoprotein and cyclooxygenase-2 (COX-2) are often increased and involved in drug resistance and poor prognosis. P-glycoprotein, in addition to drug resistance, blocks cytochrome c release, preventing apoptosis in tumor cells. Because COX-2 induces P-glycoprotein expression, we evaluated the effect of celecoxib, a specific inhibitor of COX-2 activity, on P-glycoprotein-mediated resistance to apoptosis in cell lines expressing multidrug resistant (MDR) phenotype. Experiments were done using MDR-positive and parental cell lines at basal conditions and after exposure to 10 or 50 micromol/L celecoxib. We found that 10 micromol/L celecoxib reduced P-glycoprotein, Bcl-x(L), and Bcl-2 expression, and induced translocation of Bax from cytosol to mitochondria and cytochrome c release into cytosol in MDR-positive hepatocellular carcinoma cells. This causes the activation of caspase-3 and increases the number of cells going into apoptosis. No effect was shown on parental drug-sensitive or on MDR-positive hepatocellular carcinoma cells after transfection with MDR1 small interfering RNA. Interestingly, although inhibiting COX-2 activity, 50 micromol/L celecoxib weakly increased the expression of COX-2 and P-glycoprotein and did not alter Bcl-x(L) and Bcl-2 expression. In conclusion, these results show that relatively low concentrations of celecoxib induce cell apoptosis in MDR cell lines. This effect is mediated by P-glycoprotein and suggests that the efficacy of celecoxib in the treatment of different types of cancer may depend on celecoxib concentration and P-glycoprotein expression.

P-gp localization in mitochondria and its functional characterization in multiple drug-resistant cell lines

Multidrug resistance (MDR) phenotype is characterized by the over-expression of P-glycoprotein (P-gp) on cell plasma membranes that extrudes several drugs out of cells. Cells that express the MDR phenotype are resistant to the mitochondrial related apoptosis and to several anticancer drugs. This study assessed the presence of P-gp in mitochondria and its role in parental drug-sensitive (P5) and in P5-derived MDR1 cells P1(0.5) hepatocellular carcinoma (HCC) cell lines and in drug-sensitive (PSI-2) and mdr1-transfected (PN1A) NIH/3T3 cells. By using Western blot analysis, confocal laser microscopy, measurements of Rhodamine 123 transport across mitochondrial membranes, MDR1 small interfering RNA and flow cytometry analysis, experiments indicate that P-gp is expressed in mitochondria of P1(0.5) and PN1A cells and it is functionally active. Rho 123 accumulation was largely reduced in mitochondria of P1(0.5) cells as compared to those of P5 cells; the reduced uptake of fluorescence in mitochondria of MDR cells was due to P-gp-mediated Rho 123 efflux. In conclusion, these data demonstrate that functionally active P-gp is expressed in the mitochondrial membrane of MDR-positive cells and pumps out anticancer drugs from mitochondria into cytosol. Therefore, P-gp could be involved in the protection of mitochondrial DNA from damage due to antiproliferative drugs.

Generation of novel epothilone analogs with cytotoxic activity by biotransformation.

The epothilones represent a new class of bacterial natural products with broad spectrum of antiproliferative activity against various types of human tumors and tumor cell lines. The attractive preclinical profile of epothilones has made them promising lead compounds for novel anticancer agents and has spurred a strong interest in obtaining different derivatives to fully evaluate their therapeutic potentials. We have generated a number of novel epothilone D and 10,11-dehydroepothilone D (Epo490) analogs via biotransformation using Amycolata autotrophica to alter the oxidation state of the parental compounds. The bioconverted compounds displayed different degrees of potency in cytotoxicity assays against a panel of human tumor cell lines, with 11-hydroxyepothilone D, 14-hydroxyepothilone D, and 21-hydroxyepothilone D showing comparable activity to that of epothilone D, and 21-hydroxy Epo490 being comparable to Epo490. The addition of hydroxyl group(s) seems to cause a decrease in cytotoxic activity against multiple drug resistant cell lines (with overexpressed P-glycoprotein). The compounds generated by biotransformation exert differential effects on tubulin polymerization, which correlate with their biological activities.