Drug-sensitive Parental Cell Line Research Articles

Abstract 7200: Non-cytotoxic inhibition of nuclear encoded mitochondrial protein MAGMAS resensitize treatment resistant prostate cancer cells to taxane therapy

Abstract Metastatic prostate cancer (PCa) is the leading cause of PCa-associated deaths and one of the leading causes of cancer mortality in men. Novel therapeutics, such as Prostate Specific Membrane Antigen (PSMA) ligand therapy, are emerging as effective treatment strategies for metastatic prostate cancer. However, most men succumb to Metastatic Castration Resistant Prostate Cancer (mCRPC) due to treatment resistance. MAGMAS, a 13.8 kDa mitochondrial protein, imports nuclear-encoded mitochondrial proteins into the mitochondrial matrix. MAGMAS is overexpressed in several aggressive tumors, such as breast cancer, glioblastoma, and prostate cancer. When overexpressed, MAGMAS plays a role as a cytoprotective protein by acting as a reactive oxygen species (ROS) scavenger that maintains sufficient ROS levels to promote cell proliferation but below the threshold levels that could lead to apoptosis. This study was designed to investigate the pro-survival and therapy-resistance functions of MAGMAS in treatment-resistant PCa cells. Predictively, we observed that the docetaxel (DTX)-resistant PC3-DR and DU145-DR PCa cell lines expressed significantly higher levels of MAGMAS compared to their drug-sensitive parental cell lines. We hypothesized that inhibiting Magmas reduces the aggressive properties of highly proliferative DTX-resistant metastatic PCa cell lines and re-sensitizes them to DTX. MAGMAS knockdown in DTX-resistant PC3 and DU145 cells in the presence of increasing DTX treatments enhanced sensitivity to DTX. Consistent with these results, pharmacological inhibition of MAGMAS with the novel BT#9 inhibitor in combination with increasing DTX concentrations also enhanced sensitivity to DTX in these cell lines. Additional studies using cell migration/invasion assays and tumor-sphere formation assays are underway to further establish MAGMAS as a potential therapeutic target to attenuate the aggressive properties of chemo-resistant PCa cells. Lastly, we will investigate the diagnostic potential of MAGMAS as a biomarker for advanced PCa and BT#9 as an imaging/therapeutic-specific agent for treatment-resistant tumors. Citation Format: Alfonso Duran, Kristen Whitley, Krystal Santiago, Adil Mohammed, Carlos Casiano, Bhaskar Das, Frankis Almaguel. Non-cytotoxic inhibition of nuclear encoded mitochondrial protein MAGMAS resensitize treatment resistant prostate cancer cells to taxane therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7200.

Abstract 3757: The development of MAGMAS as a potential therapeutic target in temozolomide-resistant glioma

Abstract Glioblastoma (GBM) is one of the most aggressive types of brain cancer and remains a significant challenge for drug development in oncology today. Temozolomide (TMZ) is the standard of care for GBM, and resistance to this drug is modulated partly by the DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT). Clinical studies demonstrated that low MGMT expression in GBM enhances sensitivity to TMZ and prolongs survival for patients. However, recent clinical studies indicate MGMT is a discordant biomarker since the predictive value of MGMT methylation is relatively low and there is a lack of modalities to inhibit MGMT expression. Furthermore, glioma stem cells (GSC) are another contributing factor for resistance due to tumor heterogeneity, self-renewal capacity and activation of DNA repair responses to overcome the effect of radiation and TMZ treatment. These resistance mechanisms contribute to tumor recurrence and present major impediments for the development of effective treatments. MAGMAS, 13.8 kDa mitochondria protein, is a translocase of the inner mitochondrial membrane and regulates oxidative phosphorylation. Specifically, MAGMAS regulates the ATP stimulatory activity of DNAJC19 on mtHSP70 that drives the movement of peptides through the TIM23 complex into the mitochondrial matrix. Recent evidence suggests that MAGMAS can regulate oxidative phosphorylation via interactions with the respiratory complexes present in the inner mitochondrial membrane. Previously, our lab has shown MAGMAS overexpression in malignant glioma tissues of patients and murine intracranial xenografts. Additionally, pharmacological inhibition of MAGMAS with the small molecule BT9 promoted cytotoxicity and impaired respiratory functions in malignant glioma cells demonstrating that MAGMAS is a promising potential target for GBM patients. However, further studies are needed to investigate the role of MAGMAS across the spectrum from initial tumorigenesis to disease progression and to further determine how MAGMAS is implicated during the development of treatment resistance. This study is designed to investigate the role of MAGMAS in GSCs and their contribution to TMZ resistance. We observed temozolomide-resistant (TR) U251-TR and D54 MG-TR GBM cell lines expressed significantly higher levels of MAGMAS and several stem cell markers than their drug-sensitive (S) parental cell lines. Therefore, we hypothesized that silencing MAGMAS re-sensitizes TR GBM cell lines to TMZ. We found that MAGMAS knockdown in D54-MG and U251 S/TR cells in the presence of escalating TMZ concentrations, caused enhanced sensitivity to TMZ. Consistent with these results, inhibition of MAGMAS with the novel BT9 inhibitor enhances the TMZ sensitivity in the TMZ-resistant cell lines. Additional studies of patient-derived cell lines are being utilized further to validate MAGMAS as it is an attractive target in TMZ-resistant GBM patients. Citation Format: Jennifer D. Tran, Javier J. Lepe, Maria C. Kenney, Bhaskar C. Das, Daniela A. Bota. The development of MAGMAS as a potential therapeutic target in temozolomide-resistant glioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3757.

Abstract 1754: Targeting the mitochondrial protein MAGMAS increases sensitivity to docetaxel in neuroendocrine prostate cancer cells

Abstract Metastatic prostate cancer (PCa) is the leading cause of PCa-associated deaths and one of the leading causes of cancer mortality in men. Novel therapeutics, such as Prostate Specific Membrane Antigen (PSMA) ligand therapy, are emerging as effective treatment strategies for metastatic prostate cancer. However, neuroendocrine differentiated (NED) metastatic PCa, which lacks the PSMA antigen, is deprived of effective biomarkers for disease treatment. Magmas, a 13.8 kDa mitochondrial protein, imports nuclear-encoded mitochondrial proteins into the mitochondrial matrix. Magmas is overexpressed in several aggressive tumors, such as breast cancer, glioblastoma, and prostate cancer. When overexpressed, Magmas plays a role as a cytoprotective protein by acting as a reactive oxygen species (ROS) scavenger that maintains sufficient ROS levels to promote cell proliferation, but below the threshold levels that could lead to apoptosis. This study was designed to investigate the pro-survival and therapy-resistance functions of Magmas in PCa cells expressing NED markers. Predictively, we observed that the docetaxel (DTX)-resistant PC3-DR and DU145-DR PCa cell lines expressed significantly higher levels of Magmas compared to their drug-sensitive parental cell lines. We hypothesized that inhibiting Magmas reduces the aggressive properties of highly proliferative DTX- resistant metastatic PCa cell lines and re-sensitize them to DTX. Magmas knockdown in DTX-sensitive and DTX-resistant PC3 and DU145 cells in the presence of increasing DTX treatments enhanced sensitivity to DTX. Consistent with these results, pharmacological inhibition of Magmas with the novel BT#9 inhibitor in combination with increasing DTX concentrations also enhanced sensitivity to DTX in these cell lines. Additional studies using clonogenic assays, cell migration/invasion assays, and tumorsphere formation assays are currently underway to further establish Magmas as a potential therapeutic target to attenuate the aggressive properties of chemoresistant PCa cells. Lastly, we will investigate the diagnostic potential of Magmas as a biomarker for NED and BT#9 as an imaging/therapeutic-specific agent for NED. Citation Format: Alfonso M. Durán, Christian H. Yoo, Jennifer D. Tran, Krystal Santiago, Adil S. Mohammed, Carlos A. Casiano, Frankis A. Almaguel. Targeting the mitochondrial protein MAGMAS increases sensitivity to docetaxel in neuroendocrine prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1754.

Branebrutinib (BMS-986195), a Bruton's Tyrosine Kinase Inhibitor, Resensitizes P-Glycoprotein-Overexpressing Multidrug-Resistant Cancer Cells to Chemotherapeutic Agents.

The overexpression of P-glycoprotein (P-gp/ABCB1), an ATP-binding cassette (ABC) drug transporter, often contributes to the development of multidrug resistance (MDR) in cancer cells. P-gp mediates the ATP hydrolysis-dependent efflux of a wide range of chemotherapeutic agents out of cancer cells, thereby reducing the intracellular drug accumulation and decreasing the chemosensitivity of these multidrug-resistant cancer cells. Studies with tyrosine kinase inhibitors (TKIs) in P-gp-overexpressing cells have shown that certain TKIs could reverse MDR mediated by P-gp, while some TKIs are transported by P-gp. In the present work, we explored the prospect of repositioning branebrutinib (BMS-986195), a highly selective inhibitor of Bruton’s tyrosine kinase (BTK), to resensitize P-gp-overexpressing multidrug-resistant cancer cells to chemotherapeutic agents. Our results demonstrated that branebrutinib is capable of reversing P-gp-mediated MDR at sub-toxic concentrations, most likely by directly inhibiting the drug transport function of P-gp. Our findings were supported by the result of branebrutinib stimulating the ATPase activity of P-gp in a concentration-dependent manner and the in silico study of branebrutinib binding to the substrate-binding pocket of P-gp. In addition, we found that branebrutinib is equally cytotoxic to drug-sensitive parental cell lines and the respective P-gp-overexpressing multidrug-resistant variants, suggesting that it is unlikely that the overexpression of P-gp in cancer cells plays a significant role in reduced susceptibility or resistance to branebrutinib. In summary, we discovered an additional pharmacological action of branebrutinib against the activity of P-gp, which should be investigated further in future drug combination studies.

Targeted biologic inhibition of both tumor cell-intrinsic and intercellular CLPTM1L/CRR9-mediated chemotherapeutic drug resistance

Recurrence of therapy-resistant tumors is a principal problem in solid tumor oncology, particularly in ovarian cancer. Despite common complete responses to first line, platinum-based therapies, most women with ovarian cancer recur, and eventually, nearly all with recurrent disease develop platinum resistance. Likewise, both intrinsic and acquired resistance contribute to the dismal prognosis of pancreatic cancer. Our previous work and that of others has established CLPTM1L (cleft lip and palate transmembrane protein 1-like)/CRR9 (cisplatin resistance related protein 9) as a cytoprotective oncofetal protein that is present on the tumor cell surface. We show that CLPTM1L is broadly overexpressed and accumulated on the plasma membrane of ovarian tumor cells, while weakly or not expressed in normal tissues. High expression of CLPTM1L is associated with poor outcome in ovarian serous adenocarcinoma. Robust re-sensitization of resistant ovarian cancer cells to platinum-based therapy was achieved using human monoclonal biologics inhibiting CLPTM1L in both orthotopic isografts and patient-derived cisplatin resistant xenograft models. Furthermore, we demonstrate that in addition to cell-autonomous cytoprotection by CLPTM1L, extracellular CLPTM1L confers resistance to chemotherapeutic killing in an ectodomain-dependent fashion, and that this intercellular resistance mechanism is inhibited by anti-CLPTM1L biologics. Specifically, exosomal CLPTM1L from cisplatin-resistant ovarian carcinoma cell lines conferred resistance to cisplatin in drug-sensitive parental cell lines. CLPTM1L is present in extracellular vesicle fractions of tumor culture supernatants and in patients’ serum with increasing abundance upon chemotherapy treatment. These findings have encouraging implications for the use of anti-CLPTM1L targeted biologics in the treatment of therapy-resistant tumors.

Reversal of ABCB1-related multidrug resistance by ERK5-IN-1

BackgroundInhibition of ABC transporters is considered the most effective way to circumvent multidrug resistance (MDR). In the present study, we evaluated the MDR modulatory potential of ERK5-IN-1, a potent extracelluar signal regulated kinase 5 (ERK5) inhibitor.MethodsThe cytotoxicity and MDR reversal effect of ERK5-IN-1 were assessed by MTT assay. The KBv200-inoculated nude mice xenograft model was used for the in vivo study. Doxorubicin efflux and accumulation were measured by flow cytometry. The modulation of ABCB1 activity was measured by colorimetric ATPase assay and [125I]-iodoarylazidoprazosin (IAAP) photolabeling assay. Effect of ERK5-IN-1 on expression of ABCB1 and its downstream markers was measured by PCR and/or Western blot. Cell surface expression and subcellular localization of ABCB1 were tested by flow cytometry and immunofluorescence.ResultsOur results showed that ERK5-IN-1 significantly increased the sensitivity of vincristine, paclitaxel and doxorubicin in KBv200, MCF7/adr and HEK293/ABCB1 cells, respectively. This effect was not found in respective drug sensitive parental cell lines. Moreover, in vivo combination studies showed that ERK5-IN-1 effectively enhanced the antitumor activity of paclitaxel in KBv200 xenografts without causing addition toxicity. Mechanistically, ERK5-IN-1 increased intracellular accumulation of doxorubicin dose dependently by directly inhibiting the efflux function of ABCB1. ERK5-IN-1 stimulated the ABCB1 ATPase activity and inhibited the incorporation of [125I]-iodoarylazidoprazosin (IAAP) into ABCB1 in a concentration-dependent manner. In addition, ERK5-IN-1 treatment neither altered the expression level of ABCB1 nor blocked the phosphorylation of downstream Akt or Erk1/2. No significant reversal effect was observed on ABCG2-, ABCC1-, MRP7- and LRP-mediated drug resistance.ConclusionsCollectively, these results indicated that ERK5-IN-1 efficiently reversed ABCB1-mediated MDR by competitively inhibiting the ABCB1 drug efflux function. The use of ERK5-IN-1 to restore sensitivity to chemotherapy or to prevent resistance could be a potential treatment strategy for cancer patients.

Differential microRNA profiles between fulvestrant-resistant and tamoxifen-resistant human breast cancer cells

Increasing evidence has shown that the dysregulation of microRNAs (miRNAs) is associated with drug resistance. Fulvestrant and tamoxifen represent the major endocrine drugs for the treatment of breast cancer patients, and yet little is known about the biological mechanisms of acquiring resistance to fulvestrant and tamoxifen, let alone the differences between cell lines resistant to these two drugs. Exploration of the differential miRNA profiles between these two cell lines is a useful way to further clarify these resistance mechanisms. The fulvestrant-resistant cell line (MCF7-F) and the tamoxifen-resistant cell line (MCF7-T) were established from the drug-sensitive parental MCF7 cell line using a 21-day high-dose antiestrogen induction method. Differentially expressed miRNA profiles of MCF7-F and MCF7-T were detected using microarray; then, multiple bioinformatic analyses were carried out, including protein-protein interaction network, Gene Ontology, and Kyoto Encyclopedia of Genes and Genomes pathway analysis. Compared with the parental MCF7 cell line, more miRNAs were found to be participating in the process of acquiring fulvestrant resistance than tamoxifen resistance. miR-4532, miR-486-5p, miR-138, miR-1228, and miR-3178 could be new targets for combating both fulvestrant resistance and tamoxifen resistance. miR-3188, miR-21, miR-149, and others may be associated with fulvestrant resistance, whereas miR-342 and miR-1226 may be associated with tamoxifen resistance in breast cancer cells. We found differential miRNA profiles between fulvestrant-resistant and tamoxifen-resistant breast cancer cells, but the definite mechanism involved in gaining resistance still needs further study.

P-131 - γ-H2AX, CYP3A and CYP2J2 overexpression in a drug-resistant acute myeloid leukemia cell line

Drug resistance is a major clinical problem in cancer treatment. Thus, it is important to establish cell line models to better understand and counteract this problem. The aim of this study was to establish and characterize a drug resistant acute myeloid leukemia cell line, as a sub-clone from a drug sensitive parental cell line. The resistant cell line (HL60–734 VR) was established by treating the parental sensitive cell line (HL60) with increasing concentrations of doxorubicin during several months. Dose-response curves were performed to confirm that the HL60–734 VR cell line was resistant to doxorubicin and to three other drugs (etoposide, cisplatin and daunorubicin). The resistant phenotype was not lost upon drug removal for 1 month. Resistant cells did not express the drug efflux pumps P-gp or BCRP, neither had alterations in apoptotic proteins such as Bax, XIAP, p53, Caspase-3 and Bcl-2. Resistant cells showed alterations in cell cycle profile and presented increased expression of a DNA repair protein (γ-H2AX) and also of proteins involved in drug metabolism (CYP3A and CYP2J2), suggesting that these alterations might be associated with the drug resistant phenotype in these cells.

A Screen for Drugs Showing Specific Co-Resistance or Hyper-Sensitivity Upon the Acquisition of Ara-C Resistance In AML

AbstractAbstract 1844Acute myeloid leukemia (AML) can display de novo or acquired resistance to cytosine arabinoside (Ara-C), a primary component of induction chemotherapy. We hypothesized that the changes required by the cells to become Ara-C resistant might also make them more susceptible to elimination by other drugs. We previously developed two highly resistant murine AML cell lines, B117H and B140H, by introducing increasing concentrations of Ara-C to their parental cell lines, B117P and B140P, respectively. The Ara-C resistant derivatives, B117H and B140H, can tolerate Ara-C concentrations ∼1000X that of their drug sensitive parental cell lines. We previously demonstrated that the down-regulation of deoxycytidine kinase was one mechanism of resistance in the Ara-C resistant B117H and B140H cells, a mechanism that has also been documented in human cases of refractory disease. A high throughput drug screen was conducted to determine if an increase in Ara-C resistance would result in increased sensitivity to other drugs. The screen evaluated 446 FDA approved drugs, which included a variety of drug types, including chemotherapy drugs, analgesics, steroids, antidepressants, immunosuppressives, and antipsychotics. Despite the diverse nature of the parental cell lines and their Ara-C resistant derivatives, two drugs (prednisolone and AM404), showed an increase in sensitivity in both Ara-C resistant cell lines. Follow-up drug assays were performed on both prednisolone and AM404 to establish the 50% inhibitory concentration (IC50) in all four cell lines. Both Ara-C resistant cell lines displayed a greater than 3-fold reduction in the IC50 of prednisolone as compared to their parental lines, and the IC50 of AM404 demonstrated a greater than 2-fold reduction. Prednisolone has been shown to induce differentiation in murine AML cells in vitro, and high-dose methylprednisolone has been found to induce differentiation in various subtypes of childhood AML. The mechanism of differentiation by prednisolone is theorized to be independent of glucocorticoid receptor binding. We believe this model will be useful in elucidating the role of prednisolone in leukemic differentiation, and in suggesting new mechanisms to target refractory AML cells. Disclosures:No relevant conflicts of interest to declare.

Abstract 2073: Changes in microRNA expression in response to short-term chemotherapeutic exposure is associated with transient anticancer drug resistance

Abstract MicroRNAs (miRNAs) are small, noncoding RNAs that act as posttranscriptional repressors of gene expression by binding the 3’ untranslated region of target genes. There is emerging evidence showing that miRNAs regulate diverse biologic processes including multidrug resistance (MDR). Our previous profiling study revealed that certain miRNAs are differentially expressed in multidrug resistant human T-cell leukemia cell line CEM/VM-1-5 compared to drug-sensitive parental cell line CCRF-CEM. To investigate whether these expression-altered miRNAs have a role in MDR, we ectopically expressed three miRNAs that are highly upregulated in CEM/VM-1-5 cells (hsa-miR-135b, −146b, and −196b) in CCRF-CEM cells with the use of lentiviral vectors. We found that overexpression of these miRNAs, individually or in combination, resulted in only slight increases in resistance of CCRF-CEM cells to teniposide, the agent used to select CEM/VM-1-5 cells. Moreover, we were unable to sensitize CEM/VM-1-5 cells to etoposide by suppression of hsa-miR-135b expression using LNA antisense oligonucleotide. Our data implied that these miRNAs do not confer drug resistance directly. However, it is still not clear whether these miRNAs play any role in the development of MDR. Therefore, we asked whether the expression of these miRNAs changed in CCRF-CEM cells after short exposure (3 to 48 hours) to various chemotherapeutic drugs at concentrations below their IC50-values. We observed substantial increases in hsa-miR-135b and −196b expression in cells treated with etoposide, doxorubicin and topotecan, but not in cells treated with vinblastine. These results suggested that the upregulation of these two miRNAs might be the consequence of DNA damage. Time-course and dose-response studies showed that the elevation of hsa-miR-135b and −196b correlated positively with the drug exposure time and drug concentration. Furthermore, the levels of miRNAs gradually decreased after the withdrawal of etoposide from the media following 48 h exposure to etoposide. Strikingly, the elevated expression of the miRNAs accompanied increased drug resistance. CCRF-CEM cells exposed to etoposide for 48 h were over 50-fold more resistant than control cells. The acquired drug resistance, like elevated expression of miRNAs, did not appear to be stable and became attenuated with increasing duration of the drug-free incubation. Together, our results suggest that miRNAs might play a significant role in cellular response to genotoxic agents and could be involved in the development of cancer drug resistance, but their roles in conferring resistance remain to be determined. That said, targeting miRNA expression could be a potential approach to prevent multidrug resistance. (Supported in part by 5RO1CA040570 from NCI to WTB, and in part by UIC) 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 2073.

PH-Responsive Polymeric siRNA Carriers Sensitize Multidrug Resistant Ovarian Cancer Cells to Doxorubicin via Knockdown of Polo-like Kinase 1

Small interfering RNA (siRNA)-based therapies have great potential for the treatment of debilitating diseases such as cancer, but an effective delivery strategy for siRNA is elusive. Here, pH-responsive complexes were developed for the delivery of siRNA in order to sensitize drug-resistant ovarian cancer cells (NCI/ADR-RES) to doxorubicin. The electrostatic complexes consisted of a cationic micelle used as a nucleating core, siRNA, and a pH-responsive endosomolytic polymer. Cationic micelles were formed from diblock copolymers of dimethylaminoethyl methacrylate (pDMAEMA) and butyl methacrylate (pDbB). The hydrophobic butyl core mediated micelle formation while the positively charged pDMAEMA corona enabled siRNA condensation. To enhance cytosolic delivery through endosomal release, a pH-responsive copolymer of poly(styrene-alt-maleic anhydride) (pSMA) was electrostatically complexed with the positively charged siRNA/micelle to form a ternary complex. Complexes exhibited size (30-105 nm) and charge (slightly positive) properties important for endocytosis and were found to be noncytotoxic and mediate uptake in >70% of ovarian cancer cells after 1 h of incubation. The pH-responsive ternary complexes were used to deliver siRNA against polo-like kinase 1 (plk1), a gene upregulated in many cancers and responsible for cell cycle progression, to ovarian cancer cell lines. Treatment resulted in approximately 50% reduction of plk1 gene expression in the drug-resistant NCI/ADR-RES ovarian cancer cell model and in the drug-sensitive parental cell line, OVCAR8. This knockdown functionally sensitized NCI/ADR-RES cells to doxorubicin at levels similar to OVCAR8. Sensitization occurred through a p53 signaling pathway, as indicated by caspase 3/7 upregulation following plk1 knockdown and doxorubicin treatment, and this effect could be abrogated using a p53 inhibitor. To demonstrate the potential for dual delivery from this polymer system, micelle cores were subsequently loaded with doxorubicin and utilized in ternary complexes to achieve cell sensitization through simultaneous siRNA and drug delivery from a single carrier. These results show knockdown of plk1 results in sensitization of multidrug resistant cells to doxorubicin, and this combination of gene silencing and small molecule drug delivery may prove useful to achieve potent therapeutic effects.

RAIDD expression is impaired in multidrug resistant osteosarcoma cell lines

To identify the apoptosis genes involved in the multidrug resistant phenotype of osteosarcoma. Multidrug resistant human osteosarcoma cell line (U-2 OS MR) and a drug sensitive parental cell line (U-2 OS) were both treated with paclitaxel and analyzed by the gene array containing 96 apoptosis associated genes. The different expression of the special apoptosis associated genes were further analyzed by Western blot in the multidrug resistant osteosarcoma cell lines (U-2 OS MR, KH OS R2) and the drug sensitive parental cell lines (U-2 OS, KH OS). One of the disregulated gene, RAIDD, was transfected into the multidrug resistant osteosarcoma cells for functional studies. RAIDD showed signs of significant expression in the U-2 OS cells after being treated with paclitaxel (P < 0.01). However, the induction of RAIDD did not occur in U-2 OS MR cells (P = 0.2). Subsequent analysis by Western blot confirmed the deficiency of the expression of RAIDD protein in U-2 OS MR. On the contrary, the expression of RAIDD could be significantly induced by paclitaxel and doxorubicin in U-2 OS cells as both time and dosage were deciding factors. It also demonstrated the cleavage of PARP associated with RAIDD expression in U-2 OS cells, but not however in U-2 OS MR cells after being treated with paclitaxel or doxorubicin. Similar results were found in osteosarcoma multidrug resistant cell line KH OS R2 and the drug sensitive parental cell line KH OS. Furthermore, over-expression of RAIDD in multidrug resistant cell lines could possibly reverse drug resistant phenotypes. This study indicate that impaired expression of RAIDD in drug induced apoptosis may play a role in the multidrug resistance of osteosarcoma cells.

A Novel Microarray Gene Expression Analysis of Murine AML Cell Lines Provides Clues to the Development of Drug Resistance to Ara-C

Acute myeloid leukemia (AML) has the ability to evade cell death in the presence of chemotherapeutic cocktails containing cytosine arabinoside (Ara-C). This lab previously developed two highly resistant murine AML cell lines, B117H and B140H, by introducing increasing concentrations of Ara-C to their parental cell lines, B117P and B140P, respectively. B117H and B140H can tolerate Ara-C concentrations ~1000X that of their drug sensitive parental cell lines. mRNA from all four cell lines were used in gene expression microarrays for the purpose of comparing Ara-C drug resistant murine AML cell lines with their Ara-C drug sensitive parental lines. A novel algorithm was developed to evaluate the changes in gene expression between the drug resistant and drug sensitive cells. The algorithm differed from more conventional algorithms in two key ways. First, the detection data was normalized by using ribosomal subunit 9 (Rsp9) as the normalization gene, and secondly it calculated fold change by comparing the minimum value of one population to the maximum value of the other population. The output of this algorithm was a list of genes with significant gene expression changes. These genes were next submitted to the Ingenuity Pathway Analysis (IPA) process. IPA implicated nuclear factor-κB (NFκB) in the Ara-C resistance process. Cell growth assays confirmed that the Ara-C drug resistant B117H cell line was significantly more sensitive to NFκB inhibition than its Ara-C sensitive parental cell line. This leads us to believe that the selection of Ara-C resistance may also concomitantly make some AML cells highly sensitive to killing by NFκB inhibition. This theory is being tested further through the use of drug combination assays, to determine if a synergistic or antagonistic relationship exists between Ara-C and various drugs that affect the NFκB pathway.

Modulation of function of three ABC drug transporters, P-glycoprotein (ABCB1), mitoxantrone resistance protein (ABCG2) and multidrug resistance protein 1 (ABCC1) by tetrahydrocurcumin, a major metabolite of curcumin

Many studies have been performed with the aim of developing effective resistance modulators to overcome the multidrug resistance (MDR) of human cancers. Potent MDR modulators are being investigated in clinical trials. Many current studies are focused on dietary herbs due to the fact that these have been used for centuries without producing any harmful side effects. In this study, the effect of tetrahydrocurcumin (THC) on three ABC drug transporter proteins, P-glycoprotein (P-gp or ABCB1), mitoxantrone resistance protein (MXR or ABCG2) and multidrug resistance protein 1 (MRP1 or ABCC1) was investigated, to assess whether an ultimate metabolite form of curcuminoids (THC) is able to modulate MDR in cancer cells. Two different types of cell lines were used for P-gp study, human cervical carcinoma KB-3-1 (wild type) and KB-V-1 and human breast cancer MCF-7 (wild type) and MCF-7 MDR, whereas, pcDNA3.1 and pcDNA3.1-MRP1 transfected HEK 293 and MXR overexpressing MCF7AdrVp3000 or MCF7FL1000 and its parental MCF-7 were used for MRP1 and MXR study, respectively. We report here for the first time that THC is able to inhibit the function of P-gp, MXR and MRP1. The results of flow cytometry assay indicated that THC is able to inhibit the function of P-gp and thereby significantly increase the accumulation of rhodamine and calcein AM in KB-V-1 cells. The result was confirmed by the effect of THC on [(3)H]-vinblastine accumulation and efflux in MCF-7 and MCF-7MDR. THC significantly increased the accumulation and inhibited the efflux of [(3)H]-vinblastine in MCF-7 MDR in a concentration-dependent manner. This effect was not found in wild type MCF-7 cell line. The interaction of THC with the P-gp molecule was clearly indicated by ATPase assay and photoaffinity labeling of P-gp with transport substrate. THC stimulated P-gp ATPase activity and inhibited the incorporation of [(125)I]-iodoarylazidoprazosin (IAAP) into P-gp in a concentration-dependent manner. The binding of [(125)I]-IAAP to MXR was also inhibited by THC suggesting that THC interacted with drug binding site of the transporter. THC dose dependently inhibited the efflux of mitoxantrone and pheophorbide A from MXR expressing cells (MCF7AdrVp3000 and MCF7FL1000). Similarly with MRP1, the efflux of a fluorescent substrate calcein AM was inhibited effectively by THC thereby the accumulation of calcein was increased in MRP1-HEK 293 and not its parental pcDNA3.1-HEK 293 cells. The MDR reversing properties of THC on P-gp, MRP1, and MXR were determined by MTT assay. THC significantly increased the sensitivity of vinblastine, mitoxantrone and etoposide in drug resistance KB-V-1, MCF7AdrVp3000 and MRP1-HEK 293 cells, respectively. This effect was not found in respective drug sensitive parental cell lines. Taken together, this study clearly showed that THC inhibits the efflux function of P-gp, MXR and MRP1 and it is able to extend the MDR reversing activity of curcuminoids in vivo.

Resistance to Microtubule-Stabilizing Drugs Involves Two Eventse: β-Tubulin Mutation in One Allele Followed by Loss of the Second Allele

Resistance to paclitaxel (PTX) or the epothilones (Epo) occurs via the acquisition of point mutations in β-tubulin residues important for drug-tubulin binding. We have isolated four drug-resistant clones selected with PTX or Epo A which harbor distinct β-tubulin mutations. During the development of a stable drug-resistant phenotype, early clones expressing both wild-type (wt) and mutant β-tubulin sequences exhibited a 10-fold drug resistance, while more advanced clones expressing only the mutant β-tubulin sequence exhibited 30 to 50-fold drug resistance. The drug-sensitive parental 1A9 ovarian carcinoma cell line and the drug resistant clones (1A9-A8, 1A9-PTX10 and 1A9-PTX22) were evaluated for loss of heterozygosity (LOH) for β-tubulin (6p25) by single nucleotide polymorphism (SNP) and fluorescent in situ hybridization (FISH) analyses. Functional assays such as drug-induced tubulin polymerization, cell cycle analysis by FACS, DNA sequencing for β-tubulin and mitotic index by immunofluorescence were performed to correlate the β-tubulin LOH status with drug response in the early- and late-step drug-resistant clones. Late-step drug resistant clones revealed LOH in one allele for wt b-tubulin in addition to a β-tubulin mutation in the other allele leading to increased levels of drug resistance, while the early-step clones that contained both a wt and a mutant b-tubulin allele were considerably less drug resistant. The LOH and functional assays revealed cell response that was proportional to the tubulin gene and heterozygosity status. Acquired tubulin mutations in conjunction with LOH for the wt tubulin resulted in a highly resistant phenotype, revealing a new mechanism for taxane resistance.

ABCChips as New Diagnostic Tool to Monitoring Multidrug Resistance in Human Tumor Cells to Chemotherapeutics Agents by Expression Profiling of ATP-Binding Cassette Transporter Genes.

A major problem in the treatment of tumors represents the development of resistance to chemotherapy. Many mechanisms are responsible for the failure of treatment, the main one being the activation of the ABC transporters. In the present investigation, we developed a low density DNA microarray which contains 38 ABC transporter genes. This tool has been validated with three different characterized multidrug-resistant sublines (CEM/ADR5000, HL60/AR, MCF7-CH1000) and their corresponding drug-sensitive parental cell lines (CCRF-CEM, HL60, MCF7). The multidrug-resistant cell lines used are known to overexpress either the MDR1, MRP1, or BCRP genes. Interestingly, we found not only the overexpression of these genes but also of other ABC transporter genes by using low density microarrays. These results were corroborated by quantitative real time RT-PCR. The microarray allowed the determination of the expression profile of ABC transporters in a single hybridization experiment and may, hence harbor the potential as diagnostic tool to detect drug resistance in the clinic.

Alteration in copy numbers of genes as a mechanism for acquired drug resistance.

Chemoresistance is a major obstacle for successful treatment of cancer. To identify regions of the genome associated with acquired resistance to therapeutic drugs, we conducted molecular cytogenetic analyses of 23 cancer-cell lines, each resistant to either camptothecin, cisplatin, etoposide (VP-16), Adriamycin, or 1-beta-D-arabinofuranosylcytosine, although the parental tumor lines were not. Subtractive comparative genomic hybridization studies revealed regions of gain or loss in DNA-copy numbers that were characteristic of drug-resistant cell lines; i.e., differences from their drug-sensitive parental cell lines. Thirteen ATP-binding cassette (ABC) transporter genes [ABCA3, ABCB1 (MDR1), ABCB6, ABCB8, ABCB10, ABCB11, ABCC1 (MRP1), ABCC4, ABCC9, ABCD3, ABCD4, ABCE1, and ABCF2] were amplified among 19 of the resistant cell lines examined. Three genes encoding antiapoptotic BCL-2 proteins (BCL2L2, MCL1, and BCL2L10) were also amplified and consequently overexpressed in three of the derivative lines. Down-regulation of BCL2L2 with an antisense oligonucleotide sensitized a VP-16 resistant ovarian-cancer cell line (SKOV3/VP) to VP-16. A decrease in copy numbers of genes encoding deoxycytidine kinase, DNA topoisomerase I, and DNA topoisomerase II alpha reduced their expression levels in one cytosine arabinoside-resistant line, two of three camptothecin-resistant lines, and two of five VP-16-resistant cell lines, respectively. Our results indicated that changes in DNA-copy numbers of the genes mentioned can activate or down-regulate them in drug-resistant cell lines, and that such genomic alterations might be implicated in acquired chemoresistance.

Preferential expression of a mutant allele of the amplified MDR1 (ABCB1) gene in drug-resistant variants of a human sarcoma.

Activation of the MDR1 (ABCB1) gene is a common event conferring multidrug resistance (MDR) in human cancers. We investigated MDR1 activation in MDR variants of a human sarcoma line, some of which express a mutant MDR1, which facilitated the study of allelic gene expression. Structural alterations of MDR1, gene copy numbers, and allelic expression were analyzed by cytogenetic karyotyping, oligonucleotide hybridization, Southern blotting, polymerase chain reaction, and DNA heteroduplex assays. Both chromosome 7 alterations and several cytogenetic changes involving the 7q21 locus are associated with the development of MDR in these sarcoma cells. Multistep-selected cells and their revertants contain three- to six-fold MDR1 gene amplification compared with that of the drug-sensitive parental cell line MES-SA and single-step doxorubicin-selected mutants. MDR1 gene amplification precedes the emergence of a mutant allele in cells that were coselected with doxorubicin and a cyclosporin inhibitor of P-glycoprotein (P-gp). Allele-specific oligonucleotide hybridization showed that the endogenous mutant allele was present as a single copy, with multiple copies of the normal allele. Reselection of revertant cells with doxorubicin in either the presence or the absence of the P-gp inhibitor resulted in exclusive reexpression of the mutant MDR1 allele, regardless of the presence of multiple wild-type MDR1 alleles. These data provide new insights into how multiple alleles are regulated in the amplicon of drug-resistant cancer cells and indicate that increased expression of an amplified gene can result from selective transcription of a single mutant allele of the gene.

Enhanced expression of human ABC-transporter tap is associated with cellular resistance to mitoxantrone

Multidrug resistance (MDR) phenotypes have been associated with the overexpression of various members of the superfamily of ATP binding cassette (ABC) transporters. Here we demonstrate that a member of the ABC-transporter family, the heterodimer ‘transporter associated with antigen processing’ (TAP), physiologically involved in major histocompatibility complex class I-restricted antigen presentation, is significantly overexpressed in the human gastric carcinoma cell line EPG85-257RNOV exhibiting a mitoxantrone-resistant phenotype. This tumor cell line shows an atypical MDR phenotype in the absence of ‘P-glycoprotein’ or ‘MDR-associated protein’ overexpression but with an enforced ‘breast cancer resistance protein’ expression level. Transfection of both TAP subunits encoding cDNA molecules, TAP1 and TAP2, into the drug-sensitive parental gastric carcinoma cell line EPG85-257P conferred a 3.3-fold resistance to mitoxantrone but not to alternative anti-neoplastic agents. Furthermore, cell clones transfected with both, but not singularly expressed TAP1 or TAP2, reduced cellular mitoxantrone accumulation. Taken together, the data suggest that the heterodimeric TAP complex possesses characteristics of a xenobiotic transporter and that the TAP dimer contributes to the atypical MDR phenotype of human cancer cells.

Emergence of cisplatin-resistant cells from the OVCAR-3 ovarian carcinoma cell line with p53 mutations, altered tumorigenicity, and increased apoptotic sensitivity to p53 gene replacement.

Resistance to chemotherapy commonly compromises the treatment of many advanced cancers. Evidence suggests a correlation between chemoresistance and more aggressive tumor growth, possibly through accumulation of additional genetic defects in drug-treated or resistant cells. To study this process in a human ovarian cancer model, we examined OVCAR-3 cells for acute sensitivity to cisplatin (cDDP) and subsequent emergence of drug-resistant clones following chronic cDDP exposure. Clonal cells (OVCAR-3/C-1) that displayed 20-fold reduced sensitivity to cisplatin but retained equivalent sensitivity to paclitaxel, as compared with the parental population, were isolated. The cDDP-resistant clone had growth kinetics similar to those of parental population, but when transplanted into the peritoneal cavity of nude mice, they acquired the ability to grow with the development of both ascites and solid tumor masses; such growth was not detectable after transplantation of the drug-sensitive parental cell line. C-1 cells had a p53 gene mutation (codon 266) that was not detected in the parental OVCAR-3 cell line, and infection of C-1 cells with p53-adenovirus (rAd-p53) caused greater apoptosis and gene transduction than that observed in the similarly infected parental population. rAd-p53 induced high levels of p21WAF1, p27Kip1, activated caspase 3 and apoptosis in C-1 cells, without causing major changes in bax or bcl-XL levels. Together, the results suggest that alterations in tumor growth and gene mutations characterize cDDP-resistance in OVCAR-3 cells, and viral replacement of one of these defective genes (p53) may provide an effective treatment for elimination of drug-resistant cells.