Resistant Human Cancer Research Articles

Synergistic Antitumor Effect of Triptolide and Cisplatin in Cisplatin Resistant Human Bladder Cancer Cells

We examined the synergistic antitumor effects of cisplatin combined with triptolide in cisplatin resistant T24R2 bladder cancer cells and investigated possible molecular mechanisms. T24R2 cells were treated with cisplatin and/or triptolide. Tumor cell proliferation was determined using cell counting and clonogenic assays. The combination index of the synergism between cisplatin and triptolide was calculated. The cell cycle phase and apoptosis rate were determined by flow cytometry. Apoptosis and cell cycle related protein expression were analyzed by Western blot. The synergistic cytotoxicity effect of cisplatin and triptolide combination treatment was greater than the cytotoxic effect of cisplatin or triptolide alone. Combination treatment also induced cell cycle arrest via cyclin D1 and E1 expression. Apoptosis induced by combination treatment was accompanied by increased expression of caspase-3, 8 and 9, PARP and cytochrome c. Our results suggest that triptolide synergistically enhanced the antitumor effect of cisplatin in cisplatin resistant human bladder cancer cells. Cisplatin and triptolide combination treatment may be effective for advanced bladder cancer.

POM analyses of anti-kinase activity of thirteen peptide alkaloids extracted from Zizyphus species

In continuing our programme aimed to use Petra, Osiris and Molinspiration (POM) analyses to search for potent drugs against multidrug-resistant pathogens (bacteria and fungi), a series of thirteen peptide alkaloids from Zizyphus species (PAZ) were POM analysed and evaluated for their calmodulin-dependent protein kinase-II (CDPK) inhibition. Results showed that lipophilicity and presence of (NH-CO) pharmacophore site are the major factors that governed the orientation in determining anti-Kinase-II activity. Furthermore, it was also found that some of the POM analysed PAZ have a closed pharmacophore sites which might be responsible for low bioactivity. To confirm the electronic, steric and hydrophobic requirements for future modifications, we have also carried out receptor based electrostatic analysis. Therefore, we conclude that POM analyses may prove to be a suitable method to correlate structural features of PAZ with their promising anti-Kinase-II activity and may contribute to the development of novel rigid natural analogs of most bioactive hits 13 (IC50 = 2.9 µM) against drug resistant human cancers.

Structurally diverse MDM2-p53 antagonists act as modulators of MDR-1 function in neuroblastoma.

Background:A frequent mechanism of acquired multidrug resistance in human cancers is overexpression of ATP-binding cassette transporters such as the Multi-Drug Resistance Protein 1 (MDR-1). Nutlin-3, an MDM2–p53 antagonist, has previously been reported to be a competitive MDR-1 inhibitor.Methods:This study assessed whether the structurally diverse MDM2–p53 antagonists, MI-63, NDD0005, and RG7388 are also able to modulate MDR-1 function, particularly in p53 mutant neuroblastoma cells, using XTT-based cell viability assays, western blotting, and liquid chromatography–mass spectrometry analysis.Results:Verapamil and the MDM2–p53 antagonists potentiated vincristine-mediated growth inhibition in a concentration-dependent manner when used in combination with high MDR-1-expressing p53 mutant neuroblastoma cell lines at concentrations that did not affect the viability of cells when given alone. Liquid chromatography–mass spectrometry analyses showed that verapamil, Nutlin-3, MI-63 and NDD0005, but not RG7388, led to increased intracellular levels of vincristine in high MDR-1-expressing cell lines.Conclusions:These results show that in addition to Nutlin-3, other structurally unrelated MDM2–p53 antagonists can also act as MDR-1 inhibitors and reverse MDR-1-mediated multidrug resistance in neuroblastoma cell lines in a p53-independent manner. These findings are important for future clinical trial design with MDM2–p53 antagonists when used in combination with agents that are MDR-1 substrates.

Kinetic validation of the models for P-glycoprotein ATP hydrolysis and vanadate-induced trapping. Proposal for additional steps.

P-Glycoprotein, a member of the ATP-binding cassette (ABC) superfamily, is a multidrug transporter responsible for cellular efflux of hundreds of structurally unrelated compounds, including natural products, many clinically used drugs and anti-cancer agents. Expression of P-glycoprotein has been linked to multidrug resistance in human cancers. ABC transporters are driven by ATP hydrolysis at their two cytoplasmic nucleotide-binding domains, which interact to form a closed ATP-bound sandwich dimer. Intimate knowledge of the catalytic cycle of these proteins is clearly essential for understanding their mechanism of action. P-Glycoprotein has been proposed to hydrolyse ATP by an alternating mechanism, for which there is substantial experimental evidence, including inhibition of catalytic activity by trapping of ortho-vanadate at one nucleotide-binding domain, and the observation of an asymmetric occluded state. Despite many studies of P-glycoprotein ATPase activity over the past 20 years, no comprehensive kinetic analysis has yet been carried out, and some puzzling features of its behaviour remain unexplained. In this work, we have built several progressively more complex kinetic models, and then carried out simulations and detailed analysis, to test the validity of the proposed reaction pathway employed by P-glycoprotein for ATP hydrolysis. To establish kinetic parameters for the catalytic cycle, we made use of the large amount of published data on ATP hydrolysis by hamster P-glycoprotein, both purified and in membrane vesicles. The proposed kinetic scheme(s) include a high affinity priming reaction for binding of the first ATP molecule, and an independent pathway for ADP binding outside the main catalytic cycle. They can reproduce to varying degrees the observed behavior of the protein's ATPase activity and its inhibition by ortho-vanadate. The results provide new insights into the mode of action of P-glycoprotein, and some hypotheses about the nature of the occluded nucleotide-bound state.

Tetrandrine and fangchinoline, bisbenzylisoquinoline alkaloids from Stephania tetrandra can reverse multidrug resistance by inhibiting P-glycoprotein activity in multidrug resistant human cancer cells

The overexpression of ABC transporters is a common reason for multidrug resistance (MDR) in cancer cells. In this study, we found that the isoquinoline alkaloids tetrandrine and fangchinoline from Stephania tetrandra showed a significant synergistic cytotoxic effect in MDR Caco-2 and CEM/ADR5000 cancer cells in combination with doxorubicin, a common cancer chemotherapeutic agent. Furthermore, tetrandrine and fangchinoline increased the intracellular accumulation of the fluorescent P-glycoprotein (P-gp) substrate rhodamine 123 (Rho123) and inhibited its efflux in Caco-2 and CEM/ADR5000 cells. In addition, tetrandrine and fangchinoline significantly reduced P-gp expression in a concentration-dependent manner. These results suggest that tetrandrine and fangchinoline can reverse MDR by increasing the intracellular concentration of anticancer drugs, and thus they could serve as a lead for developing new drugs to overcome P-gp mediated drug resistance in clinic cancer therapy.

MP39-05 SYNERGISTIC ANTITUMOR EFFECT OF SAHA AND CISPLATIN IN CISPLATIN RESISTANT BLADDER CANCER CELL LINE

You have accessJournal of UrologyBladder Cancer: Basic Research IV1 Apr 2014MP39-05 SYNERGISTIC ANTITUMOR EFFECT OF SAHA AND CISPLATIN IN CISPLATIN RESISTANT BLADDER CANCER CELL LINE Ha Rim Kook, Young Ju Lee, Jae Seung Yeon, Han Sol Lee, Choong Hee Noh, Kwang Mo Kim, Ki Beom Kim, Jung Keun Lee, Sangchul Lee, Seong Jin Jeong, Sung Kyu Hong, Seok-Soo Byun, Eunsik Lee, and Sang Eun Lee Ha Rim KookHa Rim Kook More articles by this author , Young Ju LeeYoung Ju Lee More articles by this author , Jae Seung YeonJae Seung Yeon More articles by this author , Han Sol LeeHan Sol Lee More articles by this author , Choong Hee NohChoong Hee Noh More articles by this author , Kwang Mo KimKwang Mo Kim More articles by this author , Ki Beom KimKi Beom Kim More articles by this author , Jung Keun LeeJung Keun Lee More articles by this author , Sangchul LeeSangchul Lee More articles by this author , Seong Jin JeongSeong Jin Jeong More articles by this author , Sung Kyu HongSung Kyu Hong More articles by this author , Seok-Soo ByunSeok-Soo Byun More articles by this author , Eunsik LeeEunsik Lee More articles by this author , and Sang Eun LeeSang Eun Lee More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2014.02.1320AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Cisplatin-based chemotherapy remains first-line treatment for advanced bladder cancer. No standard chemotherapeutic agent has been established for patients with cisplatin resistant bladder cancer. We investigated the synergistic antitumor effect of of suberoylanilide hydroxamic acid (SAHA) and cisplatin in cisplatin resistant bladder cancer cells (T24R2). METHODS The cisplatin resistant human bladder cancer cell line (T24R2) was treated with cisplatin and/or SAHA. Tumor cell proliferation was assessed by cell counting kit-8 assay and clonogenic assay. Synergism was determined by combination index. Changes in cell cycle were determined by flow cytometry. Expression of caspase-3, 8 and 9, PARP, cytochrome c, p21, Bcl-2, Bad, p27, cyclin A, cyclin D1, and cyclin E were analyzed by Western blotting. RESULTS Synergistic antitumor effect between cisplatin and SAHA was observed by cell counting kit-8 assay, clonogenic assay and confirmed with combination index less than 1.0. The underlying mechanism could be synergistic cell cycle arrest, activation of apoptotic pathway including capase-3, -8, -9 and fragmented PARP or decreased expression in anti-apoptotic Bcl-2 and increased expression in pro-apoptotic BAD. CONCLUSIONS SAHA may synergistically enhancethe antitumor effect of cisplatin and resensitize cisplatin resistant bladder cancer cells. These findings suggest the potential use of SAHA as a combination agent to enhance the antitumor effect of cisplatin in patients with advanced bladder cancer. © 2014FiguresReferencesRelatedDetails Volume 191Issue 4SApril 2014Page: e427-e428 Advertisement Copyright & Permissions© 2014MetricsAuthor Information Ha Rim Kook More articles by this author Young Ju Lee More articles by this author Jae Seung Yeon More articles by this author Han Sol Lee More articles by this author Choong Hee Noh More articles by this author Kwang Mo Kim More articles by this author Ki Beom Kim More articles by this author Jung Keun Lee More articles by this author Sangchul Lee More articles by this author Seong Jin Jeong More articles by this author Sung Kyu Hong More articles by this author Seok-Soo Byun More articles by this author Eunsik Lee More articles by this author Sang Eun Lee More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Honokiol Enhances Paclitaxel Efficacy in Multi-Drug Resistant Human Cancer Model through the Induction of Apoptosis

Resistance to chemotherapy remains a major obstacle in cancer therapy. This study aimed to evaluate the molecular mechanism and efficacy of honokiol in inducing apoptosis and enhancing paclitaxel chemotherapy in pre-clinical multi-drug resistant (MDR) cancer models, including lineage-derived human MDR (KB-8-5, KB-C1, KB-V1) and their parental drug sensitive KB-3-1 cancer cell lines. In vitro analyses demonstrated that honokiol effectively inhibited proliferation in KB-3-1 cells and the MDR derivatives (IC50 ranging 3.35±0.13 µg/ml to 2.77±0.22 µg/ml), despite their significant differences in response to paclitaxel (IC50 ranging 1.66±0.09 ng/ml to 6560.9±439.52 ng/ml). Honokiol induced mitochondria-dependent and death receptor-mediated apoptosis in MDR KB cells, which was associated with inhibition of EGFR-STAT3 signaling and downregulation of STAT3 target genes. Combined treatment with honokiol and paclitaxel synergistically augmented cytotoxicity in MDR KB cells, compared with treatment with either agent alone in vitro. Importantly, the combined treatment significantly inhibited in vivo growth of KB-8-5 tumors in a subcutaneous model. Tumor tissues from the combination group displayed a significant inhibition of Ki-67 expression and an increase in TUNEL-positive cells compared with the control group. These results suggest that targeting multidrug resistance using honokiol in combination with chemotherapy drugs may provide novel therapeutic opportunities.

Extracellular Disposal of Tumor-Suppressor miRs-145 and -34a via Microvesicles and 5-FU Resistance of Human Colon Cancer Cells

The dysregulation of microRNA (miRNA) expression causes various kinds of diseases. Especially, alterations in miRNA expression levels are frequently observed in human tumor cells and are associated with cancer pathogenesis. Earlier we established Fluorouracil (5-FU)-resistant human colon cancer DLD-1 cells (DLD-1/5FU) from parental 5-FU- sensitive DLD-1 cells. In the present study, we examined the expression of miRNA in each cell line and in its extracellular microvesicles (MVs) before and after treatment with 5-FU. The nascent RNAs of anti-oncogenic miR-34a and -145 labeled with EU in both cells were proved to be transferred into MVs in both cell lines. The levels of miR-34a and -145 in the cells and in their MVs were not largely different in the two cell lines, and a substantial amount of both miRNAs was secreted by both cell lines even in the steady-state condition. The exposure of both cell lines to 5-FU significantly increased the intracellular levels of miR-145 and miR-34a in the 5-FU-sensitive DLD-1 cells, whereas the level of neither miR was elevated in the DLD-1/5FU cells. Interestingly, the amount of miR-145 detected in the small MVs shed into the medium of the parental cells was reduced after the treatment with 5-FU. On the other hand, the intracellular expression of miR-34a in the DLD-1/5FU cells was down-regulated compared with that in the parental DLD-1 cells even in the steady-state condition. As to the miR-34a secreted into MVs, the increase in the level in DLD-1/5FU cells was greater than that in the parental DLD-1 cells after the treatment with 5-FU. Thus, the intra- and extracellular miR-145 and -34a were closely associated with 5-FU resistance, and the resistance was in part due to the enhanced secretion of miR-145 and -34a via MVs, resulting in low intracellular levels of both miRNAs.

Carbon-Ion Beams Efficiently Induce Cell Killing in X-Ray Resistant Human Squamous Tongue Cancer Cells

In order to see whether carbon ion (C-ion) beams have a biological advantage over X-rays, studies were designed to examine the effects of C-ion beams on radiosensitivity in X-ray resistant cells. Clinically relevant X-ray resistant SAS-R cells derived from human tongue cancer SAS cells were used. The cells were exposed to X-rays or Spread-Out Bragg peak (SOBP) beam C-ions. Cell survival was measured using a modified high-density survival assay. Cell survival signaling and cell death signaling were analyzed using flow cytometry. The cells were labeled with putative cancer stem cell markers such as CD44 and CD326. SAS-R cells were 1.6 times more radioresistant than SAS cells after exposure to X-rays. Cell survival was similar in each cell line after exposure to C-ion beams. SAS-R cells displayed enhanced cell survival signaling when compared to SAS cells under normal conditions. On the other hand, the phosphorylation of AKT-related proteins decreased and polycaspase activities were enhanced when cells were irradiated with C-ion beams in both cell lines. More CD44 and CD326 positive cells were seen in SAS-R cells than in SAS cells. Moreover, the marker positive cell numbers significantly decreased after exposure to C-ion beams when compared to X-rays at iso-survival doses in SAS-R cells. C-ion beams efficiently induced cell killing in X-ray resistant cells which displayed activated cell survival signaling and contained more numerous cancer stem-like cells.

Small-Molecule IAP Antagonists Sensitize Cancer Cells to TRAIL-Induced Apoptosis: Roles of XIAP and cIAPs

TNF-related apoptosis-inducing ligand (TRAIL) is a promising anticancer agent because it shows apoptosis-inducing activity in transformed, but not in normal, cells. As with most anticancer agents, however, its clinical use is restricted by either inherent or acquired resistance by cancer cells. We demonstrate here that small-molecule SMAC mimetics that antagonize the inhibitor of apoptosis proteins (IAP) potently sensitize previously resistant human cancer cell lines, but not normal cells, to TRAIL-induced apoptosis, and that they do so in a caspase-8-dependent manner. We further show that the compounds have no cytotoxicity as single agents. Also, we demonstrate that several IAP family members likely participate in the modulation of cellular sensitivity to TRAIL. Finally, we note that the compounds that sensitize cancer cells to TRAIL are the most efficacious in binding to X-linked IAP, and in inducing cellular-IAP (cIAP)-1 and cIAP-2 degradation. Our studies thus describe valuable compounds that allow elucidation of the signaling events occurring in TRAIL resistance, and demonstrate that these agents act as potent TRAIL-sensitizing agents in a variety of cancer cell lines.

Abstract C201: Transposon activation mutagenesis as a screening tool for identifying resistance to cancer therapeutics.

Abstract The development of resistance to chemotherapies represents a significant barrier to successful cancer treatment. Resistance mechanisms are complex, can involve diverse and often unexpected cellular processes, and can vary with both the underlying genetic lesion and the origin or type of tumor. For these reasons developing experimental strategies that could be used to understand, identify and predict mechanisms of resistance in different malignant cells would be a major advance. Here we describe a novel gain-of-function forward genetic approach for identifying potential mechanisms of resistance. This system uses a modified piggyBac transposon to generate libraries of mutagenized cells, each containing transposon insertions that randomly activates nearby gene expression. By combining this with high-throughput sequencing, we have performed genome-wide screens for paclitaxel resistance mechanism in a variety of cancer cell lines. Validating this approach, we identified the multidrug transporter ABCB1 as the major contributor to resistance. In addition, we identified several potential novel resistance genes. Therefore we have developed a powerful new pipeline tool to systematically discover common and tumor specific pathways of resistance in human cancers. This cost-effective approach can be readily applied to different cell lines. Moreover, its ability to identify gene interactions and to probe complex genetic context provides a significant advantage over RNAi- or cDNA-based screens. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C201. Citation Format: Li Chen, Anahita Dastur, Cyril Benes. Transposon activation mutagenesis as a screening tool for identifying resistance to cancer therapeutics. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C201.

Convergent RANK- and c-Met-Mediated Signaling Components Predict Survival of Patients with Prostate Cancer: An Interracial Comparative Study

We reported (PLoS One 6 (12):e28670, 2011) that the activation of c-Met signaling in RANKL-overexpressing bone metastatic LNCaP cell and xenograft models increased expression of RANK, RANKL, c-Met, and phosphorylated c-Met, and mediated downstream signaling. We confirmed the significance of the RANK-mediated signaling network in castration resistant clinical human prostate cancer (PC) tissues. In this report, we used a multispectral quantum dot labeling technique to label six RANK and c-Met convergent signaling pathway mediators simultaneously in formalin fixed paraffin embedded (FFPE) tissue specimens, quantify the intensity of each expression at the sub-cellular level, and investigated their potential utility as predictors of patient survival in Caucasian-American, African-American and Chinese men. We found that RANKL and neuropilin-1 (NRP-1) expression predicts survival of Caucasian-Americans with PC. A Gleason score ≥8 combined with nuclear p-c-Met expression predicts survival in African-American PC patients. Neuropilin-1, p-NF-κB p65 and VEGF are predictors for the overall survival of Chinese men with PC. These results collectively support interracial differences in cell signaling networks that can predict the survival of PC patients.

Uncovering a key to the process of metastasis in human cancers: a review of critical regulators of anoikis

Anoikis ('homelessness' in Greek) is a form of apoptosis following the detachment of cells from the appropriate extracellular matrix (Chiarugi and Giannoni in Biochem Pharmacol 76:1352-1364, 2008). Resistance to anoikis is a critical mediator of metastasis in cancer by enabling cancer cells to survive during invasion and transport in the blood and lymph. Numerous regulators and mechanisms of anoikis in human cancer have been proposed to date. Consequently, the identification of key regulators of anoikis that can be targeted to at least partially restore anoikis sensitivity in cancer cells is important in the development of therapies to treat metastatic cancer. A literature search focusing on the regulators of anoikis in human cancer was performed on the Medline, Embase and Scopus databases. Mcl-1, Cav-1, Bcl-(xL), cFLIP, 14-3-3ζ and Bit1 appear to regulate anoikis in human cancer by participating in the intrinsic apoptotic pathway, extrinsic apoptotic pathway or caspase-independent pathways. Mcl-1, Cav-1, Bcl-(xL), cFLIP and 14-3-3ζ are suppressors of anoikis, and their upregulation confers anoikis resistance to cancer cells. Bit1 is a promoter of anoikis and is downregulated to confer anoikis resistance in metastatic cancer. Anoikis is a complex process involving the crosstalk between different signalling pathways. The dysregulated expression of key regulators of anoikis that participate in these signalling pathways promotes anoikis resistance in human cancer. These regulators of anoikis might therefore be the targets for developing therapies to overcome anoikis resistance in metastatic cancer.

Multimodal Interaction with BCL-2 Family Proteins Underlies the Proapoptotic Activity of PUMA BH3

PUMA is a proapoptotic BCL-2 family member that drives the apoptotic response to a diversity of cellular insults. Deciphering the spectrum of PUMA interactions that confer its context-dependent proapoptotic properties remains a high priority goal. Here, we report the synthesis of PUMA SAHBs, structurally stabilized PUMA BH3 helices that, in addition to broadly targeting antiapoptotic proteins, directly bind to proapoptotic BAX. NMR, photocrosslinking, and biochemical analyses revealed that PUMA SAHBs engage anα1/α6 trigger site on BAX to initiate its functional activation. We further demonstrated that a cell-permeable PUMA SAHB analog induces apoptosis in neuroblastoma cells and, like expressed PUMA protein, engages BCL-2, MCL-1, and BAX. Thus, we find that PUMA BH3 is a dual antiapoptotic inhibitor and proapoptotic direct activator, and its mimetics may serve as effective pharmacologic triggers of apoptosis in resistant human cancers.

Pt(II) complexes with (N,N′) or (C,N,E)− (E = N,S) ligands: Cytotoxic studies, effect on DNA tertiary structure and structure–activity relationships

The cytotoxic activity of two series of platinum(II) complexes containing the polyfunctional imines R1–CHN–R2 [R1=phenyl or ferrocenyl unit and R2=(CH2)n–CH2–NMe2 where n=1 or 2) (1 and 2) or C6H4-2-SMe (3)] acting as a bidentate (N,N′) (4–7) or terdentate [C(phenyl or ferrocenyl),N,N′]− (8–10) or [C(ferrocenyl),N,S]− ligand (11) in front of A549 lung, MDA-MB231 breast and HCT116 colon human adenocarcinoma cell lines is reported. The results reveal that most of the platinum(II) complexes are active against the three assayed lines and compounds 6, 7 and the platinacycles 10 and 11 exhibit a remarkable antiproliferative activity, even greater than cisplatin itself, in the cisplatin resistant HCT116 human cancer cell line. Electrophoretic DNA migration studies showed that most of them modify the DNA tertiary structure in a similar way as the reference cisplatin. Solution studies of a selection of the most relevant complexes have also been performed in order to test: (a) their stability in the aqueous biological medium and/or the formation of biologically active species and (b) their proclivity to react with 9-methylguanine (9-MeG), as a model nucleobase. Computational studies at DFT level have also been performed in order to explain the different solution behaviour of the complexes and their proclivity to react with the nucleobase.

Intercellular Transfer of P-Glycoprotein from the Drug Resistant Human Bladder Cancer Cell Line BIU-87 Does Not Require Cell-to-Cell Contact

The efflux activity of transmembrane P-glycoprotein prevents various therapeutic drugs from reaching lethal concentrations in cancer cells, resulting in multidrug resistance. We investigated whether drug resistant bladder cancer cells could transfer functional P-glycoprotein to sensitive parental cells. Drug sensitive BIU-87 bladder cancer cells were co-cultured for 48 hours with BIU-87/ADM, a doxorubicin resistant derivative of the same cell line, in a Transwell® system that prevented cell-to-cell contact. The presence of P-glycoprotein in recipient cell membranes was established using fluorescein isothiocyanate, laser scanning confocal microscopy and Western blot. P-glycoprotein mRNA levels were compared between cell types. Rhodamine 123 efflux assay was done to confirm that P-glycoprotein was biologically active. The amount of P-glycoprotein protein in BIU-87 cells co-cultured with BIU-87/ADM was significantly higher than in BIU-87 cells (0.44 vs 0.25) and BIU-87/H33342 cells (0.44 vs 0.26, each p <0.001), indicating P-glycoprotein transfer. P-glycoprotein mRNA expression was significantly higher in BIU-87/ADM cells than in co-cultured BIU-87 cells (1.28 vs 0.30), BIU-87/H33342 (0.28) and BIU-87 cells (0.25, each p <0.001), ruling out a genetic mechanism. After 30 minutes of efflux, rhodamine 123 fluorescence intensity was significantly lower in BIU-87/ADM cells (5.55 vs 51.45, p = 0.004) and co-cultured BIU-87 cells than in BIU-87 cells (14.22 vs 51.45, p <0.001), indicating that P-glycoprotein was functional. Bladder cancer cells can acquire functional P-glycoprotein through a nongenetic mechanism that does not require direct cell contact. This mechanism is consistent with a microparticle mediated process.

Abstract 205: SIAH2 modulates focal adhesion and cell junction proteins in response to oncogenic ERBB/K-RAS activation to promote cancer cell dissemination, invasion and metastasis.

Abstract Oncogenic EGFR/HER2/K-RAS activation is often associated with reduced focal adhesion, altered cell junctions, increased cell motility and enhanced cancer cell dissemination, aggressive tumor growth and metastasis, cancer relapse and drug resistance in human cancer. Deciphering novel mechanisms that promote invasion/metastasis in human cancer cells in response to oncogenic ERBB/K-RAS pathway activation and finding novel means to inhibit tumor invasion and cancer metastasis remain a paramount important goal in cancer biology. SIAH, which is the human homologue of a highly conserved family of RING-domain E3 ubiquitin ligases, is a key downstream signaling component required for proper ERBB/K-RAS signal transduction. We have shown that SIAH2-insufficiency impedes proper K-RAS signal transduction, thereby obstructing tumorigenesis and metastasis. While this suggests that SIAH2 E3 ligase is critical for K-RAS-dependent oncogenesis, the exact mechanism of SIAH2 function downstream of ERBB/K-RAS signaling pathway in driving cancer cell dissemination, tumor growth and metastasis has remained unknown. In this study, we affinity purified and isolated SIAH2-protein complex from human cancer cells with oncogenic K-RAS activation. Several focal adhesion and cell junction proteins were proteomically identified as SIAH-interacting proteins by Mass Spectrometry. Bi-directional co-immunoprecipitation (co-IP) and in vivo ubiquitination assays were performed to verify and validate that these newly identified SIAH-binding partners are indeed bona fide new SIAH substrates that play important roles in cancer cell dissemination, invasion and metastasis. Our immunofluorescent staining and confocal results showed that focal adhesion, cell junction and cell motility are severely compromised in the SIAH-deficient cancer cells while in contrast, the prominent focal adhesion sites are present in the SIAH-proficient cancer cells. Importantly, ectopic expression of these focal adhesion and cell junction proteins can successfully rescue SIAH2-deficient cancer cells from the pronounced defects in focal adhesion, adherens junction, cell motility, cell death and tumor growth. Hence, these focal adhesion and cell junction proteins may represent a new class of SIAH2 substrates. Their increased expression patterns have been confirmed and verified in human pancreatic cancer and breast tumor biospecimens. We may have uncovered a new molecular mechanism of SIAH enzymatic function in human cancer cells. The discovery that SIAH2 directly modulates focal adhesion, cell junction integrity and motility may provide a novel molecular mechanism to explain the well-documented changes in cancer cell dissemination, aggressive tumor invasion and systemic metastasis in response to oncogenic EGFR/HER2/K-RAS pathway activation in human cancer. Citation Format: Minglei Bian, Yang Liao, Rebecca L. Schmidt, Zandra E. Walton, Amy H. Tang. SIAH2 modulates focal adhesion and cell junction proteins in response to oncogenic ERBB/K-RAS activation to promote cancer cell dissemination, invasion and metastasis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 205. doi:10.1158/1538-7445.AM2013-205

Abstract 990: Efficacy of various chemotherapeutic agents on 5-FU-resistant human gastric cancer cell lines and their mechanisms.

Abstract Purpose: Four 5-fluorouracil (5-FU)-resistant human gastric cancer cell lines were established by the long-term exposure of parent cell lines (MKN45, MKN-74, KATO-III, and NCI-N87) to 5-FU. The cytotoxicity and mechanisms of several chemotherapeutic agents were then investigated using 5-FU-resistant gastric cancer cell lines. Method: 5-FU-resistant cell lines were established by continuous exposure to escalating concentrations (1-5 μM) of 5-FU over a 1-year period. The sensitivities of the cell lines to chemotherapeutic agents (paclitaxel, docetaxel, cisplatin, oxaliplatin, epirubicin, and SN-38) were then evaluated using the WST-8 colorimetric assay. To elucidate the mechanisms of resistance, the gene expressions and copy numbers were measured using an Agilent Technologies whole human genome oligo DNA microarray and a CGH microarray. The protein and mRNA expressions of a target enzyme of 5-FU, thymidylate synthase (TS), were also investigated using western blotting and RT-PCR, respectively. Results: The IC50 ratios of the 5-FU-resistant cell lines were 2.6 to 15.6 times higher than those of the parent cell lines in vitro. MKN-45/5-FU showed cross-resistance to cisplatin and oxaliplatin and KATO-III/5-FU showed cross-resistance to SN-38, while MKN-74/5-FU showed collateral sensitivities to docetaxel, cisplatin, and oxaliplatin, and NCI-N87/5-FU showed collateral sensitivities to cisplatin, oxaliplatin, SN-38, and epirubicin. RT-PCR showed that TS mRNA expression was higher in the parent cell lines than in the 5-FU-resistant cell lines, and the TS protein expression level was also higher in all the resistant cell lines. For MKN-45/5-FU and KATO-III/5-FU, not only the mRNA expression level but also the TS gene copy number was increased. Interestingly, the expression of MAP3K15, which is an apoptosis-facilitating factor, increased in NCI-N87/5-FU, which showed collateral sensitivity to cisplatin, oxaliplatin, SN-38, and epirubicin. Conclusion: Analyzing the molecular mechanisms of 5-FU-resistant cell lines may be useful for the development of effective chemotherapy regimens for relapsed gastric cancer after 5-FU-based chemotherapy. Citation Format: Mamoru Nukatsuka, Takashi Kobunai, Ayako Nakamura, Kazuhiko Hayashi, Teiji Takechi. Efficacy of various chemotherapeutic agents on 5-FU-resistant human gastric cancer cell lines and their mechanisms. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 990. doi:10.1158/1538-7445.AM2013-990

Abstract 987: XCE853: A novel anticancer candidate preferentially killing drug-resistant human tumor cells.

Abstract Cancer drug pan-resistance is associated with patients having a disease progression despite receiving new treatments. Often resistance arises in two steps, initially, the tumor responds to the drug, but not all tumor cells are killed leading to a selection of tumor cells that do not respond to any current drug. The nature of the residual disease is linked to different conditions such the efflux pumps, the quiescent cells, the stem cells, the DNA repair deficiency or the EMT. Here, we have identified a family of synthetic compounds displaying a preferential antiproliferative activity on drug resistant human cancer cells. The lead compound XCE853 is 5 to 94 more potent on drug resistant cancer cell lines compared to their parental tumor cell lines (HL60/HL60-Doxorubicin; MCF7/MCF7-Doxorubicin; A2780/A2780-Cisplatin and A549/AA549-Taxotere). XCE853 induced a strong and irreversible cytolysis of MCF7-Adriamycin cells after a short exposure in vitro (250nM, 6 hours). The mechanism of action of XCE853 is independent of efflux pumps and involves an early high ROS production leading to cell death by autophagy. The antitumor activity of XCE853 is also observed using in vivo xenograft models of human NSCLC. Altogether, these data support further efforts on this drug candidate to better understand its molecular mechanism of action and the description of the most sensitive tumor types. Citation Format: Matthieu Gutmann, Jean-François Briand, Gregoire Pierre Prevost, Denis Carniato, Marc-Henry Pitty. XCE853: A novel anticancer candidate preferentially killing drug-resistant human tumor cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 987. doi:10.1158/1538-7445.AM2013-987

Abstract 3543: TOP2A is a therapeutic target for adrenocortical carcinoma .

Abstract Adrenocortical carcinoma (ACC) represents one of the most resistant human cancers to systemic chemotherapy and a major clinical challenge in the field of endocrine oncology. Recent molecular characterizations of ACC suggest new opportunities for targeted therapy. The current study aimed to evaluate expression and function of TOP2A in human adrenocortical neoplasm, and anticancer activity of agents that target TOP2A. TOP2A mRNA and protein expression levels were evaluated in 112 adrenocortical tissue samples. In vitro siRNA knockdown of TOP2A in ACC cell lines (NCI-H295R and SW13) was used to determine its effect on cellular proliferation, cell cycle, anchorage-independent growth and cellular invasion. We screened 14 TOP2A inhibitors for their anticancer activity in ACC cell lines. TOP2A mRNA expression was significantly higher in ACC than in benign and normal adrenocortical tissue samples (p &amp;lt; 0.001); likewise, TOP2A protein expression was significantly higher in ACC (p &amp;lt; 0.05). Knockdown of TOP2A gene expression decreased cell proliferation, anchorage-independent growth and invasion (p &amp;lt; 0.05). Aclarubicin had the most potent antiproliferative activity out of 11 active TOP2A inhibitors, which significantly decreased proliferation and tumor spheroid size in ACC cell lines (p&amp;lt; 0.05). Our data indicate that most TOP2A inhibitors are effective compounds for ACC, with aclarubicin having the best anticancer activity. Thus, TOP2A inhibitors should be tested in future clinical trials for patients with locally advanced and metastatic ACC. Citation Format: Meenu Jain, Lisa Zhang, Mei He, Ya-Qin Zhang, Naris Nilubol, Min Shen, Electron Kebebew. TOP2A is a therapeutic target for adrenocortical carcinoma . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3543. doi:10.1158/1538-7445.AM2013-3543