Drug-free Medium Research Articles

Abstract C102: Identify novel pathways conferring PARP inhibitor resistance in BRCA-associated breast and ovarian cancer.

Abstract Inhibitors of poly(ADP-ribose) polymerase (PARP) enzymes hold great promise in treating BRCA1/2 associated breast and ovarian cancers, which have profound homologous recombination (HR) defect. However, not all BRCA1/2 patients respond to PARP inhibitor treatment, and the majority of the responders eventually relapsed. Little is known about the resistance mechanisms that are clinical relevant. We recently developed a highly versatile insertional mutagenesis system, using transposons to insert a CMV promoter randomly into the genome and thereby activate nearby genes. We constructed genome-wide insertional libraries using both human and mouse BRCA1-deficient cancer cell lines. When these mutated cells were treated with the PARP inhibitor, resistant colonies formed at extremely low frequency. Resistant colonies were re-grown and maintained in drug-free medium for extended period of time and robust resistance was retained. Insertion sites for these colonies were then determined, many of which are near genes whose alteration in expression can up-regulate activities of MEK/ERK pathway or PI3K/AKT pathway. Next we hope to investigate the resistance mechanism. We hypothesize that resistance is mediated via decreased DNA damage, improved DNA repair, or increase in general survival pathway after PARP inhibitor treatment. We have access to tumor specimens from a recently-completed clinical trial using ABT-888 in combination with temozolomide in a BRCA1/2 carrier-enriched population. The expression level and mutational status of these genes will be surveyed and correlated with clinical response. Finally based on the insight gained from aforementioned mechanism study, we hope to find inhibitors that specifically target the resistance mechanism; by combining this inhibitor with PARP inhibitor, we could develop effective therapy to treat the subset of patients resistant to PARP inhibitor. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C102. Citation Format: Lei He, Li Chen, Saranya Ramakrishnan, Kristine Torres-Lockhart, Leif Ellisen. Identify novel pathways conferring PARP inhibitor resistance in BRCA-associated breast and ovarian cancer. [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 C102.

Increase in Resistance to Fluconazole and Itraconazole in Trichophyton rubrum Clinical Isolates by Sequential Passages In Vitro under Drug Pressure

Trichophyton rubrum, an anthropophilic dermatophyte fungus, is the predominant causative agent of superficial skin infections in human population. There are only scanty reports on drug susceptibility profiling of T. rubrum. Neither mechanisms for drug resistance development nor correlation between in vitro drug susceptibility and in vivo response to treatment is known for that species. In this study, changes in the in vitro susceptibilities to fluconazole (FLZ) and itraconazole (ITZ) among thirty T. rubrum clinical strains subjected to sequential passages in the presence or absence of the azoles were investigated. Each strain was passaged 12 times at 4-week intervals as three parallel cultures, maintained on a drug-free medium (1), and a medium containing FLZ (2) or ITZ (3) at subinhibitory concentrations. Susceptibility to FLZ and ITZ of the original strain and its 3 subcultures was determined by microdilution method. The MIC values of the two azoles remained unaltered for all T. rubrum strains tested, after 12 passages on a drug-free medium. Among the strains grown with FLZ, an increase in the MICs of FLZ and ITZ was noted in 17 (56.7 %) and 19 (63.3 %) strains, respectively. Increased MICs of ITZ and FLZ were demonstrated for 24 (80 %) and 20 (66.7 %) strains that were propagated with ITZ. The results indicate the capacity of T. rubrum to develop resistance toward the azoles after prolonged exposure to these drugs. Resistance of T. rubrum to azoles plays an important role in therapy failures and consequently contributes to persistence and chronicity of the infections.

Abstract 1621: Short-time exposure to high concentrations of sunitinib causes tumor cell death.

Abstract Background: Sunitinib, although initially developed as an antiangiogenic agent, has been shown to exert direct anti-tumor effects. Previously we demonstrated that sunitinib inhibits proliferation of various tumor cell lines in a dose-dependent manner. We here studied whether short exposure to high peak concentrations of sunitinib would induce tumor cell death. Methods: 786-O renal cancer cells were incubated with increasing concentrations of sunitinib (5, 10 and 20 μM) for various time intervals (1 hour, 3, 6, 9, 24, 48, 72 hours). At prespecified time points, sunitinib was removed and cells were subsequently allowed to grow in drug-free culture medium. Viability of these cells one week after short time sunitinib exposure and sensitivity of recovered cells after repetitive treatment with high sunitinib concentrations were determined by MTT assays. Flow cytometry with Propidium Iodide staining was employed to determine apoptosis. Results: Short-time exposure to high concentrations of sunitinib significantly induced cell death. Exposure to 20 μM of sunitinib for 9 hours provoked cell death in a manner identical to the prolonged exposure of 24, 48 or 72 hours. Flow cytometry confirmed the significant increase in cell death (60%) after 9-hour treatment with 20 μM sunitinib, compared to the untreated control (2%). Tumor cells that were repetitively treated with high concentrations of sunitinib retain their sensitivity to the drug identical to its parental cell line. Analysis of the key signaling pathways involved in sunitinib-induced cell death is ongoing. Conclusion: We here report that repetitive short-time exposure to high concentration sunitinib causes cell death without inducing resistance. Interestingly, single-dose sunitinib up to 300 mg was previously reported to be well tolerated by patients. Based on these findings, administration of high dose intermittent sunitinib may result in cytotoxic antitumor activity irrespective of angiogenesis inhibition. A phase I trial to test this hypothesis in patients with cancer will start shortly. Citation Format: Maria Rovithi, Dennis Poel, Jens Voortman, Mariette Labots, Henk Dekker, Kristy J. Gotink, Henk J. Broxterman, Henk M.W. Verheul. Short-time exposure to high concentrations of sunitinib causes tumor cell death. [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 1621. doi:10.1158/1538-7445.AM2013-1621

Abstract 5491: Marked augmentation of the polyglutamate derivatives as the basis for the enhanced activity of pralatrexate relative to methotrexate.

Abstract Pralatrexate (PDX) is a new-generation dihydrofolate reductase (DHFR) inhibitor with high affinity for the reduced folate carrier (RFC) and for folypolyglutamyl synthetase (FPGS), the enzyme that mediates the addition of glutamate moieties at the γ-carboxyl of folates and antifolates (Sirotnak et al., Cancer Chemother Pharmacol. 42:313, 1998). The polyglutamate derivatives are better accumulated and retained into the cells. Recently, PDX received FDA approval for the treatment of relapsed or refractory peripheral T-cell lymphoma. This report represents the first analysis of PDX cellular pharmacology using the tritiated derivative, comparing its properties with methotrexate (MTX) in HeLa cells. The influx Kt was determined at ∼0.5 μM in comparison to a MTX influx Ki of ∼3 μM. Consistent with RFC-targeting, [³H]PDX was a lesser substrate for the proton-coupled folate transporter (PCFT) than MTX. While the net uptake of [³H]MTX reached steady-state conditions within 30 min, net uptake of [³H]PDX continued to increase, exceeding the [³H]MTX level by a factor of 15, by 6 hrs. This difference in net uptake could be attributed to a marked increase in PDX polyglutamates. At 24 hrs, the PDX mono-, di- and triglutamate levels in the cells were 14%, 12%, and 70% of the total antifolate, respectively. When polyglutamation was suppressed, the subsequent intracellular [³H]PDX level was markedly decreased now exceeded the [³H]MTX level by a factor of only 3. The importance of PDX polyglutamation was reflected in the marked difference in growth inhibition relative to MTX when exposure to the drugs was transient. When cells were exposed continuously to PDX or MTX for six days the IC50 for the former was ∼ 1/25th the latter (7 nM vs 180 nM, respectively). However, when the exposure was limited to 6 hrs followed by growth in drug-free medium, the pharmacological advantage of PDX over MTX was markedly increased with an IC50 of 70 nM and 30 μM,respectively, now ∼ 1/450th that of MTX. Hence, in the first direct measurement of the transport and polyglutamation of PDX, the data confirm a much higher affinity for RFC relative to MTX along with a markedly greater rate of polyglutamation in cells. It is this difference in the extent of accumulation of these active polyglutamate derivatives that is the basis for the marked difference in the antitumor activities between these antifolates. The apparent low affinity of PDX for PCFT suggests that changes at the nitrogen-10 position are one determinant of the affinity of 4-amino antifolates for this transporter. Further, since PCFT is the mechanism by which folates and antifolates are absorbed across the proximal small intestine, the data suggests that the low affinity of PDX for this carrier would result in a low rate of re-absorption of this antifolate during its enterohepatic circulation, with a consequent higher level of fecal excretion, than for MTX. Citation Format: Michele Visentin, Ersin Selcuk Unal, Rongbao Zhao, I David Goldman. Marked augmentation of the polyglutamate derivatives as the basis for the enhanced activity of pralatrexate relative to methotrexate. [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 5491. doi:10.1158/1538-7445.AM2013-5491

Abstract 1650: Bone marrow microenvironment control of BCL-6 in acute lymphocytic leukemia .

Abstract BCL-6 is a proto-oncogene that encodes for a BTB/POZ-zinc-finger transcriptional repressor, classically described in the regulation of germinal center (GC) formation. Relevant to the current study BCL-6 endows GCB-cells with enhanced proliferative capacity by protecting proliferating cells from DNA damage induced apoptosis through repression of p53 (Phan 2004) and through prevention of senescence by increasing the proliferative lifespan of B-cells (Shavarts 2002). BCL-6 also represses p53 in leukemic stem cells (Hurtz 2011) and is a key survival protein in Ph+ acute lymphoblastic leukemia (ALL) cells during kinase inhibition (Duy). Based on these observations the current study investigated potential bone marrow microenvironment (BMM) influence on BCL-6 in Ph+ and Ph- ALL as the site of initiation, progression, and often relapse of aggressive disease. Utilizing an in vitro model of tumor cells grown in media alone compared to tumor cells cultured with primary human osteoblasts (Promocell) or stromal cells we explored the effects of varied cues on BCL-6 expression. Co-culture experiments suggest that microenvironment derived signals decreased BCL-6 expression. We also noted decreased BCL-6 protein when tumor cells were exposed to Ara-C. Subsequently we evaluated long term co-culture experiments in which co-cultures of stroma/tumor or ostoeblast/tumor which had been grown together for 48hrs were challenged with chemotherapy for a period of 72 hrs. This resulted in tumors cells with less BCL-6 expression compared to non-treated co-culture controls. Chemotherapy treated co-cultures were provided drug free media every 5 days to simulate a recovery following cessation of therapy to determine if residual tumor cells survived the drug treatment and their capacity to repopulate the culture. Few tumor cells where seen in culture until days 15 to 20 post-treatment at which time cell number increased dramatically, coincident with high BCL-6 expression that subsequently decreased back to control levels at days 20-35. Tumor cells recovered at the termination of co-culture were uniformly CD38+. These results suggest that BCL-6 may be very responsive to a variety of cues in the context of chemotherapy exposure when a leukemic cell resides in the BMM, potentially supporting a quiescent drug resistant population of cells, in part, through down regulation of BCL-6. However, the effect is reversible as BCL-6 is restored during a phase of proliferative repopulation in a CD 38+ subset of cells. This scenario is reminiscent of the niche's capacity to balance stem cell quiescence and proliferation during steady state hematopoiesis where stem and progenitor cells are drawn into cell cycle in a controlled manner. These preliminary observations suggest BCL-6 as a potentially important factor in maintaining quiescence and cell cycle entry in a leukemic model and draw clinical relevance based on the potential role of BCL-6 in relapse of ALL. Citation Format: William L. Slone, Zackary R. Lonergan, James Fortney, Debbie Piktel, Laura F. Gibson. Bone marrow microenvironment control of BCL-6 in acute lymphocytic leukemia . [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 1650. doi:10.1158/1538-7445.AM2013-1650

만성 골수성 백혈병 K562세포의 분화 내성 분획에서 백혈병 유지 세포의 동정

The present study investigated whether leukemia-maintaining cells reside in a differentiation-resistant fraction using a megakaryocytic differentiation model of K562 cells. Treatment with phorbol-12-myristate- 13-acetate (PMA) significantly inhibited the colony-forming efficiency of the K562 cells. At a PMA concentration of 1 nM or higher, colony was not formed, but approximately 40% of K562 cells still survived in soft agar. Approximately 70% of colony-forming cells that were isolated following the removal of PMA after exposure to the agent were differentiated after treatment with 10 nM PMA for 3 days. The differentiation rate of the colony-forming cells was gradually increased and reached about 90% 6 weeks after colony isolation, which was comparable to the level of a PMA-treated K562 control. Meanwhile, imatinib-resistant variants from the K562 cells, including K562/R1, K562/R2, and K562/R3 cells, did not show any colony-forming activity, and most imatinib-resistant variants were CD44 positive. After 4 months of culture in drug-free medium, the surface level of CD44 was decreased in comparison with primary imatinib-resistant variants, and a few colonies were formed from K562/R3 cells. In these cells, Bcr-Abl, which was lost in the imatinib-resistant variants, was re-expressed, and the original phenotypes of the K562 cells were partially recovered. These results suggest that leukemia-maintaining cells might reside in a differentiation-resistant population. Differentiation therapy to eliminate leukemia-maintaining cells could be a successful treatment for leukemia if the leukemia-maintaining cells were exposed to a differentiation inducer for a long time and at a high dose.

Microfluidic technique to measure intratumoral transport and calculate drug efficacy shows that binding is essential for doxorubicin and release hampers Doxil

Intratumoral transport and binding are important mechanisms that determine the efficacy of cancer drugs. Current drug screening methods rely heavily on monolayers of cancer cells, which overlook the contribution of tissue-level transport and binding. To quantify these factors, we developed a method that couples an in vitro, drug-delivery device containing a three-dimensional cell mass and a mathematical model of drug diffusion, binding to DNA, release from carriers, and clearance. Spheroids derived from LS174T human colon carcinoma cells were inserted into rectangular chambers to form rectangular cell masses (tissue) and subjected to continuous medium perfusion. To simulate drug delivery and clearance, the tissues were treated with doxorubicin followed by drug-free medium. To evaluate the effect of liposome encapsulation, tissues were treated with liposome-encapsulated doxorubicin (Doxil). Spatiotemporal dynamics of drug distribution and apoptosis was measured by fluorescence microscopy. The diffusivity and DNA binding constant of doxorubicin were determined by fitting experimental data to the mathematical model. Results show that an ideal combination of diffusivity, binding constant, clearance rate, and cytotoxicity contribute to the high therapeutic efficacy of doxorubicin. There was no detectable release of doxorubicin from Doxil in the tissues. The rate of doxorubicin release, evaluated by fitting experimental data to the mathematical model, was below therapeutically effective levels. These results show that despite enhanced systemic circulation obtained by liposome encapsulation, the therapeutic effect of Doxil is limited by slow intratumoral drug release. The experimental and computational methods developed here to calculate drug efficacy provide mechanisms to explain poor performance of drug candidates, and enable design of more successful cancer drugs.

SMUG1 but not UNG DNA glycosylase contributes to the cellular response to recovery from 5-fluorouracil induced replication stress

5-Fluorouracil (5-FU) is a widely utilized cancer chemotherapeutic that causes DNA damage via two mechanisms. Its active metabolite inhibits thymidylate synthase, which deprives cells of TTP and causes the introduction of uracil in DNA. Also, 5-FU is directly incorporated into DNA. Both uracil and 5-FU in DNA are recognized by uracil-DNA glycosylases (UDGs), which initiate base excision repair. UNG and SMUG1 are the two human UDGs most likely to combat the genomic incorporation of uracil and 5-FU during replication. In this study, we examined the roles of UNG and SMUG1 in the initial cellular response to 5-FU and compared continuous exposure to a 24h exposure followed by incubation in drug-free media, which mimics what occurs clinically. Loss of UNG did not alter cellular sensitivity to 5-FU in two human cell lines, despite its predominant biochemical activity for uracil and 5-FU in DNA. Loss of SMUG1 corresponded with >2-fold increase in sensitivity to 5-FU, but only with a 24h treatment followed by recovery. There was no difference between SMUG1 proficient and depleted cells following continuous exposure. We observed that 5-FU treatment induced an enhanced S-phase arrest and CHK1 activation plus an increase in the formation of strand breaks and alkali-labile sites in all sublines. However, SMUG1-depleted cells showed a prolonged S-phase arrest, a transient increase in DNA double-strand breaks following 5-FU treatment and an altered phosphorylation of CHK1 following removal of drug. Collectively, the results suggest that SMUG1 has a role in the resumption of replication following 5-FU treatment.

Autophagy As a Target for Differentiation Therapy in Acute Myeloid Leukemia.

Abstract 2464Autophagy is a catabolic cellular process whereby redundant or damaged proteins and organelles are sequestered within vesicular “autophagosomes” and degraded to reusable monomers via lysosomal hydrolases. Autophagic activity is heightened in times of cellular stress or remodeling and has recently been proposed to play an important role in cell differentiation. As Acute Myeloid Leukemia (AML) is a condition that is characterized by a differentiation block, we have investigated the importance of autophagy in the differentiation of AML cells and the consequences of pharmacologic modification of autophagy on this process.We examined autophagy in the NB4 (t15:17, PML-RARα+) Acute Promyelocytic Leukemia (APML) cell line, a well-established model of leukemic cell differentiation. Treatment of NB4 cells for 4 days with all-trans-retinoic acid (ATRA) induced differentiation evidenced by cellular morphology and confirmed by both flow cytometric and western blot detection of the CD11b and CD14 surface markers of granulocytic differentiation.During days 0 to 4 of ATRA-induced differentiation, NB4 cells progressively accumulated cytoplasmic vesicles - a characteristic feature of autophagy. LC3, GABARAP and GATE16 are cytoplasmic proteins that become lipid-conjugated during autophagy activation and incorporated into autophagic vesicles. Immune-fluorescence microscopy demonstrated a shift in the staining pattern of these proteins from a diffuse cytoplasmic pattern to a discreet punctate pattern as differentiation proceeded, consistent with incorporation into autophagic vesicles. Up-regulation of these autophagy markers during differentiation was also confirmed by western blotting.To determine the functional consequences of autophagy inhibition, we treated NB4 cells with ATRA in the presence and absence of lysosomal inhibitors E64d and Pepstatin A. Expression of CD11b and CD14 was decreased at day 4 in cells treated with ATRA/E64/Pepstatin A compared to cells treated with ATRA alone. This was also seen by western blot.We next assessed the effects of Lithium, a known inducer of autophagy, on leukemic cell differentiation. We treated high passage HL60 cells (AML cell line more resistant to ATRA-induced differentiation, PML-RARα-) with Lithium (10mM) alone and in combination with ATRA (1μM) for 4 days. Western blot analysis confirmed that Lithium treatment elevated the expression of LC3, GABARAP and GATE16. We observed early morphological features of differentiation in combination-treated cells. CD11b expression at Day 4 was increased in Lithium-treated cells compared to cells treated with ATRA alone (24.1% v 12.4%). In combination-treated cells, differentiation was even further induced (33.5% CD11b expression). Combination treatment also resulted in increased apoptosis (Annexin V flow cytometric assay) at day 4 (22.7%) compared to Lithium alone (8.4%) and to ATRA alone (6.1%). Colony-forming assays were performed to assess the proliferative capacity of cells from these treatment groups sampled at day 2 and placed in drug-free methylcellulose medium for 7 days (MethoCult H4034). Colony numbers were significantly reduced in ATRA and Lithium combination-treated cells compared to either mono-therapy or controls.We have begun to test the use of Lithium alone and in combination with ATRA on primary AML samples. Early data suggests Lithium attenuates malignant cell growth on colony-forming assay. Experiments are underway to determine the molecular mechanisms whereby lithium sensitizes cells to ATRA and promotes differentiation. Conclusion:Autophagy plays a significant role in the myeloid differentiation of APL cells. Pharmacologic inhibition of autophagy impedes ATRA-induced leukemic cell differentiation while autophagy induction in non-APL leukemia in combination with ATRA promotes differentiation, decreases the proliferative capacity of malignant cells and decreases malignant cell viability. These findings suggest that autophagy induction with readily available agents such as Lithium may represent an exciting means of re-opening differentiation pathways in AML. We are continuing to test this hypothesis in AML patient samples. Disclosures:Orfali:MSD: Unrestricted educational support, Unrestricted educational support Other; Amgen: Unrestricted educational support Other; BMS: Unrestricted educational support, Unrestricted educational support Other; Novartis: Unrestricted educational support Other; Pfizer: Unrestricted educational support, Unrestricted educational support Other.

Mitochondrial fusion: A mechanism of cisplatin-induced resistance in neuroblastoma cells?

Cisplatin induces apoptosis through different pathways. The intrinsic apoptotic pathway is mediated by mitochondria, which, as a result of cisplatin treatment, undergo morphological alterations. The aim of this study was to investigate cisplatin-induced mitochondrial functional and morphological long-term effects in neuroblastoma B50 rat cells. To this purpose, we followed evaluated different several apoptotic markers by means of flow cytometry, confocal and electron microscopy and western blotting techniques. We applied different treatment protocols based on the incubation of the neuroblastoma B50 rat cells with 40μM cisplatin: (i) for 48h and harvesting of the cells at the end of the treatment; (ii) further recovery in drug-free medium for 7 days post-treatment; (iii) conditions as in (ii) followed by re-seeding in normal medium and growth for a further 4 days. We observed apoptosis induction after the first treatment and after the recovery from cell death after long-term culture in drug-free medium. Interestingly, the latter phenomenon was characterized by mitochondrial elongation and mitochondrial protein rearrangement. In recovered and re-seeded cells, mitochondrial equilibrium moved toward fusion, possibly protecting cells from apoptosis.

Pharmacokinetic Mechanism Involved in the Prolonged High Retention of Laninamivir in Mouse Respiratory Tissues after Intranasal Administration of its Prodrug Laninamivir Octanoate

Laninamivir octanoate (LO) (Inavir; Daiichi Sankyo, Japan) is an ester prodrug of the neuraminidase inhibitor laninamivir. We previously reported that a prolonged high retention of laninamivir in mouse respiratory tissues was achieved by intranasal administration of LO. In this study, we evaluated intrapulmonary pharmacokinetics both in vivo and in vitro to investigate the potential mechanism involved in such a preferable retention. After intranasal administration of LO to mice (0.5 μmol/kg), the drug was distributed from the airway space into the lungs, and laninamivir remained in the lung at 24 hours postdose (2680 pmol/g), with a higher concentration than that in the epithelial lining fluid. The laninamivir was localized mainly on the epithelial cells of airway tracts, determined by microautoradiography using (14)C-labeled LO. In mouse airway epithelial cells, the cellular uptake and hydrolysis of LO were observed over incubation time without any apparent saturation at the highest concentration tested (1000 μM). Furthermore, after additional incubation in drug-free medium, the intracellular laninamivir was released very slowly into the medium with an estimate rate constant of 0.0707 h(-1), which was regarded as a rate-limiting step in the cellular retention. These results demonstrated that the prolonged high retention of laninamivir in the respiratory tissues was attributed to a consecutive series of three steps: uptake of LO into the airway epithelial cells, hydrolysis of LO into laninamivir by intracellular esterase(s), and limited efflux of the generated laninamivir due to its poor membrane permeability. This prodrug approach could be useful for lung-targeting drug delivery.

Evaluation of antimicrobial activity against Mycoplasma mycoides subsp. mycoides Small Colony using an in vitro dynamic dilution pharmacokinetic/pharmacodynamic model

The objectives of this study were to assess the activity of oxytetracycline (OTC), danofloxacin and tulathromycin against Mycoplasma mycoides subsp. mycoides Small Colony, the causative agent of contagious bovine pleuropneumonia, in an in vitro dynamic concentration model and to determine the concentration and/or time dependence of such activity. Time-kill assays that simulated elimination of antimicrobials from the body were performed. Initial antimicrobial concentrations corresponded to various multiples of the MIC and cultures were diluted in a stepwise fashion with either drug-free or drug-containing artificial medium to mimic administration by single-release bolus or infusion, respectively. Where appropriate, data were fitted to sigmoidal E(max) models. OTC produced no change in mycoplasma titre from the initial inoculum size, regardless of the concentration or means of drug exposure. Both danofloxacin and tulathromycin resulted in a decrease in mycoplasma titre but neither was bactericidal (99.9 % kill) over 12 h. A greater antimycoplasmal effect, defined as the change in log(10) (c.f.u. ml(-1)) over 12 h, was achieved when danofloxacin was administered as a single-release bolus, suggesting concentration-dependent activity, whereas the antimycoplasmal effect of tulathromycin was comparable following administration by single-release bolus or infusion, owing to its long half-life.

Menadione : sodium orthovanadate combination eliminates and inhibits migration of detached cancer cells.

Exposure of cancer cells to anticancer agents in cultures induces detachment of cells that are usually considered dead. These drug-induced detached cells (D-IDCs) may represent a clinical problem for chemotherapy since they may survive anoikis, enter the circulation, invade other tissues and resume proliferation, creating a metastasis, especially in tissues where the bioavailability of anticancer agents is not enough to eliminate all cancer cells. In this study we evaluated the antiproliferative effect of menadione : sodium orthovanadate (M : SO) combination on A549 lung cancer cells as well as the ability of M : SO to induce cell detachment. In addition, we followed the fate and chemosensitivity of M : SO-induced detached cells. Using transwell chambers, we found that a fraction of the M : SO-induced detached cells were viable and, furthermore, were able to migrate, re-attach, and resume proliferation when re-incubated in drug-free media. The total elimination of A549 detachment-resistant cells and M : SO-induced detached cells were successfully eliminated by equivalent M : SO concentration (17.5 μM : 17.5 μM). Thus, M : SO prevented cell migration. Similar results were obtained on DBTRG.05MG human glioma cells. Our data guarantee further studies to evaluate the in vivo occurrence of D-IDCs, their implications for invasiveness and metastasis and their sensitivity to anticancer drugs.

Stability of Tandemly Repetitive Subelement PCR Patterns in Trichophyton rubrum over Serial Passaging and with Respect to Drug Pressure

Trichophyton rubrum is the most significant agent of dermatomycoses worldwide, primarily causing tinea pedis and tinea unguium. PCR analysis of tandemly repetitive subelements (TRS) within the rDNA nontranscribed spacer region is a major tool for molecular typing of T. rubrum. The aim of this study was to investigate the stability of TRS PCR patterns by analyzing isogenic strains of T. rubrum. Twenty-seven groups of isogenic T. rubrum strains were examined, each composed of an original clinical isolate and its 3 subcultures, maintained on a drug-free medium, a medium containing fluconazole and itraconazole. TRS typing was performed for the original strains and their subcultures grown after 12 passages, at 4-week intervals, on respective media. To add more objectivity to the results, TRS typing for each of the isogenic strain was performed three times, using DNA isolated from three different colonies. Among 27 groups of isogenic strains, all but one were exclusively composed of strains with identical TRS-1 and TRS-2 PCR patterns. In one group, 3 isolates from the last, twelfth passage had identical TRS-1 PCR profiles (type 1), yet different TRS-2 PCR profiles, as compared with the original strain (type I vs. type II). The mechanism underlying the genotype switch was a deletion of a single repeat unit in the TRS-2 locus, as evidenced by sequence analysis. In the interpretation of TRS typing results, microevolutionary events need to be taken into account, urging drawing epidemiological conclusions with caution and in conjunction with other genotyping data and traditional contact tracing information.

Effects of etoposide on the proliferation of hexaploid H1 (ES) cells

Etoposide is a specific inhibitor of topoisomerase II, which is an enzyme that enables double-stranded DNA to pass through another double-stranded DNA. Topoisomerase II is a major constituent of chromosome scaffold, existing at appreciable amounts in cells. To examine the effects of etoposide on the cell cycle, hexaploid H1 (ES) cells (6H1 cells) were used with diploid H1 (ES) cells (2H1 cells) as a control. Exponentially growing 2H1 and 6H1 cells were exposed to etoposide at various concentrations, and cultured for about 60 days in L15F10 medium with leukemia inhibitory factor. With a high concentration of etoposide (1 μM), the DNA histograms showed G(2)/M accumulation, suggesting that etoposide arrested the cell cycle at the G(2)/M phase. With a low concentration of etoposide (50 nM), the cell proliferation was suppressed with a doubling time of 98.4 h for 2H1 cells and 51.6 h for 6H1 cells, and without significant alteration in DNA histograms. Time-lapse videography revealed that 6H1 cells survived in the medium containing 50 nM etoposide had a cell cycle time of 18.8 h, which was equivalent to 19.2 h of the doubling time for the 6H1 cell population in drug-free medium, suggesting that a part of the cell population died and was excluded from the cell system. It was concluded that etoposide affected the cell cycle at a wide range of concentrations.

Abstract A54: Potential mechanisms of resistance to targeted agents in human clear cell renal cell carcinoma

Abstract Targeted therapy with multiple receptor tyrosine kinase inhibitors (RTKI) has led to a substantial improvement in the standard of care for patients with advanced or metastatic clear cell renal cell carcinoma (CCRCC). We have previously reported that using a phosphoproteomics approach, Ras and Rab interactor 1 (RIN1) was identified as a potential target modulating the antiproliferative effects of sorafenib and sunitinib in a panel of human CCRCC (Proc. AACR 51: Abst. #1626, 2010). While both 10 μM sorafenib and 7.5 μM sunitinib were effective in down-regulating RIN1 protein levels in CCRCC following treatment for 48h, during subsequent recovery for 48h in drug free medium, recovery of RIN1 protein levels was observed in sorafenib but not sunitinib treated cells. mRNA expression profiling revealed that: (a) VEGF-A and HIF-1α levels were unaffected during 48h treatment with sorafenib or sunitinib or subsequent recovery; and (b) while HIF-2α levels were increased >2-fold following 48h exposure to sorafenib or sunitinib, during subsequent recovery in drug-free medium, the increased levels of HIF-2α persisted in cells treated with sorafenib but not sunitinib. Compared to CCRCC cells that were growth inhibited in vitro at concentrations of 5–10 μM sorafenib or sunitinib, in 27 patient CCRCC samples evaluated, the median mRNA levels of RIN1 was 7.2-fold lower in 22 samples (Range 1.6–22.3) and 2.2-fold higher in 5 samples (Range 1.2–27.1). Stable over-expression and down-regulation of RIN1 in CCRCC led to resistance or sensitivity respectively, to the anti-proliferative effects of sorafenib and sunitinib but not the mTOR inhibitor temsirolimus. In contrast, CCRCC cells adapted to proliferate in 4 μM everolimus exhibited a >3-fold increase in IC50, and cross-resistance to temsirolimus but not sorafenib or sunitinib. Evaluation in vitro of other targeted agents, demonstrated dose-dependent anti-proliferative effects with the PI3K/mTOR dual inhibitor BEZ235 (>80% growth inhibition at 10nM) but not the pan-PI3K inhibitor BKM120 (<30% growth inhibition at 10–500 nM) with CCRCC cells. The observed anti-proliferative effects of BEZ235 or BKM120 were comparable in CCRCC cells harboring the wild type or mutant von Hippel-Lindau gene. While BEZ235 (1–5 nM) or everolimus (100 nM) alone led to <40% growth inhibition, the combination significantly enhanced anti-proliferative effects (>85% growth inhibition) in CCRCC cells expressing resistance to everolimus. These results suggest the PI3K/mTOR dual inhibitor BEZ235 may possibly improve the treatment efficacy of mTOR inhibitors for CCRCC. The findings suggest RTKI and mTOR inhibitors have distinct mechanisms of resistance, and combinations may be useful to improve disease control of metastatic CCRCC.

β-Asarone protects PC12 cells against OGD/R-induced injury via attenuating Beclin-1-dependent autophagy

To explore the effects of β-asarone from Acorus Tatarinowii Schott on autophagy in an ischemic stroke model of PC12 cells. The ischemic stroke model of PC12 cells was made by OGD/R (2 h oxygen-glucose deprivation followed by 24 h reperfusion). Drug administration was started 1 h before OGD and last for 3 h. Then the cells were incubated in the drug-free and full culture medium under normoxic conditions for 24 h. After the treatments, Beclin-1, intracellular free calcium concentration ([Ca(2+)](i)) and mitochondrial membrane potential (MMP) were analyzed using flow cytometry. Cell viability was measured using MTT assay. Cell morphology was studied under inverted phase contrast microscope, and autophagosomes were observed under transmission electron microscope. Pretreatment with β-asarone (20, 30, or 45 μg/mL) or the calcium channel antagonist nimodipine (10 μmol/L) significantly increased the cell viability and MMP, and decreased Beclin-1 expression and [Ca(2+)](i) in OGD/R-treated PC12 cells. Under inverted phase contrast microscope, pretreatment with β-asarone or nimodipine dramatically increase the number of cells and improved the cellular morphology. Autophagosomes were found in OGD/R-treated PC12 cells as well as in drug plus OGD/R-treated PC12 cells. β-Asarone protects PC12 cells against OGD/R-induced injury partly due to attenuating Beclin-1-dependent autophagy caused by decreasing [Ca(2+)](i) and increasing MMP.

Abstract 5677: A comparative study of the interaction of the maytansinoid, DM1-Me, vinblastine, and taxol with human cells

Abstract The current interest in antimitotic compounds that interact with microtubules is caused, in part, by their clinical importance in oncology. Several microtubule-binding compounds, including Vinca alkaloids, taxanes, maytansinoids, and auristatins, are in clinical use, some additionally being evaluated as antibody conjugates. To better understand the factors that determine the potency of antibody-maytansinoid conjugates in killing cells, we examined the interaction of the maytansinoid, DM1-Me, with MCF7 cells, in comparison with vinblastine and taxol. DM1-Me was more potent in arresting mitosis than vinblastine or taxol (5- and 13-fold, respectively). We compared the levels of accumulation of these three compounds by MCF7 cells following a 24 hour exposure at the mitotic IC50 concentration and found that, at this concentration, cells accumulated comparable amounts of vinblastine and DM1-Me, but required more than a 10-fold greater concentration of taxol to achieve a similar effect. Additionally, cells exposed to each compound for 2 hours prior to being washed into drug free medium, retained most of the accumulated DM1-Me or taxol for at least 24 h, while most vinblastine was released into the medium within the first 2 h. DM1-Me and taxol had similar affinities for MCF7 cells, while that of vinblastine was 4-6-fold lower. These properties of DM1-Me, vinblastine and taxol suggest that maytansinoids are a better choice for antibody conjugates than vinblastine or taxol, a conclusion which is consistent with the experimental data and clinical experience with antibody conjugates made with compounds of these three classes. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5677. doi:1538-7445.AM2012-5677

Abstract 2852: Combination therapy: Galectin-3C and bortezomib potent anticancer activity and inhibition of angiogenesis, chemotaxis, and NF-kB activation in human multiple myeloma

Abstract Introduction Galectin-3 is a human lectin involved in cellular processes including differentiation, apoptosis, angiogenesis, neoplastic transformation, and metastasis. We evaluated galectin-3C, an N-terminally truncated form of galectin-3 that is thought to act as a dominant negative inhibitor, as a potential treatment for multiple myeloma (MM). Methods We analyzed 9 human MM cell lines tested in vitro for cell proliferation/viability; chemotaxis. The human MM cell lines used in this study were RPMI-8226, U266 (American Type Culture Collection, VA, USA), ARP-1, ARK-B, (gifts from J. Epstein, University of Arkansas for Medical Sciences, Little Rock, AR, USA). The 8226/Dox and 8226/LR-5 cell lines were kindly provided by Dr. William S. Dalton of the H. Lee Moffitt Cancer Center and Research Institute in Tampa, Florida. All MM cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) in 5% CO2 at 37 °C. The 8226/Dox cells were cultured with 40 nM doxorubicin (Sigma-Aldrich, St. Louis, MO, USA). The 8226/LR-5 cells were cultured in media containing 5 µM mephalan (Sigma-Aldrich). 8226/Dox and 8226/LR-5 cells were maintained in drug-free medium for 1 week prior to drug sensitivity assays. Human umbilical cord vascular endothelial cells (HUVEC, American Type Culture Collection, VA, USA) were maintained in EGM-2 medium (Lonza, Houston, TX, USA) and were used within 10 passages. Furthermore we used U266 MM model in the NOD/SCID mice to evaluate the efficacy of galectin-3C in single or in combination with BOR. Results Galectin-3C exhibited modest anti-proliferative effects on MM cells in vitro. Delivery of galectin-3C intravenously via an osmotic pump in a subcutaneous U266 cell NOD/SCID mouse model of MM significantly inhibited tumor growth. Bortezomib, a proteasome inhibitor approved by the F.D.A. for MM treatment, also inhibited the growth of the U266 tumors. Treatment with both agents was more effective than either alone, such that the average tumor volume of the combination group was just 14.0% of controls at day 35. We show that mechanisms by which galectin-3C facilitates the anticancer activity of BOR include inhibition of angiogenesis, chemotaxis, and NF-kB activation. Conclusion In conclusion, our study shows that in a mouse model of human MM, treatment with galectin-3C alone is efficacious, and that it enhances the anti-tumor activity of bortezomib. These findings provide the proof-of-principle to test galectin-3C clinically for human MM. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2852. doi:1538-7445.AM2012-2852

Genome Wide Adaptations of Plasmodium falciparum in Response to Lumefantrine Selective Drug Pressure

The combination therapy of the Artemisinin-derivative Artemether (ART) with Lumefantrine (LM) (Coartem®) is an important malaria treatment regimen in many endemic countries. Resistance to Artemisinin has already been reported, and it is feared that LM resistance (LMR) could also evolve quickly. Therefore molecular markers which can be used to track Coartem® efficacy are urgently needed. Often, stable resistance arises from initial, unstable phenotypes that can be identified in vitro. Here we have used the Plasmodium falciparum multidrug resistant reference strain V1S to induce LMR in vitro by culturing the parasite under continuous drug pressure for 16 months. The initial IC50 (inhibitory concentration that kills 50% of the parasite population) was 24 nM. The resulting resistant strain V1SLM, obtained after culture for an estimated 166 cycles under LM pressure, grew steadily in 378 nM of LM, corresponding to 15 times the IC50 of the parental strain. However, after two weeks of culturing V1SLM in drug-free medium, the IC50 returned to that of the initial, parental strain V1S. This transient drug tolerance was associated with major changes in gene expression profiles: using the PFSANGER Affymetrix custom array, we identified 184 differentially expressed genes in V1SLM. Among those are 18 known and putative transporters including the multidrug resistance gene 1 (pfmdr1), the multidrug resistance associated protein and the V-type H+ pumping pyrophosphatase 2 (pfvp2) as well as genes associated with fatty acid metabolism. In addition we detected a clear selective advantage provided by two genomic loci in parasites grown under LM drug pressure, suggesting that all, or some of those genes contribute to development of LM tolerance – they may prove useful as molecular markers to monitor P. falciparum LM susceptibility.