S9 Cells Research Articles

Detection and Characterization of In Vitro Payload-Containing Catabolites of Noncleavable Antibody-Drug Conjugates by High-Resolution Mass Spectrometry and Multiple Data Mining Tools.

The formation and accumulation of payload-containing catabolites (PCCs) from a noncleavable antibody-drug conjugate (ADC) in targeted and normal tissues are directly associated with the therapeutic effect and toxicity of the ADC, respectively. Understanding the PCC formation is important for supporting the payload design and facilitating preclinical evaluation of ADCs. However, detection and identification of PCCs of a noncleavable ADC are challenging due to their low concentrations and unknown structures. The main objective of this study was to develop and apply a generic liquid chromatography-high-resolution mass spectrometry (LC-HRMS) method for profiling PCCs in vitro. Noncleavable ADCs, ado-trastuzumab emtansine (T-DM1) and ADC-1, were incubated in liver lysosomes, liver S9, and/or cancer cells followed by data acquisition using LC-HRMS. Profiling PCCs mainly relied on processing LC-HRMS datasets using untargeted precise and thorough background subtraction (PATBS) processing and targeted product ion filtering (PIF). As a result, 12 PCCs of T-DM1 were detected and structurally characterized in human liver lysosomal incubation, a majority of which consisted of 4-[N-maleimidomethyl]cyclohexane-1-carboxylate (MCC)-DM1 and a few amino acids. Additionally, the incubation of ADC-1 in human, rat, and monkey liver S9 and cancer cells generated one major and three very minor PCCs, verifying the payload design. The results demonstrate that PATBS enabled the comprehensive profiling of PCCs regardless of their molecular weights, charge states, and fragmentations. As a complementary tool, PIF detected specific PCCs with superior sensitivity. The combination of the in vitro metabolism systems and the LC-HRMS method is a useful approach to profiling in vitro PCCs of noncleavable ADCs in support of drug discovery programs. SIGNIFICANCE STATEMENT: Profiling in vitro payload-containing catabolites (PCCs) of a noncleavable antibody-drug conjugate (ADC) is important for optimization of the payload design and preclinical evaluation of ADC. However, currently used analytical approaches often fail to quickly provide reliable PCC profiling results. The work introduces a new liquid chromatography high resolution mass spectrometry method for comprehensive and rapid detection and characterization of PCCs released from a noncleavable ADC in liver lysosomes and S9 incubations.

Preclinical Investigation of a Potent Geranylgeranyl Diphosphate Synthase Inhibitor

The isoprenoid biosynthesis pathway (IBP) is responsible for the synthesis of isoprenoids used for the prenylation of proteins belonging to the Ras small GTPase superfamily. Geranylgeranyl diphosphate synthase (GGDPS) is the enzyme in the IBP pathway that catalyzes the synthesis of the 20-carbon isoprenoid GGDP. The enzymes geranylgeranyl transferase (GGTase) I and II utilize GGDP as the isoprenoid substrate for their geranylgeranylation reactions. Rab proteins mediate vesicle trafficking within the cell and their activity is dependent on geranylgeranylation. Our prior work has demonstrated that agents that disrupt Rab geranylgeranylation disrupt monoclonal protein trafficking in myeloma cells, resulting in induction of the unfolded protein response (UPR) pathway and apoptosis. Because myeloma cells express near maximal levels of UPR-associated proteins to aid in the continuous production of monoclonal proteins, they are particularly sensitive to activation of the pro-apoptotic arm of the UPR. We are therefore pursuing the development of GGDPS inhibitors as a novel therapeutic strategy for myeloma. Our lead compound VSW1198 (Figure) is the most potent GGDPS inhibitor reported to date with an IC50 of 45nM against purified enzyme, high specificity for GGDPS over the related enzyme farnesyl diphosphate synthase, and measurable cellular activity at concentrations as low as 30 nM (15 ng/mL). Due to its potent activity against myeloma cells in vitro, we were interested in evaluating the metabolic stability, pharmacokinetic (PK) profile, distribution pattern, and toxicology profile of VSW1198 in preparation for in vivo efficacy studies. Single dose testing via IV administration in CD-1 mice revealed a maximum tolerated dose of 0.5 mg/kg. Doses > 1mg/kg resulted in liver toxicity that peaked around 6-7 days post-injection. Histopathological examination of the livers demonstrated centrilobular hepatocyte necrosis and apoptosis with invasion of inflammatory cells and panlobular hepatocyte swelling. Analysis of blood samples showed elevated transaminase levels without effects on bilirubin, alkaline phosphatase, lactic dehydrogenase, or albumin, consistent with the observed pattern of hepatic injury. A control compound, RAM3059, also was tested in vivo . This compound has a similar isoprenoid triazole bisphosphonate structure (Figure) but is 380-fold less potent than VSW1198 as a GGDPS inhibitor. Mice injected with RAM3059 (1.6 mg/kg IV) were grossly and histologically normal with no signs of liver toxicity, suggesting liver injury is not due to nonspecific toxicity from the triazole bisphosphonate structure. No effects on kidney function, calcium, creatine kinase, or blood counts were observed. Histopathological examination of the brains and kidneys from the treated mice revealed no abnormalities. A PK and tissue distribution study included 11 blood samples obtained over a period from 5 minutes to 7 days post-injection of VSW1198 (0.5 mg/kg IV, n=5 per time point). Tissue samples (liver, kidney, spleen, lung, heart, brain, and bone marrow) were collected at 2, 8, 24, 48, and 72 hours post-injection (n=5 per time point). VSW1198 was extracted from plasma and tissue samples, derivatized by reaction with diazomethane, and analyzed via LC-MS/MS. The maximum concentration (C0 ) is 611.0 (+ 183.3) ng/mL, the area under curve (AUC0-∞ ) is 2355.9 (+ 160.3) hr*ng/mL, and the plasma elimination half-life (t1/2 ) is 38.0 (± 10.9) hrs. Detectable concentrations of VSW1198 at 72 hours (5.0 + 1.0 ng/mL) indicate prolonged systemic exposure. Analysis of tissues samples is underway and will be reported. The metabolic stability of VSW1198 was assessed using human liver microsomes (HLM) and mouse S9 cells for phase I and phase II metabolism. In both the HLM and mouse S9 studies, VSW1198 showed complete stability while a positive control showed rapid metabolic degradation. In summary, these studies demonstrate the metabolic stability and systemic distribution of the potent GGDPS inhibitor VSW1198. Multi-dose testing and efficacy studies in a myeloma xenograft mouse model are currently underway to assess further the clinical potential of GGDPS inhibitor therapy. [Display omitted] DisclosuresHolstein:Celgene: Consultancy; Takeda: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees.

CFTR chloride channel activity modulates the mitochondrial morphology in cultured epithelial cells

The impairment of the CFTR channel activity, a cAMP-activated chloride (Cl−) channel responsible for cystic fibrosis (CF), has been associated with a variety of mitochondrial alterations such as modified gene expression, impairment in oxidative phosphorylation, increased reactive oxygen species (ROS), and a disbalance in calcium homeostasis. The mechanisms by which these processes occur in CF are not fully understood. Previously, we demonstrated a reduced MTND4 expression and a failure in the mitochondrial complex I (mCx-I) activity in CF cells. Here we hypothesized that the activity of CFTR might modulate the mitochondrial fission/fusion balance, explaining the decreased mCx-I. The mitochondrial morphology and the levels of mitochondrial dynamic proteins MFN1 and DRP1 were analysed in IB3−1 CF cells, and S9 (IB3−1 expressing wt-CFTR), and C38 (IB3−1 expressing a truncated functional CFTR) cells. The mitochondrial morphology of IB3−1 cells compared to S9 and C38 cells showed that the impaired CFTR activity induced a fragmented mitochondrial network with increased rounded mitochondria and shorter branches. Similar results were obtained by using the CFTR pharmacological inhibitors CFTR(inh)-172 and GlyH101 on C38 cells. These morphological changes were accompanied by modifications in the levels of the mitochondrial dynamic proteins MFN1, DRP1, and p(616)-DRP1. IB3−1 CF cells treated with Mdivi-1, an inhibitor of mitochondrial fission, restored the mCx-I activity to values similar to those seen in S9 and C38 cells. These results suggest that the mitochondrial fission/fusion balance is regulated by the CFTR activity and might be a potential target to treat the impaired mCx-I activity in CF.

Major Determinants of Airway Epithelial Cell Sensitivity to S. aureus Alpha-Toxin: Disposal of Toxin Heptamers by Extracellular Vesicle Formation and Lysosomal Degradation.

Alpha-toxin is a major virulence factor of Staphylococcus aureus. Monomer binding to host cell membranes results in the formation of heptameric transmembrane pores. Among human model airway epithelial cell lines, A549 cells were most sensitive toward the toxin followed by 16HBE14o- and S9 cells. In this study we investigated the processes of internalization of pore-containing plasma membrane areas as well as potential pathways for heptamer degradation (lysosomal, proteasomal) or disposal (formation of exosomes/micro-vesicles). The abundance of toxin heptamers upon applying an alpha-toxin pulse to the cells declined both in extracts of whole cells and of cellular membranes of S9 cells, but not in those of 16HBE14o- or A549 cells. Comparisons of heptamer degradation rates under inhibition of lysosomal or proteasomal degradation revealed that an important route of heptamer degradation, at least in S9 cells, seems to be the lysosomal pathway, while proteasomal degradation appears to be irrelevant. Exosomes prepared from culture supernatants of toxin-exposed S9 cells contained alpha-toxin as well as low amounts of exosome and micro-vesicle markers. These results indicate that lysosomal degradation of internalized toxin heptamers may be the most important determinant of toxin-resistance of some types of airway epithelial cells.

SOX11-induced decrease in vimentin and an increase in prostate cancer cell migration attributed to cofilin activity

SOX11 is a transcription factor in the SOX family of genes that regulate multiple cellular events by influencing the expression of key genes in developmental, physiological, and tumorigenic cells. To elucidate the role of SOX11 in prostate cancer cells, PC-3 prostate cancer cells were cloned (S6 and S9 cells) to highly express SOX11. We demonstrated that both S6 and S9 lose vimentin expression, acquiring epithelial marker proteins, which indicates the Epithelial state phenotype. S6 and S9 cells have cancer-promoting characteristics that include higher migratory properties compared with control cells. The mechanisms that are responsible for the enhanced migration are cofilin activity and keratin 18 expression. TCGA (The Cancer Genome Atlas) dataset analysis revealed that metastatic prostate cancer tumors tend to have more SOX11 gene amplification compared with primary tumors. These results suggest the tumor promotive role and epithelial protein induction of SOX11 in prostate cancer cell.

Analysis of Staphylococcus aureus proteins secreted inside infected human epithelial cells

Staphylococcus aureus, an opportunistic pathogen is able to invade into and persist inside non-professional phagocytic cells. To do so, this bacterium possesses a wide range of secreted virulence factors which enable attachment to the host as well as intracellular survival. Hence, a monitoring of virulence factors specifically produced upon internalization might reveal targets for prevention or therapy of S. aureus infections. However, previous proteome approaches enriching S. aureus from lysed host cells after infection did not cover secreted virulence factors.Therefore, we used density gradient centrifugation and mass spectrometry to identify S. aureus HG001 proteins which were secreted into compartments of infected human bronchial epithelial S9 cells. Because shotgun mass spectrometry revealed only few bacterial proteins amongst 1905 host proteins, we used highly sensitive and selective single reaction monitoring mass spectrometry as an alternative approach and quantified 37 bacterial proteins within the S. aureus containing host cell compartment 2.5 h and 6.5 h post infection. Among them were secreted bacterial virulence factors like lipases, pore forming toxins, and secreted adhesins which are usually hard to detect from infected sample material by proteomics approaches due to their low abundance. S. aureus adapted its proteome to improve its response to oxidative and cell wall stress occurring inside the host, but also, increased the amounts of some adhesins and pore-forming toxins, required for attachment and host cell lysis.

CFTR impairment upregulates c-Src activity through IL-1β autocrine signaling

Cystic Fibrosis (CF) is a disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Previously, we found several genes showing a differential expression in CFDE cells (epithelial cells derived from a CF patient). One corresponded to c-Src; its expression and activity was found increased in CFDE cells, acting as a signaling molecule between the CFTR activity and MUC1 overexpression. Here we report that bronchial IB3-1 cells (CF cells) also showed increased c-Src activity compared to ‘CFTR-corrected’ S9 cells. In addition, three different Caco-2 cell lines, each stably transfected with a different CFTR-specific shRNAs, displayed increased c-Src activity. The IL-1β receptor antagonist IL1RN reduced the c-Src activity of Caco-2/pRS26 cells (expressing a CFTR-specific shRNA). In addition, increased mitochondrial and cellular ROS levels were detected in Caco-2/pRS26 cells. ROS levels were partially reduced by incubation with PP2 (c-Src inhibitor) or IL1RN, and further reduced by using the NOX1/4 inhibitor GKT137831. Thus, IL-1β→c-Src and IL-1β→NOX signaling pathways appear to be responsible for the production of cellular and mitochondrial ROS in CFTR-KD cells. In conclusion, IL-1β constitutes a new step in the CFTR signaling pathway, located upstream of c-Src, which is stimulated in cells with impaired CFTR activity.

A multi-omics approach identifies key hubs associated with cell type-specific responses of airway epithelial cells to staphylococcal alpha-toxin.

Responsiveness of cells to alpha-toxin (Hla) from Staphylococcus aureus appears to occur in a cell-type dependent manner. Here, we compare two human bronchial epithelial cell lines, i.e. Hla-susceptible 16HBE14o- and Hla-resistant S9 cells, by a quantitative multi-omics strategy for a better understanding of Hla-induced cellular programs. Phosphoproteomics revealed a substantial impact on phosphorylation-dependent signaling in both cell models and highlights alterations in signaling pathways associated with cell-cell and cell-matrix contacts as well as the actin cytoskeleton as key features of early rHla-induced effects. Along comparable changes in down-stream activity of major protein kinases significant differences between both models were found upon rHla-treatment including activation of the epidermal growth factor receptor EGFR and mitogen-activated protein kinases MAPK1/3 signaling in S9 and repression in 16HBE14o- cells. System-wide transcript and protein expression profiling indicate induction of an immediate early response in either model. In addition, EGFR and MAPK1/3-mediated changes in gene expression suggest cellular recovery and survival in S9 cells but cell death in 16HBE14o- cells. Strikingly, inhibition of the EGFR sensitized S9 cells to Hla indicating that the cellular capacity of activation of the EGFR is a major protective determinant against Hla-mediated cytotoxic effects.

Proteomic Changes of Tissue-Tolerable Plasma Treated Airway Epithelial Cells and Their Relation to Wound Healing.

Background. The worldwide increasing number of patients suffering from nonhealing wounds requires the development of new safe strategies for wound repair. Recent studies suggest the possibility of nonthermal (cold) plasma application for the acceleration of wound closure. Methods. An in vitro wound healing model with upper airway S9 epithelial cells was established to determine the macroscopically optimal dosage of tissue-tolerable plasma (TTP) for wound regeneration, while a 2D-difference gel electrophoresis (2D-DIGE) approach was used to quantify the proteomic changes in a hypothesis-free manner and to evaluate the balance of beneficial and adverse effects due to TTP application. Results. Plasma doses from 30 s up to 360 s were tested in relation to wound closure after 24 h, 48 h, 72 h, 96 h, and 120 h, in which lower doses (30, 60, and 120 s) resulted in dose-dependent improved wound healing rate compared to untreated cells. Thereby, the 120 s dose caused significantly the best wound healing properties after 96 and 120 h. The proteome analysis combined with IPA revealed that a lot of affected stress adaptation responses are linked to oxidative stress response emphasizing oxidative stress as a possible key event in the regeneration process of epithelial cells as well as in the adaptation to plasma exposure. Further cellular and molecular functions like proliferation and apoptosis were significantly up- or downregulated by all TTP treatments but mostly by the 120 s dose. Conclusions. For the first time, we were able to show plasma effects on cellular adaptation of upper airway epithelial S9 cells improving wound healing. This is of particular interest for plasma application, for example, in the surgery field of otorhinolaryngology or internal medicine.

Metabolism of vitetrifolin D in human liver microsomes and S9 cells: Analyses by LC/TOF-MS/MS and LC-SPE-NMR

Metabolism of vitetrifolin D in human liver microsomes and S9 cells: Analyses by LC/TOF-MS/MS and LC-SPE-NMR

Influence of calcineurin on apoptosis of pre-B lymphocytes and the leukemia cells derived from pre-B lymphocytes

This study was aimed to compare the differential expressions of calcineurin (PP2B, PP3) in the mouse Pre-B cell lines (S9) and the tumor cell lines (S4C2) derived from pre-B lymphocytes, and to clarify its possible mechanism involving in the leukemia cell apoptosis. The quantitative real-time PCR was used to detect the differential expressions of H2AX-associated phosphakinase ATM, ATR, DNA-PKs, JNK1, P38 and the γ-H2AX-related phosphatase PP1, PP2A, calcineurin, PP4, PP6, PP5 between S9 and S4C2 cell lines. CCK-8 assay and flow cytometry were used to detect the effect of imatinib (IM) and cyclosporine A (CsA) on cytotoxicity and apoptosis of 2 cell lines. The Western blot was used to detect the effects of 2 drugs on apoptosis of S9 and S4C2 cell lines. The results showed that the expression level of calcineurin gene in the leukemia cell S4C2 was about 3.5 times of that in S9 cells, while the expression of other genes in these 2 kinds of cells was not significantly different. The apoptosis and toxicity of IM and CsA on S4C2 cells was significantly stronger than that on S9 cells. The expression level of calcineurin in S4C2 cells was higher than that in S9 cells.When CsA inhibited the calcineurin activity, the expression of DNA damage marker γ-H2AX in S9 cells was significantly lower than that in S4C2 cells,while the expression level of γ-H2AX between the two cell lines was no significantly different after treatment with imatinib, the expression level of γ-H2AX in S9 cells was lower than that in S4C2 cells when the two drugs were combined. It is concluded that the calcineurin plays a role of anti-apoptosis in B leukemic cells, cyclosporine A can promote the leukemia cell apoptosis.

The Effect of PnTx2-6 Protein From Phoneutria nigriventer Spider Toxin on Improvement of Erectile Dysfunction in a Rat Model of Cavernous Nerve Injury

To explore the therapeutic potential of PnTx2-6 injected 3 times a week for 4 weeks into the intracavernosal tissue in a rat model of bilateral cavernous nerve crush injury (BCNI). Eight-week-old male Sprague-Dawley rats were randomly divided into the following 6 groups (n = 5 per group): age-matched control (normal group), BCNI (injury group), post-BCNI phosphate-buffered saline injection (PBS group), post-BCNI Sf9 cell-lysate injection (N/C group), post-BCNI injection of cell lysate from S9 cells infected with wild-type recombinant baculovirus (W/T group), and post-BCNI injection of cell lysate from S9 cells infected with recombinant baculovirus containing PnTx2-6 (PnTx2-6 group). Injections were delivered 3 times a week for 4 weeks. After 4 weeks, intracavernosal pressure-to-mean arterial pressure ratio, smooth muscle and collagen content via the Masson trichrome staining, levels of neural nitric oxide synthase, phosphoendothelial nitric oxide synthase, and cyclic guanosine monophosphate were all measured. The PnTx2-6 group showed significantly higher intracavernosal pressure-to-mean arterial pressure ratio (P <.05), smooth muscle-to-collagen ratio (P <.01), expression levels of neural nitric oxide synthase, phosphoendothelial nitric oxide synthase (P <.05), and cyclic guanosine monophosphate (P <.05) than all other experimental groups. We conclude that PnTx2-6 improved erectile function and prevented muscle atrophy in a rat model of BCNI via increased synthesis of nitric oxide and cyclic guanosine monophosphate.

Disruption of interleukin-1β autocrine signaling rescues complex I activity and improves ROS levels in immortalized epithelial cells with impaired cystic fibrosis transmembrane conductance regulator (CFTR) function.

Patients with cystic fibrosis (CF) have elevated concentration of cytokines in sputum and a general inflammatory condition. In addition, CF cells in culture produce diverse cytokines in excess, including IL-1β. We have previously shown that IL-1β, at low doses (∼30 pM), can stimulate the expression of CFTR in T84 colon carcinoma cells, through NF-κB signaling. However, at higher doses (>2.5 ng/ml, ∼150 pM), IL-1β inhibit CFTR mRNA expression. On the other hand, by using differential display, we found two genes with reduced expression in CF cells, corresponding to the mitochondrial proteins CISD1 and MTND4. The last is a key subunit for the activity of mitochondrial Complex I (mCx-I); accordingly, we later found a reduced mCx-I activity in CF cells. Here we found that IB3-1 cells (CF cells), cultured in serum-free media, secrete 323±5 pg/ml of IL-1β in 24 h vs 127±3 pg/ml for S9 cells (CFTR-corrected IB3-1 cells). Externally added IL-1β (5 ng/ml) reduces the mCx-I activity and increases the mitochondrial (MitoSOX probe) and cellular (DCFH-DA probe) ROS levels of S9 (CFTR-corrected IB3-1 CF cells) or Caco-2/pRSctrl cells (shRNA control cells) to values comparable to those of IB3-1 or Caco-2/pRS26 cells (shRNA specific for CFTR). Treatments of IB3-1 or Caco-2/pRS26 cells with either IL-1β blocking antibody, IL-1 receptor antagonist, IKK inhibitor III (NF-κB pathway) or SB203580 (p38 MAPK pathway), restored the mCx-I activity. In addition, in IB3-1 or Caco-2/pRS26 cells, IL-1β blocking antibody, IKK inhibitor III or SB203580 reduced the mitochondrial ROS levels by ∼50% and the cellular ROS levels near to basal values. The AP-1 inhibitors U0126 (MEK1/2) or SP600125 (JNK1/2/3 inhibitor) had no effects. The results suggest that in these cells IL-1β, through an autocrine effect, acts as a bridge connecting the CFTR with the mCx-I activity and the ROS levels.

Staphylococcus aureus alpha-toxin mediates general and cell type-specific changes in metabolite concentrations of immortalized human airway epithelial cells.

Staphylococcus aureus alpha-toxin (Hla) is a potent pore-forming cytotoxin that plays an important role in the pathogenesis of S. aureus infections, including pneumonia. The impact of Hla on the dynamics of the metabolome in eukaryotic host cells has not been investigated comprehensively. Using 1H-NMR, GC-MS and HPLC-MS, we quantified the concentrations of 51 intracellular metabolites and assessed alterations in the amount of 25 extracellular metabolites in the two human bronchial epithelial cell lines S9 and 16HBE14o− under standard culture conditions and after treatment with sub-lethal amounts (2 µg/ml) of recombinant Hla (rHla) in a time-dependent manner. Treatment of cells with rHla caused substantial decreases in the concentrations of intracellular metabolites from different metabolic pathways in both cell lines, including ATP and amino acids. Concomitant increases in the extracellular concentrations were detected for various intracellular compounds, including nucleotides, glutathione disulfide and NAD+. Our results indicate that rHla has a major impact on the metabolome of eukaryotic cells as a consequence of direct rHla-mediated alterations in plasma membrane permeability or indirect effects mediated by cellular signalling. However, cell-specific changes also were observed. Glucose consumption and lactate production rates suggest that the glycolytic activity of S9 cells, but not of 16HBE14o− cells, is increased in response to rHla. This could contribute to the observed higher level of resistance of S9 cells against rHla-induced membrane damage.

Abstract P5-09-02: Cell culture models to study mechanisms for endocrine resistant breast cancer, new treatment options and new biomarkers

Abstract Introduction: Estrogen is the main driver of growth of the majority of breast cancers and many patients benefit from endocrine therapy. However, both innate and acquired resistance is a major problem. In order to study resistance mechanisms and to explore targeted treatment options for endocrine resistant breast cancer, we have developed in vitro cell culture models based on the estrogen receptor (ER) positive and estrogen responsive human breast cancer cell lines MCF-7 and T47D. Methods: Panels of MCF-7 and T47D sublines with acquired resistant to tamoxifen, fulvestrant and aromatase inhibitors were established by selection of colonies surviving long term treatment. Long-term estrogen deprived (LTED) MCF-7/S9 cells were developed by step-wise serum reduction until transfer to serum-free medium. The endocrine resistant sublines were analysed with respect to expression and function of the ER and the HER system, with respect to response to 195 kinase inhibitors and to second-line endocrine therapy. Results: ER expression was reduced in tamoxifen and fulvestrant resistant MCF-7 sublines, unchanged in tamoxifen resistant T47D sublines, whereas fulvestrant resistant T47D sublines were ER negative. In general, ER was functional in the resistant MCF-7 cells. Both tamoxifen and fulvestrant resistant MCF-7 sublines expressed increased level of HER1, the activated form of HER3 and activated ERK1/2, whereas HER4 was severely decreased. In fulvestrant resistant T47D sublines, HER1 and HER4 expression were decreased whereas HER2 expression was highly upregulated. The kinase inhibitor screen disclosed HER receptors as common hits in the tamoxifen and fulvestrant resistant MCF-7 sublines. Regarding T47D, c-Src and Aurora were common hits for tamoxifen and fulvestrant resistant sublines. Tamoxifen resistant MCF-7 and T47D sublines were severely growth inhibited by ER knockdown with either siRNA or fulvestrant, but complete growth arrest required combined treatment with e.g ERK1/2 inhibitor. LTED MCF-7 cells were resistant to aromatase inhibitor treatment and sensitive to both tamoxifen and fulvestrant. Conclusions: In our large panels of MCF-7 and T47D derived endocrine resistant sublines, it was a general finding that ER is the main driver of growth of tamoxifen resistant tumor cells, and also of growth of LTED MCF-7 cells. The ER down modulator fulvestrant inhibited growth of tamoxifen resistant sublines but complete growth arrest required combined treatment with e.g. a kinase inhibitor targeting the activated growth promoting pathways in the resistant cells. In fulvestrant resistant sublines, ER played only a minor role in MCF-7 sublines and no role in T47D sublines. The HER system played a major role in fulvestrant resistant MCF-7 cells, whereas Aurora B and c-Src were new candidate targets for fulvestrant resistant T47D cells. These cell culture models reflect clinical findings and will be available for collaborative studies, e.g. to discover new predictive markers to select targeted therapy for resistant tumors. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-09-02.

S. aureushaemolysin A-induced IL-8 and IL-6 release from human airway epithelial cells is mediated by activation of p38- and Erk-MAP kinases and additional, cell type-specific signalling mechanisms

Soluble virulence-associated factors of Staphylococcus aureus like haemolysin A (Hla) induce secretion of chemo/cytokines from airway epithelial cells. To elucidate the potential roles of specific signalling pathways in this response, we treated 16HBE14o-, S9 or A549 cells with recombinant Hla (rHla). In a dose-dependent manner, rHla induced secretion of IL-8 in all three cell types, but IL-6 release only in 16HBE14o- and S9 cells. rHla-mediated secretion of IL-8 and IL-6 was suppressed by pre-incubation of cells with inhibitors of Erk type or p38 MAP kinases, indicating that activation of these signalling pathways is essential for IL-8 release in all three cell types and for IL-6 release in 16HBE14o- and S9 cells. The rHla-mediated phosphorylation and activation of p38 MAP kinase seem to depend on elevations in [Ca(2+)]i, an early response in rHla-treated cells. Inhibitors of calmodulin or calcium/calmodulin-dependent kinase II attenuated rHla-mediated release of IL-8 in 16HBE14o- and A549 cells and of IL-6 in 16HBE14o- cells. This indicates that rHla may mediate simultaneous activation of calmodulin-dependent processes as additional prerequisites for chemo/cytokine secretion.However, the inhibitors of calmodulin-dependent signalling did not affect rHla-induced p38 MAP kinase phosphorylation, indicating that this pathway works in parallel with p38 MAP kinase.

The Mitochondrial Complex I Activity Is Reduced in Cells with Impaired Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Function

Cystic fibrosis (CF) is a frequent and lethal autosomal recessive disease. It results from different possible mutations in the CFTR gene, which encodes the CFTR chloride channel. We have previously studied the differential expression of genes in CF and CF corrected cell lines, and found a reduced expression of MTND4 in CF cells. MTND4 is a mitochondrial gene encoding the MTND4 subunit of the mitochondrial Complex I (mCx-I). Since this subunit is essential for the assembly and activity of mCx-I, we have now studied whether the activity of this complex was also affected in CF cells. By using Blue Native-PAGE, the in-gel activity (IGA) of the mCx-I was found reduced in CFDE and IB3-1 cells (CF cell lines) compared with CFDE/6RepCFTR and S9 cells, respectively (CFDE and IB3-1 cells ectopically expressing wild-type CFTR). Moreover, colon carcinoma T84 and Caco-2 cells, which express wt-CFTR, either treated with CFTR inhibitors (glibenclamide, CFTR(inh)-172 or GlyH101) or transfected with a CFTR-specific shRNAi, showed a significant reduction on the IGA of mCx-I. The reduction of the mCx-I activity caused by CFTR inhibition under physiological or pathological conditions may have a profound impact on mitochondrial functions of CF and non-CF cells.

Effects of the Hedgehog Inhibitor GDC-0449, Alone or in Combination with Dasatinib, on BCR-ABL-Positive Leukemia Cells

Hedgehog (Hh)-glioma-associated oncogene homolog (Gli) signaling is implicated in a large number of human cancers such as leukemia. In this study, we investigated the effects of the potent Hh antagonist GDC-0449 on the BCR-ABL-positive cell line OM9;22 and primary samples when leukemia cells were protected by a feeder cell line (S9 cells). The numbers of OM9;22 cells significantly increased with S9 cells. Treatment of OM9;22 cells with GDC-0449 caused cell growth inhibition and induced apoptosis. Moreover, GDC-0449 inhibited the colony growth of Philadelphia chromosome (Ph)-positive primary samples. We next investigated the effects of a combination of GDC-0449 and dasatinib on these cell lines. The growth inhibition typically promoted by dasatinib was significantly reduced in the presence of S9 cells. Treatment of Ph-positive leukemia cells with GDC-0449 and dasatinib in the presence of S9 caused significantly more cytotoxicity than that caused by each drug alone. Inhibition of Gli1 or Gli2 by siRNA transfection reduced the growth of the Ph-positive cell line K562 and increased cytotoxicity of dasatinib. Moreover, colony formations of Gli1 or Gli2 knockdown cells were also reduced. Data from this study suggest that administration of the Hh inhibitor GDC-0449 inhibits BCR-ABL-positive cell growth and enhances the cytotoxic effects of dasatinib in the presence of feeder cells.

Implication of NADPH Oxidases in the Early Inflammation Process Generated by Cystic Fibrosis Cells

In cystic fibrosis (CF) patients, pulmonary inflammation is a major cause of morbidity and mortality. The aim of this study was to further investigate whether oxidative stress could be involved in the early inflammatory process associated with CF pathogenesis. We used a model of CFTR defective epithelial cell line (IB3-1) and its reconstituted CFTR control (S9) cell line cultured in various ionic conditions. This study showed that IB3-1 and S9 cells expressed the NADPH oxidases (NOXs) DUOX1/2 and NOX2 at the same level. Nevertheless, several parameters participating in oxidative stress (increased ROS production and apoptosis, decreased total thiol content) were observed in IB3-1 cells cultured in hypertonic environment as compared to S9 cells and were inhibited by diphenyleneiodonium (DPI), a well-known inhibitor of NOXs; besides, increased production of the proinflammatory cytokines IL-6 and IL-8 by IB3-1 cells was also inhibited by DPI as compared to S9 cells. Furthermore, calcium ionophore (A23187), which upregulates DUOX and NOX2 activities, strongly induced oxidative stress and IL-8 and IL-6 overexpression in IB3-1 cells. All these events were suppressed by DPI, supporting the involvement of NOXs in the oxidative stress, which can upregulate proinflammatory cytokine production by the airway CFTR-deficient cells and trigger early pulmonary inflammation in CF patients.

Regulatory role of β-arrestin-2 in cholesterol processing in cystic fibrosis epithelial cells

Cystic fibrosis (CF) cells exhibit an increase in the protein expression of β-arrestin-2 (βarr2) coincident with perinuclear accumulation of free cholesterol. Arrestins are proteins that both serve as broad signaling regulators and contribute to G-protein coupled receptor internalization after agonist stimulation. The hypothesis of this study is that βarr2 is an important component in the mechanisms leading to cholesterol accumulation characteristic of CF cells. To test this hypothesis, epithelial cells stably expressing GFP-tagged βarr2 (βarr2-GFP) and respective GFP-expressing control cells (cont-GFP) were analyzed by filipin staining. The βarr2-GFP cells show a late endosomal/lysosomal cholesterol accumulation that is identical to that seen in CF cells. This βarr2-mediated accumulation is sensitive to Rp-cAMPS treatment, and depleting βarr2 expression in CF-model cells by shRNA alleviates cholesterol accumulation compared with controls. Cftr/βarr2 double knockout mice also exhibit wild-type (WT) levels of cholesterol synthesis, and WT profiles of signaling protein expression have previously been shown to be altered in CF due to cholesterol-related pathways. These data indicate a significant regulatory role for βarr2 in the development of CF-like cholesterol accumulation and give further insight into cholesterol processing mechanisms. An impact of βarr2 expression on Niemann-Pick type C-1 (NPC1)-containing organelle movement is proposed as the mechanism of βarr2-mediated alterations on cholesterol processing. It is concluded that βarr2 expression contributes to altered cholesterol trafficking observed in CF cells.