Prevalence of PD-L1 in Cervical Cancer Patients and the Potential for Combining an Immune Checkpoint Inhibitor With Lenvatinib.

BACKGROUND/AIM: Cervical cancer is the fourth most common cause of cancer-related deaths in women worldwide. The potential for targeted therapy against the immune checkpoint programmed death 1 (PD-1)/programmed death-ligand 1 (PD-L1) and receptor tyrosine kinases was examined in cervical cancer patients and cell lines. MATERIALS AND METHODS: On tissue microarrays, PD-L1 was analyzed in 123 samples of patients with cervical cancer using immunohistochemistry. In SiHa, HeLa, and CaSki cervical cancer cell lines we examined the combination of lenvatinib with a PD-1/PD-L1 inhibitor using cell viability assays, the activation of cell signaling pathway proteins using western blots and gene expression using quantitative reverse transcriptase-PCR. RESULTS: Of 113 evaluable samples, 90 (79.6%) had more than 1% PD-L1 positive cells. The single treatment with the PD-1/PD-L1 inhibitor resulted in the greatest reduction in growth for CaSki and lenvatinib in HeLa cells. In contrast, the combined treatment of lenvatinib with the PD-1/PD-L1 inhibitor demonstrated a significantly stronger impeded proliferation compared to the single treatment in all three cell lines. Moreover, the combined treatment caused significantly less phosphorylation of the signaling molecules ERK and S6 in SiHa and of S6 and STAT3 in HeLa cells but not in CaSki. All treatments diminished the mRNA levels of PD-L1, Il-8, and FGFR in SiHa cells. CONCLUSION: PD1 is frequently expressed in cervical cancer samples. Combining lenvatinib with a PD-1/PD-L1 inhibitor diminished proliferation of cervical cancer cell lines. Consequently, this combination might be a promising option to treat cervical cancer. Signaling pathways involved in tumor cell growth are blocked by the combined treatment but still a model of the underlying mechanism cannot be drawn.

Distribution of HPV Subtypes in Diverse Anogenital and Oral Samples from Women and Correlation of Infections with Neoplasia of the Cervix.

BACKGROUND: Cancers and intraepithelial lesions of different anogenital areas as well as oral cancer are associated with human papilloma virus (HPV) infections. METHODS: In this study cervical, vaginal, vulvar, anal, and oral samples were taken from 509 patients visiting our dysplasia consultation clinic. HPV genotyping was performed using the EUROArray HPV test. RESULTS: Positivity of HR HPV was found in 60.4-64.3% of anogenital and 14.6% of oral samples. HPV 16 showed the highest incidence in all investigated areas. In cervical and vaginal samples HPV 31 was detected second most, while in vulvar, anal, and oral samples HPV 53 was the second most common subtype. HPV 18 was found lower in all areas, while HPV 51, HPV 52, and HPV 73 were detected higher than expected from published data. A good concordance between cervical, vaginal and vulvar samples was examined for most of the HPV. HR HPV infection was higher in cervical cancer (CC; 91.7%) and high-grade intraepithelial squamous lesions (HSIL; 93.9%) compared to low-grade SIL (LSIL; 69.6%) and normal samples (44.8%). CONCLUSION: In addition to the well described HPV subtypes, we found others with high incidences in the investigated areas which may be evident for HSIL and CC of those areas.

Inhibitors of PD-1/PD-L1 and ERK1/2 impede the proliferation of receptor positive and triple-negative breast cancer cell lines.

PURPOSE: Triple-negative breast cancer (TNBC) is characterized by an unfavorable prognosis and missing systemic therapeutic approaches beside chemotherapy. Targeting the immune checkpoint PD-1/PD-L1 showed promising results in breast cancer and especially in TNBC. The extracellular signal-regulated kinase 1/2 (ERK1/2) is an important driver of carcinogenesis. Here, the effect of combined PD-1/PD-L1 and ERK1/2 inhibitor treatment is investigated of cell growth and intracellular impact of breast cancer cell lines. METHODS: The IC50 values of each inhibitor and the effect of combined treatment were determined in three TNBC cell lines of different subtypes and one non-TNBC cell line. Phospho-specific antibodies were used in western blot analyses to investigate an effect on ERK1/2 activation. Expressions of immune modulatory and cell cycle-associated genes were examined by quantitative reverse transcription PCR. RESULTS: Both inhibitors PD-1/PD-L1 and ERK1/2 impeded the proliferation of TNBC to a higher extent than of non-TNBC. By combined treatment, cell lines were inhibited either synergistically or additively. ERK1/2 and S6 phosphorylation were reduced and expressions of c-Fos and FosL were diminished after ERK1/2 inhibitor as single and combined treatment. Between genes involved in immune modulation, IL-8 was upregulated in TNBC cells after combined treatment. CONCLUSION: In conclusion, combination of PD-1/PD-L1 and ERK1/2 inhibitors showed favorable effects for a new therapy strategy, with better results in TNBC cell lines than in non-TNBC cells. The effects have to be validated in models that can reflect the interaction between immune and tumor cells like the situation in the tumor micro-environment.

Toward Homogenous Antibody Drug Conjugates Using Enzyme-Based Conjugation Approaches.

In the last few decades, antibody-based diagnostic and therapeutic applications have been well established in medicine and have revolutionized cancer managements by improving tumor detection and treatment. Antibodies are unique medical elements due to their powerful properties of being able to recognize specific antigens and their therapeutic mechanisms such as blocking specific pathways, antibody-dependent cellular cytotoxicity, and complement-dependent cytotoxicity. Furthermore, modification techniques have paved the way for improving antibody properties and to develop new classes of antibody-conjugate-based diagnostic and therapeutic agents. These techniques allow arming antibodies with various effector molecules. However, these techniques are utilizing the most frequently used amino acid residues for bioconjugation, such as cysteine and lysine. These bioconjugation approaches generate heterogeneous products with different functional and safety profiles. This is mainly due to the abundance of lysine and cysteine side chains. To overcome these limitations, different site-direct conjugation methods have been applied to arm the antibodies with therapeutic or diagnostics molecules to generate unified antibody conjugates with tailored properties. This review summarizes some of the enzyme-based site-specific conjugation approaches.

PSMD9 expression correlates with recurrence after radiotherapy in patients with cervical cancer.

In the current retrospective cohort study, the expression of the Proteasome 26S non-ATPase Subunit 9 (PSMD9) was investigated in 102 patients with cervical cancer. The rat homologue of PSMD9, Bridge-1, was identified as a binding protein of the transcription factors PDX-1 and E-12 via its PDZ-domain. The aim of the current study was to evaluate the prognostic or predictive value of PSMD9 expression as a biomarker for patients with cervical cancer. Tissue microarrays were constructed from formalin-fixed paraffin-embedded tissue specimens of cervical cancer and peritumoral stroma after hysterectomy and a Bridge-1 antibody was used to perform immunohistochemistry. The immunoreactions were analyzed using an immunoreactive score, which evaluated the number of positive cells as well as their intensity of PSMD9 expression. A misinterpretation of statistically significant results after multiple testing was controlled by the false discovery rate correction using the algorithm of Benjamini and Hochberg. All tumor tissues and almost all peritumoral stroma tissues expressed PSMD9. The PSMD9 expression in tumor tissues was significantly higher compared with the peritumoral stroma. PSMD9 expression correlated significantly with the expression of the proliferation marker MIB-1. Patients with stronger PSMD9 expression tended to exhibit a higher odds ratio for the recurrence of the disease in all patients (n=102) as well as in the subgroup of 47 patients having received a combined chemoradiotherapy following hysterectomy. In the group of 62 patients having that received radiotherapy following hysterectomy, which included the chemoradiotherapy patients, a higher PSMD9 expression significantly increased the odds for a recurrence to 1.983-fold even after FDR correction (P=0.0304). In conclusion, PSMD9 was indicated to be overexpressed in tumor tissues and associated with tumor cell proliferation. Therefore, PSMD9 may be useful as a tumor marker. Furthermore, increased PSMD9 overexpression may be used to predict resistance against radiation.

Analysis of HPV genotype-specific concordance between EUROArray HPV and HPV 3.5 LCD-Array Kit in cervical samples of 163 patients.

PURPOSE: Human papilloma virus (HPV) as the most common viral infection of the anogenital tract is highly associated with intraepithelial neoplasia and cancer of the cervix and other anogential regions. To date, 15 high-risk (HR-) HPV and 3 probably/possibly HR-HPVs have been found to be associated with cervical cancer. Therefore, a screening especially for HR-HPV by appropriate tests is important for detection of precancerous lesions to prevent cancer. The purpose of this study was to analyze prospectively the concordance of the EUROArray HPV genotyping assay (Euroimmun; EUROArray) and the HPV 3.5 LCD-Array Kit (Chipron; LCD-Array). METHODS: Liquid-based, clinician-collected cervical cytology samples (n = 163) from women undergoing cervical inspection at the dysplasia consultation in the colposcopy clinic at the Medical Center-University of Freiburg, Germany were analyzed. Genotype-specific agreement was assessed by Cohen's kappa statistic and McNemar's P value of significance between proportions. RESULTS: Seventeen of the HR-HPV genotypes included in both assays were detected in 42.3% and 38% of samples by EUROArray and by LCD-Array, respectively; i.e. an agreement of 92.0% and a kappa value of 0.83 could be proven between the EUROArray and the LCD-Array. In 50 of 72 samples, identical HR-HPV genotypes were analyzed (81.9%, κ = 0.47) and genotyping for HPV 16 and/or 18 was highly concordant in both tests (relative agreement 96.3%, κ = 0.88). Detection of any HR-HPV was not significantly different after comparison of EUROArray with LCD-Array. CONCLUSION: Both of the tests showed comparable results for the detection of HPV in cervical specimens and permit these assays to be suitable for routine diagnostics.

Simultaneous and Independent Dual Site-Specific Self-Labeling of Recombinant Antibodies.

Antibody-based diagnostic and therapeutic reagents armed with effector molecules such as dyes and drugs offer hope in the battle against cancer. Several site-specific conjugation methods have been developed to equip antibodies with such effector molecules, but they tend to be expensive and involve multiple reaction steps. The conjugation of two different effector molecules to a single antibody also remains a major challenge. Here we describe a simple, controlled, and robust method for the dual site-specific conjugation of an antibody with two effector molecules in a single-pot reaction using the self-labeling SNAP and CLIP protein tags. We verified the principle of the method by labeling an epidermal growth factor receptor (EGFR)-specific single-chain antibody fragment (scFv-425) simultaneously with IRDye700 and Alexa-Fluor647. This dual-labeled antibody bound to EGFR+ ovarian cancer cell lines and tissue samples with high specificity, and its phototherapeutic efficacy was confirmed by the selective killing of EGFR+ cells in vitro.

Detection and Specific Elimination of EGFR+ Ovarian Cancer Cells Using a Near Infrared Photoimmunotheranostic Approach.

PURPOSE: Targeted theranostics is an alternative strategy in cancer management that aims to improve cancer detection and treatment simultaneously. This approach combines potent therapeutic and diagnostic agents with the specificity of different cell receptor ligands in one product. The success of antibody drug conjugates (ADCs) in clinical practice has encouraged the development of antibody theranostics conjugates (ATCs). However, the generation of homogeneous and pharmaceutically-acceptable ATCs remains a major challenge. The aim of this study is to detect and eliminate ovarian cancer cells on-demand using an ATC directed to EGFR. METHODS: An ATC with a defined drug-to-antibody ratio was generated by the site-directed conjugation of IRDye®700 to a self-labeling protein (SNAP-tag) fused to an EGFR-specific antibody fragment (scFv-425). RESULTS: In vitro and ex vivo imaging showed that the ATC based on scFv-425 is suitable for the highly specific detection of EGFR+ ovarian cancer cell, human tissues and ascites samples. The construct was also able to eliminate EGFR+ cells and human ascites cells with IC50 values of 45-66 nM and 40-90 nM, respectively. CONCLUSION: Our experiments provide a framework to create a versatile technology platform for the development of ATCs for precise detection and treatment of ovarian cancer cells.

Photoimmunotheranostic agents for triple-negative breast cancer diagnosis and therapy that can be activated on demand.

Triple-negative breast cancer (TNBC) is a heterogeneous disease in which the tumors do not express estrogen receptor (ER), progesterone receptor (PgR) or human epidermal growth factor receptor 2 (HER2). Classical receptor-targeted therapies such as tamoxifen or trastuzumab are therefore unsuitable and combinations of surgery, chemotherapy and/or radiotherapy are required. Photoimmunotheranostics is a minimally invasive approach in which antibodies deliver nontoxic photosensitizers that emit light to facilitate diagnosis and produce cytotoxic reactive oxygen species to induce apoptosis and/or necrosis in cancer cells. We developed a panel of photoimmunotheranostic agents against three TNBC-associated cell surface antigens. Antibodies against epidermal growth factor receptor (EGFR), epithelial cell adhesion molecule (EpCAM) and chondroitin sulfate proteoglycan 4 (CSPG4) were conjugated to the highly potent near-infrared imaging agent/photosensitizer IRDye®700DX phthalocyanine using SNAP-tag technology achieving clear imaging in both breast cancer cell lines and human biopsies and highly potent phototherapeutic activity with IC50values of 62-165 nM against five different cell lines expressing different levels of EGFR, EpCAM and CSPG4. A combination of all three reagents increased the therapeutic activity against TNBC cells by up to 40%.

Subtypes of Triple-negative Breast Cancer Cell Lines React Differently to Eribulin Mesylate.

BACKGROUND/AIM: Diagnosis of triple-negative breast cancer (TNBC) is associated with adverse prognosis, particularly in cases of chemotherapy resistance. The goal of this analysis was to compare TNBC vs. non-TNBC cell lines and those of distinct TNBC subtypes with regard to sensitivity to eribulin in vitro. MATERIALS AND METHODS: Breast cancer cell lines were subjected to cell-viability assays, apoptosis analyses, migration and invasion experiments, and quantitative real-time polymerase chain reaction after exposure to eribulin. RESULTS: Eribulin reduced cell viability in TNBC and non-TNBC cell lines in the sub-nanomolar range. Furthermore, exposure to eribulin induced apoptosis and decreased the rate of migration and invasion. Genes known to induce malignant transformation were differentially expressed after eribulin treatment. CONCLUSION: Eribulin had a strong antiproliferative effect on breast cancer cell lines, although we did not observe a significant difference between TNBC and non-TNBC cell lines with regard to sensitivity to eribulin.

A specific photoimmunotheranostics agent to detect and eliminate skin cancer cells expressing EGFR.

PURPOSE: The term "theranostics" represents a new paradigm in medicine especially for cancer treatment. This term was coined by Funkhouser in 2002 and defines a reagent that combines therapeutic and diagnostic properties. It is widely believed that theranostics agents will have considerable impact on healthcare before, during, and after disease by improving cancer prognosis and management simultaneously. Current theranostics approaches still rely on passive tumor targeting strategies, which have scattergun effects and tend to damage both neoplastic and non-neoplastic cells. METHODS: Here we describe a simple, controlled, and efficient method to generate homogeneous photoimmunotheranostics reagents. This method combines molecular optical imaging, photodynamic therapy, and immunotherapy using SNAP-tag technology. SNAP-tag is a derivative of the O(6)-alkylguanine-DNA alkyltransferase (AGT) which has the ability to efficiently conjugate to O(6)-benzylguanine (BG) molecules under physiological conditions depending on its folding pattern. RESULTS: The theranostics agent was able to specifically recognize various epidermal growth factor receptor (EGFR)-expressing skin cancer cell lines using flow cytometry analysis and confocal microscopy and eliminate them at EC50's of 32-55 nM. CONCLUSIONS: These experiments provide a framework for using SNAP-tag technology to generate homogeneous photoimmunotheranostics reagents with unified pharmacokinetic and therapeutic profiles. Furthermore, the reagent generated in this work could be used to simultaneously monitor and suppress the growth of skin squamous carcinoma and melanoma cells expressing EGFR.

Fatty acid synthase overexpression: target for therapy and reversal of chemoresistance in ovarian cancer.

BACKGROUND: Fatty acid synthase (FASN) is crucial to de novo long-chain fatty acid synthesis, needed to meet cancer cells' increased demands for membrane, energy, and protein production. METHODS: We investigated FASN overexpression as a therapeutic and chemosensitization target in ovarian cancer tissue, cell lines, and primary cell cultures. FASN expression at mRNA and protein levels was determined by quantitative real-time polymerase chain reaction and immunoblotting and immunohistochemistry, respectively. FASN inhibition's impact on cell viability, apoptosis, and fatty acid metabolism was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium-bromide assay, cell death detection enzyme-linked immunosorbent assay, immunoblotting, and (18) F-fluoromethylcholine uptake measurement, respectively. RESULTS: Relative to that in healthy fallopian tube tissue, tumor tissues had 1.8-fold average FASN protein overexpression; cell lines and primary cultures had 11-fold-100-fold mRNA and protein overexpression. In most samples, the FASN inhibitor cerulenin markedly decreased FASN expression and cell viability and induced apoptosis. Unlike concomitant administration, sequential cerulenin/cisplatin treatment reduced cisplatin's half maximal inhibitory concentration profoundly (up to 54%) in a cisplatin-resistant cell line, suggesting platinum (re)sensitization. Cisplatin-resistant cells displayed lower (18) F-fluoro-methylcholine uptake than did cisplatin-sensitive cells, suggesting that metabolic imaging might help guide therapy. CONCLUSIONS: FASN inhibition induced apoptosis in chemosensitive and platinum-resistant ovarian cancer cells and may reverse cisplatin resistance.

Reduced hemopexin levels in peritoneal fluid of patients with endometriosis.

OBJECTIVE: To study altered hemopexin concentrations in peritoneal fluid (PF) samples from patients with endometriosis. Recent data implicate a role of altered iron metabolism in endometriosis patients. Hemopexin is the major transport protein for heme. Like iron, heme exposure to the epithelial surface can provoke oxidative stress on the peritoneal epithelium. Therefore, altered hemopexin concentrations and heme scavenging in PF might play a role in the pathophysiology of endometriosis. DESIGN: Prospective explorative study. SETTING: Academic tertiary care center. PATIENT(S): Eighty symptomatic patients scheduled for laparoscopy for the diagnosis and/or therapy of endometriosis. INTERVENTION(S): Aspiration of PF samples during laparoscopy. MAIN OUTCOME MEASURE(S): Hemopexin and heme concentration in PF. RESULT(S): At laparoscopy, 47 of 80 (58.8%) patients exhibited endometriosis, and 33 (41.2%) were proven disease-free (CO). By means of ELISA significantly lower concentrations of hemopexin in the samples from patients with endometriosis (endometriosis 0.377 ± 0.16 mg/mL) compared with controls (disease-free 0.479 ± 0.20 mg/mL) could be demonstrated. Heme levels in the samples were not significantly different between groups (endometriosis 9.130 ± 6.124 µM and disease-free 9.990 ± 4.485 µM). There was no significant correlation between heme and hemopexin levels (Pearson's correlation coefficient r = -0.146). Demographic data between the groups were comparable. CONCLUSION(S): These data provide further evidence that hemopexin is significantly down-regulated in PF samples from patients with endometriosis compared with controls. This study confirms recent findings in two-dimensional gel electrophoresis demonstrating a down-regulation of hemopexin in PF from patients with endometriosis in a larger series of samples.

Breakpoint characterization of the der(19)t(11;19)(q13;p13) in the ovarian cancer cell line SKOV-3.

About 20% of ovarian carcinomas show alterations of 19p13 and/or 19q13 in the form of added extra material whose origin often is from chromosome 11. Based on earlier spectral karyotype analysis of the ovarian cancer cell line SKOV-3, which shows an unbalanced translocation der(19)t(11;19), the aim of this study was to determine the precise breakpoints of that derivative chromosome. After rough delimitation of the breakpoints of microdissected derivative chromosomes by array analysis, we designed a matrix of primers spanning 11q13.2 and 19p13.2 detecting multiple amplicons on genomic and cDNA. Sequencing the amplicons, accurate localization of both breakpoints on both chromosomes was possible and we found that exon 14 of HOOK2 from chromosome 19 and exon 2 of ACTN3 from chromosome 11 were fused in the derivative chromosome. The breakpoint in the HOOK2 gene was in an intrinsic triplet of nucleic acids leading to a shift in the ACTN3 reading frame in the derivative chromosome. This frameshift alteration should give rise to an early stop codon causing a loss of function of ACTN3. Signals in two-dimensional Western blotting exactly match to calculated molecular mass and the isoelectric point of the fusion protein.

Systematic analysis and validation of differential gene expression in ovarian serous adenocarcinomas and normal ovary.

PURPOSE: Cancer of the ovary confers the worst prognosis among women with gynecological malignancies, primarily because most ovarian cancers are diagnosed at late stage. Hence, there is a substantial need to develop new diagnostic biomarkers to enable detection of ovarian cancer at earlier stages, which would confer better prognosis. In addition, the identification of druggable targets is of substantial interest to find new therapeutic strategies for ovarian cancer. METHODS: The expression of 22,500 genes in a series of 67 serous papillary carcinomas was compared with 9 crudely enriched normal ovarian tissue samples by RNA hybridization on oligonucleotide microarrays. Multiple genes with near-uniformly expression were elevated in carcinomas of varying grade and malignant potential, including several previously described genes (e.g., MUC-1, CD9, CD24, claudin 3, and mesothelin). We performed immunohistochemical staining with antibodies against several of the proteins encoded by differentially expressed genes in an independent cohort of 71 cases of paraffin-embedded ovarian cancer samples. RESULTS: We found striking differences in EpCAM (p < 0.005), CD9 (p < 0.001), MUC-1 (p < 0.001), and claudin 3 proteins (p < 0.001) but not for mesothelin (p > 0.05) using the Mann-Whitney U test. CONCLUSIONS: Protein expression of a majority of the differentially expressed genes tested was found to be elevated in ovarian carcinomas and, as such, define potential new biomarkers or targets.

Progression-free survival in ovarian cancer is reflected in epigenetic DNA methylation profiles.

OBJECTIVE: Many patients with ovarian cancer disease relapse within 6 months after adjuvant chemotherapy, with a limited prognosis. Epigenetic modifications have been shown to play an important role in tumor development and formation. Therefore, global analysis of DNA methylation patterns might reveal specific CpG sites that correlate with progression-free interval (PFI) after therapy. METHODS: Twenty samples of advanced ovarian cancer with a predominantly serous papillary histological subtype were subjected to DNA methylation profiling. Illumina HumanMethylation27 BeadChip technology was used for simultaneous analysis of 27,578 CpG sites in >14,000 genes. RESULTS: Differential DNA methylation of various cytosines correlated with PFI. However, this becomes only significant by classification according to PFI with a cutoff of >28 months. Longer survival was associated with hypomethylation at specific CpG sites (e.g. GREB1, TGIF and TOB1) and hypermethylation in other genes (e.g. TMCO5, PTPRN and GUCY2C). Gene ontology analysis revealed that differentially methylated genes were significantly overrepresented in the categories telomere organization, mesoderm development and immune regulation. CONCLUSION: Epigenetic modifications at specific CpG sites correlate with PFI in ovarian cancer. Therefore, such analysis might be of prognostic value.

Combined treatment with TRAIL and PPARγ ligands overcomes chemoresistance of ovarian cancer cell lines.

PURPOSE: Ovarian cancer accounts for the highest mortality among all gynecological cancers, mainly due to the fast developing chemoresistance. The death ligand TRAIL induces apoptosis and is able to sensitize tumor cells to cytostatic drugs without affecting physiological tissue. Combined treatment of TRAIL and the antidiabetic acting PPARγ ligands was shown to induce apoptosis synergistically in different ovarian cancer cell lines. METHODS: To investigate feasible TRAIL-dependent inhibition of proliferation and induction of apoptosis in chemoresistant ovarian cancer cell lines, the drug- and TRAIL-sensitive HEY cell line was utilized to develop subclones with selective resistance against cisplatin, etoposide, docetaxel, paclitaxel, gemcitabine and pemetrexed, as well as against TRAIL as control cell line. Expression of the key factors of the TRAIL signaling pathway, TRAIL receptors 1-4, caspase-8, FLIP and XIAP, was analyzed before and after TRAIL treatment by immunoblotting. RESULTS: Cell proliferation experiments showed TRAIL-dependent inhibition that was further increased by combination treatment with the PPARγ ligands. Simultaneous exposure of TRAIL and the PPARγ ligands also resulted in enhanced induction of apoptosis even in partial TRAIL-resistant HEY cell lines. In the parental HEY cell line, additional treatment with the PPARγ ligands led to an increased protein expression of DR5 and a further decline of XIAP expression. CONCLUSION: Therefore, the combinational treatment with TRAIL and PPARγ ligands might be a promising experimental therapy because the PPARγ ligands, especially d15-PGJ(2), sensitize drug-resistant ovarian cancer cells to TRAIL-induced apoptosis.

The t(14,15) in mouse strain CBA/CaH-T(14;15)6Ca/J causes a break in the ADAMTS12 gene.

The mouse strain CBA/CaH-T(14;15)6Ca/J carries a homozygous balanced reciprocal translocation between mouse chromosomes 14 and 15, but the break points of this translocation have not previously been examined in detail. Using fluorescent in situ hybridization, we assigned the break point in 14qE3 to a 200-kb region devoid of any known gene. We similarly defined the break point in 15qA1 to a 27-kb region containing involving ADAMTS12. The chromosomal break likely is between exons 2 and 3 of ADAMTS12. This gene encodes a disintegrin and metalloproteinase with thrombospondin motifs, and this product plays crucial roles in both vascularization and cancer progression and has been implicated in the development of arthritis. The CBA/CaH-T(14;15)6Ca/J mouse strain likely is a suitable model for further examination of the influences of defective ADAMTS12 in various pathologic processes.

Combination of enzastaurin and pemetrexed inhibits cell growth and induces apoptosis of chemoresistant ovarian cancer cells regulating extracellular signal-regulated kinase 1/2 phosphorylation.

New strategies in the therapy for malignant diseases depend on a targeted influence on signal transduction pathways that regulate proliferation, cell growth, differentiation, and apoptosis by the activation of serine/threonine kinases. Enzastaurin (LY317615.HCl), a selective inhibitor of protein kinase Cbeta (PKCbeta), is one of these new drugs and causes inhibition of proliferation and induction of apoptosis. Pemetrexed, a multitarget inhibitor of folate pathways, is broadly active in a wide variety of solid tumors. Therefore, the effect of enzastaurin and the combination treatment with pemetrexed was analyzed when applied to the drug-sensitive ovarian cancer cell line HEY and various subclones with drug resistance against cisplatin, etoposide, docetaxel, and paclitaxel, as well as pemetrexed, and gemcitabine. In these novel chemoresistant subclones, the expression of the enzastaurin targets PKCbetaII and glycogen synthase kinase 3beta (GSK3beta) was analyzed. Exposition to enzastaurin showed various inhibitory effects on phosphorylated forms of GSK3beta and the mitogen-activated protein kinase extracellular signal-regulated kinase 1/2. Cell proliferation experiments identified the cell line-specific half-maximal inhibitory concentration values of enzastaurin and a synergistic inhibitory effect by cotreatment with the antifolate pemetrexed. Induction of apoptosis by enzastaurin treatment was investigated by Cell Death Detection ELISA and immunoblot analyses. Simultaneous treatment with pemetrexed resulted in an enhanced inhibition of proliferation and induction of apoptosis even in partial enzastaurin-resistant cells. Therefore, the combinational effect of enzastaurin and pemetrexed can have promise in clinical application to overcome the fast-growing development of resistance to chemotherapy in ovarian cancer.

A role for membrane-bound CD147 in NOD2-mediated recognition of bacterial cytoinvasion.

NOD2 is an intracellular receptor for the bacterial cell wall component muramyl dipeptide. Mutations in the leucine-rich repeat region of NOD2, which lead to an impaired recognition of muramyl dipeptide, have been associated with chronic inflammatory diseases of barrier organs such as Crohn disease, asthma and atopic eczema. In this study we identify CD147 (also known as BSG and EMMPRIN), a membrane-bound regulator of cellular migration, differentiation and inflammatory processes, as a protein interaction partner of NOD2. We demonstrate a complex influence of the CD147-NOD2 interaction on NOD2-dependent signaling responses. We show that CD147 itself acts as an enhancer of the invasion of Listeria monocytogenes, an intracellular bacterial pathogen. We propose that the CD147-NOD2 interaction serves as a molecular guide to regulate NOD2 function at sites of pathogen invasion.