Positive Human Cancer Cell Research Articles

Evaluation of PDL1 positive cancer cell-specific binding activity of recombinant anti-PDL1 scFv.

Programmed cell death-ligand 1 (PDL1) is a transmembrane protein that is characterized as an immune regulatory molecule. We recently developed a recombinant single-chain fragment of variable domain (scFv) against PDL1, which showed high binding efficiency to purified recombinant PDL1 protein. However, at that time, proof-of-concept data for the effect of scFv using PDL1-expressing cells was lacking. In this study, we conducted two kinds of cell-based immunoassays, western blotting and enzyme-linked immunosorbent assay, using anti-PDL1 scFv. The results indicate that scFv can selectively and sensitively detect PDL1 from PDL1 positive human cancer cell lines. Our findings suggest that scFv could be used as a potential PDL1 inhibitor agent and probe for cell-based immunoassays to detect PDL1.

Development of a novel humanized mouse model for improved evaluation of in vivo anti-cancer effects of anti-PD-1 antibody.

Immune checkpoint inhibitors (ICIs) have revolutionized the treatment of cancer in the clinic. Further discovery of novel drugs or therapeutic protocols that enhance efficacy requires reliable animal models that recapitulate human immune responses to ICI treatment in vivo. In this study, we utilized an immunodeficient NOG mouse substrain deficient for mouse FcγR genes, NOG-FcγR−/− mice, to evaluate the anti-cancer effects of nivolumab, an anti-programmed cell death-1 (PD-1) antibody. After reconstitution of human immune systems by human hematopoietic stem cell transplantation (huNOG-FcγR−/− mice), four different programmed death-ligand 1 (PD-L1)-positive human cancer cell lines were tested. Among them, the growth of three cell lines was strongly suppressed by nivolumab in huNOG-FcγR−/− mice, but not in conventional huNOG mice. Accordingly, immunohistochemistry demonstrated the enhanced infiltration of human T cells into tumor parenchyma in only nivolumab-treated huNOG-FcγR−/− mice. Consistently, the number of human T cells was increased in the spleen in huNOG-FcγR−/− mice by nivolumab but not in huNOG mice. Furthermore, human PD-L1 expression was strongly induced in the spleen of huNOG-FcγR−/− mice. Collectively, our results suggest that the anti-cancer effects of anti-PD-1 antibodies can be detected more clearly in NOG-FcγR−/− mice than in NOG mice.

Design, Synthesis and Evaluation of New Bioactive Oxadiazole Derivatives as Anticancer Agents Targeting Bcl-2.

A series of 2-(1H-indol-3-yl)-5-substituted-1,3,4-oxadiazoles, 4a–m, were designed, synthesized and tested in vitro as potential pro-apoptotic Bcl-2 inhibitory anticancer agents based on our previously reported hit compounds. Synthesis of the target 1,3,4-oxadiazoles was readily accomplished through a cyclization reaction of indole carboxylic acid hydrazide 2 with substituted carboxylic acid derivatives 3a–m in the presence of phosphorus oxychloride. New compounds 4a–m showed a range of IC50 values concentrated in the low micromolar range selectively in Bcl-2 positive human cancer cell lines. The most potent candidate 4-trifluoromethyl substituted analogue 4j showed selective IC50 values of 0.52–0.88 μM against Bcl-2 expressing cell lines with no inhibitory effects in the Bcl-2 negative cell line. Moreover, 4j showed binding that was two-fold more potent than the positive control gossypol in the Bcl-2 ELISA binding affinity assay. Molecular modeling studies helped to further rationalize anti-apoptotic Bcl-2 binding and identified compound 4j as a candidate with drug-like properties for further investigation as a selective Bcl-2 inhibitory anticancer agent.

Abstract 1535: CBA-1205, a novel glycoengineered humanized antibody targeting DLK-1 exhibits potent anti-tumor activity in DLK-1 expressing tumor xenograft models

Abstract Delta-like 1 homolog (DLK-1) is a type I transmembrane protein with 6 tandem EGF-like motifs in extracellular region. In fetal development, DLK-1 is expressed at high levels in undifferentiated, highly proliferative tissues, such as, liver, kidney, skeletal muscle and brain. In the fetal liver, it has reported that DLK-1 positive hepatoblasts have a differentiation capability toward hepatocyte and cholangiocyte in vitro. In adult tissues, its expression is restricted in tissues such as adrenal grand and hypophysis. On the other hand, overexpression of DLK-1 in cancers have been reported in several cancer types, such as hepatocellular carcinoma (HCC), neuroblastoma, rhabdomyosarcoma, small cell lung carcinoma, myelodysplastic syndrome. Interestingly, the expression of DLK-1 is overlapped with other hepatic progenitor cell markers such as EpCAM in HCC and DLK-1 has been suggested to be a marker of cancer stem cells in HCC. According to these selective expressions of DLK-1 in cancer tissues as well as cancer stem cells in HCC, DLK-1 is supposed to be a potential target for antibody-based therapy. CBA-1205 is a novel humanized antibody (IgG1/κ) targeting DLK-1 which was glycol-engineered by GlymaxX® to potentiate antibody-dependent cellular cytotoxicity (ADCC) activity. The purpose of this study is to evaluate the anti-tumor activity of CBA-1205 in multiple human cancer cell lines in vitro and xenograft models in vivo. In vitro, CBA-1205 showed potent ADCC activity in several DLK-1 positive human cancer cell lines for different cancer types. In vivo, CBA-1205 exhibited potent anti-tumor activity in several human cancer xenograft mouse models. In a Hep3B HCC xenograft model, we evaluated dose-dependent efficacy of CBA-1205 at a dose of 0.1, 0.3, 1, 3 and 10 mg/kg by the intraperitoneally injection. The tumor growth was inhibited in a dose-dependent manner and the inhibition rate at the dose of ≥ 1 mg/kg was more than 90% compared to isotype human IgG1 control. At 10 mg/kg, CBA-1205 treatment resulted in tumor regression in all mice and 4 complete tumor elimination out of 8 mice was observed. Single injection of 10 mg/kg CBA-1205 induced tumor apoptosis and reduced the CD31 positive tumor micro vessel at 24 and 48 h after the treatment. Similar anti-tumor activity of CBA-1205 was exhibited in other xenograft model of HCC-derived HepG2 and neuroblastoma-derived SK-N-FI, BE2-C and Kelly. In conclusion, we demonstrated that a novel glycoengineered humanized anti-DLK-1 antibody, CBA-1205, exerts potent anti-tumor activity in vitro and in vivo in multiple models and could be a novel treatment option for DLK-1 expressing cancer such as HCC. First-in-human Phase I study of CBA-1205 is planned to be initiated in Q1 2020. Citation Format: Koji Nakamura, Kota Takahashi, Izumi Sakaguchi, Zhang Lingyi, Hiroyuki Yanai, Toru Kanke. CBA-1205, a novel glycoengineered humanized antibody targeting DLK-1 exhibits potent anti-tumor activity in DLK-1 expressing tumor xenograft models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1535.

A new DNA sensor design for the simultaneous detection of HPV type 16 and 18 DNA

In this work, we designed and fabricated two new “signal-on” electrochemical DNA sensors based on a sandwich-hybridization employing pyrrolidinyl peptide nucleic acid probes. The sensors comprised of a capture PNA probe (P1) immobilized on a screen-printed carbon electrode (SPCE) and an anthraquinone-labeled signaling probe (AQ-P2) designed to be partially complementary to the target DNA either at upstream (ASU) or downstream (ASD) positions on the DNA sequence hybridized to the P1 probe. In the presence of the DNA target, the ASD sensor showed a higher signal response when compared to the ASU sensor. The ASD sensor was then applied to simultaneously detect two high-risk human papillomavirus (HPV) DNA sequences. The target DNA was detected in the range of 0.5–100 nM, and the limits of detection (LODs) of 150 and 153 pM (3SDblank/slope) were obtained for HPV types 16 and 18, respectively. This developed sensor was successfully applied to detect a PCR-amplified sample derived from HPV type 16 (SiHa) and 18 (HeLa) positive human cancer cell lines. These findings are relevant for designing effective sensors, and the developed sensor can be readily applied to detect a wide range of DNA targets.

Abstract 5570: A novel approach to targeting the IDO/TDO pathway through degradation of the immunosuppressive metabolite kynurenine

Abstract Checkpoint inhibitors have become the cornerstone for new innovation in immune-based oncology therapy. Several orthogonal immune pathways are currently being investigated to relieve suppression or boost activity of the innate and adaptive immune system. The IDO immune-metabolism pathway was recently clinically validated in melanoma in combination with checkpoint inhibition. This opens up a new approach to relieving suppressive mediators and lends credence to the tumor microenvironment containing small molecule metabolites that induce immune tolerance. Both indoleamine-pyrrole 2,3-dioxygenase 1 (IDO1) and Tryptophan 2,3-dioxygenase (TDO) enzymes metabolize tryptophan, forming Kynurenine which binds the aryl hydrocarbon receptor (AHR) in multiple innate and adaptive immune cell types causing a net immunosuppressive effect. Both enzymes are upregulated across many tumor types, however, only the IDO1 enzyme has thus far been addressed in the clinic with small molecule inhibitors. We have postulated that enzyme-mediated depletion of Kynurenine into safe and immunologically inert metabolites can alleviate tumor immunosuppression. We have cloned and characterized several bacterial Kynureninases (KYNase) which preferentially degrade Kynurenine with a >1,000 higher kcat/KM as opposed to mammalian enzymes that cleave 3-OH Kynurenine. We show that PEGylated bacterial KYNases can deplete Kynurenine produced by IDO1+, TDO+ and IDO1/TDO+ dual positive human cancer cells whereas, the IDO1 inhibitor epacadostat or TDO inhibitor 680C91 only selectively inhibited Kyn production in IDO1+ or TDO+ cells respectively. In vivo, a single subcutaneous dose of KYNase in B16F10 tumor-bearing mice was able to deplete Kynurenine in both the plasma and tumors and increase effector T-cells in the tumor. KYNase demonstrated significant tumor growth inhibition and survival benefit either as a single agent or in combination with checkpoint inhibitors (anti-PD1 or anti-CTLA4) in B16F10, CT26 and 4T1 models. Interestingly, KYNase combined with anti-PD1, showed greater efficacy than epacadostat / anti-PD1 combination in CT26 tumor bearing mice. A pharmacologically optimized human KYNase is currently moving toward clinical development for the treatment of cancers where both IDO/TDO pathways play a significant immunosuppressive role through kynurenine production. Citation Format: Michelle Zhang, Everett Stone, Todd A. Triplett, Kendra Triplett, Candice Lamb, Christos S. Karamitros, John Blazek, George Georgiou, Mark G. Manfredi. A novel approach to targeting the IDO/TDO pathway through degradation of the immunosuppressive metabolite kynurenine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5570. doi:10.1158/1538-7445.AM2017-5570

Electrochemical detection of human papillomavirus DNA type 16 using a pyrrolidinyl peptide nucleic acid probe immobilized on screen-printed carbon electrodes

An electrochemical biosensor based on an immobilized anthraquinone-labeled pyrrolidinyl peptide nucleic acid (acpcPNA) probe was successfully developed for the selective detection of human papillomavirus (HPV) type 16 DNA. A 14-mer acpcPNA capture probe was designed to recognize a specific 14 nucleotide region of HPV type 16 L1 gene. The redox-active label anthraquinone (AQ) was covalently attached to the N-terminus of the acpcPNA probe through an amide bond. The probe was immobilized onto a chitosan-modified disposable screen-printed carbon electrode via a C-terminal lysine residue using glutaraldehyde as a cross-linking agent. Hybridization with the target DNA was studied by measuring the electrochemical signal response of the AQ label using square-wave voltammetric analysis. The calibration curve exhibited a linear range between 0.02 and 12.0µM with a limit of detection and limit of quantitation of 4 and 14nM, respectively. This DNA sensing platform was successfully applied to detect the HPV type 16 DNA from a PCR amplified (240bp fragment of the L1 gene) sample derived from the HPV type 16 positive human cancer cell line (SiHa), and failed to detect the HPV-negative c33a cell line. The sensor probe exhibited very high selectivity for the complementary 14 base oligonucleotide over the non-complementary oligonucleotides with sequences derived from HPV types 18, 31 and 33. The proposed sensor provides an inexpensive tool for the early stage detection of HPV type 16, which is an important biomarker for cervical cancer.

Suppression of Ku80 Correlates with Radiosensitivity and Telomere Shortening in the U2OS Telomerase-negative Osteosarcoma Cell Line

Ku70/80 heterodimer is a central element in the nonhomologous end joining (NHEJ) DNA repair pathway, Ku80 playing a key role in regulating the multiple functions of Ku proteins. It has been found that the Ku80 protein located at telomeres is a major contributor to radiosensitivity in some telomerase positive human cancer cells. However, in ALT human osteosarcoma cells, the precise function in radiosensitivity and telomere maintenance is still unknown. The aim of this study was to investigate the effects of Ku80 depletion in the U2OS ALT cell line cell line. Suppression of Ku80 expression was performed using a vector-based shRNA and stable Ku80 knockdown in cells was verified by Western blotting. U2OS cells treated with shRNA-Ku80 showed lower radiobiological parameters (D0, Dq and SF2) in clonogenic assays. Furthermore, shRNA-Ku80 vector transfected cells displayed shortening of the telomere length and showed less expression of TRF2 protein. These results demonstrated that down-regulation of Ku80 can sensitize ALT cells U2OS to radiation, and this radiosensitization is related to telomere length shortening.

Orientation‐Controlled Conjugation of Haloalkane Dehalogenase Fused Homing Peptides to Multifunctional Nanoparticles for the Specific Recognition of Cancer Cells

In control: The ligand positioning and orientation on multifunctional nanoparticles (MNP) are controlled by using a versatile bimodular approach that exploits an 11 amino acid (U11) homing peptide genetically fused with a haloalkane dehalogenase (HALO) enzyme. The resultant nanoparticles have good targeting efficiency and selectivity toward broadly occurring urokinase plasminogen activator receptor positive human cancer cells.

Ridaifen B, a tamoxifen derivative, directly binds to Grb10 interacting GYF protein 2

Ridaifen B (RID-B) is a tamoxifen derivative that potently inhibits breast tumor growth. RID-B was reported to show anti-proliferating activity for a variety of estrogen receptor (ER)-positive human cancer cells. Interestingly, RID-B was also reported to possess higher potency than that of tamoxifen even for some ER-negative cells, suggesting an ER-independent mechanism of action. In this study, a T7 phage display screen and subsequent binding analyses have identified Grb10 interacting GYF protein 2 (GIGYF2) as a RID-B-binding protein. Using a cell-based assay, the Akt phosphorylation level mediated by GIGYF2 was found to have decreased in the presence of RID-B.

Abstract LB-347: Is PDX-1 a new universal molecular target for PDX-1 positive human cancer therapy? In vitro and in vivo evidence

Abstract Introduction: Pancreatic and duodenal homeobox factor 1 (PDX-1) is a homeodomain-containing transcription factor, expression of which is specifically restricted to the islets and some endocrine cells of the duodenum and gastric antrum in adults. However, recent studies have revealed that over-expression of PDX-1 occurs in many different types of solid human cancer specimens including pancreatic, liver, prostate, ovarian, kidney, gastric, breast, and colon cancers by immunohistochemical analysis. It is not clear whether PDX-1 could serve as a universal therapeutic target for any PDX-1 positive cancer types besides pancreatic cancer. Here we report the anti-cancer effects of PDX-1 by suppressing the expression of PDX-1 with synthetic siRNA and virus-expressed shRNA in different type of PDX-1 positive cancers in vitro and in vivo. Method and Materials: Human cancer cells (3 pancreatic, 4 breast, 2 prostate, 1 ovary, 1 liver, 1 cervical, 1 nasopharyngeal) and 2 non-cancer human cells were employed to determine the expression of hPDX-1 by quantitative real-time RT-PCR and Western Blotting. PDX-1 siRNAs targeting different human PDX-1 sites and negative control siRNA were synthesized by Dharmacon and transfections were performed ont the cell cultures mentioned above. shRNA transfections were mediated by Lentivirus and Adenovirus vectors. Apoptosis assay was carried out using Annexin V labeling kit by FACS. In vivo anti-cancer procedure: Nude mice bearing different human cancers were intra-tumorly administrated PDX-1 shRNA-expressing adenovirus (107 PFU every other 2 days for 2 weeks) and the tumor size was measured at different times and the tumor weight was weighed after another 2 weeks. Results: PDX-1 over-expression in different human cancer cell was revealed by real-time RT-PCR as compared with non-cancer control cells. By using both synthetic PDX-1 siRNA and Lentivirus mediated shRNA, PDX-1 was effectively suppressed as determined by RT-PCR and Western Blot analysis. Significant cell apoptosis was induced consequently as measured with Annexin V-FACS assay in vitro in various types of human cancers, such as pancreas, liver, prostate, ovarian, and breast cancers, but not in non-cancer control cells. After intra-tumor administrated PDX-1 shRNA-expressing adenovirus in human ovarian and prostate cancer xenograft mice in vivo, a significant tumor shrink (97.05% Inhibitory rate, p <0.05) was observed as compared with negative control and blank control. Conclusion: PDX-1 over-expression was found in various types of human cancers at different levels and silencing PDX-1 expression by a RNA interference resulted in cancer cell specific apoptosis thereby indicating the anti-cancer effects of PDX-1 in vitro and in vivo. Our results suggested that PDX-1 might be an effective and universal molecular target for various types of PDX-1 positive human cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-347.

Tumor Detection by Imaging Proteolytic Activity

The cell surface protease membrane-type serine protease-1 (MT-SP1), also known as matriptase, is often upregulated in epithelial cancers. We hypothesized that dysregulation of MT-SP1 with regard to its cognate inhibitor hepatocyte growth factor activator inhibitor-1 (HAI-1), a situation that increases proteolytic activity, might be exploited for imaging purposes to differentiate malignant from normal tissue. In this study, we show that MT-SP1 is active on cancer cells and that its activity may be targeted in vivo for tumor detection. A proteolytic activity assay with several MT-SP1-positive human cancer cell lines showed that MT-SP1 antibodies that inhibit recombinant enzyme activity in vitro also bind and inhibit the full-length enzyme expressed on cells. In contrast, in the same assay, MT-SP1-negative cancer cell lines were inactive. Fluorescence microscopy confirmed the cell surface localization of labeled antibodies bound to MT-SP1-positive cells. To evaluate in vivo targeting capability, 0.7 to 2 nmoles of fluorescently labeled antibodies were administered to mice bearing tumors that were positive or negative for MT-SP1. Antibodies localized to MT-SP1-positive tumors (n = 3), permitting visualization of MT-SP1 activity, whereas MT-SP1-negative tumors (n = 2) were not visualized. Our findings define MT-SP1 activity as a useful biomarker to visualize epithelial cancers using a noninvasive antibody-based method.

Fhit-deficient normal and cancer cells are mitomycin C and UVC resistant

To identify functions of the fragile tumour suppressor gene, FHIT, matched pairs of Fhit-negative and -positive human cancer cell clones, and normal cell lines established from Fhit −/− and +/+ mice, were stressed and examined for differences in cell cycle kinetics and survival. A larger fraction of Fhit-negative human cancer cells and murine kidney cells survived treatment with mitomycin C or UVC light compared to matched Fhit-positive cells; ∼10-fold more colonies of Fhit-deficient cells survived high UVC doses in clonigenic assays. The human cancer cells were synchronised in G1, released into S and treated with UVC or mitomycin C. At 18 h post mitomycin C treatment ∼6-fold more Fhit-positive than -negative cells had died, and 18 h post UVC treatment 3.5-fold more Fhit-positive cells were dead. Similar results were obtained for the murine −/− cells. After low UVC doses, the rate of DNA synthesis in −/− cells decreased more rapidly and steeply than in +/+ cells, although the Atr–Chk1 pathway appeared intact in both cell types. UVC surviving Fhit −/− cells appear transformed and exhibit >5-fold increased mutation frequency. This increased mutation burden could explain the susceptibility of Fhit-deficient cells in vivo to malignant transformation.

Calcitonin receptor gene expression in K562 chronic myelogenous leukemic cells

BackgroundThe peptide hormone calcitonin (CT) can significantly effect the proliferation rate of CT receptor (CTR) positive human cancer cells. We wish to identify additional human cancers expressing CTRs and assay the effects of CT on their growth rates and signal transduction pathways.ResultsThe expression of the human calcitonin receptor (hCTR) gene in the chronic myelogenous leukemia cell line K562 was examined. RT-PCR on total RNA extracted from K562 cells detected the presence of hCTR mRNA. Further analysis demonstrated that multiple hCTR isoforms were present. Incubation of K562 cells with salmon calcitonin (sCT), but not amylin, caused an increase in intracellular levels of cAMP similar to that induced by forskolin treatment. We further demonstrated that butyrate induced erythroid differentiation of K562 cells caused a significant decrease in hCTR mRNA levels. However, phorbol myristate acetate (PMA) induced megakaryocytic differentiation of these cells had no significant effect on hCTR mRNA levels. We demonstrated that exposure to various concentrations of sCT had no effect on the cellular proliferation of K562 cells in vitro.ConclusionChronic myelogenous k562 cells express multiple CTR isoforms. However, CT does not effect K562 proliferation rates. It is likely that the small increase in intracellular levels of cAMP following CT treatment is not sufficient to interfere with cellular growth.