scholarly journals The cytotoxic conjugate of highly internalizing tetravalent antibody for targeting FGFR1-overproducing cancer cells

2021 ◽  
Vol 27 (1) ◽  
Author(s):  
Marta Poźniak ◽  
Natalia Porębska ◽  
Mateusz Adam Krzyścik ◽  
Aleksandra Sokołowska-Wędzina ◽  
Kamil Jastrzębski ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Specific Binding ◽  
Drug Carrier ◽  
Growth Factor Receptor ◽  
Cytotoxic Drug ◽  
Downstream Signaling ◽  
Drug Conjugates ◽  
Oncology Research ◽  
The Impact

Abstract Background Antibody drug conjugates (ADCs) represent one of the most promising approaches in the current immuno-oncology research. The precise delivery of cytotoxic drugs to the cancer cells using ADCs specific for tumor-associated antigens enables sparing the healthy cells and thereby reduces unwanted side effects. Overexpression of fibroblast growth factor receptor 1 (FGFR1) has been demonstrated in numerous tumors and thereby constitutes a convenient molecular target for selective cancer treatment. We have recently engineered tetravalent anti-FGFR1 antibody, T-Fc, and have demonstrated that it displays extremely efficient internalization into FGFR1 producing cells, a feature highly desirable in the ADC approach. We have revealed that T-Fc mediates clustering of FGFR1, largely enhancing the uptake of FGFR1-T-Fc complexes by induction of clathrin-independent endocytic routes. The aim of this study was to obtain highly internalizing cytotoxic conjugate of the T-Fc for specific delivery of drugs into FGFR1-positive cancer cells. Methods Conjugation of the T-Fc to a cytotoxic payload, vcMMAE, was carried out via maleimide chemistry, yielding the T-Fc-vcMMAE. The specific binding of the T-Fc-vcMMAE conjugate to FGFR1 was confirmed in vitro with BLI technique. Confocal microscopy and flow cytometry were applied to determine FGFR1-dependence of the T-Fc-vcMMAE internalization. Western blot analyses of FGFR1-dependent signaling were conducted to assess the impact of the T-Fc-vcMMAE on FGFR1 activation and initiation of downstream signaling cascades. Finally, using FGFR1-negative and FGFR1-possitive cell lines, the cytotoxic potential of the T-Fc-vcMMAE was evaluated. Results We have performed the efficient conjugation of the tetravalent engineered antibody with a cytotoxic drug and generated FGFR1-specific ADC molecule, T-Fc-vcMMAE. We have demonstrated that T-Fc-vcMMAE conjugate exhibits high selectivity and affinity for FGFR1, similarly to T-Fc. Furthermore, we have shown that T-Fc constitutes an effective drug delivery vehicle as T-Fc-vcMMAE was efficiently and selectively internalized by FGFR1-producing cells leading to their death. Interestingly, we show that the efficiency of the uptake of T-Fc-vcMMAE corresponds well with the cytotoxicity of the conjugate, but doesn’t correlate with the FGFR1expression level. Conclusion Our results show that T-Fc-vcMMAE fulfills the key criteria for the successful cytotoxic drug carrier in a targeted approach against FGFR1-positive cancer cells. Furthermore, our data implicate that not solely expression level of the receptor, but rather its cellular trafficking should be taken into account for selection of suitable molecular targets and cancer models for successful ADC approach.

The Impact of Antioxidants from the Diet on Breast Cancer Cells Monitored by Raman Microspectroscopy

2018 ◽  
Vol 16 (2) ◽  
pp. 127-137
Author(s):  
Paula Sofia Coutinho Medeiros ◽  
Ana Lúcia Marques Batista de Carvalho ◽  
Cristina Ruano ◽  
Juan Carlos Otero ◽  
Maria Paula Matos Marques
Keyword(s):  
Breast Cancer ◽  
Cancer Cells ◽  
Cell Line ◽  
Breast Cancer Cells ◽  
Triple Negative ◽  
Breast Adenocarcinoma ◽  
Coumaric Acid ◽  
Human Breast ◽  
The Impact

Background: The impact of the ubiquitous dietary phenolic compound p-coumaric acid on human breast cancer cells was assessed, through a multidisciplinary approach: Combined biological assays for cytotoxicity evaluation and biochemical profiling by Raman microspectroscopic analysis in cells. </P><P> Methods: Para-coumaric acid was shown to exert in vitro chemoprotective and antitumor activities, depending on the concentration and cell line probed: a significant anti-invasive ability was detected for the triple-negative MDA-MB-231 cells, while a high pro-oxidant effect was found for the estrogen- dependent MCF-7 cells. A striking cell selectivity was obtained, with a more noticeable outcome on the triple-negative MDA-MB-231 cell line. Results: The main impact on the cellular biochemical profile was verified to be on proteins and lipids, thus justifying the compound´s anti-invasive effect and chemoprotective ability. Conclusion: p-Coumaric acid was thus shown to be a promising chemoprotective/chemotherapeutic agent, particularly against the low prognosis triple-negative human breast adenocarcinoma.

scholarly journals Covalent inhibitors of EGFR family protein kinases induce degradation of human Tribbles 2 (TRIB2) pseudokinase in cancer cells

2018 ◽  
Vol 11 (549) ◽  
pp. eaat7951 ◽  
Author(s):  
Daniel M. Foulkes ◽  
Dominic P. Byrne ◽  
Wayland Yeung ◽  
Safal Shrestha ◽  
Fiona P. Bailey ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Small Molecule ◽  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Growth Factor Receptor ◽  
Drug Repurposing ◽  
Protein Kinase Inhibitors ◽  
Cellular Mechanisms ◽  
Cellular Analysis

A major challenge associated with biochemical and cellular analysis of pseudokinases is a lack of target-validated small-molecule compounds with which to probe function. Tribbles 2 (TRIB2) is a cancer-associated pseudokinase with a diverse interactome, including the canonical AKT signaling module. There is substantial evidence that human TRIB2 promotes survival and drug resistance in solid tumors and blood cancers and therefore is of interest as a therapeutic target. The unusual TRIB2 pseudokinase domain contains a unique cysteine-rich C-helix and interacts with a conserved peptide motif in its own carboxyl-terminal tail, which also supports its interaction with E3 ubiquitin ligases. We found that TRIB2 is a target of previously described small-molecule protein kinase inhibitors, which were originally designed to inhibit the canonical kinase domains of epidermal growth factor receptor tyrosine kinase family members. Using a thermal shift assay, we discovered TRIB2-binding compounds within the Published Kinase Inhibitor Set (PKIS) and used a drug repurposing approach to classify compounds that either stabilized or destabilized TRIB2 in vitro. TRIB2 destabilizing agents, including the covalent drug afatinib, led to rapid TRIB2 degradation in human AML cancer cells, eliciting tractable effects on signaling and survival. Our data reveal new drug leads for the development of TRIB2-degrading compounds, which will also be invaluable for unraveling the cellular mechanisms of TRIB2-based signaling. Our study highlights that small molecule–induced protein down-regulation through drug “off-targets” might be relevant for other inhibitors that serendipitously target pseudokinases.

Downregulation of CCKBR Expression Inhibits the Proliferation of Gastric Cancer Cells, Revealing a Potential Target for Immunotoxin Therapy

2022 ◽  
Vol 22 ◽  
Author(s):  
Meng Li ◽  
Jiang Chang ◽  
Honglin Ren ◽  
Defeng Song ◽  
Jian Guo ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Cancer Cells ◽  
Cell Counting ◽  
Migration And Invasion ◽  
Gastric Cancer Cells ◽  
Aberrant Expression ◽  
Promising Target ◽  
The Impact

Background Increased CCKBR expression density or frequency has been reported in many neoplasms. Objective We aimed to investigate whether CCKBR drives the growth of gastric cancer (GC) and its potential as a therapeutic target of immunotoxins. Methods A lentiviral interference system was used to generate CCKBR-knockdown gastric cancer cells. Cell Counting Kit-8 and clonogenic assays were used to evaluate cell proliferation. Wound-healing and cell invasion assays were performed to evaluate cell mobility. Cell cycle was analyzed by flow cytometry. Tumor growth in vivo was investigated using a heterologous tumor transplantation model in nude mice. In addition, we generated the immunotoxin FQ17P and evaluated the combining capacity and tumor cytotoxicity of FQ17P in vitro. Results Stable downregulation of CCKBR expression resulted in reduced proliferation, migration and invasion of BGC-823 and SGC-7901 cells. The impact of CCKBR on gastric cancer cells was further verified through CCKBR overexpression studies. Downregulation of CCKBR expression also inhibited the growth of gastric tumors in vivo. Furthermore, FQ17P killed CCKBR-overexpressing GC cells by specifically binding to CCKBR on the tumor cell surface. Conclusion The CCKBR protein drives the growth, migration, and invasion of gastric cancer cells, and it might be a promising target for immunotoxin therapy based on its aberrant expression, functional binding interactions with gastrin, and subsequent internalization.

scholarly journals Spironolactone, a Classic Potassium-Sparing Diuretic, Reduces Survivin Expression and Chemosensitizes Cancer Cells to Non-DNA-Damaging Anticancer Drugs

Cancers ◽  
2019 ◽  
Vol 11 (10) ◽  
pp. 1550 ◽  
Author(s):  
Tomomi Sanomachi ◽  
Shuhei Suzuki ◽  
Keita Togashi ◽  
Asuka Sugai ◽  
Shizuka Seino ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Kinase Inhibitors ◽  
Xenograft Model ◽  
Growth Factor Receptor ◽  
Survivin Expression ◽  
Mouse Xenograft Model ◽  
Anti Cancer ◽  
Underlying Mechanisms

Spironolactone, a classical diuretic drug, is used to treat tumor-associated complications in cancer patients. Spironolactone was recently reported to exert anti-cancer effects by suppressing DNA damage repair. However, it currently remains unclear whether spironolactone exerts combinational effects with non-DNA-damaging anti-cancer drugs, such as gemcitabine and epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs). Using the cancer cells of lung cancer, pancreatic cancer, and glioblastoma, the combinational effects of spironolactone with gemcitabine and osimertinib, a third-generation EGFR-TKI, were examined in vitro with cell viability assays. To elucidate the underlying mechanisms, we investigated alterations induced in survivin, an anti-apoptotic protein, by spironolactone as well as the chemosensitization effects of the suppression of survivin by YM155, an inhibitor of survivin, and siRNA. We also examined the combinational effects in a mouse xenograft model. The results obtained revealed that spironolactone augmented cell death and the suppression of cell growth by gemcitabine and osimertinib. Spironolactone also reduced the expression of survivin in these cells, and the pharmacological and genetic suppression of survivin sensitized cells to gemcitabine and osimertinib. This combination also significantly suppressed tumor growth without apparent adverse effects in vivo. In conclusion, spironolactone is a safe candidate drug that exerts anti-cancer effects in combination with non-DNA-damaging drugs, such as gemcitabine and osimertinib, most likely through the suppression of survivin.

scholarly journals Incorporation of a Hydrophilic Spacer Reduces Hepatic Uptake of HER2-Targeting Affibody–DM1 Drug Conjugates

Cancers ◽  
2019 ◽  
Vol 11 (8) ◽  
pp. 1168
Author(s):  
Haozhong Ding ◽  
Mohamed Altai ◽  
Sara S. Rinne ◽  
Anzhelika Vorobyeva ◽  
Vladimir Tolmachev ◽  
...  
Keyword(s):  
Therapeutic Potential ◽  
Histopathological Examination ◽  
Specific Binding ◽  
Hepatic Uptake ◽  
In Vitro Cytotoxicity ◽  
Drug Conjugates ◽  
Biodistribution Studies ◽  
Development Approach

Affibody molecules are small affinity-engineered scaffold proteins which can be engineered to bind to desired targets. The therapeutic potential of using an affibody molecule targeting HER2, fused to an albumin-binding domain (ABD) and conjugated with the cytotoxic maytansine derivate MC-DM1 (AffiDC), has been validated. Biodistribution studies in mice revealed an elevated hepatic uptake of the AffiDC, but histopathological examination of livers showed no major signs of toxicity. However, previous clinical experience with antibody drug conjugates have revealed a moderate- to high-grade hepatotoxicity in treated patients, which merits efforts to also minimize hepatic uptake of the AffiDCs. In this study, the aim was to reduce the hepatic uptake of AffiDCs and optimize their in vivo targeting properties. We have investigated if incorporation of hydrophilic glutamate-based spacers adjacent to MC-DM1 in the AffiDC, (ZHER2:2891)2–ABD–MC-DM1, would counteract the hydrophobic nature of MC-DM1 and, hence, reduce hepatic uptake. Two new AffiDCs including either a triglutamate–spacer–, (ZHER2:2891)2–ABD–E3–MC-DM1, or a hexaglutamate–spacer–, (ZHER2:2891)2–ABD–E6–MC-DM1 next to the site of MC-DM1 conjugation were designed. We radiolabeled the hydrophilized AffiDCs and compared them, both in vitro and in vivo, with the previously investigated (ZHER2:2891)2–ABD–MC-DM1 drug conjugate containing no glutamate spacer. All three AffiDCs demonstrated specific binding to HER2 and comparable in vitro cytotoxicity. A comparative biodistribution study of the three radiolabeled AffiDCs showed that the addition of glutamates reduced drug accumulation in the liver while preserving the tumor uptake. These results confirmed the relation between DM1 hydrophobicity and liver accumulation. We believe that the drug development approach described here may also be useful for other affinity protein-based drug conjugates to further improve their in vivo properties and facilitate their clinical translatability.

scholarly journals Baicalin suppresses lung cancer growth by targeting PDZ-binding kinase/T-LAK cell-originated protein kinase

2019 ◽  
Vol 39 (4) ◽  
Author(s):  
Xin Diao ◽  
Danfen Yang ◽  
Yu Chen ◽  
Wentian Liu
Keyword(s):  
Lung Cancer ◽  
Protein Kinase ◽  
Cancer Cells ◽  
Ex Vivo ◽  
Lung Cancer Cells ◽  
Cancer Growth ◽  
Downstream Signaling ◽  
Lak Cell

AbstractBaicalin is the main bioactive component extracted from the traditional Chinese medicine Baical Skullcap Root, and its anti-tumor activity has been studied in previous studies. PDZ-binding kinase/T-LAK cell-originated protein kinase (PBK/TOPK), a serine/threonine protein kinase, is highly expressed in many cancer cells and stimulates the tumorigenic properties, and so, it is a pivotal target for agent to cure cancers. We reported for the first time that baicalin suppressed PBK/TOPK activities by directly binding with PBK/TOPK in vitro and in vivo. Ex vivo studies showed that baicalin suppressed PBK/TOPK activity in JB6 Cl41 cells and H441 lung cancer cells. Moreover, knockdown of PBK/TOPK in H441 cells decreased their sensitivity to baicalin. In vivo study indicated that injection of baicalin in H441 tumor-bearing mice effectively suppressed cancer growth. The PBK/TOPK downstream signaling molecules Histone H3 and ERK2 in tumor tissues were also decreased after baicalin treatment. Taken together, baicalin can inhibit proliferation of lung cancer cells as a PBK/TOPK inhibitor both in vitro and in vivo.

scholarly journals TGF-βRII Knock-down in Pancreatic Cancer Cells Promotes Tumor Growth and Gemcitabine Resistance. Importance of STAT3 Phosphorylation on S727

Cancers ◽  
2018 ◽  
Vol 10 (8) ◽  
pp. 254 ◽  
Author(s):  
Vincent Drubay ◽  
Nicolas Skrypek ◽  
Lucie Cordiez ◽  
Romain Vasseur ◽  
Céline Schulz ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
Tumor Growth ◽  
Cancer Cells ◽  
Locally Advanced ◽  
Western World ◽  
Pancreatic Cancer Cells ◽  
Stat3 Phosphorylation ◽  
The Impact

Pancreatic adenocarcinoma (PDAC) is one of the most deadly cancers in the Western world because of a lack of early diagnostic markers and efficient therapeutics. At the time of diagnosis, more than 80% of patients have metastasis or locally advanced cancer and are therefore not eligible for surgical resection. Pancreatic cancer cells also harbour a high resistance to chemotherapeutic drugs such as gemcitabine that is one of the main palliative treatments for PDAC. Proteins involved in TGF-β signaling pathway (SMAD4 or TGF-βRII) are frequently mutated in PDAC (50–80%). TGF-β signalling pathway plays antagonistic roles during carcinogenesis by initially inhibiting epithelial growth and later promoting the progression of advanced tumors and thus emerged as both tumor suppressor and oncogenic pathways. In order to decipher the role of TGF-β in pancreatic carcinogenesis and chemoresistance, we generated CAPAN-1 and CAPAN-2 cell lines knocked down for TGF-βRII (first actor of TGF-β signaling). The impact on biological properties of these TGF-βRII-KD cells was studied both in vitro and in vivo. We show that TGF-βRII silencing alters tumor growth and migration as well as resistance to gemcitabine. TGF-βRII silencing also leads to S727 STAT3 and S63 c-Jun phosphorylation, decrease of MRP3 and increase of MRP4 ABC transporter expression and induction of a partial EMT phenotype. These markers associated with TGF-β signaling pathways may thus appear as potent therapeutic tools to better treat/manage pancreatic cancer.

scholarly journals EGFRAmplification and Glioblastoma Stem-Like Cells

2015 ◽  
Vol 2015 ◽  
pp. 1-11 ◽  
Author(s):  
Katrin Liffers ◽  
Katrin Lamszus ◽  
Alexander Schulte
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Current Knowledge ◽  
Growth Factor Receptor ◽  
Model Systems ◽  
Preclinical Research ◽  
Epidermal Growth ◽  
Methodological Approaches ◽  
The Impact

Glioblastoma (GBM), the most common malignant brain tumor in adults, contains a subpopulation of cells with a stem-like phenotype (GS-cells). GS-cells can be maintainedin vitrousing serum-free medium supplemented with epidermal growth factor, basic fibroblast growth factor-2, and heparin. However, this method does not conserve amplification of the Epidermal Growth Factor Receptor (EGFR) gene, which is present in over 50% of all newly diagnosed GBM cases. GS-cells with retainedEGFRamplification could overcome the limitations of currentin vitromodel systems and contribute significantly to preclinical research on EGFR-targeted therapy. This review recapitulates recent methodological approaches to expand stem-like cells from GBM with differentEGFRstatus in order to maintain EGFR-dependent intratumoral heterogeneityin vitro. Further, it will summarize the current knowledge about the impact ofEGFRamplification and overexpression on the stem-like phenotype of GBM-derived GS-cells and different approaches to target the EGFR-dependent GS-cell compartment of GBM.

scholarly journals Spongiform Neurodegeneration-associated E3 Ligase Mahogunin Ubiquitylates TSG101 and Regulates Endosomal Trafficking

2007 ◽  
Vol 18 (4) ◽  
pp. 1129-1142 ◽  
Author(s):  
Bong Yoon Kim ◽  
James A. Olzmann ◽  
Gregory S. Barsh ◽  
Lih-Shen Chin ◽  
Lian Li
Keyword(s):  
Mammalian Cells ◽  
Growth Factor Receptor ◽  
Endosomal Trafficking ◽  
Small Interfering Rnas ◽  
Gene Encoding ◽  
Endosomal Sorting ◽  
Ubiquitin Protein Ligase ◽  
Downstream Signaling

A null mutation in the gene encoding the putative E3 ubiquitin–protein ligase Mahogunin causes spongiform neurodegeneration, a recessively transmitted prion-like disease in mice. However, no substrates of Mahogunin have been identified, and the cellular role of Mahogunin is unknown. Here, we report the identification of TSG101, a key component of the endosomal sorting complex required for transport (ESCRT)-I, as a specific Mahogunin substrate. We find that Mahogunin interacts with the ubiquitin E2 variant (UEV) domain of TSG101 via its PSAP motif and that it catalyzes monoubiquitylation of TSG101 both in vivo and in vitro. Depletion of Mahogunin by small interfering RNAs in mammalian cells disrupts endosome-to-lysosome trafficking of epidermal growth factor receptor, resulting in prolonged activation of a downstream signaling cascade. Our findings support a role for Mahogunin in a proteasome-independent ubiquitylation pathway and suggest a link between dysregulation of endosomal trafficking and spongiform neurodegeneration.

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