15 research outputs found
Development of targeted anticancer agents using novel N-alkylisatin derivatives
Targeted therapy involves selective targeting of tumour and/or tumour associated cells using agents that recognise specific biomarkers which are characteristic of the tumour cell population. The use of ligand-drug conjugates (LDCs) for targeted therapy includes agents that combine the selectivity and specificity of biomarker ligands with the high potency of chemotherapeutic drugs. This not only causes effective reduction of tumour growth, but also minimises off target cell killing and delays the development of resistance which otherwise arises from prolonged exposure to non-specific drugs.
This study focussed on the development of LDCs incorporating novel N-alkylisatin derivatives. We have previously reported the cytotoxic activity of a series of N-alkylisatin derivatives that destabilise microtubules and induce apoptotic cell death in a range of cancer cell lines. In this study, two of the most potent N-alkylisatin derivatives, 5,7-dibromo-N-(p-hydroxymethylbenzyl)isatin (NAI-1) and 5,7-dibromo-N-(pmethoxybenzyl)isatin (NAI-2), functionalised with an esterase-labile succinate (NAI-1-succ) and an imine (NAI-2-imine) functional linker respectively, were conjugated to well described tumour ligands, namely, transferrin (Tf), plasminogen activator inhibitor type 2 (PAI-2), and trastuzumab (TRZ). These were tested against various human breast carcinoma cell lines that differed in their cell surface expression of relevant targets for these ligands. Both NAI-1-succ-Tf and NAI-2-imine-Tf conjugates were selectively toxic against the cell lines tested. NAI-1-succ-Tf conjugate (IC50 0.75 μM) was greater than 10 × more active compared to the parent compound (IC50 7.67 μM) against the high Tf receptor (TfR) expressing SK-BR-3 cells after 48 h incubation. The NAI-2- imine-Tf conjugate was equipotent to the parent NAI-2 compound against cells expressing moderate to high TfR levels. As the conjugate incorporating the imine-based linker system was less selective for their target, evident by their equipotency to the parent NAI-2 and NAI-2-imine compounds, the imine-based linker system was not used in the development of other LDCs.
Previous studies performed in our laboratory found the NAI-1-succ-PAI-2 conjugate to be equivalent in efficacy to the free NAI-1 compound at 1/20th of the dose, in a metastatic, orthotopic human breast tumour xenograft mouse model. However, our laboratory has also shown that PAI-2 is rapidly excreted from the body. Therefore, to improve the pharmacokinetic and potential tumour uptake properties, a modified form of PAI-2 containing only three potential sulfhydryl sites, C161S-PAI-2, was covalently modified using polyethylene glycol molecules (PEGs) functionalised with a maleimide functional linker (mPEG) to form mPEG-C161S-PAI-2. Purification of mPEG-C161S-PAI- 2 by ion exchange was achieved after a series of optimisations and NAI-1-succ was then conjugated to the purified mPEG-C161S-PAI-2 to form mPEG-C161S-PAI-2- NAI-1-succ conjugate. In vitro studies showed mPEG-C161S-PAI-2-NAI-1-succ conjugate to be ~3.5 × more potent than the free NAI-1 and showed selectivity against high uPAR expressing breast cancer cells.
Recent studies have found over amplification of HER-2 and uPA receptors in 70 % of HER-2 amplified advanced invasive breast carcinoma cells. Further studies targeting HER-2 and uPA showed that depleting both HER-2 and uPA receptors suppressed cell growth and induced cell apoptosis in SK-BR-3 and ZR-75-1 mammary epithelial cells that over-express HER-2 and uPA receptors. In this direction, NAI-1-succ-TRZ conjugate was prepared by reacting the commercially available recombinant humanised monoclonal antibody (mAb) TRZ directed against HER-2 receptors, with a 40-fold molar excess of NAI-1-succ in sodium phosphate buffer (pH 6.0). The resultant NAI-1- succ-TRZ conjugate (IC50 0.61 μM) was ~8 × more toxic than free NAI-1 (IC50 5.19 μM) and was highly selective against high HER-2 expressing SK-BR-3 cells. This highly potent NAI-1-succ-TRZ along with NAI-1-succ-C161S-PAI-2 was utilised in a novel combination targeting approach (also called double targeting ) to specifically target metastatic breast cancer cells that over-express uPA/uPAR and HER-2 receptors. In vitro double targeting assays using these agents against high uPAR and HER-2 expressing ZR-75-1 breast cancer cells showed increased cell death. This warrants further in vivo study of NAI-1-succ-TRZ and NAI-1-succ-C161S-PAI-2 in combination in appropriate mouse models to ascertain their potential synergistic effect in reducing tumour growth compared to treatment with each drug alone. This approach is expected to result in superior efficacy over single agent therapy and due to minimal drug exposure, delay the development of drug resistance.
On the whole, this study successfully developed and proved the selective potency of 5,7-dibromo-N-(p-hydroxymethylbenzyl)isatin functionalised with an ester linker in ligand-drug conjugates, namely mPEG-C161S-PAI-2-NAI-1-succ and NAI-1-succ-TRZ in vitro. The utility of these conjugates in a novel double targeting treatment strategy to simultaneously target highly characteristic tumour-specific biomarkers in breast cancer warrants further development using appropriate animal tumour models
HER2 and uPAR cooperativity contribute to metastatic phenotype of HER2-positive breast cancer
Human epidermal growth factor receptor type 2 (HER2)-positive breast carcinoma is highly aggressive and mostly metastatic in nature though curable/manageable in part by molecular targeted therapy. Recent evidence suggests a subtype of cells within HER2-positive breast tumors that concomitantly expresses the urokinase plasminogen activator receptor (uPAR) with inherent stem cell/mesenchymal-like properties promoting tumor cell motility and a metastatic phenotype. This HER-positive/uPAR-positive subtype may be partially responsible for the failure of HER2-targeted treatment strategies. Herein we discuss and substantiate the cumulative preclinical and clinical evidence on HER2-uPAR cooperativity in terms of gene co-amplification and/or mRNA/protein co-overexpression. We then propose a regulatory signaling model that we hypothesize to maintain upregulation and cooperativity between HER2 and uPAR in aggressive breast cancer. An improved understanding of the HER2/uPAR interaction in breast cancer will provide critical biomolecular information that may help better predict disease course and response to therapy
Improved pharmacokinetic and biodistribution properties of the selective urokinase inhibitor PAI-2 (SerpinB2) by site-specific PEGylation: implications for drug delivery
Purpose Overexpression of the serine protease urokinase (uPA) is recognised as an important biomarker of metastatic disease and a druggable anticancer target. Plasminogen activator inhibitor type-2 (PAI-2/SerpinB2) is a specific uPA inhibitor with proven potential for use in targeted therapy. However, PAI-2 is rapidly cleared via the renal system which impairs tumor uptake and efficacy. Here we aimed to improve the pharmacological properties of PAI-2 by site-specific PEGylation. Methods Several cysteine to serine substitution mutants were generated for PEGylation with PEG-maleimide (size range 12-30 kDa) and the physico-chemical and biochemical properties of the PEG-PAI-2 conjugates characterised. Radiolabeled proteins were used for evaluation of blood clearance and tissue uptake profiles in an orthotopic breast tumor xenograft mouse model. Results PEGylation of the PAI-2C161S mutant gave a predominant mono-PEGylated-PAI-2 product (~90%) with full uPA inhibitory activity, despite a significant increase in hydrodynamic radius. Compared to un-PEGylated protein the plasma half-life and AUC for PEG20-PAI-2C161S were significantly increased. This translated to a 10-fold increase in tumor retention after 24 h compared to PAI-2C161S, an effect not seen in non-target organs. Conclusions Our data underscores the potential for PEG20-PAI-2C161S drug conjugates to be further developed as anti-uPA targeted therapeutics with enhanced tumor retention
Anti-cancer activity of an acid-labile N-alkylisatin conjugate targeting the transferrin receptor
We have previously reported a series of pH-sensitive imine-linked N-alkylisatin prodrugs that are stable at pH 7.4, but readily cleaved at pH 4.5. Herein, one of the most potent prodrugs, 5,7-dibromo-N-(pmethoxybenzyl) isatin (NAI), was functionalized with a para-phenylpropionic acid linker, and the resulting NAI–imine prodrug conjugated to transferrin (Tf) to form a NAI–imine–Tf conjugate. Cytotoxicity assays revealed the conjugate was equipotent to the free drug against MCF-7 breast cancer cells, with clear selectivity patterns based on TfR levels. These results suggest that this novel isatin-based cytotoxin conjugated to a tumor targeting protein via an acid-labile linker warrants further preclinical testing
Global extracellular vesicle proteomic signature defines U87-MG glioma cell hypoxic status with potential implications for non-invasive diagnostics
Purpose Glioblastoma multiforme (GBM) is the most common and lethal of primary malignant brain tumors. Hypoxia constitutes a major determining factor for the poor prognosis of high-grade glioma patients, and is known to contribute to the development of treatment resistance. Therefore, new strategies to comprehensively profile and monitor the hypoxic status of gliomas are of high clinical relevance. Here, we have explored how the proteome of secreted extracellular vesicles (EVs) at the global level may reflect hypoxic glioma cells. Methods We have employed shotgun proteomics and label free quantification to profile EVs isolated from human high-grade glioma U87-MG cells cultured at normoxia or hypoxia. Parallel reaction monitoring was used to quantify the identified, hypoxia-associated EV proteins. To determine the potential biological significance of hypoxia-associated proteins, the cumulative Z score of identified EV proteins was compared with GBM subtypes from HGCC and TCGA databases. Results In total, 2928 proteins were identified in EVs, out of which 1654 proteins overlapped with the ExoCarta EV-specific database. We found 1034 proteins in EVs that were unique to the hypoxic status of U87-MG cells. We subsequently identified an EV protein signature, "HYPSIGNATURE", encompassing nine proteins that strongly represented the hypoxic situation and exhibited close proximity to the mesenchymal GBM subtype. Conclusions We propose, for the first time, an EV protein signature that could comprehensively reflect the hypoxic status of high-grade glioma cells. The presented data provide proof-of-concept for targeted proteomic profiling of glioma derived EVs, which should motivate future studies exploring its utility in non-invasive diagnosis and monitoring of brain tumor patients
Hypoxia regulates global membrane protein endocytosis through caveolin-1 in cancer cells
Hypoxia promotes tumour aggressiveness and resistance of cancers to oncological treatment. The identification of cancer cell internalizing antigens for drug targeting to the hypoxic tumour niche remains a challenge of high clinical relevance. Here we show that hypoxia down-regulates the surface proteome at the global level and, more specifically, membrane proteome internalization. We find that hypoxic down-regulation of constitutive endocytosis is HIF-independent, and involves caveolin-1-mediated inhibition of dynamin-dependent, membrane raft endocytosis. Caveolin-1 overexpression inhibits protein internalization, suggesting a general negative regulatory role of caveolin-1 in endocytosis. In contrast to this global inhibitory effect, we identify several proteins that can override caveolin-1 negative regulation, exhibiting increased internalization at hypoxia. We demonstrate antibody-mediated cytotoxin delivery and killing specifically of hypoxic cells through one of these proteins, carbonic anhydrase IX. Our data reveal that caveolin-1 modulates cell-surface proteome turnover at hypoxia with potential implications for specific targeting of the hypoxic tumour microenvironment
The prevalence and risk factors for cognitive impairment in obesity and NAFLD
Background:. Severe obesity may be accompanied by cognitive dysfunction and NAFLD, but the associations remain unclear. We describe the prevalence and features of cognitive dysfunction and examine the associations between cognitive dysfunction and the presence and severity of NAFLD, and the associations between cognitive dysfunction and signs of other obesity-related comorbidities and neuronal damage.
Methods:. A cross-sectional study of patients with a body mass index of 35 kg/m2 underwent evaluation for bariatric surgery. They were screened for adiposity-related comorbidity and underwent a liver biopsy and basic cognitive testing with the Continuous Reaction Time test, the Portosystemic Encephalopathy Syndrome test, and the Stroop Test. A representative subgroup also underwent the Repeatable Battery for the Assessment of Neuropsychological Status (RBANS). The primary study outcome was “cognitive impairment,” defined as ≥2 abnormal basic cognitive tests and/or an abnormal RBANS. The Triggering Receptor Expressed on Myeloid Cells 2 (TREM2) served as a biomarker for neuronal damage.
Results:. We included 180 patients; 72% were women, age 46 ± 12 years, 78% had NAFLD, and 30% with NASH without cirrhosis. 8% were cognitively impaired by the basic tests and 41% by RBANS results. Most impaired were executive and short-time memory functions. There were no associations between cognitive impairment and BMI, NAFLD presence or severity, or metabolic comorbidities. Male sex (OR: 3.67, 95% CI, 1.32–10.27) and using 2 or more psychoactive medications (5.24, 95% CI, 1.34–20.4) were associated with impairment. TREM2 was not associated with cognitive impairment.
Conclusions:. Nearly half of this severely obese study cohort exhibited measurable multidomain cognitive impairment. This was not dependent on NAFLD or another adiposity comorbidity
Interplay between Caveolin-1 and body and tumor size affects clinical outcomes in breast cancer
Background: Caveolin-1 (CAV1) is associated with cholesterol-rich membrane raft domains and is a master regulator of cell signaling and membrane transport. Here, we investigated CAV1's role in cellular compartments of breast cancer in relation to signaling pathways, clinicopathological features, and clinical outcomes. Methods: CAV1 levels were evaluated with immunohistochemistry in cytoplasm of invasive tumor cells and stromal cells in tumor tissue microarrays from a cohort of 1018 breast cancer patients (inclusion 2002-2012, Sweden). Cytoplasmic and stromal CAV1 were categorized as positive/negative and strong/not strong, respectively. CAV1 expression in relation to clinical outcomes was assessed with Cox regression. Investigations into CAV1 functional pathways was conducted in the STRING, GOBO, and TCGA databases. Results: CAV1 expression was associated with non-luminal subtypes, cell cycle control, inflammation, epithelialmesenchymal transition, and the IGF/Insulin system. Generally, CAV1 was not associated with recurrence risk. Stromal CAV1's impact on recurrence risk was modified by BMI > 25 kg/m(2) (P-interaction = 0.002), waist > 80 cm (P-interaction = 0.005), and invasive tumor size (pT2/3/4) (P-interaction = 0.028). In low-risk patients only, strong stromal CAV1 significantly increased recurrence risk (HRs(adj) > 1.61). In all patients, positive cytoplasmic CAV1 conferred > 2-fold risk for contralateral disease HRadj 2.63 (95% CI 1.36-5.10). Strong stromal CAV1 conferred nearly 2-fold risk for locoregional recurrence HRadj 1.88 (95% CI 1.09-3.24). Conclusions: CAV1's prognostic impact depended on its localization, anthropometric, and tumor factors. Stromal CAV1 predicted high recurrence risk in a group of supposedly "low-risk' patients. Cytoplasmic CAV1 predicted metachronous contralateral disease. If confirmed, CAV1 could be used as treatment target and for risk stratification
Tumor antigen glycosaminoglycan modification regulates antibody-drug conjugate delivery and cytotoxicity
Aggressive cancers are characterized by hypoxia, which is a key driver of tumor development and treatment resistance. Proteins specifically expressed in the hypoxic tumor microenvironment thus represent interesting candidates for targeted drug delivery strategies. Carbonic anhydrase (CAIX) has been identified as an attractive treatment target as it is highly hypoxia specific and expressed at the cell-surface to promote cancer cell aggressiveness. Here, we find that cancer cell internalization of CAIX is negatively regulated by post-translational modification with chondroitin or heparan sulfate glycosaminoglycan chains. We show that perturbed glycosaminoglycan modification results in increased CAIX endocytosis. We hypothesized that perturbation of CAIX glycosaminoglycan conjugation may provide opportunities for enhanced drug delivery to hypoxic tumor cells. In support of this concept, pharmacological inhibition of glycosaminoglycan biosynthesis with xylosides significantly potentiated the internalization and cytotoxic activity of an antibody-drug conjugate (ADC) targeted at CAIX. Moreover, cells expressing glycosaminoglycan-deficient CAIX were significantly more sensitive to ADC treatment as compared with cells expressing wild-type CAIX. We find that inhibition of CAIX endocytosis is associated with an increased localization of glycosaminoglycan-conjugated CAIX in membrane lipid raft domains stabilized by caveolin-1 clusters. The association of CAIX with caveolin-1 was partially attenuated by acidosis, i.e. another important feature of malignant tumors. Accordingly, we found increased internalization of CAIX at acidic conditions. These findings provide first evidence that intracellular drug delivery at pathophysiological conditions of malignant tumors can be attenuated by tumor antigen glycosaminoglycan modification, which is of conceptual importance in the future development of targeted cancer treatments
Metastasis stimulation by hypoxia and acidosis-induced extracellular lipid uptake is mediated by proteoglycan-dependent endocytosis
Hypoxia and acidosis are inherent stress factors of the tumor microenvironment and have been linked to increased tumor aggressiveness and treatment resistance. Molecules involved in the adaptive mechanisms that drive stress-induced disease progression constitute interesting candidates of therapeutic intervention. Here, we provide evidence of a novel role of heparan sulfate proteoglycans (HSPG) in the adaptive response of tumor cells to hypoxia and acidosis through increased internalization of lipoproteins, resulting in a lipid-storing phenotype and enhanced tumor-forming capacity. Patient glioblastoma tumors and cells under hypoxic and acidic stress acquired a lipid droplet (LD)-loaded phenotype, and showed an increased recruitment of all major lipoproteins, HDL, LDL, and VLDL. Stress-induced LD accumulation was associated with increased spheroid-forming capacity during reoxygenation in vitro and lung metastatic potential in vivo. On a mechanistic level, we found no apparent effect of hypoxia on HSPGs, whereas lipoprotein receptors (VLDLR and SR-B1) were transiently upregulated by hypoxia. Importantly, however, using pharmacologic and genetic approaches, we show that stress-mediated lipoprotein uptake is highly dependent on intact HSPG expression. The functional relevance of HSPG in the context of tumor cell stress was evidenced by HSPG-dependent lipoprotein cell signaling activation through the ERK/MAPK pathway and by reversal of the LD-loaded phenotype by targeting of HSPGs. We conclude that HSPGs may have an important role in the adaptive response to major stress factors of the tumor microenvironment, with functional consequences on tumor cell signaling and metastatic potential