Bilikere S. Dwarakanath

The intracellular drug delivery and anti tumor activity of doxorubicin loaded poly(γ-benzyl L-glutamate)-b-hyaluronan polymersomes

We have investigated the intracellular delivery of doxorubicin (DOX) loaded poly(gamma-benzyl L-glutamate)-block-hyaluronan (PBLG-b-HYA) based polymersomes (PolyDOX) in high (MCF-7) and low (U87) CD44 expressing cancer cell models. DOX was successfully loaded into polymersomes using nanoprecipitation method and in vitro drug release pattern were achieved at pH 5.5 and 7.4 up to 10 days. Block copolymer vesicles without loaded DOX were non cytotoxic in both cells at concentration 150-650 microg/mL. Flow cytometry data suggested successful uptake of PolyDOX in cells and high accumulation was found in MCF-7 than U87 cells. Microscopy imagings revealed that in MCF-7 cells PolyDOX was more in cytoplasm and free DOX in nuclei, whereas in U87 cells free DOX was also found in the cytoplasm. Cytotoxicity of the drug was concentration and exposure time dependent. In addition, PolyDOX significantly enhanced reactive oxygen species (ROS) level in both cells. PolyDOX also suppressed growth of breast tumor on female Sprague-Dawley (SD) rats as compared to phosphate buffer saline pH 7.4 (PBS) control group. In addition reduced level of serum enzymes (LDH and CPK) by PolyDOX formulation indicated less cardiotoxicity of DOX after loading in polymersomes. Results suggest that intracellular delivery of PolyDOX was depended on the CD44 expression level in cells due to presence of hyaluronic acid on the surface of polymersomes, and could be used as a self-targeting drug delivery cargo in over-expressed CD44 glycoprotein cells of breast cancer.

Optimizing Cancer Radiotherapy with 2-Deoxy-D-Glucose

Background and Purpose:Higher rates of glucose utilization and glycolysis generally correlate with poor prognosis in several types of malignant tumors. Own earlier studies on model systems demonstrated that the nonmetabolizable glucose analog 2-deoxy-D-glucose (2-DG) could enhance the efficacy of radiotherapy in a dose-dependent manner by selectively sensitizing cancer cells while protecting normal cells. Phase I/II clinical trials indicated that the combination of 2-DG, at an oral dose of 200 mg/kg body weight (BW), with large fractions of γ-radiation was well tolerated in cerebral glioma patients. Since higher 2-DG doses are expected to improve the therapeutic gain, present studies were undertaken to examine the tolerance and safety of escalating 2-DG dose during combined treatment (2-DG + radiotherapy) in glioblastoma multiforme patients.Patients and Methods:Untreated patients with histologically proven glioblastoma multiforme (WHO criteria) were included in the study. Seven weekly fractions of 60Co γ-rays (5 Gy/fraction) were delivered to the tumor volume (presurgical CT/MRI evaluation) plus 3 cm margin. Escalating 2-DG doses (200–250–300 mg/kg BW) were administered orally 30 min before irradiation after overnight fasting. Acute toxicity and tolerance were studied by monitoring the vital parameters and side effects. Late radiation damage and treatment responses were studied radiologically and clinically in surviving patients.Results:Transient side effects similar to hypoglycemia were observed in most of the patients. Tolerance and patient compliance to the combined treatment were very good up to a 2-DG dose of 250 mg/kg BW. However, at the higher dose of 300 mg/kg BW, two out of six patients were very restless and could not complete treatment, though significant changes in the vital parameters were not observed even at this dose. No significant damage to the normal brain tissue was observed during follow-up in seven out of ten patients who received complete treatment and survived between 11 and 46 months after treatment.Conclusion:Oral administration of 2-DG combined with large fractions of radiation (5 Gy/fraction/week) is safe and could be tolerated in glioblastoma patients without any acute toxicity and late radiation damage to the normal brain. Further clinical studies to evaluate the efficacy of the combined treatment are warranted.Hintergrund und Ziel:Erhöhter Glukoseverbrauch und verstärkte Glykolyse korrelieren bei einigen Malignomformen generell mit schlechter Prognose. Eigene Untersuchungen an Modellsystemen zeigten, dass die nichtmetabolisierbare Glukoseverbindung 2-Deoxy-D-Glukose (2-DG) die Wirksamkeit der Strahlentherapie dosisabhängig verstärken kann, indem sie selektiv Krebszellen sensibilisiert, dagegen auf normale Zellen protektiv wirkt. Klinische Phase-I/II-Studien sprechen dafür, dass die Kombination von 2-DG (in einer oralen Dosis von 200 mg/kg Körpergewicht) mit großen Fraktionen einer γ-Strahlung von Patienten mit Hirntumoren gut vertragen wird. Da man erwartet, dass höhere 2-DG-Dosierungen das therapeutische Ansprechen verbessern, wurden die hier vorgestellten Untersuchungen unternommen, um an Glioblastoma-multiforme-Patienten Verträglichkeit und Sicherheit der Dosiseskalation von 2-DG in der Kombinationsbehandlung (2-DG + Radiotherapie) zu prüfen.Patienten und Methodik:In die Studie wurden nicht vorbehandelte Patienten mit histologisch belegtem Glioblastoma multiforme (WHO-Kriterien) eingeschlossen. Das Tumorvolumen (gemäß präoperativer CT/MRT-Auswertung) plus ein 3-cm-Sicherheitssaum wurden wöchentlich mit sieben Fraktionen einer 60Co-γ-Strahlung (5 Gy/Fraktion) behandelt. In ansteigender Dosierung wurde 2-DG (200–250–300 mg/kg KW) nach nächtlicher Nahrungskarenz 30 min vor der Bestrahlung verabreicht. Akuttoxizität und Verträglichkeit wurden durch Überwachung von Vitalparametern und Nebenwirkungen untersucht. Langzeit-Strahlenfolgen und Ansprechen wurden bei überlebenden Patienten radiologisch und klinisch untersucht.Ergebnisse:Bei den meisten Patienten wurden vorübergehende, Hypoglykämie-ähnliche Symptome beobachtet. Verträglichkeit und Patienten-Compliance der Kombinationsbehandlung waren bis zu einer 2-DG-Dosis von 250 mg/kg KG sehr gut; bei der höheren Dosis von 300 mg/kg KG entwickelten zwei von sechs Patienten jedoch starke Unruhe und konnten die Behandlung nicht abschließen, obwohl auch unter dieser Dosis keine signifikanten Veränderungen der Vitalparameter festgestellt wurden. Im Follow-up war keine signifikante Schädigung des normalen Hirngewebes bei sieben von zehn Patienten zu beobachten, die die komplette Behandlung erhalten und zwischen 11 und 46 Monate nach der Behandlung überlebt hatten.Schlussfolgerung:Die orale Gabe von 2-DG kombiniert mit hohen Einzelfraktionen (5 Gy/Fraktion/Woche) ist sicher und wurde von Gliom-Patienten ohne Akuttoxizität und ohne Strahlungsspätschäden im Hirngewebe vertragen. Um die Wirksamkeit der Kombinationstherapie zu prüfen, sind weitere klinische Studien erforderlich.

In vitro and In vivo Evaluation of Docetaxel Loaded Biodegradable Polymersomes

Formulation of docetaxel (DOC), a hydrophobic anticancer drug, was successfully achieved in poly(gamma-benzyl L-glutamate)-block-hyaluronan polymersomes using a simple and reproducible nanoprecipitation method. The prepared DOC loaded polymersomes (PolyDOC) was stable either in solution or in a lyophilized form, and showed controlled release behaviour over several days. PolyDOC showed high in vitro toxicity after 24 h in MCF-7 and U87 cells compared to free DOC. Biodistribution data demonstrated that (99m)Tc labelled PolyDOC t(1/2) and MRT significantly increased compared to a DOC solution (DS). In addition, PolyDOC uptake in Ehrlich Ascites Tumor (EAT) tumor bearing mice was larger at each time point compared to DS, making such a polymer vesicle formulation an efficient drug nanocarrier for improved DOC cancer therapy.

Differences in DNA Condensation and Release by Lysine and Arginine Homopeptides Govern Their DNA Delivery Efficiencies

Designing of nanocarriers that can efficiently deliver therapeutic DNA payload and allow its smooth intracellular release for transgene expression is still a major constraint. The optimization of DNA nanocarriers requires thorough understanding of the chemical and structural characteristics of the vector-nucleic acid complexes and its correlation with the cellular entry, intracellular state and transfection efficiency. L-lysine and L-arginine based cationic peptides alone or in conjugation with other vectors are known to be putative DNA delivery agents. Here we have used L-lysine and L-arginine homopeptides of three different lengths and probed their DNA condensation and release properties by using a multitude of biophysical techniques including fluorescence spectroscopy, gel electrophoresis and atomic force microscopy. Our results clearly showed that although both lysine and arginine based homopeptides condense DNA via electrostatic interactions, they follow different pattern of DNA condensation and release in vitro. While lysine homopeptides condense DNA to form both monomolecular and multimolecular complexes and show differential release of DNA in vitro depending on the peptide length, arginine homopeptides predominantly form multimolecular complexes and show complete DNA release for all peptide lengths. The cellular uptake of the complexes and their intracellular state (as observed through flow cytometry and fluorescence microscopy) seem to be controlled by the peptide chemistry. The difference in the transfection efficiency of lysine and arginine homopeptides has been rationalized in light of these observations.

Flow-Cytometric Analysis of Reactive Oxygen Species in Peripheral Blood Mononuclear Cells of Patients with Thyroid Dysfunction

Thyroid hormones are major regulators of energy metabolism and increased levels of the hormones (hyperthyroidism) results in an increase in the metabolic rate. Thyroid dysfunction causing alteration in hormone secretion leads to perturbations in the metabolic status. The hypermetabolic state may cause increased generation of reactive oxygen species (ROS), leading to oxidative stress in these patients. This study was carried out to verify our proposition by measuring the ROS in the terminally differentiated cells like the peripheral blood mononuclear cells of the patients.

COX-2, aspirin and metabolism of arachidonic, eicosapentaenoic and docosahexaenoic acids and their physiological and clinical significance

Polyunsaturated fatty acids (PUFAs) are vital for normal growth and development and physiological function of various tissues in humans. PUFAs have immunomodulatory actions in addition to their ability to modulate inflammation, vascular reactivity, neurotransmission and stem cell biology. PUFAs and their metabolites possess both pro- and anti-inflammatory properties that underlie their actions and involvement in several diseases. Aspirin, a non-steroidal anti-inflammatory drug (NSAID), possesses both cyclo-oxygenase (COX) and lipoxygenase (LOX) inhibitory action and enhances the production of anti-inflammatory lipoxin A4 {(called as epi-lipoxin A4, aspirin-triggered lipoxins (ATLs))}. In addition, at low doses aspirin may not interfere with the production of prostacyclin (PGI2). Both lipoxin A4 and PGI2 have vasodilator, platelet anti-aggregator and anti-inflammatory actions that may underlie the beneficial actions of aspirin. Paradoxically, other NSAIDs may not have the same actions as that of aspirin on PUFA metabolism. Similar anti-inflammatory compounds are formed from eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) by the action of aspirin termed as resolvins (from EPA and DHA) and protectins and maresins from DHA. PUFAs: arachidonic acid (AA), EPA and DHA and their various products modulate not only inflammation and immune response but also possess actions on various genes, nuclear factors, cyclic AMP and GMP, G-protein coupled receptors (GPRs), hypothalamic neurotransmitters, hormones, cytokines and enzymes, and interact with nitric oxide, carbon monoxide, and hydrogen sulfide to regulate their formation and action and to form new compounds that have several biological actions. These pleiotropic actions of PUFAs and their metabolites may explain their ability to play a role in several physiological actions and diseases. The big challenge is to harness these actions to prevent and manage clinical conditions.

Tumor Suppressor Protein p53 Recruits Human Sin3B/HDAC1 Complex for Down-Regulation of Its Target Promoters in Response to Genotoxic Stress

Master regulator protein p53, popularly known as the “guardian of genome” is the hub for regulation of diverse cellular pathways. Depending on the cell type and severity of DNA damage, p53 protein mediates cell cycle arrest or apoptosis, besides activating DNA repair, which is apparently achieved by regulation of its target genes, as well as direct interaction with other proteins. p53 is known to repress target genes via multiple mechanisms one of which is via recruitment of chromatin remodelling Sin3/HDAC1/2 complex. Sin3 proteins (Sin3A and Sin3B) regulate gene expression at the chromatin-level by serving as an anchor onto which the core Sin3/HDAC complex is assembled. The Sin3/HDAC co-repressor complex can be recruited by a large number of DNA-binding transcription factors. Sin3A has been closely linked to p53 while Sin3B is considered to be a close associate of E2Fs. The theme of this study was to establish the role of Sin3B in p53-mediated gene repression. We demonstrate a direct protein-protein interaction between human p53 and Sin3B (hSin3B). Amino acids 1–399 of hSin3B protein are involved in its interaction with N-terminal region (amino acids 1–108) of p53. Genotoxic stress induced by Adriamycin treatment increases the levels of hSin3B that is recruited to the promoters of p53-target genes (HSPA8, MAD1 and CRYZ). More importantly recruitment of hSin3B and repression of the three p53-target promoters upon Adriamycin treatment were observed only in p53+/+ cell lines. Additionally an increased tri-methylation of the H3K9 residue at the promoters of HSPA8 and CRYZ was also observed following Adriamycin treatment. The present study highlights for the first time the essential role of Sin3B as an important associate of p53 in mediating the cellular responses to stress and in the transcriptional repression of genes encoding for heat shock proteins or proteins involved in regulation of cell cycle and apoptosis.

Low-dose radiation therapy of cancer: role of immune enhancement

The efficacy of conventional radiation therapy, one of the most widely used treatment modalities of cancer, is limited by resistance of tumors as well as normal tissue toxicity. In the last decade, several studies have shown that protocols using low-dose radiation (LDR) are more effective in providing local tumor control with negligible normal tissue toxicity. LDR stimulates antioxidant capacity, repair of DNA damage, apoptosis and induction of immune responses, which might be collectively responsible for providing effective local tumor control. This article focuses on the immunostimulatory effects of LDR in in vivo models and its clinical efficacy, supporting the use of LDR regimens (alone or as adjuvant) as an anticancer treatment.

Transient elevation of glycolysis confers radio-resistance by facilitating DNA repair in cells

BackgroundCancer cells exhibit increased glycolysis for ATP production (the Warburg effect) and macromolecular biosynthesis; it is also linked with therapeutic resistance that is generally associated with compromised respiratory metabolism. Molecular mechanisms underlying radio-resistance linked to elevated glycolysis remain incompletely understood.MethodsWe stimulated glycolysis using mitochondrial respiratory modifiers (MRMs viz. di-nitro phenol, DNP; Photosan-3, PS3; Methylene blue, MB) in established human cell lines (HEK293, BMG-1 and OCT-1). Glucose utilization and lactate production, levels of glucose transporters and glycolytic enzymes were investigated as indices of glycolysis. Clonogenic survival, DNA repair and cytogenetic damage were studied as parameters of radiation response.ResultsMRMs induced the glycolysis by enhancing the levels of two important regulators of glucose metabolism GLUT-1 and HK-II and resulted in 2 fold increase in glucose consumption and lactate production. This increase in glycolysis resulted in resistance against radiation-induced cell death (clonogenic survival) in different cell lines at an absorbed dose of 5 Gy. Inhibition of glucose uptake and glycolysis (using fasentin, 2-deoxy-D-glucose and 3-bromopyruvate) in DNP treated cells failed to increase the clonogenic survival of irradiated cells, suggesting that radio-resistance linked to inhibition of mitochondrial respiration is glycolysis dependent. Elevated glycolysis also facilitated rejoining of radiation-induced DNA strand breaks by activating both non-homologous end joining (NHEJ) and homologous recombination (HR) pathways of DNA double strand break repair leading to a reduction in radiation-induced cytogenetic damage (micronuclei formation) in these cells.ConclusionsThese findings suggest that enhanced glycolysis generally observed in cancer cells may be responsible for the radio-resistance, partly by enhancing the repair of DNA damage.

Metabolic oxidative stress induced by a combination of 2-DG and 6-AN enhances radiation damage selectively in malignant cells via non-coordinated expression of antioxidant enzymes

Our earlier studies have shown that simultaneous inhibition of glycolysis and pentose phosphate pathway using 2-deoxy-d-glucose (2-DG, an inhibitor of glycolysis) and 6-aminonicotinamide (6-AN, an inhibitor of pentose phosphate pathway) lead to metabolic oxidative stress (MOS), resulting in radiosensitization in malignant cells. Present study was carried out to investigate the effects of 2-DG and 6-AN on intricately regulated endogenous antioxidant defense against MOS during radiosensitization by this combination. Two human tumor cell lines {Head and Neck Squamous carcinoma (KB) and Glioma (BMG-1)} and one non-malignantly transformed cell line (human embryonic kidney, HEK) were used in this study. The presence of 2-DG and 6-AN (added just before irradiation) for 4h, significantly decreased the clonogenicity and metabolic viability of KB and BMG-1 cell lines, while no significant change was seen in HEK cells. Accumulation of ROS was observed only in malignant cell lines, which displayed a compromised redox status evident from enhanced NADP(+)/NADPH and GSSG/GSH ratios and a concomitant decrease in glutathione reductase level and activity at 24h following treatment. The levels and activities of Cu, Zn-SOD and Mn-SOD increased with MOS and were accompanied by a decreased GPx and unaltered catalase activity and level. These results suggest that non-coordinated expression of antioxidant defense, besides compromised redox status, led to selective radiosensitization in the malignant cells.