Ganesh Venkatraman

Nanomedicine: towards development of patient-friendly drug-delivery systems for oncological applications.

The focus on nanotechnology in cancer treatment and diagnosis has intensified due to the serious side effects caused by anticancer agents as a result of their cytotoxic actions on normal cells. This nonspecific action of chemotherapy has awakened a need for formulations capable of definitive targeting with enhanced tumor-killing. Nanooncology, the application of nanobiotechnology to the management of cancer, is currently the most important area of nanomedicine. Currently several nanomaterial-based drug-delivery systems are in vogue and several others are in various stages of development. Tumor-targeted drug-delivery systems are envisioned as magic bullets for cancer therapy and several groups are working globally for development of robust systems.

Blood Compatibility of Iron-Doped Nanosize Hydroxyapatite and Its Drug Release

Nanosize hydroxyapatite (nHAp) doped with varying levels of Fe(3+) (Fe-nHAp of average size 75 nm) was synthesized by hydrothermal and microwave techniques. The samples were characterized for physiochemical properties by X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), inductively coupled plasma optical emission spectrometer (ICP-OES), transmission electron microscopy (TEM), vibrating sample magnetometer (VSM), mechanical and dielectric properties. The biological properties like hemocompatibility, antibacterial efficacy, in vitro bioactivity and the cell proliferation of the samples were determined. XRD pattern of the samples were of single phase hydroxyapatite. As the content of Fe(3+) increased, the crystallite size as well as crystallinity decreased along with a morphological change from spherulites to rods. The dielectric constants and Vickers hardness were enhanced on Fe(3+) doping. The VSM studies revealed that the saturation magnetization (M(s)) and retentivity (M(r)) were found to increase for Fe-nHAp. nHAp impregnated with an antibiotic as a new system for drug delivery in the treatment of chronic osteomyelitis was also attempted. The in vitro drug release with an antibiotic amoxicillin and anticancer drug 5-fluorouracil showed sustained release for the lowest concentration of Fe(3+), while with an increase in the content; there was a rapid release of the drug. The hemolytic assay of Fe(3+) doped samples revealed high blood compatibility (<5% hemolysis). The antibacterial activities of the antibiotic impregnated materials were tested against a culture of E. coli, S. epidermidis and S. aureus by agar diffusion test. The in vitro bioactivity test using simulated body fluid (SBF) showed better bone bonding ability by the formation of an apatite layer on the doped samples. The growth of the apatite layer on the samples surface has been confirmed by EDS analysis. The proliferative potential of MG63 cells by MTT assay confirmed the noncytotoxicity of the samples.

Transcriptional regulation of fibronectin by p21-activated kinase-1 modulates pancreatic tumorigenesis.

Pancreatic ductal adenocarcinoma (PDAC) is the eighth largest cause of cancer-related mortality across the world, with a median 5-year survival rate of less than 3.5%. This is partly because the molecules and the molecular mechanisms that contribute to PDAC are not well understood. Our goal is to understand the role of p21-activated kinase 1 (Pak1) signaling axis in the progression of PDAC. Pak1, a serine/threonine kinase, is a well-known regulator of cytoskeletal remodeling, cell motility, cell proliferation and cell survival. Recent reports suggest that Pak1 by itself can have an oncogenic role in a wide variety of cancers. In this study, we analyzed the expression of Pak1 in human pancreatic cancer tissues and found that Pak1 levels are significantly upregulated in PDAC samples as compared with adjacent normals. Further, to study the functional role of Pak1 in pancreatic cancer model systems, we developed stable overexpression and lentiviral short hairpin RNA-mediated knockdown (KD) clones of Pak1 and studied the changes in transforming properties of the cells. We also observed that Pak1 KD clones failed to form tumors in nude mice. By adopting a quantitative PCR array-based approach, we identified fibronectin, a component of the extracellular matrix and a mesenchymal marker, as a transcriptional target of Pak1 signaling. The underlying molecular mechanism of Pak1-mediated transformation includes its nuclear import and recruitment to the fibronectin promoter via interaction with nuclear factor-κB (NF-κB)–p65 complex. To our knowledge, this is the first study illustrating Pak1–NF-κB–p65-mediated fibronectin regulation as a potent tumor-promoting mechanism in KRAS intact model.

Synthesis and anticancer activity of novel curcumin-quinolone hybrids.

A number of new curcumin-quinolone hybrids were synthesised from differently substituted 3-formyl-2-quinolones and vanillin and their in vitro cytotoxicity was determined on a panel of representative cell lines (A549, MCF7, SKOV3 and H460) using MTT assay. The most potent compound 14, was analysed for its mode of action using various cell biology experiments. SKOV3 cells treated with compound 14 showed distorted cell morphology under phase contrast imaging and induction of apoptosis was confirmed by Annexin V/PE assay. Further experiments on generation of reactive oxygen species (ROS) and cell cycle analysis revealed that these hybrids induce apoptosis by ROS generation and arrest cell cycle progression in S and G2/M phase.

Tumor targeting using polyamidoamine dendrimer-cisplatin nanoparticles functionalized with diglycolamic acid and herceptin

Polymer mediated drug delivery system represents a novel promising platform for tumor-targeting with reduced systemic side effects and improved chemotherapeutical efficacy. In this study, we report the preparation and characterization of herceptin targeted, diglycolamic acid (DGA) functionalized polyamidoamine (PAMAM) dendrimer as a potent drug carrier for cisplatin. DGA dendrimers carrying cisplatin demonstrated enhanced anticancer activity when targeted with herceptin. In vitro cell line studies with herceptin-DGA-G4-cisplatin in HER-2 +ve and HER-2 -ve human ovarian cancer cell lines showed that these nanoparticles possessed remarkable features such as lower IC50 value, improved S-phase arrest, and enhanced apoptosis due to increased cellular uptake and accumulation than the untargeted DGA-G4-cisplatin and free cisplatin. Furthermore, in vivo results in SCID mice bearing SKOV-3 tumor xenografts, herceptin-DGA-G4-cisplatin, appeared to be more effective in inducing tumor regression as compared to free cisplatin. Collectively, these results indicate that herceptin targeted DGA functionalized PAMAM-cisplatin conjugates serve as better anti-tumor agents than individual therapeutic agents.

P21-activated kinase 1 (Pak1) signaling influences therapeutic outcome in pancreatic cancer

BACKGROUND Therapeutic resistance to gemcitabine in pancreatic ductal adenocarcinoma (PDAC) is attributed to various cellular mechanisms and signaling molecules that influence as a single factor or in combination. DESIGN In this study, utilizing in vitro p21-activated kinase 1 (Pak1) overexpression and knockdown cell line models along with in vivo athymic mouse tumor xenograft models and clinical samples, we demonstrate that Pak1 is a crucial signaling kinase in gemcitabine resistance. RESULTS Pak1 kindles resistance via modulation of epithelial-mesenchymal transition and activation of pancreatic stellate cells. Our results from gemcitabine-resistant and -sensitive cell line models showed that elevated Pak1 kinase activity is required to confer gemcitabine resistance. This was substantiated by elevated levels of phosphorylated Pak1 and ribonucleotide reductase M1 levels in the majority of human PDAC tumors when compared with normal. Delineation of the signaling pathway revealed that Pak1 confers resistance to gemcitabine by preventing DNA damage, inhibiting apoptosis and regulating survival signals via NF-κB. Furthermore, we found that Pak1 is an upstream interacting substrate of transforming growth factor β-activated kinase 1-a molecule implicated in gemcitabine resistance. Molecular mechanistic studies revealed that gemcitabine docks with the active site of Pak1; furthermore, gemcitabine treatment induces Pak1 kinase activity both in vivo and in cell-free system. Finally, results from athymic mouse tumor models illustrated that Pak1 inhibition by IPA-3 enhances the cytotoxicity of gemcitabine and brings about pancreatic tumor regression. CONCLUSION To our knowledge, this is the first study illustrating the mechanistic role of Pak1 in causing gemcitabine resistance via multiple signaling crosstalks, and hence Pak1-specific inhibitors will prove to be a better adjuvant with existing chemotherapy modality for PDAC.

Role of vitamin D on the expression of glucose transporters in L6 myotubes

Altered expression of glucose transporters is a major characteristic of diabetes. Vitamin D has evolved widespread interest in the pathogenesis and prevention of diabetes. The present study was designed to investigate the effect of vitamin D in the overall regulation of muscle cell glucose transporter expression. L6 cells were exposed to type 1 and type 2 diabetic conditions and the effect of calcitriol (1,25, dihydroxy cholicalciferol) on the expression of glucose transporters was studied by real time polymerase chain reaction (RT-PCR). There was a significant decrease in glucose transporter type 1 (GLUT1), GLUT4, vitamin D receptor (VDR), and IR expression in type 1 and 2 diabetic model compared to control group. Treatment of myoblasts with 10-7 M calcitriol for 24 h showed a significant increase in GLUT1, GLUT4, VDR, and insulin receptor (IR) expression. The results indicate a potential antidiabetic function of vitamin D on GLUT1, GLUT4, VDR, and IR by improving receptor gene expression suggesting a role for vitamin D in regulation of expression of the glucose transporters in muscle cells.

Antimicrobial activity of cationic peptides in endodontic procedures.

Objectives: The present study aimed to investigate the antimicrobial and biofilm inhibition activity of synthetic antimicrobial peptides (AMPs) against microbes such as Enterococcus faecalis, Staphylococcus aureus, and Candida albicans which are involved in endodontic infections. Materials and Methods: Agar diffusion test was done to determine the activity of peptides. The morphological changes in E. faecalis and reduction in biofilm formation after treatment with peptides were observed using scanning electron microscope. The efficacy of peptides using an ex vivo dentinal model was determined by polymerase chain reaction and confocal laser scanning microscopy. Platelet aggregation was done to determine the biocompatibility of peptides. Results: Among 11 peptides, two of the amphipathic cationic peptides were found to be highly active against E. faecalis, S. aureus, C. albicans. Efficacy results using dentinal tubule model showed significant reduction in microbial load at 400 μm depth. The peptides were also biocompatible. Conclusion: These results suggest that synthetic AMPs have the potential to be developed as antibacterial agents against microorganisms involved in dental infections and thus could prevent the spread and persistence of endodontic infections improving treatment outcomes and teeth preservation.

Molecular and Nanotechnologic Approaches to Etiologic Diagnosis of Infectious Syndromes

Infectious diseases are a major global public health problem. Multiple agents are now recognized to cause indistinguishable illnesses. The term ‘syndrome’ applies to such situations, for which early and rapid diagnosis of the infecting agent would enable prompt and appropriate therapy. Public health physicians would also get timely information on the specific etiology of the infectious syndrome, facilitating intervention at the community level in the face of outbreaks or epidemics. A variety of molecular techniques have been evaluated for rapid diagnosis of infectious syndromes. These techniques include real-time multiplex PCR, DNA microarray, loop-mediated isothermal amplification, and other similar assays. This review surveys such state-of-the-art technologies.

Phosphorylation-Dependent Regulation of the DNA Damage Response of Adaptor Protein KIBRA in Cancer Cells

ABSTRACT Multifunctional adaptor proteins encompassing various protein-protein interaction domains play a central role in the DNA damage response pathway. In this report, we show that KIBRA is a physiologically interacting reversible substrate of ataxia telangiectasia mutated (ATM) kinase. We identified the site of phosphorylation in KIBRA as threonine 1006, which is embedded within the serine/threonine (S/T) Q consensus motif, by site-directed mutagenesis, and we further confirmed the same with a phospho-(S/T) Q motif-specific antibody. Results from DNA repair functional assays such as the γ-H2AX assay, pulsed-field gel electrophoresis (PFGE), Comet assay, terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assay, and clonogenic cell survival assay using stable overexpression clones of wild-type (wt.) KIBRA and active (T1006E) and inactive (T1006A) KIBRA phosphorylation mutants showed that T1006 phosphorylation on KIBRA is essential for optimal DNA double-strand break repair in cancer cells. Further, results from stable retroviral short hairpin RNA-mediated knockdown (KD) clones of KIBRA and KIBRA knockout (KO) model cells generated by a clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 system showed that depleting KIBRA levels compromised the DNA repair functions in cancer cells upon inducing DNA damage. All these phenotypic events were reversed upon reconstitution of KIBRA into cells lacking KIBRA knock-in (KI) model cells. All these results point to the fact that phosphorylated KIBRA might be functioning as a scaffolding protein/adaptor protein facilitating the platform for further recruitment of other DNA damage response factors. In summary, these data demonstrate the imperative functional role of KIBRA per se (KIBRA phosphorylation at T1006 site as a molecular switch that regulates the DNA damage response, possibly via the nonhomologous end joining [NHEJ] pathway), suggesting that KIBRA could be a potential therapeutic target for modulating chemoresistance in cancer cells.