Sankar Jagadeeshan

Biological evaluation of 5-fluorouracil nanoparticles for cancer chemotherapy and its dependence on the carrier, PLGA.

Nanoscaled devices have great potential for drug delivery applications due to their small size. In the present study, we report for the first time the preparation and evaluation of antitumor efficacy of 5-fluorouracil (5-FU)-entrapped poly (D, L-lactic-co-glycolic acid) (PLGA) nanoparticles with dependence on the lactide/glycolide combination of PLGA. 5-FU-loaded PLGA nanoparticles with two different monomer combinations, 50-50 and 90-10 were synthesized using a modified double emulsion method, and their biological evaluation was done in glioma (U87MG) and breast adenocarcinoma (MCF7) cell lines. 5-FU-entrapped PLGA 50-50 nanoparticles showed smaller size with a high encapsulation efficiency of 66%, which was equivalent to that of PLGA 90-10 nanoparticles. Physicochemical characterization of nanoparticles using differential scanning calorimetry and X-ray diffraction suggested the presence of 5-FU in molecular dispersion form. In vitro release studies showed the prolonged and sustained release of 5-FU from nanoparticles with both the PLGA combinations, where PLGA 50-50 nanoparticles showed faster release. Nanoparticles with PLGA 50-50 combination exhibited better cytotoxicity than free drug in a dose- and time-dependent manner against both the tumor cell lines. The enhanced efficiency of PLGA 50-50 nanoparticles to induce apoptosis was indicated by acridine orange/ethidium bromide staining. Cell cycle perturbations studied using flow cytometer showed better S-phase arrest by nanoparticles in comparison with free 5-FU. All the results indicate that PLGA 50-50 nanoparticles possess better antitumor efficacy than PLGA 90-10 nanoparticles and free 5-FU. Since, studies have shown that long-term exposure of ailing tissues to moderate drug concentrations is more favorable than regular administration of higher concentration of the drug; our results clearly indicate the potential of 5-FU-loaded PLGA nanoparticles with dependence on carrier combination as controlled release formulation to multiplex the therapeutic effect of cancer chemotherapy.

Synthesis and in vitro evaluation of some isatin-thiazolidinone hybrid analogues as anti-proliferative agents.

A range of isatin-thiazolidinone hybrid analogues were synthesized and their cytotoxicity was evaluated against several cancer cell lines in vitro. The acute toxicity studies in mice models revealed that these analogues possess low systemic toxicity and are safe up to 1600mg/Kg. Among the compounds synthesized, 5-(2-nitrobenzylidene)-2-(isatin-3-azino)-thiazolidin-4-one (CI) has been shown to be the most active, highly promising compound which induced S phase arrest in cell cycle in a time dependent manner. Our initial analysis indicate that incorporation of electron withdrawing group at ortho position of the ring favors over the meta and para positions for eliciting its cytostatic effect. Overall, the in vitro biological evaluation suggests that the growth inhibitory effect of CI is promising and can be studied further.

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.

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.

Smurf2 E3 ubiquitin ligase modulates proliferation and invasiveness of breast cancer cells in a CNKSR2 dependent manner

BackgroundSmurf2 is a member of the HECT family of E3 ubiquitin ligases that play important roles in determining the competence of cells to respond to TGF- β/BMP signaling pathway. However, besides TGF-β/BMP pathway, Smurf2 regulates a repertoire of other signaling pathways ranging from planar cell polarity during embryonic development to cell proliferation, migration, differentiation and senescence. Expression of Smurf2 is found to be dysregulated in many cancers including breast cancer. The purpose of the present study is to examine the effect of Smurf2 knockdown on the tumorigenic potential of human breast cancer cells emphasizing more on proliferative signaling pathway.MethodssiRNAs targeting different regions of the Smurf2 mRNA were employed to knockdown the expression of Smurf2. The biological effects of synthetic siRNAs on human breast cancer cells were investigated by examining the cell proliferation, migration, invasion, focus formation, anchorage-independent growth, cell cycle arrest, and cell cycle and cell proliferation related protein expressions upon Smurf2 silencing.ResultsSmurf2 silencing in human breast cancer cells resulted in a decreased focus formation potential and clonogenicity as well as in vitro cell migration/invasion capabilities. Moreover, knockdown of Smurf2 suppressed cell proliferation. Cell cycle analysis showed that the anti-proliferative effect of Smurf2 siRNA was mediated by arresting cells in the G0/G1 phase, which was caused by decreased expression of cyclin D1and cdk4, followed by upregulation p21 and p27. Furthermore, we demonstrated that silencing of Smurf2 downregulated the proliferation of breast cancer cells by modulating the PI3K- PTEN-AKT-FoxO3a pathway via the scaffold protein CNKSR2 which is involved in RAS-dependent signaling pathways. The present study provides the first evidence that silencing Smurf2 using synthetic siRNAs can regulate the tumorigenic properties of human breast cancer cells in a CNKSR2 dependent manner.ConclusionsOur results therefore suggest a novel relation between Smurf2 and CNKSR2 thereby regulating AKT-dependent cell proliferation and invasion. Owing to the fact that PI3K-AKT signaling is hyperactivated in various human cancers and that Smurf2 also regulates cellular transformation, our results indicate that Smurf2 may serve as a potential molecule for targeted cancer therapy of certain tumour types including breast cancer.

Poly (D,L-lactic-co-glycolide) Nanoparticles for the Improved Therapeutic Efficacy of All-trans-retinoic Acid: A Study of Acute Myeloid Leukemia (AML) Cell Differentiation In Vitro

All-trans-retinoic acid reverses malignant cell growth and induces cell differentiation and apoptosis. Poor aqueous solubility and uncertain bioavailability are the limiting factors for using all-trans-retinoic acid for tumor therapy. The objective of present study was to encapsulate the hydrophobic drug all-trans-retinoic acid in the polymer poly (lactide-coglycolide). The encapsulation was expected to improve the bioavailability and solubility of the drug. Oil in water single emulsion solvent evaporation technique used for the preparation efficiently encapsulated about 60% of the drug. The drug release profile showed a biphasic pattern with 70% of the drug being released in first 48 hrs and the residual drug showing a slow controlled release reaching up to 8 days. The particle size of 150-200 nm as determined with TEM was ideal for tumor targeting. All-trans-retinoic acid loaded nanoparticles were efficient to induce differentiation and blocked the proliferation of HL-60 cells invitro. These studies also revealed that the dosage of drug required for the therapeutic effects have been reduced efficiently. Our studies thereby demonstrate that Poly (lactide-co-glycolide) based nanoparticles may be efficient for parenteral administration of the drug.

Snail-Modulated MicroRNA 493 Forms a Negative Feedback Loop with the Insulin-Like Growth Factor 1 Receptor Pathway and Blocks Tumorigenesis

ABSTRACT In this study, we have identified one microRNA, microRNA 493 (miR-493), which could simultaneously and directly regulate multiple genes downstream of the insulin-like growth factor 1 receptor (IGF1R) pathway, including IGF1R, by binding with complementary sequences in the 3′ untranslated region (UTR) of mRNAs of IGF1R, insulin receptor substrate 1 (IRS1), and mitogen-activated protein kinase 1 (MAPK1), thereby potentiating their inhibitory function at multiple levels in development and progression of cancers. This binding was further confirmed by pulldown of miR with AGO-2 antibody. Further, results from head and neck samples showed that miR-493 levels were significantly downregulated in tumors, with a concomitant increase in the expression of IGF1R and key downstream effectors. Functional studies from miR-493 overexpression cells and nude-mouse models revealed the tumor suppressor functions of miR-493. Regulation studies revealed that Snail binds to the miR-493 promoter and represses it. We found the existence of a dynamic negative feedback loop in the regulation of IGF1R and miR-493 mediated via Snail. Our study showed that nicotine treatment significantly decreases the levels of miR-493—with a concomitant increase in the levels of Snail—an indication of progression of cells toward tumorigenesis, reestablishing the role of tobacco as a major risk factor for head and neck cancers and elucidating the mechanism behind nicotine-mediated tumorigenesis.

Threonine 209 phosphorylation on RUNX3 by Pak1 is a molecular switch for its dualistic functions

P21 Activated Kinase 1 (Pak1), an oncogenic serine/threonine kinase, is known to have a significant role in the regulation of cytoskeleton and cellular morphology. Runx3 was initially known for its role in tumor suppressor function, but recent studies have reported the oncogenic role of Runx3 in various cancers. However, the mechanism that controls the paradoxical functions of Runx3 still remains unclear. In this study, we show that Runx3 is a physiologically interacting substrate of Pak1. We identified the site of phosphorylation in Runx3 as Threonine 209 by mass spectrometry analysis and site-directed mutagenesis, and further confirmed the same with a site-specific antibody. Results from our functional studies showed that Threonine 209 phosphorylation in Runx3 alters its subcellular localization by protein mislocalization from the nucleus to the cytoplasm and subsequently converses its biological functions. This was further supported by in vivo tumor xenograft studies in nude mouse models which clearly demonstrated that PANC-28 cells transfected with the Runx3-T209E clone showed high tumorigenic potential as compared with other clones. Our results from clinical samples also suggest that Threonine 209 phosphorylation by Pak1 could be a potential therapeutic target and of great clinical relevance with implications for Runx3 inactivation in cancer cells where Runx3 is known to be oncogenic. The findings presented in this study provide evidence of Runx3-Threonine 209 phosphorylation as a molecular switch in dictating the tissue-specific dualistic functions of Runx3 for the first time.

Targeting p21 activated kinase 1 (Pak1) to PAKup Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is one of the leading causes of cancer-related deaths in advanced countries and is on the rise in developing countries. It is a devastating disease with a median five-year survival rate of only 8% [1]. The fundamental reason for such a poor survival rate is that so far no clinically effective targeted therapeutics have been developed for this disease [2]. This situation calls for the need to focus on discovering novel signaling molecules that could lead to PDAC, and also to define the mechanistic role of these molecules in causing cancer progression, metastasis, and therapeutic resistance. The signaling molecules as well as their molecular mechanisms that contribute to the transformation of normal pancreatic cells to pancreatic adenocarcinoma cells have been widely studied [3]. Approximately more than 95% of human pancreatic cancers carry oncogenic RAS mutations, specifically in K-RAS at codon 12 [4]. This has been shown to erratically activate Pak1. P21 activated kinase 1 (Pak1)—a serine/threonine kinase that orchestrates cytoskeletal remodeling and cell motility—has been shown to function as downstream nodule for various oncogenic signaling pathways that promote cell proliferation, regulate apoptosis, and accelerate mitotic abnormalities, resulting in tumor formation and invasiveness. Recent reports showed that Pak1 per se is overexpressed and plays a crucial role in transformation in a wide variety of cancers and also causes drug resistance [5,6]. Despite the remarkable increase in information about the biology of human Pak1 in various malignancies since its discovery, the contribution and direct role of Pak1 signaling to the etiology of pancreatic cancer remains elusive, and this represents one of the core areas to focus on. As a consequence of this, Pak1 expression pattern in human pancreatic cancer tissues was evaluated and it was found that Pak1 levels are significantly high in PDAC tissue [4,7]. Overexpression and downregulated Pak1 cell line model systems revealed explicit changes in transforming properties, and Pak1 diminished cells failed to form tumors in athymic mice [7]. Quantitative PCR array-based approach identified fibronectin (FN), a component of the extracellular matrix, as a transcriptional target of Pak1 signaling. Further, in vitro studies revealed that Pak1 per se nuclear Pak1 contributes to invasive EMT phenotype via modulating NF-κB-p65-FN nexus in pancreatic cancer cells [7]. This is one of the first molecular studies to examine the role of Pak1 pathway in the involvement of pancreatic cancer. Tumor cell–stroma interactions are being increasingly recognized as important determinants of pancreatic tumor cell fate. It is well established that pancreatic cancer cells recruit pancreatic stellate cells (PSCs) via fibrogenic and mitogenic factors, which promote PSC activation, fibrosis, and ECM remodeling capability [8]. As Pak1 modulates EMT markers like FN, E-cadherin, and vimentin, it is speculated that Pak1 might also play a role in activating PSCs, thereby causing desmoplasia, which will eventually lead to chemoresistance (Figure 1). On the other hand, Pak1 being an established survival signaling molecule may also protect pancreatic cancer cells from adverse effects of chemotherapy via multiple signaling crosstalks. Therefore, combinatorial therapeutic strategies capable of managing these detrimental effects of Pak1 need to be tailored to effectively handle pancreatic cancer [9]. Also, it was reported that targeting RAC-Pak1 signaling in RASdriven cancers using Pak1 deletion or Pak1 inhibitor could efficiently regress tumor growth in vivo [10]. As the earlier pharmacologic inhibitors of Pak1 are not very specific, new Pak1 selective inhibitors such as FRAX-597, G-5555, Novartis compound 3 etc. are been synthesized and are under evaluation for clinical efficacy [11–13]. In addition, as suggested by Baker et al. [14], it would be worthwhile to identify a reliable Pak1 substrate-specific biomarker for Pak1 inhibitor antitumor activity. This will aid in highthroughput screening of inhibitors specific and selective to Pak1 on a reliable model system. We hope that all these strategies to handle Pak1 in tumor cells will eventually pave the way to PAKup pancreatic cancer to a certain extent.

Molecular Mechanism of Regulation of MTA1 Expression by Granulocyte Colony-stimulating Factor

Parkinson disease (PD) is a neurodegenerative disorder with loss of dopaminergic neurons of the brain, which results in insufficient synthesis and action of dopamine. Metastasis-associated protein 1 (MTA1) is an upstream modulator of tyrosine hydroxylase (TH), the rate-limiting enzyme in dopamine synthesis, and hence MTA1 plays a significant role in PD pathogenesis. To impart functional and clinical significance to MTA1, we analyzed MTA1 and TH levels in the substantia nigra region of a large cohort of human brain tissue samples by Western blotting, quantitative PCR, and immunohistochemistry. Our results showed that MTA1 and TH levels were significantly down-regulated in PD samples as compared with normal brain tissue. Correspondingly, immunohistochemistry analysis for MTA1 in substantia nigra sections revealed that 74.1% of the samples had a staining intensity of <6 in the PD samples as compared with controls, 25.9%, with an odds ratio of 8.54. Because of the clinical importance of MTA1 established in PD, we looked at agents to modulate MTA1 expression in neuronal cells, and granulocyte colony-stimulating factor (G-CSF) was chosen, due to its clinically proven neurogenic effects. Treatment of the human neuronal cell line KELLY and acute 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mouse model with G-CSF showed significant induction of MTA1 and TH with rescue of phenotype in the mouse model. Interestingly, the observed induction of TH was compromised on silencing of MTA1. The underlying molecular mechanism of MTA1 induction by G-CSF was proved to be through induction of c-Fos and its recruitment to the MTA1 promoter.