Ila Sri Bharati

Fos responses of dopamine neurons to sociosexual stimuli in male zebra finches

Dopamine (DA) is produced in numerous brain areas and influences a wide variety of social behaviors, but very few data are available to establish the socially-relevant response properties of most DA populations, which comprise eight cell groups numbered A8-A15. Anatomically, these DA populations are evolutionarily conserved, and all have been identified in both birds and mammals. We now report the Fos responses of tyrosine hydroxylase-immunoreactive (TH-ir; putatively dopaminergic) neurons in the A8-A15 cell groups of male zebra finches following exposure to a control condition or one of six different social stimuli: a heterospecific male, conspecific male, fighting in a mate competition paradigm (which includes both male and female stimuli), a courtship interaction without physical contact, a courtship interaction with physical contact but no mounting, and a courtship interaction with mounting. We found that the DA cell groups exhibit distinctive profiles of responsiveness to social stimuli. Fos induction in A8, A9, A10 and midbrain A11 neurons increased significantly in response to a variety of conspecific stimuli, but not heterospecific stimuli. In contrast, Fos induction in the preoptic A14 neurons was observed specifically in response to sexual interactions, and Fos induction in hypothalamic A11 neurons appears to primarily reflect the performance of courtship singing. Infundibular A12 neurons, which may be involved in stress-related processes, showed the highest level of TH+Fos colocalization in control subjects. This colocalization decreased in response to all conspecific stimuli except fighting, and did not decrease following exposure to a heterospecific male.

Chronic lymphocytic leukemia cells receive RAF-dependent survival signals in response to CXCL12 that are sensitive to inhibition by sorafenib

The chemokine CXCL12, via its receptor CXCR4, promotes increased survival of chronic lymphocytic leukemia (CLL) B cells that express high levels of ζ-chain-associated protein (ZAP-70), a receptor tyrosine kinase associated with aggressive disease. In this study, we investigated the underlying molecular mechanisms governing this effect. Although significant differences in the expression or turnover of CXCR4 were not observed between ZAP-70(+) and ZAP-70(-) cell samples, CXCL12 induced greater intracellular Ca(2+) flux and stronger and more prolonged phosphorylation of extracellular signal-regulated kinase (ERK) and mitogen-activated protein kinase/ERK kinase (MEK) in the ZAP-70(+) CLL cells. The CXCL12-induced phosphorylation of ERK and MEK in ZAP-70(+) CLL cells was blocked by sorafenib, a small molecule inhibitor of RAF. Furthermore, ZAP-70(+) CLL cells were more sensitive than ZAP-70(-) CLL cells to the cytotoxic effects of sorafenib in vitro at concentrations that can readily be achieved in vivo. The data suggest that ZAP-70(+) CLL cells may be more responsive to survival factors, like CXCL12, that are elaborated by the leukemia microenvironment, and this sensitivity could be exploited for the development of new treatments for patients with this disease. Moreover, sorafenib may have clinical activity for patients with CLL, particularly those with ZAP-70(+) CLL.

Lenalidomide inhibits the proliferation of CLL cells via a cereblon/ p21 WAF1/Cip1 -dependent mechanism independent of functional p53

Lenalidomide has demonstrated clinical activity in patients with chronic lymphocytic leukemia (CLL), even though it is not cytotoxic for primary CLL cells in vitro. We examined the direct effect of lenalidomide on CLL-cell proliferation induced by CD154-expressing accessory cells in media containing interleukin-4 and -10. Treatment with lenalidomide significantly inhibited CLL-cell proliferation, an effect that was associated with the p53-independent upregulation of the cyclin-dependent kinase inhibitor, p21(WAF1/Cip1) (p21). Silencing p21 with small interfering RNA impaired the capacity of lenalidomide to inhibit CLL-cell proliferation. Silencing cereblon, a known molecular target of lenalidomide, impaired the capacity of lenalidomide to induce expression of p21, inhibit CD154-induced CLL-cell proliferation, or enhance the degradation of Ikaros family zinc finger proteins 1 and 3. We isolated CLL cells from the blood of patients before and after short-term treatment with low-dose lenalidomide (5 mg per day) and found the leukemia cells were also induced to express p21 in vivo. These results indicate that lenalidomide can directly inhibit proliferation of CLL cells in a cereblon/p21-dependent but p53-independent manner, at concentrations achievable in vivo, potentially contributing to the capacity of this drug to inhibit disease-progression in patients with CLL.

Novel anti-glioblastoma agents and therapeutic combinations identified from a collection of FDA approved drugs

BackgroundGlioblastoma (GBM) is a therapeutic challenge, associated with high mortality. More effective GBM therapeutic options are urgently needed. Hence, we screened a large multi-class drug panel comprising the NIH clinical collection (NCC) that includes 446 FDA-approved drugs, with the goal of identifying new GBM therapeutics for rapid entry into clinical trials for GBM.MethodsScreens using human GBM cell lines revealed 22 drugs with potent anti-GBM activity, including serotonergic blockers, cholesterol-lowering agents (statins), antineoplastics, anti-infective, anti-inflammatories, and hormonal modulators. We tested the 8 most potent drugs using patient-derived GBM cancer stem cell-like lines. Notably, the statins were active in vitro; they inhibited GBM cell proliferation and induced cellular autophagy. Moreover, the statins enhanced, by 40-70 fold, the pro-apoptotic activity of irinotecan, a topoisomerase 1 inhibitor currently used to treat a variety of cancers including GBM. Our data suggest that the mechanism of action of statins was prevention of multi-drug resistance protein MDR-1 glycosylation. This drug combination was synergistic in inhibiting tumor growth in vivo. Compared to animals treated with high dose irinotecan, the drug combination showed significantly less toxicity.ResultsOur data identifies a novel combination from among FDA-approved drugs. In addition, this combination is safer and well tolerated compared to single agent irinotecan.ConclusionsOur study newly identifies several FDA-approved compounds that may potentially be useful in GBM treatment. Our findings provide the basis for the rational combination of statins and topoisomerase inhibitors in GBM.

In vitro and in vivo anticancer effects of mevalonate pathway modulation on human cancer cells

Background:The increasing usage of statins (the 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors) has revealed a number of unexpected beneficial effects, including a reduction in cancer risk.Methods:We investigated the direct anticancer effects of different statins approved for clinical use on human breast and brain cancer cells. We also explored the effects of statins on cancer cells using in silico simulations.Results:In vitro studies showed that cerivastatin, pitavastatin, and fluvastatin were the most potent anti-proliferative, autophagy inducing agents in human cancer cells including stem cell-like primary glioblastoma cell lines. Consistently, pitavastatin was more effective than fluvastatin in inhibiting U87 tumour growth in vivo. Intraperitoneal injection was much better than oral administration in delaying glioblastoma growth. Following statin treatment, tumour cells were rescued by adding mevalonate and geranylgeranyl pyrophosphate. Knockdown of geranylgeranyl pyrophosphate synthetase-1 also induced strong cell autophagy and cell death in vitro and reduced U87 tumour growth in vivo. These data demonstrate that statins main effect is via targeting the mevalonate synthesis pathway in tumour cells.Conclusions:Our study demonstrates the potent anticancer effects of statins. These safe and well-tolerated drugs need to be further investigated as cancer chemotherapeutics in comprehensive clinical studies.

Sorafenib-Induced Apoptosis of Chronic Lymphocytic Leukemia Cells Is Associated with Downregulation of RAF and Myeloid Cell Leukemia Sequence 1 (Mcl-1)

We have previously shown that sorafenib, a multikinase inhibitor, exhibits cytotoxic effects on chronic lymphocytic leukemia (CLL) cells. Because the cellular microenvironment can protect CLL cells from drug-induced apoptosis, it is important to evaluate the effect of novel drugs in this context. Here we characterized the in vitro cytotoxic effects of sorafenib on CLL cells and the underlying mechanism in the presence of marrow stromal cells (MSCs) and nurselike cells (NLCs). One single dose of 10 µmol/L or the repeated addition of 1 µmol/L sorafenib caused caspase-dependent apoptosis and reduced levels of phosphorylated B-RAF, C-RAF, extracellular signal-regulated kinase (ERK), signal transducer and activator of transcription 3 (STAT3) and myeloid cell leukemia sequence 1 (Mcl-1) in CLL cells in the presence of the microenvironment. We show that the RAF/mitogen-activated protein kinase kinase (MEK)/ERK pathway can modulate Mcl-1 expression and contribute to CLL cell viability, thereby associating sorafenib cytotoxicity to its impact on RAF and Mcl-1. To evaluate if the other targets of sorafenib can affect CLL cell viability and contribute to sorafenib-mediated cytotoxicity, we tested the sensitivity of CLL cells to several kinase inhibitors specific for these targets. Our data show that RAF and vascular endothelial growth factor receptor (VEGFR) but not KIT, platelet-derived growth factor receptor (PDGFR) and FMS-like tyrosine kinase 3 (FLT3) are critical for CLL cell viability. Taken together, our data suggest that sorafenib exerts its cytotoxic effect likely via inhibition of the VEGFR and RAF/MEK/ERK pathways, both of which can modulate Mcl-1 expression in CLL cells. Furthermore, sorafenib induced apoptosis of CLL cells from fludarabine refractory patients in the presence of NLCs or MSCs. Our results warrant further clinical exploration of sorafenib in CLL.

High-efficiency liposomal encapsulation of a tyrosine kinase inhibitor leads to improved in vivo toxicity and tumor response profile

Staurosporine (STS) is a potent pan-kinase inhibitor with marked activity against several chemotherapy-resistant tumor types in vitro. The translational progress of this compound has been hindered by poor pharmacokinetics and toxicity. We sought to determine whether liposomal encapsulation of STS would enhance antitumor efficacy and reduce toxicity, thereby supporting the feasibility of further preclinical development. We developed a novel reverse pH gradient liposomal loading method for STS, with an optimal buffer type and drug-to-lipid ratio. Our approach produced 70% loading efficiency with good retention, and we provide, for the first time, an assessment of the in vivo antitumor activity of STS. A low intravenous dose (0.8 mg/kg) inhibited U87 tumors in a murine flank model. Biodistribution showed preferential tumor accumulation, and body weight data, a sensitive index of STS toxicity, was unaffected by liposomal STS, but did decline with the free compound. In vitro experiments revealed that liposomal STS blocked Akt phosphorylation, induced poly(ADP-ribose) polymerase cleavage, and produced cell death via apoptosis. This study provides a basis to explore further the feasibility of liposomally encapsulated STS, and potentially related compounds for the management of resistant solid tumors.

In silico modeling predicts drug sensitivity of patient-derived cancer cells

BackgroundGlioblastoma (GBM) is an aggressive disease associated with poor survival. It is essential to account for the complexity of GBM biology to improve diagnostic and therapeutic strategies. This complexity is best represented by the increasing amounts of profiling (“omics”) data available due to advances in biotechnology. The challenge of integrating these vast genomic and proteomic data can be addressed by a comprehensive systems modeling approach.MethodsHere, we present an in silico model, where we simulate GBM tumor cells using genomic profiling data. We use this in silico tumor model to predict responses of cancer cells to targeted drugs. Initially, we probed the results from a recent hypothesis-independent, empirical study by Garnett and co-workers that analyzed the sensitivity of hundreds of profiled cancer cell lines to 130 different anticancer agents. We then used the tumor model to predict sensitivity of patient-derived GBM cell lines to different targeted therapeutic agents.ResultsAmong the drug-mutation associations reported in the Garnett study, our in silico model accurately predicted ~85% of the associations. While testing the model in a prospective manner using simulations of patient-derived GBM cell lines, we compared our simulation predictions with experimental data using the same cells in vitro. This analysis yielded a ~75% agreement of in silico drug sensitivity with in vitro experimental findings.ConclusionsThese results demonstrate a strong predictability of our simulation approach using the in silico tumor model presented here. Our ultimate goal is to use this model to stratify patients for clinical trials. By accurately predicting responses of cancer cells to targeted agents a priori, this in silico tumor model provides an innovative approach to personalizing therapy and promises to improve clinical management of cancer.

Enhanced anti-tumor immune responses and delay of tumor development in human epidermal growth factor receptor 2 mice immunized with an immunostimulatory peptide in poly(D,L-lactic-co-glycolic) acid nanoparticles

IntroductionCancer vaccines have the potential to induce curative anti-tumor immune responses and better adjuvants may improve vaccine efficacy. We have previously shown that Hp91, a peptide derived from the B box domain in high-mobility group box protein 1 (HMGB1), acts as a potent immune adjuvant.MethodIn this study, Hp91 was tested as part of a therapeutic vaccine against human epidermal growth factor receptor 2 (HER2)-positive breast cancer.ResultsFree peptide did not significantly augment immune responses but, when delivered in poly(D,L-lactic-co-glycolic) acid nanoparticles (PLGA-NPs), robust activation of dendritic cells (DCs) and increased activation of HER2-specific T cells was observed in vitro. Vaccination of HER2/neu transgenic mice, a mouse breast cancer model that closely mimics the immune modulation and tolerance in some breast cancer patients, with Hp91-loaded PLGA-NPs enhanced the activation of HER2-specific cytotoxic T lymphocyte (CTL) responses, delayed tumor development, and prolonged survival.ConclusionsTaken together these findings demonstrate that the delivery of the immunostimulatory peptide Hp91 inside PLGA-NPs enhances the potency of the peptide and efficacy of a breast cancer vaccine.