Ritu Aneja

Publications of Ritu Aneja

• Molecular Cycloencapsulation Augments Solubility and Improves Therapeutic Index of Brominated Noscapine in Prostate Cancer Cells.

  Authors: Jitender Madan, Bharat Baruah, Mulpuri Nagaraju, Mohamed O Abdalla, Clayton Yates, Timothy Turner, Vijay Rangari, Donald Hamelberg, Ritu Aneja

  Molecular pharmaceutics. 04/2012;

  We have previously shown that a novel microtubule-modulating noscapinoid, EM011 (9-Br-Nos), displays potent anticancer activity by inhibition of cellular proliferation and induction of apoptosis inWe have previously shown that a novel microtubule-modulating noscapinoid, EM011 (9-Br-Nos), displays potent anticancer activity by inhibition of cellular proliferation and induction of apoptosis in prostate cancer cells and preclinical mice models. However, physicochemical and cellular barriers encumber the development of viable formulations for future clinical translation. To circumvent these limitations, we have synthesized EM011-cyclodextrin inclusion complexes to improve solubility and enhance therapeutic index of EM011. Phase solubility analysis indicated that EM011 formed a 1:1 stoichiometric complex with β-CD and methyl-β-CD, with a stability constant (K(c)) of 2.42 × 10(-3) M and 4.85 × 10(-3) M, respectively. Fourier transform infrared spectroscopy suggested the penetrance of either a O-CH(2) or OCH(3)-C(6)H(4)-OCH(3) moiety of EM011 in the β-CD or methyl-β-CD cavity. In addition, multifarious techniques, namely, differential scanning calorimetry, powder X-ray diffraction, scanning electron microscopy, NMR spectroscopy, and computational studies validated the cage complex of EM011 with β-CD and methyl-β-CD. Moreover, rotating frame overhauser enhancement spectroscopy showed that the H(a) proton of the OCH(3)-C(6)H(4)-OCH(3) moiety was in close proximity with H3 proton of the β-CD or methyl-β-CD cavity. Furthermore, we found that the solubility of EM011 in phosphate buffer saline (pH 7.4) was enhanced by ∼11 fold and ∼21 fold upon complexation with β-CD and methyl-β-CD, respectively. The enhanced dissolution of the drug CD-complexes in aqueous phase remarkably decreased their IC(50) to 28.5 μM (9-Br-Nos-β-CD) and 12.5 μM (9-Br-Nos-methyl-β-CD) in PC-3 cells compared to free EM011 (∼200 μM). This is the first report to demonstrate the novel construction of cylcodextrin-based nanosupramolecular vehicles for enhanced delivery of EM011 that warrants in vivo evaluation for the superior management of prostate cancer.

• Cep70 contributes to angiogenesis by modulating microtubule rearrangement and stimulating cell polarization and migration.

  Authors: Xingjuan Shi, Min Liu, Dengwen Li, Jun Wang, Ritu Aneja, Jun Zhou

  Cell cycle (Georgetown, Tex.). 04/2012; 11(8).

  Centrosomal proteins intricately regulate diverse microtubule-mediated cellular activities, including cell polarization and migration. However, the direct participation of these proteins inCentrosomal proteins intricately regulate diverse microtubule-mediated cellular activities, including cell polarization and migration. However, the direct participation of these proteins in angiogenesis, which involves vascular endothelial cell migration from preexisting blood vessels, remains elusive. Here we show that the centrosomal protein Cep70 is necessary for angiogenic response in mice. This protein is also required for tube formation and capillary sprouting in vitro from vascular endothelial cells. Wound healing and transwell assays reveal that Cep70 plays a significant role in endothelial cell migration. Depletion of Cep70 results in severe defects in membrane ruffling and centrosome reorientation, indicating a requirement for this protein in cell polarization. In addition, Cep70 is critically involved in microtubule rearrangement in response to the migratory stimulus. Our data further demonstrate that Cep70 is important for Cdc42 and Rac1 activation to promote angiogenesis. These findings thus establish Cep70 as a crucial regulator of the angiogenic process and emphasize the significance of microtubule rearrangement and cell polarization and migration in angiogenesis.

• In silico inspired design and synthesis of a novel tubulin-binding anti-cancer drug: folate conjugated noscapine (Targetin).

  Authors: Pradeep K Naik, Manu Lopus, Ritu Aneja, Surya N Vangapandu, Harish C Joshi

  Journal of computer-aided molecular design. 12/2011; 26(2):233-47.

  Our screen for tubulin-binding small molecules that do not depolymerize bulk cellular microtubules, but based upon structural features of well known microtubule-depolymerizing colchicine andOur screen for tubulin-binding small molecules that do not depolymerize bulk cellular microtubules, but based upon structural features of well known microtubule-depolymerizing colchicine and podophyllotoxin, revealed tubulin binding anti-cancer property of noscapine (Ye et al. in Proc Natl Acad Sci USA 95:2280-2286, 1998). Guided by molecular modelling calculations and structure-activity relationships we conjugated at C9 of noscapine, a folate group-a ligand for cellular folate receptor alpha (FRα). FRα is over-expressed on some solid tumours such as ovarian epithelial cancers. Molecular docking experiments predicted that a folate conjugated noscapine (Targetin) accommodated well inside the binding cavity (docking score -11.295 kcal/mol) at the interface between α- and β-tubulin. The bulky folate moiety of Targetin is extended toward lumen of microtubules. The binding free energy (ΔG (bind)) computed based on molecular mechanics energy minimization was -221.01 kcal/mol that revealed favourable interaction of Targetin with the receptor. Chemical synthesis, tubulin-binding experiments, and anti-cancer activity in vitro corroborate fully well with the molecular modelling experiments. Targetin binds tubulin with a dissociation constant (K (d) value) of 149 ± 3.0 μM and decreases the transition frequencies between growth and shortening phases of microtubule assembly dynamics at concentrations that do not alter the total polymer mass. Cancer cells in general were more sensitive to Targetin compared with the founding compound noscapine (IC(50) in the range of 15-40 μM). Quite strikingly, ovarian cancer cells (SKOV3 and A2780), known to overexpress FRα, were much more sensitive to targetin (IC(50) in the range of 0.3-1.5 μM).

• Near infrared active heptacyanine dyes with unique cancer-imaging and cytotoxic properties.

  Authors: Maged Henary, Vaishali Pannu, Eric A Owens, Ritu Aneja

  Bioorganic & medicinal chemistry letters. 11/2011; 22(2):1242-6.

  Three near-infrared fluorescent heptacarbocyanine dyes have been synthesized using a facile one-pot synthetic approach. The reaction methodology afforded a mixture of three symmetric and unsymmetricThree near-infrared fluorescent heptacarbocyanine dyes have been synthesized using a facile one-pot synthetic approach. The reaction methodology afforded a mixture of three symmetric and unsymmetric heptacyanines containing various N-indolenine substituents, a dicarbocyclic acid (DA), a monoester (ME), and a diester (DE). These compounds were isolated, purified, characterized and biologically investigated for tumor cell cytotoxicity and uptake selectivity. Using cell viability and in vitro proliferation assays, we found that the esterified dyes (monoester, ME and diester, DE) were selectively cytotoxic to cancer cells and spared normal fibroblast cells. Additionally, confocal fluorescence imaging confirmed selective uptake of these dyes in cancer cells, thus suggesting tumor cell targeting.

• Polyphenol-rich sweet potato greens extract inhibits proliferation and induces apoptosis in prostate cancer cells in vitro and in vivo.

  Authors: Prasanthi Karna, Sushma R Gundala, Meenakshi V Gupta, Shahab A Shamsi, Ralphenia D Pace, Clayton Yates, Satya Narayan, Ritu Aneja

  Carcinogenesis. 09/2011; 32(12):1872-80.

  Sweet potato (Ipomoea batatas) leaves or greens, extensively consumed as a vegetable in Africa and Asia, are an excellent source of dietary polyphenols such as anthocyanins and phenolic acids. Here,Sweet potato (Ipomoea batatas) leaves or greens, extensively consumed as a vegetable in Africa and Asia, are an excellent source of dietary polyphenols such as anthocyanins and phenolic acids. Here, we show that sweet potato greens extract (SPGE) has the maximum polyphenol content compared with several commercial vegetables including spinach. The polyphenol-rich SPGE exerts significant antiproliferative activity in a panel of prostate cancer cell lines while sparing normal prostate epithelial cells. Mechanistically, SPGE perturbed cell cycle progression, reduced clonogenic survival, modulated cell cycle and apoptosis regulatory molecules and induced apoptosis in human prostate cancer PC-3 cells both in vitro and in vivo. SPGE-induced apoptosis has a mitochondrially mediated component, which was attenuated by pretreatment with cyclosporin A. We also observed alterations of apoptosis regulatory molecules such as inactivation of Bcl2, upregulation of BAX, cytochrome c release and activation of downstream apoptotic signaling. SPGE caused DNA degradation as evident by terminal deoxynucleotidyl transferase-mediated dUTP-nick-end labeling (TUNEL) staining of increased concentration of 3'-DNA ends. Furthermore, apoptotic induction was caspase dependent as shown by cleavage of caspase substrate, poly (adenosine diphosphate-ribose) polymerase. Oral administration of 400 mg/kg SPGE remarkably inhibited growth and progression of prostate tumor xenografts by ∼69% in nude mice, as shown by tumor volume measurements and non-invasive real-time bioluminescent imaging. Most importantly, SPGE did not cause any detectable toxicity to rapidly dividing normal tissues such as gut and bone marrow. This is the first report to demonstrate the in vitro and in vivo anticancer activity of sweet potato greens in prostate cancer.

• Benefits of whole ginger extract in prostate cancer.

  Authors: Prasanthi Karna, Sharmeen Chagani, Sushma R Gundala, Padmashree C G Rida, Ghazia Asif, Vibhuti Sharma, Meenakshi V Gupta, Ritu Aneja

  The British journal of nutrition. 08/2011; 107(4):473-84.

  It is appreciated far and wide that increased and regular consumption of fruits and vegetables is linked with noteworthy anticancer benefits. Extensively consumed as a spice in foods and beveragesIt is appreciated far and wide that increased and regular consumption of fruits and vegetables is linked with noteworthy anticancer benefits. Extensively consumed as a spice in foods and beverages worldwide, ginger (Zingiber officinale Roscoe) is an excellent source of several bioactive phenolics, including non-volatile pungent compounds such as gingerols, paradols, shogaols and gingerones. Ginger has been known to display anti-inflammatory, antioxidant and antiproliferative activities, indicating its promising role as a chemopreventive agent. Here, we show that whole ginger extract (GE) exerts significant growth-inhibitory and death-inductory effects in a spectrum of prostate cancer cells. Comprehensive studies have confirmed that GE perturbed cell-cycle progression, impaired reproductive capacity, modulated cell-cycle and apoptosis regulatory molecules and induced a caspase-driven, mitochondrially mediated apoptosis in human prostate cancer cells. Remarkably, daily oral feeding of 100 mg/kg body weight of GE inhibited growth and progression of PC-3 xenografts by approximately 56 % in nude mice, as shown by measurements of tumour volume. Tumour tissue from GE-treated mice showed reduced proliferation index and widespread apoptosis compared with controls, as determined by immunoblotting and immunohistochemical methods. Most importantly, GE did not exert any detectable toxicity in normal, rapidly dividing tissues such as gut and bone marrow. To the best of our knowledge, this is the first report to demonstrate the in vitro and in vivo anticancer activity of whole GE for the management of prostate cancer.

• CEP70 protein interacts with γ-tubulin to localize at the centrosome and is critical for mitotic spindle assembly.

  Authors: Xingjuan Shi, Xiaoou Sun, Min Liu, Dengwen Li, Ritu Aneja, Jun Zhou

  The Journal of biological chemistry. 07/2011; 286(38):33401-8.

  Deregulation of the mitotic spindle has been implicated in genomic instability, an important aspect of tumorigenesis and malignant transformation. To ensure the fidelity of chromosome transmission,Deregulation of the mitotic spindle has been implicated in genomic instability, an important aspect of tumorigenesis and malignant transformation. To ensure the fidelity of chromosome transmission, the mitotic spindle is assembled by exquisite mechanisms and orchestrated by centrosomes in animal cells. Centrosomal proteins especially are thought to act coordinately to ensure accurate spindle formation, but the molecular details remain to be investigated. In this study, we report the molecular characterization and functional analysis of a novel centrosomal protein, Cep70. Our data show that Cep70 localizes to the centrosome throughout the cell cycle and binds to the key centrosomal component, γ-tubulin, through the peptide fragments that contain the coiled-coil domains. Our data further reveal that the centrosomal localization pattern of Cep70 is dependent on its interaction with γ-tubulin. Strikingly, Cep70 plays a significant role in the organization of both preexisting and nascent microtubules in interphase cells. In addition, Cep70 is necessary for the organization and orientation of the bipolar spindle during mitosis. These results thus report for the first time the identification of Cep70 as an important centrosomal protein that interacts with γ-tubulin and underscore its critical role in the regulation of mitotic spindle assembly.

• Second generation benzofuranone ring substituted noscapine analogs: synthesis and biological evaluation.

  Authors: Ram Chandra Mishra, Prasanthi Karna, Sushma Reddy Gundala, Vaishali Pannu, Richard A Stanton, Kamlesh Kumar Gupta, M Hope Robinson, Manu Lopus, Leslie Wilson, Maged Henary, Ritu Aneja

  Biochemical pharmacology. 07/2011; 82(2):110-21.

  Microtubules, composed of α/β tubulin heterodimers, represent a validated target for cancer chemotherapy. Thus, tubulin- and microtubule-binding antimitotic drugs such as taxanes and vincas areMicrotubules, composed of α/β tubulin heterodimers, represent a validated target for cancer chemotherapy. Thus, tubulin- and microtubule-binding antimitotic drugs such as taxanes and vincas are widely employed for the chemotherapeutic management of various malignancies. Although quite successful in the clinic, these drugs are associated with severe toxicity and drug resistance problems. Noscapinoids represent an emerging class of microtubule-modulating anticancer agents based upon the parent molecule noscapine, a naturally occurring non-toxic cough-suppressant opium alkaloid. Here we report in silico molecular modeling, chemical synthesis and biological evaluation of novel analogs derived by modification at position-7 of the benzofuranone ring system of noscapine. The synthesized analogs were evaluated for their tubulin polymerization activity and their biological activity was examined by their antiproliferative potential using representative cancer cell lines from varying tissue-origin [A549 (lung), CEM (lymphoma), MIA PaCa-2 (pancreatic), MCF-7 (breast) and PC-3 (prostate)]. Cell-cycle studies were performed to explore their ability to halt the cell-cycle and induce subsequent apoptosis. The varying biological activity of these analogs that differ in the nature and bulk of substituent at position-7 was rationalized utilizing predictive in silico molecular modeling.

• Rational design, synthesis and biological evaluations of amino-noscapine: a high affinity tubulin-binding noscapinoid.

  Authors: Pradeep K Naik, Biswa Prasun Chatterji, Surya N Vangapandu, Ritu Aneja, Ramesh Chandra, Srinivas Kanteveri, Harish C Joshi

  Journal of computer-aided molecular design. 05/2011; 25(5):443-54.

  Noscapine and its derivatives are important microtubule-interfering agents shown to have potent anti-tumor activity. The binding free energies (ΔG (bind)) of noscapinoids computed using linearNoscapine and its derivatives are important microtubule-interfering agents shown to have potent anti-tumor activity. The binding free energies (ΔG (bind)) of noscapinoids computed using linear interaction energy (LIE) method with a surface generalized Born (SGB) continuum solvation model were in agreement with the experimental ΔG (bind) with average root mean square error of 0.082 kcal/mol. This LIE-SGB model guided us in designing a novel derivative of noscapine, amino-noscapine [(S)-3-((R)-9-amino-4-methoxy-6-methyl-5,6,7,8-tetrahydro [1, 3] dioxolo[4,5-g]isoquinolin-5-yl)-6,7-dimethoxy isobenzo-furan-1(3H)-one] that has higher tubulin binding activity (predicted ΔG (bind) = -6.438 kcal/mol and experimental ΔG (bind) = -6.628 kcal/mol) than noscapine, but does not significantly change the total extent of the tubulin subunit/polymer ratio. The modes of interaction of amino-noscapine with the binding pocket of tubulin involved three hydrogen bonds and are distinct compared to noscapine which involved only one hydrogen bond. Also the patterns of non-bonded interactions are albeit different between both the lignads. The 'blind docking' approach (docking of ligand with different binding sites of a protein and their evaluations) as well as the reasonable accuracy of calculating ΔG (bind) using LIE-SGB model constitutes the first evidence that this class of compounds binds to tubulin at a site overlapping with colchicine-binding site or close to it. Our results revealed that amino-noscapine has better anti-tumor activity than noscapine.

• Long-circulating poly(ethylene glycol)-grafted gelatin nanoparticles customized for intracellular delivery of noscapine: preparation, in-vitro characterization, structure elucidation, pharmacokinetics, and cytotoxicity analyses.

  Authors: Jitender Madan, Neerupma Dhiman, Satish Sardana, Ritu Aneja, Ramesh Chandra, Anju Katyal

  Anti-cancer drugs. 04/2011; 22(6):543-55.

  Noscapine, the tubulin-binding anticancer agent, when administered orally, requires high ED(50) (300-600 mg/kg), whereas intravenous administration (10 mg/kg) results in rapid elimination of the drugNoscapine, the tubulin-binding anticancer agent, when administered orally, requires high ED(50) (300-600 mg/kg), whereas intravenous administration (10 mg/kg) results in rapid elimination of the drug with a half-life of 0.39 h. Hence, the development of long-circulating injectable nanoparticles can be an interesting option for designing a viable formulation of noscapine for anticancer activity. Noscapine-enveloped gelatin nanoparticles and poly(ethylene glycol)-grafted gelatin nanoparticles were constructed and characterized. Data indicate that smooth and spherical shaped nanoparticles of 127 ± 15 nm were engineered with maximum entrapment efficiency of 65.32 ± 3.81%. Circular dichroism confirms that nanocoacervates retained the α-helical content of gelatin in ethanol whereas acetone favored the formation of a random coil. Moreover, the Fourier transform infrared and powder X-ray diffraction pattern prevents any significant change in the noscapine-loaded gelatin nanoparticles in comparison with individual components. In-vitro release kinetic data suggest a first-order release of noscapine (85.1%) from gelatin nanoparticles with a release rate constant of 7.611×10(-3). It is to be noted that there is a 1.43-fold increase in the area under the curve up to the last sampling point for the noscapine-loaded poly(ethylene glycol)-grafted gelatin nanoparticles over the noscapine-loaded gelatin nanoparticles and a 13.09-fold increase over noscapine. Cytotoxicity analysis of the MCF-7 cell line indicated that the IC(50) value of the noscapine-loaded poly(ethylene glycol)-grafted gelatin nanoparticles was equivalent to 20.8 μmol/l, which was significantly (P<0.05) lower than the IC(50) value of the noscapine-loaded gelatin nanoparticles (26.3 μmol/l) and noscapine (40.5 μmol/l).Noscapine-loaded poly(ethylene glycol)-grafted gelatin nanoparticles can be developed as a promising therapeutic agent for the management of breast cancer.

• Drugs that target dynamic microtubules: a new molecular perspective.

  Authors: Richard A Stanton, Kim M Gernert, James H Nettles, Ritu Aneja

  Medicinal research reviews. 03/2011; 31(3):443-81.

  Microtubules have long been considered an ideal target for anticancer drugs because of the essential role they play in mitosis, forming the dynamic spindle apparatus. As such, there is a wide varietyMicrotubules have long been considered an ideal target for anticancer drugs because of the essential role they play in mitosis, forming the dynamic spindle apparatus. As such, there is a wide variety of compounds currently in clinical use and in development that act as antimitotic agents by altering microtubule dynamics. Although these diverse molecules are known to affect microtubule dynamics upon binding to one of the three established drug domains (taxane, vinca alkaloid, or colchicine site), the exact mechanism by which each drug works is still an area of intense speculation and research. In this study, we review the effects of microtubule-binding chemotherapeutic agents from a new perspective, considering how their mode of binding induces conformational changes and alters biological function relative to the molecular vectors of microtubule assembly or disassembly. These "biological vectors" can thus be used as a spatiotemporal context to describe molecular mechanisms by which microtubule-targeting drugs work.

• Regulation of Tat acetylation and transactivation activity by the microtubule-associated deacetylase HDAC6.

  Authors: Lihong Huo, Dengwen Li, Xiaoou Sun, Xingjuan Shi, Prasanthi Karna, Wei Yang, Min Liu, Wentao Qiao, Ritu Aneja, Jun Zhou

  The Journal of biological chemistry. 01/2011; 286(11):9280-6.

  Reversible acetylation of Tat is critical for its transactivation activity toward HIV-1 transcription. However, the enzymes involved in the acetylation/deacetylation cycles have not been fullyReversible acetylation of Tat is critical for its transactivation activity toward HIV-1 transcription. However, the enzymes involved in the acetylation/deacetylation cycles have not been fully characterized. In this study, by yeast two-hybrid assay, we have discovered the histone deacetylase HDAC6 to be a binding partner of Tat. Our data show that HDAC6 interacts with Tat in the cytoplasm in a microtubule-dependent manner. In addition, HDAC6 deacetylates Tat at Lys-28 and thereby suppresses Tat-mediated transactivation of the HIV-1 promoter. Inactivation of HDAC6 promotes the interaction of Tat with cyclin T1 and leads to an increase in Tat transactivation activity. These findings establish HDAC6 as a Tat deacetylase and support a model in which Lys-28 deacetylation decreases Tat transactivation activity through affecting the ability of Tat to form a ribonucleoprotein complex with cyclin T1 and the transactivation-responsive RNA.

• DNA polymerase β as a novel target for chemotherapeutic intervention of colorectal cancer.

  Authors: Aruna S Jaiswal, Sanjeev Banerjee, Ritu Aneja, Fazlul H Sarkar, David A Ostrov, Satya Narayan

  PloS one. 01/2011; 6(2):e16691.

  Chemoprevention presents a major strategy for the medical management of colorectal cancer. Most drugs used for colorectal cancer therapy induce DNA-alkylation damage, which is primarily repaired byChemoprevention presents a major strategy for the medical management of colorectal cancer. Most drugs used for colorectal cancer therapy induce DNA-alkylation damage, which is primarily repaired by the base excision repair (BER) pathway. Thus, blockade of BER pathway is an attractive option to inhibit the spread of colorectal cancer. Using an in silico approach, we performed a structure-based screen by docking small-molecules onto DNA polymerase β (Pol-β) and identified a potent anti-Pol-β compound, NSC-124854. Our goal was to examine whether NSC-124854 could enhance the therapeutic efficacy of DNA-alkylating agent, Temozolomide (TMZ), by blocking BER. First, we determined the specificity of NSC-124854 for Pol-β by examining in vitro activities of APE1, Fen1, DNA ligase I, and Pol-β-directed single nucleotide (SN)- and long-patch (LP)-BER. Second, we investigated the effect of NSC-124854 on the efficacy of TMZ to inhibit the growth of mismatch repair (MMR)-deficient and MMR-proficient colon cancer cell lines using in vitro clonogenic assays. Third, we explored the effect of NSC-124854 on TMZ-induced in vivo tumor growth inhibition of MMR-deficient and MMR-proficient colonic xenografts implanted in female homozygous SCID mice. Our data showed that NSC-124854 has high specificity to Pol-β and blocked Pol-β-directed SN- and LP-BER activities in in vitro reconstituted system. Furthermore, NSC-124854 effectively induced the sensitivity of TMZ to MMR-deficient and MMR-proficient colon cancer cells both in vitro cell culture and in vivo xenograft models. Our findings suggest a potential novel strategy for the development of highly specific structure-based inhibitor for the prevention of colonic tumor progression.

• LHRH-conjugated lytic peptides directly target prostate cancer cells.

  Authors: Clayton Yates, Starlette Sharp, Jacqueline Jones, Daphne Topps, Mathew Coleman, Ritu Aneja, Jesse Jaynes, Timothy Turner

  Biochemical pharmacology. 01/2011; 81(1):104-10.

  Prostate cancer is the second leading cause of cancer deaths among men. For patients with hormone-refractory disease, few treatments are available once the tumor has metastasized beyond the prostate.Prostate cancer is the second leading cause of cancer deaths among men. For patients with hormone-refractory disease, few treatments are available once the tumor has metastasized beyond the prostate. In the present study, two conjugated lytic peptide sequences (named JCHLHRH and JC21LHRH) were designed to target luteinizing hormone-releasing hormone receptors (LHRH-R). Our results indicate that human prostate cancer cell lines were sensitive to both LHRH-conjugated and non-conjugated lytic peptides, with IC(50) concentrations for LNCaP cells, 4.4 and 9.1μM; for DU-145 cells, 4.8 and 5.7μM; and for PC-3 cells, 4.4 and 8.2μM, respectively. JCHLHRH and JC21LHRH were nontoxic to normal primary human prostate epithelial cells or to bone marrow stromal cells in co-culture. There were morphological changes in PC-3 cells after 3h of exposure to either peptide; after 6h, there were significant reductions in cell numbers. Exposure of PC-3 cells for 24h to either JCHLHRH or JC21LHRH blocked their growth over 3 days. Since JCHLHRH and JC21LHRH have specificity for and anti-proliferative activity against tumor cells, and low toxicity for normal prostate cells, these peptides could serve as a new type of therapy for prostate cancer.

• Synergistic antimicrotubule therapy for prostate cancer.

  Authors: Vaishali Pannu, Prasanthi Karna, Hari Krishna Sajja, Deep Shukla, Ritu Aneja

  Biochemical pharmacology. 11/2010; 81(4):478-87.

  Prostate cancer has been widely viewed as a chemoresistant neoplasm. Perhaps, the most prevalent antimicrotubule strategy involves docetaxel administration at its maximum-tolerated dose (MTD).Prostate cancer has been widely viewed as a chemoresistant neoplasm. Perhaps, the most prevalent antimicrotubule strategy involves docetaxel administration at its maximum-tolerated dose (MTD). Although the goal is to obtain total eradication of cancer cells, debilitating toxicities are presented by docetaxel therapy, including myelosuppression, immunosuppression, gastrointestinal toxicity and peripheral neuropathy. In addition, solubility limitations necessitate infusion of high-doses intravenously once or twice a week followed by a rest period, which allows recovery of normal proliferating cells to counter-balance efficacy. An emerging notion is that more of a toxic drug at its MTD is not necessarily better. It is likely that combinatorial antimicrotubule therapy with drugs occupying different sites on tubulin may enhance efficacy while reducing toxicity. Here we show that bromonoscapine (EM011), a microtubule-modulating noscapine analog, displays synergism with docetaxel as seen by cell viability and proliferation assays. Cell-cycle data demonstrated that lower dose-levels of docetaxel (25nM) in combination with EM011 caused an additive increase in proapoptotic activity. Since docetaxel alone caused severe mitotic arrest followed by mitotic slippage and endoreduplication, we strategized a sequential treatment regime that involved initial pretreatment with docetaxel followed by addition of EM011 to maximize mitotic arrest and subsequent apoptosis. In vivo studies with docetaxel and EM011 in combination showed a marked inhibition of tumor growth compared to docetaxel or EM011 as single-agents. Our studies suggest the potential usefulness of EM011 in the clinic to enhance docetaxel activity. This would reduce toxicity, thus improving the quality of life of docetaxel-treated patients.

• Enhanced noscapine delivery using uPAR-targeted optical-MR imaging trackable nanoparticles for prostate cancer therapy.

  Authors: Mohamed O Abdalla, Prasanthi Karna, Hari Krishna Sajja, Hui Mao, Clayton Yates, Timothy Turner, Ritu Aneja

  Journal of controlled release : official journal of the Controlled Release Society. 10/2010; 149(3):314-22.

  The tubulin-binding anticancer activity of noscapine, an orally available plant-derived anti-tussive alkaloid, has been recently identified. Noscapine inhibits tumor growth in nude mice bearing humanThe tubulin-binding anticancer activity of noscapine, an orally available plant-derived anti-tussive alkaloid, has been recently identified. Noscapine inhibits tumor growth in nude mice bearing human xenografts of hematopoietic, breast, lung, ovarian, brain and prostate origin. Despite its nontoxic attributes, significant elimination of the disease has not been achieved, perhaps since the bioavailability of noscapine to tumors saturates at an oral dose of 300 mg/kg body weight. To enable the selective and specific delivery of noscapine to prostate cancer cells, we have engineered a multifunctional nanoscale delivery vehicle that takes advantage of urokinase plasminogen activator receptor (uPAR) overexpression in prostate cancer compared to normal prostate epithelia and can be tracked by magnetic resonance imaging (MRI) and near-infrared (NIR) imaging. Specifically, we employed the human-type 135 amino-acid amino-terminal fragment (hATF) of urokinase plasminogen activator (uPA), a high-affinity natural ligand for uPAR. Noscapine (Nos) was efficiently adsorbed onto the amphiphilic polymer coating of uPAR-targeted nanoparticles (NPs). Nos-loaded NPs were uniformly compact-sized, stable at physiological pH and efficiently released the drug at pH 4 to 5 within a span of 4h. Our results demonstrate that these uPAR-targeted NPs were capable of binding to the receptor and were internalized by PC-3 cells. uPAR-targeted Nos-loaded NPs enhanced intracellular noscapine accumulation as evident by the ~6-fold stronger inhibitory effect on PC-3 growth compared to free noscapine. In addition, Nos-loaded iron oxide NPs maintained their T2 MRI contrast effect upon internalization into tumor cells owing to their significant susceptibility effect in cells. Thus, our data provide compelling evidence that these optically and magnetic resonance imaging (MRI)-trackable uPAR-targeted NPs may offer a great potential for image-directed targeted delivery of noscapine for the management of prostate cancer.

• Induction of reactive oxygen species-mediated autophagy by a novel microtubule-modulating agent.

  Authors: Prasanthi Karna, Susu Zughaier, Vaishali Pannu, Robert Simmons, Satya Narayan, Ritu Aneja

  The Journal of biological chemistry. 06/2010; 285(24):18737-48.

  Autophagy is being increasingly implicated in both cell survival and death. However, the intricate relationships between drug-induced autophagy and apoptosis remain elusive. Here we demonstrate thatAutophagy is being increasingly implicated in both cell survival and death. However, the intricate relationships between drug-induced autophagy and apoptosis remain elusive. Here we demonstrate that a tubulin-binding noscapine analog, (R)-9-bromo-5-((S)-4,5-dimethoxy-1,3-dihydroisobenzofuran-1-yl)-4-methoxy-6-methyl-5,6,7,8-tetrahydro-[1,3]-di-oxolo[4,5-g]isoquinoline (Red-Br-nos), exerts a novel autophagic response followed by apoptotic cell death in human prostate cancer PC-3 cells. Red-Br-nos-induced autophagy was an early event detectable within 12 h that displayed a wide array of characteristic features including double membranous vacuoles with entrapped organelles, acidic vesicular organelles, and increased expression of LC3-II and beclin-1. Red-Br-nos-triggered release of reactive oxygen species (ROS) and attenuation of ROS by tiron, a ROS scavenger, reduced the sub-G(1) population suggesting ROS-dependent apoptosis. Abrogation of ROS also reduced autophagy indicating that ROS triggers autophagy. Pharmacological and genetic approaches to inhibit autophagy uncovered the protective role of Red-Br-nos-induced autophagy in PC-3 cells. Direct effects of the drug on mitochondria viz. disruption of normal cristae architecture and dissipation of mitochondrial transmembrane potential revealed a functional link between ROS generation, autophagy, and apoptosis induction. This is the first report to demonstrate the protective role of ROS-mediated autophagy and induction of caspase-independent ROS-dependent apoptosis in PC-3 cells by Red-Br-nos, a member of the noscapinoid family of microtubule-modulating anticancer agents.

• A novel microtubule-modulating agent induces mitochondrially driven caspase-dependent apoptosis via mitotic checkpoint activation in human prostate cancer cells.

  Authors: Ritu Aneja, Tohru Miyagi, Prasanthi Karna, Tucker Ezell, Deep Shukla, Meenakshi Vij Gupta, Clayton Yates, Sreenivasa R Chinni, Haiyen Zhau, Leland W K Chung, Harish C Joshi

  European journal of cancer (Oxford, England : 1990). 03/2010; 46(9):1668-78.

  Hormone-refractory prostate cancer, its skeletal metastasis and complications remain a therapeutic challenge. Here we show that treatment withHormone-refractory prostate cancer, its skeletal metastasis and complications remain a therapeutic challenge. Here we show that treatment with (S)-3-((R)-9-bromo-4-methoxy-6-methyl-5,6,7,8-tetrahydro-[1,3]dioxolo[4,5-g]isoquinolin-5-yl)-6,7-dimethoxyiso-benzofuran-1(3H)-one (EM011), the brominated analogue of a plant-derived non-toxic antitussive alkaloid, noscapine, achieved significant inhibition of hormone-refractory human prostate cancer implanted intratibially in the bone as shown by non-invasive, real-time bioluminescent imaging of tumour growth in nude mice. Mechanistically, in vitro data suggested that the antiproliferative and proapoptotic effects of EM011 in human prostate cancer cell lines were through blockade of cell-cycle progression by impairing the formation of a bipolar spindle apparatus. The G2/M arrest was accompanied by activation of the mitotic checkpoint, a pre-requisite for induction of optimal apoptosis. Attenuation of mitotic checkpoint by siRNA duplexes led to a reduction in mitotic arrest and subsequent apoptosis. Our results further demonstrated participation of an intrinsic mitochondrially mediated apoptotic pathway that ultimately triggered caspase-driven EM011-induced apoptosis. EM011 did not exert any detectable toxicity in normal tissues with frequently dividing cells such as the gut and bone marrow. Thus, these data warrant further evaluation of EM011 for the management of prostate cancer.

• Synthesis and characterization of noscapine loaded magnetic polymeric nanoparticles.

  Authors: Mohamed O Abdalla, Ritu Aneja, Derrick Dean, Vijay Rangari, Albert Russell, Jessie Jaynes, Clayton Yates, Timothy Turner

  Journal of magnetism and magnetic materials. 01/2010; 322(2):190-196.

  The delivery of noscapine therapies directly to the site of the tumor would ultimately allow higher concentrations of the drug to be delivered, and prolong circulation time in vivo to enhance theThe delivery of noscapine therapies directly to the site of the tumor would ultimately allow higher concentrations of the drug to be delivered, and prolong circulation time in vivo to enhance the therapeutic outcome of this drug. Therefore, we sought to design magnetic based polymeric nanoparticles for the site directed delivery of noscapine to invasive tumors. We synthesized Fe(3)O(4) nanoparticles with an average size of 10 ± 2.5 nm. These Fe(3)O(4) NPs were used to prepare noscapine loaded magnetic polymeric nanoparticles (NMNP) with an average size of 252 ± 6.3 nm. Fourier transform infrared (FT-IR) spectroscopy showed the encapsulation of noscapine on the surface of the polymer matrix. The encapsulation of the Fe(3)O(4) NPs on the surface of the polymer was confirmed by elemental analysis. We studied the drug loading efficiency of polylactide acid (PLLA) and poly (L-lactide acid-co-gylocolide) (PLGA) polymeric systems of various molecular weights. Our findings revealed that the molecular weight of the polymer plays a crucial role in the capacity of the drug loading on the polymer surface. Using a constant amount of polymer and Fe(3)O(4) NPs, both PLLA and PLGA at lower molecule weights showed higher loading efficiencies for the drug on their surfaces.

• Potent anti-inflammatory activity of novel microtubule-modulating brominated noscapine analogs.

  Authors: Susu Zughaier, Prasanthi Karna, David Stephens, Ritu Aneja

  PloS one. 01/2010; 5(2):e9165.

  Noscapine, a plant-derived, non-toxic, over-the-counter antitussive alkaloid has tubulin-binding properties. Based upon the structural resemblance of noscapine to colchicine, a tubulin-bindingNoscapine, a plant-derived, non-toxic, over-the-counter antitussive alkaloid has tubulin-binding properties. Based upon the structural resemblance of noscapine to colchicine, a tubulin-binding anti-inflammatory drug, noscapine and its semi-synthetic brominated analogs were examined for in vitro anti-inflammatory activity. Brominated noscapine analogs were found to inhibit cytokine and chemokine release from macrophage cell lines but did not affect cell viability. Brominated noscapine analogs demonstrated anti-inflammatory properties in both TLR- and non-TLR induced in vitro innate immune pathway inflammation models, mimicking septic and sterile infection respectively. In addition, electron microscopy and immunoblotting data indicated that these analogs induced robust autophagy in human macrophages. This study is the first report to identify brominated noscapines as innate immune pathway anti-inflammatory molecules.