Harish C Joshi

Publications of Harish C Joshi

• Copper(I) mediated facile synthesis of potent tubulin polymerization inhibitor, 9-amino-α-noscapine from natural α-noscapine.

  Authors: Naresh K Manchukonda, Balasubramanian Sridhar, Pradeep K Naik, Harish C Joshi, Srinivas Kantevari

  Bioorganic & medicinal chemistry letters. 02/2012; 22(8):2983-7.

  Facile synthesis of natural α-noscapine analogue, 9-amino-α-noscapine, a potent inhibitor of tubulin polymerization for cancer therapy, is achieved via copper(I) iodide mediated in situ aromaticFacile synthesis of natural α-noscapine analogue, 9-amino-α-noscapine, a potent inhibitor of tubulin polymerization for cancer therapy, is achieved via copper(I) iodide mediated in situ aromatic azidation and reduction of 9-bromo-α-noscapine (obtained by bromination of natural α-noscapine) with NaN(3) in DMSO at 130°C in the presence of l-proline as an amino acid promoter. The protocol developed here avoided isolation of 9-azido-α-noscapine and did not cleave the sensitive C-C bond between two heterocyclic phthalide and isoquinoline units.

• 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).

• Chemoprevention of familial adenomatous polyposis by bromo-noscapine (EM011) in the Apc(Min/+) mouse model.

  Authors: Shiwang Li, Amr M Ghaleb, Jing He, Usha Bughani, Agnieszka B Bialkowska, Vincent W Yang, Harish C Joshi

  International journal of cancer. Journal international du cancer. 11/2011;

  Germline mutation of the tumor suppressor gene, adenomatous polyposis coli (APC), is responsible for familial adenomatous polyposis (FAP) with nearly 100% risk for colon cancer at an early age.Germline mutation of the tumor suppressor gene, adenomatous polyposis coli (APC), is responsible for familial adenomatous polyposis (FAP) with nearly 100% risk for colon cancer at an early age. Although FAP is involved in only 1% of all colon cancer cases, over 80% of sporadic cancers harbor somatic mutations of APC. We show here that bromo-noscapine (EM011), a rationally designed synthetic derivative of a natural nontoxic tubulin-binding alkaloid-noscapine, that reduces the dynamics of microtubules, causes a reversible G(2) /M arrest in wild type murine embryonic fibroblasts (MEFs), but an aberrant exit from a brief mitotic block, followed by apoptosis in MEFs after APC deletion with small interfering RNA. Furthermore, both β-catenin levels and activity fell to half the original levels with a concomitant reduction of cell proliferation-inducing cyclin D1, c-Myc, and induction of cytostatic protein p21 before caspase-3 activation. Additionally, we show a statistically significant reduction in the number of newly emerging intestinal polyps (to 35% compared with untreated mice) as well as the mean size of polyps (to 42% compared with untreated mice) in EM011-treated Apc(Min/+) mice as compared to their sham-treated control littermates. The remaining polyps in the EM011 treated group of Apc(Min/+) mice showed evidence of elevated apoptosis as revealed by immunohistochemistry. We failed to detect any evidence of histopathological and hematological toxicities following EM011 treatment. Taken together, our data are persuasive that a clinical trial of EM011 is possible for the prevention/amelioration of polyposis in FAP patients.

• Rational design of novel anti-microtubule agent (9-azido-noscapine) from quantitative structure activity relationship (QSAR) evaluation of noscapinoids.

  Authors: Seneha Santoshi, Pradeep K Naik, Harish C Joshi

  Journal of biomolecular screening. 10/2011; 16(9):1047-58.

  An anticough medicine, noscapine [(S)-3-((R)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], was discovered in the authors'An anticough medicine, noscapine [(S)-3-((R)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], was discovered in the authors' laboratory as a novel type of tubulin-binding agent that mitigates polymerization dynamics of microtubule polymers without changing overall subunit-polymer equilibrium. To obtain systematic insight into the relationship between the structural framework of noscapine scaffold and its antitumor activity, the authors synthesized strategic derivatives (including two new ones in this article). The IC(50) values of these analogs vary from 1.2 to 56.0 µM in human acute lymphoblastic leukemia cells (CEM). Geometrical optimization was performed using semiempirical quantum chemical calculations at the 3-21G* level. Structures were in agreement with nuclear magnetic resonance analysis of molecular flexibility in solution and crystal structures. A genetic function approximation algorithm of variable selection was used to generate the quantitative structure activity relationship (QSAR) model. The robustness of the QSAR model (R(2) = 0.942) was analyzed by values of the internal cross-validated regression coefficient (R(2) (LOO) = 0.815) for the training set and determination coefficient (R(2) (test) = 0.817) for the test set. Validation was achieved by rational design of further novel and potent antitumor noscapinoid, 9-azido-noscapine, and reduced 9-azido-noscapine. The experimentally determined value of pIC(50) for both the compounds (5.585 M) turned out to be very close to predicted pIC(50) (5.731 and 5.710 M).

• Molecular modelling and competition binding study of Br-noscapine and colchicine provide insight into noscapinoid-tubulin binding site.

  Authors: Pradeep K Naik, Seneha Santoshi, Ankit Rai, Harish C Joshi

  Journal of molecular graphics & modelling. 06/2011; 29(7):947-55.

  We have previously discovered the tubulin-binding anti-cancer properties of noscapine and its derivatives (noscapinoids). Here, we present three lines of evidence that noscapinoids bind at or nearWe have previously discovered the tubulin-binding anti-cancer properties of noscapine and its derivatives (noscapinoids). Here, we present three lines of evidence that noscapinoids bind at or near the well studied colchicine binding site of tubulin: (1) in silico molecular docking studies of Br-noscapine and noscapine yield highest docking score with the well characterised colchicine-binding site from the co-crystal structure; (2) the molecular mechanics-generalized Born/surface area (MM-GB/SA) scoring results ΔΔG(bind-cald) for both noscapine and Br-noscapine (3.915 and 3.025 kcal/mol) are in reasonably good agreement with our experimentally determined binding affinity (ΔΔG(bind-Expt) of 3.570 and 2.988 kcal/mol, derived from K(d) values); and (3) Br-noscapine competes with colchicine binding to tubulin. The simplest interpretation of these collective data is that Br-noscapine binds tubulin at a site overlapping with, or very close to colchicine-binding site of tubulin. Although we cannot rule out a formal possibility that Br-noscapine might bind to a site distinct and distant from the colchicine-binding site that might negatively influence the colchicine binding to tubulin.

• 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.

• Noscapinoids with anti-cancer activity against human acute lymphoblastic leukemia cells (CEM): a three dimensional chemical space pharmacophore modeling and electronic feature analysis.

  Authors: Pradeep K Naik, Seneha Santoshi, Harish C Joshi

  Journal of molecular modeling. 04/2011; 18(1):307-18.

  We have identified a new class of microtubule-binding compounds-noscapinoids-that alter microtubule dynamics at stoichiometric concentrations without affecting tubulin polymer mass. Noscapinoids showWe have identified a new class of microtubule-binding compounds-noscapinoids-that alter microtubule dynamics at stoichiometric concentrations without affecting tubulin polymer mass. Noscapinoids show great promise as chemotherapeutic agents for the treatment of human cancers. To investigate the structural determinants of noscapinoids responsible for anti-cancer activity, we tested 36 structurally diverse noscapinoids in human acute lymphoblastic leukemia cells (CEM). The IC(50) values of these noscapinoids vary from 1.2 to 56.0 μM. Pharmacophore models of anti-cancer activity were generated that identify two hydrogen bond acceptors, two aromatic rings, two hydrophobic groups, and one positively charged group as essential structural features. Additionally, an atom-based quantitative structure-activity relationship (QSAR) model was developed that gave a statistically satisfying result (R(2) = 0.912, Q(2) = 0.908, Pearson R = 0.951) and effectively predicts the anti-cancer activity of training and test set compounds. The pharmacophore model presented here is well supported by electronic property analysis using density functional theory at B3LYP/3-21*G level. Molecular electrostatic potential, particularly localization of negative potential near oxygen atoms of the dimethoxy isobenzofuranone ring of active compounds, matched the hydrogen bond acceptor feature of the generated pharmacophore. Our results further reveal that all active compounds have smaller lowest unoccupied molecular orbital (LUMO) energies concentrated over the dimethoxy isobenzofuranone ring, azido group, and nitro group, which is indicative of the electron acceptor capacity of the compounds. Results obtained from this study will be useful in the efficient design and development of more active noscapinoids.

• 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.

• Recent Patents Reveal Microtubules as Persistent Promising Target for Novel Drug Development for Cancers.

  Authors: Usha Bughani, Shiwang Li, Harish C Joshi

  Recent patents on anti-infective drug discovery. 12/2009;

  Conventionally, the successful targets for the drug development in cancer range from the DNA damage, replication, signal transduction pathways, hormones, cytokines, anti-angiogenic agents, andConventionally, the successful targets for the drug development in cancer range from the DNA damage, replication, signal transduction pathways, hormones, cytokines, anti-angiogenic agents, and radio/photo-sensitizers. They dominate the therapeutic arena after the initial debulking surgery. More recently, tubulin, the primary constituent of microtubules (MTs), has made a fairly successful debut in the therapeutic armamentarium. Tubulin binding drugs come in two classes: that depolymerize microtubules and that over-polymerize and bundle them. Microtubule (MT) binding drugs are in some ways superior in nature primarily because of their less debilitating side effects when compared to the generalized DNA metabolism targeting agents, and many new promising patents are being funneled into the drug development pipeline. Nevertheless, many of these relatively new agents still face challenges relating to their delivery methods, bioavailability, toxicities, and the inevitable resistance shared by all chemotherapeutics. Finally, we disclose a new genre of anti-MT drugs, noscapinoids that have just begun climbing the clinical trials ladder. The lead compound, noscapine, is a plant derived, orally available, minimally-toxic (if at all) agent that has shown phenomenal promise in the preclinical experimentation and Phase-I clinical trial. A rational approach based upon the precise molecular model of the tubulin-noscapine complex is bound to inspire novel and better therapeutic analogs in future.

• Non-toxic melanoma therapy by a novel tubulin-binding agent.

  Authors: Ritu Aneja, Seneshaw Asress, Neerupma Dhiman, Anshumali Awasthi, Padmashree C G Rida, Sudarshan K Arora, Jun Zhou, Jonathan D Glass, Harish C Joshi

  International journal of cancer. Journal international du cancer. 08/2009;

  (S)-3-((R)-9-bromo-4-methoxy-6-methyl-5,6,7,8-tetrahydro-[1,3]dioxolo[4,5-g]isoquino-lin-5-yl)-6,7-dimethoxyisobenzofuran-1(3H)-one (EM011) is a tubulin-binding agent with significant anticancer(S)-3-((R)-9-bromo-4-methoxy-6-methyl-5,6,7,8-tetrahydro-[1,3]dioxolo[4,5-g]isoquino-lin-5-yl)-6,7-dimethoxyisobenzofuran-1(3H)-one (EM011) is a tubulin-binding agent with significant anticancer activity. Here we show that EM011 modulates microtubule dynamics at concentrations that do not alter the total polymer mass of tubulin. In particular, EM011 decreases the transition frequencies between growth and shortening phases and increases the duration microtubules spend in an idle 'pause' state. Using B16LS9 murine melanoma cells, we show that EM011 briefly arrests cell-cycle progression at the G2/M phase by formation of multiple aster spindles. An aberrant mitotic exit without cytokinesis then occurs, leading to the accumulation of abnormal multinucleated cells prior to apoptosis. Our pharmacokinetic studies conformed to a linear dose-response relationship upto 150 mg/kg. However, non-linearity was observed at 300 mg/kg. In a syngeneic murine model of subcutaneous melanoma, better antitumor responses were seen at 150 mg/kg compared to 300 mg/kg of EM011. Unlike currently available chemotherapeutics, EM011 is non-toxic to normal tissues and most importantly, does not cause any immunosuppression and neurotoxicity. Our data thus warrant a clinical evaluation of EM011 for melanoma therapy. (c) 2009 UICC.

• 9-bromonoscapine-induced mitotic arrest of cigarette smoke condensate-transformed breast epithelial cells.

  Authors: Aruna S Jaiswal, Ritu Aneja, Shahnjayla K Connors, Harish C Joshi, Asha S Multani, Sen Pathak, Satya Narayan

  Journal of cellular biochemistry. 03/2009;

  In the present investigation, we determined the chemotherapeutic efficacy of 9-bromonoscapine (Br-Nos), a more potent noscapine analog, on MCF10A, spontaneously immortalized human normal breastIn the present investigation, we determined the chemotherapeutic efficacy of 9-bromonoscapine (Br-Nos), a more potent noscapine analog, on MCF10A, spontaneously immortalized human normal breast epithelial cells and MCF10A-CSC3, cigarette smoke condensate (CSC)-transformed cells. The results from cytogenetic analysis showed that Br-Nos induced polyploidy and telomeric association in MCF10A-CSC3 cells, while MCF10A cells remained unaffected. Our immunofluorescence data further demonstrated that MCF10A-CSC3 cells were susceptible to mitotic catastrophe on exposure to Br-Nos and failed to recover after drug withdrawal. MCF10A-CSC3 cells exhibited Br-Nos-induced aberrant multipolar spindle formation, which irreversibly impaired the alignment of replicated chromosome to the equatorial plane and finally culminated in cell death. Although MCF10A cells upon Br-Nos treatment showed bipolar spindles with some uncongressed chromosomes, these cells recovered fairly well after drug withdrawal. Our flow-cytometry analysis data reconfirmed that MCF10A-CSC3 cells were more susceptible to cell death compared to MCF10A cells. Furthermore, our results suggest that decreased levels of cdc2/cyclin B1 and cdc2 kinase activity are responsible for Br-Nos-induced mitotic cell arrest leading to cell death in MCF10A-CSC3 cells. This study thus explores the underlying mechanism of Br-Nos-induced mitotic catastrophe in CSC-transformed MCF10A-CSC3 cells and its potential usefulness as a chemotherapeutic agent for prevention of cigarette smoke-induced breast cancer growth. J. Cell. Biochem. (c) 2009 Wiley-Liss, Inc.

• Parkin regulates Eg5 expression by Hsp70 ubiquitination-dependent inactivation of the c-Jun NH2-terminal kinase.

  Authors: Min Liu, Ritu Aneja, Xiaodong Sun, Songbo Xie, Hongxia Wang, Xiaojing Wu, Jin-Tang Dong, Minggang Li, Harish C Joshi, Jun Zhou

  The Journal of biological chemistry. 11/2008;

  Eg5 is a motor protein of the kinesin family that is critical for spindle assembly during mitosis and has recently been implicated in tumorigenesis. It is largely unknown how Eg5 expression isEg5 is a motor protein of the kinesin family that is critical for spindle assembly during mitosis and has recently been implicated in tumorigenesis. It is largely unknown how Eg5 expression is regulated in cells. In this study, we present the first evidence that cellular Eg5 level is downregulated by Parkin, an E3 ubiquitin ligase well known for its role in the development of Parkinson's disease. Our data show that Parkin does not trigger Eg5 protein degradation through the ubiquitin-proteasome pathway. Instead, Parkin represses Eg5 gene transcription by blocking c-Jun binding to the activator protein 1 site present in the Eg5 promoter. Our data further show that Parkin inactivates the c-Jun NH2-terminal kinase (JNK), resulting in decreased phosphorylation of c-Jun. The inactivation of JNK is further mediated by multiple mono-ubiquitination of Hsp70. Importantly, both the ubiquitination of Hsp70 and the subsequent inactivation of the JNK-c-Jun pathway are crucial for Parkin to downregulate Eg5 expression. These results thus uncover a novel function for Parkin in modulating the expression of Eg5 through the Hsp70-JNK-c-Jun signaling pathway.

• Multidrug resistance-associated protein-overexpressing teniposide-resistant human lymphomas undergo apoptosis by a tubulin-binding agent.

  Authors: Ritu Aneja, Min Liu, Clayton Yates, Jinmin Gao, Xin Dong, Binfei Zhou, Surya N Vangapandu, Jun Zhou, Harish C Joshi

  Cancer research. 04/2008; 68(5):1495-503.

  Several DNA- and microtubule-binding agents are used to manage hematologic malignancies in the clinic. However, drug resistance has been a challenge, perhaps due to a few surviving cancer stem cells.Several DNA- and microtubule-binding agents are used to manage hematologic malignancies in the clinic. However, drug resistance has been a challenge, perhaps due to a few surviving cancer stem cells. Toxicity is another major impediment to successful chemotherapy, leading to an impoverished quality of life. Here, we show that a semisynthetic nontoxic tubulin-binding agent, 9-bromonoscapine (EM011), effectively inhibits growth and regresses multidrug resistance-associated protein (MRP)-overexpressing teniposide-resistant T-cell lymphoma xenografts and prolongs longevity. As expected, teniposide treatment failed to regress teniposide-resistant xenografts, rather, treated mice suffered tremendous body weight loss. Mechanistically, EM011 displays significant antiproliferative activity, perturbs cell cycle progression by arresting mitosis, and induces apoptosis in teniposide-resistant lymphoblastoid T cells both in vitro and in vivo. EM011-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, translocation of BAX to the mitochondrial membrane, cytochrome c release, and activation of downstream apoptotic signaling. EM011 caused DNA degradation as evident by terminal deoxynucleotidyl transferase-mediated dUTP-biotin end labeling staining of the increased concentration of 3'-DNA ends. Furthermore, the apoptotic induction was caspase dependent as shown by cleavage of the caspase substrate, poly(ADP)ribose polymerase. In addition, EM011 treatment caused a suppression of natural survival pathways such as the phosphatidylinositol-3'-kinase/Akt signaling. These preclinical findings suggest that EM011 is an excellent candidate for clinical evaluation.

• Nonimmunosuppressive chemotherapy: EM011-treated mice mount normal T-cell responses to an acute lymphocytic choriomeningitis virus infection.

  Authors: Ritu Aneja, Vandana Kalia, Rafi Ahmed, Harish C Joshi

  Molecular cancer therapeutics. 12/2007; 6(11):2891-9.

  Myelosuppression and associated immunosuppression are major problems in cancer chemotherapy. Thus, infection remains a significant source of morbidity and mortality during chemotherapy of cancerMyelosuppression and associated immunosuppression are major problems in cancer chemotherapy. Thus, infection remains a significant source of morbidity and mortality during chemotherapy of cancer patients. Viral infections, particularly herpes simplex virus, varicella zoster virus, and cytomegalovirus, result either due to reactivation of latent viruses or new infections as sequelae of chemotherapy and debilitated cell-mediated immunity. Ultimately, the resolution of these infections can only be achieved after the control of malignancy and regaining the patient's ability to mount adequate immune responses. We show here that EM011, a tubulin-binding, nontoxic, orally available anticancer agent, does not alter absolute CD4(+), CD8(+), B220(+), and NK1.1(+) cell counts in immunocompetent mice. More importantly, EM011 treatment at tumor-suppressive dosages (300 mg/kg) does not suppress cell-mediated immune responses in mice experimentally challenged with acute lymphocytic choriomeningitis virus infection, in that mice mount robust virus-specific CD8(+) and CD4(+) T-cell immune responses while maintained on daily drug treatment. Thus, CD8(+) and CD4(+) T-cell expansion and acquisition of effector functions is not perturbed by EM011 treatment. These data provide compelling evidence to support the nonimmunosuppressive nature of EM011 therapy and provide strong impetus for combining chemotherapy with immunotherapy as a novel anticancer strategy.

• Preclinical pharmacokinetics and bioavailability of noscapine, a tubulin-binding anticancer agent.

  Authors: Ritu Aneja, Neerupma Dhiman, Jyoti Idnani, Anshumali Awasthi, Sudershan K Arora, Ramesh Chandra, Harish C Joshi

  Cancer chemotherapy and pharmacology. 12/2007; 60(6):831-9.

  BACKGROUND: Noscapine, a naturally occurring antitussive phthalideisoquinoline alkaloid, is a tubulin-binding agent currently in Phase I/II clinical trials for anticancer therapy. Unlike currentlyBACKGROUND: Noscapine, a naturally occurring antitussive phthalideisoquinoline alkaloid, is a tubulin-binding agent currently in Phase I/II clinical trials for anticancer therapy. Unlike currently available antimitotics such as taxanes and vincas, noscapine is water-soluble, well tolerated, and shows no detectable toxicity. OBJECTIVE: The goal was to develop a simple, sensitive, quantitative, selective, and less time-consuming high-performance liquid chromatography (HPLC) method for determination of noscapine and to study its pharmacokinetics in mice models. METHOD: Noscapine was extracted from mice plasma using the protein-precipitation method and detected using a reversed-phase C8 column with mobile phase consisting of 35% acetonitrile and 65% ammonium acetate buffer (pH 4.5) at 232 nm wavelength. Pharmacokinetic studies of noscapine were performed in mice following intravenous bolus at 10 mg/kg and oral administrations at 75, 150, and 300 mg/kg. RESULTS: The standard curves for noscapine estimation were linear between 390 and 50,000 ng/ml (lower limit of quantification was 390 ng/ml) and the recovery was approximately 80%. Following 10 mg/kg intravenous dose, mean plasma concentrations of 7.88 microg/ml were achieved at 5 min in mice and declined with undetectable levels at 4 h. The mean total body clearance was 4.78 l/h. The mean volume of distribution (V (d)) was 5.05 l. Non-compartmental analysis yielded the mean area under the plasma concentration-time curve (AUC) for noscapine as 53.42, 64.08, and 198.35 h microg/ml reaching maximum plasma concentrations (C (max)) of 12.74, 23.24, and 46.73 microg/ml at a t (max) of 1.12, 1.50, and 0.46 h at the linearly increasing dose levels. CONCLUSION: A rapid and simple HPLC/UV method for the quantification of noscapine in plasma has been developed to study pharmacokinetics of noscapine at tumor-suppressive doses in the mouse. Since orally available anticancer drugs are rare, therefore, noscapine, an innocuous agent, having a mean oral bioavailability of 31.5% over the studied dose range merits its further advancement in humans for anticancer therapy.

• PO(2)-dependent differential regulation of multidrug resistance 1 gene expression by the c-Jun NH2-terminal kinase pathway.

  Authors: Min Liu, Dengwen Li, Ritu Aneja, Harish C Joshi, Songbo Xie, Chao Zhang, Jun Zhou

  The Journal of biological chemistry. 07/2007; 282(24):17581-6.

  Hypoxia-induced multidrug resistance 1 (MDR1) gene expression is known to be mediated by c-Jun NH(2)-terminal kinase (JNK) activation. However, the molecular mechanisms underlying this action of JNKHypoxia-induced multidrug resistance 1 (MDR1) gene expression is known to be mediated by c-Jun NH(2)-terminal kinase (JNK) activation. However, the molecular mechanisms underlying this action of JNK remain elusive. On the contrary, there has been increasing evidence for a negative correlation of JNK activity with MDR1 expression under normoxic conditions. Here, we present evidence that the JNK pathway represses MDR1 expression in normoxia and activates MDR1 expression in hypoxia. Our data show that JNK pathway-induced MDR1 repression in normoxia is mediated by increased c-Jun binding to activator protein 1 site, located in the MDR1 promoter, and requires the activity of histone deacetylase 5. In contrast, JNK pathway-induced MDR1 activation in hypoxia is independent of the activator protein 1 site. Rather, this action is dependent on increased hypoxia-inducible factor 1 (HIF1) binding to the hypoxia response element in the MDR1 promoter, which is promoted by the interaction of HIF1alpha with c-Jun in the nucleus and requires the activity of the p300/CBP (CREB-binding protein) coactivator.

• p53 and p21 determine the sensitivity of noscapine-induced apoptosis in colon cancer cells.

  Authors: Ritu Aneja, Amr M Ghaleb, Jun Zhou, Vincent W Yang, Harish C Joshi

  Cancer research. 05/2007; 67(8):3862-70.

  We have previously discovered the naturally occurring antitussive alkaloid noscapine as a tubulin-binding agent that attenuates microtubule dynamics and arrests mammalian cells at mitosis viaWe have previously discovered the naturally occurring antitussive alkaloid noscapine as a tubulin-binding agent that attenuates microtubule dynamics and arrests mammalian cells at mitosis via activation of the c-Jun NH(2)-terminal kinase pathway. It is well established that the p53 protein plays a crucial role in the control of tumor cell response to chemotherapeutic agents and DNA-damaging agents; however, the relationship between p53-driven genes and drug sensitivity remains controversial. In this study, we compared chemosensitivity, cell cycle distribution, and apoptosis on noscapine treatment in four cell lines derived from the colorectal carcinoma HCT116 cells: p53(+/+) (p53-wt), p53(-/-) (p53-null), p21(-/-) (p21-null), and BAX(-/-) (BAX-null). Using these isogenic variants, we investigated the roles of p53, BAX, and p21 in the cellular response to treatment with noscapine. Our results show that noscapine treatment increases the expression of p53 over time in cells with wild-type p53 status. This increase in p53 is associated with an increased apoptotic BAX/Bcl-2 ratio consistent with increased sensitivity of these cells to apoptotic stimuli. Conversely, loss of p53 and p21 alleles had a counter effect on both BAX and Bcl-2 expression and the p53-null and p21-null cells were significantly resistant to the antiproliferative and apoptotic effects of noscapine. All but the p53-null cells displayed p53 protein accumulation in a time-dependent manner on noscapine treatment. Interestingly, despite increased levels of p53, p21-null cells were resistant to apoptosis, suggesting a proapoptotic role of p21 and implying that p53 is a necessary but not sufficient condition for noscapine-mediated apoptosis.

• Synthesis and biological evaluation of a cyclic ether fluorinated noscapine analog.

  Authors: Ritu Aneja, Surya N Vangapandu, Harish C Joshi

  Bioorganic & medicinal chemistry. 01/2007; 14(24):8352-8.

  We present here a novel semi-synthetic cyclic ether fluorinated noscapine analog (CEFNA) that shows potent antiproliferative and anticancer activity in both hormone-responsive (MCF-7) and hormoneWe present here a novel semi-synthetic cyclic ether fluorinated noscapine analog (CEFNA) that shows potent antiproliferative and anticancer activity in both hormone-responsive (MCF-7) and hormone non-responsive (MDA-MB-231) breast cancer cells. Interestingly, it is also effective against MCF-7/Adr, an adriamycin-resistant variant of MCF-7 cells. Immunofluorescence experiments showed numerous micronuclei, indicative of apoptotic cell death triggered by this novel analog. Mechanistically, CEFNA exerts a strong antimitotic effect as revealed by cell-cycle studies that show a dose-dependent increase in G2/M population preceding a rising sub-G1 population, suggesting apoptosis.

• Treatment of hormone-refractory breast cancer: apoptosis and regression of human tumors implanted in mice.

  Authors: Ritu Aneja, Jun Zhou, Binfei Zhou, Ramesh Chandra, Harish C Joshi

  Molecular cancer therapeutics. 10/2006; 5(9):2366-77.

  Following surgery, the hormone dependence of breast tumors is exploited for therapy using antagonists such as tamoxifen, although occasional hormone-resistant clones do appear. AnotherFollowing surgery, the hormone dependence of breast tumors is exploited for therapy using antagonists such as tamoxifen, although occasional hormone-resistant clones do appear. Another chemotherapeutic strategy uses microtubule inhibitors such as taxanes. Unfortunately, these agents elicit toxicities such as leukocytopenia, diarrhea, alopecia, and peripheral neuropathies and are also associated with the emergence of drug resistance. We have previously described a tubulin-binding, natural compound, noscapine, that was nontoxic and triggered apoptosis in many cancer types albeit at 10 mumol/L or higher concentrations depending on the cell type. We now show that a synthetic analogue of noscapine, 9-bromonoscapine, is approximately 10-fold to 15-fold more potent than noscapine in inhibiting cell proliferation and induces apoptosis following G2-M arrest in hormone-insensitive human breast cancers (MDA-MB-231). Furthermore, a clear loss of mitochondrial membrane potential, release of cytochrome c, activation of the terminal caspase-3, and the cleavage of its substrates such as poly(ADP-ribose) polymerase, suggest an intrinsic apoptotic mechanism. Taken together, these data point to a mitochondrially mediated apoptosis of hormone-insensitive breast cancer cells. Human tumor xenografts in nude mice showed significant tumor volume reduction and a surprising increase in longevity without signs of obvious toxicity. Thus, our data provide compelling evidence that 9-bromonoscapine can be useful for the therapy of hormone-refractory breast cancer.

• Synthesis of microtubule-interfering halogenated noscapine analogs that perturb mitosis in cancer cells followed by cell death.

  Authors: Ritu Aneja, Surya N Vangapandu, Manu Lopus, Vijaya G Viswesarappa, Neerupma Dhiman, Akhilesh Verma, Ramesh Chandra, Dulal Panda, Harish C Joshi

  Biochemical pharmacology. 09/2006; 72(4):415-26.

  We have previously identified the naturally occurring non-toxic antitussive phthalideisoquinoline alkaloid, noscapine as a tubulin-binding agent that arrests mitosis and induces apoptosis. Here weWe have previously identified the naturally occurring non-toxic antitussive phthalideisoquinoline alkaloid, noscapine as a tubulin-binding agent that arrests mitosis and induces apoptosis. Here we present high-yield efficient synthetic methods and an evaluation of anticancer activity of halogenated noscapine analogs. Our results show that all analogs display higher tubulin-binding activity than noscapine and inhibit proliferation of human cancer cells (MCF-7, MDA-MB-231 and CEM). Surprisingly, the bromo-analog is approximately 40-fold more potent than noscapine in inhibiting cellular proliferation of MCF-7 cells. The ability of these analogs to inhibit cellular proliferation is mediated by cell cycle arrest at the G2/M phase, in that all analogs except 9-iodonoscapine, caused selective mitotic arrest with a higher efficiency than noscapine followed by apoptotic cell death as shown by immunofluorescence and quantitative FACS analyses. Furthermore, our results reveal the appearance of numerous fragmented nuclei as evidenced by DAPI staining. Thus, our data indicate a great potential of these compounds for studying microtubule-mediated processes and as chemotherapeutic agents for the management of human cancers.