Vaishali Pannu

Induction of Reactive Oxygen Species-mediated Autophagy by a Novel Microtubule-modulating Agent

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 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-G1 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 noscapinoid triggers apoptosis by inducing spindle multipolarity via centrosome amplification and declustering

We have previously shown that a non-toxic noscapinoid, EM011 binds tubulin without altering its monomer/polymer ratio. EM011 is more active than the parent molecule, noscapine, in inducing G2/M arrest, inhibiting cellular proliferation and tumor growth in various human xenograft models. However, the mechanisms of mitotic-block and subsequent cell death have remained elusive. Here, we show that EM011-induced attenuation of microtubule dynamics was associated with impaired association of microtubule plus-end tracking proteins, such as EB1 and CLIP-170. EM011 treatment then led to the formation of multipolar spindles containing ‘real’ centrioles indicating drug-induced centrosome amplification and persistent centrosome declustering. Centrosome amplification was accompanied by an upregulation of Aurora A and Plk4 protein levels, as well as a surge in the kinase activity of Aurora A, suggesting a deregulation of the centrosome duplication cycle. Cell-cycle phase-specific experiments showed that the ‘cytotoxicity-window’ of the drug encompasses the late S-G2 period. Drug-treatment, excluding S-phase, not only resulted in lower sub-G1 population but also attenuated centrosome amplification and spindle multipolarity, suggesting that drug-induced centrosome amplification is essential for maximal cell death. Subsequent to a robust mitotic arrest, EM011-treated cells displayed diverse cellular fates suggesting a high degree of intraline variation. Some ‘apoptosis-evasive’ cells underwent aberrant cytokinesis to generate rampant aneuploidy that perhaps contributed to drug-induced cell death. These data indicate that spindle multipolarity induction by means of centrosome amplification has an exciting chemotherapeutic potential that merits further investigation.

Second generation benzofuranone ring substituted noscapine analogs: Synthesis and biological evaluation

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 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.

Hydroxychavicol, a betel leaf component, inhibits prostate cancer through ROS-driven DNA damage and apoptosis

Dietary phytochemicals are excellent ROS-modulating agents and have been shown to effectively enhance ROS levels beyond toxic threshold in cancer cells to ensure their selective killing while leaving normal cells unscathed. Here we demonstrate that hydroxychavicol (HC), extracted and purified from Piper betel leaves, significantly inhibits growth and proliferation via ROS generation in human prostate cancer, PC-3 cells. HC perturbed cell-cycle kinetics and progression, reduced clonogenicity and mediated cytotoxicity by ROS-induced DNA damage leading to activation of several pro-apoptotic molecules. In addition, HC treatment elicited a novel autophagic response as evidenced by the appearance of acidic vesicular organelles and increased expression of autophagic markers, LC3-IIb and beclin-1. Interestingly, quenching of ROS with tiron, an antioxidant, offered significant protection against HC-induced inhibition of cell growth and down regulation of caspase-3, suggesting the crucial role of ROS in mediating cell death. The collapse of mitochondrial transmembrane potential by HC further revealed the link between ROS generation and induction of caspase-mediated apoptosis in PC-3 cells. Our data showed remarkable inhibition of prostate tumor xenografts by ~72% upon daily oral administration of 150mg/kg bw HC by quantitative tumor volume measurements and non-invasive real-time bioluminescent imaging. HC was well-tolerated at this dosing level without any observable toxicity. This is the first report to demonstrate the anti-prostate cancer efficacy of HC in vitro and in vivo, which is perhaps attributable to its selective prooxidant activity to eliminate cancer cells thus providing compelling grounds for future preclinical studies to validate its potential usefulness for prostate cancer management.

Induction of robust de novo centrosome amplification, high-grade spindle multipolarity and metaphase catastrophe: a novel chemotherapeutic approach

Centrosome amplification (CA) and resultant chromosomal instability have long been associated with tumorigenesis. However, exacerbation of CA and relentless centrosome declustering engender robust spindle multipolarity (SM) during mitosis and may induce cell death. Recently, we demonstrated that a noscapinoid member, reduced bromonoscapine, (S)-3-(R)-9-bromo-5-(4,5-dimethoxy-1,3-dihydroisobenzofuran-1-yl)-4-methoxy-6-methyl-5,6,7,8-tetrahydro-[1,3]dioxolo-[4,5-g]isoquinoline (Red-Br-nos), induces reactive oxygen species (ROS)-mediated autophagy and caspase-independent death in prostate cancer PC-3 cells. Herein, we show that Red-Br-nos induces ROS-dependent DNA damage that resulted in high-grade CA and SM in PC-3 cells. Unlike doxorubicin, which causes double-stranded DNA breaks and chronic G2 arrest accompanied by ‘templated’ CA, Red-Br-nos-mediated DNA damage elicits de novo CA during a transient S/G2 stall, followed by checkpoint abrogation and mitotic entry to form aberrant mitotic figures with supernumerary spindle poles. Attenuation of multipolar phenotype in the presence of tiron, a ROS inhibitor, indicated that ROS-mediated DNA damage was partly responsible for driving CA and SM. Although a few cells (∼5%) yielded to aberrant cytokinesis following an ‘anaphase catastrophe’, most mitotically arrested cells (∼70%) succumbed to ‘metaphase catastrophe,’ which was caspase-independent. This report is the first documentation of rapid de novo centrosome formation in the presence of parent centrosome by a noscapinoid family member, which triggers death-inducing SM via a unique mechanism that distinguishes it from other ROS-inducers, conventional DNA-damaging agents, as well as other microtubule-binding drugs.

Synergistic antimicrotubule therapy for prostate cancer.

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.

Centrosome-declustering drugs mediate a two-pronged attack on interphase and mitosis in supercentrosomal cancer cells

Classical anti-mitotic drugs have failed to translate their preclinical efficacy into clinical response in human trials. Their clinical failure has challenged the notion that tumor cells divide frequently at rates comparable to those of cancer cells in vitro and in xenograft models. Given the preponderance of interphase cells in clinical tumors, we asked whether targeting amplified centrosomes, which cancer cells carefully preserve in a tightly clustered conformation throughout interphase, presents a superior chemotherapeutic strategy that sabotages interphase-specific cellular activities, such as migration. Herein we have utilized supercentrosomal N1E-115 murine neuroblastoma cells as a test-bed to study interphase centrosome declustering induced by putative declustering agents, such as Reduced-9-bromonoscapine (RedBr-Nos), Griseofulvin and PJ-34. We found tight ‘supercentrosomal’ clusters in the interphase and mitosis of ~80% of patients’ tumor cells with excess centrosomes. RedBr-Nos was the strongest declustering agent with a declustering index of 0.36 and completely dispersed interphase centrosome clusters in N1E-115 cells. Interphase centrosome declustering caused inhibition of neurite formation, impairment of cell polarization and Golgi organization, disrupted cellular protrusions and focal adhesion contacts—factors that are crucial prerequisites for directional migration. Thus our data illustrate an interphase-specific potential anti-migratory role of centrosome-declustering agents in addition to their previously acknowledged ability to induce spindle multipolarity and mitotic catastrophe. Centrosome-declustering agents counter centrosome clustering to inhibit directional cell migration in interphase cells and set up multipolar mitotic catastrophe, suggesting that disbanding the nuclear–centrosome–Golgi axis is a potential anti-metastasis strategy.

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

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 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.

Quantitative multi-parametric evaluation of centrosome declustering drugs: centrosome amplification, mitotic phenotype, cell cycle and death

Unlike normal cells, cancer cells contain amplified centrosomes and rely on centrosome clustering mechanisms to form a pseudobipolar spindle that circumvents potentially fatal spindle multipolarity (MP). Centrosome clustering also promotes low-grade chromosome missegregation, which can drive malignant transformation and tumor progression. Putative ‘centrosome declustering drugs’ represent a cancer cell-specific class of chemotherapeutics that produces a common phenotype of centrosome declustering and spindle MP. However, differences between individual agents in terms of efficacy and phenotypic nuances remain unexplored. Herein, we have developed a conceptual framework for the quantitative evaluation of centrosome declustering drugs by investigating their impact on centrosomes, clustering, spindle polarity, cell cycle arrest, and death in various cancer cell lines at multiple drug concentrations over time. Surprisingly, all centrosome declustering drugs evaluated in our study were also centrosome-amplifying drugs to varying extents. Notably, all declustering drugs induced spindle MP, and the peak extent of MP positively correlated with the induction of hypodiploid DNA-containing cells. Our data suggest acentriolar spindle pole amplification as a hitherto undescribed activity of some declustering drugs, resulting in spindle MP in cells that may not have amplified centrosomes. In general, declustering drugs were more toxic to cancer cell lines than non-transformed ones, with some exceptions. Through a comprehensive description and quantitative analysis of numerous phenotypes induced by declustering drugs, we propose a novel framework for the assessment of putative centrosome declustering drugs and describe cellular characteristics that may enhance susceptibility to them.

KIFCI, a novel putative prognostic biomarker for ovarian adenocarcinomas: delineating protein interaction networks and signaling circuitries.

BackgroundAmplified centrosomes in cancers are recently garnering a lot of attention as an emerging hub of diagnostic, prognostic and therapeutic targets. Ovarian adenocarcinomas commonly harbor supernumerary centrosomes that drive chromosomal instability. A centrosome clustering molecule, KIFC1, is indispensable for the viability of extra centrosome-bearing cancer cells, and may underlie progression of ovarian cancers.MethodsCentrosome amplification in low- and high- grade serous ovarian adenocarcinomas was quantitated employing confocal imaging. KIFC1 expression was analyzed in ovarian tumors using publically-available databases. Associated grade, stage and clinical information from these databases were plotted for KIFC1 gene expression values. Furthermore, interactions and functional annotation of KIFC1 and its highly correlated genes were studied using DAVID and STRING 9.1.ResultsClinical specimens of ovarian cancers display robust centrosome amplification and deploy centrosome clustering to execute an error-prone mitosis to enable karyotypic heterogeneity that fosters tumor progression and aggressiveness. Our in silico analyses showed KIFC1 overexpression in human ovarian tumors (n = 1090) and its upregulation associated with tumor aggressiveness utilizing publically-available gene expression databases. KIFC1 expression correlated with advanced tumor grade and stage. Dichotomization of KIFC1 levels revealed a significantly lower overall survival time for patients in high KIFC1 group. Intriguingly, in a matched-cohort of primary (n = 7) and metastatic (n = 7) ovarian samples, no significant differences in KIFC1 expression were detectable, suggesting that high KIFC1 expression may serve as a marker of metastases onset. Nonetheless, KIFC1 levels in both primary and matched metastatic sites were significantly higher compared to normal tissue . Ingenuity based network prediction algorithms combined with pre-established protein interaction networks uncovered several novel cell-cycle related partner genes on the basis of interconnectivity, illuminating the centrosome clustering independent agenda of KIFC1 in ovarian tumor progression.ConclusionsOvarian cancers display amplified centrosomes, a feature of aggressive tumors. To cope up with the abnormal centrosomal load, ovarian cancer cells upregulate genes like KIFC1 that are known to induce centrosome clustering. Our data underscore KIFC1 as a putative biomarker that predicts worse prognosis, poor overall survival and may serve as a potential marker of onset of metastatic dissemination in ovarian cancer patients.