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Dulal Panda

Publications and source records attributed to Dulal Panda.

At least 19 recordsLinked to original sources
PubMed2006

Dietary antioxidant curcumin inhibits microtubule assembly through tubulin binding.

Curcumin, a component of turmeric, has potent antitumor activity against several tumor types. However, its molecular target and mechanism of antiproliferative activity are not clear. Here, we identified curcumin as a novel antimicrotubule agent. We have examined the effects of curcumin on cellular microtubules and on reconstituted microtubules in vitro. Curcumin inhibited HeLa and MCF-7 cell proliferation in a concentration-dependent manner with IC(50) of 13.8 +/- 0.7 microm and 12 +/- 0.6 microm, respectively. At higher inhibitory concentrations (> 10 microm), curcumin induced significant depolymerization of interphase microtubules and mitotic spindle microtubules of HeLa and MCF-7 cells. However, at low inhibitory concentrations there were minimal effects on cellular microtubules. It disrupted microtubule assembly in vitro, reduced GTPase activity, and induced tubulin aggregation. Curcumin bound to tubulin at a single site with a dissociation constant of 2.4 +/- 0.4 microm and the binding of curcumin to tubulin induced conformational changes in tubulin. Colchicine and podophyllotoxin partly inhibited the binding of curcumin to tubulin, while vinblastine had no effect on the curcumin-tubulin interactions. The data together suggested that curcumin may inhibit cancer cells proliferation by perturbing microtubule assembly dynamics and may be used to develop efficacious curcumin analogues for cancer chemotherapy.

Antioxidants↗
PubMed2006

Synthesis and evaluation of alpha-hydroxymethylated conjugated nitroalkenes for their anticancer activity: inhibition of cell proliferation by targeting microtubules.

The Morita-Baylis-Hillman (MBH) type reaction of a variety of aromatic and heteroaromatic conjugated nitroalkenes with formaldehyde in the presence of stoichiometric amounts of imidazole and catalytic amounts (10 mol %) of anthranilic acid at room temperature provided the corresponding hydroxymethylated derivatives in moderate to good yield. The parent nitroalkenes and their MBH adducts were subsequently screened for their anticancer activity. Some of the MBH adducts were found to inhibit cervical cancer (HeLa) cell proliferation at low micromolar concentrations with half-maximal inhibitory concentrations in the range of 1-2 microM. The antiproliferative activity of 3-((E)-2-nitrovinyl)furan and three potent MBH adducts, namely, hydroxymethylated derivatives of 3-((E)-2-nitrovinyl)thiophene, 1-methoxy-4-((E)-2-nitrovinyl)benzene, and 1,2-dimethoxy-4-((E)-2-nitrovinyl)benzene was correlated well with their antimicrotubule activity. At their effective concentration range, the tested compounds perturbed the organization of mitotic spindle microtubules and chromosomes. In the presence of hydroxymethylated nitroalkenes, abnormal bipolar or multipolar mitotic spindles were apparent. Interphase microtubules were found to be significantly depolymerized at relatively higher concentrations of the tested compounds. These compounds inhibited tubulin assembly into microtubules in vitro by binding to tubulin at a site distinct from the vinblastine and colchicine binding sites. The compounds reduced the intrinsic tryptophan fluorescence of tubulin and the fluorescence of tubulin-1-anilinonaphthalene-8-sulfonic acid (ANS) complex indicating that they induced conformational changes in the tubulin. The results suggest that hydroxymethylated nitroalkenes exert their antiproliferative activity at least in part by depolymerizing cellular microtubules through tubulin binding and indicate that hydroxymethylated nitroalkenes are promising lead compounds for cancer therapy.

Alkenes↗
PubMed2006

Effects of pH and ionic strength on the assembly and bundling of FtsZ protofilaments: a possible role of electrostatic interactions in the bundling of protofilaments.

Assembly, bundling and stability of FtsZ protofilaments are important for the formation and functioning of the cytokinetic Z-ring during bacterial division. We found that the bundling of FtsZ protofilaments decreased strongly with increasing pH from 6.0 to 7.9, while the assembly of FtsZ monomers did not decrease considerably. In addition, the disassembly of FtsZ protofilaments was strongly suppressed at pH 6.0 as compared to the elevated pHs. The far-UV circular dichroism spectra of the native FtsZ and the tryptophan emission spectra of mutated FtsZ (Y371W) did not change by increasing pH from 6 to 7.9 indicating that the structure of FtsZ was not altered significantly. Further, the inhibition of bundling of FtsZ protofilaments predominantly, and the inhibition of assembly to a lesser extent by salt indicated that electrostatic interactions are important for the assembly and bundling of FtsZ protofilaments. These observations are supported by the results of computational docking of Escherichia coli dimer structure. The results suggest that the basic intracellular pH (7.4-7.8) of E. coli may play a role in regulating the assembly dynamics of FtsZ in the Z-ring by reducing protofilament stability and bundling in bacterial cytoplasm.

Escherichia coli↗
PubMed2006

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

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 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.

Antineoplastic Agents↗
PubMed2006

Antimitotic sulfonamides inhibit microtubule assembly dynamics and cancer cell proliferation.

Several sulfonamides have antitumor activities and are currently undergoing clinical evaluation for the treatment of cancer. In this study, we have elucidated the antiproliferative mechanism of action of five indole sulfonamides. The indole sulfonamides inhibited the polymerization of microtubule protein into microtubules in vitro. In addition, three representative derivatives, ER-68378 (2), ER-68384 (4) and ER-68394 (5), suppressed the dynamic instability behavior at the plus ends of individual steady-state microtubules in vitro. The analogues inhibited HeLa cell proliferation with half-maximal inhibitory concentrations in the range of 6-17 microM. The compounds blocked cell cycle progression at mitosis. At their lowest effective antimitotic concentrations, they depolymerized the spindle microtubules and disorganized the chromosomes but did not affect the microtubules in interphase cells. However, at relatively high concentrations, interphase microtubules were also depolymerized by these sulfonamides. Furthermore, all five compounds were found to induce apoptosis in the cells in association with the phosphorylation of bcl-2. The results suggest that the indole sulfonamides inhibit cell proliferation at mitosis by perturbing the assembly dynamics of spindle microtubules and that they can kill cancer cells by inducing apoptosis through the bcl-2-dependent pathway.

Aminophenols↗
PubMed2006

Rational design of the microtubule-targeting anti-breast cancer drug EM015.

We studied in silico docking of noscapine onto tubulin, combined with calculations of surface charge, pi-pi, van der Waals, and hydrogen bonding interactions, to rationally design a new compound, EM015. This tubulin-binding semisynthetic compound is a selective and potent anti-breast cancer agent and displays a 20-fold lower IC(50) against many tumor cells compared with our founding compound, (S)-6,7-dimethoxy-3-((R)-4-methoxy-6-methyl-5,6,7,8-tetrahydro[1,3]-dioxolo-[4,5-g]isoquinolin-5-yl)isobenzo-furan-1(3H)-one (noscapine). Furthermore, EM015 is also effective against a variety of drug-resistant cells. Surprisingly, the cell cycle profile of nontumorigenic normal cells is not affected. Many antimicrotubule cancer drugs in clinic today, particularly taxanes and Vincas, face challenges including frequent visits to the hospital for prolonged i.v. infusions, toxicities, and tumor recurrences due to drug resistance. EM015, on the other hand, is orally available, regresses breast tumor xenografts in nude mice models, and increases longevity. Furthermore, we have failed to observe any detectable toxicity in tissues, such as liver, kidney, spleen, lung, heart, and brain, as well as neurons, which are common targets of antimicrotubule drug therapy. Thus, EM015 has a great promise in the clinic.

Animals↗
PubMed2006

Development of a novel nitro-derivative of noscapine for the potential treatment of drug-resistant ovarian cancer and T-cell lymphoma.

We have shown previously that an antitussive plant alkaloid, noscapine, binds tubulin, displays anticancer activity, and has a safe pharmacological profile in humans. Structure-function analyses pointed to a proton at position-9 of the isoquinoline ring that can be modified without compromising tubulin binding activity. Thus, many noscapine analogs with different functional moieties at position-9 were synthesized. Those analogs that kill human cancer cells resistant to other antimicrotubule agents, vincas and taxanes, were screened. Here, we present one such analog, 9-nitro-noscapine (9-nitro-nos), which binds tubulin and induces apoptosis selectively in tumor cells (ovarian and T-cell lymphoma) resistant to paclitaxel, vinblastine, and teniposide. 9-Nitro-nos treatment at doses as high as 100 microM did not affect the cell cycle profile of normal human fibroblasts. This selectivity of 9-nitro-nos for cancer cells represents a unique edge over the other available antimitotics. 9-Nitro-nos perturbs the progression of cell cycle by mitotic arrest, followed by apoptotic cell death associated with increased caspase-3 activation and appearance of terminal deoxynucleotidyl transferase dUTP nick-end labeling-positive cells. Thus, we conclude that 9-nitro-nos has great potential to be a novel therapeutic agent for ovarian and T-cell lymphoma cancers, even those that have become drug-resistant to currently available chemotherapeutic drugs.

Antimitotic Agents↗
PubMed2006

The benzophenanthridine alkaloid sanguinarine perturbs microtubule assembly dynamics through tubulin binding. A possible mechanism for its antiproliferative activity.

Sanguinarine has been shown to inhibit proliferation of several types of human cancer cell including multidrug-resistant cells, whereas it has minimal cytotoxicity against normal cells such as neutrophils and keratinocytes. By analyzing the antiproliferative activity of sanguinarine in relation to its effects on mitosis and microtubule assembly, we found that it inhibits cancer cell proliferation by a novel mechanism. It inhibited HeLa cell proliferation with a half-maximal inhibitory concentration of 1.6 +/- 0.1 microM. In its lower effective inhibitory concentration range, sanguinarine depolymerized microtubules of both interphase and mitotic cells and perturbed chromosome organization in mitotic HeLa cells. At concentrations of 2 microM, it induced bundling of interphase microtubules and formation of granular tubulin aggregates. A brief exposure of HeLa cells to sanguinarine caused irreversible depolymerization of the microtubules, inhibited cell proliferation, and induced cell death. However, in contrast with several other microtubule-depolymerizing agents, sanguinarine did not arrest cell cycle progression at mitosis. In vitro, low concentrations of sanguinarine inhibited microtubule assembly. At higher concentrations (> 40 microM), it altered polymer morphology. Further, it induced aggregation of tubulin in the presence of microtubule-associated proteins. The binding of sanguinarine to tubulin induces conformational changes in tubulin. Together, the results suggest that sanguinarine inhibits cell proliferation at least in part by perturbing microtubule assembly dynamics.

Alkaloids↗
PubMed2006

2-Methoxyestradiol suppresses microtubule dynamics and arrests mitosis without depolymerizing microtubules.

2-Methoxyestradiol (2ME2), a metabolite of estradiol-17beta, is a novel antimitotic and antiangiogenic drug candidate in phase I and II clinical trials for the treatment of a broad range of tumor types. 2ME2 binds to tubulin at or near the colchicine site and inhibits the polymerization of tubulin in vitro, suggesting that it may work by interfering with normal microtubule function. However, the role of microtubule depolymerization in its antitumor mechanism of action has been controversial. To determine the mechanism by which 2ME2 induces mitotic arrest, we analyzed its effects on microtubule polymerization in vitro and its effects on dynamic instability both in vitro and in living MCF7 cells. In vitro, 2ME2 (5-100 micromol/L) inhibited assembly of purified tubulin in a concentration-dependent manner, with maximal inhibition (60%) at 200 micromol/L 2ME2. However, with microtubule-associated protein-containing microtubules, significantly higher 2ME2 concentrations were required to depolymerize microtubules, and polymer mass was reduced by only 13% at 500 micromol/L 2ME2. In vitro, dynamic instability was inhibited at lower concentrations. Specifically, 4 micromol/L 2ME2 reduced the mean growth rate by 17% and dynamicity by 27%. In living interphase MCF7 cells at the IC50 for mitotic arrest (1.2 micromol/L), 2ME2 significantly suppressed the mean microtubule growth rate, duration and length, and the overall dynamicity, consistent with its effects in vitro, and without any observable depolymerization of microtubules. Taken together, the results suggest that the major mechanism of mitotic arrest at the lowest effective concentrations of 2ME2 is suppression of microtubule dynamics rather than microtubule depolymerization per se.

2-Methoxyestradiol↗
PubMed2005

Sanguinarine blocks cytokinesis in bacteria by inhibiting FtsZ assembly and bundling.

Bacterial diseases are among the leading causes of human death. The development of antibiotic resistance greatly contributes to the high mortality rate, and thus, the discovery of antibacterial drugs with novel mechanisms of action is needed. In this study, we found that sanguinarine, a benzophenanthridine alkaloid, strongly induced filamentation in both Gram-positive and Gram-negative bacteria and prevented bacterial cell division by inhibiting cytokinesis. Sanguinarine did not perturb the membrane structure in Escherichia coli. However, it perturbed the cytokinetic Z-ring formation in E. coli. In addition, sanguinarine strongly reduced the frequency of the occurrence of Z rings/micrometer of Bacillus subtilis length but did not alter the number of nucleoids/micrometer of cell length. The results suggested that sanguinarine inhibited cytokinesis in B. subtilis by inhibiting Z-ring formation without affecting nucleoid segregation. Sanguinarine inhibited the assembly of purified FtsZ and reduced the bundling of FtsZ protofilaments in vitro. Further, the interaction of sanguinarine to FtsZ was investigated using size-exclusion chromatography, an extrinsic fluorescent probe 1-anilinonaphthalene-8-sulfonic acid, and tryptophan fluorescence of mutated FtsZ (Y371W). Sanguinarine was found to bind to FtsZ with a dissociation constant of 18-30 microM. The results together show that sanguinarine inhibits bacterial division by perturbing FtsZ assembly dynamics in the Z ring and provide evidence in support of the hypothesis that the assembly and bundling of FtsZ play a critical role in bacterial cytokinesis. The results suggest that sanguinarine may be used as a lead compound to develop FtsZ-targeted antibacterial agents.

Alkaloids↗
PubMed2005

Deuterium oxide promotes assembly and bundling of FtsZ protofilaments.

The assembly and bundling of FtsZ protofilaments play an important role during bacterial cell division. Deuterium oxide (D2O) is known to have strong stabilization effects on the assembly dynamics of several proteins including tubulin, a homologue of FtsZ. Here, we found that D2O enhanced the light-scattering intensity of the assembly reaction, increased sedimentable polymer mass, and induced bundling of FtsZ protofilaments. D2O also increased the stability of FtsZ polymers under challenged GTP conditions and suppressed dilution-induced disassembly of protofilaments. D2O enhances the assembly parameters of FtsZ and microtubules albeit differently. For example, D2O induced bundling of FtsZ protofilaments, whereas it did not induce bundling of microtubules in vitro. In addition, D2O strongly suppressed the GTP hydrolysis rate of microtubules, but it had no effect on the initial rate of GTP hydrolysis of the FtsZ assembly. D2O (80%) also increased the helical content of FtsZ by 25% compared to the helical content of FtsZ in aqueous buffer. D2O was shown to reduce the binding of 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid (bis-ANS) to tubulin. In contrast, we found that D2O strongly enhanced the binding of bis-ANS to FtsZ. The results indicated that D2O promotes assembly and bundling of FtsZ protofilaments by increasing hydrophobic interactions between the protofilaments. The results also suggest that the phosphate release rather than the on-site GTP hydrolysis is the rate-limiting step of the GTP turnover reaction.

Circular Dichroism↗
PubMed2005

Unfolding pathways of human serum albumin: evidence for sequential unfolding and folding of its three domains.

Human serum albumin (HSA) contains three alpha-helical domains (I-III). The unfolding process of these domains was monitored using covalently bound fluorescence probes; domain I was monitored by N-(1-pyrene)maleimide (PM) conjugated with cys-34, domain II was monitored by the lone tryptophan residue and domain III was followed by p-nitrophenyl anthranilate (NPA) conjugated with Tyrosine-411 (Tyr-411). Using domain-specific probes, we found that guanidium hydrochloride-induced unfolding of HSA occurred sequentially. The unfolding of domain II preceded that of domain I and the unfolding of domain III followed that of domain I. In addition, the domains I and III refolded within the dead time of the fluorescence recovery experiment while the refolding of domain II occurred slowly. The results suggest that individual domain of a multi-domain protein can fold and unfold sequentially.

Circular Dichroism↗
PubMed2005

Resveratrol inhibits type II phosphatidylinositol 4-kinase: a key component in pathways of phosphoinositide turn over.

Resveratrol has anti-inflammatory, cardio protective and cancer chemopreventive properties. The molecular targets for resveratrol in early signaling cascades are not well understood. Resveratrol inhibits type II PtdIns 4-kinase but not PtdIns 3-kinase activity in vitro. Resveratrol directly binds to the enzyme with a Kd of 7.2 microM. Kinetic studies show that resveratrol competes with PtdIns binding. Inhibition of PtdIns 4-kinase activity by resveratrol/phenylarsine oxide reduces Jurkat cell adhesion to matrigel/fibronectin coated surfaces, suggesting a role for type II PtdIns 4-kinase in lymphocyte infiltration to the sites of inflammation.

1-Phosphatidylinositol 4-Kinase↗
PubMed2005

Kinetic suppression of microtubule dynamic instability by griseofulvin: implications for its possible use in the treatment of cancer.

The antifungal drug griseofulvin inhibits mitosis strongly in fungal cells and weakly in mammalian cells by affecting mitotic spindle microtubule (MT) function. Griseofulvin also blocks cell-cycle progression at G(2)/M and induces apoptosis in human tumor cell lines. Despite extensive study, the mechanism by which the drug inhibits mitosis in human cells remains unclear. Here, we analyzed the ability of griseofulvin to inhibit cell proliferation and mitosis and to affect MT polymerization and organization in HeLa cells together with its ability to affect MT polymerization and dynamic instability in vitro. Griseofulvin inhibited cell-cycle progression at prometaphase/anaphase of mitosis in parallel with its ability to inhibit cell proliferation. At its mitotic IC(50) of 20 muM, spindles in blocked cells displayed nearly normal quantities of MTs and MT organization similar to spindles blocked by more powerful MT-targeted drugs. Similar to previously published data, we found that very high concentrations of griseofulvin (>100 microM) were required to inhibit MT polymerization in vitro. However, much lower drug concentrations (1-20 microM) strongly suppressed the dynamic instability behavior of the MTs. We suggest that the primary mechanism by which griseofulvin inhibits mitosis in human cells is by suppressing spindle MT dynamics in a manner qualitatively similar to that of much more powerful antimitotic drugs, including the vinca alkaloids and the taxanes. In view of griseofulvin's lack of significant toxicity in humans, we further suggest that it could be useful as an adjuvant in combination with more powerful drugs for the treatment of cancer.

Animals↗
PubMed2005

A natural osmolyte trimethylamine N-oxide promotes assembly and bundling of the bacterial cell division protein, FtsZ and counteracts the denaturing effects of urea.

Assembly of FtsZ was completely inhibited by low concentrations of urea and its unfolding occurred in two steps in the presence of urea, with the formation of an intermediate [Santra MK & Panda D (2003) J Biol Chem278, 21336-21343]. In this study, using the fluorescence of 1-anilininonaphthalene-8-sulfonic acid and far-UV circular dichroism spectroscopy, we found that a natural osmolyte, trimethylamine N-oxide (TMAO), counteracted the denaturing effects of urea and guanidium chloride on FtsZ. TMAO also protected assembly and bundling of FtsZ protofilaments from the denaturing effects of urea and guanidium chloride. Furthermore, the standard free energy changes for unfolding of FtsZ were estimated to be 22.5 and 28.4 kJ.mol(-1) in the absence and presence of 0.6 M TMAO, respectively. The data are consistent with the view that osmolytes counteract denaturant-induced unfolding of proteins by destabilizing the unfolded states. Interestingly, TMAO was also found to affect the assembly properties of native FtsZ. TMAO increased the light-scattering signal of the FtsZ assembly, increased sedimentable polymer mass, enhanced bundling of FtsZ protofilaments and reduced the GTPase activity of FtsZ. Similar to TMAO, monosodium glutamate, a physiological osmolyte in bacteria, which induces assembly and bundling of FtsZ filaments in vitro[Beuria TK, Krishnakumar SS, Sahar S, Singh N, Gupta K, Meshram M & Panda D (2003) J Biol Chem278, 3735-3741], was also found to counteract the deleterious effects of urea on FtsZ. The results together suggested that physiological osmolytes may regulate assembly and bundling of FtsZ in bacteria and that they may protect the functionality of FtsZ under environmental stress conditions.

Anilino Naphthalenesulfonates↗
PubMed2004

Antimitotic antifungal compound benomyl inhibits brain microtubule polymerization and dynamics and cancer cell proliferation at mitosis, by binding to a novel site in tubulin.

The antifungal agent benomyl [methyl-1-(butylcarbamoyl)-2-benzimidazolecarbamate] is used throughout the world against a wide range of agricultural fungal diseases. In this paper, we investigated the interaction of benomyl with mammalian brain tubulin and microtubules. Using the hydrophobic fluorescent probe 1-anilinonaphthalene-8-sulfonic acid, benomyl was found to bind to brain tubulin with a dissociation constant of 11.9 +/- 1.2 microM. Further, benomyl bound to at a novel site, distinct from the well-characterized colchicine and vinblastine binding sites. Benomyl altered the far-UV circular dichroism spectrum of tubulin and reduced the accessibility of its cysteine residues to modification by 5,5'-dithiobis-2-nitrobenzoic acid, indicating that benomyl binding to tubulin induces a conformational change in the tubulin. Benomyl inhibited the polymerization of brain tubulin into microtubules, with 50% inhibition occurring at a concentration of 70-75 microM. Furthermore, it strongly suppressed the dynamic instability behavior of individual brain microtubules in vitro as determined by video microscopy. It reduced the growing and shortening rates of the microtubules but did not alter the catastrophe or rescue frequencies. The unexpected potency of benomyl against mammalian microtubule polymerization and dynamics prompted us to investigate the effects of benomyl on HeLa cell proliferation and mitosis. Benomyl inhibited proliferation of the cells with an IC(50) of 5 microM, and it blocked mitotic spindle function by perturbing microtubule and chromosome organization. The greater than expected actions of benomyl on mammalian microtubules and mitosis together with its relatively low toxicity suggest that it might be useful as an adjuvant in cancer chemotherapy.

Animals↗