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Tapas K Das Gupta

Publications and source records attributed to Tapas K Das Gupta.

At least 19 recordsLinked to original sources
PubMed2006

Bacterial proteins and CpG-rich extrachromosomal DNA in potential cancer therapy.

Bacterial proteins such as azurin and Laz have recently been shown to enter preferentially to cancer cells and kill them by multiple mechanisms. Historically, bacterial DNA, particularly the unmethylated CpG dinucleotides, have been shown to trigger activation of specific Toll-like receptors (TLRs) in immune cells, leading to various cytokine and chemokine production that allows cancer cell death and their regression. However, the enhanced release of specific protein or extrachromosomal DNA by bacteria in response to exposure to cancer cells has not been previously demonstrated. In this review, we discuss how an opportunistic, extracellular pathogenic bacterium, Pseudomonas aeruginosa, senses the presence of cancer cells and releases a specific protein or extrachromosomal DNA with antitumor activity for inhibition of cancer cell growth.

Amino Acid Sequence↗
PubMed2006

Disrupting the entry barrier and attacking brain tumors: the role of the Neisseria H.8 epitope and the Laz protein.

Azurin is a periplasmic 128 amino acid protein in Pseudomonas aeruginosa, termed Paz, which has been shown to enter preferentially and induce apoptosis in cancer cells such as human melanoma or breast cancer. Its effectiveness against brain tumors such as glioblastomas has not been studied. The meningitis-causing bacterium Neisseria meningitidis also harbors an azurin-like protein. Unlike all other known azurins, Neisserial azurin, termed Laz, is surface-exposed and has in its N-terminal region a 39 amino acid epitope called H.8. Upstream of this H.8 moiety is a lipobox that results in the truncation of the protein at the N-terminal cysteine residue with modification by a lipid group. No function of Laz is known. We demonstrate that while Paz is deficient in entering glioblastoma cells and exhibits low cytotoxicity, Laz is much more proficient in entering glioblastoma cells and shows a higher level of cytotoxicity. When the Neisserial H.8 moiety containing the lipobox is fused in frame with Paz either in its N-terminal (H.8-Paz) or in its C-terminal (Paz-H.8), both had high cytotoxicity for glioblastoma cells and a higher level of internalization. When expressed in E. coli, H.8-Paz was much more exposed on the surface than Paz-H.8. The replacement of the Laz N-terminal cysteine residue involved in acylation with an alanine residue abolished the surface display, but had no effect on cytotoxicity or entry in glioblastoma cells, suggesting a role of the H.8 moiety, but not its lipidation, in disrupting the entry barrier in brain tumor cells.

Azurin↗
PubMed2006

Azurin, Plasmodium falciparum malaria and HIV/AIDS: inhibition of parasitic and viral growth by Azurin.

Azurin, a member of a family of copper-containing proteins involved in electron transfer called cupredoxins, demonstrates structural features similar to the variable domains of the immunoglobulin superfamily members. An azurin-like protein called Laz with an additional N-terminal 39 amino acid peptide known as H.8 epitope is present on the surface of gonnococci and meningococci. We demonstrate that azurin, Laz and H.8-azurin can bind to the C-terminal cleavage product MSP1-19 of merozoite surface protein 1 (MSP1) of the malarial parasite Plasmodium falciparum and significantly reduce parasitemia. Azurin and Laz also bound strongly to HIV-1 gp120. Interestingly, azurin could not only bind to gp120 but also to the dendritic cell-specific adhesion receptor DC-SIGN, mimicking the functionality of the intercellular adhesion molecule ICAM-3 with which it also binds avidly. Furthermore, these three proteins significantly suppressed HIV-1 growth in peripheral blood mononuclear cells and such suppression appeared to be occurring at an entry stage in the infection process. The presence of both antimalarial and antiretroviral activity in azurin, H.8-azurin and Laz makes these proteins, or peptides derived from them, potential therapeutic agents in the treatment of malaria, HIV-1 infections or coinfections with both P. falciparum and HIV-1.

Acquired Immunodeficiency Syndrome↗
PubMed2005

Human cytochrome c enters murine J774 cells and causes G1 and G2/M cell cycle arrest and induction of apoptosis.

Cytochrome c is well known as a carrier of electrons during respiration. Current evidence indicates that cytochrome c also functions as a major component of apoptosomes to induce apoptosis in eukaryotic cells as well as an antioxidant. More recently, a prokaryotic cytochrome c, cytochrome c(551) from Pseudomonas aeruginosa, has been shown to enter in mammalian cells such as the murine macrophage-like J774 cells and causes inhibition of cell cycle progression. Much less is known about such functions by mammalian cytochromes c, particularly the human cytochrome c. We now report that similar to P. aeruginosa cytochrome c(551), the purified human cytochrome c protein can enter J774 cells and induce cell cycle arrest at the G(1) to S phase, as well as at the G(2)/M phase at higher concentrations. Unlike P. aeruginosa cytochrome c(551) which had no effect on the induction of apoptosis, human cytochrome c induces significant apoptosis and cell death in J774 cells, presumably through inhibition of the cell cycle at the G(2)/M phase. When incubated with human breast cancer MCF-7 and normal mammary epithelial cell line MCF-10A1 cells, human cytochrome c entered in both types of cells but induced cell death only in the normal MCF-10A1 cells. The ability of human cytochrome c to enter J774 cells was greatly reduced at 4 degrees C, suggesting energy requirement in the entry process.

Apoptosis↗
PubMed2005

Internalization of bacterial redox protein azurin in mammalian cells: entry domain and specificity.

Azurin is a member of a group of copper-containing redox proteins called cupredoxins. Different cupredoxins are produced by different aerobic bacteria as agents of electron transfer. Recently, we demonstrated that azurin enters into J774 and several types of cancer cells leading to the induction of apoptosis. We now demonstrate that azurin is internalized in J774 or cancer cells in a temperature-dependent manner. Azurin shows preferential entry into cancer compared with normal cells. An 28-amino-acid fragment of azurin fused to glutathione S-transferase (GST) or the green fluorescent protein (GFP), which are incapable of entering mammalian cells by themselves, can be internalized in J774 or human melanoma or breast cancer cells at 37 degrees C, but not at 4 degrees C. Competition experiments as well as studies with inhibitors such as cytochalasin D suggest that azurin may enter cells, at least in part, by a receptor-mediated endocytic process. The 28-amino-acid peptide therefore acts as a potential protein transduction domain (PTD), and can be used as a vehicle to transport cargo proteins such as GST and GST-GFP fusion proteins. Another member of the cupredoxin family, rusticyanin, that has also been shown to enter J774 and human cancer cells and exert cytotoxicity, does not demonstrate preferential entry for cancer cells and lacks the structural features characteristic of the azurin PTD.

Amino Acid Sequence↗
PubMed2004

A novel RARbeta isoform directed by a distinct promoter P3 and mediated by retinoic acid in breast cancer cells.

Retinoids regulate gene transcription through activating retinoic acid receptors (RARs)/retinoic X receptors (RXRs). Of the three RAR receptors (alpha, beta, and gamma), RARbeta has been considered a tumor suppressor gene. Here, we identified a novel RARbeta isoform-RARbeta5 in breast epithelial cells, which could play a negative role in RARbeta signaling. Similar to RARbeta2, the first exon (59 bp) of RARbeta5 is RARbeta5 isoform specific, whereas the other exons are common to all of the RARbeta isoforms. The first exon of RARbeta5 does not contain any translation start codon, and therefore its protein translation begins at an internal methionine codon of RARbeta2, lacking the A, B, and part of C domain of RARbeta2. RARbeta5 protein was preferentially expressed in estrogen receptor-negative breast cancer cells and normal breast epithelial cells that are relatively resistant to retinoids, whereas estrogen receptor-positive cells that did not express detectable RARbeta5 protein were sensitive to retinoid treatment, suggesting that this isoform may affect the cellular response to retinoids. RARbeta5 isoform is unique among all of the RARs, because a corresponding isoform was not detectable for either RARalpha or RARgamma. RARbeta5 mRNA was variably expressed in normal and cancerous breast epithelial cells. Its transcription was under the control of a distinct promoter P3, which can be activated by all-trans-retinoic acid (atRA) and other RAR/RXR selective retinoids in MCF-7 and T47D breast cancer cells. We mapped the RARbeta5 promoter and found a region -302/-99 to be the target region of atRA. In conclusion, we identified and initially characterized RARbeta5 in normal, premalignant, and malignant breast epithelial cells. RARbeta5 may serve as a potential target of retinoids in prevention and therapy studies.

Amino Acid Sequence↗
PubMed2004

Rusticyanin, a bacterial electron transfer protein, causes G1 arrest in J774 and apoptosis in human cancer cells.

During acid mine drainage, Acidithiobacillus ferrooxidans, a nonpathogenic, acidophilic, lithotrophic bacterium, utilizes rusticyanin to transfer electrons for the oxidation of Fe(2+) to Fe(3+) for deriving its energy. No other function of rusticyanin is known. We demonstrate that purified rusticyanin enters mammalian cells inducing either inhibition of cell cycle progression or caspase-8 mediated apoptosis. Treatment of human melanoma cells with rusticyanin allowed significant generation of reactive oxygen species and active caspase-8, leading to cell death. The ability of rusticyanin to modulate mammalian cell death might be relevant to a role of this cupredoxin in protecting At. ferrooxidans from eukaryotic predators in the environment.

Acidithiobacillus↗
PubMed2004

Regulation of mammalian cell growth and death by bacterial redox proteins: relevance to ecology and cancer therapy.

Recent evidence indicates that bacterial redox proteins such as cupredoxins and cytochromes, that are normally involved in electron transfer during respiration, can enter mammalian cells and induce either apoptosis or inhibition of cell cycle progression. Such proteins have also been shown to demonstrate a good deal of specificity for entry and induction of cytotoxic effects in cancer cells, allowing both in vitro cell death and in vivo inhibition of cancer progression. An alteration in the hydrophobicity of the bacterial redox proteins can lead to a switch from apoptosis to growth arrest and vice versa through modulation of the intracellular levels of tumor suppressors. The preferential entry and cytotoxicity of these redox proteins in cancer cells raises interesting questions about the presence of other bacterial proteins that may affect cell cycle at the G(2)/M phase, thereby potentially arresting cancer growth. The intracellular localization of the bacterial redox proteins in nonpathogenic soil bacteria similarly raises questions about their possible role in allowing various nonpathogenic soil bacteria to defend themselves from environmental predators by inducing cytotoxicity when engulfed in large numbers. A new role of the redox proteins in soil bacteria in maintaining an ecological balance among the predators and preys is proposed.

Animals↗
PubMed2004

Modulation of mammalian cell growth and death by prokaryotic and eukaryotic cytochrome c.

Cytochrome c(551), an 8,685-Da haem-containing protein, is known to be involved in electron transfer during dissimilative denitrification by Pseudomonas aeruginosa. Both cytochrome c(551) and copper-containing redox protein azurin have been used in vitro as partners in electron transfer. Azurin has been reported to induce apoptosis in macrophages and cancer cells. We now report that, unlike azurin, cytochrome c(551), termed Cyt c(551), has very little ability to induce apoptosis in J774 cell line-derived macrophages but demonstrates significant inhibition of cell cycle progression in such cells. A mutant form of Cyt c(551), V23DI59E, has significantly reduced ability to inhibit cell cycle progression but demonstrates a higher level of apoptosis-inducing activity in macrophages, compared with WT Cyt c(551). Interestingly, the WT Cyt c(551), but not the mutant form, significantly enhances the level of tumor suppressor protein p16(Ink4a), a known inhibitor of cell cycle progression whereas the mutant form seems to form a complex with tumor suppressor protein p53, thereby enhancing its intracellular level to some extent. Eukaryotic cytochromes such as horse and bovine cytochrome c have also been shown to induce apoptosis but not inhibition of cell cycle progression in J774 cells, thus signifying a role of this redox protein in entry to, and in the induction of, cell death in mammalian cells.

Amino Acid Sequence↗
PubMed2004

Bacterial cupredoxin azurin as an inducer of apoptosis and regression in human breast cancer.

Azurin, a copper-containing redox protein released by the pathogenic bacterium Pseudomonas aeruginosa, is highly cytotoxic to the human breast cancer cell line MCF-7, but is less cytotoxic toward p53-negative (MDA-MB-157) or nonfunctional p53 cell lines like MDD2 and MDA-MB-231. The purpose of this study was to investigate the underlying mechanism of the action of bacterial cupredoxin azurin in the regression of breast cancer and its potential chemotherapeutic efficacy. Azurin enters into the cytosol of MCF-7 cells and travels to the nucleus, enhancing the intracellular levels of p53 and Bax, thereby triggering the release of mitochondrial cytochrome c into the cytosol. This process activates the caspase cascade (including caspase-9 and caspase-7), thereby initiating the apoptotic process. Our results indicate that azurin-induced cell death stimuli are amplified in the presence of p53. In vivo injection of azurin in immunodeficient mice harboring xenografted human breast cancer cells in the mammary fat pad leads to statistically significant regression (85%, P = 0.0179, Kruskal-Wallis Test) of the tumor. In conclusion, azurin blocks breast cancer cell proliferation and induces apoptosis through the mitochondrial pathway both in vitro and in vivo, thereby suggesting a potential chemotherapeutic application of this bacterial cupredoxin for the treatment of breast cancer.

Antineoplastic Agents↗
PubMed2004

Apoptosis or growth arrest: Modulation of tumor suppressor p53's specificity by bacterial redox protein azurin.

The tumor suppressor protein p53 is known to induce either apoptosis or growth arrest depending on cellular background. We have previously reported that a bacterial redox protein azurin induces apoptosis in J774 cell line-derived macrophages whereas a site-directed mutant M44KM64E azurin shows very little cytotoxicity and fails to induce apoptosis in J774 cells. We now report that purified M44KM64E mutant azurin protein can enter both J774 cells as well as the human breast cancer MCF-7 cells. Entry of M44KM64E mutant azurin in J774 cells causes strong inhibition of cell-cycle progression at the G1 to S phase and a higher level of transcription of the p21 gene. Corresponding to high p21 levels, the levels of cyclins and cyclin-dependent kinases were greatly lowered in M44KM64E mutant azurin-treated J774 cells. Interestingly, M44KM64E mutant azurin protein failed to elicit inhibition of cell-cycle progression in MCF-7 cells, presumably because of mutation at the retinoblastoma tumor suppressor protein that allows functional E2F formation in MCF-7 cells even in the presence of high intracellular p21 level. Thus, the WT azurin induces apoptosis but little inhibition of cell-cycle progression whereas the M44KM64E mutant azurin is deficient in the induction of apoptosis but mediates strong inhibition of cell-cycle progression, demonstrating the role of a single bacterial protein and its hydrophobic patch in modulating two important functions of p53.

Apoptosis↗
PubMed2003

Melanoma cells can tolerate high levels of transcriptionally active endogenous p53 but are sensitive to retrovirus-transduced p53.

Malignant melanomas are frequently characterized by elevated levels of wild-type p53, suggesting that p53 function could be suppressed by a mechanism different from p53 mutation. We analysed the functionality of the p53-signaling pathway in a panel of seven human melanoma cell lines consisting of one p53-deficient line, two lines with mutant p53, and four lines expressing wild-type p53. Only lines with wild-type p53 were characterized by elevated levels of endogenous p21, high activity of p53-responsive reporters and accumulation of p53 in response to genotoxic stress, common properties of functional p53. The presence of wild-type p53 was associated with depletion or loss of p14ARF and p16 expression. The levels of p33ING1b and p24ING1c, two major products of Ing1 locus and putative coregulators of p53, were elevated in all cell lines tested; however, ectopic expression of either ING1 isoform had no effect on cell proliferation. All lines retained expression of Apaf-1, and all but one remained sensitive to ectopic expression of retrovirus-transduced p53. Our data indicate that regardless of abnormally high levels of p53 in melanomas, their p53 remains competent in transactivation of its targets, and, if highly overexpressed, capable of growth inhibition. Hence, the p53 pathway in malignant melanomas can be considered for pharmacological targeting and anticancer gene therapy.

Apoptotic Protease-Activating Factor 1↗
PubMed2003

Efficacy and mechanism of action of 1alpha-hydroxy-24-ethyl-cholecalciferol (1alpha[OH]D5) in breast cancer prevention and therapy.

It is now well established that the active metabolite of vitamin D3, 1alpha,25(OH)2D3, regulates cell growth and differentiation in various in vitro cancer models. However, its clinical use is precluded due to its hypercalcemic activity in vivo. Hence, several less calcemic vitamin D analogs have been synthesized and evaluated for their chemopreventive and therapeutic efficacy in experimental carcinogenesis models. A novel analog of vitamin D3, 1alpha-hydroxy-24-ethyl-cholecalciferol (1alpha[OH]D5), has currently been under investigation in our laboratory for its application in breast cancer prevention and therapy. 1alpha(OH)D5 had been shown to inhibit development of estrogen- and progesterone-dependent ductal lesions as well as steroid hormone-independent alveolar lesions in a mammary gland organ culture (MMOC) model. Moreover, the inhibitory effect was more significant if 1alpha(OH)D5 was present during the promotional phase of the lesion development. The growth inhibitory effect of 1alpha(OH)D5 has also been manifested in several breast cancer cell lines, including BT-474 and MCF-7. Breast cancer cell lines that responded to 1alpha(OH)D5 treatment were vitamin D receptor positive (VDR+). Vitamin D receptor-negative (VDR-) cell lines, such as MDA-MB-231 and MDA-MB-435, did not show growth inhibition upon incubation with 1alpha(OH)D5. This suggests the requirement of VDR in 1alpha(OH)D5-mediated growth effects. Interestingly, breast cancer cells that were VDR+ as well as estrogen receptor positive (ER+) showed cell cycle arrest and apoptosis, while VDR+ but ER- cells (UISO-BCA-4 breast cancer cells) showed enhanced expression of various differentiation markers with la(OH)D5 treatment. Transcription and expression of estrogen-inducible genes, progesterone receptor (PR) and trefoil factor 1 (pS2), were significantly down-regulated in ER+ BT-474 cells with 1alpha(OH)D5 treatment. This implies a differential effect of 1alpha(OH)D5 on ER+ vs. ER- cells. Additionally, comparison between the effects of 1alpha(OH)D5 on normal vs. transformed cells indicated that 1alpha(OH)D5 does not suppress cell prolifera-

Animals↗
PubMed2003

Predicting residual lymph node basin disease in melanoma patients with sentinel lymph node metastases.

BACKGROUND: The incidence of residual occult disease in nonsentinel lymph nodes (NSLN) after a positive sentinel lymph node (SLN) biopsy in patients with melanoma is relatively low. The purpose of this study is to identify factors that may be predictive of occult NSLN metastases after positive SLN biopsy. METHODS: Fifty-six consecutive melanoma patients with positive sentinel nodes who subsequently underwent complete lymph node dissection (CLND) were evaluated. RESULTS: Only the number of positive SLN predicted the status on NSLN by univariate (P = 0.008) and multivariate (P = 0.028) analyses. None of the other variables (characteristics of SLN metastases, number of draining nodal basins, age, sex, thickness, Clark level, ulceration, number of mitoses/mm(2), histological subtype, and location of the primary) significantly predicted CLND results. CONCLUSIONS: Identifying patients with residual lymph node basin disease remains difficult. Thus, lymph node dissection should be performed in all patients after positive sentinel node biopsy.

Adolescent↗
PubMed2003

Targeting colon cancer cells with genistein-17.1A immunoconjugate.

We have shown that genistein, a major component of soy, has anti-colon cancer effects in vitro. These effects are attainable at high concentrations that are difficult to achieve in the serum. The purpose of this study was to enhance the activity of genistein against colon cancer cells by coupling it to 17.1A. The monoclonal antibody 17.1A recognizes an epithelial membrane antigen that is overexpressed in colon cancer. Synthesis of Gen-17.1A was achieved by photochemical conjugation using sulfa-SANPAH. Its purity was evaluated by SDS-PAGE. Binding of Gen-17.1A to SW-620 and HT-29 cells was shown using flow cytometry. Internalization was demonstrated by FITC-labeling. Gen-17.1A induced apoptosis in colon cancer cells as evidenced by the acridine orange/ethidium bromide staining method. Gen-17.1A significantly inhibited colon cancer cell growth in vitro and in vivo. Our findings suggest that conjugating genistein to 17.1A monoclonal antibody enhances its effects against colon cancer cells.

Animals↗
PubMed2002

Bacterial redox protein azurin, tumor suppressor protein p53, and regression of cancer.

The use of live bacteria in the treatment of cancer has a long and interesting history. We report the use of a purified bacterial redox protein, azurin, that enters human cancer (melanoma UISO-Mel-2) cells and induces apoptosis. The induction of apoptosis occurs readily in melanoma cells harboring a functional tumor suppressor protein p53, but much less efficiently in p53-null mutant melanoma (UISO-Mel-6) cells. A redox-negative mutant form of azurin (M44K/M64E) demonstrates much less cytotoxicity to the UISO-Mel-2 cells than the wild-type protein. Azurin has been shown to be internalized in UISO-Mel-2 cells and is localized predominantly in the cytosol and in the nuclear fraction. In the p53-null UISO-Mel-6 cells, azurin is localized only in the cytosol. Thus, intracellular trafficking of azurin to the nucleus is p53-dependent. Azurin forms a complex with p53, thereby stabilizing it and raising its intracellular level in cytosolic, mitochondrial, and nuclear fractions. Corresponding to an increasing level of p53, an inducer of apoptosis, the level of Bax also increases in mitochondria, allowing significant release of mitochondrial cytochrome c into the cytosol, thus initiating the onset of apoptosis. The M44K/M64E mutant form of azurin, deficient in cytotoxicity, is also deficient in forming a complex with p53 and is less efficient in stabilizing p53 than wild-type azurin. Azurin has been shown to allow regression of human UISO-Mel-2 tumors xenotransplanted in nude mice and may potentially be used in cancer treatment.

Animals↗