ABSTRACT
Breast cancer is the leading cause of cancer deaths among women. Paclitaxel (PTX), an important breast cancer medicine, exhibits reduced bioavailability and therapeutic index due to high hydrophobicity and indiscriminate cytotoxicity. PTX encapsulation in one-level active targeting overcomes such barriers, but enhances toxicity to normal tissues with cancer-similar expression profiles. This research attempted to overcome this challenge by increasing selectivity of cancer cell targeting while maintaining an ability to overcome traditional pharmacological barriers. Thus, a multi-core, multi-targeting construct for tumor specific delivery of PTX was fabricated with (i) an inner-core prodrug targeting the cancer-overexpressed cathepsin B through a cathepsin B-cleavable tetrapeptide that conjugates PTX to a poly(amidoamine) dendrimer, and (ii) the encapsulation of this prodrug (PGD) in an outer core of a RES-evading, folate receptor (FR)-targeting liposome. Compared to traditional FR-targeting PTX liposomes, this sequentially active-targeted dendrosome demonstrated better prodrug retention, an increased cytotoxicity to cancer cells (latter being true when FR and cathepsin B activities were both at moderate-to-high levels) and higher tumor reduction. This research may eventually evolve a product platform with reduced systemic toxicity inherent with traditional chemotherapy and localized toxicity inherent to single-target nanoplatforms, thereby allowing for better tolerance of higher therapeutic load in advanced disease states.
Subject(s)
Antineoplastic Agents, Phytogenic/therapeutic use , Breast Neoplasms/drug therapy , Drug Delivery Systems , Nanostructures , Paclitaxel/therapeutic use , Animals , Antineoplastic Agents, Phytogenic/administration & dosage , Cell Line, Tumor , Female , Humans , Liposomes , Paclitaxel/administration & dosageABSTRACT
Triple-negative breast cancer (TNBC), the most aggressive breast cancer subtype, occurs in younger women and is associated with poor prognosis. Gain-of-function mutations in TP53 are a frequent occurrence in TNBC and have been demonstrated to repress apoptosis and up-regulate cell cycle progression. Even though TNBC responds to initial chemotherapy, resistance to chemotherapy develops and is a major clinical problem. Tumor recurrence eventually occurs and most patients die from their disease. An urgent need exists to identify molecular-targeted therapies that can enhance chemotherapy response. In the present study, we report that targeting PELP1, an oncogenic co-regulator molecule, could enhance the chemotherapeutic response of TNBC through the inhibition of cell cycle progression and activation of apoptosis. We demonstrate that PELP1 interacts with MTp53, regulates its recruitment, and alters epigenetic marks at the target gene promoters. PELP1 knockdown reduced MTp53 target gene expression, resulting in decreased cell survival and increased apoptosis upon genotoxic stress. Mechanistic studies revealed that PELP1 depletion contributes to increased stability of E2F1, a transcription factor that regulates both cell cycle and apoptosis in a context-dependent manner. Further, PELP1 regulates E2F1 stability in a KDM1A-dependent manner, and PELP1 phosphorylation at the S1033 residue plays an important role in mediating its oncogenic functions in TNBC cells. Accordingly, depletion of PELP1 increased the expression of E2F1 target genes and reduced TNBC cell survival in response to genotoxic agents. PELP1 phosphorylation was significantly greater in the TNBC tumors than in the other subtypes of breast cancer and in the normal tissues. These findings suggest that PELP1 is an important molecular target in TNBC, and that PELP1-targeted therapies may enhance response to chemotherapies.
Subject(s)
Co-Repressor Proteins/metabolism , E2F1 Transcription Factor/metabolism , Mutation , Transcription Factors/metabolism , Triple Negative Breast Neoplasms/genetics , Triple Negative Breast Neoplasms/metabolism , Tumor Suppressor Protein p53/genetics , Antineoplastic Agents/pharmacology , Apoptosis , Cell Line, Tumor , Co-Repressor Proteins/antagonists & inhibitors , Co-Repressor Proteins/genetics , Drug Resistance, Neoplasm/drug effects , Female , Gene Knockdown Techniques , Humans , Phosphorylation , Transcription Factors/antagonists & inhibitors , Transcription Factors/genetics , Triple Negative Breast Neoplasms/drug therapy , Tumor Suppressor Protein p53/metabolismABSTRACT
Breast cancer is the second most common cause of cancer-related deaths in women. Chemotherapy is an important treatment modality, and paclitaxel (PTX) is often the first-line therapy for its metastatic form. The two most notable limitations related to PTX-based treatment are the poor hydrophilicity of the drug and the systemic toxicity due to the drug's nonspecific and indiscriminate distribution among the tissues. The present work describes an approach to counter both challenges by designing a conjugate of PTX with a hydrophilic macromolecule that is coupled through a biocleavable linker, thereby allowing for active targeting to an enzyme significantly upregulated in cancer cells. The resultant strategy would allow for the release of the active ingredient preferentially at the site of action in related cancer cells and spare normal tissue. Thus, PTX was conjugated to the hydrophilic poly(amdioamine) [PAMAM] dendrimer through the cathepsin B-cleavable tetrapeptide Gly-Phe-Leu-Gly. The PTX prodrug conjugate (PGD) was compared to unbound PTX through in vitro evaluations against breast cancer cells and normal kidney cells as well as through in vivo evaluations using xenograft mice models. As compared to PTX, PGD demonstrated a higher cytotoxicity specific to cell lines with moderate-to-high cathepsin B activity; cells with comparatively lower cathepsin B activity demonstrated an inverse of this relationship. Regression analysis between the magnitude of PGD-induced cytotoxic increase over PTX and cathepsin B expression showed a strong, statistically significant correlation (r(2) = 0.652, p < 0.05). The PGD conjugate also demonstrated a markedly higher tumor reduction as compared to PTX treatment alone in MDA-MB-231 tumor xenograft models, with PGD-treated tumor volumes being 48% and 34% smaller than PTX-treated volumes at weeks 2 and 3 after treatment initiation.
Subject(s)
Breast Neoplasms/drug therapy , Paclitaxel/pharmacology , Prodrugs/pharmacology , Up-Regulation/drug effects , Animals , Antineoplastic Agents, Phytogenic/pharmacology , Breast Neoplasms/genetics , Cathepsin B/pharmacology , Cell Line, Tumor , Cell Survival/drug effects , Cell Survival/genetics , Dendrimers/pharmacology , Female , Humans , Mice , Oligopeptides/pharmacology , Up-Regulation/geneticsABSTRACT
Proline, Glutamic acid-, and Leucine-rich Protein 1 (PELP1) is a proto-oncogene that modulates estrogen receptor (ER) signaling. PELP1 expression is upregulated in breast cancer, contributes to therapy resistance, and is a prognostic marker of poor survival. In a subset of breast tumors, PELP1 is predominantly localized in the cytoplasm and PELP1 participates in extranuclear signaling by facilitating ER interactions with Src and phosphoinositide 3-kinase (PI3K). However, the mechanism by which PELP1 extranuclear actions contributes to cancer progression and therapy resistance remains unclear. In this study, we discovered that PELP1 cross-talked with the serine/threonine protein kinase mTOR and modulated mTOR signaling. PELP1 knockdown significantly reduced the activation of mTOR downstream signaling components. Conversely, PELP1 overexpression excessively activated mTOR signaling components. We detected the presence of the mTOR signaling complex proteins in PELP1 immunoprecipitates. mTOR-targeting drugs (rapamycin and AZD8055) significantly reduced proliferation of PELP1-overexpressed breast cancer cells in both in vitro and in vivo xenograft tumor models. MCF7 cells that uniquely retain PELP1 in the cytoplasm showed resistance to hormonal therapy and mTOR inhibitors sensitized PELP1cyto cells to hormonal therapy in xenograft assays. Notably, immunohistochemical studies using xenograft tumors derived from PELP1 overexpression model cells showed increased mTOR signaling and inhibition of mTOR rendered PELP1-driven tumors to be highly sensitive to therapeutic inhibition. Collectively, our data identified the PELP1-mTOR axis as a novel component of PELP1 oncogenic functions and suggest that mTOR inhibitor(s) will be effective chemotherapeutic agents for downregulating PELP1 oncogenic functions.
Subject(s)
Breast Neoplasms/genetics , Cell Proliferation/genetics , Co-Repressor Proteins/metabolism , TOR Serine-Threonine Kinases/metabolism , Transcription Factors/metabolism , Breast Neoplasms/drug therapy , Breast Neoplasms/pathology , Co-Repressor Proteins/genetics , Drug Resistance, Neoplasm/genetics , Female , Gene Expression Regulation, Neoplastic , Gene Knockdown Techniques , Humans , MCF-7 Cells , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Mas , Receptors, Estrogen/metabolism , Signal Transduction/genetics , TOR Serine-Threonine Kinases/antagonists & inhibitors , TOR Serine-Threonine Kinases/genetics , Transcription Factors/geneticsABSTRACT
Proteasome inhibition is associated with substantial antitumor effects in preclinical models of multiple myeloma (MM) as well as in patients. However, results of recent clinical trials to evaluate the effect of the proteasome inhibitor Bortezomib (Velcade(®), also called PS-341) in MM patients have shown limited activity when used as a single agent. This underscores the need to find new efficacious and less toxic proteasome inhibitors. Recently, carfilzomib was approved for the treatment of refractory/relapsed MM and several new agents have been introduced into the clinic, including marizomib and MLN9708, and trials investigating these second-generation proteasome inhibitors have demonstrated promising results. We have recently synthesized a novel proteasome inhibitor, BU-32, and tested its growth inhibitory effects in different human MM cells including RPMI8226, MM.1S, MM.1R, and U266. In this study, we evaluate the efficacy of the novel proteasome inhibitor BU-32 (NSC D750499) using an in vitro MM model. BU-32 exhibits strong cytotoxicity in a panel of MM cell lines--RPMI8226, MM1S, MM1R, and U266. In addition, we demonstrate by proteasome inhibition assay that BU-32 potently inhibits the chymotryptic- and caspase-like activities of the 26S proteasome. We further show from Annexin V-FITC binding studies that BU-32, like Bortezomib, induces apoptosis in a panel of MM cell lines but the effect is more pronounced with BU-32-treated cells. Invasion assay with the MM.1S cell line indicates that BU-32 inhibits the invasiveness of myeloma cells. Results from our studies using real-time PCR array analyses show that BU-32 effectively downregulates an array of angiogenesis and inflammatory markers. Our results suggest that BU-32 might be a potential chemotherapeutic agent with promising antitumor activity for the treatment of MM.
Subject(s)
Multiple Myeloma/drug therapy , Proteasome Inhibitors/pharmacology , Pyrazoles/pharmacology , Animals , Apoptosis/drug effects , Caspase Inhibitors/pharmacology , Caspases/metabolism , Cell Line, Tumor , Cell Proliferation/drug effects , Chymotrypsin/antagonists & inhibitors , Chymotrypsin/metabolism , Down-Regulation/drug effects , Humans , Mice , Multiple Myeloma/enzymology , Multiple Myeloma/pathologyABSTRACT
Multiple myeloma (MM) is an incurable neoplasm characterized by devastating and progressive bone destruction. Standard chemotherapeutic agents have not been effective at significantly prolonging the survival of MM patients and these agents are typically associated with often severe, dose-limiting side effects. There is great need for methods to target the delivery of novel, effective cytotoxic agents specifically to bone, where myeloma cells reside. We have synthesized and evaluated the effects of the bone-targeted proteasome inhibitors PS-341-BP-1, PS-341-BP-2 and MG-262-BP on cell proliferation using the mouse 5TGM1 and human RPMI 8226 cell lines in vitro. The compounds exhibit strong cytotoxicity on MM cell lines and reduce the number of viable cells in a dose dependent manner.
Subject(s)
Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Multiple Myeloma/drug therapy , Multiple Myeloma/therapy , Proteasome Inhibitors/chemistry , Proteasome Inhibitors/pharmacology , Animals , Antineoplastic Agents/chemical synthesis , Bone Density Conservation Agents/chemical synthesis , Bone Density Conservation Agents/chemistry , Bone Density Conservation Agents/pharmacology , Bone and Bones/drug effects , Bone and Bones/pathology , Boronic Acids/chemical synthesis , Boronic Acids/chemistry , Boronic Acids/pharmacology , Cell Line, Tumor , Cell Proliferation/drug effects , Diphosphonates/chemical synthesis , Diphosphonates/chemistry , Diphosphonates/pharmacology , Humans , Mice , Multiple Myeloma/pathology , Proteasome Inhibitors/chemical synthesisABSTRACT
Recent work has identified changes in the metabolism of the aromatic amino acid tyrosine as a risk factor for diabetes and a contributor to the development of liver cancer. While these findings could suggest a role for tyrosine as a direct regulator of the behavior of cells and tissues, evidence for this model is currently lacking. Through the use of RNAi and genetic mutants, we identify tatn-1, which is the worm ortholog of tyrosine aminotransferase and catalyzes the first step of the conserved tyrosine degradation pathway, as a novel regulator of the dauer decision and modulator of the daf-2 insulin/IGF-1-like (IGFR) signaling pathway in Caenorhabditis elegans. Mutations affecting tatn-1 elevate tyrosine levels in the animal, and enhance the effects of mutations in genes that lie within the daf-2/insulin signaling pathway or are otherwise upstream of daf-16/FOXO on both dauer formation and worm longevity. These effects are mediated by elevated tyrosine levels as supplemental dietary tyrosine mimics the phenotypes produced by a tatn-1 mutation, and the effects still occur when the enzymes needed to convert tyrosine into catecholamine neurotransmitters are missing. The effects on dauer formation and lifespan require the aak-2/AMPK gene, and tatn-1 mutations increase phospho-AAK-2 levels. In contrast, the daf-16/FOXO transcription factor is only partially required for the effects on dauer formation and not required for increased longevity. We also find that the controlled metabolism of tyrosine by tatn-1 may function normally in dauer formation because the expression of the TATN-1 protein is regulated both by daf-2/IGFR signaling and also by the same dietary and environmental cues which influence dauer formation. Our findings point to a novel role for tyrosine as a developmental regulator and modulator of longevity, and support a model where elevated tyrosine levels play a causal role in the development of diabetes and cancer in people.
Subject(s)
Caenorhabditis elegans Proteins/genetics , Caenorhabditis elegans/growth & development , Longevity/genetics , Metabolic Networks and Pathways/genetics , Tyrosine Transaminase/genetics , Tyrosine/genetics , Animals , Animals, Genetically Modified , Caenorhabditis elegans/genetics , Forkhead Transcription Factors , Gene Expression Regulation, Developmental , Green Fluorescent Proteins , Humans , Insulin/metabolism , Insulin-Like Growth Factor I/metabolism , Larva/genetics , Larva/growth & development , Larva/metabolism , Mutation , RNA Interference , Receptor, Insulin/metabolism , Transcription Factors/genetics , Tyrosine/metabolismABSTRACT
Metastatic breast cancer is a life-threatening stage of cancer and is the leading cause of death in advanced breast cancer patients. Estrogen signaling and the estrogen receptor (ER) are implicated in breast cancer progression, and the majority of the human breast cancers start out as estrogen dependent. Accumulating evidence suggests that ER signaling is complex, involving coregulatory proteins and extranuclear actions. ER-coregualtory proteins are tightly regulated under normal conditions with miss expression primarily reported in cancer. Deregulation of ER coregualtors or ER extranuclear signaling has potential to promote metastasis in ER-positive breast cancer cells. This review summarizes the emerging role of ER signaling in promoting metastasis of breast cancer cells, discusses the molecular mechanisms by which ER signaling contributes to metastasis, and explores possible therapeutic targets to block ER-driven metastasis.
ABSTRACT
Breast cancer metastasis is a major clinical problem. The molecular basis of breast cancer progression to metastasis remains poorly understood. PELP1 is an estrogen receptor (ER) coregulator that has been implicated as a proto-oncogene whose expression is deregulated in metastatic breast tumors and whose expression is retained in ER-negative tumors. We examined the mechanism and significance of PELP1-mediated signaling in ER-negative breast cancer progression using two ER-negative model cells (MDA-MB-231 and 4T1 cells) that stably express PELP1-shRNA. These model cells had reduced PELP1 expression (75% of endogenous levels) and exhibited less propensity to proliferate in growth assays in vitro. PELP1 downregulation substantially affected migration of ER-negative cells in Boyden chamber and invasion assays. Using mechanistic studies, we found that PELP1 modulated expression of several genes involved in the epithelial mesenchymal transition (EMT), including MMPs, SNAIL, TWIST, and ZEB. In addition, PELP1 knockdown reduced the in vivo metastatic potential of ER-negative breast cancer cells and significantly reduced lung metastatic nodules in a xenograft assay. These results implicate PELP1 as having a role in ER-negative breast cancer metastasis, reveal novel mechanism of coregulator regulation of metastasis via promoting cell motility/EMT by modulating expression of genes, and suggest PELP1 may be a potential therapeutic target for metastatic ER-negative breast cancer.
Subject(s)
Breast Neoplasms/genetics , Breast Neoplasms/pathology , Carcinoma, Ductal, Breast/genetics , Carcinoma, Ductal, Breast/pathology , Co-Repressor Proteins/physiology , Transcription Factors/physiology , Animals , Breast Neoplasms/metabolism , Carcinoma, Ductal, Breast/metabolism , Cell Adhesion/drug effects , Cell Adhesion/genetics , Cell Proliferation/drug effects , Co-Repressor Proteins/antagonists & inhibitors , Co-Repressor Proteins/genetics , Co-Repressor Proteins/metabolism , Female , Gene Expression Regulation, Neoplastic/drug effects , Gene Knockdown Techniques , Humans , Mice , Mice, Nude , Neoplasm Metastasis , Proto-Oncogene Mas , RNA, Small Interfering/pharmacology , Receptors, Estrogen/metabolism , Transcription Factors/antagonists & inhibitors , Transcription Factors/genetics , Transcription Factors/metabolism , Tumor Cells, Cultured , Xenograft Model Antitumor AssaysABSTRACT
PURPOSE: Ovarian cancer remains a major threat to women's health, partly due to difficulty in early diagnosis and development of metastases. A critical need exists to identify novel targets that curb the progression and metastasis of ovarian cancer. In this study, we examined whether the nuclear receptor coregulator PELP1 (proline-, glutamic acid-, leucine-rich protein-1) contributes to progression and metastatic potential of ovarian cancer cells and determined whether blocking of the PELP1 signaling axis had a therapeutic effect. EXPERIMENTAL DESIGN: Ovarian cancer cells stably expressing PELP1-shRNA (short hairpin RNA) were established. Fluorescent microscopy, Boyden chamber, invasion assays, wound healing, and zymography assays were performed to examine the role of PELP1 in metastasis. Expression analysis of the model cells was conducted using tumor metastasis microarray to identify PELP1 Target genes. Therapeutic potential of PELP1-siRNA in vivo was determined using a nanoliposomal formulation of PELP1-siRNA-DOPC (1,2-dioleoyl-sn-glycero-3-phosphatidylcholine) administered systemically in a xenograft model. RESULTS: PELP1 knockdown caused cytoskeletal defects and significantly affected the migratory potential of ovarian cancer cells. Microarray analysis revealed that PELP1 affected the expression of selective genes involved in metastasis including Myc, MTA1, MMP2, and MMP9. Zymography analysis confirmed that PELP1 knockdown caused a decrease in the activation of matrix metalloproteases (MMP) 2 and MMP9. Compared with control siRNA-DOPC-treated mice, animals injected with PELP1-siRNA-DOPC had 54% fewer metastatic tumor nodules, exhibited a 51% reduction in tumor growth and an 84% reduction in ascites volume. CONCLUSION: The results suggest that PELP1 signaling axis is a potential druggable target and liposomal PELP1-siRNA-DOPC could be used as a novel drug to prevent or treat ovarian metastasis.
Subject(s)
Co-Repressor Proteins/genetics , Ovarian Neoplasms/genetics , RNA Interference , RNA, Small Interfering/genetics , Transcription Factors/genetics , Xenograft Model Antitumor Assays , Animals , Blotting, Western , Cell Line, Tumor , Cell Proliferation , Co-Repressor Proteins/metabolism , Cytoskeleton/metabolism , Female , Gene Expression Profiling , Humans , Liposomes/administration & dosage , Liposomes/chemistry , Matrix Metalloproteinase 2/genetics , Matrix Metalloproteinase 2/metabolism , Matrix Metalloproteinase 9/genetics , Matrix Metalloproteinase 9/metabolism , Mice , Mice, Nude , Neoplasm Metastasis , Oligonucleotide Array Sequence Analysis , Ovarian Neoplasms/pathology , Ovarian Neoplasms/therapy , Phosphatidylcholines/chemistry , RNA, Small Interfering/chemistry , Reverse Transcriptase Polymerase Chain Reaction , Transcription Factors/metabolism , Tumor BurdenABSTRACT
INTRODUCTION: Proteasome inhibition provides an attractive approach to cancer therapy and may have application in the treatment of breast cancer. However, results of recent clinical trials to evaluate the effect of the proteasome inhibitor Bortezomib (Velcade, also called PS-341) in metastatic breast cancer patients have shown limited activity when used as a single agent. This underscores the need to find new and more efficacious proteasome inhibitors. In this study, we evaluate the efficacy of the novel proteasome inhibitor BU-32 (NSC D750499-S) using in vitro and in vivo breast cancer models. METHODS: We have recently synthesized a novel proteasome inhibitor (BU-32) and tested its growth inhibitory effects in different breast cancer cells including MCF-7, MDA-MB-231, and SKBR3 by in vitro cytotoxicity and proteasomal inhibition assays. The apoptotic potential of BU32 was tested using flow cytometry and analyzing cell cycle regulatory proteins. In vivo tumor xenograft studies for solid tumor as well as tumor metastasis were conducted using MDA-MB-231-GFP cells. RESULTS: We report for the first time that BU-32 exhibits strong cytotoxicity in a panel of cell lines: MDA-MB-231 (IC50 = 5.8 nM), SKBR3 (IC50 = 5.7 nM) and MCF-7 cells (IC50 = 5.8 nM). It downregulates a wide array of angiogenic marker genes and upregulates apoptotic markers, including Bid and Bax. Incubation of MDA-MB-231 cells with BU-32 results in the accumulation of cell cycle inhibitor proteins p21 and p27 and stabilization of the tumor suppressor protein p53. Studies in in vivo solid tumor and metastasis models show significant effect with a 0.06 mg/kg dose of BU-32 and marked reduction in tumor burden in the skeleton. CONCLUSIONS: We have shown that BU-32 is effective in cultured breast cancer cells and in breast cancer xenografts. The results suggest its potential benefit in breast cancer treatment.
Subject(s)
Breast Neoplasms/drug therapy , Protease Inhibitors/pharmacology , Proteasome Inhibitors , Pyrazoles/pharmacology , Animals , Apoptosis/drug effects , Bone Neoplasms/drug therapy , Bone Neoplasms/enzymology , Bone Neoplasms/genetics , Bone Neoplasms/secondary , Boronic Acids/pharmacology , Bortezomib , Breast Neoplasms/enzymology , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Cell Growth Processes/drug effects , Cell Line, Tumor , Chymotrypsin/antagonists & inhibitors , Chymotrypsin/metabolism , Female , Gene Expression Regulation, Neoplastic/drug effects , Humans , Mice , Mice, Inbred BALB C , Mice, Nude , NF-kappa B/biosynthesis , NF-kappa B/genetics , Proteasome Endopeptidase Complex/metabolism , Pyrazines/pharmacology , Xenograft Model Antitumor AssaysABSTRACT
In visceral leishmaniasis (VL), oxidative assault on erythrocytes perturbs their cellular environment and makes them vulnerable to premature hemolysis. In this study, we assessed the contribution of oxidation-induced modifications of hemoglobin and membrane protein band 3 in the reduced survival of red cells in VL. Oxidative transformation of oxyhemoglobin to hemichrome enhanced its interaction with erythrocyte membrane in the infected animals. Association between denatured globin and band 3 contributed to the formation of insoluble copolymer of macromolecular dimension. Disulfide bonding appeared to be necessary in the making of high molecular weight aggregates during copolymerization. Hemichrome induced clustering of band 3 promoted generation of epitopes on erythrocyte cell surface. This provided a signal favoring immunologic recognition of redistributed band 3 by autologous IgG followed by erythrophagocytosis. An eventual outcome of the sequence of events pointed to early removal of affected red cells from circulation during the disease.
Subject(s)
Anion Exchange Protein 1, Erythrocyte/metabolism , Erythrocytes/metabolism , Hemoglobins/metabolism , Hemolysis/physiology , Leishmaniasis, Visceral/metabolism , Oxidation-Reduction , Phagocytosis/physiology , Animals , Cricetinae , Cricetulus , Erythrocyte Membrane/metabolism , Erythrocytes/cytology , Hemeproteins/metabolism , Macrophages/metabolismABSTRACT
Flavonoids are a broad class of plant phenolics that are known to possess a well-established protective effect against membrane lipoperoxidative damages. Oxidative damage of erythrocytes has been implicated in the reduced survival of erythrocytes during leishmanial infection. This study reveals the efficacy of five naturally occurring flavonoids in arresting the development of anemia during the postinfection period. Among the compounds studied, quercetin was most successful in inhibiting the oxidation of proteins and lipids on the red cell membranes of infected animals. Apart from its antianemic property, quercetin also seemed to be highly potent in lowering the parasite load in the spleen. Combination therapy of quercetin with the antileishmanial drug stibanate produced a better decay of .OH in the erythrocytes of the infected animals compared to that induced by quercetin or drug treatment alone. Similar results were obtained in successful prevention of proteolytic degradation resulting in an aversion to early lysis of red cells after simultaneous treatment with quercetin and stibanate. Subsequent studies demonstrated the therapeutic efficacy of the combination treatment in the abatement of both anemia and parasitemia under the diseased condition.
Subject(s)
Anemia/drug therapy , Antioxidants/therapeutic use , Leishmaniasis, Visceral/drug therapy , Quercetin/therapeutic use , Anemia/complications , Animals , Cricetinae , Erythrocyte Membrane/metabolism , Hemoglobins/metabolism , Hydroxyl Radical/metabolism , Leishmania donovani/isolation & purification , Leishmaniasis, Visceral/complications , Leishmaniasis, Visceral/parasitology , Membrane Proteins/metabolism , Mesocricetus , Osmotic Pressure , Oxidative StressABSTRACT
Membrane destabilization in erythrocytes plays an important role in the premature hemolysis and development of anemia during visceral leishmaniasis (VL). Marked degradation of the anion channel protein band 3 is likely to allow modulation of anion flux across the red cell membrane in infected animals. The present study describes the effect of structural modification of band 3 on phosphate transport in VL using (31)P NMR. The result showed progressive decrease in the rate and extent of phosphate transport during the post-infection period. Interdependence between the intracellular ionic levels seems to be a determining factor in the regulation of anion transport across the erythrocyte membrane in control and infected conditions. Infection-induced alteration in band 3 made the active sites of transport more susceptible to binding with amino reactive agents. Inhibition of transport by oxidation of band 3 and subsequent reversal by reduction using dithiothreitol suggests the contribution of sulfhydryl group in the regulation of anion exchange across the membrane. Quantitation of sulfhydryl groups in the anion channel protein showed the inhibition to be closely related to the decrease of sulfhydryl groups in the infected hamsters. Downregulation of phosphate transport during leishmanial infection may be ascribed to the sulfhydryl modification of band 3 resulting in the impaired functioning of this protein under the diseased condition.