Renal cell carcinoma (RCC) is resistant to traditional cancer therapies, and metastatic RCC (mRCC) is incurable. The shortcomings in current therapeutic options for patients with mRCC provide the rationale for the development of novel treatment protocols. TNF-related apoptosis-inducing ligand (TRAIL) has proven to be a potent inducer of tumor cell death in vitro and in vivo, and a number of TRAIL death receptor agonists (recombinant TRAIL or TRAIL death receptor-specific mAb) has been developed and tested clinically. Unfortunately the clinical efficacy of TRAIL has been underwhelming and is likely due to a number of possible mechanisms that render tumors resistant to TRAIL, prompting the search for drugs that increase tumor cell susceptibility to TRAIL. The objective of this study was to determine the effectiveness of combining the diterpene triepoxide triptolide, or its water-soluble prodrug, Minnelide, with TRAIL receptor agonists against RCC in vitro or in vivo, respectively. TRAIL-induced apoptotic death of human RCC cells was increased in the presence of triptolide. The triptolide-induced sensitization was accompanied by increased TRAIL-R2 (DR5) and decreased HSP70 expression. In vivo treatment of mice bearing orthotopic RCC (Renca) tumors showed the combination of Minnelide and agonistic anti-DR5 mAb significantly decreased tumor burden and increased animal survival compared to either therapy alone. Our data suggest triptolide/Minnelide sensitizes RCC cells to TRAIL-induced apoptosis through altered TRAIL death receptor and heat shock protein expression.
TRAIL; renal cell carcinoma; triptolide; apoptosis; death receptor
Despite significant progress in diagnostics and therapeutics, over fifty thousand patients die from colorectal cancer annually. Hence there is urgent need for new lines of treatment. Triptolide, a natural compound isolated from the Chinese herb Tripterygium wilfordii, is effective against multiple cancers. We have synthesized a water soluble analog of triptolide, named Minnelide, which is currently in phase I trial against pancreatic cancer. The aims of the current study were to evaluate whether triptolide/Minnelide is effective against colorectal cancer and to elucidate the mechanism by which triptolide induces cell death in colorectal cancer.
Efficacy of Minnelide was evaluated in subcutaneous xenograft and liver metastasis model of colorectal cancer. For mechanistic studies colon cancer cell lines HCT116 and HT29 were treated with triptolide and the effect on viability, caspase activation, annexin positivity, lactate dehydrogenase(LDH) release and cell cycle progression was evaluated. Effect of triptolide on E2F transcriptional activity, mRNA levels of E2F dependent genes, E2F1-Rb binding and proteins levels of regulator of G1-S transition was also measured. DNA binding of E2F1 was evaluated by chromatin immunoprecipitation assay.
Triptolide decreased colon cancer cell viability in a dose- and time-dependent fashion. Minnelide markedly inhibited the growth of colon cancer in the xenograft and liver metastasis model of colon cancer and more than doubles the median survival of animals with liver metastases from colon cancer. Mechanistically we demonstrate that at low concentrations, triptolide induces apoptotic cell death but at higher concentrations it induces cell cycle arrest. Our data suggest that triptolide is able to induce G1 cell cycle arrest by inhibiting transcriptional activation of E2F1. Our data also show that triptolide downregulates E2F activity by potentially modulating events downstream of DNA binding.
Triptolide and Minnelide are effective against colon cancer in multiple pre-clinical models.
Triptolide; Minnelide; colon cancer; cell cycle; apoptosis; cell death; E2F transcription factor
Pancreatic cancer is the 10th leading cause of all new cancer cases for men and the fourth leading cause of death across genders, having very poor prognosis and survival rates. The current standard of care Gemcitabine fails to add any survival benefit for this disease (www.cancer.gov). Though the incidence of pancreatic cancer is found to be higher in developed countries, the aggressive biology of the cancer, its high rate of recurrence and chemo-resistance make it a formidable disease in all parts of the globe. Hepatocellular carcinoma (HCC) or liver cancer, on the other hand affects almost 750,000 people world wide with 84% of the cases coming from underdeveloped or developing countries. Our studies show that Minnelide, a water soluble pro-drug of triptolide (active compound from a chinese herb) is very effective against a number of malignant diseases. The current study discusses the efficacy of this compound in pancreatic and liver cancer.
Pancreatic cancer has a five year survival rate of less than 5%, partly due to limited chemotherapeutic options, thereby highlighting the need for novel therapies. Triptolide, a diterpene triepoxide derived from a Chinese herb has shown great promise in preclinical testing against pancreatic cancer using immune compromised animals.
In this study, we tested the ability of triptolide to induce cell death in cell lines derived from a primary tumor and adjacent liver metastases of immuno-competent animals (KRasG12D; Trp52R172H; Pdx-1 Cre (KPC)). Both cell lines were more aggressive in their ability to form tumors when compared to other pancreatic cancer cell lines, and showed constitutive activation of the NFkB pathway. Triptolide induced apoptotic cell death in both cell lines, as evidenced by decreased cell viability and increased caspase 3/7 activity, Annexin V positivity, and increased TUNEL positivity in tumors from KPC animals treated with Minnelide. Additionally, triptolide decreased levels of HSP70, its transcription factor HSF1, and the anti-apoptotic proteins Bcl-xL, Bcl-2 and Mcl-1, known to be up-regulated in pancreatic cancer.
The ability of triptolide to cause cell death in cell lines derived from immune-competent animals further validates its potential as a novel agent against pancreatic cancer.
Pancreatic Cancer; Genetically engineered mouse model; Triptolide; Cell death
Pancreatic Ductal Adenocarcinoma (PDAC) is a devastating disease hallmarked by limited patient survival. Resistance to chemotherapy, a major cause of treatment failure in PDAC patients, is often attributed to Cancer Stem Cells (CSCs). Pancreatic CSCs are a small subset of quiescent cells within a tumor represented by surface markers like CD133. These cells are responsible not only for tumor recurrence, but also poor prognosis based on their “stem-like” characteristics. At present, conventional therapy is directed towards rapidly dividing PDAC cells and thus fails to target the CSC population.
MIA PaCa-2, S2-013 and AsPC-1 were treated with 12.5 nM triptolide (12 T cells) for 7 days. The surviving cells were recovered briefly in drug-free growth media and then transferred to Cancer Stem cell Media (CSM). As a control, untreated cells were also transferred to CSM media (CSM). The 12 T and CSM cells were tested for stemness properties using RNA and protein markers. Low numbers of CSM and 12 T cells were implanted subcutaneously in athymic nude mice to study their tumorigenic potential. 12 T and CSM cells were sorted for CD133 expression and assayed for their colony forming ability and sphere forming ability. Invasiveness of 12 T cells, CSM and MIA PaCa-2 were compared using Boyden chamber assays.
Treated 12 T cells displayed increased expression of the surface marker CD133 and the drug transporter ABCG2 compared to untreated cells (CSM cells). Both 12 T and CSM cells formed subcutaneous tumors in mice confirming their tumor-initiating properties. When tested for invasion, 12 T cells had increased invasiveness compared to CSM cells. CD133+ cells in both CSM and 12 T showed greater colony and sphere forming ability compared to CD133− cells from each group. Consistent with these data, when injected subcutaneously in mice, CD133− cells from CSM or 12 T did not form any tumors whereas CD133+ cells from both groups showed tumor formation at a very low cell number. Despite pre-exposure to triptolide in 12 T CD133+ cells, treatment of tumors formed by these cells with Minnelide, a triptolide pro-drug, showed significant tumor regression.
Our results indicated that triptolide enhanced and enriched the “stemness” in the PDAC cell lines at a low dose of 12.5 nM, but also resulted in the regression of tumors derived from these cells.
Electronic supplementary material
The online version of this article (doi:10.1186/s12943-015-0470-6) contains supplementary material, which is available to authorized users.
The tumor necrosis factor related apoptosis-inducing ligand (TRAIL) causes cancer cell death, but many cancers, including pancreatic cancer, are resistant to TRAIL therapy. A combination of TRAIL and the diterpene triepoxide, triptolide, is effective in inducing pancreatic cancer cell death. Triptolide increases levels of death receptor DR5 and decreases the pro-survival FLICE-like inhibitory protein (c-FLIP), which contribute to the activation of caspase-8. This combination further causes both lysosomal and mitochondrial membrane permeabilization, resulting in cell death. Our study provides a mechanism by which triptolide sensitizes TRAIL resistant cells, which may become a novel therapeutic strategy against pancreatic cancer.
TRAIL; pancreatic cancer; triptolide; apoptosis; death receptor
Pancreatic adenocarcinoma is the fourth leading cause for cancer-related mortality with a survival rate of less than 5%. Late diagnosis and lack of effective chemotherapeutic regimen contribute to these grim survival statistics. Relapse of any tumor is largely attributed to the presence of tumor-initiating cells (TIC) or cancer stem cells (CSC). These cells are considered as hurdles to cancer therapy as no known chemotherapeutic compound is reported to target them. Thus, there is an urgent need to develop a TIC-targeted therapy for pancreatic cancer.
We isolated CD133+ cells from a spontaneous PDAC mouse model and studied both surface expression, molecular markers of pancreatic TICs. We also studied tumor initiation properties by implanting low numbers of CD133+ cells in immune competent mice. Effect of Minnelide, a drug currently under Phase I clinical trial, was studied on the tumors derived from the CD133+ cells.
Our study showed for the first time that CD133+ population demonstrated all the molecular markers for pancreatic TIC. These cells initiated tumors in immunocompetent mouse models and showed increased expression of pro-survival and pro-invasive proteins compared to the CD133− non-TIC population. Our study further showed that Minnelide, was very efficient in downregulating both CD133− and CD133+ population in the tumors, resulting in a 60% decrease in tumor volume compared to the untreated ones.
As Minnelide is currently under Phase I clinical trial, its evaluation in reducing tumor burden by decreasing TIC as well as non-TIC population suggests its potential as an effective therapy.
Triptolide; Minnelide; pancreatic tumor initiating cells; CD133; NF-kB
CD133 has been implicated as a cancer stem cell (CSC) surface marker in several malignancies including pancreatic cancer. However, the functional role of this surface glycoprotein in the cancer stem cell remains elusive. In this study, we determined that CD133 overexpression induced “stemness” properties in MIA-PaCa2 cells along with increased tumorigenicity, tumor progression, and metastasis in vivo. Additionally, CD133 expression induced epithelial-mesenchymal transition (EMT) and increased in vitro invasion. EMT induction and increased invasiveness were mediated by NF-κB activation, as inhibition of NF-κB mitigated these effects. This study showed that CD133 expression contributes to pancreatic cancer “stemness,” tumorigenicity, EMT induction, invasion, and metastasis.
CD133; Pancreatic Cancer; invasion; metastasis; NF-kB
Osteosarcoma is the most common bone cancer in children and adolescents with a five-year survival rate of about 70%. In this study, we have evaluated the preclinical therapeutic efficacy of the novel synthetic drug, Minnelide, a prodrug of triptolide on osteosarcoma. Triptolide was effective in significantly inducing apoptosis in all osteosarcoma cell lines tested but had no significant effect on the human osteoblast cells. Notably, Minnelide treatment significantly reduced tumor burden and lung metastasis in the orthotopic and lung colonization models. Triptolide/Minnelide effectively downregulated the levels of pro-survival proteins such as heat shock proteins, cMYC, survivin and targets NF-κB pathway.
Triptolide; Minnelide; osteosarcoma; heat shock proteins; NF-κB
Nucleotide-sugar transporters (NSTs) transport activated sugars (e.g. UDP-GlcNAc) from the cytosol to the lumen of the endoplasmic reticulum or Golgi apparatus where they are used to make glycoproteins and glycolipids. UDP-Glc is an important component of the N-glycan-dependent quality control (QC) system for protein folding. Because Entamoeba has this QC system while Giardia does not, we hypothesized that transfected Giardia might be used to identify the UDP-Glc transporter of Entamoeba. Here we show Giardia membranes transport UDP-GlcNAc and have apyrases, which hydrolyze nucleoside-diphosphates to make the antiporter nucleoside-monophosphate. The only NST of Giardia (GlNst), which we could identify, transports UDP-GlcNAc in transfected Saccharomyces and is present in perinuclear and peripheral vesicles and increases in expression during encystation. Entamoeba membranes transport three nucleotide-sugars (UDP-Gal, UDP-GlcNAc, and UDP-Glc), and Entamoeba has three NSTs, one of which has been shown previously to transport UDP-Gal (EhNst1). Here we show recombinant EhNst2 transports UDP-Glc in transfected Giardia, while recombinant EhNst3 transports UDP-GlcNAc in transfected Saccharomyces. In summary, all three NSTs of Entamoeba and the single NST of Giardia have been molecularly characterized, and transfected Giardia provide a new system for testing heterologous UDP-Glc transporters.
Giardia; Entamoeba; Nucleotide sugar transporter; Apyrase; transfection; N-glycan-dependent quality control of protein folding
Pancreatic ductal adenocarcinoma (PDAC), one of the deadliest malignancies, is resistant to current chemotherapies. We previously showed that triptolide inhibits PDAC cell growth in vitro and blocks metastatic spread in vivo. Triptolide downregulates heat shock protein 70 (HSP70), a molecular chaperone upregulated in several tumor types. This study investigates the mechanism by which triptolide inhibits HSP70. As microRNAs (miRNAs) are becoming increasingly recognized as negative regulators of gene expression, we tested whether triptolide regulates HSP70 via miRNAs. Here we show that triptolide, as well as quercetin but not gemcitabine, upregulated miR-142-3p in PDAC cells (MIA PaCa-2, Capan-1, and S2-013). Ectopic expression of miR-142-3p inhibited cell proliferation, measured by Electric Cell-substrate Impedance Sensing, and decreased HSP70 expression, measured by real-time PCR and immunoblotting, compared with controls. We demonstrated that miR-142-3p directly binds to the 3’UTR of HSP70, and that this interaction is important as HSP70 overexpression rescued miR-142-3p-induced cell death. We found that miR-142-3p regulates HSP70 independently of heat shock factor 1. Furthermore, Minnelide, a water soluble prodrug of triptolide, induced the expression of miR-142-3p in vivo. This is the first description of an miRNA-mediated mechanism of HSP70 regulation in cancer, making miR-142-3p an attractive target for PDAC therapeutic intervention.
pancreatic cancer; triptolide; miR-142-3p; gene expression profiling; HSP70
Minnelide, a pro-drug of triptolide, has recently emerged as a potent anticancer agent. The precise mechanisms of its cytotoxic effects remain unclear.
Cell viability was studied using CCK8 assay. Cell proliferation was measured real-time on cultured cells using Electric Cell Substrate Impedence Sensing (ECIS). Apoptosis was assayed by Caspase activity on cultured lung cancer cells and TUNEL staining on tissue sections. Expression of pro-survival and anti-apoptotic genes (HSP70, BIRC5, BIRC4, BIRC2, UACA, APAF-1) was estimated by qRTPCR. Effect of Minnelide on proliferative cells in the tissue was estimated by Ki-67 staining of animal tissue sections.
In this study, we investigated in
vitro and in
vivo antitumor effects of triptolide/Minnelide in non-small cell lung carcinoma (NSCLC). Triptolide/Minnelide exhibited anti-proliferative effects and induced apoptosis in NSCLC cell lines and NSCLC mouse models. Triptolide/Minnelide significantly down-regulated the expression of pro-survival and anti-apoptotic genes (HSP70, BIRC5, BIRC4, BIRC2, UACA) and up-regulated pro-apoptotic APAF-1 gene, in part, via attenuating the NF-κB signaling activity.
In conclusion, our results provide supporting mechanistic evidence for Minnelide as a potential in NSCLC.
Pancreatic cancer is one of the most lethal human malignancies, with an all-stage 5-year survival of <5%, mainly due to lack of effective available therapies. Cancer cell survival is dependent upon up-regulation of the pro-survival response, mediated by anti-apoptotic proteins such as Mcl-1.
Here we show that over-expression of Mcl-1 in pancreatic patient tumor samples is linked to advancement of the disease. We have previously shown that triptolide, a diterpene triepoxide, is effective both in vitro and in vivo, in killing pancreatic cancer cells. Decrease of Mcl-1 levels, either by siRNA or by treatment with triptolide results in cell death. Using pancreatic cancer cell lines, we have shown that miR-204, a putative regulator of Mcl-1, is repressed in cancer cell lines compared to normal cells. Over-expression of miR-204, either by a miR-204 mimic, or by triptolide treatment results in a decrease in Mcl-1 levels, and a subsequent decrease in cell viability. Using luciferase reporter assays, we confirmed the ability of miR-204 to down-regulate Mcl-1 by directly binding to the Mcl-1 3’ UTR. Using human xenograft samples treated with Minnelide, a water soluble variant of triptolide, we have shown that miR-204 is up-regulated and Mcl-1 is down-regulated in treated vs. control tumors.
Triptolide mediated miR-204 increase causes pancreatic cancer cell death via loss of Mcl-1.
Pancreatic cancer; miR 204; Mcl-1; Triptolide; Cell death
Pancreatic cancer is one of the most lethal human malignancies with an all-stage 5-year survival frequency of <5%, which highlights the urgent need for more effective therapeutic strategies. We have previously shown that triptolide, a diterpenoid, is effective against pancreatic cancer cells in vitro as well as in vivo. However, triptolide is poorly soluble in water, limiting its clinical use. We therefore synthesized a water-soluble analog of triptolide, named Minnelide. The efficacy of Minnelide was tested both in vitro and in multiple independent yet complementary in vivo models of pancreatic cancer: an orthotopic model of pancreatic cancer using human pancreatic cancer cell lines in athymic nude mice, a xenograft model where human pancreatic tumors were transplanted into severe combined immunodeficient mice, and a spontaneous pancreatic cancer mouse model (KRasG12D; Trp53R172H; Pdx-1Cre). In these multiple complementary models of pancreatic cancer, Minnelide was highly effective in reducing pancreatic tumor growth and spread, and improving survival. Together, our results suggest that Minnelide shows promise as a potent chemotherapeutic agent against pancreatic cancer, and support the evaluation of Minnelide in clinical trials against this deadly disease.
MUC1 is a type I transmembrane glycoprotein aberrantly overexpressed in various cancer cells including pancreatic cancer. The cytosolic end of MUC1 (MUC1-c) is extensively involved in a number of signaling pathways. MUC1-c is reported to inhibit apoptosis in a number of cancer cells, but the mechanism of inhibition is unclear.
Expression of MUC1-c was studied in the pancreatic cancer cell line MIAPaCa-2 at the RNA level by using qRTPCR and at the protein level by Western blotting. MUC1-c expression was inhibited either by siRNA or by a specific peptide inhibitor, GO-201. Effect of MUC1-c inhibition on viability and proliferation and lysosomal permeabilization were studied. Association of MUC1-c with HSP70 was detected by co-immunoprecipitation of MUC1-c and HSP70. Localization of MUC1-c in cellular organelles was monitored by immunofluorescence and with immuno- blotting by MUC1-c antibody after subcellular fractionation.
Inhibition of MUC1-c by an inhibitor (GO-201) or siRNA resulted in reduced viability and reduced proliferation of pancreatic cancer cells. Furthermore, GO-201, the peptide inhibitor of MUC1-c, was effective in reducing tumor burden in pancreatic cancer mouse model. MUC1-c was also found to be associated with HSP70 in the cytosol, although a significant amount of MUC1 was also seen to be present in the lysosomes. Inhibition of MUC1 expression or activity showed an enhanced Cathepsin B activity in the cytosol, indicating lysosomal permeabilization. Therefore this study indicates that MUC1-c interacted with HSP70 in the cytosol of pancreatic cancer cells and localized to the lysosomes in these cells. Further, our results showed that MUC1-c protects pancreatic cancer cells from cell death by stabilizing lysosomes and preventing release of Cathepsin B in the cytosol.
Entamoeba histolytica, the protist that causes amebic dysentery and liver abscess, has a truncated Asn-linked glycan (N-glycan) precursor composed of seven sugars (Man5GlcNAc2). Here, we show that glycoproteins with unmodified N-glycans are aggregated and capped on the surface of E. histolytica trophozoites by the antiretroviral lectin cyanovirin-N and then replenished from large intracellular pools. Cyanovirin-N cocaps the Gal/GalNAc adherence lectin, as well as glycoproteins containing O-phosphodiester-linked glycans recognized by an anti-proteophosphoglycan monoclonal antibody. Cyanovirin-N inhibits phagocytosis by E. histolytica trophozoites of mucin-coated beads, a surrogate assay for amebic virulence. For technical reasons, we used the plant lectin concanavalin A rather than cyanovirin-N to enrich secreted and membrane proteins for mass spectrometric identification. E. histolytica glycoproteins with occupied N-glycan sites include Gal/GalNAc lectins, proteases, and 17 previously hypothetical proteins. The latter glycoproteins, as well as 50 previously hypothetical proteins enriched by concanavalin A, may be vaccine targets as they are abundant and unique. In summary, the antiretroviral lectin cyanovirin-N binds to well-known and novel targets on the surface of E. histolytica that are rapidly replenished from large intracellular pools.
We are interested in asparagine-linked glycans (N-glycans) of Plasmodium falciparum and Toxoplasma gondii, because their N-glycan structures have been controversial and because we hypothesize that there might be selection against N-glycans in nucleus-encoded proteins that must pass through the endoplasmic reticulum (ER) prior to threading into the apicoplast. In support of our hypothesis, we observed the following. First, in protists with apicoplasts, there is extensive secondary loss of Alg enzymes that make lipid-linked precursors to N-glycans. Theileria makes no N-glycans, and Plasmodium makes a severely truncated N-glycan precursor composed of one or two GlcNAc residues. Second, secreted proteins of Toxoplasma, which uses its own 10-sugar precursor (Glc3Man5GlcNAc2) and the host 14-sugar precursor (Glc3Man9GlcNAc2) to make N-glycans, have very few sites for N glycosylation, and there is additional selection against N-glycan sites in its apicoplast-targeted proteins. Third, while the GlcNAc-binding Griffonia simplicifolia lectin II labels ER, rhoptries, and surface of plasmodia, there is no apicoplast labeling. Similarly, the antiretroviral lectin cyanovirin-N, which binds to N-glycans of Toxoplasma, labels ER and rhoptries, but there is no apicoplast labeling. We conclude that possible selection against N-glycans in protists with apicoplasts occurs by eliminating N-glycans (Theileria), reducing their length (Plasmodium), or reducing the number of N-glycan sites (Toxoplasma). In addition, occupation of N-glycan sites is markedly reduced in apicoplast proteins versus some secretory proteins in both Plasmodium and Toxoplasma.
Cryptosporidium parvum oocysts, which are spread by the fecal-oral route, have a single, multilayered wall that surrounds four sporozoites, the invasive form. The C. parvum oocyst wall is labeled by the Maclura pomifera agglutinin (MPA), which binds GalNAc, and the C. parvum wall contains at least two unique proteins (Cryptosporidium oocyst wall protein 1 [COWP1] and COWP8) identified by monoclonal antibodies. C. parvum sporozoites have on their surface multiple mucin-like glycoproteins with Ser- and Thr-rich repeats (e.g., gp40 and gp900). Here we used ruthenium red staining and electron microscopy to demonstrate fibrils, which appear to attach or tether sporozoites to the inner surface of the C. parvum oocyst wall. When disconnected from the sporozoites, some of these fibrillar tethers appear to collapse into globules on the inner surface of oocyst walls. The most abundant proteins of purified oocyst walls, which are missing the tethers and outer veil, were COWP1, COWP6, and COWP8, while COWP2, COWP3, and COWP4 were present in trace amounts. In contrast, MPA affinity-purified glycoproteins from C. parvum oocysts, which are composed of walls and sporozoites, included previously identified mucin-like glycoproteins, a GalNAc-binding lectin, a Ser protease inhibitor, and several novel glycoproteins (C. parvum MPA affinity-purified glycoprotein 1 [CpMPA1] to CpMPA4). By immunoelectron microscopy (immuno-EM), we localized mucin-like glycoproteins (gp40 and gp900) to the ruthenium red-stained fibrils on the inner surface wall of oocysts, while antibodies to the O-linked GalNAc on glycoproteins were localized to the globules. These results suggest that mucin-like glycoproteins, which are associated with the sporozoite surface, may contribute to fibrils and/or globules that tether sporozoites to the inner surface of oocyst walls.
Chromatin in the nucleus of all eukaryotes is organized into a system of loops and domains. These loops remain fastened at their bases to the fundamental framework of the nucleus, the matrix or the scaffold. The DNA sequences which anchor the bases of the chromatin loops to the matrix are known as Scaffold/Matrix Attachment Regions or S/MARs. Though S/MARs have been studied in yeast and higher eukaryotes and they have been found to be associated with gene organization and regulation of gene expression, they have not been reported in protists like Giardia. Several tools have been discovered and formulated to predict S/MARs from a genome of a higher eukaryote which take into account a number of features. However, the lack of a definitive consensus sequence in S/MARs and the randomness of the protozoan genome in general, make it a challenge to predict and identify such sequences from protists.
Here, we have analysed the Giardia genome for the probable S/MARs predicted by the available computational tools; and then shown these sequences to be physically associated with the nuclear matrix. Our study also reflects that while no single computational tool is competent to predict such complex elements from protist genomes, a combination of tools followed by experimental verification is the only way to confirm the presence of these elements from these organisms.
This is the first report of S/MAR elements from the protozoan parasite Giardia lamblia. This initial work is expected to lay a framework for future studies relating to genome organization as well as gene regulatory elements in this parasite.
O-Linked N-acetylglucosaminyltransferase (OGT) catalyzes the transfer of a single GlcNAc to the Ser or Thr of nucleocytoplasmic proteins. OGT activity, which may compete with that of kinases, is involved in signaling in animals and plants, and abnormalities in OGT activities have been associated with type 2 diabetes. Here, we show that ogt genes that predict enzymes with characteristic tetratricopeptide repeats and a spindly domain are present in some protists (Giardia, Cryptosporidium, Toxoplasma, and Dictyostelium) but are absent from the majority of protists examined (e.g., Plasmodium, Trypanosoma, Entamoeba, and Trichomonas). Similarly, ogt genes are present in some fungi but are absent from numerous other fungi, suggesting that secondary loss is an important contributor to the evolution of ogt genes. Nucleocytosolic extracts of Giardia and Cryptosporidium show OGT activity, and recombinant Giardia and Cryptosporidium OGTs are active in yeast and bacteria, respectively. These results suggest the possibility that O-GlcNAc modification of nucleocytosolic proteins also has function(s) in simple eukaryotes.
Cryptosporidium; evolution; Giardia; O-GlcNAc transferase; recombinant expression
Infections with Giardia lamblia, Entamoeba histolytica, and Trichomonas vaginalis, which cause diarrhea, dysentery, and vaginitis, respectively, are each treated with metronidazole. Here we show that Giardia, Entamoeba, and Trichomonas have oxygen-insensitive nitroreductase (ntr) genes which are homologous to those genes that have nonsense mutations in metronidazole-resistant Helicobacter pylori isolates. Entamoeba and Trichomonas also have nim genes which are homologous to those genes expressed in metronidazole-resistant Bacteroides fragilis isolates. Recombinant Giardia, Entamoeba, and Trichomonas nitroreductases used NADH rather than the NADPH used by Helicobacter, and two recombinant Entamoeba nitroreductases increased the metronidazole sensitivity of transformed Escherichia coli strains. Conversely, the recombinant nitroimidazole reductases (NIMs) of Entamoeba and Trichmonas conferred very strong metronidazole resistance to transformed bacteria. The Ehntr1 gene of the genome project HM-1:IMSS strain of Entamoeba histolytica had a nonsense mutation, and the same nonsense mutation was present in 3 of 22 clinical isolates of Entamoeba. While ntr and nim mRNAs were variably expressed by cultured Entamoeba and Trichomonas isolates, there was no relationship to metronidazole sensitivity. We conclude that microaerophilic protists have bacterium-like enzymes capable of activating metronidazole (nitroreductases) and inactivating metronidazole (NIMs). While Entamoeba and Trichomonas displayed some of the changes (nonsense mutations and gene overexpression) associated with metronidazole resistance in bacteria, these changes did not confer metronidazole resistance to the microaerophilic protists examined here.
The dolichol-linked oligosaccharide Glc3Man9GlcNAc2-PP-Dol is the in vivo donor substrate synthesized by most eukaryotes for asparagine-linked glycosylation. However, many protist organisms assemble dolichol-linked oligosaccharides that lack glucose residues. We have compared donor substrate utilization by the oligosaccharyltransferase (OST) from Trypanosoma cruzi, Entamoeba histolytica, Trichomonas vaginalis, Cryptococcus neoformans, and Saccharomyces cerevisiae using structurally homogeneous dolichol-linked oligosaccharides as well as a heterogeneous dolichol-linked oligosaccharide library. Our results demonstrate that the OST from diverse organisms utilizes the in vivo oligo saccharide donor in preference to certain larger and/or smaller oligosaccharide donors. Steady-state enzyme kinetic experiments reveal that the binding affinity of the tripeptide acceptor for the protist OST complex is influenced by the structure of the oligosaccharide donor. This rudimentary donor substrate selection mechanism has been refined in fungi and vertebrate organisms by the addition of a second, regulatory dolichol-linked oligosaccharide binding site, the presence of which correlates with acquisition of the SWP1/ribophorin II subunit of the OST complex.