The recombinant beta subunit of C-phycocyanin inhibits cell proliferation and induces apoptosis

C-Phycocyanin (C-PC) from blue-green algae has been reported to have various pharmacological characteristics, including antiinflammatory and anti-tumor activities. In this study, we expressed the beta-subunit of C-PC (ref to as C-POP) in Escherichia coli. We found that the recombinant C-PC/beta has anti-cancer properties. Under the treatment of 5 mu M of the recombinant C-PC/beta, four different cancer cell lines accrued high proliferation inhibition and apoptotic induction. Substantially, a lower response occurred in non-cancer cells. We investigated the mechanism by which C-PC/beta inhibits cancer cell proliferation and induces apoptosis. We found that the C-PC/beta interacts with membrane-associated beta-tubulin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Under the treatment of the C-PC/beta, depolymerization of microtubules and actin-filaments were observed. The cells underwent apoptosis with an increase in caspase-3, and caspase-8 activities. The cell cycle was arrested at the G0/G1 phase under the treatment of C-PC/beta. In addition, the nuclear level of GAPDH decreased significantly. Decrease in the nuclear level of GAPDH prevents the cell cycle from entering into the S phase. Inhibition of cancer cell proliferation and induction of apoptosis may potentate the C-POP as a promising cancer prevention or therapy agent. (c) 2006 Elsevier Ireland Ltd. All rights reserved.

Differential and spermatogenic cell-specific expression of DMRT1 during sex reversal in protogynous hermaphroditic groupers

DMRT1 has been suggested to play different roles in sex determination and gonad differentiation, because different expression patterns have been reported among different vertebrates. The groupers, since their gonads first develop as ovary and then reverse into testis, have been thought as good models to study sex differentiation and determination. In this study, we cloned the full-length cDNAs of DMRT] gene from orange-spotted grouper (Epinephelus coioides), and prepared corresponding anti-EcDMRT1] antiserum to study the relationship of DMRT] to sex reversal. One important finding is that the grouper DMRT] is not only differentially expressed in different stage gonads, but also restricted to specific stages and specific cells of spermatogenesis. Grouper DMRT1 protein exists only in spermatogonia, primary spermatocytes and secondary spermatocytes, but not in the supporting Sertoli cells. Moreover, we confirmed that EcSox3 is expressed not only in oogonia and different stage oocytes, but also in Sertoli cells and spermatogonia, and EcSox9 is expressed only in Sertoli cells. The data suggested that grouper DMRT1 might be a more specific sex differentiation gene for spermatogenesis, and play its role at the specific stages from spermatogonia to spermatocytes. In addition, no introns were found in the grouper DMRT1, and no duplicated DMRT1, genes were detected. The finding implicates that the intronless DMRT1 that is able to undergo rapid transcriptional turnover might be a significant gene for stimulating spermatogenesis in the protogynous hermaphroditic gonad. (c) 2006 Published by Elsevier Ireland Ltd.

A novel anti-tumor protein extracted from Meretrix meretrix Linnaeus induces cell death by increasing cell permeability and inhibiting tubulin polymerization

Discovery and development of new pharmaceuticals from marine organisms are attracting increasing interest. Several agents derived from marine organisms are under preclinical and clinical evaluation as potential anticancer drugs. We extracted and purified a novel anti-tumor protein from the coelomic fluid of Meretrix meretrix Linnaeus by ammonium sulphate fractionation, ion exchange and hydrophobic interaction chromatography. The molecular weight of the highly purified protein, designated MML, was 40 kDa as determined by SDS-PAGE analysis. MML exhibited significant cytotoxicity to several cancer cell types, including human hepatoma BEL-7402, human breast cancer MCF-7 and human colon cancer HCT116 cells. However, no inhibitory effect was found when treating murine normal fibroblasts NIH3T3 and benign human breast MCF-10A cells with MML. The cell death induced by MML was characterized by cell morphological changes. The induction of apoptosis of BEL-7402 cells by MML was weak by DNA ladder assay. The possible mechanisms of its anti-tumor effect might be the changes in cell membrane permeability and inhibition of tubulin polymerization. MML may be developed as a novel, highly selective and effective anti-cancer drug.

Analysis of three marine fish cell lines by RAPD assay

We tested the applicability of the random amplified polymorphic deoxyribonucleic acid (RAPD) analysis for identification of three marine fish cell lines FG. SPH, and RSBK and as a possible tool to detect cross-contamination. Sixth commercial 10-mer RAPD primers were tested on the cell lines and on samples collected from individual fish. The results obtained showed that the cell lines could be identified to the correspondent species on the basis of identical Patterns produced by 35-48% of the primers tested. the total mean similarity indices for cell lines versus correspondent species of individual fish ranged from 0.825 to 0.851. indicating the existence of genetic variation in these cell lines in relation to the species of their origin. Also, four primers, which gave a monomorphic hand pattern within species/line, but different among the species/line, were obtained. These primers can be useful for identification of these cell lines and for characterization of the genetic variation of these cell lines in relation to the species of their origin. This supported the use of RAPD analysis as an effective tool in species identification and cross-contamination test among different cell lines.

Construction of shuttle, expression vector of human tumor necrosis factor alpha (hTNF-alpha) gene and its expression in a cyanobacterium, Anabaena sp. PCC 7120

The construction of the shuttle, expression vector of human tumor necrosis factor alpha (hTNF-alpha) gene and its expression in a cyanobacterium Anabaena sp. PCC 7120 was reported. The 700-bp hTNF cDNA fragments have been recovered from plasmid pRL-rhTNF, then inserted downstream of the promoter PpsbA in the plasmid pRL439. The resultant intermediary plasmid pRL-TC has further been combined with the shuttle vector pDC-8 to get the shuttle, expression vector pDC-TNF. The expression of the rhTNF gene in Escherichia coil has been analyzed by SDS-PAGE and thin-layer scanning, and the results show that the expressed TNF protein with these two vectors is 16.9 percent (pRL-TC) and 15.0 percent (pDC-TNF) of the total proteins in the cells, respectively, while the expression level of TNF gene in plasmid pRL-rhTNF is only 11.8 percent. Combined with the participation of the conjugal and helper plasmids, pDC-TNF has been introduced into Anabaena sg PCC 7120 by triparental conjugative transfer, and the stable transgenic strains have been obtained. The existence of the introduced plasmid pDC-TNF in recombinant cyanobacterial cells has been demonstrated by the results of the agarose electrophoresis with the extracted plasmid samples and Southern blotting with alpha-(32)p labeled hTNF cDNA probes, while the expression of the hTNF gene in Anabaena sp. PCC 7120 has been confirmed by the results of Western blotting with extracted protein samples and human TNF-alpha monoclonal antibodies. The cytotoxicity assays using the mouse cancer cell line L929 proved the cytotoxicity of the TNF in the crude extracts from the transgenic cyanobacterium Anabaena sp. PCC 7120.

Design, synthesis and development of novel indolocarbazole derivatives as potential anti-cancer agents

This thesis describes work carried out on the design of new routes to a range of bisindolylmaleimide and indolo[2,3-a]carbazole analogs, and investigation of their potential as successful anti-cancer agents. Following initial investigation of classical routes to indolo[2,3-a]pyrrolo[3,4-c]carbazole aglycons, a new strategy employing base-mediated condensation of thiourea and guanidine with a bisindolyl β-ketoester intermediate afforded novel 5,6-bisindolylpyrimidin-4(3H)-ones in moderate yields. Chemical diversity within this H-bonding scaffold was then studied by substitution with a panel of biologically relevant electrophiles, and by reductive desulfurisation. Optimisation of difficult heterogeneous literature conditions for oxidative desulfurisation of thiouracils was also accomplished, enabling a mild route to a novel 5,6-bisindolyluracil pharmacophore to be developed within this work. The oxidative cyclisation of selected acyclic bisindolyl systems to form a new planar class of indolo[2,3-a]pyrimido[5,4-c]carbazoles was also investigated. Successful conditions for this transformation, as well as the limitations currently prevailing for this approach are discussed. Synthesis of 3,4-bisindolyl-5-aminopyrazole as a potential isostere of bisindolylmaleimide agents was encountered, along with a comprehensive derivatisation study, in order to probe the chemical space for potential protein backbone H-bonding interactions. Synthesis of a related 3,4-arylindolyl-5-aminopyrazole series was also undertaken, based on identification of potent kinase inhibition within a closely related heterocyclic template. Following synthesis of approximately 50 novel compounds with a diversity of H-bonding enzyme-interacting potential within these classes, biological studies confirmed that significant topo II inhibition was present for 9 lead compounds, in previously unseen pyrazolo[1,5-a]pyrimidine, indolo[2,3-c]carbazole and branched S,N-disubstituted thiouracil derivative series. NCI-60 cancer cell line growth inhibition data for 6 representative compounds also revealed interesting selectivity differences between each compound class, while a new pyrimido[5,4-c]carbazole agent strongly inhibited cancer cell division at 10 µM, with appreciable cytotoxic activity observed across several tumour types.

Synthesis and evaluation of novel azaindolocarbazole derivatives as cancer chemotherapeutics

This thesis details the design and implementation of novel chemical routes towards a series of highly propitious 7-azaindolyl derivatives of the indolocarbazole (ICZ) and bisindolylmaleimide (BIM) families, with subsequent evaluation for use as cancer chemotherapeutic agents. A robust synthetic strategy was devised to allow the introduction of a 7-azaindolyl moiety into our molecular template. This approach allowed access to a wide range of β-keto ester and β-keto nitrile intermediates. Critical analysis identified F-ring modulation as a major theme towards the advancement of ICZ and BIM derivatives in drug therapy. Thus, the employment of cyclocondensation methodology furnished a number of novel aminopyrazole, isoxazolone, pyrazolone and pyrimidinone analogues, considerably widening the scope of the prevalent maleimide functionality. Photochemical cyclisation provided for the first reported aza ICZ containing a six-membered F-ring. Another method towards achieving the aza ICZ core involved use of a Perkin-type condensation approach, with chemical elaboration of the headgroup instigated post-aromatisation. Subsequent use of a modified Lossen rearrangement allowed access to further analogues containing a six-membered F-ring. Extensive screening of the novel aza ICZ and BIM derivatives was carried out against the NCI-60 cancer cell array, with nine prospective candidates selected for continued biological evaluation. From these assays, a number of compounds were shown to inhibit cancer cell growth at concentrations of below 10 nM. Indeed, the most active aza ICZ tested is currently under assessment by the Biological Evaluation Committee of the NCI due to excellent antiproliferative activity demonstrated across the panel of cell lines, with a mean GI50 of 34 nM, a mean total growth inhibition (TGI) of 4.6 μM and a mean cytotoxicity (LC50) of 63.1 μM. Correlation to known topoisomerase I (topo I) inhibitors was revealed by COMPARE analysis, and subsequent topo I-mediated DNA cleavage assays showed inhibitory activity below 1 μM for several derivatives.

Molecular and cellular aspects of the SREBP pathway

The SREBP (sterol response element binding proteins) transcription factors are central to regulating de novo biosynthesis of cholesterol and fatty acids. The SREBPs are regulated by retention or escape from the ER to the Golgi where they are proteolytically cleaved into active forms. The SREBP cleavage activating protein (SCAP) and the INSIG proteins are essential in this regulatory process. The aim of this thesis is to further characterise the molecular and cellular aspects surrounding regulation of SREBP processing. SREBP and SCAP are known to interact via their carboxy-terminal regulatory domains (CTDs) but this interaction is poorly characterised. Significant steps were achieved in this thesis towards specific mapping of the interaction site. These included cloning and over expression and partial purification of tagged SREBP1 and SREBP2 CTDs and probing of a SCAP peptide array with the CTDs. Results from the SREBP2 probing were difficult to interpret due to insolubility issues with the protein, however, probing with SREBP1 revealed five potential binding sites which were detected reproducibly. Further research is necessary to overcome SREBP2 insolubility issues and to confirm the identified SREBP1 interaction site(s) on SCAP. INSIG1 has a central role in regulating SREBP processing and in regulating stability of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR), a rate limiting enzyme in cholesterol biosynthesis. There are two protein isoforms of human INSIG1 produced through the use of two in-frame alternative start sites. Bioinformatic analysis indicated that the presence of two in-frame start sites within the 5-prime region of INSIG1 mRNA is highly conserved and that production of two isoforms of INSIG1is likely a conserved event. Functional differences between these two isoforms were explored. No difference in either the regulation of SREBP processing or HMGCR degradation between the INSIG1 isoforms was observed and the functional significance of the two isoforms is as yet unclear. The final part of this thesis focused on enhancing the cytotoxicity of statins by targeted inhibition of SREBP processing by oxysterols. Statins have significant potential as anti-cancer agents as they inhibit the activity of HMGCR leading to a deficiency in mevalonate which is essential for cell survival. The levels of HMGCR fluctuate widely due to cholesterol feedback of SREBP processing. The relationship between sterol feedback and statin mediated cell death was investigated in depth in HeLa cells. Down regulation of SREBP processing by sterols significantly enhanced the efficacy of statin mediated cell death. Investigation of sterol feedback in additional cancer cell lines showed that sterol feedback was absent in cell lines A- 498, DU-145, MCF-7 and MeWo but was present in cell lines HT-29, HepG2 and KYSE-70. In the latter inhibition of SREBP processing using oxysterols significantly enhanced statin cytotoxicity. The results indicate that this approach is valid to enhance statin cytotoxicity in cancer cells, but may be limited by deregulation of SREBP processing and off target effects of statins, which were observed for some of the cancer cell lines screened.

Integrative Genomics Reveals a Role for GNA13 in Lymphomagenesis

Lymphomas comprise a diverse group of malignancies derived from immune cells. High throughput sequencing has recently emerged as a powerful and versatile method for analysis of the cancer genome and transcriptome. As these data continue to emerge, the crucial work lies in sorting through the wealth of information to hone in on the critical aspects that will give us a better understanding of biology and new insight for how to treat disease. Finding the important signals within these large data sets is one of the major challenges of next generation sequencing.

In this dissertation, I have developed several complementary strategies to describe the genetic underpinnings of lymphomas. I begin with developing a better method for RNA sequencing that enables strand-specific total RNA sequencing and alternative splicing profiling in the same analysis. I then combine this RNA sequencing technique with whole exome sequencing to better understand the global landscape of aberrations in these diseases. Finally, I use traditional cell and molecular biology techniques to define the consequences of major genetic alterations in lymphoma.

Through this analysis, I find recurrent silencing mutations in the G alpha binding protein GNA13 and associated focal adhesion proteins. I aim to describe how loss-of-function mutations in GNA13 can be oncogenic in the context of germinal center B cell biology. Using in vitro techniques including liquid chromatography-mass spectrometry and knockdown and overexpression of genes in B cell lymphoma cell lines, I determine protein binding partners and downstream effectors of GNA13. I also develop a transgenic mouse model to study the role of GNA13 in the germinal center in vivo to determine effects of GNA13 deletion on germinal center structure and cell migration.

Thus, I have developed complementary approaches that span the spectrum from discovery to context-dependent gene models that afford a better understanding of the biological function of aberrant events and ultimately result in a better understanding of disease.

Copper signaling axis as a target for prostate cancer therapeutics.

Previously published reports indicate that serum copper levels are elevated in patients with prostate cancer and that increased copper uptake can be used as a means to image prostate tumors. It is unclear, however, to what extent copper is required for prostate cancer cell function as we observed only modest effects of chelation strategies on the growth of these cells in vitro. With the goal of exploiting prostate cancer cell proclivity for copper uptake, we developed a "conditional lethal" screen to identify compounds whose cytotoxic actions were manifested in a copper-dependent manner. Emerging from this screen was a series of dithiocarbamates, which, when complexed with copper, induced reactive oxygen species-dependent apoptosis of malignant, but not normal, prostate cells. One of the dithiocarbamates identified, disulfiram (DSF), is an FDA-approved drug that has previously yielded disappointing results in clinical trials in patients with recurrent prostate cancer. Similarly, in our studies, DSF alone had a minimal effect on the growth of prostate cancer tumors when propagated as xenografts. However, when DSF was coadministered with copper, a very dramatic inhibition of tumor growth in models of hormone-sensitive and of castrate-resistant disease was observed. Furthermore, we determined that prostate cancer cells express high levels of CTR1, the primary copper transporter, and additional chaperones that are required to maintain intracellular copper homeostasis. The expression levels of most of these proteins are increased further upon treatment of androgen receptor (AR)-positive prostate cancer cell lines with androgens. Not surprisingly, robust CTR1-dependent uptake of copper into prostate cancer cells was observed, an activity that was accentuated by activation of AR. Given these data linking AR to intracellular copper uptake, we believe that dithiocarbamate/copper complexes are likely to be effective for the treatment of patients with prostate cancer whose disease is resistant to classical androgen ablation therapies.

Neutralizing BAFF/APRIL with atacicept prevents early DSA formation and AMR development in T cell depletion induced nonhuman primate AMR model.

Depletional strategies directed toward achieving tolerance induction in organ transplantation have been associated with an increased incidence and risk of antibody-mediated rejection (AMR) and graft injury. Our clinical data suggest correlation of increased serum B cell activating factor/survival factor (BAFF) with increased risk of antibody-mediated rejection in alemtuzumab treated patients. In the present study, we tested the ability of BAFF blockade (TACI-Ig) in a nonhuman primate AMR model to prevent alloantibody production and prolong allograft survival. Three animals received the AMR inducing regimen (CD3-IT/alefacept/tacrolimus) with TACI-Ig (atacicept), compared to five control animals treated with the AMR inducing regimen only. TACI-Ig treatment lead to decreased levels of DSA in treated animals at 2 and 4 weeks posttransplantation (p < 0.05). In addition, peripheral B cell numbers were significantly lower at 6 weeks posttransplantation. However, it provided only a marginal increase in graft survival (59 ± 22 vs. 102 ± 47 days; p = 0.11). Histological analysis revealed a substantial reduction in findings typically associated with humoral rejection with atacicept treatment. More T cell rejection findings were observed with increased graft T cell infiltration in atacicept treatment, likely secondary to the graft prolongation. We show that BAFF/APRIL blockade using concomitant TACI-Ig treatment reduced the humoral portion of rejection in our depletion-induced preclinical AMR model.

MiR-215 Is Induced Post-transcriptionally via HIF-Drosha Complex and Mediates Glioma-Initiating Cell Adaptation to Hypoxia by Targeting KDM1B.

The hypoxic tumor microenvironment serves as a niche for maintaining the glioma-initiating cells (GICs) that are critical for glioblastoma (GBM) occurrence and recurrence. Here, we report that hypoxia-induced miR-215 is vital for reprograming GICs to fit the hypoxic microenvironment via suppressing the expression of an epigenetic regulator KDM1B and modulating activities of multiple pathways. Interestingly, biogenesis of miR-215 and several miRNAs is accelerated post-transcriptionally by hypoxia-inducible factors (HIFs) through HIF-Drosha interaction. Moreover, miR-215 expression correlates inversely with KDM1B while correlating positively with HIF1α and GBM progression in patients. These findings reveal a direct role of HIF in regulating miRNA biogenesis and consequently activating the miR-215-KDM1B-mediated signaling required for GIC adaptation to hypoxia.

Rapamycin Interferes With Postdepletion Regulatory T Cell Homeostasis and Enhances DSA Formation Corrected by CTLA4-Ig.

Previously, we demonstrated that alemtuzumab induction with rapamycin as sole maintenance therapy is associated with an increased incidence of humoral rejection in human kidney transplant patients. To investigate the role of rapamycin in posttransplant humoral responses after T cell depletion, fully MHC mismatched hearts were transplanted into hCD52Tg mice, followed by alemtuzumab treatment with or without a short course of rapamycin. While untreated hCD52Tg recipients acutely rejected B6 hearts (n = 12), hCD52Tg recipients treated with alemtuzumab alone or in conjunction with rapamycin showed a lack of acute rejection (MST > 100). However, additional rapamycin showed a reduced beating quality over time and increased incidence of vasculopathy. Furthermore, rapamycin supplementation showed an increased serum donor-specific antibodies (DSA) level compared to alemtuzumab alone at postoperation days 50 and 100. Surprisingly, additional rapamycin treatment significantly reduced CD4(+) CD25(+) FoxP3(+) T reg cell numbers during treatment. On the contrary, ICOS(+) PD-1(+) CD4 follicular helper T cells in the lymph nodes were significantly increased. Interestingly, CTLA4-Ig supplementation in conjunction with rapamycin corrected rapamycin-induced accelerated posttransplant humoral response by directly modulating Tfh cells but not Treg cells. This suggests that rapamycin after T cell depletion could affect Treg cells leading to an increase of Tfh cells and DSA production that can be reversed by CTLA4-Ig.

Salvianolic Acid B Inhibits Growth of Cervical Cancer Cells in vitro via Induction of Apoptosis through the Extrinsic Pathway

In 2014 alone, over 12,000 women are expected to be diagnosed with cervical cancer. Of these women who are diagnosed, about 3,909 will result in death. Despite developments in prevention methods, cervical cancer remains a major health concern for women. Growing evidence suggests that Salvianolic acid B (Sal B), a major component of the Chinese herb Danshen, may inhibit cancer cell growth and help fight against cervical cancer. This study characterizes the potential of Sal B as a cervical cancer drug through in vitro testing on HeLa cells. We hypothesized that application of Sal B to HeLa cells will result in decreased cell viability and increased apoptosis in a dose dependent manner. HeLa cells were treated with varying concentrations of Sal B: 25µM, 50µM, 100µM, and 200µM. Cell viability was determined through colony formation assay, cell death ELISA, and nuclear morphology. An inhibitor study was also conducted for further apoptosis pathway analysis. Colony formation assay demonstrated a significant decrease in cell viability with increasing concentrations of Sal B with 75% viability at 50µM down to 0% viability at 200µM. Cell death ELISA and the analysis of nuclear morphology via Hoechst staining reported significant levels of apoptosis at concentrations equal to 50µM and greater. Furthermore, experiments using caspase inhibitors indicated that Sal B’s apoptotic effects are caspase-8 dependent. In conclusion, our results demonstrate that Sal B inhibits cancer cell growth by a mechanism that involves apoptosis induction through the extrinsic pathway.

Role of hypoxia and hypoxia-inducible factor-1 in tumour immune escape

Previous studies revealed that, upon exposure to hypoxia, tumour cells acquire resistance to the cytolytic activity of IL-2-activated lymphocytes. The MHC class I chain-related (MIC) molecules – comprised of MICA and MICB – are ligands for the activating NKG2D receptor on Natural Killer (NK) and CD8+ T cells. MIC-NKG2D interactions lead to the activation of NK and CD8+ T cells and the subsequent lysis of the tumour cells. The study also showed that the mechanism of the hypoxia-mediated immune escape involves the shedding of MIC, specifically MICA, from the tumour cell surface. The objective of the present study was to determine whether the shedding of MICA requires the expression of hypoxia inducible factor-1 (HIF-1), a transcription factor that regulates cellular adaptations to hypoxia. Exposure to hypoxia (0.5% O2 vs. 20% O2) led to the shedding of MIC from the surface of MDA-MB-231 human breast cancer cells and DU-145 human prostate cancer cells as determined by flow cytometry. Knockdown of HIF-1α mRNA using siRNA technology resulted in inhibition of HIF-1α accumulation under hypoxic conditions as determined by Western blot analysis. Parallel study revealed that knockdown of HIF-1α also blocked the shedding of MICA from the surface of MDA-MB-231 cells exposed to hypoxia. These results indicate that HIF-1 is required for the hypoxia-mediated shedding of MICA and, consequently, that HIF-1 may play an important role in tumour immune escape. Ongoing studies aim to determine the HIF-1 target genes involved in the shedding of MICA under hypoxia.