Characterization and Use of Folate Receptor Isoforms for Targeting of Epithelial and Myeloid Cells

ABSTRACT CHARACTERIZATION AND USE OF FOLATE RECEPTOR ISOFORMS FOR TARGETING OF EPITHELIAL AND MYELOID CELLS by Sreya Biswas The University of Wisconsin-Milwaukee, 2016 Under the Supervision of Professor Douglas A. Steeber Folate receptor (FR) is a GPI-anchored glycoprotein with high binding affinity for folic acid. FR has two membrane-associated isoforms, α and β, that are overexpressed on epithelial and myeloid tumors, respectively. Normal cells may also exhibit FR expression at very low levels but interestingly, FR-α on normal cells is restricted to the apical surface i.e., away from the blood stream. This differential expression and orientation of the FR-α isoform on tumor cells has been exploited to selectively target and deliver conjugates (e.g., drugs, nanoparticles, liposomes) to tumor cells without harming neighboring healthy cells. However, the functions and use of FR-β as a potential target have not been explored, and its functions on myeloid cells remain largely unknown. Therefore, we investigated the functions of FR-β to determine its potential as a target in myeloid malignancies using a human myelomonocytic leukemia cell line, U937. FR-α studies were conducted using a murine epithelial breast carcinoma cell line, 4T1, and tumors harvested from 4T1 tumor-bearing mice. The isoforms were found overexpressed on tumor cells and tissues, both in vitro and in vivo, with no expression observed on corresponding normal cells. Studies conducted using folic acid-fluorochrome conjugates to determine intracellular receptor fate indicated that FR-β was not internalized into cells unlike FR-α. However, both isoforms exhibited strong binding to folic acid conjugates (e.g., fluorochromes, nanoparticles) thus indicating that they could be selectively targeted using folic acid-dependent methods. We also determined the potential of a novel histone deacetylase (HDAC) inhibitor (HDACi) as an anti-cancer agent that could be used along with folic acid for achieving better selectivity in targeting tumor cells. Preliminary studies showed that Compound 5 (Cpd5) is stable with strong anti-proliferative activities against human tumor cells. Cpd5 was also able to reduce the rate of 4T1 tumor growth in mice without inducing systemic toxicity in the animals. In addition, Cpd5 exhibited desirable pharmacokinetic properties and showed direct effects on acetylation levels of histone proteins. These studies not only provide insights into the functional differences associated with FR-α and FR-β isoforms, but also highlight the potential of future targeting strategies utilizing these to target both epithelial and myeloid malignancies with improved selectivity

Improved production of cytotoxic thailanstatins A and D through metabolic engineering of Burkholderia thailandensis MSMB43 and pilot scale fermentation

Thailanstatin A (TST-A) is a potent antiproliferative natural product discovered by our group from Burkholderia thailandensis MSMB43 through a genome-guided approach. The limited supply of TST-A, due to its low titer in bacterial fermentation, modest stability and very low recovery rate during purification, has hindered the investigations of TST-A as an anticancer drug candidate. Here we report the significant yield improvement of TST-A and its direct precursor, thailanstatin D (TST-D), through metabolic engineering of the thailanstatin biosynthetic pathway in MSMB43. Deletion of tstP, which encodes a dioxygenase involved in converting TST-A to downstream products including FR901464 (FR), resulted in 58% increase of the TST-A titer to 144.7 ± 2.3 mg/L and 132% increase of the TST-D titer to 14.6 ± 0.5 mg/L in the fermentation broth, respectively. Deletion of tstR, which encodes a cytochrome P450 involved in converting TST-D to TST-A, resulted in more than 7-fold increase of the TST-D titer to 53.2 ± 12.1 mg/L in the fermentation broth. An execution of 90 L pilot-scale fed-batch fermentation of the tstP deletion mutant in a 120-L fermentor led to the preparation of 714 mg of TST-A with greater than 98.5% purity. The half-life of TST-D in a phosphate buffer was found to be at least 202 h, significantly longer than that of TST-A or FR, suggesting superior stability. However, the IC50 values of TST-D against representative human cancer cell lines were determined to be greater than those of TST-A, indicating weaker antiproliferative activity. This work enabled us to prepare sufficient quantities of TST-A and TST-D for our ongoing translational research

Radiosensitizing effect of ellagic acid on growth of Hepatocellular carcinoma cells: an in vitro study

Abstract Failure of treatment for cancer in clinic by radio/chemotherapy is generally attributed to tumour resistance. Therefore, it is important to develop strategies to increase the cytotoxicity of tumour cells by radiation in combination with unique tumour selective cytotoxic agents. We evaluated the potential of ellagic acid (EA) as an enhancer of oxidative stress in cancer cells. HepG2 cells were treated with EA (10 µM) for 12 h prior to exposure of single 7.5 Gy dose of irradiation. Treatment of HepG2 cells with EA and gamma radiation showed increased reactive oxygen species generation, up regulation of p53 protein expression, decreased survival markers level like p-Akt, p-NF-kB and p-STAT3 which were significantly higher after radiation treatment alone. We also found that combination treatment increased G2/M phase cell population, decreased IL-6, COX–2 and TNF-α expression and caused a loss in mitochondrial membrane potential with decreased level of angiogenesis marker MMP-9. Over expression of Bax and activation of caspase 3 indicated the apoptosis of the cells. The results provided a strong unique strategy to kill cancer cells HepG2, using less radiation dose along with effective pro-oxidant dose of EA

SIV Infection Is Associated with Transient Acute-Phase Steatosis in Hepatocytes In Vivo

Metabolic-dysfunction-associated fatty liver disease (MAFLD) is a major cause of morbidity and mortality in HIV-infected individuals, even those receiving optimal antiretroviral therapy. Here, we utilized the SIV rhesus macaque model and advanced laparoscopic techniques for longitudinal collection of liver tissue to elucidate the timing of pathologic changes. The livers of both SIV-infected (N = 9) and SIV-naïve uninfected (N = 8) macaques were biopsied and evaluated at four time points (weeks −4, 2, 6, and 16–20 post-infection) and at necropsy (week 32). SIV DNA within the macaques’ livers varied by over 4 logs at necropsy, and liver SIV DNA significantly correlated with SIV RNA in the plasma throughout the study. Acute phase liver pathology (2 weeks post-infection) was characterized by evidence for fat accumulation (microvesicular steatosis), a transient elevation in both AST and cholesterol levels within the serum, and increased hepatic expression of the PPARA gene associated with cholesterol metabolism and beta oxidation. By contrast, the chronic phase of the SIV infection (32 weeks post-infection) was associated with sinusoidal dilatation, while steatosis resolved and concentrations of AST and cholesterol remained similar to those in uninfected macaques. These findings suggest differential liver pathologies associated with the acute and chronic phases of infection and the possibility that therapeutic interventions targeting metabolic function may benefit liver health in people newly diagnosed with HIV

Genomics-Guided Discovery of Thailanstatins A, B, and C As Pre-mRNA Splicing Inhibitors and Antiproliferative Agents from <i>Burkholderia thailandensis</i> MSMB43

Mining the genome sequence of <i>Burkholderia thailandensis</i> MSMB43 revealed a cryptic biosynthetic gene cluster resembling that of FR901464 (<b>4</b>), a prototype spliceosome inhibitor produced by <i>Pseudomonas</i> sp. No. 2663. Transcriptional analysis revealed a cultivation condition in which a regulatory gene of the cryptic gene cluster is adequately expressed. Consequently, three new compounds, named thailanstatins A (<b>1</b>), B (<b>2</b>), and C (<b>3</b>), were isolated from the fermentation broth of <i>B. thailandensis</i> MSMB43. Thailanstatins are proposed to be biosynthesized by a hybrid polyketide synthase–nonribosomal peptide synthetase pathway. They differ from <b>4</b> by lacking an unstable hydroxyl group and by having an extra carboxyl moiety; those differences endow thailanstatins with a significantly greater stability than <b>4</b> as tested in phosphate buffer at pH 7.4. <i>In vitro</i> assays showed that thailanstatins inhibit pre-mRNA splicing as potently as <b>4</b>, with half-maximal inhibitory concentrations in the single to sub-μM range. Cell culture assays indicated that thailanstatins also possess potent antiproliferative activities in representative human cancer cell lines, with half-maximal growth inhibitory concentrations in the single nM range. This work provides new chemical entities for research and development and new structure–activity information for chemical optimization of related spliceosome inhibitors

Therapeutic neutralizing monoclonal antibody administration protects against lethal yellow fever virus infection

Yellow fever virus (YFV) is a reemerging global health threat, driven by several factors, including increased spread of the mosquito vector and rapid urbanization. Although a prophylactic vaccine exists, vaccine hesitancy, supply deficits, and distribution difficulties leave specific populations at risk of severe YFV disease, as evidenced by recent outbreaks in South America. To establish a treatment for patients with severe YFV infection, we tested 37 YFV-specific monoclonal antibodies isolated from vaccinated humans and identified two capable of potently neutralizing multiple pathogenic primary YFV isolates. Using both hamster and nonhuman primate models of lethal YFV infection, we demonstrate that a single administration of either of these two potently neutralizing antibodies during acute infection fully controlled viremia and prevented severe disease and death in treated animals. Given the potential severity of YFV-induced disease, our results show that these antibodies could be effective in saving lives and fill a much-needed void in managing YFV cases during outbreaks