Joseph A. Burlison

The Yersinia pestis siderophore, yersiniabactin, and the ZnuABC system both contribute to zinc acquisition and the development of lethal septicaemic plague in mice.

Bacterial pathogens must overcome host sequestration of zinc (Zn2+), an essential micronutrient, during the infectious disease process. While the mechanisms to acquire chelated Zn2+ by bacteria are largely undefined, many pathogens rely upon the ZnuABC family of ABC transporters. Here we show that in Yersinia pestis, irp2, a gene encoding the synthetase (HMWP2) for the siderophore yersiniabactin (Ybt) is required for growth under Zn2+‐deficient conditions in a strain lacking ZnuABC. Moreover, growth stimulation with exogenous, purified apo‐Ybt provides evidence that Ybt may serve as a zincophore for Zn2+ acquisition. Studies with the Zn2+‐dependent transcriptional reporter znuA::lacZ indicate that the ability to synthesize Ybt affects the levels of intracellular Zn2+. However, the outer membrane receptor Psn and TonB as well as the inner membrane (IM) ABC transporter YbtPQ, which are required for Fe3+ acquisition by Ybt, are not needed for Ybt‐dependent Zn2+ uptake. In contrast, the predicted IM protein YbtX, a member of the Major Facilitator Superfamily, was essential for Ybt‐dependent Zn2+ uptake. Finally, we show that the ZnuABC system and the Ybt synthetase HMWP2, presumably by Ybt synthesis, both contribute to the development of a lethal infection in a septicaemic plague mouse model.

Activation of the Proapoptotic Bcl-2 Protein Bax by a Small Molecule Induces Tumor Cell Apoptosis

ABSTRACT The proapoptotic Bcl-2 protein Bax by itself is sufficient to initiate apoptosis in almost all apoptotic paradigms. Thus, compounds that can facilitate disruptive Bax insertion into mitochondrial membranes have potential as cancer therapeutics. In our study, we have identified small-molecule compounds predicted to associate with the Bax hydrophobic groove by a virtual-screen approach. Among these, one lead compound (compound 106) promotes Bax-dependent but not Bak-dependent apoptosis. Importantly, this compound alters Bax protein stability in vitro and promotes the insertion of Bax into mitochondria, leading to Bax-dependent permeabilization of the mitochondrial outer membrane. Furthermore, as a single agent, compound 106 inhibits the growth of transplanted tumors, probably by inducing apoptosis in tumors. Our study has revealed a compound that activates Bax and induces Bax-dependent apoptosis, which may lead to the development of new therapeutic agents for cancer.

Tumor targeted mesoporous silica-coated gold nanorods facilitate detection of pancreatic tumors using Multispectral optoacoustic tomography

Multispectral optoacoustic tomography (MSOT) is an emerging imaging technology that offers several advantages over traditional modalities, particularly in its ability to resolve optical contrast at depth on the microscopic scale. While potential applications include the early detection of tumors below clinical thresholds set by current technology, the lack of tumor-specific contrast agents limits the use of MSOT imaging. Therefore, we constructed highly stable nano-contrast agents by coating gold nanorods (GNRs) with either polyacrylic acid (PAA) or aminefunctionalized mesoporous silica (MS). Syndecan-1, which has been shown to target insulin-like growth factor 1 receptor (IGF1-R) (upregulated in pancreatic tumors), was conjugated on the surface of PAA-coated GNRs (PAA-GNRs) or MS-coated GNRs (MS-GNRs) to create tumor-targeted nanoparticles. In vitro, tumor targeting of nanoparticles was assessed with flow cytometry. In S2VP10L cells (positive for IGF1-R), the syndecan-1 MS-GNRs (Syndecan-MS-GNRs) demonstrated an increase in OA signal, 10x, compared to syndecan-1 PAAGNRs (Syndecan-PAA-GNRs). Minimal binding was observed in MiaPaca-2 cells (negative for IGF1-R). In vivo, tumor specific targeting of Syndecan-MS-GNRs was evaluated using a murine orthotopic pancreatic cancer model. The Syndecan- MS-GNRs demonstrated significantly greater accumulation within pancreatic tumors than in off-target organs such as the liver. Mice implanted with the IGF1-R negative MiaPaca-2 cells did not demonstrate specific tumor targeting. In summary, we report that targeted nano-contrast agents (Syndecan-MS-GNRs) can successfully detect orthotopic pancreatic tumors with minimum off-target binding in vivo using MSOT.

N-(3-oxo-acyl) homoserine lactone inhibits tumor growth independent of Bcl-2 proteins.

Pseudomonas aeruginosa produces N-(3-oxododecanoyl)-homoserine lactone (C12) as a quorum-sensing molecule for bacterial communication. C12 has also been reported to induce apoptosis in various types of tumor cells. However, the detailed molecular mechanism of C12-triggerred tumor cell apoptosis is still unclear. In addition, it is completely unknown whether C12 possesses any potential therapeutic effects in vivo. Our data indicate that, unlike most apoptotic inducers, C12 evokes a novel form of apoptosis in tumor cells through inducing mitochondrial membrane permeabilization independent of both pro- and anti-apoptotic Bcl-2 proteins. Importantly, C12 inhibits tumor growth in animals regardless of either pro- or anti-apoptotic Bcl-2 proteins. Furthermore, opposite to conventional chemotherapeutics, C12 requires paraoxonase 2 (PON2) to exert its cytotoxicity on tumor cells in vitro and its inhibitory effects on tumor growth in vivo. Overall, our results demonstrate that C12 inhibits tumor growth independent of both pro- and anti-apoptotic Bcl-2 proteins, and through inducing unique apoptotic signaling mediated by PON2 in tumor cells.

N‐(3‐Oxo‐acyl)‐homoserine lactone induces apoptosis primarily through a mitochondrial pathway in fibroblasts

N‐(3‐Oxododecanoyl)‐l‐homoserine lactone (C12) is produced by Pseudomonas aeruginosa to function as a quorum‐sensing molecule for bacteria–bacteria communication. C12 is also known to influence many aspects of human host cell physiology, including induction of cell death. However, the signalling pathway(s) leading to C12‐triggered cell death is (are) still not completely known. To clarify cell death signalling induced by C12, we examined mouse embryonic fibroblasts deficient in “initiator” caspases or “effector” caspases. Our data indicate that C12 selectively induces the mitochondria‐dependent intrinsic apoptotic pathway by quickly triggering mitochondrial outer membrane permeabilisation. Importantly, the activities of C12 to permeabilise mitochondria are independent of activation of both “initiator” and “effector” caspases. Furthermore, C12 directly induces mitochondrial outer membrane permeabilisation in vitro. Overall, our study suggests a mitochondrial apoptotic signalling pathway triggered by C12, in which C12 or its metabolite(s) acts on mitochondria to permeabilise mitochondria, leading to activation of apoptosis.

Abstract 1500: Syndecan-1 targeted mesoporous silica-coated gold nanorods act as theranostic agents for in vivo detection of orthotopic pancreatic tumors using multispectral optoacoustic tomography

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Detection of pancreatic cancer is especially challenging in comparison to many cancers due to severe limitations of both contrast agent delivery to pancreatic tumors and limitations of traditional imaging techniques such as poor resolution and low depth penetration. Theranostic nanoparticles encompassing both therapeutic and diagnostic capabilities can overcome limitations associated with conventional cancer diagnosis and therapy. In this study, we compared mesoporous silica coated gold nanorods (MS-GNR) and polyacrylic acid coated gold nanorods (PAA-GNR) targeted to tumor cells via Syndecan-1 ligand for detection of orthotopic pancreatic cancer in vivo. Because one of the major difficulties for translation of theranostic nanoparticles to the clinic is an inability track performance of nanoparticles in vivo, especially at depths required of orthotopic tumors, we will overcome this impediment by utilizing multispectral optoacoustic tomography (MSOT). Multispectral optoacoustic tomography provides high optical contrast images at a microscale resolution and reasonable penetration depth by combining the advantages of optical (high sensitivity) and ultrasound (increased depth of penetration). Our results indicate that the Syndecan-1 MS-GNR were superior to Syndecan-1 PAA-GNR, untargeted MS-GNR, or untargeted PAA-GNR as contrast agents to identify pancreatic cancer in vivo via MSOT. The Syndecan-1 MS-GNR particles also resulted in reduced off-target accumulation compared to Syndecan-1 PAA-GNR. This study is among the first to evaluate biodistribution of ligand-targeted nanoparticles in the context of orthotopic pancreatic cancer using multispectral optoacoustic tomography. The MS GNR can be utilized as theranostic platform due to ability of mesoporous silica to encapsulate drug and easy surface modification with cancer recognizing motif peptide. Citation Format: Anil Khanal, Charles W. Kimbrough, Nichola C. Garbett, Joseph A. Burlison, William E. Grizzle, Lacey R. McNally. Syndecan-1 targeted mesoporous silica-coated gold nanorods act as theranostic agents for in vivo detection of orthotopic pancreatic tumors using multispectral optoacoustic tomography. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1500. doi:10.1158/1538-7445.AM2015-1500

Abstract 4108: Efficacy of ellagic acid and its major urolithin metabolites in inhibiting growth of prostate cancer cells

Pomegranate juice, in the last several years, has gained tremendous attention for its proven ability to slow down prostate cancer as evident by reduced prostate-specific antigen doubling time. However, a bioactive compound responsible for this action has not yet been confirmed and thus, limited its chemo-preventive/therapeutic potential. Pharmacokinetic studies of oral consumption of pomegranate juice in human have shown partial hydrolysis of ellagitannins, major component in the juice, into ellagic acid which is subsequently converted to various urolithin metabolites by the gut microbial digestion. Urolithins are shown to have extended plasma half-life and are detected in urine up to 48 after oral consumption of pomegranate juice. We investigated the potential of ellagic acid and major urolithin metabolites; urolithin A and urolithin B, that were synthesized in our laboratory, in inhibiting the growth and survival of androgen-independent prostate cancer cells, PC-3 and DU-145 cells in vitro. We found significantly high dose and time-dependent inhibition of growth and proliferation of both cell lines by all three compounds tested. Ellagic acid was found to be the most potent inhibitor as evident by significantly lower IC50 of 8.17 µM and 1.57µM respectively for DU-145 and PC-3 cells after 48 hours. Urolithin B was more efficient than Urolithin A against DU-145 cells (IC50 of 17.42µM vs. 73.78µM) while it was reversed in PC-3 cells (IC50 of 92.25 µM vs. 36.96 µM). Similarly, these compounds also induced substantial levels of dose-dependent apoptosis in either cell lines, however, with variable sensitivity. Western blot analysis of signaling proteins to elucidate the molecular mechanisms of observed effects correlated with altered levels of proteins that mediate cell-cycle regulation. P21, an inhibitor of CDK2-CyclinA/E complex that regulates protein elongation and cell-cycle progression from G1 to S phase was highly upregulated in compound-treated PC-3 cells and DU-145 cells, while cyclinD1 which regulates G1 to S phase transition in consort with CDK4 was significantly decreased in treated cells in comparison to untreated and vehicle-treated control. Studies are underway to understand the synergistic effects between these compounds, in vitro and in vivo. Taken together, our results indicates EA and its gut metabolites are significant anti-survival and anti-proliferation candidates against prostate cancer cells and hold a tremendous chemo-therapeutic and chemo-preventive potential, for its high efficacy and natural, low toxic source. Citation Format: Anil Poudel, Manicka V. Vadhanam, Joseph Burlison. Efficacy of ellagic acid and its major urolithin metabolites in inhibiting growth of prostate cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4108. doi:10.1158/1538-7445.AM2014-4108