Search

Bacterial resistance to antibiotics has made it necessary to resort to using antibacterial drugs that have considerable toxicities. Here, we show that conjugation of vancomycin-loaded nanoparticles with the cyclic 9-amino-acid peptide CARGGLKSC (CARG), identified via phage display on Staphylococcus aureus (S. aureus) bacteria and through in vivo screening in mice with S. aureus-induced lung infections, increases the antibacterial activity of the nanoparticles in S. aureus-infected tissues and reduces the systemic dose needed, minimizing side effects. CARG binds specifically to S. aureus bacteria but not Pseudomonas bacteria in vitro, selectively accumulates in S. aureus-infected lungs and skin of mice but not in non-infected tissue and Pseudomonas-infected tissue, and significantly enhances the accumulation of intravenously injected vancomycin-loaded porous silicon nanoparticles bearing CARG in S. aureus-infected mouse lung tissue. The targeted nanoparticles more effectively suppress staphylococcal infections in vivo relative to equivalent doses of untargeted vancomycin nanoparticles or of free vancomycin. The therapeutic delivery of antibiotic-carrying nanoparticles bearing peptides targeting infected tissues may help combat difficult-to-treat infections. Nanoparticles carrying an antibiotic and conjugated with a peptide identified via phage display that binds specifically to Staphylococcus aureus effectively suppress staphylococcal infections in vivo.

Anti-angiogenic and vascular disrupting therapies rely on the dependence of tumors on new blood vessels to sustain tumor growth. We previously reported a potent vascular disrupting agent, a theranostic nanosystem consisting of a tumor vasculature-homing peptide (CGKRK) fused to a pro-apoptotic peptide [D(KLAKLAK)2] coated on iron oxide nanoparticles. This nanosystem showed promising therapeutic efficacy in glioblastoma (GBM) and breast cancer models. However, complete control of the tumors was not achieved, and some tumors became non-responsive to the treatment. Here we examined the non-responder phenomenon in an aggressive MCF10-CA1a breast tumor model. In the treatment-resistant tumors we noted the emergence of CD31-negative patent neovessels and a concomitant loss of tumor homing of the nanosystem. In vivo phage library screening in mice bearing non-responder tumors showed that compared to untreated and treatment-sensitive tumors, treatment sensitive tumors yield a distinct landscape of vascular homing peptides characterized by over-representation of peptides that target αv integrins. Our approach may be generally applicable to the development of targeted therapies for tumors that have failed treatment.

CD44, the primary receptor for hyaluronic acid, plays an important role in tumor growth and metastasis. CD44-hyaluronic acid interactions can be exploited for targeted delivery of anticancer agents specifically to cancer cells. Although various splicing variants of CD44 are expressed on the plasma membrane of cancer cells, the hyaluronic acid binding domain (HABD) is highly conserved among the CD44 splicing variants. Using a novel two-step process, we have identified monothiophosphate-modified aptamers (thioaptamers) that specifically bind to the CD44's HABD with high affinities. Binding affinities of the selected thioaptamers for the HABD were in the range of 180-295 nM, an affinity significantly higher than that of hyaluronic acid (K(d) above the micromolar range). The selected thioaptamers bound to CD44 positive human ovarian cancer cell lines (SKOV3, IGROV, and A2780) but failed to bind the CD44 negative NIH3T3 cell line. Our results indicated that thio substitution at specific positions of the DNA phosphate backbone results in specific and high-affinity binding of thioaptamers to CD44. The selected thioaptamers will be of great interest for further development as a targeting or imaging agent for the delivery of therapeutic payloads for cancer tissues.

Shear-resistant adhesion and extravasation of disseminated cancer cells at the target organ is a crucial step in hematogenous metastasis. We found that the vascular adhesion molecule E-selectin preferentially promoted the shear-resistant adhesion and transendothelial migration of the estrogen receptor (ER)–/CD44+ hormone-independent breast cancer cells, but not of the ER+/CD44-/low hormone-dependent breast cancer cells. Coincidentally, CD44+ breast cancer cells were abundant in metastatic lung and brain lesions in ER– breast cancer, suggesting that E-selectin supports hematogenous metastasis of ER–/CD44+ breast cancer. In an attempt to prevent hematogenous metastasis through the inhibition of a shear-resistant adhesion of CD44+ cancer cells to E-selectin-expressing blood vessels on the premetastatic niche, an E-selectin targeted aptamer (ESTA) was developed. We demonstrated that a single intravenous injection of ESTA reduced metastases to a baseline level in both syngeneic and xenogeneic forced breast cancer metastasis models without relocating the site of metastasis. The effect of ESTA was absent in E-selectin knockout mice, suggesting that E-selectin is a molecular target of ESTA. Our data highlight the potential application of an E-selectin antagonist for the prevention of hematogenous metastasis of ER–/CD44+ breast cancer.

The involvement of E-selectin in cancer has long been recognized based on histopathological studies, and the important role of E-selectin in cancer progression and metastasis was further reinforced by a number of basic researches. Both the solid state and soluble forms of E-selectin have been tested as a possible biomarker for different cancer types for detection as well as for monitoring. While a number of studies have suggested the potential usefulness of E-selectin as a biomarker, the results from clinical trials are somewhat controversial and it has not been translated into clinical utility. It appears that the use of E-selectin as a single biomarker for diagnosis and prognosis may require more careful evaluation. The utility of E-selectin as a biomarker in conjunction with other biomarkers that are relevant to the biological cascade for E-selectin or are critical for patient prognosis may open up a new venue to further establish E-selectin as a cancer biomarker.

RNA interference (RNAi) is a powerful approach for silencing genes associated with a variety of pathologic conditions; however, in vivo RNAi delivery has remained a major challenge due to lack of safe, efficient, and sustained systemic delivery. Here, we report on a novel approach to overcome these limitations using a multistage vector composed of mesoporous silicon particles (stage 1 microparticles, S1MP) loaded with neutral nanoliposomes (dioleoyl phosphatidylcholine, DOPC) containing small interfering RNA (siRNA) targeted against the EphA2 oncoprotein, which is overexpressed in most cancers, including ovarian. Our delivery methods resulted in sustained EphA2 gene silencing for at least 3 weeks in two independent orthotopic mouse models of ovarian cancer following a single i.v. administration of S1MP loaded with EphA2-siRNA-DOPC. Furthermore, a single administration of S1MP loaded with-EphA2-siRNA-DOPC substantially reduced tumor burden, angiogenesis, and cell proliferation compared with a noncoding control siRNA alone (SKOV3ip1, 54%; HeyA8, 57%), with no significant changes in serum chemistries or in proinflammatory cytokines. In summary, we have provided the first in vivo therapeutic validation of a novel, multistage siRNA delivery system for sustained gene silencing with broad applicability to pathologies beyond ovarian neoplasms. Cancer Res; 70(9); 3687–96. ©2010 AACR.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive neoplastic diseases and is virtually incurable. The molecular mechanisms that contribute to the intrinsic resistance of PDAC to various anticancer therapies are not well understood. Recently, we have observed that several drug-resistant and metastatic tumors and tumor cell lines expressed elevated levels of tissue transglutaminase (TG2). Because PDAC exhibits inherent resistance to various drugs, we determined the constitutive expression of TG2 in 75 PDAC and 12 PDAC cell lines. Our results showed that 42 of 75 (56%) PDAC tumor samples expressed higher basal levels of TG2 compared with the normal pancreatic ducts [odds ratio (OR), 2.439; P = 0.012]. The increased expression of TG2 in PDAC was strongly associated with nodal metastasis (OR, 3.400; P = 0.017) and lymphovascular invasion (OR, 3.055; P = 0.045). Increased expression of TG2 was also evident in all 12 cell lines examined. The elevated expression of TG2 in PDAC cell lines was associated with gemcitabine resistance and increased invasive potential. Overexpression of catalytically active or inactive (C277S mutant) TG2 induced focal adhesion kinase (FAK) activation and augmented invasive functions in the BxPC-3 cell line. Conversely, down-regulation of TG2 by small interfering RNA attenuated FAK phosphorylation. Immunoprecipitation and confocal microscopy data revealed that TG2 was associated with FAK protein in PDAC cells. The activated FAK colocalized with TG2 at focal adhesion points. These results show for the first time that elevated expression of TG2 can induce constitutive activation of FAK and thus may contribute to the development of drug resistance and invasive phenotypes in PDAC. (Cancer Res 2006; 66(21): 10525-33)

The transcription factor nuclear factor-κB (NF-κB) plays an important role in regulating cell growth, apoptosis, and metastatic functions. Constitutive activation of NF-κB has been observed in various cancers; however, molecular mechanisms resulting in such activation remain elusive. Based on our previous results showing that drug-resistant and metastatic cancer cells have high levels of tissue transglutaminase (TG2) expression and that this expression can confer chemoresistance to certain types of cancer cells, we hypothesized that TG2 contributes to constitutive activation of NF-κB. Numerous lines of evidence showed that overexpression of TG2 is linked with constitutive activation of NF-κB. Tumor cells with overexpression of TG2 exhibited increased levels of constitutively active NF-κB. Activation of TG2 led to activation of NF-κB; conversely, inhibition of TG2 activity inhibited activation of NF-κB. Similarly, ectopic expression of TG2 caused activation of NF-κB, and inhibition of expression of TG2 by small interfering RNA abolished the activation of NF-κB. Our results further indicated that constitutive NF-κB reporter activity in pancreatic cancer cells is not affected by dominant-negative IκBα. Additionally, coimmunoprecipitation and confocal microscopy showed that IκBα is physically associated with TG2. Lastly, immunohistochemical analysis of pancreatic ductal carcinoma samples obtained from 61 patients further supported a strong correlation between TG2 expression and NF-κB activation/overexpression (P = 0.0098, Fisher's exact test). We conclude that TG2 induces constitutive activation of NF-κB in tumor cells via a novel pathway that is most likely independent of IκBα kinase. Therefore, TG2 may be an attractive alternate target for inhibiting constitutive NF-κB activation and rendering cancer cells sensitive to anticancer therapies. (Cancer Res 2006; 66(17): 8788-95)

Active targeting of a drug carrier to a specific target site is crucial to provide a safe and efficient delivery of therapeutics and imaging contrast agents. E-selectin expression is induced on the endothelial cell surface of vessels in response to inflammatory stimuli but is absent in the normal vessels. Thus, E-selectin is an attractive molecular target, and high affinity ligands for E-selectin could be powerful tools for the delivery of therapeutics and/or imaging agents to inflamed vessels. In this study, we identified a thiophosphate modified aptamer (thioaptamer, TA) against E-selectin (ESTA-1) by employing a two-step selection strategy: a recombinant protein-based TA binding selection from a combinatorial library followed by a cell-based TA binding selection using E-selectin expressing human microvascular endothelial cells. ESTA-1 selectively bound to E-selectin with nanomolar binding affinity (K(D) = 47 nM) while exhibiting minimal cross reactivity to P- and L-selectin. Furthermore, ESTA-1 binding to E-selectin on the endothelial cells markedly antagonized the adhesion (over 75% inhibition) of sLe(x) positive HL-60 cells at nanomolar concentration. ESTA-1 also bound specifically to the inflamed tumor-associated vasculature of human carcinomas derived from breast, ovarian, and skin but not to normal organs, and this binding was highly associated with the E-selectin expression level. Similarly, intravenously injected ESTA-1 demonstrated distinct binding to the tumor vasculature in a breast cancer xenograft model. Together, our data substantiates the discovery of a thioaptamer (ESTA-1) that binds to E-selectin with high affinity and specificity, thereby highlighting the potential application of ESTA-1 for E-selectin targeted delivery.

E-selectin (CD62E, ELAM-1 or LECAM-2) is a calcium dependent cell surface glycoprotein that mediates an adhesion of leukocytes on inflamed endothelium through recognition of specific carbohydrate ligands, sialyl Lewis X (sLeX) and sialyl Lewis A (sLeA). E-selectin expression is transcriptionally induced by NF-κB and AP-1 in response to inflammatory cytokines such as IL-1 and TNF-alpha. Accordingly, elevated E-selectin expression was reported in many types of acute and chronic inflammatory diseases including cancer. Aberrant homing of leukocytes to tumor results in tumor progression and local invasion. In addition, E-selectin and its counter ligands have also been reported to trigger the initial adhesive step of circulating cancer cells to endothelial cells. Therefore, blockade of E-selectin is a promising therapeutic strategy to control pathological infiltration of leukocytes and/or metastatic cancer cells. Thioaptamers (TA) are single stranded DNA molecules with thio substitution in the phosphate backbone, which provide stability against nucleases. Thioaptamer can fold into tertiary structures and bind with high affinity and specificity to a target molecule, and thus suitable for in vivo applications. In this study, we aimed to identify thioaptamer ligands that bind to E-selectin with high affinity and specificity. To achieve this goal, we used two-step selection strategies, recombinant protein based thioaptamer selection from a combinatorial thioaptamer library that contains 10E14 random sequences, followed by a cell based binding to tetracycline inducible E-selectin endothelial cells. We selected 14 different groups of thioaptamer candidates following 10 iterative cycles of binding to recombinant E-selectin protein. Using Tet-on inducible E-selectin endothelial cells, we identified the lead three TA candidates that bind to E-selectin on the surface of endothelial cells at high affinity (nM range) and specificity. In addition, the TA bound to tumor associated vasculature that express E-selectin. Moreover, the TA selected demonstrated significant inhibition of leukocyte adhesion to E-selectin positive endothelial cells at low nanomolar concentrations. In summary, we identified antagonistic TA against E-selectin that binds to endothelial cells and interferes with leukocyte bindings at nM range. Potential applications of this TA could be for 1) inflamed tumor vasculature selective targeted delivery of therapeutic molecules or drug delivery carriers 2) the prevention of pathological diapedesis of leukocytes and/or metastatic cancer cells. We are currently investigating potential clinical applications of the TA. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5712.