Ruth A. Modzelewski

Antiproliferative Effects of 1α,25-Dihydroxyvitamin D3 and Vitamin D Analogs on Tumor-Derived Endothelial Cells

Although there is abundant evidence that 1α,25-dihydroxyvitamin D3 [1,25-(OH)2D3] inhibits the growth of several cancer cell types, inhibition of angiogenesis may also play a role in mediating the antitumor effects of 1,25-(OH)2D3. We examined the ability of 1,25-(OH)2D3 to inhibit the growth of tumor-derived endothelial cells (TDECs) and normal endothelial cells and to modulate angiogenic signaling. 1,25-(OH)2D3 inhibited the growth of TDECs from two tumor models at nanomolar concentrations, but was less potent against normal aortic or yolk sac endothelial cells. The vitamin D analogs Ro-25-6760, EB1089, and ILX23-7553 were also potent inhibitors of TDEC proliferation. Furthermore, the combination of 1,25-(OH)2D3 and dexamethasone had greater activity than either agent alone. 1,25-(OH)2D3 increased vitamin D receptor and p27Kip1 protein levels in TDECs, whereas phospho-ERK1/2 and phospho-Akt levels were reduced. These changes were not observed in normal aortic endothelial cells. In squamous cell carcinom...

GENE THERAPY OF CARCINOMA USING ULTRASOUND-TARGETED MICROBUBBLE DESTRUCTION

When microbubble contrast agents are loaded with genes and systemically injected, ultrasound-targeted microbubble destruction (UTMD) facilitates focused delivery of genes to target tissues. A mouse model of squamous cell carcinoma was used to test the hypothesis that UTMD would specifically transduce tumor tissue and slow tumor growth when treated with herpes simplex virus thymidine kinase (TK) and ganciclovir. UTMD-mediated delivery of reporter genes resulted in tumor expression of luciferase and green fluorescent protein (GFP) in perivascular areas and individual tumor cells that exceeded expression in control tumors (p=0.02). The doubling time of TK-treated tumors was longer than GFP-treated tumors (p=0.02), and TK-treated tumors displayed increased apoptosis (p=0.04) and more areas of cellular drop-out (p=0.03). These data indicate that UTMD gene therapy can transduce solid tumors and mediate a therapeutic effect. UTMD is a promising nonviral method for targeting gene therapy that may be useful in a spectrum of tumors.

A novel approach for the identification of unique tumor vasculature binding peptides using an E. coli peptide display library.

AbstractBackground: Tumor neovascularization is necessary for continued tumor growth and metastasis. During the process of endothelial cell (EC) recruitment and tumor infiltration, specific molecular markers unique for this interaction are expressed on the EC surface. Targeting these molecular markers would, in effect, allow for specific tumor targeting. Tripeptide sequence motifs have previously been reported that will bind to angiogenic tumor ECs. These sequences were identified from in vivo phage peptide display libraries. The purpose of this study was to use a more simplified bacterial peptide display library in an in vitro system to seek out peptide motifs with unique binding to tumor microvasculature. Methods: FliTrx™ is a bacterial peptide display library containing the entire repertoire of possible random dodecapeptides expressed on the flagella tip of E. coli. Two EC populations were used for the screening process, Matrigel invading cells (MAGIC) and tumor–derived endothelial cells (TDEC). MAGIC are obtained from ECs that infiltrate a subcutaneous fibroblast growth factor–containing Matrigel deposit, and TDEC are ECs selectively obtained from tumor vasculature. FliTrx cells were incubated with MAGIC at 4°C to remove any potential clones displaying peptides that will bind to nonspecific EC surface targets. The non–binding cells were then incubated with TDEC, allowing for clones displaying potential binding peptides to bind tumor specific targets on TDECs. The bacterial population was then expanded and this “panning” process was carried out a total of five times. Peptide insert sequences from 100 bacterial colonies were analyzed for potential repetitive peptide motifs. Results: Recurring peptide sequences were detected that were 3–mers (13 sequences) and 4–mers (4 sequences). Of the 3–mers, four repeated 3 times, whereas none of the 4–mers repeated more than twice. All of the repeated sequences were basic in charge, and arginine was the most commonly seen amino acid. A tripeptide basic–basic–nonpolar amino acid arrangement was the most prevalent charge sequence in all repetitive motifs (17 repeat sequences). Two test peptides showed TDEC binding specificity, and both conformed to the basic–basic–nonpolar motif. Conclusions: We report peptide sequences derived from panning an in vitro system designed to detect tumor–EC specific markers. These putative motifs may serve as molecular determinants for a novel therapeutic modality aimed at specifically targeting tumors through tumor angiogenic vessels.

Pharmacokinetics of 1α,25-Dihydroxyvitamin D3 in Normal Mice after Systemic Exposure to Effective and Safe Antitumor Doses

Objective: Calcitriol, 1α,25-dihydroxyvitamin D3 (1,25-D3) has potent antiproliferative effects and potentiates the antitumor activity of many other cytotoxic drugs. 1,25-D3 plasma pharmacokinetic (PK) parameters associated with antitumor activity in experimental animal models are unknown. The objective of this study was to determine plasma calcitriol PK in normal mice at doses of calcitriol which are active in suppressing tumor growth. Methods: Plasma 1,25-D3 PK were examined in normal C3H/HeJ mice after a single intraperitoneal dose of 0.125 or 0.5 µg 1,25-D3/mouse. PK blood samples were collected from groups of 5–9 mice at each time point up to 24 h after 1,25-D3 administration. Plasma 1,25-D3 concentrations were measured by radioimmunoassy. Plasma 1,25-D3 concentration diurnal variation was determined in blood samples from untreated animals collected in the morning (9:00–11:00 a.m.) and in the evening (4:00–9:00 p.m.). Results: Median baseline plasma 1,25-D3 concentration measured in the morning and in the evening were 0.082 ng/ml (CI 95%, 0.076–0.099) and 0.067 ng/ml (CI 95%, 0.058–0.075), respectively (p = 0.004). After 0.125 and 0.5 µg dosing, peak plasma 1,25-D3 concentrations (Cpmax) were 12.0 ng/ml (CI 95%, 10.8–12.6) and 41.6 ng/ml (CI 95%, 40.8–53.6), respectively. The corresponding areas under the curve (AUC0–>24 h) were 47.0 (CI 95%, 43.2–51.1) and 128.0 (CI 95%, 127.0–130.0) ng·h/ml. No dose-related changes in time to Cpmax and apparent total plasma clearance were observed. Conclusions: These results demonstrate diurnal variation in baseline plasma 1,25-D3 concentrations in mice. Plasma 1,25-D3 PK in mice receiving doses that are effective in slowing tumor growth, inducing cell cycle arrest and apoptosis, and potentiating taxanes and platinum analogue antitumor activity are at least 5–10 times higher than those easily achieved and nontoxic in patients receiving high-dose intermittent oral therapy.

Calcitriol (1,25-dihydroxycholecalciferol) selectively inhibits proliferation of freshly isolated tumor-derived endothelial cells and induces apoptosis

Calcitriol (1,25-dihydroxycholecalciferol) has antiproliferative and/or proapoptotic effects on many cell types and the glucocorticoid dexamethasone enhances these effects. We have shown that calcitriol modulates several key signaling proteins involved in differentiation, proliferation and apoptosis in tumor-derived murine endothelial cells (TDEC) and that these effects were not seen with endothelial cells isolated similarly from normal tissues. In the present study, TDEC and mouse embryonic yolk sac endothelial cells (MYSEC) were treated with calcitriol and followed over time for an effect. MYSEC were utilized as ‘normal’ control endothelial cells because they were more primitive, being isolated from a highly neovascular tissue, and had a similar morphology without the stimulus of the tumor microenvironment. The vitamin D receptor (VDR) is present in TDEC and MYSEC, and was upregulated in calcitriol-treated TDEC and MYSEC; dexamethasone further increased VDR expression following 48 h of treatment. The modulatory effects on signaling proteins were maximal by treatment for 48 h; phospho-Erk, phospho-Akt, p21 and bcl-2 were decreased in treated TDEC with the induction of p27 but there were no effects on MYSEC. After 48 h increased apoptosis was seen in treated TDEC by annexin V labeling with caspase-3 cleavage and decreased levels of poly(ADP-ribose) polymerase, but no effects were seen in MYSEC. Cell cycle analysis showed increased G₀/G1 arrest and an increase in the apoptotic sub-G1 peak in treated TDEC but similar effects were not seen in MYSEC following 48-hour treatment. Proliferation assays were utilized and TDEC demonstrated decreased proliferation compared to normal endothelial cells at 48 h. To determine whether or not the VDR signaling was impaired in MYSEC, we performed the 24-hydroxylase (CYP24) promoter-luciferase reporter assay. CYP24 is a key enzyme involved in the breakdown of vitamin D. VDR signaling was intact in both cell types and calcitriol induced CYP24 mRNA expression in MYSEC but not in TDEC. Taken together, despite similar levels of VDR expression and intact signaling in both cell types, calcitriol selectively inhibits proliferation and induces apoptosis in TDEC with no effect on MYSEC. Thus calcitriol exerts differential effects on TDEC compared to normal cells.

Anti-proliferative effects of calcitriol on endothelial cells derived from two different microenvironments

Calcitriol (1,25-dihydroxycholecalciferol), the active form of Vitamin D, is anti-proliferative in tumor cells and tumor-derived endothelial cells (TDEC). However, endothelial cells isolated from normal tissues as cell lines or freshly isolated cells or from implanted Matrigel plugs (MDEC) are relatively resistant. Both TDEC and MDEC express similar amounts of Vitamin D receptor (VDR) protein. Although the VDR from TDEC has higher binding affinity for calcitriol than those from MDEC, VDR in both cell types translocates to the nucleus and transactivates the 24-hydroxylase promoter-luciferase construct. Calcitriol selectively inhibits the growth of TDEC but not MDEC by inducing G(0)/G(1) cell cycle arrest and by promoting apoptosis. This selectivity appears to be related to 24-hydroxylase (CYP24) expression. Calcitriol significantly induced CYP24 expression in MDEC but not in TDEC and inhibition of CYP24 activity in MDEC restores their sensitivity to calcitriol. These findings indicate that the induction of CYP24 expression differs in endothelial cells isolated from different microenvironments (TDEC versus MDEC) and that this distinction contributes to selective calcitriol-mediated growth inhibition in these cell types.

Synergistic enhancement by interleukin-1 α of cisplatin-mediated antitumor activity in RIF-1 tumor-bearing C3H/HeJ mice

Administration of interleukin-1 α (IL-1 α) plus certain cytotoxic drugs causes substantially greater clonogenic tumor-cell kill and tumor-regrowth delay than does treatment with either agent alone. IL-1 α itself has little effect on tumor growth despite its ability to induce acute hemorrhagic necrosis, restrict tumor blood flow, and cause microvascular injury in a variety of murine model systems. To investigate further IL-1 αs ability to enhance the antitumor activity of cytotoxic drugs, we initiated studies to examine the effect of IL-1 α on cisplatin (cDDP)-mediated cytotoxicity using the RIF-1 tumor system. The antitumor activity of IL-1 α and cDDP was quantitated through standard clonogenic tumor-cell survival assays, a tumor hemorrhagic necrosis assay and tumor-regrowth delay studies, with the interaction between IL-1 α and cDDP being analyzed through median dose-effect. In vitro, IL-1 α had no enhancing effect on the cDDP-mediated tumorcell kill. For examination of the in vivo efficacy of this regimen. RIF-1 tumor-bearing C3H/HeJ mice (14 days postimplantation) were treated concurrently with single i.p. injections of IL-1 α and/or cDDP at various doses. The increased clonogenic tumor-cell kill obtained with IL-1 α /cDDP was dose-dependent, with significant enhancement by IL-1 α being observed (P<0.001), even at the lowest doses tested (2 mg/kg and 6 μg/kg for cDDP and IL-1 α, respectively), but it did not correlate with an increase in tumor hemorrhage. Using median dose-effect analysis, this interaction was determined to be strongly synergistic. When treated animals were monitored for long-term antitumor effects, combinations with IL-1 α significantly increased the tumor-regrowth delay and decreased the fractional tumor volume (P<0.001). These results demonstrate that IL-1 α synergistically enhances cDDP mediated in vivo antitumor activity and suggest that the combination of IL-1 α and cDDP may have potential therapeutic application in the design of effective treatment modalities for cancer.

Gene therapy and endothelial cell targeting for cancer.

The endothelium represents a potentially critical target for gene therapy because of its anatomical location and its importance in the viability in both normal and malignant tissues. Protecting the endothelium of normal tissues, such as the lungs, from the toxic effects of current antineoplastic agents and the destruction of the tumor vasculature are reasonable goals. As a target, however, the endothelium continues to represent a significant challenge. While gene delivery to cultured endothelial cells is possible, improved delivery systems are required, as well as cell-specific promoters, before in vivo gene therapy to important endothelial populations can be accomplished.

Ultrasound contrast microbubbles targeted to tumor angiogenesis specifically bind tumor-derived endothelial cells

Angiogenesis is a key phenomenon in the continued growth of clinically-significant tumors. Endothelial cells (ECs) of tumor vasculature are phenotypically dissimilar from those in normal tissues, and are characterized by altered expression of molecular markers on the EC surface. Various peptides have been identified that specifically bind to tumor angiogenic endothelium, including the tripeptide Arg-Arg-Leu (MW3). Previously, we have shown that lipid-based ultrasound contrast microbubbles (MBs) can be specifically targeted to EC inflammatory markers. Here, we hypothesized that MBs targeted via MW3 would specifically adhere to tumor angiogenic ECs in vitro. Microbubbles were conjugated via avidin/biotin bridging chemistry to cyclic peptides containing either MW3 or glycine control tripeptides. In a parallel plate chamber, MBs were perfused across coverslips of cultured human coronary artery ECs (HCAECs) or mouse tumor-derived ECs (TDECs) (3 min; wall shear rate 100s/sup -1/) and washed. Adhesion to ECs was quantified in 20 random microscopic fields per coverslip. MW3-MB adherence was 3 to 6-fold greater than glycine-MB (p<0.01). MW3-MB adherence to TDECs was 3 times greater than to HCAECs (p<0.01). These data demonstrate that MW3-MBs specifically adhere to tumor angiogenic ECs in vitro, potentially offering a means for noninvasive functional imaging of tumor neovascularization and therapeutic tumor targeting.

Inter-institutional development of a poster-based cancer biology learning tool.

There is a paucity of African-American Cancer researchers. To help address this, an educational collaboration was developed between a Comprehensive Cancer Center and a distant undergraduate biology department at a minority institution that sought to teach students introductory cancer biology while modeling research culture. A student-centered active learning curriculum was established that incorporated scientific poster presentations and simulated research exercises to foster learning of cancer biology. Students successfully mined primary literature for supportive data to test cancer-related hypotheses. Student feedback indicated that the poster project substantially enhanced depth of understanding of cancer biology and laid the groundwork for subsequent laboratory work. This inter-institutional collaboration modeled the research process while conveying facts and concepts about cancer.