Preferential accumulation within tumors and in vivo imaging by functionalized luminescent dendrimer lanthanide complexes.

We have created a dendrimer complex suitable for preferential accumulation within liver tumors and luminescence imaging by substituting thirty-two naphthalimide fluorophores on the surface of the dendrimer and incorporating eight europium cations within the branches. We demonstrate the utility and performance of this luminescent dendrimer complex to detect hepatic tumors generated via direct subcapsular implantation or via splenic injections of colorectal cancer cells (CC531) into WAG/RijHsd rats. Luminescence imaging of the tumors after injection of the dendrimer complex via hepatic arterial infusion revealed that the dendrimer complex can preferentially accumulate within liver tumors. Further investigation indicated that dendrimer luminescence in hepatic tumors persisted in vivo. Due to the incorporation of lanthanide cations, this luminescence agent presents a strong resistance against photobleaching. These studies show the dendrimer complex has great potential to serve as an innovative accumulation and imaging agent for the detection of metastatic tumors in our rat hepatic model.

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.

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.

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.

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 G(0)/G(1) arrest and an increase in the apoptotic sub-G(1) 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.

Ultrasonic imaging of tumor angiogenesis using contrast microbubbles targeted via the tumor-binding peptide arginine-arginine-leucine.

Endothelial cells (EC) of angiogenic tumor vasculature are characterized by altered expression of molecular markers on their surface. Numerous peptides have been identified that specifically bind tumor angiogenic endothelium, including the tripeptide arginine-arginine-leucine (RRL). We hypothesized that ultrasound contrast microbubbles (MB) targeted via linkage with RRL would specifically adhere to tumor angiogenic endothelium versus normal myocardium, and that this selective adhesion could be detected ultrasonically. Microbubbles were conjugated to cyclic peptides containing either RRL (RRL-MB) or a glycine control sequence (control-MB). As measured in a parallel plate flow chamber, in vitro adhesion of RRL-MBs was three times greater to cultured tumor-derived ECs than to normal ECs (P < 0.01), demonstrating selective binding of RRL-MBs to tumor endothelium. Mice bearing s.c. Clone C or PC3 tumors were given i.v. injections of fluorescent RRL to show in vivo localization to tumor vasculature or were ultrasonically imaged following i.v. injections of targeted contrast MBs. Ultrasound images showed strong RRL-MB contrast enhancement within the tumors but not the control tissue myocardium. Control-MBs caused minimal enhancement in either tissue. Quantitative acoustic videointensity was significantly greater for the tumors than the hearts (5 +/- 1 versus 0.5 +/- 1 intensity units; P = 0.001). These data show that ultrasound contrast MBs targeted to tumor vasculature via RRL preferentially adhere to tumor versus normal vasculature and that this selective adherence can be detected with ultrasound. Targeted microbubbles may thus offer a noninvasive contrast-enhanced ultrasound imaging technique for the functional imaging of tumor neovascularization, and may have further implications for therapeutic tumor targeting.

Pharmacokinetics of 1alpha,25-dihydroxyvitamin D3 in normal mice after systemic exposure to effective and safe antitumor doses.

OBJECTIVES: Calcitriol, 1alpha,25-dihydroxyvitamin D(3) (1,25-D(3)) has potent antiproliferative effects and potentiates the antitumor activity of many other cytotoxic drugs. 1,25-D(3) 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-D(3) PK were examined in normal C3H/HeJ mice after a single intraperitoneal dose of 0.125 or 0.5 microg 1,25-D(3)/mouse. PK blood samples were collected from groups of 5-9 mice at each time point up to 24 h after 1,25-D(3) administration. Plasma 1,25-D(3) concentrations were measured by radioimmunoassay. Plasma 1,25-D(3) 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-D(3) 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 microg dosing, peak plasma 1,25-D(3) concentrations (Cp(max)) 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 (AUC(0->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 Cp(max) and apparent total plasma clearance were observed. CONCLUSIONS: These results demonstrate diurnal variation in baseline plasma 1,25-D(3) concentrations in mice. Plasma 1,25-D(3) 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.

Antiproliferative effects of 1alpha,25-dihydroxyvitamin D(3) and vitamin D analogs on tumor-derived endothelial cells.

Although there is abundant evidence that 1alpha,25-dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)] 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)(2)D(3.) We examined the ability of 1,25-(OH)(2)D(3) to inhibit the growth of tumor-derived endothelial cells (TDECs) and normal endothelial cells and to modulate angiogenic signaling. 1,25-(OH)(2)D(3) 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)(2)D(3) and dexamethasone had greater activity than either agent alone. 1,25-(OH)(2)D(3) increased vitamin D receptor and p27(Kip1) 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 carcinoma and radiation-induced fibrosarcoma-1 cells, 1,25-(OH)(2)D(3) treatment caused a reduction in the angiogenic signaling molecule, angiopoietin-2. In conclusion, 1,25-(OH)(2)D(3) and its analogs directly inhibit TDEC proliferation at concentrations comparable to those required to inhibit tumor cells. Further, 1,25-(OH)(2)D(3) modulates cell cycle and survival signaling in TDECs and affects angiogenic signaling in cancer cells. Thus, our work supports the hypothesis that angiogenesis inhibition plays a role in the antitumor effects of 1,25-(OH)(2)D(3).