Expression, localization and polymorphisms of the nuclear receptor PXR in Barrett's esophagus and esophageal adenocarcinoma.

BACKGROUND: The continuous exposure of esophageal epithelium to refluxate may induce ectopic expression of bile-responsive genes and contribute to the development of Barrett's esophagus (BE) and esophageal adenocarcinoma. In normal physiology of the gut and liver, the nuclear receptor Pregnane × Receptor (PXR) is an important factor in the detoxification of xenobiotics and bile acid homeostasis. This study aimed to investigate the expression and genetic variation of PXR in reflux esophagitis (RE), Barrett's esophagus (BE) and esophageal adenocarcinoma. METHODS: PXR mRNA levels and protein expression were determined in biopsies from patients with adenocarcinoma, BE, or RE, and healthy controls. Esophageal cell lines were stimulated with lithocholic acid and rifampicin. PXR polymorphisms 25385C/T, 7635A/G, and 8055C/T were genotyped in 249 BE patients, 233 RE patients, and 201 controls matched for age and gender. RESULTS: PXR mRNA levels were significantly higher in adenocarcinoma tissue and columnar Barrett's epithelium, compared to squamous epithelium of these BE patients (P<0.001), and RE patients (P=0.003). Immunohistochemical staining of PXR showed predominantly cytoplasmic expression in BE tissue, whereas nuclear expression was found in adenocarcinoma tissue. In cell lines, stimulation with lithocholic acid did not increase PXR mRNA levels, but did induce nuclear translocation of PXR protein. Genotyping of the PXR 7635A/G polymorphism revealed that the G allele was significantly more prevalent in BE than in RE or controls (P=0.037). CONCLUSIONS: PXR expresses in BE and adenocarcinoma tissue, and showed nuclear localization in adenocarcinoma tissue. Upon stimulation with lithocholic acid, PXR translocates to the nuclei of OE19 adenocarcinoma cells. Together with the observed association of a PXR polymorphism and BE, this data implies that PXR may have a function in prediction and treatment of esophageal disease.

Bile acid-stimulated expression of the farnesoid X receptor enhances the immune response in Barrett esophagus.

OBJECTIVES: Barrett's esophagus (BE) is a premalignant condition of the esophagus. It is a consequence of mucosal injury from chronic gastroesophageal reflux in which bile acids are an important toxic component. The farnesoid X receptor (FXR) is a nuclear receptor involved in the regulation of bile acid synthesis, transport, and absorption. FXR activation is also involved in the induction of the innate immune response. This suggests that FXR is involved in the pathogenesis and the inflammation seen in BE. METHODS: mRNA levels of FXR and the FXR-regulated genes, ileal bile acid-binding protein (IBABP), small heterodimer partner (SHP), and chemokines interleukin (IL)-8 and macrophage inflammatory protein 3 alpha (MIP3 alpha), were determined by real time-polymerase chain reaction (RT-PCR). Protein expression was determined by immunohistochemistry. RESULTS: FXR was not expressed in squamous epithelium of healthy subjects (N = 7), but was present in both squamous and columnar epithelium of BE patients. Compared to the squamous epithelium of BE patients, their columnar epithelium displayed a 2.3-fold (P= 0.02) increase in FXR mRNA. Also, IBABP (2.2-fold; P= 0.0029), SHP (2.7-fold; P= 0.007), IL-8 (1.5-fold; P= 0.04), and MIP3 alpha (1.7-fold; P= 0.019) transcription levels were increased. Exposure of esophageal cell line TE7 to deoxycholic acid (DCA) resulted in a similar induction. The induction was abolished by the FXR antagonist guggulsterone. CONCLUSIONS: Expression levels of the bile acid receptor FXR, the bile acid metabolism genes IBABP and SHP, and the chemokines IL-8 and MIP3 alpha are increased in Barrett's epithelium. The in vitro induction of FXR by DCA suggests that bile acids can actively induce the inflammatory response in BE by recruiting immune cells.

Anticancer activity of targeted proapoptotic peptides.

UNLABELLED: Tumor-induced angiogenesis can be targeted by RGD (Arg-Gly-Asp) peptides, which bind to alpha(v)beta(3)-receptors upregulated on angiogenic endothelial cells. RGD-containing peptides are capable of inducing apoptosis through direct activation of procaspase-3 to caspase-3 in cells. Additionally, tumor cells overexpressing somatostatin receptors can be targeted by somatostatin analogs. Radiolabeled somatostatin analogs are successfully used to image and treat such tumors via receptor-targeted scintigraphy and therapy. We combined these 2 peptides, RGD and somatostatin, to synthesize a new hybrid peptide, RGD-diethylenetriaminepentaacetic acid (DTPA)-octreotate (c(Arg-Gly-Asp-D-Tyr-Asp)-Lys(DTPA)-D-Phe-c(Cys-Tyr-D-Trp-Lys-Thr-Cys)-Thr). An earlier study showed that tumor-bearing rats had high receptor-specific uptake of RGD-(111)In-DTPA-octreotate in somatostatin receptor subtype 2-positive tissues and tumors. Furthermore, RGD-(111)In-DTPA-octreotate showed a pronounced tumoricidal effect, which is probably the result of increased apoptosis, as is shown by an increased caspase-3 activity after incubation with (111)In-labeled RGD-DTPA-octreotate in comparison with the 2 monopeptides (111)In-DTPA-RGD and (111)In-DTPA-Tyr(3)-octreotate. In this study, we evaluated the biodistributions of RGD-(111)In-DTPA-octreotate and (125)I-RGD-octreotate and investigated the caspase-3 activation of the unlabeled compound RGD-DTPA-octreotate in vitro. METHODS: Biodistribution studies on tumor-bearing rats were performed with RGD-(111)In-DTPA-octreotate and (125)I-RGD-octreotate. The apoptotic activity, by activation of caspase-3 with RGD-DTPA-octreotate and RGD-octreotate, was examined using colorimetric and immunocytochemical assays. RESULTS: The radiolabeled compound, RGD-(111)In-DTPA-octreotate, showed high uptake and retention in the rats in which rat pancreatic CA20948 tumor had been implanted. A major drawback was high renal uptake. In vitro, the unlabeled peptide RGD-DTPA-octreotate induced a significant increase in caspase-3 levels in various cell lines in comparison with RGD and Tyr(3)-octreotate (P < 0.01). Caspase-3 activation was time dependent. To alter the elimination route, we examined the biodistribution of radioiodinated RGD-octreotate without DTPA [c(Arg-Gly-Asp-D-Tyr-Asp)-D-Phe-c(Cys-Tyr-D-Trp-Lys-Thr-Cys)-Thr], as a model of unlabeled RGD-octreotate, in tumor-bearing rats. (125)I-RGD-octreotate showed a much lower renal uptake than did RGD-(111)In-DTPA-octreotate. Furthermore, the affinity of RGD-octreotate increased in comparison with RGD-DTPA-octreotate (values of 1.4 x 10(-8) mol/L vs. 9.4 x 10(-8) mol/L, respectively, for inhibitory concentration of 50%). Finally, RGD-octreotate was still able to activate caspase-3, as was indicated with immunocytochemistry. CONCLUSION: Because of the high renal uptake, RGD-(111)In-DTPA-octreotate is unsuitable for radionuclide therapy. However, the unlabeled peptides, RGD-DTPA-octreotate and RGD-octreotate, also induced an increase in caspase-3 levels, indicating the therapeutic potential of this compound. Thus, the development of hybrid molecules can become a new approach in the treatment of cancer.

111In-labelled somatostatin analogues in a rat tumour model: somatostatin receptor status and effects of peptide receptor radionuclide therapy.

PURPOSE: Peptide receptor scintigraphy with the radioactive somatostatin analogue 111In-DTPA-octreotide is a sensitive and specific technique to show in vivo the presence of somatostatin receptors on various tumours. Since 111In emits not only gamma rays but also therapeutic Auger and internal conversion electrons with a medium to short tissue penetration (0.02-10 microm and 200-550 microm, respectively), 111In-DTPA-octreotide is also being used for peptide receptor radionuclide therapy (PRRT). In this study we investigated the therapeutic effects of 111In-DTPA-octreotide in tumours of various sizes. Regrowth of a tumour despite PRRT with 111In-DTPA-octreotide may be due to the lack of crossfire from 111In, whereby any possible receptor-negative tumour cell can multiply. We therefore also investigated the somatostatin receptor status of the tumour before and after PRRT. METHODS: The radiotherapeutic effects of different doses of 111In-DTPA-octreotide in vivo were investigated in Lewis rats bearing small (< or = 1 cm2) or large (> or = 8 cm2) somatostatin receptor-positive rat pancreatic CA20948 tumours expressing the somatostatin receptor subtype 2 (sst2). In addition, the somatostatin receptor density on the tumour after injection of a therapeutic labelled somatostatin analogue was investigated when the tumour was either declining in size or regrowing after an initial reduction in size. To initiate a partial response of the tumour (so that regrowth would follow) and not a complete response, a relatively low dose was administered. RESULTS: Impressive radiotherapeutic effects of 111In-labelled octreotide were observed in this rat tumour model. Complete responses (up to 50%) were found in the animals bearing small (< or 1 cm2) tumours after at least three injections of 111 MBq or a single injection of 370 MBq 111In-DTPA-octreotide, leading to a dose of 6.3-7.8 mGy/MBq (1-10 g tumour). In the rats bearing the larger (> or = 8 cm2) tumours, the effects were much less pronounced and only partial responses were achieved in these groups. Clear sst2 expression was found in the control as well as in the treated tumours. A significantly higher tumour receptor density (p<0.001) was found when the tumours regrew after an initial decline in size after low-dose PRRT in comparison with the untreated tumours. CONCLUSION: Therapy with 111In-labelled somatostatin analogues is feasible but should preferably start as early as possible during tumour development. One might also consider the use of radiolabelled somatostatin analogues in an adjuvant setting after surgery of somatostatin receptor-positive tumours in order to eradicate occult metastases. We showed that PRRT led to an increase in the density of somatostatin receptors when the tumours regrew after an initial decline in size because of PRRT. Upregulation of the somatostatin receptor may lead to higher uptake of radiolabelled peptides in therapeutic applications, which would probably make repeated injections of radiolabelled peptides more effective.

Induction of apoptosis with hybrids of Arg-Gly-Asp molecules and peptides and antimitotic effects of hybrids of cytostatic drugs and peptides.

The presence of a high density of somatostatin receptors (SSRs) on human tumors forms the basis for the successful visualization of primary tumors and their metastases using radiolabeled somatostatin analogs. In recent years somatostatin analogs, coupled to beta-emitting radioisotopes, have been successfully applied in the treatment of patients with metastatic SSR-positive neuroendocrine tumors. This concept of targeting SSR-expressing tumors using peptide receptor radionuclide therapy may also apply to the use of somatostatin analogs coupled to chemotherapeutic compounds. Evidence for the effectiveness of such cytotoxic somatostatin analogs as antitumor agents has been provided in a significant number of studies in experimental tumor models. In addition to cytotoxic somatostatin analogs, somatostatin analogs coupled to peptides containing arginine, glycine, and aspartate and coupled to paclitaxel have been synthesized. Here we discuss the development of the different cytotoxic somatostatin analogs and their antitumor effects in vitro and in vivo in experimental models.

Increased cell death after therapy with an Arg-Gly-Asp-linked somatostatin analog.

UNLABELLED: Receptor-targeted scintigraphy and radionuclide therapy with radiolabeled somatostatin analogs are successfully applied for somatostatin receptor-positive tumors. The synergistic effects of an apoptosis-inducing factor, for example, the Arg-Gly-Asp (RGD) motif, can increase the radiotherapeutic efficacy of these peptides. Hence, the tumoricidal effects of the hybrid peptide RGD-diethylaminetriaminepentaacetic acid (DTPA)-Tyr3-octreotate (cyclic[c](Arg-Gly-Asp-D-Tyr-Asp)-Lys(DTPA)-D-Phe-c(Cys-Tyr-D-Trp-Lys-Thr-Cys)-Thr), hereafter referred to as RGD-DTPA-octreotate, were evaluated in comparison with those of RGD (c(Arg-Gly-Asp-D-Tyr-Asp)) and Tyr3-octreotate (D-Phe-c(Cys-Tyr-D-Trp-Lys-Thr-Cys)-Thr). METHODS: The therapeutic effects of RGD-111In-DTPA-octreotate, 111In-DTPA-RGD, and 111In-DTPA-Tyr3-octreotate were investigated with various cell lines by use of a colony-forming assay, and caspase-3 activity was also determined. RESULTS: Tumoricidal effects were found with 111In-DTPA-RGD, 111In-DTPA-Tyr3-octreotate, and RGD-111In-DTPA-octreotate, in order from least effective to most effective. Also, the largest increase in caspase-3 levels was found with RGD-111In-DTPA-octreotate. CONCLUSION: RGD-111In-DTPA-octreotate has more pronounced tumoricidal effects than 111In-DTPA-RGD and 111In-DTPA-Tyr3-octreotate, because of increased apoptosis, as indicated by increased caspase-3 activity.

Low-dose-rate irradiation by 131I versus high-dose-rate external-beam irradiation in the rat pancreatic tumor cell line CA20948.

AIM: The rat pancreatic CA20948 tumor cell line is widely used in receptor-targeted preclinical studies because many different peptide receptors are expressed on the cell membrane. The response of the tumor cells to peptide radionuclide therapy, however, is dependent on the cell line's radiosensitivity. Therefore, we measured the radiosensitivity of the CA20948 tumor cells by using clonogenic survival assays after high-energy external-beam radiotherapy (XRT) in vitro. It can, however, be expected that results of high-dose-rate XRT are not representative for those after low-dose-rate radionuclide therapy (RT), such as peptide-receptor radionuclide therapy. Therefore, we compared clonogenic survival in vitro in CA20948 tumor cells after increasing doses of XRT or RT, the latter using (131)I. METHODS: Survival of CA20948 cells was investigated using a clonogenic survival assay after RT by incubation with increasing amounts of (131)I, leading to doses of 1-10 Gy after 12 days of incubation (maximum dose rate, 0.92 mGy/min), or with doses of 1-10 Gy using an X-ray machine (dose rate, 0.66 Gy/min). Colonies were scored after a 12-day-incubation period. Also, the doubling time of this cell line was calculated. RESULTS: We observed a dose-dependent reduction in tumor-cell survival, which, at low doses, was similar for XRT and RT. For high-dose-rate XRT, the quadratic over linear component ratio (alpha/beta) for CA20948 was 8.3 Gy, whereas that ratio for low-dose-rate RT was calculated to be 86.5 Gy. The calculated doubling time of CA20948 cells was 22 hours. CONCLUSIONS: Despite the huge differences in dose rate, RT tumor cell-killing effects were approximately as effective as those of XRT at doses of 1 and 2 Gy, the latter being the common daily dose given in fractionated external-beam therapies. At higher doses, RT was less effective than XRT.

Radiolabeled RGD-DTPA-Tyr3-octreotate for receptor-targeted radionuclide therapy.

The aim of this study was to develop and investigate a radiopeptide for the treatment of cancers which overexpress cell surface somatostatin receptors. The new radiopharmaceutical is composed of a somatostatin receptor-targeting peptide, a chelator (DTPA) to enable radiolabeling, and an apoptosis-inducing RGD (arginine-glycine-aspartate) peptide moiety. The receptor-targeting peptide portion of the molecule, Tyr3-octreotate, is specific for the somatostatin subtype-2 cell surface receptor (sst2), which is overexpressed on many tumor cells. Because of the rapid endocytosis of the somatostatin receptor, the entire molecule can thus be internalized, allowing the RGD portion to activate intracellular caspases, which in turn promotes apoptosis. In this paper, we present the synthesis and the in vitro and in vivo tumor binding and internalization characteristics of this hybrid peptide. In vitro internalization into sst2-positive tumor cells of the radiolabeled hybrid peptide appeared to be a rapid process and could be blocked by an excess of unlabeled octreotide, indicating an sst2-specific process. Tumor uptake in vivo in rats of radiolabeled RGD-DTPA-Tyr3-octreotate was in agreein vitro data and similar to that of radiolabeled DOTA-Tyr3-octreotate. The combined molecule is expected to significantly enhance the therapeutic efficacy of the somatostatin-based agent.

Tyr3-octreotide and Tyr3-octreotate radiolabeled with 177Lu or 90Y: peptide receptor radionuclide therapy results in vitro.

Somatostatin analogs promising for peptide receptor scintigraphy (PRS) and peptide receptor radionuclide therapy (PRRT) are D-Phe-c(Cys-Tyr-D-Trp-Lys-Thr-Cys)-Thr(ol) (Tyr 3-octreotide) and D-Phe-c(Cys-Tyr-D-Trp-Lys-Thr-Cys)-Thr (tyr3-octreotate). For radiotherapeutic applications these peptides are being labeled with the beta(-) particle emitters 177Lu or 90Y. We evaluated the therapeutic effects of these analogs chelated with tetra-azacyclododecatatro-acetic acid (DOTA) and labeled with 90Y or 177Lu in an in vitro colony-forming assay using the rat pancreatic tumor cell line CA20948. Furthermore, we investigated the effects of incubation time, radiation dose, and specific activity of [177Lu-DOTA]-D-Phe1-c (Cys-Tyr-D-Trp-Lys-Thr-Cys)-Thr (177Lu-octreotate). 177Lu-octreotate could reduce tumor growth to 100% cell kill and effects were dependent on radiation dose, incubation time, and specific activity used. Similar concentrations of 177Lu-DOTA, which is not bound to the cells, had a less pronounced effect on the tumor cell survival. Both tyr3-octreotide and tyr3-octreotate labeled with either 177Lu or 90Y, using DOTA as chelator, were able to control tumor growth in a dose-dependent manner. In all concentrations used radiolabeled tyr3-octreotate had a higher tumor kill compared to radiolabeled tyr3-octreotide, labeled with 177Lu or 90Y. This is in accordance with the higher affinity of tyr3-octreotate for the subtype 2 (sst2)-receptor compared to tyr3-octreotide, leading to a higher amount of cell-associated radioactivity, resulting in a significantly higher tumor radiation dose. In conclusion, tyr3-octreotate labeled with 177Lu or 90Y is the most promising analog for PRRT.

Anti-tumor effect and increased survival after treatment with [177Lu-DOTA0,Tyr3]octreotate in a rat liver micrometastases model.

Peptide receptor scintigraphy with [(111)In-DTPA(0)]octreotide (a stabilized radiolabeled somatostatin (SS) analogue, OctreoScan) is widely used for the visualization and staging of somatostatin receptor-positive tumors. The application of likewise somatostatin analogues as vehicle for the deliverance of radionuclides to somatostatin receptor-positive targets are now in use for peptide receptor-targeted radionuclide therapy (PRRT). Currently preclinical and clinical investigation are ongoing trying to find the optimal combination of radionuclide and ligand. The anti-tumoral effects of such combinations, like [90Y-DOTA degrees, Tyr(3)]octreotide and [(177)Lu-DOTA degrees, Tyr(3)]octreotate, on SSR-positive solid tumors have been reported. In this study we present the anti-tumor effects of (177)Lu-DOTA-tate on: a) a single SSR-positive cell model and b) on a SSR-positive tumor in a rat liver micrometastatic model, mimicking disseminated disease. (177)Lu-DOTA-tate showed anti-tumoral effects in both cases and significant survival in the PRRT-treated rats. (177)Lu-DOTA-tate is a very promising new treatment modality for SSR-positive tumors, including disseminated disease.

Peptide receptor radionuclide therapy in vitro using [111In-DTPA0]octreotide.

UNLABELLED: Peptide receptor radionuclide therapy (PRRT) using [(111)In-DTPA(0)]octreotide (where DTPA is diethylenetriaminepentaacetic acid) is feasible because, besides gamma-radiation, (111)In emits both therapeutic Auger and internal conversion electrons having a tissue penetration of 0.02-10 and 200-500 micro m, respectively. The aim of this study was to investigate the therapeutic effects of [(111)In-DTPA(0)]octreotide in a single-cell model including the effects of incubation time, radiation dose, and specific activity of [(111)In-DTPA(0)]octreotide. Finally, we discriminated between the effects of the Auger electrons and internal conversion electrons in PRRT. METHODS: An in vitro, colony-forming assay to study cell survival after PRRT using the sst subtype 2-positive rat pancreatic tumor cell line CA20948 was developed. RESULTS: In this in vitro system [(111)In-DTPA(0)]octreotide can control tumor growth to 0% survival, and the effects were dependent on incubation time, radiation dose, and specific activity used. Similar concentrations of (111)In-DTPA, which is not internalized into sst-positive tumor cells like [(111)In-DTPA(0)]octreotide, did not influence tumor survival. Excess unlabeled octreotide (10(-6) mol/L) could decrease tumor cell survival to 60% of control; the addition of radiolabeled peptide ([(111)In-DTPA(0)]octreotide [10(-9) mol/L] + 10(-6) mol/L octreotide) did not further decrease survival. CONCLUSION: These in vitro studies show that the therapeutic effect of (111)In is dependent on internalization, enabling the Auger electrons with their very short particle range to reach the nucleus. Our results also indicate that the PRRT effects were receptor mediated.