Anticancer activity of modified somatostatin analogs
Antitumor activiteit van gemodificeerde somatostatine analogen
There has been an exponential growth in the development of radiolabeled peptides for diagnosis and therapeutic applications in oncology. Peptides radiolabeled with .-emitters can be used to visualize receptor-positive cells in vivo. In 1994 111In-DTPA-octreotide (OctreoScan®) received FDA approval as the first product of its kind, and to this day remains the “Gold Standard” in somatostatin receptor scintigraphy. Research continued in this area by introducing peptide receptor radionuclide therapy (PRRT) using Auger electron (111In) and ß particle (90Y and 177Lu) emitting radionculides. The aim of this thesis was firstly to receive further insight in the therapeutic potential of somatostatin labeled analogs in vitro and in vivo. Secondly, we wanted to improve the potential of PRRT with somatostatin analogs by introducing the combination with an apoptosis-inducing factor. Since the response of tumor cells to PRRT is dependent on their radiosensitivity we determined the radiosensitivity of CA20948 tumor cells, rat pancreatic tumor cells expressing the somatostatin receptor (sst), by using clonogenic survival assays after high-dose-rate external-beam radiotherapy (XRT) in vitro. It could be expected however that results of high-dose-rate XRT are not representative for those after low-dose-rate radionuclide therapy (RT), such as PRRT. Therefore, we compared clonogenic survival in vitro in CA20948 tumor cells after increasing doses of XRT or RT, the latter using 131I (Chapter 2). 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 (a/ß) for CA20948 was 8.3 Gy, whereas that ratio for low-dose-rate RT was calculated to be 86.5 Gy. So, 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. In chapter 3 the therapeutic potential of 111In-DTPA-octreotide was evaluated. 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 µm, 200-550 µm, respectively), 111In-DTPA-octreotide is also being used for PRRT. First (Chapter 3.1), the therapeutic effects of 111In-DTPA-octreotide in a single-cell model were investigated. In this single-cell model we discriminated between the effects of the Auger electrons and internal conversion electrons in PRRT. In this in vitro system 111In-DTPA-octreotide could completely control tumor growth. The effects were dependent on incubation time, radiation dose, and specific activity used. Similar concentrations of 111In-DTPA, which is not internalized into somatostatin-positive tumor cells, did not influence tumor survival. Excess unlabeled octreotide (10-6 mol/L) could decrease tumor cell survival to 60% of control; the addition of the radiolabeled peptide 111In-DTPA-octreotide (10-9 mol/L) plus an excess of unlabeled (10-6 mol/L) octreotide, did not further decrease survival. So, these in vitro studies show that the therapeutic effect of 111In 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. Subsequently, we investigated the radiotherapeutic effect of different doses of 111In-DTPA-octreotide in vivo in Lewis rats bearing small (= 1 cm2) or larger (= 8 cm2) somatostatin receptor-positive rat pancreatic CA20948 tumors. In addition, we investigated the somatostatin receptor density on the tumors before and after radionuclide therapy (Chapter 3.2). The results showed impressive radiotherapeutic effects of 111In-labeled octreotide in this rat tumor model. Cure (up to 50%) was found in the animals bearing small tumors 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 tumor). In the rats bearing the larger tumors the effects were much less pronounced and only partial responses were reached in these groups. A possible explanation for the various responses found is the increase of somatostatin receptor-negative cells, due to the lack of crossfire. We therefore investigated the somatostatin receptor expression before and after radionuclide therapy with an In-111 labeled somatostatin analog. In this study we found a clear somatostatin receptor subtype 2 (sst2) expression both in the control and in the tumors treated with 111In. A significantly higher tumor receptor density was found when the tumor re-grew after an initial decline in size after low dose PRRT in comparison with untreated tumors. We concluded that radionuclide-therapy with 111In-labeled somatostatin analogs is feasible but should preferably start as early as possible during tumor development. One might also consider the use of radiolabeled somatostatin analogs in an adjuvant setting after surgery of somatostatin receptor-positive tumors to eradicate occult metastases. Finally we showed that PRRT led to a significant increase in somatostatin receptor density, when the tumors re-grew after an initial decline in size after PRRT. The increase in the somatostatin receptor density will lead to a higher uptake of the radiolabeled peptides in therapeutic applications, making repeated injections of radiolabeled peptides more effective. In chapter 4 the therapeutic effects of the somatostatin analogs Tyr3-octreotide and Tyr3-octreotate radiolabeled with the ß- particle emitters 177Lu or 90Y were evaluated in an in vitro colony-forming assay using the rat pancreatic tumor cell line CA20948 (Chapter 4.1). 177Lu-DOTA-Tyr3-octreotate could reduce tumor growth with 100% and effects were dependent on radiation dose, incubation time, and specific activity used. Similar concentrations of 177Lu-DOTA, which does not bind to the tumor cells, had a less pronounced effect on 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 showed a higher percentage tumor cell kill compared to radiolabeled Tyr3-octreotide, labeled with 177Lu or 90Y. This is in accordance with the higher affinity of Tyr3-octreotate for the sst2-receptor compared to Tyr3-octreotide, leading to a higher amount of cell-associated radioactivity, resulting in a significantly higher tumor radiation dose. So, Tyr3-octreotate labeled with 177Lu or 90Y is the most promising analog for PRRT. Subsequently, the anti-tumor effects of 177Lu-DOTA-Tyr3-octreotate on sst receptor positive CA20948 micrometastases in the liver were investigated (Chapter 4.2). Rats treated with 177Lu-DOTA-Tyr3-octreotate showed a significantly better survival in a rat liver micrometastic model setting, making it a very promising new treatment modality for sst receptor positive disseminated disease. In chapter 5 a study is described that aimed to develop and evaluate a new radiopharmaceutical for the treatment of cancers that overexpress sst2. 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. Biodistribution studies showed that the receptor-targeting peptide portion of the molecule, Tyr3-octreotate, binds specifically to the sst2. 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. Internalization experiments in vitro 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 of radiolabeled RGD-DTPA-octreotate in vivo in rats was in agreement with the in vitro data and comparable to that of radiolabeled Tyr3-octreotate. A drawback of the new hybrid compound was high renal uptake and retention of radioactivity, limiting the therapeutic dose that can be administered, as the kidneys are critical organs in radionuclide therapy using somatostatin analogs labeled with beta particle emitting radionuclides (Chapter 5.1). Tumoricidal effects of the hybrid peptide RGD-DTPA-octreotate were evaluated in comparison with those of RGD and Tyr3-octreotate in an in vitro colony-forming assay, the compounds were all radiolabeled to 111In. Subsequently caspase-3 activation was determined (Chapter 5.2). Tumoricidal effects were found for 111In-DTPA-RGD < 111In-DTPA-Tyr3-octreotate < RGD-111In-DTPA-octreotate. Also, the highest increase in caspase-3 levels was found with RGD-111In-DTPA-octreotate. Concluding, RGD-111In-DTPA-octreotate has a more pronounced tumoricidal effect than 111In-DTPA-RGD and 111In-DTPA-Tyr3-octreotate, because of increased apoptosis as indicated by increased caspase-3 activity. In chapter 5.3 we further evaluated the biodistribution of RGD-111In-DTPA-octreotate and 125I-RGD-octreotate, and investigated the tumoricidal effect of the unlabeled compound RGD-DTPA-octreotate in vitro. Since the biodistribution showed a very high renal uptake we studied the therapeutic effects of the unlabeled hybrid peptide, RGD-DTPA-octreotate, in vitro in the CA20948, AR42J and the CHO cell line transfected with sst2, which showed that RGD-DTPA-octreotate induced a significant increase in caspase-3 levels in comparison with RGD and Tyr3-octreotate in all cell lines. Subsequently, we examined the biodistribution of iodinated RGD-octreotate, without the presence of the chelator DTPA, in order to change the elimination route from the body (from renal clearance to more hepatic clearance). 125I-RGD-octreotate showed indeed a much lower renal uptake in comparison with RGD-111In-DTPA-octreotate. Furthermore, the affinity of RGD-octreotate increased in comparison with RGD-DTPA-octreotate (IC50 values of 1.4×10-8M vs 9.4×10-8M respectively). Finally, RGD-octreotate, without the chelator DTPA, was also able to activate caspase-3 as was indicated with immunocytochemistry. So, this concept of targeting somatostatin receptor-expressing tumors using peptide receptor radionuclide therapy might also apply for the use of somatostatin analogs coupled to chemotherapeutic compounds, which is further described in Chapter 5.4. The development of hybrid molecules that combine targeting and an effector function, such as apoptosis, can be a new approach in the treatment of cancer.