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Effect of naive and radiolabeled rhTRAIL on the cervical cancer xenografts in mice Background: There is a need for novel treatments of advanced cervical cancer. We investigated the utility of recombinant human TNF-related apoptosis-inducing ligand (rhTRAIL), a molecule capable of inducing apoptosis in cancer cells, for the therapy of CasKi cervical cancer xenografts in nude mice. Results: CasKi cells proved to be sensitive in vitro to rhTRAIL with an IC50 of 120 ng/ml. 125I-tagged rhTRAIL specifically accumulated in CasKi tumors in mice with the highest uptake of 9.4% ID/g at 2 h post-injection. Both naive and 200 µCi 188Re-tagged rhTRAIL administered in the amount of 0.35 mg/kg body weight significantly retarded CasKi tumor growth to the same extent in mice without the side effects of cisplatin chemotherapeutic control. Conclusion: rhTRAIL is a promising novel agent for treatment of advanced cervical cancer. More than 500,000 cases of cervical cancer are reported annually worldwide and 200,000 women a year are killed by this disease [1]. Standard treatment of locally advanced disease consists of radiotherapy combined with cisplatin. Overall survival following standard chemoradiation is 66–79% at 5 years, thus indicating the need for more effective and less toxic treatments (reviewed in [2]). The death receptors (DR) of the TNF superfamily represent potential targets for promoting apoptosis in cancer [3]. TNF-related apoptosisinducing ligand (TRAIL) induces apoptosis by binding to the DR4 and DR5. Recombinant human (rh) TRAIL is a 168 amino acid polypeptide (MW 19.6 kDa), consisting of the TNF homologous portion of the extracellular domain of the full length TRAIL/Apo2L protein. In earlier studies, its serum half-life was reported to be around 30 min [4]. rhTRAIL is being currently tested in patients with noncervical cancers with some encouraging results (reviewed in [2]). Though the presence of DR4 and DR5 has been demonstrated in large numbers of tumor specimens from cervical cancer patients [5], no clinical trials using TRAIL in cervical cancer have been reported so far. Human cervical cancer cell lines have been exposed to rhTRAIL and their sensitivity to rhTRAIL-induced apoptosis has been analyzed. CasKi cells were found to be very sensitive to rhTRAIL, HeLa cells demonstrated intermediate sensitivity and SiHa cells were resistant to TRAIL [6–11]. Importantly, the rhTRAIL amounts that induced apoptosis in cervical cancer cell lines were not toxic to normal cervical cells in vitro [12]. Despite this wealth of in vitro
experimental data, to the best of our knowledge, no studies of the effects of rhTRAIL alone or in combination with chemo/radiation on cervical cancer xenografts have been reported so far. The possibility of radiolabeling rhTRAIL with a therapeutic radionuclide to potentially enhance rhTRAIL efficacy has not been described in literature either. 188Re radionuclide is a powerful b emitter with a short physical half-life of 16.9 h, which has been successfully used in our and other laboratories for antibody-based experimental therapies of cervical cancer [13] and for clinical trials in patients with leukemia [14] and metastatic melanoma [15]. Here, we describe the first experiments in treating mice bearing CasKi xenografts with naive and 188Re-labeled rhTRAIL, showing the efficacy and safety of this treatment in comparison with the standard chemotherapy.
10.4155/TDE.13.137 © 2014 Future Science Ltd
Ther. Deliv. (2014) 5(2), 139–147
Xing Guo Wang1, Thomas Jandl2 , Ekaterina Dadachova* ‡2 & Ekaterina Revskaya ‡2 The Faculty of Life Sciences, Hubei University, Wuhan, China 2 Department of Radiology, Albert Einstein College of Medicine, 1695A Eastchester Road, Bronx, NY 10461, USA *Author for correspondence: Tel.: +1 718 405 8485 Fax: +1 718 405 8457 E-mail: ekaterina.dadachova@ einstein.yu.edu 1
‡
Authors contributed equally
Materials & methods Materials, cells & radiolabeling rhTRAIL was purchased from PeproTech (NJ, USA), and cis-diamminedichloroplatinum (II) (cisplatin) was purchased from Sigma-Aldrich (MO, USA). Human HPV16-positive 2A3 head and neck cancer cells are maintained in our laboratory and were grown as described in [13]. Human cervical cancer CasKi cells and A2058 human melanoma cells were purchased from American Type Culture Collection (VA, USA) and grown as described in [13,16], respectively. 125I was obtained from Perkin Elmer (Ma, USA). 188 Re was eluted from a 188W/188Re generator procured from Isotopen Technologies Garching (Garching, Germany). For biodistribution experiments, rhTRAIL was radiolabeled with ISSN 2041-5990
139
Research Article | Wang, Jandl, Dadachova & Revskaya Key Terms Apoptosis: Process of
programmed cell death that may occur in both multicellular or unicellular organisms.
Therapeutic radionuclide: Radionuclide emitting cytocidal b or a radiation in the process of its radioactive decay.
Biodistribution: Process of
the quantification of uptake of the radiolabeled or differently labeled compounds in the organs of an animal or a human.
Xenografts bearing nude mice: Mice with a
compromised immune system (lack of thymus and absence of T cells), which if injected with human cancer cells will not reject them but will allow for the tumors to develop.
Ki67 marker: Ki67 protein is a
cellular marker for proliferation. Ki67 protein can be detected during all active phases of the cell cycle (G1, S, G2 and mitosis) but is absent in the resting cells (G 0).
I using IodoBeads (Pierce, Il, USA) according to the manufacturer’s instructions. For therapy, rhTRAIL was radiolabeled with 188Re by pretreatment of rhTRAIL with dithiothreitol (Sigma Aldrich) followed by combining rhTRAIL with reduced 188Re according to the method for protein labeling with 188Re described in [17]. 125
In vitro
toxicity studies with unlabeled rhTRAIL To ascertain the toxicity of rhTRAIL towards CasKi cells, in vitro toxicity studies were performed using the tetrazolium dye (2,3)-bis[2-methoxy-4-nitro-5-sulphenyl]-(2H)-terazolium-5-carboxanilide (XTT) assay as described in with some modifications [18]. A2058 human melanoma cells and 2A3 human head and neck cancer cells were used as controls. The cells were grown at 10,000 cells/well in 96-well plates overnight, followed by addition of rhTRAIL in phosphate buffered saline (PBS) at the concentration of 0–1500 ng/ml for 24 h. Wells were then washed, fresh media added along with 50 µl XTT (Sigma) at 1 mg/ml in PBS, and 4 µl menadione (Sigma) at 1 mM in acetone. Cells were incubated for another 3 h, and the absorbance at 492 nm was read. All conditions were done in triplicate, and the experiment was performed twice. In vitro
toxicity of 188Re-rhTRAIL To ascertain that 188Re-rhTRAIL preserved its toxicity towards CasKi cells postradiolabeling and to investigate if the attachment of 188Reradiolabel increased the cytotoxicty of rhTRAIL, we performed a crystal violet assay, which measures cellular ability to proliferate. Monolayers of CasKi cells with up to 10,000 cells per well were grown for 24 h in 96-well plates, then exposed for 24–48 h to either 200 ng/ml unlabeled rhTRAIL, 20 µCi/200 ng 188Re-rhTRAIL or left untreated. Monolayers were then washed and fixed with 100% ethanol, and 5% crystal violet was added for 30 min, as described in [18]. The crystal violet solution was removed and the cells were washed repeatedly in water. A total of 100 µl ethanol was added to the wells to solubilize the crystal violet, 50 µl were removed and the optical density at 595 was measured. All conditions were done in triplicate. Biodistribution
of 125I-rhTRAIL in nude mice bearing CasKi xenografts All animal studies were carried out in accordance with the guidelines of the Institute for Animal Studies at the Albert Einstein College 140
Ther. Deliv. (2014) 5(2)
of Medicine. 6- to 8-week-old athymic Nu/Nu balb/c nude mice were purchased from Charles River Laboratories (MA, USA). A total of 10 million CasKi cells were mixed with 80% Matrigel and injected subcutaneously into the right flank of each mouse. When the tumors reached 0.4–0.6 cm in diameter, the xenografts bearing nude mice were randomized into groups of four and injected with 0.35 mg/kg body weight (7 µg per mouse) 125I-rhTRAIL (25 µCi) intravenously. The mice were sacrificed at 2, 4 and 24 h, their major organs and tumors were removed, blotted from blood, weighed, counted in a g counter (Wallac, Turku, Finland), and the percentage of injected dose per gram tissue (ID/g, %) was calculated. Therapy
of CasKi tumors in nude mice with naive and 188Re-rhTRAIL For the therapy experiments, nude mice with tumors of 0.4–0.6 cm in diameter were randomized into groups of seven, and treated intravenously with the single injection of the following agents: 0.35 mg/kg body weight (7 µg per mouse) naive rhTRAIL; 200 µCi/7 µg 188Re-rhTRAIL; 5 mg/kg cisplatin (100 µg per mouse); or 100 µl PBS. The tumor size was measured every 3–4 days with electronic calipers (Pro-max Sylvac System IP67, Fowler Tools and Instruments; MA, USA) in 3D and the tumor volume was calculated as a product of three dimensions divided by two. The mice were observed for 28 days, at the completion of the experiment the mice were sacrificed, their tumors removed and analyzed histologically for Ki67 marker and apoptosis. Immunohistological
quantification of tumor tissue for dividing cells (Ki67 marker) & apoptotic cells (TUNEL) At the end of the 28-day observation period, mice were sacrificed and each tumor was harvested, fixed in 10% neutral-buffered formalin, and paraffin embedded. Sections of the paraffin embedded samples (5 µm thick) were deparaffinized and antigens retrieved by immersion in 100°C citrate buffer for 20 min. For TUNEL DNA strand-breaks analysis, slides were treated according to the manufacturer’s instructions of the TumorTACS™ in situ Apoptosis Detection Kit (Trevigen, MD, USA). For the Ki67 staining, slides were peroxidase blocked with 2% H2O2 in Tris-buffered saline, nonspecific binding was blocked with goat serum in tris-buffered saline. Endogenous biotin was blocked with avidin/ biotin blocking kit (Vector Labs, CA, USA). future science group
Effect of naive & radiolabeled rhTRAIL on the cervical cancer xenografts in mice Primary antibody for Ki67 (Clone SP6; Thermo Scientific, IL, USA) was applied in a wet chamber overnight at 4°C. Secondary biotin coupled anti-rabbit-IgG (Vector Labs, CA, USA) was incubated for 1 h at room temperature. VECTASTAIN® Elite ABC kit (Vector Labs, CA, USA) and Metal Enhanced DAB Substrate Kit (Thermo Scientific, MA, USA) were used according to the manufacturer’s instructions. Slides were counterstained with hematoxylin (Sigma). Statistical
analyses The statistical analyses of the differences in tumor volumes in various groups, as well as of the number of the Ki67 and TUNEL positive cells in the tumors, were performed using Student’s t-test with Prism software (GraphPad
Software, CA, USA). A p value of
Effect of naive and radiolabeled rhTRAIL on the cervical cancer xenografts in mice Background: There is a need for novel treatments of advanced cervical cancer. We investigated the utility of recombinant human TNF-related apoptosis-inducing ligand (rhTRAIL), a molecule capable of inducing apoptosis in cancer cells, for the therapy of CasKi cervical cancer xenografts in nude mice. Results: CasKi cells proved to be sensitive in vitro to rhTRAIL with an IC50 of 120 ng/ml. 125I-tagged rhTRAIL specifically accumulated in CasKi tumors in mice with the highest uptake of 9.4% ID/g at 2 h post-injection. Both naive and 200 µCi 188Re-tagged rhTRAIL administered in the amount of 0.35 mg/kg body weight significantly retarded CasKi tumor growth to the same extent in mice without the side effects of cisplatin chemotherapeutic control. Conclusion: rhTRAIL is a promising novel agent for treatment of advanced cervical cancer. More than 500,000 cases of cervical cancer are reported annually worldwide and 200,000 women a year are killed by this disease [1]. Standard treatment of locally advanced disease consists of radiotherapy combined with cisplatin. Overall survival following standard chemoradiation is 66–79% at 5 years, thus indicating the need for more effective and less toxic treatments (reviewed in [2]). The death receptors (DR) of the TNF superfamily represent potential targets for promoting apoptosis in cancer [3]. TNF-related apoptosisinducing ligand (TRAIL) induces apoptosis by binding to the DR4 and DR5. Recombinant human (rh) TRAIL is a 168 amino acid polypeptide (MW 19.6 kDa), consisting of the TNF homologous portion of the extracellular domain of the full length TRAIL/Apo2L protein. In earlier studies, its serum half-life was reported to be around 30 min [4]. rhTRAIL is being currently tested in patients with noncervical cancers with some encouraging results (reviewed in [2]). Though the presence of DR4 and DR5 has been demonstrated in large numbers of tumor specimens from cervical cancer patients [5], no clinical trials using TRAIL in cervical cancer have been reported so far. Human cervical cancer cell lines have been exposed to rhTRAIL and their sensitivity to rhTRAIL-induced apoptosis has been analyzed. CasKi cells were found to be very sensitive to rhTRAIL, HeLa cells demonstrated intermediate sensitivity and SiHa cells were resistant to TRAIL [6–11]. Importantly, the rhTRAIL amounts that induced apoptosis in cervical cancer cell lines were not toxic to normal cervical cells in vitro [12]. Despite this wealth of in vitro
experimental data, to the best of our knowledge, no studies of the effects of rhTRAIL alone or in combination with chemo/radiation on cervical cancer xenografts have been reported so far. The possibility of radiolabeling rhTRAIL with a therapeutic radionuclide to potentially enhance rhTRAIL efficacy has not been described in literature either. 188Re radionuclide is a powerful b emitter with a short physical half-life of 16.9 h, which has been successfully used in our and other laboratories for antibody-based experimental therapies of cervical cancer [13] and for clinical trials in patients with leukemia [14] and metastatic melanoma [15]. Here, we describe the first experiments in treating mice bearing CasKi xenografts with naive and 188Re-labeled rhTRAIL, showing the efficacy and safety of this treatment in comparison with the standard chemotherapy.
10.4155/TDE.13.137 © 2014 Future Science Ltd
Ther. Deliv. (2014) 5(2), 139–147
Xing Guo Wang1, Thomas Jandl2 , Ekaterina Dadachova* ‡2 & Ekaterina Revskaya ‡2 The Faculty of Life Sciences, Hubei University, Wuhan, China 2 Department of Radiology, Albert Einstein College of Medicine, 1695A Eastchester Road, Bronx, NY 10461, USA *Author for correspondence: Tel.: +1 718 405 8485 Fax: +1 718 405 8457 E-mail: ekaterina.dadachova@ einstein.yu.edu 1
‡
Authors contributed equally
Materials & methods Materials, cells & radiolabeling rhTRAIL was purchased from PeproTech (NJ, USA), and cis-diamminedichloroplatinum (II) (cisplatin) was purchased from Sigma-Aldrich (MO, USA). Human HPV16-positive 2A3 head and neck cancer cells are maintained in our laboratory and were grown as described in [13]. Human cervical cancer CasKi cells and A2058 human melanoma cells were purchased from American Type Culture Collection (VA, USA) and grown as described in [13,16], respectively. 125I was obtained from Perkin Elmer (Ma, USA). 188 Re was eluted from a 188W/188Re generator procured from Isotopen Technologies Garching (Garching, Germany). For biodistribution experiments, rhTRAIL was radiolabeled with ISSN 2041-5990
139
Research Article | Wang, Jandl, Dadachova & Revskaya Key Terms Apoptosis: Process of
programmed cell death that may occur in both multicellular or unicellular organisms.
Therapeutic radionuclide: Radionuclide emitting cytocidal b or a radiation in the process of its radioactive decay.
Biodistribution: Process of
the quantification of uptake of the radiolabeled or differently labeled compounds in the organs of an animal or a human.
Xenografts bearing nude mice: Mice with a
compromised immune system (lack of thymus and absence of T cells), which if injected with human cancer cells will not reject them but will allow for the tumors to develop.
Ki67 marker: Ki67 protein is a
cellular marker for proliferation. Ki67 protein can be detected during all active phases of the cell cycle (G1, S, G2 and mitosis) but is absent in the resting cells (G 0).
I using IodoBeads (Pierce, Il, USA) according to the manufacturer’s instructions. For therapy, rhTRAIL was radiolabeled with 188Re by pretreatment of rhTRAIL with dithiothreitol (Sigma Aldrich) followed by combining rhTRAIL with reduced 188Re according to the method for protein labeling with 188Re described in [17]. 125
In vitro
toxicity studies with unlabeled rhTRAIL To ascertain the toxicity of rhTRAIL towards CasKi cells, in vitro toxicity studies were performed using the tetrazolium dye (2,3)-bis[2-methoxy-4-nitro-5-sulphenyl]-(2H)-terazolium-5-carboxanilide (XTT) assay as described in with some modifications [18]. A2058 human melanoma cells and 2A3 human head and neck cancer cells were used as controls. The cells were grown at 10,000 cells/well in 96-well plates overnight, followed by addition of rhTRAIL in phosphate buffered saline (PBS) at the concentration of 0–1500 ng/ml for 24 h. Wells were then washed, fresh media added along with 50 µl XTT (Sigma) at 1 mg/ml in PBS, and 4 µl menadione (Sigma) at 1 mM in acetone. Cells were incubated for another 3 h, and the absorbance at 492 nm was read. All conditions were done in triplicate, and the experiment was performed twice. In vitro
toxicity of 188Re-rhTRAIL To ascertain that 188Re-rhTRAIL preserved its toxicity towards CasKi cells postradiolabeling and to investigate if the attachment of 188Reradiolabel increased the cytotoxicty of rhTRAIL, we performed a crystal violet assay, which measures cellular ability to proliferate. Monolayers of CasKi cells with up to 10,000 cells per well were grown for 24 h in 96-well plates, then exposed for 24–48 h to either 200 ng/ml unlabeled rhTRAIL, 20 µCi/200 ng 188Re-rhTRAIL or left untreated. Monolayers were then washed and fixed with 100% ethanol, and 5% crystal violet was added for 30 min, as described in [18]. The crystal violet solution was removed and the cells were washed repeatedly in water. A total of 100 µl ethanol was added to the wells to solubilize the crystal violet, 50 µl were removed and the optical density at 595 was measured. All conditions were done in triplicate. Biodistribution
of 125I-rhTRAIL in nude mice bearing CasKi xenografts All animal studies were carried out in accordance with the guidelines of the Institute for Animal Studies at the Albert Einstein College 140
Ther. Deliv. (2014) 5(2)
of Medicine. 6- to 8-week-old athymic Nu/Nu balb/c nude mice were purchased from Charles River Laboratories (MA, USA). A total of 10 million CasKi cells were mixed with 80% Matrigel and injected subcutaneously into the right flank of each mouse. When the tumors reached 0.4–0.6 cm in diameter, the xenografts bearing nude mice were randomized into groups of four and injected with 0.35 mg/kg body weight (7 µg per mouse) 125I-rhTRAIL (25 µCi) intravenously. The mice were sacrificed at 2, 4 and 24 h, their major organs and tumors were removed, blotted from blood, weighed, counted in a g counter (Wallac, Turku, Finland), and the percentage of injected dose per gram tissue (ID/g, %) was calculated. Therapy
of CasKi tumors in nude mice with naive and 188Re-rhTRAIL For the therapy experiments, nude mice with tumors of 0.4–0.6 cm in diameter were randomized into groups of seven, and treated intravenously with the single injection of the following agents: 0.35 mg/kg body weight (7 µg per mouse) naive rhTRAIL; 200 µCi/7 µg 188Re-rhTRAIL; 5 mg/kg cisplatin (100 µg per mouse); or 100 µl PBS. The tumor size was measured every 3–4 days with electronic calipers (Pro-max Sylvac System IP67, Fowler Tools and Instruments; MA, USA) in 3D and the tumor volume was calculated as a product of three dimensions divided by two. The mice were observed for 28 days, at the completion of the experiment the mice were sacrificed, their tumors removed and analyzed histologically for Ki67 marker and apoptosis. Immunohistological
quantification of tumor tissue for dividing cells (Ki67 marker) & apoptotic cells (TUNEL) At the end of the 28-day observation period, mice were sacrificed and each tumor was harvested, fixed in 10% neutral-buffered formalin, and paraffin embedded. Sections of the paraffin embedded samples (5 µm thick) were deparaffinized and antigens retrieved by immersion in 100°C citrate buffer for 20 min. For TUNEL DNA strand-breaks analysis, slides were treated according to the manufacturer’s instructions of the TumorTACS™ in situ Apoptosis Detection Kit (Trevigen, MD, USA). For the Ki67 staining, slides were peroxidase blocked with 2% H2O2 in Tris-buffered saline, nonspecific binding was blocked with goat serum in tris-buffered saline. Endogenous biotin was blocked with avidin/ biotin blocking kit (Vector Labs, CA, USA). future science group
Effect of naive & radiolabeled rhTRAIL on the cervical cancer xenografts in mice Primary antibody for Ki67 (Clone SP6; Thermo Scientific, IL, USA) was applied in a wet chamber overnight at 4°C. Secondary biotin coupled anti-rabbit-IgG (Vector Labs, CA, USA) was incubated for 1 h at room temperature. VECTASTAIN® Elite ABC kit (Vector Labs, CA, USA) and Metal Enhanced DAB Substrate Kit (Thermo Scientific, MA, USA) were used according to the manufacturer’s instructions. Slides were counterstained with hematoxylin (Sigma). Statistical
analyses The statistical analyses of the differences in tumor volumes in various groups, as well as of the number of the Ki67 and TUNEL positive cells in the tumors, were performed using Student’s t-test with Prism software (GraphPad
Software, CA, USA). A p value of
