RESUMEN
In many tumors, including prostate cancer, anti-apoptotic members of the Bcl-2 family are overexpressed and cause cell death resistance, which is a typical hallmark of cancer. Different therapeutic approaches, therefore, aim to restore the death mechanisms for enhanced apoptosis. Our recombinant immunotoxin D7(VL-VH)-PE40 is composed of the scFv D7(VL-VH) against the prostate-specific membrane antigen (PSMA) on the surface of prostate cancer cells and of the cytotoxic domain of the bacterial toxin Pseudomonas Exotoxin A (PE40). Since Pseudomonas Exotoxin A-based immunotoxins are known to preferentially inhibit the expression of the anti-apoptotic protein Mcl-1, the rationale was to test our immunotoxin in combination with the BH3 mimetic ABT-737, which specifically inhibits Bcl-2, Bcl-xl, and Bcl-w for enhanced induction of apoptosis in prostate cancer cells. The immunotoxin showed high and specific binding and cytotoxicity against PSMA expressing prostate cancer cells marked by a direct inhibition of Mcl-1. The combination of the immunotoxin with a subtoxic concentration of ABT-737 caused additive or even synergistic effects, which were based on an enhanced apoptosis induction as detected by poly(ADP-ribose) polymerase (PARP) and Caspase-3 cleavage in Western blot. Our study shows that the combination therapy of immunotoxin plus ABT-737 is a promising approach for the future treatment of advanced prostate cancer to improve therapeutic efficacy and to reduce adverse side effects.
Asunto(s)
Compuestos de Bifenilo/administración & dosificación , Inmunotoxinas/administración & dosificación , Nitrofenoles/administración & dosificación , Fragmentos de Péptidos/administración & dosificación , Neoplasias de la Próstata/terapia , Proteínas Proto-Oncogénicas/administración & dosificación , Sulfonamidas/administración & dosificación , ADP Ribosa Transferasas/administración & dosificación , Apoptosis/efectos de los fármacos , Toxinas Bacterianas/administración & dosificación , Línea Celular Tumoral , Sinergismo Farmacológico , Exotoxinas/administración & dosificación , Humanos , Masculino , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Piperazinas/administración & dosificación , Neoplasias de la Próstata/metabolismo , Factores de Virulencia/administración & dosificación , Exotoxina A de Pseudomonas aeruginosaRESUMEN
BACKGROUND/AIM: Reports on over-expression of the epidermal growth factor receptor (EGFR) in bladder cancer and its function in tumorigenesis have suggested to target this antigen. MATERIALS AND METHODS: We generated the targeted toxin EGF-PE40 consisting of the human epidermal growth factor (EGF) as the binding domain and PE40, a truncated version of Pseudomonas Exotoxin A, as the toxin domain. EGF-PE40 was tested on EGFR-expressing bladder cancer cells in view of binding via flow cytometry, and cytotoxicity via WST viability assay. Induction of apoptosis was examined by western blot. RESULTS: The targeted toxin specifically triggered cytotoxicity in the bladder cancer cells with 50% inhibitory concentration (IC50) values in the low nanomolar or picomolar range, and was about 1,250- to 1,500-fold more cytotoxic than the EGFR inhibitor erlotinib. Cytotoxicity of EGF-PE40 was based on the induction of apoptosis. CONCLUSION: EGF-PE40 represents a promising candidate for the future treatment of bladder cancer.
Asunto(s)
Antineoplásicos/química , Antineoplásicos/farmacología , Factor de Crecimiento Epidérmico/química , Neoplasias de la Vejiga Urinaria/tratamiento farmacológico , ADP Ribosa Transferasas/química , Animales , Toxinas Bacterianas/química , Células CHO , Línea Celular Tumoral , Cricetulus , Factor de Crecimiento Epidérmico/genética , Factor de Crecimiento Epidérmico/metabolismo , Receptores ErbB/genética , Receptores ErbB/metabolismo , Exotoxinas/química , Humanos , Factores de Virulencia/química , Exotoxina A de Pseudomonas aeruginosaRESUMEN
Background: Upregulation of anti-apoptotic Bcl-2 proteins in advanced prostate cancer leads to therapeutic resistance by prevention of cell death. New therapeutic approaches aim to target the Bcl-2 proteins for the restoration of apoptosis. Methods: The immunotoxin hD7-1(VL-VH)-PE40 specifically binds to the prostate specific membrane antigen (PSMA) on prostate cancer cells and inhibits protein biosynthesis. It was tested with respect to its effects on the expression of anti-apoptotic Bcl-2 proteins. Combination with the BAD-like mimetic ABT-737 was examined on prostate cancer cells and 3D spheroids and in view of tumor growth and survival in the prostate cancer SCID mouse xenograft model. Results: The immunotoxin led to a specific inhibition of Mcl-1 and Bcl2A1 expression in PSMA expressing target cells. Its combination with ABT-737, which inhibits Bcl-2, Bcl-xl, and Bcl-w, led to an induction of the intrinsic apoptotic pathway and to a synergistic cytotoxicity in prostate cancer cells and 3D spheroids. Furthermore, combination therapy led to a significantly prolonged survival of mice bearing prostate cancer xenografts based on an inhibition of tumor growth. Conclusion: The combination therapy of anti-PSMA immunotoxin plus ABT-737 represents the first tumor-specific therapeutic approach on the level of Bcl-2 proteins for the induction of apoptosis in prostate cancer.
RESUMEN
BACKGROUND: We generated humanized/de-immunized immunotoxins targeting the prostate-specific membrane antigen (PSMA) and tested their cytotoxic activity against prostate cancer cells in vitro. MATERIALS AND METHODS: The humanized/de-immunized version of our murine anti-PSMA single-chain antibody fragment (scFv) D7, termed hD7-1(VL-VH), was ligated to the 40-kDa toxin domain of Pseudomonas aeruginosa exotoxin A (PE40), and to the deimmunized 24-kDa toxin domains PE24 or PE24mut. The immunotoxins designated as hD7-1(VL-VH)-PE40, hD7-1(VL-VH)-PE24 and hD7-1(VL-VH)-PE24mut were bacterially expressed and purified by affinity chromatography. Binding and cytotoxicity were examined by flow cytometry and viability assay, respectively. RESULTS: All immunotoxins revealed strong binding to prostate cancer cells expressing PSMA and specific cytotoxicity, with half-maximal inhibitory concentration values in the picomolar range. CONCLUSION: We successfully created powerful anti-PSMA immunotoxins with reduced immunogenicity for further clinical development and application against advanced prostate cancer.
Asunto(s)
Antígenos de Superficie/inmunología , Glutamato Carboxipeptidasa II/inmunología , Inmunotoxinas/farmacología , Neoplasias de la Próstata/inmunología , ADP Ribosa Transferasas/genética , ADP Ribosa Transferasas/inmunología , Animales , Toxinas Bacterianas/genética , Toxinas Bacterianas/inmunología , Línea Celular Tumoral , Escherichia coli/genética , Exotoxinas/genética , Exotoxinas/inmunología , Humanos , Masculino , Ratones , Neoplasias de la Próstata/tratamiento farmacológico , Factores de Virulencia/genética , Factores de Virulencia/inmunología , Exotoxina A de Pseudomonas aeruginosaRESUMEN
BACKGROUND: Cisplatin-based chemotherapy is the treatment of choice for advanced bladder cancer. Since many tumor cells show inherent or acquired cisplatin resistance, research is needed to improve the therapeutic efficacy. Since the analgesic methadone is discussed as being a sensitizer for chemotherapy, we tested its effects on the cisplatin treatment of bladder cancer cells. MATERIALS AND METHODS: T24 and HT-1376 bladder cancer cells were incubated with cisplatin in combination with methadone. Cytotoxicity was examined using the WST-1 viability assay and induction of apoptosis was analyzed via phase-contrast microscopy, flow cytometry, and western blot analysis. RESULTS: Methadone was shown to enhance the cytotoxic effects of cisplatin on T24 cells based on the induction of apoptosis. In contrast, HT-1376 cells were identified as non-responders to methadone. CONCLUSION: Methadone could act as a chemosensitizer in the future treatment of advanced bladder cancer. Further research is needed to identify the underlying molecular mechanisms.