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Background: Fibroblast growth factor receptor 1 (FGFR1) plays a crucial role in carcinogenesis. Exploring the combination of the novel humanized monoclonal anti-FGFR1 antibody OM-RCA-01 and immunotherapy was intriguing due to involvement of FGFR1 in mechanisms of resistance to checkpoint inhibitors. Materials and methods: Lung cancer A549, exhibiting distinct levels of FGFR1 expression, were cultured in basic FGF medium with OM-RCA-01 supplementation. The efficacy of antibody monotherapy was validated in a lung cancer xenograft study. To investigate whether OM-RCA-01 could enhance the efficacy of immunotherapy in vitro and in vivo, mixed lymphocyte reaction/Staphylococcal enterotoxin B assays and FGFR1/programmed death-ligand 1-positive patient-derived xenograft model were established. Results: The antibody effectively suppressed receptor phosphorylation, resulting in inhibited cell proliferation. OM-RCA-01 led to a substantial delay in tumor growth compared to non-specific immunoglobulin G in a xenograft study. The median tumor volume was 1048.5 mm3 and 2174 mm3 in the study and vehicle groups, respectively, representing a twofold difference in favor of the anti-FGFR1 antibody. In vitro, the combination of nivolumab and OM-RCA-01 resulted in higher levels of interferon gamma and interleukin-2 release compared with nivolumab alone. In vivo, pembrolizumab in combination with OM-RCA-01 produced a greater inhibitory effect on tumor growth compared with vehicle and pembrolizumab alone. The curve plateaued, indicating minimal tumor growth from day 16 onwards in the combination group. The OM-RCA-01 demonstrated no toxicity, even at therapeutic doses or higher doses. Conclusions: Our preclinical studies demonstrate that OM-RCA-01 exhibits robust activity with minimal toxicity. Combining an anti-FGFR1 antibody with a checkpoint inhibitor may enhance the efficacy of both drugs. However, further studies are needed to elucidate the mechanism of this interaction.
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BACKGROUND: The Akt/mammalian target of rapamycin (mTOR) signalling pathway serves as a critical regulator of cellular growth, proliferation and survival. Akt aberrant activation has been implicated in carcinogenesis and anticancer therapy resistance. Piperlongumine (PL), a natural alkaloid present in the fruit of the Long pepper, is known to exhibit notable anticancer effects. Here we investigate the impact of PL on Akt/mTOR signalling. METHODS: We examined Akt/mTOR signalling in cancer cells of various origins including prostate, kidney and breast after PL treatment. Furthermore, cell viability after concomitant treatment with PL and the autophagy inhibitor, Chloroquine (CQ) was assessed. We then examined the efficacy of in vivo combination treatment using a mouse xenograft tumour model. RESULTS: We demonstrate for the first time that PL effectively inhibits phosphorylation of Akt target proteins in all tested cells. Furthermore, the downregulation of Akt downstream signalling resulted in decrease of mTORC1 activity and autophagy stimulation. Using the autophagy inhibitor, CQ, the level of PL-induced cellular death was significantly increased. Moreover, concomitant treatment with PL and CQ demonstrated notable antitumour effect in a xenograft mouse model. CONCLUSIONS: Our data provide novel therapeutic opportunities to mediate cancer cellular death using PL. As such, PL may afford a novel paradigm for both prevention and treatment of malignancy.
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Apoptosis/efectos de los fármacos , Autofagia/efectos de los fármacos , Dioxolanos/farmacología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Animales , Neoplasias de la Mama/tratamiento farmacológico , Carcinoma de Células Renales/tratamiento farmacológico , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Cloroquina/farmacología , Femenino , Células HEK293 , Humanos , Neoplasias Renales/tratamiento farmacológico , Células MCF-7 , Masculino , Diana Mecanicista del Complejo 1 de la Rapamicina , Ratones , Complejos Multiproteicos/antagonistas & inhibidores , Trasplante de Neoplasias , Fosforilación/efectos de los fármacos , Neoplasias de la Próstata/tratamiento farmacológico , Proteínas Proto-Oncogénicas c-akt/efectos de los fármacos , Especies Reactivas de Oxígeno , Transducción de Señal/efectos de los fármacos , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Serina-Treonina Quinasas TOR/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The X-linked inhibitor of apoptosis (XIAP), the most potent member of the inhibitor of apoptosis protein (IAP) family of endogenous caspase inhibitors, blocks the initiation and execution phases of the apoptotic cascade. As such, XIAP represents an attractive target for treating apoptosis-resistant forms of cancer. Here, we demonstrate that treatment with the membrane-permeable zinc chelator, N,N,N',N',-tetrakis(2-pyridylmethyl) ethylenediamine (TPEN) induces a rapid depletion of XIAP at the post-translational level in human PC-3 prostate cancer cells and several non-prostate cell lines. The depletion of XIAP is selective, as TPEN has no effect on the expression of other zinc-binding members of the IAP family, including cIAP1, cIAP2 and survivin. The downregulation of XIAP in TPEN-treated cells occurs via proteasome- and caspase-independent mechanisms and is completely prevented by the serine protease inhibitor, Pefabloc. Finally, our studies demonstrate that TPEN promotes activation of caspases-3 and -9 and sensitizes PC-3 prostate cancer cells to TRAIL-mediated apoptosis. Taken together, our findings indicate that zinc-chelating agents may be used to sensitize malignant cells to established cytotoxic agents via downregulation of XIAP.
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Apoptosis/efectos de los fármacos , Quelantes/farmacología , Neoplasias de la Próstata/patología , Ligando Inductor de Apoptosis Relacionado con TNF/farmacología , Proteína Inhibidora de la Apoptosis Ligada a X/deficiencia , Zinc/farmacología , Caspasas/metabolismo , Línea Celular Tumoral , Cobre/farmacología , Ensayos de Selección de Medicamentos Antitumorales , Activación Enzimática/efectos de los fármacos , Etilaminas/farmacología , Humanos , Espacio Intracelular/efectos de los fármacos , Espacio Intracelular/metabolismo , Masculino , Modelos Biológicos , Neoplasias de la Próstata/enzimología , Inhibidores de Proteasas/farmacología , Complejo de la Endopetidasa Proteasomal/metabolismo , Estructura Terciaria de Proteína , Piridinas , Pirimidinas/farmacología , ARN Interferente Pequeño/metabolismo , Sulfonas/farmacología , Proteína Inhibidora de la Apoptosis Ligada a X/química , Zinc/metabolismoRESUMEN
Expression of DNA sequences, related to MMTV env gene, in peripheral blood lymphocytes, which was strictly specific for human mammary carcinoma, has been previously reported. These sequences (homologous to env gene site coding for MMTV gp52 envelope antigen) expressed in T cells can play the key role in virus infection transmission and propagation. In order to elucidate the possible routes of env MMTV-homologous sequences expression, we tried to induced it in donot T lymphocytes by various methods: hormone and virus treatment (related genome "saving" at the expense of the added virus envelope), T cell culturing with conA, interferon-2, and 5-azacytidine. RT-PCR with primers specific for the gp52-coding area of MMTV env gene showed expression of env-homologous sequences in donor T cells cultured in medium with 5-azacytidine. Indirect immunofluorescence with monospecific serum to MMTV gp52 detected gp52 analogous genes only in cultures with 5-azacytidine but not other agents. We therefore suggested that MMTV env-homologous sequences in donors are situated in the methylated promoter zone. Expression of these sequences in T cells, specific for human mammary carcinoma, can be due to demethylation of the promoter and induction of env-homologous sequences to the level of translation of gp52 analogous antigens or by initial location of some of the expressed sequences in the demethylated zone of the genome.
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Azacitidina/farmacología , Genes env/efectos de los fármacos , Virus del Tumor Mamario del Ratón/genética , Linfocitos T/efectos de los fármacos , Animales , Antígenos Virales de Tumores , Células Cultivadas , Femenino , Expresión Génica/efectos de los fármacos , Humanos , Interferones/farmacología , Masculino , Neoplasias Mamarias Experimentales/tratamiento farmacológico , Neoplasias Mamarias Experimentales/genética , Ratones , Reacción en Cadena de la Polimerasa , Homología de Secuencia de Ácido Nucleico , Células Tumorales CultivadasRESUMEN
Expression of DNA sequences homologous to sequences of env gene of mouse mammary tumor virus (MMTV) in the lymphocytes of patients with breast cancer and in subjects at a high risk of breast cancer has been reported. Antigen analogous to envelope protein gp52, product of MMTV env gene, is detected in T lymphocytes of virtually all patients with breast cancer and extremely rarely in T cells of controls, where its expression is confined to B cells. For explaining such unexpected results, we studied the molecular basis of this antigen synthesis. Specific PCR products were obtained using primers to gp52-coding region of MMTV env gene. One of them (957 nucleotides) was used as a probe for hybridization of DNA and RNA from lymphocytes of patients with breast cancer and controls. This sequence was hybridized with 90% frequency with genome DNA of breast cancer patients and with 85% frequency with genome RNA of such patients, which is almost 4-fold more than in the controls (patients with gynecological tumors or donors). These results correlate with the frequency of detection of the studied antigen in patients with breast cancer and control group patients.
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Antígenos Virales de Tumores/genética , Neoplasias de la Mama/genética , Genes Virales , Genes env , Virus de la Leucemia Murina/genética , Secuencia de Bases , Southern Blotting , Estudios de Casos y Controles , Cartilla de ADN , Predisposición Genética a la Enfermedad , Humanos , Hibridación de Ácido Nucleico , Reacción en Cadena de la Polimerasa , ARN Viral/genéticaRESUMEN
Previously we reported that HMCAg related to gp52, env product of MMTV, was a specific marker of human breast cancer (BC). This antigen was expressed not only in BC tissue and patients' sera, but in peripheral T- and B-cells. HMCAg was found in 21.4% of healthy donors, but only in the fraction rich in B-cells. We searched for env-homologous sequences in BC patients by Northern and Southern dot-blot hybridization of plasmid clones containing env MMTV with total RNA and DNA of peripheral blood lymphocytes of BC patients, gynecological patients with hormone-dependent tumors, and donors. Transcription of DNA sequences related to env MMTV was revealed in 91.3% of BC patients, 22.2% gynecological patients, and 27.2% donors. In all cases except 1 donor and 1 gynecological patient this transcription correlated with HMCAg expression in lymphoid cells from the same subjects detected by indirect immunofluorescence with anti-gp52 MMTV rabbit serum. Analysis of in vitro translation products with poly(A) RNA as a template and immunoblotting method showed that at least one of the polypeptides formed corresponded to HMCAg by molecular weight and immunological reactivity, which fact appears to explain the correlation described above.