RESUMEN
AIM: To assess the synergistic actions of lidamycin (LDM) and chloroquine (CQ), a lysosomal enzyme inhibitor, in human non-small cell lung cancer (NSCLC) cells, and to elucidate the potential mechanisms. METHODS: Human NSCLC cell lines A549 and H460 were treated with CQ and/or LDM. Cell proliferation was analyzed using MTT assay, and apoptosis was quantified using flow cytometry. Western blotting was used to detect the protein levels of caspase 3, PARP, Bcl-2, Bax, p53, LC3-I and LC3-II. A H460 cell xenograft model in BALB/c nude mice was used to evaluate the anticancer efficacy of CQ and LDM in vivo. RESULTS: Both LDM and CQ concentration-dependently suppressed the proliferation of A549 and H460 cells in vitro (the IC50 values of LDM were 1.70 ± 0.75 and 0.043 ± 0.026 nmol/L, respectively, while the IC50 values of CQ were 71.3 ± 6.1 and 55.6 ± 12.5 µmol/L, respectively). CQ sensitized both NSCLC cell lines to LDM, and the majority of the coefficients of drug interaction (CDIs) for combination-doses were less than 1. The ratio of apoptosis of H460 cells induced by a combined treatment of CQ and LDM (77.0% ± 5.2%) was significantly higher than those caused by CQ (23.1% ± 4.2%) or by LDM (65.1% ± 4.1%) alone. Furthermore, the combined treatment markedly increased the cleaved PARP and cleaved caspase 3 in H460 cells, which were partly reversed by pretreatment with the caspase inhibitor zVAD.fmk. zVAD.fmk also partially reversed the inhibitory effect of the combination treatment on the proliferation of H460 cells. The combination therapy group had a notable increase in expression of Bax and a very slight decrease in expression of Bcl-2 and p53 protein. LDM alone scarcely affected the level of LC3-II in H460 cells, but slightly reduced CQ-induced LC3-II expression. 3-MA, an autophagy inhibitor also sensitized H460 cells to LDM. In nude mice bearing H460 cell xenograft, administration of LDM (25 µg/kg, iv) and CQ (60 mg/kg, ip) suppressed tumor growth by 57.14% and 73.02%, respectively. CONCLUSION: The synergistic anticancer effect of LDM and CQ in vitro results from activation of a caspase-dependent and p53-independent apoptosis pathway as well as inhibition of cytoprotective autophagy.
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Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Neoplasias Pulmonares/tratamiento farmacológico , Aminoglicósidos/administración & dosificación , Animales , Antineoplásicos/administración & dosificación , Apoptosis/efectos de los fármacos , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Caspasa 3/metabolismo , Línea Celular Tumoral , Cloroquina/administración & dosificación , Sinergismo Farmacológico , Enediinos/administración & dosificación , Femenino , Humanos , Neoplasias Pulmonares/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Proteínas Asociadas a Microtúbulos/metabolismo , Poli(ADP-Ribosa) Polimerasas/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Proteína X Asociada a bcl-2/metabolismoRESUMEN
The proteasome inhibitor bortezomib has been applied successfully to treat multiple myeloma (MM). Its synergistic effects with other anticancer drugs have been studied widely. In the present study, it was found that lidamycin (LDM), a member of the enediyne antibiotic family, showed much more potent cytotoxicity than bortezomib to MM cell lines: U266 and SKO-007. Here, we investigated the potential synergy of bortezomib and LDM on MM cells. The results showed that cotreatment of bortezomib and LDM synergistically induced cytotoxicity and apoptosis in MM cell lines, followed by enhanced caspase-3 cleavage and degradation of poly-ADP-ribose polymerase together with the decreased nuclear factor-κB protein. These two drugs synergistically induced apoptosis, which was associated with enhanced activation of two mitogen-activated protein kinases: p38 mitogen-activated protein kinase and c-Jun NH(2)-terminal kinase. Moreover, bortezomib plus LDM synergistically induced apoptosis was also associated with downregulation of extracellular signal-regulated kinase, and induction of endoplasmic reticulum stress response. Overall, our results indicate that the combined regimen of bortezomib and LDM might be a potential therapeutic remedy for the treatment of MM.
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Aminoglicósidos/farmacología , Antineoplásicos/farmacología , Ácidos Borónicos/farmacología , Enediinos/farmacología , Mieloma Múltiple/tratamiento farmacológico , Pirazinas/farmacología , Aminoglicósidos/administración & dosificación , Antineoplásicos/administración & dosificación , Apoptosis/efectos de los fármacos , Ácidos Borónicos/administración & dosificación , Bortezomib , Caspasa 3/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Sinergismo Farmacológico , Enediinos/administración & dosificación , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , FN-kappa B/metabolismo , Poli(ADP-Ribosa) Polimerasas/metabolismo , Pirazinas/administración & dosificación , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
BACKGROUND: The outlook for patients with refractory and relapsed acute lymphocytic leukaemia (ALL) is poor. CD22 is highly expressed in patients with ALL. Inotuzumab ozogamicin is a CD22 monoclonal antibody conjugated to the toxin calecheamicin. We did a phase 2 study to assess the efficacy of this antibody. METHODS: We recruited patients at the MD Anderson Cancer Center, Houston, TX, USA, between June, 2010, and March, 2011. Adults and children with refractory and relapsed ALL were eligible. Ten adults were treated before enrolment of children started. Patients were given 1·8 mg/m(2) inotuzumab ozogamicin intravenously over 1 h every 3-4 weeks (the first three adults and three children received 1·3 mg/m(2) in the first course). The primary endpoint was overall response (complete response or marrow complete response with no recovery of platelet count or incomplete recovery of neutrophil and platelet counts). Analysis was done by intention to treat. This study is registered, number NCT01134575. FINDINGS: 49 patients were enrolled and treated. Median age was 36 years (range 6-80). CD22 was expressed in more than 50% of blasts in all patients. The median number of courses was two (range one to five) and the median time between courses was 3 weeks (range 3-6). Nine (18%) patients had complete response, 19 (39%) had marrow complete response, 19 (39%) had resistant disease, and two (4%) died within 4 weeks of starting treatment. The overall response rate was 57% (95% CI 42-71). The most frequent adverse events during course one of treatment were fever (grade 1-2 in 20 patients, grade 3-4 in nine), hypotension (grade 1-2 in 12 patients, grade 3 in one), and liver-related toxic effects (bilirubin: grade 1-2 in 12 patients, grade 3 in two; raised aminotransferase concentration: grade 1-2 in 27 patients, grade 3 in one). INTERPRETATION: Inotuzumab ozogamicin shows promise as a treatment for refractory and relapsed ALL. FUNDING: Pfizer.
Asunto(s)
Anticuerpos Monoclonales Humanizados/administración & dosificación , Anticuerpos Monoclonales/administración & dosificación , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Lectina 2 Similar a Ig de Unión al Ácido Siálico/inmunología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Aminoglicósidos/administración & dosificación , Anticuerpos Monoclonales/efectos adversos , Anticuerpos Monoclonales Humanizados/efectos adversos , Niño , Enediinos/administración & dosificación , Humanos , Inotuzumab Ozogamicina , Persona de Mediana Edad , Estadificación de Neoplasias , Leucemia-Linfoma Linfoblástico de Células Precursoras/epidemiología , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Análisis de Supervivencia , Estados UnidosRESUMEN
Lidamycin (LDM, also known as C-1027) as an anti-cancer agent inhibits growth in a variety of cancer cells by inducing apoptosis and cell cycle arrest. In this study we demonstrated that inhibition of mouse embryonic carcinoma (EC) cell growth using LDM at low concentrations can be attributed to a loss of the cell's self-renewal capability but not to apoptosis or cell death, which can be correlated to the down-regulation of embryonic stem (ES) cell-like genes Oct4, Sox2 and c-Myc. MTT assays showed that LDM inhibited the growth of mouse P19 EC cells in a time- and dose-dependent manner. The EC cells exposed to a low dose (0.01 nM) of LDM lost their capability to generate colonies, as evidenced by the colony forming assay. Flow cytometer analyses demonstrated that LDM induced G1 arrest in exposed EC cells without apoptosis. Real-time qPCR, Western blotting and immunocytochemistry revealed that Oct4, Sox2 and c-Myc were down-regulated in LDM-exposed EC cells, but not adriamycin (ADM)-exposed cells. Furthermore, a combination of the low dose of LDM and ADM significantly reduced the proliferation of the cancer cells than single-agent treatment. This suggested that synergy of ADM and LDM improved chemotherapy. Taking together, our results indicate that LDM can reduce the capability for self-renewal that mouse EC cells possess through the repression of ES cell-like genes, thereby inhibiting carcinoma cell growth. This data also suggests that LDM might have potential for application in CSC-based therapy and be a useful tool for studying ES cell pluripotency and differentiation.
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Aminoglicósidos/farmacología , Antibióticos Antineoplásicos/farmacología , Células Madre de Carcinoma Embrionario/efectos de los fármacos , Enediinos/farmacología , Aminoglicósidos/administración & dosificación , Animales , Antibióticos Antineoplásicos/administración & dosificación , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo/efectos de los fármacos , Doxorrubicina/farmacología , Sinergismo Farmacológico , Células Madre de Carcinoma Embrionario/metabolismo , Enediinos/administración & dosificación , Citometría de Flujo , Humanos , Ratones , Factor 3 de Transcripción de Unión a Octámeros/genética , Proteínas Proto-Oncogénicas c-myc/genética , Factores de Transcripción SOXB1/genética , Factores de TiempoRESUMEN
OBJECTIVE: Lidamycin (LDM) can be dissociated to an apoprotein (LDP) and an active enediyne chromophore (AE). The detached AE can reassemble with its LDP-containing fusion protein to endow the latter with potent antitumor activity. However, the reassembly of AE with LDP is affected by several factors. Our aim was to optimize the assembly efficiency of the AE with a LDP-containing fusion protein and investigate the influence of several factors on the assembly efficacy. METHODS: A method based on RP-HPLC was developed to analyze the assembly rate, and an orthogonal experimental design L(9) (3(4)) was used to investigate the effects of temperature, assembly time, pH and molecular ratio of LDP-containing fusion protein to AE on the assembly rate. Furthermore, the determined optimum conditions for the assembly rate of the LDP-containing fusion protein with AE were applied and evaluated. RESULTS: A calibration curve based on the LDM micromolar concentration against the peak-area of AE by HPLC was obtained. The order in which individual factors in the orthogonal experiment affected the assembly rate were temperature>time>pH>molar ratio of AE to protein and all were statistically significant (P<0.01). The optimal assembly conditions were temperature at 10°C, time of 12 h, pH 7.0, and the molar ratio of AE: protein of 5:1. The assembly rate of AE with a LDP-containing fusion protein was improved by 23% after condition optimization. CONCLUSION: The assembly rate of chromophore of lidamycin with its LDP-containing fusion protein was improved after condition optimization by orthogonal design, and the optimal conditions described herein should prove useful for the development of this type of LDP-containing fusion protein.
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Aminoglicósidos/síntesis química , Antibióticos Antineoplásicos/síntesis química , Apoproteínas/química , Enediinos/síntesis química , Proteínas Recombinantes de Fusión/química , Anticuerpos de Cadena Única/química , Aminoglicósidos/administración & dosificación , Aminoglicósidos/química , Aminoglicósidos/farmacología , Antibióticos Antineoplásicos/administración & dosificación , Antibióticos Antineoplásicos/química , Antibióticos Antineoplásicos/farmacología , Línea Celular Tumoral , Supervivencia Celular , Cromatografía Líquida de Alta Presión , Diseño de Fármacos , Enediinos/administración & dosificación , Enediinos/química , Enediinos/farmacología , HumanosRESUMEN
This study is to investigate the inhibitory effect of lidamycin (LDM) and its combination with methotrexate (MTX) on lung metastasis of fibrosarcoma by bioluminescence imaging in athymic mice. A stable luciferase transfected HT-1080 cell line was constructed and the capability to establish experimental lung metastasis in athymic mice was confirmed. The optical imaging system was applied to evaluate the formation of lung metastasis in vivo. In addition, metastatic nodules were counted for the evaluation of inhibition rates. As shown, the fluorescent intensity of luciferase-transfected HT-1080 cells was colinear with the cell population and the minimal detected cell population was 100 cells/well. Optical imaging showed that the fluorescent intensity of treated group was apparently lower than that of the control. The inhibition rates of lung metastasis by LDM alone at 0.025 mg x kg(-1) and 0.05 mg x kg(-1) were 53.9% and 75.9%, respectively, while that of MTX alone at 0.5 mg x kg(-1) was 70.2%. The combination of LDM at 0.025 mg x kg(-1) and MTX at 0.5 mg x kg(-1) showed an inhibition rate of 88.7%. The coefficient of drug interaction (CDI) was 0.82. The results herein demonstrated that LDM alone had strong anti-metastasis effect on human fibrosarcoma HT-1080 and the inhibition efficacy is strengthened when combined with MTX.
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Aminoglicósidos/administración & dosificación , Antibióticos Antineoplásicos/administración & dosificación , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Enediinos/administración & dosificación , Fibrosarcoma/patología , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/secundario , Animales , Antimetabolitos Antineoplásicos/administración & dosificación , Línea Celular Tumoral , Sinergismo Farmacológico , Femenino , Humanos , Mediciones Luminiscentes , Pulmón/patología , Neoplasias Pulmonares/patología , Metotrexato/administración & dosificación , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Distribución Aleatoria , Transfección , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
This study is to investigate inhibitory effects of lidamycin (LDM) on the proliferation of HERG K+ channel highly expressing cancer cells and its synergy with anticancer drugs. MTT assay was used to examine the inhibitory effects of lidamycin combined with various anticancer drugs on the proliferation of human lung cancer A549 cells, human colon cancer HT-29 cells and herg-stably-transfected A549 cells. Using the xenograft model of subcutaneously transplanted HT-29 in nude mice, inhibitory effect was appraised in vivo. The coefficient of drug interaction (CDI) was used to evaluate the synergistic effect of drug combination. LDM significantly inhibited the proliferation ofA549 cells and HT-29 cells with IC50 values of 2.14 and 4.64 ng mL(-1), respectively. The efficacy in HT-29 cells with high HERG potassium expression level is less potent than that in A549 cells with low expression level. In terms of IC50 values, LDM suppressed the growth of herg-stably-transfected A549 cells less potently than pCDNA3.1-stably-transfected A549 cells. There existed synergistic effects in the combinations of fluorouracil (5-FU) and LDM, doxorubicin (DOX) and LDM, or hydroxycamptothecine (HCPT) and LDM. CDI values of the combinations of 5-FU and LDM were more than 0.75. CDI values of LDM and DOX were more than 0.70, but some CDI values of LDM and HCPT were less than 0.70. As for the CDI values, synergistic effects of the combination of LDM and HCPT were the most potent of the three groups. There is no relationship between the inhibitory effect of the growth of cancer cells by 5-FU and HERG potassium expression level. HERG expression level negatively correlated with inhibitory effect on the proliferation of cancer cells by DOX. HERG expression levels and chemosensitivity were positively correlated for HCPT. In the model of subcutaneously xenograft transplanted HT-29 in vivo, LDM and/or HCPT effectively inhibited the growth of HT-29 in nude mice, and the optimum CDI of the combination of LDM and HCPT was less than 1. HERG expression level negatively correlates the chemosensitivity of cancer cells to LDM. There exist synergistic effects in vitro and in vivo in the combination of LDM and HCPT, which inhibitory effects of the proliferation of cancer cells positively modulated by HERG potassium expression level. HERG K+ channel may become a target of combined therapy for choosing anticancer drugs.
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Aminoglicósidos/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Proliferación Celular/efectos de los fármacos , Enediinos/farmacología , Canales de Potasio Éter-A-Go-Go/metabolismo , Neoplasias Pulmonares/patología , Aminoglicósidos/administración & dosificación , Animales , Antibióticos Antineoplásicos/administración & dosificación , Antibióticos Antineoplásicos/farmacología , Antineoplásicos Fitogénicos/administración & dosificación , Camptotecina/administración & dosificación , Camptotecina/análogos & derivados , Línea Celular Tumoral , Doxorrubicina/administración & dosificación , Sinergismo Farmacológico , Canal de Potasio ERG1 , Enediinos/administración & dosificación , Fluorouracilo/administración & dosificación , Células HT29 , Humanos , Neoplasias Pulmonares/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
This study is to investigate the effect and its possible mechanisms of lidamycin (LDM) combined with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in human non-small cell lung cancer (NSCLC) cells. MTT assay was used to determine the growth inhibition of the two ingredients on H460 cells. Apoptosis was examined by Annexin V-FITC/PI staining, flow cytometry assay and DNA-specific dye Hoechst 33342 staining. The level of TRAIL receptor and apoptosis-associated protein expression was detected by Western blotting analysis. The results showed that the IC50 value of LDM and TRAIL for H460 cells was 4.603 x 10(-10) mol x L(-1) and 915.3 ng x mL(-1) respectively, but the IC50 value of LDM was 3.064 x 10(-11) mol x L(-1) and 1.611 x 10(-11) mol x L(-1) when different concentrations of LDM was combined with 50 and 100 ng x mL(-1) TRAIL respectively. And the CDI value was less than 1. The apoptosis ratios also increased in the combination group relative to the single-agent treatment and the untreated control. Furthermore, the induction of the cleavage of PARP and the activation of Caspase-3 and Caspase-8 by the combination were more effective than LDM or TRAIL alone. At last, the level of death receptor 5 (DR5) expressions increased in a dose-dependent manner and time-related pattern. The data indicate that LDM inhibits the growth of H460 cells in vitro. DR5 induction contributes to enhancement of TRAIL-induced apoptosis by LDM in human non-small lung cancer cells.
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Aminoglicósidos/farmacología , Apoptosis/efectos de los fármacos , Carcinoma de Pulmón de Células no Pequeñas/patología , Enediinos/farmacología , Neoplasias Pulmonares/patología , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Ligando Inductor de Apoptosis Relacionado con TNF/farmacología , Aminoglicósidos/administración & dosificación , Antibióticos Antineoplásicos/administración & dosificación , Antibióticos Antineoplásicos/farmacología , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Caspasa 3/metabolismo , Caspasa 8/metabolismo , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Enediinos/administración & dosificación , Humanos , Neoplasias Pulmonares/metabolismo , Poli(ADP-Ribosa) Polimerasas/metabolismo , Ligando Inductor de Apoptosis Relacionado con TNF/administración & dosificaciónRESUMEN
BACKGROUND: Neocarzinostatin is a potent antitumor drug consisting of an enediyne chromophore and a protein carrier. METHODS: We characterized an intermediate in the equilibrium unfolding pathway of aponeocarzinostatin, using a variety of biophysical techniques including 1-anilino-8-napthalene sulfonate binding studies, size-exclusion fast protein liquid chromatography, intrinsic tryptophan fluorescence, circular dichroism, and 1H-15N heteronuclear single quantum coherence spectroscopy. RESULTS: The partially unfolded protein is in molten globule-like state, in which approximately 60% and approximately 20% tertiary and secondary structure is disrupted respectively. Despite lacking a fully coordinated tertiary structure for assembling a functional binding cleft, the protein in molten globule-like state is still able to fully protect the labile chromophore. Titration of chromophore leads the partially denatured apoprotein to fold into its native state. CONCLUSION: These findings bring insight into conserving mechanism of neocarzinostatin under harsh environment, where even the partially denatured apoprotein exhibits protective effect, confirming the superiority of the drug carrier.
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Antineoplásicos/administración & dosificación , Apoproteínas/química , Portadores de Fármacos/química , Enediinos/administración & dosificación , Cinostatina/química , Antineoplásicos/química , Sitios de Unión , Dicroismo Circular , Enediinos/química , Guanidina/farmacología , Modelos Moleculares , Conformación Proteica , Pliegue de ProteínaRESUMEN
Pancreatic ductal adenocarcinoma (PDAC) is a refractory malignant tumor with poor prognosis, limited chemotherapeutic efficacy, and only about 5% of 5-year survival rate. We generated a dual-targeting ligand-based lidamycin (DTLL) to investigate its efficacy against pancreatic cancer after preparing its precursor, DTLP. DTLP was shown specifically binding to EGFR and HER2 on cell surface, followed by endocytosis into cytoplasm of pancreatic cancer cells. DTLL significantly promoted apoptosis and cell cycle arrest at G2/M stages and inhibited cell proliferation. Pancreatic tumors of either MIA-paca-2 cell line-derived (CDX) or patient-derived xenograft (PDX) mouse models were significantly regressed in response to DTLL. It suggested that DTLL might be a highly potent bispecific antibody-drug conjugate (ADC)-like agent for pancreatic cancer therapy. LDM is known to function as an antitumor cytotoxic agent by its induction of DNA damage in cancer cells, therefore, DTLL, as its derivative, also showed similar cytotoxicity. However, we found that DTLL might reverse the AKT/mTOR feedback activation induced by LDM at the first time. The results from both in vitro and in vivo experiments suggested that DTLL enhanced DNA damage via EGFR/HER2-dependent blockage of PI3K/AKT/mTOR and PD-L1 signaling pathways in cancer cells, leading to the inhibition of cell proliferation and immunosurveillance escape from pancreatic tumor. Our studies on DTLL functional characterization revealed its novel mechanisms on internal enhancement of DNA damage and implied that DTLL might provide a promising targeted therapeutic strategy for pancreatic cancer.
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Aminoglicósidos/administración & dosificación , Antibióticos Antineoplásicos/administración & dosificación , Anticuerpos Biespecíficos/administración & dosificación , Antígeno B7-H1/antagonistas & inhibidores , Enediinos/administración & dosificación , Inmunoconjugados/administración & dosificación , Neoplasias Pancreáticas/tratamiento farmacológico , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Animales , Antígeno B7-H1/inmunología , Antígeno B7-H1/metabolismo , Línea Celular Tumoral , Daño del ADN , Receptores ErbB/metabolismo , Femenino , Humanos , Ratones Endogámicos BALB C , Ratones Desnudos , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/efectos de los fármacos , Serina-Treonina Quinasas TOR/inmunología , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
Purpose: Adverse reactions reported in patients treated with antibody-calicheamicin conjugates such as gemtuzumab ozogamicin (Mylotarg) and inotuzumab ozogamicin include thrombocytopenia and sinusoidal obstruction syndrome (SOS). The objective of this experimental work was to investigate the mechanism for thrombocytopenia, characterize the liver injury, and identify potential safety biomarkers.Experimental Design: Cynomolgus monkeys were dosed intravenously at 6 mg/m2/dose once every 3 weeks with a nonbinding antibody-calicheamicin conjugate (PF-0259) containing the same linker-payload as gemtuzumab ozogamicin and inotuzumab ozogamicin. Monkeys were necropsied 48 hours after the first administration (day 3) or 3 weeks after the third administration (day 63).Results: PF-0259 induced acute thrombocytopenia (up to 86% platelet reduction) with nadirs on days 3 to 4. There was no indication of effects on megakaryocytes in bone marrow or activation of platelets in peripheral blood. Microscopic evaluation of liver from animals necropsied on day 3 demonstrated midzonal degeneration and loss of sinusoidal endothelial cells (SECs) associated with marked platelet accumulation in sinusoids. Liver histopathology on day 63 showed variable endothelial recovery and progression to a combination of sinusoidal capillarization and sinusoidal dilation/hepatocellular atrophy, consistent with early SOS. Among biomarkers evaluated, there were early and sustained increases in serum hyaluronic acid (HA) that correlated well with serum aspartate aminotransferase and liver microscopic changes, suggesting that HA may be a sensitive diagnostic marker of the liver microvascular injury.Conclusions: These data support the conclusion that target-independent damage to liver SECs may be responsible for acute thrombocytopenia (through platelet sequestration in liver sinusoids) and development of SOS. Clin Cancer Res; 23(7); 1760-70. ©2016 AACR.
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Aminoglicósidos/administración & dosificación , Anticuerpos Monoclonales Humanizados/administración & dosificación , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Trombocitopenia/patología , Aminoglicósidos/efectos adversos , Aminoglicósidos/química , Animales , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales/efectos adversos , Anticuerpos Monoclonales Humanizados/efectos adversos , Células Endoteliales/efectos de los fármacos , Células Endoteliales/patología , Enediinos/administración & dosificación , Enediinos/química , Gemtuzumab , Humanos , Ácido Hialurónico/sangre , Inmunoconjugados/administración & dosificación , Inmunoconjugados/efectos adversos , Inotuzumab Ozogamicina , Hígado/efectos de los fármacos , Macaca fascicularis , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Trombocitopenia/inducido químicamenteRESUMEN
Recent studies have revealed that the epidermal growth factor receptor (EGFR) and insulin-like growth factor-1 receptor (IGF-1R) are overexpressed in various types of human tumors and are attractive targets for anticancer drugs. In the present study, the expression of EGFR and IGF-1R in esophageal squamous cell carcinoma (ESCC) and adjacent normal tissues in a tissue microarray was firstly detected by immunohistochemical staining. In addition, their co-overexpression was observed in 48 out of 75 (64%) patients. Based on the findings, the antitumor activity of an EGFR/IGF-1R bispecific and enediyne-energized fusion protein EGF-LDP-IGF-AE, which we constructed recently by fusing two ligands (EGF and IGF-1) with an enediyne antibiotic lidamycin (LDM), on ESCC were evaluated. Binding assay indicated that the EGF-LDP-IGF protein bound to esophageal cancer cells, and then internalized into the cytoplasm. In vitro, the enediyneenergized fusion protein EGF-LDP-IGF-AE exhibited extremely potent cytotoxicity to ESCC cells with IC50 values between 10-10 and 10-15 mol/l. In vivo, EGF-LDPIGF-AE also markedly suppressed the growth of human KYSE450 xenografts by 75.1% when administered at 0.3 mg/kg in a nude mouse model, and its efficacy was significantly higher than that of LDM (at maximum tolerated dosage) and mono-specific counterparts. In addition, EGF-LDP-IGF-AE arrested cell cycle progression and it concentration-dependently induced cell apoptosis as well as inhibited the activation of EGFR/IGF-1R and two major downstream signaling pathways (PI3K/AKT and RAS/MAPK). These data imply the potential clinical application of EGF-LDP-IGF-AE for ESCC patients with EGFR and/or IGF-1R overexpression.
Asunto(s)
Carcinoma de Células Escamosas/tratamiento farmacológico , Receptores ErbB/genética , Neoplasias Esofágicas/tratamiento farmacológico , Receptor IGF Tipo 1/genética , Proteínas Recombinantes de Fusión/administración & dosificación , Aminoglicósidos/administración & dosificación , Aminoglicósidos/química , Animales , Apoptosis/efectos de los fármacos , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patología , Proliferación Celular/efectos de los fármacos , Enediinos/administración & dosificación , Enediinos/química , Factor de Crecimiento Epidérmico/química , Factor de Crecimiento Epidérmico/genética , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/patología , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Factor I del Crecimiento Similar a la Insulina/química , Factor I del Crecimiento Similar a la Insulina/genética , Ligandos , Masculino , Ratones , Unión Proteica , Proteínas Recombinantes de Fusión/genética , Transducción de Señal/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: Rituximab is widely used in clinical setting for the treatment of B malignant lymphoma and has achieved remarkable success. However, in most patients, the disease ultimately relapses and become resistant to rituximab. To overcome the limitation, there is still a need to find novel strategy for improving therapeutic efficacy. OBJECTIVE: To construct genetically engineered antibody anti-CD19(Fab)-LDM, and verify the anticancer activity targeted toward B-lymphoma. METHODS: The anticancer activity of anti-CD19(Fab)-LDM in vitro and in vivo was examined. In vitro, the binding activity and internalization of anti-CD19(Fab)-LDP were measured. Using comet assay and apoptosis, the cytotoxicity of energized fusion proteins was observed. From in vivo experiments, targeting of therapeutic effect and anticancer efficacy bythe fusion protein was verified. RESULTS: Data showed that anti-CD19(Fab)-LDM does not only binding the cell surface but is also internalized into the cell. The energized fusion proteins anti-CD19(Fab)-LDM can induce DNA damage. Furthermore, significant in vivo therapeutic efficacy was observed. CONCLUSION: The present study demonstrated that the genetically engineered antibody anti-CD19(Fab)-LDM exhibited enhanced cytotoxicity compared to LDM alone. One of the most powerful advantages of anti-CD19(Fab)-LDM, however, is that it can be internalized within the cells and carry out cytotoxic effects. Therefore, anti-CD19(Fab)-LDM may be as a useful targeted therapy for B-cell lymphoma.
Asunto(s)
Aminoglicósidos/farmacología , Antibióticos Antineoplásicos/farmacología , Anticuerpos Monoclonales/farmacología , Antígenos CD19/inmunología , Enediinos/farmacología , Linfoma de Células B/fisiopatología , Proteínas Recombinantes de Fusión/farmacología , Aminoglicósidos/administración & dosificación , Animales , Antibióticos Antineoplásicos/administración & dosificación , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales/química , Apoptosis , Línea Celular Tumoral , Supervivencia Celular , Relación Dosis-Respuesta a Droga , Portadores de Fármacos , Enediinos/administración & dosificación , Femenino , Xenoinjertos , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Proteínas Recombinantes de Fusión/administración & dosificaciónRESUMEN
AIMS: The aim of this study is to explore the antitumor efficacy of lidamycin (LDM) against human multiple myelomas (MM). MATERIALS AND METHODS: Human MM RPMI 8226 cells and the xenograft model in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice were used to examine the antitumor activity of LDM. RESULTS: Notably, LDM markedly suppressed the growth of human MM RPMI 8226 xenograft in NOD/SCID mice. In vitro, there was a significant reduction in cell proliferation after treatment with LDM. The overall growth inhibition correlated with the increase of apoptotic cells. The apoptosis-related proteins including caspase-3, 7, and 9 were activated, and poly adenosine diphosphate-ribose polymerase was cleaved. Further investigation revealed that cellular Bcl-2 and survivin decreased, whereas the level of Bax increased in the LDM-treated cells. CONCLUSIONS: LDM is highly effective against the growth of MM xenograft in NOD/SCID mice. The potent apoptosis.inducing effect of LDM may be mediated through caspase. and mitochondria.dependent pathway.
Asunto(s)
Aminoglicósidos/administración & dosificación , Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Enediinos/administración & dosificación , Mieloma Múltiple/tratamiento farmacológico , Animales , Caspasas/biosíntesis , Caspasas/genética , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Ratones , Ratones Endogámicos NOD , Mieloma Múltiple/patología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
AIM: To study the mechanism of inhibition of basic fibroblast growth factor (bFGF) related signal transduction by lidamycin in cancer cells. METHODS: MTT assay was used to determine the growth inhibitory effect of lidamycin (LDM) and adriamycin (ADR) in several cancer cell lines. The inhibition of bFGF bound to its receptor by LDM was measured with [125I]-bFGF binding assay. Intracellular Ca2+ stimulated by bFGF was measured by Fura-3. The formation of bFGF-receptor immune complex and the inhibitory effect of LDM on the activity of PKC isoenzymes induced by bFGF in cancer cells were identified by Western blotting analysis. RESULTS: LDM displayed extremely potent growth inhibitory effect on several cancer cell lines in a dose-dependent manner. A comparison of the IC50 values showed that the effect of LDM was 1000-fold more potent than that of ADR. LDM blocked the specific binding of [125I]-bFGF to rat lung membranes with an IC50 value of 2.0 x 10(-4) nmol x L(-1). As detected by anti-FGFR specific antibody, LDM inhibited the formation of bFGF-receptor immune complex. bFGF induced cytosolic Ca2+ response was obstructed by pretreatment with 10 nmol x L(-1) LDM. Immunoblotting demonstrated that LDM inhibited the activity of PKC isoenzymes in cancer cells stimulated with bFGF. CONCLUSION: The blocking of bFGF receptors in the signal transduction pathway may be involved in the effect of LDM on cancer cells.
Asunto(s)
Aminoglicósidos/farmacología , Antibióticos Antineoplásicos/farmacología , Enediinos/farmacología , Factor 2 de Crecimiento de Fibroblastos/farmacología , Proteína Quinasa C/metabolismo , Aminoglicósidos/administración & dosificación , Animales , Antibióticos Antineoplásicos/administración & dosificación , Neoplasias de la Mama/patología , Calcio/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Doxorrubicina/farmacología , Enediinos/administración & dosificación , Femenino , Células HT29 , Humanos , Proteínas de la Membrana/metabolismo , Unión Proteica , Ratas , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Transducción de SeñalRESUMEN
Gelatinases play important roles in tumour invasion and metastasis and are thus considered promising targets for cancer therapy. In this study, a new single-chain variable fragment (scFv)-based fusion protein Fv-LDP, composed of the anti-gelatinases scFv and lidamycin apoprotein (LDP), was prepared, and its combination with angiogenesis inhibitor Endostar was then investigated. The fusion protein Fv-LDP specifically bound to various tumour cells, and its binding capability to human pulmonary giant cell carcinoma (PG) cells was higher than that of LDP. Fv-LDP inhibited the expression and secretion of gelatinases and could be internalized into tumour cells via endocytosis. Fv-LDP also suppressed the growth of human hepatoma cells and murine hepatoma 22 transplanted in Kunming mice in various degrees. In addition, Endostar could enhance the synergistic or additive inhibition of Fv-LDP on the growth, migration or invasion of human hepatoma cells shown by a colony formation assay and a transwell-based migration or invasion assay, respectively. In vivo, Fv-LDP/Endostar combination showed a significantly synergistic effect on the growth of a human hepatoma xenograft, with an inhibition rate of 80.8% compared with the Fv-LDP (44.1%) or Endostar (8.9%)-treated group. The above-mentioned results indicate that the fusion protein Fv-LDP is effective against transplantable hepatoma in mice and human hepatoma xenografts in athymic mice. Moreover, Endostar can potentiate the inhibition effect of Fv-LDP on the growth of human hepatoma cells and xenografts. These data will provide a new combined strategy for improving the therapeutic efficacy of treatments for hepatoma or other gelatinase-overexpressing tumours.
Asunto(s)
Aminoglicósidos/farmacología , Carcinoma Hepatocelular/tratamiento farmacológico , Endostatinas/farmacología , Enediinos/farmacología , Neoplasias Hepáticas/tratamiento farmacológico , Anticuerpos de Cadena Única/farmacología , Aminoglicósidos/administración & dosificación , Inhibidores de la Angiogénesis/administración & dosificación , Inhibidores de la Angiogénesis/farmacología , Animales , Apoproteínas/administración & dosificación , Apoproteínas/farmacología , Carcinoma de Células Gigantes/tratamiento farmacológico , Carcinoma de Células Gigantes/enzimología , Carcinoma de Células Gigantes/patología , Carcinoma Hepatocelular/enzimología , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Sinergismo Farmacológico , Endostatinas/administración & dosificación , Enediinos/administración & dosificación , Femenino , Gelatinasas/metabolismo , Humanos , Neoplasias Hepáticas/enzimología , Neoplasias Hepáticas/patología , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/enzimología , Neoplasias Pulmonares/patología , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Proteínas Recombinantes , Anticuerpos de Cadena Única/administración & dosificación , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
PURPOSE: Capecitabine (CAP), a prodrug, needs to be converted to 5-fluorouracil by several key enzymes, including thymidine phosphorylase (TP). To improve the therapeutic index, potentiation of antitumor activity of CAP is required. In this study, we explored whether lidamycin (LDM), an enediyne anticancer antibiotic, can induce synergistic antitumor effects in combination with CAP in murine breast cancer in vitro and in vivo. METHODS: Using MTT, cell migration and invasion, siRNA knockdown, and Western blot assays, the in vitro synergistic effects of LDM plus CAP on 4T1(LUC) cells were evaluated, and the mechanism of this synergy was explored. For in vivo model of orthotopic implantation model of 4T1(LUC) cells, optical molecular imaging system was utilized to evaluate the growth of primary tumor and metastasis. To further understand the mechanism of action of the LDM/CAP combination, immunohistochemistry analysis was carried out to detect thymidine phosphorylase induction and ERK signaling. RESULTS: As determined by MTT and transwell assay, LDM enhanced the inhibitory effects of CAP on cancer cell proliferation, migration, and invasion. Western blot showed that this synergistic effect was attributed to the up-regulated expression of TP induced by LDM. Knocking down TP impaired the synergistic anti-proliferative effect of LDM and CAP. Furthermore, our data suggested that LDM-induced up-regulation of TP both in vitro and in vivo is associated with ERK activation, because the inhibition of ERK activity by ERK inhibitor U0126 abrogated LDM-induced TP up-regulation. In animal models, LDM plus CAP potently inhibited primary tumor growth as well as lung metastasis compared with control or single-agent-treated group. CONCLUSIONS: LDM can potentiate the antitumor effects of CAP on breast cancer line. The synergistic effects suggest that the combination of LDM and CAP is an innovative antitumor strategy for breast cancer therapy.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Neoplasias Pulmonares/prevención & control , Neoplasias Mamarias Experimentales/tratamiento farmacológico , Timidina Fosforilasa/genética , Regulación hacia Arriba/efectos de los fármacos , Aminoglicósidos/administración & dosificación , Animales , Western Blotting , Capecitabina , Línea Celular Tumoral , Desoxicitidina/administración & dosificación , Desoxicitidina/análogos & derivados , Sinergismo Farmacológico , Enediinos/administración & dosificación , Inducción Enzimática/efectos de los fármacos , Femenino , Fluorouracilo/administración & dosificación , Fluorouracilo/análogos & derivados , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Neoplasias Pulmonares/secundario , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Neoplasias Mamarias Experimentales/patología , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Timidina Fosforilasa/biosíntesisRESUMEN
The overexpression of tissue factor (TF) observed in numerous cancer cells and clinical samples of human cancers makes TF an ideal target for cancer therapy. The purpose of this study is to develop a TF-targeting energized fusion protein hlFVII-LDP-AE, which is composed of a human Factor VII light chain (hlFVII) as the targeting domain conjugated to the cytotoxic antibiotic lidamycin (LDM, LDP-AE) as the effector domain. The potential efficacy of hlFVII-LDP-AE for cancer therapy was tested in vitro by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and colony formation assays and in vivo with a BALB/c nude mouse xenograft model of human liver cancer line HepG2. The inhibitory concentration (IC(50)) value of hlFVII-LDP-AE varied from 0.15 to 0.64 nM for the various human tumor lines. hlFVII-LDP-AE showed a tumor growth inhibition rate of 90.6% at the dose of 0.6 mg/kg in in vivo animal experiments. The mechanism through which hlFVII-LDP-AE inhibits tumor growth also was determined by Hoechst 33342 staining and Tdt-mediated dUTP nick-end labeling (TUNEL) assay. hlFVII-LDP-AE causes tumor cell death through inducing chromatin condensation and cleavage of genomic DNA. These findings suggest that the hlFVII-LDP-AE protocol is efficacious and tolerated in the mouse model of human liver cancer HepG2 and has clinical applicability for treating cancer patients.
Asunto(s)
Aminoglicósidos/administración & dosificación , Aminoglicósidos/farmacocinética , Enediinos/administración & dosificación , Enediinos/farmacocinética , Factor VII/administración & dosificación , Factor VII/farmacocinética , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/metabolismo , Tromboplastina/metabolismo , Animales , Línea Celular Tumoral , Sistemas de Liberación de Medicamentos , Femenino , Células Hep G2 , Humanos , Etiquetado Corte-Fin in Situ , Ratones , Ratones Desnudos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Tumor-targeted delivery of a potent cytotoxic agent, calicheamicin, using its immunoconjugates is a clinically validated therapeutic strategy. Rituximab is a human CD20-specific chimeric antibody extensively used in B-NHL therapy. We investigated whether conjugation to calicheamicin can improve the anti-tumor activity of rituximab against human B-cell lymphoma (BCL) xenografts in preclinical models. BCL cells were cultured with rituximab or its calicheamicin conjugates and their in vitro growth was monitored. BCL cells were injected s.c. to establish localized xenografts in nude mice or i.v. to establish disseminated BCL in severe combined immunodeficient (scid) mice. I.p. treatment with rituximab or its calicheamicin conjugates was initiated and its effect on s.c. BCL growth or survival of mice with disseminated BCL was monitored. Conjugation of calicheamicin to rituximab vastly enhanced its growth inhibitory activity against BCL in vitro. Conjugation to calicheamicin had no deleterious effect on the effector functional activity of rituximab. Calicheamicin conjugated to rituximab with an acid-labile linker exhibited greater anti-tumor activity against s.c. BCL xenografts and improved survival of mice with disseminated BCL over that of unconjugated rituximab. Anti-tumor activities of rituximab conjugated to calicheamicin via an acid-stable linker were similar to that of unconjugated rituximab. Superior anti-tumor efficacy exhibited by a calicheamicin immunoconjugate of rituximab with an acid-labile linker over that of rituximab demonstrates the therapeutic potential of CD20-specific antibody-targeted chemotherapy strategy in the treatment of B-NHL.