RESUMEN
BACKGROUND: Glioblastoma (GBM) is the most common primary brain tumor in adults. Despite extensive research and clinical trials, median survival post-treatment remains at 15 months. Thus, all opportunities to optimize current treatments and improve patient outcomes should be considered. A recent retrospective clinical study found that taking TMZ in the morning compared to the evening was associated with a 6-month increase in median survival in patients with MGMT-methylated GBM. Here, we hypothesized that TMZ efficacy depends on time-of-day and O6-Methylguanine-DNA Methyltransferase (MGMT) activity in murine and human models of GBM. METHODS AND RESULTS: In vitro recordings using real-time bioluminescence reporters revealed that GBM cells have intrinsic circadian rhythms in the expression of the core circadian clock genes Bmal1 and Per2, as well as in the DNA repair enzyme, MGMT. Independent measures of MGMT transcript levels and promoter methylation also showed daily rhythms intrinsic to GBM cells. These cells were more susceptible to TMZ when delivered at the daily peak of Bmal1 transcription. We found that in vivo morning administration of TMZ also decreased tumor size and increased body weight compared to evening drug delivery in mice bearing GBM xenografts. Finally, inhibition of MGMT activity with O6-Benzylguanine abrogated the daily rhythm in sensitivity to TMZ in vitro by increasing sensitivity at both the peak and trough of Bmal1 expression. CONCLUSION: We conclude that chemotherapy with TMZ can be dramatically enhanced by delivering at the daily maximum of tumor Bmal1 expression and minimum of MGMT activity and that scoring MGMT methylation status requires controlling for time of day of biopsy.
Asunto(s)
Neoplasias Encefálicas , Glioblastoma , Humanos , Animales , Ratones , Glioblastoma/tratamiento farmacológico , Glioblastoma/genética , Glioblastoma/patología , Temozolomida/farmacología , Temozolomida/uso terapéutico , Dacarbazina/uso terapéutico , Antineoplásicos Alquilantes/farmacología , Antineoplásicos Alquilantes/uso terapéutico , O(6)-Metilguanina-ADN Metiltransferasa/genética , Estudios Retrospectivos , Factores de Transcripción ARNTL/genética , Factores de Transcripción ARNTL/metabolismo , Metilación , Enzimas Reparadoras del ADN/genética , Enzimas Reparadoras del ADN/metabolismo , Metilasas de Modificación del ADN/genética , Metilasas de Modificación del ADN/metabolismo , Metilación de ADN , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismoRESUMEN
BACKGROUND: Inherent resistance to radio/chemotherapy is one of the major reasons for early recurrence, treatment failure, and dismal prognosis of glioblastoma. Thus, the identification of resistance driving regulators as prognostic and/or predictive markers as well as potential vulnerabilities for combined modality treatment approaches is of pivotal importance. METHODS: We performed an integrative analysis of treatment resistance and DNA damage response regulator expression in a panel of human glioblastoma cell lines. mRNA expression levels of 38 DNA damage response regulators were analyzed by qRT-PCR. Inherent resistance to radiotherapy (single-shot and fractionated mode) and/or temozolomide treatment was assessed by clonogenic survival assays. Resistance scores were extracted by dimensionality reduction and subjected to correlation analyses with the mRNA expression data. Top-hit candidates with positive correlation coefficients were validated by pharmacological inhibition in clonogenic survival assays and DNA repair analyses via residual γH2AX/53BP1-foci staining. RESULTS: Inherent resistance to single-shot and similarly also to fractionated radiotherapy showed strong positive correlations with mRNA expression levels of known vulnerabilities of GBM, including PARP1, NBN, and BLM, as well as ATR and LIG4-two so far underestimated targets. Inhibition of ATR by AZD-6738 resulted in robust and dose-dependent radiosensitization of glioblastoma cells, whereas LIG4 inhibition by L189 had no noticeable impact. Resistance against temozolomide showed strong positive correlation with mRNA expression levels of MGMT as to be expected. Interestingly, it also correlated with mRNA expression levels of ATM, suggesting a potential role of ATM in the context of temozolomide resistance in glioblastoma cells. ATM inhibition exhibited slight sensitization effects towards temozolomide treatment in MGMT low expressing glioblastoma cells, thus encouraging further characterization. CONCLUSIONS: Here, we describe a systematic approach integrating clonogenic survival data with mRNA expression data of DNA damage response regulators in human glioblastoma cell lines to identify markers of inherent therapy resistance and potential vulnerabilities for targeted sensitization. Our results provide proof-of-concept for the feasibility of this approach, including its limitations. We consider this strategy to be adaptable to other cancer entities as well as other molecular data qualities, and its upscaling potential in terms of model systems and observational data levels deserves further investigation.
Asunto(s)
Neoplasias Encefálicas , Glioblastoma , Antineoplásicos Alquilantes/uso terapéutico , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/terapia , Línea Celular Tumoral , Quimioradioterapia , Terapia Combinada , Metilasas de Modificación del ADN/genética , Metilasas de Modificación del ADN/metabolismo , Metilasas de Modificación del ADN/uso terapéutico , Enzimas Reparadoras del ADN/genética , Enzimas Reparadoras del ADN/metabolismo , Enzimas Reparadoras del ADN/uso terapéutico , Glioblastoma/tratamiento farmacológico , Glioblastoma/terapia , Humanos , ARN Mensajero/genética , Temozolomida/farmacología , Temozolomida/uso terapéutico , TranscriptomaRESUMEN
Banxia xiexin decoction (BXXX) is a classic preparation used to treat gastrointestinal diseases, and also has certain therapeutic effects on gastrointestinal tumors. BXXX has been reported to regulate the expression of proteins associated with drug resistance and sensitivity in tumors, and thus, the aim of the present study was to investigate the mechanisms of BXXX drug sensitivity in gastric cancer (GC). The expression levels of programmed cell death 1 ligand 1 (PDL1), 6OmethylguanineDNA methyltransferase (MGMT) and STAT3 were immunohistochemically detected in the cancer and adjacent noncancer tissues of patients with GC, and in vitro experimentation was conducted using drugresistant and sensitive GC cells. The expression levels of PDL1, MGMT and STAT3 were determined using reverse transcriptionquantitative PCR. Different concentrations of BXXX drug serum were used to treat the cells and the cellular inhibition rate was assessed using a Cell Counting Kit8 assay. Flow cytometry was used to detect apoptosis, and western blot analysis was used to detect the expression levels of IL6, IFNγ, JAK/STAT3 pathway proteins, PDL1 and MGMT. The association between PDL1 and MGMT protein expression levels was subsequently assessed via coimmunoprecipitation. Furthermore, in vivo studies were conducted following the establishment of a drugresistant tumorbearing mouse model, where GC tumor size was assessed under different treatment conditions, and western blot analysis was used to detect the expression of related pathway proteins. The expression levels of PDL1, MGMT and STAT3 were significantly increased in GC tissues, GC cells and cisplatinresistant cells. Furthermore, BXXX inhibited the proliferation of drugresistant cells and promoted the inhibitory effects of chemotherapeutic drugs on drugresistant cells. BXXX also inhibited the expression levels of IL6, IFNγ and JAK/STAT3 pathway proteins, as well as the expression levels of PDL1 and MGMT. Colivelin, an activator of STAT3, reversed the effects of BXXX on drugresistant GC cells, and significantly reversed the effect of BXXX on PDL1 expression. In conclusion, BXXX was found to influence the drug sensitivity of GC cells by regulating the expression of MGMT. This process functions viaPDL1, which was itself mediated by IL6/JAK/STAT3 signaling.
Asunto(s)
Antígeno B7-H1/genética , Metilasas de Modificación del ADN/genética , Enzimas Reparadoras del ADN/genética , Medicamentos Herbarios Chinos/farmacología , Transducción de Señal/efectos de los fármacos , Neoplasias Gástricas/genética , Proteínas Supresoras de Tumor/genética , Adulto , Anciano , Animales , Antígeno B7-H1/metabolismo , Línea Celular Tumoral , Metilasas de Modificación del ADN/metabolismo , Enzimas Reparadoras del ADN/metabolismo , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Interleucina-6/metabolismo , Quinasas Janus/metabolismo , Masculino , Ratones Endogámicos BALB C , Ratones Desnudos , Persona de Mediana Edad , Factor de Transcripción STAT3/genética , Factor de Transcripción STAT3/metabolismo , Transducción de Señal/genética , Neoplasias Gástricas/tratamiento farmacológico , Neoplasias Gástricas/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto/métodosRESUMEN
Colorectal cancer is a common cancer worldwide and reduced expression of the DNA repair endonuclease XPF (xeroderma pigmentosum complementation group F) is associated with colorectal cancer. Bacopa monnieri extracts were previously found to exhibit chemical-genetic synthetic lethal effects in a Saccharomyces cerevisiae model of colorectal cancer lacking Rad1p, a structural and functional homologue of human XPF. However, the mechanisms for B. monnieri extracts to limit proliferation and promote an apoptosis-like event in RAD1 deleted yeast was not elucidated. Our current analysis has revealed that B. monnieri extracts have the capacity to promote mutations in rad1∆ cells. In addition, the effects of B. monnieri extracts on rad1∆ yeast is linked to disruption of the vacuole, similar to the mammalian lysosome. The absence of RAD1 in yeast sensitizes cells to the effects of vacuole disruption and the release of proteases. The combined effect of increased DNA mutations and release of vacuolar contents appears to induce an apoptosis-like event that is dependent on the meta-caspase Yca1p. The toxicity of B. monnieri extracts is linked to sterol content, suggesting saponins may be involved in limiting the proliferation of yeast cells. Analysis of major constituents from B. monnieri identified a chemical-genetic interaction between bacopasaponin C and rad1∆ yeast. Bacopasaponin C may have potential as a drug candidate or serve as a model for the development of analogs for the treatment of colorectal cancer.
Asunto(s)
Bacopa/química , Enzimas Reparadoras del ADN/metabolismo , Endonucleasas/metabolismo , Glicósidos/farmacología , Extractos Vegetales/farmacología , Proteínas de Saccharomyces cerevisiae/metabolismo , Triterpenos/farmacología , Vacuolas/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Enzimas Reparadoras del ADN/deficiencia , Enzimas Reparadoras del ADN/genética , Endonucleasas/deficiencia , Endonucleasas/genética , Glicósidos/química , Extractos Vegetales/química , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Triterpenos/química , Vacuolas/metabolismoRESUMEN
The 2016 World Health Organization classification of central nervous system tumor firstly introduces molecular diagnosis to glioma, while the molecular features of adult thalamic gliomas (ATGs) in a relatively large sample have not been reported. We aimed at exploring molecular characteristics in ATGs. The data of 97 and 575 newly diagnosed ATGs and superficial gliomas (SGs) patients were collected, and we performed a comparative analysis of molecular characteristics between them. We analyzed expressions of molecules as follow: H3 K27M, isocitrate dehydrogenase1 (IDH1), Ki-67, O6-Methylguanine-DNA methyltransferase (MGMT) promoter, EGFR, p53, ATRX, GFAP, Oligo2, PTEN, MGMT, and MMP9 by immunohistochemistry. Direct gene sequencing was performed to test the H3 K27M, IDH1, and TERT promoter mutation. The median age at diagnosis of ATGs was 36.0 years, and majority of them were high-grade glioma. We found a significant difference in H3 K27M mutation (P = 0.003), IDH1 mutation (P < 0.001), MGMT promoter methylation (P = 0.005), and Ki67 > 0.1 (P < 0.001) between ATGs and SGs. The statuses of IDH1 (P < 0.001), MGMT promoter (P < 0.001), and Ki67 (P < 0.001) were significantly different between these two groups in lower-grade gliomas. And statuses of IDH1 (P < 0.001), Ki67 (P < 0.001), and EGFR (P = 0.032) were different between these two groups in high-grade gliomas. Only Ki67 > 0.1 was differentially expressed between lower- and high-grade gliomas in ATGs (P = 0.014). The high occurrence of H3 K27M mutation and Ki67 > 0.1, rare occurrence of IDH1 mutation, and MGMT promoter methylation in ATGs suggested that ATGs may be a distinct type of glioma entity.
Asunto(s)
Neoplasias Encefálicas/metabolismo , Glioma/metabolismo , Tálamo/metabolismo , Adulto , Anciano , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Metilación de ADN , Metilasas de Modificación del ADN/genética , Metilasas de Modificación del ADN/metabolismo , Enzimas Reparadoras del ADN/genética , Enzimas Reparadoras del ADN/metabolismo , Receptores ErbB/genética , Receptores ErbB/metabolismo , Femenino , Proteína Ácida Fibrilar de la Glía/genética , Proteína Ácida Fibrilar de la Glía/metabolismo , Glioma/genética , Glioma/patología , Humanos , Isocitrato Deshidrogenasa/genética , Isocitrato Deshidrogenasa/metabolismo , Antígeno Ki-67/genética , Antígeno Ki-67/metabolismo , Masculino , Metaloproteinasa 9 de la Matriz/genética , Metaloproteinasa 9 de la Matriz/metabolismo , Persona de Mediana Edad , Mutación , Fosfohidrolasa PTEN/genética , Fosfohidrolasa PTEN/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Proteína Nuclear Ligada al Cromosoma X/genética , Proteína Nuclear Ligada al Cromosoma X/metabolismoRESUMEN
XPA (Xeroderma pigmentosum complementation group A) is a core scaffold protein that plays significant roles in DNA damage verification and recruiting downstream endonucleases in the nucleotide excision repair (NER) pathway. Here, we present the 2.81 Å resolution crystal structure of the DNA-binding domain (DBD) of human XPA in complex with an undamaged splayed-arm DNA substrate with a single pair of non-complementary nucleotides. The structure reveals that two XPA molecules bind to one splayed-arm DNA with a 10-bp duplex recognition motif in a non-sequence-specific manner. XPA molecules bind to both ends of the DNA duplex region with a characteristic ß-hairpin. A conserved tryptophan residue Trp175 packs against the last base pair of DNA duplex and stabilizes the conformation of the characteristic ß-hairpin. Upon DNA binding, the C-terminal last helix of XPA would shift towards the minor groove of the DNA substrate for better interaction. Notably, human XPA is able to bind to the undamaged DNA duplex without any kinks, and XPA-DNA binding does not bend the DNA substrate obviously. This study provides structural basis for the binding mechanism of XPA to the undamaged splayed-arm DNA with a single pair of non-complementary nucleotides.
Asunto(s)
Daño del ADN , ADN/química , Modelos Moleculares , Proteína de la Xerodermia Pigmentosa del Grupo A/química , Aminoácidos , Sitios de Unión , Enzimas Reparadoras del ADN/química , Enzimas Reparadoras del ADN/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Humanos , Sustancias Macromoleculares/química , Sustancias Macromoleculares/metabolismo , Modelos Biológicos , Conformación Molecular , Unión Proteica , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Relación Estructura-Actividad , Factor de Transcripción TFIIH/química , Factor de Transcripción TFIIH/metabolismo , Proteína de la Xerodermia Pigmentosa del Grupo A/metabolismoRESUMEN
PURPOSE: A previous study has identified that XRCC4-like factor (XLF) is a potential target to overcome resistance to 5-fluorouracil (5-Fu) and oxaliplatin (OXA) in colorectal cancer (CRC). The purpose of this study is to develop potent XLF inhibitors to chemoresistance in CRC. METHODS: Virtual screening was adopted to identify novel XLF-binding compounds by initially testing 6800 molecules in Chemical Entities of Biological Interest library. Hit compounds were further validated by Western blot assay. Cell sensitivity to 5-Fu and OXA was measured using sulforhodamine B assay. The effect of XLF inhibitor on DNA repair efficiency was evaluated by comet assay, fluorescent-based nonhomologous end joining (NHEJ) and homologous recombination (HR) reporter assays. DNA-binding activity of NHEJ key factors was examined by chromatin fractionation assay. RESULTS: We identified G3, a novel and potent XLF inhibitor (IC50 0.47±0.02 µM). G3 induced XLF protein degradation in CRC cells. Significantly, G3 improved cell sensitivity to 5-Fu and OXA in chemoresistant CRC cell lines. Mechanistically, G3 depleted XLF expression, severely compromised NHEJ efficiency by up to 65% and inhibited NHEJ key factor assembly on DNA. G3 also inhibited HR efficiency in a time-dependent manner. CONCLUSION: These results suggest that G3 overcomes 5-Fu and OXA resistance in CRC cells by inhibiting XLF expression. Thus, XLF is a promising target and its inhibitor G3 is a potential candidate for treatment of chemoresistant CRC patients.
Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Productos Biológicos/farmacología , Neoplasias Colorrectales/tratamiento farmacológico , Enzimas Reparadoras del ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/antagonistas & inhibidores , Antineoplásicos Fitogénicos/química , Antineoplásicos Fitogénicos/aislamiento & purificación , Productos Biológicos/química , Productos Biológicos/aislamiento & purificación , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Reparación del ADN , Enzimas Reparadoras del ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Ensayos de Selección de Medicamentos Antitumorales , Células HCT116 , Humanos , Simulación del Acoplamiento Molecular , Estructura Molecular , Relación Estructura-Actividad , Células Tumorales CultivadasRESUMEN
Adult thalamic glioblastomas (GBM) are uncommon tumors with limited available molecular data. One of the reported molecular alterations in these tumors is the H3K27M mutation. It has been documented that H3K27M mutation is found in a high proportion of pediatric thalamic gliomas. In this study, we have analyzed the molecular alterations exclusive to adult thalamic GBM. This is a 6 years retrospective study of adult thalamic GBM patients who underwent surgical decompression of the tumor. Clinical data were obtained from the case records. Immunohistochemistry (IHC) was performed on the tumors using antibodies directed against the gene products of R132H mutant isocitrate dehydrogenase 1 (IDH1), alpha-thalassemia/mental retardation X-linked (ATRX), p53, H3K27M, H3K27me3, and V600E mutant BRAF. Molecular analyses were carried out to detect other IDH1 and IDH2 mutations, O6 -methylguanine-DNA-methyltransferase gene (MGMT) promoter methylation, and epidermal growth factor gene (EGFR) and telomerase reverse transcriptase gene (TERT) promoter mutations. A total of 42 cases of adult thalamic GBM were studied. The mean age of presentation was 42 years with age range of 19-58 years. Male predominance was noted. All the tumors were IDH wild-type, BRAF (V600E)-immunonegative and unmethylated for MGMT promoter. H3K27M immunopositivity was noted in 60% of tumors. Of these 33.3% were from older adults above the age of 50 years. Of the H3K27M-immunopositive cases, ATRX loss of expression was seen in 32%, p53 immunopositivity in 24% and EGFR amplification in 12%. Higher frequency of TERT promoter mutations was noted in H3K27M-immunonegative cases (58.8%) compared to immunopositive cases (20%). Ours is one of the few studies elucidating the molecular alterations exclusive to adult thalamic GBM. We show a high frequency of H3K27M immunopositivity, suggestive of its mutational status in these tumors, including in older adults.
Asunto(s)
Neoplasias Encefálicas/metabolismo , Glioblastoma/metabolismo , Histonas/metabolismo , Tálamo/metabolismo , Adulto , Metilasas de Modificación del ADN/metabolismo , Enzimas Reparadoras del ADN/metabolismo , Receptores ErbB/metabolismo , Femenino , Humanos , Inmunohistoquímica , Isocitrato Deshidrogenasa/metabolismo , Masculino , Persona de Mediana Edad , Estudios Retrospectivos , Telomerasa/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Proteína Nuclear Ligada al Cromosoma X/metabolismo , Adulto JovenRESUMEN
Radiotherapy is one of the leading treatments for clinical cancer therapy. External beam radiotherapy has been proposed as an adjuvant treatment for patients bearing differentiated thyroid cancer refractory to conventional therapy. Our purpose was to study the combined effect of HDAC inhibitors (HDACi) and ionizing irradiation in thyroid cancer cell lines (Nthy-ori 3-1, WRO, TPC-1 and 8505c). HDACi radiosensitized thyroid cancer cells as evidenced by the reduction of survival fraction, whereas they had no effect in the normal cells. HDACi enhanced radiation-induced cell death in WRO cells. Gamma-H2AX foci number increased and persisted long after ionizing exposure in the HDACi-treated cells (WRO and TPC-1). Moreover, the expression of the repair-related gene Ku80 was differentially modulated only in the cancer cells, by the compounds at the protein and/or mRNA levels. We present in vitro evidence that HDACi can enhance the radiosensitivity of human thyroid cancer cells.
Asunto(s)
Ácido Butírico/farmacología , Inhibidores de Histona Desacetilasas/farmacología , Tolerancia a Radiación/efectos de los fármacos , Neoplasias de la Tiroides/patología , Ácido Valproico/farmacología , Puntos de Control del Ciclo Celular/efectos de los fármacos , Puntos de Control del Ciclo Celular/efectos de la radiación , Muerte Celular/efectos de los fármacos , Muerte Celular/efectos de la radiación , Línea Celular Tumoral , Daño del ADN , Enzimas Reparadoras del ADN/genética , Enzimas Reparadoras del ADN/metabolismo , Rayos gamma , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de la radiación , Histonas/metabolismo , Humanos , Tolerancia a Radiación/efectos de la radiación , Neoplasias de la Tiroides/genéticaRESUMEN
BACKGROUND/AIM: The aim of the present study was to investigate the radio-sensitizing efficacy of curcumin, a traditional Chinese medicine (TCM) on colon cancer cells in vitro and in vivo. MATERIALS AND METHODS: Human colon cancer HT-29 cells were treated with curcumin (2.5 µM), irradiation (10 Gy) and the combination of irradiation and curcumin. Cell proliferation was assessed using the MTT assay. Apoptotic cells were detected by Annexin V-PE/7-AAD analysis. PCR was performed to determine differential-expression profiling of 95 DNA-repair genes in irradiated cells and cells treated with both irradiation and curcumin. Differentially-expressed genes were confirmed by Western blotting. In vivo radio-sensitizing efficacy of curcumin was assessed in a xenograft mouse model of HT-29 colon cancer. Curcumin was administrated daily by intraperitoneal injection at 20 mg/kg/dose. Mice received irradiation (10 Gy) twice weekly. Apoptosis of the cancer cells following treatment was determined by TUNEL staining. RESULTS: Irradiation induced proliferation inhibition and apoptosis of HT-29 cells in vitro. Concurrent curcumin treatment sensitized the HT-29 tumor to irradiation (p<0.01). DNA repair-related genes CCNH and XRCC5 were upregulated and LIG4 and PNKP downregulated by the combination of curcumin and irradiation compared with irradiation alone (p<0.05). Combined treatment of curcumin and irradiation resulted in a significantly greater tumor-growth inhibition and apoptosis compared to irradiation treatment alone (p<0.01). CONCLUSION: Curcumin sensitizes human colon cancer in vitro and in vivo to radiation. Downregulation of LIG4 and PNKP and upregulation of XRCC5 and CCNH DNA-repair-related genes were involved in the radio-sensitizing efficacy of curcumin in colon cancer.
Asunto(s)
Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/radioterapia , Curcumina/farmacología , Curcumina/uso terapéutico , Fármacos Sensibilizantes a Radiaciones/farmacología , Fármacos Sensibilizantes a Radiaciones/uso terapéutico , Animales , Apoptosis/efectos de los fármacos , Apoptosis/efectos de la radiación , Proliferación Celular/efectos de los fármacos , Proliferación Celular/efectos de la radiación , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Ciclina H/genética , Ciclina H/metabolismo , ADN Ligasa (ATP)/genética , ADN Ligasa (ATP)/metabolismo , Reparación del ADN/genética , Enzimas Reparadoras del ADN/genética , Enzimas Reparadoras del ADN/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de la radiación , Células HT29 , Humanos , Autoantígeno Ku/genética , Autoantígeno Ku/metabolismo , Medicina Tradicional China , Ratones Endogámicos BALB C , Ratones Desnudos , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Carga Tumoral/efectos de los fármacosRESUMEN
A strategy to suppress the expression of the DNA repair enzyme O6methylguanineDNA methyltransferase (MGMT) by inhibition of Wnt/ßcatenin signaling may be useful as a novel treatment for pituitary adenoma. Previous studies have reported that Tanshinone IIA (TSA), a major quinone compound isolated from Salvia miltiorrhiza, had antitumor effects. However, whether TSA has antitumor effects against pituitary adenoma and whether the mechanisms are associated with the Wnt/ßcatenin/MGMT pathway remains to be clarified. In the present study, TSA treatment caused apoptosis in AtT20 cells in a concentrationdependent manner, as demonstrated by cell viability reduction, phophatidylserine externalization detected by Annexin V staining and mitochondrial membrane potential disruption detected by JC1 staining, which were associated with activation of caspase3 and DNA fragmentation detected by TUNEL in AtT20 cells. Tcell factor (TCF)lymphoidenhancing factor (LEF) reporter activity was determined by dual luciferase reporter assay and the interaction between ßcatenin and TCF4 were detected using a coimmunoprecipitation kit. The results indicated TSA treatment increased ßcatenin phosphorylation, inhibited ßcatenin nuclear translocation, reduced ßcatenin/TCF4 complex formation and TCFLEF luciferase reporter activity, and subsequently reduced the expression of cyclin D1 and MGMT. Notably, overexpression of MGMT in ßcatenin knock down AtT20 cells abrogated the TSAmediated effects in AtT20 cells. In conclusion, TSA induced apoptosis via inhibition of Wnt/ßcatenindependent MGMT expression, which may provide novel insights into the understanding of the mechanism of the antitumor effects of Salvia miltiorrhiza.
Asunto(s)
Abietanos/farmacología , Antineoplásicos Fitogénicos/farmacología , Corticotrofos/efectos de los fármacos , Metilasas de Modificación del ADN/genética , Enzimas Reparadoras del ADN/genética , Regulación Neoplásica de la Expresión Génica , Salvia miltiorrhiza/química , Proteínas Supresoras de Tumor/genética , beta Catenina/genética , Abietanos/aislamiento & purificación , Animales , Antineoplásicos Fitogénicos/aislamiento & purificación , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Corticotrofos/metabolismo , Corticotrofos/patología , Fragmentación del ADN , Metilasas de Modificación del ADN/antagonistas & inhibidores , Metilasas de Modificación del ADN/metabolismo , Enzimas Reparadoras del ADN/antagonistas & inhibidores , Enzimas Reparadoras del ADN/metabolismo , Relación Dosis-Respuesta a Droga , Humanos , Factor de Unión 1 al Potenciador Linfoide/genética , Factor de Unión 1 al Potenciador Linfoide/metabolismo , Ratones , Extractos Vegetales/química , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Factor de Transcripción 4/genética , Factor de Transcripción 4/metabolismo , Proteínas Supresoras de Tumor/antagonistas & inhibidores , Proteínas Supresoras de Tumor/metabolismo , Vía de Señalización Wnt/efectos de los fármacos , beta Catenina/antagonistas & inhibidores , beta Catenina/metabolismoRESUMEN
Unmethylated O6-methylguanine-DNA-methyltransferase (MGMT) promoter leads to Temozolomide (TMZ) resistance in most of the glioblastoma multiforme (GBM) patients. We previously investigated the synergistic effect of Olea europaea leaf extract (OLE) on TMZ cytotoxicity through modulating microRNA expression. To date, knowledge about the effect of OLE on MGMT methylation is insufficient. The aim of the current study was to evaluate the potential modulating effect of OLE on the TMZ response of GBM tumors through MGMT methylation. Exposure to 1 mg/mL OLE caused a significant induction of CpG island methylation in the MGMT gene using Methyl quantitative PCR assay (P < 0.001). In WST-1 analysis, the use of 350 µM TMZ plus 1 mg/mL OLE significantly increased the TMZ response of MGMT unmethylated cells (P = 0.003). Using the comet assay, the impact of 1 mg/mL OLE plus 350 µM TMZ on the formation of DNA strand breaks was significantly higher than that of 450 µM TMZ alone (P < 0.001) and Western blot analysis revealed that, when cells are treated with 1-mg/mL OLE, the total p53 protein levels tended to decrease. The results presented in this study uniquely demonstrated that OLE synergistically increased the TMZ response of GBM tumors by regulating MGMT gene methylation and p53 expression. However, further studies to validate our findings are required.
Asunto(s)
Metilación de ADN , Metilasas de Modificación del ADN/metabolismo , Enzimas Reparadoras del ADN/metabolismo , Dacarbazina/análogos & derivados , Glioblastoma/tratamiento farmacológico , Extractos Vegetales/farmacología , Proteína p53 Supresora de Tumor/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Anciano , Línea Celular Tumoral , Ensayo Cometa , Islas de CpG , Daño del ADN , Metilasas de Modificación del ADN/genética , Enzimas Reparadoras del ADN/genética , Dacarbazina/uso terapéutico , Relación Dosis-Respuesta a Droga , Resistencia a Antineoplásicos/genética , Femenino , Humanos , Concentración 50 Inhibidora , Masculino , Persona de Mediana Edad , Olea/química , Hojas de la Planta/química , Regiones Promotoras Genéticas , Temozolomida , Proteína p53 Supresora de Tumor/genética , Proteínas Supresoras de Tumor/genéticaRESUMEN
The majority of colon tumors are driven by aberrant Wnt signaling in intestinal stem cells, which mediates an efficient route toward initiating intestinal cancer. Natural lipophilic polyphenols and long-chain polyunsaturated fatty acids (PUFAs) generally suppress Wnt- and NF-κB- (nuclear factor-κ light-chain enhancer of activated B-cell) related pathways. However, the effects of these extrinsic agents on colonic leucine-rich repeat-containing G-protein-coupled receptor 5-positive (Lgr5+) stem cells, the cells of origin of colon cancer, have not been documented to date. Therefore, we examined the effect of n-3 PUFA and polyphenol (curcumin) combination on Lgr5+ stem cells during tumor initiation and progression in the colon compared with an n-6 PUFA-enriched control diet. Lgr5-EGFP-IRES-creERT2 knock-in mice were fed diets containing n-6 PUFA (control), n-3 PUFA, n-6 PUFA+curcumin or n-3 PUFA+curcumin for 3 weeks, followed by 6 azoxymethane (AOM) injections, and terminated 17 weeks after the last injection. To further elucidate the effects of the dietary bioactives at the tumor initiation stage, Lgr5+ stem cells were also assessed at 12 and 24 h post AOM injection. Only n-3 PUFA+curcumin feeding reduced nuclear ß-catenin in aberrant crypt foci (by threefold) compared with control at the progression time point. n-3 PUFA+curcumin synergistically increased targeted apoptosis in DNA-damaged Lgr5+ stem cells by 4.5-fold compared with control at 12 h and maximally reduced damaged Lgr5+ stem cells at 24 h, down to the level observed in saline-treated mice. Finally, RNAseq analysis indicated that p53 signaling in Lgr5+ stem cells from mice exposed to AOM was uniquely upregulated only following n-3 PUFA+curcumin cotreatment. These novel findings demonstrate that Lgr5+ stem cells are uniquely responsive to external dietary cues following the induction of DNA damage, providing a therapeutic strategy for eliminating damaged Lgr5+ stem cells to reduce colon cancer initiation.
Asunto(s)
Ciclo Celular , Neoplasias del Colon/patología , Dieta , Receptores Acoplados a Proteínas G/metabolismo , Células Madre/citología , Focos de Criptas Aberrantes/metabolismo , Animales , Apoptosis/efectos de los fármacos , Azoximetano , Carcinogénesis/efectos de los fármacos , Carcinogénesis/metabolismo , Carcinogénesis/patología , Carcinógenos , Ciclo Celular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Quimioprevención , Colon/efectos de los fármacos , Colon/metabolismo , Colon/patología , Neoplasias del Colon/metabolismo , Curcumina/farmacología , Roturas del ADN de Doble Cadena/efectos de los fármacos , Metilasas de Modificación del ADN/metabolismo , Enzimas Reparadoras del ADN/metabolismo , Ácidos Grasos Omega-3 , Aceites de Pescado/farmacología , Proteínas Fluorescentes Verdes/metabolismo , Ratones , Regeneración/efectos de los fármacos , Factores de Riesgo , Transducción de Señal/efectos de los fármacos , Células Madre/efectos de los fármacos , Células Madre/metabolismo , Fracciones Subcelulares/efectos de los fármacos , Fracciones Subcelulares/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Proteínas Supresoras de Tumor/metabolismo , beta Catenina/metabolismoRESUMEN
Lasers emit light beams with specific characteristics, in which wavelength, frequency, power, fluence, and emission mode properties determine the photophysical, photochemical, and photobiological responses. Low-intensity lasers could induce free radical generation in biological tissues and cause alterations in macromolecules, such as DNA. Thus, the aim of this work was to evaluate excision repair cross-complementing group 1 (ERCC1) and excision repair cross-complementing group 2 (ERCC2) messenger RNA (mRNA) expression in biological tissues exposed to low-intensity lasers. Wistar rat (n = 28, 4 for each group) skin and muscle were exposed to low-intensity red (660 nm) and near-infrared (880 nm) lasers at different fluences (25, 50, and 100 J/cm(2)), and samples of these tissues were withdrawn for RNA extraction, cDNA synthesis, and gene expression evaluation by quantitative polymerase chain reaction. Laser exposure was in continuous wave and power of 100 mW. Data show that ERCC1 and ERCC2 mRNA expressions decrease in skin (p < 0.001) exposed to near-infrared laser, but increase in muscle tissue (p < 0.001). ERCC1 mRNA expression does not alter (p > 0.05), but ERCC2 mRNA expression decreases in skin (p < 0.001) and increases in muscle tissue (p < 0.001) exposed to red laser. Our results show that ERCC1 and ERCC2 mRNA expression is differently altered in skin and muscle tissue exposed to low-intensity lasers depending on wavelengths and fluences used in therapeutic protocols.
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Expresión Génica/efectos de la radiación , Terapia por Luz de Baja Intensidad , ARN Mensajero/metabolismo , Animales , Reparación del ADN , Enzimas Reparadoras del ADN/genética , Enzimas Reparadoras del ADN/metabolismo , Rayos Infrarrojos , Rayos Láser , Masculino , Músculo Esquelético/enzimología , Músculo Esquelético/efectos de la radiación , ARN Mensajero/genética , Ratas , Ratas Wistar , Piel/enzimología , Piel/efectos de la radiación , Proteína de la Xerodermia Pigmentosa del Grupo D/genética , Proteína de la Xerodermia Pigmentosa del Grupo D/metabolismoRESUMEN
DNA 3'-phosphatase takes an important role in DNA damage repair, replication and recombination. Here, we present a novel label-free fluorescent assay for T4 polynucleotide kinase/phosphatase (T4 PNKP) activity and its inhibitor screening by using poly(thymine)-templated fluorescent copper nanoparticles (CuNPs) as a fluorescent indicator. In this assay, we designed a simple T-rich hairpin primer with a 3'-phosphoryl end, which can serve as both the substrate for T4 PNKP and DNA template for the formation of fluorescent CuNPs. Once the phosphorylated hairpin primer was hydrolyzed by T4 PNKP, the resulting hairpin primer with a 3'-hydroxyl end was immediately elongated to form a long double-strand product by DNA polymerase, which prohibited the formation of fluorescent CuNPs due to the lack of poly T single-stranded DNA template. This new strategy provides a sensitive, selective, and cost-effective manner for T4 PNKP analysis, which holds a great potential in the study of DNA damage repair mechanisms.
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Cobre/química , Enzimas Reparadoras del ADN/antagonistas & inhibidores , Enzimas Reparadoras del ADN/metabolismo , Evaluación Preclínica de Medicamentos/métodos , Pruebas de Enzimas/métodos , Fosfotransferasas (Aceptor de Grupo Alcohol)/antagonistas & inhibidores , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Polímeros/química , Timina/química , Biocatálisis , Línea Celular Tumoral , Inhibidores Enzimáticos/farmacología , Humanos , Nanopartículas del Metal/química , FosforilaciónRESUMEN
OBJECTIVE: The majority of prostate cancers require androgen hormones for growth, and androgen ablation is an important part of the systemic treatment of advanced prostate cancer. Nevertheless, most of these cancers eventually relapse as they become less sensitive to androgen ablation and anti-androgen treatment. Elucidating the molecular events that are responsible for the conversion of androgen-sensitive cancers to androgen-refractory tumors may reveal new therapeutic opportunities. METHODS: In the present study, we investigated nine androgen-sensitive and nine androgen-refractory prostate cancer samples to evaluate the expression levels of 10 selected proteins that have been implicated in oncogenesis and cancer progression. RESULTS: Our immunohistochemical data show that three of the investigated proteins (i.e., minichromosome maintenance-2, methylguanine-DNA methyltransferase, and androgen receptor) are expressed at significantly different levels in the androgen-refractory cancer samples than in the androgen-sensitive tumors, whereas the expression levels of the seven other studied proteins (i.e., ß-catenin, p27, p21, p16, Ki67, hypoxia-inducible factor 1 alpha, and geminin) are not significantly different regarding the two groups. CONCLUSIONS: Our data suggest that the increased expression of minichromosome maintenance-2 and decreased expression of methylguanine-DNA methyltransferase related to androgen receptor are indicative of the androgen-refractory stage in prostate cancer. Further studies are required to determine whether these expression changes play a causative role in the transition of androgen-sensitive to androgen-refractory prostate cancer.
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Adenocarcinoma , Antagonistas de Andrógenos , Próstata , Neoplasias de la Próstata Resistentes a la Castración , Neoplasias de la Próstata , Receptores Androgénicos/metabolismo , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Adenocarcinoma/terapia , Anciano , Antagonistas de Andrógenos/metabolismo , Antagonistas de Andrógenos/farmacología , Metilasas de Modificación del ADN/metabolismo , Enzimas Reparadoras del ADN/metabolismo , Progresión de la Enfermedad , Geminina/metabolismo , Humanos , Inmunohistoquímica , Masculino , Componente 2 del Complejo de Mantenimiento de Minicromosoma/metabolismo , Estadificación de Neoplasias , Próstata/metabolismo , Próstata/patología , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Neoplasias de la Próstata/terapia , Neoplasias de la Próstata Resistentes a la Castración/metabolismo , Neoplasias de la Próstata Resistentes a la Castración/patología , Neoplasias de la Próstata Resistentes a la Castración/terapia , Transducción de Señal , Resección Transuretral de la Próstata/métodos , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Alkylating agents are present in food and tobacco smoke, but are also used in cancer chemotherapy, inducing the DNA lesion O (6)-methylguanine. This critical adduct is repaired by O (6)-methylguanine-DNA methyltransferase (MGMT), resulting in MGMT inactivation and degradation. In the present study, we analyzed the effects of the natural disulfide compound lipoic acid (LA) on MGMT in vitro and in colorectal cancer cells. We show that LA, but not its reduced form dihydrolipoic acid, potently inhibits the activity of recombinant MGMT by interfering with its catalytic Cys-145 residue, which was partially reversible by N-acetyl cysteine. Incubation of HCT116 colorectal cancer cells with LA altered their glutathione pool and caused a decline in MGMT activity. This was mirrored by LA-induced depletion of MGMT protein, which was not attributable to changes in MGMT messenger RNA levels. Loss of MGMT protein coincided with LA-induced autophagy, a process resulting in lysosomal degradation of proteins, including presumably MGMT. LA-stimulated autophagy in a p53-independent manner as revealed by the response of isogenic HCT116 cell lines. Knockdown of the crucial autophagy component beclin-1 and chemical inhibitors blocked LA-induced autophagy, but did not abrogate LA-triggered MGMT degradation. Concomitant with MGMT depletion, LA pretreatment resulted in enhanced O (6)-methylguanine levels in DNA. It also increased the cytotoxicity of the alkylating anticancer drug temozolomide in temozolomide-resistant colorectal cancer cells. Taken together, our study showed that the natural compound LA inhibits MGMT and induces autophagy. Furthermore, LA enhanced the cytotoxic effects of temozolomide, which makes it a candidate for a supplement in cancer therapy.
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Autofagia/efectos de los fármacos , Neoplasias Colorrectales/tratamiento farmacológico , Metilasas de Modificación del ADN/metabolismo , Enzimas Reparadoras del ADN/metabolismo , Ácido Tióctico/farmacología , Proteínas Supresoras de Tumor/metabolismo , Animales , Antineoplásicos Alquilantes/farmacología , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Cisteína/metabolismo , Metilasas de Modificación del ADN/antagonistas & inhibidores , Metilasas de Modificación del ADN/genética , Reparación del ADN/efectos de los fármacos , Enzimas Reparadoras del ADN/antagonistas & inhibidores , Enzimas Reparadoras del ADN/genética , Dacarbazina/análogos & derivados , Dacarbazina/farmacología , Resistencia a Antineoplásicos/efectos de los fármacos , Femenino , Glutatión/metabolismo , Guanina/análogos & derivados , Guanina/metabolismo , Células HCT116/efectos de los fármacos , Humanos , Masculino , Ratones Endogámicos BALB C , Terapia Molecular Dirigida , Temozolomida , Ácido Tióctico/análogos & derivados , Proteínas Supresoras de Tumor/antagonistas & inhibidores , Proteínas Supresoras de Tumor/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
UNLABELLED: In neuroblastoma, MYCN genomic amplification and segmental chromosomal alterations including 1p or 11q loss of heterozygocity and/or 17q gain are associated with progression and poor clinical outcome. Segmental alterations are the strongest predictor of relapse and result from unbalanced translocations attributable to erroneous repair of chromosomal breaks. Although sequence analysis of affected genomic regions suggests that these errors arise by nonhomologous end-joining (NHEJ) of DNA double-strand breaks (DSB), abnormalities in NHEJ have not been implicated in neuroblastoma pathogenesis. On this basis, the hypothesis that an error-prone mechanism of NHEJ is critical for neuroblastoma cell survival was tested. Plasmid-based DSB repair assays demonstrated efficient NHEJ activity in human neuroblastoma cells with repair products that were error-prone relative to nontransformed cells. Neuroblastoma cells derived from tumorigenic neuroblastic phenotypes had differential DNA repair protein expression patterns compared with nontumorigenic cells. Tumorigenic neuroblastoma cells were deficient in DNA ligase IV (Lig4) and Artemis (DCLRE1C), mediators of canonical NHEJ. Conversely, enzymes required for an error-prone alternative NHEJ pathway (alt-NHEJ), DNA Ligase IIIα (Lig3), DNA Ligase I (Lig1), and PARP1 protein were upregulated. Inhibition of Lig3 and Lig1 led to DSB accumulation and cell death, linking alt-NHEJ to cell survival in neuroblastoma. Neuroblastoma cells demonstrated sensitivity to PARP1 inhibition (PARPi) that paralleled PARP1 expression. In a dataset of human neuroblastoma patient tumors, overexpression of genes encoding alt-NHEJ proteins associated with poor survival. IMPLICATIONS: These findings provide an insight into DNA repair fidelity in neuroblastoma and identify components of the alt-NHEJ pathway as promising therapeutic targets.
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Roturas del ADN de Doble Cadena , Reparación del ADN por Unión de Extremidades , Neuroblastoma/genética , Línea Celular Tumoral , Supervivencia Celular , Roturas del ADN de Doble Cadena/efectos de los fármacos , Reparación del ADN por Unión de Extremidades/efectos de los fármacos , Enzimas Reparadoras del ADN/genética , Enzimas Reparadoras del ADN/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Inhibidores Enzimáticos/farmacología , Regulación Neoplásica de la Expresión Génica , Humanos , Neuroblastoma/tratamiento farmacológico , Neuroblastoma/patología , Poli(ADP-Ribosa) Polimerasa-1 , Poli(ADP-Ribosa) Polimerasas/metabolismo , PronósticoRESUMEN
The circadian clock is a global regulatory system that interfaces with most other regulatory systems and pathways in mammalian organisms. Investigations of the circadian clock-DNA damage response connections have revealed that nucleotide excision repair, DNA damage checkpoints, and apoptosis are appreciably influenced by the clock. Although several epidemiological studies in humans and a limited number of genetic studies in mouse model systems have indicated that clock disruption may predispose mammals to cancer, well-controlled genetic studies in mice have not supported the commonly held view that circadian clock disruption is a cancer risk factor. In fact, in the appropriate genetic background, clock disruption may instead aid in cancer regression by promoting intrinsic and extrinsic apoptosis. Finally, the clock may affect the efficacy of cancer treatment (chronochemotherapy) by modulating the pharmacokinetics and pharmacodynamics of chemotherapeutic drugs as well as the activity of the DNA repair enzymes that repair the DNA damage caused by anticancer drugs.
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Antineoplásicos/administración & dosificación , Relojes Circadianos , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Animales , Antineoplásicos/farmacocinética , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Daño del ADN/efectos de los fármacos , Reparación del ADN/efectos de los fármacos , Enzimas Reparadoras del ADN/metabolismo , Cronoterapia de Medicamentos , Humanos , Neoplasias/epidemiología , Neoplasias/metabolismo , Factores de RiesgoRESUMEN
To better understand the molecular basis of the enhanced cell killing effected by the combined modality of paclitaxel and ²¹²Pb-trastuzumab (Pac/²¹²Pb-trastuzumab), gene expression in LS-174T i.p. xenografts was investigated 24 h after treatment. Employing a real time quantitative PCR array (qRT-PCR array), 84 DNA damage response genes were quantified. Differentially expressed genes following therapy with Pac/²¹²Pb-trastuzumab included those involved in apoptosis (BRCA1, CIDEA, GADD45α, GADD45γ, GML, IP6K3, PCBP4, PPP1R15A, RAD21, and p73), cell cycle (BRCA1, CHK1, CHK2, GADD45α, GML, GTSE1, NBN, PCBP4, PPP1R15A, RAD9A, and SESN1), and damaged DNA repair (ATRX, BTG2, EXO1, FEN1, IGHMBP2, OGG1, MSH2, MUTYH, NBN, PRKDC, RAD21, and p73). This report demonstrates that the increased stressful growth arrest conditions induced by the Pac/²¹²Pb-trastuzumab treatment suppresses cell proliferation through the regulation of genes which are involved in apoptosis and damaged DNA repair including single and double strand DNA breaks. Furthermore, the study demonstrates that ²¹²Pb-trastuzumab potentiation of cell killing efficacy results from the perturbation of genes related to the mitotic spindle checkpoint and BASC (BRCA1-associated genome surveillance complex), suggesting cross-talk between DNA damage repair and the spindle damage response.