RESUMO
We previously reported the association of HSPA1A and HSPB1 with high-grade astrocytomas, suggesting that these proteins might be involved in disease outcome and response to treatment. With the aim to better understand the resistance/susceptibility processes associated to temozolomide (TMZ) treatment, the current study was performed in three human malignant glioma cell lines by focusing on several levels: (a) apoptotic index and senescence, (b) DNA damage, and (c) interaction of HSPB1 with players of the DNA damage response. Three human glioma cell lines, Gli36, U87, and DBTRG, were treated with TMZ evaluating cell viability and survival, apoptosis, senescence, and comets (comet assay). The expression of HSPA (HSPA1A and HSPA8), HSPB1, O6-methylguanine-DNA methyltransferase (MGMT), MLH1, and MSH2 was determined by immunocytochemistry, immunofluorescence, and Western blot. Immunoprecipitation was used to analyze protein interaction. The cell lines exhibited differences in viability, apoptosis, and senescence after TMZ administration. We then focused on Gli36 cells (relatively unstudied) which showed very low recovery capacity following TMZ treatment, and this was related to high DNA damage levels; however, the cells maintained their viability. In these cells, MGMT, MSH2, HSPA, and HSPB1 levels increased significantly after TMZ administration. In addition, MSH2 and HSPB1 proteins appeared co-localized by confocal microscopy. This co-localization increased after TMZ treatment, and in immunoprecipitation analysis, MSH2 and HSPB1 appeared interacting. In contrast, HSPB1 did not interact with MGMT. We show in glioma cells the biological effects of TMZ and how this drug affects the expression levels of heat shock proteins (HSPs), MGMT, MSH2, and MLH1. In Gli36 cells, the results suggest that interactions between HSPB1 and MSH2, including co-nuclear localization, may be important in determining cell sensitivity to TMZ.
Assuntos
Apoptose/efeitos dos fármacos , Enzimas Reparadoras do DNA/metabolismo , Dacarbazina/análogos & derivados , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteínas de Choque Térmico/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Antineoplásicos Alquilantes/farmacologia , Linhagem Celular Tumoral , Dano ao DNA/efeitos dos fármacos , Dacarbazina/farmacologia , Glioma/patologia , Proteínas de Choque Térmico HSC70/metabolismo , Proteínas de Choque Térmico HSP27/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Humanos , Imuno-Histoquímica , Chaperonas Moleculares , Proteína 1 Homóloga a MutL , Proteína 2 Homóloga a MutS/metabolismo , Proteínas Nucleares/metabolismo , O(6)-Metilguanina-DNA Metiltransferase/metabolismo , TemozolomidaRESUMO
Mismatch repair (MMR) deficiency and higher expression levels of heat shock proteins (Hsps) have been implicated with drug resistance to topoisomerase II poisons (doxorubicin) and to platinum compounds (cisplatin). This study was designed to determine individual influences of doxorubicin and cisplatin treatment on the expression of Hsp27, Hsp70, hMLH1 and hMSH2 proteins and in the DNA damage status in peripheral blood lymphocytes (PBLs). In addition, we studied whether these proteins and the DNA damage correlated with the survival of cancer patients. PBLs from 10 healthy donors and 25 cancer patients (before and after three cycles of chemotherapy) were exposed to in vitro treatments: C (control), HS (heat shock at 42 degrees C), Do or Pt (doxorubicin or cisplatin alone), and HS+Do or HS+Pt (heat shock+doxorubicin or heat shock+cisplatin). PBLs were collected at time 0 (T0: immediately after drug treatment) and after 24h of repair (T24). Hsp27, Hsp70, hMLH1 and hMSH2 were studied by immunocytochemistry and the DNA damage by alkaline comet assay. Immunofluorescence studies and confocal microscopy revealed that hMLH1 and hMSH2 colocalized with Hsp27 and Hsp72 (inducible form of Hsp70). hMLH1 and hMSH2 were significantly induced by Pt and HS+Pt at T24 in cancer patients, but only modestly influenced by Do. Cancer patients presented higher basal expression of total and nuclear Hsp27 and Hsp70 than controls, and these proteins were also increased by HS, Do and HS+Do. The Hsp70 induction by Pt and HS+Pt was noted in cancer patients, especially nuclear Hsp70. In cancer patients, basal DNA damage was slightly higher than in healthy persons; and after Pt and HS+Pt treatments, DNA migration and number of apoptotic cells were higher than controls. Hsps accomplished a cytoprotective function in pre-chemotherapy PBLs (HS before Do or Pt), but not in post-chemotherapy samples. In Pt-treated patients the ratio N/C (nuclear/cytoplasmic) of Hsp27 was related to disease free survival and overall survival, and hMSH2 correlated with overall survival. The results point to the utility of these molecules and of the comet assay as possible predictive markers.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Resistencia a Medicamentos Antineoplásicos , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico/metabolismo , Proteína 2 Homóloga a MutS/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias/tratamento farmacológico , Proteínas Nucleares/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/análise , Adolescente , Adulto , Antineoplásicos/uso terapêutico , Biomarcadores/análise , Biomarcadores/metabolismo , Núcleo Celular/metabolismo , Criança , Pré-Escolar , Cisplatino/uso terapêutico , Citoplasma/metabolismo , DNA/análise , Dano ao DNA , Reparo de Erro de Pareamento de DNA , Doxorrubicina/uso terapêutico , Feminino , Proteínas de Choque Térmico HSP27 , Proteínas de Choque Térmico HSP70/análise , Proteínas de Choque Térmico/análise , Humanos , Lactente , Linfócitos/química , Linfócitos/metabolismo , Masculino , Chaperonas Moleculares , Proteína 1 Homóloga a MutL , Proteína 2 Homóloga a MutS/análise , Proteínas de Neoplasias/análise , Neoplasias/mortalidade , Proteínas Nucleares/análise , PrognósticoRESUMO
Drug resistance is considered the main impediment to successful cancer chemotherapy. The quest for a method useful to predict individual responses to chemotherapy prior to treatment is highly desired. This study was designed to determine the individual influences of doxorubicin and cisplatin on the degree of DNA damage, DNA repair and hMSH2 and the hMLH1 protein expression in peripheral blood lymphocytes (PBL) and their correlations with the clinical response. PBL were obtained from 25 cancer patients (pre- and post-chemotherapy) and from 10 healthy persons, cultured and exposed to doxorubicin or cisplatin. Cells were collected at T0 (immediately after drug treatment) and 24h after damage (T24). The alkaline comet assay was employed to assess the DNA damage and repair function, and immunocytochemistry to study hMLH1 and hMSH2 expression. Clinical response was evaluated after three cycles of chemotherapy. Pre-chemotherapy PBL from cancer patients showed significantly higher levels of basal DNA damage than healthy persons, with appreciable interindividual variations between them. The in vivo administration of antineoplasic drugs was accompanied by significant DNA damage, and an increased in the number of apoptotic cells. Cancer patients with complete response showed a high number of apoptotic cells. The DNA migration increased at T0 and at T24 in cisplatin-treated patients, reflecting a decreased rate of cisplatin adducts repair than that observed in healthy individuals. The ability to repair DNA lesions in doxorubicin-damaged cells was very similar between healthy individuals and cancer patients. Cisplatin-treated patients that died by the disease showed lower DNA migration than the mean value. The expression of hMLH1 and hMSH2 was practically identical between healthy individuals and cancer patients. Nevertheless, chemotherapy induced a depletion mostly of hMLH1. In 83% of cisplatin-treated patients with CR the hMLH1 and hMSH2 expression at T24 was higher than the mean. In this pilot study the alkaline comet assay offered information about the amount of DNA damage and the DNA repair status in PBL from individual patients and this seems useful in predicting the response to chemotherapy.
Assuntos
Antineoplásicos/uso terapêutico , Dano ao DNA/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Linfócitos/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Proteínas Adaptadoras de Transdução de Sinal , Adolescente , Adulto , Antibióticos Antineoplásicos/farmacologia , Proteínas de Transporte/metabolismo , Criança , Pré-Escolar , Cisplatino/farmacologia , Ensaio Cometa , Doxorrubicina/farmacologia , Feminino , Humanos , Lactente , Linfócitos/metabolismo , Masculino , Pessoa de Meia-Idade , Proteína 1 Homóloga a MutL , Proteína 2 Homóloga a MutS/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Projetos PilotoRESUMO
This study was designed to assess whether the chemotherapeutic drug paclitaxel can induce DNA damage in peripheral blood lymphocytes of human healthy donors, and to evaluate if such damage could be repaired. Venous blood was collected by routine venipuncture, the lymphocytes were isolated and cultured and then treated with 100nM, 500nM, 10microM, and 30microM of taxol for 4h. The alkaline comet assay technique was used to quantify the level of DNA damage and the DNA repair in lymphocytes. A significant increase in DNA damage was achieved when the cells were incubated with paclitaxel concentrations of 10microM or above. To test the DNA repair capability, the lymphocytes were allowed to recover for 2, 4, 6, and 24h. The DNA damage was almost completely repaired after 24h of incubation demonstrating a time-dependent repair capability. In conclusion, we demonstrate that paclitaxel induces DNA damage in peripheral blood lymphocytes and that this damage can be repaired.