Zinc chelation: a metallothionein 2A's mechanism of action involved in osteosarcoma cell death and chemotherapy resistance

Abstract

Osteosarcoma is the most common primary tumor of bone occurring in children and adolescents. The histological response to chemotherapy represents a key clinical factor related to survival. We previously showed that statins exhibit antitumor effects in vitro, inducing apoptotic cell death, reducing cell migration and invasion capacities and strengthening cytotoxic effects in combination with standard drugs. Comparative transcriptomic analysis between control and statin-treated cells revealed strong expression of several genes, including metallothionein (MT) 2A. MT2A overexpression by lentiviral transduction reduced bioavailable zinc levels, an effect associated with reduced osteosarcoma cell viability and enhanced cell differentiation. In contrast, MT2A silencing did not modify cell viability but strongly inhibited expression of osteoblastic markers and differentiation process. MT2A overexpression induced chemoresistance to cytotoxic drugs through direct chelation of platinum-containing drugs and indirect action on p53 zinc-dependent activity. In contrast, abrogation of MT2A enhanced cytotoxic action of chemotherapeutic drugs on osteosarcoma cells. Finally, clinical samples derived from chemonaive biopsies revealed that tumor cells expressing low MT2A levels correspond to good prognostic (good responder patients with longer survival rate), whereas high MT2A levels were associated with adverse prognosis (poor responder patients). Taken together, these data show that MT2A contributes to chemotherapy resistance in osteosarcoma, an effect partially mediated by zinc chelation. The data also suggest that MT2A may be a potential new prognostic marker for osteosarcoma sensitivity to chemotherapy.

Figure 1

Atorvastatin increases MT2A expression in osteosarcoma cells. SaOS2 and U2OS cells were incubated in the presence or absence of atorvastatin (10 μM) for 24 h. (a) MT2A mRNA levels were evaluated by RT-qPCR. (b) MT2A and housekeeping gene β-actin protein levels were assessed by western blot analysis. (c) Free intracellular zinc content was evaluated by fluorimetry. Results are expressed as mean±S.D. *P<0.05 versus untreated cells

Figure 2

MT2A overexpression reduced osteosarcoma cell viability. SaOS2 and U2OS cell lines were transduced with an integrative lentiviral vector encoding the complete coding sequence of MT2A (LV-MT2A). (a) MT2A mRNA levels were evaluated by RT-qPCR. (b) MT2A protein levels were evaluated by western blot analysis. (c) Cell viability was evaluated using the MTT test. (d) Relative percentage of viable, necrotic and apoptotic cells were evaluated by acridine orange/ethidium bromide staining. (e) Cell replication was evaluated using a BrdU incorporation assay. (f) Apoptotic cell proportion (sub-G0/G1) was evaluated by flow cytometry. (g) Bax and Bcl-2 protein levels were evaluated by western blot analysis. Results are expressed as mean±S.D. and arbitrary units relative to parental cells. *P<0.05 versus untreated cells

Figure 3

MT2A overexpression modulates osteosarcoma cell viability through zinc chelation. SaOS2 and U2OS parental cells were incubated with increasing doses of Zn chelator TPEN. (a) Cell viability was evaluated using the MTT test. (b) SaOS2 and U2OS parental and MT2A-overexpressing cells were incubated with increasing doses of ZnCl₂. Free intracellular zinc content was evaluated by fluorimetry. (c) SaOS2 and U2OS MT2A-overexpressing cells were incubated with increasing doses of ZnCl₂. Parental cells were maintained in normal medium. Cell replication was evaluated using a BrdU incorporation assay. (d) SaOS2 and U2OS parental cells were incubated in chelex-treated medium supplemented with increasing doses of ZnCl₂. Cell replication was evaluated using a BrdU incorporation assay. Results are expressed as mean±S.D. *P<0.05 versus untreated cells

Figure 4

MT2A overexpression modulates cell response to cytotoxic agents. (a) SaOS2 parental and MT2A-overexpressing cells were incubated with or without cisplatin or transplatin for 48 h. Cell viability was evaluated using the MTT test. (b) U2OS parental and MT2A-overexpressing cells were incubated with or without cisplatin or transplatin for 48 h. Cell viability was evaluated using the MTT test. (c) SaOS2 parental and MT2A-overexpressing cells were incubated with or without doxorubicin or vincristine. Cell viability was evaluated using the MTT test. (d) U2OS parental and MT2A-overexpressing cells were incubated with or without doxorubicin or vincristine. Cell viability was evaluated using the MTT test. Results are expressed as mean±S.D. and as arbitrary units relative to corresponding untreated cells. *P<0.05 versus untreated cells; #P<0.05 versus parental cells

Figure 5

MT2A modulates p53 conformation-dependent activity. (a) Relative content of active form of P53 was evaluated by immunoprecipitation using a conformational-dependent antibody. (b) P21 protein levels were evaluated in U2OS parental and MT2A-overexpressing cells by western blot analysis. (c,d) P73 and P63 protein levels were evaluated in U2OS parental and MT2A-overexpressing cells by western blot analysis. (e) MT2A protein levels were evaluated in U2OS cells incubated in the presence or absence of doxorubicin (100 ng/ml). (f) Relative content of active form of P53 was evaluated by immunoprecipitation using a conformational-dependent antibody

Figure 6

MT2A silencing amplifies osteosarcoma cell response to cytotoxic agents. U2OS cell line was transduced with an integrative lentiviral vector encoding a specific shRNA sequence targeting MT2A (sh-MT2A). (a) MT2A mRNA levels were evaluated by RT-qPCR in U2OS parental and MT2A-silenced cells. (b) MT2A protein levels were evaluated by western blot analysis in U2OS parental and MT2A-silenced cells. (c) U2OS parental and MT2A-silenced cells were incubated with increasing doses of ZnCl₂. Free intracellular zinc content was evaluated by fluorimetry. (d) U2OS parental and MT2A-silenced cells were incubated with or without cisplatin, transplatin, doxorubicin or vincristine for 48 h. Cell viability was evaluated using the MTT test. (e) Relative amounts of doxorubicin combined to DNA were evaluated by flow cytometry into parental, silenced and overexpressing U2OS cells. (f) Relative amounts of Hoechst 33342 bound to DNA were evaluated by flow cytometry into parental, silenced and overexpressing U2OS cells. Results are expressed as mean±S.D. *P<0.05 versus untreated cells; #P<0.05 versus parental cells

Figure 7

Prognostic value of MT2A mRNA expression in osteosarcoma. (a) MT2A mRNA levels were evaluated by RT-qPCR, in biopsies from good and poor responders. Results were expressed as arbitrary unit (AU). Mean values of each group are indicated as black bars. *P<0.05 versus untreated cells. (b) Kaplan–Meier survival curves from patients with low MT2A mRNA level or patients with highest MT2A mRNA level