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Potent antitumor activity of oncolytic adenovirus expressing Beclin-1 via induction of autophagic cell death in leukemia.

Tong Y, You L, Liu H, Li L, Meng H, Qian Q, Qian W - Oncotarget (2013)

Bottom Line: Here, we investigated whether a strategy that combines the oncolytic effects of an adenoviral vector with simultaneous expression of Beclin-1, an autophagy gene, offers a therapeutic advantage for leukemia.We also showed that SG511-BECN strongly inhibited the growth of leukemic progenitors in vitro.Our results suggest that infection of leukemia cells with an oncolytic adenovirus overexpressing Beclin-1 can induce significant autophagic cell death and provide a new strategy for the elimination of leukemic cells via a unique mechanism of action distinct from apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Institute of Hematology, the First Affiliated Hospital, College of Medicine, Zhejiang University, P.R. China.

ABSTRACT
An attractive strategy among adenovirus-based oncolytic systems is to design adenoviral vectors to express pro-apoptotic genes, in which this gene-virotherapy approach significantly enhances tumor cell death by activating apoptotic pathways. However, the existence of cancer cells with apoptotic defects is one of the major obstacles in gene-virotherapy. Here, we investigated whether a strategy that combines the oncolytic effects of an adenoviral vector with simultaneous expression of Beclin-1, an autophagy gene, offers a therapeutic advantage for leukemia. A Beclin-1 cDNA was cloned in an oncolytic adenovirus with chimeric Ad5/11 fiber (SG511-BECN). SG511-BECN treatment induced significant autophagic cell death, and resulted in enhanced cell killing in a variety of leukemic cell lines and primary leukemic blasts. SG511-BECN effects were seen in chronic myeloid leukemia and acute myeloid leukemia with resistance to imatinib or chemotherapy, but exhibited much less cytotoxicity on normal cells. The SG511-BECN-induced autophagic cell death could be partially reversed by RNA interference knockdown of UVRAG, ATG5, and ATG7. We also showed that SG511-BECN strongly inhibited the growth of leukemic progenitors in vitro. In murine leukemia models, SG511-BECN prolonged the survival and decreased the xenograft tumor size by inducing autophagic cell death. Our results suggest that infection of leukemia cells with an oncolytic adenovirus overexpressing Beclin-1 can induce significant autophagic cell death and provide a new strategy for the elimination of leukemic cells via a unique mechanism of action distinct from apoptosis.

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SG511-BECN induces autophagy in primary leukemic cells to preferentially inhibit CFU-L formation(A) Freshly isolated leukemia cells from CML patients (n=15) were cultured in methylcellulose medium in the presence of SG511, or SG511-BECN at an MOI of 50. After 12 days, colonies were scored. *, P>0.05 vs. Control; # P<0.01 vs. SG511. (B) Leukemic cells from 12 patients with AML were treated with the indicated vectors at an MOI of 50 before analysis of effects on colony formation. *, P<0.001 vs. Control; # P<0.001 vs. SG511. (C) Bone marrow cells obtained from 4 healthy volunteers were treated with the indicated viruses, and colonies were counted after 12 days. For all panels, values represent the mean of experiments in triplicate. (D) The primary leukemic cells from a CML patient in blast crisis (case 1) were infected with the indicated viruses at an MOI of 50, cell lysate was immunoblotted with anti-Beclin-1, anti-LC3, and anti-â-actin antibodies at day 2 of the experiment. (E) The primary blasts were treated with SG511-BECN (50 MOI), or SG511-BECN plus chloroquine (25 ìM) for 48 h. Autophagic flux was determined using Western blotting.
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Figure 4: SG511-BECN induces autophagy in primary leukemic cells to preferentially inhibit CFU-L formation(A) Freshly isolated leukemia cells from CML patients (n=15) were cultured in methylcellulose medium in the presence of SG511, or SG511-BECN at an MOI of 50. After 12 days, colonies were scored. *, P>0.05 vs. Control; # P<0.01 vs. SG511. (B) Leukemic cells from 12 patients with AML were treated with the indicated vectors at an MOI of 50 before analysis of effects on colony formation. *, P<0.001 vs. Control; # P<0.001 vs. SG511. (C) Bone marrow cells obtained from 4 healthy volunteers were treated with the indicated viruses, and colonies were counted after 12 days. For all panels, values represent the mean of experiments in triplicate. (D) The primary leukemic cells from a CML patient in blast crisis (case 1) were infected with the indicated viruses at an MOI of 50, cell lysate was immunoblotted with anti-Beclin-1, anti-LC3, and anti-â-actin antibodies at day 2 of the experiment. (E) The primary blasts were treated with SG511-BECN (50 MOI), or SG511-BECN plus chloroquine (25 ìM) for 48 h. Autophagic flux was determined using Western blotting.

Mentions: To determine whether SG511-BECN virus is effective against primary leukemia cells, we tested the clonogenic capacity of primary blasts isolated from patients with CML in chronic phase or myeloid blast crisis with imatinib-resistant disease, and AML patients in newly diagnosed or relasped disease. The characteristics of these cases are shown in Table 1. Primary cells were exposed to the different viruses at an MOI of 50 and subjected to blast colony assays. Results showed that exposure of primary cells to SG511 virus had relatively little effect on clonogenic potential. However, in both AML (Fig. 4A) and CML (Fig. 4B), SG511-BECN resulted in a pronounced reduction in colony formation compared to SG511 (P<0.01, and P<0.001, respectively). In contrast, the ability of normal specimens (n=4) to form colonies was not substantially affected by treatment with SG511 or SG511-BECN, respectively (Fig. 4C). Further, a dramatic increase in the Beclin-1 expression and conversion of LC3-I to LC3-II was observed in primary cells treated with SG511-BECN (Fig. 4D). Using chloroquine, an alkalinizing lysosomotropic drug, autophagic flux was determined in primary leukemic cells obtained from a CML patient. Results showed that SG511-BECN-induced LC3-II accumulation was apparently augmented in cells exposed to chloroquine (Fig. 4E), suggesting efficient autophagic flux. Collectively, these data suggest that SG511-BECN significantly impairs leukemic but not normal hematopoietic progenitor-cell function.


Potent antitumor activity of oncolytic adenovirus expressing Beclin-1 via induction of autophagic cell death in leukemia.

Tong Y, You L, Liu H, Li L, Meng H, Qian Q, Qian W - Oncotarget (2013)

SG511-BECN induces autophagy in primary leukemic cells to preferentially inhibit CFU-L formation(A) Freshly isolated leukemia cells from CML patients (n=15) were cultured in methylcellulose medium in the presence of SG511, or SG511-BECN at an MOI of 50. After 12 days, colonies were scored. *, P>0.05 vs. Control; # P<0.01 vs. SG511. (B) Leukemic cells from 12 patients with AML were treated with the indicated vectors at an MOI of 50 before analysis of effects on colony formation. *, P<0.001 vs. Control; # P<0.001 vs. SG511. (C) Bone marrow cells obtained from 4 healthy volunteers were treated with the indicated viruses, and colonies were counted after 12 days. For all panels, values represent the mean of experiments in triplicate. (D) The primary leukemic cells from a CML patient in blast crisis (case 1) were infected with the indicated viruses at an MOI of 50, cell lysate was immunoblotted with anti-Beclin-1, anti-LC3, and anti-â-actin antibodies at day 2 of the experiment. (E) The primary blasts were treated with SG511-BECN (50 MOI), or SG511-BECN plus chloroquine (25 ìM) for 48 h. Autophagic flux was determined using Western blotting.
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Figure 4: SG511-BECN induces autophagy in primary leukemic cells to preferentially inhibit CFU-L formation(A) Freshly isolated leukemia cells from CML patients (n=15) were cultured in methylcellulose medium in the presence of SG511, or SG511-BECN at an MOI of 50. After 12 days, colonies were scored. *, P>0.05 vs. Control; # P<0.01 vs. SG511. (B) Leukemic cells from 12 patients with AML were treated with the indicated vectors at an MOI of 50 before analysis of effects on colony formation. *, P<0.001 vs. Control; # P<0.001 vs. SG511. (C) Bone marrow cells obtained from 4 healthy volunteers were treated with the indicated viruses, and colonies were counted after 12 days. For all panels, values represent the mean of experiments in triplicate. (D) The primary leukemic cells from a CML patient in blast crisis (case 1) were infected with the indicated viruses at an MOI of 50, cell lysate was immunoblotted with anti-Beclin-1, anti-LC3, and anti-â-actin antibodies at day 2 of the experiment. (E) The primary blasts were treated with SG511-BECN (50 MOI), or SG511-BECN plus chloroquine (25 ìM) for 48 h. Autophagic flux was determined using Western blotting.
Mentions: To determine whether SG511-BECN virus is effective against primary leukemia cells, we tested the clonogenic capacity of primary blasts isolated from patients with CML in chronic phase or myeloid blast crisis with imatinib-resistant disease, and AML patients in newly diagnosed or relasped disease. The characteristics of these cases are shown in Table 1. Primary cells were exposed to the different viruses at an MOI of 50 and subjected to blast colony assays. Results showed that exposure of primary cells to SG511 virus had relatively little effect on clonogenic potential. However, in both AML (Fig. 4A) and CML (Fig. 4B), SG511-BECN resulted in a pronounced reduction in colony formation compared to SG511 (P<0.01, and P<0.001, respectively). In contrast, the ability of normal specimens (n=4) to form colonies was not substantially affected by treatment with SG511 or SG511-BECN, respectively (Fig. 4C). Further, a dramatic increase in the Beclin-1 expression and conversion of LC3-I to LC3-II was observed in primary cells treated with SG511-BECN (Fig. 4D). Using chloroquine, an alkalinizing lysosomotropic drug, autophagic flux was determined in primary leukemic cells obtained from a CML patient. Results showed that SG511-BECN-induced LC3-II accumulation was apparently augmented in cells exposed to chloroquine (Fig. 4E), suggesting efficient autophagic flux. Collectively, these data suggest that SG511-BECN significantly impairs leukemic but not normal hematopoietic progenitor-cell function.

Bottom Line: Here, we investigated whether a strategy that combines the oncolytic effects of an adenoviral vector with simultaneous expression of Beclin-1, an autophagy gene, offers a therapeutic advantage for leukemia.We also showed that SG511-BECN strongly inhibited the growth of leukemic progenitors in vitro.Our results suggest that infection of leukemia cells with an oncolytic adenovirus overexpressing Beclin-1 can induce significant autophagic cell death and provide a new strategy for the elimination of leukemic cells via a unique mechanism of action distinct from apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Institute of Hematology, the First Affiliated Hospital, College of Medicine, Zhejiang University, P.R. China.

ABSTRACT
An attractive strategy among adenovirus-based oncolytic systems is to design adenoviral vectors to express pro-apoptotic genes, in which this gene-virotherapy approach significantly enhances tumor cell death by activating apoptotic pathways. However, the existence of cancer cells with apoptotic defects is one of the major obstacles in gene-virotherapy. Here, we investigated whether a strategy that combines the oncolytic effects of an adenoviral vector with simultaneous expression of Beclin-1, an autophagy gene, offers a therapeutic advantage for leukemia. A Beclin-1 cDNA was cloned in an oncolytic adenovirus with chimeric Ad5/11 fiber (SG511-BECN). SG511-BECN treatment induced significant autophagic cell death, and resulted in enhanced cell killing in a variety of leukemic cell lines and primary leukemic blasts. SG511-BECN effects were seen in chronic myeloid leukemia and acute myeloid leukemia with resistance to imatinib or chemotherapy, but exhibited much less cytotoxicity on normal cells. The SG511-BECN-induced autophagic cell death could be partially reversed by RNA interference knockdown of UVRAG, ATG5, and ATG7. We also showed that SG511-BECN strongly inhibited the growth of leukemic progenitors in vitro. In murine leukemia models, SG511-BECN prolonged the survival and decreased the xenograft tumor size by inducing autophagic cell death. Our results suggest that infection of leukemia cells with an oncolytic adenovirus overexpressing Beclin-1 can induce significant autophagic cell death and provide a new strategy for the elimination of leukemic cells via a unique mechanism of action distinct from apoptosis.

Show MeSH
Related in: MedlinePlus