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Combination of adenoviral virotherapy and temozolomide chemotherapy eradicates malignant glioma through autophagic and apoptotic cell death in vivo.

Ulasov IV, Sonabend AM, Nandi S, Khramtsov A, Han Y, Lesniak MS - Br. J. Cancer (2009)

Bottom Line: Recent studies suggest that the cytopathic effect elicited by these vectors is mediated through autophagy, a form of programmed cell death.Western blot analysis showed increased expression of BAX and p53, decreased expression of BCL2 and elevated level of APG5.Treatment with TMZ followed by CRAd-Survivin-pk7 (CRAd-S-pk7) led to a significant over-expression of autophagy markers, acidic vesicular organelles and light-chain 3 (LC3).

View Article: PubMed Central - PubMed

Affiliation: The Brain Tumor Center, The University of Chicago Pritzker School of Medicine, Chicago, IL 60637, USA.

ABSTRACT
Conditionally replicative adenoviruses (CRAds) represent a novel treatment strategy for malignant glioma. Recent studies suggest that the cytopathic effect elicited by these vectors is mediated through autophagy, a form of programmed cell death. Likewise, temozolomide (TMZ), a chemotherapeutic agent used for the treatment of malignant gliomas, also triggers autophagic cell death. In this study, we examined the potential to combine the two treatments in the setting of experimental glioma. In vitro, pretreatment with TMZ followed by CRAd-Surivin-pk7 enhanced cytotoxicity against a panel of glioma cell lines. Western blot analysis showed increased expression of BAX and p53, decreased expression of BCL2 and elevated level of APG5. Treatment with TMZ followed by CRAd-Survivin-pk7 (CRAd-S-pk7) led to a significant over-expression of autophagy markers, acidic vesicular organelles and light-chain 3 (LC3). These results were further evaluated in vivo, in which 90% of the mice with intracranial tumours were long-term survivors (>100 days) after treatment with TMZ and CRAd-S-pk7 (P<0.01). Analysis of tumours ex vivo showed expression of both LC3 and cleaved Caspase-3, proving that both autophagy and apoptosis are responsible for cell death in vivo. These results suggest that combination of chemovirotherapy offers a powerful tool against malignant glioma and should be further explored in the clinical setting.

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Induction of autophagy in U87MG cells treated with TMZ followed by CRAd-S-pk7 infection. Effect of combined treatment of U87MG cells was detected by (A) light microscopy, (B) western blot, (C) membrane potential, (D) flow cytometry and (E) LDH toxicity. (A) Decrease in cell density and morphological changes associated with treatment with TMZ followed by CRAd-S-pk7. (B) Modulation of pro-apoptotic, anti-apoptotic and autophagic proteins in response to treatment with TMZ (100 μM), CRAd-S-pk7 (100 vp per cell) or combination (TMZ and CRAd-S-pk7) as determined by Western blot analysis. Pretreatment of U87MG cells with TMZ followed by CRAd-S-pk7 infection showed over-expression of p53, Bax and APG5 proteins and downregulation of Puma, Noxa, BNIP3 and BCL-2 proteins. There was no evidence of cleaved Caspase-3. (C) To show that mitochondrial pathway is not activated, we measured mitochondrial potential changes. TMZ, CRAd-S-pk7 and combination group did not induce significant changes in Δψ. (D) Autophagy was determined by staining with acridine orange (AO) and α-LC3B antibody followed by flow cytometry analysis. Experiment was performed in triplicates and the mean of two independent experiments is shown here. (E) Effect of Bafilomycin A1 (BAF-A1) and 3-MA treatments on co-treatment induced toxicity. Cells were pretreated with 3-MA, BAF-A1 or vehicle control for 12 h before exposure to TMZ, CRAd-S-pk7 or TMZ followed by CRAd-S-pk7. Figure summarises data from two independent experiments each having six replicates per condition. (*), (**) and (***) P<0.05. (*), (**) and (***) P-value determined by comparing mock vs TMZ alone, CRAd-S-pk7 alone or combination TMZ then CRAd-S-pk7, respectively.
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fig2: Induction of autophagy in U87MG cells treated with TMZ followed by CRAd-S-pk7 infection. Effect of combined treatment of U87MG cells was detected by (A) light microscopy, (B) western blot, (C) membrane potential, (D) flow cytometry and (E) LDH toxicity. (A) Decrease in cell density and morphological changes associated with treatment with TMZ followed by CRAd-S-pk7. (B) Modulation of pro-apoptotic, anti-apoptotic and autophagic proteins in response to treatment with TMZ (100 μM), CRAd-S-pk7 (100 vp per cell) or combination (TMZ and CRAd-S-pk7) as determined by Western blot analysis. Pretreatment of U87MG cells with TMZ followed by CRAd-S-pk7 infection showed over-expression of p53, Bax and APG5 proteins and downregulation of Puma, Noxa, BNIP3 and BCL-2 proteins. There was no evidence of cleaved Caspase-3. (C) To show that mitochondrial pathway is not activated, we measured mitochondrial potential changes. TMZ, CRAd-S-pk7 and combination group did not induce significant changes in Δψ. (D) Autophagy was determined by staining with acridine orange (AO) and α-LC3B antibody followed by flow cytometry analysis. Experiment was performed in triplicates and the mean of two independent experiments is shown here. (E) Effect of Bafilomycin A1 (BAF-A1) and 3-MA treatments on co-treatment induced toxicity. Cells were pretreated with 3-MA, BAF-A1 or vehicle control for 12 h before exposure to TMZ, CRAd-S-pk7 or TMZ followed by CRAd-S-pk7. Figure summarises data from two independent experiments each having six replicates per condition. (*), (**) and (***) P<0.05. (*), (**) and (***) P-value determined by comparing mock vs TMZ alone, CRAd-S-pk7 alone or combination TMZ then CRAd-S-pk7, respectively.

Mentions: On the basis of the finding that pretreatment with TMZ followed by CRAd-S-pk7 (combination therapy) leads to an enhanced cytotoxic effect in all cell lines tested, we investigated the mechanism behind this phenomenon in the U87MG cell line. Consistent with the cell integrity assay (Figure 1), treatment with TMZ followed by CRAd-S-pk7 led to a cytopathic effect characterised by a decrease in cell density and morphological changes (Figure 2A). To assess the possibility of cell death by activation of apoptosis, we evaluated the expression of proteins BAX, BIK, BAD, p53 and Caspase-3 (both cleaved and uncleaved), among other pro-apoptotic proteins, which have been implicated in the induction of apoptosis by adenovirus (Verma et al, 2001; Nahle et al, 2002; Zhang et al, 2003; Subramanian et al, 2007), as well as anti-apoptotic proteins BCL-2 and BCL-XL (Shimazu et al, 2007) (Figure 2B). It is interesting to note that the expression pattern of some of these proteins points towards a pro-apoptotic stage triggered by treatment with TMZ followed by CRAd-S-pk7. This is suggested by the increase in expression of p53 and BAX, and a decrease in BCL-2 expression. On the other hand, the fact that the expression of BAD and PUMA was decreased, the lack of change in expression of BID and NOXA, and the absence of Caspase-3 cleavage along with absence of mitochondrial depolarisation (Figure 2C) do not support the hypothesis of classical apoptosis as the definitive mechanism for the observed cytotoxic effect elicited by this TMZ and CRAd-S-pk7 combination.


Combination of adenoviral virotherapy and temozolomide chemotherapy eradicates malignant glioma through autophagic and apoptotic cell death in vivo.

Ulasov IV, Sonabend AM, Nandi S, Khramtsov A, Han Y, Lesniak MS - Br. J. Cancer (2009)

Induction of autophagy in U87MG cells treated with TMZ followed by CRAd-S-pk7 infection. Effect of combined treatment of U87MG cells was detected by (A) light microscopy, (B) western blot, (C) membrane potential, (D) flow cytometry and (E) LDH toxicity. (A) Decrease in cell density and morphological changes associated with treatment with TMZ followed by CRAd-S-pk7. (B) Modulation of pro-apoptotic, anti-apoptotic and autophagic proteins in response to treatment with TMZ (100 μM), CRAd-S-pk7 (100 vp per cell) or combination (TMZ and CRAd-S-pk7) as determined by Western blot analysis. Pretreatment of U87MG cells with TMZ followed by CRAd-S-pk7 infection showed over-expression of p53, Bax and APG5 proteins and downregulation of Puma, Noxa, BNIP3 and BCL-2 proteins. There was no evidence of cleaved Caspase-3. (C) To show that mitochondrial pathway is not activated, we measured mitochondrial potential changes. TMZ, CRAd-S-pk7 and combination group did not induce significant changes in Δψ. (D) Autophagy was determined by staining with acridine orange (AO) and α-LC3B antibody followed by flow cytometry analysis. Experiment was performed in triplicates and the mean of two independent experiments is shown here. (E) Effect of Bafilomycin A1 (BAF-A1) and 3-MA treatments on co-treatment induced toxicity. Cells were pretreated with 3-MA, BAF-A1 or vehicle control for 12 h before exposure to TMZ, CRAd-S-pk7 or TMZ followed by CRAd-S-pk7. Figure summarises data from two independent experiments each having six replicates per condition. (*), (**) and (***) P<0.05. (*), (**) and (***) P-value determined by comparing mock vs TMZ alone, CRAd-S-pk7 alone or combination TMZ then CRAd-S-pk7, respectively.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
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fig2: Induction of autophagy in U87MG cells treated with TMZ followed by CRAd-S-pk7 infection. Effect of combined treatment of U87MG cells was detected by (A) light microscopy, (B) western blot, (C) membrane potential, (D) flow cytometry and (E) LDH toxicity. (A) Decrease in cell density and morphological changes associated with treatment with TMZ followed by CRAd-S-pk7. (B) Modulation of pro-apoptotic, anti-apoptotic and autophagic proteins in response to treatment with TMZ (100 μM), CRAd-S-pk7 (100 vp per cell) or combination (TMZ and CRAd-S-pk7) as determined by Western blot analysis. Pretreatment of U87MG cells with TMZ followed by CRAd-S-pk7 infection showed over-expression of p53, Bax and APG5 proteins and downregulation of Puma, Noxa, BNIP3 and BCL-2 proteins. There was no evidence of cleaved Caspase-3. (C) To show that mitochondrial pathway is not activated, we measured mitochondrial potential changes. TMZ, CRAd-S-pk7 and combination group did not induce significant changes in Δψ. (D) Autophagy was determined by staining with acridine orange (AO) and α-LC3B antibody followed by flow cytometry analysis. Experiment was performed in triplicates and the mean of two independent experiments is shown here. (E) Effect of Bafilomycin A1 (BAF-A1) and 3-MA treatments on co-treatment induced toxicity. Cells were pretreated with 3-MA, BAF-A1 or vehicle control for 12 h before exposure to TMZ, CRAd-S-pk7 or TMZ followed by CRAd-S-pk7. Figure summarises data from two independent experiments each having six replicates per condition. (*), (**) and (***) P<0.05. (*), (**) and (***) P-value determined by comparing mock vs TMZ alone, CRAd-S-pk7 alone or combination TMZ then CRAd-S-pk7, respectively.
Mentions: On the basis of the finding that pretreatment with TMZ followed by CRAd-S-pk7 (combination therapy) leads to an enhanced cytotoxic effect in all cell lines tested, we investigated the mechanism behind this phenomenon in the U87MG cell line. Consistent with the cell integrity assay (Figure 1), treatment with TMZ followed by CRAd-S-pk7 led to a cytopathic effect characterised by a decrease in cell density and morphological changes (Figure 2A). To assess the possibility of cell death by activation of apoptosis, we evaluated the expression of proteins BAX, BIK, BAD, p53 and Caspase-3 (both cleaved and uncleaved), among other pro-apoptotic proteins, which have been implicated in the induction of apoptosis by adenovirus (Verma et al, 2001; Nahle et al, 2002; Zhang et al, 2003; Subramanian et al, 2007), as well as anti-apoptotic proteins BCL-2 and BCL-XL (Shimazu et al, 2007) (Figure 2B). It is interesting to note that the expression pattern of some of these proteins points towards a pro-apoptotic stage triggered by treatment with TMZ followed by CRAd-S-pk7. This is suggested by the increase in expression of p53 and BAX, and a decrease in BCL-2 expression. On the other hand, the fact that the expression of BAD and PUMA was decreased, the lack of change in expression of BID and NOXA, and the absence of Caspase-3 cleavage along with absence of mitochondrial depolarisation (Figure 2C) do not support the hypothesis of classical apoptosis as the definitive mechanism for the observed cytotoxic effect elicited by this TMZ and CRAd-S-pk7 combination.

Bottom Line: Recent studies suggest that the cytopathic effect elicited by these vectors is mediated through autophagy, a form of programmed cell death.Western blot analysis showed increased expression of BAX and p53, decreased expression of BCL2 and elevated level of APG5.Treatment with TMZ followed by CRAd-Survivin-pk7 (CRAd-S-pk7) led to a significant over-expression of autophagy markers, acidic vesicular organelles and light-chain 3 (LC3).

View Article: PubMed Central - PubMed

Affiliation: The Brain Tumor Center, The University of Chicago Pritzker School of Medicine, Chicago, IL 60637, USA.

ABSTRACT
Conditionally replicative adenoviruses (CRAds) represent a novel treatment strategy for malignant glioma. Recent studies suggest that the cytopathic effect elicited by these vectors is mediated through autophagy, a form of programmed cell death. Likewise, temozolomide (TMZ), a chemotherapeutic agent used for the treatment of malignant gliomas, also triggers autophagic cell death. In this study, we examined the potential to combine the two treatments in the setting of experimental glioma. In vitro, pretreatment with TMZ followed by CRAd-Surivin-pk7 enhanced cytotoxicity against a panel of glioma cell lines. Western blot analysis showed increased expression of BAX and p53, decreased expression of BCL2 and elevated level of APG5. Treatment with TMZ followed by CRAd-Survivin-pk7 (CRAd-S-pk7) led to a significant over-expression of autophagy markers, acidic vesicular organelles and light-chain 3 (LC3). These results were further evaluated in vivo, in which 90% of the mice with intracranial tumours were long-term survivors (>100 days) after treatment with TMZ and CRAd-S-pk7 (P<0.01). Analysis of tumours ex vivo showed expression of both LC3 and cleaved Caspase-3, proving that both autophagy and apoptosis are responsible for cell death in vivo. These results suggest that combination of chemovirotherapy offers a powerful tool against malignant glioma and should be further explored in the clinical setting.

Show MeSH
Related in: MedlinePlus