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Clusterin inhibition using OGX-011 synergistically enhances zoledronic acid activity in osteosarcoma.

Lamoureux F, Baud'huin M, Ory B, Guiho R, Zoubeidi A, Gleave M, Heymann D, Rédini F - Oncotarget (2014)

Bottom Line: The combined effects of OGX-011 and ZOL were investigated in vitro on cell growth, viability, apoptosis and cell cycle repartition of ZOL-sensitive or -resistant human OS cell lines (SaOS2, U2OS, MG63 and MNNG/HOS).These biologic events were accompanied by decreased expression of HSPs, MDR1 and HSF1 transcriptional activity.These results indicate that ZOL-mediated induction of CLU can be attenuated by OGX-011, with synergistic effects on delaying progression of osteosarcoma.

View Article: PubMed Central - PubMed

Affiliation: Université de Nantes, Nantes atlantique universités, Laboratoire de Physiopathologie de la Résorption Osseuse et Thérapie des Tumeurs Osseuses Primitives, Nantes F-44035, France. INSERM, UMR 957, Nantes F-44035, France. LUNAM Université. Equipe labellisée LIGUE 2012, Nantes, cedex.

ABSTRACT

Purpose: Despite recent improvements in therapeutic management of osteosarcoma, ongoing challenges in improving the response to chemotherapy warrants new strategies still needed to improve overall patient survival. Among new therapeutic approaches, zoledronic acid (ZOL) represents a promising adjuvant molecule to chemotherapy to limit the osteolytic component of bone tumors. However, ZOL triggers the elevation of heat shock proteins (Hsp), including Hsp27 and clusterin (CLU), which could enhance tumor cell survival and treatment resistance. We hypothesized that targeting CLU using siRNA or the antisense drug, OGX-011, will suppress treatment-induced CLU induction and enhance ZOL-induced cell death in osteosarcoma (OS) cells.

Methods: The combined effects of OGX-011 and ZOL were investigated in vitro on cell growth, viability, apoptosis and cell cycle repartition of ZOL-sensitive or -resistant human OS cell lines (SaOS2, U2OS, MG63 and MNNG/HOS).

Results: In OS cell lines, ZOL increased levels of HSPs, especially CLU, in a dose- and time-dependent manner by mechanism including increased HSF1 transcription activity. The OS resistant cells to ZOL exhibited higher CLU expression level than the sensitive cells. Moreover, CLU overexpression protects OS sensitive cells to ZOL-induced cell death by modulating the MDR1 and farnesyl diphosphate synthase expression. OGX-011 suppressed treatment-induced increases in CLU and synergistically enhanced the activity of ZOL on cell growth and apoptosis. These biologic events were accompanied by decreased expression of HSPs, MDR1 and HSF1 transcriptional activity. In vivo, OGX-011, administered 3 times a week (IP, 20mg/kg), potentiated the effect of ZOL (s.c; 50µg/kg), significantly inhibiting tumor growth by 50% and prolonging survival in MNNG/HOS xenograft model compared to ZOL alone.

Conclusion: These results indicate that ZOL-mediated induction of CLU can be attenuated by OGX-011, with synergistic effects on delaying progression of osteosarcoma.

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Related in: MedlinePlus

OGX-011 potentiates ZOL activity in MNNG/HOS osteosarcoma xenograft modelMice were treated twice a week with 50μg/kg ZOL and 15mg/kg OGX-011 starting when tumors were palpable as described in M&M. The mean tumor volume (A) and individual tumor volume (B) were compared between the 3 groups ± SEM (n=8). ***, p<0.001. C, in Kaplan-Meier curve, cancer-specific survival was compared between the 3 groups over a 46-d period. ***, p<0.001. D, tumors were collected and CLU, Ki67, and cleaved-caspase-3 were evaluated by immunohistochemical analysis. Specimens were scored and estimated in percentage of positive cells. The control group corresponds to the mice bearing tumor that did not receive any treatment. This group only received vehicle. * p<0.05; *** p<0.001.
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Figure 5: OGX-011 potentiates ZOL activity in MNNG/HOS osteosarcoma xenograft modelMice were treated twice a week with 50μg/kg ZOL and 15mg/kg OGX-011 starting when tumors were palpable as described in M&M. The mean tumor volume (A) and individual tumor volume (B) were compared between the 3 groups ± SEM (n=8). ***, p<0.001. C, in Kaplan-Meier curve, cancer-specific survival was compared between the 3 groups over a 46-d period. ***, p<0.001. D, tumors were collected and CLU, Ki67, and cleaved-caspase-3 were evaluated by immunohistochemical analysis. Specimens were scored and estimated in percentage of positive cells. The control group corresponds to the mice bearing tumor that did not receive any treatment. This group only received vehicle. * p<0.05; *** p<0.001.

Mentions: The effects of combined treatment with OGX-011 and ZOL were evaluated in HOS-MNNG xenograft tumors. Athymic mice were injected with human osteosarcoma HOS-MNNG cells in paratibial site. Once tumors were palpable, mice were randomly assigned to vehicle control, ZOL + ScrB ASO, or ZOL + OGX-011 groups. In a first in vivo experiment, we confirmed that ScrB and OGX-011 do not have effect on tumor growth compared with tumor control group (Supp. Fig. 5A and B). In a second experiment, all animals treated with ZOL + OGX-011 (n=8) had significant delays in tumor growth compared with other groups starting at day 15 (respectively 196 mm3 for ZOL + OGX-011 versus 451.3 mm3 for control and 404.9 mm3 for ZOL + ScrB ASO) and after 36 days (respectively 997.2 mm3 for ZOL + OGX-011 versus 2023.6 mm3 for control and 1954.2 mm3 for ZOL + ScrB ASO; ***, p<0.001; Fig. 5A). Until day 27, ZOL + ScrB shows marginal but not significant decrease of tumor growth compared with control group (respectively 1368.5 mm3 and 1649.9 mm3 at day 27). Moreover, at individual level, 0 out of 8 mice developed tumor with volume over 1000mm3 at day 30, while all mice in control and ZOL + ScrB ASO groups exhibited tumor volume over 1000mm3 (Fig. 5B). Overall survival was significantly prolonged in mice treated with combined ZOL + OGX-011 (p<0.001; Fig. 5C). By day 42, all mice died or were euthanized due to high tumor burden in control and in ZOL + ScrB groups as compared with the combined ZOL + OGX-011 group, where all mice were still alive after 46 days. These data demonstrate that targeting CLU using OGX-011 potentiates the effects of ZOL to significantly inhibit tumor growth and prolongs survival in osteosarcoma xenograft model.


Clusterin inhibition using OGX-011 synergistically enhances zoledronic acid activity in osteosarcoma.

Lamoureux F, Baud'huin M, Ory B, Guiho R, Zoubeidi A, Gleave M, Heymann D, Rédini F - Oncotarget (2014)

OGX-011 potentiates ZOL activity in MNNG/HOS osteosarcoma xenograft modelMice were treated twice a week with 50μg/kg ZOL and 15mg/kg OGX-011 starting when tumors were palpable as described in M&M. The mean tumor volume (A) and individual tumor volume (B) were compared between the 3 groups ± SEM (n=8). ***, p<0.001. C, in Kaplan-Meier curve, cancer-specific survival was compared between the 3 groups over a 46-d period. ***, p<0.001. D, tumors were collected and CLU, Ki67, and cleaved-caspase-3 were evaluated by immunohistochemical analysis. Specimens were scored and estimated in percentage of positive cells. The control group corresponds to the mice bearing tumor that did not receive any treatment. This group only received vehicle. * p<0.05; *** p<0.001.
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Figure 5: OGX-011 potentiates ZOL activity in MNNG/HOS osteosarcoma xenograft modelMice were treated twice a week with 50μg/kg ZOL and 15mg/kg OGX-011 starting when tumors were palpable as described in M&M. The mean tumor volume (A) and individual tumor volume (B) were compared between the 3 groups ± SEM (n=8). ***, p<0.001. C, in Kaplan-Meier curve, cancer-specific survival was compared between the 3 groups over a 46-d period. ***, p<0.001. D, tumors were collected and CLU, Ki67, and cleaved-caspase-3 were evaluated by immunohistochemical analysis. Specimens were scored and estimated in percentage of positive cells. The control group corresponds to the mice bearing tumor that did not receive any treatment. This group only received vehicle. * p<0.05; *** p<0.001.
Mentions: The effects of combined treatment with OGX-011 and ZOL were evaluated in HOS-MNNG xenograft tumors. Athymic mice were injected with human osteosarcoma HOS-MNNG cells in paratibial site. Once tumors were palpable, mice were randomly assigned to vehicle control, ZOL + ScrB ASO, or ZOL + OGX-011 groups. In a first in vivo experiment, we confirmed that ScrB and OGX-011 do not have effect on tumor growth compared with tumor control group (Supp. Fig. 5A and B). In a second experiment, all animals treated with ZOL + OGX-011 (n=8) had significant delays in tumor growth compared with other groups starting at day 15 (respectively 196 mm3 for ZOL + OGX-011 versus 451.3 mm3 for control and 404.9 mm3 for ZOL + ScrB ASO) and after 36 days (respectively 997.2 mm3 for ZOL + OGX-011 versus 2023.6 mm3 for control and 1954.2 mm3 for ZOL + ScrB ASO; ***, p<0.001; Fig. 5A). Until day 27, ZOL + ScrB shows marginal but not significant decrease of tumor growth compared with control group (respectively 1368.5 mm3 and 1649.9 mm3 at day 27). Moreover, at individual level, 0 out of 8 mice developed tumor with volume over 1000mm3 at day 30, while all mice in control and ZOL + ScrB ASO groups exhibited tumor volume over 1000mm3 (Fig. 5B). Overall survival was significantly prolonged in mice treated with combined ZOL + OGX-011 (p<0.001; Fig. 5C). By day 42, all mice died or were euthanized due to high tumor burden in control and in ZOL + ScrB groups as compared with the combined ZOL + OGX-011 group, where all mice were still alive after 46 days. These data demonstrate that targeting CLU using OGX-011 potentiates the effects of ZOL to significantly inhibit tumor growth and prolongs survival in osteosarcoma xenograft model.

Bottom Line: The combined effects of OGX-011 and ZOL were investigated in vitro on cell growth, viability, apoptosis and cell cycle repartition of ZOL-sensitive or -resistant human OS cell lines (SaOS2, U2OS, MG63 and MNNG/HOS).These biologic events were accompanied by decreased expression of HSPs, MDR1 and HSF1 transcriptional activity.These results indicate that ZOL-mediated induction of CLU can be attenuated by OGX-011, with synergistic effects on delaying progression of osteosarcoma.

View Article: PubMed Central - PubMed

Affiliation: Université de Nantes, Nantes atlantique universités, Laboratoire de Physiopathologie de la Résorption Osseuse et Thérapie des Tumeurs Osseuses Primitives, Nantes F-44035, France. INSERM, UMR 957, Nantes F-44035, France. LUNAM Université. Equipe labellisée LIGUE 2012, Nantes, cedex.

ABSTRACT

Purpose: Despite recent improvements in therapeutic management of osteosarcoma, ongoing challenges in improving the response to chemotherapy warrants new strategies still needed to improve overall patient survival. Among new therapeutic approaches, zoledronic acid (ZOL) represents a promising adjuvant molecule to chemotherapy to limit the osteolytic component of bone tumors. However, ZOL triggers the elevation of heat shock proteins (Hsp), including Hsp27 and clusterin (CLU), which could enhance tumor cell survival and treatment resistance. We hypothesized that targeting CLU using siRNA or the antisense drug, OGX-011, will suppress treatment-induced CLU induction and enhance ZOL-induced cell death in osteosarcoma (OS) cells.

Methods: The combined effects of OGX-011 and ZOL were investigated in vitro on cell growth, viability, apoptosis and cell cycle repartition of ZOL-sensitive or -resistant human OS cell lines (SaOS2, U2OS, MG63 and MNNG/HOS).

Results: In OS cell lines, ZOL increased levels of HSPs, especially CLU, in a dose- and time-dependent manner by mechanism including increased HSF1 transcription activity. The OS resistant cells to ZOL exhibited higher CLU expression level than the sensitive cells. Moreover, CLU overexpression protects OS sensitive cells to ZOL-induced cell death by modulating the MDR1 and farnesyl diphosphate synthase expression. OGX-011 suppressed treatment-induced increases in CLU and synergistically enhanced the activity of ZOL on cell growth and apoptosis. These biologic events were accompanied by decreased expression of HSPs, MDR1 and HSF1 transcriptional activity. In vivo, OGX-011, administered 3 times a week (IP, 20mg/kg), potentiated the effect of ZOL (s.c; 50µg/kg), significantly inhibiting tumor growth by 50% and prolonging survival in MNNG/HOS xenograft model compared to ZOL alone.

Conclusion: These results indicate that ZOL-mediated induction of CLU can be attenuated by OGX-011, with synergistic effects on delaying progression of osteosarcoma.

Show MeSH
Related in: MedlinePlus