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Schwann cell autophagy induced by SAHA, 17-AAG, or clonazepam can reduce bortezomib-induced peripheral neuropathy.

Watanabe T, Nagase K, Chosa M, Tobinai K - Br. J. Cancer (2010)

Bottom Line: A tractable system to evaluate combination drugs for use with bortezomib is essential to enable continuing clinical benefit from this drug.To then monitor aggresome formation as a result of proteasome inhibition and the activation of chaperone-mediated autophagy (CMA), we performed double-labelling immunofluorescent analyses of a cellular aggregation-prone protein marker.Aggresome formation was interrupted by VCR, whereas combination treatments with bortezomib involving suberoylanilide hydroxamic acid, 17-allylamino-17-demethoxy-geldanamycin, or clonazepam appear to facilitate the disposal of unfolded proteins via CMA, inducing HSP70 and lysosome-associated membrane protein type 2A (LAMP-2A).

View Article: PubMed Central - PubMed

Affiliation: Hematology Division, National Cancer Center Hospital, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, Japan. takawata@ncc.go.jp

ABSTRACT

Background: The proteasome inhibitor bortezomib has improved the survival of patients with multiple myeloma but bortezomib-induced peripheral neuropathy (BiPN) has emerged as a serious potential complication of this therapy. Animal studies suggest that bortezomib predominantly causes pathological changes in Schwann cells. A tractable system to evaluate combination drugs for use with bortezomib is essential to enable continuing clinical benefit from this drug.

Methods: Rat schwannoma cells were pretreated with vincristine (VCR), histone deacetylase inhibitors, anticonvulsants, or a heat-shock protein 90 (HSP90) inhibitor. To then monitor aggresome formation as a result of proteasome inhibition and the activation of chaperone-mediated autophagy (CMA), we performed double-labelling immunofluorescent analyses of a cellular aggregation-prone protein marker.

Results: Aggresome formation was interrupted by VCR, whereas combination treatments with bortezomib involving suberoylanilide hydroxamic acid, 17-allylamino-17-demethoxy-geldanamycin, or clonazepam appear to facilitate the disposal of unfolded proteins via CMA, inducing HSP70 and lysosome-associated membrane protein type 2A (LAMP-2A).

Conclusions: This schwannoma model can be used to test BiPN-reducing drugs. The present data suggest that aggresome formation in Schwann cells is a possible mechanism of BiPN, and drugs that induce HSP70 or LAMP-2A have the potential to alleviate this complication. Combination clinical trials are warranted to confirm the relevance of these observations.

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Vincristine (VCR) abrogates aggresome formation and in a combination treatment with bortezomib augments the exocytosis of endogenous misfolded proteins. (A) Peripheral myelin protein 22 (PMP22) is homogeneously distributed throughout the cytoplasm of RT4-D6P2T cells before treatment with bortezomib (left panel). After treatment with bortezomib (Bzb), PMP22 appears to undergo retrograde transport towards the MTOC where it forms perinuclear aggresomes (arrows, middle panel, green signals) and colocalises with γ-tubulin (arrows, middle panel, red signals). A merged image of both fluorophores is shown in the far right panel (yellow signal). (B) Following pretreatment with VCR, a microtubule depolymerisation agent, PMP22 signals are evident at multiple sites in a granular pattern of aggregates throughout the cytoplasm, most notably in the perikaryon. Cells pretreated with (C) suberoylanilide hydroxamic acid (SAHA), a known histone deacetylase inhibitor (HDACi), or (D) clonazepam (CZP), an anticonvulsant, and (E) 17-allylamino-17-demethoxy-geldanamycin (17-AAG), a HSP90 inhibitor, fail to form aggresomes, but instead form rounded structures outside of the cell (arrowheads), which are smaller than the perinuclear aggresomes. (F) Pretreatment with valproic acid (VPA) causes the appearance of similar rounded structures outside of the cells (arrowheads) in addition to juxtanuclear aggresomes (arrows).
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fig2: Vincristine (VCR) abrogates aggresome formation and in a combination treatment with bortezomib augments the exocytosis of endogenous misfolded proteins. (A) Peripheral myelin protein 22 (PMP22) is homogeneously distributed throughout the cytoplasm of RT4-D6P2T cells before treatment with bortezomib (left panel). After treatment with bortezomib (Bzb), PMP22 appears to undergo retrograde transport towards the MTOC where it forms perinuclear aggresomes (arrows, middle panel, green signals) and colocalises with γ-tubulin (arrows, middle panel, red signals). A merged image of both fluorophores is shown in the far right panel (yellow signal). (B) Following pretreatment with VCR, a microtubule depolymerisation agent, PMP22 signals are evident at multiple sites in a granular pattern of aggregates throughout the cytoplasm, most notably in the perikaryon. Cells pretreated with (C) suberoylanilide hydroxamic acid (SAHA), a known histone deacetylase inhibitor (HDACi), or (D) clonazepam (CZP), an anticonvulsant, and (E) 17-allylamino-17-demethoxy-geldanamycin (17-AAG), a HSP90 inhibitor, fail to form aggresomes, but instead form rounded structures outside of the cell (arrowheads), which are smaller than the perinuclear aggresomes. (F) Pretreatment with valproic acid (VPA) causes the appearance of similar rounded structures outside of the cells (arrowheads) in addition to juxtanuclear aggresomes (arrows).

Mentions: We next examined whether endogenous misfolded proteins destined to be processed by the ubiquitin-proteasome system could be induced to aggregate and undergo retrograde transport towards the microtubule-organising center (MTOC) upon proteasome inhibition. To accomplish this, we employed the cellular marker PMP22, a short-lived glycoprotein present in Schwann cells (Fortun et al, 2003). Following bortezomib treatment, PMP22 showed a distinct juxtanuclear and rounded appearance and colocalised with γ-tubulin to form aggresomes (Figure 2A), as previously reported (Fortun et al, 2003). Interestingly, treatments with VCR completely abrogated the bortezomib-induced accumulation of PMP22, which was instead observed as numerous spots in the perikaryon (Figure 2B).


Schwann cell autophagy induced by SAHA, 17-AAG, or clonazepam can reduce bortezomib-induced peripheral neuropathy.

Watanabe T, Nagase K, Chosa M, Tobinai K - Br. J. Cancer (2010)

Vincristine (VCR) abrogates aggresome formation and in a combination treatment with bortezomib augments the exocytosis of endogenous misfolded proteins. (A) Peripheral myelin protein 22 (PMP22) is homogeneously distributed throughout the cytoplasm of RT4-D6P2T cells before treatment with bortezomib (left panel). After treatment with bortezomib (Bzb), PMP22 appears to undergo retrograde transport towards the MTOC where it forms perinuclear aggresomes (arrows, middle panel, green signals) and colocalises with γ-tubulin (arrows, middle panel, red signals). A merged image of both fluorophores is shown in the far right panel (yellow signal). (B) Following pretreatment with VCR, a microtubule depolymerisation agent, PMP22 signals are evident at multiple sites in a granular pattern of aggregates throughout the cytoplasm, most notably in the perikaryon. Cells pretreated with (C) suberoylanilide hydroxamic acid (SAHA), a known histone deacetylase inhibitor (HDACi), or (D) clonazepam (CZP), an anticonvulsant, and (E) 17-allylamino-17-demethoxy-geldanamycin (17-AAG), a HSP90 inhibitor, fail to form aggresomes, but instead form rounded structures outside of the cell (arrowheads), which are smaller than the perinuclear aggresomes. (F) Pretreatment with valproic acid (VPA) causes the appearance of similar rounded structures outside of the cells (arrowheads) in addition to juxtanuclear aggresomes (arrows).
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Related In: Results  -  Collection

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fig2: Vincristine (VCR) abrogates aggresome formation and in a combination treatment with bortezomib augments the exocytosis of endogenous misfolded proteins. (A) Peripheral myelin protein 22 (PMP22) is homogeneously distributed throughout the cytoplasm of RT4-D6P2T cells before treatment with bortezomib (left panel). After treatment with bortezomib (Bzb), PMP22 appears to undergo retrograde transport towards the MTOC where it forms perinuclear aggresomes (arrows, middle panel, green signals) and colocalises with γ-tubulin (arrows, middle panel, red signals). A merged image of both fluorophores is shown in the far right panel (yellow signal). (B) Following pretreatment with VCR, a microtubule depolymerisation agent, PMP22 signals are evident at multiple sites in a granular pattern of aggregates throughout the cytoplasm, most notably in the perikaryon. Cells pretreated with (C) suberoylanilide hydroxamic acid (SAHA), a known histone deacetylase inhibitor (HDACi), or (D) clonazepam (CZP), an anticonvulsant, and (E) 17-allylamino-17-demethoxy-geldanamycin (17-AAG), a HSP90 inhibitor, fail to form aggresomes, but instead form rounded structures outside of the cell (arrowheads), which are smaller than the perinuclear aggresomes. (F) Pretreatment with valproic acid (VPA) causes the appearance of similar rounded structures outside of the cells (arrowheads) in addition to juxtanuclear aggresomes (arrows).
Mentions: We next examined whether endogenous misfolded proteins destined to be processed by the ubiquitin-proteasome system could be induced to aggregate and undergo retrograde transport towards the microtubule-organising center (MTOC) upon proteasome inhibition. To accomplish this, we employed the cellular marker PMP22, a short-lived glycoprotein present in Schwann cells (Fortun et al, 2003). Following bortezomib treatment, PMP22 showed a distinct juxtanuclear and rounded appearance and colocalised with γ-tubulin to form aggresomes (Figure 2A), as previously reported (Fortun et al, 2003). Interestingly, treatments with VCR completely abrogated the bortezomib-induced accumulation of PMP22, which was instead observed as numerous spots in the perikaryon (Figure 2B).

Bottom Line: A tractable system to evaluate combination drugs for use with bortezomib is essential to enable continuing clinical benefit from this drug.To then monitor aggresome formation as a result of proteasome inhibition and the activation of chaperone-mediated autophagy (CMA), we performed double-labelling immunofluorescent analyses of a cellular aggregation-prone protein marker.Aggresome formation was interrupted by VCR, whereas combination treatments with bortezomib involving suberoylanilide hydroxamic acid, 17-allylamino-17-demethoxy-geldanamycin, or clonazepam appear to facilitate the disposal of unfolded proteins via CMA, inducing HSP70 and lysosome-associated membrane protein type 2A (LAMP-2A).

View Article: PubMed Central - PubMed

Affiliation: Hematology Division, National Cancer Center Hospital, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, Japan. takawata@ncc.go.jp

ABSTRACT

Background: The proteasome inhibitor bortezomib has improved the survival of patients with multiple myeloma but bortezomib-induced peripheral neuropathy (BiPN) has emerged as a serious potential complication of this therapy. Animal studies suggest that bortezomib predominantly causes pathological changes in Schwann cells. A tractable system to evaluate combination drugs for use with bortezomib is essential to enable continuing clinical benefit from this drug.

Methods: Rat schwannoma cells were pretreated with vincristine (VCR), histone deacetylase inhibitors, anticonvulsants, or a heat-shock protein 90 (HSP90) inhibitor. To then monitor aggresome formation as a result of proteasome inhibition and the activation of chaperone-mediated autophagy (CMA), we performed double-labelling immunofluorescent analyses of a cellular aggregation-prone protein marker.

Results: Aggresome formation was interrupted by VCR, whereas combination treatments with bortezomib involving suberoylanilide hydroxamic acid, 17-allylamino-17-demethoxy-geldanamycin, or clonazepam appear to facilitate the disposal of unfolded proteins via CMA, inducing HSP70 and lysosome-associated membrane protein type 2A (LAMP-2A).

Conclusions: This schwannoma model can be used to test BiPN-reducing drugs. The present data suggest that aggresome formation in Schwann cells is a possible mechanism of BiPN, and drugs that induce HSP70 or LAMP-2A have the potential to alleviate this complication. Combination clinical trials are warranted to confirm the relevance of these observations.

Show MeSH
Related in: MedlinePlus