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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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Bortezomib induces aggresome formation at the microtubule-organising centres (MTOCs) of Schwann cells. (A) In untreated RT4-D6P2T cells, γ-tubulin is distributed homogeneously throughout the cytoplasm (left panel). In bortezomib (Bzb)-treated cells, aggresomes form as distinct pericentriolar structures (arrows) with weak staining in the cytoplasm (right panel). Insets in the right panel show the juxtanuclear rounded structures evident at higher magnification. (B) Untreated RT4-D6P2T cells contain dynein (red), which is distributed homogeneously in the cytoplasm with predominant localisation in the perinuclear region, and vimentin (green), which is distributed diffusely throughout the cytoplasm and above the nuclei. (C) In bortezomib-treated cells, dynein (red) and vimentin (green) appear as rounded structures at the MTOC (arrows) and are colocalised in the region adjacent to the nuclei (yellow signals in the merged image of both fluorochrome channels. Bar, 20 μ. −Bzb, untreated; +Bzb, bortezomib treated.
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fig1: Bortezomib induces aggresome formation at the microtubule-organising centres (MTOCs) of Schwann cells. (A) In untreated RT4-D6P2T cells, γ-tubulin is distributed homogeneously throughout the cytoplasm (left panel). In bortezomib (Bzb)-treated cells, aggresomes form as distinct pericentriolar structures (arrows) with weak staining in the cytoplasm (right panel). Insets in the right panel show the juxtanuclear rounded structures evident at higher magnification. (B) Untreated RT4-D6P2T cells contain dynein (red), which is distributed homogeneously in the cytoplasm with predominant localisation in the perinuclear region, and vimentin (green), which is distributed diffusely throughout the cytoplasm and above the nuclei. (C) In bortezomib-treated cells, dynein (red) and vimentin (green) appear as rounded structures at the MTOC (arrows) and are colocalised in the region adjacent to the nuclei (yellow signals in the merged image of both fluorochrome channels. Bar, 20 μ. −Bzb, untreated; +Bzb, bortezomib treated.

Mentions: A diffuse expression pattern of γ-tubulin, a protein that adheres to the centrosome (Dictenberg et al, 1998), was observed in the cytoplasm of RT4-D6P2T cells. Following a 3-h treatment with 40 n bortezomib, however, γ-tubulin staining in the cytoplasm became weak and coalesced to form round structures in the juxtanuclear area (Figure 1A). Similarly, the dynein and vimentin proteins became rounded and colocalised in region adjacent to the nucleus after exposure to bortezomib (Figure 1C).


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)

Bortezomib induces aggresome formation at the microtubule-organising centres (MTOCs) of Schwann cells. (A) In untreated RT4-D6P2T cells, γ-tubulin is distributed homogeneously throughout the cytoplasm (left panel). In bortezomib (Bzb)-treated cells, aggresomes form as distinct pericentriolar structures (arrows) with weak staining in the cytoplasm (right panel). Insets in the right panel show the juxtanuclear rounded structures evident at higher magnification. (B) Untreated RT4-D6P2T cells contain dynein (red), which is distributed homogeneously in the cytoplasm with predominant localisation in the perinuclear region, and vimentin (green), which is distributed diffusely throughout the cytoplasm and above the nuclei. (C) In bortezomib-treated cells, dynein (red) and vimentin (green) appear as rounded structures at the MTOC (arrows) and are colocalised in the region adjacent to the nuclei (yellow signals in the merged image of both fluorochrome channels. Bar, 20 μ. −Bzb, untreated; +Bzb, bortezomib treated.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2990589&req=5

fig1: Bortezomib induces aggresome formation at the microtubule-organising centres (MTOCs) of Schwann cells. (A) In untreated RT4-D6P2T cells, γ-tubulin is distributed homogeneously throughout the cytoplasm (left panel). In bortezomib (Bzb)-treated cells, aggresomes form as distinct pericentriolar structures (arrows) with weak staining in the cytoplasm (right panel). Insets in the right panel show the juxtanuclear rounded structures evident at higher magnification. (B) Untreated RT4-D6P2T cells contain dynein (red), which is distributed homogeneously in the cytoplasm with predominant localisation in the perinuclear region, and vimentin (green), which is distributed diffusely throughout the cytoplasm and above the nuclei. (C) In bortezomib-treated cells, dynein (red) and vimentin (green) appear as rounded structures at the MTOC (arrows) and are colocalised in the region adjacent to the nuclei (yellow signals in the merged image of both fluorochrome channels. Bar, 20 μ. −Bzb, untreated; +Bzb, bortezomib treated.
Mentions: A diffuse expression pattern of γ-tubulin, a protein that adheres to the centrosome (Dictenberg et al, 1998), was observed in the cytoplasm of RT4-D6P2T cells. Following a 3-h treatment with 40 n bortezomib, however, γ-tubulin staining in the cytoplasm became weak and coalesced to form round structures in the juxtanuclear area (Figure 1A). Similarly, the dynein and vimentin proteins became rounded and colocalised in region adjacent to the nucleus after exposure to bortezomib (Figure 1C).

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