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Intracellular clusterin negatively regulates ovarian chemoresistance: compromised expression sensitizes ovarian cancer cells to paclitaxel

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Tumor Biology

Abstract

Understanding the molecular events that lead to paclitaxel (TX) resistance is necessary to identify effective means to prevent chemoresistance. Previously, results from our lab revealed that secretory clusterin (CLU) form positively mediates TX response in ovarian cancer cells. Thus, we had interest to study the role of another non-secreted form (intracellular clusterin (i-CLU)) in chemo-response. Here, we provide evidences that i-CLU form localizes mainly in the nucleus and differentially expressed in the TX-responsive KF cells, versus TX-resistant, KF-TX, ovarian cancer cells and negatively regulate cellular chemo-response. I-CLU was cloned, by deleting the secretion-leading signaling peptide from full-length CLU cDNA, and transiently over-expressed in OVK-18 cells. Forced expression of truncated i-CLU was mainly detectable in the nuclei and significantly reduced cellular growth, accumulating cells in G1 phase which finally died through apoptosis. Importantly, compromised expression of i-CLU under an inducible promoter was tolerated and did not induce apoptosis but sensitized ovarian cancer cells to TX. We then demonstrated that this sensitization mechanism was cell cycle independent and relied on i-CLU/Ku70 binding probably due to controlling the free amount of Ku70 available for DNA repair in the nucleus. Results from CLU immunehistochemistry in ovarian tumor tissues verified the retardation of nuclear CLU staining in the recurrent tumor even though their primary counterparts showed nuclear CLU staining. Thus, the controversial data on CLU function in chemo-response/resistance may be explained by a shift in the pattern of CLU expression and intracellular localization as well when tumor acquires chemoresistance.

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Abbreviations

TX:

Paclitaxel

i-CLU:

Intracellular clusterin

s-CLU:

Secretory clusterin

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Acknowledgments

We thank Dr. Takahiko Kobayashi, Dr. Masaki Suzuki, and Dr. Shoichi Inoue for their technical advices. This study was supported in part by a grant-in-aid (no. 18390442) from the Ministry of Education, Science, Sports, and Culture of Japan; SB acknowledges AICR UK grant no. 06-711 and FIL 2008 from University of Parma, Italy.

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Authors and Affiliations

  1. Department of Gynecology and Obstetrics, Graduate School of Medicine, Hokkaido University, Nishi Ku, Kita-15, Nishi-7, 060-8638, Sapporo, Japan

    Mohamed Kamel Hassan, Hidemichi Watari & Noriaki Sakuragi

  2. Biotechnology Department, School of Science, Port Said University, Port Said, Egypt

    Mohamed Kamel Hassan

  3. Department of Molecular and Integrative Physiology, University of Kansas Medical Center, Kansas City, KS, USA

    Lane Christenson

  4. Department of Medicina Sperimentale, University of Parma, Parma, Italy

    Saverio Bettuzzi

  5. INBB (Istituto Nazionale Biostrutture e Biosistemi), Rome, Italy

    Saverio Bettuzzi

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  1. Mohamed Kamel Hassan
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  2. Hidemichi Watari
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  3. Lane Christenson
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  4. Saverio Bettuzzi
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  5. Noriaki Sakuragi
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Correspondence to Mohamed Kamel Hassan.

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Fig. 1

This figure is the western blot analysis result showing the effect of the PNGase (deglycosylase) enzyme on CLU. Whole cell lysate was extracted, treated either with PNGase or vehicle, fractionated, and blotted with anti-CLU Ab. The blot shows that, 60 kDa band had been shifted to about 50 kDa while 40 kDa band had shifted to about 35 kDa. This observation means that 60 kDa is glycosylated while the original non-glycosylated protein is about 35 kDa (DOC 195 kb)

Fig. 2

Flowcytometric analysis of KF cells after i-CLU induction. The above figures shows the flow cytometry analysis and conclusion of its results of KF cells 12, 24, and 48 h right after i-CLU induction. Cells were treated by Dox for the induction purpose and were collected at the indicated time. Cellular populations were calculated and histogram was developed. Although there were no prominent cell deaths until 2 days after treatments, there was some increase in the G1 population and some reduction in S and G2/M phase populations (DOC 268 kb)

The movie shows OVK-18 cells over-expressing i-CLU under inducible promoter in the presence of Dox. Cells were monitored for 48 h starting 36 h after the addition of Dox. The movie shows the reduction of cellular viability until anoikis-like cell death (WMV 6,404 kb)

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Hassan, M.K., Watari, H., Christenson, L. et al. Intracellular clusterin negatively regulates ovarian chemoresistance: compromised expression sensitizes ovarian cancer cells to paclitaxel. Tumor Biol. 32, 1031–1047 (2011). https://doi.org/10.1007/s13277-011-0207-0

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