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Tumor Biology

, 32:1031 | Cite as

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

  • Mohamed Kamel HassanEmail author
  • Hidemichi Watari
  • Lane Christenson
  • Saverio Bettuzzi
  • Noriaki Sakuragi
Research Article

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.

Keywords

Intracellular clusterin Paclitaxel Ovarian cancer Chemoresistance 

Abbreviation

TX

Paclitaxel

i-CLU

Intracellular clusterin

s-CLU

Secretory clusterin

Notes

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.

Conflicts of interest

None.

Supplementary material

13277_2011_207_MOESM1_ESM.doc (195 kb)
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)
13277_2011_207_MOESM2_ESM.doc (268 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)
Supplementary movie

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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Copyright information

© International Society of Oncology and BioMarkers (ISOBM) 2011

Authors and Affiliations

  • Mohamed Kamel Hassan
    • 1
    • 2
    Email author
  • Hidemichi Watari
    • 1
  • Lane Christenson
    • 3
  • Saverio Bettuzzi
    • 4
    • 5
  • Noriaki Sakuragi
    • 1
  1. 1.Department of Gynecology and Obstetrics, Graduate School of MedicineHokkaido UniversitySapporoJapan
  2. 2.Biotechnology Department, School of SciencePort Said UniversityPort SaidEgypt
  3. 3.Department of Molecular and Integrative PhysiologyUniversity of Kansas Medical CenterKansas CityUSA
  4. 4.Department of Medicina SperimentaleUniversity of ParmaParmaItaly
  5. 5.INBB (Istituto Nazionale Biostrutture e Biosistemi)RomeItaly

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