Silencing Expression of the Clusterin/Apolipoprotein J Gene in Human Cancer Cells Using Small Interfering RNA Induces Spontaneous Apoptosis, Reduced Growth Ability, and Cell Sensitization to Genotoxic and Oxidative Stress

Abstract

Clusterin/Apolipoprotein J (CLU) is a heterodimeric ubiquitously expressed secreted glycoprotein that is implicated in several physiological processes and is differentially expressed in many severe physiological disturbances, including tumor formation and in vivo cancer progression. Despite extensive efforts, clarification of CLU's biological role has been exceptionally difficult and its precise function remains elusive. Short RNA duplexes, referred to as small interfering RNAs (siRNAs), provide a new approach for the elucidation of gene function in human cells. Here, we describe siRNA-mediated CLU gene silencing in osteosarcoma and prostate human cancer cells and illustrate that CLU mRNA is amenable to siRNA-mediated degradation. Our data demonstrate that CLU knockdown in human cancer cells induces significant reduction of cellular growth and higher rates of spontaneous endogenous apoptosis. Moreover, CLU knockdown cancer cells were significantly sensitized to both genotoxic and oxidative stress induced by chemotherapeutic drugs and H 2 O 2 , respectively. These effects were more pronounced in cell lines that express high endogenous steady-state levels of the CLU protein and occur through hyperactivation of the cellular apoptotic machinery. Overall, our results reveal that, in the distinct cellular contexts of the osteosarcoma and prostate cancer cells assayed, CLU is a central molecule in cell homeostasis that exerts a cytoprotective function. The described CLU-specific siRNA oligonucleotides that can potently silence CLU gene expression may thus prove valuable agents during antitumor therapy or at other pathological conditions where CLU has been implicated.

Challenge and promise: roles for clusterin in pathogenesis, progression and therapy of cancer

“…A number of studies 1,5,13,16,17 have demonstrated that overexpression of the full-length CLU mRNA that would produce, depending on where the translation would start from and in this case, nCLU, acts as a prodeath signal, inhibiting cell growth and survival. However, other studies 2,6,8,10,[17][18][19] have shown that CLU expression may exert cytoprotective properties. We favour the recent proposal that tumour cell survival is connected with overexpression of sCLU and loss of nCLU.…”

“…2,4 These early apparently ambiguous functions appear to be attributed to the existence of two different but related CLU protein isoforms, a glycosylated as well as an apparently nonglycosylated form that are produced in the cell by alternative splicing. These two forms of the CLU protein can be immunologically distinguished.…”

“…13 sCLU expression is initiated by translation from the full-length CLU mRNA, wherein the first AUG codon is translated into the 49 kDa sCLU precursor protein ( Figure 1a). 2,13 This protein is directed to the endoplasmic reticulum by a leader signalling sequence. As the protein is transported towards the Golgi, this form is cleaved at the a and b site and heavily glycosylated.…”

“…[1][2][3][4] It is involved in numerous physiological processes important for carcinogenesis and tumour growth, including apoptotic cell death, cell cycle regulation, DNA repair, cell adhesion, tissue remodelling, lipid transportation, membrane recycling, and immune system regulation. [1][2][3] Increased CLU mRNA and protein levels have been consistently detected in various tissues undergoing stress, including heart, brain, liver, kidney, breast, and retinal tissues both in vivo and in vitro. Early studies seemed to establish that CLU gene expression was a marker of apoptotic cell loss.…”

Apolipoprotein C1 promotes prostate cancer cell proliferation in vitro

“…Additionally, other apolipoproteins, such as apolipoprotein J/clusterin (CLU) and ApoE, may also be involved in the progression of human tumors. Trougakos et al 30. showed that silencing of CLU suppressed cell growth and enhanced spontaneous apoptosis in osteosarcoma and PCa cells.…”