Multiple pathways regulating the anti-apoptotic protein clusterin in breast cancer

Cancer chemotherapy inhibits tumor growth, in part, by triggering apoptosis, and anti-apoptotic proteins reduce the effectiveness of chemotherapy. Clusterin, a chaperone-like protein that binds to apoptotic and DNA repair proteins, is induced by chemotherapy and promotes tumor cell survival. Histone deacetylase inhibitors (HDIs) such as sodium butyrate and suberoylanilide hydroxamic acid (SAHA) are pharmacological agents that induce differentiation and apoptosis in cancer cells by altering chromatin structure, and we have found that combinations of chemotherapeutic drugs such as doxorubicin and HDIs efficiently induce apoptosis, even though they paradoxically induce high levels of clusterin. The hyper-expressed form of clusterin localizes to mitochondria, inhibits cytochrome c release, and is inhibited by the proteasome. When HDIs are used as single agents, clusterin suppresses cytochrome c release and apoptosis. However, doxorubicin/HDI-induced apoptosis is not inhibited by clusterin, and clusterin-resistant apoptosis corresponds with markers of the extrinsic/receptor-mediated apoptotic pathway. Thus, chemotherapy-HDI combinations are capable of overcoming an innate anti-apoptotic pathway of tumor cells, suggesting that chemotherapy-HDI combinations have potential for treating advanced stage breast cancer.     

A novel anti-proliferative property of clusterin in prostate cancer cells

Clusterin is a ubiquitous secretory glycoprotein that is known to suppress certain forms of apoptosis. Since apoptosis and proliferation are two opposing cellular events, it remains unclear if clusterin has any effect on cellular proliferation. The objective of the present study was to examine the effects of clusterin on proliferation in a prostate cancer cell line, LNCaP. We found that clusterin inhibited EGF-mediated proliferation in these cells, as measured by (3)H-thymidine incorporation and by cell counting. Clusterin did not bind with EGF nor did it block phosphorylation of the EGF receptor. Treatment of LNCaP cells with EGF resulted in a transient increase in the expression of both c-Fos and c-Jun. Addition of clusterin to these cultures significantly down-regulated the protein level of c-Fos, but not c-Jun. These results demonstrated a novel biological role for clusterin. Clusterin is not only anti-apoptotic but also anti-proliferative. The anti-proliferative event maybe associated with a down-regulation of c-Fos.     

Interaction of caveolin-1 with Ku70 inhibits Bax-mediated apoptosis

Caveolin-1, the structural protein component of caveolae, acts as a scaffolding protein that functionally regulates signaling molecules. We show that knockdown of caveolin-1 protein expression enhances chemotherapeutic drug-induced apoptosis and inhibits long-term survival of colon cancer cells. In vitro studies demonstrate that caveolin-1 is a novel Ku70-binding protein, as shown by the binding of the scaffolding domain of caveolin-1 (amino acids 82-101) to the caveolin-binding domain (CBD) of Ku70 (amino acids 471-478). Cell culture data show that caveolin-1 binds Ku70 after treatment with chemotherapeutic drugs. Mechanistically, we found that binding of caveolin-1 to Ku70 inhibits the chemotherapeutic drug-induced release of Bax from Ku70, activation of Bax, translocation of Bax to mitochondria and apoptosis. Potentiation of apoptosis by knockdown of caveolin-1 protein expression is greatly reduced in the absence of Bax expression. Finally, we found that overexpression of wild type Ku70, but not a mutant form of Ku70 that cannot bind to caveolin-1 (Ku70 Φ→A), limits the chemotherapeutic drug-induced Ku70/Bax dissociation and apoptosis. Thus, caveolin-1 acts as an anti-apoptotic protein in colon cancer cells by binding to Ku70 and inhibiting Bax-dependent cell death.     

Clusterin overexpression is responsible for the anti-apoptosis effect in a mouse neuroblastoma cell line,B103

The functional role of clusterin in apoptosis was examined using flow cytometry. Clusterin cDNA was transfected into the mouse neuroblastoma cell line, B103, in order to determine if clusterin overexpression inhibits apoptosis. The increased clusterin expression level in the B103 cells tended to suppress the apoptotic index. This suggests an association of clusterin gene expression with apoptosis inhibition. These results support the conclusion that clusterin expression in B103 cells has an anti-apoptotic influence.     

Shikonin selectively induces apoptosis in human prostate cancer cells through the endoplasmic reticulum stress and mitochondrial apoptotic pathway

Background

Despite the recent progress in screening and therapy, a majority of prostate cancer cases eventually attain hormone refractory and chemo-resistant attributes. Conventional chemotherapeutic strategies are effective at very high doses for only palliative management of these prostate cancers. Therefore chemo-sensitization of prostate cancer cells could be a promising strategy for increasing efficacy of the conventional chemotherapeutic agents in prostate cancer patients. Recent studies have indicated that the chemo-preventive natural agents restore the pro-apoptotic protein expression and induce endoplasmic reticulum stress (ER stress) leading to the inhibition of cellular proliferation and activation of the mitochondrial apoptosis in prostate cancer cells. Therefore reprogramming ER stress-mitochondrial dependent apoptosis could be a potential approach for management of hormone refractory chemoresistant prostate cancers. We aimed to study the effects of the natural naphthoquinone Shikonin in human prostate cancer cells.

Results

The results indicated that Shikonin induces apoptosis in prostate cancer cells through the dual induction of the endoplasmic reticulum stress and mitochondrial dysfunction. Shikonin induced ROS generation and activated ER stress and calpain activity. Moreover, addition of antioxidants attenuated these effects. Shikonin also induced the mitochondrial apoptotic pathway mediated through the enhanced expression of the pro-apoptotic Bax and inhibition of Bcl-2, disruption of the mitochondrial membrane potential (MMP) followed by the activation of caspase-9, caspase-3, and PARP cleavage.

Conclusion

The results suggest that shikonin could be useful in the therapeutic management of hormone refractory prostate cancers due to its modulation of the pro-apoptotic ER stress and mitochondrial apoptotic pathways.

Electronic supplementary material

The online version of this article (doi:10.1186/s12929-015-0127-1) contains supplementary material, which is available to authorized users.     

Differential regulation of the clusterin gene by Ha-ras and c-myc oncogenes and during apoptosis

Clusterin (ApoJ) is an extracellular glycoprotein expressed during processes of tissue differentiation and regression that involve programmed cell death (apoptosis). Increased clusterin expression has also been found in tumors, however, the mechanism underlying this induction is not known. Apoptotic processes in tumors could be responsible for clusterin gene activation. Alternatively, oncogenic mutations could modulate signal transduction, thereby inducing the gene. We examined the response of the rat clusterin gene to two oncogenes, Ha-ras and c-myc, in transfected Rat1 fibroblasts. While c-myc overexpression did not modify clusterin gene activity, the Ha-ras oncogene produced a seven to tenfold repression of clusterin mRNA; this down-regulation was also observed in the presence of c-myc. Since no induction of the clusterin gene was observed by the two oncogenes, we tested the alternative mechanism involving apoptosis. Growth factor withdrawal induced apoptosis, as shown by DNA degradation and micronuclei formation in the floating cells. Concomittantly we observed a three to tenfold increase in the amount of clusterin mRNA in the adhering cells of Rat1 and the c-myc transformed cell lines, and a weaker induction in the Ha-ras transformed cell line. On the basis of our results, we suggest that clusterin gene induction in the vital cells is produced by signaling molecules that are generated by the apoptotic cells. We conclude that apoptotic processes, not oncogenic mutations, are responsible for increased clusterin expression in tumors.     

Molecular mechanisms of resveratrol (3,4,5-trihydroxy-trans-stilbene) and its interaction with TNF-related apoptosis inducing ligand (TRAIL) in androgen-insensitive prostate cancer cells

Although resveratrol, an active ingredient derived from grapes and red wine, possesses chemopreventive properties against several cancers, the molecular mechanisms by which it inhibits cell growth and induces apoptosis have not been clearly understood. Here, we examined the molecular mechanisms of resveratrol and its interactive effects with TRAIL on apoptosis in prostate cancer PC-3 and DU-145 cells. Resveratrol inhibited cell viability and colony formation, and induced apoptosis in prostate cancer cells. Resveratrol downregulated the expression of Bcl-2, Bcl-X_L and survivin and upregulated the expression of Bax, Bak, PUMA, Noxa, and Bim, and death receptors (TRAIL-R1/DR4 and TRAIL-R2/DR5). Treatment of prostate cancer cells with resveratrol resulted in generation of reactive oxygen species (ROS), translocation of Bax to mitochondria and subsequent drop in mitochondrial membrane potential, release of mitochondrial proteins (cytochrome c, Smac/DIABLO, and AIF) to cytosol, activation of effector caspase-3 and caspase-9, and induction of apoptosis. Resveratrol-induced ROS production, caspase-3 activity and apoptosis were inhibited by N-acetylcysteine. Bax was a major proapoptotic gene mediating the effects of resveratrol as Bax siRNA inhibited resveratrol-induced apoptosis. Resveratrol enhanced the apoptosis-inducing potential of TRAIL, and these effects were inhibited by either dominant negative FADD or caspase-8 siRNA. The combination of resveratrol and TRAIL enhanced the mitochondrial dysfunctions during apoptosis. These properties of resveratrol strongly suggest that it could be used either alone or in combination with TRAIL for the prevention and/or treatment of prostate cancer.     

Protein kinase C-ε protects MCF-7 cells from TNF-mediated cell death by inhibiting Bax translocation

We have previously shown that protein kinase Cε (PKCε) acts as an antiapoptotic protein and protects breast cancer MCF-7 cells from tumor necrosis factor-α (TNF)-mediated apoptosis. In the present study, we have investigated the mechanism by which PKCε inhibits TNF-induced cell death. Overexpression of wild-type PKCε (WT-PKCε) in MCF-7 cells decreased TNF-induced mitochondrial depolarization. Depletion of Bax by small interfering RNA (siRNA) attenuated TNF-induced cell death. Overexpression of PKCε in MCF-7 cells decreased dimerization of Bax and its translocation to the mitochondria. Knockdown of PKCε using siRNA induced Bax dimerization and mitochondrial translocation. PKCε was coimmunoprecipitated with Bax in MCF-7 cells. These results suggest that PKCε mediates its antiapoptotic effect partly by preventing activation and translocation of Bax to the mitochondria.     

Human nuclear clusterin mediates apoptosis by interacting with Bcl-XL through C-terminal coiled coil domain

Clusterin (CLU), a glycoprotein, is involved in apoptosis, producing two alternatively spliced isoforms in various cell types. The pro-apoptotic CLU appears to be a nuclear isoform (nuclear clusterin; nCLU), and the secretory CLU (sCLU) is thought to be anti-apoptotic. The detailed molecular mechanism of nCLU as a pro-apoptotic molecule has not yet been clear. In the current study, overexpressed nCLU induced apoptosis in human kidney cells. Biochemical studies revealed that nCLU sequestered Bcl-XL via a putative BH3 motif in the C-terminal coiled coil (CC2) domain, releasing Bax, and promoted apoptosis accompanied by activation of caspase-3 and cytochrome c release. These results suggest a novel mechanism of apoptosis mediated by nCLU as a pro-apoptotic molecule.     

Salinomycin-induced apoptosis of human prostate cancer cells due to accumulated reactive oxygen species and mitochondrial membrane depolarization

The anticancer activity of salinomycin has evoked excitement due to its recent identification as a selective inhibitor of breast cancer stem cells (CSCs) and its ability to reduce tumor growth and metastasis in vivo. In prostate cancer, similar to other cancer types, CSCs and/or progenitor cancer cells are believed to drive tumor recurrence and tumor growth. Thus salinomycin can potentially interfere with the end-stage progression of hormone-indifferent and chemotherapy-resistant prostate cancer. Androgen-responsive (LNCaP) and androgen-refractive (PC-3, DU-145) human prostate cancer cells showed dose- and time-dependent reduced viability upon salinomycin treatment; non-malignant RWPE-1 prostate cells were relatively less sensitive to drug-induced lethality. Salinomycin triggered apoptosis of PC-3 cells by elevating the intracellular ROS level, which was accompanied by decreased mitochondrial membrane potential, translocation of Bax protein to mitochondria, cytochrome c release to the cytoplasm, activation of the caspase-3 and cleavage of PARP-1, a caspase-3 substrate. Expression of the survival protein Bcl-2 declined. Pretreatment of PC-3 cells with the antioxidant N-acetylcysteine prevented escalation of oxidative stress, dissipation of the membrane polarity of mitochondria and changes in downstream molecular events. These results are the first to link elevated oxidative stress and mitochondrial membrane depolarization to salinomycin-mediated apoptosis of prostate cancer cells.     

Clusterin activates survival through the phosphatidylinositol 3-kinase/Akt pathway

Clusterin is, in its major form, a secreted heterodimeric disulfide-linked glycoprotein (75-80 kDa). It was first linked to cell death in the rat ventral prostate after androgen deprivation. Recent studies have demonstrated that overexpression of clusterin in prostatic cells protects them against tumor necrosis factor-alpha (TNFalpha)-induced apoptosis. However the details of this survival mechanism remain undefined. Here, we investigate how clusterin prevents cells from undergoing TNFalpha-induced apoptosis. We established a double-stable prostatic cell line for inducible clusterin by using the Tet-On gene expression system. We demonstrated that 50% of the cells overexpressing clusterin escaped from TNFalpha- and actinomycin D-induced cell death. Moreover we demonstrated that the incubation of MLL cells with conditioned medium containing the secreted clusterin or the supplementation of purified clusterin in the extracellular medium decreased the TNFalpha-induced apoptosis significantly. This extracellular action implicates megalin, the putative membrane receptor for clusterin to mediate survival. Indeed clusterin overexpression up-regulated the expression of megalin and induced its phosphorylation in a dose-dependent manner. We interestingly showed that clusterin overexpression is associated with the up-regulation of the phosphorylation of Akt. Activated Akt induced the phosphorylation of Bad and caused a decrease of cytochrome c release. These results enable us to pinpoint one mechanism by which secreted clusterin favors survival in androgen-independent prostate cancer cells, implicating its receptor megalin and Akt survival pathway.     

Apoptosis of metastatic prostate cancer cells by a combination of cyclin-dependent kinase and AKT inhibitors

Effective treatments for advanced prostate cancer are much needed. Toward this goal, we show apoptosis and impaired long-term survival of androgen-independent prostate cancer cells (PC3 and PC3 derivatives) co-treated with the cyclin-dependent kinase (CDK) inhibitor roscovitine and an AKT inhibitor (LY294002 or API-2). Apoptosis of PC3 cells by the drug combination required caspase-9 but not caspase-8 activity and thus is mitochondria-dependent. Roscovitine reduced amounts of the caspase inhibitor XIAP, and API-2 increased amounts of the BH3-only protein Bim. PC3 cells apoptosed when co-treated with API-2 and either cdk9 siRNA, dominant-negative cdk9, or the cdk9 inhibitor DRB; they did not apoptose when co-treated with API-2 and XIAP siRNA. Bax accumulated in mitochondria in response to API-2, whereas release of cytochrome c from mitochondria required both API-2 and roscovitine. We suggest that roscovitine elicits events that activate Bax once it translocates to mitochondria and that inactivation of cdk9 signals these events and the down-regulation of XIAP. Collectively, our data show apoptosis of prostate cancer cells by a drug combination and identify Bax activation as a basis of cooperation.     

Swainsonine promotes apoptosis in human oesophageal squamous cell carcinoma cells <Emphasis Type="BoldItalic">in vitro</Emphasis> and <Emphasis Type="BoldItalic">in vivo</Emphasis> through activation of mitochondrial pathway

Swainsonine, a natural indolizidine alkaloid, has been reported to have antitumour effects, and can induce apoptosis in human gastric and lung cancer cells. In the present study, we evaluated the antitumour effects of swainsonine on several oesophageal squamous cell carcinoma cells and investigated relative molecular mechanisms. Swainsonine treatment inhibited the growth of Eca-109, TE-1 and TE-10 cells in a concentration-dependent manner as measured by MTT assay. Morphological observation, DNA laddering detection and flow cytometry analysis demonstrated that swainsonine treatment induced Eca-109 cell apoptosis in vitro. Further results showed that swainsonine treatment up-regulated Bax, down-regulated Bcl-2 expression, triggered Bax translocation to mitochondria, destructed mitochondria integrity and activated mitochondria-mediated apoptotic pathway, followed by the release of cytochrome c, which in turn activated caspase-9 and caspase-3, promoted the cleavage of PARP, resulting in Eca-109 cell apoptosis. Moreover, swainsonine treatment inhibited Bcl-2 expression, promoted Bax translocation, cytochrome c release and caspase-3 activation in xenograft tumour cells, resulting in a significant decrease of tumour volume and tumour weight in the swainsonine-treated xenograft mice groups compared with that in the control group. Taken together, this study demonstrated that swainsonine inhibited Eca-109 cells growth through activation of mitochondria-mediated caspase-dependent pathway.