Induction of apoptosis and enhancement of chemosensitivity in human prostate cancer LNCaP cells using bispecific antisense oligonucleotide targeting Bcl‐2 and Bcl‐xL genes
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OBJECTIVE To determine whether a specifically designed bispecific ( Bcl‐2/Bcl‐xL ) antisense oligonucleotide (ASO) induces apoptosis and enhances chemosensitivity in human prostate cancer LNCaP cells, as Bcl‐2 and Bcl‐xL are both anti‐apoptotic genes associated with treatment resistance and tumour progression in many malignancies, including prostate cancer. MATERIALS AND METHODS Inhibition of Bcl‐2 and Bcl‐xL expression by the bispecific ASO was evaluated using real‐time reverse transcription‐polymerase chain reaction and Western blotting, while growth inhibition and induction of apoptosis were analysed by a crystal violet assay, flow cytometry and Western blotting of apoptosis‐relevant proteins. The effect of combined treatment with bispecific ASO and chemotherapy or small‐interference RNA (siRNA) targeting the clusterin gene was also investigated. RESULTS Bispecific ASO reduced Bcl‐2 and Bcl‐xL expression in LNCaP cells in a dose‐dependent manner. There was cell growth inhibition, increases in the sub‐G0–G1 fraction, and cleavage of caspase‐3 and poly(ADP‐Ribose) polymerase proteins in LNCaP cells after bispecific ASO treatment. Interestingly, Bcl‐2 / Bcl‐xL bispecific ASO treatment also resulted in the down‐regulation of Mcl‐1 and up‐regulation of Bax. The sensitivity of LNCaP cells to mitoxantrone, docetaxel or paclitaxel was significantly increased, reducing the 50% inhibitory concentration by 45%, 80% or 90%, respectively. Furthermore, the apoptotic induction by Bcl‐2 / Bcl‐xL bispecific ASO was synergistically enhanced by siRNA‐mediated inhibition of clusterin, a cytoprotective chaperone that interacts with and inhibits activated Bax. CONCLUSIONS These findings support the concept of the targeted suppression of Bcl‐2 anti‐apoptotic family members using multitarget inhibition strategies for prostate cancer, through the effective induction of apoptosis.Keywords:
Clusterin
Bcl-xL
Mitoxantrone
Variants in the clusterin gene have been associated with Alzheimer's disease (AD) through replicated genome-wide studies, but the underlying mechanisms remain unknown. In this study the association of the AD clusterin common risk polymorphism rs93318
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Abstract: Expression of the glycoprotein clusterin is markedly increased following tissue injury. One function of clusterin is to promote cell interactions which are perturbed in these pathologic settings. Clusterin causes cell aggregation and adhesion in vitro yet the molecular mechanism for this effect is not known. In order to identify the active site(s) of clusterin, 34 peptides, each 15 amino acid residues in length, were synthesized from hydrophilic regions of human clusterin. When studied individually, none of the peptides caused aggregation of LLC‐PK1 cells, a porcine renal epithelial cell line. However, two out of the 34 peptides inhibited clusterin‐induced cell aggregation in a dose‐dependent manner. Scrambled versions of these two ‘active’ peptides did not inhibit cell aggregation. Seven peptides promoted cell adhesion. In conclusion, these findings provide evidence for novel amino acid sequences mediating clusterin‐induced renal cell interactions.
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clusterin은 모든 포유류의 체액에 존재하는 이종 이량성 당단백질이며, 지금까지 연구된 거의 모든 종류의 세포에서 유도 및 발현된다. 또한 다기능성 단백질로 면역계, 남성생식계, 중추신경계에 관여하며, 보체, 지방수송, 분비 등의 기능에도 관여하는 것으로 알려져 있다. 특히 clusterin이 구강 내에 유리되면 구강 내 세균을 응집시켜 여러 가지 구강병소 발현에 영향을 미칠 수 있다. 이에 스트레스, 임신, 당뇨, 간염과 같은 전신질환자의 타액 내 clusterin의 발현유무를 확인하여, clusterin의 역할을 구명함으로써, 전신질환과 구강질환 간의 상호관계를 보다 과학적으로 확인하는데 기초를 마련하고자 본 실험을 시행하였다. 먼저 타액에서 clusterin의 발현유무를 확인하기 위하여, 스트레스, 임신, 당뇨, 간염과 같은 전신증상을 가지고 있는 실험군과 정상군을 설정한 후 각각의 전 타액 및 이하선의 순수 타액을 채취하고, clusterin의 양성 표식자로사용하기 위하여 정상인의 혈청을 채취하였다. 스트레스군은 발치 직전의 환자와 평소 스트레스를 호소하는 본과에 내원 환자를 선택하였다. 채취한 표본을 가열군과 비가열군으로 분류한 후 원심 분리하여 상층액 만을 분리한 후 각각의 표본에 western blot분석법을 시행하여 전신 질환과 관련된 타액 내 clusterin의 발현 유무를 관찰하였다. 1.타액과 혈청의 경우 비가열처리 군에서 단백질의 끌림 현상이 관찰되었으나,가열처리 군에서는 선명하게 관찰 되었다. 2. 가열처리한 사람의 전 타액에서 전신질환자군 및 정상군 중 스트레스군 일부와 임신군에서 clusterin이 관찰되었다. 3. 가열처리한 사람의 이하선 순수 타액에서는 전신질환자군 및 정상군 모두에서 clusterin이 발현되지 않았다. 사람의 타액 실험 중 스트레스, 임신, 당뇨, 간염과 같은 전신증상을 가지고 있는 실험군의 전 타액을 이용하여 clusterin의 변화를 관찰한 결과, 가열처리를 함에 따라 단백질의 끌림 현상이 해결되었던 것으로 보아 타액 중의 clusterin을 관찰하기 위해서는 가열처리가 필수적이라고 생각된다. 또한 통법에 의한 western blot을 통하여 실시하였을 때 clusterin이 발현되지 않았던 것은 전신질환과 관련된 타액 내의 clusterin의 분비가 매우 미약하거나 또는 다른 변수에 의하여 변성되는 것이 아닌가 생각하였다. 그러나 침전과정을 통하여 단백질의 양을 크게 늘려 관찰하였을 때, 일부 전 타액에서 clusterin의 발현을 관찰할 수 있었던 것으로 보아 전신상태의 변화에 따라 소량이나마 존재한다는 것을 확인하였고, 향후 구강 내로 분비된 clusterin이 구강 병소나 구강 내 미생물에 어떠한 영향을 미치는지의 연구가 수행되어야 할 것으로 생각된다.
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Advanced prostate cancer is treated with androgen deprivation, but most patients eventually progress and need new therapy. Recent genomic/exomic sequencing identified SPOP as the most frequently mutated gene in 6% - 15% of prostate cancer. Based on the function of SPOP as a ubiquitin ligase in protein degradation, it was hypothesized that loss-of-function mutations of SPOP led to accumulation of SPOP substrates that enhance androgen receptor activity and facilitate prostate cancer formation. SPOP substrates could thus be potential targets for treatment of androgen-sensitive prostate cancer. PubMed and BLAST search identified that Gli, SRC-3, and AWP1 are SPOP substrates, and that inhibition of PRK-1, a binding partner of AWP1, by lestaurtinib suppressed androgen receptor activity. LNCaP, PC3, DU145 and 22RV1 prostate cancer cells were used to evaluate the effect of lestaurtinib. LNCaP cells, an androgen-sensitive prostate cancer cell line, were the most sensitive. SRC-3 protein decreased when LNCaP cells were treated with lestaurtinib; whereas PRK-1 increased in nucleus after lestaurtinib treatment. These data suggest that lestaurtinib modulates SRC-3 and PRK-1 to induced cell death in androgen-sensitive prostate cancer, and could be a useful agent for future development for prostate cancer with SPOP mutations.
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Eur J Clin Invest 2010; 40 (10): 893–902 Abstract Background Clusterin (Apolipoprotein J), a plasma protein with cytoprotective and complement-inhibiting activities, localizes in the infarcted heart during myocardial infarction (MI). Recently, we have shown a protective effect of exogenous clusterin in vitro on ischaemically challenged cardiomyocytes independent of complement. We therefore hypothesized that intravenous clusterin administration would reduce myocardial infarction damage. Methods Wistar rats undergoing experimental MI, induced by 40 min ligation of a coronary vessel, were treated with either clusterin (n = 15) or vehicle (n = 13) intravenously, for 3 days post-MI. After 4 weeks, hearts were analysed. The putative role of megalin, a clusterin receptor, was also studied. Results Administration of human clusterin significantly reduced both infarct size (with 75 ± 5%) and death of animals (23% vehicle group vs. 0% clusterin group). Importantly, histochemical analysis showed no signs of impaired wound healing in the clusterin group. In addition, significantly increased numbers of macrophages were found in the clusterin group. We also found that the clusterin receptor megalin was present on cardiomyocytes in vitro which, however, was not influenced by ischaemia. Human clusterin co-localized with this receptor in vitro, but not in the human heart. In addition, using a megalin inhibitor, we found that clusterin did not exert its protective effect on cardiomyocytes through megalin. Conclusions Our results thus show that clusterin has a protective effect on cardiomyocytes after acute myocardial infarction in vivo, independent of its receptor megalin. This indicates that clusterin, or a clusterin derivate, is a potential therapeutic agent in the treatment of MI.
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Objective Cloning of some specific genes related to the prostate cancer. Methods Specimen from a patient with prostate cancer and from a normal adult were studied by the improved mRNA differential display and the differentially expressed sequence-tags were cloned,sequenced and analized.Reverse transcriptive-polymerase chain reaction(RT-PCR) was used to examine the expression level of Clusterin in prostate cancer tissues,prostate cancer cell line and normal prostate tissues. Results Significant difference was observed between the two kinds of tissues in gene expression and seven differentially expressed sequence-tags were found through analysis in GenBank.Among them,five were proved to be new gene tags,one shared 100% homology with Clusterin,which was highly expressed in the prostate cancer tissue and the other one shared 97% homology with superoxide dismutase 1 (SOD1),which was highly expressed in the normal prostate tissue.It was proved by RT-PCR that to compare with the internal marker gene β-actin,the expression level of clusterin in prostate cancer is much higher than that of normal prostate. Conclusions Five new gene tags,clusterin and SOD1 were found to be differentially expressed between tissues of prostate cancer and normal prostate and may play important roles in carcinogenesis and development of prostate cancer.
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Androgen-sensitive prostate cancer cells turn androgen resistant through complex mechanisms that involve dysregulation of apoptosis. We investigated the role of antiapoptotic Bcl-xL in the progression of prostate cancer as well as the interactions of Bcl-xL with proapoptotic Bax and Bak in androgen-dependent and -independent prostate cancer cells. Immunohistochemical analysis was used to study the expression of Bcl-xL in a series of 139 prostate carcinomas and its association with Gleason grade and time to hormone resistance. Expression of Bcl-xL was more abundant in prostate carcinomas of higher Gleason grades and significantly associated with the onset of hormone-refractory disease. In vivo interactions of Bcl-xL with Bax or Bak in untreated and camptothecin-treated LNCaP and PC3 cells were investigated by means of coimmunoprecipitation. In the absence of any stimuli, Bcl-xL interacts with Bax and Bak in androgen-independent PC3 cells but only with Bak in androgen-dependent LNCaP cells. Interactions of Bcl-xL with Bax and Bak were also evidenced in lysates from high-grade prostate cancer tissues. In LNCaP cells treated with camptothecin, an inhibitor of topoisomerase I, the interaction between Bcl-xL and Bak was absent after 36 h, Bcl-xL decreased gradually and Bak increased coincidentally with the progress of apoptosis. These results support a model in which Bcl-xL would exert an inhibitory effect over Bak via heterodimerization. We propose that these interactions may provide mechanisms for suppressing the activity of proapoptotic Bax and Bak in prostate cancer cells and that Bcl-xL expression contributes to androgen resistance and progression of prostate cancer.
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