In LNCaP cells enhanced expression of the androgen receptor compensates for Bcl-2 suppression by antisense oligonucleotides (original) (raw)
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Medical oncology (Northwood, London, England), 2013
Antisense oligonucleotides (oligos) have been employed against prostate cancer models in both in vivo and in vitro systems. While most target growth factors or their receptors, other oligos are directed against inhibitors of apoptosis or mediators of androgen action. Those which suppress bcl-2 activity (in prostate cancer patients) have even reached clinical trials. We evaluated a set of oligos which targeted and comparably suppressed the expression of bcl-2, an apoptosis inhibitory protein. Our first study reported that LNCaP cells were adapted by suppression of caspase-3 (a promoter of apoptosis). In this study we evaluated additional proteins associated with tumor progression and found the expression of the androgen receptor, its p300 and IL-6 co-activators, as well as v-myc (oncogenic) and (unexpectedly) tumor suppressor p53 genes to be enhanced. We conclude that oligo treatment directed against bcl-2, intended to stimulate apoptosis, can be evaded through compensatory changes i...
Therapeutic advances in urology, 2011
Antisense oligonucleotides (oligos) have been employed against in vivo and in vitro prostate cancer models targeting growth stimulatory gene products. While most oligos have targeted growth factors or their receptors, others have been directed against inhibitors of apoptosis and mediators of androgen action. In LNCaP cells we evaluated a set of oligos which targeted and comparably suppressed the expression of the apoptosis inhibitor protein bcl-2. LNCaP cells adapted to this restoration of apoptosis with an enhanced expression of the androgen receptor (AR) suggesting an increased sensitivity to androgens. In a continuation of this study, we now evaluate the expression of p300, an AR coactivating protein expressed in the later stages of prostate cancer. In previous experiments, monospecific and bispecific oligos directed against bcl-2 suppressed both the targeted bcl-2 protein (an inhibitor of apoptosis) and the nontargeted caspase-3 (a promoter of apoptosis), potentially negating th...
Antisense oligonucleotides (oligos) have been evaluated for treating prostate cancer in both in vivo and in vitro models. Although most oligos contain a single mRNA binding site, our laboratory evaluates bi-specific oligos directed towards two proteins. This study evaluates the growth inhibition in vitro of the LNCaP cell line employing mono-and bispecific oligos directed against BCL-2 [the second binding site was directed against the epidermal growth factor receptor (EGFR)]. These oligos were administered with lipofectin as part of a nanoparticle delivery system. Additionally, g, the expression of five apoptosis regulatory proteins, BCL-2, bax, caspase-3, clusterin and AKT-1 was evaluated by RT-PCR.
In vivo (Athens, Greece)
Antisense oligonucleotides (oligos) have been employed against prostate cancer in both in vivo and in vivo models. While most oligos contain a single mRNA binding site, our laboratory has developed bispecifics directed towards two. Previous work has determined that when oligos are used to suppress the expression of individual proteins in highly regulated physiologic processes, additional proteins can be affected. These non-targeted (non-specific effects) regulators can compensate for proteins specifically targeted, reverse the intended effect and promote tumor growth. To evaluate specific and compensatory non-specific effects on growth inhibition of LNCaP cells employing mono- and bispecific oligos directed against BCL-2, LNCaP cells were incubated in the presence of oligos specifically directed against BCL-2 [the second binding site was directed against epidermal growth factor receptor (EGFR)] and compared to lipofectin containing controls. Significant, but comparable, growth inhib...
Cancer Science & Research, 2019
Almost 25 years ago we first employed antisense oligonucleotides (oligos) against human derived prostatic LNCaP cells using both in vitro [1] and in vivo [2] models; initially targeting the epidermal growth factor receptor, and its ligand, transforming growth factor-alpha. Similar studies and results were found when we treated in vitro and in vivo breast cancer models [3]. Clinically, oligos have been employed to treat cancer patients (including those with prostate tumors), targeting apoptosis inhibitors (bcl-2, clusterin) in attempts to restore tumor chemo-[4] or radio-sensitivity [5]. Yet, in spite of these patient trials and additional advances in early detection of prostate cancer, its treatment has not greatly improved in recent years. In the last few years gene therapy and newly discovered immune checkpoint blockade has given some indication that they could provide some additional improvement, particularly when administered following surgery, chemotherapy or irradiation.
Open Journal of Apoptosis, 2015
Previously we have shown that when LNCaP cells are treated with antisense oligonucleotides (oligos) directed against BCL-2, compensatory changes in non-targeted genes take place in attempts to restore apoptosis and promote tumor aggressiveness. In addition to the inhibition of BCL-2, we find that the apoptosis promoter caspase-3 activity is suppressed, the transcription activity of STAT-3 is enhanced, while other regulators (bax, clusterin, AKT-1) associated with mitochondrial regulated apoptosis and caspase cascade are either unchanged or undetectable. We now evaluate proteins associated with the second pathway of apoptosis activation mediated by direct signal transduction involving fas, fas-ligand (a tumor necrosis factor-like cell surface receptor aka CD95), as well as the similar programmed death cell surface receptor (PD-1) and its respective ligand (PD-1L). This study evaluates the growth inhibition of in vitro propagating LNCaP cells employing mono-and bispecific oligos directed against BCL-2 [the second binding site was directed against the epidermal growth factor receptor (EGFR)]; and employing RT-PCR. The expression of these four proteins was evaluated. Expression of fas-ligand, PD-1 and PD-L1 were all significantly enhanced, whereas fas itself was undetectable. This suggests that in addition to pathways associated with the mitochondrial pathway of apoptosis, compensatory changes occur in the direct signal * Corresponding author. M. Rubenstein et al. 2 transduction pathway of this process. In addition to alterations in androgen sensitivity, growth factor expression and oncogene expression, these data suggest that suppressive BCL-2 therapy involves multiple pathways, including those involved with immune targeting and cytotoxicity and must be taken into account to make gene therapy more efficacious.
Enliven: Journal of Genetics, Molecular and Cellular Biology, 2014
Antisense oligonucleotides (oligos) have been evaluated for treating prostate cancer in both in vivo and in vitro models. Although most oligos contain a single mRNA binding site, our laboratory evaluates bi-specific oligos directed towards two proteins. This study evaluates the growth inhibition in vitro of the LNCaP cell line employing mono-and bispecific oligos directed against BCL-2 [the second binding site was directed against the epidermal growth factor receptor (EGFR)]. These oligos were administered with lipofectin as part of a nanoparticle delivery system. Additionally, g, the expression of five apoptosis regulatory proteins, BCL-2, bax, caspase-3, clusterin and AKT-1 was evaluated by RT-PCR. LNCaP prostate tumor cells were incubated in the presence of oligos specifically directed against BCL-2 and their effect was compared to lipofectin containing controls. Significant, but comparable, growth inhibition was produced by both mono-and bi-specific forms. Employing RT-PCR to determine BCL-2 expression, we found that the greatest amount of mRNA suppression approached 100% for each type of oligo with mono-specific MR 4 (directed only against BCL-2) equal 100%; and bispecifics MR 24 and MR 42 , being 86% and 100% respectively. Based upon both inhibition of cell growth and BCL-2 expression, bi-specific antisense oligos directed against EGFR and BCL-2 mRNAs are at least as effective as a mono-specific directed solely towards BCL-2. In an effort to determine a compensatory response by cells needed to evade apoptosis in the presence of BCL-2 suppression, the levels of mRNA encoding non-targeted bax, caspase-3, clusterin and AKT-1 were evaluated. Suppression of the apoptosis inhibitor (BCL-2) in LNCaP cells did not alter either bax or clusterin expression. However, the expression of non-targeted caspase-3 (an apoptosis promoter) was suppressed and the expression of non-targeted AKT-1 (an apoptosis inhibitor) was enhanced. This suggests that tumor variants can resist apoptosis through the altered expression of non-targeted regulators of apoptosis. If BCL-2 suppression was to be clinically targeted by antisense oligos, similar experiments may be helpful in identifying genes with a similar function. Therefore, our laboratory evaluates bi-specific oligos which target two distinct proteins (which are able to regulate different pathways and do not share sequence homology). These bi-specifics differ from those targeting genes which share sequence homology (BCL-2 and BCL-xL) [1] or the OGX-225 oligo which targets (three) structurally related insulin like growth factor binding proteins [2]. We have demonstrated that the addition of a second binding site does not affect the activity of the first and that dual binding sites can simultaneously be directed against genes involved in either a single growth promoting autocrine loop [3] or towards those of even different regulatory pathways [4-7]. While most assessments of activity quantitate the inhibition of in vitro cell growth, more specific methods use the polymerase chain reaction (PCR) to measure specific protein encoding mRNA. Previously we have shown that in LNCaP cells a single mono-and two bispecific oligos directed towards BCL-2 increase the expression
The Open Access Journal of Science and Technology, 2013
Antisense oligonucleotides (oligos) have been employed against in vivo and in vitro prostate cancer models. While most oligos target growth factors or their receptors, others are directed against inhibitors of apoptosis or mediators of androgen activity. In previous experiments, mono-and bispecific oligos directed against bcl-2 suppressed both the targeted bcl-2 protein (an inhibitor of apoptosis) and non-targeted caspase-3 (a promoter of apoptosis), potentially negating the effect of therapeutic bcl-2 inhibition. Subsequently we reported that AR and p300 expression were significantly enhanced by these oligos. In a continuation of this study, we now report that the expression of another androgen receptor co-stimulatory protein, CREB binding protein (CREBBP), is not similarly increased. These data suggest that oligo treatment directed against bcl-2 can be evaded through compensatory increases in AR and p300 expression. Increased AR and p300 expression may transition the tumor to a more dedifferentiated and aggressive phenotype. However, not all co-stimulating proteins (CREBBP) are involved, and this may be important when controlling unanticipated (compensatory) effects of gene therapy.
Molecular Cancer Therapeutics, 2005
Bcl-2 and Bcl-xL are associated with treatment resistance and progression in many cancers, including prostate cancer. The objective of this study was to determine whether a novel bispecific antisense oligonucleotide targeting both Bcl-2 and Bcl-xL induces apoptosis and enhances chemosensitivity in androgen-independent PC3 prostate cancer cells. An antisense oligonucleotide with complete sequence identity to Bcl-2 and three-base mismatches to Bcl-xL selected from five antisense oligonucleotides targeting various regions with high homology between Bcl-2 and Bcl-xL was found to be the most potent inhibitor of both Bcl-2 and Bcl-xL expression in PC3 cells. This selected Bcl-2/Bcl-xL bispecific antisense oligonucleotide reduced mRNA and protein levels in a dose-dependent manner, reducing Bcl-2 and Bcl-xL protein levels to 12% and 19%, respectively. Interestingly, Mcl-1 was down-regulated as well, although levels of Bax, Bad, or Bak were not altered after treatment with this bispecific an...