Mixed-backbone Oligonucleotide Research Articles

Abstract 3441: Silencing glucosylceramide synthase using MBO-asGCS eliminates tumor metastasis

Abstract Disseminated metastasis is often observed in cancer patients fail in chemotherapy. Tumor metastasis is known to rely on the interaction of cancer cells and the extracellular matrix components but the molecular mechanism by which drug resistance promotes tumor metastasis is still not clear. Glucosylceramide synthase (GCS) that is a limiting enzyme for ceramide glycosylation and regulates glycosphingolipid (GSL) synthesis can cause drug resistance. Hence we report that GCS is a determinant of metastatic properties of drug-resistant cancer cells, and silencing GCS eliminates tumor metastasis. Our recent study found that the metastatic potency of drug-resistant cancer cells depended on the overexpression of GCS. Compared with drug-sensitive MCF-7 cells, the wound healing, matrigel attachment and MMP-9 activity significantly was 1.5 fold faster, 3 fold more and 2 folds higher respectively in the drug resistant MCF-7/Dox that also had 39-fold higher GCS protein level and 2-fold higher GCS enzyme activity. Introduction of GCS gene enhanced the metastatic potency of MCF-7 cells, because its matrigel attachment and invasion potential significantly increased by 2-fold and 1.5-fold after transfection. In contrast, silencing GCS by using mixed-backbone oligonucleotide against GCS (MBO-asGCS, 100 nM) substantially diminished the metastatic potency of MCF-7/Dox cells, as the wound healing, matrigel attachment and invasion reduced by 2-fold, 1.5-fold and 3-fold respectively. MBO-asGCS treatments eliminated lung metastasis of MCF-7/Dox tumors. Further mechanistic studies disclosed that silencing GCS by MBO-asGCS decreased its expression and enzyme activity and also decreased the levels of globo-series glycosphingolipids (GSLs) in GSL-enriched microdomain located on the plasma membrane. Sequentially, silencing GCS decreased the expression levels of uPA, MMP-9, VEGF and FGF-2 that actively mediated the migration and invasion of cancer cells. This study thus indicates that GCS is determinant for the metastatic potency of drug-resistant cancer cells and silencing GCS by using MBO-asGCS is an effective approach to target the metastasis of tumor with drug resistance. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3441. doi:1538-7445.AM2012-3441

Abstract 1470: Depletion of glycosphingolipids with MBO-asGCS eliminates cancer cell metastasis

Abstract Tumor metastasis, particularly multi-organ metastasis often causes failure of cancer treatment. It is well-known that the interaction of tumor cells with extracellular matrix (ECM) determines the metastatic ability of cancer cells; however, it is not clear whether depletion of glycosphingolipids (GSLs), a major component of GSL-enriched microdomain on plasma membrane is an approach to eliminate tumor metastasis. Glucosylceramide synthase (GCS) that converts ceramide to glucosylceramide is a limiting enzyme controlling GSL synthesis. We found that overexpression of GCS enhanced metastatic ability of human breast cancer cells. In MCF-7/Dox cells, GCS protein levels were increased 39 folds and GCS activity were increased 2 folds, as compared to MCF-7 cells. The wound healing, Matrigel attachment and MMP-9 activity in MCF-7/Dox cells increased 1.5 folds, 3 folds, and 2 folds respectively compared to MCF-7 cells. Introduction of GCS gene overexpressed GCS protein and significantly increased wound healing and Matrigel attachment by 2 folds and cell invasion 1.5 folds in MCF-7 cells. MBO-asGCS is a mixed backbone oligonucleotide that substantially suppresses GCS expression. Silencing of GCS with MBO-asGCS (100 nM) decreased the wound healing by 2 folds, Matrigel attachment by 1.5 folds and cell invasion by 3 folds in MCF/Dox cells. These results were also observed in MCF-7/DOX cells treated with d-PDMP, a GCS inhibitor. Mechanistic study indicated that overexpression of GCS increased GSLs, particular globo-series GSLs and enhanced VEGF and uPA to promote the mobility, cellular attachment and invasion of cancer cells. As GCS overexpression is tightly associated with drug-resistant cancer cells, this study may suggest that depletion of GSLs with MBO-asGCS is an effective approach to eliminate the metastasis of cancers that even fail in conventional chemotherapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1470. doi:10.1158/1538-7445.AM2011-1470

Abstract 4365: Glucosylceramide synthase promotes cancer stem cells accumulating in chemotherapy

Abstract Cancer stem cells initiate tumorigenesis and are the causes of metastasis and relapse. Cancer stem cells, rather than other differentiated cells in tumors display resistance to cytotoxicity of anticancer drugs, but we do not know whether by-product of chemotherapy can induce cancer stem cells leading to chemotherapy failure. Glucosylceramide synthase (GCS) is a limiting enzyme regulating the synthesis of glycosphingolipids that play an essential role in the maintenance of embryonic stem cells. The purpose of this study was to identify and characterize the effect of ceramide glycosylation in the formation and maintenance of breast cancer stem cells (BCSCs). We have found that GCS overexpression was interrelated to the increment BCSCs and drug resistance in human breast cancer MCF-7 cell lines after doxorubicin induction. In MCF-7/Dox (doxorubicin-resistant) cells, GCS protein was increased by 39-fold accompanied with enhanced enzyme activity, as compared to wild-type MCF-7 cells. The BCSCs with CD24-/CD44+/ESA+ phenotype was increased by 5-fold in MCF-7/Dox cells as compared to MCF-7 cells. Silencing GCS by using mixed-backbone oligonucleotide (MBO-asGCS) significantly decreased the numbers of BCSCs more than 2-fold in MCF-7/Dox cells. In the soft agar colony formation assay, MCF-7/Dox cells showed significantly higher colonies formed as compare to the MCF-7 cells. BCSCs sorted from MCF-7/Dox cells displayed significantly higher GCS enzyme activity more than 3-fold and formed 2-fold more colonies, as compare with other non-stem cell subsets. The BCSCs cells showed significantly higher tumorigenicity and metastasibility as compared to non-stem cells (CD24-/CD44-) in athymic nude mice. More interestingly, doxorubicin treatment (1 mg/kg, once a week, and 40 days) considerably increased BCSCs 2-fold in tumors, and MBO-asGCS treatment eliminated the tumor growth and reduced metastasis due to reduction of BCSCs more than 2-fold in vivo. Comparison of glycosphingolipids and gene profile of stem cells indicated GCS or globo-series glycosphingolipids upregulates FGF1, FGFR1, ALDH2, ISL1, MSX1, SIGMAR1, PPARD and downregulates CXCL12, FRAT1, CCND1 to accumulate BCSCs during the course of chemotherapy. These results, for the first time, demonstrate that ceramide glycosylation by GCS accumulates BCSCs formation and causes aggressive tumor progression. Silencing of GCS that eliminates BCSCs can prevent and treat drug-resistant tumors and relapse. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4365. doi:10.1158/1538-7445.AM2011-4365

Abstract 4282: The role of glycosphingolipids in the formation of cancer stem cells and drug resistance

Abstract Tumorigenesis and malignant progression of cancers rely on a small subset of cells known as cancer stem cells (CSCs). In addition to forming primary tumors, metastasis and relapse, CSCs always display resistance to cytotoxins and this resistant feature protects CSCs during the course of chemotherapy. Glucosylceramide synthase (GCS) is a limited enzyme catalyzing the first glycosylation in glycosphingolipids synthesis. Glycosphingolipids are likely to play an essential role in maintaining the stemness of stem cells, since that deletion of GCS induces apoptosis of embryonic stem cell and stops embryo development in GCS knockout mice. The stage-specific embryonic antigens (SSEA-3, SSEA-4) are glycosphingolipids and serve as common pluripotent markers for human embryonic stem cells. In the present study, we have found that GCS modulates the formation and maintenance of breast cancer stem cells. GCS overexpression was interrelated to the increase of breast cancer stem cells (BCSCs) and drug resistance in human breast cancer MCF-7 cell lines after doxorubicin induction. In MCF-7/Dox (doxorubicin-resistant) cells, GCS protein was increased by 39-fold accompanied with enhanced enzyme activity, as compared to wild-type MCF-7 cell. Analyzed by using flow cytometry and immunostaining, the BCSCs with CD44+/CD24-/ESA+ phenotype were increased by 5-fold, and 3-fold in MCF-7/Dox and NCI-ADR/RES cell lines, as compared to MCF-7 cell lines, respectively. In BCSCs, GCS enzyme activity was 2-fold greater than other populations sorted from MCF-7/Dox. Silencing GCS by using mixed-backbone oligonucleotide (MBO-asGCS) significantly decreased the numbers of BCSCs in MCF-7/Dox cells. We observed tumorigenesis of NCI/ADR-RE/GCS (GCS overexpressed) and NCI/ADR-RE/asGCS (GCS silenced) in athymic nude mice. Aggressive tumors were found in all mice inoculated with NCI/ADR-RES and NCI/ADR-RE/GCS cells that were BCSC-enriched; however, no tumor or metastasis was observed in mice injected with NCI-ADR/asGCS cells. Furthermore, MBO-asGCS treatment (1 mg/kg) significantly decreased the numbers of BCSCs isolated from tumors of NCI-ADR/RES, as compared with control groups. Furthermore, BCSCs after MBO-asGCS treatment were more sensitive to doxorubicin. These results, for the first time, demonstrate that glycosphingolipid is involved in the regulation of cancer stem cell formation. Silencing GCS eliminates BCSCs, that can reverses drug resistance as well as prevent tumor relapse. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4282.

Abstract 2569: Disruption of ceramide glycosylation restores p53-dependent apoptosis in p53 mutant drug-resistant cancer cells: Alternative splicing determines the feature of p53 activity

Abstract Mutation of tumor suppressor p53 is frequently detected in many different types of cancers. p53 mutants acts as oncogenic proteins preventing programmed cell death and promoting cancer progression. Restoration of p53 that is to increase the expression of wild-type p53 and to eliminate mutant expression can substantially increase the efficacy of chemotherapy and stop malignant progression. However, incomplete understanding the molecular mechanism underlying p53 gene regulation limits the success in p53 restoration. We, for the first time, report that disruption of ceramide glycosylation restores p53 dependent-apoptosis in p53 mutant drug-resistant cancer cells. A lose of 21 base-pair in the mRNA of exon-5 can result in the accumulation of mutant p53 and cells insensitiveness to induced-apoptosis in human NCI/ADR-RE ovarian cancer cells. We found that treatment of mixed-backbone oligonucleotide against human glucosylceramide synthase (MBO-asGCS) increased the levels of wild-type p53 mRNA (containing the 21 bp in exon-5) and protein of wild-type p53 in dose-dependent manner (50-200 nM). In response to doxorubicin-induced DNA damage, phosphorylated p53 (Ser15 in the exon-5) was significantly increased, that in turn increased the protein levels of p21 and Bax, and apoptosis (52% vs. 3%) after MBO-asGCS treatment compared to control in NCI/ADR-RE cells. Silencing of GCS using MBO-asGCS (1 mg/kg) restored wild-type p53 expression and substantially sensitizes NCI/ADR-RE tumors to doxorubicin-induced apoptosis in vivo (10 mice per group), as compared to MBO-scramble control or other treatments. In contrast, introduction of GCS gene in NCI-ADR-RE cells reduces wild-type p53 expression and p53-depedent apoptosis. Further mechanic studies indicated that increased ceramide after GCS silencing modulates the function of SRp30a (ASF/SF2) regulating alternative pre-mRNA splicing. Increasing endogenous ceramide by inhibition of its glycosylation using PDMP or enhanced its production using sphingomylinase promoted wild-type p53 expression at the levels of mRNA, protein and phosphorylation; decreasing ceramide by inhibition of ceramide synthesis using fumonisin (FB1) repressed wild-type p53 expression. Present study indicates that cancer cells marked with mutant p53 phenotype are capable of expressing functional p53; ceramide glycosylation is one molecular mechanism that shifts expression to wild-type form of p53 at alternative splicing of post-transcription processing. MBO-asGCS, a specific agent silencing GCS restores p53-dependent apoptosis in cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2569.

Glucosylceramide synthase upregulates MDR1 expression in the regulation of cancer drug resistance through cSrc and β-catenin signaling

BackgroundDrug resistance is the outcome of multiple-gene interactions in cancer cells under stress of anticancer agents. MDR1 overexpression is most commonly detected in drug-resistant cancers and accompanied with other gene alterations including enhanced glucosylceramide synthase (GCS). MDR1 encodes for P-glycoprotein that extrudes anticancer drugs. Polymorphisms of MDR1 disrupt the effects of P-glycoprotein antagonists and limit the success of drug resistance reversal in clinical trials. GCS converts ceramide to glucosylceramide, reducing the impact of ceramide-induced apoptosis and increasing glycosphingolipid (GSL) synthesis. Understanding the molecular mechanisms underlying MDR1 overexpression and how it interacts with GCS may find effective approaches to reverse drug resistance.ResultsMDR1 and GCS were coincidently overexpressed in drug-resistant breast, ovary, cervical and colon cancer cells; silencing GCS using a novel mixed-backbone oligonucleotide (MBO-asGCS) sensitized these four drug-resistant cell lines to doxorubicin. This sensitization was correlated with the decreased MDR1 expression and the increased doxorubicin accumulation. Doxorubicin treatment induced GCS and MDR1 expression in tumors, but MBO-asGCS treatment eliminated "in-vivo" growth of drug-resistant tumor (NCI/ADR-RES). MBO-asGCS suppressed the expression of MDR1 with GCS and sensitized NCI/ADR-RES tumor to doxorubicin. The expression of P-glycoprotein and the function of its drug efflux of tumors were decreased by 4 and 8 times after MBO-asGCS treatment, even though this treatment did not have a significant effect on P-glycoprotein in normal small intestine. GCS transient transfection induced MDR1 overexpression and increased P-glycoprotein efflux in dose-dependent fashion in OVCAR-8 cancer cells. GSL profiling, silencing of globotriaosylceramide synthase and assessment of signaling pathway indicated that GCS transfection significantly increased globo series GSLs (globotriaosylceramide Gb3, globotetraosylceramide Gb4) on GSL-enriched microdomain (GEM), activated cSrc kinase, decreased β-catenin phosphorylation, and increased nuclear β-catenin. These consequently increased MDR1 promoter activation and its expression. Conversely, MBO-asGCS treatments decreased globo series GSLs (Gb3, Gb4), cSrc kinase and nuclear β-catenin, and suppressed MDR-1 expression in dose-dependent pattern.ConclusionThis study demonstrates, for the first time, that GCS upregulates MDR1 expression modulating drug resistance of cancer. GSLs, in particular globo series GSLs mediate gene expression of MDR1 through cSrc and β-catenin signaling pathway.

A New Mixed-Backbone Oligonucleotide against Glucosylceramide Synthase Sensitizes Multidrug-Resistant Tumors to Apoptosis

Enhanced ceramide glycosylation catalyzed by glucosylceramide synthase (GCS) limits therapeutic efficiencies of antineoplastic agents including doxorubicin in drug-resistant cancer cells. Aimed to determine the role of GCS in tumor response to chemotherapy, a new mixed-backbone oligonucleotide (MBO-asGCS) with higher stability and efficiency has been generated to silence human GCS gene. MBO-asGCS was taken up efficiently in both drug-sensitive and drug-resistant cells, but it selectively suppressed GCS overexpression, and sensitized drug-resistant cells. MBO-asGCS increased doxorubicin sensitivity by 83-fold in human NCI/ADR-RES, and 43-fold in murine EMT6/AR1 breast cancer cells, respectively. In tumor-bearing mice, MBO-asGCS treatment dramatically inhibited the growth of multidrug-resistant NCI/ADR-RE tumors, decreasing tumor volume to 37%, as compared with scrambled control. Furthermore, MBO-asGCS sensitized multidrug-resistant tumors to chemotherapy, increasing doxorubicin efficiency greater than 2-fold. The sensitization effects of MBO-asGCS relied on the decreases of gene expression and enzyme activity of GCS, and on the increases of C18-ceramide and of caspase-executed apoptosis. MBO-asGCS was accumulation in tumor xenografts was greater in other tissues, excepting liver and kidneys; but MBO-asGCS did not exert significant toxic effects on liver and kidneys. This study, for the first time in vivo, has demonstrated that GCS is a promising therapeutic target for cancer drug resistance, and MBO-asGCS has the potential to be developed as an antineoplastic agent.

Antisense targeting protein kinase A type I as a drug for integrated strategies of cancer therapy.

We have studied the role of protein kinase A (PKA) and its type I isoform (PKAI) in the transduction of mitogenic signaling, apoptosis, and angiogenesis. We have contributed to the development of selective inhibitors of PKAI, including a hybrid DNA/RNA mixed backbone oligonucleotide (AS-PKAI). We, and others, have demonstrated that AS-PKAI has a cooperative antitumor effect with a selected class of cytotoxic drugs and with radiotherapy in vitro and in vivo and that these effects can also be obtained following oral adinistration. Previously, we developed a series of therapeutic models based on the pleiotropic role played by PKAI in cell proliferation, apoptosis, and angiogenesis. On the basis of our former demonstration of functional and structural interactions of PKAI and the activated epidermal growth factor receptor (EGFR), we have shown that the combined blockade of both signaling molecules by AS-PKAI and either the monoclonal antibody C225 (erbitux) or the small molecule ZD1839 (gefitinib), results in a marked cooperative antitumor effect in a variety of human tumor models. A further cooperative antitumor effect can be obtained when AS-PKAI is used in combination with both EGFR inhibitors and either cytotoxic drugs or radiotherapy. The antitumor activity is associated with inhibition of growth factors and angiogenic factors production and to induction of apoptosis. In light of the recently demonstrated role of PKAI on the bcl-2-dependent apoptotic pathway, we have recently shown a synergistic antitumor, antiangiogenic, and proapoptotic effect of AS-PKAI in combination with antisense bcl-2 (oblimersen) or with a bispecific bcl-2/bcl-xL second generation antisense. A connection between COX-2, EGFR and PKAI was established, and we demonstrated that the combination of AS-PKAI with gefitinib and a COX-2 inhibitor, all adminstered orally, can result in a potent antitumor and antiangiogenic activity. These studies support the development of AS-PKAI as a novel anticancer agent and suggest its potentially relevant role when integrated with conventional treatments and/or other signaling inhibitors in novel therapeutic strategies.

Antisense therapy targeting MDM2 oncogene in prostate cancer: Effects on proliferation, apoptosis, multiple gene expression, and chemotherapy.

This study was undertaken to investigate the role of mouse double minute 2 (MDM2) oncogene in prostate cancer growth and the potential of MDM2 as a target for prostate cancer therapy. An antisense anti-human-MDM2 mixed-backbone oligonucleotide was tested in human prostate cancer models with various p53 statuses, LNCaP (p53wt/wt), DU145 (p53mt/mt), and PC3 (p53null). In a dose- and time-dependent manner, it specifically inhibited MDM2 expression and modified expression of several genes, at both mRNA and protein levels. In LNCaP cells, p53, p21, Bax, and hypophosphorylated retinoblastoma tumor suppressor protein (pRb) levels increased, whereas Bcl2, pRb protein, and E2F transcription factor 1 (E2F1) levels decreased. In DU145 cells, p21 levels were elevated and E2F1 levels decreased, although mutant p53, Rb, and Bax levels remained unchanged. In PC3 cells, MDM2 inhibition resulted in elevated p21, Bax, and pRb levels and decreased ppRb and E2F1 levels. In all three cell lines, MDM2 inhibition reduced cell proliferation, induced apoptosis, and potentiated the effects of the chemotherapeutic agents 10-hydroxycamptothecin and paclitaxel. The anti-MDM2 oligonucleotide showed antitumor activity and increased therapeutic effectiveness of paclitaxel in both LNCaP and PC3 xenografts, causing changes in gene expression similar to those seen in vitro. In summary, this study demonstrates that MDM2 has a role in prostate cancer growth via p53-dependent and p53-independent mechanisms and that multiple genes are involved in the process. MDM2 inhibitors such as second-generation antisense oligonucleotides have a broad spectrum of antitumor activities in human cancers regardless of p53 status, providing novel approaches to therapy of human prostate cancer.

(2′‐O‐Methyl‐RNA)‐3′‐PNA Chimeras: A New Class of Mixed Backbone Oligonucleotide Analogues with High Binding Affinity to RNA.

The automated on-line synthesis of DNA-3′-PNA chimeras 1–4 and (2′-O-methyl-RNA)-3′-PNA chimeras 5–8 is described, in which the 3′-terminal part of the oligonucleotide is linked to the N-terminal part of the PNA via N-(ω-hydroxyalkyl)-N-[(thymin-1-yl)acetyl]glycine units (alkyl=Et, Ph, Bu, and pentyl). By means of UV thermal denaturation, the binding affinities of all chimeras were directly compared by determining their Tm values in the duplex with complementary DNA and RNA. All investigated DNA-3′-PNA chimeras and (2′-O-methyl-RNA)-3′-PNA chimeras form more-stable duplexes with complementary DNA and RNA than the corresponding unmodified DNA. Interestingly, a N-(3-hydroxypropyl)glycine linker resulted in the highest binding affinity for DNA-3′-PNA chimeras, whereas the (2′-O-methyl-RNA)-3′-PNA chimeras showed optimal binding with the homologous N-(4-hydroxybutyl)glycine linker. The duplexes of (2′-O-methyl-RNA)-3′-PNA chimeras and RNA were significantly more stable than those containing the corresponding DNA-3′-PNA chimeras. Surprisingly, we found that the charged (2′-O-methyl-RNA)-3′-PNA chimera with a N-(4-hydroxybutyl)glycine-based unit at the junction to the PNA part shows the same binding affinity to RNA as uncharged PNA. Potential applications of (2′-O-methyl-RNA)-3′-PNA chimeras include their use as antisense agents acting by a RNase-independent mechanism of action, a prerequisite for antisense-oligonucleotide-mediated correction of aberrant splicing of pre-mRNA.

Experimental therapy of human prostate cancer by inhibiting MDM2 expression with novel mixed-backbone antisense oligonucleotides: in vitro and in vivo activities and mechanisms.

MDM2 oncogene is overexpressed in many human cancers including prostate cancer and MDM2 levels are associated with poor prognosis. This study was undertaken to investigate the functions of MDM2 oncogene in prostate cancer growth and the value of MDM2 as a drug target for prostate cancer therapy by inhibiting MDM2 expression. Antisense anti-human-MDM2 mixed-backbone oligonucleotide and its mismatch control were tested in in vitro and in vivo human prostate cancer models (LNCaP, DU 145, and PC-3) for anti-tumor activity. Targeted gene products and related proteins were analyzed and the anti-tumor activity was determined when the oligonucleotides were used alone or in combination with cancer therapeutics. The antisense oligonucleotide specifically inhibited MDM2 expression in a dose- and time-dependent manner, resulting in significant anti-tumor activity in vitro and in vivo. In LNCaP cells, p53 and p21 levels were elevated. The antisense oligonucleotide also potentiated the effects of p53 activation and p21 induction by chemotherapeutic agents 10-hydroxycamptothecin, adriamycin, 5-fluorouracil, and paclitaxel. In DU145 cells, following inhibition of MDM2 expression, p21 levels were elevated although p53 levels remained unchanged. In both cell lines, the antisense oligonucleotide inhibited tumor cell growth and induced apoptosis in vitro. In a dose-dependent manner, the antisense oligonucleotide showed anti-tumor activity in nude mice bearing DU145 or PC-3 xenografts. It significantly increased therapeutic effectiveness of the chemotherapeutic agent irinotecan and slightly improved the effects of paclitaxel and Rituxan. These results indicate that MDM2 has a role in prostate tumor growth through both p53-dependent and p53-independent mechanisms, indicating that MDM2 inhibitors have a broad spectrum of anti-tumor activities in human prostate cancers regardless of p53 status.

(2′‐O‐Methyl‐RNA)‐3′‐PNA Chimeras: A New Class of Mixed Backbone Oligonucleotide Analogues with High Binding Affinity to RNA

The automated on-line synthesis of DNA-3′-PNA chimeras 1–4 and (2′-O-methyl-RNA)-3′-PNA chimeras 5–8 is described, in which the 3′-terminal part of the oligonucleotide is linked to the N-terminal part of the PNA via N-(ω-hydroxyalkyl)-N-[(thymin-1-yl)acetyl]glycine units (alkyl=Et, Ph, Bu, and pentyl). By means of UV thermal denaturation, the binding affinities of all chimeras were directly compared by determining their Tm values in the duplex with complementary DNA and RNA. All investigated DNA-3′-PNA chimeras and (2′-O-methyl-RNA)-3′-PNA chimeras form more-stable duplexes with complementary DNA and RNA than the corresponding unmodified DNA. Interestingly, a N-(3-hydroxypropyl)glycine linker resulted in the highest binding affinity for DNA-3′-PNA chimeras, whereas the (2′-O-methyl-RNA)-3′-PNA chimeras showed optimal binding with the homologous N-(4-hydroxybutyl)glycine linker. The duplexes of (2′-O-methyl-RNA)-3′-PNA chimeras and RNA were significantly more stable than those containing the corresponding DNA-3′-PNA chimeras. Surprisingly, we found that the charged (2′-O-methyl-RNA)-3′-PNA chimera with a N-(4-hydroxybutyl)glycine-based unit at the junction to the PNA part shows the same binding affinity to RNA as uncharged PNA. Potential applications of (2′-O-methyl-RNA)-3′-PNA chimeras include their use as antisense agents acting by a RNase-independent mechanism of action, a prerequisite for antisense-oligonucleotide-mediated correction of aberrant splicing of pre-mRNA.

Antisense oligonucleotide targeted to RIα subunit of cAMP-dependent protein kinase (GEM231) enhances therapeutic effectiveness of cancer chemotherapeutic agent irinotecan in nude mice bearing human cancer xenografts: in vivo synergistic activity, pharmacokinetics and host toxicity

The RIalpha-subunit of cAMP-dependent protein kinase (PKA) is overexpressed in various human cancers and PKA has been suggested to be a potential target for cancer therapy. We have shown an antisense oligonucleotide with advanced chemistry (mixed-backbone oligonucleotide) targeted to PKA RIalpha-subunit (GEM231) to have anti-tumor activity in vitro and in vivo. In the present study, we demonstrated synergistic effects between the anti-PKA antisense oligonucleotide and the clinically used anticancer agent irinotecan, using nude mouse models of human cancers of colon (LS174T and DLD-1), breast (MCF-7), prostate (DU-145 and PC-3) and lung (H1299). To elucidate the underlying mechanisms, in vivo pharmacokinetics of irinotecan was determined following pre-treatment of oligo GEM 231 in CD-1 mice and nude mice bearing LS174T xenografts. GEM 231 increased tissue uptake of irinotecan. However, no significant change in host toxicity was observed following combination treatment of irinotecan and GEM231 compared with irinotecan alone. These results suggest that GEM231 have a role in irinotecan metabolism and its antitumor activity, providing a basis for future development of this oligonucleotide as a chemosensitizer for irinotecan-based therapy.

Anti-Tumor Efficacy of a Novel Antisense Anti-MDM2 Mixed-Backbone Oligonucleotide in Human Colon Cancer Models: p53-Dependent and p53-Independent Mechanisms

The MDM2 oncogene is amplified or overexpressed in many human cancers and MDM2 levels are associated with poor prognosis. MDM2 not only serves as a negative regulator of p53 but also has p53-independent activities. This study investigates the functions of the MDM2 oncogene in colon cancer growth and the potential value of MDM2 as a drug target for cancer therapy, by inhibiting MDM2 expression with an antisense anti-human-MDM2 oligonucleotide. The selected antisense mixed-backbone oligonucleotide was evaluated for its in vitro and in vivo antitumor activity in human colon cancer models: LS174T cell line containing wild-type p53 and DLD-1 cell line containing mutant p53. The levels of MDM2, p53 and p21 proteins were quantified by Western blot analysis. In vitro antitumor activity was found in both cell lines, resulting from specific inhibition of MDM2 expression. In vivo antitumor activity of the oligonucleotide occurred in a dose-dependent manner in both models and synergistically or additive therapeutic effects of MDM2 inhibition and the cancer chemotherapeutic agents 10-hydroxycamptothecin and 5-fluorouracil were also observed. These results suggest that MDM2 have a role in tumor growth through both p53-dependent and p53- independent mechanisms. We speculate that MDM2 inhibitors have a broad spectrum of antitumor activities in human cancers regardless of p53 status. This study should provide a basis for future development of anti-MDM2 antisense oligonucleotides as cancer therapeutic agents used alone or in combination with conventional chemotherapeutics.

Detection of a mixed-backbone oligonucleotide (GEM 231) in liver and tumor tissues by capillary electrophoresis

A simple, rapid and sensitive method has been developed and validated for the analysis of a mixed-backbone oligonucleotide (GEM 231) in tumor tissues. The analysis was performed using a capillary electrophoresis (CE) system with UV detection. An extended light path (bubble cell) capillary column of 64.5 cm (effective length 56 cm)×50 μm I.D. is used as the separation column. The optimized chromatographic conditions were background electrolyte: sodium borate buffer (60 m M, pH 9.1), electrokinetic injection: 10 s, applied voltage: 30 kV, detection at λ=210 nm. A linear relationship was observed between the peak area and the amount of GEM 231 in the range of 1.0–1000 μg/ml. The lower detection limit of the drug was 100 pg with an average recovery of about 75±5%. The inter-day and intra-day relative standard deviations were <10%. Assay validation studies revealed that CE method is reproducible and specific for the determination of GEM 231 in tissue homogenates with a run time of less than 5 min.

A Short Phosphodiester Window Is Sufficient to Direct RNase H-Dependent RNA Cleavage by Antisense Peptide Nucleic Acid

The potential pharmacologic benefits of using peptide nucleic acid (PNA) as an antisense agent are tempered by its incapacity to activate RNase H. The mixed backbone oligonucleotide (ON) (or gapmer) approach, in which a short internal window of RNAse H-competent residues is embedded within an RNase H-incompetent ON has not been applied previously to PNA because PNA and DNA hybridize to RNA with very different helical structures, creating structural perturbations at the two PNA-DNA junctions. It is demonstrated here for the first time that a short internal phosphodiester window within a PNA is sufficient to evoke the RNase H-dependent cleavage of a targeted RNA and to abrogate translation elongation in a well-characterized in vitro assay.

Biodistribution and metabolism of a mixed backbone oligonucleotide (GEM 231) following single and multiple dose administration in mice.

Biodistribution and metabolism of a mixed backbone oligonucleotide (MBO), GEM 231, targeted to the RIalpha subunit of protein kinase A has been studied in normal and tumor xenografted mice. The study has been carried out using [35S]-labeled MBO following single and multiple administrations of doses varying from 2 to 50 mg/kg. MBO showed wide tissue distribution following intravenous and subcutaneous administration. The highest concentration of MBO was in the kidney and liver. The general disposition of MBO was followed by digitized autoradiographic pictures of tumored mice and further confirmed wide tissue disposition and also showed defined intratumor uptake of MBO. Multiple dose administration showed increased disposition in the majority of the tissues/organs, with the exception of the kidneys. Analysis of the extracted MBO by polyacrylamide gel electrophoresis (PAGE) showed the presence of primarily intact MBO along with its degraded forms. Based on our radioactivity levels, the primary route of excretion was in urine, analysis of which showed mainly degraded forms of MBO.

Vesicular stomatitis virus as model system for studies of antisense oligonucleotide translation arrest

The focus of this chapter is neither to describe the morphology and ecology of vesicular stomatitis virus (VSV) or the Rhabdoviridae nor to claim that VSV is a suitable target for antisense drug development. This chapter presents some of the characteristics of the molecular physiology of this virus and how they can be exploited to create a model system in which fundamental studies of antisense action may be studied. In vitro RNase H-mediated cleavage of an mRNA target has been demonstrated to reflect efficiency in cell culture systems in many cases. Most of the new chemistries, however, do not elicit RNase H activation. This major hurdle can be circumvented by two strategies: (1) restoring nucleolytic activity by the conjugation of suitable pendant groups or by incorporating an RNase H-competent window (mixed backbone oligonucleotide) or (2) increasing the efficiency of steric blockade. In this respect, the simple assay described in this chapter has the advantage of discriminating true translation arrest from target RNA destruction. Because natural mRNAs are used in this assay, both untranslated and coding regions can be examined. Finally, the simultaneous translation of three mRNAs provides a convenient and, important, internal control.

Antitumor activity and pharmacokinetics of a mixed-backbone antisense oligonucleotide targeted to the RIalpha subunit of protein kinase A after oral administration.

Overexpression of the RIalpha subunit of cAMP-dependent protein kinase (PKA) has been demonstrated in various human cancers. PKA has been suggested as a potential target for cancer therapy. The goal of the present study was to evaluate an anti-PKA antisense oligonucleotide (mixed-backbone oligonucleotide) as a therapeutic approach to human cancer treatment. The identified oligonucleotide inhibited the growth of cell lines of human colon cancer (LS174T, DLD-1), leukemia (HL-60), breast cancer (MCF-7, MDA-MB-468), and lung cancer (A549) in a time-, concentration-, and sequence-dependent manner. In a dose-dependent manner, the oligonucleotide displayed in vivo antitumor activity in severe combined immunodeficient and nude mice bearing xenografts of human cancers of the colon (LS174T), breast (MDA-MB-468), and lung (A549). The routes of drug administration were intraperitoneal and oral. Synergistic effects were found when the antisense oligonucleotide was used in combination with the cancer chemotherapeutic agent cisplatin. The pharmacokinetics of the oligonucleotide after oral administration of (35)S-labeled oligonucleotide into tumor-bearing mice indicated an accumulation and retention of the oligonucleotide in tumor tissue. This study further provides a basis for clinical studies of the antisense oligonucleotide targeted to the RIalpha subunit of PKA (GEM 231) as a cancer therapeutic agent used alone or in combination with conventional chemotherapy.

Mixed backbone oligonucleotides: improvement in oligonucleotide-induced toxicity in vivo.

Mixed backbone oligonucleotides: improvement in oligonucleotide-induced toxicity in vivo.