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Results for “antisense targeting”

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PubMed2026

A First-in-Japanese Phase 1, Double-Blind, Placebo-Controlled, Parallel-Cohort Study of Sefaxersen, an Antisense Oligonucleotide Targeting Complement Factor B, in Healthy Participants.

Increased activity in the complement alternative pathway (AP) plays a key role in diseases such as IgA nephropathy (IgAN). This first-in-Japanese double-blind Phase 1 study investigated the pharmacokinetics (PK), pharmacodynamics (PD), safety, and tolerability of sefaxersen (RO7434656), an antisense oligonucleotide targeting complement factor B messenger RNA. Healthy participants were randomized equally into four cohorts: placebo or sefaxersen 20, 40, or 70 mg. The PK, PD, and safety endpoints were monitored throughout the study and during the 90-day follow-up period. All 24 participants completed the study, with no new safety signals or clinically meaningful changes in blood chemistry, electrocardiogram, or vital signs observed. Plasma sefaxersen concentration demonstrated a biphasic PK profile, characterized by an initial rapid decline followed by a slow elimination. Sefaxersen decreased PD markers related to the complement AP selectively, without affecting the classical pathway, in a dose-dependent manner, and the PD effects persisted over 2 to 3 months. Sefaxersen was well tolerated by healthy Japanese participants, with a manageable safety profile. These findings support the inclusion of Japanese patients with IgAN in the global Phase 3 study (IMAGINATION, NCT05797610).

Humans↗

PubMed2006

[Experimental study of antisense oligodeoxynucleotide targeting survivin gene for cisplatin resistant human lung adeno-carcinoma xenograft in nude mice].

OBJECTIVE: To explore the feasibility of antisense oligodeoxynucleotide (ASODN) targeting survivin gene for cisplatin resistant human lung adeno-carcinoma xenograft in nude mice. METHODS: Cisplatin resistant cell lines A549/CDDP were cultured routinely with RPMI1640 medium. A549/CDDP cells were subcutaneously implanted in nude mice to establish cisplatin resistant xenograft animal models. After survivin ASODN mediated by cytofectin was directly injected into xenograft in 5 places. The volumes and weight of tumor mass were detected, respectively, and then tumor growth inhibitory rate and tumor growth index were calculated. Reverse transcription-polymerase chain reaction (RT-PCR) and immunochemohistology assay were performed to detect the expression level of survivin mRNA and protein. RESULTS: In mice treated with single ASODN, the tumor growth inhibitory rate and tumor growth index was 35.4% and 4.23+/-0.4456. The difference of the tumor growth inhibitory rate and tumor growth index between blank control group and ASODN group was significant (P<0.05). While combined ASODN with cisplatin,the anticancer efficacy was far more significant and the tumor growth inhibitory rate was enhanced to 63.7%. The tumor growth index, however, reduced to 1.700+/-0.436, which was obviously significant,compared with the cisplatin group and other controls (P<0.05). The anticancer efficacy was even more obvious than that of ASODN group (P<0.05). Significant down-regulation of survivin mRNA and protein level expression in tumor tissues of ASODN group and ASODN and cisplatin group was detected by RT-PCR and immunochemohistology assay, respectively (P<0.05). CONCLUSION: Survivin ASODN mediated by cytofectin can inhibit the cisplatin resistant tumor growth by direct intra-tumoral injection. The anticancer efficacy may be associated with the down regulation of survivin expression. ASODN targeting survivin gene can be a supportive therapy to cisplatin resistant lung cancer, while the clinical effective values need further exploration.

Adenocarcinoma↗

PubMed1994

Cationic liposomes mediated delivery of antisense oligonucleotides targeted to HPV 16 E7 mRNA in CaSki cells.

The "high risk" types 16 and 18 of human papillomavirus (HPV) are involved in the etiology of genital squamous cell carcinoma. The early genes 6 and 7 (E6-E7) of these viruses code for the major transforming proteins, capable of inducing cell transformation alone or acting synergistically with other oncogenes. Antisense oligonucleotides, recently applied to inhibit the functions of a number of cellular and viral proteins, might provide the basis for a new therapeutic strategy against HPV-induced malignancies. We studied the proliferation of CaSki cells by the MTT assay after their exposure to HPV 16 E7 mRNA antisense oligonucleotides with and without cationic liposomes (containing dimethyldioctadecylammonium bromide DDAB, and dioleylphosphatidylethanolamine, DOPE). Unmodified oligonucleotides (either 12- or 23-mers) did not have any effect on either CaSki cell proliferation or morphology when compared with the untreated cells. The cellular uptake of oligonucleotides was significantly enhanced by the cationic liposomes as assessed by confocal laser scanning microscopy (CLSM). The cationic liposomes were toxic to the cells as demonstrated by the reduced cell number and altered cell morphology. Only a slight reduction of the cell proliferation was seen when antisense 12-mer was protected from its 3'- and 5'-ends with thiolate and FITC, respectively. Both the 12- and the 23-mers with the cationic liposomes inhibited cell proliferation, the inhibitory effect being longer with the 23-mer. Overall, the MTT assay was less sensitive than light microscopy to reveal the toxic effects on CaSki cells. The results suggest that antisense oligonucleotides targeted to HPV 16 E7 mRNA can be introduced into CaSki cells with cationic liposomes.

Base Sequence↗

PubMed1995

Inhibition of chronic myelogenous leukemia cells harboring a BCR-ABL B3A2 junction by antisense oligonucleotides targeted at the B2A2 junction.

Chronic myeloid leukemia (CML) is a hematopoietic stem cell disorder characterized by the BCR-ABL hybrid gene. Two types of hybrid BCR-ABL mRNA have been found, B2A2 and B3A2. As the BCR-ABL rearrangement is specific to leukemic cells, selective inhibition of leukemic cell growth by BCR-ABL antisense oligonucleotides (ASO) has been reported in vitro for CML patients and cell lines. However, controversial results have been obtained from preclinical studies using anti-BCR-ABL ASO, as nonspecific inhibition of leukemic cell growth was evidenced in some cases. B3 exon secondary structure was deduced from its sequence and found to be a loop. According to this predictive structure of exon B3, a 56-mer antisense oligonucleotide targeting the polypurine bases from the B2A2 junction was devised which would inhibit proliferation (MTT assay) of B3A2 junction cell lines (K562 and a murine cell line Ba/F3 transfected with the B3A2 junctional sequence). This ASO had a hairpin-like secondary structure and was found to be much more resistant to the action of nucleases than control 18-mer standard oligonucleotides. Hybridization to its target mRNA occurs via formation of a triplex structure. A concentration of 5 microM of specific 56-mer B2A2 ASO was necessary to demonstrate 50% optical density (OD) reduction for K562 cell line and Ba/F3 transformed by B3A2 cDNA. Sense and non-sense 56-mer sequence or 18-mer linear ASO showed no effect for these concentrations. Western blot showed a partial inhibition of P210 protein; expression of P145abl remains unchanged. The 56-mer ASO also inhibited the proliferation of B2A2 junction cell line BV173 at the same concentration and showed no effect on the HL60 cell line used as control.

Base Sequence↗

PubMed2002

Inward rectifier K(+) current in human bronchial smooth muscle cells: inhibition with antisense oligonucleotides targeted to Kir2.1 mRNA.

Inward rectifier K(+) (Kir) channels play an important role in forming membrane potential and then modulating muscle tone in certain types of smooth muscles. In cultured human bronchial smooth muscle cells (hBSMCs), Kir current was identified using whole-cell voltage clamp techniques and explored by using RT-PCR analysis of mRNA, Western blotting, and antisense oligonucleotide methods to block the synthesis of Kir channel protein. The K(+) current with strong inward rectification and high K(+) ion selectivity was observed. The current was unaffected by 4-aminopyridine, glibenclamide, and charybdotoxin, and hardly inhibited by tetraethylammonium, but was potently inhibited by extracellular Ba(2+). The IC(50) value of external Ba(2+) was approximately 1.3 microm. RT-PCR analysis of mRNA showed transcripts for Kir2.1, but not Kir1.1, Kir2.2, or Kir2.3. Treatment of cells with antisense oligonucleotides targeted to Kir2.1 resulted in a decrease in the current density of the Kir current and Kir protein expression, as compared with the mismatch-treated cells, whereas the current density of 4-AP-sensitive K(+) currents (K(V)) remained unaffected. The application of Ba(2+) markedly depolarized the membrane. These results demonstrate that Kir channel is present in human bronchial smooth muscle cells, and the Kir2.1 gene encodes the Kir channel protein in these cells.

Bronchi↗

PubMed1993

Inhibitory effects of antisense oligodeoxynucleotides targeting c-myc mRNA on smooth muscle cell proliferation and migration.

Smooth muscle cell (SMC) proliferation and migration play pivotal roles in restenosis following angioplasty. c-myc is an immediate early response gene induced by various mitogens, and several lines of evidence derived from experiments using transformed or hematopoietic cell lines, or transgenic mice, suggest its protein product plays a role in numerous signaling transduction pathways, including those modulating cell division. We therefore reasoned that a strategy employing oligodeoxynucleotides (ODNs) complementary to c-myc mRNA (antisense ODNs) might be potent inhibitors of SMC proliferation and, perhaps, of SMC migration. To evaluate this concept, we tested several antisense ODNs targeted to c-myc mRNA (15- or 18-mer ODNs complementary to different c-myc mRNA sequences) by introducing them individually into the medium of cultured rat aortic SMCs. Phosphoroamidate-modified ODNs were employed to retard degradation. Antisense ODNs inhibited, in a concentration-dependent manner, SMC proliferation and SMC migration. Maximal inhibitory effect was 50% for proliferation and > 90% for migration. These effects were associated with decreased SMC expression of c-myc-encoded protein by Western immunoblotting and immunocytochemical staining. ODNs with the same nucleotides but a scrambled sequence caused no effect. These results indicate that the c-myc gene product is involved in the signal transduction pathways mediating SMC proliferation and migration in the in vitro model we employed. The results also suggest a potential role of antisense strategies designed to inhibit c-myc expression for the prevention of coronary restenosis.

Animals↗

PubMed1998

Potent inhibition of respiratory syncytial virus replication using a 2-5A-antisense chimera targeted to signals within the virus genomic RNA.

The 2-5A system is a recognized mechanistic component of the antiviral action of interferon. Interferon-induced 2-5A synthetase generates 2-5A, which, in turn, activates the latent constitutive RNase L that degrades viral RNA. Chemical conjugation of 2-5A to an antisense oligonucleotide can target the 2-5A-dependent RNase L to the antisense-specified RNA and effect its selective destruction. Such a 2-5A-antisense chimera (NIH351) has been developed that targets a consensus sequence within the respiratory syncytial virus (RSV) genomic RNA. NIH351 was 50- to 90-fold more potent against RSV strain A2 than was ribavirin, the presently approved drug for clinical management of RSV infection. It was similarly active against a variety of RSV strains of both A and B subgroups and possessed a cell culture selectivity index comparable to ribavirin. In addition, the anti-RSV activity of NIH351 was shown to be virus-specific and a result of a true antisense effect, because a scrambled nucleotide sequence in the antisense domain of NIH351 caused a significant decrease in antiviral activity. The 2-5A system's RNase L was implicated in the mechanism of action of NIH351 because a congener with a disabled 2-5A moiety was of greatly reduced anti-RSV effectiveness. These findings represent an innovative approach to the control of RSV replication.

Adenine Nucleotides↗

PubMed2005

[Antisense oligonucleotide targeting survivin induces apoptosis of renal clear-cell carcinoma cells and enhances their sensitivity to epirubicin in vitro].

OBJECTIVE: To investigate the effect of antisense oligonucleotide (ASODN) targeting survivin on the apoptosis and proliferation of renal cancer cell line 786-O and enhancement of its sensitivity to epirubicin. METHODS: ASODN targeting survivin was designed and constructed. Cultured cells were divided into 6 groups: control group, liposome group, sense oligonucleotide (SODN) group, 600 nmol/L ASODN group, and 600 nmol/L ASODN combined with epirubicin group. After transfected for 24 h, cultured cells were harvested to carry on the next tests. Cell morphological changes were examined by transmission electron microscopy. Survivin protein was detected by immunohistochemical method. Apoptosis index (AI) and proliferation index (PI) were examined by flow cytometry. RESULTS: Morphological abnormalities of cells were observed in ASODN transfected groups. Expression of survivin in ASODN groups were significantly decreased compared with that in the control group, liposomes group and SODN group. AI of ASODN groups was significantly higher than that in other groups. PI of ASODN groups was significantly lower than that in other groups. The PI of ASODN combined with epirubicin group was (35.7 +/- 1.67)%, but (9.3 +/- 0.34)% or (8.5 +/- 0.21)% in liposomes group or SODN group that had combined with epirubicin. The ASODN group achieved the strongest effects to enhance apoptosis in comparison with control group (P < 0.05), while SODN did not cause statistically significant change (P > 0.05). CONCLUSION: The expression of survivin protein in the renal clear cell carcinoma cell line 786-O is downregulated by survivin ASODN. ASODN targeting survivin induces apoptosis and inhibits proliferation of 786-O cells. Inhibition of survivin enhances sensitivity of 786-O to epirubicin.

Antibiotics, Antineoplastic↗

PubMed2004

Effect of antisense RNA targeting polo-like kinase 1 on cell cycle and proliferation in A549 cells.

BACKGROUND: Expression of polo-like kinase 1 (Plk1) is elevated in lung cancer and has been proposed as having prognostic value and related to resistance to chemotherapy and radiation. In addition, Plk1 has several functions in mitotic progression. In this study, the authors investigated the effect of Plk1 depletion on cell cycle progression and proliferation in A549 cells, a lung cancer cell line. METHODS: A recombinant plasmid containing antisense RNA targeting Plk1 (pcDNA3-Plk1) was transfected into A549 cells. Reverse transcription-polymerase chain reaction and Western blot were used to examine Plk1 gene expression. Cell proliferation was evaluated by direct cell counting and bromodeoxyuridine (BrdU) labelling. Cell cycle and apoptosis were examined by flow cytometry. Expression of alpha-tubulin was detected by immunofluorescence, and the inhibition rate (IR) by chemotherapeutic agents was determined by 3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyl-tetrazolium bromide assay. RESULTS: After transfection into A549 cells, pcDNA3-Plk1 reduced Plk1 mRNA by 46.75% for 24 hours and by 61.84% for 48 hours. Plk1 protein was significantly decreased simultaneously (P < 0.05). Abnormal morphological changes of cells and growth inhibition were observed in pcDNA3-Plk1 transfected groups. The BrdU labelling index was 25.59% 48 hours after transfection, which was significantly lower than that of the control groups (P < 0.05). Forty-eight hours after transfection, there showed absence of microtubule polymerization and spindle abnormalities in staining for alpha-tubulin. A549 cells showed a strong G2/M arrest and apoptosis 72 hours post transfection. IR of vinorelbine in pcDNA3-Plk1 transfected groups was significantly higher than that of the other groups (P < 0.05, respectively). CONCLUSIONS: Plk1 depletion interferes with spindle formation, induces cell cycle arrest and apoptosis, and consequently inhibits cell proliferation in A549 cells. Moreover, it sensitizes lung cancer cells to chemotherapy.

Apoptosis↗

PubMed2005

[Effect of antisense RNA targeting Polo-like kinase 1 on cell cycle of lung cancer cell line A549].

BACKGROUND & OBJECTIVE: Expression of polo-like kinase 1 (Plk1), which has several functions in mitotic progression, is elevated in lung adenocarcinoma cell line A549. It suggests that over-expression of Plk1 relates to chemotherapy- and radiotherapy-resistance, and poor prognosis of lung cancer patients. This study was to investigate the effect of down-regulation of Plk1 on cell cycle of A549 cells using the technique of antisense RNA. METHODS: Antisense RNA targeting Plk1 (pcDNA3.0-Plk1, pc3.0P) was designed and constructed, and transfected into A549 cells. Reverse transcription-polymerase chain reaction (RT-PCR) and Western blot were used to examine expression of Plk1 gene. Cell proliferation was evaluated by BrdU stain; cell cycle changes and apoptosis were examined by flow cytometry; expression of alpha-tubulin was detected by immunofluorescence. RESULTS: mRNA level of Plk1 was significantly lower in pc3.0P-transfected A549 cells than in control cells (P < 0.05). pc3.0P reduced Plk1 mRNA in A549 cells by 46.75% 24 h after transfection, and by 61.84% 48 h after transfection. Protein level of Plk1 was also decreased after transfection. Positive rate of BrdU stain in experimental cells was only 25.59% 48 h after transfection, which was significantly lower than that in control cells (P < 0.05). A549 cells showed a strong G(2)/M arrest and apoptosis 72 h after transfection. alpha-tubulin staining showed absence of microtubule connection,and spindle abnormalities. CONCLUSION: Down-regulation of Plk1 interferes spindle formation, and inhibits cell proliferation, induces cell cycle arrest and apoptosis.

Adenocarcinoma↗

PubMed2001

Tumor growth inhibition in vivo and G2/M cell cycle arrest induced by antisense oligodeoxynucleotide targeting thymidylate synthase.

Chemotherapeutic agents targeting thymidylate synthase (TS) are effective against human tumors. Efficacy is limited by drug resistance, often mediated by TS overexpression. Treatment of HeLa cells in vitro with an antisense oligodeoxynucleotide (ODN 83) targeting human TS mRNA reduces TS mRNA and protein levels, inhibits cell proliferation, and sensitizes cells to TS-targeting drugs (Ferguson et al., 1999). The present study investigates the mechanism by which ODN 83 inhibits cell proliferation and examines its antitumor efficacy in vivo. ODN 83 treatment did not induce apoptosis in HeLa cells in vitro but caused accumulation of cells at G2/M. In contrast, TS-targeting chemotherapeutics arrest at G1 or S. Antisense down-regulation reduced TS mRNA levels in human colon cancer (HT29) cells by 40% in vitro, resulted in G2/M arrest, and reduced proliferation without enhanced cell death. Growth of HT29 tumors in immunocompromised mice was significantly inhibited when antisense ODN 83 treatment began promptly after tumor implantation and was accompanied by a 40% reduction in TS protein levels. Growth of tumors allowed to reach 400 mm3 prior to ODN administration was unaffected by antisense ODN 83. Radiolabeled ODNs were localized to the tumor periphery but evenly distributed in normal tissue. Thus, down-regulation of TS mRNA and protein by antisense ODN treatment exerts a novel G2/M cell cycle block without increasing cell death and inhibits HT29 tumor cell growth in vivo. Antisense ODN 83 may be an effective therapy for colon carcinoma, alone or in combination with TS-targeting cytotoxic drugs.

Animals↗

PubMed2005

[Antisense RNA targeting survivin enhances the chemosensitivity of LOVO/Adr cells to taxotere].

OBJECTIVE: To investigate the ability of antisense RNA eukaryotic expression plasmid pcDNA3.0/survivin targeting survivin gene to inhibit survivin expression and enhance the sensitivity to taxotere in multidrug resistant colon carcinoma cell line LOVO/Adr. METHODS: The antisense RNA eukaryotic plasmid pcDNA3.0/survivin was transfected into LOVO/Adr cells by lipofectamine. The expression of survivin mRNA was measured using RT-PCR. After treated with taxotere, MTT assay and flow cytometry were used to evaluate the proliferation inhibition and apoptosis of LOVO/Adr cells. RESULTS: The expression of survivin mRNA in LOVO/Adr cells transfected with pcDNA3.0/survivin was down-regulated in a time- dependent manner. The inhibitory rate of taxotere (0.5 micromol/L) was (37.3 +/- 2.9)% in pcDNA3.0/survivin transfected cells, significantly higher than (21.9 +/- 2.3)% and (21.1 +/- 1.9)% in pcDNA3.0 transfected and untransfected control cells respectively (P< 0.01). The apoptosis rate of taxotere was (28.7 +/- 1.7)% in pcDNA3.0/survivin transfected cells,significantly higher than (13.4 +/- 1.6)% and (14.3 +/- 1.8)% in pcDNA3.0 transfected and untransfected cells respectively. CONCLUSION: The antisense RNA eukaryotic expression plasmid pcDNA3.0/survivin could down-regulate the expression of survivin gene and enhance the chemosensitivity of LOVO/Adr cells to taxotere, which may provide a novel therapy for colon carcinoma.

Apoptosis↗

PubMed2000

The in vitro and in vivo pharmacology of antisense oligonucleotides targeted to murine Stat6.

OBJECTIVE AND DESIGN: This study was designed to establish whether phosphorothioate (PS) antisense oligonucleotides (AS-ODN) targeted to Stat6 were active in vivo in a mouse model of active sensitisation. MATERIALS: Female Balb/c mice (6-8) per group were used for in vivo study. TREATMENT: Mice were treated with active PS AS-ODNs determined in initial in vitro studies. Compounds were dosed daily (3-30mg/kg i.v.) over the course of sensitisation to ovalbumin. METHODS: Stat6 mRNA and protein levels were determined in the spleen after treatment (quantitative northern and western analysis respectively), in addition to serum IgE (ELISA). ANOVA was used to determine any significant differences between groups. RESULTS: Both of the AS-ODNs tested in vivo, down regulated Stat6 mRNA and protein levels in the spleen by 40-50% although there was no effect on serum IgE. These treatments also induced splenomegaly in vivo and caused splenocyte proliferation in vitro. CONCLUSIONS: The AS-ODNs used can down regulate Stat6 mRNA and protein although not sufficiently to influence IgE-levels. These effects are likely to be complicated in vivo by the immune-stimulation evident as splenomegaly.

Animals↗

PubMed2000

Resistance to taxanes is induced by c-erbB-2 overexpression in human MCF-10A mammary epithelial cells and is blocked by combined treatment with an antisense oligonucleotide targeting type I protein kinase A.

We have tested the sensitivity of human MCF-10A mammary epithelial cells and of their transformed derivatives overexpressing an activated c-Ha-ras gene (MCF-10A Ha-ras cells), the c-erbB-2 gene (MCF-10A c-erbB-2 cells) or both genes (MCF-10A HE cells) to different cytotoxic drugs. As compared with parental MCF-10A cells, the transformed cells exhibited an increased sensitivity to topoisomerase I- and topoisomerase II-inhibitors, and to platinum-derivatives with a 2- to 10-fold reduction in IC(50) values. A remarkable difference in sensitivity was observed following treatment with taxanes. While MCF-10A Ha-ras cells showed an increased sensitivity, MCF-10A c-erbB-2 and MCF-10A HE cells exhibited a relative resistance to taxol and taxotere, with an approximately 3.5- to 6.5-fold higher IC(50) as compared with MCF-10A cells suggesting that c-erbB-2 overexpression has a dominant effect compared with an activated c-Ha-ras gene. The type I cAMP-dependent protein kinase (PKAI) is overexpressed in cancer cells. Inhibition of PKAI by antisense oligonucleotides targeting its RIalpha regulatory subunit results in cancer cell growth inhibition. To evaluate the effect of blocking PKAI on MCF-10A cell sensitivity to taxanes, we treated these cells with taxol or taxotere in combination with a PKAI antisense oligonucleotide. Treatment with this agent, but not with a control scramble sequence, was able to overcome the effect of c-erbB-2 overexpression on MCF-10A cell sensitivity to taxol and taxotere, with a 20- to 40-fold shift in the IC(50) values for the 2 drugs.

Antineoplastic Agents↗

PubMed1992

Increased specificity for antisense oligodeoxynucleotide targeting of RNA cleavage by RNase H using chimeric methylphosphonodiester/phosphodiester structures.

One of the inherent problems in the use of antisense oligodeoxynucleotides to ablate gene expression in cell cultures is that the stringency of hybridization in vivo is not subject to control and may be sub-optimal. Consequently, phosphodiester or phosphorothioate antisense effectors and non-targeted cellular RNA may form partial hybrids which are substrates for RNase H. Such processes could promote the sequence dependent inappropriate effects recently reported in the literature. We have attempted to resolve this problem by using chimeric methylphosphonodiester/phosphodiester oligodeoxynucleotides. In contrast to the extensive RNA degradation observed with all-phosphodiester oligodeoxynucleotides, highly modified chimeric antisense effectors displayed negligible, or undetectable, cleavage at non-target sites without significantly impaired activity at the target site. We also note that all of the all-phosphodiester oligodeoxynucleotides tested demonstrated inappropriate effects, and that such undesirable activity could vary widely between different sequences.

Amino Acid Sequence↗

PubMed2004

Synergistic antitumor activity by combined treatment with gemcitabine and antisense oligodeoxynucleotide targeting clusterin gene in an intravesical administration model against human bladder cancer kotcc-1 cells.

PURPOSE: We investigated whether antisense (AS) oligodeoxynucleotide (ODN) targeting the clusterin gene enhances the cytotoxic effect of gemcitabine in human bladder cancer KoTCC-1 cells in vitro and in vivo, and evaluated the usefulness of the combined administration of AS clusterin ODN and gemcitabine using an intraperitoneal tumor cell injection model. MATERIALS AND METHODS: The cytotoxic effect of combined treatment with AS clusterin ODN and gemcitabine on in vitro KoTCC-1 growth was examined using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The in vivo growth inhibitory effects of combined AS clusterin ODN and gemcitabine therapy on subcutaneous KoTCC-1 tumor was also examined. The intraperitoneal tumor cell injection model, which mimics intravesical administration therapy against bladder cancer, was used to evaluate the efficacy of combined AS clusterin ODN and gemcitabine therapy. RESULTS: AS clusterin ODN treatment of KoTCC-1 cells significantly enhanced gemcitabine chemosensitivity in a dose dependent manner, decreasing gemcitabine IC50 by approximately 90%. In vivo systemic administration of AS clusterin ODN and gemcitabine significantly decreased subcutaneous KoTCC-1 tumor volume compared with scramble control ODN plus gemcitabine. Furthermore, combined administration of AS clusterin ODN plus gemcitabine resulted in significantly delayed formation of hemorrhagic ascites compared with scramble control ODN plus gemcitabine in an intraperitoneal tumor cell injection model. CONCLUSIONS: These findings suggest that AS clusterin ODN may be useful for enhancing the cytotoxicity of gemcitabine in patients with bladder cancer, particularly as a novel therapeutic strategy for intravesical instillation therapy.

Administration, Intravesical↗

PubMed2005

Mcl-1 overexpression in hepatocellular carcinoma: a potential target for antisense therapy.

BACKGROUND/AIMS: Recently, the anti-apoptotic Mcl-1 protein has been reported as a resistance factor in various types of cancer. Here we investigated the presence of Mcl-1 protein in hepatocellular carcinoma (HCC) tissues and its potential role as a molecular drug target for HCC therapy. METHODS: HCC specimens of 149 patients were examined by immunohistochemistry for Mcl-1 expression. Antisense oligonucleotides (ASO) targeting Mcl-1 were evaluated as monotherapy and in combination with cisplatin in the HCC cell lines HepG2 and Snu398. Protein regulation, cell viability, and apoptosis were assessed by western blotting, cell counting, and FACS analysis. RESULTS: Mcl-1 protein is overexpressed in 51% of all cases irrespective of underlying disease. Targeting Mcl-1 by ASO specifically downregulated Mcl-1 protein expression and led to significant dose and time dependent single agent activity in HCC cells characterized by increased apoptosis and decreased cell viability. No significant target regulation or cell death was observed for control oligonucleotide treatment. Upon combination with cisplatin, Mcl-1 ASO revealed a significant chemosensitizing effect. CONCLUSIONS: Mcl-1 is overexpressed in half of HCC-tissues. ASO targeting Mcl-1 revealed a prominent single agent and chemosensitizing activity against HCC in vitro. Targeting Mcl-1 might qualify as a promising novel approach in HCC therapy.

Apoptosis↗

PubMed2005

Targeting TGF-beta1 increases hepatocyte growth factor (HGF/SF) levels in external auditory canal cholesteatoma (EACC) epithelial cell culture.

OBJECTIVE: The transforming growth factor (TGF)-beta 1 is known to have pro- and anti-angiogenic actions. Hepatocyte growth factor/scatter factor (HGF/SF) antagonizes TGF-beta1 by stabilizing SMAD transcriptional co-repressor TGIF. HGF/SF is a multifunctional polypeptide with morphogenic, motogenic, angiogenic, and proliferative capabilities. We assume HGF to be a pivotal factor during the pathogenesis of the external auditory canal cholesteatoma (EACC). In this study, we investigate the effect of antisense targeting TGF-beta1 on HGF/SF levels in the epithelial EACC-culture. MATERIALS: For 48 h, epithelial EACC cell culture was incubated with 3 and 6 mumol antisense targeting TGF-beta1, respectively. Levels of HGF/SF were determined and normalized to cellular protein. Untreated EACC cell culture and scrambled TGF-beta1-antisense served as control. In the second experiment, EACC cells were incubated with rh TGF-beta1 (2 and 4 ng/ml) for 48 h and HGF/SF was determined. RESULTS: After incubation with 3 mumol TGF-beta1-antisense, the average level of HGF/SF was measured at 43.68 pg/ml. Incubation with 6 micromol TGF-beta1-antisense showed 64.95 pg/ml. In untreated EACC (control), the average level of HGF/SF after 48 was 34.55 pg/ml. Incubation with scrambled TGF-beta1 oligonucleotide showed an average HGF/SF level of 34.41 (3 micromol) and 35.66 (6 micromol), respectively. The difference between the scrambled antisense and the targeting antisense TGF-beta1 was significant (p<0.05). After incubation with 2 ng/ml TGF-beta1, the HGF/SF levels were at 22.16 pg/ml. TGF-beta1, 4 ng/ml, resulted in 15.33 pg/ml of HGF/SF. The difference of the levels of HGF/SF after incubation with exogenous TGF-beta1 was significant (p<0.05). CONCLUSION: In this study, levels of HGF/SF increased in the epithelial EACC cell culture after incubation with 3 and 6 mumol antisense TGF-beta1 oligonucleotides depending on the concentration of the antisense. In reverse, TGF-beta1 acted as inhibiting cytokine on HGF/SF levels. In conclusion, TGF-beta1 may be a useful therapeutic agent for managing EACC.

Blotting, Western↗