Simultaneous blockage of different EGF-like growth factors results in efficient growth inhibition of human colon carcinoma xenografts

A majority of human colon carcinomas coexpress the epidermal growth factor (EGF)-related peptides transforming growth factor alpha (TGFalpha), amphiregulin (AR) and CRIPTO-1 (CR). We have synthesized novel, antisense mixed backbone oligonucleotides (AS MBOs) directed against TGFalpha, AR and CR. We screened the EGF-related AS MBOs for their ability to inhibit the anchorage independent growth of GEO human colon carcinoma cells. The MBOs that showed a high in vitro efficacy were then used for in vivo experiments. TGFalpha, AR and CR AS MBOs were able to inhibit the growth of GEO tumor xenografts in nude mice in a dose-dependent manner. Furthermore, the AS MBOs were able to specifically inhibit the expression of the target mRNAs and proteins in the tumor xenografts. A more significant tumor growth inhibition was observed when mice were treated with a combination of the three AS MBOs as compared to treatment with a single AS MBO. Finally, tumors from mice treated with TGFalpha, AR and CR AS MBOs showed a significant reduction of microvessel count, as compared with tumors from untreated mice or from mice treated with a single AS MBO. These data suggest that combinations of AS oligonucleotides directed against different growth factors might represent a novel, experimental therapy approach of colon carcinomas.     

Antitumor effect and potentiation of cytotoxic drugs activity in human cancer cells by ZD-1839 (Iressa), an epidermal growth factor receptor-selective tyrosine kinase inhibitor.

Transforming growth factor alpha (TGF-alpha) is an autocrine growth factor for human cancer. Overexpression of TGF-alpha and its specific receptor, the epidermal growth factor receptor (EGFR), is associated with aggressive disease and poor prognosis. The EGFR has been proposed as a target for anticancer therapy. Compounds that block ligand-induced EGFR activation have been developed. ZD-1839 (Iressa) is a p.o.-active, quinazoline derivative that selectively inhibits the EGFR tyrosine kinase and is under clinical development in cancer patients. The antiproliferative activity of ZD-1839 alone or in combination with cytotoxic drugs differing in mechanism(s) of action, such as cisplatin, carboplatin, oxaliplatin, paclitaxel, docetaxel, doxorubicin, etoposide, topotecan, and raltitrexed, was evaluated in human ovarian (OVCAR-3), breast (ZR-75-1, MCF-10A ras), and colon cancer (GEO) cells that coexpress EGFR and TGF-alpha. ZD-1839 inhibited colony formation in soft agar in a dose-dependent manner in all cancer cell lines. The antiproliferative effect was mainly cytostatic. However, treatment with higher doses resulted in a 2-4-fold increase in apoptosis. A dose-dependent supra-additive increase in growth inhibition was observed when cancer cells were treated with each cytotoxic drug and ZD-1839. The combined treatment markedly enhanced apoptotic cell death induced by single-agent treatment. ZD-1839 treatment of nude mice bearing established human GEO colon cancer xenografts revealed a reversible dose-dependent inhibition of tumor growth because GEO tumors resumed the growth rate of controls at the end of the treatment. In contrast, the combined treatment with a cytotoxic agent, such as topotecan, raltitrexed, or paclitaxel, and ZD-1839 produced tumor growth arrest in all mice. Tumors grew slowly for approximately 4-8 weeks after the end of treatment, when they finally resumed a growth rate similar to controls. GEO tumors reached a size not compatible with normal life in all control mice within 4-6 weeks and in all single agent-treated mice within 6-8 weeks after GEO cell injection. In contrast, 50% of mice treated with ZD-1839 plus topotecan, raltitrexed, or paclitaxel were still alive 10, 12, and 15 weeks after cancer cell injection, respectively. These results demonstrate the antitumor effect of this EGFR-selective tyrosine kinase inhibitor and provide a rationale for its clinical evaluation in combination with cytotoxic drugs.     

EGFR antisense treatment of human HNSCC cell lines down-regulates VEGF expression and endothelial cell migration

Overexpression of the epidermal growth factor receptor (EGFR) is thought to play a key role in the development of head and neck squamous cell carcinoma (HNSCC) primarily through its effect on promoting uncontrolled cell proliferation. Blocking EGFR ligand binding might also inhibit angiogenesis and down-regulate the production of angiogenic factors. Angiogenesis is increased in various human tumors, including head and neck squamous cell carcinoma (HNSCC), and correlates with tumor progression and metastasis. The vascular endothelial growth factor (VEGF) is thought to be the most important angiogenic factor. We determined whether VEGF antisense oligonucleotide treatment can decrease angiogenic activity of HNSCC cell lines in vitro. By using a 21-mer antisense phosphorothioate oligonucleotide targeting the translation start site of human EGFR mRNA, we examined modulation of VEGF expression in cell line supernatants by capture ELISA, and in cell lysates by Western blotting. Human umbilica vein endothelial cells (HUVEC) were grown in conditioned medium produced from the treated tumor cells. Endothelial cell migration was measured using a modified Boyden chamber. EGFR antisense oligonucleotide treatment resulted in a significant reduction of VEGF protein expression compared to sense oligonucleotide control. Addition of conditioned medium from EGFR antisense-treated tumor cells resulted in decreased endothelial cell migration. In conclusion, therapeutic strategies targeting EGFR signaling in head and neck cancer might have an antitumor effect mediated in part by inhibition of tumor angiogenesis.     

The effect of epidermal growth factor receptor antisense morpholino oligomer on non-small cell lung cancer cell line

Overexpression of the epidermal growth factor receptor (EGFR) has been identified as a common component of various cancer types including lung cancer. Recently morpholino oligonucleotides appeared as a promising modification for antisense applications with few toxic effects and their stability. We investigated the effect of EGFR antisense morpholino oligomer on non-small cell lung cancer (NSCLC) cell line by evaluating EGFR mRNA, protein product and cell proliferation. The EGFR antisense morpholino oligomer was designed to target the translation start site in the EGFR mRNA. The four base-mismatch morphlino oligomer was designed as a control for EGFR antisense morpholino oligomer. These morpholino oligomers were introduced into NCI-H125 cell line which showed overexpression of EGFR. The EGFR mRNA and protein expression were quantified by real time RT-PCR and ELISA, respectively. The significant repression in both EGFR mRNA and protein expression was observed for three days after single treatment with EGFR antisense morpholino oligomer. Furthermore, the growth of NCI-H125 cell line was significantly inhibited with treatment by EGFR antisense morpholino oligomer. Our results indicate that EGFR antisense morpholino oligomer represses the EGFR expression at both mRNA and protein level and inhibits the proliferation of NSCLC cell line suggesting that it may be a promising strategy as one of antisense therapies for NSCLC.     

Antisense epidermal growth factor receptor delivered by adenoviral vector blocks tumor growth in human gastric cancer.

Epidermal growth factor receptor (EGFR) protein overexpression is commonly found in human gastric cancer, and its gene amplification is known to correlate with poor prognosis in gastric cancer patients. With regard to therapy trials targeting EGFR, it has been reported that stable transfection of EGFR antisense or treatment with antibody against EGFR results in growth suppression of human cancer cells that express high levels of EGFR. We have designed an adenovirus-expressing antisense EGFR and have investigated its effect on the growth of gastric cancer in vitro and in vivo. Following infection with EGFR antisense RNA-expressing adenovirus (Ad-EAS), the cell surface EGFR protein levels of infected cancer cells were markedly reduced, and the in vitro growth of Ad-EAS-infected cells was significantly inhibited relative to control-infected cells in all three gastric cancer cell lines (AGS, KKLS, and MKN28) studied here (P < .0002). In a nude mouse subcutaneous tumor system, in vivo tumor growth of MKN28 was significantly inhibited after Ad-EAS treatment, and inhibition on day 48 was 93% by volume compared with that of untreated controls. These results suggest that an adenoviral vector system targeting the down-regulation of EGFR could be a good candidate for the therapy of gastric cancers that overexpress EGFR.     

Potential roles of antisense therapy in the molecular targeting of genes involved in cancer (review)

Recent advances in antisense methods targeting genes involved in cell proliferation, angiogenesis, and apoptosis provide a potential anticancer effect alone as well as in combination with drugs, and antisense therapy may be useful in overcoming drug resistance and increasing survival in patients with advanced cancer including those with solid tumors. In particular, antisense to Bcl-2 comprises a most promising therapy and is being tested in combination with anticancer drugs in randomized phase III trials for chronic lymphocytic leukemia, multiple myeloma, and malignant melanoma. The targeting of apoptosis-related proteins is promising for enhancing the effect of cancer chemotherapy. The molecular mechanism by which anticancer drugs induce apoptosis has been identified as mitochondrial dysfunction mediated by the release of cytochrome c. Modulation of multiple antiapoptotic signaling pathways involving Bcl-2 and Akt, which are related to growth factor-stimulated signal transduction in cell survival, is essential for enhancement of the cytotoxic effect of anticancer drugs. Herein, we review the current status of antisense therapy and its potential for enhancing anticancer drug-induced apoptosis.     

Synergistic growth inhibition and induction of apoptosis by a novel mixed backbone antisense oligonucleotide targeting CRIPTO in combination with C225 anti-EGFR monoclonal antibody and 8-Cl-cAMP in human GEO colon cancer cells.

We have evaluated the antiproliferative effect of a novel mixed backbone antisense oligonucleotide generated against the 5'-coding region of the human CRIPTO mRNA in GEO human colon cancer cells. We have also evaluated the effects of this anti-CRIPTO antisense oligonucleotide in combination with a chimeric anti-human epidermal growth factor receptor (EGFR) monoclonal antibody (MAb C225) and with 8-Cl-cAMP, a cAMP analog that specifically inhibits type I protein kinase A (PKAI), since a functional EGFR-driven autocrine pathway is operative and PKAI is overexpessed in GEO colon cancer cells. Treatment with a single agent at low doses determined a 15-35% growth inhibition. A synergistic antiproliferative effect was observed when combinations of two agents were used with a co-operativity quotient ranging between 1.5 and 2.2. Furthermore, the combined treatment with all three drugs caused an almost complete suppression of the ability of GEO cells to form colonies in soft agar. We next evaluated whether any combination of 8-Cl-cAMP, the anti-CRIPTO antisense oligonucleotide and MAb C225 could induce programmed cell death in GEO cells. Treatment with each agent alone at all doses tested did not cause DNA fragmentation. The treatment with any combination of two agents was not able to induce apoptosis. In contrast, treatment with all three compounds determined an approximately three-fold increase in DNA fragmentation. In conclusion, the combination of selective antineoplastic agents directed against different but related key signal tranduction pathways efficiently inhibits cell growth and causes apoptosis in human colorectal cancer cells.     

Antisense therapy for cancer--the time of truth

The recent acceleration in the identification and characterisation of new molecular targets for cancer and the limited effectiveness of conventional treatment strategies has focused considerable interest on the development of new types of anticancer agents. These new drugs are hoped to be highly specific for malignant cells with a favorable side-effect profile due to well-defined mechanisms of action. Antisense oligonucleotides are one such class of new agent--they are short, synthetic stretches of DNA which hybridise with specific mRNA strands that correspond to target genes. By binding to the mRNA, the antisense oligonucleotides prevent the sequence of the target gene being converted into a protein, thereby blocking the action of the gene. Several genes known to be important in the regulation of apoptosis, cell growth, metastasis, and angiogenesis, have been validated as molecular targets for antisense therapy. Furthermore, new targets are rapidly being uncovered through coordinated functional genomics and proteomics initiatives. Phosphorothioate oligonucleotides are the current gold standard for antisense therapy; they have acceptable physical and chemical properties and show reasonable resistance to nucleases. Recently, new generations of these phosphorothioate oligonucleotides that contain 2'-modified nucleoside building blocks to enhance RNA binding affinity and decrease indirect toxic effects have been developed. Antisense therapeutics are, after decades of difficulties, finally close to fulfilling their promise in the clinic.     

Antitumor activity of sequential treatment with topotecan and anti-epidermal growth factor receptor monoclonal antibody C225.

Epidermal growth factor (EGF)-related proteins such as transforming growth factor alpha (TGF-alpha) control cancer cell growth through autocrine and paracrine pathways. Overexpression of TGF-alpha and/or its receptor (EGFR) has been associated with a more aggressive disease and a poor prognosis. The blockade of EGFR activation has been proposed as a target for anticancer therapy. Monoclonal antibody (MAb) C225 is an anti-EGFR humanized chimeric mouse MAb that is presently in Phase II clinical trials in cancer patients. Previous studies have suggested the potentiation of the antitumor activity of certain cytotoxic drugs, such as cisplatin and doxorubicin, in human cancer cell lines by treatment with anti-EGFR antibodies. We have evaluated in human ovarian, breast, and colon cancer cell lines, which express functional EGFR, the antiproliferative activity of MAb C225 in combination with topotecan, a cytotoxic drug that specifically inhibits topoisomerase I and that has shown antitumor activity in these malignancies. A dose-dependent supraadditive increase of growth inhibition in vitro was observed when cancer cells were treated with topotecan and MAb C225 in a sequential schedule. In this respect, the cooperativity quotient, defined as the ratio between the actual growth inhibition obtained by treatment with topotecan followed by MAb C225 and the sum of the growth inhibition achieved by each agent, ranged from 1.2 to 3, depending on drug concentration and cancer cell line. Treatment with MAb C225 also markedly enhanced apoptotic cell death induced by topotecan. For example, in GEO colon cancer cells, 5 nM topotecan, followed by 0.5 microg/ml MAb C225, induced apoptosis in 45% cells as compared with untreated cells (6%) or to 5 nM topotecan-treated cells (22%). Treatment of mice bearing established human GEO colon cancer xenografts with topotecan or with MAb C225 determined a transient inhibition of tumor growth because GEO tumors resumed the growth rate of untreated tumors at the end of the treatment period. In contrast, an almost complete tumor regression was observed in all mice treated with the two agents in combination. This determined a prolonged life span of the mice that was significantly different as compared with controls (P < 0.001), to MAb C225-treated group (P < 0.001), or to the topotecan-treated group (P < 0.001). All mice of the topotecan plus MAb C225 group were the only animals alive 14 weeks after tumor cell injection. Furthermore, 20% of mice in this group were still alive after 19 weeks. The combined treatment with MAb C225 and topotecan was well tolerated by mice with no signs of acute or delayed toxicity. These results provide a rationale for the evaluation of the anticancer activity of the combination of topoisomerase I inhibitors and anti-EGFR blocking MAbs in clinical trials.     

Antitumor activity of ZD6474, a vascular endothelial growth factor receptor tyrosine kinase inhibitor, in human cancer cells with acquired resistance to antiepidermal growth factor receptor therapy.

PURPOSE: The epidermal growth factor receptor (EGFR) autocrine signaling pathway is involved in cancer development and progression. EGFR inhibitors such as C225 (cetuximab), a chimeric human-mouse anti-EGFR monoclonal antibody, and ZD1839 (gefitinib), a small molecule EGFR-selective tyrosine kinase inhibitor, are in advanced clinical development. The potential emergence of cancer cell resistance in EGFR-expressing cancers treated with EGFR inhibitors could determine lack of activity of these drugs in some cancer patients. Vascular endothelial growth factor (VEGF) is secreted by cancer cells and plays a key role in the regulation of tumor-induced endothelial cell proliferation and permeability. ZD6474 is a small molecule VEGF flk-1/KDR (VEGFR-2) tyrosine kinase inhibitor that also demonstrates inhibitory activity against EGFR tyrosine kinase. EXPERIMENTAL DESIGN: The antitumor activity of ZD1839, C225, and ZD6474 was tested in athymic mice bearing human GEO colon cancer xenografts. GEO cell lines resistant to EGFR inhibitors were established from GEO xenografts growing in mice treated chronically with ZD1839 or C225. Expression of EGFR was evaluated by flow cytometry. Expression of various proteins involved in intracellular cell signaling was assessed by Western blotting. Tumor growth data were evaluated for statistical significance using the Student's t test. All Ps were two-sided. RESULTS: Although chronic administration of optimal doses of C225 or ZD1839 efficiently blocked GEO tumor growth in the majority of mice, tumors slowly started to grow within 80-90 days, despite continuous treatment. In contrast, continuous treatment of mice bearing established GEO xenografts with ZD6474 resulted in efficient tumor growth inhibition for the entire duration of dosing (up to 150 days). ZD6474 activity was also determined in mice pretreated with ZD1839 or C225. When GEO growth was apparent after 4 weeks of treatment with EGFR inhibitors, mice were either re-treated with EGFR inhibitors or treated with ZD6474. GEO tumor growth was blocked only in mice treated with ZD6474, whereas tumor progression was observed in mice re-treated with C225 or ZD1839. GEO tumors growing during treatment with C225 or with ZD1839 were established as cell lines (GEO-C225-RES and GEO-ZD1839-RES, respectively). Cell membrane-associated EGFR expression was only slightly reduced in these cell lines compared with parental GEO cells. Western blotting revealed no major change in the expression of the EGFR ligand transforming growth factor alpha of bcl-2, bcl-xL, p53, p27, MDM-2, akt, activated phospho-akt, or mitogen-activated protein kinase. However, both GEO-C225-RES and GEO-ZD1839-RES cells exhibited a 5-10-fold increase in activated phospho-mitogen-activated protein kinase and in the expression of cyclooxygenase-2 and of VEGF compared with GEO cells. GEO-C225-RES and GEO-ZD1839-RES growth as xenografts in nude mice was not significantly affected by treatment with either C225 or ZD1839 but was efficiently inhibited by ZD6474. CONCLUSIONS: Long-term treatment of GEO xenografts with selective EGFR inhibitors results in the development of EGFR inhibitor-resistant cancer cells. Growth of EGFR inhibitor-resistant tumors can be inhibited by ZD6474. These data indicate that inhibition of VEGF signaling has potential as an anticancer strategy, even in tumors that are resistant to EGF inhibitors.     

Antisense oligonucleotides directed against p53 have antiproliferative effects unrelated to effects on p53 expression.

Antisense oligonucleotides targeting p53 have been hailed as a potentially new technique for treating patients with cancer, and there have been encouraging reports of good patient tolerance in vivo and of antiproliferative effects in vitro. However, evidence is lacking that these oligonucleotides are acting via an antisense interaction to modulate p53 expression. We examined a phosphorothioate antisense oligonucleotide, directed against exon 10 of the TP53 gene, and a chimaeric phosphorothioate-phosphodiester oligonucleotide directed against the p53 translation initiation codon. Both failed to specifically suppress p53 protein production in a cell-free assay system or to have any effect on mutant p53 expression by human pancreatic cancer cell lines. Antiproliferative effects were apparent, especially with the phosphorothioate antisense oligonucleotide, but this was independent of the p53 status of the cells (mutant, wild-type or absent) and also occurred with the control (sense and randomised) oligonucleotides. The most dramatic antiproliferative effects were seen with the ''control'' phosphorothioate oligonucleotides. These findings suggest that the antiproliferative effects of some antisense oligonucleotides may be unrelated to expression of the gene they have been designed to target.     

Antitumor activity of combined treatment of human cancer cells with ionizing radiation and anti-epidermal growth factor receptor monoclonal antibody C225 plus type I protein kinase A antisense oligonucleotide.

Recent studies have suggested that selective inhibition of mitogenic pathways may improve the antitumor activity of ionizing radiation. The epidermal growth factor receptor (EGFR) is overexpressed and is involved in autocrine growth control in the majority of human carcinomas. Protein kinase A type I (PKAI) plays a key role in neoplastic transformation and is overexpressed in cancer cells in which an EGFR autocrine pathway is activated. We used two specific inhibitors of EGFR and PKAI that are under clinical evaluation in cancer patients: C225, an anti-EGFR chimeric human-mouse monoclonal antibody (MAb); and a mixed-backbone antisense oligonucleotide targeting the PKAI RIalpha subunit (PKAI AS). We tested in human colon cancer (GEO) and ovarian cancer (OVCAR-3) cell lines the antiproliferative activity of MAb C225 and/or PKAI AS in combination with ionizing radiation. In vivo antitumor activity was evaluated in nude mice bearing established GEO xenografts. Dose-dependent inhibition of soft agar growth was observed in both cancer cell lines with ionizing radiation, C225, or PKAI AS oligonucleotide. A cooperative antiproliferative effect was obtained when cancer cells were treated with ionizing radiation followed by MAb C225 or PKAI AS oligonucleotide. This effect was observed at all doses tested in both GEO and OVCAR-3 cancer cell lines. A combination of the three treatments at the lowest doses produced an even greater effect than that observed when two modalities were combined. Treatment of mice bearing established human GEO colon cancer xenografts with radiotherapy (RT), MAb C225, or PKAI AS oligonucleotide produced dose-dependent tumor growth inhibition that was reversible upon treatment cessation. A potentiation of the antitumor activity was observed in all mice treated with RT in combination with MAb C225 or PKAI AS oligonucleotide. Long-term GEO tumor growth regression was obtained following treatment with ionizing radiation in combination with MAb C225 plus PKAI AS oligonucleotide, which produced a significant improvement in survival compared with controls (P < 0.001), the RT-treated group (P < 0.001), or the group treated with MAb C225 plus PKAI AS oligonucleotide (P < 0.001). All mice of the RT + MAb C225 + PKAI AS group were alive 26 weeks after tumor cell injection. Furthermore, 50% of mice in this group were alive and tumor-free after 35 weeks. This study provides a rationale for evaluating in cancer patients the combination of ionizing radiation and selective drugs that block EGFR and PKAI pathways.     

Antisense treatment against Ki-67 mRNA inhibits proliferation and tumor growth in vitro and in vivo

The Ki-67 protein is tightly regulated and depends on the proliferative status of a cell. It is present in the nuclei of proliferating cells but absent in resting cells. Since transformation of malignant cells is frequently associated with high cell proliferation and since proliferation is tightly associated with the Ki-67 protein labeling index, this antigen may represent a potential target for cancer therapy. In the present study we determined the ability of a phosphorothioate antisense oligodeoxyribonucleotide (ODN) targeted against Ki-67 mRNA to inhibit tumor cell proliferation specifically in cell culture, in multicellular 3-dimensional spheroids (MCS) and in subcutaneous murine tumor models. Antisense treatment of 1 myeloid and different epithelial tumor cell lines in suspension and monolayer culture, respectively, resulted in specific reduction of Ki-67 mRNA and protein, inhibition of proliferation and increased apoptotic cell death. Multicellular human bladder carcinoma spheroids lost their 3-dimensional structure and underwent cell death after incubation with antisense oligonucleotides. The growth of subcutaneous syngeneic prostatic (p = 0.05) and transitional cell tumors (p = 0.001) in immunocompetent mice was significantly inhibited in antisense-treated animals. From these findings we conclude that antisense inhibition of Ki-67 protein expression may be a rational approach in anticancer therapy.     

Contiguous Four-guanosine Sequence in c-myc Antisense Phosphorothioate Oligonucleotides Inhibits Cell Growth on Human Lung Cancer Cells: Possible Involvement of Cell Adhesion Inhibition

A contiguous four-guanosine (4G) sequence in c-myc antisense phosphorothioate oligonucleotides caused an antiproliferative effect in smooth muscle cells. To investigate the antiproliferative effect of c-myc antisense oligonucleotides on human lung cancer cell lines, we synthesized oligonucleotides of various lengths and sequences, focusing on the contiguous four-guanosine (4G) sequence. While a c-myc antisense oligonucleotide (20AS1 (4G)) targeted to the translation initiation codon of c-myc mRNA inhibited cell growth of A549 cells by 69% at 10 μM, a scrambled oligonucleotide (20SCR1 (4G)) containing the contiguous four-guanosine (4G) sequence also inhibited cell growth by 72% at the same dose. Although treatment with either 20AS1 (4G) or 20SCR1 (4G) inhibited cell adhesion by 70% at 10 μM, expression of c-myc protein was significantly suppressed only by 20AS1 (4G) (62%), and was only weakly inhibited by 20SCR1 (4G) (32%). Furthermore, a small cell lung carcinoma cell line, Lu65, which can grow in suspension form, was highly resistant to 20AS1 (4G) treatment (IC₅₀>20 μM). These results suggest that the cell growth inhibition by C-myc antisense oligonucleotides containing the contiguous four-guanosine (4G) sequence was possibly correlated with inhibition of cell adhesion, but not with inhibition of c-myc protein expression, via a sequence-specific non-antisense mechanism.     

Single-injection ornithine decarboxylase-directed antisense therapy using atelocollagen to suppress human cancer growth

BACKGROUND: Substantial evidence supports a direct role of ornithine decarboxylase (ODC) in the development and maintenance of human tumors. Although antisense oligonucleotide therapy targeting various genes are useful for cancer treatment, 1 of the major limitations is the problem of delivery. A novel antisense oligonucleotide delivery method is described that allows prolonged sustainment and release of ODC antisense oligonucleotides in vivo using atelocollagen. METHODS: The effect of ODC antisense oligonucleotides in the atelocollagen on cell growth of gastrointestinal cancer (MKN 45 and COLO201) and rhabdomyosarcoma (RD) was studied in vitro using a cell-counting method with a hemocytometer. In vivo, the effect of intratumoral, intramuscular, and intraperitoneal single administration of ODC antisense oligonucleotides in the atelocollagen on tumor growth of MKN45, COLO201, and RD cells was studied. ODC activity and polyamine contents were measured. RESULTS: In vitro, ODC antisense oligonucleotides in the atelocollagen remarkably suppressed MKN45, COLO201, and RD cell growth. A single administration of antisense oligonucleotides in the atelocollagen via 3 routes remarkably suppressed the growth of MKN45, COLO201, and RD tumor over a period of 35-42 days. CONCLUSIONS: As various human cancers significantly express ODC, the results strongly suggest that this new antisense method may be of considerable value for treatment of human cancers.     

Abrogation of VEGF expression in human head and neck squamous cell carcinoma decreases angiogenic activity in vitro and in vivo

Angiogenesis is increased in various human cancers, including head and neck squamous cell carcinoma (HNSCC), and correlates with tumor progression and metastasis. Vascular endothelial growth factor (VEGF) has been shown to be a key regulator of angiogenesis. We determined whether VEGF antisense oligonucleotide treatment can decrease angiogenic activity of HNSCC cell lines in vitro and of HNSCC xenografts in vivo. Established human HNSCC cell lines were screened for VEGF expression at both mRNA and protein levels. By using a 21-mer antisense phosphorothioate oligonucleotide targeting the translation start site of human VEGF mRNA, we examined modulation of VEGF expression in cell line supernatants by capture ELISA, and in cell lysates by Western blotting. Human umbilica vein endothelial cells (HUVEC) were grown in conditioned medium produced from the treated tumor cells. Endothelial cell (EC) proliferation was determined by cell count and EC migration was measured using a modified Boyden chamber. Mice with HNSCC xenografts were treated with PBS, VEGF antisense or sense oligonucleotides (10 mg/kg; i.p. injection), respectively and tumor volumes were measured for 5 weeks. VEGF antisense oligonucleotide treatment resulted in a significant reduction of VEGF protein expression compared to sense control. Although the growth rate of the tumor cell lines was not affected, addition of conditioned medium from VEGF antisense-treated tumor cells resulted in decrease of endothelial cell proliferation and migration. VEGF antisense oligonucleotide treatment of HNSCC xenografts resulted in a significant tumor growth suppression. These results suggest that downmodulation of VEGF using antisense oligonucleotides may be a potential therapy for the inhibition of angiogenesis in HNSCC.     

The epidermal growth factor receptor signaling network in head and neck carcinogenesis and implications for targeted therapy

Improved understanding of the molecular signaling pathways that mediate cellular transformation has led to the development of novel strategies for the treatment of cancer. The epidermal growth factor receptor (EGFR), a transmembrane protein with intrinsic tyrosine kinase activity, transduces important signals from the surface of epithelial cells to the intracellular domain. Aberrant signaling through EGFR plays a key role in the carcinogenesis of squamous cell carcinomas of the head and neck (SCCHN). SCCHN tend to express high levels of EGFR, and the degree of expression correlates with poor clinical outcome. Since EGFR is present at much higher levels in cancerous lesions than in normal epithelial tissue, the receptor has been implicated as a highly specific therapeutic target for the treatment of SCCHN. EGFR can be abrogated at the extracellular level using either monoclonal antibodies or toxin conjugates that compete with the natural ligand at the binding site of the receptor, and targeting of the EGFR intracellular domain has been achieved by specific inhibitors of tyrosine kinase activity. Antisense strategies use synthesized DNA or RNA oligonucleotides to block the translation of the mRNA sequences that code for the production of the EGFR or other proteins with a role in EGFR-mediated cell signaling. Clinical evaluation of EGFR-specific monoclonal antibodies and tyrosine kinase inhibitors has demonstrated limited toxicity in SCCHN patients, and concurrent administration with standard cytotoxic therapies has produced additive or synergistic antitumor effects.     

A novel antisense oligonucleotide targeting survivin expression induces apoptosis and sensitizes lung cancer cells to chemotherapy

Survivin, an inhibitor of apoptosis protein, deserves attention as a selective target for cancer therapy because it lacks expression in differentiated adult tissues but is expressed in a variety of human tumors. We designed 20-mer phosphorothioate antisense oligonucleotides targeting different regions of survivin mRNA and investigated their ability to down-regulate survivin mRNA and induce apoptosis in the lung adenocarcinoma cell line A549. Oligonucleotide 4003, which targets nucleotides 23-251 of survivin mRNA, was identified as the most potent compound. As measured by real-time PCR, 4003 down-regulated survivin mRNA in a dose-dependent manner with an IC50 of 200 nM. Its maximum effect was achieved at a concentration of 400 nM, at which mRNA was down-regulated by 70%. As revealed by increased caspase-3-like protease activity, nuclear condensation and fragmentation, and trypan blue uptake, treatment with 4003 induced apoptosis and sensitized tumor cells to the chemotherapeutic agent etoposide. Oligonucleotide 4003 did not reduce the viability of normal blood leukocytes with marginal levels of survivin mRNA.     

The role of epidermal growth factor receptor in head and neck squamous cell carcinoma

Growth factor receptors play a crucial role in the cell proliferation pathways involved in the development of cancer. One such receptor, the epidermal growth factor receptor (EGFR), is upregulated in many types of human tumors, particularly head and neck squamous cell carcinoma (HNSCC). EGFR overexpression in HNSCC has been the basis for investigation of therapeutic strategies that target EGFR. EGFR-blocking methods under evaluation involve immunotoxins, monoclonal antibodies, EGFR-specific tyrosine kinase inhibitors, and antisense approaches. These molecular targeting tactics have produced a number of agents that are currently in various stages of preclinical investigation, along with clinical trials to assess their potential as anticancer treatments.