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Purpose: Immunostimulating Toll-like receptor 9 (TLR9) agonists cause antitumor activity interfering also with cancer proliferation and angiogenesis by mechanisms still incompletely understood. We hypothesized that modified TLR9 agonists could impair epidermal growth factor receptor (EGFR) signaling and, by this means, greatly enhance EGFR inhibitors effect, acting on both the receptor targeting and the immunologic arm., Experimental Design: We used a novel second-generation, modified, immunomodulatory TLR9 agonist (IMO), alone and in combination with the anti-EGFR monoclonal antibody cetuximab or tyrosine kinase inhibitor gefitinib, on the growth of GEO and cetuximab-resistant derivatives GEO-CR colon cancer xenografts. We have also evaluated the expression of several proteins critical for cell proliferation, apoptosis, and angiogenesis, including EGFR, mitogen-activated protein kinase, Akt, bcl-2, cyclooxygenase-2, vascular endothelial growth factor, and nuclear factor-kappaB., Results: IMO inhibited GEO growth and signaling by EGFR and the other proteins critical for cell proliferation and angiogenesis. IMO plus the anti-EGFR antibody cetuximab synergistically inhibited tumor growth, signaling proteins, and microvessel formation. EGFR signaling inhibition by IMO is relevant because IMO cooperated also with EGFR tyrosine kinase inhibitor gefitinib in GEO tumors, while it was inactive against GEO-CR xenografts. On the other hand, IMO boosted the non-EGFR-dependent cetuximab activity, causing a cooperative antitumor effect in GEO-CR cells. Finally, combination of IMO, cetuximab and chemotherapeutic irinotecan eradicated the tumors in 90% of mice., Conclusion: IMO interferes with EGFR-related signaling and angiogenesis and has a synergistic antitumor effect with EGFR inhibitors, especially with cetuximab, boosting both the EGFR dependent and independent activity of this agent. Moreover, this therapeutic strategy could be translated in patients affected by colorectal cancer.

Purpose: Glioblastoma multiforme is an aggressive disease in which vascular endothelial growth factor (VEGF) and the EGF receptor (EGFR) are implicated in tumor growth, relapse, and resistance to radiotherapy and chemotherapy. The VEGF receptors VEGFR-1 (flt-1) and VEGFR-2 (KDR), typically present on endothelial cells, have also been identified in human glioblastoma tissues and cell lines. In addition, EGFR is dysregulated in the majority of human glioblastomas and EGFR overexpression correlates with shorter survival. We have investigated the antitumor and antiangiogenic effect of ZD6474, an inhibitor of both VEGFR and EGFR signaling as a single agent and in combination with ionizing radiation., Experimental Design: We have used ZD6474 and/or ionizing radiation in human glioblastoma cell lines D54 and U251 in vitro and in nude mice bearing established xenografts. The effects of treatment on tumor blood vessels and protein expression were evaluated by Western blot and immunohistochemistry., Results: As single agents, ionizing radiation and ZD6474 caused a dose-dependent inhibition of soft agar growth in D54 and U251 cell lines, whereas a cooperative effect was obtained in combination. Treatment of mice bearing D54 xenografts with either ZD6474 or radiotherapy alone caused tumor growth inhibition that was reversible upon treatment cessation. A cooperative and long-lasting inhibition of tumor growth was obtained with ZD6474 in combination with concomitant radiotherapy. The antiproliferative effect was accompanied by inhibition of VEGF protein expression and inhibition of angiogenesis as measured by vessel counting., Conclusion: This study shows the antitumor activity of ZD6474 in combination with ionizing radiation in glioblastoma both in vitro and in vivo, and provides a scientific rationale to evaluate ZD6474 alone or in combination with radiotherapy in patients affected by this disease.

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.

In the present paper, we investigated the relationship between the growth inhibitory effects of recombinant interferon-alpha2b (rIFN-alpha2b) and poly (ADPR) polymerase-1 (PARP-1) activity in the human squamous KB cancer cell line. Growth inhibition of the KB cells mediated by 1000 IU/ml of rIFN-alpha2b was accompanied by a transient rise in PARP-1 specific activity 24 h after rIFN-alpha2b treatment, confirmed by both the increase of intracellular poly (ADP-ribose) content and the PARP-1 auto-modification level. At longer times of incubation, the onset of apoptosis accompanied KB cell growth inhibition, as demonstrated by both flow cytometry and western-blotting analysis showing an 89 kDa apoptotic fragment of PARP-1. Moreover, pretreatment of the cells with the PARP-1 inhibitor, 3-aminobenzamide (3-ABA), at non-cytotoxic concentrations (1 mM), reduced the cell-growth inhibition, cell-cycle perturbation and apoptosis caused by rIFN-alpha2b. Taken together, these results strongly suggest that PARP-1 may be directly involved in the effects of rIFN-alpha2b in the KB cancer cell line.

Purpose: Epidermal growth factor receptor (EGFR) and protein kinase A type I(PKAI) play an important role in the control of cancer cell growth and angiogenesis. Inhibitors of EGFR and PKAI have antitumor activity in vitro and in vivo in a variety of tumor types, and some of these agents are active after oral administration. Increasing evidence shows that cyclooxygenase (COX)-2 also plays a role in promoting cancer cell proliferation and angiogenesis. COX-2 expression can be induced by EGFR activation and is regulated by cAMP and PKA. Combination of an EGFR inhibitor with a nonselective COX-1/COX-2 inhibitor prevents the development of intestinal cancer in nude mice. Therefore, we investigated whether any cooperative antitumor effect can be obtained by the combined blockade of COX-2, EGFR, and PKAI., Experimental Design: The COX-2 inhibitor SC-236 was combined with the selective EGFR tyrosine kinase inhibitor ZD1839 (Iressa) and the DNA/RNA-mixed backbone oligonucleotide AS-PKAI to study their effect on human cancer growth and angiogenesis, measuring vascular endothelial growth factor (VEGF) and basic fibroblast growth factor expression and vessel formation, in vitro and after oral administration of these agents in mice., Results: A cooperative effect was observed with SC-236 in combination with either ZD1839 or AS-PKAI, as well as with all three agents together, on the proliferation of human colon and breast cancer cells in soft agar at doses that were ineffective for each agent alone. The antiproliferative effect was accompanied by inhibition of COX-2 expression. Moreover, combination of SC-236 with either agent or the triple combination markedly reduced VEGF secretion in the conditioned medium and completely suppressed VEGF and basic fibroblast growth factor expression. In nude mice bearing human colon cancer xenografts, a low, noninhibitory dose of SC-236 with ZD1839 and AS-PKAI, all given p.o., caused a dramatic cooperative antitumor effect, with no histological evidence of tumor in 60% of mice 5 weeks after treatment withdrawal, at which time all mice were alive. Moreover, analysis of tumor specimens revealed inhibition of vessel formation and expression of COX-2 and VEGF., Conclusions: This is the first demonstration that three novel agents blocking multiple signaling pathways, in absence of cytotoxic drugs, may have a potent antitumor and antiangiogenic activity after oral administration. Because all agents are under clinical evaluation, our results provide a rationale to translate this feasible therapeutic strategy into a clinical setting.

Purpose: This study investigated whether the functional and structural interactions between epidermal growth factor receptor (EGFR), protein kinase AI (PKAI), and bcl-2/bcl-xL could be exploited to obtain cooperative antitumor effects against models of human colon and breast cancer., Experimental Design: Antisense bcl-2/bcl-xL (4625), antisense PKAI (AS-PKAI), and ZD1839 ("Iressa"), a selective EGFR tyrosine kinase inhibitor, were administered as single agents and in combination against GEO colon and ZR-75-1 breast cancer cell lines in vitro and to mice bearing s.c. GEO human tumor xenografts in vivo. Effects on growth inhibition, vascular endothelial growth factor secretion, and induction of apoptosis were assessed., Results: Antisense bcl-2/bcl-xL inhibited the growth of GEO and ZR-75-1 cells in vitro, reducing bcl-2 and bcl-xL expression and vascular endothelial growth factor secretion. Supra-additive growth inhibition and apoptosis induction were observed when 4625 was combined with ZD1839 or AS-PKAI. Combining all three agents resulted in a complete growth inhibitory effect in vitro. Antisense bcl-2/bcl-xL, AS-PKAI, and ZD1839 administered in vivo as single agents caused growth inhibition of GEO xenografts. Combining all three agents caused a marked and sustained effect, with 50% growth inhibition and 50% of mice tumor free 5 weeks after treatment withdrawal. The combination was well tolerated., Conclusions: The combination of 4625, AS-PKAI, and ZD1839 resulted in a strong antiproliferative, proapoptotic, and antiangiogenic response, suggestive of a functional interaction between EGFR, PKAI, and bcl-2/bcl-xL and providing a rationale for the selection of specific molecular treatments for the development of therapeutic strategies. Iressa is a trademark of the AstraZeneca group of companies.

Purpose: The epidermal growth factor receptor (EGFR) is expressed in the majority of human epithelial cancers and has been implicated in the development of cancer cell resistance to cyotoxic drugs and to ionizing radiation., Experimental Design: We used ZD1839, a selective small molecule EGFR tyrosine kinase inhibitor currently in clinical development. We tested the antiproliferative and the proapoptotic activity of ZD1839 in combination with ionizing radiation in human colon (GEO), ovarian (OVCAR-3), non-small cell lung (A549 and Calu-6), and breast (MCF-7 ADR) cancer cell lines. The antitumor activity of this combination was also tested in nude mice bearing established GEO colon cancer xenografts., Results: With ionizing radiation or ZD1839, a dose-dependent growth inhibition was observed in all of the cancer cell lines growing in soft agar. A cooperative antiproliferative and proapoptotic effect was obtained when cancer cells were treated with ionizing radiation followed by ZD1839. This effect was accompanied by inhibition in the expression of the antiapoptotic proteins bcl-xL and bcl-2, and by a suppression of the activated (phosphorylated) form of akt protein. Treatment of mice bearing established human GEO colon cancer xenografts with radiotherapy (RT) resulted in a dose-dependent tumor growth inhibition that was reversible upon treatment cessation. Long term GEO tumor growth regressions were obtained after RT in combination with ZD1839. This resulted in a significant improvement in survival of these mice as compared with the control group (P < 0.001), the RT-treated group (P < 0.001), or the ZD1839-treated group (P < 0.001). The only mice alive 10 weeks after tumor cell injection were in the RT-plus-ZD1839 group. Furthermore, 10% of mice in this group were alive and tumor-free after 26 weeks. Similar results were obtained in mice bearing established human A549 lung adenocarcinoma xenografts. Finally, the combined treatment with RT plus ZD1839 was accompanied by a significant potentiation in the inhibition of transforming growth factor alpha, vascular epidermal growth factor, and basic fibroblast growth factor expression in cancer cells, which resulted in significant antiangiogenic effects as determined by immunohistochemical count of neovessels within the GEO tumors., Conclusion: This study provides a rationale for evaluating in cancer patients the combination of ionizing radiation and selective EGFR tyrosine kinase inhibitors such as ZD1839.

At present, no effective therapy is available for hepatocellular carcinoma, when local treatments have failed. We reported the results obtained with prolonged, ultra-low-dose (1 MIU/d until progression), subcutaneous interleukin-2 (IL-2) in a series of 18 consecutive patients (14 men and 4 women, median age 66 years, range 49-82 years) with advanced, histologically proven HCC on liver cirrhosis. During a median follow-up time of 19.5 months, two complete responses (11.1%), lasting 35 and 46 months, respectively, and one partial response (5.5%) were recorded (overall response rate: 16.6%; 95% CI: 0-33.8%). Thirteen patients (72.3%; 95% CI: 61.6-82.7) had stable disease lasting at least 4 months; 1 of these patients obtained a complete response on lung metastases. Median time to progression was 15.3 months (95% CI: 10-33). Median overall survival was 24.5 months (95% CI: 12-43). Two patients (11.1%) progressed during therapy. Toxicity was only local (usually pain and pomphus in the site of injection). Low-dose IL-2 can be considered an active and well-tolerated treatment for unresectable hepatocellular carcinoma. Future studies on large numbers of patients are necessary to confirm these results.

Constitutive bcl-2 overexpression increases the tumorigenic and metastatic potential of doxorubicin-resistant, estrogen-independent, MCF-7 ADR human breast cancer cells. We evaluated the sensitivity to taxanes (paclitaxel, docetaxel and IDN 5109) of 2 bcl-2-overexpressing MCF-7 ADR clones and control neomycin-transfected MCF-7 ADR neo cells. The 2 bcl-2-overexpressing MCF-7 ADR clones were relatively resistant to all 3 taxanes, whereas the MCF-7 ADR neo cells were relatively resistant to paclitaxel and docetaxel, but sensitive to IDN 5109. We found that both MCF-7 ADR neo and bcl-2-overexpressing MCF-7 ADR clones express high levels of the epidermal growth factor receptor (EGFR) and its ligand, transforming growth factor-alpha (TGF-alpha). Therefore, we tested the growth inhibitory effect of ZD1839 (Iressa, AstraZeneca, Macclesfield, UK), an orally active, selective EGFR tyrosine kinase inhibitor (EGFR-TKI) that is in clinical development. ZD1839 inhibited the growth in soft agar of all 3 clones in a dose-dependent manner (IC(50) of approximately 0.1 microm). This effect was accompanied by a dose-dependent inhibition of EGFR tyrosine autophosphorylation and of the production of TGF-alpha, basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF). To determine whether the blockade of EGFR signaling might affect the sensitivity of bcl-2-overexpressing MCF-7 ADR cells to taxanes, cells were treated with ZD1839 in combination with paclitaxel, docetaxel or IDN 5109, and dose-dependent cooperative growth inhibition as well as apoptosis potentiation were observed. Combined treatment with IDN 5109 and ZD1839 also resulted in a significant inhibition of bcl-2 expression in bcl-2-overexpressing MCF-7 ADR cells. These results demonstrate the ability of ZD1839 to overcome taxane resistance in a model of hormone-independent, multidrug-resistant, human breast cancer., (Copyright 2002 Wiley-Liss, Inc.)

Background: Chronic subcutaneous rIL-2 at low doses produces long-lasting immunomodulatory effects and is considered an effective treatment for renal cell carcinoma with marginal activity in malignant melanoma and colorectal cancer., Patients and Methods: In this study we evaluated, by Minnesota Multiphasic Personality Inventory (MMPI), the psychological changes induced by rIL-2 in 10 patients with advanced tumors., Results: After 3 months of rIL-2 treatment, 80% of the patients had a significantly increased score on the clinical scale of depression (D) and psychasthenia (Pt) (p<0.01), 70% on the scale of conversion hysteria (Hy) and 60% on the scales of schizophrenia (Sc) and psychopathic deviate (Pd), (p<0.05). These MMPI changes were however not paralleled by disease progression or clinical-overt psychological disease., Conclusion: These findings demonstrate that low-dose rIL-2 is a psychoactive treatment, which deserves psychological monitoring The MMPI is feasible for the evaluation of subclinical psychological modifications induced by cytokine immunotherapy.

Purpose: Protein kinase A type I (PKAI) and the epidermal growth factor receptor (EGFR) play a role in neoplastic transformation and interact with each other in transducing mitogenic signals. We developed different PKAI and EGFR inhibitors, demonstrating their cooperation with cytotoxic drugs and the therapeutic potential of the combined blockade of PKAI and EGFR. In this study, we investigated the effect of orally active PKAI and EGFR inhibitors in combination with a novel taxane., Experimental Design: We combined a hybrid PKAI antisense oligonucleotide sequence (AS-PKAI), the EGFR inhibitor ZD1839 (Iressa), and the taxane IDN5109, studying their effect on human cancer growth, apoptosis, and angiogenesis and measuring vascular endothelial growth factor (VEGF) expression and vessel formation in vitro and after oral administration in nude mice., Results: We demonstrated cooperative growth inhibitory and proapoptotic effects and inhibition of VEGF expression with any combination of two drugs and a marked synergistic effect when all three agents were combined. Oral administration of AS-PKAI, ZD1839, and IDN5109 in combination to nude mice caused a remarkable antitumor effect with no histological evidence of tumors in 50% of mice 5 weeks after treatment withdrawal, accompanied by complete suppression of vessel formation and VEGF expression., Conclusion: This is the first demonstration of the cooperative antitumor and antiangiogenic activity of three novel agents that block multiple signaling pathways after oral administration. Because all agents are under clinical evaluation in cancer patients, our results provide a rationale to translate this feasible therapeutic strategy in a clinical setting.

We have constructed a series of 22 phosphorothioate 20-mer antisense oligonucleotides directed against different regions of the human (EGFR) mRNA. Treatment with EGFR antisense oligonucleotides showed a dose-dependent inhibition of human GEO colon cancer cell growth in soft agar. Western blot analysis demonstrated a significant reduction in EGFR expression after treatment with each EGFR antisense oligonucleotide. The ability to inhibit GEO anchorage-independent growth, however, varied among the EGFR antisense sequences with an IC(50) ranging between 0.5 and 3.5 microM. Two of these antisense oligonucleotides targeting the regions between 2457-2476 and 614-4633 bases of the human EGFR mRNA have been modified as hybrid DNA/RNA mixed backbone oligonucleotides (MBO) to examine their anticancer properties in vivo. The 2 EGFR antisense MBOs retained the same biological properties of the fully phosphorothioate EGFR antisense oligonucleotides targeting the same EGFR mRNA sequences, such as blocking EGFR synthesis, inhibiting cell growth and enhancing programmed cell death in human cancer cell lines that express functional EGFRs. Furthermore, a potentiation in the growth inhibitory effect on GEO cancer cells was observed after treatment with these EGFR antisense MBOs in combination with cytotoxic drugs, including cisplatin, doxorubicin, paclitaxel, or topotecan. These results show the antiproliferative activity of specific EGFR antisense oligonucleotides and allow to identify novel EGFR antisense MBOs that deserve further evaluation as potential selective anticancer agents alone or in combination with cytotoxic drugs in human carcinomas that express functional EGFRs., (Copyright 2001 Wiley-Liss, Inc.)

The transforming growth factor-alpha/epidermal growth factor receptor (TGF-alpha-EGFR) autocrine pathway, which is involved in the development and the progression of human epithelial cancers, controls, in part, the production of angiogenic factors. These angiogenic factors, including vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF), are secreted by cancer cells to stimulate normal endothelial cell growth through paracrine mechanisms. ZD1839 (Iressa) is a p.o.-active, selective EGFR-tyrosine kinase inhibitor (TKI) in clinical trials in cancer patients. In this study, we evaluated the antiangiogenic and antitumor activity of ZD1839 in human colon (GEO, SW480, and CaCo2), breast (ZR-75-1 and MCF-7 ADR), ovarian (OVCAR-3), and gastric (KATO III and N87) cancer cells that coexpress TGF-alpha and EGFR. ZD1839 treatment determined a dose- and time-dependent growth inhibition accompanied by the decrease of VEGF, bFGF and TGF-alpha production in vitro. Treatment of immunodeficient mice bearing well-established, palpable GEO xenografts with ZD1839 determined a cytostatic dose-dependent tumor growth inhibition. Immunohistochemical analysis of GEO tumor xenografts after ZD1839 treatment revealed a significant dose-dependent reduction of TGF-alpha, bFGF, and VEGF expression in cancer cells and of neoangiogenesis, as determined by microvessel count. Furthermore, the antitumor activity of ZD1839 was potentiated in combination with the cytotoxic drug paclitaxel in GEO tumor xenografts. Tumor regression was observed in all mice after treatment with ZD1839 plus paclitaxel, and it was accompanied by a significant potentiation in inhibition of TGF-alpha, VEGF, and bFGF expression with a few or no microvessels. Furthermore, 6 of 16 mice bearing well-established, palpable GEO xenografts had no histological evidence of GEO tumors at the end of treatment with ZD1839 plus paclitaxel. These results demonstrate that the antitumor effect of ZD1839 is accompanied by inhibition in the production of autocrine and paracrine growth factors that sustain autonomous local growth and facilitate angiogenesis, and that this effect can be potentiated by the combined treatment with certain cytotoxic drugs, such as paclitaxel.

MDM2 is over-expressed in several human tumors. Its product is a negative-feedback regulator of p53, which interferes with the control of cell proliferation and apoptosis, interacting not only with p53 but also with retinoblastoma (Rb) and E2F. Moreover, mutations in the ARF-Ink4a locus may also allow MDM2 to override p53 functions. In this study, we have used a novel oligonucleotide anti-sense MDM2, with mixed-backbone structure and demonstrate that it causes inhibition of MDM2 expression, induction of both p53 and p21/WAF1 expression and a dose-dependent, growth-inhibitory effect in human GEO colon-cancer cells. We also show that anti-sense MDM2 has a co-operative growth-inhibitory effect with different classes of cytotoxic drugs acting by different mechanisms. Moreover, anti-sense MDM2 induces apoptosis and markedly enhances the apoptotic activity of different cytotoxic drugs. Finally, we show that anti-sense MDM2 has anti-tumor activity in vivo in nude mice bearing GEO xenografts and potentiates the anti-tumor effect of cytotoxic drugs. Indeed, despite the short treatment period, the combination of anti-sense MDM2 and cytotoxic drugs causes a marked delay in tumor growth and prolongation of mice survival, lasting several months after treatment cessation. The anti-tumor effect is associated with inhibition of MDM2 expression in tumor specimens of animals treated with anti-sense MDM2, alone or in combination with a cytotoxic drug. Our results provide the rationale for development of a novel mixed-backbone anti-sense MDM2 into a clinical setting in therapeutic combination strategies with conventional cytotoxic drugs., (Copyright 2000 Wiley-Liss, Inc.)

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.

Somatostatin analogs are promising agents in the treatment of medullary thyroid carcinoma. We have evaluated the effects of the slow release somatostatin analog lanreotide in combination with interferon-alpha2b in seven patients with advanced and symptomatic medullary thyroid carcinoma. The frequency and intensity of daily flushing episodes and bowel movements, the intensity of fatigue, weight, performance status, calcitonin levels, and change in tumor masses were recorded before and during treatment. No objective complete or partial responses were recorded. However, disease stabilization and minor tumor regression were observed in three of seven and two of seven patients, respectively. The number and intensity of bowel movements and flushing episodes decreased in five of six and two of two patients, respectively. Decrease in fatigue and improvement in performance status were observed in five of seven and six of seven patients, respectively. Weight gain was recorded in three of four patients. Plasma levels of calcitonin decreased significantly in six of seven patients. Clinical benefit, evaluated by a structured algorithm, was achieved in six of seven patients and was coupled with a decrease of 50% or more in serum calcitonin levels in three of seven patients. In conclusion, the combination of lanreotide with interferon had a major impact on clinical symptoms and was well tolerated.

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., (Copyright 2000 Wiley-Liss, Inc.)

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.

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.

Background: The expression of epidermal growth factor receptor (EGFR) and type I cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKAI) is associated with neoplastic transformation. By use of human renal cancer cell lines (i.e., 769-P, ACHN, A498, and SW839), we investigated the antiproliferative activity and the antitumor activity of an anti-EGFR humanized chimeric mouse monoclonal antibody, MAb C225, and a novel mixed backbone 18-mer antisense oligonucleotide, HYB 190, that targets expression of the RIalpha regulatory subunit of PKAI., Methods: The antiproliferative activity of MAb C225 and oligonucleotide HYB 190, alone or in combination, on different renal cancer cell lines was determined by monitoring cell growth in soft agar. In addition, the induction of apoptosis by treatment with the anti-EGFR antibody and/or antisense PKAI oligonucleotides was evaluated by flow cytometric analysis of fragmented DNA. The antitumor activity of MAb C225 and oligonucleotide HYB 190 was determined in athymic mice bearing established ACHN tumor xenografts. Cell proliferation and tumor growth data were evaluated for statistical significance using Student's t test; reported P values are two-sided., Results: MAb C225 and oligonucleotide HYB 190 inhibited colony formation in soft agar in a dose-dependent manner for all renal cancer cell lines tested. We observed a potentiation of growth inhibition and induction of apoptosis when 769-P cells and ACHN cells were treated with both agents. Combination treatment with MAb C225 and oligonucleotide HYB 190 caused regression of ACHN tumor xenografts, whereas single-agent treatment only delayed tumor growth., Conclusion: The combination of anti-EGFR MAb C225 and ited cooperative antiproliferative effects and cooperative antitumor effects on EGFR and PKAI-expressing human renal cancer cell lines.

A phase II clinical trial of subcutaneous recombinant Interleukin 2 (rIL-2) given by 5 days pulses followed by a 9 days rest has been performed in patients affected by renal cell carcinoma, malignant melanoma and colorectal cancer. A total of 25 patients entered the study, completed at least six courses of treatment, and were evaluable for toxicity and response to treatment. This schedule of subcutaneous rIL-2 was well tolerated and no World Health Organization grade 3 side effects were observed. A 33.3% response rate was recorded in patients affected by renal cell carcinoma, although no major responses were achieved in patients with malignant melanoma and colorectal cancer. A durable increase of natural killer activity retained by poeripheral blood mononuclear cells was demonstrated in these patients and was paralleled by increased serum levels of interferon gamma and tumor necrosis factor a without changes of circulating interleukin-1d. It is concluded that this schedule of pulse administration of subcutaneous rIL-2 has antitumor activity in renal cell carcinoma and produces durable biomodulatory effects.

8-Chloro-cAMP (8-Cl-cAMP) is a novel agent that is able to inhibit the growth of a wide variety of cancer cell types in vitro and in vivo and, at doses devoid of toxicity, to achieve plasma concentrations in cancer patients in a range effective for cancer cell growth inhibition. In this study, we have demonstrated that 8-Cl-cAMP, at a dose causing mild or no growth inhibition, synergistically increased the growth-inhibitory effect of paclitaxel or cisplatin in a wide series of cell lines including human breast, lung, ovary, colon, and head carcinomas and melanoma. A similar effect was also observed with another taxane, docetaxel, and with the platinum-derivative carboplatin. 8-Cl-cAMP also markedly enhanced apoptotic cell death induced by each cytotoxic drug. A cooperative antitumor effect was also observed in vivo, because treatment with paclitaxel followed by 8-Cl-cAMP markedly inhibited the growth of GEO human colon cancer xenografts as compared to paclitaxel alone without signs of toxicity. These data demonstrate that 8-Cl-cAMP synergistically increases the antiproliferative activity of taxanes and platinum-derived compounds and provide a rationale to use 8-Cl-cAMP in combination with taxanes and platinum-derived compounds.

Protein kinase A type I plays a key role in neoplastic transformation, conveying mitogenic signals of different growth factors and oncogenes. Inhibition of protein kinase A type I by antisense oligonucleotides targeting its RIalpha regulatory subunit results in cancer cell growth inhibition in vitro and in vivo. A novel mixed backbone oligonucleotide HYB 190 and its mismatched control HYB 239 were tested on soft agar growth of several human cancer cell types. HYB 190 demonstrated a dose-dependent inhibition of colony formation in all cell lines whereas the HYB 239 at the same doses caused a modest or no growth inhibition. A noninhibitory dose of each mixed backbone oligonucleotide was used in OVCAR-3 ovarian and GEO colon cancer cells to study whether any cooperative effect may occur between the antisense and a series of cytotoxic drugs acting by different mechanisms. Treatment with HYB 190 resulted in an additive growth inhibitory effect with several cytotoxic drugs when measured by soft agar colony formation. A synergistic growth inhibition, which correlated with increased apoptosis, was observed when HYB 190 was added to cancer cells treated with taxanes, platinum-based compounds, and topoisomerase II selective drugs. This synergistic effect was also observed in breast cancer cells and was obtained with other related drugs such as docetaxel and carboplatin. Combination of HYB 190 and paclitaxel resulted in an accumulation of cells in late S-G2 phases of cell cycle and marked induction of apoptosis. A cooperative effect of HYB 190 and paclitaxel was also obtained in vivo in nude mice bearing human GEO colon cancer xenografts. These results are the first report of a cooperative growth inhibitory effect obtained in a variety of human cancer cell lines by antisense mixed backbone oligonucleotide targeting protein kinase A type I-mediated mitogenic signals and specific cytotoxic drugs.

We have demonstrated that anti-sense phosphorothioate oligodeoxynucleotides (AS S-oligos) directed against the EGF-like growth factors CRIPTO (CR), amphiregulin (AR) or transforming-growth-factor-alpha(TGFalpha) mRNA, are equipotent in their ability to inhibit the growth of human colon-carcinoma GEO cells. In this study, we evaluated the effect of combinations of these AS S-oligos and conventional anti tumor drugs, such as 5-fluorouracil (5-FU), adriamycin (ADR), mitomycin C (MIT) and cis-platinum (CDDP), on GEO cell growth. Dose-dependent growth inhibition was observed by treatment either with AS S-oligos or with anti-tumor drugs, using a clonogenic assay. Furthermore, an additive growth inhibitory effect occurred when GEO cells were exposed to the AS S-oligos after treatment with different concentrations of either 5-FU, MIT, ADR or CDDP. For example, treatment of GEO cells with a combination of low concentrations of 5-FU and any of the 3 AS S-oligos resulted in up to 70% growth inhibition. However, treatment of GEO cells with AS S-oligos before exposure to 5-FU or CDDP resulted in reduced efficacy of both drugs. Flow-cytometric analysis of DNA content demonstrated that treatment with the AS S-oligos caused a slight reduction of the percentage of cells in the S-phase of the cell cycle. These data suggest that combinations of AS S-oligos directed against EGF-related growth factors and of conventional anti-tumor drugs may result in efficient inhibition of colon-carcinoma cell growth.

8-Chloro-cyclic AMP (8-Cl-cAMP) is a cAMP analogue that specifically down-regulates type I protein kinase A, a signaling protein directly involved in cell proliferation and neoplastic transformation, and that causes growth inhibition in a variety of human cancer cell types. In this report, we have investigated the effects of 8-Cl-cAMP on the expression of several growth factors in human colon (GEO and LS174T) and breast (MDA-MB468) cancer cell lines. 8-Cl-cAMP treatment caused in the three cancer cell lines a significant dose- and time-dependent inhibition in the expression of various endogenous autocrine growth factors, such as transforming growth factor alpha, amphiregulin, and CRIPTO, and of two angiogenic factors, such as vascular endothelial growth factor and basic fibroblast growth factor, at both the mRNA and protein levels. Furthermore, 8-Cl-cAMP treatment markedly inhibited the ability of all three cell lines to invade a basement membrane matrix in a chemoinvasion assay. Finally, 8-Cl-cAMP-induced inhibition of GEO tumor growth in nude mice was accompanied by a significant suppression of transforming growth factor alpha, amphiregulin, CRIPTO, basic fibroblast growth factor, and vascular endothelial growth factor production by the tumor cells, and of neoangiogenesis, as detected by factor VIII staining of host blood cells. These results demonstrate that 8-Cl-cAMP is a novel anticancer drug that inhibits the production of various autocrine and paracrine tumor growth factors that are important in sustaining autonomous local growth and facilitate invasion and metastasis.

Background: Epidermal growth factor (EGF)-related proteins, such as transforming growth factor-alpha (TGF-alpha), control cancer cell growth through hormonal pathways (i.e., autocrine [hormone acts on cell that produces it] and paracrine [hormone acts on nearby cells] pathways). Overexpression of TGF-alpha and/or its receptor (EGFR) has been detected in human cancers. The blockade of EGFR activation by the use of anti-EGFR monoclonal antibodies (MAbs) has been proposed as a potential anticancer therapy. The type I cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKAI) is generally overexpressed in human cancer cells and is involved in neoplastic transformation. Inhibition of PKAI by selective cAMP analogues, such as 8-chloro-cAMP (8-CI-cAMP), induces growth inhibition in various human cancer cell lines., Purpose: On the basis of our previous observations of a cooperative anti-proliferative effect of anti-EGFR Mab 528 and 8-Cl-cAMP in human cancer cell lines in vitro, we evaluated the anticancer activity in vivo of the combination of an anti-EGFR MAb (MAb C225) and 8-Cl-cAMP., Methods: Athymic mice were injected subcutaneously with 10(7) human colon carcinoma GEO cells. After 7 days, when established tumor xenografts of 0.30-0.35 cm3 were detectable, 10-15 mice per group were treated intraperitoneally twice weekly with different doses of 8-Cl-cAMP and/or MAb C225. Cancer cell expression of various growth factors was evaluated by immunohistochemical analysis in tumors obtained from control and treated mice. Data were evaluated for statistical significance using the Student's t test and the Mantel-Cox logrank test. All P values represent two-sided tests of statistical significance., Results: A 5-week treatment with low doses of 8-Cl-cAMP (0.5 mg/dose) and MAb C225 (0.25 mg/dose) blocked GEO tumor growth (compared with that in control mice; P < .00001) and suppressed cancer cell production of autocrine growth factors, such as TGF-alpha, amphiregulin, and CRIPTO, and of angiogenic (promotes new blood vessel formation) factors, such as vascular endothelial growth factor and basic fibroblast growth factor, with no signs of toxicity. Control and 8-Cl-cAMP (0.5 mg/dose)-treated mice died within 9-10 weeks after tumor cell injection. In MAb C225 (0.25 mg/dose)-treated mice, GEO tumors resumed a growth rate comparable to that in control animals within 3 weeks following the end of treatment and the mice died between 11 and 20 weeks after tumor cell injection. GEO tumor growth was significantly delayed in the MAb C225 plus 8-Cl-cAMP treatment group (P < .00001) and was accompanied by a prolonged survival of mice (P < .00001) as compared with the control group., Conclusions: Long-term treatment with a combination of agents that selectively inhibit two intracellular signal-transduction enzymes, such as the PKAI serine-threonine kinase and the EGFR tyrosine kinase, has anticancer activity in vivo, reflected by suppression of tumor proliferation and angiogenesis, with no signs of toxicity., Implications: Since these inhibitors of intracellular mitogenic (growth-stimulating) signaling have a different mechanism(s) of action and do not antagonize the effects of cytotoxic therapy, a combination of anti-EGFR MAb C225 and 8-Cl-cAMP should be investigated as a nontoxic, long-term treatment for cancer patients following chemotherapy.

In attempt to develop a new chemotherapeutic regimen including carboplatin (CBDCA), epirubicin (EPI), and VP-16 in extensive small cell lung cancer, with a higher dose intensity compared with previous experience of our group, we determined the maximum tolerated dose (MTD) of VP-16 when administered in association with CBDCA (300 mg/ m2, i.v., day 1) and EPI (75 mg/m2, i.v., day 1), recycling chemotherapy every 3 weeks, with the support of granulocyte-colony-stimulating factor (G-CSF). A total of 15 patients received three dose levels of VP-16 (mg/m2, i.v., daily on days 1-3): 100 (three patients), 120 (six), and 140 (six). G-CSF was administered subcutaneously at the dose of 5 micrograms/kg/day on days 6-15 of each chemotherapy course. The MTD was established at 140 mg/m2 and myelotoxicity, grade 4 neutropenia with death for sepsis in one case and grade 3 thrombocytopenia in three cases, was dose limiting. The recommended dose of VP-16 for a phase II study is 140 mg/m2.

Bryostatin 1 interferes with protein kinase C (PKC) signaling which is involved in the activation of human and murine cytotoxic T lymphocytes, and in the growth and differentiation of tumor cells. Bryostatin 1 has immunomodulating and antitumor properties as demonstrated by preclinical and clinical studies. Here we report that bryostatin 1 increases the susceptibility to lymphokine activated killers and modifies the pattern of beta 1 integrin expression of human tumor cells. On the basis of these results the use of bryostatin 1 in combination with immunostimulating cytokines such as interleukin-2 in the treatment of human cancer is suggested.

8-Chloro-cyclic AMP (8-Cl-cAMP), a site-selective cAMP analogue, is a specific inhibitor of type I cAMP-dependent protein kinase (PKAI) and induces growth inhibition in several human and rodent tumor cell lines. The anti-epidermal growth factor receptor (EGFR) mAb 528 is a blocking antibody able to inhibit the in vitro and in vivo growth of several human cancer cell lines that express functional EGFRs. Since enhanced levels of PKAI are generally found in tumor cells and an increase in PKAI expression is induced by transformation through a transforming growth factor alpha/EGFR autocrine pathway, we have evaluated whether treatment with mAb 528 in combination with 8-Cl-cAMP may have an additive or synergistic growth inhibitory effect on human cancer cells. A dose-dependent inhibition of monolayer cell growth was observed in two human colon cancer cell lines (GEO and CBS) and in a human breast cancer cell line (MDA-468) by treatment with either mAb 528 or 8-Cl-cAMP with 50% inhibitory concentration of 2-10 microgram/ml or 20-25 micrometer, respectively. The combined treatment with low noninhibitory doses of mAb 528 (0.25 microgram/ml) and with 8-Cl-cAMP had a more than additive growth inhibitory effect with a 3- to 5-fold reduction in the 8-Cl-cAMP 50% inhibitory concentration in all cell lines tested. This combined treatment was similarly effective in inhibiting the soft agar cloning efficiency of GEO cells. 8-Cl-cAMP treatment of GEO cells induced a dose-dependent increase in cell membrane-associated EGFRs with a maximum 3- to 4-fold increase within 48-72 h of treatment. These results suggest that a double blockade of the PKAI serine-threonine kinase-dependent and of the EGFR tyrosine kinase-dependent pathways is potentially useful in cancer therapy.

We have shown that a mutant derivative of Chinese hamster ovary CHO-K1 cells, ADR-5, which shows hypersensitivity to topoisomerase II (topo II)-inhibitory drugs, is cross-sensitive to the site-selective cyclic AMP analogue 8-chloro-cyclic AMP. We tested the hypothesis that overexpression of the type I alpha regulatory subunit of protein kinase A may represent a common element conferring hypersensitivity to both topo II inhibitors and 8-chloro-cyclic AMP in ADR-5 cells. We have demonstrated that ADR-5 cells overexpress RI alpha protein, compared to parental CHO-K1 cells. Moreover, retroviral vector-mediated transfer of the RI alpha gene into CHO-K1 cells was able to confer a drug-hypersensitive phenotype similar to that exhibited by ADR-5 cells. Analysis of topo II protein levels and activity revealed no differences between parental and infected cells, suggesting that protein kinase A may be involved in the downstream processing of topo II-mediated events.

Background: The epidermal growth factor (EGF-R) receptor is an important growth regulator of epithelial cancer cells, and is presently considered a tumor-associated antigen (TAA) which is overexpressed by several human cancers and barely detectable in most normal tissues. Since TAA density at the tumor cell surface is a critical factor regulating the efficiency of immunotargeting procedures, a therapeutic advantage may derive from the pharmacologic enhancement of membrane expression of such antigens on tumor cells., Materials and Methods: Utilizing a panel of different human cancer cell lines of epithelial derivation, we have investigated in the in vitro effects of 5-aza-2'-deoxycytidine (5azaCdR), an antineoplastic agent able to induce gene activation and phenotypic modulation, on the surface expression of EGF-R by tumor cells., Results: 5azaCdR (10-1000 nM) induced growth inhibition, in the absence of acute cell kill, on KB (human oropharyngeal carcinoma), LoVo and the drug-resistant clone LoVo-DX (colon carcinoma) and A549 (lung adenocarcinoma) cell lines, along with a significant enhancement of EGF-R expression at the tumor cell surface. A single 24 h pulse of 5azaCdR, followed by 96 h of culture in drug-free medium, induced 50% growth inhibition on KB cells at a concentration (IC50) of 500 nM, on A549 (IC50 = 490 nM), LoVo (IC50 = 400 nM) and LoVo-DX (IC50 = 100 nM) cell lines. Under these conditions the specific binding of 125I-EGF was significantly upregulated at the surface of growth-inhibited cancer cells. Scatchard analysis of EGF-binding data revealed no changes in the Kd of EGF-R for its ligand in 5azaCdR-treated tumor cells and demonstrated a significant increase in the number of both the high- and low-affinity EGF-binding sites on KB cells, while only one class of EGF-binding site was detectable on A549, LoVo and LoVo-DX tumor cell lines before and after exposure to 5azaCdR. The EGF-R upregulation induced by 5azaCdR was paralleled by the increased binding of the anti-EGF-R monoclonal antibody (MAb) 108.1 on the surface of cancer cells. Finally, the rate of endocytosis of the anti-EGF-R MAb by KB cells was not modified by drug treatment, indicating that exposure to 5azaCdR does not hamper MAb internalization by the tumor cells. This latter represents an essential process for the cytotoxic effects of immunoconjugate drugs or toxins., Conclusions: We suggest a role for 5azaCdR in enhancing the efficacy of therapeutic approaches involving the use of anti-EGF-R immunoconjugated for the imaging and the treatment of human epithelial neoplasias.

Intestinal function during cytotoxic therapy was studied in nine patients with nonintestinal lymphoma. An early, marked, and rapidly reversible increase in passive permeability, as disclosed by the lactulose test, was observed without concomitant changes in active absorption or in intestinal histology. The possibility of a toxic effect by cytotoxic drugs on intestinal intracellular junctions is discussed.