Senior Investigator Experimental Pharmacology Unit Experimental Oncology Department National Institute of Tumours Fondazione G. Pascale Via Mariano Semmola 80131 Naples Italy Phone: 00390815903595 Fax: 00390815903813 Cell.: 00393475939575 email: michele.caraglia@fondazionepascale.it michele.caraglia@alice.it
michele.caraglia@fondazionepascale.it
Name and Surname: Michele Caraglia Place and date of birth: Naples 18/04/1966 Marriage status: married with two sons Academic Career
Education and Training (include degrees and post-doctoral training)
1990- Degree in Medicine with magna cum laude, University Federico II of Naples, Italy.
1991-1992 Guest researcher in the Laboratory of IIGB, CNR, Naples
1994- Expecialization in Oncology with magna cum laude, University Federico II of Naples, Italy.
1995-1996 Guest researher in the Leukemia Unit of CRO, Aviano, Italy
1997-1998 Guest researcher at the University Tor Vergata, Biology Dept.
1999-2002 Ph D degree in Cellular Biochemistry at the Department of Biochemistry and Biophysis, Second University of Naples, Naples, Italy.
2002-2004 Guest researcher at the Department of Biochemistry and Biophysis, Second University of Naples, Naples, Italy.
1 Sept. 2004-present Senior Investigator of the Experimental Pharmacology Unit of the National Institute of Tumours of Naples
Expertise field and main projects
Experimental degree thesis on the study of EGF-R expression modulation induced by IFN in human squamous cancer cells (SCC).
Study of the synthesis of EGF-R protein in cells exposed to IFN or 8ClcAMP in SCC
Study of the effects of cytotoxic drugs on EGFR, transferrin receptor and NGFR expression
Study of the activation status of ras in response to pharmacological treatments in SCC – escape mechanisms
Study of the effects of cytokines on the protein synthesis – escape mechanisms
Potentiation of the antitumour activity of cytotkines and of inhibitors of isoprenylation – overcoming the escape mechanisms in SCC
Potentiation of the antitumour activity of cytotkines and of inhibitors of isoprenylation – overcoming the escape mechanisms in prostate cancer
Potentiation of the antitumour activity of cytotkines and of inhibitors of isoprenylation – overcoming the escape mechanisms in prostate cancer
1997-present – Professor at the School for the Specialty in Biochemistry and Clinical Chemistry of the Second University of Naples.
AWARDS AND PERSONAL GRANTS
27/10/1992 – Annual grant from “Fondazione Marinelli” for the research in Molecular Oncology..
20/12/1993 – Three year grant (1993-1996) form the Italian Association for Cancer Research for a Research on the Homeostatic mechanisms of tumour cells in response to cytokines.
1996 – First prize from the Italian Association of Medical Oncology for the best communication presented at the XIV National Congress of the Association in Milan.
2002- First Prize for the best Communication presented at the Scentific Meeting of the School of Medicine of the Second University of Naples, Naples, Italy.
FINANCIAL SUPPORTS AND COOPERATIONS
2004-2005 Financial support received from Novartis for the research project entitled: EFFECTS OF ZOLEDRONATE ON EGF-DEPENDENT SIGNAL TRANSDUCTION AND ON APOPTOSIS OF PANCREAS AND PROSTATE ADENOCARCINOMA CELLS.
2005 Financial support received from Janssen for the research project entitled:: NEW INTEGRATED THERAPEUTIC STRATEGIES IN THE TREATMENT OF HORMONE-REFRACTORY PROSTATE ADENOCARCINOMA
2005-2007 Financial support received from Italian Association for Cancer Research for the research project entitled: INHIBITION OF ISOPRENYLATION AND OF ACETYLATION AS A NEW STRATEGY FOR THE THERAPY OF PROSTATE CANCER.
2005-2007 Financial support received from Italian Ministry of Health for a project on the study of isoprenylation inhibition in prostate cancer.
Reviewer activity for the following peer reviewed international journals.
- Clinical Endocrinology - FEBS Journal - Leukemia and Lymphoma - Oncogene - Oncology - Phytochemistry - Journal of Interferon and Cytokine Research - Amino Acids - Cell Biology International - International Journal of Cancer - British Journal of Haematology - Cancer Research - Clinical and Experimental Metastases - Journal of Cellular Physiology
Editorial Board Member
- Cancer Cell International - Recent Patents in Anti-Cancer Drug Discovery - World Journal of Experimental Medicine - International Journal of Oncology
Abstract: Advanced chemorefractory thymic epithelial tumors still represent a challenge in clinical oncology. A rationale-based therapeutic approach targeting a key pathway should represent the ideal solution in a neoplasm that can over-express Epidermal Growth Factor Receptor (EGFR) in the epithelial component. On the basis of these considerations, two patients with metastatic heavily pretreated disease were evaluated for EGFR expression in the primitive tumor, being considered this data as a basis for an anti EGFR treatment with the monoclonal antibody cetuximab which targets EGFR. A strong EGFR expression was revealed by immunohistochemistry in the two cases considered, thus the patients received cetuximab and reported a partial response as assessed by Computed Tomography (CT), Positron Emission Tomography (PET) and fused PET-CT after three months of therapy. Therefore, both patients are still on therapy. This preliminary experience suggests that cetuximab may be a useful therapeutic choice in advanced pre-treated thymic tumors.
Abstract: Zoledronic acid (ZOL) has proved activity in bone metastases from prostate cancer through inhibition of mevalonate pathway and of prenylation of intracellular proteins. We have reported that ZOL synergizes with R115777 farnesyltransferase inhibitor (FTI, Zarnestra) in inducing apoptosis and growth inhibition on epidermoid cancer cells. Here, we have studied the effects of the combination of these agents in prostate adenocarcinoma models and, specifically, on androgen-independent (PC3 and DU145) and -dependent (LNCaP) prostate cancer cell lines. We have found that ZOL and R115777 were synergistic in inducing both growth inhibition and apoptosis in prostate adenocarcinoma cells. These effects were paralleled by disruption of Ras-->Erk and Akt survival pathways, consequent decreased phosphorylation of both mitochondrial bcl-2 and bad proteins, and caspase activation. Finally, ZOL/R115777 combination induced cooperative effects also in vivo on tumor growth inhibition of prostate cancer xenografts in nude mice with a significant survival increase. These effects were paralleled by enhanced apoptosis and inactivation of both Erk and Akt. In conclusions, the combination between ZOL and FTI leads to enhanced anti-tumor activity in human prostate adenocarcinoma cells likely through a more efficacious inhibition of ras-dependent survival pathways and consequent bcl-related proteins-dependent apoptosis.
Abstract: A correlation between regulation of cell proliferation and polyamine metabolism is described. The latter can enter protein synthesis through the modification of eukaryotic initiation factor 5A (eIF5A) and the formation of the peculiar amino acid hypusine. Specific inhibitors of hypusine formation induce apoptosis that can be potentiated by the combination with cytokines such as interferonalpha (IFNalpha) that itself decreases hypusine synthesis. We have also demonstrated that the concomitant treatment of cancer cells with IFNalpha and the protein synthesis inhibitor fusion protein TGFalpha/Pseudomonas Aeruginosa toxin synergize in inducing cancer cell growth inhibition. Another way used by polyamines to induce apoptosis is the generation of intracellular oxidative stress through the interaction with bovine serum amine oxidase (BSAO). This enzyme used simultaneously to spermine induces apoptosis, necrosis, inhibition of cell proliferation and inhibition of DNA and protein synthesis in several cell types. The enzymatic oxidation products of polyamine, H(2)O(2) and aldehyde(s) cause these effects. We have recently found that the cytotoxicity of anti-cancer agents, either etoposide or docetaxel, in cancer cells is potentiated in the presence of BSAO/Spermine. In conclusion, polyamine metabolites could be useful in the design of new therapeutic strategies.
Abstract: BACKGROUND: Brain metastases (BM) represent one of the most frequent complications related to cancer, and their treatment continues to evolve. We have evaluated the activity, toxicity and the impact on Quality of Life (QoL) of a concomitant treatment with whole brain radiotherapy (WBRT) and Temozolomide (TMZ) in patients with brain metastases from solid tumors in a prospective Simon two stage study. METHODS: Fifty-nine patients were enrolled and received 30 Gy WBRT with concomitant TMZ (75 mg/m2/day) for ten days, and subsequently TMZ (150 mg/m2/day) for up to six cycles. The primary end points were clinical symptoms and radiologic response. RESULTS: Five patients had a complete response, 21 patients had a partial response, while 18 patients had stable disease. The overall response rate (45%) exceeded the target activity per study design. The median time to progression was 9 months. Median overall survival was 13 months. The most frequent toxicities included grade 3 neutropenia (15%) and anemia (13%), and only one patient developed a grade 4 thrombocytopenia. Age, Karnofsky performance status, presence of extracranial metastases and the recursive partitioning analysis (RPA) were found to be predictive factors for response in patients. Overall survival (OS) and progression-free survival (PFS) were dependent on age and on the RPA class. CONCLUSION: We conclude that this treatment is well tolerated, with an encouraging objective response rate, and a significant improvement in quality of life (p < 0.0001) demonstrated by FACT-G analysis. All patients answered the questionnaires and described themselves as 'independent' and able to act on their own initiatives. Our study found a high level of satisfaction for QoL, this provides useful information to share with patients in discussions regarding chemotherapy treatment of these lesions.
Abstract: Imatinib, a tyrosine kinase inhibitor directed against the enzymatic domain of KIT protein, was found to produce dramatic clinical responses in metastatic gastrointestinal stromal tumors (GISTs). However, resistance usually develops thus determining treatment failure. The present study was performed to analyse the expression of somatostatin receptor (SSTR) subtypes, modulators of tissue transglutaminase, in a series of GISTs and leiomyosarcomas by immunohistochemistry to identify a new potential therapeutic target. Sixteen cases (8 males and 8 females, age range: 38-73; 11 GISTs, 4 leiomyosarcomas, 1 leiomyoma) were studied. Immunohistochemical detection of the relevant SSTRs was performed on paraffin-embedded tissue sections, stained with polyclonal antibodies directed against the five somatostatin receptor subtypes. We found 7 out of 16 (44%) tumors expressing all SSTRs and 14 out of 16 (87%) tumors positive for at least 3 subtypes. SSTR2(A) was the most represented subtype in the tumors studied, being expressed in approximately 70% of cases exhibiting an intense labeling in most of these cases. The significant expression of SSTRs shown in this series of GISTs and gastrointestinal leiomyosarcomas suggests a potential therapeutic target to be explored alone and/or in combination with other therapeutic agents in the setting of refractory GI stromal tumors.
Abstract: We investigated the ability of the histone deacetylase (HDAC) inhibitor trichostatin A (TSA) to interact with gemcitabine (GEM) in inducing pancreatic cancer cell death. The combined treatment with TSA and GEM synergistically inhibited growth of four pancreatic adenocarcinoma cell lines and induced apoptosis. This effect was associated with the induction of reactive oxygen species (ROS) by GEM, increased expression of the pro-apoptotic BIM gene by both TSA and GEM and downregulation of the 5'-nucleotidase UMPH type II gene by TSA. The expression of other genes critical for GEM resistance (nucleoside transporters, deoxycytidine kinase, cytidine deaminase, and ribonucleotide reductase genes) was not affected by TSA. The functional role of ROS in cell growth inhibition by GEM was supported by (i) a significantly reduced GEM-associated growth inhibition by the free radical scavenger N-acetyl-l-cysteine, and (ii) a positive correlation between the basal level of ROS and sensitivity to GEM in 10 pancreatic cancer cell lines. The functional role of both Bim and 5'-nucleotidase UMPH type II in cell growth inhibition by TSA and GEM was assessed by RNA interference assays. In vivo studies on xenografts of pancreatic adenocarcinoma cells in nude mice showed that the association of TSA and GEM reduced to 50% the tumour mass and did not cause any apparent form of toxicity, while treatments with TSA or GEM alone were ineffective. In conclusion, the present study demonstrates a potent anti-tumour activity of TSA/GEM combination against pancreatic cancer cells in vitro and in vivo, strongly supporting the use of GEM in combination with an HDAC inhibitor for pancreatic cancer therapy.
Abstract: BACKGROUND: Unusual sites of metastasis to the parotid gland and face are reported in patients with colorectal cancer, but the localization to both sites at the same time has never been described so far. CASE REPORT: Here, we describe the case of a 76-year-old woman with a T3N1M0 G2 rectal adenocarcinoma, treated with preoperative chemoradiotherapy performed at suboptimal dosages due to unacceptable toxicity. At the end of the program, the re-staging demonstrated the presence of metastasis in both the left parotid gland and subcutaneously on the frontal region of her face while the primary locoregional tumour manifestation was radiologically down-staged (reduction in N staging from N1 to N0). The patient did not respond to any other treatment and died due to disease progression 15 months after the diagnosis. CONCLUSIONS: Metastatic tumours in the parotid gland are uncommon with a higher incidence of primary sites of the head and neck. Parotid involvement of rectal adenocarcinoma is also extremely rare, and concomitant involvement of both the parotid gland and the face was not previously described. In this case, we cannot rule out the hypothesis that the delay of surgical removal of the primary tumour and/or a specific action of concomitant chemoradiotherapy on the tumour cell phenotype could promote cancer cell spreading to unusual sites.
Abstract: We have reported previously that interferon-alpha (IFN-alpha) induces apoptosis that is counteracted by an epidermal growth factor (EGF) --> Ras --> extracellular signal-regulated kinase (ERK)-dependent survival response in human epidermoid cancer KB cells. We have studied the effects of the cytokine on the cAMP-dependent pathway in these cells. A decrease in the intracellular cAMP levels was recorded in KB cells treated with IFN-alpha, whereas forskolin induced an increase in the production of cAMP that was reduced in the presence of IFN-alpha, suggesting a reduction in the activity of adenylate cyclase (AC) induced by IFN-alpha. These effects were paralleled by significant change in the expression of some AC catalytic subunit(s) and by reduction in the activity of protein kinase A (PKA). 8-Br-cAMP completely antagonized the reduction of PKA activity induced by IFN-alpha, whereas PKA inhibitor KT5720 enhanced the reduction of the enzyme activity induced by IFN-alpha. We have found that IFN-alpha induced a decrease in cAMP response element binding protein (CREB) phosphorylation without changes in its total expression. The concomitant treatment with IFN-alpha and 8-Br-cAMP potentiated and KT5720 counteracted apoptosis induced by IFN-alpha alone. In conclusion, these data suggest that the decrease in AC/cAMP pathway activity is a survival response to the apoptosis induced by IFN-alpha. Therefore, this pathway could represent a target to enhance the antitumor activity of IFN-alpha.
Abstract: Interferon alpha (IFNalpha) induces both apoptosis and a counteracting epidermal growth factor Erk-dependent survival response in cancer cells. In this report, IFNalpha increased eukaryotic elongation factor 1A (eEF-1A) protein expression by inhibition of eEF-1A degradation via a proteasome-dependent pathway. The reduction of the expression level of eEF-1A by RNA interference enhanced the apoptosis induced by IFNalpha on the same cells. Moreover, IFNalpha induced the phosphorylation of both serine and threonine in eEF-1A. These effects were paralleled by an increased co-immunoprecipitation and colocalization of eEF-1A with C-Raf. The suppression of C-Raf kinase activity with the inhibitor BAY 43-9006 completely antagonized the increase of both eEF-1A phosphorylation and expression and of C-Raf/eEF-1A colocalization induced by IFNalpha and enhanced apoptosis and eEF-1A ubiquitination. Cell transfection with the mutated K48R ubiquitin increased EF-1A expression and desensitized tumor cells to the modulating effects of IFNalpha. The dynamic simulation of 3Dstructure of eEF-1A identified putative serine and threonine phosphorylation sites. In conclusion, the interaction between eEF-1A and C-Raf increases eEF-1A stability and induces a survival activity.
Abstract: OBJECTIVE: A combination regimen of temozolomide (TMZ) and pegylated liposomal doxorubicin has been evaluated in the treatment of brain metastases from solid tumours. STUDY DESIGN: Nineteen consecutive patients (pts) have been enrolled in a prospective phase II trial and treated with TMZ 200 mg/m2 (days 1-5) and pegylated liposomal doxorubicin 35 mg/m2 (day 1) every 28 days. The study was prospectively projected according to the Simon's two-stage optimal design. RESULTS: Major toxicities have been grade III neutropenia and thrombocytopenia in one patient (pt) and grade III erythrodisesthesia in two pts. Three pts achieved a complete response (CR) and four a partial response (PR), for an overall response rate of 36.8% (95% CI: 19.1-59.2), which exceeded the target activity in the study design. A significant improvement in quality of life was demonstrated by FACT-G analysis. The median Progression Free Survival (PFS) was 5.5 (95% CI: 2.7-8.2) months while the median Overall Survival (OS) was 10.0 months (95% CI: 6.3-13.7). CONCLUSIONS: The TMZ/pegylated liposomal doxorubicin regimen was well tolerated with an encouraging activity in brain metastases from solid tumours.
Abstract: Bone metastases are common in patients with many types of cancer, especially breast and prostate cancer--in which the incidence is approximately 70% among patients with advanced metastatic disease. Aminobisphosphonates (NBPs) have entered clinical practice in the treatment of bone metastases from several neoplasms, including breast and prostate adenocarcinoma, as a result of their anti-resorption properties. However, evidence has accumulated on the direct anti-tumour effects of NBPs. This review describes the metabolic pathways that are putative molecular targets of NBPs and that are involved in the prenylation processes of several intracellular small GTP-binding proteins (ras family related proteins). The latter regulate the intracellular survival and proliferative pathways of tumour cells and could be the intracellular molecular targets of the NBPs responsible for the direct anti-cancer effects, even if definitive conclusions cannot be drawn at present. Different mechanisms have been reported to account for the anti-neoplastic action of NBPs, including: the induction of apoptosis; cell cycle perturbations; and anti-invasive, anti-migration and anti-angiogenic effects. Moreover, this review describes the most important clinical studies that demonstrate the activity of NBPs in preventing skeletal-related events induced by bone metastases. The main pharmacokinetic pitfalls of NBPs are described, and methods of overcoming these pitfalls through the use of liposome vehicles are proposed. Finally, the principal pre-clinical studies on the interaction between NBPs and other biological agents are also described; these studies may enable reductions in the in vivo NBP concentrations required to achieve anti-tumour activity. To date, however, the real molecular targets of NBPs are not completely known and new technological platforms are required in order to detect them and to develop new anti-cancer strategies based on the use of NBPs.
Abstract: Target-based therapy has been a promising anti-cancer strategy in the preclinical setting, but its efficacy is still limited in clinical practice. The latter was probably due to the lack of identification of molecular targets in order to predict clinical response and for the existence of multiple survival compensatory downstream pathways. Therefore, the use of downstream targets could be useful in order to avoid these overcoming pathways. One of these targets is Raf-kinase. In this review we describe the structure and functions of the components of Raf-kinase family and their relevance in proliferation and survival of tumor cells. Moreover, we illustrate the signal transduction pathways regulated by Raf-kinases. The main preclinical and clinical results obtained with the use of the Raf-kinase inhibitor BAY 43-9006 or sorafenib are also described. The multi-target function of sorafenib is also explained and the disclosure of new therapeutic opportunities based on the dual inhibition of cancer proliferation and neo-angiogenesis is discussed. In conclusion, Raf-kinase appears an appealing therapeutic target, even it other preclinical and clinical studies are warranted in order to evaluate the activity of sorafenib both in monotherapy and in combination with other agents.
Abstract: For patients with malignant bone disease, bisphosphonate therapy is the standard treatment. Preclinical and preliminary clinical data suggest that bisphosphonates have direct or indirect antitumor effects: they affect growth-factor release, cancer-cell adhesion, invasion and viability, angiogenesis, and apoptosis of cancer cells. These effects might be enhanced through co-administration with chemotherapy agents, biological agents, or both. We survey the biochemical pathways and molecular targets of bisphosphonates, and discuss the molecular mechanisms of these antitumor effects, as well as the documented antineoplastic preclinical effects of bisphosphonates used in combination with cytotoxic and biological drugs. Moreover, the positive interactions between bisphosphonates and farnesyltransferase inhibitors, KIT receptor tyrosine kinase inhibitors (e.g. imatinib mesylate) and cyclo-oxygenase-2 inhibitors are discussed in relation to their potential synergistic and additive effects. We briefly discuss identification of new molecular targets of bisphosphonates from genomic and proteomic analysis, and highlight the cellular consequences of drug-related enzyme inhibition.
Abstract: Cell proliferation, differentiation, and survival are regulated by a number of extracellular hormones, growth factors, and cytokines in complex organisms. The transduction of the signals by these factors from the outside to the nucleus often requires the presence of small intracellular proteins (i.e. ras and other small G proteins) that are linked to the plasma membrane through a isoprenyl residue that functions as hydrophobic anchor. Isoprenylation is a complex process regulated by different enzymatic steps that could represent potential molecular targets for anti-cancer strategies. In the present paper the different transduction pathways regulated by some isoprenylated proteins such as ras and other small G proteins are described. Moreover, the molecular mechanisms of the isoprenylation process and the mode of action of the different isoprenylation inhibitors are discussed with attention to statins, farnesyltransferase inhibitors (FTI) and aminobisphosphonates. The role of different candidate targets in the determination of anti-tumour effects by FTIs is also described in order to define potential molecular markers predictor of clinical response. On the basis of several preclinical data, new strategies based on multi-step enzyme inhibition or on target prioritization are proposed in order to enhance the anti-tumour activity of agents inhibiting isoprenylation. Finally, a summary of the principal data on clinical trials based on the use of FTIs and statins is given. In conclusion, the inhibition of isoprenylation is an attractive, but still not completely investigated therapeutic alternative that requires optimization for the translation in the current treatment of neoplasms.
Abstract: The exposure to non-thermal microwave electromagnetic field (MW-EMF) at 1.95 MHz, a frequency used in mobile communication, affects the refolding kinetics of eukaryotic proteins (Mancinelli et al., 2004). On these basis we have evaluated the in vivo effect of MW-EMF in human epidermoid cancer KB cells. We have found that MW-EMF induces time-dependent apoptosis (45% after 3 h) that is paralleled by an about 2.5-fold decrease of the expression of ras and Raf-1 and of the activity of ras and Erk-1/2. Although also the expression of Akt was reduced its activity was unchanged likely as a consequence of the increased expression of its upstream activator PI3K. In the same experimental conditions an about 2.5-fold increase of the ubiquitination of ras and Raf-1 was also found and the addition for 12 h of proteasome inhibitor lactacystin at 10 microM caused an accumulation of the ubiquitinated isoforms of ras and Raf-1 and counteracted the effects of MW-EMF on ras and Raf-1 expression suggesting an increased proteasome-dependent degradation induced by MW-EMF. The exposure of KB cells to MW-EMF induced a differential activation of stress-dependent pathway with an increase of JNK-1 activity and HSP70 and 27 expression and with a reduction of p38 kinase activity and HSP90 expression. The overexpression of HSP90 induced by transfection of KB cells with a plasmid encoding for the factor completely antagonized the apoptosis and the inactivation of the ras --> Erk-dependent survival signal induced by MW-EMF. Conversely, the inhibition of Erk activity induced by 12 h exposure to 10 mM Mek-1 inhibitor U0126 antagonized the effects induced by HSP90 transfection on apoptosis caused by MW-EMF. In conclusion, these results demonstrate for the first time that MW-EMF induces apoptosis through the inactivation of the ras --> Erk survival signaling due to enhanced degradation of ras and Raf-1 determined by decreased expression of HSP90 and the consequent increase of proteasome dependent degradation.
Abstract: Interferon-alpha (IFNalpha) is a recombinant protein widely used in the therapy of several neoplasms such as myeloma, renal cell carcinoma, epidermoid cervical and head and neck tumors, and melanoma. IFNalpha, the first cytokine to be produced by recombinant DNA technology, has emerged as an important regulator of cancer cell growth and differentiation, affecting cellular communication and signal transduction pathways. However, the way by which tumor cell growth is directly suppressed by IFNalpha is not well known. Wide evidence exists on the possibility that cancer cells undergo apoptosis after the exposure to the cytokine. Here we will review the consolidate signal transducer and activator of transcription (STAT)-dependent mechanism of action of IFNalpha. We will discuss data obtained by us and others on the triggering of the stress-dependent kinase pathway induced by IFNalpha and its correlations with the apoptotic process. The regulation of the expression of proteins involved in apoptosis occurrence will be also described. In this regard, IFNalpha is emerging as a post-translational controller of the intracellular levels of the apoptosis-related protein tissue transglutaminase (tTG). This new way of regulation of tTG occurs through the modulation of their proteasome-dependent degradation induced by the cytokine. Until today, inconsistent data have been obtained regarding the clinical effectiveness of IFNalpha in the therapy of solid tumors. In fact, the benefit of IFNalpha treatment is limited to some neoplasms while others are completely or partially resistant. The mechanisms of tumor resistance to IFNalpha have been studied in vitro. The alteration of JAK-STAT components of the IFNalpha-induced signaling, can be indeed a mechanism of resistance to IFN. However, we have recently described a reactive mechanism of protection of tumor cells from the apoptosis induced by IFNalpha dependent on the epidermal growth factor (EGF)-mediated Ras/extracellular signal regulated kinase (Erk) signaling. The involvement of the Ras-->Erk pathway in the protection of tumor cells from the apoptosis induced by IFNalpha is further demonstrated by both Ras inactivation by RASN17 transfection and mitogen extracellular signal regulated kinase 1 (Mek-1) inhibition by exposure to PD098059. These data strongly suggest that the specific disruption of the latter could be a useful approach to potentiate the antitumour activity of IFNalpha against human tumors based on the new mechanistic insights achieved in the last years.
Abstract: The multi-drug combination of oxaliplatin (OXA), 5-Fluorouracil (5-FU) and leucovorin (LF) is currently considered as the gold standard treatment for metastatic colorectal carcinoma. In previous studies, we have studied a chemotherapy regimen containing gemcitabine (GEM), OXA, LF, and 5-FU (named GOLF regimen) that has shown a good safety profile and highly significant anti-tumor activity. In the present study, we have investigated on the anti-tumour mechanisms of GOLF in human colon cancer HT-29 and WiDr cell lines. We have found that GOLF induced growth inhibition that was largely caused by apoptosis differently from other combinations. Moreover, the different drugs composing GOLF were highly synergistic in inducing growth inhibition. Apoptosis induced by GOLF combination was paralleled by PARP cleavage and caspase 9 and 3 activation that were not recorded in the other combinations. An about 85% decrease of the activity of Erk and Akt was found in GOLF-treated cells. These effects were likely due to decreased expression of the upstream activator Raf-1 and of Akt itself, respectively. The intracellular levels of these signalling components can be post-translationally regulated by ubiquitin-dependent degradation through proteasome. Therefore, we have evaluated the expression of some chaperone components and we have found that GOLF did not affect the expression of both heat shock protein (HSP) 90 and 27 but induced an about 90% increase of HSP70 levels suggesting the inactivation of the multi-chaperone complex. Moreover, an about 4-fold increase of the ubiquitination of Raf-1 was also found and the addition for 12 h of 10 microM proteasome inhibitor lactacystin caused an accumulation of the ubiquitinated isoforms of Raf-1. In conclusions, GOLF was a combination highly synergistic in inducing both growth inhibition and apoptosis of colon cancer cells. These effects likely occurred through the disruption of critical survival pathways and the inactivation of multi-chaperone complex.
Abstract: The administration of mineral sulphur water is an alternative experimental approach for the treatment of rheumatic diseases, such as osteoarthritis (OA), that cause the degeneration of bone and cartilage and sufferance to the patients. Chondroitin sulfate (CS) is a symptomatic slow acting nutropeucital agent currently used in molecular therapy of OA. Therefore, we have studied the role and efficacy of the selective soil paste from the mineral sulphur enriched spring (mud)-therapy alone or in combination with CS in the treatment of OA. The study was performed on 40 C57 Black 6N mice, an experimental model which spontaneously develop an osteoarthritic process. The animals were divided in 4 groups and were treated with the single agents or with the combination. After 30 days of treatment all the mice were sacrificed and right knees and blood were collected. It was found that CS determined a reduction of radiological and histological features of chondrodegeneration and that mud-therapy increased the effects of CS in the animal group treated with the combination. However, the effects of thermal therapy alone were not statistically significant. Since OA is characterized by an increase of the production of nitric oxide (NO) by chondrocytes in extracellular matrix with its consequent elevation in serum and synovial fluid, we have evaluated the effects of the treatments on serum NO levels. CS alone induced a statistically significant reduction of NO serum levels (90+/-13 micromM vs 219+/-60 microM of control group, P<0.05) while mud-therapy alone induced a not statistically significant reduction of serum NO (170+/-62 microM, P>0.05). However, the latter strongly potentiated the decrease of serum NO induced by CS (31+/-1.5 microM) with a high statistical significance if compared to both the control group (P<0.01) and the CS-treated group (P<0.05). In conclusion, this study demonstrates that mud-therapy with sulphur mineral water could represent an important phase of the therapeutic strategy of OA. This experimental strategy could integrate and potentiate the standard pharmacological tools. Moreover, we have set a valid experimental in vivo model for the study of the thermal effects on the development of OA.
Abstract: Docetaxel (Taxotere, DTX) is a promoter of apoptosis in cancer cells. Since cytotoxic mechanisms of DTX are not yet fully understood, we have investigated the effects of DTX on apoptosis and ras-->Erk-mediated signal transduction in human epidermoid KB, colon HT-29 and breast HCC1937 cancer cells. We have found that the exposure to 0.78 or 1.56 or 2.5 ng/ml DTX for 48 h induced apoptosis and growth inhibition in about 50 % of KB, HCC1937 and HT-29 cell population, respectively. In these experimental conditions, PARP and caspase 3 cleavage was also showed in all cell lines. KB and HCC1937 cells express a wild type p53 while HT-29 display a mutated form. Interestingly, we have found that DTX reduces the expression of mutated p53 in HT-29 and increases the expression of wild type in KB and HCC1937 cells. Moreover, DTX reduces ubiquitination of the wild type p53 in KB and HCC1937 cells and increases the ubiquitin-conjugated form of mutated p53 in HT-29 cells. Furthermore, exposure of cancer cells to DTX for 48 h increases the expression and activity of Ras and up-regulates Raf-1 and the phosphorylated isoforms of Erk-1/2. On the bases of these data, we have hypothesized that the increased activity of the ras-->erk-dependent pathway induced by DTX could be a protective signalling from the apoptosis caused by the drug. Therefore, we have used R115777, a farnesyl transferase inhibitor that inactivates ras, in combination with DTX. The combined treatment with DTX and R115777 resulted in a strong synergism in growth inhibition in the three cell lines. These data suggest the use of the combination in these therapeutic settings even if further experiments are required for the clinical translation.
Abstract: We have previously reported that interferon-alpha (IFNalpha) induces apoptosis and EGF can antagonize this effect in human epidermoid cancer KB cells. Since apoptosis occurs together with cytoskeleton reorganization we have evaluated if IFNalpha and EGF could modulate cell remodeling in our experimental conditions. We have found that 48 h 1,000 IU/ml IFNalpha induced structural reorganization of stress fibers and membrane delocalization and partial capping of the actin severing protein gelsolin. The transfection of KB cells with both a wild type (WT) or a C-terminal truncated form of gelsolin caused overexpression of the protein and an increase of both the spontaneous and IFNalpha-induced apoptosis and cell cytoskeletal modifications. In fact, after 48 h of treatment IFNalpha induced 45% of apoptotic cell death in parental cells while an approximately 80% of cell population was apoptotic in transfected cells. These effects occurred together with an increase of the expression and consequent degradation of gelsolin. Again the addition of EGF to IFNalpha-treated transfected cells caused a recovery of the apoptosis. Notably, IFNalpha and EGF did not modify the expression of other molecules associated to cytoskeleton such as focal adhesion kinase and vinculin. In the same experimental conditions IFNalpha induced also gelsolin cleavage that occurred together with caspase-3 activation and release of cytochrome c. All these effects were antagonized by the exposure of IFNalpha-treated KB to 10 nM EGF for the last 12 h. Moreover, the specific inhibition of caspase-3 with 20 microM DEVD completely abrogated apoptosis and gelsolin cleavage induced by IFNalpha. In conclusion, our data are the first demonstration that IFNalpha can induce morphological cell changes that are peculiar of apoptosis onset through the caspase-3-mediated cleavage of gelsolin. Furthermore, we have demonstrated that EGF is able to antagonize these effects through the inhibition of caspase-3 activation.
Abstract: Pamidronate (PAM) and zoledronic acid (ZOL) are aminobisphosphonates (BPs) able to affect the isoprenylation of intracellular small G proteins. We have investigated the antitumor activity of BPs and R115777 farnesyl transferase inhibitor (FTI) against epidermoid cancer cells. In human epidermoid head and neck KB and lung H1355 cancer cells, 48 h exposure to PAM and ZOL induced growth inhibition (IC(50) 25 and 10 microM, respectively) and apoptosis and abolished the proliferative and antiapoptotic stimuli induced by epidermal growth factor (EGF). In these experimental conditions, ZOL induced apoptosis through the activation of caspase 3 and a clear fragmentation of PARP was also demonstrated. A strong decrease of basal ras activity and an antagonism on its stimulation by EGF was recorded in the tumor cells exposed to BPs. These effects were paralleled by impaired activation of the survival enzymes extracellular signal regulated kinase 1 and 2 (Erk-1/2) and Akt that were not restored by EGF. Conversely, farnesol induced a recovery of ras activity and antagonized the proapoptotic effects induced by BPs. The combined treatment with BPs and R115777 resulted in a strong synergism both in growth inhibition and apoptosis in KB and H1355 cells. The synergistic activity between the drugs allowed BPs to produce tumor cell growth inhibition and apoptosis at in vivo achievable concentrations (0.1 micromolar for both drugs). Moreover, the combination was highly effective in the inhibition of ras, Erk and Akt activity, while farnesol again antagonized these effects. In conclusion, the combination of BPs and FTI leads to enhanced antitumor activity at clinically achievable drug concentrations that resides in the inhibition of farnesylation-dependent survival pathways and warrants further studies for clinical translation.
Abstract: Interferon-alpha (IFNalpha) is a recombinant protein widely used in the therapy of several neoplasms such as myeloma, renal cell carcinoma, epidermoid cervical and head and neck tumours and melanoma. IFNalpha, the first cytokine to be produced by recombinant DNA technology, has emerged as an important regulator of cancer cell growth and differentiation, affecting cellular communication and signal transduction pathways. However, the way by which tumour cell growth is directly suppressed by IFNalpha is not well known. Wide evidence exists on the possibility that cancer cells undergo apoptosis after the exposure to the cytokine. Here we will review the consolidate Signal transducer and activator of transcription (STAT)-dependent mechanism of action of IFNalpha and the supposed mechanism of apoptosis induction by IFNalpha. We will discuss data obtained by us and others on the triggering of the stress-dependent kinase pathway and on the modulation of protein synthesis machinery induced by IFNalpha and their correlations with the apoptotic process. Until today, inconsistent data have been obtained regarding the clinical effectiveness of IFNalpha in the therapy of solid tumours. In fact, the benefit of IFNalpha treatment is limited to some neoplasms while others are completely or partially resistant. The mechanisms of tumour resistance to IFNalpha have been studied in vitro. The alteration of JAK- Signal transducer and activator of transcription components of the IFNalpha-induced signalling, can be indeed a mechanism of resistance to IFN and cross talks between IFNalpha and survival signals has been also described. However, we have recently described a reactive mechanism of protection of tumour cells from the apoptosis induced by IFNalpha dependent on the epidermal growth factor (EGF)-mediated Ras/extracellular signal regulated kinase (Erk) signalling. The involvement of the Ras->Erk pathway in the protection of tumour cells from the apoptosis induced by IFNalpha is further demonstrated by both Ras inactivation by RASN17 transfection and mitogen extracellular signal regulated kinase 1 (Mek-1) inhibition by exposure to PD098059. These data strongly suggest that the specific disruption of the latter could be a useful approach to potentiate the antitumour activity of IFNalpha against human tumours based on the new mechanistic insights achieved in the last years.
Abstract: Interferon-alpha (IFNalpha) is a recombinant protein widely used in the therapy of several neoplasms such as myeloma, renal cell carcinoma, epidermoid cervical and head and neck tumours and melanoma. IFNalpha, the first cytokine to be produced by recombinant DNA technology, has emerged as an important regulator of cancer cell growth and differentiation, affecting cellular communication and signal transduction pathways. However, the way by which tumour cell growth is directly suppressed by IFNalpha is not well known. Wide evidence exists on the possibility that cancer cells undergo apoptosis after the exposure to the cytokine. Here we will discuss data obtained by us and others on the post-translational regulation of the expression of proteins involved in the occurrence of apoptotic process such as tissue transglutaminase (tTG) or in the modulation of cell cycle such as the cyclin-dependent kinase inhibitor p27. This new way of regulation of p27 and tTG occurs through the modulation of their proteasome-dependent degradation induced by the cytokine. We will also review the involvement of protein synthesis machinery in the induction of cell growth inhibition by IFNalpha. In details, we will describe the effects of IFNalpha on the expression and activity of the protein kinase dependent from dsRNA (PKR) and on the eukaryotic initiation factor of protein synthesis 5A (eIF-5A) and their correlations with the regulation of cancer cell growth. These data strongly suggest that the antitumour activity of IFNalpha against human tumours could involve still unexplored mechanisms based on post-translational and translational control of the expression of proteins that regulate cell proliferation and apoptosis.
Abstract: The mechanisms of tumor cell resistance to interferon-alpha (IFNalpha) are at present mostly unsolved. We have previously demonstrated that IFNalpha induces apoptosis on epidermoid cancer cells and EGF antagonizes this effect. We have also found that IFNalpha-induced apoptosis depends upon activation of the NH(2)-terminal Jun kinase-1 (Jnk-1) and p(38) mitogen-activated protein kinase, and that these effects are also antagonized by EGF. At the same time, IFNalpha increases the expression and function of the epidermal growth factor receptor (EGF-R). Here we report that the apoptosis induced by IFNalpha occurs together with activation of caspases 3, 6 and 8 and that EGF also antagonizes this effect. On the basis of these results, we have hypothesized that the increased EGF-R expression and function could represent an inducible survival response that might protect tumor cells from apoptosis caused by IFNalpha via extracellular signal regulated kinase 1 and 2 (Erk-1/2) cascades. We have found an increased activity of Ras and Raf-1 in IFNalpha-treated cells. Moreover, IFNalpha induces a 50% increase of the phosphorylated isoforms and enzymatic activity of Erk-1/2. We have also demonstrated that the inhibition of Ras activity induced by the transfection of the dominant negative Ras plasmid RASN17 and the inhibition of Mek-1 with PD098059 strongly potentiates the apoptosis induced by IFNalpha. Moreover, the selective inhibition of this pathway abrogates the counteracting effect of EGF on the IFNalpha-induced apoptosis. All these findings suggest that epidermoid tumor cells counteract the IFNalpha-induced apoptosis through a survival pathway that involves the hyperactivation of the EGF-dependent Ras->Erk signalling. The selective targeting of this pathway appears to be a promising approach in order to enhance the antitumor activity of IFNalpha.
Abstract: The addition of 2500 i.u./ml interferon alpha (IFNalpha) for 48 h induced apoptosis, and caused an approx. 4-fold increase in the activity and expression of tissue transglutaminase (tTG), in human lung cancer H1355 cells. However, the increase in mRNA levels for tTG was just 1.6-fold. On the basis of these data, we investigated whether tTG levels may be regulated through regulation of its degradation via ubiquitination. It was found that 2500 i.u./ml IFNalpha induced a time-dependent decrease in tTG ubiquitination. On the other hand, addition of the proteasome inhibitor lactacystin led to accumulation of the ubiquitinated form of the enzyme and to a consequent increase in its expression. Treatment of the cells with the two agents combined antagonized the accumulation of the ubiquitinated isoforms of tTG induced by lactacystin and caused a potentiation of tTG expression. Moreover, the tTG inducer retinoic acid was also able to cause increased expression and ubiquitination of tTG in H1355 cells. The addition of monodansylcadaverine (a tTG inhibitor) to IFNalpha-treated H1355 cells completely antagonized growth inhibition and apoptosis induced by the cytokine. In conclusion, we demonstrate for the first time that tTG is ubiquitinated and degraded by a proteasome-dependent pathway. Moreover, IFNalpha can, at least in part, induce apoptosis through the modulation of this pathway.
Abstract: Interferon-alpha (IFNalpha) can induce apoptosis, a process regulated by a complex network of cell factors. Among these, eukaryotic initiation factor-5A (eIF-5A) is peculiar because its activity is modulated by the post-translational formation of the amino acid hypusine. Here we report the effects of IFNalpha and epidermal growth factor (EGF) on apoptosis and eIF-5A activity in human epidermoid oropharyngeal KB and lung H1355 cancer cells. We found that 48-h exposure to 1000 and 2000 IU/ml IFNalpha induced about 50% growth inhibition and apoptosis in H1355 and KB cells, respectively, and the addition of EGF completely antagonized this effect. When IFNalpha induced apoptosis, a hyperactivation of MEK-1 and ERK signalling and a decrease of the hypusine-containing form and, thus, of eIF-5A activity were recorded. The latter effect was again antagonized by the addition of EGF to IFNalpha-pretreated cells, probably through the activation of the EGF-->ERK-dependent pathway, since the addition of the specific MEK-1 inhibitor PD098059 abrogated the recovery of intracellular hypusine content induced by EGF in IFNalpha-pretreated cancer cells. Subsequently, we evaluated if the hypusine synthesis inhibitor (and eIF-5A inactivator) N1-guanyl-1,7-diaminoheptane (GC7) synergized with IFNalpha in the induction of cell growth inhibition and apoptosis. The analysis of the isobologram of IFNalpha and GC7 demonstrated a strong synergism between the two drugs in inducing cell growth inhibition. We also found that GC7 and IFNalpha had a synergistic effect on apoptosis. These data suggest that the apoptosis induced by IFNalpha could be regulated by eIF-5A that, therefore, could represent a useful target for the potentiation of IFNalpha antitumor activity.
Abstract: Bisphosphonates (BPs) are an emerging class of drugs mostly used in the palliative care of cancer patients. We investigated the in vitro activity of the most potent antiresorptive BP, zoledronic acid (ZOL), on the growth and survival of three human pancreatic cancer (PC) cell lines (BxPC-3, CFPAC-1 and PANC-1). Pancreatic cancer frequently has a dysregulated p21(ras) pathway and therefore appears to be a suitable target for BPs that interfere with the prenylation of small GTP-binding proteins such as p21(ras). We found that ZOL induces growth inhibition (IC(50):10-50 micro M) and apoptotic death of PC cells. The proapoptotic effect was correlated to cleavage/activation of caspase-9 and poly(ADP)-ribose polymerase, but not of caspase-3. Moreover, we studied the p21(ras) signalling in cells exposed to ZOL and detected a reduction of p21(ras) and Raf-1 content and functional downregulation of the terminal enzyme ERK/MAPkinase and of the pKB/Akt survival pathway. Finally, we observed that ZOL induces significant cytoskeletal rearrangements. In conclusion, we demonstrated that ZOL induces growth inhibition and apoptosis on PC cells and interferes with growth and survival pathways downstream to p21(ras). These findings might be relevant for expanding application of BPs in cancer treatment.
Abstract: It has been reported that theophylline induces growth inhibition and apoptosis in tumour cells. We report that theophylline induces growth inhibition and apoptosis of several human epithelial tumour cells with an IC:50 of 2.5 mM after 48 h of exposure. Moreover, 2.5 mM theophylline induces the accumulation of cancer cells in S-phase of the cell cycle with a concomitant reduction in the percentage of tumour cells in G(1)/G(0) phase. These effects are paralleled by cytoskeletal remodelling with a consequent redistribution of actin fibers and shape change as demonstrated by fluorescence microscopy. The apoptotic death of tumour cells occurs together with an increase in the expression and activity of the pro-apoptotic enzyme tissue transglutaminase (tTGase). All these effects are promptly antagonized by the specific PKA inhibitor KT5720, suggesting the involvement of cAMP intracellular elevation and, consequently, PKA activation. On the other hand, growth inhibition and tTGase expression and activity are potentiated by retinoic acid, a tTGase inducer. Therefore, a mechanistic model of theophylline action and anti-tumour strategies based on the concomitant use of theophylline and agents that potentiate tTGase activity can be hypothesized.
Abstract: In the past years, the attention of scientists has mainly focused on the study of the genetic information and alterations that regulate eukaryotic cell proliferation and that lead to neoplastic transformation. An increasing series of data are emerging about the involvement of the initiation phase of translational processes in the control of cell proliferation. In this paper we review the novel insights on the biochemical and molecular events leading to the initiation and its involvement in cell proliferation and tumourigenesis. We describe the structure, regulation and proposed functions of the eukaryotic initiation factor 5A (eIF-5A) focusing the attention on its involvement in the regulation of apoptosis and cell proliferation. Moreover, we describe the modulation of its activity (through the reduction of hypusine synthesis) in apoptosis induced either by tissue transglutaminase or interferon a. Finally, we propose eIF-5A as an additional target of anti-cancer strategies.
Abstract: In the past years, the attention of scientists has focused mainly on the study of the genetic information and alterations that regulate eukaryotic cell proliferation and that lead to neoplastic transformation. All therapeutic strategies against cancer are, to date, directed at DNA either with cytotoxic drugs or gene therapy. Little or no interest has been aroused by protein synthesis mechanisms. However, an increasing body of data is emerging about the involvement of translational processes and factors in control of cell proliferation, indicating that protein synthesis can be an additional target for anticancer strategies. In this paper we review the novel insights on the biochemical and molecular events leading to protein biosynthesis and we describe their involvement in cell proliferation and tumorigenesis. A possible mechanistic explanation is given by the interactions that occur between protein synthesis machinery and the proliferative signal transduction pathways and that are therefore suitable targets for indirect modulation of protein synthesis. We briefly describe the molecular tools used to block protein synthesis and the attempts made at increasing their efficacy. Finally, we propose a new multimodal strategy against cancer based on the simultaneous intervention on protein synthesis and signal transduction.
Abstract: 8-Cl-cAMP, a cAMP analogue that antagonizes type I cAMP-dependent protein kinase, is a novel anti-tumor agent presently under investigation in clinical trials. Herein we report the effects of this agent on epidermal growth factor receptor expression and degradation in human KB cancer cells. Exposure to 10 microM 8-Cl-cAMP for 48 h induced a 65% increase in epidermal growth factor receptor surface expression while the receptor synthesis was 22-fold enhanced. Analysis of epidermal growth factor-dependent receptor internalization in 8-Cl-cAMP-treated cells showed a higher endocytosis rate as well as an accelerated degradation which occurred together with an increased receptor ubiquitination. The enhanced degradation of epidermal growth factor receptor correlated with the lack of epidermal growth factor-induced proliferation and mitogen-activated protein kinase stimulation. The disregulation of epidermal growth factor receptor internalization and ubiquitin-dependent degradation could underlay a new mechanism of the anti-tumor activity of 8-Cl-cAMP suggesting its combination with agents that disrupt epidermal growth factor receptor signalling.
Abstract: We have demonstrated that interferon-alpha2-recombinant (IFNalpha) at growth inhibitory concentrations enhances the expression and signalling activity of the epidermal growth factor receptor (EGF-R) in human epidermoid carcinoma KB cells. Here we report that KB cells exposed to IFNalpha underwent apoptotic cell death and this effect was antagonized by EGF. We have also found that IFNalpha enhanced the expression of heat shock proteins (HSP) HSP-70, HSP-90 and HSP-27 and activated the NH2-terminal Jun kinase-1 (JNK-1) and p38 mitogen activated protein kinase, the target enzymes of a stress-dependent intracellular transduction pathway. Moreover, the overexpression of the wild-type JNK-1, obtained through plasmid transfection of KB cells, induced apoptosis which was potentiated by the exposure of wild-type JNK-1 (JNK-1wt)-transfected cells to IFNalpha. All these effects were neutralized by the addition of EGF to parental and JNK-1wt-transfected KB cells exposed to IFNalpha. In conclusion, EGF has a protective effect on KB cells from apoptosis while antagonizing a stress response elicited by IFNalpha and targeted on the stress pathway terminal kinases.
Abstract: Eukaryotic translation initiation factor 5A (eIF-5A) is the only cell protein that contains the unusual basic amino acid hypusine [N epsilon-(4-amino-2-hydroxybutyl)lysine]. Hypusine is formed by the transfer of the butylamine portion from spermidine to the epsilon-amino group of a specific lysine residue of eIF-5A precursor and the subsequent hydroxylation at carbon 2 of the incoming 4-aminobutyl moiety. Agents that reduce cell hypusine levels inhibit the growth of mammalian cells. These observations suggest that hypusine is crucial for proliferation and transformation of eukaryotic cells. Here we have studied whether the inhibition of hypusine synthesis can potentiate the anti-cancer activity of the anti-tumour agents interferon-alpha (IFN alpha) and cytosine arabinoside (ara-C). We have found that IFN alpha increased epidermal growth factor receptor (EGF-R) expression, but reduced S phase and proliferative marker expression in human epidermoid KB cells and that this effect was antagonised by epidermal growth factor (EGF). Growth inhibition induced by IFN alpha was paralleled by decreased hypusine synthesis and, when EGF counteracted anti-proliferative effects, a reconstitution of hypusine levels was recorded. We also studied the effects of IFN alpha on the cytotoxicity of the recombinant toxin TP40 which inhibits elongation factor 2, another step of protein synthesis, through EGF-R binding and internalisation; IFN alpha induced an about 27-fold increase of TP40 cytotoxicity in KB cells. Ara-C, another antineoplastic agent commonly used in haematologic malignancies, induced both apoptosis and iron depletion in human acute myeloid leukaemic cells. The combination of ara-C and of the iron chelator desferioxamine, a strong inhibitor of hypusine synthesis, had a synergistic activity on apoptosis in these cells. The data strongly suggest that the post-translational modifications of eIF-5A could be a suitable target for the potentiation of the activity of anti-cancer agents.
Abstract: We investigated whether changes in iron metabolism and the transferrin receptor (TRF-R) expression were involved in the antileukaemic effects of arabinoside cytosine (ara-C). Treatment with 100 nM ara-C for 48h reduced thymidine uptake and increased the surface expression of the TRF-R on leukaemic blasts derived from 13/16 (81%) patients and on the HL-60 and U-937 cell lines. Whereas intracellular non-haem iron was strongly depleted 24 h after ara-C addition, TRF-R up-regulation and recovery of intracellular non-haem iron concentration occurred together after a longer exposure of the cultured cells to the drug. Since iron is an essential regulator of cell proliferation we have evaluated the effects of the combination between ara-C and the iron chelator desferioxamine (DSF) on the growth of HL-60 and U-937 cells. We found that desferioxamine strongly potentiated the effects of ara-C on leukaemic cell growth inhibition and apoptosis. This is the first report of a positive interaction between ara-C and an iron chelator in terms of antileukaemic effects.
Abstract: We previously found that interferon alpha2 recombinant (IFNalpha) increases the expression of epidermal growth factor receptor (EGF-R) in the human epidermoid cancer KB cell line. Here we report the effects of IFNalpha and epidermal growth factor (EGF) on KB cell cycle kinetics. IFNalpha (1000 i.u./ml) for 48 h decreased the S-phase fraction and diminished the expression of Ki67 and proliferating cell nuclear antigen on KB cells. Incubation of IFNalpha-treated KB cells with 10 nM EGF for 12 h reversed these effects. We then studied several biochemical markers of cell proliferation. Ornithine decarboxylase activity was decreased to about one-tenth by IFNalpha and partly restored by EGF. Hypusine is contained only in eukaryotic initiation factor 5A and its levels are correlated with cell proliferation. IFNalpha decreased hypusine synthesis by 75%; exposure of cells to EGF for 12 h restored hypusine synthesis almost completely. We also studied the effects of IFNalpha on the cytotoxicity of the recombinant toxin TP40, which inhibits elongation factor 2 through EGF-R binding and internalization. IFNalpha greatly enhanced the TP40-induced inhibition of protein synthesis in KB cells. In conclusion, IFNalpha, which affects protein synthesis machinery and increases EGF-R expression, enhances the tumoricidal activity of TP40 and hence could be useful in the setting of anti-cancer therapy.
Abstract: We have demonstrated that anticancer drugs at cytostatic concentrations enhance the expression and function of epidermal growth factor (EGF-R) and transferrin (TRF-R) receptors on human tumor cells. We hypothesized that these effects could represent a protective response of tumor cells to sublethal antiproliferative stimuli which could lead to enhanced growth potential. 72 hours exposure of human melanoma GLL-19 cells to 1,000 nM ara-C induced growth inhibition and increased the number of EGF-R, TRF-R and nerve growth factor receptor (NGF-R) on cell surface. Enhanced expression of beta 3 integrins CD49a, CD49c and CD49e, av integrin CD51, beta 3 integrin CD61, CD58/LFA3 and collagen IV and laminin was also detected in ara-C-treated GLL-19 cells. These changes at the tumor cell surface were paralleled by increased in vitro adhesion, invasive potential and clonogenic growth in soft agar and in vivo tumor formation. A more aggressive tumor cell phenotype is induced in human melanoma cells after transient exposure to cytostatic concentrations of ara-C.
Abstract: The molecular mechanisms underlying the growth inhibition of human tumor cells induced by recombinant interferon-alpha (IFN alpha) are mostly unknown. It has been proposed that this effect could be related to down-regulation and/or impaired function of peptide growth factor receptors (PGF-Rs) in tumor cells exposed to IFN alpha. However, we have previously described that IFN alpha-induced growth inhibition of human epidermoid carcinoma cells is paralleled by up-regulation of epidermal growth factor receptor (EGF-R). Here we report that an increase in EGF-R synthesis is detectable after 3 hr of exposure to cytostatic concentration of IFN alpha in epidermoid KB tumor cells. In these experimental conditions IFN alpha does not depress and even potentiates EGF-R function. IFN alpha-treated KB cells retain sensitivity to the cytotoxic activity of the anti-EGF-R 225 monoclonal antibody (MAb), which acts through receptor blockade, and are sensitized to the growth-promoting effect of EGF. EGF-induced tyrosine (tyr) phosphorylation both of total cellular protein extracts and of the immunoprecipitated EGF-R is increased in IFN alpha-treated cells. We conclude that a cross-talk between IFN alpha and EGF occurs in KB cells since IFN alpha, at cytostatic concentration, potentiates the effects mediated by the EGF-R.
Abstract: We have demonstrated that interferon-alpha (IFN alpha) upregulates the epidermal growth factor receptor (EGF-R) on human epidermoid carcinoma cells. Here we report that IFN alpha induces growth inhibition and upregulation of transferrin receptor (TRF-R) on epidermoid cancer KB cells. IFN alpha does not alter TRF-R affinity for its ligand and induces a two-fold increase of TRF binding sites. IFN alpha does not modify receptor internalization and cycling. Intracellular iron levels are known to regulate TRF-R expression: we have, therefore, evaluated whether changes in the iron content could be determined by IFN alpha. Iron levels are transiently increased after addition of fresh growth medium in untreated controls but not in KB cells exposed for 48 h to IFN alpha. Iron depletion is however completely reversed 24 h later when maximal TRF-R upregulation occurs in IFN alpha-treated cells. We suggest that IFN alpha-induced iron depletion elicits a homeostatic cellular response through upregulation of TRF-R.
Abstract: 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.
Abstract: Membrane receptors for peptide growth factor receptors (PGF-R) play a crucial role in the regulation of cancer cell proliferation and may behave as tumor associated antigens (TAA), which are currently regarded as specific targets for immunodetection and immunotherapy of human cancer. PGF-R are often more expressed by tumor cells than by normal counterparts and, by analogy to TAA, their surface expression may be regulated by cytokines. Moreover, the biological functions and specific ligands of most PGF-R are presently well elucidated as opposed to the great majority of TAA. PGF-R may, therefore, represent ideal cellular targets for at least two different therapeutic approaches: (i) naked or conjugated monoclonal antibodies and (ii) genetically engineered fusion proteins composed of PGF-R physiological ligands linked to genetically modified bacterial toxins. To date, clinical studies based on targeting of receptors for epidermal growth factor and interleukin-2 on tumor cells have been performed. Information from such studies suggests that PGF-R as well as TAA targeting strategies are clinically feasible, but that they still have to be optimized. A variety of host and tumor factors which affect targeting of neoplastic cells have been recently identified. For instance, it has been demonstrated that the antigenic density of the targeted molecule at the tumor cell surface is an important factor. In this view upregulation of PGF-R on cancer cells could be of major clinical advantage in immunotargeting. It has been reported that several cytokines and chemical compounds can induce PGF-R modulation on tumor cells. This paper reviews therapeutic opportunities related to the pharmacologic modulation of PGF-R expression. In addition a mechanistic hypothesis regarding PGF-R upregulation induced by cytostatic drugs and cytokines is proposed.
Abstract: We report that cytosine arabinoside (Ara-C), a cytosine analogue that at low doses causes phenotypical changes on human leukemia cells in vitro and in vivo, induces growth inhibition of oropharyngeal cancer KB and lung adenocarcinoma A549 cell lines. An increase in the number of epidermal growth factor and transferrin receptors (EGFR, TrfR) is induced by Ara-C on these cells. Maximal EGFR up-regulation occurs 96 h after the beginning of Ara-C exposure while maximal TrfR up-regulation is detected 24 h later. These effects occur without changes in the affinity of EGFR and TrfR for their ligands. Two classes of EGF-binding sites with a Kd of 0.055 nM and 2.3 nM respectively, and one class of transferrin-binding sites with a Kd of about 4 nM are detected on both untreated and Ara-C-treated KB cells. [3H]Thymidine uptake is clearly stimulated on KB cells by nanomolar concentrations of EGF and transferrin, whereas in Ara-C-treated cells [3H]thymidine uptake is not increased by EGF and transferrin under conditions where maximal EGFR and TrfR up-regulation occurs. The enhanced EGF and transferrin binding is paralleled by a twofold increase of in vitro targeting of Ara-C-treated KB and A549 cells with anti-EGFR 108.1 mAb and anti-TrfR OKT9 mAb. We propose that Ara-C could provide a new approach for the improvement of the therapeutic index of anti-EGFR and anti-TrfR immunoconjugates.
Abstract: Unregulated or increased expression of epidermal growth factor receptor (EGF-R) is a common event in neoplastic transformation; modulation of such a receptor by physiological agents could be, therefore, of clinical interest. We have studied the binding ability, the availability at cell surface, and the synthesis of EGF-R in the A431 and KB human epidermoid cancer cell lines after treatment with recombinant alpha-interferon (IFN-alpha). After 48 h of treatment, IFN-alpha induces, in both cell lines, growth inhibition and enhances class I major histocompatibility HLA complex expression, which is a common marker of IFN action. [125I]EGF total binding assessed after 48 h of treatment with IFN-alpha shows a dose-dependent upregulation of EGF-R binding capacity. Saturation plots of the binding data show that IFN-alpha treatment does not dramatically alter the affinity of the EGF-R and indicate that IFN-alpha only increases the number of low affinity receptors. We show that this effect is due to a specific increase in the synthesis of the receptor protein, as assessed by immunoprecipitation of [35S]methionine-labeled cell extracts. Electron microscopy analysis has confirmed an increase of EGF-R proteins at cell surface without major changes in the morphology of the cells. Taken together, these results indicate that IFN-alpha consistently induces both the binding capacity and the synthesis of EGF-R in human epidermoid cancer cells and suggest the use of such a mechanism for new anticancer therapies.
