Role of TGF-alpha in the progression of diabetic kidney disease.

Transforming growth factor-alpha (TGFA) has been shown to play a role in experimental chronic kidney disease associated with nephron reduction, while its role in diabetic kidney disease (DKD) is unknown. We show here that intrarenal TGFA mRNA expression, as well as urine and serum TGFA, are increased in human DKD. We used a TGFA neutralizing antibody to determine the role of TGFA in two models of renal disease, the remnant surgical reduction model and the uninephrectomized (uniNx) db/db DKD model. In addition, the contribution of TGFA to DKD progression was examined using an adeno-associated virus approach to increase circulating TGFA in experimental DKD. In vivo blockade of TGFA attenuated kidney disease progression in both nondiabetic 129S6 nephron reduction and Type 2 diabetic uniNx db/db models, whereas overexpression of TGFA in uniNx db/db model accelerated renal disease. Therapeutic activity of the TGFA antibody was enhanced with renin angiotensin system inhibition with further improvement in renal parameters. These findings suggest a pathologic contribution of TGFA in DKD and support the possibility that therapeutic administration of neutralizing antibodies could provide a novel treatment for the disease.

ß-ionone inhibits persistent preneoplastic lesions during the early promotion phase of rat hepatocarcinogenesis: TGF-α, NF-κB, and p53 as cellular targets.

Dietary isoprenic derivatives such as ß-ionone (ßI) are a promising class of chemopreventive agents. In this study, cellular aspects of ßI protective activities during early hepatocarcinogenesis were evaluated. Male Wistar rats were submitted to "resistant hepatocyte" model and then received daily 16 mg/100 g body weight (b.w.) of ßI (ßI group) or only 0.25 mL/100 g b.w. of corn oil (vehicle, control group [CO]) during 4 wk, specifically during early promotion phase. Compared to controls, ßI inhibited (P < 0.05) the development of persistent preneoplastic lesions (pPNL), considered to be potential hepatocellular carcinoma (HCC) progression sites, and increased remodeling PNL (rPNL) (P < 0.05) that tend to regress to a normal phenotype. Increased ßI hepatic levels (P < 0.05), in the ßI group, were associated with its chemopreventive actions. Compared to control rats, ßI reduced the frequency of both pPNL and rPNL positive for tumor growth factor (TGF)-α (P < 0.05), reduced the frequency of pPNL stained for p65 (nuclear factor-kappaB; NF-κB) (P < 0.05), and reduced the frequency of pPNL positive for cytoplasmic p53 (P < 0.05). Our data demonstrated that ßI targets TGF-α, NF-κB, and p53 in initial phases of hepatocarcinogenesis and specifically inhibits PNL with increased probability to progress to HCC. This isoprenoid may represent a chemopreventive agent of choice for HCC control.

Inhibitors/antagonists of TGF-ß system in kidney fibrosis.

Renal fibrosis is a major hallmark of chronic kidney disease, regardless of the initial causes, and prominent renal fibrosis predicts poor prognosis for renal insufficiency. Transforming growth factor (TGF)-ß plays a pivotal role in the progression of renal fibrosis, and therapeutic interventions targeting TGF-ß have been successful and well tolerated in animal models. However, these interventions might have adverse effects by inducing systemic inflammation due to the strong bifunctional role of TGF-ß (pro-fibrotic and anti-inflammatory). This review of the current literature focuses on the inhibitors/antagonists of TGF-ß, and discusses possible therapeutic approaches targeting them, describing the effectiveness of orally active bone morphogenetic protein 7 mimetics in reversing established fibrosis. It will conclude with a brief discussion of possible future directions for research.

Peroxisome proliferator activated receptor-α agonists suppress transforming growth factor-α-induced matrix metalloproteinase-9 expression in human keratinocytes.

The peroxisome proliferator-activated receptors (PPARs), PPARα, PPARδ, and PPARγ, are nuclear ligand-activated transcription factors that are best known as regulators of glucose and lipid metabolism. PPARα agonists have been shown to have profound anti-inflammatory and antiproliferative effects on human keratinocytes, and are important for maintaining homeostasis of the skin. To better characterize the mechanisms underlying these phenomena, we analysed the effects of PPARα agonists on transforming growth factor (TGF)-α-induced expression of matrix metalloproteinase (MMP)-9. MMP-9 expression in keratinocytes has previously been linked to inflammatory skin diseases, regenerative skin mechanisms, and tumour development and metastasis. We found that PPARα agonists effectively inhibited TGF-α-induced MMP-9 expression in human keratinocytes via a post-transcriptional mechanism, revealing a novel and important aspect of the anti-inflammatory and anticarcinogenic action of these compounds.

Altered proTGFα/cleaved TGFα ratios offer new therapeutic strategies in renal carcinoma.

BACKGROUND: Treatment of renal cancer has significantly improved with the arrival to the clinic of kinase inhibitors and immunotherapies. However, the disease is still incurable in advanced stages. The fact that several approved inhibitors for kidney cancer target receptor tyrosine kinases (RTKs) suggests that these proteins play a critical role in the pathophysiology of the disease. Based on these precedents, we decided to explore whether RTKs other than those targeted by approved drugs, contribute to the development of kidney cancer. METHODS: The activation status of 49 RTKs in 44 paired samples of normal and tumor kidney tissue was explored using antibody arrays, with validation by western blotting. Genetic and pharmacologic approaches were followed to study the biological implications of targeting the epidermal growth factor receptor (EGFR) and its ligand Transforming Growth Factor-α (TGFα). RESULTS: Activation of the EGFR was found in a substantial number of tumors. Moreover, kidney tumors expressed elevated levels of TGFα. Down-regulation of EGFR or TGFα using RNAi or their pharmacological targeting with blocking antibodies resulted in inhibition of the proliferation of in vitro cellular models of renal cancer. Importantly, differences in the molecular forms of TGFα expressed by tumors and normal tissues were found. In fact, tumor TGFα was membrane anchored, while that expressed by normal kidney tissue was proteolytically processed. CONCLUSIONS: The EGFR-TGFα axis plays a relevant role in the pathophysiology of kidney cancer. This study unveils a distinctive feature in renal cell carcinomas, which is the presence of membrane-anchored TGFα. That characteristic could be exploited therapeutically to act on tumors expressing transmembrane TGFα, for example, with antibody drug conjugates that could recognize the extracellular region of that protein.

Estradiol, progesterone, and transforming growth factor alpha regulate insulin-like growth factor binding protein-3 (IGFBP3) expression in mouse endometrial cells.

Insulin-like growth factor 1 (IGF1) is involved in the proliferation of mouse and rat endometrial cells in a paracrine or autocrine manner. Insulin-like growth factor binding protein-3 (IGFBP3) modulates actions of IGFs directly or indirectly. The present study aimed to determine whether IGFBP3 is involved in the regulation of proliferation of mouse endometrial cells. Mouse endometrial epithelial cells and stromal cells were isolated, and cultured in a serum free medium. IGF1 stimulated DNA synthesis by endometrial epithelial and stromal cells, and IGFBP3 inhibited IGF1-induced DNA synthesis. Estradiol-17beta (E2) decreased the Igfbp3 mRNA level in endometrial stromal cells, whereas it increased the Igf1 mRNA level. Transforming growth factor alpha (TGFalpha) significantly decreased IGFBP3 expression at both the mRNA and secreted protein levels in endometrial stromal cells. Progesterone (P4) did not affect the E2-induced down-regulation of Igfbp3 mRNA expression in endometrial stromal cells, although P4 alone increased Igfbp3 mRNA levels. The present findings suggest that in mouse endometrial stromal cells E2 enhances IGF1 action through enhancement of IGF1 synthesis and reduction of IGFBP3 synthesis, and that TGFalpha affects IGF1 actions through modulation of IGFBP3 levels.

Multigene targeting of signal transduction pathways for the treatment of breast and prostate tumors: comparison between combination therapies employing bispecific oligonucleotides with either Rapamycin or Paclitaxel.

Previous studies have demonstrated that monospecific antisense oligonucleotides (oligos) directed against mRNA encoding proteins associated with tumor growth, death, and survival are efficacious against breast and prostate tumors. Targeted proteins, associated with different signal transduction pathways, have included transforming growth factor-alpha [TGF-alpha (MR(1))], its binding site the epidermal growth factor receptor [EGFR (MR(2))] sharing sequence homology to the breast cancer prognostic marker Her-2/neu, an apoptosis inhibiting protein [bcl-2 (MR(4))], and the androgen receptor [AR (MR(5))]. In attempts to enhance antisense therapy, recent reports describe how two of the binding sites mentioned above can be sequentially placed within a single complementary (bispecific) strand and administered either in the presence or absence of additional therapeutic agents. When tested against breast and prostate tumor cell lines specific differences were noted: MCF-7 breast cancer cells were more receptive to the inhibitory effects of monospecific oligos, whereas PC-3 and LNCaP prostate cells were particularly responsive to bispecifics. In an effort to identify agents which enhance the activity of oligos and which possess less toxicity than traditionally employed chemotherapeutics, Rapamycin, an immunosuppressive agent known to regulate tumor growth and signal transduction mediated by the mTOR receptor, is compared to paclitaxel in combination therapy employing monospecific or bispecific oligos. Bispecifics were constructed recognizing the binding sites for TGF-alpha and EGFR mRNA [TGF-alpha/EGFR (MR(12)) and EGFR/TGF-alpha (MR(21))]; another pair recognized binding sites for EGFR and bcl-2 [EGFR/bcl-2 (MR(24)) and bcl-2/EGFR (MR(42))]; while a third pair employed only against the LNCaP prostate cell line recognized bcl-2 and the androgen receptor [bcl-2/AR (MR4(45)) and AR/bcl-2 (MR(54))]. Oligo pairs differ in their 5'-3' linear binding site orientations, and were tested in vitro against MCF-7 breast and PC-3 and LNCaP prostate tumor cell lines. Following cell attachment, incubations were done for 2 days with the agents followed by 2 days in their absence. Five experiments evaluated the effect of monospecific or bispecific antisense oligos in combination with an LD(50) dosage of either Rapamycin or paclitaxel and led to the conclusion that although these agents act via different mechanisms, they are comparable in effectiveness.

A novel mechanism of resistance to epidermal growth factor receptor antagonism in vivo.

Epidermal growth factor receptor (EGFR) is widely expressed in a number of solid tumors including colorectal cancers. Overexpression of this receptor is one means by which a cell can achieve positive signals for survival and proliferation; another effective means is by constitutive activation of EGFR. We have elucidated the role of constitutive EGFR signaling in malignant progression by stably transfecting colon cancer cells with a human transforming growth factor-alpha cDNA (a ligand for EGFR) under repressible control by tetracycline. We show that constitutive expression of transforming growth factor-alpha and its subsequent constitutive activation of EGFR allows for cancer cell survival in response to environmental stress in vitro and in vivo as well. The reversal of constitutive EGFR activation results in the loss of downstream mitogen-activated protein kinase and Akt activation, and a reduction in xenograft size that is associated with decreased proliferation and increased apoptosis. We used CI-1033, a small molecule antagonist of EGFR, to dissect an activation pathway that shows the ability of ERBb2 to activate Akt, but not Erk in the face of EGFR antagonism. This novel escape mechanism is a possible explanation of why anti-EGFR therapies have shown disappointing results in clinical trials.

Blockage of TGF-α Induced by Spherical Silica Nanoparticles Inhibits Epithelial-Mesenchymal Transition and Proliferation of Human Lung Epithelial Cells.

Background. Xuanwei City in Yunnan province has been one of the towns with highest lung cancer mortality in China. The high content of amorphous silica in the bituminous coal from Xuanwei of Yunnan is mainly present as irregular and spherical silica nanoparticles (SiNPs). It has been reported that silica nanoparticles in bituminous coal correlated with the high incidence of lung cancer in Xuanwei. To explore the role and mechanism of SiNPs in the tumorigenesis of lung cancer in Xuanwei, human mononuclear cells (THP-1) and human bronchial epithelial cells (BEAS-2B) were cocultured in a transwell chamber. Combined with Benzo[a]pyrene-7, 8-dihydrodiol-9, and 10-epoxide (BPDE), SiNPs could significantly promote the proliferation and Epithelial-Mesenchymal Transition (EMT) and inhibit apoptosis of BEAS-2B cells and induce the release of TGF-α from THP-1 cells. After neutralizing TGF-α with antibody, the proliferation and EMT were decreased and enhanced apoptosis of BEAS-2B cells. Furthermore, the results showed that TGF-α in the sera of patients with lung adenocarcinoma in Xuanwei were significantly higher than in patients with benign pulmonary lesions in Xuanwei and those with lung adenocarcinoma in outside of Xuanwei of Yunnan. Taken together, our study found that SiNPs promoted the proliferation and EMT of BEAS-2B cells by inducing the release of TGF-α from THP-1 cells.

Treatment of MCF-7 breast cancer cells employing mono- and bispecific antisense oligonucleotides having binding specificity toward proteins associated with autocrine regulated growth and BCL-2.

Antisense oligonucleotides (oligos) against transforming growth factor-alpha (TGF-alpha) (MR1) and its binding site, the epidermal growth factor receptor (EGFR) (MR2), are efficacious against the UACC 897 breast, PC-3 and LNCaP prostate, and T98G glioblastoma tumor lines in both in vitro and in vivo studies. Oligos against the anti-apoptosis protein bcl-2 (MR4) are also efficient against PC-3 and LNCaP tumors in similar in vitro experiments. To enhance activity, and also to introduce a derivative type of multifunctional oligo into this field, "bispecifics" were constructed containing two truncated complementary DNA sequences (from either MR1 or MR2) designed to bind targeted mRNA about their respective AUG initiation codons, and/or a similar sequence adjacent to the AUG site of mRNA encoding bcl-2. Tandem pairs of bispecifics were constructed: The first had complementary sequences for TGF-alpha and EGFR mRNA, but differed in 5' to 3' tandem orientation (TGF-alpha/EGFR [MR12] and EGFR/TGF-alpha [MR21] sequences); a second pair had binding sites associated with EGFR and bcl-2, also differing in orientation (EGFR/bcl-2 [MR24] and bcl-2/EGFR [MR42]). In studies targeting PC-3 and LNCaP cells, bispecifics demonstrated significant in vitro activity, and the second pair was significantly better than the original monospecifics. These studies are now extended to the MCF-7 breast cancer model in order to determine whether these particular bispecifics have similar anti-breast cancer activity and if they are significantly better than monospecific oligos from which they were derived. We conclude that bispecific oligos significantly inhibit MCF-7 growth, however, in contrast to results obtained with PC-3 and LNCaP, the monospecific oligos directed against EGFR and bcl-2 have significantly greater activity than the bispecifics targeting a combination of TGF-alpha, EGFR, or bcl-2. These data suggest that the relative activities of oligos, whether mono- or bispecific, change with tumor type. Bispecific oligos which target different proteins, possibly those which regulate estrogen utilization, may be more effective against MCF-7 cells and warrant additional investigation, particularly if co-administered with traditional chemotherapeutics.

Inhibition of Proliferation and Induction of Apoptosis by Thymoquinone via Modulation of TGF Family, p53, p21 and Bcl-2α in Leukemic Cells.

INTRODUCTION: Adult T-cell leukemia (ATL) is an aggressive form of malignancy caused by human T- cell lymphotropic virus 1 (HTLV-1). Currently, there is no effective treatment for ATL. Thymoquinone has been reported to have anti-cancer properties. OBJECTIVE: The aim of this study is to investigatthe effects of TQ on proliferation, apoptosis induction and the underlying mechanism of action in both HTLV-1 positive (C91-PL and HuT-102) and HTLV-1 negative (CEM and Jurkat) malignant T-lymphocytes. MATERIALS AND METHODS: Cells were incubated with different thymoquinone concentrations for 24h. Cell cytotoxicity was assayed using the CytoTox 96® Non-Radioactive Cytotoxicity Assay Kit. Cell proliferation was determined using CellTiter 96® Non-Radioactive Cell Proliferation. Cell cycle analysis was performed by staining with propidium iodide. Apoptosis was assessed using cell death ELISA kit. The effect of TQ on p53, p21, Bcl-2 protein expression was determined using Western blot analysis while TGF mRNA expression was determined by RT-PCR. RESULTS: At non-cytotoxic concentrations of TQ, it resulted in the inhibition of proliferation in a dose dependent manner. Flow cytometric analysis revealed a shift in the cell cycle distribution to the PreG1 phase which is a marker of apoptosis. Also TQ increase DNA fragmentation. TQ mediated its anti-proliferative effect and apoptosis induction by an up-regulation of TGFß1, p53 and p21 and a down-regulation of TGF-α and Bcl-2α. CONCLUSION: Thymoquinone presents antiproliferative and proapoptotic effects in ATL cells. For this reason, further research is required to investigate its possible application in the treatment of ATL.

Combination chemotherapy employing bispecific antisense oligonucleotides having binding sites directed against an autocrine regulated growth pathway and bcl-2 for the treatment of prostate tumors.

In previous studies we demonstrated that antisense oligonucleotides (oligos) against transforming growth factor-alpha (TGF-alpha [MR1]), its binding site the epidermal growth factor receptor (EGFR [MR2]), and the anti-apoptosis protein bcl-2 (MR4) are efficacious against prostate tumors. In recent reports we also describe how two of these mRNA directed binding sites can be synthesized sequentially within a single linear complementary strand and administered either in the presence or absence of additional therapeutic agents. In these continuing experiments "bispecific" oligo pairs were further evaluated in the presence or absence of Cytoxan, Taxol, or DES. One oligo pair recognized the binding sites for TGF-alpha and EGFR mRNA (TGF-alpha/EGFR [MR12] and EGFR/TGF-alpha [MR21]); another pair recognized binding sites for EGFR and bcl-2 (EGFR/bcl-2 [MR24] and bcl-2/EGFR [MR42]). Oligo pairs differ in their linear 5' to 3' binding site orientations, and were tested in vitro against PC-3 and LNCaP prostate tumor cell lines. Following cell attachment, incubations were for 2 days with the agents followed by 2 days in their absence. When tested against PC-3 cells and combined with LD50 Cytoxan, MR2, MR4, MR24, MR42 significantly inhibited 47.3, 45.7, 68.3, and 64.9%; with LD50 Taxol MR2, MR4, MR24, MR42 significantly inhibited 49.8, 45.8, 64.1, and 59.2%; and with LD50 DES MR2, MR4, MR24, MR42 significantly inhibited 66.6, 67.6, 64.3, and 67.2% respectively. Each agent significantly increased the inhibition produced by either oligo alone.LNCaP cells were also incubated with mono- and bispecific oligos in either the presence or absence of chemotherapeutics. MR2, MR4, MR24, MR42 produced significant inhibitions of 57.4, 58.4, 69.4, and 68.6% with LD50 Cytoxan; 70.4, 70.1, 73.6, and 74.0% with LD50 Taxol; and 49.8, 50.1, 59.6, and 53.9%, respectively with LD50 DES.A complete PC-3 experiment compared MR1, MR2, MR4, MR12, MR21, MR24 and MR42, in the presence of LD50 Cytoxan. Each oligo combined with Cytoxan significantly inhibited: MR1 by 51.0, MR2 by 55.0, MR4 by 58.0; MR12 by 56.0; MR21 by 61.1, MR24 by 65.5 and MR42 by 66.0%. Bispecifics directed against two different pathways, MR24, and MR42 were the most effective.A complete LNCaP experiment compared the same series of oligos also in the presence of LD50 Cytoxan. Each oligo combined with Cytoxan significantly inhibited: MR1 by 49.0, MR2 by 50.0, MR4 by 53.0; MR12 by 52.0; MR21 by 58.6, MR24 by 53.9 and MR42 by 58.0%.

Bispecific antisense oligonucleotides having binding sites directed against an autocrine regulated growth pathway and bcl-2 for the treatment of prostate tumors.

Antisense oligonucleotides (oligos) against transforming growth factor-alpha (TGF-alpha) (MR1) and its binding site, the epidermal growth factor receptor (EGFR) (MR2), are efficacious against PC-3 and LNCaP prostate tumors. To enhance activity and aid in simultaneous delivery, "bispecific" 39-mer oligos were constructed containing portions of both MR1 and MR2 sequences. The first pair contained truncated sequences recognizing TGF-alpha and EGFR mRNA binding sites, about their respective AUG initiation codons. These bispecifics differ in their 5' to 3' tandem orientation (TGF-alpha/EGFR [MR12] and EGFR/TGF-alpha [MR21] sequences). A second pair was constructed having complementary sequences for EGFR and bcl-2 (EGFR/bcl-2 [MR24] and bcl-2/EGFR [MR42]). All bispecifics were tested in vitro against PC-3 and LNCaP prostate tumor cells, and compared to mono-specific oligos from which they were derived. The purpose of this study was: (1) to evaluate bispecific antitumor activity; (2) to identify dominant sequences; (3) to identify effects of binding site orientation; and (4) to determine whether bispecifics are more effective when targeting one versus different growth-dependent pathways. Comparisons were made between oligos tested against either PC-3 or LNCaP cells incubated for 2 d with the agents followed by 2 d in their absence. The first PC-3 cell experiment demonstrated that bispecific MR12 and MR21 oligos are at least as effective as their mono-specific counterparts and that the MR21 bispecific orientation is more effective than the MR1 mono-specific by 64% (p = 0.014). It also suggested that the sequence directed against EGFR contributed most to bispecific activity, particularly in the MR21 orientation. In a second PC-3 study a second bispecific pair of 37-mer oligos was constructed containing bases complementary to mRNA encoding EGFR and the apoptosis-associated protein bcl-2 (MR4). MR24 was constructed with the EGFR complementary site at the 5' end (EGFR/bcl-2), and MR42, containing the opposite orientation (bcl-2/EGFR). Each contained the dominant EGFR activity identified previously. MR1, MR2, MR4, MR12, MR21, MR24, and MR42 (1X and 2X in concentration) were cultured with cells and compared to controls. Each oligo significantly inhibited growth of PC-3 cells. MR42 was most effective and significantly better than MR1 (p = 0.0128), MR2 (p = 0.021), MR4 (p = 0.0002), and MR12 (p = 0.0032). 2X MR24 and 2X MR42 were better than their 1X concentration counterparts, but the differences were not significant. In a similar experiment MR1, MR2, MR4, MR12, MR21, MR24, and MR42 were cultured with LNCaP cells and compared to lipofectin-containing controls. Each oligo significantly inhibited the growth of LNCaP cells. Again, MR42 was most effective and significantly better than MR2 (p = 0.021) and MR4 (p = 0.038). MR24 was significantly better than MR2 (p = 0.048). Bispecific oligos are a significant advance in antisense technology and could play a role in treating prostate cancer, particularly if combined with traditional chemotherapeutics.

Epidermal growth factor receptor-related protein inhibits cell growth and induces apoptosis of BxPC3 pancreatic cancer cells.

Dysregulation of the epidermal growth factor receptor (EGFR) signaling network has been frequently reported in pancreatic cancer. Inhibition of EGFR was associated with antitumor effects in both in vitro and in vivo studies of pancreatic cancer. We have previously reported the isolation and characterization of an EGFR-related protein (ERRP), which seems to be a negative regulator of EGFR. In the present investigation, we tested our hypothesis whether recombinant ERRP could be an effective inhibitor of growth of BxPC3 pancreatic cancer cells. Cell growth and apoptosis were measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and apoptosis ELISA assay, respectively, in the presence and absence of recombinant ERRP in BxPC3 cells. To evaluate activation of EGFR and its downstream signaling events, levels of phospho-EGFR, phospho-AKT, and phospho-extracellular signal-regulated kinase (phospho-ERK) were determined by Western blot analysis. NF-kappaB activity was measured by electrophoretic mobility shift assay. Our data show, for the first time, that ERRP inhibits the growth of BxPC3 cells in a dose- and time-dependent manner. The EGF or transforming growth factor (TGF)-alpha-induced stimulation of cell growth and activation of EGFR was also inhibited by ERRP. These changes were accompanied by a concomitant attenuation of activation of mitogen-activated protein (MAP) kinases, AKT, and NF-kappaB. ERRP also induced apoptosis as evidenced by increased poly(ADP-ribose) polymerase cleavage and reduction in procaspase3. From these results, we conclude that ERRP is a potent inhibitor of growth of BxPC-3 pancreatic cancer cells, which could be due to attenuation of EGFR cellular signaling processes. We also suggest that ERRP could be a potential therapeutic agent for pancreatic cancer.

Cetuximab in advanced non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) remains a major problem in the western civilization and developing countries. Since most patients with NSCLC have advanced disease at diagnosis, to date, chemotherapy, with third-generation platinum-based doublets, represents the standard of care. Advances in the knowledge of tumour biology and mechanisms of oncogenesis has granted the singling out of several molecular targets for NSCLC treatment. Epidermal growth factor receptor (EGFR), a member of ErbB family, is one of the most studied target. Cetuximab is a chimeric (human-murine) monoclonal antibody directed against the extracellular domain of the EGFR that blocks ligand (TGF-alpha, EGF) access to the receptor. In the present paper we discuss about the activity, tolerability and efficacy of cetuximab, the EGFR monoclonal blocking antibody with the largest amount of clinical data being available on the treatment of advanced NSCLC.

Orotic acid, nucleotide-pool imbalance, and liver-tumor promotion: a possible mechanism for the mitoinhibitory effects of orotic acid in isolated rat hepatocytes.

This study was designed to determine the possible mechanism by which orotic acid exerts its mitoinhibitory effect on rat hepatocytes in primary culture. Orotic acid inhibited, dose-dependently DNA synthesis in hepatocytes induced by epidermal growth factor, transforming growth factor alpha, hepatocyte growth factor, acidic fibroblast growth factor, or plasma from rats exposed to various liver cell-proliferative stimuli, such as two-thirds partial hepatectomy, lead nitrate, cyproterone acetate, ethylene dibromide, or a diet deficient in choline. Further, orotic acid inhibited DNA synthesis even when added 24 h after the hepatocytes were primed with transforming growth factor alpha. Taken together, these results suggested that the target site may not be at the level of the growth-factor receptor and receptor-mediated early events. In a preliminary experiment, orotic acid inhibited the expression of the ribonucleoside diphosphate reductase gene. Exposure to orotic acid results in an imbalance in nucleotide pools characterized by an increase in uridine nucleotides and a decrease in adenosine nucleotides. It is hypothesized that this imbalance in nucleotide pools inhibits the expression of the ribonucleoside diphosphate reductase gene and, therefore, is a likely target for the mitoinhibitory effect of orotic acid.

Altered gene expression of c-myc, epidermal growth factor receptor, transforming growth factor-alpha, and c-erb-B2 in an immortalized human breast epithelial cell line, HMT-3522, is associated with decreased growth factor requirements.

Activation of protooncogenes and constitutive secretion of autocrine growth factors are thought to be involved in the uncontrolled growth of cancer cells. We have attempted to elucidate the role of oncogenes and growth factors in the premalignant progression of human breast epithelial cells by using an immortalized, nontumorigenic, near-diploid human mammary epithelial cell line, HMT-3522, derived from a fibrocystic lesion and established in our laboratory. During propagation in tissue culture, the growth factor requirements of the HMT-3522 cells decreased simultaneously with an amplification and overexpression of the c-myc protooncogene. Other protooncogenes related to human breast cancer were unaltered with regard to gene copy number and expression. In passage 118, in which the most important growth factor still was epidermal growth factor (EGF), we were able to isolate an EGF-independent subline (S2). The EGF independence of S2 was accompanied by an overexpression of the mRNAs for epidermal growth factor receptor (EGF-R), transforming growth factor-alpha, and c-erb-B2 as compared to the EGF-dependent subline (S1). Moreover, by application of a blocking anti-EGF-R antibody, growth of S2 cells in EGF-free medium was inhibited significantly, indicating that EGF-R was involved in an autocrine loop probably with transforming growth factor-alpha as ligand. Neither the late passages of S1 cells nor S2 cells were tumorigenic after subcutaneous transplantation to athymic mice. Our results indicate that c-myc amplification and overexpression are correlated with a decreased requirement for growth factors. Even when these alterations are combined with immortalization and EGF independence, they are insufficient for malignant transformation of these human breast epithelial cells.

Alterations in transforming growth factor-alpha and -beta production and cell responsiveness during the progression of MCF-7 human breast cancer cells to estrogen-autonomous growth.

Hormonal management of breast cancer is confounded by an almost inevitable progression of cell growth from a steroid-regulated to a steroid-autonomous state. We have experimentally induced this progression in the estrogen growth-responsive MCF-7 human breast cancer cell line by long-term culture in the absence of steroids. After an initial period (10-12 weeks) of slowed growth in response to steroid deprivation, rapid, steroid-independent growth rates were consistently established. In these cells, which contained 3-fold elevated, functional estrogen receptor levels (as determined by induction of PgR and transactivation of a transiently transfected estrogen-responsive gene construct), antiestrogens still effectively suppressed cell proliferation, although estrogens only minimally increased the proliferation rate. Depletion of steroids from the growth media also resulted in a marked (70-80%) transient decrease in transforming growth factor (TGF) alpha mRNA and TGF-alpha protein production at 2 weeks that was followed by a progressive, partial return to the initial parental TGF-alpha mRNA and protein levels. In contrast, the mRNAs for TGF-beta 1, -beta 2, and -beta 3 and bioactive TGF-beta proteins transiently increased (3-10-fold) at 2 to 10 weeks of steroid deprivation and then returned by 24 weeks to the lower levels of the parental MCF-7 cells. These results suggest that the cells acquired steroid-independent means to regulate the production of these peptides. The long-term steroid-deprived sublines showed a loss of regulation of proliferation by TGF-alpha or anti-TGF-alpha antibodies and a 10-fold decrease in sensitivity to the growth-suppressive effects of TGF-beta 1, despite little change in receptor levels for these factors. The diminished contributions of TGF-alpha and TGF-beta s to the regulation of cell proliferation in long-term steroid-deprived MCF-7 breast cancer cells suggest that the TGFs do not act as major growth regulators in these estrogen-autonomous sublines. However, the marked, transient alterations in the levels of these growth factors indicate that they may play a role in the events which accompany the progression from estrogen-responsive to estrogen-autonomous growth. In addition, continued exposure to estrogen may be needed for the long-term maintenance of cell responsiveness to these TGFs.

Glucocorticoids coordinately disrupt a transforming growth factor alpha autocrine loop and suppress the growth of 13762NF-derived Con8 rat mammary adenocarcinoma cells.

We have demonstrated previously that the synthetic glucocorticoid dexamethasone suppresses the growth of Con8 rat mammary tumor cells, which are derived from the 13762NF transplantable, hormone-responsive rat mammary adenocarcinoma. Dexamethasone inhibited [3H]thymidine incorporation into Con8 cells at high cell density under both serum and serum-free conditions. Fractionation in nonreducing sodium dodecyl sulfate-polyacrylamide gels of proteins secreted from dexamethasone-treated and untreated Con8 mammary tumor cells revealed two size classes of glucocorticoid inhibited mitogenic activities; a larger M(r) 27,000-33,000 and a smaller M(r) 5,000-12,000 activity. Both size classes of mitogens restimulated the growth of glucocorticoid-suppressed Con8 cells suggesting that they can act in an autocrine fashion. The smaller mitogen was identified as transforming growth factor alpha (TGF-alpha) since this activity competed with 125I-epidermal growth factor (EGF) for EGF receptor binding and was selectively immunodepleted with monoclonal TGF-alpha antibodies but not with EGF antibodies. Western blots and radioreceptor assay of Con8-secreted proteins revealed that glucocorticoids inhibited the production of a M(r) 5500 immunoreactive TGF-alpha protein by 10-fold. Consistent with a steroid effect on the level of TGF-alpha production, rather than on its activity, the specific mitogenic activities of the TGF-alpha s secreted by dexamethasone-treated and untreated Con8 cells were identical to that of recombinant human TGF-alpha. Treatment of intact cells with suramin, which dissociates ligand-receptor complexes, revealed that the EGF receptor-mediated mitogenic response is functional in both glucocorticoid-treated and untreated cells. Taken together, our results demonstrate that glucocorticoids suppress Con8 mammary tumor cell growth and disrupt a potential TGF-alpha autocrine loop which results in a dramatic reduction in the level of extracellular TGF-alpha.

SRC family kinases mediate epidermal growth factor receptor ligand cleavage, proliferation, and invasion of head and neck cancer cells.

Head and neck squamous cell carcinomas (HNSCCs) are characterized by up-regulation of the epidermal growth factor receptor (EGFR). We previously reported that a gastrin-releasing peptide/gastrin-releasing peptide receptor (GRP/GRPR) autocrine growth pathway is activated early in HNSCC carcinogenesis. GRP can induce rapid phosphorylation of EGFR and p42/44 mitogen-activated protein kinase (MAPK) activation in part via extracellular release of transforming growth factor alpha (TGF-alpha) by matrix metalloproteinases (MMPs). It has been reported that Src family kinases are activated by G-protein-coupled receptors (GPCRs), followed by downstream EGFR and MAPK activation. To further elucidate the mechanism of activation of EGFR by GRP in HNSCC, we investigated the role of Src family kinases. Blockade of Src family kinases using an Src-specific tyrosine kinase inhibitor A-419259 decreased GRP-induced EGFR phosphorylation and MAPK activation. GRP also failed to induce MAPK activation in dominant-negative c-Src-transfected HNSCC cells. Invasion and growth assays showed that c-Src was required for GRP-induced proliferation or invasion of HNSCC cells. In addition to TGF-alpha release, GRP induced amphiregulin, but not EGF, secretion into HNSCC cell culture medium, an effect that was blocked by the MMP inhibitor marimastat. TGF-alpha and amphiregulin secretion by GRP stimulation also was inhibited by blockade of Src family kinases. These results suggest that Src family kinases contribute to GRP-mediated EGFR growth and invasion pathways by facilitating cleavage and release of TGF-alpha and amphiregulin in HNSCC.