Chlorogenic Acid Improves the Regorafenib Effects in Human Hepatocellular Carcinoma Cells.

Chlorogenic acid (CGA) is a polyphenol present in many human dietary foods. Several studies indicated a beneficial role of CGA in the prevention of cancer and an enhancement of chemotherapy when combined with CGA in the treatment of human hepatocarcinoma (HCC). Drug toxicity, resistance and subsequent disease progression represent a problem in HCC management, although treatment with the multikinase inhibitor Regorafenib improved overall survival. This study focused on the evaluation of the effects of combined treatment using both low Regorafenib concentrations and CGA as natural compound in HCC cells. The analysis of cell proliferation by Ki67 staining and cell cycle progression showed that CGA enhanced Regorafenib-mediated cell growth inhibition. Moreover, CGA potentiated the apoptotic effect of Regorafenib by the activation of the pro-apoptotic Annexin V, Bax and Caspase 3/7 and the inhibition of anti-apoptotic Bcl2 and Bcl-xL. Combined treatments were also effective in inhibiting cell motility. The mechanisms underlying the positive effects of combining CGA and Regorafenib were also addressed and an increased inhibition of MAPK (mitogen-activated protein kinase)and PI3K/Akt/mTORC (phosphatidylinositol-3-kinase (PI3K)/Akt and the mammalian target of rapamycin (mTOR) signaling was observed. Overall, these data demonstrated that co-treatment with Regorafenib and CGA enhanced Regorafenib action, reducing its cytotoxicity in HCC cells. In conclusion, this drug combination could be considered as a safe and more effective approach in HCC therapy.

Platelet extracts induce growth, migration and invasion in human hepatocellular carcinoma in vitro.

BACKGROUND: Thrombocytopenia has been reported to be associated with small size HCCs, and thrombocytosis to be associated with large size HCCs. The aim was to examine the effects of platelets in relation to HCC cell growth. METHODS: The effects of time-expired pooled normal human platelets were examined on human HCC cell line growth and invasion. RESULTS: Blood platelet numbers increased with increasing HCC tumor size and portal vein invasion. Platelet extracts enhanced cell growth in 4 human HCC cell lines, as well as cell migration, medium AFP levels and decreased apoptosis. Cell invasion was significantly enhanced, using a Matrigel-coated trans-well membrane and 3D (Real-Time Imaging) invasion assay. Western blots showed that platelets caused enhanced phospho-ERK and phospho-JNK signaling and anti-apoptotic effect with increase of Bcl-xL (anti-apoptotic marker) and decrease of Bid (pro-apoptotic marker) levels. Their growth effects were blocked by a JNK inhibitor. CONCLUSIONS: Platelets stimulated growth and invasion of several HCC cell lines in vitro, suggesting that platelets or platelet growth factors could be a potential pharmacological target.

Antagonism of sorafenib and regorafenib actions by platelet factors in hepatocellular carcinoma cell lines.

BACKGROUND: Platelets are frequently altered in hepatocellular carcinoma (HCC) patients. Platelet lysates (hPL) can enhance HCC cell growth and decrease apoptosis. The aims were to evaluate whether hPL can modulate the actions of sorafenib or regorafenib, two clinical HCC multikinase antagonists. METHODS: Several human HCC cell lines were grown in the presence and absence of sorafenib or regorafenib, with or without hPL. Growth was measured by MTT assay, apoptosis was assessed by Annexin V and by western blot, and autophagy and MAPK growth signaling were also measured by western blot, and migration and invasion were measured by standard in vitro assays. RESULTS: Both sorafenib and regorafenib-mediated inhibition of cell growth, migration and invasion were all antagonized by hPL. Drug-mediated apoptosis and decrease in phospho-ERK levels were both blocked by hPL, which also increased anti-apoptotic phospho-STAT, Bax and Bcl-xL levels. Preliminary data, obtained with epidermal growth factor (EGF) and insulin-like growth factor-I (IGF-I), included in hPL, revealed that these factors were able to antagonized sorafenib in a proliferation assay, in particular when used in combination. CONCLUSIONS: Platelet factors can antagonize sorafenib or regorafenib-mediated growth inhibition and apoptosis in HCC cells. The modulation of platelet activity or numbers has the potential to enhance multikinase drug actions.

Fluoro-Sorafenib (Regorafenib) effects on hepatoma cells: growth inhibition, quiescence, and recovery.

To evaluate the growth-inhibitory properties of the potent multi-kinase antagonist Regorafenib (Fluoro-Sorafenib), which was synthesized as a more potent Sorafenib, a Raf inhibitor and to determine whether similar mechanisms were involved, human hepatoma cell lines were grown in the presence or absence of Regorafanib and examined for growth inhibition. Western blots were performed for Raf targets, apoptosis, and autophagy. Regorafenib inhibited growth of human Hep3B, PLC/PRF/5, and HepG2 cells in a concentration- and time-dependent manner. Multiple signaling pathways were altered, including MAP kinases phospho-ERK and phospho-JNK and its target phospho-c-Jun. There was evidence for apoptosis by FACS, cleavage of caspases and increased Bax levels; as well as induction of autophagy, as judged by increased Beclin-1 and LC3 (II) levels. Prolonged drug exposure resulted in cell quiescence. Full growth recovery occurred after drug removal, unlike with doxorubicin chemotherapy. Regorafenib is a potent inhibitor of cell growth. Cells surviving Regorafenib treatment remain viable, but quiescent and capable of regrowth following drug removal. The reversibility of tumor cell growth suppression after drug removal may have clinical implications.

Effects of low concentrations of regorafenib and sorafenib on human HCC cell AFP, migration, invasion, and growth in vitro.

Sorafenib was shown in clinical trial to enhance survival in hepatocellular carcinoma (HCC) patients, but with minimal tumor shrinkage. To correlate several indices of HCC growth at various drug concentrations, HCC cells were grown in various low concentrations of two multikinase inhibitors, regorafenib (Stivarga) and sorafenib (Nexavar) and their effects were examined on alpha-fetoprotein (AFP), cell growth, migration, and invasion. In two AFP positive human HCC cell lines, AFP was inhibited at 0.1-1 µM drug concentrations. Cell migration and invasion were also inhibited at similar low drug concentrations. However, 10-fold higher drug concentrations were required to inhibit cell growth in both AFP positive and negative cells. To investigate this concentration discrepancy of effects, cells were then grown for prolonged times and sub-cultured in low drug concentrations and then their growth was re-tested. The growth in these drug-exposed cells was found to be slower than cells without prior drug exposure and they were also more sensitive to subsequent drug challenge. Evidence was also found for changes in cell signaling pathways in these slow-growth cells. Low multikinase inhibitor concentrations thus modulate several aspects of HCC cell biology.

The role of miRNA-133b and its target gene SIRT1 in FAP-derived desmoid tumor.

Signaling pathways have a key role in driving the uncontrolled development of familial adenomatous polyposis (FAP)- associated and sporadic desmoid tumors (DTs). The relationship between the Wnt/b-catenin signaling pathway and DTs has been extensively studied, but no reliable biomarkers able to detect their histological subtype have been identified for the accurate diagnosis. In this study we studied the differences in miRNA expression between sporadic (20 patients) and FAP-associated DTs (7 patients) using microarray confirmed by quantitative PCR (qPCR). The analysis showed 19 dysregulated miRNAs. Among them miR-133b levels were significantly lower in FAP-associated DT than in sporadic DT. Therefore, two mRNAs, associated to miR-133b and ß-catenin expression, the SIRT1 and ELAVL1were analyzed. The qPCR analysis showed that SIRT1 mRNA levels were significantly up-regulated in FAP-associated DT than in sporadic DT, whereas no differences in ELAVL1 expression was observed between these two DT types. In addition, a negative correlation was observed between miR-133b and SIRT1 in FAP-associated DTs, but not in sporadic DTs. The miR-133b-SIRT1-ß-catenin axis may represent a novel mechanism underlying progression of FAP-associated DT.

Apoptosis in normal ovaries of women with and without endometriosis.

The aim of this study was to evaluate the factors predisposing to implants of endometriotic lesions in normal ovarian cortexes of women with and without endometriosis by assessing the expression of pro-apoptotic and anti-apoptotic factors and follicular density. Ovarian biopsies were performed during laparoscopy in 18 patients with endometrioma and in 10 healthy women. Detection of apoptosis was performed with terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling assay. p53 and BCL2 proteins were assessed by immunohistochemistry. Quantitative real-time polymerase chain reaction was performed to evaluate BAX , BAK , BCL2 , BCL-XL , survivin and beta-actin ( ACTB ) expression. The p53 protein was positive in a significantly higher number of secondary follicles, whereas the B-cell chronic lymphocytic leukaemia/lymphoma 2 (BCL2) protein was positive in all follicles in unaffected tissue of endometriotic women, compared with the controls. Overexpression of the BCL2 and survivin genes and a decreased BAX and BAK gene expression were observed in the endometriotic group although only the difference in survivin expression was significant (P = 0.016). The BCL2 / BAX ratio showed an increased value in the ovarian cortex in controls compared with endometriosis patients. In conclusion, the reduction of apoptosis in unaffected tissue in women with endometriosis suggests that they may be predisposed to develop endometriosis.

Effects of Lactobacillus rhamnosus GG on proliferation and polyamine metabolism in HGC-27 human gastric and DLD-1 colonic cancer cell lines.

Previous in vitro and in vivo studies have suggested that lactobacilli can exert antiproliferative effects on the gastrointestinal epithelium. However, their role in affecting the cellular proliferative mechanisms is not completely clear. The aim of this study was to investigate the effects of increasing concentrations of Lactobacillus rhamnosus strain GG (L. GG) homogenate on cell growth and proliferation (by MTT, [3H]-thymidine incorporation and polyamine biosynthesis) in neoplasms originating from different gastrointestinal tracts. Thus, HGC-27 human gastric cancer cells and DLD-1 human colonic adenocarcinoma cells were evaluated. Besides, in order to verify which bacterial fraction was involved in the antiproliferative effects, the cytoplasm and cell wall extracts were tested separately. Gastric HGC-27 and colonic DLD-1 cells showed significant differences in their proliferative behavior, in particular in their polyamine profile and biosynthesis. Notwithstanding, one and the other proved to be sensitive to the growth inhibition by the highest concentrations of bacterial homogenate. Both HGC-27 and DLD-1 cells were resistant to the bacterial cell wall fractions, whereas increasing cytoplasm fraction concentrations induced an evident antiproliferative effect. These data suggest that cytoplasm extracts could be the responsible for L. GG action on proliferation in these two cell lines from gastric and colonic neoplasms.

Estrogen receptor (ER) and ER-related receptor expression in normal and atrophic human vagina.

OBJECTIVES: The estrogen level decline in menopausal status is involved in physiological alterations of different human tissues including vaginal mucosa. In this study, we have evaluated the estrogen receptor (ER) and estrogen receptor-related receptor (ERR) expression in tissue samples of posterior vaginal wall obtained from pre- and post-menopausal women. METHODS: The nuclear receptor expression was determined by quantitative real-time PCR (qPCR). RESULTS: The qPCR results showed the presence of the three isoforms of the ERR family (ERRalpha, ERRbeta and ERRgamma) that were coexpressed with ERs in all vaginal tissue samples examined. The ERRalpha and ERRgamma mRNA levels decreased from normal vagina of the pre-menopausal women to atrophic vaginal tissue in post-menopausal women. This trend was also observed for the ERbeta subtype. CONCLUSIONS: The ERRs, such as ERs, are present in human vagina at the mRNA level and the cessation of ovarian estrogen secretion, that is the key event during the post-menopause, may be linked to ERbeta, ERRalpha and ERRgamma mRNA decline in human vaginal mucosa. These findings may provide a biological rationale for the clinical susceptibility of the post-menopausal vagina to local estrogen treatment.

Strong enhancement by IGF1-R antagonists of hepatocellular carcinoma cell migration inhibition by Sorafenib and/or vitamin K1.

PURPOSE: Emerging evidence indicates that combining Sorafenib with vitamin K1 (VK1) may result in a synergistic inhibition of hepatocellular carcinoma (HCC) cell migration and proliferation. Despite this synergy, its benefits may be limited due to drug resistance resulting from cross-talk with the tumor microenvironment. Insulin-like growth factor-1 (IGF1) signaling acts as an important modulator of HCC cell growth, motility and drug resistance. Therefore, we aimed to explore the effects of Sorafenib in combination with VK1 and/or IGF1-R antagonists on HCC cells. METHODS: Scratch wound migration assays were performed to assess the motility of HCC-derived PLC/PRF/5, HLF and Hep3B cells. The synergistic, additive or antagonistic effects of Sorafenib, VK1 and IGF1-R antagonists on HCC cell motility were assessed using CompuSyn software. The effects mediated by these various compounds on HCC cytoskeleton organization were evaluated using DyLight 554 Phalloidin staining. Proliferation and migration-associated signaling pathways were analyzed in PLC/PRF/5 cells using Erk1/2 and Akt activation kits and Western blotting (Mek, JNK, Akt, Paxillin and p38), respectively. RESULTS: The effects of the IGF1-R antagonists GSK1838705A and OSI-906 on HCC cell migration inhibition after Sorafenib and/or VK1 administration, individually or in combination, were evaluated. We found a synergistic effect in PLC/PRF/5, HLF and Hep3B cells for combinations of fixed doses of GSK1838705A or OSI-906 together with different doses of Sorafenib and/or VK1. The levels of synergy were found to be stronger at higher Sorafenib and/or VK1 concentrations and lower or absent at lower concentrations, with some variation among the different cell lines tested. In addition, we found that in PLC/PRF/5 and HLF cells IGF1-R blockage strongly enhanced the reduction and redistribution of F-actin induced by Sorafenib and/or VK1 through alterations in the phosphorylation levels of some of the principal proteins involved in the MAPK signaling cascade, which is essential for cell migration. CONCLUSIONS: Our results indicate that modulation of the efficacy of Sorafenib through combinations with VK1 and/or IGF1-R antagonists results in synergistic inhibition of HCC cell migration.

High degradation and no bioavailability of artichoke miRNAs assessed using an in vitro digestion/Caco-2 cell model.

Although the cross-kingdom transfer of vegetable miRNAs (miRNAs) in mammalian species, including humans, is still controversial, recent studies have rejected this theory. Based on these recent studies, we hypothesized that artichoke-derived miRNAs (cca-miRNAs) are not adsorbed into human intestinal cells after cooking and in vitro digestion. In order to test this hypothesis, we evaluated miRNA (cca-miRNAs) in the edible part of globe artichokes (head portion), after cooking and digestion by an in vitro digestion system. The cca-miRNA levels were analyzed by real-time PCR (RT-qPCR), and those that withstood cooking and digestion conditions were further analyzed for their bioavailability using an in vitro system (Caco-2/TC7 cell clone). We detected 20 cca-miRNAs after cooking, 5 of which were statistically down-regulated in comparison with uncooked samples. Only 4 cca-miRNAs were found after in vitro digestion. By using scanning electron microscopy (SEM), we also evaluated the extracellular vesicles (EVs) in homogenized artichoke as possible miRNA transporters. However, approximately 81% were degraded after cooking, while the remaining EVs had changed shape from round to elliptical. Finally, we detected no cell-free cca-miRNAs, miRNAs bound to protein complex, and no cca-miRNAs encapsulated in EVs inside Caco-2 cells or in basolateral medium after bioavailability experiments. In conclusion, the data from the present study agrees with recent findings that the human small intestine does not uptake dietary miRNAs from raw or cooked artichoke heads.

IGF-1R tyrosine kinase inhibitors and Vitamin K1 enhance the antitumor effects of Regorafenib in HCC cell lines.

The recent RESORCE trial showed that treatment with Regorafenib after Sorafenib failure provided a significant improvement in overall survival in HCC patients. Preclinical and clinical trial data showed that Regorafenib is a more potent drug than Sorafenib. In this study we aimed at improving Regorafenib actions and at reducing its toxicity, by targeting parallel pathways or by combination with Vitamins K (VKs). We investigated the effects of Regorafenib administrated at low concentrations and in combination with either VK1 and/or with GSK1838705A or OSI-906, two IGF1-R inhibitors, on HCC cell growth and motility. Our results showed that both IGF1-R inhibitors potentiated the antiproliferative and pro-apoptotic effects of Regorafenib and/or VK1 in HCC cell lines. Moreover we provide evidence that the combined treatment with IG1-R antagonists and Regorafenib (and/or VK1) also caused a significant reduction and depolymerization of actin resulting in synergistic inhibition exerted on cell migration. Thus, simultaneous blocking of MAPK and PI3K/Akt cascades with IGF1-R inhibitors plus Regorafenib could represent a more potent approach for HCC treatment.

Human microRNA expression in sporadic and FAP-associated desmoid tumors and correlation with beta-catenin mutations.

Desmoid tumors (DT) are rare, benign, fibroblastic neoplasm with challenging histological diagnosis. DTs can occur sporadically or associated with the familial adenomatous polyposis coli (FAP). Most sporadic DTs are associated with ß-catenin gene (CTNNB1) mutations, while mutated APC gene causes FAP disease. microRNAs (miRNAs) are involved in many human carcinogenesis.The miRNA profile was analyzed by microarray in formalin-fixed, paraffin-embedded (FFPE) specimens of 12 patients (8 sporadic, 4 FAP-associated) and 4 healthy controls. One hundred and one mRNAs resulted dysregulated, of which 98 in sporadic DTs and 8 in FAP-associated DTs, 5 were shared by both tumors. Twenty-six miRNAs were then validated by RT-qPCR in 23 sporadic and 7 FAP-associated DT samples matched with healthy controls. The qPCR method was also used to evaluate the CTNNB1 mutational status in sporadic DTs. The correlation between sporadic DTs and miRNA expression showed that miR-21-3p increased in mutated versus wild-type DTs, while miR-197-3p was decreased. The mRNA expression of Tetraspanin3 and Serpin family A member 3, as miR-21-3p targets, and L1 Cell Adhesion Molecule, as miR-197-3p target, was also evaluate. CTNNB1 mutations associated to miRNA dysregulation could affect the genesis and the progression of this disease and help histological diagnosis of sporadic DTs.

Peroxisome proliferator-activated receptor gamma and spermidine/spermine N1-acetyltransferase gene expressions are significantly correlated in human colorectal cancer.

BACKGROUND: The peroxisome proliferator-activated receptor gamma (PPARgamma) is a transcription factor that regulates adipogenic differentiation and glucose homeostasis. Spermidine/spermine N1-acetyltransferase (SSAT) and ornithine decarboxylase (ODC) are key enzymes involved in the metabolism of polyamines, compounds that play an important role in cell proliferation. While the PPARgamma role in tumour growth has not been clearly defined, the involvement of the altered polyamine metabolism in colorectal carcinogenesis has been established. In this direction, we have evaluated the PPARgamma expression and its relationship with polyamine metabolism in tissue samples from 40 patients operated because of colorectal carcinoma. Since it is known that the functional role of K-ras mutation in colorectal tumorigenesis is associated with cell growth and differentiation, polyamine metabolism and the PPARgamma expression were also investigated in terms of K-ras mutation. METHODS: PPARgamma, ODC and SSAT mRNA levels were evaluated by reverse transcriptase and real-time PCR. Polyamines were quantified by high performance liquid chromatography (HPLC). ODC and SSAT activity were measured by a radiometric technique. RESULTS: PPARgamma expression, as well as SSAT and ODC mRNA levels were significantly higher in cancer as compared to normal mucosa. Tumour samples also showed significantly higher polyamine levels and ODC and SSAT activities in comparison to normal samples. A significant and positive correlation between PPARgamma and the SSAT gene expression was observed in both normal and neoplastic tissue (r = 0.73, p < 0.0001; r = 0.65, p < 0.0001, respectively). Moreover, gene expression, polyamine levels and enzymatic activities were increased in colorectal carcinoma samples expressing K-ras mutation as compared to non mutated K-ras samples. CONCLUSION: In conclusion, our data demonstrated a close relationship between PPARgamma and SSAT in human colorectal cancer and this could represent an attempt to decrease polyamine levels and to reduce cell growth and tumour development. Therefore, pharmacological activation of PPARgamma and/or induction of SSAT may represent a therapeutic or preventive strategy for treating colorectal cancer.

The Effects of Chronic Lifelong Activation of the AHR Pathway by Industrial Chemical Pollutants on Female Human Reproduction.

Environmental chemicals, such as heavy metals, affect female reproductive function. A biological sensor of the signals of many toxic chemical compounds seems to be the aryl hydrocarbon receptor (AHR). Previous studies demonstrated the environmental of heavy metals in Taranto city (Italy), an area that has been influenced by anthropogenic factors such as industrial activities and waste treatments since 1986. However, the impact of these elements on female fertility in this geographic area has never been analyzed. Thus, in the present study, we evaluated the AHR pathway, sex steroid receptor pattern and apoptotic process in granulosa cells (GCs) retrieved from 30 women, born and living in Taranto, and 30 women who are living in non-contaminated areas (control group), who were undergoing in vitro fertilization (IVF) protocol. In follicular fluids (FFs) of both groups the toxic and essential heavy metals, such as chromiun (Cr), Manganese (Mn), iron (Fe), cobalt (Co), nickel (Ni), copper (Cu), zinc (Zn), cadmium (Cd) and lead (Pb), were also analyzed. Higher levels of Cr, Fe, Zn and Pb were found in the FFs of the women from Taranto as compared to the control group, as were the levels of AHR and AHR-dependent cytochrome P450 1A1 and 1B1; while CYP19A1 expression was decreased. The anti-apoptotic process found in the GCs of women fromTaranto was associated with the highest levels of progesterone receptor membrane component 1 (PGRMC1), a novel progesterone receptor, the expression of which is subjected to AHR activated by its highest affinity ligands (e.g., dioxins) or indirectly by other environmental pollutants, such as heavy metals. In conclusion, decreased production of estradiol and decreased number of retrieved mature oocytes found in women from Taranto could be due to chronic exposure to heavy metals, in particular to Cr and Pb.

Oestrogen receptor-related receptor alpha (ERRalpha) and oestrogen receptors (ERalpha and ERbeta) exhibit different gene expression in human colorectal tumour progression.

We have investigated the presence of oestrogen receptor-related (ERR) mRNA in human colorectal tumour tissues and adjacent normal mucosa by reverse transcriptase and nested-polymerase chain reaction. ERRalpha was found in 100% of the patients and ERRgamma in approximately 30% while ERRbeta was not detected at all. The multiplex PCR analysis showed elevated levels of ERRalpha mRNA in tumour tissue compartment as compared to normal mucosa, whereas ERRgamma mRNA was found in lower levels but in both tissue compartments. In contrast, oestrogen receptor (ERalpha and ERbeta) mRNA levels were shown to be decreased in tumour tissues. A positive correlation was observed between ERalpha and ERbeta and between ERalpha and ERRalpha, respectively, in normal mucosa but not in tumour tissue. ERRalpha expression in tumour tissues significantly increased from TNM stages II to IV, whereas both ERs progressively declined. These findings suggest that ERRalpha, as well as the two ERs, might play a critical role in the progression of the colorectal cancer.

Expression analysis of a subset of coregulators and three nuclear receptors in human colorectal carcinoma.

In human colorectal tissue samples, the gene expressions of 4 coactivators, p300, pCAF, TIF-2 and TRAP 220, and 7 corepressors, N-CoR, REA, MTA1, MTA1L1, HDAC1, HDAC2 and HDAC3, linked to estrogen receptors (ER), were revealed by traditional RT-PCR. Cofactors ERalpha, ERbeta and ERRalpha mRNA levels were then measured in 40 tumor tissue samples matched with respective normal mucosa by real-time PCR. The decline of mRNA levels of all coactivators and the increase of NCoR, HDAC1, HDAC2 and MTA1 were observed from normal to tumor tissue, whereas REA, HDAC3 and MTA1L1 expressions were similar in both tissue compartments. The gene expression of ERbeta correlated with those of p300, TIF-2 and REA in normal mucosa, and with that of REA in tumor tissue only. No association was found between ERalpha and coregulators and between each coregulator and different clinical parameters. Our findings suggest that the co-induction of ERbeta and some cofactors may play an important role during the development of human colorectal carcinoma.

Investigations on cellular proliferation induced by zearalenone and its derivatives in relation to the estrogenic parameters.

Many man-made chemicals (pesticides) and naturally occurring compounds (mycotoxins and phytoestrogens) can enter the food chain and bind to estrogen receptors (ERs). Mycotoxins, including zearalenone (ZEA) and its derivatives, can occur worldwide in cereals and cause several health disorders. In order to characterize the estrogenic activity of zearalenone and its derivatives (alpha-zearalenol (alpha-ZEA), beta-zearalenol (beta-ZEA), alpha-zearalanol (alpha-ZAL) and beta-zearalanol (beta-ZAL)), the proliferation of ER-positive (MCF-7) and ER-negative (MDA-MB-231) human breast cancer cell lines was measured. After exposure at levels ranging from 0.1 pM to 0.1microM, cell proliferation (E-screen assay) was evaluated by MTT test through estrogenic parameters. On the MCF-7 cell line, estrogenic concentration that induced 50% cellular proliferation (EC(50)) of beta-zearalenol was statistically higher (5.2 x 10(-3)microM) than those of other zearalenone-related compounds, in agreement with other authors. All mycotoxins showed similar estrogenic parameters, with the exception of alpha-zearalenol that induced a higher proliferative effect (PE=2.6) and relative proliferative potency (RPP=7). Since MCF-7 contains both ERalpha and ERbeta-positive cells, at the mRNA and protein level, the estrogenic activity induced by mycotoxins may be ER-mediated, particularly through ERalpha that was the predominant ER subtype in these cells. A partial antagonism of mycotoxin-related estrogenic proliferation was seen when tamoxifen was used, confirming a receptor-dependent estrogenic response. MDA-MB-231 cells did not show ERs and after exposure to mycotoxins or 17beta-estradiol marginal PE values related to growth variability of MDA-MB-231 were found. Further studies are needed to understand in human tissues the mechanisms of action of ZEA and its derivatives that may be found as contaminants in the human diet.

Resistance to multikinase inhibitor actions mediated by insulin like growth factor-1.

BACKGROUND: Blood platelet numbers are correlated with growth and aggressiveness of several tumor types, including hepatocellular carcinoma (HCC). We previously found that platelet lysates (hPLs) both stimulated HCC cell growth and migration, and antagonized the growth-inhibitory and apoptotic effects of Regorafenib, multikinase growth inhibitor, on HCC cell lines. We evaluated the effects of human insulin-like growth factor-1 (IGF1), a mitogen contained in platelets, on the Regorafenib-mediated growth inhibition. METHODS: An Elisa kit was used to evaluate hPL IGF1 concentrations. The effects of IGF1 on cell proliferation were assessed with MTT assay and analysis of cell cycle progression. Apoptosis assays, scratch assay and Transwell assay were performed to measure apoptosis, cell migration and invasion respectively. Western blots were performed by standard protocols. RESULTS: IGF1 antagonized growth inhibition exerted by Regorafenib on HCC cell lines. Moreover the mitogen blocked Regorafenib-induced apoptosis and decreased the rate of cell migration and invasion. The IGF1 effects were in turn antagonized by actions of a potent IGF1 receptor inhibitor, GSK1838705A, showing that the IGF1 receptor was involved in the mechanisms of IGF1-mediated blocking of Regorafenib action. GSK1838705A also partially blocked the effects of hPLs in antagonizing Regorafenib-mediated growth inhibition, showing that IGF1 was an important component of hPL actions. CONCLUSIONS: These results show that IGF1 antagonized Regorafenib-mediated growth, migration and invasion inhibition, as well as the drug-mediated induction of apoptosis in HCC cells and reinforce the idea that microenvironmental factors can influence cancer drug actions.