Adriana Amaro

Direct inhibition of hexokinase activity by metformin at least partially impairs glucose metabolism and tumor growth in experimental breast cancer

Emerging evidence suggests that metformin, a widely used anti-diabetic drug, may be useful in the prevention and treatment of different cancers. In the present study, we demonstrate that metformin directly inhibits the enzymatic function of hexokinase (HK) I and II in a cell line of triple-negative breast cancer (MDA-MB-231). The inhibition is selective for these isoforms, as documented by experiments with purified HK I and II as well as with cell lysates. Measurements of 18F-fluoro-deoxyglycose uptake document that it is dose- and time-dependent and powerful enough to virtually abolish glucose consumption despite unchanged availability of membrane glucose transporters. The profound energetic imbalance activates phosphorylation and is subsequently followed by cell death. More importantly, the “in vivo” relevance of this effect is confirmed by studies of orthotopic xenografts of MDA-MB-231 cells in athymic (nu/nu) mice. Administration of high drug doses after tumor development caused an evident tumor necrosis in a time as short as 48 h. On the other hand, 1 mo metformin treatment markedly reduced cancer glucose consumption and growth. Taken together, our results strongly suggest that HK inhibition contributes to metformin therapeutic and preventive potential in breast cancer.

Metformin Impairs Glucose Consumption and Survival in Calu-1 Cells by Direct Inhibition of Hexokinase-II

The anti-hyperglycaemic drug metformin has important anticancer properties as shown by the direct inhibition of cancer cells proliferation. Tumor cells avidly use glucose as a source for energy production and cell building blocks. Critical to this phenotype is the production of glucose-6-phosphate (G6P), catalysed by hexokinases (HK) I and II, whose role in glucose retention and metabolism is highly advantageous for cell survival and proliferation. Here we show that metformin impairs the enzymatic function of HKI and II in Calu-1 cells. This inhibition virtually abolishes cell glucose uptake and phosphorylation as documented by the reduced entrapment of 18F-fluorodeoxyglucose. In-silico models indicate that this action is due to metformin capability to mimic G6P features by steadily binding its pocket in HKII. The impairment of this energy source results in mitochondrial depolarization and subsequent cell death. These results could represent a starting point to open effective strategies in cancer prevention and treatment.

Exogenous hormonal regulation in breast cancer cells by phytoestrogens and endocrine disruptors.

Malaria is one of the deadliest diseases on the planet affecting about 50% of the population worldwide. It is a leading cause of morbidity and mortality in the developing world. Plasmodium falciparum, a tiny parasite is the major cause of malaria and is possibly the most dangerous stow-away in history. Malaria has become a major economic concern to some of the tropical and sub-tropical countries. Though a number of antimalarials have been developed from plants as such or their semi-synthetic analogues, there is again an alarming situation of drug resistance against most of the antimalarial drugs. Plants have been an excellent source of antimalarial compounds. There are several plant leads exhibiting antimalarial activity better than the existing drugs. A systematic evaluation of these plant based leads is the need of the time to develop safe, effective and affordable new antimalarials. The present review is an update of plant based antimalarial agents.

Metformin Temporal and Localized Effects on Gut Glucose Metabolism Assessed Using 18F-FDG PET in Mice

In the course of metformin treatment, staging abdominal cancer lesions with 18F-FDG PET images is often hindered by the presence of a high bowel radioactivity. The present study aimed to verify the mechanism underlying this phenomenon. Methods: Fifty-three mice were submitted to dynamic acquisitions of 18F-FDG kinetics under fasting conditions. Three small-animal PET scans were obtained over a 4-mo study period. The animals were subdivided into 4 groups according to the following metformin administration protocol: group 1, untreated mice (n = 15); group 2, mice exposed to metformin treatment (750 mg/kg/d) for the 48 h before each PET study (pulsed, n = 10); group 3, mice treated for the whole study period (prolonged, n = 10); and group 4, mice in which prolonged treatment was interrupted 48 h before PET (interrupted, n = 8). The rate constant of 18F-FDG uptake was estimated by Patlak analysis. At the end of the study, the ileum and colon were harvested, washed, and counted ex vivo. Two further groups, of 5 animals each, were included to evaluate the effect of prolonged metformin treatment on phosphorylated adenosine monophosphate (AMP)–activated protein kinase (pAMPK) form and gene expression for thioredoxin-interacting protein (TXNIP). Results: Pulsed treatment did not modify gut tracer retention with respect to the untreated group. Conversely, prolonged treatment induced a progressive increase in 18F-FDG uptake that selectively involved the colonic wall, without any significant contamination of bowel content. This effect persisted after a complete drug washout in the interrupted group. These responses were paralleled by increased pAMPK availability and by reduced expression of TXNIP messenger RNA in colonic enterocytes exposed to prolonged metformin treatment. Conclusion: Metformin causes a selective increase in colonic 18F-FDG uptake. This effect appears after a relatively long period of treatment and persists soon after drug washout. Accordingly, the increased bowel glucose metabolism reflects a biologic response to chronic metformin treatment characterized by increased levels of pAMPK and reduced levels of TXNIP.

Endocrine disruptor agent nonyl phenol exerts an estrogen-like transcriptional activity on estrogen receptor positive breast cancer cells.

Several substances widely dispersed in the environment including hormones, industrial by-products and pollutants exert hormone like activity affecting steroid-responsive physiological systems. These compounds, named endocrine disruptors, are suspected to affect the mammalian reproductive system. However it is still unclear whether these substances are able to elicit estrogen like activity at the low concentrations encountered in the environment. Here we compare the effects of the endocrine disruptor nonylphenol with the effects elicited by 17-β-estradiol on gene transcription in the human breast cancer cell line MCF7. The correlation of the nonylphenol induced gene expression alterations with a reference profile of estradiol treated cells shows that nonylphenol at a concentration of 100 nM exerts a significant effect on estrogen responsive gene transcription in MCF7 cells. Most of the genes regulated by 17-β-estradiol respond to the nonylphenol in the same direction though to a much lesser extent. Molecular modeling of the potential interaction of nonylphenol with the estrogen receptor α shows that nonylphenol is likely to bind to the estrogen receptor α.

The biology of uveal melanoma

Uveal melanoma (UM), a rare cancer of the eye, is distinct from cutaneous melanoma by its etiology, the mutation frequency and profile, and its clinical behavior including resistance to targeted therapy and immune checkpoint blockers. Primary disease is efficiently controlled by surgery or radiation therapy, but about half of UMs develop distant metastasis mostly to the liver. Survival of patients with metastasis is below 1 year and has not improved in decades. Recent years have brought a deep understanding of UM biology characterized by initiating mutations in the G proteins GNAQ and GNA11. Cytogenetic alterations, in particular monosomy of chromosome 3 and amplification of the long arm of chromosome 8, and mutation of the BRCA1-associated protein 1, BAP1, a tumor suppressor gene, or the splicing factor SF3B1 determine UM metastasis. Cytogenetic and molecular profiling allow for a very precise prognostication that is still not matched by efficacious adjuvant therapies. G protein signaling has been shown to activate the YAP/TAZ pathway independent of HIPPO, and conventional signaling via the mitogen-activated kinase pathway probably also contributes to UM development and progression. Several lines of evidence indicate that inflammation and macrophages play a pro-tumor role in UM and in its hepatic metastases. UM cells benefit from the immune privilege in the eye and may adopt several mechanisms involved in this privilege for tumor escape that act even after leaving the niche. Here, we review the current knowledge of the biology of UM and discuss recent approaches to UM treatment.

IGF1 regulates PKM2 function through Akt phosphorylation

Pyruvate kinase M2 (PKM2) acts at the crossroad of growth and metabolism pathways in cells. PKM2 regulation by growth factors can redirect glycolytic intermediates into key biosynthetic pathway. Here we show that IGF1 can regulate glycolysis rate, stimulate PKM2 Ser/Thr phosphorylation and decrease cellular pyruvate kinase activity. Upon IGF1 treatment we found an increase of the dimeric form of PKM2 and the enrichment of PKM2 in the nucleus. This effect was associated to a reduction of pyruvate kinase enzymatic activity and was reversed using metformin, which decreases Akt phosphorylation. IGF1 induced an increased nuclear localization of PKM2 and STAT3, which correlated with an increased HIF1α, HK2, and GLUT1 expression and glucose entrapment. Metformin inhibited HK2, GLUT1, HIF-1α expression and glucose consumption. These findings suggest a role of IGFIR/Akt axis in regulating glycolysis by Ser/Thr PKM2 phosphorylation in cancer cells.

ADAM10 correlates with uveal melanoma metastasis and promotes in vitro invasion

Uveal melanoma (UM) is a rare ocular tumor that may lead to deadly metastases in 50% of patients. A disintegrin and metalloproteinase (ADAM)10, ADAM17, and the HGF‐receptor c‐Met support invasiveness in different tumors. Here, we report that high ADAM10, MET, and, to a lesser extent, ADAM17 gene expression correlates with poor progression‐free survival in UM patients (hazard ratio 2.7, 2.6, and 1.9, respectively). About 60% of primary UM expresses c‐Met and/or ADAM10 proteins. Four UM cell lines display high levels of ADAM10 and ADAM17, which constitutively cleave c‐Met, inducing the release of soluble c‐Met. ADAM10/17 pharmacological inhibition or gene silencing reduces c‐Met shedding, but has limited impact on surface c‐Met, which is overexpressed. Importantly, ADAM10 silencing inhibits UM cell invasion driven by FCS or HGF, while ADAM17 silencing has a limited effect. Altogether our data indicate that ADAM10 has a pro‐invasive role and may contribute to UM progression.

Molecular evolution of colorectal cancer: from multistep carcinogenesis to the big bang

Colorectal cancer is characterized by exquisite genomic instability either in the form of microsatellite instability or chromosomal instability. Microsatellite instability is the result of mutation of mismatch repair genes or their silencing through promoter methylation as a consequence of the CpG island methylator phenotype. The molecular causes of chromosomal instability are less well characterized. Genomic instability and field cancerization lead to a high degree of intratumoral heterogeneity and determine the formation of cancer stem cells and epithelial–mesenchymal transition mediated by the TGF-β and APC pathways. Recent analyses using integrated genomics reveal different phases of colorectal cancer evolution. An initial phase of genomic instability that yields many clones with different mutations (big bang) is followed by an important, previously not detected phase of cancer evolution that consists in the stabilization of several clones and a relatively flat outgrowth. The big bang model can best explain the coexistence of several stable clones and is compatible with the fact that the analysis of the bulk of the primary tumor yields prognostic information.

A highly invasive subpopulation of MDA-MB-231 breast cancer cells shows accelerated growth, differential chemoresistance, features of apocrine tumors and reduced tumorigenicity in vivo

The acquisition of an invasive phenotype is a prerequisite for metastasization, yet it is not clear whether or to which extent the invasive phenotype is linked to other features characteristic of metastatic cells. We selected an invasive subpopulation from the triple negative breast cancer cell line MDA-MB-231, performing repeated cycles of preparative assays of invasion through Matrigel covered membranes. The invasive sub-population of MDA-MB-231 cells exhibits stronger migratory capacity as compared to parental cells confirming the highly invasive potential of the selected cell line. Prolonged cultivation of these cells did not abolish the invasive phenotype. ArrayCGH, DNA index quantification and karyotype analyses confirmed a common genetic origin of the parental and invasive subpopulations and revealed discrete structural differences of the invasive subpopulation including increased ploidy and the absence of a characteristic amplification of chromosome 5p14.1-15.33. Gene expression analyses showed a drastically altered expression profile including features of apocrine breast cancers and of invasion related matrix-metalloproteases and cytokines. The invasive cells showed accelerated proliferation, increased apoptosis, and an altered pattern of chemo-sensitivity with lower IC50 values for drugs affecting the mitotic apparatus. However, the invasive cell population is significantly less tumorigenic in orthotopic mouse xenografts suggesting that the acquisition of the invasive capacity and the achievement of metastatic growth potential are distinct events.