Stefano Iotti

Coumarin derivatives as potential antitumor agents: Growth inhibition, apoptosis induction and multidrug resistance reverting activity

A small library of coumarins, carrying butynyl-amino chains, was synthesized continuing our studies in the field of MDR reverting ageEnts and in order to obtain multipotent agents to combat malignancies. In particular, the reported anticancer and chemopreventive natural product 7-isopentenyloxycoumarin was linked to different terminal amines, selected on the basis of our previously reported results. The anticancer behaviour and the MDR reverting ability of the new compounds were evaluated on human colon cancer cells, particularly prone to develop the MDR phenotype. Some of the new derivatives showed promising effects, directly acting as cytotoxic compounds and/or counteracting MDR phenomenon. Compound 1e emerged as the most interesting of this series, showing a multipotent biological profile and suggesting that conjugation of an appropriate coumarin core with a properly selected butynyl-amino chain allows to obtain novel hybrid molecules endowed with improved inxa0vitro antitumor activity.

Magnesium homeostasis in colon carcinoma LoVo cells sensitive or resistant to doxorubicin

Neoplastic cells accumulate magnesium, an event which provides selective advantages and is frequently associated with TRPM7overexpression. Little is known about magnesium homeostasis in drug-resistant cancer cells. Therefore, we used the colon cancer LoVo cell model and compared doxorubicin-resistant to sensitive cells. In resistant cells the concentration of total magnesium is higher while its influx capacity is lower than in sensitive cells. Accordingly, resistant cells express lower amounts of the TRPM6 and 7, both involved in magnesium transport. While decreased TRPM6 levels are due to transcriptional regulation, post-transcriptional events are involved in reducing the amounts of TRPM7. Indeed, the calpain inhibitor calpeptin markedly increases the levels of TRPM7 in resistant cells. In doxorubicin-sensitive cells, silencing TRPM7 shifts the phenotype to one more similar to resistant cells, since in these cells silencing TRPM7 significantly decreases the influx of magnesium, increases its intracellular concentration and increases resistance to doxorubicin. On the other hand, calpain inhibition upregulates TRPM7, decreases intracellular magnesium and enhances the sensitivity to doxorubicin of resistant LoVo cells. We conclude that in LoVo cells drug resistance is associated with alteration of magnesium homeostasis through modulation of TRPM7. Our data suggest that TRPM7 expression may be an additional undisclosed player in chemoresistance.

Nanoscale quantification of intracellular element concentration by X-ray fluorescence microscopy combined with X-ray phase contrast nanotomography

We present here a correlative X-ray microscopy approach for quantitative single cell imaging of molar concentrations. By combining the elemental content provided by X-ray fluorescence microscopy and the morphology information extracted from X-ray phase nanotomography, we determine the intracellular molarity distributions. This correlative method was demonstrated on a freeze-dried human phagocytic cell to obtain the absolute elemental concentration maps of K, P, and Fe. The cell morphology results showed a very good agreement with atomic-force microscopy measurements. This work opens the way for non-destructive single cell chemical analysis down to the sub-cellular level using exclusively synchrotron radiation techniques. It will be of high interest in the case where it is difficult to access the morphology using atomic-force microscopy, for example, on frozen-hydrated cells or tissues.

Single cell versus large population analysis: cell variability in elemental intracellular concentration and distribution

AbstractThe quantification of elemental concentration in cells is usually performed by analytical assays on large populations missing peculiar but important rare cells. The present article aims at comparing the elemental quantification in single cells and cell population in three different cell types using a new approach for single cells elemental analysis performed at sub-micrometer scale combining X-ray fluorescence microscopy and atomic force microscopy. The attention is focused on the light element Mg, exploiting the opportunity to compare the single cell quantification to the cell population analysis carried out by a highly Mg-selective fluorescent chemosensor. The results show that the single cell analysis reveals the same Mg differences found in large population of the different cell strains studied. However, in one of the cell strains, single cell analysis reveals two cells with an exceptionally high intracellular Mg content compared with the other cells of the same strain. The single cell analysis allows mapping Mg and other light elements in whole cells at sub-micrometer scale. A detailed intensity correlation analysis on the two cells with the highest Mg content reveals that Mg subcellular localization correlates with oxygen in a different fashion with respect the other sister cells of the same strain.n Graphical abstractSingle cells or large population analysis this is the question!

The different expression of TRPM7 and MagT1 impacts on the proliferation of colon carcinoma cells sensitive or resistant to doxorubicin

The processes leading to anticancer drug resistance are not completely unraveled. To get insights into the underlying mechanisms, we compared colon carcinoma cells sensitive to doxorubicin with their resistant counterpart. We found that resistant cells are growth retarded, and show staminal and ultrastructural features profoundly different from sensitive cells. The resistant phenotype is accompanied by the upregulation of the magnesium transporter MagT1 and the downregulation of the ion channel kinase TRPM7. We demonstrate that the different amounts of TRPM7 and MagT1 account for the different proliferation rate of sensitive and resistant colon carcinoma cells. It remains to be verified whether they are also involved in the control of other “staminal” traits.

p53-dependent and p53-independent anticancer activity of a new indole derivative in human osteosarcoma cells

Osteosarcoma (OS) is the most common primary malignant tumor of bone, occurring most frequently in children and adolescents. The mechanism of formation and development of OS have been studied for a long time. Tumor suppressor pathway governed by p53 gene are known to be involved in the pathogenesis of osteosarcoma. Moreover, loss of wild-type p53 activity is thought to be a major predictor of failure to respond to chemotherapy in various human cancers. In previous studies, we described the activity of a new indole derivative, NSC743420, belonging to the tubulin inhibitors family, capable to induce apoptosis and arrest of the cell cycle in the G2/M phase of various cancer cell lines. However, this molecule has never been tested on OS cell line. Here we address the activity of NSC743420 by examine whether differences in the p53 status could influence its effects on cell proliferation and death of OS cells. In particular, we compared the effect of the tested molecule on p53-wild type and p53-silenced U2OS cells, and on SaOS2 cell line, which is null for p53. Our results demonstrated that NSC743420 reduces OS cell proliferation by p53-dependent and p53-independent mechanisms. In particular, the molecule induces proliferative arrest that culminate to apoptosis in SaOS2 p53-null cells, while it brings a cytostatic and differentiating effect in U2OS cells, characterized by the cell cycle arrest in G0/G1 phase and increased alkaline phosphatase activity.

Effects of supplementation with different Mg salts in cells: is there a clue?

The differing bioavailability of magnesium salts remains an open question, both at the cellular and systemic level. However, this issue is relevant for identifying the most effective magnesium supplement. We compared the effects of three widely used magnesium salts: MgSO4, MgCl2 and Mg pidolate, on the proliferation of four human cell types: promyelocytic leukaemia HL60, osteoblast-like Saos-2 and U-2 OS, and endothelial cells from the umbilical vein. The three magnesium salts had no effect on endothelial and leukemic cell growth, but magnesium pidolate impaired cell growth in osteoblast-like cells. In particular, in Saos-2 cells, 1xa0mM pidolate induced a slight accumulation of cells in the G0/G1 phase of the cell cycle and, in parallel, an early rise in intracellular calcium and a late decrease in intracellular magnesium content. Interestingly, when cultured in 5xa0mM magnesium pidolate, Saos-2 cells grew as fast as the controls. Moreover, intracellular magnesium and calcium concentrations did not vary. These results suggest a lower bioavailability of magnesium pidolate in osteoblast-like cells.

Actomyosin interaction at low ATP concentrations

In vitro motility assay (IVMA) experiments were performed to analyze the movement of actin filaments sliding on a pavement of myosin molecules at different [ATP] and [ADP]. In standard experimental conditions at [ATP]xa0=xa02xa0mM, about 80% of the actin filaments move in unloaded conditions with a constant velocity. However, a fraction of at least 20% static actin filaments is always present. The accepted explanation is the occurrence of damaged “rigor”-like myosin heads that do not undergo the normal ATP-dependent cycling motion. However, in a series of IVMA experiments performed at different [ATP] we observed that the mobility of actin filaments increased with lowering [ATP]. We investigated the influence of [ATP] on the number of mobile actin filaments. IVMA experiments were performed at controlled nucleotide concentrations and the percentage of mobile filaments accurately determined by specific operator-guided software. The value of ΔGATP involved was determined. Results showed that the number of mobile actin filaments sliding on type 2B heavy meromyosin isoform (2B HMM) increased at very low [ATP] accompanied by less negative ΔGATP values. Similar results were obtained by increasing [ADP]. Performing experiments at the same [ATP] with different myosin types, we found a higher number of mobile actin filaments on slow type 1 HMM with respect to type 2B HMM while the highest number of mobile actin filaments was found on single-head myosin (S1 fraction). We also found that [ATP] did not influence the percentage of mobile actin filaments sliding on S1. Our results reveal novel aspects of actomyosin interaction.

Repeatability and reproducibility of intracellular molar concentration assessed by synchrotron-based x-ray fluorescence microscopy

Elemental analysis of biological sample can give information about content and distribution of elements essential for human life or trace elements whose absence is the cause of abnormal biological function or development. However, biological systems contain an ensemble of cells with heterogeneous chemistry and elemental content; therefore, accurate characterization of samples with high cellular heterogeneity may only be achieved by analyzing single cells. Powerful methods in molecular biology are abundant, among them X-Ray microscopy based on synchrotron light source has gaining increasing attention thanks to its extremely sensitivity. However, reproducibility and repeatability of these measurements is one of the major obstacles in achieving a statistical significance in single cells population analysis. In this study, we compared the elemental content of human colon adenocarcinoma cells obtained by three distinct accesses to synchrotron radiation light.

Combined use of X-ray fluorescence microscopy, phase contrast imaging for high resolution quantitative iron mapping in inflamed cells

X-ray fluorescence microscopy (XRFM) is a powerful technique to detect and localize elements in cells. To derive information useful for biology and medicine, it is essential not only to localize, but also to map quantitatively the element concentration. Here we applied quantitative XRFM to iron in phagocytic cells. Iron, a primary component of living cells, can become toxic when present in excess. In human fluids, free iron is maintained at 10-18 M concentration thanks to iron binding proteins as lactoferrin (Lf). The iron homeostasis, involving the physiological ratio of iron between tissues/secretions and blood, is strictly regulated by ferroportin, the sole protein able to export iron from cells to blood. Inflammatory processes induced by lipopolysaccharide (LPS) or bacterial pathoge inhibit ferroportin synthesis in epithelial and phagocytic cells thus hindering iron export, increasing intracellular iron and bacterial multiplication. In this respect, Lf is emerging as an important regulator of both iron and inflammatory homeostasis. Here we studied phagocytic cells inflamed by bacterial LPS and untreated or treated with milk derived bovine Lf. Quantitative mapping of iron concentration and mass fraction at high spatial resolution is obtained combining X-ray fluorescence microscopy, atomic force microscopy and synchrotron phase contrast imaging.