High-performance thin-layer chromatography for the evaluation of voacamine intracellular concentration related to its cytotoxic effect.

Previous investigations demonstrated that pretreatment with non-cytotoxic concentrations of voacamine had a chemosensitizing effect on cultured multidrug resistant osteosarcoma cells exposed to doxorubicin; whereas when used alone at high concentrations voacamine induced apoptosis-independent cell death on both sensitive and resistant cells. To gain insight into the mechanism of action of voacamine at the subcellular level, we developed an analytical high-performance thin-layer chromatography technique to assess the intracellular content of voacamine that could be correlated with the induction of cell death and consequent morphological and ultrastructural changes. The results of the quantitative analysis not only did allow us to measure both the amount of unmodified voacamine molecules (determined by the method) and the amount of molecules which reacted with cellular components (undetectable), but also to confirm the findings of our previous studies and support the validity of this method.

The combined treatment with chloroquine and the enzymatic oxidation products of spermine overcomes multidrug resistance of melanoma M14 ADR2 cells: a new therapeutic approach.

It has been confirmed that multidrug resistant (MDR) melanoma cells (M14 ADR2) are more sensitive than their wild-type counterparts (M14 WT) to H2O2 and aldehydes, the products of bovine serum amine oxidase (BSAO)-catalyzed oxidation of spermine. The metabolites formed by BSAO and spermine are more toxic, in M14 cells, than exogenous H2O2 and acrolein, even though their concentration is lower during the initial phase of incubation due to their more gradual release than the exogenous products. Binding of BSAO to the cell membrane and release of the reaction products of spermine into the immediate vicinity of the cells, or directly into the cells, may explain the apparently paradoxical phenomenon.

Voacamine modulates the sensitivity to doxorubicin of resistant osteosarcoma and melanoma cells and does not induce toxicity in normal fibroblasts.

In previous studies it has been demonstrated that the plant alkaloid voacamine (1), used at noncytotoxic concentrations, enhanced the cytotoxicity of doxorubicin and exerted a chemosensitizing effect on cultured multidrug-resistant (MDR) U-2 OS-DX osteosarcoma cells. The in vitro investigations reported herein gave the following results: (i) the chemosensitizing effect of 1, in terms of drug accumulation and cell survival, was confirmed using SAOS-2-DX cells, another MDR osteosarcoma cell line; (ii) compound 1 enhanced the cytotoxic effect of doxorubicin also on the melanoma cell line Me30966, intrinsically drug resistant and P-glycoprotein-negative; (iii) at the concentrations used to sensitize tumor cells, 1 was not cytotoxic to normal cells (human fibroblasts). These findings suggest possible applications of voacamine (1) in integrative oncologic therapies against resistant tumors.

Structural and functional alterations of cellular components as revealed by electron microscopy.

Scanning (SEM) and transmission electron microscopy (TEM) are two fundamental microscopic techniques widely applied in biological research for the study of ultrastructural cell components. With these methods, especially TEM, it is possible to detect and quantify the morphological and ultrastructural parameters of intracellular organelles (mitochondria, Golgi apparatus, lysosomes, peroxisomes, endosomes, endoplasmic reticulum, cytoskeleton, nucleus, etc.) in normal and pathological conditions.

Guidelines for the use and interpretation of assays for monitoring autophagy.

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding.