Three-dimensional cell culture: a breakthrough in vivo.

Cell culture is an important tool for biological research. Two-dimensional cell culture has been used for some time now, but growing cells in flat layers on plastic surfaces does not accurately model the in vivo state. As compared to the two-dimensional case, the three-dimensional (3D) cell culture allows biological cells to grow or interact with their surroundings in all three dimensions thanks to an artificial environment. Cells grown in a 3D model have proven to be more physiologically relevant and showed improvements in several studies of biological mechanisms like: cell number monitoring, viability, morphology, proliferation, differentiation, response to stimuli, migration and invasion of tumor cells into surrounding tissues, angiogenesis stimulation and immune system evasion, drug metabolism, gene expression and protein synthesis, general cell function and in vivo relevance. 3D culture models succeed thanks to technological advances, including materials science, cell biology and bioreactor design.

Combination of the mTOR inhibitor RAD001 with temozolomide and radiation effectively inhibits the growth of glioblastoma cells in culture.

The present in vitro study aimed to assess the effects of combining the mTOR inhibitor RAD001 and temozolomide (TMZ) together with irradiation by either low-linear energy transfer (LET) radiation (γ-rays) or high-LET radiation (fast neutrons) on the growth and cell survival of the human glioblastoma cell line U-87. We observed a strong decrease in cell proliferation along with a concomitant increase in cell death as a function of the radiation dose. As expected, high-LET radiation was more effective and induced more sustained damage to DNA than low-LET radiation. While RAD001 in association with TMZ induced autophagic cell death, additional combination with either type of radiation did not further increase its occurrence. On the contrary, apoptosis remained at a low level in all experimental groups.

The mTOR inhibitor RAD001 potentiates autophagic cell death induced by temozolomide in a glioblastoma cell line.

We have studied the consequences of the combination of the mammalian target of rapamycin (mTOR) inhibitor RAD001 and temozolomide on the growth and cell death of the glioblastoma cell line U-87 in vitro. A progressive decrease of cell proliferation was recorded with increasing concentrations of temozolomide, which was markedly reinforced and prolonged by the addition of RAD001. While this combination treatment resulted in only a low level of apoptosis, it led to a pronounced enhancement of autophagic cell death. When combined with γ-ray irradiation, a significant reinforcement of the overall cytotoxicity was obtained, suggesting the efficacy of such a multipronged approach for the treatment of glioblastoma. RAD001 strongly contributes to the reinforcement of temozolomide-induced autophagy, which appears to represent a major form of cell death in glioblastoma. The association of such combined chemotherapies with radiotherapy could be useful for the management of these hard-to-treat malignancies.

Novel pharmacological modulators of autophagy and therapeutic prospects.

INTRODUCTION: Autophagy is an intracellular process of self-digestion involving the lysosomal degradation of cytoplasmic organelles and macromolecules. It occurs at low basal levels to perform housekeeping functions and is dramatically augmented upon nutrient depletion or exposure to other stresses, thus maintaining cellular homeostasis and energy balance and providing cytoprotective responses to adverse conditions. Mounting evidence that autophagy malfunction contributes to the pathogenesis of diverse human diseases has stimulated efforts to identify pharmacological agents that modulate autophagy in potentially beneficial ways. Here, we review the progresses accomplished toward this goal in recent years, as reflected by the patent literature. AREAS COVERED: Patent applications published from 2008 to mid-2012 that pertain to the pharmacological modulation of autophagy are reviewed and their potential therapeutic utilities are discussed. EXPERT OPINION: Of 40 patents related to autophagy, 21 claim novel enhancers or inhibitors of autophagy. One of the most promising applications of these compounds concerns cancer therapy, a few of them being already considered for clinical evaluation. Further work is, however, needed to identify compounds that target unique molecular effectors/regulators of autophagy to selectively modulate its various stages in different tissues and to design therapeutic interventions applicable to a broad variety of dysfunctional autophagy-associated disorders.

The mTOR inhibitor RAD001 augments radiation-induced growth inhibition in a hepatocellular carcinoma cell line by increasing autophagy.

Treatment of hepatocellular carcinoma (HCC) is a major concern for physicians as its response to chemotherapy and radiotherapy remains generally poor, due, in part, to intrinsic resistance to either form of treatment. We previously reported that an irradiation with fast neutrons, which are high-linear energy transfer (LET) particles, massively induced autophagic cell death in the human HCC SK-Hep1 cell line. In the present study, we tested the capacity of the mammalian target of rapamycin (mTOR) inhibitor RAD001 to augment the cytotoxicity of low and high-LET radiation in these cells. As mTOR is a key component in a series of pathways involved in tumor growth and development, it represents a potential molecular target for cancer treatment. Results indicate that RAD001, at clinically relevant nanomolar concentrations, enhances the efficacy of both high- and low-LET radiation in SK-Hep1 cells, and that the induction of autophagy may account for this effect. However, fast neutrons were found to be more efficient at reducing tumor cell growth than low-LET radiation.