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Even though commercialized anticancer drugs are now produced by pharmaceutical companies, most of them were originally obtained from natural sources, and more particularly from plants. Indeed, many structurally diverse compounds isolated from plants or marine flora have been purified and synthesized for their anticancer bioactivity. Among these, several molecules belong to the class of anticancer drugs which target the microtubule cytoskeleton, either by stabilizing it or destabilizing it. To characterize the activity of these drugs and to understand in which physiological context they are more likely to be used as therapeutic agents, it is necessary to fully determine their interaction with tubulin. Understanding the molecular basis of their effects on microtubule cytoskeleton is an important step in designing analogs with greater pharmacological activity and with fewer side effects. In addition, knowing the molecular mechanism of action of each drug that is already used in chemotherapy protocols will also help to find strategies to circumvent resistance. By taking examples of known anti-tubulin plant derived drugs, we show how identification of microtubule targeting agents and further characterization of their activity can be achieved combining biophysical and biochemical approaches. We also illustrate how continuing in depth study of molecules with already known primary mechanisms of action can lead to the discovery of new targets or biomarkers which can open new perspectives in anticancer strategies. [ABSTRACT FROM AUTHOR]

Tumour drug-resistant ABCB1 gene expression is regulated at the chromatin level through epigenetic mechanisms. We examined the effects of the histone deacetylase inhibitor trichostatin A (TSA) on ABCB1 gene expression in small cell lung carcinoma (SCLC) drug-sensitive (H69WT) or etoposide-resistant (H69VP) cells. We found that TSA induced an increase in ABCB1 expression in drug-sensitive cells, but strongly decreased it in drug-resistant cells. These up- and downregulations occurred at the transcriptional level. Protein synthesis inhibition reduced these modulations, but did not completely suppress them. Differential temporal patterns of histone acetylation were observed at the ABCB1 promoter: increase in H4 acetylation in both cell lines, but different H3 acetylation with a progressive increase in H69WT cells but a transient one in H69VP cells. ABCB1 regulations were not related with the methylation status of the promoter −50GC, −110GC, and Inr sites, and did not result in further changes to these methylation profiles. Trichostatin A treatment did not modify MBD1 binding to the ABCB1 promoter and similarly increased PCAF binding in both H69 cell lines. Our results suggest that in H69 drug-resistant SCLC cell line TSA induces downregulation of ABCB1 expression through a transcriptional mechanism, independently of promoter methylation, and MBD1 or PCAF recruitment.British Journal of Cancer (2007) 97, 562–573. doi:10.1038/sj.bjc.6603914 www.bjcancer.com Published online 31 July 2007 [ABSTRACT FROM AUTHOR]

Abstract: A selective analysis of adsorbed mitoxantrone (MTX) was performed by surface-enhanced Raman scattering (SERS) at the range of cellular membrane. Disruption of the membrane fluidity was carried out to appraise changes in membrane adsorption of MTX and drug uptake in sensitive (HCT-116 S) and resistant BCRP/MXR (HCT-116 R) cells. Based on spectral MTX modifications, micro-SERS spectroscopy discriminated clearly drug adsorption phenomena on plasma membrane from drug in solution. A 3-fold higher SERS intensity of MTX for HCT-116 R was observed concluding to a higher drug adsorption on resistant membrane. The increase of membrane fluidity with benzyl alcohol (BA) or chloroform (CF) resulted in a 3-fold decrease of MTX adsorption on HCT-116 R, exclusively. BA and CF improved intracellular accumulation of MTX (e.g., 823 and 191pmol MTX/106 HCT-116 R incubated with or without BA). At 4°C, drug accumulation measurements showed a decrease of MTX permeability in resistant membrane (42pmol MTX/106 cells), restored with fluidizers (e.g., 342pmol MTX/106 cells with BA). Fluorescence confocal microscopy involved an exclusive MTX emission around the plasma membrane of resistant cells whereas fluidizers increased the intracellular uptake of MTX in both cell lines at the same time with less drug emission around the plasma membrane. Changes of the membrane structure of resistant cells should modify both drug adsorption and membrane permeation. [Copyright &y& Elsevier]

Surface-enhanced Raman scattering (SERS) spectroscopy was applied to analyze mitoxantrone (MTX) adsorption on the plasma membrane microenvironment of sensitive (HCT-116 S) or BCRP/MXR-type resistant (HCT-116 R) cells. The addition of silver colloid to MTX-treated cells revealed an enhanced Raman scattering of MTX. Addition of extracellular DNA induced a total extinction of MTX Raman intensity for both cell lines, which revealed an adsorption of MTX on plasma membrane. A threefold higher MTX Raman intensity was observed for HCT-116 R, suggesting a tight MTX adsorption in the plasma membrane microenvironment. Fluorescence confocal microscopy confirmed a relative MTX emission around plasma membrane for HCT-116 R. After 30 min at 4 °C, a threefold decrease of the MTX Raman scattering was observed for HCT-116 R, contrary to HCT-116 S. Permeation with benzyl alcohol revealed a threefold decrease of membrane MTX adsorption on HCT-116 R, exclusively. This additional MTX adsorption should correspond to the drug bound to an unstable site on the HCT-116 R membrane. This study showed that SERS spectroscopy could be a direct method to reveal drug adsorption to the membrane environment of living cells. [Copyright &y& Elsevier]