NAD+ and SIRT3 control microtubule dynamics and reduce susceptibility to antimicrotubule agents.

Nicotinamide adenine dinucleotide (NAD(+)) is an endogenous enzyme cofactor and cosubstrate that has effects on diverse cellular and physiologic processes, including reactive oxygen species generation, mitochondrial function, apoptosis, and axonal degeneration. A major goal is to identify the NAD(+)-regulated cellular pathways that may mediate these effects. Here we show that the dynamic assembly and disassembly of microtubules is markedly altered by NAD(+). Furthermore, we show that the disassembly of microtubule polymers elicited by microtubule depolymerizing agents is blocked by increasing intracellular NAD(+) levels. We find that these effects of NAD(+) are mediated by the activation of the mitochondrial sirtuin sirtuin-3 (SIRT3). Overexpression of SIRT3 prevents microtubule disassembly and apoptosis elicited by antimicrotubule agents and knockdown of SIRT3 prevents the protective effects of NAD(+) on microtubule polymers. Taken together, these data demonstrate that NAD(+) and SIRT3 regulate microtubule polymerization and the efficacy of antimicrotubule agents.

Isolation of breast cancer and gastric cancer circulating tumor cells by use of an anti HER2-based microfluidic device.

Circulating tumor cells (CTCs) have emerged as a reliable source of tumor cells, and their concentration has prognostic implications. CTC capture offers real-time access to cancer tissue without the need of an invasive biopsy, while their phenotypic and molecular interrogation can provide insight into the biological changes of the tumor that occur during treatment. The majority of the CTC capture methods are based on EpCAM expression as a surface marker of tumor-derived cells. However, EpCAM protein expression levels can be significantly down regulated during cancer progression as a consequence of the process of epithelial to mesenchymal transition. In this paper, we describe a novel HER2 (Human Epidermal Receptor 2)-based microfluidic device for the isolation of CTCs from peripheral blood of patients with HER2-expressing solid tumors. We selected HER2 as an alternative to EpCAM as the receptor is biologically and therapeutically relevant in several solid tumors, like breast cancer (BC), where it is overexpressed in 30% of the patients and expressed in 90%, and gastric cancer (GC), in which HER2 presence is identified in more than 60% of the cases. We tested the performance of various anti HER2 antibodies in a panel of nine different BC cell lines with varying HER2 protein expression levels, using immunoblotting, confocal microscopy, live cells imaging and flow cytometry analyses. The antibody associated with the highest capture efficiency and sensitivity for HER2 expressing cells on the microfluidic device was the one that performed best in live cells imaging and flow cytometry assays as opposed to the fixed cell analyses, suggesting that recognition of the native conformation of the HER2 extracellular epitope on living cells was essential for specificity and sensitivity of CTC capture. Next, we tested the performance of the HER2 microfluidic device using blood from metastatic breast and gastric cancer patients. The HER2 microfluidic device exhibited CTC capture in 9/9 blood samples. Thus, the described HER2-based microfluidic device can be considered as a valid clinically relevant method for CTC capture in HER2 expressing solid cancers.