Matrix metalloproteinases (MMPs) are secretory endopeptidases. They have been associated with invasion by cancer-cell and metastasis. Previous studies have demonstrated that proteolytic activity could be detected using fluorescence resonance energy transfer (FRET) with mutants of GFP. To monitor MMP activity, we constructed vectors that encoded a MMP Substrate Site (MSS) between YFP and CFP. In vitro, YFP-MSS-CFP can be used to detect MMP activity and 1,10-phenathroline inhibition of MMP activity. In living cells, MMPs are secreted proteins and act outside of the cell, and therefore YFP-MSS-CFPdisplay was anchored on the cellular surface to detect extracellular MMP. A pDisplay-YC vector expressing the YFP-MSS-CFPdisplay on the cellular surface was transfected into MCF-7 cells that expressed low levels of MMP. Efficient transfer of energy from excited CFP to YFP within the YFP-MSS-CFPdisplay molecule was observed, and real-time FRET was declined when MCF-7 was incubated with MMP2. However, no such transfer of energy was detected in the YFP-MSS-CFPdisplay expressing MDA-MB 435s cells, in which high secretory MMP2 were expressed. The FRET sensor YFP-MSS-CFPdisplay can sensitively and reliably monitor MMP activation in living cells and can be used for high-throughput screening of MMP inhibitors for anti-cancer treatments.
The single-cell Raman spectra of human Burkitt's lymphoma cells (CA46) including cells treated with different doses of paclitaxel and controls without paclitaxel can be detected by confocal micro-Raman spectroscopy. It shows that the Raman bands at 1094 cm–1assigned to the symmetric stretching vibration mode of O–P–O in the DNA backbone, 1338 cm–1and 1578 cm–1due to adenine and guanine of DNA all decrease in intensity with increasing drug dose. On the contrary, the intensity of peaks at 1257 cm–1due to characteristic vibration ofa-helix of Amide III and 1658 cm–1due to characteristic vibration ofa-helix of Amide I both increases with increasing drug dose. Multivariate statistical methods, such as Principle Components Analysis (PCA) and Linear Discriminant Analysis (LDA) were employed to discriminate normal lymphoma cells (CA46) and cells treated with different doses of paclitaxel. It was found that the sensitivity and specificity of differentiating the treated and untreated cell groups increase with drug doses and approach 100% for the high drug dose, consistent with the perception that the cytotoxicity increases with drug dose. These results suggest that Raman spectroscopy combined with multivariate analysis could become a useful tool for assessing the cytotoxicity of drugs such as paclitaxel on human lymphoma cells.
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