The Role Of The Cholesterol Metabolite, 27-Hydroxycholesterol, In Colon Cancer Cells

Colorectal Cancer (CRC) is a major health concern with more than 600,000 deaths worldwide each year. Several risk factors for CRC exist including a diet rich in fat and cholesterol. However, the link between cholesterol and CRC is controversial. It is possible that oxysterols, which are active cholesterol metabolites, may correlate better with CRC than cholesterol itself. The most abundant oxysterol in the plasma is 27-hydroxycholesterol (27-OHC), which is a ligand for the nuclear receptors estrogen receptor (ER) and liver-x-receptor (LXR). 27-OHC has been shown to be involved in breast and prostate cancers, but its role in colon cancer remains unexplored. Therefore, we sought to determine the role of 27-OHC in colon cancer cell lines. We also determined the impact of 27-OHC on cellular differentiation through measurement of the protein SLFN12. Using two colon cancer cell lines (Caco2 and SW620), an MTT assay and CyQUANT® proliferation assays, an LDH cytotoxicity assay, a TUNEL assay, western blotting, immunofluorescence, and a scratch assay, we determined 27-OHCâs effects on cellular proliferation, migration, cytotoxicity, and apoptosis. We also determined the role of ER and LXR in cellular proliferation. We found that treatment of Caco2 and SW620 cells with 27-OHC induced a decrease in cell proliferation without a detectable amount of cellular cytotoxicity or apoptosis. Additionally, we found that the observed decrease in cell proliferation is independent of ER and LXR, as 27-OHC did not activate the LXR target genes ABCG1 and ABCA1 nor lead to the nuclear localization of ER. Additionally, we found that 27-OHC leads to a decrease in the phosphorylated and activated form of AKT, a molecule involved in cell cycle progression, protein synthesis, and cellular survival. We also found that 27-OHC increased cellular migration through a scratch assay. 27-OHC decreased the epithelial marker E-cadherin without changes to the mesenchymal markers N-cadherin. In order for cells to migrate, they need to stop proliferating. In conclusion, our data shows that 27-OHC treatment leads to an increase in cellular migration through a loss of proliferation

Quality Control Methods for Optimal BCR-ABL1 Clinical Testing in Human Whole Blood Samples

Reliable breakpoint cluster region (BCR)–Abelson (ABL) 1 measurement is essential for optimal management of chronic myelogenous leukemia. There is a need to optimize quality control, sensitivity, and reliability of methods used to measure a major molecular response and/or treatment failure. The effects of room temperature storage time, different primers, and RNA input in the reverse transcription (RT) reaction on BCR-ABL1 and β-glucuronidase (GUSB) cDNA yield were assessed in whole blood samples mixed with K562 cells. BCR-ABL1 was measured relative to GUSB to control for sample loading, and each gene was measured relative to known numbers of respective internal standard molecules to control for variation in quality and quantity of reagents, thermal cycler conditions, and presence of PCR inhibitors. Clinical sample and reference material measurements with this test were concordant with results reported by other laboratories. BCR-ABL1 per 103 GUSB values were significantly reduced (P = 0.004) after 48-hour storage. Gene-specific primers yielded more BCR-ABL1 cDNA than random hexamers at each RNA input. In addition, increasing RNA inhibited the RT reaction with random hexamers but not with gene-specific primers. Consequently, the yield of BCR-ABL1 was higher with gene-specific RT primers at all RNA inputs tested, increasing to as much as 158-fold. We conclude that optimal measurement of BCR-ABL1 per 103 GUSB in whole blood is obtained when gene-specific primers are used in RT and samples are analyzed within 24 hours after blood collection