Doxycycline at Low Concentrations Could Influence Your Experimental Results When Working With Prostate Cancer Cell Lines

at Low Concentrations Could Influence Your Experimental Results When Working With Cell Doxycycline is a tetracycline derivate commonly used in various inducible expression systems to study the function of different proteins. In this study, we show that 22Rv1 and PC3, two different, commonly used prostate cancer cell lines are very sensitive to this antibiotic and addition of the drug at inductive concentrations can affect cells in ways that can be misinterpreted as effects from the protein studied. Therefore, proper controls are very important in order to avoid conclusions drawn by artifacts caused by experimental conditions.

Doxycycline is a tetracycline derivate commonly used in various inducible expression systems to study the function of different proteins. In this study, we show that 22Rv1 and PC3, two different, commonly used prostate cancer cell lines are very sensitive to this antibiotic and addition of the drug at inductive concentrations can affect cells in ways that can be misinterpreted as effects from the protein studied. Therefore, proper controls are very important in order to avoid conclusions drawn by artifacts caused by experimental conditions. the concentration of Tet System approved fetal bovine serum was reduced from 10% to 0.5%.Doxycycline (BioNordika) was added at concentrations indicated in the figures.

TetOn 3G Expression System in 22Rv1
The TetOn3G expression system was used to over express  Note: 22Rv1 (A) and PC3 (B) cells were incubated with increasing concentrations of doxycycline (dox) as indicated in the figure, for 72 h before cell proliferation was measured using an ELISA reader. Absorbance was measured at 590nm, and reference wavelength was set to 650nm. The experiment was done in triplicate and the individual values are plotted in the figure as a scatter plot with mean and SD shown with bars.

Migration and Invasion Assay
For migration and invasion assays, 3.5x10522Rv1 cells, were seeded in 5 ml volume into T25 flasks (Sarstedts) and pre-

Statistical Analysis
Statistical significance was determined in multiple t-tests using the Holm Sidak method, with α=5.000% on the transfected 22Rv1 cells shown in (Figures 2A & 2C). For the non-transfected 22Rv1 cells, shown in ( Figures 2B & 2D), we used an unpaired t-test. All analysis were performed using the GraphPad Prism version 6.07 for Windows, Graph Pad Software, La Jolla California USA, www. graphpad.com

Results
Doxycycline has an effect on cell proliferation in 22Rv1 and PC3 cell lines as can be seen in (Figure 1). No difference is observed between cells grown in the presence of 10ng/ml doxycycline compared to the negative control. At 100ng/ml and 1000ng/ml you can see an increase in the variation of the proliferation rate which seems to increase with more dense cultures (data not shown). Doxycycline reduces the growth rate but do not affect the viability of the cells ( Table 1)  had impact on the migratory ability of the cells (Figure 2A). A trend of increased invasion of the cells could also be seen ( Figure 2C).
But when we looked at the results from the 22Rv1 cells transfected with the regulatory plasmid, a control usually not included, we got quite concerned as those cells seemed to be as affected as the   [2,7], but in those studies higher concentrations of the tetracycline was used. In the work of Ahler et al [2] it is obvious that various cell lines differ in their sensitivity towards the drug, but interestingly, the most sensitive cell line they tested was LNCaP, also derived from human prostate cancer cells.
Doxycycline and other tetracyclines have also been tested in cancer therapy [8].

Conclusion
Doxycycline induced expression systems are commonly used.
Some cell lines are very sensitive to also very low concentrations of doxycycline, therefore it is of uttermost importance to include the correct controls before interpreting the results obtained.