Differential proteome analysis of human embryonic kidney cell line (HEK-293) following mycophenolic acid treatment

Abstract

Background: Mycophenolic acid (MPA) is widely used as a post transplantation medicine to prevent acute organ rejection. In the present study we used proteomics approach to identify proteome alterations in human embryonic kidney cells (HEK-293) after treatment with therapeutic dose of MPA. Following 72 hours MPA treatment, total protein lysates were prepared, resolved by two dimensional gel electrophoresis and differentially expressed proteins were identified by QTOF-MS/MS analysis. Expressional regulations of selected proteins were further validated by real time PCR and Western blotting.

Results: The proliferation assay demonstrated that therapeutic MPA concentration causes a dose dependent inhibition of HEK-293 cell proliferation. A significant apoptosis was observed after MPA treatment, as revealed by caspase 3 activity. Proteome analysis showed a total of 12 protein spots exhibiting differential expression after incubation with MPA, of which 7 proteins (complement component 1 Q subcomponent-binding protein, electron transfer flavoprotein subunit beta, cytochrome b-c1 complex subunit, peroxiredoxin 1, thioredoxin domain-containing protein 12, myosin regulatory light chain 2, and profilin 1) showed significant increase in their expression. The expression of 5 proteins (protein SET, stathmin, 40S ribosomal protein S12, histone H2B type 1 A, and histone H2B type 1-C/E/F/G/I) were down-regulated. MPA mainly altered the proteins associated with the cytoskeleton (26%), chromatin structure/dynamics (17%) and energy production/conversion (17%). Both real time PCR and Western blotting confirmed the regulation of myosin regulatory light chain 2 and peroxiredoxin 1 by MPA treatment. Furthermore, HT-29 cells treated with MPA and total kidney cell lysate from MMF treated rats showed similar increased expression of myosin regulatory light chain 2.

Conclusion: The emerging use of MPA in diverse pathophysiological conditions demands in-depth studies to understand molecular basis of its therapeutic response. The present study identifies the myosin regulatory light chain 2 and peroxiredoxin 1 along with 10 other proteins showing significant regulation by MPA. Further characterization of these proteins may help to understand the diverse cellular effects of MPA in addition to its immunosuppressive activity.

Figure 1

Inhibition of HEK-293 cells proliferation by MPA treatment. The cell proliferation was determined after 72 hr of treatment with different doses of MPA (0-100 μmol/L) using BrdU colorimetric based method. Results are shown as percentage of control (DMSO treated) and represent four independent experiments.

Figure 2

Differential protein expression after incubation of HEK-293 cells with MPA. Total protein lysate from DMSO and MPA treated cells was separated by 2-D gel electrophoresis and silver stained. Encircled differentially regulated proteins spots were identified using Q-TOF MS/MS analysis. The figure shows exemplary 2-DE gels of DMSO and MPA treated HEK-293 cells.

Figure 3

Functional classification of regulated proteins. Biological functions were assigned using online KOGnitor NCBI (http://www.ncbi.nlm.nih.gov/COG/grace/kognitor.html) software.

Figure 4

Differential expression of Prdx1 and MLC2 by MPA treatment. (a) Selected areas in the silver stained gels showing differential expression of Prdx1 and MLC2. Delta 2D software was used for densitrometric analysis. The quantification of the level of expression (% volume) in MPA treated cells and control cells (DMSO) is illustrated as a bar chart with the mean and SD of four separate experiments (*p < 0.05). (b) Expression patterns of Prdx1 and MLC2 genes determined by real-time PCR. The relative expression of Prdx1 and MLC2 mRNA in the treated samples was determined as a fold change compared with control samples using the comparative threshold cycle (C_T) method (2^-ΔΔC_T) as described in materials and methods part. Results shown are representative of four independent experiments. EF-2 was used to normalize the values. The boxes represent range in variation statistics and the lines across the boxes represent the medians and the whiskers extend to the highest and lowest values. Significance was calculated using the Mann-Whitney-U test (*p < 0.05) (c) Effect of MPA treatment on Prdx1 and MLC2 protein expression. Protein extracts from MPA and DMSO treated cells were Western blotted using specific antibodies against Prdx1 and MLC2. Densitometric analysis was done using Lab image version 2.71 software. β tubulin signal was used to control the equal protein load. The experiments were repeated four times and error bars represent ± SD (**p < 0.005).

Figure 5

Expression of MLC2 in MMF treated rat kidney lysate and HT-29 cells. Protein lysate was prepared and immunoblotted for MLC2 as described in method section. β tubulin was used to show equal protein load. Lab image software was used for quantification of protein bands. Four independent experiments were performed and results presented as mean ± SD (**p < 0.005).

Figure 6

Measurement of MPA induced caspase-3 activity. Cells were treated with MPA and DMSO for 72 h. Protein extracts from each was measured for caspase-3 activity. Five independent experiments were performed and results presented as mean absorbance ± SD (**p < 0.005).