Studies on the functional consequences of cyclin D2 downregulation in VPA- treated PCa

Witt et al. (2013) could show that VPA treatment of PCa cells specifically led to a re-expression of cyclin D2. Because the cyclin D2 promoter region is hypermethylated cyclin D2 expression is downregulated in PCa. Treatment of PCa cells with the histone deacetylase inhibitor VPA induced increased acetylation of the cyclin D2 promoter and consequently re-expression of cyclin D2 (Witt et al., 2013). Witt et al. could also show that VPA treatment of PCa cells reduced the ability of the cells to proliferate, a characteristic of cancer cells. To study the possible connection between the increased cyclin D2 expression and the reduced proliferation of PCa cells after VPA treatment an experiment was conducted where human and mouse PCa cells (LNCaP, DU145, PC-3 and 2E) were first transfected with two different cyclin D2-specific siRNAs and at the following day transfected cells were treated with different concentrations of VPA (1mM and 5mM). Simultaneously, the proliferation rate was measured by a MTT assay. Cyclin D2 siRNA treatment was supposed to prevent the VPA-induced re-expression of cyclin D2 and should thereby lead to an increase in the proliferation in case cyclin D2 would be responsible for the VPA-induced reduced proliferation of PCa cells. Since the efficiency of the three mouse cyclin D2-specific siRNAs had already been tested by western blot on proteins isolated from mouse NIH/3T3 cells transfected with the siRNAs (see Fig. 3.28A), the human cyclin D2-specific siRNAs (siRNA A, B, C) still had to be tested regarding their efficient downregulation of cyclin D2. Therefore, the human colon epithelial cancer cell line CaCo-2, which is known to express human cyclin D2, were transfected with the human cyclin D2-specific siRNAs and 120 hours later total amount of protein was isolated and immunoblotted with a cyclin D2-specific antibody.

Human cyclin D2 siRNA A and B led to a sufficient downregulation of cyclin D2 whereas siRNA C led only to a minor downregulation of cyclin D2 as compared to the luciferase control transfected cells (Fig. 3.36). For the following study of simultaneous cyclin D2 siRNA treatment and VPA treatment only human cyclin D2 specific siRNA A and B were used.

Fig. 3.36: Testing the efficiency of three different human cyclin D2-specific siRNAs in CaCo2 cells. Human colon epithelial cancer cells CaCo-2 were transfected with three different cyclin D2-specific siRNAs (A, B, C) and 120 hours later protein was isolated from the cells. Whole protein lysate was immunoblotted using a cyclin D2-specific antibody. Transfection with either siRNA A or siRNA B led to an efficient cyclin D2 downregulation as compared to the luciferase control transfected cells. Transfection with siRNA C had only minor effects on cyclin D2 downregulation.

Immunoblotting of HSC70 ensured equal protein loading.

After testing the efficiency of the human cyclin D2-specific siRNAs human and mouse PCa cells (LNCaP, DU145, PC-3 and 2E) were transfected with two different cyclin D2 specific siRNAs (LNCaP, DU145, PC-3 cells: human-specific siRNAs A and B; 2E cells: mouse-specific siRNAs B and C) and controls were transfected with luciferase. The following day, the transfected cells were treated with different concentrations of VPA (1mM and 5mM) before the proliferation rate was measured by a MTT assay for 96 consecutive hours (Fig 3.37).

Control cells that were transfected only with the siRNA but were not treated with VPA exhibited a reduced proliferation rate as compared to luciferase transfected cells during the four day study.

In PC-3 and LNCaP cells this effect was time-dependent. An increased proliferation rate as compared to the control was observed in 2E cells after 48 and 96 hours and in LNCaP cells during the first two days, although this was a minor effect (Fig.37, left panel). Upon VPA treatment of all four cell lines their proliferation rate decreased further as compared to the controls in a concentration-dependent manner (only shown four one time point (48 hours), Fig.37, right panel). The only exception here was observed in PC-3 cells transfected with siRNA B and treated with 1mM VPA, where the proliferation rate slightly increased.

Here it was expected, that cells which have a reduced cyclin D2 expression due to siRNA-transfection would have an increased or comparable proliferation rate to controls upon VPA treatment. The known increased cyclin D2 expression after VPA treatment should be prevented by the siRNA which should lead to an increased proliferation rate in case cyclin D2 is directly responsible for the decreased proliferation rate observed in 2E cells (Witt et al., 2013). But contrary to the expectation, VPA treatment of the siRNA-transfected cells further decreased the proliferation rate. This could be due to the fact that downregulation of cyclin D2 expression via siRNA is not sufficient to prevent VPA-induced re-expression of cyclin D2. This assumption is

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supported by the observation from a quantitative real-time PCR analysis which showed that cyclin D2 siRNA-transfected 2E cells still exhibited an increased cyclin D2 expression upon VPA treatment as compared to controls by using quantitative real-time PCR analysis (data not shown).

Fig. 3.37: Analysis of the proliferation rate in VPA-treated PCa cells with a reduced cyclin D2 expression. The mouse PCa cells 2E and the human PCa cells PC-3, LNCaP and DU145 were transfected with two different cyclin D2-specific siRNAs (human: siRNA A and B; mouse: siRNA B and C). Cells transfected with luciferase served as control. The day following transfection the cells were treated with either 1mM or 5mM VPA. Controls were not treated with VPA. Simultaneously, PMS/MTS solution was added to the cells and two hours later the measurement of the proliferation rate started by a MTT assay, which was conducted for 96 consecutive hours. Here, the proliferation rate for transfected control cells (no VPA treatment) over the time course is depicted on the left panel. The human PCa cell lines transfected with either one of the two siRNAs exhibit a reduced proliferation rate as compared to luciferase-transfected cells, which was time-dependent in PC-3 and LNCaP cells. In 2E cells, after 48 and 96 hours and in LNCaP cells during the first 48 hours the proliferation rate is slightly increased as compared to controls. On the right panel, the proliferation rate for the four cell lines upon VPA treatment is represented for one time point, namely after 48 hours. In all cell lines analyzed, the proliferation rate decreased further upon VPA treatment in a concentration-dependent manner as compared to untreated controls. Solely in PC-3 cells transfected with siRNA B and treated with 1mM VPA a slight increase in the proliferation rate was observed. This experiment was conducted once.