Pharmacological inhibition of cyclin dependent kinase 2 inhibits proliferation and pro-fibrotic features of Hepatic Stellate Cells

Background & Aims:

Liver fibrosis represents a common and difficult clinical challenge of worldwide importance. It occurs as response to chronic liver diseases induced e.g. by hepatitis C virus (HCV) infection, alcohol abuse or non-alcoholic steatohepatitis (NASH) and is characterized by regenerative proliferation of hepatocytes and the accumulation of extracellular collagen produced by Hepatic Stellate Cells (HSCs). Resting HSCs can be activated by liver damage and subsequent inflammatory processes resulting in transdifferentiation towards matrix-producing myofibroblasts. This process involves cell cycle re-entry and proliferation of the normally quiescent HSCs. The cell cycle is regulated by cyclins and associated cyclin-dependent kinases (Cdks). Cdk2 is thought to mediate the entry and progression through the phase of DNA synthesis (S-phase) in complex with E-and A-type cyclins. Pharmacological pan-Cdk inhibitors (e.g. Roscovitine, also targeting Cdk2) are tested in clinical phase II trials against non-small cell lung cancer and breast cancer with limited toxicity and side effects. The aim of this study was to evaluate the benefit of the second generation pan-Cdk inhibitor CR8 for treatment of liver fibrosis. To this end the anti-fibrotic effects of CR8 on HSCs and its potential anti-regenerative effects on hepatocytes were addressed.

Methods:

In this pilot study, continuous proliferating and activated human (LX-2) and murine (GRX) HSC cell lines were treated with increasing concentrations of (S)-CR8. In parallel, murine primary hepatocytes were isolated and also treated with (S)-CR8 with or without mitogen stimulation (i.e. epidermal growth factor (EGF)/insulin). Markers of proliferation, DNA damage and HSC activation were examined on transcriptional and post-transcriptional levels by quantitative real-time PCR, immunoblotting, immunofluorescence and flow cytometry.

Results:

(S)-CR8 treatment resulted in a reduction of proliferation, activation and collagen deposition in both HSC cell lines. Interestingly, human HSCs were more sensitive towards low doses of CR8 than their murine counterparts. Response towards (S)-CR8 was characterized by induction of apoptotic cell death in both HSC cell lines as determined by a downregulation of anti-apoptotic mediators and activation of caspase-3. As one underlying mechanism we identified the induction of DNA damage and subsequent cell cycle arrest in both cell lines. Importantly, (S)-CR8 dosages leading to anti-fibrotic effects in HSCs did not affect cell cycle activity and survival in mitogen-activated primary hepatocytes.

Conclusion:

In summary, our data showed that pharmacological pan-Cdk inhibition restricts the pro-fibrotic properties of HSCs, while preserving regeneration capacity of hepatocytes under the same conditions. Therefore, CR8 and related compounds might be promising therapeutic agents for the treatment of liver fibrosis in vivo.