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Targeting ROCK1/2 blocks cell division and induces mitotic catastrophe in hepatocellular carcinoma
Hua Wu1, Yuyuan Chen1, Bin Li1
1School of Pharmacy, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.
Background:
Rho-Associated kinases ROCK1 and ROCK2 have been extensively investigated in the pathogenesis of cardiovascular disease. However, their roles are not fully understood in carcinogenesis. In this study, we investigated whether ROCK1 or ROCK2 is required for the survival and growth of hepatocellular carcinoma (HCC) cells and underlying mechanism.
Methods:
ROCKs expression was determined in human HCC tissue and cell lines using qRT-PCR, western blotting, and immunohistochemistry (IHC). Cell growth and proliferation were assayed using cell counting kit-8 (CCK-8) and EdU incorporation assay. Cell cycle and apoptosis analysis were performed using flow cytometry. HCC cell division or mitosis was observed using a confocal microscope and a time relapse fluorescence microscope. Inhibitory role of targeting ROCK1/2 on HCC was assayed in both xenograft and primary HCC mouse models.
Results:
Both ROCK1 and ROCK2 are over-expressed in human HCC tissues and cell lines. Knockdown of ROCK1 or ROCK2 inhibited HCC cell growth. Pharmacological inactivation of ROCK1/2 with Fasudil further blocked the growth and survival of HCC both in vitro and in vivo. Mechanically, Fasudil induces cell cycle arrest in HCC cells, but not apoptosis. Instead, Fasudil treatment led to mitotic catastrophe in HCC cells, characterized with the multipolar and asymmetric mitosis, and disassociated stress fibers. Knockdown of cofilin restored the cell morphology and division, and reduced the mitotic catastrophe induced by Fasudil.
Conclusions:
Both ROCK1 and ROCK2 are required for HCC cell division and growth. Targeting ROCK1 or ROCK2 rather than both can serve as a potential approach for HCC treatment and may reduce the side effects.
Insights
Rho-associated kinases ROCK1 and ROCK2 are vital for hepatocellular carcinoma (HCC) cell division and growth. Targeting either ROCK1 or ROCK2 offers a promising therapeutic strategy for HCC, potentially minimizing side effects.
Area of Science:
- Oncology
- Cell Biology
- Molecular Medicine
Background:
- Rho-associated kinases (ROCK1 and ROCK2) are implicated in cardiovascular disease but their role in cancer, specifically hepatocellular carcinoma (HCC), remains unclear.
- Investigating the necessity of ROCK1/ROCK2 for HCC cell survival and growth is crucial for understanding carcinogenesis.
- Understanding the underlying mechanisms of ROCK1/ROCK2 in HCC is essential for developing targeted therapies.
Purpose of the Study:
- To determine if ROCK1 or ROCK2 are essential for the survival and proliferation of hepatocellular carcinoma (HCC) cells.
- To elucidate the molecular mechanisms by which ROCK1/ROCK2 influence HCC progression.
- To evaluate the therapeutic potential of targeting ROCK1/ROCK2 in HCC models.
Main Methods:
- ROCK1 and ROCK2 expression levels were quantified in HCC tissues and cell lines via qRT-PCR, western blotting, and IHC.
- Cell growth, proliferation, cell cycle, and apoptosis were assessed using CCK-8, EdU, and flow cytometry.
- The in vitro and in vivo effects of ROCK1/ROCK2 inhibition (using Fasudil) on HCC were evaluated in mouse models, with detailed observation of cell division and mitosis.
Main Results:
- ROCK1 and ROCK2 were found to be overexpressed in human HCC tissues and cell lines.
- Inhibition of ROCK1 or ROCK2, either by knockdown or pharmacological treatment with Fasudil, significantly suppressed HCC cell growth and survival.
- Fasudil induced cell cycle arrest and mitotic catastrophe in HCC cells, characterized by abnormal mitosis and disrupted stress fibers, without inducing apoptosis.
Conclusions:
- ROCK1 and ROCK2 play critical roles in hepatocellular carcinoma cell division and overall growth.
- Targeting individual ROCK1 or ROCK2 pathways presents a potential therapeutic strategy for HCC, possibly with fewer side effects than targeting both.
- Further research into selective ROCK1/ROCK2 inhibition could lead to novel HCC treatments.
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