Antitumor Activity of Simvastatin in Preclinical Models of Mantle Cell Lymphoma

Abstract

Background: Mantle cell lymphoma (MCL) is a rare and aggressive subtype of B-cell non-Hodgkin lymphoma that remains incurable with standard therapy. Statins are well-tolerated, inexpensive, and widely prescribed as cholesterol-lowering agents to treat hyperlipidemia and to prevent cardiovascular diseases through the blockage of the mevalonate metabolic pathway. These drugs have also shown promising anti-cancer activity through pleiotropic effects including the induction of lymphoma cell death. However, their potential use as anti-MCL agents has not been evaluated so far.

Aim: The present study aimed to investigate the activity of simvastatin on MCL cells.

Methods: We evaluated the cytotoxicity of simvastatin in MCL cell lines by CellTiter-Glo and lactate dehydrogenase (LDH) release assays. Cell proliferation and mitotic index were assessed by direct cell recounting and histone H3-pSer10 immunostaining. Apoptosis induction and reactive oxygen species (ROS) generation were evaluated by flow cytometry. Cell migration and invasion properties were determined by transwell assay. The antitumoral effect of simvastatin in vivo was evaluated in a chick embryo chorioallantoic membrane (CAM) MCL xenograft model.

Results: We show that treatment with simvastatin induced a 2 to 6-fold LDH release, inhibited more than 50% of cell proliferation, and enhanced the caspase-independent ROS-mediated death of MCL cells. The effective impairment of MCL cell survival was accompanied by the inhibition of AKT and mTOR phosphorylation. Moreover, simvastatin strongly decreased MCL cell migration and invasion ability, leading to a 55% tumor growth inhibition and a consistent diminution of bone marrow and spleen metastasis in vivo.

Conclusion: Altogether, these data provide the first preclinical insight into the effect of simvastatin against MCL cells, suggesting that this agent might be considered for repurpose as a precise MCL therapy.

Keywords: cell death; cell proliferation; mantle cell lymphoma; statins.

Figure 1

MCL cytotoxicity, proliferation impairment and cell death induced by simvastatin. (A) Cell viability of MCL cell lines in the presence or absence of different doses of simvastatin; (B) cytotoxicity rates assessed by LDH release of MCL cell lines in presence or absence of 5 μM, 10 μM and 20 μM simvastatin; (C) Western blot evaluation of phospho-AKT and phospho-mTOR levels after the treatment with 5 μM, 10 μM and 20 μM simvastatin, or vehicle. Full western blot images can be seen in Figure S4. (D) Proliferation rate assessed by MCL cell counting after the treatment with increasing doses of simvastatin; (E) proliferation rate assessed by phospho-Histone 3 (red) immunofluorescence of MCL cell lines in the presence or absence of 5 μM, 10 μM or 20 μM simvastatin. Nuclei were counterstained with DAPI (blue); (F) mitochondrial transmembrane potential (ΔΨm) after cell treatment with 5 μM, 10 μM or 20 μM simvastatin or vehicle; (G) apoptosis rate measured by AnnexinV-FITC/PI staining after cell treatment with 5 μM, 10 μM or 20 μM simvastatin or vehicle, previously treated or not with 10 μM of the pan-caspase inhibitor, Q-VD-OPh hydrate; (H) ROS generation measured by DHE (dihydroethidium) staining in MCL cells lines treated with 5 μM, 10 μM or 20 μM simvastatin, or vehicle. Values are expressed as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, when compared to control group.

Figure 2

MCL migration and invasion ability, tumor growth and metastasis inhibited by simvastatin treatment. (A) Cell migration index of MCL cell lines exposed to 10 μM of simvastatin or vehicle for 24 h; (B) cell invasion index of MCL cell lines exposed to 10 μM simvastatin or vehicle for 24 h; (C) scheme depicting the chick embryo chorioallantoic membrane (CAM) model; (D) representative pictures of engrafted MCL tumors treated with 10 μM simvastatin or vehicle on day 16 of embryonic development. The dotted line delimitates the tumor; (E) tumor weight on day 16 of embryonic development after the treatment with 10 μM simvastatin or vehicle; (F) H&E and immunohistochemical (IHC) detection of CD20 and H3-pSer10 in tissue sections from CAM-tumor specimens dosed with 10 μM simvastatin or vehicle; (G) metastasis evaluation measured by qPCR-based Alu-sequence presence into embryo’s bone marrow and spleen. Values are expressed as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, when compared to the control group.