Sorafenib inhibits lymphoma xenografts by targeting MAPK/ERK and AKT pathways in tumor and vascular cells

Abstract

The anti-lymphoma activity and mechanism(s) of action of the multikinase inhibitor sorafenib were investigated using a panel of lymphoma cell lines, including SU-DHL-4V, Granta-519, HD-MyZ, and KMS-11 cell lines. In vitro, sorafenib significantly decreased cell proliferation and phosphorylation levels of MAPK and PI3K/Akt pathways while increased apoptotic cell death. In vivo, sorafenib treatment resulted in a cytostatic rather than cytotoxic effect on tumor cell growth associated with a limited inhibition of tumor volumes. However, sorafenib induced an average 50% reduction of tumor vessel density and a 2-fold increase of necrotic areas. Upon sorafenib treatment, endothelial and tumor cells from SU-DHL-4V, Granta-519, and KMS-11 nodules showed a potent inhibition of either phospho-ERK or phospho-AKT, whereas a concomitant inhibition of phospho-ERK and phospho-AKT was only observed in HD-MyZ nodules. In conclusion, sorafenib affects the growth of lymphoid cell lines by triggering antiangiogenic mechanism(s) and directly targeting tumor cells.

Figure 1. Antiproliferative and apoptotic effects of sorafenib.

Following incubation with increasing doses of sorafenib, viable cell counts and apoptotic cell death were assessed by annexin-V/PI double staining and flow cytometry analysis. (A) Viable cell counts upon exposure to 0 (▪), 5 (□) and 10 (•) µM sorafenib. Viable cells are expressed as fold increase (FI) of annexin-V⁻/PI⁻ cells after 24 and 48 hours of incubation with sorafenib as compared to day 0. (B) Cell death following 24 (□) or 48 (▪) hours sorafenib exposure. Percentages of cell death include annexin-V⁺/PI⁻ plus annexin-V⁺/PI⁺ plus annexin-V⁻/PI⁺ cells. Values refer to four independent experiments. * p≤0.001, ° p≤0.01, and ≠ p≤0.05, compared to controls.

Figure 2. Mechanism of sorafenib-induced apoptosis.

SU-DHL-4V, Granta-519, HD-MyZ and KMS-11 cells were treated with sorafenib (10 µM) for 48 hours. (A) Cytosolic proteins were then separated by SDS-PAGE and analyzed by immunoblotting with anti-caspases-8, -9, -3, and anti-PARP. CF, indicates cleaved fragments. (B) Loss of mitochondrial potential was measured using TMRE staining and flow cytometry. * p≤0.001, compared to controls. (C) Representative dot plots of mitochondrial membrane depolarization in untreated and sorafenib-treated cell lines.

Figure 3. Sorafenib treatment induced changes in survival signals in NHL cells.

(A) To assess the effects of sorafenib, cells were exposed for 2 hours to control vehicle (DMSO) or sorafenib (10 µM). Shown are data from one of two independent experiments. The chemiluminescence signal intensity of individual spots was analyzed using the open source imaging software ImageJ (http://rsb.info.nih.gov/ij/). The assays were conducted in duplicate. A ratio of signal intensity (sorafenib∶control) was calculated for each of the four replicates (two duplicates per assay) and transformed into a log value (base 10). * p≤0.001, and ° p≤0.01, compared to controls. (B) Shown are data from one of two independent experiments. Representative proteome profiles of control and sorafenib-treated cell lines. The position of the antibodies (double spots for each antibody) relative to the relevant protein kinases is shown. The chemiluminescence signal intensity of individual spots was analyzed using the open source imaging software ImageJ (http://rsb.info.nih.gov/ij/). 1 = positive control; 2 = p70 S6 kinase; 3 = Akt pan; 4 = Akt 2; 5 = Akt 1; 6 = GSK-3 α/β; 7 = p38 α; 8 = JNK pan; 9 = ERK2; and 10 = ERK1. (C) Immunoblots of extracts from SU-DHL-4V, Granta-519, HD-MyZ and KMS-11 cells treated with control vehicle (DMSO) or sorafenib (10 µM) for the indicated time periods showed consistent downregulation of p-38α phospho-ERK, phospho-MEK, phospho-Akt, phospho-S6, phospho- GSK-3 α/β and Mcl-1. Equal protein loading was confirmed by blotting for β-actin.

Figure 4. Effect of sorafenib on tumor growth.

(A) NOD/SCID mice bearing SC tumor nodules 100 mg in weight were randomly assigned to receive a 15-day treatment with sorafenib (▪) (90 mg/kg/day, 5 days per week over 3 weeks) or control vehicle (DMSO) (•). Mice were checked twice weekly for tumor appearance, tumor dimensions, body weight, and toxicity. Mean (± SEM) values refer to at least two independent experiments, using 5 mice per experiment. Treatment initiation is indicated by horizontal black lines. * p≤0.001 and ° p≤0.01 compared to controls. (B) Ki-67 staining of lymphoma tumor treated with sorafenib (90 mg/kg/day, 5 days) or control vehicle (DMSO). In the Ki-67-stained section, brown staining represents positive signals within the tumors (blue cells are the negative, living cells). Objective lens, original magnification: 0.75 NA dry objective, 20×. Scale bar: 50 µm.

Figure 5. Effect of sorafenib on tumor vasculature.

(A) Mice treated with sorafenib (90 mg/kg) or control vehicle (DMSO) were in vivo biotinylated with sulfo-NHS-LC-biotin, and tumor vasculature was revealed by staining sections with Alexa Fluor 488-streptavidin (upper panel, objective lens, original magnification: 1.0 NA oil objective, 40×). CD31-stained tumor paraffin sections are shown for comparison (lower panel, objective lens, original magnification: 0.75 NA dry objective, 20×). (B) Quantification of endothelial area on entire tissue sections was achieved with ImageJ software. The reduction of vessel density detected in sorafenib-treated nodules was calculated by assessing only viable areas of tissue sections while excluding necrotic areas. The boxes extend from the 25^th to the 75^th percentiles, the lines indicate the median values, and the whiskers indicate the range of values.

Figure 6. Effects of sorafenib on pericytes.

Mice treated with sorafenib (90 mg/kg/die, 5 days) or control vehicle (DMSO) were in vivo biotinylated with sulfo-NHS-LC-biotin. SU-DHL-4V, Granta-519, HD-MyZ and KMS-11 tumor vasculature was revealed by staining sections with Alexa Fluor 568-streptavidin (red). Tumor sections were stained with NG-2 (green) followed by AlexaFluor 488-conjugated secondary antibody for indirect immunofluorescent detection of pericytes. Nuclei were detected with DAPI nuclear dye (blue). Representative images are shown. Objective lens, original magnification: 1.0 NA oil objective, 40×. Scale bar: 100 µm.

Figure 7. Sorafenib-induced inhibition of Akt and ERK phosphorylation in tumor and endothelial cells.

SU-DHL-4V, Granta-519, HD-MyZ and KMS-11 tumor nodules growing subcutaneously in mice treated with sorafenib (90 mg/kg) or control vehicle (DMSO) for 5 days were excised 3 hours after the last treatment, fixed in formalin and embedded in paraffin. Tumor sections were double-stained with CD31 (red) and phospho-ERK 1/2 (green) or phospho-Akt (green) followed by the appropriate AlexaFluor 568- or 488-conjugated secondary antibody for indirect immunofluorescent detection of the corresponding antigen. Nuclei were detected with TO-PRO-3 nuclear dye (blue). Arrows indicate phospho-ERK 1/2 or pospho-Akt expression by endothelial cells; arrowheads indicate phospho-ERK 1/2 or pospho-Akt expression by tumor cells. Representative images are shown. Objective lens, original magnification: 1.0 NA oil objective, 40×. Scale bar: 50 µm.

Figure 8. Sorafenib induces in vivo tumor necrosis.

(A) H&E and TUNEL staining of lymphoma tumor treated with sorafenib (90 mg/kg) or control vehicle (DMSO). In the TUNEL-stained section, brown staining represents positive signals within the tumors (blue cells are the negative, living cells). Objective lens, original magnification: 0.08 NA dry objective, 2×. (B) Quantification of necrotic areas on entire tissue sections using ImageJ software. Percentage of necrosis was calculated by the following formula: (necrotic area/total tissue area)×100. The boxes extend from the 25^th to the 75^th percentiles, the lines indicate the median values, and the whiskers indicate the range of values.