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, 127 (12), 2949-58

Vitamin K Enhancement of Sorafenib-Mediated HCC Cell Growth Inhibition in Vitro and in Vivo

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Vitamin K Enhancement of Sorafenib-Mediated HCC Cell Growth Inhibition in Vitro and in Vivo

Gang Wei et al. Int J Cancer.

Abstract

The multikinase inhibitor sorafenib is the first oral agent to show activity against human hepatocellular carcinoma (HCC). Apoptosis has been shown to be induced in HCC by several agents, including sorafenib as well as by the naturally occurring K vitamins (VKs). As few nontoxic agents have activity against HCC growth, we evaluated the activity of sorafenib and VKs, both independently and together on the growth of HCC cells in vitro and in vivo. We found that when VK was combined with sorafenib, the concentration of sorafenib required for growth inhibition was substantially reduced. Conversely, VK enhanced sorafenib effects in several HCC cell lines on growth inhibition. Growth could be inhibited at doses of VK plus sorafenib that were ineffective with either agent alone, using vitamins K1, K2 and K5. Combination of VK1 plus sorafenib induced apoptosis on FACS, TUNEL staining and caspase activation. Phospho-extracellular signal-regulated kinase (ERK) levels were decreased as was myeloid cell leukemia sequence 1 (Mcl-1), an ERK target. Sorafenib alone inhibited growth of transplantable HCC in vivo. At subeffective sorafenib doses in vivo, addition of VK1 caused major tumor regression, with decreased phospho-ERK and Mcl-1 staining. Thus, combination of VK1 plus sorafenib strongly induced growth inhibition and apoptosis in rodent and human HCC and inhibited the RAF/mitogen-activated protein kinase kinase/ERK pathway. VK1 alone activated PKA, a mediator of inhibitory Raf phosphorylation. Thus, each agent can antagonize Raf: sorafenib as a direct inhibitor and VK1 through inhibitory Raf phosphorylation. As both agents are available for human use, the combination has potential for improving sorafenib effects in HCC.

Figures

Figure 1
Figure 1. Inhibition of HCC cell growth by Sorafenib plus vitamin K
A, Growth inhibition of PLC/PRF/5 cells by Sorafenib 2.5 μM, VK1 or the combination (upper graph). The inset shows the growth inhibitory actions of each agent alone. The lower graph shows the growth inhibitory effects of vitamin K1 addition to various Sorafenib concentrations. B. Growth inhibition of Hep 3B, Hep G2, Hep 40 and JM1 cells by Sorafenib, VK1 or the combination. C. Inhibition of PLC/PRF/5 cell growth by Sorafenib, with or without VK1, VK2 or VK5. Each experimental point was done in triplicate, repeated three times and expressed mean SD. P value was determined by student’s t test. D. Normalized isobologram. The combination of varying concentrations of Sorafenib and a fixed concentration of VK1 of 50 μM. Calculations as described in Refs and .
Figure 2
Figure 2. Induction of apoptosis by combination Sorafenib plus vitamin K1
A: TUNEL staining. PLC/PRF/5 cells were treated with vitamin K1 (50 μM), Sorafenib (2.5 μM), or combination vitamin K1 (50 μM) plus Sorafenib (2.5 μM), or pre-treated with caspase inhibitor for 2 hr and then incubated with vitamin K1 plus Sorafenib. TUNEL-stained cells were observed at 40X magnification. B. PLC/PRF/5 cells were treated under the same conditions as in A, above. Floating and adherent cells were harvested at 36 hours and analyzed by flow cytometry. C: Quantitation of cell death in Fig. 2B.
Figure 3
Figure 3. Activation of the extrinsic caspase death pathway by combination Sorafenib plus vitamin K1
A: Cleaved PARP and caspase-3 detection 36 hr after treatment with vitamin K1 (50 μM) plus Sorafenib (2.5 μM) in PLC/PRF/5 cells; but no cleavage was observed after vitamin K1 or low concentrations of Sorafenib alone. B: Combination treatment with Sorafenib plus vitamin K1 did not effect on caspase-9 activity, but procaspase-8 was activated in the same lysates. C: Sorafenib plus vitamin K1 decreased the expression of Mcl-1. Cells were lysed and 20 μg of soluble protein were separated by electrophoresis by SDS-PAGE gel electrophoresis. Protein levels were detected by Western blot analysis. Actin was used as loading control.
Figure 4
Figure 4. Inhibition of phospho-ERK and induction of apoptosis
A: Combination treatment with Sorafenib plus vitamin K1 decreased phospho-ERK levels in PLC/PRF/5 hepatoma cell. B: Low concentration Sorafenib (2.5 μM) plus vitamin K1 (50 μM) inhibited phospho-ERK and Mcl-1 levels. Cells were isolated after 36 hours treatment and subjected to SDS-PAGE gel electrophoresis, followed by immunoblotting to determine protein expression.
Figure 5
Figure 5. Cellular effects of Sorafenib or vitamin K1 alone
A: Concentration-dependent inhibition of phospho-ERK and Mcl-1 levels by Sorafenib. B: Vitamin K1 effects on phospho-ERK, phospho-PKA, phospho-Raf (ser43) and phospho-Raf (ser259) levels. C: PKA inhibitor (H-89) antagonizes vitamin k1 induced phophorylation of PKA and Raf. Cells were lysed after 36 hours treatment and 20 μg of soluble protein were separated by electrophoresis by SDS-PAGE gel electrophoresis. Protein levels were detected by Western blot analysis.
Figure 6
Figure 6. Inhibition of HCC tumor growth in vivo by Sorafenib plus vitamin K1
A: After injection of JM1 hepatoma cells into the liver via a portal vein tributary, rats were treated with either solvent control, vitamin K1, Sorafenib or combination vitamin K 1 plus Sorafenib. They were then sacrificed and whole livers were isolated as described in materials and methods. A tiny tumor in combination group is labeled with arrow. B: Representative histology and histochemistry slides from the tumors and livers stained by H and E and for phospho-ERK , Mcl-1 and Phospho-PKA. Original magnification × 10. C: The in vivo JM-1 cell transplantable hepatoma experiment was divided into 4 groups, (1) vehicles, (2) Sorafenib (3) Vitamin k1 and (4) combined Sorafenib/Vitamin K1 treatment. The compounds were injected intra-peritoneally daily for 8 consecutive days. Three days after the last injection, the animals were sacrificed and the tumors were excised. The ratio of total weight of the tumors and liver from each rat liver was counted. The results are expressed as the mean ± the standard deviation.

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