HCC typically arises on the basis of cirrhosis and responds poorly to conventional cytotoxic chemotherapy. The latter is often poorly tolerated by a liver that has been damaged by chronic viral or ethanol exposure. This has led to a search for novel approaches to therapy, including the targeting of the EGF receptor (erlotinib), VEGF (bevacizumab) or the VEGF receptor (Sorafenib, Sunitinib), and the convergent RAF/MEK/ERK pathways by new therapeutic agents. The RAF/MEK/ERK cascade is one of the principal RAS-regulated pathways. Raf expression has been reported to be increased in human HCC (36
) and Sorafenib was synthesized to molecularly target RAF in this vital pathway, and has been shown to have antitumor activity against renal cell cancer (37
) and HCC (35
). Sorafenib seems less effective for treating other types of cancer, although preliminary data have shown some activity for Sorafenib in pancreas cancer (8
) and some other tumor types (9
). However, it also has many toxicities (40
) and a variable but large percent of patients need to be dose-reduced or stop taking the drug for this reason. This was the stimulus for our search for agents that could be combined with Sorafenib to enhance its HCC growth-inhibitory actions, given that it can enhance overall survival in HCC patients, albeit by only a few weeks (10
). Given that vitamin K1 appears to be without toxicity in adult humans and that vitamin K can enhance HCC cell PKA phosphorylation, and phospho-PKA in turn has been shown to phosphorylate and modulate Raf activity in HCC cells (26
), we examined whether these 2 agents might have additive or super-additive effects on HCC cell growth.
We previously found preliminary evidence that addition of vitamin K1 could enhance Sorafenib-mediated apoptosis in HCC cells in vitro (43
). The present studies examined this combination in greater detail and begin to elucidate the mechanism(s) responsible for this super-additive phenomenon. We show here for the first time, that combination of vitamin K1 with low and clinically relevant concentrations of Sorafenib inhibited growth and induced apoptosis in several human HCC cell lines in vitro and in a rodent HCC cell line, both in vitro and in vivo. This finding has clinical possibilities, as it suggests that the combination might be a candidate for clinical application, either to permit use of lower and less toxic Sorafenib doses, or to add to standard Sorafenib doses in order to enhance clinical responses, that have so far been meager (10
Prior studies have shown that Sorafenib in the 10 μmol/L range, which is at pharmacologically achievable concentrations, induced cell death in human leukemic cells (44
), HCC cells (45
) and pancreatic cancer cells (46
). The results of the present study, show that low concentrations of Sorafenib (2.5 μM) or vitamin K1 (50 μM) when used as a single agent, did not cause growth inhibition or apoptosis However, treatment of HCC cells with low concentrations of both Sorafenib (2.5 μM) plus vitamin K1 (50 μM) resulted in cell growth inhibition and caused apoptosis (-), as well as significantly inhibiting the phosphorylation of ERK (). Therefore, vitamin K1 seems to add to Sorafenib inhibition of the MEK/ERK pathway. Apoptosis induced by Sorafenib plus vitamin K1 was caspase-dependent, since pre-treatment with pan-caspase inhibitor could dramatically block the apoptosis induced by Sorafenib plus vitamin K1. The apoptotic signaling pathways are generally divided into two types: the extrinsic, or death receptor pathway and the intrinsic or mitochondrial pathway (47
). The extrinsic pathway involves cell surface death receptors, such as tumor necrosis factor or Fas, which upon binding of their ligands, initiate signaling to activate caspase-8, which cleaves caspase-3 directly to induce apoptosis. The intrinsic pathway involves mitochondrial changes and triggers the release of cytochrome C, which in turn activates caspase-9 and then caspase-3 (48
). Our findings suggest that a central mechanism of Sorafenib plus vitamin K1 mediated apoptosis in HCC cells, involves activation of the extrinsic apoptosis pathway.
It was recently reported that activated ERK plays an important role in controlling levels of the anti-apoptotic protein Mcl-1 (33
). Here, we demonstrated that actions of Sorafenib or vitamin K1 (at very high concentrations) alone can result in decreased phospho-ERK levels, but vitamin K1 at low concentrations significantly added to low and minimally effective concentrations of Sorafenib to result in inhibition of the MEK/ERK pathway, a decrease in Mcl-1 levels and induction of apoptosis. Sorafenib alone at higher concentrations could cause a decrease in Mcl-1 levels, but much lower concentrations of Sorafenib also caused this, when vitamin K1 was present ( and ).
Tumors were grown in the animal livers after injection into the mesenteric vein under direct visualization. Vitamin K1 alone had minimal effects at the doses used. As reported for HCC in other models (39
) Sorafenib could inhibit tumor growth. However, when sub-effective doses of Sorafenib were used, addition of vitamin K was able to cause complete disappearance of tumors, as judged both direct inspection of the excised livers () and on microscopic examination (). Furthermore, we found that the tumors from treated animals had decreased staining for both phospho-ERK and Mcl-1, consistent with the western blot results from treated cells in culture.
There mechanisms for the growth-inhibitory effects of vitamin K1 are not yet clear. We considered this combination after reports that K vitamin actions can result in phosphorylation and enhancement of PKA activity (20
). Since PKA has been shown to mediate inhibitory phosphorylation of Raf, the key target of Sorafenib, it seemed possible that the combination might cause increased growth inhibition by each agent inhibiting Raf through different mechanisms: Sorafenib as a direct Raf inhibitor and vitamin K1 by inducing inhibitory Raf phosphorylation. Preliminary evidence is presented for the latter.
In conclusion, our data indicates that: 1) combination Sorafenib plus vitamin K1 can decrease the concentrations of Sorafenib that were needed to inhibit HCC cell growth and induce apoptosis; 2) vitamin K1 enhanced Sorafenib- mediated inhibition of the MEK/ERK pathway and activation of caspase pathway; 3) the possible mechanisms involved in Sorafenib plus vitamin K1 induced apoptosis of HCC cells involve the caspase-dependent extrinsic apoptotic pathway, likely via inhibition of Mcl-1; 4) Sorafenib-mediated tumor cell growth inhibition in vivo was enhanced by the non toxic vitamin K1.