Pancreas cancer is often a systemic disease by virtue of its metastases, which respond poorly to conventional cytotoxic chemotherapy. This has led to a search for novel approaches to therapy, including the targeting of the EGF receptor (Erlotinib) and the RAF/MEK/ERK pathways by new therapeutic agents. The RAF/MEK/ERK cascade, one of the principal RAS-regulated pathways, is hyper-stimulated in pancreas cancers with activating K-ras mutations. Sorafenib, with its molecular targeting on RAF in this vital pathway, has been shown to have antitumor activity against renal cell cancer (25
) and HCC (27
). However, Sorafenib is less effective for treating other types of cancer, although preliminary data have shown some activity for Sorafenib in pancreas cancer also (16
). This was the stimulus to our search for agents that could be combined with Sorafenib to enhance its pancreas cancer growth-inhibitory actions.
We previously showed that combination of vitamin K1 with Sorafenib could sensitize HCC cell lines to Sorafenib actions and enhance Sorafenib-mediated apoptosis in vitro and in vivo (29
). The present studies attempted to determine whether Sorafenib plus vitamin K1 could also result in enhanced cell growth inhibition in pancreas cancer cell lines and, if so, to elucidate the mechanism(s) responsible for this 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 pancreas cancer cells in vitro. This finding has clinical implications, as it suggests that the combination might be a candidate for therapy in pancreas cancer patients, especially since each component is currently used in patients in other settings.
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 (30
), HCC cells (28
) and pancreatic cancer cells (32
). 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 induce growth inhibition or apoptosis or inhibit phosphorylation of MEK/ERK, although high doses of either can inhibit phospho-ERK levels and increase phospho-c-Jun levels (). However, treatment of pancreas cancer cells with low concentrations of both Sorafenib (2.5 µM) plus vitamin K1 (50 µM) resulted in cell growth inhibition and apoptosis ( and ), as well as significantly inhibiting the phosphorylation of MEK and ERK (). Therefore, vitamin K1 seems to add to Sorafenib in inhibiting the MEK/ERK pathway. The 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 (33
). 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 (34
). Our findings suggest that one central mechanism of Sorafenib plus vitamin K1 mediated apoptosis in pancreas cancer cells, involves activation of the extrinsic apoptosis pathway.
Fas signaling plays a fundamental role in induction of extrinsic apoptosis in a wide variety of different types of cancer cell (35
). Increased expression of FasL signal transduction activates caspase-8 and triggers apoptosis. Fas ligand has been shown to be a target gene for c-Jun, primarily in lymphoid and neuronal cell types (37
) and there are recent reports on c-Jun transcriptional regulation of Fas gene expression (38
). Another interesting aspect of our studies is that we show the importance of JNK, c-Jun and Fas signaling in Sorafenib plus vitamin K induced apoptosis. C-Jun NH2-terminal kinase (JNK) belongs to the superfamily of MAP kinases that are involved in the regulation of cell proliferation, differentiation and apoptosis. The activation of JNK leads to apoptosis, which is dependent on the stimuli and cell type involved in such activation (40
). Prolonged activation of JNK has been observed in human breast, gastric, prostate and ovarian cancer cells with apoptosis-inducing compounds. JNK has also been recognized as a key mediator of a variety of cell fates (42
) and was initially identified as a protein kinase involved in the transactivation of c-Jun by phosphorylation (43
) and activated apoptotic signaling through the up-regulation of pro-apoptotic genes through the transactivation of specific transcription factors such as c-Jun. FasL has been shown to be a target gene for c-Jun primarily in lymphoid neuronal cell type (36
). Similarly, we found that Sorafenib plus vitamin K1 activated JNK and increased phosphorylation of c-Jun and increased FasL levels. Transcriptional regulation of Fas ligand has also been observed in ovarian cancer cells undergoing apoptosis following cisplatin treatment (44
). Furthermore, our data showed that a JNK inhibitor reduced the phosphorylation levels of JNK and c-Jun and decreased the levels of FasL that were induced by Sorafenib plus vitamin K1 (). The apoptosis induced by Sorafenib plus vitamin K1 was also partially antagonized by the JNK inhibitor, supporting the idea that the increase in the JNK levels that were induced by our combination, were involved in their growth-inhibitory mechanisms. On the other hand, other signaling pathways also might contribute to the cell growth inhibition and apoptosis which were induced Sorafenib plus vitamin K1.
Recently, it has been found that FasL mediated death signal is abrogated by the mitogen-activated protein kinase (MAPK/ERK)-mediated signals (45
). In the FasL-mediated apoptosis process, ERK activity inhibits the caspase-8 cleavage (46
). This strongly suggests that MEK/ERK-mediated signals can suppress apoptosis and the source of this anti-apoptotic activity may exist at the level of caspase-8 activation. Here, we demonstrated that Sorafenib or vitamin K1 alone can inhibit phospho-MEK and phosphor-ERK levels, when used at high concentrations, but vitamin K1 at low concentrations significantly added to Sorafenib-mediated inhibition of the MEK/ERK pathway and induction of apoptosis via the extrinsic pathway. After treatment with the MEK inhibitor U0126 alone, we found the expected inhibition of phospho-ERK levels, but also found augmented activity of caspase-8 (). Thus, the combination of Sorafenib plus vitamin K1 resulted in the simultaneous decrease in levels of phospho-MEK and phospho-ERK MAPKs, while increasing the levels of phospho-JNK MAPK and its downstream targets, phospho-c-Jun and FasL. Simultaneous and opposite effects on different MAPKs have been previously reported with different agents (47
). The mechanisms underlying that selectivity were not elucidated. These opposite effects, which both result in enhanced apoptosis, are summarized in .
The mechanism of combination Sorafenib and vitamin k1 seems to involve inhibition of MEK/ERK activity and activation of JNK/c-Jun. Modulation of both of these pathways results in activation of caspase-8, ultimately leading to apoptotic cell death.
In conclusion, our data indicates that: 1) combination Sorafenib plus vitamin K1 can decrease the concentrations of Sorafenib that were needed to inhibit pancreas cancer cell growth and induce apoptosis; 2) vitamin K1 seems to be additive with Sorafenib in inhibiting the MEK/ERK pathway and activating caspase activity; 3) the possible mechanisms involved in Sorafenib plus vitamin K1 induced apoptosis of pancreas cancer cells involve both caspase-dependent extrinsic, as well as JNK/c-Jun-dependent upregulation of FasL-mediated apoptosis.