In the present study with human HCC cell lines we provide evidence that a well standardized aqueous extract from wood and bark of L.obtusiloba exerts direct and non-direct anti-neoplastic effects via attenuated IGF-1R- and NF-κB-signaling.
Initially, we examined the effects of a standardized active extract of L.obtusiloba on the proliferation of well characterized human HCC cell lines with poorly differentiated SK-Hep1 considered more aggressive than the other three used. L.obtusiloba extract blocked the growth of the HCC cells in a dose dependent manner with a physiologically relevant IC50 of ~100 μg/ml (Figure 1A) [31, 32]. In addition, L.obtusiloba extract inhibited tumor cell invasion (Figure 1C). Here, SK-Hep1 cells rather than the well differentiated HepG2, Hep3B and Huh-7 cells were more sensitive to L.obtusiloba extract. Thus, in conjunction with the induction of apoptosis in all four cell lines (Figure 1B), L.obtusiloba extract exerts three primary prerequisites for the treatment of cancer [35, 36].
Aberrant growth and apoptosis regulation in carcinogenesis is mediated by growth factor receptors such as IGF-1R which therefore represents an attractive therapeutic target [8, 37] and all of the four cell lines investigated are known to express the IGF-1R . Since HCC is characterized by strong neo-angiogenesis  with VEGF as its main mediator we investigated the upstream IGF-1/IGF-1R signal transduction and the expression of VEGF via induction of HIF-1α . L.obtusiloba extract blocked the basal and IGF-1-induced protein expression of HIF-1α and VEGF accompanied by decreased phosphorylation of Akt, Stat3 and Erk. (Figure 2, Tables 1, 2, 3). Since a forced activation of Akt, Stat3 and Erk was shown to protect from apoptosis and to induce VEGF expression [40, 41], our results suggest that a decreased activation of the IGF-1/IGF-1R-axis due to L.obtusiloba extract treatment contributes to its apoptosis-inducing effects and might be a reason for the reduced expression of VEGF and HIF-1α in HCC cells treated with L.obtusiloba extract [11, 12]. These findings are in accordance with studies using extracts from green tea describing a decreased expression of VEGF and HIF-1α accompanied by a block of PI3K/Akt-signaling in HCC cells .
IGF-1R signaling also impacts the expression of the transcription factor PPARγ which in turn modulates the expression of other angiogenesis-regulating proteins like COX-2 and iNOS. The implication of PPARγ in carcinogenesis is still debated. Some data show anti-tumor effects of PPARγ ligands. However, these effects could also be independent of PPARγ activation and in addition the usage of PPARγ antagonists also exerts anticancer effects . In contrast to PPARγ, several studies clearly show a positive correlation between the expression of COX-2 and iNOS and HCC progression, e.g. indicated as enhanced microvessel density in HCC . While COX-2 impacts growth and progression of HCC and its inhibition suppressed HCC-associated angiogenesis in vitro and in vivo , iNOS is a key enzyme in generating nitric oxide, thus modulating tumorigenesis by regulating tumor cell proliferation, survival and migration, as well as angiogenesis, drug resistance and DNA repair [5, 46].
In line with previous reports [47, 48], L.obtusiloba extract reduced the expression of COX-2 and iNOS (Table 2). Notably, poorly differentiated SK-Hep1 cells were susceptible to IGF-1 and inhibition of IGF-1 by L.obtusiloba extract. A similar result was obtained for the expression of PPARγ (Table 2). We therefore conclude that downregulation of COX 2 and iNOS by L.obtusiloba extract is mediated by diminished expression of PPARγ.
Beside PPARγ, IGF-R-signaling, through different upstream pathways, could trigger the activation of the transcription factor NF-κB  which likewise regulates COX-2 and iNOS and plays a role in viral hepatitis, chronic liver disease including fibrosis and cirrhosis and in HCC [24, 50] and is spontaneously activated in HCC cells . Inhibition of NF-κB reduced proliferation and invasion as well as expression of VEGF in HCC cells and sensitized the cells to sorafenib induced cell death .
As shown in Figure 3, L.obtusiloba extract markedly reduced the transcriptional activity of NF-κB in Hep3B, Huh-7 and SK-Hep1 cells and to a lesser extent in HepG2 cells. Thus, downregulation of COX-2 and iNOS by L.obtusiloba extract is mediated by diminished expression of PPARγ and due to a reduced transcriptional activity of NF-κB. Since NF-κB activity supports cell survival or entails anti-apoptotic effects [23, 24, 49], the inhibition of NF-κB by L.obtusiloba extract might contribute to the apoptosis inducing effects of the extract in the cancer cells (Figure 1B).
In summary, our findings in vitro strongly suggest L.obtusiloba extract as a specific compound to suppress tumor cell growth and migration and to induce apoptosis in aggressive, poorly differentiated human tumor cells via attenuation of NF-κB transcriptional activity and IGF-1R signaling. Further, the expression of key proteins in regulation of angiogenesis was reduced due to L.obtusiloba extract treatment. Due to its good physiological compatibility, in Korea L.obtusiloba extract is traditionally applied in humans to treat chronic inflammatory diseases of the liver . Thus, our in vitro results are in line with and add more scientific strength to the traditional use of L.obtusiloba extract in treatment for chronic liver disease including HCC.
Regarding biologically active compounds in the extract several studies describe the isolation and structural characterization of drugs from Lindera obtusiloba [29, 30, 52]. In this line, preliminary data of us suggest that lignans such as sesamin or episesamin might contribute to the anti-fibrotic and anti-tumor effects of L.obtusiloba extract (not shown).
Complemental to the anti-fibrogenic, anti-inflammatory and anti-adipogenic efficacy of L.obtusiloba extract [31, 32], our results suggest the use of an inflammation-associated tumor model of HCC to assess all aspects of the anti-tumor effects of L.obtusiloba extract in vivo.