In this study we investigated the apoptotic potential of the whole mistletoe extract viscumTT in comparison to its constituents viscum and TT. ViscumTT very effectively inhibited cell proliferation and synergistically induced apoptosis in osteosarcoma cells in vitro in a time- and dose-dependent manner. Our in vitro data correlate with the assumption that whole plant extracts are often more potent in anti-cancer activity than the single ingredients [
32,
33]. The mechanisms of action of the individual constituents are still unclear. We and others were able to demonstrate a caspase-dependent apoptosis induction with involvement of CASP8, CASP9 and CASP3 [
19,
34]. Bantel
et al. proved the activation of CASP8 without any death receptor signalling [
17], which supported the assumption that apoptosis is mainly mediated by the mitochondrial pathway. CASP3 [
35] and cytochrome c [
31] can also activate CASP8 in a feedback mechanism. Further cleavage of BID by CASP8 triggers a stronger apoptotic response by an intrinsic mechanism. We observed activation of CASP3, CASP8 and CASP9 with additional loss of mitochondrial membrane potential, BID cleavage and cytochrome c release after viscumTT treatment. TT treatment led to no cytochrome c release in Saos-2 although CASP9 was activated (Figs.
2 and
3). Both cell lines are almost resistant to viscum after 24 h and in 143B cells CASP9 was interestingly not affected. Older studies of our group observed CASP9 and CASP8 activation after viscum treatment in different leukaemia cell lines [
18,
19]. However, after addition of TT CASP9 was strongly activated in both cell lines. These results support the statement that the anti-cancer effect is often dependent on cell line and tumor entity. In addition, different OA derivatives induce apoptosis directly mediated by CASP8 in various cancer cells [
23,
36,
37]. Apoptosis induction by VAE alters apoptosis associated protein levels of the BCL2 family proteins BAX and BCL2 [
38,
39]. This is in line with our results. p53 interacts directly with BCL-2 family members [
40] and regulates the expression of BAX and BCL2 in apoptosis [
41,
42]. p53 was down-regulated by all three extracts in 143B. In hepatocarcinoma cells [
38] and human lymphocytes [
39] down-regulation of p53 was also observed. Saos-2 cells are null mutants for p53 but apoptosis was also induced after treatment. These results suggest the assumption that apoptosis induction is p53 independent in these osteosarcoma. Cell lines. Furthermore, VAEs led to reduction in several IAP family members such as XIAP and BIRC5 in Ewing sarcoma (Twardziok et al. accepted)), AML [
19], colon cancer and epidermoidal cancer [
43]. This is in line with our present results. The abilities of IAPs to inhibit caspases, assemble pro-apoptotic protein complexes and mediate expression of anti-apoptotic proteins, make them promising targets in cancer treatment [
44]. Interestingly, CASP3 is able to degrade its own inhibitor (XIAP) which again enhances CASP3 activity and consequently induces apoptosis [
45]. In addition, XIAP and BIRC5 are also affected by derivatives of OA [
46,
47] and BA [
48]. It is evident from our results that viscumTT combines the anti-cancer effects cell type independently, but seems to modulate the mechanism of apoptosis derived from triterpene acids and mistletoe lectins. The mechanism of the synergistic action of viscumTT could not yet be decoded. Recently, it was shown that after a short incubation time uptake of ML by 143B cells after viscum treatment only occurred after addition of TT [
49]. Our data suggest that ML uptake needs more time in 143B cells because after 24 h an anti-proliferative effect was observed. However, this observation leads to the hypothesis that triterpenic acids improve the uptake of viscum, which could also be one explanation for the synergistic effect. Triterpenic acids are able to embed in cell membranes, which leads to their disturbance [
50,
51], and interact with anionic phospholipids [
52]. Further investigations of this are necessary. In the future, reduction of side effects and overcoming of resistances during chemotherapy will be of increasing interest. Some studies were even able to show an enhancement of cytotoxicity when chemotherapy was combined with VAE in vitro [
28]. An optimal anti-tumor effect was demonstrated in K562 leukaemia cells after co-treatment with VAE and sub-apoptotic doxorubicin concentrations [
27]. Also, VP16 led to an enhanced apoptosis induction after combined incubation with ML-I [
17]. In our study we observed an additional synergism when viscum, TT and viscumTT were co-treated with Doxo, 4OOH and VP16 in 143B cells. In Saos-2 cells the enhanced apoptotic effect was additive rather than synergistic. The synergistic apoptosis induction after co-treatment with viscum, TT and viscumTT and 4OOH was demonstrated here for the first time. Generally, hight potential of apoptosis induction, also in combination with classical chemotherapeutic drugs, promises importance as additional therapy in osteosarcoma.