In spite of aggressive treatment protocols including high-dose chemotherapy and wide surgical resection, the long-term survival of patients with localized disease remains between 60-70% during the last two decades. Although maximal dose escalation of conventional chemotherapy has been utilized, there is still no significant gain in clinical outcome. The use of conventional antitumor drugs, such as doxorubicin and methotrexate, is usually limited due to their systemic toxicity and lack of specificity. Furthermore, no effective standard second-line chemotherapeutic agent has been identified in local or distant relapsed osteosarcoma. Many reports have emphasized that use of dietary bioactive compounds is becoming an alternative, safe, and desirable approach to controlling and treating cancer. Our previous studies have shown that FKB exhibits cytotoxic potency against mesenchymal tumors, including synovial sarcoma and uterine leiomyosarcoma[9, 12]. The results presented here confirm that FKB could inhibits proliferation of human osteosarcoma cells in vitro via G2/M arrest and leads to a robust induction of apoptosis.
We further evaluated the regulatory mechanism for the apoptotic effect of FKB in osteosarcoma cells. Investigations have shown that apoptosis is controlled by both mitochondrial and membrane death receptor pathways. Previous reported research showed that the mechanisms through which FKB induces apoptosis depend primarily on mitochondrial damage[8, 20]. The pro-survival protein Bcl-2, combined with Bax, can regulate apoptosis via homologous and heterogeneous complexes. Bax induces the release of cytochrome c and activates the Bax-initiated mitochondria pathway and the capsese 3-dependent apoptotic pathway. Bcl-2 inhibits the realease of cytochrome c against Bax. The disturbance of Bcl-2/Bax protein ratio has been recognized as a factor contributing to the FKB-induced apoptosis[6, 20]. In the present study, the increase in Bax and decrease in Bcl-2 was observed in both OS cell lines. Also activity level of caspase-3 was found to increase incrementally with escalating doses. The extrinsic pathway is initiated by the binding of transmembrane death receptors, including Fas, DR5 and TNFR receptors. Activation of Fas receptor leads to receptor clustering and formation of a death-inducing signaling complex, which results in the activation of procapase-8. Then active caspase-8 can then go on trigger the apoptotic caspase cascade. Fas expression may be triggered by FKB treatment and may account for independent activation of caspase-9. Puma is a critical mediator of p53-dependent and p53-independent apoptosis induced by a wide variety of stimuli, including deregulated oncogene expression, toxins, growth factor/cytokine withdrawal, and infection. It has been suggested that Puma can also sponsor apoptosis by directly activating Bax in some cells. Data from the present study suggests that FKB-induced apoptosis is mediated by both mitochondrial and membrane death receptor pathways.
Many conventional anticancer treatments at least partly damage the DNA of cells without specific selectivity selective for cancer cells. Anticancer insights derived from cell cycle research has given birth to the idea of cell cycle G2 checkpoint abrogation as a cancer specific therapy. Several studies have revealed that FKB induce G2/M arrest[12, 20, 24]. In current study, significant G2/M arrest by FKB in osteosarcoma cells was confirmed by synchronized cell cycle analysis. Further mechanism was explored. The cell cycle blockade was associated with reduction in Cyclin B1 and Cdc25C and increase in Myt1, and phosphorylation-cdc2. During G2, the Cdc2/Cyclin B complex is kept inactive by phosphorylation by the kinase Myt1. At the onset of mitosis, both residues are dephosphorylated by Cdc25C. Repression of Cyclin B1 and Cdc2 enforces the G2/M arrest. Inhibitory phosphorylation of Cdc2 is essential for the p53-independent G2 arrest that occurs in response to DNA damage, and is dependent on the protein kinases Atm and Atr. The Cdc2 is inactivated by Atm and Atr through increasing phosphorylation of the residues tyrosine 15, which cause G2 arrest in response to DNA damage. We found that FKB did not change the expression level of p53 (Data not shown). Hence, p53-independent G2 arrest may be the main mechanism in FKB-induced cell cycle block.
Results of motility and invasion assays encouraged the potential use of FKB as a new candidate for anticancer therapy against migration and invasion of osteosarcoma cells. Inhibition of motility and invasion with dose-dependent manner was observed in 143B and Saos-2 cell lines. To further explore the exact expression of FKB-induced inhibition of invasion and migration, we performed a gelatin zymography assay to detect the activities of MMP-2 and MMP-9 in 143B cells. The results showed that FKB notably down-regulated activities and protein levels of MMP-2 and MMP-9 in a dose-dependent manner. One of the major characteristics of cancer cell metastasis is altered adhesion ability between cells and the extracellular matrix which is associated with invasion and migration of tumor cells. MMPs are overexpressed in the metastatic tumor cells and have been shown to be involved in the invasion and metastasis of various tumor cells. High MMP-9 expression was observed in pretreatment osteosarcoma tumor samples and in most metastatic lesions, leading to the speculation that MMP-9 is associated with the micrometastatic behavior of osteosarcoma. It is well-established that inhibitions of MMP enzyme activity are early targets for preventing cancer metastasis. Both MMP-2 and MMP-9 are involved with the invasive metastatic potential of tumor cells. The current resutls clearly showed that FKB inhibited the migration and invasion of 143B and Saos-2 cells in vitro, which may account for its inhibitory effect on tumor metastasis. Here we found the protein activity of MMP-2 and MMP-9, which are involved in degradation of extracellular matrix and play vital roles in cancer cell migration and invasion.
Any discussion surrounding novel therapeutics must include concerns regarding untoward side effects. The toxicity is an important feature to be considered when a compound is used for treatment, especially for chemopreventive purposes. In order to investigate the potential toxic effect on the resident normal bone marrow mesenchysmal stem cells, we used murine bone marrow cells to study possible toxicity. Notably, the bone marrow cells were quite significantly less sensitive to the FKB, thereby suggesting a preferential toxicity on tumor cells. Compared with adriamycin, FKB showed a considerably lower toxicity on bone marrow cells in the colony formation assay (Figure 5C). FKB was found to have potent hepatocellular toxin. However, the LD50 for the two normal liver cell lines was 5- and 10-fold greater than the IC50 identified in the current experiment for osteosarcoma cell lines. Based on the in vitro results, FKB showed chemotherapeutic effect on tumor cells with significant less toxic effect on normal cells, suggesting its potential use in chemoprevention of OS. Nonetheless, characterization of in vivo toxicities related to FKB is highly warranted.