Gambogic acid is a major ingredient of gamboge, a brownish-orange resin exuded from the Asian Garcinia hanburryi tree. The resin has been used as Chinese traditional medicine in China for hundreds of years to treat cancers with little side effect. Recent experiments have demonstrated that GA exhibits a general inhibitory effect against various tumor cell lines [4–6]. However, little information is available in the literature about the effect and mechanism of this compound on the growth of human oral squamous cell carcinomas. In this study, we investigated the effects of GA on the growth of human OSCC cell lines. Our results demonstrate that GA significantly inhibits the viability of OSCC cells and induces their apoptosis in a time- and dose-dependent manner. These results suggest that GA could be a potential chemotherapeutic agent, like many other plant-derived natural compounds .
The transcription factor, NF-kappa B, is well established as a regulator of genes encoding cytokines, cytokine receptors and cell adhesion molecules that drive immune and inflammatory responses. In recent years, NF-kappa B activation has been linked to many aspects of tumorigenesis[13, 14], including the control of apoptosis, cell cycle, differentiation, cell adhesion, cell migration and angiogenesis. Five NF-kappa B family members have been identified in mammalian cells, including p105/p50 (NF-kappa B1), p100/p52 (NF-kappa B2), p65 (REL A), c-REL and RELB, which associate with one another to form various heterodimeric and homodimeric combinations. These proteins are regulated by inhibitors that bind to the inhibitory subunit of the NF-kappa B (I-kappa B) family of ankyrin domain-containing proteins, which include I-kappa B α, I-kappa B β, I-kappa B γ and I-kappa B ε. In a classic NF-kappa B signaling pathway, NF-kappa B resides in the cytoplasm in an inactive state as a heterotrimer consisting of p50, p65 and I-kappa B α subunits. Most carcinogens, inflammatory agents and tumor promoters have been shown to activate NF-kappa B. In response to an activation signal, the I-kappa B α subunit is phosphorylated at serine residues 32 and 36, ubiquitinated at lysine residues 21 and 22, and degraded through the proteasome pathway, thus exposing the nuclear localization signals on the p50-p65 heterodimer. The p65 subunit is then phosphorylated, leading to nuclear translocation and binding to a specific sequence in DNA, which, in turn, results in gene transcription. NF-kappa B regulates the expression of several genes whose products are involved in anti-apoptosis such as Bcl-xL, cIAP and TRAF[16, 17].
It has been found that many chemotherapeutic agents, including taxol, doxorubicin, etoposide, cisplatin and tamoxifen, can activate NF-kappa B. The activation of NF-kappa B can lead to resistance to apoptosis induced by chemotherapeutic agents. Thus, while activating apoptosis, the same agent can also activate NF-kappa B[19, 20], which can prevent apoptosis and block the ability of therapeutic agents to induce cell death. The recognized method of GA's antitumor effect is its ability to bind and interact with the transferrin receptor, and thus induce cell apoptosis. It is also suggested that GA has the ability to repress telomerase activity, which inhibits the proliferation of tumor cells. In this study, we examined the effect of GA on the activation of NF-kappa B in OSCC cells. The results clearly show that GA stimulates nuclear translocation of NF-kappa B p65, up-regulates the DNA binding activity of NF-kappa B, and thus activates the NF-kappa B signaling pathway. Considering the role of NF-kappa B in preventing apoptosis, we could hypothesize that, when treated with GA, an apoptosis inducer, OSCC cells activate the NF-kappa B signaling pathway to resist the apoptosis-inducing effect. This process is similar to the chemotherapy resistance caused by other chemotherapeutic agents.
The pivotal role of the NF-kappa B pathway in the inhibition of cell apoptosis, strongly suggests that NF-kappa B inhibitors would be useful in cancer therapy. In recent years, it has been reported that some agents, mostly plant-derived products, with tumor chemopreventative and chemotherapeutic effects suppress the activation of NF-kappa B. Although some of these agents are known to induce apoptosis, the most likely therapeutic consequence of NF-kappa B inhibition is a reduced apoptotic threshold. Therefore, a reasonable strategy is to use a NF-kappa B inhibitor as an adjuvant with other modalities to sensitize tumor cells to chemotherapeutic agents.
Celastrol is a novel compound that has demonstrated the ability to inhibit cancer progression and down-regulate NF-kappa B activity. It is also a natural product extracted from the traditional Chinese medicine "Thunder of God Vine". Celastrol has been shown to decrease NF-kappa B activity in prostate cancer and leukemia cells[23, 24]. However, it had not previously been evaluated as a potential chemotherapeutic sensitizer for head and neck or other solid tumors. Our results show that a low dose of celastrol (less than 2 μM) had no significant cytotoxic effect on OSCC cells. However, celastrol had a significant synergistic effect on GA-induced apoptosis in OSCC cells. Our combined results show that celastrol decreases NF-kappa B activity stimulated by GA and then decreases the apoptotic threshold of OSCC cells and sensitizes cells to GA-induced apoptosis by inhibiting the NF-kappa B signaling pathway.
In most cases, the efficacy of chemotherapeutic agents is limited due to toxicity and side effects. Therefore, increasing the potency of chemotherapeutic agents remains a worthy goal. Our results show that GA demonstrates antitumor and apoptosis-inducing abilities in oral squamous cell carcinoma cells. GA activates the NF-kappa B pathway while inducing apoptosis, thus blunting its own antitumor effect. Celastrol has a synergistic effect on the GA-induced apoptosis in oral cancer cells by inhibiting the NF-kappa B activity stimulated by GA.