Gambogenic Acid Enhances Chemosensitivity of Multiple Myeloma to Bortezomib through p53/ROS/p38 MAPK Apoptotic Pathway

Background: Multiple myeloma (MM) is the second most prevalent hematological cancer (10%) with a five-year survival rate of only 49%. Approval of bortezomib, the first in-class proteasome inhibitor, has significantly improved outcomes of MM in the last few years; however, the almost inevitable refractory or resistance has made a cure of MM remains out of reach. Identification and validation of novel effective targeted agents are necessary. As one of the natural compounds isolated from Traditional Chinese Medicine gamboge, gambogenic acid (GNA) is a very promising antitumor agent and has been proved to have more potent anticancer effect and less systemic toxicity according to early investigations. In our previous studies, we have demonstrated that GNA could synergistically potentiate bortezomib-induced apoptosis of MM. In this study, our aim was to further explore and understand the concrete apoptotic action mechanism of GNA and bortezomib combination treatment against MM in pre-clinical models.

Methods: We performed pre-clinical studies in myeloma cell lines and mouse models treated by GNA and bortezomib, alone and in combination. Cell counting kit-8 (CCK-8) assay, combination index (CI) isobologram, flow cytometry (FCM), western blot, xenograft tumor models, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), immunohistochemistry (IHC) and oxidation-sensitive fluorescent dye carboxy-DCFDA were applied in this study. The thiol antioxidant N-acetylcysteine (NAC) and p38 MAPK inhibitor SB203580 were used to detect the effect of GNA and bortezomib combination therapy on ROS, p53 and p38 MAPK.

Results: Both p53 wild type cell line MM.1S and mutant type cell line RPMI 8226 myeloma cell lines were sensitive to GNA therapy. Interestingly, GNA only enhanced the chemosensitivity of bortezomib on p53 wild type cell line MM.1S. Both 0.51μM GNA combined with 2.3nM bortezomib and 0.90μM GNA combined with 4.0nM bortezomib to MM.1S cells showed a more markedly apoptotic rate of MM compared with that of the single agent group of each accordingly (p <0.01; p <0.01), and the apoptosis was presented in a dosage- and time-dependent manner. Increased apoptosis of MM.1S cells was found via the activation of PARP cleavage, p53, Caspase-3 cleavage and Bax and inhibition of Bcl-2 expression. An increased antitumor effect of combination therapy of GNA and bortezomib on MM.1S xenograft models were also observed. A significant generation of ROS was detected after exposure of GNA and bortezomib in MM.1S cells. The expression level of phospho-p38MAPK was upregulated by combination therapy compared with that of single agent (p<0.05; p<0.05). To characterize the mechanisms of action of GNA in combination with bortezomib, we pretreated MM.1S cells with NAC and SB203580. We found that the apoptosis of MM.1S cells could be inhibited by both NAC and SB203580 (p<0.01; p<0.01). However, the generation of ROS could only be inhibited by NAC (p<0.05), and the elevated expression of p53 could be inhibited neither by NAC nor SB203580. These results indicated that p53/ROS/p38 MAPK pathway is a pre-requisite for GNA to accomplish the enhancement of chemosensitivity of MM to bortezomib.

Conclusions: We demonstrate that GNA could potentiate apoptosis of MM induced by bortezomib in vivo and in vitro, and p53/ROS/p38 MAPK pathway plays a central role of the apoptotic action. Thus, our data provide a persuasive rationale for the successful utilization of dual GNA and bortezomib for chemotherapy in MM patients in the future.