PLX4032 shows prominent inhibition of BRAFV600E melanoma cell proliferation by arresting the cells at G1-phase of the cell cycle; however, when melanoma cells acquire resistance to the drug, PLX4032 is not able to control the cell proliferation (ref). A large body of evidence shows that melanomas with acquired resistance to PLX4032 have deregulation at the checkpoints important for orchestrating cell cycle progression, and CDK inhibitors show promising results in preclinical models of melanoma (ref). Here we demonstrated that the triterpene glucoside CUMA significantly suppresses the growth of BRAFV600E mutant melanoma with acquired resistance to PLX4032 in vitro and in vivo. CUMA effectively induced G2/M cell cycle arrest and inhibited proliferation of A375-R cells, in part through inhibition of the CDK1/cyclin B1 complex important for the transition of G2/M, and the level of the CDC25C and its active form essential for promoting the transition to M-phase (Table 1, Figure 2).
It is known that during ER stress, IRE1? acts as a switch between cell survival and cell death. In hepatoma cells, overexpression of IRE1? inhibited cell growth and repression of IRE1? inhibited ER stress-related apoptosis (Li et al., 2012). In our study, we observed that the increased expression of IRE1? was accompanied by increased cleavage of PARP (Figure 5A), suggesting that A375-R cell apoptosis might be the consequence of CUMA-induced ER stress. Further, CUMA treatment increased the levels of the pro-apoptotic molecule Bim which is mainly involved in ER stress-induced apoptosis (Figure 3C). However, further studies, such as knockdown of ER stress sensors (e.g.
, IRE1?) in A375-R cells are needed to investigate whether the apoptotic cell death caused by CUMA is mainly through the ER stress response pathways. The role of autophagy in cancer is complex. It is pro-survival by clearing the damaged intracellular components and providing nutrients for facilitating cancer cell growth, or cell damaging when excessive autophagy leads to irreversible cellular function impairment (refs). We found that inhibition of autophagy with the well-known autophagy inhibitors 3-MA and CQ did not alter the antiproliferative effect of CUMA on A375-R (Figure S4B). However, our immunoblotting results showed that the CUMA-induced conversion of LC3B was reduced when co-treated with an ER stress inhibitor (4-PBA) (Figure 5F). These data implied that activation of autophagy in A375-R might be provoked by the CUMA-induced ER stress.
On the other hand, prolonged incubation with CUMA led to A375-R apoptosis as observed by activation of apoptotic hallmarks, caspase 3, caspase 7 and PARP; in addition, the anti-apoptotic protein Bcl-2 involved in mitochondria intrinsic cell death was decreased (Figure 3C). We have observed that CUMA treatment induced a 1.5 fold increase in ROS levels (data not shown); however, it is not clear whether this phenomenon will lead to mitochondrial damage and activation of apoptosis in PLX4032 resistant melanoma cells; this point that will need further investigation. Reactivation of RAF/MEK/ERK signaling is the central mechanism that leads to acquired resistance in BRAFV600E mutant melanoma, and treatment fails in 50% of melanoma patients (ref). In our mechanistic study, we observed that CUMA does not suppress the activity of MEK and ERK (Figure S4A). However, we found that CUMA is effective on A2058 BRAFV600E melanoma cells which are intrinsically resistant to BRAF inhibitors (Table 1), suggesting that CUMA might work by a different mechanism than BRAF and MEK inhibitors to suppress tumor growth. The significance of CUMA is highlighted by the inhibition of the growth of A375-R tumors with acquired resistance to PLX4032 in animals. CUMA significantly inhibited cell proliferation and angiogenesis in the tumor tissues, and induction of tumor cell apoptosis was also observed (Figure 4E), which is in good agreement with the data obtained from in vitro assays for the CUMA inhibitory effect against PLX4032 resistant A375-R cells.
This study is the first to demonstrate the pharmacological activities of CUMA against drug resistant BRAF mutant melanoma. Many natural products with substantial inhibitory activities in cancer cell models display very weak inhibitory activities in vivo as a consequence of their unfavorable pharmacokinetics. We observed that CUMA exhibited much less activity in inhibiting A375-R tumor growth by intraperitoneal injection, but it showed a potent dose-dependent inhibitory effect when administered by oral gavage. It may be worth further elucidating the pharmacokinetic mechanism of CUMA in animals to identify the potential bioactive metabolites derived from CUMA.