PLX4032 has a prominent inhibition on BRAFV600E melanoma cell proliferation by arresting the cells at G1-phase of the cell cycle, however, when melanoma cell acquire resistance to the drug, PLX4032 is not able to control the cell proliferation ****(ref).
A large body of evidence show that melanomas with acquired resistance to PLX4032 have deregulation in the checkpoints important for orchestrating cell cycle progression, and CDK inhibitors show promising results in preclinical models of melanoma (Valerie Kedinger1, 2013, Yadav, 2014, Azimi, 2018). Here we demonstrate that 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 CDK1/cyclin B1 complex important for the transition of G2/M, and decreasing the levels of the CDC25C and its active form essential for promoting the transition to M-phase. 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.
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, 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 the ER stress induced apoptosis. However, further studies, such as knockdown of ER stress sensors (e.g., IRE1?) in A375-R cells are needed to answer whether the apoptotic cell death caused by CUMA is mainly through the ER stress response pathways.
Autophagy role in cancer is complex, pro-survival by clearing the damaged intracellular components and providing nutrients and energy 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 in 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, prolong 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 the mitochondria intrinsic cell death was decreased (Figure 3).
We have observed that CUMA treatment induced 1.5 fold increase in ROS levels (data not shown), however, it is not clear whether this phenomenon will lead to mitochondria damage and activation of apoptosis in PLX4032 resistant melanoma cells that need further investigation. Reactivation of RAF/MEK/ERK ***** signaling is the well-characterized central mechanism that leads to acquired resistance in BRAFV600E mutant melanoma, and 50% of melanoma patients are under treatment failure (ref).
In our mechanistic study, we observed that CUMA is not suppressing the activity of MEK and ERK (Figure S4). However, we found that CUMA is effective to A2058 BRAFV600E melanoma cells which are intrinsically resistant to BRAF inhibitors***, 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 animal.
CUMA significantly inhibited cell proliferation and angiogenesis in the tumor tissues, and induction of tumor cell apoptosis was also observed (Figure 4E) that are 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 have observed that CUMA exhibited much less activities in inhibiting A375-R tumor growth by intraperitoneal injection, but it shows potent inhibitory effect by oral fed administration and in dose-dependent manner. It maybe worthy for further elucidation of pharmacokinetic mechanism of CUMA in animal to identify the potential bioactive metabolites derived from CUMA.