Natural products have been recognized in therapeutic drugs for many years as an essential source of active substances. Among them, Ursolic acid has already been exhibited as a strong anti-carcinoma candidate by mounting evidence. Nevertheless, the effect of UA on LSCC remains uncovered. In this study, we thoroughly investigated UA’s role in the activity of countering LSCC from two aspects in a laryngeal carcinoma cell line. On one hand, we discovered that Ursolic acid potently inhibited HEp-2 cell proliferation using cell viability assay and clonogenic growth assay. By cell viability assay, we find 20 µg/mL, or 4.38 µmol/L UA is sufficient to hinder cell growth of HegG2 by half. This concatenation is close to that of some clinical drugs like tamoxifen used in cell-line models (17). We further verified this anti-proliferating effect was via checking G2 and M phase arrest in cell cycle progression, which indicates a cellular molecular change at G2/M phase was implicated. This antiproliferative profile is also exemplified by Lin’s (18) and Yang’s (19) report. Moreover, Lin’s team proved that suppression of Cyclin B1 could be the regulator of the cell cycle arrest caused by UA in colon adenocarcinoma cells (19).
On the other, we exploited multiple methods to investigate the apoptosis profile of HEp-2 cells. Apoptosis is regulated by two major pathways, the extrinsic pathway which is mediated by the transduction of extracellular death ligand signaling, and the intrinsic pathway, also referred to as the mitochondrial pathway, which is governed by a caspase cascade (20). And herein, we observed a substantial increase in Annexin V- or JC-1-stained positive cells, which demonstrates the flipped membrane during apoptosis occurred in HEp-2 cells that had been exposed to UA for 24 h. This cytometry data preliminarily confirmed UA induced apoptosis of HEp-2 through mitochondrial pathway. We further verified this mechanism by detecting primary molecules in intrinsic apoptosis pathway cascade. Intrinsic apoptosis cascade is mediated by antagonism between prosurvival and proapoptotic members of the Bcl-2 family and subsequent Capspase family (21). Our data showed treatment with UA decreased prosurvial Bcl-2 level and augmented proapoptotic Bax level. And this molecular change allowed downstream cleavage of the apoptotic initiator, Caspase3 and Caspase9 protein. Together, these data verified UA induces HEp-2 apoptosis via an intrinsic pathway.
In order to determine the molecular mechanism of UA on cell growth inhibition and apoptosis, we turned our interested into intracellular ROS level, which we believe is likely to be affected by the addition of UA, because ROS plays a pivotal role in mediating these two phenomena (16). We proved that ROS synthesis was markedly stimulated by UA in HEp-2 cells. This finding is also exemplified in the work undertaken in other cells (22–24), indicating the elevation of ROS level induced by UA in vitro might be more common than expected. However, this finding is inconsistent with results from in vivo experiments, as Ursolic acid inhibits interactive NOX4/ROS in liver fibrosis mice (23), reduced ROS production stimulated by LPS in ApoE−/− mice (25) and ROS accumulation in C. elegans serotonin-deficient mutants (26). The controversy of UA’s effect on ROS regulation between in vitro and in vivo data might be due to the difference of malignancy of the cell types and the diversity of pharmacokinetics process. More work needs to be done to explain the controversies found in vitro and in vivo models.
In addition, in attempt to identify the exact mechanism for the increase of ROS synthesis stimulated by UA in HEp-2 cells, we analyzed a class of typical signaling pathways such as ERK, JNK, p38, PI3K/Akt, NF-κB and STAT/JAK signaling, as they are all reported to mediate ROS-induced cell damage (27). Interestingly, we found that it is not a mere but all signaling pathways that are strongly impeded, which indicates the underlying mechanism is attributed to a strategy of multitarget inhibition. This theory can be evidenced by some previous findings. Kim reported that UA inhibits JAK2/STAT3 pathway in colorectal cancer cells (28). Prasad has long established UA inhibits JNK signaling (24). And Li found PI3K/Akt and NF-κB pathway was inhibited in vitro and in vivo respectively (25, 29). Besides, Park finds that Wnt/β-catenin is the mediator in the apoptosis in prostate cancer cells (30). Although above reports elaborated versatile mechanisms from various cellular signaling pathways, none of them analyzed the UA’s antiproliferative and proapoptotic profile from a multitarget perspective. Yet our finding is consistent with Lin’s work, which exhibits UA’s multitarget feature in a cancer angiogenesis model (31). Given that these signaling pathways have extensive crosstalks to each other and interacts with other molecules as well (32), it is believed that a wider range of screening for signaling pathways is demanded to further verify the certain mechanism.
In conclusion, we characterized the natural-occurring compound Ursolic acid as a potent indictor to LSCC in HEp-2 cell model. Through a body of signaling activation and ROS stimulation, it suppresses LSCC cell growth and provokes LSCC apoptosis. We hold that this compound may be a promising candidate for clinical application, as its multitarget strategy represent a novel and effective approach to hinder LSCC.