In the current study, we evaluated the anti-HCC activity of B5G9 on four HCC cell lines, including HepG2, Bel-7402 and Hep3B cells (p53-null), and multidrug-resistant HepG2/ADM cells. B5G9 showed comparable cytotoxicity in the four hepatoma cell lines, which indicates the potential of B5G9 in the treatment of various types of HCC cells, regardless of p53 status. B5G9 also exhibited outstanding anti-proliferation activity on HepG2 tumour xenografts without significant toxicity, whereas 23-HBA had a minimal effect.
ROS generation is considered the key mechanism for the anti-cancer activity of BA and 23-HBA [
27,
28,
53]. However, the source of ROS was unclear. In the Human Oxidative Stress Plus PCR Array, we surprisingly found that B5G9-induced mitochondria-specific SOD2 upregulation was completely inhibited by antioxidants. The results indicated that the mitochondria might undergo oxidative stress. This hypothesis was confirmed by the co-localization of H
2DCFDA and Mito-Tracker as well as MitoSOX Red staining. We found that 23-HBA had a similar but weaker effect, which could explain why B5G9 had greater anti-HCC activity than that of 23-HBA; however, B5G9 may have a different underlying mechanism of mitochondrial dysfunction. Moreover, we noted that B5G9 just induced a slight increase of mitochondrial ROS and apoptosis in normal liver LO2 cells compare to HepG2 cells (Additional file
2: Figure S1A&B) which indicated its selective cytotoxicity against HCC cells. These results also implied the important role of mitochondrial ROS in B5G9-induced apoptosis. In addition to mitochondria, NADPH oxidase, XO, LOX, COX and cytochrome p450 contribute to ROS overproduction, but the results showed that inhibitors of these enzymes failed to abolish B5G9-induced ROS production. All these data indicate that mitochondria are the major site of B5G9-induced ROS production. Inhibition of mitochondrial complex activity (especially complex I and III) can induce ROS production [
54,
55]. We further measured mitochondrial complex activities upon B5G9 treatment by cell-based assay. As shown in Additional file
2: Figure S1C, B5G9 treatment (0.5, 1 and 3 h) had no effect on activities of mitochondrial complex I, III and V. In our study, B5G9-induced mitochondrial-ROS burst was observed at 3 h. These data indicate that B5G9-induced mitochondrial-ROS overload was probably not a result of mitochondrial complex complex inhibition. The mechanisms underlying B5G9-induced mitochondrial-ROS burst are complicated and need be explored in our future study. Recently, BA was found to induce ROS production in hepatocellular carcinoma via a p53-dependent p66
shc/miR-21-Sod2 pathway [
53]. However, B5G9 could also induce oxidative stress in Hep3B cells (Additional file
2: Figure S1D), indicating that a p53-independent mechanism underlying the ROS burst induced by B5G9 must exist. Wolfgang Wick found that BA-induced ROS generation required new protein synthesis [
27], but this was not observed in our studies because cycloheximide (CHX), an inhibitor of
de novo protein synthesis, could not inhibit the ROS induced by B5G9 (Additional file
2: Figure S1E&F). This result was also confirmed by the fact that five genes (HMOX1, SPINK1, COX-2, DUSP1 and SOD2) were significantly upregulated by B5G9, and their upregulation was a consequence of ROS production (Fig.
4). Dual-specificity phosphatase 1 (DUSP1) is a negative regulator of MAPKs that dephosphorylates both the threonine/serine and tyrosine residues of the substrates to facilitate tumourigenesis [
56]. In human HCC, the activated RAS-MAPK cascade leads to ERK activation, which results in the phosphorylation of its inhibitor, DUSP1. Then, the phosphorylated DUSP1 is degraded by the ubiquitin proteasome system [
57]. Hence, DUSP1 is negatively correlated with ERK activity and acts as a negative regulator of HCC development [
58]. Thus, DUSP1 upregulation may be a promising approach in HCC therapy. In our study, we found that the DUSP1 mRNA level was substantially upregulated; however, the role of DUSP1 in B5G9-induced HepG2 cell death requires further study.
It has been frequently reported that BA as well as its derivatives activated the mitochondrial apoptotic pathway. Unfortunately, few studies have examined the requirement of mitochondria for their anti-cancer effects. To gain insight into this issue, we established a mitochondrial DNA-depleted ρ0 HepG2 cell line. B5G9 only slightly increased the fluorescence of MitoSOX Red in ρ0 HepG2 cells, and ρ0 HepG2 cells were less sensitive to the B5G9 treatment compared with wt HepG2 cells. These phenomena are partly due to the incomplete efficiency of mitochondrial DNA depletion. Nevertheless, these results indicate that mitochondria play a crucial role in B5G9-induced cell death. MPT is a key event in the mitochondrial apoptotic pathway. BA-induced MPT was a consequence of the opening of the mitochondrial PT pore, which was abolished by cyclosporine A (CsA) or bongkrekic acid [
59,
60]. In contrast to these data, we found that cyclosporine failed to alleviate B5G9-induced MPT and cell death (Additional file
2: Figure S1G&H). Recently, a novel mechanism associated with mitochondrial cardiolipin was discovered. BA induced cancer cell death by modifying the biosynthesis of cardiolipin . This modification was accomplished by the inhibition of the activity of steroyl-CoA-desaturase [
61]. Moreover, BA could also induce MPT by directly interacting with the mitochondrial membrane to change the permeability of the cardiolipin film [
62]. These results suggested that piperazidine may enhance the mitochondrial cardiolipin modification effect, and this should be further investigated.