Anti-proliferative and pro-apoptotic effects of BD

It is known that dysregulation of cell cycle leading to uncontrolled cell proliferation and apoptosis evasion are hallmarks of all cancer cell types [29, 38–41]. Indeed, through various cell proliferation assays like Sulforhodamine B assay, MTT assay, and Cell Counting Kit 8 assay, BD has been established to exhibit anti-proliferative properties against pancreatic cancer cells (PANC-1, SW1990, CAPAN-1) [29, 37], lung cancer cells [A549, NCI-H292, non-small cell lung cancer (NSCLC) H460] [28, 42, 43], chronic myeloid leukemia (K562) [44], breast cancer cells (MDA-MB-231) [30], hepatocellular carcinoma cells (Bel7404, HepG2, Hep3B, Huh7, PLC) [45, 46], and osteosarcoma cells (MNNG/HOS, U-2OS, MG-63, Saos-2) [47]. In all of the investigated cancer cell lines, there was increased activation of pro-apoptotic proteins like B-cell lymphoma 2 (Bcl-2) associated protein (Bax) and Bak and downregulation of anti-apoptotic proteins like Bcl-2 and myeloid cell leukemia 1 (Mcl-1) [48], all of which are tightly linked to cellular proliferation and apoptotic pathways like phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of Rapamycin (mTOR), c-Jun N-terminal kinase (JNK), mitogen-activated protein kinases (MAPK), and canonical Wnt signaling pathways (Figure 1) [49–52]. The anti-proliferative effects of BD and IC₅₀ values on the aforementioned in vitro cancer cell lines and in vivo cancer models are summarized in Table 1 and Table 2, respectively. It has been noticed that the anti-proliferative effect of BD varied in a time-dependent and dose-dependent manner in all of the experiments. Importantly, no significant toxicity of BD was observed against normal control cell lines.

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Figure 1. 

Mechanisms of BD for its anticancer effect in human cancers. BD treatment suppressed the AKT/ERK and activate the JNK signaling cascades to induce apoptosis of cancer cells. BD caused apoptosis of cancer cells by regulating ROS and mitochondrial proteins

Effect of BD against triple-negative breast cancer (TNBC)

TNBC is characterized by the absence or down-regulation of estrogen receptors, progesterone receptors, and human epidermal growth factor 2 receptors [53–55]. Due to its aggressiveness, malignancy, and distant recurrence, TNBC is usually accompanied by a poor clinical prognosis and short life expectancy [55, 56]. To date, the best therapeutic treatment for TNBC is restricted only to chemotherapy, surgical tumor removal, and limited clinical drugs which are still being evaluated in clinical trials [55, 57–59]. It is therefore imperative to uncover new sources of adjuvant or stand-alone treatment for TNBC, be it to combat TNBC or to alleviate discomfort from standard chemotherapy and surgical procedures. Luo et al. [30], have investigated the inhibitory effects of BD on MDA-MB-231 (TNBC) cells. They investigated the role of PI3K/AKT signaling in tumor metastasis with low doses of BD that does not affect cell viability as it was previously reported that the PI3K/AKT signaling pathway was implicated, albeit in colorectal cancer cells [60]. It was found that BD significantly decreased PI3K expression and AKT phosphorylation in a dose-dependent manner while total AKT levels remained constant, leading them to conclude that the PI3K/AKT signaling pathway was implicated in tumor metastasis of TNBC. While the role of PI3K/AKT signaling in cell proliferation was not clearly established in the study, it has long known that the PI3K/AKT signaling pathway regulates cellular proliferation, cell cycle, and apoptosis. Its dysregulation is also implicated in various in vitro cancer models, where PI3K and it’s downstream molecular targets are constitutively active, leading to uninhibited cell proliferation and cell growth [61]. Further, it was also found that the PI3K/AKT/mTOR pathway is frequently activated in TNBC [62]. With knowledge from existing literature, there is a high possibility that anti-proliferative and pro-apoptotic effects of the BD on TNBC could be mediated through the PI3K/AKT pathway. However, we can only hypothesize at best since it was never explicitly investigated in the context of proliferation and apoptosis.

Effect of BD against pancreatic adenocarcinoma

Pancreatic adenocarcinoma comprises 95% of the diagnosed pancreatic cancer cases, with other cases being rarer forms of neuroendocrine cancers [63, 64]. Patients with pancreatic cancer have worse prognosis due to its aggressive, metastatic, and drug-resistance nature [65, 66]. Chemotherapeutic agents such as gemcitabine, 5-fluorouracil, and capecitabine are commonly administered for patients with pancreatic adenocarcinoma. To date, gemcitabine remains the chemotherapeutic agent of choice [64, 67], but acquired-gemcitabine resistance of pancreatic adenocarcinoma cells is the unsolved mystery [68–70]. Natural bioactive compounds like camptothecin (DNA topoisomerase I poison) could also be used as a chemotherapeutic agent but it showed only low therapeutic efficacy with severe toxicity [71]. It has been previously demonstrated that Brucea javanica fruit extract was able to exert cytotoxic and pro-apoptotic effects against pancreatic adenocarcinoma cell lines PANC1, SW1990, and CAPAN-1 [37, 72]. In a follow-up study in 2009 by the same team, BD was used directly instead of Brucea javanica fruit extract. It was shown that there was a BD dose-dependent apoptogenic effect on PANC-1 cells following Annexin V-PI double staining. Western blot analysis was also congruent with the results of the Annexin V-PI assay, whereby induction of caspase 3 and 8, enhancement of pro-apoptotic protein Bak, and decrease in anti-apoptotic protein Bcl-2 were seen. Further investigation elucidated that BD-induced cellular apoptosis was mediated through the necessary activation of the p38-MAPK signaling pathway as prior incubation with p38-MAPK inhibitor SB203580 attenuated caspase activation and DNA fragmentation.

MAPK participates in signaling pathways that are crucial for the maintenance of normal cellular processes like cell-cycle progression, cell proliferation, and apoptosis [73–77]. p38-MAPK constitutes one of the four subgroups within the MAPK family and respond to a wide range of extracellular stimuli such as stress or growth factor stimulation [78]. It is known that p38-MAPK activation could induce apoptosis and cell-cycle arrest, leading to tumor suppression. Conversely, inactivation, or down-regulation of p38-MAPK increases the tumorigenic potential of cells as DNA damage accumulates with each successive cell cycle [79]. Downstream molecular targets of p38-MAPK include c-Jun, signal transducers and activator of transcription 1 (STAT1), and p53 proteins amongst an abundance of other proteins, all of which are involved in regulating the balance between cellular proliferation and apoptosis [79–84].

It was proposed in the study that BD could have participated in both extrinsic and intrinsic apoptotic pathways. In the intrinsic pathway, BD would have acted as an extracellular stress signal which activates the p38-MAPK pathway. In the extrinsic pathway, BD would have acted as a ligand to membrane death receptors. No discrimination was made between those two apoptotic pathways when it came to examining the expression of caspase 3, caspase 8, and caspase 9 is known to be implicated in both mitochondrial-mediated apoptosis and receptor-mediated apoptosis [85].

Effect of BD against chronic myeloid leukemia

Chronic myeloid leukemia is a myeloproliferative malignancy that is usually caused by a chromosomal rearrangement event between chromosome 22 and chromosome 9 to form the Philadelphia chromosome [86, 87]. It is characterized by over-expression of a fusion oncoprotein BCR-ABL1 that acts as a constitutively active defective tyrosine kinase, implicating downstream signaling pathways such as PI3K/AKT/mTOR, Janus kinase (JAK)/STAT and Ras protein family (Ras)/mitogen-activated protein kinase kinase (MEK), all of which are crucial in maintaining normal cellular proliferation and apoptosis [86, 88]. Conventional therapeutic agents like imatinib, nilotinib, dasatinib, ponatinib, and bosutinib, therefore, act as tyrosine kinase inhibitors to prevent constitutive activation of the receptor tyrosine kinases in CML [86, 89–91].

BD was found to induce cellular apoptosis via the intrinsic mitochondrial-apoptotic pathway to K562 cells in a time-dependent manner [44, 80]. The intrinsic apoptosis pathway is characterized by a loss of membrane potential leading to the release of cytochrome c (cyt c) to the cytosol. Cyt c then binds to apoptotic peptidase activating factor 1 and caspase-9 to form an apoptosome, which cleaves caspase 3 to trigger apoptosis [44, 92]. It was postulated that the PI3K/AKT and Ras/Raf/extracellular signal-regulated kinase (ERK) pathways were upstream targets of BD in the study. Indeed, phosphorylated AKT and phosphorylated ERK levels were attenuated in cells treated with BD. However, since ERK activation need not always lead to pro-survival signals as ERK activation was reported to exhibit pro-apoptotic functions [93, 94], ERK activity must be interpreted in context. It remains unclear whether BD acted as a receptor tyrosine kinase inhibitor prior to PI3K and ERK activation or as an activator of phosphatases to PI3K and ERK.

Effect of BD against NSCLC

NSCLC constitute the majority of lung cancer cases in the world [95–100]. The effects of BD on the NSCLC cell lines A549, NCI-H292, and H460 has been investigated by different groups and reported that BD-induced intrinsic cellular apoptosis was mediated through the MAPK/JNK pathway [28, 42, 43]. Similar to p38-MAPKs, JNKs belong to the MAPK superfamily and regulate important biological processes like cellular proliferation and apoptosis. Phosphorylation of JNK will translocate it to the nucleus, where it phosphorylates c-Jun and forms activator protein 1, a transcription factor that is involved in the expression of pro-apoptotic proteins in both extrinsic and intrinsic apoptosis pathways [101]. In addition to apoptosis, BD was able to enhance autophagic flux in cell lines A549 and NCI-H292, another cellular process that can inhibit tumorigenesis and cancerous cell proliferation [28, 43].

Furthermore, it was highlighted that JNK activation and subsequent cellular apoptosis and autophagy was mediated mostly through BD-induced reactive oxygen species (ROS) production. Pre-treatment of cells with N-acetylcysteine, an antioxidant, before BD administration abolished apoptosis and autophagy almost entirely [28]. ROS are constantly being produced by cellular metabolic processes, and they are removed by antioxidant proteins [102]. Failure to maintain the dynamic equilibrium between ROS production and ROS elimination would result in oxidative stress to the cell [102, 103]. To this effect, ROS can activate different MAPK pathways to elicit different cellular responses. Herein, it is widely recognized that ROS oxidizes Trx (an antioxidant protein) to dissociate from apoptosis signal-regulating kinase 1 (ASK-1) (MAP3K5, another member of the MAPK family). The active ASK-1 would then activate downstream JNK pathways [102, 103]. BD also demonstrated the same effects in vivo, at least for A549 and NCI-H292 cell lines [28, 43].

Effect of BD against osteosarcoma

Osteosarcoma is a relatively rare type of primary bone malignancy that occurs mostly during adolescence and young adulthood [104]. While tumors can be treated efficiently with traditional chemotherapy, increasingly resistant cancer stem cells (CSCs) pose a significant threat to recurrent cancer progression [104]. This highlights the need to develop agents that could target CSCs specifically in addition to proliferating tumors [105–108]. In this light, the effect of BD against osteosarcoma cell lines MNNG/HOS and U-2OS was explored by Wang et al. [47]. It was demonstrated that BD-induced inhibition of cellular proliferation was through modulating key proteins involved in cell cycle progression and promoting apoptosis. There was significant downregulation of Cyclin D1, CDK4, and CDK2 expression. Cyclin D1 is vital in regulating the transition from G1 to S phase, and over-expression of cyclin D1 has been associated with tumorigenesis [47]. Increased proapoptotic protein expression was also detected for cells treated with BD. The repression of the JAK2/STAT3 signaling pathway was seen to be implicated for cells with BD treatment, with particular attention being paid to the upregulation of phosphatase Tyrosine-protein phosphatase non-receptor type 6 (SHP1) that negatively regulates STAT3 [47].

Most notably however was that BD treatment was able to decrease the proportion of stem-like osteosarcoma cells and impaired the self-renewal ability of osteosarcoma stem cells [47]. This was quantified through the downregulation of biomarkers via flow cytometry and Western blot analysis of multiple CSC markers like CD133 and stem cell markers like SOX2, Oct-4, and Nanog (Figure 2) [47].

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Figure 2. 

Mechanisms of BD for anti-metastatic effect in human malignancies. BD results in increased expression of E-cadherin and decreased expression of vimentin and N-cadherin leading to decreased EMT. BD treatment leads to decreased expression of OCT4 and SOX2 to eradicate the CSCs. BD exposure attenuates the expression of MMP2 and MMP9 to inhibit migration and invasion of cancer cells

Effect of BD against hepatocellular carcinoma (HCC)

HCC is one of the most common malignancies in the world [109–111]. To date, only a few targeted therapies have found to be effective against HCC and natural products may also exhibit their diverse anti-cancer actions against this malignancy [112–114]. Hence, there is a need for the development of cheaper and more encompassing therapeutic options. Two modes of action of BD-induced inhibition cellular proliferation were uncovered. In one study, BD suppresses proliferation via β-catenin/JAG1 pathways [45]. BD was demonstrated, both in vivo and in vitro, to induce proteasomes that degraded β-catenin and active β-catenin and pro-survival proteins from the transcriptional activities of active β-catenin from the canonical Wnt/β-catenin signaling pathway [45], which includes the JAG1 protein [115]. JAG1 in turn acts as a ligand to the canonical Notch signaling pathway, in which over-expression of JAG1 and/or aberrant activation of the Notch pathway is associated with cancer [116].

In a separate study using Hep3B, PLC, HepG2, Huh7, and Bel7407, BD suppressed cellular proliferation via miR-95 expression and induction of pro-apoptotic protein CUGBP2 [46]. There is growing evidence that miRNAs are capable of effecting oncogenic or tumor-suppressive functions, dysregulation of which have significant influence over tumorigenesis and its underlying cellular processes [117]. The oncogenic function of miR-95 is implicated in a wide range of cancers like osteosarcoma, recurrent NSCLC, and HCC, where down-regulation of miR-95 has been found to suppress tumor growth [38, 118]. BD was suggested as a negative regulator of the promoter of miR-95, whereby BD treatment decreased miR-95 expression. Using bioinformatics analysis via TargetScan and PicTar, it was further identified that the 3’ untranslated region of CUGBP2 mRNA was a direct downstream target for miR-95, and miR-95 binding interfered with CUGBP2 translation [119]. While CUGBP2 was previously shown to induce apoptosis in colon cancer cells via active stabilization and translation inhibition of pro-survival protein Mcl-1 [120], the mechanism of CUGBP2 within HCC has not been elucidated.