Glioblastomas are the most common and aggressive brain tumors [1
]. The patients with malignant glioblastomas usually have poor prognosis [2
]. The median survival rate of glioblastoma patients is about 12 months. Temozolomide (TMZ) is the first-line chemotherapeutic drug for therapy of malignant glioblastomas [3
]. Nevertheless, TMZ may induce drug tolerance to high-grade glioblastomas, especially in recurrent patients [4
]. Thus, the chemoresistance is a serious issue for therapy of human glioblastomas. To build up a novel and effective strategy for treatment of human glioblastomas, it is very critical to discover new chemotherapeutic drugs that can overcome TMZ-induced drug tolerance.
Honokiol (2-(4-hydroxy-3-prop-2-enyl-phenyl)-4-prop-2-enyl-phenol), a small-molecule bisphenol polyphenol, is a major bioactive constituent of the traditional Chinese medicine Magnolia officinalis
]. Amorati et al. demonstrated that the hydroxyl group of the second phenol possesses better chemical reactivity with peroxyl radicals [6
]. Honokiol can effectively treat a variety of diseases, including anxiety and nervous disturbances, thrombotic stroke, typhoid fever, and dermatologic disorders [5
]. Drug resistance to therapy in cancer is multifaceted and challenged until now. Interestingly, Tian et al. demonstrated that honokiol could synergize chemotherapeutic drugs in multidrug resistant breast cancer cells via apoptotic and programmed necrotic death [7
]. A previous study used pharmacogenomics and molecular docking approaches to supplementary show epidermal growth factor receptor (EGFR)-transfected tumor cells were collaterally sensitive to honokiol compared with wild type cells [8
]. Recently, honokiol is reported to be a promising natural compound in overcoming acquired resistance to cetuximab, a monoclonal antibody against EGFR used for treatment of head and neck squamous cell carcinoma and metastatic colorectal cancer [9
]. As a result, targeting drug resistance by using honokiol alone or combined with other chemotherapy agents can provide de novo therapeutic strategies.
A previous study reported low toxicity of honokiol to normal human astrocytes and murine cerebrovascular endothelial cells [10
]. The blood-brain barrier (BBB) is the major limitation for therapy of brain diseases [11
]. Notably, honokiol was shown to pass through the BBB in vitro and in vivo [10
]. Our laboratory reported the benefits of honokiol to induce apoptosis of neuroblastoma cells and glioblastoma cells via an intrinsic mitochondria-dependent pathway [10
]. Moreover, the molecular mechanisms were confirmed through a p53/phosphoinositide 3-kinases (PI3K)/mammalian target of rapamycin (mTOR) mechanism and an endoplasmic reticular stress/extracellular signal-regulated kinases (ERK)1/2 pathway in neuroblastoma cells and glioblastoma cells, respectively [13
]. In addition, autophagy induced by cancer therapy frequently contributes to cancer cell survival [15
]. The effects of honokiol on autophagy of neuroblastoma cells and glioblastoma cells were further identified [12
]. Furthermore, cancer stemness is the other critical cause for drug resistance [16
]. Previous studies presented the potential of honokiol on suppressing sphere formation and xenograft growth of oral cancer stem cells [17
]. Thus, honokiol has the potential for treatment of drug-resistant glioblastomas.
Antiapoptosis of cancer cells against chemotherapy is the other important reason for chemoresistance [19
]. Extrinsic and intrinsic pathways are involved in cell apoptosis. In an extrinsic pathway, caspase-8 is activated following binding of extracellular cytotoxic Fas ligand to its death receptor [20
]. In contrast, activation of capase-9 by release of mitochondrial cytochrome c to the cytoplasm can trigger apoptosis via an intrinsic mechanism [20
]. Recently, we have shown that honokiol could synergistically improve TMZ-induced killing to human malignant glioblastoma cells through a mitochondrion-dependent apoptotic mechanism [22
]. Hence, caspase-8 and caspase-9 are two typical molecules specifically triggering cell apoptosis through an extrinsic death ligand-dependent mechanism and an intrinsic mitochondria-dependent pathway, respectively [20
]. Based on previous studies, honokiol is able to kill glioblastoma cells by inducing autophagic and apoptotic insults. Elucidating the apoptotic mechanism is crucial for clinical application of honokiol for treatment of drug-resistant glioblastomas. Therefore, this study was aimed to further evaluate the effects of honokiol on the drug-tolerant glioblastoma cells and the possible mechanisms, especially in the caspases-8/-9-involed apoptotic pathways.
In this study, we have shown that honokiol could kill the drug-resistant glioblastoma cells. Glioblastomas are major solid brain tumors [1
]. Until now, administration of TMZ may lead to drug resistance, especially for high-grade glioblastomas and recurrent patients [4
]. In the present investigation, we successfully prepared human drug-tolerant U87-MG-R9 glioblastoma cells from human U87-MG cells as our experimental model. In drug discovery for therapy of brain tumors, the BBB is a significant limitation and challenge [10
]. Fascinatingly, our previous study has demonstrated that honokiol could pass through the BBB and possess low toxicity to normal human HA-h astrocytes and mouse cerebrovascular endothelial cells [10
]. Furthermore, we reported the benefits of propofol on killing human U87-MG and U373MG and murine GL-261 glioblastoma cells, as well as neuroblastoma cells. Recently, we demonstrated the synergistic effects of honokiol on TMZ-induced insults to glioblastoma cells [22
]. In this study, we further verify the welfares of honokiol to efficiently kill human TMZ-resistant glioblastoma cells. Human malignant glioblastomas are very aggressive and recurrent [2
]. Most of glioblastoma patients are drug-resistant and recurrent [3
]. Cancer stemness and autophagy are two key causes for drug resistance. A previous study reported the suppressive effects of honokiol on sphere formation and xenograft growth of oral cancer stem cells [17
]. In addition, honokiol can induce autophagic insults to neuroblastoma cells and glioblastoma cells [13
]. As a result, our study attractively suggested that honokiol has potential welfares for therapy of drug-resistant glioblastoma patients.
Rapid proliferation is an important characteristic of malignant glioblastoma cells [1
]. Furthermore, rapid tumor growth because of speedy cell proliferation is one of major features and reasons illuminating the casual recurrence and poor prognoses of human malignant glioblastomas [26
]. Herein, we demonstrated that honokiol could descend proliferation of human drug-resistant glioblastoma cells. The honokiol-induced suppression in proliferation of human TMZ-resistant glioblastoma cells discloses the other potential benefits for therapy of malignant glioblastomas. In tumor microenvironments, low oxygen conditions are broadly present [27
]. Our previous study showed that under extended hypoxic stress, proliferation of human U87-MG cells was significantly repressed [28
]. A previous study has also reported that hypoxia is able to activate a self-protective mechanism against glioblastoma proliferation [29
]. Consequently, honokiol can prevent tumor cell proliferation in a hypoxic microenvironment and then suppress tumor growth.
Honokiol can activate caspase-3 and caspase-6 in human drug-resistant glioblastoma cells. Caspase-3, a protease in the process of intrinsic and extrinsic apoptosis, is activated by caspase-9 [12
]. Sequentially, treatment with honokiol enhanced caspase-6 activity, a downstream target of caspase-3, in human drug-resistant glioblastoma cells. Activations of caspase-9 and capse-3 are essential for proteolytic maturation of caspase-6 [30
]. After activation, caspase-3 and caspase-6 can cleave cellular crucial proteins such as lamin and nuclear mitotic apparatus proteins to affect cell functions [31
]. Our previous study has shown that honokiol could stimulate cascade activations of caspase-9 and -3, G1 phase arrest, and cell apoptosis in human TMZ-sensitive glioblastoma cells [12
]. In the present study, honokiol could increase levels of cleaved caspase-3 and activity of this proteinase. Our previous and present studies displayed the helpful effects of honokiol on killing drug-sensitive and -resistant glioblastoma cells through cascade activation of the caspase proteases.
Honokiol can kill human drug-resistant glioblastoma cells via an apoptotic pathway due to a main activation of caspase-9. Our present study revealed that honokiol could induce caspase activation and DNA fragmentation in human TMZ-resistant glioblastoma cells. In addition, treatment with honokiol led to cell shrinkage and cell cycle arrest at the sub-G1 phase. Characteristically, a shrunken morphology, caspase activation, DNA fragmentation, and cell cycle arrest at the sub-G1 phase are significant features of cells undergoing apoptosis [32
]. As a result, honokiol can meaningfully kill human drug-resistant glioblastoma cells via an apoptotic pathway. Antiapoptosis of cancer cells against chemotherapy is another important cause resulting in drug resistance [19
]. Jeong et al. reported the anticancer effects of honokiol on human glioblastoma cells via induction of cell apoptosis [33
]. Moreover, a previous study also demonstrated the effects of honokiol on triggering apoptosis of glioblastoma cells via p53/p21-mediated cell cycle arrest at the G1 phase [13
]. Zhang et al. stated that honokiol induced apoptosis of U87-MG cells via activation of p38MAPK [34
]. In addition, our previous study further showed that honokiol can repress growth of human glioblastomas by inducing cell apoptosis due to activating the p53/cyclin D1/CDK6/CDK4/E2F1-dependent mechanism [12
]. Caspase-8 and -9 are two typical molecules explaining the extrinsic and intrinsic mechanisms of cell apoptosis [20
]. In this study, we showed that exposure to honokiol triggered more activation of caspase-9 than caspase-8 in human drug-resistant glioblastoma cells. In addition, our data mining in TCGA cohort further verified higher expression of caspase-9 mRNA in human glioblastomas. In addition, levels of caspase-9 protein in human glioblastomas were enlarged compared to human meningioma tissues. Interestingly, knocking-down caspase-9 concurrently lowered honokiol-induced caspase-6 activation, DNA damage, and cell apoptosis. As a result, caspase-9 plays a major role in honokiol-induced apoptotic insults to human drug-resistant glioblastoma cells.
Drug resistance to therapy in cancer is multifaceted and challenged until now. Antiapoptosis of cancer cells against chemotherapy is an important reason for chemoresistance. Honokiol is a multifunctional antiangiogenic and antitumor agent [5
]. Compared with wild type cells, Saeed et al. reported that that the EGFR-transfected tumor cells were collaterally sensitive to honokiol [8
]. In multidrug resistant breast cancer cells, honokiol can synergize chemotherapeutic drugs to induce apoptotic and programmed necrotic cell death [7
]. Lately, honokiol is reported to be a promising natural compound in overcoming acquired resistance to cetuximab, a monoclonal antibody against EGFR used for treatment of head and neck squamous cell carcinoma and metastatic colorectal cancer [9
]. Our previous study has demonstrated synergistic effects of honokiol with TMZ to kill human glioblastoma cells [23
]. In this study, we further showed a major role of caspase-9 in mediating honokiol-induced apoptotic insults to human drug-resistant glioblastoma cells. Therefore, honokiol can be a drug candidate for treatment of glioblastoma patients with chemoresistance.