Contributed equally
The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potent anticancer agent possessing the ability to induce apoptosis in various cancer cells but not in non-malignant cells. However, certain type of cancer cells are resistant to TRAIL-induced apoptosis and some acquire resistance after the first treatment. So development of an agent that can reduce or avoid resistance in TRAIL-induced apoptosis has garnered significant attention. The present study evaluated the anticancer potential of hispolon in TRAIL-induced apoptosis and indicated hispolon can sensitize cancer cells to TRAIL. As the mechanism of action was examined, hispolon was found to activate caspase-3, caspase-8 and caspase-9, while downregulating the expression of cell survival proteins such as cFLIP, Bcl-2 and Bcl-xL and upregulating the expression of Bax and truncated Bid. We also found hispolon induced death receptors in a non-cell type-specific manner. Upregulation of death receptors by hispolon was found to be p53-independent but linked to the induction of CAAT enhancer binding protein homologous protein (CHOP). Overall, hispolon was demonstrated to potentiate the apoptotic effects of TRAIL through downregulation of anti-apoptotic proteins and upregulation of death receptors linked with CHOP and pERK elevation.
According to National Vital Statistics System (2013), cancer is the second major cause of death in US. Despite improvements in diagnostic techniques, clinical intervention and increased public concern, the prevalence of cancer in developed countries continues to rise (
The tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL) was first identified based on its sequence homology to TNF and CD95L (
Hispolon, a phenol compound isolated from
In the present study, the ability of hispolon to modulate TRAIL-induced apoptosis in human colon cancer cells was investigated, as well as the subsequent mechanism of action. Hispolon was found to enhance TRAIL-induced apoptosis through the upregulation of pro-apoptotic proteins, downregulation of cell survival proteins and upregulation of death receptors.
Hispolon was kindly provied by Dr B.B. Aggarwal, MD Anderson Cancer Center. Soluble recombinant human TRAIL was purchased from PeproTech (Rocky Hill, NJ, USA). Penicillin, streptomycin, Dulbecco's modified Eagle's medium, RPMI-1640 and fetal bovine serum were obtained from Invitrogen (Carlsbad, CA, USA). Soluble antibodies against Bcl-2, c-FLIP, Bcl-xL, DR4, Bid, Bax, CAAT enhancer binding protein homologous protein (CHOP), p53, procaspase-3 and procaspase-8 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The DR5 antibody was purchased from ProSci, Inc. (Poway, CA, USA), anti XIAP antibody was from BD Biosciences. Antibodies against caspase-9 and cleaved caspase-8 were purchased from Cell Signaling Technology Inc. (Danvers, MA, USA). Mouse monoclonal anti-β-actin antibody was purchased from Sigma (St. Louis, MO, USA).
Human colon adenocarcinoma HCT-116, embryonic kidney carcinoma A293, multiple myeloma U-266 cells were obtained from American Type Culture Collection (Manassas, VA, USA). Human myeloid leukemia KBM-5 cells were kindly supplied by Dr Nicholas Donato (University of Michigan Comprehensive Cancer Center). Human colon cancer cell line HCT-116 was cultured in McCoy's 5A medium supplemented with 10% fetal calf serum and penicillin/streptomycin (Invitrogen). KBM-5 cells were cultured in Iscove's modified Dulbecco's medium with 15% fetal bovine serum. U-266 cells were cultured in RPMI-1640 with 10% fetal bovine serum, and A293 cells were cultured in Dulbecco's modified Eagle's medium, 100 U/ml penicillin and 100 mg/ml streptomycin.
To measure apoptosis of cells, we used the live/dead assay (Invitrogen), which assesses intracellular esterase activity and plasma membrane integrity. It is a two color fluorescence assay that simultaneously examines live and dead cells. The details of this assay were described before (
The effects of hispolon on the cytotoxic potential of TRAIL were detected by measuring mitochondrial dehydrogenase activity using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) as the substrate. This assay is based on the conversion of soluble MTT to purple colored insoluble formazan by mitochondrial dehydrogenases of viable cells. To examine the synergy between hispolon and TRAIL, cells were treated with hispolon alone (25
The effect of hispolon on the level of protein expression was studied by western blot analysis. The prepared whole cell extract was separated by SDS-PAGE. The separated proteins on the acrylamide gel were electro-transferred onto nitrocellulose membrane, stained with specific antibodies, and detected by an ECL regent (GE Healthcare, Pittsburgh, PA, USA).
HCT116 cells were treated with hispolon and washed with 1X PBS supplemented with 0.5% bovine serum albumin (BSA) after detachment with EDTA. Then cells were stained with phycoerythrin (PE)-conjugated mouse monoclonal anti-human DR4 and DR5 (clone 69036 and 71908, respectively) (R&D Systems, Minneapolis, MN, USA) for 45 min at 4°C according to the manufacturer's instructions before washing and resuspension in a fluorescence-activated cell sorting buffer (1X PBS + 0.5% BSA). The cells were analyzed by flow cytometry using an excitation wavelength of 488 nm.
Hispolon enhancement of TRAIL-induced apoptosis was examined and detected by live/dead assay in human colon cancer cells. As shown in
TRAIL is known to mediate apoptosis through the activation of caspase-8, caspase-9 and caspase-3, so the effect of hispolon on activation of these caspases and poly(ADP-ribose) polymerase (PARP) cleavage enhanced by TRAIL in HCT-116 cells was investigated. Although neither TRAIL nor hispolon had significant effect on the activation of these caspases or on cleavage of PARP, treatment of cells with combination TRAIL and hispolon enhanced activation of all caspases and ensuing PARP cleavage (
Hispolon modulation of anti-apoptotic proteins linked to TRAIL resistance in human colon cancer cells was examined. Results indicate the expression of c-FLIP, Bcl-2 and Bcl-xL was strongly downregulated in a concentration-dependent manner, while the downregulation of XIAP was not visible (
Whether hispolon regulates the expression of pro-apoptotic proteins, Bid and Bax, was examined. Results showed that hispolon upregulated the expression of Bax in a concentration- and time-dependent manner in HCT-116 cells (
To understand further possible mechanisms of regulation of TRAIL-induced apoptosis by hispolon, the effect of hispolon on expression of the death receptor was investigated in human colon cancer cells. As shown in
We investigated whether upregulation of death receptor by hispolon is specific to human colon cancer cells. As shown in
CHOP has been previously linked with the upregulation of DR5 expression (
Because p53 is known to induce the TRAIL receptor, hispolon modulation of the expression of p53 in HCT-116 cells was investigated. As shown in
Various studies reported the importance of MAPK activation in TRAIL receptor induction (
TRAIL is a very promising cytokine molecule for anticancer therapy, but it has a limitation caused by the resistance developed by certain cancer types. The development of agents that sensitize cancer cells to TRAIL is important for the success of the promising TRAIL-based cancer therapy. In the present study, we showed that hispolon enhances TRAIL-induced apoptosis of human colon cancer cells and investigate the molecular mechanism of sensitizing human cancer cells to TRAIL.
Hispolon was identified to obviously enhance the TRAIL-induced apoptosis in HCT-116, human colon cancer cells, through live/dead and MTT assays. HCT-116 belongs to type II cells, classified based on how apoptosis induction is employed upon DISC activation (
The expression of Bcl-2 and Bcl-xL was also downregulated by hispolon. These proteins have been linked to suppression of apoptosis by TRAIL, frequently found in TRAIL resistance in a variety of cancer cells including colon (
We further found that hispolon significantly induced the expression of both the TRAIL receptors, DR4 and DR5. We also demonstrated the upregulation of death receptors on the cell surface by hispolon. Moreover, hispolon-induced death receptor upregulation is not tissue-specific, which means hispolon can be applied in treatment to various cancer patients as a combination therapy with TRAIL. A few agents have been shown to upregulate the death receptor in human colon cancer cells including capsazepine (
The molecular mechanism of DR4 and DR5 induction in colon cancer cells was also investigated. Numerous mechanisms have been suggested for induction of this death receptor, including ROS generation, p53 induction and NF-κB, DNA damage-inducible transcript 3 (DDIT3), peroxisome proliferator-activated receptor and MAPK activation (
Another mechanism, induction of apoptosis through p53 was also investigated because of the importance of this pathway in the response to cell stress such as chemotherapy and radiotherapy. In this pathway, the upregulation of p53 is essential for an apoptosis event, however, hispolon could not induce p53 in HCT-116 cells. In other words, hispolon-induced apoptosis is not involved in the extrinsic apoptosis pathway, which corresponds to the type II classification of HCT-116.
Overall, our studies provide strong evidence that hispolon could potentiate TRAIL-induced apoptosis hypothetically through ROS-, ERK- and CHOP-mediated upregulation of death receptors. The enhancement of apoptosis by hispolon was also shown to be related to the upregulation of cell survival proteins and the upregulation of pro-apoptotic proteins. Hispolon combined with TRAIL may be a good candidate for anticancer therapy, however, further studies using animal models are needed to realize this combination anticancer therapy.
The present study was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and Future Planning (NRF-2013R1A1A1062064).
Hispolon enhances TRAIL-induced cell death in HCT-116, human colon cancer cells. (A) Cells were treated with hispolon (25
Hispolon regulates anti-apoptotic and pro-apoptotic protein expression. (A and B) For determining concentration-dependent regulation of anti-apoptotic proteins, HCT 116 cells were pretreated with the indicated concentration of hispolon for 24 h. For determining time-dependent modulation of anti-apoptotic proteins, cells were treated with 25
Hispolon upregulates DR4 and DR5 expression. (A and B) HCT 116 cells were treated with indicated concentrations of hispolon for 24 h (A) or with 25
Hispolon upreuglates CHOP and activates ERK. (A) Cells were treated with the indicated concentration of hispolon for 24 h. The whole cell extracts were analyzed by western blot analysis using antibodies against CHOP and p53. The same blots were stripped and reprobed with β-actin to verify equal protein loading. (B) Cells were treated with 25