Introduction
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/Apo2L) is a member of the tumor necrosis factor (TNF) superfamily and known to induce apoptosis of cancer cells without significant toxicity toward normal cells (1–3). The TRAIL induces apoptosis through an extrinsic pathway by binding to the death receptors (DR) 4 and DR5. Activation of DR4 or DR5 recruits Fas-associated death domain (FADD) protein and procaspase-8, thereby forming a death-inducing signaling complex (DISC), which leads to activation of the caspase cascade (1,4). Caspase activation can be suppressed by inhibitors of apoptosis protein (IAP) family members, as well as by anti-apoptotic B-cell lymphoma 2 (Bcl-2) family proteins (5). Application of TRAIL to cancer treatment is currently under intensive clinical evaluation. Especially, human recombinant TRAIL or high-affinity agonist monoclonal antibodies against DRs are now in phase I/II clinical trials (6–9). However, recent studies have shown that many types of cancer cells are resistant to the apoptotic effects of TRAIL including colorectal cancer (CRC) cells (10). TRAIL resistance in CRC cells can occur at several steps in signaling cascade such as deficient receptor redistribution to the membrane, mutation of caspase-8, cellular fas-associated death domain-like interleukin-1-β-converting enzyme-inhibitory protein (cFLIP) expression, Bax deficiency, or through X-linked inhibitor of apoptosis protein (XIAP) expression have been reported (11–15). Therefore, overcoming TRAIL resistance is a major challenge for the development of effective TRAIL-based therapeutic strategies.
Parthenolide (PT), a natural product, has been used for the treatment of fever and inflammatory disease. It is well known to inhibit interleukin-1 (IL-1) and tumor necrosis factor-α-mediated nuclear factor-κB (NF-κB) activation, which is responsible for its inflammatory activity (16–18). For over two decades, it is known that anticancer property of PT is through induction of apoptotic cell death in a number of human cancer cells (19–23). Apoptotic effect of PT is associated with inhibition of NF-κB and the activator of transcription 3 (STAT3), enhanced oxidative stress, and mitochondria-mediated apoptosis (19,20,24–26). In our previous studies, we found that PT can be a potential chemopreventive and therapeutic agent for CRC treatment by inducing apoptosis through mitochondrial dysfunction and inhibition of angiogenesis (20,27).
In recent years, various investigations of combined therapy using PT were reported; PT sensitizes cancer cells to the non-steroidal anti-inflammatory drugs (NSAIDs), anticancer drug toxicity and radiation (25,28–33). Previously we have shown that combination of PT and 5-FU can overcome 5-FU resistance in human CRC cells and that intra-peritoneal injection of PT and 5-FU significantly inhibits tumor growth in the xenograft model (34). However, the effect and functional role of PT on TRAIL-induced apoptosis in CRC cells has not been reported.
In this study, we evaluated effects of combination therapy with PT and TRAIL on TRAIL-sensitive and -insensitive human CRC cells to gain insight into a potential treatment for CRC, especially TRAIL-resistant CRC. We also investigated the molecular mechanism of enhancing sensitivity of TRAIL by PT.
Materials and methods
Chemicals and reagents
PT and z-VAD-FMK were from Calbiochem (San Diego, CA, USA). TRAIL was purchased from Pepprotech (Rocky Hill, NJ, USA). Parthenolide was dissolved in dimethylsulfoxide (DMSO; Sigma, St. Louis, MO, USA) to a concentration of 100 μM and stored in the dark at −20°C. Annexin-V-FITC and propidium iodide (PI) were purchased from Invitrogen (Eugene, OR, USA). Hoechst 33258 was from Sigma. Levels of DR4 and DR5 protein were analyzed using a respective specific antibody from ProSci Inc. (San Diego, CA, USA). Other antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
Cell culture and treatment
Human CRC cell lines, HT-29 and HCT 116 cells (American Type Culture Collection, Rockville, MD, USA) were employed as TRAIL-resistant and TRAIL-sensitive CRC cells, respectively. The cells were cultured in RPMI-1640 medium supplemented with 10% FBS, 100 U penicillin and 100 U streptomycin. For treatment of cells with PT or TRAIL or PT plus TRAIL, cells were sub-cultured in RPMI-1640 medium without FBS for 12 h. PT and TRAIL in the stock were diluted with FBS-free medium to achieve designated concentrations. Same concentration of DMSO was applied to the cells as a control.
Cell viability assay
Human CRC cells were plated at a density of 1.0×104 cells per well in 96-well plates. Cells were treated with PT and/or TRAIL for 24 h, the medium was removed, and 200 μl of fresh medium plus 20 μl of 3-(4, 5-dimethylthiazol-2yl)-2, 5-diphenyltetrazolium bromide (MTT, 2.5 mg dissolved in 50 μl of dimethylsulfoxide, Sigma) were added to each well. After incubation for 4 h at 37°C, the culture medium containing MTT was withdrawn and 200 μl of dimethylsulfoxide (DMSO) was added, followed by shaking until the crystals were dissolved. Viable cells were detected by measuring absorbance at 570 nm using a microplate reader (Molecular Devices, Sunnyvale, CA, USA).
Annexin-V-fluorescein staining
After being incubated with single or dual agent for 24 h, the cells were trypsinized, collected, washed with ice-cold PBS, suspended in a 500 μl Annexin V binding buffer containing 5 μl of Annexin V-FITC, and incubated for 15 min at room temperature in the dark. The fluorescence was measured using a BD LSR flow cytometer and processed with CellQuest software for analysis.
Cell cycle and sub-G1 analysis
Cell cycle and sub-G1 distribution were determined by staining of DNA with propidium iodide (PI; Sigma-Aldrich) (Ex/Em = 488 nm/617 nm). PI is a fluorescent biomolecule that can be used to stain DNA. In brief, 1×106 cells were incubated with single or dual agents for 24 h. Cells were then washed with phosphate-buffered saline (PBS) and fixed in 70% ethanol overnight. Cells were washed again with PBS and then incubated with PI (10 μg/ml) with simultaneous treatment of RNase at 37°C for 1 h. The percentage of cells in different phases of the cell cycle or having sub-G1 DNA content was measured with a BD LSR flow cytometer and analyzed using CellQuest software.
Hoechst 33258 staining
Apoptotic feature of cancer cells was assessed by determining DNA condensation using Hoechst 33258. The cells were treated with single or dual agents for 24 h and then stained with Hoechst 33258 (1 μg/ml) at 37°C for 10 min. Nuclear morphology was examined under a Confocal Laser Scanning Microscope (Carl Zeiss, Germany) to identify cells undergoing apoptosis.
Quantification of death receptor expression on cell surface
In order to quantify the cell surface expression of death receptors, DR4 and DR5, cells were harvested by trypsinization, washed in PBS and incubated for 30 min a 4°C with phycoerythrin (PE)-conjugated monoclonal anti-human DR4 and DR5 antibody (eBioscences, San Diego, CA, USA). Non-immune mouse IgG was used as the negative control. The fluorescence was measured using a BD LSR flow cytometer and processed with CellQuest software for analysis.
Cell extraction and western blotting
After being incubated with single or dual agent for 24 h, the cells were collected, washed twice with PBS, and then lysed for 30 min on ice in a lysis buffer (50 mM Tris-HCl pH 8.0, 150 mM EDTA, 1% Triton X-100, 0.5% SDS and protease inhibitor cocktail). The protein concentration in cell lysates was measured by using Protein Quantification kit from Bio-Rad. Total 50 μg proteins were loaded onto an SDS-PAGE gel. After transferring and blocking, the membrane was probed with various antibodies (anti-DR4, anti-DR5, anti-Bcl2, anti-Bax, anti-cytochrome C, anti-p53, anti-caspase-3, anti-caspase-8, anti-caspase-9 and anti-actin antibody). The binding of antibody to antigen was detected by using enhanced ECL prime (Amersham, UK), captured, and analyzed by the Las-3000 luminescent Image Analyzer (Fuji Film, Tokyo, Japan).
Statistical analysis
The data are presented as the mean ± standard error (SE) of at least three independent experiments done in duplicate. Representative blots are shown. The data were entered into the Microsoft Excel 5.0, and SPSS software was used to perform the two-tailed t-tests or the analysis of the variance, where appropriate. P-value <0.05 was considered significant.
Results
TRAIL sensitivity of human colorectal cancer cells and effect of PT on TRAIL-induced cell death
A panel of 4 human colorectal cancer cell lines (HT-29, SW480, SW620 and HCT116) were screened for TRAIL sensitivity by determining viability at various concentrations (0, 5, 10, 25, 50 or 100 ng/ml) for 24 h using the MTT assay. Of the 4 cell lines, 3 showed inhibition of viability in a dose-dependent manner to TRAIL treatment (Fig. 1A). Especially, HCT116 sensitively responded to TRAIL, showing ~70% of growth inhibition at 100 ng/ml concentration. On the contrary, treatment with TRAIL induced marginal cell death in HT-29 cells. These results indicate that HT-29 cells are TRAIL-resistant and HCT116 cells are TRAIL-sensitive.
To determine the synergistic effect of PT on TRAIL-induced cell death, HT-29 and HCT116 cells were incubated in the absence or presence of PT (10 μM) and TRAIL (with 5, 25 and 40 ng/ml) for 24 h. Co-treatment of PT with TRAIL significantly increased death of not only HCT116 cells but also HT-29 cells, suggesting that PT may sensitize HT-29 cells to TRAIL (Fig. 1B).
Effect of PT on TRAIL-induced apoptosis
To ascertain the above observations, Annexin V analysis was performed using FACScan. We found that treatment of HT-29 cells with PT and TRAIL alone induced 15.29 and 8.6% apoptosis in HT-29 cells, respectively, and 14.77 and 22.01% in HCT116 cells, respectively. In agreement with cell growth inhibition, treatment with TRAIL plus PT dramatically increased the Annexin V-positive cells (41.86%) by 5-fold than treatment with TRAIL, indicating that PT promotes TRAIL-induced apoptosis in HT-29 cells. Similar combination effect on apoptotic cell death was found in HCT116 cells (Fig. 2A).
We also evaluated cell cycle modifications induced by PT and TRAIL on TRAIL-resistant and -sensitive cells. After 24-h incubation with PT or PT plus TRAIL, cells were analyzed by PI staining using flow cytometric analysis (FCM). Treatment with PT and/or TRAIL resulted in the presence of a sub-G1 population, suggestive of apoptotic cell death. Similarly, FCM revealed that PT significantly enhanced TRAIL-induced apoptosis >3-fold in HT-29 cells (8.07 versus 27.77%). In addition, higher sub-G1 arrested cells were detected.
HCT116 cells treated with PT plus TRAIL were arrested at higher level than single treatment in sub-G1 stage of the cell cycle. Peaks accounting for 11.34 and 8.07% of HT-29 cells and 5.6 and 18.05% of HCT116 cells of the overall cell population were detectable in treated with PT or TRAIL, respectively. In addition, a peak accounting for 27.77% was observed in treated with combination of HT-29 and HCT116 cells, indicating that combination treatment dramatically promotes apoptosis (Fig. 2B).
To understand the mechanism of cell death induced by combination treatment, apoptotic nuclear morphology was evaluated after Hoechst 33258 staining. After treatment with single agent, HT-29 cells were regular in morphology and grew fully in patches and were confluent (Fig. 2C). However, treatment with PT and TRAIL together, HT-29 cells exhibited apoptotic characteristics, such as cell shrinkage, nuclear condensation and fragmentation. In HCT116 cells, treatment with TRAIL or PT plus TRAIL exhibited apoptotic nuclear morphologies while treatment with PT alone showed regular nuclear morphology.
Effect of PT on the expression of death receptor in CRC cells
In order to ascertain the mechanism by which PT sensitizes CRC cells to TRAIL, we investigated the level of expression of death receptors on the cell surface. Levels of DR4 and DR5 expression were analyzed by flow cytometry using PE conjugated anti-human DR4 and DR5 antibody (Fig. 3A). Cell surface expression of DR4 (green line) and DR5 (pink line) was found in both of cell lines. Interestingly, the level of DR5 expression was significantly higher than the DR4 level. Moreover, the level of DR5 expression on HCT116 cells was much higher than the level on HT-29 cells. Supporting the data, western blotting results were well correlated with flow cytometry analysis (Fig. 3B).
In addition, effects of PT on DR4 and DR5 expression in HT-29 and HCT116 cells were analyzed by western blotting. Treatment with PT (10 μM) markedly changed the level of DR5 protein in both cell lines in a time-dependent manner (Fig. 3C). The PT treatment increased the level of DR5 protein up to 6 h in HT-29 cells and 3 h in HCT116 cells but the effect disappeared after that. On the contrary, the level of DR4 protein did not change in either cell line after treatment with PT.
We also investigated whether treatment with PT increases surface expression of the TRAIL receptors. In these experiments, cells treated with PT (10 μM) for 3 h were incubated for 30 min with anti-human DR4 or DR5 antibody conjugated with PE and analyzed by flow cytometry. The PT treatment did not induce surface expression of DR4, while the cell surface expression of DR5 were increased in both cell lines (Fig. 3D). Together, these observations suggest that TRAIL sensitivity is influenced by the level of DR5 expression.
PT enhances TRAIL-induced apoptosis via activation of caspase cascade
Many anticancer agents are capable of initiating caspase activation and inducing apoptotic cell death (35). The effects of PT, TRAIL or PT plus TRAIL on caspase activation in TRAIL-resistant and -sensitive cells were examined. Western blot analysis of HT-29 cells treated with PT plus TRAIL revealed that levels of cleaved caspase-3, -8 and -9 were significantly increased compared the levels in the cells treated PT or TRAIL alone (Fig. 4). Although, in HCT116 cells, TRAIL alone had effects on the activation of the caspase (-3, -8 and -9) cleavage, the combination treatment significantly increased the activation of caspases.
Western blot analysis of HCT116 cells treated with TRAIL or PT plus TRAL showed that levels of cleaved caspase-3, -8 and -9 were significantly increased compared to the levels of control and PT treated cells. In addition, the decrease in the levels of caspase-3, -8 and -9 in cells treated with PT plus TRAIL was significantly blocked by pretreatment of a general caspase inhibitor, Z-VAD-FMK (Fig. 4).
PT enhances TRAIL-induced expression of proteins involved in apotosis
To evaluate the mechanisms responsible for apoptosis by combination of TRAIL and PT, we examined the expression level of several pro-apoptosis and anti-apoptosis proteins in TRAIL-resistant and -sensitive cells, HT-29 and HCT116 cells, respectively. Western blot analysis showed that the level of Bcl-2 in both cell lines was significantly decreased by treatment with PT plus TRAIL compared to the level in the cells treated with PT or TRAIL alone. In contrast, the expression of Bax was significantly increased by treatment with PT plus TRAIL compared to the level in the cells treated with PT or TRAIL alone (Fig. 4, first and second panel).
One of the consequences following the changes of Bcl-2 family members is the dissipation of mitochondrial potential and release of the mitochondrial proapoptotic protein, cytochrome C. Treatment with PT plus TRAIL significantly increased the release of cytochrome C of both of cell lines compared to that of control and single drug treated cells (Fig. 4, third panel).
In addition, the p53 gene, which is inactivated in a majority of human cancers, has been recognized as a hallmark of apoptosis (36). The level of p53 was significantly increased by treatment with PT plus TRAIL compared to the level in cells treated with PT or TRAIL alone (Fig. 4, fourth panel). These results indicated that the apoptosis induced by the combination of TRAIL and PT may be associated with the mitochondrial pathway.
Discussion
In CRC, chemotherapy is currently used to reduce tumor recurrence and prolong survival. However, due to drug resistance, investigations of new chemotherapy strategies are required. Compared to other cancer cells, CRC cells show increased resistance to chemotherapeutic agents (37). The molecular mechanisms of drug resistance in colon cancer cells are still unknown.
Drug combination therapies play a prominent role in overcoming of drug resistance in cancer treatment. Exploration of the molecular mechanisms underlying the synergistic effects achieved by a drug combination would fuel efforts to rationally develop combination therapeutics that could significantly improve patient outcomes in cancer.
Recent studies have demonstrated that PT has anticancer activity and induces apoptosis through wide range of intracellular signals in cancer cells (22,38). We have also found that PT induces apoptosis through mitochondrial dysfunction in human CRC cells and inhibits tumor growth and angiogenesis in a CRC xenograft model (20). PT is considered to be a promising candidate as a new type of chemotherapeutic agent for cancer treatment. It would be interesting to investigate whether treatment with PT alone or in combination with an anticancer drug can overcome drug resistance. In this study, we found that combination treatment with PT and TRAIL resulted in the increase of apoptosis via caspase activation and that PT enhanced TRAIL sensitivity by upregulation of DR5 in TRAIL-resistant and -sensitive cells. These observations indicate that combination of TRAIL and PT may overcome TRAIL resistance in CRC and be an effective therapeutic strategy for patients with CRC.
Interactions between TRAIL and PT have been examined in a limited number of preclinical studies. A study in breast cancer cells has demonstrated that PT reverses resistance of breast cancer cells to TRAIL through c-Jun N-terminal kinase (JNK) activation (39). PT also sensitizes hepatocellular carcinoma cells to TRAIL through inhibition of STAT3 (28). However, the effect of PT on TRAIL-induced apoptosis remains to be understood. Therefore, in this study, we focused on the mechanism of apoptosis induced by PT, evaluating regulation of TRAIL receptors and mitochondrial apoptotic pathway using TRAIL-resistant and -sensitive human CRC cells.
Our results showed that TRAIL inhibited growth of HCT116 cells (a TRAIL-sensitive cell line) in a dose-dependent manner; however, the inhibition did not occur in TRAIL-resistant HT-29 cells. These results are very similar to the findings reported on the TRAIL sensitivity of human CRC cells (40–42). Interestingly, our results also indicated that combination of PT and TRAIL reduced cell growth and increased apoptotic cell death of not only TRAIL-sensitive cells but also TRAIL-resistant cells.
TRAIL mediates apoptotic cell death through enhancing expression of the death receptors DR4 and DR5, which are expressed on the surface of cancer cells (43,44). The binding of TRAIL to DR4 and DR5 leads to activation of caspases, which in turn cleaves and activates executioner caspases that mediate apoptosis (45,46). Several studies have provided evidence that DR upregulation is a promising strategy for sensitizing TRAIL resistance in cancer cells (47–51). Garcinol, a polyisoprenylated benzophenone derivative, can potentiate TRAIL-induced apoptotic cell death of human CRC cell through upregulation of DR4 and DR5 (47). Quercetin, a ubiquitous bioactive plant flavonoid, enhances TRAIL-induced apoptotic cell death in prostate cancer cells via expression of DR5 (48,49). Snake venom toxin from Vipera lebetina turanica sensitizes cancer cells to TRAIL through upregulation of death receptors and downregulation of survival proteins (51). In the present study, our data indicated that PT markedly increased the expression of DR5 protein at early stage, while PT did not affect the expression of DR4. In particular, the effect of PT on the expression of DRs at various time-points has not been evaluated. Moreover, analysis by flow cytometry permitted to establish that PT upregulated cell surface expression of DR5 and that PT was ineffective on DR4 expression. These findings suggest that upregulation of surface expression of DR5 is the prominent event by which PT sensitizes human CRC cells to TRAIL-induced apoptosis.
Many anticancer agents are capable of initiating activation of caspase cascade and inducing apoptotic cell death (52). Caspase-3 and -9, terminal factor of apoptosis, exist as an inactive precursor in cytoplasm, which is activated during apoptosis and takes part in apoptosis induced by multiple factors. Moreover, caspase-8 activation is the initial step in the TRAIL-mediated caspase activation cascade, and lack of caspase-8 expression has been reported to cause TRAIL resistance (53). To understand the mechanism by which PT and TRAIL induce apoptosis, we examined its effect on the activation and cleavage of these caspases. The results showed that the levels of cleaved form of caspase-3, -8 and -9 were increased by combination treatment with PT and TRAIL especially in TRAIL-resistant cells, decreasing the levels of pro-forms. The cleavage of caspases was prevented by pretreatment with the pancaspase inhibitor Z-VAD-FMK. Our findings suggest that PT and TRAIL-induced apoptosis is mediated by enhancing the apoptotic sensitivity to TRAIL via caspase-dependent pathway.
Previous studies have reported that the Bcl-2 family members play an important role in PT action in CRC. The mitochondrial pathway is regulated by the Bcl-2 family, which is divided into two groups, the anti-apoptotic members (Bcl-2 and Bcl-xl) and pro-apoptotic members (Bax, BAD, BAK and Bid) (54,55). Level of Bcl-2 is often enhanced in tumors, with a possible substitution of Bcl-2 by Bcl-xL in the most aggressive tumors (56,57). Mutation in Bax has been reported in some colon cancers, the majority of which have a defect in DNA mismatch repair which is readily detected by mutations in repetitive sequences (58). Therefore, the regulation of Bcl-2 and Bax expression has an important role in chemotherapy, and it could be a measure of chemotherapeutic effect. In this study, the results showed that expression level of Bcl-2 in HCT116 and HT-29 cells treated with PT plus TRAIL was decreased while the level of Bax was increased. These results demonstrate that resistance of TRAIL CRC cells (HCT116 cells) is overcome by combining with PT and that the combination treatment-induced apoptosis is under the control of the mitochondrial pathway.
The tumor suppressor gene p53 is involved in G1 growth arrest by inducing the cyclin-dependent kinase inhibitor p21 and also in apoptosis through transactivation of the pro-apoptotic Bax gene in response to DNA-damage (58,59). The p53 gene, which is inactivated in a majority of human cancers, has been proposed as an accurate indicator of response of CRC to anticancer drug (36). In the present study, that results showed that the level of p53 was enhanced by combination treatment with PT and TRAIL in TRAILresistant and -sensitive cells.
In conclusion, we investigated effects of combination of PT and TRAIL on cell growth and apoptotic cell death using TRAIL-resistant and -sensitive human CRC cells. Treatment with PT dramatically increased the surface expression of DR5 protein in both cell types. Moreover, combination of PT and TRAIL upregulated expression of proteins involved in the mitochondrial apoptotic pathway and increased caspase activation. Taken together, these results suggest that PT sensitizes CRC cells resistant to TRAIL, therefore, we believe that combined treatment with PT and TRAIL could represent a new therapeutic strategy for CRC treatment.