Proteasome Inhibitor MG132 Enhances Cisplatin-Induced Apoptosis in Osteosarcoma Cells and Inhibits Tumor Growth
Although cisplatin has been shown to be an integral part of chemotherapy regimen in osteosarcoma (OS) treat- ment, toxicity issues and chemoresistance have hindered therapeutic development for OS. Exploring novel combination therapy methods is needed to circumvent the limitations of cisplatin alone. The proteasome inhibi- tor MG132 has shown antitumor effects in many solid tumors. However, little is known about its effects in combination with cisplatin in OS cells. In this study, we examined the effects of MG132 in combination with cisplatin in human OS cells (MG-63 and HOS). MG132 and cisplatin were applied to OS cells, respectively or jointly. The results demonstrated that MG132 markedly inhibited cell viability in a dose- and time-dependent manner, whereas viability of osteoblast cells was not affected, suggesting a selective toxicity of MG132 to cancerous cells. Mechanistically, MG132 arrested cells in the G /M phase in association with increased p21waf1 and induced cell apoptosis, which was accompanied by cleaved PARP. In addition to its apoptotic effect alone, MG132 significantly enhanced cisplatin-induced apoptosis in OS cells. Furthermore, cell viability of the com- bined application of 10 µM MG132 and 5 µg/ml cisplatin was markedly inhibited compared to that of the individual application. These events were accompanied by the downregulation of NF-B, mitochondrial anti- apoptotic protein Bcl-xL, and PI3K/Akt, which play a key role in cell survival. Finally, combination treatment of MG132 and cisplatin showed more antiproliferative effect than the single treatment in OS xenograft models. In summary, we concluded that MG132 interacted synergistically with cisplatin, which raised the possibility that combining the two drugs may represent a novel strategy in OS.
INTRODUCTION
Osteosarcoma (OS) is the most prevalent malignant bone tumor, mainly accounting for 56% of malignant bone cancers and 6% of all cancers in children and young adults1. The 5-year survival rate has increased to approxi- mately 70% with standard treatment, including a combi- nation of resection of the primary tumor, radiotherapy, and multiple chemotherapeutic agents2,3. Cisplatin is a DNA damage-inducing agent that is widely used for the treatment of solid tumors4. Most tumor cells are sensitive to the apoptotic effects induced by cisplatin. However, toxicity and drug resistance associated with chemo- therapy are major impediments affecting its efficacy. Induction of apoptosis requires additional treatment with other chemotherapeutic agents that may damage normal cells. Therefore, novel, safe, and more effective adjuvant treatments are needed to complement current treatments and improve overall survival. The ubiquitin–proteasome pathway is involved in the degradation of regulatory proteins that govern DNA repair, signal transduction, cell differentiation, and apo- ptosis5. Therefore, the proteasome represents a novel target for cancer therapy. OS cells have been reported to undergo apoptosis when treated with proteasome inhibitors6–8. Recent studies have reported that a variety of tumor cells can be sensitized to cisplatin-induced apo- ptosis by combining with proteasome inhibitors such as bortezomib9,10. In contrast to bortezomib, sensitivity of OS cells to MG132 and its synergistic effect with other agents have not been extensively studied.
In our study, we investigated the sensitivity of OS cells to MG132 and examined the efficacy of combination therapy with cisplatin and MG132. We demonstrated that MG132 potently inhibited OS cell proliferation, whereas viability of osteoblast cells was not affected. Mechanistically, MG132 induced G2/M arrest and cell apoptosis in OS cells. Furthermore, we found that MG132 significantly enhanced cisplatin-induced cell apoptosis and markedly inhibited cell viability when combined with cisplatin. We then demonstrated that the synergis- tic effects were accompanied by the downregulation of NF-B and PI3K/Akt, which play a key role in cell survival. Furthermore, we found a synergistic antitumor effect of combined treatment with MG132 and cisplatin in xenograft models. Human OS cell lines (MG-63 and HOS) and noncan- cerous osteoblast hFOB 1.19 cells were purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, P.R. China). All cell lines were maintained at 37C in a humidified incubator with 5% CO2 in Eagle’s minimum essential medium (Gibco Life Technologies, Grand Island, NY, USA), supplemented with 10% (v/v) fetal bovine serum (FBS; Gibco, Rock- ville, MD, USA), 100 U/ml penicillin, and 100 µg/ml streptomycin (Sigma-Aldrich, St. Louis, MO, USA).
Cisplatin was obtained from MCE (HaoYuan Chem- express, Shanghai, P.R. China) and was dissolved in dimethyl sulfoxide (DMSO) at 1 mM. MG132 was pur- chased from Sigma Chemical Co. (St. Louis, MO, USA) and was dissolved in DMSO at 1 mM.Proteins were extracted from cells using radioimmuno- precipitation assay (RIPA) lysis buffer containing protease inhibitor cocktail tablets (Roche, Mannheim, Germany) for 30 min on ice. Equivalent amounts of 20 µg of proteins were separated using 10% SDS-PAGE electrophoresis and transferred to nitrocellulose membranes. After block- ing with 5% skim milk in Tween–Tris-buffered saline (TBST) for 1 h, the membranes were incubated with specific primary antibodies against poly(ADP-ribose) polymerase (PARP), p21waf1, Bcl-xL, Akt, p-Akt, -actin, or GAPDH (Cell Signaling Technology, San Jose, CA, USA) at 4C overnight. Subsequently, the membranes were washed with TBST three times and incubated with horseradish peroxidase-conjugated secondary antibodies (1:5,000; Cell Signaling Technology) for 1 h at room tem- perature. Protein bands were visualized by enhanced Super Signal WestPico chemiluminescent (Pierce, Rockford, IL, USA); the relative protein expression levels were evalu- ated using Quantity One software.The sensitivity of OS and osteoblast cells to MG132 and cell proliferation were detected using cell counting kit-8 (CCK-8; Dojindo Molecular Technologies, Kumamoto, Japan).
Briefly, cells were seeded into 96-well plates at a density of 5 103 cells/well in 100 µl of medium in sep- tuple and exposed to varying concentrations of MG132 and/or cisplatin or DMSO for the indicated times. At each time point, 10 µl of CCK-8 was added into each well. After 1 h of incubation, the absorbance at 450 nm was measured using a microplate reader (iMark; Bio-Rad Laboratories, Inc., Hercules, CA, USA), and a cell growth curve was produced. Sensitivity of cells was calculated as a percentage: (AMG132/ADMSO) 100.Cell cycle distribution was evaluated by DNA content analysis using propidium iodide (PI) staining. After treat- ing with DMSO or 10 µM MG132 for 24 h, cells were detached by trypsin digestion, washed with phosphate- buffered saline (PBS), and fixed with ice-cold 70% eth- anol at −20C overnight, and then stained with 50 µg/ ml PI in the presence of RNase A at room temperature for 1 h. Intracellular DNA content was analyzed using a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA). For each sample, 10,000 events were counted. The fractions of the cells in the G1, S, and G2 phases were analyzed using CELL Quest software. The experiment was repeated three times.Treated cells were digested into suspension of single cells with EDTA-free trypsin and then washed twice with cold PBS. Cell apoptosis was evaluated by flow cytom- etry using an Annexin-V–FITC Apoptosis Detection Kit (KeyGen Biotech Co. Roche, Nanjing, P.R. China) accord- ing to the manufacturer’s instructions.
FACSCalibur flow cytometer was used to quantify the level of cell apoptosis.The morphology of OS cells treated with DMSO, 10 µM MG132, and/or 5 µg/ml cisplatin for 24 h was directly observed with optical microscopy.For nuclear extraction, cells were washed with cold PBS and suspended in hypotonic lysis buffer for 15 min. The cells were then lysed with 10% NP-40 and centrifuged; the supernatants containing the cytoplasmic extracts were removed and stored. The nuclear extracts were resuspended in hypertonic extraction buffer (10 mM HEPES, 0.42 M NaCl, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM phenyl- methylsulfonyl fluoride, 1 mM dithiothreitol plus cock- tail) and mixed intermittently for 40 min. Subsequently, the extracts were centrifuged, and the supernatants containing nuclear proteins were obtained. The protein concentrationwas measured by bicinchoninic acid (BCA) assay. A total of 10 µg of nuclear extracts was used to determine nuclear factor B (NF-B) activity using the NF-B p65 ELISA Kit (Enzo-Stressgen, Farmingdale, NY, USA) according to the manufacturer’s instructions. Unlabeled wild-type consensus NF-B oligonucleotide (50-fold) was used as a competitor for NF-B binding to monitor the specific- ity of the assay. The mutated consensus oligonucleotide should have no effect on NF-B binding and served as the negative control.Male BALB/c nude mice (aged 4 to 6 weeks old) were maintained under specific pathogen-free conditions.
The animal study was approved by the Institutional Animal Ethics Committee of the Affiliated Hospital of Hubei Polytechnic University. HOS cells (1 107) were sus- pended in 200 µl of PBS and subcutaneously injected into the right flanks of nude mice. When the tumors were visible, the mice were randomly divided into four groups (n = 5): control (0.2 ml of PBS), MG132 (2 mg/kg), cis- platin (2 mg/kg), and combination group (MG132: 2 mg/ kg + 2 mg/kg). The mice were intraperitoneally injectedwith the above reagents every 3 days for 15 days. Mice body weights were measured once a week and were used as an indicator of the systemic toxicity of the treatment. Tumor volume was measured every 2 days using a caliper and calculated by the formula volume = (length width2)/2. At the end, mice were sacrificed, and tumors were removed and weighed.Statistical analysis was performed using the SPSS 13.0 software (IBM, Armonk, NY, USA). Student’s t-tests were carried out to make a comparison between differ- ent groups. Data are expressed as mean SD. A value of p 0.05 was considered statistically significant.
RESULTS
To investigate if MG132 had any cytotoxic activity against OS (MG-63 and HOS) and osteoblast cells (hFOB 1.19), cells were treated with 0.5, 1, 5, 10, and20 µM MG132 for 12, 24, 36, 48, 60, and 72 h. As shownin Figure 1, MG132 markedly reduced the viability of MG-63 (Fig. 1A) and HOS (Fig. 1B) cells in a dose- and time-dependent manner. MG132 did not have a signifi- cant effect on the survival of osteoblast hFOB 1.19 cells (Fig. 1C). Next, we examined whether inhibition of cell proliferation was also accompanied with MG132-induced cell apoptosis and cell cycle arrest.MG132 Induced Cell G2/M Arrest and Apoptosis in OS CellsTo explore the potential mechanism by which MG132 suppressed OS cell growth, we conducted flow cytom- etry analysis to monitor the alteration of the cell cycle distribution. The results showed that MG132 elicited an increase in OS cells at the G2 phase (8.2% to 45.3% in MG-63; 7.5% to 40.7% in HOS) and a decrease at the S phase (39.4% to 14.4% in MG-63; 45.4% to 24.1%in HOS) and the G1 phase (52.4% to 40.4% in MG-63; 47.1% to 35.2% in HOS) compared with DMSO (Fig. 2A and B). Furthermore, the apoptotic rate of OS cells increased significantly to 15% compared with the control 3% in MG-63 and 2.8% to 14% in HOS cells (Fig. 2C and D). To confirm the induction of MG132-mediated cell cycle arrest and apoptosis in OS cells, we carried out Western blot to examine the expression of p21waf1 and cleaved PARP.
The accumulation of p21waf1 and cleaved forms of PARP were detected in cell lines treated with MG132 (Fig. 2E). These results indicated that suppres- sion of cell growth by MG132 is partially attributable to increased G2 phase arrest and cell apoptosis. We next examined the effect of MG132 on cisplatin-induced cyto- toxicity in OS cells.MG132 Increased Cisplatin-Induced Cytotoxicity and Growth InhibitionDMSO-treated MG-63 and HOS cells were attached to a dish under the inverted phase-contrast microscope. The cells appeared fusiform and angular (Fig. 3A). Parts of the cells became round and small with 10 µM MG132 or 5 µg/ml cisplatin (Fig. 3B and C). With the combination of MG132 and cisplatin, most of the cells became non- adherent and suspended in the culture medium (Fig. 3D). We then conducted a cell viability assay to evaluate the synergistic effects of MG132 with cisplatin. As shown in Figure 3E and F, more inhibition of cell growth was induced by the combination of MG132 and cisplatin than by either one alone.NF-B increases expression of diverse antiapoptotic molecules such as Bcl-xL. One of the major effects of proteasome inhibitors is the reduction of NF-B activ- ity.
Besides NF-B, the PI3K/Akt pathway also plays a critical role in cancer cell survival. To assess the mecha- nism of the synergistic effects, we explored the effect of MG132 on these pathways. Using Western blot analysis, we detected a significant decrease in Bcl-xL and PI3K/ Akt in OS cells 24 h after MG132 treatment (Fig. 4A). As assessed by the ELISA assay, MG132 also inhibited nearly 50% of the NF-B activity (Fig. 4B).Following the in vitro experiments, we performed HOS xenografts in nude mice. As shown in Figure 5A, treat- ment with MG132 or cisplatin alone resulted in a mod- est but suppressed tumor growth. These inhibitory events became more significant when we applied combined injection of MG132 and cisplatin. We also discovered a significantly decreased tumor weight in the combined treatment group compared with mice in the MG132/ cisplatin/PBS treatment group (Fig. 5B). In this model, none of the treatment regimens produced any obvious loss of body weight, which may serve as a sign of low toxicity (Fig. 5C).
DISCUSSION
The use of surgery and adjuvant chemotherapy has proven successful for the treatment of OS. Despite a high rate of long-term cure (65%–70%), OS is still the main reason for cancer-related mortality in children and ado- lescents. With its positive effects against numerous types of solid tumors, cisplatin has been one of the most com- monly used first-line chemotherapy drugs for the regular treatment of OS11. However, occurrence of drug resis- tance and considerable side effects make it imperative to develop less toxic and more effective approaches to overcome these limitations12,13. Herein we presented the first study on the combined treatment of MG132 and cis- platin in OS. Identification of signals and effective agents that pro- mote cell apoptosis and sensitize resistant cells may pro- vide clues for developing novel therapeutic strategies for OS therapy14,15. Ubiquitin–proteasome-mediated intracel- lular protein degradation is involved in numerous cellular processes including cell cycle, signal transduction, apo- ptosis, and transcription. Pharmacological inhibition of proteasome activity by small-molecule inhibitors exerts antitumor efficacy in vivo and induces apoptosis in tumor cells in vitro16,17. Bortezomib (also known as PS-341) has been reported as an antitumor reagent for hematological malignancies and numerous tumors18 by inducing MAPK pathway activity and apoptosis7,19. Gene expression sig- natures associated with sensitivity to bortezomib, impact of bortezomib on cancer stem cells, and its potent role in inducing endoplasmic reticulum (ER) stress responses have also been widely studied20–22. Proteasome inhibitors not only have antitumor effects but can also be combined with other chemotherapy drugs for better outcomes. Previous studies reported the effects of bortezomib on cisplatin-induced apoptosis in various types of tumor cells9,10. Epoxomicin, another proteasome inhibitor, was shown to sensitize resistant OS cells to TRAIL-induced apoptosis and significantly increase caspase 3, 8, and 9 activities.
MG132 is a peptide aldehyde proteasome inhibitor extracted from a Chinese medicinal plant. By covalently binding to the active site of the subunits, MG132 inhib- its 20S proteasome activity24,25. Yang et al provided con- vincing evidence to suggest an essential role for MG132 in inducing cell cycle arrest and apoptosis26. Recently, it was shown that MG132 enhances TRAIL-induced apo- ptosis and inhibits the invasion of OS cells27. Findings of previous studies have indicated that MG132 enhances cisplatin-induced apoptosis in esophageal squamous cancer cells and ovarian carcinoma cells in vitro and in vivo28,29. However, the effects of MG132-related lethality and its synergistic effect with cisplatin in OS cells are not fully defined. In this present study, we indicated that MG132 exhibited significant inhibitory effects on the growth of OS cells MG-63 and HOS in a dose- and time-dependent manner, whereas it had no toxicity to osteoblast hFOB
1.19 cells. MG132 also induced G2/M cell cycle arrest, which was associated with accumulation of the CDK inhibitor p21waf1, and cell apoptosis in MG-63 and HOS cells. Some studies had reported that combination of protea- some inhibitors and chemotherapy drugs could improve sensitivity to chemo drugs, not including the combina- tion of MG132 and cisplatin in OS. We then addressed these issues in the present study. Exposure of OS cells to MG132 combined with cisplatin resulted in a marked increase in the cell apoptosis compared with a single agent. Cell viability assay showed that the combination of MG132 and cisplatin may have stronger inhibitory effect than the single application group. The results from the OS xenograft studies also showed that MG132 exhibited synergistic inhibitory effects with cisplatin on the growth of OS cells in vivo.
NF-B and PI3K/Akt pathways play pivotal roles in tumor cell survival30. NF-B activation depends on phos- phorylation and ubiquitination of its inhibitory protein IB-. Proteasome inhibitors can stabilize IB- by inhi- biting the proteasome activity and ultimately inhibit the activity of NF-B. In agreement, we found that MG132 decreased the nuclear activity of NF-B, causing a reduc- tion in Bcl-xL, which is a classical NF-B-regulated mitochondrial antiapoptosis protein. Furthermore, Akt phosphorylation was also significantly inhibited by M132. However, there are two main mechanisms of cytotoxicity due to proteasome activity inhibition in cancer cells, including NF-B and a terminal unfolded protein response (UPR) due to enhanced ER stress. More studies will be needed to determine the precise molecular
mechanism involved.
In conclusion, we demonstrated that the proteasome inhibitor MG132 has important effects on OS cells by inhibiting cell growth and inducing cell apoptosis. In addition to the inhibitory activity, MG132 synergized with cisplatin in OS cells by inducing more cell apoptosis and suppressing cell/tumor growth in vivo and in vitro. This was mediated by the Leupeptin inhibition of NF-B and PI3K/ Akt. Considering that side effects and chemoresistance are the main obstacles in OS treatment, MG132 might be an effective adjuvant agent of classic chemotherapeutics.