The ubiquitin-proteasome system and autophagy-lysosome system are 2 major protein degradation pathways in eukaryotic cells, which are tightly linked to cancer. Proteasome inhibitors have been approved in clinical use against hematologic malignancies, but their application in solid tumors is uncertain. Moreover, the role of autophagy after proteasome inhibition is controversial.
Two proteasome inhibitors, 2 autophagy inhibitors, and 3 hepatocellular carcinoma (HCC) cell lines were investigated in the current study. In vitro, cell proliferation was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, cell apoptosis was evaluated by flow cytometry analysis of annexin-V/propidium iodide staining, and autophagy was evaluated by green fluorescent protein-light chain 3 (GFP-LC3) redistribution and LC3 Western blot analysis. In vivo, Ki-67 staining was used to detect cell proliferation, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) staining was used to detect apoptosis, and electron microscopy and p62 immunohistochemical staining were used to detect autophagy.
Proteasome inhibitors suppressed proliferation, induced apoptosis, and activated autophagy in HCC cell lines in vitro, and autophagy exerted a protective role after proteasome inhibition. In vivo, anticancer effects of bortezomib on the MHCC-97H orthotopic model (human HCC cells) were different from the effects observed on the Huh-7 subcutaneous model (human HCC cells). The autophagy inhibitor chloroquine interacted synergistically with bortezomib to suppress proliferation and induce apoptosis in both tumor models.
Hepatocellular carcinoma (HCC) is the fifth most frequent cancer and the third most common cause of death from cancer worldwide.1 Surgical resection, liver transplantation, and ablative therapies can be used as curative treatments for early stage disease, but pharmacotherapy is considered to be the final and main treatment option for patients with advanced HCC. Unfortunately, the therapeutic response to traditional chemotherapy is poor; thus, the identification of novel agents and therapeutic strategies is of the utmost importance for patients with advanced HCC.
Increased protein synthesis and degradation, both of which are required for aggressive tumor growth, are hallmarks of cancer.2 There are 2 major intracellular pathways of protein degradation in eukaryotic cells: the ubiquitin-proteasome system (UPS) and the autophagy-lysosome system (autophagy). UPS-mediated proteolysis consists of 2 steps: ubiquitination and proteasome-mediated degradation,3 which affect cell-cycle regulation proteins, tumor suppressors, oncogenes, transcription factors, and proapoptotic and antiapoptotic proteins. Thus, specific chemical inhibitors of the proteasome have emerged as effective antitumor drugs.4 The proteasome inhibitor bortezomib is now approved by the US Food and Drug Administration (FDA) and is in clinical use for the treatment of refractory multiple myeloma and mantle cell lymphoma. Several studies have identified a link between abnormal UPS activity and HCC progression. Plasma proteasome levels are considered be a reliable marker of malignant transformation of cirrhosis,5 whereas the ubiquitin ligase subunits SKP2 (S-phase kinase-associated protein 2) and CKS1 (C-terminal Src kinase 1) are associated with the prognosis of HCC,6 and ubiquitin-conjugating enzyme E2C gene is overexpressed in HCC.7 On the basis of these findings, we hypothesized that proteasome inhibitors may be attractive agents for the treatment of HCC.
Autophagy is an evolutionarily conserved, intracellular self-defense mechanism characterized by the formation of double-membraned autophagic vesicles, in which long-lived, aggregated, misfolded proteins and damaged organelles are sequestered and subsequently degraded through fusion with lysosomes. Autophagy generally functions to maintain cellular homeostasis through nutrition recycling and protein quality control.8 Increasing evidence suggests that autophagy may be activated during chemotherapies.9 Recent studies on the role of autophagy have highlighted advances in the pharmacologic manipulation of autophagy pathways as a therapeutic strategy for cancer.10, 11 However, whether such autophagy contributes to tumor cell death or is a mechanism of resistance remains uncertain and may vary, depending on stimulus type, nutrient availability, organism development, and apoptotic status.12
In this study, we evaluated the proapoptotic and antiproliferative effects of 2 proteasome inhibitors, carbobenzoxy-Leu-Leu-leucinal (MG-132) and bortezomib, on 3 HCC cell lines. Furthermore, we investigated the abilities of these proteasome inhibitors to induce autophagy and the effect of autophagy on HCC cell destiny.
MATERIALS AND METHODS
MG-132, chloroquine (CQ), carbobenzoxy-valyl-alanyl-aspartyl-(O-methyl)-fluoromethylketone (Z-VAD-fmk) (a cell-permeant pan caspase inhibitor), and 3 methyladenine (3-MA) were purchased from Sigma-Aldrich (St. Louis, Mo); bortezomib was purchased from LC Laboratories (Woburn, Mass). MG-132, Z-VAD-fmk, and bortezomib were dissolved in 100% dimethyl sulfoxide and diluted with Dulbecco modified eagle medium to the desired concentration with a final dimethyl sulfoxide concentration of 0.1% for the in vitro studies. CQ and 3-MA were dissolved in phosphate-buffered saline and diluted with Dulbecco modified eagle medium to the desired concentration.
Apoptotic cells were evaluated in vitro by annexin-V-fluorescein isothiocyanate (FITC) and propidium iodide using Annexin-V/FITC Apoptosis Detection Kit I (BD Pharmingen, San Jose, Calif) according to the manufacturer's protocol. Stained cells were then analyzed with a FACS Calibur Flow Cytometer (BD Biosciences), and the data were analyzed using FlowJo software (Tree Star Inc., Ashland, Ore).
Autophagy was assessed using green fluorescent protein-light chain 3 (GFP-LC3) redistribution and LC3 mobility shift. Redistribution of GFP-LC3 was detected using a laser confocal microscope. The number of GFP-LC3–positive dots per cell was determined in 3 independent experiments. Five randomly selected fields representing 20 cells were counted. For the LC3 mobility shift assay, cells were lysed with M-PER (Pierce Chemical Company, Rockford, Ill) and then subjected to immunoblot analysis with an antibody against LC3 (Cell Signaling Technology, Inc., Beverly, Mass).
Gene Knockdown by RNA Interference
Autophagy-related 5 homolog (Atg5) RNA interference was accomplished by transfecting MHCC-97H (human HCC cells), PLC/PRF/5 (a hepatoma cell line), and Huh-7 cells (a human HCC cell line) with specific small interfering RNA (siRNA) duplexes. Primers for the Atg5 targeting sense and the negative control sense were purchased from GenePharma Company (Shanghai, China). SiRNAs were transfected using the Lipofectamine 2000 transfection reagent (Invitrogen Corporation, Carlsbad, Calif) according to the manufacturer's protocol.
Western Blot Analysis
Briefly, the proteins from total cell lysates were separated by standard sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to polyvinylidene difluoride membranes (Millipore Corporation, Billerica, Mass). The membranes were washed, blocked, and incubated with the primary antibody anti-Atg5, anti-Beclin-1, or anti-LC3 (Cell Signaling Technology, Inc.) in a 1:1000 dilution for 16 hours at 4°C followed by incubation with horseradish peroxidase-conjugated secondary antibodies. The reactions were detected by enhanced chemiluminescence assay.
Orthotopic/Subcutaneous Tumor Model
Briefly, 5 × 106 cells were injected subcutaneously into the upper left flank region of nude mice. When the tumor reached 10 mm in length, it was minced into small pieces of equal volume (2 × 2×2 mm3). MHCC-97H tumor tissues were transplanted into the livers of 24 nude mice, and Huh-7 tissues were transplanted subcutaneously into another 24 mice. Three days later, the mice were randomly divided into 4 groups and received the following treatments through intraperitoneal injection (n = 6 in each group): 1) control group, saline 4 times weekly; 2) CQ group, 60 mg/kg 4 times weekly; 3) bortezomib group, 0.3 mg/kg twice weekly; and 4) combined group, CQ and bortezomib combination. The mice were killed on day 35. At necropsy, tumor volumes were measured for the largest diameter (a) and the smallest diameter (b) and were calculated as follows: V = a × b2/2. Immunoreactivity was analyzed using Ki-67 and p62 (Abcam, Boston, Mass) for tumor tissue. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) staining was performed using an In Situ Apoptosis Detection Kit (Oncor, Gaithersburg, Md) according to the manufacturer's instructions.
All data are reported as means ± standard deviations. Comparisons of quantitative data were analyzed using Student t tests between 2 groups or by 1-way analyses of variance for multiple groups. Differences were considered significant if the P value was < .05. All analyses were performed using SPSS statistical software (version 16.0; SPSS, Inc., Chicago, Ill).
Proteasome Inhibitors Suppress Proliferation and Induce Apoptosis in Hepatocellular Carcinoma Cell Lines
A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay demonstrated that both MG-132 and bortezomib reduced cell viability in a time-dependent and concentration-dependent manner (Fig. 1A). MHCC-97H cells had partial resistance to proteasome inhibitors with the most gradual dose-response curves, and treatment with 5 μM MG-132 or 500 nM bortezomib for 60 hours resulted in approximately 30% to 40% growth inhibition. PLC/PRF/5 cells were moderately reactive to proteasome inhibitors, and treatment with 2 μM MG-132 or 200 nM bortezomib for 48 hours led to 50% growth inhibition. Huh-7 cells were the most sensitive of the 3 tested cell types, and the dose-response curves revealed similar growth inhibition patterns at 36 hours and 60 hours. Treatment with 4 μM MG-132 and 200 nM bortezomib for 36 hours resulted in almost 50% growth inhibition. On the basis of these results, the following conditions were used in subsequent in vitro experiments: 5 μM MG-132 or 500 nM bortezomib for 60 hours in MHCC-97H cells, 2 μM MG-132 or 200 nM bortezomib for 48 hours in PLC/PRF/5 cells, and 4 μM MG-132 or 200 nM bortezomib for 36 hours in Huh-7 cells.
Flow cytometric analysis demonstrated that the percentage of annexin-V-positive cells increased significantly in MG-132-treated and bortezomib-treated cells compared with control cells (Fig. 1B). The apoptotic indices (percentage of annexin-V-positive population) in MHCC-97H cells were 33% ± 4% and 34% ± 2% after MG-132 and bortezomib treatment, respectively (Fig. 1C). The corresponding apoptotic indices were 45% ± 13% and 43% ± 9%, respectively, for PLC/PRF/5 cells and 44% ± 11% and 46% ± 10%, respectively, for Huh-7 cells. The apoptotic index decreased significantly in all 3 HCC cell lines after cotreatment with the universal caspase inhibitor Z-VAD-fmk (50 μM), suggesting that the induction of apoptosis was at least partially caspase dependent.
Proteasome Inhibitors Activate Autophagy in Hepatocellular Carcinoma Cell Lines
First, we first examined dynamic changes in Beclin-1 and LC3 after proteasome inhibition. Levels of both Beclin-1 and LC3-II proteins increased significantly after treatment of the cells with proteasome inhibitors at the specific concentrations mentioned above for 0 hours, 3 hours, 6 hours, 12 hours, or 24 hours, and the increases were especially evident at 6 hours and 12 hours (Fig. 2A).
Second, 3 HCC cell lines with stable GFP-LC3 expression were established and analyzed using confocal microscopy. The control group for each cell line demonstrated mostly diffuse cytoplasmic staining of GFP-LC3 with very few punctae. Small GFP-LC3–positive dots were apparent in the perinuclear region in all 3 cell lines after treatment with either proteasome inhibitor for 12 hours (Fig. 2B). In addition, GFP-LC3 signals revealed a transition from a diffuse cytoplasmic pattern to a punctated membrane pattern, suggesting the localization of LC3 to autophagosomes. Coincubation with the lysosomal protease inhibitor CQ, which blocks the final steps of autophagic degradation, resulted in a marked increase in the number and size of autolysosomes with a much stronger accumulation of GFP-LC3 dots. In contrast, 3-MA, which inhibits autophagosome formation, significantly decreased the number and size of visible dots in the treated cells and redistributed GFP-LC3 to the cytoplasm (Fig. 2C).
The role of proteasome inhibitor-induced autophagy in cell survival or cell death in HCC cells was examined by blocking autophagy with 3-MA or CQ in cells that were exposed to proteasome inhibitors. The combination of either proteasome inhibitor with CQ or 3-MA markedly increased the percentage of annexin-V-positive cells compared with treatment using either single agent (Fig. 3), whereas CQ or 3-MA alone had only a slight effect on cell death.
The effects of autophagy on apoptosis were investigated further using genetic modification with Atg5-specific siRNA and negative control siRNA. Western blot analyses indicated that Atg5 expression in HCC cells was silenced successfully compared with the negative control at 48 hours after siRNA transfection (Fig. 4A). Atg5 knockdown by siRNA resulted in a significant decrease in the induction of autophagosomes by proteasome inhibitors detected by GFP-LC3 analysis (data not shown). Furthermore, Atg5 siRNA-transfected cells had significantly increased vulnerability to proteasome inhibitors compared with mock and negative control siRNA-transfected cells (Fig. 4B,C).
Combined Inhibition of Proteasome and Autophagy Pathways Suppresses Hepatocellular Carcinoma Growth In Vivo
Unexpectedly, the anticancer effects of bortezomib on the HCC tumor models in vivo were dramatically different from those demonstrated in vitro. Tumor volume decreased significantly after bortezomib treatment in the MHCC-97H orthotopic model compared with the control group (1987 ± 53 mm3 vs 3097 ± 404 mm3; P < .05) (Fig. 5A). However, bortezomib produced no significant antitumor activity in the Huh-7 subcutaneous model, in which the mean tumor volume was 863 ± 290 mm3 in the control group versus 790 ± 230 mm3 in the bortezomib group (P = .85) (Fig. 5B).
It is noteworthy that the combination of bortezomib with CQ was associated with a significant decrease in tumor volume in both nude mouse models compared with animals that were treated with either agent alone. In the MHCC-97H orthotopic model, the mean tumor volume in the combination group was 980 ± 206 mm3, which was almost 50% less than that in the bortezomib group (P < .05) (Fig. 5A). Although single-agent bortezomib produced no obvious antitumor activity in the Huh-7 subcutaneous model, the mean tumor volume when bortezomib was combined with CQ was only 421 ± 100 mm3 (P < .05) (Fig. 5B). Furthermore, treatment with CQ alone had no effect on tumor growth in either tumor model.
Bortezomib Interacts Synergistically With Chloroquine to Suppress Proliferation and Induce Apoptosis in Hepatocellular Carcinoma In Vivo
TUNEL staining demonstrated that bortezomib alone resulted in a moderate increase in apoptosis, with apoptotic indexes of 5.1% ± 0.4% in the MHCC-97H orthotopic model and 3.9 ± 0.5% in the Huh-7 subcutaneous model (Fig. 6A,B). Therefore, the proapoptotic effect of bortezomib was greater in the MHCC-97H model than in the Huh-7 model. Similarly, Ki-67 staining also indicated a significant reduction in proliferative activity in the bortezomib-treated MHCC-97H orthotopic model compared with the control group (40.5% ± 4% vs 62.6% ± 1.7%; P < .05), but not in the Huh-7 subcutaneous model (76.5% ± 9.7% vs 83.6% ± 3.3%; P = .59).
Although CQ alone produced no obvious proapoptotic or antiproliferative effects in vivo, the combination of bortezomib with CQ increased proapoptotic activity. The apoptotic index in the MHCC-97H orthotopic model increased from 5.1% ± 0.4% to 8.2% ± 1% (P < .05), whereas the apoptotic index in the Huh-7 subcutaneous model increased from 3.9% ± 0.5% to 11.3% ± 3.1% (P < .05). Combination therapy also increased the proliferative inhibition: The proliferation index in MHCC-97H tumors decreased from 40.5% ± 4% (bortezomib) to 20.3% ± 0.7% (combination; P < .05), whereas the proliferative index in Huh-7 tumors decreased from 76.5 ± 9.7% to 36.8 ± 9.6% (P < .05).
To determine whether bortezomib could effectively activate and whether CQ could block autophagy in vivo, we investigated the effects of these drugs on the expression of p62 (Fig. 6A,B), which accumulates during defective autophagy. Expression levels of p62 were decreased to 0.66 in MHCC-97H tumors and to 0.81 in Huh-7 tumors after bortezomib treatment, suggesting autophagy influx in vivo. However, the combination of bortezomib with CQ caused p62 expression levels to increase to 1.37 (MHCC-97H) and 1.27 (Huh-7), demonstrating that the decrease in p62 accumulation by autophagy activation after bortezomib treatment could be blocked by CQ.
Transmission electron microscopy further indicated the dramatic accumulation of autophagic vesicles that contained electron-dense, undigested materials in the CQ group, suggesting a role for CQ in the degradation of autophagosomes/lysosome fusion blockage (Fig. 6C). The formation of multiple autophagosomes in cells in the bortezomib-treated group indicated the induction of autophagy, whereas the presence of masses of condensed chromatin and apoptotic bodies indicated an increase of apoptosis in the combination group.
Apoptosis resistance is a major obstacle to successful chemotherapy in patients with advanced HCC and is correlated with the metastatic potential of tumor cells.13 Conquering this issue, thus, is a key goal in HCC treatment. Metabolic disorders recently have become a recognized feature of cancer; therefore, targeting protein degradation pathways may represent a new therapeutic strategy.14 We examined the effects of proteasome inhibitors on HCC by observing the role of inhibition of UPS and the interlinked autophagy system on HCC apoptosis and proliferation, both in vitro and in vivo.
Consistent with most other in vitro studies,15, 16 proteasome inhibitors demonstrated antiproliferative and proapoptotic effects in all 3 tested HCC cell lines, and proliferation suppression and apoptosis induction were inversely proportional to the different metastatic potentials of the cells (MHCC-97H>PLC/PRF/5>Huh-7). In vivo, the apoptotic index of bortezomib in an MHCC-97H orthotopic model was 5%, whereas the proliferation index decreased by 20%, resulting in a reduction in mean tumor volume of 20%. This suggests that proteasome inhibitors (such as bortezomib), which have been approved by the FDA for hematologic malignancies, also may play a potential therapeutic role in solid tumors.
We were surprised to observe that the apoptotic index of bortezomib in the Huh-7 subcutaneous model was only 3.9%, and no obvious antiproliferative effects or tumor regression were observed. Excluding other possible mechanisms of molecular resistance,17, 18 we assume that the contrasting results for the MHCC-97H and Huh-7 models in vitro and in vivo may be attributable to the different tumor model types. Previous research on the association between bortezomib efficacy and tumor architecture, pharmacokinetics, and pharmacodynamics demonstrated that a lack of broad bortezomib activity in solid tumors was associated with insufficient tumor perfusion.19 It was reported that injection of bortezomib directly into human tumors implanted into mice resulted in a 70% reduction in the volume of the tumors in 40% of samples.20 Therefore, the definite anticancer effects of proteasome inhibitors on the 3 HCC cell lines in vitro may be caused by perfect drug exposure, like the experience in hematologic malignancies. However, in the in vivo models, the orthotopic tumor (MHCC-97H) had a double blood supply from the hepatic artery and the portal vein, suggesting that bortezomib exposure and local concentrations may have been much higher than in the subcutaneous Huh-7 model after intraperitoneal injection.
It was believed initially that UPS and autophagy were independent systems associated with different degradation mechanisms, substrates, and signaling pathways and with different physiologic characteristics. However, recent evidence suggests that they comprise 2 complementary and interlinked protein degradation pathways. Ubiquitinated proteins can be degraded by selective autophagy through p62 docking protein,21 and the autophagic protein LC3 can be processed by the 20S proteasome.22 In addition, proteasome inhibitors induce misfolded proteins and protein aggregates to accumulate in the endoplasmic reticulum, leading to endoplasmic reticulum stress,23 which, in turn, activates autophagy, resulting in the removal of potentially toxic, damaged proteins.24 In the current study, 2 key autophagy genes (Beclin-1 and LC3) were up-regulated by proteasome inhibitors in a time-dependent manner from 0 hours to 24 hours in the HCC cell lines. In addition, confocal microscopy provided visible evidence that GFP-LC3 punctae were increased significantly in all 3 cell lines after proteasome inhibitor treatment, indicating the activation of autophagy after proteasome inhibition. Autophagy detection in vivo has been a challenging problem for many years. However, studies have indicated that p62, also called sequestosome 1, binds directly to a central component of the autophagy machinery of LC3; thus, p62 accumulates in mouse models of defective autophagy.25 Therefore, we evaluated p62 staining in histologic sections and observed that bortezomib alone reduced p62 levels in both MHCC-97H and Huh-7 tumor models. Furthermore, p62 expression increased significantly after combined treatment with bortezomib and the autophagy inhibitor CQ. These results were consistent with the electron microscopic findings, suggesting that immunohistochemical staining for p62 may be a reliable marker of autophagy in vivo.
Although cell death resulting from progressive cellular consumption has been attributed to unrestrained autophagy, leading to the belief that autophagy is a nonapoptotic form of programmed cell death, most evidence supports autophagy as a survival pathway required for cellular viability.26 Treatment with both 3-MA and CQ, as well as siRNA knockdown of the essential autophagy gene Atg5, all potentiated proteasome inhibitor-induced cell death in vitro. Combined treatment with bortezomib and CQ resulted in a marked increase in the number of TUNEL-positive tumor cells compared with tumors that were treated with bortezomib alone. These findings indicate that autophagy activation in HCC directly contributes to the survival of cancer cells treated with proteasome inhibitors.
Targeting the protein metabolism system is revealing increased validity and multiplicity as a promising cancer therapy strategy. Disruption of the UPS by bortezomib has been introduced for the clinical treatment of hematologic malignancies, and bortezomib as an adjuvant combined with other chemotherapeutic agents also has demonstrated promising clinical efficacy in some solid tumors.27 However, to date, single proteasome inhibitor agents have proven unsatisfactory for solid tumors in clinical trials.28 The results of the current study suggest that efficient tumor perfusion, drug exposure, and autophagy inhibition can enhance the efficacy of proteasome inhibitors on HCC. The FDA-approved antimalarial agent CQ has been used safely for several decades for malaria prophylaxis and may be useful in combination with proteasome inhibitors for the treatment of HCC. Furthermore, locoregional therapy, such as hepatic arterial infusion, may improve drug exposure.
To our knowledge, the drug dose in combined therapy with bortezomib and CQ for HCC has not been reported previously. In clinical studies, patients with myeloma who received bortezomib alone at a dose of 1.3 mg/m2 on days 1, 4, 8, and 11 followed by a 10-day rest period. However, recently, Takeda Oncology and the FDA suggested these patients with moderate or severe hepatic impairment should receive bortezomib at reduced starting doses and should be monitored closely for toxicities, because bortezomib is metabolized by liver enzymes and its exposure is increased in these patients. HCC is always concomitant with cirrhosis and/or hepatitis B or C virus infection, which is compromised in liver function. Moreover, we also expected that combined therapy would further reduce drug dose and improve efficacy. Consequently, the decreased dose of bortezomib (0.3 mg/kg [0.9 mg/m2] twice weekly), which is widely adopted in in vivo research, was used in our study.
In conclusion, this study offers a novel strategy for enhancing cancer control in HCC through integration of the UPS and autophagy systems. Simultaneous targeting of the proteasome and autophagy pathways may represent a promising method for treating HCC.
This work is supported by the National Natural Science Foundation of China (N.30801103, N.81001056), the National Key Sci-Tech Project (2008ZX10002-019), the Shanghai Rising-Star Program (09QA1401100, 11QA1401200), the Shanghai Morning Light Program (10CG02) and the PhD Programs Foundation of the Ministry of Education of China (200802461028, 20100071120063).