Background
The 26S proteasome is a multi-subunit enzymatic complex composed of a barrel-shaped 20S core region with catalytic activity adjacent to a 19S regulatory complex [
1]. Recent investigations have revealed that the ubiquitin-proteasome pathway plays a key role in regulating the homeostasis of cellular proteins involved in cell cycle regulation, cell survival, and apoptosis. Therapeutic targeting of the proteasome pathway with the specific inhibitor bortezomib has been successful in selectively inducing apoptosis in mesothelioma and a variety of other human cancer cells, with tolerable toxicity to normal cells and tissues [
2‐
4]. Importantly, bortezomib has received US FDA approval for the treatment of patients with multiple myeloma (MM) and mantle cell lymphoma [
5].
However, cancer cell resistance to bortezomib-mediated apoptosis may limit the successful application of bortezomib as a cancer therapeutic agent. Although bortezomib shows much stronger anti-tumor activity in MM than in solid tumors, approximately 50% of MMs do not respond to this medication [
6,
7]. Moreover, many patients with MM who initially responded to bortezomib ultimately relapse with bortezomib-refractory disease [
8], suggesting that even in the cancer exhibiting the best treatment response, bortezomib resistance remains a significant obstacle to treatment efficacy.
Bortezomib (PS-341, or Velcade) is a dipeptidyl boronic acid that reversely inhibits 20S proteasome activity. In MM, the transcriptional regulatory protein nuclear factor κB (NFκB) has been proposed as a major target of bortezomib [
4,
9,
10]. Bortezomib blocks the degradation of IκB, a cytoplasmic NFκB inhibitory protein, effectively reducing NFκB translocation from the cytoplasm to the nucleus and blocking its transcriptional regulatory activity. Given the established roles of NFκB in angiogenesis, cell invasion, oncogenesis, proliferation, and inhibition of apoptosis, inhibition of this important transcription factor is widely regarded as an attractive strategy of cancer therapy and a primary mechanism of bortezomib anti-tumor activity in MM cells [
4,
9,
10]. Moreover, as a proteasome inhibitor, bortezomib is able to overcome chemoresistance or induce chemosensitization by inhibiting the NFκB functions that are typically activated by conventional chemotherapeutic agents [
9,
10]. Beyond NFκB inhibition, bortezomib also induces the intracellular unfolded protein response (UPR) [
11] and stabilizes the expression of the proapoptotic genes p53 [
12], Bim [
13], or noxa [
14], indirectly contributing to bortezomib anti-tumor activity.
Although progress has made in defining bortezomib mechanisms of action, mechanisms of bortezomib resistance in cancer are not well understood. In one early study, heat shock protein 27 (HSP27) was shown to play an important role in bortezomib resistance [
15]. Recently, evidence was reported supporting a relationship between proteasome subunit β5 (PSMB5) expression and bortezomib resistance. Bortezomib is a reversible inhibitor that primarily targets PSMB5, which is responsible for the chymotrypsin activity of the 26S proteasome. Several studies focused on acute myeloid leukemia, lymphoma, and MM have shown that a series of bortezomib-adapted cell lines developed from the above malignancies exhibit higher PSMB5 expression at the both RNA and protein levels than the respective parental bortezomib-sensitive cells [
16‐
19]. Further investigation demonstrated that inhibition of PSMB5 expression partially restored bortezomib sensitivity in resistant cells [
18].
In the present study, a novel bortezomib resistant cell line was developed from the mesothelioma cell line I-45. Our results suggest that UPR evasion together with reduced pro-apoptotic gene induction accounted for bortezomib resistance in this new bortezomib-adapted mesothelioma cell line.
Discussion
In the present study, we developed a bortezomib-resistant cell line (I-45-BTZ-R) from a bortezomib-sensitive mesothelioma cell line (I-45). I-45-BTZ-R cells showed no cross-resistance with the common chemotherapeutic drugs cisplatin, 5-fluorouracil, and doxorubicin. Moreover, we observed that the bortezomib-adapted I-45-BTZ-R cells exhibited decreased growth kinemics as compared to the parental I-45 cells. We also found that I-45-BTZ-R cells did not over express the proteasome subunit PSMB5 as compared with the parental I-45 cells. In addition, 40 nM bortezomib induced similar inhibition of proteasome activity in the bortezomib-adapted cells and the parental I-45 cells, but significantly reduced accumulation of ubiquitinated protein accumulation. Further studies revealed that relatively low doses of bortezomib did not induce UPR in the bortezomib-adapted cells, while higher doses induced UPR with concomitant cell death, as evidenced by higher protein expression of the mitochondrial chaperone Bip and the ER stress-related pro-apoptotic gene CHOP. Bortezomib treatment of I-45-BTZ-R cells also failed to induce the accumulation of the pro-apoptotic genes p53, Mcl-1S, and noxa. These results suggest that evading UPR together with reduced induction of pro-apoptotic gene expression accounts for bortezomib resistance in these bortezomib-adapted mesothelioma cells.
Bortezomib is a reversible proteasome inhibitor that primarily targets the PSMB5 subunit, which is responsible for 26S proteasome chymotrypsin activity. Several research groups have recently developed bortezomib-resistant cells representing different type of cancer, including acute myeloid leukemia, lymphoma, and MM [
16‐
19]. Most of the reported bortezomib-adapted cells have shown higher PSMB5 RNA and protein expression as compared to the respective parental bortezomib-sensitive cells. A missense point mutation has been reported in a highly conserved PSMB5 bortezomib-binding pocket and that siRNA-mediated reduction of PSMB5 expression restored bortezomib sensitivity in the bortezomib-resistant cell line [
18]. In line with higher PSMB5 expression, most reported bortezomib-adapted cells showed increased proteasome activity as compared to their respective parental cell lines [
16‐
19]. This increased activity has been used as a basis for explaining increased cell survival following a lethal challenge with proteasome inhibition. In the present study, over expression of proteasome subunit PSMB5 was not observed. We also did not find any mutations by DNA sequencing in the coding region of PSMB5 in both of the cell lines (data not shown). Accordingly, the bortezomib-adapted cells and I-45 cells showed the same degree of bortezomib-induced proteasome inhibition. I-45-BTZ-R cells showed much less accumulation of ubiquitinated proteins following bortezomib treatment. There are two possible explanations for this observation. First, an alternate protease pathway may compensate for reduced proteasome function. For example, in lymphoma, continuous inhibition of proteasome activity selected for proteasome inhibitor-resistant cells with lower proteasome activity, but higher expression of TPPII, which effectively replaced certain proteasome functions [
20,
21]. However, TPPII was not upregulated in I-45-BTZ-R cells (data not shown) and inhibition of TPPII activity using the specific inhibitor AAF-cmk had very little effect on the sensitivity of I-45-BTZ-R cells to bortezomib (Figure
2D). However, we could not exclude the possibility that the activity of other, as-yet unidentified proteases compensated for reduced proteasome function in I-45-BTZ-R cells. Second, the slower growth of I-45-BTZ-R cells may have induced a general decrease in protein synthesis, resulting in a reduction in the number of ubiquitinated proteins. This possibility may also partially explain our observation that relatively low concentrations of bortezomib did not induce ER stress and UPR in I-45-BTZ-R cells.
The ER is a eukaryotic organelle critical to the production and modification of one third of all cellular proteins. In the ER lumen, excessive accumulation of misfolded or oxidized proteins induced by ER stress leads to induction of the UPR, a protective mechanism that initially restores the luminal folding capacity of the ER, but will ultimately trigger cell death if the protective mechanism is overwhelmed. During ER stress, increased concentrations of protein chaperones, particularly Bip, can limit protein aggregation inside the ER and inhibit general protein synthesis in order to reduce the ER-Golgi network workload and the cellular damage induced by ER stress [
22,
24,
25]. CHOP, another protein marker of ER stress, functions to mediate the execution of programmed cell death [
22,
24,
25]. It has been reported that bortezomib induces apoptosis in MM [
11] and head and neck squamous carcinoma cells [
26] by activating ER stress concurrent with upregulation of Bip and CHOP. In the present study, we observed upregulation of Bip and CHOP in the bortezomib-sensitive cell line I-45 following bortezomib treatment, indicating bortezomib was able to induce ER stress in this mesothelioma cell line. However, low doses of bortezomib did not induce ER stress and apoptosis in the bortezomib-resistant cell line I-45-BTZ-R. Since proteasome inhibition resulting in the accumulation of ubiquitinated proteins is thought to induce ER stress and the UPR, reduced accumulation of ubiquitinated proteins in I-45-BTZ-R cells may have prevented ER stress induction and UPR-mediated cell death. Reduced accumulation of ubiquitinated proteins may have also decreased the stabilization of the pro-apoptotic proteins p53, Mcl-1S, and noxa in I-45-BTZ-R cells, thus further limiting bortezomib-induced apoptosis.
In agreement with the results of the present study, most other reported bortezomib-adapted cells did not exhibit much cross-resistance to most chemotherapeutic drugs [
16,
18]. These observations indicate that cancer cell resistance to bortezomib treatment can be overcome by most other therapies. Therefore, even though combination therapy using bortezomib together with chemotherapeutics does not show synergistic efficacy, such therapeutic approaches may still benefit patients through a reduction in the development of bortezomib resistance. Although expression of the multi-drug resistance proteins MDR1 and MRP1 was not detected in either I-45 or I-45-BTZ-R cell lines, we demonstrated that bortezomib can enter I-45-BTZ-R cells and access to the proteasome complex through direct assessment of proteasome activity. I-45-BTZ-R cells, which exhibit an approximately 30% decrease in basal proteasome activity as compared to I-45 cells, showed the same degree of proteasome inhibition following bortezomib treatment at doses that readily killed I-45-cells but spared I-45-BTZ-R cells.
Methods
Cell Culture and Reagents
The human sarcomatoid type mesothelioma cell line I-45 expressing wild type p53 was kindly provided by Dr. J. Testa (Fox Chase Institute, Philadelphia, PA). Cells were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum, glutamine, and antibiotics. Cells were cultured at 37°C in a humidified incubator containing a 5% CO2 atmosphere. Bortezomib was kindly provided by Millennium: The Takeda Oncology Company (Cambridge, MA) and was dissolved in phosphate buffered saline (PBS) to make a stock concentration of 100 μM.
Cell Viability Assay
Cells were seeded at a density of 5000 cells/well in 96-well plates one day before exposure to various treatments. Following treatment, cell viability was determined using an XTT cell viability assay (Cell Proliferation Kit II, Roche Molecular Biochemicals, Indianapolis, IN) according to the manufacturer's protocol and as previously described [
27].
Cell Growth Assay
Cells were seeded in 20 cm cell culture dishes at a density of 1 × 106 cells per dish. Cells were trypsinized at days 2, 4, 6, or 8 and stained with trypan blue. Viable cells were counted under a microscope using a hemocytometer.
Western Blot Analysis
Western blot analysis was performed as described previously [
27]. Rabbit polyclonal antibodies against PARP, caspase-3, Bcl-x
L, Bax, Bip, HSP27, or CHOP were purchased from Cell Signaling (Beverly, MA). Rabbit polyclonal anti-human p53 and Mcl-1 antibodies were provided by Santa Cruz Biotechnology (Santa Cruz, CA). A mouse monoclonal anti-human noxa antibody was purchased from Calbiochem (San Diego, CA). Mouse monoclonal antibodies against human 20S proteasome subunits β1, β2, β5 and anti-ubiquitin (FK2H) antibodies were obtained from Biomol (Plymouth Meeting, PA).
Small Interfering RNA Transfection
CHOP expression was silenced using a pool of four small interfering RNAs (siRNAs) directed against CHOP mRNA (Dharmacon, Lafayette, CO). Cells were transfected with 100 nM of CHOP-specific siRNA or siCONTROL Non-Targeting Pool siRNAs (Dharmacon) using the transfection reagent Dharma FECT 1 (Dharmacon) according to the manufacturer's protocol. Cells were cultured for 48 hours, and then treated simultaneously with bortezomib for an additional 72 hours.
Flow Cytometry
Cells were trypsinized, washed once with cold PBS, and then fixed with 70% ethanol overnight at 4°C. Fixed cells were suspended in PBS containing 25 μg/mL propidium iodide (Roche Diagnostics, Indianapolis, IN) and 10 μg/mL RNase A (Sigma-Aldrich, St. Louis, MO) at 3°C for 30 minutes. Flow cytometry analysis for cell cycle distribution and determination of the sub-G1 apoptotic cell population was performed as previously described [
27].
Proteasome chymotrypsin-like activity assay
Cells treated with bortezomib and untreated control cells were lysed in 20 mM Tris-HCl buffer (pH7.6) by repeated freezing in liquid nitrogen and thawing in a 37°C water bath. Cell lysate chymotrypsin-like activity was determined by measuring the release of the fluorophore 7-amido-4-methylcoumarin (AMC) from 10 μM of the substrate N-succinyl-Leu-Val-Tyr-7 (LLVY) amido-4-methylcoumarin (Sigma-Aldrich). Fluorescence was measured on a Flexstation microplate fluorometer (Molecular Devices, Sunnyvale, CA, USA) at excitation/emission wavelengths of 380/440 nm.
Statistical Analysis
Differences among treatment groups were assessed by analysis of variance using PRISM 4 software. P values of = 0.05 were regarded as significant.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
LZ and WRS conceived the study, coordinated its design and execution, and drafted the manuscript. LZ, CP and YC performed the cell culture, cell viability assays, immunoblots and siRNA assays. XC and JEL were involved in the overall design of the study and helped draft the manuscript. All authors read and approved the final manuscript.