Characterization of bortezomib-adapted I-45 mesothelioma cells

Abstract

Background: Bortezomib, a proteasome-specific inhibitor, has emerged as a promising cancer therapeutic agent. However, development of resistance to bortezomib may pose a challenge to effective anticancer therapy. Therefore, characterization of cellular mechanisms involved in bortezomib resistance and development of effective strategies to overcome this resistance represent important steps in the advancement of bortezomib-mediated cancer therapy.

Results: The present study reports the development of I-45-BTZ-R, a bortezomib-resistant cell line, from the bortezomib-sensitive mesothelioma cell line I-45. I-45-BTZ-R cells showed no cross-resistance to the chemotherapeutic drugs cisplatin, 5-fluorouracil, and doxorubicin. Moreover, the bortezomib-adapted I-45-BTZ-R cells had decreased growth kinemics and did not over express proteasome subunit beta5 (PSMB5) as compared to parental I-45 cells. I-45-BTZ-R cells and parental I-45 cells showed similar inhibition of proteasome activity, but I-45-BTZ-R cells exhibited much less accumulation of ubiquitinated proteins following exposure to 40 nm bortezomib. Further studies revealed that relatively low doses of bortezomib did not induce an unfolded protein response (UPR) in the bortezomib-adapted cells, while higher doses induced UPR with concomitant cell death, as evidenced by higher expression of the mitochondrial chaperone protein Bip and the endoplasmic reticulum (ER) stress-related pro-apoptotic protein CHOP. In addition, bortezomib exposure did not induce the accumulation of the pro-apoptotic proteins p53, Mcl-1S, and noxa in the bortezomib-adapted cells.

Conclusion: These results suggest that UPR evasion, together with reduced pro-apoptotic gene induction, accounts for bortezomib resistance in the bortezomib-adapted mesothelioma cell line I-45-BTZ-R.

Figure 1

Characterization of the bortezomib-adapted cell line I-45-BTZ-R. A: I-45 and I-45-BTZ-R cells were treated with bortezomib (3.13 nM to 400 nM) for 72 hours. Cell viability was determined after treatment using the XTT assay. Control cells were treated with PBS and their viability was set as 100%. Values are the mean ± SD of triplicate assays from two experiments. B: I-45 and I-45-BTZ-R cells were treated with 40 nM bortezomib for 24, 48, or 72 hours. Caspase-3 and PARP activation (cleavage) were analyzed by Western blot analysis. C: I-45 and I-45-BTZ-R cells were treated with bortezomib (25 nM to 200 nM) for 72 hours. Percentages of sub-G1 cells and cell cycle distribution were determined by flow cytometry analysis. Values are the mean ± SD of two experiments. D: I-45 and I-45-BTZ-R cells were seeded into 20 cm cell culture dishes (1 × 10⁶ cells per dish). At days 2, 4, 6, or 8, the cells were trypsinized and stained with trypan blue. Viable cells were counted under a microscope using a hemocytometer. Values are the mean ± SD of three experiments.

Figure 2

Expression of proteasome subunit proteins, proteasome activity, and ubiquitinated protein accumulation. A: I-45 and I-45-BTZ-R cells were treated with 40 nM bortezomib for 24, 48, or 72 hours. Expression of the proteasome subunits β1, β2, and β5 was analyzed by Western blot analysis. B: I-45 and I-45-BTZ-R cells were treated with 40 nM bortezomib for 8, 24, or 30 hours. Proteasome chymotrypsin-like activity was determined by measuring the release of the fluorophore 7-amido-4-methylcoumarin (AMC) from the substrate N-succinyl-Leu-Val-Tyr-7 (LLVY) amido-4-methylcoumarin. Values are the mean ± SD of three experiments. * p < 0.05 as compared to untreated I-45 cells. C: I-45 and I-45-BTZ-R cells were treated with 40 nM bortezomib for 24, 48, or 72 hours. Expression of ubiquinated proteins were analyzed by Western blot analysis. D: I-45-BTZ-R cells were treated with 5, 10, or 20 μM AAF-CMK with or without 40 nM bortezomib for 48 hours. Cell viability was determined following treatment using the XTT assay. Control cells were treated with PBS and their viability was set as 100%. Values are the mean ± SD of triplicate assays from two experiments.

Figure 3

Sensitivity of I-45 and I-45-BTZ-R cells to cisplatin. I-45 and I-45-BTZ-R cells were treated with cisplatin (0.63 μM to 40 μM) for 72 hours. Cell viability was determined following treatment using the XTT assay. Control cells were treated with PBS and their viability was set as 100%. Values are the mean ± SD of triplicate assays from two experiments.

Figure 4

ER stress activation and UPS induction. A: I-45 and I-45-BTZ-R cells were treated with 40 nM bortezomib for 24, 48, or 72 hours. Expression of Bip, CHOP, or HSP27 was analyzed by Western blot analysis. B: I-45 and I-45-BTZ-R cells were treated with 50,100, 200, or 400 nM bortezomib for 48 hours. Expression of Bip, CHOP, or noxa, and cleavage of caspase-3 and PARP was analyzed by Western blot analysis.

Figure 5

Effects of siRNA inhibition of CHOP expression on bortezomib-induced changes in I-45-BTZ-R cell viability. A: I-45-BTZ-R cells were transfected with 100 nM CHOP-specific siRNA or control siRNA. After 48 hours, cells were treated with 200 nM bortezomib for an additional 48 hours, and then CHOP protein expression was determined by Western blot analysis. B: I-45-BTZ-R cells were treated as described above, and then cell viability was determined using the XTT assay. Values are the mean ± SD of three experiments. * p < 0.05 as compared to treatment with bortezomib following transfection with control siRNA.

Figure 6

Bcl-2 family protein expression following the bortezomib treatment. I-45 and I-45-BTZ-R cells were treated with 40 nM bortezomib for 24, 48, or 72 hours. Expression of Bcl-xL, Mcl-1, Bax, noxa, or p53 was determined by Western blot analysis.