Proteasome inhibitor PSI induces apoptosis in human mesothelioma cells
Introduction
Malignant mesothelioma (MM) can grow with either epithelioid, sarcomatoid, or mixed phenotypes. The sarcomatoid phenotype is associated with a shorter survival rate and frequently grows in a more aggressive way. The diagnosis of MM is rarely made at such a stage that treatment can be curative. Although various conventional regimens are recommended, the average survival rate improves by only a few months.
The 20S proteasome is an intracellular multicatalytic complex, which together with the two regulators PA28 (also called proteasome 11S) and PA700 (also called proteasome 19S) forms the 26S proteasome. This structure is responsible for degrading a number of different polypeptides important for cell cycle progression and apoptosis, such as tumor suppressors (p53, p21), cyclins, nuclear factor kappa B (NFκB), and mitosis-regulating proteins [1], [2], [3], [4]. The subunits of the proteasome are structurally conserved through evolution [5], and complete loss-of-function mutations in the subunits are lethal, indicating that normal proteasome function plays an essential role in normal cell physiology.
A main mechanism of action of antineoplastic agents is their ability to induce apoptosis. Although the proteasome is present in all cells, it should be noted that proteasome inhibition causes preferential apoptosis in neoplastic cells, as compared to their benign counterparts [6]. Evidence is mounting to indicate that proteasome inhibitors, such as the peptide aldehyde PSI, selectively inhibit the chymotrypsin-like activity of the proteasome and suppress the proliferation of human cancer cells by causing apoptosis and/or cell cycle arrest [7], [8], [9], [10]. Caspases are commonly activated as mediators of apoptosis [8], [11], [12], but the primary trigger of this effect is disputed. Proteasome inhibition may be beneficial in tumor therapy, not just by the induction of apoptosis, but by other mechanisms as well. Recently, it has been found that inhibition of the proteasome also counteracts angiogenesis [13].
Despite these advances, however, the effects of proteasome inhibition and possible development of apoptosis have not been studied in MM cells. In this study, we evaluated the effects of proteasome inhibitors on several MM cell lines and other cancer cell lines. We found that proteasome inhibitors reduced cell viability by inducing cell apoptosis, as shown by cell morphology, caspase 3 degradation and flow cytometry. The PSI-induced apoptosis differed between the two MM phenotypes in harmony with our previous finding that several proteasome components are differentially expressed in the respective phenotype [14], unpublished data. Thus subunits from both the α and β portions of the 20S complex and from the PA28 activator were overexpressed in epithelioid MM cells. In case such an overexpression gives the cells growth advantage, then this suggests that proteasome inhibitors may be of value in the treatment of MM, primarily with effects on tumor cells with epithelioid morphology.
Section snippets
Cell culture
The study was performed with seven human cell lines. The STAV-AB and STAV-FCS MM cell lines were provided by Dr J. Klominek (Huddinge, Sweden) and ZL34, M14K, and M28K MM cell lines by Dr K. Linnainmaa (Helsinki, Finland). The two adenocarcinomas—A549 (lung cancer) and MCF7 (breast cancer)—were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA). Epithelioid MM cells (STAV-AB) were grown in RPMI 1640-based medium supplemented with 10% (v/v) human AB serum but without
The effects of proteasome inhibitors on proliferation of MM and other cell lines
The proliferation rates varied only slightly between the cell lines, the doubling time being 34±1.4 h in the epithelioid STAV-AB sub-line, and 41±0.4 h in the STAV-FCS sub-line.
Treatment with PSI reduced the viability of the two MM cell sub-lines to different extents in a time- and concentration-dependent manner (Fig. 1). Thus exposure to PSI for 24 h resulted in an IC50 of 4.01±0.25 μM on STAV-AB cells, but 16.31±2.4 μM of the same drug was needed for a similar reduction in cell numbers on STAV-FCS
Acknowledgements
This work was supported by the Swedish Cancer Fund (grant no. 2485) and the Swedish Heart and Lung Fund.
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