Cancer Letters

Cancer Letters

Volume 232, Issue 2, 8 February 2006, Pages 161-169
Cancer Letters

Proteasome inhibitor PSI induces apoptosis in human mesothelioma cells

https://doi.org/10.1016/j.canlet.2005.02.022Get rights and content

Abstract

Malignant mesothelioma is an increasingly common tumor with an almost 100% mortality rate. It is refractory to conventional treatment. We have previously shown with SSH and microarray that the mRNA expression level of proteasome is higher in epithelioid mesothelioma cell lines than in sarcomatoid ones. This study evaluates the differential apoptotic effect of proteasome inhibitors on both of these mesothelioma sub-lines. Proteasome inhibitors show substantial anti-tumor activity in some tumor cells in vitro and in vivo, but the effects on mesothelioma cells has not been studied. The viability of mesothelioma cells was reduced in a dose- and time-dependent manner by the proteasome inhibitors tested; PSI was effective with a low dose, but higher concentrations were needed for calpain inhibitor I. The epithelioid mesothelioma cells are more sensitive to the inhibitors than the sarcomatoid ones, their IC50 after 24 h of treatment with PSI being 4 and 16 μm, respectively. Other mesothelioma cell lines show similar sensitivity. PSI seemed to decrease mesothelioma viability by inducing apoptosis, as verified by cell morphology, Western blotting analysis of caspase 3 cleavage, and flow-cytometric analysis. In conclusion, PSI, a representative agent that reduces viability and induces apoptosis of mesothelioma cells, might be useful in the treatment of patients with mesothelioma, especially of epithelioid phenotype.

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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