Doxorubicin anti-tumor mechanisms include Hsp60 post-translational modifications leading to the Hsp60/p53 complex dissociation and instauration of replicative senescence

Highlights

• Low doxorubicin doses induce replicative senescence in human lung mucoepidermoid carcinoma cells.

• Cells treated with doxorubicin show a reduction of Hsp60 levels and an increase in its acetylation.

• Hsp60 acetylation may induce the destabilization of the pro-tumor Hsp60/p53 complex in these cells.

• p53 may exert its tumor suppressive function by activating p53-dependent cell senescence pathway via the induction of p21.

Abstract

The chaperone Hsp60 is pro-carcinogenic in certain tumor types by interfering with apoptosis and with tumor cell death. In these tumors, it is not yet known whether doxorubicin anti-tumor effects include a blockage of the pro-carcinogenic action of Hsp60. We found a doxorubicin dose-dependent viability reduction in a human lung mucoepidermoid cell line that was paralleled by the appearance of cell senescence markers. Concomitantly, intracellular Hsp60 levels decreased while its acetylation levels increased. The data suggest that Hsp60 acetylation interferes with the formation of the Hsp60/p53 complex and/or promote its dissociation, both causing an increase in the levels of free p53, which can then activate the p53-dependent pathway toward cell senescence. On the other hand, acetylated Hsp60 is ubiquitinated and degraded and, thus, the anti-apoptotic effect of the chaperonin is abolished with subsequent tumor cell death. Our findings could help in the elucidation of the molecular mechanisms by which doxorubicin counteracts carcinogenesis and, consequently, it would open new roads for the development of cancer treatment protocols targeting Hsp60.

Introduction

Replicative senescence (RS) or cellular senescence has been described as a state reached by normal mammalian fibroblasts cultured in vitro after a limited number of divisions [1]. In this state, senescent cells cannot divide and become un-responsive to growth signaling and resistant to apoptosis. Senescent cells show a flattened and enlarged shape and an increase in senescence-associated β-galactosidase (SA-β-gal) activity [2]. Cytoskeletal proteins may be involved in RS, for instance vimentin, since this protein is highly expressed in senescent fibroblasts [3].

Since cancer cells proliferate indefinitely, a requisite for their immortalization must be bypassing the physiological program that leads to RS. Abundant data support the notion that RS is a natural barrier against tumorigenesis [5], [5]. Stress-induced premature senescence is a program executed by cells in response to chemotherapy, and RS induced by DNA-damaging anticancer drugs is one of the key determinants of successful chemotherapy [6].

Heat shock proteins (Hsps) are highly expressed in a variety of cancer cells and are essential to their survival contributing to tumor cell propagation, metastasis, and protection against apoptosis [7], [8]. Several Hsps function as molecular chaperones for other proteins to prevent their aggregation after environmental stress, for example. Molecular chaperones participate in the response to anti-cancer drugs [9], [10] and are intensively studied as therapeutic targets and as diagnostic and/or prognostic markers in many types of cancer, for example breast cancer, osteosarcomas [11], ovarian carcinoma [12], and pancreatic carcinoma [13]. As stated above, the senescence program seems to represent one of the major breaks on cancer emergence and it has been demonstrated that high levels of chaperones play an important role in suppressing the senescent program, keeping the p53 signaling under control and thus allowing cancer cells to proliferate [14]. In fact, specific down-regulation of Hsp70 leads to rapid senescence of various cancer cell lines [15], and human neuroblastoma cells displayed senescence-like characteristics after inhibition of Hsp90 with tanespimycin (17AAG) [16]. The mitochondrial chaperonin Hsp60 also named HSPD1 [17] was found in multiple subcellular sites and function in the folding and intracellular trafficking of many proteins [18], [19]. Hsp60 has been found elevated in a large number of human carcinomas, which opens novel perspectives for cancer diagnosis and therapy targeting Hsp60 [20], [21]. The chaperonin can activate the immune system [22] and can have both, pro-survival and pro-death functions, depending on tissue, cell type, and apoptosis inducers [23].

Senescence and apoptosis are alternative cell fates: in some cases apoptosis is a response to intense stress while senescence is a consequence of mild damage [4]. For example, doxorubicin, an anthracycline antitumor drug widely used in clinical chemotherapy, induces senescence at low doses and apoptosis at high doses in breast cancer cells and in neonatal rat cardiomyocytes [24], [25], [26]. Hsp60 over-expression suppressed doxorubicin-induced apoptosis in cardiomyocytes [27], and doxorubicin-induced apoptosis in HeLa cells triggering Hsp60 up-regulation [23]. To the best of our knowledge no data are available that would show a direct participation of Hsp60 in RS. Here we investigated if sub-apoptotic doses of doxorubicin have an impact on the tumor-favoring Hsp60 action and, thereby, produces anti-tumor effects via the induction of RS in a human lung mucoepidermoid cell line.