Abstract
The lack of knowledge about molecular pathology of uterine sarcomas with a representation of 3–7% of all malignant uterine tumors prevents the establishment of effective therapy protocols. Here, we explored advanced therapeutic options to the previously discovered antitumorigenic effects of the histone deacetylase (HDAC) inhibitor suberoylanilide hydroxamic acid (SAHA) by combined treatment with the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/Apo-2L). In addition, we investigated the uterine sarcoma cell lines, MES-SA and ESS-1, regarding the underlying molecular mechanisms of SAHA and TRAIL-induced apoptosis and their resistance towards TRAIL. Compared to single SAHA or TRAIL treatment, the combination of SAHA with TRAIL led to complete cell death of both tumor cell lines after 24 to 48 hours. In contrast to single SAHA treatment, apoptosis occured faster and was more pronounced in ESS-1 cells than in MES-SA cells. Induction of SAHA- and TRAIL-induced apoptosis was accompanied by upregulation of the intrinsic apoptotic pathway via reduction of mitochondrial membrane potential, caspase-3, -6, and -7 activation, and PARP cleavage, but was also found to be partially caspase-independent. Apoptosis resistance was caused by reduced expression of caspase-8 and DR 4/TRAIL-R1 in ESS-1 and MES-SA cells, respectively, due to epigenetic silencing by DNA hypermethylation of gene promoter sequences. Treatment with the demethylating agent 5-Aza-2'-deoxycytidine or gene transfer therefore restored gene expression and increased the sensitivity of both cell lines against TRAIL-induced apoptosis. Our data provide evidence that deregulation of epigenetic silencing by histone acetylation and DNA hypermethylation might play a fundamental role in the origin of uterine sarcomas. Therefore, tumor growth might be efficiently overcome by a cytotoxic combinatorial treatment of HDAC inhibitors with TRAIL.
Highlights
Uterine sarcomas consist of several distinct histiological subtypes and are rare entities as they comprise only 3–7% of all uterine cancers but account for 20% of deaths [1]
As TRAIL acts in a similar way as tumor necrosis factor (TNF)-a by binding to the cell membrane receptors DR4/TRAIL-R1 and/or DR5/TRAIL-R2 and by transmitting an apoptotic signal via their cytoplasmic death domain [20], we investigated whether a suberoylanilide hydroxamic acid (SAHA)/TRAIL combination treatment enhances cell death in uterine sarcoma cell lines
Even a TRAIL concentration as low as 5 ng/ml caused significant growth inhibition in comparison to untreated, or SAHA treated control cells, indicating a strong complementary interaction between the two compounds. These effects largely increased with higher TRAIL concentrations and were more effective for endometrial stromal sarcomas (ESS)-1 than for MES-SA cells after 24 hours. 3 mM SAHA and 100 ng/ml TRAIL were established as a final concentration for all further experiments performed as cell viability was reduced to about 9% for ESS-1 cells, or to about 38% in MES-SA cells, respectively
Summary
Uterine sarcomas consist of several distinct histiological subtypes and are rare entities as they comprise only 3–7% of all uterine cancers but account for 20% of deaths [1]. Patients with unresectable advanced uterine sarcomas have a very poor prognosis because no effective chemotherapeutic protocols exist [4]. One reason for this might originate in the lack of information regarding molecular pathogenetic mechanisms of these tumors. Due to the rareness of the disease only few tumors have so far been characterized at the molecular level. There are currently hardly any established primary human uterine sarcoma cell lines available, in particular for ESS that can be used to investigate disease mechanisms and potential therapies
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