Proteomics Research. Depsipeptide Mechanisms Of Killing Tumor Cells

Histone acetylation is a reversible posttranslational modification regulated by histone acetyltransferase (HAT) and histone deacetylase (HDAC). Acetylation ofε-lysine in the N-terminal regions of histone proteins relaxes the chromatin by neutralizing the positive charge, thereby enhancing access of transcription factors to promoter regions of DNA. HDAC has been identified as target proteins for cancer therapy since its overexpression in the tumor cells facilitates cell growth and proliferation by inhibiting the expression of tumor suppressor genes.Histone deacetylase inhibitors (HDACi) are new generation of chemotherapeutics, which significantly inhibit cell proliferation and induce differentiation and apoptosis of tumor cells. Depsipeptide (FK228, FR901228) is a new kind of HDACi which displays cytotoxicity in many kinds of tumor cells. Furthermore, the phase I/II clinical trials of FK228 in both hematological malignancies and solid tumors have acquired encouraging results. Though FK228 shows significant anticancer activity both in vitro and in vivo, the mechanism of FK228-induced cytotoxicity remains largely undefined. The previous studies indicate that FK228 kills tumor cells by restoring the expression of tumor suppressor genes and inducing the degradation of many kinds of oncoproteins, so the alteration of target proteins plays a key role in mediating FK228-induced cytotoxicity. In this study, we identified the proteins with significant change between control and FK228-treated H322 cells by using two dimensional electriphoresis (2-DE) and mass spectrometry (MS). In addition, based on the identification of target proteins regulated by FK228, we also evaluated the role of several identified proteins in determining HDACi sensitivity in tumor cells. Therefore, this study would provide useful guide for FK228 in the therapy of many kind of cancers.Results and Conclusions1. FK228 induces time-dependent growth arrest and apoptosis in tumor cellsIn this study, MTT assay was used to evaluate the effect of FK228 on cell growth and viability. The results indicate that FK228 time-dependently inhibited cell growth and survival in non-small cell lung cancer (H322 and H1299) cells and prostate cancer (LNCaP and PC-3) cells. The effect of FK228 on suppressing cell viability was also confirmed by the inhibition of colony formation induced by FK228 in lung cancer cells. FK228 induced time-dependent cleavage of caspase-3 and PARP in both lung and prostate cancer cells, demonstrating FK228 induces apoptosis via activating caspase pathway. FK228-induced apoptosis was also confirmed by Annexin-V staining assays. In addition, H322 and LNCaP cells are more sensitive than H1299 and PC-3 cells to FK228-induced cytotoxicity.2. Proteomic assay to identify the differentially expressed proteins induced by FK228In order to identify the target proteins of FK228, the protein profile of H322 cells in respond to FK228 treatment was determined by 2-DE and MALDI-TOF-MS. In total, 35 proteins with significant alteration between control and FK228-treated H322 cells were identified, including 20 down-regulated protein spots and 15 up-regulated spots. Among 27 identified proteins, the alteration of TrxR, HSP27, CRT, GRB2, G3BP, HMGB1, FBP1, hnRNP K, PKM2 and RCN1 determined by proteomic assays was further confirmed by RT-PCR and Western blotting. The results indicate that the alteration of most proteins determined by proteomic assays was consistent with that determined by RT-PCR and Western blotting, demonstrating that the results of proteomic assays accurately represented for FK228-induced cytotoxicity. According to the biological function, the identified proteins were classified into five clusters, including signal transduction, transcriptional regulation, metabolism, cytoskeleton and protein folding, biosynthesis and degradation. Furthermore, most identified proteins were regulated in the manner that facilitated FK228-induced cytotoxicity. 3. Identification of target proteins determining HDACi sensitivityAs H322 and LNCaP cells are more sensitive than H1299 and PC-3 cells to FK228-induced cytotoxicity, we evaluated whether TrxR, HSP27, CRT and GRB2 were involved in determining FK228 sensitivity in tumor cells. The results indicate that FK228 decreased TrxR and HSP27 in HDACi-sensitive (H322 and LNCaP) cells, but increased TrxR and had no significant effect on HSP27 in HDACi-resistant (H1299 and PC-3) tumor cells. FK228 increased the expression of CRT and GRB2 in both HDACi-sensitive and resistant tumor cells, demonstrating CRT and GRB2 had no correlation with HDACi sensitivity. Collectively, TrxR and HSP27 were involved in determining HDACi sensitivity for their differential expression.4. The role of HSP27 in regulating FK228-induced apoptosisHSP27 is a chaperone protein with significant anticancer activity and was identified as candidate protein determining HDACi sensitivity in tumor cells, so we further investigated the role of HSP27 in regulating FK228-induced cytotoxicity. HSP27 was overexpressed in H322 cells by the transfection of HSP27 plasmid, but overexpression of HSP27 did not inhibit FK228-induced cleavage of PARP. Compared with non-silencing RNA, HSP27 siRNA significantly depleted HSP27 in H1299 cells, but the depletion of HSP27 did not facilitate FK228-induced apoptosis. Collectively, HSP27 has no effect on FK228-induced cytotoxicity and is not the target protein determining HDACi sensitivity.5. The correlation of TrxR and HDACi sensitivityTrxR is a reductase which efficiently scavenges cellular ROS by reducing its substrate protein Trx indirectly or H2O2 directly, so it plays a key role in protecting cells from apoptosis induced by oxidative stress. However, it is not clear whether TrxR display its function via reducing Trx in this study, so we determined the expression of TrxR and Trx in HDACi-sensitive and resistant tumor cells after FK228 treatment. The results indicate that FK228 time-dependently downregulated TrxR in H322 cells, but upregulated it in H1299 cells at 24 hours in culture. Trx was time-dependently depleted in H1299 cells, but slightly increased in H322 cells. In addition, the alteration of TrxR reducing activity was consistent with that of TrxR protein level. Therefore, TrxR but not Trx was involved in determining HDACi sensitivity.6. The role of FK228 in regulating ROS accumulation and DNA damageTrxR is an antioxidant protein and protects cells from oxidative damage by scavenging cellular ROS. As FK228 differentially regulated TrxR in HDACi-sensitive and resistant tumor cells, we investigated the effect of FK228 on ROS accumulation and DNA damage. The results indicate that FK228 induced time-dependent ROS accumulation and DNA damage in both H322 and H1299 cells. Furthermore, H322 cells are more sensitive than H1299 cells. As ROS accumulation and DNA damage were consistent with FK228-induced apoptosis, which might play a key role in mediating FK228-induced cytotoxicity.7. The role of TrxR in regulating FK228-induced ROS accumulation and apoptosisAs the alteration of TrxR was consistent with HDACi sensitivity, so we further investigated the role of TrxR in regulating FK228-induced ROS accumulation and apoptosis. Compared to non-silencing siRNA, TrxR siRNA significantly depleted TrxR in H1299 cells. Moreover, the depletion of TrxR facilitated FK228-induced ROS accumulation and apoptosis, so TrxR is a new target protein determining HDACi sensitivity.In conclusion, we first identified the protein profile of H322 cells in respond to FK228 treatment. In total, 27 different proteins were identified and classified into five cluster, including signal transduction, transcription regulation, metabolism, skeleton and protein biosynthesis, folding and degradation. Among the identified proteins, TrxR and HSP27 altered in the manner consistent with HDACi sensitivity, but HSP27 is not the target protein determining HDACi sensitivity since either overexpression or depletion of HSP27 had no effect on FK228-induced apoptosis. The alteration of TrxR was consistent with the sensitivity of tumor cells to FK228-induced ROS accumulation, DNA damage and apoptosis. In addition, the depletion of TrxR facilitated FK228-induced ROS accumulation and apoptosis, so TrxR is a new target protein determining HDACi sensitivity.