DAPK plays an important role in panobinostat-induced autophagy and commits cells to apoptosis under autophagy deficient conditions

The histone deacetylase inhibitor (HDACi) LBH589 has been verified as an effective anticancer agent. The identification and characterization of new targets for LBH589 action would further enhance our understanding of the molecular mechanisms involved in HDACi therapy. The role of the tumor suppressor death-associated protein kinase (DAPK) in LBH589-induced cytotoxicity has not been investigated to date. Stable DAPK knockdown (shRNA) and DAPK overexpressing (DAPK+++) cell lines were generated from HCT116 wildtype colon cancer cells. LBH589 inhibited cell proliferation, reduced the long-term survival, and up-regulated and activated DAPK in colorectal cancer cells. Moreover, LBH589 significantly suppressed the growth of colon tumor xenografts and in accordance with the in vitro studies, increased DAPK levels were detected immunohistochemically. LBH589 induced a DAPK-dependent autophagy as assessed by punctuate accumulation of LC3-II, the formation of acidic vesicular organelles, and degradation of p62 protein. LBH589-induced autophagy seems to be predominantly caused by DAPK protein interactions than by its kinase activity. Caspase inhibitor zVAD increased autophagosome formation, decreased the cleavage of caspase 3 and PARP but didn’t rescue the cells from LBH589-induced cell death in crystal violet staining suggesting both caspase-dependent as well as caspase-independent apoptosis pathways. Pre-treatment with the autophagy inhibitor Bafilomycin A1 caused caspase 3-mediated apoptosis in a DAPK-dependent manner. Altogether our data suggest that DAPK induces autophagy in response to HDACi-treatment. In autophagy deficient cells, DAPK plays an essential role in committing cells to HDACi-induced apoptosis.     

组蛋白去乙酰化酶抑制剂－FK228对人非小细胞肺癌A549细胞纺锤体检查点的影响

HDACi—FK228是一种新型抗肿瘤药物,但其作用机制研究的尚未十分明确,为进一步阐明FK228杀伤肿瘤细胞的机制,该文应用流式细胞术检测FK228对非小细胞肺癌A549细胞周期的影响：应用免疫荧光染色和免疫印迹检测FK228对检查点蛋白Bub1及BubR1定位和表达的影响;采用纺锤体检查点功能实验检测FK228对细胞检查点功能的影响。结果提示,FK228处理24h后,G2／M期细胞比例由6．35％增至19．91％;FK228能够抑制检查点蛋白Bub1及BubR1着丝粒定位并上调两种蛋白表达：纺锤体检查点功能实验提示对照组经Nocodazole或Taxol处理后G2／M期细胞比例最高分别达35．74％及29．24％,而FK228处理组经上述两种药物处理后各时间点G2／M期细胞所占比例皆明显减少（最高所占比例分别仅为7．13％及6．03％）,失去了随时相点的动态变化,提示FK228抑制了A549细胞纺锤体检查点在监测张力和黏附上的功能。该研究证实FK228能够通过影响纺锤体检查点蛋白着丝粒定位以及抑制纺锤体检查点的功能,进而干涉肿瘤细胞有丝分裂过程,有助于阐明HDACi杀伤肿瘤细胞的新机制。     

Analysis of apoptosis regulatory genes altered by histone deacetylase inhibitors in chronic lymphocytic leukemia cells

Histone deacetylases (HDACs) play a key role in the regulation of acetylation status not only of histones but also of many other non-histone proteins involved in cell cycle regulation, differentiation or apoptosis. Therefore, histone deacetylase inhibitors (HDACi) have emerged as promising anticancer agents. Herein, we report the characterization of apoptosis in B-cell chronic lymphocytic leukemia (CLL) induced by two HDACi, Kendine 92 and SAHA. Both inhibitors induce dose-, time- and caspase-dependent apoptosis through the mitochondrial pathway. Interestingly, Kendine 92 and SAHA show a selective cytotoxicity for B lymphocytes and induce apoptosis in CLL cells with mutated or deleted TP53 as effectively as in tumor cells harboring wild-type TP53. The pattern of apoptosis-related gene and protein expression profile has been characterized. It has shown to be irrespective of TP53 status and highly similar between SAHA and Kendine 92 exposure. The balance between the increased BAD, BNIP3L, BNIP3, BIM, PUMA and AIF mRNA expression levels, and decreased expression of BCL-W, BCL-2, BFL-1, XIAP and FLIP indicates global changes in the apoptosis mRNA expression profile consistent with the apoptotic outcome. Protein expression analysis shows increased levels of NOXA, BIM and PUMA proteins upon Kendine 92 and SAHA treatment. Our results highlight the capability of these molecules to induce apoptosis not only in a selective manner but also in those cells frequently resistant to standard treatments. Thus, Kendine 92 is a novel HDACi with anticancer efficacy for non-proliferating CLL cells.     

p53-dependent inhibition of TrxR1 contributes to the tumor-specific induction of apoptosis by RITA

Thioredoxin reductase 1 (TrxR1) is a key regulator in many redox-dependent cellular pathways, and is often overexpressed in cancer. Several studies have identified TrxR1 as a potentially important target for anticancer therapy. The low molecular weight compound RITA (NSC 652287) binds p53 and induces p53-dependent apoptosis. Here we found that RITA also targets TrxR1 by non-covalent binding, followed by inhibition of its activity in vitro and by inhibition of TrxR activity in cancer cells. Interestingly, a novel ~130 kDa form of TrxR1, presumably representing a stable covalently linked dimer, and an increased generation of reactive oxygen species (ROS) were induced by RITA in cancer cells in a p53-dependent manner. Similarly, the gold-based TrxR inhibitor auranofin induced apoptosis related to oxidative stress, but independently of p53 and without apparent induction of the ~130 kDa form of TrxR1. In contrast to the effects observed in cancer cells, RITA had no impact on TrxR or ROS formation in normal fibroblasts (NHDF). The inhibition of TrxR1 can sensitize tumor cells to agents that induce oxidative stress and may directly trigger cell death. Thus, our results suggest that a unique p53-dependent effect of RITA on TrxR1 in cancer cells might synergize with p53-dependent induction of pro-apoptotic genes and oxidative stress, thereby leading to a robust induction of cancer cell death, without affecting non-transformed cells.     

A Quantitative Proteomic Analysis Uncovers the Relevance of CUL3 in Bladder Cancer Aggressiveness

To identify aggressiveness-associated molecular mechanisms and biomarker candidates in bladder cancer, we performed a SILAC (Stable Isotope Labelling by Amino acids in Cell culture) proteomic analysis comparing an invasive T24 and an aggressive metastatic derived T24T bladder cancer cell line. A total of 289 proteins were identified differentially expressed between these cells with high confidence. Complementary and validation analyses included comparison of protein SILAC data with mRNA expression ratios obtained from oligonucleotide microarrays, and immunoblotting. Cul3, an overexpressed protein in T24T, involved in the ubiquitination and subsequent proteasomal degradation of target proteins, was selected for further investigation. Functional analyses revealed that Cul3 silencing diminished proliferative, migration and invasive rates of T24T cells, and restored the expression of cytoskeleton proteins identified to be underexpressed in T24T cells by SILAC, such as ezrin, moesin, filamin or caveolin. Cul3 immunohistochemical protein patterns performed on bladder tumours spotted onto tissue microarrays (n = 284), were associated with tumor staging, lymph node metastasis and disease-specific survival. Thus, the SILAC approach identified that Cul3 modulated the aggressive phenotype of T24T cells by modifying the expression of cytoskeleton proteins involved in bladder cancer aggressiveness; and played a biomarker role for bladder cancer progression, nodal metastasis and clinical outcome assessment.     

Revealing PACMA 31 as a new chemical type TrxR inhibitor to promote cancer cell apoptosis

Thioredoxin reductase (TrxR) is a pivotal regulator of redox homeostasis, while dysregulation of redox homeostasis is a hallmark for cancer cells. Thus, there is considerable potential to inhibit the aberrantly upregulated TrxR in cancer cells to discover selective cancer therapeutic agents. Nevertheless, the structural types of TrxR inhibitors presented currently are still relatively limited. We herein report that PACMA 31, previously reported to inhibit protein disulfide isomerase (PDI), is a potent TrxR inhibitor. PACMA 31 possesses a pharmacophore scaffold that is structurally different from the announced TrxR inhibitors and exhibits effective cytotoxicity against cervical cancer cells. Our results reveal that PACMA 31 selectively inhibits TrxR over the related glutathione reductase (GR) and in the presence of reduced glutathione (GSH). Further studies with mutant enzyme and molecular docking suggest that the propynamide fragment of PACMA 31 interacts covalently with the selenocysteine residue of TrxR. Moreover, PACMA 31 effectively and selectively curbs TrxR activity in cells and further stimulates the production of reactive oxygen species (ROS) at low micromolar concentrations, which in turn triggers the accumulation of oxidized thioredoxin (Trx) and GSSG in cells. Follow-up studies demonstrate that PACMA 31 targets TrxR in cells to induce oxidative stress-mediated cancer cell apoptosis. Our results provide a new structural type of TrxR inhibitor that may serve as a useful probe for investigating the biology of TrxR-implicated pathways, and uncover a new target of PACMA 31 that contributes to it becoming a candidate for cancer treatment.     

Gambogic acid induces apoptosis in hepatocellular carcinoma SMMC-7721 cells by targeting cytosolic thioredoxin reductase

The thioredoxin reductase (TrxR) isoenzymes, TrxR1 in cytosol or nucleus and TrxR2 in mitochondria, are essential mammalian selenocysteine (Sec)-containing flavoenzymes with a unique C-terminal -Gly-Cys-Sec-Gly active site. TrxRs are often overexpressed in a number of human tumors, and the reduction of their expression in malignant cells reverses tumor growth, making the enzymes attractive targets for anticancer drug development. Gambogic acid (GA), a natural product that has been used in traditional Chinese medicine for centuries, demonstrates potent anticancer activity in numerous types of human cancer cells and has entered phase II clinical trials. We discovered that GA may interact with TrxR1 to elicit oxidative stress and eventually induce apoptosis in human hepatocellular carcinoma SMMC-7721 cells. GA primarily targets the Sec residue in the antioxidant enzyme TrxR1 to inhibit its Trx-reduction activity, leading to accumulation of reactive oxygen species and collapse of the intracellular redox balance. Importantly, overexpression of functional TrxR1 in cells attenuates the cytotoxicity of GA, whereas knockdown of TrxR1 sensitizes cells to GA. Targeting of TrxR1 by GA thus discloses a previously unrecognized mechanism underlying the biological action of GA and provides useful information for further development of GA as a potential agent in the treatment of cancer.     

Analysis of apoptosis regulatory genes altered by histone deacetylase inhibitors in chronic lymphocytic leukemia cells

Histone deacetylases (HDACs) play a key role in the regulation of acetylation status not only of histones but also of many other non-histone proteins involved in cell cycle regulation, differentiation or apoptosis. Therefore, histone deacetylase inhibitors (HDACi) have emerged as promising anticancer agents. Herein, we report the characterization of apoptosis in B-cell chronic lymphocytic leukemia (CLL) induced by two HDACi, Kendine 92 and SAHA. Both inhibitors induce dose-, time- and caspase-dependent apoptosis through the mitochondrial pathway. Interestingly, Kendine 92 and SAHA show a selective cytotoxicity for B lymphocytes and induce apoptosis in CLL cells with mutated or deleted TP53 as effectively as in tumor cells harboring wild-type TP53. The pattern of apoptosis-related gene and protein expression profile has been characterized. It has shown to be irrespective of TP53 status and highly similar between SAHA and Kendine 92 exposure. The balance between the increased BAD, BNIP3L, BNIP3, BIM, PUMA and AIF mRNA expression levels, and decreased expression of BCL-W, BCL-2, BFL-1, XIAP and FLIP indicates global changes in the apoptosis mRNA expression profile consistent with the apoptotic outcome. Protein expression analysis shows increased levels of NOXA, BIM and PUMA proteins upon Kendine 92 and SAHA treatment. Our results highlight the capability of these molecules to induce apoptosis not only in a selective manner but also in those cells frequently resistant to standard treatments. Thus, Kendine 92 is a novel HDACi with anticancer efficacy for non-proliferating CLL cells.     

Downregulation of uridine-cytidine kinase like-1 decreases proliferation and enhances tumor susceptibility to lysis by apoptotic agents and natural killer cells

Natural killer (NK) cells target and kill tumor cells by direct anti-tumor cytotoxicity. NK lytic-associated molecule (NKLAM) is a protein involved in this cytolytic function. Acting as an E3 ubiquitin ligase, NKLAM binds to and ubiquitinates a novel protein, uridine-cytidine kinase like-1 (UCKL-1), targeting it for degradation. However, UCKL-1’s function in tumor cell survival and NK cell cytotoxicity is unknown. UCKL-1’s homology to uridine kinases and over expression in tumor cells suggests a role for UCKL-1 in tumor growth and/or survival. We propose that NKLAM and UCKL-1 interact in the tumor cell, where degradation of UCKL-1 leads to increased tumor cell apoptosis. Here we use RNA interference to downregulate UCKL-1 expression in K562 erythroleukemia cells. It was seen that downregulation of UCKL-1 initiated apoptosis and slowed the cell cycle, resulting in lower growth in the small interfering UCKL-1 RNA treated K562 cell culture. In addition, the chemotherapeutic agent staurosporine was seen to be more effective in inducing cell death by apoptosis in UCKL-1 depleted K562 cells compared with controls. We also found that UCKL-1 depleted K562 cells were more susceptible to NK mediated cytolysis than controls. These results indicate a role for UCKL-1 in tumor cell survival and suggest possible therapeutic potential of UCKL-1 inhibitors in cancer treatment.     

Proteomic study identified HSP 70 kDa protein 1A as a possible therapeutic target, in combination with histone deacetylase inhibitors, for lymphoid neoplasms

Histone deacetylase inhibitors (HDACi) demonstrate possible anticancer activities in various malignancies including lymphoid neoplasms. However, the anticancer effects of HDACi are often limited, and combination therapy with other drugs has been undertaken to improve the outcome of patients. Here we conducted proteomic investigation of 33 lymphoid cell lines to identify novel therapeutic targets for enhancing the effects of HDACi. Using the proteomic data in our published 2D-DIGE database, we examined the proteins associated with resistance to valproic acid (VPA). The lymphoid neoplasm cell lines in the database were grouped according to their sensitivity to VPA treatment. A comparative proteomic study of the cell line groups resulted in the identification of 10 protein spots, whose intensity was associated with chemosensitivity. Among the identified proteins, HSPA1A showed higher expression in cell lines with resistance to VPA, and the results were validated by Western blotting. In vitro experiments demonstrated that treatment with KNK-437, an inhibitor of HSPA1A, enhanced the cytotoxic effects of VPA, as well as vorinostat, in the lymphoid neoplasm cell line. Treatment with KNK-437 facilitated the apoptotic effects of VPA. In conclusion, we identified HSPA1A as a possible therapeutic target, in combination with HDACi, for lymphoid neoplasms.     

Proteomic analysis of cervical cancer cells treated with suberonylanilide hydroxamic acid

Suberonylanilide hydroxamic acid (SAHA) is an orally administered histone deacetylase inhibitor (HDACI) that has shown significant antitumour activity in a variety of tumour cells. To identify proteins involved in its antitumour activity, we utilized a proteomic approach to reveal protein expression changes in the human cervical cancer cell line HeLa following SAHA treatment. Protein expression profiles were analysed by 2-dimensional polyacrylamide gel electrophoresis (2-DE) and protein identification was performed on a MALDI-Q-TOF MS/MS instrument. As a result, a total of nine differentially expressed proteins were visualized by 2-DE and Coomassie brilliant blue (CBB) staining. Further, all the changed proteins were positively identified via mass spectrometry (MS)/MS analysis. Of these, PGAM1 was significantly downregulated in HeLa cells after treatment with SAHA. Moreover, PGAM1 has been proven to be downregulated in another cervical cancer cell line (CaSki) by western blot analysis. Together, using proteomic tools, we identified several differentially expressed proteins that underwent SAHA-induced apoptosis. These changed proteins may provide some clues to a better understanding of the molecular mechanisms underlying SAHA-induced apoptosis in cervical cancer.