p53-dependent and p53-independent anticancer effects of different histone deacetylase inhibitors

Background:

Histone deacetylase inhibitors (HDACi) are promising antineoplastic agents, but their precise mechanisms of actions are not well understood. In particular, the relevance of p53 for HDACi-induced effects has not been fully elucidated. We investigated the anticancer effects of four structurally distinct HDACi, vorinostat, entinostat, apicidin and valproic acid, using isogenic HCT-116 colon cancer cell lines differing in p53 status.

Methods:

Effects were assessed by MTT assay, flow-cytometric analyses of propidium iodide uptake, mitochondrial depolarisation and cell-cycle distribution, as well as by gene expression profiling.

Results:

Vorinostat was equally effective in p53 wild-type and null cells, whereas entinostat was less effective in p53 null cells. Histone deacetylase inhibitors treatment suppressed the expression of MDM2 and increased the abundance of p53. Combination treatments showed that vorinostat enhanced the cytotoxic activity of TRAIL and bortezomib, independent of the cellular p53 status. Investigations into the effects of an inhibitor of the sirtuin class of HDAC, tenovin-1, revealed that tenovin-1-mediated cell death hinged on p53.

Conclusion:

These results demonstrate that vorinostat activates p53, but does not require p53 for inducing its anticancer action. Yet they also demonstrate that entinostat-induced cytotoxic effects partially depend on p53, indicating that different HDACi have a different requirement for p53.     

A sulfate polysaccharide/TNF‐related apoptosis‐inducing ligand (TRAIL) complex for the long‐term delivery of TRAIL in poly(lactic‐co‐glycolic acid) (PLGA) microspheres

Objectives The aim was to develop a long‐term delivery system for Apo2 ligand/tumour necrosis factor‐related apoptosis‐inducing ligand (TRAIL) without chemical modification (such as pegylation). Methods A nanocomplex system between the positively charged TRAIL and the negatively charged chondroitin sulfate (CS) (CS/TRAIL) was designed and applied in poly(lactide‐co‐glycolide) (PLGA) microspheres (MSs). Key findings A nanocomplex of approximately 200 nm was easily formed in a weight ratio of 2 TRAIL to CS (TC2) at pH 5.0. The cytotoxicity of CS/TRAIL against HeLa cells was similar to that of native TRAIL. The complex also had higher loading efficiency (above 95%) in PLGA MSs prepared by the multi‐emulsion method than that of native TRAIL. The release behaviour of TRAIL from the PLGA MSs was monitored. Although the release of TRAIL from native TRAIL‐loaded PLGA MSs (TMS0) was almost complete after 3 days, TC2‐loaded PLGA MSs (TMS2) showed sustained TRAIL release without an initial burst for 10 days. The released TRAIL from TMS2 led to cytotoxicity accompanied by massive apoptosis of cancer cells. TMS2 significantly inhibited tumour growth in an in‐vivo xenograft model in mice, without any loss of body weight after treatment. Conclusions From the results, we concluded that TC‐loaded PLGA MSs have the potential for long‐term delivery of TRAIL without side effects.     

Cell death pathways – potential therapeutic targets

Programmed cell death and its morphological manifestation termed apoptosis is a conserved pathway that appears to operate in all multicellular organisms. During embryonic development, cell death is essential for successful organogenesis, and apoptosis also operates in adult organisms to maintain normal cellular homeostasis. The removal of disordered cells by a controlled cellular mechanism is especially critical in long-lived mammals that must integrate multiple physiological as well as pathological death signals. Gain- and loss-of-function models of genes in the core apoptotic pathway suggest that perturbation of cellular homeostasis can be a primary pathogenic event that results in disease. Indeed, there is now compelling evidence that insufficient apoptosis can manifest as cancer or autoimmunity, whereas accelerated cell death is evident in acute and chronic degenerative diseases, further highlighting the fact that deregulation of cell death pathways has major health implications. Not surprisingly, during the past 25 years a huge endeavour aimed at unravelling this fundamental biological process has led to major advances in our understanding of cell death pathways. Therapeutic strategies to manipulate apoptosis have immense potential and this review highlights several potentially viable drug targets for modulating cell death that have been discovered from the elegant work of many scientists in elucidating the protein components and key regulators of apoptosis signalling pathways.     

siRNA腺病毒载体沉默MEKK3基因促进TRAIL诱导肝癌HCC-9204细胞凋亡

目的研究抑制丝裂原活化蛋白激酶/细胞外信号调节激酶-激酶3（MEKK3）基因表达促进TRAIL诱导人肝癌HCC-9204细胞凋亡的作用。方法构建人MEKK3基因的siRNA重组腺病毒载体,转导人肝癌HCC-9204细胞,以RTPCR和印迹法检测MEKK3 mRNA和蛋白的表达,筛选并建立稳定沉默MEKK3基因表达的人肝癌HCC-9204细胞株,以流式细胞仪检测TRAIL诱导细胞凋亡的情况。结果①成功构建并鉴定表达人MEKK3基因的siRNA重组腺病毒载体。②建立MEKK3基因有效且稳定沉默的人肝癌HCC-9204细胞株。③500 g/LTRAIL处理MEKK3基因沉默的HCC-9204细胞株,细胞凋亡率较野生HCC-9204细胞株显著提高。结论本研究成功建立有效且稳定沉默MEKK3基因表达的人肝癌HCC-9204细胞株模型,初步证实MEKK3基因表达与TRAIL诱导肝癌细胞凋亡的敏感性相关。     

Synergistic induction of TRAIL-mediated apoptosis by anisomycin in human hepatoma cells via the BH3-only protein Bid and c-Jun/AP-1 signaling pathway

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the TNF super-family, and it has been shown that many human cancer cell lines are refractory to TRAIL-induced cell death. However, the molecular mechanisms underlying resistance are unclear. In the present study, we show that TRAIL-resistance is reversed in human hepatoma cells by anisomycin, which is known to inhibit protein synthesis and induce ribotoxic stress. Synergistic induction of apoptosis in cells treated with anisomycin plus TRAIL was associated with activation of caspases and cleavage of Bid, a pro-apoptotic BH3-only protein. Silencing of Bid expression by small interfering RNA (siRNA) significantly attenuated the loss of mitochondrial membrane potential (MMP, Δψ_m) and significantly increased induction of apoptosis in cells treated with anisomycin and TRAIL, confirming that Bid cleavage is required for the response. In addition, c-Jun/AP-1 was rapidly activated upon stimulation with anisomycin; however, the knockdown of c-Jun/AP-1 expression by c-Jun siRNA markedly reduced anisomycin plus TRAIL-induced loss of MMP and apoptosis. Taken together, the findings show that anisomycin sensitizes TRAIL-mediated hepatoma cell apoptosis via the mitochondria-associated pathway, involving the cleavage of Bid and activation of the c-Jun/AP-1 pathway, indicating that this compound can be used as an anti-tumor agent in combination with TRAIL.     

High expression of crystallin αB represents an independent molecular marker for unfavourable ovarian cancer patient outcome and impairs TRAIL‐ and cisplatin‐induced apoptosis in human ovarian cancer cells

Dysregulated apoptotic pathways are regarded as major reasons for chemoresistance development as a particular challenge in ovarian cancer therapy. In search of molecular factors affecting human ovarian cancer cell apoptosis and, consequently, patient survival, we examined tumors of 103 platinum‐/taxane‐treated ovarian cancer patients by mRNA‐array hybridization, qPCR, and immunohistochemistry. We identified high expression of crystallin αB (CRYAB), a proposed negative regulator of tumor necrosis factor‐related apoptosis inducing ligand (TRAIL)‐mediated apoptosis. By Kaplan Meier analysis, this factor turned out to be significantly associated with poor patient outcome [overall survival (OS) p = 0.001, recurrence‐free survival (RFS) p = 0.003]. Elevated hazard ratios (HR) were estimated with regard to OS (HR = 2.11, 95% CI 1.10–4.06) and RFS (HR = 1.92, 95% CI 1.07–3.47) in multivariable analyses. These associations were confirmed in independent, publicly available mRNA data comprising 431 patients for OS (p < 0.001) and 413 for RFS (p < 0.001)_. Our findings were validated by studying apoptotic events in cultured human ovarian cancer cells which were stably transfected to express elevated CRYAB levels. These data emphasized the crucial role of CRYAB in human ovarian cancer biology since TRAIL‐ as well as cisplatin‐induced apoptosis was significantly impaired as a function of enhanced CRYAB expression. Taken together, we identified CRYAB as an independent biomarker for unfavourable outcome of human ovarian cancer patients. Since TRAIL is currently tested as anti‐cancer drug and large proportions of the present patient cohort displayed low CRYAB levels in their tumors, CRYAB may enable the selection of patient subgroups benefiting most from TRAIL‐containing therapy.     

Targeting apoptosis proteins in hematological malignancies

The apoptotic machinery plays a key role in hematopoietic cell homeostasis. Terminally differentiated cells are eliminated, at least in part, by apoptosis, whereas part of the apoptotic machinery, including one or several caspases, is required to go through very specific steps of the differentiation pathways. A number of hematological diseases involve a deregulation of this machinery, which in most cases is a decrease in cell sensitivity to pro-apoptotic signals through over-expression of anti-apoptotic molecules. In some situations however, e.g. in the erythroid lineage of low grade myelodysplastic syndromes, cell sensitivity to apoptosis is increased in a death receptor-dependent manner and cell death pathways are inhibited only when these diseases progress into high grade and acute leukemia. Therapeutic strategies targeting the apoptotic machinery specifically block cell death inhibitors that are over-expressed in transformed cells, mainly Bcl-2-related proteins and Inhibitor of Apoptosis Proteins (IAPs). Another strategy is the activation of the extrinsic pathway to apoptosis, mainly through the death receptor agonist Tumor necrosis factor-Related Apoptosis Inducing Ligand (TRAIL) or agonistic antibodies targeting TRAIL receptors. The use of inhibitors of death receptors could make sense when these receptors are involved in excessive cell death or activation of survival pathways. Most of the drugs targeting apoptotic pathways introduced in clinics have demonstrated their tolerability. Their efficacy, either alone or in combination with other drugs such as demethylating agents and histone deacetylase inhibitors, is currently tested in both myeloid and lymphoid hematological diseases.     

PMA对Jurkat细胞株中可溶型分泌和膜型TRAIL表达的调节

目的:探讨乙酰肉豆蔻佛波酯(PMA)对Jurkat细胞可溶型TNF相关的凋亡诱导配体(solubletumornecrosisfac tor-relatedapoptosis inducingigand,sTRAIL)分泌和膜型TRAIL(membraneboundTRAIL,mTRAIL)表达的调节,以及二者的细胞毒作用。方法:分别采用ELISA及间接免疫荧光染色和流式细胞术分析,检测sTRAIL的分泌和mTRAIL的表达水平;用4h51Cr释放试验检测sTRAIL和mTRAIL对靶细胞Raji的细胞毒作用。结果:PMA刺激24h后jurkat细胞sTRAIL分泌和mTRAIL的表达均增加,sTRAIL分泌在刺激后48h达峰值,mTRAIL表达峰值在60h。两型TRAIL都具有对Raji细胞的细胞毒作用。结论:PKC活化剂PMA可上调Jurkat细胞sTRAIL分泌和mTRAIL的表达,两型TRAIL分子都具有杀伤靶细胞的细胞毒作用。     

Alpha-Tocopheryl succinate: toxicity and lack of anti-tumour activity in immuno-competent mice.

Alpha-tocopheryl succinate (α-TOS), an analogue of vitamin E (VitE), inhibits peritoneal human malignant mesoethelioma xenograft development in immuno-compromised mice via the induction of apoptosis of tumour cells [Tomasetti, M., Gellert, N., Procopio, A., Neuzil, J., 2004. A vitamin E analogue suppresses malignant mesothelioma in a preclinical model: a future drug against a fatal neoplastic disease? Int. J. Cancer 109, 641–642]. We tested the effect of systemic α-TOS treatment in our immuno-competent and syngeneic murine mesothelioma model. VitE analogues such as α-TOS have been developed for clinical use as supplements mainly for the treatment of VitE deficiency and are considered safe and non-toxic when taken orally. In our murine model of mesothelioma α-TOS was not only ineffective at inhibiting established tumour development at the published doses, but resulted in severe side effects characterized by both behavioural changes, intra-peritoneal abnormalities and the destruction of T cells. Toxicity of α-TOS has not been reported to date perhaps due to a lack of studies conducted in fully immuno-competent hosts. Our results suggest that the translation of animal studies to clinical treatment with α-TOS requires careful consideration.     

凋亡诱导配体TRAIL与细胞膜脂筏形成的关系

目的: 探讨肿瘤坏死因子相关凋亡诱导配体(TRAIL)与细胞膜微结构域脂筏的关系。方法: 采用间接免疫荧光流式细胞术, 分析K562细胞表面TRAIL分子的表达。用FITC标记的霍乱毒素B亚单位 (CTx)对K562细胞的脂筏进行染色; 用抗FITC单克隆抗体(mAb)交联脂筏后, 以兔抗TRAIL分子抗体及Cy3标记的羊抗兔抗体进行间接免疫荧光染色, 激光共聚焦显微镜分析TRAIL分子与脂筏微结构域的关系。结果: FITC -CTx和抗FITCmAb可使脂筏发生交联。脂筏交联的同时TRAIL分子发生聚集。动态观察表明, 抗FITC抗体作用 20min后, 脂筏发生交联, 作用 30min时交联最明显; 随着脂筏的交联, TRAIL分子发生聚集。抗FITC抗体作用 40min后, 脂筏交联程度减弱, 且TRAIL分子逐渐被排除于脂筏之外。结论: 抗FITC抗体可用于CTx-FITC脂筏染色后脂筏交联的研究。TRAIL分子可能与细胞膜脂筏微结构域有关。