Reactive oxygen species up-regulate p53 and Puma; a possible mechanism for apoptosis during combined treatment with TRAIL and wogonin

Background and purpose:

Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) triggers apoptotic death in a variety of cancer cells without marked toxicity to most normal cells. We previously reported that wogonin, a potent anticancer agent from a Chinese herb, up-regulates p53 in prostate cancer cells. In this study, the effects of combinations of TRAIL and wogonin on a human prostate cancer cell line LNCaP, resistant to TRAIL, was evaluated for evidence of synergy in triggering apoptosis.

Experimental approach:

Western blot assay and the ‘comet’ assay were used to study the underlying mechanisms of cell death and search for any mechanisms of enhancement of TRAIL-induced apoptosis in the presence of wogonin.

Key results:

During combined treatment with wogonin and TRAIL, cytotoxicity, poly(ADP-ribose) polymerase cleavage and caspase activation were associated with up-regulation of p53 through DNA damage and reactive oxygen species (ROS) generation. N-acetylcysteine (NAC), an antioxidant, inhibited ROS generation and synergistic interaction between TRAIL and wogonin. Experimental results in human colon cancer HCT116 cells demonstrated that p53-dependent Puma up-regulation played an important role; deficiency in either p53 or Puma prevented wogonin-enhanced TRAIL-induced apoptosis.

Conclusions and implications:

The present studies suggest that wogonin enhances TRAIL-induced cytotoxicity through up-regulation of p53 and Puma, mediated by ROS.     

Cardamonin sensitizes tumour cells to TRAIL through ROS- and CHOP-mediated up-regulation of death receptors and down-regulation of survival proteins

BACKGROUND AND PURPOSE

TNF-related apoptosis-inducing ligand (TRAIL) is currently in clinical trials as a treatment for cancer, but development of resistance is a major drawback. Thus agents that can overcome resistance to TRAIL are urgently needed. Cardamonin (2′,4′-dihydroxy-6′-methoxychalcone) has been shown to affect cell growth by modulating various cell signalling pathways. Hence, we investigated the effect of cardamonin on the actions of TRAIL.

EXPERIMENTAL APPROACH

The effect of cardamonin on TRAIL was measured by plasma membrane integrity, phosphatidylserine exposure, mitochondrial activity, and activation of caspase-8, caspase-9, and caspase-3 in human colon cancer cells.

KEY RESULTS

Cardamonin potentiated TRAIL-induced apoptosis and this correlated with up-regulation of both the TRAIL death receptor (DR) 4, 5 at mRNA and protein levels. TRAIL-decoy receptor DcR1 was down-regulated by cardamonin. Induction of DRs by cardamonin occurred in a variety of cell types. Gene silencing of the DRs by small interfering RNA (siRNA) abolished the effect of cardamonin on TRAIL-induced apoptosis, suggesting that sensitization was mediated through the DR. Induction of the DR by cardamonin was p53-independent but required CCAAT/enhancer binding protein homologous protein (CHOP); cardamonin induced CHOP, and its silencing by siRNA eliminated the induction of DR5. Cardamonin increased the production of reactive oxygen species (ROS) and quenching ROS abolished its induction of receptors and enhancement of TRAIL-induced apoptosis. Cardamonin also decreased the expression of various cell survival proteins.

CONCLUSIONS AND IMPLICATIONS

Cardamonin potentiates TRAIL-induced apoptosis through ROS-CHOP-mediated up-regulation of DRs, decreased expression of decoy receptor and cell survival proteins. Thus, cardamonin has the potential to make TRAIL more effective as an anticancer therapy.     

TRAIL receptor signaling and therapeutics

Importance of the field: TNF-related apoptosis-inducing ligand (TRAIL) is a member of the TNF family of cytokines, which can induce apoptotic cell death in a variety of tumor cells by engaging specific death receptors, TRAIL-R1 and TRAIL-R2, while having low toxicity towards normal cells. There is interest in cancer therapy inducing cell death by activation of the death-receptor-mediated apoptotic pathway while avoiding decoy-receptor-mediated neutralization of the signal. This has led to the development of a number of receptor-specific TRAIL-variants and agonistic antibodies. Some of these soluble recombinant TRAIL and agonist antibodies targeting TRAIL-R1 and/or TRAIL-R2 are progressing in clinical trials. In addition, TRAIL-resistant tumors can be sensitized to TRAIL by a combination of TRAIL or agonistic antibodies with chemotherapeutic agents, targeted small molecules or irradiation.

Areas covered in this review: Recent advances in developing TRAIL or its agonist receptor antibodies in cancer therapy. We also discuss combination therapies in overcoming TRAIL resistance in cancer cells.

What the reader will gain: Knowledge of current clinical trials, the promise and obstacles in the future development of therapies affecting TRAIL signaling pathways.

Take home message: Cancer therapeutics targeting the TRAIL/TRAIL receptor signaling pathway hold great promise for molecularly targeted pro-apoptotic anti-cancer therapy.     

TRAIL in cancer therapy: present and future challenges

Since its identification in 1995, TNF-Related apoptosis-inducing ligand (TRAIL) has sparked growing interest in oncology due to its reported ability to selectively trigger cancer cell death. In contrast to other members of the TNF superfamily, TRAIL administration in vivo is safe. The relative absence of toxic side effects of this naturally occurring cytokine, in addition to its antitumoural properties, has led to its preclinical evaluation. However, despite intensive investigations, little is known in regards to the mechanisms underlying TRAIL selectivity or efficiency. An appropriate understanding of its physiological relevance, and of the mechanisms controlling cancer cells escape from TRAIL-induced cell death, will be required to optimally use the cytokine in clinics. The present review focuses on recent advances in the understanding of TRAIL signal transduction and discusses the existing and future challenges of TRAIL-based cancer therapy development.     

TRAIL的诱导肿瘤细胞凋亡与临床

TRAIL是最近发现的属肿瘤坏死因子家族新成员 ,它能选择性地杀伤多种肿瘤细胞而对正常细胞没有细胞毒性。它的作用机制主要通过细胞膜的表面死亡受体DR4和DR5介导细胞的凋亡信号 ,激活细胞内的多种诱导细胞凋亡蛋白的表达。主要的途径是通过TRAIL与死亡受体结合后促进DISC的形成和caspase8的激活 ,然后启动非依赖线粒体的凋亡途径和线粒体的凋亡途径。非线粒体凋亡途径通过激活的csapase8直接激活caspase3,从而启动细胞的凋亡。线粒体依赖途径通过激活的tBID导致细胞色素C的释放 ,与Apaf1和caspase9形成凋亡小体导致细胞的凋亡。通过以往的研究表明 ,非融合表达的TRAIL能明显诱导多种肿瘤细胞的凋亡 ,对正常人和猴的肝细胞没有细胞毒性 ,提示非融合表达的人TRAIL蛋白可能是未来最有发展前途的抗肿瘤药物     

Targeting TRAIL in the treatment of cancer: new developments

Introduction: While apoptosis is critical for maintaining homeostasis in normal cells, defective apoptosis contributes to the survival of cancer cells. TNF-related apoptosis-inducing ligand (TRAIL)-targeted therapy has attracted significant effort for treating cancer, but the clinical results have revealed limitations. The authors review the current status of development of TRAIL-targeted therapy with an outlook towards the future.

Areas covered: Recombinant human proteins, small molecules and agonistic monoclonal antibodies targeting death receptors that trigger TRAIL-mediated apoptosis are covered in this article. The authors review both intrinsic and extrinsic apoptotic pathways, highlighting how the apoptosis serves as a promising therapeutic target. They also review different categories of TRAIL pathway targeting agents and provide a brief overview of clinical trials using these agents. The authors discuss the limitations of conventional approaches for targeting the TRAIL pathway as well as future directions.

Expert opinion: The development of better combination partners for pro-apoptotic TRAIL pathway modulators including novel agents inhibiting anti-apoptotic molecules or targeting alternative resistance pathways may improve the chances for anti-tumor responses in the clinic. Developing predictive biomarkers via circulating tumor cells/DNA, apoptosis signal products, and genetic signatures/protein biomarkers from tumor tissue are also suggested as future directions.     

TRAIL and curcumin codelivery nanoparticles enhance TRAIL-induced apoptosis through upregulation of death receptors

Active targeting nanoparticles were developed to simultaneously codeliver tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and Curcumin (Cur). In the nanoparticles (TRAIL-Cur-NPs), TRAIL was used as both active targeting ligand and therapeutic agent, and Cur could upregulate death receptors (DR4 and DR5) to increase the apoptosis-inducing effects of TRAIL. Compared with corresponding free drugs, TRAIL-Cur-NPs group showed enhanced cellular uptake, cytotoxicity and apoptosis induction effect on HCT116 colon cancer cells. In addition, in vivo anticancer studies suggested that TRAIL-Cur-NPs had superior therapeutic effect on tumors without obvious toxicity, which was mainly due to the high tumor targeting and synergistic effect of TRAIL and Cur. The synergistic mechanism of improved antitumor efficacy was proved to be upregulation of DR4 and DR5 in tumor cells induced by Cur. Thus, the prepared codelivery nanoparticles may have potential applications in colorectal cancer therapy.     

TRAIL联合阿霉素的体外抗肿瘤活性

目的探讨肿瘤坏死因子相关的凋亡诱导配体 (TRAIL)与阿霉素联合应用的体外抗肿瘤活性。方法将TRAIL与阿霉素单独及联合应用于体外培养的骨肉瘤OS 732细胞 ,观察其对肿瘤细胞的促凋亡作用。结果 5 0 μg·L-1的TRAIL与 5mg·L-1阿霉素合用于OS 732细胞 2 4h后 ,采用MTT(四甲基偶氮唑盐 )法测细胞抑制率为 85 4 7% ,明显高于单用TRAIL(5 0 μg·L-1)时的9 6 8%及单用阿霉素 (5mg·L-1)时的 18 4 1% (P <0 0 1)。倒置相差显微镜、荧光显微镜及电镜下的细胞超微结构观察均显示 ,合用比单用有更大的肿瘤杀伤效应。结论TRAIL与阿霉素联合应用可明显促进肿瘤细胞的凋亡     

TRAIL及其受体在咖啡因抑制肝癌细胞系HepG2增殖中的作用

目的探讨TRAIL及其受体在咖啡因(caffeine)抑制肝癌细胞系HepG2增殖中的作用。方法HepG2细胞分别经caffeine、TRAIL及caffeine+TRAIL作用24h,采用MTT法检测HepG2细胞增殖抑制情况,根据中效原理进行联合用药效应评价;流式细胞仪检测细胞凋亡和细胞周期分布;Westernblot法检测caffeine作用不同时间HepG2细胞中TRAIL受体相关蛋白的表达。结果在1.25～20mmol.L-1浓度范围内,caffeine明显抑制HepG2细胞增殖;在0.01275～0.2040μmol.L-1浓度范围内,TRAIL可明显抑制HepG2细胞增殖。Caffeine联合TRAIL在多数效应范围内的合用指数小于1,具有协同作用。Caffeine5mmol.L-1和TRAIL0.0510μmol.L-1联合用药组HepG2细胞凋亡率明显高于各单独用药组,且两者联合用药对HepG2细胞周期具有明显的影响,使G0/G1期细胞比例明显增加,S期及G2/M期细胞比例明显减少;caffeine5mmol.L-1作用HepG2细胞24h时,其DR4及DR5的表达量明显增加,而DcR1和DcR2的表达无改变。结论TRAIL在caffeine抑制HepG2细胞增殖过程中具有一定的协同作用,其机制可能与caffeine上调HepG2细胞表面DR4、DR5的表达,联合TRAIL后能够进一步诱导凋亡及调节细胞周期有关。     

Recent advances in targeting regulators of apoptosis in cancer cells for therapeutic gain

Apoptosis is a fundamental cellular death process that is essential for normal tissue homeostasis, whose deregulation is associated with several human disease states, including cancer. Increased understanding of cancer biology has led to the hypothesis that although cancer cells are inherently resistant to the engagement of apoptosis due to the deregulation of molecular components of core apoptotic machinery or of survival signalling cascades, they are primed to die as a result of microenvironmental and oncogenic proapoptotic stress. Recently, deeper insight into the molecular regulation of apoptosis and, specifically, into its deregulation in cancer has led to the development of promising therapies to restore apoptosis and enable selective tumour cell kill. It is hoped that these mechanism-based therapies will exhibit less problematic toxicity profiles than those of conventional agents. Moreover, the development of tailored therapies directed at malignancies bearing specific alterations in apoptotic or survival signalling components may be used in combination approaches to overcome the resistance to other forms of treatment.     

TRAIL基因修饰间充质干细胞治疗恶性肿瘤研究进展

间充质干细胞（mesenchymal stem cells,MSCs）是一种具有自我更新及多向分化潜能的干细胞。MSCs具有对多种肿瘤组织归巢及定向迁移的特性,可以将其作为治疗肿瘤药物的载体,用来治疗无法通过手术去除的肿瘤;同时,MSCs自身参与重塑肿瘤微环境,影响其侵袭、增殖和转移等生物学行为,并且具有抗炎及损伤组织修复的能力。肿瘤坏死因子相关凋亡配体（TRAIL/Apo2L）作为诱导细胞凋亡的肿瘤坏死因子家族成员之一,可通过结合肿瘤细胞表面两个特异性死亡受体（DR4、DR5）,特异激活肿瘤细胞凋亡。TRAIL基因修饰的MSCs（TRAIL-MSCs）可定位至肿瘤组织并抑制原发肿瘤及转移瘤的增殖、促进其凋亡,有望用于多种实体肿瘤及血液肿瘤的靶向治疗。综述了TRAIL-MSCs治疗肿瘤的研究进展,并讨论了TRAIL-MSCs联合放化疗对肿瘤的治疗效果,为该疗法在肿瘤治疗领域的应用提供理论依据。     

肿瘤坏死因子相关凋亡诱导配体在消化系统肿瘤治疗中的研究进展

肿瘤坏死因子相关凋亡诱导配体（TRAIL）可通过特异性识别肿瘤细胞死亡受体而诱导并启动多种凋亡机制,发挥抑制肿瘤的增殖、浸润、转移的作用,但TRAIL对食管癌、胃癌、大肠癌等消化系统恶性肿瘤治疗并不敏感。TRAIL可通过联合使用某些小分子化合物,或通过调节凋亡相关蛋白的表达等来提高其抑癌的能力,然而其具体作用机制尚不清楚。就肿瘤坏死因子相关凋亡诱导配体在消化系统肿瘤治疗中的研究进展进行综述。     

伊立替康联合TRAIL对胃癌细胞SGC7901凋亡的影响

目的探讨伊立替康和肿瘤坏死因子相关凋亡诱导配体(TRAIL)对胃癌SGC7901细胞凋亡的影响并初步探讨其作用机制。方法不同浓度的伊立替康和/或200μg·L-1TRAIL作用于人胃癌细胞SGC7901细胞,MTT检测细胞增殖能力;流式细胞术检测细胞凋亡;TRAIL和/或伊立替康作用SGC7901细胞后,Western-blot检测蛋白表达。结果 200μg·L-1的TRAIL对胃癌细胞的增殖抑制率为18.46%,TRAIL(200μg·L-1)联合伊立替康(28.67mg·L-1)引起明显的增殖抑制和细胞凋亡(P<0.05)。TRAIL(200μg·L-1)单药和伊立替康(28.67mg·L-1)单药都没有改变Bax、Caspase-8蛋白的表达,而两者联用增加了Bax、Caspase-8蛋白的表达。结论伊立替康通过增加Bax、Caspas-8蛋白表达来增强TRAIL诱导的胃癌SGC7901细胞凋亡。     

蛇床子素对TRAIL抗乳腺癌活性的影响及其作用机制

目的 探讨中药活性成分蛇床子素对抗肿瘤药物TRAIL抗乳腺癌活性的影响并研究其机制。方法 用蛇床子素联合TRAIL体外治疗乳腺癌细胞系BT-20,MTT法检测肿瘤细胞的细胞活力,Annexin V/PI染色检测肿瘤细胞的凋亡,免疫共沉淀法检测RIP1-FADD-caspase-8复合物的形成。western blot法检测BT-20细胞caspase-8的活化及蛇床子素对BT-20细胞cIAP2蛋白表达的影响。结果 联用蛇床子素显著提高TRAIL对BT-20细胞活力的抑制率和凋亡诱导活性。免疫共沉淀及western blot结果发现联合蛇床子素后,TRAIL治疗的BT-20细胞内的RIP1-FADD-caspase-8复合物水平及caspase-8的活化程度显著提高。Western blot结果发现蛇床子素对BT-20细胞内的cIAP2蛋白表达有抑制作用。在BT-20细胞中转染cIAP2表达质粒后,蛇床子素对TRAIL抗肿瘤活性的促进作用受到抑制。结论 蛇床子素通过促进死亡受体复合物的形成增强TRAIL对乳腺癌细胞的凋亡诱导效应。     

大肠上皮恶性转化不同阶段p53表达与细胞凋亡的关系

目的：观察大肠腺癌,腺瘤恶性变区及非恶变区细胞凋亡及其调控基因ｐ５３的表达状态,探讨它们在大肠上皮恶性转化进程中的作用及二者的关系。方法：利用ＤＮＡ缺口末端标记技术,ｐ５３蛋白免疫组化染色及双重染色技术,原位观察２７例大肠腺癌及２１例瘤恶变标本中凋亡细胞和ｐ５３阳性表达细胞的密度与分布,以８例非种瘤大肠粘膜作为对照。结果：腺瘤非恶变区凋亡密度分别高于腺癌（Ｐ〈０．０１）、腺瘤恶变区（Ｐ〈０．０１）     

SAHA (vorinostat) facilitates functional polymer-based gene transfection via upregulation of ROS and synergizes with TRAIL gene delivery for cancer therapy

Abstract

Non-viral gene delivery is an attractive approach for the treatment of many diseases including cancer, benefiting from its safety and large-scale production concerns. However, the relatively low transfection efficacy compared with viral vectors restricts the clinical applications of non-viral gene vectors. Reactive oxygen species (ROS) triggered charge reversal polymers (named B-PDEAEA) presented improved transfection efficacy, because of fast release of plasmid DNA responding to enhanced oxidative stress in cancer cells. But inadequate dissociation can still occur owing to the insufficient intracellular ROS generation. Here, we report SAHA (vorinostat), which is a clinical histone deacetylase inhibitor and anticancer drug, induces the ROS accumulation in cancer cells, and facilitates the charge reversal process of B-PDEAEA and the cellular dissociation of the delivered gene from the vectors. As a result, SAHA remarkably increases the gene transfection efficacy in an ROS-dependent manner. Importantly, SAHA synergizes with B-PDEAEA mediated therapeutic gene TNF-related apoptosis-inducing ligand (TRAIL) delivery in inducing apoptosis of cancer cells. These findings support the first concept of improving the gene delivery efficacy of stimuli-responsive vectors through upregulating the cellular ROS via an FDA approved anticancer agent. Additionally, combination of SAHA and TRAIL gene therapy could be a potential strategy for cancer treatment.     

Crocetin induces cytotoxicity and enhances vincristine-induced cancer cell death via p53-dependent and -independent mechanisms

Aim:

To investigate the anticancer effect of crocetin, a major ingredient in saffron, and its underlying mechanisms.

Methods:

Cervical cancer cell line HeLa, non-small cell lung cancer cell line A549 and ovarian cancer cell line SKOV3 were treated with crocetin alone or in combination with vincristine. Cell proliferation was examined using MTT assay. Cell cycle distribution and sub-G₁ fraction were analyzed using flow cytometric analysis after propidium iodide staining. Apoptosis was detected using the Annexin V-FITC Apoptosis Detection Kit with flow cytometry. Cell death was measured based on the release of lactate dehydrogenase (LDH). The expression levels of p53 and p21^WAF1/Cip1 as well as caspase activation were examined using Western blot analysis.

Results:

Treatment of the 3 types of cancer cells with crocetin (60-240 μmol/L) for 48 h significantly inhibited their proliferation in a concentration-dependent manner. Crocetin (240 μmol/L) significantly induced cell cycle arrest through p53-dependent and -independent mechanisms accompanied with p21^WAF1/Cip1 induction. Crocetin (120-240 μmol/L) caused cytotoxicity in the 3 types of cancer cells by enhancing apoptosis in a time-dependent manner. In the 3 types of cancer cells, crocetin (60 μmol/L) significantly enhanced the cytotoxicity induced by vincristine (1 μmol/L). Furthermore, this synergistic effect was also detected in the vincristine-resistant breast cancer cell line MCF-7/VCR.

Conclusion:

Ccrocetin is a potential anticancer agent, which may be used as a chemotherapeutic drug or as a chemosensitizer for vincristine.     

miR-144通过靶向TIGAR调控胃癌细胞增殖与凋亡及自噬的分子机制

目的研究miR-144对胃癌细胞增殖、凋亡和自噬的影响和机制。方法将miR-144 mimics、mimics control分别转染胃癌细胞,依次标记为mimics-NC组、miR-144 mimics组。将miR-144 mimics分别与pcDNA3.1、pcDNA3.1-TIGAR共转染至胃癌细胞,依次标记为miR-144 mimics+NC组、miR-144 mimics+TIGAR组。正常培养的胃癌细胞标记为Control组。以噻唑蓝法检测细胞增殖,以流式细胞术测定细胞凋亡,以Western blot检测凋亡蛋白caspase-3和自噬蛋白Beclin1、LC3Ⅱ/Ⅰ表达变化,以生物信息学软件预测miR-144的靶基因可能为TIGAR,利用荧光素酶报告系统鉴定靶向关系。结果 mimics-NC组和miR-144 mimics组的细胞增殖能力分别为0.36±0.05,0.20±0.02,细胞凋亡率分别为3.05±0.34,13.84±1.47,caspase-3表达水平分别为0.22±0.04,0.43±0.05, Beclin1分别为0.38±0.06,0.73±0.07,LC3Ⅱ分别为0.19±0.03,0.39±0.05,差异均有统计学意义(均P<0.05)。miR-144 mimics+NC组和miR-144 mimics+TIGAR组的细胞增殖能力分别为0.21±0.04,0.32±0.03,细胞凋亡率分别为12.04±1.25,5.98±0.73,caspase-3表达水平分别为0.40±0.03,0.24±0.05,Beclin1分别为0.60±0.05,0.46±0.06,LC3Ⅱ分别为0.34±0.06,0.20±0.03,差异均有统计学意义(均P<0.05)。结论 miR-144靶向抑制TIGAR表达降低胃癌细胞增殖能力,并诱导胃癌细胞凋亡和自噬。