Induction and regulation of tumor necrosis factor-related apoptosis-inducing ligand/Apo-2 ligand-mediated apoptosis in renal cell carcinoma

The lack of effective therapy for disseminated renal cell carcinoma (RCC) has stimulated the search for novel treatments including immunotherapeutic strategies. However, poor therapeutic responses and marked toxicity associated with immunological agents has limited their use. The tumor necrosis factor family member tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)/Apo-2 ligand induces apoptosis in a variety of tumor cell types, while having little cytotoxic activity against normal cells. In this study the activation and regulation of TRAIL-induced apoptosis and TRAIL receptor expression in human RCC cell lines and pathologic specimens was examined. TRAIL induced caspase-mediated apoptotic death of RCC cells with variable sensitivities among the cell lines tested. Compared with TRAIL-sensitive RCC cell lines (A-498, ACHN, and 769-P), the TRAIL-resistant RCC cell line (786-O) expressed lesser amounts of the death-inducing TRAIL receptors, and greater amounts of survivin, an inhibitor of apoptosis. Incubation of 786-O with actinomycin D increased the expression of the death-inducing TRAIL receptors and, concomitantly, decreased the intracellular levels of survivin, resulting in TRAIL-induced apoptotic death. The link between survivin and TRAIL regulation was confirmed when an increase in TRAIL resistance was observed after overexpression of survivin in the TRAIL-sensitive, survivin-negative RCC line A-498. These findings, along with our observation that TRAIL receptors are expressed in RCC tumor tissue, suggest that TRAIL may be useful as a therapeutic agent for RCC and that survivin may partially regulate TRAIL-induced cell death.     

Adenoviral vector expressing CYLD augments antitumor activity of TRAIL by suppression of NF-kappaB survival signaling in hepatocellular carcinoma

CYLD is a tumor suppressor gene related to cylindroma and is negative regulator of NF-kappaB. However, antitumor effect of CYLD has not been reported. The activation of NF-kappaB induced by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) renders hepatocellular carcinoma (HCC) resistant to TRAIL-mediated cell apoptosis. Here we described that the adenoviral vector expressing CYLD (Ad/hTERT-CYLD) augmented the cytotoxicity of TRAIL in HCC cells by negatively regulating NF-kappaB activity since CYLD could reverse the ubiquitination of TNF receptor-associated factor 2 (TRAF2) and interact with the IkappaB kinasegamma (IKKgamma). The combined treatment of Ad/hTERT-CYLD and a conditionally replicating adenovirus carrying TRAIL gene (ZD55-TRAIL) induced rapid and potent apoptosis in HCC cells, characterized by activation of caspase-3, caspase-8, PARP and the reduction of X-linked inhibitor of apoptosis protein (XIAP). In animal study, the combined treatment could eradicate the BEL7404 xenograft tumors. In contrast, treatment with Ad/hTERT-CYLD or ZD55-TRAIL alone achieved less antitumor effect. In conclusion: CYLD inhibits TRAIL-mediated NF-kappaB activation and enhances the sensitivity of HCC cells to TRAIL-triggered apoptosis. The combined delivery of Ad/hTERT-CYLD and ZD55-TRAIL may be a new useful strategy for HCC or other tumor cells with enhanced NF-kappaB activity.     

Concomitant use of Ad5/35 chimeric oncolytic adenovirus with TRAIL gene and taxol produces synergistic cytotoxicity in gastric cancer cells

Chimeric adenoviral vectors possessing fiber derived from human adenovirus subgroup B (Ad35) have been developed for their high infection efficiency in cell types which are refractory to adenovirus serotype 5 (Subgroup C). The present study constructed an E1B-deleted chimeric oncolytic adenovirus, SG235-TRAIL, which carries a human TRAIL gene expression cassette and whose fiber shaft and knob domains are from serotype Ad35. It was found that SG235-TRAIL preferentially replicated in gastric cancer cell lines, SGC-7901 and BGC-823 compared to in normal human fibroblast BJ cells. Also, when compared with a replication-deficient chimeric vector Ad5/35-TRAIL, SG235-TRAIL mediated a higher level of the transgene expression via viral replication in the cancer cells. Further, because of the more efficient cell-entry and infection, SG235-TRAIL induced stronger cell apoptosis than the Ad5 CRAD vector, ZD55-TRAIL. In addition, SG235-TRAIL in combination with the chemotherapeutic drug, taxol, produced a synergistic cytotoxic effect in cancer cells in vitro without causing significant toxicity to normal cells. In the gastric tumor xenograft mouse model, intratumoral SG235-TRAIL injection produced a significant antitumor effect 14 days after treatment. Pathological examination demonstrated TRAIL expression and associated apoptosis in majority of SG235-TRAIL-treated tumor cells. These results suggest that SG235-TRAIL is a potential novel, efficient anti-cancer agent, and in combination with taxol, it would be even more useful with considerably low toxic side effects.     

Inhibition of histone deacetylase class I but not class II is critical for the sensitization of leukemic cells to tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis

From work done largely on derived cell lines, it has been suggested that tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) might be a therapeutic target for many forms of malignancy. However, use of primary tumor cells, including chronic lymphocytic leukemic (CLL) cells, has shown inherent resistance to TRAIL. Although the molecular basis for this resistance remains unknown, treatment with histone deacetylase inhibitors (HDACi) often sensitizes resistant cells to TRAIL-induced apoptosis. We used structurally diverse HDACi to ascertain which HDAC needs to be inhibited for the sensitization. Inhibition of HDAC class I but not class II is required for sensitization to TRAIL-induced apoptosis of CLL cells and various cell lines. Using different HDACi together with small interfering RNA for HDAC1, HDAC2, HDAC3, and HDAC6, we report that inhibition of HDAC1 and HDAC2 but not HDAC3, HDAC6, and HDAC8 are primarily responsible for sensitization to TRAIL-induced apoptosis. Based on these data and our previous studies, we propose that a clinical trial in CLL is warranted using a combination of a selective HDACi that inhibits HDAC1 and/or HDAC2 together with a form of TRAIL that signals through TRAIL receptor 1.     

受体依赖性TRAIL重组腺病毒对人肺癌细胞凋亡的诱导作用

目的：观察重组TRAIL腺病毒（Ad5TRAIL及Ad5F35TRAIL）对人非小细胞肺癌（nonsmallcell lung cancer,NSCLC）原代培养细胞和细胞株的凋亡诱导作用,探讨两种AdTRAIL用于肺癌基因治疗的价值。方法：采用流式细胞术检测人肺癌细胞系A549、Z793、QG56和NCIH520及10例原代培养肺癌细胞中CAR和CD46的表达水平;分别以Ad5TRAIL及Ad5F35TRAIL重组腺病毒按MOI 10和50感染上述细胞,48 h后Annexin VFITC双标法流式细胞术检测细胞的早期凋亡。结果：A549、Z793、QG56和NCIH520 4株肺癌细胞中,CD46的表达均明显高于CAR表达。Z793、QG56细胞对Ad5TRAIL和Ad5F35TRAIL的作用较敏感, MOI=10感染后凋亡率分别为（11.76±2.10）%、（15.96±2.89）%和（6.05±158）%、（10.11±1.26）%,显著高于对照组\[（2.33±0.37）%和(5.95±1.89）%,P<0.05\];MOI=50感染时NCIH520细胞凋亡率分别为（12.89±3.2）%和（9.08±1.35）%,与对照组（7.04±2.17）%相比差异无统计学意义（P>0.05）;Ad5TRAIL和Ad5F35TRAIL均不能诱导A549细胞凋亡。10例原代肺癌细胞CD46表达也明显较CAR高;Ad5TRAIL或Ad5F35TRAIL 感染后,5例的原代肺癌细胞检测到凋亡;与Ad5TRAIL相比,Ad5F35TRAIL诱导的凋亡率更高。结论：两种TRAIL重组腺病毒对非小细胞肺癌细胞均有凋亡诱导作用,Ad5F35TRAIL的作用强于Ad5TRAIL,更适合于非小细胞肺癌的基因治疗。     

桧木醇对肾癌786-O细胞增殖及相关细胞因子的影响

目的从体外水平研究桧木醇(hinokitio1)的抗肾癌作用,探讨其作用机制。方法采用MTT法和流式细胞术检测桧木醇对肾癌786-O细胞增殖的抑制作用和促凋亡作用的影响;采用Western-blot检测桧木醇对人肾癌786-O细胞Caspase-3剪切体、LC3和P62蛋白表达水平的影响。以3-MA验证其抗癌作用与自噬作用的关系。结果桧木醇对肾癌786-O细胞增殖有显著抑制作用,主要是通过激活Caspase途径对细胞凋亡进行诱导。同时桧木醇可以对肾癌786-O细胞进行诱导自噬发生,使LC3蛋白的表达量出现显著上调,而P62蛋白表达则显著下调。结论桧木醇能够显著抑制肾癌786-O细胞的增殖,而且可以通过过度激活细胞自噬促进肾癌细胞凋亡。     

Complete elimination of colorectal tumor xenograft by combined manganese superoxide dismutase with tumor necrosis factor-related apoptosis-inducing ligand gene virotherapy

Manganese superoxide dismutase (MnSOD) is a latent tumor suppressor gene. To investigate the therapeutic effect of MnSOD and its mechanisms, a replication-competent recombinant adenovirus with E1B 55-kDa gene deletion (ZD55) was constructed, and human MnSOD and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) genes were inserted to form ZD55-MnSOD and ZD55-TRAIL. ZD55-MnSOD exhibited an inhibition in tumor cell growth approximately 1,000-fold greater than Ad-MnSOD. ZD55-TRAIL was shown to induce the MnSOD expression in SW620 cells. Accordingly, by the combined use of ZD55-MnSOD with ZD55-TRAIL (i.e., "dual gene virotherapy"), all established colorectal tumor xenografts were completely eliminated in nude mice. The evidence exists that the MnSOD overexpression led to a slower tumor cell growth both in vitro and in vivo as a result of apoptosis caused by MnSOD and TRAIL overexpression after adenoviral transduction. Our results showed that the production of hydrogen peroxide derived from MnSOD dismutation activated caspase-8, which might down-regulate Bcl-2 expression and induce Bax translocation to mitochondria. Subsequently, Bax translocation enhanced the release of apoptosis-initiating factor and cytochrome c. Cytochrome c finally triggered apoptosis by activating caspase-9 and caspase-3 in apoptotic cascade. Bax-mediated apoptosis seems to be dependent on caspase-8 activation because the inhibition of caspase-8 prevented Bid processing and Bax translocation. In conclusion, our dual gene virotherapy completely eliminated colorectal tumor xenografts via enhanced apoptosis, and this novel strategy points toward a new direction of cancer treatment.     

The topoisomerase I inhibitor topotecan increases the sensitivity of prostate tumor cells to TRAIL/Apo-2L-induced apoptosis

PURPOSE.:TRAIL/Apo-2L is cytotoxic against numerous prostate tumor cell lines; however, some lines are more resistant than others. Identification of an agent that increases prostate tumor cell sensitivity to TRAIL/Apo-2L would prove valuable for TRAIL/Apo-2L-mediated tumor therapy. Thus, we examined the effect of combining five clinically approved chemotherapeutic agents with TRAIL/Apo-2L for treating prostate tumor cells. METHODS: Four human prostate tumor cell lines were initially tested for TRAIL/Apo-2L sensitivity. Subsequent studies examined whether the TRAIL/Apo-2L-induced killing of DU-145 cells was augmented in the presence of the chemotherapeutic molecules, as measured by annexin V-FITC/propidium iodide staining. Furthermore, caspase 8 activation and BID cleavage were examined by immunoblotting. RT-PCR and flow cytometry were performed to monitor TRAIL-R1 and TRAIL-R2 levels after chemotherapeutic treatment. RESULTS: DU-145 cells were the least responsive of the prostate tumor cell lines tested to TRAIL/Apo-2L-induced death. Surprisingly, only topotecan, a topoisomerase I inhibitor, when used in combination with rTRAIL/Apo-2L led to significant apoptosis of DU-145 cells, as measured by caspase 8 activation, BID cleavage, and annexin V-FITC/PI staining. Topotecan alone had little to no toxicity on the DU-145 cells. Furthermore, the increase in TRAIL/Apo-2L sensitivity following topotecan treatment correlated with increased expression of TRAIL-R1 and TRAIL-R2 and decreased intracellular levels of the antiapoptotic protein survivin. CONCLUSIONS: Our results define a promising direction for alternative therapies against androgen-independent prostate cancers. The sensitivity of DU-145 cells to TRAIL/Apo-2L was dramatically increased when combined with topotecan, suggesting that low-dose topotecan treatment to upregulate TRAIL-R1 and TRAIL-R2 and downregulate survivin, followed by TRAIL/Apo-2L administration, may be a viable therapy for treating cancer of the prostate.     

Sorafenib (BAY 43-9006) inhibits tumor growth and vascularization and induces tumor apoptosis and hypoxia in RCC xenograft models

Purpose

New research findings have revealed a key role for vascular endothelial growth factor (VEGF) in the stimulation of angiogenesis in clear cell renal carcinoma (RCC) which is a highly vascularized and treatment-resistant tumor. Sorafenib (BAY 43-9006, Nexavar^®) is a multi-kinase inhibitor which targets receptor tyrosine and serine/threonine kinases involved in tumor progression and tumor angiogenesis. The effect of sorafenib on tumor growth and tumor histology was assessed in both ectopic and orthotopic mouse models of RCC.

Methods

Sorafenib was administered orally to mice bearing subcutaneous (SC, ectopic) or sub-renal capsule (SRC, orthotopic) tumors of murine (Renca) or human (786-O) RCC. Treatment efficacy was determined by measurements of tumor volume and tumor growth delay. In mechanism of action studies, using the 786-O and Renca RCC tumor models, the effect of sorafenib was assessed after dosing for 3 or 5 days in the SC models and 21 days in the SRC models. Inhibition of tumor angiogenesis was assessed by measuring level of CD31 and α-smooth muscle actin (αSMA) staining by immunohistochemistry (IHC). The effect of sorafenib on MAPK signaling, cell cycle progression and cell proliferation was also assessed by IHC by measuring levels of phospho-ERK, phospho-histone H3 and Ki-67 staining, respectively. The extent of tumor apoptosis was measured by terminal deoxynucleotidyl transferase-mediated nick-end labeling (TUNEL) assays. Finally, the effects of sorafenib on tumor hypoxia was assessed in 786-O SC model by injecting mice intravenously with pimonidazole hydrochloride 1 h before tumor collection and tumor sections were stained with a FITC-conjugated Hypoxyprobe antibody.

Results

Sorafenib produced significant tumor growth inhibition (TGI) and a reduction in tumor vasculature of both ectopic and orthotopic Renca and 786-O tumors, at a dose as low as 15 mg/kg when administered daily. Inhibition of tumor vasculature was observed as early as 3 days post-treatment, and this inhibition of angiogenesis correlated with increased level of tumor apoptosis (TUNEL-positive) and central necrosis. Consistent with these results, a significant increase in tumor hypoxia was also observed 3 days post-treatment in 786-O SC model. However, no significant effect of sorafenib on phospho-ERK, phospho-histone H3 or Ki-67 levels in either RCC tumor model was observed.

Conclusion

Our results show the ability of sorafenib to potently inhibit the growth of both ectopically- and orthotopically-implanted Renca and 786-O tumors. The observed tumor growth inhibition and tumor stasis or stabilization correlated strongly with decreased tumor angiogenesis, which was due, at least in part, to inhibition of VEGF and PDGF-mediated endothelial cell and pericyte survival. Finally, sorafenib-mediated inhibition of tumor growth and angiogenesis occurred at concentrations equivalent to those achieved in patients in the clinic.     

Pretreatment with paclitaxel enhances apo-2 ligand/tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis of prostate cancer cells by inducing death receptors 4 and 5 protein levels

We have demonstrated that Apo-2 ligand (Apo-2L)/tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis of human prostate cancer PC-3, DU145, and LNCaP cells in a dose-dependent manner, with PC-3 cells displaying the greatest sensitivity to Apo-2L/TRAIL. Susceptibility of the prostate cancer cell types to Apo-2L/TRAIL-induced apoptosis did not appear to correlate with the levels of the Apo-2L/TRAIL receptors death receptor (DR) 4 (TRAIL receptor 1) or DR5 (TRAIL receptor 2), decoy receptor (DcR) 1 and DcR2, Flame-1, or the inhibitors of apoptosis proteins family of proteins. Apo-2L/TRAIL-induced apoptosis of PC-3 cells was associated with the processing of caspase-8, caspase-10, and the proapoptotic Bid protein, resulting in the cytosolic accumulation of cytochrome c as well as the processing of procaspase-9 and procaspase-3. Cotreatment with the caspase-8 inhibitor z-IETD-fmk or DR4:Fc significantly inhibited Apo-2L/TRAIL-induced apoptosis. Treatment with paclitaxel or taxotere increased DR4 and/or DR5 protein levels (up to 8-fold) without affecting the protein levels of DcR1 and DcR2, Apo-2L/TRAIL, Fas, or Fas ligand. Up-regulation of DR4 and DR5 was not preceded by the induction of their mRNA levels but was inhibited by cotreatment with cycloheximide. Importantly, sequential treatment of PC-3, DU145, and LNCaP cells with paclitaxel followed by Apo-2L/TRAIL induced significantly more apoptosis than Apo-2L/TRAIL treatment alone (P < 0.01). This was also associated with greater processing of procaspase-8 and Bid, as well as greater cytosolic accumulation of cytochrome c and the processing of caspase-3. These findings indicate that up-regulation of DR4 and DR5 protein levels by treatment with paclitaxel enhances subsequent Apo-2L/TRAIL-induced apoptosis of human prostate cancer cells.     

结肠癌TRAIL耐药细胞株的建立及其耐药机制的初步研究

背景与目的以人结肠癌DLD1细胞株为载体,探讨恶性肿瘤获得性TRAIL基因耐药的可能机制。材料与方法用重组腺病毒介导的TRAIL基因(Ad/gTRAIL)反复处理人结肠癌DLD1细胞,得到耐药细胞群DLD1-TRAIL/R。通过MTT比色法和蛋白电泳,检测耐药细胞对Ad/gTRAIL杀伤作用的敏感性及其凋亡通路中信号分子的表达情况,分析其获得性耐药的可能机制。结果DLD1-TRAIL/R细胞对Ad/gTRAIL和重组TRAIL蛋白处理耐药,但对腺病毒介导的Bax基因处理仍然敏感。耐药细胞内Bcl-XL的表达明显升高,caspase-8的表达显著下降,未见明显的caspase-8活性裂解形式。结论人结肠癌DLD1细胞株经Ad/gTRAIL反复处理后产生特异性针对TRAIL基因的耐药,其发生机制可能与Bcl-XL表达上调和caspase-8表达下调有关。     

Tumor-derived tumor necrosis factor-alpha promotes progression and epithelial-mesenchymal transition in renal cell carcinoma cells

Pro-inflammatory cytokines and chemokines are involved in promoting tumorigenesis by facilitating tumor proliferation and metastasis. The serum levels of interleukin (IL)-6, IL-1β, and tumor necrosis factor-alpha (TNF-α) are significantly elevated in patients with renal cell carcinoma (RCC). However, the mechanisms of how these cytokines participate in the progression of RCC remains unknown. In the present study, we investigated the effects of tumor-derived cytokines on invasion and the epithelial-mesenchymal transition (EMT) of RCC cells. We found that expression of IL-1β, IL-6, TNF-α, hypoxia-inducible factor-alpha (HIF-1α), and matrix metalloproteinase-2 (MMP2) were significantly elevated in high malignancy A498 cells compared to low malignancy 786-O cells. The invasion ability of A498 was three-fold higher than that of 786-O cells. The invasiveness of 786-O cells was markedly enhanced by adding conditioned medium derived from A498 cells. This phenomenon was significantly inhibited by immunodepletion of TNF-α followed by MMP2, IL-6, or IL-1β from A498 conditioned medium. Synergistic inhibition was also noted after simultaneous immunodepletion of TNF-α, IL-1β, and IL-6. RCC cell lines with higher malignancy produced more TNF-α, which was correlated with their stronger invasive ability. The invasiveness of 786-O cells was significantly promoted by TNF-α in a dose-dependent manner. Moreover, TNF-α induced the EMT of 786-O cells by repressing E-cadherin, promoting vimentin expression, and activating MMP9 activity. Our findings demonstrate that pro-inflammatory cytokines, especially TNF-α, can enhance invasion and the EMT of renal cancer cells, which provides a therapeutic target to prevent and treat advanced RCC. ( Cancer Sci 2008; 99: 905–913)     

Gene-viro-therapy targeting liver cancer by a dual-regulated oncolytic adenoviral vector harboring IL-24 and TRAIL

Cancer-targeting gene-viro-therapy is a promising cancer therapeutic strategy that strengthens the antitumor effect of oncolytic viruses by expressing an inserted foreign antitumor gene. To achieve liver cancer targeting and to improve the safety of the ZD55 vector (a widely-used E1B55KD gene-deleted oncolytic adenoviral vector (OV), we previously constructed), we designed a novel OV named Ad·AFP·D55 that selectively replicates in hepatocellular carcinoma (HCC) cells by replacing the E1A promoter with the liver-cancer specific α-Fetoprotein (AFP) promoter based on the ZD55 vector. We found that the oncolytic adenoviruses Ad·AFP·D55-IL-24 and Ad·AFP·D55-TRAIL express tumor-suppressor gene interleukin-24 (IL-24) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), respectively, significantly suppressed the HCC cell growth in vitro by inducing apoptosis by the caspase-8 and mitochondria-dependent caspase-9 signaling pathways. Furthermore, the combined treatment of Ad·AFP·D55-IL-24 and Ad·AFP·D55-TRAIL showed strong antitumor effects in vivo by significantly inhibiting the tumor growth in HCC HuH-7 cell xenograft mice, and markedly increasing animal survival rate. Therefore, this novel HCC cell-targeting OV carrying tumor-suppressor genes may provide a promising approach for liver cancer gene therapy.     

The histone deacetylase inhibitors depsipeptide and MS-275, enhance TRAIL gene therapy of LNCaP prostate cancer cells without adverse effects in normal prostate epithelial cells

Gene therapy of cancer using adenovirus as a single treatment modality has met limited success and efforts to enhance therapeutic outcomes have included combination of gene therapy with chemotherapy. The goal of this study was to investigate which chemotherapeutic agents may be suitable for combination with gene therapy of prostate cancer. Using an adenovirus expressing green fluorescent protein (GFP), we determined the effect of cisplatin, gemcitabine, doxorubicin, depsipeptide and MS-275 on adenoviral infectivity and transgene expression in LNCaP cells. We found that the two histone deacetylase inhibitors (HDACi), depsipeptide and MS-275, and to a lesser extent doxorubicin, increased infectivity and transgene expression. However, only the HDACi selectively increased infectivity in LNCaP cells while doxorubicin increased infectivity to a greater extent in normal prostate epithelial cells (PrEC). The increase in infectivity but not transgene expression correlated to increased surface expression of coxsackie and adenovirus receptor (CAR). Increased transgene expression following infection with an adenovirus expressing tumor necrosis factor-related apoptosis inducing ligand (TRAIL) was observed only in LNCaP cells treated with depsipeptide or MS-275. Combination of TRAIL gene therapy with HDACi but not doxorubicin resulted in increased induction of apoptosis in LNCaP cells. In contrast, apoptosis was not enhanced by HDACi in normal PrEC. These results suggest that combination of HDACi with adenoviral TRAIL gene therapy may be a new therapeutic approach for the treatment of prostate cancer that warrants further investigation.