Enhanced antitumor efficacy of a novel fiber chimeric oncolytic adenovirus expressing p53 on hepatocellular carcinoma

Oncolytic adenoviruses may offer a new treatment option and improve the prognosis for patients with hepatocellular carcinoma (HCC). However, the antitumor efficacy of oncolytic adenoviruses on HCC cells is compromised due to low expression of the adenovirus serotype 5 (Ad5) receptor on the target cells. In this study we showed that all HCC cell lines and clinical samples expressed high level of CD46, the receptor for Adenovirus 35 (Ad35) and constructed new fiber chimeric oncolytic adenoviruses with or without a p53 gene expression cassette, SG635-p53 and SG635, respectively. These variants were derived from the previously described Ad5 vectors SG600-p53 and SG600 by replacing the Ad5 fiber with a chimeric Ad5/35 fiber. It was found that the 5/35 fiber chimeric adenovirus vector (Ad5/35-EGFP) demonstrated significantly improved transduction in all tested HCC cell lines compared with the Ad5 vector (Ad5-EGFP). The new fiber chimeric oncolytic adenoviruses produced more progeny viruses in HCC cells than did the Ad5-based viruses but replicated weakly in normal fibroblast BJ cells. In addition, SG635-p53 mediated a higher level of transgenic expression than SG600-p53 in Hep3B and Huh7 cells and showed a markedly enhanced antitumor effect on HCC cells in vitro compared with SG635 or SG600-p53 without causing significant cytotoxicity to normal cells. Antitumor activity of SG635-p53 was shown in Hep3B subcutaneous xenograft tumor models following intratumoral injection, resulting in significant inhibition of tumor growth and prolonged survival of animals. Our data suggest that SG635-p53, as a fiber chimeric oncolytic adenovirus in combination with p53 expression, may serve as a novel, promising and safe anticancer agent for the treatment of HCC.     

Different susceptibility of osteosarcoma cell lines and primary cells to treatment with oncolytic adenovirus and doxorubicin or cisplatin

Despite improvements in treatment regimens for osteosarcoma (OS) patients, survival rate has not increased over the last two decades. New treatment modalities are therefore warranted. Preclinical results with conditionally replicative adenoviruses (CRAds) to treat OS are promising. One type of CRAd that was effective against OS cells is Ad5-Delta24RGD. In other types of cancer, CRAds have been shown to interact synergistically with chemotherapeutic agents. Chemotherapy for OS often includes doxorubicin and cisplatin. Therefore, we explored combination treatment of OS cell lines and primary OS cell cultures with Ad5-Delta24RGD and doxorubicin or cisplatin. On OS cell lines, combination treatment was additive to synergistic. Surprisingly, however, on seven of eight primary OS samples no such combination effects were observed. In contrast, in many cases chemotherapy even inhibited CRAd-mediated cell killing. The inhibitory effect of doxorubicin on Ad5-Delta24RGD in primary OS cells appeared to correlate with slow cell growth rate; reduced viral replication and absence of chemotherapy-induced G2 cell cycle arrest. Our results point to the possibility that, at least for OS, virotherapy and chemotherapy should best not be performed simultaneously. In general, our work underscores the importance of testing new genetic anticancer agents and treatment regimens on primary cancer specimens.     

Macrophage depletion combined with anticoagulant therapy increases therapeutic window of systemic treatment with oncolytic adenovirus

Liver tropism of systemically delivered adenoviruses (Ad) represents a considerable challenge for their use as anticancer therapeutics. More than 90% of i.v. injected Ad is rapidly taken up by the liver leading to hepatotoxicity, reduced virus uptake by target tumor tissue, and diminished therapeutic efficacy. The lack of clinical activity of systemically given oncolytic Ad demands for better understanding and improvement of virus pharmacokinetics. We studied the effects of Ad "detargeting" from liver macrophages (Kupffer cells) and hepatocytes on toxicity and anticancer efficacy using a nonattenuated oncolytic Ad expressing enhanced green fluorescent protein-firefly luciferase fusion protein (Ad-EGFPLuc). Kupffer cell depletion before i.v. injection of Ad-EGFPLuc increased transgene expression in the liver 40.7-fold on day 3 after the injection indicating compensatory enhancement of hepatocyte transduction due to increased bioavailability of the virus. Pretreatment of mice with the anticoagulant drug warfarin to block blood factor-dependent binding of the virus to hepatocytes markedly reduced luciferase expression in the liver and mediated the corresponding decrease of hepatotoxicity in mice with intact and depleted liver macrophages. Combined depletion of Kupffer cells and pretreatment with warfarin before a single i.v. injection of Ad-EGFPLuc significantly reduced tumor growth and prolonged survival of nude mice bearing subcutaneous xenografts of aggressive human hepatocellular carcinoma. The improved antitumor activity correlated with enhanced transgene expression and virus spread in the tumors. These data suggest that detargeting oncolytic Ad from liver macrophages and hepatocytes is an effective strategy to increase the therapeutic window for therapy against disseminated tumor sites.     

Serotype chimeric oncolytic adenovirus coding for GM‐CSF for treatment of sarcoma in rodents and humans

Sarcomas are a relatively rare cancer, but often incurable at the late metastatic stage. Oncolytic immunotherapy has gained attention over the past years, and a wide range of oncolytic viruses have been delivered via intratumoral injection with positive safety and promising efficacy data. Here, we report preclinical and clinical results from treatment of sarcoma with oncolytic adenovirus Ad5/3‐D24‐GMCSF (CGTG‐102). Ad5/3‐D24‐GMCSF is a serotype chimeric oncolytic adenovirus coding for human granulocyte‐macrophage colony‐stimulating factor (GM‐CSF). The efficacy of Ad5/3‐D24‐GMCSF was evaluated on a panel of soft‐tissue sarcoma (STS) cell lines and in two animal models. Sarcoma specific human data were also collected from the Advanced Therapy Access Program (ATAP), in preparation for further clinical development. Efficacy was seen in both in vitro and in vivo STS models. Fifteen patients with treatment‐refractory STS (13/15) or primary bone sarcoma (2/15) were treated in ATAP, and treatments appeared safe and well‐tolerated. A total of 12 radiological RECIST response evaluations were performed, and two cases of minor response, six cases of stable disease and four cases of progressive disease were detected in patients progressing prior to virus treatment. Overall, the median survival time post treatment was 170 days. One patient is still alive at 1,459 days post virus treatment. In summary, Ad5/3‐D24‐GMCSF appears promising for the treatment of advanced STS; a clinical trial for treatment of refractory injectable solid tumors including STS is ongoing.© 2013 UICC     

LRIG1 combined with cisplatin enhances bladder cancer lesions via a novel pathway

One aspect of chemotherapy insensitivity and resistance results from induction of epidermal growth factor receptor (EGFR) internalization and initial DNA damage repair in response to DNA-damaging stimuli, such as cisplatin (CDDP). Previously, we found that leucine-rich repeats and immunoglobulin-like domains 1 (LRIG1), as one of the natural ligands of EGFR, could combine with and down-regulate the expression of EGFR in bladder cancer cells. This finding interested us and we hypothesized that LRIG1 could be a novel candidate for facilitating cisplatin-induced bladder cancer cell lesions. To investigate this further, we overexpressed LRIG1 with an adenovirus vector in EJ/T24 bladder cancer cells and investigated total EGFR, nuclear expression of phosphorylated EGFR (pEGFR) and cell lesions with exposure to CDDP. CDDP-induced nuclear pEGFR levels accumulated with time and were decreased by LRIG1 overexpression. LRIG1-transduced cells treated with CDDP had more severe DNA damage, cellular apoptosis, growth inhibition and reversal of invasion. These preclinical studies indicate that LRIG1 may represent a new therapeutic approach to improve the response of bladder cancer to chemotherapy through a novel pathway.     

Evaluation of a selectively oncolytic adenovirus for local and systemic treatment of cervical cancer

Treatment options for disseminated cervical cancer remain inadequate. Here, we investigated a strategy featuring Ad5-Delta 24 RGD, an oncolytic adenovirus replication-competent selectively in cells defective in the Rb-p16 pathway, such as most cervical cancer cells. The viral fiber contains an alpha(v)beta(3) and alpha(v)beta(5) integrin-binding RGD-4C motif, allowing coxsackie-adenovirus receptor-independent infection. These integrins have been reported to be frequently upregulated in cervical cancer. Oncolysis of cervical cancer cells was similar to a wild-type control in vitro. In an animal model of cervical cancer, the therapeutic efficacy of Ad5-Delta 24 RGD could be demonstrated for both intratumoral and intravenous application routes. Biodistribution was determined following intravenous administration to mice. Further preclinical safety data were obtained by demonstrating lack of replication of the agent in human peripheral blood mononuclear cells. These results suggest that Ad5-Delta 24 RGD could be useful for local or systemic treatment of cervical cancer in patients with disease resistant to currently available modalities.     

培美曲塞联合顺铂与长春瑞滨联合顺铂治疗晚期乳腺癌对比观察

目的:比较培美曲塞联合顺铂与长春瑞滨联合顺铂(NP)治疗蒽环类和紫杉类耐药晚期乳腺癌临床疗效和毒副反应。方法:58例对蒽环类和紫杉类耐药晚期乳腺癌患者随机分为两组,分别给予培美曲塞联合顺铂30例与NP方案28例至少治疗两周期后进行评价。结果:培美曲塞联合顺铂组总有效率(RR)CR+PR=53.3%,疾病控制率(DCR)CR+PR+SD=83.3%,NP方案组总有效率(RR)CR+PR=50.0%,疾病控制率(DCR)CR+PR+SD=78.6%。两组疗效比较无显著性差异(P>0.05)。主要毒副反应为骨髓抑制和胃肠道反应。Ⅲ-Ⅳ度白细胞减少发生率:培美曲塞联合顺铂组8例(26.7%),NP方案组11例(39.3%),Ⅲ-Ⅳ度胃肠道反应率:培美曲塞联合顺铂组3例(10.0%),NP方案组6例(21.4%),培美曲塞联合顺铂Ⅲ-Ⅳ度毒副反应明显低于NP方案组,两组相比有显著性差异(P<0.05)。结论:培美曲塞联合顺铂与NP方案均是治疗蒽环类和紫杉类耐药晚期乳腺癌有效的方案。前者血液学及消化道反应毒性较轻,患者耐受性好,值得临床进一步推广。     

Selective effects of a fiber chimeric conditionally replicative adenovirus armed with hep27 gene on renal cancer cell

Adenoviruses mediated cancer gene therapies are widely investigated and show a promising effect on cancer treatment. However, efficient gene transfer varies among different cancer cell lines based on the expression of coxsakie adenovirus receptor (CAR). Hep27, a member of dehydrogenase/reductase (SDR) family, can bind to Mdm2, resulting in the attenuation of Mdm2-mediated p53 degradation. Here we constructed a fiber chimeric adenovirus carrying hep27 gene (F5/35-ZD55-Hep27), in which the fiber protein of 5-serotype adenovirus (Ad5) was substituted by that of 35-serotype adenovirus (Ad35), aiming to facilitate the infection for renal cancer cells and develop the role of hep27 in cancer therapy. We evaluated the CAR and CD46 (a membrane cofactor protein for Ad35) expression in four kinds of renal cancer cells and assessed the relationship between receptors and infection efficiency. 5/35 fiber-modified adenovirus had a much promising infectivity compared with Ad5-based vector in renal cancer cells. F5/35-ZD55-Hep27 had enhanced antitumor activity against human renal cancer cells compared to the other groups. Further, hep27 mediated p53 and cleaved-PARP upregulation and mdm2 downregulation was involved and caused increased apoptosis. Moreover, F5/35-ZD55-Hep27 significantly suppressed tumor growth in subcutaneous renal cancer cell xenograft models. Our data demonstrated that 5/35 fiber-modified adenovirus F5/35-ZD55-Hep27 transferred into renal cancers efficiently and increased p53 to induce cancer cell apoptosis. Thus 5/35 fiber-modified adenoviral vector F5/35-ZD55-Hep27 might a promising vector and antitumor reagent for renal cancer gene therapy.     

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.     

Computer-based identification of a novel LIMK1/2 inhibitor that synergizes with salirasib to destabilize the actin cytoskeleton

Neurofibromin regulates cell motility via three distinct GTPase pathways acting through two different domains, the Ras GTPase-activating protein-related domain (GRD) and the pre-GRD domain. First, the GRD domain inhibits Ras-dependent changes in cell motility through the mitogen activated protein cascade. Second, it also regulates Rho-dependent (Ras-independent) changes by activating LIM kinase 2 (LIMK2), an enzyme that phosphorylates and inactivates cofilin (an actin-depolymerizing factor). Third, the pre-GRD domain acts through the Rac1 GTPase, that activate the P21 activated kinase 1 (PAK1)-LIMK1-cofilin pathway. We employed molecular modeling to identify a novel inhibitor of LIMK1/2. The active sites of an ephrin-A receptor (EphA3) and LIMK2 showed marked similarity (60%). On testing a known inhibitor of EphA3, we found that it fits to the LIMK1/2-ATP binding site and to the latter's substrate-binding pockets. We identified a similar compound, T56-LIMKi, and found that it inhibits LIMK1/2 kinase activities. It blocked the phosphorylation of cofilin which led to actin severance and inhibition of tumor cell migration, tumor cell growth, and anchorage-independent colony formation in soft agar. Because modulation of LIMK by neurofibromin is not affected by the Ras inhibitor Salirasib, we examined the combined effect of Salirasib and T56-LIMKi each of which can affect cell motility by a distinct pathway. We found that their combined action on cell proliferation and stress-fiber formation in neurofibromin-deficient cells was synergistic. We suggest that this drug combination may be developed for treatment of neurofibromatosis and cancer.     

转录靶向性溶瘤腺病毒治疗肺癌研究的现状与思考

目的:总结分析国内外应用转录靶向性溶瘤腺病毒治疗肺癌的进展和面临的困难。方法:以"腺病毒、肺癌、启动子"为关键词,检索CNKI及PubMed数据库,年限为2000-2010,相关文献72篇,其中中文文献16篇,英文56篇。纳入标准:1)以腺病毒为载体治疗肺癌;2)用肺癌基因启动子取代腺病毒复制必须基因启动子。根据纳入标准分析23篇文献。结果:转录靶向性溶瘤腺病毒是应用肿瘤特异性启动子调控腺病毒复制必须基因的表达,使腺病毒只在特定的肿瘤细胞中复制、扩增,从而能最大程度地靶向杀伤肿瘤细胞,而不杀伤正常细胞。可应用于肺癌的肿瘤特异性启动子有10余种,用这些特异性启动子取代腺病毒复制起始基因启动子后,腺病毒复制必须基因序列因受其特异性调控,只在肺癌细胞中复制、扩增,从而最大程度地靶向杀伤肺癌细胞,不杀伤正常细胞;同时,腺病毒载体的优越性在于其能保证启动子和治疗基因安全、稳定、高效地在肺癌组织中表达。这使得靶向性溶瘤腺病毒在肺癌治疗研究中取得了显著进展,但这些研究多集中于体外实验及动物体内实验,临床应用尚面临诸多困难。结论:转录靶向性溶瘤腺病毒为肺癌的治疗提供了新思路,如能突破目前研究中所面临的种种困难,进一步全面、深入研究肺癌的发病机制,探寻特异性更强的肺癌启动子,增强溶瘤腺病毒的靶向性,降低毒副反应,可望广泛应用于临床。     

塞来昔布联合携带白细胞介素24基因的溶瘤腺病毒对人类膀胱尿路上皮癌T24细胞的作用

目的 评估塞来昔布联合携带白细胞介素24(IL-24)基因的溶瘤腺病毒(ZD55-IL-24)对人类膀胱尿路上皮癌T24细胞的选择性杀伤效能.方法 荧光显微镜观察ZD55-增强型绿色荧光蛋白(EGFP)对细胞的转染效率.以人类脐静脉内皮细胞(HUVEC)作为对照.RT-PCR法测IL-24 mRNA在细胞中的表达.MTT法测塞来昔布联合ZD55-IL-24对肿瘤细胞选择性抑制作用.流式细胞术检测细胞凋亡情况.结果 ZD55-EGFP感染细胞后,在相同时间下T24细胞中的荧光强度明显高于HUVEC细胞.ZD55-IL-24转染后在相同时间内T24细胞组IL-24 mRNA表达量均高于HUVEC组(t=-12.54,P＜ 0.01).ZD55-IL-24与塞来昔布联合对T24细胞的抑制率最高(P＜0.01).T24细胞各组的细胞增殖抑制率也明显高于HUVEC组(F=251.35,P＜ 0.01).联合用药组的T24细胞凋亡率也明显高于单独用药组;T24细胞组凋亡率明显高于HUVEC组(P＜ 0.001).结论 塞来昔布与ZD55-IL-24联合用药能最大程度地抑制T24细胞的增殖,而对正常细胞影响轻微.这种抑制作用可能与细胞凋亡有关.     

替吉奥胶囊联合顺铂治疗晚期胃癌的临床疗效

目的探讨晚期胃癌患者采用替吉奥胶囊联合顺铂治疗的近期疗效和不良反应。方法选取2013年1月至2015年1月间收治的60例晚期胃癌患者(初治或复治),采用数字抽样法随机分为观察组和对照组,每组30例。观察组患者采用替吉奥胶囊联合顺铂治疗,对照组患者给予单纯使用替吉奥胶囊治疗,比较两组患者的近期疗效和不良反应。结果观察组患者近期疗效(56.7%)显著高于对照组(40.0%),差异有统计学意义(P<0.05)。观察组患者的临床受益率(73.3%)显著高于对照组(56.7%),差异有统计学意义(P<0.05)。在口腔黏膜炎、腹泻、恶心呕吐、白细胞减少、眩晕、肝功能损害、肾功能损害等不良反应方面,观察组患者的Ⅲ~Ⅵ度不良反应发生率与对照组比较,差异无统计学意义(P>0.05)。结论替吉奥胶囊联合顺铂治疗方案的疗效可靠,且不良反应较少,安全性较高。     

NVB联合顺铂治疗蒽环类和/或紫杉类化疗后的晚期乳腺癌

目的观察去甲长春花碱(vinorelbine,NVB)联合顺铂(cisplatin,DDP)治疗用过蒽环类和/或紫杉类的转移性乳腺癌的疗效及不良反应.方法采用NVB25mg/m2 d1,5,DDP80～100mg/m2(d1～3),每3周重复1次.结果有效率为42.3%(11/26),均为PR.主要不良反应是白细胞减少(Ⅲ、Ⅳ度发生率为44.4%).结论NVB联合DDP治疗转移性乳腺癌有较好的疗效,且对蒽环类和/或泰素治疗失败的患者亦有较好的疗效,不良反应可耐受.     

NM23-H1 expression of head and neck squamous cell carcinoma in association with the response to cisplatin treatment

We recently reported that low NM23-H1 expression of head and neck squamous cell carcinoma (HNSCC) correlated with poor patients'' prognosis. Growing evidence has indicated that high tumor NM23-H1 expression contributes to a good response to chemotherapy. Therefore, we investigated the role of NM23-H1 in susceptibility of HNSCC cells to cisplatin and its clinical significance, as well as the in vitro study for validation was performed. Using immunohistochemistry, we analyzed NM23-H1 expression in surgical specimens from 46 HNSCC patients with cervical metastases receiving surgery and adjuvant chemoradiotherapy. Low tumor NM23-H1 expression correlated with locoregional recurrence of HNSCC following postoperative cisplatin-basedtherapy (p = 0.056) and poor patient prognosis (p = 0.001). To validate the clinical observation and the effect of NM23-H1 on cisplatin cytotoxicity, we established several stable clones derived from a human HNSCC cell line (SAS) by knockdown and overexpression. Knockdown of NM23-H1 attenuated the chemosensitivity of SAS cells to cisplatin, which was associated with reduced cisplatin-induced S-phase accumulation and downregulation of cyclin E1 and A. Overexpression of NM23-H1 reversed these results, indicating the essential role of NM23-H1 in treatment response to cisplatin. NM23-H1 may participate in HNSCC cell responses to cisplatin and be considered a potential therapeutic target.     

DP与IP方案二线治疗进展期胃癌临床观察

目的 进展期胃癌(advanced gastric cancer,AGC)的一线化疗方案主要以氟尿嘧啶类及顺铂为基础,一旦出现肿瘤进展,二线治疗疗效欠佳.本研究主要观察多西他赛联合顺铂(DP)以及伊立替康联合顺铂(IP)二线治疗AGC的有效性和安全性.方法 回顾性分析2011-01-01-2014-01-31入住于皖南医学院弋矶山医院肿瘤内科的94例既往接受以氟尿嘧啶类药物为基础的一线化疗方案治疗失败的AGC患者,分为DP和IP组.DP组51例,多西他赛(docetaxel,TXT) 35 mg/m2,静脉滴入,d1、d8;顺铂(cisplatin,DDP) 20 mg/m2,静脉滴入,d1～d4;21 d为1个周期.IP组43例,伊立替康(irinotecan,CPT-11) 65 mg/m2,静脉滴入,d 1、d8;DDP 20 mg/m2,静脉滴入,d1～d4;21 d为1个周期.化疗过程中注意观察不良反应,根据不良反应的程度调整药物用量.每2个周期评价疗效.结果 94例患者均可评价疗效.DP组完全缓解(complete response,CR)1例(1.96％),部分缓解(partial response,PR) 13例(25.49％),稳定(stable disease,SD) 15例(29.41％),进展(progressive disease,PD) 22例(43.14％),客现有效率(objective response rate,ORR)为27.45％,疾病控制率(disease control rate,DCR)为56.86％,中位无进展生存时间(median progression-freesurvival,mPFS) 5.1个月,中位总生存时间(median overall survival,mOS)8.4个月.IP组PR 9例(20.93％),SD 13例(30.23％),PD 21例(48.84％),ORR为20.93％,DCR为51.16％,mPFS 4.5个月,mOS 7.6个月.2组间ORR(x2-0.063,P=0.467)、DCR(x2 =0.305,P=0.581)、mPFS(x2=0.320,P=0.571)和mOS(x2 =0.436,P=0.509)均差异无统计学意义;2组间临床分期较早者疗效明显优于临床分期较晚者,P=0.009;ECOG评分较好者疗效明显优于ECOG评分较差者,P=0.009.常见的不良反应主要为骨髓抑制、胃肠道反应、口腔黏膜炎和脱发等.DP组的脱发和口腔黏膜炎发病率高于IP组,差异有统计学意义,Z值分别为-7.854和-2.726,P值分别为＜0.001和0.006;IP组的腹泻及胆碱能综合征发病率远高于DP组,差异有显著统计学意义,Z值分别为=5.648和-2.741,P值分别为＜0.001和0.006;2组血液学毒性类似,多为Ⅰ～Ⅱ度,可耐受,IP组血小板下降发病率高于DP组,但差异无统计学意义,Z=-1.777,P=0.076.结论 DP和IP方案二线治疗以氟尿嘧啶类药物为基础的一线化疗失败的AGC疗效相当,不良反应轻,患者可耐受,不失为较好的挽救方案.