去泛素化酶USP9X在etoposide促结肠癌细胞SW620凋亡中的作用研究

目的:探讨USP9X在etoposide促结肠癌细胞SW620凋亡中的作用和可能的机制。方法:设计并合成USP9X的shRNA和对照shcontrol,采用半定量RT-PCR和Western blot检测干扰效率。结肠癌细胞SW620分别转染USP9X-shRNA和shcontrol后用etoposide处理,检测MCL1的蛋白水平变化并通过c-PARP的水平检测细胞的凋亡水平。结果:半定量RT-PCR和Western blot检测显示在SW620细胞中USP9X-shRNA能够显著降低USP9X的mRNA和蛋白的表达,抑制率达70%,该条shRNA可以作为有效干扰序列进行后续实验。20μg/mL etoposide处理24 h后,经c-PARP的水平检测发现感染USP9X-shRNA的SW620细胞比对照组细胞的凋亡率显著增加。结论:以RNA干扰技术沉默USP9X基因可增加结肠癌细胞SW620对etoposide的敏感性,显著增加etoposide诱导的结肠癌SW620细胞的凋亡,推测USP9X基因可能成为结肠癌基因治疗的一个新靶点。

Etoposide通过线粒体路径诱导Hela细胞凋亡

目的研究Etoposide诱导Hela细胞凋亡的分子机制.方法 Etoposide处理含有10%胎牛血清DMEM培养液培养的Hela细胞;Caspase-3活性检测试剂盒检测Etoposide处理Hela细胞Caspase-3活性;激光共聚焦显微镜和Western blot技术检测细胞色素C从线粒体的释放.结果 Etoposide能够导致Hela细胞凋亡;Etoposide处理Hela细胞内的Caspase-3活性增加;Etoposide诱导细胞色素C从线粒体释放到细胞质.结论 Etoposide通过线粒体路径诱导Hela细胞凋亡.

Etoposide经由caspase依赖途径诱导RGC-5细胞凋亡

目的研究etoposide诱导视网膜神经节细胞RGC-5细胞死亡的分子机制。方法建立etoposide诱导的RGC-5细胞死亡模型,应用APOPercentageTM及原位TUNEL染色确定RGC-5细胞的死亡方式,并进而利用Westernblot检测细胞内活化caspase-3及PARP-1的变化情况,并通过广谱caspase抑制剂间接证明是否有caspase依赖途径的激活。结果相对高浓度的etoposide(1～10μmol/L)可以迅速降低RGC-5细胞的活性并诱导死亡;APOPercentageTM及原位TUNEL染色确定etoposide是以凋亡的方式诱导RGC-5细胞死亡;Westernblot显示etoposide诱导后的RGC-5细胞中caspase-3被激活并伴有PARP-1的降解片段出现;广谱caspase抑制剂Z-VAD-fmk可以保护etoposide诱导的RGC-5细胞,提高细胞活性。结论Etoposide经由caspase依赖途径诱导RGC-5细胞凋亡。

Preparation of stealthy etoposide proliposomes and the pharmacokinetics in rabbits

The objectives of the present study were to prepare stealthy etoposide proliposomes and study the pharmacokinetics in rabbits. Blank stealthy liposomes were prepared by film dispersion method. Stealthy etoposide liposomes were prepared by using the ammonium sulfate gradient loading procedure. Vacuum freeze-drying technique was used to dry stealthy etoposide liposomes. Encapsulation efficiency of stealthy etoposide proliposomes was determined by Sephadex chromatography. The morphology was observed by transmission electronic microscope. The particle size and zeta potential were measured by using electrophoretic light scattering technology. The pharmacokinetics in rabbits was evaluated by comparison with etoposide injection and conventional liposomes, respectively. Mean encapsulation efficiency of stealthy etoposide proliposomes was 83.92% ± 3.65% （n = 3）. The liposomes were round or oval. Mean particle size was （124.5 ±26.9） nm, and zeta potential was （-39.50 ±1.04） mV. Following intravenous injection administration at a dose of 1.5 mg/kg etoposide, the three kinds of etoposide preparations were fitted with the two-compartment model. T1/2 β and A UC values of stealthy etoposide proliposomes were （19.26 ± 3.16） h and （26.04 ±3.53） μg/h/mL, respectively. T1/2 β and AUC values of etoposide injection were （0.94 ± 0.21） h and （0.98 ± 0.26） μg/h/mL, respectively. T1/2β and AUC values of conventional liposomes were （7.99 ± 1.36） h and （11.65 ± 1.70） μg/h/mL, respectively. Results indicated that the stealthy etoposide proliposomes could significantly extend the duration of etoposide in blood circulation.

Altered expression of nuclear matrix proteins in etoposide induced apoptosis in HL-60 cells

The events of cell death and the expression of nuclear matrix protein (NMP) have been investigated in a promyelocytic leukemic cell line HL-60 induced with etoposide. By means of TUNEL assay, the nuclei displayed a characteristic morphology change, and the amount of apoptotic cells increased early and reached maximun about 39% after treatment with etoposide for 2 h. Nucleosomal DNA fragmentation was observed after treatment for 4 h. The morphological change of HL-60 cells, thus, occurred earlier than the appearance of DNA ladder. Total nuclear matrix proteins were analyzed by 2-dimensional gel electrophoresis. Differential expression of 59 nuclear matrix proteins was found in 4 h etoposide treated cells. Western blotting was then performed on three nuclear matrix acssociated proteins, PML, HSC70 and NuMA. The expression of the suppressor PML protein and heat shock protein HSC70 were significantly upregulated after etoposide treatment, while NuMA, a nuclear mitotic apparatus protein, was down regulated. These results demonstrate that significant biochemical alterations in nuclear matrix proteins take place during the apoptotic process.

Anticancer Drugs (V)─—Synthesis of Etoposide Derivatives

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Comparing irinotecan/cisplatin with etoposide/cisplatin in patients with ED-SCLC: A meta-analysis of efficacy and toxicity

Objective： Irinotecan in combination with cisplatin for extensive-stage disease small-ceU lung cancer （ED-SCLC） patients has gained wide interest. Varying results for this treatment underpin the need for a synthesis of evidence. Methods： We conducted a literature-based meta-analysis to quantify the magnitude of the benefit comparing irinotecan in combination with cisplatin （IP） with etoposide in combination with cisplatin （EP） in ED-SCLC patients. The primary outcome was overall survival （OS） and progression-free survival （PFS）; secondary outcomes included overall response rate, 1- and 2-year survival rates, disease control rate and toxicity. Results： Four trials including 1,541 patients were identified in the analysis. No positive results （P〈0.05） were seen： OS （HR=0.85, CI95%=0.71-1.01; P=-0.08） with high heterogeneity （Chi2=7.76, dr=-3 [P=-0.05]; I2=61%）, PFS （HR=0.91, CI95%=0.74-1.28; P=-0.36） with high heterogeneity （Chi2=11.96, df=3 [P=-0.008]; I2=75%）, overall response rate（OR=1.16; CI95%=0.79-1.70; P=0.45）, disease control rate （OR=1.01; CI95%=0.74-1.38; P=0.95）, 1-year survival rate （OR = 1.30; CI95%=0.98-1.72; P=0.07） and 2-year survival rate （OR=1.97; CI95%=0.95-4.09; P=-0.07）. Fewer patients who received IP suffered severe hematologic toxicities （grade≥3）, such as neutropenia, thrombocytopenia and leucopenia. However, severe non-hematologic toxicities （grade≥3）, such as diarrhea, nausea, vomiting, fatigue, anorexia, and dehydration, were more common among patients who received IP. Conclusion： IP does not lengthen the overall survival or progression-free survival compared with EP in patients with ED-SCLC Fewer patients receiving IP had grade ≥ 3 hematological toxicities of nentropenia, leucopenia and thrombocytopenia, but more had grade≥3 diarrhea, nausea, vomiting, fatigue, anorexia and dehydration.

Cyclophosphamide and Etoposide as a Salvage Treatment in Metastatic Osteosarcoma Patients

Background and Objective: Osteosarcoma is a rare bone cancer with approximately 30% - 35% of patients who will relapse either systemically or locally, with the lung being the commonest site of relapse. The objective of this trial was to evaluate the efficacy of cyclophosphamide and etoposide, in treatment of metastatic osteosarcoma patients progressed after one or more chemotherapy lines, with the progression free survival and treatment response as the primary endpoints, while the secondary endpoints were overall survival and treatment toxicity. Patients and Methods: Twenty seven metastatic osteosarcoma patients were enrolled into this trial and received cyclophosphamide and etoposide chemotherapy. Cyclophosphamide was given at a dose of 500 mg/m2 per day, I.V for 5 days and etoposide (100 mg/m2 per day I.V for 5 days). Response was assessed after 3 cycles according to Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1. Chemotherapy Toxicity was graded according to the Common Terminology Criteria for Adverse Events (CTCAE). Results: The median overall survival time and progression-free survival were 12 months and 8 months, respectively. Four patients (14.8%) achieved partial response;14 patients (51.9%) had stationary disease (SD);and 9 (33.3%) expressed tumor progression. Hematologic toxicity was the main toxicity. None of the patients had G4 or life threatening toxicities. Conclusion: The combination of cyclophosphamide and etoposide represents an efficient and tolerable treatment option for patients with metastatic osteosarcoma.

Etoposide-based Combination Chemotherapy in Malignant Histiocytosis

We investigated the responses of patients with malignant histiocytosis (MH) to the treatment of epotoside-based regimen.Of 11 evaluable cases,9(81. 8%)achieved complete remission and 1 partial remission.7 complete remission cases (77. 7%) received only one therapeutic course.The side effects were mild and welltolerated. We conclude that etoposide-containing regimen is highly effective and can be used as first-line treatment for MH and is worthy of further study.

Effect of etoposide plus thalidomide as maintenance therapy on progression-free survival of elderly patients with advanced non-small cell lung cancer

Objective The aim of the study was to evaluate the efficacy and safety of etoposide plus thalidomide as maintenance therapy for elderly patients with advanced non-small cell lung cancer（NSCLC） without disease progression after first-line chemotherapy.Methods After four to six cycles of platinum-based first-line therapy, 64 elderly patients with advanced NSCLC without disease progression who were treated in the General Hospital of Shenyang Military Region（China） from 2014 to 2016 were enrolled in this study. According to the different maintenance treatment methods, patients were divided as having received etoposide plus thalidomide therapy（treatment group, n = 32） and best supportive care（control group, n = 32）. Disease control and progression-free survival（PFS） were compared between the two groups. Results The recent curative effect objective response rates of the treatment group and the control group were 31.3% and 3.1%, respectively, and the disease control rates were 71.9% and 31.3%, respectively. The Kaplan-Meier survival curves of the two groups were significantly different（χ2 = 26.532, P = 0.001）. The median PFS for the treatment group and control group was 6.0 months [95% confidence interval（CI） = 4.3–7.9 months] and 3.2 months（95% CI = 2.6–3.8 months）, respectively. The side effects in the treatment group included hematologic abnormalities, gastrointestinal toxicity, and impaired liver function, which were relieved after symptomatic support therapy and drug withdrawal.Conclusion Etoposide plus thalidomide as maintenance therapy is associated with a significantly longer PFS with tolerable toxicity for elderly patients with advanced NSCLC.AcknowledgementThe authors would like to thank Liu Zhongzheng for his technical assistance.

Etoposide sensitizes CT26 colorectal adenocarcinoma to radiation therapy in BALB/c mice

AIM： To investigate the combined effect of etoposide and radiation on CT26 colorectal adenocarcinoma implanted into BALB/c mice. METHODS： We evaluated the radiosensitizing effect of etoposide on CT26 colorectal adenocarcinoma in a syngeneic animal model. BALB/c mice were subcutaneously implanted with CT26 cells and divided into four groups： Gonlyol （intra-peritoneal saline×2） group, etoposide （5 mg/kg intra-peritoneally×2） group, radiation therapy （RT 5 Gy×2 fractions） group, and combination therapy with etoposide （5 mg/kg intra-peritoneally 1 h before radiation） group. RESULTS： Tumor growth was significantly inhibited by RT and combination therapy. The effect of combination therapy was better than that of RT. No significant changes were noted in body weight, plasma alanine aminotransferase, or creatinine in any group. The leukocyte count significantly but transiently decreased in the RT and combination therapy groups, but not in the etoposide and control groups. There was no skin change or hair loss in the RT and combination therapy groups. CONCLUSION： Etoposide can sensitize CT26 colorectal adenocarcinoma in BALB/c mice to RT without significant toxicity.

Etoposide Induces Mitochondria-Associated Apoptotic Cell Death in Human Gastric Carcinoma Cells

Recent observations indicate that the resistance of apoptosis is an important process of tumor metastasis and metastases are the cause of 90% of human cancer death. Etoposide, a semisynthetic derivative of the podophyllotoxins, is a clinically used anti-cancer reagent, but the effects of it on metastatic gastric carcinoma cells are totally unknown. In this study, etoposide induced apoptotic cell death in human gastric adenocarcinoma cell line SGC-7901, derived from metastatic lymph nodes, as evidenced by the analysis of DNA fragmentation, apoptotic body formation, caspase activation, and apoptosis specific changes in cell morphology is demonstrated. The depolarization of mitochondrial membrane and the release of cytochrome c were most early events in etoposide treated SGC-7901 cells, and were followed by caspase-3 activation and PARP cleavage. Caspase-8 activation was not detected under the same condition. Thus, it was proposed that etoposide induces caspase-associated apoptotic cell death in human metastatic gastric carcinoma, which is initiated by mitochondrial cytochrome c release.

Treatment of etoposide capsule combined with cisplatin or carboplatin in elderly patients with small cell lung cancer

We aimed to explore the efficacy and safety of etoposide capsule combined with cisplatin or carboplatin in the treatment of elderly patients with small cell lung cancer （SCLC）. Methods： From October 2011 to November 2013, 32 elderly patients （71-79 years old） with histopathologically confirmed SCLC in General Hospital of Shenyang Military Region （China） were enrolled in the research. The patients were administrated with lastet capsule 150-175 mg, dl-5, combined with cisplatin 20 mg/m^2 dl-3 or carbopiatin AUC = 5, applied over 2 days. Twenty-one days were 1 treatment cycle. Results：After treatments, 2 cases acquired complete response （CR）, 19 cases acquired partial response （PR）, 8 cases acquired stable disease （SD）, and 3 cases had progression of disease （PD）. The objective response rate was 65.6% （21/32）, disease control rate was 90.6% （29/32）. The median time of progression-free survival （PFS） was 6.9 months, the median survival time was 14.0 months, and 1 year survival rate was 62.4%. The main adverse reactions of 1/11 leukopenia and gastrointestinal reaction were observed. Conclusion： Etoposide capsule combined with cisplatin or carboplatin therapy have curative effect and good tolerance in elderly patients with SCLC.

Etoposide-induced apoptosis results in chromosome breaks within the <i>AF</i>9 gene: Its implication in chromosome rearrangement in leukaemia

Treatment with etoposide (VP-16) has been associated with translocation of the mixed lineage leukaemia (MLL) gene seen in treatment-related acute myeloid leukaemia (t-AML). Among the different partner genes, AF9 is the most common partner gene of MLL. AF9 shares similar structural element with the MLL gene. Various mechanisms of translocation have been proposed for the MLL gene, including apoptosis, particularly the apoptotic nuclease. In the current study, we show that VP-16 induced cleavage of the AF9 gene in both leukaemic cells and cultured normal blood cell. All the breakpoints were mapped within the BCR1 of the AF9 gene. AF9 cleavages in leukaemic cells were abolished by pre-treatment with caspase inhibitor (Z-DEVD-FMK), suggesting the involvement of caspase-activated DNase (CAD). The absence of AF9 cleavage in K562 cells further supported the involvement of apoptosis. However, AF9 cleavages in cultured normal blood cell were not eliminated by caspase inhibitor. The possible role of CAD and other apoptotic nucleases/effectors that could be involved in AF9 translocation are discussed.

依托扑沙(ETOPOSIDE)

由英国Bristol-Myers药厂于1983年在美国上市。为半合成鬼臼素。虽然鬼臼素本身有抗肿瘤作用,但依托扑沙是迄今为止所有鬼臼素衍生物中单剂应用时对呼吸道肿瘤治疗作用最好的化合物。主要应用于呼吸道肿瘤的治疗,尤其是应用于已进行过手术、放疗或其它化疗的病人。 [化学名称]4’-去甲鬼臼素-9-[4,6-O-(R)]-乙叉-β-D-吡喃葡糖苷 (1)4’-Demethylpipodophylloto-xin-9-[4,6- O-(R)]-ethylidene-β-D-glucopyranoside (2)5-(4.6-OO-Ethylidene-β-D-glucopyranosyl)oxy)-2,5,5a,6,8a,9-he-xahydro-9-(4-hydroxy-3,5-dimeth-oxyphenyl)furo〔3’,4’:6,7)

Accelerated Hypofractionated Radiotherapy and Concurrent Etoposide/Cisplatin in Patients with Limited-Disease SCLC (LD-SCLC)

Background: The optimal TRT dose/fraction for LD-SCLC remains debatable, and due to increasing number of population in Egypt and number of patients as well, so reducing the duration of radiation therapy is favored. This study was conducted using etoposide and cisplatin (EP) concurrently with accelerated hypofractionated TRT to evaluate the response and toxicity of this protocol in the treatment of patients with limited-disease small cell lung cancer (LD-SCLC). Patients and Methods: Thirty patients with previously untreated LD-SCLC were enrolled into this study between June 2012 and February 2015. All patients received etoposide 100 mg/m2 days 1 to 3 and cisplatin 25 mg/m2 days 1 to 3 with start of accelerated hypofractionated thoracic radiation therapy on first day of the second cycle of chemotherapy of 55 Gy, 2.5 Gy/fraction over 30 days. Chemotherapy was given 4 - 6 cycles. Prophylactic cranial irradiation 25 Gy/10 fractions were given for patients who achieved complete remission. Results: The median age was 60 years;28 patients (93%) were men. ECOG PS was 0 in 5 (17%) patients and 1 in 12 (40%) patients. Four (13%) patients achieved a complete response (CR), 17 (57%) patients achieved a partial response (PR), while 7 patients (23%) had progressive disease (PD), and the ORR was 90%. The median survival time was 26.4 months. The median PFS was 16.7 months. Among the hematologic toxicities neutropenia was the most prevalent toxicity and it was evident as grade 3 - 4 in 12 (40%) patients. Grade 3 - 4 Asthenia was the most prevalent nonhematological toxicity, in 12 (40%) patients;esophagitis occurred in 7 (23%) patients. No treatment-related deaths (due to sepsis or bleeding) were reported in the study. Conclusion: Using etoposide and cisplatin concurrently with accelerated hypofractionated thoracic radiation therapy for the treatment of patients with LD-SCLC showed an encouraging outcome and acceptable toxicity and warrants further research.

Allogeneic and autologous stem cell transplantation with busulfan, cyclophosphamide, and etoposide conditioning therapy for relapsed/refractory non-Hodgkin lymphoma

The optimal stem cell transplantation (SCT) conditioning therapy for relapsed/refractory non-Hodgkin lymphoma (NHL) is not clearly defined. In a retrospective analysis, we examined 25 patients with “high risk” relapsed/refractory NHL who received busulfan, cyclophosphamide, and etoposide (Bu/Cy/VP16) conditioning with autologous or allogeneic SCT. The majority of patients had aggressive histology and 52% had primary refractory NHL. Furthermore, 48% of patients had chemotherapy-resistant disease at the time of SCT. Fifty-six percent of patients underwent allogeneic SCT, while 44% had autologous SCT. The median engraftment time for neutrophils and platelets was 13.5 and 14 days, respectively. The 100-day treatment-related mortality (TRM) was 16%, while the 2-year non-relapse mortality (NRM) rate was also 16%. At a median follow-up of 15 months, the estimated 2-year disease-free survival (DFS) rate was 64% (95% confidence interval (CI): 36%-82%) and the estimated 2-year overall survival (OS) was 69% (95% CI: 40%-86%). Furthermore, the 2-year disease-specific survival (DSS) rate was 73% (95% CI: 40%-90%). Using Cox proportional hazard modeling, the International Prognostic Index at time of relapse predicted DFS and OS. Altogether, Bu/Cy/VP16 was associated with early TRM;however, late toxicities (including NRM) were uncommon resulting in relatively good survival rates in a high-risk relapsed/refractory NHL population.

A Randomized Controlled Clinical Study of Irinotecan/cisplatin Regimen and Etoposide/cisplatin Regimen in the First-line Treatment of Small Cell Lung Cancer

Objective:This paper mainly study on the effects of irinotecan/cisplatin and etoposide/cisplatin regimens in the treatment of small cell lung cancer.Methods:50 cases of small cell lung cancer patients in our hospital were divided into control group and experimental group and administered with etoposide/cisplatin and irinotecan/cisplatin regimen,and the treatment effects of the two regimens were compared statistically.Results:After treatment,both groups achieved high treatment efficiency,and the incidence of toxic side effects was low,with no significant difference(P>0.05);serological ABCG2 and FGFBP1 level indicators were better than the control group,both showing significant differences(P<0.05).Conclusions:Irinotecan has achieved better improvement in serological indicators in the first-line treatment of small cell lung cancer,with no significant difference in short-term treatment efficiency.

BET inhibitors enhance the anti-cancer effect of etoposide by suppressing the MRN-ATM axis in the DNA damage response

Etoposide is widely used for cancer chemotherapy in the clinic.However,long-term etoposide treatment can lead to adverse effects or drug resistance.To improve the situation,we evaluated the therapeutic efficiency of etoposide combined with inhibitors of bromodomain and extraterminal(BET)family proteins,which have recently emerged as novel anti-cancer targets due to their critical roles in cancer development.Firstly,we showed BRD4,one of the main targets of BET inhibitors,was involved in DNA damage response(DDR)via the homologous recombination(HR)repair pathway.