Metformin targets gastric cancer stem cells

Gastric cancer is the third leading cause of cancer-related deaths worldwide and has still a poor prognosis. Therefore, new therapeutic strategies are needed: among them, targeting cancer stem cells (CSCs) could offer new opportunities. The aim of our study was to evaluate the anti-tumoural effect of metformin on gastric cancer in vitro and in vivo and especially, to determine whether this molecule could target the gastric CSCs. Metformin effects were evaluated on the proliferation and tumourigenic properties of the gastric CSCs from patient-derived primary tumour xenografts (PDXs) and cancer cell lines (MKN45, AGS and MKN74) in vitro in conventional 2 dimensional (2D) and in 3 dimensional (3D) culture systems, in which only CSCs are able to form tumourspheres and in mouse xenograft models in vivo. Metformin induced a cell cycle arrest, which decreased cell proliferation in the 2D cultures. In a 3D culture system, metformin decreased the number of tumourspheres, revealing its capacity to target the CSCs. This effect was confirmed by the study of the expression of CSC markers (CD44 and Sox2) and differentiation markers (Kruppel-like factor 4 and MUC5AC), which were decreased or increased in response to metformin, respectively. Finally, in vivo treatment of PDXs with metformin led to a tumour growth delay and decreased the self-renewal ability of the CSCs. These results suggest that the use of metformin could represent an efficient strategy to inhibit tumour growth by targeting gastric CSCs.     

Suppression of the ATP-binding cassette transporter ABCC4 impairs neuroblastoma tumour growth and sensitises to irinotecan in vivo

The ATP-binding cassette transporter ABCC4 (multidrug resistance protein 4, MRP4) mRNA level is a strong predictor of poor clinical outcome in neuroblastoma which may relate to its export of endogenous signalling molecules and chemotherapeutic agents. We sought to determine whether ABCC4 contributes to development, growth and drug response in neuroblastoma in vivo. In neuroblastoma patients, high ABCC4 protein levels were associated with reduced overall survival. Inducible knockdown of ABCC4 strongly inhibited the growth of human neuroblastoma cells in vitro and impaired the growth of neuroblastoma xenografts. Loss of Abcc4 in the Th-MYCN transgenic neuroblastoma mouse model did not impact tumour formation; however, Abcc4-null neuroblastomas were strongly sensitised to the ABCC4 substrate drug irinotecan. Our findings demonstrate a role for ABCC4 in neuroblastoma cell proliferation and chemoresistance and provide rationale for a strategy where inhibition of ABCC4 should both attenuate the growth of neuroblastoma and sensitise tumours to ABCC4 chemotherapeutic substrates.     

Proliferative activity of early ovarian clear cell adenocarcinoma depends on association with endometriosis

OBJECTIVE: To investigate differences in the biological characteristics of ovarian clear cell adenocarcinoma based on the presence/absence of endometriosis and tumor proliferative activity. METHODS: Stage I ovarian clear cell adenocarcinoma patients were divided into groups with and without endometriosis, and immunohistochemical expression of proliferating cell nuclear antigen was determined in surgical specimens. Then xenograft models of human ovarian clear cell adenocarcinoma with or without human ectopic endometrium were created in severe combined immunodeficiency mice, and tumor growth was assessed from the wet weight and the bromodeoxyuridine uptake. Furthermore, a xenograft model of human endometriosis was made with or without ovarian clear cell adenocarcinoma and cytokine production was investigated. RESULTS: The proliferating cell nuclear antigen labeling index was significantly lower in the tumors of patients with endometriosis compared to the tumors of patients without endometriosis. In tumor-bearing mice, the tumor weight and bromodeoxyuridine uptake were both significantly lower when ovarian clear cell adenocarcinoma was associated with endometriosis than in its absence. Release of transforming growth factor-beta1 and interleukin-6 from the ectopic human endometrium was greater in the presence of clear cell adenocarcinoma than without it, and transforming growth factor-beta1 levels showed a significant difference. CONCLUSION: The proliferative activity of early ovarian clear cell adenocarcinoma seems to depend on the association of this cancer with endometriosis. When endometriosis is associated with ovarian clear cell adenocarcinoma, there is a change of its cytokine production that may inhibit tumor growth.     

Increased Ras GTPase activity is regulated by miRNAs that can be attenuated by CDF treatment in pancreatic cancer cells

Ras gene is frequently mutated, and also associated with increased Ras expression and its GTPase activity (activity) in pancreatic cancer (PC), which could in part be due to deregulated expression of microRNAs (miRNAs) contributing to tumor aggressiveness. Here we report, for the first time, that Ras expression and its activity were significantly higher in MIAPaCa-2 cells compared to COLO-357 and BxPC-3 cell lines, which was correlated with loss of let-7 family and miR-143 expression in MIAPaCa-2 cells compared to COLO-357 and BxPC-3 cells. Whereas the expression of miR-21, a frequently up-regulated miRNA in solid tumors was up-regulated in MIAPaCa-2 cells and it was correlated with increased Ras expression and its activity. The miRNAs, let-7i and miR-143 was found to target Ras, and forced re-expression of let-7i and miR-143 inhibited Ras activity, cell proliferation and colony formation in vitro. We also found that the treatment of cells in vitro or treatment of MIAPaCa-2 induced tumors in vivo with CDF, a novel synthetic analog of curcumin, led to the re-expression of let-7 and miR-143, and down-regulated miR-21 expression, which was consistent with attenuation of Ras expression and its activity. Moreover, re-expression of let-7iin vivo resulted in decreased tumor growth and Ras activity. These results suggest that the loss of expression of let-7 and miR-143, and increased expression of miR-21 leads to increased expression of Ras and its GTPase activity, which could be attenuated by CDF treatment and, thus CDF could become a novel therapeutic agent for the treatment of PC.     

siRNA介导的间皮素基因沉默对卵巢癌裸鼠腹腔移植瘤生长的影响

目的:研究间皮素(mesothelin, MSLN)基因被沉默后的卵巢癌细胞SKOV3-MSLN-shRNA增殖能力的变化,以及MSLN基因沉默对裸鼠卵巢癌腹腔移植瘤生长的影响.方法:利用针对MSLN基因的siRNA慢病毒液和对照组慢病毒液分别感染SKOV3细胞,建立SKOV3-MSLN-shRNA细胞和SKOV3-MSLN-neg细胞的稳定转染株;以SKOV3细胞作空白对照,用平板克隆形成法和细胞计数法检测3组细胞的增殖能力.应用3组细胞分别建立裸鼠卵巢癌腹腔移植瘤模型,2周后处死裸鼠,观察每组动物的成瘤率及每只裸鼠的肿瘤质量、肿瘤数目和肿瘤种植部位等.结果:SKOV3-MSLN-shRNA细胞的体外增殖能力明显低于SKOV3-MSLN-neg细胞和SKOV3细胞,差异有统计学意义(P<0.05).SKOV3-MSLN-shRNA组裸鼠成瘤率为60%,而2个对照组SKOV3-MSLN-neg和SKOV3细胞的裸鼠成瘤率均为100%,差异无统计学意义(P＞0.05).SKOV3-MSLN-shRNA组动物的肿瘤数目、肿瘤质量及肿瘤种植部位明显低于2个对照组 (P<0.05).结论:MSLN基因沉默可以降低卵巢癌SKOV3细胞的体外增殖能力,同时可以抑制卵巢癌裸鼠腹腔移植瘤的生长和种植.     

SAC重组腺相关病毒载体的构建及其对前列腺癌CAM移植瘤的抑制作用

目的 构建携带NT4-SAC-HA2-TAT融合基因的重组腺相关病毒载体,并感染前列腺癌鸡胚绒毛尿囊膜(CAM)移植瘤,观察目的基因前列腺凋亡反应基因 4(par-4)选择性癌细胞凋亡结构域(SAC)的表达及其对CAM移植瘤的生长抑制作用。方法 利用磷酸钙共沉淀法将包装质粒pAAV/Ad、腺病毒辅助质粒pFG140及重组穿梭质粒pSSCMV/NT4-SAC-HA2-TAT共转染293细胞,制备重组腺相关病毒(rAAV), 斑点杂交法检测病毒滴度。取孵育9d龄健康鸡胚,CAM接种前列腺癌细胞系PC 3细胞,建立前列腺癌的CAM移植瘤模型,接种3d后挑选成瘤鸡胚随机分为3组：空白对照组(PBS),空病毒组(AAV)和重组病毒组(rAAV),观察瘤体生长情况,上述处理4d后测量比较不同组移植瘤体积,并切片染色病理检查。免疫荧光法检测移植瘤中SAC融合肽的表达。结果 酶切鉴定结果证实成功构建pSSCMV/NT4-SAC-HA2-TAT, 斑点杂交法检测重组病毒滴度约为3.34×1010～3.34×1011cfu/mL。免疫荧光法证实SAC融合肽表达。移植瘤能在CAM上生长,可见瘤周围丰富毛细血管汇集,rAAV组瘤体显著缩小,病理HE染色可见瘤细胞局限及侵袭抑制。结论 成功构建SAC重组腺相关病毒载体并感染前列腺癌CAM移植瘤,重组病毒表达分泌的SAC融合肽对前列腺癌CAM移植瘤具有诱导细胞凋亡、抑制肿瘤细胞侵袭性增殖等作用。     

不同剂量贝伐单抗在荷结肠癌细胞裸鼠中对伊立替康分布的影响

目的 观察不同剂量贝伐单抗对荷结肠癌细胞裸鼠中伊立替康分布的影响。方法 接种人结肠癌DLD-1细胞的裸鼠24只,随机分为4组。对照组：0.9%氯化钠溶液+伊立替康;实验1组：2.5 mg/kg贝伐单抗联合伊立替康治疗;实验2组：5 mg/kg贝伐单抗联合伊立替康治疗;实验3组：10 mg/kg贝伐单抗联合伊立替康治疗。观察不同组经处理后的肿瘤大小,外周血及肿瘤组织内伊立替康浓度的差异。结果 对照组及3个实验组间肿瘤体积差异比较无统计学意义。外周血中伊立替康的浓度在3个实验组中均明显高于对照组,且浓度随着贝伐单抗剂量增加而增加[（432.33±104.76）、（409.69±267.15）、（719.21±253.00）vs. （299.69±83.63）ng/ml]。其中实验3组与实验2组、对照组的差异有统计学意义（P=0.045, 0.010）。肿瘤组织内伊立替康的浓度在3个实验组均明显低于对照组,其中实验3组与对照组的差异有统计学意义（P=0.047）。结论 贝伐单抗的治疗能改变伊立替康在荷结肠癌细胞裸鼠中分布,并与贝伐单抗的剂量有关。     

A novel antibody-like TCRγδ-Ig fusion protein exhibits antitumor activity against human ovarian carcinoma

TCRγ9δ2(OT3) is a tumor-specific TCR with an unique complementarity-determining region 3 (CDR3) sequence, referred to as OT3, in its δ2 chain. This region was identified in tumor-infiltrating lymphocytes (TILs) from human ovarian epithelial carcinoma. We demonstrated that TCRγ9δ2(OT3)-Fc, a fusion protein composed of the complete extracellular domains of the γ9 and δ2 chains linked to the Fc domains of human IgG1, exhibited successful binding to multiple human carcinoma cell lines. In vitro, TCRγ9δ2(OT3)-Fc mediated cell killing via antibody-dependent cellular cytotoxicity (ADCC) in a dose-dependent manner. In vivo, TCRγ9δ2(OT3)-Fc significantly inhibited tumor growth and enhanced survival in human ovarian carcinoma xenograft models. Our findings suggest that the TCRγ9δ2(OT3)-Fc fusion protein possesses both the antigen-recognition properties of TCR γδ and the Fc-mediated effector functions of the antibody.     

Antitumor activities of the targeted multi-tyrosine kinase inhibitor lenvatinib (E7080) against RET gene fusion-driven tumor models

RET gene fusions are recurrent oncogenes identified in thyroid and lung carcinomas. Lenvatinib is a multi-tyrosine kinase inhibitor currently under evaluation in several clinical trials. Here we evaluated lenvatinib in RET gene fusion-driven preclinical models. In cellular assays, lenvatinib inhibited auto-phosphorylation of KIF5B-RET, CCDC6-RET, and NcoA4-RET. Lenvatinib suppressed the growth of CCDC6-RET human thyroid and lung cancer cell lines, and as well, suppressed anchorage-independent growth and tumorigenicity of RET gene fusion-transformed NIH3T3 cells. These results demonstrate that lenvatinib can exert antitumor activity against RET gene fusion-driven tumor models by inhibiting oncogenic RET gene fusion signaling.