Selective ALK inhibitor alectinib with potent antitumor activity in models of crizotinib resistance

The clinical efficacy of the ALK inhibitor crizotinib has been demonstrated in ALK fusion-positive NSCLC; however, resistance to crizotinib certainly occurs through ALK secondary mutations in clinical use. Here we examined the efficacy of a selective ALK inhibitor alectinib/CH5424802 in models of crizotinib resistance. Alectinib led to tumor size reduction in EML4-ALK-positive xenograft tumors that failed to regress fully during the treatment with crizotinib. In addition, alectinib inhibited the growth of some EML4-ALK mutant-driven tumors, including the G1269A model. These results demonstrated that alectinib might provide therapeutic opportunities for crizotinib-treated patients with ALK secondary mutations.     

Activation of AMP-activated protein kinase sensitizes lung cancer cells and H1299 xenografts to erlotinib

Objectives

The therapeutic scheme for non-small cell lung cancer (NSCLC) patients can be improved if adapted to the individual response. For example, 60–70% of adenocarcinoma patients show response to EGFR-tyrosine kinase inhibitors in the presence of mutated EGFR. We searched for additional target molecules involved in the action of the EGFR-tyrosine kinase inhibitor erlotinib in the absence of EGFR mutations, which might be suitable for combinatorial therapy approaches.

Materials and Methods

Erlotinib-response associated proteins were investigated in patient-derived NSCLC mouse xenografts by reverse-phase protein array technology (RPPA) and Western blotting. A combinatorial treatment approach was carried out in NSCLC cell lines and H1299 mouse xenografts, and subsequently analyzed for consequences in cell growth and signal transduction.

Results

AMP-activated protein kinase (AMPK) expression was increased in erlotinib responders before and after treatment. In a combinatorial approach, activation of AMPK by A-769662 and erlotinib treatment showed a synergistic effect in cell growth reduction and apoptosis activation in H1299 cells compared to the single drugs. AMPK pathway analyses revealed an effective inhibition of mTOR signaling by drug combination. In H1299 xenografts, the tumor size was significantly decreased after combinatorial treatment.

Conclusion

Our results suggest that AMPK activation status affects response to erlotinib in distinct lung tumor models.     

全长型脾酪氨酸激酶基因转染对人喉鳞癌裸鼠移植瘤的生长抑制作用

目的 探讨全长型脾酪氨酸激酶[full-length spleen tylosine kinase,SYK(L)]基因转染对人喉鳞状细胞癌Hep-2细胞裸鼠移植瘤生长的影响。方法 建立人喉鳞癌Hep-2细胞裸鼠皮下移植瘤模型,分为SYK(L)重组质粒转染组、阴性对照质粒转染组和生理盐水组,分别向瘤体内注射SYK(L)重组质粒pIRES2-EGFP-SYK(L)及脂质体混合物、阴性对照质粒pIRES2-EGFP及脂质体混合物和生理盐水,观察肿瘤生长情况。通过实时荧光定量聚合酶链式反应(quantitative real time fluorescence polymerase chain reaction,Q-RT-PCR)、Western blot法检测各组瘤体内的SYK(L) mRNA及蛋白表达水平。结果 SYK(L)重组质粒转染组的肿瘤平均体积为367.61±81.23mm³,明显小于阴性对照质粒转染组900.20±131.41mm³及生理盐水组930.71±143.73mm³,差异有统计学意义(F=99.91,P<0.01);Q-RT-PCR结果显示SYK(L)重组质粒转染组瘤体内mRNA相对表达量为2.268±0.075,明显高于阴性对照质粒转染组(1.212±0.025)及生理盐水组(1.175±0.031)(F=102.01,P<0.01);Western blot法检测结果显示SYK(L)重组质粒转染组瘤体内相对蛋白含量(0.834±0.021)明显高于阴性对照质粒转染组(0.243±0.041)和生理盐水组(0.301±0.039),差异有统计学意义(F=104.13,P<0.01)。结论 全长型脾酪氨酸激酶基因转染对人喉鳞癌裸鼠移植瘤有明显的生长抑制作用,有望成为基因治疗喉癌的新靶点及途径。     

苏拉明对肺腺癌小鼠移植瘤生长的影响及其机制

背景与目的:有研究表明,苏拉明对恶性肿瘤有明显抑制作用,但有关其对肺腺癌体内抗瘤作用的报道很少。本实验观察苏拉明对肺腺癌LA795细胞的T739小鼠移植瘤生长和转移的抑制作用,并探讨其作用机制。方法:将32只接种LA795肺腺癌细胞T739的小鼠随机分成4组:对照组、顺铂组、苏拉明组、联合组(苏拉明加顺铂组),每组8只。用药16d,观察肿瘤生长情况,于接种后24d处死各组小鼠,取出双肺并剥离皮下肿瘤,计算出肺转移发生率,计数各组小鼠肺表面转移结节数并算出肺表面结节转移抑制率;收集移植瘤标本,称重和测量体积,免疫组化和图像分析系统定量检测肿瘤组织中表皮生长因子受体(epidermal growth factor receptor,EGFR)、P-选择素和增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)的表达。结果:顺铂组、苏拉明组、联合组肿瘤的生长明显受到抑制,瘤重明显低于对照组,其抑瘤率分别为34.9%、23.8%、57.3%;苏拉明组及联合组与对照组和顺铂组相比肺转移发生率、肺表面转移结节数明显下降(P<0.05),而顺铂组与对照组比较差异则无统计学意义。EGFR和P-选择素的表达对照组高于顺铂组、苏拉明组、联合组,对照组、顺铂组、苏拉明组、联合组EGFR表达的灰度值分别为157.7±6.1、130.7±5.9、110.3±5.8、89.2±5.4,P-选择素表达的灰度值分别为134.5±5.7、117.9±5.1、96.2±5.4、78.3±4.5,各用药组与对照组相比差异均有统计学意义(P<0.05或P<0.01);对照组、顺铂组、苏拉明组、联合组增殖指数(S phase fraction,SPF)分别为(89.7±3.8)%、(68.8±4.0)%、(65.2±4.2)%、(51.3±4.2)%,各用药组SPF都比对照组低,差异有统计学意义(P<0.01)。结论:苏拉明可明显抑制肺腺癌细胞在小鼠体内的生长和转移,其作用机制可能为通过调控肿瘤细胞中的EGFR和P-选择素表达,从而抑制肿瘤细胞增殖和减少肿瘤细胞的粘附而防止转移。     

Adoptive T cell transfer augments IL-2 mediated tumour regression in a HNSCC xenograft nude mouse model

The potential of cytokine gene therapy using naked plasmid DNA encoding human IL-2 cDNA in a xenograft nude mouse model was investigated. To overcome the T cell deficiency in nude mice, Adoptive T Cell Transfer [ATCT] into tumour bearing nude mice was performed. The migration of T cells was tracked in vivo using CFSE labeling. IL-2 in circulation was observed till day 15 with the levels reaching a peak by day 3. Significant activation of transferred T cells (CD69+, CD25+) as well as significant increase in levels of NK cells (CD49b+) was seen in animals receiving IL-2. Tumour regression was observed in the IL-2+ ATCT group.     

Efficacy of Hsp90 inhibition for induction of apoptosis and inhibition of growth in cervical carcinoma cells in vitro and in vivo

Purpose Heat shock protein 90 (Hsp90) is a conserved chaperone involved in crucial signaling events in normal and malignant cells. Previous research suggests that tumor cells are particularly dependent on Hsp90 for survival as well as malignant progression. Hsp90 inhibitors which are derivates of the natural compound geldanamycin, such as the orally bioavailable 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (17-DMAG), are currently being tested in clinical trials and small molecule inhibitors are in development. In this study we investigated the response of a panel of cervical carcinoma cell lines in vitro and in vivo to determine potential factors that might influence the sensitivity towards Hsp90 inhibition. Methods Cell viability, proliferation and drug-induced changes on Hsp90 chaperoned “client” factors were examined with focus on G2/M cell cycle regulators, and a comparison with immortalized and normal keratinocytes was performed. ME180 and CaSki cells were grown as subcutaneous xenografts in mice treated with 6–10 mg/kg 17-DMAG by oral gavage 2×/day on a chronic schedule. Tissue concentrations of 17-DMAG were measured by high performance liquid chromatography. Results Cell death during abnormal mitosis was observed within 48 h after treatment start. ME180 and CaSki showed more cell death at this time point than SiHa and HeLa, and higher levels of pre-treatment Akt activity. IC₅₀ values ranged between 17 and 37 nanoM geldanamycin (MTS). Keratinocytes were at least as sensitive as carcinoma cells. All cell lines responded with an increase of the G2/M fraction. Despite in vitro effectiveness and tissue concentrations of 1 μM, only a limited tumor growth reduction was observed with 17-DMAG given close to the maximum tolerated dose level. Lower levels of Hsp90 protein, a lower Akt activity and signs of tissue hypoxia were observed in xenografts compared to cell cultures. Conclusions We show here that Hsp90 inhibition effectively induces apoptosis and growth arrest in cervical carcinoma cells in vitro. Mitotic catastrophe was identified as one mechanism of cell death. In contrast, a limited efficacy of 17-DMAG was observed in subcutaneous xenograft models. Induction of a heat shock response has previously been implicated in resistance towards Hsp90 inhibition. Additional factors might be (1) an altered abundance and/or activity of primary (Hsp90) and secondary (e.g., Akt) target(s), (2) a narrow therapeutic range of 17-DMAG by oral application and (3) response-modifying factors within the tumor environment. The further development of synthetic Hsp90 inhibitors with increased therapeutic window is warranted.     

Aurora kinase a inhibitor MLN8237 suppresses pancreatic cancer growth

Pancreatic ductal adenocarcinoma (PDAC) is notorious for high mortality due to limited options of appropriate chemotherapy drugs. Here we report that Aurora kinase-A expression is elevated in both human and mouse PDAC samples. MLN8237, an inhibitor of Aurora kinase-A, efficiently reduced the proliferation and motility of PDAC cells in vitro as well as tumor growth in orthotropic xenograft model and genetic pancreatic cancer animal models (p53/LSL/Pdx-Cre mice) in vivo. MLN8237 exhibited tumor inhibitory effect through inhibiting proliferation and migration, and inducing apoptosis and senescence. These results provide the molecular basis for a novel chemotherapy strategy for PDAC patients.     

脑膜瘤孕激素受体检测与米非司酮的抗肿瘤作用及相关性初探

目的 寻找和开发治疗脑膜瘤的有效药物。方法 应用裸鼠肾包膜下法建立人脑膜瘤动物模型。采用三种不同剂量的米非司酮在动物模型建立后皮下注射用药，28天后将各组间肿瘤生长率作统计学分析。原始标本用免疫组化SP法检测孕激素受体（PR）。结果 中剂量组和高剂量组与对照组之间有显著性差异（P＜0．01），低剂量组与对照组之间无显著性差异（P＞0．05）。7例标本经免疫组化检测，4例PR阳性，但米非司酮对PR阴性组与PR阳性组的作用差异不显著（P＞0．05）。结论 米非司酮对人脑膜瘤有明显的生长抑制作用，可能是通过阻断PR或其他受体而抑制了脑膜瘤细胞的增殖。     

供者特异性抗原诱导异种免疫耐受的初步研究

目的：为探讨异种免疫耐受的可能性。方法：以豚鼠－大鼠心脏移植建立超急排斥反应模型,在眼镜蛇毒因子（ＣＶＦ,３０Ｕ／ｋｇ）及环磷酰胺（ＣｙＰ）的覆盖下,将供者特异性或非特异性血管内皮细胞以４．０×１０６个／只,注入受者的门静脉,２周后行颈部心脏移植。结果：移植供心存活时间获得延长至（４６．１±５）ｍｉｎ,加用ＣｙＰ后延长尤为明显（５４．５±９．７ｍｉｎ）,同时血管内皮细胞溶解率下降,ＣｙＰ对移植物存活也有一定的延长作用（３４．６±８．７ｍｉｎ）,而非特异性血管内皮细胞则无此作用。结论：在豚鼠－大鼠异种移植模型中,供者特异性血管内皮细胞经门静脉径路注射可诱导受者产生一定程度的免疫耐受。这种耐受的产生机制尚需进一步研究。