Ectopic expression of von Hippel-Lindau tumor suppressor induces apoptosis in 786-O renal cell carcinoma cells and regresses tumor growth of 786-O cells in nude mouse

The von Hippel-Lindau (VHL) is a known tumor suppressor that binds to alpha-subunits of hypoxia-inducible factors and induces ubiquitin-mediated degradation of the protein in an oxygen-dependent manner. VHL is also involved in the regulation of tumor angiogenesis, glycolysis, cell cycle regulation, and apoptosis. In the present study, we showed that ectopic expression of VHL induces apoptosis in renal cell carcinoma 786-O cells which contain only the mutant VHL, evidenced by TUNEL assay and DAPI staining. Furthermore, biochemical studies indicated that expression of VHL in 786-O cells results in both PARP and CPP32 cleavage, suggesting that VHL-induced apoptosis in 786-O cells is caspase dependent. Moreover, we also observed that apoptosis induced by ectopic VHL expression was associated with up-regulation of p27 as well as Bax, implicating the roles of these two proteins in VHL-induced apoptosis. The up-regulation of p27 and Bax by VHL was specific since we did not detect any changes in the level of other apoptotic factors including Fas and Bcl2 by the expression of VHL. We next examined the effect of VHL expression on the tumor growth of 786-O renal cell carcinoma cells in nude mouse. The results showed that injection of Ad.VHL adenovirus regresses the tumor growth of 786-O cells in nude mouse. The analysis by TUNEL assay as well as DAPI staining of 786-O tumors injected with Ad.VHL showed clear evidence of apoptosis. These results suggest that ectopic VHL expression induces apoptotic response in 786-O VHL mutant cells both in vitro and in vivo.     

Development and preclinical studies of 64Cu-NOTA-pertuzumab F(ab′)2 for imaging changes in tumor HER2 expression associated with response to trastuzumab by PET/CT

We previously reported that microSPECT/CT imaging with ¹¹¹In-labeled pertuzumab detected decreased HER2 expression in human breast cancer (BC) xenografts in athymic mice associated with response to treatment with trastuzumab (Herceptin). Our aim was to extend these results to PET/CT by constructing F(ab′)₂ of pertuzumab modified with NOTA chelators for complexing ⁶⁴Cu. The effect of the administered mass (5–200 µg) of ⁶⁴Cu-NOTA-pertuzumab F(ab′)₂ was studied in NOD/SCID mice engrafted with HER2-positive SK-OV-3 human ovarian cancer xenografts. Biodistribution studies were performed in non-tumor bearing Balb/c mice to predict radiation doses to normal organs in humans. Serial PET/CT imaging was conducted on mice engrafted with HER2-positive and trastuzumab-sensitive BT-474 or trastuzumab-insensitive SK-OV-3 xenografted mice treated with weekly doses of trastuzumab. There were no significant effects of the administered mass of ⁶⁴Cu-NOTA-pertuzumab F(ab′)₂ on tumor or normal tissue uptake. The predicted total body dose in humans was 0.015 mSv/MBq, a 3.3-fold reduction compared to ¹¹¹In-labeled pertuzumab. MicroPET/CT images revealed specific tumor uptake of ⁶⁴Cu-NOTA-pertuzumab F(ab′)₂ at 24 or 48 h post-injection in mice with SK-OV-3 tumors. Image analysis of mice treated with trastuzumab showed 2-fold reduced uptake of ⁶⁴Cu-NOTA-pertuzumab F(ab′)₂ in BT-474 tumors after 1 week of trastuzumab normalized to baseline, and 1.9-fold increased uptake in SK-OV-3 tumors after 3 weeks of trastuzumab, consistent with tumor response and resistance, respectively. We conclude that PET/CT imaging with ⁶⁴Cu-NOTA-pertuzumab F(ab′)₂ detected changes in HER2 expression in response to trastuzumab while delivering a lower total body radiation dose compared to ¹¹¹In-labeled pertuzumab.     

Hyperthermia Induces Apoptosis of 786-O Cells through Suppressing Ku80 Expression

Hyperthermia as an anticancer method has been paid increasing attention in recent years. Several studies have shown that hyperthermia can kill tumor cells by inducing apoptosis. However, the underlying molecular mechanisms of hyperthermia-induced apoptosis are largely unknown. To investigate the effects and molecular mechanism of hyperthermia on the apoptosis in renal carcinoma 786-O cells, we firstly examined apoptosis and Ku expression in 786-O cell line treated with heat exposure (42°C for 0-4 h). The results showed that hyperthermia induced apoptosis of 786-O cells, and suppressed significantly Ku80 expression, but not Ku70 expression. Next, we knock-down Ku80 in 786-O cells, generating stable cell line 786-O-shKu80, and detected apoptosis, cell survival and cell cycle distribution. Our data showed higher apoptotic rate and lower surviving fraction in the stable cell line 786-O-shKu80 compared with those in control cells, exposed to the same heat stress (42°C for 0-4 h). Moreover, the results also showed suppression of Ku80 led to G2/M phase arrest in the stable cell line 786-O-shKu80 following heat treatment. Together, these findings indicate that Ku80 may play an important role in hyperthermia-induced apoptosis and heat-sensitivity of renal carcinoma cells through influencing the cell cycle distribution.     

Assessment of a novel VEGF targeted agent using patient-derived tumor tissue xenograft models of colon carcinoma with lymphatic and hepatic metastases

The lack of appropriate tumor models of primary tumors and corresponding metastases that can reliably predict for response to anticancer agents remains a major deficiency in the clinical practice of cancer therapy. It was the aim of our study to establish patient-derived tumor tissue (PDTT) xenograft models of colon carcinoma with lymphatic and hepatic metastases useful for testing of novel molecularly targeted agents. PDTT of primary colon carcinoma, lymphatic and hepatic metastases were used to create xenograft models. Hematoxylin and eosin staining, immunohistochemical staining, genome-wide gene expression analysis, pyrosequencing, qRT-PCR, and western blotting were used to determine the biological stability of the xenografts during serial transplantation compared with the original tumor tissues. Early passages of the PDTT xenograft models of primary colon carcinoma, lymphatic and hepatic metastases revealed a high degree of similarity with the original clinical tumor samples with regard to histology, immunohistochemistry, genes expression, and mutation status as well as mRNA expression. After we have ascertained that these xenografts models retained similar histopathological features and molecular signatures as the original tumors, drug sensitivities of the xenografts to a novel VEGF targeted agent, FP3 was evaluated. In this study, PDTT xenograft models of colon carcinoma with lymphatic and hepatic metastasis have been successfully established. They provide appropriate models for testing of novel molecularly targeted agents.     

Evaluation of 64Cu radiolabeled anti-hPD-L1 Nb6 for positron emission tomography imaging in lung cancer tumor mice model

Recently, we selected a novel anti-hPD-L1-specific HCAb named Nb6 with high affinity (EC₅₀ = 0.65 ng/mL) for potential hPD-L1 targeted non-invasive PET imaging. In this research, Nb6 was conjugated with the bifunctional chelator NCS-Bz-NOTA ((2-[(4-Isothiocyanophenyl) methyl]-1,4,7-triazacy-clononane-1,4,7-triacetic acid)) and further labeled with radio-nuclide ⁶⁴Cu. ⁶⁴Cu-NOTA-Nb6 was prepared with over 95% labeling yield, over 99% radiochemical purity and 14–16 GBq/μmol specific activity after PD-10 column purification. It shows good stability in 0.01 M PBS and 5% HSA solutions. ⁶⁴Cu-NOTA-Nb6 has a high binding affinity to 3.60 nM which was tested by human lung adenocarcinoma A549 cell lines. Tumor lesion can be clearly observed from 20 h to 38 h by Micro-PET equipment after ⁶⁴Cu-NOTA-Nb6 administration. The study revealed that ⁶⁴Cu-NOTA-Nb6 has good lesion detection ability, high ratios between tumor and non-tumor signal and can specifically target A549 xenografted tumor model. Taken together of good stability, high binding affinity, and tumor detection ability, ⁶⁴Cu labeled Nb6 is a promising radio-tracer in diagnosing of hPD-L1 overexpression tumor, supposed to monitor PD-L1overexpression tumor progression and guide targeted therapy with PET molecular imaging.     

Induction of apoptosis by magnolol via the mitochondrial pathway and cell cycle arrest in renal carcinoma cells

Magnolol (Mag), an effective natural compound isolated from the stem bark of Magnolia officinalis, was found to have the potential for antitumor activity by inducing apoptosis in tumor cells. However, the effect of Mag on renal carcinoma cells and its molecular mechanism are unexplored. Our study provided evidence that Mag induced apoptosis in 786-O and OS-RC-2?cell lines via the mitochondrial pathway and cell cycle arrest. In this work, we found that Mag induced morphological changes and inhibited the proliferation of 786-O and OS-RC-2?cells in a dose- and time-dependent manner but exerted no notable inhibitory effects on normal human renal proximal tubular (HK-2) cells. Treatment with Mag suppressed the migration and invasion ability of renal carcinoma cells. Moreover, Mag caused the openness of mPTP, the accumulation of intracellular ROS and decreased △Ψm, leading to mitochondrial dysfunction. However, pretreatment with the antioxidant N-acetyl cysteine (NAC) reversed the apoptosis induced by Mag and decreased the generation of ROS. In addition, the increased proportion of the G1/G0 phase indicated that Mag caused cell cycle arrest. Further analyses suggested that magnolol-induced apoptosis was related to the abnormal expression of p53, Bax, Bcl-2, cytochrome c and caspase activation. Together, the results above revealed that Mag had antitumor effects in renal carcinoma cells via ROS production as well as cell cycle arrest and the apoptotic mitochondrial pathway was suppressed in part by NAC.     

RAD001 (everolimus) inhibits tumour growth in xenograft models of human hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is the fifth most common malignancy worldwide and highly resistant to available chemotherapies. Mammalian target of rapamycin (mTOR) functions to regulate protein translation, angiogenesis and cell cycle progression in many cancers including HCC. In the present study, subcutaneous patient-derived HCC xenografts were used to study the effects of an mTOR inhibitor, RAD001 (everolimus), on tumour growth, apoptosis and angiogenesis. We report that oral administration of RAD001 to mice bearing patient-derived HCC xenografts resulted in a dose-dependent inhibition of tumour growth. RAD001-induced growth suppression was associated with inactivation of downstream targets of mTOR, reduction in VEGF expression and microvessel density, inhibition of cell proliferation, up-regulation of p27^Kip1 and down-regulation of p21^Cip1/Waf1, Cdk-6, Cdk-2, Cdk-4, cdc-25C, cyclin B1 and c-Myc. Our data indicate that the mTOR pathway plays an important role in angiogenesis, cell cycle progression and proliferation of liver cancer cells. Our study provides a strong rationale for clinical investigation of mTOR inhibitor RAD001 in patients with HCC.     

Immuno-PET Imaging of HER3 in a Model in which HER3 Signaling Plays a Critical Role

HER3 is overexpressed in various carcinomas including colorectal cancer (CRC), which is associated with poor prognosis, and is involved in the development of therapy resistance. Thus, an in vivo imaging technique is needed to evaluate the expression of HER3, an important therapeutic and diagnostic target. Here, we report successful HER3 PET imaging using a newly generated anti-human HER3 monoclonal antibody, Mab#58, and a mouse model of a HER3-overexpressing xenograft tumor. Furthermore, we assessed the role of HER3 signaling in CRC cancer tissue-originated spheroid (CTOS) and applied HER3 imaging to detect endogenous HER3 in CTOS-derived xenografts. Cell binding assays of ⁸⁹Zr-labeled Mab#58 using the HER3-overexpressing cell line HER3/RH7777 demonstrated that [⁸⁹Zr]Mab#58 specifically bound to HER3/RH7777 cells (K_d = 2.7 nM). In vivo biodistribution study in mice bearing HER3/RH7777 and its parent cell xenografts showed that tumor accumulation of [⁸⁹Zr]Mab#58 in HER3/RH7777 xenografts was significantly higher than that in the control from day 1 to day 4, tending to increase from day 1 to day 4 and reaching 12.2 ± 4.5%ID/g. Radioactivity in other tissues, including the control xenograft, decreased or remained unchanged from day 1 to day 6. Positron emission tomography (PET) in the same model enabled clear visualization of HER3/RH7777 xenografts but not of RH7777 xenografts. CTOS growth assay and signaling assay revealed that CRC CTOS were dependent on HER3 signaling for their growth. In PET studies of mice bearing a CRC CTOS xenograft, the tumor was clearly visualized with [⁸⁹Zr]Mab#58 but not with the ⁸⁹Zr-labeled control antibody. Thus, tumor expression of HER3 was successfully visualized by PET with ⁸⁹Zr-labeled anti-HER3 antibody in CTOS xenograft-bearing mice, a model that retains the properties of the patient tumor. Non-invasive targeting of HER3 by antibodies is feasible, and it is expected to be useful for cancer diagnosis and treatment.     

Synthesis and evaluation of 18F-hexafluorophosphate as a novel PET probe for imaging of sodium/iodide symporter in a murine C6-glioma tumor model

Noninvasive imaging of iodide uptake via the sodium/iodide symporter (NIS) has received great interest for evaluation of thyroid cancer and reporter imaging of NIS-expressing viral therapies. In this study, we investigate ¹⁸F-labeled hexafluorophosphate (HFP or PF₆^?) as a high-affinity iodide analog for NIS imaging. ¹⁸F-HFP was synthesized by radiofluorination of phosphorus pentafluoride·N-methylpyrrolidine complex and evaluated in human NIS (hNIS)-expressing C6 glioma cells and a C6 glioma xenograft mouse model. ¹⁸F-HFP was obtained in radiochemical yield of 10?±?5%, radiochemical purity of >96% and specific radioactivity of 604?±?18?MBq/µmol. Specific uptake of ¹⁸F-HFP and high affinity of ¹⁹F-HFP were observed in hNIS+ C6-glioma cells. PET imaging showed robust uptake of ¹⁸F-HFP in NIS-expressing tissues (thyroid, stomach, and hNIS+ C6 glioma xenografts), and the uptake of ¹⁸F-HFP was blocked by NaClO₄ pretreatment. Specific accumulation in hNIS-expressing xenograft (hNIS+) was observed relative to isogenic control tumor (hNIS?). Clearance of ¹⁸F-HFP was predominantly through renal excretion. The biodistribution showed consistent results with PET imaging. Minimal bone uptake was observed over 2?h period post-injection, indicating excellent in vivo stability of ¹⁸F-HFP. Although improvement in specific radioactivity is desirable, the results indicate that ¹⁸F-HFP is a promising candidate radiotracer for further evaluation for NIS imaging.     

[18F]-Fluorodeoxyglucose Positron Emission Tomography/Computed Tomography of LAPC4-CR Castration-Resistant Prostate Cancer Xenograft Model in Soft Tissue Compartments

Preclinical xenograft models have contributed to advancing our understanding of the molecular basis of prostate cancer and to the development of targeted therapy. However, traditional preclinical in vivo techniques using caliper measurements and survival analysis evaluate the macroscopic tumor behavior, whereas tissue sampling disrupts the microenvironment and cannot be used for longitudinal studies in the same animal. Herein, we present an in vivo study of [¹⁸F]-fluorodeoxyglucose (FDG) positron emission tomography (PET)/computed tomography (CT) designed to evaluate the metabolism within the microenvironment of LAPC4-CR, a unique murine model of castration-resistant prostate cancer. Mice bearing LAPC4-CR subcutaneous tumors were administered [¹⁸F]-FDG via intravenous injection. After a 60-minute distribution phase, the mice were imaged on a PET/CT scanner with submillimeter resolution; and the fused PET/CT images were analyzed to evaluate tumor size, location, and metabolism across the cohort of mice. The xenograft tumors showed [¹⁸F]-FDG uptake that was independent of tumor size and was significantly greater than uptake in skeletal muscle and liver in mice (Wilcoxon signed-rank P values of .0002 and .0002, respectively). [¹⁸F]-FDG metabolism of the LAPC4-CR tumors was 2.1 ± 0.8 ID/cm³*wt, with tumor to muscle ratio of 7.4 ± 4.7 and tumor to liver background ratio of 6.7 ± 2.3. Noninvasive molecular imaging techniques such as PET/CT can be used to probe the microenvironment of tumors in vivo. This study showed that [¹⁸F]-FDG-PET/CT could be used to image and assess glucose metabolism of LAPC4-CR xenografts in vivo. Further work can investigate the use of PET/CT to quantify the metabolic response of LAPC4-CR to novel agents and combination therapies using soft tissue and possibly bone compartment xenograft models.     

Noninvasive Assessment of Tumor VEGF Receptors in Response to Treatment with Pazopanib: A Molecular Imaging Study

Vascular endothelial growth factor (VEGF) and its receptors (VEGFRs) drive angiogenesis, and several VEGFR inhibitors are already approved for use as single agents or in combination with chemotherapy. Although there is a clear benefit with these drugs in a variety of tumors, the clinical response varies markedly among individuals. Therefore, there is a need for an efficient method to identify patients who are likely to respond to antiangiogenic therapy and to monitor its effects over time. We have recently developed a molecular imaging tracer for imaging VEGFRs known as scVEGF/^99mTc; an engineered single-chain (sc) form of VEGF radiolabeled with technetium Tc 99m (^99mTc). After intravenous injection, scVEGF/^99mTc preferentially binds to and is internalized by VEGFRs expressed within tumor vasculature, providing information on prevalence of functionally active receptors. We now report that VEGFR imaging readily detects the effects of pazopanib, a small-molecule tyrosine kinase inhibitor under clinical development, which selectively targets VEGFR, PDGFR, and c-Kit in mice with HT29 tumor xenografts. Immunohistochemical analysis confirmed that the changes in VEGFR imaging reflect a dramatic pazopanib-induced decrease in the number of VEGFR-2⁺/CD31⁺ endothelial cells (ECs) within the tumor vasculature followed by a relative increase in the number of ECs at the tumor edges. We suggest that VEGFR imaging can be used for the identification of patients that are responding to VEGFR-targeted therapies and for guidance in rational design, dosing, and schedules for combination regimens of antiangiogenic treatment.     

Synthesis and evaluation of an 18F-labeled boramino acid analog of aminosuberic acid for PET imaging of the antiporter system xC−

In this study, we synthesized ¹⁸F-ASu-BF₃, a close boramino acid analog of 5-[¹⁸F]fluoro-aminosuberic acid (¹⁸F-ASu), via ¹⁸F-¹⁹F isotope exchange reaction and evaluated its potential for imaging with positron emission tomography (PET). ¹⁸F-ASu-BF₃ was stable in mouse plasma and taken up into PC3 prostate cancer cells via the system x_C^? amino acid transporter. The continuous use of isoflurane for anesthesia during dynamic imaging acquisition slowed down the excretion of ¹⁸F-ASu-BF₃ and enabled visualization of PC3 tumor xenografts in mice. In contrast, no tumor visualization was observed from static images of ¹⁸F-BF₃-ASu due to its rapid renal excretion mediated in part by the organic anion transporter. Our data indicate that the pharmacokinetics of amino acids could be altered after being converted into their boramino acid analogs. Therefore, care should be taken when using the boramino acid strategy to design and prepare ¹⁸F-labeled tracers for imaging amino acid transporters/receptors with PET.     

IGFBP3通过癌旁脂肪细胞促进肾癌细胞生长与转移的作用研究

目的:探讨肾透明细胞癌的分泌蛋白IGFBP3对癌旁脂肪细胞分化的作用及通过脂肪细胞促进肾透明癌细胞生长与转移的作用。方法:通过肾细胞癌的基因数据库发现过表达的基因IGFBP3,免疫组化和RT-PCR确认IGFBP3在标本中的表达。RT-PCR和Western Blot检测IGFBP3对脂肪细胞分化成熟特征标志物表达的影响。以过表达IGFBP3的786-O细胞为模型,Western Blot检测IGFBP3的促脂肪细胞分化作用与TGFβ-smad1/5/8及TGFβ-p38MAPK信号通路的关系。将过表达IGFBP3的786-O细胞与脂肪细胞共培养得到条件培养基,通过油红染色检测条件培养的肾癌细胞中脂滴含量,迁移实验和CCK8实验分别检测脂肪细胞对肾癌细胞侵袭性及增殖的影响。结果:相较于正常组,肾癌标本中IGFBP3的表达增加(P=0.017)。IGFBP3使脂肪细胞分化成熟相关标志物PPARγ、PGC1α、c/EBPα、Prdm16、UCP1表达增加。以IGFBP3处理脂肪细胞时,可以增加TGFβ下游蛋白表达水平,30 min后p-smad1/5/8表达增加(P=0.024),60 min后p-p38MAPK表达明显增加(P=0.013)。条件培养后的786-O细胞内的脂滴形成增加(P=0.028),脂肪细胞促进786-O细胞的增殖和迁移能力。结论:IGFBP3是肾透明细胞癌中过度表达的蛋白,能够促进前脂肪细胞分化,其机制主要通过激活TGFβ通路中的smad1/5/8、p38MAPK。成熟的脂肪细胞能够促进肾癌细胞质脂滴形成,并且促进肿瘤的增殖、提高肿瘤的侵袭性。     

Acyldepsipeptides inhibit the growth of renal cancer cells through G1 phase cell cycle arrest

Acyldepsipeptides are a group of potent antibiotics discovered in the secondary metabolites of Streptomyces species. However, besides the function of antibiotics, no other activities have been reported about these important compounds so far. In the course of searching the natural products as chemotherapeutic agents for renal cell carcinoma, we found that ADEP1, a major metabolic component of Streptomyces hawaiiensis NRRL 15010, could effectively inhibit the growth of 786-O, 769-P, and ACHN renal carcinoma cells in MTT assay. Flow cytometric analysis demonstrated that ADEP1 could block the cell cycle arrested at G1 phase. Moreover, it was found that ADEP1 down-regulated the expressions of cyclin D1, CDK4 and PCNA and inhibited activity of MAPK–ERK pathway by detection of decreased expression of phosphorylated ERK1/2 and c-Fos in 786-O and 769-P cells by Western blotting. To our knowledge, this is the first report concerning to the antitumor activities of acyldepsipeptides. Based on these results, ADEP1 may become a promising lead compound to be developed a novel chemotherapeutic agent for treatment of renal carcinoma.     

Anti-tumor effects of fusion vaccine prepared by renal cell carcinoma 786-O cell line and peripheral blood dendritic cells of healthy volunteers in vitro and in human immune reconstituted SCID mice

Dendritic cells (DC), as professional antigen presenting cells, play the central role in the process of body initiating the anti-tumor immunity, and the study on DC anti-tumor vaccine has become heated in recent years. In this study, we used polyethylene glycol (PEG) to induce renal cell carcinoma (RCC) 786-O cell line fused with peripheral blood DC of healthy volunteers, and discuss the biological characteristics of fusion vaccine and its anti-tumor effects in vitro and in human immune reconstituted SCID mice model of RCC. The study found that PEG could effectively induce cell fusion, and the expressions of CD86 and HLA-DR in fusion vaccine group were significantly up-regulated compared with the DC control group; the secretion of IL-12 was much higher and longer than that of the control; the functions of dendritic cell-tumor fusion vaccine to stimulate the proliferation of allogenic T lymphocytes and to kill RCC786-O cells in vitro were significantly higher than those of the control group, and after the killing, apoptosis body was observed in the target cells; after the injection of fusion vaccine into human immune reconstituted SCID mice model of RCC786-O via vena caudalis, the volume of mice tumor was reduced significantly, proliferation index of tumor cells decreased obviously compared with that of the control group, and more hemorrhage and putrescence focuses presented, accompanying large quantity of lymphocytes soakage. The results of this experimental study shows that fusion vaccine of RCC786-O cell line and DC can significantly stimulate the proliferation of allogenic T cells and specifically inhibit and kill RCC cells in vitro and in vivo, which makes the DC-RCC786-O fusion vaccine a possible new way of effective RCC immunotherapy.