Characterization of sphere cells derived from a patient-derived xenograft model of lung adenocarcinoma treated with ionizing radiation.

PURPOSE: Cancer stem cells (CSCs) are relatively resistant to radiation compared to their non-tumorigenic progeny. Ionizing radiation (IR) can expand the pool of CSCs that leads to more aggressive cancers, but the reason underlying CSC-induced cancer aggressiveness after radiation therapy remains unclear. To understand this, we investigated the phenotypic and molecular characteristics of sphere cells formed from IR-treated patient-derived xenograft (PDX) lung adenocarcinoma tumors. MATERIALS AND METHODS: After treatment with various modes of IR, we collected tumors from PDX mice and successfully obtained sphere cells. To compare tumorigenicity, we performed migration, invasion, and mouse transplantation assays with sphere cells from each group. To investigate the molecular features, we used a cDNA microarray and compared gene expression among groups. RESULTS AND CONCLUSIONS: Tumorigenicity assays revealed that sphere cells from 2- or 5-Gy IR-treated tumors more aggressive than sphere cells from non-IR treated tumors. Microarray results showed that SERPIB4 and CCL2 were upregulated in sphere cells from IR-treated tumors compared to that in sphere cells from non-IR treated tumors. Interestingly, these genes are related to immune reactions in cancer. Taken together, our results suggest that the aggressiveness of sphere cells obtained after IR treatment is related to resistance, and provide new opportunities for exploring targeted therapies to overcome common radioresistance.

Nanoparticulated docetaxel exerts enhanced anticancer efficacy and overcomes existing limitations of traditional drugs.

Nanoparticulation of insoluble drugs improves dissolution rate, resulting in increased bioavailability that leads to increased stability, better efficacy, and reduced toxicity of drugs. Docetaxel (DTX), under the trade name Taxotere™, is one of the representative anticancer chemotherapeutic agents of this era. However, this highly lipophilic and insoluble drug has many adverse effects. Our novel and widely applicable nanoparticulation using fat and supercritical fluid (NUFS™) technology enabled successful nanoscale particulation of DTX (Nufs-DTX). Nufs-DTX showed enhanced dissolution rate and increased aqueous stability in water. After confirming the preserved mechanism of action of DTX, which targets microtubules, we showed that Nufs-DTX exhibited similar effects in proliferation and clonogenic assays using A549 cells. Interestingly, we observed that Nufs-DTX had a greater in vivo tumor growth delay effect on an A549 xenograft model than Taxotere™, which was in agreement with the improved drug accumulation in tumors according to the biodistribution result, and was caused by the enhanced permeability and retention (EPR) effect. Although both Nufs-DTX and Taxotere™ showed negative results for our administration dose in the hematologic toxicity test, Nufs-DTX showed much less toxicity than Taxotere™ in edema, paralysis, and paw-withdrawal latency on a hot plate analysis that are regarded as indicators of fluid retention, peripheral neuropathy, and thermal threshold, respectively, for toxicological tests. In summary, compared with Taxotere™, Nufs-DTX, which was generated by our new platform technology using lipid, supercritical fluid, and carbon dioxide (CO2), maintained its biochemical properties as a cytotoxic agent and had better tumor targeting ability, better in vivo therapeutic effect, and less toxicity, thereby overcoming the current hurdles of traditional drugs.

Multifunctional hollow gold nanoparticles designed for triple combination therapy and CT imaging.

Hollow gold nanoparticles (HGNP) are a novel class of hybrid metal nanoparticles whose unique optical and morphological properties have spawned new applications including more effective cancer therapy. The shell thickness of HGNPs can tune the surface plasmon resonance to the near infrared light, resulting in photothermal ablation of tumors with optimal light penetration in tissue. The hollow cavity within a HGNP is able to accommodate a high payload of chemotherapeutic agents. They have also been used for enhancing radiosensitization in tumors during radiotherapy due to the high X-ray absorption capability of gold particles. However, no report has yet been published that utilize HGNPs for the triple combination therapy and CT imaging. In this study, we synthesized HGNPs which exhibit better response to radiation for therapy and imaging and demonstrated the effects of combined chemotherapy, thermal and radiotherapy. This combination strategy presented delayed tumor growth by 4.3-fold and reduced tumor's weight by 6.8-fold compared to control tumors. In addition, we demonstrated the feasibility of HGNP as a CT imaging agent. It is expected that translating these capabilities to human cancer patients could dramatically increase the antitumor effect and potentially overcome resistance to chemotherapeutic agents and radiation.

Novel peptides functionally targeting in vivo human lung cancer discovered by in vivo peptide displayed phage screening.

Discovery of the cancer-specific peptidic ligands have been emphasized for active targeting drug delivery system and non-invasive imaging. For the discovery of useful and applicable peptidic ligands, in vivo peptide-displayed phage screening has been performed in this study using a xenograft mouse model as a mimic microenvironment to tumor. To seek human lung cancer-specific peptides, M13 phage library displaying 2.9 × 10(9) random peptides was intravenously injected into mouse model bearing A549-derived xenograft tumor through the tail vein. Then the phages emerged from a course of four rounds of biopanning in the xenograft tumor tissue. Novel peptides were categorized into four groups according to a sequence-homology phylogenicity, and in vivo tumor-targeting capacity of these peptides was validated by whole body imaging with Cy5.5-labeled phages in various cancer types. The result revealed that novel peptides accumulated only in adenocarcinoma lung cancer cell-derived xenograft tissue. For further confirmation of the specific targeting ability, in vitro cell-binding assay and immunohistochemistry in vivo tumor tissue were performed with a selected peptide. The peptide was found to bind intensely to lung cancer cells both in vitro and in vivo, which was efficiently compromised with unlabeled phages in an in vitro competition assay. In conclusion, the peptides specifically targeting human lung cancer were discovered in this study, which is warranted to provide substantive feasibilities for drug delivery and imaging in terms of a novel targeted therapeutics and diagnostics.

A cisplatin­incorporated liposome that targets the epidermal growth factor receptor enhances radiotherapeutic efficacy without nephrotoxicity.

Radiotherapy (RT) is one of the major modalities for non­small cell lung cancer (NSCLC), but its efficacy is often compromised by cellular resistance caused by various mechanisms including the overexpression of epidermal growth factor receptor (EGFR). Although cis­diamminedichloroplatinum(Ⅱ) (cisplatin, CDDP) has been well characterized as an effective radiosensitizer, its clinical application is limited by its severe nephrotoxic effects. In our current study, we developed a CDDP­incorporated liposome (LP) conjugated with EGFR antibodies (EGFR:LP­CDDP) and evaluated its potential to radiosensitize EGFR­overexpressing cells without exerting nephrotoxic effects. EGFR:LP­CDDP showed higher cytotoxicity than non­targeting liposomal CDDP (LP­CDDP) in the cells expressing EGFR in vitro. In an A549 cell­derived xenograft tumor mouse model, increased delays in tumor growth were observed in the mice treated with a combination of EGFR:LP­CDDP and radiation. Notably, the EGFR:LP­CDDP­treated animals showed no differences in body weight loss, survival rates of nephrotoxicity compared with untreated control mice. In contrast, the use of CDDP caused lower body weights and poorer survival outcomes accompanied by a significant level of nephrotoxicity [e.g., decreased kidney weight, increased blood urea nitrogen (BUN) and creatinine, and pathological change]. These findings suggest the feasibility of using EGFR:LP­CDDP to radiosensitize cells in a targeted manner without inducing nephrotoxic effects. This compound may therefore have clinical potential as part of a tailored chemoradiotherapy strategy.

Ibulocydine sensitizes human cancers to radiotherapy by induction of mitochondria-mediated apoptosis.

BACKGROUND AND PURPOSE: Ibulocydine (IB), a novel prodrug of CDK inhibitor, has been reported to have anti-cancer effect in human hepatoma cells. In order to address its feasibility as a radiosensitizer to improve radiotherapeutic efficacy for human cancers, this study was designed. MATERIAL AND METHODS: Human cancer cells of lung and colon were treated with IB and/or radiotherapy (RT). The cellular effects were assessed by CCK-8, clonogenic, flow cytometric, and western blotting assays. In vivo radiotherapeutic efficacy was evaluated using the xenograft mouse model. RESULTS: Combined treatment of IB and RT significantly reduced viability and survival fraction of the cells. Apoptotic cell death accompanied with activation of caspases, decrease in Bcl-2/Bax expression, loss of mitochondrial membrane potential (MMP) leading to release of cytochrome c into cytosol was observed. Recovery of Bcl-2 expression level by introducing Bcl-2 expressing plasmid DNA compromised the loss of MMP and apoptosis induced by IB and RT. In vivo therapeutic efficacy of combined treatment was verified in the xenograft mouse model, in which tumor growth was markedly delayed by RT with IB. CONCLUSIONS: IB demonstrated the property of sensitizing human cancer cells to RT by induction of mitochondria-mediated apoptosis, suggesting that IB deserves to be applied for chemoradiotherapy.

Use of macrophages to deliver therapeutic and imaging contrast agents to tumors.

Drug targeting to tumors with limited toxicity and enhanced efficacy of drug is one of the important goals for cancer treatment pharmaceutics. Monocytes/macrophages are able to migrate to tumor sites across the blood barriers by acting as Trojan horses carrying drug cargoes. Taking this advantage, we have intended to develop an efficient administration system using a biologically active carrier of mouse peritoneal macrophage bearing liposomal doxorubicin (macrophage-LP-Dox). We expect that this system could improve the cancer therapeutic efficacy through deeper penetration into tumor even hypoxic region behind tumor blood vessel. We first confirmed that macrophages containing iron oxides could migrate and infiltrate into tumors effectively by MR imaging. Next, we showed that doxorubicin (Dox) encapsulated with liposomes (LP-Dox) was successfully loaded into macrophages, in which the biological activity of macrophage and cytotoxicity of Dox against tumor cells were well preserved. Delivery of Dox into tumor tissue by systemic administration of macrophage-LP-Dox was verified in both subcutaneous and metastasis xenograft tumor models. Importantly, the effective inhibition of in vivo tumor growth was proved with this system. Our results provide the feasibility of macrophages-LP-drug as an active biocarrier for the enhancement of therapeutic effects in cancer treatment and open new perspectives for the active delivery of drugs.

MET-dependent cancer invasion may be preprogrammed by early alterations of p53-regulated feedforward loop and triggered by stromal cell-derived HGF.

MET, a receptor protein tyrosine kinase activated by hepatocyte growth factor (HGF), is a crucial determinant of metastatic progression. Recently, we have identified p53 as an important regulator of MET-dependent cell motility and invasion. This regulation occurs via feedforward loop suppressing MET expression by miR-34-dependent and -independent mechanisms. Here, by using Dicer conditional knockout, we provide further evidence for microRNA-independent MET regulation by p53. Furthermore, we show that while MET levels increase immediately after p53 inactivation, mutant cells do not contain active phosphorylated MET and remain non-invasive for a long latency period at contrary to cell culture observations. Evaluation of mouse models of ovarian and prostate carcinogenesis indicates that formation of desmoplastic stroma, associated production of HGF by stromal cells and coinciding MET phosphorylation precede cancer invasion. Thus, initiation mutation of p53 is sufficient for preprogramming motile and invasive properties of epithelial cells, but the stromal reaction may represent a critical step for their manifestation during cancer progression.

Local mesenchymal stem/progenitor cells are a preferential target for initiation of adult soft tissue sarcomas associated with p53 and Rb deficiency.

The cell of origin and pathogenesis of the majority of adult soft tissue sarcomas (STS) remains poorly understood. Because mutations in both the P53 and RB tumor suppressor genes are frequent in STS in humans, we inactivated these genes by Cre-loxP-mediated recombination in mice with floxed p53 and Rb. Ninety-three percent of mice developed spindle cell/pleomorphic sarcomas after a single subcutaneous injection of adenovirus carrying Cre-recombinase. Similar to human STS, these sarcomas overexpress Cxcr4, which contributes to their invasive properties. Using irradiation chimeras generated by transplanting bone marrow cells from mice carrying either the Rosa26StoploxPLacZ or the Z/EG reporter, as well as the floxed p53 and Rb genes, into irradiated p53loxP/loxPRbloxP/loxP mice, it was determined that sarcomas do not originate from bone marrow-derived cells, such as macrophages, but arise from the local resident cells. At the same time, dermal mesenchymal stem cells isolated by strict plastic adherence and low levels of Sca-1 expression (Sca-1low, CD31negCD45neg) have shown enhanced potential for malignant transformation according to soft agar, invasion, and tumorigenicity assays, after the conditional inactivation of both p53 and Rb. Sarcomas formed after transplantation of these cells have features typical for undifferentiated high-grade pleomorphic sarcomas. Taken together, our studies indicate that local Sca-1low dermal mesenchymal stem/progenitor cells are preferential targets for malignant transformation associated with deficiencies in both p53 and Rb.

Mouse models for cancer stem cell research.

The cancer stem cell concept assumes that cancers are mainly sustained by a small pool of neoplastic cells, known as cancer stem cells or tumor initiating cells, which are able to reproduce themselves and produce phenotypically heterogeneous cells with lesser tumorigenic potential. Cancer stem cells represent an appealing target for development of more selective and efficient therapies. However, direct testing of the cancer stem cell concept and assessment of its therapeutic implications in human cancers have been complicated by the use of immunocompromised mice. Genetically defined immunocompetent autochthonous mouse models of human cancer provide a valuable tool to address this problem. Furthermore, they allow for a better understanding of the relevance of mechanisms controlling normal stem cell compartment to carcinogenesis. Advantages and disadvantages of some of the existing mouse models are reviewed, and future challenges in cancer stem cell research are outlined.

Core-shell silica nanoparticles as fluorescent labels for nanomedicine.

Progress in biomedical imaging depends on the development of probes that combine low toxicity with high sensitivity, resolution, and stability. Toward that end, a new class of highly fluorescent core-shell silica nanoparticles with narrow size distributions and enhanced photostability, known as C dots, provide an appealing alternative to quantum dots. Here, C dots are evaluated with a particular emphasis on in-vivo applications in cancer biology. It is established that C dots are nontoxic at biologically relevant concentrations, and can be used in a broad range of imaging applications including intravital visualization of capillaries and macrophages, sentinel lymph node mapping, and peptide-mediated multicolor cell labeling for real-time imaging of tumor metastasis and tracking of injected bone marrow cells in mice. These results demonstrate that fluorescent core-shell silica nanoparticles represent a powerful novel imaging tool within the emerging field of nanomedicine.

Genotoxicity and toxicological effects of acrylamide on reproductive system in male rats.

The toxicity of acrylamide was evaluated through mutagenicity of Salmonella, chromosome aberration of Chinese hamster lung fibroblasts, micronucleus formation in mice and reproductive toxicity in rats. Based on Ames test, acrylamide showed mutagenic potency for strains TA98 and TA100. Moreover, both chromosomal aberration assay and micronucleus assay indicated that acrylamide might have genotoxic potency; the chromosomal aberration frequencies were observed to be proportional to acrylamide concentrations of 5-50 mM, and acrylamide significantly increased micronuclei in peripheral blood cells of mice at doses of higher than 72.5 mg/kg. Male rats were treated with acrylamide at doses of 0, 5, 15, 30, 45, or 60 mg/kg/day for 5 consecutive days, and the toxicity of acrylamide was observed. In the group treated with the highest dose of acrylamide (60 mg/kg/day), the loss of body weight and reduced testis weight were observed. Also the epididymides weights were reduced significantly in all the groups treated with acrylamide. The number of sperms in cauda epididymidis decreased significantly in an acrylamide dose-dependent manner. Rats treated with 60 mg/kg/day of acrylamide showed several histopathological lesions in the seminiferous tubules. There were thickening and multiple layering of the tubular endothelium, and the formation of many multinucleated giant cells in seminiferous tubules. Taken together, acrylamide not only causes the genotoxicity of eukaryotic cells and mice but also shows the toxicological effects on reproductive system in male rats.

Toxicological effects of acrylamide on rat testicular gene expression profile.

Toxicological effects of acrylamide on differential gene expression profile of rat testis were evaluated. Acrylamide induced morphological sperm defects, and decreased sperm concentration in cauda epididymis. Serum testosterone level and Leydig cell viability were also decreased dose-dependently, which resulted in decreased spermatogenesis. Acrylamide-induced histopathological lesions, such as formation of multinucleated giant cells and vacuolation, and numerous apoptotic cells were observed in seminiferous tubules. cDNA microarray analysis revealed that genes related to testicular-functions, apoptosis, cellular redox, cell growth, cell cycle, and nucleic acid-binding were up/down-regulated in testes isolated from acrylamide-treated group (60 mg/kg/day). Acrylamide toxicity appears to increase Leydig cell death and perturb gene expression levels, contributing to sperm defects and various abnormal histopathological lesions including apoptosis in rat testis.