Establishment of a patient-derived xenograft mouse model of pleomorphic leiomyosarcoma.

Soft tissue sarcomas are difficult to treat using chemotherapy owing to a current deficiency in candidate drugs for specific targets. Screening candidate compounds and analyzing therapeutic targets in sarcomas is insufficient, given the lack of an appropriate human sarcoma animal model to accurately evaluate their efficacy, as well as the lack of an adequate technical protocol for efficient transplantation and engraftment of sarcoma specimens in patient-derived xenograft (PDX) models. Accordingly, in this study, we sought to identify the optimal type of sarcoma and develop a protocol for generating a PDX model. We characterized a PDX mouse model using histopathological and immunohistochemical analyses to determine whether it would show pathological characteristics similar to those of human sarcomas. We achieved engraftment of one of the 10 transplanted sarcoma specimens, the xenografted tumor of which exhibited massive proliferation. Histologically, the engrafted sarcoma foci resembled a primary tumor of pleomorphic leiomyosarcoma and maintained their histological structure in all passages. Moreover, immunohistochemical analysis revealed the expression of specific markers of differentiation to smooth muscle, which is consistent with the features of leiomyosarcoma. We thus demonstrated that our pleomorphic leiomyosarcoma PDX mouse model mimics at least one aspect of human sarcomas, and we believe that this model will facilitate the development of novel therapies for sarcomas.

An improved mouse orthotopic bladder cancer model exhibiting progression and treatment response characteristics of human recurrent bladder cancer.

Nonmuscle-invasive (superficial) bladder cancer is generally treated via surgical removal, followed by adjuvant therapy (bacillus Calmette-Guerin). However, bladder cancer can often recur, and in a substantial number of recurrent cases, the cancer progresses and metastasizes. Furthermore, residual microtumors following excision may lead to an increased risk of recurrence. An in vivo model mimicking the pattern of urinary bladder microtumor regrowth may provide an effective experimental system for improving postsurgical treatment outcomes. A mouse bladder cancer model established using orthotopic transplant of UM-UC-3 human urinary bladder carcinoma cells has been established, however, to the best of our knowledge, no report has investigated sequential histological changes, including early-phase changes and treatment responses in bladder cancer. In the present study, the efficiency of the model was optimized and the sequential changes were examined using histopathology and in situ imaging. The therapeutic effects of cisplatin (CDDP) and gemcitabine (GEM) were also examined, which are drugs that are often used for follow-up chemotherapy. Tumor-seeding efficiency reached 90-100%, with muscle layer and bladder lumen invasion occurring in ~21 days, using the following modifications: i) Shallow catheter insertion to mitigate bladder wall damage; ii) bladder pretreatment using prewarmed trypsin, followed by light urethral clamping and body temperature maintenance for more efficient removal of transitional epithelium; and iii) seeding with UM-UC-3 cells (rather than HT1376, 5637 or T24 tumor cells) in a medium supplemented with Matrigel. Transplant with UM-UC-3 cells resulted in isolated microlesions that progressed into tumors, invading the bladder lumen and muscle layer to the serosal surface. Tumor growth was markedly reduced by weekly intravenous injections of CDDP and partially suppressed by GEM. Therefore, this model is reliable, and pathological progression and treatment responses recapitulate the features of recurrent human bladder cancer.

FF-10832 enables long survival via effective gemcitabine accumulation in a lethal murine peritoneal dissemination model.

Chemotherapy has been the treatment of choice for unresectable peritoneal dissemination; however, it is difficult to eradicate such tumors because of poor drug delivery. To solve this issue, we developed FF-10832 as liposome-encapsulated gemcitabine to maintain a high concentration of gemcitabine in peritoneal tumors from the circulation and ascites. A syngeneic mouse model of peritoneal dissemination using murine Colon26 cell line was selected to compare the drug efficacy and pharmacokinetics of FF-10832 with those of gemcitabine. Despite the single intravenous administration, FF-10832 treatment enabled long-term survival of the lethal model mice as compared with those treated with gemcitabine. Pharmacokinetic analysis clarified that FF-10832 could achieve a more effective gemcitabine delivery to peritoneal tumors owing to better stability in the circulation and ascites. The novel liposome-encapsulated gemcitabine FF-10832 may be a curative therapeutic tool for cancer patients with unresectable peritoneal dissemination via the effective delivery of gemcitabine to target tumors.

Dedifferentiated retroperitoneal liposarcoma spontaneously occurring in an aged SD rat.

Liposarcoma is a rare neoplasm in rats and is characterized by the presence of lipoblasts containing multiple cytoplasmic vacuoles. We encountered a rare type of liposarcoma in a male SD (Crj:CD(SD)IGS) rat during a long-term study to gather background data. At necropsy at 105 weeks of age, there was a large amount of fatty tissue covering the mesentery, pancreas, and retroperitoneum; a white nodule in the right kidney; and paleness of the liver. Microscopically, the tumor had a well-differentiated component and dedifferentiated high-grade component. Immunohistochemical and electron microscopic examinations revealed that the pleomorphic tumor cells retained the characteristics of lipoblasts. Distant or disseminated metastasis was also confirmed in various organs. A liposarcoma with these histological features is extremely rare in rats, and this is the first report of a highly metastatic dedifferentiated type of liposarcoma originating from the abdominal fat tissue in a rat.

Investigation into metastatic processes and the therapeutic effects of gemcitabine on human pancreatic cancer using an orthotopic SUIT-2 pancreatic cancer mouse model.

Prognosis of pancreatic cancer is poor, thus the development of novel therapeutic drugs is necessary. During preclinical studies, appropriate models are essential for evaluating drug efficacy. The present study sought to determine the ideal pancreatic cancer mouse model for reliable preclinical testing. Such a model could accurately reflect human pancreatic cancer phenotypes and predict future clinical trial results. Systemic pathology analysis was performed in an orthotopic transplantation model to prepare model mice for use in preclinical studies, mimicking the progress of human pancreatic cancer. The location and the timing of inoculated cancer cell metastases, pathogenesis and cause of fatality were analyzed. Furthermore, the efficacy of gemcitabine, a key pancreatic cancer drug, was evaluated in this model where liver metastasis and peritoneal dissemination occur. Results indicated that the SUIT-2 orthotopic pancreatic cancer model was similar to the phenotypic sequential progression of human pancreatic cancer, with extra-pancreatic invasion, intra-peritoneal dissemination and other hematogenous organ metastases. Notably, survival was prolonged by administering gemcitabine to mice with metastasized pancreatic cancer. Furthermore, the detailed effects of gemcitabine on the primary tumor and metastatic tumor lesions were pathologically evaluated in mice. The present study indicated the model accurately depicted pancreatic cancer development and metastasis. Furthermore, the detailed effects of pancreatic cancer drugs on the primary tumor and on metastatic tumor lesions. We present this model as a potential new standard for new drug development in pancreatic cancer.

Role of the uridine/cytidine kinase 2 mutation in cellular sensitiveness toward 3'-ethynylcytidine treatment of human cancer cells.

A nucleosidic medicine, 1-(3-C-ethynyl-ß-D-ribo-pentofuranosyl)cytosine [3'-ethynylcytidine (ECyd)], is a potent inhibitor of RNA polymerase I and shows anticancer activity to various human solid tumors in vitro and in vivo. ECyd is phosphorylated to 3'-ethyntlcytidine 5'-monophosphate by uridine/cytidine kinase 2 (UCK2) and subsequently further to diphosphate and triphosphate (3'-ethyntlcytidine 5'-diphosphate, 3'-ethyntlcytidine 5'-triphosphate). 3'-Ethyntlcytidine 5'-triphosphate is an active metabolite that can inhibit RNA polymerase I competitively, causing cancer cell death. Here, to identify the UCK2 mutation for detecting responder or nonresponder to ECyd, we investigated the relationship between point mutation of the UCK2 gene and response to ECyd in various human solid tumors. We identified several functional point mutations including the splice-site mutation of the UCK2 gene IVS5+5 G>A. In addition, we found that the IVS5+5 G>A variant generates an aberrant mRNA transcript, namely, truncated mRNA was produced and normal mRNA levels were markedly decreased in the ECyd-resistant cancer cell line HT1080. We concluded that these findings strongly suggest that the IVS5+5 G>A variant would affect the expression level of the UCK2 transcript, resulting in decreased sensitivity to ECyd.

Association of RNase L with a Ras GTPase-activating-like protein IQGAP1 in mediating the apoptosis of a human cancer cell-line.

Mammalian intracellular ribonuclease L (RNase L) is a latent endoribonuclease that functions against viral infections as an apoptosis-inducing protein, and its activity requires intracellular 5'-end-triphosphorylated-2',5' oligoadenylates (2-5A) as an activator. Previously, we showed that RNase L can be activated in human cancer cell line HT1080 by an RNA polymerase I inhibitor, 1-(3-C-ethynyl-ß-D-ribo-pentofuranosyl)cytosine (3'-ethynylcytidine; ECyd). In ECyd-treated cells, knockdown of the RNase L resulted in a marked decrease in c-jun N-terminal kinase (JNK) phosphorylation, thereby inhibiting apoptosis. We investigate RNase L binding partners by focused proteomic approach using immunoprecipitation with anti-RNase L IgG and mass spectrometry. We found that the IQ motif-containing Ras GTPase-activating-like protein 1 (IQGAP1) can associate with RNase L, and that phosphorylation occurs on the IQGAP1. ECyd-induced JNK phosphorylation and apoptosis were inhibited when IQGAP1 was knocked down with a small interfering RNA. These results raise the interesting possibility that the RNase L-IQGAP1 association may regulate JNK phosphorylation in RNase L-madiated apoptosis. It is likely IQGAP1 works as a regulator in apoptosis.

Molecular mechanisms of apoptosis induced by 3'-ethynylcytidine.

1-(3-C-Ethynyl-beta-D-ribo-pentofuranosyl)cytosine (3'-Ethynylcytidine; ECyd), a ribonucleoside analog, has a potent cytotoxic activity against cancer cells. We have investigated the cancer-cell death induced by ECyd, focusing on its molecular mechanisms. In ECyd-treated cells, RNase L is activated and involved in c-jun NH(2)-terminal kinase (JNK) phosphorylation, followed by induction of mitochondria-dependent apoptosis. The mechanism of JNK phophorylation by RNase L was unknown. To investigate the mechanism, we performed the identification of RNase L-binding partners by proteomic approach using co-immunoprecipitation and mass spectrometry. We found that RNase L was associated with a protein (we named it Protein-190). At the same time, we observed that Protein-190 was amply phosphorylated. Furthermore, the participation of Protein-190 in the ECyd-induced apoptosis was supported by a knockdown experiment using small interfering RNA (siRNA). Thus, the number of ECyd-induced apoptotic cells was drastically decreased when Protein-190 was knocked-down. These results indicated Protein-190 as a regulator in apoptosis, and provide the possibility for a new clinical target in cancer chemotherapy.

Role of RNase L in apoptosis induced by 1-(3-C-ethynyl-beta-D-ribo-pentofuranosyl)cytosine.

PURPOSE: 1-(3-C-Ethynyl-beta-D: -ribo-pentofuranosyl)cytosine (ECyd), a ribonucleoside analog, has a potent cytotoxic activity against cancer cells. The present studies have been performed to elucidate the overall mechanisms of ECyd-induced apoptotic cell death. METHODS: Cultured cells of mouse mammary carcinoma FM3A and human fibrosarcoma HT 1080 lines were used. The efficacy of RNA synthesis inhibition by ECyd was assessed by kinetic analysis using nuclei isolated from FM3A cells. RNA status in ECyd-treated cells was investigated by Northern blots, and the cleavage sites of RNA were identified by rapid amplification of 5' cDNA ends (5'-RACE). The effect of protein functions on the ECyd-induced apoptotic pathway was analyzed by siRNA and immunohistochemical techniques. Apoptotic cells were detected by TdT-mediated dUTP-biotin Nick End Labeling (TUNEL) assay. RESULTS: ECyd induces inhibition of RNA synthesis in vitro and in vivo, which appears to be a major cause for the apoptosis. It is known that ECyd is converted inside the cell into its 5'-triphosphate (ECTP). We have now found in test-tube experiments that ECTP strongly inhibits the activity of RNA polymerase I by competing with CTP. In the absence of robust RNA synthesis, the cellular RNAs would be destined to break down. RNase L was found to be playing a role in the breakdown: thus, the 28S rRNA-fragmentation pattern observed for the ECyd-treated cells was very similar to that observable in an in vitro treatment of the 28S ribosomes with RNase L. Association of RNase L with the cytotoxic action of ECyd was confirmed by use of the siRNA-mediated suppression of the cellular RNase L. Thus, the cells in which the RNase L was knocked-down were highly resistant to the cytotoxic action of ECyd. Further events, downstream of the RNase L action that can lead to the eventual apoptosis, would conceivably involve the phosphorylation of c-jun N-terminal kinase and subsequent decrease in mitochondrial membrane-potential. Evidence to support this flow of events was obtained by siRNA-experiments. CONCLUSION: The results from this study demonstrated that RNase L is activated after the inhibition of RNA polymerase, and induces mitochondria-dependent apoptotic pathway. We propose this new role for RNase L in the apoptotic mechanism. These findings may open up the possibility of finding new targets for anticancer agents.

Synthesis and silencing properties of siRNAs possessing lipophilic groups at their 3'-termini.

Short-interfering RNAs (siRNAs) conjugated with lipophilic groups at their 3'-termini were synthesized. The properties of the synthesized siRNAs were examined in detail, and it was found that at low concentrations, their silencing abilities were dependent on the positions of the modifications and the types of organic molecules attached. Although the modification of siRNAs with palmitic acid or oleic acid at the 3'-end slightly reduced their silencing activities, siRNAs had enough abilities to induce RNAi at 10 nM concentrations. On the other hand, the modification of siRNAs with cholesterol at the 3'-end of the passenger strand was tolerated; however, the modification at the guide strand significantly reduces its silencing activity. The siRNAs modified with the lipophilic groups did not possess ability to penetrate the plasma membranes of HT-1080 cells without the transfection reagent. However, the results described in this report will aid in designing novel siRNAs with cell membrane-permeable molecules.

Gene expression profiles of necrosis and apoptosis induced by 5-fluoro-2'-deoxyuridine.

5-Fluoro-2'-deoxyuridine (FUdR), a potent anticancer agent, exerts its effects by inhibiting thymidylate synthase, an essential machinery for DNA synthesis in cell proliferation. Also, cell death is caused by FUdR, primarily due to an imbalance in the nucleotide pool resulting from this enzyme inhibition. We have investigated the cancer cell death induced by FUdR, focusing on its molecular mechanisms. Using mouse mammary tumor FM3A cell lines, the original clone F28-7 and its variant F28-7-A cells, we previously reported an interesting observation that FUdR induces a necrotic morphology in F28-7, but induces, in contrast, an apoptotic morphology in F28-7-A cells. In the present study, to understand the molecular mechanisms underlying these differential cell deaths, i.e., necrosis and apoptosis, we investigated the gene expression changes occurring in these processes. Using the cDNA microarray technology, we found 215 genes being expressed differentially in the necrosis and apoptosis. Further analysis revealed differences between these cell lines in terms of the expressions of both a cluster of heat shock protein (HSP)-related genes and a cluster of apoptosis-related genes. Notably, inhibition of HSP90 in F28-7 cells caused a shift from the FUdR-induced necrosis into apoptosis. These findings are expected to lead to a better understanding of this anticancer drug FUdR for its molecular mechanisms and also of the general biological issue, necrosis and apoptosis.

Association of nuclear membrane protein lamin B1 with necrosis and apoptosis in cell death induced by 5-fluoro-2'-deoxyuridine.

We report that anticancer 5-fluoro-2 '-deoxyuridine (FUdR) shows cytotoxicity against mouse cancer cell line FM3A, using a progeny clone F28-7 and its variant F28-7-A. In this process, the cell-death morphology is different between F28-7 and F28-7-A cells, that is, necrosis in F28-7 but apoptosis in F28-7-A cells. In the proteomic analysis of these cells before their exposure to FUdR, the nuclear inner-membrane protein lamin B1 is up-regulated in F28-7 but not in F28-7-A, suggesting that lamin B1 may possess a function to regulate the morphology of cell-death. A knockdown of lamin B1 expression in F28-7 cells was performed by use of the small interfering RNA technique, resulting in a decrease of the lamin B1-expression level down to the level in F28-7-A. Remarkably, the FUdR-induced death morphology of this knocked-down F28-7 was apoptosis, definitely different from the necrosis that occurs in the FUdR-treated original F28-7. Thus, the swelling feature for the necrosis was no longer observable, and instead cell shrinkage typical of apoptosis took place in almost all the cells examined. This finding suggests a new role for lamin B1 as a regulator in cell death.

A novel apoptotic pathway of 3'-Ethynylcytidine(ECyd) involving the inhibition of RNA synthesis--the possibility of RNase L activated pathway as a target of ECyd.

RNase L functions as a tumor suppressor protein due to its role in apoptosis during viral infections and various agents treatment. RNase L-mediated apoptosis is accompanied by cytochrome c release from mitochondria and requires caspase-3 activity. It was reported that RNase L is involved in JNK activation during viral infections, and that JNK is essential for apoptosis in response to RNase L activation. However, the proximal signals of RNase L that trigger JNK activation have not as yet been identified, and it is possible that the interactions between RNase L and other proteins play an important role in the RNase L-JNK apoptotic signal pathway. To investigate this hypothesis, we attempted to identify the proteins associated with RNase L using coimmunoprecipitation. In this study, we found that RNase L was associated with other proteins. Here we identified the protein X by LC-MS/MS analysis. We demonstrated that this association was increased in the presence of activated RNase L. Moreover, Protein X was phosphorylated during the activation of RNase L by treatment with cytotoxic agent, ECyd, 1-(3-C-ethynyl-beta-D-ribo-pentofuranosyl) cytosine and 2-5A. To reveal the role of protein X in RNase L-mediated apoptosis, we decreased the level of protein X by a small interfering RNA (siRNA). As a result, protein X deficient cells became resistant to the apoptosis mediated by RNase L, suggesting that protein X is related to RNase L-mediated apoptosis following JNK activation. Therefore, in this study, we report the identification of a novel protein, protein X that transduces between RNase L and JNK signals.

An apoptotic pathway of 3'-Ethynylcytidine(ECyd) involving the inhibition of RNA synthesis mediated by RNase L.

RNase L is an endoribonuclease that requires 2'-5' oligoadenylate to cleave single-stranded RNA. Although the antiviral effects of RNase L are well known because of its viral RNA degradation activity recently but it has been suggested that RNase L is concerned in mitochondrial-caspase dependent apoptotic signaling pathway induced by a number of anticancer agents. Moreover, it has variety of functions including translation and transcription of proteins. In this report, we found that 1-(3-C-ethynyl-beta-D-ribo-pentofuranosyl) cytosine (ECyd), which inhibits RNA synthesis through competitive inhibition of RNA polymerase I induced 28S rRNA fragmentation. The cleavage pattern of rRNA induced by ECyd was similar and the cleavage sites were identical to those cleaved by RNase L. Additionaly, apoptosis induced by ECyd was elevated following the protein expression of RNase L in the tumor cells when treated with IFN-alpha2a which was known to induce RNase L expression. To identify the role of RNase L in apoptosis induced by ECyd, we detected the decreased level of RNase L by several folds in the tumor cell lines through a small interfering RNA (siRNA). These results indicated that RNase L might integrate apoptotic signals induced by ECyd and provide the possibility to be a novel clinical target for cancer chemotherapy.

Inhibitory mechanisms of 1-(3-C-ethynyl-beta-D-ribopentofuranosyl)uracil (EUrd) on RNA synthesis.

1-(3-C-ethynyl-beta-D-ribo-pentofuranosyl)uracil (EUrd) is an antimetabolite that strongly inhibits RNA synthesis and shows a broad antitumor activity in vitro and in vivo. In mouse mammary tumor FM3A cells, EUrd is sequentially phosphorylated to its 5'-triphosphate, EUTP, a major metabolite, and the RNA synthesis is inhibited proportionally to its intracellular accumulation. To study the inhibitory mechanisms of EUrd on RNA synthesis, we have performed the kinetic analysis of EUTP on RNA polymerization using isolated nuclei RNA synthesis was inhibited competitively by EUTP. The inhibition constant, Ki was much lower than the Km value of UTP (Ki value of EUTP, 84 nM; Km value of UTP, 13 microM), indicating that the high affinity of EUTP could contribute to the specific inhibition of RNA synthesis. As a result of RNA synthesis inhibition, EUrd, but not ara-C, induced shrinkage of nucleoli, which are the main sites for RNA synthesis in FM3A cells. Thus, the strong affinity of EUTP to RNA polymerase and specific inhibition of RNA synthesis could contribute to its antitumor effect. EUrd is expected to be a new antitumor drug, possessing a strong inhibitory effect on the synthesis of RNA.

Synthesis of novel siRNAs having thymidine dimers consisting of a carbamate or a urea linkage at their 3' overhang regions and their ability to suppress human RNase L protein expression.

In order to examine the effect of modifications at the 3' overhang regions of short interfering RNAs (siRNAs) on their gene-silencing activities, we designed and synthesized novel siRNAs having thymidine dimers consisting of a carbamate or a urea linkage at their 3' overhang regions. Suppression of human RNase L protein expression by these siRNAs was analyzed by immunoblot with RNase L-specific antibody. It was found that, at 24 h post-transfection, the modified siRNAs having the thymidine dimers with the carbamate and urea linkage suppress the protein expression 78 and 37 times more efficiently than that with the natural phosphodiester linkage, respectively. Furthermore, the siRNA containing the carbamate linkage was 37 times more resistant to nucleolytic degradation by snake venom phosphodiesterase than the siRNA consisting of the natural phosphodiester linkage. Thus, the RNA duplexes having the thymidine dimers with the carbamate or urea linkage at their 3' overhang regions will be promising candidates for novel siRNA molecules to down-regulate protein expression.

Anticancer mechanisms of 1-(3-C-ethynyl-beta-D-ribo-pentofuranosyl) cytosine (ECyd, TAS-106).

We investigated the molecular mechanisms of cell death induced by 1-(3-C-ethynyl-beta-D-ribo-pentofuranosyl)cytosine (ECyd, TAS-106), a potent inhibitor of RNA synthesis, using mouse mammary tumor FM3A cells and human fibrosarcoma HT1080 cells. ECyd induced the characteristics of apoptosis on these cells, such as morphological changes, DNA fragmentations and caspase-3-like protease activation. General caspases inhibitor, Z-Asp-CH2-DCB inhibited cell death. Interestingly, we also found that ECyd induced rRNA fragmentation. The cleavage pattern of rRNA resembled in that mediated by RNase L. On the other hands, it was suggested that caspase-1, 3, 8 and 9 concerned with ECyd-induced apoptosis through mitochondria. ECyd-induced rRNA fragmentation was inhibited by general caspases inhibitor (Z-Asp-CH2-DCB) and caspase-5 inhibitor (Z-WEHD-fmk). So it is clear that caspase-5 (ICErel III/TY), member of ICE (Interleukin-1 beta-converting enzyme) protease, activated pathway concerned with ECyd-induced rRNA fragmentation. These results indicate that antitumor mechanisms of ECyd are involved in caspase-dependent activation of RNase L. rRNA fragmentation may occur one of the death events, as a result of inhibition of RNA synthesis and play an important role in the antitumor activity of ECyd.