Establishment and characterization of TK-ALCL1: a novel NPM-ALK-positive anaplastic large-cell lymphoma cell line.

TK-ALCL1, a novel anaplastic lymphoma kinase (ALK)-positive anaplastic large-cell lymphoma (ALK+ ALCL) cell line, was established from the primary tumor site of a 59-year-old Japanese male patient. The immune profile of TK-ALCL1 corresponds to that seen typically in primary ALCL cells, i.e., positive for ALK, CD30, EMA, and CD4, but negative for CD2, CD3, CD5, CD8a, and EBV-related antigens. The rearrangement of the T cell receptor-gamma locus shows that TK-ALCL1 is clonally derived from T-lineage lymphoid cells. FISH and RT-PCR analysis revealed that TK-ALCL1 has the nucleophosmin (NPM)-ALK fusion transcript, which is typical for ALK+ ALCL cell lines. When TK-ALCL1 was subcutaneously inoculated into 6-week-old BALB/c Rag2-/-/Jak3-/- (BRJ) mice, it formed tumor masses within 4-6 weeks. Morphological, immunohistochemical, and molecular genetic investigations confirmed that the xenograft and the original ALCL tumor were identical. The ALK inhibitors Alectinib and Lorlatinib suppressed proliferation in a dose-dependent manner. Thus, TK-ALCL1 provides a useful in vitro and in vivo model for investigation of the biology of ALK+ ALCL and of novel therapeutic approaches targeting ALK.

Novel Midkine Inhibitor Induces Cell Cycle Arrest and Apoptosis in Multiple Myeloma.

BACKGROUND/AIM: Multiple myeloma (MM), the second most common hematological malignancy, is characterized by the accumulation of malignant plasma cells within the bone marrow. Despite various drug classes for MM treatment, it remains incurable, necessitating novel and efficacious agents. This study aims to explore the anti-cancer activity of a midkine inhibitor, iMDK (C21H13FN2O2S), in myeloma cell lines. MATERIALS AND METHODS: This study assessed the antiproliferative activity using the MTT assay. Cell cycle and apoptosis were evaluated using flow cytometry. To further investigate the inhibitory mechanism, western blotting was used to detect cell cycle-related proteins, pro-apoptotic proteins, and anti-apoptotic proteins. RESULTS: iMDK inhibits MM cell proliferation in a dose- and time-dependent manner, inducing cell cycle arrest and apoptosis. The reduction in Cdc20 expression by iMDK treatment leads to G2/M phase cell cycle arrest. Furthermore, iMDK down-regulates anti-apoptotic proteins (Bcl-2, Bcl-xL, Mcl-1, and c-FLIP), thereby activating both intrinsic and extrinsic apoptosis pathways. CONCLUSION: iMDK could be a potential candidate for MM treatment.

The IL-17-IL-17RA axis is required to promote osteosarcoma progression in mice.

Osteosarcoma is rare but is the most common bone tumor. Diagnostic tools such as magnetic resonance imaging development of chemotherapeutic agents have increased the survival rate in osteosarcoma patients, although 5-year survival has plateaued at 70%. Thus, development of new treatment approaches is needed. Here, we report that IL-17, a proinflammatory cytokine, increases osteosarcoma mortality in a mouse model with AX osteosarcoma cells. AX cell transplantation into wild-type mice resulted in 100% mortality due to ectopic ossification and multi-organ metastasis. However, AX cell transplantation into IL-17-deficient mice significantly prolonged survival relative to controls. CD4-positive cells adjacent to osteosarcoma cells express IL-17, while osteosarcoma cells express the IL-17 receptor IL-17RA. Although AX cells can undergo osteoblast differentiation, as can patient osteosarcoma cells, IL-17 significantly inhibited that differentiation, indicating that IL-17 maintains AX cells in the undifferentiated state seen in malignant tumors. By contrast, IL-17RA-deficient mice transplanted with AX cells showed survival comparable to wild-type mice transplanted with AX cells. Biopsy specimens collected from osteosarcoma patients showed higher expression of IL-17RA compared to IL-17. These findings suggest that IL-17 is essential to maintain osteosarcoma cells in an undifferentiated state and could be a therapeutic target for suppressing tumorigenesis.

Bradykinin deficiency causes high blood pressure in mice.

Bradykinin has a wide variety of physiological functions, including vasodilation and blood pressure reduction. However, the physiological roles of bradykinin are not fully understood. We used the CRISPR/Cas9 method to generate BKdelK1 and BKdelK2 mutant mice, targeting the BK portion of mouse kininogen1 and kininogen2 genes, respectively. The BKdelK1 and BKdelK2 mutant mice had about 50% reductions in plasma low molecular weight kininogen and trypsin-released BK, compared to wild mice. Both BKdelK1 and BKdelK2 mice had significantly elevated systolic blood pressure compared to WT mice. These results suggest that plasma LKNG is a source of KNG in the vascular kallikrein-kinin system and contributes to maintaining lower systolic blood pressure.

Shikonin Induces ROS-Dependent Apoptosis Via Mitochondria Depolarization and ER Stress in Adult T Cell Leukemia/Lymphoma.

Adult T cell leukemia/lymphoma (ATLL) is an aggressive T-cell malignancy that develops in some elderly human T-cell leukemia virus (HTVL-1) carriers. ATLL has a poor prognosis despite conventional and targeted therapies, and a new safe and efficient therapy is required. Here, we examined the anti-ATLL effect of Shikonin (SHK), a naphthoquinone derivative that has shown several anti-cancer activities. SHK induced apoptosis of ATLL cells accompanied by generation of reactive oxygen species (ROS), loss of mitochondrial membrane potential, and induction of endoplasmic reticulum (ER) stress. Treatment with a ROS scavenger, N-acetylcysteine (NAC), blocked both loss of mitochondrial membrane potential and ER stress, and prevented apoptosis of ATLL cells, indicating that ROS is an upstream trigger of SHK-induced apoptosis of ATLL cells through disruption of the mitochondrial membrane potential and ER stress. In an ATLL xenografted mouse model, SHK treatment suppressed tumor growth without significant adverse effects. These results suggest that SHK could be a potent anti-reagent against ATLL.

Establishment and characterization of a novel cancer stem-like cell of cholangiocarcinoma.

Cholangiocarcinoma (CCA) is an aggressive malignant tumor of bile duct epithelia. Recent evidence suggests the impact of cancer stem cells (CSC) on the therapeutic resistance of CCA; however, the knowledge of CSC in CCA is limited due to the lack of a CSC model. In this study, we successfully established a stable sphere-forming CCA stem-like cell, KKU-055-CSC, from the original CCA cell line, KKU-055. The KKU-055-CSC exhibits CSC characteristics, including: (1) the ability to grow stably and withstand continuous passage for a long period of culture in the stem cell medium, (2) high expression of stem cell markers, (3) low responsiveness to standard chemotherapy drugs, (4) multilineage differentiation, and (5) faster and constant expansive tumor formation in xenograft mouse models. To identify the CCA-CSC-associated pathway, we have undertaken a global proteomics and functional cluster/network analysis. Proteomics identified the 5925 proteins in total, and the significantly upregulated proteins in CSC compared with FCS-induced differentiated CSC and its parental cells were extracted. Network analysis revealed that high mobility group A1 (HMGA1) and Aurora A signaling through the signal transducer and activator of transcription 3 pathways were enriched in KKU-055-CSC. Knockdown of HMGA1 in KKU-055-CSC suppressed the expression of stem cell markers, induced the differentiation followed by cell proliferation, and enhanced sensitivity to chemotherapy drugs including Aurora A inhibitors. In silico analysis indicated that the expression of HMGA1 was correlated with Aurora A expressions and poor survival of CCA patients. In conclusion, we have established a unique CCA stem-like cell model and identified the HMGA1-Aurora A signaling as an important pathway for CSC-CCA.

Diminishing acetyl-CoA carboxylase 1 attenuates CCA migration via AMPK-NF-κB-snail axis.

Cholangiocarcinoma (CCA), a cancer of the biliary tract, is a significant health problem in Thailand. Reprogramming of cellular metabolism and upregulation of lipogenic enzymes have been revealed in CCA, but the mechanism is unclear. The current study highlighted the importance of acetyl-CoA carboxylase 1 (ACC1), a rate-limiting enzyme in de novo lipogenesis, on CCA migration. ACC1 expression in human CCA tissues was determined by immunohistochemistry. The results demonstrated that increased ACC1 was related to the shorter survival of CCA patients. Herein, ACC1-deficient cell lines (ACC1-KD) were generated by the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (cas9) system and were used for the comparative study. The ACC1 levels in ACC1-KD were 80-90 % lower than in parental cells. Suppression of ACC1 significantly reduced intracellular malonyl-CoA and neutral lipid contents. Two-fold growth retardation and 60-80 % reduced CCA cell migration and invasion were observed in ACC1-KD cells. The reduced 20-40 % of intracellular ATP levels, AMPK activation, lowered NF-κB p65 nuclear translocation, and snail expression were emphasized. Migration of ACC1-KD cells was restored by supplementation with palmitic acid and malonyl-CoA. Altogether, the importance of rate-limiting enzyme in de novo fatty acid synthesis, ACC1, and AMPK-NF-κB-snail axis on CCA progression was suggested herein. These might be the novel targets for CCA drug design. (ACC1, AMPK, Cholangiocarcinoma, De novo lipogenesis, NF-κB, Palmitic acid).

Dihydroartemisinin Induced Apoptosis and Synergized With Chemotherapy in Pleural Effusion Lymphoma Cells.

BACKGROUND/AIM: Primary effusion lymphoma (PEL) is a rare aggressive B-cell lymphoma associated with HHV-8. With a median survival of fewer than six months, the prognosis of the disease with current standard therapies is usually dismal. Dihydroartemisinin (DHA) is a derivative of artemisinin, originally designed as an antimalarial drug. Several studies have shown that this compound also demonstrates anti-cancer activity in various types of cancer, including hematologic malignancies. MATERIALS AND METHODS: Anti-proliferation activity of DHA on 5 PEL cell lines was assessed by MTT assay. Cell cycle arrest was determined by propidium iodide staining and flow cytometry analysis. DHA-induced PEL apoptosis was shown by annexin V/PI staining and western blotting for cleaved caspases 3, 8, and 9. An inhibitory effect on PEL growth was evaluated in a PEL-xenograft mouse model. A synergistic effect of DHA and doxorubicin combination treatment was shown in vitro. RESULTS: DHA showed anti-proliferative activity on PEL and induced caspase-dependent apoptosis in a time- and dose-dependent manner. DHA-induced cell death appeared to be triggered by increased levels of reactive oxygen species (ROS). N-acetylcysteine treatment inhibited DHA-induced ROS elevation and suppressed expression of cleaved caspases leading to significantly reduced PEL apoptosis. DHA treatment also demonstrated an inhibitory effect on PEL cell growth in an in-vivo xenograft model. Moreover, we found that a combination treatment of DHA and doxorubicin, the standard chemotherapy drug for PEL, demonstrated a synergistic effect on PEL cell lines. CONCLUSION: DHA is a potentially effective candidate drug for PEL treatment.

Cepharanthine inhibits dengue virus production and cytokine secretion.

Dengue virus (DENV) infection is a public health problem in tropical and subtropical regions. It can cause a spectrum of clinical manifestations ranging from mild dengue fever (DF) to severe dengue haemorrhagic fever (DHF) and potentially life-threatening disease including dengue shock syndrome (DSS). Severe DENV infection is caused by high viral load and cytokine storm in dengue-infected patients. Currently, there is no specific antiviral drug for DENV infection. An anti-DENV agent that demonstrates inhibitory effects on both DENV replication and cytokine secretion is urgently needed. In this study, cepharanthine (CEP), which is an anti-inflammatory, anti-HIV, and anti-tumor compound isolated from Stephania cepharantha Hayata, was tested for inhibition of DENV infection. We investigated the efficacy of CEP to inhibit DENV infection, replication, and cytokine production. The inhibitory effect of CEP treatment was studied in DENV-infected human chronic myeloid leukemia (K562) cells. The levels of DENV E protein and DENV production were determined by flow cytometry and FFU assay, respectively. CEP treatment significantly reduced viral E protein and viral production in all DENV-1, 2, 3, 4 serotypes. In addition, CEP treatment reduced the IL-6 proinflammatory cytokine production in DENV-infected A549 cells. Taken together, CEP has inhibitory effects on DENV infection specifically at the initial viral replication states and proinflammatory cytokine secretion, and is a promising candidate for further development as an anti-DENV treatment.

A novel tdTomato transgenic mouse model to visualize FAP-positive cancer-associated fibroblasts.

Fibroblast activation protein (FAP) generally shows low or undetectable expression in most normal tissues but is highly expressed in fibroblasts in almost all carcinomas. FAP is one of the potential molecules to detect activated fibroblasts and has multiple roles in tumour progression. We generated transgenic mice that specifically expressed tdTomato along with FAP promoter activity. Coculturing a mouse gastric cancer cell line and FAP-tdTomato transgenic mouse-derived fibroblasts showed that tdTomato expression was elevated in the cocultured fibroblasts. Moreover, stomach wall transplanted tumours in mice also showed FAP-tdTomato expression in fibroblasts of the stomach and each metastatic legion. These results indicated that FAP-tdTomato expression in fibroblasts was elevated by stimulation through the interaction with cancer cells. Functionally, collagen production was increased in FAP/tdTomato-positive fibroblasts cocultured with mouse cancer cells. These FAP-tdTomato transgenic mice have the potential to be used to investigate real-time FAP dynamics and the importance of FAP expression in tumour development.

An oncolytic virus as a promising candidate for the treatment of radioresistant oral squamous cell carcinoma.

We evaluated the usefulness of an oncolytic virus (Suratadenoturev; OBP-301) against radioresistant oral squamous cell carcinoma. We confirmed the expression of human telomerase reverse transcriptase and the coxsackievirus and adenovirus receptor in cell lines. Also, we examined the potential presence in a patient who has received existing therapy that is amenable to treatment with OBP-301. We evaluated: (1) the antitumor effects of OBP-301 alone and in combination with radiotherapy on radioresistant cell lines, (2) the molecular mechanism underlying the radiosensitizing effect and cell death increased by the combination therapy, and (3) the antitumor effect of the combination therapy in vivo using xenograft models (a radioresistant cell line-derived xenograft in mouse and a patient-derived xenograft). Human telomerase reverse transcriptase and the coxsackievirus and adenovirus receptor were expressed in all cell lines. OBP-301 decreased the proliferative activity of these cell lines in a concentration-dependent manner, and significantly enhanced the antitumor effect of irradiation. Phosphorylated STAT3 and its downstream molecules, which correlated with apoptosis and autophagy, showed significant changes in expression after treatment with OBP-301. The combination therapy exerted a significant antitumor effect versus radiotherapy alone in both xenograft models. Combination of OBP-301 with radiotherapy exerts a synergistic effect and may represent a promising treatment for radioresistant oral squamous cell carcinoma.

Potential use of EGFR-targeted molecular therapies for tumor suppressor CYLD-negative and poor prognosis oral squamous cell carcinoma with chemoresistance.

BACKGROUND: Tumor suppressor CYLD dysfunction by loss of its expression, triggers malignant transformation, especially drug resistance and tumor invasion/metastasis. Although loss of CYLD expression is significantly associated with poor prognosis in a large variety of tumors, no clinically-effective treatment for CYLD-negative cancer patients is available. METHODS: We focused on oral squamous cell carcinoma (OSCC), and sought to develop novel therapeutic agents for CYLD-negative cancer patients with poor prognosis. CYLD-knockdown OSCC cells by using CYLD-specific siRNA, were used to elucidate and determine the efficacy of novel drug candidates by evaluating cell viability and epithelial-mesenchymal transition (EMT)-like change. Therapeutic effects of candidate drug on cell line-derived xenograft (CDX) model and usefulness of CYLD as a novel biomarker using patient-derived xenograft (PDX) model were further investigated. RESULTS: CYLD-knockdown OSCC cells were resistant for all currently-available cytotoxic chemotherapeutic agents for OSCC, such as, cisplatin, 5-FU, carboplatin, docetaxel, and paclitaxel. By using comprehensive proteome analysis approach, we identified epidermal growth factor receptor (EGFR), a receptor tyrosine kinase, played key roles in CYLD-knockdown OSCC cells. Indeed, cell survival rate in the cisplatin-resistant CYLD-knockdown OSCC cells was markedly inhibited by treatment with clinically available EGFR tyrosine kinase inhibitors (EGFR-TKIs), such as gefitinib. In addition, gefitinib was significantly effective for not only cell survival, but also EMT-like changes through inhibiting transforming growth factor-ß (TGF-ß) signaling in CYLD-knockdown OSCC cells. Thereby, overall survival of CYLD-knockdown CDX models was significantly prolonged by gefitinib treatment. Moreover, we found that CYLD expression was significantly associated with gefitinib response by using PDX models. CONCLUSIONS: Our results first revealed that EGFR-targeted molecular therapies, such as EGFR-TKIs, could have potential to be novel therapeutic agents for the CYLD-negative OSCC patients with poor prognosis.

Midkine inhibitor (iMDK) induces apoptosis of primary effusion lymphoma via G2/M cell cycle arrest.

Primary effusion lymphoma (PEL) is an aggressive B-cell non-Hodgkin lymphoma in immunocompromised individuals such as AIDS patients. PEL shows a poor prognosis (median survival time < 6 months) compared with other AIDS-related lymphomas, and is generally resistant to conventional treatments. Novel drugs for PEL treatment are required. Midkine inhibitor (iMDK) was previously found to suppress midkine protein expression. Interestingly, iMDK suppressed cell proliferation in PEL cell lines in a time- and dose-dependent manner, regardless of midkine gene expression. We examined the mechanism of iMDK on PEL. Importantly, iMDK strongly induced cell cycle arrest at the G2/M phase within 12 h of incubation and suppressed the p-CDK1 protein level, which is associated with the cell cycle checkpoint at G2/M, resulting in mitotic catastrophe with observation of multipolar division. After mitotic catastrophe, iMDK-treated PEL showed apoptosis with caspase-3, - 8, and - 9 activation at 24 h incubation. However, iMDK showed no effects on viral protein-activated signaling pathways such as JAK-STAT, PI3K-Akt and NF-κB, and HHV-8/KSHV gene expression in PEL. These results indicate that iMDK is a novel CDK1 inhibitor and a promising lead compound for PEL chemotherapy treatment.

Elotuzumab, a potential therapeutic humanized anti-SLAMF7 monoclonal antibody, enhances natural killer cell-mediated killing of primary effusion lymphoma cells.

Primary effusion lymphoma (PEL) is a rare aggressive B-cell non-Hodgkin's lymphoma with no optimal treatment. Signaling lymphocytic activation molecule-F7 (SLAMF7, CD319), a type I transmembrane glycoprotein highly expressed in multiple myeloma (MM), represents a promising target for mAb-based immunotherapy. SLAMF7 also expresses on several hematopoietic lineages including NK cells. Elotuzumab (Elo), a humanized antibody targeting SLAMF7, is approved by FDA for MM treatment. In this study, we analyzed the expression of SLAMF7 on seven PEL cell lines. All PEL cells and NK cells showed high expression of SLAMF7. NK cells were enriched from PBMCs of healthy donors by MACS and expanded by co-culturing with MHC-class I negative K562 cells in the presence of IL-2 and IL-15. Expanded NK cells showed direct killing, and Elo demonstrated potent ADCC against PEL in an Effector:Target (E:T) dependent manner. Surface expression of CD107a on NK cells also increased in the process of ADCC. We also examined SLAMF7 expression of NK subpopulations and found that the CD56+CD16+ NK subpopulation demonstrated the highest SLAMF7 expression. Full-length-Elo but not F(ab')2-Elo exerts direct engagement to the expressing SLAMF7 on NK cells, promotes CD107a expression, and further augments NK cytotoxicity toward PEL. Elo enhanced survival of PEL-bearing immunodeficient mice with adoptive transfer of human NK cells. Taken together, our results show that NK cells play roles in PEL killing, and Elo causes ADCC/SLAMF7 ligation to boost NK cytotoxicity against PEL, offering promising preclinical evidence of Elo as a therapeutic monoclonal antibody treatment for PEL.

Efficacy and mechanism of the anti-CD38 monoclonal antibody Daratumumab against primary effusion lymphoma.

Primary effusion lymphoma (PEL) is a rare, aggressive B cell non-Hodgkin's lymphoma of the body cavities with malignant effusions. The prognosis is poor, and no optimal treatment has been established. CD38 is a type II transmembrane glycoprotein known to overexpress in multiple myeloma (MM). Daratumumab (DARA), a human CD38-targeting monoclonal antibody (mAb), is approved for MM treatment. In this study, we found expression of CD38 on PEL cells and assessed the anti-PEL activity of DARA. We found that both KHYG-1 and N6 (CD16-transfected KHYG-1) NK cell lines showed direct killing activity against PEL cells with induction of CD107a, and NK-mediated cytotoxicity by N6NK (CD16+) cells increased with DARA treatment. We confirmed direct NK activity and antibody-dependent cell cytotoxicity (ADCC) by expanded NK cells, indicating that DARA has high ADCC activity. We elucidated the antibody-dependent cell phagocytosis (ADCP) by using human monocyte-derived macrophages (MDMs) and mouse peritoneal macrophages. DARA also showed potent complement-dependent cytolysis (CDC) toward PEL. DARA also induced PEL cell death in the presence of a cross-linking antibody. Moreover, treatment with DARA inhibited tumor growth in a PEL xenograft mouse model. These results provide preclinical evidence that Ab targeting of CD38 could be an effective therapeutic strategy for the treatment of PEL.

Homophilic Interaction of CD147 Promotes IL-6-Mediated Cholangiocarcinoma Invasion via the NF-κB-Dependent Pathway.

Cholangiocarcinoma (CCA), an aggressive cancer of bile ducts, is a well-known chronic inflammation-related disease. The major impediment in CCA treatment is limited treatment options for advanced disease; hence, an alternative is urgently required. The role of CD147 on cytokine production has been observed in inflammation-related diseases, but not in CCA. Therefore, this study was focused on CD147-promoting proinflammatory cytokine production and functions. Proinflammatory cytokine profiles were compared between CD147 expressing CCA cells and CD147 knockout cells (CD147 KO). Three cytokines, namely interleukin (IL)-6, IL-8, and granulocyte-monocyte colony-stimulating factor (GM-CSF), were dramatically diminished in CD147 KO clones. The involvement of the CD147-related cytokines in CCA invasion was established. CD147-promoted IL-6, IL-8, and GM-CSF secretions were regulated by NF-κB nuclear translocation, Akt activation, and p38 phosphorylation. CD147-fostering IL-6 production was dependent on soluble CD147, CD147 homophilic interaction, and NF-κB function. The overexpression of specific genes in CCA tissues compared to normal counterparts emphasized the clinical importance of these molecules. Altogether, CD147-potentiated proinflammatory cytokine production leading to CCA cell invasion is shown for the first time in the current study. This suggests that modulation of CD147-related inflammation might be a promising choice for advanced CCA treatment.

Antitumor effect of acanthoic acid against primary effusion lymphoma via inhibition of c-FLIP.

Acanthoic acid (AA) is an active substance that is extracted from Croton oblongifolius Roxb., a traditional plant in Thailand. The antiinflammatory effect of AA on NF-κB pathway has been exclusively reported, however, its anticancer effect is still lacking. PEL is a B cell lymphoma that is mostly found in HIV patients. The prognosis and progression of PEL patients are terribly poor with a median survival time less than 6 months, so the new effective treatment is urgently needed. In this study, we found that AA effectively inhibited PEL cell proliferation with IC50s at 120-130 µM in well-representative cells, while the IC50s of AA in PBMC were higher (>200 µM). AA increased percentages of Annexin V/PI positive cells, whereas adding of caspase inhibitor (Q-VD-OPh) prevented AA-induced cell death. The antiapoptotic protein, c-FLIP, was downregulated by AA which leading to the activation of caspase-8 and -3. Combination of AA and TRAIL dramatically enhanced apoptotic cell death. In PEL xenograft model, AA at the dose of 250 mg/kg effectively inhibited PEL tumor growth without detectable toxicities assessed by mice weight and appearance.

Mucin-producing hamster cholangiocarcinoma cell line, Ham-2, possesses the aggressive cancer phenotypes with liver and lung metastases.

Cholangiocarcinoma (CCA) is an aggressive bile duct cancer. Opisthorchis viverrini (O. viverrini) infection is a significant cause of CCA in the Greater Mekong subregion. Currently, there is no standard chemotherapeutic regimen for CCA. A unique hamster carcinogenesis model of O. viverrini-associated CCA was established. Molecular targets identified from the hamster CCA-comparative model are valuable for target identification and validation. Hamster CCA was induced by the administration of O. viverrini metacercariae and N-nitrosodimethylamine. Hamster-derived cancer cells were isolated and continuously cultured for more than 6 months. Ham-2 cell line was established and characterized in vitro and in vivo. Ham-2 exhibited chromosome hyperploidy. A comparative study with previously established cell line, Ham-1, demonstrated that Ham-2 acquired slower growth, higher adhesion, higher migration, and resistance to doxorubicin and 5-fluorouracil (5-FU). In BALB/c Rag-2/Jak3 double-deficient (BRJ) mice, Ham-2 subcutaneous transplantation formed mucin-producing cancers, which morphologically resemble human tubular cholangiocarcinoma. Intravenous-injected Ham-2 established the metastatic nodules in the lungs and livers of BRJ mice. Altogether, a new hamster cholangiocarcinoma cell line, Ham-2, which acquired more aggressive phenotypes in vitro and in vivo, was established. This cell line might be a valuable tool for comparative drug target identification and validation.

Five-(Tetradecyloxy)-2-furoic Acid Alleviates Cholangiocarcinoma Growth by Inhibition of Cell-cycle Progression and Induction of Apoptosis.

BACKGROUND/AIM: Cholangiocarcinoma (CCA), a biliary cancer, is a health problem worldwide. The major problem in CCA treatment presents limited options. To date, targeting cancer metabolism is a promising anti-cancer strategy. To elucidate the functional importance of lipid metabolism in CCA, de novo lipogenesis was inhibited using 5-(tetradecyloxy)-2-furoic acid (TOFA), an acetyl CoA carboxylase inhibitor. MATERIALS AND METHODS: Anti-proliferative effects of TOFA were determined both in vitro and in vivo. Its inhibitory effect on cell-cycle and apoptosis was investigated by flow cytometry and western blot analysis of relevant markers. RESULTS: TOFA inhibited CCA cell growth, induced cell-cycle progression accompanied by apoptosis in a dose-dependent manner. Induction of p21, and caspase-3, -8, and -9 cleavages, while down-regulation of cyclin B1 and cyclin D1 were observed in TOFA-treated cells. The therapeutic potential was demonstrated in vivo. CONCLUSION: De novo lipogensis is essential for CCA cell growth and is an alternative target for CCA treatment.

Establishment of Nude Mice Lacking NK Cells and Their Application for Human Tumor Xenografts.

OBJECTIVE: Nude mice are used as a recipient for human tumor cell xenografts. However, the success rate of xenotransplantation is unsatisfactory due to high natural killer (NK) activity. To overcome this limitation, we established nude mice with no NK cells, and compared the transplantation efficiency with that in nude mice. METHODS: BALB/c Nude Jak3-/- (Nude-J) mice were established by crossing BALB/c Nude mice and BALB/c Jak-3-/- mice. Hematopoietic malignant cell lines (BCBL-1 and Z138) were implanted subcutaneously to compare the tumorigenicity in Nude-J mice with Nude Rag-2/Jak3 double deficient (Nude RJ) mice and nude mice. RESULTS: Nude-J mice showed complete loss of NK and T lymphocytes, whereas B lymphocytes remained. Both BCBL-1 and Z138 human lymphoid malignant cell lines formed almost the same sizes of subcutaneous tumors in Nude-J and Nude RJ mice, whereas they formed no or only small tumors in nude mice. Splenocytes from Nude-J mice showed no cytotoxic activity in vitro. CONCLUSION: Nude-J mice can be a valuable tool for human tumor cell transplantation studies.
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