LM3 Cells Research Articles

Cisplatin induces apoptosis of hepatocellular carcinoma LM3 cells via down-regulation of XIAP.

Cisplatin is an important anti-cancer drug. However, the molecular mechanism of cisplatin on inhibition of the proliferation of liver cancer cells is unclear. Thus, we aimed to investigate the regulatory role of cisplatin on the growth and apoptosis of hepatoma LM3 cells. LM3 cells were treated with cisplatin (2 μmol/L) for 48 h. MTT assay, flow cytometry, and caspase-3 activity assay were used to detect the growth, proliferation and apoptosis of LM3 cells. Western blot was applied to detect the expression of the inhibitor of apoptosis protein, XIAP. siRNA was used to knockdown the level of XIAP followed by cisplatin (2 μmol/L) treatment, and then the apoptosis of LM3 cells was measured. The treatment of cisplatin significantly inhibited the growth but induced the apoptosis of LM3 cells. Cisplatin also downregulated the expression of XIAP. The downregulation of XIAP by using siRNA enhanced the apoptosis of LM3 cells induced by cisplatin. Downregulation of XIAP enhanced the proapoptotic effect of cisplatin on LM3 cells, suggesting that XIAP might be used as a potential target in the treatment of liver cancer.

Kindlin-2 promotes hepatocellular carcinoma invasion and metastasis by increasing Wnt/\u03b2-catenin signaling

BackgroundKindlin-2 is a member of the focal adhesion protein family that regulates invasion and metastasis in multiple malignancies; however, little is known about the role of Kindlin-2 in hepatocellular carcinoma (HCC) progression.MethodsImmunohistochemistry was used to investigate Kindlin-2 expression in 177 pairs of human HCC and adjacent liver tissue samples. The role of Kindlin-2 in the in vitro invasion and migration of HCC cell lines was evaluated in MHCC97H, LM3 and SMMC7721 cells. Microarray expression analysis was applied to explore the molecular mechanism through which Kindlin-2 promoted HCC progression. Quantitative real-time PCR and Western blotting were performed to verify the microarray results.ResultsHigh Kindlin-2 expression was found to significantly correlate with aggressive HCC clinicopathological features including tumor encapsulation, microvascular invasion, extrahepatic metastasis and poor prognosis. In vitro, Kindlin-2 knockout or knockdown inhibited HCC cell adhesion, migration and invasion, while ectopic Kindlin-2 expression promoted these processes. Importantly, Kindlin-2 activated Wnt/β-catenin signaling and increased β-catenin expression, especially levels of non-phosphorylated β-catenin, as well as two Wnt/β-catenin signaling pathway targets, Axin2 and MMP7. Kindlin-2 also induced a change in the expression profile of HCC cells, suggesting the cells underwent epithelial-mesenchymal transition. For example, the expression of the epithelial marker E-cadherin was downregulated, while the mesenchymal markers Vimentin, N-cadherin and Snail were upregulated.ConclusionKindlin-2 promotes HCC invasion, metastasis and epithelial-mesenchymal transition through Wnt/β-catenin signaling.

Characterization of Cladosporols from the Marine Algal-Derived Endophytic Fungus Cladosporium cladosporioides EN-399 and Configurational Revision of the Previously Reported Cladosporol Derivatives.

Four new cladosporol derivatives, cladosporols F-I (1-4), the known cladosporol C (5), and its new epimer, cladosporol J (6), were isolated and identified from the marine algal-derived endophytic fungus Cladosporium cladosporioides EN-399. Their structures were determined by detailed interpretation of NMR and MS data, and the absolute configurations were established on the basis of TDDFT-ECD and OR calculations. The configurational assignment of cladosporols F (1) and G (2) showed that the previously reported absolute configuration of cladosporol A and all the related cladosporols need to be revised from (4'R) to (4'S). Compounds 1-6 showed antibacterial activity against Escherichia coli, Micrococcus luteus, and Vibrio harveyi with MIC values ranging from 4 to 128 μg/mL. Compound 3 showed significant cytotoxicity against A549, Huh7, and LM3 cell lines with IC50 values of 5.0, 1.0, and 4.1 μM, respectively, and compound 5 showed activity against H446 cell line with IC50 value of 4.0 μM.

The effect of immune checkpoint inhibitors on lung metastases of osteosarcoma

Background/purposeThe prognosis of patients with metastases remains unsatisfactory in certain pediatric solid tumors. In this study, we evaluated the efficacy of immune checkpoint inhibitors against such metastases using a murine model of osteosarcoma. MethodsMurine osteosarcoma LM8 cells were transplanted subcutaneously into C3H mice. The primary tumor lesion was surgically resected 11 days after transplantation. Two hundred micrograms of three antibodies (anti-PD-1, anti-PD-L1, and anti-OX-86) or an isotype antibody were administered intraperitoneally on post-transplantation days 11, 14, 18, and 21. Survival curves were plotted by the Kaplan-Meier method and compared with the log-rank test. Computed tomography (CT) scans were performed on day 11 after tumor transplantation (pre-therapy) and on day 25 (post-therapy). For pathology, 3 mice from each group were euthanized on days 11, 22, and 33 after tumor transplantation. ResultsThe antibody-treated group had a significantly longer survival time compared with the control group (p = 0.002). Both the CT scan and pathological results revealed suppression of metastatic tumor proliferation in the treatment group as compared with the control group. ConclusionsThese results suggest that immune checkpoint inhibitors may be an innovative therapy for lung metastases of advanced pediatric solid tumors.

MicroRNA-98-5p Inhibits Cell Proliferation and Induces Cell Apoptosis in Hepatocellular Carcinoma via Targeting IGF2BP1.

Some microRNAs (miRs) have been demonstrated to play promoting or tumor-suppressing roles in the development and progression of hepatocellular carcinoma (HCC). However, the regulatory mechanism of miR-98-5p in HCC still remains largely unclear. In the present study, our data showed that miR-98-5p was significantly downregulated in 84 cases of HCC tissues compared to the matched adjacent nontumor tissues. In addition, downregulation of miR-98-5p was associated with tumor size, portal vein tumor embolus, node metastasis, and clinical stage in HCC. HCC patients with low expression of miR-98-5p showed a shorter survival time compared with those with high miR-98-5p levels. Moreover, the expression of miR-98-5p was also reduced in HCC cell lines (HepG2, Hep3B, LM3, and SMCC7721) compared to the normal liver cell line THLE-3. Overexpression of miR-98-5p significantly decreased LM3 cell growth by inducing cell cycle arrest at the G1 stage and cell apoptosis. Insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) was then identified as a novel target gene of miR-98-5p, and its protein expression was negatively regulated by miR-98-5p in LM3 cells. Overexpression of IGF2BP1 eliminated the effects of miR-98-5p overexpression on the proliferation, cell cycle, and apoptosis of LM3 cells. Finally, we found that IGF2BP1 was upregulated in HCC, and its expression was negatively correlated to miR-98-5p levels. In summary, we demonstrate that miR-98-5p could inhibit HCC cell proliferation while inducing cell apoptosis, partly at least, via inhibition of its target gene IGF2BP1, and we suggest that miR-98-5p may become a promising therapeutic candidate for HCC treatment.

Abstract 842: Breast cancer: Relationship between GPC3 and EMT process

Abstract Epithelial-mesenchymal transition (EMT) is a process whereby epithelial cells acquire mesenchymal fibroblast-like properties and display reduced intracellular adhesion and increased motility. Glypican-3 (GPC3) is a heparan sulfate proteoglycan associated with cancer. We have established that GPC3 regulates the EMT process underwent by breast cancer cells. However, cellular and molecular events underlying this modulation are not completely described. At the present work we analyzed the effect of GPC3 on cell adhesion and on the expression of molecules associated with this property. In addition, molecular regulators of EMT were also studied. We employed a murine breast cancer cell line (LM3: GPC3-) as well as human lines (MDA-MB231: GPC3- and MCF-7: GPC3+). We have blocked GPC3 expression in MCF-7 cells, while it was overexpressed in MDA-MB231 and LM3 ones. It is know that EMT depends on a reduction in cell adhesion. We determined that GPC3 overexpression induced an increase in the homotypic adhesion of MDA-MB231 and LM3 cells, although GPC3 silencing did not lead significant changes in the organization of MCF-7 spheroids. GPC3 was also able to modulate the adhesion to extracellular matrix components. GPC3 overexpressing cells were more adherent to fibronectin than controls, but no changes were observed in their adhesion to laminin. E-Cadherin, a molecule involved in homotypic adhesion, is considered a suppressor of invasion and growth of many epithelial cancers. Here, we have corroborated by WB that GPC3 overexpression induces the E-Cadherin upregulation in LM3 and MDA-MB231 cells, whereas GPC3 silencing inhibits the expression of this epithelial marker in MCF-7 cells. We also confirmed the called "cadherin switch", since N-Cadherin was downregulated in GPC3 overexpressing cells and upregulated in GPC3 silenced ones. β-Catenin is a key downstream effector in the Wnt pathway. We confirmed that GPC3 expressing breast cancer cells exhibit a canonical Wnt signaling inhibition. β-Catenin also activates Slug. Similar to Snail, Slug binds to the E-Cadherin promoter to repress transcription. By WB and qPCR we demonstrated that GPC3 induces a decrease in Slug and Snail levels. Surprisingly, Twist expression was higher in GPC3 expressing cells. It was reported that Slug is able to represses integrins expression. However, β1-Integrin levels were decreased in GPC3 overexpressing MDA-MB231 cells, while they were increased in GPC3 blocked MCF-7 sublines. Our results suggest the key role of GPC3 in the EMT regulation. GPC3 would modulate cell adhesion and expression of E-Cadherin, N-Cadherin and β1-Integrin. In addition, GPC3 would regulate the expression of different EMT molecules (as Slug, Snail and Twist) as well as signaling pathways involved in that process (like canonical Wnt). The set and balance of these regulations would result in the reversal of EMT, reinforcing the function of GPC3 as an inhibitor of mammary tumor progression. Citation Format: Lilian F. Castillo, Gisela V. Novack, Elisa Bal de Kier Joffé, María G. Peters. Breast cancer: Relationship between GPC3 and EMT process [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 842. doi:10.1158/1538-7445.AM2017-842

Abstract 4701: Blocking FSTL1 ameliorates immunity against osteosarcoma

Abstract Purposes: Osteosarcoma is a rare malignant tumor of bone in children and young adults, and it is very hard to successfully control the systemic metastases and life-threatening health problems more than a decade after the completion of the care due to the intensiveness of the conventional treatments including surgical resection of the primary tumor and chemotherapy. Therefore, non/low-toxic but advanced treatments have been expected for a long while. We recently found that a BMP family FSTL1 is highly and functionally expressed in murine and human osteosarcoma cell lines, and systemically expands pluripotent mesenchymal stem/stromal cells (MSCs), which play a central role in amplification of tumor heterogeneity that is consequence of reciprocal evolution together with the tumor microenvironment, including induction of immune suppression and dysfunction, desmoplasia, and angiogenesis. FSTL1 and its induced MSCs also directly confer higher invasive and metastatic properties on tumor cells leading to further cancer progression. In this study, we evaluated antitumor efficacy induced by anti-FSTL1 mAb on murine osteosarcoma models, and also compared the therapeutic efficacy with those induced by immune checkpoint inhibitory mAbs (ICIs). Results: We used two syngeneic tumor models that were subcutaneously implanted with murine osteosarcoma NHOS cells in BALB/c mice, or LM3 cells in C3H mice. These mice were intraperitoneally injected with anti-FSTL1 mAbs (10mg/kg) twice on day 5 and day 8 after tumor implantation, and were sacrificed for immunological analysis of tumor-infiltrating cells, spleen cells and bone marrow cells on day 15. In both osteosarcoma models, CD45- MSCs as well as other immunosuppressive CD4+Foxp3+ Tregs and CD11b+Gr1+ MDSCs systemically increased, indicating impairment of antitumor immunity in the mice. Treatment with the anti-FSTL1 mAb significantly induced potent tumor-specific CTLs through reduction of MSCs, resulting in tumor-free in some of the treated mice. Although the antitumor efficacy was similar to those induced by ICIs, the mode of action was totally distinct because MSCs and the MSC-inducible exhausted CD8+PD1+Tim3+ T cells were reduced only in the anti-FSTL1-treated mice, but not in the ICI-treated mice. Conclusions: These results suggest that blocking FSTL1 properly reprograms immunity toward elimination of osteosarcoma by reducing the tumor-supportive MSCs. Anti-FSTL1 mAb may be a promising drug for treating osteosarcoma more moderately but effectively in clinical settings. Citation Format: Chie Kudo-Saito, Yamato Ogiwara, Kazunori Aoki. Blocking FSTL1 ameliorates immunity against osteosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4701. doi:10.1158/1538-7445.AM2017-4701

Motile hepatocellular carcinoma cells preferentially secret sugar metabolism regulatory proteins via exosomes.

Exosomes are deliverers of critically functional proteins, capable of transforming target cells in numerous cancers, including hepatocellular carcinoma (HCC). We hypothesize that the motility of HCC cells can be featured by comparative proteome of exosomes. Hence, we performed the super-SILAC-based MS analysis on the exosomes secreted by three human HCC cell lines, including the non-motile Hep3B cell, and the motile 97H and LM3 cells. More than 1400 exosomal proteins were confidently quantified in each MS analysis with highly biological reproducibility. We justified that 469 and 443 exosomal proteins represented differentially expressed proteins (DEPs) in the 97H/Hep3B and LM3/Hep3B comparisons, respectively. These DEPs focused on sugar metabolism-centric canonical pathways per ingenuity pathway analysis, which was consistent with the gene ontology analysis on biological process enrichment. These pathways included glycolysis I, gluconeogenesis I and pentose phosphate pathways; and the DEPs enriched in these pathways could form a tightly connected network. By analyzing the relative abundance of proteins and translating mRNAs, we found significantly positive correlation between exosomes and cells. The involved exosomal proteins were again focusing on sugar metabolism. In conclusion, motile HCC cells tend to preferentially export more sugar metabolism-associated proteins via exosomes that differentiate them from non-motile HCC cells.

Abstract 4585: Radiation combined with checkpoint blockades enhanced antitumor efficacy for osteosarcoma

Abstract Osteosarcoama is one of the common malignancies at bone in children and adolescent. Recent study demonstrated that combination of anti-PD-L1 and anti-CTLA-4 antibodies provided grate control of metastatic osteosarcoma. However, a strong local treatment strategy including surgery or high precision radiation therapy is necessary to elucidate osteosarcoma. Radiation therapy plays an important role in local control for malignant tumors but previous studies demonstrated that radiation enhanced immune response in which not only local tumor regression at irradiated sites but also regression of metastatic tumor outside the radiation field were observed. Although this phenomenon, so called the abscopal effect, is rarely seen, recent studies demonstrated that combination of X-ray irradiation with checkpoint blockades provided higher probability of the abscopal effect for some kind of tumors. However, the effect of x-ray irradiation combined with checkpoint blockade on the abscopal effect for osteosarcoma has been totally unknown. We investigated whether local X-ray irradiation combined with the anti-PD-L1 and anti-CTLA-4 antibodies enhances local and distant antitumor efficacy for osteosarcoma. LM8 mouse osteosarcoma cells were inoculated into both legs of C3H mice. Mice were treated by 10 Gy X-ray irradiation alone to the tumor in the one side leg (RAD group) at day 12, 150 ug of anti-PD-L1 and anti-CTLA-4 antibodies (P1C4 group) at days 9, 12, and 15, or those combination (COMB group). Administration of anti-PD-L1 and anti-CTLA-4 antibodies provided tumor growth delay or complete response at day 37 for about 20% of the mice. X-ray irradiation strongly inhibited tumor growth at irradiated tumor but not in unirradiated tumor. On the other hand, the combination therapy provided the strongest tumor growth inhibition not only at irradiated tumor but also at unirradiated tumor for about 89% of the mice. Accordingly, lung metastasis in mice in COMB group was strongly reduced by 97% with the significant survival benefit compared with the mice in P1C4 group. Flow cytometric analysis revealed that mice in COMB group significantly recruited CD8 tumor infiltrating lymphocytes with moderate reduction of regularly T cells (Tregs), thereby increasing the CD8/Treg ratio. Furthermore, quantitative real time PCR showed significant induction of PD-L1 on irradiated LM8 cells in vitro. The radiation-induced upregulations of PD-L1, B7-1, and B7-2, the ligands of CTLA-4, were also confirmed by flow cytometry, indicating that the enhanced efficacy anti-PDL1 and CTLA-4 antibodies may associate with these upregulations by radiation. These results suggest that X-ray irradiation contributes to the enhancement of the efficacy for the distant metastasis as well as local control in the treatment of anti-PD-L1 and anti-CTLA-4 antibodies for osteosarcoma. Our data provide a rational to establish a new therapeutic strategy and start up a clinical trial for osteosarcoma. Citation Format: Yutaka Takahashi, Tomohiro Yasui, Keisuke Tamari, Kazumasa Minami, Masahiko Koizumi, Yuji Seo, Fumiaki Isohashi, Keisuke Ohtani, Ryosuke Kambe, Kazuhiko Ogawa. Radiation combined with checkpoint blockades enhanced antitumor efficacy for osteosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4585. doi:10.1158/1538-7445.AM2017-4585

Inhibitory effects of low-intensity pulsed ultrasound sonication on the proliferation of osteosarcoma cells.

To date, there is limited data on the biological effects of low-intensity pulsed ultrasound (LIPUS) on primary malignant bone tumors. The purpose of the present study was to investigate the antitumor effects of LIPUS on osteosarcoma cells. The effects of LIPUS on cell viability, induction of apoptosis, mitochondrial membrane potential and intracellular signaling molecules in the LM8 osteosarcoma cell line were investigated. LIPUS inhibited cell viability (P=0.0022) and mitochondrial membrane potential (P=0.0019) in LM8 cells. Flow cytometry analysis and terminal deoxynucleotidyl transferase dUTP nick end labeling staining revealed significantly higher numbers of apoptotic (P<0.0001) and necrotic cells (P=0.0091) compared with cells without treatment. LIPUS treatment significantly increased phosphorylated Akt (P<0.0001) and IκBα (P=0.0001) levels, and reduced phosphorylated mitogen-activated protein kinase 7 (P<0.0001) and phosphorylated checkpoint kinase 1 (P=0.0008) levels. These results suggest that LIPUS is a non-invasive adjuvant therapy that is able to inhibit cellular proliferation in osteosarcoma cells.

Bisphenol A Inhibits Cell Proliferation and Reduces the Motile Potential of Murine LM8 Osteosarcoma Cells.

The aim of this study was to examine the effect of bisphenol A (BPA) on the proliferation and motility potential of murine LM8 osteosarcoma cells. LM8 cells were treated for 3 days with or without 80 μM BPA. The effect of BPA on cell proliferation was determined by DNA measurement in the cultures and 5-bromo-2'-deoxyuridine (BrdU) incorporation study. Ethanol-fixed cells were stained with hematoxylin-eosin (H&E) to visualize cell morphology. Cell motility was assayed using inserts with uncoated membranes in invasion chambers. Expression of cell division cycle 42 (CDC42) was determined by immunofluorescence staining and western blotting. BPA reduced the DNA content of cultures and the number of BrdU-positive cells. BPA induced a change in morphology from cuboidal with multiple filopodia on the cell surface to spindle-shaped with a smooth cell surface. BPA-treated cells expressed less CDC42 and were less motile than untreated cells. BPA inhibited DNA replication and cell proliferation. BPA inhibited filopodia formation and motile potential by inhibiting CDC42 expression in LM8 cells.

Actinidia chinensis Planch root extract inhibits cholesterol metabolism in hepatocellular carcinoma through upregulation of PCSK9

Actinidia chinensis Planch root extract (acRoots) is a traditional Chinese medicine with anti-tumor efficacy. To investigate the mechanisms responsible for this activity, we examined the effects of acRoots on cholesterol metabolism in hepatocellular carcinoma (HCC). mRNA chip analysis was used to identify the metabolic genes regulated by acRoots. The effects of acRoots on cholesterol synthesis and uptake were evaluated by measuring intracellular cholesterol levels and 3,3′-dioctadecylindocarbocyanine-labeled low-density lipoprotein (Dil-LDL) uptake. Expression of metabolic genes was analyzed using quantitative reverse transcription PCR, western blotting, and flow cytometry. acRoots reduced the viability of LM3 and HepG2 cells at 5 mg/mL and HL-7702 cells at 30 mg/mL. Gene expression profiling revealed that treatment with acRoots altered expression of genes involved in immune responses, inflammation, proliferation, cell cycle control, and metabolism. We also confirmed that acRoots enhances expression of PCSK9, which is important for cholesterol metabolism. This resulted in decreased LDL receptor expression, inhibition of LDL uptake by LM3 cells, decreased total intracellular cholesterol, and reduced proliferation. These effects were promoted by PCSK9 overexpression and rescued by PCSK9 knockdown. Our data demonstrate that acRoots is a novel anti-tumor agent that inhibits cholesterol metabolism though a PCSK9-mediated signaling pathway.

Pulsed Electromagnetic Field Stimulation Promotes Anti-cell Proliferative Activity in Doxorubicin-treated Mouse Osteosarcoma Cells.

We aimed to investigate the synergistic effects of pulsed electromagnetic field (PEMF) and doxorubicin therapy in a mouse osteosarcoma cell line (LM8 cells) in vitro. The effects of PEMF (5 mT, 200 Hz) of different durations and doxorubicin on the proliferative activity of LM8 cells were measured by the MTT assay. Apoptotic-related factors such as cell-cycle phase, mitochondrial membrane potential, and caspase 3/7 activity were investigated using 4',6-diamidino-2-phenylindole staining and apoptosis kits. Identification of intracellular signaling molecules induced by the combination was comprehensively explored using a stress and apoptosis-related protein array kit. PEMF enhanced the inhibition of cell proliferation mediated by doxorubicin but did not affect the cell cycle, mitochondrial membrane potential, or doxorubicin-induced G2/M arrest. The combination of PEMF and doxorubicin altered a few signaling molecules. PEMF tended to reduce the doxorubicin-induced decrease of phosphorylated BAD, while reducing the increased expression of total IĸB and phosphorylated-CHK1 induced by doxorubicin. Our results indicate that combination of PEMF and doxorubicin could be a novel chemotherapeutic strategy.

Protein phosphatase 2A Cα regulates proliferation, migration, and metastasis of osteosarcoma cells

Osteosarcoma is the most frequent primary bone tumor. Serine/threonine protein phosphatase 2A (PP2A) participates in regulating many important physiological processes, such as cell cycle, growth, apoptosis, and signal transduction. In this study, we examined the expression and function of PP2A Cα in osteosarcoma cells. PP2A Cα expression was expected to be higher in malignant osteosarcoma tissues. PP2A Cα expression level and PP2A activity was higher in malignant osteosarcoma LM8 cells compared with that in primary osteoblasts and in the osteoblast-like cell line MC3T3-E1. Okadaic acid, an inhibitor of PP2A, reduced cell viability and induced apoptosis in LM8 cells. PP2A Cα-knockdown LM8 cells (shPP2A) exhibited less striking filopodial and lamellipodial structures than that in original LM8 cells. Focal adhesion kinase phosphorylation and NF-κB activity decreased in shPP2A-treated cells. Sensitivity to serum deprivation-induced apoptosis increased in shPP2A-treated cells, accompanied by a lower expression level of anti-apoptotic BCL-2 in these cells. Reduction of PP2A Cα resulted in a decrease in the migration ability of LM8 cells in vitro. Reduction in PP2A Cα levels in vivo suppressed proliferation and metastasis in LM8 cells. PP2A Cα expression was also higher in human osteosarcoma MG63 and SaOS-2 cells than that in primary osteoblasts and MC3T3-E1 cells, and reduction in PP2A Cα levels suppressed the cell proliferation rate and migration ability of MG63 cells. These results indicate that PP2A Cα has a critical role in the proliferation and metastasis of osteosarcoma cells; therefore, its inhibition could potentially suppress the malignancy of osteosarcoma cells.

Influence of ezrin-shRNA in combination with HSP70 on the apoptosis and proliferation of osteosarcoma cells.

Ezrin and heat shock protein (HSP)70 have been reported to regulate cell apoptosis and tumor development of osteosarcoma. However, there has not been reported the synergy effect of knocking down ezrin and overexpressing HSP70. In the present study, two vectors, pGFP-V-RS-shRNA and pGFP-V-RS-shRNA-HSP70, were constructed and transfected into LM8 cells [denoted as small hairpin (sh)RNA group and dual group, respectively]. The apoptosis rates in these two transfected groups were significantly higher than those in the control group (empty vector) (P=0.036), while significantly lower proliferation rates were observed in these two groups (P=0.023). The cytotoxic T lymphocyte activity on target LM8 tumor cells in the dual group was significantly higher than in other groups, with cytotoxicity as high as 55.56±2.10%. Further studies revealed that the transfection of ezrin-shRNA/HSP70 also suppressed tumor formation in vivo in nude mice. A lower cluster of differentiation (CD)4/CD8 ratio was detected in the tumor formed by injecting cells in the dual group (P=0.006). Furthermore, the serum level of interleukin-4 in the dual group was significantly decreased, while the serum level of interferon-γ was significantly increased, compared with the other two groups (P=0.004). Simultaneously knocking down ezrin and overexpressing HSP70 promotes cellular apoptosis and suppresses the proliferation of osteosarcoma cells in vitro, and enhances the tumor killing effects of HSP70-induced immune killing.

PU-H71, a novel Hsp90 inhibitor, as a potential cancer-specific sensitizer to carbon-ion beam therapy.

PU-H71, a heat shock protein 90 (Hsp90) inhibitor, has yielded therapeutic efficacy in many preclinical models and is currently in clinical trials. Carbon-ion radiotherapy (CIRT) has provided successful tumor control; however, there is still room for improvement, particularly in terms of tumor-specific radiosensitization. The Hsp90 inhibitor PU-H71 has been shown to sensitize tumor cells to X-ray radiation. A murine osteosarcoma cell line (LM8) and a normal human fibroblast cell line (AG01522) were treated with PU-H71 before X-ray, 14- or 50-keV/µm carbon-ion beam (C-ion) irradiation. Cell survival and protein expression were evaluated with colony formation and western blot, respectively. Treatment with PU-H71 alone was shown to be non-toxic to both cell lines; however, PU-H71 was shown to significantly sensitize LM8 cells to not only X-ray, but also to C-ion irradiation, while only a minimal sensitizing effect was observed in AG01522 cells. PU-H71 treatment was found to suppress the protein expression levels of Rad51 and Ku70, which are associated with the homologous recombination pathway and the non-homologous end-joining pathway of double-strand break repair. The findings reported here suggest that PU-H71 could be a promising radiosensitizer for CIRT.

Abstract 3536: Rapamycin in combination with gemcitabine reduced viability, growth and metastasis of the murine osteosarcoma cell line LM8 in vitro and in vivo

Abstract Introduction Rapamycin is an mTOR inhibitor with anti-tumor activity in several human cancers. However, the efficacy of this compound in osteosarcoma has not been fully elucidated. It acts downstream in the PI3K pathway and impedes cell growth, cell proliferation, cell motility, cell survival, protein synthesis and transcription. Here, we assessed the anti-tumor activity of rapamycin. We had previously reported that gemcitabine reduced viability, growth and metastasis of osteosarcoma cell lines. In this study, we investigated whether gemcitabine could enhance the anti-tumor effects of rapamycin in the murine osteosarcoma cell line LM8 in vitro and in vivo. Results In the LM8 cell line, rapamycin synergized with gemcitabine to reduce cell proliferation as determined by the WST assay and to induce apoptosis as analyzed by flow cytometry using the annexin V and 7AAD staining method. Rapamycin at doses &amp;gt;10 μM showed significant cytotoxicity. By combining with gemcitabine, the amount of rapamycin required for apoptosis was reduced to 1 μM. Autophagy was evaluated with the Cyto-ID® Autophagy detection kit. Not only rapamycin but also gemcitabine induced autophagy in the LM8 cell line. However, no synergistic effects were observed. Cell mobility was evaluated by migration assay using a trans-well system (LM8 cells in the upper layer and conditioned medium in the lower chamber). Rapamycin synergized with gemcitabine to reduce LM8 cell mobility. In C3H mice inoculated subcutaneously with LM8, we observed frequent metastasis to the lung. Treatment of the mice with rapamycin reduced the size of the primary tumor; elevated levels of apoptosis were observed. Moreover, rapamycin in combination with gemcitabine had additive effects on the primary tumor and almost no metastatic lesions in the lung were seen. Consequently, combination therapy prolonged the overall survival of tumor-bearing mice. Conclusions Rapamycin synergized with gemcitabine to reduce cell proliferation, and to induce apoptosis, autophagy and cell mobility of the LM8 murine osteosarcoma cell line in vitro. Rapamycin in combination with gemcitabine exhibited synergistic antitumor effects. The combination prolonged overall survival and reduced the number of lung metastases in osteosarcoma-bearing mice. Rapamycin has been used to treat osteosarcoma in clinical trials and gemcitabine combined with docetaxel chemotherapy has shown good clinical results. Rapamycin combined with gemcitabine chemotherapy might be a novel and promising therapeutic approach to the treatment of osteosarcoma. Citation Format: Takashi Ando, Jiro Ichikawa, Nobutaka Sato, Tetsuro Ohba, Kensuke Koyama, Tetsuo Hagino, Hirotaka Haro. Rapamycin in combination with gemcitabine reduced viability, growth and metastasis of the murine osteosarcoma cell line LM8 in vitro and in vivo. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3536.

MiR-503 inhibits hepatocellular carcinoma cell growth via inhibition of insulin-like growth factor 1 receptor.

MicroRNAs (miRs) have been demonstrated to play key roles in the development and progression of hepatocellular carcinoma (HCC). However, the regulatory mechanism of miR-503 in HCC has not been fully uncovered. In this study, we found that miR-503 was significantly downregulated in HCC tissues compared to nontumorous liver tissues. Moreover, lower miR-503 levels were associated with the malignant progression of HCC, and the expression of miR-503 was also decreased in several common HCC cell lines compared to normal human liver cell line THLE-3. Overexpression of miR-503 inhibited proliferation but induced apoptosis of LM3 and HepG2 cells. Bioinformatical analysis and luciferase reporter assay further identified insulin-like growth factor 1 receptor (IGF-1R) as a novel target of miR-503 in 293T cells. Moreover, overexpression of miR-503 led to a significant decrease in the protein levels of IGF-1R, while knockdown of miR-503 enhanced its protein levels in LM3 and HepG2 cells. Besides, overexpression of IGF-1R reversed the effects of miR-503-mediated HCC cell proliferation and apoptosis, indicating that IGF-1R acts as a downstream effector of miR-503 in HCC cells. Furthermore, IGF-1R was found to be significantly upregulated in HCC tissues compared to nontumorous liver tissues. In addition, the mRNA levels of IGF-1R were inversely correlated to the miR-503 levels in the HCC tissues. Thus, we demonstrate that miR-503 inhibits the proliferation and induces the apoptosis of HCC cells, partly at least, by directly targeting IGF-1R, and suggest that IGF-1R may serve as a promising target for the treatment of HCC.

CCL11-induced eosinophils inhibit the formation of blood vessels and cause tumor necrosis.

We previously demonstrated that IL-18 and CCL11 were highly expressed in an NFSA tumor cell line that showed limited angiogenesis and severe necrosis. However, IL-18 was not responsible for the immune cell accumulation and necrosis. Here, we attempted to clarify the relevance of CCL11 in angiogenesis and tumor formation. We established CCL11-overexpressing MS-K cell clones (MS-K-CCL11) to assess the role of CCL11 in immune cell accumulation and angiogenesis. The MS-K-CCL11 cells did not form tumors in mice. MS-K-CCL11-conditioned medium (CM) and recombinant CCL11 induced macrophage and eosinophil differentiation from bone marrow cells. The MS-K-CCL11-CM effectively recruited the differentiated eosinophils. Furthermore, the eosinophils damaged the MS-K, NFSA and endothelial cells in a dose-dependent manner. Administration of an antagonist of CCR3, a CCL11 receptor, to NFSA tumor-bearing mice restored the blood vessel formation and blocked the eosinophil infiltration into the NFSA tumors. Furthermore, other CCL11-overexpressing LM8 clones were established, and their tumor formation ability was reduced compared to the parental LM8 cells, accompanied by increased eosinophil infiltration, blockade of angiogenesis and necrosis. These results indicate that CCL11 was responsible for the limited angiogenesis and necrosis by inducing and attracting eosinophils in the tumors.

TR4 nuclear receptor enhances the cisplatin chemo-sensitivity via altering the ATF3 expression to better suppress HCC cell growth.

Early studies indicated that TR4 nuclear receptor (TR4) may play a key role to modulate the prostate cancer progression, its potential linkage to liver cancer progression, however, remains unclear. Here we found that higher TR4 expression in hepatocellular carcinoma (HCC) cells might enhance the efficacy of cisplatin chemotherapy to better suppress the HCC progression. Knocking down TR4 with TR4-siRNA in HCC Huh7 and Hep3B cells increased cisplatin chemotherapy resistance and overexpression of TR4 with TR4-cDNA in HCC LM3 and SNU387 cells increased cisplatin chemotherapy sensitivity. Mechanism dissection found that TR4 might function through altering the ATF3 expression at the transcriptional level to enhance the cisplatin chemotherapy sensitivity, and interrupting ATF3 expression via ATF3-siRNA reversed TR4-enhanced cisplatin chemotherapy sensitivity in HCC cells. The in vivo HCC mouse model using xenografted HCC LM3 cells also confirmed in vitro cell lines data showing TR4 enhanced the cisplatin chemotherapy sensitivity. Together, these results provided a new potential therapeutic approach via altering the TR4-ATF3 signals to increase the efficacy of cisplatin to better suppress the HCC progression.