Human Fibrosarcoma HT1080 Cells Research Articles

Abstract 3161: TPX-0005, an ALK/ROS1/TRK inhibitor, overcomes multiple resistance mechanisms by targeting SRC/FAK signaling

Abstract ALK, ROS1, and TRK inhibitors have achieved marked efficacy in treating cancer patients expressing abnormal ALK, ROS1 or NTRK genes. However, the initial success of these therapies is rapidly overshadowed with the development of acquired resistance. In addition, approximately 30-40% of ALK + or ROS1+ NSCLC patients fail to respond to initial crizotinib treatment, representing intrinsic resistance. Target gene amplification, acquired resistance mutations, bypass signaling, epithelial-mesenchymal transition (EMT) and metastasis are the common resistance mechanisms. None of the current ALK, ROS1 or TRK inhibitors can overcome bypass or EMT-based resistance when applied as a single agent therapy. TPX-0005 is a potent kinase inhibitor against WT and mutated ALK, ROS1 and TRK family kinases, especially the clinically significant solvent front mutations. At clinically relevant concentrations, TPX-0005 also inhibits JAK2, SRC and FAK that are important targets in modulating multiple resistance mechanisms. H2228 lung cancer cell line endogenously expresses EML4-ALK v3 and was reported to be resistant to ALK inhibitor TAE684. Upregulation of the bypass signaling kinase EGFR, EMT marker vimentin, and cancer stem-like marker CD44 was reported in H2228 cells, likely leading to intrinsic resistance to ALK inhibitors. In the anti-proliferation assay, both crizotinib and ceritinib were resistant in H2228 cells with IC50 values around 1000 nM, however, TPX-0005 was able to overcome the intrinsic resistance with an IC50 of 100 nM. In H2228 cells, TPX-0005 suppressed the phosphorylation of SRC, FAK and paxillin with IC50 values in a range of 80-100 nM, and downregulated the expression of EGFR, CD44, and vimentin with IC50 values around 100 nM. In addition, TPX-0005 inhibited the phosphorylation of the oncogenic transcription/translation factor YB-1 with an IC50 value around 100 nM in H2228 cells. YB-1 is involved in many aspects of gene expression control that lead to tumor cell growth and drug resistance, including modulation of EGFR upregulation, EMT, and cancer stemness. Therefore, it was postulated that inhibition of SRC/FAK by TPX-0005 suppressed the phosphorylation of YB-1, leading to the downregulation of EGFR, CD44 and vimentin, and eventually to anti-proliferation effect on H2228 cells. TPX-0005 demonstrated an in vitro anti-metastatic activity by inhibiting cell migration in both H2228 cells and HT1080 human fibrosarcoma cells. Taken together, the potent kinase inhibitory activities against SRC/FAK signaling provide a unique polypharmacology profile to TPX-0005 for combatting multiple resistance mechanisms simultaneously, including a broad spectrum of acquired mutations, bypass signaling, EMT, cancer stemness, and metastasis. Citation Format: Dayong Zhai, Wei Deng, John Huang, Evan Rogers, J. Jean Cui. TPX-0005, an ALK/ROS1/TRK inhibitor, overcomes multiple resistance mechanisms by targeting SRC/FAK signaling [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 3161. doi:10.1158/1538-7445.AM2017-3161

Abstract 1044: Development of a miRNA-based signature to predict human cancer metastasis

Abstract Metastasis is the primary cause of death for cancer patients, and invasive cancer cell migration is required at multiple steps during metastasis (e.g. intravasation and extravasation). Since microRNAs (miRNAs) have been implicated as key regulators of metastatic spread of cancer cells, we sought to develop a miRNA signature that can be used to predict cancer metastasis. We hypothesized that the miRNAs that are functionally required for invasive cell migration could serve as biomarkers to predict human cancer metastasis. We developed an intravital imaging-based approach combined with NGS to screen for miRNAs that are required for invasive cell migration of human HT1080 fibrosarcoma cells. The screen-identified miRNAs were validated in a panel of in vitro and in vivo assays for invasive migration. Microarray analysis identified genes downregulated in transfected HT1080 cells. Publically available databases were used to correlate the expression of screen-identified miRNAs and the progression of multiple human cancers. We used a qRT-PCR approach combined with machine learning to develop a miRNA signature to predict metastasis in a 66-patient cohort of prostate cancer. We identified over twenty novel miRNAs that regulate directional cancer invasion. Microarray analysis in HT1080 cells revealed that the altered expression of metastasis-regulating miRNAs is associated with 50% reduction in gene expression of migration and adhesion gene network components such as integrin a4, CDC42, and transgelin). We evaluated the potential of screen-identified miRNAs to serve as biomarkers to predict cancer metastasis. Using publically available databases, we found that majority of screen-identified miRNAs are dysregulated in multiple human cancer types (e.g. prostate, breast, ovarian and lung) and the expression of these miRNAs correlate directly with patient disease progression. We analyzed the expression of the top two screen-identified miRNAs in plasma samples from the PCa patient cohort. A signature was generated using a weighted K-nearest neighbor algorithm that provided a ROC area under the curve of 0.79 for predicting metastatic disease. We identified a panel of novel metastasis-regulating miRNAs that is functionally involved in human cancer metastasis. These miRNAs have the potential to serve as both biomarkers to predict metastasis and potentially as therapeutic targets to block metastasis. Citation Format: Lian Willetts, Konstantin Stoletov, Juan Jovel, Emma Woolner, John D. Lewis. Development of a miRNA-based signature to predict human cancer metastasis [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 1044. doi:10.1158/1538-7445.AM2017-1044

Cellular uptake of proMMP-2:TIMP-2 complexes by the endocytic receptor megalin/LRP-2

Matrix metalloproteinases (MMPs) are regulated at multiple transcriptional and post-transcriptional levels, among which receptor-mediated endocytic clearance. We previously showed that low-density lipoprotein receptor-related protein-1 (LRP-1) mediates the clearance of a complex between the zymogen form of MMP-2 (proMMP-2) and tissue inhibitor of metalloproteinases, TIMP-2, in HT1080 human fibrosarcoma cells. Here we show that, in BN16 rat yolk sac cells, proMMP-2:TIMP-2 complex is endocytosed through a distinct LRP member, megalin/LRP-2. Addition of receptor-associated protein (RAP), a natural LRP antagonist, caused accumulation of endogenous proMMP-2 and TIMP-2 in conditioned media. Incubation with RAP also inhibited membrane binding and cellular uptake of exogenous iodinated proMMP-2:TIMP-2. Moreover, antibodies against megalin/LRP-2, but not against LRP-1, inhibited binding of proMMP-2:TIMP-2 to BN16 cell surface. BIAcore analysis confirmed direct interaction between the complex and megalin/LRP-2. Conditional renal invalidation of megalin/LRP-2 in mice resulted in accumulation of proMMP-2 and TIMP-2 in their urine, highlighting the physiological relevance of the binding. We conclude that megalin/LRP-2 can efficiently mediate cell-surface binding and endocytosis of proMMP-2:TIMP-2 complex. Therefore megalin/LRP-2 can be considered as a new actor in regulation of MMP-2 activity, an enzyme crucially involved in many pathological processes.

Identification of Small Molecule Translesion Synthesis Inhibitors That Target the Rev1-CT/RIR Protein-Protein Interaction.

Translesion synthesis (TLS) is an important mechanism through which proliferating cells tolerate DNA damage during replication. The mutagenic Rev1/Polζ-dependent branch of TLS helps cancer cells survive first-line genotoxic chemotherapy and introduces mutations that can contribute to the acquired resistance so often observed with standard anticancer regimens. As such, inhibition of Rev1/Polζ-dependent TLS has recently emerged as a strategy to enhance the efficacy of first-line chemotherapy and reduce the acquisition of chemoresistance by decreasing tumor mutation rate. The TLS DNA polymerase Rev1 serves as an integral scaffolding protein that mediates the assembly of the active multiprotein TLS complexes. Protein-protein interactions (PPIs) between the C-terminal domain of Rev1 (Rev1-CT) and the Rev1-interacting region (RIR) of other TLS DNA polymerases play an essential role in regulating TLS activity. To probe whether disrupting the Rev1-CT/RIR PPI is a valid approach for developing a new class of targeted anticancer agents, we designed a fluorescence polarization-based assay that was utilized in a pilot screen for small molecule inhibitors of this PPI. Two small molecule scaffolds that disrupt this interaction were identified, and secondary validation assays confirmed that compound 5 binds to Rev1-CT at the RIR interface. Finally, survival and mutagenesis assays in mouse embryonic fibroblasts and human fibrosarcoma HT1080 cells treated with cisplatin and ultraviolet light indicate that these compounds inhibit mutagenic Rev1/Polζ-dependent TLS in cells, validating the Rev1-CT/RIR PPI for future anticancer drug discovery and identifying the first small molecule inhibitors of TLS that target Rev1-CT.

5-(Furan-2-yl)-4-(3,4,5-trimethoxyphenyl)-3H-1,2-dithiol-3-one oxime (6f), a new synthetic compound, causes human fibrosarcoma HT-1080 cell apoptosis by disrupting tubulin polymerisation and inducing G2/M arrest.

In the current study, we synthesized a series of new compounds targeting tubulin and tested their anti-proliferative activities. Among these new synthetic com-pounds, 5-(furan-2-yl)-4-(3,4,5-trimethoxyphenyl)-3H-1,2-dithiol-3-one oxime (6f) exhibited significant anti-proliferative activity against different human cancer cell lines including human gastric adenocarcinoma SGC-7901, human non-small cell lung cancer A549, and human fibrosarcoma HT-1080. As a result, 6f was selected to further test the sensitivity to different cancer cell lines including human cervical cancer cell line HeLa, human breast cancer cell line MCF-7, non-small cell lung cancer cell line A549, human liver carcinoma cell line HepG-2, human oral squamous cell carcinoma cell lines KB, SGC-7901 and HT-1080. Among these cell lines, HT-1080 and HeLa are the most sensitive. Therefore, HT-1080 was selected to further explore the properties of anti-proliferative activity and the underlying mechanisms. Our data proved that 6f exhibited strong anti-proliferative effects against HT-1080 cells in a time- and dose-dependent manner. We showed that the growth inhibitory effect of 6f in HT-1080 cells was related with microtubule depolymerisation. Molecular docking studies revealed that 6f interacted and bound efficiently with the colchicine-binding site of tubulin. In addition, 6f treatment induced G2/M cell cycle arrest dose-dependently and subsequently induced cell apoptosis. Western blot study indicated that upregulation of cyclin B1 and p-cdc2 was related with G2/M arrest. 6f-induced cell apoptosis was associated with both mitochondrial and death receptor pathway. In conclusion, our data showed that 6f, among the newly synthetic compounds, exhibited highest anti-proliferative activity by disrupting the microtubule polymerisation, causing G2/M arrest and subsequently inducing cell apoptosis in HT-1080 cells. Hence, 6f is a promising microtubule depolymerising agent for the treatment of various cancers especially human fibrosarcoma.

Inhibitory effect of blue light emitting diode on migration and invasion of cancer cells.

The aim of this study was to determine the effects and molecular mechanism of blue light emitting diode (LED) in tumor cells. A migration and invasion assay for the metastatic behavior of mouse colon cancer CT-26 and human fibrosarcoma HT-1080 cells was performed. Cancer cell migration-related proteins were identified by obtaining a 2-dimensional gel electrophoresis (2-DE) in total cellular protein profile of blue LED-irradiated cancer cells, followed by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) analysis of proteins. Protein levels were examined by immunoblotting. Irradiation with blue LED inhibited CT-26 and HT-1080 cell migration and invasion. The anti-metastatic effects of blue LED irradiation were associated with inhibition of matrix metalloproteinase (MMP)-2 and MMP-9 expression. P38 MAPK phosphorylation was increased in blue LED-irradiated CT-26 and HT-1080 cells, but was inhibited after pretreatment with SB203580, a specific inhibitor of p38 MAPK. Inhibition of p38 MAPK phosphorylation by SB203580 treatment increased number of migratory cancer cells in CT-26 and HT-1080 cells, indicating that blue LED irradiation inhibited cancer cell migration via phosphorylation of p38 MAPK. Additionally blue LED irradiation of mice injected with CT-26 cells expressing luciferase decreased early stage lung metastasis compared to untreated control mice. These results indicate that blue LED irradiation inhibits cancer cell migration and invasion in vitro and in vivo.

Triterpenes with Anti-invasive Activity from Sclerotia of Inonotus obliquus

The methanolic extract [inhibition (%): 61.2±3.8 (p<0.01) at 100 μg/mL] and its EtOAc-soluble fraction [inhibition (%): 82.5±1.7 (p<0.01) at 100 μg/mL1 from the sclerotia of Inonotus obliquus collected in Japan significantly inhibited invasion of human fibrosarcoma HT1080 cells through matrigel-coated filters. In addition, the methanolic extract significantly inhibited lung tumor formation fifteen days after injection of BI6F10 melanoma cells in mice [inhibition (%) 66.1 ± 12:8 (p < 0.05) at 500 mg/kg/d, p.o.]. Lanostane-type triterpenes were isolated as the common principal constituents from Japanese and Russian . obliquus. Furtheremore, we examine the inhibitory effects of the constituents on the invasion of HT 1080 cells. Interestingly, 3β-hydroxylanosta-8,24-dien- 21-al [inhibition (%) 37.9 ± 3.0 (p < 0.05) at 30 μM] significantly inhibited the invasion, and no cytotoxic effect at 30 μM was observed.

MicroRNA-29 Family Suppresses the Invasion of HT1080 Human Fibrosarcoma Cells by Regulating Matrix Metalloproteinase 2 Expression.

Matrix metalloproteinase 2 (MMP2) is a potent protumorigenic, proangiogenic, and prometastatic enzyme that is overexpressed in metastatic cancer. Although there have been various studies on the MMP2 gene, further studies of regulatory factors are required to achieve inhibition of MMP2 enzyme activities. MicroRNAs (miRNAs) play key roles in tumor metastasis. However, the specific functions of miRNAs in metastasis are unclear. In this study, we assessed the function of the microRNA-29 family (miR-29s) in HT1080 human fibrosarcoma cells and examined the regulatory mechanisms of these miRNAs on MMP2 activation. Using miRanda, TargetScan, and PicTar databases, miR-29s were identified as candidate miRNAs targeting MMP2. Gain-of-function studies showed that overexpression of miR-29s could inhibit the invasion of HT1080 cells, suggesting their tumor-suppressive roles in HT1080 cells. In addition, dual luciferase reporter assays indicated that miR-29s could inhibit the expression of the luciferase gene containing the 3'-untranslated region of MMP2 mRNA. Ectopic expression of miR-29s down-regulated the expression of MMP2. Moreover, ectopic expression of miR-29s reduced MMP2 enzyme activity. These results suggested that miR-29s could decrease the invasiveness of HT1080 cells by modulating MMP2 signaling. Taken together, our results demonstrated that miR-29s may serve as therapeutic targets to control tumor metastasis.

Bleb Formation in Human Fibrosarcoma HT1080 Cancer Cell Line Is Positively Regulated by the Lipid Signalling Phospholipase D2 (PLD2)

Blebs are spherical plasma membrane protrusions formed when the membrane detaches from the underlying cortex as a result of actomyosin contractility-powered increase of hydrostatic pressure in the cytoplasm. Different tumour cells metastasize using blebbing as alternative mode of migration by squeezing through pre-existing pores in the extracellular matrix (ECM). This study investigated the role of the lipid signalling phospholipases D1 and D2 (PLD1/PLD2) in bleb formation in human fibrosarcoma HT1080 cell line in the extracellular matrix, and reports that pharmacological inhibition of PLD1 and PLD2 with a potent universal PLD inhibitor potently inhibited bleb formation in HT1080 cells embedded in three-dimensional (3D) matrigel matrix. Use of smartpool small interfering RNAs (siRNAs) that target PLD1 and PLD2 isoforms at four different sequences revealed that PLD2, but not PLD1 is involved in blebbing of HT1080 cells. Furthermore, we demonstrate that PLD2-mediated bleb formation is via the PA-LPAR-Rho-ROCK signalling pathway. Thus, PLD2 is a promising therapeutic target in combating metastasis of cancers of fibrous connective tissues.

Anti-tumor and anti-invasion effects of a combination of 4-methylumbelliferone and ionizing radiation in human fibrosarcoma cells.

Hyaluronan (HA) is a major component of the extracellular matrix that is synthesized in excess in cancer tissues. 4-methylumbelliferone (MU) inhibits the synthesis of HA and is closely related to the invasion and metastasis of cancer. However, the effects of MU in conjunction with cancer radiotherapy remain unknown. The present study assessed the anti-tumor and anti-invasion effects of the concomitant use of ionizing radiation (IR) and 100 µM MU on human fibrosarcoma HT1080 cells. Cell viability and cellular invasion potency assays were performed. There was a greater decrease in the viability of cells cultured with a combination of 2 Gy IR and MU compared with untreated control cells. In addition, cell cycle distribution analysis demonstrated that a higher proportion of these cells were in the sub-G1 phase and higher fractions of annexin-V positive, propidium iodide positive cells (i.e., apoptotic cells) were observed. HA concentration in the 2 Gy irradiated culture was similar to that in the non-irradiated control culture, however, it significantly decreased following the administration of both MU alone and 2 Gy IR with MU. Furthermore, treatment with 2 Gy IR and MU resulted in a significant decrease in the invasion rate and matrix metalloproteinase (MMP)-2 and MPP-9 expression. Taken together, these results suggest that the administration of MU with 2 Gy IR is effective at reducing HA production, cell invasion and the metastatic potential of cancer cells.

Gliotoxin from the marine fungus Aspergillus fumigatus induces apoptosis in HT1080 fibrosarcoma cells by downregulating NF-κB

Gliotoxin has been recognized as an immunosuppressive agent for a long time. Recently, it was reported to have antitumor properties. However, the mechanisms by which it inhibits tumors remain unclear. Here, we showed that gliotoxin isolated from the marine fungus Aspergillus fumigatus inhibited proliferation and induced apoptosis in HT1080 human fibrosarcoma cells. Gliotoxin repressed phosphorylation-dependent degradation of IκB-α, an antagonist of nuclear factor kappa B (NF-κB), which is a known tumor-promoting factor. This coincided with a decrease in nuclear import of NF-κB, suggesting its signaling activity was impaired. Moreover, gliotoxin increased intracellular reactive oxygen species (ROS). Since ROS have been known to inhibit NF-κB, this may also contribute to gliotoxin’s antitumorigenic effects. These results suggest that gliotoxin suppressed the activation of NF-κB by inhibiting phosphorylation and degradation of IκB-α and by increasing ROS, which resulted in apoptosis of HT1080 cells. Cumulatively, gliotoxin is a promising candidate antagonist of NF-κB, and it should be investigated for its possible use as a selective inhibitor of human fibrosarcoma cells.

Chemotherapy Drug Induced Discoordination of Mitochondrial Life Cycle Detected by Cardiolipin Fluctuation.

Chemotherapy drugs have been prescribed for the systemic treatment of cancer. We selected three chemotherapy drugs, including methotrexate, mitomycine C and vincristine to inhibit the proliferation of HT1080 human fibrosarcoma cells in S, G2 and M phases of the cell cycle respectively. These chemotherapy drugs showed significant toxicity and growth inhibition to the cancer cells measured by MTT assay. After treated with a 50% inhibitory dosage for 48 hours, these cancer cells showed significant accumulation of cardiolipin (CL), which was a reverse trend of the nutritional deficiency induced arrest at G1 phase. The quantity of each CL species was further semi-quantitated by HPLC-ion trap mass spectrometer. Methotraxate treatment caused unique increases of acyl chain length on CL, which were the opposite of the serum starvation, mitomycine C and vincristine treatments. Although mitomycine C and vincristine have different mechanisms to induce cell cycle arrest, these two drugs displayed similar effects on decreasing chain length of CL. Continuation of CL synthesis during cell cycle arrest indicated the chemotherapy drugs resulting in the discoordination of the mitochondrial life cycle from the cell cycle and thus caused the accumulation of CL. These finding reveals that the pre-remodeling nascent CL accumulates during the methotraxate induced arrest; however, the post-remodeling mature CL accumulates during the mitomycine C and vincristine induced arrest after the synthesis phase.

Biological study of the effect of water soluble [N-(2-hydroxybenzyl)-L-aspartato] gallium complexes on breast carcinoma and fibrosarcoma cells.

Two water soluble gallium complexes described as [Ga(III)LCl], where L is the deprotonated form of N-2-hydroxybenzyl aspartic acid derivatives, were synthesized and characterized by (1)H NMR, (13)C NMR, FT-IR, mass spectrometry, and elemental analysis. The 2-(5-chloro-2-hydroxybenzylamino)succinic acid derivative (GS2) has been found to be a promising anticancer drug candidate. This compound was found to be more cytotoxic against human breast carcinoma MDA-MB231 and fibrosarcoma HT-1080 cell lines than the unsubstituted derivative and GaCl3. GS2 was able to induce apoptosis through downregulation of AKT phosphorylation, G2M arrest in cell cycle, and caspase 3/7 pathway. This gallium complex was found to induce an increase in mitochondrial ROS level in HT-1080 cells but not in MDA-MB231 cells. This suggests that the mechanism of action of GS2 would not be mediated by the drug-induced oxidative stress but probably by directly and indirectly inhibiting the AKT cell-signaling pathway.

Abstract 2127: Identification of a novel NAMPT inhibition resistance mechanism utilizing the de novo NAD+ biosynthesis pathway

Abstract Targeting NAD(H) metabolism is being interrogated as a novel anti-cancer strategy. Nicotinamide phoshophoribosyltransferase (NAMPT) was found to be overexpressed in tumor cells, and plays a key role in the replenishment of the NAD(H) pool in cells. In this study we have used a commercially available NAMPT inhibitor, GMX1778, which exerts a cytotoxic effect by decreasing the cellular level of NAD(H). Through serial selection with increasing sub-lethal concentrations of GMX1778, we derived a GMX1778-resistant HT1080 human fibrosarcoma cell line (HT1080-GMX). The IC50 value for GMX1778-induced cell killing of the resistant cell line was 100-fold greater than that determined for the parental cell line. No significant change in the level of expression of NAMPT was observed in HT1080 vs. HT1080-GMX cells. However, a key enzyme in the de novo NAD(H) synthesis pathway known as quinolinate phosphoribosyl transferase (QPRT) was significantly upregulated as validated by Western blot and bDNA analysis. Knockdown of QPRT partially restored the sensitivity to GMX1778 exposure in HT1080-GMX cells, but did not affect the parental cells. The cytotoxicity of GMX1778 can be partially rescued with exogenous quinolinic acid, which permits NAD(H) repletion via QPRT in the de novo pathway, in HT1080-GMX cells but not HT1080 parental cells. Overexpression of QPRT in HT1080 cells did not fully rescue GMX-induced cytotoxicity and strongly suggests that there are additional mechanisms of resistance in the HT1080-GMX cell line beyond QPRT overexpression. Exome sequencing of the NAMPT gene in the HT1080-GMX resistant lines identified a single mutation of amino acid residue 18 from tyrosine to cysteine [NAMPT(Y18C)] located in the first exon of one allele. Tyrosine 18 is located near the active site of NAMPT and associates with NAMPT small molecule inhibitors through a pi stacking interaction, as determined by studies of the enzyme's crystal structure. Overexpression of the NAMPT mutant Y18C in HT1080 cells significantly reduced the sensitivity to GMX1778 exposure, but overexpression of a NAMPT Y18F mutant had no effects compared to the parental line. Based on our results, activation of an alternate NAD(H) biosynthesis pathway via QPRT or the Y18C mutation within the NAMPT coding sequence can both be contributors to resistance in this line. The characterization of resistance inducing up-regulation of other sources of NAD(H) such as de novo synthesis or mutations in NAMPT may be useful in developing next-generation NAMPT inhibitors that are less affected by known resistance mechanisms. Disclosures: All authors are employees of AbbVie. The design, study conduct, and financial support for this research was provided by AbbVie. AbbVie participated in the interpretation of data, review, and approval of the publication. Citation Format: jun guo, Chris Tse, William N. Pappano. Identification of a novel NAMPT inhibition resistance mechanism utilizing the de novo NAD+ biosynthesis pathway. [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 2127.

5-Azacytidine regulates matrix metalloproteinase-9 expression, and the migration and invasion of human fibrosarcoma HT1080 cells via PI3-kinase and ERK1/2 pathways.

Abnormal methylation of promoter CpG islands is one of the hallmarks of cancer cells, and is catalyzed by DNA methyltransferases. 5-azacytidine (5-aza C), a methyltransferase inhibitor, can cause demethylation of promoter regions of diverse genes. Epigenetic processes contribute to the regulation of matrix metalloproteinase (MMP) expression. However, little is known about the mechanisms and effects of 5-aza C on the invasive and migratory capacities of human fibrosarcoma HT1080 cells. In the present study, we found that 5-aza C induces MMP-9 activity, as determined by zymography. HT1080 cell proliferation was determined following 5-aza C administration by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Cell cycle was examined by flow cytometry. 5-aza C treatment inhibited cell proliferation without affecting cell viability. Furthermore, 5-aza C significantly promoted migration and invasion of HT1080 cells. 5-aza C treatment enhanced phosphorylation of extracellular signal-regulated kinase (ERK) and phosphoinositide (PI)3-kinase/Akt, and their inhibitors blocked MMP-9 activity induction, and cellular invasion and migration. Together, these findings suggest that promoter methylation may be one of the mechanisms modulating MMP-9 levels in HT1080 cells, and that 5-aza C-induced MMP-9 production is associated with the activation of ERK and PI3-kinase/Akt signaling pathways.

Isolation and Structure Elucidation of Cytotoxic Saccharothriolides D to F from a Rare Actinomycete Saccharothrix sp. and Their Structure-Activity Relationship.

Three new 10-membered macrolides, saccharothriolides D-F (1-3), were isolated from a rare actinomycete, Saccharothrix sp. A1506. The planar structures were determined from analysis of extensive NMR and HR-ESI-MS data, and the absolute configurations were established by ECD spectroscopy analysis. Saccharothriolides D (1) and E (2) were determined to be C-2 epimers of saccharothriolides A (4) and B (5), respectively. Saccharothriolide F (3) was identified to be a demethylated congener of saccharothriolides D (1) and A (4) at the C-2 position. The availability of compounds 1-6 enabled a structure-activity relationship study that revealed the importance of the phenolic hydroxy group at C-2″ and the stereochemistry of C-2 for the inhibition of human fibrosarcoma HT1080 cell growth.

Experimental Investigation of Honey as Wound Repair Enhancer by In Vitro Time-Lapse Microscopy

Honey is widely used for treating wounds, burns and ulcers, due to its antimicrobial and anti-inflammatory properties. Recently, it has been shown that it can also promote tissue repair. However, the mechanisms of action in the wound healing process are still far from full comprehension. In this work we propose an experimental methodology to investigate the potential role of honey as wound repair enhancer, by analyzing in vitro cell motility and proliferation, that are the main mechanisms involved in the wound repair. Our methodological approach is based on the well-assessed in vitro Wound Healing (WH) assay, coupled with Time-Lapse Microscopy (TLM) and image analysis techniques. Our methodology was applied to test the potential healing activities of several kinds of honeys and mixtures of selected honeys, on monolayers of HT1080 human fibrosarcoma cells. The honeys were also characterized for their antimicrobial activity. Overall, this work is focused on raising new therapeutic interest in the valuable natural product honey.

Type I collagen aging impairs discoidin domain receptor 2-mediated tumor cell growth suppression.

Tumor cells are confronted to a type I collagen rich environment which regulates cell proliferation and invasion. Biological aging has been associated with structural changes of type I collagen. Here, we address the effect of collagen aging on cell proliferation in a three-dimensional context (3D).We provide evidence for an inhibitory effect of adult collagen, but not of the old one, on proliferation of human fibrosarcoma HT-1080 cells. This effect involves both the activation of the tyrosine kinase Discoidin Domain Receptor 2 (DDR2) and the tyrosine phosphatase SHP-2. DDR2 and SHP-2 were less activated in old collagen. DDR2 inhibition decreased SHP-2 phosphorylation in adult collagen and increased cell proliferation to a level similar to that observed in old collagen.In the presence of old collagen, a high level of JAK2 and ERK1/2 phosphorylation was observed while expression of the cell cycle negative regulator p21CIP1 was decreased. Inhibition of DDR2 kinase function also led to an increase in ERK1/2 phosphorylation and a decrease in p21CIP1 expression. Similar signaling profile was observed when DDR2 was inhibited in adult collagen. Altogether, these data suggest that biological collagen aging could increase tumor cell proliferation by reducingthe activation of the key matrix sensor DDR2.

Salinomycin causes migration and invasion of human fibrosarcoma cells by inducing MMP-2 expression via PI3-kinase, ERK-1/2 and p38 kinase pathways.

Salinomycin (SAL) is a polyether ionophore antibiotic that has recently been shown to regulate a variety of cellular responses in various human cancer cells. However, the effects of SAL on metastatic capacity of HT1080 human fibrosarcoma cells have not been elucidated. We investigated the effect of SAL on migration and invasion, with emphasis on the expression and activation of matrix metalloproteinase (MMP)-2 in HT1080 human fibrosarcoma cells. Treatment of SAL promoted the expression and activation of MMP-2 in a dose- and time-dependent manner, as detected by western blot analysis, gelatin zymography, and real-time polymerase chain reaction. SAL also increased metastatic capacities, as determined by an increase in the migration and invasion of cells using the wound healing assay and the invasion assay, respectively. To confirm the detailed molecular mechanisms of these effects, we measured the activation of phosphoinositide3 kinase (PI3-kinase) and mitogen-activated protein kinase (MAPK)s (ERK-1/2 and p38 kinase), as detected by the phosphorylated proteins through western blot analysis. SAL treatment increased the phosphorylation of Akt and MAPKs. Inhibition of PI3-kinase, ERK-1/2, and p38 kinase with LY294002, PD98059, and SB203580, respectively, in the presence of SAL suppressed the metastatic capacity by reducing MMP-2 expression, as determined by gelatin zymography. Our results indicate that the PI3-kinase and MAPK signaling pathways are involved in migration and invasion of HT1080 through induction of MMP-2 expression and activation. In conclusion, SAL significantly increases the metastatic capacity of HT1080 cells by inducing MMP-2 expression via PI3-kinase and MAPK pathways. Our results suggest that SAL may be a potential agent for the study of cancer metastatic capacities.

An essential role for functional lysosomes in ferroptosis of cancer cells.

Pharmacological challenges to oncogenic Ras-expressing cancer cells have shown a novel typeof cell death, ferroptosis, which requires intracellular iron. In the present study, we assessed ferroptosis following treatment of human fibrosarcoma HT1080 cells with several inhibitors of lysosomal activity and found that they prevented cell death induced by the ferroptosis-inducing compounds erastin and RSL3. Fluorescent analyses with a reactive oxygen species (ROS) sensor revealed constitutive generation of ROS in lysosomes, and treatment with lysosome inhibitors decreased both lysosomal ROS and a ferroptotic cell-death-associated ROS burst. These inhibitors partially prevented intracellular iron provision by attenuating intracellular transport of transferrin or autophagic degradation of ferritin. Furthermore, analyses with a fluorescent sensor that detects oxidative changes in cell membranes revealed that formation of lipid ROS in perinuclear compartments probably represented an early event in ferroptosis. These results suggest that lysosomal activity is involved in lipid ROS-mediated ferroptotic cell death through regulation of cellular iron equilibria and ROS generation.