Human Fibrosarcoma Cell Line HT1080 Research Articles

Frequency-Dependent Antioxidant Responses in HT-1080 Human Fibrosarcoma Cells Exposed to Weak Radio Frequency Fields.

This study explores the complex relationship between radio frequency (RF) exposure and cancer cells, focusing on the HT-1080 human fibrosarcoma cell line. We investigated the modulation of reactive oxygen species (ROS) and key antioxidant enzymes, including superoxide dismutase (SOD), peroxidase, and glutathione (GSH), as well as mitochondrial superoxide levels and cell viability. Exposure to RF fields in the 2-5 MHz range at very weak intensities (20 nT) over 4 days resulted in distinct, frequency-specific cellular effects. Significant increases in SOD and GSH levels were observed at 4 and 4.5 MHz, accompanied by reduced mitochondrial superoxide levels and enhanced cell viability, suggesting improved mitochondrial function. In contrast, lower frequencies like 2.5 MHz induced oxidative stress, evidenced by GSH depletion and increased mitochondrial superoxide levels. The findings demonstrate that cancer cells exhibit frequency-specific sensitivity to RF fields even at intensities significantly below current safety standards, highlighting the need to reassess exposure limits. Additionally, our analysis of the radical pair mechanism (RPM) offers deeper insight into RF-induced cellular responses. The modulation of ROS and antioxidant enzyme activities is significant for cancer treatment and has broader implications for age-related diseases, where oxidative stress is a central factor in cellular degeneration. The findings propose that RF fields may serve as a therapeutic tool to selectively modulate oxidative stress and mitochondrial function in cancer cells, with antioxidants playing a key role in mitigating potential adverse effects.

Release of Exosomal PD-L1 in Bone and Soft Tissue Sarcomas and Its Relationship to Radiotherapy.

(1) Background: Exosomal PD-L1 has garnered attention owing to its role in instigating systemic immune suppression. The objective of this study is to elucidate whether bone and soft tissue sarcoma cells possess the capacity to secrete functionally active exosomal PD-L1 and whether radiotherapy (RT) induces the exosomal PD-L1 release. (2) Methods: Human osteosarcoma cell line 143B and human fibrosarcoma cell line HT1080 were utilized. Exosomes were isolated from the culture medium and blood via ultracentrifugation. The expression of PD-L1 on both tumor cells and exosomes was evaluated. The inhibitory effect on PBMC was employed to assess the activity of exosomal PD-L1. Post radiotherapy, changes in PD-L1 expression were compared. (3) Results: Exosomal PD-L1 was detected in the culture medium of tumor cells but was absent in the culture medium of PD-L1 knockout cells. Exosomal PD-L1 exhibited an inhibitory effect on PBMC activation. In tumor-bearing mice, human-derived exosomal PD-L1 was detected in the bloodstream. Following radiotherapy, tumor cells upregulated PD-L1, and human-derived exosomal PD-L1 were detected in the bloodstream. (4) Conclusions: Exosomal PD-L1 emanates from bone and soft tissue sarcoma cells and is disseminated into the circulatory system. The levels of PD-L1 in tumor cells and the release of exosomal PD-L1 were augmented after irradiation with RT.

Pacidusin B isolated from Phyllanthus acidus triggers ferroptotic cell death in HT1080 cells

Cancer cells generally exhibit ‘iron addiction’ phenotypes, which contribute to their vulnerability to ferroptosis inducers. Ferroptosis is a newly discovered form of programmed cell death caused by iron-dependent lipid peroxidation. In the present study, pacidusin B, a dichapetalin-type triterpenoid from Phyllanthus acidus (L.) Skeels (Euphorbiaceae), induces ferroptosis in the HT1080 human fibrosarcoma cell line. Cells treated with pacidusin B exhibited the morphological characteristic ‘ballooning’ phenotype of ferroptosis. The biochemical hallmarks of ferroptosis were also observed in pacidusin B-treated cells. Both oxidative stress and ER stress play significant roles in pacidusin B-induced ferroptosis. The activation of the PERK-Nrf2-HO-1 signaling pathway led to iron overload, while inhibition of GPX4 further sensitized cancer cells to ferroptosis. Furthermore, the molecular docking study showed that pacidusin B docked in the same pocket in xCT as the ferroptosis inducer erastin. These results revealed that pacidusin B exerts anticancer effects via inducing ER-mediated ferroptotic cell death.Graphical

Several first-line anti-hypertensives act on fibrosarcoma progression and PD1ab blockade therapy.

Patients are typically diagnosed with both hypertension and fibrosarcoma. Medical oncologists must prescribe suitable anti-hypertensive medications while considering anti-tumor drugs. Recently, immunotherapy has become prominent in cancer treatment. Nonetheless, it is unknown what role anti-hypertensive medications will play in immunotherapy. We examined the effects of six first-line anti-hypertensive medications on programmed cell death protein 1 antibody (PD1ab) in tumor treatment using a mouse model of subcutaneous fibrosarcoma. The drugs examined were verapamil, losartan, furosemide, spironolactone, captopril, and hydrochlorothiazide (HCTZ). The infiltration of CD8+ T cells was examined by immunohistochemistry. Additionally, several in vitro and in vivo assays were used to study the effects of HCTZ on human fibrosarcoma cancer cells to explore its mechanism. Verapamil suppressed tumor growth and showed an improved effect on the tumor inhibition of PD1ab. Captopril did not affect tumor growth but brought an unexpected benefit to PD1ab treatment. In contrast, spironolactone andfurosemide showed no effect on tumor growth but had an offset effect on the PD1ab therapy. Consequently, the survival time of mice was also significantly reduced. Notably, losartan and HCTZ, especially HCTZ, promoted tumor growth and weakened the effect of PD1ab treatment. Consistent results were observed in vivo and in vitro using the human fibrosarcoma cell line HT1080. We determined that the Solute Carrier Family 12 Member 3 (SLC12A3), a known target of HCTZ, may be the principal factor underlying its effect-enhancing properties through mechanism studies employing The Cancer Genome Atlas (TCGA) data and in vivo and in vitro assays. Verapamil and captopril potentiated the anti-tumor effect of PD1ab, whereas spironolactone andfurosemide weakened the effect of PD1ab on tumor inhibition. Alarmingly, losartan and HCTZ promoted tumor growth and impaired the effect of PD1ab. Furthermore, we preliminarily found that HCTZ may promote tumor progression through SLC12A3. Based on this study, futher mechanism researches and clinical trials should be conducted in the future.

Inhibition of nonsense-mediated mRNA decay reduces the tumorigenicity of human fibrosarcoma cells.

Nonsense-mediated mRNA decay (NMD) is a eukaryotic RNA decay pathway with roles in cellular stress responses, differentiation, and viral defense. It functions in both quality control and post-transcriptional regulation of gene expression. NMD has also emerged as a modulator of cancer progression, although available evidence supports both a tumor suppressor and a pro-tumorigenic role, depending on the model. To further investigate the role of NMD in cancer, we knocked out the NMD factor SMG7 in the HT1080 human fibrosarcoma cell line, resulting in suppression of NMD function. We then compared the oncogenic properties of the parental cell line, the SMG7-knockout, and a rescue cell line in which we re-introduced both isoforms of SMG7. We also tested the effect of a drug inhibiting the NMD factor SMG1 to distinguish NMD-dependent effects from putative NMD-independent functions of SMG7. Using cell-based assays and a mouse xenograft tumor model, we showed that suppression of NMD function severely compromises the oncogenic phenotype. Molecular pathway analysis revealed that NMD suppression strongly reduces matrix metalloprotease 9 (MMP9) expression and that MMP9 re-expression partially rescues the oncogenic phenotype. Since MMP9 promotes cancer cell migration and invasion, metastasis and angiogenesis, its downregulation may contribute to the reduced tumorigenicity of NMD-suppressed cells. Collectively, our results highlight the potential value of NMD inhibition as a therapeutic approach.

The Distinct Roles of LKB1 and AMPK in p53-Dependent Apoptosis Induced by Cisplatin.

Liver kinase B1 (LKB1) is a serine/threonine protein kinase that acts as a key tumor suppressor protein by activating its downstream kinases, such as AMP-activated protein kinase (AMPK). However, the regulatory actions of LKB1 and AMPK on DNA damage response (DDR) remain to be explored. In this study, we investigated the function of LKB1 in DDR induced by cisplatin, a representative DNA-damaging agent, and found that LKB1 stabilizes and activates p53 through the c-Jun N-terminal kinase (JNK) pathway, which promotes cisplatin-induced apoptosis in human fibrosarcoma cell line HT1080. On the other hand, we found that AMPKα1 and α2 double knockout (DKO) cells showed enhanced stabilization of p53 and increased susceptibility to apoptosis induced by cisplatin, suggesting that AMPK negatively regulates cisplatin-induced apoptosis. Moreover, the additional stabilization of p53 and subsequent apoptosis in AMPK DKO cells were clearly canceled by the treatment with the antioxidants, raising the possibility that AMPK suppresses the p53 activation mediated by oxidative stress. Thus, our findings unexpectedly demonstrate the reciprocal regulation of p53 by LKB1 and AMPK in DDR, which provides insights into the molecular mechanisms of DDR.

KIF20A promotes the development of fibrosarcoma via PI3K-Akt signaling pathway

Adult fibrosarcoma is an aggressive subtype of soft tissue sarcoma (STS), in which high expression of KIF20A indicates a poor prognosis. However, the precise role of KIF20A in fibrosarcoma progression remains unknown. In this study, we initially examined KIF20A expression and function in the human fibrosarcoma cell line HT-1080. The results showed that KIF20A was highly expressed in HT-1080, knockdown of KIF20A impaired cell proliferation, migration, invasion and induced G2/M arrest and cell apoptosis. Transcriptome study suggested that PI3K-Akt signal pathway was involved in these biological changes. We confirmed that PI3K-Akt and NF-κB signaling pathways were impaired after the down-regulation of KIF20A, which can be reversed by the Akt activator SC79 in HT-1080 in vitro. In a xenograft mouse model, knockdown of KIF20A inhibited tumor growth, Ki67 expression and liver metastasis. Taken together, our results suggested that KIF20A promoted fibrosarcoma progression via PI3K-Akt signaling pathway and might be a potential therapeutic target for fibrosarcoma.

Analysis of Active Compounds Using Target Protein Cofilin-Cucurbitacins in Cytotoxic Plant Bryonia cretica.

We examined a two-step target protein binding strategy that uses cofilin as the target protein to analyze the active constituents in Bryonia cretica. In the first step, we prepared the target protein, and used it to analyze the compounds binding to it in the second step. We used the methanolic extract of B. cretica as a library of possible active compounds. We conducted LC–MS analysis using information from our previous study. The peaks in the HPLC profile were identified as cucurbitacin D, isocucurbitacin D, and cucurbitacin I. As far as we know, there is no known study of the activity of isocucurbitacin D in this research field. Therefore, we examined the effects of isocucurbitacin D on cell proliferation and cofilin protein in human fibrosarcoma cell line HT1080 to confirm the effectiveness of this strategy. The cytotoxicity assay, the fibrous/globular actin ratio assay, and the immunoblotting analysis revealed that isocucurbitacin D showed a cytotoxic effect with disruption of target protein cofilin. The target protein binding strategy is a direct and straightforward method for finding new drug seeds from crude sources, such as natural plant extracts.

DNA-Directed Assembly of a Cell-Responsive Biohybrid Interface for Cargo Release.

The development of a DNA-based cell-responsive biohybrid interface that can be used for spatially confined release of molecular cargo is reported. To this end, tailored DNA-protein conjugates are designed as gatekeepers that can be specifically cleaved by matrix metalloproteases (MMPs), which are secreted by many cancer cells. These gatekeepers can be installed by DNA hybridization on the surface of mesoporous silica nanoparticles (MSNs). The MSNs display another orthogonal DNA oligonucleotide that can be exploited for site-selective immobilization on solid glass surfaces to yield micropatterned substrates for cell adhesion. Using the human fibrosarcoma cell line HT1080 that secretes MMPs, it is demonstrated that the biohybrid surface is specifically modified by the cells to release both MSN-bound gatekeeper proteins and the encapsulated cargo peptide KLA. In view of the enormously high modularity of the system presented here, this approach promising for applications in drug delivery, tissue engineering, or other areas of nanobiotechnology is considered.

Microrna-145 Inhibits EMT and Fibrosarcoma Cell Proliferation, Migration and Invasion by Targeting HMGA2

1.1 Aim: Fibrosarcoma is a rare malignant tumor of subcutaneous tissue characterized by slow infiltrative growth and a high rate of local recurrence. The participation of microRNAs (miRNAs/miRs) in the pathogenesis of human diseases and the vital role of miR-145 in different types of cancer. However, the role of miR-145 in fibrosarcoma remains unclear. The present study aimed to investigate the effect of miR-145 in the progression of fibrosarcoma and its potential application as a new therapeutic target. 1.2. Methods: The present study screened out a differential gene named high mobility group protein A2 (HMGA2) via bioinformatics analysis. In the present study, the gene expression profiles of fibrosarcoma cells and normal cells were downloaded from GEO Datasets GSE10021 and GSE1774. The human fibrosarcoma cell line HT1080 were transfected with miR-145 mimics, mimic negative control (NC), pCMV6 vector, overexpression plasmid vector of HMGA2, (si)RNA-HMGA2 or siRNA-NC. Reverse transcription-quantitative PCR and western blot analysis were performed to examine the mRNA (miR-145) and protein (HMGA2, N-cadherin, Vimentin and E-cadherin) expression levels in HT1080 cells. Cell Counting Kit-8, wound healing and Transwell assays were used to examine the TH1080 cell viability, proliferation and migratory capacities, respectively. Bioinformatics prediction and luciferase reporter assay were performed to investigate the relationship between miR145 and its potential target gene (HMGA2). 1.3. Results: The results demonstrated that overexpression of HMGA2 promoted cell proliferation, migration and invasion of HT1080 fibrosarcoma cells, whereas HMGA2 knockdown had no effect on the proliferation of HT1080 fibrosarcoma cells. However, overexpression of miR-145 reversed the facilitating effects of HMGA2 in tumor progression via epithelial-to-mesenchymal transition (EMT) modulation. Mechanistically, miR-145 exerted its inhibitory role in fibrosarcoma by directly binding to the 3’-untranslated region of HMGA2. 1.4. Conclusion: miR-145 plays an inhibitory role in fibrosarcoma progression by directly suppressing HGMA2 expression and EMT progression. Thus, miR-145 may act as a potential therapeutic target for malignant fibrosarcoma therapy

Pro-apoptotic functions of TRAF2 in p53-mediated apoptosis induced by cisplatin.

Tumor necrosis factor receptor-associated factor 2 (TRAF2) is an essential component of tumor necrosis factor-α (TNF-α) signaling that regulates nuclear factor-κB (NF-κB) and c-Jun N-terminal kinase (JNK) pathways, and compelling evidence has demonstrated that TRAF2 suppresses TNF-α-induced cytotoxicity. On the other hand, it has been reported that oxidative stress-induced cytotoxicity is potentiated by TRAF2, indicating that TRAF2 both positively and negatively regulates stress-induced cytotoxicity in a context-specific manner. However, the causal role of TRAF2 in DNA damage response (DDR) remains to be explored. In this study, we assessed the function of TRAF2 in DDR induced by cisplatin, a representative DNA-damaging agent, and found that TRAF2 exerts pro-apoptotic activity through p53-dependent mechanisms at least in human fibrosarcoma cell line HT1080. TRAF2 deficient cells exhibit significant resistance to cell death induced by cisplatin, accompanied by the reduction of both p53 protein level and caspase-3 activation. Moreover, cisplatin-induced JNK activation was attenuated in TRAF2-deficient cells, and pharmacological inhibition of JNK signaling suppressed p53 stabilization. These results suggest that TRAF2 promotes p53-dependent apoptosis by activating the JNK signaling cascade in HT1080 cells. Thus, our data demonstrate a novel function of TRAF2 in cisplatin-induced DDR as a pro-apoptotic protein.

Preparation and biological evaluation of a novel pH-sensitive poly (β-malic acid) conjugate for antitumor drug delivery.

Poly (β‑malic acid), referred to as PMLA, has been synthesized and introduced as a polymeric drug carrier due to its desirable biological properties. In the present study, a novel pH‑sensitive polymer‑drug conjugate based on PMLA, PMLA‑Hz‑doxorubicin (DOX), was prepared, and another conjugate, PMLA‑ami‑DOX, was synthesized as a comparison. The structures, conjugation efficiency, and drug release properties of the prodrugs were determined. The cytotoxicity and cell uptake were assessed using the HT1080 human fibrosarcoma cell line as an invitro cell model. The release of DOX in the two conjugates were pH‑dependent in PBS buffer at a pH of 5.6, 6.0, 6.8 and 7.4. The quantity of drug released increased with the decrease in pH, and PMLA‑ami‑DOX released twice as much as PMLA‑Hz‑DOX (12h). The cytotoxicity of PMLA‑Hz‑DOX at pH 7.4 was lower than that of free DOX and increased with the decrease in pH, indicating that the cytotoxicity of PMLA‑Hz‑DOX was pH‑sensitive. Flow cytometry and confocal experiments confirmed the efficiency of the PMLA‑Hz‑DOX conjugate. Therefore, bonding DOX to PMLA via an acid‑sensitive hydrazone bond may be used to reduce its toxic side effects on normal tissues while responding to tumor pH and releasing the drug.

The effect of hyperthermia on the DNA damage response induced by γ-rays, as determined through in situ cell tracking.

Hyperthermia (HT) acts as a cancer treatment by direct cell killing, radiosensitization, and promotion of tumor reoxygenation. The sensor proteins of the DNA damage response (DDR) are the direct targets of HT. However, the spatiotemporal properties of sensor proteins under HT are still unclear. Therefore, investigating the impact of HT on sensor proteins is of great importance. In the present study, the human fibrosarcoma cell line HT1080 stably transfected with 53BP1-GFP [the DDR protein 53BP1 fused to green fluorescent protein (GFP)] was used to investigate the real-time cellular response to DNA double-strand breaks (DSBs) induced by γ-rays. Using live-cell imaging combined with HT treatment, the spatiotemporal properties of the 53BP1 protein were directly monitored and quantitatively studied. We found that HT could delay and decrease the formation of 53BP1 ionizing radiation–induced foci (IRIF). Moreover, through the in situ tracking of individual IRIF, it was found that HT resulted in more unrepaired IRIF over the period of observation compared with IR alone. Additionally, the unrepaired IRIF had a larger area, higher intensity, and slower repair rate. Indeed, almost every cell treated with HT had unrepaired IRIF, and the majority of these IRIF increased in area individually, while the rest increased in area by the merging of adjacent IRIF. In summary, our study demonstrated that HT could perturb the primary event in the DDR induced by IR, and this may have important implications for cancer treatment and heat radiosensitization.

Genetically modified human type II collagen for N- and C-terminal covalent tagging

Collagen is the predominant structural protein in vertebrates, where it contributes to connective tissues and the ECM; it is also widely used in biomaterials and tissue engineering. Dysfunction of this protein and its processing can lead to a wide variety of developmental disorders and connective tissue diseases. Recombinantly engineering the protein is challenging due to post-translational modifications generally required for its stability and secretion from cells. Introducing end labels into the protein is problematic, because the N- and C-termini of the physiologically relevant tropocollagen lie internal to the initially flanking N- and C-propeptide sequences. Here, we introduce mutations into human type II procollagen in a manner that addresses these concerns and purify the recombinant protein from a stably transfected HT1080 human fibrosarcoma cell line. Our approach introduces chemically addressable groups into the N- and C-telopeptide termini of tropocollagen. Simultaneous overexpression of formylglycine generating enzyme (FGE) allows the endogenous production of an aldehyde tag in a defined, substituted sequence in the N terminus of the mutated collagen, whereas the C-terminus of each chain presents a sulfhydryl group from an introduced cysteine. These modifications are designed to enable specific covalent end-labelling of collagen. We find that the doubly mutated protein folds and is secreted from cells. Higher order assembly into well-ordered collagen fibrils is demonstrated through transmission electron microscopy. Chemical tagging of thiols is successful; however, background from endogenous aldehydes present in wild-type collagen has thus far obscured the desired specific N-terminal labelling. Strategies to overcome this challenge are proposed.

Synthesis and anticancer activity of novel quinazolinone-based rhodanines

BackgroundRhodanines and quinazolinones have been reported to possess various pharmacological activities.ResultsA novel series of twenty quinazolinone-based rhodanines were synthesized via Knoevenagel condensation between 4-[3-(substitutedphenyl)-3,4-dihydro-4-oxoquinazolin-2-yl)methoxy]substituted-benzaldehydes and rhodanine. Elemental and spectral analysis were used to confirm structures of the newly synthesized compounds. The newly synthesized compounds were biologically evaluated for in vitro cytotoxic activity against the human fibrosarcoma cell line HT-1080 as a preliminary screen using the MTT assay.ConclusionsAll the target compounds were active, displaying IC50 values roughly in the range of 10–60 µM. Structure–activity relationship study revealed that bulky, hydrophobic, and electron withdrawing substituents at the para-position of the quinazolinone 3-phenyl ring as well as methoxy substitution on the central benzene ring, enhance cytotoxic activity. The four most cytotoxic compounds namely, 45, 43, 47, and 37 were further tested against two human leukemia cell lines namely, HL-60 and K-562 and showed cytotoxic activity in the low micromolar range with compound 45 being the most active, having IC50 values of 1.2 and 1.5 μM, respectively. Interestingly, all four compounds were devoid of cytotoxicity against normal human fibroblasts strain AG01523, indicating that the synthesized rhodanines may be selectively toxic against cancer cells. Mechanistic studies revealed that the most cytotoxic target compounds exhibit pro-apoptotic activity and trigger oxidative stress in cancer cells.

On-command on/off switching of progenitor cell and cancer cell polarized motility and aligned morphology via a cytocompatible shape memory polymer scaffold.

Invitro biomaterial models have enabled advances in understanding the role of extracellular matrix (ECM) architecture in the control of cell motility and polarity. Most models are, however, static and cannot mimic dynamic aspects of invivo ECM remodeling and function. To address this limitation, we present an electrospun shape memory polymer scaffold that can change fiber alignment on command under cytocompatible conditions. Cellular response was studied using the human fibrosarcoma cell line HT-1080 and the murine mesenchymal stem cell line C3H/10T1/2. The results demonstrate successful on-command on/off switching of cell polarized motility and alignment. Decrease in fiber alignment causes a change from polarized motility along the direction of fiber alignment to non-polarized motility and from aligned to unaligned morphology, while increase in fiber alignment causes a change from non-polarized to polarized motility along the direction of fiber alignment and from unaligned to aligned morphology. In addition, the findings are consistent with the hypothesis that increased fiber alignment causes increased cell velocity, while decreased fiber alignment causes decreased cell velocity. On-command on/off switching of cell polarized motility and alignment is anticipated to enable new study of directed cell motility in tumor metastasis, in cell homing, and in tissue engineering.

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.

DNA-hypomethylating agent, 5'-azacytidine, induces cyclooxygenase-2 expression via the PI3-kinase/Akt and extracellular signal-regulated kinase-1/2 pathways in human HT1080 fibrosarcoma cells.

The cytosine analogue 5'-azacytidine (5'-aza) induces DNA hypomethylation by inhibiting DNA methyltransferase. In clinical trials, 5'-aza is widely used in epigenetic anticancer treatments. Accumulated evidence shows that cyclooxygenase-2 (COX-2) is overexpressed in various cancers, indicating that it may play a critical role in carcinogenesis. However, few studies have been performed to explore the molecular mechanism underlying the increased COX-2 expression. Therefore, we tested the hypothesis that 5'-aza regulates COX-2 expression and prostaglandin E2 (PGE2) production. The human fibrosarcoma cell line HT1080, was treated with various concentrations of 5'-aza for different time periods. Protein expressions of COX-2, DNA (cytosine-5)-methyltransferase 1 (DNMT1), pAkt, Akt, extracellular signal-regulated kinase (ERK), and phosphorylated ERK (pERK) were determined using western blot analysis, and COX-2 mRNA expression was determined using RT-PCR. PGE2 production was evaluated using the PGE2 assay kit. The localization and expression of COX-2 were determined using immunofluorescence staining. Treatment with 5'-aza induces protein and mRNA expression of COX-2. We also observed that 5'-aza-induced COX-2 expression and PGE2 production were inhibited by S-adenosylmethionine (SAM), a methyl donor. Treatment with 5'-aza phosphorylates PI3-kinase/Akt and ERK-1/2; inhibition of these pathways by LY294002, an inhibitor of PI3-kinase/Akt, or PD98059, an inhibitor of ERK-1/2, respectively, prevents 5'-aza-induced COX-2 expression and PGE2 production. Overall, these observations indicate that the hypomethylating agent 5'-aza modulates COX-2 expression via the PI3-kinase/Akt and ERK-1/2 pathways in human HT1080 fibrosarcoma cells.

Hydrophilic extract from Posidonia oceanica inhibits activity and expression of gelatinases and prevents HT1080 human fibrosarcoma cell line invasion

Posidonia oceanica (L.) Delile is an endemic Mediterranean sea-grass distributed in the infralittoral zones, where it forms meadows playing a recognized ecological role in the coastal marine habitat. Although its use as a traditional herbal remedy is poorly documented, recent literature reports interesting pharmacological activities as antidiabetic, antioxidant and vasoprotective. Differently from previous literature, this study presents a hydrophilic extraction method that recovers metabolites that may be tested in biological buffers. We showed for the first time in the highly invasive HT1080 human fibrosarcoma cell line that our hydrophilic extract from P. oceanica was able to strongly decrease gene and protein expression of gelatinases MMP-2 and MMP-9 and to directly inhibit in a dose-dependent manner gelatinolytic activity in vitro. Moreover, we have revealed that our extract strongly inhibited HT1080 cell migration and invasion. Biochemical analysis of the hydrophilic extract showed that catechins were the major constituents with minor contribution of gallic acid, ferulic acid and chlorogenic plus a fraction of uncharacterized phenols. However, if each individual compound was tested independently, none by itself was able to induce a direct inhibition of gelatinases as strong as that observed in total extract, opening up new routes to the identification of novel compounds. These results indicate that our hydrophilic extract from P. oceanica might be a source of new pharmacological natural products for treatment or prevention of several diseases related to an altered MMP-2 and MMP-9 expression.

In vitro and in vivo bioactivities of aqueous and ethanol extracts from Helicteres angustifolia L. root

Ethnopharmacological relevanceHelicteres angustifolia L. (H. angustifolia L.) has been used as traditional medicine in the treatment of cancer in China and Laos. Its medical benefits, however, are still lacking of scientific evidence. Two extracts successively obtained from the root of H. angustifolia L., namely the aqueous root extract (ARE) and the ethanolic root extract (ERE), were used to evaluate the antioxidant and anticancer activities in vitro, and the antitumor efficacy of ARE was examined in vivo, respectively. Materials and methodsARE and ERE were extracted successively from H. angustifolia L. root with water and ethanol. In vitro antioxidant activities were assessed by radicals scavenging assay, ferrous chelating assay and reducing power assay. In vitro anticancer activities of ARE and ERE were evaluated by their cytotoxic effects against three human cancer cell lines. In addition, the anti-tumor activities of ARE in vivo were assessed by using Ht1080 (human fibrosarcoma cell line Ht1080) tumor xenografts mice. BALB/c nude mice were orally administrated with 200mg/kg/d of ARE. The tumor inhibition rate was determined on day 42 after treatment by using histopathology analysis of the tumor tissues. Furthermore, relevant biochemical parameters in blood were analyzed to monitor their cytotoxic effect. ResultsIn vitro assays indicated that ARE possessed relatively higher antioxidant and anticancer activities than ERE, with IC50 values of 82.31±9.62, 62.50±6.99, and 127.49±2.9μg/mL against DLD-1, A549, and HepG2 cells, respectively. In vivo tumor inhibition experiments suggested that ARE possessed significant antitumor efficacy in BALB/c nude mice with a tumor inhibition rate of 49.83±14.38% (p<0.05) and little toxicity was observed to the host. ConclusionARE from H. angustifolia L. possessed high antioxidant activities is active against liver cancer HepG2, lung cancer A549 and colon cancer DLD-1 cells in vitro and tumor xenografts bearing BALB/c nude mice in vivo. Further studies on elucidation of the mechanisms involved and isolation of the active components may provide more valuable information for the development of functional products from H. angustifolia L. and their application in cancer treatment.