Human Fibrosarcoma HT1080 Cells Research Articles

Target irradiation induced bystander effects between stem-like and non stem-like cancer cells

Tumors are heterogeneous in nature and consist of multiple cell types. Among them, cancer stem-like cells (CSCs) are suggested to be the principal cause of tumor metastasis, resistance and recurrence. Therefore, understanding the behavior of CSCs in direct and indirect irradiations is crucial for clinical radiotherapy. Here, the CSCs and their counterpart non stem-like cancer cells (NSCCs) in human HT1080 fibrosarcoma cell line were sorted and labeled, then the two cell subtypes were mixed together and chosen separately to be irradiated via a proton microbeam. The radiation-induced bystander effect (RIBE) between the CSCs and NSCCs was measured by imaging 53BP1 foci, a widely used indicator for DNA double strand break (DSB). CSCs were found to be less active than NSCCs in both the generation and the response of bystander signals. Moreover, the nitric oxide (NO) scavenger c-PTIO can effectively alleviate the bystander effect in bystander NSCCs but not in bystander CSCs, indicating a difference of the two cell subtypes in NO signal response. To our knowledge, this is the first report shedding light on the RIBE between CSCs and NSCCs, which might contribute to a further understanding of the out-of-field effect in cancer radiotherapy.

Huaier aqueous extract induces apoptosis of human fibrosarcoma HT1080 cells through the mitochondrial pathway.

In recent years, aqueous extract of Trametes robiniophila Murr. (Huaier), a traditional Chinese medicine, has been frequently used in China for complementary cancer therapy. However, the mechanisms underlying its anticancer effects have yet to be elucidated. The present study aimed to evaluate the ability of Huaier extract to inhibit proliferation, promote apoptosis and suppress mobility in the fibrosarcoma HT1080 cell line in vitro. The cells were treated with gradient doses of Huaier extract at concentrations of 0, 4, 8 or 16 mg/ml for 24, 48 or 72 h. The cell viability and motility were measured in vitro using MTT, invasive, migration and scratch assays. The distribution of the cell cycle and the extent of cellular apoptosis were analyzed by flow cytometry. The apoptotic pathways were detected using a mitochondrial membrane potential transition assay and western blotting. The results revealed that the cellular viability decreased significantly with increasing concentrations of Huaier extract. In addition, cell invasiveness and migration were also suppressed significantly. It was demonstrated that Huaier extract induced G2 cell-cycle arrest and cellular apoptosis in a time- and dose-dependent manner. The decreased mitochondrial membrane potential, the downregulation of B-cell lymphoma 2 and pro-caspase-3, and upregulation of Bcl-2-associated X protein, cleaved caspase-9 and caspase-3 suggested that Huaier extract induced the apoptosis of HT1080 cells through the mitochondrial pathway. The results of the present study indicate that Huaier extract is a potential complementary agent for the treatment of fibrosarcoma.

Antioxidant Peptide Derived fromSpirulina maximaSuppresses HIF1α-Induced Invasive Migration of HT1080 Fibrosarcoma Cells

Hypoxia causes the malignant progression of tumor cells; hence, it has been considered a central issue that must be addressed for effective cancer therapy. The initiation of tumor metastasis requires invasive cell migration. Here, we show that an antioxidant peptide derived fromSpirulina maximasuppresses hypoxia-induced invasive migration of HT1080 human fibrosarcoma cells. HT1080 cells treated with a hypoxia-inducing agent, CoCl2, exhibited an increase in invasive migration and intracellular reactive oxygen species (ROS), which is associated with an increase in the expression of hypoxia-induced factor 1α(HIF1α) accompanied by the activation of PI3K/Akt and ERK1/2. The inhibition of PI3K/Akt and ERK1/2 with specific inhibitors diminished the CoCl2-induced increase in HIF1αexpression and invasive cell migration. Moreover, CoCl2-induced HIF1αexpression was associated with an increase in the expression of molecules downstream ofβ-integrin, such as N-cadherin, vimentin, andβ-catenin. Therefore, theS. maximapeptide effectively attenuated the CoCl2-induced ROS generation and downregulated the HIF1αsignaling pathway involving PI3K/Akt, ERK1/2, andβ-integrin in cells. These results suggest that theS. maximaantioxidant peptide downregulates the HIF1αsignaling pathway necessary for hypoxia-induced invasive migration of HT1080 cells by attenuating intracellular ROS.S. maximapeptide may be an effective constituent in antitumor progression products.

Abstract A06: The role of the epidermal growth factor receptor (EGFR) in primary tumor cell escape and development of an intravasation-sustaining vasculature

Abstract Metastasis of malignant cells is regarded as the leading cause of cancer-related deaths. Metastasis is a complex process whereby a subset or individual cancer cells disseminate from the primary tumor to distant/secondary sites. Intravasation, which is the entry of escaping tumor cells into the vasculature, is a critical and rate-limiting early event in the metastatic cascade. The complex process of intravasation is difficult to model and quantify in vivo, and therefore it remains the least studied step of the metastatic cascade. To study intravasation, we employ the chick embryo model involving grafting of human cancer cells on the chorioallantoic membrane (CAM), the highly vascularized tissue capable of supporting primary tumor growth and development of angiogenic vasculature. The epidermal growth factor receptor (EGFR) is known to play an important role in cancer progression and EGFR-induced signaling has been associated with tumor invasion and metastasis. In this study, we investigated the specific roles of EGFR in the early steps of tumor metastasis such as escape of cancer cells from the primary tumor and development of angiogenic vasculature, the two critical events required for tumor cell intravasation. For this purpose we employed the EGFR-overexpressing highly disseminating variant of human fibrosarcoma HT-1080 cell line, HT-hi/diss, in several independent in vivo CAM models. In the spontaneous metastasis model, siRNA silencing of EGFR significantly reduced the levels of HT-hi/diss intravasation, suggesting a role of EGFR overexpression in tumor cell escape and/or development of intravasation-sustaining vasculature. We have demonstrated that EGFR silencing prevented HT-hi/diss cell escape from the primary microtumors in the intramesodermal model, linking EGFR-regulated cell motility to tumor cell intravasation. We further investigated the role of EGFR in development of tumor-associated vasculature. By using our newly developed topical microtumor model, we have demonstrated that both control and EGFR-silenced cells attract comparable numbers of angiogenic vessels converging to developing primary microtumors. However, epifluorescence microscopy revealed dramatic difference in the microarchitecture of blood vessels within the two types of microtumors. Whereas control microtumors exhibited well-developed networks of intratumoral blood vessels with lumen diameter of 15-30 µm, EGFR-silenced microtumors contained only thin blood vessels with lumen diameter less than 15 µm. This lack of appropriately dilated intratumoral vasculature in EGFR-silenced tumors directly correlated with a significant decrease in vascular permeability concomitant with the suppression of tumor cell intravasation. These data highlight a specific aspect of EGFR functionality that is different from its well-established role in transcriptional regulation of genes required for tumor growth and suggest a novel mechanism whereby tumor cell EGFR regulates microenvironmental cues for induction, development, and functionality of an intravasation-sustaining vasculature. Citation Format: Petra Minder, Elena I. Deryugina, James P. Quigley. The role of the epidermal growth factor receptor (EGFR) in primary tumor cell escape and development of an intravasation-sustaining vasculature. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr A06. doi:10.1158/1538-7445.CHTME14-A06

3-(3-Hydroxy-4-methoxyphenyl)-4-(3,4,5-trimethoxyphenyl)-1,2,5-selenadiazole (G-1103), a novel combretastatin A-4 analog, induces G2/M arrest and apoptosis by disrupting tubulin polymerization in human cervical HeLa cells and fibrosarcoma HT-1080 cells

Microtubule is a popular target for anticancer drugs. In this study, we describe the effect 3-(3-hydroxy-4-methoxyphenyl)-4-(3,4,5-trimethoxyphenyl)-1,2,5-selenadiazole (G-1103), a newly synthesized analog of combretastatin A-4 (CA-4), showing a strong time- and dose-dependent anti-proliferative effect on human cervical cancer HeLa cells and human fibrosarcoma HT-1080 cells. We demonstrated that the growth inhibitory effects of G-1103 in HeLa and HT-1080 cells were associated with microtubule depolymerization and proved that G-1103 acted as microtubule destabilizing agent. Furthermore, cell cycle analysis revealed that G-1103 treatment resulted in cell cycle arrest at the G2/M phase in a time-dependent manner with subsequent apoptosis induction. Western blot analysis revealed that down-regulation of cdc25c and up-regulation of cyclin B1 was related with G2/M arrest in HeLa and HT-1080 cells treatment with G-1103. In addition, G-1103 induced HeLa cell apoptosis by up-regulating cleaved caspase-3, Fas, cleaved caspase-8 expression, which indicated that G-1103 induced HeLa cell apoptosis was mainly associated with death receptor pathway. However, G-1103 induced HT-1080 cell apoptosis by up-regulating cleaved caspase-3, Fas, cleaved caspase-8, Bax and cleaved caspase-9 expression and down-regulating anti-apoptotic protein Bcl-2 expression, which indicated that G-1103 induced HT-1080 cell apoptosis was associated with both mitochondrial and death receptor pathway. Taken together, all the data demonstrated that G-1103 exhibited its antitumor activity through disrupting the microtubule assembly, causing cell cycle arrest and consequently inducing apoptosis in HeLa and HT-1080 cells. Therefore, the novel compound G-1103 is a promising microtubule inhibitor that has great potentials for therapeutic treatment of various malignancies.

Activation of apoptosis by caspase-3-dependent specific RelB cleavage in anticancer agent-treated cancer cells: Involvement of positive feedback mechanism

DTCM-glutarimide (DTCM-G) is a newly found anti-inflammatory agent. In the course of experiments with lymphoma cells, we found that DTCM-G induced specific RelB cleavage. Anticancer agent vinblastine also induced the specific RelB cleavage in human fibrosarcoma HT1080 cells. The site-directed mutagenesis analysis revealed that the Asp205 site in RelB was specifically cleaved possibly by caspase-3 in vinblastine-treated HT1080 cells. Moreover, the cells stably overexpressing RelB Asp205Ala were resistant to vinblastine-induced apoptosis. Thus, the specific Asp205 cleavage of RelB by caspase-3 would be involved in the apoptosis induction by anticancer agents, which would provide the positive feedback mechanism.

Galangin and kaempferol suppress phorbol-12-myristate-13-acetate-induced matrix metalloproteinase-9 expression in human fibrosarcoma HT-1080 cells.

Matrix metalloproteinase (MMP)-9 degrades type IV collagen in the basement membrane and plays crucial roles in several pathological implications, including tumorigenesis and inflammation. In this study, we analyzed the effect of flavonols on MMP-9 expression in phorbol-12-myristate-13-acetate (PMA)-induced human fibrosarcoma HT-1080 cells. Galangin and kaempferol efficiently decreased MMP-9 secretion, whereas fisetin only weakly decreased its secretion. Galangin and kaempferol did not affect cell viability at concentrations up to 30 μM. Luciferase reporter assays showed that galangin and kaempferol decrease transcription of MMP-9 mRNA. Moreover, galangin and kaempferol strongly reduce IκBα phosphorylation and significantly decrease JNK phosphorylation. These results indicate that galangin and kaempferol suppress PMA-induced MMP-9 expression by blocking activation of NF-κB and AP-1. Therefore, these flavonols could be used as chemopreventive agents to lower the risk of diseases involving MMP-9.

Scutellarein inhibits cancer cell metastasis in vitro and attenuates the development of fibrosarcoma in vivo.

Fibrosarcoma is an aggressive and highly metastatic cancer of the connective tissue, for which effective therapeutic methods are limited. Recently, there has been a renewed interest in small molecular compounds from natural products in the treatment of cancer. In the present study, we investigated the compound, scutellarein, extracted from the perennial herb Scutellaria lateriflora, and it was found to possess anticancer potential. Cell proliferation assay and cell cycle analysis revealed that the proliferation rate of HT1080 human fibrosarcoma cells was significantly suppressed by treatment with scutellarein through the induction of apoptosis. Moreover, an in vivo experiment using Balb/c nude mice revealed that the volume and weight of the tumors were markedly reduced following treatment with scutellarein. We also analyzed the effects of scutellarein on the markers of metastasis, using the HT1080 cells. The results indicated that scutellarein potently inhibited cell migration, invasion and the expression and activity of matrix metalloproteinase (MMP)-2, -9 and -14. Furthermore, MMP activation and cell survival were suppressed due to the scutellarein-mediated downregulation of nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) activation. In conclusion, our data suggest that scutellarein has the ability to attenuate the development of fibrosarcoma and inhibit cancer cell metastasis.

Separation of replication and transcription domains in nucleoli.

In mammalian cells, active ribosomal genes produce the 18S, 5.8S and 28S RNAs of ribosomal particles. Transcription levels of these genes are very high throughout interphase, and the cell needs a special strategy to avoid collision of the DNA polymerase and RNA polymerase machineries. To investigate this problem, we measured the correlation of various replication and transcription signals in the nucleoli of HeLa, HT-1080 and NIH 3T3 cells using a specially devised software for analysis of confocal images. Additionally, to follow the relationship between nucleolar replication and transcription in living cells, we produced a stable cell line expressing GFP-RPA43 (subunit of RNA polymerase I, pol I) and RFP-PCNA (the sliding clamp protein) based on human fibrosarcoma HT-1080 cells. We found that replication and transcription signals are more efficiently separated in nucleoli than in the nucleoplasm. In the course of S phase, separation of PCNA and pol I signals gradually increased. During the same period, separation of pol I and incorporated Cy5-dUTP signals decreased. Analysis of single molecule localization microscopy (SMLM) images indicated that transcriptionally active FC/DFC units (i.e. fibrillar centers with adjacent dense fibrillar components) did not incorporate DNA nucleotides. Taken together, our data show that replication of the ribosomal genes is spatially separated from their transcription, and FC/DFC units may provide a structural basis for that separation.

Abstract 4027: Traditional Chinese Medicine herbal mixture LQ prevents cyclophosphamide-induced metastasis and toxicity

Abstract Cyclophosphamide (CTX) can enhance metastasis (Cancer Res. 66, 303-306, 2006; Cancer Res. 68, 516-520, 2008) and has toxic side effects such as immune-suppression, weight loss, and hair loss. In the present study, the traditional Chinese medicine (TCM) herbal mixture LQ (J. Cell. Biochem. 114, 2131-2137, 2013) was tested to determine if it could enhance CTX efficacy and reduce side effects. HT1080 human fibrosarcoma cells, labeled with GFP in the nucleus and RFP in the cytoplasm, were injected into the epigastric cranialis vein of nude mice that were pre-treated with CTX and LQ. Lewis lung carcinoma (LLC)-RFP cells were injected into the tail vein of mice that were pre-treated with CTX and LQ. Efficacy was monitored with dynamic subcellular imaging of trafficking of cancer cells in blood vessels (Cancer Res. 68, 516-520, 2008) and imaging metastasis to the lung. LQ significantly reduced CTX-induced lung metastasis and cancer-cell adhesion to the inner wall of blood vessels. LQ also reduced kidney toxicity induced by CTX. Treatment with LQ induced hair growth after CTX administration. This result demonstrates that LQ provides safe and effective prevention of metastasis and toxicity in combination with chemotherapeutic agents such as CTX. Citation Format: Lei Zhang, Chengyu Wu, Lingna Li, Yong Zhang, Robert M. Hoffman. Traditional Chinese Medicine herbal mixture LQ prevents cyclophosphamide-induced metastasis and toxicity. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4027. doi:10.1158/1538-7445.AM2014-4027

Abstract LB-69: Rab11 mediated focal adhesion turnover in sarcoma cell migration

Abstract Cell migration is central for many physiological and pathological processes, including embryogenesis, tissue repair, inflammatory responses, and cancer metastasis. Focal adhesion (FA) turnover has been demonstrated to play important role in controlling cell migration. Recently, FA disassembly was shown to be regulated by endocytosis during cell migration. Rabs are GTPase proteins that participate in various cellular vesicle transports; however, whether Rab proteins play the role in mediating vesicle transport for FA formation is not clear. This study is examining whether Rab11 mediated endosome recycling regulates FA formation during cell migration. Rab11 Wild-type, dominant negative or shRNA were transfected into human HT1080 fibrosarcoma cells, the cell motility was determined by trans-well assay and random migration assay, the localization of Rab11 and focal adhesion molecules were monitored by confocal microscopy. Results showed the Rab11 dominant negative form or shRNA transfection inhibited chemoattractive cell migration, however, only the Rab11 shRNA transfection inhibited random cell migration. Rab11 was also found colocalized with recycled β1 integrin and focal adhesion kinase and affected focal adhesion formation. Taken together, the results suggested Rab11 affected cell migration by regulating FAK dynamics and the activity of Rab11 is required for external stimulation. The interaction of Rab11 with associated focal adhesion molecules will be further investigated. Citation Format: Ling-Yi Kao, Yi-Che Wu, Wei-Ting Chao. Rab11 mediated focal adhesion turnover in sarcoma cell migration. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-69. doi:10.1158/1538-7445.AM2014-LB-69

Probing single-tumor cell interactions with different-age type I collagen networks by synchrotron-based Fourier transform infrared microspectroscopy

We report here on a first study using synchrotron radiation-based Fourier transform infrared microspectroscopy and imaging to investigate HT1080 human fibrosarcoma cells grown onto different-aged type I collagen networks. Spectral images were analyzed with k-means and fuzzy C-means (FCM) clustering algorithms. K-means delineated tumor cells from their surrounding collagen networks and the latter as a function of age mainly due to specific changes in the sugar absorption region. The FCM analysis gave a better nuance of the spectral images. A progression of the biochemical information was observed upon going from the cellular compartments to the pericellular contact regions and to the intact collagens of the different age groups. Two spectral markers based on sugar and protein bands via the intensity ratio (I1032/I1655) and band area ratio (Asugar/Aamide II), showed an increase in advanced glycation endproducts (AGEs) with age. A clear-separation of the three age groups was obtained for spectra originating from the peripheral contact areas mainly due to changes in protein band intensities. The above-described markers decreased to constant levels for the three conditions indicating a masking of the biochemical information. These results hold promises to better understand the impact of age on tumor progression processes while highlighting new markers of the tumor cell invasion front.

Inhibitory effect of the carnosine-gallic acid synthetic peptide on MMP-2 and MMP-9 in human fibrosarcoma HT1080 cells.

Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases that degrade extracellular matrix components and play important roles in a variety of biological and pathological processes such as malignant tumor metastasis and invasion. In this study, we constructed carnosine-gallic acid peptide (CGP) to identify a better MMP inhibitor than carnosine. The inhibitory effects of CGP on MMP-2 and MMP-9 were investigated in the human fibrosarcoma (HT1080) cell line. As a result, CGP significantly decreased MMP-2 and MMP-9 expression levels without a cytotoxic effect. Moreover, CGP may inhibit migration and invasion in HT1080 cells through the urokinase plasminogen activator (uPA)-uPA receptor signaling pathways to inhibit MMP-2 and MMP-9. Based on these results, it appears that CGP may play an important role in preventing and treating several MMP-2 and MMP-9-mediated health problems such as metastasis.

Actinopyrone D, a new downregulator of the molecular chaperone GRP78 from Streptomyces sp.

A new downregulator of the molecular chaperone GRP78, actinopyrone D, was isolated together with a known related compound, PM050463, from Streptomyces sp. RAG92. The molecular formula of actinopyrone D was established as C25H36O4 by high-resolution FAB-MS. NMR spectroscopic analysis revealed the structure of actinopyrone D, which consists of an α-methoxy-γ-pyrone ring and a C17 side chain containing a cis olefin moiety. Actinopyrone D and PM050463 dose-dependently inhibited 2-deoxyglucose-induced luciferase expression in HT1080 human fibrosarcoma cells transfected with a luciferase reporter plasmid containing the GRP78 promoter. Actinopyrone D inhibited GRP78 protein expression and induced cell death under endoplasmic reticulum stress.

A novel herbal medicine, KIOM-C, induces autophagic and apoptotic cell death mediated by activation of JNK and reactive oxygen species in HT1080 human fibrosarcoma cells.

KIOM-C was recently demonstrated to have anti-metastatic activity in highly malignant cancer cells via suppression of NF-κB-mediated MMP-9 activity. In addition, it was reported to be effective for clearance of the influenza virus by increasing production of anti-viral cytokines, such as TNF-α and IFN-γ, and efficacious in the treatment of pigs suffering from porcine circovirus-associated disease (PCVAD). In this study, we investigated whether KIOM-C induces cancer cell death and elucidated the underlying anti-cancer mechanisms. In addition, we examined whether KIOM-C oral administration suppresses in vivo tumor growth of HT1080 cells in athymic nude mice. We initially found that KIOM-C at concentrations of 500 and 1000 µg/ml caused dose- and time-dependent cell death in cancer cells, but not normal hepatocytes, to approximately 50% of control levels. At the early stage of KIOM-C treatment (12 h), cells were arrested in G1 phase, which was accompanied by up-regulation of p21 and p27, down-regulation of cyclin D1, and subsequent increases in apoptotic and autophagic cells. Following KIOM-C treatment, the extent of caspase-3 activation, PARP cleavage, Beclin-1 expression, and LC3-II conversion was remarkably up-regulated, but p62 expression was down-regulated. Phosphorylation of AMPK, ULK, JNK, c-jun, and p53 was increased significantly in response to KIOM-C treatment. The levels of intracellular ROS and CHOP expression were also increased. In particular, the JNK-specific inhibitor SP600125 blocked KIOM-C-induced ROS generation and CHOP expression almost completely, which consequently almost completely rescued cell death, indicating that JNK activation plays a critical role in KIOM-C-induced cell death. Furthermore, daily oral administration of 85 and 170 mg/kg KIOM-C efficiently suppressed the tumorigenic growth of HT1080 cells, without systemic toxicity. These results collectively suggest that KIOM-C efficiently induces cancer cell death by both autophagy and apoptosis via activation of JNK signaling pathways, and KIOM-C represents a safe and potent herbal therapy for treating malignancies.

Synthesis and antiproliferative effects of amino-modified perillyl alcohol derivatives.

Two series of amino-modified derivatives of (S)-perillyl alcohol were designed and synthesized using (S)-perillaldehyde as the starting material. These derivatives showed increased antiproliferative activity in human lung cancer A549 cells, human melanoma A375-S2 cells and human fibrosarcoma HT-1080 cells comparing with that of (S)-perillyl alcohol. Among these derivatives, compounds VI5 and VI7 were the most potent agents, with the IC50s below 100 μM. It was demonstrated that the antiproliferative effect of VI5 was mediated through the induction of apoptosis in A549 cells.

In Situ Generation of Tunable Porosity Gradients in Hydrogel‐Based Scaffolds for Microfluidic Cell Culture

Compared with preformed anisotropic matrices, an anisotropic matrix that allows users to alter its properties and structure in situ after synthesis offers the important advantage of being able to mimic dynamic in vivo microenvironments, such as in tissues undergoing morphogenesis or in wounds undergoing tissue repair. In this study, porous gradients are generated in situ in a hydrogel comprising enzymatically crosslinked gelatin hydroxyphenylpropionic acid (GTN-HPA) conjugate and carboxylmethyl cellulose tyramine (CMC-TYR) conjugate. The GTN-HPA component acts as the backbone of the hydrogel, while CMC-TYR acts as a biocompatible sacrificial polymer. The hydrogel is then used to immobilize HT1080 human fibrosarcoma cells in a microfluidic chamber. After diffusion of a biocompatible cellulase enzyme through the hydrogel in a spatially controlled manner, selective digestion of the CMC component of the hydrogel by the cellulase gives rise to a porosity gradient in situ instead of requiring its formation during hydrogel synthesis as with other methods. The influence of this in situ tunable porosity gradient on the chemotactic response of cancer cells is subsequently studied both in the absence and presence of chemoattractant. This platform illustrates the potential of hydrogel-based microfluidics to mimic the 3D in vivo microenvironment for tissue engineering and diagnostic applications.

Lysophosphatidic acid receptor-5 negatively regulates cell motile and invasive activities of human sarcoma cell lines

LPA signaling via LPA receptors [LPA receptor-1 (LPA1)-LPA6] mediates the several cellular responses in cancer cells, including cell motility and invasion. In the present study, to investigate a role of LPA5 in the cell motile and invasive activities of sarcoma cells, LPAR5 knockdown (HOSL5 and HT1080L5) cells were generated from human osteosarcoma HOS and fibrosarcoma HT1080 cells, respectively. In cell motility assays with cell culture inserts, HOSL5 and HT1080L5 cells indicated the high cell motile activities, compared with control cells. The cell invasive activities of HOSL5 and HT1080L5 cells were significantly higher than those of control cells. Moreover, the activities of matrix metalloproteinase (MMP)-2 and MMP-9 were measured by gelatin zymography. MMP-2 was significantly activated in HOSL5 cells, but not MMP-9. The elevated activities of MMP-2 and MMP-9 were found in HT1080L5 cells, in comparison with control cells. These results suggest that LPA signaling via LPA5 negatively regulates the cell motile and invasive activities of human sarcoma cells.

Photodynamic anti-cancer effects of fullerene [C60]–PEG complex on fibrosarcomas preferentially over normal fibroblasts in terms of fullerene uptake and cytotoxicity

A water-soluble complex of fullerene [C60]:polyethylene glycol (PEG) (1:350 wt/wt) (C60-PEG), but not PEG alone, was found in the present study by ESR/DMPO spin-trap method to generate hydroxyl radicals 6.5-fold as abundant as the non-irradiation level, when irradiated with visible light (400-600 nm, 140 J/cm(2): 450-fold as intense as in average outdoor), but not to generate without irradiation. At 3 h after irradiation with C60-PEG, human fibrosarcoma cells HT1080 were obviously degenerated together with diminished microvilli, cell shrinkage and cell fragmentation as observed by SEM and were shown either for increased cytotoxicity by dual stains with calcein-AM and propidium iodide or for nuclear condensation and fragmentation by Hoechst 33342 stain, any of which were, in contrast, scarcely changed in normal human fibroblastic cells DUMS16 derived from the same connective tissue type as HT1080 cells. Under the conditions, the maximum intracellular uptake amount was more abundant for HT1080 cells than for DUMS16 cells, either by immunostain/fluorography using polyclonal antibody against fullerene [C60], or by HPLC method indicating the 2.4-fold preferential uptake of C60-PEG into HT1080 cells, suggested to greater phagocytotic ability characteristic of cancer cells, over DUMS16 cells being non-macrophage-like normal cells. Thus, C60-PEG is expected as a photosensitizer for photodynamic therapy with scarce side effects to normal cells and preferential reactive oxygen species generation in cancer cells.

Cell-penetrating peptide-conjugated lipid nanoparticles for siRNA delivery

Lipid nanoparticles (LNP) modified with cell-penetrating peptides (CPP) were prepared for the delivery of small interfering RNA (siRNA) into cells. Lipid derivatives of CPP derived from protamine were newly synthesized and used to prepare CPP-decorated LNP (CPP-LNP). Encapsulation of siRNA into CPP-LNP improved the stability of the siRNA in serum. Fluorescence-labeled siRNA formulated in CPP-LNP was efficiently internalized into B16F10 murine melanoma cells in a time-dependent manner, although that in LNP without CPP was hardly internalized into these cells. In cells transfected with siRNA in CPP-LNP, most of the siRNA was distributed in the cytoplasm of these cells and did not localize in the lysosomes. Analysis of the endocytotic pathway indicated that CPP-LNP were mainly internalized via macropinocytosis and heparan sulfate-mediated endocytosis. CPP-LNP encapsulating siRNA effectively induced RNA interference-mediated silencing of reporter genes in B16F10 cells expressing luciferase and in HT1080 human fibrosarcoma cells expressing enhanced green fluorescent protein. These data suggest that modification of LNP with the protamine-derived CPP was effective to facilitate internalization of siRNA in the cytoplasm and thereby to enhance gene silencing.