Dysregulation of intercellular communication in vitro and in vivo via extracellular vesicles secreted by pancreatic duct adenocarcinoma cells and generated under the influence of the AG9 elastin peptide-conditioned microenvironment.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy with poor prognosis due to its highly metastatic profile. Intercellular communication between cancer and stromal cells via extracellular vesicles (EVs) is crucial for the premetastatic microenvironment preparation leading to tumour metastasis. This study shows that under the influence of bioactive peptides derived from the extracellular matrix microenvironment, illustrated here by the AG-9 elastin-derived peptide (EDP), PDAC cells secrete more tumour-derived EVs. Compared to PDAC-derived EVs, tumour-derived EVs resulting from AG-9 treatment (PDAC AG-9-derived EVs) significantly stimulated cell proliferation. At constant amount, tumour-derived EVs were similarly taken up by PDAC and HMEC-1 cells. Tumour-derived EVs stimulated cell proliferation, migration, proteinase secretion, and angiogenesis. Bioluminescence imaging allowed tumour-derived EV/FLuc+ tracking in vivo in a PDAC mouse model. The biodistribution of PDAC AG-9-derived EVs was different to PDAC-derived EVs. Our results demonstrate that the microenvironment, through EDP release, may not only influence the genesis of EVs but may also affect tumour progression (tumour growth and angiogenesis), and metastatic homing by modifying the in vivo biodistribution of tumour-derived EVs. They are potential candidates for targeted drug delivery and modulation of tumour progression, and they constitute a new generation of therapeutic tools, merging oncology and genic therapy.

A multimodal imaging study to highlight elastin-derived peptide pro-tumoral effect in a pancreatic xenograft model.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is highly malignant with a very poor prognosis due to its silent development and metastatic profile with a 5-year survival rate below 10%. PDAC is characterised by an abundant desmoplastic stroma modulation that influences cancer development by extracellular matrix/cell interactions. Elastin is a key element of the extracellular matrix. Elastin degradation products (EDPs) regulate numerous biological processes such as cell proliferation, migration and invasion. The aim of the present study was to characterise for the first time the effect of two EDPs with consensus sequences "GxxPG" and "GxPGxGxG" (VG-6 and AG-9) on PDAC development. The ribosomal protein SA (RPSA) has been discovered recently, acting as a new receptor of EDPs on the surface of tumour cells, contributing to poor prognosis. METHODS: Six week-old female Swiss nude nu/nu (Nu(Ico)-Foxn1nu) mice were subcutaneously injected with human PDAC MIA PaCa-2/eGFP-FLuc+ cells, transduced with a purpose-made lentiviral vector, encoding green fluorescent protein (GFP) and Photinus pyralis (firefly) luciferase (FLuc). Animals were treated three times per week with AG-9 (n = 4), VG-6 (n = 5) or PBS (n = 5). The influence of EDP on PDAC was examined by multimodal imaging (bioluminescence imaging (BLI), fluorescence imaging (FLI) and magnetic resonance imaging (MRI). Tumour volumes were also measured using a caliper. Finally, immunohistology was performed at the end of the in vivo study. RESULTS: After in vitro validation of MIA PaCa-2 cells by optical imaging, we demonstrated that EDPs exacerbate tumour growth in the PDAC mouse model. While VG-6 stimulated tumour growth to some extent, AG-9 had greater impact on tumour growth. We showed that the expression of the RPSA correlates with a possible effect of EDPs in the PDAC model. Multimodal imaging allowed for longitudinal in vivo follow-up of tumour development. In all groups, we showed mature vessels ending in close vicinity of the tumour, except for the AG-9 group where mature vessels are penetrating the tumour reflecting an increase of vascularisation. CONCLUSIONS: Our results suggest that AG-9 strongly increases PDAC progression through an increase in tumour vascularisation.

F4, a collagen XIX-derived peptide, inhibits tumor angiogenesis through αvß3 and α5ß1 integrin interaction.

We previously demonstrated that F4 peptide (CNPEDCLYPVSHAHQR) from collagen XIX was able to inhibit melanoma cell migrationin vitro and cancer progression in a mouse melanoma model. The aim of the present work was to study the anti-angiogenic properties of F4 peptide. We demonstrated that F4 peptide inhibited VEGF-induced pseudo-tube formation on Matrigel by endothelial cells and endothelial sprouting in a rat aortic ring assay. By affinity chromatography, we identified αvß3 and α5ß1 integrins as potential receptors for F4 peptide on endothelial cell surface. Using solid phase assays, we proved the direct interaction between F4 and both integrins. Taken together, our results demonstrate that F4 peptide is a potent antitumor agent inhibiting both angiogenesis and tumor cell migration.

Extracellular Vesicle-Dependent Cross-Talk in Cancer-Focus on Pancreatic Cancer.

Extracellular vesicles (EVs) like exosomes and shed microvesicles are generated by many different cells. However, among all the cells, cancer cells are now recognized to secrete more EVs than healthy cells. Tumor-derived EVs can be isolated from biofluids such as blood, urine, ascitic fluid, and saliva. Their numerous components (nucleic acids, proteins, and lipids) possess many pleiotropic functions involved in cancer progression. The tumor-derived EVs generated under the influence of tumor microenvironment play distant roles and promote cellular communication by directly interacting with different cells. Moreover, they modulate extracellular matrix remodeling and tumor progression. Tumor-derived EVs are involved in pre-metastatic niche formation, dependent on the EV-associated protein receptors, and in cancer chemoresistance as they transfer drug-resistance-related genes to recipient cells. Recent advances in preclinical and clinical fields suggest their potential use as biomarkers for diagnosis and prognosis as well as for drug delivery in cancer. In this Review, we discuss EV characteristics and pro-tumor capacities, and highlight the future crucial impact of tumor-derived EVs in pancreatic cancer diagnosis and prognosis.

Angiogenesis Inhibition by a Short 13 Amino Acid Peptide Sequence of Tetrastatin, the α4(IV) NC1 Domain of Collagen IV.

Angiogenesis is defined as the formation of new capillaries by sprouting from the pre-existing microvasculature. It occurs in physiological and pathological processes particularly in tumor growth and metastasis. α1, α2, α3, and α6 NC1 domains from type IV collagen were reported to inhibit tumor angiogenesis. We previously demonstrated that the α4 NC1 domain from type IV collagen, named Tetrastatin, inhibited tumor growth in a mouse melanoma model. The inhibitory activity was located in a 13 amino acid sequence named QS-13. In the present paper, we demonstrate that QS-13 decreases VEGF-induced-angiogenesis in vivo using the Matrigel plug model. Fluorescence molecular tomography allows the measurement of a 65% decrease in Matrigel plug angiogenesis following QS-13 administration. The results are confirmed by CD31 microvessel density analysis on Matrigel plug slices. QS-13 peptide decreases Human Umbilical Vein Endothelial Cells (HUVEC) migration and pseudotube formation in vitro. Relevant QS-13 conformations were obtained from molecular dynamics simulations and docking. A putative interaction of QS-13 with α5ß1 integrin was investigated. The interaction was confirmed by affinity chromatography, solid phase assay, and surface plasmon resonance. QS-13 binding site on α5ß1 integrin is located in close vicinity to the RGD binding site, as demonstrated by competition assays. Collectively, our results suggest that QS-13 exhibits a mighty anti-angiogenic activity that could be used in cancer treatment and other pathologies with excessive angiogenesis such as hemangioma, psoriasis or diabetes.

TRPM7/RPSA Complex Regulates Pancreatic Cancer Cell Migration.

Pancreatic ductal adenocarcinoma (PDAC) is a malignancy with a very poor prognosis due to highly metastatic profile. Cell migration is an essential step of the metastatic cascade allowing cancer cells to spread toward target tissues. Recent studies strongly suggest that bioactive elastin peptides, also named elastokines or elastin-derived peptides (EDPs), are released in the extracellular microenvironment during tumoral remodeling of the stroma. EDPs stimulate cancer cell migration by interacting with their membrane receptor, ribosomal protein SA (RPSA). Others membrane proteins like ion channels are also involved in cancer cell migration. It has been recently shown that the transient receptor potential melastatin-related 7 (TRPM7) channel regulates PDAC cell migration and invasion. The objective of this work was to study the effect of EDPs on TRPM7 channel in human pancreatic cancer cells. We showed that EDPs promote MIA PaCa-2 cell migration using Boyden chamber assay. Cells transfected with a siRNA targeting TRPM7 were not able to migrate in response to EDPs indicating that TRPM7 regulated cell migration induced by these peptides. Moreover, EDPs were able to stimulate TRPM7 currents recorded by Patch-Clamp. Finally, we showed that TRPM7 channels and RPSA receptors are colocalized at the plasma membrane of human pancreatic cancer cells. Taken together, our data suggest that TRPM7/RPSA complex regulated human pancreatic cancer cell migration. This complex may be a promising therapeutic target in PDAC.

Immunotherapy in non-small-cell lung cancer: from targeted molecules to resistance patterns.

Immunotherapies are now considered as a pillar of non-small-cell lung cancer treatment. The main targets of immune-checkpoint inhibitors (ICI) are programmed cell death 1/programmed cell death ligand 1 and cytotoxic T-lymphocyte antigen 4, aiming at restoring antitumor immunity. Despite durable responses observed in some patients, all patients do not benefit from the treatment and almost all responders ultimately relapse after some time. In this review, we discuss the biomarkers that could be used to predict response to ICI, the current indications of ICI in non-small-cell lung cancer, the mechanisms inducing tumor-cell intrinsic or extrinsic resistance to ICI and finally, the potential treatment response monitoring.

Tumor Microenvironment: Extracellular Matrix Alterations Influence Tumor Progression.

The tumor microenvironment (TME) is composed of various cell types embedded in an altered extracellular matrix (ECM). ECM not only serves as a support for tumor cell but also regulates cell-cell or cell-matrix cross-talks. Alterations in ECM may be induced by hypoxia and acidosis, by oxygen free radicals generated by infiltrating inflammatory cells or by tumor- or stromal cell-secreted proteases. A poorer diagnosis for patients is often associated with ECM alterations. Tumor ECM proteome, also named cancer matrisome, is strongly altered, and different ECM protein signatures may be defined to serve as prognostic biomarkers. Collagen network reorganization facilitates tumor cell invasion. Proteoglycan expression and location are modified in the TME and affect cell invasion and metastatic dissemination. ECM macromolecule degradation by proteases may induce the release of angiogenic growth factors but also the release of proteoglycan-derived or ECM protein fragments, named matrikines or matricryptins. This review will focus on current knowledge and new insights in ECM alterations, degradation, and reticulation through cross-linking enzymes and on the role of ECM fragments in the control of cancer progression and their potential use as biomarkers in cancer diagnosis and prognosis.

Cadmium exposure enhances cell migration and invasion through modulated TRPM7 channel expression.

Cadmium is a xenobiotic involved in neoplastic transformation. Cadmium enters the cells through divalent cation transporters including the Transient Receptor Potential Melastatin-related 7 (TRPM7) which is known to be involved in cancer cell fate. This work aimed to study the role of TRPM7 in neoplastic transformation induced by cadmium exposure in non-cancer epithelial cells. Non-cancer epithelial cells were chronically exposed to low-dose of cadmium. TRPM7 expression and function were studied by Western-Blot, Patch-Clamp and calcium and magnesium imaging. Finally, cell migration and invasion were studied by Boyden chamber assays. Chronic cadmium exposure induced TRPM7 overexpression and increased the membrane currents (P < 0.001). Cells exposed to cadmium had higher intracellular calcium and magnesium levels (P < 0.05). TRPM7 silencing restored calcium levels but strongly decreased intracellular magnesium concentration (P < 0.001). Moreover, cadmium exposure enhanced both cell migration and invasion, but TRPM7 silencing strongly decreased these features (P < 0.001). Furthermore, mammary epithelial cells exposed to cadmium became rounded and had less cell-to-cell junctions. Cadmium exposure decreased epithelial markers while the mesenchymal ones were increased. Importantly, TRPM7 silencing was able to reverse these phenotypic modifications (P < 0.05). To summarize, our data show that chronic cadmium exposure enhanced TRPM7 expression and activity in non-cancer epithelial cells. TRPM7 overexpression induced intracellular magnesium increase and stimulated cell migration and invasion. These neoplastic properties could be linked to a TRPM7-dependent epithelial-to-mesenchymal transition reprogramming in cell exposed to cadmium. These findings provide new insights into the regulation of cell fates by cadmium exposure.

AG-9, an Elastin-Derived Peptide, Increases In Vitro Oral Tongue Carcinoma Cell Invasion, through an Increase in MMP-2 Secretion and MT1-MMP Expression, in a RPSA-Dependent Manner.

Oral tongue squamous cell carcinoma is one of the most prevalent head and neck cancers. During tumor progression, elastin fragments are released in the tumor microenvironment. Among them, we previously identified a nonapeptide, AG-9, that stimulates melanoma progression in vivo in a mouse melanoma model. In the present paper, we studied AG-9 effect on tongue squamous cell carcinoma invasive properties. We demonstrated that AG-9 stimulates cell invasion in vitro in a modified Boyen chamber model. It increases MMP-2 secretion, analyzed by zymography and MT1-MMP expression, studied by Western blot. The stimulatory effect was mediated through Ribosomal Protein SA (RPSA) receptor binding as demonstrated by SiRNA experiments. The green tea-derived polyphenol, (-)-epigallocatechin-3-gallate (EGCG), was previously shown to bind RPSA. Molecular docking experiments were performed to compare the preferred areas of interaction of AG-9 and EGCG with RPSA and suggested overlapping areas. This was confirmed by competition assays. EGCG abolished AG-9-induced invasion, MMP-2 secretion, and MT1-MMP expression.

Tumour cell blebbing and extracellular vesicle shedding: key role of matrikines and ribosomal protein SA.

BACKGROUND: Carcinogenesis occurs in elastin-rich tissues and leads to local inflammation and elastolytic proteinase release. This contributes to bioactive matrix fragment (Matrikine) accumulation like elastin degradation products (EDP) stimulating tumour cell invasive and metastatic properties. We previously demonstrate that EDPs exert protumoural activities through Hsp90 secretion to stabilised extracellular proteinases. METHODS: EDP influence on cancer cell blebbing and extracellular vesicle shedding were examined with a videomicroscope coupled with confocal Yokogawa spinning disk, by transmission electron microscopy, scanning electron microscopy and confocal microscopy. The ribosomal protein SA (RPSA) elastin receptor was identified after affinity chromatography by western blotting and cell immunolocalisation. mRNA expression was studied using real-time PCR. SiRNA were used to confirm the essential role of RPSA. RESULTS: We demonstrate that extracellular matrix degradation products like EDPs induce tumour amoeboid phenotype with cell membrane blebbing and shedding of extracellular vesicle containing Hsp90 and proteinases in the extracellular space. EDPs influence intracellular calcium influx and cytoskeleton reorganisation. Among matrikines, VGVAPG and AGVPGLGVG peptides reproduced EDP effects through RPSA binding. CONCLUSIONS: Our data suggests that matrikines induce cancer cell blebbing and extracellular vesicle release through RPSA binding, favouring dissemination, cell-to-cell communication and growth of cancer cells in metastatic sites.

Conformation-dependent binding of a Tetrastatin peptide to αvß3 integrin decreases melanoma progression through FAK/PI3K/Akt pathway inhibition.

Tetrastatin, a 230 amino acid sequence from collagen IV, was previously demonstrated to inhibit melanoma progression. In the present paper, we identified the minimal active sequence (QKISRCQVCVKYS: QS-13) that reproduced the anti-tumor effects of whole Tetrastatin in vivo and in vitro on melanoma cell proliferation, migration and invasion. We demonstrated that QS-13 binds to SK-MEL-28 melanoma cells through the αvß3 integrin using blocking antibody and ß3 integrin subunit siRNAs strategies. Relevant QS-13 conformations were extracted from molecular dynamics simulations and their interactions with αVß3 integrin were analyzed by docking experiments to determine the binding areas and the QS-13 amino acids crucial for the binding. The in silico results were confirmed by in vitro experiments. Indeed, QS-13 binding to SK-MEL-28 was dependent on the presence of a disulfide-bound as shown by mass spectroscopy and the binding site on αVß3 was located in close vicinity to the RGD binding site. QS-13 binding inhibits the FAK/PI3K/Akt pathway, a transduction pathway that is largely involved in tumor cell proliferation and migration. Taken together, our results demonstrate that the QS-13 peptide binds αvß3 integrin in a conformation-dependent manner and is a potent antitumor agent that could target cancer cells through αVß3.

Structural characterization and in vivo pro-tumor properties of a highly conserved matrikine.

Elastin-derived peptides (EDPs) exert protumor activities by increasing tumor growth, migration and invasion. A number of studies have highlighted the potential of VGVAPG consensus sequence-derived elastin-like polypeptides whose physicochemical properties and biocompatibility are particularly suitable for in vivo applications, such as drug delivery and tissue engineering. However, among the EDPs, the influence of elastin-derived nonapeptides (xGxPGxGxG consensus sequence) remains unknown. Here, we show that the AGVPGLGVG elastin peptide (AG-9) present in domain-26 of tropoelastin is more conserved than the VGVAPG elastin peptide (VG-6) from domain-24 in mammals. The results demonstrate that the structural features of AG-9 and VG-6 peptides are similar. CD, NMR and FTIR spectroscopies show that AG-9 and VG-6 present the same conformation, which includes a mixture of random coils and ß-turn structures. On the other hand, the supraorganization differs between peptides, as demonstrated by AFM. The VG-6 peptide gathers in spots, whereas the AG-9 peptide aggregates into short amyloid-like fibrils. An in vivo study showed that AG-9 peptides promote tumor progression to a greater extent than do VG-6 peptides. These results were confirmed by in vitro studies such as 2D and 3D proliferation assays, migration assays, adhesion assays, proteinase secretion studies and pseudotube formation assays to investigate angiogenesis. Our findings suggest the possibility that the AG-9 peptide present in patient sera may dramatically influence cancer progression and could be used in the design of new, innovative antitumor therapies.

A new gallium complex inhibits tumor cell invasion and matrix metalloproteinase MMP-14 expression and activity.

In this study, we investigated the effect of [N-(5-chloro-2-hydroxyphenyl)-l-aspartato] chlorogallate (GS2), a new water soluble gallium complex, on cell invasion and on the expression and activity of matrix metalloproteinases (MMPs) in human metastatic HT-1080 fibrosarcoma and MDA-MB 231 breast carcinoma cells. The effect on cell invasion was studied using a modified Boyden chamber coated with a type-I collagen. We analyzed the effect of GS2 on MMP-2, MMP-9, and MMP-14 via zymography and enzymatic assay using high affinity fluorogenic substrates. The expression of MMP mRNA was analyzed via qRT-PCR. GS2 induced a decrease in cell invasion. A dose-dependent inhibition effect was observed on the activities of MMP-2, MMP-9, and MMP-14 with the IC50 values of 168, 82, and 20 µM, respectively. A decrease in the expression of MMP-14 mRNA was observed in both cell lines, whereas the expression of MMP-2 and MMP-9 mRNA was decreased only in the MDA-MB231 cells. Data obtained for the expression of MMP-14 mRNA were confirmed via Western blotting. In fact, MMP-14 expression was decreased in the presence of GS2. Overall, these data show that GS2 is a promising compound for anti-invasive and anticancer therapy.

The Transient Receptor Potential Melastatin 7 Channel Regulates Pancreatic Cancer Cell Invasion through the Hsp90α/uPA/MMP2 pathway.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy with a very poor prognosis. There is an urgent need to better understand the molecular mechanisms that regulate PDAC cell aggressiveness. The transient receptor potential melastatin 7 (TRPM7) is a nonselective cationic channel that mainly conducts Ca2+ and Mg2+. TRPM7 is overexpressed in numerous malignancies including PDAC. In the present study, we used the PANC-1 and MIA PaCa-2 cell lines to specifically assess the role of TRPM7 in cell invasion and matrix metalloproteinase secretion. We show that TRPM7 regulates Mg2+ homeostasis and constitutive cation entry in both PDAC cell lines. Moreover, cell invasion is strongly reduced by TRPM7 silencing without affecting the cell viability. Conditioned media were further studied, by gel zymography, to detect matrix metalloproteinase (MMP) secretion in PDAC cells. Our results show that MMP-2, urokinase plasminogen activator (uPA), and heat-shock protein 90α (Hsp90α) secretions are significantly decreased in TRPM7-deficient PDAC cells. Moreover, TRPM7 expression in human PDAC lymph node metastasis is correlated to the channel expression in primary tumor. Taken together, our results show that TRPM7 is involved in PDAC cell invasion through regulation of Hsp90α/uPA/MMP-2 proteolytic axis, confirming that this channel could be a promising biomarker and possibly a target for PDAC metastasis therapy.

Tomentosin Induces Telomere Shortening and Caspase-Dependant Apoptosis in Cervical Cancer Cells.

Tomentosin, a natural sesquiterpene lactone purified from of Inula viscosa L., was investigated for its anti-proliferative, telomere shortening, and apoptotic effects on human cervical cancer HeLa and SiHa cell lines. Tomentosin was found to inhibit the growth of SiHa and HeLa cell lines in dose and time-dependent manner (IC50 values of 7.10 ± 0.78 µM and 5.87 ± 0.36 µM, respectively after 96 h of treatment). As evidenced by TTAGGG telomere length assay, tomentosin target specifically the telomeric overhang lengthening. This was confirmed by the evaluation of the cytotoxic effects of tomentosin in the foetal fibroblast Wi38 and JW10 cells which were derived from Wi38 and express hTERT, the telomerase catalytic subunit. We found that JW10 cells are 4.7-fold more sensitive to tomentosin which argues for telomere as its specific target. Furthermore, we found that tomentosin mediate this cytotoxic effect by inducing apoptosis and cell cycle arrest at G2/M phase. Morphological features of treated cells, as evidenced by Hoechst 33324 staining, revealed that the cytotoxic effect was due to induction of apoptosis. This was accompanied by pro-caspase-3 cleavage, an increase in caspase-3 activity and a cleavage of poly (ADP-ribose) polymerase (PARP). Moreover, tomentosin induced a decrease in mitochondrial membrane potential (ΔΨm) and an increase in reactive oxygen species (ROS), accompanied by a decrease in Bcl-2 expression. This indicates that tomentosin-induced apoptosis may involve a mitochondria-mediated signaling pathway. This study provides the first evidence that tomentosin targets telomere machinery and induces apoptosis in cervical cancer cells. The molecular mechanism underlying tomentosin-induced apoptosis may involve a mitochondria-mediated signaling pathway. J. Cell. Biochem. 118: 1689-1698, 2017. © 2016 Wiley Periodicals, Inc.

Type XIX collagen: A new partner in the interactions between tumor cells and their microenvironment.

Type XIX collagen is a minor collagen that is associated with the basement membrane zone that belongs to the FACIT family (Fibril-Associated Collagens with Interrupted Triple helices). The FACIT family is composed of type IX, XII, XIV, XVI, XX, XXI, XXII and XIX collagens, which share many highly conserved structural motifs: a short NC1 domain, a thrombospondin-like N-terminal domain (TSPN), and numerous cysteine residues. The main role of FACITs is to ensure the integrity and stability of the extracellular matrix and its fibrillar collagen network by regulating the formation and size of the collagen fibrils. Type XIX collagen was discovered in a human rhabdomyosarcoma cell line. The collagen α1(XIX) chain is composed of 5 triple-helical domains (COL) interrupted by 6 non-triple-helical (NC) domains with a short, C-terminal, 19 amino acid non-collagenous domain (NC1). This collagen is involved in the differentiation of muscle cells, central nervous system development, and formation of the esophagus. Type XIX collagen is associated with the basement membrane zone, like type XVIII and XV collagens. Its short NC1(XIX) C-terminal domain inhibits the migration and invasion of melanoma cells. It also exerts a strong anti-angiogenic effect by inhibiting MMP-14 and VEGF expression. NC1(XIX) binding to αvß3 integrin decreases the phosphorylation of proteins involved in the FAK (Focal Adhesion Kinase)/PI3K (PhosphoInositide 3-Kinase)/Akt (protein kinase B)/mTOR (Mammalian Target Of Rapamycin) pathway. On the other hand, NC1(XIX) induces an increase in GSK3ß activity by decreasing its level of phosphorylation. The inhibition of this pathway could explain the anti-tumor properties of the NC1(XIX) domain.

Aging decreases collagen IV expression in vivo in the dermo-epidermal junction and in vitro in dermal fibroblasts: possible involvement of TGF-ß1.

Collagen IV is a major component of the dermo-epidermal junction (DEJ). To study expression of collagen IV upon aging in the DEJ and dermal fibroblasts isolated from the same patients. A model of senescent fibroblasts was developed in order to identify biological compounds that might restore the level of collagen IV. Skin fragments of women (30 to 70 years old) were collected. Localisation of collagen IV expression in the DEJ was studied by immunofluorescence. Fibroblast collagen IV expression was studied by real-time PCR, ELISA, and western blotting. Premature senescence was simulated by exposing fibroblasts to subcytotoxic H2O2 concentrations. Collagen IV decreased in the DEJ and fibroblasts relative to age. TGF-ß1 treatment significantly increased collagen IV gene and protein expression in fibroblasts and restored expression in the model of senescence. Addition of TGF-ß1-neutralizing antibody to fibroblast cultures decreased collagen IV expression. Taken together, the results suggest that the decrease in collagen IV in the DEJ, relative to age, could be due to a decrease in collagen IV expression by senescent dermal fibroblasts and may involve TGF-ß1 signalling.

Inula Viscosa Extracts Induces Telomere Shortening and Apoptosis in Cancer Cells and Overcome Drug Resistance.

Telomerase is activated in human papillomavirus (HPV) positive cervical cancer and targeting telomeres offers a novel anticancer therapeutic strategy. In this study, the telomere targeting properties, the cytotoxic as well as the pro-apoptotic effects of hexane (IV-HE) and dichloromethane (IV-DF) fractions from Inula viscosa L. extracts were investigated on human cervical HeLa and SiHa cancer cells. Our data demonstrate that IV-HE and IV-DF extracts were able to inhibit cell growth in HeLa and SiHa cells in a dose-dependent manner and studied resistant cell lines exhibited a resistance factor less than 2 when treated with the extracts. IV-HE and IV-DF extracts were able to inhibit telomerase activity and to induce telomere shortening as shown by telomeric repeat amplification protocol and TTAGGG telomere length assay, respectively. The sensitivity of fibroblasts to the extracts was increased when telomerase was expressed. Finally, IV-HE and IV-DF were able to induce apoptosis as evidenced by an increase in annexin-V labeling and caspase-3 activity. This study provides the first evidence that the IV-HE and IV-DF extracts from Inula viscosa L. target telomeres induce apoptosis and overcome drug resistance in tumor cells. Future studies will focus on the identification of the molecules involved in the anticancer activity.

The anti-tumor NC1 domain of collagen XIX inhibits the FAK/ PI3K/Akt/mTOR signaling pathway through αvß3 integrin interaction.

Type XIX collagen is a minor collagen associated with basement membranes. It was isolated for the first time in a human cDNA library from rhabdomyosarcoma and belongs to the FACITs family (Fibril Associated Collagens with Interrupted Triple Helices). Previously, we demonstrated that the NC1 domain of collagen XIX (NC1(XIX)) exerts anti-tumor properties on melanoma cells by inhibiting their migration and invasion. In the present work, we identified for the first time the integrin αvß3 as a receptor of NC1(XIX). Moreover, we demonstrated that NC1(XIX) inhibits the FAK/PI3K/Akt/mTOR pathway, by decreasing the phosphorylation and activity of the major proteins involved in this pathway. On the other hand, NC1(XIX) induced an increase of GSK3ß activity by decreasing its degree of phosphorylation. Treatments targeting this central signaling pathway in the development of melanoma are promising and new molecules should be developed. NC1(XIX) seems to have the potential for the design of new anti-cancer drugs.