Cancer-associated fibroblasts induce high mobility group box 1 and contribute to resistance to doxorubicin in breast cancer cells.
ABSTRACT: BACKGROUND: Cancer-associated fibroblasts and high mobility group box 1 (HMGB1) protein have been suggested to mediate cancer progression and chemotherapy resistance. The role of such fibroblasts in HMGB1 production in breast cancer is unclear. This study aimed to investigate the effects of cancer-associated fibroblasts on HMGB1 expression in breast cancer cells and its role in chemotherapeutic response. METHODS: Breast cancer-associated fibroblasts (BCFs) and non-tumor-associated fibroblasts (NTFs) were isolated from human breast cancers or adjacent normal tissues and established as primary cultures in vitro. After confirmation of the activated status of these fibroblasts, conditioned-media (CM) were collected and applied to MDA-MB-231 human triple negative breast cancer cells. The levels of intracellular and extracellular HMGB1 were measured by real-time PCR and/or Western blot. The response of BCF-CM-pre-treated cancer cells to doxorubicin (Dox) was compared with those pre-treated with NTF-CM or control cultures. The effect of an HMGB1 neutralizing antibody on Dox resistance induced by extracellular HMGB1 from non-viable Dox-treated cancer cells or recombinant HMGB1 was also investigated. RESULTS: Immunocytochemical analysis revealed that BCFs and NTFs were alpha-smooth muscle actin (ASMA) positive and cytokeratin 19 (CK19) negative cells: a phenotype consistent with that of activated fibroblasts. We confirmed that the CM from BCFs (but not NTFs), could significantly induce breast cancer cell migration. Intracellular HMGB1 expression was induced in BCF-CM-treated breast cancer cells and also in Dox-treated cells. Extracellular HMGB1 was strongly expressed in the CM after Dox-induced MDA-MB-231 cell death and was higher in cells pre-treated with BCF-CM than NTF-CM. Pre-treatment of breast cancer cells with BCF-CM induced a degree of resistance to Dox in accordance with the increased level of secreted HMGB1. Recombinant HMGB1 was shown to increase Dox resistance and this was associated with evidence of autophagy. Anti-HMGB1 neutralizing antibody significantly reduced the effect of extracellular HMGB1 released from dying cancer cells or of recombinant HMGB1 on Dox resistance. CONCLUSIONS: These findings highlight the potential of stromal fibroblasts to contribute to chemoresistance in breast cancer cells in part through fibroblast-induced HMGB1 production.
Project description:Biomass conversion factors (BCFs, defined as the ratios of tree components (i.e. stem, branch, foliage and root), as well as aboveground and whole biomass of trees to growing stock volume, Mg m-3) are considered as important parameters in large-scale forest biomass carbon estimation. To date, knowledge of possible sources of the variation in BCFs is still limited at large scales. Using our compiled forest biomass dataset of China, we presented forest type-specific values of BCFs, and examined the variation in BCFs in relation to forest type, stand development and environmental factors (climate and soil fertility). BCFs exhibited remarkable variation across forest types, and also were significantly related to stand development (especially growing stock volume). BCFs (except Stem BCF) had significant relationships with mean annual temperature (MAT) and mean annual precipitation (MAP) (P<0.001). Climatic data (MAT and MAP) collectively explained 10.0-25.0% of the variation in BCFs (except Stem BCFs). Moreover, stronger climatic effects were found on BCFs for functional components (i.e. branch, foliage and root) than BCFs for combined components (i.e. aboveground section and whole trees). A general trend for BCFs was observed to decrease and then increase from low to high soil fertility. When qualitative soil fertility and climatic data (MAT and MAP) were combined, they explained 14.1-29.7% of the variation in in BCFs (except Stem BCFs), adding only 4.1-4.9% than climatic data used. Therefore, to reduce the uncertainty induced by BCFs in forest carbon estimates, we should apply values of BCFs for a specified forest type, and also consider climatic and edaphic effects, especially climatic effect, in developing predictive models of BCFs (except Stem BCF).
Project description:BACKGROUND:Brain metastases are a major cause of death in patients with metastatic breast cancer. While surgical resection and radiation therapy are effective treatment modalities, the majority of patients will succumb from disease progression. We have developed a novel therapy for brain metastases that delivers athermal radiofrequency electromagnetic fields that are amplitude-modulated at breast cancer specific frequencies (BCF). METHODS:27.12?MHz amplitude-modulated BCF were administered to a patient with a breast cancer brain metastasis by placing a spoon-shaped antenna on the anterior part of the tongue for three one-hour treatments every day. In preclinical models, a BCF dose, equivalent to that delivered to the patient's brain, was administered to animals implanted with either brain metastasis patient derived xenografts (PDXs) or brain-tropic cell lines. We also examined the efficacy of combining radiation therapy with BCF treatment. Additionally, the mechanistic underpinnings associated with cancer inhibition was identified using an agnostic approach. FINDINGS:Animal studies demonstrated a significant decrease in growth and metastases of brain-tropic cell lines. Moreover, BCF treatment of PDXs established from patients with brain metastases showed strong suppression of their growth ability. Importantly, BCF treatment led to significant and durable regression of brain metastasis of a patient with triple negative breast cancer. The tumour inhibitory effect was mediated by Ca2+ influx in cancer cells through CACNA1H T-type voltage-gated calcium channels, which, acting as the cellular antenna for BCF, activated CAMKII/p38 MAPK signalling and inhibited cancer stem cells through suppression of ?-catenin/HMGA2 signalling. Furthermore, BCF treatment downregulated exosomal miR-1246 level, which in turn decreased angiogenesis in brain environment. Therefore, targeted growth inhibition of breast cancer metastases was achieved through CACNA1H. INTERPRETATION:We demonstrate that BCF, as a single agent or in combination with radiation, is a novel treatment approach to the treatment of brain metastases. This paradigm shifting modality warrants further clinical trials for this unmet medical need.
Project description:BACKGROUND: Cancer-associated fibroblasts (CAFs) activated by tumour cells are the predominant type of stromal cells in breast cancer tissue. The reciprocal effect of CAFs on breast cancer cells and the underlying molecular mechanisms are not fully characterised. METHODS: Stromal fibroblasts were isolated from invasive breast cancer tissues and the conditioned medium of cultured CAFs (CAF-CM) was collected to culture the breast cancer cell lines MCF-7, T47D and MDA-MB-231. Neutralising antibody and small-molecule inhibitor were used to block the transforming growth factor-? (TGF-?) signalling derived from CAF-CM, which effect on breast cancer cells. RESULTS: The stromal fibroblasts isolated from breast cancer tissues showed CAF characteristics with high expression levels of ?-smooth muscle actin and SDF1/CXCL12. The CAF-CM transformed breast cancer cell lines into more aggressive phenotypes, including enhanced cell-extracellular matrix adhesion, migration and invasion, and promoted epithelial-mesenchymal transition (EMT). Cancer-associated fibroblasts secreted more TGF-?1 than TGF-?2 and TGF-?3, and activated the TGF-?/Smad signalling pathway in breast cancer cells. The EMT phenotype of breast cancer cells induced by CAF-CM was reversed by blocking TGF-?1 signalling. CONCLUSION: Cancer-associated fibroblasts promoted aggressive phenotypes of breast cancer cells through EMT induced by paracrine TGF-?1. This might be a common mechanism for acquiring metastatic potential in breast cancer cells with different biological characteristics.
Project description:BACKGROUND:Human platelets (PLT) and PLT-extracellular vesicles (PEV) released upon thrombin activation express receptors that interact with tumour cells and, thus, can serve as a delivery platform of anti-cancer agents. Drug-loaded nanoparticles coated with PLT membranes were demonstrated to have improved targeting efficiency to tumours, but remain impractical for clinical translation. PLT and PEV targeted drug delivery vehicles should facilitate clinical developments if clinical-grade procedures can be developed. METHODS:PLT from therapeutic-grade PLT concentrate (PC; N?>?50) were loaded with doxorubicin (DOX) and stored at -?80?°C (DOX-loaded PLT) with 6% dimethyl sulfoxide (cryopreserved DOX-loaded PLT). Surface markers and function of cryopreserved DOX-loaded PLT was confirmed by Western blot and thromboelastography, respectively. The morphology of fresh and cryopreserved naïve and DOX-loaded PLT was observed by scanning electron microscopy. The content of tissue factor-expressing cancer-derived extracellular vesicles (TF-EV) present in conditioned medium (CM) of breast cancer cells cultures was measured. The drug release by fresh and cryopreserved DOX-loaded PLT triggered by various pH and CM was determined by high performance liquid chromatography. The thrombin activated PEV was analyzed by nanoparticle tracking analysis. The cellular uptake of DOX from PLT was observed by deconvolution microscopy. The cytotoxicities of DOX-loaded PLT, cryopreserved DOX-loaded PLT, DOX and liposomal DOX on breast, lung and colon cancer cells were analyzed by CCK-8 assay. RESULTS:15~36?×?106 molecules of DOX could be loaded in each PLT within 3 to 9?days after collection. The characterization and bioreactivity of cryopreserved DOX-loaded PLT were preserved, as evidenced by (a) microscopic observations, (b) preservation of important PLT membrane markers CD41, CD61, protease activated receptor-1, (c) functional activity, (d) reactivity to TF-EV, and (e) efficient generation of PEV upon thrombin activation. The transfer of DOX from cryopreserved PLT to cancer cells was achieved within 90?min, and stimulated by TF-EV and low pH. The cryopreserved DOX-loaded PLT formulation was 7~23-times more toxic to three cancer cells than liposomal DOX. CONCLUSIONS:Cryopreserved DOX-loaded PLT can be prepared under clinically compliant conditions preserving the membrane functionality for anti-cancer therapy. These findings open perspectives for translational applications of PLT-based drug delivery systems.
Project description:The current study aimed at preparing AgNPs and three different core-shell silver/polymeric NPs composed of Ag core and three different polymeric shells: polyvinyl alcohol (PVA), polyethylene glycol (PEG) and polyvinylpyrrolidone (PVP). Thereafter, the core/shell NPs were loaded with a chemotherapeutic agent doxorubicin (DOX). Finally, the cytotoxic effects of the different core-shell Ag/polymeric NPs-based combinatorial therapeutics were tested in-vitro against breast cancer (MCF-7) and human fibroblast (1BR hTERT) cell lines. AgNPs, Ag/PVA and Ag/PVP NPs were more cytotoxic to MCF-7 cells than normal fibroblasts, as well as DOX-Ag, DOX-Ag/PVA, DOX-Ag/PEG and DOX-Ag/PVP nanocarriers (NCs). Notably, low dosage of core-shell DOX-loaded Ag/polymeric nanocarriers (NCs) exhibited a synergic anticancer activity, with DOX-Ag/PVP being the most cytotoxic. We believe that the prepared NPs-based combinatorial therapy showed a significant enhanced cytotoxic effect against breast cancer cells. Future studies on NPs-based combinatorial therapy may aid in formulating a novel and more effective cancer therapeutics.
Project description:Cancer-associated fibroblast (CAF)-specific proteins serve as both prognostic biomarkers and targets for anticancer drugs. In this study, we investigated the role of NGFI-A-binding protein (NAB)2 derived from CAFs in the progression of head and neck squamous cell carcinoma (HNSCC). Patient-derived HNSCC and paired metastatic lymph node tissues were examined for NAB2 expression by immunohistochemistry. Primary CAF cultures were established from HNSCC patient tissue, with paired non-tumor fibroblasts (NTFs) serving as a control. CAF or NTF was used to evaluate the effect of NAB2 on HNSCC progression using FaDu cell spheroids and an in vivo mouse xenograft model. NAB2 was detected in interstitial CAFs in primary and metastatic lymph node tissues of HNSCC patients. NAB2 mRNA and protein levels were higher in CAFs as compared to paired NTFs. Conditioned medium (CM) of NAB2-overexpressing CAFs increased the invasion of FaDu spheroids in the Matrigel invasion assay as compared to CM of NTF. Co-injection of NAB2-overexpressing CAFs with FaDu spheroids into mice enhanced the growth of tumors. These data suggest that NAB2-overexpressing CAFs promotes HNSCC progression and is a potential therapeutic target for preventing HNSCC metastasis.
Project description:Doxorubicin (DOX) induces dose-dependent cardiotoxicity in part due to its ability to induce oxidative stress. We showed that loss of multidrug resistance-associated protein 1 (Abcc1/Mrp1) potentiates DOX-induced cardiac dysfunction in mice in vivo Here, we characterized DOX toxicity in cultured cardiomyocytes (CM) and cardiac fibroblasts (CF) derived from C57BL wild type (WT) and Mrp1 null (Mrp1-/-) neonatal mice. CM accumulated more intracellular DOX relative to CF but this accumulation did not differ between genotypes. Following DOX (0.3-4??M), Mrp1-/- CM, and CF, especially CM, showed a greater decrease in viability and increased apoptosis and DNA damage, demonstrated by higher caspase 3 cleavage, poly (ADP-ribose) polymerase 1 (PARP) cleavage and phosphorylated histone H2AX (?H2AX) levels versus WT cells. Saline- and DOX-treated Mrp1-/- cells had significantly higher intracellular GSH and GSSG compared with WT cells (P?<?.05), but the redox potential (Eh) of the GSH/GSSG pool did not differ between genotypes in CM and CF, indicating that Mrp1-/- cells maintain this major redox couple. DOX increased expression of the rate-limiting GSH synthesis enzyme glutamate-cysteine ligase catalytic (GCLc) and regulatory subunits (GCLm) to a significantly greater extent in Mrp1-/- versus WT cells, suggesting adaptive responses to oxidative stress in Mrp1-/- cells that were inadequate to afford protection. Expression of extracellular superoxide dismutase (ECSOD/SOD3) was lower (P?<?.05) in Mrp1-/- versus WT CM treated with saline (62%?± 8% of WT) or DOX (43% ± 12% of WT). Thus, Mrp1 protects CM in particular and CF against DOX-induced toxicity, potentially by regulating extracellular redox states.
Project description:In order to avoid the instability and quick separation between emulsifier and drug in the interventional chemoembolization, an injectable low molecular weight peptide gel (LMWG) was prepared to localize ethanol and chemotherapeutic for <i>in situ</i> synergistic therapy. The formation mechanism, rheological property and morphology of the LMWG were investigated by NMR, UV-vis, MS and SEM. The interaction between gelator and anticancer drug doxorubicin hydrochloride (DOX) was evaluated by fluorescence spectroscopy and its contribution on drug loading properties was demonstrated. The gel was non-toxic to both 3T3 fibroblasts and 4T1 breast cancer cells. DOX as well as ethanol were encapsulated in the gel and injected in breast cancer bearing mice with low drug dose (2.5 mg/kg body weight). The LMWG surrounded tumors act as a depot for ethanol release and release DOX to induce the apoptosis of cancer cells. With the combination of percutaneous ethanol injection (PEI) and chemotherapy, the DOX loaded LMWG exhibited great significance in necrosis of tumor tissue and exciting tumor inhibition efficiency.
Project description:Binary transition metal oxides (BTMOs) have been explored as promising candidates in rechargeable lithium-ion battery (LIB) anodes due to their high specific capacity and environmental benignity. Herein, 2D ultrathin NiCo<sub>2</sub>O<sub>4</sub> nanosheets vertically grown on a biomass-derived carbon fiber substrate (NCO NSs/BCFs) were obtained by a facile synthetic strategy. The BCF substrate has superior flexibility and mechanical strength and thus not only offers a good support to NCO NSs/BCFs composites, but also provides high-speed paths for electron transport. Furthermore, 2D NiCo<sub>2</sub>O<sub>4</sub> nanosheets grown vertically present a large contact area between the electrode and the electrolyte, which shortens the ions/electrons transport distance. The nanosheets structure can effectively limit the volume change derived from Li<sup>+</sup> insertion and extraction, thus improving the stability of the electrode material. Therefore, the synthesized self-supporting NCO NSs/BCFs electrode displays excellent electrochemical performance, such as a large reversible capacity of 1128 mA·h·g<sup>-1</sup> after 80 cycles at a current density of 100 mA·g<sup>-1</sup> and a good rate capability of 818.5 mA·h·g<sup>-1</sup> at 1000 mA·g<sup>-1</sup>. Undoubtedly, the cheap biomass carbon source and facile synthesis strategy here described can be extended to other composite materials for high-performance energy-storage and conversion devices.