Hypoxia in Breast Cancer-Scientific Translation to Therapeutic and Diagnostic Clinical Applications.

Breast cancer has been the leading cause of female cancer deaths for decades. Intratumoral hypoxia, mainly caused by structural and functional abnormalities in microvasculature, is often associated with a more aggressive phenotype, increased risk of metastasis and resistance to anti-malignancy treatments. The response of cancer cells to hypoxia is ascribed to hypoxia-inducible factors (HIFs) that activate the transcription of a large battery of genes encoding proteins promoting primary tumor vascularization and growth, stromal cell recruitment, extracellular matrix remodeling, cell motility, local tissue invasion, metastasis, and maintenance of the cancer stem cell properties. In this review, we summarized the role of hypoxia specifically in breast cancer, discuss the prognostic and predictive value of hypoxia factors, potential links of hypoxia and endocrine resistance, cancer hypoxia measurements, further involved mechanisms, clinical application of hypoxia-related treatments and open questions.

Toosendanin-induced apoptosis in colorectal cancer cells is associated with the κ-opioid receptor/ß-catenin signaling axis.

Developing new drugs for killing colorectal cancer (CRC) cells is urgently needed. Here, we explored the antitumor effects of toosendanin (TSN) in CRC, as well as explored its antitumor mechanisms and direct targets. Cell proliferation and apoptosis were analyzed by CCK8, colony formation, real-time cell impedance and flow cytometry. The signaling pathway and Wnt activity were analyzed by Wnt luciferase activity assay, quantitative real-time PCR and western blot. The interaction between TSN and the κ-opioid receptor was analyzed by a molecular docking simulation. BALB/c nude mice were used to detect the effects of TSN on tumor growth in vivo. We found that TSN inhibited proliferation, induced G1 phase arrest and caused caspase-dependent apoptosis in both 5-FU-sensitive and 5-FU-resistant CRC cells. Moreover, TSN effectively inhibited CRC growth in vivo. In terms of the mechanism, TSN inhibited Wnt/ß-catenin signaling in CRC cells, and the molecular docking results showed that TSN could bind to κ-opioid receptors directly. Additionally, TSN-induced apoptosis and ß-catenin decline were both reversed by the selective κ-opioid receptor agonist U50,488H. Our data demonstrate that TSN-induced apoptosis in CRC cells is associated with the κ-opioid receptor/ß-catenin signaling axis, and TSN has promising potential as an antitumor agent for CRC treatment.

Targeted destruction of cancer stem cells using multifunctional magnetic nanoparticles that enable combined hyperthermia and chemotherapy.

Cancer stem cells (CSCs) have been implicated in cancer recurrence and therapy resistance. Therefore, a CSC-targeted therapy that disrupts the maintenance and survival of CSCs may offer an effective approach in killing tumor cells in primary tumors and preventing the metastasis caused by CSCs. Nanoparticles (NPs)-based thermotherapy and/or chemotherapy are promising therapeutic methods for cancer treatment. Methods: A silica-based multifunctional NP system was present, which encapsulated a chemotherapeutic agent and magnetic cores and coated with a specific antibody against the lung CSCs. The efficacy of this novel therapeutic strategy was systematically studied both in vitro and in vivo by simultaneous activating the combined thermotherapy and chemotherapy via CSC-targeted NPs. Results: These NPs were systematically administered and activated for targeted chemotherapy and thermotherapy by using an externally applied alternating magnetic field (AMF). The antibody-modified NPs targeted to lung CSCs with enhanced cellular uptake in vitro and extended accumulation in tumor in vivo. Up to 98% of lung CSCs was killed in vitro with 30-min application of AMF, due to the combined effects of hyperthermia and chemotherapeutic drug treatment. In in vivo models, this combined therapy significantly suppressed tumor growth and metastasis in lung CSC xenograft-bearing mice, with minimal side effects and adverse effects. Conclusion: With good biocompatibility and targeting capability, the nanodrug delivery system may offer a promising clinical platform for the combined thermotherapy and chemotherapy. This work demonstrated the feasibility of developing multifunctional nanomedicine targeting CSCs for effective cancer treatment.

Electrotaxis of tumor-initiating cells of H1975 lung adenocarcinoma cells is associated with both activation of stretch-activated cation channels (SACCs) and internal calcium release.

The metastatic potential of cancer cells is related to their migratory ability, which is influenced by in vivo microenvironment possessing specific physiochemical factors including electric properties. In the present study, we isolated two different subsets of lung adenocarcinoma H1975 cells, as side population (SP) and main population (MP). SP cells were demonstrated to have cancer stem cell characteristics. Using a microscale device to provide physiological direct-current electric field (dcEF), we investigated the electrotactic responses of the SP and MP cells. The results showed that both SP and MP cells exhibited enhanced cathodal migration ability with actin reorganization and transient intracellular calcium ions ([Ca2+]i) increase under dcEF stimulation. For SP cells, the treatment of either stretch-activated cation channels (SACCs) inhibitor or the blockage of intracellular Ca2+ release could partially inhibited dcEF-activated [Ca2+]i increase, and the concomitant treatment led to a complete inhibition. For MP cells, SACCs activation was entirely responsible for EF-activated increase of [Ca2+]i. All these results suggested that that intracellular Ca2+ activation may be associated with cancer cell tumorigenicity and metastasis.

Chemoresistant lung cancer stem cells display high DNA repair capability to remove cisplatin-induced DNA damage.

BACKGROUND AND PURPOSE: The persistence of lung cancer stem cells (LCSCs) has been proposed to be the main factor responsible for the recurrence of lung cancer as they are highly resistant to conventional chemotherapy. However, the underlying mechanisms are still unclear. EXPERIMENTAL APPROACH: We examined the cellular response of a human LCSC line to treatment with cisplatin, a DNA-damaging anticancer drug that is used extensively in the clinic. We compared the response to cisplatin of LCSCs and differentiated LCSCs (dLCSCs) by determining the viability of these cells, and their ability to accumulate cisplatin and to implement genomic and transcription-coupled DNA repair. We also investigated the transcription profiles of genes related to drug transport and DNA repair. KEY RESULTS: LCSCs were found to be more stem-like, and more resistant to cisplatin-induced cytotoxicity than dLCSCs, confirming their drug resistance properties. LCSCs accumulated less cisplatin intracellularly than dLCSCs and showed less DNA damage, potentially due to their ability to down-regulate AQP2 and CTR1. The results of the transcription-coupled repair of cisplatin-DNA cross-links indicated a higher level of repair of DNA damage in LCSCs than in dLCSCs. In addition, LCSCs showed a greater ability to repair cisplatin-DNA interstrand cross-links than dLCSCs; this involved the activation of various DNA repair pathways. CONCLUSIONS AND IMPLICATIONS: Our results further clarify the mechanism of cisplatin resistance in LCSCs in terms of reduced cisplatin uptake and enhanced ability to implement DNA repairs. These findings may aid in the design of the next-generation of platinum-based anticancer drugs.

Microfluidic Platform for Studying Chemotaxis of Adhesive Cells Revealed a Gradient-Dependent Migration and Acceleration of Cancer Stem Cells.

Recent studies reveal that solid tumors consist of heterogeneous cells with distinct phenotypes and functions. However, it is unclear how different subtypes of cancer cells migrate under chemotaxis. Here, we developed a microfluidic device capable of generating multiple stable gradients, culturing cells on-chip, and monitoring single cell migratory behavior. The microfluidic platform was used to study gradient-induced chemotaxis of lung cancer stem cell (LCSC) and differentiated LCSC (dLCSC) in real time. Our results showed the dynamic and differential response of both LCSC and dLCSC to chemotaxis, which was regulated by the ß-catenin dependent Wnt signaling pathway. The microfluidic analysis showed that LCSC and dLCSC from the same origin behaved differently in the same external stimuli, suggesting the importance of cancer cell heterogeneity. We also observed for the first time the acceleration of both LCSC and dLCSC during chemotaxis caused by increasing local concentration in different gradients, which could only be realized through the microfluidic approach. The capability to analyze single cell chemotaxis under spatially controlled conditions provides a novel analytical platform for the study of cellular microenvironments and cancer cell metastasis.

Activation of multiple signaling pathways during the differentiation of mesenchymal stem cells cultured in a silicon nanowire microenvironment.

Mesenchymal stem cells (MSC) offer an optimal source for bone tissue engineering due to their capability of undergoing multilineage differentiation, where the mechanical properties of the microenvironment of MSCs are vital for osteochondral formation. However, the mechanisms of how mechanical and microenvironmental cues control osteogenesis and chondrogenesis are yet to be elucidated. In this study, we investigated the effects of vertically aligned silicon nanowire (SiNW) array on the differentiation of MSCs and the associated molecular mechanisms involved in osteogenesis and chandrogenesis. The results showed that the microenvironment of SiNW array activated a number of mechanosensitive pathways (including Integrin, TGF-ß/BMP, Akt, MAPK, Insulin, and Wnt pathways) in MSCs, which converged to stimulate the osteogenesis and chondrogenesis via the Ras-Raf-MEK-ERK cascade. FROM THE CLINICAL EDITOR: This study reports on the mechanisms and microenvironmental influence of osteogenesis and chondrogenesis by mesenchymal stem cells interacting with vertically aligned silicon nanowire scaffolds.

Herbal formula Astragali Radix and Rehmanniae Radix exerted wound healing effect on human skin fibroblast cell line Hs27 via the activation of transformation growth factor (TGF-ß) pathway and promoting extracellular matrix (ECM) deposition.

Astragali Radix (AR) and Rehmanniae Radix (RR) have long been used in traditional Chinese Medicine and as the principal herbs in treating diabetic foot ulcer. In this study, we investigated the effect of NF3, which comprises of AR and RR in the ratio of 2:1(w/w), on skin fibroblast cell migration and the activation of selected genes and proteins related to wound healing. Human skin fibroblast cell line Hs27 was treated with NF3 at 4 mg/ml for 24h, and in vitro scratch wound healing and quantitative cell migration assays were performed, respectively. The expression of transformation growth factor (TGF-ß1) and bone morphogenetic protein 6 (BMP6) in Hs27 cells with or without NF3 treatment was analyzed by western blot analysis. In addition, the expression of a panel of genes involved in human TGF-ß signaling pathway was analyzed in Hs27 cells upon NF3 treatment (4 mg/ml, 24 h) by quantitative real-time PCR (qRT-PCR). Furthermore, the expression of several genes and proteins associated with ECM synthesis was investigated by qRT-PCR analysis or/and ELISA techniques. The results suggested that NF3 promoted the migration of human skin fibroblast cells. Western blot analysis demonstrated that NF3 up-regulated TGF-ß1 and BMP-6 synthesis. qRT-PCR analysis revealed that the expression of 26 genes in Hs27 cells was changed upon NF3 induction, including TGF-ß superfamily ligands and down stream effectors genes, and genes involved in TGF/Smad pathway, and Ras/MAPK (non-Smad) pathway. Among the extracellular matrix (ECM)-related molecules, it was found that NF3 up-regulated the expression of type I and III collagens, fibronectin as well as TIMP-1, and down-regulated the MMP-9 expression in skin fibroblast cells. This study demonstrated that herb formula NF3 could enhance skin fibroblast cell migration and activated genes involved in TGF-ß1 pathway. NF3 could regulate gene transcription for extracellular matrix synthesis via the Smad pathway, and gene transcription for cell motility via the Ras/MAPK (non-Smad) pathway.

Transcriptional profiling of human skin fibroblast cell line Hs27 induced by herbal formula Astragali Radix and Rehmanniae Radix.

ETHNOPHARMACOLOGICAL RELEVANCE: The herbs Astragali Radix (AR) and Rehmanniae Radix (RR) have long been used in traditional Chinese Medicine and serve as the principal herbs in treating diabetic foot ulcer. AIM OF THE STUDY: Chinese herbal formulus comprising Astragali Radix (AR) and Rehmanniae Radix (RR) have been shown to improve the healing of diabetic foot ulcer through enhancing the viability of primary fibroblasts in diabetic patients suffering insulin resistance. Our previous study demonstrated that the herbal formula NF3 comprising of AR and RR in the ratio of 2:1 was effective in promoting wound healing in diabetic rats, and in vitro data indicated that the wound healing effects of NF3 might be due to the regulation and coordination of inflammation, angiogenesis and tissue regeneration. However, the underlying molecular mechanism has not been well investigated. In this study, we investigated the cellular and molecular effects of the herbal formula NF3 on human skin fibroblast cells. MATERIALS AND METHODS: Human skin fibroblast cells Hs27 were treated with NF3 ranging from 0 to 8 mg/ml for 24h, and the cells without NF3 treatment were used as control. Cell proliferation assay and cell cycle analysis were performed. Transcriptional profiles of Hs27 cells upon NF3 treatment were acquired by using a human cDNA microarray containing 10,000 genes, and the signaling pathways differentially regulated by NF3 were identified and analyzed. RESULTS: NF3 promoted Hs27 cell proliferation and cell cycle progression. Microarray analysis revealed that 116 genes were differentially expressed upon NF3 treatment. Functional analysis of the genes indicated that NF3 mainly activated Wnt and angiogenesis related pathways, which are directly related to cell proliferation, angiogenesis, extracellular matrix (ECM) formation and inflammation during the process of wound healing. CONCLUSION: This study provides insight into the molecular mechanism of how the herbal formula Astragali Radix and Rehmanniae Radix may serve as potential therapeutics for wound healing.

Danshen-Gegen decoction exerts proliferative effect on rat cardiac myoblasts H9c2 via MAPK and insulin pathways.

ETHNOPHARMACOLOGICAL RELEVANCE: Danshen (root of Salvia miltiorrhiza) and Gegen (roots of Pueraria lobata) are traditional Chinese medicines that have been used in combination for cardiovascular disease treatment. AIM OF THE STUDY: The present study was performed to investigate the effect of Danshen-Gegen decoction on rat myocardium cell line H9c2 and the possible molecular mechanisms. MATERIALS AND METHODS: Rat heart myocardium H9c2 cells were treated with or without Danshen-Gegen decoction (DG) ranging from 10 to 1000µg/ml for 24h. Cell viability was measured by Alarma blue assay and cell proliferation assay was performed by BrdU Cell Proliferation ELISA kit. The activation of mitogen-activated protein kinase and insulin pathways was analyzed by Luminex technology and the growth factors and cytokine expression of H9c2 cells induced by DG was evaluated by protein array. Moreover, a rat functional specific cDNA microarray was constructed to study the gene expression profiles of H9c2 cells upon the DG treatment at 50µg/ml for 24h. RESULTS: DG promoted H9c2 cell viability and cell proliferation at dose-dependent manner within the range between 0 and 250µg/ml. A Bio-Plex assay kit (Bio-Rad Bioscience) was used to detect the expression level of phosphoprotein as well as total proteins involved in the MAPK and insulin pathways. Significant phosphorylation of ERK, c-Jun, JNK, p38, AKT, IGF-IR, IRS-1and I kappa B were observed after DG treatment at 2h or 4h. A rat cytokine antibody array was used to detect and quantify 22 growth factors and cytokines in samples collected from the control and DG treated H9c2 cells. In the category of growth factors, GM-CSF, CNIF and b-NGF were stimulated by DG, while the expression of TIMP-1 was suppressed. For cytokine expression, it was found that DG stimulated three interleukin subclasses, IL-1α, 1X and 6, respectively. However, the expression of pro-inflammatory factors such as TNF-α and IFN-γ were down-regulated significantly. Moreover, the microarray analysis revealed that DG significantly up-regulated anti-apoptosis related genes such as Cdkn2c and Ppp3ca, and several cardiovascular disease suppressers and anti-inflammatory mediators; on the other hand, pro-apoptotic related genes including Caspase and Tnf-α were down-regulated by DG. Based on the results, a tentative scheme was proposed to show that the activation of the MAPK and insulin pathways are involved in the bioactive effect of Danshen-Gegen decoction on cardiomyocytes. CONCLUSION: Our study suggested that Danshen-Gegen decoction has proliferative effect on myocardium cells via MAPK and insulin signaling pathways. The molecular mechanism of the action may include the up-regulation of IRS/AKT and JNK pathways as well as the inhibition of TNF and p38 pathways.