The role of the atypical chemokine receptor CCX-CKR in progression and metastasis of cancer.

The significance of chemokine receptors CCR7, CCR9 and their ligands CCL19, CCL21, and CCL25 in various types of cancer including mammary carcinoma and melanoma has been highlighted over the last decade. The atypical chemokine receptor CCK-CKR is a high affinity receptor for these chemokine ligands but rather than inducing classical downstream signalling events promoting migration, it instead sequesters and targets its ligands for degradation. Therefore, CCX-CKR has been proposed to regulate chemokine bioavailability in vivo. This putative function of CCX-CKR to regulate the levels of pro-tumourigenic chemokines initially led to the hypothesis that local and systemic regulation of chemokine levels by CCX-CKR influences tumour growth and metastasis in vivo, and ultimately, targeting of CCX-CKR could be an effective cancer therapy. Three broad approaches were taken to investigate the role of CCX-CKR in tumour progression and metastasis including overexpression of the receptor on tumour cells, deletion from the mouse host and receptor expression knockdown in tumour cells. The results revealed that overexpression of CCX-CKR on 4T1.2 mouse mammary carcinoma cells inhibits orthotopic tumour growth. However, this effect could not be correlated with chemokine scavenging in vivo and was not attributed to host adaptive immunity from experiments performed during the course of the current study. On the other hand, overexpression of CCX-CKR on 4T1.2 cells also resulted in enhanced spontaneous metastasis and haematogenous metastasis in vivo. In vitro characterisation of tumourigenicity of 4T1.2 cells revealed that overexpression of CCX-CKR rendered them more invasive, less adherent to the ECM and to each other and more resistant to anoikis. These are established characteristics of cells which have undergone EMT and indeed, CCX-CKR overexpressing cells showed a typical expression pattern of EMT markers. In contrast, when endogenous expression of CCX-CKR is deleted in the mouse host, growth and metastasis of E0771 mammary carcinoma and B16 melanoma are inhibited, which is accompanied by elevated levels of CCX-CKR ligands in tumours and relevant naïve tissues from CCX-CKR-deleted mice. Similarly, shRNA-mediated knockdown of endogenous CCX-CKR from B16 melanoma cells leads to the rejection of primary and secondary tumours. This effect is attributed to elevated levels of CCX-CKR ligands and CCR7⁺ and CCR9⁺ leukocytes in tumour tissues, which resulted in an overall enhancement of the host anti-tumour immune response. Consistent with these observations, growth of CCX-CKR knockdown tumours was comparable to that of control tumours in CCR7-deleted mice indicating host CCR7 dependency of CCX-CKR-mediated rejection of B16 melanoma. Together, findings from this study revealed important insights into the complex role of CCX-CKR in cancer progression and highlights CCX-CKR as a novel target for the development of more effective anti-melanoma therapies and potentially for the treatment of other types of cancer which affect millions of people worldwide.Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 201

Cell cycle, energy metabolism and DNA repair pathways in cancer cells are suppressed by Compound Kushen Injection

Abstract Background In this report we examine candidate pathways perturbed by Compound Kushen Injection (CKI), a Traditional Chinese Medicine (TCM) that we have previously shown to alter the gene expression patterns of multiple pathways and induce apoptosis in cancer cells. Methods We have measured protein levels in Hep G2 and MDA-MB-231 cells for genes in the cell cycle pathway, DNA repair pathway and DNA double strand breaks (DSBs) previously shown to have altered expression by CKI. We have also examined energy metabolism by measuring [ADP]/[ATP] ratio (cell energy charge), lactate production and glucose consumption. Our results demonstrate that CKI can suppress protein levels for cell cycle regulatory proteins and DNA repair while increasing the level of DSBs. We also show that energy metabolism is reduced based on reduced glucose consumption and reduced cellular energy charge. Results Our results validate these pathways as important targets for CKI. We also examined the effect of the major alkaloid component of CKI, oxymatrine and determined that it had no effect on DSBs, a small effect on the cell cycle and increased the cell energy charge. Conclusions Our results indicate that CKI likely acts through the effect of multiple compounds on multiple targets where the observed phenotype is the integration of these effects and synergistic interactions

Multiple functions of CXCL12 in a syngeneic model of breast cancer

Abstract Background A growing body of work implicates chemokines, in particular CXCL12 and its receptors, in the progression and site-specific metastasis of various cancers, including breast cancer. Various agents have been used to block the CXCL12-CXCR4 interaction as a means of inhibiting cancer metastasis. However, as a potent chemotactic factor for leukocytes, CXCL12 also has the potential to enhance anti-cancer immunity. To further elucidate its role in breast cancer progression, CXCL12 and its antagonist CXCL12(P2G) were overexpressed in the syngeneic 4T1.2 mouse model of breast carcinoma. Results While expression of CXCL12(P2G) significantly inhibited metastasis, expression of wild-type CXCL12 potently inhibited both metastasis and primary tumor growth. The effects of wild-type CXCL12 were attributed to an immune response characterized by the induction of CD8+ T cell activity, enhanced cell-mediated cytotoxicity, increased numbers of CD11c+ cells in the tumor-draining lymph nodes and reduced accumulation of myeloid-derived suppressor cells in the spleen. Conclusions This study highlights the need to consider carefully therapeutic strategies that block CXCL12 signaling. Therapies that boost CXCL12 levels at the primary tumor site may prove more effective in the treatment of metastatic breast cancer.</p

Sequences of leaf UniTransModels from Astragalus membranaceus (Huangqi)

This FASTA format file contains sequences of Unique Transcription Models (UniTransModel) from the leaf tissue of Astragalus membranaceus (Huangqi) using PacBio Iso-Seq technology

Data from: Long read reference genome-free reconstruction of a full-length transcriptome from Astragalus membranaceus reveals transcript variants involved in bioactive compound biosynthesis

Astragalus membranaceus, also known as Huangqi in China, is one of the most widely used medicinal herbs in Traditional Chinese Medicine. Traditional Chinese Medicine formulations from Astragalus membranaceus have been used to treat a wide range of illnesses, such as cardiovascular disease, type 2 diabetes, nephritis and cancers. Pharmacological studies have shown that immunomodulating, anti-hyperglycemic, anti-inflammatory, antioxidant and antiviral activities exist in the extract of Astragalus membranaceus. Therefore, characterising the biosynthesis of bioactive compounds in Astragalus membranaceus, such as Astragalosides, Calycosin and Calycosin-7-O-β-D-glucoside, is of particular importance for further genetic studies of Astragalus membranaceus. In this study, we reconstructed the Astragalus membranaceus full-length transcriptomes from leaf and root tissues using PacBio Iso-Seq long reads. We identified 27 975 and 22 343 full-length unique transcript models in each tissue respectively. Compared with previous studies that used short read sequencing, our reconstructed transcripts are longer, and are more likely to be full-length and include numerous transcript variants. Moreover, we also re-characterised and identified potential transcript variants of genes involved in Astragalosides, Calycosin and Calycosin-7-O-β-D-glucoside biosynthesis. In conclusion, our study provides a practical pipeline to characterise the full-length transcriptome for species without a reference genome and a useful genomic resource for exploring the biosynthesis of active compounds in Astragalus membranaceus

Sequences of leaf lncRNAs from Astragalus membranaceus (Huangqi)

This FASTA format file contains sequences of long non-coding RNAs (lncRNA) from the leaf tissue of Astragalus membranaceus (Huangqi) using PacBio Iso-Seq technology

The effect of compound kushen injection on cancer cells: Integrated identification of candidate molecular mechanisms.

Traditional Chinese Medicine (TCM) preparations are often extracts of single or multiple herbs containing hundreds of compounds, and hence it has been difficult to study their mechanisms of action. Compound Kushen Injection (CKI) is a complex mixture of compounds extracted from two medicinal plants and has been used in Chinese hospitals to treat cancer for over twenty years. To demonstrate that a systematic analysis of molecular changes resulting from complex mixtures of bioactives from TCM can identify a core set of differentially expressed (DE) genes and a reproducible set of candidate pathways. We used in vitro cancer models to measure the effect of CKI on cell cycle phases and apoptosis, and correlated those phenotypes with CKI induced changes in gene expression. We treated two cancer cell lines with or without CKI and assessed the resulting phenotypes by employing cell viability and proliferation assays. Based on these results, we carried out high-throughput transcriptome data analysis to identify genes and candidate pathways perturbed by CKI. We integrated these differential gene expression results with previously reported results and carried out validation of selected differentially expressed genes. CKI induced cell-cycle arrest and apoptosis in the cancer cell lines tested. In these cells CKI also altered the expression of 363 core candidate genes associated with cell cycle, apoptosis, DNA replication/repair, and various cancer pathways. Of these, 7 are clinically relevant to cancer diagnosis or therapy, 14 are cell cycle regulators, and most of these 21 candidates are downregulated by CKI. Comparison of our core candidate genes to a database of plant medicinal compounds and their effects on gene expression identified one-to-one, one-to-many and many-to-many regulatory relationships between compounds in CKI and DE genes. By identifying genes and promising candidate pathways associated with CKI treatment based on our transcriptome-based analysis, we have shown that this approach is useful for the systematic analysis of molecular changes resulting from complex mixtures of bioactives

Sequences of root lncRNAs from Astragalus membranaceus (Huangqi)

This FASTA format file contains sequences of long non-coding RNAs (lncRNA) from the root tissue of Astragalus membranaceus (Huangqi) using PacBio Iso-Seq technology

Exame papanicolaou em gestantes: conhecimento dos enfermeiros atuantes em unidades de atenção primária à saúde Pap smears in pregnant women: knowledge of nurses working in units of primary health care

OBJETIVO: Investigar o conhecimento dos enfermeiros sobre o exame ginecológico papanicolaou realizado em gestantes atendidas em Unidades de Atenção Primária à Saúde (UAPS), de Fortaleza-CE. MÉTODOS: Estudo descritivo, transversal, realizado em três UAPS, com amostra de 27 enfermeiros. Os dados foram obtidos por meio de questionário e para a análise utilizou-se o programa epi-info. RESULTADOS: Os dados mostraram que a maioria dos enfermeiros, 17 (62,97%), não realiza o exame ginecológico nas gestantes. Dos enfermeiros que realizam, 3 (7,4%) fazem a coleta de forma incorreta. No que se refere a participação em capacitações sobre pré-natal e exame ginecológico, 24 (88,8%) afirmaram ter participado anteriormente. CONCLUSÃO: Dessa forma, fazem-se necessárias capacitações sistemáticas e eficazes com intuito de reformular as práticas assistencialistas que se encontram estabelecidas nos programas de saúde da família