// Dhanya K. Nambiar 1,2 , Gagan Deep 1,3 , Rana P. Singh 2 , Chapla Agarwal 1,3 and Rajesh Agarwal 1,3 1 Department of Pharmaceutical Sciences, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of Colorado Anschutz Medical Campus, Aurora, CO, USA 2 School of Life Sciences, Jawaharlal Nehru University, India 3 University of Colorado Cancer Center, Aurora, CO, USA Correspondence: Rajesh Agarwal, email: // Keywords : Prostate cancer, lipogenesis, chemoprevention, phytochemicals, AMPK, SREBP1 Received : August 16, 2014 Accepted : September 15, 2014 Published : September 16, 2014 Abstract Prostate cancer (PCA) kills thousands of men every year, demanding additional approaches to better understand and target this malignancy. Recently, critical role of aberrant lipogenesis is highlighted in prostate carcinogenesis, offering a unique opportunity to target it to reduce PCA. Here, we evaluated efficacy and associated mechanisms of silibinin in inhibiting lipid metabolism in PCA cells. At physiologically achievable levels in human, silibinin strongly reduced lipid and cholesterol accumulation specifically in human PCA cells but not in non-neoplastic prostate epithelial PWR-1E cells. Silibinin also decreased nuclear protein levels of sterol regulatory element binding protein 1 and 2 (SREBP1/2) and their target genes only in PCA cells. Mechanistically, silibinin activated AMPK, thereby increasing SREBP1 phosphorylation and inhibiting its nuclear translocation; AMPK inhibition reversed silibinin-mediated decrease in nuclear SREBP1 and lipid accumulation. Additionally, specific SREBP inhibitor fatostatin and stable overexpression of SREBP1 further confirmed the central role of SREBP1 in silibinin-mediated inhibition of PCA cell proliferation and lipid accumulation and cell cycle arrest. Importantly, silibinin also inhibited synthetic androgen R1881-induced lipid accumulation and completely abrogated the development of androgen-independent LNCaP cell clones via targeting SREBP1/2. Together, these mechanistic studies suggest that silibinin would be effective against PCA by targeting critical aberrant lipogenesis.
<p>A. Terminal Tumor volume of MOC2 WT/KO-Gal1 tumors in Rag2-/- IL2rg-/- mice. B. Gating strategy used for analyses of PMN-MDSCs and M-MDSCs in tumor and lung tissues. C. Unsupervised tSNE clustering of live cells in MOC2 WT/KO-Gal1 tumor showing the proportion of M-MDSC, PMN-MDSC, and CD8+ T cells. D. Visual representation of the spleens isolated from either naïve mice or mice bearing WT- or KO-Gal1 tumors. E. Comparison of MDSC enrichment scores between smaller T1-2 and larger T3-4 tumors in the HNSCC TCGA cohort. The student’s t-test was used to assess the significance of the difference between the two groups. F. Heat map showing gene expression clustering based on the MDSC enrichment score in the HNSCC TCGA cohort. Differentially expressed genes (DEGs) were defined by a false discovery rate smaller than 0.05 and fold change higher than 1.5. G. Representative images showing low and high staining Gal1 or LOX1 (OLR1, established marker of PMN-MDSCs) using immunohistochemistry (IHC). H. Table showing the distribution of tumors expressing high or low Gal1 and high or low LOX1 (PMN-MDSCs) in 81 patients with oral cavity tumors. Gal1 and LOX1 staining distribution was analyzed using a chi-square test and was not statistically significant.</p>
Abstract Engaging innate immune pathways is emerging as a productive way of achieving durable anti-tumor responses. However, systemic administration of these therapies can result in toxicity, deemed to be particularly problematic when combined with current standard-of-care cytotoxic treatments such as radiotherapy. Increasing the therapeutic window of radiotherapy may be achieved by using targeted therapies, however, few pre-clinical studies investigate both tumor and normal tissue responses in detail. Here we show that targeting innate immune receptor C5aR1 improves tumor radiation response while reducing radiation-induced normal tissue toxicity, thereby increasing the therapeutic window. Genetically or pharmacologically targeting C5aR1 increases both IL-10 expression in the small intestine and IL-10 secretion by tumor cells. Increased IL-10 attenuates RelA phosphorylation and increases apoptosis in tumor cells, leading to improved radiation responses in murine models. Of note, these radiosensitizing effects are tumor-specific since, in the gastrointestinal tract, targeting C5aR1 instead results in decreased crypt cell apoptosis reduced signs of histological damage and improved survival following total abdominal irradiation in mice. Furthermore, the potent and orally active C5aR1 inhibitor, PMX205, improves tumor radiation responses even in a context of reduced/absent CD8+ T cell infiltration. These data indicate that PMX205 can modulate cancer-cell intrinsic functions to potentiate anti-tumor radiation responses even in tumors displaying features of T-cell deficiency or exclusion. Finally, using a preclinical murine model allowing the simultaneous assessment of tumor and normal tissue radiation responses, we show that PMX205 treatment reduces histological and functional markers of small-bowel toxicity while affording a positive tumor response. Our data, therefore, suggest that targeting C5aR1 could be a promising approach for increasing the therapeutic window of radiotherapy.
<p>Supplementary Figure S1: Proportion of total NK cell in the tumor microenvironment of 138 pancreatic cancer patients based on the CIBERSORTx analysis of TCGA. Supplementary Figure S2: Immunohistochemical staining of NK cells (Red circle) using NKp46. Supplementary Figure S3: Pan02 tumor in C57BL/6 mice as a syngeneic mouse model. Supplementary Figure S4: Representative flow cytometry gating strategy for NK cells (a) and percent of NK cells relative to immune cells in the C3aR antagonist-treated and vehicle-treated tumors (b). Supplementary Figure S5: Flow cytometry gating strategy for B cells (B220+), CD4+ T cells, CD8+ T cells, macrophages (Gr1-CD11b+F4/80+) and myeloid derived suppressor cells (MDSC) (Gr1+CD11b+) in the spleen, vehicle-treated (control) tumor and C3aR antagonist-treated tumor. Supplementary Figure S6: C3aR antagonism does not impact immune cell infiltration in the spleen of vehicle-treated and C3aR antagonist-treated mice. Supplementary Figure S7: Depletion of NK cells and CD8+ T cells and confirmatory analysis of depletion and survival of Pan02 tumor-bearing mice.</p>
<p>Somatic mutation of NFE2L2 leads to Nrf2 activation and promotes intrinsic radiation resistance in human head and neck squamous cell carcinoma (HNSCC).</p>
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