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Genomic DNA is folded into a higher-order structure that regulates transcription and maintains genomic stability. Although progress has been made on understanding biochemical characteristics of epigenetic modifications in cancer, the in-situ higher-order folding of chromatin structure during malignant transformation remains largely unknown. Here, using optimized stochastic optical reconstruction microscopy (STORM) for pathological tissue (PathSTORM), we uncover a gradual decompaction and fragmentation of higher-order chromatin folding throughout all stages of carcinogenesis in multiple tumor types, and prior to tumor formation. Our integrated imaging, genomic, and transcriptomic analyses reveal functional consequences in enhanced transcription activities and impaired genomic stability. We also demonstrate the potential of imaging higher-order chromatin disruption to detect high-risk precursors that cannot be distinguished by conventional pathology. Taken together, our findings reveal gradual decompaction and fragmentation of higher-order chromatin structure as an enabling characteristic in early carcinogenesis to facilitate malignant transformation, which may improve cancer diagnosis, risk stratification, and prevention. Aberrant chromatin structure is often found in cancer. Here, the authors optimise super-resolution microscopy for pathological tissue and discovered a significant decompaction of chromatin folding in early carcinogenesis prior to tumour formation.

A subset of human prostate cancer exhibits increased de novo synthesis of fatty acids, but the molecular driver(s) of this metabolic abnormality remains obscure. This study demonstrates a novel metabolic function of c-Myc (Myc) in regulation of fatty acid synthesis. The role of Myc in regulation of fatty acid synthesis was investigated by: (a) interrogation of the prostate cancer The Cancer Genome Atlas (TCGA) dataset, (b) chromatin immunoprecipitation, and (c) determination of the expression of fatty acid synthesis enzymes and targeted metabolomics using a mouse model and human specimens. The expression of MYC was positively associated with that of key fatty acid synthesis genes including ACLY , ACC1 , and FASN in prostate cancer TCGA dataset. Chromatin immunoprecipitation revealed Myc occupancy at the promoters of ACLY , ACC1 , and FASN . Prostate-specific overexpression of Myc in Hi-Myc transgenic mice resulted in overexpression of ACLY, ACC1, and FASN proteins in neoplastic lesions and increased circulating levels of total free fatty acids. Targeted metabolomics confirmed increased circulating levels of individual fatty acids in the plasma of Hi-Myc mice and human subjects when compared to corresponding controls. Immunohistochemistry also revealed a positive and statistically significant association in expression of Myc with that of ACC1 in human prostate adenocarcinoma specimens. We propose that Myc-regulated fatty acid synthesis is a valid target for therapy and/or prevention of prostate cancer.

Withaferin A (WA), a promising anticancer constituent of Ayurvedic medicinal plant Withania somnifera, inhibits growth of MDA-MB-231 and MCF-7 human breast cancer cells in culture and MDA-MB-231 xenografts in vivo in association with apoptosis induction, but the mechanism of cell death is not fully understood. We now demonstrate, for the first time, that WA-induced apoptosis is mediated by reactive oxygen species (ROS) production due to inhibition of mitochondrial respiration. WA treatment caused ROS production in MDA-MB-231 and MCF-7 cells, but not in a normal human mammary epithelial cell line (HMEC). The HMEC was also resistant to WA-induced apoptosis. WA-mediated ROS production as well as apoptotic histone-associated DNA fragment release into the cytosol was significantly attenuated by ectopic expression of Cu,Zn-superoxide dismutase in both MDA-MB-231 and MCF-7 cells. ROS production resulting from WA exposure was accompanied by inhibition of oxidative phosphorylation and inhibition of complex III activity. Mitochondrial DNA-deficient Rho-0 variants of MDA-MB-231 and MCF-7 cells were resistant to WA-induced ROS production, collapse of mitochondrial membrane potential, and apoptosis compared with respective wild-type cells. WA treatment resulted in activation of Bax and Bak in MDA-MB-231 and MCF-7 cells, and SV40 immortalized embryonic fibroblasts derived from Bax and Bak double knockout mouse were significantly more resistant to WA-induced apoptosis compared with fibroblasts derived from wild-type mouse. In conclusion, the present study provides novel insight into the molecular circuitry of WA-induced apoptosis involving ROS production and activation of Bax/Bak.

Benzyl isothiocyanate (BITC), a constituent of edible cruciferous vegetables, inhibits growth of breast cancer cells but the mechanisms underlying growth inhibitory effect of BITC are not fully understood. Here, we demonstrate that BITC treatment causes FoxO1-mediated autophagic death in cultured human breast cancer cells. The BITC-treated breast cancer cells (MDA-MB-231, MCF-7, MDA-MB-468, BT-474, and BRI-JM04) and MDA-MB-231 xenografts from BITC-treated mice exhibited several features characteristic of autophagy, including appearance of double-membrane vacuoles (transmission electron microscopy) and acidic vesicular organelles (acridine orange staining), cleavage of microtubule-associated protein 1 light chain 3 (LC3), and/or suppression of p62 (p62/SQSTM1 or sequestosome 1) expression. On the other hand, a normal human mammary epithelial cell line (MCF-10A) was resistant to BITC-induced autophagy. BITC-mediated inhibition of MDA-MB-231 and MCF-7 cell viability was partially but statistically significantly attenuated in the presence of autophagy inhibitors 3-methyl adenine and bafilomycin A1. Stable overexpression of Mn-superoxide dismutase, which was fully protective against apoptosis, conferred only partial protection against BITC-induced autophagy. BITC treatment decreased phosphorylation of mTOR and its downstream targets (P70s6k and 4E-BP1) in cultured MDA-MB-231 and MCF-7 cells and MDA-MB-231 xenografts, but activation of mTOR by transient overexpression of its positive regulator Rheb failed to confer protection against BITC-induced autophagy. Autophagy induction by BITC was associated with increased expression and acetylation of FoxO1. Furthermore, autophagy induction and cell growth inhibition resulting from BITC exposure were significantly attenuated by small interfering RNA knockdown of FoxO1. In conclusion, the present study provides novel insights into the molecular circuitry of BITC-induced cell death involving FoxO1-mediated autophagy.

D, L-Sulforaphane (SFN), a synthetic racemic analog of broccoli constituent L-sulforaphane, is a highly promising cancer chemopreventive agent with in vivo efficacy against chemically-induced as well as oncogene-driven cancer in preclinical rodent models. Cancer chemopreventive effect of SFN is characterized by G(2)/M phase cell cycle arrest, apoptosis induction, and inhibition of cell migration and invasion. Moreover, SFN inhibits multiple oncogenic signaling pathways often hyperactive in human cancers, including nuclear factor-κB, Akt, signal transducer and activator of transcription 3, and androgen receptor. The present study was designed to determine the role of Notch signaling, which is constitutively active in many human cancers, in anticancer effects of SFN using prostate cancer cells as a model. Exposure of human prostate cancer cells (PC-3, LNCaP, and/or LNCaP-C4-2B) to SFN as well as its naturally-occurring thio-, sulfinyl-, and sulfonyl-analogs resulted in cleavage (activation) of Notch1, Notch2, and Notch4, which was accompanied by a decrease in levels of full-length Notch forms especially at the 16- and 24-hour time points. The SFN-mediated cleavage of Notch isoforms was associated with its transcriptional activation as evidenced by RBP-Jk-, HES-1A/B- and HEY-1 luciferase reporter assays. Migration of PC-3 and LNCaP cells was decreased significantly by RNA interference of Notch1 and Notch2, but not Notch4. Furthermore, SFN-mediated inhibition of PC-3 and LNCaP cell migration was only marginally affected by knockdown of Notch1 and Notch2. Strikingly, SFN administration to Transgenic Adenocarcinoma of Mouse Prostate transgenic mice failed to increase levels of cleaved Notch1, cleaved Notch2, and HES-1 proteins in vivo in prostatic intraepithelial neoplasia, well-differentiated carcinoma or poorly-differentiated prostate cancer lesions. These results indicate that Notch activation is largely dispensable for SFN-mediated inhibition of cell migration, which should be viewed as a therapeutic advantage as Notch activation is frequent in human prostate cancers.

Phenethyl isothiocyanate (PEITC) is a promising cancer chemopreventive component of edible cruciferous vegetables with in vivo efficacy against prostate cancer in experimental rodents. Cancer chemopreventive response to PEITC is characterized by its ability to inhibit multiple oncogenic signaling pathways, including nuclear factor-κB, Akt, and androgen receptor. The present study demonstrates, for the first time, that PEITC treatment activates Notch signaling in malignant as well as normal human prostate cells. Exposure of human prostate cancer cells (LNCaP, PC-3, and DU145) and a normal human prostate epithelial cell line (PrEC) to PEITC resulted in cleavage (active form) of Notch1 and Notch2, and increased transcriptional activity of Notch. In PC-3 and LNCaP cells, PEITC treatment caused induction of Notch ligands Jagged1 and Jagged2 (PC-3), overexpression of γ-secretase complex components Presenilin1 and Nicastrin (PC-3), nuclear enrichment of cleaved Notch2, and/or up-regulation of Notch1, Notch2, Jagged1, and/or Jagged2 mRNA. PEITC-induced apoptosis in LNCaP and PC-3 cells was significantly attenuated by RNA interference of Notch2, but not by pharmacological inhibition of Notch1. Inhibition of PC-3 and LNCaP cell migration resulting from PEITC exposure was significantly augmented by knockdown of Notch2 protein as well as pharmacological inhibition of Notch1 activation. Nuclear expression of cleaved Notch2 protein was significantly higher in PC-3 xenografts from PEITC-treated mice and dorsolateral prostates from PEITC-fed TRAMP mice compared with respective control. Because Notch signaling is implicated in epithelial-mesenchymal transition and metastasis, the present study suggests that anti-metastatic effect of PEITC may be augmented by a combination regimen involving a Notch inhibitor.

Organosulfur compounds from garlic effectively inhibit growth of transplanted as well as spontaneous cancers in preclinical animal models without any adverse side effects. However, the mechanisms underlying anticancer effect of this class of compounds are not fully understood. This study reports, for the first time, that garlic organosulfide diallyl trisulfide (DATS) inhibits estrogen receptor-α (ER-α) activity in human breast cancer cells. Exposure of MCF-7 and T47D cells to DATS resulted in downregulation of ER-α protein, which peaked between 12- and 24-h post-treatment. DATS was relatively more effective in suppressing ER-α protein expression compared with its mono and disulfide analogs. The 17β-estradiol (E2)-induced expression of pS2 and cyclin D1, ER-α target gene products, was also decreased in the presence of DATS. Downregulation of ER-α protein expression resulting from DATS treatment was accompanied by a decrease in nuclear levels of ER-α protein, ER - α mRNA suppression, and inhibition of ERE2e1b-luciferase reporter activity. DATS-mediated inhibition of cell viability and apoptosis induction were not affected in the presence of E2. In agreement with these results, ectopic expression of ER-α in MDA-MB-231 cell line failed to confer any protection against cell proliferation inhibition or apoptosis induction resulting from DATS exposure. DATS treatment caused a decrease in protein levels of peptidyl-prolyl cis – trans isomerase (Pin1), and overexpression of Pin1 partially attenuated ER-α downregulation by DATS. DATS-induced apoptosis was modestly but significantly augmented by overexpression of Pin1. In conclusion, this study identifies ER-α as a novel target of DATS in mammary cancer cells.

[...]they are of interest, as they could provide alternatives to the existing chemotherapeutic agents. The aim of this Research Topic was to publish original research and review articles in this area to gain the current knowledge and future perspective of using phytochemicals in cancer therapy and prevention with reduced side effect. Studies have shown the roles of hesperidin, a flavonoid phytochemical available in the skins of citrus species, to maintain blood pressure, cholesterol levels, to have anti-allergic, antiviral and anticancer effects (Muhammad et al., 2019).

The present study offers novel insights into the molecular circuitry of accelerated in vivo tumor growth by Notch2 knockdown in triple-negative breast cancer (TNBC) cells. Therapeutic vulnerability of Notch2-altered growth to a small molecule (withaferin A, WA) is also demonstrated. MDA-MB-231 and SUM159 cells were used for the xenograft studies. A variety of technologies were deployed to elucidate the mechanisms underlying tumor growth augmentation by Notch2 knockdown and its reversal by WA, including Fluorescence Molecular Tomography for measurement of tumor angiogenesis in live mice, Seahorse Flux analyzer for ex vivo measurement of tumor metabolism, proteomics, and Luminex-based cytokine profiling. Stable knockdown of Notch2 resulted in accelerated in vivo tumor growth in both cells reflected by tumor volume and/or latency. For example, the wet tumor weight from mice bearing Notch2 knockdown MDA-MB-231 cells was about 7.1-fold higher compared with control ( P  < 0.0001). Accelerated tumor growth by Notch2 knockdown was highly sensitive to inhibition by a promising steroidal lactone (WA) derived from a medicinal plant. Molecular underpinnings for tumor growth intensification by Notch2 knockdown included compensatory increase in Notch1 activation, increased cellular proliferation and/or angiogenesis, and increased plasma or tumor levels of growth stimulatory cytokines. WA administration reversed many of these effects providing explanation for its remarkable anti-cancer efficacy. Notch2 functions as a tumor growth suppressor in TNBC and WA offers a novel therapeutic strategy for restoring this function.

Purpose Present study was undertaken to elucidate the mechanism of cellular responses to D,L-sulforaphane (SFN), a highly promising cancer chemopreventive agent. Methods Mitochondrial DNA deficient Rho-0 variants of LNCaP and PC-3 cells were generated by culture in the presence of ethidium bromide. Apoptosis was assessed by analysis of cytoplasmic histone-associated DNA fragmentation and activation of caspase-3. Immunoblotting was performed to determine the expression of apoptosis- and cell cycle-regulating proteins. Generation of reactive oxygen species (ROS), mitochondrial membrane potential (MMP), and cell cycle distribution were measured by flow cytometry. Results The Rho-0 variants of LNCaP and PC-3 cells were significantly more resistant to SFN-induced ROS generation, apoptotic DNA fragmentation, disruption of MMP, cytosolic release of cytochrome c, and G2/M phase cell cycle arrest compared with corresponding wild-type cells. SFN-induced autophagy, which serves to protect against apoptotic cell death in PC-3 and LNCaP cells, was also partially but markedly suppressed in Rho-0 variants compared with wild-type cells. SFN statistically significantly inhibited activities of mitochondrial respiratory chain enzymes in LNCaP and PC-3 cells. Conclusion These results indicate, for the first time, that mitochondria-derived ROS serve to initiate diverse cellular responses to SFN exposure in human prostate cancer cells.