Cytosine deaminase (CDA) is a prodrug mediating enzyme converting 5-flurocytosine into 5-flurouracil with profound broad-range anticancer activity towards various cell lines. Availability, molecular stability, and catalytic efficiency are the main limiting factors halting the clinical applications of this enzyme on prodrug and gene therapies, thus, screening for CDA with unique biochemical and catalytic properties was the objective. Thermotolerant/ thermophilic fungi could be a distinctive repertoire for enzymes with affordable stability and catalytic efficiency. Among the recovered thermotolerant isolates, Aspergillus niger with optimal growth at 45 °C had the highest CDA productivity. The enzyme was purified, with purification 15.4 folds, molecular mass 48 kDa and 98 kDa, under denaturing and native PAGE, respectively. The purified CDA was covalently conjugated with dextran with the highest immobilization yield of 75%. The free and CDA-dextran conjugates have the same optimum pH 7.4, reaction temperature 37 °C, and pI 4.5, and similar response to the inhibitors and amino acids suicide analogues, ensuring the lack of effect of dextran conjugation on the CDA conformational structure. CDA-Dextran conjugates had more resistance to proteolysis in response to proteinase K and trypsin by 2.9 and 1.5 folds, respectively. CDA-Dextran conjugates displayed a dramatic structural and thermal stability than the free enzyme, authenticating the acquired structural and catalytic stability upon dextran conjugation. The thermal stability of CDA was increased by about 1.5 folds, upon dextran conjugation, as revealed from the half-life time (T1/2). The affinity of CDA-conjugates (Km 0.15 mM) and free CDA (Km 0.22 mM) to deaminate 5-fluorocytosine was increased by 1.5 folds. Upon dextran conjugation, the antiproliferative activity of the CDA towards the different cell lines “MDA-MB, HepG-2, and PC-3” was significantly increased by mediating the prodrug 5-FC. The CDA-dextran conjugates strongly reduce the tumor size and weight of the Ehrlich cells (EAC), dramatically increase the titers of Caspase-independent apoptotic markers PARP-1 and AIF, with no cellular cytotoxic activity, as revealed from the hematological and biochemical parameters.

Background Matricellular proteins comprising matrisome and adhesome are responsible for structure integrity and interactions between cells in the tumour microenvironment of breast cancer. Changes in the gene expression of matrisome and adhesome augment metastasis. Since inflammatory breast cancer (IBC) is characterized by high metastatic behavior. Herein we compared the gene expression profile of matrisome and adhesome in non-IBC and IBC in fresh tissue and ex-vivo patients derived explants (PDEs), we also compared the secretory inflammatory mediators of PDEs in non-IBC and IBC to identify secretory cytokines participate in cross-talk between cells via interactions with matrisome and adhisome. Methods Fifty patients (31 non-IBC; 19 IBC) were enrolled in the present study. To test their validation in clinical studies, PDEs were cultured as an ex-vivo model. Gene expression and cytokine array were used to identify candidate genes and cytokines contributing to metastasis in the examined fresh tissues and PDEs. Bioinformatics analysis was applied on identified differentially expressed genes (DEGs) using GeneMANIA and Metascape gene annotation and analysis resource to identify pathways involved in IBC metastasis. Results Normal and cancer fresh tissues and PDEs of IBC were characterized by overexpression of CDH1 and MMP14 and downregulation of CTNNA1 and TIMP1 compared to non-IBC. The secretome of IBC cancer PDEs is characterized by significantly high expression of interleukin 6 (IL-6), and monocyte chemoattractant protein-1 (MCP-1/CCL2) compared to non-IBC. Conclusion Genes expressed by adhisome and matrisome play a significant role in IBC metastasis and should be considered novel target therapy.

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Background: Many studies suggested that the pathogenesis of inflammatory breast cancer (IBC) is related to inflammatory manifestations and changes in tumor microenvironment (TME). IBC characterized by significantly higher infiltration of tumor associated macrophages (TAMs). TAMs contribute to the metastatic process in IBC patients via secreting many cytokines such as TNF, IL-6, IL-8, and IL-10 that enhance invasion and angiogenesis. To understand the role of TAMs in IBC, there is a need to firstly understand how IBC TME affect the polarization of tumor infiltrated monocytes via using sensitive and accurate analytical tool. Synchrotron FTIR microspectroscopy (SR-µFTIR) is a highly advanced analytical approach has the ability to detect any biochemical changes even prior to any morphological changes. Herein we will use SR-µFTIR to study the biochemical changes in tumor infiltrating monocytes after stimulation by the secretome of non-IBC and IBC breast cancer cells. Methods: ten breast cancer patients were enrolled in this study (5 IBC and 5 non-IBC). tumor infiltrated monocytes were isolated from patient’s blood samples which collected during modified radical mastectomy surgery. The isolated monocytes were stimulated with the secretome of MCF7, MDA-MB-231 and SUM149 cell line. Synchrotron FTIR Microspectroscopy (SR-FTIRM) single cell analysis was done at SESAME light source. The obtained raw spectral data were processed and analyzed using OMNIC©, PeakFit© V4.12, and Unscrambler© X v.10.4 software. Results: The obtained FTIR spectra showed very intense absorption bands in the region of 1700- 1500 cm-1 attributed to the amide I (ν(C=O), & νAS (C-N), δ (N-H) and amide II ν(C-N), δ (N-H) proteins bands, with a strong broad signal in the 3600-3200 cm-1 related to the νN-H Amides A and B. In addition, three peaks of different intensity and area were detected in the lipids region of the vCH2 and vCH3 stretching modes positioned within the 3000 - 2800 cm-1 range. Curve fitting assignment clearly demonstrate some differences between the three stimulated monocytes in both amide I and II, and lipids regions. The Principal Component Analysis (PCA) scores plot of the second derivative spectra of the amide region explain 77% of the variance, with 63% explained by PC1 and 14% explained by PC2. On the other hand, the PCA scores plot of the lipid region explain a total of 92% of the variance with PC1 explaining 72% and PC2 explaining 20%. Conclusion: The obtained results showed that there is different effect of non-IBC and IBC cells on the polarization of tumor infiltrating monocytes even with the cell surface markers look the same and from this, we may start to understand different roles of TAMs in IBC microenvironment. Citation Format: Hossam T. Mohamed, Gihan Kamel, Aya A. El-Sharkawy, Noura El-Husseiny, Mohamed El-Shinawi, Mona M. Mohamed. Synchrotron Fourier-transform infrared microspectroscopy: Characterization of tumor infiltrating monocytes stimulated by the secretome of inflammatory and non-inflammatory breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6132.

Aim: The previously reported dual histone deacetylase type II (HDAC II) / topoisomerase type I (Topo I) inhibitors suffer pharmacokinetic limitations because of their huge molecular weights. Materials & methods: We report the design and synthesis of a smarter novel set of uracil-linked Schiff bases (19–30) as dual HDAC II/Topo I inhibitors keeping the essential pharmacophoric features. Cytotoxicity of all compounds was assessed against three cancer cell lines. Studies of their effects on the apoptotic BAX and antiapoptotic BCL2 genes, molecular docking studies, and absorption, distribution, metabolism and excretion studies were conducted. Results: Compounds 22, 25 and 30 exhibited significant activities. The bromophenyl derivative 22 displayed the best selectivity index, with IC50 values against HDAC II and Topo I of 1.12 and 13.44 μM, respectively. Conclusion: Compound 22 could be considered a lead HDAC II/Topo I inhibitor.

Inflammatory breast cancer (IBC) is a highly aggressive phenotype of breast cancer that is characterized by a high incidence early metastasis. We previously reported a significant association of human cytomegalovirus (HCMV) DNA in the carcinoma tissues of IBC patients but not in the adjacent normal tissues. HCMV-infected macrophages serve as “mobile vectors” for spreading and disseminating virus to different organs, and IBC cancer tissues are highly infiltrated by tumor-associated macrophages (TAMs) that enhance IBC progression and promote breast cancer stem cell (BCSC)-like properties. Therefore, there is a need to understand the role of HCMV-infected TAMs in IBC progression. The present study aimed to test the effect of the secretome (cytokines and secreted factors) of TAMs derived from HCMV+ monocytes isolated from IBC specimens on the proliferation, invasion, and BCSC abundance when tested on the IBC cell line SUM149. HCMV+ monocytes were isolated from IBC patients during modified radical mastectomy surgery and tested in vitro for polarization into TAMs using the secretome of SUM149 cells. MTT, clonogenic, invasion, real-time PCR arrays, PathScan Intracellular Signaling array, and cytokine arrays were used to characterize the secretome of HCMV+ TAMs for their effect on the progression of SUM149 cells. The results showed that the secretome of HCMV+ TAMs expressed high levels of IL-6, IL-8, and MCP-1 cytokines compared to HCMV- TAMs. In addition, the secretome of HCMV+ TAMs induced the proliferation, invasion, colony formation, and expression of BCSC-related genes in SUM149 cells compared to mock untreated cells. In addition, the secretome of HCMV+ TAMs activated the phosphorylation of intracellular signaling molecules p-STAT3, p-AMPKα, p-PRAS40, and p-SAPK/JNK in SUM149 cells. In conclusion, this study shows that the secretome of HCMV+ TAMs enhances the proliferation, invasion, colony formation, and BCSC properties by activating the phosphorylation of p-STAT3, p-AMPKα, p-PRAS40, and p-SAPK/JNK intracellular signaling molecules in IBC cells.