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Tumor-associated macrophages (TAMs) have been shown to both aid and hinder tumor growth, with patient outcomes potentially hinging on the proportion of M1, pro-inflammatory/growth-inhibiting, to M2, growth-supporting, phenotypes. Strategies to stimulate tumor regression by promoting polarization to M1 are a novel approach that harnesses the immune system to enhance therapeutic outcomes, including chemotherapy. We recently found that nanotherapy with mesoporous particles loaded with albumin-bound paclitaxel (MSV-nab-PTX) promotes macrophage polarization towards M1 in breast cancer liver metastases (BCLM). However, it remains unclear to what extent tumor regression can be maximized based on modulation of the macrophage phenotype, especially for poorly perfused tumors such as BCLM. Here, for the first time, a CRISPR system is employed to permanently modulate macrophage polarization in a controlled in vitro setting. This enables the design of 3D co-culture experiments mimicking the BCLM hypovascularized environment with various ratios of polarized macrophages. We implement a mathematical framework to evaluate nanoparticle-mediated chemotherapy in conjunction with TAM polarization. The response is predicted to be not linearly dependent on the M1:M2 ratio. To investigate this phenomenon, the response is simulated via the model for a variety of M1:M2 ratios. The modeling indicates that polarization to an all-M1 population may be less effective than a combination of both M1 and M2. Experimental results with the CRISPR system confirm this model-driven hypothesis. Altogether, this study indicates that response to nanoparticle-mediated chemotherapy targeting poorly perfused tumors may benefit from a fine-tuned M1:M2 ratio that maintains both phenotypes in the tumor microenvironment during treatment.

Triple-negative breast cancer is one of the most aggressive breast cancer. The first therapeutic option is chemotherapy, often based on anthracycline as doxorubicin. However, chemotherapy efficacy is limited in by the presence of P-glycoprotein (Pgp), a membrane transporter protein that effluxes doxorubicin, reducing its cellular accumulation and toxicity. Inhibiting Pgp activity with effective and non-toxic products is still an open challenge. In this work, we demonstrated that the natural product Glabratephrin (Glab), a prenylated flavonoid from Tephrosia purpurea with a unique chemical structure, increased doxorubicin accumulation and cytotoxicity in triple negative breast cancer cells with high levels of Pgp, characterized by both acquired or intrinsic resistance to doxorubicin. Glab also reduced the growth of Pgp-expressing tumors, without adding significant extra-toxicities to doxorubicin treatment. Interestingly, Glab did not change the expression of Pgp, but it reduced the affinity for Pgp and the efflux of doxorubicin, as suggested by the increased Km and the reduced Vmax. In silico molecular docking predicted that Glab binds two residues (phenylalanine 322, glutamine 721) localized in the transmembrane domains of Pgp, facing the extracellular environment. Moreover, site-directed mutagenesis identified glycine 185 as a critical residue mediating the reduced catalytic efficacy of Pgp elicited by Glab. We propose Glab as an effective and safe compound able to reverse doxorubicin resistance mediated by Pgp in triple negative breast cancers, opening the way to a new combinatorial approach that may improve chemotherapy efficacy in the most refractory and aggressive breast cancer.

In the current medical era, spirooxindole motif stands out as a privileged heterospirocyclic scaffold that represents the core for a wide range of bioactive naturally isolated products (such as Strychnofoline and spirotryprostatins A and B) and synthetic compounds. Interestingly, no much attention has been paid to develop spirooxindole derivatives with dual antioxidant and anticancer activities. In this context, a series of spirooxindoles 6a-p was examined for their anticancer effect towards HepG2 hepatocellular carcinoma and PC-3 prostate cancer cell lines. Spirooxindole 6a was found to be an efficient anti-proliferative agent towards both HepG2 and PC-3 cells (IC50 = 6.9 and 11.8 µM, respectively). Afterwards, spirooxindole 6a was assessed for its apoptosis induction potential in HepG2 cells, where its pro-apoptotic impact was approved via the significant elevation in the Bax/Bcl-2 ratio and the expression levels of caspase-3

A polysaccharide characterized as galactomannan (GMann) with a molecular weight of 117.76 kDa was isolated from the aqueous extract of Caesalpinia gilliesii (C. gilliesii) seeds then assessed for antiproliferative potential against human hepatocellular carcinoma cell line (HepG2). Further, HCC was induced in Wister albino rats by Diethylnitrosamine (DEN) ip injection (200 mg/kg bw), and CCl4 orally (2 ml/kg bw) for two months then subjected to GMann orally treatment (2 mg/kg bw) for one month. In results, isolated GMann is constituted of sugars (89.99 ± 2.3%), moisture (6.89 ± 0.45%), ash (0.06 ± 0.2%), and protein (2.81%) and composed mainly of mannose and galactose in ratio M/G 3.79. In vitro study, data revealed a concentration-dependent potency of GMann to induce cell death of HepG2 cells with IC50 value of 0.375 µg/ml. Mechanistic studies revealed the potential of GMann to arrest cell cycle at G2/M phase with induction of apoptosis. Biochemical results in vivo showed a significant reduction in serum transaminases (ALT and AST) as well as hepatic malondialdehyde (MDA) and nitric oxide (NOx). Molecular analysis declared a significant down-regulation in mRNA gene expression of both nuclear factor kappa-B (NF-κB) and tumor necrosis factor (TNF-α). Furthermore, a significant down-regulation in the cellular oncogene-fos (C-fos) and marked up-regulation in Glycogen synthase kinase-3 (GSK-3β) level were observed. These results were supported with histopathological investigation. Whereas GMann improved inflammatory and apoptotic markers, it could be a promising new therapeutic agent for HCC suppression and this warrant further development as a possible drug candidate for HCC.

Triple-negative breast cancer (TNBC) is a highly aggressive malignancy with limited treatment options due to its heterogeneity and the lack of well-defined molecular targets. In our endeavour towards the development of novel anti-TNBC agents, herein we report a one-pot three-component synthesis of novel spirooxindoles 6a–p, and evaluation of their potential anti-proliferative activity towards TNBC MDA-MB-231 cells. Spirooxindoles 6a, 6e and 6i emerged as the most potent analogues with IC50 = 6.70, 6.40 and 6.70 µM, respectively. Compounds 6a and 6e induced apoptosis in MDA-MB-231 cells, as evidenced by the up-regulation of the Bax and down-regulation of the Bcl-2, besides boosting caspase-3 levels. Additionally, 6e displayed significant increase in the percent of annexin V-FITC positive apoptotic cells from 1.34 to 44%. Furthermore, spirooxindoles 6e and 6i displayed good inhibitory activity against EGFR (IC50 = 120 and 150 nM, respectively). Collectively, these data demonstrated that 6e might be a potential lead compound for the development of effective anti-TNBC agents.

Artificially designed miRNAs mimics and inhibitors that specifically target known oncogenes have attracted significant research attention. Herein, we aimed to explore whether MIR-375, MIR-145, and MIR-224 are involved in induction of apoptosis of CRC cells by regulating apoptosis-mediating genes MTDH, MAP3K1, PDK1, BAX, and BCL-XL. MTT assay was used to assess cell growth. Apoptosis was determined in terms of caspase activity measurement and phosphatidylserine detection using annexin V staining by flow cytometry. Quantitative real time PCR, Western blotting, and luciferase reporter assay were carried out to validate genes regulation and targeting by miRNAs. We found that ectopic expression of MIR-375 and MIR-145, and inhibition of MIR-224 can decrease cell growth and induce cell ability to undergo early apoptosis. At mRNA level, transfected cells displayed down-regulation of MTDH, PDK1 and BCL-XL, while BAX and MAP3K1 were up-regulated. Protein expression of MTDH was decreased in cells transfected with MIR-145 mimic and MIR-224 inhibitor but remained unchanged in MIR-375 mimic-transfected cells. Furthermore, MAP3K1 protein expression exibited a decreased level after MIR-375 transient expression with no significant change after MIR-145 mimic or MIR-224 inhibitor transfection. Luciferase reporter assay revealed that MIR-375 and MIR-145 can bind to 3'UTR of MTDH, supporting that MTDH is directly targeted by both miRNAs. Similarly, MAP3K1 was found to be directly regulated by MIR-375. The study concluded that the expression modulation of tumor suppressors MIR-375 and MIR-145, and oncomiR MIR-224 have the ability to induce apoptosis of CRC cells through regulation of apoptosis mediating genes MTDH, MAP3K1, PDK1, BCL-XL and BAX.

Euphorbia species are rich in diterpenes. A solvent extraction of Euphorbia sanctae-catharinae, a species indigenous to the Southern Sinai of Egypt, afforded several premyrsinane diterpenoids (1–4) as well as previously reported metabolites (5–13) that included three flavonoids. Isolated compounds were chemically characterized by spectroscopic analysis. Identified compounds were bioassayed for anti-proliferative activity in vitro against colon (Caco-2) and lung (A549) tumor cell lines. Compound 9 exhibited robust anti-proliferative activity against A549 cells (IC50 = 3.3 µM). Absolute configurations for 8 versus 9 were determined by experimental and TDDFT-calculated electronic circular dichorism (ECD) spectra.

Diterpenoids salvimulticanol (1) and salvimulticaoic acid (2) together with known diterpenoid (3-6) were isolated from Salvia multicaulis. Structures were elucidated by spectroscopic techniques including HRESIMS as well as 1D-, and 2D-NMR. In-vitro cytotoxicity was assayed against human cancer cell lines. As several metabolites exhibited activity against drug-resistance lines, compounds were screened against a panel of human drug-sensitive and multidrug-resistant cancer lines. A proposed biosynthetic pathway for these new diterpenoids (1-2) as well as the cytotoxic structure-activity relationship of all identified compounds were discussed. Compound 1 and 6 showed the most potent cytotoxicity with IC50 11.58 and 4.13 towards leukemia cell lines CCRF-CEM and CEM-ADR5000, respectively.

Multidrug resistance (MDR) in various kinds of cancers represents a true obstacle which hinders the successes of most of current available chemotherapies. ATP-binding cassette (ABC) trasporter proteins have been shown to contribute to the majority of MDR in various types of malignancies. c-myc has recently been reported to participate, at least partly, in MDR to some types of cancers. This study aimed to test whether c-myc could play a role, solely or with coordination with other ABCs, in the resistance of HepG2 cells to doxorubicin (Dox). MDR has been induced in wild-type HepG2 and has been verified both on gene and protein levels. Various assays including efflux assays as well as siRNA targeting ABCB1 and c-myc have been employed to explore the role of both candidate molecules in MDR in HepG2. Results obtained, with regard to ABCB1 silencing on HepG2/Dox cells, have shown that ABCB1-deficient cells exhibited a significant reduction in ABCC1 expression as compared to ABCB1-sufficient cells. However, these cells did not show a significant reduction in other tested ABCs (ABCC5 and ABCC10) while c-myc silencing had no significant effect on any of the studied ABCs. Moreover, silencing of ABCB1 on HepG2 significantly increased fluorescent calcein retention in HepG2 cells as compared to the control cells while downregulation of c-myc did not have any effect on fluorescent calcein retention. Altogether, this work clearly demonstrates that c-myc has no role in MDR of HepG2 to Dox which has been shown to be ABCB1-mediated in a mechanism which might involve ABCC1.

MicroRNAs (miRNAs) trigger a two-layer regulatory network directly or through transcription factors and their co-regulators. Unlike miR-375, the role of miR-145 and miR-224 in inhibiting or driving cancer cell migration is controversial. This study is a step towards addressing the potential of miR-375, miR-145 and miR-224 expression modulation to inhibit colorectal carcinoma (CRC) cells migration in vitro through regulation of non-target genes VEGFA, TGFβ1, IGF1, CD105 and CD44. Transwell migration assay results revealed a significant subdue of migration ability of cells transfected with miR-375 and miR-145 mimics and miR-224 inhibitor. Real time PCR data showed that expression of VEGFA, TGFβ1, IGF1, CD105 and CD44 was downregulated as a consequence of exogenous re-expression of miR-375 and inhibition of miR-224. On the other hand, ectopic expression of miR-145 did not affect VEGFA, TGFβ1 and CD44 expression, while it elevated CD105 and suppressed IGF1 expression. MAP4K4, a predicted target of miR-145, was validated as a target that could play a role in miR-145-mediated regulation of migration. At mRNA level, no change was observed in expression of MAP4K4 in cells with restored expression of miR-145, while western blotting analysis revealed a 25% reduction of protein level. By applying luciferase reporter assay, a significant decrease in luciferase activity was observed, supporting that miR-145 directly target 3' UTR of MAP4K4. The study highlighted the involvement of non-target genes VEGFA, TGFβ1, IGF1, CD105 and CD44 in mediating anti- and pro-migratory effect of miR-375 and miR-224, respectively, and validated MAP4K4 as a direct target of anti-migratory miR-145.

Forty-four endophytic fungal isolates obtained from marine sponge, Hyrtios erectus, were evaluated and screened for their hydrolase activities. Most of the isolates were found to be prolific producers of hydrolytic enzymes. Only 11 isolates exhibited maximum cellular contents of lipids, rhamnolipids, and protein in the fungal isolates under the isolation numbers MERVA5, MERVA22, MERVA25, MERVA29, MERVA32, MERVA34, MERV36, MERVA39, MERVA42, MERVA43, and MERVA44. These isolate extracts exhibit the highest reducing activities against carbohydrate-metabolizing enzymes including α-amylase, α-glucosidase, β-glucosidase, β-glucuronidase, and tyrosinase. Consequently, based on morphological and cultural criteria, as well as sequence information and phylogenetic analysis, these isolates could be identified and designated as Penicillium brevicombactum MERVA5, Arthrinium arundinis MERVA22, Diaporthe rudis MERVA25, Aspergillus versicolor MERVA29, Auxarthron alboluteum MERVA32, Dothiorella sarmentorum MERVA34, Lophiostoma sp. MERVA36, Fusarium oxysporum MERVA39, Penicillium chrysogenum MERVA42, Penicillium polonicum MERVA43, and Trichoderma harzianum MERVA44. The endophytic fungal species, D. rudis MERVA25, P. polonicum MERVA43, Lophiostoma sp. MERVA36, A. alboluteum MERVA32, T. harzianum MERVA44, F. oxysporum MERVA39, A. versicolor MERVA29, and P. chrysogenum MERVA42 extracts, showed significant hepatitis C virus (HCV) inhibition. Moreover, D. sarmentorum MERVA34, P. polonicum MERVA43, and T. harzianum MERVA44 extracts have the highest antitumor activity against human hepatocellular carcinoma cells (HepG2).

While select eudesmane sesquiterpenes exhibit anti-neoplastic activity, tumor-inhibition for costic-acids has not been established. Here biological activity of 3-oxo-γ-costic acid (1), previously isolated from Chiliadenus montanus, as well as new sesquiterpenes (2–5) and the known derivative, 3-oxoeudesma-1,4,11(13)-trien-7-1061αH-l2-oic acid (6), all produced from 1 by the fungus Athelia rolfsii, are reported. Structures were elucidated using MS and NMR spectroscopy with activity-screening utilizing human colon- and lung-tumor lines, Caco-2 and A549 respectively. Compound 1 exhibited anti-proliferative activity against Caco-2 (IC50 39 µM) and 2 was active against A549 (IC50 74 µM) suggesting therapeutic potential for the original substrate and a bio-transformed product.

Background The multidrug resistance (MDR) phenotype encounters a major challenge to the success of established chemotherapy in cancer patients. We hypothesized that cytotoxic medicinal plants with novel phytochemicals can overcome MDR and kill MDR-cells with similar efficacy as drug sensitive cells. Purpose We evaluated plant extracts from an unexplored ecosystem in Egypt with unusual climate and nutrient conditions for their activity against sensitive and multidrug-resistant cancer cell lines. Material and methods/study design Methylene chloride: methanol (1:1) and methanol: H2O (7:3) extracts of 40 plants were prepared resulting in a sum of 76 fraction containing compounds with varying polarity. The resazurin reduction assay was employed to evaluate the cytotoxicity of these extracts on five matched pairs of drug-sensitive and their drug-resistant cell lines. Flow cytometry and Western blotting was used to determine cell cycle analyses, apoptosis, and autophagy. Reactive oxygen species (ROS) were measured spectrophotometrically. Results Extracts derived from Withania obtusifolia (WO), Jasonia candicans (JC), Centaurea lippii (CL), and Pulicaria undulata (PU) were the most active ones among 76 extracts from 40 Egyptian medicinal plants. They showed a significant reduction of cell viability on drug-sensitive CCRF-CEM leukemia cell line with IC50 values less than 7 µg/ml. Low cross-resistance degrees were observed in multidrug-resistant CEM/ADR5000 cells towards CL (1.82-fold) and JC (6.09-fold). All other drug-resistant cell lines did not reveal cross-resistance to the four extracts. Further mechanistic assessment have been studied for these four extracts. Conclusion The methylene chloride: methanol (1:1) fractions of WO, JC, CL, and PU are promising cytotoxic extracts that could be used to combat MDR cancer cells through different cell death pathways.

Aims To investigate effect of metallic nanoparticles, silver (AgNPs) and gold nanoparticles (AuNPs) as antitumor treatment in vitro against human breast cancer cells (MCF-7) and their associated mechanisms. This could provide new class of engineered nanoparticles with desired physicochemical properties and may present newer approaches for therapeutic modalities to breast cancer in women. Materials and methods A human breast cancer cell line (MCF-7) was used as a model of cells. Metallic nanoparticles were characterized using UV-visible spectra and transmission electron microscopy (TEM). Cytotoxic effects of metallic nanoparticles on MCF-7 cells were followed by colorimetric SRB cell viability assays, microscopy, and cellular uptake. Nature of cell death was further investigated by DNA analysis and flow cytometry. Results Treatment of MCF-7 with different concentrations of 5-10nm diameter of AgNPs inhibited cell viability in a dose-dependent manner, with IC50 value of 6.28μM, whereas treatment of MCF-7 with different concentrations of 13-15nm diameter of AuNPs inhibited cell viability in a dose-dependent manner, with IC50 value of 14.48μM. Treatment of cells with a IC50 concentration of AgNPs generated progressive accumulation of cells in the S phase of the cell cycle and prevented entry into the M phase. The treatment of cells with IC50 concentrations of AuNPs similarly generated progressive accumulation of cells in sub-G1 and S phase, and inhibited the entrance of cells into the M phase of the cell cycle. DNA fragmentation, as demonstrated by electrophoresis, indicated induction of apoptosis. Conclusions Our engineered silver nanoparticles effectively inhibit the proliferation of human breast carcinoma cell line MCF-7 in vitro at high concentration (1000 μM) through apoptotic mechanisms, and may be a beneficial agent against human carcinoma but further detailed study is still needed.

Background Egyptians recognized the healing power of herbs and used them in their medicinal formulations. Nowadays, “Attarin” drug shops and the public use mainly the Unani medicinal system for treatment of their health problems including improvement of memory and old age related diseases. Numerous medicinal plants have been described in old literature of Arabic traditional medicine for treatment of Alzheimer’s disease (AD) (or to strengthen memory). Methods In this study, some of these plants were evaluated against three different preliminary bioassays related to AD to explore the possible way of their bio-interaction. Twenty three selected plants were extracted with methanol and screened in vitro against acetylcholinesterase (AChE) and cycloxygenase-1 (COX-1) enzymes. In addition, anti-oxidant activity using DPPH was determined. Results Of the tested plant extracts; Adhatoda vasica and Peganum harmala showed inhibitory effect on AChE at IC50 294 μg/ml and 68 μg/ml respectively. Moreover, A. vasica interacted reversibly with the enzyme while P. harmala showed irreversible inhibition. Ferula assafoetida (IC50 3.2 μg/ml), Syzygium aromaticum (34.9 μg/ml) and Zingiber officinalis (33.6 μg/ml) showed activity against COX-1 enzyme. Potent radical scavenging activity was demonstrated by three plant extracts Terminalia chebula (EC50 2.2 μg/ml), T. arjuna (3.1 μg/ml) and Emblica officinalis (6.3 μg/ml). Conclusion Interestingly, differential results have been obtained which indicate the variability of the mode of actions for the selected plants. Additionally, the reversible interaction of A. vasica against AChE and the potent activity of F. assafoetida against COX-1 make them effective, new and promising agents for treatment of AD in the future, either as total extracts or their single bioactive constituents.

Background The Egyptian government is considering embarking on a new wave of health sector reform. Although primary care is seen as central to the anticipated reforms, little is known about the current morbidity and utilization patterns in Egyptian publicly funded primary care. We conducted this survey study of patient encounters to describe the demographic characteristics of patients attending publicly-funded primary care practices, the relative frequency of conditions encountered in these practices, and the rates of drug prescription, investigation and referral. Method Cross-sectional survey of twelve primary care practices and 2458 patient consultations. Additional secondary data were collected from five of the twelve practices for preventive services provided at these practices i.e. immunizations, family planning and ante-natal care. Results 54% of the attendances were for people below the age of twenty, of which 54% were females. In patients above the age of twenty, women accounted for 73% of consultations. Upper respiratory tract infection was the most common reason for encounter, accounting for 24% of the presentations, followed by gastroenteritis (10%), intestinal parasites (5%), and lower respiratory tract infections (5%). Over 97% of patients were prescribed at least one drug, whereas investigation and referral rates were low (15% and 5% respectively). When the analysis was repeated for practices where data on both curative and preventive services were available (5 practices and 2146 consultations), substantial proportions of patients were found to seek care for immunizations (25%), family planning (12%), and ante-natal care (11%). Conclusion Most patients utilizing primary care practices in Egypt seek care for minor and preventive services with relatively few consultations for more serious conditions. There is also a pattern of prescribing drugs to most primary care patients which may reflect over-prescribing by primary care doctors.