Salvimulticanol from Salvia multicaulis suppresses LPS-induced inflammation in RAW264.7 macrophages: in vitro and in silico studies.

Sustained inflammatory responses can badly affect several vital organs and lead to chronic inflammation-related disorders, such as atherosclerosis, pneumonia, rheumatoid arthritis, obesity, diabetes, Alzheimer's disease, and cancers. Salvia multicaulis is one of the widely distributed plants that contains several biologically active phytochemicals and diterpenoids with anti-inflammatory effects. Therefore, finding alternative and safer natural plant-extracted compounds with good curative anti-inflammatory efficiencies is an urgent need for the clinical treatment of inflammation-related diseases. In the current study, S. multicaulis Vahl was used to extract and isolate two compounds identified as salvimulticanol and candesalvone B methyl ester to examine their effects against inflammation in murine macrophage RAW264.7 cells that were induced by lipopolysaccharide (LPS). Accordingly, after culturing RAW264.7 cells and induction of inflammation by LPS (100 ng/ml), cells were exposed to different concentrations (9, 18, 37.5, 75, and 150 µM) of each compound. Then, Griess assay for detection of nitric oxide (NO) levels and western blotting for the determination of inducible nitric oxide synthase (iNOS) expression were performed. Molecular docking and molecular dynamics (MD) simulation studies were employed to investigate the anti-inflammatory mechanism. Our obtained results validated that the level of NO was significantly decreased in the macrophage cell suspensions as a response to salvimulticanol treatment in a dose-dependent manner (IC50: 25.1 ± 1.2 µM) as compared to the methyl ester of candesalvone B which exerted a weaker inhibition (IC50: 69.2 ± 3.0 µM). This decline in NO percentage was comparable with a down-regulation of iNOS expression by western blotting. Salvimulticanol strongly interacted with both the Toll-like receptor 4 (TLR4)/myeloid differentiation factor 2 (MD-2) complex and the inhibitor of nuclear factor kappa-B (NF-κB) kinase subunit beta (IKKß) to disrupt their inflammatory activation due to the significant hydrogen bonds and effective interactions with amino acid residues present in the target proteins' active sites. S.multicaulis is a rich natural source of the aromatic abietane diterpenoid, salvimulticanol, which exerted a strong anti-inflammatory effect through targeting iNOS and diminishing NO production in LPS-induced RAW264.7 cells in a mechanism that is dependent on the inhibition of TLR4-MD-2 and IKKß as activators of the classical NF-κB-mediated inflammatory pathway.

Restoring microRNA-34a overcomes acquired drug resistance and disease progression in human breast cancer cell lines via suppressing the ABCC1 gene.

PURPOSE: Breast cancer is one of the leading types of cancer diagnosed in women. Despite the improvements in chemotherapeutic cure strategies, drug resistance is still an obstacle leading to disease aggressiveness. The small non-coding RNA molecules, miRNAs, have been implicated recently to be involved as regulators of gene expression through the silencing of mRNA targets that contributed to several cellular processes related to cancer metastasis. Hence, the present study aimed to investigate the beneficial role and mechanism of miRNA-34a-based gene therapy as a novel approach for conquering drug resistance mediated by ATP-binding cassette (ABC) transporters in breast cancer cells, besides exploring the associated invasive behaviors. MATERIAL AND METHODS: Bioinformatics tools were used to predict miRNA ABC transporter targets by tracking the ABC transporter pathway. After the establishment of drug-resistant breast cancer MCF-7 and MDA-MB-231 sublines, cells were transfected with the mimic or inhibitor of miRNA-34a-5p. The quantitative expression of genes involved in drug resistance was performed by QRT-PCR, and the exact ABC transporter target specification interaction was confirmed by dual-luciferase reporter assay. Furthermore, flow cytometric analysis was utilized to determine the ability of miRNA-34a-treated cells against doxorubicin uptake and accumulation in cell cycle phases. The spreading capability was examined by colony formation, migration, and wound healing assays. The apoptotic activity was estimated as well. RESULTS: Our findings firstly discovered the mechanism of miRNA-34a-5p restoration as an anti-drug-resistant molecule that highly significantly attenuates the expression of ABCC1 via the direct targeting of its 3'- untranslated regions in resistant breast cancer cell lines, with a significant increase of doxorubicin influx by MDA-MB-231/Dox-resistant cells. Additionally, the current data validated a significant reduction of metastatic potentials upon miRNA-34a-5p upregulation in both types of breast cancer-resistant cells. CONCLUSION: The ectopic expression of miRNA-34a ameliorates the acquired drug resistance and the migration properties that may eventually lead to improved clinical strategies and outcomes for breast cancer patients. Additionally, miRNA-34a could be monitored as a diagnostic/prognostic biomarker for resistant conditions.

Anti-proliferative effect of melatonin in human hepatoma HepG2 cells occurs mainly through cell cycle arrest and inflammation inhibition.

Hepatocellular carcinoma (HCC) is the major lethal primary liver malignant worldwide. Although, melatonin has various antitumor bioactivities; there is a requirement for more investigations to elucidate the not discussed effects, and the controversial responses of the treatment with melatonin on targets mediated in HCC. To achieve the aim of the present study, HCC-HepG2 cells were treated with different concentrations of melatonin at various time intervals. The selected minimal proliferation inhibition doses of melatonin were then incubated with cells to examine the arresting effect of melatonin on dividing cells using flow cytometry. Furthermore, the molecular patterns of genes that contributed to apoptosis, drug resistance development, antioxidation, and melatonin crossing were quantified by qRT-PCR. Additionally, the Human inflammation antibody array membrane (40 targets) was used to check the anti-inflammatory effect of melatonin. Our results validated that, melatonin shows anti-proliferative action through preserving cells in G0/G1 phase (P < 0.001) that is associated with a highly significant increase in the expression level of the P53 gene (P < 0.01). On contrary, as a novelty, our data recorded decreases in expression levels of genes involved in the pro-apoptotic pathway; with a significant increase (P < 0.05) in the expression level of an anti-apoptotic gene, Bcl2. Interestingly, we detected observed increases in the expression levels of genes responsible for conferring drug resistance including ABCB1, ABCC1, and ABCC5. Our study proved the anti-inflammatory activity of 1 mM melatonin in HCC-HepG2 cells. Accordingly, we can conclude that melatonin facilitates the anti-proliferation of cells at doses of 1 mM, and 2.5 mM after 24 h. This action is initiated through cell cycle arrest at G0/G1 phase via increasing the expression of P53, but independently on apoptosis. Collectively, melatonin is an effective anti-inflammatory and anti-proliferative promising therapy for the treatment of HCC. However, its consumption should be cautious to avoid the development of drug resistance and provide a better treatment strategy.

Anti-drug resistance, anti-inflammation, and anti-proliferation activities mediated by melatonin in doxorubicin-resistant hepatocellular carcinoma: in vitro investigations.

Hepatocellular carcinoma (HCC) is the major life-threatening primary liver malignancy in both sexes all over the world. Unfortunately, the majority of patients are diagnosed at later stages because HCC does not elicit obvious symptoms during its early incidence. Consequently, most individuals escape the first-line HCC treatments and are treated with chemotherapy. Regrettably, the therapeutic outcomes for those patients are usually poor because of the development of multidrug resistance phenomena. Furthermore, most anti-HCC therapies cause severe undesired side effects that notably interfere with the life quality of such patients. Accordingly, there is an important need to search for an alternative therapeutic drug or adjuvant which is more efficient with safe or even minimal side effects for HCC treatment. Melatonin was recently reported to exert intrinsic antitumor activity in different cancers. However, the regulatory pathways underlying the antitumor activity of melatonin are poorly understood in resistant liver cells. Furthermore, a limited number of studies have addressed the therapeutic role of melatonin in HCC cells resistant to doxorubicin chemotherapy. In this study, we investigated the antitumor effects of melatonin in doxorubicin-resistant HepG2 cells and explored the regulatory pivotal targets underlying these effects. To achieve our aim, an MTT assay was used to calculate the 50% inhibitory concentration of melatonin and evaluate its antiproliferative effect on resistant cells. Additionally, qRT-PCR was used to quantify genes having a role in drug resistance phenotype (ABCB1, ABCC1, ABCC2, ABCC3, ABCC4, ABCC5, and ABCG2); apoptosis (caspases-3, and -7, Bcl2, Bax, and p53); anti-oxidation (NRF2); expression of melatonin receptors (MT1, MT2, and MT3); besides, programmed death receptor PD-1 gene. The active form of the caspase-3 enzyme was estimated by ELISA. A human inflammatory antibody membrane array was employed to quantify forty inflammatory factors expressed in treated cells. We observed that melatonin inhibited the proliferation of doxorubicin-resistant HepG2 cells in a dose-dependent manner after 24-h incubation time with a calculated IC50 greater than 10 mM (13.4 mM), the expression levels of genes involved in drug resistance response (ABCB1, ABCC1, ABCC5, and ABCG2) were downregulated. Also, the expression of caspase-3, Caspase-7, NRF2, and p53 genes were expressed at higher levels as compared to control (DMSO-treated cells). An active form of caspase-3 was confirmed by ELISA. Moreover, the anti-inflammatory effect of melatonin was detected through the calculated fold change to control which was reduced for various mediators that have a role in the inflammation pathway. The current findings introduce melatonin as a promising anti-cancer treatment for human-resistant HCC which could be used in combination with current chemotherapeutic regimens to improve the outcome and reduce the developed multidrug resistance.

The diagnostic significance of circulating miRNAs and metabolite profiling in early prediction of breast cancer in Egyptian women.

OBJECTIVE: Breast cancer (BC) is one of the most commonly diagnosed solid malignancies in women worldwide. PURPOSE: Finding new non-invasive circulating diagnostic biomarkers will facilitate the early prediction of BC and provide valuable insight into disease progression and response to therapy using a safe and more accessible approach available every inspection time. Therefore, our present study aimed to investigate expression patterns of potentially circulating biomarkers that can differentiate well between benign, malignant, and healthy subjects. METHODS: To achieve our target, quantitative analyses were performed for some circulating biomarkers which have a role in the proliferation and tumor growth, as well as, glutamic acid, and human epidermal growth receptor 2 (HER2) in blood samples of BC patients in comparison to healthy controls using qRT-PCR, liquid chromatography/mass spectrometry (LC/MS/MS), and ELISA. RESULTS: Our findings showed that the two miRNAs (miRNA-145, miRNA-382) were expressed at lower levels in BC sera than healthy control group, while miRNA-21 was expressed at higher levels in BC patients than control subjects. Area under ROC curves of BC samples revealed that AUC of miRNA-145, miRNA-382, miRNA-21, and glutamic acid was evaluated to equal 0.99, 1.00, 1.00 and 1.00, respectively. Besides, there was a significantly positive correlation between miRNA-145 and miRNA-382 (r = 0.737), and a highly significant positive correlation between miRNA-21 and glutamic acid (r = 0.385). CONCLUSION: Based on our results, we conclude that the detection of serum miRNA-145, -382 and -21 as a panel along with glutamic acid, and circulating HER2 concentrations could be useful as a non-invasive diagnostic profiling for early prediction of breast cancer in Egyptian patients. It can provide an insight into disease progression, discriminate between malignancy and healthy control, and overcome the use limitations (low sensitivity and specificity, repeated risky exposure, and high cost) of other detecting tools, including mammography, magnetic resonance imaging, and ultrasound.

Importance of immunoglobulin therapy for COVID-19 patients with lymphocytopenia.

BACKGROUND: The global coronavirus disease 2019 (COVID-19) was announced as pandemic by the World Health Organization (WHO). With the increased number of infected and dead victims daily all over the world, it becomes necessary to stop or overcome its rapid spread.Main bodyAlthough the production of vaccine or even specified effective anti-virus may take about six months to a year, intravenous immunoglobulin (IVIg) may be clinically used as a safe treatment to save and improve the quality of life of patients with a variety of immunodeficiency diseases such as lymphocytopenia, which is a common clinical feature in COVID-19. CONCLUSION: Through the current review, it was concluded that this passive immunization may promote the immunity to better fight against the virus, so the survival of the patients could be kept longer. The efficacy of immunotherapy with IVIg would be greater if the immune IgG antibodies were collected from convalescent plasma therapy.

Crosstalk between NRF2 and Dicer through metastasis regulating MicroRNAs; mir-34a, mir-200 family and mir-103/107 family.

Oxidative stress due to generation of reactive oxygen species (ROS) can cause damage to cellular proteins, lipids and DNA, which is one of crucial causes responsible for cancer. Nuclear factor erythroid 2 [NF-E2]-related factor 2 (NRF2) is a transcription factor of a variety of antioxidant and cytoprotective enzymes, so that it reduces the levels of damaging ROS in the cell. Over expression of NRF2 in cancer cells can enhance cancer progression, confer resistance to chemo and radiotherapy, and metastasis through the process of epithelial-to mesenchymal transition (EMT); which is a hallmark of cancer-related death. Dicer, a key component of the microRNAs biogenesis, is a ribonuclease enzyme which involves in maturation of microRNAs that have a role in distinct steps of metastasis cascade. Moreover, Dicer was found to be regulated by ROS/NRF2 interaction to contribute to activation of DNA damage repair mechanism. In addition, Dicer is directly reduced by mir-103/107 family that confers migratory capacity through down-regulation of mir-200 family (mir-200b/mir-200c/mir-429). Mir-200c and mir-34a were predicted to target the repressor of NRF2; Sirt1. On the other hand, mir-200a and mir-141 (mir-200 family) were detected to regulate NRF2 expression. This review highlights the regulation of redox homeostasis that is mediated by NRF2 could be modulated by metastasis regulating microRNAs under the control of Dicer. In addition, NRF2 may indirectly control DNA damage repair and microRNAs processing machinery through the crosstalk between NRF2 and Dicer. Understanding such interrelations could provide and shed light on the significance of microRNA-based therapies that will improve the action of clinically used cancer treatments.

The Significance of HCV Viral Load in the Incidence of HCC: a Correlation Between Mir-122 and CCL2.

BACKGROUND: Hepatocellular carcinoma (HCC) is the fifth most common cancer, the third leading cause of cancer deaths worldwide with over 500,000 people affected. It is a major cause of death in patients with chronic hepatitis C virus (HCV) infection. Overwhelming lines of epidemiological evidence have indicated that persistent infection with HCV is a major risk for the development of HCC. Although a proportion of patients with a chronic hepatitis C virus infection progress to HCC, the peak incidence of HCC associated with HCV infection has not yet occurred. AIM: This review aimed to assess the impact of hepatitis C viral load on the development of HCC as a correlation between mir-122 and, the key factor in fibrogenesis, CCL2. CONCLUSION: According to the detailed explanation of the role of mir-122 and CCL2 in HCV and HCC and the evidence of the inverse correlation between them, it may be concluded that HCV may affect mir-122 expression level of the hepatocytes with different patterns depending on the viral genotype. Collectively, HCV viral load alone is not sufficient to predict the HCC development and progression. Besides the quantitative evaluation of the HCV, mir-122 and CCL2 determinations should also be taken into consideration.

Possible Role of microRNA-122 in Modulating Multidrug Resistance of Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is a hypervascular primary liver cancer characterized by rapid progression, besides, resistance to traditional chemotherapeutic agents. It has been shown that microRNAs play critical roles in regulation of tumor cell sensitivity to drugs through modulating the expression of genes involved in drug transport. The present study investigated whether restoration of miR-122 in HCC cells could alter the cell cycle distribution and the expression of multidrug resistance (MDR)-related genes (ABCB1, ABCC1, ABCG2 and ABCF2). After overexpression of miR-122 in HepG2 cells treated or untreated with doxorubicin doses, total RNAs and protein extracts were isolated for application of QRT-PCR and western blotting techniques. Moreover, cell cycle distribution was monitored by flow cytometry. Our results revealed that, the over expression of miR-122 in HepG2 cells treated or untreated with doxorubicin could modulate the sensitivity of cells to chemotherapeutic drug through downregulation of MDR-related genes, ABCB1 and ABCF2. Interpretation of cell cycle distribution revealed that, the anti-proliferative effect of miR-122 is associated with the accumulation of cells in G0/G1 phase. Moreover, treatment with miR-122 and doxorubicin resulted in high percentage of HCC cells in G0/G1 phase. Taken together, our findings revealed that, overexpression of miR-122 inhibited HCC cell growth by inducing cell cycle arrest and this arrest is associated with down-regulation of MDR-related genes.