Epithelial-mesenchymal transition-related signaling pathways in gastric Cancer cells distinctively respond to long-term experimental ketosis.

BACKGROUND: The interrelationship between cellular metabolism and the epithelial-to-mesenchymal transition (EMT) process has made it an interesting topic to investigate the adjuvant effect of therapeutic diets in the treatment of cancers. However, the findings are controversial. In this study, the effects of glucose limitation along and with the addition of beta-hydroxybutyrate (bHB) were examined on the expression of specific genes and proteins of EMT, Wnt, Hedgehog, and Hippo signaling pathways, and also on cellular behavior of gastric cancer stem-like (MKN-45) and non-stem-like (KATO III) cells. METHODS AND RESULTS: The expression levels of chosen genes and proteins studied in cancer cells gradually adopted a low-glucose condition of one-fourth, along and with the addition of bHB, and compared to the unconditioned control cells. The long-term switching of the metabolic fuels successfully altered the expression profiles and behaviors of both gastric cancer cells. However, the results for some changes were the opposite. Glucose limitation along and with the addition of bHB reduced the CD44+ population in MKN-45 cells. In KATO III cells, glucose restriction increased the CD44+ population. Glucose deprivation alleviated EMT-related signaling pathways in MKN-45 cells but stimulated EMT in KATO III cells. Interestingly, bHB enrichment reduced the beneficial effect of glucose starvation in MKN-45 cells, but also alleviated the adverse effects of glucose restriction in KATO III cells. CONCLUSIONS: The findings of this research clearly showed that some controversial results in clinical trials for ketogenic diet in cancer patients stemmed from the different signaling responses of various cells to the metabolic changes in a heterogeneous cancer mass.

A Narrative Review on Therapeutic Potentials of Watercress in Human Disorders.

Watercress (WC) is an aquatic vegetable that belongs to the Brassicaceae family, and it often grows near water. In traditional medicine, WC is a known remedy for hypercholesterolemia, hyperglycemia, hypertension, arthritis, bronchitis, diuresis, odontalgia, and scurvy. It also acts as an antiestrogenic and can be used as a nutritional supplement. It has been reported that these therapeutic effects are due to primary metabolites such as isothiocyanates, glucosinolates, polyphenols (flavonoids, phenolic acids, and proanthocyanidins), vitamins (B1, B2, B3, B6, E, and C), terpenes (including carotenoids), and bioelements which exist in this plant. Many pharmacological studies confirm the antioxidant, antibacterial, anticancer, antipsoriatic, anti-inflammatory, cardioprotective, renoprotective, hepatoprotective, and antigenotoxicity effects of WC. The consumption of WC extract can be useful in reducing the complications of hypercholesterolemia and hyperglycemia. Furthermore, the extract of WC could markedly augment the antioxidant enzymes such as superoxide dismutase and catalase activity. Interestingly, consumption of food rich in polyphenols such as WC extract can help reduce oxidative stress, DNA damage, and cancer susceptibility. Several studies also showed that WC extract significantly reduced liver injury as a result of cholestatic hepatic injury, gamma radiation, arsenic, and acetaminophen-induced hepatotoxicity. In this review, the researchers focus on the phytochemical and biochemical characterizations of WC and its therapeutic effects in the treatment of human diseases.

Plasma Cytokines Profile in Subjects with Alzheimer's Disease: Interleukin 1 Alpha as a Candidate for Target Therapy.

Background: Alzheimer's disease (AD) is the main cause of the neurodegenerative disorder, which is not detected unless the cognitive deficits are manifested. An early prediagnostic specific biomarker preferably detectable in plasma and hence non-invasive is highly sought-after. Various hypotheses refer to AD, with amyloid-beta (Aß) being the most studied hypothesis and inflammation being the most recent theory wherein pro-and anti-inflammatory cytokines are the main culprits. Materials and Methods: In this study, the cognitive performance of AD patients (n=39) was assessed using mini-mental state examination (MMSE), AD assessment scale-cognitive subscale (ADAS-cog), and clinical dementia rating (CDR). Their neuropsychiatric symptoms were evaluated through neuropsychiatric inventory-questionnaire (NPI-Q). Moreover, plasma levels of routine biochemical markers, pro-/anti-inflammatory cytokines such as tumor necrosis factor α (TNF-α), interleukin-1 α (IL-1α), IL-1ß, IL-2, IL-4, IL-6, IL-8, IL-12p70, IL-10, Interferon-gamma, chemokines, including prostaglandin E2 (PGE-2), monocyte chemoattractant protein-1, interferon gamma-induced protein 10, Aß peptide species (42, 40) and Transthyretin (TTR) were measured. Results: Our results revealed that Aß 42/40 ratio and TTR were correlated (r=0.367, P=0.037). IL-1α was directly correlated with ADAS-cog (r=0.386, P=0.017) and Aß 40 (r=0.379, P=0.019), but was inversely correlated with IL-4 (r=-0.406, P=0.011). Negative correlations were found between MMSE and PGE2 (r=-0.405, P=0.012) and TNF-α/ IL-10 ratio (r=-0.35, P=0.037). CDR was positively correlated with both PGE2 (r=0.358, P=0.027) and TNF-α (r=0.416, P=0.013). There was a positive correlation between NPI-caregiver distress with CDR (r=0.363, P=0.045) and ADAS-cog (r=0.449, P=0.019). Conclusion: Based on the observed correlation between IL-1α, as a clinical moiety, and ADAS-cog, as a clinical manifestation of AD, anti-IL-1α therapy in AD could be suggested.

Long-Term Glucose Restriction with or without ß-Hydroxybutyrate Enrichment Distinctively Alters Epithelial-Mesenchymal Transition-Related Signalings in Ovarian Cancer Cells.

The beneficial impacts of the ketogenic diet and metabolic reprograming were recently reported for ovarian cancer patients. In this study, the effects of glucose restriction with or without beta-hydroxybutyrate (bHB) enrichment were studied in drug-resistant CD133high A2780CP and CD133low SK-OV-3 ovarian cancer cells to scrutinize the impact of experimental ketosis on ATP production, epithelial to mesenchymal transition (EMT), and related signaling pathways including Wnt, Hippo, and Hedgehog. Cells were adapted and maintained for a month with restricted levels of glucose (250 mg/l) with or without the therapeutic concentration of bHB (5 mM). Quantitative PCR, Western blot analysis, flow cytometry, chemiluminescence, and wound healing assay were used in this study. Glucose restriction and bHB enrichment reduced the stemness marker and diminished In Vitro migration in both cell lines. Glucose restriction significantly reduced ATP levels in both cells, but bHB enrichment was partially compensated for the ATP levels solely in SK-OV-3 cells. Glucose restriction mainly inhibited the Wnt pathway in the CD133high A2780CP cells, but the Hedgehog pathway was the main target in CD133low SK-OV-3 cells. In Conclusion, Prior targeted evaluations of key genes' expression would help to predict the distinctive impacts of metabolic fuels and to optimize the efficacy of ketogenic diets.

Effects of Quinacrine on Expression of Hippo signaling Pathway Components (LATS1, LATS2, and YAP) in Human Breast Cancer Stem Cells.

OBJECTIVE: The Hippo signaling pathway has important role in the pathogenesis of some tumors. Breast cancer is the most prevalent cancer among females in the world. In recent years, various articles referred to inhibiting effect of quinacrine, a derivative of 9-aminoacridine, on the growth of several types of cancer cells. In this study, we evaluated the effect of quinacrine on expression of LATS1, LATS2, and YAP genes of the Hippo signaling pathway and YAP level in human breast cancer stem cells (MDA-MB 231 cell line). This cell line of breast cancer expresses the triple negative characteristics. METHODS: MDA-MB 231 cells was treated with 0.5 µM of quinacrine for 3 days. The dose was selected using MTT assays. The expression of genes was quantified by Real-time PCR. The protein expression was performed by Western blotting. Significance of observations were checked by means of Mann-Whitney test using p.

Evaluation of the protective potential of hydroalcoholic extract of Thymus daenensis on acetaminophen-induced nephrotoxicity in rats.

BACKGROUND: Acetaminophen (APAP) is an antinociceptive and antipyretic drug that can be useful in therapeutic doses, although it can cause serious damage to the kidney if used overdose. The current study aimed to evaluate the protective effect of Thymus daenensis (TD) extract on APAP-induced kidney damage in rats. METHODS: Thirty female Wistar rats were randomly divided into 5 groups: control, APAP (3 g/kg), TD (500 mg/kg), APAP + TD (500 mg/kg), and APAP + N- acetylcysteine (140 mg/kg). The APAP groups received APAP on the 6th day and the rats were sacrificed on the 7th day. Plasma levels of creatinine (Cr) and urea were measured. Ferric reducing antioxidant power (FRAP), nitric oxide (NO) metabolite, total thiol (T-SH), tumor necrosis factor-α (TNF-α) and antioxidant enzymes activity of superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT) were measured in kidney tissue. The gene expression of TNF-α was also measured by real-time PCR. The histological examination of kidney tissue was also performed. RESULTS: Results showed that urea, Cr and FRAP markers markedly elevated in the APAP rats compared with the control group. There was a significant decrease in T-SH levels in the APAP animals in comparison with the control group. CAT activity also augmented in the APAP group compared to the control group. Urea and Cr levels were significantly decreased in the APAP + TD group in comparison with the APAP group. The administration of TD extract significantly increased the SOD enzyme activity. Histological findings were improved in the group treated with TD extract. CONCLUSION: In general, the results indicate that TD extract can protect against APAP-induced nephrotoxicity by improving biochemical, histological and antioxidant effects. However, more studies are required to determine the mechanism of this extract.

The effects of rosmarinic acid on oxidative stress parameters and inflammatory cytokines in lipopolysaccharide-induced peripheral blood mononuclear cells.

Rosmarinic acid (RA) is a potential herbal medicine and has received considerable attention due to its strong antioxidant properties. The aim of this study is to investigate the impact of RA on inflammation and oxidative stress induced by lipopolysaccharide (LPS) in peripheral blood mononuclear cells (PBMCs). PBMCs were pre-treated with various contents of RA (20, 40, 80 µM) for 24 h, then, stimulated with LPS (10 ng/ml) for more 6 h. ELISA and Real-time PCR were done to detect the levels of IL-6, TNF-α, COX-2, IL-1ß and IL-10. Western blot was done to investigate the phosphorylated amounts of P65-NF-κB and JNK. Inflammatory cytokines and oxidant-antioxidant parameters were determined by colorimetric and ELISA methods. The results indicated that LPS augmented the protein levels of IL-6, TNF-α, and IL-1ß cytokines as well as the mRNA levels of IL-6, TNF-α, IL-1ß, COX-2, and IL-10 cytokines in in PBMCs. However, pretreatment with RA could reduce the impact of LPS on inflammatory markers. In addition, RA inhibited P65-NF-κB and JNK phosphorylation. LPS also caused a decrease in antioxidant enzymes, total thiol, and total antioxidant capacity as well as an increment in malondialdehyde and nitric oxide metabolite contents that RA abrogated them. Collectively, our finding demonstrated that RA ameliorates LPS-induced inflammation in PBMCs. RA reduces oxidative stress by preventing lipid peroxidation and nitric oxide production as well as restarting the activity of the GPx and SOD enzymes. Furthermore, our findings indicated that RA was able to protect PBMCs from inflammation via inhibiting the NF-κB and JNK MAPK pathways. This evidence shows a promising therapeutic role for RA in inflammatory status.

Evaluation of Impacts of Cellular Metabolism on the Migration of Ovarian Cancer Cells by Two in Vitro Assays: A Method Comparison Study.

BACKGROUND: Alteration of metabolic pathways in cancer cells can intensely modulate their migration as an important step in invasion and metastasis. Ketogenic diet showed some contradictory results in cancer patients. In this study the impact of metabolic reprogramming of A2780CP as a model of ovarian cancer stem-like cells on cell migration by two in vitro methods: wound healing and soft agar colony-forming assays. MATERIALS AND METHODS: short term and long term metabolic reprogramming were done by restriction of glucose to 250mg/L with or without enrichment with beta-hydroxybutyrate (5 milimolar) for 48 hours and 30 days, respectively. Wound healing assay was done and the wound ratio was calculated for 24 and 48 hours. Soft agar colony formation assay was also done in treated and control cells. For method comparison, ten biological replicates were analyzed in triplicate. RESULTS: Migration of A2780CP ovarian cancer stem-like cells were significantly alleviated by long term glucose restriction but no significant changes were observed in short term study. Beta-hydroxybutyrate enrichment did not produce significant impacts on glucose restriction in short or long term studies. CONCLUSION: The results of colony formation in soft agar and wound or scratch healing assay were in good correlation and convergence which could be used interchangeably in the investigation of metabolic reprogramming in cancer cells.

Histone Butyrylation/ Acetylation Remains Unchanged in Triple Negative Breast Cancer Cells after a Long Term Metabolic Reprogramming.

BACKGROUND: Altered metabolism is one of the hallmarks of the cancer cells which reciprocally interrelate with epigenetic processes, such as post-translational histone modifications to maintain their desired gene expression profiles. The role of beta-hydroxybutyrate as a ketone body in cancer cell biology and histone modifications are reported. The present study aimed to evaluate the impacts of long-term metabolic reprogramming via glucose restriction and beta-hydroxybutyrate treatment on histone acetylation and butyrylation in MDA-MB231 cells as a model of triple negative stem-like breast cancer. METHODS: For long-term treatment, cells were set up in three groups receiving DMEM with restricted glucose (250 mg/L), DMEM with restricted glucose but enriched with five millimolar beta-hydroxybutyrate and DMEM with standard glucose (1gL) and investigated for a month. Histone modifications, including H3 acetylation and butyrylation, were investigated by immunoblotting after an acid extraction of the histone proteins. RESULTS AND CONCLUSION: Neither beta-hydroxybutyrate enrichment nor glucose restriction elicited a significant effect on the butyrylation or acetylation level of histone H3 upon a long-term treatment. Metabolic plasticity of cancer cells, mainly stem-like triple negative breast cancer cells alleviate or neutralize the impact of long-term metabolic reprogramming via restriction of glucose and histone modifications enrichment. These results shed new light upon the mechanism of controversial efficacy of ketogenic diets in clinical trials.

Preparation and Characterization of a Monoclonal Antibody Against Morphine.

A monoclonal antibody (MAb) was produced by immunization of a BALB/c mouse with a conjugated morphine C6-hemisuccinated derivative (MHS) to cationized bovine serum albumin (cBSA). The hybridoma clones were screened by indirect ELISA using MHS-BSA. The best hybridoma clone was subcloned thrice by limiting dilution. This hybridoma was found to be of IgG2b class and subclass and contained lambda light chain. The affinity of the MAb to morphine was obtained 2.8×10(9) M(-1). The titer of the cell culture supernatant was at least 1:800. The MAb was cross-reacted with codeine (100%) and apomorphine (16.5%), but not with heroin, naloxone, naltrexone, or papaverine. Morphine was conjugated to HRP using a mixed anhydride method and a direct competitive ELISA was designed using anti-morphine MAb. The assay was sensitive over the 50 ng/mL to 5 µg/mL concentration range. In conclusion, this MAb is useful for the development of immunoassays to measure morphine in urine.

Characteristics of 26 kDa antigen of H. Pylori by Monoclonal Antibody.

Alkylhydroperoxide reductase (AhpC, the 26 kDa antigen) is one of the abundant antioxidant enzymes in Helicobacter pylori and seems to have a good potential for use in development of immunoassays to detect H. pylori infection in clinical specimens. This study aimed to investigate some properties of this antigen by the produced monoclonal antibodies. Five established hybridoma cell lines secreting monoclonal antibodies (MAbs) against 26 kDa antigen of H. pylori were cultivated and MAbs were purified by affinity chromatography. Subsequently, MAbs were conjugated with biotin, and different combinations of capture and tracer antibodies used in sandwich ELISA. Immunoblotting of bacterial extracts were performed to estimate aggregation status of the antigen. Release of antigen from the cultivated bacteria on solid media was examined by sandwich ELISA, and also, existence of interference in fecal extract was investigated by immunoblotting and sandwich ELISA. Our findings showed that the MAbs against 26 kDa antigen of H. pylori could recognize three bands of nearly 25 kDa, 50 kDa, and 75 kDa in immunoblotting. This study also indicated presence of more antigens in the culture medium around the bacteria than the bacterial extract itself. The results of sandwich ELISA and immunoblotting on fecal extracts suggest the presence of interfering agents that prevent detection of antigen by antibody in ELISA but not in immunoblotting. In this study the oligomerization of the 26 kDa antigen, presence of interfering agents in stool matrix, and release of antigen to outside of bacteria, were demonstrated.

Development of a nanogold-based immunochromatographic assay for detection of morphine in urine using the Amor-HK16 monoclonal antibody.

A simple, rapid competitive immunochromatography (ICG) strip test was developed to detect morphine in urine samples using a monoclonal antibody produced in-house and conjugated to gold nanoparticles. Hybridoma cells were cultured and the Amor-HK16 monoclonal antibody against morphine was obtained from the supernatant after purification by salting out and passing through a Protein G-Agarose affinity column. Morphine was obtained from morphine sulfate and a C6-hemisuccinate derivative of morphine was prepared, conjugated to bovine serum albumin, and immobilized to a nitrocellulose membrane as the test line. Goat anti-mouse antibody was used as a binder in the control line in the detection zone of the strip. Colloidal gold particles of diameter approximately 20 nm were prepared and conjugated to the monoclonal antibody. The detection limit of the test strip was found to be 2000 ng/mL of morphine in urine samples. Reliability was determined by performing the ICG test on 103 urine samples and comparing the results with those obtained by thin-layer chromatography. The sensitivity of the test was 100%, and the analysis time for the assay was approximately 5 min. The new ICG method was adequately sensitive and accurate for the rapid screening of morphine in urine.