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Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer that lacks specific targets such as estrogen, progesterone, and HER2 receptors. TNBC affects one in eight women in the United States, making up 15-20% of breast cancer cases. Patients with TNBC can develop resistance to chemotherapy over time, leading to treatment failure. Therefore, finding other options like natural products is necessary for treatment. The advantages of using natural products sourced from plants as anticancer agents are that they are less toxic, more affordable, and have fewer side effects. These products can modulate several cellular processes of the tumor microenvironment, such as proliferation, migration, angiogenesis, cell cycle arrest, and apoptosis. The phosphatidyl inositol 3-kinase (PI3K)-AKT signaling pathway is an important pathway that contributes to the survival and growth of the tumor microenvironment and is associated with these cellular processes. This current study examined the anticancer effects of fucoxanthin, a marine carotenoid isolated from brown seaweed, in the MDA-MB-231 and MDA-MB-468 TNBC cell lines. The methods used in this study include a cytotoxic assay, PI3K-AKT signaling pathway PCR arrays, and Wes analysis. Fucoxanthin (6.25 µM) + TNF-α (50 ng/mL) and TNF-α (50 ng/mL) showed no significant effect on cell viability compared to the control in both MDA-MB-231 and MDA-MB-468 cells after a 24 h treatment period. PI3K-AKT signaling pathway PCR array studies showed that in TNF-α-stimulated (50 ng/mL) MDA-MB-231 and MDA-MB-468 cells, fucoxanthin (6.25 µM) modulated the mRNA expression of 12 genes, including FOXO1 , RASA1 , HRAS , MAPK3 , PDK2 , IRS1 , EIF4EBP1 , EIF4B , PTK2 , TIRAP , RHOA , and ELK1 . Additionally, fucoxanthin significantly downregulated the protein expression of IRS1, EIF4B, and ELK1 in MDA-MB-231 cells, and no change in the protein expression of EIF4B and ELK1 was shown in MDA-MB-468 cells. Fucoxanthin upregulated the protein expression of RHOA in both cell lines. The modulation of the expression of genes and proteins of the PI3K-AKT signaling pathway may elucidate fucoxanthin's effects in cell cycle progression, apoptotic processes, migration, and proliferation, which shows that PI3K-AKT may be the possible molecular mechanism for fucoxanthin's effects. In conclusion, the results obtained in this study elucidate fucoxanthin's molecular mechanisms and indicate that fucoxanthin may be considered a promising candidate for breast cancer-targeted therapy.

The lack of hormone receptors in triple negative breast cancer (TNBC) is associated with the inefficacy of anti-estrogen chemotherapies, leaving fewer options for patient treatment and higher mortality rates. Additionally, as with numerous types of inflammatory breast cancer, infiltration of tumor associated macrophages and other leukocyte sub-populations within the tumor inevitably lead to aggressive, chemo-resistant, metastatic and invasive types of cancer which escape immune surveillance. These processes are orchestrated by the release of potent cytokines, including TNFα, IL-6 and CCL2 from the stroma, tumor and immune cells within the tumor microenvironment. The present study evaluated apigenin modulating effects on the pro-inflammatory activating action of TNFα in TNBC MDA-MB-468 cells, derived from an African American woman. Initially, cell viability was determined to establish an optimal sub-lethal dose of TNFα and apigenin in MDA-MB-468 cells. Subsequently, various treatments effects were evaluated using whole transcriptomic analysis of mRNA and long intergenic non-coding RNA with Affymetrix HuGene-2.1-st human microarrays. Gene level differential expression analysis was conducted on 48,226 genes where TNFα caused significant upregulation of 53 transcripts and downregulation of 11 transcripts. The largest upward differential shift was for CCL2 [+61.86 fold change (FC); false discovery rate (FDR), P<0.0001]; which was down regulated by apigenin (to +10.71 FC vs. Control; FDR P-value <0.001), equivalent to an 83% reduction. Several TNFα deferentially upregulated transcripts were reduced by apigenin, including CXCL10, C3, PGLYRP4, IL22RA2, KMO, IL7R, ROS1, CFB, IKBKe, SLITRK6 (a checkpoint target) and MMP13. Confirmation of CCL2 experimentally induced transcript alterations was corroborated at the protein level by ELISA assays. The high level of CCL2 transcript in the cell line was comparable to that in our previous studies in MDA-MB-231 cells. The differential effects of TNFα were corroborated by ELISA, where the data revealed a >10-fold higher releasing rate of CCL2 in MDA-MB-468 cells compared with in MDA-MB-231 cells, both of which were attenuated by apigenin. The data obtained in the present study demonstrated a high level of CCL2 in MDA-MB-468 cells and a possible therapeutic role for apigenin in downregulating TNFα-mediated processes in these TNBC cells., (Copyright: © Bauer et al.)

Background: Prostate cancer is the second most common cause of mortality. Gallic acid (GA) is a natural polyphenol, and we tested its in-vitro cytotoxicity after 24 h in prostate cancer LNCaP cells., Materials and Methods: GA autoxidation was measured fluorimetrically for H(2)O(2), and O(2)(•-) radicals by chemiluminescence. Intracellular reactive oxygen species (ROS) levels were detected with 2',7'-dichlorodihydrofluorescein diacetate. Cytotoxicity was evaluated by crystal-violet, while apoptosis and mitochondrial membrane potential were determined by flow cytometry. Cytochrome c release was detected by enzyme-linked immunosorbent assay, and caspase-8, -9 and -3 activities were measured calorimetrically., Results: GA autoxidation produced significant levels of H(2)O(2) and O2.-. Increased intracellular ROS levels with GA were reduced by N-acetyl-L-cysteine (NAC) and L-glutathione (GSH). Cells were protected against GA cytotoxicity when pretreated with increasing levels of superoxide dismutase/catalase mixture, NAC, or GSH for 3 h. The number of apoptotic cells increased with GA dose. GA caused mitochondrial potential loss, cytochrome c release, and activation of caspases 3, 8 and 9., Conclusion: The ROS-dependent apoptotic mechanism of GA kills malignant cells effectively; it is likely that GA could be a good anticancer agent.

Diallyl disulfide (DADS), a garlic organosulfur compound, has been researched as a cancer prevention agent; however, the role of DADS in the suppression of cancer initiation in nonneoplastic cells has not been elucidated. To evaluate DADS inhibition of early carcinogenic events, MCF-10A cells were pretreated (PreTx) with DADS followed by the ubiquitous carcinogen benzo(a)pyrene (BaP), or cotreated (CoTx) with DADS and BaP for up to 24 h. The cells were evaluated for changes in cell viability/proliferation, cell cycle, induction of peroxide formation, and DNA damage. BaP induced a statistically significant increase in cell proliferation at 6 h, which was attenuated with DADS CoTx. PreTx with 6 and 60 μM of DADS inhibited BaP-induced G2/M arrest by 68% and 78%, respectively. DADS, regardless of concentration or method, inhibited BaP-induced extracellular aqueous peroxide formation within 24 h. DADS attenuated BaP-induced DNA single-strand breaks at all time points through both DADS Pre- and CoTx, with significant inhibition for all treatments sustained after 6 h. DADS was effective in inhibiting BaP-induced cell proliferation, cell cycle transitions, reactive oxygen species, and DNA damage in a normal cell line, and thus may inhibit environmentally induced breast cancer initiation.

Phencyclidine (PCP) is a non-competitive N-methyl-d-aspartate (NMDA) receptor antagonist and exposing the developing brain to PCP has been shown to cause deficits in neurobehavioral functions. In the present study we tested the effects of PCP, as an NMDA receptor inhibitor, on the neuronal differentiation and biogenic amines levels including norepinephrine (NE), epinephrine, dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), serotonin (5-HT), and 5-hydroxyindole-3-acetic acid (5-HIAA) in the rat pheochromocytoma (PC12) cells. After PC12 cells were differentiated with nerve growth factor (NGF) in the presence of PCP, NMDA binding kinetics, biogenic amines analysis and NMDA receptor protein expression assay were conducted. The results showed that NMDA receptor binding activities were significantly increased after differentiated with NGF in PC12 cells. B(max) values were increased in differentiated cells by four-folds, whereas K(d) values were not changed. All of biogenic amines were significantly increased in differentiated cells. On the other hand, PCP at 50 and 100 microM inhibited neuronal differentiation in a dose-dependent manner in NGF-stimulated PC12 cells without affecting cell viability. PCP treatment during differentiation significantly reduced NMDA binding activity and biogenic amine levels. Western blotting analysis revealed that NMDA receptor protein expression was significantly higher in NGF-differentiated cells and PCP treatment decreased the expression of NMDA receptor proteins. These results indicate that NMDA receptor functions and monoaminergic nervous systems are significantly stimulated during NGF-induced differentiation. PCP suppresses neuronal outgrowth and hampers neuronal functions possibly by inhibiting NMDA receptor functions and biogenic amine production, implying the suppressive effects of PCP exposure on neuronal developments.

Although the etiology of Parkinson's disease (PD) is not fully understood, there are numerous studies that have linked the increased risk for developing PD to pesticides exposure including paraquat (PQ). Moreover, the exposure to a combination of compounds or chemical mixtures has been suggested to further increase this risk. In the current study, the effects of PQ on the nigrostriatal dopaminergic system in male C57BL6 mice exposed to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) were examined to assess the impact of toxic substance mixtures exposure on neurochemical and behavioral changes. In this study, a low non-toxic dose of MPTP (10mg/kg) was injected once a day for 5 days and was followed by PQ (7 mg/kg) once a day for 6 days (subacute protocol) or once a week for 10 weeks (chronic protocol). The results from the subacute protocol showed that PQ reduced the turnover of dopamine (DA) as indicated by a 21% and a 22.3% decrease in dihydroxyphenyl acetic acid (DOPAC), homovanillic acid and increased S-adenosyl methionine/S-adenosyl homocysteine index (SAM/SAH) by 100%. However, the administration of PQ to MPTP primed mice resulted in the decrease of DOPAC, HVA, DA, by 35.8%, 35.2% and 22.1%, respectively. In addition, PQ decreased the total number of movements (TM) by 28% but MPTP plus PQ decreased TM by 41%. The SAM/SAH index showed that MPTP increased methylation by 33.3%, but MPTP plus PQ increased methylation by 81%. In the chronic protocol, the data showed that MPTP administration did not affect DA, DOPAC, and HVA levels. The administration of PQ led to significant decrease in DOPAC, HVA, and TD by 31.6%, 19.9%, and 21.2% respectively with no effect on DA levels. The MPTP plus PQ group showed reduced DA, DOPAC, HVA, and total distance traveled by 58.4%, 82.8%, 55.8%, and 83.9%, respectively. Meanwhile, PQ administration caused a reduction in tyrosine hydroxylase immunoreactivity in the substantia nigra, and this effect was more pronounced in MPTP pretreated mice. It was concluded from this study that prior treatment with MPTP potentiated the effects of PQ in reducing DA, DOPAC, HVA, TH immunoreactivity, locomotor activity, and increasing the methylation index. The enhanced effects of PQ following MPTP administration further support the role of toxic substance mixtures in causing Parkinson's disease.

In case of injury or disease, microglia are recruited to the site of the pathology and become activated as evidenced by morphological changes and expression of pro-inflammatory cytokines. Evidence suggests that microglia proliferate by cell division to create gliosis at the site of pathological conditions such as the amyloid plaques in Alzheimer's disease and the substantia nigra of Parkinson's disease patients. The hyperactivation of microglia contributes to neurotoxicity. In the present study we tested the hypothesis that anti-inflammatory compounds modulate the progression of cell cycle and induce apoptosis of the activated cells. We investigated the effects of ibuprofen (non-steroidal anti-inflammatory drug) and apigenin (a flavonoid with anti-inflammatory and anti-proliferative properties) on the cell cycle of the murine microglial cell line BV-2. The findings indicate that apigenin-induced cell cycle arrest preferentially in the G2/M phase and ibuprofen caused S phase arrest. The binding of annexin V-FITC to the membranes of cells which indicates the apoptotic process were examined, whereas the DNA was stained with propidium iodide. Both apigenin and ibuprofen induced apoptosis significantly in early and late stages. The induction of apoptosis by ibuprofen and apigenin was confirmed using TUNEL assay, revealing that 25 microM apigenin and 250 microM ibuprofen significantly increased apoptosis in BV-2 cells. The results from the present study suggest that anti-inflammatory compounds might inhibit microglial proliferation by modulating the cell cycle progression and apoptosis.

In many cases, development of insulin resistance has been linked to obesity and may contribute to mechanism of aging. The role of diet, irrespective of degree of obesity, in modulating insulin resistance and development of age degeneration disease remains uncertain. Lowered blood glucose levels are commonly associated with diet restriction (DR), which is an intervention shown to successfully retard aging and age associated disease. The effects of DR on blood glucose and insulin resistance were measured in yellow obese (A(vy)/A), lean black (a/a) mice and in another common inbred strain (B6C3F1) (at three different ages). The yellow obese mice become diabetic as a result of an insulin receptor defect which is not clearly understood. Insulin responses and radioinsulin binding were assayed in yellow obese and lean black mice fed either ad libitum (AL) or DR diets (YAL, BAL, YDR and YAL, respectively) at four different circadian intervals. The B6C3F1 controls were fed either AL (CAL) or DR (CDR) and measures were made at six circadian stages and three different ages. Within 23 days, DR produced a significant loss in body weight and a time-dependent 22-55% reduction in basal blood glucose levels in the yellow obese mice. Additionally, exogenous insulin produced circadian stage dependent (at the time of food intake) reductions in blood glucose in the YDR animals that were not present in YAL animals. (125)I-Insulin binding in liver was increased nearly 2-fold in YDR and BDR mice during the time of day that animals were active and eating. (125)I-Insulin binding was two-fold-higher in CDR mice at 4, 12 and >24 months of age. Binding decreased as a function of age in both the CAL and CDR animals. However, even in the >24 month group the CDR animals were found to have levels of binding that were as high as those found in younger CAL liver. The mechanism of action appears to be through resolution of insulin resistance by modulating an insulin receptor defect.

We examined the influences of dietary restriction (DR) on the circadian profile of liver catalase (CAT), glutathione peroxidase (GPx), and interacting systems required for removal of H2O2 (support systems), in 18-week old female Fischer 344 rats fed 60% of their ad libitum (AL) diet for six weeks. Food was presented to the DR animals during the early light-span. Regardless of diet, enzyme levels were generally consistent with circadian patterns. In CR animals, maximum activities often occurred at the time of food presentation. CAT and GPx activities generally were significantly higher in DR animals than in AL animals at the time of feeding. When assessing glucose-6-phosphate dehydrogenase (G6PDH) activity using saturating substrate (NADP(+)) concentrations, higher activities were seen at all times of day in the AL animals; however, when activity was measured in the presence of lower (i.e., physiologic) NADP(+) concentrations, the reverse was true. In contrast, glutathione reductase (GR) activity was not influenced by DR. Cytosolic levels of NADPH peaked and were higher in DR than in AL rodents prior to feeding. NADH levels were not influenced by diet, but did manifest a significant circadian pattern with a maximum occurring toward the middle of the dark span. These data suggest that even at a young age and following only a relatively brief duration of DR, there exists an enhanced enzymatic capability in rats subjected to DR to remove free radicals generated as a consequence of normal oxidative metabolism. Further, these data support emerging trends suggesting metabolic regulation of antioxidant defense systems in response to free radical generation.

Novel steroidal antiinflammatory antedrugs, 11 beta,20-dihydroxy-3,20-dioxo-3'-(ethoxycarbonyl)-isoxazolino[16,17 - d]pregna-1,4-diene (2a) and 9-fluoro-11 beta,20-dihydroxy-3,20-dioxo-3'-ethoxy- carbonylisoxazolino[16,17-d]pregna-1,4-diene (2b) were prepared in 97% yield via 1,3-dipolar cycloaddition of carbethoxyformonitrile (CEFNO) to 11 beta,21-dihydroxy-3,20-dioxopregna-1,4,-16-triene (1a) and 11 beta,21-dihydroxy-3,20-dioxo-9-fluoropregna-1,4,16-triene (1b), respectively, which were prepared via five steps from prednisolone and 9-fluoroprednisolone, respectively. The treatment of steroids 2a and 2b with acetic anhydride in pyridine led to the corresponding 21-acetates 3a and 3b, respectively, in 95% yield. Dose-response profiles of the croton oil-induced ear edema bioassay in rats were used to calculate the following ID50 values (nmol/ear resulting in a 50% reduction of edema): prednisolone (P), 540 nmol; 2b, 135 nmol; and 3b, 101 nmol. Inhibition of edema did not exceed 50% following application of either 2a or 3a. Relative potency calculations indicated that 2b was 4-fold and 3b 5.3-fold more potent than the parent compound P when applied topically. No significant adverse systemic effects were seen following treatments with 3b. These results suggest that C-9-fluorination, side-chain hydroxy group esterification, and [16 alpha,17 alpha-d]-3'-carbethoxyisoxazoline additions to the conventional steroid P improve topical antiinflammatory activity without concomitant increases in adverse systemic activity.

Male Sprague-Dawley rats weighing approximately 225 g and maintained under controlled lighting and temperature conditions, were used in this experiment. Animals were given morphine (10 mg/kg, i.p.) either at 10:00 hr or at 22:00 hr. One hour later, treated animals with their proper control were sacrificed and the mucosa of the stomach, duodenum, ileum and colon were separated and assayed for acetylcholinesterase (AChE) and choline acetyltransferase (ChAT) activities. Data obtained indicate that morphine administration during the light phase resulted in a significant increase (p less than 0.01) in AChE activity in the colon, with no significant effect on the stomach, duodenum or the ileum. There was also significant increase (p less than 0.05) in ChAT activity in the stomach and the duodenum as a result of morphine administration during the light phase, with no effect on other tissues studied. During the dark phase, there were no significant changes observed in AChE activity of all tissues examined following morphine administration. However, the determination of ChAT activity during the dark phase, following morphine treatment, showed a significant increase (p less than 0.01) in the duodenum and the ileum, with a significant decline (P less than 0.05) in the colon. Results obtained from this study indicate that gastrointestinal changes seen after morphine administration could be due to changes in the activity of the cholinergic enzymes and these changes may be diurnally controlled.

Male Sprague-Dawley rats maintained under controlled lighting and temperature conditions were used in this experiment. Animals were exposed to acute cold (4 degrees C) and immobilization stress for one hour, exposed to cold stress for 7 days (chronic stress) or treated with corticosterone (2 mg/kg) 1 hr prior to sacrificing. Animals with their proper controls were sacrificed and the stomach, duodenum, ileum and colon were separated and assayed for choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) activities. The data obtained indicated that exposure to acute stress resulted in significant decline in ChAT activity in all tissues studied. The administration of corticosterone resulted in significant decline in ChAT activity in all tissues studied except for the duodenum. Meanwhile, the exposure to chronic stress did not have any significant effect on ChAT activity. On the other hand, acute stress caused significant increase in AChE activity in all tissues studied except for the ileum and stomach. The duodenal AChE activity of stressed animals increased thirty-fold when compared to control. The administration of glucocorticoids significantly reduced AChE in all tissues studied, except for the duodenum and stomach where there was thirty-two-fold increase as compared to the control levels. The exposure to chronic stress also caused significant increase in AChE of all tissues studied, except for the colon. The results of this experiments indicate that the duodenal AChE is extremely sensitive to stress or glucocorticoids and that stress induced changes in the cholinergic enzymes of the gastrointestinal tract may be mediated by adrenal steroids.

Male Sprague-Dawley rats weighing between 180 and 220 g and maintained under controlled lighting and temperature conditions were used in this experiment. Animals were given ethanol (3 g/kg, p.o.) 24 h after fasting. One group was given ethanol at 10.00 h (light phase) and the other at 22.00 h (dark phase). One hour later, the treated animals with their proper controls were sacrificed and the mucosa of the stomach, duodenum, ileum and colon were separated and assayed for choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) activities. Data obtained indicate that the administration of ethanol resulted in significant decline (p less than 0.01) in ChAT activity in the stomach and the colon during the light phase. A significant increase (p less than 0.01) in ChAT activity was also noted in the ileum during the dark phase. There was a significant decrease (p less than 0.01) in AChE activity in the stomach during the dark phase. The administration of ethanol also resulted in a significant decline in AChE activity (p less than 0.05) in the duodenum and the colon (p less than 0.01). The results obtained indicate that the gastrointestinal changes caused by ethanol administration may be related to changes in the cholinergic enzymes of the mucosa of the gastrointestinal tract.

Male Sprague-Dawley rats maintained under controlled environmental conditions were used. Choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) activities were determined in the cerebral cortex, bulbus olfactorius, midbrain, hypothalamus, hippocampus, cerebellum, pons and medulla oblongata of control rats and rats treated with apomorphine (15 mg/kg, i.p.) after a single dose or after a second dose administered 24 h later at 10.00 or 22.00 h. Results of this experiment indicate that the repeated administration of apomorphine at 10.00 h was associated with rapid development of tolerance to the hypothermic action of this drug. A single injection of apomorphine resulted in significant (p less than 0.01) decrease of AChE and significant increase of ChAT activity (p less than 0.01) in the cortex, hypothalamus and pons. There were no significant differences between tolerant and control animals in the activities of AChE or ChAT in the bulbus olfactorius, cerebral cortex, midbrain, pons or medulla. On the other hand, repeated administration of apomorphine at 22.00 h was not associated with tolerance to the hypothermic action of this drug. A single injection of apomorphine at 22.00 h resulted in significant (p less than 0.01) increase in AChE activity of the midbrain, hippocampus, and the medulla oblongata with no significant changes in the cerebral cortex and bulbus olfactorius. There was a significant decline (p less than 0.01) of ChAT activity of the hypothalamus, hippocampus, cerebral cortex and the medulla oblongata (p less than 0.05). Meanwhile, there was a significant (p less than 0.05) increase of ChAT activity of the midbrain with no significant changes in the cerebellum, pons and the bulbus olfactorius.(ABSTRACT TRUNCATED AT 250 WORDS)

In this study, male Sprague-Dawley rats maintained under controlled environmental conditions were used. Choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) activities were determined in cerebral cortex, bulbus olfactorius, midbrain, hypothalamus, hippocampus, cerebellum, pons and medulla oblongata in control rats and rats treated with morphine (10 mg/kg) for 1 or 2 days. Repeated administration of morphine was associated with a decline in the degree of analgesia produced. Significant increase (p less than 0.01) in AChE activity of the medulla oblongata was observed following morphine administration for 1 or 2 days. A single injection of morphine resulted in a significant decline (p less than 0.01) in ChAT activity of hypothalamus, cerebellum and medulla oblongata. However, no such decline could be observed after 2 consecutive daily injections of morphine. In the cerebral cortex there was a significant decline (p less than 0.01) in ChAT activity after the second administration of morphine. These findings indicate that the changes in the responsiveness of the brain cholinergic enzymes following repeated morphine administration may in part explain the rapid development of tolerance to the analgesic effect of morphine.