The inhibitory effects of plumbagin on the NF-қB pathway and CCL2 release in racially different triple-negative breast cancer cells.

Breast cancer (BC) is the second leading cause of death among women in the US, and its subtype triple-negative BC (TNBC) is the most aggressive BC with poor prognosis. In the current study, we investigated the anticancer effects of the natural product plumbagin (PL) on racially different TNBC cells. The PL effects were examined in two TNBC cell lines: MDA-MB-231 (MM-231) and MDA-MB-468 (MM-468), representing Caucasian Americans and African Americans, respectively. The results obtained indicate that PL inhibited cell viability and cell proliferation and induced apoptosis in both cell lines. Notably, MM-468 cells were 5-fold more sensitive to PL than MM-231 cells were. Testing PL and Taxol® showed the superiority of PL over Taxol® as an antiproliferative agent in MM-468 cells. PL treatment resulted in an approximately 20-fold increase in caspase-3 activity with 3 µM PL in MM-468 cells compared with an approximately 3-fold activity increase in MM-231 cells with 8 µM PL. Moreover, the results indicate a higher sensitivity to PL in MM-468 cells than in MM-231 cells. The results also show that PL downregulated CCL2 cytokine expression in MM-468 cells by 30% compared to a 90% downregulation in MM-231 cells. The ELISA results confirmed the array data (35% vs. 75% downregulation in MM-468 and MM-231 cells, respectively). Moreover, PL significantly downregulated IL-6 and GM-CSF in the MM-231 cells. Indeed, PL repressed many NF-қB-regulated genes involved in the regulation of apoptosis, proliferation, invasion, and metastasis. The compound significantly downregulated the same genes (BIRC3, CCL2, TLR2, and TNF) in both types of cells. However, PL impacted five more genes in MM-231 cells, including BCL2A1, ICAM1, IKBKE, IL1ß, and LTA. In conclusion, the data obtained in this study indicate that the quinone compound PL could be a novel cancer treatment for TNBC in African American women.

The attenuating effects of plumbagin on pro-inflammatory cytokine expression in LPS-activated BV-2 microglial cells.

Activated microglial cells produce the pro-inflammatory mediators such as nitric oxide (NO) and cytokines. The excessive release of these mediators can lead to neurodegenerative diseases, such as Alzheimer's disease (AD) and Parkinson's disease (PD). Inhibition of the release of these pro-inflammatory molecules may prevent or halt the progression of these diseases. Plumbagin (PL), a naphthoquinone compound in the roots of the traditional medicinal plant Plumbago zeylanica L., showed anti-inflammatory effects on macrophages. However, PL effects on activated microglia remain unknown. In the present study, PL has been examined for its anti-inflammatory effect on LPS - activated microglial BV-2 cells. In this study, NO and iNOS expression were investigated in BV-2 microglial cells in the presence of PL or the selective iNOS inhibitor L-N6-(1-iminoethyl) lysine (L-NIL). The results obtained indicate that PL was >30-fold potent than L-NIL in inhibiting NO production with an IC50 of 0.39µM. Our immunofluorescence study confirmed the ability of PL to significantly inhibit iNOS expression in the activated microglia. Furthermore, the extracellular microglial pro-inflammatory cytokine expression in the presence of 2µM of PL was detected, quantified, and validated using cytokine antibody protein arrays and quantitative ELISA. The results obtained showed that PL significantly downregulated the expression of many cytokines including IL-1α, G-CSF, IL-12 p40/p70, MCP-5, MCP-1, and IL-6. In conclusion, PL potency in attenuating multiple pro-inflammatory agents indicates its potential to be used for neurodegenerative diseases.

Inhibitory effects of cigarette smoke on glial inducible nitric oxide synthase and lack of protective properties against oxidative neurotoxins in vitro.

Epidemiological studies consistently report an inverse correlation between cigarette smoking and associated risk for Parkinson's disease (PD). The degeneration of dopaminergic neurons may involve the toxic metabolic products of glial cell monoamine oxidase (MAO) and inducible nitric oxide synthase (iNOS). This study evaluates the direct protective effects of cigarette smoke (CS) against potential neurotoxic products of MAO, such as 1-methyl-4-phenylpyridinium (MPP+), 6-hydroxydopamine (6-OHDA) and hydrogen peroxide (H2O2) in brain neuroblastoma. Moreover, the effects of CS were also evaluated on endotoxin/cytokine activated glioma iNOS protein expression and MAO enzyme activity. Cigarette smoke condensates (CSCs) were acquired from Marlboro 20 Class A and Kentucky 2R4F reference research (2R4F) cigarettes. The CSCs did not protect against 6-OHDA or H2O2 toxicity in neuroblastoma, and exhibited a very mild protective effect [approximately 10%] against MPP+. Neither CSC demonstrated antioxidant capability, but conversely contained high concentration of NO2-. Paradoxically, in glioma cells, iNOS protein expression and endogenous enzymatic NO2- production were significantly blocked by both CSCs. Both CSCs also inhibited glioma MAO-A and MAO-B [1.4.3.4]. Kinetic analysis indicated that 2R4F-CSC displayed competitive inhibition and the Marlboro-CSC exerted potent competitive and non-competitive inhibition. In conclusion, these data suggest that cigarette smoke does not appear to directly protect against the toxicity of the selected neurotoxins. In contrast, CS exerts pronounced effects on glia, whereby its presence can simultaneously attenuate cytokine induction of iNOS and MAO.

Prenatal Cocaine Exposure Affects Postnatal Dopaminergic Systems in Various Regions of the Rat Braina.

Pregnant female Sprague-Dawley rats were injected once daily with either 40 mg/kg cocaine hydrochloride or 0.9% saline from gestation day (GD)12 to GD 21. On postnatal day (PND)30, male offspring were sacrificed and fresh tissue from the striatum (ST) and nucleus accumbens (NA) was dissected for assessment of dopamine (DA) receptor affinity, DA uptake and DA release. 10-6 M cocaine inhibited [3H]-DA uptake in ST tissue, whereas 10-4 and 10-5 M cocaine inhibited [3H]-DA uptake in the NA tissue of postnatally exposed cocaine offspring verses saline-treated controls (p <0.05). DA release stimulated by 10-6 M amphetamine was significantly reduced in both the ST (p <0.001) and NA (p <0.01) of postnatal offspring exposed to cocaine in utero compared with saline controls. In utero cocaine exposure did not influence offspring ST or NA dopamine 1 (D1) dissociation constant (Kd) or receptor density (Bmax). However, the treatment group experienced a significant increase of binding affinity for the ST D2 receptor with no change in D2 Bmax. The treatment group also experienced no change in D2 receptor binding affinity or number of binding sites in the NA. These results show that in utero exposure to cocaine results in altered postnatal dopaminergic function.

Repeated Administration of Cocaine Alters Dopamine Uptake and Release in the Striatum Nucleus Accumbensa.

Male Sprague-Dawley rats were administered 25 mg/kg, intraperitoneally (i.p.) cocaine-HCl twice daily for 14 consecutive days (total of 50 mg/kg), while control animals received an equivalent volume of 0.9% saline. After three days of withdrawal, the animals were sacrificed for dissection of striatal (STR) and nucleus accumbens (NA) brain regions. The treated group demonstrated a dose-dependent reduction for in vitro cocaine inhibition of [3H]dopamine (DA) uptake in the NA tissue verses controls. There were no significant differences amongst the treated and control groups for in vitro cocaine inhibition of [3H]DA in the STR. In vitro d-amphetamine (1, 5 and 10 µM)-stimulated DA release from STR tissue was not significantly different between the treated and the control groups. However, there was a significant decline in basal STR DA release and a significant enhancement of d-amphetamine (1 and 5 µM)-stimulated DA release in the NA for the treatment group versus controls. The results from the present study indicates sensitization to cocaine is primarily related to DA uptake and release in the NA.