Administration Of Plumbagin Research Articles

Oral administration of plumbagin is beneficial in in vivo models of Duchenne muscular dystrophy through control of redox signaling

Duchenne muscular dystrophy (DMD) is a progressive muscle-wasting disease. Recently approved molecular/gene treatments do not solve the downstream inflammation-linked pathophysiological issues such that supportive therapies are required to improve therapeutic efficacy and patients' quality of life. Over the years, a plethora of bioactive natural compounds have been used for human healthcare. Among them, plumbagin, a plant-derived analog of vitamin K3, has shown interesting potential to counteract chronic inflammation with potential therapeutic significance.In this work we evaluated the effects of plumbagin on DMD by delivering it as an oral supplement within food to dystrophic mutant of the fruit fly Drosophila melanogaster and mdx mice. In both DMD models, plumbagin show no relevant adverse effect. In terms of efficacy plumbagin improved the climbing ability of the dystrophic flies and their muscle morphology also reducing oxidative stress in muscles. In mdx mice, plumbagin enhanced the running performance on the treadmill and the muscle strength along with muscle morphology. The molecular mechanism underpinning these actions was found to be the activation of nuclear factor erythroid 2-related factor 2 pathway, the re-establishment of redox homeostasis and the reduction of inflammation thus generating a more favorable environment for skeletal muscles regeneration after damage.Our data provide evidence that food supplementation with plumbagin modulates the main, evolutionary conserved, mechanistic pathophysiological hallmarks of dystrophy, thus improving muscle function in vivo; the use of plumbagin as a therapeutic in humans should thus be explored further.

Plumbagin rescues the granulosa cell’s pyroptosis by reducing WTAP-mediated N6-methylation in polycystic ovary syndrome

The survival of ovary granulosa cells (GC) is critical in the initiation and progression of polycystic ovary syndrome (PCOS) in females. Here, we found that the PCOS process is accompanied by massive GC pyroptosis resulting from Caspase-1 inflammasome activation. Administration of plumbagin, an effective compound isolated from plant medicine, can prevent the pyroptosis of GC and the onset of PCOS. Mechanistic study indicates the over-activation of the inflammasome in GC is due to the upregulation of WTAP, a key regulator of the RNA N6-methylase complex. WTAP mediates the mRNA N6-methylation of NLRP3 inflammasome component ASC and enhances ASC RNA stability, which results in the overactivation of the inflammasome in GCs from the PCOS model. Plumbagin treatment suppresses the WTAP-mediated N6-methylation of ASC mRNA and reduces the pyroptosis of GCs. This study supports the profound potential of plumbagin in PCOS treatment.Graphical

Plumbagin Attenuates Depression-Like Behavior in Mice by Modulating Dopamine Levels

This research was done to investigate the antidepressant properties of plumbagin using a chronic mild stress induced depression model in experimental animals. Albino mice of either sex were given 3 weeks of chronic mild stress and their antidepressant properties were assessed using the tail suspension test and sucrose preference test. The period of immobility in the tail suspension test and the percentage preference for sucrose solution were both recorded and compared. Plumbagin was given in three doses (4, 8, and 16 mg/kg). The immobility time in the tail suspension test was significantly decreased in the animals that received plumbagin administration during the three phases of the chronic mild stress when compared to control. Further, plumbagin administration dose dependently increased the percentage preference for sucrose solution on par with the standard antidepressant, imipramine. The spontaneous locomotion count was significantly higher in the animals that received a high dose of plumbagin. Further, a high dose of plumbagin significantly reverses the chronic mild stress induced plasma corticosterone and nitrile levels. Additionally, a high level of dopamine was found in the prefrontal and nucleus accumbens parts of the brain. To summarize, plumbagin, in addition to its antioxidant characteristics, has potential antidepressant action at high doses, potentially by raising the dopamine levels.

Effects of Plumbagin on Proliferation, Invasion and Migration of Glioma Cell Line U-118

To observe the inhibitory effect of plumbagin on the invasion and migration of glioma cells U-118 and to explore the possible mechanism of its action. U-118 cells were cultured in medium with different concentrations of plumbagin and the cells were counted and morphological changes were observed under the microscope after 24 h of incubation and the cell proliferation was detected by 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyl tetrazolium bromide, the effect of plumbagin on cell migration was detected by cell scratching assay and the effect of cell invasion was detected by Transwell method. Western blotting was used to detect the expression of matrix metalloproteinase-2, matrix metalloproteinase-9 and p21 protein. The results showed that the morphology of U-118 cells could be changed by plumbagin and it was dependent on the concentration; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide results showed that plumbagin significantly inhibited the growth of U-118 cells and its inhibitory ability was positively correlated with the concentration; scratch assay results showed that the planar motility of cells decreased significantly with the increase of plumbagin concentration; Transwell Western blotting results showed that plumbagin administration could inhibit the expression of matrix metalloproteinase-2 and matrix metalloproteinase-9 proteins in U-118 cells, but significantly increased the expression of P21 and there was a positive correlation with the concentration. Plumbagin can inhibit the proliferation of U-118 cells and suppress cell invasion and the mechanism may be related to the inhibition of matrix metalloproteinase-2, matrix metalloproteinase-9 and p21 expression.

Plumbagin Prevents Secretory Diarrhea by Inhibiting CaCC and CFTR Channel Activities.

Secretory diarrhea, which primarily originates through intestinal pathogens and viruses, is a health burden in many regions worldwide. Enterocyte Cl− channels, as the final step in enterotoxin-induced fluid secretion, constitute an attractive class of targets for diarrhea therapy. Chloride channel inhibitors have become a new class of candidates for antisecretion and anti-intestinal motility agents. In the present study, we identified plumbagin as a transmembrane protein 16A (TMEM16A) inhibitor in a cell-based fluorescence-quenching assay, and the IC50 value was ∼12.46 µM. Short-circuit current measurements showed that plumbagin reversibly inhibited the Eact-induced Cl− current on the apical side of TMEM16A-transfected Fischer rat thyroid (FRT) cells with no significant effect on cytoplasmic Ca2+ signaling. Notably, plumbagin also inhibited the activity of intestinal epithelial calcium-activated chloride channel (CaCC) and cystic fibrosis transmembrane conductance regulator (CFTR) in both HT-29 cells and mouse colons, but had no effects on the activity of the Na+-K+ ATPase or K+ channels. In in vivo experiments, the administration of plumbagin reduced both Escherichia coli heat-stable enterotoxin (STa)- and cholera toxin (CT)-induced intestinal fluid secretion. In neonatal mouse models of CT- and rotavirus infection-induced diarrhea, 0.4 µg plumbagin inhibited secretory diarrhea by >40% and 50%, respectively, without affecting intestinal epithelial integrity or the rotaviral infection. In addition, plumbagin exerted inhibitory effects on the vasoactive intestinal peptide (VIP)-, prostaglandin E2 (PGE2)-, and 5-hydroxytryptamine (5-HT)-stimulated Cl− currents. In the evaluations of intestinal motility, plumbagin significantly delayed intestinal motility and inhibited intestinal smooth muscle contractility without an evident impact on contractive frequency. Collectively, our results indicate that plumbagin inhibits both Ca2+- and cAMP-activated Cl− channels, accounting for the mechanisms of plumbagin inhibition of chloride secretion and intestinal motility. Thus, plumbagin can be a lead compound in the treatment of CT-induced, Traveler’s, and rotaviral diarrhea, as well as other types of secretory diarrhea that result from excessive intestinal fluid secretion and increased intestinal peristalsis.

Evaluation of wound healing activity of plumbagin in diabetic rats

This study was performed to evaluate the antidiabetic and wound healing activity of plumbagin in diabetic rats by macroscopical, biochemical, histological, immunohistochemical and molecular methods. Percentage of wound closure and contraction was delayed in diabetic rats when compared to non-diabetic group. There was significant reduction in period of epithelialization, collagen and protein content. Serum insulin level was significantly lowered together with increase in glucose level in diabetic rats. Lipid levels were increased significantly with concomitant decrease in HDL level. The mRNA levels of Nrf2, collagen-1, TGF-β and α-SMA were significantly lowered whereas Keap-1 levels were increased in diabetic rats. The level of lipid peroxides was increased while the levels of antioxidants were lowered significantly. ELISA results reveal upregulated levels of inflammatory markers. Western blot result shows upregulated levels of CD68 and CD163 proteins in wound area of diabetic rats. Histopathological observation revealed increased inflammatory cells infiltration in diabetic control. Immunofluorescent staining and immunohistochemical analysis also displayed delayed wound healing in diabetic groups. Diabetic rats treated with 10% and 20% plumbagin showed increased epithelialization, collagen deposition, increased serum insulin level and increased antioxidant status. Lipid peroxides and lipid levels were lowered significantly with increase in HDL level. Inflammatory markers were lowered, and growth factors expressions were increased markedly. Thus, the results of the study indicated that plumbagin administration could improve wound healing activity and could serve as a potent antidiabetic and anti-inflammatory agent.

Plumbagin, a vitamin K3 analogue ameliorate malaria pathogenesis by inhibiting oxidative stress and inflammation.

Plumbagin, a vitamin K3 analogue is the major active constituent in several plants including root of Plumbago indica Linn. This compound has been shown to exhibit a wide spectrum of pharmacological activities. The present investigation was to evaluate the ameliorative effects of plumbagin (PL) against severe malaria pathogenesis due to involvement of oxidative stress and inflammatory response in Plasmodium berghei infected malaria in mice. Malaria pathogenesis was induced by intra-peritoneal injection of P. berghei infected red blood cells into the Swiss albino mice. PL was administered orally at doses of 3, 10 and 30mg/kg/day following Peter's 4day suppression test. Oral administration of PL showed significant reduction of parasitaemia and increase in mean survival time. PL treatment is also attributed to significant increase in the blood glucose and haemoglobin level when compared with vehicle-treated infected mice. Significant inhibition in level of oxidative stress and pro-inflammation related markers were observed in PL treated group. The trend of inhibition in oxidative stress markers level after oral treatment of PL was MPO > LPO > ROS in organ injury in P. berghei infected mice. This study showed that plumbagin is able to ameliorate malaria pathogenesis by augmenting anti-oxidative and anti-inflammatory mechanism apart from its effect on reducing parasitaemia and increasing mean survival time of malaria-induced mice.

Research of the inhibitory effect and the mechanisms of plumbagin on large cell lung cancer NL9980 cell line

Objective To investigate the effects of plumbagin on human NL9980 large-cell lung cancer cells and the role of interleukin-6/signal transducers and activators of transcription 3 (IL-6/STAT3) signaling pathway in the anticancer procedure of plumbagin. Methods Different concentrations of plumbagin were administrated in NL9980 cell line. Inhibitory effects of plumbagin on NL9980 cells were analyzed using methyl thiazol tetrazolium (MTT) assay. Groups of different drug doses (5.0, 7.5, 10.0 μmol/L) and blank control (without administration of plumbagin) were set with simple random sampling method. Cells were collected after 24 h of administration. Expression of IL-6 and STAT3 and the downstream genes B-cell lymphoma/leukemia-2 (bcl-2), vascular endothelial growth factor (VEGF), and Cyclin D1 were assessed by real-time quantitative polymerase chain reaction (Real-time PCR). Enzyme linked immunosorbent assay (ELISA) was used to measure secreted IL-6 levels and western blotting to measure intracellular phospho-STAT3 levels. Results Plumbagin significantly inhibited NL9980 cells via suppressing IL-6/STAT3 signaling. Expression of all genes was inhibited by plumbagin in a dose-dependent manner (F=32.170, P=0.000). As the concentration of plumbagin increased, the expression of IL-6 mRNA decreased significantly (F=173.067, P=0.000) to 0.50±0.02, 0.44±0.1, 0.34±0.04 respectively and IL-6 protein decreased to 4.71±0.02, 4.08±0.14, 2.01±0.07 significantlly (F=1 805.177, P=0.000). Besides, the expression of STAT3 mRNA (0.86±0.08, 0.72±0.03, 0.52±0.06, F=230.918, P=0.000) and protein (0.65±0.19, 0.45±0.22, 0.31±0.11, F=17.332, P=0.000) were reduced with the increase of concentration of plumbagin. Compared with the control group, expression of downstream genes bcl-2, VEGF and Cyclin D1 mRNA were decreased significantly after administration of plumbagin (F=33.698, P=0.000; F=337.743, P=0.000; F=180.136, P=0.000), and declined gradually as drug concentration increased. Moreover, bcl-2, VEGF, and Cyclin D1 mRNA levels positively correlated with levels of IL-6 and STAT3 gene expressions. Conclusion IL-6-mediated STAT3 suppression decreased bcl-2, VEGF, and Cyclin D1 expression. Thus, plumbagin may be an effective therapy for large-cell lung cancer that acts by targeting the IL-6/STAT3 signaling pathway. Key words: Plumbagin; Interleukin-6; Signal transducers and activators of transcription 3; Large cell lung cancer

Plumbagin suppresses chronic periodontitis in rats via down-regulation of TNF-α, IL-1β and IL-6 expression.

Chronic periodontitis (CP) is one of the most common oral diseases, which causes alveolar bone absorption and tooth loss in adults. In this study we aimed to investigate the potential of plumbagin (PL), a widely-investigated active compound extracted from the traditional Chinese herb Plumbago zeylanica L in treating CP. Human periodontal ligament stem cells (PDLSCs) were used for in vitro studies, whereas an animal model of CP was established in SD rats by ligation+Porphyromonas gingivalis (Pg) stimulation. The rats were injected with PL (2, 4, and 6 mg·kg-1·d-1, ip) for 4 weeks. Treatment of PDLSCs with TNF-α (10 ng/mL) markedly stimulated the expression of the proinflammatory cytokines TNF-α, IL-1β and IL-6, as well as the chemokines CCL-2 and CCL-5, which were dose-dependently suppressed by co-treatment with PL (1.25-5 μmol/L). Furthermore, PL (3.75 μmol/L) markedly suppressed TNF-α-induced activation of the MAPK, NF-κB and JAK/STAT signaling pathways in PDLSCs. In consistence with the in vitro studies, PL administration significantly decreased the expression of TNF-α, IL-1β and IL-6 in gingiva of the rat with CP, with the dosage 4 mg·kg-1·d-1 showing the best anti-inflammatory effect. Moreover, PL administration decelerated bone destruction in the rat with CP, evidenced by the aveolar bone loss (ABL) and H&E staining results. In conclusion, PL suppresses CP progression in rats by downregulating the expressions of TNF-α, IL-1β and IL-6 and inhibiting the MAPK, NF-κB and JAK/STAT signaling pathways.

Plumbagin inhibits neuronal apoptosis, intimal hyperplasia and also suppresses TNF-α/NF-κB pathway induced inflammation and matrix metalloproteinase-2/9 expression in rat cerebral ischemia.

Cerebral ischemic damage and infarction are well documented in stroke, which is presenting a foremost health concern globally with very high mortality and morbidity rates. Mechanisms that are associated with excitotoxicity, inflammation and oxidative stress are found to be critically involved in ischemic damage. Adverse effects of current therapies are imposing the need in development of neuroprotective agents that are very effective. To explore this we experimentally induced ischemic brain injury and investigated the effects of plumbagin. Induction of cerebral infarction and ischemia-reperfusion (I/R) was done by middle cerebral artery occlusion (MCAO) in Sprague-Dawley rats. Plumbagin (50, 100 or 200 mg/kg b.wt) was intragastrically administered for 9 days before ischemia induction and an hour prior on the day of ischemic insult. Plumbagin treatment attenuated pulmonary edema, neuronal apoptosis and reduced cerebral infarct volume. Cleaved caspase-3 and apoptotic cascade protein expressions were regulated. Overproduction of pro-inflammatory cytokines (TNF-α, IL-1β and IL-6) and nitric oxide (NO) following I/R were reduced. Prior plumbagin administration had down-regulated NF-κB signalling and MMP-2 and MMP-9 expression. Overall, the results reveal the potent neuroprotective efficacy of plumbagin against I/R-induced brain injury via effectively modulating apoptotic pathways, MMPs and neuro-inflammatory cascades.

Chemosuppressive effect of plumbagin on human non-small lung cancer cell xenotransplanted zebrafish.

Plumbagin (5-hydroxy-2-methyl-1,4-napthoquinone) derived from Plumbago species is a potential anti-tumour agent. Plumbagin has been tested for anti-cancer activity in vitro and in vivo using mice model. To study the tumour suppressing efficacy of plumbagin using zebrafish model. Human Non-small lung cancer cell line were cultured in vitro and transplanted in to zebrafish. The development of tumour was confirmed by performing histology. The tumour was then allowed to progress in vivo and the fishes were administered with plumbagin orally for three continuous days. The tumour suppression capacity was monitored subsequently using transcriptosome analysis. The pixel integrated density obtained was converted into relative gene expression using IBM SPSS. The administration of plumbagin had an ability to suppress tumour and the size of the tumour were relatively lesser when compared with the control sample; it has also increased p53 gene expression. The study helps to conclude that plumbagin is an effective anti-tumour agent against human cancer cells based on the study in vivo in zebrafish.

Plumbagin Alleviates Capillarization of Hepatic Sinusoids In Vitro by Downregulating ET-1, VEGF, LN, and Type IV Collagen.

Critical roles for liver sinusoidal endothelial cells (LSECs) in liver fibrosis have been demonstrated, while little is known regarding the underlying molecular mechanisms of drugs delivered to the LSECs. Our previous study revealed that plumbagin plays an antifibrotic role in liver fibrosis. In this study, we investigated whether plumbagin alleviates capillarization of hepatic sinusoids by downregulating endothelin-1 (ET-1), vascular endothelial growth factor (VEGF), laminin (LN), and type IV collagen on leptin-stimulated LSECs. We found that normal LSECs had mostly open fenestrae and no organized basement membrane. Leptin-stimulated LSECs showed the formation of a continuous basement membrane with few open fenestrae, which were the features of capillarization. Expression of ET-1, VEGF, LN, and type IV collagen was enhanced in leptin-stimulated LSECs. Plumbagin was used to treat leptin-stimulated LSECs. The sizes and numbers of open fenestrae were markedly decreased, and no basement membrane production was found after plumbagin administration. Plumbagin decreased the levels of ET-1, VEGF, LN, and type IV collagen in leptin-stimulated LSECs. Plumbagin promoted downregulation of ET-1, VEGF, LN, and type IV collagen mRNA. Altogether, our data reveal that plumbagin reverses capillarization of hepatic sinusoids by downregulation of ET-1, VEGF, LN, and type IV collagen.

Plumbagin exerts protective effects in nucleus pulposus cells by attenuating hydrogen peroxide-induced oxidative stress, inflammation and apoptosis through NF-κB and Nrf-2.

Plumbagin, one of the constituents responsible for the various biological activities of Plumbagozeylanica has been demonstrated to possess antioxidant activity, which may inhibit lipid peroxidation in a dose- and time-dependent manner. In the present study, we aimed to examine the protective effects of plumbagin as well as the underlying mechansim through which plumbagin attenuates hydrogen peroxide(H2O2)-induced oxidative stress in nucleus pulposus cells(NPCs). For this purpose, the NPCs were incubated with fresh medium containing H2O2 (200µM) at 37˚C in a humidified 5%CO2 atmosphere for 6h, and cultured with various concentrations of plumbagin (0, 0.5, 1, 2, 5, 10 and 20µM). Treatment with plumbagin significantly increased the viability of the H2O2-exposed NPCs in a dose‑dependent manner. Moreover, plumbagin significantly reduced the generation of reactive oxygen species, lipid peroxidation, as well as the levels of tumor necrosis factor-α(TNF-α), interleukin(IL)-1β and IL-6 in the H2O2‑exposed NPCs. Glutathione(GSH) content, as well as the activity of catalase(CAT), superoxide dismutase(SOD) and glutathione peroxdiase(GSH-Px) were increased. We found that the administration of plumbagin significantly inhibited the activity of caspase-9 and-3, and downregulated NF-κB expression and upregulated Nrf-2 expression in the H2O2-exposed NPCs. Taken together, these findings suggest that plumbagin exerts neuroprotective effects in NPCs by attenuating H2O2‑induced oxidative stress, inflammation and apoptosis through mediating the expression of NF-κB and Nrf-2.

Structural simulation of adenosine phosphate via plumbagin and zoledronic acid competitively targets JNK/Erk to synergistically attenuate osteoclastogenesis in a breast cancer model.

The treatment of breast cancer-induced osteolysis remains a challenge in clinical settings. Here, we explored the effect and mechanism of combined treatment with zoledronic acid (ZA) and plumbagin (PL), a widely investigated component derived from Plumbago zeylanica, against breast cancer-induced osteoclastogenesis. We found that the combined treatment with PL and ZA suppressed cell viability of precursor osteoclasts and synergistically inhibited MDA-MB-231-induced osteoclast formation (combination index=0.28) with the abrogation of recombinant mouse receptor activator of nuclear factor-κB ligand (RANKL)-induced activation of NF-κB/MAPK (nuclear factor-κB/mitogen-activated protein kinase) pathways. Molecular docking suggested a putative binding area within c-Jun N-terminal kinase/extracellular signal-regulated kinase (JNK/Erk) protease active sites through the structural mimicking of adenosine phosphate (ANP) by the spatial combination of PL with ZA. A homogeneous time-resolved fluorescence assay further illustrated the direct competitiveness of the dual drugs against ANP docking to phosphorylated JNK/Erk, contributing to the inhibited downstream expression of c-Jun/c-Fos/NFATc-1 (nuclear factor of activated T cells, cytoplasmic, calcineurin-dependent 1). Then, in vivo testing demonstrated that the combined administration of PL and ZA attenuated breast cancer growth in the bone microenvironment. Additionally, these molecules prevented the destruction of proximal tibia, with significant reduction of tartrate-resistant acid phosphatase (TRAcP)-positive osteoclast cells and potentiation of apoptotic cancer cells, to a greater extent when combined than when the drugs were applied independently. Altogether, the combination treatment with PL and ZA could significantly and synergistically suppress osteoclastogenesis and inhibit tumorigenesis both in vitro and in vivo by simulating the spatial structure of ANP to inhibit competitively phosphorylation of c-Jun N-terminal kinase/extracellular signal-regulated kinase (JNK/Erk).

Plumbagin Protects Against Spinal Cord Injury-induced Oxidative Stress and Inflammation in Wistar Rats through Nrf-2 Upregulation.

Spinal cord injury causes post-traumatic degeneration through series of biochemical events. This study aims to evaluate the possible protective mechanism of Plumbagin against Spinal cord injury induced oxidative stress and inflammation. Plumbagin is a potent antioxidant and shows anti-carcinogenic, anti-inflammatory and analgesic activities. However, its exact molecular mechanism of action has yet to be explored. We tested the effects of Plumbagin on spinal cord injury induced ROS generation and lipid peroxidation content in wistar rats. Additionally, the expression of 2 important transcription factors NF-κB and Nrf-2 was investigated. Plumbagin treatment significantly ameliorated oxidative stress through inhibition of ROS and lipid peroxidation with a concomitant increase in antioxidant status. Western blot analysis revealed enhanced nuclear levels of Nrf-2, while NF-κB expression was suppressed during Plumbagin administration. Enzyme linked immunosorbent assay for pro-inflammatory cytokines (TNF-α, IL-1β) showed a significant downregulation followed by Plumbagin treatment in spinal cord injury rats. Taken together, the data suggests potential and novel role of Plumbagin in cytoprotection by modulating NF-κB and Nrf-2 levels against spinal cord injury.

Plumbagin attenuates cancer cell growth and osteoclast formation in the bone microenvironment of mice.

To investigate the effects of plumbagin, a naphthoquinone derived from the medicinal plant Plumbago zeylanica, on human breast cancer cell growth and the cancer cell-induced osteolysis in the bone microenvironment of mice. Human breast cancer cell subline MDA-MB-231SA with the ability to spread and grow in the bone was tested. The cell proliferation was determined using the CCK-8 assay. Apoptosis was detected with Annexin V/PI double-labeled flow cytometry. Red fluorescent protein-labeled MDA-MB-231SArfp cells were injected into the right tibia of female BALB/c-nu/nu mice. Three days after the inoculation, the mice were injected with plumbagin (2, 4, or 6 mg/kg, ip) 5 times per week for 7 weeks. The growth of the tumor cells was monitored using an in vivo imaging system. After the mice were sacrificed, the hind limbs were removed for radiographic and histological analyses. Plumbagin (2.5-20 μmol/L) concentration-dependently inhibited the cell viability and induced apoptosis of MDA-MB-231SA cells in vitro (the IC50 value of inhibition of cell viability was 14.7 μmol/L). Administration of plumbagin to breast cancer bearing mice delayed the tumor growth by 2-3 weeks and reduced the tumor volume by 44%-74%. The in vivo imaging study showed that plumbagin dose-dependently inhibited MDA-MB-231SArfp cell growth in bone microenvironment. Furthermore, X-ray images and micro-CT study demonstrated that plumbagin reduced bone erosion area and prevented a decrease in bone tissue volume. Histological studies showed that plumbagin dose-dependently inhibited the breast cancer cell growth, enhanced the cell apoptosis and reduced the number of TRAcP-positive osteoclasts. Plumbagin inhibits the cell growth and induces apoptosis in human breast cancer cells in mice bone microenvironment, leading to significant reduction in osteolytic lesions caused by the tumor cells.

Plumbagin, a Vitamin K3 Analogue, abrogates Lipopolysaccharide-Induced Oxidative Stress, Inflammation and Endotoxic Shock via NF-κB Suppression

Plumbagin has been reported to modulate cellular redox status and suppress NF-κB. In the present study, we investigated the effect of plumbagin on lipopolysaccharide (LPS)-induced endotoxic shock, oxidative stress and inflammatory parameters in vitro and in vivo. Plumbagin inhibited LPS-induced nitric oxide, TNF-α, IL-6 and prostaglandin-E2 production in a concentration-dependent manner in RAW 264.7 cells without inducing any cell death. Plumbagin modulated cellular redox status in RAW cells. Plumbagin treatment significantly reduced MAPkinase and NF-κB activation in macrophages. Plumbagin prevented mice from endotoxic shock-associated mortality and decreased serum levels of pro-inflammatory markers. Plumbagin administration ameliorated LPS-induced oxidative stress in peritoneal macrophages and splenocytes. Plumbagin also attenuated endotoxic shock-associated changes in liver and lung histopathology and decreased the activation of ERK and NF-κB in liver. These findings demonstrate the efficacy of plumbagin in preventing LPS-induced endotoxemia and also provide mechanistic insights into the anti-inflammatory effects of plumbagin.

Plumbagin, a plant derived natural agent inhibits the growth of pancreatic cancer cells in in vitro and in vivo via targeting EGFR, Stat3 and NF-κB signaling pathways.

Pancreatic cancer (PC) is the most aggressive malignant disease, ranks as the fourth most leading cause of cancer-related death among men and women in the United States. We present here that plumbagin (PL), a quinoid constituent isolated from the roots of the medicinal plant Plumbago zeylanica L, inhibits the growth of PC cells both in vitro and in vivo model systems. PL treatment induces apoptosis and inhibits cell viability of PC cells (PANC1, BxPC3 and ASPC1). In addition, i.p. administration of PL (2 mg/kg body weight, 5 days a week) in severe combined immunodeficiency (SCID) mice beginning 3 days after ectopic implantation of PANC1 cells resulted in a significant (P < 0.01) inhibition of both tumor weight and volume. PL treatment inhibited (1) constitutive expression of epidermal growth factor receptor (EGFR), pStat3Tyr705 and pStat3Ser727, (2) DNA binding of Stat3 and (3) physical interaction of EGFR with Stat3, in both cultured PANC1 cells and their xenograft tumors. PL treatment also inhibited phosphorylation and DNA-binding activity of NF-κB in both cultured PC cells (PANC1 and ASPC1) and in PANC1 cells xenograft tumors. Downstream target genes (cyclin D1, MMP9 and Survivin) of Stat3 and NF-κB were similarly inhibited. These results suggest that PL may be used as a novel therapeutic agent against human PC. Published 2012 Wiley-Liss, Inc. This article is a US Government work, and, as such, is in the public domain in the United States of America.

Abstract PR-06: PKC∈-activated Stat3 and subsequent AR interaction are potential molecular targets for prevention of late-stage prostate cancer

Abstract Prostate cancer (PCa) is the second most common malignancy in men and the second leading cause of cancer-related deaths in the Western World. Androgen ablation therapy remains the gold standard for the treatment of PCa. Despite the initial success of androgen ablation therapy, resistance to anti-androgen therapy manifests by progression to castration-resistant prostate cancer (CRPC). CRPC is the end stage that accounts for the majority of PCa patient deaths. An understanding of the mechanisms linked to the emergence of CRPC is the key to the prevention of this disease. Overwhelming evidence supports the concept that development of CRPC is usually related to continued activation of androgen receptor (AR). Several mechanisms are proposed for androgen-dependent (AD) and androgen-independent (AI) activation of AR in CRPC. Gene amplification and mutations in AR are frequently observed in recurrent PCa, which may account for the hypersensitivity of the AR to low castrate level of androgens and altered ligand specificity. In the AR-dependent pathway, the functions of AR are modified significantly and involve AR interaction with many co-activators and co-repressors. Increased AR activity in CRPC is perhaps caused by cross talk of AR with multiple intracellular signaling cascades. We present here that PKC∈ and Stat3, which correlate with the aggressiveness of PCa, may play roles in both AI AR activation and promotion of PCa cell survival for emergence and progression of CRPC. Stat3 has two conserved amino acid residues (Tyr705 and Ser727), which have to be phosphorylated for maximum transcriptional activity. PKC∈, which is constitutively activated in PCa, mediates phosphorylation of both Stat3Tyr705 and Stat3Ser727. Specific observations are our finding that PKC∈ deletion in TRAMP mice, which prevents PCa development and metastasis, inhibits the expression levels of IL-6, pStat3Ser727 and pStat3Tyr705. PKC∈ overexpression is sufficient to transform AD LNCaP cells into an AI variant. We further explored the possibility that PKC∈− activated Stat3 is a non-androgen activator of AR. In this experiment, human PCa LNCaP (AD) cells were infected with adenoviral vector containing PKC∈ or empty vector; 48 h later, cells were harvested and whole cell lysates were prepared. The effect of PKC∈ overexpression on the interaction of Stat3 with AR was determined by performing reciprocal immunoprecipitation/blotting experiments. Overexpression of PKC∈ increased the interaction of Stat3 with AR. We also found similar interaction of Stat3 with AR in human PCa C4–2 (AI) cells treated with IL-6. To determine the link of PKC∈ in Stat3 interaction with AR and the growth of C4–2 cells, we used plumbagin (PL). PL is a unique plant-derived chemopreventive agent, which selectively inhibits expression of PKC∈. PL administration delayed the growth of C4–2 cells xenograft tumors as determined by both tumor weight and volume. In a reciprocal immunoprecipation/blotting experiments, PL inhibited Stat3-AR interaction. We conclude that: 1) PKC∈-mediated Stat3Ser727 phosphorylation and subsequent Stat3-AR interaction are essential components of the emergence and progression of CRPC, 2) Progression of CRPC is the result of cross-talk of PKC∈-Stat3 signaling with other signaling pathways (AKT and MAPK) which activate AR as well as promote cell survival and 3) PKC∈-activated Stat3 and subsequent AR interaction are potential molecular targets for prevention of late stage PCa. Support: NIH Grant CA138761. Citation Information: Cancer Prev Res 2011;4(10 Suppl):PR-06.

Plumbagin, a novel Nrf2/ARE activator, protects against cerebral ischemia

Many phytochemicals function as noxious agents that protect plants against insects and other damaging organisms. However, at subtoxic doses, the same phytochemicals may activate adaptive cellular stress response pathways that can protect cells against a variety of adverse conditions. We screened a panel of botanical pesticides using cultured human and rodent neuronal cell models, and identified plumbagin as a novel potent activator of the nuclear factor E2-related factor 2 (Nrf2)/antioxidant response element (ARE) pathway. In vitro, plumbagin increases nuclear localization and transcriptional activity of Nrf2, and induces the expression of the Nrf2/ARE-dependent genes, such as heme oxygenase 1 in human neuroblastoma cells. Plumbagin specifically activates the Nrf2/ARE pathway in primary mixed cultures from ARE-human placental alkaline phosphatase reporter mice. Exposure of neuroblastoma cells and primary cortical neurons to plumbagin provides protection against subsequent oxidative and metabolic insults. The neuroprotective effects of plumbagin are abolished by RNA interference-mediated knockdown of Nrf2 expression. In vivo, administration of plumbagin significantly reduces the amount of brain damage and ameliorates-associated neurological deficits in a mouse model of focal ischemic stroke. Our findings establish precedence for the identification and characterization of neuroprotective phytochemicals based upon their ability to activate adaptive cellular stress response pathways.