Plumbagin induces apoptotic and autophagic cell death through inhibition of the PI3K/Akt/mTOR pathway in human non-small cell lung cancer cells.

Plumbagin (PLB) has shown anti-cancer activity but the mechanism is unclear. This study has found that PLB has a potent pro-apoptotic and pro-autophagic effect on A549 and H23 cells. PLB arrests cells in G2/M phase, and increases the intracellular level of reactive oxygen species in both cell lines. PLB dose-dependently induces autophagy through inhibition of PI3K/Akt/mTOR pathway as indicated by reduced phosphorylation of Akt and mTOR. Inhibition or induction of autophagy enhances PLB-induced apoptosis. There is crosstalk between PLB-induced apoptosis and autophagy. These findings indicate that PLB initiates both apoptosis and autophagy in NSCLC cells through coordinated pathways.

Current and emerging therapeutic strategies for preventing inflammation and aggrecanase-mediated cartilage destruction in arthritis

Arthritis is a multifactorial disease for which current therapeutic intervention with high efficacy remains challenging. Arthritis predominately affects articular joints, and cartilage deterioration and inflammation are key characteristics. Current therapeutics targeting inflammatory responses often cause severe side effects in patients because of the systemic inhibition of cytokines or other global immunosuppressive activities. Furthermore, a lack of primary response or failure to sustain a response to treatment through acquired drug resistance is an ongoing concern. Nevertheless, treatments such as disease-modifying anti-rheumatic drugs, biological agents, and corticosteroids have revealed promising outcomes by decreasing pain and inflammation in patients and in some cases reducing radiographic progression of the disease. Emerging and anecdotal therapeutics with anti-inflammatory activity, alongside specific inhibitors of the A Disintegrin-like And Metalloproteinase domain with Thrombospondin-1 repeats (ADAMTS) cartilage-degrading aggrecanases, provide promising additions to current arthritis treatment strategies. Thus, it is paramount that treatment strategies be optimized to increase efficacy, reduce debilitating side effects, and improve the quality of life of patients with arthritis. Here, we review the current strategies that attempt to slow or halt the progression of osteoarthritis and rheumatoid arthritis, providing an up-to-date summary of pharmaceutical treatment strategies and side effects. Importantly, we highlight their potential to indirectly regulate ADAMTS aggrecanase activity through their targeting of inflammatory mediators, thus providing insight into a mechanism by which they might inhibit cartilage destruction to slow or halt radiographic progression of the disease. We also contrast these with anecdotal or experimental administration of statins that could equally regulate ADAMTS aggrecanase activity and are available to arthritis sufferers worldwide. Finally, we review the current literature regarding the development of synthetic inhibitors directed toward the aggrecanases ADAMTS4 and ADAMTS5, a strategy that might directly inhibit cartilage destruction and restore joint function in both rheumatoid arthritis and osteoarthritis.

Effect of lactoferrin protein on red blood cells and macrophages: mechanism of parasite-host interaction

BACKGROUND: Lactoferrin is a natural multifunctional protein known to have antitumor, antimicrobial, and anti-inflammatory activity. Apart from its antimicrobial effects, lactoferrin is known to boost the immune response by enhancing antioxidants. Lactoferrin exists in various forms depending on its iron saturation. The present study was done to observe the effect of lactoferrin, isolated from bovine and buffalo colostrum, on red blood cells (RBCs) and macrophages (human monocytic cell line-derived macrophages THP1 cells). METHODS: Lactoferrin obtained from both species and in different iron saturation forms were used in the present study, and treatment of host cells were given with different forms of lactoferrin at different concentrations. These treated host cells were used for various studies, including morphometric analysis, viability by MTT assay, survivin gene expression, production of reactive oxygen species, phagocytic properties, invasion assay, and Toll-like receptor-4, Toll-like receptor-9, and MDR1 expression, to investigate the interaction between lactoferrin and host cells and the possible mechanism of action with regard to parasitic infections. RESULTS: The mechanism of interaction between host cells and lactoferrin have shown various aspects of gene expression and cellular activity depending on the degree of iron saturation of lactoferrin. A significant increase (P<0.05) in production of reactive oxygen species, phagocytic activity, and Toll-like receptor expression was observed in host cells incubated with iron-saturated lactoferrin when compared with an untreated control group. However, there was no significant (P>0.05) change in percentage viability in the different groups of host cells treated, and no downregulation of survivin gene expression was found at 48 hours post-incubation. Upregulation of the Toll-like receptor and downregulation of the P-gp gene confirmed the immunomodulatory potential of lactoferrin protein. CONCLUSION: The present study details the interaction between lactoferrin and parasite host cells, ie, RBCs and macrophages, using various cellular processes and expression studies. The study reveals the possible mechanism of action against various intracellular pathogens such as Toxoplasma, Plasmodium, Leishmania, Trypanosoma, and Mycobacterium. The presence of iron in lactoferrin plays an important role in enhancing the various activities taking place inside these cells. This work provides a lot of information about targeting lactoferrin against many parasitic infections which can rule out the exact pathways for inhibition of diseases caused by intracellular microbes mainly targeting RBCs and macrophages for their survival. Therefore, this initial study can serve as a baseline for further evaluation of the mechanism of action of lactoferrin against parasitic diseases, which is not fully understood to date.

EpCAM Aptamer-siRNA chimera targets and regress epithelial cancer

Epithelial cell adhesion molecule (EpCAM), a cancer stem cell (CSC) marker is over expressed in epithelial cancers and in retinoblastoma (RB). We fabricated an EpCAM targeting aptamer-siRNA chimera and investigated its anti-tumor property and EpCAM intracellular domain (EpICD) mediated signaling in epithelial cancer. The anti-tumor efficacy of EpCAM aptamer-siEpCAM chimera (EpApt-siEp) was evaluated by qPCR, northern and Western blotting in WERI-Rb1- RB cell line, primary RB tumor cells and in MCF7- breast cancer cell line. Anti-tumor activity of EpApt-siEp was studied in vivo using epithelial cancer (MCF7) mice xenograft model. The mechanism and pathways involved in the anti-tumor activity was further studied using protein arrays and qPCR. EpApt-siEp chimera was processed in vitro by dicer enzyme. Treatment of the WERI-Rb1 and MCF7 cells with EpApt-siEp revealed statistically significant down regulation of EpCAM expression (P<0.005) and concomitant reduction in cellular proliferation. In primary RB cells cultured from RB tumors, EpApt-siEp silenced EpCAM, significantly inhibited (P<0.01) cell proliferation and induced cytotoxicity. Knockdown of EpICD expressed in RB primary tumors led to repression of pluripotency markers, SOX2, OCT4, NANOG, and CD133. In vivo studies showed complete tumor growth regression without any toxicity in animals (P<0.001) and tumor tissues showed significant downregulation (P<0.05) of EpCAM, MRP1, ABCG2, stathmin, survivin and upregulation of ATM (P<0.05) leading to apoptosis by intrinsic pathway with minor alteration in cytokines. Our results revealed that EpApt-siEp potentially eradicated EpCAM positive cancer cells through CSC marker suppression and apoptosis, while sparing normal EpCAM negative adjacent cells.

Locked nucleic acid modified bi-specific aptamer-targeted nanoparticles carrying survivin antagonist towards effective colon cancer therapy

We investigated the anti-cancer activity of alginate coated chitosan nanoparticles (CHNP) encapsulating cell-permeable dominant negative survivin (SR9) with locked nucleic acid (LNA) aptamers targeting EpCAM and nucleolin (termed as "nanobullets") in vitro (2D and 3D cell culture models) and in vivo (colon cancer mouse xenograft model). We incorporated three LNA modifications in each sequence in order to enhance the stability of these aptamers. Confocal microscopy revealed binding of the LNA-aptamers to their specific markers with high affinity. The muco-adhesive nanobullets showed 6-fold higher internalization in cancer cells when compared to non-cancerous cells, suggesting a tumour specific uptake. A higher intensity of nanobullets was observed in both the periphery and the core of the multicellular tumour spheroids compared to non-targeted CHNP-SR9. The nanobullets were found to be the highly effective as they led to a 2.26 fold (p < 0.05) reduction at 24 h and a 4.95 fold reduction (p ≤ 0.001) in the spheroid size at 72 h. The tumour regression was 4 fold higher in mice fed on a nanobullet diet when compared to a control diet. The nanobullets were able to show a significantly high apoptotic (p ≤ 0.0005) and necrotic index in the tumour cell population (p ≤ 0.005) when compared to void NPs. Therefore, our nanoparticles have shown highly promising results and therefore deliver a new conduit towards the approach of cancer-targeted nanodelivery. This journal is

Oral administration of encapsulated bovine lactoferrin protein nanocapsules against intracellular parasite Toxoplasma gondii

Toxoplasma gondii is a deadly intracellular parasite known to reside in every nucleated cell and known to cause severe complications in immunocompromised host. Standard drugs are cost effective and cause side effects, therefore, there is a necessity for a new drug molecule with immunomodulatory potential. Lactoferrin (Lf) is a natural milk protein, which has shown antimicrobial properties in its nanoformulation using alginate chitosan calcium phosphate bovine lactoferrin nanocapsules (AEC-CCo-CP-bLf-NCs). The present study was aimed to analyze and compare the effect of bovine Lf (bLf) in its native as well as nanoformulation (AEC-CCo-CP-bLf-NC) against coccidian parasite T. gondii. In vitro analysis has shown a significant increase in nitric oxide production and low parasitemia in in vitro cell culture model. In vivo BALB/c mice model have been used to develop human toxoplasmosis model. After treatment with NCs it has substantially increased the bioavailability of the protein and showed comparatively increased levels of reactive oxygen species, nitric oxide production, and Th1 cytokine which helped in parasite clearance. The mechanism of action of NCs has been clarified by immunoreactivity analysis, which showed accumulation of Lf in macrophages of various visceral organs, which is the site of parasite multiplication. Effect of NCs has significantly decreased (P<0.05) the parasite load in various organs and helped survival of mice till day 25 postinfection. Fe metabolism inside the mice has been found to be maintained even after administration of mono form of Lf, this indicates novelty of Lf protein. From the present study we concluded that nanoformulation did not reduce the therapeutic potential of Lf protein; however, nanoformulation has enhanced the stability of the protein and shown anti-toxoplasmal activity. Our study presents for the first time nanoformulation of Lf protein against Toxoplasma, which has advantages over the standard drug therapy without any side effects.

Metabolites derived from the tropical seagrass Thalassia testudinum are bioactive against pathogenic Labyrinthula sp

Temperate and tropical seagrasses are susceptible to wasting disease outbreaks caused by pathogenic protists of the genus Labyrinthula. Even though there is an increasing awareness of the environmental conditions that influence the etiology of seagrass-. Labyrinthula disease dynamics, the biochemical basis of seagrass defense responses, in particular chemical defenses, is still vastly understudied. Using an in vitro bioassay, we provide evidence that previously characterized phenolic and potentially novel, undescribed non-phenolic metabolites derived from Thalassia testudinum Banks ex Konig exhibit anti-labyrinthulid activity. All phenolic compounds tested displayed dose-dependent behavior and selected combinations interacted synergistically. The flavone glycoside thalassiolin B was roughly 20-100 times more active than any phenolic acid tested. Based upon values reported in the literature, it was calculated that infected specimens of T. testudinum contain natural concentrations of phenolic acids that are consistently greater than what is required to inhibit Labyrinthula growth. This suggests that while there may be an ample supply of phenolic-based derivatives available to inhibit Labyrinthula growth, they may not be readily bio-accessible.Using a bioactivity-guided approach, a semi-purified chemical fraction from T. testudinum was found to contain anti-labyrinthulid activity. 1H NMR spectra for this fraction lacked aromatic hydrogen signals, suggesting that the bioactive compound was non-aromatic in nature. Furthermore, the LC-MS fragmentation patterns were suggestive of the presence of glycosylated natural products of an unknown structural class. This has the potential to provide a foundation for future chemical investigations.