Zinc Acts Synergistically with Berberine for Enhancing Its Efficacy as an Anti-cancer Agent by Inducing Clusterin-Dependent Apoptosis in HT-29 Colorectal Cancer Cells.

It is now widely accepted that anti-cancer medications are most effective when administered in combination. Zinc is an essential micronutrient whilst berberine is a well-known natural phytochemical, both having multiple molecular mechanisms of action. The present study aimed to determine the combinatorial effect of zinc and berberine on the human adenocarcinoma HT-29 cancer cell line. The anti-proliferative activity of berberine and zinc was determined by cell viability and colony-forming assays. The combination index and drug reduction index values of zinc and berberine co-treatments were estimated by suitable software. Flow cytometry was used to analyse cell cycle distribution and Annexin V/PI staining. The expression of apoptosis and zinc signalling markers were analysed by RT-qPCR and immunoblot analysis. Berberine decreased the viability of colon cancer cells in a dose-dependent manner whilst zinc alone had no significant influence on it. However, zinc and berberine co-treatment resulted in a synergistic anti-cancer action which was demonstrated by G2/M phase arrest of cell growth at a lower dose of berberine. Annexin V assay demonstrated that the synergistic impact of zinc and berberine boosted the number of apoptotic cells. Gene expression analysis at both transcriptional and translational levels showed the upregulation of apoptotic (caspase-3 and caspase-8) and a zinc-sensing receptor (GPR39) gene with concomitant downregulation of anti-apoptotic genes like proliferating cell nuclear antigen (PCNA) and clusterin. Our findings showed that the combination of zinc and berberine has synergistic anti-cancer efficacy and thus could be used as a potential chemopreventive option for colon cancer.

Decellularized xenogenic cartilage extracellular matrix (ECM) scaffolds for the reconstruction of osteochondral defects in rabbits.

The use of decellularized native allogenic or xenogenic cartilaginous extracellular matrix (ECM) biomaterials is widely expanding in the fields of tissue engineering and regenerative medicine. In this study, we aimed to develop an acellular, affordable, biodegradable, easily available goat conchal cartilaginous ECM derived scaffolding biomaterial for repair and regeneration of osteochondral defects in rabbits. Cartilages harvested from freshly collected goat ears were decellularized using chemical agents, namely, hypotonic-hypertonic (HH) buffer and Triton X-100 solution, separately. The morphologies and ultrastructure orientations of the decellularized cartilages remained unaltered in spite of complete cellular loss. Furthermore, when the acellular cartilaginous ECMs were cultured with murine mesenchymal stem cells (MSCs) (C3H10T1/2 cells), cellular infiltration and proliferation were thoroughly monitored using SEM, DAPI and FDA stained images, whereas the MTT assay proved the biocompatibility of the matrices. The increasing amounts of secreted ECM proteins (collagen and sGAG) indicated successful chondrogenic differentiation of the MSCs in the presence of the treated cartilage samples. In vivo biocompatibility studies showed no significant immune response or tissue rejection in the treated samples but tissue necrosis in control samples after 3 months. Upon implantation of the constructs in rabbits' osteochondral defects for 3 months, the histological and micro-CT evaluation revealed significant enhancement and regeneration of neocartilage and subchondral bony tissues. The IGF-1 loaded cartilaginous constructs showed comparatively better healing response after 3 months. Our results showed that decellularized xenogenic cartilaginous biomaterials preserved the bioactivity and integrity of the matrices that also favored in vitro stem cell proliferation and chondrogenic differentiation and enabled osteochondral regeneration, thus paving a new way for articular cartilage reconstruction.

Betel leaf extract and its major component hydroxychavicol promote osteogenesis and alleviate glucocorticoid-induced osteoporosis in rats.

Piper betle leaves possess several ethnomedicinal properties and are immensely used in traditional medicinal practices in regions of Asian and African subcontinents. However, their effects in treating skeletal complications are least known. In this study, we evaluated cellular and molecular effects of betel leaf extract (BLE) and its major phytoconstituent, hydroxychavicol (HCV) in promoting osteogenesis in vitro and alleviating glucocorticoid induced osteoporosis (GIO) in vivo. Both BLE and HCV markedly stimulated osteoblast differentiation of C3H10T1/2 cells with increased expression of RUNX2 and osteopontin through the GSK-3ß/ß-catenin-signaling pathway. Also, oral administration of BLE and HCV in GIO rats resulted in restoration of bone mass and tissue microarchitecture. Thus, with our findings we conclude that BLE and HCV promote osteogenesis of C3H10T1/2 cells via the GSK-3ß/ß-catenin pathway and alleviate GIO in rats.

3-D macro/microporous-nanofibrous bacterial cellulose scaffolds seeded with BMP-2 preconditioned mesenchymal stem cells exhibit remarkable potential for bone tissue engineering.

Bone repair using BMP-2 is a promising therapeutic approach in clinical practices, however, high dosages required to be effective pose issues of cost and safety. The present study explores the potential of low dose BMP-2 treatment via tissue engineering approach, which amalgamates 3-D macro/microporous-nanofibrous bacterial cellulose (mNBC) scaffolds and low dose BMP-2 primed murine mesenchymal stem cells (C3H10T1/2 cells). Initial studies on cell-scaffold interaction using unprimed C3H10T1/2 cells confirmed that scaffolds provided a propitious environment for cell adhesion, growth, and infiltration, owing to its ECM-mimicking nano-micro-macro architecture. Osteogenic studies were conducted by preconditioning the cells with 50 ng/mL BMP-2 for 15 min, followed by culturing on mNBC scaffolds for up to three weeks. The results showed an early onset and significantly enhanced bone matrix secretion and maturation in the scaffolds seeded with BMP-2 primed cells compared to the unprimed ones. Moreover, mNBC scaffolds alone were able to facilitate the mineralization of cells to some extent. These findings suggest that, with the aid of 'osteoinduction' from low dose BMP-2 priming of stem cells and 'osteoconduction' from nano-macro/micro topography of mNBC scaffolds, a cost-effective bone tissue engineering strategy can be designed for quick and excellent in vivo osseointegration.

Berberine reverses epithelial-mesenchymal transition and modulates histone methylation in osteosarcoma cells.

Osteosarcoma is the most frequently occurring cancer in children as well as young adolescents and the metastatic forms worsen this condition to a further great extent. The metastatic dissemination of cancer cells is often acquired through a process of epithelial-mesenchymal transition (EMT). Since, phytochemicals have attracted intense interest in recent years due to their diverse pharmacological effects, in the present study, we investigated if berberine, a naturally occurring isoquinoline quaternary alkaloid, could modulate the EMT in osteosarcoma cells. Our experimental studies showed that berberine reduced cell viability, colony formation, wound healing ability and migration of osteosarcoma cells. Also, berberine significantly reduced the expression of matrix metalloproteinase (MMP)-2, suggesting its inhibitory action on the matrix metalloproteinases that are required for cancer cell invasion. The significant reduction in the expression of vimentin, N-cadherin, fibronectin and increased expression of E-cadherin further suggested its role in the inhibition of EMT in osteosarcoma cells. The downregulation of H3K27me3, as well as the decreased expression of the histone methyl transferase enzyme EZH2, further substantiated the fact that the plant alkaloid can be used as an epigenetic modulator in the treatment of osteosarcoma. In conclusion, our findings suggest that berberine inhibits proliferation and migration of osteosarcoma cells and most importantly reverses EMT along with modulation of key epigenetic regulators.

Metabolite profiling and wound-healing activity of Boerhavia diffusa leaf extracts using in vitro and in vivo models.

Boerhavia diffusa is a perennial herb belonging to the Nyctaginaceae family. This plant has been widely used in Indian traditional medicinal system to cure several human ailments. However, traditional use of this plant in the treatment and management of wounds has not been validated by any comprehensive scientific study. The present study was aimed to explore the in vitro and in vivo wound healing potential of methanol extract (ME) and chloroform extract (CE) from B. diffusa leaf and subsequent identification of the bioactive metabolites, which might be responsible for enhancement of wound healing property of the extracts. The study included in vitro cell viability and wound scratch assays as well as in vivo excision wound assays in rat models. Both ME and CE were analysed for their antioxidant properties and phenolics content. The gas chromatography-mass spectrometry analyses were performed for identification of bioactive metabolites present in the ME and CE. ME of B. diffusa leaf significantly enhanced viability and migration of human keratinocyte cells (HaCaT) as compared to the untreated and CE-treated groups. The topical application of ME of B. diffusa leaf in excision wound model significantly decreased the wound area by the 14th day (91%) as compared to control (22%) (p < 0.05). The GC-MS analysis revealed the presence of caffeic acid, ferulic acid and D-pinitol as the major bioactive metabolites in ME. These results suggest that ME of B. diffusa possesses significant wound healing potential, where D-pinitol and caffeic acid served as the lead constituent metabolites responsible for the healing.

Kaempferol alleviates palmitic acid-induced lipid stores, endoplasmic reticulum stress and pancreatic ß-cell dysfunction through AMPK/mTOR-mediated lipophagy.

Kaempferol, a natural flavonoid, has the beneficial effects of preserving pancreatic ß-cell mass and function, but its action on ß-cell lipid metabolism still remains elusive. Recently, autophagy has been reported to play a major role in lipid metabolism in various cell types, but its role in pancreatic ß-cell's lipid metabolism is rarely reported. Here, we investigated the role of kaempferol-induced autophagy in inhibition of lipid stores, ER stress and ß-cell dysfunction in palmitic acid-challenged RIN-5F cells and isolated pancreatic islets. The lipid-lowering effect of kaempferol was determined by Oil Red O staining, triglyceride assay, BODIPY labeling, RT-PCR and immunoblot analysis of PLIN2 (the lipid droplet coat protein) expression. Further, the involvement of AMPK/mTOR-mediated lipophagy was established by pharmacological and genetic inhibitors of autophagy and AMPK. The co-localization studies of lipid droplets with autophagosomes/lysosomes by BODIPY-MDC-LysoTracker co-staining, LC3/BODIPY labeling and LC3/PLIN2 double immunolabeling further strengthened the findings. Kaempferol treatment exhibited decreased lipid stores and increased co-localization of lipid droplets with autophagosomes and lysosomes in palmitic acid-challenged ß-cells. Moreover, inhibition of autophagy led to decreased co-localization and increased lipid droplets accumulation. Kaempferol-induced alleviation of ER stress and ß-cell dysfunctions was established by immunoblot analysis of CHOP-10 (a key mediator of cell death in response to ER stress) and insulin content/secretion analysis respectively. Together, these findings suggest that kaempferol prevents ectopic lipid accumulation and ER stress, thus restoring ß-cell function through AMPK-mediated lipophagy. The current data implies that kaempferol may be a potential therapeutic candidate to prevent obesity-linked diabetic complications.

Antifungal and Anti-Biofilm Activity of Essential Oil Active Components against Cryptococcus neoformans and Cryptococcus laurentii.

Cryptococcosis is an emerging and recalcitrant systemic infection occurring in immunocompromised patients. This invasive fungal infection is difficult to treat due to the ability of Cryptococcus neoformans and Cryptococcus laurentii to form biofilms resistant to standard antifungal treatment. The toxicity concern of these drugs has stimulated the search for natural therapeutic alternatives. Essential oil and their active components (EO-ACs) have shown to possess the variety of biological and pharmacological properties. In the present investigation the effect of six (EO-ACs) sourced from Oregano oil (Carvacrol), Cinnamon oil (Cinnamaldehyde), Lemongrass oil (Citral), Clove oil (Eugenol), Peppermint oil (Menthol) and Thyme oil (thymol) against three infectious forms; planktonic cells, biofilm formation and preformed biofilm of C. neoformans and C. laurentii were evaluated as compared to standard drugs. Data showed that antibiofilm activity of the tested EO-ACs were in the order: thymol>carvacrol>citral>eugenol=cinnamaldehyde>menthol respectively. The three most potent EO-ACs, thymol, carvacrol, and citral showed excellent antibiofilm activity at a much lower concentration against C. laurentii in comparison to C. neoformans indicating the resistant nature of the latter. Effect of the potent EO-ACs on the biofilm morphology was visualized using scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM), which revealed the absence of extracellular polymeric matrix (EPM), reduction in cellular density and alteration in the surface morphology of biofilm cells. Further, to realize the efficacy of the EO-ACs in terms of human safety, cytotoxicity assays and co-culture model were evaluated. Thymol and carvacrol as compared to citral were the most efficient in terms of human safety in keratinocyte- Cryptococcus sp. co-culture infection model suggesting that these two can be further exploited as cost-effective and non-toxic anti-cryptococcal drugs.