Piperlongumine induces ROS mediated apoptosis by transcriptional regulation of SMAD4/P21/P53 genes and synergizes with doxorubicin in osteosarcoma cells.

Piperlongumine is a herbal drug, with well-known anti-microbial and anti-neoplastic properties. The anti-carcinogenic potential of piperlongumine has been extensively explored for breast, colorectal, lungs, pancreatic, prostate, and oral carcinoma. However, a few numbers of studies are available on its bio-activity in osteosarcoma. Therefore, the present study aimed at exploring the therapeutic potential and possible mechanisms of action of piperlongumine in three human osteosarcoma cell lines in-vitro. The cytotoxicity of piperlongumine was determined by MTT assay, which shows dose and time-dependent inhibition of MG-63, 143B and KHOS/NP cells. Piperlongumine arrest the cells in G2/M phase of cell cycle and increases reactive oxygen species production, which possibly leads to lethal oxidative stress and apoptosis. Piperlongumine treatment significantly upregulated the expression of genes BAX, P21, P53, and SMAD4; while the BCL-2, SURVIVIN, TNFA, and NFKB genes expression was found down-regulated. Furthermore, piperlongumine exposure inhibited the migration of osteosarcoma cells as the expression of migration marker genes CDH2, CTNNB1, FN1, and TWIST were found to be down-regulated. The drug combination studies show the synergistic effect of piperlongumine with the conventional chemotherapeutic drug doxorubicin in osteosarcoma cells. Taken together, the above results suggest that PL displays anticancer properties against osteosarcoma and can be used as a therapeutic agent for osteosarcoma treatment in clinical settings.

One Pot Synthesis of PEGylated Bimetallic Gold-Silver Nanoparticles for Imaging and Radiosensitization of Oral Cancers.

BACKGROUND: Radiotherapy is an important treatment modality for many types of head and neck squamous cell carcinomas. Nanomaterials comprised of high atomic number (Z) elements are novel radiosensitizers enhance radiation injury by production of free radicals and subsequent DNA damage. Gold nanoparticles are upcoming as promising radiosensitizers due to their high (Z) biocompatibility, and ease for surface engineering. Bimetallic nanoparticles have shown enhanced anticancer activity compared to monometallic nanoparticles. MATERIALS AND METHODS: PEG-coated Au-Ag alloy nanoparticles (BNPs) were synthesized using facile one pot synthesis techniques. Size of ~50±5nm measured by dynamic light scattering. Morphology, structural composition and elemental mapping were analyzed by electron microscopy and SAXS (small-angle X-ray scattering). The radiosensitization effects on KB oral cancer cells were evaluated by irradiation with 6MV X-rays on linear accelerator. Nuclear damage was imaged using confocal microscopy staining cells with Hoechst stain. Computed tomography (CT) contrast enhancement of BNPs was compared to that of the clinically used agent, Omnipaque. RESULTS: BNPs were synthesized using PEG 600 as reducing and stabilizing agent. The surface charge of well dispersed colloidal BNPs solution was -5mV. Electron microscopy reveals spherical morphology. HAADF-STEM and elemental mapping studies showed that the constituent metals were Au and Ag intermixed nanoalloy. Hydrodynamic diameter was ~50±5nm due to PEG layer and water molecules absorption. SAXS measurement confirmed BNPs size around 35nm. Raman shift of around 20 cm-1 was observed when BNPs were coated with PEG. 1H NMR showed extended involvement of - OH in synthesis. BNPs efficiently enter cytoplasm of KB cells and demonstrated potent in vitro radiosensitization with enhancement ratio ~1.5-1.7. Imaging Hoechst-stained nuclei demonstrated apoptosis in a dose-dependent manner. BNPs exhibit better CT contrast enhancement ability compared to Omnipaque. CONCLUSION: This bimetallic intermix nanoparticles could serve a dual function as radiosensitizer and CT contrast agent against oral cancers, and by extension possibly other cancers as well.

Ursolic acid disturbs ROS homeostasis and regulates survival-associated gene expression to induce apoptosis in intestinal cancer cells.

Ursolic acid is a natural compound possessing several therapeutic properties including anticancer potential. In present study, cytotoxic and antimetastatic properties of ursolic acid were investigated in intestinal cancer cell lines INT-407 and HCT-116. The cells growth and number were decreased in a dose- and time-dependent manner in both the cell lines. It also increases reactive oxygen species levels in the cells in order to induce apoptosis. Ursolic acid was found to be a significant inhibitor of cancer cells migration and gene expression of migration markers FN1, CDH2, CTNNB1 and TWIST was also downregulated. Ursolic acid treatment downregulated the gene expression of survival factors BCL-2, SURVIVIN, NFKB and SP1, while upregulated the growth-restricting genes BAX, P21 and P53. These results indicate that ursolic acid has anticancer and antimetastatic properties against intestinal cancer. These properties could be beneficial in cancer treatment and could be used as complementary medicine.

Piperlongumine induces ROS mediated cell death and synergizes paclitaxel in human intestinal cancer cells.

Piperlongumine (PL), a herbal drug extracted from long pepper (Piper longum L), is known for its anti-inflammatory and anti-cancer properties. Although, its anti-cancer potential has been evaluated in cancer models like breast, pancreatic, gastric, hepatocellular and lung carcinoma, there is no report on its bio-activity evaluation in intestinal cancers. Here, we report the anti-neoplastic potential of PL against human intestinal carcinoma in-vitro and its possible mechanisms of action. Cytotoxicity studies demonstrate that PL inhibits cell proliferation of INT-407 and HCT-116 cells in a concentration and time-dependent manner. Also, PL elevated the levels of intracellular reactive oxygen species, which may lead to lethal oxidative stress, mitochondrial dysfunction, and nuclear fragmentation. Remarkably, P53, P21, BAX, and SMAD4 were significantly upregulated after PL treatment whereas; BCL2 and SURVIVIN were down-regulated. Moreover, the combination study also shows the synergistic effect of PL with the current chemotherapeutic drug paclitaxel. These findings suggest that PL possesses anti-neoplastic properties in intestinal cancer cells.

Response of Salmonella Typhi to bile-generated oxidative stress: implication of quorum sensing and persister cell populations.

Salmonella Typhi can chronically persist within the gallbladder of patients suffering from gallbladder diseases. This study, intended to improve our understanding of bacterial mechanisms underlying bile adaptation, revealed that bile, which is a bactericidal agent, led to the generation of reactive oxygen species in S Typhi. Salmonella Typhi in response showed a significant increase in the production of anti-oxidative enzymes, namely superoxide dismutase and catalase. The work reports that the quorum-sensing (QS) system of S Typhi regulates the level of these enzymes during oxidative stress. In support of these observations, the quorum-sensing mutant of S Typhi was found to be sensitive to bile with significantly lower levels of anti-oxidant enzymes compared to other clinical isolates. Furthermore the addition of exogenous cell-free extracts (CFEs) of S Typhi containing the quorum-sensing signalling molecule significantly increased the levels of these enzymes within the mutant. Interestingly the CFE addition did not significantly restore the biofilm-forming ability of the mutant strain when compared with the wild-type. In the presence of ciprofloxacin and ampicillin, S Typhi formed persister cells which increased >3-fold in the presence of bile. Thus the QS-system of S Typhi aids in oxidative stress management, and enhanced persister cell populations could assist chronic bacterial persistence within the gallbladder.

Role of gold nanoparticles as drug delivery vehicles for chondroitin sulfate in the treatment of osteoarthritis.

Osteoarthritis is a disease which is characterized by joint pain, swelling and stiffness. Articular cartilage has limited self-repair capacity due to its avascular and aneural nature. In this work, we show the use of gold nanoparticles (AuNps) for enhancing the delivery of chondroitin sulfate (CS), a drug used in the treatment of osteoarthritis (OA). AuNps were synthesized and were characterized by transmission electron microscopy (TEM), ultraviolet-visible (UV-Vis) spectroscopy, Fourier transform infrared spectroscopy (FTIR), and X-Ray diffraction analysis. AuNps were combined with CS (AuNps-CS) and their effect on primary goat chondrocytes was studied using MTT assay, Hoechst staining, production of glycosaminoglycan and collagen. Cell viability studies by MTT revealed that AuNps-CS stimulate cell proliferation. A two-fold increase in GAG and collagen production was observed in presence of AuNps-CS combination as compared to native CS, indicating that this combination stimulates chondrocyte proliferation and enhances extracellular matrix production (ECM). Hence, this study exhibits the potential of AuNps as a carrier of CS for treatment of osteoarthritis.

Efficacy of supermacroporous poly(ethylene glycol)-gelatin cryogel matrix for soft tissue engineering applications.

Three dimensional scaffolds synthesized using natural or synthetic polymers act as an artificial niche for cell adherence and proliferation. In this study, we have fabricated cryogels employing blend of poly (ethylene glycol) (PEG) and gelatin using two different crosslinkers like, glutaraldehyde and EDC-NHS by cryogelation technique. Synthesized matrices possessed interconnected porous structure in the range of 60-100 µm diameter and regained their original length after 90% compression without deformation. Visco-elastic behavior was studied by rheology and unconfined compression analysis, elastic modulus of these cryogels was observed to be >10(5)Pa which showed their elasticity and mechanical strength. TGA and DSC also showed the stability of these cryogels at different temperatures. In vitro degradation capacity was analyzed for 4 weeks at 37°C. IMR-32, C2C12 and Cos-7 cells proliferation and ECM secretion on PEG-gelatin cryogels were observed by SEM and fluorescent analysis. In vitro biocompatibility was analyzed by MTT assay for the period of 15 days. Furthermore, cell proliferation efficiency, metabolic activity and functionality of IMR-32 cells were analyzed by neurotransmitter assay and DNA quantification. The cell-matrix interaction, elasticity, mechanical strength, stability at different temperatures, biocompatible, degradable nature showed the potentiality of these cryogels towards soft tissue engineering such as neural, cardiac and skin.

Three-dimensional supermacroporous carrageenan-gelatin cryogel matrix for tissue engineering applications.

A tissue-engineered polymeric scaffold should provide suitable macroporous structure similar to that of extracellular matrix which can induce cellular activities and guide tissue regeneration. Cryogelation is a technique in which appropriate monomers or polymeric precursors frozen at sub-zero temperature leads to the formation of supermacroporous cryogel matrices. In this study carrageenan-gelatin (natural polymers) cryogels were synthesized by using glutaraldehyde and 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride and N-hydroxysuccinimide (EDC-NHS) as crosslinking agent at optimum concentrations. Matrices showed large and interconnected pores which were in the range of 60-100 µm diameter. Unconfined compression analysis showed elasticity and physical integrity of all cryogels, as these matrices regained their original length after 90% compressing from the original size. Moreover Young's modulus was found to be in the range of 4-11 kPa for the dry cryogel sections. These cryogels also exhibited good in vitro degradation capacity at 37 °C within 4 weeks of incubation. Supermacroporous carrageenan-gelatin cryogels showed efficient cell adherence and proliferation of Cos-7 cells which was examined by SEM. PI nuclear stain was used to observe cell-matrix interaction. Cytotoxicity of the scaffolds was checked by MTT assay which showed that cryogels are biocompatible and act as a potential material for tissue engineering and regenerative medicine.

Study on Salmonella Typhi occurrence in gallbladder of patients suffering from chronic cholelithiasis-a predisposing factor for carcinoma of gallbladder.

Cholelithiasis is frequently associated with carcinoma of gallbladder, and the presence of Salmonella Typhi in gallbladder of patients suffering from cholelithiasis is implicated as a predisposing factor for carcinogenesis. This study was conducted on patients suffering from chronic cholelithiasis from a region in North India-endemic area for enteric fever with high incidence of gallstones and gallbladder cancer. Since culture studies rarely reveal the chronic Salmonella Typhi persistence, we use PCR assay to specifically amplify the H1-d flagellin gene sequence homologous with Salmonella Typhi. Seven cases (17.5%), none of which were positive for culture, showed positive PCR results for Salmonella Typhi, 4 (10%) of which were tissue, 2 bile (5%), and 1 gallstone (2.5%). The chronic existence of Salmonella Typhi in gallbladder disease was confirmed. Thus, the study would indicate the importance of vaccination so as to prevent chronic infection and need for early diagnostic tools to prevent any further complications.

Proliferation of chondrocytes on a 3-d modelled macroporous poly(hydroxyethyl methacrylate)-gelatin cryogel.

Tissue-engineering constructs should be designed to mimic the native tissue environment for cells, the scaffold matching to stiffness and strength of the tissues while maintaining an interconnected porous network and a reasonable porosity. This study presents a new single-step protocol for synthesis of a poly(hydroxyethyl methacrylate)-poly(ethylene glycol) diacrylate-gelatin (HPG) macroporous polymeric scaffold with well-controlled porous structure and good mechanical strength. The pore size of these matrices lies in the range of 30 to 100 µm with an average pore diameter of 80 µm and with an interconnected pore structure as analyzed by scanning electron microscopy. Further, interconnectivity was also confirmed by high solvent uptake capacity, as the cryogel reached its equilibrium within 2 min. The gels also showed substantial mechanical integrity, i.e., the average compressive modulus was 32.73 ± 2.36 kPa at 15% compression of their original length. The degree of weight loss of these cryogels was found to be approx. 88% within 8 weeks of incubation in PBS (pH 7.4) at 37°C. Physio-chemically optimized cryogel was further evaluated for in vitro growth and proliferation of isolated primary goat chondrocytes up to 3 weeks. The cell adherence on cryogel was examined by SEM analysis, while cell-matrix interaction was examined by 4-6-diamidino-2-phenylindole and propidium iodide staining. Furthermore, the cell compatibility and proliferation was evaluated using the MTT assay. Increase in total cellular metabolic activity was observed as shown by continuous increase in glycosaminoglycan and collagen contents with time. Collagen type-I and type-II gene expression analysed for over 3 weeks by RT-PCR showed the prominent expression of collagen type-II. These results suggest the use of synthesised cryogel scaffold as a matrix for chondrocyte attachment and proliferation in 3-D environment and as a delivery system in cartilage-tissue engineering.

Proliferation of myoblast skeletal cells on three-dimensional supermacroporous cryogels.

Cardiac and skeletal muscle tissue engineering provides a smart approach to overcome problems associated with organ transplantation and cardiac tissue and also lays a platform for superior alternative approaches in muscle regeneration. The aim of the study was to demonstrate cryogel scaffold potential in the field of skeletal muscle and cardiac tissue engineering. Poly-hydroxyethyl methacrylate (pHEMA)-gelatin cryogel scaffold was synthesized using cryogelation technique and such a designed material is being reported first time. Rheology study of the pHEMA-gelatin (HG) suggested that the cryogel scaffolds were stable at different temperatures and phase angle remained constant in both dry and wet state. HG cryogel was able to bear increased stress without leading to deformation. Monitoring the hydration of HG scaffold showed shift from a stiff to a more pliable material and upon continuing hydration, shear modulus remained constant with no further change observed. However, the change in phase angle <0.24 masculine indicates a gradual increase in stiffness of the material over time. Scaffold synthesised using such polymer combinations gave cells a native environment for proliferation and surface stiffness have shown to help in differentiation of the cells. Myoskeletal cell lines were cultured on these scaffolds to check the biocompatibility and cell proliferation. Alamar blue assay performed over a period of 3 weeks analysed the metabolic activity of cells which showed more than 60% increase in the total cellular activity. DNA content of cells was found to be directly related to number of cells present at a given time point and this was found to have increased by more than 50% in 3 weeks. Since in 3-D scaffold the surface area is more in comparison to 2-D, hence better cell proliferation is observed. Hoechst and DAPI staining showed tubular structure and alignment of the cells during formation of the tubules shows promising cellular response to the cryogel matrix. The mechanical strength, stiffness and elastic measurements of the scaffold indicated potential application of these materials for skeletal and cardiac tissue engineering.