Nanoencapsulation of Docetaxel Induces Concurrent Apoptosis and Necroptosis in Human Oral Cancer Cells (SCC-9) via TNF-α/RIP1/RIP3 Pathway.

Human oral squamous cell carcinoma is the sixth most frequent malignant cancer, with an unacceptably high death rate that affects people's health. Albeit, there are several clinical approaches for diagnosing and treating oral cancer they are still far from ideal. We previously synthesised and characterised the docetaxel nanoformulation (PLGA-Dtx) and discovered that docetaxel nanoencapsulation may suppress oral cancer cells. The goal of this study was to figure out the mechanism involved in the suppression of oral cancer cell proliferation. We discovered that PLGA-Dtx inhibited SCC-9 cell growth considerably as compared to free docetaxel (Dtx), and that the viability of SCC-9 cells treated with PLGA-Dtx was decreased dose-dependently. MTT assay showed that PLGA-Dtx selectively inhibited the growth of PBMCs from oral cancer patients while sparing PBMCs from normal healthy controls. Further, flow cytometry analysis showed that PLGA-Dtx induced apoptosis and necroptosis in SCC-9 cells. G2/M cell cycle arrest has been confirmed on exposure of PLGA-Dtx for 24 h in SCC-9 cells. Interestingly, western blot investigation found that PLGA-Dtx increased the amounts of necroptic proteins and apoptosis-related proteins more efficiently than Dtx. Furthermore, PLGA-Dtx was more effective in terms of ROS generation, and mitochondrial membrane potential depletion. Pretreatment with necroptosis inhibitor Nec-1 efficiently reversed the ROS production and further recover MMP caused by PLGA-Dtx. Overall, this study revealed a mechanistic model of therapeutic response for PLGA-Dtx in SCC-9 cells and proposed its potency by inducing cell death via activation of concurrent apoptosis and necroptosis in SCC-9 cells via TNF-α/RIP1/RIP3 and caspase-dependent pathway.

The Anti-Tumor and Immunomodulatory Effects of PLGA-Based Docetaxel Nanoparticles in Lung Cancer: The Potential Involvement of Necroptotic Cell Death through Reactive Oxygen Species and Calcium Build-Up.

Taxanes, microtubule stabilizing agents, are extensively used in the treatment of non-small cell lung cancer (NSCLC). However, their clinical effectiveness remains restricted owing to significant adverse effects and drug resistance. Nanotechnology may guide chemotherapeutic drugs directly and selectively to malignant cells, improving their therapeutic efficiency. In the present study, we synthesized polylactic-co-glycolic acid (PLGA) based nanoparticles encapsulating docetaxel and evaluated their efficacy in non-small cell lung carcinoma (A549) cells and primary immune cells derived from humans. Docetaxel-PLGA nanoparticles (PLGA-Dtx) were synthesized and characterized using distinct methods. Moreover, the cytotoxicity of free docetaxel (Dtx) and Dtx-conjugated nanoparticles (PLGA-Dtx) was studied in A549 cells and peripheral blood mononuclear cells derived from humans. Furthermore, annexin V-FITC/PI staining was used to assess the mode of cell death. Additionally, human peripheral blood mononuclear cells (PBMCs) were used for assessing the associated immune response and cytokine profile following PLGA-Dtx treatment. Spherical PLGA-Dtx nanoparticles with a 150 ± 10 nm diameter and 70% encapsulation efficiency (EE) were synthesized. The MTT assay showed that the IC50 of PLGA-Dtx nanoparticles was significantly lower than free docetaxel in A549 cells. Cytotoxicity data also revealed the selective nature of PLGA-Dtx with no significant effects in normal human bronchial epithelial cells (BEAS-2B) and PBMCs derived from healthy donors. Interestingly, PLGA-Dtx exerted an improved effect and tempted both apoptosis and necroptosis, as evidenced by annexin V and propidium iodide-positive cells. Further, PLGA-Dtx-exposed A549 cells showed increased Cas-3, Cas-9, RIP-1, and RIP-3, indicating apoptosis and necroptosis. An increased pro-inflammatory response manifested from the enhancement of IFN-γ and TNF-α in PLGA-Dtx-exposed PBMCs, posed by the occurrence of necroptosis and the immune stimulatory effect of PLGA-Dtx. In conclusion, PLGA-Dtx has a selective anticancer potential and better immunostimulatory potential. Therefore, PLGA-Dtx may be useful for the treatment of non-small cell lung carcinoma.

Fungal mediated biotransformation reduces toxicity of arsenic to soil dwelling microorganism and plant.

Rhizospheric and plant root associated microbes generally play a protective role against arsenic toxicity in rhizosphere. Rhizospheric microbial interaction influences arsenic (As) detoxification/mobilization into crop plants and its level of toxicity and burden. In the present investigation, we have reported a rhizospheric fungi Aspergillus flavus from an As contaminated rice field, which has capability to grow at high As concentration and convert soluble As into As particles. These As particles showed a reduced toxicity to soil dwelling bacteria, fungi, plant and slime mold. It does not disrupt membrane potential, inner/outer membrane integrity and survival of the free N2 fixating bacteria. In arbuscular mycorrhiza like endophytic fungi Piriformospora indica, these As particles does not influence mycelial growth and plant beneficial parameters such as phosphate solubilizing enzyme rAPase secretion and plant root colonization. Similarly, it does not affect plant growth and chlorophyll content negatively in rice plant. However, these As particles showed a poor absorption and mobilization in plant. These As particle also does not affect attachment process and survival of amoeboid cells in slime mold, Dictyostelium discoideum. This study suggests that the process of conversion of physical and chemical properties of arsenic during transformation, decides the toxicity of arsenic particles in the rhizospheric environment. This phenomenon is of environmental significance, not only in reducing arsenic toxicity but also in the survival of healthy living organism in arsenic-contaminated rhizospheric environment.

Nitrogen-dependent metabolic regulation of lipid production in microalga Scenedesmus vacuolatus.

Microalga Scenedesmus vacuolatus exhibited maximum growth, protein and carbohydrate contents at 10.0 mM concentration of nitrate, 1.0 mM of glutamate nitrogen and at C/N ratio (12 mM acetate+10 mM nitrate). However, these cell constituents showed the highest values in the C+N grown cells, but the lipid content was found to be the highest glutamate grown cells. FTIR analysis of Lipid/Carbohydrate and Lipid/Protein ratio and flow cytometric analysis of neutral lipids revealed higher lipid content in the glutamate grown cells than in the nitrogen starved, nitrate and C+N grown cells. The nitrate reductase activity was the highest in the C+ N grown cells and the lowest activity was found in the glutamate grown cells. A corollary of these results suggested that suppression of nitrogen assimilatory system, whether by glutamate or by nitrogen deprivation, was the most suitable physiological condition for enhanced lipid synthesis and biofuel production in microalgal cells.

Arsenic, Cadmium, and Lead Like Troglitazone Trigger PPARγ-Dependent Poly (ADP-Ribose) Polymerase Expression and Subsequent Apoptosis in Rat Brain Astrocytes.

We previously demonstrated that arsenic, cadmium, and lead mixture at environmentally relevant doses induces astrocyte apoptosis in the developing brain. Here, we investigated the mechanism and contribution of each metal in inducing the apoptosis. We hypothesized participation of transcription factor, peroxisome proliferator-activated receptor gamma (PPARγ), reported to affect astrocyte survival. We treated cultured rat astrocytes with single metals and their combinations and performed apoptosis assay and measured PPARγ expression levels. We found that cadmium demonstrated maximum increase in PPARγ as well as apoptosis, followed by arsenic and then lead. Interestingly, we observed that the metals mimicked PPARγ agonist, troglitazone, and enhanced PPARγ-transcriptional activity. Co-treatment with PPARγ-siRNA or PPARγ-antagonist, GW9662, suppressed the astrocyte apoptosis, suggesting a prominent participation of PPARγ in metal(s)-induced astrocyte loss. We explored PPARγ-transcriptional activity and identify its target gene in apoptosis, performed in silico screening. We spotted PPARγ-response elements (PPREs) within poly(ADP-ribose) polymerase (PARP) gene, and through gel-shift assay verified metal(s)-mediated increased PPARγ binding to PARP-PPREs. Chromatin-immunoprecipitation and luciferase-reporter assays followed by real-time PCR and Western blotting proved PPRE-mediated PARP expression, where cadmium contributed most and lead least, and the effects of metal mixture were comparable with troglitazone. Eventually, dose-dependent increased cleaved-PARP/PARP ratio confirmed astrocyte apoptosis. Additionally, we found that PPARγ and PARP expressions were c-Jun N-terminal kinases and cyclin-dependent kinase5-dependent. In vivo treatment of developing rats with the metals corroborated enhanced PPARγ-dependent PARP and astrocyte apoptosis, where yet again cadmium contributed most. Overall, our study enlightens a novel PPARγ-dependent mechanism of As-, Cd-, and Pb-induced astrocyte apoptosis.

Intricatinol synergistically enhances the anticancerous activity of cisplatin in human A549 cells via p38 MAPK/p53 signalling.

Platinum containing drugs are widely used to treat advanced lung carcinomas. However, their clinical success is still limited due to severe side effects, and drug resistance. Alternative approaches are warranted to augment efficacy of platinum based chemotherapeutic drugs with minimal side effects. Intricatinol (INT), a homoisoflavonoid, has been shown to possess anti-tubercular, antioxidant, hypoglycaemic, and hypolipidemic activity. However, its anticancer activity largely remains unknown. In the present study, we have evaluated anticancer potential of INT alone or in combination with cisplatin (CIS) in non-small cell lung carcinoma (A549) cells. Treatment with INT alone reduced the viability of A549 cells in a dose-dependent manner. Interestingly, the combination of low doses of INT and CIS exerted a synergistic effect and induced apoptosis as evident by DNA fragmentation and Annexin V positive cells. Enhanced Bax:Bcl-2 ratio, loss of Δψm, cytochrome c release, cleavage of caspase 3 and PARP1 strongly corroborated our findings. Further, increased expression of p53, p38 MAPK and their phosphorylated counterparts, loss of clonogenicity and reduced migration potential were also recorded with INT + CIS treatment. Most interestingly, INT could not induce any significant cell death in primary mouse embryonic fibroblasts (MEFs). Moreover, no additive or synergistic effect was noted with INT + CIS in MEFs under similar treatment conditions. In conclusion, INT has a selective anticancer potential and could synergize cytotoxicity of CIS. Therefore, the combination of INT and CIS may serve as an effective anticancer strategy for the treatment of non-small cell lung carcinoma.

Dynamin-related Protein 1 Inhibition Mitigates Bisphenol A-mediated Alterations in Mitochondrial Dynamics and Neural Stem Cell Proliferation and Differentiation.

The regulatory dynamics of mitochondria comprises well orchestrated distribution and mitochondrial turnover to maintain the mitochondrial circuitry and homeostasis inside the cells. Several pieces of evidence suggested impaired mitochondrial dynamics and its association with the pathogenesis of neurodegenerative disorders. We found that chronic exposure of synthetic xenoestrogen bisphenol A (BPA), a component of consumer plastic products, impaired autophagy-mediated mitochondrial turnover, leading to increased oxidative stress, mitochondrial fragmentation, and apoptosis in hippocampal neural stem cells (NSCs). It also inhibited hippocampal derived NSC proliferation and differentiation, as evident by the decreased number of BrdU- and ß-III tubulin-positive cells. All these effects were reversed by the inhibition of oxidative stress using N-acetyl cysteine. BPA up-regulated the levels of Drp-1 (dynamin-related protein 1) and enhanced its mitochondrial translocation, with no effect on Fis-1, Mfn-1, Mfn-2, and Opa-1 in vitro and in the hippocampus. Moreover, transmission electron microscopy studies suggested increased mitochondrial fission and accumulation of fragmented mitochondria and decreased elongated mitochondria in the hippocampus of the rat brain. Impaired mitochondrial dynamics by BPA resulted in increased reactive oxygen species and malondialdehyde levels, disruption of mitochondrial membrane potential, and ATP decline. Pharmacological (Mdivi-1) and genetic (Drp-1siRNA) inhibition of Drp-1 reversed BPA-induced mitochondrial dysfunctions, fragmentation, and apoptosis. Interestingly, BPA-mediated inhibitory effects on NSC proliferation and neuronal differentiations were also mitigated by Drp-1 inhibition. On the other hand, Drp-1 inhibition blocked BPA-mediated Drp-1 translocation, leading to decreased apoptosis of NSC. Overall, our studies implicate Drp-1 as a potential therapeutic target against BPA-mediated impaired mitochondrial dynamics and neurodegeneration in the hippocampus.

Oxidative Stress Biomarkers in the Freshwater Fish, Heteropneustes fossilis (Bloch) Exposed to Sodium Fluoride: Antioxidant Defense and Role of Ascorbic Acid.

The present study highlights fluoride -induced toxicity and the protective role of ascorbic acid in the liver and ovary of freshwater fish, Heteropneustis fossilis. The fish specimens were exposed to different concentrations (35 mg F/L and 70 mg F/L) of fluoride. Parameters related to oxidative stress were studied at the end of the experiment. The biomarkers selected for the study were thiobarbituric acid reactive substances for assessing the extent of lipid peroxidation (LPO) and antioxidant defense system such as reduced glutathione (GSH), superoxide dismutase (SOD) catalase (CAT) glutathione peroxidase (GPx), and glutathione S-transferase (GST) activities. The fluoride exposure significantly elevated the level of LPO, CAT, SOD, and GST in the tissues of treated group as well as modulated the activities of GSH and level of GPx after exposure as compared to the control. A significant decrease in GPx activity was found in these tissues suggesting that fluoride exposure increases the level of free radical, as well as CAT activity. Pre- and post treatment with ascorbic acid decreased the LPO, SOD, CAT, GST level, and increased GSH, GPx levels in the liver and ovary.