Alpha Tocopherol Loaded Polymeric Nanoparticles: Preparation, Characterizations, and In Vitro Assessments Against Oxidative Stress in Spinal Cord Injury Treatment.

Spinal cord injury (SCI) is characterized by mechanical injury or trauma to the spinal cord. Currently, SCI treatment requires extremely high doses of neuroprotective agents, which in turn, causes several adverse effects. To overcome these limitations, the present study focuses on delivery of a low but effective dose of a naturally occurring antioxidant, α-tocopherol (α-TP). Calcium alginate nanoparticles (CA-NP) and poly D,L-lactic-co-glycolic acid nanoparticles (PLGA-NP) prepared by ionotropic gelation and solvent evaporation technique had particle size of 21.9 ± 11.19 and 152.4 ± 10.6 nm, respectively. Surface morphology, surface charge, as well as particle size distribution of both nanoparticles were evaluated. Entrapment of α-TP into CA-NP and PLGA-NP quantified by UPLC showed entrapment efficiency of 4.00 ± 1.63% and 76.6 ± 11.4%, respectively. In vitro cytotoxicity profiles on human astrocyte-spinal cord (HA-sp) showed that blank CA-NP at high concentrations reduced the cell viability whereas blank PLGA-NP showed relatively safer cytotoxic profiles. In addition, PLGA nanoparticles encapsulated with α-TP (α-TP-PLGA-NP) in comparison to α-TP alone at high concentrations were less toxic. Pretreatment of HA-sp cells with α-TP-PLGA-NP showed two-fold higher anti-oxidative protection as compared to α-TP alone, when oxidative stress was induced by H2O2. In conclusion, CA-NP were found to be unsuitable for treatment of SCI due to their cytotoxicity. Comparatively, α-TP-PLGA-NP were safer and showed high degree of protection against oxidative stress than α-TP alone.

Sophorolipid: a glycolipid biosurfactant as a potential therapeutic agent against COVID-19.

Biosurfactants are natural surfactants produced by a variety of microorganisms. In recent years, biosurfactants have garnered a lot of interest due to their biomedical and pharmaceutical applications. Sophorolipids are glycolipid types of biosurfactants produced by selected nonpathogenic yeasts. In addition to the detergent activity (reduction in surface and interfacial tension), which is commonly utilized by biomedical applications, sophorolipids have shown some unique properties such as, antiviral activity against enveloped viruses, immunomodulation, and anticancer activity. Considering their antiviral activity, the potential of sophorolipids as an antiviral therapy for the treatment of COVID-19 is discussed in this review. Being a surfactant molecule, sophorolipid could solubilize the lipid envelope of SARS-CoV-2 and inactivate it. As an immunomodulator, sophorolipid could attenuate the cytokine storm caused by the SARS-CoV-2 upon infection, and inhibit the progression of COVID-19 in patients. Sophorolipids could also be used as an effective treatment strategy for COVID-19 patients suffering from cancer. However, there is limited research on the use of sophorolipid as a therapeutic agent for the treatment of cancer and viral diseases, and to modulate the immune response. Nevertheless, the multitasking capabilities of sophorolipids make them potential therapeutic candidates for the bench-to-bedside research for the treatment of COVID-19.

Regulation of Prdx6 by Nrf2 Mediated Through aiPLA2 in White Matter Reperfusion Injury.

Hypoxia and reperfusion produces overproduction of ROS (reactive oxygen species), which may lead to mitochondrial dysfunction leading to cell death and apoptosis. Here, we explore the hypothesis that Prdx6 protects the spinal cord white matter from hypoxia-reperfusion injury and elucidate the possible mechanism by which Prdx6 elicits its protective effects. Briefly, rats were deeply anesthetized with isoflurane. A 30-mm section of the spinal cord was rapidly removed and placed in cold Ringer's solution (2-4 °C). The dissected dorsal column was exposed to hypoxia with 95% N2 and 5% CO2 and reperfusion with 95% O2 and 5% CO2. The expression of Prdx6 significantly upregulated in white matter after hypoxia compared to the sham group, whereas reperfusion caused a gradual decrease in Prdx6 expression after reperfusion injury. For the first time, our study revealed the novel expression and localized expression of Prdx6 in astrocytes after hypoxia, and possible communication of astrocytes and axons through Prdx6. The gradual increase in Nrf2 expression suggests a negative regulation of Prdx6 through Nrf2 signaling. Furthermore, inhibition of aiPLA2 activity of Prdx6 by MJ33 shows that the regulation of Prdx6 by Nrf2 is mediated through aiPLA2 activity. The present study uncovers a differential distribution of Prdx6 in axons and astrocytes and regulation of Prdx6 in hypoxia-reperfusion injury. The low levels of Prdx6 in reperfusion injury lead to increased inflammation and apoptosis in the white matter; therefore, the results of this study suggest that Prdx6 has a protective role in spinal hypoxia-reperfusion injury.

Neuroprotective effects of Riluzole and Curcumin in human astrocytes and spinal cord white matter hypoxia.

Damage to the spinal cord (SC) can result in irreversible impairments or complete loss of motor, sensory, and autonomic functions. Riluzole, a sodium channel-blocker and glutamate inhibitor, is in preclinical use for SC injury (SCI), and curcumin is an intracellular calcium inhibitor that attenuates glutamate-induced neurotoxicity. As riluzole and curcumin have different mechanisms to protect against SCI, we aimed to investigate the neuroprotective effects of a combination of riluzole and curcumin in human astrocytes and white matter injury (WMI) model of SCI. Our data show that a combination of riluzole (1 µM) and curcumin (1 µM) was effective in inhibiting hydrogen peroxide (H2O2)-induced oxidative dress in astrocytes derived from human SC, however, curcumin alone showed a significant inhibition. In addition, our results demonstrated that curcumin alone downregulates the hypoxia-induced expression of HIF-1, GFAP, and NF-H proteins in WMI, whereas riluzole alone and in combination with curcumin remained ineffective in changing the expression of these proteins. Contrarily, after inhibiting Ca2+ influx with EGTA, riluzole alone and in combination with curcumin significantly downregulated hypoxia-induced expression of GFAP and NF-H. After analysis of caspase 9 and cleaved caspase 9, we observed that curcumin and riluzole both inhibit apoptosis significantly, whereas their combination remains ineffective. Furthermore, we observed that neuroprotective effects of curcumin and riluzole are mediated through Nrf2/HO-1 signaling. In conclusion, our results demonstrate that curcumin and riluzole protect astrocytes from oxidative stress and white matter from hypoxia. However, their combination is not beneficial to reduce hypoxia-induced astrocytosis, axonal damage, and apoptosis. From our results, it is evident that curcumin is more effective in reducing WMI than riluzole.

Curcumin Protects against White Matter Injury through NF-κB and Nrf2 Cross Talk.

Inflammation and oxidative stress play a central role in the pathogenesis of white matter injury (WMI). Curcumin (Cur), a polyphenolic compound, exhibits anti-inflammatory and anti-oxidant effects on several conditions. The objective of this study was to investigate neuroprotective effects of Cur on WMI and explore its underlying mechanisms of action. Sprague-Dawley rats were subjected to the removal of white matter from the dorsal column of the spinal cord. Dorsal columns were randomly divided into three groups: Sham (Ringer's solution bubbled with 95% O2 and 5% CO2), hypoxia (Hyp; Ringer's solution bubbled with 95% N2 and 5% CO2 for 1 h), and Cur-treated (Hyp+Cur; Ringer's solution bubbled with 95% N2 and 5% CO2 for 1 h in the presence of 50 µM Cur). For NF-κB inhibition experiments, dorsal columns were incubated with 50 µM BAY 11-7082 (BAY) for 30 min in 95% O2 and 5% CO2 prior to 1-h incubation with 50 µM Cur in 95% N2 and 5% CO2. Our data show that Cur inhibited hypoxia-induced HIF1-α expression and tissue damage by demonstrating the improved morphology of astrocytes and remarkable reduction in vacuolation. Cur also inhibited the hypoxia-induced upregulation of glial fibrillary acidic protein (GFAP) and neurofilament-H (NF-H) after hypoxia and downregulated the expression of pro-inflammatory cytokines such as tumor necrosis factor alpha (TNF-α) and interleukin 1 (IL-1). Terminal dexynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)-assay analysis showed that Cur effectively attenuated apoptosis in white matter. In addition, we demonstrated that Cur exerted its neuroprotective effect through cross talk between nuclear factor kappa-light-chain-enhancer of activated B (NF-κB) and nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathways. In conclusion, our results indicate that treatment with Cur inhibited the hypoxia, inflammation and apoptosis associated with WMI. Further, the Nrf-2 pathway inhibits NF-κB activation by preventing IkB degradation and increasing HO-1 expression, which in turn reduces reactive oxygen species (ROS) and as a result NF-κB activation is suppressed. Similarly, NF-κB-mediated transcription reduces Nrf2 activation by reducing anti-oxidant response element (ARE) gene and free CREB binding protein by competing with Nrf2 for CBP thus inhibiting the Nrf-2 activation.

Depletion of mitochondrial protease OMA1 alters proliferative properties and promotes metastatic growth of breast cancer cells.

Metastatic competence of cancer cells is influenced by many factors including metabolic alterations and changes in mitochondrial biogenesis and protein homeostasis. While it is generally accepted that mitochondria play important roles in tumorigenesis, the respective molecular events that regulate aberrant cancer cell proliferation remain to be clarified. Therefore, understanding the mechanisms underlying the role of mitochondria in cancer progression has potential implications in the development of new therapeutic strategies. We show that low expression of mitochondrial quality control protease OMA1 correlates with poor overall survival in breast cancer patients. Silencing OMA1 in vitro in patient-derived metastatic breast cancer cells isolated from the metastatic pleural effusion and atypical ductal hyperplasia mammary tumor specimens (21MT-1 and 21PT) enhances the formation of filopodia, increases cell proliferation (Ki67 expression), and induces epithelial-mesenchymal transition (EMT). Mechanistically, loss of OMA1 results in alterations in the mitochondrial protein homeostasis, as reflected by enhanced expression of canonic mitochondrial unfolded protein response genes. These changes significantly increase migratory properties in metastatic breast cancer cells, indicating that OMA1 plays a critical role in suppressing metastatic competence of breast tumors. Interestingly, these results were not observed in OMA1-depleted non-tumorigenic MCF10A mammary epithelial cells. This newly identified reduced activity/levels of OMA1 provides insights into the mechanisms leading to breast cancer development, promoting malignant progression of cancer cells and unfavorable clinical outcomes, which may represent possible prognostic markers and therapeutic targets for breast cancer treatment.

Pre and post treatment with curcumin and resveratrol protects astrocytes after oxidative stress.

The two most studied polyphenolic compounds, curcumin (Cur) and resveratrol (Res), have been reported to protect oxidative damage of astrocytes. The present study is designed to examine the comparative anti-oxidative effect of Cur and Res on astrocytes by studying their potential to protect H2O2 induced oxidative stress at 4 h and 24 h time exposure. The effect of Cur and Res on cell viability, ROS production, inflammation and astrogliosis was compared. The effect of these two on Nrf2 expression and its translocation to nuclear compartment was investigated. The results showed that both Cur and Res significantly increase astrocytes survival after oxidative stress at both time points, however, Res demonstrated better effect on cell viability than the Cur. Res, showing significant inhibition of ROS production at both time points. Cur displayed significant inhibition of ROS production at 4 h, suggesting that Cur is more active on ROS inhibition in the earlier phase of insult. Comparing the expression of NF-κB, Cur showed better anti-inflammatory action on NF-κB while Res did not have any effect of NF-κB expression at 4 h. Interestingly, Cur showed an upregulation of nuclear Nrf2 expression at 24 h whereas Res displayed no effect after 24 h incubation. Both Cur and Res inhibited the H2O2 induced translocation of Nrf2 into nucleus. In conclusion, based on our observation, we found that Cur and Res both protected astrocytes from oxidative stress. In addition, we observed that Cur is most effective in early hours of insult while Res is effective in late hours suggesting that Res may or may not have immediate effect on astrocytes.

Curcumin alleviates oxidative stress and mitochondrial dysfunction in astrocytes.

Oxidative stress plays a critical role in various neurodegenerative diseases, thus alleviating oxidative stress is a potential strategy for therapeutic intervention and/or prevention of neurodegenerative diseases. In the present study, alleviation of oxidative stress through curcumin is investigated in A172 (human glioblastoma cell line) and HA-sp (human astrocytes cell line derived from the spinal cord) astrocytes. H2O2 was used to induce oxidative stress in astrocytes (A172 and HA-sp). Data show that H2O2 induces activation of astrocytes in dose- and time-dependent manner as evident by increased expression of GFAP in A172 and HA-sp cells after 24 and 12h respectively. An upregulation of Prdx6 was also observed in A172 and HA-sp cells after 24h of H2O2 treatment as compared to untreated control. Our data also showed that curcumin inhibits oxidative stress-induced cytoskeleton disarrangement, and impedes the activation of astrocytes by inhibiting upregulation of GFAP, vimentin and Prdx6. In addition, we observed an inhibition of oxidative stress-induced inflammation, apoptosis and mitochondria fragmentation after curcumin treatment. Therefore, our results suggest that curcumin not only protects astrocytes from H2O2-induced oxidative stress but also reverses the mitochondrial damage and dysfunction induced by oxidative stress. This study also provides evidence for protective role of curcumin on astrocytes by showing its effects on attenuating reactive astrogliosis and inhibiting apoptosis.

Physical Intimacy of Breast Cancer Cells with Mesenchymal Stem Cells Elicits Trastuzumab Resistance through Src Activation.

The development of resistance to trastuzumab is a major obstacle for lasting effective treatment of patients with ErbB2-overexpressing tumors. Here, we demonstrate that the physical contact of breast cancer cells with mesenchymal stem cells (MSCs) is a potential modulator of trastuzumab response by activation of nonreceptor tyrosine kinase c-Src and down regulation of phosphatase and tensin homolog (PTEN). Using an in vitro patterned breast cancer/MSC co-culture model, we find that the presence of MSCs results in Src activation that is missing in cancer cells monoculture, transwell co-culture, and cells treated with MSCs conditioned media. Interestingly, the co-culture model also results in PTEN loss and activation of PI3K/AKT pathway that has been demonstrated as fundamental proliferative and survival pathways in clinical settings. To our knowledge, this is the first report that showed PTEN loss without the use of chemical inhibitors, matrix stiffness, or silencing RNAs. In addition, breast cancer cells in co-culture with MSCs conferred trastuzumab resistance in vitro as observed in the lack of inhibition of proliferative and migrative properties of the cancer cells. Our findings show that MSCs are potent mediators of resistance to trastuzumab and might reveal targets to enhance trastuzumab efficacy in patients.

Breast Cancer/Stromal Cells Coculture on Polyelectrolyte Films Emulates Tumor Stages and miRNA Profiles of Clinical Samples.

In this study, we demonstrate a method for controlling breast cancer cells adhesion on polyelectrolyte multilayer (PEM) films without the aid of adhesive proteins/ligands to study the role of tumor and stromal cell interaction on cancer biology. Numerous studies have explored engineering coculture of tumor and stromal cells predominantly using transwell coculture of stromal cells cultured onto coverslips that were subsequently added to tumor cell cultures. However, these systems imposed an artificial boundary that precluded cell-cell interactions. To our knowledge, this is the first demonstration of patterned coculture of tumor cells and stromal cells that captures the temporal changes in the miRNA signature as the breast tumor develops through various stages. In our study we used synthetic polymers, namely poly(diallyldimethylammonium chloride) (PDAC) and sulfonated poly(styrene) (SPS), as the polycation and polyanion, respectively, to build PEMs. Breast cancer cells attached and spread preferentially on SPS surfaces while stromal cells attached to both SPS and PDAC surfaces. SPS patterns were formed on PEM surfaces, by either capillary force lithography (CFL) of SPS onto PDAC surfaces or vice versa, to obtain patterns of breast cancer cells and patterned cocultures of breast cancer and stromal cells. In this study, we utilized cancer cells derived from two different tumor stages and two different stromal cells to effectively model a heterogeneous tumor microenvironment and emulate various tumor stages. The coculture model mimics the proliferative index (Ki67 expression) and tumor aggressiveness (HER-2 expression) akin to those observed in clinical tumor samples. We also demonstrated that our patterned coculture model captures the temporal changes in the miRNA-21 and miRNA-34 signature as the breast tumor develops through various stages. The engineered coculture platform lays groundwork toward precision medicine wherein patient-derived tumor cells can be incorporated within our in vitro models to identify potential pathways and drug treatment regimens for individual patients.

In vitro evaluation of PEGylated mesoporous MgFe2O4 magnetic nanoassemblies (MMNs) for chemo-thermal therapy.

A size tunable synthesis of mesoporous MgFe2O4 magnetic nanoassemblies (MMNs) through a PEG-diacid mediated polyol method is reported. The PEG-diacid coated MMNs exhibit a significant specific surface area of 92 m2 g-1 and saturation magnetization of 57 emu g-1. These MMNs exhibit a very good colloidal stability in PBS (pH 7.4) with nonappreciable cytotoxicity in mouse fibroblast (L929) and cervical cancer (HeLa) cells. We demonstrate the potential of MMNs as an integrated nanosystem for drug delivery and magnetic hyperthermia (MHT) through in vitro studies. 80% loading efficiency of doxorubicin (DOX) has been achieved due to the highly negative surface charge and mesoporous nature of MMNs. It is observed that 65-70% of HeLa cells undergo apoptosis through DNA fragmentation after 24 h of incubation with DOX loaded MMNs. MHT alone induces the death of 40-45% of cells, whereas the synergistic effect of a combination of DOX and MHT leads to the death of about 90% of cells. Our results show that MHT significantly increases the therapeutic efficacy of DOX to induce more apoptosis in cancer cells. Hence, a combination of MHT with chemotherapy makes MMNs a powerful multimodal system for synergistic chemo-thermal cancer therapy.

Expression of estrogen receptor co-regulators SRC-1, RIP140 and NCoR and their interaction with estrogen receptor in rat uterus, under the influence of ormeloxifene.

Ormeloxifene binds competitively to ERs and antagonizes estrogen-induced gene expression in the uterus. However its detailed molecular mechanisms are not well understood. Present study was aimed to examine the changes in expression pattern of co-regulatory proteins SRC-1 (co-activator), RIP140 and NCoR (co-repressors) and their interaction with ERalpha in rat uterus under the influence of ormeloxifene (Orm) and tamoxifen (Tam). Adult ovariectomized rats were treated with estradiol (E(2)) (5 microg/100g), or Orm or Tam (200 microg/100g, s.c.) alone or along with E(2), for 3 days. RT-PCR analysis of uterine RNA and immunoblotting of uterine extracts revealed that expression of SRC-1, RIP140 and NCoR was insensitive to E(2) or Orm or Tam treatment. Direct protein-protein interaction experiments using co-immunoprecipitation revealed that E(2)-induced the interaction of ERalpha with co-activator SRC-1. In rats given Orm alone or along with E(2), there was a significant reduction in E(2)-induced effect on ERalpha-SRC-1 interaction. In case of ERbeta and SRC-1, Orm reduced interaction only in the absence of E(2). Interaction of RIP140 or NCoR with ERalpha was found to be more in rats treated with Orm along with E(2) as compared to that in E(2)-treated rats whereas no such recruitment was found in Tam treated rats. Interaction of RIP140 with ERbeta was insensitive to Orm or Tam treatment whereas the interaction of NCoR with ERalpha and ERbeta was increased in Orm treated rats. Ormeloxifene also showed inhibitory effects on uterine ER-ERE binding and estrogen-induced expression of progesterone receptor. Taken together, these findings demonstrate that ormeloxifene antagonizes ERalpha-mediated transcription by inhibiting the recruitment of SRC-1 and inducing the recruitment of RIP140 and NCoR.

Modulation of AP-1 mediated estrogenic response by ormeloxifene in rat uterus.

Ormeloxifene is a selective estrogen receptor modulator that exerts antiestrogenic effects and thereby inhibits growth in uterus. The present study was undertaken to examine the AP-1 protein interaction with AP-1 enhancer DNA elements in rat uterus in vivo and in vitro with a view to explore the modulation of estrogen action mediated via alternative pathway under the influence of ormeloxifene (Orm). In addition, the changes in expression of c-fos and c-jun transcription factors and mRNA expression of growth factor (IGF-1) were investigated with a view to assess the AP-1 mediated transcription. Ovariectomizedoung adult rats were administered with estradiol-17beta (5 microg/100 g body weight) or Orm (200 microg/100 g body weight) or vehicle for 3 days and sacrificed on fourth day. Electrophoretic mobility shift assay using uterine nuclear fraction from various treatment groups demonstrated that Orm caused a significant reduction in E2 induced AP-1 DNA binding. In vitro study revealed that Orm promotes AP-1 complex formation whereas its 7-hydroxy derivative inhibits it significantly. Uterine expression of c-fos and c-jun was increased significantly in Orm treated rats as compared to vehicle treated rats. However, the expression of c-fos and c-jun was decreased in rats receiving Orm plus E2. Semi-quantitative RT-PCR analysis revealed that mRNA expression of IGF-1 was increased in E2 treated group as compared to control group whereas reduced expression was observed in Orm treated rats as compared to E2 treated rats. The uterine weight and IGF-1 mRNA showed similar pattern, indicating that IGF-1 is involved in regulation of uterine weight. These results indicate that 7-hydroxy ormeloxifene (an active metabolite of Orm) is a potent antagonist at AP-1 sites. It inhibits the function of AP-1 transcription factors rather than their expression as evident by downregulation of mRNA expression of AP-1 regulated gene IGF-1, thereby inhibits proliferation in rat uterus. Study suggested a non-classicalregulation of estrogen action on uterus by ormeloxifene.

Modulation of estrogen receptor transactivation and estrogen-induced gene expression by ormeloxifene-a triphenylethylene derivative.

The study was aimed to investigate the interaction of D,L-ormeloxifene (Orm), a triphenylethylene and its hydroxy derivative with estrogen receptor subtypes alpha and beta, its influence on ERE-driven transcriptional activation and progesterone receptor expression. In competitive binding experiments using human recombinant ERalpha and ERbeta, Orm showed interaction with both ER subtypes, with more selectivity and higher affinity towards ERalpha (8.8%) as compared to ERbeta (3%). In case of 7-hydroxy derivative, the relative binding affinity for both ERs was enhanced several folds. Orm showed lower Ki, i.e. higher affinity for ERalpha (250 nM) than for ERbeta (750 nM). It was observed that Orm promoted the formation of ER-ERE complexes in uterine tissue extract whereas its hydroxy derivative showed inhibitory effects. Transient co-transfection assay in COS-1 cells using ERE-luciferase reporter construct, revealed that Orm showed estrogenic response whereas its hydroxy-derivative was potent antiestrogen at ERalpha at transcription level. In immature rats, Orm (2 mg/kg) was associated with less increase in uterine weight and in luminal epithelial cell height than E2 or Tam. Orm also induced the expression of PR mRNA but the expression level was significantly less than estradiol treated group. These results suggest that ER-ERE complexes formed under the influence of 7-hydroxy Orm appear to be transcriptionally less effective hence antagonizing the E2-regulated gene expression in this target tissue.