Cytotoxicity Of Curcumin Research Articles

Precise Electrical Detection of Curcumin Cytotoxicity in Human Liver Cancer Cells

Curcumin is a polyphenol extracted from the roots of Curcuma plants that exerts potential anticancer effects. However, owing to its low toxicity, curcumin is known to be effective only at high doses. Here, we report the highly sensitive assessment of curcumin toxicity in human liver cancer cells, which was facilitated using a fabricated conductive platform and an electrochemical detection method. To identify the best platform for assessing cell viability, both electrochemical deposition time (0–150 s) and RGD peptide concentrations (0–0.1 mg/mL) were varied. Unlike other cell types, liver cancer cells showed the highest electrical signals without the modification of cell adhesion peptides. With 120 s of gold deposition under peptide-free conditions, 10,000 cells/chip were detectable within a linear range of 10,000–500,000 cells. Notably, even at low concentrations (e.g., 20 µM), curcumin could inhibit liver cancer cell-specific electrical signals by 45%, which would be undetectable in conventional colorimetric assay (CCK-8). The half-maximal inhibitory concentration (IC50) of curcumin for HepG2 cells was 23.63 µM, which indicates 3.71-fold higher sensitivity than that determined in the CCK-8 assay (IC50 = 87.88 µM). Hence, the developed conductive cell culture platform, in combination with the electrochemical detection method, could serve as a promising tool for detecting toxicity and/or anticancer activity of food-derived compounds in cancer/normal cells.

The Combination Treatment of Curcumin and Probucol Protects Chondrocytes from TNF-α Induced Inflammation by Enhancing Autophagy and Reducing Apoptosis via the PI3K-Akt-mTOR Pathway.

Osteoarthritis (OA) is a chronic joint disease characterized by cholesterol accumulation in chondrocytes, cartilage degeneration, as well as extracellular matrix (ECM) destruction, and joint dysfunction. Curcumin, a chemical that can reduce cholesterol levels in OA patients, also can inhibit the progression of OA. However, a high concentration of curcumin may also trigger apoptosis in normal chondrocytes. Besides curcumin, probucol that is found can also effectively decrease the cholesterol level in OA patients. Considering that high cholesterol is a risk factor of OA, it is speculated that the combination treatment of curcumin and probucol may be effective in the prevention of OA. To investigate the possible effects of such two chemicals on OA pathophysiology, chondrocyte apoptosis and autophagy behavior under inflammatory cytokine stress were studied, and specifically, the PI3K-Akt-mTOR signaling pathway was studied. Methods. Cell proliferation, colony formation, and EdU assay were performed to identify the cytotoxicity of curcumin and probucol on chondrocytes. Transwell assay was conducted to evaluate chondrocyte migration under TNF-α inflammation stress. Immunofluorescence, JC-1, flow cytometry, RT-PCR, and western blot were used to investigate the signal variations related to autophagy and apoptosis in chondrocytes and cartilage. A histological study was carried out on OA cartilage. Glycosaminoglycan (GAG) release was determined to evaluate the ECM degradation under stress. Results. Compared with a single intervention with curcumin or probucol, a combined treatment of these two chemicals is more effective in terms of protecting chondrocytes from stress injury induced by inflammatory cytokines. The promoted protection may be attributed to the inhibition of apoptosis and the blockage of the autophagy-related PI3K/Akt/mTOR pathway. Such results were also verified in vitro by immunofluorescence staining of OA chondrocytes and in vivo by immunohistochemistry staining of cartilage. Besides, in vivo studies also showed that when applied in combination, curcumin and probucol could block the PI3K-AKT-mTOR signaling pathway; promote COL-II expression; suppress P62, MMP-3, and MMP-13 expression; and inhibit TNF-α-stimulated cartilage degradation. Moreover, the combined medication could help reduce the release of ECM GAGs in OA cartilage and alleviate the severity of OA. Conclusion. A combined treatment of curcumin and probucol could be used to protect chondrocytes from inflammatory cytokine stress via inhibition of the autophagy-related PI3K/Akt/mTOR pathway both in vitro and in vivo, which might be of potential pharmaceutical value for OA prevention and therapy.

Preparation of hyaluronic acid-decorated mixed nanomicelles for targeted delivery of hydrophobic drugs to CD44-overexpressing cancer cells

Most of the employed methods for preparation of targeted nanoparticles containing hydrophobic herbal drugs have multiple surface modifications with time-consuming steps. The present research was aimed to develop a facile method for preparation of hyaluronic acid (HA)-decorated mixed nanomicelles loaded with curcumin (as a hydrophobic drug model) to provide an efficient drug delivery system for targeted therapy of breast cancer cells with high expression of CD44 receptor. To this end, curcumin was first encapsulated in the hydrophobic core of Pluronic F127/didecyldimethylammonium bromide (PD) mixed nanomicelles using thin-film hydration method. Then, negatively charged HA was coated on the positively charged surface of PD mixed nanomicelles via electrostatic interactions. The drug loading and entrapment efficiency of the targeted nanomicelles were 2.8% and 95.1%, respectively. The average hydrodynamic size of the prepared nanomicelles before and after coating with HA were 19.8 and 35.8 nm, respectively. Moreover, in vitro cytotoxicity analyses showed that, HA-coated PD (HA-PD) mixed nanomicelles can enhance the cytotoxicity of curcumin against MDA-MB-231 cancer cells compared to non-targeted ones (PD mixed nanomicelles), and free curcumin. The IC50 concentrations of free curcumin, curcumin-loaded PD mixed nanomicelles, and curcumin-loaded HA-PD mixed nanomicelles were 4.11, 3.20, and 2.83 μg/mL, respectively, after 48 h incubation with MDA-MB-231 cancer cells. Our results suggest that, curcumin-loaded HA-PD mixed nanomicelles may be considered as a promising targeted anticancer drug delivery system for breast cancer therapy and/or delivering other hydrophobic drugs to different kinds of cancer cells with CD44-receptor overexpression.

Antineoplastic effect of a novel nanosized curcumin on cutaneous T cell lymphoma.

Cutaneous T cell lymphomas (CTCLs) are a group of heterogeneous, life-threatening, extra-nodal and lymphoproliferative T cell neoplasms. Since chronic inflammation serves a key role in CTCL progression, curcumin, a natural pigment with proven anti-inflammatory and antineoplastic properties, as well as minimal toxicity, may be used as a therapeutic agent. In the present study, two formulations of curcumin (standard ethanolic and a Pluronic®P-123/F-127 micellar solution) were compared regarding their cytotoxic efficacy and speed of internalization in three CTCL cell lines, namely HuT-78, HH and MJ. In addition, the modulating effect of curcumin on selected proteins involved in the proliferation and progression of the disease was determined. The results indicated the superiority of the Pluronic®P-123/F-127 micellar curcumin over the standard ethanol solution in terms of cellular internalization efficiency as determined by spectrophotometric analysis. Notably, the presence of commonly used media components, such as phenol red, may interfere when interpreting the cytotoxicity of curcumin, due to their overlapping absorbance peaks. Therefore, it was concluded that phenol red-free media are superior over media with phenol red in order to correctly measure the cytotoxic efficacy and cell penetration of curcumin. Depending on the cell line, the IC50 values of micellar curcumin varied from 29.76 to 1.24 µΜ, with HH cells demonstrating the highest sensitivity. This cell line had the lowest expression levels of the Wilms' tumor-1 transcription factor. Performing western blot analyses of treated and untreated CTCL cells, selective signal transduction changes were recorded for the first time, thus making curcumin nano-formulation an attractive and prospective option with therapeutic relevance for CTCL as a rare orphan disease.

Curcumin and Photobiomodulation in Chronic Viral Hepatitis and Hepatocellular Carcinoma.

Immune modulation is a very modern medical field for targeting viral infections. In the race to develop the best immune modulator against viruses, curcumin, as a natural product, is inexpensive, without side effects, and can stimulate very well certain areas of the human immune system. As a bright yellow component of turmeric spice, curcumin has been the subject of thousands of scientific and clinical studies in recent decades to prove its powerful antioxidant properties and anticancer effects. Curcumin has been shown to influence inter- and intracellular signaling pathways, with direct effects on gene expression of the antioxidant proteins and those that regulate the immunity. Experimental studies have shown that curcumin modulates several enzyme systems, reduces nitrosative stress, increases the antioxidant capacity, and decreases the lipid peroxidation, protecting against fatty liver pathogenesis and fibrotic changes. Hepatitis B virus (HBV) affects millions of people worldwide, having sometimes a dramatic evolution to chronic aggressive infection, cirrhosis, and hepatocellular carcinoma. All up-to-date treatments are limited, there is still a gap in the scientific knowledge, and a sterilization cure may not yet be possible with the removal of both covalently closed circular DNA (cccDNA) and the embedded HBV DNA. With a maximum light absorption at 420 nm, the cytotoxicity of curcumin as photosensitizer could be expanded by the intravenous blue laser blood irradiation (IVBLBI) or photobiomodulation in patients with chronic hepatitis B infection, Hepatitis B e-antigen (HBeAg)-positive, noncirrhotic, but nonresponsive to classical therapy. Photobiomodulation increases DNA repair by the biosynthesis of complex molecules with antioxidant properties, the outset of repairing enzyme systems and new phospholipids for regenerating the cell membranes. UltraBioavailable Curcumin and blue laser photobiomodulation could suppress the virus and control better the disease by reducing inflammation/fibrosis and stopping the progression of chronic hepatitis, reversing fibrosis, and diminishing the progression of cirrhosis, and decreasing the incidence of hepatocellular carcinoma. Photodynamic therapy with blue light and curcumin opens new avenues for the effective prevention and cure of chronic liver infections and hepatocellular carcinoma. Blue laser light and UltraBioavailable Curcumin could be a new valuable alternative for medical applications in chronic B viral hepatitis and hepatocarcinoma, saving millions of lives.

Dual-functional liposomes for curcumin delivery and accelerating silk fibroin hydrogel formation.

The administration of a drug-loaded implantable hydrogel at the tumor site after surgical resection is a viable approach to prevent the local recurrence or metastasis. Dimyristoyl glycerophosphorylglycerol (DMPG)-based liposomes were developed for inducing the rapid gelation of silk fibroin (SF) and delivering an anticancer drug, curcumin. Curcumin was loaded in the liposomes and the stability of curcumin was enhanced. The gelation time of liposome-induced SF hydrogels ranged from 3min to more than 6h. The biological activity of liposome-SF hydrogels was evaluated in vitro using L929 fibroblasts and MDA-MB-231 breast cancer cells. The release of curcumin can inhibit the growth of cancer cells. Both cells cultured on the surface of the hydrogels loaded with curcumin displayed low cell survival due to the combination of low cell attachment and cytotoxicity of curcumin. Liposome-SF hydrogels show potential as a sealant administered at the tumor site to eliminate residual cancer cells after tumor removal.

Abstract 5276: Secretory pathway calcium ATPase 2 (SPCA2) promotes cell survival and chemoresistance in receptor positive breast cancer cells

Abstract Introduction: Dysregulation of the Ca2+ toolkit has profound consequences for tumor growth and metastasis, raising hopes for novel avenues of therapeutic intervention. The Secretory Pathway Ca2+-ATPase Isoform 2 (SPCA2) has a dual function in sequestering Ca2+ and Mn2+ into secretory stores as well as eliciting their entry into cells by activating plasma membrane ion channels. We have previously showed that SPCA2 is implicated in breast cancer progression, microcalcifications, epithelial-mesenchymal transition, and metastasis. Methods and Results: High SPCA2 expression was associated with poor overall survival in receptor positive breast cancer patients (n=548, P =8.7e-0.6; KM Plotter). Depletion of SPCA2 (SPCA2 KD) in MCF-7 cells significantly reduced cell proliferation (P < 0.05). Analysis of the cell cycle (PI staining, Flow cytometry) revealed a significant increase (P < 0.05) in the number of cells in the G0/G1 phase, and significant increase in apoptosis (via TUNEL assay) (P < 0.05) in SPCA2 KD cells compared to control. Microarray analysis showed the p53 pathway is the most significantly upregulated pathway (P < 0.05) in SPCA2 KD cells compared to control. P53 knockout abrogated SPCA2 mediated cell cycle arrest and apoptosis. Depletion of SPCA2 increased reactive oxygen species (ROS, measured by DCFDA dye and MitoSox Red using flow cytometry) leading to DNA damage (confocal microscopy), and activation of ATM/ATR pathway (Immunoblotting, P < 0.05). As a consequence, SPCA2 KD cells showed specific hypersensitivity to DNA damaging agents used in breast cancer therapy (carboplatin, radiation, and doxorubicin) (determined by MTT assay) (P < 0.05). Thus SPCA2 is a novel molecular target in breast cancer therapy. Curcumin is currently being evaluated in clinical trials of breast cancer. We show that curcumin causes dose-dependence inhibition of SPCA2 expression (P < 0.05) in multiple receptor-positive cell lines, and that forced overexpression of SPCA2 decreased curcumin cytotoxicity. Interestingly, we did not observe these results with SPCA1, the housekeeping SPCA isoform, revealing isoform-specific function of Golgi calcium pumps in breast cancer subtypes. Conclusion: These novel findings point to a causal link between SPCA2, breast cancer progression, and chemoresistance to DNA damaging agents. Decrease of SPCA2 expression by curcumin may have therapeutic potential in treating receptor positive breast cancer. Citation Format: Monish Ram Makena, Donna Dang, Allatah Mekile, Phillip Buckhaults, Rajini Rao. Secretory pathway calcium ATPase 2 (SPCA2) promotes cell survival and chemoresistance in receptor positive breast cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5276.

Curcumin encapsulated colloidal amphiphilic block co-polymeric nanocapsules: colloidal nanocapsules enhance photodynamic and anticancer activities of curcumin.

Curcumin-based novel colloidal nanocapsules were prepared from amphiphilic poly(ethylene oxide)-block-poly(propylene oxide)-block-poly(ethylene oxide) (F108). These colloidal nanocapsules appeared as spherical particles with size ranging between 270 and 310 nm. Curcumin fluorescence spectra exhibited an aggregation-induced 23 nm red-shift of the emission maximum in addition to the enhancement of the fluorescence quantum yield in these nanocapsules. The cytotoxicity of curcumin and colloidal nanocapsules was assessed using human derived immortalized cell lines (A549 and A375 cells) in the presence and absence of light irradiation. The nanocapsules exhibited a >30-fold decrease in IC50, suggesting enhanced anticancer activity associated with curcumin encapsulation. Higher toxicity was also reported in the presence of light irradiation (as shown by the IC50 data), indicating their potential for future application in photodynamic therapy. Finally, A375 cells treated with curcumin and the nanocapsules showed a significant increase in single- and/or double-strand DNA breaks upon exposure to light, indicating promising biological effects.

The Role of Curcumin on Apoptosis through The RASSF1A and Bax Pathways in Breast Cancer

Curcumin has been reported with an in vitro the cytotoxic effect on several human cancer cells. However, reports on the mode of action and detail mechanism of curcumin in breast cancer disease are limited. Hence, curcumin’s effect on the human breast cancer cell line MCF-7 and MDA-MB-468 was investigated. The MCF-7 and MDA-MB-468 breast cancer cells line were given curcumin in several doses. The anti-proliferation activity of curcumin was determined using the MTS cell viability test and caspase-3 activity was used to detect apoptosis using flowcytometry. The expression of Ras-association domain family 1 isoform A (RASSF1A) and Bax protein in cells was evaluated by ELISA analysis. Kruskal-Wallis followed by the Mann-Whitney test and the Spearman correlation tests were used to asses correlation among RASSF1A, Bax, and caspase-3. Cytotoxicity of curcumin on MCF-7 was lower than that of MDA-MB-468 (75.73 μg/mL and 380.79 μg/mL). The concentration of curcumin at 80 μg/mL induced apoptosis mainly through the intrinsic pathway by caspase-3 activation. Curcumin also showed an anti-proliferative activity as shown by the increase of RASSF1A and Bax protein. Curcumin mediates anti-proliferative and apoptotic effect through the activation of RASSF1A and Bax. Our research data adds information about the role of curcumin in epigenetic events through RASSF1A protein.Keywords: Bax, caspase-3, curcumin, MCF-7, MDA-MB-468, RASSF1A

Cytotoxicity evaluation of curcumin-loaded affibody-decorated liposomes against breast cancerous cell lines

Curcumin is known as an effective anticancer herbal medicine but unfortunately, its bioavailability is poor which necessitate efforts for developing more efficient and specific delivery systems. Human epidermal growth factor receptor 2 (HER 2) due to its overexpression in various types of cancers, is demonstrated to be a good candidate as a target for anticancer therapy. In this study, cytotoxicity of curcumin encapsulated in ZHER2:342 Affibody-decorated liposome was investigated against SKBR3 and MCF-7 cancerous cell lines. Curcumin-containing liposomes were prepared from soybeans lecetin and cholesterol by thin-film hydration method. Affibody ZHER2:342 molecules via C-terminal cysteine residue were conjugated covalently to the prepared liposomes. Particle size analysis was performed using atomic force microscopy (AFM) and dynamic light scattering (DLS). Curcumin loading was measured using UV-Vis spectrophotometry and cytotoxic activity of curcumin formulations against cancerous cell lines was investigated by MTT assay. Induction of apoptosis was investigated using flow cytometry through Annexin V staining. Particle analysis showed the formation of spherical liposomes with a mean diameter of about 150 nm. Cytotoxic activity of curcumin was improved by its encapsulation in both liposomes and affibody-decorated liposomes. The Annexin V staining indicated the induction of apoptosis by affibody-decorated liposomes in both MCF-7 and SKBR3 cells. Decoration of curcumin-loaded liposomes with affibody ZHER2:342 may improve curcumin apoptotic function independently of HER2 expression level.

Self-assembled formation of chondroitin sulfate-based micellar nanogel for curcumin delivery to breast cancer cells

Nanogel based drug delivery systems have been broadly used for cancer treatment. In this research, octadecylamine was grafted to chondroitin sulfate using three different mole ratios (10, 20, and 30) and named CS-ODA1, 2, and 3, respectively. The amide bond formation between chondroitin sulfate and octadecylamine was confirmed by 1H-nuclear magnetic resonance (HNMR) in the CS-ODA3 sample; therefore, further analysis was performed on this sample. Curcumin was loaded at defined Cur/CS-ODA ratios (5, 10 and 15%) and CS-ODA3 with 10% curcumin was selected for further experiments due to more entrapment efficiency (79.56% ± 5.56). In vitro release profile of the curcumin loaded nanogels showed >80% release after 70 h. In addition, the results of MTT analysis on the MCF-7 cell line showed almost no toxicity toward blank nanogels, while curcumin-loaded nanogels induced significant death after 24 h. In the end, analysis of the cell cycle using MCF-7 cells also confirmed the cytotoxicity of curcumin loaded nanogels. This study also showed that the presence of curcumin loaded chondroitin sulfate nanogels could successfully increase cellular uptake in comparison with free curcumin. The synthesized nanogels containing curcumin are expected to be effective for further studies in cancer treatment.

Nanoceria-curcumin conjugate: Synthesis and selective cytotoxicity against cancer cells under oxidative stress conditions

A water- and alcohol-soluble cerium oxide-curcumin conjugate was obtained by co-evaporation with poly(N-vinylpyrrolidone) (PVP). A nanocomposite consisting of hybrid organic-inorganic particles was stable in a wide range of pH values. Its properties were evaluated using nine cell lines: normal (MDBK, ST, Vero) and malignant (L929, T98G, HEp-2, A549, RIN-m5F, Hep G2). PVP-stabilised nanoceria was shown to inhibit autoxidation of curcumin, to enhance curcumin photostability, to promote bioaccumulation and to affect curcumin cytotoxicity and photocytotoxicity, depending on cell type, being more toxic to cancer cells in a selective manner. Under the conditions of UVA/UVC or H2O2-induced oxidative stress, the nanoceria-PVP-curcumin (NPC) conjugate was found to possess a selective cytotoxicity: it caused drastic inhibition of metabolic activity or a decrease in the total number of tumour cells, while in non-transformed cultures under the same conditions, the nanoceria-PVP-curcumin conjugate protected cells from these damaging factors. The NPC-conjugate, unlike curcumin itself, demonstrated a photosensitising effect in tumour cell cultures, while protecting non-transformed cultures from the damaging effects of UV radiation or oxidative stress. Based on the results obtained, we strongly believe that this novel hybrid material has enhanced characteristics compared to other curcumin formulations, and can be considered as a potent drug for biomedical applications, including cancer therapy.

Curcumin Encapsulated Lecithin Nanoemulsions: An Oral Platform for Ultrasound Mediated Spatiotemporal Delivery of Curcumin to the Tumor

Systemic toxicity caused by conventional chemotherapy is often regarded as one of the major challenges in the treatment of cancer. Over years, the trigger-based modality has gained much attention as it holds the spatiotemporal control over release and internalization of the drug. In this article, we are reporting an increase in the anti-tumor efficacy of curcumin due to ultrasound pulses. MDA MB 231 breast cancer and B16F10 melanoma cells were incubated with lecithin-based curcumin encapsulated nanoemulsions and exposed to ultrasound in the presence and absence of microbubble. Ultrasound induced sonoporation enhanced the cytotoxicity of curcumin in MDA MB 231 and B16F10 cancer cells in the presence of microbubble by 100- and 64-fold, respectively. To study the spatiotemporal delivery of curcumin, we developed B16F10 melanoma subcutaneous tumor on both the flanks of C57BL/6 mice but only the right tumor was exposed to ultrasound. Insonation of the right tumor spatially enhanced the cytotoxicity and enabled the substantial regression of the right tumor compared to the unexposed left tumor which grew continuously in size. This study showed that the ultrasound has the potential to target and increase the drug’s throughput to the tumor and enable effective treatment.

Cytotoxicity of curcumin derivatives in ALK positive non-small cell lung cancer

Non-small cell lung cancer with ALK rearrangements can be targeted effectively with ALK inhibitors such as crizotinib. However, cancer progression typically occurs within a year as drug resistance develops. One strategy to overcome this drug resistance is to determine if novel cytotoxic agents retain the ability to kill lung cancer cells that have developed ALK inhibitor resistance. We therefore examined curcumin, a drug with anticancer properties, and 2 s-generation curcumin derivatives (1-methyl-3,5-bis[(E)-4-pyridyl) methylidene]-4-piperidone (RL66) and 1-isopropyl-3,5-bis[(pyridine-3-yl) methylene]piperidin-4-one (RL118)) in lung cancer cell lines. The cytotoxicity of curcumin, RL66, and RL118 were tested in both ALK+ lung cancer cells (H3122), crizotinib resistant ALK+ cells (CR-H3122) and ALK− lung cancer cells (A549), both alone and in combination with crizotinib. ALK+ cells were 2-3x more sensitive to RL66 and RL118 than ALK− cells, with the drugs’ eliciting IC50 values in the range of 0.7–1 μM in H3122 cells. Retained cytotoxic potency of the curcumin derivatives in crizotinib resistant cells indicated that mechanisms of resistance to the two drug types are independent, with resistance to ALK inhibitors not necessarily causing cross-resistance to curcumin derivatives. This was further corroborated by drug combination analysis where the effect of the drugs in combination was consistent with Bliss additivity, consistent with independent targets for crizotinib and curcumin derivatives. Results from Western blotting showed that RL118 (2 μM) inhibited p-ALK/ALK by ~50%, which was not as potent as the 90% inhibition elicited by crizotinib (0.25 μM). Since this is the primary mechanism of crizotinib cytotoxicity this provides further evidence of independent mechanisms of toxicity.

Curcumin inhibits LPS-induced neuroinflammation by promoting microglial M2 polarization via TREM2/ TLR4/ NF-κB pathways in BV2 cells

Microglia mediate multiple facets of neuroinflammation, which plays a double-edged role in various brain diseases via distinct microglial phenotypes (deleterious M1 and neuroprotective M2). Therefore, the inhibition of overactivated inflammatory M1 microglia by switching to the protective M2 phenotype appears to be a potential therapeutic strategy in neuroinflammatory disorders. Curcumin has been shown to exhibit anti-inflammatory and neuroprotective activities. The present study investigated the potential effects of curcumin on microglial M1/M2 polarization and elucidated the possible molecular mechanisms of action in vitro. In this study, the BV2 microglial cell line was pretreated with different curcumin concentrations in the presence or absence of lipopolysaccharide (LPS) to assess the anti-inflammatory efficacy of curcumin based on the morphological and inflammatory changes. The cytotoxicity of curcumin for BV2 cells was evaluated using the CCK-8 assay. Further, the effect of curcumin concentrations on LPS-induced BV2 cells was studied. The morphological changes were observed using an optical microscope and immunofluorescent staining. Nitric oxide (NO) expression was determined using the Griess reagent. The expression of cytokines and inflammatory mediators was also measured by ELISA, qRT-PCR, flow cytometry, and immunofluorescence. Western blot analysis was used to determine the levels of triggering receptor expressed on myeloid cells 2 (TREM2), toll-like receptor 4 (TLR4), nuclear factor-kappa B (NF-κB) p65, p-NF-κB p65, IκB, and p-IκB expression. Results showed that curcumin concentrations less than 10 μM did not induce any detectable cytotoxicity but decreased BV2 cell viability up to 20 μM. Curcumin inhibited LPS-induced microglial activation. Curcumin treatment switched the M1 pro-inflammatory phenotype to the M2 anti-inflammatory phenotype by decreasing the expression of M1 markers (i.e., iNOS, IL-1β, IL-6, and CD16/32) and elevating the expression of M2 markers (i.e., arginase 1, IL-4, IL-10, and CD206). Interestingly, curcumin attenuated the activation of TLR4/NF-κB pathways and the downregulation of TREM2 expression in LPS-activated BV2 cells. Collectively, these results suggest that curcumin significantly alleviates LPS-induced inflammation by regulating microglial (M1/M2) polarization by reducing the imbalance of TREM2 and TLR4 and balancing the downstream NF-κB activation.

PH‐Sensitive Poly(ethylene glycol)/Poly(ethoxyethyl glycidyl ether) Block Copolymers: Synthesis, Characterization, Encapsulation, and Delivery of a Hydrophobic Drug

AbstractCurcumin is a natural polyphenolic compound known for its numerous pharmacological properties. However, its low water solubility and instability at neutral pH are serious drawbacks preventing its use as an oral drug. Well‐defined amphiphilic poly(ethylene glycol)‐block‐poly(ethoxyethyl glycidyl ether) (PEG‐b‐PEEGE) block copolymers carrying acid‐labile acetal groups are synthesized by anionic ring‐opening polymerization and investigated as potential pH‐sensitive nano‐carriers for delivery of curcumin to cancer cells. The nanoparticles, resulting from copolymer self‐assembly in aqueous media, are characterized by dynamic light scattering and cryo‐transmission electron microscopy. The nanoparticles’ stabilities are evaluated in three different phosphate buffers (pH = 7.2, 6.4, and 5.3). The stability decreases at lower pH and a complete disappearance of the nanoparticles is noticed after 4 days at pH 5.3. Curcumin is encapsulated in hydrophobic core of mPEG40‐b‐PEEGE25 nanoparticles allowing significant enhancements of curcumin solubility in water and lifetime at neutral pH. In vitro curcumin release is studied at different pH by UV‐spectroscopy and high‐performance liquid chromatography (HPLC). The cytotoxicity of curcumin and curcumin encapsulated in micelles is evaluated by cell viability 3‐(4,5‐Dimethyl‐2‐thiazolyl)‐2,5‐diphenyl‐2H‐tetrazolium bromide (MTT) assay on MDA‐MB‐231 human breast cancer cells.

Inhibition of TLR4/TRIF/IRF3 Signaling Pathway by Curcumin in Breast Cancer Cells.

Toll-like receptor 4 (TLR4) is over-expressed in breast tumors and thus contributing to the tumor progression and metastasis. Natural products have drawn attention in cancer immunotherapy due to their various biological activities. Curcumin is well investigated in different types of cancer. However, the mechanisms underlying its anti-inflammatory actions have not been extensively elucidated. For this purpose, we explored the inhibitory effects of curcumin on lipopolysaccharide (LPS)-induced TLR4 dependent TRIF signaling pathway in two subtypes of breast cancer cell lines (MCF-7 and MDA-MB-231) in this study. In this context, the cytotoxicity of curcumin and LPS alone and the combination of curcumin with LPS on these cells was evaluated by WST-1 assay. The expression level of TLR4 and the release of type I interferon (IFN) levels were determined after treatment with curcumin and/or LPS by RT-PCR and ELISA analysis, respectively. Furthermore, the subcellular localization of TLR4 and interferon regulatory factor 3 (IRF3) were detected by immunofluorescence analysis. Curcumin treatment suppressed breast cancer cells viabilities and the activation of TLR4-mediated TRIF signaling pathway by the downregulation of TLR4 and IRF3 expression levels and the inhibition of type I IFN (IFN-α/β) levels induced by LPS. However, curcumin was more efficient in MDA- MB-231 cells than MCF-7 cells owing to its greater inhibitory efficacy in the LPS- enhanced TLR4 signaling pathway. Furthermore, IFN-α/β levels induced by TLR4 and IRF3 were decreased in these cells following curcumin treatment. Consequently, these results demonstrated that the activation of LPS stimulated TLR4/TRIF/IRF3 signaling pathway was mediated by curcumin in breast cancer cells, in vitro. However, more studies are necessary to examine the curcumin's anti-inflammatory activities on TLR4/MyD88/NF-κB as well as other signaling pathways downstream of TLRs in breast cancer.

An In Vivo Study for Targeted Delivery of Curcumin in Human Triple Negative Breast Carcinoma Cells Using Biocompatible PLGA Microspheres Conjugated with Folic Acid.

Among the different types of polymeric vehicles, (PLGA) is biodegradable and has emerged as promising tool for the delivery of cancer therapeutics. The salient features of PLGA micro carriers include prolonged circulation time, increased tumor localization and biodegradability and effectiveness of the therapeutics. We have synthesized PLGA microspheres where curcumin can be loaded and thereby increases its bioavailability. The cytotoxicity of curcumin (PLGA@CCM) microspheres was evaluated on triple negative breast cancer (TNBC) cell lines. They were found to induce apoptosis by perturbing the mitochondrial membrane potential. PLGA@CCM@FA induces apoptosis in human triple negative breast cancer cells by up-regulating Cleaved caspase-3 and down regutes p-AKT. The in-vivo study in BALB/C mice model exhibited more tumor regression in case of PLGA@CCM@FA microspheres. Our results suggests that these microspheres can be an effective vehicle for delivery of hydrophobic drugs to the folate over expressed cancer cells.

Curcumin-Loaded Mesoporous Silica Nanoparticles Markedly Enhanced Cytotoxicity in Hepatocellular Carcinoma Cells.

Curcumin, a natural polyphenol extracted from a perennial herb Curcuma longa has been verified for many physiological activities such as anti-oxidant, anti-inflammatory, and anti-tumor properties. The direct use of curcumin cytotoxicity studies are limited due to its unstable chemical structure, low bioavailability, easy oxidation, and degradation by ultraviolet (UV) light etc. Trying to overcome this problem, silica-encapsulated curcumin nanoparticles (SCNP) and chitosan with silica co-encapsulated curcumin nanoparticles (CSCNP) were prepared by silicification and biosilicification methods, respectively, and encapsulated curcumin within it. We investigated the antitumor properties of SCNP and CSCNP on different tumor cell lines. Scanning electron microscopy (SEM) analysis revealed that both SCNP and CSCNP were almost spherical in shape and the average particle size of CSCNP was 75.0 ± 14.62 nm, and SCNP was 61.7 ± 23.04 nm. The results show that CSCNP has more anti-oxidant activity as compared to curcumin and SCNP. The higher cytotoxicity towards different cancerous cell lines was also observed in CSCNP treated tumor cells. It was noted that the SCNP and CSCNP has a high percentage of IC50 values in Hep G2 cells. The encapsulation of curcumin improved instability, antioxidant activity, and antitumor activity. Our results demonstrated that nanoencapsulation of curcumin with silica and chitosan not only increase curcumin stability but also enhance its cytotoxic activity on hepatocellular carcinoma cells. On the basis of these primary studies, the curcumin-loaded nanoparticles appear to be promising as an innovative therapeutic material for the treatment of tumors.

Interactive anticancer effect of nanomicellar curcumin and galbanic acid combination therapy with some common chemotherapeutics in colon carcinoma cells.

In the current investigation, we aimed to study the combined cytotoxicity of curcumin, as a nanomicellar formulation, and galbanic acid (Gal), dissolved in DMSO against the murine C26 and human Caco-2 colon carcinoma cells. Further, curcumin potential for cisplatin and doxorubicin (Dox) co-therapy was studied. The combined cytotoxic effect of these phytochemicals at varying dose ratios were examined using the MTT colorimetric assay. Moreover, the time-dependent toxicity of curcumin, cisplatin, Dox, and pegylated liposomal Dox (Doxil) was determined. The interactive anti-proliferative behavior of these compounds was examined using the CompuSyn software. Nanomicellar curcumin showed considerable cytotoxicity in C26 cells 24 hr post-treatment. Co-treatment of cells with curcumin nanomicelles: Gal had a synergistic effect in C26 (at 10:1 molar ratio), and Caco-2 (at 1:5 molar ratio) cell lines in cell cultures. Nanomicellar curcumin showed strong and mild synergistic inhibitory effects in C26 cells when co-administered with Doxil and cisplatin, respectively. Curcumin nanomicelles and Gal had a synergistic effect in C26 and Caco-2 cell lines. It is speculated that nanomicellar curcumin shows synergistic cancer cell killing if administered 24-hr post-injection of Doxil and cisplatin.