In vivo Anti-Inflammatory and Immunomodulatory Activity of Soft Coral Nephthea sp. from Southeast Sulawesi.

<b>Background and Objective:</b> <i>Nephthea</i> sp., has various biological activities. The study on anti-inflammatory and immunomodulatory of <i>Nephthea</i> sp., from Southeast Sulawesi is still limited. Hence, this study aims to determine the content of secondary metabolite compounds and their pharmacological activities including anti-inflammatory and immunomodulatory. <b>Materials and Methods:</b> <i>Nephthea</i> sp., was collected from Saponda Island and extracted using ethyl acetate. The chemical contents were analyzed by a phytochemical screening test, anti-inflammatory activity by xylene-induced ear edema and immunomodulatory activity using macrophage phagocytic activity (SPA) in experimental animals. <b>Results:</b> The ethyl acetate extract of <i>Nephthea</i> sp., contains flavonoids and steroids. According to the result obtained, the ethyl acetate extract of <i>Nephthea</i> sp., exhibited anti-inflammatory and immunomodulatory activity <i>in vivo</i>. The EAN 0.2 demonstrated the highest potency and showed no significant difference compared to diclofenac sodium at a concentration of 0.15 mg mL¹ (p>0.05) with the highest percentage edema inhibition as in xylene-induced ear edema. In addition, EAN 0.2 exhibited a similar result in increasing SPA compared to control (p>0.05). Both assays showed significant differences with negative control in this study (p<0.05). <b>Conclusion:</b> Soft coral <i>Nephthea</i> sp., can be a potential candidate as an anti-inflammatory and immunomodulatory agent.

Safflower (Carthamus tinctorius Linn.) Inhibits Cell Proliferation and Induces Apoptotic in Breast Cancer Cell Lines T47D.

<b>Background and Objective:</b> Safflower (<i>Carthamus tinctorius</i> Linn.) is one of the medicinal plants that contain secondary metabolites that have the potential to as anti-cancer by inducing apoptosis. This study aims to determine the content of secondary metabolite compounds and the induction activity of apoptosis from ethanol extract of safflower in the T47D breast cancer cell line. <b>Materials and Methods:</b> Safflower was extracted using 96% ethanol and assayed for phytochemical screening, cytotoxic tests by cell counting kit-8 to determine inhibitory concentration and apoptosis induction activity by flow cytometry to determine the ability of samples induce the programmed cell cancer in death. The data collected was analyzed with the PRISM GraphPad version. <b>Results:</b> The ethanol extract of safflower contains flavonoid compounds, alkaloids, saponins, tannins and terpenoids. The results of the anticancer activity test showed an IC₅₀ value of 479 µg mL¹ and the best percentage of apoptosis at a concentration of 200 µg mL¹ was 16.61% at the beginning of apoptosis and 10.52% at the end of apoptosis. <b>Conclusion:</b> The safflower can be developed as a breast anticancer agent that works through the induction of apoptosis to improve the effectiveness of breast cancer treatment.

Formulation optimization of sterilized xanthones-loaded nanoemulgels and evaluation of their wound healing activities.

Xanthones (XTs) are bioactive compounds found in mangosteen trees (Garcinia mangostana Linn.). They are used as an active ingredient in various health products. However, there is a lack of data of their application in wound healing. In particular, the topical products of XTs for wound healing; they should be sterilized to minimize the risks of wound infection from contaminated microorganisms. This study thus aimed to optimize the formulation of sterilized XTs-loaded nanoemulgel (XTs-NE-G) and to investigate their wound healing activities. The XTs-NE-Gs were prepared by mixing various gels containing sodium alginate (Alg) and Pluronic F127 (F127) into a XTs-nanoemulsion (NE) concentrate according to the face-centered central composite design. The results showed that the optimized XTs-NE-G was A5-F3 containing 5% w/w Alg and 3% w/w F127. It enhanced the proliferation-, migration rates of skin fibroblasts (HFF-1 cells) with an optimal viscosity. After blending the XTs-NE concentrate and the gel that was previously sterilized by a membrane filtration and an autoclaving technique, respectively, the sterilized A5-F3 was obtained. The sterilized A5-F3 still had effective bioactivities towards the HFF-1 cells. It promoted re-epithelialization, collagen deposition and inflammation suppression in the mice' wounds. It could thus be accepted for further investigation in clinical studies.

Alpha-mangostin as an inhibitor of GSK3ß in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is a breast cancer subtype that does not express the estrogen receptor, the progesterone receptor, or the human epidermal growth factor receptor 2 and that is characterized by high invasiveness, high metastatic potential, and poor prognosis. TNBC lacks receptors and hence cannot be treated by using targeted therapies; as such, the therapeutic potential of Indonesian herbal plants against this disease is worth exploring. Herein, we explore the molecular docking and the molecular dynamics simulations of α-mangostin on glycogen synthase kinase 3ß (GSK3ß; PDB ID: 4ACC). Our findings reveal that α-mangostin has a weaker binding affinity to GSK3ß than the native ligand (-8.22 kcal/mol), while the latter binds to GSK3ß with a stronger binding affinity of -8.92 kcal/mol. According to the binding site analysis, the hydrogen bonds of the native ligand on Asp133 and Arg141, while α-mangostin only appeared to form a hydrogen bond on the enzyme's Asp133. On the other hand, α-mangostin shares similar docking sites with the native ligand (namely, Ile62, Phe67, Val70, and Thr138), thus leading to the conclusion that the native ligand and α-mangostin might share a similar molecular mechanism. The molecular dynamics simulation by using the molecular mechanics Poisson-Boltzmann and surface area (MM-PBSA) calculations' method shows that α-mangostin maintains a better affinity (with a value of ΔGTotal at -114.463 kJ/mol) as compared with the native ligand (with a respective ΔGTotal value of -75.158 kJ/mol). Our findings are suggestive of α-mangostin possessing a valuable potential as an anti-TNBC agent through GSK3ß inhibition.Communicated by Ramaswamy H. Sarma.

Signaling Pathways and Natural Compounds in Triple-Negative Breast Cancer Cell Line.

Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer, having a poor prognosis and rapid metastases. TNBC is characterized by the absence of estrogen, progesterone, and human epidermal growth receptor-2 (HER2) expressions and has a five-year survival rate. Compared to other breast cancer subtypes, TNBC patients only respond to conventional chemotherapies, and even then, with limited success. Shortages of chemotherapeutic medication can lead to resistance, pressured index therapy, non-selectivity, and severe adverse effects. Finding targeted treatments for TNBC is difficult owing to the various features of cancer. Hence, identifying the most effective molecular targets in TNBC pathogenesis is essential for predicting response to targeted therapies and preventing TNBC cell metastases. Nowadays, natural compounds have gained attention as TNBC treatments, and have offered new strategies for solving drug resistance. Here, we report a systematic review using the database from Pubmed, Science Direct, MDPI, BioScince, Springer, and Nature for articles screening from 2003 to 2022. This review analyzes relevant signaling pathways and the prospect of utilizing natural compounds as a therapeutic agent to improve TNBC treatments in the future.

Optimization of Production Parameters for Andrographolide-Loaded Nanoemulsion Preparation by Microfluidization and Evaluations of Its Bioactivities in Skin Cancer Cells and UVB Radiation-Exposed Skin.

Andrographolide (AG) is an active compound isolated from Andrographis paniculata (Family Acanthaceae). Although it possesses beneficial bioactivities to the skin, there is insufficient information of its applications for treatment of skin disorders due to low water solubility leading to complications in product development. To overcome the problem, an AG-loaded nanoemulsion (AG-NE) was formulated and prepared using a microfluidization technique. This study aimed to investigate the effect of pressure and the number of homogenization cycles (factors) on droplet size, polydispersity index and zeta potential of AG-NE (responses) and to determine the effect of AG-NE on skin cancer cells and UVB irradiation-induced skin disorders in rats. Relationships between factors versus responses obtained from the face-centered central composite design were described by quadratic models. The optimum value of parameters for the production of optimized AG-NE (Op-AG-NE) were 20,000 psi of pressure and 5 homogenization cycles. Op-AG-NE showed promising cytotoxicity effects on the human malignant melanoma- (A375 cells) and non-melanoma cells (A-431 cells) via apoptosis induction with a high selectivity index and also inhibited intracellular tyrosinase activity in the A375 cells. Op-AG-NE could reduce melanin index and healed UVB irradiation exposed skin. Op-AG-NE thus had potential for treatment of skin cancers and skin disorders from exposure to UVB radiation.

Suppression of Proinflammatory Cytokines by Etlingera alba (A.D.) Poulsen Rhizome Extract and Its Antibacterial Properties.

Etlingera alba is one of the Etlingera plants that has not been studied intensively. Plants that belong to the same genus have similar constituents and pharmacological activities. Thus, we aim to investigate the chemical composition and pharmacological activities, namely, anti-inflammatory and antibacterial properties, of E. alba rhizome extract (EA). The chemical constituent was detected using the test tube method. The inflammatory model rats were obtained by inducing them with 1% carrageenan, and their palm edema volume and cytokine levels, namely, IL-6, IL-12, and TNF-α, were measured. Antibacterial activity was performed with broth microdilution. The phytochemical screening of EA was detecting alkaloids, flavonoids, tannins, steroids, and phenols. The EA has anti-inflammatory activity by reducing the palms' edema volume and cytokine levels (IL-6, IL-12, and TNF-α), and the optimal concentration was 400 mg/kg body weight (BW). On the other hand, EA also exhibited antibacterial properties against E. coli and S. enterica. In conclusion, similar to other Etlingera plants, EA also demonstrates pharmacological activities, namely, anti-inflammatory and antibacterial properties.

12(S)-HETE induces lymph endothelial cell retraction in vitro by upregulation of SOX18.

Metastasising breast cancer cells communicate with adjacent lymph endothelia, intravasate and disseminate through lymphatic routes, colonise lymph nodes and finally metastasize to distant organs. Thus, understanding and blocking intravasation may attenuate the metastatic cascade at an early step. As a trigger factor, which causes the retraction of lymph endothelial cells (LECs) and opens entry ports for tumour cell intravasation, MDA-MB231 breast cancer cells secrete the pro-metastatic arachidonic acid metabolite, 12S-hydroxy-5Z,8Z,10E,14Z-eicosatetraenoic acid [12(S)-HETE]. In the current study, treatment of LECs with 12(S)-HETE upregulated the expression of the transcription factors SRY-related HMG-box 18 (SOX18) and prospero homeobox protein 1 (PROX1), which determine endothelial development. Thus, whether they have a role in LEC retraction was determined using a validated intravasation assay, small interfering RNA mediated knockdown of gene expression, and mRNA and protein expression analyses. Specific inhibition of SOX18 or PROX1 significantly attenuated in vitro intravasation of MDA-MB231 spheroids through the LEC barrier and 12(S)-HETE-triggered signals were transduced by the high and low affinity receptors, 12(S)-HETE receptor and leukotriene B4 receptor 2. In addition, the current findings indicate that there is crosstalk between SOX18 and nuclear factor κ-light-chain-enhancer of activated B cells, which was demonstrated to contribute to MDA-MB231/lymph endothelial intravasation. The present data demonstrate that the endothelial-specific and lymph endothelial-specific transcription factors SOX18 and PROX1 contribute to LEC retraction.

Apigenin and Luteolin Attenuate the Breaching of MDA-MB231 Breast Cancer Spheroids Through the Lymph Endothelial Barrier in Vitro.

Flavonoids, present in fruits, vegetables and traditional medicinal plants, show anticancer effects in experimental systems and are reportedly non-toxic. This is a favorable property for long term strategies for the attenuation of lymph node metastasis, which may effectively improve the prognostic states in breast cancer. Hence, we studied two flavonoids, apigenin and luteolin exhibiting strong bio-activity in various test systems in cancer research and are readily available on the market. This study has further advanced the mechanistic understanding of breast cancer intravasation through the lymphatic barrier. Apigenin and luteolin were tested in a three-dimensional (3-D) assay consisting of MDA-MB231 breast cancer spheroids and immortalized lymph endothelial cell (LEC) monolayers. The 3-D model faithfully resembles the intravasation of breast cancer emboli through the lymphatic vasculature. Western blot analysis, intracellular Ca2+ determination, EROD assay and siRNA transfection revealed insights into mechanisms of intravasation as well as the anti-intravasative outcome of flavonoid action. Both flavonoids suppressed pro-intravasative trigger factors in MDA-MB231 breast cancer cells, specifically MMP1 expression and CYP1A1 activity. A pro-intravasative contribution of FAK expression in LECs was established as FAK supported the retraction of the LEC monolayer upon contact with cancer cells thereby enabling them to cross the endothelial barrier. As mechanistic basis, MMP1 caused the phosphorylation (activation) of FAK at Tyr397 in LECs. Apigenin and luteolin prevented MMP1-induced FAK activation, but not constitutive FAK phosphorylation. Luteolin, unlike apigenin, inhibited MMP1-induced Ca2+ release. Free intracellular Ca2+ is a central signal amplifier triggering LEC retraction through activation of the mobility protein MLC2, thereby enhancing intravasation. FAK activity and Ca2+ levels did not correlate. This implicates that the pro-intravasative contribution of FAK and of Ca2+ release in LECs was independent of each other and explains the better anti-intravasative effects of luteolin in vitro. In specific formulations, flavonoid concentrations causing significant anti-intravasative effects, can certainly be achieved in vivo. As the therapeutic strategy has to be based on permanent flavonoid treatment both the beneficial and adverse effects have to be investigated in future studies.

Intravasation of SW620 colon cancer cell spheroids through the blood endothelial barrier is inhibited by clinical drugs and flavonoids in vitro.

Mechanisms how colorectal cancer (CRC) cells penetrate blood micro-vessel endothelia and metastasise is poorly understood. To study blood endothelial cell (BEC) barrier breaching by CRC emboli, an in vitro assay measuring BEC-free areas underneath SW620 cell spheroids, so called "circular chemorepellent induced defects" (CCIDs, appearing in consequence of endothelial retraction), was adapted and supported by Western blotting, EIA-, EROD- and luciferase reporter assays. Inhibition of ALOX12 or NF-κB in SW620 cells or BECs, respectively, caused attenuation of CCIDs. The FDA approved drugs vinpocetine [inhibiting ALOX12-dependent 12(S)-HETE synthesis], ketotifen [inhibiting NF-κB], carbamazepine and fenofibrate [inhibiting 12(S)-HETE and NF-κB] significantly attenuated CCID formation at low µM concentrations. In the 5-FU-resistant SW620-R/BEC model guanfacine, nifedipine and proadifen inhibited CCIDs stronger than in the naïve SW620/BEC model. This indicated that in SW620-R cells formerly silent (yet unidentified) genes became expressed and targetable by these drugs in course of resistance acquisition. Fenofibrate, and the flavonoids hispidulin and apigenin, which are present in medicinal plants, spices, herbs and fruits, attenuated CCID formation in both, naïve- and resistant models. As FDA-approved drugs and food-flavonoids inhibited established and acquired intravasative pathways and attenuated BEC barrier-breaching in vitro, this warrants testing of these compounds in CRC models in vivo.

Fenofibrate inhibits tumour intravasation by several independent mechanisms in a 3-dimensional co-culture model.

Lymph node metastasis of breast cancer is a clinical marker of poor prognosis. Yet, there exist no therapies targeting mechanisms of intravasation into lymphatics. Herein we report on an effect of the antidyslipidemic drug fenofibrate with vasoprotective activity, which attenuates breast cancer intravasation in vitro, and describe the potential mechanisms. To measure intravasation in a 3-dimensional co-culture model MDA-MB231 and MCF-7 breast cancer spheroids were placed on immortalised lymphendothelial cell (LEC) monolayers. This provokes the formation of circular chemorepellent induced defects (CCIDs) in the LEC barrier resembling entry ports for the intravasating tumour. Furthermore, the expression of adhesion molecules ICAM-1, CD31 and FAK was investigated in LECs by western blotting as well as cell-cell adhesion and NF-κB activity by respective assays. In MDA-MB231 cells the activity of CYP1A1 was measured by EROD assay. Fenofibrate inhibited CCID formation in the MDA-MB231/LEC- and MCF-7/LEC models and the activity of NF-κB, which in turn downregulated ICAM-1 in LECs and the adhesion of cancer cells to LECs. Furthermore, CD31 and the activity of FAK were inhibited. In MDA-MB231 cells, fenofibrate attenuated CYP1A1 activity. Combinations with other FDA-approved drugs, which reportedly inhibit different ion channels, attenuated CCID formation additively or synergistically. In summary, fenofibrate inhibited NF-κB and ICAM-1, and inactivated FAK, thereby attenuating tumour intravasation in vitro. A combination with other FDA-approved drugs further improved this effect. Our new concept may lead to a novel therapy for cancer patients.