Combination of Autophagy and Stem Cell Enhancing Properties of Natural Product Extracts in Human Dermal Papilla Stem Cells.

BACKGROUND/AIM: Dermal papilla (DP) stem cells are known for their remarkable regenerative capacity, making them a valuable model for assessing the effects of natural products on cellular processes, including stemness, and autophagy. MATERIALS AND METHODS: Autophagy and stemness characteristics were assessed using real-time RT-PCR to analyze mRNA levels, along with immunofluorescence and western blot techniques for protein level evaluation. RESULTS: Butterfly Pea, Emblica Fruits, Kaffir Lime, and Thunbergia Laurifolia extracts induced autophagy in DP cells. Kaffir Lime-treated cells exhibited increase in the OCT4, NANOG, and SOX2 mRNA (6-, 5, and 5.5-fold, respectively), and protein levels (4-, 3-, and 1.5-fold, respectively). All extracts activated the survival protein kinase B (Akt) in DP cells. CONCLUSION: Natural products are a promising source for promoting hair growth by rejuvenating hair stem cells.

Effects of collagen, chitosan and mixture on fibroblast responses and angiogenic activities in 2D and 3D in vitro models.

Despite accumulating evidences have demonstrated the potential of collagen and chitosan on tissue repair, it remains unclear on their combination effects. Here, we examined the regenerative effects of single collagen, chitosan and their mixture on fibroblasts and endothelial cells at cellular levels. The results showed that fibroblast responses, as indicated by high proliferative rate, increased spheroid diameter and migrated area existing from spheroid edge, and decreased wound area, were significantly promoted by either collagen or chitosan stimulation. Similarly, both collagen and chitosan resulted in increased endothelial cell proliferation and migration with accelerated tube-like network formation and upregulated VE-cadherin expression, although collagen strongly provided this effect. While the 1:1 mixture (100:100 µg/mL of chitosan to collagen) treatment caused a reduction in fibroblast viability, the lower ratio of chitosan (1:10 mixture; 10:100 µg/mL) did not produce any impact on both fibroblast and endothelial cell viabilities. The 1:10 mixture also significantly enhanced the additional effects on fibroblast responses and angiogenic activities as shown by higher endothelial growth, proliferation and migration with accelerated capillary-like network formation than those treated with the single substance. Further investigation of signaling proteins found that collagen significantly increased expressions of p-Fak, p-Akt and Cdk5 whereas chitosan upregulated p-Fak and Cdk5 expressions. Comparing to the single treatments, p-Fak, p-Akt and Cdk5 were higher expressed in the 1:10 mixture. These observations indicate that proper collagen-chitosan mixture provides the combination effects on fibroblast responses and angiogenic activities when a high concentration of collagen is used, possibly through Fak/Akt and Cdk5 signaling pathways. Therefore, this study helps to define the clinical use of collagen and chitosan as promising biomaterials for tissue repair.

Cordycepin attenuates migration and invasion of HSC-4 oral squamous carcinoma cells through autophagy-dependent FAK/Akt and MMP2/MMP9 suppression.

Background/purpose: Cordycepin has been proposed anti-cancer effects, however, it is unclear whether and how cordycepin affects oral squamous carcinoma cell (OSCC) migration and invasion. This study aimed to investigate the effect of cordycepin on migration and invasion of OSCC (HSC-4 cells), and its underlying mechanism. Materials and methods: Cell viability was measured with MTT assay. Migrative and invasive abilities were determined by scratch wound healing, agarose spot and transwell invasion assays, respectively. Monodasylcadaverine (MDC) staining, immunofluorescence staining of LC3 and RT-PCR evaluated the gene expression of LC3 and p62 were applied to investigate autophagy. MMP2 and MMP9 gene expression and activity were examined by RT-PCR and gelatin zymography. Expression of caspase 3, cleaved caspase 3, FAK, p-FAK, Akt and p-Akt was determined by Western blot. Results: Cordycepin significantly inhibited HSC-4 cell migration and invasion in a concentration-dependent manner. Cordycepin treatment caused an induction of autophagy, as evidenced by increased MDC fluorescence intensity and MDC positive cells, and upregulated expression level of LC3 gene. In addition, inhibition of autophagy by chloroquine (CQ) significantly abolished cordycepin-inhibited HSC-4 cell migration and invasion, demonstrating that cordycepin-inhibited migration and invasion was mediated by autophagy. Mechanistic studies showed that cordycepin significantly suppressed FAK and Akt phosphorylation, and MMP2 and MMP9 activities. Conversely, CQ pre-incubation significantly restored its expression and activity in cordycepin-treated cells. Conclusion: Cordycepin induces autophagy to suppress FAK and Akt phosphorylation, and MMP2 and MMP9 activity, which responsible for the attenuation of HSC-4 cell migration and invasion.

Investigation of mechanisms underlying the inhibitory effects of metformin against proliferation and growth of neuroblastoma SH-SY5Y cells.

Besides being anti-diabetic drug, metformin also has anti-proliferation and growth in several tumors; however, details of possible mechanism have not been elucidated. Here, we investigated the effects of metformin in neuroblastoma which has been termed as extra-cranial solid tumor that is due to a differentiation block with more stemness. The results showed that 5 mM metformin inhibited cell cycle progression at G0/G1 phase. Metformin also induced morphological differentiation of neuroblastoma into neuron-like phenotypes by which upregulation of MAP2, ß-tubulin III and tyrosine hydroxylase expressions with no significant difference to retinoic acid (RA)-treated cells. We also tested proliferative, growth and self-renewal ability after neuroblastoma being differentiated by metformin for 24 h. The proliferative rate, sizes and numbers of colonies and spheroids were significantly reduced in differentiated neuroblastoma compared to undifferentiated neuroblastoma. A significant increase of ROS and ADP/ATP ratio with decreased mitochondrial membrane potential (MMP) were observed in metformin-treated cells, indicating mitochondrial biogenesis and metabolic change during metformin-mediated differentiation. The further studies exhibited that p-Erk1/2 and Cdk5 levels were reduced in metformin treatment whereas using PD98095 and roscovitine, selectively inhibited Erk1/2 and Cdk5, respectively, significantly increased neurite length and MAP2 expression. In addition, cell proliferation was decreased by cell cycle arrested at G0/G1 phase. Taken together, this study suggests the inhibitory effects of metformin against proliferation and growth of neuroblastoma due to induced morphological differentiation may be through Erk1/2 and Cdk5 pathways. Therefore, metformin might be eventually considered as a differentiation agent for neuroblastoma treatment in term of differentiation therapy.

Effects of sonication and ultrasound on properties and bioactivities of liposomes loaded with hydrolyzed collagen from defatted sea bass skin conjugated with epigallocatechin gallate.

Hydrolyzed collagen (HC) from defatted sea bass skin conjugated with 3% epigallocatechin gallate (EGCG) was prepared and the resulting HC-EGCG conjugate at various levels (0.25%-2%, w/v) was loaded into liposome. The obtained liposomes were subjected to sonication (S). Liposome loaded with 1% conjugate showed the highest encapsulation efficiency (EE) (p < .05). When the ultrasound-assisted process (UAP) at different amplitudes (20% and 40%) and times (2, 5, 10, and 15 min) were implemented, the highest EE of conjugate-loaded liposome was found at 20% amplitude for 2 min (p < .05). When S-liposome and UAP-liposome were lyophilized, decreasing EE of both samples was observed (p < .05). Lyophilized UAP-liposome had higher stability than lyophilized S-liposome during storage at 25℃ for 28 days. Additionally, antioxidant activity in the gastrointestinal track model system (GIMs) and digest obtained from GIMs were higher for UAP-liposome (p < .05). Therefore, liposome can be used for the delivery of conjugate. PRACTICAL APPLICATIONS: HC from defatted sea bass skin is considered to possess several bioactivities, especially skin nourishment and bone strengthening. Nevertheless, antioxidant activity, related to the treatment of several ailments, is still low for HC. Thus, grafting of HC with polyphenol such as EGCG via free radical method can be used for the enhancement of the antioxidant activity of HC. Although the resulting conjugate has augmented activity, it is unstable during storage and in the gastrointestinal digestion system. Liposome is a promising means to stabilize the conjugate under harsh condition, especially with the aid of the UAP. Thus, liposome loaded with conjugate having the reduced size has higher antioxidant activity with increased stability, which can have a wider range of applications.

Suppressing Cdk5 Activity by Luteolin Inhibits MPP+-Induced Apoptotic of Neuroblastoma through Erk/Drp1 and Fak/Akt/GSK3ß Pathways.

Parkinson's disease (PD) is characterized by the progressive degeneration of dopaminergic neurons. The cause of PD is still unclear. Oxidative stress and mitochondrial dysfunction have been linked to the development of PD. Luteolin, a non-toxic flavonoid, has become interested in an alternative medicine, according to its effects on anti-oxidative stress and anti-apoptosis, although the underlying mechanism of luteolin on PD has not been fully elucidated. This study aims to investigate whether luteolin prevents neurotoxicity induction by 1-methyl-4-phenylpyridinium iodide (MPP+), a neurotoxin in neuroblastoma SH-SY5Y cells. The results reveal that luteolin significantly improved cell viability and reduced apoptosis in MPP+-treated cells. Increasing lipid peroxidation and superoxide anion (O2-), including mitochondrial membrane potential (Δψm) disruption, is ameliorated by luteolin treatment. In addition, luteolin attenuated MPP+-induced neurite damage via GAP43 and synapsin-1. Furthermore, Cdk5 is found to be overactivated and correlated with elevation of cleaved caspase-3 activity in MPP+-exposed cells, while phosphorylation of Erk1/2, Drp1, Fak, Akt and GSK3ß are inhibited. In contrast, luteolin attenuated Cdk5 overactivation and supported phosphorylated level of Erk1/2, Drp1, Fak, Akt and GSK3ß with reducing in cleaved caspase-3 activity. Results indicate that luteolin exerts neuroprotective effects via Cdk5-mediated Erk1/2/Drp1 and Fak/Akt/GSK3ß pathways, possibly representing a potential preventive agent for neuronal disorder.

Luteolin attenuates migration and invasion of lung cancer cells via suppressing focal adhesion kinase and non-receptor tyrosine kinase signaling pathway.

BACKGROUND/OBJECTIVES: Non-small cell lung cancer is mostly recognized among other types of lung cancer with a poor prognosis by cause of chemotherapeutic resistance and increased metastasis. Luteolin has been found to decrease cell metastasis. However, its underlying mechanisms remain unresolved. The objective of this study was to examine the effect (and its mechanism) of luteolin on the migration and invasion of human non-small cell lung cancer A549 cells. MATERIALS/METHODS: Cell viability was investigated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Wound healing and transwell assays were evaluated to assess migration and invasion, respectively. Western blot analysis and immunofluorescence were further performed to investigate the role of luteolin and its mechanisms of action. RESULTS: Administration with up to 40 µM luteolin showed no cytotoxic activity on lung cancer A549 cells or non-cancer MRC-5 cells. Additionally, luteolin at 20-40 µM significantly suppressed A549 cells' migration, invasion, and the formation of filopodia in a concentration-dependent manner at 24 h. This is similar with western blot analysis, which revealed diminished the phosphorylated focal adhesion kinase (pFAK), phosphorylated non-receptor tyrosine kinase (pSrc), Ras-related C3 botulinum toxin substrate 1 (Rac1), cell division control protein 42 (Cdc42), and Ras homolog gene family member A (RhoA) expression levels. CONCLUSIONS: Overall, our data indicate that luteolin plays a role in controlling lung cancer cells' migration and invasion via Src/FAK and its downstream Rac1, Cdc42, and RhoA pathways. Luteolin might be considered a promising candidate for suppressing invasion and metastasis of lung cancer cells.

Metformin Inhibit Cervical Cancer Migration by Suppressing the FAK/Akt Signaling Pathway.

BACKGROUND: Metformin, an antidiabetic drug, has been previously reported to have anti-cancer activities. However, its role in the control of cancer cell migration remains elusive. METHODS: To examine the possible effect of metformin on migration of cervical cancer cells. The related mechanisms were further determined by immunocytochemistry and Western's blotting assay. RESULTS: The results showed that metformin treatment substantially inhibited the migration ability of cervical cancer cells. Consistently, the filopodia and lamellipodia formation were depleted after exposure to metformin. The suppression of migration mediated through the regulatory proteins such as focal adhesion kinase (FAK), ATP-dependent tyrosine kinase (Akt), Rac1 and RhoA after metformin treatment. CONCLUSION: Metformin displays antimigration effects in cervical cancer cells by inhibiting filopodia and lamellipodia formation through the suppression of FAK, Akt and its downstream Rac1 and RhoA protein. We propose that metformin could be a novel potential candidate as an antimetastatic cancer drug in the cervical cancer management.

ECa 233 Suppresses LPS-Induced Proinflammatory Responses in Macrophages via Suppressing ERK1/2, p38 MAPK and Akt Pathways.

A current anti-inflammatory agent often targets the prevention of inflammatory disorder development. The standardized Centella asiatica ECa 233 extract has been previously reported for anti-inflammatory effect. This study aimed to investigate its anti-inflammatory effect and mechanisms of ECa 233 in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages, through 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, nitric oxide (NO) assay, reactive oxygen species (ROS) production assay, enzyme-linked immunosorbent assay (ELISA) and Western blot analysis. Our results found that ECa 233 significantly inhibited LPS-stimulated pro-inflammatory mediators production including ROS, NO and prostaglandin E2 (PGE2), and pro-inflammatory cytokines, including tumor necrosis factor (TNF)-α and interleukin (IL)-1ß without cytotoxicity. In addition, ECa 233 downregulated not only the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), but also the activation of nuclear factor-kappa B (NF-κB), activated protein kinase B (Akt), extracellular signal-regulated kinase (ERK1/2) and p38 mitogen-activated protein kinases (MAPK) induced by LPS. The inhibition of LPS-induced inflammation due to ECa 233 offered an opportunity as a tentatively potential candidate for the prevention and treatment of inflammatory diseases.

Metformin Promotes Neuronal Differentiation via Crosstalk between Cdk5 and Sox6 in Neuroblastoma Cells.

Metformin has recently emerged as a key player in promotion of neuroblastoma differentiation and neurite outgrowth. However, molecular mechanisms of how metformin promotes cellular differentiation have not yet been fully elucidated. In this study, we investigated how metformin promotes cell differentiation, via an inhibition of cell proliferation, by culturing SH-SY5Y neuroblastoma cells with or without metformin. Pretreatment with reactive oxygen species (ROS) scavenger, NAC, revealed that ROS plays a crucial role in induction of cell differentiation. Cell differentiation was observed under various morphological criteria: extension of neuritic processes and neuronal differentiation markers. Treatment with metformin significantly increased neurite length, number of cells with neurite, and expression of neuronal differentiation markers, ß-tubulin III and tyrosine hydroxylase (TH) compared with untreated control. Further investigation found that metformin significantly decreased Cdk5 but increased Sox6 during cell differentiation. Analysis of the mechanism underlying these changes using Cdk5 inhibitor, roscovitine, indicated that expressions of Cdk5 and Sox6 corresponded to metformin treatment. These results suggested that metformin produces neuronal differentiation via Cdk5 and Sox6. In addition, phosphorylated Erk1/2 was decreased while phosphorylated Akt was increased in metformin treatment. Taken together, these findings suggest that metformin promotes neuronal differentiation via ROS activation through Cdk5/Sox6 crosstalk, relating to Erk1/2 and Akt signaling.

Induction of keratinocyte migration by ECa 233 is mediated through FAK/Akt, ERK, and p38 MAPK signaling.

Centella asiatica is widely considered the most important medicinal plant for treating and relieving skin diseases. Recently developed standardized extract of Centella asiatica ECa 233 has demonstrated positive effects on wound healing of incision and burn wound in rats. However, knowledge associated with wound healing mechanism of ECa 233 was scare. Therefore, this study aimed to investigate the effect and underlying molecular mechanisms of ECa 233 on the migration of a human keratinocyte cell line (HaCaT) using scratch wound healing assay. Formation of filopodia, a key protein in cell migration as well as signaling pathways possibly involved were subsequently assessed. It was found that HaCaT cell migration was significantly enhanced by ECa 233 in a concentration- and time-dependent manner. The filopodia formations were accordingly increased in exposure to ECa 233 at concentrations of 0.1-100 µg/ml. Furthermore, ECa 233 was found to significantly upregulate the expression of Rac1 and RhoA and to induce phosphorylation of FAK and Akt as well as ERK and p38 MAPK. Taken all together, it is suggestive that ECa 233 induces cell migration and subsequently promotes wound healing activity, through the activation of FAK, Akt, and MAPK signaling pathways thereby supporting the role of ECa 233 to be further developed for the clinical treatment of wound.

Barakol-induced apoptosis in P19 cells through generation of reactive oxygen species and activation of caspase-9.

ETHNOPHARMACOLOGICAL RELEVANCE: Barakol, an anxiolytic agent isolated from Senna siamea leaves which has been traditionally used for producing natural sleep, has been described as toxic to patients. AIM OF THE STUDY: The aim of current study was to investigate the molecular mechanism of barakol-induced toxicity in mouse embryonal carcinoma P19 cell model. MATERIALS AND METHODS: XTT assay was used to determine cell viability in P19 cells treated with barakol. Apoptotic cells were detected by Hoechst 33342 staining. Intracellular reactive oxygen species (ROS) generation was analyzed by flow cytometry using a fluorescent dye, DCFH-DA. Detection of apoptotic protein expression in P19 cells was performed by Western blot analysis. Caspase-9 activity was measured using a fluorescent immunosorbent enzyme assay kit. RESULTS: Treatment with barakol decreased cell viability in a concentration- and time-dependent manner with an IC(50) value of 1.5mM in 24-h treated cells. A Hoechst 33342 assay revealed that barakol cytotoxicity was due to a significant increase in the number of apoptotic cells. Different scavengers to characterize ROS were utilized and revealed that hydroxyl radicals played a major role in ROS-induced apoptosis in barakol-treated cells. Western blot analysis demonstrated that barakol-induced apoptosis was mediated by the increase in expression ratio of Bax/Bcl-2. Furthermore, increase in caspase-9 activity after exposure to barakol for 24h was also observed. Pretreatment of cells with N-acetyl-l-cysteine (NAC) attenuated intracellular ROS generation, the Bax/Bcl-2 protein expression, and apoptosis. CONCLUSIONS: The mechanism of barakol-mediated toxicity in P19 cells is mainly associated with the ROS generation, followed by the imbalance of the Bax/Bcl-2 ratio, and caspase-9 activation leading to apoptotic cell death. Pretreatment of cells with NAC could antagonize the toxicity produced by barakol.

Effect of nicotine and polyaromtic hydrocarbons on cerebral endothelial cells.

The present study was designed to investigate the effect of nicotine and polyaromatic hydrocarbon compounds on cerebral endothelial cells (CECs). Nicotine treatments from 15 min to 5h did not cause any changes in the expression and localization of principal junctional proteins. One day of treatment with a relatively high concentration of nicotine induced a decrease in the expression of the tight junction protein ZO-1, occludin, and the adherens junction protein, cadherin. Treatment with 3 x 10(-5)M phenanthrene for 24h caused a redistribution of occludin from the Triton X-100 insoluble to the Triton X-100 soluble fraction. Transendothelial electrical resistance was not significantly affected by 24h treatments with nicotine, methylanthracene or phenanthrene. However, 24h nicotine treatment increased transendothelial permeability in CECs exposed to oxidative stress. Both nicotine and phenanthrene were able to regulate the expression of a large number of proteins as revealed by 2D electrophoresis. Our experiments suggest that tobacco smoking may affect the junctional complex of CECs, and that this effect is enhanced by oxidative stress.