Protective Effect of Curcumin on the Tight Junction Integrity and Cellular Senescence in Human Retinal Pigment Epithelium of Early Diabetic Retinopathy.

Diabetic retinopathy (DR) is a secondary complication of diabetes that can lead to visual impairment and blindness. The retinal pigment epithelium (RPE) is a monolayer of pigment cells that forms the blood-retinal barrier (BRB) via tight junction (TJ) proteins and plays a crucial role in the physiological function of the retina. Hyperglycemia induces RPE death and BRB breakdown, which accelerates the process of DR. Curcumin, an active extract of Curcuma longa , has anti-inflammatory, antioxidant, antiapoptotic, and neuroprotective properties. However, the effect of Curcumin on the BRB under high glucose conditions remains unknown. This study aimed to investigate the protective effects of Curcumin on RPE physiology in vitro and in vivo . Curcumin significantly alleviated cell viability inhibition under high glucose conditions. Moreover, high glucose reduced extracellular signal-regulated kinase and Akt pathways activation to diminish RPE cell growth but reversed by Curcumin treatment. Curcumin protected not only TJ integrity but also retinoid regeneration through TJ proteins and isomerase modulation in diabetic retina. Furthermore, Curcumin decreased the expression of angiogenic factor to inhibit retinal neovascularization. Finally, Curcumin treatment markedly reduced apoptosis during hyperglycemia. In conclusion, Curcumin can alleviate the progression of DR by promoting RPE survival, TJ integrity, retinoid isomerase activity, RPE senescence inhibition, and neovascularization. Therefore, Curcumin exhibits high potential for use as a therapeutic agent for early DR.

Downregulation of VEGFR2 signaling by cedrol abrogates VEGF­driven angiogenesis and proliferation of glioblastoma cells through AKT/P70S6K and MAPK/ERK1/2 pathways.

Cedrol is a sesquiterpene alcohol isolated from Cedrus atlantica, which has been traditionally used in aromatherapy and has anticancer, antibacterial and antihyperalgesic effects. One characteristic of glioblastoma (GB) is the overexpression of vascular endothelial growth factor (VEGF), which induces a high degree of angiogenesis. Although previous studies have reported that cedrol inhibits GB growth by inducing DNA damage, cell cycle arrest and apoptosis, its role in angiogenesis remains unclear. The aim of the present study was to investigate the effects of cedrol on VEGF-induced angiogenesis of human umbilical vein endothelial cells (HUVECs). HUVECs were treated with 0-112 µM cedrol and 20 ng/ml VEGF for 0-24 h, and then anti-angiogenic activation of cedrol was determined by MTT assay, wound healing assay, Boyden chamber assay, tube formation assay, semi-quantitative reverse transcription-PCR and western blotting. These results demonstrated that cedrol treatment inhibited VEGF-induced cell proliferation, migration and invasion in HUVECs. Furthermore, cedrol prevented VEGF and DBTRG-05MG GB cells from inducing capillary-like tube formation in HUVECs and decreased the number of branch points formed. Moreover, cedrol downregulated the phosphorylation of VEGF receptor 2 (VEGFR2) and the expression levels of its downstream mediators AKT, ERK, VCAM-1, ICAM-1 and MMP-9 in HUVECs and DBTRG-05MG cells. Taken together, these results demonstrated that cedrol exerts anti-angiogenic effects by blocking VEGFR2 signaling, and thus could be developed into health products or therapeutic agents for the prevention or treatment of cancer and angiogenesis-related diseases in the future.

The alleviation effects of n-butylidenephthalide on apoptosis, senescence, and tight junction impairment of retinal pigment epithelium by activating Nrf-2/HO-1 signaling pathway in early diabetic retinopathy.

AIMS: Diabetic retinopathy (DR) is a common complication of diabetes that causes visual impairment and blindness in adults. This study aimed to explore the protective effects of n-Butylidenephthalide (BP) on hyperglycemia-treated RPE in vitro and in vivo. MAIN METHODS: C57BL/6 mice were injected with STZ by intraperitoneal to induce early DR and orally administrated with 2 mg/kg BP every day for twelve weeks. Body weight and blood glucose were measured once a week. The level of retina damage was determined by TUNEL assay and H&E staining. The outer blood-retinal barrier integrity and RPE65 expression of retina were evaluated by immunofluorescence. In in vitro study, ARPE-19 cells were long-term cultured with high glucose and BP for 8 days and studied for cell survival, tight junction integrity, RPE65 expression, angiogenic factors, mitochondria membrane potential (MMP), and ROS by MTT assay, Western blot, ß-galactosidase staining, immunofluorescence, JC-1, or DCFH-DA. KEY FINDINGS: The results indicate that BP suppressed the hyperglycemic effect and maintained retina anatomy normalization, as well as protected RPE cell survival, tight junction integrity, and RPE65 expression in vitro and in vivo. In vitro results showed BP stimulated high glucose-treated ARPE-19 cell proliferation and suppressed senescence via ERK pathway. Numerous ROS production and MMP imbalance were prevented by BP through Nrf-2/HO-1 pathway. BP inhibited high glucose-induced RPE neovascularization by VEGF dysregulation. SIGNIFICANCE: BP significantly protected tight junction integrity and RPE cellular physiology through ERK/Nrf-2/HO-1 pathway to prevent DR progression. Thus, BP has great potential to be developed therapeutic agents or adjuvants for DR.

Patchouli alcohol induces G0 /G1 cell cycle arrest and apoptosis in vincristine-resistant non-small cell lung cancer through ROS-mediated DNA damage.

BACKGROUND: Lung cancer, especially non-small cell lung cancer (NSCLC), is one of the leading causes of cancer-related deaths worldwide. Vincristine (VCR) is a chemotherapeutic agent for lung cancers; however, its effectiveness is limited by side effects and the development of drug resistance. Patchouli alcohol (PA), from Pogostemon cablin extract, is known to possess anti-inflammatory and anticancer properties. In this study, we investigated the role of PA in inducing reactive oxygen species (ROS)-mediated DNA damage in A549 and VCR-resistant A549/V16 NSCLC cells. METHODS: The anticancer potential of PA was studied using the MTT assay, colony formation, flow cytometry analysis, western blotting, DCFDA staining, immunofluorescence staining, and TUNEL assay techniques. RESULTS: The intracellular ROS levels were enhanced in PA-treated cells, activating the CHK1 and CHK2 signaling pathways. PA further inhibited proliferation and colony-forming abilities and induced cell cycle arrest at the G0 /G1 phase by regulating p53/p21 and CDK2/cyclin E1 expression. Moreover, PA increased the percentage of cells in the subG1 phase and induced apoptosis by activating the Bax/caspase-9/caspase-3 intrinsic pathway. In addition, drug resistance (p-glycoprotein) and cancer stem cell (CD44 and CD133) markers were downregulated after PA treatment. Furthermore, combining PA and cisplatin exhibited synergistic inhibitory activity in A549 and A549/V16 cells. CONCLUSIONS: PA induced ROS-mediated DNA damage, triggered cell cycle arrest and apoptosis, attenuated drug resistance and cancer stem cell phenotypes, and synergistically inhibited proliferation in combination with cisplatin. These findings suggest that PA has the potential to be used for the treatment of NSCLC with or without VCR resistance.

The effects of patchouli alcohol and combination with cisplatin on proliferation, apoptosis and migration in B16F10 melanoma cells.

Melanoma is a highly metastatic cancer with a low incidence rate, but a high mortality rate. Patchouli alcohol (PA), a tricyclic sesquiterpene, is considered the main active component in Pogostemon cablin Benth, which improves wound healing and has anti-tumorigenic activity. However, the pharmacological action of PA on anti-melanoma remains unclear. Thus, the present study aimed to investigate the role of PA in the proliferation, cell cycle, apoptosis and migration of melanoma cells. These results indicated that PA selectively inhibited the proliferation of B16F10 cells in a dose- and time-dependent manner. It induced cell cycle arrest at the G0 /G1 phase and typical morphological changes in apoptosis, such as chromatin condensation, DNA fragmentation and apoptotic bodies. In addition, PA reduced the migratory ability of B16F10 cells by upregulating E-cadherin and downregulating p-Smad2/3, vimentin, MMP-2 and MMP-9 expression. PA was also found to strongly suppress tumour growth in vivo. Furthermore, PA combined with cisplatin synergistically inhibited colony formation and migration of B16F10 cells and attenuated the development of resistance to treatment. Therefore, the results of this study indicate that PA may play a pivotal role in inducing apoptosis and reducing the migration of melanoma cells, and may thus be a potential candidate for melanoma treatment.

Cedrus atlantica extract exerts antiproliferative effect on colorectal cancer through the induction of cell cycle arrest and apoptosis.

Cedrus atlantica is a tree species found in Morocco with many clinical benefits in genitourinary, musculoskeletal, and skin systems. Previous studies have reported that extracts of Cedrus atlantica have antioxidant, antimicrobial, and anticancer properties. However, its role in colorectal cancer (CRC) remains unclear. The present study investigated the effects and underlying mechanisms of Cedrus atlantica extract (CAt) using HT-29 (human colorectal adenocarcinoma) and CT-26 CRC cell lines. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed to measure cell viability. Flow cytometry analysis and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay were used to study the cell cycle and cell apoptosis, respectively. The expression of cell cycle and apoptosis-associated proteins was detected by western blotting or immunohistochemical (IHC) staining. CAt showed significant inhibitory effects on the proliferation of HT-29 and CT-26 cells, and combined with the clinical drug, 5-fluorouracil (5-FU), exhibited synergistic effects. CAt induced cell cycle arrest at the G0/G1 phase through the upregulation of p53/p21 and the downregulation of cyclin-dependent kinases (CDKs)/cyclins. In addition, CAt-treated cells exhibited chromatin condensation, DNA fragmentation, and apoptotic bodies, which are typical characteristics of apoptosis activated via both the extrinsic (Fas ligand (FasL)/Fas/caspase-8) and intrinsic (Bax/caspase-9) pathways. Importantly, CAt suppressed tumor progression and prolonged the life span of mice within a well-tolerated dose. Therefore, our findings provide novel insights into the use of CAt for the treatment of CRC.

Cedrus atlantica Extract Suppress Glioblastoma Growth through Promotion of Genotoxicity and Apoptosis: In Vitro and In Vivo Studies.

Glioblastoma (GBM) is the most common malignant primary brain tumor in humans, exhibiting highly infiltrative growth and drug resistance to conventional chemotherapy. Cedrus atlantica (CAt) extract has been shown to decrease postoperative pain and inhibit the growth of K562 leukemia cells. The aim of this study was to assess the anti-GBM activity and molecular mechanism of CAt extract in vitro and in vivo. The results showed that CAt extract greatly suppressed GBM cells both in vitro and in vivo and enhanced the survival rate in subcutaneous and orthotopic animal models. Moreover, CAt extract increased the level of ROS and induced DNA damage, resulting in cell cycle arrest at the G0/G1 phase and cell apoptosis. Western blotting results indicated that CAt extract regulates p53/p21 and CDK4/cyclin D1 protein expression and activates extrinsic and intrinsic apoptosis. Furthermore, CAt extract enhanced the cytotoxicity of Temozolomide and decreased AKT/mTOR signaling by combination treatment. In toxicity assays, CAt extract exhibited low cytotoxicity toward normal cells or organs in vitro and in vivo. CAt extract suppresses the growth of GBM by induction of genotoxicity and activation of apoptosis. The results of this study suggest that CAt extract can be developed as a therapeutic agent or adjuvant for GBM treatment in the future.

Pogostemon cablin Triggered ROS-Induced DNA Damage to Arrest Cell Cycle Progression and Induce Apoptosis on Human Hepatocellular Carcinoma In Vitro and In Vivo.

The purpose of the study was to elucidate the anti-hepatoma effects and mechanisms of Pogostemon cablin essential oils (PPa extract) in vitro and in vivo. PPa extract exhibited an inhibitory effect on hepatocellular carcinoma (HCC) cells and was less cytotoxic to normal cells, especially normal liver cells, than it was to HCC cells, exerting a good selective index. Additionally, PPa extract inhibited HCC cell growth by blocking the cell cycle at the G0/G1 phase via p53 dependent or independent pathway to down regulated cell cycle regulators. Moreover, PPa extract induced the FAS-FASL-caspase-8 system to activate the extrinsic apoptosis pathway, and it increased the bax/bcl-2 ratio and reduced ΔΨm to activate the intrinsic apoptosis pathway that might be due to lots of reactive oxygen species (ROS) production which was induced by PPa extract. In addition, PPa extract presented to the potential to act synergistically with sorafenib to effectively inhibit HCC cell proliferation through the Akt/mTOR pathway and reduce regrowth of HCC cells. In an animal model, PPa extract suppressed HCC tumor growth and prolonged lifespan by reducing the VEGF/VEGFR axis and inducing tumor cell apoptosis in vivo. Ultimately, PPa extract demonstrated nearly no or low system-wide, physiological, or pathological toxicity in vivo. In conclusion, PPa extract effectively inhibited HCC cell growth through inducing cell cycle arrest and activating apoptosis in vitro and in vivo. Furthermore, PPa extract exhibits less toxicity toward normal cells and organs than it does toward HCC cells, which might lead to fewer side effects in clinical applications. PPa extract may be developed into a clinical drug to suppress tumor growth or functional food to prevent HCC initiation or chemoprotection of HCC recurrence.

Cedrol suppresses glioblastoma progression by triggering DNA damage and blocking nuclear translocation of the androgen receptor.

Glioblastoma (GBM) is the most common and aggressive primary brain tumor with great invasiveness and resistance to chemotherapy, which presents a treatment challenge. In this study, we investigated the antitumor effect of Cedrol, a sesquiterpene alcohol isolated from Cedrus atlantica, against GBM cells in vitro and in vivo. Cedrol was found to potently inhibit cell growth and induce intracellular ROS generation and DNA damage response. In addition, Cedrol induced significant G0/G1 cell cycle arrest and cell apoptosis via the extrinsic (Fas/FasL/Caspase-8) and intrinsic (Bax/Bcl-2/Caspase-9) pathways. In addition, Cedrol had a synergistic effect with temozolomide (TMZ) and reduced drug resistance by blockage of the AKT/mTOR pathway. Cedrol suppressed tumor growth in both orthotopic and xenograft GBM animal models with low or no short-term acute toxicity or long-term accumulative toxicity. In a molecular docking study, Cedrol targeted the androgen receptor (AR), and reduced DHT-mediated AR nuclear translocation, downstream gene KLK3/TMPRSS2 expression and cell proliferation. Our study demonstrates that Cedrol may be a potential candidate for drug development for single or combination treatment with TMZ in GBM therapy.

Cedrol, a Sesquiterpene Alcohol, Enhances the Anticancer Efficacy of Temozolomide in Attenuating Drug Resistance via Regulation of the DNA Damage Response and MGMT Expression.

Glioblastoma (GBM) is a common and aggressive brain tumor with a median survival of 12-15 months. Temozolomide (TMZ) is a first-line chemotherapeutic agent used in GBM therapy, but the occurrence of drug resistance limits its antitumor activity. The natural compound cedrol has remarkable antitumor activity and is derived from Cedrus atlantica. In this study, we investigated the combined effect of TMZ and cedrol in GBM cells in vitro and in vivo. The TMZ and cedrol combination treatment resulted in consistently higher suppression of cell proliferation via regulation of the AKT and MAPK signaling pathways in GBM cells. The combination treatment induced cell cycle arrest, cell apoptosis, and DNA damage better than either drug alone. Furthermore, cedrol reduced the expression of proteins associated with drug resistance, including O6-methlyguanine-DNA-methyltransferase (MGMT), multidrug resistance protein 1 (MDR1), and CD133 in TMZ-treated GBM cells. In the animal study, the combination treatment significantly suppressed tumor growth through the induction of cell apoptosis and decreased TMZ drug resistance. Moreover, cedrol-treated mice exhibited no significant differences in body weight and improved TMZ-induced liver damage. These results imply that cedrol may be a potential novel agent for combination treatment with TMZ for GBM therapy that deserves further investigation.