Apoptotic Cell Population Research Articles

Effect of Acacia concinna Extract on Apoptosis Induction Associated with Endoplasmic Reticulum Stress and Modulated Intracellular Signaling Pathway in Human Colon HCT116 Cancer Cells.

Colorectal cancer (CRC) stands as one of the most prevalent cancer types and among the most frequent causes of cancer-related death globally. Acacia concinna (AC) is a medicinal and edible plant that exhibits a multitude of biological properties, including anticancer properties. This study aimed to investigate the impact of the AC extract on apoptosis induction and the underlying mechanisms associated with this effect in KRAS-mutated human colon HCT116 cells. The effect of AC extract on cell cytotoxicity was evaluated using MTT assay. Nuclear morphological changes were visualized with Hoechst 33342 staining, while mitochondrial membrane potential (MMP) was assessed via JC-1 staining. Flow cytometry was employed for cell cycle analysis, and intracellular ROS levels were determined using DCFH-DA staining. The results showed that HCT116 cells exposed to AC extract showed reduced cell growth and prompted apoptosis, as indicated by an increase in chromatin condensation, apoptotic bodies, the sub-G1 apoptotic cell population, and disrupted MMP. Expression levels of apoptosis mediator proteins determined by Western blot analysis showed an increase in pro-apoptotic proteins (Bak and Bax) while decreasing anti-apoptotic proteins (Bcl-2, Bcl-xL, and Mcl-1), leading to caspase-7 activation and PARP inactivation. AC extract was also found to enhance intracellular reactive oxygen species (ROS) levels and stimulate endoplasmic reticulum (ER) stress. Furthermore, AC extract increases the phosphorylation of ERK1/2, p38, and c-Jun while downregulating PI3K, Akt, β-catenin, and their downstream target proteins. These results demonstrate that AC extract could inhibit cancer cell growth via ROS-induced ER stress associated with apoptosis and regulate the MAPK, PI3K/Akt, and Wnt/β-catenin signaling pathways in HCT116 cells. Therefore, AC extract may be a novel candidate for natural anticancer resources for colon cancer treatment.

Naphthoindole-2-carboxamides as a lipophilic chemotype of hetarene-anthraquinones potent against P-gp resistant tumor cells

The acquisition of multidrug resistance (MDR) to chemotherapy is a major obstacle to successful cancer treatment. Aiming to improve the potency of anthraquinone-derived antitumor compounds against MDR cancer cells, we employed a rational design approach to develop new heteroarene-fused anthraquinones. Shifting the carboxamide group in the naphtho[2,3-f]indole scaffold from the 3-position to 2 increased the lipophilicity and P-glycoprotein (P-gp) binding of the derivatives, potentially enhancing their ability to circumvent P-gp-mediated MDR. To validate the computations, we developed a scheme for heterocyclization into esters of naphtho[2,3-f]indole-2-carboxylic acid, based on the 5-endo-dig cyclization of 2-alkynyl-3-amino-1,4-dimethoxyanthraquinone under mild basic conditions using tetra-n-butylammonium fluoride (TBAF). The synthesized naphthoindole-2-carboxamides, particularly compound 1a bearing (S)-3-aminopyrrolidine in the carboxamide fragment, demonstrated the highest antiproliferative activity. Most importantly, 1a suppressed the growth of the P-gp-positive K562/4 leukemia tumor cell line (resistance index = 2.4), while its 3-isomer LCTA-2640 and Dox did not (RI = 125 and 140, respectively). Studies of intracellular uptake and distribution showed that 1a, unlike its 3-substituted isomer, effectively accumulated in resistant tumor cells, confirming the correlation between in silico and experimental data. The lead compound 1a interacts with DNA duplex and inhibits topoisomerase 1 but does not induce oxidative stress. Treatment with 1a increases the population of apoptotic cells in both K562 and K562/4 sublines, regardless of the cell cycle phase. Taken together, this work provides an interesting example of how a little modification in chemical structure can lead to striking differences in antitumor properties. In conclusion, we have identified a potent class of compounds that offer distinct advantages in combating resistant tumor cells.

Effects of inorganic phosphate on stem cells isolated from human exfoliated deciduous teeth

Calcium phosphate-based materials (CaP) are introduced as potential dental pulp capping materials for deciduous teeth. The present study investigated the influence of inorganic phosphate (Pi) on regulating stem cells isolated from human exfoliated deciduous teeth (SHED). SHEDs were treated with Pi. Cell cycle progression and apoptosis were examined using flow cytometry analysis. Osteo/odontogenic and adipogenic differentiation were analyzed using alizarin red S and oil red O staining, respectively. The mRNA expression profile was investigated using a high-throughput RNA sequencing technique. Pi increased the late apoptotic cell population while cell cycle progression was not altered. Pi upregulated osteo/odontoblastic gene expression and enhanced calcium deposition. Pi-induced mineralization was reversed by pretreatment of cells with Foscarnet, or p38 inhibitor. Pi treatment inhibited adipogenic differentiation as determined by decreased PPARγ expression and reduced intracellular lipid accumulation. Bioinformatic analysis of gene expression profiles demonstrated several involved pathways, including PI3K/AKT, MAPK, EGFR, and VEGF signaling. In conclusion, Pi enhanced osteo/odontogenic but inhibited adipogenic differentiation in SHED.

Prorocentin-5: A Cytotoxic Polyketide from the Benthic Marine Dinoflagellate Prorocentrum lima.

Prorocentrin-5 (1) was isolated from the benthic marine dinoflagellate Prorocentrum lima. A combination of NMR spectroscopy, quantum chemical calculations, and chemical reactions was then employed to elucidate its molecular structure, including the configurations of all stereogenic centers. In cytotoxicity assays, prorocentin-5 exhibited potent activity against the HCT-116 and Neuro2a cell lines, with IC50 values of 4.4 and 2.8 μM, respectively. Furthermore, 1 increased the apoptotic cell population and induced cell cycle arrest, leading to the accumulation of cells in the S or G2/M phase and an accompanying decrease in the G0/G1 phase in HCT-116, Neuro2a, and HepG2 cells.

TongGuanWan Alleviates Doxorubicin- and Isoproterenol-Induced Cardiac Hypertrophy and Fibrosis by Modulating Apoptotic and Fibrotic Pathways.

Heart failure, a major public health issue, often stems from prolonged stress or damage to the heart muscle, leading to cardiac hypertrophy. This can progress to heart failure and other cardiovascular problems. Doxorubicin (DOX), a common chemotherapy drug, and isoproterenol (ISO), a β-adrenergic agonist, both induce cardiac hypertrophy through different mechanisms. This study investigates TongGuanWan (TGW,), a traditional herbal remedy, for its effects on cardiac hypertrophy and fibrosis in DOX-induced H9c2 cells and ISO-induced mouse models. TGW was found to counteract DOX-induced increases in H9c2 cell surface area (n = 8, p < 0.01) and improve biomarkers like ANP (n = 3, p < 0.01)) and BNP (n = 3, p < 0.01). It inhibited the MAPK pathway (n = 4, p < 0.01) and GATA-4/calcineurin/NFAT-3 signaling, reduced inflammation by decreasing NF-κB p65 translocation, and enhanced apoptosis-related factors such as caspase-3 (n = 3, p < 0.01), caspase-9 (n = 3, p < 0.01), Bax (n = 3, p < 0.01), and Bcl-2 (n = 3, p < 0.01). Flow cytometry showed TGW reduced apoptotic cell populations. In vivo, TGW reduced heart (n = 8~10, p < 0.01), and left ventricle weights (n = 6~7), cardiac hypertrophy markers (n = 3, p < 0.01), and perivascular fibrosis in ISO-induced mice, with Western blot analysis confirming decreased levels of fibrosis-related factors like fibronectin, α-SMA (n = 3, p < 0.05), and collagen type I (n = 3, p < 0.05). These findings suggest TGW has potential as a therapeutic option for cardiac hypertrophy and fibrosis.

P53 genotype-independent anticancer effects of olibanum extract and 11-keto-beta-boswellic acid on human colorectal cancer cells

BackgroundColorectal cancer (CRC) is the third most common cancer, and CRC-related mortality is increasing annually. A key regulator of the cell cycle and cell death, the p53 gene, is frequently found in patients with CRC as mutants. Since tumors with abnormal p53 sometimes show resistance to anticancer drugs, it is important to develop anticancer drugs according to genetic characteristics. Although evidence shows that boswellic acids (BAs) might be potential anticancer agents, the anticancer effects and mechanisms involved in their activities in CRC are unclear. Methods and ResultsHere, we investigated whether olibanum (Boswellia serrata Roxb.) extract and 11-keto-β-boswellic acid exerted cytotoxic effects against p53 wildtype, p53 mutated-, and p53-deleted human CRC cell lines. Results show that 11-keto-β-boswellic acid considerably reduced cell viability and induced cell cycle arrest in HCT116, HT29, and SW1417 cells. And increase of apoptotic cell population and morphological changes in apoptotic cells induced by 11-keto-β-boswellic acid were observed. The 11-keto-β-boswellic acid mediated cell cycle arrest and apoptosis were accompanied by changes in the expression of factors linked to apoptosis, regardless of p53 genotype. In addition, 11-keto-β-boswellic acid-treated cells dissociated from their spheroidal structure and appeared in an irregular form. ConclusionsAlthough further investigations are required to fully understand the underlying mechanisms, these results may provide insight into the potential of using olibanum extract and 11-keto-β-boswellic acid as therapeutic agents, with no difference in sensitivity depending on the p53 genotype.

3-Aminobenzamide-linked multifunctional nanoparticles enhances anticancer activity of low-dose cisplatin chemotherapy in lung adenocarcinoma

Chemotherapy is one of the main treatment methods for cancer patients, but its effectiveness is limited by drug resistance. Combining a chemotherapeutic agent with targeted molecular therapy may improve the curative effect of the chemotherapeutic agent. In this study, we investigate the efficacy of combining a 3-Aminobenzamide (3AB)-linked multifunctional platform with low-dose cisplatin chemotherapy aiming to modulate poly [ADP-ribose] polymerase 1 (PARP1) function in DNA repair to increase cytotoxic activity of the platinum-based cisplatin. The structure of the synthesized nanoplatforms was characterized by several physicochemical techniques, including dynamic light scattering (DLS), Fourier-transform infrared spectroscopy (FT-IR), scanning electron microscopy (SEM) and an in vitro pH-dependent release study. Cellular uptake experiments demonstrated preferentially targeted delivery of nanoparticles in lung carcinoma A549 cells, whereas the cellular uptake capacity was minimal in normal lung BEAS-2B cells. On the other hand, cytotoxicity experiments showed a reduction of cancer cell viability compared to free formulations. Furthermore, the combination treatment was examined by detecting the loss of mitochondrial membrane potential and the apoptotic cell population, confirming the treatment's functional involvement in apoptosis. Soft agar colony formation and cell invasion tests were also performed to detect the cancer cell's tumorigenic potential, confirming the synergistic effect of this combination in the reduction of tumorigenicity. Moreover, we analyzed the expression profiles of three candidate genes, which play important roles in cancer initiation, promotion and progression. Cell biology experiments indicated that this novel combination treatment possesses significant synergy between 3AB and low-dose cisplatin and is promising for development as an antitumor treatment for lung cancer.

Modified Jiaoqi Powder enhances epithelial autophagy against TNF-triggered apoptosis in chronic ulcerative colitis

BackgroundA vicious cycle of dysregulated intestinal epithelial cell death, intestinal barrier defect, and subsequent inflammation response is core to chronic ulcerative colitis (UC). Modified Jiaoqi Powder (MJQP), a traditional Chinese medicine formula, has been clinically applied to treat chronic relapsing type and chronic persistent type of UC . Nevertheless, the underlying mechanism of MJPQ in chronic UC remains unknown. PurposeThe present study aimed to demonstrate the favorable effect and potential molecular mechanism of MJQP in chronic UC. MethodsThe chemical components of MJQP and MJQP drug serum were identified by LC-MS/MS. The curative effect of MJQP was evaluated in a well-established DSS-induced chronic UC mice model by measuring body weight, colon length, disease activity index (DAI) and histological scores . Serum cytokines, including interleukin (IL)-1β, IL-12, IL-13, IL-4, tumor necrosis factor-alpha (TNF-α) and INF-γ were measured using enzyme-linked immunosorbent assay. Western blotting, immunofluorescence, and MTT assay were used to analyze the effects of MJQP on colonic barrier function in chronic UC mice and human epithelial cell lines. TUNEL assay, western blotting, and flow cytometry were used to examine the related apoptosis indicators. An electron microscope was used to observe autophagosomes and autolysosomes, while western blotting and immunofluorescence were used to detect autophagy-associated proteins. Network pharmacology was used to predict potential targets and pathways of MJQP in UC. Finally, the TNF pathway-related proteins were detected by immunohistochemistry and western blotting. ResultsMJQP administration prevented the UC progression with faster weight gain, longer colon length, lower histological scores and DAI, and up/down- regulation of inflammatory factors. The expressions of tight junction proteins ki67 and E-cadherin increased dose-dependently after MJQP intervention. Moreover, MJQP treatment promoted the viability of NCM460 and Caco2 cells in a concentration-dependent manner. MJQP dose-dependently decreased the proportion of TUNEL-positive cells and attenuated the pro-apoptotic proteins cleaved-caspase 8 and cleaved-caspase 3 in colonic tissues. Flow cytometry also showed that MJQP dose-dependently decreased the apoptotic cell population of LPS-induced NCM460 and Caco2 cells. Electron microscopy revealed that autophagosomes and autolysosomes were significantly improved in the MJQP-treated groups. Additionally, autophagy-related proteins were significantly expressed after MJQP treatment. Network pharmacological analysis predicted that MJQP may alleviate chronic UC by affecting TNF-related signaling pathways and promoting intestinal epithelial cell proliferation. As anticipated, the TNF pathway-associated proteins were attenuated dose-dependently in colonic tissues after MJQP treatment. ConclusionThese results provide novel therapeutic strategies, indicating that MJQP may be a promising candidate treatment for chronic UC by promoting epithelial barrier restitution by enhancing epithelial autophagy against TNF–mediated apoptosis.

From Sea to Science: Coral Aquaculture for Sustainable Anticancer Drug Development.

Marine natural products offer immense potential for drug development, but the limited supply of marine organisms poses a significant challenge. Establishing aquaculture presents a sustainable solution for this challenge by facilitating the mass production of active ingredients while reducing our reliance on wild populations and harm to local environments. To fully utilize aquaculture as a source of biologically active products, a cell-free system was established to target molecular components with protein-modulating activity, including topoisomerase II, HDAC, and tubulin polymerization, using extracts from aquaculture corals. Subsequent in vitro studies were performed, including MTT assays, flow cytometry, confocal microscopy, and Western blotting, along with in vivo xenograft models, to verify the efficacy of the active extracts and further elucidate their cytotoxic mechanisms. Regulatory proteins were clarified using NGS and gene modification techniques. Molecular docking and SwissADME assays were performed to evaluate the drug-likeness and pharmacokinetic and medicinal chemistry-related properties of the small molecules. The extract from Lobophytum crassum (LCE) demonstrated potent broad-spectrum activity, exhibiting significant inhibition of tubulin polymerization, and showed low IC50 values against prostate cancer cells. Flow cytometry and Western blotting assays revealed that LCE induced apoptosis, as evidenced by the increased expression of apoptotic protein-cleaved caspase-3 and the populations of early and late apoptotic cells. In the xenograft tumor experiments, LCE significantly suppressed tumor growth and reduced the tumor volume (PC3: 43.9%; Du145: 49.2%) and weight (PC3: 48.8%; Du145: 7.8%). Additionally, LCE inhibited prostate cancer cell migration, and invasion upregulated the epithelial marker E-cadherin and suppressed EMT-related proteins. Furthermore, LCE effectively attenuated TGF-β-induced EMT in PC3 and Du145 cells. Bioactivity-guided fractionation and SwissADME validation confirmed that LCE's main component, 13-acetoxysarcocrassolide (13-AC), holds greater potential for the development of anticancer drugs.

Anti-psoriatic potential of medicinal plants, Alstonia scholaris, Wrightia tinctoria, and Solanum xanthocarpum, using human HaCaT keratinocytes by multi-parametric analysis

Ethnopharmacological relevancePsoriasis, a widespread skin condition impacting over 100 million individuals globally, is characterised by uncontrolled hyperproliferation of keratinocytes, abnormal apoptosis, and excessive secretion of inflammatory cytokines and angiogenic factors. Traditional use of Alstonia scholaris (L.) R.Br., Wrightia tinctoria (Roxb.) R.Br. and Solanum xanthocarpum Schrad. & Wendl. in Ayurveda and Siddha medicinal systems have shown promising anti-inflammatory and wound-healing properties. However, underlying mechanisms of their phytoactivity in addressing psoriasis-like skin inflammation on human keratinocytes remain largely unexplored. Aim of the studyThe study was aimed to investigate anti-psoriatic potential of ethyl acetate and ethanolic extracts of A. scholaris, W. tinctoria and S. xanthocarpum in human keratinocyte cell line (HaCaT). Material and methodsEthyl acetate and ethanolic extracts of A. scholaris (ASEA and ASE), W. tinctoria (WTEA and WTE) and S. xanthocarpum (SXEA and SXE) were first subjected to phytochemical screening through high-performance liquid chromatography (HPLC) using their marker compound loganin, kaempferol and chlorogenic acid, respectively. The proliferation inhibition efficiency of these extracts was measured using MTT assay on HaCaT cell line. Subsequently, the apoptotic effect of these extracts on HaCaT cell line was determined by JC-1 and Annexin V assays. Furthermore, IL-8 and RANTES levels were measured in TNF-alpha-induced HaCaT cell line post-treatment with these extracts to determine their anti-inflammatory properties. ResultsASEA, ASE, WTEA, WTE, SXEA and SXE significantly inhibited proliferation of keratinocytes (HaCaT cells) and resulted in the induction of apoptotic markers (mitochondrial membrane potential and phosphatidyl serine externalization). Additionally, pro-inflammatory markers (IL-8 and RANTES levels) were downregulated in HaCaT cells.The anti-proliferative effects were particularly distinct at higher concentrations (200 μg/mL), with inhibition rates reaching over 85% for W. tinctoria and S. xanthocarpum extracts. In apoptotic assays, notable increases in late apoptotic or necrotic cell populations and significant losses in mitochondrial membrane potential were observed. All extracts markedly reduced the secretion of inflammatory mediators IL-8 and RANTES. ConclusionAll three plants exerted an anti-psoriatic effect at the cellular level via multiple parameters (anti-proliferative, pro-apoptotic, anti-inflammatory effect). This study provides insight into the mechanism of action of ASEA, ASE, WTEA, WTE, SXEA and SXE and highlights their promising potential for development as herbal therapeutic agents for psoriasis. It emphasizes the need for further pharmacological evaluation and toxicological studies of these extracts.

Yadanziolide A Inhibits Proliferation and Induces Apoptosis of Hepatocellular Carcinoma via JAK-STAT Pathway: A Preclinical Study.

Liver cancer is a significant global health concern, prompting the search for innovative therapeutic solutions. Yadanziolide A (Y-A), a natural derivative of Brucea javanica, has emerged as a promising candidate for cancer treatment; however, its efficacy and underlying mechanisms in liver cancer remain incompletely understood. In this study, we conducted a comprehensive evaluation of Y-A's effects on liver cancer cells using a range of in vitro assays and an orthotopic liver cancer mouse model. Our findings reveal that Y-A exerts dose-dependent cytotoxic effects on liver cancer cells, significantly inhibiting proliferation, migration, and invasion at concentrations ≥ 0.1 μM. Furthermore, Y-A induces apoptosis, as evidenced by increased apoptotic cell populations and apoptosome formation. In vivo studies confirm that Y-A inhibits tumor growth and reduces liver damage in mouse models. Mechanistically, Y-A targets the TNF-α/STAT3 pathway, inhibiting STAT3 and JAK2 phosphorylation, thereby activating apoptotic pathways and suppressing tumor cell growth. These results suggest that Y-A has promising anticancer activity and potential utility in liver cancer therapy.

Oxidative and carbonyl stress induced AMD and Codonopsis lanceolata ameliorates AMD via controlling oxidative and carbonyl stress

Age-related macular degeneration (AMD) is one of the leading causes of blindness. AMD is currently incurable; the best solution is to prevent its occurrence. To develop drugs for AMD, it is crucial to have a model system that mimics the symptoms and mechanisms in patients. It is most important to develop safer and more effective anti-AMD drug. In this study, the dose of A2E and the intensity of blue light were evaluated to establish an appropriate atrophic in vitro model of AMD and anti-AMD effect and therapeutic mechanism of Codonopsis lanceolata. The experimental groups included a control group an AMD group treated with A2E and blue light, a lutein group treated with 25 μM lutein after AMD induction, and three groups treated with different doses of C. lanceolata (10, 20, and 50 μg/mL) after AMD induction. Intrinsic apoptotic pathway (Bcl-2 family), anti-oxidative system (Keap1/Nrf2/HO-1 antioxidant response element), and anti-carbonyl effect (4-hydroxynonenal [4-HNE]) were evaluated using immunofluorescence, MTT, TUNEL, FACS, and western blotting analyses. A2E accumulation in the cytoplasm of ARPE-19 cells depending on the dose of A2E. Cell viability of ARPE-19 cells according to the dose of A2E and/or blue light intensity. The population of apoptotic or necrotic cells increased based on the A2E dose and blue light intensity. Codonopsis lanceolata dose-dependently prevented cell death which was induced by A2E and blue light. The antiapoptotic effect of that was caused by activating Keap1/Nrf2/HO-1 pathway, suppressing 4-HNE, and modulating Bcl-2 family proteins like increase of antiapoptotic proteins such as Bcl-2 and Bcl-XL and decrease of proapoptotic protein such as Bim. Based on these findings, 30 μM A2E and 20 mW/cm2 blue light on adult retinal pigment epithelium-19 cells was an appropriate condition for AMD model and C. lanceolata shows promise as an anti-AMD agent.

Uncovering SIRT3 and SHMT2-dependent pathways as novel targets for apigenin in modulating colorectal cancer: In vitro and in vivo studies

Despite significant advances in the treatment of colorectal cancer (CRC), identification of novel targets and treatment options are imperative for improving its prognosis and survival rates. The mitochondrial SIRT3 and SHMT2 have key roles in metabolic reprogramming and cell proliferation. This study investigated the potential use of the natural product apigenin in CRC treatment employing both in vivo and in vitro models and explored the role of SIRT3 and SHMT2 in apigenin-induced CRC apoptosis. The role of SHMT2 in CRC patients’ survival was verified using TCGA database. In vivo, apigenin treatment restored the normal colon appearance. On the molecular level, apigenin augmented the immunohistochemical expression of cleaved caspase-3 and attenuated SIRT3 and SHMT2 mRNA expression CRC patients with decreased SHMT2 expression had improved overall and disease-free survival rates. In vitro, apigenin reduced the cell viability in a time-dependent manner, induced G0/G1 cell cycle arrest, and increased the apoptotic cell population compared to the untreated control. Mechanistically, apigenin treatment mitigated the expression of SHMT2, SIRT3, and its upstream long intergenic noncoding RNA LINC01234 in CRC cells. Conclusively, apigenin induces caspase-3-dependent apoptosis in CRC through modulation of SIRT3-triggered mitochondrial pathway suggesting it as a promising therapeutic agent to improve patient outcomes.

Zosterabisphenone B, a new diarylheptanoid heterodimer from the seagrass Zostera marina, induces apoptosis cell death in colon cancer cells and reduces tumour growth in mice.

Colorectal cancer (CRC) is one of the most common malignant tumours worldwide. Diarylheptanoids, secondary metabolites isolated from Zostera marina, are of interest in natural products research due to their biological activities. Zosterabisphenone B (ZBP B) has recently been shown to inhibit the viability of CRC cells. The aim of this study was to investigate the therapeutic potential of ZBP B for targeting human CRC cells. Cell viability was determined using the MTT assay. Flow cytometry and Western blot analyses were used to assess apoptosis and autophagy. A CRC xenograft model was used to evaluate the in vivo effect of ZBP B. No cytotoxic effect on HCEC cells was observed in the in vitro experiments. ZBP B caused morphological changes in HCT116 colon cancer cells due to an increase in early and late apoptotic cell populations. Mechanistically, ZBP B led to an increase in cleaved caspase-3, caspase-8, caspase-9, PARP and BID proteins and a decrease in Bcl-2 and c-Myc proteins. In the xenograft model of CRC, ZBP B led to a reduction in tumour growth. These results indicate that ZBP B exerts a selective cytotoxic effect on CRC cells by affecting apoptotic signalling pathways and reducing tumour growth in mice. Taken together, our results suggest that ZBP B could be a lead compound for the synthesis and development of CRC drugs.

Fumaria vaillantii extract protects PC12 cells against neurotoxicity induced by 6-OHDA.

Parkinson's disease is a neurological disorder caused by the loss of dopaminergic neurons in the midbrain. Various mechanisms are involved in the incidence of the disease including oxidative stress. Several herbs and natural products may interfere with the oxidative-stress pathway due to their antioxidant effects. Herein, we aimed to investigate the neuroprotective role of F. vaillantii extract on Parkinson's in vitro and in vivo model owing to the presence of the bioactive agents with antioxidant properties. In vitro experments showed that 6-hydroxydopamine could induce toxicity in PC12 cells. The impact of F. vaillantii extract on cell viability was measured by using MTT assay. Nuclear morphological changes were qualitatively evaluated employing Hoechst staining. The antioxidant activity of the extract was determined by ROS and lipid peroxidation assays. Tyrosine hydroxylase protein expression was measured by western blotting in PC12 cells. For in vivo study, movement parameters were evaluated. The results indicated that 75 µΜ of 6-OHDA induced 50% toxicity in PC12 cells for 24h. Following post-treatment with F. vaillantii extract (0.1mg/ml) for 72h, we observed that the extract effectively prevented cell toxicity induced by 6-OHDA and reduced the apoptotic cell population. Furthermore, the extract attenuated the ROS level, lipid peroxidation and increased protein expression of TH after 72h of treatment. In addition, oral administration of 300mg/kg of F. vaillantii extract for 14 days improved locomotor activity, catalepsy, bradykinesia, motor coordination and reduced the apomorphine-caused rotation in 6-OHDA- induced Parkinson's disease-like symptoms in male rats. The present study suggests a protective role for the extract of F. vaillantii against oxidative stress-induced cell damage in the PC12 cells exposed to neurotoxin 6-OHDA which was verified in in vivo model by reducing the motor defects induced by 6-OHDA. This extract could be a promising therapeutic agent for the prevention of PD progression.

#170 Vasoconstriction Inhibiting Factor: An endogenous inhibitor of vascular calcification as calcimimetic of calcium-sensing receptor

Abstract Background and Aims Patients with chronic kidney disease (CKD), have an increased risk of cardiovascular disease due to the massively accelerated calcification they develop. Vascular calcification is a highly regulated process mediated by different inducers and inhibitors. Recently, the peptide ‘vasoconstriction inhibiting factor’ (VIF) was isolated from bovine adrenal glands and has been described as an inhibitor of the angiotensin II-induced vasoconstriction. Angiotensin II inhibits calcium deposition, but VIF effect on vascular calcification is still unknown. In the present study, VIF has been characterised on vascular calcification assays. Method The effect of VIF was analysed in vitro in human aortic smooth muscle cells (hAoSMC) and ex vivo in rat aortic rings, both cultivated under high phosphate concentrations. VIF was also studied in vivo in rats treated with vitamin D and nicotine (calcification model). Results VIF inhibits calcium deposition in all the models studied. Furthermore, in hAoSMC VIF reduces the production of ROS and the initiation of diverse cascades in the cells, like activation of inflammatory cytokines and MAPK kinases, which in turn trigger the expression of various genes involved in the development of vascular calcification. Furthermore, in presence of VIF the population of apoptotic cells, directly linked to vascular calcification, is decreased. Calcium-sensing receptor (CaSR) has been found as VIF binding partner, pointing to VIF as a new calcimimetic of this receptor, that inhibits vascular calcification by increasing the production of carboxylated Matrix Gla Proteins (cMGP). Conclusion In conclusion, VIF is a new potent endogenous inhibitor of vascular calcification acting as a calcimimetic of CaSR, that leads to an increase cMGP production. This finding represents a basis for a new target for the prevention and therapy of patients with increased vascular calcification and shows an encouraging perspective for the future.

18‑α‑glycyrrhetinic acid induces apoptosis in gingival fibroblasts exposed to phenytoin.

Phenytoin (PHT)-induced gingival overgrowth is caused by the increased proliferation and reduced apoptosis of gingival fibroblasts in inflammatory gingiva. Licorice has long been used as a component of therapeutic preparations. It inhibits cell proliferation, induces cell apoptosis and has anti-inflammatory effects. 18-α-glycyrrhetinic acid (18α-GA), the active compound in licorice, promotes apoptosis in various types of cells. The present study determined whether 18α-GA affects apoptosis in gingival fibroblasts exposed to PHT. The present study aimed to establish a basis for the therapeutic application of 18α-GA to treat the gingival overgrowth induced by PHT. Human gingival fibroblasts from healthy donors were cultured to semi-confluence and then stimulated in serum-free DMEM containing PHT with or without 18α-GA for subsequent experiments. Apoptotic cells were detected by ELISA. Analysis of the distribution of cell cycle phases and the apoptotic cell population was performed by flow cytometry. The expression levels of mRNAs and proteins of apoptotic regulators were measured using reverse transcription-quantitative PCR and western blotting, respectively. Caspase (CASP) activities were assessed by an ELISA. Treatment with 18α-GA markedly increased the number of apoptotic cells, reduced BCL2 mRNA expression, increased CASP2 and receptor (TNFRSF)-interacting serine-threonine kinase 1 (RIPK1) domain containing adaptor with death domain, Fas (TNFRSF6)-associated via death domain, RIPK1, tumor necrosis factor receptor superfamily; member 1A, TNF receptor-associated factor 2, CASP2, CASP3 and CASP9 mRNA expression, and also upregulated the protein expression levels and activities of caspase-2, caspase-3 and caspase-9. These results demonstrated that 18α-GA induced apoptosis through the activation of the Fas and TNF pathways in the death receptor signaling pathway in gingival fibroblasts treated with PHT. 18α-GA exhibited therapeutic potential for the treatment of PHT-induced gingival overgrowth.

Determination of the therapeutic effects of apigenin on chronic myeloid leukemia stem cells; a mechanistic approach

Aims: Chronic myeloid leukemia (CML) is a type of leukemia characterized by the Philadelphia (Ph) chromosome encoding the BCR-ABL protein. Significant success has been achieved in the treatment of CML with the development of agents that target this protein and inhibit its activity, but the resistance that develops in patients against these drugs limits absolute success. One of the main reasons for the resistance problem is the ineffectiveness of current therapies for CML stem cells. Apigenin (4',5,7- trihydroxyflavone) is a flavanoid found in various vegetables and fruits and has the effects of suppressing proliferation and inducing apoptosis in different cancer types. In this study, we aimed to determine the therapeutic potential of apigenin on K562 CML stem cells. Methods: The effects of apigenin on the proliferation of K562 CML stem cells were determined by an XTT cell proliferation assay. Apoptotic effects of apigenin on K562 CML stem cells were determined by changes in mitochondrial membrane potential (MZP), caspase-3 enzyme activity, and the Annexin-V method using flow cytometry. Results: The proliferation of K562 CML stem cells exposed to increasing doses of Apigenin (1-40 nM) for 48 hours decreased dose-dependently, and the IC50 value of Apigenin was calculated at 3 nM. Compared to the control group, an increase in caspase-3 enzyme activity was calculated in stem cells treated with apigenin at the same doses. Disruptions in the mitochondrial membrane potential of Apigenin-treated CML stem cells and Annexin-V assay results also showed that Apigenin dose-dependently induced apoptosis and caused significant increases in the apoptotic cell population. Conclusion: It was shown for the first time in this study that apigenin may have therapeutic effects on CML stem cells. It was determined that this effect of apigenin was achieved by triggering caspase-3 enzyme activity and disruptions in the mitochondrial membrane.

Scopoletin alleviates high glucose-induced toxicity in human renal proximal tubular cells via inhibition of oxidative damage, epithelial-mesenchymal transition, and fibrogenesis.

Recent years of evidence suggest the crucial role of renal tubular cells in developing diabetic kidney disease. Scopoletin (SCOP) is a plant-based coumarin with numerous biological activities. This study aimed to determine the effect of SCOP on renal tubular cells in developing diabetic kidney disease and to elucidate mechanisms. In this study, SCOP was evaluated in vitro using renal proximal tubular (HK-2) cells under hyperglycemic conditions to understand its mechanism of action. In HK-2 cells, SCOP alleviated the high glucose-generated reactive oxygen species (ROS), restored the levels of reduced glutathione, and decreased lipid peroxidation. High glucose-induced alteration in the mitochondrial membrane potential was markedly restored in the SCOP-treated cells. Moreover, SCOP significantly reduced the high glucose-induced apoptotic cell population in the Annexin V-FITC flow cytometry study. Furthermore, high glucose markedly elevated the mRNA expression of fibrotic and extracellular matrix (ECM) components, namely, transforming growth factor (TGF)-β, alfa-smooth muscle actin (α-SMA), collagen I, and collagen III, in HK-2 cells compared to the untreated cells. SCOP treatment reduced these mRNA expressions compared to the high glucose-treated cells. Collagen I and TGF-β protein levels were also significantly reduced in the SCOP-treated cells. Further findings in HK-2 cells revealed that SCOP interfered with the epithelial-mesenchymal transition (EMT) in the high glucose-treated HK-2 cells by normalizing E-cadherin and downregulating the vimentin and α-SMA proteins. In conclusion, SCOP modulates the high glucose-generated renal tubular cell oxidative damage and accumulation of ECM components and may be a promising molecule against diabetic nephropathy.

Polyphenolic dendrimers as carriers of anticancer siRNA

Dendrimers have emerged as an important group of nanoparticles to transport drugs, DNA, or RNA into target cells in cancer and other diseases. Various functional modifications can be imposed on dendrimers to increase the efficacy and specificity in delivering their cargo to the target cells and decrease their toxicity. In the present work, we evaluated the potential of carbosilane polyphenolic dendrimers modified with caffeic acid (CA) and polyethylene glycol (PEG) to deliver proapoptotic Mcl-1 and Bcl-2 siRNAs to A549 cancer cells. Dendrimers formed stable complexes with siRNAs as assessed by transmission electron microscopy and gel electrophoresis. Modification of dendrimers with PEG reduced the size and the zeta potential of dendrimer/siRNA complexes. The presence of PEG caused a red shift of the CD spectrum, and this effect was the more pronounced, the higher the dendrimer/siRNA ratio was. The nanocomplexes were internalized by A549. All studied dendrimer/siRNA formulations inhibited tumor cell migration and adhesion and caused an increase in the population of early apoptotic cells. Among four tested dendrimers, the polyphenolic compound containing two caffeic acid moieties complexed with siRNA demonstrated the lowest polydispersity index and showed an excellent transfection profile. In conclusion, this dendrimer are a promising candidate for the delivery of siRNA into cancer cells in further in vivo studies.