Activation Of Mitochondrial Pathway Research Articles

Flavonoid-Rich Extract of Oldenlandia diffusa (Willd.) Roxb. Inhibits Gastric Cancer by Activation of Caspase-Dependent Mitochondrial Apoptosis.

To evaluate the apoptosis and cycle arrest effects of Oldenlandia diffusa flavonoids on human gastric cancer cells, determine the action mechanisms in association with the mitochondrial dependent signal transduction pathway that controls production of reactive oxygen species (ROS), and evaluate the pharmacodynamics of a mouse xenotransplantation model to provide a reference for the use of flavonoids in prevention and treatment of gastric cancer. Flavonoids were extracted by an enzymatic-ultrasonic assisted method and purified with D-101 resin. Bioactive components were characterized by high-performance liquid chromatography. Cell lines MKN-45, AGS, and GES-1 were treated with different concentrations of flavonoids (64, 96, 128, 160 µg/mL). The effect of flavonoids on cell viability was evaluated by MTT method, and cell nuclear morphology was observed by Hoechst staining. The apoptosis rate and cell cycle phases were measured by flow cytometry, the production of ROS was detected by laser confocal microscope, the mitochondrial membrane potential (MMP) were observed by fluorescence microscope, and the expression of apoptotic proteins related to activation of mitochondrial pathway were measured by immunoblotting. MKN-45 cells were transplanted into BALB/c nude mice to establish a xenograft tumor model. Hematoxylin and eosin staining was used to reveal the subcutaneous tumor tissue. The tumor volume and tumor weight were measured, the expression levels of proliferation markers proliferating cell nuclear antigen (PCNA) and Ki-67 were detected by immunohistochemistry, and the expression levels of CA72-4 were measured by enzyme linked immunosorbent assay. Oldenlandia diffusa flavonoids inhibited proliferation of MKN-45 and AGS human gastric cancer cells, arrested the cell cycle in G1/S phase, induced accumulation of ROS in the process of apoptosis, and altered MMP. In addition, flavonoids increased Apaf-1, Cleaved-Caspase-3, and Bax, and decreased Cyclin A, Cdk2, Bcl-2, Pro-Caspase-9, and Mitochondrial Cytochrome C (P<0.05). The MKN-45 cell mouse xenotransplantation model further clarified the growth inhibitory effect of flavonoids towards tumors. The expression levels of PCNA and Ki-67 decreased in each flavonoid dose group, the expression level of CA72-4 decreased (P<0.05). Flavonoids derived from Oldenlandia diffusa can inhibit proliferation and induce apoptosis of human gastric cancer cells by activating the mitochondrial controlled signal transduction pathway.

Effect of Physical Activity/Exercise on Oxidative Stress and Inflammation in Muscle and Vascular Aging.

Functional status is considered the main determinant of healthy aging. Impairment in skeletal muscle and the cardiovascular system, two interrelated systems, results in compromised functional status in aging. Increased oxidative stress and inflammation in older subjects constitute the background for skeletal muscle and cardiovascular system alterations. Aged skeletal muscle mass and strength impairment is related to anabolic resistance, mitochondrial dysfunction, increased oxidative stress and inflammation as well as a reduced antioxidant response and myokine profile. Arterial stiffness and endothelial function stand out as the main cardiovascular alterations related to aging, where increased systemic and vascular oxidative stress and inflammation play a key role. Physical activity and exercise training arise as modifiable determinants of functional outcomes in older persons. Exercise enhances antioxidant response, decreases age-related oxidative stress and pro-inflammatory signals, and promotes the activation of anabolic and mitochondrial biogenesis pathways in skeletal muscle. Additionally, exercise improves endothelial function and arterial stiffness by reducing inflammatory and oxidative damage signaling in vascular tissue together with an increase in antioxidant enzymes and nitric oxide availability, globally promoting functional performance and healthy aging. This review focuses on the role of oxidative stress and inflammation in aged musculoskeletal and vascular systems and how physical activity/exercise influences functional status in the elderly.

A cold-water polysaccharide-protein complex from Grifola frondosa exhibited antiproliferative activity via mitochondrial apoptotic and Fas/FasL pathways in HepG2 cells

Grifola frondosa (G. frondosa) is widely known for its anti-tumor potential, which has been demonstrated by numerous scientific researches. In this study, two water soluble polysaccharide-protein complexes were extracted from G. frondosa at 4 °C (GFG-4) and 100 °C (GFG-100) and purified. Compared with GFG-100, GFG-4 had a higher protein content and molecular weight. The main monosaccharides of GFG-4 and GFG-100 were rhamnose, glucose, and galactose, with an approximate ratio of 3.00: 1.00: 0.86 and 2.85: 1.00: 0.94, respectively. The Fourier transform infrared spectra indicated that the two polysaccharide-protein complexes displayed characteristic functional groups of polysaccharides and proteins, and mainly contain pyranose ring with α-glycosidic linkage. Atomic force microscope images showed that both GFG-4 and GFG-100 exhibited straight chains, and GFG-4 possessed a relatively abundant fraction of branched chains. Intriguingly, GFG-4 showed a stronger antiproliferative activity against HepG2 cells than GFG-100. The mechanisms were further investigated by quantitative real-time PCR and western blot, it found that GFG-4 inhibited the proliferation of HepG2 cells mainly through the intrinsic activation of mitochondrial pathway and the Fas/FasL-mediated Caspase-8/-3 pathway. Conclusively, G. frondosa cold-water extracted polysaccharide-protein complexes could be used as a functional food for preventing or treating hepatocellular carcinoma.

Obesity-Associated Neuropathy: Recent Preclinical Studies and Proposed Mechanisms.

Significance: The prevalence of metabolic syndrome (MetS) and associated obesity has increased in recent years, affecting millions worldwide. One of the most common complications of obesity is damage to the peripheral nerve system, referred to as neuropathy. The lack of disease-modifying therapy for this complication is largely due to a poor understanding of the complex neurobiology underlying neuropathy. Recent preclinical studies suggest that in addition to glucotoxic events, other mechanisms, including lipid signaling, microbiome, or inflammation, may be viable targets to prevent nerve damage and neuropathic pain in obesity. Recent Advances: Clinical and preclinical studies using diet-induced obesity rodent models have identified novel interventions that improve neuropathy. Notably, mechanistic studies suggest that lipid, calcium signaling, and inflammation are converging pathways. Critical Issues: In this review, we focus on interventions and their mechanisms that are shown to ameliorate neuropathy in MetS obese models, including: (i) inhibition of a sensory neuron population, (ii), modification of dietary components, (iii) activation of nuclear and mitochondrial lipid pathways, (iv) exercise, and (v) modulation of gut microbiome composition and their metabolites. Future Directions: These past years, novel research increased our knowledge about neuropathy in obesity and discovered the involvement of nonglucose signaling. More studies are necessary to uncover the interplay between complex metabolic pathways in the peripheral nerve system of obese individuals. Further mechanistic studies in preclinical models and humans are crucial to create single- or multitarget interventions for this complex disease implying complex metabolic phenotyping. Antioxid. Redox Signal. 37, 597-612.

Ivermectin-Induced Apoptotic Cell Death in Human SH-SY5Y Cells Involves the Activation of Oxidative Stress and Mitochondrial Pathway and Akt/mTOR-Pathway-Mediated Autophagy.

Ivermectin (IVM) could cause potential neurotoxicity; however, the precise molecular mechanisms remain unclear. This study explores the cytotoxicity of IVM in human neuroblastoma (SH-SY5Y) cells and the underlying molecular mechanisms. The results show that IVM treatment (2.5–15 μM) for 24 h could induce dose-dependent cell death in SH-SY5Y cells. Compared to the control, IVM treatment significantly promoted the production of ROS, mitochondrial dysfunction, and cell apoptosis. IVM treatment also promoted mitophagy and autophagy, which were charactered by the decreased expression of phosphorylation (p)-Akt and p-mTOR proteins, increased expression of LC3II, Beclin1, ATG5, PINK, and Pakin1 proteins and autophagosome formation. N-acetylcysteine treatment significantly inhibited the IVM-induced production of ROS and cell death in SH-SY5Y cells. Autophagy inhibitor (e.g., 3-methyladenine) treatment significantly inhibited IVM-induced autophagy, oxidative stress, and cell apoptosis. Taken together, our results reveal that IVM could induce autophagy and apoptotic cell death in SH-SY5Y cells, which involved the production of ROS, activation of mitochondrial pathway, and inhibition of Akt/mTOR pathway. Autophagy inhibition improved IVM-induced oxidative stress and apoptotic cell death in SH-SY5Y cells. This current study provides new insights into understanding the molecular mechanism of IVM-induced neurotoxicity and facilitates the discovery of potential neuroprotective agents.

A Novel Imidazo[1,2-a]pyridine Compound Reduces Cell Viability and Induces Apoptosis of HeLa Cells by p53/Bax-Mediated Activation of Mitochondrial Pathway.

Despite emerging research on new treatment strategies, chemotherapy remains one of the most important therapeutic modalities for cancers. Imidazopyridines are important targets in organic chemistry and, given their numerous applications, they are worthy of attention. The objective of this study was to design and synthesize a novel series of imidazo[1,2-a]pyridine-derived compounds and investigate their antitumor effects and the underlying mechanisms. Imidazo[1,2-a]pyridine-derived compounds were synthesized with new strategies and conventional methods. The antitumor activities of the new compounds were evaluated by MTT assay. Flow cytometry and immunofluorescence were performed to examine the effects of the most effective antiproliferative compound on cell apoptosis. Western blot analysis was used to assess the expression of apoptotic proteins. Fifty-two new imidazo[1,2-a]pyridine compounds were designed and successfully synthesized. The compound, 1-(imidazo[1,2-a]pyridin-3-yl)-2-(naphthalen-2-yl)ethane-1,2-dione, named La23, showed high potential for suppressing the viability of HeLa cells (IC50 15.32 μM). La23 inhibited cell proliferation by inducing cell apoptosis, and it reduced the mitochondrial membrane potential of HeLa cells. Moreover, treatment with La23 appeared to increase the expression of apoptotic-related protein P53, Bax, cleaved caspase-3, and cytochrome c at a low concentration range. The novel imidazo[1,2-a]pyridine compound, La23, was synthesized and it suppressed cell growth by inducing cell apoptosis via the p53/Bax mitochondrial apoptotic pathway.

Experimental hyperthermia: expression of proteins involved in the regulation of ovarian corpus luteum apoptosis in the acute and recovery periods

Background.Heat shock effects can initiate apoptosis of oocytes and corpus luteum cells in mammalian ovaries. During folliculogenesis, follicular apoptosis is regulated by Bcl-2 and BAX proteins which are key effectors of granular cell death. Mechanisms of disruption of the ovarian corpus luteum development under heat stress remain largely unclear.Aim of the research: to identify the expression features of anti-apoptotic Bad and proapoptotic Bcl-2 proteins in the rat ovarian luteocytes in the acute (by day 3) and recovery (by days 7 and 14) periods after a single exposure of experimental hyperthermia (EH) (rectal temperature 43.5 °C).Materials and methods. The expression of Bad and Bcl-2 was determined immunohistochemically using an indirect two-stage streptavidin-biotin method.Results. On day 3 after EH, the expression areas of both Bad and Bcl-2 increased 2-fold, but the ratio of Bcl-2/Bad areas did not change, indicating that the intensity of apoptosis along the mitochondrial pathway in luteocytes in the acute period was maintained within physiological values. On day 7, the Bad and Bcl-2 expression areas remained at the level of day 3, but the Bcl-2/Bad index decreased, indicating the activation of the apoptosis internal pathway in the ovarian corpus luteum cells. By day 14, the protein expression areas decreased (Bad – by 1.7 times, Bcl-2 – by 3.2 times) compared to the acute period, and the Bcl-2/Bad index decreased by 2 times compared to the control and the acute period group.Conclusion. The observed predominance of proapoptotic Bad protein over antiapoptotic Bcl-2 in luteocytes on day 14 after EH indicates the anti-apoptotic protection violation, which leads to the apoptosis mitochondrial pathway activation of the latter. A decrease in Bcl-2 expression can be regarded as a manifestation of the defective luteocytes removal mechanism and the body’s desire to normalize the ovarian-uterine cycle disrupted by high temperature exposure.

Semisynthesis and Non-Small-Cell Lung Cancer Cytotoxicity Evaluation of Germacrane-Type Sesquiterpene Lactones from Elephantopus scaber

Two series of germacrane-type sesquiterpene lactones were produced by semisynthetic modulation of scaberol C, which was prepared by a standard chemical transformation from an Elephantopus scaber extract. Their inhibition activities against non-small-cell lung cancer cells were screened, and preliminary structure-activity relationships were also established. Among them, monomeric analog 1u and dimeric analog 3d exhibited superior anti-non-small-cell lung cancer cytotoxic potencies with IC50 values of 4.3 and 0.7 μM against A549 cells, respectively, and were more active than cisplatin and the standard sesquiterpene lactones, parthenolide and scabertopin. Further studies revealed that compounds 1u and 3d cause G2/M phase arrest and induce apoptosis through the activation of mitochondrial pathways in A549 cells. Collectively, the results obtained suggest that compounds 1u and 3d are promising anti-non-small-cell lung cancer lead compounds.

Minimal Change Disease Is Associated with Mitochondrial Injury and STING Pathway Activation.

We hypothesized that minimal change disease (MCD) pathogenesis may be associated with mitochondrial injury, and that the degree of mitochondrial injury at the time of diagnosis may serve as a valuable prognostic marker. We compared urinary mitochondrial DNA (mtDNA) at the time of diagnosis in patients with MCD and age- and sex-matched healthy controls (MHC) (n = 10 each). We analyzed the site and signal intensity of immunohistochemical (IHC) staining of stimulator of interferon genes (STING) using kidney tissues at the time of diagnosis in patients with MCD. Patients with MCD were divided into high (n = 6) and low-intensity (n = 14) subgroups according to the signal intensity. Urinary mtDNA levels were elevated in the MCD groups more than in the MHC group (p < 0.001). Time-averaged proteinuria and frequency of relapses during the follow-up period were higher in the high-intensity than in the low-intensity subgroup (1.18 ± 0.54 vs. 0.57 ± 0.45 g/day, p = 0.022; and 0.72 ± 0.60 vs. 0.09 ± 0.22 episodes/year, p = 0.022, respectively). Mitochondrial injury may be associated with MCD pathogenesis, and the signal intensity of STING IHC staining at the time of diagnosis could be used as a valuable prognostic marker in MCD.

Cisplatin induces damage of auditory cells: Possible relation with dynamic variation in calcium homeostasis and responding channels

AimsThe present study was aimed to explore the possible mechanism(s) underlying the action of cisplatin on auditory cells of mice in vitro, with special attention given to the dynamic variation in calcium homeostasis and responding channels. MethodsThe apoptosis of auditory cells was tested by flow cytometry and TUNEL staining. The expressions of inositol 1,4,5-trisphosphate receptors (IP3R), voltage-dependent anion channel 1 (VDAC1), phosphorylated protein kinase R-like ER kinase (p-PERK), activating transcription factor 6 (ATF6), caspase-12, bcl-2, bax, cleaved caspase-9, cleaved caspase-3, beclin-1 and light chain 3β (LC3B) were measured by immunofluorescence or Western blotting. The calcium variations in subcellular structures were evaluated by Rhod-2 AM and Mag-Fluo-4 AM staining. The colocalization ratio between IP3R and beclin-1 was determined by immunocytochemistry. ResultsWe found that cisplatin exposure induced the apoptosis of HEI-OC1 cells and hair cells (HCs) in a caspase-3 dependent manner. This apoptotic process was attributed to the activation of endoplasmic reticulum (ER) stress and mitochondrial pathway and, meanwhile, accompanied by variation in calcium homeostasis and responding channels. Interestingly, we also observed that IP3R might dissociate from beclin-1 to motivate autophagy under the cisplatin insult. ConclusionsOverall, the findings from this work indicate that cisplatin leads to auditory cell damage of mice in vitro, which is closely relevant to dynamic variation in calcium homeostasis and responding channels in subcellular structure.

Regulation of ROS-Dependent JNK Pathway by 2'-Hydroxycinnamaldehyde Inducing Apoptosis in Human Promyelocytic HL-60 Leukemia Cells.

The present study demonstrated that 2′-hydroxycinnamaldehyde (2′-HCA) induced apoptosis in human promyelocytic leukemia HL-60 cells through the activation of mitochondrial pathways including (1) translocation of Bim and Bax from the cytosol to mitochondria, (2) downregulation of Bcl-2 protein expression, (3) cytochrome c release into the cytosol, (4) loss of mitochondrial membrane potential (ΔΨm), and (5) caspase activation. 2′-HCA also induced the activation of c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase1/2 (ERK1/2) in HL-60 cells. The pharmacological and genetic inhibition of JNK effectively prevented 2′-HCA-induced apoptosis and activator protein-1 (AP-1)-DNA binding. In addition, 2′-HCA resulted in the accumulation of reactive oxygen species (ROS) and depletion of intracellular glutathione (GSH) and protein thiols (PSH) in HL-60 cells. NAC treatment abrogated 2′-HCA-induced JNK phosphorylation, AP-1-DNA binding, and Bim mitochondrial translocation, suggesting that oxidative stress may be required for 2′-HCA-induced intrinsic apoptosis. Xenograft mice inoculated with HL-60 leukemia cells demonstrated that the intraperitoneal administration of 2′-HCA inhibited tumor growth by increasing of TUNEL staining, the expression levels of nitrotyrosine and pro-apoptotic proteins, but reducing of PCNA protein expression. Taken together, our findings suggest that 2′-HCA induces apoptosis via the ROS-dependent JNK pathway and could be considered as a potential therapeutic agent for leukemia.

The Protective Role of E-64d in Hippocampal Excitotoxic Neuronal Injury Induced by Glutamate in HT22 Hippocampal Neuronal Cells.

Epilepsy is the most common childhood neurologic disorder. Status epilepticus (SE), which refers to continuous epileptic seizures, occurs more frequently in children than in adults, and approximately 40–50% of all cases occur in children under 2 years of age. Conventional antiepileptic drugs currently used in clinical practice have a number of adverse side effects. Drug-resistant epilepsy (DRE) can progressively develop in children with persistent SE, necessitating the development of novel therapeutic drugs. During SE, the persistent activation of neurons leads to decreased glutamate clearance with corresponding glutamate accumulation in the synaptic extracellular space, increasing the chance of neuronal excitotoxicity. Our previous study demonstrated that after developmental seizures in rats, E-64d exerts a neuroprotective effect on the seizure-induced brain damage by modulating lipid metabolism enzymes, especially ApoE and ApoJ/clusterin. In this study, we investigated the impact and mechanisms of E-64d administration on neuronal excitotoxicity. To test our hypothesis that E-64d confers neuroprotective effects by regulating autophagy and mitochondrial pathway activity, we simulated neuronal excitotoxicity in vitro using an immortalized hippocampal neuron cell line (HT22). We found that E-64d improved cell viability while reducing oxidative stress and neuronal apoptosis. In addition, E-64d treatment regulated mitochondrial pathway activity and inhibited chaperone-mediated autophagy in HT22 cells. Our findings indicate that E-64d may alleviate glutamate-induced damage via regulation of mitochondrial fission and apoptosis, as well as inhibition of chaperone-mediated autophagy. Thus, E-64d may be a promising therapeutic treatment for hippocampal injury associated with SE.

Combination of oxytetracycline and quinocetone synergistically induces hepatotoxicity via generation of reactive oxygen species and activation of mitochondrial pathway

Oxytetracycline (OTC) and Quinocetone (QCT) are antimicrobials, whose residues have been found in food and environment. These two are sometimes used simultaneously in livestock and aquaculture, potentially resulting in the simultaneous consumption of multi-antimicrobials by consumers. However, the combined toxic effects of this phenomenon have yet to be addressed. Since the liver is a major target of both OTC and QCT, we tested their hepatotoxic effect using both cell cultures and animal models. Results showed that the QCT (5–25 μM) or OTC (20–100 μM) treatments alone caused dose-dependent reductions in cell numbers, while their combination strongly further enhanced inhibitory effects. Mechanistically, the combination enhanced the generation of reactive oxygen species (ROS) and activated mitochondrial cell death pathways. It also showed that the combination of OTC (800 mg/kg, i.g., 5d) and QCT (5000 mg/kg, i.g., 5d) resulted in significantly enhanced liver toxicity in C57BL/6N mice, the serum alanine transaminase (ALT) and aspartate transaminase (AST) were significantly increased by the OTC/QCT. These findings indicate the necessity of considering the combined toxicity of these two antimicrobials in safety assessments.

STAT3-mediated activation of mitochondrial pathway contributes to antitumor effect of dihydrotanshinone I in esophageal squamous cell carcinoma cells

BACKGROUNDEsophageal squamous cell carcinoma (ESCC) is one of the most common malignancies with a poor prognosis, and its treatment remains a great challenge. Dihydrotanshinone I (DHTS) has been reported to exert antitumor effect in many cancers. However, the role of DHTS in ESCC remains unclear. AIMTo investigate the antitumor effect of DHTS in ESCC and the underlying mechanisms.METHODSCCK-8 assay and cell cycle analysis were used to detect proliferation and cell cycle in ESCC cells. Annexin V-PE/7-AAD double staining assay and Hoechst 33258 staining were used to detect apoptosis in ESCC cells. Western blot was used to detect the expression of proteins associated with the mitochondrial pathway. Immunofluorescence was used to detect the expression of phosphorylated STAT3 (pSTAT3) in DHTS-treated ESCC cells. ESCC cells with STAT3 knockdown and overexpression were constructed to verify the role of STAT3 in DHTS induced apoptosis. A xenograft tumor model in nude mice was used to evaluate the antitumor effect of DHTS in vivo.RESULTSAfter treatment with DHTS, the proliferation of ESCC cells was inhibited in a dose- and time-dependent manner. Moreover, DHTS induced cell cycle arrest in the G0/1 phase. Annexin V-PE/7-AAD double staining assay and Hoechst 33258 staining revealed that DHTS induced obvious apoptosis in KYSE30 and Eca109 cells. At the molecular level, DHTS treatment reduced the expression of pSTAT3 and anti-apoptotic proteins, while increasing the expression of pro-apoptotic proteins in ESCC cells. STAT3 knockdown in ESCC cells markedly promoted the activation of the mitochondrial pathway while STAT3 overexpression blocked the activation of the mitochondrial pathway. Additionally, DHTS inhibited tumor cell proliferation and induced apoptosis in a xenograft tumor mouse model.CONCLUSIONDHTS exerts antitumor effect in ESCC via STAT3-mediated activation of the mitochondrial pathway. DHTS may be a novel therapeutic agent for ESCC.

Plumbagin induces testicular damage via mitochondrial-dependent cell death

Different aspects of reproductive functions are regulated by mitochondrial-controlled events. This study investigated the effect of plumbagin (PL) on testicular mitochondria with a view to unravelling the mechanism of the antifertility potential of plumbagin in testis of healthy rats. Thirty-two male Wistar strain albino rats were randomly allocated into four groups of eight animals each. The control or healthy group received orally 0.1 % DMSO while animals in the remaining three groups received 2.5 mg PL/kg bdwt, 5.0 mg PL/kg bdwt and 10 mg PL/kg bdwt, respectively, for 14 days. In study two, twenty-four male Wistar rats were randomly divided into three (3) groups and were orally administered 0.1% DMSO (control), 30 and 100 mg/kg PL, respectively once daily for 72 h. Rat testis mitochondria were isolated using differential centrifugation. The mitochondrial Permeability Transition (mPT) pore, mitochondrial ATPase (mATPase) activity and mitochondrial lipid peroxidation were assessed spectrophotometrically. Expression of apoptotic proteins (p53, Bax, Bcl-2) and the release of cytochrome c were determined by immunochemical technique. Reproductive receptors (FSH, PR), the expression of aromatase, Testis Specific Kinase-1 {TESK-1} were quantified by RT-PCR. The various doses of plumbagin (2.5, 5.0 and 10 mg/kg bdwt) induced opening of the testicular mPT pore by 2, 5 and 8 folds, respectively, after 14 days of oral administration. These doses of plumbagin also caused enhancement of mATPase activity, elevated generation of mLPO as well as increases in the concentrations of caspases 9 and 3. Sperm analysis revealed that these doses of PL also caused significant decreases in sperm count and motility and increased sperm abnormalities compared to control. Interestingly, these effects were accompanied by dose-dependent expressions of the Bak, p53 and cytochrome c release. Conversely, the abundance of anti‐apoptotic Bcl-2 protein decreased relative to control. The levels of transcripts of FSH and progesterone receptors as well as TESK-1 and aromatase decreased significantly relative to control. Furthermore, PL strongly inhibited p53-MDM2 compared to control. Altogether, these findings show that plumbagin damages testicular cells through the activation of mitochondrial pathway involving the p53 protein network.

Feiyanning Formula Induces Apoptosis of Lung Adenocarcinoma Cells by Activating the Mitochondrial Pathway.

Feiyanning formula (FYN) is a traditional Chinese medicine (TCM) prescription used for more than 20 years in the treatment of lung cancer. FYN is composed of Astragalus membranaceus, Polygonatum sibiricum, Atractylodes macrocephala, Cornus officinalis, Paris polyphylla, and Polistes olivaceous, etc. All of them have been proved to have anti-tumor effect. In this study, we used the TCM network pharmacological analysis to perform the collection of compound and disease target, the prediction of compound target and biological signal and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. It was found that the activation of mitochondrial pathway might be the molecular mechanism of the anti-lung cancer effect of FYN. The experimental results showed that FYN had an inhibitory effect on the growth of lung cancer cells in a dose-dependent and time-dependent manner. Moreover, FYN induced G2/M cell cycle arrest and apoptotic cell death as early as 6 h after treatment. In addition, FYN significantly induced mitochondrial membrane depolarization and increased calreticulin expression. Metabolomics analysis showed the increase of ATP utilization (assessed by a significant increase of the AMP/ATP and ADP/ATP ratio, necessary for apoptosis induction) and decrease of polyamines (that reflects growth potential). Taken together, our study suggested that FYN induced apoptosis of lung adenocarcinoma cells by promoting metabolism and changing the mitochondrial membrane potential, further supporting the validity of network pharmacological prediction.

Pro-Apoptotic Activity of Artichoke Leaf Extracts in Human HT-29 and RKO Colon Cancer Cells.

(1) Background: Cynara cardunculus L. subsp. scolymus (L.) Hegi, popularly known as artichoke, is an herbaceous plant belonging to the Asteraceae family. Artichoke leaf extracts (ALEs) have been widely used in traditional medicine because of their hepatoprotective, cholagogic, hypoglycaemic, hypolipemic and antibacterial properties. ALEs are also recognized for their antioxidative and anti-inflammatory activities. In this study, we evaluated the cytotoxic, genotoxic, and apoptotic activities, as well as effect on cell growth of ALEs on human colon cancer HT-29 and RKO cells. HT-29 and RKO cells exhibit a different p53 status: RKO cells express the wild-type protein, whereas HT-29 cells express a p53-R273H contact mutant. (2) Methods: Four different ALEs were obtained by sequential extraction of dried artichoke leaves; ALEs were characterized for their content in chlorogenic acid, cynaropicrin, and caffeoylquinic acids. HT-29 and RKO cells were used for in vitro testing (i.e., cytotoxicity and genotoxicity assessment, cell cycle analysis, apoptosis induction). (3) Results: Two out of the four tested ALEs showed marked effects on cell vitality toward HT-29 and RKO tumour cells. The effect was accompanied by a genotoxic activity exerted at a non-cytotoxic concentrations, by a significant perturbation of cell cycle (i.e., with increase of cells in the sub-G1 phase), and by the induction of apoptosis. (4) Conclusions: ALEs rich in cynaropicrin, caffeoylquinic acids, and chlorogenic acid showed to be capable of affecting HT-29 and RKO colon cancer cells by inducing favourable biological effects: cell cycle perturbation, activation of mitochondrial dependent pathway of apoptosis, and the induction of genotoxic effects probably mediated by the induction of apoptosis. Taken together, these results weigh in favour of a potential cancer chemotherapeutic activity of ALEs.

Mitochondria's Role in the Maintenance of Cancer Stem Cells in Glioblastoma.

Glioblastoma (GBM), one of the deadliest primary brain malignancies, is characterized by a high recurrence rate due to its limited response to existing therapeutic strategies such as chemotherapy, radiation therapy, and surgery. Several mechanisms and pathways have been identified to be responsible for GBM therapeutic resistance. Glioblastoma stem cells (GSCs) are known culprits of GBM resistance to therapy. GSCs are characterized by their unique self-renewal, differentiating capacity, and proliferative potential. They form a heterogeneous population of cancer stem cells within the tumor and are further divided into different subpopulations. Their distinct molecular, genetic, dynamic, and metabolic features distinguish them from neural stem cells (NSCs) and differentiated GBM cells. Novel therapeutic strategies targeting GSCs could effectively reduce the tumor-initiating potential, hence, a thorough understanding of mechanisms involved in maintaining GSCs’ stemness cannot be overemphasized. The mitochondrion, a regulator of cellular physiological processes such as autophagy, cellular respiration, reactive oxygen species (ROS) generation, apoptosis, DNA repair, and cell cycle control, has been implicated in various malignancies (for instance, breast, lung, and prostate cancer). Besides, the role of mitochondria in GBM has been extensively studied. For example, when stressors, such as irradiation and hypoxia are present, GSCs utilize specific cytoprotective mechanisms like the activation of mitochondrial stress pathways to survive the harsh environment. Proliferating GBM cells exhibit increased cytoplasmic glycolysis in comparison to terminally differentiated GBM cells and quiescent GSCs that rely more on oxidative phosphorylation (OXPHOS). Furthermore, the Warburg effect, which is characterized by increased tumor cell glycolysis and decreased mitochondrial metabolism in the presence of oxygen, has been observed in GBM. Herein, we highlight the importance of mitochondria in the maintenance of GSCs.

Protease Inhibitor Anti-HIV, Lopinavir, Impairs Placental Endocrine Function.

Protease Inhibitors (PI e.g., ritonavir (RTV) and lopinavir (LPV)) used to treat pregnant mothers infected by HIV induce prematurity and endocrine dysfunctions. The maintenance of pregnancy relies on placental hormone production (human Chorionic Gonadotrophin (hCG) and progesterone (P4)). Those functions are ensured by the villous trophoblast and are mainly regulated by the Unfolded Protein Response (UPR) pathway and mitochondria. We investigated, in vitro, if PI impair hCG and P4 production and the potential intracellular mechanisms involved. Term villous cytotrophoblast (VCT) were cultured with or without RTV or LPV from 6 to 48 h. VCT differentiation into syncytiotrophoblast (ST) was followed measuring hCG and P4 secretion. We evaluated the expression of P4 synthesis partners (Metastatic Lymph Node 64 (MLN64), cholesterol side-chain cleavage (P450SCC), Hydroxy-delta-5-Steroid Dehydrogenase and 3 Beta-and steroid delta-isomerase 1 (HSD3B1)), of mitochondrial pro-fusion factors (Mitofusin 2 (Mfn2), Optic Atrophy 1 (OPA1)) and of UPR factors (Glucose-Regulated Protein 78 (GRP78), Activating Transcription Factor 4 (ATF4), Activating Transcription Factor 6 (ATF6), spliced X-box Binding Protein 1 (sXBP1)). RTV had no significant effect on hCG and P4 secretion, whereas lopinavir significantly decreased both secretions. LPV also decreased P450SCC and HSD3B1 expression, whereas it increased Mfn2, GRP78 and sXBP1 expression in ST. RTV has no effect on the endocrine placenta. LPV impairs both villous trophoblast differentiation and P4 production. It is likely to act via mitochondrial fusion and UPR pathway activation. These trophoblastic alterations may end in decreased P4 levels in maternal circulation, inducing prematurity.

Benzo(a)pyrene induces MUC5AC expression through the AhR/mitochondrial ROS/ERK pathway in airway epithelial cells

ObjectivesBenzo(a)pyrene (BaP) is a ubiquitous air pollutants, and BaP exposure leads to a risk of respiratory diseases. The oversecretion of airway mucus and high expression of mucin 5AC (MUC5AC) are associated with common respiratory disorders caused by air pollution. We aimed to investigate the effect of BaP on MUC5AC expression, especially the mechanisms by which BaP induces MUC5AC gene expression. MethodsThe human airway epithelial cell NCI-H292 was used to test the effects of BaP on the expression of MUC5AC in vitro. MUC5AC mRNA and protein expression were assessed with real-time quantitative PCR, immunochemistry, and western blotting. A luciferase assay was conducted to detect the activity of the promoter. The total cellular ROS and mitochondrial ROS were measured by corresponding probes. Small-interfering RNAs were used for gene silencing. AhR-overexpressing cell lines were constructed by transfection with AhR overexpression lentivirus. ResultsWe found that BaP stimulation upregulated the MUC5AC mRNA and protein levels and activated the ERK pathway. Suppressing ERK with U0126 (an ERK inhibitor) or knocking down ERK with siRNA decreased BaP-induced MUC5AC expression. The luciferase activity transfected with the MUC5AC promoter and cAMP-response element (CRE) was increased after BaP treatment, whereas CREB siRNA suppressed the BaP-induced overexpression of MUC5AC. In addition, BaP increased mitochondrial ROS production, and Mito-TEMP, a mitochondrial ROS inhibitor, inhibited BaP-induced MUC5AC expression and ERK activation. BaP increased the mRNA levels of CYP1A1 and CYP1B1, while Alizarin, a CYP1s inhibitor, suppressed the effects of BaP, including the MUC5AC overexpression, ERK activation and mitochondrial ROS generation. BaP induced the translocation of aryl hydrocarbon receptor (AhR) from the cytoplasm to the nucleus. SiRNA-mediated knockdown or chemical inhibition of AhR decreased the BaP-induced expression of MUC5AC, while the overexpression of AhR significantly enhanced the BaP-induced expression of MUC5AC. ITE, an endogenous AhR ligand, also upregulated the mRNA and protein expression of MUC5AC. Furthermore, resveratrol treatment inhibited the BaP-induced MUC5AC overexpression, AhR translocation, mitochondrial ROS production and ERK pathway activation. ConclusionHere, we highlighted the crucial role of AhR/mitochondrial ROS/ERK pathway activation in BaP-induced MUC5AC overexpression and identified resveratrol as a promising drug to reduce BaP-induced MUC5AC overexpression.