Scoparone induces autophagic cell death via the PAK1/AKT axis in colorectal cancer.

Colorectal cancer (CRC) is one of most common malignancies worldwide, yet curative therapy remains a clinical challenge. Here, we demonstrate that scoparone (Scop), a traditional Chinese medicine monomer, inhibits the growth of CRC cells both in vitro and in vivo. Further studies found that Scop treatment induces complete autophagic flux in CRC cells, while inhibition of autophagy markedly represses the antiproliferative activities of Scop, suggesting an antitumour property of Scop-induced autophagy in CRC. Mechanistically, Scop induced autophagy initiation by reducing P21-activated kinase 1 (PAK1) expression and subsequently repressing the AKT/mTOR signaling pathway. Collectively, our study suggests that Scop is a potential anti-CRC therapeutic option and provides an underlying molecular mechanism for its antitumour effect in CRC.

Usenamine A induces apoptosis and autophagic cell death of human hepatoma cells via interference with the Myosin-9/actin-dependent cytoskeleton remodeling.

BACKGROUND: Hepatocellular carcinoma (HCC) is a major cause of cancer-associated mortality worldwide. Myosin-9's role in HCC and the anti-HCC effect of the drugs targeting Myosin-9 remain poorly understood so far. Candidate antitumor agents obtained from natural products have attracted worldwide attention. Usenamine A is a novel product, which was first extracted in our laboratory from the lichen Usnea longissima. According to published reports, usenamine A exhibits good antitumor activity, while the mechanisms underlying its antitumor effects remain to be elucidated. PURPOSE: The present study investigated the anti-hepatoma effect of usenamine A and the underlying molecular mechanisms, along with evaluating the therapeutic potential of targeting Myosin-9 in HCC. METHODS: The CCK-8, Hoechst staining, and FACS assays were conducted in the present study to investigate how usenamine A affected the growth and apoptosis of human hepatoma cells. Moreover, TEM, acridine orange staining, and immunofluorescence assay were performed to explore the induction of autophagy by usenamine A in human hepatoma cells. The usenamine A-mediated regulation of protein expression in human hepatoma cells was analyzed using immunoblotting. MS analysis, SPR assay, CETSA, and molecular modeling were performed to identify the direct target of usenamine A. Immunofluorescence assay and co-immunoprecipitation assay were conducted to determine whether usenamine A affected the interaction between Myosin-9 and the actin present in human hepatoma cells. In addition, the anti-hepatoma effect of usenamine A was investigated in vivo using a xenograft tumor model and the IHC analysis. RESULTS: The present study initially revealed that usenamine A could suppress the proliferation of HepG2 and SK-HEP-1 cells (hepatoma cell lines). Furthermore, usenamine A induced cell apoptosis via the activation of caspase-3. In addition, usenamine A enhanced autophagy. Moreover, usenamine A administration could dramatically suppress the carcinogenic ability of HepG2 cells, as evidenced by the nude mouse xenograft tumor model. Importantly, it was initially revealed that Myosin-9 was a direct target of usenamine A. Usenamine A could block cytoskeleton remodeling through the disruption of the interaction between Myosin-9 and actin. Myosin-9 participated in suppressing proliferation while inducing apoptosis and autophagy in response to treatment with usenamine A. In addition, Myosin-9 was revealed as a potential oncogene in HCC. CONCLUSIONS: Usenamine A was initially revealed to suppress human hepatoma cells growth by interfering with the Myosin-9/actin-dependent cytoskeleton remodeling through the direct targeting of Myosin-9. Myosin-9 is, therefore, a promising candidate target for HCC treatment, while usenamine A may be utilized as a possible anti-HCC therapeutic, particularly in the treatment of HCC with aberrant Myosin-9.

Pomiferin targets SERCA, mTOR, and P-gp to induce autophagic cell death in apoptosis-resistant cancer cells, and reverses the MDR phenotype in cisplatin-resistant tumors in vivo.

Drug resistance in cancer has been classified as innate resistance or acquired resistance, which were characterized by apoptotic defects and ABC transporters overexpression respectively. Therefore, to preclude or reverse these resistance mechanisms could be a promising strategy to improve chemotherapeutic outcomes. In this study, a natural product from Osage Orange, pomiferin, was identified as a novel autophagy activator that circumvents innate resistance by triggering autophagic cell death via SERCA inhibition and activation of the CaMKKß-AMPK-mTOR signaling cascade. In addition, pomiferin also directly inhibited the P-gp (MDR1/ABCB1) efflux and reversed acquired resistance by potentiating the accumulation and efficacy of the chemotherapeutic agent, cisplatin. In vivo study demonstrated that pomiferin triggered calcium-mediated tumor suppression and exhibited an anti-metastatic effect in the LLC-1 lung cancer-bearing mouse model. Moreover, as an adjuvant, pomiferin potentiated the anti-tumor effect of the chemotherapeutic agent, cisplatin, in RM-1 drug-resistant prostate cancer-bearing mouse model by specially attenuating ABCB1-mediated drug efflux, but not ABCC5, thereby promoting the accumulation of cisplatin in tumors. Collectively, pomiferin may serve as a novel effective agent for circumventing drug resistance in clinical applications.

Targeting regulated cell death with plant natural compounds for cancer therapy: A revisited review of apoptosis, autophagy-dependent cell death, and necroptosis.

Regulated cell death (RCD) refers to programmed cell death regulated by various protein molecules, such as apoptosis, autophagy-dependent cell death, and necroptosis. Accumulating evidence has recently revealed that RCD subroutines have several links to many types of human cancer; therefore, targeting RCD with pharmacological small-molecule compounds would be a promising therapeutic strategy. Moreover, plant natural compounds, small-molecule compounds synthesized from plant sources, and their derivatives have been widely reported to regulate different RCD subroutines to improve potential cancer therapy. Thus, in this review, we focus on updating the intricate mechanisms of apoptosis, autophagy-dependent cell death, and necroptosis in cancer. Moreover, we further discuss several representative plant natural compounds and their derivatives that regulate the above-mentioned three subroutines of RCD, and their potential as candidate small-molecule drugs for the future cancer treatment.

Hernandezine induces autophagic cell death in human pancreatic cancer cells via activation of the ROS/AMPK signaling pathway.

Hernandezine (Her) is a bisbenzylisoquinoline alkaloid extracted from the traditional Chinese herbal medicine Thalictrum glandulosissimum. Evidence shows that Her is a natural agonist of adenosine monophosphate (AMP)-activated protein kinase (AMPK) and induces apoptosis and autophagy in tumor cells. In this study, we investigated the role of autophagy in Her-induced cell death in human pancreatic cancer cell lines. We showed that Her dose-dependently suppressed cell proliferation, promoted autophagy and induced autophagic death in pancreatic ductal adenocarcinoma (PDAC) cell lines Capan-1 and SW1990. The IC50 values of Her in inhibition of Capan-1 and SW1990 cells were 47.7 µM and 40.1 µM, respectively. Immunoblotting showed that Her (1-40 µM) promoted the conversion of LC3-I to LC3-II, and Her exerted concentration-dependent and time-dependent effects on autophagy activation in PDAC cells. In transmission electron microscopy and fluorescence image analysis, we found that autophagic vacuoles were significantly increased in Her-treated cells. Knockdown of ATG5, a key gene in the autophagy pathway, alleviated the activation of autophagy by Her. These results demonstrated that Her induced autophagy in PDAC cells. Intensely activated autophagy could promote cell death. The autophagy inhibitors, BafA1 and HCQ significantly inhibited Her-induced cell death, implying that Her induced autophagic cell death in PDAC cells. Moreover, we showed that Her activated autophagy by increasing the phosphorylation of AMPK and decreasing the phosphorylation of mTOR/p70S6K. Knockdown of AMPKα relieves the autophagic cell death induced by Her. Furthermore, Her concentration-dependently enhanced reactive oxygen species (ROS) generation in PDAC cells. Antioxidants could reduce the phosphorylation of AMPK and suppress autophagic cell death induced by Her. Our study provides evidence for the development of Her as a therapeutic agent for the treatment of pancreatic cancer.

Selaginella tamariscina Inhibits Glutamate-Induced Autophagic Cell Death by Activating the PI3K/AKT/mTOR Signaling Pathways.

Glutamate-induced neural toxicity in autophagic neuron death is partially mediated by increased oxidative stress. Therefore, reducing oxidative stress in the brain is critical for treating or preventing neurodegenerative diseases. Selaginella tamariscina is a traditional medicinal plant for treating gastrointestinal bleeding, hematuria, leucorrhea, inflammation, chronic hepatitis, gout, and hyperuricemia. We investigate the inhibitory effects of Selaginella tamariscina ethanol extract (STE) on neurotoxicity and autophagic cell death in glutamate-exposed HT22 mouse hippocampal cells. STE significantly increased cell viability and mitochondrial membrane potential and decreased the expression of reactive oxygen species, lactate dehydrogenase release, and cell apoptosis in glutamate-exposed HT22 cells. In addition, while glutamate induced the excessive activation of mitophagy, STE attenuated glutamate-induced light chain (LC) 3 II and Beclin-1 expression and increased p62 expression. Furthermore, STE strongly enhanced the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) phosphorylation activation. STE strongly inhibited glutamate-induced autophagy by activating the PI3K/Akt/mTOR signaling pathway. In contrast, the addition of LY294002, a PI3K/Akt inhibitor, remarkably suppressed cell viability and p-Akt and p62 expression, while markedly increasing the expression of LC3 II and Beclin-1. Our findings indicate that autophagy inhibition by activating PI3K/Akt/mTOR phosphorylation levels could be responsible for the neuroprotective effects of STE on glutamate neuronal damage.

Diterpene promptly executes a non-canonical autophagic cell death in doxorubicin-resistant lung cancer.

16-hydroxycleroda-3,13-dien-15,16-olide (HCD) has antitumor activity reported in numerous types of cancers. However, the efficacy of HCD treatment in non-small-cell lung cancer (NSCLC) cells and doxorubicin-resistant (Dox-R)-NSCLC cells remains to be unraveled. The underlying anti-cancer mechanism of HCD on Dox-R and Dox-sensitive (Dox-S) of A549 cells was also investigated. Cytotoxicity of HCD against two cell lines (Dox-S and Dox-R) were determined via MTT assay, flow cytometry, and Western blot. A further examination of its anti-cancer efficacy was performed in A549-bearing xenograft mice via orthotopic intratrachea (IT) inoculation, which showed that HCD could arrest both Dox-S and Dox-R cells at G2/M phase without altering the sub-G1 cycle along with increasing of cleaved-PARP. HCD downregulated the mTOR/Akt/PI3K-p85 and PI3K-ClassIII/Beclin-1 signals and upregulated p62/LC3-I/II expressions to further confirm that the cell autophagy of NSCLC cells after being HCD-induced. Morphological observations of mouse lung sections illustrated that fewer cancer cells accumulated close to the trachea while less neoplastic activities were found in HCD orthotopic treated mice without liver, kidney, and spleen toxicity. Lastly, Dox, HCD, and target therapy medicines of EGFR and ALK were nicely docked with EGFR, ALK, and mTOR. Conclusively, HCD was demonstrated the chemotherapeutic potential regardless of Dox-R and Dox-S cells, suggesting natural autophagic inducer HCD provides a promising lead compound for new drug discovery and development of lung cancer therapies.

Overcoming Basal Autophagy, Kangai Injection Enhances Cisplatin Cytotoxicity by Regulating FOXO3a-Dependent Autophagic Cell Death and Apoptosis in Human Lung Adenocarcinoma A549/DDP Cells.

Cisplatin resistance is one of the major obstacles in the treatment of nonsmall cell lung cancer (NSCLC). Kangai injection (KAI), a Chinese herbal medicine, has been used in tumors as adjuvant treatment, but its exact antitumor mechanism is still unclear. In this study, we first demonstrated that cisplatin-resistant A549/DDP cells showed a higher level of basal autophagy in response to cisplatin treatment with increasing autophagic protein expression levels of Beclin 1, p62, and LC3 compared to cisplatin-sensitive A549/DDP cells; then, we assessed the antitumor effect of KAI in cisplatin-resistant lung adenocarcinoma A549/DDP cells. Our results showed that KAI exhibited direct cytotoxic and chemosensitizing effects in A549/DDP cells. Combining KAI with cisplatin promoted A549/DDP cell apoptosis, which was confirmed by cell cycle arrest, condensed nuclear chromatin, annexin V fluorescein isothiocyanate/propidium iodide (Annexin V-FITC/PI) staining, and apoptosis-related protein expression. In addition, combining KAI with cisplatin induced autophagic cell death in A549/DDP cells with a high level of basal autophagy, as indicated by an increase in LC3 spot count, an accumulation of Beclin 1 and LC3 II, and reduced p62 protein expression. We also found that the apoptosis and autophagic cell death induced by cotreatment of KAI and cisplatin in A549/DDP cells were FOXO3a-dependent as indicated by decreased p-FOXO3a expression and increased FOXO3a nuclear localization, respectively. Furthermore, the FOXO3a gene knockdown assay further confirmed that KAI enhanced cisplatin cytotoxicity in A549/DDP cells with a high level of basal autophagy by inducing apoptosis and autophagic cell death in a FOXO3a-dependent manner. These findings suggest that the combination of KAI and cisplatin might support the potential clinical treatment as a novel strategy to overcome cisplatin resistance.

Berberine Induces Autophagic Cell Death by Inactivating the Akt/mTOR Signaling Pathway.

The incidence of skin cancer has been increasing over the past decades, and melanoma is considered highly malignant because of its high rate of metastasis. Plant-derived berberine, an isoquinoline quaternary alkaloid, has been reported to possess multiple pharmacological effects against various types of cancer cells. Therefore, we treated melanoma B16F10 cells with berberine to induce cell death and understand the cell death mechanisms. The berberine-treated cells showed decreased cell viability, according to berberine concentration. However, western blot analysis of apoptosis-related marker proteins showed that the expression of Bcl-2, an apoptosis inhibitory protein, and the Bcl-2/Bax ratio were increased. Therefore, by adding 3-methyladenine to the berberine-treated cells, we investigated whether the reduced cell viability was due to autophagic cell death. The results showed that 3-methyladenine restored the cell viability decreased by berberine, suggesting autophagy. To clarify autophagic cell death, we performed transmission electron microscopy analysis, which revealed the presence of autophagosomes and autolysosomes in the cells after treatment with berberine. Next, by analyzing the expression of autophagy-related proteins, we found an increase in the levels of light chain 3A-II and Atg12-Atg5 complex in the berberine-treated cells. We then assessed the involvement of the Akt/mTOR signaling pathway and found that berberine inhibited the expression of phosphorylated Akt and mTOR. Our data demonstrated that berberine induces autophagic cell death by inactivating the Akt/mTOR signaling pathway in melanoma cells and that berberine can be used as a possible target for the development of anti-melanoma drugs.

Qili Qiangxin, a compound herbal medicine formula, alleviates hypoxia-reoxygenation-induced apoptotic and autophagic cell death via suppression of ROS/AMPK/mTOR pathway in vitro.

OBJECTIVE: Qili Qiangxin (QLQX), a compound herbal medicine formula, is used effectively to treat congestive heart failure in China. However, the molecular mechanisms of the cardioprotective effect are still unclear. This study explores the cardioprotective effect and mechanism of QLQX using the hypoxia-reoxygenation (H/R)-induced myocardial injury model. METHODS: The main chemical constituents of QLQX were analyzed using high-performance liquid chromatography-evaporative light-scattering detection. The model of H/R-induced myocardial injury in H9c2 cells was developed to simulate myocardial ischemia-reperfusion injury. Apoptosis, autophagy, and generation of reactive oxygen species (ROS) were measured to assess the protective effect of QLQX. Proteins related to autophagy, apoptosis and signalling pathways were detected using Western blotting. RESULTS: Apoptosis, autophagy and the excessive production of ROS induced by H/R were significantly reduced after treating the H9c2 cells with QLQX. QLQX treatment at concentrations of 50 and 250 µg/mL caused significant reduction in the levels of LC3II and p62 degradation (P < 0.05), and also suppressed the AMPK/mTOR signalling pathway. Furthermore, the AMPK inhibitor Compound C (at 0.5 µmol/L), and QLQX (250 µg/mL) significantly inhibited H/R-induced autophagy and apoptosis (P < 0.01), while AICAR (an AMPK activator, at 0.5 mmol/L) increased cardiomyocyte apoptosis and autophagy and abolished the anti-apoptotic effect of QLQX. Similar phenomena were also observed on the expressions of apoptotic and autophagic proteins, demonstrating that QLQX reduced the apoptosis and autophagy in the H/R-induced injury model via inhibiting the AMPK/mTOR pathway. Moreover, ROS scavenger, N-Acetyl-L-cysteine (NAC, at 2.5 mmol/L), significantly reduced H/R-triggered cell apoptosis and autophagy (P < 0.01). Meanwhile, NAC treatment down-regulated the ratio of phosphorylation of AMPK/AMPK (P < 0.01), which showed a similar effect to QLQX. CONCLUSION: QLQX plays a cardioprotective role by alleviating apoptotic and autophagic cell death through inhibition of the ROS/AMPK/mTOR signalling pathway.

ACT001 inhibits pituitary tumor growth by inducing autophagic cell death via MEK4/MAPK pathway.

ACT001, derived from traditional herbal medicine, is a novel compound with effective anticancer activity in clinical trials. However, little is known regarding its role in pituitary adenomas. Here, we demonstrated that ACT001 suppressed cell proliferation and induced cell death of pituitary tumor cells in vitro and in vivo. ACT001 was also effective in suppressing the growth of different subtypes of human pituitary adenomas. The cytotoxic mechanism ACT001 employed was mainly related to autophagic cell death (ACD), indicated by autophagosome formation and LC3-II accumulation. In addition, ACT001-mediated inhibitory effect decreased when either ATG7 was downregulated or cells were cotreated with autophagy inhibitor 3-methyladenine (3-MA). RNA-seq analysis showed that mitogen-activated protein kinase (MAPK) pathway was a putative target of ACT001. Specifically, ACT001 treatment promoted the phosphorylation of JNK and P38 by binding to mitogen-activated protein kinase kinase 4 (MEK4). Our study indicated that ACT001-induced ACD of pituitary tumor cells via activating JNK and P38 phosphorylation by binding with MEK4, and it might be a novel and effective anticancer drug for pituitary adenomas.

Falcarindiol Stimulates Apoptotic and Autophagic Cell Death to Attenuate Cell Proliferation, Cell Division, and Metastasis through the PI3K/AKT/mTOR/p70S6K Pathway in Human Oral Squamous Cell Carcinomas.

Human oral squamous cell carcinomas (OSCCs) have high cancer mortality and a 5-year survival rate lower than that of most other carcinomas. New therapeutic strategies are required for the treatment and prevention against OSCCs. An approach to cancer therapy using plant-derived natural compounds has been actively in progress as a trend. Falcarindiol (FALC), or its isolated form Ostericum koreanum Kitagawa (O. koreanum), is present in many food and dietary plants, especially in carrots, and this compound has a variety of beneficial effects. However, biological activity of FALC has not been reported in OSCCs yet. This study aimed to demonstrate the antitumor effects of FALC against OSCCs, YD-10B cells. In this study, FALC was selected as a result of screening for compounds isolated from various natural products in YD-10B cells. FALC suppressed cell growth, and FALC-induced apoptotic cell death was mainly accompanied by the dephosphorylation of PI3K, AKT, mTOR, and p70S6K. The apoptotic cell death was also associated with autophagy as evidenced by the expression of Beclin-1, the conversion of LC3-II, and the formation of autophagosome. FALC-induced autophagy was accompanied by MAPKs including ERK1/2 and p38. Furthermore, FALC caused the antimetastatic effects by inhibiting the migration and invasion of YD-10B cells. Taken together, the findings suggest the potential value of FALC as a novel candidate for therapeutic strategy against OSCCs.

Qili Qiangxin, a compound herbal medicine formula, alleviates hypoxia-reoxygenation-induced apoptotic and autophagic cell death via suppression of ROS/AMPK/mTOR pathway in vitro / 中西医结合学报

OBJECTIVE@#Qili Qiangxin (QLQX), a compound herbal medicine formula, is used effectively to treat congestive heart failure in China. However, the molecular mechanisms of the cardioprotective effect are still unclear. This study explores the cardioprotective effect and mechanism of QLQX using the hypoxia-reoxygenation (H/R)-induced myocardial injury model.@*METHODS@#The main chemical constituents of QLQX were analyzed using high-performance liquid chromatography-evaporative light-scattering detection. The model of H/R-induced myocardial injury in H9c2 cells was developed to simulate myocardial ischemia-reperfusion injury. Apoptosis, autophagy, and generation of reactive oxygen species (ROS) were measured to assess the protective effect of QLQX. Proteins related to autophagy, apoptosis and signalling pathways were detected using Western blotting.@*RESULTS@#Apoptosis, autophagy and the excessive production of ROS induced by H/R were significantly reduced after treating the H9c2 cells with QLQX. QLQX treatment at concentrations of 50 and 250 μg/mL caused significant reduction in the levels of LC3II and p62 degradation (P < 0.05), and also suppressed the AMPK/mTOR signalling pathway. Furthermore, the AMPK inhibitor Compound C (at 0.5 μmol/L), and QLQX (250 μg/mL) significantly inhibited H/R-induced autophagy and apoptosis (P < 0.01), while AICAR (an AMPK activator, at 0.5 mmol/L) increased cardiomyocyte apoptosis and autophagy and abolished the anti-apoptotic effect of QLQX. Similar phenomena were also observed on the expressions of apoptotic and autophagic proteins, demonstrating that QLQX reduced the apoptosis and autophagy in the H/R-induced injury model via inhibiting the AMPK/mTOR pathway. Moreover, ROS scavenger, N-Acetyl-L-cysteine (NAC, at 2.5 mmol/L), significantly reduced H/R-triggered cell apoptosis and autophagy (P < 0.01). Meanwhile, NAC treatment down-regulated the ratio of phosphorylation of AMPK/AMPK (P < 0.01), which showed a similar effect to QLQX.@*CONCLUSION@#QLQX plays a cardioprotective role by alleviating apoptotic and autophagic cell death through inhibition of the ROS/AMPK/mTOR signalling pathway.

Apigenin Induces Autophagy and Cell Death by Targeting EZH2 under Hypoxia Conditions in Gastric Cancer Cells.

Hypoxia is a major obstacle to gastric cancer (GC) therapy and leads to chemoresistance as GC cells are frequently exposed to the hypoxia environment. Apigenin, a flavonoid found in traditional medicine, fruits, and vegetables and an HDAC inhibitor, is a powerful anti-cancer agent against various cancer cell lines. However, detailed mechanisms involved in the treatment of GC using APG are not fully understood. In this study, we investigated the biological activity of and molecular mechanisms involved in APG-mediated treatment of GC under hypoxia. APG promoted autophagic cell death by increasing ATG5, LC3-II, and phosphorylation of AMPK and ULK1 and down-regulating p-mTOR and p62 in GC. Furthermore, our results show that APG induces autophagic cell death via the activation of the PERK signaling, indicating an endoplasmic reticulum (ER) stress response. The inhibition of ER stress suppressed APG-induced autophagy and conferred prolonged cell survival, indicating autophagic cell death. We further show that APG induces ER stress- and autophagy-related cell death through the inhibition of HIF-1α and Ezh2 under normoxia and hypoxia. Taken together, our findings indicate that APG activates autophagic cell death by inhibiting HIF-1α and Ezh2 under hypoxia conditions in GC cells.

Isoangustone A induces autophagic cell death in colorectal cancer cells by activating AMPK signaling.

Phytochemicals, especially flavonoids, have been widely investigated for their diversified pharmacological activities including anticancer activities. Previously we identified isoangustone A from licorice-derived compounds as a potent inducer of cell death. In the present study, the exact mechanism by which isoangustone A induced cell death was further investigated, with autophagy as an indispensible part of this process. Isoangustone A treatment activated autophagic signaling and induced a complete autophagic flux in colorectal cancer cells. Knockdown of ATG5 or pre-treatment with autophagy inhibitors significantly reversed isoangustone A-induced apoptotic signaling and loss of cell viability, suggesting autophagy plays an important role in isoangustone A-induced cell death. Isoangustone A inhibited Akt/mTOR signaling, and overexpressing of a constitutively activated Akt mildly suppressed isoangustone A-induced cell death. More importantly, isoangustone A inhibited cellular ATP level and activated AMPK, and pre-treatment with AMPK inhibitor or overexpression of dominant negative AMPKα2 significantly reversed isoangustone A-induced autophagy and cell death. Further study shows isoangustone A dose-dependently inhibited mitochondrial respiration, which could be responsible for isoangustone A-induced activation of AMPK. Finally, isoangustone A at a dosage of 10 mg/kg potently activated AMPK and autophagic signaling in and inhibited the growth of SW480 human colorectal xenograft in vivo. Taken together, induction of autophagy through activation of AMPK is an important mechanism by which isoangustone A inhibits tumor growth, and isoangustone A deserves further investigation as a promising anti-cancer agent.

Artemisia kruhsiana leaf extract induces autophagic cell death in human prostate cancer cells.

Some species of Artemisia have been reported to induce apoptosis and autophagy, but little is known of the apoptotic and autophagic effects of the stems and leaves of Artemisia kruhsiana Bess. (AkB). This study was conducted to investigate the antioxidant and anti-autophagic effects of the methanol extracts of the stems (EAkBs) and leaves (EAkBl) of AkB on human prostate cancer PC-3 cells. The antioxidant effects of EAkBs and EAkBl were measured using in vitro total flavonoid and total phenolic assays and a free radical scavenging assay. The effects of EAkBl on cell viability, apoptosis, autophagy, intracellular reactive oxygen species (ROS) generation and protein expression levels were also investigated. EAkBl was found to induce apoptosis, autophagy, and intracellular ROS generation in PC-3 cells. In terms of protein levels, EAkBl reduced phospho (p)-protein kinase B (AKT)/AKT, p-mammalian target of rapamycin (mTOR)/mTOR, B-cell lymphoma 2 (Bcl-2)/Bcl-2-associated X protein (Bax) ratios, and the activations of beclin 1/ß-actin and microtubule-associated protein 1A/1B-light chain 3 (LC3) II/LC3 I ratios in PC-3 cells. The results of this study indicate EAkBl has antioxidant and anticancer effects on prostate cancer cells, and that these effects are associated with suppressions of p-AKT, p-mTOR, Bcl-2, and Bax, and the activations of beclin 1 and LC3. Our results indicate EAkBl has potential as a treatment for prostate cancer.

Suppression of Metacaspase- and Autophagy-Dependent Cell Death Improves Stress-Induced Microspore Embryogenesis in Brassica napus.

Microspore embryogenesis is a biotechnological process that allows us to rapidly obtain doubled-haploid plants for breeding programs. The process is initiated by the application of stress treatment, which reprograms microspores to embark on embryonic development. Typically, a part of the microspores undergoes cell death that reduces the efficiency of the process. Metacaspases (MCAs), a phylogenetically broad group of cysteine proteases, and autophagy, the major catabolic process in eukaryotes, are critical regulators of the balance between cell death and survival in various organisms. In this study, we analyzed the role of MCAs and autophagy in cell death during stress-induced microspore embryogenesis in Brassica napus. We demonstrate that this cell death is accompanied by the transcriptional upregulation of three BnMCA genes (BnMCA-Ia, BnMCA-IIa and BnMCA-IIi), an increase in MCA proteolytic activity and the activation of autophagy. Accordingly, inhibition of autophagy and MCA activity, either individually or in combination, suppressed cell death and increased the number of proembryos, indicating that both components play a pro-cell death role and account for decreased efficiency of early embryonic development. Therefore, MCAs and/or autophagy can be used as new biotechnological targets to improve in vitro embryogenesis in Brassica species and doubled-haploid plant production in crop breeding and propagation programs.

Traditional Uyghur medicine Quercus infectoria galls water extract triggers apoptosis and autophagic cell death in colorectal cancer cells.

BACKGROUND: The water extract of Quercuse infectoria galls (QIG) is the active ingredient of Uyghur medicine Xipayi Kui Jie'an (KJA) which has promising therapeutic effects on Ulcerative Colitis (UC) as an alternative medicine. Considering the relationship between UC and the development of colorectal cancer (CRC), the present work aims to explore the direct anti-CRC activity of QIG extract. METHODS: CCK8 assay and flow cytometry were used to detect cytotoxicity and apoptosis. Transmission electron microscopy (TEM), flow cytometry, laser confocal and western blotting were performed to examine autophagy. We also adopted Reactive Oxygen Assay kit, as well as transwell and wound healing tests to study the underlying mechanism of QIG against CRC cells. RESULTS: First, we found that QIG extract could suppress the viability of CRC cells and trigger caspases-dependent apoptosis. Subsequently, we proved for the first time that QIG extract also triggered autophagic cell death in CRC cells, which together with apoptosis contributed to the cytotoxic effect on CRC cells. Further investigation revealed that QIG-induced cytotoxicity associated with intracellular ROS accumulation which could suppress the AKT/mTOR signaling pathway, and then induce autophagy and inhibit cell growth. Besides, Erk signaling pathway was also involved in the process of autophagic cell death. Moreover, QIG extract also influenced EMT process and inhibited CRC cell migration. CONCLUSION: Altogether, this study provides a basis for the utilization of QIG as an alternative medicine for CRC prevention and treatment.

SH003 activates autophagic cell death by activating ATF4 and inhibiting G9a under hypoxia in gastric cancer cells.

In gastric cancer (GC), hypoxia is one of the greatest obstacles to cancer therapy. In this present study, we report that SH003, an herbal formulation, induces ER stress via PERK-ATF4-CHOP signaling in GC. SH003-mediated ER stress inhibits G9a, a histone methyltransferase, by reducing STAT3 phosphorylation and activates autophagy, indicating to the dissociation of Beclin-1 and autophagy initiation from Bcl-2/Beclin-1 complex. However, the inhibition of PERK and CHOP inhibited SH003-induced cell death and autophagy activation. Moreover, targeting autophagy using specific siRNAs of LC3B or p62 or the autophagy inhibitor 3-MA also inhibited SH003-induced cell death in GC. Interestingly, SH003 induces BNIP3-mediated autophagic cell death under hypoxia than normoxia in GC. These findings reveal that SH003-induced ER stress regulates BNIP3-induced autophagic cell death via inhibition of STAT3-G9a axis under hypoxia in GC. Therefore, SH003 may an important tumor therapeutic strategy under hypoxia-mediated chemo-resistance.

Punicalagin Regulates Apoptosis-Autophagy Switch via Modulation of Annexin A1 in Colorectal Cancer.

Punicalagin (PU), a polyphenol extracted from pomegranate (Punica granatum) husk is proven to have anti-cancer effects on different types of cancer including colorectal cancer (CRC). Its role in modulating endogenous protein as a means of eliciting its anti-cancer effects, however, has not been explored to date. Hence, this study aimed to investigate the role of PU in modulating the interplay between apoptosis and autophagy by regulating Annexin A1 (Anx-A1) expression in HCT 116 colorectal adenocarcinoma cells. In the study, selective cytotoxicity, pro-apoptotic, autophagic and Anx-A1 downregulating properties of PU were shown which indicate therapeutic potential that this polyphenol has against CRC. Autophagy flux analysis via flow cytometry showed significant autophagosomes degradation in treated cells, proving the involvement of autophagy. Proteome profiling of 35 different proteins in the presence and absence of Anx-A1 antagonists in PU-treated cells demonstrated a complex interplay that happens between apoptosis and autophagy that suggests the possible simultaneous induction and inhibition of these two cell death mechanisms by PU. Overall, this study suggests that PU induces autophagy while maintaining basal level of apoptosis as the main mechanisms of cytotoxicity via the modulation of Anx-A1 expression in HCT 116 cells, and thus has a promising translational potential.