Vesicular Organelles Research Articles

Effects of different autophagy inhibitors on sensitizing KG-1 and HL-60 leukemia cells to chemotherapy.

A little number of current autophagy inhibitors may have beneficial effects on the acute myeloid leukemia (AML) patients. However, there is a strong need to figure out which settings should be activated or inhibited in autophagy pathway to prevail drug resistance and also to improve current treatment options in leukemia. Therefore, this study aimed to compare the effects of well-known inhibitors of autophagy (as 3-MA, BafA1, and HCQ) in leukemia KG-1 and HL-60 cells exposed to arsenic trioxide (ATO) and/or all-trans retinoic acid (ATRA). Cell proliferation and cytotoxicity of cells were examined by MTT assay. Autophagy was studied by evaluating the development of acidic vesicular organelles, and the autophagosomes formation was investigated by acridine orange staining and transmission electron microscopy. Moreover, the gene and protein expressions levels of autophagy markers (ATGs, p62/SQSTM1, and LC-3B) were also performed by qPCR and western blotting, respectively. The rate of apoptosis and cell cycle were evaluated using flow cytometry. We compared the cytotoxic and apoptotic effects of ATO and/or ATRA in both cell lines and demonstrated that some autophagy markers upregulated in this context. Also, it was shown that autophagy blockers HCQ and/or BafA1 could potentiate the cytotoxic effects of ATO/ATRA, which were more pronounced in KG-1 cells compared to HL-60 cell line. This study showed the involvement of autophagy during the treatment of KG-1 and HL-60 cells by ATO/ATRA. This study proposed that therapy of ATO/ATRA in combination with HCQ can be considered as a more effective strategy for targeting leukemic KG-1 cells.

Ginsenoside Rh2 represses autophagy to promote cervical cancer cell apoptosis during starvation

BackgroundCancer cells through autophagy-mediated recycling to meet the metabolic demands of growth and proliferation. The steroidal saponin 20(S)-ginsenoside Rh2 effectively inhibits the growth and survival of a variety of tumor cell lines and animal models, but the effects of Rh2 on autophagy remain elusive.MethodsCell viability was measured by CCK-8 (cell counting kit-8) assays. Apoptosis, ROS generation and mitochondrial membrane potential were analyzed by flow cytometry. Western blot analyses were used to determine changes in protein levels. Morphology of apoptotic cells and autophagosome accumulation were analyzed by DAPI staining and transmission electron microscopy. Autophagy induction was monitored by acidic vesicular organelle staining, EGFP-LC3 and mRFP-GFP-LC3 transfection. Atg7 siRNA and autophagy regulator was used to assess the effect of autophagy on apoptosis induced by G-Rh2.ResultsIn this study, we found that low concentration G-Rh2 attenuated cancer cell growth and induced apoptosis upon serum-free starvation. Caspase 3 inhibitors failed to block apoptosis in G-Rh2-treated cells, indicating a caspase-independent mechanism. G-Rh2-treated cells in serum-deprived conditions showed impaired mitochondrial function, increased release and nuclear translocation of apoptosis-inducing factor, but little changes in the mitochondrial and cytoplasmic distributions of cytochrome C. Annexin A2 overexpression in 293T cells inhibited G-Rh2-induced apoptosis under serum-starved conditions. Meanwhile, G-Rh2 reduced lysosomal activity and inhibited the fusion of autophagosome and lysosome, leading to a block of autophagic flux. Knockdown Atg7 significantly inhibited autophagy and triggered AIF-induced apoptosis in serm free condition. The autophagy inducer significantly decreased the apoptosis levels of G-Rh2-treated cells in serum-free conditions.ConclusionsUnder nutrient deficient conditions, G-Rh2 represses autophagy in cervical cancer cells and enhanced apoptosis through an apoptosis-inducing factor mediated pathway.

Novel SIRT Inhibitor, MHY2256, Induces Cell Cycle Arrest, Apoptosis, and Autophagic Cell Death in HCT116 Human Colorectal Cancer Cells.

We examined the anticancer effects of a novel sirtuin inhibitor, MHY2256, on HCT116 human colorectal cancer cells to investigate its underlying molecular mechanisms. MHY2256 significantly suppressed the activity of sirtuin 1 and expression levels of sirtuin 1/2 and stimulated acetylation of forkhead box O1, which is a target protein of sirtuin 1. Treatment with MHY2256 inhibited the growth of the HCT116 (TP53 wild-type), HT-29 (TP53 mutant), and DLD-1 (TP53 mutant) human colorectal cancer cell lines. In addition, MHY2256 induced G0/G1 phase arrest of the cell cycle progression, which was accompanied by the reduction of cyclin D1 and cyclin E and the decrease of cyclin-dependent kinase 2, cyclin-dependent kinase 4, cyclin-dependent kinase 6, phosphorylated retinoblastoma protein, and E2F transcription factor 1. Apoptosis induction was shown by DNA fragmentation and increase in late apoptosis, which were detected using flow cytometric analysis. MHY2256 downregulated expression levels of procaspase-8, -9, and -3 and led to subsequent poly(ADP-ribose) polymerase cleavage. MHY2256-induced apoptosis was involved in the activation of caspase-8, -9, and -3 and was prevented by pretreatment with Z-VAD-FMK, a pan-caspase inhibitor. Furthermore, the autophagic effects of MHY2256 were observed as cytoplasmic vacuolation, green fluorescent protein-light-chain 3 punctate dots, accumulation of acidic vesicular organelles, and upregulated expression level of light-chain 3-II. Taken together, these results suggest that MHY2256 could be a potential novel sirtuin inhibitor for the chemoprevention or treatment of colorectal cancer or both.

The In Vitro and In Vivo Anticancer Properties of Chalcone Flavokawain B through Induction of ROS-Mediated Apoptotic and Autophagic Cell Death in Human Melanoma Cells.

Simple SummaryMelanoma is the most dangerous type of skin cancer that develops from the pigment-producing cells known as melanocytes. One of the primary causes of melanoma is ultraviolet light (UV) exposure in those with low levels of the skin pigment melanin. Flavokawain B (FKB) is a naturally occurring chalcone, which is known to possess anti-proliferative pharmacological activity on various cancer cells. However, the effect of FKB on the anti-melanoma pharmacological role has not been investigated. Therefore, in this study, we explored the anti-melanoma properties of FKB on human melanoma cells and the cell death mechanisms that were mediated through the induction of reactive oxygen species (ROS) were investigated via in vitro and in vivo approaches. Our study results support promising application prospects of FKB in the treatment of human melanoma cancer.Melanoma is the most prevalent type of skin cancer with high mortality rates. This study demonstrates the in vitro and in vivo anticancer properties of chalcone flavokawain B (FKB) induced ROS-mediated apoptosis and autophagy in human melanoma (human epithelial melanoma cell line A375 and/or human skin lymph node derived melanoma cell line A2058) cells. Cell viability was calculated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and the expression patterns of various apoptosis, autophagy-associated proteins were determined by Western blot methods. Annexin V was detected by flow cytometry, whereas acidic vesicular organelles (AVOs) and intracellular ROS levels were measured by fluorescence microscopy. The in vivo anticancer properties of FKB were evaluated by xenografting the A375 cells into nude mice. The results convey that FKB inhibited cell viability, B-Raf proto-oncogene, serine/threonine kinase (BRAF)/extracellular signal-regulated kinase (ERK) expression in human melanoma cells. Caspase-3 activation, poly (ADP-ribose) polymerase (PARP) cleavage pathway, and Bcl2 associated X (Bax)/B-cell lymphoma 2 (Bcl-2) dysregulation were involved in the execution of apoptosis. Moreover, FKB-induced autophagy was observed through increased microtubule-associated protein 1A/1B-light chain 3B (LC3-II) accumulation and AVOs formation, which was also associated with an increase in sequestosome 1 (SQSTM1/p62), decreased protein kinase B (AKT)/mammalian target of rapamycin (mTOR) expressions, and dysregulated Beclin-1/Bcl-2 levels. Autophagy inhibitors [3-methyladenine (3-MA)/chloroquine (CQ)] and LC3 silencing suppressed FKB-induced apoptosis by decreasing caspase-3 in melanoma cells. The antioxidant N-acetylcysteine (NAC) diminished FKB-induced apoptotic and autophagic cell death. However, the inhibition of apoptosis decreased FKB-induced autophagy (LC3-I/II). The in vivo study confirmed that FKB inhibited melanoma growth in A375-xenografted nude mice. This study concluded that FKB is critically associated with the execution and generation of ROS-modulated apoptotic and autophagic cell death of melanoma cells. FKB also repressed tumor growth in xenografted nude mice. Therefore, flavokawain B might be a potential anti-tumor agent in human melanoma treatment.

Vesicular ATP-binding cassette transporters in human disease: relevant aspects of their organization for future drug development

Vesicular ATP-binding cassette transporters in human disease: relevant aspects of their organization for future drug development

BW373U86 upregulates autophagy by inhibiting the PI3K/Akt pathway and regulating the mTOR pathway to protect cardiomyocytes from hypoxia-reoxygenation injury.

The purpose of this study was to explore the protective effect of BW373U86 (a δ-opioid receptor (DOR) agonist) on ischemia-reperfusion (I/R) injury in rat cardiomyocytes and its underlying mechanism. Primary rat cardiomyocytes were cultured and pretreated with BW373U86 for intervention. The cardiomyocytes were cultured under the condition of 94% N2 and 5% CO2 for 24 h to perform hypoxia culture and conventionally cultured for 12 h to perform reoxygenation culture. The cell viability of cardiomyocytes was detected by an MTT assay (Sigma-Aldrich). The autophagy lysosome levels in cardiomyocytes were evaluated by acidic vesicular organelles with dansylcadaverine (MDC) staining (autophagy test kit, Kaiji Biology, kgatg001). The protein expression levels of LC3, p62, and factors in the PI3K/Akt/mTOR signaling pathway were detected by Western blot. Pretreatment with BW373U86 could improve the cell viability of cardiomyocytes with hypoxia-reoxygenation (H/R) injury (p < 0.05). Interestingly, after coculture of BW373U86 and PI3K inhibitor (3-methyladenine), the protein expression levels of p-Akt in cardiomyocytes were markedly increased in comparison with those in the BW373U86 group (p < 0.05). However, there were no significant differences in the protein expression levels of mTOR between the coculture group and the BW373U86 group (p > 0.05). BW373U86 upregulated autophagy to protect cardiomyocytes from H/R injury, which may be related to the PI3K/Akt/m TOR pathway.

Berberine, a natural alkaloid sensitizes human hepatocarcinoma to ionizing radiation by blocking autophagy and cell cycle arrest resulting in senescence.

To study the radiosensitizing potential of Berberine and the underlying mechanism in human hepatocarcinoma (HepG2) cells. HepG2 cells were challenged with X-rays in combination with Berberine treatment and several in vitro assays were performed. Alteration in cell viability was determined by MTT assay. Changes in intracellular ROS levels, mitochondrial membrane potential/mass, intracellular acidic vesicular organelles as well as cell cycle arrest and apoptotic cell death were analysed by flow cytometry. Induction of autophagy was assessed by staining the cells with Monodansylcadaverine/Lysotracker red dyes and immunoblotting for LC3I/II and p62 proteins. Phase-contrast/fluorescence microscopy was employed to study mitotic catastrophe and senescence. Cellular senescence was confirmed by immunoblotting for p21 levels and ELISA for Interleukin-6. X-rays+Berberine had a synergistic effect in reducing cell proliferation accompanied by a robust G2/M arrest. Berberine-mediated radiosensitization was associated with elevated levels of LC3II and p62 suggesting blocked autophagy that was followed by mitotic catastrophe and senescence. Treatment of cells with X-rays+Berberine resulted in increased oxidative stress, hyperpolarized mitochondria with increased mitochondrial mass and reduced ATP levels. The study expands the understanding of the pharmacological properties of Berberine and its applicability as a radiosensitizer towards treating liver cancer.

Non-thermal plasma induces apoptosis accompanied by protective autophagy via activating JNK/Sestrin2 pathway

Although non-thermal plasma (NTP) has been proved to be an effective way to kill cancer cells, the mechanism of NTP-induced cancer cell death is still not clear. In this study, we found that NTP exposure caused reactive oxygen species generation and apoptosis in A549 and MDA-MB-231 cells. Meanwhile, NTP treatment also induced autophagy, as evidenced by the formation of acidic vesicular organelles and conversion of LC3-I to LC3-II. Suppression of autophagy by chloroquine significantly increased NTP-induced cell death, indicating that NTP-induced autophagy acted in a protective role from apoptosis. Furthermore, NTP treatment significantly increased Sestrin2 (Sesn2) expression and activated the JNK signaling pathway. Knocking down Sesn2 with special siRNA enhanced NTP-induced cell death, while pretreatment with JNK inhibitor abolished the increase of Sesn2 and LC3 formation, and promoted cell apoptosis induced by NTP. These indicated that the JNK/Sesn2 pathway was involved in autophagy and apoptosis induced by NTP. These findings provided evidence that supplement with autophagy inhibition might be a useful strategy to improve the tumor cell killing effect of NTP.

Combined treatment of menadione and calcitriol increases the antiproliferative effect by promoting oxidative/nitrosative stress, mitochondrial dysfunction, and autophagy in breast cancer MCF-7 cells

The aim of this study was to determine new insights into the molecular mechanisms involved in the antiproliferative action of menadione + calcitriol (MEN+D) on MCF-7 cells. After 24 h, MEN+D inhibited the cell growth but was not observed with each single treatment. The combined drugs reduced the mitochondrial respiration at that time, as judged by an increase in the proton leak and a decrease in the ATP generation and coupling efficiency. At longer times, 48 or 96 h, either D or MEN reduced the proliferation, but the effect was higher when both drugs were used together. The combined treatment increased the superoxide anion ([Formula: see text]) and nitric oxide (NO•) contents as well as acidic vesicular organelles (AVOs) formation. The percentage of cells showing the lower mitochondrial membrane potential (ΔΨm) was highly increased by the combined therapy. LC3-II protein expression was enhanced by any treatment. In conclusion, the antiproliferative action of MEN+D involves oxidative/nitrosative stress, mitochondrial alteration, and autophagy. This combined therapy could be useful to treat breast cancer cells because it inhibits multiple oncogenic pathways more effectively than each single agent.

Flavonoids Identification and Pancreatic Beta-Cell Protective Effect of Lotus Seedpod.

Oxidative stress is highly associated with the development of diabetes mellitus (DM), especially pancreatic beta-cell injury. Flavonoids derived from plants have caused important attention in the prevention or treatment of DM. Lotus seedpod belongs to a traditional Chinese herbal medicine and has been indicated to possess antioxidant, anti-age, anti-glycative, and hepatoprotective activities. The purpose of this study was to demonstrate the pancreatic beta-cell protective effects of lotus seedpod aqueous extracts (LSE) against oxidative injury. According to HPLC/ESI-MS-MS method, LSE was confirmed to have flavonoids derivatives, especially quercetin-3-glucuronide (Q3G). In vitro, LSE dose-dependently improved the survival and function of rat pancreatic beta-cells (RIN-m5F) from hydrogen peroxide (H2O2)-mediated loss of cell viability, impairment of insulin secretion, and promotion of oxidative stress. LSE showed potential in decreasing the H2O2-induced occurrence of apoptosis. In addition, H2O2-triggered acidic vesicular organelle formation and microtubule-associated protein light chain 3 (LC3)-II upregulation, markers of autophagy, were increased by LSE. Molecular data explored that antiapoptotic and autophagic effects of LSE, comparable to that of Q3G, might receptively be mediated via phospho-Bcl-2-associated death promoter (p-Bad)/B-cell lymphoma 2 (Bcl-2) and class III phosphatidylinositol-3 kinase (PI3K)/LC3-II signal pathway. In vivo, LSE improved the DM symptoms and pancreatic cell injury better than metformin, a drug that is routinely prescribed to treat DM. These data implied that LSE induces the autophagic signaling, leading to protect beta-cells from oxidative stress-related apoptosis and injury.

A promising therapeutic combination for metastatic prostate cancer: Chloroquine as autophagy inhibitor and palladium(II) barbiturate complex

Autophagy is a catabolic process for cells that can provide energy sources and allows cancer cells to evade cell death. Therefore, studies on the combination of autophagy inhibitors with drugs are increasing as a new treatment modality in cancer. Previously, we reported the anti-tumor activity of a Palladium (Pd)(II) complex against different types of cancer in vitro and in vivo. Chloroquine (CQ), the worldwide used anti-malarial drug, has recently been focused as a chemosensitizer in cancer treatment. The aim of this study was to investigate the efficacy of a combined treatment of these agents that work through different mechanisms to provide an effective treatment modality for metastatic prostate cancer that is certainly fatal. Metastatic prostate cancer cell lines (PC-3 and LNCaP) were treated with Pd (II) complex, CQ, and their combination. The combination enhanced apoptosis by increasing phosphatidylserine translocation and pro-apoptotic proteins. Apoptosis was confirmed by the use of apoptosis inhibitor. The formation of acidic vesicular organelles (AVOs) was observed by acridine orange staining in fluorescence microscopy. The Pd (II) complex increased AVOs formation in prostate cancer cells and CQ-pretreatment has potentiated this effect. Importantly, treatment with CQ suppressed the pro-survival function of autophagy, which might have contributed to enhanced cytotoxicity. In addition, PI3K/AKT/mTOR-related protein expressions were altered after the combination of treatments. Our results suggest that combination treatment enhances apoptotic cell death possibly via the inhibition of autophagy, and may therefore be regarded as a novel and better approach for the treatment of metastatic prostate cancer.

The interplay between autophagy and apoptosis mediates toxicity triggered by synthetic cathinones in human kidney cells

Synthetic cathinones abuse remains a serious public health problem. Kidney injury has been reported in intoxications associated with synthetic cathinones, but the molecular mechanisms involved have not been explored yet. In this study, the potential in vitro nephrotoxic effects of four commonly abused cathinone derivatives, namely pentedrone, 3,4-dimethylmethcatinone (3,4-DMMC), methylone and 3,4-methylenedioxypyrovalerone (MDPV), were assessed in the human kidney HK-2 cell line. All four derivatives elicited cell death in a concentration- and time-dependent manner, in the following order of potency: 3,4-DMMC >> MDPV > methylone ≈ pentedrone. 3,4-DMMC and methylone were selected to further elucidate the mechanisms behind synthetic cathinones-induced cell death. Both drugs elicited apoptotic cell death and prompted the formation of acidic vesicular organelles and autophagosomes in HK-2 cells. Moreover, the autophagy inhibitor 3-methyladenine significantly potentiated cell death, indicating that autophagy may serve as a cell survival mechanism that protects renal cells against synthetic cathinones toxicity. Both drugs triggered a rise in reactive oxygen and nitrogen species formation, which was completely prevented by antioxidant treatment with N‑acetyl‑L‑cysteine or ascorbic acid. Importantly, these antioxidant agents significantly aggravated renal cell death induced by cathinone derivatives, most likely due to their autophagy-blocking properties. Taken together, our results support an intricate control of cell survival/death modulated by oxidative stress, apoptosis and autophagy in synthetic cathinones-induced renal injury.

Apigetrin induces extrinsic apoptosis, autophagy and G2/M phase cell cycle arrest through PI3K/AKT/mTOR pathway in AGS human gastric cancer cell

Apigetrin is a flavonoid glycoside phytonutrient derived from fruits and vegetables that is well known for a variety of biological activities such as antioxidant and anti-inflammatory activities. In the current study, we determined the effect of apigetrin on AGS gastric cancer cell. Apigetrin reduced cancer cell proliferation and induced G2/M phase cell cycle arrest by regulating cyclin B1, cdc25c and cdk1 protein expression in AGS cell. Apigetrin treatment caused apoptotic cell death in AGS cells, characterized by the accumulation of apoptosis portion, cleavage of caspase-3 and poly ADP-ribose polymerase (PARP). Apigetrin-treated cells increased the expression of extrinsic apoptosis pathway proteins and mRNA. However, intrinsic apoptosis pathway related proteins were not altered. In addition, AGS cells treated with apigetrin increased autophagic cell death, featured by the formation of autophagic vacuole and acidic vesicular organelles. Autophagy marker proteins, such as LC3B-II and beclin-1, were increased, and p62, an autophagy flux marker protein, was also increased by endoplasmic reticulum stress. Also, the phosphorylation of PI3K/AKT/mTOR pathway proteins and its downstream targets in apigetrin-treated AGS cells was identified to be decreased. Taken together, these data suggest that apigetrin-treated AGS cells induced G2/M phase cell cycle arrest, extrinsic apoptosis and autophagic cell death through PI3K/AKT/mTOR pathway, which can lead to the inhibition of gastric cancer development. Thus, our findings strongly indicate that apigetrin is a basic natural derived compound that could be used as a nutrient source with potential anticancer activities against gastric cancer.

Biological effects of selective COX-2 inhibitor NS398 on human glioblastoma cell lines

BackgroundCyclooxygenase-2 (COX-2), an inflammation-associated enzyme, has been implicated in tumorigenesis and progression of glioblastoma (GBM). The poor survival of GBM was mainly associated with the presence of glioma stem cells (GSC) and the markedly inflammatory microenvironment. To further explore the involvement of COX-2 in glioma biology, the effects of NS398, a selective COX-2 inhibitor, were evaluated on GSC derived from COX-2 expressing GBM cell lines, i.e., U87MG and T98G, in terms of neurospheres’ growth, autophagy, and extracellular vesicle (EV) release.MethodsNeurospheres’ growth and morphology were evaluated by optical and scanning electron microscopy. Autophagy was measured by staining acidic vesicular organelles. Extracellular vesicles (EV), released from neurospheres, were analyzed by transmission electron microscopy. The autophagic proteins Beclin-1 and LC3B, as well as the EV markers CD63 and CD81, were analyzed by western blotting. The scratch assay test was used to evaluate the NS398 influence on GBM cell migration.ResultsBoth cell lines were strongly influenced by NS398 exposure, as showed by morphological changes, reduced growth rate, and appearance of autophagy. Furthermore, the inhibitor led to a functional change of EV released by neurospheres. Indeed, EV secreted by NS398-treated GSC, but not those from control cells, were able to significantly inhibit adherent U87MG and T98G cell migration and induced autophagy in recipient cells, thus leading to effects quite similar to those directly caused by NS398 in the same cells.ConclusionDespite the intrinsic diversity and individual genetic features of U87MG and T98G, comparable effects were exerted by the COX-2 inhibitor NS398 on both GBM cell lines. Overall, our findings support the crucial role of the inflammatory-associated COX-2/PGE2 system in glioma and glioma stem cell biology.

Effects of ICI 182,780, an ERα and ERβ antagonist, and G-1, a GPER agonist, on autophagy in breast cancer cells

ABSTRACTObjectiveTo investigate if ICI 182,780 (fulvestrant), a selective estrogen receptor alpha/beta (ERα/ERβ) antagonist, and G-1, a selective G-protein-coupled receptor (GPER) agonist, can potentially induce autophagy in breast cancer cell lines MCF-7 and SKBr3, and how G-1 affects cell viability.MethodsCell viability in MCF-7 and SKBr3 cells was assessed by the MTT assay. To investigate the autophagy flux, MCF-7 cells were transfected with GFP-LC3, a marker of autophagosomes, and analyzed by real-time fluorescence microscopy. MCF-7 and SKBr3 cells were incubated with acridine orange for staining of acidic vesicular organelles and analyzed by flow cytometry as an indicator of autophagy.ResultsRegarding cell viability in MCF-7 cells, ICI 182,780 and rapamycin, after 48 hours, led to decreased cell proliferation whereas G-1 did not change viability over the same period. The data showed that neither ICI 182,780 nor G-1 led to increased GFP-LC3 puncta in MCF-7 cells over the 4-hour observation period. The cytometry assay showed that ICI 182,780 led to a higher number of acidic vesicular organelles in MCF-7 cells. G-1, in turn, did not have this effect in any of the cell lines. In contrast, ICI 182,780 and G-1 did not decrease cell viability of SKBr3 cells or induce formation of acidic vesicular organelles, which corresponds to the final step of the autophagy process in this cell line.ConclusionThe effect of ICI 182,780 on increasing acidic vesicular organelles in estrogen receptor-positive breast cancer cells appears to be associated with its inhibitory effect on estrogen receptors, and GPER does notseem to be involved. Understanding these mechanisms may guide further investigations of these receptors’ involvement in cellular processes of breast cancer resistance.

PD10-02 THE MTORC1/2 DUAL INHIBITOR AZD2014 WITH POTENT ANTITUMOR ACTIVITY IN DOCETAXEL SENSITIVE/RESISTANT PROSTATE CANCER IN VITRO

You have accessJournal of UrologyProstate Cancer: Advanced (including Drug Therapy) I (PD10)1 Apr 2020PD10-02 THE MTORC1/2 DUAL INHIBITOR AZD2014 WITH POTENT ANTITUMOR ACTIVITY IN DOCETAXEL SENSITIVE/RESISTANT PROSTATE CANCER IN VITRO Senmao Li, Shuai Hu, Wei Yu, and Jie Jin* Senmao LiSenmao Li More articles by this author , Shuai HuShuai Hu More articles by this author , Wei YuWei Yu More articles by this author , and Jie Jin*Jie Jin* More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000000844.02AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: The mammalian target of rapamycin (mTOR) is a vital effect factor in regulating cell growth, survival and autophagy via direct interaction with mTORC1 (mTOR complex 1) and mTORC2 (mTOR complex 2). The mTOR signaling pathway is abnormally activated in prostate cancer and may play an important role in prostate cancer progression and drug resistance.The purpose of this study was to investigate the effects of mTORC1/2 dual inhibitor AZD2014 on docetaxel-sensitive and resistant human prostate cancer cell lines. METHODS: In vitro, using Dimethyl sulfoxide(DMSO) and rapamycin as controls,the effects of AZD2014 on the activation state of mTOR signaling pathway in docetaxel-sensitive (C4-2、CWR22Rv1)and resistant (C4-2-Doc-R、 CWR22Rv1-Doc-R)human prostate cancer cell lines were examined by Western blot.The effects of AZD2014 on cell proliferation, cell cycle and cell apoptosis were detected by CCK8 assay, flow cytometry and apoptosis assay.The effects of AZD2014 on cell invasion, migration, autophagy and EMT were determined by Transwell assay, wound healing assay, Acidic vesicular organelles staining assay, immunofluorescence and Western blot assay. RESULTS: We found that in docetaxel-sensitive and resistant human prostate cancer cell lines, rapamycin only inhibited the phosphorylation of 4EBP1 but had no significant effect on the AKT phosphorylation level; AZD2014 could inhibite 4EBP1 phosphorylation more strongly than rapamycin and can significantly inhibit the phosphorylation of AKT.Meanwhile, AZD2014 lead to more profound apoptosis, proliferation suppression, cell cycle arrest and autophagy in all PCa cells when compared with rapamycin. Moreover, AZD2014 was more effcacious than rapamycin in inhibiting the migration, invasion and EMT progression of all PCa cells. CONCLUSIONS: These data highlight an important role for AZD2014 in the regulation of multiple biological functions in PCa and indicate the potential more effcacious antitumor ability of AZD2014 than rapamycin. AZD2014 is a potent dual inhibitor of mTORC1 and mTORC2 that simultaneously blocks mTORC1/2 signaling in docetaxel-sensitive and resistant human prostate cancer cell lines and prevents feedback activation of AKT signaling, thus showing significant anti-tumor effects, including inhibition of proliferation, induction of apoptosis, cycle arrest and autophagy, inhibition of migration, invasion and EMT. This study provides a reliable theoretical basis for the clinical application of AZD2014 in both docetaxel-sensitive and resistant human prostate cancer. Source of Funding: National Natural Science Foundation(NO.81770755,81570683) © 2020 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 203Issue Supplement 4April 2020Page: e244-e244 Advertisement Copyright & Permissions© 2020 by American Urological Association Education and Research, Inc.MetricsAuthor Information Senmao Li More articles by this author Shuai Hu More articles by this author Wei Yu More articles by this author Jie Jin* More articles by this author Expand All Advertisement PDF downloadLoading ...

Anticancer Effects of Antihypertensive L-Type Calcium Channel Blockers on Chemoresistant Lung Cancer Cells via Autophagy and Apoptosis.

PurposeHypertension and cancer are frequently found comorbidity occurring in same individual. This study was intended to evaluate the anticancer effects of commonly used antihypertensive medications and chemotherapy on chemoresistant lung cancer cells.MethodsCalcium channel blockers (CCBs), including Verapamil, Diltiazem, and Nifedipine, either alone or combined with docetaxel (DOC) or vincristine (VCR) were used to treat A549 lung adenocarcinoma chemoresistant sublines. Cell viability was determined by MTT assay, and colony formation assay was used to demonstrate the long-term effect of CCBs on proliferation of the sublines. Apoptosis was evaluated by Annexin V assay and autophagy intensity was quantitated from acidic vesicular organelle formation. Pan-caspase inhibitor, shATG5 interference and chloroquine were applied to study the roles of Verapamil on apoptosis and autophagy, with related proteins verified by Western blot analysis.ResultsResults show that 10 μM of Verapamil and Diltiazem, but not Nifedipine, differentially induce autophagy in DOC-resistant or VCR-resistant A549 cells, respectively. When CCBs are combined with DOC or VCR to treat the sublines, 10 μM of Verapamil induces autophagy more significantly than Diltiazem and Nifedipine, respectively, in DOC-resistant (54.91±0.76, 18.03±0.69, 7.05±0.30) or VCR-resistant A549 (32.41±1.04, 21.51±0.63, 7.14±0.24) cells. Inhibition of apoptosis by pan-caspase inhibitor partly reduced cell death indicates association of caspase-dependent cell death but with persistence of autophagy. Inhibition of autophagy by interfering ATG5 expression reduced c-PARP level and apoptotic cells suggest a pro-death role of autophagy. Chloroquine treatment enhanced autophagosome accumulation and cell death but with reduced c-PARP level suggests that mechanism of caspase-independent cell death also contributes to Verapamil/chemotherapy-induced anticancer effects. ConclusionVerapamil combined with DOC or VCR induces chemoresistant lung cancer cells to death through autophagy burst and apoptosis more strongly than Diltiazem and Nifedipine. Administering Verapamil or Diltiazem individually with chemotherapy, but not Nifedipine, can be considered in lung cancer patients with hypertension.

TiO2 nanoparticles enhance the chemotherapeutic effects of 5-fluorouracil in human AGS gastric cancer cells via autophagy blockade

AimsNanoparticles (NPs)-based drugs have been recently introduced to improve the efficacy of current therapeutic strategies for the treatment of cancer; however, the molecular mechanisms by which a NP interacts with cellular systems still need to be delineated. Here, we utilize the autophagic potential of TiO2 NPs for improving chemotherapeutic effects of 5-fluorouracil (5-FU) in human AGS gastric cells. Materials and methodsCell growth and viability were determined by trypan blue exclusion test and MTT assay, respectively. Vesicular organelles formation was evaluated by acridine orange staining of cells. Cell cycle and apoptosis were monitored by flow cytometry. Reactive oxygen species (ROS) level were measured by DCHF-DA staining. Autophagy was examined by q-PCR and western blotting. Molecular docking was used for studying NP interaction with autophagic proteins. Key findingsTiO2 NPs increase ROS production, impair lysosomal function and subsequently block autophagy flux in AGS cells. In addition, the autophagy blockade induced by non-toxic concentrations of TiO2 NPs (1 μg/ml) can promote cytotoxic and apoptotic effects of 5-FU in AGS cells. SignificanceThese results confirm the beneficial effects of TiO2 NPs in combination with chemotherapy in in vitro model of gastric cancer, which may pave the way to develop a possible solution to circumvent chemoresistance in cancer.

The Biofunctional Effects of Mesima as a Radiosensitizer for Hepatocellular Carcinoma.

The tropical basidiomycete fungus Phellinus linteus (Mesima) exhibits anti-tumor, anti-angiogenic, and immunomodulatory properties in various cancers including prostate, colon, and lung cancer along with melanoma by, for example, inducing apoptosis or cell cycle arrest. However, whether medina also facilitates treatment of hepatocellular carcinoma (HCC), the third global cause of cancer deaths, remains unknown. Here, we examined its potential as a radiosensitizer in HCC radiotherapy using human HCC Hep3B and HepG2 cell lines and xenograft tumors. Mesima pretreatment significantly enhanced HCC cell radiosensitivity in vitro and the combination of mesima + radiation treatment significantly reduced xenograft tumor growth and size in vivo compared to those with single treatments. Mechanistically, mesima significantly enhanced radiotherapy efficiency by inhibiting tumor cell survival through inducing apoptosis (assessed via annexin V), impairing cell cycle regulation (shown by flow cytometry), and reducing radiation-induced DNA damage repair (measured via γ-H2AX foci). Combination treatment also facilitated autophagic cell death beyond that from single treatments (assessed by quantifying stained acidic vesicular organelles), and diminished tumor cell metastatic potentials (shown by wound and Transwell assays). These findings support the synergistic anti-tumor effects of mesima combined with radiation and suggest scientific evidence for mesima as a radiosensitizer in HCC.

Anticancer effects of Calotropis procera latex extract in mcf-7 breast cancer cells

Background: Calotropis procera is a wild growing medicinal plant with many pharmacological properties, arising mainly from its latex, which contains many biologically active compounds, including cardiac glycosides. Objectives: The present study was conducted to isolate a cardiac glycosidal (CG) extract from the latex of C. procera and to assess its potential in inducing anticancer effects on breast cancer cells (MCF-7). Materials and Methods: Cytotoxicity was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay and morphological changes observations. The generation of intracellular reactive oxygen species (ROS) was evaluated both qualitatively and quantitatively. Flow cytometry technique was used to evaluate apoptosis and autophagy was determined by fluorescence microscopy and Western blotting. Results: The extract significantly (P < 0.05) inhibited the proliferation of MCF-7 cells and that this effect increased in line with concentration. Systemic changes in the morphology of treated cells when compared with control cells were observed. ROS levels were increased by about 1.5 and 1.95-fold at the highest concentration of 75 μg/ml after 12 and 24 h of treatment, respectively. A significant (P < 0.05) increase in the percentage of early and late apoptotic cells were recorded. Autophagy induction in treated MCF-7 was confirmed with the presence of acidic vesicular organelles. Finally, the change in the intracellular localization of light chain 3 (LC3) protein was determined by Western blotting using primary antibodies. A maximum of a 1.76-fold increase was observed in the expression level of the LC3 marker protein. Conclusion: These findings suggest that CG extract increased the levels of intracellular ROS resulting in the induction of cytotoxicity, apoptosis, and autophagy in MCF-7 cells.