Accumulation Of Acidic Vesicular Organelles Research Articles

Autophagy in antitumor activity of aloin for breast cancer cells compared with doxorubicin

Breast cancer is the most commonly diagnosed cancer and is one of the leading causes of cancer mortality in women worldwide. Natural product compounds have attracted significant attention for their potent effects against human cancers. Aloin, a natural phytochemical anthraquinone glycoside extracted from Aloe sp., has been previously reported for its antitumor activity. Autophagy is a highly conserved process that mediates the degradation of dysfunctional cellular components, such as senescent proteins and organelles. In the present study, we verified the involvement of autophagy in tolerance to aloin, especially in breast cancer cells with negative estrogen receptors, and as an alternative pathway to promote cell death in cells expressing mutant p53 status, which often limits the efficacy and accounts for resistance to chemotherapy. We studied the effect of aloin on 2 types of breast cancer cell lines, estrogen receptor-positive (T47D) and triple negative (MDA-MB231), and compared to an anthraquinone analog, doxorubicin (Dox) as a reference compound. Aloin inhibited the cell growth of both T47D and MDA-MB231 cancer cells, in a time- and dose-dependent manner with a more pronounced effect in the 72 h exposure regimen, and in the ERα+ breast cell line. The autophagic activity of aloin was emphasized by the formation of autophagosomes and autolysophagosomes, as early and late autophagic compartments, respectively, as well as the accumulation of acidic vesicular organelles in the tumor cells. Also, upregulation in the protein expression of some marker genes of autophagy such as beclin 1 and LC3BII/LC3I, and conversely down-regulation in pmTOR and p62 was recorded. The results suggest that autophagy can be regarded as one of the mechanistic modes of aloin cytotoxicity in breast cancer cells that evade apoptosis through genetic mutations in p53.

The PIP4K2 inhibitor THZ-P1-2 exhibits antileukemia activity by disruption of mitochondrial homeostasis and autophagy

The treatment of acute leukemia is challenging because of the genetic heterogeneity between and within patients. Leukemic stem cells (LSCs) are relatively drug-resistant and frequently relapse. Their plasticity and capacity to adapt to extracellular stress, in which mitochondrial metabolism and autophagy play important roles, further complicates treatment. Genetic models of phosphatidylinositol-5-phosphate 4-kinase type 2 protein (PIP4K2s) inhibition have demonstrated the relevance of these enzymes in mitochondrial homeostasis and autophagic flux. Here, we uncovered the cellular and molecular effects of THZ-P1-2, a pan-inhibitor of PIP4K2s, in acute leukemia cells. THZ-P1-2 reduced cell viability and induced DNA damage, apoptosis, loss of mitochondrial membrane potential, and the accumulation of acidic vesicular organelles. Protein expression analysis revealed that THZ-P1-2 impaired autophagic flux. In addition, THZ-P1-2 induced cell differentiation and showed synergistic effects with venetoclax. In primary leukemia cells, LC-MS/MS-based proteome analysis revealed that sensitivity to THZ-P1-2 is associated with mitochondrial metabolism, cell cycle, cell-of-origin (hematopoietic stem cell and myeloid progenitor), and the TP53 pathway. The minimal effects of THZ-P1-2 observed in healthy CD34+ cells suggest a favorable therapeutic window. Our study provides insights into the pharmacological inhibition of PIP4K2s targeting mitochondrial homeostasis and autophagy, shedding light on a new class of drugs for acute leukemia.

Engineered Nanoparticles as Potential Therapeutics for Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is a malignancy associated with poor prognosis. Particularly, older patients suffer greatly from the standard chemotherapy and have a 5‐year survival of only 4%. Hence, new therapeutic agents with higher specificity and lower general cytotoxicity are urgently required.We have discovered that positively charged amino‐functionalized polystyrene nanoparticles (NP‐PS+) induce accumulation of acidic vesicular organelles with elevated pH and impaired processing of procathepsin B leading to mTOR inhibition, activation of autophagy, and induction of caspase‐dependent apoptosis in leukemia cells, but not in normal human macrophages. The antileukemic effect of NP‐PS+ was also preserved in vivo, where NP‐PS+ inhibited proliferation and induced apoptosis in leukemia xenografts grown on chick chorioallantoic membranes.Similar to polystyrene particles, amino‐functionalized gold nanoparticles (NP‐Au+) exhibited selective cytotoxicity towards AML cell lines as well as primary patient‐derived AML cells. Thus, NP‐Au+ particles induced cell death in primary human leukemia cells and reduced their colony‐forming potential, whereas normal hematopoietic cells remained unaffected by the treatment with NP‐Au+. NP‐Au+ targeted specifically the oxidative mitochondrial respiration, which is, different to normal hematopoietic cells, the main source of energy production in AML blasts and leukemic stem cells. Different to NP‐Au+, conventional chemotherapeutics such ascytarabine act in a cell cycle‐dependent manner and target only on proliferating AML blasts but not on quiescent leukemic stem cells. In agreement with the in vitro data, NP‐Au+ exhibited antileukemic efficacy against primary human AML xenografted into mice applied either as monotherapy or as a cytarabine combination regimen in the absence of detectable adverse events.Thus, this engineered nanomaterial that targets particularly resistant quiescent leukemic stem cells holds great promise as a novel nanotherapeutic for the treatment of acute myeloid leukemia independent of its cytogenetic profile.

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.

Autophagy Inhibition Potentiates Ruxolitinib-Induced Apoptosis in JAK2V617F Cells

Introduction: Autophagy is a paradoxical and evolutionarily conserved cellular process that is fundamental to eliminating harmful intracellular components and allowing cells to adapt to nutrient starvation or stress conditions. Ruxolitinib, a selective JAK1/2 inhibitor approved by the FDA for treatment of intermediate and high-risk primary myelofibrosis and polycythemia vera patients, provides clinical benefits, but fail to eliminate the myeloproliferative neoplasm (MPN)-initiating cells. Frequency of mutations in autophagy-related genes has recently been reported in myeloid neoplasms (Visconte et al. Leukemia 2017). Considering that JAK2V617F mimics constitutive growth factors signal and leads to mTOR/PI3K activation, we hypothesized that ruxolitinib induces autophagy as a mechanism of resistance. Aims: To investigate the effects of ruxolitinib treatment on autophagy-related genes and cellular processes, the potential benefit of autophagy inhibitors plus ruxolitinib in JAK2V617F cells, and to verify frequency and clinical impact of autophagy-related genes mutations in MPN patients. Methods: SET2 cells (JAK2V617F-positive) were treated with increasing doses or exposure to ruxolitinib and subjected to protein extraction or autophagy analysis. Ruxotilinib (300 nM) and autophagy inhibitors (3-methyladenine [1 mM], bafilomycin A1 [10 nM] and chloroquine [20 μM]) were used in monotherapy or in combination. The expression of 79 autophagy-related genes was investigated in SET2 cells upon ruxolitinib treatment by RNA-seq (data obtained from Meyer et al. Cancer Cell 2015) and validated by qPCR and Western blot. Autophagy was evaluated by acridine orange staining and LC3BI/II or SQSTM1/p62 consumption, apoptosis by annexin V/PI staining and caspase 3 cleavage, and mitochondrial damage by JC-1 staining. Mutations in 124 autophagy-related genes were investigated by whole genome sequencing in a cohort of 65 MPN patients from a single Institution. ANOVA and Bonferroni post-test and Mann-Whitney test were used as appropriated. Kaplan-Meyer curves, log-rank test, and cox regression analysis were applied for survival analysis. Results: Ruxolitinib treatment promoted an accumulation of acidic vesicular organelles and consumption of LC3BI/II and SQSTM1/p62 proteins, characterizing autophagy induction. Using a 1.3-fold cutoff in both directions, treatment with ruxolitinib modulated the expression of 26 out of 79 autophagy-related genes investigated. BCL2 and BCL2L1 downregulation, BAX upregulation and stable BECN1 expression were validated, which impacted BCL2/BAX and BCL2/BECN1 ratios. In SET2 cells, ruxolitinib treatment reduced important autophagy regulators in a dose-dependent manner, including STAT3Y705, STAT5Y694, mTORS2448, p70S6KT421/S424 and 4EBP1T70 phosphorytation, and BCL2 and BCL-XL expression. All autophagy inhibitors significantly suppressed the ruxolitinib-induced autophagy, being bafilomycin A1 (reduction of 89%) and chroloquine (reduction of 70%) more efficient than 3-methyladenine (reduction of 10%) (all p<0.05). Autophagy pharmacological inhibition significantly increased ruxolitinib-induced apoptosis (all p<0.05) and accumulation of cells containing damaged mitochondria (all p<0.05). Mutations in autophagy-related genes were found in 25% (16 out of 65) of MPN patients and was associated with increased age (p<0.05). TP53 mutations were found in 6 patients and ARSA, TGFB1, APP, PPP2R3A, BECN1, SQSTM1, HGS, TNFSF13B, ATG2A, STAT3, ATG9B, WAC or PRKACB mutations were found in one case each. Mutated patients presented JAK2 (n=14), CALR (n=1) or MPL (n=1) mutations. Median overall survival for autophagy-related genes mutated and wild-type patients were 20 months vs. not reached (p=0.066), respectively. In multivariate analysis using mutations in TP53 only, mutations in other autophagy-related genes, and DIPSS as confounders, only DIPSS independently predicted overall survival (p<0.001). Conclusions: Ruxolitinib induced autophagy in JAK2V617F cells through modulation of mTOR/p70S6K/4EBP1 and STAT/BCL2 axis, which potentially represent a mechanism of resistance to apoptosis. Our results indicate that combination of ruxolitinib with autophagy pharmacological inhibitors, especially chloroquine, may be a promissory strategy to improving apoptosis induction in JAK2V617F cells. DisclosuresNo relevant conflicts of interest to declare.

Silencing of cadherin-17 enhances apoptosis and inhibits autophagy in colorectal cancer cells.

Cadherin-17 (CDH17), a structurally unique member of the non-classical cadherin family, is associated with poor survival, cell proliferation, and metastasis in colorectal cancer. However, the role of CDH17 in the apoptosis and autophagy of colorectal cancer cells remains unclear. Here, we aimed to investigate the effect of CDH17 knockdown on autophagy and apoptosis in colorectal cancer cells. We inhibited CDH17 expression in KM12SM and KM12C colorectal cancer cells by RNA interference and found that silencing of CDH17 significantly inhibited cell viability and increased apoptosis in KM12SM and KM12C cells. In addition, silencing of CDH17 significantly increased the expression of cleaved caspase-3 and Bax and decreased the expression of Bcl-2. Concurrently, silencing of CDH17 significantly inhibited the conversion of LC3-I to LC3-II and decreased the formation of LC3+ autophagic vacuoles and the accumulation of acidic vesicular organelles, indicating that autophagy was significantly inhibited in KM12SM and KM12C cells. Additionally, treatment with the autophagy-specific activator rapamycin attenuated apoptosis in CDH17-knockdown cells and as indicated by decreased caspase-3 activity, decreased expression of cleaved caspase-3 and Bax, and increased expression of Bcl-2. In conclusion, CDH17 silencing induced apoptosis and inhibited autophagy in KM12SM and KM12C cells, and this autophagy protected the cells from apoptotic cell death.

Clostridium difficile toxin B induces autophagic cell death in colonocytes.

Toxin B (TcdB) is a major pathogenic factor of Clostridum difficile. However, the mechanism by which TcdB exerts its cytotoxic action in host cells is still not completely known. Herein, we report for the first time that TcdB induced autophagic cell death in cultured human colonocytes. The induction of autophagy was demonstrated by the increased levels of LC3‐II, formation of LC3+ autophagosomes, accumulation of acidic vesicular organelles and reduced levels of the autophagic substrate p62/SQSTM1. TcdB‐induced autophagy was also accompanied by the repression of phosphoinositide 3‐kinase (PI3K)/Akt/mechanistic target of rapamycin (mTOR) complex 1 activity. Functionally, pharmacological inhibition of autophagy by wortmannin or chloroquine or knockdown of autophagy‐related genes Beclin 1, Atg5 and Atg7 attenuated TcdB‐induced cell death in colonocytes. Genetic ablation of Atg5, a gene required for autophagosome formation, also mitigated the cytotoxic effect of TcdB. In conclusion, our study demonstrated that autophagy serves as a pro‐death mechanism mediating the cytotoxic action of TcdB in colonocytes. This discovery suggested that blockade of autophagy might be a novel therapeutic strategy for C. difficile infection.

Abstract 3304: Inhibition of autophagy enhances mitochondria-mediated apoptosis in malignant mesothelioma cells by cisplatin and sulforaphane

Abstract Drug resistance to current chemotherapy regimens has been considered a cell survival adaptive response in malignant mesothelioma cells. Therefore, new strategies to enhance the apoptotic signal and overcome resistance to chemotherapeutics are necessary to improve treatment outcomes for this deadly disease. Here, the effect of sulforaphane, an isothiocyanate compound derived from glucoraphanin present in cruciferous vegetables, on enhancing anticancer role of cisplatin was investigated in malignant mesothelioma cells. At concentrations showing little toxicity in normal human mesothelial MeT-5A cells, the combined treatment with both compounds exhibited synergistic growth-inhibiting and apoptosis-promoting effects, as demonstrated in a series of pro-apoptotic events including reactive oxygen species (ROS) accumulation, loss of mitochondrial membrane potential, up-regulation of p53, increased Bax/Bcl-2 ratio, activation of caspase-3, and the occurrence of a sub-G0/G1 peak with an increase in the cells with pyknotic and fragmented nuclei, Annexin V-PE(+), and G2/M phase transition delay in the cell cycle. The levels of phosphorylation of both Akt and mTOR were decreased by the combined treatment, which was accompanied by a significant increase in the level of autophagosomal marker protein LC3-II and the accumulation of acidic vesicular organelles. Furthermore, scavenging of ROS by antioxidant N-acetylcysteine inhibited both the apoptosis and autophagy, while the inhibition of autophagy by bafilomycin A1 potentiated apoptotic cell death following the combination treatment of sulforaphane and cisplatin, indicating the cytoprotective role of autophagy counteracts apoptosis of malignant mesothelioma cells. Considering the pro-oxidant-based combinational approach, our data provide a rationale for targeting cytoprotective autophagy as a potential therapeutic strategy for malignant mesothelioma. Citation Format: Yoon Jin Lee, David M. Lee, Hae Nam, Sang Lee. Inhibition of autophagy enhances mitochondria-mediated apoptosis in malignant mesothelioma cells by cisplatin and sulforaphane [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3304. doi:10.1158/1538-7445.AM2017-3304

Abstract 3300: Inhibition of autophagy by 3-MA enhances the effect of ursolic acid-induced apoptosis in hepatoma SK-Hep-1 cells

Abstract Purpose The aim of this study is to investigate the autophagic effect of Ursolic Acid (UA) on hepatoma SK-Hep-1 cells. Methods The cytotoxicity of UA in hepatoma SK-Hep-1 cells was measured by MTT assay, the accumulation of acidic vesicular organelles was measured by FACS, the protein expression of LC3-II was detected by immunofluorescence staining, and the autophagic protein expression was evaluated by western blotting. Hepatoma SK-Hep-1 cells treated with a combination of autophagy inhibitor 3-Methyladenine (3-MA) and UA, then use Annexin V/PI Double Staining and western blotting to detect apoptosis and apoptotic protein levels. Statistical Analyses Data was expressed as the mean ± standard deviation and were compared using the Student’s t-test. A p value of &amp;lt;0.05 was considered to indicate statistical significance. All statistical analyses were performed using GraphPad Prism software, version 4.0 (GraphPad Software, Inc., La Jolla, CA, USA). Preliminary Results SK-Hep-1 cells treated with different concentrations of UA (0, 20, 40, and 60 μM). Regarding the results, the half-maximal inhibitory concentration (IC50) was 45.9 and 36.6 μM for 24 and 48 h, respectively. The FACS results showed that when increase the dose of UA could increase the percentages of autophagic populations. The anti-autophagic proteins such as Beclin, Atg-5, Atg-7, and LC3-II, in our results showed that UA decreased the protein expression of mTOR, PI3K, and p62 and PI3K/Akt/mTOR expression. Immunocytochemistry staining showed that SK-Hep-1 cells treated with UA increased the protein expression of LC3-II. The combination treatment of 3-MA and UA significantly increased the apoptotic cell death as monitored by increased protein expression of Caspase-3 and PAPR. Conclusions UA could induced autophagy in hepatoma SK-Hep-1 cells through inhibition of mTOR and PI3K signaling pathways. SK-Hep-1 cells treated with UA exhibited LC3-І/LC3-II conversion, increased protein expression levels of Beclin, Atg-7, and Atg-5 along with reduced protein expression levels of p62. UA-induced apoptosis was enhanced by the treatment of autophagy inhibitor 3-MA. Our findings may assist in the development of novel chemotherapeutic agents for the treatment of malignant types of liver cancer. Citation Format: Chuang Wan-Ling, Lin Ping-Yi, Chen Yao-Li. Inhibition of autophagy by 3-MA enhances the effect of ursolic acid-induced apoptosis in hepatoma SK-Hep-1 cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3300. doi:10.1158/1538-7445.AM2017-3300

Salinomycin induces endoplasmic reticulum stress‑mediated autophagy and apoptosis through generation of reactive oxygen species in human glioma U87MG cells.

Salinomycin is a polyether ionophore antibiotic that has recently been shown to induce cell apoptosis in human cancer cells displaying multiple mechanisms of drug resistance. In the present study, we explored the impact of salinomycin on the apoptosis and autophagy as well as the correlation between those effects and endoplasmic reticulum (ER) stress molecular mechanisms in human glioma U87MG cells. Apoptosis, autophagy and reactive oxygen species (ROS) were analyzed using flow cytometry. In addition, expression levels of apoptosis-, autophagy-and ER stress-related proteins were determined by western blotting. The results showed that salinomycin induced apoptosis, ER stress and autophagy in glioma cancer cell lines. In addition, salinomycin also induced ROS generation, and the ROS scavenger N-acetyl-L-cysteine was found to inhibit the salinomycin-induced apoptosis, ER stress and autophagy. The inhibition of ER stress with 4-phenylbutyric acid depressed salinomycin-induced apoptosis and autophagy. Salinomycin increased the expression of autophagy marker protein, LC3B, and accumulation of acidic vesicular organelles. Furthermore, pre-treatment with the autophagy inhibitor 3-methyladenine showed potential in increasing the apoptosis rate induced by salinomycin in the U87MG cells. Taken together, these results revealed that salinomycin induced apoptosis and autophagy via ER stress mediated by ROS, suggesting that ER stress by salinomycin plays a dual function in both promoting and suppressing cell death.

Pro-oxidant activity of sulforaphane and cisplatin potentiates apoptosis and simultaneously promotes autophagy in malignant mesothelioma cells.

Sulforaphane (SFN) is an isothiocyanate compound derived from glucoraphanin, which is found in cruciferous vegetables, and has been heralded as a chemopreventive and/or chemotherapeutic agent. The present study investigated the effects of SFN on enhancing the anticancer role of cisplatin (cis-dichlorodiammineplatinum; CDDP) in H‑28 malignant mesothelioma cells. At concentrations demonstrating limited toxicity in MeT‑5A normal human mesothelial cells, combination treatment with the two compounds exhibited synergistic growth‑inhibiting and apoptosis‑promoting activities, as demonstrated by a series of proapoptotic events, including reactive oxygen species accumulation, loss of mitochondrial membrane potential, upregulation of p53 expression, increased B‑cell lymphoma 2 (Bcl‑2) associated X protein/Bcl‑2 ratio, activation of caspase‑3, the occurrence of a sub‑G0/G1 peak and an increase in cells with pyknotic and fragmented nuclei, Annexin V‑phycoerythrin‑positive staining and G2/M phase‑transition delay in the cell cycle. The phosphorylation levels of Akt and mammalian target of rapamycin were reduced by the combination treatment, which was accompanied by a significant increase in the level of autophagosomal marker protein microtubule‑associated protein 1 light chain 3B‑II and the accumulation of acidic vesicular organelles. Pretreatment with the antioxidant N‑acetylcysteine attenuated both apoptosis and autophagy, whereas inhibition of autophagy by bafilomycin A1 potentiated apoptotic cell death following the combination treatment with SFN and CDDP. Considering the pro‑oxidant‑based combinational approach, the results of the present study provide a rationale for targeting cytoprotective autophagy as a potential therapeutic strategy for malignant mesothelioma.

Salinomycin simultaneously induces apoptosis and autophagy through generation of reactive oxygen species in osteosarcoma U2OS cells

Salinomycin, a polyether antibiotic, acts as a highly selective potassium ionophore. It was reported to anticancer activity on various cancer cell lines. In this study, salinomycin was examined on apoptosis and autophagy through generation of reactive oxygen species (ROS) in osteosarcoma U2OS cells. Apoptosis, autophagy, mitochondrial membrane potential (MMP) and ROS were analyzed using flow cytometry. Also, expressions of apoptosis- and autophagy-related proteins were determined by western blotting. As a result, salinomycin triggered apoptosis of U2OS cells, which was accompanied by change of MMP and cleavage of caspases-3 and poly (ADP-ribose) polymerase. And salinomycin increased the expression of autophagy-related protein and accumulation of acidic vesicular organelles (AVO). Salinomycin-induced ROS production promotes both apoptosis and autophagy, as evidenced by the result that treatment of N-acetyl-l-cysteine (NAC), a ROS scavenger, attenuated both apoptosis and autophagy. In addition, inhibition of autophagy by 3-methyladenine (3 MA) enhanced the salinoymcin-induced apoptosis. Taken together, these results suggested that salinomycin-induced autophagy, as a survival mechanism, might be a potential strategy through ROS regulation in cancer therapy.

Sodium Butyrate Induces Endoplasmic Reticulum Stress and Autophagy in Colorectal Cells: Implications for Apoptosis.

PurposeButyrate, a short-chain fatty acid derived from dietary fiber, inhibits proliferation and induces cell death in colorectal cancer cells. However, clinical trials have shown mixed results regarding the anti-tumor activities of butyrate. We have previously shown that sodium butyrate increases endoplasmic reticulum stress by altering intracellular calcium levels, a well-known autophagy trigger. Here, we investigated whether sodium butyrate-induced endoplasmic reticulum stress mediated autophagy, and whether there was crosstalk between autophagy and the sodium butyrate-induced apoptotic response in human colorectal cancer cells.MethodsHuman colorectal cancer cell lines (HCT-116 and HT-29) were treated with sodium butyrate at concentrations ranging from 0.5–5mM. Cell proliferation was assessed using MTT tetrazolium salt formation. Autophagy induction was confirmed through a combination of Western blotting for associated proteins, acridine orange staining for acidic vesicles, detection of autolysosomes (MDC staining), and electron microscopy. Apoptosis was quantified by flow cytometry using standard annexinV/propidium iodide staining and by assessing PARP-1 cleavage by Western blot.ResultsSodium butyrate suppressed colorectal cancer cell proliferation, induced autophagy, and resulted in apoptotic cell death. The induction of autophagy was supported by the accumulation of acidic vesicular organelles and autolysosomes, and the expression of autophagy-associated proteins, including microtubule-associated protein II light chain 3 (LC3-II), beclin-1, and autophagocytosis-associated protein (Atg)3. The autophagy inhibitors 3-methyladenine (3-MA) and chloroquine inhibited sodium butyrate induced autophagy. Furthermore, sodium butyrate treatment markedly enhanced the expression of endoplasmic reticulum stress-associated proteins, including BIP, CHOP, PDI, and IRE-1a. When endoplasmic reticulum stress was inhibited by pharmacological (cycloheximide and mithramycin) and genetic (siRNA targeting BIP and CHOP) methods, the induction of BIP, PDI, IRE1a, and LC3-II was blocked, but PARP cleavage was markedly enhanced.DiscussionTaken together, these results suggested that sodium butyrate-induced autophagy was mediated by endoplasmic reticulum stress, and that preventing autophagy by blocking the endoplasmic reticulum stress response enhanced sodium butyrate-induced apoptosis. These results provide novel insights into the anti-tumor mechanisms of butyric acid.

Autophagy Plays a Cytoprotective Role During Cadmium-Induced Oxidative Damage in Primary Neuronal Cultures.

Cadmium (Cd) induces significant oxidative damage in cells. Recently, it was reported that autophagy could be induced by Cd in neurons. However, little is known about the role of reactive oxygen species (ROS) during Cd-induced autophagy. In our study, we examined the cross-talk between ROS and autophagy by using N-acetyl cysteine (NAC, an antioxidant) and chloroquine (CQ, a pharmacological inhibitor of autophagy) in a primary rat neuronal cell cultures. We observed accumulation of acidic vesicular organelles and the increased expression of endogenous protein light chain 3 (LC3) in Cd-treated neurons, revealing that Cd induced a high level of autophagy. Moreover, increased levels of ROS were observed in neurons treated with Cd, showing that ROS accumulation was closely associated with neuron's exposure to Cd. Furthermore, we found that autophagy was inhibited by using CQ and/or NAC with further aggravation of mitochondrial damage, lactate dehydrogenase (LDH) leakage and hypoploid apoptotic cell number in Cd-treated neurons. These results proved that autophagy has a cytoprotective role during Cd-induced toxicity in neurons, and it can prevent the oxidative damage. These findings may enable the development of novel therapeutic strategies for neurological diseases.

MHY218-induced apoptotic cell death is enhanced by the inhibition of autophagy in AGS human gastric cancer cells.

We previously reported the anticancer effects of MHY218, which is a hydroxamic acid derivative, in HCT116 human colon cancer cells. In the present study, the involvement of autophagy in the MHY218-induced apoptotic cell death of AGS human gastric cancer cells was investigated. MHY218 treatment induced growth inhibition and apoptotic cell death in a concentration- and time-dependent manner. The induction of apoptosis was confirmed by observations of decreased viability, DNA fragmentation, and an increase in late apoptosis and sub-G1 DNA, which were detected with a flow cytometric analysis. Western blot analyses showed that MHY218 treatment resulted in decreased protein levels of procaspase-8, -9, and -3; cleavage of poly(ADP-ribose) polymerase (PARP); and alterations in the ratio of Bax/Bcl-2 protein expression. Apoptosis induced by MHY218 was involved in the activation of caspase-8, -9, and -3, and it was blocked by the addition of Z-VAD‑FMK, a pan-caspase inhibitor. In addition, autophagy-inducing effects of MHY218 were indicated by cytoplasmic vacuolation, the accumulation of acidic vesicular organelles, the appearance of green fluorescent protein-light-chain 3 (LC3) punctate dots, and increased levels of Beclin-1 and LC3-II protein expression. Pretreatment with the autophagy inhibitors LY294002, 3-methyladenine, chloroquine, and bafilomycin A1 enhanced the induction of apoptosis by MHY218, and this was accompanied by an increase in PARP cleavage. Taken together, these results provide new insights into the role of MHY218 as a potential antitumor agent. The combination of MHY218 with an autophagy inhibitor might be a useful candidate for the chemoprevention and/or treatment of gastric cancer.

Sann-Joong-Kuey-Jian-Tang induces autophagy in HepG2 cells via regulation of the phosphoinositide-3 kinase/Akt/mammalian target of rapamycin and p38 mitogen-activated protein kinase pathways.

Sann-Joong-Kuey-Jian-Tang (SJKJT), a traditional Chinese medicine, was previously reported to induce autophagy and inhibit the proliferation of the human HepG2 hepatocellular carcinoma cell line via an extrinsic pathway. In the present study, the effects of SJKJT-induced autophagy and the cytotoxic mechanisms mediating these effects were investigated in HepG2 cells. The cytotoxicity of SJKJT in the HepG2 cells was evaluated using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The results demonstrated that the half-maximal inhibitory concentration of SJKJT was 2.91 mg/ml at 24 h, 1.64 mg/ml at 48 h and 1.26 mg/ml at 72 h. The results of confocal fluorescence microscopy indicated that SJKJT resulted in the accumulation of green fluorescent protein-LC3 and vacuolation of the cytoplasm. Flow cytometric analysis revealed the accumulation of acidic vesicular organelles. Furthermore, western blot analysis, used to determine the expression levels of autophagy-associated proteins, demonstrated that the HepG2 cells treated with SJKJT exhibited LC3B-I/LC3B-II conversion, increased expression levels of Beclin, Atg-3 and Atg-5 and reduced expression levels of p62 and decreased signaling of the phosphoinositide-3 kinase/Akt/mammalian target of rapamycin and the p38 mitogen-activated protein kinase pathways. Taken together, these findings may assist in the development of novel chemotherapeutic agents for the treatment of malignant types of liver cancer.

Apigenin-induced apoptosis is enhanced by inhibition of autophagy formation in HCT116 human colon cancer cells

Apigenin (4',5,7-trihydroxyflavone) is a natural flavonoid, shown to have chemopreventive and/or anticancer properties in a variety of human cancer cells. The involvement of autophagy in apigenin-induced apoptotic cell death of HCT116 human colon cancer cells was investigated. Apigenin induced suppression of cell growth in a concentration-dependent manner in HCT116 cells. Flow cytometric analyses indicated that apigenin resulted in G2/M phase arrest. This flavone also suppressed the expression of both cyclin B1 and its activating partners, Cdc2 and Cdc25c, whereas the expression of cell cycle inhibitors, such as p53 and p53-dependent p21(CIP1/WAF1), was increased after apigenin treatment. Apigenin induced poly (ADP-ribose) polymerase (PARP) cleavage and decreased the levels of procaspase-8, -9 and -3. In addition, the apigenin-treated cells exhibited autophagy, as characterized by the appearance of autophagosomes under fluorescence microscopy and the accumulation of acidic vesicular organelles by flow cytometry. Furthermore, the results of the western blot analysis revealed that the levels of LC3-II, the processed form of LC3-I, was increased by apigenin. Treatment with the autophagy inhibitor 3-methyladenine (3-MA) significantly enhanced the apoptosis induced by apigenin, which was accompanied by an increase in the levels of PARP cleavage. These results indicate that apigenin has apoptosis- and autophagy-inducing effects in HCT116 colon cancer cells. Autophagy plays a cytoprotective role in apigenin-induced apoptosis, and the combination of apigenin and an autophagy inhibitor may be a promising strategy for colon cancer control.

Autophagy inhibition enhances apigenin-induced apoptosis in human breast cancer cells.

Apigenin (4',5,7-trihydroxyflavone) is a member of the flavone subclass of flavonoids present in fruits and vegetables. The involvement of autophagy in the apigenin-induced apoptotic death of human breast cancer cells was investigated. Cell proliferation and viability were assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and clonogenic assays. Flow cytometry, fluorescent staining and Western blot analysis were employed to detect apoptosis and autophagy, and the role of autophagy was assessed using autophagy inhibitors. Apigenin dose- and time-dependently repressed the proliferation and clonogenic survival of the human breast cancer T47D and MDA-MB-231 cell lines. The death of T47D and MDA-MB-231 cells was due to apoptosis associated with increased levels of Caspase3, PARP cleavage and Bax/Bcl-2 ratios. The results from flow cytometry and fluorescent staining also verified the occurrence of apoptosis. In addition, the apigenin-treated cells exhibited autophagy, as characterized by the appearance of autophagosomes under fluorescence microscopy and the accumulation of acidic vesicular organelles (AVOs) by flow cytometry. Furthermore, the results of the Western blot analysis revealed that the level of LC3-II, the processed form of LC3-I, was increased. Treatment with the autophagy inhibitor, 3-methyladenine (3-MA), significantly enhanced the apoptosis induced by apigenin, which was accompanied by an increase in the level of PARP cleavage. Similar results were also confirmed by flow cytometry and fluorescence microscopy. These results indicate that apigenin has apoptosis- and autophagy-inducing effects in breast cancer cells. Autophagy plays a cyto-protective role in apigenin-induced apoptosis, and the combination of apigenin and an autophagy inhibitor may be a promising strategy for breast cancer control.

Synthesized Pheophorbide a‐mediated photodynamic therapy induced apoptosis and autophagy in human oral squamous carcinoma cells

Pheophorbide a (Pa) is a chlorine-based photosensitizer derived from an ethnopharmacological herb, and our group recently synthesized Pa by the removal of a magnesium ion and a phytyl group from chlorophyll-a. In this study, the effect of photodynamic therapy (PDT) with synthesized Pa was examined in a human oral squamous cell carcinoma (OSCC) cells. Cells were treated with PDT with Pa, and reactive oxygen species (ROS) and mitochondrial membrane potential [ΔΨ (m)] were examined. Apoptosis was measured using annexin V staining and immunoblot. Autophagy was characterized by the increase in LC3B-II and the formation of autophagosome and acidic vesicular organelles (AVOs). Pa-PDT inhibited the proliferation of OSCC cells in a dose-dependent manner. Pa-PDT increased the number of apoptotic cells by inactivating ERK pathway. Pa-PDT also induced autophagy in OSCC cells evidenced by the increased levels of LC3 type II expression and the accumulation of AVOs. The inhibition of autophagy enhanced Pa-PDT-mediated cytotoxicity through an increase in necrosis. These results suggest that synthesized Pa-PDT exerts anti-tumor effects by inducing apoptosis and autophagy and provide novel evidence that Pa-PDT induces autophagy, and autophagy inhibition enhances Pa-PDT-mediated necrosis in OSCC cells.

Inhibition of autophagy by 3-methyladenine protects 1321N1 astrocytoma cells against pyocyanin- and 1-hydroxyphenazine-induced toxicity

Central nervous system (CNS) infections due to Pseudomonas aeruginosa are difficult to treat and have a high mortality rate. Pyocyanin, a virulence factor produced by P. aeruginosa, has been shown to be responsible for the majority of P. aeruginosa's pathogenicity in mammalian cells. Several lines of evidence in respiratory cells suggest that this damage is primarily mediated by pyocyanin's ability to generate ROS and deplete host antioxidant defense mechanisms. However, it has yet to be established whether pyocyanin or 1-hydroxyphenazine have potential toxicity to the CNS. Therefore, the aim of this study was to compare the CNS toxicity of pyocyanin and 1-hydroxyphenazine in vitro and to provide insight into mechanisms that underlie this toxicity using 1321N1 astrocytoma cells. To achieve this, we investigated the contribution of oxidative stress and other mediators of cell death including autophagy, senescence and apoptosis. We show that oxidative stress is not a primary mediator of pyocyanin (0-100μM) and 1-hydroxyphenazine (0-100μM) induced toxicity in 1321N1 cells. Instead, our results suggest that autophagy may play a central role. The autophagy inhibitor 3-methyladenine (5mM) protected 1321N1 astrocytoma cells against both pyocyanin and 1-hydroxyphenazine-induced cell injury and increased accumulation of acidic vesicular organelles, a hallmark of autophagy. Furthermore, apoptosis and senescence events may be secondary to autophagy in pyocyanin and 1-hydroxyphenazine-mediated cell injury. In conclusion, this study provides the first evidence on mechanisms underlying the toxicity of both pyocyanin and 1-hydroxyphenazine to astrocytoma cells and provides novel evidence suggesting that this toxicity may be mediated by the formation of acidic vesicular organelles, a hallmark of autophagic cell death.