Investigating the multifaceted cooperation of autophagy, PI3K/AKT signaling pathways, and INPP4B gene in de novo acute myeloid leukemia patients.

BACKGROUND: Acute myeloid leukemia (AML) has been the most prevalent form of acute leukemia among adults, and it has been associated with poor survival rates over the last four decades. Understanding the processes involved in leukemogenesis, particularly autophagy and signaling pathways, can provide critical insights into their roles in disease development, risk assessment, and potential therapeutic interventions. This study investigated gene expression changes, focusing on MAP1LC3B and BECN1, related to autophagy, as well as PI3KCA and AKT1 in the PI3K-AKT pathway, and INPP4B, which regulates this signaling cascade. METHODS: We collected blood samples from 21 AML patients and 9 healthy volunteers. Gene expression was analyzed through qPCR following RNA extraction and cDNA synthesis. Statistical analysis encompassed t-tests, ANOVA, and correlation coefficients. RESULTS: AML patients exhibited significantly increased MAP1LC3B gene expression (****P < 0.0001; fold change = 11.9) and significantly reduced levels of INPP4B (****P < 0.0001; fold change = 0.026), AKT1 (*P < 0.05; fold change = 0.59), and PI3KCA (****P < 0.0001; fold change = 0.16) compared to healthy controls. However, BECN1 gene expression did not significantly differ between the two groups. Additionally, noteworthy correlations were observed between INPP4B and BECN1 (r = 0.57; P = 0.006) and BECN1 and PI3KCA (r = 0.61; P = 0.003) in AML patients. CONCLUSIONS: This study highlights variations in leukemogenesis pathways, exemplified by increased MAP1LC3B expression and diminished expression of regulatory genes in specific AML cases. These findings contribute to our comprehension of the molecular mechanisms underlying AML and may inform future diagnostic and therapeutic approaches.

The therapeutic effect of hesperetin on doxorubicin-induced testicular toxicity: Potential roles of the mechanistic target of rapamycin kinase (mTOR) and dynamin-related protein 1 (DRP1).

Clinical utilization of doxorubicin (DOX), which is a commonly used chemotherapeutic, is restricted due to toxic effects on various tissues. Using hesperetin (HST), an antioxidant used in Chinese traditional medicine protects testis against DOX-induced toxicity although the molecular mechanisms are not well-known. The study was aimed to examine the possible role of the mechanistic target of rapamycin kinase (mTOR) and dynamin 1-like dynamin-related protein 1 (DRP1) in the therapeutic effects of HST on the DOX-induced testicular toxicity. Rats were divided into Control, DOX, DOX + HST, and HST groups (n = 7). Single-dose DOX (15 mg/kg) was administered intraperitoneally and HST (50 mg/kg) was administered by oral gavage every other day for 28 days. Total antioxidant status (TAS), histopathological evaluations, immunohistochemistry, and gene expression level detection analyses were performed. Histopathologically, DOX-induced testicular damage was ameliorated by HST treatment. DOX reduced testicular TAS levels and increased oxidative stress markers, 8-Hydroxy-deoxyguanosine (8-OHdG), and 4-Hydroxynonenal (4-HNE). Also, upregulated mTOR and DRP1 expressions with DOX exposure were decreased after HST treatment in the testis (p < 0.05). On the other hand, DOX-administration downregulated miR-150-5p and miR-181b-2-3p miRNAs, targeting mTOR and mRNA levels of beclin 1 (BECN1) and autophagy-related 5 (ATG5), autophagic markers. Furthermore, these levels were nearly similar to control testis samples in the DOX + HST group (p < 0.05). The study demonstrated that HST may have a therapeutic effect on DOX-induced testicular toxicity by removing reactive oxygen species (ROS) and by modulating the mTOR and DRP1 expressions, which have a critical role in regulating the balance of generation/elimination of ROS.

Selenium inhibits growth of trastuzumab-resistant human breast cancer cells via downregulation of Akt and beclin-1.

The response rate to treatment with trastuzumab (Tz), a recombinant humanized anti-HER2 monoclonal antibody, is only 12-34% despite demonstrated effectiveness on improving the survival of patients with HER2-positive breast cancers. Selenium has an antitumor effect against cancer cells and can play a cytoprotective role on normal cells. This study investigated the effect of selenium on HER2-positive breast cancer cells and the mechanism in relation to the response of the cells to Tz. HER2-positive breast cancer cell lines, SK-BR-3 as trastuzumab-sensitive cells, and JIMT-1 as Tz-resistant cells were treated with Tz and sodium selenite (selenite). Cell survival rates and expression of Her2, Akt, and autophagy-related proteins, including LC3B and beclin 1, in both cell lines 72 h after treatment were evaluated. Significant cell death was induced at different concentrations of selenite in both cell lines. A combined effect of selenite and Tz at 72 h was similar to or significantly greater than each drug alone. The expression of phosphorylated Akt (p-Akt) was decreased in JIMT-1 after combination treatment compared to that after only Tz treatment, while p-Akt expression was increased in SK-BR-3. The expression of beclin1 increased particularly in JIMT-1 after only Tz treatment and was downregulated by combination treatment. These results showed that combination of Tz and selenite had an antitumor effect in Tz-resistant breast cancer cells through downregulation of phosphorylated Akt and beclin1-related autophagy. Selenite might be a potent drug to treat Tz-resistant breast cancer by several mechanisms.

Vitamin D promotes autophagy in AML cells by inhibiting miR-17-5p-induced Beclin-1 overexpression.

MicroRNA (miR)-17-5p has been investigated in many diseases as a regulator of disease progression and is highly expressed in acute myeloid leukemia (AML). However, potential mechanisms underlying the function of miR-17-5p in AML need more elucidation. MiR-17-5p expression was augmented, while 25(OH)D3 and Beclin-1 levels were decreased in AML patients with the highest risk for disease progression. MiR-17-5p, 25(OH)D3 and Beclin-1 were determined to be clinically important in AML based on ROC curve analysis. Higher miR-17-5p expression as well as lower 25(OH)D3 and Beclin-1 expression were relevant with poor prognosis in AML. In addition, miR-17-5p was negatively correlated with and bound to BECN1. Vitamin D was found to diminish cell proliferation and enhance autophagy. Finally, through rescue assays, miR-17-5p facilitated the ability of cell proliferation, inhibited autophagy and apoptosis by modulating Beclin-1 in HL-60 cells following the treatment of 4 µM vitamin D. Vitamin D promoted autophagy in AML cells by modulating miR-17-5p and Beclin-1.

MicroRNA-204-5p regulates apoptosis by targeting Bcl2 in rat ovarian granulosa cells exposed to cadmium†.

This study aimed to investigate whether cadmium (Cd) cytotoxicity in rat ovarian granulosa cells (OGCs) is mediated through apoptosis or autophagy and to determine the role of microRNAs (miRNAs) in Cd cytotoxicity. To test this hypothesis, rat OGCs were exposed to 0, 10, and 20 µM CdCl2 in vitro. As the Cd concentration increased, OGC apoptosis increased. In addition, Cd promoted apoptosis by decreasing the mRNA and protein expression levels of inhibition of B-cell lymphoma 2 (Bcl2). However, under our experimental conditions, no autophagic changes in rat OGCs were observed, and the mRNA and protein expression levels of the autophagic markers microtubule-associated protein 1 light chain 3 alpha (Map1lc3b) and Beclin1 (Becn1) were not changed. Microarray chip analysis, miRNA screening, and bioinformatics approaches were used to further explore the roles of apoptosis regulation-related miRNAs. In total, 19 miRNAs putatively related to Cd-induced apoptosis in rat OGCs were identified. Notably, miR-204-5p, which may target Bcl2, was identified. Then, rat OGCs were cultured in vitro and used to construct the miR-204-5p-knockdown cell line LV2-short hairpin RNA (shRNA). LV2-shRNA cells were exposed to 20 µM Cd for 12 h, and the mRNA and protein expression levels of Bcl2 were increased. Our findings suggest that Cd is cytotoxic to rat OGCs, and mitochondrial apoptosis rather than autophagy mediates Cd-induced damage to OGCs. Cd also affects apoptosis-related miRNAs, and the underlying apoptotic mechanism may involve the Bcl2 gene.

The beclin 1 interactome: Modification and roles in the pathology of autophagy-related disorders.

Beclin 1 a yeast Atg6/VPS30 orthologue has a significant role in autophagy process (Macroautophagy) and protein sorting. The function of beclin 1 depends on the interaction with several autophagy-related genes (Atgs) and other proteins during the autophagy process. The role mediated by beclin 1 is controlled by various conditions and factors. Beclin 1 is regulated at the gene and protein levels by different factors. These regulations could subsequently alter the beclin 1 induced autophagy process. Therefore, it is important to study the components of beclin 1 interactome and factors affecting its expression. Expression of this gene is differentially regulated under different conditions in different cells or tissues. So, the regulation part is important to study as beclin 1 is one of the candidate genes involved in diseases related to autophagy dysfunction. This review focuses on the functions of beclin 1, its interacting partners, regulations at gene and protein level, and the role of beclin 1 interactome in relation to various diseases along with the recent developments in the field.

Endoplasmic reticulum stress potentiates the autophagy of alveolar macrophage to attenuate acute lung injury and airway inflammation.

Endoplasmic reticulum (ER) stress has been reported to play a role in acute lung injury (ALI), yet the in-depth mechanism remains elusive. This study aims to investigate the effect of ER stress-induced autophagy of alveolar macrophage (AM) on acute lung injury (ALI) and airway inflammation using mouse models. ALI models were induced by intranasal instillation of lipopolysaccharide (LPS). The lung weight/body weight (LW/BW) ratio and excised lung gas volume (ELGV) in each group were measured. Mouse bronchoalveolar lavage fluid (BALF) was collected for cell sorting and protein concentration determination. Expression of tumor necrosis factor α (TNF-α) and interleukin-6 (IL-6) in lung tissues and BALF was also detected. Mouse AMs were isolated to observe the autophagy. Expression of GRP78, PERK, LC3I, LC3II and Beclin1 was further determined. The results indicated that tunicamycin (TM) elevated GRP78 and PERK expression of AMs in ALI mice in a dose-dependent manner. Low dosage of TM abated LC3I expression, increased LC3II and Beclin1 expression, triggered ER stress and AM autophagy, and alleviated pathological changes of AMs in ALI mice. Also, in ALI mice, low dosage of TM attenuated goblet cell proliferation of tracheal wall, and declined LW/BW ratio, ELGV, total cells and neutrophils, protein concentrations in BALF, and IL-6 and TNF-α expression in lung tissues and BALF. Collectively, this study suggests that a low dosage of TM-induced ER stress can enhance the autophagy of AM in ALI mice models, thus attenuating the progression of ALI and airway inflammation.

LncRNA-MALAT1 influences myocardial infarction by regulating miR-30a/beclin-1 pathway.

OBJECTIVE: Long non-coding ribonucleic acid-metastasis-associated lung adenocarcinoma transcript 1 (lncRNA-MALAT1) has been confirmed as a key factor involving in various physiological and pathological processes. The present study aims to investigate whether lncRNA-MALAT1 affects the process of myocardial infarction (MI) in rats by regulating the micro RNA (miR-30a)/Beclin-1 (BECN1) pathway. MATERIALS AND METHODS: Twelve healthy male Sprague-Dawley rats were randomly selected and equally divided into sham-operation group and MI group. In MI group, a rat model of acute MI (AMI) was established by ligating the left anterior descending coronary artery. Rats in sham-operation group were set as the control. The messenger RNA (mRNA) expression levels of lncRNA-MALAT1, miR-30a, and BECN1 in the infarcted myocardial tissues were detected via real-time fluorescence quantitative polymerase chain reaction (qRT-PCR). The rat myocardial cell line H9c2 was cultured in vitro and then transfected with vectors of lncRNA-MALAT1, miR-30a, and BECN1. After that, qRT-PCR was performed to measure mRNA levels of lncRNA-MALAT1, miR-30a, and BECN1 in H9c2 cells. The protein level of BECN1 in cells was determined via Western blotting (WB) assay. RESULTS: Expression levels of lncRNA-MALAT1 and BECN1 in the rat myocardium of MI group were up-regulated markedly (p<0.01), while miR-30a was down-regulated notably (p<0.01) compared with those in sham-operation group. After H9c2 cells were transfected with overexpression vectors of lncRNA-MALAT1 or miR-30a, expression levels of miR-30a (p<0.01) and BECN1 (p<0.01) were remarkably down-regulated, respectively. CONCLUSIONS: LncRNA-MALAT1 up-regulates the expression of BECN1 by binding to miR-30a, thereby increasing the level of cell autophagy after MI.

Autophagy inhibition via Becn1 downregulation improves the mesenchymal stem cells antifibrotic potential in experimental liver fibrosis.

Liver fibrosis (LF) is the result of a vicious cycle between inflammation-induced chronic hepatocyte injury and persistent activation of hepatic stellate cells (HSCs). Mesenchymal stem cell (MSC)-based therapy may represent a potential remedy for treatment of LF. However, the fate of transplanted MSCs in LF remains largely unknown. In the present study, the fate and antifibrotic effect of MSCs were explored in a LF model induced by CCl4 in mouse. Additionally, MSCs were stimulated in vitro with LF-associated factors, tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), and transforming growth factor-ß1 (TGF-ß1), to mimic the LF microenvironment. We unveiled that MSCs exhibited autophagy in response to the LF microenvironment through Becn1 upregulation both in vivo and in vitro. However, autophagy suppression induced by Becn1 knockdown in MSCs resulted in enhanced antifibrotic effects on LF. The improved antifibrotic potential of MSCs may be attributable to their inhibitory effects on T lymphocyte infiltration, HSCs proliferation, as well as production of TNF-α, IFN-γ, and TGF-ß1, which may be partially mediated by elevated paracrine secretion of PTGS2/PGE2 . Thus, autophagy manipulation in MSCs may be a novel strategy to promote their antifibrotic efficacy.

MicroRNA-206 inhibition and activation of the AMPK/Nampt signalling pathway enhance sevoflurane post-conditioning-induced amelioration of myocardial ischaemia/reperfusion injury.

This study aimed to investigate the effect of miR-206 on apoptosis and autophagy of cardiomyocytes in ischaemia/reperfusion (I/R) injured rats treated with sevoflurane post-conditioning (SP) through the AMPK/Nampt signalling pathway. Rat models of myocardial I/R injury were established. The combination of SP, miR-206 inhibitor, AMPK activator AICAR and inhibitor Compound C was induced in rats, and their effects on I/R injury were determined with detection of malondialdehyde (MDA), hydroxyproline (HYP) and superoxide dismutase (SOD) levels in cardiomyocytes, autophagy, and apoptosis. We also conducted experiments to determine p62, Beclin1, Bax and Bcl-2 expression and the mRNA and expression pattern of AMPK/Nampt signalling pathway-related genes. Myocardial I/R injured rats revealed decreased SOD activity and elevated MDA content, autophagy and apoptosis. With the combined performance of SP, miR-206 inhibitor and AMPK activator AICAR, the rats presented higher SOD level and lower MDA and HYP levels, suppressed autophagy and apoptosis. Meanwhile, miR-206 inhibition also contributed to elevated expression of Nampt and the extent of AMPK phosphorylation, increased Bcl-2/Bax ratio, but degraded expression of p62 and Beclin1. Collectively, inhibition of miR-206 could activate the AMPK/Nampt signalling pathway, thus protecting against myocardial I/R injury on the basis of SP.

Merkel cell polyomavirus oncoproteins induce microRNAs that suppress multiple autophagy genes.

Viruses can inhibit host autophagy through multiple mechanisms, and evasion of autophagy plays an important role in immune suppression and viral oncogenesis. Merkel cell polyomavirus (MCPyV) T-antigens are expressed and involved in the pathogenesis of a large proportion of Merkel cell carcinoma (MCC). Yet, how MCPyV induces tumorigenesis is not fully understood. Herein, we show that MCPyV T-antigens induce miR-375, miR-30a-3p and miR-30a-5p expressions, which target multiple key genes involved in autophagy, including ATG7, SQSTM1 (p62) and BECN1. In MCC tumors, low expression of ATG7 and p62 are associated with MCPyV-positive tumors. Ectopic expression of MCPyV small T-antigen and truncated large T-antigen (LT), but not the wild-type LT, resulted in autophagy suppression, suggesting the importance of autophagy evasion in MCPyV-mediated tumorigenesis. Torin-1 treatment induced cell death, which was attenuated by autophagy inhibitor, but not pan-caspase inhibitor, suggesting a potential role of autophagy in promoting cell death in MCC. Conceptually, our study shows that MCPyV oncoproteins suppress autophagy to protect cancer cells from cell death, which contribute to a better understanding of MCPyV-mediated tumorigenesis and potential MCC treatment.

[The role of autophagy in PM2.5-induced inflammation in human nasal epithelial cells].

Objective: To explore the role of autophagy in PM2.5-induced inflammation in human nasal epithelial cells and related mechanism. Methods: Human nasal epithelial cells were exposed to different concentration of PM2.5 for different times, and the expression levels of microtubule-associated protein-1 light chain-3 Ⅱ (LC3 Ⅱ) and Beclin1 proteins were measured by Western blot. The typical autophagosome and autolysosome were observed by using transmission electron microscopy (TEM). To observe autophagic flux, mRFP-GFP-LC3 plasmid was transfected to nasal epithelial cells and the punctate staining of mRFP-GFP-LC3 were determined by confocal laser scanning microscope. The expression of inflammatory cytokines interleukin 6 (IL-6) and tumor necrosis factor-α (TNF-α) in cell culture supernatant were assessed by enzyme-linked immunosorbent assay (ELISA). To assess the role of autophagy in PM2.5-mediated inflammation, autophagy related gene Atg5 and Beclin-1 were silenced by siRNA knockdown, and inflammatory cytokines were analyzed.GraphPad Prism 6.0 was used for statistical analysis. Results: PM2.5 exposure increased the expression of LC3 Ⅱ and Beclin-1 proteins in a dose- (in PM2.5 group with concentration of 0, 15, 30, 60, 120 µg/ml, the expression of LC3 Ⅱ was 0.021±0.001(x±s), 0.037±0.002, 0.058±0.005, 0.075±0.006, 0.085±0.004, respectively, F=126.8, P<0.05; the expression of Beclin-1 was 0.002±0.000, 0.003±0.000, 0.005±0.000, 0.007±0.001, 0.008±0.001, respectively, F=137.3, P<0.05) and time-dependent manner (in PM2.5 group with exposure time of 0, 3, 6, 12, 24 h, the expression of LC3Ⅱ was 0.160±0.007, 0.222±0.003, 0.251±0.015, 0.483±0.029, 0.585±0.035, respectively, F=215.3, P<0.05; the expression of Beclin-1 was 0.059±0.002, 0.080±0.002, 0.087±0.002, 0.183±0.007, 0.228±0.005, respectively, F=137.3, P<0.05) in human nasal epithelial cells. TEM analysis showed typical autophagosome and autolysosome in cells after PM2.5 exposure for 24 h. PM2.5 significantly increased the number of yellow and red dots representing autophagosomes and autolysosomes respectively, indicating autophagic flux was elevated. Moreover, PM2.5 enhanced the secretion of inflammatory cytokines such as IL-6 and TNF-α, which was dramatically prevented by Atg5-siRNA and Beclin-1-siRNA. Conclusion: Autophagy plays an important role in PM2.5-caused inflammation response in nasal epithelial cells, which can induce release of inflammatory factors such as IL-6 and TNF-α and advance the inflammatory reaction.

Involvement of HuR in the serum starvation induced autophagy through regulation of Beclin1 in breast cancer cell-line, MCF-7.

Starvation is a cellular stress that induces autophagy, a conserved cellular self-digestion mechanism that allows cells to degrade and recycle damaged proteins and organelles. The present study illustrated that during serum deprivation, Beclin1, a crucial gene that is essential for autophagosome formation in autophagy, gets controlled post-transcriptionally in breast cancer cell-line MCF-7. RNA affinity chromatography and co-immunoprecipitation confirmed the association of HuR with 3'-UTR of beclin1 mRNA. After cytosolic translocation, HuR enhances beclin1 protein synthesis in response to serum starvation by enhancing the association of beclin1 mRNA to the polysomes. Partial silencing of HuR resulted in reduction of beclin1 expression both at mRNA and protein levels, which in turn decreased starvation-induced autophagic flux. Thus, in conclusion, fine-tuning of beclin1 gene expression at post-transcriptional level by HuR is one of the key regulatory mechanisms of starvation induced autophagy in breast cancer cell-line, MCF-7.

Autophagy related markers (Beclin-1 and ATG4B) are strongly expressed in Wilms' tumor and correlate with favorable histology.

BACKGROUND: Wilms' tumor treatment has achieved great success in the last decade. Nevertheless, some cases still fail to respond to the current multimodality therapy. These cases fall mainly in the unfavorable histology group with very few belonging to the favorable histology group. In recent years, autophagy manipulation whether inhibition or stimulation has been shown to affect cancer cell behavior and has emerged as a novel mechanism to improve cancer cell response to currently used therapeutic regimens. OBJECTIVE: The current study aimed to investigate the expression of autophagy related markers (ATG4B and Beclin1) in WT, its association with the different clinic-pathological parameters and its impact on patient survival. METHODS: Twenty-one formalin fixed paraffin embedded (FFPE) WT specimens were immunohistochemically stained using autophagy related markers; Beclin-1 and ATG4B. All clinical, radiological and follow up data were retrieved from the patient records. RESULTS: All specimens showed positive expression of both Beclin-1 and ATG4B. The staining score for Beclin1 varied between 50 and 300, and its expression was significantly associated with favorable histology (p=0.007). Similarly, ATG4B expression was significantly higher in favorable histology tumors compared to unfavorable histology (p=0.046). A statistically significant positive correlation between Beclin-1 and ATG4B expression was observed. The cumulative disease-free survival in patients with favorable histology was significantly higher compared to patients with unfavorable histology (p=0.0027). CONCLUSIONS: Beclin-1 and ATG4B expression were both found to be statistically significant discriminators of survival. Collectively these findings suggest that the expression of autophagy-related markers is associated with a favorable histology and could predict better survival in these patients.

Prognostic and clinicopathological value of Beclin-1 expression in hepatocellular carcinoma: a meta-analysis.

BACKGROUND: The abnormal expression of Beclin-1 has recently been investigated in a variety of tumors. However, previous studies have obtained contradicting results regarding the clinical and prognostic value of Beclin-1 in hepatocellular carcinoma (HCC). We performed a meta-analysis to clarify the prognostic value of Beclin-1 and its correlations with clinical pathological parameters in HCC. METHODS: Relevant studies were systematically retrieved from PubMed, EMBASE, China National Knowledge Infrastructure (CNKI), Wan Fang and Chinese VIP databases. We used the Newcastle-Ottawa scale (NOS) to estimate the quality of the involved studies. RESULTS: Ten eligible studies with 1086 HCC patients were included in this study. Our results showed that decreased Beclin-1 expression in HCC related to histological grade [poor-undifferentiated vs. well-moderate: odds ratio (OR) = 2.34, 95% confidence interval (CI) = 1.65-3.32, P < 0.00001]. The pooled hazard ratio (HR) (HR = 1.43, 95% CI = 1.17-1.75, P = 0.0004) indicated that decreased Beclin-1 expression correlated with poor overall survival (OS). CONCLUSIONS: This meta-analysis indicated that decreased Beclin-1 expression might relate to poor differentiation and unfavorable outcome in HCC.

Expression of autophagy related genes in chronic lymphocytic leukemia is associated with disease course.

Autophagy leads cells to different fates in various cell types and under diverse contexts. Chronic lymphocytic leukemia (CLL), an incurable hematologic neoplasm, has highly variable course and its heterogeneity prompts interest in exploring autophagic trajectories in CLL. We detected the mRNA levels of two autophagy-related genes, BECN1 and ATG5, assessed the association between expression levels and clinical characteristics, and did survival analysis. One hundred and six patients with CLL and fifty healthy controls were enrolled in the present study. CLL samples were found higher expression levels of BECN1 and ATG5 mRNA compared with healthy controls. Further confirmation at the protein level performed in a small cohort of patients, which included Beclin1, ATG5 and LC3-II showed the same trend. What's more, high expression at the mRNA level correlated with early Binet stage, isolated 13q deletion and negative CD38, which were associated with favor prognosis, suggesting that autophagy differs in CLL due to the presence of heterogeneity and high levels of these two genes may reflect better outcomes. Survival analysis did show patients with high expression of ATG5 mRNA had longer treatment free survival from the date of sampling.

Inhibition of autophagy potentiates the apoptosis-inducing effects of photodynamic therapy on human colon cancer cells.

BACKGROUND: Photodynamic therapy (PDT) has been reported to be a promising therapy for colon cancer because of its substantial safety features and its ability to induce a systematic reaction rather than local effects on the focal lesion in the intestine. Autophagy and apoptosis play important roles in the response to PDT. However, the role of autophagy after PDT treatment has not yet been clarified. METHODS: In this study, we investigated the relationship between apoptosis and autophagy in porphyrin IX (PpIX)-mediated PDT (PpIX-PDT) in HCT116 colon cancer cells. PpIX-PDT decreased cell viability in a concentration- and light dose-dependent manner. RESULTS: PpIX-PDT results in nuclear condensation, increased the expression of Caspase-3, Bax, and PARP, and decreased expression of Bcl-2. PpIX-PDT also induces the double membrane autophagosome, up-regulates LC3B, Atg7, Beclin-1, and Bcl-2 expression and down-regulates P62 expression. Inhibition of autophagy using chloroquine (CQ) or Atg7 knockdown with a shRNA enhances apoptotic cell death. Based on these findings, autophagy plays a self-protective role in HCT116 cells in response to PpIX-PDT treatment. DISCUSSION: Both autophagy and apoptosis were induced by PpIX-PDT in HCT116 cells, and the inhibition of autophagy strengthened the proapoptotic effect of PpIX-PDT. Thus, the appropriate modulation of autophagy may be as a potential therapeutic target for colon cancer cells treated with PpIX-PDT.

MicroRNA expression profiles in non­epithelial ovarian tumors.

Ovarian germ cell tumors (OGCTs) and sex cord stromal tumors (SCSTs) are rare gynecologic tumors that are derived from germ and stromal cells, respectively. Unlike their epithelial counterparts, molecular pathogenesis of these tumor types is still poorly understood. Here, we characterized microRNA (miRNA) expression profiles of 9 OGCTs (2 malignant and 7 benign) and 3 SCSTs using small RNA sequencing. We observed significant miRNA expression variations among the three tumor groups. To further demonstrate the biological relevance of our findings, we selected 12 miRNAs for validation in an extended cohort of 16 OGCTs (9 benign and 7 malignant) and 7 SCSTs by reverse transcription-quantitative polymerase chain reaction. Higher expression of miR­373­3p, miR­372­3p and miR­302c­3p and lower expression of miR­199a­5p, miR­214­5p and miR­202­3p were reproducibly observed in malignant OGCTs as compared to benign OGCTs or SCSTs. Comparing with benign OGCTs, miR­202c­3p and miR­513c­5p were more abundant in SCSTs. Additionally, we examined Beclin 1 (BECN1), a target of miR­199a­5p, in the clinical samples using western blot analysis. Our results show that BECN1 expression was higher in malignant OGCTs than benign OGCTs, which is concordant with their lower miR­199a­5p expression. This study suggests that these miRNAs may have potential value as tumor markers and implications for further understanding the molecular basis of these tumor types.

The Autophagy-Related Beclin-1 Protein Requires the Coiled-Coil and BARA Domains To Form a Homodimer with Submicromolar Affinity.

Beclin-1 (BECN1) is an essential component of macroautophagy. This process is a highly conserved survival mechanism that recycles damaged cellular components or pathogens by encasing them in a bilayer vesicle that fuses with a lysosome to allow degradation of the vesicular contents. Mutations or altered expression profiles of BECN1 have been linked to various cancers and neurodegenerative diseases. Viruses, including HIV and herpes simplex virus 1 (HSV-1), are also known to specifically target BECN1 as a means of evading host defense mechanisms. Autophagy is regulated by the interaction between BECN1 and Bcl-2, a pro-survival protein in the apoptotic pathway that stabilizes the BECN1 homodimer. Disruption of the homodimer by phosphorylation or competitive binding promotes autophagy through an unknown mechanism. We report here the first recombinant synthesis (3-5 mg/L in an Escherichia coli culture) and characterization of full-length, human BECN1. Our analysis reveals that full-length BECN1 exists as a soluble homodimer (KD ∼ 0.45 µM) that interacts with Bcl-2 (KD = 4.3 ± 1.2 µM) and binds to lipid membranes. Dimerization is proposed to be mediated by a coiled-coil region of BECN1. A construct lacking the C-terminal BARA domain but including the coiled-coil region exhibits a homodimer KD 3.5-fold weaker than that of full-length BECN1, indicating that both the BARA domain and the coiled-coil region of BECN1 contribute to dimer formation. Using site-directed mutagenesis, we show that residues at the C-terminus of the coiled-coil region previously shown to interact with the BARA domain play a key role in dimerization and mutations weaken the interface by ∼5-fold.

Mechanisms of Action of Vitamin D as Supplemental Therapy for Pneumocystis Pneumonia.

The combination of trimethoprim and sulfamethoxazole (TMP-SMX) is the most effective regimen for therapy of Pneumocystis pneumonia (PCP). As many patients with PCP are allergic or do not respond to it, efforts have been devoted to develop alternative therapies for PCP. We have found that the combination of vitamin D3 (VitD3) (300 IU/kg/day) and primaquine (PMQ) (5 mg/kg/day) was as effective as TMP-SMX for therapy of PCP. In this study, we investigated the mechanisms by which vitamin D enhances the efficacy of PMQ. C57BL/6 mice were immunosuppressed by CD4+ cell depletion, infected with Pneumocystismurina for 8 weeks, and then treated for 9 days with the combination of VitD3 and PMQ (VitD3-PMQ) or with TMP-SMX or PMQ to serve as controls. The results showed that vitamin D supplementation increased the number of CD11c+ cells, suppressed the production of proinflammatory cytokines (tumor necrosis factor alpha [TNF-α], gamma interferon [IFN-γ], and interleukin-6 [IL-6]) and inducible nitric oxide synthase (iNOS), and enhanced the expression of genes related to antioxidation (glutathione reductase and glutamate-cysteine ligase modifier subunit), antimicrobial peptides (cathelicidin), and autophagy (ATG5 and beclin-1). These results suggest that the main action of vitamin D is enhancing the ability of the host to defend against Pneumocystis infection.