Intensive expression of UNC-51-like kinase 1 is a novel biomarker of poor prognosis in patients with esophageal squamous cell carcinoma.

The serine/threonine kinase UNC-51-like kinase 1 (ULK1) plays an essential role in autophagosome formation, although the exact molecular mechanism is unknown. The present study was first to investigate the clinical and prognostic significance of ULK1 in esophageal squamous cell carcinoma (ESCC). Protein and mRNA levels of ULK1 in normal esophageal epithelial cells, ESCC cell lines, paired ESCC lesions and the adjacent noncancerous tissues were examined using western blot and real-time RT-PCR. The results showed that only the ULK1 protein level was upregulated in ESCC samples compared with normal esophageal cells and tissues. Also, we found that protein stabilization of ULK1 was higher in ESCC cell lines. Furthermore, immunohistochemical staining of ULK1 was performed on the tissue microarray containing 248 ESCC and 51 normal esophageal tissues. A total of 70.2% ESCC specimens showed intensive expression of ULK1 in contrast to the undetectable expression of ULK1 in normal esophageal tissues. Statistical analysis revealed that ULK1 expression was significantly correlated with T status (P = 0.048). Moreover, patients with higher ULK1 expression were associated with shorter overall survival time. Multivariate analysis suggested that ULK1 expression and N status (P < 0.001) were independent prognostic indicators for the survival of patients. Functional studies showed that suppression of ULK1 expression in ESCC cell lines by specific small interfering RNA resulted in inhibition of cell proliferation and induction of apoptosis under starvation conditions. These findings provide evidence that ULK1 represents a novel and clinically useful biomarker for ESCC patients and plays an important role during the progression of ESCC.

Chloroquine enhances the cytotoxicity of topotecan by inhibiting autophagy in lung cancer cells.

Although the anti-malaria drug chloroquine (CQ) has been shown to enhance chemotherapy and radiation sensitivity in clinical trials, the potential mechanisms underlying this enhancement are still unclear. Here, we examined the relevant mechanisms by which the multipotent CQ enhanced the cytotoxicity of topotecan (TPT). The lung cancer cell line A549 was treated with TPT alone or TPT combined with CQ at non-cytotoxic concentrations. Cell viability was assessed using the MTT assay. The percentage of apoptotic cells and the presence of a side population of cells were both determined by flow cytometry. Autophagy and the expression of Bcl-2 family proteins were examined by Western blotting. The accumulation of YFP-LC3 dots and the formation of acidic vesicular organelles were examined by confocal microscopy. CQ sensitized A549 cells to TPT and enhanced TPT-induced apoptosis in a Bcl-2 family protein-independent fashion. CQ inhibited TPT-induced autophagy, which modified the cytotoxicity of TPT. However, CQ failed to modify the transfer of TPT across the cytoplasmic membrane and did not increase lysosomal permeability. This study showed that CQ at non-cytotoxic concentrations potentiated the cytotoxicity of TPT by interfering with autophagy, implying that CQ has significant potential as a chemotherapeutic enhancer.

[Effect of canopy condition on Machilus nanmu seedling configuration and biomass allocation]. / æž—å† çŽ¯å¢ƒå¯¹æ¶¦æ¥ å¹¼è‹—æž„åž‹å’Œç”Ÿç‰©é‡åˆ†é çš„å½±å“.

The adaptation strategy of seedlings plays a decisive role in population regeneration. Machilus nanmu is a tree species belonging to Lauraceae, which is national class Ⅱ protected species and one of the dominant species in the evergreen broadleaved forest in Jinyun Mountain, Chongqing. Therefore, it is of great significance to understand the adaptation strategies of M. nanmu seedlings to maintain population regeneration and protect the biodiversity of evergreen broadleaved forest. We studied the temporal dynamics of early adaptation strategy of M. nanmu in Jinyun Mountain in Chongqing and its response to heterogeneous habitats from the perspective of morphology and biomass allocation. The seedlings of M. nanmu were classified into different age stages (stage 1: 1-3 a; stage 2: 4-6 a; stage 3: 7-9 a) under different canopy environments (gap/understory). Stem configurations (except branch angle) and leaf inclination angle of M. nanmu seedlings in the gap were significantly greater than understory at stages 2 and 3. Root configurations (except root diameter) and leaf area were significantly greater than that in the understory at stages 1 and 2. Specific leaf area in the gap was significantly smaller than understory at all three stages. Across all the conditions, biomass distribution was dominated by leaves. From the stage 2, stem biomass distribution of M. nanmu seedlings in the gap was increased, while root biomass distribution was decreased. There was no significant variation in root biomass of M. nanmu seedlings in the understory. The coordination among different organs of M. nanmu seedlings would help their adaptation to different habitats. Root and leaf of M. nanmu seedlings in the gap were significantly correlated, with the correlation changing from positive to negative as the age increased. While in the understory, there was significant positive correlation between root and stem, but no correlation between root and leaf. The slope of SMA equation of branch weight and branch length had significant difference under different canopy environments only at stage 3, while the slope of SMA equation of leaf and root biomass and configuration had no significant difference. Most of the SMA equation intercepts between biomass and configuration differed significantly at stage 2.

CUL3 (cullin 3)-mediated ubiquitination and degradation of BECN1 (beclin 1) inhibit autophagy and promote tumor progression.

Macroautophagy/autophagy plays an important role during the development of human cancer. BECN1 (beclin 1), a core player in autophagy regulation, is downregulated in many kinds of malignancy. The underlying mechanism, however, has not been fully illuminated. Here, we found that CUL3 (cullin 3), an E3 ubiquitin ligase, could interact with BECN1 and promote the K48-linked ubiquitination and degradation of this protein; In addition, CUL3 led to a decrease in autophagic activity through downregulating BECN1. We also found that KLHL38 was a substrate adaptor of the CUL3 E3 ligase complex-mediated ubiquitination and degradation of BECN1. In breast and ovarian cancer, CUL3 could promote the proliferation of tumor cells, and the expression of CUL3 was related to poor prognosis in patients. Our study reveals the underlying mechanism of BECN1 ubiquitination and degradation that affects autophagic activity and subsequently leads to tumor progression, providing a novel therapeutic strategy that regulates autophagy to combat cancer.Abbreviations: ATG: autophagy-related BECN1: beclin 1 CHX: cycloheximide CoIP: co-immunoprecipitation CUL3: cullin 3 IP: immunoprecipitation MS: mass spectrometry PtdIns3K: phosphatidylinositol 3-kinase UPS: ubiquitin-proteasome system.

A novel sesquiterpene Hirsutanol A induces autophagical cell death in human hepatocellular carcinoma cells by increasing reactive oxygen species.

BACKGROUND AND OBJECTIVE: Hirsutanol A is a novel sesquiterpene compound purified from fungus chondrostereum sp in Sarcophyton tortuosum. Its pharmacologic effect has not been reported yet. This study aimed to investigate cytotoxic effect of Hirsutanol A on hepatocellular carcinoma (HCC) cells and its mechanism. METHODS: Hep3B cells were treated with different concentrations of Hirsutanol A. Cell proliferation was detected by MTT assay. The protein expression of LC3 was determined by Western blot. The generation of reactive oxygen species (ROS) was monitored by flow cytometry. RESULTS: Hirsutanol A significantly inhibited proliferation of Hep3B cells with 50% inhibition concentrations (IC50) of 14.54, 6.71, and 3.59 micromol/L when exposed to Hirsutanol A for 24, 48, and 72 h, respectively. Incubation of Hep3B cells with Hirsutanol A markedly increased the level of ROS and the autophagy marker MAP-LC3 conversion from type I to type II. Pre-incubation with an antioxidant N-acetyl cystein (NAC) decreased the level of ROS, and reduced MAP-LC3 I-II conversion, and suppressed cell death. Blocking autophagy with a specific autophagy inhibitor 3-methyladenine (3-MA), the cytotoxic effect of this compound was attenuated. CONCLUSION: Hirsutanol A has potent cytotoxic effect, and can induce autophagic cell death via increasing ROS production.

CaMKII-mediated Beclin 1 phosphorylation regulates autophagy that promotes degradation of Id and neuroblastoma cell differentiation.

Autophagy is a degradative pathway that delivers cellular components to the lysosome for degradation. The role of autophagy in cell differentiation is poorly understood. Here we show that CaMKII can directly phosphorylate Beclin 1 at Ser90 to promote K63-linked ubiquitination of Beclin 1 and activation of autophagy. Meanwhile, CaMKII can also promote K63-linked ubiquitination of inhibitor of differentiation 1/2 (Id-1/2) by catalyzing phosphorylation of Id proteins and recruiting TRAF-6. Ubiquitinated Id-1/Id-2 can then bind to p62 and be transported to autolysosomes for degradation. Id degradation promotes the differentiation of neuroblastoma cells and reduces the proportion of stem-like cells. Our study proposes a mechanism by which autophagic degradation of Id proteins can regulate cell differentiation. This suggests that targeting of CaMKII and the regulation of autophagic degradation of Id may be an effective therapeutic strategy to induce cell differentiation in neuroblastoma.

ATM-mediated PTEN phosphorylation promotes PTEN nuclear translocation and autophagy in response to DNA-damaging agents in cancer cells.

PTEN (phosphatase and tensin homolog), a tumor suppressor frequently mutated in human cancer, has various cytoplasmic and nuclear functions. PTEN translocates to the nucleus from the cytoplasm in response to oxidative stress. However, the mechanism and function of the translocation are not completely understood. In this study, topotecan (TPT), a topoisomerase I inhibitor, and cisplatin (CDDP) were employed to induce DNA damage. The results indicate that TPT or CDDP activates ATM (ATM serine/threonine kinase), which phosphorylates PTEN at serine 113 and further regulates PTEN nuclear translocation in A549 and HeLa cells. After nuclear translocation, PTEN induces autophagy, in association with the activation of the p-JUN-SESN2/AMPK pathway, in response to TPT. These results identify PTEN phosphorylation by ATM as essential for PTEN nuclear translocation and the subsequent induction of autophagy in response to DNA damage.

DC120, a novel AKT inhibitor, preferentially suppresses nasopharyngeal carcinoma cancer stem-like cells by downregulating Sox2.

Side population (SP) contains cancer stem-like cells (CSLCs). In this study, we characterized SP cells from nasopharyngeal carcinoma (NPC) cell lines and found that SP cells had a higher self-renewal ability in vitro and greater tumorigenicity in vivo. The AKT pathway was activated in NPC SP cells. DC120, a 2-pyrimidyl-5-amidothiazole inhibitor of the ATP binding site of AKT, inhibited phosphorylation of FKHRL1 and GSK-3ß. DC120 inhibited SP fraction, the sphere-forming ability in vitro and growth of primary xenografts as well as secondary xenografts' tumor recurrence. This inhibition was accompanied by reduced expression of stem-related gene Sox2 due to induction of p27 and miR-30a. A combination of DC120 and CDDP more effectively inhibited NPC cells compared with monotherapy in vitro and in vivo. Clinical evaluation of DC120 is warranted.

Upregulation of sestrin 2 expression via JNK pathway activation contributes to autophagy induction in cancer cells.

JNK signaling functions to induce defense mechanisms that protect organisms against a variety of different situations. The sestrin 2 gene, a p53-regulated member of the sestrins family, which lead to AMPK-dependent inhibition of TOR signaling, emerges as a novel player in autophagy induction. However, the relationship between JNK pathway, autophagy induction and sestrin 2 expression remains elusive. In the present study, we identify JNK as a regulator of autophagy in nasopharyngeal carcinoma cell lines CNE1 and CNE2 exposed to excisanin A or serum deprivation and demonstrate that activation of JNK can cause upregulation of sestrin 2 expression, which could be blocked by specific siRNAs directed against JNK1/2 or c-Jun. Chromatin immunoprecipitation and luciferase reporter analysis revealed that c-Jun was transcriptionally involved in the regulation of sestrin 2. Furthermore, knockdown of sestrin 2 by siRNAs similarly inhibited autophagy induction. Moreover, silencing the expression of autophagy related gene ATG5 or sestrin 2 significantly decreases cell death induced by excisanin A. Our results therefore identify JNK as a novel mediator of sestrin 2 expression, which plays a key role in autophagy induction following anticancer therapies in cancers.

Dual phosphoinositide 3-kinase/mammalian target of rapamycin inhibitor NVP-BEZ235 has a therapeutic potential and sensitizes cisplatin in nasopharyngeal carcinoma.

Phosphoinositide 3-kinase (PI3K)/AKT/mammalian target of rapamycin inhibitor (mTOR) pathway is often constitutively activated in human tumor cells and thus has been considered as a promising drug target. To ascertain a therapeutical approach of nasopharyngeal carcinoma (NPC), we hypothesized NVP-BEZ235, a novel and potent imidazo[4,5-c] quinolone derivative, that dually inhibits both PI3K and mTOR kinases activities, had antitumor activity in NPC. Expectedly, we found that NVP-BEZ235 selectively inhibited proliferation of NPC cells rather than normal nasopharyngeal cells using MTT assay. In NPC cell lines, with the extended exposure, NVP-BEZ235 selectively inhibited proliferation of NPC cells harboring PIK3CA mutation, compared to cells with wild-type PIK3CA. Furthermore, exposure of NPC cells to NVP-BEZ235 resulted in G1 growth arrest by Propidium iodide uptake assay, reduction of cyclin D1and CDK4, and increased levels of P27 and P21 by Western blotting, but negligible apoptosis. Moreover, we found that cisplatin (CDDP) activated PI3K/AKT and mTORC1 pathways and NVP-BEZ235 alleviated the activation by CDDP through dually targeting PI3K and mTOR kinases. Also, NVP-BEZ235 combining with CDDP synergistically inhibited proliferation and induced apoptosis in NPC cells. In CNE2 and HONE1 nude mice xenograft models, orally NVP-BEZ235 efficiently attenuated tumor growth with no obvious toxicity. In combination with NVP-BEZ235 and CDDP, there was dramatic synergy in shrinking tumor volumes and inducing apoptosis through increasing Noxa, Bax and decreasing Mcl-1, Bcl-2. Based on the above results, NVP-BEZ235, which has entered phase I/II clinical trials in patients with advanced solid tumors, has a potential as a monotherapy or in combination with CDDP for NPC treatment.

The inhibition of autophagy sensitises colon cancer cells with wild-type p53 but not mutant p53 to topotecan treatment.

BACKGROUND: Topotecan produces DNA damage that induces autophagy in cancer cells. In this study, sensitising topotecan to colon cancer cells with different P53 status via modulation of autophagy was examined. METHODOLOGY/PRINCIPAL FINDINGS: The DNA damage induced by topotecan treatment resulted in cytoprotective autophagy in colon cancer cells with wild-type p53. However, in cells with mutant p53 or p53 knockout, treatment with topotecan induced autophagy-associated cell death. In wild-type p53 colon cancer cells, topotecan treatment activated p53, upregulated the expression of sestrin 2, induced the phosphorylation of the AMPKα subunit at Thr172, and inhibited the mTORC1 pathway. Furthermore, the inhibition of autophagy enhanced the anti-tumour effect of topotecan treatment in wild-type p53 colon cancer cells but alleviated the anti-tumour effect of topotecan treatment in p53 knockout cells in vivo. CONCLUSIONS/SIGNIFICANCE: These results imply that the wild-type p53-dependent induction of cytoprotective autophagy is one of the cellular responses that determines the cellular sensitivity to the DNA-damaging drug topotecan. Therefore, our study provides a potential therapeutic strategy that utilises a combination of DNA-damaging agents and autophagy inhibitors for the treatment of colon cancer with wild-type p53.

ON-III inhibits erbB-2 tyrosine kinase receptor signal pathway and triggers apoptosis through induction of Bim in breast cancer cells.

ErbB-2 gene encodes tyrosine kinase receptor p185(neu). Overexpression of erbB-2 plays a key role in tumorigenesis or progression such as breast cancer and ovarian cancer. Our previous study showed that ON-III (2',4'-dihydroxy-6'-methoxy-3',5'-dimethylchalcone) extracted from Traditional Chinese Medicine Cleistocaly xoperculatus dry flower could inhibit KDR tyrosine kinase phosphorylation and tumor growth in vivo. In this study, we reported that ON-III repressed tyrosine phosphorylation of erbB-2 without reduced erbB-2 receptor expression in MDA-MB-453 cells. Activation of mitogen-activated protein kinase (MAPK) and AKT, downstream molecules of erbB-2-mediated signal transduction pathway, was inhibited following exposure to ON-III. ON-III induced apoptosis in breast cancer cells as determined by caspase-3 and PARP cleavage. Also, ON-III upregulated the expression of proapoptotic BH3-only Bcl-2 family member Bim. Bim siRNA could inhibit ON-III-mediated apoptosis in MDA-MB-453 cells. It concludes that ON-III inhibits erbB-2 tyrosine kinase phosphorylation, shuts down its downstream pathway and triggered apoptosis via induction of Bim. These results suggest that ON-III is a potential novel anti-cancer agent for erbB-2-overexpressing cancer.