The role of Atg5 gene in tumorigenesis under autophagy deficiency conditions.

Autophagy is a self-recycling machinery to maintain cellular homeostasis by degrading harmful materials in the cell. Autophagy-related gene 5 (Atg5) is required for autophagosome maturation. However, the role of Atg5 in tumorigenesis under autophagy deficient conditions remains unclear. This study focused on the autophagy-independent role of Atg5 and the underlying mechanism in tumorigenesis. We demonstrated that knockout of autophagy-related genes including Atg5, Atg7, Atg9, and p62 in mouse embryonic fibroblast (MEF) cells consistently decreased cell proliferation and motility, implying that autophagy is required to maintain diverse cellular functions. An Atg7 knockout MEF (Atg7-/- MEF) cell line representing deprivation of autophagy function was used to clarify the role of Atg5 transgene in tumorigenesis. We found that Atg5-overexpressed Atg7-/-MEF (clone A) showed increased cell proliferation, colony formation, and migration under autophagy deficient conditions. Accordingly, rescuing the autophagy deficiency of clone A by overexpression of Atg7 gene shifts the role of Atg5 from pro-tumor to anti-tumor status, indicating the dual role of Atg5 in tumorigenesis. Notably, the xenograft mouse model showed that clone A of Atg5-overexpressed Atg7-/- MEF cells induced temporal tumor formation, but could not prolong further tumor growth. Finally, biomechanical analysis disclosed increased Wnt5a secretion and p-JNK expression along with decreased ß-catenin expression. In summary, Atg5 functions as a tumor suppressor to protect the cell under normal conditions. In contrast, Atg5 shifts to a pro-tumor status under autophagy deprivation conditions.

Degradative autophagy regulates the homeostasis of miRnas to control cancer development.

Macroautophagy/autophagy acts as an anti-tumor mechanism in early cancer stages but promotes growth in established tumors. Similarly, miRNAs function as tumor suppressors or oncogenes, depending on their target genes. This reciprocal relationship between autophagy and miRNAs is a well-studied area, primarily focused on how miRNAs regulate autophagy-related genes. Our research provides innovative insights into how autophagy selectively controls miRNAs. For instance, MIR224 is preferentially degraded within autophagosomes, leading to the upregulation of SMAD4 and suppressing hepatocellular carcinoma (HCC) tumorigenesis. Conversely, autophagy positively regulates MIR449A by degrading EP300/p300 to activate FOXO1 and facilitate MIR449A transcription in colorectal cancer (CRC). In conclusion, our findings reveal the role of autophagy in maintaining the cellular balance of two miRNAs to mitigate tumorigenic stresses and highlight that autophagy-regulated miRNA profiles may serve as diagnostic and therapeutic markers for cancer development.

Synthetic lethality in human bladder cancer cells by curcumin via concurrent Aurora A inhibition and autophagy induction.

Combination therapies to induce mixed-type cell death and synthetic lethality have the potential to overcome drug resistance in cancer. In this study, we demonstrated that the curcumin-enhanced cytotoxicity of cisplatin/carboplatin in combination with gemcitabine was associated with Aurora A suppression-mediated G2/M arrest, and thus apoptosis, as well as MEK/ERK-mediated autophagy in human bladder cancer cells. Animal study data confirmed that curcumin combined with cisplatin/gemcitabine reduced tumorigenesis of xenograft in mice and this phenomenon was associated with elevated expressions of p-ERK and reduced p-Aurora A in tumors. Gene analyses using data repositories further revealed that reduced Aurora A expression alone did not significantly elevate the sensitivity of human bladder carcinoma cells to these anticancer drugs. Unlike other major cancer types, human bladder urothelial carcinoma tissue coexpressed higher AURKA and lower MAP1LC3B than normal tissue, and reduced Aurora A and induction of autophagy have been clinically associated with a better prognosis in patients with early but not advanced stage bladder cancer. Therefore, our results suggest that treatment strategies can utilize the synthetic lethal pair to concurrently suppress oncogenic Aurora A and induce autophagy by coadministrating curcumin with anticancer drugs for early-stage bladder cancer with high expression of Aurora A.

Secretory autophagy-promoted cargo exocytosis requires active RAB37.

RAB37 GTPase regulates cargo exocytosis by cycling between an inactive GDP-bound form and an active GTP-bound form. We reveal that RAB37 simultaneously regulates autophagy activation and tissue inhibitor of metalloproteinase 1 (TIMP1) secretion in lung cancer cells under starvation conditions. TIMP1, an inflammatory cytokine, is a known inhibitory molecule of matrix metalloproteinases matrix metalloproteinase 9 and suppresses the mobility of lung cancer cells both in vitro and in vivo through conventional exocytosis under serum-free conditions. Notably, we disclosed that secretory autophagy participates in TIMP1 secretion in a RAB37- and Sec22b-dependent manner. Sec22b, a SNARE family protein, participates in vesicle and membrane fusion of secretory autophagy. Knockdown of Sec22b decreased TIMP1 secretion and cell motility but did not affect cell proliferation under starvation conditions. We confirmed that starvation-activated RAB37 accompanied by Sec22b is essential for secretory autophagy to further enhance TIMP1 exocytosis. We further use an off-label drug amiodarone to demonstrate that autophagy induction facilitates TIMP1 secretion and suppresses the motility and metastasis of lung cancer cells in a RAB37-dependent manner in the lung-to-lung mouse model. In conclusion, we demonstrated that the RAB37 activation plays a pivotal regulatory role in secretory autophagy for TIMP1 secretion in lung cancer.Abbreviations: ATG: autophagy-related gene; GDP: guanosine diphosphate; GTP: guanosine triphosphate; LC3: microtubule-associated protein 1A/1B-light chain 3; SNARE: soluble N-ethylmaleimide-sensitive-factor attachment protein receptor; TIMP1: tissue inhibitor matrix metalloproteinase 1.

Formosanin C suppresses cancer cell proliferation and migration by impeding autophagy machinery.

Formosanin C (FC) is a natural compound extracted from Paris formosana Hayata with anticancer activity. FC induces both autophagy and apoptosis in human lung cancer cells. FC-induced depolarization of mitochondrial membrane potential (MMP) may trigger mitophagy. In this study, we clarified the effect of FC on autophagy, mitophagy, and the role of autophagy in FC-related cell death and motility. We found FC caused the continuous increase of LC3 II (representing autophagosomes) from 24 to 72 h without degradation after treatment of lung and colon cancer cells, indicating that FC blocks autophagic progression. In addition, we confirmed that FC also induces early stage autophagic activity. Altogether, FC is not only an inducer but also a blocker of autophagy progression. Moreover, FC increased MMP accompanied by overexpression of COX IV (mitochondria marker) and phosphorylated Parkin (p-Parkin, mitophagy marker) in lung cancer cells, but no colocalization of LC3 with COX IV or p-Parkin was detected under confocal microscopy. Moreover, FC could not block CCCP (mitophagy inducer)-induced mitophagy. These results imply that FC disrupts mitochondria dynamics in the treated cells, and the underlying mechanism deserves further exploration. Functional analysis reveals that FC suppresses cell proliferation and motility through apoptosis and EMT-related pathway, respectively. In conclusion, FC acts as an inducer as well as a blocker of autophagy that results in cancer cell apoptosis and decreased motility. Our findings shed the light on the development of combined therapy with FC and clinical anticancer drugs for cancer treatment.

Secretory autophagy promotes RAB37-mediated insulin secretion under glucose stimulation both in vitro and in vivo.

High blood glucose is one of the risk factors for metabolic disease and INS (insulin) is the key regulatory hormone for glucose homeostasis. Hypoinsulinemia accompanied with hyperglycemia was diagnosed in mice with pancreatic ß-cells exhibiting autophagy deficiency; however, the underlying mechanism remains elusive. The role of secretory autophagy in the regulation of metabolic syndrome is gaining more attention. Our data demonstrated that increased macroautophagic/autophagic activity leads to induction of insulin secretion in ß-cells both in vivo and in vitro under high-glucose conditions. Moreover, proteomic analysis of purified autophagosomes from ß-cells identified a group of vesicular transport proteins participating in insulin secretion, implying that secretory autophagy regulates insulin exocytosis. RAB37, a small GTPase, regulates vesicle biogenesis, trafficking, and cargo release. We demonstrated that the active form of RAB37 increased MAP1LC3/LC3 lipidation (LC3-II) and is essential for the promotion of insulin secretion by autophagy, but these phenomena were not observed in rab37 knockout (rab37-/-) cells and mice. Unbalanced insulin and glucose concentration in the blood was improved by manipulating autophagic activity using a novel autophagy inducer niclosamide (an antihelminthic drug) in a high-fat diet (HFD)-obesity mouse model. In summary, we reveal that secretory autophagy promotes RAB37-mediated insulin secretion to maintain the homeostasis of insulin and glucose both in vitro and in vivo.

Secretory autophagy promotes Rab37-mediated exocytosis of tissue inhibitor of metalloproteinase 1.

BACKGROUND: Rab37-mediated exocytosis of tissue inhibitor of metalloproteinase 1 (TIMP1), an inflammatory cytokine, under serum-depleted conditions which leads to suppression of lung cancer cell metastasis has been reported. Starvation is also a stimulus of autophagic activity. Herein, we reveal that starvation activates Rab37 and induces autophagy. METHODS: We used an overexpression/knockdown system to determine the relationship between autophagy and Rab37 in vitro and in vivo. The autophagy activity was detected by immunoblotting, transmission electron microscope, autophagosome purification, and immunofluorescence under the confocal microscope. Lung-to-lung metastasis mouse model was used to clarify the role of autophagy and Rab37 in lung cancer. Clinical lung cancer patient specimens and an online big database were analyzed. RESULTS: Initially, we demonstrated that active-form Rab37 increased LC3-II protein level (the marker of autophagosome) and TIMP1 secretion. Accordingly, silencing of Rab37 gene expression alleviated Rab37 and LC3-II levels as well as TIMP1 secretion, and induction of autophagy could not increase TIMP1 exocytosis under such conditions. Moreover, silencing the Atg5 or Atg7 gene of lung cancer cells harboring active-mutant Rab37 (Q89L) led to decreased autophagy activity and TIMP1 secretion. In the lung-to-lung metastasis mouse model, increased TIMP1 expression accompanied by amiodarone-induced autophagy led to decreased tumor nodules and cancer cell metastasis. These phenomena were reversed by silencing the Atg5 or Atg7 gene. Notably, increasing autophagy activity alone showed no effect on TIMP1 secretion under either Rab37 or Sec22b silencing conditions. We further detected colocalization of LC3 with either Rab37 or TIMP1, identified Rab37 and Sec22b proteins in the purified autophagosomes of the lung cancer cells harboring the active-form Rab37 gene, and confirmed that these proteins are involved in the secretion of TIMP1. We reveal that autophagic activity was significantly lower in the tumors compared to the non-tumor parts and was associated with the overall lung cancer patient survival rate. CONCLUSIONS: We are the first to report that autophagy plays a promoting role in TIMP1 secretion and metastasis in a Rab37-dependent manner in lung cancer cells and the lung-to-lung mouse model.

Vulnerability of Triple-Negative Breast Cancer to Saponin Formosanin C-Induced Ferroptosis.

Targeting ferritin via autophagy (ferritinophagy) to induce ferroptosis, an iron- and reactive oxygen species (ROS)-dependent cell death, provides novel strategies for cancer therapy. Using a ferroptosis-specific inhibitor and iron chelator, the vulnerability of triple-negative breast cancer (TNBC) MDA-MB-231 cells to ferroptosis was identified and compared to that of luminal A MCF-7 cells. Saponin formosanin C (FC) was revealed as a potent ferroptosis inducer characterized by superior induction in cytosolic and lipid ROS formation as well as GPX4 depletion in MDA-MB-231 cells. The FC-induced ferroptosis was paralleled by downregulation of ferroportin and xCT expressions. Immunoprecipitation and electron microscopy demonstrated the involvement of ferritinophagy in FC-treated MDA-MB-231 cells. The association of FC with ferroptosis was strengthened by the results that observed an enriched pathway with differentially expressed genes from FC-treated cells. FC sensitized cisplatin-induced ferroptosis in MDA-MB-231 cells. Through integrated analysis of differentially expressed genes and pathways using the METABRIC patients' database, we confirmed that autophagy and ferroptosis were discrepant between TNBC and luminal A and that TNBC was hypersensitive to ferroptosis. Our data suggest a therapeutic strategy by ferroptosis against TNBC, an aggressive subtype with a poor prognosis.

Autophagy Upregulates miR-449a Expression to Suppress Progression of Colorectal Cancer.

Many studies reported that microRNAs (miRNAs) target autophagy-related genes to affect carcinogenesis, however, autophagy-deficiency-related miRNA dysfunction in cancer development remains poorly explored. During autophagic progression, we identified miR-449a as the most up-regulated miRNA. MiR-449a expression was low in the tumor parts of CRC patient specimens and inversely correlated with tumor stage and metastasis with the AUC (area under the curve) of 0.899 and 0.736 as well as poor overall survival rate, indicating that miR-449a has the potential to be a prognostic biomarker. In the same group of CRC specimens, low autophagic activity (low Beclin 1 expression and high p62 accumulation) was detected, which was significantly associated with miR-449a expression. Mechanistic studies disclosed that autophagy upregulates miR-449a expression through degradation of the coactivator p300 protein which acetylates the transcription factor Forkhead Box O1 (FoxO1). Unacetylated FoxO1 translocated to the nucleus and bound to the miR-449a promoter to drive gene expression. Either activation of autophagy by the inducer or overexpression of exogenous miR-449a decreases the expression of target gene LEF-1 and cyclin D1, which lead to decreased proliferation, colony formation, migration, and invasion of CRC cells. Autophagy-miR-449a-tartet genes mediated suppression of tumor formation was further confirmed in the xenograft mouse model. In conclusion, this study reveals a novel mechanism wherein autophagy utilizes miR-449a-LEF1-cyclin D1 axis to suppress CRC tumorigenesis. Our findings open a new avenue toward prognosis and treatment of CRC patients by manipulating autophagy-miR-449a axis.

The Autophagosomes Containing Dengue Virus Proteins and Full-Length Genomic RNA Are Infectious.

Autophagic machinery is involved in selective and non-selective recruitment as well as degradation or exocytosis of cargoes, including pathogens. Dengue virus (DENV) infectioninduces autophagy that enhances virus replication and vesicle release to evade immune systemsurveillance. This study reveals that DENV2 induces autophagy in lung and liver cancer cells andshowed that DENV2 capsid, envelope, NS1, NS3, NS4B and host cell proinflammatory high mobilitygroup box 1 (HMGB1) proteins associated with autophagosomes which were purified by gradientcentrifugation. Capsid, NS1 and NS3 proteins showing high colocalization with LC3 protein in thecytoplasm of the infected cells were detected in the purified double-membrane autophagosome byimmunogold labeling under transmission electron microscopy. In DENV infected cells, the levels ofcapsid, envelope, NS1 and HMGB1 proteins are not significantly changed compared to the dramaticaccumulation of LC3-II and p62/SQSTM1 proteins when autophagic degradation was blocked bychloroquine, indicating that these proteins are not regulated by autophagic degradation machinery.We further demonstrated that purified autophagosomes were infectious when co-cultured withuninfected cells. Notably, these infectious autophagosomes contain DENV2 proteins, negativestrandand full-length genomic RNAs, but no viral particles. It is possible that the infectivity ofthe autophagosome originates from the full-length DENV RNA. Moreover, we reveal that DENV2promotes HMGB1 exocytosis partially through secretory autophagy. In conclusion, we are the firstto report that DENV2-induced double-membrane autophagosomes containing viral proteins andfull-length RNAs are infectious and not undergoing autophagic degradation. Our novel findingwarrants further validation of whether these intracellular vesicles undergo exocytosis to becomeinfectious autophagic vesicles.

Role and mechanism of action of circular RNA and laryngeal cancer.

Laryngeal squamous cell carcinoma (LSCC), is the second most common malignant cancer of the head and neck after oral cavity cancer. Laryngeal cancer is associated with huge socio-economic burden worldwide. Studies have widely explored the role of non-coding RNAs, especially microRNAs and long non-coding RNAs in pathogenesis of laryngeal cancer. In addition, several studies have explored the mechanism and function of circRNAs. CircRNAs has higher stability and more extensive function models, including combining miRNA as sponge, modifying transcription, and even regulating protein translation have been developed. Therefore, circRNAs is applied as an excellent diagnostic tool and a promising candidate for development of cancer therapies. This study reviews the biogenesis and function of circRNAs, explores potential mechanism of circRNAs in LSCC, and implications and challenges in LSCC research.

Effect of Fe3O4 Nanomaterial-Based MRI on the Efficacy of One-Sided Butterfly Sinus Lesions in the Nose.

Fe3O4 materials have many crystal forms, and the morphology of their nanomaterials is more diverse. Under the influence of nano-effects, they exhibit unique physical and chemical properties. The synthesized Fe3O4 materials with different morphologies have different properties. They are widely used in related fields such as electromagnetics, chemical engineering, catalysis, sensors, acoustics, medicine, environmental protection and so on. Choosing the appropriate contrast agent to display the anatomical characteristics of diseased and normal tissue is an important thing before using MRI (Magnetic Resonance Imaging) technology. At present, Gd-DTPA is a commonly used magnetic contrast agent in clinical practice, but Gd-DTPA has obvious shortcomings, such as short circulation time, non-specific distribution, and high price. In comparison, the superparamagnetic iron oxide nanocrystals have a high specific distribution in living tissues, mainly concentrated in reticular endothelial cells-rich tissues and organs, such as liver, spleen, lymph nodes and bone marrow. This study analyzed the imaging characteristics of 21 patients with unilateral benign sphenoid sinus disease who were confirmed by nasal endoscopic surgery and postoperative pathology. Among the 21 patients with unilateral benign sphenoid sinus lesions, 7 cases of them had cysts. Those with altered sphenoid sinus cavity expansion may be accompanied by thinning of bone resorption. MRI manifested as long T1 and long T2 irregular spherical signal shadows. 8 cases of them are fungal lesions. MRI showed mixed signals are in the sphenoid sinus, the flaky exudation showed a high signal, and the fungal nodule showed a low T2WI signal. In the 3 cases of cerebrospinal fluid rhinorrhea, MRI showed a line between the high-signal shadow of the cerebrospinal fluid and the high-signal liquid shadow in the sinuses. High signal and shadow are connected, and other tissues (brain tissue, nasal mucosa) show low signal. Fe3O4-based MRI technology can improve imaging quality and diagnostic efficiency.

Pterostilbene Sensitizes Cisplatin-Resistant Human Bladder Cancer Cells with Oncogenic HRAS.

Analysis of various public databases revealed that HRAS gene mutation frequency and mRNA expression are higher in bladder urothelial carcinoma. Further analysis revealed the roles of oncogenic HRAS, autophagy, and cell senescence signaling in bladder cancer cells sensitized to the anticancer drug cisplatin using the phytochemical pterostilbene. A T24 cell line with the oncogenic HRAS was chosen for further experiments. Indeed, coadministration of pterostilbene increased stronger cytotoxicity on T24 cells compared to HRAS wild-type E7 cells, which was paralleled by neither elevated apoptosis nor induced cell cycle arrest, but rather a marked elevation of autophagy and cell senescence in T24 cells. Pterostilbene-induced autophagy in T24 cells was paralleled by inhibition of class I PI3K/mTOR/p70S6K as well as activation of MEK/ERK (a RAS target) and class III PI3K pathways. Pterostilbene-induced cell senescence on T24 cells was paralleled by increased pan-RAS and decreased phospho-RB expression. Coadministration of PI3K class III inhibitor 3-methyladenine or MEK inhibitor U0126 suppressed pterostilbene-induced autophagy and reversed pterostilbene-enhanced cytotoxicity, but did not affect pterostilbene-elevated cell senescence in T24 cells. Animal study data confirmed that pterostilbene enhanced cytotoxicity of cisplatin plus gemcitabine. These results suggest a therapeutic application of pterostilbene in cisplatin-resistant bladder cancer with oncogenic HRAS.

Regulation of autophagy, glucose uptake, and glycolysis under dengue virus infection.

We previously reported that dengue virus (DENV)-induced autophagy plays a promoting role in viral replication and pathogenesis both in vitro and in vivo. Although it is known that DENV infection increases glycolysis, which promotes viral replication, the role of glucose metabolism together with autophagic activity in DENV replication remains unclear. In this study, we reveal that DENV2 infection increased autophagic activity, glucose uptake, protein levels of glucose transporter-1 (GLUT1), and glycolysis rate-limiting enzyme hexokinase-2 (HK2) in cells. Furthermore, the protein levels of LC3-II and HK2 were increased in the brain tissues of the DENV2-infected suckling mice. However, DENV2 infection decreased ATP level and showed no effect on mRNA expression of HK2 and phosphofructokinase, as well as lactate production, indicating that DENV2-regulated glycolytic flux occurs at the post-transcriptional level and is lactate pathway-independent. Moreover, amiodarone-induced autophagic activity, glucose uptake, HK2 level, and viral titer were reversed by the autophagy inhibitor spautin-1 or silencing of Atg5 gene expression. Intriguingly, blocking of glycolysis, HK2 protein level, and viral titer were accordingly decreased, but autophagic activity was increased, suggesting the existence of another regulation mechanism that influences the relationship between glycolysis and autophagy. This is the first report to reveal that DENV2-induced autophagy positively regulates glycolysis and viral replication in vitro and in vivo. Our findings open a new avenue wherein metabolic modulation could be used as a target for the treatment of DENV infection.

Saponin Formosanin C-induced Ferritinophagy and Ferroptosis in Human Hepatocellular Carcinoma Cells.

Ferroptosis, a recently discovered form of iron-dependent cell death, requires an increased level of lipid-reactive oxygen species (ROS). Ferritinophagy, a ferritin degradation pathway, depends on a selective autophagic cargo receptor (NCOA4). By screening various types of natural compounds, formosanin C (FC) was identified as a novel ferroptosis inducer, characterized by attenuations of FC-induced viability inhibition and lipid ROS formation in the presence of ferroptosis inhibitor. FC also induced autophagic flux, evidenced by preventing autophagic marker LC3-II degradation and increasing yellow LC3 puncta in tandem fluorescent-tagged LC3 (mRFP-GFP) reporter plasmid (ptfLC3) transfected cells when combined with autophagic flux inhibitor. It is noteworthy that FC-induced ferroptosis and autophagic flux were stronger in HepG2 cells expressing higher NCOA4 and lower ferritin heavy chain 1 (FTH1) levels, agreeing with the results of gene expression analysis using CTRP and PRISM, indicating that FTH1 expression level exhibited a significant negative correlation with the sensitivity of the cells to a ferroptosis inducer. Confocal and electron microscopy confirmed the pronounced involvement of ferritinophagy in FC-induced ferroptosis in the cells with elevated NCOA4. Since ferroptosis is a non-apoptotic form of cell death, our data suggest FC has chemotherapeutic potential against apoptosis-resistant HCC with a higher NCOA4 expression via ferritinophagy.

Growth Inhibition of Nasopharyngeal Carcinoma Cells Mediated by p53 Gene-Containing Nanolipid Composites.

To study the growth inhibition and cell cycle changes in nasopharyngeal carcinoma (CNE1) cells after transfection with p53 gene. A mixture of nano-liposomes and plasmid containing p53 was used for transfecting CNE1 cells. Cellular apoptosis was examined after transfection using the CCK-8 reagent method with flow cytometry. The results showed that a ratio of nanoliposome/p-ORF-GFP of 3.5:1 showed the highest transfection efficiency in CNE1 cells. The cells transfected with a mixture of composites in this proportion showed significant apoptosis of up to 50-70%. In addition, we observed that cell cycle changes-measured using flow cytometry-as well as cellular apoptosis were accelerated after administration of composites. The CCK-8 kit was used to determine the viability of nano-liposome-encapsulated p53 transfected cells. In vitro experiments showed that the combination significantly inhibited the growth of CNE1 cells with an inhibition rate of approximately 63.8%. Therefore, the nanocomposites have a significant effect on inhibiting the growth of CNE1 cells. Through the investigation of apoptosis and cell cycle changes in CNE1 cells we found that the nanoliposome-encapsulated p53 gene can inhibit growth in these cells, and might therefore serve as a novel treatment strategy for adjuvant treatment of nasopharyngeal carcinoma and ca also reduce incompatibility issues with radiotherapy and chemotherapy. This method can also provide technical and theoretical support for the development of novel drugs.

Negative Pressure Wound Therapy With Chymotrypsin Irrigation: A Maximal Implant Retention Procedure Treating the Exposure/Infection of Titanium Mesh in Cranioplasty.

This preliminary study aims to investigate the effects of a maximal implant retention procedure. The authors retrospectively reviewed the use of negative pressure wound therapy with chymotrypsin irrigation treating implant infection/exposure in titanium mesh cranioplasty by comparing patients with titanium mesh totally retained, partially removed, or totally removed according to the evaluation during the surgery. Negative pressure wound therapy with chymotrypsin irrigation was applied 5 days after the surgery. The negative pressure was set at -125 to -150 mmHg. A total of 21 patients were included, 4 patients treated with titanium mesh totally removed; 3 patients treated with titanium mesh partially removed; and 14 patients treated with U-shape debridement with titanium mesh preserved completely. However, 1 patient in the U-shape group required a second debridement to remove all implant. Negative pressure wound therapy with chymotrypsin irrigation is a novel procedure and could be used to treat implant-related infection without the exchange of implant.

Curcumin functions as a MEK inhibitor to induce a synthetic lethal effect on KRAS mutant colorectal cancer cells receiving targeted drug regorafenib.

Curcumin, a major yellow pigment and spice in turmeric and curry, has been demonstrated to have an anticancer effect in human clinical trials. Mutation of KRAS has been shown in 35%-45% of colorectal cancer, and regorafenib has been approved by the US FDA to treat patients with colorectal cancer. Synthetic lethality is a type of genetic interaction between two genes such that simultaneous perturbations of the two genes result in cell death or a dramatic decrease of cell viability, while a perturbation of either gene alone is not lethal. Here, we reveal that curcumin significantly enhanced the growth inhibition of regorafenib in human colorectal cancer HCT 116 cells (KRAS mutant) to a greater extent than in human colorectal cancer HT-29 cells (KRAS wild-type), producing an additive or synergistic effect in HCT 116 cells and causing an antagonistic effect in HT-29 cells. Flow cytometric analysis showed that the addition of curcumin elevated apoptosis and greatly increased autophagy in HCT 116 cells but not in HT-29 cells. Mechanistically, curcumin behaved like MEK-specific inhibitor (U0126) to enhance regorafenib-induced growth inhibition, apoptosis and autophagy in HCT 116 cells. Our data suggest that curcumin may target one more gene other than mutant KRAS to enhance regorafenib-induced growth inhibition (synthetic lethality) in colorectal cancer HCT 116 cells, indicating a possible role of curcumin in regorafenib-treated KRAS mutant colorectal cancer.

Degradative autophagy selectively regulates CCND1 (cyclin D1) and MIR224, two oncogenic factors involved in hepatocellular carcinoma tumorigenesis.

Overexpressed CCND1 (cyclin D1) is associated with hepatocellular carcinoma (HCC) and we used 147 tumor tissue samples from HCC patients and 3 murine models to reveal an inverse correlation between low autophagic activity and high CCND1 expression. These 2 phenomena in combination correlated with poor overall survival in HCC patients. Mechanistic analysis showed that activated autophagy triggered CCND1 ubiquitination followed by SQSTM1 (sequestosome 1)-mediated selective phagophore recruitment, autophagosome formation, fusion with a lysosome, and degradation. Functional studies revealed that autophagy-selective degradation of CCND1 suppresses DNA synthesis, cell proliferation, and colony, and liver tumor formation by arresting the cell cycle at the G1 phase. Most importantly, diverse pharmacological inducers (rapamycin and amiodarone) effectively suppress tumor growth in orthotopic liver tumor and subcutaneous tumor xenograft models. In conclusion, we have demonstrated a link between degradative autophagy and the cell cycle regulator CCND1, and have discovered the underlying mechanism by which the autophagic degradation machinery regulates the turnover of the cell-cycle regulator CCND1, which in turn affects HCC tumorigenesis. Abbreviations: CCDN1: cyclin D1; HBV: hepatitis B virus; HCC: hepatocellular carcinoma; HCV: hepatitis C virus; SQSTM1: sequestosome 1.

Hepatocellular carcinoma-related cyclin D1 is selectively regulated by autophagy degradation system.

Dysfunction of degradation machineries causes cancers, including hepatocellular carcinoma (HCC). Overexpression of cyclin D1 in HCC has been reported. We previously reported that autophagy preferentially recruits and degrades the oncogenic microRNA (miR)-224 to prevent HCC. Therefore, in the present study, we attempted to clarify whether cyclin D1 is another oncogenic factor selectively regulated by autophagy in HCC tumorigenesis. Initially, we found an inverse correlation between low autophagic activity and high cyclin D1 expression in tumors of 147 HCC patients and three murine models, and these results taken together revealed a correlation with poor overall survival of HCC patients, indicating the importance of these two events in HCC development. We found that increased autophagic activity leads to cyclin D1 ubiquitination and selective recruitment to the autophagosome (AP) mediated by a specific receptor, sequestosome 1 (SQSTM1), followed by fusion with lysosome and degradation. Autophagy-selective degradation of ubiquitinated cyclin D1 through SQSTM1 was confirmed using cyclin D1/ubiquitin binding site (K33-238 R) and phosphorylation site (T286A) mutants, lentivirus-mediated silencing autophagy-related 5 (ATG5), autophagy-related 7 (ATG7), and Sqstm1 knockout cells. Functional studies revealed that autophagy-selective degradation of cyclin D1 plays suppressive roles in cell proliferation, colony, and liver tumor formation. Notably, an increase of autophagic activity by pharmacological inducers (amiodarone and rapamycin) significantly suppressed tumor growth in both the orthotopic liver tumor and subcutaneous tumor xenograft models. Our findings provide evidence of the underlying mechanism involved in the regulation of cyclin D1 by selective autophagy to prevent tumor formation. CONCLUSION: Taken together, our data demonstrate that autophagic degradation machinery and the cell-cycle regulator, cyclin D1, are linked to HCC tumorigenesis. We believe these findings may be of value in the development of alternative therapeutics for HCC patients. (Hepatology 2018;68:141-154).