Knockdown of NADK promotes LUAD ferroptosis via NADPH/FSP1 axis.

BACKGROUND: Lung cancer is a serious threat to human health and is the first leading cause of cancer death. Ferroptosis, a newly discovered form of programmed cell death associated with redox homeostasis, is of particular interest in the lung cancer, given the high oxygen environment of lung cancer. NADPH has reducing properties and therefore holds the potential to resist ferroptosis. Resistance to ferroptosis exists in lung cancer, but the role of NADK in regulating ferroptosis in lung cancer has not been reported yet. METHODS: Immunohistochemistry (IHC) was used to analyse the expression of NADK in 86 cases of lung adenocarcinoma(LUAD) and adjacent tissues, and a IHC score was assigned to each sample. Chi-square and kaplan-meier curve was performed to analyse the differences in metastasis and five-year survival between the two groups with NADK high or low scores. Proliferation of NADK-knockdown LUAD cell lines was detected in vivo and vitro. Furthermore, leves of ROS, MDA and Fe2+ were measured to validate the effect and mechanism of NADK on ferroptosis in LUAD. RESULTS: The expression of NADK was significantly evaluated in LUAD tissues as compared to adjacent non-cancerous tissues. The proliferation of NADK-knockdown cells was inhibited both in vivo and vitro, and increasing levels of intracellular ROS, Fe2+ and lipid peroxide products (MDA) were observed. Furthermore, NADK-knockdown promoted the ferroptosis of LUAD cells induced by Erastin/RSL3 by regulating the level of NADPH and the expression of FSP1. Knockdown of NADK enhanced the sensitivities of LUAD cells to Erastin/RSL3-induced ferroptosis by regulating NADPH level and FSP1 expression. CONCLUSIONS: NADK is over-expressed in LUAD patients. Knockdown of NADK inhibited the proliferation of LUAD cells both in vitro and in vivo and promotes the Erastin/RSL3-induced ferroptosis of LUAD cells by down-regulating the NADPH/FSP1 axis.

Mechanism study of oleanolic acid derivative, K73-03, inducing cell apoptosis in hepatocellular carcinoma.

Oleanolic acid (3ß-hydroxyolean-12-en-28-oic acid, OA) is a kind of pentacyclic triterpene, which widely distributes in nature. OA possesses a powerful anti-cancer effect; however, its low solubility limits its bioavailability and application. In this study, a new OA derivative, K73-03, was used to determine its effect on liver cancer cells and detailed molecular mechanisms. Here, we show that K73-03 may lead to the disorder of mitochondria in HepG2 cells, leading to excessive ROS production and apoptosis in cells. Meanwhile, K73-03 could induce cell apoptosis by inhibiting JAK2/STAT3 pathway and NF-κB/P65 pathway. Collectively, this study may provide a preliminary basis for further cancer treatment of hepatocellular carcinoma.

Protective effects of phosphocreatine on human vascular endothelial cells against hydrogen peroxide-induced apoptosis and in the hyperlipidemic rat model.

Phosphocreatine (PCr) has been shown to have a cardio-protective effect during cardiopulmonary resuscitation (CPR). However, little is known about its impact on atherosclerosis. In this study, we first evaluated the pharmacological effects of PCr on antioxidative defenses and mitochondrial protection against hydrogen peroxide (H2O2) induced human umbilical vascular endothelial cells (HUVECs) damage. Then we investigated the hypolipidemic and antioxidative effects of PCr on hyperlipidemic rat model. Via in vitro studies, H2O2 significantly reduced cell viability and increased apoptosis rate of HUVECs, while pretreatment with PCr abolished its apoptotic effect. PCr could reduce the generation of ROS induced by H2O2. Moreover, PCr could increase the activity of SOD and the content of NO, as well as decrease the activity of LDH and the content of MDA. PCr could also antagonize H2O2-induced up-regulation of Bax, cleaved-caspase3, cleaved-caspase9, and H2O2-induced down-regulation of Bcl-2 and p-Akt/Akt ratio. In addition, PCr reduced U937 cells' adhesion to H2O2-stimulated HUVECs. Via in vivo study, PCr could decrease MDA, TC, TG and LDL-C levels in hyperlipidemic rats. Finally, different-concentration PCr could increase the leaching of TC, HDL, and TG from fresh human atherosclerotic plaques. In conclusion, PCr could suppress H2O2-induced apoptosis in HUVECs and reduce hyperlipidemia through inhibiting ROS generation and modulating dysfunctional mitochondrial system, which might be an effective new therapeutic strategy to further prevent atherosclerosis.

GPX2 promotes EMT and metastasis in non-small cell lung cancer by activating PI3K/AKT/mTOR/Snail signaling axis.

Lung cancer, with non-small cell lung cancer (NSCLC) being the main subtype, is the leading cause of cancer death worldwide, which is mainly due to the cancer metastasis. Glutathione peroxidase 2 (GPX2), an antioxidant enzyme, is involved in tumor progression and metastasis. Nevertheless, the role of GPX2 in NSCLC metastasis has not been clarified. In this study, we found that GPX2 expression was elevated in NSCLC tissues and high GPX2 expression was correlated with poor prognosis in patients with NSCLC. In addtion, GPX2 expression was related to the patient's clinicopathological features, including lymph node metastasis, tumor size, and TNM stage. Overexpression of GPX2 promoted epithelial-mesenchymal transition (EMT), migration, and invasion of NSCLC cells in vitro. Knockdown of GPX2 showed the opposite effects in vitro and inhibited the metastasis of NSCLC cells in nude mice. Furthermore, GPX2 reduced reactive oxygen species (ROS) accumulation and activated the PI3K/AKT/mTOR/Snail signaling axis. Therefore, our results indicate that GPX2 promotes EMT and metastasis of NSCLC cells by activating the PI3K/AKT/mTOR/Snail signaling axis via the removal of ROS. GPX2 may be an effective diagnostic and prognostic biomarker for NSCLC.

Long Noncoding RNA VESTAR Regulates Lymphangiogenesis and Lymph Node Metastasis of Esophageal Squamous Cell Carcinoma by Enhancing VEGFC mRNA Stability.

Lymph node metastasis is one of the most malignant clinical features in patients with esophageal squamous cell carcinoma (ESCC). Understanding the mechanism of lymph node metastasis will provide treatment strategies for patients with ESCC. Long noncoding RNAs (lncRNA) play a critical role in the development and progression of human cancers. However, the role and mechanism of lncRNAs in lymph node metastasis remain largely unknown. Here we show that VEGFC mRNA stability-associated long noncoding RNA (VESTAR) is involved in lymph node metastasis of ESCC. VESTAR was overexpressed in ESCC tissues and was predictive of poor prognosis in patients with ESCC. In ESCC, NXF1 and SRSF3 facilitated nuclear export of VESTAR to the cytoplasm, which was associated with lymph node metastasis. Depletion of VESTAR inhibited ESCC-associated lymphangiogenesis and lymphatic metastasis. Mechanistically, VESTAR directly bound and stabilized VEGFC mRNA. VESTAR also interacted with HuR, a positive regulator of VEGFC mRNA stability, and increased HuR binding to VEGFC mRNA. Our study reveals a novel lncRNA-guided mechanism of lymph node metastasis in ESCC and may provide a potential target for treatment of ESCC lymphatic metastasis. SIGNIFICANCE: These findings illustrate the lncRNA-guided regulation of VEGFC mRNA stability via direct RNA-RNA interactions, highlighting a therapeutic target for patients with ESCC with lymphatic metastasis.

Nogo-B receptor is required for stabilizing TGF-ß type I receptor and promotes the TGF-ß1-induced epithelial-to-mesenchymal transition of non-small cell lung cancer.

Background and Objective: Metastasis is the leading cause of death in patients with advanced non-small cell lung cancer (NSCLC), and epithelial-mesenchymal transition (EMT) is a crucial event in the metastasis of NSCLC. Our previous works demonstrated that NgBR promoted EMT in NSCLC. However, the molecular mechanism was unclear. Methods: TGF-ß1 was used to induce EMT process of NSCLC cells. The biological functions of NgBR in promoting TGF-ß1-induced NSCLC metastasis were studied by gain- and loss-of-function assays both in vitro and in vivo. The underlying mechanisms were studied using molecular biology assays. Results: We found that knockdown of NgBR inhibited TGF-ß1-induced cell migration and invasion in NSCLC cells. In contrast, NgBR overexpression promoted TGF-ß1-induced EMT of A549 cells. Mechanically, we found that knockdown of NgBR facilitated ubiquitination and degradation of TßRI, leading to downregulation of TßRI expression in NSCLC cells. Moreover, we confirmed a positive correlation between NgBR and TßRI in NSCLC tissues. Conclusions: Our findings provide a novel role of NgBR in modulating TGF-ß1-induced EMT and propose NgBR as a new therapeutic target for treating NSCLC patients.

lncRNA TM4SF1-AS1 Activates the PI3K/AKT Signaling Pathway and Promotes the Migration and Invasion of Lung Cancer Cells.

PURPOSE: Metastasis is a crucial cause of the high mortality in patients with lung cancer. Long non-coding RNAs (lncRNAs) are emerging as important players in the development and progression of human cancers. Here, we aimed to identify metastasis-associated lncRNA and to study its roles in the migration and invasion of lung cancer cells. MATERIALS AND METHODS: We screened differentially expressed lncRNAs between high- and low-metastatic lung cancer cell lines by using microarray and identified the target lncRNA TM4SF1-AS1. The effect of the TM4SF1-AS1 on the invasion and migration was evaluated through the wound healing experiment and transwell assay. The expression of related genes was assessed by RNA sequence and Western blotting. RESULTS: TM4SF1-AS1 was highly expressed in high metastatic lung cancer cell line, and it was also significantly up-regulated in lymph node metastatic lung cancer and was associated with lymph node metastasis. Overexpression of TM4SF1-AS1 promoted the migration and invasion of lung cancer cells. Overexpression of TM4SF1-AS1 decreased the expression of E-Cadherin and increased the expression of Vimentin, Snail and Twist, while knockdown of TM4SF1-AS1 exhibited the opposite trend. Furthermore, RNA sequence analysis revealed that some signaling pathways, including PI3K/AKT signaling pathway, were enriched upon TM4SF1-AS1 overexpression. Western blotting further confirmed that the PI3K/AKT signaling pathway was activated by TM4SF1-AS1. CONCLUSION: This study illustrates that TM4SF1-AS1 promotes the migration and invasion of lung cancer cells by activating the PI3K/AKT signaling pathway. TM4SF1-AS1 might be a novel target of molecular treatment for lung cancer.

Long non-coding RNA LOC100133669 promotes cell proliferation in oesophageal squamous cell carcinoma.

OBJECTIVES: LOC100133669 is a lncRNA whose function during tumorigenesis remains unclear now. Thus, we aimed to explore its clinical significance and function in oesophageal squamous cell carcinoma (ESCC). MATERIALS AND METHODS: ISH was used to detect LOC100133669 expression in ESCC tissues. The full-length LOC100133669 was identified by using RACE assay. Subcellular distribution of LOC100133669 was examined by nuclear/cytoplasmic RNA fractionation and qPCR. The role of LOC100133669 in ESCC cell growth was determined by colony formation, MTT and flow cytometry experiments in vitro, as well as xenograft tumour experiment in vivo. RNA pull-down assay was performed to find LOC100133669-interacted protein, which was further examined by RIP, IP, Western blot and rescue experiments. RESULTS: LOC100133669 was upregulated in ESCC tissues compared with adjacent non-tumour tissues. High LOC100133669 expression was associated with poor prognosis of patients with ESCC. We defined LOC100133669 to be 831 nt in length and mainly localized in the cytoplasm of ESCC cells. Knockdown of LOC100133669 inhibited ESCC cell proliferation and cell cycle progression, while overexpression of LOC100133669 showed the opposite effects. Furthermore, LOC100133669 could bind to Tim50 and upregulated its protein level through inhibiting ubiquitination. Overexpression of Tim50 in part abolished the LOC100133669 depletion-caused inhibitory effect on ESCC cell proliferation. CONCLUSIONS: LOC100133669 plays an oncogenic role in ESCC and may serve as a promising diagnostic marker and therapeutic target for ESCC patients.

GADD45G Interacts with E-cadherin to Suppress the Migration and Invasion of Esophageal Squamous Cell Carcinoma.

BACKGROUND/AIMS: Esophageal squamous cell carcinoma (ESCC) is one of the most prevalent cancers with poor prognosis. Metastasis is the leading cause of cancer-related deaths. The growth arrest and DNA damage-inducible 45 gamma (GADD45G) has been reported to correlate with survival, invasion, and metastasis of ESCC. This study was aimed to investigate the role and mechanism of GADD45G in ESCC cell migration and invasion. METHODS: Both the effects of GADD45G and its need for E-cadherin to function on ESCC cell migration and invasion were determined through loss- and gain-of-function approaches via Transwell assays. The interaction between GADD45G and E-cadherin was detected by GST-pull down and IP assays. The expression of E-cadherin upon GADD45G overexpression was evaluated by RT-qPCR and western blot. The level of E-cadherin in cytoplasmic, nuclear, and membrane fractions was examined by western blot following subcellular fractionation. RESULTS: Knockdown of GADD45G increased the migration and invasion abilities of KYSE150 cells, while overexpression of GADD45G showed the opposite effects on YES2 and KYSE30 cells. GADD45G could interact with E-cadherin and enhanced its membrane level. Knockdown of E-cadherin abolished the inhibitory effects of GADD45G on ESCC cell migration and invasion. Intriguingly, dimer-dissociating mutant of GADD45G could not interact with E-cadherin and almost lost its ability to suppress the ESCC cell migration and invasion. CONCLUSIONS: This study reveals a novel role for GADD45G in inhibiting the ESCC cell migration and invasion, which will provide a new insight in understanding the ESCC metastatic mechanism.

Dihydroartemisinin and doxorubicin co-loaded Soluplus®-TPGS mixed micelles: formulation characterization, cellular uptake, and pharmacodynamic studies.

Clinically, co-delivery of chemotherapeutics has been limited by poor water-solubility and severe systemic toxicity. This study was aimed at integrating the merits of combination chemotherapy and mixed micellar technology and demonstrating the anticancer potential of doxorubicin (DOX) and dihydroartemisinin (DHA) co-loaded Soluplus®-TPGS mixed micellar system. In this study, physiochemically stable multidrug loaded mixed micelles were successfully prepared, encapsulation efficiencies of DOX and DHA were as high as 90%, and the average diameter of the micelles was 64.27 nm. The cellular uptake of DOX from the mixed micelles increased by 1.3 and 1.2 times for MCF-7 and MCF-7/ADR cell lines, respectively. The micelles were more cytotoxic than free DHA-DOX. Surprisingly, the co-loaded mixed micelles exhibited higher antitumor activity, while the systemic toxicity was reduced during the treatment. Therefore, the DOX and DHA mixed micelle might be a potential, effective, and less toxic drug-delivery system for cancer therapy.

PDIA6 modulates apoptosis and autophagy of non-small cell lung cancer cells via the MAP4K1/JNK signaling pathway.

BACKGROUND: Non-small cell lung cancer (NSCLC) is the most common type of lung cancer with a poor prognosis. We previously found that protein disulfide isomerase family 6 (PDIA6) is upregulated in lung squamous cell carcinoma (LSCC). This study aimed to elucidate the clinical relevance, biological functions, and molecular mechanisms of PDIA6 in NSCLC. METHODS: The expression of PDIA6 in NSCLC was assessed using the TCGA database, western blotting, and immunohistochemistry. Correlations of PDIA6 expression with clinicopathological and survival features were evaluated. The functions of PDIA6 in regulating NSCLC cell growth, apoptosis, and autophagy were investigated using gain-and loss-of-function strategies in vitro or in vivo. The underlying molecular mechanisms of PDIA6 function were examined by human phospho-kinase array and co-immunoprecipitation. FINDINGS: PDIA6 expression was upregulated in NSCLC compared with adjacent normal tissues, and the higher PDIA6 expression was correlated with poor prognosis. PDIA6 knockdown decreased NSCLC cell proliferation and increased cisplatin-induced intrinsic apoptosis, while PDIA6 overexpression had the opposite effects. In addition, PDIA6 regulated cisplatin-induced autophagy, and this contributed to PDIA6-mediated apoptosis in NSCLC cells. Mechanistically, PDIA6 reduced the phosphorylation levels of JNK and c-Jun. Moreover, PDIA6 interacted with MAP4K1 and inhibited its phosphorylation, ultimately inhibiting the JNK/c-Jun signaling pathway. INTERPRETATION: PDIA6 is overexpressed in NSCLC and inhibits cisplatin-induced NSCLC cell apoptosis and autophagy via the MAP4K1/JNK/c-Jun signaling pathway, suggesting that PDIA6 may serve as a biomarker and therapeutic target for NSCLC patients. FUND: National Natural Science Foundation of China and Institutions of higher learning of innovation team from Liaoning province.

Triptolide interferes with XRCC1/PARP1-mediated DNA repair and confers sensitization of triple-negative breast cancer cells to cisplatin.

Triptolide is a natural compound isolated from the Tripterygium wilfordii, which possesses anti-inflammatory and anti-tumor activities. Triptolide reportedly inhibits RNA polymerase II-mediated transcription and ATM activities to interfere with DNA repair. However, the roles of triptolide in DNA repair are still largely unknown. Triple negative breast cancer cells (TNBC) are insensitive to targeted anti-tumoral drugs, thus DNA damage chemotherapeutic drugs are the available treatments used in clinic, while the drug resistance of TNBC causes the challenge for successful cure. In this study, we investigated the efficiency of cisplatin in combination with triptolide in treatment of TNBC. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay shows triptolide suppresses the growth of two triple-negative breast cancer cells, BT549 and MDA-MB-231. Triptolide induces DNA breaks and arrests TNBC in the cell cycle S phase, and sensitizes TNBC to cisplatin. Western blot analysis shows triptolide down-regulated the levels of PARP1 and XRCC1, and slightly decreases the levels of RAD51. The results demonstrate triptolide interferes with single strand-break and base excision repair. The over-expressed PARP1/XRCC1 help the TNBC to resist triptolide. Based on these results, we conclude triptolide confers sensitization of TNBC to cisplatin via interference with XRCC1/PARP1-mediated base excision repair.

Nogo-B receptor promotes epithelial-mesenchymal transition in non-small cell lung cancer cells through the Ras/ERK/Snail1 pathway.

Nogo-B receptor (NgBR) is a specific receptor of Nogo-B that regulates vascular remodeling and angiogenesis. Previously, we found that NgBR promotes the membrane translocation and activation of Ras in breast cancer cells and enhances the chemoresistance of hepatocellular carcinoma cells to 5-fluorouracil. However, the role of NgBR in lung cancer has not yet been elucidated. In the present study, we found that NgBR knockdown inhibited epithelial-mesenchymal transition (EMT) in non-small cell lung cancer (NSCLC) cells in vitro and metastasis of NSCLC cells in vivo. In contrast, NgBR overexpression promoted EMT in and lung metastasis of NSCLC cells. At the molecular level, NgBR modulated the expression of EMT-related proteins and enhanced the protein expression of Snail1, a crucial transcription factor that represses epithelial cell protein marker E-cadherin. Moreover, we found that NgBR overexpression promoted the membrane localization of Ras and activation of downstream MEK/ERK signaling pathway and that NgBR knockdown by using a specific shRNA inversely affected the expression of EMT-related proteins in NSCLC cells. Thus, our results provide novel insights on the regulatory role of NgBR in the metastasis of NSCLC that should be investigated further for developing a therapeutic strategy for treating patients with NSCLC.

Quantitative Proteomic Profiling the Molecular Signatures of Annexin A5 in Lung Squamous Carcinoma Cells.

Lung cancer remains the leading cancer killer around the world. It's crucial to identify newer mechanism-based targets to effectively manage lung cancer. Annexin A5 (ANXA5) is a protein kinase C inhibitory protein and calcium dependent phospholipid-binding protein, which may act as an endogenous regulator of various pathophysiological processes. However, its molecular mechanism in lung cancer remains poorly understood. This study was designed to determine the mechanism of ANXA5 in lung cancer with a hope to obtain useful information to provide a new therapeutic target. We used a stable isotope dimethyl labeling based quantitative proteomic method to identify differentially expressed proteins in NSCLC cell lines after ANXA5 transfection. Out of 314 proteins, we identified 26 and 44 proteins that were down- and up-regulated upon ANXA5 modulation, respectively. The IPA analysis revealed that glycolysis and gluconeogenesis were the predominant pathways modulated by ANXA5. Multiple central nodes, namely HSPA5, FN1, PDIA6, ENO1, ALDOA, JUP and KRT6A appeared to occupy regulatory nodes in the protein-protein networks upon ANXA5 modulation. Taken together, ANXA5 appears to have pleotropic effects, as it modulates multiple key signaling pathways, supporting the potential usefulness of ANXA5 as a potential target in lung cancer. This study might provide a new insight into the mechanism of ANXA5 in lung cancer.

Elevated eukaryotic elongation factor 2 expression is involved in proliferation and invasion of lung squamous cell carcinoma.

Eukaryotic elongation factor 2 (EF2), is a critical enzyme solely responsible for catalyzing the translocation of the elongated peptidyl-tRNA from the A to P sites of the ribosome during the process of protein synthesis. EF2 is found to be highly expressed in a variety of malignant tumors and is correlated with cancer cell progression and recurrence. The present study was designed to uncover the function of EF2 on lung squamous cell carcinoma (LSCC) cancer cell growth and progression. Our results from clinical tissue studies showed that EF2 protein was significantly overexpressed in LSCC tissues, compared with the adjacent normal lung tissues, which was confirmed by western blotting and tissue microarray. Forced expression of EF2 resulted in the enhancement of lung squamous carcinoma NCI-H520 cells growth through promotion of G2/M progression in cell cycle, activating Akt and Cdc2/Cyclin B1. In nude mice cancer xenograft model, overexpression of EF2 significantly facilitated cell proliferation in vivo. Furthermore, forced expression of EF2 in the cells increased the capabilities of migration and invasion by changing the expressions of EMT-related proteins and genes. These results provided novel insights into the role of EF2 in tumorigenesis and progression in LSCC. EF2-targeted therapy could become a good strategy for the clinical treatment of LSCC.

PDIA6 promotes the proliferation of HeLa cells through activating the Wnt/ß-catenin signaling pathway.

Protein disulfide isomerase family 6 (PDIA6) belongs to the protein disulfide isomerase (PDI) family, which function as isomerases and molecular chaperones. PDIA6 has recently been shown to promote the proliferation and growth of various types of human cancer cells; however the underlying molecular mechanism remains elusive. Here, we report that PDIA6 enhances the proliferation of HeLa cells through activation of the Wnt/ß-catenin signaling pathway. Ectopic overexpression of PDIA6 in HeLa cells led to increased cell proliferation accompanied with accelerated cell cycle progression. Further mechanistic investigation demonstrated that overexpression of PDIA6 resulted in decreased phosphorylation of ß-catenin at Ser45 and Ser33/Ser37/Thr41, while increased ß-catenin nuclear accumulation, and upregulation of Wnt/ ß-catenin signaling target genes cyclinD1 and c-myc, which was abolished by ubiquitin-proteasome inhibitor MG132. These results demonstrated that PDIA6 overexpression promoted the proliferation of HeLa cells by suppressing the phosphorylation of ß-catenin, thereby inhibiting the degradation of ß-catenin through the ubiquitin-proteasome pathway.

PRP19 upregulation inhibits cell proliferation in lung adenocarcinomas by p21-mediated induction of cell cycle arrest.

Precursor messenger RNA processing factor 19 (PRP19) is known to be a critical component of the eukaryotic spliceosomal machinery and DNA damage repair system, the deregulation of which leads to many disease conditions. In many human cancers, PRP19 expression is upregulated, but its functional significance and corresponding underlying mechanisms remain to be addressed. Focusing on lung carcinomas, PRP19 upregulation was achieved by plasmid transfection into A549 adenocarcinoma cells. The transfected cells were then subjected to several in vitro and in vivo assays following in situ assessment of the protein in paired clinical lung tissues. We report that PRP19 expression is elevated in lung carcinoma tissues compared to non-tumor tissues. Following its upregulation, PRP19 repressed cell proliferation and tumor growth by upregulating the expression of the cell cycle arrest protein p21.