The interaction between apigenin and PKM2 restrains progression of colorectal cancer.

Apigenin, a flavonoid that widely existed in vegetables and fruits, possesses anticarcinogenic, low toxicity, and no mutagenic properties, suggesting that apigenin is a potential therapeutic agent for tumors. However, the underlying anti-cancer molecular target of apigenin is still unclear. Therefore, to reveal the direct target and amino acid site of apigenin against colorectal cancer is the focus of this study. In the present study, the results proved that the anti-CRC activity of apigenin was positively correlated with pyruvate kinase M2 (PKM2) expression, characterized by the inhibition of cell proliferation and increase of apoptotic effects induced by apigenin in LS-174T cells of knock down PKM2. Next, pull-down and MALDI-TOF/TOF analysis determined that apigenin might interact directly with PKM2 in HCT-8 cells. Further, the study confirmed that lysine residue 433 (K433) was a key amino acid site for PKM2 binding to apigenin. Apigenin restricted the glycolysis of LS-174T and HCT-8 cells by targeting the K433 site of PKM2, thereby playing an anti-CRC role in vivo and in vitro. Meanwhile, apigenin markedly attenuated tumor growth without any adverse effects. Taken together, these findings reveal that apigenin is worthy of consideration as a promising PKM2 inhibitor for the prevention of CRC.

Long-term outcomes of endoscopic papillary large-balloon dilation (12-15 mm) with or without limited sphincterotomy for removal of bile duct stones.

BACKGROUND: Limited endoscopic sphincterotomy with large balloon dilation (ES-LBD) and endoscopic papillary large-balloon dilation (EPLBD) have been proven safe and effective for removal of bile duct stones. However, the long-term outcomes are not clear. The aim of this study was to assess the long-term outcomes of EPLBD (12-15 mm) with or without limited sphincterotomy for removal of common bile duct (CBD) stones. METHODS: Patients with EPLBD or ES-LBD referred for the removal of bile-duct stones between June 2008 and August 2020 were retrospectively reviewed. Complete stone clearance, endoscopic retrograde cholangiopancreatography (ERCP)-related adverse events, and late biliary complications during long-term follow-up were analyzed. RESULTS: Basic patient characteristics were not significantly different between the groups that underwent EPLBD (n = 168) and ES-LBD (n = 57). EPLBD compared with ES-LBD resulted in similar outcomes in terms of overall successful stone removal (99.4% vs. 100%, P = 1.00) and ERCP-related adverse events (7.7% vs. 5.3%, P = 0.77). The mean duration of the follow-up were 113.6 months and 106.7 months for patients with EPLBD and ES-LBD, respectively (P = 0.13). There was no significant difference between EPLBD and ES-LBD in the incidence of stone recurrence [20 (11.9%) vs. 9 (15.8%); P = 0.49]. Multivariate analysis showed that a diameter of CBD ≥ 15 mm (OR = 3.001; 95% CI: 1.357-6.640; P = 0.007) was an independent risk factor for stone recurrence. CONCLUSIONS: The application of a large balloon (12-15 mm) via EPLBD is an effective and safe alternative to ES-LBD for extraction of large CBD stones. Endoscopic sphincterotomy prior to EPLBD may be unnecessary. A diameter of CBD ≥ 15 mm is a risk factor of stone recurrence.

Characterization of cell cycle and apoptosis in Chinese hamster ovary cell culture using flow cytometry for bioprocess monitoring.

Chinese hamster ovary (CHO) cells are by far the most important mammalian cell lines used for producing antibodies and other therapeutic proteins. It is critical to fully understand their physiological conditions during a bioprocess in order to achieve the highest productivity and the desired product quality. Flow cytometry technology possesses unique advantages for measuring multiple cellular attributes for a given cell and examining changes in cell culture heterogeneity over time that can be used as metrics for enhanced process understanding and control strategy. Flow cytometry-based assays were utilized to examine the progression of cell cycle and apoptosis in three case studies using different antibody-producing CHO cell lines in both fed-batch and perfusion bioprocesses. In our case studies, we found that G0/G1 phase distribution and early apoptosis accumulation responded to subtle changes in culture conditions, such as pH shifting or momentary glucose depletion. In a perfusion process, flow cytometry provided an insightful understanding of the cell physiological status under a hypothermic condition. More importantly, these changes in cell cycle and apoptosis were not detected by a routine trypan blue exclusion-based cell counting and viability measurement. In summary, integration of flow cytometry into bioprocesses as a process analytical technology tool can be beneficial for establishing optimum process conditions and process control.

Shikonin Inhibits Cholangiocarcinoma Cell Line QBC939 by Regulating Apoptosis, Proliferation, and Invasion.

This study was designed to clarify whether Shikonin causes proliferation, apoptosis, and invasion in cholangiocarcinoma cells and to investigate the mechanism of action. QBC939 cells were cultured with different doses of Shikonin, and then 3-(4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium assay was used to detect cell viability. Apoptosis of cells was detected using flow cytometry with Annexin V/propidium iodide (PI) assay after being stained with Hoechst 33242. The role of Shikonin on the invasive and metastasis ability was detected using Transwell invasion assay. Real-time polymerase chain reaction and Western blotting were used to detect the expression of caspase-3, caspase-8, epidermal growth factor receptor (EGFR), and matrix metalloproteinase (MMP)-9. Shikonin inhibited proliferation and invasive ability of QBC939 cells in a dose-dependent manner; at the same time, apoptosis of cells was also observed in a concentration-dependent fashion. Moreover, Annexin V/PI assay and Transwell invasion assay results indicated that Shikonin induced apoptosis and invasion inhibitory probably due to upregulation of caspase-3 and caspase-8 expression and downregulation of MMP-9 and EGFR expression in a concentration-dependent fashion. Shikonin could enhance apoptosis and inhibit proliferation and invasion of QBC939 cells; such biological behaviors mainly occurred via upregulating the expression of caspase-3 and caspase-8 and downregulating the expression of MMP-9 and EGFR.

Tuning monoclonal antibody galactosylation using Raman spectroscopy-controlled lactic acid feeding.

A key aspect of large-scale production of biotherapeutics is a well-designed and consistently-executed upstream cell culture process. Process analytical technology tools provide enhanced monitoring and control capabilities to support consistent process execution, and also have potential to aid in maintenance of product quality at desired levels. One such tool, Raman spectroscopy, has matured as a useful technique to achieve real-time monitoring and control of key cell culture process attributes. We developed a Raman spectroscopy-based nutrient control strategy to enable dual control of lactate and glucose levels for a fed-batch CHO cell culture process for monoclonal antibody (mAb) production. To achieve this, partial least squares-based chemometric models for real-time prediction of glucose and lactate concentrations were developed and deployed in feedback control loops. In particular, feeding of lactic acid post-metabolic shift was investigated based on previous work that has shown the impact of lactate levels on ammonium as well as mAb product quality. Three feeding strategies were assessed for impact on cell metabolism, productivity, and product quality: bolus-fed glucose, glucose control at 4 g/L, or simultaneous glucose control at 4 g/L and lactate control at 2 g/L. The third feeding strategy resulted in a significant reduction in ammonium levels (68%) while increasing mAb galactosylation levels by approximately 50%. This work demonstrated that when deployed in a cell culture process, Raman spectroscopy is an effective technique for simultaneous control of multiple nutrient feeds, and that lactic acid feeding can have a positive impact on both cell metabolism and mAb product quality.

SnoN Antagonizes the Hippo Kinase Complex to Promote TAZ Signaling during Breast Carcinogenesis.

SnoN regulates multiple signaling pathways, including TGF-ß/Smad and p53, and displays both pro-oncogenic and anti-oncogenic activities in human cancer. We have observed previously that both its intracellular localization and expression levels are sensitive to cell density, suggesting that it may crosstalk with Hippo signaling. Here we report that, indeed, SnoN interacts with multiple components of the Hippo pathway to inhibit the binding of Lats2 to TAZ and the subsequent phosphorylation of TAZ, leading to TAZ stabilization. Consistently, SnoN enhances the transcriptional and oncogenic activities of TAZ, and reducing SnoN decreases TAZ expression as well as malignant progression of breast cancer cells. Interestingly, SnoN itself is downregulated by Lats2 that is activated by the Scribble basolateral polarity protein. Thus, SnoN is a critical component of the Hippo regulatory network that receives signals from the tissue architecture and polarity to coordinate the activity of intracellular signaling pathways.

Compensatory induction of MYC expression by sustained CDK9 inhibition via a BRD4-dependent mechanism.

CDK9 is the kinase subunit of positive transcription elongation factor b (P-TEFb) that enables RNA polymerase (Pol) II's transition from promoter-proximal pausing to productive elongation. Although considerable interest exists in CDK9 as a therapeutic target, little progress has been made due to lack of highly selective inhibitors. Here, we describe the development of i-CDK9 as such an inhibitor that potently suppresses CDK9 phosphorylation of substrates and causes genome-wide Pol II pausing. While most genes experience reduced expression, MYC and other primary response genes increase expression upon sustained i-CDK9 treatment. Essential for this increase, the bromodomain protein BRD4 captures P-TEFb from 7SK snRNP to deliver to target genes and also enhances CDK9's activity and resistance to inhibition. Because the i-CDK9-induced MYC expression and binding to P-TEFb compensate for P-TEFb's loss of activity, only simultaneously inhibiting CDK9 and MYC/BRD4 can efficiently induce growth arrest and apoptosis of cancer cells, suggesting the potential of a combinatorial treatment strategy.

Ski regulates Hippo and TAZ signaling to suppress breast cancer progression.

Ski, the transforming protein of the avian Sloan-Kettering retrovirus, inhibits transforming growth factor-ß (TGF-ß)/Smad signaling and displays both pro-oncogenic and anti-oncogenic activities in human cancer. Inhibition of TGF-ß signaling is likely responsible for the pro-oncogenic activity of Ski. We investigated the mechanism(s) underlying the tumor suppressor activity of Ski and found that Ski suppressed the activity of the Hippo signaling effectors TAZ and YAP to inhibit breast cancer progression. TAZ and YAP are transcriptional coactivators that can contribute to cancer by promoting proliferation, tumorigenesis, and cancer stem cell expansion. Hippo signaling activates the the Lats family of kinases, which phosphorylate TAZ and YAP, resulting in cytoplasmic retention and degradation and inhibition of their transcriptional activity. We showed that Ski interacted with multiple components of the Hippo pathway to facilitate activation of Lats2, resulting in increased phosphorylation and subsequent degradation of TAZ. Ski also promoted the degradation of a constitutively active TAZ mutant that is not phosphorylated by Lats, suggesting the existence of a Lats2-independent degradation pathway. Finally, we showed that Ski repressed the transcriptional activity of TAZ by binding to the TAZ partner TEAD and recruiting the transcriptional co-repressor NCoR1 to the TEAD-TAZ complex. Ski effectively reversed transformation and epithelial-to-mesenchyme transition in cultured breast cancer cells and metastasis in TAZ-expressing xenografted tumors. Thus, Ski inhibited the function of TAZ through multiple mechanisms in human cancer cells.

LARP7 suppresses P-TEFb activity to inhibit breast cancer progression and metastasis.

Transcriptional elongation by RNA polymerase (Pol) II is essential for gene expression during cell growth and differentiation. The positive transcription elongation factor b (P-TEFb) stimulates transcriptional elongation by phosphorylating Pol II and antagonizing negative elongation factors. A reservoir of P-TEFb is sequestered in the inactive 7SK snRNP where 7SK snRNA and the La-related protein LARP7 are required for the integrity of this complex. Here, we show that P-TEFb activity is important for the epithelial-mesenchymal transition (EMT) and breast cancer progression. Decreased levels of LARP7 and 7SK snRNA redistribute P-TEFb to the transcriptionally active super elongation complex, resulting in P-TEFb activation and increased transcription of EMT transcription factors, including Slug, FOXC2, ZEB2, and Twist1, to promote breast cancer EMT, invasion, and metastasis. Our data provide the first demonstration that the transcription elongation machinery plays a key role in promoting breast cancer progression by directly controlling the expression of upstream EMT regulators.

Metabolic profiling reveals PAFAH1B3 as a critical driver of breast cancer pathogenicity.

Many studies have identified metabolic pathways that underlie cellular transformation, but the metabolic drivers of cancer progression remain less well understood. The Hippo transducer pathway has been shown to confer malignant traits on breast cancer cells. In this study, we used metabolic mapping platforms to identify biochemical drivers of cellular transformation and malignant progression driven through RAS and the Hippo pathway in breast cancer and identified platelet-activating factor acetylhydrolase 1B3 (PAFAH1B3) as a key metabolic driver of breast cancer pathogenicity that is upregulated in primary human breast tumors and correlated with poor prognosis. Metabolomic profiling suggests that PAFAH1B3 inactivation attenuates cancer pathogenicity through enhancing tumor-suppressing signaling lipids. Our studies provide a map of altered metabolism that underlies breast cancer progression and put forth PAFAH1B3 as a critical metabolic node in breast cancer.

Ether lipid generating enzyme AGPS alters the balance of structural and signaling lipids to fuel cancer pathogenicity.

Aberrant lipid metabolism is an established hallmark of cancer cells. In particular, ether lipid levels have been shown to be elevated in tumors, but their specific function in cancer remains elusive. We show here that the metabolic enzyme alkylglyceronephosphate synthase (AGPS), a critical step in the synthesis of ether lipids, is up-regulated across multiple types of aggressive human cancer cells and primary tumors. We demonstrate that ablation of AGPS in cancer cells results in reduced cell survival, cancer aggressiveness, and tumor growth through altering the balance of ether lipid, fatty acid, eicosanoid, and fatty acid-derived glycerophospholipid metabolism, resulting in an overall reduction in the levels of several oncogenic signaling lipids. Taken together, our results reveal that AGPS, in addition to maintaining ether lipids, also controls cellular utilization of fatty acids, favoring the generation of signaling lipids necessary for promoting the aggressive features of cancer.

EphB3 suppresses non-small-cell lung cancer metastasis via a PP2A/RACK1/Akt signalling complex.

Eph receptors are implicated in regulating the malignant progression of cancer. Here we find that despite overexpression of EphB3 in human non-small-cell lung cancer, as reported previously, the expression of its cognate ligands, either ephrin-B1 or ephrin-B2, is significantly downregulated, leading to reduced tyrosine phosphorylation of EphB3. Forced activation of EphB3 kinase in EphB3-overexpressing non-small-cell lung cancer cells inhibits cell migratory capability in vitro as well as metastatic seeding in vivo. Furthermore, we identify a novel EphB3-binding protein, the receptor for activated C-kinase 1, which mediates the assembly of a ternary signal complex comprising protein phosphatase 2A, Akt and itself in response to EphB3 activation, leading to reduced Akt phosphorylation and subsequent inhibition of cell migration. Our study reveals a novel tumour-suppressive signalling pathway associated with kinase-activated EphB3 in non-small-cell lung cancer, and provides a potential therapeutic strategy by activating EphB3 signalling, thus inhibiting tumour metastasis.

RACK1 promotes non-small-cell lung cancer tumorigenicity through activating sonic hedgehog signaling pathway.

Non-small-cell lung cancer (NSCLC) is a deadly disease due to lack of effective diagnosis biomarker and therapeutic target. Much effort has been made in defining gene defects in NSCLC, but its full molecular pathogenesis remains unexplored. Here, we found RACK1 (receptor of activated kinase 1) was elevated in most NSCLC, and its expression level correlated with key pathological characteristics including tumor differentiation, stage, and metastasis. In addition, RACK1 activated sonic hedgehog signaling pathway by interacting with and activating Smoothened to mediate Gli1-dependent transcription in NSCLC cells. And silencing RACK1 dramatically inhibited in vivo tumor growth and metastasis by blocking the sonic hedgehog signaling pathway. These results suggest that RACK1 represents a new promising diagnosis biomarker and therapeutic target for NSCLC.

RACK1 suppresses gastric tumorigenesis by stabilizing the ß-catenin destruction complex.

BACKGROUND & AIMS: Dysregulation of Wnt signaling has been involved in gastric tumorigenesis by mechanisms that are not fully understood. The receptor for activated protein kinase C (RACK1, GNB2L1) is involved in development of different tumor types, but its expression and function have not been investigated in gastric tumors. METHODS: We analyzed expression of RACK1 in gastric tumor samples and their matched normal tissues from 116 patients using immunohistochemistry. Effects of knockdown with small interfering RNAs or overexpression of RACK1 in gastric cancer cell lines were evaluated in cell growth and tumor xenograft. RACK1 signaling pathways were investigated in cells and zebrafish embryos using immunoblot, immunoprecipitation, microinjection, and in situ hybridization assays. RESULTS: Expression of RACK1 was reduced in gastric tumor samples and correlated with depth of tumor infiltration and poor differentiation. Knockdown of RACK1 in gastric cancer cells accelerated their anchorage-independent proliferation in soft agar, whereas overexpression of RACK1 reduced their tumorigenicity in nude mice. RACK1 formed a complex with glycogen synthase kinase Gsk3ß and Axin to promote the interaction between Gsk3ß and ß-catenin and thereby stabilized the ß-catenin destruction complex. On stimulation of Wnt3a, RACK1 repressed Wnt signaling by inhibiting recruitment of Axin by Dishevelled 2 (Dvl2). Moreover, there was an inverse correlation between expression of RACK1 and localization of ß-catenin to the cytoplasm/nucleus in human gastric tumor samples. CONCLUSIONS: RACK1 negatively regulates Wnt signaling pathway by stabilizing the ß-catenin destruction complex and act as a tumor suppressor in gastric cancer cells.

Tumor initiating cells in esophageal squamous cell carcinomas express high levels of CD44.

BACKGROUND: Esophageal Squamous Cell Carcinoma (ESCC) is a major subtype of esophageal cancer causing significant morbility and mortality in Asia. Mechanism of initiation and progression of this disease is unclear. Tumor initiating cells (TICs) are the subpopulation of cells which have the ability to self-renew, as well as, to drive initiation and progression of cancer. Increasing evidence has shown that TICs exist in a variety of tumors. However, the identification and characterization of TICs in esophageal carcinoma has remained elusive. METHODOLOGY/PRINCIPAL FINDINGS: to identify TICs in ESCC, ESCC cell lines including two primary cells were used for screening suitable surface marker. Then colony formation assay, drug resistant assay and tumorigenicity assay in immune deficient mice were used to characterize TICs in ESCC. We found that just the CD44 expression correlated with tumorigenicity in ESCC cell lines. And then induced differentiation of ESCC cells by all-trans retinoic acid treatment led to decreased expression of CD44. The FACS isolated cell subpopulations with high CD44 expression showed increased colony formation and drug resistance in vitro, as well as significantly enhanced tumorigenicity in NOD/SICD mice, as compared to the low expressing CD44 ESCC cells. CONCLUSIONS/SIGNIFICANCE: our study has discovered a novel TIC surface marker, CD44, which can be utilized to enrich efficiently the TICs in ESCC. These findings will be useful for further studies of these cells and exploring therapeutic approaches.

EphB3 is overexpressed in non-small-cell lung cancer and promotes tumor metastasis by enhancing cell survival and migration.

Eph receptors, the largest subfamily of transmembrane tyrosine kinase receptors, have been increasingly implicated in various physiologic and pathologic processes, and the roles of the Eph family members during tumorigenesis have recently attracted growing attention. Until now, research on EphB3 function in cancer is limited to focusing on tumor suppression by EphB receptors in colorectal cancer. However, its function in other types of cancer remains poorly investigated. In this study, we explored the function of EphB3 in non-small-cell lung cancer (NSCLC). We found that the expression of EphB3 was significantly upregulated in clinical samples and cell lines, and the expression level correlated with the patient pathologic characteristics, including tumor size, differentiation, and metastasis. Overexpression of EphB3 in NSCLC cell lines accelerated cell growth and migration and promoted tumorigenicity in xenografts in a kinase-independent manner. In contrast, downregulation of EphB3 inhibited cell proliferation and migration and suppressed in vivo tumor growth and metastasis. Furthermore, we showed that silencing of EphB3 inhibited cell growth by reducing DNA synthesis and caspase-8-mediated apoptosis and suppressed cell migration by increasing accumulation of focal adhesion formation. Taken together, our findings suggest that EphB3 provides critical support to the development and progression of NSCLC by stimulating cell growth, migration, and survival, thereby implicating EphB3 as a potential therapeutic target in NSCLC.

Overexpression of degenerative spermatocyte homolog 1 up-regulates the expression of cyclin D1 and enhances metastatic efficiency in esophageal carcinoma Eca109 cells.

Cyclin D1 plays a pivotal role in cell-cycle transition through G1 phase. In this article, we found that Degenerative Spermatocyte Homolog 1 (DEGS1) up-regulated the expression of cyclin D1 and the activation of transcription factor NF-kappaB was essential for DEGS1-induced cyclin D1 production. Forced expression of DEGS1 in Esophageal carcinoma cell line Eca109 cells increased their ability of cell migration and significantly induced tumor metastasis in nude mice, whereas RNA interference-mediated knockdown of DEGS1 cells significantly inhibited cell migration in vitro, as well as tumor metastasis in vivo. Our results demonstrated that expression of DEGS1 up-regulated the expression of cyclin D1 and enhanced the efficiency of tumor metastasis.

Expression of soluble, biologically active recombinant human endostatin in Escherichia coli.

Endostatin, a 20kDa C-terminal fragment of collagen XVIII, is a potent anti-angiogenic protein and inhibitor of tumor growth. Recombinant endostatin was prepared from Escherichia coli deposited as insoluble, inactive inclusion bodies. In the present study, we produced soluble and biologically active recombinant human endostatin (rhEndostatin) in E. coli by employing both co-expression of the molecular chaperones and lower temperature fermentation. Two groups of chaperones Trigger factor and GroEL-GroES (GroEL/ES), DnaK-DnaJ-GrpE and GroEL/ES, were co-expressed, respectively, with rhEndostatin at different temperatures (37, 25, and 16 degrees C). It revealed that low temperature or molecular chaperones alone could enhance the production of active rhEndostatin; meanwhile, combinational employment of low temperature cultivation (16 degrees C) together with co-expression of DnaK-DnaJ-GrpE and GroEL/ES was more effective to prevent aggregation of rhEndostatin. The production of soluble rhEndostatin was about 36 mg/L, and at least 16 mg of rhEndostatin was purified from 1L flask culture. The purified rhEndostatin specifically inhibited the proliferation of endothelial cell-bovine capillary endothelial cell in a dose-dependent manner, and it showed potent anti-angiogenic capability on the chorioallantoic membrane of chick embryo in vivo. Our study provides a feasible and convenient approach to produce soluble and biologically active rhEndostatin.