Cisplatin Disrupts Proteasome Bounce-Back Effect through Suppressing ZEB1/Nfe2l1 in Cholangiocarcinoma.

BACKGROUND: Bortezomib (BTZ) is a powerful proteasome inhibitor that has been approved for the treatment of haematologic malignancies. Its effectiveness has been assessed against different types of solid tumours. BTZ is ineffective in most solid tumours because of drug resistance, including cholangiocarcinoma, which is associated with a proteasome bounce-back effect. However, the mechanism through which proteasome inhibitors induce the proteasome bounce-back effect remains largely unknown. METHODS: Cholangiocarcinoma cells were treated with BTZ, cisplatin, or a combination of both. The mRNA levels of Nfe2l1 and proteasome subunit genes (PSMA1, PSMB7, PSMD1, PSMD11, PSMD14, and PSME4) were determined using quantitative real time polymerase chain reaction (qPCR). The protein levels of nuclear factor-erythroid 2-related factor 1 (Nfe2l1) and proteasome enzyme activity were evaluated using western blotting and proteasome activity assays, respectively. Transcriptome sequencing was performed to screen for potential transcription factors that regulate Nfe2l1 expression. The effect of zinc finger E-box-binding homeobox 1 (ZEB1) on the expression of Nfe2l1 and proteasome subunit genes, as well as proteasome enzyme activity, was evaluated after the knockdown of ZEB1 expression with siRNA before treatment with BTZ. The transcriptional activity of ZEB1 on the Nfe2l1 promoter was detected using dual-luciferase reporter gene and chromatin immunoprecipitation assays. Cell viability was measured using the cell counting kit-8 (CCK-8) assay and cell apoptosis was assessed using western blotting and flow cytometry. RESULTS: Cisplatin treatment of BTZ-treated human cholangiocarcinoma cell line (RBE) suppressed proteasome subunit gene expression (proteasome bounce-back) and proteasomal enzyme activity. This effect was achieved by reducing the levels of Nfe2l1 mRNA and protein. Our study utilised transcriptome sequencing to identify ZEB1 as an upstream transcription factor of Nfe2l1, which was confirmed using dual-luciferase reporter gene and chromatin immunoprecipitation assays. Notably, ZEB1 knockdown using siRNA (si-ZEB1) hindered the expression of proteasome subunit genes under both basal and BTZ-induced conditions, leading to the inhibition of proteasomal enzyme activity. Furthermore, the combination treatment with BTZ, cisplatin, and si-ZEB1 significantly reduced the viability of RBE cells. CONCLUSIONS: Our study uncovered a novel mechanism through which cisplatin disrupts the BTZ-induced proteasome bounce-back effect by suppressing the ZEB1/Nfe2l1 axis in cholangiocarcinoma. This finding provides a theoretical basis for developing proteasome inhibitor-based strategies for the clinical treatment of cholangiocarcinoma and other tumours.

Silibinin ameliorates cisplatin-induced acute kidney injury via activating Nfe2l1-mediated antioxidative response to suppress the ROS/MAPK signaling pathway.

Cisplatin, a first-line chemotherapeutic agent commonly used to treat various solid tumors, induce severe adverse effects, especially nephrotoxicity, which largely limits its clinical application. However, the currently used measures to prevent nephrotoxicity are not ideal owing to the mechanisms underlying cisplatin-induced nephrotoxicity are not comprehensively understood. Herein, we examined the effects of silibinin on cisplatin-induced nephrotoxicity and found that silibinin exerted cytoprotection effects during cisplatin treatment in HEK293 cells and in a cisplatin-induced acute kidney injury (AKI) model. Mechanistically, silibinin ameliorated cisplatin-induced AKI via decreasing ROS-mediated MAPK signaling pathway activation, which was confirmed using the inhibitor N-acetylcysteine. Moreover, the protective effect of silibinin against cisplatin-induced ROS generation through the antioxidant transcription factor nuclear factor-erythroid 2-related factor 1 (Nfe2l1), rather than Nfe2l2, mediates HO1 expression. Furthermore, interference with the abundance of Nfe2l1 using siRNA or an overexpression plasmid enhanced or decreased the effect of cisplatin-induced apoptosis, respectively, in HEK293 cells. Interestingly, Nfe2l1 protein stability was more sensitive to cisplatin than that of Nfe2l2. More importantly, the mechanism that silibinin activates Nfe2l1-mediated antioxidant responses was confirmed in a cisplatin-induced AKI model. Silibinin rescued cisplatin-induced Nfe2l1 inhibition by regulating its transcription and post-translational modifications. Taken together, our results reveal a novel mechanism by which silibinin ameliorates cisplatin-induced AKI via activating Nfe2l1-mediated antioxidative response, which provides a new insights to protect patients receiving cisplatin-based cancer treatment against AKI.

Activation of the membrane-bound Nrf1 transcription factor by USP19, a ubiquitin-specific protease C-terminally anchored in the endoplasmic reticulum.

The membrane-bound transcription factor Nrf1 (encoded by Nfe2l1) is activated by sensing glucose deprivation, cholesterol abundance, proteasomal inhibition and oxidative stress and then mediates distinct signaling responses to maintain cellular homeostasis. Herein, we found that Nrf1 stability and transactivity are both enhanced by USP19, a ubiquitin-specific protease tail-anchored in the endoplasmic reticulum (ER) through its C-terminal transmembrane domain. Further experiments revealed that USP19 directly interacts with Nrf1 in proximity to the ER and topologically acts as a deubiquitinating enzyme to remove ubiquitin moieties from this protein, which allow it to circumvent potential proteasomal degradation. This USP19-mediated effect takes place only after Nrf1 is retro-translocated by p97 out of the ER membrane to dislocate the cytoplasmic side. Conversely, knockout of USP19 causes significant decreases in the abundance of Nrf1 and the entrance of its active isoform into the nucleus, which result in the downregulation of its target proteasomal subunits and a modest reduction in USP19-/--derived tumor growth in xenograft mice when compared with wild-type controls. Altogether, these results demonstrate that USP19 serves as a novel mechanistic modulator of Nrf1, but not Nrf2, thereby enabling Nrf1 to be rescued from the putative ubiquitin-directed ER-associated degradation pathway. In turn, our additional experimental evidence has revealed that transcriptional expression of endogenous USP19 and its promoter-driven reporter genes is differentially regulated by Nrf2, as well by Nrf1, at distinct layers within a complex hierarchical regulatory network.

Circulating tumor DNA: a noninvasive biomarker for tracking ovarian cancer.

Ovarian cancer is the fifth leading cause of cancer-related mortality in women worldwide. Despite the development of technologies over decades to improve the diagnosis and treatment of patients with ovarian cancer, the survival rate remains dismal, mainly because most patients are diagnosed at a late stage. Traditional treatment methods and biomarkers such as cancer antigen-125 as a cancer screening tool lack specificity and cannot offer personalized combinatorial therapy schemes. Circulating tumor DNA (ctDNA) is a promising biomarker for ovarian cancer and can be detected using a noninvasive liquid biopsy. A wide variety of ctDNA applications are being elucidated in multiple studies for tracking ovarian carcinoma during diagnostic and prognostic evaluations of patients and are being integrated into clinical trials to evaluate the disease. Furthermore, ctDNA analysis may be used in combination with multiple "omic" techniques to analyze proteins, epigenetics, RNA, nucleosomes, exosomes, and associated immune markers to promote early detection. However, several technical and biological hurdles impede the application of ctDNA analysis. Certain intrinsic features of ctDNA that may enhance its utility as a biomarker are problematic for its detection, including ctDNA lengths, copy number variations, and methylation. Before the development of ctDNA assays for integration in the clinic, such issues are required to be resolved since these assays have substantial potential as a test for cancer screening. This review focuses on studies concerning the potential clinical applications of ctDNA in ovarian cancer diagnosis and discusses our perspective on the clinical research aimed to treat this daunting form of cancer.

Modulating serine palmitoyltransferase-deoxysphingolipid axis in cancer therapy.

A schematic illustration is given regarding serine restriction on tumor growth. Once the cellular abundance of serine decreased or alanine accumulated, the serine palmitoyltransferase (SPT) alternatively conjugates alanine and palmitoyl-CoA to form 3-keto-intermediates, which is rapidly converted to 1-deoxysphinganine and further metabolized to 1-deoxydihydroceramide (1-DeoxyDHCER) and 1-deoxyceramide (1-DeoxyDHCER), so that to exert cytotoxicity for tumor suppression.

Lipid Metabolism in Tumor-Associated Macrophages.

Macrophages are essential components of the immune system in tumors. It can be recruited and educated to two mainly polarized subpopulations (M1-like and M2-like) of tumor-associated macrophages (TAMs) to display anti-tumor or protumor function during the tumor occurrence and progression. Reprogramming of metabolism, especially lipid metabolism, is a typical characteristic of TAMs polarization, which was confirmed recently as a vital target for tumor therapy. However, the relationship between TAMs and lipid metabolism is still obscure in the past decade. In this review, we will first introduce the historical aspects of TAMs, and then discuss the correlation of main lipids (triglycerides, cholesterol, and phospholipids) to TAMs activation and summarize the mechanisms by which lipid metabolism mediated tumor escape the immunological surveillance as well as currently available drugs targeting these mechanisms. We hope that this chapter will give a better understanding of lipid metabolism in TAMs for those who are interested in this field, and lay a foundation to develop novel strategies for tumor therapy by targeting lipid metabolism.

Synergism and Antagonism of Two Distinct, but Confused, Nrf1 Factors in Integral Regulation of the Nuclear-to-Mitochondrial Respiratory and Antioxidant Transcription Networks.

There is hitherto no literature available for explaining two distinct, but confused, Nrf1 transcription factors, because they shared the same abbreviations from nuclear factor erythroid 2-related factor 1 (also called Nfe2l1) and nuclear respiratory factor (originally designated α-Pal). Thus, we have here identified that Nfe2l1Nrf1 and α-PalNRF1 exert synergistic and antagonistic roles in integrative regulation of the nuclear-to-mitochondrial respiratory and antioxidant transcription profiles. In mouse embryonic fibroblasts (MEFs), knockout of Nfe2l1-/- leads to substantial decreases in expression levels of α-PalNRF1 and Nfe2l2, together with TFAM (mitochondrial transcription factor A) and other target genes. Similar inhibitory results were determined in Nfe2l2-/- MEFs but with an exception that both GSTa1 and Aldh1a1 were distinguishably upregulated in Nfe2l1-/- MEFs. Such synergistic contributions of Nfe2l1 and Nfe2l2 to the positive regulation of α-PalNRF1 and TFAM were validated in Keap1-/- MEFs. However, human α-PalNRF1 expression was unaltered by hNfe2l1α-/- , hNfe2l2-/-ΔTA , or even hNfe2l1α-/-+siNrf2, albeit TFAM was activated by Nfe2l1 but inhibited by Nfe2l2; such an antagonism occurred in HepG2 cells. Conversely, almost all of mouse Nfe2l1, Nfe2l2, and cotarget genes were downexpressed in α-PalNRF1+/- MEFs. On the contrary, upregulation of human Nfe2l1, Nfe2l2, and relevant reporter genes took place after silencing of α-PalNRF1, but their downregulation occurred upon ectopic expression of α-PalNRF1. Furtherly, Pitx2 (pituitary homeobox 2) was also identified as a direct upstream regulator of Nfe2l1 and TFAM, besides α-PalNRF1. Overall, these across-talks amongst Nfe2l1, Nfe2l2, and α-PalNRF1, along with Pitx2, are integrated from the endoplasmic reticulum towards the nuclear-to-mitochondrial communication for targeting TFAM, in order to finely tune the robust balance of distinct cellular oxidative respiratory and antioxidant gene transcription networks, albeit they differ between the mouse and the human. In addition, it is of crucial importance to note that, in view of such mutual interregulation of these transcription factors, much cautions should be severely taken for us to interpret those relevant experimental results obtained from knockout of Nfe2l1, Nfe2l2, α-Pal or Pitx2, or their gain-of-functional mutants.

GSK­3ß inhibition promotes doxorubicin­induced apoptosis in human cholangiocarcinoma cells via FAK/AKT inhibition.

Cholangiocarcinoma (CCA) is the most common type of malignant tumor of the bile duct and is characterized by high morbidity and mortality; it is difficult to diagnose in the early stages and responds poorly to current conventional radiotherapy and chemotherapy. The present study investigated the role of GSK­3ß signaling on the anticancer effects of doxorubicin in human CCA cells. Blocking GSK­3ß enhanced the sensitivity of human CCA cells to doxorubicin (Dox)­induced apoptosis, which was accompanied by decreased AKT and focal adhesion kinase (FAK) activity. Moreover, inhibiting GSK­3ß using 6­bromoindirubin­3'­oxime, CHIR99021 or small interfering RNA decreased phosphorylation of FAK and AKT, and promoted apoptosis of Dox­induced human CCA cells. Moreover, FAK inhibition suppressed AKT activity independently of phosphoinositide 3­kinase activity. These results indicated that GSK­3ß protects human CCA cells against Dox­induced apoptosis via sustaining FAK/AKT activity.

Glucose Starvation-Induced Rapid Death of Nrf1α-Deficient, but Not Nrf2-Deficient, Hepatoma Cells Results from Its Fatal Defects in the Redox Metabolism Reprogramming.

Metabolic reprogramming exists in a variety of cancer cells, with the most relevance to glucose as a source of energy and carbon for survival and proliferation. Of note, Nrf1 was shown to be essential for regulating glycolysis pathway, but it is unknown whether it plays a role in cancer metabolic reprogramming, particularly in response to glucose starvation. Herein, we discover that Nrf1α-/- hepatoma cells are sensitive to rapid death induced by glucose deprivation, such cell death appears to be rescued by Nrf2 interference, but HepG2 (wild-type, WT) or Nrf2-/- cells are roughly unaffected by glucose starvation. Further evidence revealed that Nrf1α-/- cell death is resulted from severe oxidative stress arising from aberrant redox metabolism. Strikingly, altered gluconeogenesis pathway was aggravated by glucose starvation of Nrf1α-/- cells, as also accompanied by weakened pentose phosphate pathway, dysfunction of serine-to-glutathione synthesis, and accumulation of reactive oxygen species (ROS) and damages, such that the intracellular GSH and NADPH were exhausted. These demonstrate that glucose starvation leads to acute death of Nrf1α-/- , rather than Nrf2-/- , cells resulting from its fatal defects in the redox metabolism reprogramming. This is owing to distinct requirements of Nrf1 and Nrf2 for regulating the constructive and inducible expression of key genes involved in redox metabolic reprogramming by glucose deprivation. Altogether, this work substantiates the preventive and therapeutic strategies against Nrf1α-deficient cancer by limiting its glucose and energy demands.

Nrf1 Is Endowed with a Dominant Tumor-Repressing Effect onto the Wnt/ß-Catenin-Dependent and Wnt/ß-Catenin-Independent Signaling Networks in the Human Liver Cancer.

Our previous work revealed that Nrf1α exerts a tumor-repressing effect because its genomic loss (to yield Nrf1α-/- ) results in oncogenic activation of Nrf2 and target genes. Interestingly, ß-catenin is concurrently activated by loss of Nrf1α in a way similar to ß-catenin-driven liver tumor. However, a presumable relationship between Nrf1 and ß-catenin is not yet established. Here, we demonstrate that Nrf1 enhanced ubiquitination of ß-catenin for targeting proteasomal degradation. Conversely, knockdown of Nrf1 by its short hairpin RNA (shNrf1) caused accumulation of ß-catenin so as to translocate the nucleus, allowing activation of a subset of Wnt/ß-catenin signaling responsive genes, which leads to the epithelial-mesenchymal transition (EMT) and related cellular processes. Such silencing of Nrf1 resulted in malgrowth of human hepatocellular carcinoma, along with malignant invasion and metastasis to the lung and liver in xenograft model mice. Further transcriptomic sequencing unraveled significant differences in the expression of both Wnt/ß-catenin-dependent and Wnt/ß-catenin-independent responsive genes implicated in the cell process, shape, and behavior of the shNrf1-expressing tumor. Notably, we identified that ß-catenin is not a target gene of Nrf1, but this CNC-bZIP factor contributes to differential or opposing expression of other critical genes, such as CDH1, Wnt5A, Wnt11A, FZD10, LEF1, TCF4, SMAD4, MMP9, PTEN, PI3K, JUN, and p53, each of which depends on the positioning of distinct cis-regulatory sequences (e.g., ARE and/or AP-1 binding sites) in the gene promoter contexts. In addition, altered expression profiles of some Wnt/ß-catenin signaling proteins were context dependent, as accompanied by decreased abundances of Nrf1 in the clinic human hepatomas with distinct differentiation. Together, these results corroborate the rationale that Nrf1 acts as a bona fide dominant tumor repressor, by its intrinsic inhibition of Wnt/ß-catenin signaling and relevant independent networks in cancer development and malignant progression.

Topovectorial mechanisms control the juxtamembrane proteolytic processing of Nrf1 to remove its N-terminal polypeptides during maturation of the CNC-bZIP factor.

The topobiological behaviour of Nrf1 dictates its post-translational modification and its ability to transactivate target genes. Here, we have elucidated that topovectorial mechanisms control the juxtamembrane processing of Nrf1 on the cyto/nucleoplasmic side of endoplasmic reticulum (ER), whereupon it is cleaved and degraded to remove various lengths of its N-terminal domain (NTD, also refolded into a UBL module) and acidic domain-1 (AD1) to yield multiple isoforms. Notably, an N-terminal ~12.5-kDa polypeptide of Nrf1 arises from selective cleavage at an NHB2-adjoining region within NTD, whilst other longer UBL-containing isoforms may arise from proteolytic processing of the protein within AD1 around PEST1 and Neh2L degrons. The susceptibility of Nrf1 to proteolysis is determined by dynamic repositioning of potential UBL-adjacent degrons and cleavage sites from the ER lumen through p97-driven retrotranslocation and -independent pathways into the cyto/nucleoplasm. These repositioned degrons and cleavage sites within NTD and AD1 of Nrf1 are coming into their bona fide functionality, thereby enabling it to be selectively processed by cytosolic DDI-1/2 proteases and also partiality degraded via 26S proteasomes. The resultant proteolytic processing of Nrf1 gives rise to a mature ~85-kDa CNC-bZIP transcription factor, which regulates transcriptional expression of cognate target genes. Furthermore, putative ubiquitination of Nrf1 is not a prerequisite necessary for involvement of p97 in the client processing. Overall, the regulated juxtamembrane proteolysis (RJP) of Nrf1, though occurring in close proximity to the ER, is distinctive from the mechanism that regulates the intramembrane proteolytic (RIP) processing of ATF6 and SREBP1.

Mechanisms controlling the multistage post-translational processing of endogenous Nrf1α/TCF11 proteins to yield distinct isoforms within the coupled positive and negative feedback circuits.

To gain a better understanding of the multistep processing of Nrf1 to yield various isoforms with confused molecular masses, we herein establish a generally acceptable criterion required for identification of its endogenous full-length proteins and derivative isoforms expressed differentially in distinct experimental cell lines. Further work has been focused on the molecular mechanisms that dictate the successive post-translational modifications (i.e. glycosylation by OST, deglycosylation by NGLY, and ubiquitination by Hrd1) of this CNC-bZIP protein and its proteolytic processing to give rise to multiple proteoforms. Several lines of experimental evidence have demonstrated that the nascent Nrf1α/TCF11 polypeptide (non-glycosylated) is transiently translocated into the endoplasmic reticulum (ER), in which it becomes an inactive glycoprotein-A, and is folded in a proper topology within and around membranes. Thereafter, dynamic repositioning of the ER-resident domains in Nrf1 glycoprotein is driven by p97-fueled retrotranslocation into extra-ER compartments. Therein, Nrf1 glycoprotein is allowed for deglycosylation digestion by glycosidases into a deglycoprotein-B and its progressive proteolytic processing by cytosolic DDI-1/2 and proteasomes so as to generate N-terminally-truncated protein-C/D. This processing is accompanied by removal of a major N-terminal ~12.5-kDa polypeptide from Nrf1α. Interestingly, our present study has further unraveled that there exist coupled positive and negative feedback circuits between Nrf1 and cognate target genes, including those encoding its regulators p97, Hrd1, DDI-1 and proteasomes. These key players are differentially or even oppositely involved in diverse cellular signaling responses to distinct extents of ER-derived proteotoxic and oxidative stresses induced by different concentrations of proteasomal inhibitors.

Nach Is a Novel Subgroup at an Early Evolutionary Stage of the CNC-bZIP Subfamily Transcription Factors from the Marine Bacteria to Humans.

Normal growth and development, as well as adaptive responses to various intracellular and environmental stresses, are tightly controlled by transcriptional networks. The evolutionarily conserved genomic sequences across species highlights the architecture of such certain regulatory elements. Among them, one of the most conserved transcription factors is the basic-region leucine zipper (bZIP) family. Herein, we have performed phylogenetic analysis of these bZIP proteins and found, to our surprise, that there exist a few homologous proteins of the family members Jun, Fos, ATF2, BATF, C/EBP and CNC (cap'n'collar) in either viruses or bacteria, albeit expansion and diversification of this bZIP superfamily have occurred in vertebrates from metazoan. Interestingly, a specific group of bZIP proteins is identified, designated Nach (Nrf and CNC homology), because of their strong conservation with all the known CNC and NF-E2 p45 subunit-related factors Nrf1 and Nrf2. Further experimental evidence has also been provided, revealing that Nach1 and Nach2 from the marine bacteria exert distinctive functions, when compared with human Nrf1 and Nrf2, in the transcriptional regulation of antioxidant response element (ARE)-battery genes. Collectively, further insights into these Nach/CNC-bZIP subfamily transcription factors provide a novel better understanding of distinct biological functions of these factors expressed in distinct species from the marine bacteria to humans.

Nrf1D Is the First Candidate Secretory Transcription Factor in the Blood Plasma, Its Precursor Existing as a Unique Redox-Sensitive Transmembrane CNC-bZIP Protein in Hemopoietic and Somatic Tissues.

Among multiple distinct isoforms, Nrf1D is synthesized from a de novo translation of an alternatively-spliced transcript of Nrf1 mRNA, as accompanied by a naturally-occurring deletion of its stop codon-flanking 1466 nucleotides. This molecular event leads to the generation of a reading frameshift mutation, which results in a constitutive substitution of the intact Nrf1's C-terminal 72 amino acids (aa, covering the second half of the leucine zipper motif to C-terminal Neh3L domain) by an additional extended 80-aa stretch to generate a unique variant Nrf1D. The C-terminal extra 80-aa region of Nrf1D was herein identified to be folded into a redox-sensitive transmembrane domain, enabling it to be tightly integrated within the endoplasmic reticulum (ER) membranes. Notably, the salient feature of Nrf1D enables it to be distinguishable from prototypic Nrf1, such that Nrf1D is endowed with a lesser ability than wild-type Nrf1 to mediate target gene expression. Further evidence has also been presented revealing that both mRNA and protein levels of Nrf1D, together with other isoforms similar to those of Nrf1, were detected to varying extents in hemopoietic and somatic tissues. Surprisingly, we found the existence of Nrf1D-derived isoforms in blood plasma, implying that it is a candidate secretory transcription factor, albeit its precursor acts as an integral transmembrane-bound CNC-bZIP protein that entails dynamic topologies across membranes, before being unleashed from the ER to enter the blood.

A Naturally-Occurring Dominant-Negative Inhibitor of Keap1 Competitively against Its Negative Regulation of Nrf2.

Transcription factor Nrf2 (nuclear factor-erythroid 2-related factor 2) is a master regulator of antioxidant and/or electrophile response elements (AREs/EpREs)-driven genes involved in homeostasis, detoxification, and adaptation to various stresses. The cytoprotective activity of Nrf2, though being oppositely involved in both cancer prevention and progression, is critically controlled by Keap1 (Kelch-like ECH-associated protein 1), which is an adaptor subunit of Cullin 3-based E3 ubiquitin ligase and also is a key sensor for oxidative and electrophilic stresses. Here, we first report a novel naturally-occurring mutant of Keap1, designated Keap1ΔC, which lacks most of its C-terminal Nrf2-interacting domain essential for inhibition of the cap'n'collar (CNC) basic-region leucine zipper (bZIP) factor. This mutant Keap1ΔC is yielded by translation from an alternatively mRNA-spliced variant lacking the fourth and fifth exons, but their coding sequences are retained in the wild-type Keap1 locus (with no genomic deletions). Although this variant was found primarily in the human highly-metastatic hepatoma (MHCC97H) cells, it was widely expressed at very lower levels in all other cell lines examined. Such Keap1ΔC retains no or less ability to inhibit Nrf2, so that it functions as a dominant-negative competitor of Keap1 against its inhibition of Nrf2 due to its antagonist effect on Keap1-mediated turnover of Nrf2 protein.

Salubrinal Enhances Doxorubicin Sensitivity in Human Cholangiocarcinoma Cells Through Promoting DNA Damage.

Cholangiocarcinoma (CCA) is a highly malignant and aggressive tumor of the bile duct that arises from epithelial cells. Chemotherapy is an important treatment strategy for CCA patients, but its efficacy remains limited due to drug resistance. Salubrinal, an inhibitor of eukaryotic translation initiation factor 2 alpha (eIF2α), has been reported to affect antitumor activities in cancer chemotherapy. In this study, the authors investigated the effect of salubrinal on the chemosensitivity of doxorubicin in CCA cells. They showed that doxorubicin induces CCA cell death in a dose- and time-dependent manner. Doxorubicin triggers reactive oxygen species (ROS) generation and induces DNA damage in CCA cells. In addition, ROS inhibitor N-acetylcysteine (NAC) pretreatment inhibits doxorubicin-induced CCA cell death. Importantly, these data demonstrate a synergistic death induction effect contributed by the combination of salubrinal and doxorubicin in CCA cells. It is notable that salubrinal promotes doxorubicin-induced ROS production and DNA damage in CCA cells. Taken together, these data suggest that salubrinal enhances the sensitivity of doxorubicin in CCA cells through promoting ROS-mediated DNA damage.

Compound C induces protective autophagy in human cholangiocarcinoma cells via Akt/mTOR-independent pathway.

Compound C, a well-known inhibitor of AMP-activated protein kinase (AMPK), has been reported to exert antitumor activities in some types of cells. Whether compound C can exert antitumor effects in human cholangiocarcinoma (CCA) remains unknown. Here, we demonstrated that compound C is a potent inducer of cell death and autophagy in human CCA cells. Autophagy inhibitors increased the cytotoxicity of compound C towards human CCA cells, as confirmed by increased LDH release, and PARP cleavage. It is notable that compound C treatment increased phosphorylated Akt, sustained high levels of phosphorylated p70S6K, and decreased mTOR regulated p-ULK1 (ser757). Based on the data that blocking PI3K/Akt or mTOR had no apparent influence on autophagic response, we suggest that compound C induces autophagy independent of Akt/mTOR signaling in human CCA cells. Further study demonstrated that compound C inhibited the phosphorylation of JNK and its target c-Jun. Blocking JNK by SP600125 or siRNA suppressed autophagy induction upon compound C treatment. Moreover, compound C induced p38 MAPK activation, and its inhibition promoted autophagy induction via JNK activation. In addition, compound C induced p53 expression, and its inhibition attenuated compound C-induced autophagic response. Thus, compound C triggers autophagy, at least in part, via the JNK and p53 pathways in human CCA cells. In conclusion, suppresses autophagy could increase compound C sensitivity in human CCA.