Melatonin regulates cancer migration and stemness and enhances the anti-tumour effect of cisplatin.

Melatonin, a lipophilic hormone released from the pineal gland, has oncostatic effects on various types of cancers. However, its cancer treatment potential needs to be improved by deciphering its corresponding mechanisms of action and optimising therapeutic strategy. In the present study, melatonin inhibited gastric cancer cell migration and soft agar colony formation. Magnetic-activated cell sorting was applied to isolate CD133+ cancer stem cells. Gene expression analysis showed that melatonin lowered the upregulation of LC3-II expression in CD133+ cells compared to CD133- cells. Several long non-coding RNAs and many components in the canonical Wnt signalling pathway were altered in melatonin-treated cells. In addition, knockdown of long non-coding RNA H19 enhanced the expression of pro-apoptotic genes, Bax and Bak, induced by melatonin treatment. Combinatorial treatment with melatonin and cisplatin was investigated to improve the applicability of melatonin as an anticancer therapy. Combinatorial treatment increased the apoptosis rate and induced G0/G1 cell cycle arrest. Melatonin can regulate migration and stemness in gastric cancer cells by modifying many signalling pathways. Combinatorial treatment with melatonin and cisplatin has the potential to improve the therapeutic efficacy of both.

WITHDRAWN: JUN and ATF3 are deficient in prostate cancer patients and their delivery in vivo via lipid nanoparticles has therapeutic efficacy by enhancing immune surveillance.

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Modulation of lncRNA H19 enhances resveratrol-inhibited cancer cell proliferation and migration by regulating endoplasmic reticulum stress.

The phytoalexin resveratrol exhibits anti-tumour activity in many types of cancer. In this study, we showed that resveratrol suppressed the survival of gastric tumour cells both in vivo and in vitro. Resveratrol promoted apoptosis, autophagy and endoplasmic reticulum (ER) stress in a dose-dependent manner. RNA-seq analysis showed that multiple cell death signalling pathways were activated after resveratrol treatment, while the use of ER stress activators (tunicamycin and thapsigargin) in combinatorial with resveratrol led to further inhibition of cancer cell survival. Results also showed that resveratrol altered the expression of several long non-coding RNAs (lncRNAs), including MEG3, PTTG3P, GAS5, BISPR, MALAT1 and H19. Knockdown of H19 in resveratrol-treated cells further enhanced the effects of resveratrol on apoptosis, ER stress and cell cycle S-phase arrest. Furthermore, the migratory ability of resveratrol-treated cells was dramatically decreased after H19 knockdown. In conclusion, resveratrol inhibited cancer cell survival, while knockdown of lncRNA H19 resulted in increased sensitivity to resveratrol therapy.

Developing metabolic gene signatures to predict intrahepatic cholangiocarcinoma prognosis and mining a miRNA regulatory network.

BACKGROUND: Metabolic disturbance is closely correlated with intrahepatic cholangiocarcinoma (IHCC), and we aimed to identify metabolic gene marker for the prognosis of IHCC. METHODS: We obtained expression and clinical data from 141 patients with IHCC from public databases. Prognostic metabolic genes were selected using univariate Cox regression analysis. Unsupervised cluster analysis was applied to identify IHCC subtypes, and CIBERSORT was used for immune infiltration analysis of different subtypes. Then, the metabolic gene signature was screened using multivariate Cox regression analysis and the LASSO algorithm. The prognostic potential and regulatory network of the metabolic gene signature were further investigated. RESULTS: We screened 228 prognosis-related metabolic genes. Based on their expression levels, IHCC samples were divided into two subtypes, which showed significant differences in survival and immune cell infiltration. After LASSO analysis, eight metabolic genes including CYP19A1, SCD5, ACOT8, SRD5A3, MOGAT2, PFKFB3, PPARGC1B, and RPL17 were identified as the optimal genes for the prognosis signature. The prognostic model had excellent predictive abilities, with areas under the receiver-operating characteristic curves over 0.8. A nomogram model was also established based on two independent prognostic clinical factors (pathologic stage and prognostic model), and the generated calibration curves and c-indexes determined its excellent accuracy and discriminative ability to predict 1- and 5-year survival status (c-indexes>0.7). Finally, we found that miR-26a-5p, miR-27a-3p, and miR-27b-3p were the upstream regulators that mediate the involvement of gene signatures in metabolic pathways. CONCLUSION: We developed eight metabolic gene signatures to predict IHCC prognosis and proposed potential upstream regulatory axes of gene signatures.

Melatonin inhibiting the survival of human gastric cancer cells under ER stress involving autophagy and Ras-Raf-MAPK signalling.

Melatonin exhibits antitumour activities in the treatment of many human cancers. In the present study, we aimed to improve the therapeutic potential of melatonin in gastric cancer. Our results confirmed that melatonin dose-dependently suppressed the proliferation and necrosis, and increased G0/G1 phase arrest, apoptosis, autophagy and endoplasmic reticulum (ER) stress. The Ras-Raf-MAPK signalling pathway was activated in cells after melatonin treatment. RNA-seq was performed and GSEA analysis further confirmed that many down-regulated genes in melatonin-treated cells were associated with proliferation. However, GSEA analysis also indicated that many pathways related to metastasis were increased after melatonin treatment. Subsequently, combinatorial treatment was conducted to further investigate the therapeutic outcomes of melatonin. A combination of melatonin and thapsigargin increased the apoptotic rate and G0/G1 cell cycle arrest when compared to treatment with melatonin alone. Melatonin in combination with thapsigargin triggered the increased expression of Bip, LC3-II, phospho-Erk1/2 and phospho-p38 MAPK. In addition, STF-083010, an IRE1a inhibitor, further exacerbated the decrease in survival rate induced by combinatorial treatment with melatonin and thapsigargin. Collectively, melatonin was effective in gastric cancer treatment by modifying ER stress.

lncRNA MALAT1 participates in metformin inhibiting the proliferation of breast cancer cell.

In recent years, the repurposing of conventional and chemotherapeutic drugs is recognized as an alternative strategy for health care. The main purpose of this study is to strengthen the application of non-oncological drug metformin on breast cancer treatment in the perspective of epigenetics. In the present study, metformin was found to inhibit cell proliferation, promote apoptosis and induce cell cycle arrest in breast cancer cells at a dose-dependent manner. In addition, metformin treatment elevated acH3K9 abundance and decreased acH3K18 level. The expression of lncRNA MALAT1, HOTAIR, DICER1-AS1, LINC01121 and TUG1 was up-regulated by metformin treatment. In metformin-treated cells, MALAT1 knock-down increased the Bax/Bcl2 ratio and enhanced p21 but decreased cyclin B1 expression. The expression of Beclin1, VDAC1, LC3-II, CHOP and Bip was promoted in the cells received combinatorial treatment of metformin and MALAT1 knock-down. The reduced phosphorylation of c-Myc was further decreased in the metformin-treated cells in combination with MALAT1 knock-down than metformin treatment alone. Taken together, these results provide a promising repurposed strategy for metformin on cancer treatment by modulating epigenetic modifiers.

B-Cell Receptor-Associated Protein 31 Regulates the Expression of Valosin-Containing Protein Through Elf2.

BACKGROUND/AIMS: B-cell receptor-associated protein 31 (Bap31) is an evolutionarily conserved, ubiquitously expressed, polytopic integral membrane protein in the endoplasmic reticulum (ER) that is involved in the regulation of apoptosis, protein transport and degradation. Patients with Bap31 mutations exhibit symptoms similar to those exhibited by patients with central nervous system (CNS) diseases, such as deafness, dystonia, and intellectual disability. The present study aimed to investigate the function of Bap31 in CNS diseases by identifying a CNS disease-related gene regulated by Bap31 and exploring the underlying molecular mechanism. METHODS: ShRNA-Bap31 and siRNA-Bap31 were used to knockdown Bap31 in N2a cells, and real-time PCR was performed to detect the mRNA levels of genes involved in CNS diseases. Western blot analyses were used to examine the protein levels of the candidate gene (valosin-containing protein, VCP) both in vivo and in vitro. The functions of Bap31 and VCP in mediating the degradation of the hyper-unstable mutant of cystic fibrosis trans-membrane conductance regulator (CFTRΔF508) were studied. Moreover, real-time PCR, Western blot and dual luciferase reporter analyses were conducted to investigate the molecular mechanism by which Bap31 regulates the expression levels of VCP. RESULTS: VCP was identified as a candidate gene based on its differential expression in N2a cells following both shRNA- and siRNA-mediated knockdown of Bap31. Both the mRNA and protein levels of VCP were regulated by Bap31 in vivo and in vitro. In the ER-associated degradation (ERAD) pathway, Bap31 also regulated VCP expression and caused differences in the binding quantities of CFTRΔF508 and VCP. Furthermore, a transcription factor of VCP (E74-like factor 2, Elf2) was regulated by Bap31, and Elf2 mediated the changes in VCP transcription and expression in cells with altered Bap31 expression. CONCLUSION: These results indicate that Bap31 regulates the expression of VCP possibly via Elf2 and support the potential molecular function of Bap31 in CNS diseases.

Barriers for Deriving Transgene-Free Pig iPS Cells with Episomal Vectors.

To date no authentic embryonic stem cell (ESC) line or germline-competent-induced pluripotent stem cell (iPSC) line has been established for large animals. Despite this fact, there is an impression in the field that large animal ESCs or iPSCs are as good as mouse counterparts. Clarification of this issue is important for a healthy advancement of the stem cell field. Elucidation of the causes of this failure in obtaining high quality iPSCs/ESCs may offer essential clues for eventual establishment of authentic ESCs for large animals including humans. To this end, we first generated porcine iPSCs using nonintegrating replicating episomal plasmids. Although these porcine iPSCs met most pluripotency criteria, they could neither generate cloned piglets through nuclear transfer, nor contribute to later stage chimeras through morula injections or aggregations. We found that the reprogramming genes in iPSCs could not be removed even under negative selection, indicating they are required to maintain self-renewal. The persistent expression of these genes in porcine iPSCs in turn caused differentiation defects in vivo. Therefore, incomplete reprogramming manifested by a reliance on sustained expression of exogenous-reprogramming factors appears to be the main reason for the inability of porcine iPSCs to form iPSC-derived piglets.

Valproic Acid Improves Porcine Parthenogenetic Embryo Development Through Transient Remodeling of Histone Modifiers.

BACKGROUND/AIMS: Parthenogenetic embryos are useful in many applications, such as being an alternative source of embryonic stem cells that would avoid ethical problems. Aberrance in epigenetic reprogramming is considered the major reason for the developmental failure of parthenogenetic embryos. Many histone deacetylase inhibitors have been shown to improve the reprogramming of stem cells and embryos. Here, the relationship between histone modification and parthenogenetic embryonic development was explored. METHODS: Valproic acid (VPA) treatment was applied during the culture of parthenogenetic embryos. The abundance of histone modifiers was examined by immunofluorescence and quantified by Image-pro software. RESULTS: The acH3K9 level in in vitro fertilized embryos was significantly higher than parthenogenetic embryos. VPA treatment improved both the blastocyst formation rate and the acH3K9 level in parthenogenetic embryos. The signal intensities of acH4K5 and H3K4me2 were also enhanced in VPA treated embryos. The H3K27me2 level was decreased in the VPA treated embryos at the 2-cell stage. However, the enhancement in the acH3K9, acH4K5 and H3K4me2 level, or the decrease in the H3K27me2 level disappeared shortly after VPA withdrawal. CONCLUSION: Optimizing histone modifications for a short time following activation was sufficient to enhance the in vitro development of parthenogenetic embryos.

Expression of Cre recombinase in alveolar epithelial cells of the AQP2-Cre transgenic mini-pigs.

BACKGROUND/AIMS: Optimal use of Cre mediated recombination in conditional animal models depends on well characterized Cre driver lines. Unfortunately, some Cre driver lines exhibit unexpected expression patterns hindering their utility in Cre/loxP systems. Thus, systematic assessment of new Cre lines is essential for generating useful Cre driver lines for future studies. METHODS: Here, we describe a Cre Transgenic (Tg) mini-pig line in which the expression of Cre is directed by a 3-kb 5' fragment of the kidney-specific aquaporin 2 (AQP2); however, the AQP2-Cre Tg mini-pig line exhibits expression of Cre in alveolar epithelial cells (AECs) instead of collecting duct cells. The specificity of the AQP2-Cre plasmid was validated in vitro, and indicating that the AQP2-Cre was specifically expressed in the transfected LLC-PK1 cells. Absolute quantitative real-time PCR (qRT-PCR) and inverse PCR were performed to determine the copy numbers and integration sites of the AQP2-Cre transgene. Relative qRT-PCR was performed to evaluate variation in Cre expression levels over time. RESULTS: Our data indicated that this AQP2-Cre Tg mini-pig line exhibits stable expression of Cre recombinase over time and in subsequent generations, even though the AQP2-Cre transgene was segregated and reduced in subsequent generations. CONCLUSION: Combined with our previous studies of the activity of this Cre, we conclude that this Cre Tg mini-pig line will provide a reliable tool for generating lung-specific gene targeting mini-pig models, thereby allowing the investigation of gene functions in lung development and studying the molecular mechanisms of human lung disease.

Generation of AQP2-Cre transgenic mini-pigs specifically expressing Cre recombinase in kidney collecting duct cells.

The important differences in physiological parameters and anatomical characteristics of the kidney between humans and mice make it difficult to replicate the precise progression of human renal cystic diseases in gene modification mouse models. In contrast to mice, pigs are a better animal model of human diseases, as they are more similar in terms of organ size, structure, and physiological parameters. Here, we report the generation and initial examination of an AQP2-Cre transgenic (Tg) Chinese miniature (mini)-pig line that expresses Cre recombinase exclusively in kidney collecting duct cells. An 8-kb fragment of the mini-pig aquaporin 2 (AQP2) 5'-flanking region was utilized to direct Cre expression in Tg mini-pigs. Two Tg mini-pigs were generated by pig somatic cell nuclear transfer and both carried the entire coding sequence of Cre recombinase. RT-PCR and western blotting analysis revealed that Cre recombinase was uniquely expressed in the kidney, while immunohistochemical studies located its expression in kidney collecting duct cells. Furthermore, six integration sites and 12-14 copies of the Cre gene were detected in various tissues by high-efficiency thermal asymmetric interlaced PCR and absolute quantitative real-time PCR, respectively. Combined with previous studies of Cre recombinase activity, we believe that this AQP2-Cre Tg mini-pig line will be a useful tool to generate kidney collecting duct cell-specific gene knockout mini-pig models, thereby allowing the investigation of gene functions in kidney development and the mechanisms of human renal cystic disease.

Genomic imprinting analysis of Igf2/H19 in porcine cloned fetuses using parthenogenetic somatic cells as nuclear donors.

To gain insight into parthenogenesis in pigs, we report for the first time that using parthenogenetic somatic cells as nuclear donors (PSCNT), the porcine parthenogenetic fetus can develop to gestational day 39. Weight and morphological analysis revealed that PSCNT fetuses were smaller and developmentally retarded when compared to normally fertilized controls. Quantitative gene expression analysis indicated that in PSCNT fetuses, H19 was over-expressed, whereas Igf2 was significantly reduced (p < 0.05) compared with their controls. In addition, bisulfite-sequencing PCR results demonstrated that H19 differentially DNA methylated regions (DMRs) were hypomethylated in PSCNT fetuses, while Igf2 DMRs were hypermethylated in both PSCNT and control fetuses. Our results suggest that extended development of the porcine parthenogenetic fetus can be accomplished using PSCNT and that abnormal DNA methylation of H19 DMRs might contribute to the critical barrier of parthenogenesis in pigs.

Elevated expression of vascular endothelial growth factor (VEGF) 120 in parthenogenetic porcine placentas.

Most mammalian parthenogenetic embryos are unable to develop to term due to placental defects, potentially caused by decreased vasculogenesis and angiogenesis of the parthenogenetic placenta. Here we have compared the expression status of vascular endothelial growth factor (VEGF) and angiopoietin family members between normally developing and parthenogenetic porcine placentas. The result showed significantly reduced expression of these genes but elevated expression of VEGF 120 in the parthenogenetic porcine placenta (p < 0.05). We postulate that the abnormal expression levels of VEGF and angiopoietin family members and, especially, the elevated expression of VEGF 120 observed in parthenogenetic porcine placentas are related to the early miscarriage of parthenogenetic embryos in pigs.

Pluripotent-related gene expression analyses in single porcine recloned embryo.

The developmental ability among embryos produced by three different techniques were examined: there were no significant differences in the developmental rate in porcine embryos produced by in vitro fertilization (IVF) and first generation of somatic cell nucleus transfer (SCNT), but the developmental rate dropped sharply at the 2- to four-cell stage in recloned (second generation of SCNT) embryos. In most recloned embryos, Oct4 and Klf4 were under-expressed at all stages, whereas Sox2 and Nanog were over-expressed at the two-cell stage. In contrast, Nanog was absent in IVF and SCNT embryos at the two-cell stage. The recloned embryos were treated with valproic acid to enhance developmental capacity and this led to an increase in the rate of blastocyst formation and total cell number compared with the findings for untreated recloned embryos (29.8 vs. 12.4 %, 39 vs. 25, respectively, p < 0.05).

Aberrant expression of Xist in aborted porcine fetuses derived from somatic cell nuclear transfer embryos.

Cloned pigs generated by somatic cell nuclear transfer (SCNT) show a greater ratio of early abortion during mid-gestation than normal controls. X-linked genes have been demonstrated to be important for the development of cloned embryos. To determine the relationship between the expression of X-linked genes and abortion of cloned porcine fetuses, the expression of X-linked genes were investigated by quantitative real-time polymerase chain reaction (q-PCR) and the methylation status of Xist DMR was performed by bisulfate-specific PCR (BSP). q-PCR analysis indicated that there was aberrant expression of X-linked genes, especially the upregulated expression of Xist in both female and male aborted fetuses compared to control fetuses. Results of BSP suggested that hypomethylation of Xist occurred in aborted fetuses, whether male or female. These results suggest that the abnormal expression of Xist may be associated with the abortion of fetuses derived from somatic cell nuclear transfer embryos.

Engineered CARD11-PIK3R3 T-cell therapies as weapons of cancer mass destruction.

Garcia et al. discover a novel immunotherapy approach by engineering naturally occurring mutations in therapeutic T cells to strongly elevate anti-tumor activity. The authors identify a gene fusion, CARD11-PIK3R3, to increase activator protein 1 and nuclear factor-κB signaling, interleukin-2 production, and tumor death in vitro and in vivo .

Liquid-liquid phase separation in diseases.

Liquid-liquid phase separation (LLPS), an emerging biophysical phenomenon, can sequester molecules to implement physiological and pathological functions. LLPS implements the assembly of numerous membraneless chambers, including stress granules and P-bodies, containing RNA and protein. RNA-RNA and RNA-protein interactions play a critical role in LLPS. Scaffolding proteins, through multivalent interactions and external factors, support protein-RNA interaction networks to form condensates involved in a variety of diseases, particularly neurodegenerative diseases and cancer. Modulating LLPS phenomenon in multiple pathogenic proteins for the treatment of neurodegenerative diseases and cancer could present a promising direction, though recent advances in this area are limited. Here, we summarize in detail the complexity of LLPS in constructing signaling pathways and highlight the role of LLPS in neurodegenerative diseases and cancers. We also explore RNA modifications on LLPS to alter diseases progression because these modifications can influence LLPS of certain proteins or the formation of stress granules, and discuss the possibility of proper manipulation of LLPS process to restore cellular homeostasis or develop therapeutic drugs for the eradication of diseases. This review attempts to discuss potential therapeutic opportunities by elaborating on the connection between LLPS, RNA modification, and their roles in diseases.

An unresectable and metastatic intrahepatic cholangiocarcinoma with EML4-ALK rearrangement achieving partial response after first-line treatment with ensartinib: a case report.

Systemic chemotherapies are the primary treatment options for patients with unresectable and metastatic intrahepatic cholangiocarcinoma (ICC), but the effectiveness of current systemic therapies is limited. The development of targeted-therapy has changed the treatment landscape of ICC, and comprehensive genome sequencing of advanced cholangiocarcinoma patients could be beneficial to identify potential targets to guide individualized treatment. Herein, we reported an unresectable and metastatic ICC patient who detected EML4-ALK rearrangement in peripheral blood, which was later confirmed on tissue-based testing, and achieved partial response (PR) after first-line treatment with ensartinib. This case suggests that the liquid biopsy is of clinical value for unresectable or metastatic ICC, and the discovery of rare molecular targets provides new therapeutically approaches for advanced ICC patients.

Isolation and culture of embryonic stem-like cells from pig nuclear transfer blastocysts of different days.

This study was conducted to establish pig embryonic stem (ES)-like cell lines from nuclear transfer blastocysts. A green fluorescent protein (GFP)-expressing cell line was used as the source of donor cells injected into the enucleated oocytes. Blastocysts were collected at D5 (the fifth day), D7 (the seventh day) and D9 (the ninth day). Differential staining was used to assay the viability and development of blastocysts from the 3 days. The number of inner cell mass (ICM) cells increased from 1.83 ± 0.8 (D5) to 5.37 ± 1.2 (D7) to 7.56 ± 1.5 (D9). The expression profiles of embryonic stem (ES) cell factors (OCT4, SOX2, KLF4 and c-MYC) correlated best with the undifferentiated ES state and were identified by qPCR. The expression of the four factors was increased from D5 to D7, whereas the expression decreased from D7 to D9. We tried to isolate ES-like cells from these embryos. However, ES-like cells from the D7 blastocysts grew slowly and expressed alkaline phosphatase. The cells from the D9 blastocysts grew rapidly but did not express alkaline phosphatase. ES-like cells were not isolated from the D5 blastocysts. These results show that the cells from the D7 embryos are pluripotent but grow slowly. The cells from the D9 embryos grow rapidly but start to lose pluripotency.

Remodeling tumor microenvironment with natural products to overcome drug resistance.

With cancer incidence rates continuing to increase and occurrence of resistance in drug treatment, there is a pressing demand to find safer and more effective anticancer strategy for cancer patients. Natural products, have the advantage of low toxicity and multiple action targets, are always used in the treatment of cancer prevention in early stage and cancer supplement in late stage. Tumor microenvironment is necessary for cancer cells to survive and progression, and immune activation is a vital means for the tumor microenvironment to eliminate cancer cells. A number of studies have found that various natural products could target and regulate immune cells such as T cells, macrophages, mast cells as well as inflammatory cytokines in the tumor microenvironment. Natural products tuning the tumor microenvironment via various mechanisms to activate the immune response have immeasurable potential for cancer immunotherapy. In this review, it highlights the research findings related to natural products regulating immune responses against cancer, especially reveals the possibility of utilizing natural products to remodel the tumor microenvironment to overcome drug resistance.