Molecular mechanism of vimentin nuclear localization associated with the migration and invasion of daughter cells derived from polyploid giant cancer cells.

BACKGROUND: Polyploid giant cancer cells (PGCCs), a specific type of cancer stem cells (CSCs), can be induced by hypoxic microenvironments, chemical reagents, radiotherapy, and Chinese herbal medicine. Moreover, PGCCs can produce daughter cells that undergo epithelial-mesenchymal transition, which leads to cancer recurrence and disseminated metastasis. Vimentin, a mesenchymal cell marker, is highly expressed in PGCCs and their daughter cells (PDCs) and drives migratory persistence. This study explored the molecular mechanisms by which vimentin synergistically regulates PGCCs to generate daughter cells with enhanced invasive and metastatic properties. METHODS: Arsenic trioxide (ATO) was used to induce the formation of PGCCs in Hct116 and LoVo cells. Immunocytochemical and immunohistochemical assays were performed to determine the subcellular localization of vimentin. Cell function assays were performed to compare the invasive metastatic abilities of the PDCs and control cells. The molecular mechanisms underlying vimentin expression and nuclear translocation were investigated by real-time polymerase chain reaction, western blotting, cell function assays, cell transfection, co-immunoprecipitation, and chromatin immunoprecipitation, followed by sequencing. Finally, animal xenograft experiments and clinical colorectal cancer samples were used to study vimentin expression in tumor tissues. RESULTS: Daughter cells derived from PGCCs showed strong proliferative, migratory, and invasive abilities, in which vimentin was highly expressed and located in both the cytoplasm and nucleus. Vimentin undergoes small ubiquitin-like modification (SUMOylation) by interacting with SUMO1 and SUMO2/3, which are associated with nuclear translocation. P62 regulates nuclear translocation of vimentin by controlling SUMO1 and SUMO2/3 expression. In the nucleus, vimentin acts as a transcription factor that regulates CDC42, cathepsin B, and cathepsin D to promote PDC invasion and migration. Furthermore, animal experiments and human colorectal cancer specimens have confirmed the nuclear translocation of vimentin. CONCLUSION: P62-dependent SUMOylation of vimentin plays an important role in PDC migration and invasion. Vimentin nuclear translocation and overexpressed P62 of cancer cells may be used to predict patient prognosis, and targeting vimentin nuclear translocation may be a promising therapeutic strategy for metastatic cancers.

Cdc42 Regulates the Expression of Cytoskeleton and Microtubule Network Proteins to Promote Invasion and Metastasis of Progeny Cells Derived from CoCl2-induced Polyploid Giant Cancer Cells.

Purpose: Our previous studies have shown that CoCl2 can induce the formation of polyploid giant cancer cells (PGCCs) and PGCCs could produce progeny cells via asymmetric division. In this study, the molecular mechanism by which PGCCs generate progeny cells with high invasion and migration abilities was explored. Methods: In this study, PGCCs induced by CoCl2 produced progeny cells via asymmetric division, which was observed dynamically using laser scanning confocal microscopy. Cell cycle in LoVo and Hct116 before and after CoCl2 treatment was analyzed by flow cytometry. Cell function experiments, co-immunoprecipitation, mass spectrometry analysis, ML141 treatment, western blotting, and siRNA transfection experiments were used to demonstrate that Cdc42/PAK1 was involved in the regulation of cytoskeleton expression. The proliferation, migration, and invasion abilities of PGCCs and progeny cells were compared in PGCCs and progeny cells with and without inhibiting the expression of Cdc42 and PAK1. Results: G2/M phase arrest appeared in CoCl2-treated LoVo and Hct116 cells. After CoCl2 treatment, an increased expression of Cdc42 and PAK1 led to a decrease in the expression of stathmin and an increase in the expression of phosphorylated stathmin, which is located in the nucleus of PGCCs and progeny cells. PTPN14 negatively regulates the expression of PAK1 and p38MAPK. Low levels of PTPN14 expression, a downstream regulatory protein of stathmin, endows progeny tumor cells generated by PGCCs with the ability to invade and metastasize. The expression of PKA1α, cathepsin B, and D increased in CoCl2-treated cells compared with that in the control cells, associated with the infiltration and migration of PGCCs with their progeny cells. Conclusion: CoCl2-induced overexpression of Cdc42 plays a critical role in increasing the infiltration and migration abilities of PGCCs and progeny cells by regulating cytoskeleton protein expression.

Clinicopathological Significances and Prognostic Value of PPFIA4 in Colorectal Cancer.

Purpose: The PPFIA gene family (PPFIA1, PPFIA2, PPFIA3, and PPFIA4) is associated with multiple human diseases, particularly malignant tumors. However, the expression and prognostic value of the PPFIA family in human colorectal cancers (CRCs) have not been reported. Materials and methods: In this study, several databases, including Oncomine, UALCAN, and the cancer cell line encyclopedia, were used to compare differences in PPFIA1, PPFIA2, PPFIA3, and PPFIA4 expression between normal colon samples and CRCs. The expression levels of these four proteins were used to evaluate the survival of patients with CRC, as determined by the Cancer Genome Atlas Program (TCGA) portal and gene expression profiling interactive analysis (GEPIA) databases. Western blotting and reverse transcription-polymerase chain reaction were performed to detect protein and mRNA levels of PPFIA1, PPFIA3, and PPFIA4, respectively. Immunohistochemical (IHC) staining was used to detect the correlation between PPFIA4 expression and the degree of CRC malignancy. Furthermore, potential miRNAs targeting PPFIA4 in CRCs were studied and confirmed. Results: Bioinformatic analysis showed that the mRNA levels of PPFIA1, PPFIA3, and PPFIA4 were higher in CRC tissue samples than in normal colon tissue. Both mRNA and protein expression of PPFIA1, PPFIA3, and PPFIA4 were increased in the CRC cell lines LoVo and Hct116 compared with the normal colon epithelial cell line. Only PPFIA4 was associated with the prognosis of patients with CRC, which was confirmed by TCGA portal and GEPIA. IHC staining confirmed that the expression of PPFIA4 was higher in CRC tissues than in normal colon tissues and also increased in poorly differentiated CRC tissues and lymph node metastatic foci in comparison with well-differentiated CRC tissues and moderately differentiated CRC tissues. Functional annotation enrichment analysis indicated that the top 100 genes co-expressed with PPFIA4 were enriched in the G-protein coupled peptide receptor activity, leukotrience B4 receptor activity, and peroxisome proliferator-activated receptors and hypoxia-inducible factor-1 signaling pathways. In addition, miR-485-5p negatively regulates the expression of PPFIA4. Conclusion: PPFIA4 expression is associated with the development of CRCs and may be a novel potential prognostic marker for human CRCs.

A rational foundation for micheliolide-based combination strategy by targeting redox and metabolic circuit in cancer cells.

Accumulating evidence has supported that targeting oxidative stress and metabolic alterations of cancer is an effective strategy to combat cancer. We previously reported that Dimethylaminomicheliolide (DMAMCL) and its active metabolite micheliolide (MCL) can cause oxidative stress and cell death in leukemia and glioblastoma. However, the detailed mechanism underlying MCL or DMAMCL triggered oxidative stress remains elusive. Herein, using leukemia HL60 cells and glioblastoma U118MG cells as models, we found that MCL-induced oxidative stress is mainly mediated by reduced glutathione (GSH). Overproduced reactive oxygen species (ROS) can lead to oxidative damage to mitochondrial, impairing the ability of the tricarboxylic acid (TCA) cycle and causing dysfunction of mitochondrial respiratory chain. On the other hand, the depletion of GSH activates GSH biosynthesis pathway and has possibility to give rise to more GSH to scavenge ROS in cancer cells. Targeting this redox and metabolic circuit, we identified L-buthionine sulfoximine (BSO), an inhibitor in GSH biosynthesis, as an agent that can enhance MCL regimen to inhibit GSH compensatory event and thereby further facilitate cancer cell oxidative stress. Together, these results illustrate that targeting redox and metabolic pathway by MCL/DMAMCL combination with BSO is a potent therapeutic intervention for the treatments of glioblastoma and acute-myelocytic leukemia.

Laparoscopic Pelvic Exenteration with Bladder Sparing for Men with Locally Advanced Rectal Cancer (with Video).

OBJECTIVE: The aim of this article was to introduce a fascial space priority approach for laparoscopic pelvic exenteration (PE) with bladder-sparing for men with locally advanced rectal cancer. METHODS: We present a video of bladder-sparing laparoscopic PE with fascial space priority approach in a 70-year old man. The systematic de-arterialization of the prostate on the basis of complete separation of the avascular lateral pelvic spaces is introduced in detail. RESULTS: The operation time was 360 min and the estimated intraoperative blood loss was 50 mL. The postoperative course was uneventful and the patient was discharged on postoperative day 14. Histopathological examination showed all margins to be tumor-free. CONCLUSIONS: Bladder-sparing laparoscopic PE using a fascial space priority approach is a feasible and safe procedure that can be performed in well-selected patients following neoadjuvant chemoradiotherapy. Extensive multivisceral resection is possible without a permanent stoma.

An Improved NGS Library Construction Approach Using DNA Isolated from Human Cancer Formalin-Fixed Paraffin-Embedded Samples.

Identification of genomic alterations from formalin-fixed paraffin-embedded (FFPE) samples using next-generation sequencing (NGS) is very important for cancer-targeted therapy today. To achieve a higher efficiency and shorter turn-around time for NGS library preparation, here, we compared NGS library preparation processes and outcomes with three commercial library construction methods and two hybridization capture methods thus, developed an improved NGS library construction approach. This improved approach took advantage of both methods and resulted in a higher output from the same input DNA, including higher library construction success rate, higher probe capture rate, and shorter turn-around time. Using this approach, targeted region libraries could be constructed within only 1 day for FFPE samples; therefore, this approach has potential applications of NGS in routine clinical tests. Anat Rec, 302:941-946, 2019. © 2018 Wiley Periodicals, Inc.

SMAD7 rs4939827 variant contributes to colorectal cancer risk in Chinese population.

A genome-wide association study identified a common genetic variant rs4939827 at 18q21 in SMAD7 to be related with colorectal cancer (CRC) risk with OR=1.2 and P =7.80E-28. Until recently, several meta-analysis studies have been conducted, and reported significant association between rs4939827 and CRC risk. However none of these studies evaluated the potential association between rs4939827 and CRC risk in Chinese population. In this study, we evaluated this association by a meta-analysis using 12077 samples including 5816 CRC cases and 6261 controls. In the end, we identified the T allele of rs4939827 to be significantly related with an increase CRC risk (P=2.22E-05, OR=1.14, 95% CI 1.07-1.21) in Chinese population.

miR-659-3p is involved in the regulation of the chemotherapy response of colorectal cancer via modulating the expression of SPHK1.

Colorectal cancer (CRC) is one of most prevalent malignant diseases worldwide. Metastasis and chemo-resistance are the two prominent death-related factors of CRCs. Thus, it is urgent to understand the mechanism responsible for the chemo-resistant properties of CRC and develop new therapeutic methods. Here, we found that the expression of miR-659-3p was significantly reduced in cisplatin (CDDP)-resistant HT29 and LOVO colorectal cancer cells and in CDDP-resistant clinical colorectal cancer samples compared with respective CDDP-sensitive counterparts. Sphingosine kinase 1 (SPHK1) is a direct target of miR-659-3p in colorectal cancer cells, and it is negatively regulated by miR-659-3p. We found that anti-miR-659-3p could increase the IC50 of CDDP in parental HT29 and LOVO colorectal cancer cells; additionally, miR-659-3p mimics decreased the IC50 of CDDP in HT29/CDDP and LOVO/CDDP colorectal cancer cells. Furthermore, we showed that the miR-659-3p/SPHK1 pathway was involved in the regulation of chemotherapy responses of colorectal cancer cells in vivo. In all, our findings suggest a new mechanism involved in the regulation of the chemotherapy response of CRC and might provide new targets for CRC prevention and treatment.

A Meta-analysis on the Effect of Ulinastatin on Serum Levels of C-Reactive Protein, Interleukin 6, and Tumor Necrosis Factor Alpha in Asian Patients with Acute Pancreatitis.

OBJECTIVES: We aimed to investigate the influence of ulinastatin (UTI) on the serum levels of C-reactive protein (CRP), interleukin 6 (IL-6), and tumor necrosis factor alpha (TNF-α) in Asian patients with acute pancreatitis (AP) by performance of a meta-analysis. METHODS: Two investigators independently searched 11 databases, including PUBMED, EBSCO, Ovid, SpringerLink, Wiley, Web of Science, Cochrane Library, Wanfang database, China National Knowledge Infrastructure (CNKI), Chinese Journal Full-text Database, and China Biomedicine Database. The full-text articles were screened and the data were extracted using a standardized data extraction form. All statistical analyses were conducted with Stata software, version 12.0 (Stata Corporation, College Station, TX). RESULTS: A total of 94 studies were initially retrieved, and 10 studies containing 424 Asian patients with AP were ultimately enrolled in this meta-analysis. The results revealed that the serum levels of CRP, IL-6, and TNF-α in Asian AP patients significantly decreased after UTI therapy (CRP: standardized mean difference [SMD] = 3.26, 95% confidence interval [CI] = 1.69-4.83, p < 0.001; IL-6: SMD = 5.92, 95% CI = 2.09-9.75, p = 0.002; TNF-α: SMD = 4.07, 95% CI = 0.79-7.35, p = 0.015). CONCLUSION: The results of this meta-analysis suggest that UTI can effectively depress the serum levels of CRP, IL-6, and TNF-α in Asian patients with AP, and thereby inhibit inflammation.

Combination therapy with molecular hydrogen and hyperoxia in a murine model of polymicrobial sepsis.

Sepsis is the most common cause of death in intensive care units. Some studies have found that hyperoxia may be beneficial to sepsis. However, the clinical use of hyperoxia is hindered by concerns that it could exacerbate organ injury by increasing free radical formation. Recently, it has been suggested that molecular hydrogen (H2) at low concentration can exert a therapeutic antioxidant activity and effectively protect against sepsis by reducing oxidative stress. Therefore, we hypothesized that combination therapy with H2 and hyperoxia might afford more potent therapeutic strategies for sepsis. In the present study, we found that inhalation of H2 (2%) or hyperoxia (98%) alone improved the 14-day survival rate of septic mice with moderate cecal ligation and puncture (CLP) from 40% to 80% or 70%, respectively. However, combination therapy with H2 and hyperoxia could increase the 14-day survival rate of moderate CLP mice to 100% and improve the 7-day survival rate of severe CLP mice from 0% to 70%. Moreover, moderate CLP mice showed significant organ damage characterized by the increases in lung myeloperoxidase activity, lung wet-to-dry weight ratio, protein concentration in bronchoalveolar lavage, serum biochemical parameters (alanine aminotransferase, aspartate aminotransferase, creatinine, and blood urea nitrogen), and organ histopathological scores (lung, liver, and kidney), as well as the decrease in PaO2/FIO2 ratio at 24 h, which was attenuated by either H2 or hyperoxia alone. However, combination therapy with H2 and hyperoxia had a more beneficial effect against lung, liver, and kidney damage of moderate or severe CLP mice. Furthermore, we found that the beneficial effect of this combination therapy was associated with the decreased levels of oxidative product (8-iso-prostaglandin F2α), increased activities of antioxidant enzymes (superoxide dismutase and catalase) and anti-inflammatory cytokine (interleukin 10), and reduced levels of proinflammatory cytokines (high-mobility group box 1 and tumor necrosis factor α) in serum and tissues. Therefore, combination therapy with H2 and hyperoxia provides enhanced therapeutic efficacy via both antioxidant and anti-inflammatory mechanisms and might be potentially a clinically feasible approach for sepsis.

Modulation of the number and functions of endothelial progenitor cells by interleukin 1ß in the peripheral blood of pigs: involvement of p38 mitogen-activated protein kinase signaling in vitro.

BACKGROUND: Endothelial progenitor cells (EPCs) have therapeutic potential for the treatment of organ ischemia following trauma or sepsis, frequently associated with inflammatory conditions. We aimed to investigate the effects of interleukin 1ß (IL-1ß) on the properties of EPCs and explore its possible relationship with p38 mitogen-activated protein kinase (MAPK). METHODS: EPCs were isolated from peripheral blood of a porcine model and were characterized. Effects of IL-1ß on cell number, proliferation, migration, adhesion, and angiogenic function of EPCs were evaluated in a time- and dose-dependent manner. The activity of p38 MAPK in EPCs was measured by Western blot. Moreover, the effects of SB203580, a specific p38 MAPK inhibitor, on levels of p38 MAPK phosphorylation and the number and functions of EPCs under IL-1ß conditions were examined. RESULTS: Incubation of EPCs with IL-1ß (5 ng/mL) for 5 days and with IL-1ß (0.05-50 ng/mL) for 48 hours induced a significant reduction in EPC numbers and proliferation, respectively (p < 0.01 vs. control cells). The capacities for migration, adhesion, and angiogenic function of EPCs were also reduced in a time- and dose-dependent manner. IL-1ß induced dose- and time-dependent activation of p38 MAPK in EPCs. Moreover, inhibition of p38 MAPK by SB203580 significantly increased the total number of EPCs by twofold as compared with the IL-1ß-alone group (p < 0.01) and blocked the ability of IL-1ß to impair the functional response of EPCs. CONCLUSION: These results demonstrate that there is a negative cause-effect relationship between IL-1ß and EPCs. Thus, IL-1ß inhibits EPC proliferation, migration, adhesion, and tube formation by a mechanism, which involves p38 MAPK signaling in regulating the number and functions of EPCs in vitro.

Novel 4-azasteroidal N-glycoside analogues bearing sugar-like D ring: synthesis and anticancer activities.

A series of novel N-glycoside analogues with 4-azasteroid moiety bearing sugar-like D ring were conveniently synthesized by constructing the core dihydropyran ring embedded in 4-azasteroidal skeleton which was prepared from 4-aza-5α-androst-3,17-dione 1 in four steps. The structure of 6b were unambiguously proved by the appropriate X-ray structural analysis. Anticancer activity was found for all of the analogues with purinyl moiety against breast cancer (MCF-7), human neuroblastoma (SK-N-SH), cervical cancer cell (HeLa) and prostatic cancer (PC-3), while the analogue 7 containing 1,2,4-triazole heterocycle as the nucleobase was inactive against all of the tested cancer cell lines. The biology results showed the purinyl moiety attached to the pyran ring of 6a-d, substituent at 6'-position of purine base and introduction of a halogen atom at 2'-position of 6'-chloropurine had obviously effect on the evaluated anticancer activity.

[Changes in endothelial progenitor cells and their implications in multiple organ dysfunction syndrome caused by trauma in pigs].

OBJECTIVE: To investigate characteristics of changes in bone marrow endothelial progenitor cells (EPCs) and implications on multiple organ dysfunction syndrome (MODS) as a result of trauma. METHODS: Eighteen mini-pigs were randomized into two groups: MODS group (n=9) and control group (n=9). The animal models of MODS were reproduced by "two-hit" injury with hemorrhagic shock and lipopolysaccharide (LPS) injection. Bone marrow and peripheral blood of them were collected at five time points: normal condition (T1), before injection of LPS (T2), and 0 (T3), 24 (T4) and 48 hours (T5) after injection of LPS. Erythrocytic lysate was added to the samples, and the number of leucocytes in every sample was counted. The rate of EPCs in each sample was determined by flow cytometry. Number of EPCs in bone marrow and peripheral blood were calculated, and the results were analyzed statistically. RESULTS: The number of EPCs (x10(6)/L) in bone marrow of control group at T1-5 was 7.64+/-0.68, 7.32+/-0.55, 7.58+/-1.13, 7.77+/-0.70, and 7.88+/-0.84, respectively, and in peripheral blood control group was 3.54+/-0.26, 4.06+/-0.64, 3.74+/-0.55, 3.61+/-0.37, and 3.98+/-0.63, respectively. The number of EPCs (x10(6)/L) in bone marrow in the experimental group was 7.45+/-1.55, 6.58+/-0.80, 11.27+/-1.20, 10.88+/-1.15, and 8.36+/-2.88, respectively. The number of EPCs (x10(6)/L) in peripheral blood in the experimental group was 3.21+/-0.48, 8.71+/-2.04, 5.98+/-0.77, 1.27+/-0.91, and 2.14+/-0.96, respectively. The number of EPCs in bone marrow of experimental group was larger than that of control group at T3, T4, T5. The number of EPCs in the experimental group in peripheral blood was larger than that of control group at T2, T3, T4, T5. The number of EPCs in bone marrow was larger than that in peripheral blood at every time point (all P<0.05). CONCLUSION: The number of EPCs in peripheral blood elevates sharply in the earlier period, then plummeted quickly during MODS after a trauma. While the number of EPCs in bone marrow descends mildly at first, then rises obviously. Along with the aggravation of MODS, a declination of EPCs in bone marrow emerges. The change in bone marrow EPCs plays an important role in recovery of MODS.

[Influences of the phosphorylation of p38 mitogen activated protein kinase on gene expression of tumor necrosis factor-alpha in multiple organ dysfunction syndrome in pigs].

OBJECTIVE: To investigate that the phosphorylation of the p38 mitogen activated protein kinase (p38MAPK) influences gene expression of tumor necrosis factor-alpha (TNF-alpha) in multiple organ dysfunction syndrome (MODS) in pigs. METHODS: Thirty pigs were divided into MODS group and control group, and an animal model of MODS of "two-hit" injury, including hemorrhagic shock and endotoxemia, was reproduced. The content of p38MAPK's phosphorylation was assessed with Western blotting. TNF-alpha mRNA in peripheral blood monocytes was assayed with real time-polymerase chain reaction (RT-PCR). TNF-alpha was monitored in the peripheral blood plasma with enzyme linked immunosorbent assay (ELISA). RESULTS: Phosphorylation of p38MAPK was obviously increased in extent, which enhanced gene expression of TNF-alpha and then secretion of TNF-alpha by the peripheral blood mononuclear cell in MODS, and the differences were statistically significant compared with that of control group (P<0.05 or P<0.01). CONCLUSION: p38MAPK's phosphorylation is important in pathogenesis of MODS, and phosphorylation of p38MAPK can enhance TNF-alpha mRNA transcription and secretion of TNF-alpha from peripheral blood mononuclear cells, which is the mechanism of increased TNF-alpha in MODS.