PAK4-relevant proliferation reduced by cell autophagy via p53/mTOR/p-AKT signaling.

Background: P21-activated kinase 4 (PAK4) involves in cell proliferation in cancer and mutually regulates with p53, a molecule is demonstrated to control cell autophagy by mammalian target of rapamycin (mTOR)/protein kinase B (AKT) signaling. Since the signaling exhibits an association with PAK family members in cell autophagy, it implies that PAK4-relevant proliferation may be impacted by autophagy via p53 with a lack of evidence in cancer cells. Methods: In this research, transient and stable PAK4-knockdown human hepatocarcinoma cell lines (HepG2) were constructed by transfection of PAK4-RNA interference (RNAi) plasmid and lentivirus containing PAK4-RNAi plasmid, respectively. We investigated cell proliferation using methyl thiazolyl tetrazolium (MTT) and Cell Counting Kit 8 (CCK8) assays, cell cycle by flow cytometry (FCM) and cell autophagy by monodansylcadaverine (MDC) staining and autophagic biomarker's expression, and detected the expressions of p53, mTOR, phosphorylated-AKT (p-AKT) and AKT by immunofluorescence and western blot to explore the mechanism. Results: We successfully constructed transient and stable PAK4-knockdown HepG2 cell lines, and detected dysfunction of the cells' proliferation. An increased expression of p53, as a molecule of cell-cycle-surveillance on G1/S phase, was demonstrated in the cells although the cell cycle blocked at G2/M. And then, we detected increased autophagosome and autophagic biomarker LC3-II, and decreased expressions in p-AKT and mTOR. Conclusions: The proliferation is reduced in PAK4-knockdown HepG2 cells, which is relative to not only cell cycle arrest but also cell autophagy, and p53/mTOR/p-AKT signaling involves in the cell progress. The findings provide a new mechanism on PAK4 block in cancer therapy.

Dynamic changes in the migratory microbial components of colon tissue during different periods of sepsis in an LPS-induced rat model.

Previous studies have shown that bacterial translocation may play an important role in worsening gastrointestinal injury during sepsis. However, the dynamics of specific microbiota components in intestinal tissues at different sepsis stages remain unclear. Rats receiving intraperitoneal lipopolysaccharide (LPS) were sacrificed at 12 h and 48 h post-injection. Routine blood, serum cytokines, and microbiota in colon tissue, colonic contents, and lung tissue at different time points were assessed. Migratory microbial components in colonic tissue at 12 h and 48 h post-LPS were identified using source tracking, characteristic component identification, and abundance difference analyses. Colonic tissue microbiota changed dynamically over time after LPS injection, involving translocation of microbial components from colon contents and lung tissue at different time points. Bacteria migrating to colon tissue at 12 h sepsis were mainly from colonic contents, while those at 48 h were predominantly from the lung tissue. The migratory microbial components in colon tissue were widely associated with blood indicators and colonizing genus abundance and microbiota functionality in colon tissue. In this study, the temporal dynamics of bacterial translocation from various sources into colon tissues at different sepsis progression stages were characterized for the first time, and the species composition of these migrating microbes was delineated. These bacterial migrants may contribute to the pathophysiological processes in sepsis through direct interactions or indirectly by modulating colonic microbiota community structure and function.

Dynamic alterations in the lung microbiota in a rat model of lipopolysaccharide-induced acute lung injury.

The lung microbiota have been found to be substantially altered in numerous pulmonary disorders, and crosstalk between the host pathophysiology and lung microbiota plays critical roles in the regulation of disease states. The aim of this study was to investigate dynamic changes in the lung microbiota during different stages of acute lung injury and acute respiratory distress syndrome (ALI/ARDS). Rats receiving an intraperitoneal administration of lipopolysaccharide (LPS) were sacrificed at 12 and 48 h after injection, and the hematological parameters, serum cytokine levels, and histological characteristics of the lung tissue and lung microbiota were assessed. After LPS injection, along with fluctuations of systemic cytokine levels and the onset and regression of pulmonary edema, the diversity, components, and functionalities of the pulmonary microbiota underwent significant dynamic changes. The volatility of the α-diversity indices narrowed after LPS injection, and the indices significantly decreased 48 h later. The abundance of 18 genera and functionality of adenosine triphosphate-binding cassette (ABC) transporters, pentose phosphate, and bacterial chemotaxis pathways were found to significantly differ between specified time points. Several significant correlations between the components and functionalities of the lung microbiota and indicative symptoms of ALI/ARDS were also observed. Brevibacterium was correlated with cytokines tumor necrosis factor (TNF)-α, interleukin (IL)-10, and IL-6 and with hematological percentage of neutrophils (NEU%); Wnt, Notch, and chronic myeloid leukemia signaling pathways were correlated with IL-1ß; mitogen-activated protein kinase (MAPK) signaling pathway-yeast was correlated with IL-10; and the pathways of ascorbate and aldarate metabolism and basal transcription factors were correlated with platelet-related indicators. The correlations between the lung microbiota and indicative symptoms of ALI/ARDS identified in this study support further investigation into the underlying mechanism of host-microbiota interactions during lung injury and repair.

Uncovering the anti-NSCLC effects and mechanisms of gypenosides by metabolomics and network pharmacology analysis.

ETHNOPHARMACOLOGICAL RELEVANCE: Lung cancer is the chief reason of cancer death worldwide, and non-small cell lung cancer (NSCLC) make up the majority of lung cancers. Gypenosides are the main active constituents from Gynostemma pentaphyllum. Previous studies showed that they were used to remedy many cancers. The effect of gypenosides on NSCLC has never been studied from the perspective of network pharmacology and metabolomics. The mechanism is still not clear and remains to be explored. AIM OF THE STUDY: To explore the anti-NSCLC activity and mechanism of gypenosides in A549 cells. MATERIAL/METHODS: Gypenosides of G. pentaphyllum were detected by HPLC-MS. The cytotoxicity was detected by MTT assay. The migration, cell cycle and apoptosis of gypenosides were studied by wound healing assay, JC-1 assay and flow cytometry. The mechanism of gypenosides on NSCLC was studied by metabolomics and network pharmacology. Some key proteins and pathways were further confirmed by Western blot. RESULTS: Eleven gypenosides were detected by HPLC-MS. Gypenosides could suppress the proliferation of A549 cells, inhibit the migration of A549 cells, induce apoptosis and arrest cell cycle in G0/G1 phase. Metabolomics and network pharmacology approach revealed that gypenosides might affect 17 metabolite related proteins by acting on 9 candidate targets (STAT3, VEGFA, EGFR, MMP9, IL2, TYMS, FGF2, HPSE, LGALS3), thus resulting in the changes of two metabolites (uridine 5'-monophosphate, D-4'-Phosphopantothenate) and two metabolic pathways (pyrimidine metabolism; pantothenate and CoA biosynthesis). Western blotting indicated that gypenosides might inhibit A549 cells through MMP9, STAT3 and TYMS to indirectly affect the pathways of pyrimidine metabolism, pantothenate and CoA biosynthesis. CONCLUSIONS: This study revealed that metabolomics combined with network pharmacology was conducive to understand the anti-NSCLC mechanism of gypenosides.

Prevalence of human papillomavirus in archival head and neck cancer in the eastern Inner Mongolian Autonomous Region, China.

OBJECTIVE: To investigate the prevalence of human papillomavirus (HPV) in archival head and neck cancer (HNC) collected from the Tong-Liao area, which is located in east Inner Mongolia, China. METHODS: The presence of HPV in 54 HNCs and 25 benign biopsies was detected and the sequence variation of the E6 gene in HPV-positive samples was analyzed to determine their lineage/sublineage classification. RESULTS: HPV was detected in only 4 out of 54 HNCs and no benign biopsies were found to be HPV-positive. After further p16INK4a immunostaining, only 3 cases of HNC were positive for both HPV and p16INK4a. Phylogenetic analysis of the isolated E6 gene shows that the HPV 16, HPV 31, and HPV 58 isolated in this study belong to lineage A. CONCLUSIONS: The prevalence of HPV in HNC from this area is very low. The lineage/sublineage classification of the 3 HPV types in HNC in this area is consistent with the previous reported data of HPV lineage distribution in cervical cancer within China.

Evaluation of the Associations Between Cervical Microbiota and HPV Infection, Clearance, and Persistence in Cytologically Normal Women.

The aim of this study was to investigate the associations between cervical microbiota and different human papillomavirus (HPV) infection statuses in cytologically normal women. The cervical microbiota of HPV-positive or -negative women with a normal cytologic diagnosis was characterized and compared using 16S rDNA-based high-throughput sequencing, and the differences in cervical microbiota associated with new acquisition, persistence, and clearances of HPV genotypes were analyzed via one-year follow-up. The results showed that the cervical microbial richness of HPV-positive women was lower than for HPV-negative women, and the difference was more significant in the postmenopausal group relative to the premenopausal group. Ureaplasma parvum and related taxa were associated with baseline HPV positivity, while Brochothrix, Diplorickettsia, Ezakiella, Faecalibacterium, and Fusobacterium genera and their related taxa and Pseudomonas aeruginosa were associated with baseline HPV negativity. For HPV-positive women, the baseline abundance of Actinomyces was negatively associated with new HPV infection, Alloprevotella tannerae, Prevotella nigrescens, and Prevotella oulorum; and Dialister invisus were positively associated with new HPV-type infection within the year of follow-up. Lactobacillus delbrueckii was found to be negatively associated with persistent HPV infection and 9 taxa belonging to Prevotella, Dialister, and Lachnospiraceae were found to be positively associated with persistence, and/or negatively associated with clearance of HPV types. We also observed 10 novel taxa associated with the clearance/persistence of HPV that had not been reported elsewhere. Those taxa associated with different infection statuses of HPV could be used as a biomarker to help predict the risk of developing persistent HPV infection.

p21-activated kinase 4 as a switch between caspase-8 apoptosis and NF-κB survival signals in response to TNF-α in hepatocarcinoma cells.

PAK4 is overexpressed in a variety of human cancers and considered a promising candidate for therapeutic target. However, its functions remain poorly understood, especially in liver carcinogenesis which could be triggered by inflammation. In the present study, endogenous PAK4 was knockdown using siRNA in HepG2 and SK-Hep1 cells. The two cell lines performed reduced cell viability, altered cell cycle composed of decreased S and arrest in G2, and apoptosis. Meanwhile, expression of NF-κB p65 in the nuclei and caspase-8 activity did not show significant differences from control. However, after treating cells with TNF-α, an inflammatory cytokine, we investigated repressed nuclear expression and localization of NF-κB p65, and induced apoptosis with increased caspase-8 activity in PAK4-knockdown cells. The findings revealed that ablation of PAK4 inhibited cell viability via blocking cell cycle and progressing apoptosis. The apoptosis was partially dependent upon caspase-8 concomitant with attenuated NF-κB survival signal due to stimulus of TNF-α. It suggests that PAK4 as target is a switch between caspase-8 apoptosis and NF-κB survival signals induced by TNF-α in hepatocarcinoma cells.

Mutation analysis underlying the downregulation of the thyroid hormone receptor ß1 gene in the Chinese breast cancer population.

PURPOSE: There are a growing number of reports suggesting that the aberrant expression and mutation of the thyroid hormone receptor ß1 (TRß1) gene is associated with the development of human neoplasms. However, its exact role in the pathogenesis of breast cancer remains elusive. In the present study, we analyzed the mRNA expression and mutations of the TRß1 gene in the Chinese breast cancer population. METHODS: The expression of TRß1 mRNA was examined by real-time quantitative reverse transcription polymerase chain reaction, and mutations in the TRß1 gene in the hotspot region that spans exons 7-10 were analyzed by polymerase chain reaction single-strand conformation polymorphism and automated DNA sequencing. RESULTS: TRß1 mRNA expression was significantly reduced in all 105 breast cancer specimens examined. A total of 20 samples showed truncating mutations within the exons 7-10 of the TRß1 gene, where eight cases harbored a frame shift mutation (five cases of c.850insA in exon 7 and three cases c.1028delA in exon 8), whereas missense mutations were observed in 12 breast cancer cases. The 20 cases with mutation in the TRß1 gene showed a reduction in TRß1 mRNA expression compared with that observed in matched normal tissues. The mutation was also correlated with menopausal stage and estrogen receptor status. CONCLUSION: The findings of the present study suggest that the aberrant expression and mutations of the TRß1 gene are associated with the development of breast cancer and that the mutations in the TRß1 gene partly serve as the underlying mechanism for TRß1 inactivation in the Chinese breast cancer population.

Loss of heterozygosity in thyroid hormone receptor beta in invasive breast cancer.

BACKGROUND: Loss of heterozygosity (LOH) on chromosome arm 3p, where the gene of thyroid hormone receptor beta (THRB) is located, has been reported in breast cancer. Although some studies performed in vitro have suggested that THRB could act as a tumor suppressor in breast cancer development, there is still no unequivocal evidence to support this. METHODS: To determine the role of LOH in breast tumor development, the LOH of THRB and its proximal microsatellite markers D3S1293, D3S3659, D3S3700, D3S2307 and D3S2336 was investigated in a genomic region spanning ~3.3 Mb in tumor specimens and in corresponding normal tissues of 74 invasive breast cancer patients. The association was analyzed between LOH in microsatellite markers and clinicopathological characteristics. RESULTS: LOH was detected in D3S1293 (36.7%), THRB (59.4%), D3S3659 (37.5%) and D3S3700 (55.2%) among the informative cases, while LOH was not detected in D3S2307 and D3S2336. Cases exhibited LOH of 52.8%-71.4% if any 2 markers were combined and analyzed out of the first 4 microsatellite markers. LOH in THRB was associated with negative estrogen receptor (ER), negative progesterone receptor (PR), both negative estrogen receptor and progesterone receptor (HR) and human epidermal growth factor receptor-2 (HER2) and lymph node metastasis (p = 0.0001, p = 0.005, p = 0.001 and p = 0.018). The association with negative PR in LOH in THRB and/or D3S1293 was pronounced (p<0.0001). LOH in D3S3700 showed an association with lymph node metastasis (p = 0.014). This association was enhanced if D3S3700 was combined with THRB or D3S3659 (p = 0.0004, p = 0.0002). CONCLUSIONS: LOH in THRB and its proximal microsatellite markers may play a role in tumorigenesis and development in invasive breast cancer.

Meta-analysis on the relationship between HLA-DRBl gene polymorphism and cervical cancer in Chinese population.

AIM: To determine the association between HLA-DRB1 haplotypes and risk of cervical cancer in unselected and samples from Chinese ethnicities. METHODS: A comprehensive search for articles from their inception to April 1st, 2013 was conducted from PubMed, Medline, Elsevier Science, Springer Link, Cochrane Library database, China biology medical literature database (CBM),China National Knowledge Infrastructure (CNKI),VIP,and Chinese literature database(Wang fang). A total of 1596 patients with cervical cancer and 2048 controls from the 12 studies on the relationship between gene polymorphism of HLA-DRB l and cervical cancer were performed and data were analyzed and processed using Review Manager 5.0 and Stata 11.0. RESULTS: Among the 13 family alleles, two (DRB1*03 and DRB1*08) were found to be negatively associated with cervical cancer in all studies or in Uighur subgroups, and two (DRB1*10 and DRB1*15) were positively associated with in all studies or in Uighur subgroups. Among the 25 specific alleles, six (DRB1*0301, *0403,*0404, *0803, *1312 and *1502) were associated with an increased risk cervical cancer in all studies. No significant association was established for other HLA-DRB1 family alleles and specific alleles. Ethnicity partially explained the race influence of DRB1*12, DRB1*14, DRB1*0301, DRB1*0403, DRB1*0404, DRB1*0803, DRB1*1312 and DRB1*1502 phenotypes. CONCLUSION: Our results support the hypothesis that the HLA-DRB1 family alleles and specific alleles might influence the susceptibility or resistance to cervical cancer, suggesting that immune regulation may play a key role in this disease, although further investigations are still needed.

Down-regulation of thyroid hormone receptor ß1 gene expression in gastric cancer involves promoter methylation.

Hypermethylation has been shown in the promoter region of the thyroid hormone receptor ß1 (TRß1) gene in several human tumors. However, its role in gastric cancer formation is still unclear. In the study, we analyzed mRNA expression of TRß1 gene using real-time quantitative PCR (qPCR). A quantitative methylation-specific PCR (Q-MSP) assay was used to determine the methylation status of the TRß1 gene promoter region in 46 pair-matched gastric neoplastic and adjacent non-neoplastic tissues. The results showed that TRß1 mRNA expression was significantly reduced in gastric cancer specimens. The methylation of promoter of TRß1 gene in gastric cancer tissues was significantly higher than in adjacent normal tissues. Promoter hypermethylation of the TRß1 gene correlated with tumor infiltration, lymph node metastasis, and distant metastasis, but it was not associated with other clinicopathological characteristics. We treated gastric cancer cell lines MKN-45, MKN-28, SGC-7901, NCI-N87, and SNU-1 with 5-Aza-2-deoxycytidine (5-Aza-dC). The results showed the expression of TRß1 mRNA was increased in MKN-45, MKN-28, SGC-7901, but not increased in NCI-N87 and SNU-1. These results suggest that the TRß1 gene plays important roles in the development of gastric cancer partially through epigenetic mechanisms.

[Bortezomib combined with autologous peripheral blood hematopoietic stem cell transplantation for therapy of patients with multiple myeloma].

This study was aimed to evaluate the therapeutic efficacy of bortezomib combined with autologous peripheral blood hematopoietic stem cell transplantation (autoPBSCT) for patients with multiple myeloma (MM). 5 patients underwent autologous hematopoietic stem cell transplantation. Bortezomib treatment was supplied for patients before autoPBSCT and in the conditioning of transplantation, it was also used in maintaining treatment. Patients with transplantation adopted bortezomib plus melphalan conditioning regimen. The number of infused MNC and number of CD34(+) cells were 4.06×10(8) (4.09×10(8) - 4.37×10(8))/kg and 3.98×10(6) (2.49×10(6) - 8.2×10(6))/kg respectively. The results showed that hematopoiesis was reconstituted in 5 patients, with a neutrophil cell count more than 0.5×10(9)/L at day 14 (13 - 25 days) after transplantation and platelet count more than 50×10(9)/L at day 28 (21 - 41 days) after transplantation. Transplantation-associated death was not observed. 5 patients were disease-free survival. In conclusion, treatment of bortezomib combined with autologous peripheral hematopoietic stem cell transplantation is an effective method for patients with multiple myeloma. Use of bortezomib after transplantation might still be favourable to MM patients, for survival prolongation and life quality improvement.

Aberrant methylation of the THRB gene in tissue and plasma of breast cancer patients.

The thyroid hormone receptors (TR) have three major isoforms, TRalpha1, TRalpha2, and TRbeta1; these are ligand-dependent nuclear transcription factors. THRB, the gene encoding TRbeta1, is considered a potential cancer suppressor. The mechanism of its inactivation is not yet clear. Aberrant silencing of THRB in breast cancer tissue and plasma by promoter hypermethylation was investigated in the present study. Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to examine THRB mRNA expression in the breast cancer tissues. Methylation-specific polymerase chain reaction (MSP) combined with nested PCR was used to determine the methylation status of the THRB gene promoter region in 40 cancer tissue and 40 plasma samples from breast cancer patients. Methylation status of MSP product in plasma was also evaluated by direct sequencing. The expression of THRB mRNA in breast cancer tissues was lower than that in the normal tissues; hypermethylation was found in 32 of 40 breast cancer tissues (80%) and in 28 of 40 plasma samples (70%). Loss of THRB gene expression was associated with the CpG island hypermethylation of promoter regions. THRB gene CpG island methylation was not related to clinical pathologic parameters. Sequencing results were identical to agarose gel electrophoresis results. The present results indicate that hypermethylation of THRB as an alternative gene silencing mechanism is highly prevalent in breast cancer. Methylated tumor-specific DNA may serve as a plasma biomarker for prognosis in patients with breast cancer.