Analysis of the impact of immunotherapy efficacy and safety in patients with gastric cancer and liver metastasis

BACKGROUND Gastric cancer(GC)is the fifth most common type of cancer and has the fourth highest death rate among all cancers.There is a lack of studies examining the impact of liver metastases on the effectiveness of immunotherapy in individuals diagnosed with GC.AIM To investigate the influence of liver metastases on the effectiveness and safety of immunotherapy in patients with advanced GC.METHODS This retrospective investigation collected clinical data of patients with advanced stomach cancer who had immunotherapy at our hospital from February 2021 to January 2023.The baseline attributes were compared using either the Chi-square test or the Fisher exact probability method.The chi-square test and Kaplan-Meier survival analysis were employed to assess the therapeutic efficacy and survival duration in GC patients with and without liver metastases.RESULTS The analysis comprised 48 patients diagnosed with advanced GC,who were categorized into two groups:A liver metastasis cohort(n=20)and a non-liver metastatic cohort(n=28).Patients with liver metastasis exhibited a more deteriorated physical condition compared to those without liver metastasis.The objective response rates in the cohort with metastasis and the cohort without metastasis were 15.0%and 35.7%(P>0.05),respectively.Similarly,the disease control rates in these two cohorts were 65.0%and 82.1%(P>0.05),respectively.The median progression-free survival was 5.0 months in one group and 11.2 months in the other group,with a hazard ratio of 0.40 and a significance level(P)less than 0.05.The median overall survival was 12.0 months in one group and 19.0 months in the other group,with a significance level(P)greater than 0.05.CONCLUSION Immunotherapy is less effective in GC patients with liver metastases compared to those without liver metastasis.

An Improved Strategy for Efficient Expression and Purification of Soluble HIV-1 Tat Protein in E.coli

Although the endogenous function of Tat has been elucidated in the past twenty years, the study of its exogenous activity has been hampered due to the difficulty of large scale preparation of the active Tat protein. To express the full-length Tat protein in E.coli, the tat gene was cloned from an HIV infected patient by overlapping PCR. Rare codon usage analysis showed that rare E.coli codons, especially consecutive rare codons for Arg, account for 14% (14 of 101) rare E.coli codons in the tat gene. The expression of the HIV-1 tat gene was verified to be very poor in strain BL21 (DE3) due to the abundance of rare codons; however, tat gene expression was found to be very efficient in the host strain of Rosetta-gami B (DE3), which was supplemented with six rare tRNAs for Arg, Leu, Ile and Pro. Subsequent purification revealed that the proteins are soluble and unusually, the tagged Tat can form dimers independent of cystine disulfide bonds. The purity, integrity and molecular weight of the Tat protein were demonstrated by MALDI-TOF mass spectrometry. Reporter gene activating assay was further confirmed by investigating the transactivation activity of the recombinant Tat protein. Our improved strategy for efficient high level expression and purification of soluble Tat protein has paved the way to fully investigate its exogenous function.

Hepatocyte growth factor gene therapy prevents radiation-induced liver damage

AIM: To transfer human HGF gene into the liver of rats by direct electroporation as a means to prevent radiationinduced liver damage.METHODS: Rat whole liver irradiation model was accomplished by intra-operative approach. HGF plasmid was injected into liver and transferred by electroporation using a pulse generator. Control rats (n = 8) received electrogene therapy (EGT) vehicle plasmid and another 8rats received HGF-EGT 100 μg 48 h before WLIR.Expression of HGF in liver was examined by RT-PCR and ELISA methods. Apoptosis was determined by TUNEL assay. Histopathology was evaluated 10 wk after whole liver irradiation.RESULTS: Marked decrease of apoptotic cells and downregulation of transforming growth factor-beta 1 (TGF-β1)mRNA were observed in the HGF-EGT group 2 d after liver irradiation compared to control animals. Less evidence of radiation-induced liver damage was observed morphologically in liver specimen 10 wk after liver irradiation and longer median survival time was observed from HGF-EGT group (14 wk) compared to control rats (5 wk). (P = 0.031).CONCLUSION: For the first time it has been demonstrated that HGF-EGT would prevent liver from radiation-induced liver damage by preventing apoptosis and down-regulation of TGF-β1.

Partners of patients with ulcerative colitis exhibit a biologically relevant dysbiosis in fecal microbial metacommunities

AIM To investigate alterations in the fecal microbiome using 16 S r RNA amplicon sequencing in couples in the same cohabitation environment.METHODS Fecal samples were collected from eight ulcerative colitis(UC) patients and their healthy partners at Lishui People's Hospital, Zhejiang Province, China. DNA was extracted and the variable regions V3 and V4 of the 16 S r RNA genes were PCR amplified using a two-step protocol. Clear reads were clustered into operational taxonomic units(OTUs) at the 97% sequence similarity level using UCLUST v1.2.22. The Wilcoxon rank-sumtest(R v3.1.2) was used to compare inter-individual differences. Differences with a P value < 0.05 were considered statistically significant.RESULTS Fecal microbial communities were more similar among UC patients than their healthy partners(P = 0.024). UC individuals had a lower relative abundance of bacteria belonging to the Firmicutes, especially Blautia, Clostridium, Coprococcus and Roseburia(P < 0.05). Microbiota dysbiosis was detected in UC patients and their healthy partners. Relevant genera included Akkermansiam, Bacteroides, Escherichia, Lactobacillales, Klebsiella and Parabacteroides. The enriched pathways in fecal samples of UC patients were related to lipid and nucleotide metabolism. Additionally, the pathways involved in membrane transport and metabolism of cofactors and vitamins were more abundant in the healthy partners.CONCLUSION Our results suggested that the microbial composition might be affected in healthy partners cohabiting with UC patients, especially in terms of microbiota dysbiosis.

Digital gene expression profiling analysis of A549 cells cultured with PM10 in moxa smoke

Background:Moxibustion is a traditional Chinese medicine therapy to cure diseases by fumigating meridians or affected parts via burning of moxa floss.Moxa smoke(MS)is one of the key factors in moxibustion.In this study,we adopted digital gene expression profiling,a next-generation gene sequencing technology,to investigate the effect of MS,inhalable particulate matter(PM10),on human lung adenocarcinoma A549 cells.Methods:The effects of MS PM10 on A549 cells,over different treatment durations were investigated in different groups:the 4-h group(4-h MS group and 4-h control group)and the 20-h group(20-h MS group and 20-h control group).Samples collected from the four groups were stored at
80C for subsequent digital gene expression analysis.The differentially expressed genes(DEGs),identified after PM10 treatment,were screened,and their expression patterns analyzed by cluster analysis,Gene Ontology term enrichment,and Kyoto Encyclopedia of Genes and Genomes pathway analysis.Results:Compared with two control groups,1109 DEGs were identified after 4 h of MS intervention and 3565 DEGs were found after 20 h of MS intervention,respectively.Compared with that after 4-h intervention,2149 DEGs were identified after 20-h intervention.Cluster analysis demonstrated that PM10 can significantly inhibit cell cycle process with the prolongation of intervention time.Significant pathway enrichment analysis showed that MS PM10 can inhibit A549 cell cycle process at all phases.When MS PM10 exposure time prolongs,the inhibitory effect on cell cycle process becomes more obvious.Conclusion:MS PM10 has many biological activities,and may cause differential expression of genes involved in various biological processes.Nevertheless,further research on MS is warranted for better understanding of the mechanistic details.

p53-dependent upregulation of PIG3 transcription by γ-ray irradiation and its interaction with KAP1 in responding to DNA damage

PIG3 (p53-inducible gene 3), originally identified as one of a set of genes induced by p53 before the onset of apoptosis, was assumed to contribute to early cellular response to DNA damage. Here, we studied the relation between p53 status and the increased expression of PIG3 by ionizing radiation (IR), and the related clues regarding the involvement of PIG3 in the cellular response to IR-induced DNA damage signaling. We demonstrated that the pentanucleotide microsatellite sequence was responsible for the p53-dependent induction of PIG3 transcription after irradiation, while sequence upstream of PIG3 promoter could maintain the basal level of expression which was not inducible by irradiation. The interaction of PIG3 and the KRAB-ZFP-associated protein 1 (KAP1), a DNA damage response protein, was revealed. PIG3 nucleus foci were formed 15 min after γ-ray irradiation, and which were found to partially colocalize with the phospho-KAP-1 foci as well as γ-H2AX foci. Although the lac operator tagged EGFP based reporter system revealed that PIG3 does not remodel chromatin in large scale in the cells under normal growing condition, it indeed prompted the chromatin relaxation in the cellular response to DNA damage signaling. All these data suggest that PIG3 is involved in IR-induced DNA damage response, and which maybe partially attribute to its interaction with KAP1.

siRNA-mediated silencing of Cockayne Cyndrome group B gene potentiates radiation-induced apoptosis and antiproliferative effect in HeLa cells

Background Cockayne syndrome （CS） is a rare human genetic disorder characterized by increased UV sensitivity, developmental abnormalities and premature aging. Cells isolated from individuals with CS have a defect in transcription-coupled DNA repair. Despite the repair defect, there is no any increased risk of spontaneous or UV-induced cancer for CS individuals. The strategy of RNA interfering was used here to explore the potential radiosensitizing and anticancer activity of targeting CS group B （CSB） gene. Methods The vectors encoding CSB-specific siRNAs were constructed by inserting duplex siRNA encoding oligonucleotides into the plasmid P^silencer TM 3.1. The cell lines expressing the CSB-siRNA were generated from HeLa cells transfected with the above vectors. Colony-forming ability was used to assay cell survival. Cell cycle was analyzed by FACScan flow cytometry. The apoptosis was measured by detecting the accumulation of sub-G1 population as well as by fluorescence staining assay. Reverse transcriptase polymerase chain reaction （RT-PCR） was used to semi-quantify mRNA expression. Protein level was detected by Western blotting analysis. Results Two constructs encoding CSB-specific siRNA were generated, both of them resulted in remarkable suppression on CSB expression in HeLa cells, and led to an increased sensitivity to T-ray and UV light. siRNA-mediated silencing of CSB decreased cell proliferation rate, increased spontaneous apoptosis as well as the occurrence of UV- or cisplatin-induced apoptosis by 2 to 3.5 fold. A significant S phase blockage and a remarkable reduction of G1 population were induced in control HeLa cells at 18 hours after being exposed to 10 J/m^2 of UV light. The S phase blockage was also observed in UV-irradiated CSB-siRNA transfected HeLa cells, but the extent of increased S phase population was lower than that in the UV-irradiated control cells. No or a relative weak reduction on G1 phase population was observed in UV-irradiated CSB-siRNA transfected HeLa cells. In addition, siRNA-mediated silencing of CSB promoted the elimination of G2/M phase cells after UV light radiation. Conclusions siRNA-mediated silencing of CSB causes cells to proliferate more slowly, sensitize cells to genotoxicants, and modify UV radiation-induced cell cycle changes, siRNA-mediated inactivation of CSB could be an attractive strategy for ameliorating cancer therapy, which can be fulfilled via the combination of gene therapy and sensitization of radiotherapy or chemotherapy.

PIG3 downregulation enhances the radiosensitivity of NSCLC cells by promoting G_(2)/M cell cycle arrest and apoptosis

Objective:To investigate the mechanism of p53-induced gene 3(PIG3)-regulation of radioresistance in human non-small cell lung cancer(NSCLC)cells,in order to explore new biomarkers and therapeutic targets to combat radioresistance and improve the 5-year survival rate.Methods:The PIG3 gene was knocked down in A549 cells using siRNA,and was overexpressed in H1299 cells using a PIG3 expression plasmid.After confirming PIG3 knockdown and overexpression through the Western blot analysis,the radiosensitivity,DNA damage,cell cycle distribution,and apoptosis in these cells were analyzed using colony formation assay,immunofluorescence staining forγH2AX,and flow cytometry,respectively.Results:PIG3 silencing markedly increased the radiosensitivity of NSCLC cells,with radiosensitization ratios of 1.12 and 1.25.Compared with the corresponding negative control,PIG3 knockdown significantly enhanced G2/M phase arrest(siNC:26.12±2.50,siPIG3#1:34.98±4.19,siPIG3#2:37.79±3.53,P<0.05),promoted radiation-induced apoptosis(siNC:14.61±1.85,siPIG3#1:17.26±1.14,siPIG3#2:20.70±2.04,P<0.05),and reduced the number ofγ-H2AX foci 0.5,1,and 2 h after radiation(P<0.05).Conversely,PIG3 overexpression markedly decreased the radiosensitivity of NSCLC cells,as evidenced by the reduction of G2/M phase arrest(NC:33.18±2.11 vs.PIG3:24.21±3.09,P<0.05)and apoptosis(NC:15.49±0.56 vs.PIG3:12.79±0.29,P<0.05),and increased DNA damage(P<0.05).Conclusions:PIG3 downregulation increases the radiosensitivity of NSCLC cells,and PIG3-upregulation leads to the progression in radioresistance.Therefore,PIG3 is a potential target for radiotherapy for NSCLC.

Genome-wide identification of DNA-PKcs-associated RNAs by RIP-Seq

Dear Editor,The DNA-dependent protein kinase catalytic subunit(DNA-PKcs)forms a serine/threonine protein kinase complex with the Ku heterodimer(Ku70/Ku80)and plays an important role in the DNA damage response(DDR)and maintenance of genomic stability through nonhomologous end joining(NHEJ),wherein the Ku heterodimer recognizes and binds broken DNA ends,facilitating the recruitment and activation of DNA-PKcs.1 Activated DNA-PKcs phosphorylates and alters the function of factors that mediate NHEJ,including DNA-PKcs itself.2 In addition,DDR-independent roles of DNA-PKcs have been demonstrated.3 Studies further identified DNA-PKcs as a modulator of cancer-associated pathways distinct from DNA repair,including hypoxia,metabolism,the inflammatory response,and transcriptional regulation.