Expression of cell cycle regulator p57^(kip2), cyclinE protein and proliferating cell nuclear antigen in human pancreatic cancer: An immunohistochemical study

AIM： To investigate the effects of p57^kip2, cyclinE protein and proliferating cell nuclear antigen （PCNA） on occurrence and progression of human pancreatic cancer. METHODS： The expression of p57^kip2, cyclinE protein and PCNA in tumor tissues and adjacent tissues from 32 patients with pancreatic cancer was detected by SP immunohistochemical technique. RESULTS： The positive expression rate of p57^kip2 protein in tumor tissues was 46.9%, which was lower than that in adjacent pancreatic tissues （χ^2 = 5.317, P〈0.05）. p57^kip2 protein positive expression remarkably correlated with tumor cell differentiation （P〈0.05）, but not with lymph node metastasis （P〉0.05）. The positive expression rate of cyclinE protein in tumor tissues was 68.8%, which was higher than that in adjacent pancreatic tissues （χ^2 = 4.063, P〈0.05）. CyclinE protein positive expression significantly correlated with tumor cell differentiation and lymph node metastasis （P〈0.05）. The positive expression rate of PCNA in the tumor tissues was 71.9%, which was higher than that in adjacent pancreatic tissues （χ^2 = 5.189, P〈0.05）. PCNA positive expression remarkably correlated with tumor cell differentiation and lymph node metastasis （P〈0.05）. CONCLUSION： The decreased expression of p57^kip2 and/or overexpression of cyclinE protein and PCNA may contribute to the occurrence and progression of pancreatic cancer. p57^kip2, cyclinE protein, and PCNA play an important role in occurrence and progression of pancreatic cancer.

Correlation of Seven Biological Factors(Hsp90α,p53,MDM2,Bcl-2,Bax,Cytochrome C,and Cleaved caspase3)with Clinical Outcomes of ALK+ Anaplastic Large-cell Lymphoma

Objective To explore correlation of seven apoptosis-related proteins （Hsp9Oa, p53, MDM2, Bcl-2, Bax, Cytochrome C, and Cleaved caspase3） with clinical outcomes of ALK＋ anaplastic large-cell lymphoma （ALCL）. Methods Using immunohistochemistry and immunofluorescence double staining methods, the expressions of these seven apoptosis-associated proteins were studied to clarify their relationship with clinical outcomes of 36 ALK＋ and 25 ALK- systemic ALCL patients enrolled between 1996 and 2006. The relationship of these apoptosis-regulating proteins with NPM-ALK status was also evaluated with the tyrosine inhibitor herbimycin A （HA） in vitro by immunocytochemistry, Western blotting and flow cytometric assays. Results The presence of Hsp90a-, MDM2-, Bax-, Cytochrome C, and Cleaved caspase3-positive tumor cells was found significantly different in ALK＋ and ALK- ALCLs , which was correlated with highly favorable clinical outcome. The Bcl-2- and p53-positive tumor cells were found in groups of patients with unfavorable prognosis. Inhibition of NPM-ALK by HA could reactivate the p53 protein and subsequent apoptosis-related proteins and therefore induced apoptosis in ALK＋ ALCL cells. Conclusion Our results suggest that these seven proteins might be involved in apoptosis regulation and associated with clinical outcome of ALK＋ systemic ALCLs. We also reveal a dynamic chain relation that NPM-ALK regulates p53 expression and subsequent apoptosis cascade in ALK＋ ALCLs.

Special AT-rich sequence-binding protein 2 acts as a negative regulator of stemness in colorectal cancer cells

AIM To find the mechanisms by which special AT-rich sequence-binding protein 2(SATB2) influences colorectal cancer(CRC) metastasis.METHODS Cell growth assay, colony-forming assay, cell adhesion assay and cell migration assay were used to evaluate the biological characteristics of CRC cells with gain or loss of SATB2. Sphere formation assay was used to detect the self-renewal ability of CRC cells. The m RNA expression of stem cell markers in CRC cells with upregulated or downregulated SATB2 expression was detected by quantitative real-time polymerase chain reaction. Chromatin immunoprecipitation(Ch IP) was used to verify the binding loci of SATB2 on genomic sequences of stem cell markers. The Cancer Genome Atlas(TCGA) database and our clinical samples wereanalyzed to find the correlation between SATB2 and some key stem cell markers.RESULTS Downregulation of SATB2 led to an aggressive phenotype in SW480 and DLD-1 cells, which was characterized by increased migration and invasion abilities. Overexpression of SATB2 suppressed the migration and invasion abilities in SW480 and SW620 cells. Using sequential sphere formation assay to detect the selfrenewal abilities of CRC cells, we found more secondary sphere formation but not primary sphere formation in SW480 and DLD-1 cells after SATB2 expression was knocked down. Moreover, most markers for stem cells such as CD133, CD44, AXIN2, MEIS2 and NANOG were increased in cells with SATB2 knockdown and decreased in cells with SATB2 overexpression. Ch IP assay showed that SATB2 bound to regulatory elements of CD133, CD44, MEIS2 and AXIN2 genes. Using TCGA database and our clinical samples, we found that SATB2 was correlated with some key stem cell markers including CD44 and CD24 in clinical tissues of CRC patients.CONCLUSION SATB2 can directly bind to the regulatory elements in the genetic loci of several stem cell markers and consequently inhibit the progression of CRC by negatively regulating stemness of CRC cells.

Expression of caspase-3 and TRAIL receptors in CD4^+ and CD8^+ T cells of SLE patients

Objective: To study the expression of caspase-3 and tumor necrosis factor-related apoptosisinducing ligand (TRAIL) receptors in the CD4+ and CD8+ T cells of systemic lupus enythematosus (SLE) patients. Methods: RT-PCR was used to analyze the expression of caspase-3 and TRAIL receptors in CD4+ and CD8+ T cells of SLE patients and normal subjects. Results: The death domain-containing TRAIL-R1/R2 as well as 'decoy' TRAIL-R3/R4 were co-expressed in majority of CD4+ and CD8+ T cells in both SLE patients and normal subjects. The CD8+ T cells from SLE patients showed significantly higher expression of caspase-3 and TRAIL-R2 than those from normal subjects,and the expression was correlated with the activity of the disease. Conclusion: The TRAIL-R2 signal pathway might contribute to the apoptosis of T cells in SLE.

Construction and analysis of tree models for chromosomal classification of diffuse large B-cell lymphomas

AIM: To construct tree models for classification of diffuse large B-cell lymphomas (DLBCL) by chromosome copy numbers, to compare them with cDNA microarray classification, and to explore models of multi-gene, multi-step and multi-pathway processes of DLBCL tumorigenesis. METHODS: Maximum-weight branching and distancebased models were constructed based on the comparative genomic hybridization (CGH) data of 123 DLBCL samples using the established methods and software of Desper et al . A maximum likelihood tree model was also used to analyze the data. By comparing with the results reported in literature, values of tree models in the classification of DLBCL were elucidated. RESULTS: Both the branching and the distance-based trees classified DLBCL into three groups. We combined the classification methods of the two models and classified DLBCL into three categories according to their characteristics. The first group was marked by +Xq, +Xp, -17p and +13q; the second group by +3q, +18q and +18p; and the third group was marked by -6q and +6p. This chromosomal classification was consistent with cDNA classification. It indicated that -6q and +3q were two main events in the tumorigenesis of lymphoma. CONCLUSION: Tree models of lymphoma established from CGH data can be used in the classification of DLBCL. These models can suggest multi-gene, multistep and multi-pathway processes of tumorigenesis. Two pathways, -6q preceding +6q and +3q preceding+18q, may be important in understanding tumorigenesis of DLBCL. The pathway, -6q preceding +6q, may have a close relationship with the tumorigenesis of non-GCB DLBCL.

Induction of epithelial-mesenchymal transition (EMT) in human hepatocellular carcinoma after radiotherapy

Objective: Epithelial-mesenchymal transition (EMT) is a critical early event for the invasion and metastasis of many carcinomas. In the present study, we examined EMT markers in the residual cancer cells of hepatocellular carcinoma (HCC) after radiotherapy. Methods: Eight patients with large HCC who underwent hepatectomy with preoperative radiothera- py were studied. The expressions of E-cadherin and vimentin were determined immunohistochemically in the residual cancer cells of HCC following radiotherapy, and also in the pre-radiotherapy biopsy cancer cells. Results: Histological analysis showed that some residual cancer cells of HCC displayed an elongated spindle or fibroblast-like shape. The expression of E- cadherin was markedly reduced or negative in the spindle residual cancer cells, but the expression of vimentin significantly in- duced. However, the above changes were not found in the pre-radiotherapy biopsy cancer cells. Conclusion: EMT is induced in the residual cancer cells of HCC following radiotherapy, which may facilitate the systemic dissemination of cancer cells.

Metastasis-associated long noncoding RNAs in gastrointestinal cancer: Implications for novel biomarkers and therapeutic targets

Long non-coding RNAs(lnc RNAs), a newly discovered class of nc RNA molecules, have been widely accepted as crucial regulators of various diseases including cancer. Increasing numbers of studies have demonstrated that lnc RNAs are involved in diverse physiological and pathophysiological processes, such as cell cycle progression, chromatin remodeling, gene transcription, and posttranscriptional processing. Aberrant expression of lnc RNAs frequently occurs in gastrointestinal cancer and plays emerging roles in cancer metastasis. In this review, we focus on and outline the regulatory functions of recently identified metastasis-associated lnc RNAs, and evaluate the p o t e n t i a l r o l e s o f l n c R N A s a s n o v e l d i a g n o s t i c biomarkers and therapeutic targets in gastrointestinal cancer.

Glycogen Synthase Kinase 3β Inhibitor (2'Z,3'E)-6-Bromo-indirubin-3'-Oxime Enhances Drug Resistance to 5-Fluorouracil Chemotherapy in Colon Cancer Cells

Objective： To explore the effects and mechanism of glycogen synthase kinase 3β （GSK-313） inhibitor （2＇Z,3＇E）-6-bromo-indirubin-3＇-oxime （BIO） on drug resistance in colon cancer cells. Methods： The colon cancer SW480 and SW620 cells were treated with BIO, 5-fluorouracil （5-FU） and BIO/5-FU, separately. Cell cycle distribution, apoptosis level and efflux ability of rhodamine 123 （Rh123） were detected by flow cytometry. The protein expressions of P-glycoprotein （P-gp）, multidrug resistance protein 2 （MRP2）, thymidylate synthase （TS）, β-catenin, E2F-1 and βcl-2 were detected by Western blot. β-catenin and P-gp were stained with double immunofluorescence and observed under a confocal microscope. Results： BIO up-regulated β-catenin, P-gp, MRP2 and TS, enhanced the efflux ability of Rh123, decreased Bcl-2 protein and gave the opposite effect to E2F-1 protein in SW480 and SW620 ceils. Furthermore, BIO significantly inhibited cell apoptosis, increased S and G2/M phase cells, and reduced the cell apoptosis induced by 5-FU in SW480 cells, whereas the effects were slight or not obvious in SW620 cells. Conclusion： GSK-3β was involved in drug resistance regulation, and activation of β-catenin and inhibition of E2F-1 may be the most responsible for the enhancement of 5-FU chemotherapy resistance induced by GSK-β inhibitor β10 in colon cancer.

Mixed extragonadal germ cell tumor arising from the prostate： a rare combination

Dear Editor, We report a rare case of a mixed germ cell tumor （GCT） that most likely arose from the prostate and included four components： an immature teratoma, a seminoma, an embryonal carcinoma and an endodermal sinus tumor （EST）.

Cellular zinc metabolism and zinc signaling:from biological functions to diseases and therapeutic targets

Zinc metabolism at the cellular level is critical for many biological processes in the body.A key observation is the disruption of cellular homeostasis,often coinciding with disease progression.As an essential factor in maintaining cellular equilibrium,cellular zinc has been increasingly spotlighted in the context of disease development.Extensive research suggests zinc’s involvement in promoting malignancy and invasion in cancer cells,despite its low tissue concentration.This has led to a growing body of literature investigating zinc’s cellular metabolism,particularly the functions of zinc transporters and storage mechanisms during cancer progression.Zinc transportation is under the control of two major transporter families:SLC30(ZnT)for the excretion of zinc and SLC39(ZIP)for the zinc intake.Additionally,the storage of this essential element is predominantly mediated by metallothioneins(MTs).This review consolidates knowledge on the critical functions of cellular zinc signaling and underscores potential molecular pathways linking zinc metabolism to disease progression,with a special focus on cancer.We also compile a summary of clinical trials involving zinc ions.Given the main localization of zinc transporters at the cell membrane,the potential for targeted therapies,including small molecules and monoclonal antibodies,offers promising avenues for future exploration.

Screening and regulatory network analysis of survival-related genes of patients with colorectal cancer

The purpose of this study was to screen key survival-related genes from patients with colorectal cancer and explore signal transduction network of the involved genes.In a previous study,survival-related genes of patients with colorectal cancer were selected by colorectal cancer-related expression data GSE17538 using the Significance Analysis of Microarrays(SAM3.01)software,and 235 genes related to the survival of patients with colorectal cancer were obtained.Therefore,the following screening and analysis were conducted on these 235 genes in this study.First,the enrichment analysis of transcription factor binding sites was conducted on the 235 genes.Genes with more than seven transcription factor binding sites were screened.Then,these genes and upregulated genes in colorectal cancer were intersected.Finally,survival analysis and regulatory network analysis were conducted on the screened genes.This allowed clarification of the relationship between these genes and the survival of patients with colorectal cancer and the signaling network involving these genes in the cell signal transduction network of colorectal cancer.Through the above analysis,six upregulated genes in colorectal cancer related to the survival of colorectal cancer patients and highly regulated by transcription factors were selected,namely STX2,PODXL,KLK6,GRB10,EHBP1 and CREB5.These genes are involved in signal regulatory networks related to colorectal cancer metastasis-related signaling pathways.Therefore,the survival of patients with colorectal cancer is closely correlated with colorectal cancer metastasis.The six survival-related genes affect the survival of patients by regulating colorectal cancer metastasis-associated signaling pathways.

Potential antitumor mechanisms of phenothiazine drugs

In this study,three kinds of phenothiazine drugs were analyzed to explore their potential antitumor mechanisms.First,target proteins that could interact with chlorpromazine,fluphenazine and trifluoperazine were predicted.Then,the target proteins of the three drugs were intersected.Cell signaling pathway enrichment and related disease enrichment were conducted for the intersected proteins to extract the enrichment categories associated with tumors.By regulation network analysis of the protein interactions,the mechanisms of action of these target proteins in tumor tissue were clarified,thus confirming the potential antitumor mechanisms of the phenothiazine drugs.The final results of cell signaling pathway enrichment and related disease enrichment showed that the categories with the highest score were all found in tumors.Target proteins belonging to the tumor category included signaling pathway members such as Wnt,MAPK and retinoic acid receptor.Moreover,another target protein,MAPK8,could indirectly act on target proteins CDK2,IGF1R,GSK3B,RARA,FGFR2 and MAPK10,thereby affecting tumor cell division and proliferation.Therefore,phenothiazine drugs may have potential antitumor effects,and tumorassociated target proteins play important roles in the process of cell signaling transduction cascades.

Potentiating CD8^(+) T cell antitumor activity by inhibiting PCSK9 to promote LDLR-mediated TCR recycling and signaling

Metabolic regulation has been proven to play a critical role in T cell antitumor immunity.However,cholesterol metabolism as a key component of this regulation remains largely unexplored.Herein,we found that the low-density lipoprotein receptor(LDLR),which has been previously identified as a transporter for cholesterol,plays a pivotal role in regulating CD8+T cell antitumor activity.Besides the involvement of cholesterol uptake which is mediated by LDLR in T cell priming and clonal expansion,we also found a non-canonical function of LDLR in CD8+T cells:LDLR interacts with the T-cell receptor(TCR)complex and regulates TCR recycling and signaling,thus facilitating the effector function of cytotoxic T-lymphocytes(CTLs).Furthermore,we found that the tumor microenvironment(TME)downregulates CD8+T cell LDLR level and TCR signaling via tumor cell-derived proprotein convertase subtilisin/kexin type 9(PCSK9)which binds to LDLR and prevents the recycling of LDLR and TCR to the plasma membrane thus inhibits the effector function of CTLs.Moreover,genetic deletion or pharmacological inhibition of PCSK9 in tumor cells can enhance the antitumor activity of CD8+T cells by alleviating the suppressive effect on CD8+T cells and consequently inhibit tumor progression.While previously established as a hypercholesterolemia target,this study highlights PCSK9/LDLR as a potential target for cancer immunotherapy as well.

Differences and similarities between extremely severe oligozoospermia and cryptozoospermia in intracytoplasmic sperm injection

Patients with extremely severe oligozoospermia （ESO） and cryptozoospermia （CO） are suitable using intracytoplasmic sperm injection （ICSI） as an infertility treatment. However, some andrologists are confused to distinguish ESO and CO in clinic diagnose. This study was designed for the first time to evaluate and compare patients with ESO and CO to determine whether these are useful clinical distinctions. A total of 270 infertile men in our center were classified into four groups as Group nonobstruction azoospermia （NOA, n = 44）, Group ESO （n = 78）, Group CO （n = 40）, and Group obstruction azoospermia （OA, n = 108）. Comparisons of the volume of bilateral testes, the level of follicle stimulating hormone （FSH） and inhibin B were obtained in four groups. Then comparisons of fertilization rates, cleavage rate, and excellent embryos rate were obtained when couples performed ICSh All indexes （volume of bilateral testis, level of FSH and inhibin B） in Groups ESO and CO were no difference, while Groups OA versus NOA, OA versus ESO, and OA versus CO were significant differences （P 〈 0.05）. The rates of fertilization were no differences in Groups ESO and CO while Groups OA versus ESO, OA versus CO were significant differences （P 〈 0.05）. Therefore, the spermatogenic functions in patients with CO and ESO were similar, better than NOA but worse than OA. However, it would be helpful to evaluate their spermatogenesis using testicular biopsies, especially accompanied azoospermia in clinical practice.

Depression accelerates gastric cancer invasion and metastasis by inducing a neuroendocrine phenotype via the catecholamine/β_(2)-AR/MACC1 axis

Background:Depression is a common,easily ignored,accompanied disease of gastric cancer(GC)patients and is often observed with elevated plasma catecholamine levels.Depression frequently promotes GC progression and leads to poor clinical outcomes;however,the molecular mechanisms underlying depression-induced GC progression remain poorly understood.We aimed to study the effects of depression on GC progression and explore possible mechanisms mediating the action of depression-associated catecholamines on GC.Methods:Depression states of GC patients were graded using the Patient Health Questionnaire-9,and plasma catecholamine levels were examined by high performance liquid chromatography coupled with tandem mass spectrometry.Migrative and invasive GC cells were examined using transwell assays,and metastatic GC niches were imaged using bioluminescence technology in a depression mouse model established with chronic unpredictable mild stress.Mouse depression-like behaviors were assessed through sucrose preference,forced swimming,and tail suspension tests.Characteristics of the neuroendocrine phenotype were observed via RT-PCR,Western blotting,flow cytometry,and transmission electron microscopy.Results:Fifty-one GC patients(age:53.61±1.79 years;cancer duration:3.71±0.33 months;depression duration:2.37±0.38 months;male-to-female ratio:1.55:1)were enrolled in the study.Depression grade was significantly higher in GC patients showing higher plasma levels of catecholamines(epinephrine:P=0.018;noradrenaline:P=0.009),higher oncogene metastasis-associated in colon cancer-1(MACC1)level(P=0.018),and metastasis(P<0.001).Further,depression-associated catecholamine specifically bound to the beta-2 adrenergic receptor(β_(2)-AR)and upregulated MACC1 expression,and thus promoting neuroendocrine phenotypic transformation through direct binding betweenMACC1 and synaptophysin.Eventually,the neuroendocrine phenotypic transformation accelerated GC invasion in vitro and metastasis in vivo.However,β_(2)-AR antagonist ICI-118,551 or MACC1 silencing effectively blocked the catecholamineinduced neuroendocrine phenotypic transformation and eliminated depressionenhanced GC migration and invasion.Moreover,β_(2)-AR blocking or MACC1 silencing prevented GC metastasis attributed to a neuroendocrine phenotype in a depression mouse model.Conclusions:Catecholamine-induced neuroendocrine phenotypes of GC cells led to depression-accelerated GC invasion and metastasis via the β_(2)-AR/MACC1 axis,while β_(2)-AR antagonist or MACC1 silencing could reverse it,showing promising potential therapeutic strategies for improving the outcome of GC patients with comorbid depression.

Genomic evolution during locoregional recurrence in colorectal cancer determined by whole-exome sequencing: a retrospective observational study

Objective:The genomic landscapes of metastatic colorectal cancer(mCRC)have been extensively studied;however,the genetic mechanisms underlying the locoregional recurrence(LR)of CRC remain unclear.The objective of our study was to investigate genomic evolution during LR in CRC using high-throughput sequencing.Methods:Twenty-three CRC patients with matched primary and LR tissues were recruited from Nanfang Hospital and Zhejiang Cancer Hospital between January 2011 and December 2018.The last date of follow-up was March 2020.Tissue samples were analyzed by whole-exome sequencing and the genomic profiles were depicted by single nucleotide variation,mutational signature,copy number variation,clonal architecture,and other features.The evolutionary process was speculated with comparison of the genetic variations between primary and LR lesions.The disseminating clusters from primary to LR lesions were identified by variant allele frequency dynamics.Furthermore,the early-recurrent biomarker was explored by comparing the indel signature between early-and late-recurrent patients.The study was approved by the Institutional Review Board of Nanfang Hospital of Southern Medical University(approval No.2020010)on September 11,2020.Results:The results highlighted distinct origins of LR between patients with high microsatellite instability and microsatellite stability.LR lesions evolved independently in patients with high microsatellite instability,while LR lesions were highly clonally related to the primary lesions in patients with microsatellite stability.Late-acquired variations in LR lesions encompassed a wide range of driver genes involved in histone methylation,DNA replication,T cell activation,PDCD1 gain,and LMNA loss.Furthermore,clonal analysis of the disseminating cells identified a dominant polyclonal seeding pattern during LR.The indel signature ID4 was associated with significantly shorter disease-free survival in patients with relapsed CRC according to a public dataset.Conclusion:These findings pose a challenge for the development of new approaches targeting the interactions of multiple clones in the establishment of LR and in terms of optimizing the clinical management of susceptible patients.

A preliminary pathological study on human allotransplantation

Objective: To observe the survival of hand allograft under the state of immunosuppression and the pathological changes of rejection in the recovery process. Methods: The biopsies of the skin, nerve, muscle, tendon and bone tissue of hand allografts during different stages from 1 day to 7 months after operation were observed using routine histological technique. Results: No significant changes due to rejection in skin, nerve, muscle and bone tissue were observed. But different degrees of weak rejective changes were found on the wall of blood vessels; in the muscle and nerve the reactions were markedly stronger than those found in skin tissues. Conclusions: The rejection in deep tissues should be monitored in controlling the rejection of hand allograft.

Spleen tyrosine kinase expression and its correlation with necrosis and high-risk histopathologic features in retinoblastoma

To the Editor:Retinoblastoma(RB)is the most common primary malignant intra-ocular tumor in children.It is thought to be initiated in response to biallelic RB1 inactivation and loss of functional RB protein.Spleen tyrosine kinase(SYK)plays different roles in the regulation of immunomodulatory signaling and cell proliferation in multiple malignancies,indicating either poor or favorable prognosis.A previous study showed that SYK is the fifth most significant gene and the only up-regulated kinase gene in RB according to the results derived from whole-genome sequencing,and SYK is also required for tumor cell survival.[1]However,the specific role of SYK in RB is still poorly understood.In this study,we examined SYK expression in RB and analyzed its relevance to necrosis and histopathologic high-risk factors(HRFs).