Polo-like kinase 1 as a biomarker predicts the prognosis and immunotherapy of breast invasive carcinoma patients

Invasive breast carcinoma(BRCA)is associated with poor prognosis and high risk of mortality.Therefore,it is critical to identify novel biomarkers for the prognostic assessment of BRCA.Methods:The expression data of polo-like kinase 1(PLK1)in BRCA and the corresponding clinical information were extracted from TCGA and GEO databases.PLK1 expression was validated in diverse breast cancer cell lines by quantitative real-time polymerase chain reaction(qRT-PCR)and western blotting.Single sample gene set enrichment analysis(ssGSEA)was performed to evaluate immune infiltration in the BRCA microenvironment,and the random forest(RF)and support vector machine(SVM)algorithms were used to screen for the hub infiltrating cells and calculate the immunophenoscore(IPS).The RF algorithm and COX regression model were applied to calculate survival risk scores based on the PLK1 expression and immune cell infiltration.Finally,a prognostic nomogram was constructed with the risk score and pathological stage,and its clinical potential was evaluated by plotting calibration charts and DCA curves.The application of the nomogram was further validated in an immunotherapy cohort.Results:PLK1 expression was significantly higher in the tumor samples in TCGA-BRCA cohort.Furthermore,PLK1 expression level,age and stage were identified as independent prognostic factors of BRCA.While the IPS was unaffected by PLK1 expression,the TMB and MATH scores were higher in the PLK1-high group,and the TIDE scores were higher for the PLK1-low patients.We also identified 6 immune cell types with high infiltration,along with 11 immune cell types with low infiltration in the PLK1-high tumors.A risk score was devised using PLK1 expression and hub immune cells,which predicted the prognosis of BRCA patients.In addition,a nomogram was constructed based on the risk score and pathological staging,and showed good predictive performance.Conclusions:PLK1 expression and immune cell infiltration can predict post-immunotherapy prognosis of BRCA patients.

Interleukin-1 receptor associated kinase 2 is a functional downstream regulator of complement factor D that controls mitochondrial fitness in diabetic cardiomyopathy

Diabetic cardiomyopathy is a disorder of the cardiac muscle that affects patients with diabetes.The exact mechanisms underlying diabetic cardiomyopathy are mostly unknown,but several factors have been implicated in the pathogenesis of the disease and its progression towards heart failure,including endothelial dysfunction,autonomic neuropathy,metabolic alterations,oxidative stress,and alterations in ion homeostasis,especially calcium transients[1].In Military Medical Research,Jiang et al.[2]sought to determine the functional role of complement factor D(Adipsin)in the pathophysiology of diabetic cardiomyopathy.

Diabetes and high-glucose could upregulate the expression of receptor for activated C kinase 1 in retina

BACKGROUND Diabetic retinopathy(DR)is a major ocular complication of diabetes mellitus,leading to visual impairment.Retinal pigment epithelium(RPE)injury is a key component of the outer blood retinal barrier,and its damage is an important indicator of DR.Receptor for activated C kinase 1(RACK1)activates protein kinase C-ε(PKC-ε)to promote the generation of reactive oxygen species(ROS)in RPE cells,leading to apoptosis.Therefore,we hypothesize that the activation of RACK1 under hypoxic/high-glucose conditions may promote RPE cell apoptosis by modulating PKC-ε/ROS,thereby disrupting the barrier effect of the outer blood retinal barrier and contributing to the progression of DR.AIM To investigate the role and associated underlying mechanisms of RACK1 in the development of early DR.METHODS In this study,Sprague-Dawley rats and adult RPE cell line-19(ARPE-19)cells were used as in vivo and in vitro models,respectively,to explore the role of RACK1 in mediating PKC-εin early DR.Furthermore,the impact of RACK1 on apoptosis and barrier function of RPE cells was also investigated in the former model.RESULTS Streptozotocin-induced diabetic rats showed increased apoptosis and upregulated expression of RACK1 and PKC-εproteins in RPE cells following a prolonged modeling.Similarly,ARPE-19 cells exposed to high glucose and hypoxia displayed elevated mRNA and protein levels of RACK1 and PKC-ε,accompanied by an increases in ROS production,apoptosis rate,and monolayer permeability.However,silencing RACK1 significantly downregulated the expression of PKC-εand ROS,reduced cell apoptosis and permeability,and protected barrier function.CONCLUSION RACK1 plays a significant role in the development of early DR and might serve as a potential therapeutic target for DR by regulating RPE apoptosis and barrier function.

MicroRNA-298 determines the radio-resistance of colorectal cancer cells by directly targeting human dual-specificity tyrosine(Y)-regulated kinase 1A

BACKGROUND Radiotherapy stands as a promising therapeutic modality for colorectal cancer(CRC);yet,the formidable challenge posed by radio-resistance significantly undermines its efficacy in achieving CRC remission.AIM To elucidate the role played by microRNA-298(miR-298)in CRC radio-resistance.METHODS To establish a radio-resistant CRC cell line,HT-29 cells underwent exposure to 5 gray ionizing radiation that was followed by a 7-d recovery period.The quantification of miR-298 levels within CRC cells was conducted through quantitative RT-PCR,and protein expression determination was realized through Western blotting.Cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and proliferation by clonogenic assay.Radio-induced apoptosis was discerned through flow cytometry analysis.RESULTS We observed a marked upregulation of miR-298 in radio-resistant CRC cells.MiR-298 emerged as a key determinant of cell survival following radiation exposure,as its overexpression led to a notable reduction in radiation-induced apoptosis.Intriguingly,miR-298 expression exhibited a strong correlation with CRC cell viability.Further investigation unveiled human dual-specificity tyrosine(Y)-regulated kinase 1A(DYRK1A)as miR-298’s direct target.CONCLUSION Taken together,our findings underline the role played by miR-298 in bolstering radio-resistance in CRC cells by means of DYRK1A downregulation,thereby positioning miR-298 as a promising candidate for mitigating radioresistance in CRC.

Crosslink among cyclin-dependent kinase 9,ATP binding cassette transporter G2 and Beclin 1 in colorectal cancer

Colorectal cancer(CRC)ranks third in the number of cancers mainly because of the inability to diagnose it at an early stage.The pathogenesis of CRC is complicated,which is the result of the complex interaction of multiple genetic and environmental factors.Currently,one of the main treatments for CRC is chemotherapy.But the primary cause of CRC treatment failure is drug resistance.The expression of cyclin-dependent kinase 9(CDK9)was correlated with elevated autophagy levels in colon cancer,and high expression of CDK9 indicates a poor prognosis in CRC.The incidence of autophagy and the expressions of Beclin 1 and ATP binding cassette transporter G2 are different in left and right colon cancer,and autophagy may be involved in the occurrence of chemotherapy resistance.In this article,the roles of CDK9,ATP binding cassette transporter G2 and Beclin 1 in CRC were elucidated,emphasizing the linkages among them and providing potential therapeutic targets of CRC.

Present and prospect of transarterial chemoembolization combined with tyrosine kinase inhibitor and PD-1 inhibitor for unresectable hepatocellular carcinoma

In this editorial,we comment on the article(World J Gastrointest Oncol 2024;16:1236-1247),which is a retrospective study of transarterial chemoembolization(TACE)combined with multi-targeted tyrosine kinase inhibitor(TKI)and programmed cell death protein-1(PD-1)inhibitor for the treatment of unresectable hepatocellular carcinoma(HCC).Herein,we focus specifically on the mechanisms of this triple therapy,administration sequence and selection of each medication,and implications for future clinical trials.Based on the interaction mechanisms between medications,the triple therapy of TACE+TKI+PD-1 is proposed to complement the deficiency of each monotherapy,and achieve synergistic antitumor effects.Although this triple therapy has been evaluated by several retrospective trials,it is still controversial whether the triple therapy achieves better clinical benefits,due to the flawed study design and heterogeneity in medications.In addition,the administration sequence,which may greatly affect the clinical benefit,needs to be fully considered at clinical decision-making for obtaining better prognosis.We hope that this editorial could contribute to the design and optimization of future trials.

Bibliometric analysis of phosphoglycerate kinase 1 expression in breast cancer and its distinct upregulation in triple-negative breast cancer

BACKGROUND Phosphoglycerate kinase 1(PGK1)has been identified as a possible biomarker for breast cancer(BC)and may play a role in the development and advancement of triple-negative BC(TNBC).AIM To explore the PGK1 and BC research status and PGK1 expression and mecha-nism differences among TNBC,non-TNBC,and normal breast tissue.METHODS PGK1 and BC related literature was downloaded from Web of Science Core Co-llection Core Collection.Publication counts,key-word frequency,cooperation networks,and theme trends were analyzed.Normal breast,TNBC,and non-TNBC mRNA data were gathered,and differentially expressed genes obtained.Area under the summary receiver operating characteristic curves,sensitivity and specificity of PGK1 expression were determined.Kaplan Meier revealed PGK1’s prognostic implication.PGK1 co-expressed genes were explored,and Gene Onto-logy,Kyoto Encyclopedia of Genes and Genomes,and Disease Ontology applied.Protein-protein interaction networks were constructed.Hub genes identified.RESULTS PGK1 and BC related publications have surged since 2020,with China leading the way.The most frequent keyword was“Expression”.Collaborative networks were found among co-citations,countries,institutions,and authors.PGK1 expression and BC progression were research hotspots,and PGK1 expression and BC survival were research frontiers.In 16 TNBC vs non-cancerous breast and 15 TNBC vs non-TNBC datasets,PGK1 mRNA levels were higher in 1159 TNBC than 1205 non-cancerous breast cases[standardized mean differences(SMD):0.85,95%confidence interval(95%CI):0.54-1.16,I²=86%,P<0.001].PGK1 expression was higher in 1520 TNBC than 7072 non-TNBC cases(SMD:0.25,95%CI:0.03-0.47,I²=91%,P=0.02).Recurrence free survival was lower in PGK1-high-expression than PGK1-low-expression group(hazard ratio:1.282,P=0.023).PGK1 co-expressed genes were concentrated in ATP metabolic process,HIF-1 signaling,and glycolysis/gluconeogenesis pathways.CONCLUSION PGK1 expression is a research hotspot and frontier direction in the BC field.PGK1 may play a strong role in promoting cancer in TNBC by mediating metabolism and HIF-1 signaling pathways.

Overexpression of mitogen-activated protein kinase phosphatase-1 in endothelial cells reduces blood-brain barrier injury in a mouse model of ischemic stroke

Ischemic stroke can cause blood-brain barrier(BBB)injury,which worsens brain damage induced by stroke.Abnormal expression of tight junction proteins in endothelial cells(ECs)can increase intracellular space and BBB leakage.Selective inhibition of mitogen-activated protein kinase,the negative regulatory substrate of mitogen-activated protein kinase phosphatase(MKP)-1,improves tight junction protein function in ECs,and genetic deletion of MKP-1 aggravates ischemic brain injury.However,whether the latter affects BBB integrity,and the cell type-specific mechanism underlying this process,remain unclear.In this study,we established an adult male mouse model of ischemic stroke by occluding the middle cerebral artery for 60 minutes and overexpressed MKP-1 in ECs on the injured side via lentiviral transfection before stroke.We found that overexpression of MKP-1 in ECs reduced infarct volume,reduced the level of inflammatory factors interleukin-1β,interleukin-6,and chemokine C-C motif ligand-2,inhibited vascular injury,and promoted the recovery of sensorimotor and memory/cognitive function.Overexpression of MKP-1 in ECs also inhibited the activation of cerebral ischemia-induced extracellular signal-regulated kinase(ERK)1/2 and the downregulation of occludin expression.Finally,to investigate the mechanism by which MKP-1 exerted these functions in ECs,we established an ischemic stroke model in vitro by depriving the primary endothelial cell of oxygen and glucose,and pharmacologically inhibited the activity of MKP-1 and ERK1/2.Our findings suggest that MKP-1 inhibition aggravates oxygen and glucose deprivation-induced cell death,cell monolayer leakage,and downregulation of occludin expression,and that inhibiting ERK1/2 can reverse these effects.In addition,co-inhibition of MKP-1 and ERK1/2 exhibited similar effects to inhibition of ERK1/2.These findings suggest that overexpression of MKP-1 in ECs can prevent ischemia-induced occludin downregulation and cell death via deactivating ERK1/2,thereby protecting the integrity of BBB,alleviating brain injury,and improving post-stroke prognosis.

三氧化二砷对结肠癌SW-480细胞凋亡及polo-like kinase-1基因表达的影响

目的观察三氧化二砷对人结肠癌细胞SW-480并探讨其作用机理。方法采用不同浓度的三氧化二砷作用结肠癌SW-480细胞后,采用MTT法检测细胞的恶性增殖,采用琼脂糖凝胶电泳和流式细胞仪检测凋亡及细胞周期情况,采用定量PCR检测polo-likekinase-1(PLK1)mRNA水平。结果三氧化二砷能抑制结肠癌细胞的恶性增殖,且与浓度相关。三氧化二砷能诱导结肠癌细胞凋亡,且呈浓度依赖性。流式细胞仪检测发现,三氧化二砷将细胞阻滞在G2/M期。三氧化二砷能下调结肠癌PLK1mRNA水平,且呈药物浓度和作用时间依赖性。结论三氧化二砷有效地抑制结肠癌SW-480细胞增殖,其机制可能与其下调PLK1表达,从而诱导凋亡有关。

Death-associated protein kinase 1 is associated with cognitive dysfunction in major depressive disorder

We previously showed that death-associated protein kinase 1(DAPK1)expression is increased in hippocampal tissue in a mouse model of major depressive disorde and is related to cognitive dysfunction in Alzheimer's disease.In addition,depression is a risk factor for developing Alzheimer's disease,as well as an early clinical manifestation of Alzheimer's disease.Meanwhile,cognitive dysfunction is a distinctive feature of major depressive disorder.Therefore,DAPK1 may be related to cognitive dysfunction in major depressive disorder.In this study,we established a mouse model of major depressive disorder by housing mice individually and exposing them to chronic,mild,unpredictable stressors.We found that DAPK1 and tau protein levels were increased in the hippocampal CA3 area,and tau was hyperphosphorylated at Thr231,Ser262,and Ser396 in these mice.Furthermore,DAPK1 shifted from axonal expression to overexpression on the cell membrane.Exercise and treatment with the antidepressant drug citalopram decreased DAPK1 expression and tau protein phosphorylation in hippocampal tissue and improved both depressive symptoms and cognitive dysfunction.These results indicate that DAPK1 may be a potential reason and therapeutic target of cognitive dysfunction in major depressive disorder.

RNAi沉默Polo-like kinase-1基因表达对胰腺癌细胞增殖的影响

目的:探讨polo-like kinase-1(PLK1)基因在胰腺癌细胞中的作用.方法:采用PLK1小干扰核糖核酸分子(small interfering RNA,siRNA)转染人胰腺癌Mi- aPaCa-2细胞后,分别采用实时定量PCR和Western blot检测PLK1基因mRNA和蛋白表达水平,观察PLK1 siRNA转染对胰腺癌细胞体内外增殖的影响.于转染不同时间后收集细胞,分别采用琼脂糖凝胶电泳和TUNEL方法检测胰腺癌细胞凋亡情况.结果:胰腺癌MiaPaCa-2细胞经siRNA转染处理后,PLK1 mRNA和蛋白表达水平明显下降(P<0.05).PLK1基因siRNA可明显抑制癌细胞体外生长(P<0.05)和体内裸鼠模型增殖(P<0.05).细胞凋亡检测发现,DNA电泳出现明显的梯度图谱,且与浓度相关(r=0.836,P<0.05).TUNEL结果显示,转染组癌细胞凋亡指数明显增加,且呈时间和浓度依赖性(r= 0.875,P<0.05).结论:PLK1 siRNA转染可明显抑制胰腺癌细胞增殖,其机制可能与诱导细胞凋亡有关.

Tousled-like kinase 1 promotes gastric cancer progression by regulating the tumor growth factor-beta signaling pathway

BACKGROUND The role of Tousled-like kinase 1(TLK1)in in gastric cancer(GC)remains unclear.AIM To investigate the expression,biological function,and underlying mechanisms of TLK1 in GC.METHODS We measured TLK1 protein expression levels and localized TLK1 in GC cells and tissues by western blot and immunofluorescence,respectively.We transfected various GC cells with lentiviruses to create TLK1 overexpression and knockdown lines and established the functional roles of TLK1 through in vitro colony formation,5-ethynyl-2`-deoxyuridine,and Transwell assays as well as flow cytometry.We applied bioinformatics to elucidate the signaling pathways associated with TLK1.We performed in vivo validation of TLK1 functions by inducing subcutaneous xenograft tumors in nude mice.RESULTS TLK1 was significantly upregulated in GC cells and tissues compared to their normal counterparts and was localized mainly to the nucleus.TLK1 knockdown significantly decreased colony formation,proliferation,invasion,and migration but increased apoptosis in GC cells.TLK1 overexpression had the opposite effects.Bioinformatics revealed,and subsequent experiments verified,that the tumor growth factor-beta signaling pathway was implicated in TLK1-mediated GC progression.The in vivo assays confirmed that TLK1 promotes tumorigenesis in GC.CONCLUSION The findings of the present study indicated that TLK1 plays a crucial role in GC progression and is,therefore,promising as a therapeutic target against this disease.

Inhibition of Cyclin F Promotes Cellular Senescence through Cyclin-dependent Kinase 1-mediated Cell Cycle Regulation

Objective Kidney renal clear cell carcinoma(KIRC)is a common renal malignancy that has a poor prognosis.As a member of the F box family,cyclin F(CCNF)plays an important regulatory role in normal tissues and tumors.However,the underlying mechanism by which CCNF promotes KIRC proliferation still remains unclear.Methods Bioinformatics methods were used to analyze The Cancer Genome Atlas(TCGA)database to obtain gene expression and clinical prognosis data.The CCK8 assay,EdU assay,and xenograft assay were used to detect cell proliferation.The cell senescence and potential mechanism were assessed by SA-β-gal staining,Western blotting,as well as ELISA.Results Our data showed that CCNF was highly expressed in KIRC patients.Meanwhile,downregulation of CCNF inhibited cell proliferation in vivo and in vitro.Further studies showed that the reduction of CCNF promoted cell senescence by decreasing cyclin-dependent kinase 1(CDK1),increasing the proinflammatory factors interleukin(IL)-6 and IL-8,and then enhancing the expression of p21 and p53.Conclusion We propose that the high expression of CCNF in KIRC may play a key role in tumorigenesis by regulating cell senescence.Therefore,CCNF shows promise as a new biomarker to predict the clinical prognosis of KIRC patients and as an effective therapeutic target.

Deleted in liver cancer 1 suppresses the growth of prostate cancer cells through inhibiting Rho-associated protein kinase pathway

Objective:Deleted in liver cancer 1(DLC1)is a GTPase-activating protein that is reported as a suppressor in certain human cancers.However,the detailed biological function of DLC1 is still unclear in human prostate cancer(PCa).In the present study,we aimed to explore the function of DLC1 in PCa cells.Methods:Silencing and overexpression of DLC1 were induced in an androgen-sensitive PCa cell line(LNCaP)using RNA interference and lentiviral vector transduction.The Cell Counting Kit-8 assay was performed to determine cell proliferation.The cell cycle was examined by performing a propidium iodide staining assay.Results:Our results indicated that DLC1 overexpression markedly suppressed the proliferation and cell cycle progression of LNCaP cells.Moreover,DLC1 expression was negatively correlated with Rho-associated protein kinase(ROCK)expression in LNCaP cells.Importantly,this study showed that the ROCK inhibitor Y27632 restored the function of DLC1 in LNCaP cells and reduced the tumorigenicity of LNCaP cells in vivo.Conclusion:Our results indicated that DLC1 overexpression markedly suppressed the proliferation and cell cycle progression of PCa cells and negatively correlated with ROCK expression in PCa cells and tissue.

Suppressing high mobility group box-1 release alleviates morphine tolerance via the adenosine5'-monophosphate-activated protein kinase/heme oxygenase-1 pathway

Opioids,such as morphine,are the most potent drugs used to treat pain.Long-term use results in high tolerance to morphine.High mobility group box-1(HMGB1) has been shown to participate in neuropathic or inflammatory pain,but its role in morphine tolerance is unclear.In this study,we established rat and mouse models of morphine tolerance by intrathecal injection of morphine for 7 consecutive days.We found that morphine induced rat spinal cord neurons to release a large amount of HMGB1.HMGB1 regulated nuclear factor κB p65 phosphorylation and interleukin-1β production by increasing Toll-like receptor 4receptor expression in microglia,thereby inducing morphine tolerance.Glycyrrhizin,an HMGB1 inhibito r,markedly attenuated chronic morphine tole rance in the mouse model.Finally,compound C(adenosine 5’-monophosphate-activated protein kinase inhibitor) and zinc protoporphyrin(heme oxygenase-1 inhibitor)alleviated the morphine-induced release of HMGB1 and reduced nuclear factor κB p65 phosphorylation and interleukin-1β production in a mouse model of morphine tolerance and an SH-SY5Y cell model of morphine tole rance,and alleviated morphine tolerance in the mouse model.These findings suggest that morphine induces HMGB1 release via the adenosine 5’-monophosphate-activated protein kinase/heme oxygenase-1 signaling pathway,and that inhibiting this signaling pathway can effectively reduce morphine tole rance.

Mammalian Ste20-like kinase 1 inhibition as a cellular mediator of anoikis in mouse bone marrow mesenchymal stem cells

BACKGROUND The low survival rate of mesenchymal stem cells(MSCs)caused by anoikis,a form of apoptosis,limits the therapeutic efficacy of MSCs.As a proapoptotic molecule,mammalian Ste20-like kinase 1(Mst1)can increase the production of reactive oxygen species(ROS),thereby promoting anoikis.Recently,we found that Mst1 inhibition could protect mouse bone marrow MSCs(mBMSCs)from H 2 O 2-induced cell apoptosis by inducing autophagy and reducing ROS production.However,the influence of Mst1 inhibition on anoikis in mBMSCs remains unclear.AIM To investigate the mechanisms by which Mst1 inhibition acts on anoikis in isolated mBMSCs.METHODS Poly-2-hydroxyethyl methacrylate-induced anoikis was used following the silencing of Mst1 expression by short hairpin RNA(shRNA)adenovirus transfection.Integrin(ITGs)were tested by flow cytometry.Autophagy and ITGα5β1 were inhibited using 3-methyladenine and small interfering RNA,respe-ctively.The alterations in anoikis were measured by Terminal-deoxynucleoitidyl Transferase Mediated Nick End Labeling and anoikis assays.The levels of the anoikis-related proteins ITGα5,ITGβ1,and phospho-focal adhesion kinase and the activation of caspase 3 and the autophagy-related proteins microtubules associated protein 1 light chain 3 II/I,Beclin1 and p62 were detected by Western blotting.RESULTS In isolated mBMSCs,Mst1 expression was upregulated,and Mst1 inhibition significantly reduced cell apoptosis,induced autophagy and decreased ROS levels.Mechanistically,we found that Mst1 inhibition could upregulate ITGα5 and ITGβ1 expression but not ITGα4,ITGαv,or ITGβ3 expression.Moreover,autophagy induced by upregulated ITGα5β1 expression following Mst1 inhibition played an essential role in the protective efficacy of Mst1 inhibition in averting anoikis.CONCLUSION Mst1 inhibition ameliorated autophagy formation,increased ITGα5β1 expression,and decreased the excessive production of ROS,thereby reducing cell apoptosis in isolated mBMSCs.Based on these results,Mst1 inhibition may provide a promising strategy to overcome anoikis of implanted MSCs.

Overexpression of polo-like kinase1 predicts a poor prognosis in hepatocellular carcinoma patients

AIM： To elucidate the role of overexpressed polo-like kinasel （PLK1）in hepatocellular carcinoma （HCC）. METHODS： We prospectively collected clinicopathological, immunohistochemical and semi-quantitative reverse transcription-polymerase chain reaction （RT-PCR） data from 135 HCC patients undergoing successful hepatectomy. The correlations between PLK1 mRNA expression and clinicopathologic variables were analyzed by Mann-Whitney U test. Prognostic factors were identified by univariate and multivariate analyses. RESULTS： Immunohistochemical results showed overexpression of PLK1 was mainly found in tumor tissues compared with tumor-free tissue. A similar mRNA result was obtained by semi-quantitative RT-PCR. A total of 111 samples were positive for PLK1 mRNA expression. The positive expression was correlated with venous invasion, tumor nodules and Edmondson grade. Furthermore, 1, 3, 5-year survival rates in the positive expression group were significantly lower than the negative control group. Multivariate analysis showed that positive PLK1 expression was an independent risk factor for HCC. CONCLUSION： PLK1 could be a potential biomarker for diagnosis and therapy for HCC.

Polo-like kinase 1,a new therapeutic target in hepatocellular carcinoma

AIM： To investigate the role of polo-like kinase 1 （PLK1） as a therapeutic target for hepatocellular carcinoma （HCC）. METHODS： PLK1 gene expression was evaluated in HCC tissue and HCC cell lines. Gene knockdown with short-interfering RNA （siRNA） was used to study PLK1 gene and protein expression using real-time reverse transcription polymerase chain reaction （RT-PCR） and Western blotting, and cell proliferation using 3-（4,5-dim ethylthiazol-2-yl）-5-（3-carboxymethoxyphenyl）-2（4-sulf ophenyl）-2H-tetrazolium （MTS） and bromodeoxyuridine （BrdU） assays. Apoptosis was evaluated using the terminal deoxynucleotidyl transferase dUTP nick end label- ing （TUNEL） assay, and caspase-inhibition assay. Huh-7 cells were transplanted into nude mice and co-cultured with PLK1 siRNA or control siRNA, and tumor progres- sion was compared with controls. RESULTS： RT-PCR showed that PLK1 was overexpre- ssed 12-fold in tumor samples compared with controls, and also was overexpressed in Huh-7 cells, siRNA against PLK1 showed a reduction in PLK1 gene and protein expression of up to 96% in Huh-7 cells, and a reduction in cell proliferation by 68% and 92% in MTS and BrdU cell proliferation assays, respectively. There was a 3-fold increase in apoptosis events, and TUNEL staining and caspase-3 assays suggested that this was caspase-independent. The pan-caspase inhibitor Z-VAD-FMK was unable to rescue the apoptotic cells. Immnofluorescence co-localized endonuclease-G to fragmented chromosomes, implicating it in apoptosis. Huh-7 cells transplanted subcutaneously into nude mice showed tumor regression in siPLKl-treated mice, but not in controls. CONCLUSION： Knockdown of PLK1 overexpression in HCC was shown to be a potential therapeutic target, leading to apoptosis through the endonuclease-G path- way.

Dual targeting of Polo-like kinase 1 and baculoviral inhibitor of apoptosis repeat-containing 5 in TP53-mutated hepatocellular carcinoma

BACKGROUND Hepatocellular carcinoma(HCC),often diagnosed at advanced stages without curative therapies,is the fifth most common malignant cancer and the second leading cause of cancer-related mortality.Polo-like kinase 1(PLK1)is activated in the late G2 phase of the cell cycle and is required for entry to mitosis.Interestingly,PLK1 is overexpressed in many HCC patients and is highly associated with poor clinical outcome.Baculoviral inhibitor of apoptosis repeatcontaining 5(BIRC5)is also highly overexpressed in HCC and plays key roles in this malignancy.AIM To determine the expression patterns of PLK1 and BIRC5,as well as their correlation with p53 mutation status and patient clinical outcome.METHODS The expression patterns of PLK1 and BIRC5,and their correlation with p53 mutation status or patient clinical outcome were analyzed using a TCGA HCC dataset.Cell viability,cell apoptosis,and cell cycle arrest assays were conducted to investigate the efficacy of the PLK1 inhibitors volasertib and GSK461364 and the BIRC5 inhibitor YM155,alone or in combination.The in vivo efficacy of volasertib and YM155,alone or in combination,was assessed in p53-mutated Huh7-derived xenograft models in immune-deficient NSIG mice.RESULTS Our bioinformatics analysis using a TCGA HCC dataset revealed that PLK1 and BIRC5 were overexpressed in the same patient subset and their expression was highly correlated.The overexpression of both PLK1 and BIRC5 was more frequently detected in HCC with p53 mutations.High PLK1 or BIRC5 expression significantly correlated with poor clinical outcome.PLK1 inhibitors(volasertib and GSK461364)or a BIRC5 inhibitor(YM155)selectively targeted Huh7 cells with mutated p53,but not HepG2 cells with wild-type p53.The combination treatment of volasertib and YM155 synergistically inhibited the viability of Huh7 cells via apoptotic pathway.The efficacy of volasertib and YM155,alone or in combination,was validated in vivo in a Huh7-derived xenograft model.CONCLUSION PLK1 and BIRC5 are highly co-expressed in p53-mutated HCC and inhibition of both PLK1 and BIRC5 synergistically compromises the viability of p53-mutated HCC cells in vitro and in vivo.

Effect of Antisense RNA Targeting Polo-like Kinase 1 on Cell Growth in A549 Lung Cancer Cells

In order to investigate the effect of Polo-like kinase-1 （Plk1） depletion on cell cycle progression and cell growth in lung cancer cells, a recombinant plasmid containing antisense RNA targeting Plk1 （pcDNA3-Plk1） was transfected into A549 cells by lipofectine. RT-PCR and Western-blot were used to detect the Plk1 gene expression. Cell proliferation was evaluated by direct cell counting and bromodeoxyuridine （BrdU） labeling. Cell cycle distribution and apoptosis were examined by flow cytometry, and the inhibition rate （IR） by vinorebline （NVB） was determined by MTF assay. The results showed that after transfection of pcDNA3-Plk1 into A549 cells, the expression levels of Plk1 mRNA and protein were greatly decreased. In pcDNA3-Plk1 transfected groups, abnormal morphological changes of cells and growth inhibition were observed, and the BrdU labeling index was significantly lower than in the control groups （P〈0.05）. Cells in pcDNA3-Plk1 transfected groups were arresed in G2/M phase and apoptosis was detectable 72 h post transfection. IR induced by vinorebline in pcDNA3-Plk1 transfected groups was significantly higher than in other groups. These data suggested that antisense RNA targeting Plk1 could suppress the Plk1 expression, and therefore, significantly inhibit cell proliferation and induce cell cycle arrest and apoptosis. Moreover, it sensitized lung cancer cells to chemotherapy.