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.

Expression of cyclin-dependent kinase inhibitor 2A 16,tumour protein 53 and epidermal growth factor receptor in salivary gland carcinomas is not associated with oncogenic virus infection

It is known that human papillomavirus （HPV） infection can cause squamous cell neoplasms at several sites, such as cervix uteri carcinoma and oral squamous carcinoma. There is little information on the expression of HPV and its predictive markers in tumours of the major and minor salivary glands of the head and neck. We therefore assessed oral salivary gland neoplasms to identify associations between HPV and infection-related epidermal growth factor receptor （EGFR）, cyclin-dependent kinase inhibitor 2A （CDKN2A/p16） and tumour protein p53 （TP53）. Formalin-fixed, paraffin-embedded tissue samples from oral salivary gland carcinomas （n=51） and benign tumours （n=26） were analysed by polymerase chain reaction （PCR） analysis for several HPV species, including high-risk types 16 and 18. Evaluation of EGFR, CDKN2A, TP53 and cytomegalovirus （CMV） was performed by immunohistochemistry. Epstein-Barr virus （EBV） was evaluated by EBV-encoded RNA in situ hybridisation. We demonstrated that salivary gland tumours are not associated with HPV infection. The expression of EGFR, CDKN2A and TP53 may be associated with tumour pathology but is not induced by HPV. CMV and EBV were not detectable. In contrast to oral squamous cell carcinomas, HPV, CMV and EBV infections are not associated with malignant or benign neoplastic lesions of the salivary glands.

Cyclin E、CDK_2和p21^(WAF1)在食管上皮癌变过程中的表达及意义

目的 探讨食管上皮癌变过程中细胞周期调控因子cyclin E、CDK2和p21WAF1的表达状况及其意义。方法 应用免疫组化SP法和原位杂交方法分别检测48例食管癌组织、31例非典型增生组织和17例正常食管粘膜中cyclin E、CDK2和p21WAF1蛋白及mRNA表达。应用半定量RT PCR和Western blot检测22例新鲜食管癌及相应癌旁组织的mRNA和蛋白表达。结果 从食管正常粘膜、非典型增生组织到癌组织,cyclin E和CDK2蛋白和mRNA阳性表达率逐渐上升,差异具有统计学意义(P<0.01或P<0.05)。食管癌组织中cyclin E、CDK2和p21WAF1蛋白及mRNA高表达,与癌旁组织或切缘正常食管粘膜有显著性差异(P<0.01)。cyclin E、CDK2和p21WAF1 基因表达显著正相关(P<0.01 或P<0.05)。结论 食管上皮癌变过程中,细胞周期相关基因cyclin E和CDK2表达逐渐增强。cyclin E基因表达异常是食管癌变过程中的早期事件。p21WAF1 基因在食管癌中高表达,可能与细胞周期调控的反馈机制有关。

Promoter methylation status of hMLH1,MGMT,and CDKN2A/p16 in colorectal adenomas

AIM:To investigate aberrant DNA methylation of CpG islands and subsequent low-or high-level DNA microsatellite instability(MSI)which is assumed to drive colon carcinogenesis. METHODS:DNA of healthy individuals,adenoma(tu-bular or villous/tubulovillous)patients,and colorectal carcinoma patients who underwent colonoscopy was used for assessing the prevalence of aberrant DNA methylation of human DNA mismatch repair gene mutator L homologue 1(hMLH1),Cyclin-dependent kinase inhibitor 2A(CDKN2A/p16),and O-6-methylguanine DNA methyltransferase(MGMT),as well as their rela- tion to MSI. RESULTS:The frequency of promoter methylation for each locus increased in the sequence healthy tissue/adenoma/carcinoma.MGMT showed the highest frequency in each group.MGMT and CDKN2A/p16 presented a statistically significant increase in promoter methylation between the less and more tumorigenic forms of colorectal adenomas(tubular vs tubullovillous and villous adenomas).All patients with tubulovillous/villous adenomas,as well as all colorectal cancer patients,showed promoter methylation in at least one of the examined loci.These findings suggest a potentially crucial role for methylation in the polyp/adenoma to cancer progres- sion in colorectal carcinogenesis.MSI and methylation seem to be interdependent,as simultaneous hMLH1, CDKN2A/p16,and MGMT promoter methylation was present in 8/9 colorectal cancer patients showing the MSI phenotype. CONCLUSION:Methylation analysis of hMLH1,CD- KN2A/p16,and MGMT revealed specific methylation profiles for tubular adenomas,tubulovillous/villous adenomas,and colorectal cancers,supporting the use of these alterations in assessment of colorectal tumorigenesis.

MiR-451 inhibits proliferation of esophageal carcinoma cellline EC9706 by targeting CDKN2D and MAP3K1

AIM To investigate the underlying molecularmechanisms of miR-451 to inhibit proliferation ofesophageal carcinoma cell line EC9706.METHODS： Assays for cell growth, apoptosis andinvasion were used to evaluate the effects of miR-451expression on EC cells. Luciferase reporter and Westernblot assays were used to test whether cyclin-dependentkinase inhibitor 2D （CDKN2D） and MAP3K1 act as majortargets of miR-451.RESULTS： The results showed that CDKN2D andMAP3K1 are direct targets of miR-451. CDKN2D andMAP3K1 overexpression reversed the effect of miR-451.MiR-451 inhibited the proliferation of EC9706 bytargeting CDKN2D and MAP3K1.CONCLUSION： These findings suggest that miR-451might be a novel prognostic biomarker and a potentialtarget for the treatment of esophageal squamous cellcarcinoma in the future.

Comprehensive proteomic signature and identification of CDKN2A as a promising prognostic biomarker and therapeutic target of colorectal cancer

BACKGROUND The carcinogenesis of colorectal cancer(CRC)involves many different molecules and multiple pathways,and the specific mechanism has not been elucidated until now.Existing studies on the proteomic signature profiles of CRC are relatively limited.Therefore,we herein aimed to provide a more comprehensive proteomic signature profile and discover new prognostic markers and therapeutic targets by performing proteomic analysis of CRC and paired normal tissues.AIM To investigate the proteomic signature and identify novel protein prognostic biomarkers of CRC.METHODS Cancer tissues and paired normal tissues were collected from 48 patients who underwent surgical removal at the China-Japan Friendship Hospital from January 2020 to June 2021.Data independent acquisition(DIA)quantitative proteomic analysis was performed using high-performance liquid chromatography–mass spectrometry/mass spectrometry(nano-UHPLC–MS/MS)to identify differen tially expressed proteins,among which those with a P adj value(t test,BH correction)<0.05 and an absolute fold change(|log2FC|)>2 were identified as potential markers.Differentially expressed proteins were selected by bioinformatics analysis and validated by immunohistochemical tissue microarrays,and their association with prognosis was further analyzed with the Gene Expression Profiling Interactive Analysis database to identify prognostic protein biomarkers of CRC.RESULTS Significantly differential protein expression was observed between cancer tissues and normal tissues.Compared with normal tissues,1115 proteins were upregulated and 705 proteins were downregulated in CRC based on P adj<0.05 and|log2FC|>2,and bioinformatics analysis revealed that the differentially expressed proteins were involved in multiple biological processes associated with tumorigenesis,including ribosome biogenesis in eukaryotes,focal adhesion,extracellular matrix-receptor interactions and other tumor metabolism processes.Moreover,cyclin-dependent kinase inhibitor 2A(CDKN2A)expression was markedly upregulated in CRC,as validated by immunohistochemistry(0.228 vs 0.364,P=0.0044),and was significantly enriched in tumor proliferation and signal transduction pathways such as the cell cycle and p53 signaling pathways.High CDKN2A expression was significantly correlated with poor prognosis(P=0.021).These results demonstrated that CDKN2A functions as a driver of CRC.CONCLUSION Our study provides a comprehensive proteomic signature of CRC and highlights CDKN2A as a potential powerful prognostic marker and precision therapeutic target.

Anti-proliferative effects of a small molecule inhibitor of CDKAT7519 on chronic myeloid leukemia (CML) cells through haltingthe transition of cells from G2/M phase of the cell cycle

Pathogenesis of chronic myeloid leukemia(CML)has mostly been studied with regard to the oncogenic role of BCR/ABL fusion,however,recent disclosures have declared that the challenges with the treatment of CML patients would not be resolved until the role of other aberrancies is ignored.Given the involvement of cyclin-dependent kinases(CDKs)in the pathogenesis of CML,the present study aimed to investigate the effects of a multi-CDK inhibitor AT7519 on BCR/ABL-harboring CML-derived K562 cells.Our results showed that AT7519 effectively reduced the survival of K562 and induced its anti-proliferative effect through the induction of G2/M arrest due to elevated p21 and p27.The resulting data also revealed that either direct or indirect suppression of c-Myc using specific c-Myc inhibitor 10058-F4 and selective PI3K inhibitor CAL-101 resulted in a superior cytotoxicity,suggesting that the activation of PI3K pathway could attenuate antileukemic effects of the inhibitor,at least partly,through a c-Mycdependent mechanism.To the best of our knowledge,to date,no study has addressed the effect of autophagy on CML cell response to AT7519,and,herein,we proposed for the first time that the suppression of autophagy boosted AT7519 cytotoxicity against K562.Overall,we suggested that selective CDK inhibitor AT7519 exerted antileukemic effect against CML cells and propose a novel therapeutic application for the inhibitor either as a single agent or in combination with c-Myc and/or PI3K inhibitors.

Expressions of miRNA29 target genes CCND2 and CDK6 in cervical cancer

Objective: To study the expression of miRNA29 target genes recombinant cyclin D2 (CCND2) and cyclin-dependent kinase 6 (CDK6) in cervical squamous cell carcinoma tissues and their relationship with clinicopathological factors. Methods: Levels of mRNA of CCND2 and CDK6 in cervical squamous cell carcinoma tissues, cervical intraepithelial neoplasia (CIN) tissues and normal cervical tissues were detected by reverse transcription-polymerase chain reaction (RT-PCR). Results: There was a statistical difference in the expression of CCND2 (one of miRNA29 target genes) in normal cervical tissues, CIN tissues and cervical squamous cell carcinoma tissues (H = 29.27, p = .00), but there was no statistical difference in the expression of CDK6 (one of miRNA29 target genes) in them (H = 2.76, p = .25). CCND2 was positively correlated to CDK6 in CIN tissues (r = 0.58, p < .05). Conclusions: CCND2, one of miRNA29 target genes, may be involved in the occurrence and development of cervical cancer, but CDK6 is less relevant to the occurrence and development of cervical cancer;CCND2 and CDK6 may play a synergistic role in the occurrence and development of CIN.

Human cytomegalovirus RNA2.7 inhibits RNA polymeraseⅡ(PolⅡ)Serine-2 phosphorylation by reducing the interaction between PolⅡand phosphorylated cyclin-dependent kinase 9(pCDK9)

Human cytomegalovirus(HCMV)is a ubiquitous pathogen belongs to betaherpesvirus subfamily.RNA2.7 is a highly conserved long non-coding RNA accounting for more than 20%of total viral transcripts.In our study,functions of HCMV RNA2.7 were investigated by comparison of host cellular transcriptomes between cells infected with HCMV clinical strain and RNA2.7 deleted mutant.It was demonstrated that RNA polymeraseⅡ(PolⅡ)-dependent host gene transcriptions were significantly activated when RNA2.7 was removed during infection.A145 nt-in-length motif within RNA2.7 was identified to inhibit the phosphorylation of PolⅡSerine-2(PolⅡS2)by reducing the interaction between PolⅡand phosphorylated cyclin-dependent kinase 9(pCDK9).Due to the loss of PolⅡS2 phosphorylation,cellular DNA pre-replication complex(pre-RC)factors,including Cdt1 and Cdc6,were significantly decreased,which prevented more cells from entering into S phase and facilitated viral DNA replication.Our results provide new insights of HCMV RNA2.7 functions in regulation of host cellular transcription.

Xylocydine(5-氨甲酰基-4-氨基-6-溴-7-(β-L-呋喃木糖)吡咯[2,3-d]嘧啶)的合成

Xylocydine(5-氨甲酰基-4-氨基-6-溴-7-(β-L-呋喃木糖)吡咯[2,3-d]嘧啶)是一种新型的CDK(细胞周期蛋白依赖性激酶)抑制剂,具有较高的抗癌活性.以四氰乙烯和L-木糖为原料,首先分别合成了5-氰基-4-氨基-6-溴吡咯[2,3-d]嘧啶和1-O-乙酰基-2,3,5-三-O-苯甲酰基-L-呋喃木糖,然后将它们通过糖苷化反应、苯甲酰基水解和氰基氧化反应,合成得到了Xylocydine,并通过1HNMR、13C NMR对其结构进行了表征.

Long non-coding RNA H19 promotes proliferation inhepatocellular carcinoma cells via H19/miR-107/CDK6 axis

Hepatocellular carcinoma (HCC) is the leading cause of cancer death worldwide;nevertheless, currenttherapeutic options are limited or ineffective for many patients. Therefore, elucidation of molecular mechanisms inHCC biology could yield important insights for the intervention of novel therapies. Recently, various studies havereported dysregulation of long non-coding RNAs (lncRNAs) in the initiation and progression of HCC, including H19;however, the biological function of H19 in HCC remains unclear. Here, we show that knockdown of H19 disruptedHCC cell growth, impaired the G1-to-S phase transition, and promoted apoptosis, while overexpression of H19yielded the opposite results. Screening for expression of cell cycle-related genes revealed a significant downregulationof CDK6 at both RNA and protein levels upon H19 suppression. Bioinformatic analysis of the H19 sequence and the3′ untranslated region (3′ UTR) of CDK6 transcripts showed several binding sites for microRNA-107 (miR-107), andthe dual luciferase reporter assay confirmed their direct interaction with miR-107. Consistently, blockage of miR-107activity alleviated the growth suppression phenotypes induced by H19 downregulation, suggesting that H19 serves asa molecular sponge for miR-107 to promote CDK6 expression and cell cycle progression. Together, this studydemonstrates a mechanistic function of H19 in driving the proliferation of HCC cells and suggests H19 suppressionas a novel approach for HCC treatment.

Synergistic Effects of Targeting Survivin and CDK1 on Nasopharyngeal Carcinoma <i>in Vitro</i>and <i>in Vivo</i>

Background: To explore the impact of pU6-based tandem survivin and CDK1-specific short hairpin RNA on the biological behaviors of CNE-2 nasopharyngeal carcinoma cells in vitro and in vivo. Patients and Methods: The vectors of pU6-survivinshRNA, pU6-CDK1shRNA and pU6-survivinshRNA-CDK1shRNA were constructed and transfected into CNE-2 cells with Lipofectamine TM 2000, respectively. The mRNAs and proteins of CDK1 and survivin were determined by RT-PCR and Western blotting, accordingly. MTT assay was employed to evaluate the proliferation of CNE-2 cells, and flow cytometry was performed to determine the apoptosis of CNE-2 cells. The effects of interfering survivin and CDK1 on tumorigenesis were evaluated by tumor xenografts experiments. Results: Effective plasmids were successfully constructed knocking down survivin and/or CDK1. The proliferation inhibition of CNE-2 cells by pU6-survivinshRNA-CDK1shRNA (32.5%) was higher than that of by pU6-survivinshRNA (25.6%) and pU6-CDK1shRNA (15.6%), and apoptosis in CNE-2 cells simultaneously interfering survivin and CDK1 (15.2%) dramatically increased when compared to those of interfering survivin (5.4%) or CDK1 (4.7%) alone. Furthermore, simultaneously interfering survivin and CDK1 is more effective than interfering alone component in inhibiting tumor growth of fBalb/C nude mice xenografted with CNE-2 cells. Conclusion: The results altogether indicate that interfering survivin and CDK1simutaneously can produce synergistic effects of anti-nasopharyngeal carcinoma, which could be a potential therapeutic method.

EXPRESSION OF CYCLIN D1 AND CDK4 IN OSTEOSARCOMA OF THE JAWS

Objective: To analyze cyclin D1 and cyclin-dependent kinase 4 (CDK4) expression and their significance in osteosarcoma of the jaws. Methods: Immunohistochemical ABC method was used to detect the expression of cyclin D1 and CDK4 in 20 cases of osteosarcoma and 8 cases of osteochondroma of the jaws. Results: The positive rates of cyclin D1 and CDK4 were 65% (13/20) and 60% (12/20), respectively. There was significant positive correlation between cyclin D1 and CDK4 expression (γs=0.48, P<0.05). Both cyclin D1 and CDK4 were present in 1/8 (12.5%) osteochondroma. The positive rate was remarkably different between osteosarcoma and osteochondroma (P<0.05). Conclusion: Cyclin D1 and CDK4 are overexpressed in osteosarcoma of the jaws and closely related to its occurrence and development.

Insights into the Use of CDK 4/6 Inhibitors in Patients with HR-positive Advanced or Metastatic Breast Cancer

Hormone receptor(HR)-positive breast cancer(BC)is the most common subtype of BC and some patients with such tumors experience recurrences.Endocrine-based therapy(ET)(e.g.,tamoxifen,aromatase inhibitors(AIs),and fulvestrant)that has improved outcomes in such patients represents the initial therapy for women with HR-positive/human epidermal growth factor receptor 2(HER2)-negative BC(considering no evidence of visceral crisis).However,the resistance to ET can occur in almost 50%of HR-positive BCs.In order to improve outcomes of patients with HR-positive metastatic BC,new treatment strategies are required.One such therapy is the new class of medications,cyclin-dependent kinase(CDK)4/6 inhibitors,that have improved the outcomes in such patients(both endocrine-sensitive and endocrine-resistant).This article presents evidence from the main clinical trials,which led to the approval of palbociclib,ribociclib,and abemaciclib.These three CDK 4/6 inhibitors have shown a significant improvement of the progression-free survival(PFS)in patients with HR-positive/HER2-negative metastatic BC when used in combination with selected ETs.In addition,some important patient management considerations,when choosing a particular CDK 4/6 inhibitor for an individual patient are presented.Furthermore,a need to find biomarkers for CDK 4/6 inhibitor sensitivity,efficacy,and resistance,to be able to precisely select the best patientcandidates for this treatment is highlighted.

Identification of Novel CDK9 Inhibitors with Better Inhibitory Activity and Higher Selectivity for Cancer Treatment by an Effective Two-Stage Virtual Screening Strategy

The aberrant overexpression of cyclin-dependent kinase 9 (CDK9) in cancer cells results in the loss of proliferative control, making it an attractive therapeutic target for various cancers. However, the highly structural similarity between CDK9 and CDK2 makes the development of novel selective CDK9 inhibitors a challenging task and thus limits their clinical applications. Here, an effective two-stage virtual screening strategy was developed to identify novel CDK9 inhibitors with better inhibitory activity and higher selectivity. The first screening stage aims to select potential compounds with better inhibitory activity than Roniciclib, one of the most effective CDK9 inhibitors, through reliable structure-based pharmacophoric virtual screening and accurate molecular docking analyses. The second stage employs a very detailed visual inspection process, in which several structural criteria describing the major difference between the binding pockets of CDK9 and CDK2 are taken into consideration, to identify compounds with higher selectivity than CAN508, one of the CDK9 inhibitors with distinguished selectivity. Finally, three compounds (NCI207113 from NCI database and TCM0004 and TCM3282 from TCM database) with better inhibitory activity and higher selectivity were successfully identified as novel CDK9 inhibitors. These three compounds also display excellent binding stabilities, great pharmacokinetic properties and low toxicity in MD simulations and ADMET predictions. Besides, the results of binding free energy calculations suggest that enhancing van der Waals interaction and nonpolar solvation energy and/or reducing polar solvation energy can significantly improve the binding affinity of these CDK9 inhibitors. Their clinical potentials to serve as anticancer drug candidates can be further evaluated through a series of in vitro/in vivo bioassays in the future. To the best of our knowledge, this is the first attempt to identify novel CDK9 inhibitors with both better inhibitory activity and higher selectivity through an effective two-stage virtual screening strategy.

Expressions of cyclin E, cyclin dependent kinase 2 and p57^(KIP2) in human gastric cancer

Objective To investigate the expressions of cyclin E, cyclin dependent kinase 2(CDK-2)and cyclin-dependent kinase inhibitor p57 KIP2 in human gastric cancer, and to evaluate the relationships between protein levels and clinicopathological parameters. Methods Western blot was used to measure the expressions of cyclin E, CDK-2 and p57 KIP2 proteins in the surgically resected gastric carcinoma, adjacent normal mucosa and metastatic lymph nodes from 36 patients. Results Cyclin E and CDK-2 protein levels were higher in gastric cancer tissues in comparison with normal tissues (P<0.05). Overexpression of cyclin E was correlated with lymph node involvement, poor histological grade and serosa invasion (P<0.05). Overexpression of CDK-2 was correlated with lymph nodes involvement (P<0.05). No statistically significant difference between cyclin E and CDK-2 expression was found when samples were stratified according to tumor size (P>0.05). Expression of cyclin E and CDK-2 showed a positive linear correlation (r=0.451, P=0.01). Protein levels of p57 KIP2 were lower in gastric cancer tissues than in the normal mucosa (P<0.05). Decreased expression of p57 KIP2 was correlated with lymph node involvement (P<0.05). No statistically significant difference in p57 KIP2 expression was found when sample were stratified according to tumor size, histological grade or serosa invasion (P>0.05). In metastatic lymph nodes, expression of cyclin E was increased and the expression of p57 KIP2 decreased. Conclusion Overexpressions of cyclin E, CDK-2 and downregulated expression of p57 KIP2 may play important roles in tumorigenesis and metastatic potential of gastric cancer.

Cell cycle regulation and anticancer drug discovery

Cellular growth,development,and differentiation are tightly controlled by a conserved biological mechanism:the cell cycle.This cycle is primarily regulated by cyclin-dependent kinase(CDK)-cyclin complexes,checkpoint kinases,and CDK inhibitors.Deregulation of the cell cycle is a hallmark of the transformation of normal cells into tumor cells.Given its importance in tumorigenesis,several cell cycle inhibitors have emerged as potential therapeutic drugs for the treatment of cancers-both as singleagent therapy and in combination with traditional cytotoxic or molecular targeting agents.In this review,we discuss the mechanisms underlying cell cycle regulation and present small-molecule anticancer drugs that are under development,including both pan-CDK inhibitors and CDK4/6-selective inhibitors.In addition,we provide an outline of some promising CDK inhibitors currently in preclinical and clinical trials that target cell cycle abnormalities in various cancers.

Cdk2 acts upstream of mitochondrial permeability transition during paclitaxel-induced apoptosis

Sequential activation of cyclin-dependent kinases(Cdks)controls mammalian cell cycle.Here we demonstrate that the upregulation of cyclin-dependent kinase 2(Cdk2)activity coincides with the loss of mitochondrial membrane potential(MMP)in paclitaxel-induced apoptosis.Ectopic expression of the dominant negative Cdk2(Cdk2-dn)and a specific Cdk2 inhibitor,p21WAF1/CIP1,effectively suppresses the loss of MMP,the release of cytochrome c,and subsequent activation of caspase-3 in paclitaxel-treated cells.Whereas forced activation of Cdk2 by overexpression of cyclin A dramatically promotes these events.We further show that Cdk2 activation status does not interfere with a procedure that lies downstream of cytochrome c release induced by Bax protein.These findings suggest that Cdk2 kinase can regulate apoptosis at earlier stages than mitochondrial permeability transition and cytochrome c release.

Repurposed benzydamine targeting CDK2 suppresses the growth of esophageal squamous cell carcinoma

Esophageal squamous cell carcinoma (ESCC) is one of the leading causes of cancer death worldwide. It is urgent to develop new drugs to improve the prognosis of ESCC patients. Here, we found benzydamine, a locally acting non-steroidal anti-inflammatory drug, had potent cytotoxic effect on ESCC cells. Benzydamine could suppress ESCC proliferation in vivo and in vitro. In terms of mechanism, CDK2 was identified as a target of benzydamine by molecular docking, pull-down assay and in vitro kinase assay. Specifically, benzydamine inhibited the growth of ESCC cells by inhibiting CDK2 activity and affecting downstream phosphorylation of MCM2, c-Myc and Rb, resulting in cell cycle arrest. Our study illustrates that benzydamine inhibits the growth of ESCC cells by downregulating the CDK2 pathway.

Cdk5 knocking out mediated by CRISPR-Cas9 genome editing for PD-L1 attenuation and enhanced antitumor immunity

Blocking the programmed death-ligand 1(PD-L1)on tumor cells with monoclonal antibody therapy has emerged as powerful weapon in cancer immunotherapy.However,only a minority of patients presented immune responses in clinical trials.To develop an alternative treatment method based on immune checkpoint blockade,we designed a novel and efficient CRISPR-Cas9 genome editing system delivered by cationic copolymer aPBAE to downregulate PD-L1 expression on tumor cells via specifically knocking out Cyclin-dependent kinase 5(Cdk5)gene in vivo.The expression of PD-L1 on tumor cells was significantly attenuated by knocking out Cdk5,leading to effective tumor growth inhibition in murine melanoma and lung metastasis suppression in triple-negative breast cancer.Importantly,we demonstrated that aPBAE/Cas9-Cdk5 treatment elicited strong T cell-mediated immune responses in tumor microenvironment that the population of CD8^+T cells was significantly increased while regulatory T cells(Tregs)was decreased.It may be the first case to exhibit direct in vivo PD-L1 downregulation via CRISPR-Cas9 genome editing technology for cancer therapy.It will provide promising strategy for preclinical antitumor treatment through the combination of nanotechnology and genome engineering.