Can cyclin-dependent kinase 4/6 inhibitors convert inoperable breast cancer relapse to operability? A case report

BACKGROUND Pathological complete response(pCR) is rare in hormone receptor-positive(HR+)HER2-negative breast cancer(BC) treated with either endocrine therapy(ET) or chemotherapy. Radical resection of locoregional relapse, although potentially curative in some cases, is challenging when the tumor invades critical structures.The oral cyclin-dependent kinase 4/6 inhibitor palbociclib in combination with ET has obtained a significant increase in objective response rates and progression-free survival in patients with advanced BC and is now being evaluated in the neoadjuvant setting. We present a clinical case of a patient with an inoperable locoregional relapse of HR+ HER2-negative BC who experienced p CR after treatment with palbociclib.CASE SUMMARY We report the clinical case of a 60-year-old patient who presented with an inoperable locoregional relapse of HR+, HER2-negative BC 10 years after the diagnosis of the primary tumor. During a routine follow-up visit, breast magnetic resonance imaging and positron emission tomography/computed tomography revealed a 4-cm lesion in the right subclavicular region, infiltrating the chest wall and extending to the subclavian vessels, but without bone or visceral involvement. Treatment was begun with palbociclib plus letrozole, converting the disease to operability over a period of 6 mo. Surgery was performed and a p CR achieved. Of note, during treatment the patient experienced a very uncommon toxicity characterized by burning tongue and glossodynia associated with dysgeusia, paresthesia, dysesthesia, and xerostomia. A reduction in the dose of palbociclib did not provide relief and treatment with the inhibitor was thus discontinued, resolving the tongue symptoms. Laboratory exams were unremarkable. Given that this was a late relapse, the tumor was classified asendocrine-sensitive, a condition associated with high sensitivity to palbociclib.CONCLUSION This case highlights the potential of the cyclin-dependent kinase 4/6 inhibitor plus ET combination to achieve pCR in locoregional relapse of BC, enabling surgical resection of a lesion initially considered inoperable.

Cyclin-dependent kinase 5 is required for suppressing D1-dependent signaling mediated through muscarinic 4 in isolated medium spiny neurons

OBJECTIVE Previous studies have demonstrated acetylcholine muscarinic 4(M4) receptor regulates DARPP-32 phosphorylation at Thr75 in isolated medium spiny neurons(MSNs),indicating antagonistic mechanism with D1 dependent signal cascade,but the exact molecular mechanisms remain unclearly.In this study,we investigated the roles of M4 receptor in modulation D1 dependent signal to integrate striatal DA inputs in isolated MSNs.METHODS(1)Lentivirus technology was employed to genetically knock down the M4 receptor of MSNs;(2) Apomorphine(APO),acts as a dopamine receptor agonist,while SCH23390,acts as a selective antagonist for D1,were used to study the pharmacologically profiles with D1 receptor stimulation or blockade,respectively.Then the no subtype-selective muscarinic agonist oxotremorine M(OX) were used to show that mAchRs activation,in order to dissect the particular function of M4,a selective M4 antagonist,MT3 was used;(3) Intracellular cAMP production of MSNs was measured by using time resolved fluorescence resonance energy transfer detection method;(4) Laser confocal was used to explore the expression of M4 and D1 in MSNs;(5) Immunofluorescence cytochemistry and Western blotting were used to confirm the alteration of signaling molecular including P-CREB,DARPP-32 P-Thr34,DARPP-32 P-Thr75,cyclin-dependent kinase 5(CDK5) as wel as p25/35,which are involved in DA-dependent signaling modulations.RESULTS Firstly,TR-FRET assay revealed APO(10-2 mol·L^(-1))significantly increased the level of intracellular cAMP(vs control,n=3,P<0.01),also Western blotting results showed that APO(10-6 mol · L^(-1))increased DARPP-32 Thr34 phosphorylation(vs control,n=3,P<0.01),and these effect were reversed by D1 receptor antagonist SCH23390(vs APO,n=3,P<0.01).Interestingly,we confirmed that OX(10-6 mol · L^(-1)) down-regulated APO-induced DARPP-32 Thr34 phosphorylation(vs APO,n=3,P<0.01),due to its effects on DARPP-32 phosphorylation at Thr75.The results presented the antagonistic mechanism of mAchRs stimulation with D1 dependent signal cascade in MSNs.Meanwhile,OX(10-7,10-6 and10^(-5) mol·L^(-1)) stimulated DARPP-32 phosphorylation at Thr75,and simultaneously up regulated P25/35 and CDK5 activity(vs control,n=3,P<0.01) by using Western blotting assay.Furthermore,roscovitine(10^(-5) mol · L^(-1)),acts as a CDK5 inhibitor,suppressed CDK5 activity(vs control,n=10,P<0.01),and fully inhibited OX-induced DARPP-32 Thr75 phosphorylation(vs OX,n=10,P<0.01).More important,pretreated with roscovitine(10^(-5) mol·L^(-1)),the effect of APO on DARPP-32 Thr34 phosphorylation was potentiated(vs APO,n=3,P<0.05).The result presented CDK5 is required in suppression of APO on DARPP-32 Thr34 phosphorylation mediated through mAchRs stimulation.In addition,laser confocal results showed that the CDK5 up-regulation was mostly confined to MSNs co-expressing M4,which means that M4 participated in CDK5-mediated phosphorylation of DARPP-32 at Thr75.Consistently,immunofluorescence and Western blotting results confirmed that both genetic knockdown and pharmacologic inhibition of M4 receptors with MT3(10-7 mol · L^(-1)) down-regulated the OX-induced the expression of CDK5(vs OX,n=3,P<0.01) and P25/35(vs OX,n=3,P<0.01)in isolated MSNs.CONCLUSION M4 receptor may play an important role in antagonistic regulation D1 dependent signaling,in which CDK5 is required for suppressing D1-DARPP-32 Thr34 phosphorylation in isolated medium spiny neurons.

Expression of cyclin-dependent protein kinase 5 in the hippocampus of vascular dementia mice after cerebral ischemia and reperfusion

BACKGROUND： The p25-activated cyclin-dependent protein kinase 5 （Cdk5） may induce neuronal cell death and cause the development of dementia following cerebral ischemia and reperfusion. OBJECTIVE： To observe changes in the expression of Cdk5 and p25 in hippocampal tissue of vascular dementia mice at different time points following cerebral ischemia and reperfusion. DESIGN, TIME AND SETTING： A randomized, controlled animal experiment was performed in the clinical trial center of Hebei Provincial People＇s Hospital between September 2007 and October 2008. MATERIALS： Cdk5 rabbit anti-mouse polyclonal antibody, p35 rabbit anti-mouse polyclonal antibody, and β-actin mouse monoclonal antibody were purchased from Santa Cruz Biotechnology, Inc., USA; horseradish peroxidase-labeled goat anti-rabbit IgG and horseradish peroxidase-labeled goat anti-mice IgG were offered by Beijing Zhongshan Geldenbridye Biotechnology Co.,Ltd., China; the protein quantitative kit was produced by Applygen Gene Technology Corp., Beijing, China; cDNA reverse transcription and PCR amplification reagents were products of TianGen＆ Biotech （Beijing） Co.,Ltd., China. METHODS： One hundred and sixty male Kunming mice were randomly divided into two groups： a sham-operated group （n = 65） and a model group （n = 95）. Vascular dementia was induced with three periods of transient ischemia and reperfusion of the bilateral common carotid arteries. In the sham-operated group, the bilateral common carotid arteries were not blocked. MAIN OUTCOME MEASURES： Behavioral tests were done at four and six weeks post surgery. Pathological changes in the hippocampal CA1 region were observed with hematoxylin-eosin staining Cdk5 mRNA expression was examined by RT-PCR, and Western blots were used to evaluate Cdk5 and p25 expression. Learning and memory performance were assayed using the Morris water maze. RESULTS： Vascular dementia reduced learning and memory performance at 4 and 6 weeks post surgery. Vascular dementia also caused severe, time-dependent neuronal damage and death in the hippocampal CA1 region. Dementia induction also increased mRNA and protein expression of Cdk5 and p25 at both 4 and 6 weeks after surgery. CONCLUSION： Cdk5/p25 is involved in the development of vascular dementia in mice following cerebral ischemia and reperfusion.

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.

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.

Abemaciclib, a Recent Novel FDA-Approved Small Molecule Inhibiting Cyclin-Dependant Kinase 4/6 for the Treatment of Metastatic Breast Cancer: A Mini-Review

Abemaciclib (Verzerio®) is a cell cycle inhibitor of both CDK4 and CDK6. In 2017, abemaciclib was approved by the Food and Drug Administration (FDA) and, in 2018 by the European Medicines Agency (EMA) for the treatment of postmenopausal women with hormone receptor positive (HR+), human epidermal growth factor receptor 2 negative (HER2−) advanced breast cancer. In this mini-review, we provide a series of information for respectively their targets and its selectivity, results on preclinical trial, clinical phase I, II and III trials, and some perspectives. We also describe the batch and flow steps used for the synthesis of this cancer drug.

Expression of cyclin-dependent kinases in HL-60 cells during differentiation induced by retinoic acid

This study was designed to investigate the relationship of the expression of cyclin-dependent kinases (CDKs) with theeffects of all-trans retinoic acid (ATRA) on the proliferation of HL-cells. HL-60 cells were treated with ATRA for 1-4 d. Then thecapacity of DNA Synthesis was evaluated with 3H-TdR incorporation and the expression of cyclin E, cyclin D, CDK2 and CDK4protein determined with immunocytochemical staining. In addition, the expression Of CDC2, CDK2 and CDK4 mRNA was deter-mined with in situ hybridization. It was found that ATRA suppressed the proliferation of HL-60 cells and decreased their capacityof DNA synthesis to result in a down-regulation of the expression of cyclin E, cyclin D and CDC2 without comcomittant suppressionon the expression of CDK2 and CDK4. It is concluded that the effects of ATRA on the proliferation of HL-60 cells may be relatedto the down-regulation of the expression of cyclin E, cyclin D and CDC2.

Synthesis of 5-substituted benzyl-2,4-diamino pyrimidine derivatives as c-Fms kinase inhibitors

A serials of novel 5-substituted benzyl-2,4-diamino pyrimidine derivatives have been synthesized and evaluated as inhibitors of c-Fms kinase by the standard MTT method.The results showed that compound 15,5-[3-methoxy-4-（pyridine-3-yl）benzyl]-2,4-diamino pyrimidine,had an IC50 of 1.45μmol/L in inhibiting the proliferation of M-CSF-dependent myeloid leukemia cells in mice （NFS-60）,which was similar with GW2580,a selective inhibitor of c-Fms kinase.

Role of Toll-like receptor 4 and Janus kinase and signal transducer and activator of transcription signal transduction pathway in sepsis-induced brain damage

The Janus kinase and signal transducer and activator of transcription （JAK/STAT） signal transduction pathway is involved in sepsis-induced functional damage to the heart, liver, kidney, and other organs. However, the cellular and molecular mechanisms underlying sepsis-induced brain damage remain elusive. In the present study, we found severe loss of neurons in the hippocampal CA1 region in rats with sepsis-induced brain damage following intraperitoneal injection of endotoxin, The expression of toll-like receptor 4, tumor necrosis factor a, and interleukin-6 was significantly increased in brain tissues following lipopolysaccharide exposure. AG490 （JAK2 antagonist） and rapamycin （STAT3 antagonist） significantly reduced neuronal loss and suppressed the increased expression of toll-like receptor 4, tumor necrosis factor a, and interleukin-6 in the hippocampal CA1 region in sepsis-induced brain damaged rats. Overall, these data suggest that blockade of the JAK/STAT signal transduction pathway is neuroprotective in sepsis-induced brain damage via the inhibition of toll-like receptor 4, tumor necrosis factor a, and interleukin-6 exoression.

Downregulation of Aquaporin 4 Expression through Extracellular Signal-regulated Kinases1/2 Activation in Cultured Astrocytes Following Scratch-injury

Objective To investigate the role of extracellular signal-regulated kinase1/2（ERK1/2） pathway in the regulation of aquaporin 4（AQP4） expression in cultured astrocytes after scratch-injury. Methods The scratch-injury model was produced in cultured astrocytes of rat by a 10-μL plastic pipette tip. The morphological changes of astrocytes and lactate dehydrogenase（LDH） leakages were observed to assess the degree of scratch-injury. AQP4 expression was detected by immunofluorescence staining and Western blot, and phosphorylated-ERK1/2（p-ERK1/2） expression was determined by Western blot. To explore the effect of ERK1/2 pathway on AQP4 expression in scratch-injured astrocytes, 10 μmol/L U0126（ERK1/2 inhibitor） was incubated in the medium at 30 min before the scratch-injury in some groups. Results Increases in LDH leakage were observed at 1, 12, and 24 h after scratch-injury, and AQP4 expression was reduced simultaneously. Decrease in AQP4 expression was associated with a significant increase in ERK1/2 activation. Furthermore, pretreatment with U0126 blocked both ERK1/2 activation and decrease in AQP4 expression induced by scratch-injury. Conclusion These results indicate that ERK1/2 pathway down-regulates AQP4 expression in scratch-injured astrocytes, and ERK1/2 pathway might be a novel therapeutic target in reversing the effects of astrocytes that contribute to traumatic brain edema.

EIL-4 protects the B-cell lymphoma cell line CH31 from anti-lgM-induced growth arrest and apoptosis： contribution of the PI-3 kinase/AKT pathway

Interleukin-4 （IL-4） promotes lymphocyte survival and protects primary lymphomas from apoptosls. Previous studies reported differential requirements for the signal transducer and activator of transcription 6 （STAT6） and IRS2/phosphati-dylinositol 3 kinase （PI-3K） signaling pathways in mediating the IL-4-induced protection from Fas-mediated apoptosis. In this study, we characterized IL-4-activated signals that suppress anti-IgM-mediated apoptosis and growth arrest of CH31, a model B-cell lymphoma line. In CH31, anti-IgM treatment leads to the loss of mitochondrial membrane poten-tial, phospho-Akt, phospho-CDK2, and c-myc protein. These losses are followed by massive induction of p27^Kip1 protein expression, cell cycle arrest, and apoptosis. Strikingly, IL-4 treatment prevented or reversed these changes. Furthermore, IL-4 suppressed the activation of caspases 9 and 3, and, in contrast to previous reports, induced the phosphorylation （de-activation） of BAD. IL-4 treatment also induced expression of BclxL, a STAT6-dependent gene. Pharmacologic inhibitors and dominant inhibitory forms of PI-3K and Akt abrogated the anti-apoptotic function of IL-4. These results suggest that the IL-4 receptor activates several signaling pathways, with the Akt pathway playing a major role in suppression of the apoptotic program activated by anti-IgM.

SPOC domain-containing protein 1 regulates the proliferation and apoptosis of human spermatogonial stem cells through adenylate kinase 4

BACKGROUND Spermatogonial stem cells(SSCs)are the origin of male spermatogenesis,which can reconstruct germ cell lineage in mice.However,the application of SSCs for male fertility restoration is hindered due to the unclear mechanisms of proliferation and self-renewal in humans.AIM To investigate the role and mechanism of SPOC domain-containing protein 1(SPOCD1)in human SSC proliferation.METHODS We analyzed publicly available human testis single-cell RNA sequencing(RNAseq)data and found that SPOCD1 is predominantly expressed in SSCs in the early developmental stages.Small interfering RNA was applied to suppress SPOCD1 expression to detect the impacts of SPOCD1 inhibition on SSC proliferation and apoptosis.Subsequently,we explored the target genes of SPOCD1 using RNA-seq and confirmed their role by restoring the expression of the target genes.In addition,we examined SPOCD1 expression in some non-obstructive azoospermia(NOA)patients to explore the correlation between SPOCD1 and NOA.RESULTS The uniform manifold approximation and projection clustering and pseudotime analysis showed that SPOCD1 was highly expressed in the early stages of SSC,and immunohistological results showed that SPOCD1 was mainly localized in glial cell line-derived neurotrophic factor family receptor alpha-1 positive SSCs.SPOCD1 knockdown significantly inhibited cell proliferation and promoted apoptosis.RNA-seq results showed that SPOCD1 knockdown significantly downregulated genes such as adenylate kinase 4(AK4).Overexpression of AK4 in SPOCD1 knockdown cells partially reversed the phenotypic changes,indicating that AK4 is a functional target gene of SPOCD1.In addition,we found a significant downregulation of SPOCD1 expression in some NOA patients,suggesting that the downregulation of SPOCD1 may be relevant for NOA.CONCLUSION Our study broadens the understanding of human SSC fate determination and may offer new theories on the etiology of male infertility.

The Akt/glycogen synthase kinase-3β pathway participates in the neuroprotective effect of interleukin-4 against cerebral ischemia/reperfusion injury

Interleukin-4(IL-4) has a protective effect against cerebral ischemia/reperfusion injury. Animal experiments have shown that IL-4 improves the short-and long-term prognosis of neurological function. The Akt(also called protein kinase B, PKB)/glycogen synthase kinase-3β(Akt/GSK-3β) signaling pathway is involved in oxidative stress, the inflammatory response, apoptosis, and autophagy. However, it is not yet clear whether the Akt/GSK-3β pathway participates in the neuroprotective effect of IL-4 against cerebral ischemia/reperfusion injury. In the present study, we established a cerebral ischemia/reperfusion mouse model by middle cerebral artery occlusion for 60 minutes followed by a 24-hour reperfusion. An IL-4/anti-IL-4 complex(10 μg) was intraperitoneally administered 30 minutes before surgery. We found that administration of IL-4 significantly alleviated the neurological deficits, oxidative stress, cell apoptosis, and autophagy and reduced infarct volume of the mice with cerebral ischemia/reperfusion injury 24 hours after reperfusion. Simultaneously, IL-4 activated Akt/GSK-3β signaling pathway. However, an Akt inhibitor LY294002, which was injected at 15 nmol/kg via the tail vein, attenuated the protective effects of IL-4. These findings indicate that IL-4 has a protective effect on cerebral ischemia/reperfusion injury by mitigating oxidative stress, reducing apoptosis, and inhibiting excessive autophagy, and that this mechanism may be related to activation of the Akt/GSK-3β pathway. This animal study was approved by the Animal Ethics Committee of Renmin Hospital of Wuhan University, China(approval No. WDRY2017-K037) on March 9, 2017.

Inositol 1,4,5-trisphosphate 3-kinases:functions and regulations

Inositol 1,4,5-trisphosphate 3-kinase (IP3 3-kinase/IP3K) plays an important role in signal transduction in animal cellsby phosphorylating inositol 1,4,5-trisphosphate (IP3) to inositol 1,3,4,5-tetrakisphosphate (IP4). Both IP3 and IP4 arecritical second messengers which regulate calcium (Ca2+) homeostasis. Mammalian IP3Ks are involved in many biologicalprocesses, including brain development, memory, learning and so on. It is widely reported that Ca2+ is a canonicalsecond messenger in higher plants. Therefore, plant IP3K should also play a crucial role in plant development. Recently,we reported the identification of plant IP3K gene (AtIpk2β/AtIP3K) from Arabidopsis thaliana and its characterization.Here, we summarize the molecular cloning, biochemical properties and biological functions of IP3Ks from animal, yeastand plant. This review also discusses potential functions of IP3Ks in signaling crosstalk, inositol phosphate metabolism,gene transcriptional control and so on.

1-methyl-4-phenylpyridinium ion induces endoplasmic reticulum stress through glycogen synthase kinase-3 beta activation in PC12 cells

1-methyl-4-phenylpyridinium ion （MPP^＋） induces endoplasmic reticulum stress and activates caspase-12 in PC12 cells, leading to neuronal apoptosis. However, the underlying molecular mechanism remains unknown. The present study investigated the regulatory effects of nerve growth factor （Akt activator） and lithium chloride （glycogen synthase kinase-3β inhibitor） on the endoplasmic reticulum stress signaling pathway. The results revealed that MPP＋ induced expression of Bip and C/EBP homologous protein. The upregulation of Bip and C/EBP homologous protein, as well as the decreased pro-caspase-12 level induced by MPP^＋ were inhibited by pretreatment of the nerve growth factor or lithium chloride. These results suggest that the phosphatidylinositol 3 kinase-Aktglycogen synthase kinase-3β pathway is involved in MPP-induced endoplasmic reticulum stress.

Sarcoidosis mimicking metastases in an echinoderm microtubuleassociated protein-like 4 anaplastic lymphoma kinase positive nonsmall-lung cancer patient:A case report

BACKGROUND Rearrangements of the anaplastic lymphoma kinase(ALK)gene(ALK-positive)represent an oncogenic driver in approximately 3%-5%of non-small-lung cancer(NSCLC)patients.Sarcoidosis is a multisystem disease,and its reported incidence in Asia is 1 or less per 100000 people per year.The co-occurrence of sarcoidosis and ALK-positive NSCLC is rare,and ALK-positive lung cancer is likely to spread quickly.Therefore,the co-occurrence of sarcoidosis is more easily misdiagnosed as metastatic lung cancer by radiological examination.CASE SUMMARY A 50-year-old man had a nodule in the left superior lobe,many small nodules in left superior and right lungs,and enlarged bilateral hilar,mediastinal,and right supraclavicular lymph nodes.Computed tomography-guided pulmonary biopsy of the nodule in the left superior lobe revealed echinoderm microtubuleassociated protein-like 4 gene-ALK positive NSCLC with concomitant noncaseating granuloma.This patient was treated with crizotinib.Thirty days later,a chest computed tomography scan revealed a dramatic decrease in the size of the left superior lobe nodule;however,the lesions in the right lung progressed.The right supraclavicular lymph nodes showed granulomas,and no tumor cells were identified in the specimens. The angiotensin-converting enzyme level was high.After 1 wk of methylprednisolone treatment, a significant response of all lesionswas revealed. Following radical resection of the lung cancer, noncaseatinggranulomas were observed in both lung tissues and lymph nodes, which resultedin a diagnosis of echinoderm microtubule-associated protein-like 4-ALK positiveNSCLC accompanied with sarcoidosis.CONCLUSIONOur experience illustrates that pathological evidence is needed to confirmmetastatic disease, especially when some suspected metastatic lesions arenegative for malignancy.

Synthesis of New 2-Phenylamino-4<i>H</i>-chromene-3-carbonitrile Derivatives and Their Effects on Tumor Cell Lines and against Protein Kinases

The synthesis of 2-phenylimino-4H-chromene-3-carbonitriles 6(a-d) in good overall yields using an efficient and practical methodology in 3 steps has been implemented in this present work. The first step was a heterocyclization between 2-hydroxybenzaldehyde 1 and propanedinitrile 2 which produced 2-iminocoumarin 3 which was submitted to nitrogen/nitrogen displacement in the presence of aromatic primary amine 4. In the third step, reduction of 5 led to the desired 2-phenylimino-4H-chromene-3-carbonitriles 6. Compounds 5(a-d) and 6(a-d) were evaluated for their potential in vitro cytotoxicity against six selected tumor cell lines (Huh7-D12, Caco2, MDA-MB231, HCT 116, PC3 and NCI-H727) and tested for their protein kinase inhibition on eight selected protein kinases. Among them, compounds 5c and 6b exhibited inhibition on HsCK1e (5c: 44% and 6b: 42% at 1 μM) and 5c for cytotoxicity on PC3 cell lines (63% at 25 μM).

Polyglutamylase activity of tubulin tyrosine ligase-like 4 is negatively regulated by the never in mitosis gene A family kinase never in mitosis gene A-related kinase 5

BACKGROUND Tubulins,building blocks of microtubules,are modified substrates of diverse post-translational modifications including phosphorylation,polyglycylation and polyglutamylation.Polyglutamylation of microtubules,catalyzed by enzymes from the tubulin tyrosine ligase-like(TTLL)family,can regulate interactions with molecular motors and other proteins.Due to the diversity and functional importance of microtubule modifications,strict control of the TTLL enzymes has been suggested.AIM To characterize the interaction between never in mitosis gene A-related kinase 5(NEK5)and TTLL4 proteins and the effects of TTLL4 phosphorylation.METHODS The interaction between NEK5 and TTLL4 was identified by yeast two-hybrid screening using the C-terminus of NEK5(a.a.260–708)as bait and confirmed by immunoprecipitation.The phosphorylation sites of TTLL4 were identified by mass spectrometry and point mutations were introduced.RESULTS Here,we show that NEK5 interacts with TTLL4 and regulates its polyglutamylation activity.We further show that NEK5 can also interact with TTLL5 and TTLL7.The silencing of NEK5 increases the levels of polyglutamylation of proteins by increasing the activity of TTLL4.The same effects were observed after the expression of the catalytically inactive form of NEK5.This regulation of TTLL4 activity involves its phosphorylation at Y815 and S1136 amino acid residues.CONCLUSION Our results demonstrate,for the first time,the regulation of TTLL activity through phosphorylation,pointing to NEK5 as a potential effector kinase.We also suggest a general control of tubulin polyglutamylation through NEK family members in human cells.

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.

基于FAERS数据库的周期蛋白依赖性激酶4/6抑制剂血液毒性真实世界研究

目的 基于美国美国食品药品管理局(FDA)的不良事件报告系统(FAERS)分析3种周期蛋白依赖性激酶4/6(CDK4/6)抑制剂上市后的不良事件(AEs)信号,为临床用药安全提供参考。方法 提取FAERS数据库2015年第一季度至2022年第一季度共29个季度AEs,利用报告比值比法(ROR)和比例报告比值法(PRR)对CDK4/6抑制剂AEs进行数据挖掘。结果 CDK4/6抑制剂相关性血液毒性报告共有7 872份,各抑制剂血液毒性AEs占总AEs比例依次为哌柏西利(80.31%)>瑞博西利(15.36%)>阿贝西利(4.33%)。血液毒性常见中性粒细胞减少和贫血。哌柏西利(2 982/6 322,47.17%)和瑞博西利(613/1 209,50.70%)致中性粒细胞减少的报告占比较阿贝西利(117/341,34.31%)更高,血液毒性主要发生在药物开始使用后60 d内(1 630,61.86%),哌柏西利中位时间最长,且用药90 d后仍有32.9%的患者存在血液毒性,不同CDK4/6抑制剂血液毒性临床表现及发生强度存在差异。结论 哌柏西利、阿贝西利、瑞博西利均会导致明显的血液毒性,其中阿贝西利致血液毒性报告最少,但要警惕阿贝西利致贫血后导致死亡的风险。用药后的2个月内密切监测全血细胞计数,关注中性粒细胞、血红蛋白等水平,警惕CDK4/6抑制剂相关血液AEs的发生。