C-Phycocyanin elicited antitumor efficacy via cell-cycle arrest,apoptosis induction,and invasion inhibition in esophageal squamous cell carcinoma

Objectives: Mounting evidence has demonstrated that C-Phycocyanin (C-PC) exhibits marked antitumor activity in a wide type of tumors, such as pancreas cancer, breast carcinoma, lung cancer, and colon cancer. The current study aimed to confirm the antitumor efficacy of C-PC in esophageal squamous cell carcinoma (ESCC).

Methods: The efficacy of C-PC was evaluated against the proliferation of ESCC cell lines EC9706 and EC1 by CCK-8 kit and in a mice model of ESCC EC9706. Cell cycle and apoptosis were investigated by flow cytometry, and cell invasion was determined via transwell chamber. Protein expression was examined by Western blots.

Results: We found that C-PC exhibited anti-proliferation ability in a time-dependent manner and a dose-dependent manner in ESCC EC9706 and EC1 cells. Besides, C-PC markedly arrested cell cycle in the G0/G1 phase, induced cell apoptosis and suppressed cell invasion ability in both EC9706 and EC1 cells (p?<?.01). Notably, C-PC evoked the elevations of Bax, PARP, and cleaved-caspase-3 protein, but reduced cyclin D1, CDK4, Bcl-2, MMP-2, and MMP-9 expression levels. Further investigation from in vivo experiment revealed that C-PC displayed significant antitumor efficacy in the xenografted EC9706 model.

Conclusions: Our data presented herein suggest C-PC exerts antitumor efficacy in ESCC.     

Downregulation of beta1,4-galactosyltransferase 1 inhibits CDK11(p58)-mediated apoptosis induced by cycloheximide

Cyclin-dependent kinase 11 (CDK11; also named PITSLRE) is part of the large family of p34(cdc2)-related kinases whose functions appear to be linked with cell cycle progression, tumorigenesis, and apoptotic signaling. The mechanism that CDK11(p58) induces apoptosis is not clear. Some evidences suggested beta1,4-galactosyltransferase 1 (beta1,4-GT 1) might participate in apoptosis induced by CDK11(p58). In this study, we demonstrated that ectopically expressed beta1,4-GT 1 increased CDK11(p58)-mediated apoptosis induced by cycloheximide (CHX). In contrast, RNAi-mediated knockdown of beta1,4-GT 1 effectively inhibited apoptosis induced by CHX in CDK11(p58)-overexpressing cells. For example, the cell morphological and nuclear changes were reduced; the loss of cell viability was prevented and the number of cells in sub-G1 phase was decreased. Knock down of beta1,4-GT 1 also inhibited the release of cytochrome c from mitochondria and caspase-3 processing. Therefore, the cleavage of CDK11(p58) by caspase-3 was reduced. We proposed that beta1,4-GT 1 might contribute to the pro-apoptotic effect of CDK11(p58). This may represent a new mechanism of beta1,4-GT 1 in CHX-induced apoptosis of CDK11(p58)-overexpressing cells.     

CDK11p58 protein kinase activity is associated with Bcl-2 down-regulation in pro-apoptosis pathway

CDK11^p58, a G2/M-specific protein kinase, has been shown to be associated with apoptosis in many cell lines, with largely unknown mechanisms. Our previous study proved that CDK11^p58-enhanced cycloheximide (CHX)-induced apoptosis in SMMC-7721 hepatocarcinoma cells. Here we report for the first time that ectopic expression of CDK11^p58 down-regulates Bcl-2 expression and its Ser70, Ser87 phosphorylation in CHX-induced apoptosis in SMMC-7721 cells. Overexpression of Bcl-2 counteracts the pro-apoptotic activity of CDK11^p58. Furthermore, we confirm that the kinase activity of CDK11^p58 is essential to the down-regulation of Bcl-2 as well as apoptosis. Taken together, these results demonstrate that CDK11^p58 down-regulates Bcl-2 in pro-apoptosis pathway depending on its kinase activity, which elicits survival signal in hepatocarcinoma cells.     

Increased expression of CDK11p58 and cyclin D3 following spinal cord injury in rats

Protein kinases are critical signalling molecules for normal cell growth and development. CDK11^p58 is a p34^cdc2-related protein kinase, and plays an important role in normal cell cycle progression. However its distribution and function in the central nervous system (CNS) lesion remain unclear. In this study, we mainly investigated the protein expression and cellular localization of CDK11 during spinal cord injury (SCI). Western blot analysis revealed that CDK11^p58 was not detected in normal spinal cord. It gradually increased, reached a peak at 3 day after SCI, and then decreased. The protein expression of CDK11^p58 was further analyzed by immunohistochemistry. The variable immunostaining patterns of CDK11^p58 were visualized at different periods of injury. Double immunofluorescence staining showed that CDK11 was co-expressed with NeuN, CNPase and GFAP. Co-localization of CDK11/active caspase-3 and CDK11/proliferating cell nuclear antigen (PCNA) were detected in some cells. Cyclin D3, which was associated with CDK11^p58 and could enhance kinase activity, was detected in the normal and injured spinal cord. The cyclin D3 protein underwent a similar pattern with CDK11^p58 during SCI. Double immunofluorescence staining indicated that CDK11 co-expressed with cyclin D3 in neurons and glial cells. Coimmunoprecipitation further showed that CDK11^p58 and cyclin D3 interacted with each other in the damaged spinal cord. Thus, it is likely CDK11^p58 and cyclin D3 could interact with each other after acute SCI. Another partner of CDK11^p58 was β-1,4-galactosyltransferase 1 (β-1,4-GT 1). The co-localization of CDK11/β-1,4-GT 1 in the damaged spinal cord was revealed by immunofluorescence analysis. The cyclin D3-CDK4 complexes were also present by coimmunoprecipitation analysis. Taken together, these data suggested that both CDK11 and cyclin D3 may play important roles in spinal cord pathophysiology. The authors Yuhong Ji and Feng Xiao contributed equally to this work.     

A uniform procedure for the purification of CDK7/CycH/MAT1, CDK8/CycC and CDK9/CycT1

We have established a uniform procedure for the expression and purification of the cyclin-dependent kinases CDK7/CycH/MAT1, CDK8/CycC and CDK9/CycT1. We attach a His₆-tag to one of the subunits of each complex and then co-express it together with the other subunits in Spodoptera frugiperda insect cells. The CDK complexes are subsequently purified by Ni²⁺-NTA and Mono S chromatography. This approach generates large amounts of active recombinant kinases that are devoid of contaminating kinase activities. Importantly, the properties of these recombinant kinases are similar to their natural counterparts (Pinhero et al. 2004, Eur J Biochem 271:1004–14). Our protocol provides a novel systematic approach for the purification of these three (and possibly other) recombinant CDKs. Published: August 18, 2004.     

HIF-1α involvement in low temperature and anoxia survival by a freeze tolerant insect

Protein kinases are important signalling molecules critical for normal cell growth and development. CDK11^p58 is a p34^cdc2-related protein kinase, and plays an important role in normal cell cycle progression. In this study, we mainly characterized the protein expression of CDK11^p58 during postnatal development in mouse testes and examined the cellular localization of CDK11^p58 and cyclinD3, which was associated with CDK11^p58 in mammalian cells. Western blot analysis revealed that CDK11^p58 was present in the early stages of development. It gradually increased and reached a peak in adult testes. The protein expression of CDK11^p58 was further analysed by immunohistochemistry due to its developmentally regulated expression. The variable immunostaining patterns of CDK11^p58 were visualized during different developmental periods and, in adult mouse, different stages of seminiferous tubules. CDK11^p58 expression was detected in proliferating germ cells in the early stages of developing testes. In adult testes, the protein was expressed in pachytene primary spermatocytes from stage VII to XI of spermatogenesis and in postmeiotic spermatids in all stages at different levels. The colocalization of CDK11^p58 and cyclinD3 in the adult testeis was revealed by immunofluorescence analysis. (Mol Cell Biochem 270: 99–106, 2005)     

CDK1 inhibition sensitizes normal cells to DNA damage in a cell cycle dependent manner

Cyclin-dependent kinase 1 (CDK1) orchestrates the transition from the G2 phase into mitosis and as cancer cells often display enhanced CDK1 activity, it has been proposed as a tumor specific anti-cancer target. Here we show that the effects of CDK1 inhibition are not restricted to tumor cells but can also reduce viability in non-cancer cells and sensitize them to radiation in a cell cycle dependent manner.

Radiosensitization by the specific CDK1 inhibitor, RO-3306, was determined by colony formation assays in three tumor lines (HeLa, T24, SQ20B) and three non-cancer lines (HFL1, MRC-5, RPE). Initial results showed that CDK1 inhibition radiosensitized tumor cells, but did not sensitize normal fibroblasts and epithelial cells in colony formation assays despite effective inhibition of CDK1 signaling. Further investigation showed that normal cells were less sensitive to CDK1 inhibition because they remained predominantly in G1 for a prolonged period when plated in colony formation assays. In contrast, inhibiting CDK1 a day after plating, when the cells were going through G2/M phase, reduced their clonogenic survival both with and without radiation. Our finding that inhibition of CDK1 can damage normal cells in a cell cycle dependent manner indicates that targeting CDK1 in cancer patients may lead to toxicity in normal proliferating cells. Furthermore, our finding that cell cycle progression becomes easily stalled in non-cancer cells under normal culture conditions has general implications for testing anti-cancer agents in these cells.     

Molecular and phylogenetic analysis of PCR-amplified cyclin-dependent kinase (CDK) family sequences from representatives of the earliest available lineages of eukaryotes

Cyclin-dependent kinase (CDK) and cell division control (CDC2) sequences are strongly conserved among eukaryotes and may complement the use of other sequence families in eukaryotic phylogenetic inference. We synthesized degenerate PCR primers to amplify the catalytic region of CDK homologs in representatives of the earliest available lineages of eukaryotes. CDK family sequence-based, maximum-likelihood distance measurements with neighbor joining, and Fitch-Margoliash least-squares analyses produced unrooted dendrograms that included protists, yeasts, and higher eukaryotes. Bootstrap confidence estimates supported CDK-based phylogenetic groupings among the protists, fungi, and vertebrates although resolution within these groups was often insignificant. However, Trichomonas vaginalis and Giardia lamblia exhibited two of the most divergent CDK-like sequences consistent with rRNA-phylogenetic inference of early divergence of these eukaryotic lineages. In the evolution from unicellular to multicellular organisms, a constellation of amino acid residues aligning with the human, CDK N-terminal sheet may have undergone abrupt replacement.Abbreviations CDC cell division control - CDK cyclin-dependent kinase family - PCR polymerase chain reaction Correspondence to: D.E. Riley     

CDK11<Superscript>p58</Superscript> Promotes Rat Astrocyte Inflammatory Response via Activating p38 and JNK Pathways Induced by Lipopolysaccharide

In response to a variety of neural damages in the CNS, quiescent astrocytes become reactive astrocytes. Astrocytes are the major glial subtype and are important effectors that participate in the pathogenesis of numerous neural disorders, including trauma, stroke, aging, and developmental, genetic, idiopathic or acquired neurodegenerative diseases. CDK11^p58 (Cyclin-dependent kinases 11 protein 58/PITSLRE) is a p34cdc2-related protein kinase that plays an important role in normal cell cycle progression. In the process of LPS stimulus, the expression of CDK11^p58 in astrocytes was increased. Induced CDK11^p58 was parallel to astrocyte inflammatory response. Knockdown of CDK11^p58 by small-interfering RNAs (siRNAs) reduced the LPS-induced astrocyte inflammatory response, while overexpression CDK11^p58 enhanced the process. CDK11^p58 exerted its functions via activating p38 and JNK MAPK pathways. This study delineates that CDK11^p58 may be a significant regulatory factor for host defenses in central nervous system (CNS) inflammation.     

Bone marrow-derived mesenchymal stem cells promote cell proliferation of multiple myeloma through inhibiting T cell immune responses via PD-1/PD-L1 pathway

Objectives: This study aims to explore the effect of bone marrow mesenchymal stem cells (BMSCs) on multiple myeloma (MM) development and the underlying mechanism.

Materials and Methods: BMSCs from C57BL/6 J mice were isolated and the third passage was used for subsequent experiments. Additionally, a series of in vitro transwell coculture assays were performed to explore the effects of BMSCs on the proliferation of MM cells 5TGM1 and CD4⁺ T cells. Furthermore, a 5TGM1-induced MM mice model was established. Moreover, PD-L1 shRNA was transfected into BMSCs to investigate whether PD-1/PD-L1 pathway involved in BMSCs-mediated regulation of T cells and MM growth.

Results: Data revealed that BMSCs significantly promoted 5TGM1 proliferation in a dose-dependent manner. Furthermore, BMSCs administration exerted stimulatory effects on MM development in terms of shortening the mouse survival rate, promoting tumor growth, and enhancing inflammatory infiltration in the MM model mice. Moreover, BMSCs decreased the percentage of Th1 and Th17 cells, whereas increased that of Th2 and Treg cells. Their corresponding cytokines of these T cell subsets showed similar alteration in the presence of BMSCs. Additionally, BMSCs significantly suppressed CD4⁺ T cell proliferation. We also found that PD-L1 shRNA inhibited 5TGM1 proliferation likely through activation of CD4⁺ T cells. Further in vivo experiments confirmed that PD-L1 inhibition attenuated BMSCs-induced MM growth, inflammation infiltration and imbalance of Th1/Th2 and Th17/Treg.

Conclusion: In summary, our findings demonstrated that BMSCs promoted cell proliferation of MM through inhibiting T cell immune responses via PD-1/PD-L1 pathway.     

Altered expression of G1/S phase cell cycle regulators in placental mesenchymal stromal cells derived from preeclamptic pregnancies with fetal-placental compromise

Herein, we evaluated whether Placental Mesenchymal Stromal Cells (PDMSCs) derived from normal and Preeclamptic (PE) placentae presented differences in the expression of G1/S-phase regulators p16^INK4A, p18^INK4C, CDK4 and CDK6. Finally, we investigated normal and PE-PDMSCs paracrine effects on JunB, Cyclin D1, p16^INK4A, p18^INK4C, CDK4 and CDK6 expressions in physiological term villous explants.

PDMSCs were isolated from physiological (n = 20) and PE (n = 24) placentae. Passage three normal and PE-PDMSC and conditioned media (CM) were collected after 48h. Physiological villous explants (n = 60) were treated for 72h with normal or PE-PDMSCs CM. Explants viability was assessed by Lactate Dehydrogenase Cytotoxicity assay. Cyclin D1 localization was evaluated by Immuofluorescence (IF) while JunB, Cyclin-D1 p16^INK4A, p18^INK4C, CDK4 and CDK6 levels were assessed by Real Time PCR and Western Blot assay.

We reported significantly increased p16^INK4A and p18^INK4C expression in PE- relative to normal PDMSCs while no differences in CDK4 and CDK6 levels were detected. Explants viability was not affected by normal or PE-PDMSCs CM. Normal PDMSCs CM increased JunB, p16^INK4 and p18^INK4C and decreased Cyclin-D1 in placental tissues. In contrast, PE-PDMSCs CM induced JunB downregulation and Cyclin D1 increase in placental explants. Cyclin D1 IF staining showed that CM treatment targeted mainly the syncytiotrophoblast.

We showed Cyclin D1-p16INK4A/p18INK4C altered pathway in PE-PDMSCs demonstrating an aberrant G1/S phase transition in these pathological cells. The abnormal Cyclin D1-p16INK4A/p18INK4C expression in explants conditioned by PE-PDMSCs media suggest a key contribution of mesenchymal cells to the altered trophoblast cell cycle regulation typical of PE pregnancies with fetal-placental compromise.     

Identification and characterization of the BmCyclin L1-BmCDK11A/B complex in relation to cell cycle regulation

Cyclin proteins are the key regulatory and activity partner of cyclin-dependent kinases (CDKs), which play pivotal regulatory roles in cell cycle progression. In the present study, we identified a Cyclin L1 and 2 CDK11 2 CDK11 splice variants, CDK11A and CDK11B, from silkworm, Bombyx mori. We determined that both Cyclin L1 and CDK11A/B are nuclear proteins, and further investigations were conducted to elucidate their spatiofunctional features. Cyclin L1 forms a complex with CDK11A/B and were co-localized to the nucleus. Moreover, the dimerization of CDK11A and CDK11B and the effects of Cyclin L1 and CDK11A/B on cell cycle regulation were also investigated. Using overexpression or RNA interference experiments, we demonstrated that the abnormal expression of Cyclin L1 and CDK11A/B leads to cell cycle arrest and cell proliferation suppression. Together, these findings indicate that CDK11A/B interacts with Cyclin L1 to regulate the cell cycle.     

The non-canonical functions of p27Kip1 in normal and tumor biology

p27^Kip1 was first discovered as a key regulator of cell proliferation. The canonical function of p27^Kip1 is inhibition of cyclin-dependent kinase (CDK) activity. In addition to its initial identification as a CDK inhibitor, p27^Kip1 has also emerged as an intrinsically unstructured, multifunctional protein with numerous non-canonical, CDK-independent functions that exert influence on key processes such as cell cycle regulation, cytoskeletal dynamics and cellular plasticity, cell migration, and stem-cell proliferation and differentiation. Many of these non-canonical functions, depending on the cell-specific contexts such as oncogenic activation of signaling pathways, have the ability to turn pro-oncogenic in nature and even contribute to tumor-aggressiveness and metastasis. This review discusses the various non-canonical, CDK-independent mechanisms by which p27^Kip1 functions either as a tumor-suppressor or tumor-promoter.     

Effect of lyophilization on liposomal encapsulation of salmon calcitonin

Purpose: The intent of this work was to assess the impact of lyophilization on the encapsulation of salmon calcitonin (sCT) into liposomes.

Methods: Four different liposomal formulations were investigated, i.e. DPPC:Chol:DSPE-PEG₂₀₀₀ (75:20:5 and 65:30:5) and DPPC:Chol (80:20 and 66.7:33.3). Lipid films were prepared and hydrated with loading buffer containing sCT and different concentrations of the cryoprotectant, trehalose dihydrate. The liposomes were lyophilized, reconstituted and extruded to obtain small unilamellar vesicles. Non-encapsulated sCT was separated by gel filtration. Non-lyophilized formulations and liposomes lyophilized without the cryoprotectant were used as controls. Liposomes were analyzed for particle size, polydispersity index, zeta-potential and encapsulation efficiency. ³¹P-NMR (phosphorous nuclear magnetic resonance spectroscopy) was performed on selected formulations.

Results: Post-lyophilization, no significant change in particle sizes and zeta-potentials were noted, regardless of the presence or absence of the cryoprotectant. Encapsulation efficiencies, however, increased following lyophilization, in both PEGylated (lyophilization control batch) and non-PEGylated liposomes (cryoprotectant batches only). ³¹P-NMR revealed the presence of two distinct vesicle populations – liposomes and micelles – in PEGylated formulation. The presence of micelles might be responsible for the observed encapsulation enhancement of sCT in the PEGylated formulation.

Conclusions: Lyophilization resulted in an increase in encapsulation efficiency of sCT in PEGylated liposomes, even in the absence of a cryoprotectant, due to presence of micellar vesicles.     

Characterization of Plasmodium falciparum cdc2-related kinase and the effects of a CDK inhibitor on the parasites in erythrocytic schizogony

The cell cycle of Plasmodium is unique among major eukaryotic cell cycle models. Cyclin-dependent kinases (CDKs) are thought to be the key molecular switches that regulate cell cycle progression in the parasite. However, little information is available about Plasmodium CDKs. The present study was performed to investigate the effects of a CDK inhibitor, olomoucine, on the erythrocytic growth of Plasmodium falciparum. This agent inhibited the growth of the parasite at the trophozoite/schizont stage. Furthermore, we characterized the Plasmodium CDK homolog, P. falciparum cdc2-related kinase-1 (Pfcrk-1), which is a potential target of olomoucine. We synthesized a functional kinase domain of Pfcrk-1 as a GST fusion protein using a wheat germ protein expression system, and examined its phosphorylation activity. The activity of this catalytic domain was higher than that of GST-GFP control, but the same as that of a kinase-negative mutant of Pfcrk-1. After the phosphatase treatment, the labeling of [γ-³²P]ATP was abolished. Recombinant human cyclin proteins were added to these kinase reactions, but there were no differences in activity. This report provides important information for the future investigation of Plasmodium CDKs.     

Functional specialization of chordate CDK1 paralogs during oogenic meiosis

Cyclin-dependent kinases (CDKs) are central regulators of eukaryotic cell cycle progression. In contrast to interphase CDKs, the mitotic phase CDK1 is the only CDK capable of driving the entire cell cycle and it can do so from yeast to mammals. Interestingly, plants and the marine chordate, Oikopleura dioica, possess paralogs of the highly conserved CDK1 regulator. However, whereas in plants the 2 CDK1 paralogs replace interphase CDK functions, O. dioica has a full complement of interphase CDKs in addition to its 5 odCDK1 paralogs. Here we show specific sub-functionalization of odCDK1 paralogs during oogenesis. Differential spatiotemporal dynamics of the odCDK1a, d and e paralogs and the meiotic polo-like kinase 1 (Plk1) and aurora kinase determine the subset of meiotic nuclei in prophase I arrest that will seed growing oocytes and complete meiosis. Whereas we find odCDK1e to be non-essential, knockdown of the odCDK1a paralog resulted in the spawning of non-viable oocytes of reduced size. Knockdown of odCDK1d also resulted in the spawning of non-viable oocytes. In this case, the oocytes were of normal size, but were unable to extrude polar bodies upon exposure to sperm, because they were unable to resume meiosis from prophase I arrest, a classical function of the sole CDK1 during meiosis in other organisms. Thus, we reveal specific sub-functionalization of CDK1 paralogs, during the meiotic oogenic program.     

LIM kinase-2 induces programmed necrotic neuronal death via dysfunction of DRP1-mediated mitochondrial fission

Although the aberrant activation of cell cycle proteins has a critical role in neuronal death, effectors or mediators of cyclin D1/cyclin-dependent kinase 4 (CDK4)-mediated death signal are still unknown. Here, we describe a previously unsuspected role of LIM kinase 2 (LIMK2) in programmed necrotic neuronal death. Downregulation of p27^Kip1 expression by Rho kinase (ROCK) activation induced cyclin D1/CDK4 expression levels in neurons vulnerable to status epilepticus (SE). Cyclin D1/CDK4 complex subsequently increased LIMK2 expression independent of caspase-3 and receptor interacting protein kinase 1 activity. In turn, upregulated LIMK2 impaired dynamic-related protein-1 (DRP1)-mediated mitochondrial fission without alterations in cofilin phosphorylation/expression and finally resulted in necrotic neuronal death. Inhibition of LIMK2 expression and rescue of DRP1 function attenuated this programmed necrotic neuronal death induced by SE. Therefore, we suggest that the ROCK-p27^Kip1-cyclin D1/CDK4-LIMK2-DRP1-mediated programmed necrosis may be new therapeutic targets for neuronal death.