P27 Kip1 Expression Research Articles

Combination of FGFR4 inhibitor Blu9931 and 5-fluorouracil effects on the biological characteristics of colorectal cancer cells.

The aim of this study was to explore the effects of single agent treatments and combination of Blu9931 and 5-fluorouracil (5-FU) on the biological characteristics of colorectal cancer cells and its mechanism. Blu9931 is the first selective small molecule inhibitor of the fibroblast growth factor receptor4 (FGFR4) and exquisitely selective for FGFR4 versus other FGFR family members and all other kinases. The colorectal cancer cells HCT116 and SW620 with high expression of FGFR4 were selected for a series of functional tests including cell viability, cell proliferation, apoptosis and cell cycle detection. Western blotting was used to detect the expression of related molecules including signal pathway (STAT3), apoptosis (cleaved caspase‑3), cell cycle (cyclinD1 and P27kip1) and epithelial-mesenchymal transition (E-cadherin and vimentin) in HCT116 and SW620 cells used as single and combination treatments of 5‑FU and Blu9931. The cell viability gradually decreased when the concentration of 5‑FU and Blu9931 increased. Blu9931 can inhibit FGFR4 protein expression while 5‑FU cannot, as assessed by western blot analysis. The single agent treatment and combinations of 5‑FU and Blu9931 arrest cell cycle (P<0.05), increased p27kip1 expression and reduced cyclinD1 expression. The single agent treatment and combinations of 5‑FU and Blu9931 inhibited EMT. Furthermore, the combination of 5‑FU and Blu9931 has a synergistic effect in reducing colorectal cancer cell proliferation and preventing cell cycle. Taken together, this study provides the first evidence that Blu9931 functions as a FGFR4-selective inhibitor in colorectal cancer (CRC) cells, and Blu9931 may be a new targeted drug.

Betulinic acid induces apoptosis by regulating PI3K/Akt signaling and mitochondrial pathways in human cervical cancer cells.

Betulinic acid (BA), a potential anticancer compound, could induce apoptosis in human cervical cancer (HeLa) cells, but its mechanism has yet to be fully elucidated. The present study was focused on deciphering the detailed molecular mechanism of BA-induced apoptosis. In the present study, results indicated that BA was highly effective against HeLa cells via induction of time-dependent apoptosis, and the authors demonstrated that the BA treatment acted through downregulating a phosphatidylinositol 3-kinase (PI3K) subunit and suppressing the Akt phosphorylation at Thr308 and Ser473 after increasing the generation of intracellular reactive oxygen species. Then, BA induced cell cycle arrest at the G0/G1 phase, which was consistent with the cell cycle-related protein results in which BA significantly enhanced the expression of p27Kip and p21Waf1/Cip1 in HeLa cells. This target-specific inhibition was associated with mitochondrial apoptosis, as reflected by the increased expression of Bad and caspase-9, the generation of reactive oxygen species (ROS) and the decline in mitochondrial membrane potential. Moreover, preincubation of the cells with glutathione (antioxidant) blocked the process of apoptosis, prevented the phosphorylation of downstream substrates. These results established that ROS acted as a key factor to effect apoptosis by BA treatment in HeLa cells. Therefore, these findings demonstrated that BA induced apoptosis in HeLa cells by downregulating the expression of PI3K/Akt signaling molecules via ROS, and triggering a mitochondrial pathway.

Protective effects of aloperin on monocroline-induced pulmonary hypertension via regulation of Rho A/Rho kinsase pathway in rats

Pulmonary hypertension (PH) is fatal disease which closely involves Rho A/ Rho kinsase (ROCK) pathway. Aloperine is a main active alkaloid extracted from Sophora alopecuroides, which is a traditional Chinese herbal medicine that has been used widely. However, the effects of this alkaloid on pulmonary hypertension and its mechanisms remain unclear. Therefore, this study is designed to investigate whether aloperine has protective effects on PH induced by monocrotaline, whether these effects may be related to regulation of RhoA/ROCK pathway in rats.Pulmonary hypertension was induced by monocrotaline (60mg/kg), and subsequently oral administration of aloperine (25, 50, 100mg/kg/day) for 21 days. At the end of the experiment, rats were underwent hemodynamic and morphologic assessments. At same time, the expression of Rho A, ROCK1, ROCK2, as well as activities of ROCK in the lung of rat has been detected. Afterwards, the expression of p27kip1, Bax, Bcl-2, which was the downstream proliferation and apoptosis factors of ROCK, were tested.The result indicted that aloperine treatment showed significantly improvement in hemodynamic and pathomorphologic data. Moreover, the reduction in expression of Rho A, ROCK1, ROCK2, and suppression in activities of ROCK were found in rat lungs after aloperine treatment. Furthermore, aloperine also alleviated the MCT-induced changes of p27kip1, Bax and Bcl-2.In summary, this study indicates that aloperine have protective effects on monocrotaline-induced PH. And these effects may be partially related to RhoA/ROCK pathway. Thus, aloperine could be considered a possible therapeutic strategy for PH.

NADPH oxidase 5 (NOX5)-induced reactive oxygen signaling modulates normoxic HIF-1α and p27Kip1 expression in malignant melanoma and other human tumors.

NADPH oxidase 5 (NOX5) generated reactive oxygen species (ROS) have been implicated in signaling cascades that regulate cancer cell proliferation. To evaluate and validate NOX5 expression in human tumors, we screened a broad range of tissue microarrays (TMAs), and report substantial overexpression of NOX5 in malignant melanoma and cancers of the prostate, breast, and ovary. In human UACC‐257 melanoma cells that possesses high levels of functional endogenous NOX5, overexpression of NOX5 resulted in enhanced cell growth, increased numbers of BrdU positive cells, and increased γ‐H2AX levels. Additionally, NOX5‐overexpressing (stable and inducible) UACC‐257 cells demonstrated increased normoxic HIF‐1α expression and decreased p27Kip1 expression. Similarly, increased normoxic HIF‐1α expression and decreased p27Kip1 expression were observed in stable NOX5‐overexpressing clones of KARPAS 299 human lymphoma cells and in the human prostate cancer cell line, PC‐3. Conversely, knockdown of endogenous NOX5 in UACC‐257 cells resulted in decreased cell growth, decreased HIF‐1α expression, and increased p27Kip1 expression. Likewise, in an additional human melanoma cell line, WM852, and in PC‐3 cells, transient knockdown of endogenous NOX5 resulted in increased p27Kip1 and decreased HIF‐1α expression. Knockdown of endogenous NOX5 in UACC‐257 cells resulted in decreased Akt and GSK3β phosphorylation, signaling pathways known to modulate p27Kip1 levels. In summary, our findings suggest that NOX5 expression in human UACC‐257 melanoma cells could contribute to cell proliferation due, in part, to the generation of high local concentrations of extracellular ROS that modulate multiple pathways that regulate HIF‐1α and networks that signal through Akt/GSK3β/p27Kip1.

Application of galangin, an active component of Alpinia officinarum Hance (Zingiberaceae), for use in drug-eluting stents

In clinical pathology, stent interposition is used to treat vascular disease but can lead to restenosis. Drug-eluting stents (DES) are most commonly used to suppress restenosis but can also have side effects. Therefore, we investigated the anti-proliferative effect and its possible target in vitro and in vivo. We found that Alpinia officinarum Hance (AO) extract efficiently inhibited VSMC proliferation by arresting the transition from the G0/G1 to the S phase via the up-regulation of p27KIP1 expression. Galangin (GA) was determined to be a significant component of this extract, with the same anti-proliferative activity as the raw extract. Immunoblotting and immunofluorescence staining showed that both the AO extract and GA targeted the up-regulation of p27KIP1 expression. Therefore, we next examined the effect of these compounds in a cuff-injured neointimal hyperplasia model in vivo. In this animal model, both the AO extract and GA completely suppressed the neointima formation, and this inhibitory effect was also demonstrated to target the up-regulation of p27KIP1, including the suppression of proliferating cell nuclear antigen expression. Our findings indicate that AO extract and GA have a potent anti-proliferative activity, targeting the up-regulation of p27 expression. Thus, GA may represent an alternative medicine for use in DES.

Staphylococcal enterotoxin C2 mutant drives T lymphocyte activation through PI3K/mTOR and NF-ĸB signaling pathways

Staphylococcal enterotoxin C2 (SEC2), a superantigen, causes rapid clonal expansion of lymphocytes and secretion of T cell growth factors, leading to a severe inflammatory response within tissues. Although previous studies have shown that ST-4, a SEC2 mutant with enhanced recognition of Vβ regions of T-cell receptors (TCRVβ), can activate an increased number of T cells and produce more cytokines than SEC2. However, the signaling mechanisms of SEC2/ST-4-mediated immune activation have not been addressed. In this study, we showed that the phosphatidylinositide-3-kinase (PI-3K) inhibitor LY294002, mammalian target of rapamycin (mTOR) inhibitor rapamycin, and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) inhibitor Bay11-7085 could suppress SEC2/ST-4-induced proliferation, CD69/CD25 expression, cell-cycle progression, and IL-2 production in BALB/c mouse splenocytes. In addition, we observed significantly upregulated expression of p70S6K, cyclin E, cyclin D3, and NF-ĸB/p65, but downregulated expression of p27kip during SEC2/ST-4-driven T cells activation. However, SEC2/ST-4-induced changes in cell cycle and PI3K/mTOR signaling were significantly relieved by either LY294002 or rapamycin, and the induction of NF-ĸB/p65 induced was significantly downregulated by Bay11-7085. Moreover, we found that IL-2 secretion was positively associated with p65 expression in a time- and dose-dependent manner. Taken together, our findings demonstrate the involvement of PI3K/mTOR and NF-κB signaling pathways in SEC2/ST-4-induced T cell activation. ST-4 intensifies PI3K/mTOR and NF-ĸB signaling transduction, ultimately leading to enhance T cell activation. These results provide a theoretical mechanism for future immunotherapy using ST-4.

Synergistic effect of melatonin and ghrelin in preventing cisplatin-induced ovarian damage via regulation of FOXO3a phosphorylation and binding to the p27Kip1 promoter in primordial follicles.

Premature ovarian failure during chemotherapy is a serious problem for young women with cancer. To preserve the fertility of these patients, approaches to prevent chemotherapy-induced ovarian failure are needed. In a previous study, we reported that melatonin treatment prevents the depletion of the dormant follicle pool via repression of the simultaneous activation of dormant primordial follicles by cisplatin. However, melatonin's protective effect was only partial and thus insufficient. In this study, we found that the hormone ghrelin enhances the protective effect of melatonin against cisplatin-induced ovarian failure in mouse model. Co-administration of melatonin and ghrelin more effectively prevented cisplatin-induced follicle disruption. Simultaneous treatment with melatonin and ghrelin almost restored the number of primordial follicles and the corpus luteum in cisplatin-treated ovaries, compared with single administration. We found melatonin and ghrelin receptors on the cell membrane of premature oocytes of primordial follicles. In addition, melatonin and ghrelin co-administration inhibited the cisplatin-induced phosphorylation of PTEN and FOXO3a that induces cytoplasmic translocation of FOXO3a. Inhibition of FOXO3a phosphorylation by melatonin and ghrelin increased the binding affinity of FOXO3a for the p27Kip1 promoter in primordial follicles. Co-administration of melatonin and ghrelin in cisplatin-treated ovaries restored the expression of p27Kip1 , which is critical for retention of the dormant status of primordial follicles. In conclusion, these findings suggest that melatonin and ghrelin co-administration is suitable for use as a fertoprotective adjuvant therapy during cisplatin chemotherapy in young female cancer patients.

Heat shock protein 70 downregulation inhibits proliferation, migration and tumorigenicity in hepatocellular carcinoma cells.

The overexpression of heat shock protein 70 (HSP70), a major stress-inducible heat shock protein, has been identified to enhance the proliferation, survival, invasion and metastasis of diverse types of human cancer. However, its role in hepatocellular carcinoma (HCC) remains poorly understood. The present study demonstrated that HSP70 expression was higher in tested HCC cell lines, compared with the normal hepatocyte LO2, and the suppression of HSP70 significantly inhibited the proliferation of SMMC-7721 and Hep3B cells. The growth inhibitory effect was mediated by cell cycle arrest at the G1/S phase with reduced cyclin D1 and increased p27Kip1 expression. Furthermore, HSP70 knockdown significantly inhibited the migration and invasion abilities of HCC cells. In conclusion, HSP70 is a key regulator involved in the proliferation, migration and invasion of HCC, and it may be used as a potential therapeutic target for HCC.

Identification of LEFTY as a molecular marker for ovarian clear cell carcinoma.

To identify proteins involved in ovarian clear cell carcinoma (OCCCa), shotgun proteomics analysis was applied using formalin-fixed and paraffin-embedded samples of ovarian carcinoma. Analysis of 1521 proteins revealed that 52 were differentially expressed between four OCCCa and 12 non-OCCCa samples. Of the highly expressed proteins in OCCCa, we focused on left-right determination factor (LEFTY), a novel member of the transforming growth factor-β superfamily. In 143 cases of ovarian epithelial carcinoma including 99 OCCCas and 44 non-OCCCas, LEFTY expression at both mRNA and protein levels was significantly higher in OCCCas compared with non-OCCCas, with the mRNA expression of LEFTY1 being predominant compared to that of LEFTY2. OCCCa cells stably overexpressing LEFTY1 showed reduced cell proliferation, along with decreased pSmad2 expression, and also either displayed an activated p53/p21waf1 pathway or increased p27kip1 expression, directly or indirectly. Moreover, the treatment of stable cell lines with cisplatin led to increased apoptotic cells, together with the inhibition of protein expression of a pSmad2-mediated X-linked inhibitor of apoptosis and a decreased bcl2/bax ratio. Blocking LEFTY1 expression with a specific short hairpin RNA inhibited cisplatin-induced apoptosis, probably through the increased expression of both XIAP and bcl2, but not bax. In clinical samples, a significantly higher number of apoptotic cells and lower Ki-67 labeling indices were observed in OCCCas with a high LEFTY score relative to those with a low score. These findings suggest that LEFTY may be an excellent OCCCa-specific molecular marker, which has anti-tumor effects in altering cell proliferation and cellular susceptibility to apoptosis.

Skp2 expression exhibits a negative correlation with P27Kip1 in lungs of SD rat stress model induced by lipopolysaccharide

In order to investigate action mechanism of Skp2 in SD rat stress model continuously challenged with LPS, not only the histopathological changes but also the distribution and expression of Skp2 and P27Kip1 were detected in lungs from SD rats by HE and immunohistochemistry staining respectively. According to present results, Skp2 and P27Kip1 mianly distributed in the epithelia of respiratory bronchioles, pseudostratified columnar ciliated epithelia of bronchus and vascular endothelial, a few were located at alveolar wall. As well, area, IOD and IOD/area of Skp2 observably increased in LPS-treated rats (P less than 0.05 or Pless than 0.01), but IOD and IOD/area of P27Kip1 apparently decreased in LPS-treated rats (P less than 0.05 or P less than 0.01). In conclusion, moderate pathological changes existed in lungs from SD rat stress model challenged by LPS. Moreover, Skp2 high level was negatively correlated with P27Kip1 low expression in lungs, it is indicated that Skp2 probably became a key indicator during stress disease process

Morphological Features and Immunohistochemical Expression of p57Kip2 in Early Molar Pregnancies and Their Relations to the Progression to Persistent Trophoblastic Disease.

BackgroundAlthough the morphological features characteristic of products of conception specimens including molar pregnancies are well described, substantial histopathological similarities are observed between the different entities, especially in cases of early pregnancies. Furthermore, there are no current solid criteria that could predict cases with progression to persistent gestational trophoblastic disease. In this study, we aimed to determine the most specific histopathological and immunohistochemical features required for accurate diagnosis that can reliably predict the clinical behavior.MethodsSixty-five cases of products of conception were reviewed clinically and pathologically, and any progression to persistent gestational trophoblastic disease (GTD), if present, was noted. Pathological assessment of the archival material included re-cut sections of 5 μm in thickness, routine staining with hematoxylin and eosin and immunohistochemical staining of p57Kip2.ResultsCertain histopathological criteria were found to be significant in differentiation between complete hydatidiform mole (CHM) and partial hydatidiform mole including villous shape and outline, villous trophoblast hyperplasia, and atypia in extravillous trophoblasts. There were no significant differences in any morphological or immunohistochemical features between cases with or without subsequent development of GTD.ConclusionsHistopathological diagnosis of molar pregnancy remains problematic especially in early gestation. Their diagnosis should be stated after a constellation of specific histopathological criteria in order not to miss CHM. p57Kip2 immunohistochemistry is of great value in diagnosis of cases that had equivocal morphology by histopathological examination. However, there were no significant features to predict cases that subsequently developed persistent GTD.

Murine Aortic Crush Injury: An Efficient In Vivo Model of Smooth Muscle Cell Proliferation and Endothelial Function.

Arterial reconstruction, whether angioplasty or bypass surgery, involves iatrogenic trauma causing endothelial disruption and vascular smooth muscle cell (VSMC) proliferation. Common murine models study small vessels such as the carotid and femoral arteries. Herein we describe an in vivo system in which both VSMC proliferation and endothelial barrier function can be simultaneously assessed in a large vessel. We studied the infrarenal aortic response to injury in C57BL/6 mice. The aorta was injured from the left renal vein to the aortic bifurcation by 30 transmural crushes of 5-seconds duration with a cotton-tipped applicator. Morphological changes were assessed with conventional histology. Aorta wall thickness was measured from the luminal surface to the adventitia. EdU integration and counter staining with DAPI and alpha-actin was used to demonstrate VSMC proliferation. Activation of ERK1/2, a known moderator of intimal hyperplasia formation, was determined by Western Blot analysis. The effect of inflammation was determined by immunohistochemistry for B-cells, T-cells, and macrophages. En face sections of endothelium were visualized with scanning electron microscopy (SEM). Endothelial barrier function was determined with Evans Blue staining. Transmural injury resulted in aortic wall thickening. This injury induced VSMC proliferation, most prominently at 3 days after injury, and early activation of ERK1/2 and decreased p27kip1 expression. Injury did not result in increased B-cells, T-cells, or macrophages infiltration in the vessel wall. Injury caused partial endothelial cell denudation and loss of cell-cell contact. Injury resulted in a significant loss of endothelial barrier function, which returned to baseline after seven days. The murine transmural blunt aortic injury model provides an efficient system to simultaneously study both VSMC proliferation and endothelial barrier function in a large vessel.

Maternal exposure to polybrominated diphenyl ether (BDE-209) during lactation affects germ cell survival with altered testicular glucose homeostasis and oxidative status through down-regulation of Cx43 and p27Kip1 in prepubertal mice offspring.

BDE-209, a congener of polybrominated diphenyl ethers (PBDEs), is mainly used as flame retardant and accounts for over 82% of total PBDE usage. PBDEs have recently been detected in human milk and cord blood. BDE-209 possesses weak estrogenic/anti-estrogenic properties and evokes hyperglycemia through impaired glucose homeostasis in rat liver. However, little is known of the effect of lactational exposure to BDE-209 on germ cell survival in relation to testicular glucose homeostasis, estradiol and oxidative status during prepubertal period. The present study, therefore, evaluated the effect of maternal exposure to BDE-209 during lactation on above-mentioned parameters with reference to Cx43 and p27Kip1 in prepubertal Parkes (P) mice offspring. Lactating female P mice were orally gavaged with 500, and 700mg/kg body weight of BDE-209 in corn oil from postnatal day (PND) 0 to PND 28; male pups were euthanized at PND 21 and 28. Maternal exposure to BDE-209 during lactation increased germ cell apoptosis with altered expressions of various cell survival and apoptotic markers along with decreased expression of Cx43 and p27Kip1 in prepubertal mice offspring. Testicular glucose and lactate concentrations were markedly reduced in these pups with down-regulation in GLUT3 and GLUT8 expressions and decreased LDH activity. Rise in oxidative stress in testes with increased estrogen level in these pups was also noted. In conclusion, our results suggest that maternal BDE-209 exposure during lactation affects germ cell survival with altered testicular glucose homeostasis and oxidative status through down-regulation of Cx43 and p27Kip1 in prepubertal mice offspring.

CyclinD1 and p57kip2 as biomarkers in differentiation, metastasis and prognosis of gastric cardia adenocarcinoma.

ObjectiveThis study aims to investigate the expression and significance of p57kip2 and cyclinD1 in gastric cardia adenocarcinoma (GCA). p57kip2 is a negative regulator in the cell cycle. On the contrary, cyclinD1 is a positive regulator of cell cycle progression.MethodsThirty-two cases of GCA tissues and adjacent non-cancerous tissues were collected for this study. Immunohistochemistry and fluorescence qualitative PCR was used to determine the level of p57kip2 and cyclinD1 in GCA and its adjacent non-cancerous tissues. Furthermore, the correlation between the mRNA/protein and GCA clinical pathologic parameters were analyzed, and the relationship of p57kip2 and cyclinD1 in GCA were also evaluated.ResultsThe expression of p57kip2 significantly lower in GCA (P = 0.036), and there was a significant correlation in the different degrees of differentiation (P < 0.05). Furthermore, median survival time was 41 months for patients with high mRNA expression of p57kip2. This was longer compared to patients with low mRNA expression of P57kip2 (37 months, X2 = 4.788, P = 0.029).The expression of cyclinD1 was significantly higher in GCA(P = 0.002), and was significant correlated to clinical stage(P<0.05). Median survival time was 34 months in patients with high mRNA expression of cyclinD1, which was shorter than in patients with low expression of cyclinD1 mRNA (41 months, X2 = 4.071, P = 0.044). The protein expression of p57kip2 was not correlated to the protein expression of cyclinD1 (P = 0.55).ConclusionThe expression of p57kip2 and cyclinD1 are likely to suppress or promote the tumorigenesis and progression of GCA.

Aberrant Level of Skp2 and p27KIP1 in Intraductal Proliferative Lesions is Associated with Tumorigenesis

ABSTRACTBreast cancer is one of the leading causes of cancer-related death in women worldwide. Here we aimed to examine the expression status of S-phase kinase-associated protein 2 (Skp2) and p27KIP1, and assess the significance of Skp2 plus p27KIP1 expression in patients with intraductal proliferative lesions, including ductal carcinoma in situ (DCIS) and atypical ductal hyperplasia (ADH). Skp2 and p27KIP1 mRNA levels in DCIS, ADH, flat epithelial atypia, and usual ductal hyperplasia (UDH) were evaluated by quantitative real-time reverse transcription polymerase chain reaction and protein expression was evaluated immunohistochemically in 60 fresh tissues and 120 paraffin-embedded tissues from patients with the four subtypes above. We found that the protein and mRNA level of Skp2 were significantly increased in DCIS and ADH as compared with that in UDH. In contrast, p27KIP1 protein and mRNA levels were reduced. Based on the above findings, abnormal levels of Skp2 and p27KIP1 have probably been involved in the pathogenesis of ADH and DCIS. Thus, Skp2 and p27KIP1 may serve as important diagnosis markers.

Novel role of Rac-Mid1 signaling in medial cerebellar development.

Rac signaling impacts a relatively large number of downstream targets; however, few studies have established an association between Rac pathways and pathological conditions. In the present study, we generated mice with double knockout of Rac1 and Rac3 (Atoh1-Cre;Rac1flox/flox;Rac3-/- ) in cerebellar granule neurons (CGNs). We observed impaired tangential migration at E16.5, as well as numerous apoptotic CGNs at the deepest layer of the external granule layer (EGL) in the medial cerebellum of Atoh1-Cre;Rac1flox/flox;Rac3-/- mice at P8. Atoh1-Cre;Rac1flox/flox;Rac3-/- CGNs differentiated normally until expression of p27kip1 and NeuN in the deep EGL at P5. Primary CGNs and cerebellar microexplants from Atoh1-Cre;Rac1flox/flox;Rac3-/- mice exhibited impaired neuritogenesis, which was more apparent in Map2-positive dendrites. Such findings suggest that impaired tangential migration and final differentiation of CGNs have resulted in decreased cerebellum size and agenesis of the medial internal granule layer, respectively. Furthermore, Rac depleted/deleted cells exhibited decreased levels of Mid1 and impaired mTORC1 signaling. Mid1 depletion in CGNs produced mild impairments in neuritogenesis and reductions in mTORC1 signaling. Thus, a novel Rac-signaling pathway (Rac1-Mid1-mTORC1) may be involved in medial cerebellar development.

Rp58 and p27kip1 coordinate cell cycle exit and neuronal migration within the embryonic mouse cerebral cortex

BackgroundDuring the development of the mammalian cerebral cortex, newborn postmitotic projection neurons are born from local neural stem cells and must undergo radial migration so as to position themselves appropriately to form functional neural circuits. The zinc finger transcriptional repressor Rp58 (also known as Znf238 or Zbtb18) is critical for coordinating corticogenesis, but its underlying molecular mechanism remains to be better characterised.FindingsHere, we demonstrate that the co-expression of Rp58 and the cyclin dependent kinase inhibitor (CDKI) p27kip1 is important for E14.5-born cortical neurons to coordinate cell cycle exit and initiate their radial migration. Notably, we find that the impaired radial positioning of Rp58-deficient cortical neurons within the embryonic (E17.5) mouse cortex, as well as their multipolar to bipolar transition from the intermediate zone to the cortical plate can be restored by forced expression of p27kip1 in concert with suppression of Rnd2, a downstream target gene of Rp58. Furthermore, the restorative effects of p27kip1 and Rnd2 abrogation are reminiscent of suppressing RhoA signalling in Rp58-deficient cells.ConclusionsOur findings demonstrate functional interplay between a transcriptional regulator and a CDKI to mediate neuroprogenitor cell cycle exit, as well as to promote radial migration through a molecular mechanism consistent with suppression of RhoA signalling.

All‐Trans‐Retinoic Acid Stimulates Overexpression of Tumor Protein D52 (TPD52, Isoform 3) and Neuronal Differentiation of IMR‐32 Cells

Tumor protein D52 (TPD52), a proto-oncogene is overexpressed in a variety of epithelial carcinomas and plays an important role in cell proliferation, migration, and cell death. In the present study we found that the treatment of IMR-32 neuroblastoma (NB) cells with retinoic acid (RA) stimulates an increase in expression of TPD52. TPD52 expression is detectable after 72 h, can be maintained till differentiation of NB cells suggesting that TPD52 is involved in differentiation. Here, we demonstrate that TPD52 is essential for RA to promote differentiation of NB cells. Our results show that exogenous expression of EGFP-TPD52 in IMR-32 cells resulted cell differentiation even without RA. RA by itself and with overexpression of TPD52 can increase the ability of NB cells differentiation. Interestingly, transfection of IMR-32 cells with a specific small hairpin RNA for efficient knockdown of TPD52 attenuated RA induced NB cells differentiation. Transcriptional and translational level expression of neurotropic (BDNF, NGF, Nestin) and differentiation (β III tubulin, NSE, TH) factors in NB cells with altered TPD52 expression and/or RA treatment confirmed essential function of TPD52 in cellular differentiation. Furthermore, we show that TPD52 protects cells from apoptosis and arrest cell proliferation by varying expression of p27Kip1, activation of Akt and ERK1/2 thus promoting cell differentiation. Additionally, inhibition of STAT3 activation by its specific inhibitor arrested NB cells differentiation by EGFP-TPD52 overexpression with or without RA. Taken together, our data reveal that TPD52 act through activation of JAK/STAT signaling pathway to undertake NB cells differentiation induced by RA. J. Cell. Biochem. 118: 4358-4369, 2017. © 2017 Wiley Periodicals, Inc.

Gastrin induces multidrug resistance via the degradation of p27Kip1 in the gastric carcinoma cell line SGC7901.

Multidrug resistance (MDR) is one of the major reasons for the failure of chemotherapy-based gastric carcinoma (GC) treatments, hence, biologically based therapies are urgently needed. Gastrin (GAS), a key gastrointestinal(GI) hormone, was found to be involved in tumor formation, progression, and metastasis. In this study, quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemical staining analysis revealed a high level of expression of GAS in drug-insensitive GC tissues (P<0.01) and similar results were revealed in GC cell lines SGC7901 and its multidrug-resistant variants SGC7901/VCR and SGC7901/ADR. We constructed a eukaryotic expression vector pCDNA3.1(+)/GAS for GAS overexpression and recombinant lentiviral vectors for specific siRNA (siGAS). Transfection of pCDNA3.1(+)/GAS increased (P<0.05) while transfection of siGAS (P<0.05) and co-treated with paclitaxel(TAX) and vincristine (VCR) combination (TAX-VCR) decreased (P<0.01) the cell viability of SGC7901, SGC7901/VCR and SGC7901/ADR. Apoptosis rates of SGC7901/VCR and SGC7901/ADR were reduced by pCDNA3.1(+)/GAS and increased by siGAS (P<0.05). The apoptosis rates of SGC7901/VCR, SGC7901/ADR and SGC7901 were all upregulated (P<0.01) when cells were co-treated with a combination of siGAS and TAX-VCR. Additionally, siGAS significantly downregulated the expression of Bcl-2 and multidrug-resistant associate protein (MRP1) and P-glycoprotein (Pgp) (P<0.05) in SGC7901/VCR and SGC7901/ADR cells. Moreover, GAS overexpression in SGC7901 cells significantly inhibited p27Kip1 expression but increased phosphorylation levels of p27Kip1 on Thr(187) and Ser(10) sites (P<0.05), as well as increasing nuclear accumulation of S-phase kinase-associated protein 2 (Skp2) and cytoplasmic accumulation of the Kip1 ubiquitination-promoting complex (KPC) (P<0.05). Silencing of Skp2 blocked the promoting effects of pCDNA3.1(+)/GAS on viability, the expression of MRP1 and Pgp and the inhibitory effects of pCDNA3.1(+)/GAS on apoptosis. In conclusion, we suggest that GAS contributes to the emergence of MDR of SGC7901 cells via the degradation of p27Kip1.

Angiogenic and lymphangiogenic profiles in histological variants of papillary thyroid carcinoma.

Papillary thyroid carcinoma (PTC) is a well‑differentiated tumor that occurs in several histological variants whose biological behaviors remain unclear. Angiogenesis and lymphangiogenesis are critical processes that enable tumor progression. The aim of this study was to evaluate the angiogenic and lymphangiogenic phenotypes of PTC, considering the differences between histological variants. Angiogenic and lymphangiogenic profiles were analyzed by determining microvascular density (MVD) and lymphatic vessel density (LVD) in 73 cases of PTC, using immunohistochemistry. To assess the biological markers involved in blood and lymph vessel formation, the expression of vascular endothelial growth factor (VEGF), cyclooxygenase 2 (COX‑2), and p27kip1 (p27) was determined. MVD was significantly higher in patients with high‑risk PTC and in those with local extrathyroidal and vascular invasion. Positive VEGF expression was strongly associated with high MVD and age‑related tumor enlargement. The presence of lymph vessel invasion was associated with the expression of either VEGF or COX‑2. The analysis of angiogenesis and lymphangiogenesis in different histological variants of PTC revealed elevated LVD rather than MVD in the follicular variant of PTC (FV‑PTC).Lower MVD was observed in FV‑PTC relative to the classic variant of PTC (CV‑PTC). The frequency of VEGF‑positive tumors was higher in CV‑PTC than in FV‑PTC. A significant association between COX‑2 and p27 expression was observed in FV‑PTC but not in CV‑PTC. These results suggest that VEGF, COX‑2, and p27 may be important biological markers that determine the angiogenic and lymphangiogenic potentials of PTC, particularly between the follicular and classic variants.