Mechanism Of Cell Cycle Arrest Research Articles

S-Phase Cell Cycle Arrest, Apoptosis, and Molecular Mechanisms of Aplasia Ras homolog Member I–Induced Human Ovarian Cancer SKOV3 Cell Lines

ObjectiveAplasia Ras homolog member I (ARHI) is associated with human ovarian cancer (HOC) growth and proliferation; however, the mechanisms are unclear. The purpose of this study was to investigate ARHI effects in HOC SKOV3 cells.MethodsWe transfected SKOV3 cells with PIRES2-EGFP-ARHI and measured growth inhibition rates, cell cycle distribution, apoptosis rates, and expression of P-STAT3 (phosphorylated signal transduction and activators of transcription 3) and P-ERK (phosphorylated extracellular signal regulated protein kinase).ResultsOur data showed significant inhibition of growth, significantly increased S-phase arrest and apoptosis rates, and reduction of P-STAT3 and P-ERK1/2 expression levels.ConclusionsWe propose the mechanism may involve ARHI-induced phosphorylation of ERK1/2 and STAT3 protein kinases, thereby blocking proliferation signaling pathways, to induce HOC SKOV3 apoptosis.

The expression of cell cycle related proteins PCNA, Ki67, p27 and p57 in normal and preeclamptic human placentas.

Placenta is a transitional area making many physiological activities between mother and fetus and therefore, it is a critical organ influencing the outcome of pregnancy. Fetal growth is directly related to placental development. Accurate placental development depends on coordinated action of trophoblasts' proliferation, differentiation and invasion. Information on cell cycle related proteins that control these events is limited and how they are affected in preeclampsia is not fully understood yet. Therefore, in this study, in order to understand the role of cell cycle regulators in preeclamptic placentas we aimed to determine the spatio-temporal immunolocalizations of cell cycle regulators in preeclamptic and normal human term placentas. Term placentas were obtained from women diagnosed with preeclampsia and from normal pregnancies with informed consent following cesarean deliveries. Placental samples were stained via immunohistochemistry with PCNA, Ki67, p27, p57, vimentin and cytokeratin 7 antibodies and were examined by light microscopy. PCNA and Ki67 staining intensities significantly increased in villous parts, significantly decreased in basal plates of PE group and did not change in chorionic plates. Staining intensities of cell cycle inhibitors p27 and p57 significantly increased in all parts of preeclamptic placentas compared to control. Placental abnormalities of preeclamptic placentas might be associated with proliferation and cell cycle arrest mechanisms' alterations occurred in preeclampsia.

United we stand not dividing: The syncytiotrophoblast and cell senescence

The multinucleate syncytiotrophoblast of the human placenta is responsible for transport functions between maternal and fetal blood supplies and is a major site of protein synthesis and steroid production. It is formed by cell fusion of the underlying cytotrophoblast cells. The nuclei of the multinucleate syncytiotrophoblast are non-mitotic yet the mechanism of cell cycle arrest in the syncytiotrophoblast is not known. The recent publication by the group of Krizhanovsky (2013), demonstrates that cell fusion induces cell senescence. The work reported the exciting finding that term placenta syncytiotrophoblast displays markers associated with cellular senescence. Cellular senescence is perhaps best known as a component of aging, a response to stress and an important factor in preventing tumor cell growth. The aforementioned study suggests myriad avenues of investigation in placental biology with intriguing possibilities to furthering our understanding of placental development and aging, health of pregnancy and placental pathologies having their origin in placental stress.

Resveratrol Differentially Regulates NAMPT and SIRT1 in Hepatocarcinoma Cells and Primary Human Hepatocytes

Resveratrol is reported to possess chemotherapeutic properties in several cancers. In this study, we wanted to investigate the molecular mechanisms of resveratrol-induced cell cycle arrest and apoptosis as well as the impact of resveratrol on NAMPT and SIRT1 protein function and asked whether there are differences in hepatocarcinoma cells (HepG2, Hep3B cells) and non-cancerous primary human hepatocytes. We found a lower basal NAMPT mRNA and protein expression in hepatocarcinoma cells compared to primary hepatocytes. In contrast, SIRT1 was significantly higher expressed in hepatocarcinoma cells than in primary hepatocytes. Resveratrol induced cell cycle arrest in the S- and G2/M- phase and apoptosis was mediated by activation of p53 and caspase-3 in HepG2 cells. In contrast to primary hepatocytes, resveratrol treated HepG2 cells showed a reduction of NAMPT enzymatic activity and increased p53 acetylation (K382). Resveratrol induced NAMPT release from HepG2 cells which was associated with increased NAMPT mRNA expression. This effect was absent in primary hepatocytes where resveratrol was shown to function as NAMPT and SIRT1 activator. SIRT1 inhibition by EX527 resembled resveratrol effects on HepG2 cells. Furthermore, a SIRT1 overexpression significantly decreased both p53 hyperacetylation and resveratrol-induced NAMPT release as well as S-phase arrest in HepG2 cells. We could show that NAMPT and SIRT1 are differentially regulated by resveratrol in hepatocarcinoma cells and primary hepatocytes and that resveratrol did not act as a SIRT1 activator in hepatocarcinoma cells.

MiR-192 Induces G2/M Growth Arrest in Aristolochic Acid Nephropathy

Aristolochic acid nephropathy is characterized by rapidly progressive tubulointerstitial nephritis culminating in end-stage renal failure and urothelial malignancy. Profibrotic effects of aristolochic acid are linked to growth arrest of proximal tubular epithelial cells; however, the underlying mechanisms are largely undetermined. miRNAs are small, endogenous, post-transcriptional regulators of gene expression implicated in numerous physiological and pathological processes. In the present study, we characterized the mechanism of aristolochic acid-induced cell cycle arrest and its regulation by miRNAs. Incubation with aristolochic acid led to profound G2/M arrest in proximal tubular epithelial cells via p53-mediated inactivation of the maturation-promoting complex, CDK1/cyclin-B1. Analysis of miRNA expression identified up-regulation of miRNAs, including miR-192, miR-194, miR-450a, and miR-542-3p. The stable overexpression of miR-192 recapitulated G2/M arrest via repression of the E3 ubiquitin ligase, murine double-minute 2, a negative regulator of p53. p53-induced transcription of p21(cip1) and growth arrest and DNA damage 45 and resulted in the inactivation and dissociation of the maturation-promoting complex. These data demonstrate a core role for miR-192 in mediating proximal tubular epithelial cell G2/M arrest after toxic injury by aristolochic acid. Because numerous studies have linked such growth arrest to fibrosis after proximal tubular epithelial cell injury, this mechanism may have widespread relevance to recovery/nonrecovery after acute kidney injury.

Malnutrition suppresses cell cycle progression of hematopoietic progenitor cells in mice via cyclin D1 down-regulation

ObjectiveProtein malnutrition (PM) often is associated with changes in bone marrow (BM) microenvironment leading to an impaired hematopoiesis; however, the mechanism involved is poorly understood. The aim of this study was to compare the cell cycle progression of hematopoietic stem cells (HSC) and hematopoietic progenitor cells (HPC) and evaluate the cell cycle signaling in malnourished mice to assess the mechanism of cell cycle arrest. MethodsC57Bl/6J mice were randomly assigned in control and malnourished groups receiving normoproteic and hypoproteic diets (12% and 2% protein, respectively) over a 5-wk period. Nutritional and hematologic parameters were assessed and BM immunophenotypic analysis was performed. Cell cycle of HPC (Lin–) and HSC (Lin–Sca-1+c-Kit+) were evaluated after 6 h of in vivo 5-bromo-2'-deoxyuridine (BrDU) incorporation. Cell cycle regulatory protein expression of HPC was assessed by Western blot. ResultsMalnourished mice showed lower levels of serum protein, albumin, glucose, insulin-like growth factor-1, insulin, and higher levels of serum corticosterone. PM also caused a reduction of BM myeloid compartment resulting in anemia and leukopenia. After 6 h of BrDU incorporation, malnourished mice showed G0-G1 arrest of HPC without changes of HSC proliferation kinetics. HPC of malnourished mice showed reduced expression of proteins that induce cell cycle (cyclin D1, cyclin E, pRb, PCNA, Cdc25a, Cdk2, and Cdk4) and increased expression of inhibitory proteins (p21 and p27) with no significant difference in p53 expression. ConclusionPM suppressed cell cycle progression mainly of HPC. This occurred via cyclin D1 down-regulation and p21/p27 overexpression attesting that BM microenvironment commitment observed in PM is affecting cell interactions compromising cell proliferation.

Immunohistochemical distribution of cell cycle proteins p27, p57, cyclin D3, PCNA and Ki67 in normal and diabetic human placentas

The placenta is a regulator organ for many metabolic activities between mother and fetus. Therefore, fetal growth is directly related to the placental development. Placental development is a series of events that depend on the coordinated action of trophoblasts' proliferation, differentiation and invasion. Studies on cell cycle related proteins which control these events are fairly limited. How placental tissue proliferation is affected by diabetes is not exactly known yet. Therefore in this study, the immunohistochemical localizations of cell cycle related proteins like PCNA, Ki67, cyclin D3, p27 and p57 in the differentiation, proliferation and apoptosis mechanisms of normal and diabetic placentas were investigated. Information on cell cycle related proteins that control these events is limited and how they are affected in diabetes mellitus is not fully understood yet. Therefore, in this study, to understand the role of cell cycle regulators in diabetic placentas we aimed to determine the spatio-temporal immunolocalizations of cell cycle regulators in diabetic and normal human term placentas. Term placentas were obtained from diabetic women and from normal pregnancies with informed consent following caesarean deliveries. Placental samples were stained via immunohistochemistry with PCNA, Ki67, cyclin D3, p27 and p57 antibodies and were examined by light microscopy. When compared to control placentas, PCNA, Ki67 and cyclin D3 staining intensities significantly increased in villous parts of diabetes group. Moreover, Ki67 and cyclin D3 stainings also significantly increased in basal plates and chorionic plate respectively. In chorionic plates, p27 and p57 staining intensities significantly decreased in diabetic group. p57 staining also significantly decreased in villous parts of diabetic placentas. Placental abnormalities seen in diabetic placentas could be associated with proliferation and cell cycle arrest mechanisms' alterations occurred in diabetes mellitus.

Role of anthocyanin-enriched purple-fleshed sweet potato p40 in colorectal cancer prevention.

Anthocyanins, the natural pigments in plant foods, have been associated with cancer prevention. However, the content of anthocyanins in staple foods is typically low and the mechanisms by which they exert anticancer activity is not yet fully defined. We selected an anthocyanin-enriched purple-fleshed sweet potato clone, P40, and investigated its potential anticancer effect in both in vitro cell culture and in vivo animal model. In addition to a high level of total phenolics and antioxidant capacity, P40 possesses a high content of anthocyanins at 7.5 mg/g dry matter. Treatment of human colonic SW480 cancer cells with P40 anthocyanin extracts at 0-40 μM of peonidin-3-glucoside equivalent resulted in a dose-dependent decrease in cell number due to cytostatic arrest of cell cycle at G1 phase but not cytotoxicity. Furthermore, dietary P40 at 10-30% significantly suppressed azoxymethane-induced formation of aberrant crypt foci in the colons of CF-1 mice in conjunction with, at least in part, a lesser proliferative PCNA and a greater apoptotic caspase-3 expression in the colon mucosal epithelial cells. These observations, coupled with both in vitro and in vivo studies reported here, suggest anthocyanin-enriched sweet potato P40 may protect against colorectal cancer by inducing cell-cycle arrest, antiproliferative, and apoptotic mechanisms.

Neferine isolated from Nelumbo nucifera enhances anti-cancer activities in Hep3B cells: Molecular mechanisms of cell cycle arrest, ER stress induced apoptosis and anti-angiogenic response

Hepatocellular carcinoma (HCC) is one of the most aggressive malignant diseases and is highly resistant to conventional chemotherapy. Neferine, a major bisbenzylisoquinoline alkaloid derived from the embryos of Nelumbo nucifera, has been reported a few physiological activities. However, the mechanisms of anticancer effects are not well understood and its detailed activities on Hep3B cells have not been determined. Our results suggest that neferine exhibited cytotoxicity against HCC Hep3B cells, but not against HCC Sk-Hep1 and THLE-3, a normal human liver cell line. In addition, consistent with the induction of G1/S phase cell population in flow cytometry, downregulation of c-Myc, cyclin D1, D3, CDK4, E2F-1, as well as dephosphorlyation of cdc2 by western blot analysis, as evidenced by the appearance of cell cycle arrest, were observed in Hep3B cells treated with neferine. Our results demonstrated neferine induced ER stress and apoptosis, acting through multiple signaling cascades by the activation of Bim, Bid, Bax, Bak, Puma, caspases-3, -6, -7, -8 and PARP, and the protein expression levels of Bip, calnexin, PDI, calpain-2 and caspase-12 were also upregulated dramatically by neferine treatment. Overexpression of GFP-LC3B by neferine resulted in a diffuse cytosolic GFP fluorescence and the strong fluorescent spots, representing autophagosomes. The significant reduction of the migration in Hep3B cells and the capillary tube-like formation of HUVECs by neferine were also determined. These observations reveal that the therapeutic potential of neferine in treating HCC Hep3B cells, containing copies of hepatitis B virus (HBV) genomes.

P-0303 - “The Early Growth Response Genes 1,2 and 3 Cause Cell Cycle Arrest and Apoptosis in Colon Cancer”

Background: The Egr family (Early Growth Response Genes) of zinc finger transcription factors, which consists of four members; Egr-1, -2, -3 and -4, have been proved to have dynamic functions in the regulation of cell growth and the immune responses. Moreover, in a number of malignancies-which is a cell growth and immune related process- it seems that Egr1 and 2 are able to induce cell cycle arrest and apoptosis. The importance of such an observation relies on the fact that -by definition- a cell becomes malignant when the cell cycle arrest mechanism is not functional.

Synthesis and anticancer activity of aminodihydroquinoline analogs: Identification of novel proapoptotic agents

A series of 2-aminodihydroquinoline analogs were synthesized and their in vitro cytotoxicities against metastatic breast adenocarcinoma cell line MDA-MB-231 were tested. Five out of 16 compounds exhibited promising activity and structure–activity relationship revealed major role of dialkylaminoethyl substituents on dihydroquinoline ring for the activity. Two compounds, 5f and 5h, presented cytotoxicity with IC50 values of about 2μM when the compounds were treated to the cells without serum. The cell proliferation was inhibited mildly when the cells cultured with serum. Flow cytometry analyses showed that those compounds arrested the cells at G2/M checkpoint when the cell cycle is active while they induce apoptosis when the cell growth is restricted due to the absence of growth factors. These results suggest the two novel compounds may have anticancer activity through cell cycle arrest and pro apoptosis mechanism.

Meis1 regulates postnatal cardiomyocyte cell cycle arrest

The neonatal mammalian heart is capable of substantial regeneration following injury through cardiomyocyte proliferation. However, this regenerative capacity is lost by postnatal day 7 and the mechanisms of cardiomyocyte cell cycle arrest remain unclear. The homeodomain transcription factor Meis1 is required for normal cardiac development but its role in cardiomyocytes is unknown. Here we identify Meis1 as a critical regulator of the cardiomyocyte cell cycle. Meis1 deletion in mouse cardiomyocytes was sufficient for extension of the postnatal proliferative window of cardiomyocytes, and for re-activation of cardiomyocyte mitosis in the adult heart with no deleterious effect on cardiac function. In contrast, overexpression of Meis1 in cardiomyocytes decreased neonatal myocyte proliferation and inhibited neonatal heart regeneration. Finally, we show that Meis1 is required for transcriptional activation of the synergistic CDK inhibitors p15, p16 and p21. These results identify Meis1 as a critical transcriptional regulator of cardiomyocyte proliferation and a potential therapeutic target for heart regeneration.

UCN-01 induces S and G2/M cell cycle arrest through the p53/p21waf1or CHK2/CDC25C pathways and can suppress invasion in human hepatoma cell lines

BackgroundUCN-01 (7-hydroxystaurosporine), a protein kinase inhibitor, has attracted a great deal of attention as a potent antitumour agent. Several clinical trials of UCN-01 alone or in combination with other agents for different tumour types are currently underway, and some of these trials have had positive results. Hepatocellular carcinoma has high incidence rates and is associated with poor prognosis and high mortality rates.MethodsThree different hepatoma cell lines (Huh7, HepG2, and Hep3B) were treated with different concentrations of UCN-01, and the anti-tumour effects of UCN-01 were evaluated. Following UCN-01 treatment, cell growth was measured using an MTT assay, cell cycle arrest was assayed using flow cytometry, and the mechanisms of cell cycle arrest and invasion inhibition were investigated through western blotting and a Matrigel invasion assay.ResultsAfter a 72-h UCN-01 treatment, the growth of different hepatoma cell lines was significantly inhibited in a dose-dependent manner, with IC50 values ranging from 69.76 to 222.74 nM. Flow cytometry results suggested that UCN-01 inhibits proliferation in the hepatoma cells by inducing S and G2/M phase arrest, but not G1/S arrest, which differs from previous reports that used other tumour cell lines. Western blot results illustrated that UCN-01 induces a G2/M phase arrest, regardless of the status of the p53/P21waf1 pathway, whereas the CHK2/CDC25C pathway and the p53/p21waf1 pathway were involved in the UCN-01-induced S phase arrest. UCN-01 remarkably inhibited Huh7 cell invasion in a time-dependent manner. Suppression of Huh7 cell invasion may be due to the down-regulation of phosphorylated β-catenin by UCN-01.ConclusionsThese findings suggest that UCN-01 induces hepatoma cell growth inhibition by regulating the p53/p21waf1 and CHK2/CDC25 pathways. Suppression of Huh7 cell invasion by UCN-01 may be due to the down-regulation of phosphorylated β-catenin. These data lend support for further studies on UCN-01 as a promising anti-HCC candidate.

Mechanisms of G1 cell cycle arrest and apoptosis in myeloma cells induced by hybrid-compound histone deacetylase inhibitor

ObjectivesHistone deacetylase (HDAC) inhibitors are new therapeutic agents, used to treat various types of malignant cancers. In the present study, we investigated the effects of Ky-2, a hybrid-compound HDAC inhibitor, on the growth of mouse myeloma cells. Materials and methodsMyeloma cells, HS-72, P3U1, and mouse normal cells were used in this study. Effect of HDAC inhibitors on cell viability was determined by WST-assay and trypan blue assay. Cell cycle was analyzed using flow cytometer. The expression of cell cycle regulatory and the apoptosis associated proteins were examined by Western blot analysis. Hoechst’s staining was used to detect apoptotic cells. ResultsOur findings showed that Ky-2 decreased the levels of HDACs, while it enhanced acetylation of histone H3. Myeloma cell proliferation was inhibited by Ky-2 treatment. Interestingly, Ky-2 had no cytotoxic effects on mouse normal cells. Ky-2 treatment induced G1-phase cell cycle arrest and accumulation of a sub-G1 phase population, while Western blotting analysis revealed that expressions of the cell cycle-associated proteins were up-regulated. Also, Ky-2 enhanced the cleavage of caspase-9 and -3 in myeloma cells, followed by DNA fragmentation. In addition, Ky-2 was not found to induce apoptosis in bcl-2 overexpressing myeloma cells. ConclusionThese findings suggest that Ky-2 induces apoptosis via a caspase-dependent cascade and Bcl-2-inhibitable mechanism in myeloma cells.

The effect of ATM kinase inhibition on the initial response of human dental pulp and periodontal ligament mesenchymal stem cells to ionizing radiation

Purpose: This study evaluates early changes in human mesenchymal stem cells (MSC) isolated from dental pulp and periodontal ligament after γ-irradiation and the effect of ataxia-telangiectasia mutated (ATM) inhibition.Methods: MSC were irradiated with 2 and 20 Gy by 60Co. For ATM inhibition, specific inhibitor KU55933 was used. DNA damage was measured by Comet assay and γH2AX detection. Cell cycle distribution and proteins responding to DNA damage were analyzed 2–72 h after the irradiation.Results: The irradiation of MSC causes an increase in γH2AX; the phosphorylation was ATM-dependent. Irradiation activates ATM kinase, and the level of p53 protein is increased due to its phosphorylation on serine15. While this phosphorylation of p53 is ATM-dependent in MSC, the increase in p53 was not prevented by ATM inhibition. A similar trend was observed for Chk1 and Chk2. The increase in p21 is greater without ATM inhibition. ATM inhibition also does not fully abrogate the accumulation of irradiated MSC in the G2-phase of the cell-cycle.Conclusions: In irradiated MSC, double-strand breaks are tagged quickly by γH2AX in an ATM-dependent manner. Although phosphorylations of p53(ser15), Chk1(ser345) and Chk2(thr68) are ATM-dependent, the overall amount of these proteins increases when ATM is inhibited. In both types of MSC, ATM-independent mechanisms for cell-cycle arrest in the G2-phase are triggered.

Tubulin‐targeting agent combination therapies: dosing schedule could matter

Tubulin-binding agents are potent cytotoxic drugs that are largely used to treat haematological malignancies and solid tumours. In this issue of British Journal of Pharmacology, doxorubicin was shown to decrease the activity of vincristine when administered simultaneously, unless cell cycle arrest mechanisms were disrupted. This observation emphasizes the need to better explore schedule-dependent antagonist effects in anticancer drug combination therapies. This article is a commentary on the research paper by Ehrhardt et al., pp. 1558-1569 of this issue. To view this paper visit http://dx.doi.org/10.1111/bph.12068.

Human rpL3 induces G₁/S arrest or apoptosis by modulating p21waf1/cip1 levels in a p53-independent manner

It is now largely accepted that ribosomal proteins may be implicated in a variety of biological functions besides that of components of the translation machinery. Many evidences show that a subset of ribosomal proteins are involved in the regulation of the cell cycle and apoptosis through modulation of p53 activity. In addition, p53-independent mechanisms of cell cycle arrest in response to alterations of ribosomal proteins availability have been described. Here, we identify human rpL3 as a new regulator of cell cycle and apoptosis through positive regulation of p21 expression in a p53-independent system. We demonstrate that the rpL3-mediated p21 upregulation requires the specific interaction between rpL3 and Sp1. Furthermore, in our experimental system, p21 overexpression leads to a dual outcome, activating the G₁/S arrest of the cell cycle or the apoptotic pathway through mitochondria, depending on its intracellular levels. It is noteworthy that depletion of p21 abrogates both effects. Taken together, our findings unravel a novel extraribosomal function of rpL3 and reinforce the proapoptotic role of p21 in addition to its widely reported ability as an inhibitor of cell proliferation.

Abstract PD09-09: The Akt inhibitor MK-2206 is an effective radio-sensitizer of p53 deficient triple negative breast cancer (TNBC) cells.

Abstract Purpose: Triple negative breast cancer (TNBC) is an aggressive tumor with a higher locoregional recurrence compared to estrogen receptor (ER) positive breast cancer. Mutations in the PI3K/Akt/mTOR pathway leading to up-regulation of Akt occur with high frequency in TNBC. Hyperactive Akt is associated with increase radiation resistance, mediated through inhibition of apoptosis and up-regulation of DNA damage-induced G2 arrest. p53 plays a key role in mediating G1 cell cycle arrest following genotoxic stress such as radiation; p53-deficient cells however must rely on alternate mechanisms for cell cycle arrest in S- and G2-phases. Inhibition of Akt therefore would be expected to act in a synthetic lethal fashion to radiosensitize p53 deficient TNBC cells by decreasing their ability to arrest in G2. Given that p53 mutations occur in 44% of TNBC this may be an effective strategy for radiosensitizing TNBC. Methods: Experiments were conducted using MDA-MB-468, MDA-MB-231 and SUM 149 cells, all well established models for p53-deficient TNBC. Cells were treated with MK-2206, a potent allosteric Akt inhibitor, alone or in combination with increasing doses of radiation from 0–8 Gy. In vitro studies preformed included; cell survival assays, MTT assay, immunoblot analysis of key proteins, FACS analysis for cell cycle, apoptosis, and gH2AX staining. Results: We found that the combination of IR and Akt inhibition by MK-2206 is synergistic. MK-2206 inhibited both endogenous and radiation-induced Akt activation and phosphorylation of its down-stream targets. Decreased clonogenic survival was seen after 2 or 24 hours pretreatment with MK-2206 as well as decreased viability by MTT assay. Cell cycle analysis demonstrates MK-2206 decreases the proportion of cells in G2/M arrest following irradiation, as well as induced a greater proportion of apoptosis. Evaluation of the DNA damage response pathway demonstrated decreased levels of total DNA-PK, as well as a delayed DNA damage response. Conclusions: These studies demonstrate that targeted inhibition of Akt effectively radiosensitizes p53 deficient TNBC cells lines, possibly through a synthetic lethal effect on the DNA damage response. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr PD09-09.

Inhibition of p21-activated kinase 4 expression suppresses the proliferation of Hep-2 laryngeal carcinoma cells via activation of the ATM/Chk1/2/p53 pathway

In the present study, we investigated the effects of the p21-activated kinase 4 (Pak4) gene on Hep-2 laryngeal carcinoma cells in vivo and in vitro. The expression of Pak4 was downregulated using small interfering RNA (siRNA). The downregulation of Pak4 decreased the proliferation and increased apoptosis and S phase arrest in Hep-2 cells in vitro. In further experiments, we determined that the S/G(2) transition was obstructed by the downregulation of Pak4 using 5‑chloro-2'‑deoxyuridine (CldU) and 5‑iodo‑2'‑deoxyuridine (IdU) double staining. A xenografted Hep-2 tumor mouse model was created by inducing human tumors with a subcutaneous (s.c.) injection of 5x10(6) Hep-2 cells into the dorsal flank region of nu/nu mice. The downregulation of Pak4 in established xenografted tumors decreased tumor size and weight. The survival rate of the mice with tumors that did not express Pak4 was significantly higher compared to the mice with tumors expressing Pak4. These results confirm the role of Pak4 as an oncogene in laryngeal carcinoma cells. To identify the mechanism of the cell cycle arrest induced by Pak4, immunohistochemical staining was performed to detect changes in cell cycle‑related proteins. The results demonstrated that p53 was activated following the downregulation of Pak4. The levels of ataxia telangiectasia mutated (ATM), the upstream protein of checkpoint kinase (Chk)1 and Chk2, also increased. Therefore, we confirmed that the mechanisms of the Pak4-induced cell cycle arrest invovlve the activation of the ATM/Chk1/2/p53 pathway. These results may prove helpful for the development of novel therapies for the treatment of laryngeal carcinoma.

Effector mechanisms of sunitinib-induced G1 cell cycle arrest, differentiation, and apoptosis in human acute myeloid leukaemia HL60 and KG-1 cells

Acute myeloid leukaemia (AML) is a heterogeneous disease with dismal outcome. Sunitinib is an orally active inhibitor of multiple tyrosine kinase receptors approved for renal cell carcinoma and gastrointestinal stromal tumour that has also been studied for AML in several clinical trials. However, the precise mechanism of sunitinib action against AML remains unclear and requires further investigation. For this purpose, this study was conducted using human AML cell lines (HL60 and KG-1) and AML patients' mononucleated cells. Sunitinib induced G1 phase arrest associated with decreased cyclin D1, cyclin D3, and cyclin-dependent kinase (Cdk)2 and increased p27(Kip1), pRb1, and p130/Rb2 expression and phosphorylated activation of protein kinase C alpha and beta (PKCα/β). Selective PKCα/β inhibitor treatment abolished sunitinib-elicited AML differentiation, suggesting that PKCα/β may underlie sunitinib-induced monocytic differentiation. Furthermore, sunitinib increased pro-apoptotic molecule expression (Bax, Bak, PUMA, Fas, FasL, DR4, and DR5) and decreased anti-apoptotic molecule expression (Bcl-2 and Mcl-1), resulting in caspase-2, caspase-3, caspase-8, and caspase-9 activation and both death receptor and mitochondria-dependent apoptosis. Taken together, these findings provide evidence that sunitinib targets AML cells through both differentiation and apoptosis pathways. More clinical studies are urgently needed to demonstrate its optimal clinical applications in AML.