Senescence-associated secretory phenotype and activation of NF-κB in splenocytes of old mice exposed to irradiation at a young age.

DNA damage-induced cellular senescence is involved in aging. We reported previously that p53+/- mice subjected to irradiation at a young age exhibited an increased number of splenic lymphocytes in the S and G2/M phases. However, the detailed nature of splenic disorders in these mice is not fully understood. In this study, we investigated the effects on molecules in splenocytes, especially on senescence factors after early exposure of mice to radiation. Mice, 8- (young) or 17-, 30-, and 41-week-old (old) p53+/- were subjected to 3-Gy whole-body irradiation. Splenocytes were prepared at 56 weeks of age. Immunoblot showed that irradiation at 8 weeks enhanced the expression and phosphorylation of p53, cyclin-dependent kinase 2, cell division cycle 6, and the MDM2 proto-oncogene in splenocytes. However, these molecules were not affected by irradiation at 17, 30, and 41 weeks of age. Similarly, irradiation at 8, but not 17, 30, or 41 weeks, induced phosphorylation of IKKα, NF-κB inhibitor alpha, and p65. Electrophoretic mobility shift assay demonstrated that active forms of NF-κB were increased. In addition, enzyme-linked immunosorbent assay showed that lipopolysaccharide-induced IL-6 production was enhanced in splenocytes of mice irradiated at 8 weeks. ATP levels were increased in splenocytes of mice irradiated at 8, but not 17, 30, or 41 weeks. CDK2 expression and p65 phosphorylation were induced in CD45R/B220+ cells from irradiated mice. Overall, irradiation induced a NF-κB-related immune response in the spleen with an increase in senescence marker proteins, such as CDKs and IL-6, which are known to be typical senescence-associated secretory phenotype factors related to stresses, such as DNA damage.

Novel [(N-alkyl-3-indolylmethylene)hydrazono]oxindoles arrest cell cycle and induce cell apoptosis by inhibiting CDK2 and Bcl-2: synthesis, biological evaluation and in silico studies.

As a continuation for our previous work, a novel set of N-alkylindole-isatin conjugates (7, 8a-c, 9 and 10a-e) is here designed and synthesised with the prime aim to develop more efficient isatin-based antitumor candidates. Utilising the SAR outputs from the previous study, our design here is based on appending four alkyl groups with different length (ethyl and n-propyl), bulkiness (iso-propyl) and unsaturation (allyl) on N-1 of indole motif, with subsequent conjugation with different N-unsubstituted isatin moieties to furnish the target conjugates. As planned, the adopted strategy achieved a substantial improvement in the growth inhibitory profile for the target conjugates in comparison to the reported lead VI. The best results were obtained with N-propylindole -5-methylisatin hybrid 8a which displayed broad spectrum anti-proliferative action with efficient sub-panel GI50 (MG-MID) range from 1.33 to 4.23 µM, and promising full-panel GI50 (MG-MID) equals 3.10 µM, at the NCI five-dose assay. Also, hybrid 8a was able to provoke cell cycle disturbance and apoptosis in breast T-47D cells as evidenced by the DNA flow cytometry and Annexin V-FITC/PI assays. Furthermore, hybrid 8a exhibited good inhibitory action against cell cycle regulator CDK2 protein kinase and the anti-apoptotic Bcl-2 protein (IC50= 0.85 ± 0.03 and 0.46 ± 0.02 µM, respectively). Interestingly, molecular docking for hybrid 8a in CDK2 and Bcl-2 active sites unveiled that N-propyl group is involved in significant hydrophobic interactions. Taken together, the results suggested conjugate 8a as a promising lead for further development and optimisation as an efficient antitumor drug.

miR-500 promotes cell proliferation by directly targetting LRP1B in prostate cancer.

Accumulating evidence suggests that miRNAs play a crucial role in the development of prostate cancer (PC); however, the role of miR-500 in PC remains poorly understood. The data presented here reveal abnormal increases in miR-500 expression in PC tissues and cell lines. Suppression of miR-500 expression significantly inhibited the proliferation of PC-3 and LnCap cells and was negatively regulative with low-density lipoprotein receptor-related protein 1B (LRP1B). Increased cell cycle arrest at the G1 stage and decreased protein expression of cyclinD1 and CDK2 was observed in response to miR-500 knockdown in PC-3 and LnCap cells, in combination with LRP1B overexpression. LRP1B was identified as a target of miR-500 and was significantly decreased in PC tissues. Taken together, these findings demonstrate that miR-500 plays an important role in the proliferation of PC cells via the inhibition of LRP1B expression.

Cyclin E1 and cyclin-dependent kinase 2 are critical for initiation, but not for progression of hepatocellular carcinoma.

E-type cyclins E1 (CcnE1) and E2 (CcnE2) are regulatory subunits of cyclin-dependent kinase 2 (Cdk2) and thought to control the transition of quiescent cells into the cell cycle. Initial findings indicated that CcnE1 and CcnE2 have largely overlapping functions for cancer development in several tumor entities including hepatocellular carcinoma (HCC). In the present study, we dissected the differential contributions of CcnE1, CcnE2, and Cdk2 for initiation and progression of HCC in mice and patients. To this end, we tested the HCC susceptibility in mice with constitutive deficiency for CcnE1 or CcnE2 as well as in mice lacking Cdk2 in hepatocytes. Genetic inactivation of CcnE1 largely prevented development of liver cancer in mice in two established HCC models, while ablation of CcnE2 had no effect on hepatocarcinogenesis. Importantly, CcnE1-driven HCC initiation was dependent on Cdk2. However, isolated primary hepatoma cells typically acquired independence on CcnE1 and Cdk2 with increasing progression in vitro, which was associated with a gene signature involving secondary induction of CcnE2 and up-regulation of cell cycle and DNA repair pathways. Importantly, a similar expression profile was also found in HCC patients with elevated CcnE2 expression and poor survival. In general, overall survival in HCC patients was synergistically affected by expression of CcnE1 and CcnE2, but not through Cdk2. Our study suggests that HCC initiation specifically depends on CcnE1 and Cdk2, while HCC progression requires expression of any E-cyclin, but no Cdk2.

Ureaplasma spp. lipid-associated membrane proteins induce human monocyte U937 cell cycle arrest through p53-independent p21 pathway.

Ureaplasma spp. are known to be associated with human genitourinary tract diseases and perinatal diseases and Ureaplasma spp. Lipid-associated membrane proteins (LAMPs) play important roles in their related diseases. However, the exact mechanism underlying pathogenesis of Ureaplasma spp. LAMPs is largely unknown. In this study, we explored the pathogenic mechanisms of Ureaplasma spp. LAMPs by elucidating their role in modulating the cell cycle and related signaling pathways in human monocytic cell U937, which is highly related to the inflammatory and protective effect in infectious diseases. We utilized the two ATCC reference strains (Ureaplasma parvum serovar 3 str. ATCC 27,815 (UPA3) and Ureaplasma urealyticum serovar 8 str. ATCC 27,618 (UUR8)) and nine clinical isolates which including both UPA and UUR to study the effects of Ureaplasma spp. LAMPs on U937 in vitro. We found that LAMPs derived from UUR8 and both UPA and UUR of clinical strains markedly inhibited the cell proliferation, while UPA3 LAMPs suppressed slightly. Besides, the result of flow cytometry analysis indicated all the Ureaplasma spp. LAMPs could arrest U937 cells in G1 phase. Next, we found that the cell cycle arrest was associated with increased levels of p53 and p21, and a concomitant decrease in the levels of CDK2, CDK4, CDK6 and cyclin E1 at both transcriptional and translational levels after treatment with LAMPs derived from UUR8 or clinical strains, while only cyclin E1 was down-regulated after treatment with UPA3 LAMPs. Further study showed that p53 down-regulation had almost no effect on the distribution of cell cycle and the expression of p21. In conclusion, this study demonstrated that LAMPs derived from UUR8 and clinical strains could inhibit the proliferation of U937 cells by inducing G1 cell cycle arrest through increasing the p21 expression in a p53-independent manner, while UPA3 LAMPs could induce the cell cycle arrest slightly. Our study could contribute to the understanding of Ureaplasma spp. pathogenesis, which has potential value for the treatment of Ureaplasma spp. infections.

Bafilomycin C1 induces G0/G1 cell-cycle arrest and mitochondrial-mediated apoptosis in human hepatocellular cancer SMMC7721 cells.

Bafilomycin C1, which was isolated from Streptomyces albolongus in our previous work, exhibited strong cytotoxicity against several cancer cell lines. This study aimed to evaluate its antitumor effect on human hepatocellular cancer SMMC7721 cells and the underlying mechanism in vitro and in vivo. MTT assay revealed that bafilomycin C1 retarded SMMC7721 cell growth and proliferation. Western blot and real-time qPCR analysis revealed that bafilomycin C1 caused partial G0/G1 phase cell-cycle arrest, downregulated the expression of cyclin D3, cyclin E1, CDK2, CDK4, and CDK6 and upregulated the expression of p21. Moreover, bafilomycin C1 caused mitochondrial membrane dysfunction through oxidative stress. Furthermore, bafilomycin C1 decreased the expression of Bcl-2; increased the expression of Bax, p53, and P-p53; and increased cleavage of caspase-9 and caspase-3, thereby inducing the intrinsic caspase-dependent apoptotic pathway. In vivo experiments in mice suggested that bafilomycin C1 suppressed tumor growth with few side effects. Cell-cycle arrest and induced apoptosis in tumor tissues in a mouse model treated with bafilomycin C1 were demonstrated by histological analyses, western blot and TUNEL. These findings indicate that bafilomycin C1 may be a promising candidate for hepatic cellular cancer therapy.

GSK-3 promotes S-phase entry and progression in C. elegans germline stem cells to maintain tissue output.

Adult C. elegans germline stem cells (GSCs) and mouse embryonic stem cells (mESCs) exhibit a non-canonical cell cycle structure with an abbreviated G1 phase and phase-independent expression of Cdk2 and cyclin E. Mechanisms that promote the abbreviated cell cycle remain unknown, as do the consequences of not maintaining an abbreviated cell cycle in these tissues. In GSCs, we discovered that loss of gsk-3 results in reduced GSC proliferation without changes in differentiation or responsiveness to GLP-1/Notch signaling. We find that DPL-1 transcriptional activity inhibits CDK-2 mRNA accumulation in GSCs, which leads to slower S-phase entry and progression. Inhibition of dpl-1 or transgenic expression of CDK-2 via a heterologous germline promoter rescues the S-phase entry and progression defects of the gsk-3 mutants, demonstrating that transcriptional regulation rather than post-translational control of CDK-2 establishes the abbreviated cell cycle structure in GSCs. This highlights an inhibitory cascade wherein GSK-3 inhibits DPL-1 and DPL-1 inhibits cdk-2 transcription. Constitutive GSK-3 activity through this cascade maintains an abbreviated cell cycle structure to permit the efficient proliferation of GSCs necessary for continuous tissue output.

The cell cycle regulator protein P16 and the cellular senescence of dental follicle cells.

Cellular senescence is a restricting factor for regenerative therapies with somatic stem cells. We showed previously that the onset of cellular senescence inhibits the osteogenic differentiation in stem cells of the dental follicle (DFCs), although the mechanism remains elusive. Two different pathways are involved in the induction of the cellular senescence, which are driven either by the cell cycle protein P21 or by the cell cycle protein P16. In this study, we investigated the expression of cell cycle proteins in DFCs after the induction of cellular senescence. The induction of cellular senescence was proved by an increased expression of ß-galactosidase and an increased population doubling time after a prolonged cell culture. Cellular senescence regulated the expression of cell cycle proteins. The expression of cell cycle protein P16 was up-regulated, which correlates with the induction of cellular senescence markers in DFCs. However, the expression of cyclin-dependent kinases (CDK)2 and 4 and the expression of the cell cycle protein P21 were successively decreased in DFCs. In conclusion, our data suggest that a P16-dependent pathway drives the induction of cellular senescence in DFCs.

LTA4H regulates cell cycle and skin carcinogenesis.

Leukotriene A4 hydrolase (LTA4H), a bifunctional zinc metallo-enzyme, is reportedly overexpressed in several human cancers. Our group has focused on LTA4H as a potential target for cancer prevention and/or therapy. In the present study, we report that LTA4H is a key regulator of cell cycle at the G0/G1 phase acting by negatively regulating p27 expression in skin cancer. We found that LTA4H is overexpressed in human skin cancer tissue. Knocking out LTA4H significantly reduced skin cancer development in the 7,12-dimethylbenz(a)anthracene (DMBA)-initiated/12-O-tetradecanoylphorbol-13-acetate (TPA)-promoted two-stage skin cancer mouse model. LTA4H depletion dramatically decreased anchorage-dependent and -independent skin cancer cell growth by inducing cell cycle arrest at the G0/G1 phase. Moreover, our findings showed that depletion of LTA4H enhanced p27 protein stability, which was associated with decreased phosphorylation of CDK2 at Thr160 and inhibition of the CDK2/cyclin E complex, resulting in down-regulated p27 ubiquitination. These findings indicate that LTA4H is critical for skin carcinogenesis and is an important mediator of cell cycle and the data begin to clarify the mechanisms of LTA4H's role in cancer development.

KIF15 promotes pancreatic cancer proliferation via the MEK-ERK signalling pathway.

BACKGROUND: Pancreatic cancer is highly malignant and characterised by rapid and uncontrolled growth. While some of the important regulatory networks involved in pancreatic cancer have been determined, the cancer relevant genes have not been fully identified. METHODS: We screened genes that may control proliferation in pancreatic cancer in seven pairs of matched pancreatic cancer and normal pancreatic tissue samples. We examined KIF15 expression in pancreatic cancer tissues and the effect of KIF15 on cell proliferation in vitro and in vivo. The mechanisms underlying KIF15 promotion of cell proliferation were investigated. RESULTS: mRNA microarray and functional analysis identified 22 genes that potentially play an important role in the proliferation of pancreatic cancer. High-content siRNA screening evaluated whether silencing these 22 genes affected proliferation of pancreatic cancer. Notably, silencing KIF15 exhibited the most potent inhibition of proliferation compared with the rest of the 22 genes. KIF15 was upregulated in human pancreatic cancer tissues, and higher KIF15 expression levels correlated with shorter patient survival times. Upregulation KIF15 promoted pancreatic cancer growth. KIF15 upregulated cyclin D1, CDK2, and phospho-RB and also promoted G1/S transition in pancreatic cancer cells. KIF15 upregulation activated MEK-ERK signalling by increasing p-MEK and p-ERK levels. MEK-ERK inhibitors successfully inhibited cell cycle progression, and PD98059 blocked KIF15-mediated pancreatic cancer proliferation in vivo and in vitro. CONCLUSIONS: This study identified KIF15 as a critical regulator that promotes pancreatic cancer proliferation, broadening our understanding of KIF15 function in tumorigenesis.

Platelet rich plasma releasate promotes proliferation of skeletal muscle cells in association with upregulation of PCNA, cyclins and cyclin dependent kinases.

Platelet rich plasma (PRP) contains various cytokines and growth factors which may be beneficial to the healing process of injured muscle. The purpose of this study is to investigate the effect and molecular mechanism of PRP releasate on proliferation of skeletal muscle cells. Skeletal muscle cells intrinsic to Sprague-Dawley rats were treated with PRP releasate. Cell proliferation was evaluated by 3-[4,5-Dimethylthiazol- 2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay and immunocytochemistry with Ki-67 stain. Flow cytometric analysis was used to evaluate the cell cycle progression. Western blot analysis was used to evaluate the protein expressions of PCNA, cyclin E1, cyclin A2, cyclin B1, cyclin dependent kinase (cdk)1 and cdk2. The results revealed that PRP releasate enhanced proliferation of skeletal muscle cells by shifting cells from G1 phase to S phase and G2/M phases. Ki-67 stain revealed the increase of proliferative capability after PRP releasate treatment. Protein expressions including cyclin A2, cyclin B1, cdk1, cdk2 and PCNA were up-regulated by PRP releasate in a dose-dependent manner. It was concluded that PRP releasate promoted proliferation of skeletal muscle cells in association with the up-regulated protein expressions of PCNA, cyclin A2, cyclin B1, cdk1 and cdk2.

R-Phycoerythrin Induces SGC-7901 Apoptosis by Arresting Cell Cycle at S Phase.

R-Phycoerythrin (R-PE), one of the chemical constituents of red algae, could produce singlet oxygen upon excitation with the appropriate radiation and possibly be used in photodynamic therapy (PDT) for cancer. Documents reported that R-PE could inhibit cell proliferation in HepG2 and A549 cells, which was significative for cancer therapy. This is due to the fact that R-PE could kill cancer cells directly as well as by PDT. However, little is known about the cytotoxicity of R-PE to the SGC-7901 cell. In this study, it has been found that R-PE could inhibit SGC-7901 proliferation and induce cell apoptosis, which was achieved by arresting the SGC-7901 cell at S phase. CyclinA, CDK2 and CDC25A are proteins associated with the S phase, and it was found that R-PE could increase the expression of cyclin A protein and decrease the expression of CDK2 and CDC25A proteins. Thus, it was concluded that R-PE reduced the CDK2 protein activated through decreasing the CDC25A factor, which reduced the formation of Cyclin-CDK complex. The reduction of Cyclin-CDK complex made the SGC-7901 cells arrest at the S phase. Therefore, R-PE induced apoptosis by arresting the SGC-7901 cell at S phase was successful, which was achieved by the expression of the CDC25A protein, which reduced the CDK2 protein actived and the formation of Cyclin-CDK complex.

Novel miR-5582-5p functions as a tumor suppressor by inducing apoptosis and cell cycle arrest in cancer cells through direct targeting of GAB1, SHC1, and CDK2.

MicroRNAs (miRNAs) play pivotal roles in tumorigenesis as either tumor suppressors or oncogenes. In the present study, we discovered and demonstrated the tumor suppressive function of a novel miRNA miR-5582-5p. miR-5582-5p induced apoptosis and cell cycle arrest in cancer cells, but not in normal cells. GAB1, SHC1, and CDK2 were identified as direct targets of miR-5582-5p. Knockdown of GAB1/SHC1 or CDK2 phenocopied the apoptotic or cell cycle arrest-inducing function of miR-5582-5p, respectively. The expression of miR-5582-5p was lower in tumor tissues than in adjacent normal tissues of colorectal cancer patients, while the expression of the target proteins exhibited patterns opposite to that of miR-5582-5p. Intratumoral injection of a miR-5582-5p mimic or induced expression of miR-5582-5p in tumor cells suppressed tumor growth in HCT116 xenografts. Collectively, our results suggest a novel tumor suppressive function for miR-5582-5p and its potential applicability for tumor control.

AMPKα1 deletion in fibroblasts promotes tumorigenesis in athymic nude mice by p52-mediated elevation of erythropoietin and CDK2.

Angiogenesis is essential for tumor development. Accumulating evidence suggests that adenosine monophosphate-activated protein kinase (AMPK), an energy sensor and redox modulator, is associated with cancer development. However, the effect of AMPK on tumor development is controversial, and whether AMPK affects tumor angiogenesis has not been resolved. We show that deletion of AMPKα1, but not AMPKα2, upregulates non-canonical nuclear factor kappa B2 (NF-κB2)/p52-mediated cyclin-dependent kinase 2 (CDK2), which is responsible for the anchorage-independent cell growth of immortalized mouse embryo fibroblasts (MEFs). Co-culture with AMPKα1 knockout MEFs (or their conditioned medium) enhances the migration and network formation of human microvascular endothelial cells, which is dependent on p52-upregulated erythropoietin (Epo). AMPKα1 deletion stimulates cellular proliferation of allograft MEFs, angiogenesis, and tumor development in athymic nu/nu mice, which is partly ameliorated by antibody-mediated Epo neutralization. Therefore, the AMPKα1-p52-Epo pathway may be involved in stromal fibroblast-mediated angiogenesis and tumorigenesis.

Adenoviral Vector-Mediated Delivery of p21WAF1/CIP1 Prevents Retinal Neovascularization in an Oxygen-Induced Retinopathy Model.

OBJECTIVE: To evaluate the inhibitory effect(s) of adenovirus (Ad) vector-mediated delivery of p21WAF1/CIP1 (Ad-p21) on retinal neovascularization (RNV) in an animal model of oxygen-induced retinopathy (OIR). METHODS: RNV was determined by examination of retinal fiat mounts and sections postnatal (P) day-17 (P17). Non-perfused retinal areas were analyzed using Image-Pro plus 6.0 software. Reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot analysis were used to measure mRNA and protein expression of p21 and cyclin-dependent kinase (CDK) 2. RESULTS: Compared with phosphate buffer saline (PBS) and Ad-NC group mice, non-perfused retinal areas, neovascularization, and number of endothelial cell nuclei breaking through the internal limiting membrane (ILM) in Ad-p21 group mice were significantly reduced. There were fewer non-perfused retinal areas in Ad-p21 mice than in either PBS or Ad-NC group mice, the differences being significant (F = 101.634; p < 0.05). Levels of p21 mRNA and protein in the Ad-p21 group had increased significantly compared with the other three groups (F = 839.664, 509.817; p < 0.05). Levels of CDK2 mRNA and protein in the Ad-p21 group decreased significantly (F = 301.858, 592.882; p < 0.05). CONCLUSIONS: Ad-p21 inhibits RNV in OIR. A potential underlying mechanism for this may be that overexpression of p21 arrests the cell cycle at the G1- to S-phase transition via inhibition of CDK2 activity.

RBEL1 is required for osteosarcoma cell proliferation via inhibiting retinoblastoma 1.

Osteosarcoma is the most common type of primary malignant tumor of the bone. However, mechanisms underlying osteosarcoma cell proliferation are poorly understood. The present study shows that RBEL1, a newly identified Rab-like GTPase, may be a key regulator of osteosarcoma cell proliferation. Knockdown of RBEL1 in osteosarcoma cells resulted in impaired colony formation and cell proliferation. Cell cycle analysis suggested that RBEL1 depletion induced G1-S arrest in osteosarcoma cells. Furthermore, it was demonstrated that retinoblastoma 1 (Rb) was upregulated and activated following RBEL1 knockdown. In addition, Rb inhibitory downstream targets, such as cyclin A2, cyclin D1, c-Myc and cyclin-dependent kinase 2, were downregulated. Rb knockdown reversed RBEL1 depletion-induced tumor suppressive effects. In conclusion, the present results suggest that RBEL1 modulates cell proliferation and G1­S transition by inhibiting Rb in osteosarcoma. These results suggest a potential therapeutic target in osteosarcoma.

Antibiotic drug tigecycline inhibits melanoma progression and metastasis in a p21CIP1/Waf1-dependent manner.

Antibiotics are common drugs with low toxicity but high effectiveness. They have been suggested to be drug candidates for cancer therapy in recent years. Here, we tried to investigate the antitumour effect of tigecycline on malignant melanoma. We showed that tigecycline dramatically inhibited cell proliferation and induced cell cycle arrest at G0/G1 phase. At the same time, tigecycline suppressed cell invasion and migration through preventing epithelial-mesenchymal transition (EMT) process. In addition, tigecycline also significantly blocked tumor growth in vivo. Expression of cell cycle-related proteins were investigated and resulted in downregulation of G1/S checkpoint proteins, such as CDK2 and Cyclin E. However, cyclin-dependent kinase inhibitor 1 (CDKN1A, p21(CIP1/Waf1)) was downregulated after tigecycline treatment, which was not conformed to its conventional function. To explain this, we overexpressed p21 in melanoma cells. We found that p21 overexpression significantly rescued tigecycline-induced cell proliferation inhibition as well as migration and invasion suppression. Taken together, our results revealed that the essential role of p21 in the inhibitory effect of tigecycline on proliferation, migration and invasion of melanoma. Tigecycline might act as a candidate therapeutic drug for treatment of patients suffering from malignant melanoma.

Cyclin-dependent kinase 2 (CDK2) is a key mediator for EGF-induced cell transformation mediated through the ELK4/c-Fos signaling pathway.

Cyclin-dependent kinase 2 (CDK2) is a known regulator in the cell cycle control of the G1/S and S/G2 transitions. However, the role of CDK2 in tumorigenesis is controversial. Evidence from knockout mice as well as colon cancer cell lines indicated that CDK2 is dispensable for cell proliferation. In this study, we found that ectopic CDK2 enhances Ras (G12V)-induced foci formation and knocking down CDK2 expression markedly decreases epidermal growth factor (EGF)-induced cell transformation mediated through the downregulation of c-fos expression. Interestingly, CDK2 directly phosphorylates ELK4 at Thr194 and Ser387 and regulates the ELK4 transcriptional activity, which serves as a mechanism to regulate c-fos expression. In addition, ELK4 is overexpressed in melanoma and knocking down the ELK4 or CDK2 expression significantly attenuated the malignant phenotype of melanoma cells. Taken together, our study reveals a novel function of CDK2 in EGF-induced cell transformation and the associated signal transduction pathways. This indicates that CDK2 is a useful molecular target for the chemoprevention and therapy against skin cancer.

Diacerein retards cell growth of chondrosarcoma cells at the G2/M cell cycle checkpoint via cyclin B1/CDK1 and CDK2 downregulation.

BACKGROUND: Chondrosarcoma is characterized for its lack of response to conventional cytotoxic chemotherapy, propensity for developing lung metastases, and low rates of survival. Research within the field of development and expansion of new treatment options for unresectable or metastatic diseases is of particular priority. Diacerein, a symptomatic slow acting drug in osteoarthritis (SYSADOA), implicates a therapeutic benefit for the treatment of chondrosarcoma by an antitumor activity. METHODS: After treatment with diacerein the growth behaviour of the cells was analyzed with the xCELLigence system and MTS assay. Cell cycle was examined using flow cytometric analysis, RT-PCR, and western blot analysis of specific checkpoint regulators. The status for phosophorylation of mitogen-activated protein kinases (MAPKs) was analyzed with a proteome profiler assay. In addition, the possible impact of diacerein on apoptosis was investigated using cleaved caspase 3 and Annexin V/PI flow cytometric analysis. RESULTS: Diacerein decreased the cell viability and the cell proliferation in two different chondrosarcoma cell lines in a dose dependent manner. Flow cytometric analysis showed a classical G2/M arrest. mRNA and protein analysis revealed that diacerein induced a down-regulation of the cyclin B1-CDK1 complex and a reduction in CDK2 expression. Furthermore, diacerein treatment increased the phosphorylation of p38α and p38ß MAPKs, and Akt1, Akt2, and Akt 3 in SW-1353, whereas in Cal-78 the opposite effect has been demonstrated. These observations accordingly to our cell cycle flow cytometric analysis and protein expression data may explain the G2/M phase arrest. In addition, no apoptotic induction after diacerein treatment, neither in the Cal-78 nor in the SW-1353 cell line was observed. CONCLUSIONS: Our results demonstrate for the first time that the SYSADOA diacerein decreased the viability of human chondrosarcoma cells and induces G2/M cell cycle arrest by CDK1/cyclin B1 down-regulation.

Magnolol inhibits growth of gallbladder cancer cells through the p53 pathway.

Magnolol, the major active compound found in Magnolia officinalis has a wide range of clinical applications due to its anti-inflammation and anti-oxidation effects. This study investigated the effects of magnolol on the growth of human gallbladder carcinoma (GBC) cell lines. The results indicated that magnolol could significantly inhibit the growth of GBC cell lines in a dose- and time-dependent manner. Magnolol also blocked cell cycle progression at G0 /G1 phase and induced mitochondrial-related apoptosis by upregulating p53 and p21 protein levels and by downregulating cyclin D1, CDC25A, and Cdk2 protein levels. When cells were pretreated with a p53 inhibitor (pifithrin-a), followed by magnolol treatment, pifithrin-a blocked magnolol-induced apoptosis and G0 /G1 arrest. In vivo, magnolol suppressed tumor growth and activated the same mechanisms as were activated in vitro. In conclusion, our study is the first to report that magnolol has an inhibitory effect on the growth of GBC cells and that this compound may have potential as a novel therapeutic agent for the treatment of GBC.