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CDK2-AP1 inhibits growth of breast cancer cells by regulating cell cycle and increasing docetaxel sensitivity in vivo and in vitro.

ABSTRACT: BACKGROUND:Cell cycle regulatory pathway is a well-established pathway mainly dependent on cyclin-dependent kinases (CDKs), which are regulated positively by cyclins and negatively by cyclin-dependent kinase inhibitors(CKIs). Cyclin-dependent kinase 2 associate protein 1(CDK2-AP1) is a specific negative regulatory protein for CDK2, is important in the cancer cell cycle. However, the function of CDK2-AP1 in breast cancer remains unclear. We designed therefore explored the effects of CDK2-AP1 on breast cancer growth and its chemo-sensitivity. METHODS:Expression of CDK2-AP1, CDK2 and CyclinD1 in 209 cases of pathological specimens using IHC staining was measured. Lost-of-function and Gain-of-function assays were used in vivo and in vitro relating to the specific role of CDK2-AP1 in breast cancer. We analyzed in vivo and in vitro the impact of CDK2-AP1 on chemotherapy sensitivity in breast cancer. RESULTS:The positive ratio of CDK2-AP1 expression was reduced successively in normal breast tissue, DCIS, invasive breast cancer and relapsed breast cancer, however, with CDK2 and CyclinD1 it was suggested that CDK2-AP1 was correlated closely with the tumorigenesis and progress, and might work as a tumor suppressor. After down-regulating CDK2-AP1 in breast cancer cells, the cell cycle was accelerated and cell proliferation enhanced. The cell cycle was arrested in G0/G1 phase and G2/M phase after up-regulating CDK2-AP1 in breast cancer cells, inhibiting cell proliferation. The expression of CDK2 and CyclinD1 changed accordingly after downregulation or upregulation of CDK2-AP1 by western blot, suggesting a role of the CDK2-AP1/CDK2/CyclinD1 cell cycle pathway in the initiation and progression of breast cancer. Similar results were obtained in animal assays. The data indicates that CDK2-AP1 can induce sensitivity to docetaxel treatment in breast cancer cells. CONCLUSIONS:CDK2-AP1 affects tumorigenesis, tumor growth and chemo-sensitivity by cell cycle regulation, which can potentially to be a therapeutical agent in breast cancer.

SUBMITTER: He X

PROVIDER: S-EPMC4279590 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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CDK2-AP1 inhibits growth of breast cancer cells by regulating cell cycle and increasing docetaxel sensitivity in vivo and in vitro.

He Xiangming X Xiang Hua H Zong Xiangyun X Yan Xuebing X Yu Yang Y Liu Guan G Zou Dehong D Yang Hongjian H

Cancer cell international 20141130 1

<h4>Background</h4>Cell cycle regulatory pathway is a well-established pathway mainly dependent on cyclin-dependent kinases (CDKs), which are regulated positively by cyclins and negatively by cyclin-dependent kinase inhibitors(CKIs). Cyclin-dependent kinase 2 associate protein 1(CDK2-AP1) is a specific negative regulatory protein for CDK2, is important in the cancer cell cycle. However, the function of CDK2-AP1 in breast cancer remains unclear. We designed therefore explored the effects of CDK2- ...[more]

PMID: 25550687

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Project description:BackgroundEven though new therapies are available against melanoma, novel approaches are needed to overcome resistance and high-toxicity issues. In the present study the anti-melanoma activity of Nicotinamide (NAM), the amide form of Niacin, was assessed in vitro and in vivo.MethodsHuman (A375, SK-MEL-28) and mouse (B16-F10) melanoma cell lines were used for in vitro investigations. Viability, cell-death, cell-cycle distribution, apoptosis, Nicotinamide Adenine Dinucleotide+ (NAD+), Adenosine Triphosphate (ATP), and Reactive Oxygen Species (ROS) levels were measured after NAM treatment. NAM anti-SIRT2 activity was tested in vitro; SIRT2 expression level was investigated by in silico transcriptomic analyses. Melanoma growth in vivo was measured in thirty-five C57BL/6 mice injected subcutaneously with B16-F10 melanoma cells and treated intraperitoneally with NAM. Interferon (IFN)-γ-secreting murine cells were counted with ELISPOT assay. Cytokine/chemokine plasmatic levels were measured by xMAP technology. Niacin receptors expression in human melanoma samples was also investigated by in silico transcriptomic analyses.ResultsNAM reduced up to 90% melanoma cell number and induced: i) accumulation in G1-phase (40% increase), ii) reduction in S- and G2-phase (about 50% decrease), iii) a 10-fold increase of cell-death and 2.5-fold increase of apoptosis in sub-G1 phase, iv) a significant increase of NAD+, ATP, and ROS levels, v) a strong inhibition of SIRT2 activity in vitro. NAM significantly delayed tumor growth in vivo (p ≤ 0.0005) and improved survival of melanoma-bearing mice (p ≤ 0.0001). About 3-fold increase (p ≤ 0.05) of Interferon-gamma (IFN-γ) producing cells was observed in NAM treated mice. The plasmatic expression levels of 6 cytokines (namely: Interleukin 5 (IL-5), Eotaxin, Interleukin 12 (p40) (IL12(p40)), Interleukin 3 (IL-3), Interleukin 10 (IL-10) and Regulated on Activation Normal T Expressed and Secreted (RANTES) were significantly changed in the blood of NAM treated mice, suggesting a key role of the immune response. The observed inhibitory effect of NAM on SIRT2 enzymatic activity confirmed previous evidence; we show here that SIRT2 expression is significantly increased in melanoma and inversely related to melanoma-patients survival. Finally, we show for the first time that the expression levels of Niacin receptors HCAR2 and HCAR3 is almost abolished in human melanoma samples.ConclusionNAM shows a relevant anti-melanoma activity in vitro and in vivo and is a suitable candidate for further clinical investigations.

Project description:BackgroundA variety of anticancer chemotherapeutics induce adverse side effects including myelotoxicity. Dried roots of Phragmites communis Trinius, Phragmitis rhizoma, have been clinically used in traditional folk medicine to relieve various symptoms like fever. In this study, we evaluated the protective effect of the aqueous extract of Phragmitis rhizoma (EPR) against docetaxel-induced myelotoxicity in vitro and in vivo.MethodsThe in vitro myelo-protective effect of EPR was evaluated using the colony forming unit (CFU) assay with hematopoietic progenitor cells. The in vivo efficacy of EPR was evaluated in myelosuppressed C57BL/6 male mice which were induced by repeated intraperitoneal injections of 30 mg/kg docetaxel for 3 times. EPR was orally administered for 4 days to docetaxel-induced myelosuppressed C57BL/6 male mice which were induced by intraperitoneal injection of 30 mg/kg docetaxel for 3 times: Group 1 (vehicle control, n = 10), Group 2 (docetaxel plus vehicle, n = 10), Group 3 (docetaxel plus EPR 30 mg/kg, n = 10), Group 4 (docetaxel plus EPR 100 mg/kg, n = 10) and Group 5 (docetaxel plus EPR 300 mg/kg, n = 10). Whole blood counts were measured automatically, and immune organs were histologically examined. Expression of immunomodulatory cytokines was measured by quantitative real-time polymerase chain reaction or enzyme-linked immunosorbent assay. The toxicity of EPR itself was evaluated in normal human cell lines including IMR-90, foreskin fibroblast and human umbilical vein endothelial cells. The hepatotoxicity of EPR was predicted by multi-parametric assays involving cell viability, caspase 3/7 activity, GSH contents and LDH leakage using the HepaRG hepatic cell line.ResultsCo-treatment of EPR or its major component, p-hydroxycinnamic acid, increased the numbers of hematopoietic CFU counts in the docetaxel-induced in vitro myelotoxicity assay system. The in vitro protective effect of EPR against docetaxel toxicity was replicated in a myelosuppressed animal model: white blood cells, neutrophils, lymphocytes and red blood cells rebounded; bone marrow niche and structural integrity of the thymus were preserved; and the expression of immune-stimulating cytokines including IL3, IL6, SCF and GM-CSF was enhanced. Furthermore, EPR and p-hydroxycinnamic acid promoted the proliferation of primary splenocytes and thymocytes. In the toxicity assays, no remarkable signs related with toxicity were observed in all tested normal human cells and HepaRG.ConclusionsEPR has the potential to ameliorate docetaxel-mediated myelotoxicity in both in vitro and in vivo models. However, the identification of the responsible active components and the precise underlying myelo-protective mechanism of EPR need to be elucidated before novel drug development using EPR can precede.

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CDK2-AP1 inhibits growth of breast cancer cells by regulating cell cycle and increasing docetaxel sensitivity in vivo and in vitro.

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CDK2-AP1 inhibits growth of breast cancer cells by regulating cell cycle and increasing docetaxel sensitivity in vivo and in vitro.

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