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Cisplatin-resistant ovarian cancer cell lines induced apoptosis time-and dose A2780-DDP compliance_1395

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Cisplatin-resistant ovarian cancer cell lines induced apoptosis time-and dose A2780-DDP compliance_1395

    Cisplatin-resistant ovarian cancer cell lines induced apoptosis time-and dose A2780/DDP compliance

     Author: Fu-chun, Li Guangyi, Liu Xiaoying, Qiu-

    Hua Lin, Liu Feng-ying, Xie Yong

     Abstract Objective To investigate the role of different

    concentrations of cisplatin at different time-sensitive and

    drug-induced apoptosis in ovarian cancer cell line variation and apoptosis pathway-related protein expression. Methods cisplatin-resistant ovarian cancer cell lines A2780/DDP and

    sensitive cell lines A2780 for the study. In cell culture, based on the use of MTT assay, in situ labeling method, routine immunohistochemistry and Western blot methods, the effects of different doses of cisplatin (5,10,20,40 μmol / L)

    and the role of different time (24 , 48,72 h) to the A2780 and A2780/DDP cell apoptosis and this dynamic process caspase

     2 (Caspase

     2), and

     3 (Caspase

     3) expression. The results of the A2780 and the cisplatin-

    induced apoptosis A2780/DDP cell lines in a time-and dose-

    compliance. Apoptosis proteins Caspase

     2 and Caspase

     3 activation enhances cisplatin-induced apoptosis, its

    expression was a right time and the cisplatin dose compliance. Conclusions (1) in cisplatin-resistant mechanism, the drug in

    vivo distribution and metabolism of the effective concentration of plasma and cellular rejection caused by the drug cisplatin into the cells, there are some obstacles may be the factors that reduce the sensitivity to cisplatin. (2) Caspase

     2 and the delayed activation of caspase

     3 protein and activity decrease are likely to affect

apoptosis-resistant A2780/DDP reasons.

     Key words cisplatin-resistant; apoptosis; Caspase

     2; Caspase

     3; human ovarian cancer

     Cisplatin

     Induced Apoptosis in Cisplatin

     resistant Ovarian Cancer Cell Line A2780/DDP on Time

     and Dose

     Dependence

     Key words: Cisplatin

     resistance; Apoptosis; Caspase

     2; Caspase

     3; Human ovarian caner

     Cisplatin-resistant ovarian cancer cells is to reduce the

    5-year survival rate of key issues. The performance of drug-

    resistant ovarian cancer cells significantly decreased the ability of spontaneous apoptosis, and malignant proliferation of unrestricted growth of cancer cells [1]. Cisplatin-induced

    apoptosis in ovarian cancer the most significant change is the cell Neijing mitochondrial apoptosis pathway activation [2]. From the point of grasping the time-and dose cisplatin-induced

    and drug-resistant ovarian cancer A2780 cell A2780/DDP changes of apoptosis to understand the upstream channels in apoptosis signal transduction from the role of Caspase

     2 protein and downstream of the implementation of the role of Caspase

     3 protein expression patterns of change will help to further understanding of mechanism of cisplatin-resistant

    ovarian cancer.

     1 Materials and methods

     1.1 The human ovarian cancer cell line

     A2780, and A2780/DDP were purchased from Tongji Hospital Cancer Center, A2780/DDP cell lines to cisplatin-resistant

    A2780 cell lines was 9.4-fold [3]. With DMEM containing 10% fetal calf serum complete medium at 37 ?, 5% CO2, relative

    humidity of 90% of the incubator.

     1.2 Main Reagents

     Cis-platinum (Batch H37021358): Shandong Qilu Pharmaceutical products, molecular weight of 300.3. Coomassie brilliant blue protein determination kit purchased from Nanjing Jiancheng Bio-Engineering Institute. Caspase

     2 (BA0587, 200μg/ml), Caspase

     3 (BA0588, 200μg/ml), and actin (200μg/ml) rabbit

    polyclonal antibodies, apoptosis detection kit (MK1020),

    ready-to-SABC immunohistochemistry staining kit (SA1022), Western blotting (Western blotting) detection kit (SA2022), DAB chromogenic kit were purchased from Wuhan Boster Company.

     1.3 the main method

     1.3.1 cisplatin on ovarian cancer cells A2780 and A2780/DDP Determination of growth inhibitory effect of MTT, a final concentration of cisplatin, respectively 0,2,5,10,20,40,80,200 μmol / L (μM), were cultured 24,48,72

    h; calculations with different concentrations of cisplatin on

    cell growth inhibition rate: inhibition rate = (control group A mean - experimental group, the mean A) / A control group mean.

     1.3.2 cisplatin on ovarian cancer cell line A2780 and A2780/DDP the results of apoptosis-inducing according to

    1.3.1, select the five kinds of cis-platinum concentrations

    (0,5,10,20,40 μmol / L) induced apoptosis, respectively 24, 48,72 h to terminate the experiment作成cell crawling piece or

    collection of cells, experimental 1.3.3 ~ 1.3.5 are here methods.

     1.3.3 Terminal deoxynucleotidyl transferase (TDT) to detect apoptosis in situ labeling and judging criteria based approach to detection kit. Positive and semi-quantitative

    criterion: the nucleus has brown granules were positive for apoptotic cells. Positive semi-quantitative methods are: per

    case (400 × microscope) Select 9 vision, that is, slide left, middle, and right from each of three areas before, during and after the three horizons, calculate the number of ovarian cancer within the field of vision and positive cells to

    calculate the percentage of cases of positive cells.

     1.3.4Caspase

     2 protein in cisplatin-induced apoptosis in ovarian cancer cells A2780 and A2780/DDP expressed in ovarian cancer cell protein extract; determination of protein concentration;

    protein SDS

     polyacrylamide gel electrophoresis; the protein samples were transferred membrane ; kit as shown by Western blot assay OK. One anti-titer of 1:400 ~ 1:500, the selection of control for the actin (50μg).

     1.3.5 cisplatin-induced apoptosis in ovarian cancer cells A2780 and A2780/DDP in Caspase

     3 protein detection and assessment criteria: by immunohistochemistry SABC method, by adding an anti-titer of

    1:100. The positive criterion: nucleus or cytoplasm showed brown to dark brown cells are positive. Positive semi-

    quantitative method as shown in the same 1.3.3.

     1.4 Statistical Methods

     Using SPSS11.0 statistical software package, on the measurement data homogeneity of variance test. Homogeneity of variance by using single-factor analysis of variance (one

     way ANOVA) tests in general are a few differences, there was significant difference in a further 22 were compared (Student

     Newman

     Keuls Test). P <0.05 for the difference was significant.

     2 Results

     2.1 of cisplatin on ovarian cancer cells A2780 and A2780/DDP growth inhibition

     Cis-platinum on the A2780 cell line growth inhibition was significant dose-time-dependent relationship: the role of cis-

    platinum, after 48h, significantly inhibited the growth of

    A2780 cells, which 20μmol / L for cisplatin 48h half of the amount of inhibitor. A2780/DDP cisplatin on cell growth inhibition is also dose-time-dependent relationship,

    characteristics: Effect of intensity of significantly weaker than A2780 cells, especially in less than 40μmol / L

    concentration effect was weaker than the corresponding doses of A2780; effect of time, has lagged far behind, 72h before there a little obvious growth inhibition. Therefore, this

study selected four different concentrations (5,10,20,40 μmol

    / L) cis-platinum in the following experiments, the

    concentration of a stimulus to induce apoptosis of A2780 and A2780/DDP. Reposted elsewhere in the paper for free download http://

     2.2TDT in situ labeling detection of apoptosis in ovarian

    cancer cells

     Cisplatin A2780 and A2780/DDP cells 72h culture, the occurrence rate of apoptotic cells was time for a dose-

    dependent, this trend is evident in the A2780 cells. (10 ~ 40) μmol / L cisplatin A2780 cells 48h and (5 ~ 40) μmol / L

    cisplatin in A2780 cells 72h, apoptosis was significantly greater than the number of its role in 24h by the same dose. Right A2780/DDP cells, only when the 20μmol / L and 40μmol /

    L cisplatin for 72h, the number of apoptotic cells was significantly more than the same dose of its role in 24h are shown in Table 1. Table 1 with cisplatin A2780 and A2780/DDP apoptosis-positive cell count results A2780 cells, P <0.05: between groups at each time; 24h group, starting from 20μM

    cisplatin group, 48h group and 72h group from 10μM cisplatin

    group, the positive cells was significantly increased. A2780/DDP cells, P <0.05:72 h group, 20μM and 40μM cisplatin

    group than in the 24h and 48h in both groups were induced by the same dose significantly increased

     2.3Western blot detection of Caspase

     2 protein expression

     Caspase

     2 gene encodes two proteins Caspase

     2L and Caspase

     2S in the two ovarian cancer cell lines expressed varying degrees, Western blot in the detection of Caspase

     2 protein expression is actually referring to 48Kda of Caspase

     2L protein changes, its 35Kda of Caspase

     2S protein did not change significantly (no special instructions, the following Caspase

     2 refers to Caspase

     2L). A2780 cells Caspase

     2 protein in cisplatin 24h and 48h, from 10μM cisplatin

    began its increased expression; with cisplatin extended to

    72h, from 5μM cisplatin began to increase performance. A2780/DDP of Caspase

     2 protein expression in cisplatin 24h and 48h between the

    expression of the various dose groups no significant change; wait cisplatin 72h, starting from 10μM cisplatin, Caspase

     2 expression began to increase, shown in Figure 1, 2.

     2.4Caspase

     3 protein in the cisplatin-induced apoptosis in ovarian

    cancer cells A2780 and A2780/DDP expression

     Caspase

     3 protein in pathological manifestations mainly cytoplasm and nucleus while coloring, especially evident nucleus coloring. Suishun in A2780 cells prolonged the role of platinum has increased significantly and dose-increase trend,

    this trend was particularly evident reflected: 24h group, from 20 μM cis-platinum-group effect, 48h group and 72h group from 10μM cisplatin group, the positive cells was significantly increased. Caspase

     3 protein expression in the A2780/DDP cells, 5μM and 10μ

    M cisplatin for the next change over time was not obvious, when the doses of cisplatin 20μM and 40μM, the role of the

    72h post-Caspase

     3 protein expression significantly increased, but this effect is still significantly lower than A2780 cells, in Table 2.

     3 Discussion

     3.1 cis-platinum-induced apoptosis in ovarian cancer

    cells A2780 and A2780/DDP the time and dose compliance

     Tumor cells to chemotherapeutic drug resistance mechanisms are manifold [4]. Study found that ovarian cancer cell line A2780 and cisplatin-resistant line A2780/DDP to

    cisplatin sensitivity of the relationship between dose and time compliance. With the prolonged cisplatin and dose

    increased, and two cell lines significantly inhibited the growth of Jun Cheng appeared timeliness and relevance of dose-

    effect. Indicate the mechanism of cisplatin resistance, drug distribution and metabolism in vivo, the effective

    concentration of serum may be reduced cisplatin sensitivity of one of the factors. As the dose increased, both inside and

    outside cells, increased drug concentration gradient into the cells tend to enhancements. This phenomenon shows that the sensitivity of ovarian cancer cells to cisplatin difference may be related to cisplatin into cells there are some obstacles.

     A Comparative Study of A2780 and A2780/DDP two cell lines was found, A2780/DDP require higher concentrations and longer action time, to get the same effect as with the A2780, the entry of drug-resistant cells may have excluded results. In clinical drug metabolism and distribution to consider the impact, if raised to the same in vitro dose-sensitive, the

    human body is difficult to tolerate its side effects. This

    phenomenon prompted targeted in the clinical treatment methods available to improve the therapeutic effect of local drug concentration. Except the dose of this factor in the later, with the role of prolonged, A2780/DDP emergence of resistance

    to cisplatin-sensitive line, even though A2780/DDP resistant to cisplatin, with the role of prolonged, this resistance would be to weaken .

     3.2 apoptosis proteins Caspase

     2 and Caspase

     3 in cisplatin-induced ovarian cancer cell line A2780 and

    the resistant cells A2780/DDP the expression of apoptosis was time-and dose compliance

     The study found that Caspase

     2L and Caspase

     2S protein in the two ovarian cancer cell lines were expressed, including Caspase

     2L protein expression, or both variations for both A2780 and A2780/DDP two cell lines the most important forms of expression, so Caspase

     2 since these two kinds of cells were positive effects [5]. In cisplatin-induced apoptosis in ovarian cancer cells were observed during the dynamic changes of Caspase

     2 protein in the case, can better reflect the activity of Caspase

     2. With cisplatin prolonged and dose increase, Caspase

     2 protein expression in a time-and dose-compliance, this

    trend is particularly visible in the A2780. Further studies showed that the time and dose dependence and under the same conditions of cisplatin-induced apoptosis in A2780 and

A2780/DDP time-and dose changes in compliance match. Caspase

     2 protein in A2780 cisplatin-sensitive and drug-resistant

    line A2780/DDP Department of play time and the effects of varying degrees, it is still possible in these two cell lines in cisplatin-induced apoptosis plays an important role in Caspase. Drug-resistant ovarian cancer cells Caspase

     2 protein activation and extent of delay reduced its upstream pathway may have been inhibited. Caspase

     3 is an important apoptosis effector proteins, which can Caspase

     2 involved in regulation of mitochondria and the subsequent changes in mitochondria react, the implementation of apoptosis command [6]. Caspase

     3 protein in the cisplatin-induced cell lines A2780 and

    A2780/DDP expression of apoptosis and Caspase

     2 protein, similar to that Caspase

     3 protein expression in a time-and dose-compliance, in

    cisplatin-sensitive cell lines prominent, and the time-and

    dose-dependence and under the same conditions of cisplatin-

    induced apoptosis in time-and dose compliance consistent with the change. Differentiated the Caspase

     3 in the resistant line A2780/DDP activity was

    significantly weaker than Caspase

     2 protein. Caspase

     3 protein in ovarian cancer cell line A2780 and A2780/DDP in diverse pathological manifestations, such as the cytoplasm, nucleus-and cytoplasm nucleus type, the most common type

    nucleus to the cytoplasm. From the side may reflect the Caspase

     3 protein in the cisplatin-induced apoptosis in the active function. Caspase

     2 and Caspase

     3 protein in the induction of cisplatin-sensitive cell

    line A2780 and the expression of apoptosis-resistant line

    A2780/DDP Juncheng time-and dose compliance, co-expression

    suggested that this may be related to two kinds of protein expression changes in the gene encoding mutation caused the loss or decrease protein activity has little to do, but with a

    number of external factors, such as access to effective drug concentration within the cell is closely related to, when exposed to sufficient quantities of drugs and the role of prolonged, these two proteins in cisplatin-resistant cells can

    be activated to play a role.

     To sum up, in cisplatin-resistant mechanisms, drug

    distribution and metabolism in vivo, the effective concentration of plasma and cellular rejection caused by the drug cisplatin into the cells, there are some obstacles may be

    the factors that reduce the sensitivity to cisplatin . Caspase

     2 and Caspase

     3 protein activation and activity may account for a delayed impact-resistant A2780/DDP apoptosis causes. In the initial treatment of ovarian cancer or that have been

    resistant to the treatment of ovarian cancer should be possible, enhance the local drug concentration or continuous application of small and medium-dose method may produce some positive results.

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