[Abstract] Objective concept Cha Xinji phenol MCF  7 breast cancer cell cycle and protein expression. MTT assay, flow cytometry, immunocytochemistry and RT  PCR methods such as concept Cha Xinji phenol MCF  7 breast cancer cell proliferation, cell cycle phase and apoptosis, CDK2 and CDK4 mRNA expression, Cyclin D1 mRNA and protein expression. 4,8,16 μmol / L OP role MCF  7 breast cancer cells at 72h, the rate of cell proliferation respectively to 107.31%, 168.06%, 62.00%, G0/G1 phase cells (52.46 ± 6.67)%, (50.19 ± 7.39)%, (67.31 ± 5.47)%, control group (55.27 ± 7.53)%, apoptosis rates were (4.84 ± 1.12)%, (6.48 ± 1.36)%, (19.26 ± 3.57)% in the control group ( 5.56 ± 1.125)%, 16μmol / L OP reduced CDK2, CDK4, Cyclin D1 mRNA and Cyclin D1 expression in cells. Conclusion 4,8 μmol / L OP promote the proliferation of breast cancer cells MCF  7, 16μmol / L OP inhibit cell proliferation and promote apoptosis, which may affect CDK2, CDK4, Cyclin D1 mRNA and Cyclin D1 expression in the cells and OP.

Key words breast cancer; octylphenol; cyclin; cyclin-dependent kinases

 Effect of Octylphenol on Cell Cycle and Cyclin Expression on MCF  7 Breast Cancer Cells

    PENG Jun  hua, ZHANG Hua  xin, ZHANG Feng, ZHAO Yong, FAN Hong  yan

    Department of Clinical Laboratory, Lanzhou General Hospital, Lanzhou Command, PLA, Lanzhou 730050, ChinaAbstract: Objective To observe the effect of octylphenol (OP) on cell proliferation, cell cycle and cyclin expression of MCF  7 breast cancer cells.Methods Using MTT test , flow cytometry, immunocytochemistry and RT  PCR to observe the changes of cell proliferation, cell cycle, apoptosis, CDK2, CDK4 and Cyclin D1 mRNA expression, Cyclin D1 protein expression of MCF  7 cells.Results When MCF  7 breast cancer cells were treated with 4,8 and 16μmol / L OP on 72h, the proliferation ratio of MCF  7 was 107.31%, 168.06% and 62.00% respectively, MCF  7 cell at G0/G1 were (52.46 ± 6.67)%, (50.19 ± 7.39)% and (67.31 ± 5.47)% respectively, while control group was (55.27 ± 7.53)%. The apoptosis ratio of MCF  7 was (4.84 ± 1.12)%, (6.48 ± 1.36)% and (19.26 ± 3.57) % respectively, and the control group was (5.56 ± 1.125)%. The expression of CDK2, CDK4 and Cyclin D1 mRNA significant decreased when MCF  7 breast cancer cells were treated with 16μmol / L OP, the expression of Cyclin D1 protein decreased too . Conclusion The proliferation of MCF  7 breast cancer cells increases when treated with 4,8 μmol / L OP, but it decreases when treated with 16μmol / L OP. These different effects on MCF  7 cells maybe related to the expressive regulation of CDK2, CDK4 and Cyclin D1.

    Key words: Breast cancer; Octylphenol; Cyclin D1; Cyclin  dependent kinases

Breast cancer is a common female malignancy, and its incidence is rising, the incidence of breast cancer genetics, estrogen and environmental factors. Octylphenol (octylphenols, OP) is an estrogenic activity of chemical products, can promote the proliferation of MCF  7 breast cancer cells at low concentrations, but high concentrations inhibit the proliferation of MCF  7 cells [1]. Closely related to cell proliferation and cell cycle. In this study, the role of the OP in MCF  7 cells to observe the change of cell proliferation and expression of CDK2, CDK4, Cyclin D1, mechanism of OP affect MCF  7 breast cancer cell proliferation, and to provide a basis for the prevention and treatment of breast cancer.

    1 Materials and methods

    1.1 Reagents

    98%,Sigma公司产品。">Octylphenol, purity> 98%, the the Sigma company’s products. Rabbit anti-human antibody Cyclin D1 (with a concentration of 1:75), goat anti-rabbit SP kit, Beijing Zhongshan product. RT  PCR kit, the Shanghai the Sangon company products. 98%,进口分装试剂,用于配制OP。">Dimethyl sulfoxide (DMSO), purity> 98%, imports dispensing reagents, for the preparation of OP.

    1.2 MCF  7 cell culture and grouping

    Human breast cancer MCF  7 breast cancer cells by the Shanghai Institute of Cell. Before the test, the breast cancer MCF  7 cells were cultured in phenol red-free DMEM/F12 24h of endogenous estrogen depleted cells, and then to the medium test. Test is divided into four groups: control group (Con), given DMSO 4μmol / L OP group (OP4) 8μmol / L OP group (OP8), 16 μmol / L OP group (OP16).

    1.3 MTT test

    See ref [2], the absorbance (A) 492nm wavelength determination of living cells, the proliferation rate (PR) = experimental group A value / A value of the control group × 100%.

    1.4 cell cycle phase detection

    Experimental group cells to process 72h, cells were harvested, fixed cold 70% ethanol, RNase digestion, propidium iodide (PI) staining, flow cytometry.

    1.5 Cyclin D1 protein expression was detected

    Cells grown on cover slips, fixed with paraformaldehyde, serum closed, plus an anti-incubated at 37 ℃ for 50 min, operating SABC kit, DAB color, dehydration, transparent, microscopic observation and photography, and data analysis.

    1.6 quantitative PCR CDK2, CDK4 and Cyclin D1 mRNA expression

    Harvested the OP at 48h cells, the one-step extraction of total RNA was reverse transcribed into cDNA using Trizol reagent. The primers were synthesized by Shanghai Sangon marked with fluorescent hybridization probes. CDK2: 5 ‘ GCT CTC ACT GGC ATTCCT CCT  3’, 5 ‘ CGA AAT GAA GGC ACT AGA GC  3’; CDK4: 5 ‘ CTA CCT CTC GAT ATG AGC CAG T  3’, 5 ‘ CAT CTG GTA GCT GTA GAT TCT G  3 ‘; Cyclin D1: 5’  GAG GAA CAG AAG TGC GAG GA  3 ‘, 5’  TCT GGA GAG GAA GCG TGT GA  3 ‘. Sampling the total cDNA 3μl, upstream and downstream of each primer, 1.5μl, join the other components of the kit, analysis with PE Gene Amp 5700 PCR for the detection instrument. The reaction conditions at 94 ° C for 40s, 60 ° C for 40s, at 72 ° C for 40s, 40 cycles.

1.7 statistical methods

    Data ± s, differences between groups one-way ANOVA using SPSS 10.0 software.

    2 Results

    2.1 octylphenol MCF  7 breast cancer cell proliferation

    4,8 μmol / L OP can promote MCF  7 breast cancer cell proliferation, 16 μmol / L OP but inhibition of cell proliferation; OP role 72h MCF  7 breast cancer cell proliferation rate was 107.31%, 168.06%, 62.00% , are shown in Table 1. Table 1 OP proliferation of MCF  7 breast cancer cells n = 3, * P <0.01 vs. Con2.2 octylphenol MCF  7 breast cancer cell cycle phase distribution

    Flow cytometry analysis showed less OP cell cycle, 16μmol / L OP MCF  7 breast cancer cells arrest at the G1/G0 phase when 72 hours, apoptosis was significantly increased, as shown in Table 2. Table 2 OP MCF  7 breast cancer cell cycle phase

    2.3 OP Cyclin D1 protein expression in MCF  7 breast cancer cells.

    16μmol / L OP significantly down-regulated the expression of Cyclin D1 in breast cancer cells MCF  7 8μmol / L OP raised MCF-7 breast cancer cells Cyclin D1 expression, as shown in Figure 1.

    2.4 OP CDK2, CDK4, Cyclin D1 mRNA expression in MCF  7 breast cancer cells

    8μmol / L OP can promote CDK2, CDK4, Cyclin D1 mRNA expression in MCF  7 breast cancer cells, high concentration (16μmol / L) OP inhibition of CDK2, CDK4, Cyclin D1 mRNA expression in the cell, as shown in Table 3. Table 3 OP CDK2, CDK4, Cyclin D1 mRNA expression in MCF  7 breast cancer cells (value)

    3 Discussion

    Breast cancer is a common malignancy of the female, a serious threat to women’s health and lives. Over the past 50 years, the incidence of breast cancer increased by 2 times, since 1940, the incidence of breast cancer in the United States rate of 1% per year. Breast cancer incidence and genetic, reproductive, estrogen, nutrition, ionizing radiation and other factors. Environmental estrogen is able to simulate or interfere with the machine endogenous estrogen synthesis, secretion, transport, combined excretion, the physiological role of exogenous chemicals. The octylphenol estrogenic activity of chemical raw materials, mainly used in the production of non-ionic surfactants, plasticizers, heat stabilizers, and widely present in the life, work environment, water, sludge, soil and food such as fish , shellfish, drinking water and beverages [3]. OP have reproductive and developmental toxicity, prepubertal rats than long-term exposure to OP cause apoptosis in adult rat sperm cells, reduce male fertility [4], [5] increased incidence of hormone-dependent tumors. The study found that low concentration (4,8 μmol / L) of OP inhibit the proliferation of MCF  7 breast cancer cells can promote the proliferation of MCF  7 breast cancer cells, high concentration (16mol / L) OP. With low concentrations previously reported (4,8 μmol / L) OP can inhibit the proliferation of breast cancer cells MDA  MB  231 inconsistent [2], which may be used in different breast cancer cell lines, with the results of previous studies consistent with the [1].

    The smooth progress of cell proliferation depends on the cell cycle, cell by mitosis, cells were split into two, the process of the G1 / S and G2 / M two Key point regulation. The study found that the OP through lowered Bcl2 and Caspase 3 expression induced Sertoli cells [6] and apoptosis in breast cancer cells MDA  MB  231. The study found that low concentrations of OP no significant effect on cell cycle phase, high concentrations of OP cell cycle arrest in G0/G1 phase, apoptosis was significantly increased, this phytoestrogen genistein MCF  7 breast cancer cell resistance stagnation in the G2 / M.

    Normal expression is closely related to the normal conduct of the cell cycle and cell cycle regulatory proteins and cyclin-dependent kinase. Cyclin D  CDK G0/G1 phase of the rate-limiting step. When growth signals to the cell cycle regulation, Cyclin D transcription speed and increase the amount and stability increase the expression of Cyclin D. Cyclin D activated CDK4, release E2F, drive the cell cycle. Cyclin D overexpression can accelerate cells through the G1 phase of the cell proliferation accelerated. Koroxenidou [7] found that the 17  hydroxyl estradiol by reducing the expression of CDK2, CDK4 and reduce the activity of CDK2, the cells arrest at the G1 / S phase. The study found high concentrations of OP role MCF  7 breast cancer cells, can down-regulate the expression of CDK2, CDK4 and Cyclin D1 mRNA and reduce Cyclin D1 protein expression, cell cycle arrest at the G1 / S phase inhibition of cell proliferation, promote cell apoptosis, OP performance cytotoxic. The low concentrations OP by upregulating of CDK2, CDK4 and Cyclin D1 mRNA expression, cell cycle progression and promoting cell proliferation, OP performance estrogenic activity.