INHIBITION OF PROLIFERATION OF HUMAN BREAST CANCER MCF-7 CELLS BY SMALL INTERFERENCE RNA AGAINST LRP16 GENE

Objective: Our previous studies have firstly demonstrated that 17β -E2 up-regulates LRP16 gene expression in human breast cancer MCF-7 cells, and ectopic expression of the LRP16 gene promotes MCF-7 cells proliferation. Here, the effects of the LRP16 gene expression on growth of MCF-7 human breast cancer cells and the mechanism were further studied by establishing two stably LRP16-inhibitory MCR-7 cell lines. Methods: Hairpin small interference RNA (siRNA) strategy, by which hairpin siRNA was released by U6 promoter and was mediated by pLPC-based retroviral vector, was adopted to knockdown endogenous LRP16 level in MCF-7 cells. And the hairpin siRNA against green fluorescence protein (GFP) was used as the negative control. The suppressant efficiency of the LRP16 gene expression was confirmed by Nothern blot. Cell proliferation assay and soft agar colony formation assay were used to determine the status of the cells proliferation. Cell cycle checkpoints including cyclin E and cyclin D1 were examined by Western blot. Results: The results from cell proliferation assays suggested that down-regulation of LRP16 gene expression is capable of inhibiting MCF-7 breast cancer cell growth and down-regulation of the LRP16 gene expression is able to inhibit anchorage-independent growth of breast cancer cells in soft agar. We also demonstrated that cyclin E and cyclin D1 proteins were much lower in the LRP16-inhibitory cells than in the control cells. Conclusion: These data suggest that LRP16 gene play an important role in MCF-7 cells proliferation by regulating the pathway of the G1/S transition and may function as an important modulator in regulating the process of tumorigenesis in human breast.

Effects of Mitofusin-2 Gene on Cell Proliferation and Chemotherapy Sensitivity of MCF-7

In order to evaluate the effect of mitofusin-2 gene （mfn2） on proliferation and chemotherapy sensitivity of human breast carcinoma cell line MCF-7 in vitro, pEGFPmfn2 plasmid carrying full length of mitofusin-2 gene was transfected, by using sofast, into MCF-7 cells. Mitofusin-2 gene expression in MCF-7 cells transfected by sofast after 48 h was detected by PCR and Western blotting, and the stable expression of GFP protein in MCF-7 cells by Western blot analysis. The proliferation of MCF-7 cells was assayed by MTT and cell counting. By using PI method, the effects of mfn2 on the cell cycle distribution of MCF-7 were measured. Annexin-Ⅴ/PI double labeling method was employed to detect the changes in apoptosis induced by chemotherapeutics before and after transfection. The results showed that the MCF-7 cells transfected with mfn2 gene could stably and highly express GFP protein. MTT assay revealed that after transfection of mfn2 cDNA, the proliferation of MCF-7 cells was significantly inhibited. DNA histogram showed that cells arrested in S phase, and the percentage of S phase cells was 42.7, 17.2 and 19.6 in mfn2 cDNA transfection group, blank plasmid transfection group and blank control group, respectively （P〈0.05）. The apoptosis ratio of the cells transfected with mfn2 gene was increased from 3.56% to 15.95%, that of the cells treated with camptothecin （CAMP） followed by mfn2 gene transfection was 69.6%, and that in blank plasmid transfection group and blank control group was 31.0% and 23.4% respectively （P〈0.05）. It was suggested that transfection of mfn2 gene could significantly inhibit the proliferation of MCF-7 cells and promote their sensitivity to CAMP with a synergic effect.

Study on Cisplatin Aggravating DNA Damage and Causing a High Apoptosis Rate on Breast Cancer MCF-7 Cells

[Objectives] To investigate the mechanism of DNA damage of cisplatin( DDP),a broad spectrum anticancer drug on breast cancer MCF-7 cells,and to study the mechanism of apoptosis induced by DDP.[Methods]MCF-7 cells were treated by DDP( 0 mg/L,2 mg/L,4 mg/L,6 mg/L,6 mg/L,and 10 mg/L) for 48 hours. MTT assay was used to detect the inhibitory effect of DDP on MCF-7 cells and IC50 value was calculated. Western blot was adopted to detect the expression of γ-H2 AX,which was the marker of DNA double stranded breaks( DSBs) and ATM( sensory molecules of DSBs),the apoptotic signal transduction molecule cleaved caspase-3,and the proteins associated with apoptosis calpain.[Results]DDP inhibited MCF-7 cell activity in a concentration-dependent manner and IC50 was 7. 57 mg/L. In contrast to the control group( without DDP treatment),MCF-7 cells with DDP treatment expressed more γ-H2 AX,ATM,cleaved caspase-3 and calpain.[Conclusions] DDP could inhibit the activity of breast cancer MCF-7 cells. Its mechanisms may be associated with inhibition of MCF-7 cell apoptosis,induction of DNA double strand breaking and the expression of pro-apoptotic protein up-regulation.

The activity of <i>Rhaphidophora pinnta</i>Lf. Schott leaf on MCF-7 cell line

Ekor naga’s leaf (Rhaphidophora pinnata (Lf) Schott) is a type of vines and climbing plant. The leaves are elongated round and hollowed inside. This plant had been using for the treatment of breast cancer. Extraction with percolation method has been done in ekor naga’s leaves with ethanol, and fractionated by nhexane, chloroform and ethyl acetate using liquid-liquid extraction (LLE). Cytotoxicity assay of ethanol extract, n-hexane fraction, chloroform fraction, ethyl acetate fraction and water fraction against MCF-7 cells were done using MTT method (3-(4,5-dimetiltiazol-2-il)-2,5-diphenyl tetrazolium bromide). Phytochemical screening results showed the presence of the compounds such as triterpenoida/steroid, alkaloid, flavonoid, tannin, and saponin. n-hexane fraction was positive for the presence of triterpenoida/steroid, chloroform fraction containing alkaloids, saponin and triterpenoid;ethyl acetate fraction contained, flavonoid, tannin, and the fraction of water indicated the presence of tannin and saponin. Secondary metabolite compounds in ethanol extract, chloroform fraction and ethyl acetate fraction gave positive results against MCF-7 cells. Cytotoxicity assay of MCF-7 cell line showed that crude ethanol extracts had 112.240 mcg/ml IC50 chloroform fraction IC50 was 59.082 mcg/ml, and ethyl acetate fraction IC50 was 812.663 mcg/ml.

Sensitivity Evaluation of Two Human Breast Cancer Cell Lines to Tamoxifen through Apoptosis Induction

Tamoxifen citrate (TAM) has been used to treat breast cancer in women for many years. The com-parative effects of TAM in inducing apoptosis were evaluated in estrogen receptor-positive (ER- positive MCF-7) and estrogen receptor-negative (ER-negative MDA-MB-231) human breast cancer cell lines in vitro in order to determine if these two cell lines differ in their sensitivity to TAM. Mi-tochondrial membrane permeability potential disruption was assessed in both cell lines by a lip-ophilic cationic dye (DePsipher assay, Trevigen, Inc.) utilizing fluorescence microscopy. Using this specific fluorochrome, we were able to associate mitochondrial membrane disruption to early, mid-, and late apoptotic cells. TAM induced cell death via apoptosis in both ER-positive and ER- negative cells, however, apoptosis induction was more pronounced in ER-positive MCF-7 compared to ER-negative MDA-MB-231 breast cancer cells. These findings may have some therapeutic use in the treatment of estrogen dependent and estrogen independent breast cancer.

The molecular interaction of ADAMTS-1 and fibulin-1 and its potential contribution to breast cancer biology

Aim: Fibulins and ADAMTSs are two families of extracellular matrix proteins implicated in key functional and pathological processes. The fact that the fibulin-1 and ADAMTS-1 proteins interact raises new questions about the roles of these extracellular matrix proteins in modulating tumor progression. Herein, we described the functional implications of the interaction between fibulin-1 and ADAMTS-1 on the behavior of breast cancer cell lines. Methods: Fibulin-1 and ADAMTS-1 were exogenously expressed in MCF-7 and MDA-MB-231 cell lines to assay the effect of their interaction in cellular properties. Results: ADAMTS-1 expression exacerbates tumor effects in terms of proliferation, invasion and mammosphere formation. In contrast, the simultaneous expression of ADAMTS-1 and fibulin-1 impairs these effects. The analysis of the expression of both proteins in human breast cancer tissue arrays provides new insights into the complex roles of fibulin-1 and ADAMTS-1 in this type of tumor. ;Conclusion: Our results suggests that the interaction between ADAMTS-1 and fibulin-1 induces a pronounced anti-tumoral effect.