Garcinol, an acetyltransferase inhibitor, suppresses proliferation of breast cancer cell line MCF-7 promoted by 17ß-estradiol.

The acetyltransferase inhibitor garcinol, a polyisoprenylated benzophenone, is extracted from the rind of the fruit of Garcinia indica, a plant found extensively in tropical regions. Anti-cancer activity has been suggested but there is no report on its action via inhibiting acetylation against cell proliferation, cell cycle progression, and apoptosis-inhibtion induced by estradiol (E2) in human breast cancer MCF-7 cells. The main purposes of this study were to investigate the effects of the acetyltransferase inhibitor garcinol on cell proliferation, cell cycle progression and apoptosis inhibition in human breast cancer MCF-7 cells treated with estrogen, and to explore the significance of changes in acetylation levels in this process. We used a variety of techniques such as CCK-8 analysis of cell proliferation, FCM analysis of cell cycling and apoptosis, immunofluorescence analysis of NF-κB/ p65 localization, and RT-PCR and Western blotting analysis of ac-H3, ac-H4, ac-p65, cyclin D1, Bcl-2 and Bcl- xl. We found that on treatment with garcinol in MCF-7 cells, E2-induced proliferation was inhibited, cell cycle progression was arrested at G0/G1 phase, and the cell apoptosis rate was increased. Expression of ac-H3, ac-H4 and NF-κB/ac-p65 proteins in E2-treated MCF-7 cells was increased, this being inhibited by garcinol but not ac- H4.The nuclear translocation of NF-κB/p65 in E2-treated MCF-7 cells was also inhibited, along with cyclin D1, Bcl-2 and Bcl-xl in mRNA and protein expression levels. These results suggest that the effect of E2 on promoting proliferation and inhibiting apoptosis is linked to hyperacetylation levels of histones and nonhistone NF-κB/ p65 in MCF-7 cells. The acetyltransferase inhibitor garcinol plays an inhibitive role in MCF-7 cell proliferation promoted by E2. Mechanisms are probably associated with decreasing ac-p65 protein expression level in the NF-κB pathway, thus down-regulating the expression of cyclin D1, Bcl-2 and Bcl-xl.

MiR-155 promotes proliferation of human breast cancer MCF-7 cells through targeting tumor protein 53-induced nuclear protein 1.

BACKGROUND: MiR-155 has emerged as an "oncomiR", which is the most significantly up-regulated miRNA in breast cancer. However, the mechanisms of miR-155 functions as an oncomiR are mainly unknown. In this study, the aims were to investigate the effects of miR-155 on cell proliferation, cell cycle, and cell apoptosis of ERalpha (+) breast cancer cells and to verify whether TP53INP1 (tumor protein 53-induced nuclear protein 1) is a target of miR-155, and tried to explore the mechanisms of miR-155 in this process. RESULTS: The expression of miR-155 is significantly higher in MCF-7 cells compared with MDA-MB-231 cells. Ectopic expression of TP53INP1 inhibits growth of MCF-7 cells by inducing cell apoptosis and inhibiting cell cycle progression. Overexpression of miR-155 increases cell proliferation and suppress cell apoptosis, whereas abrogating expression of miR-155 suppress cell proliferation and promotes cell apoptosis of MCF-7 cells. In addition, miR-155 negatively regulates TP53INP1 mRNA expression and the protein expression of TP53INP1, cleaved-caspase-3, -8, -9, and p21, and luciferase reporter reveals that TP53INP1 is targeted by miR-155. CONCLUSIONS: TP53INP1 is the direct target of miR-155. MiR-155, which is overexpressed in MCF-7 cells, contributes to proliferation of MCF-7 cells possibly through down-regulating target TP53INP1.

Effect of Trastuzumab on Notch-1 Signaling Pathway in Breast Cancer SK-BR3 Cells.

OBJECTIVE: To investigate the effects and mechanisms of trastuzumab on Notch-1 pathway in breast cancer cells, recognizing the significance of Notch-1 signaling pathway in trastuzumab resistance. METHODS: Immunocytochemistry staining and Western blotting were employed to justify the expression of Notch-1 protein in HER2-overexpressing SK-BR3 cells and HER2-non-overexpressing breast cancer MDA-MB-231 cells. Western blotting and reverse transcription PCR (RT-PCR) were used to detect the activated Notch-1 and Notch-1 target gene HES-1 mRNA expression after SK-BR3 cells were treated with trastuzumab. Double immunofluorescence staining and co-immunoprecipitation were used to analyze the relationship of Notch-1 and HER2 proteins. RESULTS: The level of Notch-1 nuclear localization and activated Notch-1 proteins in HER2-overexpressing cells were significantly lower than in HER2-non-overexpressing cells (P<0.01), and the expressions of activated Notch-1 and HES-1 mRNA were obviously increased after trastuzumab treatment (P<0.05), but HER2 expression did not change significantly for trastuzumab treating (P>0.05). Moreover, Notch-1 was discovered to co-localize and interact with HER2 in SK-BR3 cells. CONCLUSION: Overexpression of HER2 decreased Notch-1 activity by the formation of a HER2-Notch1 complex, and trastuzumab can restore the activity of Notch-1 signaling pathway, which could be associated with cell resistance to trastuzumab.

[Effect of HSP90 function inhibited on the telomerase and P53 mutant in breast cancer cells].

OBJECTIVE: To investigate the effect of heat shock protein 90 (Hsp90) function inhibited on the telomerase activity and P53 expression in human breast cancer cells, in which a specific inhibitor geldanamycin (GA) was used to inhibit Hsp90 function. METHODS: Human breast cancer cell line MDA-MB-435s was used for the study. Proliferation of MDA-MB-435s cells was measured with MTT assay. Cell cycle was analyzed by flow cytometry. The telomerase activity was measured by TRAP-ELISA assay. The protein expression of mutant P53 in cells was detected by Western blot. RESULTS: GA inhibited the proliferation of MDA-MB-435s cells on a time- and dose-depending manner. After treated with GA, MDA-MB-435s cells arrested at G2/M phase. The telomerase activity of cells was decreasing, the expression level of mutant P53 protein in MDA-MB-435s cells was reduced obviously compared with that in control cells. CONCLUSION: The telomerase activation and mutant P53 expression in MDA-MB-435s cells are related to HSP90 function, of which the inhibition with GA can decrease the telomerase activity and the protein expression of mutant P53, and further repress the proliferation of MDA-MB-435s cells.

[Effect of Janus kinase inhibitor AG490 on invasion and metastasis of human breast cancer cells].

OBJECTIVE: To investigate the effect of Janus kinase (JAK) inhibitor AG490 on invasion and metastasis of the human breast cancer cell MDA-MB-231, and to explore the regulating role of JAK-STAT3 signaling pathway when the breast cancer occurs to the invasiveness and metastasis. METHODS: The human breast cancer cell MDA-MB-231 was used as the research object, and AG490 was as Janus kinase inhibitor. The adhesion of MDA-MB-231 cell attaching to matrigel was measured with MTT assay. The invasion and metastasis potential were evaluated with transwell chamber. The P-STAT3 protein in cell was detected by Western-blot. RESULTS: Janus kinase inhibitor AG490 could make that the expression of P-STAT3 in human breast cancer cell MDA-MB-231 became weak, and that the abilities of adhesion, invasion and metastasis were also dropping down as compared with the control group (P < 0.01). CONCLUSION: JAK-STAT3 signaling pathway participates in regulating the invasion and metastasis of human breast cancer. Inhibiting the activation of JAK-STAT3 signaling pathway can suppress the invasion and metastasis of human breast cancer.

[Effects of a JAK inhibitor, AG490, on proliferation and survivin expression of breast cancer cell line MDA-MB-231].

BACKGROUND & OBJECTIVE: Many researches have proven that Survivin is expressed abundantly and is involved in tumorigenesis and prognosis of breast cancers. However, the signal transduction pathway of Survivin in breast cancer cells is still not clear. This study was to investigate the effects of AG490, a JAK enzyme inhibitor, on the expression of Survivin and on the proliferation and apoptosis of human breast cancer cell line MDA-MB-231. METHODS: MDA-MB-231 cells were treated with AG490. The protein levels of P-STAT3 and Survivin were detected by Western blot; the mRNA level of survivin was measured by reverse transcription-polymerase chain reaction (RT-PCR). Cell proliferation was detected by MTT; cell apoptosis was detected by flow cytometry (FCM). RESULTS: AG490 reduced the expression of P-STAT3 and Survivin, inhibited cell proliferation and induced apoptosis in MDA-MB-231 cells. When treated with 40 micromol/L AG490 for 24 h, the protein level of P-STAT3 was decreased from 0.74 to 0.21, and that of Survivin was decreased from 1.09 to 0.28. The "sub-G1" peak appeared and the apoptosis rate was 5.62% at 24 h, and further reached 28.81% at 48 h. CONCLUSION: AG490 could induce apoptosis and reduce the expression of Survivin in breast cancer MDA-MB-231 cells.

[Correlation of MUC1 expression to adhesion of breast cancer cell line MDA-MB-231].

BACKGROUND & OBJECTIVE: Recent researches found that an abundant production of mucin protein well correlates with tumor cell metastasis. This study was to investigate inhibitory effect of benzyl-alpha-GalNAc on production of mucin 1 (MUC1), and on adhesion and invasion of breast cancer cell line MDA-MB-231. METHODS: MDA-MB-231 cells were incubated with benzyl-alpha-GalNAc, expression of MUC1 was detected by immunohistochemistry, adhesive ability of MDA-MB-231 cells to artificial basement membrane Matrigel was measured by MTT assay. Gelatin zymography was performed to detect the secretion changes of matrix metalloproteinase-2 (MMP-2) and MMP-9. RESULTS: Compared with control cells, the tumor cells deglycosylated by benzyl-alpha-GalNAc showed lower expression of MUC1 (P < 0.05), and less adhesion to the Matrigel (P< 0.01), the secretion of MMP-2 and MMP-9 suppressed (P< 0.05). CONCLUSION: The expression of MUC1 correlates to adhesion and invasion of MDA-MB-231 cells, benzyl-alpha-GalNAc may weaken adhesion and type IV collagenase-secreting activity of MDA-MB-231 cells by inhibiting MUC1.