Genistein Inhibits HeLa Cell Growth By Suppressing Toposiomerase Ⅱα And SIRT1 Expression

Part 1 genistein inhibits HeLa cell growthObjective:Cervical cancer is a common cancer for women, according to statistics, the incidence of cervical cancer ranks as the first female cancer. Cervical cancer is possible for all ages women, but the 25-year-old to 45-year-old women most common. Mortality of cervical cancer ranks fourth in that of female cancer and the number of deaths accounted for 4% in that of cancer deaths.Genistein is an isoflavone compound from natural plants and has many physiological functions, such as estrogen action, anti-oxidation, preventing mutation, anti-infection, preventing and therapy of heart-cerebrovascular disorders, which provide its usage in health care or in treating related diseases. For genistein anticancer property, recently studies have shown that genistein could inhibit a wide range of cancer cells, it can inhibit cancer cells growth,, induce cell deferience, reverse cell drug resisdence, do advantage for other anti-cancer drugs effect. This research will investage if genistein inhibite HeLa cell growth, and to know the effect of genistein on inducing cell apoptosis and arresttingt the cell cycle.Methods:The effect of genistein on HeLa cell growth was assessed using the MTT assay to measure the cell viability and calculate the half inhibition concentration (IC50) of genistein on the cell. To verify whether the decreased cell viability of HeLa cells treated with genistein was related to apoptosis, Hoechst 33258 staining was used to detect the nuclear morphological changes. Cell cycle distribution was examined with flow cytometry. To assess the capability of genistein inducing HeLa cell apoptosis and distinguish different types of cell death, HeLa cells were double-stained with annexin V-FITC and propidium iodide (PI), and analyzed by flow cytometry.Results:MTT results showed that genistein inhibited HeLa cell growth in a dose and time dependent manner. The IC50 value of genistein on HeLa cell was calculated at 126μM for 24 h and 75μM for 48 h. By Hoechst 33258 staining, the control cells were well spread flatly and appeared uniform in chromatin density, while genistein treated cells displayed many examples of chromatin fragments or nuclear debris, which could be identified as apoptotic cells, and the percentage of apoptotic cells was 18.34±2.35% after exposure to genistein for 48 h, while that of control cells was only 1.52±0.56%. Double-stained with annexin V-FITC and propidium iodide (PI), and analyzed by flow cytometry reveal the ratio of apoptotic cells obviously increased in genistein treated cells compared to that of control groups. Cell cycle distribution was examined with flow cytometry showed that the percentage of G2/M cells increased significantly, while the percentage of G0/G1 and S cells have no significant difference with that of control group, which suggesting that the cell cycle was arrested at G2/M phase.Conclusions:genistein can inhibit HeLa cell growth and induce cell apoptosis and arrest the cell cycle at G2/M phase.Part 2 The effect of genistein on topoisomeraseⅡαexpression in HeLa cellObjective:TopoisomeraseⅡ(TopoⅡ) is a ubiquitous nuclear protein catalyzing the reaction of breakage and relinking of DNA, which plays an important role in maintenance of DNA topology required for DNA replication, transcription, and recombination of cellular genes. The two TopoⅡisoforms, TopoⅡαand TopoⅡβ, are 170 kDa and 180 kDa proteins, respectively, and have different roles in regulating cellular function. TopoⅡαshows cell-cycle specific expression during the S and G2/M phases and is essential for chromosome condensation. TopoⅡαis closely related to the occurrence, development, diagnosis, prognosis and treatment of cancer. Otherwise, TopoⅡβis associated with non-proliferating function and believed to maintain the integrity of nuclear chromatin.In recent years, TopoⅡhas become a popular target for cancer chemotherapy treatments. Genistein can inhibit a wide range of cancer cells, and the mechanism of genistein cytotoxicity is considered to involve an inhibitory effect on TopoⅡ. It is believed that genistein can bind to and stabilize the topoisomerase-DNA complex, resulting in DNA strand breaks and inducing cell apoptosis. However, the molecular events of genistein inhibiting TopoⅡexpression at the gene transcription level have not been clearly understood.It is known that the human TopoⅡαgene promoter has five functional CCAAT boxes and two GC boxes. The two GC boxes, GC1 and GC2 are located at the proximal and distal region of the TopoⅡαgene promoter, respectively. Sp1 and Sp3 can bind with similar affinities to both GC boxes by three zinc-fingers. It is believed that Sp1 and Sp3 have different functions in regulating TopoⅡαgene expression. Sp1 acts mainly as a transcriptional activator, while Sp3, consisting of three subtype proteins of 115 kDa,80 kDa, 78 kDa, respectively, can act as a transcriptional repressor binding the GC boxes competitively with Sp1. Recently, it has been shown that the GC-rich sequence is necessary for the effect of genistein on metal-binding protein, metallothioneinⅡA expression in human intestinal Caco-2 cells. Moreover, genistein can block the stimulation of VEGF gene transcription by all trans-retinoic acid through Sp1 and Sp3 sites in a human bronchioloalveolar carcinoma cell. Thus, it is likely that genistein may be able to regulate TopoⅡαgene expression through Sp1 and Sp3.The aim of the present study is to investigate the effect of genistein on TopoⅡαgene expression and to determine whether the transcription factors Sp1 and Sp3 have a role in the regulation of TopoⅡαexpression in HeLa cells.Methods:To investigate the effect of genistein on TopoⅡαexpression at gene transcription level, HeLa cells were treated with genistein for 48 h and the expression of TopoⅡα, Spl and Sp3 mRNA in HeLa cells were examined with RT-PCR. TopoⅡα, Sp1 and Sp3 protein expression were examined by Western blot assay. There are two GC boxes, GC1 and GC2 in the TopoⅡαpromoter, which the transcription factors Sp1 and Sp3 can bind to. To assess the role of Sp1 and Sp3 in the inhibitory effect of genistein on TopoⅡαexpression, we analyzed their binding ability to the two GC boxes by ChIP experiment.Results:RT-PCR results showed that genistein down-regulated TopoⅡαand Spl mRNA expression, while Sp3 mRNA expression was up-regulated in HeLa cells Western blot assay results showed that genistein down-regulated TopoⅡαand Sp1 protein expression, while Sp3 protein expression was up-regulated in HeLa cells. ChIP experiment results showed that Sp1 occupancy was reduced and Sp3 occupancy was elevated in cells treated with genistein compared to those of controls for both GC1 and GC2.Conclusions:genistein can inhibit TopoⅡαexpression, which has relation with the regulation of Specificity protein 1 and Specificity protein 3 in HeLa cell.Part 3 The effect of genistein on SIRT1 expression in HeLa cellObjective:Lysine side chains can be acetylated, methylated, ubiquitinated, sumoylated and ADP-ribosylated. These rivalling and reversible post translational modifications are regulated by a complex interplay of different enzymes. Reversible acetylation of lysine-amino groups crucially modulates protein function und cellular networks. In eukaryotic cells, acetylation is among the most common covalent modifications and ranks similar to the important master switch phosphorylation. Acetylation apparently shows a broader substrate spectrum than phosphorylation. Hundreds of proteins are known to be modified by acetylation. Acetylation can change protein characteristics and functions enormously. In general, acetylation changes the electrostatic state of lysine from positive to neutral and increases the size of the amino acid side chain. Acetylation can equally affect enzymatic activities and determine protein function at multiple levels. Comparatively little attention has been drawn to inhibitors of acetyltransferases (HATi), as HATs are rarely considered as drug targets. A reason for this could be the promising use of inhibitor of histone deacetylases (HDACi) in various diseases like leukaemia and other haematological disorders. HDACs can be grouped into two distinct families. The "classical family" of zinc-dependent HDACs are structurally related to the yeast Hdal/Rpd3 proteins, and the second one consists of the NAD+-dependent yeast Sir2 homologues. Sirtuins make use of a different mechanism of catalysis. Instead of using an electrophilic Zn2+ ion to directly hydrolyse the amide bond with water, they transfer the acetyl group to the cosubstrate NAD+ yielding two products, nicotinamide and 2-O-acetyl-ADPribose. This reaction depends on the NAD+/NADH ratio. Metabolism thus may provide a mechanism to regulate SIRTs. Silent Information Regulator 2 (Silent information regulator 2, Sir 2) was found by the Sinclair and colleagues at the Massachusetts Institute of Technology's Leonard Guarente lab in yeast, it is a NAD dependent histone deacetylase. Yeast Sir2 has relation with the chromosome end inactivation and the silencing of rDNA transcription. If content of yeast Sir2p double, it can delay replicative senescence and prolong life. Sir2 is a highly conservative gene family. Sir2-related enzymes (sirtuins, SIRT) is a Sir2 homologs, it not only in yeast, flies, but widely present in various mammalian cells. SIRT1 can delay replicative senescence, prolonging life, which is considered a longevity gene and the evidence is from caloric restriction (CR) test, in yeast, worms, mice and human CR have increased SIRT1 activity can prolong life.To date, seven human isoforms have been identified (SIRT1 to SIRT7), and SIRT1 is the most studied one. Recent reports revealed that hSIRT1 is up-regulated in tumor cell lines, suggesting that hSIRTl may be involved in contributing to tumorigenesis. Therefore, this study will research the action of genistein on SIRT1 in HeLa cell.Methods:After treatment with splitomicin, a special inhibitor of SIRT1, the effect of genistein on HeLa cell growth was assessed using the MTT assay, the capability of genistein inducing HeLa cell apoptosis was tested with Hoechst 33258 staining, and cell cycle distribute was analyzed by flow cytometry. The action of genistein on SIRT1 protein expression was detected by western blotting.Results:after pretreatment with splitomicin, MTT results showed that genistein inhibited more HeLa cells growth in a dose and time dependent manner. By Hoechst 33258 staining, after pretreatment with splitomicin the ratio of apoptotic cells obviously increased in genistein treated cells. Cell cycle distribution was examined with flow cytometry showed the percentage of S cells increased after treatment with splitomicin only, and the percentage of G2/M cells in genistein and splitomicin group is more than of splitomicin group. Genistein can inhibite SIRT1 expression, after pretreatment with splitomicin, genistein can increase SIRT1 expression, but SIRT1 protein level is still lower than that of control group.Conclusions:SIRT1 has relation with genistein inhibition on HeLa cell, and SIRT1 inhibitor can help genistein induce cell apoptosis and arrest the cell cycle at G2/M phase.