In Vitro Study On Antitumor Activity Against MCF-7 Cell Lines And Basic Mechanism Of Geraniin

Breast cancer have been one of the common cancer in women. The incidence of breast cancer is rising year by year in our country, and the incidence of coming younger in average age. So the method of treatment and prevention of breast cancer is becoming a hot spot at present. Signaling pathway is closely related with tumors. With the in-depth research of tumor cell signal transduction pathway, finding the compounds which can regulate the tumor cell signal transduction so as to achieve the aim of antitumor has become one of the assignment in the field of research new medicine.Geraniin, a polyphenols isolated from Geranium, has been found to possess a range of bioactive properties which includes antioxidant and free radical scavenging activity, antimicrobial, antiviral, anticancer and antihypertensive properties and in vitro antihyperglycaemic activity. The anticancer activity of Geraniin had been reported. However, the mechanism of Geraniin-induced apoptosis has not been reported in depth. So we investigated the anti-proliferative effect and molecular mechanisms of Geraniin on MCF-7 cells in more detail. The results were as follows:1. The markedly anti-proliferative activity of Geraniin to MCF-7 cells.The results showed that MCF-7 were the most sensitive towards Geraniin, and the IC50 value of MCF-7 was 9.94±0.65 μM after 72 h treatment. Further research showed that Geraniin inhibited the growth of MCF-7 cells in a time and dose-dependent manner. However, the IC50 values on normal cells, mouse macrophage cell line RAW264.7 was 65.69±0.83μM after 72 h treatment, respectively, much bigger than MCF-7 cells, which indicated that Geraniin was less cytotoxicity to normal cells. Moreover, after treatment of MCF-7 cells with Geraniin for 24 h, 48 h, the IC50 values was 42.32±0.23μM and 17.98±0.62 μM. The same time, after treatment of MCF-7 cells with Geraniin, we can see that cell chromatin condensation together and the number of cells had decreased.2. Geraniin could induce apoptosis of MCF-7 cells.The integrity of the inner mitochondrial membrane can be assessed by monitoring the potential gradient across the membrane using the fluorescent dye, Rh123. Representative flow cytometric results showed that Geraniin led to a dose-dependent depolarization of mitochondria. Secondly, after treatment with 10 or 25 uM Geraniin, DNA ladder appeared, but the result was not very obvious in MCF-7 cells. Finally, as detected by annexin V(+), PI(-)% cells using flow cytometry, Geraniin led to a concentration-dependent externalization of phosphatidylserine (PS), a hallmark of the apoptosis.3. Geraniin induces apoptosis in human MCF-7 cells by modulating p38 MAPK signal pathway.(1) Geraniin evoked caspase-3 activation and PARP cleavage. PARP was sheared by caspase-3, that cannot combine with DNA, thus cannot play normal function, then the cells happen apoptosis.(2) Geraniin induced phosphorylation of Bcl-2 and down-regulated the expression of this protein. Several different kinases in intracellular signaling pathways are involved in regulating the phosphorylation of Bcl-2. To determine which protein kinase was involved in Geraniin-induced Bcl-2 phosphorylation, we examined the effects of Geraniin on activation of several kinases. Geraniin treatment resulted in inhibition of Akt phosphorylation, but did not affect the phosphorylation of JNK. Geraniin induced the phosphorylation of p38 MAPK.(3) To determine whether Geraniin-induced p38 activation is causally linked to induction of apoptosis, the MCF-7 cells were treated with Geraniin in the absence or presence of the pharmacological p38 inhibitor SB203580. SB203580 diminished Geraniin-induced cytotoxicity in MCF-7 cells. Furthermore, Geraniin-induced phosphorylation of Bcl-2, down-regulation of Bcl-2 and cleavage of PARP were disrupted in the presence of SB203580.(4) To determine whether Geraniin-induced apoptosis is mediated through generation of ROS, the MCF-7 cells were treated with Geraniin in the absence or presence of the NAC. Geraniin caused pronounced intracellular ROS accumulation at 24 h, which was abolished by the addition of NAC. The addition of NAC also suppressed the Geraniin-induced cytotoxicity in MCF-7 cells. Moreover, NAC treatment abrogated Geraniin-induced phosphorylation of Bcl-2 and p38, down-regulation of Bcl-2 and cleavage of PARP in MCF-7 cells.In conclusion, the present study demonstrated that Geraniin exhibited potential anti-cancer activity in MCF-7 cells through induction of apoptosis and it had low cytotoxicity to normal cells. In a word, Geraniin induced apoptosis in MCF-7 cells via ROS-mediated p38 MAPK-dependent pathway. The results above might provide the theoretical basis for future clinical application of Geraniin treatment.