Effect Of Genistein On Growth And Apoptosis Of Colon Cancer LOVO Cells And Its Mechanisms

Objective: To study the effects of genistein and 17β-estradiol on cell growth and cell death in the cultured human colon cancer cell line LOVO. To measure the mRNA level of bcl-2 and caspase-3 gene to further investigate the effect and mechanism of estrogen receptor on death and proliferation of LOVO cells.Methods: (1) Growth inhibition of 17β-estradiol and genistein on LOVO cells was analyzed by MTT assay. (2) Microscopic structure changes of cells were observed by light microscopy. The feature of cell death was studied by Hoechst33342/PI staining, and nuclear morphological assessment of apoptotic cells and necrosis were observed by fluorescence microscopy. (3) Cell cycle and apoptosis ratio were detected by flow cytometry. (4) The mRNA expression of estrogen receptorαandβ, caspase-3 and bcl-2 were measured by reverse transcription PCR ( RT-PCR).Results: (1) The MTT assay showed that both genistein and 17β-estradiol inhibited the growth of LOVO cell in a dose and time-dependent manner. Genistein at low concentration(30μmol/L) could inhibit the proliferation of LOVO cells partially, and genistein (120μmol/L) at high concentrations, inhibition ratio of which reached 89.63% for 96 hours, had dramatic inhibitory effect on LOVO cells. 17β-estradiol (30μmol/L) at low concentration could also inhibit the proliferation of LOVO cells in time-dependent manner, but its effect of growth inhibition was weaker than that induced by 17β-estradiol at high concentration (120μmol/L) in time-dependent manner.(2) The obvious morphological changes of cells treated with 17β-estradiol or genistein were detected by light microscopy. The cells in control group were multiplied and well adherenced. Cells appeared as fusiform shape, spreaded out of the field of vision with clear nucleoli and caryocinesis phase. The number of LOVO cells treated with 60μmol/L genistein for 48h was decreased compared with the control group. With more floating ones, cells were deranged and inequality of size, shrinkage and vacuoluse, appearing karyoplasmic ratio inversion and diminution of karyokinesis phase. There occured effervescencing and descented-deuterium, while cell membrane were integrity. Moreover, the cell debris increased and background got muddy. With the increasing concentration of genistein, the change-degree of cells increased correspondingly. LOVO cells were not multiplied in 17β-estradiol group and were characterized by the presence of swelling, disrupted nuclei and many vacuolar structures. Stained with Hoechest 33342/PI, most normal alive cells were stained into blue ones with integrity cell membrane and common nucleus structure; a small quantity cells were stained into red ones with common nucleus structure. While the part of cells of in genistein group were stained into blue ones with highly concertrated chromatin and coagulated into relucent clumping called apoptotic body, and some cells got small and anomalo-formed, with nuclear fragmentation. Some of the cells were stained into red ones and appeared transparent chromatin coagulation. With the increasing concentration of genistein, the proportion of cells apoptosis increased correspondingly. But majority cells in 17β-estradiol group were stained into red ones and showed no apparent chromatin coagulation. The proportion of cells in necrosis increased corresponding to concentration of 17β-estradiol. (3) FCM result: After 48 hour incubation, cells in the genistein group appeared visible apoptosis, and the hypodiploid peak appeared before stage G1 was apoptotic peak, simultaneously, the percentage of apoptosis increased apparently. But the cells in 17β-estradiol group appeared no apoptosis, and most of cells were arrested in G2/M phase in dose-dependent manner. (4) Caspase-3 mRNA expression was poor, but the bcl-2 mRNA expression was strong in normal LOVO cells. On the contrary, caspase-3 mRNA expression were significantly up-regulated and bcl-2 mRNA expression showed a significant decrease in LOVO cells when treated with genistin for 48 hours in a dose-dependment manner.Conclusion: (1) Estrogen receptor are expressed in human colon cancer LOVO cells. Expression of estrogen receptorβare much stronger than expression of estrogen receptorα. (2) Genistein have potent growth-inhibitory effect on LOVO cells in a dose and time dependent manner, mainly through inhibiting proliferation, inducing apoptosis, altering the cell cycles and regulating the expression of poptosis related gene caspase-3 and Bcl-2.(3) 17β-estradiol can inhibit proliferation of LOVO cells in a dose-and time-dependent manner, maybe through alerting the cell cycles. 17β-estradiol can't induce apoptosis of LOVO cells, but could stimulate necrosis of LOVO cells.(4) Both genistein and 17β-estradiol can inhibit proliferation and growth of LOVO cells, however, through different pathways. The mechanism of genistein on the counteract of colon carcinoma may be involved in non- estrogen pathway.