Research On The Effect Of Genistein Against Hepatoma Carcinoma Cell

Hepatic cell carcinoma (HCC) is one of the frequent malignant tumors inChina, with an annual mortality rate as high as 14.52 per ten thousandpopulation. It is often accompanied with hepatic cirrhosis and propagates withinliver at the early stage. Because of the lack of specific symptoms and signs, it isdifficult to be detected early. Furthermore, its development is very rapid, somany patients miss the chance of surgery treatment. Ninety five percent patientsare not sensitive to radiotherapy. Systemic chemotherapy has a poor curativeeffect, which is lower than fifteen percent. All these above-mentioned matterscontribute to the rapid death of patients shortly after the onset of the disease.Therefore, it becomes an important research direction to search for drugs thatcan inhibit the growth of tumor cells and promote apoptosis but with lowtoxicity. Hino et al confirmed that apoptosis existed in all the developmentstages of hepatoma, and with the progression its growth rate is higher thanapoptosis rate. Apoptosis is an active death procedure programmed by genes.Many works showed that apoptosis is closely associated with the pathogenesisand progression of hepatoma. For this reason, it appears to become the key pointto find drugs that can induce apoptosis of neoplasm cells and have low toxicityto normal cells.Recently, it has become a hot point to search for effective but low toxic·6·drugs from natural plants. Genistein is a sort of osajin compound derived frombean and denticulatum plants, whose structure and potency are similar to that ofestrogen. Epidemiological survey suggests that both breast cancer and prostaticcarcinoma are negatively associated with the consumption of soybean products.Many components in soybean (for example soyasaponin, phytic acid andproteinase inhibitor) have anticancer effect. Isoflavone, especially genisteinmaybe are the main active component which has the anticancer effect. Genisteinhas depressant effects on cell lines of human gastric cancer, esophagealcarcinoma and colorectal cancer in different degree. In lowconcentration(<10μg/ml), genistein shows inhibitory effect in eifferent degreeagainst cancer;while in high concentration(>20μg/ml), it shows cytotoxic effect,resulting in apoptotic structural changes such as chromatin condensation,nuclear fragmentation and DNA interruption. The major anticancer mechanismof genistein lies in the following aspect: 1 inhibiting the activity of tyrosineprotein kinase (PTK);2 increasing the activity of antioxidase;3 estrogen andantiestrogen-like effect;4inducing programmed cell death;5 inhibitingangiopoiesis.In order to search for the possibility of applying genistein to the treatmentof cancer, this study adopted the MTT reduction assay (MTT), flow cytometry,electron microscope, immunohistochemistry and Western blot to investigate themechanism of genistein inhibiting hepatoma carcinoma cell proliferation,promoting apoptotic and blocking invasion.This study discovered that genistein could notably inhibit the growth ofhepatoma carcinoma cell SMMC-7721.With the increase of drug concentrationand the duration of action time, the cell proliferation speed stepped down,showing dose-effect relationship. When the concentration reached 10.0μg /ml,cell growth was remarkably inhibited. The inhibition ratio was 58.8﹪ and 65.2﹪ respectively 24 hours and 48 hours after genistein adoption when the drugconcentration was 10.0μg /ml. While when the drug concentration reached 20.0μg /ml, the inhibition ratio was 78.8﹪ and 85.4﹪ respectively 24 hours and48 hours after genistein adoption, the difference was significant compared withthat of normal control.Flow cytometry analysis showed that the G0/G1 ratio was the highest innon-drug treatment group among all groups, reaching 60.56﹪;the ratio in Sstage was 30.17﹪,the ration in G2/M stage was the lowest, only 9.23﹪.With theincrement of genistein concentration from 2.5μg /ml to 20.0μg/ml, the ratio ofG2/M raised from 9.23﹪ to 15.54﹪ after treatment of 24 hours, and raisedfrom 8.56﹪ to 18.41﹪ after treatment of 48 hours. These results indicated thatwith the increment of genistein concentration, the cells in G2/M stage increased,cells in S stage decreased and the ratio of G0/G1 having the trend of decrement.Transmission electron microscope analysis showed that SMMC-7721 cells innon-drug treatment group had clear cellular structure, having rich microvilli incellular membrane and intact nuclear membrane. The cellular nucleus wasirregular, round or ellipse, with clear nucleoli. Chromatin was well-distributedwithin the nucleus. Several glycogen granule could been seen in intracytoplasm .Cell organelles such as Golgi complex, rough endoplasmic reticulum andchondriosome were clear. After being treated with genistein 24 hours at theconcentration of 5.0 μ g /ml, chromatin in cellular nucleus becameconglomeration or lumping, with chondriosome swelling, cavitating andkytoplasm cavitating. After being treated with genistein at the concentration of10.0μg /ml 24 hours, cellular nucleus became condensed, nuclear membraneunclear, cytoblast and cytoplasm cavitating, ground cytoplasm cavitating.Twenty four hours after being treated with genistein at the concentration of20.0μg /ml, cellular nucleus became condensed, heterochromatin agglutinating,kytoplasm electron density deepening, cytoplasm obviously cavitating, apoptoticbody appearing, showing typical apoptosis changes in morphology, indicatingthat the anticancer mechanism of genistein involving the induction of cellapoptosis.Immunohistochemistry results showed that SMMC-7721 cell after beingtreated 48 hours with different concentration of genistein, the expression ofCaspases-3 and Bax protein increased with the increment of genisteinconcentration.The positive rate of Caspases-3 and Bax protein in cells of 10.0μg/ml and 20.0ug/ml genistein concentration group was remarkably differentfrom that in normal control group. The expression of p53 and Bcl-2 proteinexpression had the tendency to drop with the increment of genisteinconcentration. Compared with control group, positive rates of p53 and Bcl-2protein expression in cells of 10.0 μ g/ml and 20.0 μ g /ml genisteinconcentration groups were significantly different from that in control group.Results of Western blot showed that genistein could regulate the expressionof MMP-2,MMP-9,TIMP-2 and NF-κB in human hepatoma carcinoma cells.With the increment of genistein concentration, the expression ofMMP-2,MMP-9 and NF-κ B up-regulated and TIMP-2 down-regulated,showing that genistein probably inhibited the invision of human hepatomacarcinoma cells by means of influencing the expression ofMMP-2,MMP-9,TIMP-2 and NF-κB.Genistein has a promising applying prospect in the field of biotherapy of tumors.Theresearch provides a theoretical and experimental foundation for the clinically applyinggenistein against tumors.