| According to incomplete statistics, every year there occur about 1.6 million new cancer sufferers in our country:According to the predication of WHO, the number of cancer patients will increased to 14.7 million. Malignant tumor is one of the severe diseases harmful to human health. Liver cancer is a tumor of digestive system with high degree of malignancy, which annually causes more than 626,000 cases. The incidence of liver cancer ranks the 6th among whole malignant tumor. The death toll of liver cancer is 589,000 every year and the lethality occupies the first place next to lung carcinoma and gastric carcinoma. The mortality is higher gradually in recent years. Surgical procedures and postoperative chemotherapy and radiotherapy are the main treatments at present, but which have more side effects. Many of patients died with not liver cancer but the side effects of chemotherapy and radiotherapy. Therefore, the preparation of new-type antineoplastic agents of high efficiency and low toxicity holds the best hope for the success of tumor therapy.Sulforaphane (SFN), one of naturally occurring isothiocyanates (ITCs) found in crucifer, is the enzymatic hydrolysate of Glucosinolates, which shows the highest amount in Brassica oleracea Linnaeus var. botrytis Linnaeus. A large number of studies show that ITCs can inhibit the activity of the phase I drug metabolizing enzyme, induce the activity of the phase II drug metabolizing enzyme and decrease activation of carcinogen, which can protect against injury. ITCs show a strong chemopreventive potential. At present, SFN not only restrains tumor during different stages of carcinogenesis, but also is a potential drug in the oncotherapy. The antitumor effect of SFN is a research focus in antineoplastic research field.Though there are many theories on pathogenesis of cancer, in a word, cancer is a diseases associated to cell cycle and apoptosis deregulation. Researching the drug to regulate the cell cycle and induce apoptosis is a new way of tumor therapy. Our previous research proved that ITCs in Brassica oleracea Linnaeus var. botrytis Linnaeus could induce human hepatoma HepG-2 cells apoptosis. Further study found that SFN was the anti-hepatoma component and its content is the highest and the activity is the strongest. Therefore, based on previous research results, in this study, human hepatoma cell Line HepG-2 was chosen as research object. The objective of this article is to investigate systematically the antitumor mechanism of SFN starting from regulation of cell cycle and induction of apoptosis and to provide scientific basis for future the anti-hepatoma drug research and development.1 Study on the effect of SFN on the proliferation inhibition and apoptosis induction in HepG-2 cells1.1 Study on the effect of SFN on the proliferation inhibition in HepG-2 cells OBJECTIVE:To investigate the effect of SFN on the proliferation inhibition in HepG-2 cells. METHODS:MTT assay and SRB assay were used to determine the proliferation-inhibition ratio of SFN on HepG-2 cells and calculate IC50 and GI50; Inverted microscope was used to observe the growth situation of the cells. RESULTS:The results of MTT assay showed that SFN of different concentration (5,10,20,40 and 80μmol/L) could inhibit cell growth at 24h, 48h and 72h time points in a time and dose-dependent manner. IC50 of SFN of 24h,48h and 72h were 32.03±0.96,20.90±1.96和13.87±0.44μmol/L respectively. The results of MTT assay showed that IC50, GI50, TGI and LC50 were 21.80μmol/L,19.40μmol/L,26.68μmol/L and 61.87μmol/L, which agreed well with the results obtained by MTT assay. Under inverted microscope, the cells of control group grew adhering to the wall showing plump appearance and clear edge. After exposure to SFN, adherent cells decreased. HepG-2 cells became rounded and shrunken gradually with lower refraction. CONCLUSION:SFN can significantly inhibit the proliferation in SGC-7901 cells.1.2 Study on the effect of SFN on cell cycle arrest in HepG-2 cells OBJECTIVE:To investigate cell cycle arrest effects of SFN in HepG-2 cells. METHODS: Cells were marked by PI and FCM was used to analysis the cell cycle distribution of HepG-2 cells after exposed to SFN. RESULTS:After SFN with different concentration (10,20,40μmol/L) treated on HepG-2 cells for 48 h, the proportion of G2/M phase cell increased significantly while the proportion of G1 phase cell decreased obviously. The percentage of cells in G2/M phase was increased with increasing concentrations of SFN. The subdiploid peak was founded in 40μmol/L dosage group of SFN. CONCLUSION:SFN can arrest HepG-2 cells on G2/M phase. 1.3 Study on the effect of SFN on the inducing apoptosis in HepG-2 cells OBJECTIVE:To investigate the apoptotic effect of SFN in HepG-2 cells. METHODS:In situ detection of DNA fragments by terminal deoxyribonucleotide transferase (TdT)-mediated dUTP nick end labeling (TUNEL) was performed using the one-step TUNEL apotosis assay kit; Cells were doubly marked by Annexin V-FITC/PI, the typical morphological changes of early apoptosis in HepG-2 cells were observed with LCSM; TEM was used to observe the apoptotic morphology and the ultrastructure changes of HepG-2 cells; Cells were doubly marked by Annexin V-FITC/PI, the apoptosis rate of SGC-7901 cells was detected. RESULTS:The results of TUNEL assay showed that after SFN treated on HepG-2 cells for 48 h, under LCSM, the cell nuclear of the early apoptosis cells showed green fluorescent. The number of apoptosis cells was increased with increasing concentrations of SFN while the cells of control group were no significant coloring. Under LCSM, cells that stain positive for Annexin V-FITC (green) and negative for PI are undergoing apoptosis. Cells that stain positive for both Annexin V-FITC (green) and PI (red) are either in the end stage of apoptosis, or are undergoing necrosis. The number of early and late apoptotic cells increased in a dose-dependent manner. Under TEM, the morphological characteristics such as the clear cellularity, complete structure of organelles, the chromatin well distributing in the cell nuclear, the normal mitochondria and the microvilli on the cell membrane were commonly seen in these tumor cells of control group. After exposure to SFN, some tumor cells showed typical apoptotic morphology, including a decrease in microvilli, chromatin condensation, crescent margination of chromatin against the nuclear envelope, chromatin crescent formation, enlargement of perinuclear space, mitochondria swelling, cytoplasmic vacuolation and formation of apoptotic body. FCM analysis result showed that the apoptosis rates of HepG-2 cells treated with 10,20,40μmol/L of SFN for 48 h were 27.42%±0.43%,46.53%±0.35% and 58.92%±0.48%, which were significantly higher than that of the control (1.6%±0.06%) (P<0.01). CONCLUSION:SFN can induce apoptosis in SGC-7901 cells.2 Study on the mechanism of G2/M phase arrest and apoptosis induced by SFN in HepG-2 cells2.1 Study on the mechanism of G2/M phase arrest induced by SFN in HepG-2 cellsOBJECTIVE:To investigate the molecular mechanism of G2/M phase arrest induced by SFN in HepG-2 cells. METHODS:Western blot assay was used to investigate the G2/M phase correlative protein expression of CdKl, p-CdKl(Thrl4), cyclinBl, Cdc25C and p21. RESULTS:After treated with SFN at the dosage of 10,20,40μmol/L on HepG-2 cells for 48 h, the expression of CdKl, cyclinBl and Cdc25C protein were down-regulated, which were significantly lower than that of control group (P＜0.01). Meanwhile, the expression of p-CdKl(Thrl4) and p21 were significantly up-regulated (P＜0.05 or P＜0.01). CONCLUSION:SFN can induce G2/M phase arrest in HepG-2 cells through reducing complex formation of CdKl-cyclinBl and inhibiting the activity of CdKl or CdKl-cyclinBl by down-regulation of Cdc25C protein and up-regulation of p21 protein.2.2 Study on the mechanism of apoptosis via mitochondria pathway induced by SFN in HepG-2 cellsOBJECTIVE:To investigate the molecular mechanism of cell apoptosis via mitochondria pathway induced by SFN in HepG-2 cells. METHODS:Caspase Assay Kit was used to detect the activity of caspase-3,-8,-9 in HepG-2 cells; LCSM was used to detect the mitochondrial transmembrane potential (△Ψm) and [Ca2+]i in HepG-2 cells; FCM was used to detect the cellular concentration of ROS in HepG-2 cells; Western blot assay was used to detect the expression of Cyt-c protein; RT-PCR and Western blot assay were used to detect the expression of Bcl-2 and Bax protein and mRNA. RESULTS:After treated with different concentration of SFN for 48 h,△Ψm decreased gradually in a dose dependent manner. At the dose of 10,20 and 40μmol/L of SFN, [Ca2+]I (FI) were 45.68±10.51,88.62±16.09 and 176.33±25.14 respectively, and the intracellular ROS level were 28.74±4.59,52.90±6.61 and 90.32±5.95, which were significantly higher than that in the control group (P＜0.01). The results of RT-PCR and Western blot assay showed that SFN could down-regulate the expression of Bcl-2 mRNA and protein, in the meantime, up-regulate the expression of Bax mRNA and protein. SFN could remarkably decrease the ratio of Bcl-2/Bax and Bcl-2 mRNA/Bax mRNA compared with control group (P＜0.05 or P＜0.01). Besides, SFN could significantly up-regulate the expression of Cyt-c in a dose dependent manner compared with control group (P＜0.05). Activity kit detection result showed that SFN could increase the activities of Caspase-3,-8,-9 in a dose dependent manner, which were remarkably higher than that of control group (P＜0.05 or P＜0.01). CONCLUSION:SFN can induce the opening of MPTP by down-regulating the Bcl-2 gene expression and up-regulating the Bax gene expression, and then, improve the release of Cyt-c from mitochondria to the cytoplasm. Cyt-c can further combine with Apaf-1 and procaspase-9 which formed an apoptosome. Then Caspase-9 activates Caspase-3 which may trigger the cytological events in the terminal stage of apoptosis induced by SFN.2.3 Study on the mechanism of apoptosis via MAPK pathway induced by SFN in HepG-2 cellsOBJECTIVE:To investigate the molecular mechanism of cell apoptosis via MAPK pathway induced by SFN in HepG-2 cells. METHODS:Western blot assay was used to detect the MAPK correlative protein expression of P38, p-P38, ERK, p-ERK, JNK/SAPK and p-JNK/SAPK. RESULTS:After treated with SFN at the dosage of 10,20,40μmol/L on HepG-2 cells for 48 h, the expression of p-P38 and p-JNK protein were up-regulated, while p-ERK expression was down-regulated which showed significantly difference compared with control group (P<0.05 or P<0.01). Meanwhile, there were no significant changes on the expression of P38, ERK and JNK protein in HepG-2 cells (P>0.05). CONCLUSION:MAPK pathway plays an important role in the apoptosis induced by SFN in HepG-2 cells. SFN can induce G2/M phase arrest and cell apoptosis in HepG-2 cells through MAPK pathway.CONCLUSIONSFN has remarkably antitumor effect on Human hepatoma HepG-2 cells, and its possible mechanism are as follows:①SFN can induce G2/M phase arrest in HepG-2 cells through reducing complex formation of CdKl-cyclinBl and inhibiting the activity of CdKl or CdKl-cyclinBl by down-regulation of Cdc25C protein and up-regulation of p21 protein.②SFN can induce the opening of MPTP by down-regulating the Bcl-2 gene expression and up-regulating the Bax gene expression, and then, improve the release of Cyt-c from mitochondria to the cytoplasm. Cyt-c can further activate caspase-9 and Caspase-3 which may trigger the cytological events in the terminal stage of apoptosis induced by SFN.③SFN can activate the p38MAPK signaling pathway by up-regulating the expression of p-P38, which can induce G2/M phase arrest by deactivating of Cdc25C and Cdkl. Meanwhile, the up-regulation of p-p38 can promote the activation of caspase-8 and then activate caspase cascade by activating the fas/fasL apoptosis pathway.④SFN can inhibit the activation of ERK pathway by down-regulating p-ERK protein, which can block the expression of Bcl-2 gene and inhibit the anti-apoptosis effect of Bcl-2. This may be one of the most important mechanism of apoptosis inducing effect of SFN.⑤SFN can promote cell apoptosis via mitochandial pathway by improving the phosphorylation of JNK. At the same time, the activation of JNK pathway can promote the activation of caspase-8 and then activate caspase cascade by activating the fas/fasL apoptosis pathway. It can also stabilize the activation of p21, which can inhibit the activation of Cdkl and induce G2/M phase arrest in HepG-2 cells.INNOVATIVE POINTS OF CURRENT STUDY1. For the first time, the activation of the of G2/M DNA damage checkpoint pathway is found to be the main molecular mechanism of SFN on the G2/M phase arrest in HepG-2 cells.2. For the first time, mitochondria pathway is found to be the main approach of SFN on the induction of HepG-2 cell apoptosis, in which, down-regulation of bcl-2 gene expression and up-regulaiton of bax gene expression play a key role.3. For the first time, MAPK signaling pathway is found to be the key regulation pathway and the most important effect target of SFN in the inducion of G2/M phase arrest and apoptosis of HepG-2 cells.
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