Mechanistic Studies Of The Growth-Inhibitory Effects Of Berberine And Neferine On Human Osteosarcoma Cells

Osteosarcoma is the most common type of bone cancer in children and adolescents,although cancer in young people is rare in general.Most osteosarcomas develop between the ages of 10 and 25 when the bones are rapidly growing.Since osteosarcomas are usually resistant to ionizing radiation,its treatment primarily relies on the combination of chemotherapy and surgical removal of the tumor. Chemotherapy plays a very important role in cancer treatment.However,multidrug resistance and toxicity greatly limit its application.Great efforts have been devoted to the search for new cancer chemopreventive agents from natural products.Berberine,an isoquinoline alkaloid component in several Chinese herbs including Huanglian,has been shown to have antimicrobial,anti-inflammatory,anti-diabetic and anti-angiogenesis and cholesterol-lowering effects.A large number of studies also showed that berberine possesses anti-tumor activity,against cancer cells established from cervical,esophageal,oral,colonic,prostate cancers,leukemia,melanoma and glioblastoma.Many studies showed that berberine inhibits tumor cell growth by inducing cell cycle arrest and/or apoptosis.Some in vitro studies showed that berberine could inhibit tumor cell adhesion,migration and invasion.Recent studies also showed the anti-tumor activity of berberine in vivo.However,there are many unanswered questions such as follows.How does berberine initiate the cascade that eventually leads to cell cycle arrest and/or apoptosis? Why does berberine induce some cancer cells arrest at G1 phase and other cancer cells at G2/M phase? What is the direct target of berberine in tumor cells?Neferine(Nef) is a bis-benzylisoquinoline alkaloid isolated from the green seed embryo of Nelumbo nucifera Gaertn.Neferine has been shown to have anti-inflammation,anti-oxidant,anti-hypertensive,anti-arrhythmic anti-platelet aggregation effects.Recently,neferine has been found to be a potent agent for reversing Adriamycin resistance of tumor cells in vitro.Neferine can also enhance the anti-tumor effect of adriamycin on osteosarcoma cells in vitro.However,it is not known whether neferine has direct anti-tumor effect.We evaluated the anti-tumor effects of berberine and neferine on osteosarcoma cells and characterized the mechanisms underlying the anti-tumor effect of these two natural products.PART ONE The inhibitory effects of berberine and neferine on the proliferation of human osteosarcoma cells and normal osteoblast cells.We analyzed the inhibitory effects of berberine and neferine on osteosarcoma cell lines U2OS,Saos-2,HOS and normal osteoblast HCO by morphological observation, MTT and BrdU assays.1.Analysis of the growth-inhibitory effects of berberine and neferine on U2OS cells by MTT assay.(1) Berberine and neferine significantly inhibited growth of U2OS cells in a time- and dose-dependent manner.(2) Neferine showed a more potent inhibitory effect than berberine.Such as the viability of U2OS cells after 24 hours was 50.6%with 4μmol/L neferine and 75.7%with 135μmol/L berberine.2.Berberine reduced the proportions of cells in S phase in a dose-dependent manner,as measured by BrdU incorporation assay..3.Inhibitory effects of berberine and neferine on additional human osteosarcoma cells and normal osteoblast cells by MTT assay.(1) Both berberine and neferine exhibited significant inhibitory effects in a time- and dose- dependent manner on osteosarcoma cell lines.(2) Normal osteoblast cells HCO appeared to be more resistant than the osteosarcoma cells to berberine and neferine.Neferine promoted HCO cell survival atlowdose(1,2,3,4μmol/L).The results in this part suggested that berberine and neferine exhibited a dose-dependent growth-inhibitory on osteosarcoma cells.However,normal osteoblast cells HCO appeared to be more resistant than the osteosarcoma cells.Neferine showed a more potent inhibitory effect than berberine.PART TWO Cell cycle analysis of human osteosarcoma cells in response to berberine and neferineInduction of cell cycle arrest is one of the most important mechanisms of antitumor drugs.In this part,we studied the effect ofberberine and neferine on cell cycle progression and the underlying mechanism by flow cytometry,Real-time PCR and Western Blottinging.1.U2OS,Saos-2 and HCO cells were analyzed for their cell cycle distribution by flow eytometry treated with berberine and neferine.(1) In U2OS and HCO cells(p53 wild type) treated with berberine,the percentages of cells in G1 phase were increased at the expense of cells in S phase.While there was no change in the percentage of cells in G2/M at lower doses,the percentage of cells at G2/M was markedly increased at the highest dose tested,50μg/ml.The p53-deficient Saos-2 cells preceded into S phase in the presence of berberine.However,a great proportion of cells were arrested in G2/M.These results suggest that berberine arrests cells at G1 and/or at G2/M,depending on the concentration of berberine and on the cells it is applied to.(2) In U2OS(p53 wild type) and Saos-2(p53-deficient) cells treated with neferine,the percentages of both two cell lines in G1 phase were increased in dose dependent manner at the expense of cells in S phase.2.Responses of p53 and cell cycle effectors/regulators to berberine and neferine treatment.(1) Berberine treatment resulted in a marked decrease in the expression of cyclin E in a dose-dependent manner.The expression level of cyclin D1 decreased only at the high concentration(50μg/ml).Berberine treatment resulted in a dose-dependent enhancement of p53 in protein and mRNA level.The level of phosphorylated p53 at serine 15 was also elevated in a dose-dependent manner.Berberine treatment resulted in a dose-dependent enhancement of p21WAF1/CIP1 and p27KIP1 protein level compared with control cells.(2) Neferine caused G1 cycle arrest accompanied by an up regulation of p21WAF1/CIP1 and a down regulation of Cyclin E.There were no obvious changes of cyclin D1 and p53.Importantly,we observed that the up regulation of p21WAF1/CIP1 by neferine is independent on functional p53.3.Cell cycle arrest at G1,but not G2/M,depends on functional p53 treated with berberine.Berberine treatment of U2OS-neo cells showed an accumulation at the G1 phase.U2OS-mtp53 cells,however,experienced no obvious changes. In contrast to the berberine-induced reduction of S phase cells in U2OS-neo, the percentage of U2OS-mtp53 cells at S phase was not significantly decreased upon berberine treatment.The results above suggested that berberine induced U2OS cells arrest at G1 phase through down regulation of Cyclin E,cyclin D1 and up regulation of p21WAF1/CIP1 and p27KIP1.Cell cycle arrest at G1 in U2OS cells,but not G2/M,depends on functional p53.Neferine induced G1 arrest both in U2OS and Saos-2 cells.Neferine caused p53 independent G1 cycle arrest which was accompanied by up regulation of p21WAF 1/CIP1 and down regulation of Cyclin E in U2OS cells.PART THREE Induction of apoptosis in osteosarcoma cells by berberine and neferineIn addition to cell cycle arrest,apoptosis has also been reported to be responsible for inhibition of tumor cell proliferation.In this part,we studied the effect of berberine and neferine on human osteosarcoma and normal osteoblast cells by Annexin V-PI and TUNEL assay.And we also analyzed the mRNA levels of p53 target genes involved in apoptosis in U2OS-neo and U2OS-mtp53 cells.1.U2OS,HOS,Saos-2 and HCO cells were analyzed by TUNEL assay.After berberine treatment for 48h,the percentage of TUNEL-positive cells was increased in a dose-dependent manner in all the cell lines tested.Interestingly, the induction of apoptosis was less pronounced in the non-neoplastic cells HCO.There was no obvious difference of apoptotic cells between HCO and Saos-2 at low dose.2.The induction of apoptosis by berberine was more dramatic in U2OS-neo cells than in U2OS-mtp53 cells,suggesting that berberine-induced apoptosis was also dependent on the function of p53 in osteosarcoma cells.3.To gain further insights into the mechanism by which the activation of p53 induces apoptosis,we analyzed the mRNA levels of p53 target genes involved in apoptosis.The mRNA levels of pro-apoptotic genes BAX,PUMA and FAS were all increased in U2OS-neo cells after berberine treatment.Similarly treated U2OS-mtp53 cells,on the other hand,showed no significant increase.4.When we used Annexin V-PI assay to test apoptotic cells in U2OS cells treated with neferine.We didn't observe an increase in the percentage of apoptotic cells in neferine treated cells compared to control cells.These results indicated that berberine induced apoptosis both in osteosarcoma and normal osteoblast cells.The induction of apoptosis was less pronounced in the non-neoplastic cells than neoplastic cells.Berberine induced apoptosis by p53 dependent and p53-independent pathways.Neferine didn't cause apoptosis in U2OS cells.PART FOUR Investigation of upstream factors of cell cycle arrest and apoptosis in osteosarcoma cells treated with berberineThe results in the previous two parts showed that p53 played an important role in the inhibitory effect of berberine on human osteosarcoma cells.Berberine up regulated p53 both at mRNA and protein level.The level of phosphorylated p53 was also elevated in a dose- dependent manner.We speculated that DNA damage might be the upstream factor that activated p53 and caused cell cycle arrest and apoptosis.In this part,western blotting and immunofluorescence staining ofγ-H2AX were performed on berberine-treated osteosarcoma cells.In addition,micronucleus assay was used to evaluate whether berberine can cause the genomic instability.1.Phosphorylation of H2AX was determined by immunofluorescence staining and western blotting in berberine treated U2OS cells.Compared to untreated cells,berberine treatment led to an increased phosphorylation of H2AX in a dose-dependent manner.Foci ofγ-H2AX were abundant in the nuclei of most cells exposed to berberine. 2.The foci ofγ-H2AX were less abundant in HCO cells than in U2OS cells. Berberine was accumulated to a less extent in HCO cells than in U2OS cells, as reflected by the fluorescence intensity in the nuclei.3.The frequency of U2OS cells containing micronuclei treated with berberine was increased in a dose-dependent manner.4.Neferine treatment induced no detectable phosphorylation of H2AX as determined by immunofluorescence staining.These results suggest that berberine causes DNA damage in U2OS cells.The accumulation ofγ-H2AX in nuclei and the formation of micronuclei of U2OS cells indicate that berberine can inflict genomic lesions.However,unlike berberine, neferine did not cause DNA damage in U2OS cells.Thus,the mechanisms underlying their inhibitory effect on human osteosarcoma cells differ greatly between berberine and neferine.