Combinational Anticancer Effects And Mechanism Of The Flavone And Hydroxycamptothecin

Flavones are the most distributed in the nature and generally exist in fruits,vegetables,and traditional Chinese medicine(TCM).as well as possess various biological functions such as antioxidant,anti-inflammatory,antibacterial and antiviral.It is reported that they also have potential anti-tumor activity.However,there are few flavones applied practically in clinic for cancer treatment,which is caused by the relatively lower anticancer activities of flavanoes.How to elevate the roles of flavones in cancer treatment is the ultimate goal of this research.To solve this problem,we provide two approaches:one is combinational therapy of hydroxycamptothecin(HCPT)and flavonoids on anti-cancer,the other is to modificate the flavonoids to produce a high-efficiency and low-toxicity derivate.The major methods,results and conclusion are listed below:(1)First,we found that low doses of baicalein(BA)(15.625 μM)significantly enhanced the inhibition of HCPT on the proliferation of BGC823.MCF7.and SMMC7721 cells by MTT assay,along with CI～0.4.Then,we deteced the effects of combinational group on cell cycle and apoptosis of BGC823 cells after treatment for 48 h?by the methods of Annexin-FITC/PI double stained,PI stained,rhodamine 123 stained and western blot assays.The results indicated that the combined group increased apoptotic cells,enlarged Bax/Bcl-2 ration and the loss of mitochondrial membrane potential,as well as elevated the express levels of cleaved-casape 3/9,but had few effects on pro/cleaved-caspase,suggesting that BGC823 cells apoptosis induced by the combined group was depedent on mitochondrial rather than death receptor signal pathway.On the other hand,the combined group arrested BGC823 cells in G1 and G2 phase,as well as upregulated the express level of p21 and p53.Then,the comet assay showed that combination also strenthed the degree of DNA damages.Thus,we respectively used DCFH-DA.DHE stained and a DNA relaxtion assay to determine the alteration of reactive oxygen(ROS)and topomerase Ⅰ(Topo Ⅰ)activity,the possible inducement of DNA damage.The results showed that combinational group increased the levels of ROS in BGC823 cells,but the inhibiton of combinational group on BGC823 cells were not eliminated after pretreated with N-acetyl-cysteine(NAC),an antioxidant agent,meaning that ROS is not the synergic target of BA and HCPT.On the other hand,low doses of BA(15.625 μM)enhanced the inhibiton of HCPT on catalytic activities of Topo Ⅰ while high-dose of BA(125 μ M)also inhibited the catalytic activities of Topo Ⅰ,indicating that Topo Ⅰ may be the synergic target of BA and HCPT.Otherwise,the inhibiton rate of combinational group of 5 mg/kg BA and 3 mg/kg HCPT on BGC823 cells xenograft in nude is 67.60±6.56%,while 3 mg/kg and 9 mg/kg HCPT group is respectively 20.60±5.23%and 64.88±5.51%.(2)Then,we found that HCPT combined with other flavones such as quercetin(Qu).apigenin,luteolin and chrysin,also showed an significantly synergic anticancer effects in MCF7 cells by MTT assay,with CI<0.3.Subsequently,we explored the synergic anti-cancer mechanism of Qu and HCPTin MCF7 cells by Annexin-FITC/PI stained,rhodamine 123 stained,PI stained and western blot so on.The results determined that Qu(1-10 μM)significantly increased the inhibiton of HCPT on the proliferation of MCF7 cells and the catalytic activities of Topo Ⅰ.The combined group triggered phosophorylation of Ser139 in H2A.X,which is the marker of DNA double strands breakes,after treatment for 6 h.Meanwhile,it successively arrested cell cycle at Gl,S close to G2 and G2/M along with 12r 24 and 48 h,respectively.After MCF7 cells were seperately pre-treated with ATM or Chkl inhibitor,the cells only pretreated with Chkl inhibitor were sensitive to combined group,suggesting that combined group arreat cell cycle arrest by ATR-Chkl pathway.The express levels of p21 were up-regulated after treated with combined group for 3 h while up-regulationg of p53 stared from 6 h,indicating that there may exsit a p5 3-independent pathway to upregulate p21.Moreover,cells pretreated with pifithrina(Pft-a),a p53 inhibitor,were sensitive to HCPT rather than combiantionanl group,indicating that p53 was not resistant to combiantionanl group.The effects of combination of Qu and HCPTon apoptosis in MCF7 cells were similar to the combination of BA and HCPT in BGC823 cells.Moreover.cells pretreated with caspase inhibitor would restore the vitality inhibited by Qu and HCPTcombination.Furthermore,the inhibiton of combined group on MCF7 cells xenograft in nude with routine method is lower than that with peritumor administration,but significantly stronger than single-drug group,as well as almost equivalent to high-dose HCPT group.(3)Basically on the structural characteristics of DNA intercalator,we designed and synthesized 5 chrysin derivates.The inhibiting effects of chrysin and its derivatives on the proliferation of cancer cells Hela,BGC823,MCF-7,HepG2,and normal cells HEK-293,were evaluated by MTT assays.5-(2’-amino)phenyl-7,cyclohexanemethylchrysin(Ch-1),a unique chrysin derivate,killed Hela cells in a bluff manner,with lower toxicity in normal cells HEK293.The result of circular dichxoism spectra showed that Ch-1 could intercalate DNA while chrysin had no effects on DNA.Then,we deteced the effects of Ch-1 on cell cycle and apoptosis of Hela cells after treatment for 48 h?by the methods of Annexin-FITC/PI double stained,PI stained,rhodamine 123 and western blot assays.The results showed that 25 |iM Ch-1 arrested Hela cells in G1 phase.Meanwhile,25 μM Ch-1 induced apoptosis by enlarging Bax/Bcl-2 ration and the loss of mitochondrial membrane potential.decreasing the express levels of pro-casape 3/8/9 and Bid,and up-regulating the express levels of p53.Moreover,the cell survival of Ch-1 was restored and the alterations of apoptotic related proteins were also abolished,after pretreated with 125 μM Pft-a.Thus,Ch-1 induced the apoptosis of Hela cells dependening on p53.Otherwise,2.5-10 μM Ch-1 remarkablely enhanced the inhibiton of HCPT on Hela,MCF7 and BGC823 cells,and the synergic target may be Topo Ⅰ because Ch-1 also enhanced the inhibition of HCPT on catalytic activities of Topo Ⅰ.Additional,other chrysin derivates also enhanced the inhibiton of HCPT on MCF7 cells at 10μM,and the best is 5-(2’-amino)phenylchrysin(Ch-3).Above all,we found that low doses of flavones such as baicalein,quercetin,apigenin,luteolin and chrysin,significantly enhanced the inhibition effects of HCPT on cancer cells,and identified the synergic target was Topo Ⅰ,which provide a new combinational model for cancer therapy On the other hand,we synthesized a new DNA intercalator,Ch-1,which inhibited the proliferation of Hela cells in a bluff manner and killed the normal cells in a much higher dose,thereby providing a new anticancer drug for cervical cancer therapy.Otherwise,1-10 μM Ch-1 and other chrysin derivates also enhanced the inhibition effects of HCPT on cancer cells.