| Hepatocellular carcinoma (HCC) is the fifth most common malignant tumor andthe third most common cause of cancer-related death throughout the world, whichresults in approximately one million deaths worldwide annually. To date, the onlyeffective approaches for HCC are resection or liver transplantation, unresectable HCCare treated with radiotherapy or chemotherapy. However, chemotherapy is hamperedby its dose-limiting toxic side effects. Therefore, it is an urgent task to find out newstrategies for the treatment of HCC.Flavonoids are polyphenolic compounds present in vegetables, fruit, grains andbeverages of plant origin which have antioxidant, antimutagenic and antiproliferativeproperties. Thus, flavonoids have a potential protective role in various chronicdiseases, including common cancers, for their high potency and low toxicity.Liquiritigenin (LQ) is a kind of flavonoids with a polyphenolic structure existing in Radix glycyrrhizae, a traditional Chinese food and herbal medicine widely used forthousands of years. Our previous studies have found that LQ could significantlyinhibit the growth of human hepatocellular carcinoma SMMC-7721cells and humancervical carcinoma HeLa cells in a dose-and time-dependent manner and induceapoptosis in vitro. Furthermore, our previous researches have also demonstrated thatLQ could effectively inhibit the growth of hepatoma22tumors in mice and humancervical carcinoma HeLa cells xenografts in nude mice in vivo.In order to further explore the anti-cancer effect of LQ, this study investigatedthat LQ in combination with DOX inhibited the proliferation and induced apoptosisof human hepatocellular carcinoma HepG2cells and tumor xenografts via themitochondrial apoptotic pathway. Section1Study of liquiritigenin combined with doxorubicinon cell viability, apoptosis induction in humanhepatocellular carcinoma HepG2cells.Objective: This study investigated the effects of liquiritigenin (LQ) combined withdoxorubicin (DOX) on cell viability and apoptosis induction in human hepatocellularcarcinoma HepG2cells.Methods: After DOX (1,4μM) alone and LQ (100,200,300μM) in combinationwith DOX (1μM) treatment on HepG2cells for48h, the cell viability effect wasmeasured by the MTT-colorimetric assay; the apoptotic morphological alterations inthe nuclear chromatin of HepG2cells were observed by Hoechst33258staining; thepatterns of cell apoptosis were analyzed by flow cytometry with Annexin V and PIdouble staining.Results: The HepG2cell viability of the LQ (L, M, H) combined with DOX (L)groups (79.16%,56.91%,36.91%) was lower than the DOX (L)-treated group(90.43%)(p<0.01) in a dose-dependent manner; the cell viability of the LQ (H)combined with DOX (L) group (36.91%) was lower than the DOX (H)-treated group(48.39%). The characteristic features of apoptotic HepG2cells with a bright-bluefluorescent chromatin condensation, reduction of cell volume and nuclearfragmentation in a dose-dependent manner were observed in the LQ (L, M, H)combined with DOX (L) groups, while almost no apoptotic cells were observed in theDOX (L)-treated group. The LQ (H) combined with DOX (L) group had moreapoptotic cells than the DOX (H)-treated group. The HepG2cells apoptosis rates of the LQ (L, M, H) combined with DOX (L) groups (10.55%、22.99%、30.69%) werehigher than the DOX (L)-treated group (5.04%)(p<0.01) in a dose-dependent manner;the apoptosis rate of the LQ (H) combined with DOX (L) group (30.69%) was higherthan the DOX (H)-treated group (25.71%).Conclusion: LQ combined with DOX effectively inhibited the proliferation andinduced apoptosis of HepG2cells in a dose-dependent manner. Section2Study on apoptotic mechanisms of humanhepatocellular carcinoma HepG2cells by liquiritigenincombined with doxorubicinObjective: The aim of our study was to explore the mechanisms of LQ combinedwith DOX-induced apoptosis in human hepatocellular carcinoma HepG2cells.Methods: After DOX (1μM) alone or combined with LQ (100,200,300μM)treatment on HepG2cells for6,12h, intracellular reactive oxygen species (ROS) wasdetected by reactive oxygen species assay kit. After DOX (1μM) alone or combinedwith LQ (100,200,300μM) treatment on HepG2cells for48h, mitochondrialmembrane potential (△ψm) was detected by mitochondrial membrane potential assaykit with JC-1; voltage dependent anion channels (VDAC), cytochrome c, Caspase-9,Caspase-3, Bcl-2and Bax protein expression was analyzed by Western blot analysis.Results: The HepG2cells of the LQ (L, M, H) combined with DOX (L) groupsproduced more ROS than the DOX (L)-treated group (p<0.01) in a time-and dose-dependent manner; the LQ (H) combined with DOX (L) group produced more ROSthan the positive control (Rosup) group. The VDAC protein expression of HepG2cells in the LQ (M, H) combined with DOX (L) groups was more than the DOX (L)-treated group (p<0.01) in a dose-dependent manner; the mitochondria membranepotential (△ψm) in the LQ (L, M, H) combined with DOX (L) groups was lower thanthe DOX (L)-treated group (p<0.05) in a dose-dependent manner; the LQ (L, M, H)combined with DOX (L) groups had more cytosolic cytochrome c than the DOX (L)-treated group (p<0.01) in a dose-dependent manner. The HepG2cells of the LQ (L, M, H) combined with DOX (L) groups had less Pro-Caspase-9,-3protein expression(p<0.01, p<0.05) and more Cleaved Caspase-9,-3protein expression than the DOX(L)-treated group in a dose-dependent manner; less Bcl-2and more Bax proteinexpression in the LQ (L, M, H) combined with DOX (L) groups resulted in higherBax/Bcl-2ratios than the DOX (L)-treated group (p<0.01) in a dose-dependentmanner.Conclusion: LQ combined with DOX could produce ROS to induce apoptosis inHepG2cells via the mitochondrial apoptotic pathway. Section3Study on the anti-tumor effects of liquiritigenincombined with doxorubicin in human hepatocellularcarcinoma HepG2cells tumor xenografts in nude miceObjective: To investigate the effects of LQ combined with DOX on inhibiting thegrowth and inducing apoptosis of human hepatocellular carcinoma HepG2cellstumor xenografts in nude mice.Methods: Human hepatocellular carcinoma HepG2cells tumor xenografts model innude mice was established.40mice were randomly divided into five groups witheight mice per group as follows: control group, the DOX (1mg/kg)-treated group, theLQ (5,10,20mg/kg) combined with DOX (1mg/kg) groups. Body weights weremeasured and tumor volumes were assessed every two days throughout theexperiment. Mice were sacrificed after24days, tumor weights were measured.Tumor tissues were fixed in4%paraformaldehyde solution and embedded in paraffin,then the histological characteristics were observed by HE staining; the apoptotic cellswere detected using TUNEL assays. VDAC, cytosolic cytochrome c, Caspase-9,Caspase-3, Bcl-2and Bax protein expression in HepG2tumor tissues was analysedby Western blot analysis.Results: Human hepatocellular carcinoma HepG2cells tumor xenografts model innude mice was established successfully. The body weights of all the groups wereincreased gradually with no significant difference among all the groups. The LQ (L,M, H) combined with DOX groups had smaller HepG2tumors, lighter tumor weights(p<0.05), smaller tumor volumes (p<0.01) and the growth of tumor volumes more slowly than the DOX-treated group in a dose-dependent manner. The LQ (L, M, H)combined with DOX groups had larger necrosis areas, more TUNEL positiveapoptotic cells, higher apoptotic index (AI)(p<0.01) than the DOX-treated group in adose-dependent manner. The VDAC protein expression of HepG2tumors in the LQ(M, H) combined with DOX groups was more than the DOX-treated group (p<0.01)in a dose-dependent manner; the LQ (L, M, H) combined with DOX groups had morecytosolic cytochrome c than the DOX-treated group (p<0.05) in a dose-dependentmanner. The HepG2tumors of the LQ (L, M, H) combined with DOX groups had lessPro-Caspase-9,-3protein expression (p<0.01, p<0.05) and more Cleaved Caspase-9,-3protein expression than the DOX-treated group in a dose-dependent manner; lessBcl-2and more Bax protein expression in the LQ (L, M, H) combined with DOXgroups resulted in higher Bax/Bcl-2ratios than the DOX-treated group (p<0.05) in adose-dependent manner.Conclusion: LQ combined with DOX effectively inhibited the growth and inducedapoptosis of HepG2tumors via the mitochondrial apoptotic pathway in nude mice. |
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