Study On Anti-cancer Effects Of Sanguinarine And The Underlying Mechanisms

Purpose:The aim of study was to investigate the ant-cancer activities of sanguinarine incervical cancer and its possible mechanisms via a series of in vitro and in vivo studies.Methods:In vitro studies, two human cervical cancer cell lines, HeLa and Siha, with thefollowing methods were employed, in some cases, normal cell lines GM and ECV304were used as control:1) Cell morphology was observed by inverted microscope and livecell imaging system;2) Cell viability was examined by MTT assay;3) Colonyformation assay was also used to study the cell growth inhibition of sanguinarine;4)Scratch healing assay and the trans-well assay were employed to study the influence ofsanguinarine on the cell migration and invasion;5) Flow cytometry was applied toanalyze cell cycle progression and apoptosis induction;6) Single cell gel electrophoresis(comet assay) was used to detect DNA damage;7) Apoptosis and necrosis weremeasured by TUNEL and Annexin V-FITC cell apoptosis kits;8) ROS content andmitochondrial membrane potential was determined by commercial kits;9) Alteration ofProtein expression level was detected by Western blot assay;10) Autophagy wasexamined by MDC (monodansylcadaverin) staining;11) Autophagy and apoptosisultrastructure changes of cells were observed by transmission electron microscope(TEM).In vivo studies, a transplanted tumor model by injecting HeLa cells intosubcutaneous tissue of Babl/c mice was established. Mice were randomly assigned into4groups by intraperitoneal injection:1) saline solution as negative control;2)5mg/kgcisplatin as positive control (1/week);3)1.25mg/kg injection of sanguinarine (1/3days)and4)2.5mg/kg injection of sanguinarine (1/3days). The period time of therapies was28days, the tumor volume was measured once every other day upto another28days following sanguinarine treatment. At the end of experiments, tumors were carefullyremoved and weighted. Microvascular density in tumor tissues was measured by aninverted microscope and CD34immunostaining. The expression levels of Bcl-2, Baxand VEGF tumor tissues were detected by Western blot and the expression levels ofVEGF, EGFR and Ki67, by immunohistochemical assay, in tumor tissues. Changes ofcell ultrastructure related to autophagy and apoptosis in tumors were observed by TEM.Results:1) A dose-and time-dependent inhibition of cell proliferation by sanguinarine wasobserved in both cell lines. An IC50value for24h treatment of sanguinarine in HeLa andSiha cells was2.62±0.21μmol/L and3.07±0.23μmol/L, respectively, indicatingslightly more sensitivity in HeLa cells (P <0.05). While, sanguinarine showed lesstoxicity in normal GM and ECV304cells with an IC50value of4.93±0.22μmol/L and5.78±0.18μmol/L, respectively.2) After treatment of0,1,2and3μmol/L sanguinarine for24h, the proportion ofG2/M phase in HeLa cells was (25.21±0.66)%,(29.97±0.84)%,(37.31±0.93)%and(57.72±0.13)%, respectively. Similar a dose-dependent arrest of G2/M phase wasobserved in Siha cells after sanguinarine treatment. Consistant with cell cycle arrest,sanguinarine caused a significant down-regulation of CyclinB1, CyclinE and CDC2protein expression and an up-regulation of P21and P27protein expression.3) With24h treatment of sanguinarine, HeLa and Siha cells showed typicalapoptotic morphologic changes, including chromatin condensation and DNA fragment.Comet assay evidenced that sanguinarine increased the mean tail length and thepercentage of cells with comet tails. Sanguinarine decreased Bcl-2expression andincreased Bax expression via a dose-response manner, and consequently the ratio of Baxto Bcl-2was significantly increased (P<0.05). The expression of procaspase-3wasdecreased and AIF and Cleaved PARP were increased in HeLa and Siha cells treatedwith sanguinarine (P<0.05). The ROS content in HeLa cells treated with1μmol/Lsanguinarine markedly increased in0.5h (29.63±1.28)%compared with0h (38.4±2.90)%and reached peak in4h (58.37±6.03)%(P<0.01) following exposure tosanguinarine. Similar results were observed for ROS generation in Siha cells. On theother hand, mitochondrial transmembrane potential was decreased and the expression ofcytochrome c protein was increased in both of cell lines after sanguinarine treatment. 4) As for migration and invasion, sanguinarine significantly decreased theadherence rate and inhibited the ability to cover “cell wound” by scratch in both of celllines via a time-dependent fashion. Tran-well assay showed that HeLa cell number aftertreatment with sanguinarine at0,0.5and0.75mol/L was156±9.85,108±13.23and74.67±7.02, respectively (P <0.01). Similar observation was obtained in Siha cells withsanguinarine treatment. Additionally, a significant dose-dependent alteration of someproteins which are related to cell migration and invasion was found in both of cell linestreated with sanguinarine, i.e., a decrease level of VEGF, EGFR, PTEN, NF-κB,-catenin,-catenin and γ-catenin expression and an increased expression of E-cadherinprotein (P<0.05).5) TEM observation indicated that autophagosomes and apoptotic bodies inducedby sanguinarine in both of cell lines. However, sanguinarine-induced autophagy wasreversed by emplying a specific autophagy inhibitor,3-methylademine (3-MA). Aquantitative method of MDC staining releaved that the cell number of autophagy was149±22.65and412.67±16.80in HeLa cells as well as138±17.47and371±20.84inSiha cells after4h treatment with1and2mol/L sanguinarine (P<0.01). Sanguinarinecaused a reduced expression of Beclin1protein. In addition, a decrease of LC3-I proteincomparing with an increase of LC3-II protein were detectable after treatment withsanguinarine.6) A significant inhibition of tumor growth and volume in transplanted tumor micewere observed after therapy with sanguinarine via an intraperitoneal injection. At theend of experiment, the inhibition rate of tumor growth was29.40%,39.96%and45.16%in cisplatin,1.25mg/kg and2.5mg/kg sanguinarine, compared with that inun-treated control mice. Microvascular density in tumor tissues as evidenced by CD34immunostaining was77.33±7.04,25.00±4.90,28.33±1.70and15.67±2.05inun-treated controls, cisplatin,1.25mg/kg and2.5mg/kg sanguinarine, respectively(P<0.01). TEM observation also showed a significant change of apoptosis andautophagy in tumor tissues after therapy with sanguinarine. On the other hand,sanguinarine inhibited tumor angiogenesis possibly via down-regulating the expressionof VEGF, EGFR and NF-κB.Conclusion:In summary, a significant inhibitory proliferation in both of human cervical cancer cell lines, HeLa and Siha, was observed after treatment with sanguinarine in a dose-andtime-dependent manner. Sanguinarine-caused arrest of G2/M phase was accompaniedwith a down-regulation of CyclinB1, CyclinE, and CDC2protein and a up-regulation ofp21and p27expression. The occurrence of apoptosis and autophagy after sanguinarinetreatment was confirmed by a few methods, the underlying mechanisms may beinvolved in an increase of Bax/Bcl-2, an alteration of LC3II/LC3I expression, anactivation of caspase-3and MAPK pathway. Anti-cancer growth in vivo bysanguinarine may be related to an inhibition of tumor angiogenesis and an induction ofapoptosis and autophagy by down-regulating the expression of VEGF, EGFR andNF-κB. These novel findings indicate that sanguinarine inhibites cervical cancer growthin vitro and in vivo by multimechanisms, which suggesting that sanguinarine may serveas a potential therapeutic agent for cervical cancer.