Induction Of Apoptosis And Molecular Mechanisms After Panax Quinquefolium L Effective Parts (PQEP) Treatment On Tumor Cell

Panax quinquefolium L is one of those valuable medicines. It has been usedfor many years in china, for its wide spectrum of medicinal effects such as tonic,immunomodulatory and anti-aging activities. Many of its medicinal effects areattributed to the triterpene glycosides known as ginsinosides. It is noteworthy thatthe undesirable side effects, if any, are relatively mild and undetectable. Chemicalanalysis of ginseng revealed the presence of many ingredients;many of thesecompounds are responsible for the wide range of medicinal effects of Panaxquinquefolium L, whose cumulative effect increases their efficacy in curing thediseases. In previous study, ginsinoside Rg3, Rh2, 20(S)-Protopanaxatriol,20(S)-protopanaxadiol have been claimed to possess anti-tumor effect. Therefore,in our study, Panax quinquefolium' effective part (PQEP) including mainly theingredients such as Rg3, Rh2, PPd and PPT, were extracted from the Panaxquinquefolium leaves and combined to enhance the anti-tumor activity throughapoptotic pathways. Moreover, their molecular mechanisms were studied bymorphological and biochemical procedure.Purposes: To evaluate the anti-proliferative activity of PQEP in vivo and in vitroand to elucidate potential molecular mechanismsMethods:This study consists of three parts: 1. Animal tumor inhibition experiments invivo and PQEP–induced apoptosis in S180 tumor in mice;2. S180 tumor cellinhibition experiments in vitro;3. K562 tumor cell inhibition experiments in vitroand the study of molecular mechanisms.1. Animal tumor inhibition experiments in vivoTumor inhibition experiment in ICR mouse: mice were injected with S180,cells (2×106cells/animal) in 0.2 ml subcutaneously to the right anterior limb. Thenext day, PQEP (48mg/kg, 24mg/kg, and 12mg/kg) suspended in 0.5%carboxymethyl cellulose (CMC) was taken orally once a day. Tumor weight wasmeasured after 10-days and calculated the inhibition rate. Meanwhile, a portion ofthe tumor was cut into small pieces, fixed in 4% neutral buffered formalin forhaematoxylin and eosin (HE) staining and immuno-histochemical observation ofProliferating cell nuclear antigen (PCNA). Moreover, flow cytometry and agarosegel electrophoresis were applied to detect the change of apoptotic rate, thealteration of cell cycle and DNA fragment.2. S180 tumor cell inhibition experiments in vitroThe inhibition of PQEP on S180 tumor cells was determined by MTT [3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazo-lium bromide] assay. Themorphological changes of S180 cells treated with PQEP were observed by lightmicroscope. The effects of PQEP on the cell cycle distribution and apoptosis weredetermined using propidium iodide staining and through flow cytometry. DNAfragmentation of S180 cells was assayed by agarose gel electrophoresis. The effectsof PQEP on the expression of bcl-2 and bax mRNA and protein were detected byreal time RT-PCR and immuno-histochemical methods.3. Human chronic myelogenous leukemia (CML) K562 tumor cell inhibitionexperiments in vitroThe inhibition of PQEP on K562 tumor cells was determined by MTT assay.The morphological changes of K562 cells treated with PQEP were observed bylight microscope and fluorescence microscope. The effects of PQEP on the cellcycle distribution and apoptosis were determined using propidium iodide stainingand through flow cytometry. DNA fragmentation of K562 cells was assayed byagarose gel electrophoresis. The levels of cyclin D1, P21, P27 associated with cellcycle were measured by reverse transcription-polymerase chain reaction(RT-PCR). The effects of PQEP on the expression of bcl-2 and bax mRNA andprotein were detected by real time RT-PCR and immuno-histochemical methods.In addition, changes in mitochondrial membrane potential in K562 cells afterPQEP treatment were assayed using Rhodamine 123 and through flow cytometry.The intracellular calcium ion were evaluated by laser confuse micrograph.Furthermore, Flow cytometry and immuno-histochemical method were used totest the changes of Fas gene expression. The level of PARP associated withapoptosis was measured by western blot.Results:1. Tumor inhibition experiments in vivoPQEP had a remarkable inhibitory effect on the growth of S180 afteradministered to the tumor-bearing mice at the dose of 48mg/kg, 24mg/kg and12mg/kg. We got almost the tumor inhibitory rate around 30%~50% after threeindependent experiments. Histopathology of tumor from transplanted S180 tumormice indicated the tumor cells of untreated mice grew vigorously;however, thetumor cells from the treated groups had clear nucleus pycnosis and necrosis areasin different degree. Meanwhile, the PCNAIL was decreased after PQEP treatment.Furthermore, the results from flow cytometry analysis showed the presence of asub-Gl peak characteristic of potential apoptotic cells, meanwhile, the typicalladder bar were observed when mice were treated with different concentrationPQEP, which indicated PQEP can predominantly induce apoptosis of S180 cells.2. S180 tumor cell inhibition experiments in vitroPQEP could retard the proliferation of S180 cell dramatically. The IC50 valueat 48h was 58.83μg/ml. The typical apoptosis morphology was identified underlight microscope. Analysis of S180 cell cycle was performed through flowcytometry. In the presence of different concentration of PQEP, the cells wereblocked at G0/G1 phase, and the effect was strengthened with the increase of theconcentration. The results indicated an alteration and/or redistribution in cell cycle,which occurred during the cell proliferation by the addition of PQEP. The resultsfrom flow cytometry analysis showed the presence of a sub-Gl peak characteristicof potential apoptotic cells. Indeed, the number of cells with subdiploid DNAcontent increased with concentration to reach 20.69% at 30μg/ml. DNA fragmentwas increased in a dose-dependence manner. The typical ladder bar were observedwhen S180 cells were treated with 3~30μg/ml PQEP. Furthermore, the real timeRT-PCR and immuno-histochemical results suggested that PQEP coulddown-regulation the expression of bcl-2, while up-regulation the expression ofbax.3. K562 tumor cell inhibition experiments in vitroThe inhibitory effects of PQEP on the proliferation of K562 cells wereobserved in a dose-and time -dependent manner. The ability of the inhibition toK562 cells growth was positively correlated with the concentration and thetreatment time of PQEP. Morphological changes of PQEP-treated K562 cells wereexamined using a light and fluorescent microscopy. In the PQEP-treated cells,characteristics of apoptosis (namely, cell detachment, membrane blebbing, cellshrinkage with a condensed cytoplasm, and vesicle formation) appeared at 48hafter the addition of 30μg/ml PQEP. Apoptotic apex was observed by flowcytometry after incubation with PQEP. Apoptotic ratios of K562 cells aftertreatment with PQEP 3, 10, 30μg/ml for 48h were separately 11.08%, 20.78%,30.39%, significantly different comparing with the control. The results showedthat PQEP induced apoptosis of K562 cells in a dose-dependent relationship. Toinvestigate possible mechanisms whereby PQEP interferes with cell cycleprogression. We evaluated the cellular content of G0/G1 phase related cyclin D1,P21 and P27, They showed lower level of cyclinD1 but higher expression of P21.All are parallel with the results of cell cycle showed by flow cytometry. In ourexperiment, we used the Rhodamine 123 to monitor changes in △ψmtt induced byPQEP. K562 cells treated with PQEP exhibited a significant reduction in cellularuptake of the fluorochrome. Fas expression was lower in K562 cell, PQEP canclearly increase Fas expression of K562 cell, especially in the middle and largedosage groups, which had remarkable difference, compared with the control group.The content of calcium ion in the cells increased with the addition of PQEP, theaverage fluorescent light strength of calcium ion in K562 cell after beingprocessed by PQEP for 24h had an obvious positive correlation with the cellularapoptosis rate. Our results clearly showed a decrease in the PARP protein contentthat appeared after treatment with 10 and 30μg/ml PQEP for 24h, and the level ofbcl-2 was also decreased with an increase of bax in contrast.Conclusions:The above results suggested PQEP had significant anti-tumor activitywhatever it was in vivo or in vitro. PQEP can decrease the expression of CyclinD1and increase the expression of P21, which is related to G0/G1 arrest of the cellcycle. Meanwhile, PQEP-induced apoptosis involved in mitochondria-mediatedpathway, activated caspase-3 to trigger apoptosis, in turn, increased levels of bax,decreased of bcl-2 also enhanced apoptosis. Furthermore, PQEP can upregulatethe expression of Fas antigen, which showed apoptosis involved in deathreceptor-mediated pathway.