The Study Of The Effect And Mechanism Of Vinpocetine On Breast Cancer Cells Apoptosis In Vitro And In Vivo

Background and ObjectiveBreast cancer which occurs in lobular or ductal epithelial, is the most frequentmalignancy in the female with a rising morbidity taked over7%~10%of variousmalignancy tumors. Therefore, Many scientists of the world do a lot of research onthe origin, growth, treatment and prognosis of breast cancer.Cell-cycle plays a critical role in the pathogenesis of Breast cancer, and thereforeunderstanding the mechanism of cell cycle control in the breast cancer precursor cellshas profound implications for disease progression and treatment. Today, inhibitiedcellular proliferation and induced apoptosis is the main idea of the treatment of cancer.The adjustment or the abnormal implementation of apoptosis may lead to canceroccurrence, development and treatment prognosis so we observed the influencevinpocetine on cellular apoptosis in breast cancer cells.Vinpocetine has been widely used in the treatment of cerebrovascular disordersand its，therapeutic effects are attributable to improvement of cerebral blood flow andattenuation of neuro-impairment. Besides, vinpocetine relaxes cerebral smoothmuscle cells and hence enhances cerebral blood flow by selectively inhibitingphosphodiesterase (PDE), an enzyme that breaks down intracellular cGMP. Given theevidence that inhibition of PDE suppressed smooth muscle cell growth and survival.It is likely that the PDE-inhibiting by vinpocetine could exert effects on other celltypes, such as cancer cells, however, this likelihood has not yet been examined so far.This study that supported by the National Natural Science Foundation of China(No.30900530), we investigated the therapeutic effects of vinpocetine on breastcancer cells and found that vinpocetine inhibited cell proliferation, survival andmigration in vitro and suppressed cancer progression in vivo. Methods and ResultsPartⅠVinpocetine inhibites proliferation and includes cellularapoptosis in breast cancer cellsMethod:1. Effect of vinpocetine on cell cycle in breast cancer cellsIn the part of this study, cellular viability was measured with Cell Counting AssayKit-8in breast cancer cells, the effect of vinpocetine on cell cycle distribution inbreast cancer cells was determined by flowcytometry. In order to investigate thepossible molecular mechanisms, Western Blot was used to detect the expression ofcyclin D,cyclin E and p16INK4a.Result：1) Breast cancer cells were inhibitied cellular proliferation by dose-dependentvinpocetine detected by CCK-8assay.2) Treatment of breast cancer cells with vinpocetine was found to cause a remarkabledose-dependent accumulation of cells in G1phase followed by apoptosis,suggesting a block in the step from G1phase to S-phase.3) We grabed the results that vinpocetine-induced G1phase arrest was associated withdecreased expression of cyclin D, cyclin E and aggrandized expression of p16.2. Effect of Vinpocetine on apoptosis in breast cancer cellsIn this part of our study, cellular apoptosis was determined with annexinV-FITC/PI or Hoechst33258staining using flow cytometry and confocal system asdescribed previously, and The change of mitochondrial membrane potential, asmeasured by JC-1fluorescence staining, indicates the occurrence of apoptosis. Wefurther detected the modulated caspases activation, cytochrome C release, and theexpression of Bcl-2or Bax.1) Incubation with vinpocetine or vehicle for24h, breast cancer cells apoptosiswas analyzed by annexin V-FITC/PI double staining. We crabbed that the higerearly apoptosis rate and the higer late apoptosis rate.2) Morphological changes of apoptosis were determined by Hoechst33258staining. Hoechst33258staining revealed cell shrinkage, chromatin condensation in thenucleus with vinpocetine treated.3) JC-1stainning was employed to detect Δψm. Breast cancer cells were treatedwith vinpocetine, the intensity of green fluorescence emitted by JC-1dye wasremarkably enhanced with an increased ratio of green/red intensity, indicatingthat the mitochondrial membrane potential was decreased and mitochondrialmembrane permeability was altered.4) Vinpocetine induces breast cancer cells apoptosis by reduces Bcl-2and enhancesBax protein expression, decreases mitochondrial membrane potential, potentiatescytochrome C release and modulates caspases activation.3. Effect of Vinpocetine on breast cancer cells migrationIn this part, cell migration was analyzed using a transwell system according tothe manufacturer’s instructions. The results demonstrated that vinpocetine inhibitedBreast cancer cells migration in a concentration-dependent manner.4. Effect of Vinpocetine on Akt/STAT3/MAPKs signalingTo figure out the underlying molecular signaling pathways by which vinpocetineexerted anti-tumor effects, we examined the activity of Akt/STAT3pathway, whichhas been confirmed to be involved in the progression of many malignancies includingbreast cancer. Results of western blot assays showed that vinpocetine significantlyattenuated the phosphorylation of Akt and downstream protein STAT3. We alsoexamined the effects of vinpocetine on the activity of MAP kinases, which were alsofrequently reported to mediate tumorigenesis. In contrast, vinpocetine did notmarkedly influence the activity of Erk1/2, JNK and p38.Summary:1) Vinpocetine induces a dose-dependent apoptosis in breast cancer cells.2) Vinpocetine induces breast cancer cellulars apoptosis by reduces Bcl-2andenhances Bax protein expression, decreases mitochondrial membrane potential,potentiates cytochrome C release and modulates caspases activation.3) Vinpocetine inhibites Breast cancer cells migration in a concentration-dependent manner.4) Vinpocetine inhibites proliferation and induced apoptosis in breast cancer cellsby attenuated Akt/STAT3signaling.PartⅡ Vinpocetine suppresses growth of human breast tumorxenograft in nude miceMethod:To evaluate whether vinpocetine inhibits tumor growth in vivo, we establishedthe xenograft model of human breast cancer in nude mice and treated the mice withvinpocetine. Human breast tumor xenografts were prepared by injectingMDA-MB-231cells or MCF-7cells subcutaneously into flanks of the mice, and weretreated with vinpocetine or vehicle control.Result:1). We obtained that Vinpocetine suppressed the growth of human breast tumor invivo.2). To investigate the changes of cell apoptosis in tumour tissues，tumour biopsieswere stained with TUNEL to analyze cellular apoptosis, and observed withfluorescence microscope. We obtained that intraperitoneal (i.p.) administration of5mg/kg vinpocetine induced apoptosis of human breast cancer.3). To investigate the changes of p-Stat3or p-Akt expression in tumour tissues,tumourbiopsies were stained with Immunofluorescence staining of p-Stat3or p-Akt, andobserved with fluorescence microscope. We obtained that vinpocetineattenuates Akt/STAT3signaling of human breast tumor xenograft in nude mice.Summary:1).Vinpocetine suppresses the growth of human breast tumor and induced apoptosis invivo.2).Vinpocetine attenuates Akt/STAT3signaling in vivo.Conclusion:1). Vinpocetine induces G1phase arrest and inhibities breast cancer cells growth. 2). Vinpocetine includes cellular apoptosis and inhibities cell migration in breastcancer cells.3). Vinpocetine suppresses the growth of human breast tumor and induced apoptosisin vivo.4). Vinpocetine inhibites proliferation and induces apoptosis in breast cancer cellsby attenuated Akt/STAT3signaling but has no influence on MAPKs.