The Molecular Mechanism Of Zeylenone On Inducing Cervical Cancer Cells Apoptosis By Inhibiting PI3K/AKT/mTOR And ERK/MAPK Signaling Pathways

Objective:Zeylenone (Zey), which is isolated from Uvaria griandiflora, is a type of polyoxygenated cyclohexene. Previous studies showed that Zey exerts remarkable anti-cancer activity on many types of tumor cells. The inhibitory effect of drug-resistant tumor cells is almost equal to that of sensitive tumor cells. In addition, Zey significantly enhances the anti-tumor activity of doxorubicin. Nude mice inoculated with A549 cells (a type of human lung carcinoma) were administered intraperitoneally with Zey at a dose of 15 mg/kg to determine its inhibitory rate. The tumor-inhibition rate is 75%, which is better than that of the medicine gefitinib; at the same time, the toxicity of Zey is lower. Therefore, Zey deserves further study as a potential anti-tumor drug candidate. However, the molecular mechanism of Zey tumor inhibition is unclear. After screening a variety of solid tumor types, we choose two Zey-sensitive cervical cancer cell lines (HeLa and Caski) for use in further experiments to reveal the molecular mechanism of Zey and lay the foundation for new drug applications.Methods:1. Anti-tumor effect of Zey in vitroIn vitro, we cultured 10 human cancer cell lines, including colon cancer cells (HCT-8), cervical carcinoma cells (HeLa and Caski), hepatoma cells (HepG2), breast cancer cells (MCF-7 and MX-1), oral epidermoid carcinoma (KB), gastric cancer cells (BGC823), prostate cancer (DU-145 and PC-3) and two types of healthy cell lines including immortalized normal prostate stromal cells (WPMY-1) and immortalized normal human bladder epithelial cells (SV-HUC-1). The proliferation inhibition activities on these 12 cell lines of Zey were detected with the MTT method, which was used to calculate their IC50 values (μM).Different doses of Zey were used to treat HeLa and Caski cells; 0,3.27,6.54,13.08, 26.16 and 52.32 μM Zey and 0,1.64,3.27,6.54,13.08 and 3.27 μM were used with Hela and Caski cells, respectively, for different times (12,24,36,72,96 h). These conditions were used to determine the relationship between the inhibition effect of cell proliferation and time or dosage of Zey. A fluorescence microscope was used to observe the influence of Zey on the apoptosis morphology of cells after 24 h; DAPI staining was used to observed the effect on the form of the nucleus; a clonogenic assay tested the effects on tumor cell colony formation; flow cytometry was used to analyze the distribution of tumor cell in cell cycle phases; gelatin zymography was used to detect the protein expression of matrix metalloproteinases (MMP-2 and MMP-9) in carcinoma cells.2. Analysis of the effect of Zey on the cancer cell proteome expression by SILACSILAC culture medium was used to culture human cervical cancer HeLa cells in vitro. Isotope labeling and intervention was carried out after incubation with Zey for 24 h. Subsequently, protein were extracted and subjected to electrophoresis and mass spectrometry. Gene Ontology and Cytoscape software analyses combined with western blotting and qPCR technology were used to compare the changes in the expression of the proteome and phosphorylated proteome in cervical cancer cell HeLa before and after the treatment of Zey.3. Zey induction of tumor cell apoptosisTransmission electron microscopy was used to observe the effects of Zey on tumor cell ultrastructure. AO/EB staining was used to detect the degree of apoptotic tumor cells with Zey treatment; flow cytometry with by JC-1 dye was applied to detect the effects of Zey on tumor cell mitochondrial membrane potential; flow cytometry with an Annexin V-FITC apoptosis kit was used to detect the effects of Zey over time on the proportion of apoptotic tumor cells; DCFH-DA fluorescence was used to detect the impact of Zey on reactive oxygen species; TUNEL in situ apoptosis kits were used to confirm the proportion of apoptotic tumor cells upon Zey treatment.4. Anti-tumor molecular mechanism of Zey in vitroUsing the ligand profiler module in the Discovery Studio 3.5 package, more than 6000 types of targets were used to match pharmacophores in the PharmaDB pharmacophore library as targets of Zey.Using western blot technology, we assessed ① the protein expression in endogenous and exogenous apoptotic pathways with Zey treatment; ②the protein expression in Bcl-2 protein family with Zey treatment; ③ the kinase phosphorylation in the ERK/MAPK pathway; ④ the kinase phosphorylation in PI3K/AKT/mTOR pathway; ⑤ey target proteins in PI3K/AKT/mTOR pathway with the application of Zey and various small-molecule inhibitors.5. Anti-tumor efficacy experiments and molecular mechanism research using cervical cancer xenograft models in nude mice in vivoCcervical cancer HeLa xenograft models were investigated in nude mice. Zey was injected intraperitoneally at the dose of 7.5 mg/kg,15 mg/kg and mice were followed for 14 days to observe the influence on the weight of the human cervical cancer transplantation Tumor volume and tumor weight were used to calculate the inhibitory rate and T/C value; immunohistochemistry and western blotting were used to detect how Zey affects protein expression in signaling pathways of tumor tissue.Results:1. Anti-tumor effects of Zey in vitroWe examined the antitumor effects of Zey in vitro. After treatment of Zey, significant antitumor effects could be observed in 10 cancer cell types and the IC50 values ranged between 3.3-25.7 μM. Particularly, HeLa and Caski exhibited high sensitivity to Zey, with IC50 values of 4.2 and 3.3 μM, respectively. The inhibitory activity of Zey on the normal cells WPMYland SV-HUC-1 was significantly lower than that of Zey on cancer cell lines, with IC50 values of 81.4 and 98.4 μM, respectively. This result indicates the high selectivity of Zey on cancer cells. Experiments showed that Zey inhibits HeLa and Caski in a dose-dependent and time-dependent manner.After incubation with Zey for 24 h, HeLa and Caski cells became smaller and the adhesive abilities were decreased. Cytoskeletal collapse, cytoplasmic condensation and fragmentation, nuclear pyknosis, chromatin condensation and fragmentation could be obviously observed after Zey treatment with DAPI. In the cloning experiments, Zey showed strong inhibitory activity on colony formation. Flow cytometry showed that HeLa and Caski cells were blocked in G0/G1 phase and S phase by Zey treatment, respectively. Gelatinase spectrum testing showed that Zey could inhibit the secretion of MMP-2 and MMP-9 in HeLa and Caski cells.2. SILAC analysis for significantly altered proteins upon Zey exposureTo elucidate the anti-tumor mechanism of Zey, the Zey-regulated protein alterations were identified by SILAC quantitative proteomics. After incubated with Zey for 24 h, 229 proteins showed significantly altered protein levels, including 15 up-regulations and 214 down-regulations. Down-regulation of proteins including 14-3-3 protein theta, 14-3-3 protein zeta/delta, Bcl-2, Mcl-1, Bcl-xL, PARP1, HSP70 1A/1B, HSP90-beta, HSP70-4, HSP beta-1, HSP105, HSP90-alpha, RPL38, RPL17, RPS8, RPL7a and RPL37a, which are involved in energy metabolism, apoptosis and cell cycle arrest.3. Apoptosis assaysOn the basis of SILAC analysis, further investigations were conducted to confirm that Zey could induce apoptosis in cancer cell lines. After incubation with Zey for 24 h, apoptotic assays with transmission electron microscopic observations, AO/EB, TUNEL, JC-1, Annexin V-FITC, DCFH-DA were performed in HeLa and Caski. Obvious apoptosis morphology in HeLa and Caski cells could be observed. Apoptotic bodies were produced, the mitochondrial membrane potential decreased significantly, and a large amount of reactive oxygen species (ROS) was produced. The apoptotic levelsin HeLa and Caski cells were detected to be 83.68% and 78.00%, respectively, indicating that Zey could markedly induce apoptosis in HeLa and Caski cells.4. Anti-tumor molecular mechanism of Zey in vitroStudies of the anti-tumor mechanism of Zey showed that Zey induced tumor apoptosis by influencing endogenous and exogenous apoptosis pathways. Zey activated caspase-8, caspase-9, caspase-7, caspase-3, PARP and Bid and down-regulated FasL, Bcl-2, the Bc1-xL. Zey up-regulated Bax, Fas and FADD and induced the release of mitochondrial Cytochrome c and AIF into the cytoplasm.Pharma DB pharmacophore library of Discovery Studio 3.5 was used to search for potential targets of Zey. We found that MAPK14 is one of the identified targets, indicating that anti-tumor effects of Zey may be associated with the MAPK signal pathway.The MAPK/ERK pathway is an important signaling pathway in transmitting extracellular signals to the nuclei, and it plays a crucial role in cell proliferation, survival differentiation, apoptosis, and metabolism. Western blot results showed that the Zey could inhibit phosphorylation of ERK proteins in the MAPK/ERK signaling pathway; however, it exhibited no inhibition on the phosphorylation of the upstream proteins MEK and RAF. ERK could also be activated by the PI3K/AKT/mTOR signaling pathway. The PI3K pathway could upregulate the MAPK/ERK pathway. To test this, we supposed that Zey inhibited ERK phosphorylation by inhibiting the PI3K/AKT/mTOR signaling pathway. Western blot results showed that Zey could inhibit phosphorylation of PI3K, AKT, or mTOR in PI3K/AKT/mTOR signaling pathway.We used PI3K/AKT/mTOR signaling pathway inhibitors to further study the role of this pathway in the anti-tumor mechanism of Zey. After application of the EGFR inhibitor OSI-744, AKT inhibitor MK-2206, and mTOR inhibitor Rapamycin, we observed no interference with the Zey inhibitory effect on the PI3K/AKT/mTOR signaling pathway proteins or the ERK protein activities. Only application of PI3K inhibitor LY294002 interfered with the inhibitory effect of Zey on EGFR, PI3K, AKT, mTOR and ERK phosphorylation, and with the activation of apoptosis-related proteins Bax, Bad, Bcl-2 and caspase-3. These results provide convincing evidence that Zey may inhibit tumor-cell proliferation and induce tumor-cell apoptosis via inhibition of PI3K activity.5. Investigation of the efficacy and molecular mechanism of Zey in HeLa xenograft modelsIn vivo experiments showed that tumor inhibitory rate of Zey at a concentration of 15 mg/kg in HeLa xenograft nude mice was 83%, which is similar to that of Paclitaxel (15 mg/kg). Moreover, Zey significantly reduced expression of p-PI3K protein in tumor tissue, consistent with in vitro experiments. These results further suggest that the PI3K protein may be a target of Zey.Conclusions:1. Zey has significant inhibitory activity on a wide range of tumor cells. Particular sensitivity to Zey was observed in cervical cancer cells. Zey exhibits good selectivity for tumor cells over healthy cells.2. Zey inhibited proliferation, clone formation and invasive ability of cervical cancer cells in a time-dependent and dose-dependent manner.3. Zey suppressed tumor growth in HeLa xenograft models, with a similar tumor inhibitory rate to that of Paclitaxel.4. Zey induced cell cycle arrest and apoptosis, and produced effects on both exogenous and endogenous signaling pathways.5. The anti-tumor effect of Zey was correlated with levels of proteins involving cellular energy metabolism, cell apoptosis and the cell cycle. This study confirmed that the anti-tumor effect of Zey is closely associated with expression levels in the Bcl-2 protein family and the PI3K/AKT/mTOR and MAPK/ERK signaling pathways.6. Experiments with specific kinase inhibitors indicate that Zey may function through targeting PI3K protein, thereby inhibiting tumor-cell proliferation and inducing cell apoptosis.7. Zey is worthy of further study as a potential PI3K inhibitor.