Matrine Inhibited Proliferation In Gastric Cancer Cells

Cancer became a major public health problem in China and all other countries, its incidence and mortality had kept increasing since 1970s. According to the statistic data for the year 2005, the incidence and the mortality of gastric cancer was 54.5 per 100,000 person-year and 42.1 per 100,000 person-year respectively, positioning it to a third commonly occurring cancer in China. Multiple therapies were co-operated to gastric cancer. However the number of incidence and mortality of gastric cancer was very close, suggesting that the current therapy strategies were still far from satisfaction. Many experimental and clinic studies aimed to find new effective anticancer drugs. It was both very promising and of great importance to explore new anticancer targets from the components of Chinese traditional herbs, which were Chinese characteristic resources and had a long history in cancer treatment.Matrine was a main alkaloid component in Sophora flavescens ait, a traditional Chinese drug. Belonging to Quinolizidine Alkaloids family, it had been reported for its anticancer activity in vivo and in vitro. Matrine could inhibit the growth of many human cancer cells, including hepatic cancer cell SMMC-7721, gastric adenocarcinoma cells MKN45 and SGC7901. Some studies indicated that increased pro-apoptotic molecules of Bcl-2 family and upregulated cell cycleprotein E2F-1 were involved in Matrine induced apoptosis of cancer cells. Matrine could also promote K-562 cells differentiation and induce apoptosis in K562/vin and K562/dox, which were multidrug resistant cell lines. In an animal experiment, Matrine and 5-fluorouracil showed synergistic effects on tumor growth. Other studies revealed that the anticancer effect of Matrine was associated with the inhibition of COX-2 and the up-regulated expression of topoisomerase I . However, its exact anticancer mechanism was still unclear. Based on our previous results of Matrine in HT29 cells, we tried to explore whether Matrine inhibited tumor growth via modulating protein translation and related cell signal transduction.Translation was the final step in the flow of the genetic information. It included three distinct phases: initiation, elongation and termination. Initiation was the rate-limiting step in mRNA translation and served as the most common target of translation regulation, wherein the number, the activity and the integrality of eukaryotic initiation factors (eIFs) were very crucial. The chief event in initiation was the formation of the translation initiation complex eIF4F, which was a heterotrimeric protein complex and could bind the 7-methyl guanosine triphosphate (m~7GTP) that capped the 5'ends of all nuclear-encoded eukaryotic mRNAs. To form eIF4F complex, eIF4E interacted with eIF4G, a scaffold protein, and in turn binded to eIF4A, an ATP-dependent bi-directional RNA helicase. Two typical pathways were involved in eIF4E regulation, which were mitogen-activated protein kinase (MAPK) and Akt/mammalian target of rapamycin (mTOR). For example the well-known translation inhibitor Rapamicin was a specific inhibitor of mTOR kinase. Furthermore, a family of proteins named 4E-binding proteins (4E-BPs) acted as competitive inhibitors of the eIF4E-eIF4G interaction. When being dephosphorylated, 4E-BPs competed with eIF4G and formed a nonfunctional complex with eIF4E. While upon phosphorylation, 4E-BPs dissociated from the complex and allowed eIF4E to activate cap-dependent translation.Protein synthesis in mammalian cells was subject to rapid regulation after exposure of cells to a wide variety of growth-promoting, growth-inhibitory and stress-inducing conditions. Aberrant control of translation could result in cell transformation, and changes in the expression of key initiation factors or the signaling pathways that regulated them were often observed in tumors. The key element of translation initiation eIF4E, which was regarded as an oncogene, had received abundant attentions due to its effect on tumorgenesis. In addition to modulating global translation rates, eIF4E could also enhance the translation of some key malignancy-related proteins, such as oncoproteins and growth factors. Therefore overexpression of eIF4E had the potential to promote proliferation, to protect cells against apoptosis, and even to play a role in transformation and tumorigenesis. Many studies demonstrated that the expression of eIF4E was lower in normal tissue than that of in a series of malignant tumors, such as lung, breast, gastrointestinal cancers. The level of eIF4E was correlated with pathologic grades and clinical outcomes of those tumors.Ras was a small GTP-binding protein, which was the common upstream molecule of several signaling pathways including Raf/MEK/ERK and PI3K/Akt. Ras proteins showed varying abilities to activate the Raf/MEK/ERK. and PI3K/Akt cascades. The mutation of ras was an early event in tumorigenesis. It had been reported that approximately 30% of all human cancers exhibited ras mutations. In gastric carcinoma, the incidence of ras gene mutation was roughly 2.8% to 18.5%. Most mutation forms of ras gene identified in tumors resulted in constitutive activation of Ras and uncontrolled cell proliferation. Some reported that Ras mediated elF4E-induced malignant transformation. And the anti-apoptotic function of Ras relied on the elF4E-dependent protein synthesis.In our previous studies, up-regulations of some elFs and MAPKs mRNAs were observed in Matrine-treating HT29 cells using Affymetrix genechips. The correspondent proteins encoded by those genes were related to the function of eIF4E. The interesting phenomena provided a clue and suggested Matrine might interfere with the translation initiation. However, some important information was unknown such as the status of eIF4E phosphorylation and the activity of related proteins and kinases. We studied the interesting phenomena through three different parts.. In the first part, we measured the phosphorylation of eIFs in Matrine treating MKN45 cells; then we focused on the activation of the associated kinases and phosphatases; finally, the role of the upstream regulator Ras in the anticancer mechanism of Matrine was discussed by transfecting gastric cell AGS with an eukaryotic plasmid carrying wild H-ras (Ras-pcDNA3.1(-))Part IMatrine probably inhibited proliferation via dephosphorylation of translation initiation factors eIF4E and 4E-BP1 in gastric MNK45 cellsWe observed that Matrine could inhibit MKN45 cells proliferation in a time and dose dependent manner. The IC50 value was 0.53 mg/ml. Additionally, cell apoptosis and cell cycle were also evaluated by flow cytometry. The apoptosis rate of the Matrine-treated cells was in a dose dependent style. At the dose of 0.50 mg/ml, the apoptosis rate notably rised to 26.88%. Except for 0.50 mg/ml, the percentage of G0/G1 phase elevated in MKN45 cells. It suggested Matrine might induce G0/G1 arrest.The regulation of eIF4E and 4E-BP1 played a critical role on translation control that had an effect on cell proliferation and apoptosis. To this end, weanalyzed the activity of eIF4E in Matrine treating MKN45 cells. The results demonstrated that eIF4E were dephosphorylated in a time and dose dependent manner after treating with Matrine. Similar results were observed in eIF4E binding protein 4E-BP1. Additionally, we found an increasing level of 4E-BP1 after treating with Matrine. However, no change was observed in the phosphorylation of eIF2 α , which was important in the stability of eIF2-GDP-eIF2B complex. Together, these data showed that Matrine decreased the phosphorylation of 4E-BP1 and eIF4E.Part IIMatrine attenuated the activity of Erk1/2 MAPK to inhibit translation initiation in MNK45 cellsThere were two typical pathways, mitogen-activated protein kinase (MAPK) and Akt/mammalian target of rapamycin (mTOR), involved in eIF4E regulation. Interestingly, each dosage of Matrine could inactivate Erkl/2 effectively, which was accordant to the change of eIF4E and 4E-BP1 phosphorylation. This result indicated that Matrine could attenuate the activity of Erkl/2 MAPK to dephosphorylate eIF4E and 4E-BP1 in MNK45 cells. However, Matrine had no obvious effect on p38MAPK. Meanwhile, the changes of JNK MAPK and Akt/mTOR were not parallel with the dephosphorylation of eIF4E and 4E-BP1. We also observed that inhibition of PP2A activity by microcystin LR enhanced the dephosphorylation of 4E-BP1 by Matrine, which suggested that PP2A might involve in this process.Part IIIEffect of Matrine on growth of Ras-ectopic-expression gastric cancer cell AGSConstitutive activation of Ras pathways played a critical role ingastrointestinal cancer development and maintenance. However there was no efficient Ras inhibitor used for clinic therapy. To this end, we measured the growth inhibition of Ras-ectopic-expression cells by Matrine treatment. A wild H-ras sequence (571bp, BC095471) was cloned from Hela cells by RT-PCR and an eukaryotic expression plasmid H-Ras-pcDNA3.1(-) was reconstructed. Then the reconstructed plasmid and pcDNA3.1(-) were used for cell transfection. Considering the low transfection efficiency of MKN45 cells, we chose another gastric cancer cell line AGS instead of MKN45. The transfected AGS cells were named as H-Ras-pcDNA3.1(-)-AGS (R-AGS) and pcDNA3.1(-)-AGS (p-AGS) respectively. The R-AGS expressed a considerable higher level of Ras than that of p-AGS and AGS by western blot.MTT tests were used for detecting the proliferation inhibition by Matrine in AGS,p-AGS and R-AGS. We observed that Matrine had a dose-dependent growth inhibition in all the three cells (ANOVA, p < 0.001). At the same time the clone formation efficiencies of each transfected cell line treated by Matrine were evaluated by soft-agar culture. The clones of R-AGS and AGS were smaller than that of p-AGS and ectopic expression of Ras dramatically elevated the colony formation efficiencies of R-AGS (Univariate, P < 0.001).The clone numbers at the dose of 0.5 mg/ml matrine were much lower than the other four dosages in all three cell lines (Dunnett t test, p < 0.001). However, Matrine showed no more inhibition of the clone formation rate in R-AGS than in p-AGS and AGS (ANOVA, p>0.05). All the founding suggested that Ras might not be the target of Matrine.This part of study indicated that high dosage of Matrine could suppress the proliferation of Ras-ectopic-expression AGS cell. However no more specific inhibition was detected in R-AGS than in p-AGS and AGS by Matrine. Inconclusion, Matrine exerted its anticancer ability via a Ras-independent way.Conclusion1 Matrine inhibited the phosphorylation of eukaryotic initiation factor 4E and its inhibitor 4E-BP1 in a dose and time dependent manner, which suggested eIF4E and4E-BP1 might be new anticancer targets of Matrine.2 Matrine attenuated the activity of Erk1/2 MAPK accordant to the dephosphorylation of 4E-BP1 and eIF4E. However, p38, JNK/SAPK and Akt/mTOR were not the main upstream regulators .3 Ras might not be an anticancer target of Matrine.