The Study Of Antitumor Activity And Mechanism Of Rasfonin

With the rapid development of tumor biology, the focus of research and developmentof anticancer drugs has transfered from conventional cytotoxic drugs to targeted drugs.The ras gene is an important oncogene. Harvey ras (H-ras), Kirsten ras (K-ras) andneuroblastoma ras (N-ras) are the mainly ras genes associated with human tumors.K-ras is most closely related to human tumor. Many studies showed that there wasK-ras gene mutation in90%patients with pancreatic cancer. Mutated ras gene causesRas protein activation, elevation of Ras-GTP level in the cell, alleviation the negativeregulation of GAP, and over activation of the downstream signaling pathway,Ras-Raf-MEK-ERK, thus leading to tumor cell over proliferation and tumorigenesis.Therefore, it was a new research trend on targeting the ras signaling pathway in tumortherapy.Rasfonin is a α-pyranone-containing chemical ioslated from fungus. Preliminarystudy showed that rasfonin selectively inhibited the survival of ras-dependentBa/F3-V12cells and induced cell apoptosis, but showed no effect on non-ras-dependentcell line. It indicated that rasfonin had antitumor action on ras mutation tumor.However, due to the complex and low-yield methods of synthesis and extraction ofrasfonin, it is hard for studying the pharmacological mechanism and further researchand development of rasfonin. The lab of professor Yong-sheng Che isolated a fungus Doratomyces sp. from a soilsample collected on the Qinghai-Tibetan plateau at an altitude above4,000m, whichlargely increased the rasfonin yield. Up to now, we have obtained hundred millgramsrasfonin with purity over98%. Therefore, adequate amounts of rasfonin were obtainedto evaluate the antitumor activity and further study the mechanism of antitumor.1. The antitumor activity of rasfonin in vitro1.1The selectivity of rasfonin and its analogues on wild type and mutated ras cellslinesThe effects of rasfonin and its analogues on cell survival were observed to verify theselectivity of rasfonin on mutated ras tumor cell lines using mutated ras and wild typetumor cell lines derived from various tissues. The results showed that rasfonin and itsanalogues (A305and A306) inhibited cell survival, and the IC50of rasfonin on cellsurvival of mutated ras cancer cell was4.45-10.16μM, but it had no inhibition effect onwild type ras cell at the same concentration, which suggested the high selectivity ofrasfonin on mutated ras cancer cells lines.1.2Effects of rasfonin on proliferation of wild type and mutated ras pancreaticcancer cellsTwo pancreatic cancer cell lines, Panc-1(mutated K-ras) and BxPC-3(wild type),were used to observe the effects of rasfonin on proliferation using MTS assay and thexCELLigence system. The results showed rasfonin inhibited Panc-1cell proliferation atconcentration of5μM, while the inhibitory effect of rasfonin on BxPC-3cell was at10μM. The results indicated that rasfonin may inhibit proliferation of mutated raspancreatic cancer cells with high selectivity.1.3Effects of rasfonin on colony formation of mutated ras cancer cellsColony formation represents the survival ability of cell, which reflects thepopulation-dependent traits and the proliferation ability of cell. Cancer cells which formcolony easily have strong survival and proliferation abilities. The colony formationexperiment is a sensitively method to determine the activity of anti-cancer drugs.The mutated K-ras Panc-1and SW620cells were more easily to form colonies thanBxPC-3cell. The effects of rasfonin on colony formation of mutated ras cancer cells were observed on Panc-1and SW620cells. The results showed that rasfonin inhibitedcolony formation significantly on Panc-1and SW620cells at concentration of0.25-1μM in a dose-dependent manner. But when the concentration of rasfonin was5μM,Panc-1and SW620cells could not form visible colony at all. The results suggested thatrasfonin had obvious inhibition effects on the survival and proliferation abilities ofmutated K-ras cancer cells.1.4Effects of rasfonin on migration and invasion abilities of Panc-1cellsThe migration and invasion of cancer cells are key factors in the development andmalignancy of cancer. The effects of rasfonin on migration and invasion abilities ofPanc-1cells were studied using the xCELLigence system, a real-time, non-labeled cellanalysis system, The results showed that the mutated K-ras Panc-1cells had strongmigration and invasion abilities, while the unmutated K-ras cell, BxPC-3cells, had nomigration and invasion abilities. The positive drug, Salirasib FTS, significantly reducedthe migration and invasion abilities of Panc-1cells at concentration of50μM, andrasfonin also showed the same effects at concentration of1-15μM in a dose-dependentmanner.1.5Effect of rasfonin on early apoptosis of Panc-1cellsThe effect of rasfonin on early apoptosis of Panc-1cells was observed using flowcytometry. The results showed that rasfonin at concentration of1-5μM did not affectthe early apoptosis, and rasfonin at concentration of10μM significantly increased theapoptosis proportion to20.34%, but the apoptosis proportion was still significantlylower than that of star cytochalasin group (31.86%).In summary, rasfonin inhibited cell proliferation, survival ability, and the migrationand invasion abilities of Panc-1cells, but did not affect the early apoptosis of Panc-1cells.2. Evaluation on antitumor efficacy of rasfonin in vivoTo evaluate the antitumor efficacy of rasfonin, nude mice xenograft model of humanpancreatic cancer were used. After1week of cell implantation, the mice were randomlyseparated into5groups, control group, FTS groups (15mg/kg, every day, i.p.) andrasfonin groups (7.5,15,30mg/kg, every other day, i.p.). Rasfonin was administratedby intraperitoneal injection every other day for20days. During the experiment period (20days), the average tumor volume in model groupincreased significantly from the10th day, and the average tumor volume reached1000mm3at the20th day. FTS (15mg/kg) and rasfonin (30mg/kg) reduced tumor volumesignificantly from the10th day and the average tumor volume of rasfonin group(30mg/kg) was lower than that of FTS group at the20th day. At the end of theexperiment, the average tumor weights in rasfonin treated groups (15,30mg/kg) weremuch lower than those of model group, and tumor inhibition rates were54.3%and61.2%.The administration of FTS and rasfonin did not affect the body weights of animalsduring the experiment. But rasfonin treatment (30mg/kg) group significantly reducedspleen coefficient, which suggested the immune inhibition of rasfonin. The result ofblood routine test showed that FTS and rasfonin did not affect the number of red bloodcells and hemoglobin content, but significantly increased the number of platelets.In summary, the human pancreatic cancer Panc-1nude mice xenograft model wassuccessfully established, and rasfonin could significantly reduce the growth of xenografttumor.3. The mechanisms of action of rasfoninThe mechanisms of antitumor action of rasfonin were further studied from Ras and itssignaling pathway.3.1Effect of rasfonin on expression and activity of Ras protein in Panc-1cellsWestern blotting and pull down tests were used to analyze the expression and activityof Ras protein. The result showed that rasfonin did not affect Ras protein expression,and rasfonin decreased the activity of Ras protein caused by EGF in a dose-dependentmanner. FTS had the same effects as rasfonin.3.2Effect of rasfonin on expression of downstream protein kinases of Ras-MAPKsignaling pathway in Panc-1cellsThe expression of downstream protein kinases of Ras-MAPK signaling pathway weredetedcted to observe the effect of rasfonin on the Ras-MAPK signaling pathway inPanc-1cells. The result showed that FTS and rasfonin decreased the levels ofphospho-c-Raf, phospho-MEK1/2and phospho-ERK1/2, but did not affect the levels of nonphosphorylated MEK1/2and ERK1/2. The results indicated that rasfonin may affectthe phosphorylation of downstream protein kinases by decreasing Ras protein activity.3.3Effect of rasfonin on the expression of upstream proteins of Ras-MAPKsignaling pathway in Panc-1cellsThe effect of rasfonin on upstream proteins of Ras-MAPK signaling pathway, such asGRB2, Sos1and GAP, were studied. The results showed that FTS and rasfonin had noeffect on expression of GRB2and GAP, however, they significantly decreased theexpression of Sos1. The effects of rasfonin on activities of Sos1and GAP wereobserved using radioactive isotope labeling. The results showed that rasfonin did notaffect the activities of GEF and GAP, which suggested that the reduction of Rasfonin onRas activity may relate with reducing Sos1expression.3.4Effect of rasfonin on the activity of protein kinases in EGFR signaling pathwayThe effect of rasfonin on the activity of protein kinases in EGFR signaling pathwaywas studied to verify the selectivity of rasfonin on protein kinases. The activities of19kinds of kinases (EGFR、FGFR、PDGFRα、PDGFRβ、Flt3、Met、FAK、c-RAF、MEK1、PI3Kinase、PKBα、PKBβ、PKBγ、IKKα、mTOR、p70S6K、JNK1α1、JNK2α2、Ros) were evaluated with radioactive ligand labeling experiments. The results showedthat Rasfonin had no effect on the activity of most protein kinases, but it up-regulatedp70s6k kinase of mTOR downstream. The result further confirmed that the targets ofrasfonin may be in Ras signaling pathway.Conclusions:1. Rasfonin selectively inhibited proliferation of mutated ras tumor cell linesderived from various tissues.2. Rasfonin could inhibit the colony formation of Panc-1cells and SW620cells ina dose-dependent manner.3. Rasfonin could inhibit the migration and invasion of Panc-1cells in adose-dependent manner, but did not affect early apoptosis.4. Rasfonin could inhibit the tumor growth in nude mice xenograft model ofhuman pancreatic cancer.5. One mechanism of antitumor action of Rasfonin may be due to inhibiting Rasactivition by reducing Sos1expression, thereby affecting its downstreamMAPK protein phosphorylation. In summary, this is the first report of systematic pharmacological research ofrasfonin. The results showed that Rasfonin had antitumor effects both in mutated rastumor cell lines and nude mice xenograft model of human pancreatic cancer. Onemechanism of antitumor action of Rasfonin may be due to inhibiting Ras activition byreducing Sos1expression, thereby affecting its downstream MAPK proteinphosphorylation.