Mutiltarget Atitumor Effects Of Fluorouracil Combined With Vinorelbine

Tumor is a common and frequent disease, which threaten the human health seriously. The mortality of patient with cancer occupied the first in the developed country, and the second in the developing country. Thus, how to overcome cancer is one of the most main missions for the government, scientific research agencies, and medical institutions of each country in the recent forty years. Chemotherapy as one of four convertional therapy is the most important means. The purpose of chemotherapy is to kill tumour cell and warrant the patient'safe. However, because of lack of essential distinction in metabolism between malignant tumour cell and normal cell, at present all of the antitumor chemotherapeutics can not completely avoid the damage for normal tissue, and furthermore commonly have to withdrawa from the chemotherapy for their disadvantage. Thus, it is undoubtedly important that how to raise the sensitivity of tumors to chemotherapeutics, protect the normal tissues from the injury induced by chemotherapeutics, and reduce the toxic and side reaction to improve the efficacy of tumor treatment.In the recent years the progresses of molecular oncology, molecular pharmacology, and cancer cell biology indicate that tumor is a multigenic disease caused by at least two kind of oncogene through multi-stage changes. It not only provides considerable new target and field for the prevention and cure of cancer, but also converts the classical cytotoxic drug to neotype antitumor drug possessing multitarget effect in the investigation and development of antitumor drug. The signal conduction of solid tumor is a complicate multifactorial proteinic network system and usually blocking of single signal conduction is not enough to inhibit cancer cell growth. Furthermore, many clinical trials demonstrated that the multitarget inhibitors surpass the monotarget inhibitors in therapy of cancer. Such as the antitumor effects of ZD6474 and inhibitors of COX2 involve in several molecular targets, the combination of COX2 inhibitor with traditional chemotherapeutics can also enhance their antitumor effect.As multitarget inhibitor surpasses montarget inhibitor and combination of chemotherapeutics with different antitumor mechanism can enhance therapeutic effect through converting the montarget action to multitarget action, we can combine the chemotherapeutics, which possess known therapeutic effects and definite mechanisms, to find out their suitable dosage and an excellent prescription by orthogonal design,which reveal a synergistic antitumor effect without respective toxicity through acting on different target in tumor signal conduction to prepare a new complex multitarget inhibitor. Thus, multitarget inhibitor of tumor signal conduction is playing a more important role in tumor treatment; two or more chemotherapeutics combination will be a common schedule for tumor chemotherapy.In the present study we determined the concentration-effet relationship of fluorouracil (5-FU), gemcitabine (GEM), cisplatin (DDP), and Vinorelbine (VRB) in H₂₂ cell to observe the synergistic effect of combinations of the different drugs and dose, to find out a new prescription with multitarget antitumor effect, and to analyze their antitumor mechanism in vivo and in vitro.Part 1 Screens of best dosage and prescription of 5-FU, DDP, GEM and VRB in antitumor effectsObjective: To search a best prescription from 5-FU, DDP, GEM, VRB.Methods: (1) MTT assay was used to study concentration- effect curve of 5-FU, DDP, GEM, and VRB in H₂₂ cell, choose suitable doses (22 cell to find out a best prescription.Results: (1)The effect of each drug on proliferation in H₂₂ cell was dependet to dose, the IC50 for 5-FU, DDP, GEM, and VRB were 490.79,10.01,0.021,and 16.49μmol/L, respectively. The following dose points of inhibitory ratio was lower than 30%, 5-FU: 0.00128, 0.0064, 0.032, 0.16, 0.8μmol/L; DDP: 0.15625, 0.3125, 0.625, 1.25, 2.5, 5μmol/L; GEM: 0.000128, 0.00064, 0.0032μmol/L; VRB: 5×10-7, 5×10-6, 5×10-5, 5×10-4, 5×10-3μmol/L. (2) In all of different combinations of two, three or four drugs, the inhibitory effect of 5-FU (0.8μmol/L) +VRB (0.005μmol/L) on H₂₂ cell was the most potent, the inhibitory ratio of 5-FU (0.8μmol/L) +VRB (0.005μmol/L) on H₂₂ cell growth was 40.84%±6.17%, which is the best prescription.Conclusions: Combination of 5-FU (0.8μmol/L) +VRB (0.005μmol/L) displays significant synergism in inhibition of H₂₂ cell growth and may be the best prescription in the combination of four antitumor drugs obove.Part 2 Investigation of synergistic antitumor effect and mechanism of Fluorouracil combined with VinorelbineObjective: To investigate the antitumor effect and mechanism of 5-FU (0.8μmol/L)+VRB(0.005μmol/L) on H₂₂ cell in vitro and in vivo.Methods: (1) MTT assay was used to measure the effect of 5-FU+VRB on the H₂₂ cell activity. (2) The leak rate of LDH in culture medium and apoptotic H₂₂ cells were determined. (3) Laser confocal microscopy was applied to detect the fluorescence signal in apoptotic H₂₂ cells stained by AO/EB. (4) Apoptosis and proliferation of H₂₂ cell were determined by flow cytometry (FCM). (5)The H₂₂ mice were sacrificed and used to measure the inhibitory rate of tumor growth to evaluate the influence of 5-FU+VRB on WBC, thymus and spleen indices of H₂₂ mice.Results: (1) The inhibitory rate of 5-FU+VRB on H₂₂ cancer cells growth was higher than 5-FU or VRB (P<0.01, P<0.01), at 72h the inhibitory rate reached the peak value (47.70%±5.40%). (2)The leakage rate (LDH%) and the cytonecrosis rate (LDHn%) at different times in 5-FU+VRB group had no statistical significance compared with the other groups; apoptosis rate (LDHa%) in 5-FU+VRB group was dependent to time and was significant higher at 96h, 120h, 144h, and 168h. (3) Cellular morphology: nucleus of non-apoptotic cells appeared green fluorescence, apoptotic cells showed strong yellow fluorescence, cell shringkage, reduction in cell volume, membrane blebing, and viable apoptotic cell vesic. The cell population in 5-FU+VRB group was obviously lower than other groups. (4) Cell cycle and Apoptosis: In all of groups the peak of sub G1 appeared, but the peak of sub G1 in 5-FU+VRB group was obviously higher than other groups, the apoptosis rate calculated from sub G1 in 5-FU+VRB group (40.65%±7.44%) was higher than blank control group (16.71%±1.49%), 5-FU group (22.46%±1.90%), and VRB group (21.64%±1.98%) (P<0.05 or 0.05). The proliferation index (PI) in 5-FU+VRB group was lower than other groups (P<0.05 or 0.01). (5) The effects of 5-FU+VRB on H₂₂-bearing mice: Inhibitory rates of 5-FU+VRB high and medium doses on H₂₂ cell growth were 60.61% and 21.21%, respectively (P<0.05 or 0.01). The WBC, spleen and thymus weight was significantly lowered in 5-FU+VRB high dose group (P<0.01), but did not in the medium dose and low dose group.Conclusions: The combination of 5-FU+VRB reveals a significant synergistic antitumor effect both in vitro and in vivio, which may be related to its effect on metabolic activity, apoptosis, cell necrosis, and cell membrane integrity. But at the high dose the side reaction of 5-FU+VRB was also strengthed.