Studies On The Antitumor Effects Of Protein Glycosylation Inhibitor Tunicamycin Alone And In Combination With HER2-target Drug Herceptin

Breast cancer is one of the most common tumors for women. HER2, one kind of membrane glycoprotein, is over-expressed in many breast cancer patients. HER2targeting treatment now is an effective biological treatment in these years. The target drug Herceptin is a humanized IgG monoclonal antibody, specifically targeted against the extracellular domain of HER2. Because of its target specifity, Herceptin has no inhibitory effects on other receptors and cell signaling pathways, which easily lead to the drug resistance. So the combination treatment is a hotspot in cancer treamtment.Tunicamycin is an antibiotic which can inhibit the synthesis of N-glycans in the glycoprotein. So it can downregulate the expressions of mature receptors in the cells which can avoid the effects of cell signaling pathway when treated with Herceptin alone. At the same time, tunicamycin has no significant toxicity on the cells. So the tunicamycin can enhance the antitumor effects of Herceptin, and avoid the side effects caused by the cytotoxic drugs when used in combination.In this paper, the antitumor effects of tunicamycin alone and in combination with Herceptin agaist breast cancer through cell and animal experiments were studied, aiming at delinating the molecular mechanisms responsible for these results.1Construction of plasmid pcDNA3.1/HER2and breast cancer cell line MCF-7/HER2The cDNA of HER2was obtained from breast cancer cell MAD-MB-453by using RT-PCR and cloned to pGEM(?)-T Easy vector. After the sequence was confirmed by sequencing analysis, pGEM(?)-T Easy/HER2and plasmid pcDNA3.1were digested by NheIå'ŒXhoI, ligated with T4DNA ligase, and the recombinant plasmid pcDNA3.1/HER2was obtained. Plasmid pcDNA3.1/HER2was transfected into breast cancer cell MCF-7, and then the positive trasfection cells were selected by the motheds of G418and Western blot. MCF-7/HER2cancer cell line was constructed and could be used as target modle for the following experiments.2The antitumor effects of tunicamycin and Herceptin used alone or in combination in vitro2.1Effects on cancer cell proliferationA panel of breast cancer cell lines and an immortalized human breast epithelial cell line were used to determine the growth inhibitory effects of tunicamycin and Herceptin or their combination by CCK8. Remarkable growth inhibitory effects of tunicamycin were found in all tested breast cell lines. The IC50values of tunicamycin for all the cell lines had no relationships with the expression levels of HER2in these cells. All the tested breast cells except for BT474were not sentitive to Herceptin. Herceptin in combination with0.5μg/ml tunicamycin can significantly increase the growth inhibition effects in all these tested breast cell lines. The rates of in combination were higher than the rates of the treamtments which gave tunicamycin or Herceptin alone.What’s more, based on the above datas and the formula of CDI (coefficient of drug interaction, CDI), the CDI values of the combinated treatments with tunicamycin and Herceptin in MCF-7/HER2breast cells were less than1, indicating that tunicamycin has synergistic effect with Heceptin in the treatment of cancer.2.2Effects on the apoptosis of HER2high or low expressed cell linesFlow cytometry combined with AnnexinV-FITC/PI staining showed that tunicamycin induced apoptosis of HER2high expressed breast cell lines, MCF-7/HER2and SKBR3, dose-and time-dependently. SKBR3cells consisted of8.69%apoptotic cells after treated with0.5μg/ml tunicamycin for24h and the apoptotice cells reached to15.57%after48h. MCF-7/HER2cells consisted of7.01%apoptotic cells after treated with0.5μg/ml tunicamycin for24h and the apoptotice cells reached to13.62%after48h. Herceptin also induced apoptosis in SKBR3and MCF-7/HER2cells. When cells were treated with Hercpetin10μg/ml and100μg/ml for24h, the apoptosis rates were7.14%and12.50%in SKBR3cells,8.74%and9.49%in MCF-7/HER2cells. Combination treatment induced more intensive apoptosis compared with tunicamycin and Herceptin alone in SKBR3and MCF-7/HER2cells.0.5μg/ml tunicamycin plus10μg/ml or100μg/ml Herceptin induced12.69%and14.57%apotosis in SKBR3cells, and8.78%and13.2%in MCF-7/HER2cells.The percentage of apoptosis cells was much lower in HER2low expressed MCF-7cells, even in high dose of Herceptin. The combined use of tunicamycin and Herceptin could slightly increase the apopotosis of MCF-7cells.0.5μg/ml tunicamycin plus10μg/ml or100μg/ml Herceptin induced5.10%and5.13%apotosis in MCF-7cells, whereas the same doses of Herceptin and Tunicamycin given alone resulted in3.01%,3.40%and4.42%apoptosis, respectively.Western blot analysis showed that tunicamycin with or without Herceptin significantly increased Caspase-3activities and PARP cleavages. The decreased Bcl-2and increased Bax indicated that mitochondria-casapase cascade was responsible for tunicamycin-induced apoptosis. When MCR-7/HER2cells were treated with Herceptin alone, the Caspase-3activites, PARP cleavages and Bax expression could also be increased. However, there are no relationships beween protein expression levels and Herceptin concentration.2.3Effects on cell cycle distributionsFlow cytometry combining with PI staining showed that tunicamycin and Herceptin could induce MCF-7/HER2G1arrest. As the time and dose increased, the arrest was much more significant. When MCF-7/HER2cells were treated with0.5μg/ml tunicamycin plus10μg/ml Herceptin,72.51%of the cells were arrested in G1, compared with65.08%of0.5μg/ml tunicamycin alone and68.52%of10μg/ml Herceptin alone.As for MCF-7cell, tunicamycin could also induce the cells in G1arrest dose-dependently. However Herceptin had no effects on the cell cycle distrutions. Additionally, when the cells were treated with tunicamycin and Herceptin together, most of them were also in Gl arrest.Western blot analysis was used to evaluate the effects of tunicamycin with or without Heceptin on the expressions of cell cycle-related proteins. The results showed that for MCF-7and MCF-7/HER2cells, the expressions of cell cycle-related proteins, p21and p27, increased dose-dependently and the expression of Cyclin D1decreased. The combination treamtment was much more effective than tunicamycin treamtment alone. The decreased Cyclin D1and up-regulated p21and p27were the potent evidences for G1phase arrest. When MCR-7/HER2cells were treated with Herceptin alone, only100μg/ml Herceptin had effects on the expreesions of p21and p27, the other two treated groups (10μg/ml and50μg/ml) had no significant differences from control group. When MCF-7cells were treated with Herceptin alone, it down regulated the expreesions of p21and p27, and the expression of Cyclin D1had no significant difference from that of control group. When cells were exposed to0.5μg/ml tunicamycin plus10μg/ml Herceptin, the expressions of p21and p27were up-regulated and the expression of Cyclin Dl was inhibited compared with the group receving tunicamycin or Herceptin alone.2.4Effects on on the expressions of EGFR familyWestern blot was used to analyze the effects of tunicamycin with or without Heceptin on the expressions of EGFR family. The results showed that for MCF-7and MCF-7/HER2cells, the expressions of HER2, p-HER2, EGFR, p-EGFR and HER3were downregulated by tunicamycin dose-dependently. When MCR-7/HER2cells were treated with Herceptin alone, only the expressions of HER2, p-HER2and EGFR were downregulated. Herceptin alone had no effects on the expressions of EGFR family in MCF-7cells. When MCF-7/HER2and MCF-7cells were treated with0.5μg/ml tunicamycin plus10μg/ml Herceptin, the expressions of HER2, EGFR and HER3were down-regulated and the phosphorylations of HER2and EGFR were also inhibited, compared with the group receving tunicamycin or Herceptin alone.2.5Effects on HER2down-stream signal pathways MAPK and PI3K/Akt The effects of tunicamycin with or without Heceptin on the expressions of important proteins in EGFR related-signal pathways were analyzed by Western blot. The results showed that for MCF-7and MCF-7/HER2cells, after tunicamycin treatment, the expressions of ERK1/2, p-ERK1/2, Akt and p-Akt were downregulated dose-dependently. The effects of combination treamtment were much more obvieous than that of tunicamycin treamtment alone. The decreased ERK1/2, p-ERK1/2, Akt and p-Akt were the evidences of EGFR-related signal pathway blockage, which could cause the inhibition of proliferation, the induction of apoptosis, cell cyle arrest and anti-invasion.3The antitumor effects of tunicamycin and Herceptin used alone or in combination in vivo3.1The antitumor effects of tunicamycin on breast cancer cell SKBR3xenografts in vivoTreatment with Tunicamycin at the dose of0.005mg/kg,0.02mg/kg and0.075mg/kg inhibited the growth of human breast cancer cell SKBR3xenografts in nude mice by12.9%,16%and33.5%, respectively. The body weights of the animals showed no significant differences between the control and treated groups.Western blot was employed to analyze the effects of tunicamycin on the expression of HER2in tumor tissues. The results showed that the expression of HER2in tumor tissues was downregulated by tunicamycin dose-dependently.3.2The antitumor effects of tunicamycin on breast cancer cell MCF-7/HER2xenografts in vivoTunicamycin treatment could inhibit the growth of human breast cancer cell MCF-7/HER2xenografts in nude mice. After21days treamtments, the inhibition effects of each group were as followings:0.0075mg/kg>0.02mg/kg>0.005mg/kg, and the effects of0.005mg/kg group were the same as the Hercpetin (lOmg/kg) group. The body weight of the0.02mg/kg group with a tumor inhibition rate of27%showed no significant differences from that of control group. Although the inhibition rate of0.075mg/kg group was50%, the body weight loss was up to23%. The effects of tunicamycin on the expressions of HER2in tumor tissues were analyzed by Western blot. The results showed that the expressions of HER2in MCF-7/HER2tumor tissues were downregulated by tunicamycin. Additionally, the treamment time had effects on the expression of HER2. When exposed to tunicamycin for24h, the expressions of HER2in tumor tissues had no difference compared with that of control, but the expressions of HER2were significantly downregulated when treated with tunicamycin48h and72h. The expression of HER2in72h group was higher than in48h group, which indicated that most of the tunicamycin in vivo had been metabolized and the drug should be administered again to keep the efficacy.3.3The antitumor effects of tunicamycin and Herceptin in combination on breast cancer cell MCF-7/HER2xenografts in vivoCombination treatment of tunicamycin and Herceptin showed smaller tumor volumes than that of tunicamycin or Herceptin treatment group. Compared with the treamtment in the control group, tunicamycin (0.04mg/kg), Hercpetin (10mg/kg) and the combined treatment reduced the tumor volume of the MCF-7/HER2xenografts by47.6%,22.9%and61.9%, respectively. The combined treatment groups had no significant body weight losses compared with control. So the combination of Herceptin with tunicamycin had better antitumor effects than they are used alone, without toxicity increase.