Epigenetic Reprogramming Effects Of Bovine Oocyte Extract On Human Lung Cancer Cells

Malignant tumors are serious hazards to human health and lives.Traditional treatment is to remove tumors by surgery as well as to kill tumor cells by radiotherapy and chemotherapy. However, surgery fails to remove tiny disseminated lesions; radiotherapy and chemotherapy have the drawbacks of accelerating tumor evolution and lacking specificity, which results in severe impairment to normal cells within bodies. Therefore, it is necessary to jump out of past anti-tumor strategies of removing and killing tumor cells, seek new directions for cancer research, and provide new means for cancer treatment.Oocyte extract-mediated reprogramming is a new reprogramming method developed in recent years. It employs the extract of oocyte nuclei, cytoplasm or whole cells to incubate reversibly permeabilized somatic cells. During incubation, the reprogramming factors in the extract can enter the treated cells to change their epigenetic modifications and gene expression patterns. After the treatment, the treated cells can escape from their original development program and obtain a new developmental fate.epigenetic aberration plays impotant roles in cancer development. We consider that treatment of tumor cells with oocyte extracts may change their epigenetic modifications, rescue them from abnormal development procedures, and restore to them the ability to develop towards normal cells.In this study, extract of bovine MII, GV and parthenogenetic oocytes was used for the first time to treat H460human lung cancer cells. The reprogramming effects on human cancer cells were observed at epigenetic modification, gene expression and cell function levels.1. Bovine oocyte extract reversed CpG island hypermethylation of tumor suppressor gene promoters in H460cellsTreatment of H460human lung cancer cells with bovine MII, GV and parthenogenetic oocyte extract significantly reversed the promoter CpG island hypermethylation of tumor suppressor gene RUNX3and CDH1. The DNA methylation level of RUNX3promoter CpG island was decreased by30.44%,40.29%and33.81%by MII, GV and parthenogenetic oocyte treatment, respectively. The DNA methylation level of CDH1promoter CpG island was decreased by36.36%,42.57%and39.36%by MII, GV and parthenogenetic oocyte treatment, respectively. The demethylation effects by three kinds of oocyte extract showed no significant difference as analyzed by chi-square test. The demethylation induced by the three kinds of oocyte extract was not evenly distributed among the CpG sites within the two CpG islands; instead, it was more concentrated on certain special CpG sites. The CpG sites within transcription factor binding sequences all showed high demethylation rate. 2. Bovine oocyte extract changed histone modification patterns of tumor suppressor gene promoters in H460cellsTreatment of H460human lung cancer cells with bovine MII, GV and parthenogenetic oocyte extract all reduced the inhibiting histone modifications H3K27me3and H3K9me3at the promoter regions of tumor suppressor genes RUNX3and CDH1. MII oocyte extract displayed the strongest suppression of H3K27me3modification. The three oocyte extract had eaqual ability in reducing H3K9me3modification. MII oocyte extract had no effect on the activating modifications H3K9ac and H3K4me3at both RUNX3and CDH1promoter regions. However, GV oocyte extract increased the activating modifications H3K9ac and H3K4me3at RUNX3promoter region, as well as the activating modification H3K4me3at CDH1promoter region. Parthenogenetic oocyte extract increased the activating modification H3K4me3at both RUNX3and CDH1promoter regions.3. Bovine oocyte extract reactivated tumor suppressor gene expression in H460cellsTreatment of H460human lung cancer cells with bovine MII, GV and parthenogenetic oocyte extract all activated the expression of tumor suppressor genes RUNX3and CDH1. The transcription of RUNX3and CDH1was considerably upregulated after6h of culture, and slightly furthure upregulated after24h and48h of culture in MII oocyte extract-treated cells. The transcription of RUNX3and CDH1was moderately upregulated after6h of culture, and considerably upregulated after24h and48h of culture in GV and parthenogenetic oocyte extract-treated cells. After 48h of culture, the three groups of cells showed no significant difference in RUNX3and CDH1transcription.The protein expression of RUNX3and CDH1could be detected at24h after culture, and further enhanced at48h after culture.4. The effects of bovine oocyte extract on tumor suppressor genes showed specificityBovine MII, GV and parthenogenetic oocytes extract all showed specificity in remodeling the epigenetic modifications at tumor suppressor gene promoters and activating the transcription of tumor suppressor genes in H460cells. They caused neither demethylation of alpha satellite repeats or retroviral long terminal repeat sequence of minisatellite MS32, nor transcriptional upregulation of pluripotency genes OCT4, NANOG, KLF4and SOX2. Moreover, parthenogenetic oocyte extract increased the inhibiting histone modification H3K27me3and decreased the activating histone modification H3K9ac at the promoter region of pluripotency gene SOX2, leading to down-regulation of its expression.5. Bovine oocyte extract inhibited oncogenic ability of H460cellsBovine MII, GV and parthenogenetic oocyte extract could effectively reverse the malignant phenotype of H460human lung cancer cells. Compared with control group, the three treated groups decreased24.88%,26.42%and26.38%in proliferation,60.56%,53.18%and54.71%in anchor-independent growth,40.72%,46.93%and35.20%in migration, and78.05%,83.33%and73.81%in invasion, respectively. ConclusionTreatment of human lung cancer cells with bovine MII, GV and parthenogenetic oocyte extract demethylated the hypermethylated CpG island and remodeled the histone modifications specifically at tumor suppressor gene promoters, thereby activating the expression of tumor suppressor genes. These reprogramming effects strongly inhibited cancer cell proliferation, anchorage-independent growth, migration and invasion. These results indicate that treatment of cancer cells with bovine oocyte extract provides a new pathway for studying cancer epigenetic mechanisms, and opens up a new field for developing safe effective anti-cancer drugs.