The Preliminary Analysis Of Curative Effect Of AdFOXMlshRNA In Nasopharyngeal Carcinoma And Breast Cancer Gene Therapy

Cancer seems to be the plague of the21st century, which is known as one of themost dangerous threats to human health and life. Nasopharyngeal carcinoma (NPC)represents a significant disease burden in Southern China and Southeast Asia, withan annual incidence rate of about20cases per100,000people in endemic areas. Theincidence and mortality of NPC are at the first place among head and neck cancer.Breast cancer is the first common malignancy and is an important cause of mortalityamong women. Moreover, breast cancer remains a public-health issue on a globalscale. Surgery, chemotherapy and radiotherapy are the commonly used traditionarymethods of cancer treatment. However, there are limitations in these therapeuticapplications because of their undesirable side-effects and local recurrence anddistant metastasis. Thus, novel therapeutic targets and new approaches for cancertreatment are urgently needed.FOXM1belongs to the large family of Forkhead Box transcription factors andis involved in many important physiological processes, such as cell proliferation,cell senescence, DNA damage repair and organogenesis. The results of generousresearch have shown that FoxM1is highly expressed in various types of humanmalignancies including breast cancer. However, no information is availableregarding the role of FOXM1in human NPC. Our study found that there were strongexpression of FOXM1in clinical tissue specimens and cell lines of human malignantnasopharyngeal carcinoma and breast cancer. Furthermore, many studies indicatedthat FOXM1played a crucial role in maintaining the balance of proliferation,differentiation, apoptosis and metastasis of cancer cells. All these findings suggestthat FOXM1appears to be an attractive target for the development of novelanti-cancer therapies.Adenovirus is the most popular viral gene therapy vector in practice. Popularityof Ad vectors for therapeutic gene delivery is based on several advantages such asefficient transgene delivery and expression, transduction of both dividing andnon-dividing cells, ease of propagation to high titers, episomal persistence of the Adgenome within the nucleus with minimal risk of genomic insertional mutagenesis,and high capacity to accommodate foreign DNA. Though RNAi is one of the mostpopular molecular tools in gene therapy recently, the delivery of siRNA intomammalian cells is the critical factor for a successful application of RN Ai in gene function study and cancer gene therapy. To overcome this rate-limiting step, in thisstudy, we constructed an adenovirus-mediated shRNA expression system, named asAdFOXM1shRNA, to knock-down FOXM1expression in cancer cells and theninvestigated the preliminary curative effect of this replication-defected adenovirusvector in NPC and breast cancer gene therapy through a series of experiments. Themain results were listed as followed.Firstly, we need to grope the suitable dosage of virus used in the three cellsCNE, MDA-MB-231and ZR-75-30respectively. AdLacZ control adenovirusinfections determined that almost100%of cells were infected with the viral dosageat10plaque-forming units (pfu)/cell. And we found that the expression of FOXM1was effectively suppressed by the infection of AdFOXM1shRNA in NPC and breastcancer cells at the dosage of10pfu/cell.Secondly， we assessed the effects of AdFOXM1shRNA-mediated FOXM1silencing on cancer cell proliferation. As expected, the mRNA expression o f FOXM1and cell cycle related genes were significantly decreased inAdFOXM1shRNA-infected cells. The analysis of cell cycle progression showed thatthe cell cycle in FOXM1-depleted cells were blocked. The data suggested thatAdFOXM1shRNA was an effective reagent to inhibit the proliferation of cancercells.Thirdly, we investigated the effect of FOXM1knockdown on tumorigenesis invitro and in vivo. We performed the soft agar assays to measure the ability ofanchorage-independent growth of cancer cells infected with AdLacZ orAdFOXM1shRNA. AdFOXM1shRNA infection resulted in dramatic decrease ofcolony formation of CNE and ZR-75-30cells. And the results of animal tumor assayshowed that AdFOXM1shRNA reduced the induction of the neoplastic phenotype ofZR-75-30cells in vivo.Lastly, we generated nude mice xenografts models to determine the effect ofAdFOXM1shRNA on tumor growth kinetics in vivo. We found that tumor growthwas significantly inhibited in the group treated with AdFOXM1shRNA intratumoralinjections as compared with the control group. These results indicated thatAdFOXM1shRNA targeting FOXM1elicited a strong anti-tumor effect on NPC andbreast cancers in vivo.