Study Of Effect Of Recombinant Human Lactoferrin On Breast Cancer And Its Mechanism

Breast cancer is a common malignant tumor in females and accounts for the first indeveloped countries. The gene therapy of breast cancer is a promising strategy besidessurgery, chemotherapy and radiotherapy, which can carry tumor suppressor or otherantitumor drugs into the tumor site directly and targeted kill the tumor cells. So it gainsmany attentions.Lactoferrin (LTF) has antitumor effects and can inhibit the proliferation andmetastasis of different tumor cells, and it is regard as extendly the new antitumor drug.Previously, we reported on the construction of adenoviral vectors containing human LTFcDNA and a green fluorescent protein (GFP) which is a reported gene (ad-rhLTF), anddescribed the abundant expression of human LTF in the milk of goats and rabbits.However, the pharmacologic mechanisms of ad-rhLTF on breast cancer are still unknown.Therefore, the effects of ad-rhLTF and its mechanisms on breast cancer had beensystematically researched in this study in vivo and in vitro.In vivo experiments, mice bearing EMT6breast cancer were randomly divided intothe control group, CTX group, ad-rhLTF low dose group (108pfu/mL) and ad-rhLTF highdose group (5×10⁸pfu/mL), and then the effects of ad-rhLTF on mice bearing EMT6breast cancer and its likely mechanism were researched by using immunohistochemistrySP method, Flow cytometry (FCM), Enzyme Linked Immunosorbent (ELISA) method,Reverse Transcription-poly merase Chain Reaction (RT-PCR) and Western blot methodfrom regulating the immune function, interfering cell cycle and inducing cell apoptosis.The results were as following:After treatment of tumor mice with ad-rhLTF for14days, hLTF had the highexpression in tumor tissues and the expression of ad-rhLTF is of more obviously in highdose group (5×10⁸pfu/mL). Ad-rhLTF administration of108pfu/mL and5×10⁸pfu/mLsignificantly inhibited the tumor growth of tumor-bearing mice (p<0.01), and the tumorinhibition rate was42.80%and52.64%, respectively. Ad-rhLTF decreased the positivenumbers of mutant p53, CerbB-2and Ki-67proteins in tumor cells (p<0.01) without liver and kidney injury. By cell cycle analysis, ad-rhLTF increased tumor cells in G0/G1phage(p<0.01), decreased the percentage in S and G2/M phase (p<0.01), and appeared apoptoticpeak of Sub-G1, which demonstrated that the antitumor effect of ad-rhLTF was througharresting cell cycle. In the meantime, the expression of Bcl-2mRNA and protein werereduced (p<0.01), while the expression of Bax,Fas and caspase3were increasedcompared with the control group (p<0.01), which indicated that the mechanisms ofapoptosis likely occur through the triggering of the mitochondrial-dependent pathway anddeath-receptor pathway and then caspase3activation. It was also found that the p53mRNA expression was increased but the expression of PKB and PKC proteins werereduced in ad-rhLTF groups (p<0.01), which indicated that p53,PKB and PKC played animportant roles in the processing of apoptosis induced by ad-rhLTF.In the research of the immune function, ad-rhLTF (5×10⁸pfu/mL) significantlyincreased the thymus index and the spleen index of EMT6mice (p<0.05), but108pfu/mLad-rhLTF had no influences on them (p＞0.05). Ad-rhLTF raised the phagocytosis ofperitoneal macrophages and the activity of NK cells (p<0.05, p<0.01), and also increasedthe ability for splenocyte proliferation (p<0.01). Moreover, administrated with ad-rhLTFcould increase the proportion of CD4+T lymphocytes subsets (p<0.05) and repair the ratioof CD₄⁺/CD₈⁺T cells in peripheral blood (p<0.05), promoted the level of IL-2fromsplenocytes and the level of TNF-α from peritoneal macrophages (p<0.05) and the levelsof IL-2and TNF-α in serum were also increased (p<0.05). Besides, the expression of IL-2,TNF-α and B7mRNA in PBMC were raised (p<0.01), and neuclear factor NF-κB andAP-1proteins in mice splenocytes were also increased (p<0.01), which promoted T cellsproliferation and clone and then increased the antitumor immunity.In MCF-7cells experiments in vitro, the IC50value of ad-rhLTF was100pfu/cell in72h.72h after infection, ad-rhLTF had considerable cytotoxicity on MCF-7cells, andinhibited MCF-7cell proliferation. MCF-7cell appeared the typical characteristics ofapoptosis by light microscope, fluorescence microscope and TEM observation.Ad-rhLTF infection of MCF-7cells at100pfu/cell increased the number of apoptoticcells, and the apoptotic rate reached to31.92%(100pfu/cell) by Annexin V-FITC. MCF-7cells after ad-rhLTF infection appeared DNA "ladder" zone in the agarose gel electrophoresis and the mitochondrial membrane potential decreased. Ad-rhLTF arrestedcell cycle in G0/G1phase, and resulted in a decrease of Bcl-2mRNA and an increase inBax mRNA (p<0.01), which indicated that the mechanism of apoptosis induced byad-rhLTF was associated with the mitochondrial pathway.In the study, the anti tumor effects of ad-rhLTF and its mechanisms were researcheddeeply, which provided a theoretical basis for the application of ad-rhLTF in the field oftargeted cancer therapy.