Studies On Inhibition Effect Of Recombinate Human Lactoferrin On Oral Squamous Cell Carcinoma Tca8113 Cells And Its Mechanism

Malignant tumors are extremely harmful to human health. The incidence rate of which shows an upward trend year after year, and its mortality rate stands forefront of various diseases. In all malignant tumors, oral squamous cell carcinoma (OSCC) accounted for 2% -3%. OSCC, which occupies more than 80% of malignant tumors in oral and maxillofacial region, is one of the top ten human malignant tumors. At present, we mainly rely on surgical resection, radiotherapy and chemotherapy as conventional therapy. After analysis of oral combination therapy studies and clinical significance, Chinese scholars propose a comprehensive individualized treatment plan of oral cancer. That is, ensure the quality of patient's life while enhance tumor cure rate. Clinical and research workers in the country and abroad constantly sum up experience and improve existing treatment methods to carry out an active exploration of new ideas for cancer treatment. Nowadays, researchers promote using of natural foods as chemoprevention of oral cancer carcinogenesis. Chemoprevention aims to utilize a part of physiological activities of compounds from natural plants/animals. Chemoprevention and chemotherapy with naturally occurring compounds such as lactoferrin have become increasingly important strategies in inhibiting carcinogenesis and tumor growth.Lactoferrin (LF), an 80-kDa basic glycoprotein, is a member of the transferrin family of iron-binding proteins which was originally isolated from human milk. Human lactoferrin consists of 691 amino acid residues. The complete amino acid sequence of bovine lactoferrin has been determined to contain 689 amino acids. The structure of human lactoferrin and bovine lactoferrin is similar. The protein is present in mammalian exocrine secretions, including breast milk, tears, nasal and bronchial mucus, cervical mucus, and seminal fluid. Recombinate human lactoferrin (rhLF) is structurally and functionally equivalent to native human LF in all material respects, as demonstrated by comparison of their 3D structures, molecular weights, biologic activities and other physicochemical properties, differing only in the nature of its glycosylation. Lactoferrin has multiple known biological activities, including iron regulation, cellular growth and differentiation, antimicrobial defense, antiinflammatory activity, and cancer protection. Immunoreactivity of Lf has been detected in many human neoplasms such as adenocarcinomas of the parotid gland and prostate, breast carcinoma, thyroid tumours, renal cell carcinoma, gastric and colorectal adenomas and carcinomas , gallbladder carcinomas,astrocytomas and multiforme glioblastomas and, recently, in skin nevi and melanomas; nevertheless, in these neoplastic conditions, the functional role of LF has not been fully elucidated. LF is a multifunctional protein,Although LF was originally identified as a mucosal host defense mediator and inflammatory immune modulator, it also displays antineoplastic activity. Recently, studies have suggested chemopreventive and antitumor activity for lactoferrin as well. bLF and its digested fragments have been found to remarkably inhibit colon, esophagus, lung, and bladder carcinogenesis in rats when administered orally in the post-initiation stage. Marked inhibition of tumor growth and reduced lung colonization by B16-F10 melanoma experimental metastasis were found in mice treated with hLF injected. Added to drinking water inhibited 4-nitroquinoline 1-oxide carcinogenesis in rats. Oral bLF reduced lung colonization by colon carcinoma cells in mice. Yoo et al demonstrated lactoferrin had the ability to inhibit metastasis of primary tumors in mice with cancer. Despite the evidence that LF possesses chemopreventive activity, little is known about the mechanisms by which its antitumor activity is mediated. In order to test whether human recombinant lactoferrin has a chemotherapeutic effect on oral cancers ,we examined the effect of rhLF on Tca8113 cell lines. To study one possible mechanism of lactoferrin-mediated antitumor activity in head and neck cancer cells, we investigated how the cell cycle, proinflammatory cytokines, proangiogenics are affected by rhLF treatment.Measurement of cell proliferation by MTT: Incubated Tca8113 cells and two normal cell lines CHO and 293T were treated with rhLF at different concentration, at the same time, morphology of cells were observed with microscope. During these seven experimental groups, rhLF has no inhibitory effect to the proliferation of CHO and 293T cells. Different doses of rhLF have inhibitory effects on Tca8113 cells. The result showed that rhLF1-rhLF7 group inhibitory rates were 16%, 28%, 30%, 36%, 43%, 56%, 63%. Groups add 50 and 100μg/ml rhLF had distinct difference when compared with cell culture group without rhLF (C group) (p <0.05), and with the increase of lactoferrin concentration, inhibition effect gradually increase. 35.7μg/ml rhLF treatment group, with the culture time extended showed no increasing inhibitory effect. rhLF can induce dose-dependent growth inhibition on the oral tumor cells cultivated in vitro.Flow cytometric analysis: For cell analysis, 50μg/ml and 100μg/ml rhLF were added to medium, with augment of drug concentration, Tca8113 cells distribution in the cell cycle changed dramatically. During G 0/G1 phase, the number of cells increased, and during S and G2/M phase, cells reduced gradually. It can be seen, Tca8113 cells were arrested in the Go / Gl phase by rhLF, and there is a clear dose-effect relationship. Incubated with different concentration of rhLF for 24h, Tca8113 cells were respectively detected cyclinD1, p19, p27 gene mRNA expression, and then compared with no rhLF control group. CyclinD1 of 50μg/ml and 25μg/ml rhLF experimental groups mRNA has a statistically significant difference when compared with the control group (t = 9.107, p <0.05; t = 4.963, p <0.05). The level of p19, p27 mRNA expression did not change obviously. There was no statistically significant difference, compared with the control group (p19 :F = 0.309, p27: F = 2.573, p> 0.05).RT-PCR and Western blot are used to detect rhLF influence on angiogenic factors VEGF, bFGF mRNA and protein expression, secreted by Tca8113 cells. After 24 hours constant stimulation of rhLF, VEGF and bFGF gene PCR product of Tca8113 cells were tested. It is obvious that there are statistical significance differences between 50μg/ml rhLF experimental group and control group, p <0.05 (VEGF: t = 8.530, bFGF: t = 7.056). Accordingly VEGF and bFGF protein expression of Tca8113 cells reduced with the increased concentration of rhLF and significant difference was found when compared 50μg/ml rhLF experimental group (E group) and the control group (A group),p <0.05(VEGF:t=4.324. bFGF:t=6.396).Immunohistochemical methods were employed in normal oral mucosa (n = 10) and OSCC patients'(n = 65) tumor tissue to detect expression of proinflammatory cytokines IL-6 and IL-8 in oral squamous cell carcinoma, and there relationship with clinicopathological parameters. Quantitative analysis was made by image analysis system. The level of IL-6, IL-8 in patients with OSCC was significantly higher than normal oral mucosa. IL-6, IL-8 expression has nothing to do with tumor pathological grade, gender, age and size, but IL-6, IL-8 expression is closely related to lymph node metastasis.Adhibit immunohistochemical method, enzyme linked immunosorbent assay to detect impact of recombinant human lactoferrin on IL-6, IL-8 protein . According to the results of immunohistochemical staining, IL-6, IL-8 were expressed by Tca8113 cells. Interestingly, in 50μg/ml rhLF group, IL-6, IL-8 expression seemed to be suppressed. There are only weak expression or no expression in this group. ELISA test results: Different conditioned medium (contains rhLF 50μg/ml and100μg/ml separately) separately cultivate Tca8113 cells for 24 h, levels of IL-6, IL-8 were significantly decreased in the culture supernatant of 100μg/ml rhLF group. Compared with the normal control group, the difference was significant (p <0.05).Conclusion: Direct inhibition of cellular growth is one mechanism by which Lactoferrin may inhibit cancer growth. Lactoferrin induces direct cell cycle arrest which seemed to be modulated by down-regulation of cyclin D1. Lactoferrin can decrease cellular release of the proangiogenic cytokines, including VEGF and bFGF. Lactoferrin may also inhibit tumor mediated angiogenesis. Lactoferrin can decrease cellular release of the proinflammatory cytokines, including interleukin-6 and interleukin-8, which suppresses tumor growth.This innovative point lies in that studies of recombinant human lactoferritin of oral cancer treatment is carried out for the first time in China. Inhibitory effect of rhLF on cancer cells was investigated in vitro, and take advantage of FCM, westernblot, ELISA, immunohistochemical methods, influences of rhLF in oral squamous cell carcinoma cells such as cell cycle and cytokine expression were studied. It is a comprehensive and systematic evaluation of inhibitory effect of rhLF on oral cancer cells and its mechanism. A basis for the subsequent further animal experiments and clinical trials, new ideas and theoretical foundation for oral squamous cell carcinoma of chemotherapy , and other malignant tumors treatment are provided.