Immunohistochemical Evidence of Lactoferrin in Malignant Gastrointestinal Tumors’: A Mini Review

Lactoferrin (LF) is an 80kDa glycosylated single chain protein, constituted of ca. 700 amino acids, with high homology among species, present in milk and colostrums as well as in many body fluids, such as blood plasma, amniotic fluid, tears, saliva, semen, bile, urine [1,2]. Several functions have been attributed to LF, although the corresponding mechanisms remain still controversial [3]; it appears involved in the regulation of iron homeostasis and absorption in the bowel as well as in the antimicrobial activity against bacteria, viruses, fungi and parasites [4]. Moreover, immunomodulatory and anti-inflammatory effects of LF have been reported [3,4]. Finally, LF appears to show some enzymatic properties such as protease, DNAase, RNAase and ATPase [5].


Introduction
Lactoferrin (LF) is an 80kDa glycosylated single chain protein, constituted of ca. 700 amino acids, with high homology among species, present in milk and colostrums as well as in many body fluids, such as blood plasma, amniotic fluid, tears, saliva, semen, bile, urine [1,2]. Several functions have been attributed to LF, although the corresponding mechanisms remain still controversial [3]; it appears involved in the regulation of iron homeostasis and absorption in the bowel as well as in the antimicrobial activity against bacteria, viruses, fungi and parasites [4]. Moreover, immunomodulatory and anti-inflammatory effects of LF have been reported [3,4]. Finally, LF appears to show some enzymatic properties such as protease, DNAase, RNAase and ATPase [5].
Recently, it has been suggested that LF is involved in the regulation of some important processes, such as the cycle and the death of cells, fighting against the carcinogenesis and the development of metastases [6]. In particular, it has been hypothesized that LF inhibits cell proliferation and suppresses tumour growth, blocking the transition from G1 to S in the cell cycle of malignant cells, both in vitro and in vivo [7,8]. Therefore, to better understand the LF anticarcinogenic activity, we report herein an immunohistochemical analysis of LF in human neoplasms of different gastrointestinal district in the attempt to elucidate also its possible pathogenetic role.

Materials and Methods
We have reviewed the immunohistochemical pattern of LF distribution in 101 surgical samples obtained from a corresponding number of malignant tumours developed in the stomach (30), colon (39) and gall bladder (32). The study was conducted in accordance with Good Clinical Practice guidelines and the Declaration of Helsinki and approved by local ethics committee.
All samples have been fixed in 10% neutral formalin for 24hrs at room temperature (RT) and then embedded in paraffin at 56 °C. From each block of malignant neoplastic tissue, 4µm-thick sections were stained with haematoxylin/eosin for the microscopic evaluation, but parallel sections were cut and mounted on silanecoated glasses, then dewaxed in xylene and rehydrated in graded ethanols. Antigen retrieval was performed before adding primary antibody by heating slides placed in 0.01 M citrate buffer, pH 6.0, in a microwave oven for three cycles x 5min.
For the immunohistochemical study, sections were treated in a moist chamber at room temperature: i. with 0.1% H 2 O 2 in methanol to block the intrinsic peroxidase activity (30min); ii.
For the demonstration of peroxidase activity, the sections were incubated in darkness for 10 min with 3-3' diaminobenzidine tetra hydrochloride (Sigma Chemical Co., St Louis, MO), in the amount of 100 mg in 200 ml 0.03% hydrogen peroxide in phosphatebuffered saline (PBS). The nuclear counterstain was performed by Mayer's haemalum. Renal tubular structures within normal kidney samples as well as portions of parotid gland were utilised as additional positive controls, as elsewhere suggested [7,8]. The LF immunoreactivity demonstrated in granules of polymorphonuclear neutrophils was utilised as positive control. Finally, in order to test the inter-run variability of LF staining, the same LF-positive parotid sample was utilised in every run. To test the specificity of LF immunostaining in order to deny the possibility of non-specific reaction, serial sections of each neoplastic specimen were tested by replacing the specific antiserum by either PBS, normal rabbit serum or absorbing with excess of purified human LF from human liver and spleen (Sigma Chemical Co.) as well as with pre-absorbed primary antibody: the results obtained were negative.
Immunostained sections were estimated by light microscopy using an x20 and x40 objective lens and x10 eyepiece; the assessment of LF immunostained sections was performed on a consensus basis by two pathologists using a double-headed microscope.

Results and Discussion
All neoplastic samples, routinely stained by haematoxylin and eosin, exhibited a good morphology, confirming the histopatological diagnosis; however, parallel sections were adequately stained by LF immunohistochemistry, with an immunoreactivity generally localized in the cytoplasm but occasionally in the nucleus.
The mucous neck cells of the antrum and body of the stomach were positive for LF; moreover, an evident LF immunoreactivity was encountered in intestinal type carcinomas, whereas diffuse type ones were always unstained. A clear intense cytoplasmatic immunopositivity for LF was found in well and moderately differentiated colo-rectal adenocarcinomas as well as mucinous carcinomas, even if some undifferentiated cases were unreactive; the LF immunostaining was also encountered in neoplastic elements present in metastatic lymphnodes, when the primary cancer was stained.
In gastric and colonic cancer, a positive LF immunoreactivity was found in a variable share of adenocarcinomas, mainly represented in papillary or glandular areas, while sarcomatoid, squamous or mucinous components were negative; the number of immunostained elements as well as the staining intensity showed some differences in the context of the same tumour. (Figure 1) In the present study, we have analyzed LF immunoexpression in a series of human gastrointestinal malignant tumours, showing that LF presence was not exclusively localized to the cytoplasm, but also in the nucleus. However, the site of LF immunolocalization in both the nucleus and cytoplasm has not been considered surprising since this glycoprotein has been thought to be involved in ribosomal biogenesis and after its transport into the nucleus; LF is able to bind specific DNA sequences, thus activating transcription [9]. Moreover, we have shown that heterogeneity in LF immunoexpression between different malignancies as well as inside the same tumour were not infrequent; if this observation could reflect different cell subpopulations, the stage in the cell cycle or instead some metabolic abnormalities should be verified by methods other than morphological analysis [10][11][12][13][14][15][16][17][18][19].
The origin of LF in human malignant tumours has not yet been fully elucidated. It is well known that LF has a high affinity for iron, which has been considered an essential nutrient for cells that are dividing rapidly such as tumour cells, taking part in various metabolic processes such as oxydative phosphorylation and RNA and DNA synthesis [1,5]. Therefore, neoplastic elements should be able to produce LF in order to make a greater amount of iron available for their turnover, similarly to that elsewhere suggested [7,20]. Alternatively, the localization of LF in malignant cells may not reflect an intracellular synthesis, reflecting instead the degree of transmembranous iron transfer as the consequence of defective or functionally impaired LF-receptors, already documented on the surface of target cells as well as in human neoplastic cell lines [21].
In our casuistry, the LF immunostaining was never founded in relation to the site, grade and stage of malignant tumours, excluding thus its role as predictive or prognostic neoplastic markers. Nevertheless, the immunohistochemical evidence of LF was largely confined to differentiated carcinomatous histotypes, such as differentiated glandular carcinomas of the stomach, colon and gallbladder, while anaplastic and undifferentiated carcinomas were always unstained; consequently, it may be suggested a role for LF as marker of glandular or acinar differentiation, similarly to that already pointed out in other malignancies [20,22,23].

Conclusion
The protective effects of LF have been demonstrated on chemically induced tumors of rodents [1]; moreover, it has been previously reported that LF is able to inhibit the development of experimental metastases in mice, mainly by an increase of NK cells and T lymphocytes expressing CD8, CD4 and IFNγ [24]. Meanwhile, other potential mechanisms have been suggested regarding the role of LF in the process of human carcinogenesis, including induction of programmed cell death, prevention of angiogenesis and regulation of cell cycle protein expression [25,26]. In fact, LF is able to trigger the apoptotic process by the activation of caspases 3 and 8 as well as the FAS signaling pathway [24]; on the other hand, LF was also shown to inhibit tumour-initiated angiogenesis in vitro and in vivo, possibly by blocking endothelial function and inducing IL-18 production [25]. Moreover, it has been reported that LF promoted growth arrest either at the G1 to S transition in breast cancer cells [8] as well as at the G0-G1 checkpoint in oral and neck cancer cells [27]; finally, LF demonstrated its ability to regulate cell growth by controlling the level of retinoblastoma protein, a key tumour suppressor involved in cell cycle progression 28-30. Nevertheless, whatever was the mechanism of action of LF in tumours; we probably still require additional investigations about the opportunity for new applications of LF in cancer, mainly regarding its nutraceutical function as well as its ability to potentiate chemotherapy Nevertheless, whatever was the mechanism of action of LF in tumours we probably still require additional investigations about the opportunity for new applications of LF in cancer, mainly regarding nutraceutical function as well as ability to potentiate chemotherapy..

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