Anno XVII n. 2/01

Introduction

Previous in vivo experiments have demonstrated that pregnant uteri homogenates taken from C57BL/6 syngeneic mice during the period of embryo organogenesis inhibit dramatically Lewis tumor growth. In fact, the inoculation of different groups of C57BL/6 syngeneic mice with 1x106 Lewis Lung Carcinoma cells demonstrated that the growth of the primary tumor and the formation of pulmonary metastases were completely suppressed when the mice were treated with the pregnant uteri extracts (1). These results may indicate that pregnant uterus represents not only a nutritional organ, but also a structure of regulation and control of pathological multiplication. In this work we tested several different human tumor cell lines with mouse and pig pregnant uterus extracts, in order to:

1) in vitro confirm the previous in vivo data with mouse tissue extracts and verify pig uterine anti-proliferative activity;

2) assess whether the inhibition of cell proliferation is restricted to tumor lines or whether normal cells are also affected;

3) isolate specific fractions or substances putatively responsible for these inibitory effects;

4) assess apoptosis as a mechanism responsible for this inhibition.

Material and Methods

Pregnant uterine mucosa extracts Five human tumor cell lines of different origin (A 172 glioblastoma, A 375 melanoma, ACHN kidney carcinoma, ZR 75.1 breast carcinoma, H9 lymphoblastic leukemia) and one murine fibroblast cell line were used for these experiments. They were treated with extracts of uterine mucosa taken from 4, 5, 9 and 11 day-pregnant mice and from a 24 day-pregnant sow. Other treatments were also performed using small molecular weight fractions of the above cited extracts, further analyzed by HPLC. The proliferation curves of all cell lines after the treatments were calculated with a classical technique, i.e. the manual count with Trypan Blue, and with a colorimetric-spectrophotometric method. They were finally interpretated with statistical analyses.

Results and discussion

Fig.1A shows the mean proliferation curves of glioblastoma A 172 cells after three separate experiments with the crude mice uteri extracts at different days of pregnancy. Cell curves are significantly inhibited after the treatment with uteri extracts on the 4th day (P=0.04 after 24 hours, P= 0.012 after 48 hours), on the 5 th day (P=0.04 after 24 hours, P= 0.006 after 48 hours), on the 9 th day (P= 0.02 after 48 hours), on the 11 th day (P= 0.03 after 48 hours). Fig. 1B shows the cell proliferation curves of kidney carcinoma ACHN cells after the treatment with the crude mice uteri extracts on the 4 th and 9 th days of pregnancy. The cell proliferation curves are strongly inhibited after the treatments. Fig. 1C shows the cell proliferation curves of breast carcinoma ZR.75.1 cells after the treatment with crude mice uteri extracts on the 4 th and 9 th days of pregnancy. In all curves, a strong inhibition of cell proliferation after the treatment with pregnant uteri extracts is observed. Fig. 2A shows the mean proliferation curve of glioblastoma A172 cells after six separate experiments using crude pregnant uterine mucosa extracts of pig. The inhibition of extract-treated cell proliferation curve is about 75% after 24 hours and 45% after 48 hours compared to untreated cells. The statistical analysis (Student’s t-TEST) are highly significant (P<0.008) both after 24 and 48 hours. Fig. 2B shows the mean proliferation curve of melanoma A375 cells after two separate experiments using crude pregnant uterine mucosa extracts of pig. The inhibition of extract-treated cells proliferation curve is about 35% after 48 hours compared to untreated cells. Fig. 2C shows the mean proliferation curve of breast carcinoma ZR.75.1 cells after three separate experiments using pig crude pregnant uterine mucosa extracts. Cell curves are nearly stopped after 24 hours and 48 hours from the treatment. Statistical analysis (Student’s t-TEST) are significant (P<0.007 and P<0.047 at 24 and 48 hrs respectively). Fig. 2D shows the mean proliferation curve of NIH 3T3 murine fibroblasts after two separate experiments performed with pig pregnant uterine mucosa extracts. Untreated and treated cells proliferation curves nearly overlap . Fig. 3 shows the cell proliferation curve of lymphoblastic leukemia H9 cells after an individual experiment performed with crude pregnant uterine mucosa extracts of pig . The inhibition of extract-treated cells proliferation curves is about 80% after both 24 and 48 hours from the treatment. Fig. 4A shows the proliferation curve of glioblastoma A 172 cells line after the treatment using the 10 kDa fraction obtained from mouse pregnant uterus. The inhibition of the cell proliferation curve is about 80% after 24 hours and 40% after 48 hours from the treatment. Fig. 4B shows the mean proliferation curve of breast carcinoma ZR.75.1 cells after two separate experiments using the 10 kDa fraction obtained from pig pregnant uterine mucosa. The inhibition of fraction-treated cells proliferation curve is about 90% after 48 hour from the treatment. Fig. 5A shows an HPLC analysis of the 10 kDa fraction obtained from pig crude pregnant uterine mucosa. Four major peaks are visible, likely to correspond to 4 peptides. The HPLC profiles of both pig and mouse 5 kDa fraction (Fig. 5B and 5C) were similar and demonstrated the presence of only one major peak. Figg.6A, 6B and 6C show the cell proliferation curve of A 375 cells, ZR-75-1 cells and A 172 cells respectively, after individual treatments with pig crude pregnant uterine mucosa and the 5 kDa fraction. The 5 kDa fraction inhibited the proliferation of the three treated cells lines as well as the crude pregnant uterine mucosa extracts. At 48 hours after treatment of A172 cells, an even more marked effect on cell proliferation rate is observed by the 5 kDa fraction. Fig. 7 shows the increase of apoptosis in treated glioblastoma A172 cells after an individual experiment with pig crude uterine mucosa extracts compared to untreated cells ( zero value). In a previous work, we showed that pregnant uterus extracts inhibited the growth of tumors in vivo (1). Mice injected with Lewis lung carcinoma cells, a highly invasive line, did not develop primary tumor or metastases when treated with uterus extracts. Since the in vivo and in vitro behaviour of tumor cells with drugs is sometimes diverging, we have here investigated the effect of pregnant uterine mucosa on cultured tumor cells. First, we confirmed that mouse whole pregnant uterus extracts reduce dramatically tumor cell proliferation: in all investigated tumor cell lines, proliferation is quite arrested 48 hrs after the treatment. The cell number is even markedly reduced in breast carcinoma cells, suggesting that a massive cell death event may have occurred. Pig crude uterine mucosa extracts were less effective on tumor cell proliferation than those of mouse . The most satisfactory results were obtained with ZR.75.1, a breast carcinoma cell line, A172, a glioblastoma cell line, and H9, a T-cell lymphoma cell line. A375, a melanoma cell line, showed a slightly less significant response. Anyway, cell proliferation was always reduced but never quite arrested as we observed in the experiments with mouse extracts. It should be pointed out that murine fibroblasts NIH3T3 are not affected by the treatment with pig pregnant uterine mucosa, suggesting a somewhat fine in vivo mechanism by which an abnormally behaving cell clone is deleted. Thus, indeed pregnant uterus may be considered as a regulator of both normal and abnormal multiplication, as already suggested by our previous in vivo experiments (1) and by other authors (3,4). It may regulate cell proliferation by continuously monitoring the microenvironment surrounding the embryo: when a cell clone shows abnormous proliferation, pregnant uterus recognizes it, inducing death of all its cells. A similar mechanism, involving immunoregulatory compounds of murine placenta on activated cytotoxic T-cell clones, has already been described (5), relying on Fas-induced apoptosis (6). Although we cannot claim Fas role for inhibition of tumor cell proliferation by uterine extracts to date, an apoptosis-based mechanism may be involved, since a significative increase of nucleosome complexes in surnatant of cells 24 hours after the treatment was observed. Anyway, we cannot also exclude a role for other regulation pathways. It should be considered that the anti-proliferative effect of reproductive structures is not restricted to viviparous models, since it was also observed in vitro upon treatment of several tumor cell lines with fish embryo extracts at different stages of development (7). In our experiments with mouse whole uterus extracts, we considered uteri from different days pregnant mice. Actually, a very intriguing matter of study would be the screening of the activity of pregnant uteri extracts at different developmental stages on tumor cells behaviour. Early developmental stages, when the most decisive events of embryo differentiation and organogenesis take place, may require more efficient molecular mechanisms of embryo protection by the mother, by means of production of stage-specific regulatory molecules. Interactions between decidua and trophoblast were shown to be important for the modulation of the so-called innate immune system (8,9), and they may play a significative role in the control of proliferative events. As our data with 4, 5, 9 and 11 days-pregnant uteri extracts showed a general homogeneously strong response on tumor cell proliferation, future studies will add data from later developmental stages for a comparison. Moreover, we suggest that a low molecular weight fraction of the pregnant uterine mucosa may be in charge of inhibiting the growth of tumor cell lines; actually, the 10 kDa fraction significantly inhibited ZR.75.1 and A172 cell growth, while the 5 kDa fraction inhibited tumor cell proliferation at the same rate, or even more strongly, as the whole crude pregnant uterine mucosa extracts. Since only four peptides were distinguished by HPLC analysis in the 10 kDa fraction, and even one in the 5 kDa fraction, the inhibitory effect on tumor cells proliferation may be restricted to a very small number of molecules. HPLC of mouse and pig uterus extracts showed similar profiles, suggesting that the molecules involved in the anti-proliferative activity are shared by different species. We named the 5 kDa fraction “ Life Protecting Factor” (LPF), because its molecules may prevent both mother and embryo from life-compromising undesired proliferative events. Since the one-peptide 5 kDa fraction alone shows the most significant effects on tumor cell growth, it may be the leading LPF component, although the role of the other peptides should be better evaluated. They are present at very low concentrations in the 5 -10 kDa range, so that higher concentrations may have more evident inhibitory effects on tumor growth.. Works are in progress in order to confirm the in vitro proliferation-inhibiting function of LPF on other tumor cell lines, to isolate and characterise the individual LPF components, and to elucidate molecular pathways involved in this life-regulating mechanism. (traduzione dell'autore)

Pier Mario Biava

Fondazione per la Ricerca delle Terapie Biologiche del Cancro.

Ospedale Civile Sesto San Giovanni

Milano