- Email: [email protected]
Pineal peptide with an inhibiting effect on growth of HeLa S3 tumor cells
Peptides 24 (2003) 221–225
Pineal peptide with an inhibiting effect on growth of HeLa S3 tumor cells Petrelli Cristina∗ , Lupidi Giulio Dipartimento di Biologia Molecolare, Cellulare e Animale, Università di Camerino, Via Camerini n. 5, Camerino (MC) 62032, Italy Received 10 May 2002; accepted 24 September 2002
Abstract The aim of the present study was to assess whether a peptide fraction isolated from calf pineal glands has an effect on proliferation and morphology of HeLa S3 tumor cells. Under the experimental conditions adopted, the results showed that the peptide has a marked inhibitory effect on proliferation of HeLa S3 cells and that permeabilization with calcium phosphate of the plasmatic membrane increases this effect. Moreover, the pineal peptide affects the cytoskeletal morphology of HeLa cells by modifying the distribution of actin. The peptide is probably internalized by the cells and irreversibly modifies the cytoskeletal morphology with consequent inhibition of cellular proliferation. © 2003 Published by Elsevier Science Inc. Keywords: Permeabilization; Pineal peptide; HeLa cells
1. Introduction
2. Materials and methods
In the literature, a physiological relationship between the pineal gland and neoplastic growth has been discussed [8,10–12,14,15,17,20]. We have isolated from calf pineal mammalian glands a low molecular weight peptide of about 1200 Da, with the following aminoacid composition: glycine (one residue), threonine (one residue), lysine (one residue), alanine (six residues), glutamic acid (two residues). The purified peptide has inhibiting activity on DNA transcription in vitro [21], on RNA synthesis and proliferation in L1210 and HL60 tumoral cells [22] and on vitellogenin synthesis in Rana esculenta liver in vitro [23]. The peptide fraction we isolated appears different from those having antimitotic or antiblastic action extracted from pineal gland by other authors [1–7,20,27]. In fact, this pineal peptide inhibits the development of spontaneous and transplantable tumors, induced by chemical and ionizing radiation in mice and rats. In the present paper, we report the results of research performed on the effects of the peptide isolated in our laboratory on HeLa S3 cell proliferation, its ability to pass through the cellular membrane, and on the change in cellular morphology.
2.1. Pineal glands tissue
∗
Corresponding author. Tel.: +39-0737-403232; fax: +39-0737-636216. E-mail address: [email protected] (P. Cristina).
0196-9781/03/$ – see front matter © 2003 Published by Elsevier Science Inc. doi:10.1016/S0196-9781(03)00031-7
Pineal glands were removed from calves (from 5 to 8 months old) immediately after death, cleaned of contaminating material, wiped with filter paper, weighed and stored at −80 ◦ C until required for experimental use. 2.2. Extraction and purification of peptide Pineal aqueous extract and the isolation of a peptide fraction with inhibiting activity on DNA transcription were carried out according to Petrelli et al. [21]. The peptide purification from aqueous extract was performed using five successive purification steps: (a) ultrafiltration on SM 121-36 membrane (Mr cutoff 10,000); (b and c) gel filtration on Sephadex G 25 and gel filtration on Sephadex G 10 equilibrated and eluted with bidistilled water; (d) thin layer ascendent chromatography on aluminium sheet cellulose in t-butanol:formic acid:water (75:15:15); (e) high-performance liquid chromatography (HPLC) using a Supercosil LC 318 (particle 5 m) reverse phase column (4.6 mm × 25 cm) with precolumn (4.6 mm × 2 cm) equilibrated and eluted for 20 min with solvent A (0.1% trifluoroacetic acid in water) followed by a linear gradient (from 0 to 100%) solvent B (0.1% tricloroacetic acid in acetonitrile).
222
P. Cristina, L. Giulio / Peptides 24 (2003) 221–225
2.3. Protein and peptide determination Protein and peptide determination were performed following the procedures of Lowry et al. [18] and Nakai et al. [19], respectively. 2.4. Cell culture HeLa S3 cervical human carcinoma were used to test the growth inhibiting activity of the pineal peptide fraction obtained by HPLC. The HeLa S3 cells were grown at 37 ◦ C in a humidified atmosphere containing 5% CO2 and 95% air in Minimum Essential Medium Eagle, supplemented with 1% NEAA, 10% Newborn Bovine Serum, 2 mM l-glutamine, penicillin (100 IU/ml) and streptomycin (100 g/ml) (ICN Biomedicals, Belgium). Cell numbers were monitored by direct counting using a Coulter Counter ZM hemocytometer. Cell viability was assessed by Trypan blue exclusion. 2.5. Calcium phosphate-mediated transfection of HeLa S3 The cells were made permeable with calcium phosphate according to the method of Sambrook et al. [24]. 2.6. Fluorescence measurements Fluorescence measurements were performed using a Greg-200 ISS spectrofluorimeter thermostatted to 25 ◦ C for 20 min on plasmatic membranes. TMA-DPH (having amphiphatic properties) were used as fluorescent probes. For steady-state fluorescence anisotropy (r) measurements, the excitation and emission wavelengths were, respectively, 340 and 425 nm. The degree of TMA-DPH anisotropy was obtained by the following equation: R=
(I − I⊥ g) (I + 2I⊥ g)
Fig. 1. Effect of peptide on the proliferation over time of HeLa S3 cells in vitro, expressed as cell number/ml (×105 ). Initial inoculum: 1 × 105 cells/ml. The peptide was added at 0 time. The cells were cultured for 24, 48, and 72 h, without and with 0.5 g/100 l peptide. The error bars represent the S.D. of the mean, where n = 5.
Fig. 2. Effect of peptide on the proliferation over time of HeLa S3 cells, expressed as cell number/ml (×105 ). Initial inoculum: 1.5 × 105 cells/ml. The peptide (0.5 g/100 l) was added at 0 time in culture treated and not with calcium phosphate. The calcium phosphate was added at 0 time and removed after 24 h of cell incubation. The cells were cultured for 24, 48, and 72 h. The error bars represent the S.D. of the mean, where n = 5.
where g is an instrumental correction factor and I and I⊥ are the emission intensity polarized vertically and horizontally, respectively. Results of fluorescence were performed on at least three different samples with three measurements for each sample. Cells grown in the presence and in absence of inhibitor were washed with HEPES-NaOH 10 mM pH 7 + NaCl 0.135 M + KCl 2.5 mM + Na2 HPO4 0.33 M + glucose 1.6 mM. For fluorescence studies, washed cells were suspended in the wash buffer at a concentration of 2 × 105 cells/ml. Fluorescent probe, TMA-DPH [1-(4-trimethyl ammonium phenyl)-6-phenyl-1,3,5-hexatriene, p-toluene sulfonate], dissolved in absolute methanol was added with rapid mixing to the suspension to give a final concentration of 1 M. Control cell suspensions were treated with ethanol alone. Cells were incubated for 20 min at 25 ◦ C in the dark. After this time fluorescence polarization was performed at 25 ◦ C as previously described.
Fig. 3. Effect of peptide and taxol on the proliferation over time (24, and 48 h) of HeLa S3 cells, expressed as cell number/ml (×105 ). Initial inoculum: 1 × 105 cells/ml. The peptide (0.5 g/100 l) and taxol (10 M in DMSO) were added at 0 time. The cells were cultured without and with peptide or taxol. The error bars represent the S.D. of the mean, where n = 5.
P. Cristina, L. Giulio / Peptides 24 (2003) 221–225
2.7. Immunofluorescence preparations HeLa S3 cells were grown on coverslips and fixed with 3.7% paraformaldehyde/PBS for 8 min at room temperature. This solution was then substituted with PBS containing 0.2% Triton X-100 and, after 5 min at room temperature, the glasses were washed (three times) with PBS. The samples were then incubated with the first monoclonal antibody (anti-tubulin, Sigma Chemical Co.),
223
diluted 1:20 with PBS + BSA 1%, for 60 min at 37 ◦ C. After three washings with PBS, the cells were incubated with the second antibody, anti-rabbit immunoglobulin G, fluorescein isothiocyanate (FITC, Böringher) conjugate, diluted 1:60 with PBS + BSA 1%, for 60 min at 37 ◦ C. Finally, after three washings with PBS at room temperature, the glasses were placed on objective slides, and were readily examined by Fluorescence Microscope Olympus BH-2.
Fig. 4. (a–c) Confocal laser microscopy showing reactivity of the monoclonal antibody for tubulin against HeLa S3 cells. Confocal analysis of HeLa S3 cells cultured in the absence (a), and in the presence of pineal peptide (b) or of taxol (c) (see Section 2).
224
P. Cristina, L. Giulio / Peptides 24 (2003) 221–225
3. Results The results in Fig. 1 show that the calf pineal peptide purified by us causes an inhibition of HeLa S3 cell proliferation, as observed in other tumor cells L1210 and HL60 [22]; at 72 h the inhibition averaged 65%. To obtain information about the ability of the peptide to pass through the cell membrane, we studied the effect of cellular membrane permeabilization with calcium phosphate on proliferation of HeLa S3 cells in presence and in absence of the peptide in culture medium. The data in Fig. 2 show that the pineal peptide causes an inhibition of cellular proliferation of 80 and 64% when treated or not, respectively, with calcium phosphate. The inhibiting effect on permeabilized cells is not reversed by washing and reseeding the cells in fresh medium not containing the peptide effector. The steady-state fluorescence anisotropy measurements of plasmatic membranes as described in Section 2 did not evidence any statistically significant perturbation in HeLa S3 cells cultured in presence of pineal peptide. In the light of these results, the action of the pineal peptide on proliferation and on cytoskeletal changes in the HeLa S3 cells was compared with taxol which, as is known, irreversibly modifies the morphology of the cytoskeleton and inhibits tumor cell proliferation [9,25,26]. The data in Fig. 3 show that the presence in the culture medium of peptide (0.5 g/100 l) or taxol (10 M in DMSO) (a gift of Dr. Franco Venanzi) causes 80 or 100% inhibition of cellular proliferation, respectively. The results in Fig. 4a–c show that pineal peptide modifies the distribution of actin in the cytoskeleton of the HeLa S3 cells studied. The alteration, examined by fluorescence microscope, is quite similar to the alterations obtained with taxol. Research is in progress to clarify the cellular structure changes observed.
4. Discussion The data reported demonstrated that a peptide fraction purified from calf pineal gland strongly inhibits the growth of HeLa S3 tumor cells probably through an interaction with the cytoskeletal structure. The experiments performed with cells permeabilized by calcium phosphate seem to suggest that the peptide is able to pass through the cellular membrane: the permeabilization increases the peptide’s inhibitory activity. This result is noteworthy because in these experiments the peptide was removed from the culture medium together with the calcium phosphate after 24 h (see Section 2). On the contrary, the inhibitory effect observed with unpermeabilized cells was obtained with the peptide continuously present in the cell medium. The hypothesis that the control of cell proliferation is mediated by peptide internalization is supported also by evidence that the peptide does not cause any significant perturbation on HeLa
cells membrane, that should be expected if an interaction membrane receptor-peptide is involved. On the other hand, it should be not impossible for the pineal peptide to pass through the cellular membrane, since other Authors have demonstrated that the nonapeptide Val-Glu-Gly-Glu-Glu-Ser-Asn-Asp-Lys is internalized by several cell lines [13]. Similarly, the delta-sleep inducing peptide Trp-Ala-Gly-Gly-Asp-Ala-Ser-Gly-Glu has been reported as a membrane-permeable peptide [16]. In conclusion, the peptide probably is able to cross the plasmatic membrane and to interact with cytoskeletal constituents, to irreversibly modify the cytoskeletal morphology and to inhibit cellular proliferation, thus showing a mechanism similar to that exerted by taxol [9,25,26]. Experiments performed by washing and reseeding in fresh medium the peptide-treated cells, grown for 48 or 72 h in the absence of Ca/P, demonstrate that the inhibition caused by peptide is slowly removed. The inhibition is almost completely lost after two or three washing/reseeding processes. These experiments show that the effect caused by peptide is not cytostatic (data not shown). Therefore, on the whole, the results obtained could lead one to hypothesize that the biologic activity of the calf pineal peptide studied by us on DNA transcription in vitro in a specific system (data not shown) and a non-specific one [21,22], and in cellular cultures [22] may be an unlikely mechanism, even if we cannot exclude a direct action of the peptide on DNA nuclear transcription. References [1] Anisimov VN. Age as a risk factor in multistage carcinogenesis. In: Balducci L, Lyman GH, Ershler WB, editors. Comprehensive geriatric oncology. Amsterdam: Harwood Academic Publishers; 1998. p. 157–78. [2] Anisimov VN, Khavinson VK, Morozov VG. Carcinogenesis and ageing. IV. Effect of low-molecular weight factors of thymus pineal gland and anterior hypothalamus on the immunity tumour incidence and life span of C3H/Sn mice. Mech Ageing Dev 1982;19:245–58. [3] Anisimov VN, Loktionov AS, Khavinson VK, Morozov VG. Effect of low-molecular-weight factors of thymus and pineal gland on the life span and spontaneous tumour development in female mice of different age. Mech Ageing Dev 1989;49:245–57. [4] Anisimov VN, Khavinson VK, Morozov VG. Twenty years of study on effects of pineal peptide preparation: epithalamin in experimental gerontology and oncology. Ann NY Acad Sci 1994;719:483–93. [5] Anisimov VN, Mylnikov SV, Oparina TI, Khavinson VK. Effect of melatonin and pineal peptide preparation Epithalamin on life span and free radical oxidation in Drosophila melanogaster. Mech Ageing Dev 1997;97:81–91. [6] Anisimov VN, Mylnikov CV, Khavinson VK. Pineal peptide preparation Epithalamin increase the life span of fruit flies, mice and rats. Mech Ageing Dev 1998;103:123–32. [7] Anisimov VN, Khavinson VK, Mikhalski AI, Yashin AI. Effect of synthetic thymic and pineal peptides on biomarkers of ageing, survival and spontaneous tumour incidence in female CBA mice. Mech Ageing Dev 2001;122:41–68. [8] Aubert C, Prade M, Bohoun C. Effect de la pinéalectomie sur les tumeurs mélaniques de Hamster doré induites par l’administration (per os) d’une seule dose de 9,10-diméthyl-1,2-benzanthracène. CR Acad Sci (Paris) 1970;271:2465–6.
P. Cristina, L. Giulio / Peptides 24 (2003) 221–225 [9] Bacus SS, Gudkov AV, Lowe M, Liass L, Yung Y, Komarov AP, et al. Taxol-induced apoptosis depends on MAP kinase pathways (ERK and p38) and is independent of p53. Oncogene 2001;20:147–55. [10] Bartsch H, Bartsch C, Noteborn HPJM, Flehmig B, Ebels I, Salemink CA. Growth-inhibiting effect of crude pineal extracts on human melanoma cells in vitro is different from that of known synthetic pineal substances. J Neural Trans 1987;69:299–311. [11] Blask DE. The pineal: an oncostatic gland? In: Reiter RJ, editor. The pineal gland. New York: Raven Press; 1984. p. 253–84. [12] Blask DE, Leadem CA. Neuroendocrine aspects of neoplastic growth: a review. Neuroendocrinol Lett 1987;9(2):63–73. [13] Boraschi D, Ghiara P, Scapigliati G, Villa L, Sette A, Tagliabue A. Binding and internalization of 163–171 fragment of human IL-1. Cytokine 1992;4:201–4. [14] Carr DB, Axelrod L. The pineal gland, pineal dysfunction and cancer. Rev Endocr Relat Cancer 1981;8:13–21. [15] Das Gupta TK, Terz J. Influence of pineal gland on the growth and spread of melanoma in the hamster. Cancer Res 1967;27:1306–11. [16] Gray RA, Vander Velde DG, Burke CJ, Manning MC, Middaugh CR, Borchardt RT. Delta-sleep-inducing peptide: solution conformational studies of a membrane-permeable peptide. Biochemistry 1994;33:1323–31. [17] Lapin V. Pineal gland and malignancy. Osterr Z Onkol 1976;3:51–9. [18] Lowry OH, Rosenbrough MJ, Farr AL, Randall RJ. Protein measurement with the folin phenol reagent. J Biol Chem 1951;193:265–75.
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[19] Nakai N, Lai CY, Horecker BL. Use of fluorescamine in the chromatographic analysis of peptides from proteins. Anal Biochem 1974;58:563–70. [20] Noteborn HPJM, Bartsch H, Bartsch C, Mans DRA, Weusten JJAM, Flehmig B, et al. Partial purification of (a) low molecular weight ovine pineal compound(s) with an inhibiting effect on the growth of human melanoma cells in vitro. J Neural Transm 1988;73:135–55. [21] Petrelli C, Moretti P, Petrelli F, Barra D. Purification of a low molecular weight calf pineal peptide controlling DNA transcription in vitro. Int J Biochem 1992;41(3):170–82. [22] Petrelli C, Moretti P, Petrelli F, Bramucci M. A low molecular weight calf pineal peptide with an inhibiting effect on the growth of L1210 and HL60 cells. Cell Biol Int Rep 1992a;16(10):967–74. [23] Petrelli C, Mosconi G. A low molecular weight calf pineal peptide with inhibiting effect on vitellogenin synthesis in Rana esculenta, in vitro. Int J Biochem 2001;50(1/2). [24] Sambrook J, Fritsch EF, Maniatis T. Molecular cloning, laboratory manual. 2nd ed. New York: Cold Spring Harbor Laboratory Press; 1989. p. 16.33–.36. [25] Torres K, Horwitz SB. Mechanism of taxol-induced cell death are concentration dependent. Cancer Res 1998;58:3620–6. [26] Turner PF, Margolis RL. Taxol-induced bundling of brain-derived microtubules. J Cell Biol 1984;99:940–6. [27] Yuwiler A, Brammer GL. Neurotransmitters and peptides in the pineal gland. In: Wetterberg L, editor. Light and biological rhythms in man. Oxford: Pergamon Press; 1993. p. 133–44.
Pineal peptide with an inhibiting effect on growth of HeLa S3 tumor cells Petrelli Cristina∗ , Lupidi Giulio Dipartimento di Biologia Molecolare, Cellulare e Animale, Università di Camerino, Via Camerini n. 5, Camerino (MC) 62032, Italy Received 10 May 2002; accepted 24 September 2002
Abstract The aim of the present study was to assess whether a peptide fraction isolated from calf pineal glands has an effect on proliferation and morphology of HeLa S3 tumor cells. Under the experimental conditions adopted, the results showed that the peptide has a marked inhibitory effect on proliferation of HeLa S3 cells and that permeabilization with calcium phosphate of the plasmatic membrane increases this effect. Moreover, the pineal peptide affects the cytoskeletal morphology of HeLa cells by modifying the distribution of actin. The peptide is probably internalized by the cells and irreversibly modifies the cytoskeletal morphology with consequent inhibition of cellular proliferation. © 2003 Published by Elsevier Science Inc. Keywords: Permeabilization; Pineal peptide; HeLa cells
1. Introduction
2. Materials and methods
In the literature, a physiological relationship between the pineal gland and neoplastic growth has been discussed [8,10–12,14,15,17,20]. We have isolated from calf pineal mammalian glands a low molecular weight peptide of about 1200 Da, with the following aminoacid composition: glycine (one residue), threonine (one residue), lysine (one residue), alanine (six residues), glutamic acid (two residues). The purified peptide has inhibiting activity on DNA transcription in vitro [21], on RNA synthesis and proliferation in L1210 and HL60 tumoral cells [22] and on vitellogenin synthesis in Rana esculenta liver in vitro [23]. The peptide fraction we isolated appears different from those having antimitotic or antiblastic action extracted from pineal gland by other authors [1–7,20,27]. In fact, this pineal peptide inhibits the development of spontaneous and transplantable tumors, induced by chemical and ionizing radiation in mice and rats. In the present paper, we report the results of research performed on the effects of the peptide isolated in our laboratory on HeLa S3 cell proliferation, its ability to pass through the cellular membrane, and on the change in cellular morphology.
2.1. Pineal glands tissue
∗
Corresponding author. Tel.: +39-0737-403232; fax: +39-0737-636216. E-mail address: [email protected] (P. Cristina).
0196-9781/03/$ – see front matter © 2003 Published by Elsevier Science Inc. doi:10.1016/S0196-9781(03)00031-7
Pineal glands were removed from calves (from 5 to 8 months old) immediately after death, cleaned of contaminating material, wiped with filter paper, weighed and stored at −80 ◦ C until required for experimental use. 2.2. Extraction and purification of peptide Pineal aqueous extract and the isolation of a peptide fraction with inhibiting activity on DNA transcription were carried out according to Petrelli et al. [21]. The peptide purification from aqueous extract was performed using five successive purification steps: (a) ultrafiltration on SM 121-36 membrane (Mr cutoff 10,000); (b and c) gel filtration on Sephadex G 25 and gel filtration on Sephadex G 10 equilibrated and eluted with bidistilled water; (d) thin layer ascendent chromatography on aluminium sheet cellulose in t-butanol:formic acid:water (75:15:15); (e) high-performance liquid chromatography (HPLC) using a Supercosil LC 318 (particle 5 m) reverse phase column (4.6 mm × 25 cm) with precolumn (4.6 mm × 2 cm) equilibrated and eluted for 20 min with solvent A (0.1% trifluoroacetic acid in water) followed by a linear gradient (from 0 to 100%) solvent B (0.1% tricloroacetic acid in acetonitrile).
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P. Cristina, L. Giulio / Peptides 24 (2003) 221–225
2.3. Protein and peptide determination Protein and peptide determination were performed following the procedures of Lowry et al. [18] and Nakai et al. [19], respectively. 2.4. Cell culture HeLa S3 cervical human carcinoma were used to test the growth inhibiting activity of the pineal peptide fraction obtained by HPLC. The HeLa S3 cells were grown at 37 ◦ C in a humidified atmosphere containing 5% CO2 and 95% air in Minimum Essential Medium Eagle, supplemented with 1% NEAA, 10% Newborn Bovine Serum, 2 mM l-glutamine, penicillin (100 IU/ml) and streptomycin (100 g/ml) (ICN Biomedicals, Belgium). Cell numbers were monitored by direct counting using a Coulter Counter ZM hemocytometer. Cell viability was assessed by Trypan blue exclusion. 2.5. Calcium phosphate-mediated transfection of HeLa S3 The cells were made permeable with calcium phosphate according to the method of Sambrook et al. [24]. 2.6. Fluorescence measurements Fluorescence measurements were performed using a Greg-200 ISS spectrofluorimeter thermostatted to 25 ◦ C for 20 min on plasmatic membranes. TMA-DPH (having amphiphatic properties) were used as fluorescent probes. For steady-state fluorescence anisotropy (r) measurements, the excitation and emission wavelengths were, respectively, 340 and 425 nm. The degree of TMA-DPH anisotropy was obtained by the following equation: R=
(I − I⊥ g) (I + 2I⊥ g)
Fig. 1. Effect of peptide on the proliferation over time of HeLa S3 cells in vitro, expressed as cell number/ml (×105 ). Initial inoculum: 1 × 105 cells/ml. The peptide was added at 0 time. The cells were cultured for 24, 48, and 72 h, without and with 0.5 g/100 l peptide. The error bars represent the S.D. of the mean, where n = 5.
Fig. 2. Effect of peptide on the proliferation over time of HeLa S3 cells, expressed as cell number/ml (×105 ). Initial inoculum: 1.5 × 105 cells/ml. The peptide (0.5 g/100 l) was added at 0 time in culture treated and not with calcium phosphate. The calcium phosphate was added at 0 time and removed after 24 h of cell incubation. The cells were cultured for 24, 48, and 72 h. The error bars represent the S.D. of the mean, where n = 5.
where g is an instrumental correction factor and I and I⊥ are the emission intensity polarized vertically and horizontally, respectively. Results of fluorescence were performed on at least three different samples with three measurements for each sample. Cells grown in the presence and in absence of inhibitor were washed with HEPES-NaOH 10 mM pH 7 + NaCl 0.135 M + KCl 2.5 mM + Na2 HPO4 0.33 M + glucose 1.6 mM. For fluorescence studies, washed cells were suspended in the wash buffer at a concentration of 2 × 105 cells/ml. Fluorescent probe, TMA-DPH [1-(4-trimethyl ammonium phenyl)-6-phenyl-1,3,5-hexatriene, p-toluene sulfonate], dissolved in absolute methanol was added with rapid mixing to the suspension to give a final concentration of 1 M. Control cell suspensions were treated with ethanol alone. Cells were incubated for 20 min at 25 ◦ C in the dark. After this time fluorescence polarization was performed at 25 ◦ C as previously described.
Fig. 3. Effect of peptide and taxol on the proliferation over time (24, and 48 h) of HeLa S3 cells, expressed as cell number/ml (×105 ). Initial inoculum: 1 × 105 cells/ml. The peptide (0.5 g/100 l) and taxol (10 M in DMSO) were added at 0 time. The cells were cultured without and with peptide or taxol. The error bars represent the S.D. of the mean, where n = 5.
P. Cristina, L. Giulio / Peptides 24 (2003) 221–225
2.7. Immunofluorescence preparations HeLa S3 cells were grown on coverslips and fixed with 3.7% paraformaldehyde/PBS for 8 min at room temperature. This solution was then substituted with PBS containing 0.2% Triton X-100 and, after 5 min at room temperature, the glasses were washed (three times) with PBS. The samples were then incubated with the first monoclonal antibody (anti-tubulin, Sigma Chemical Co.),
223
diluted 1:20 with PBS + BSA 1%, for 60 min at 37 ◦ C. After three washings with PBS, the cells were incubated with the second antibody, anti-rabbit immunoglobulin G, fluorescein isothiocyanate (FITC, Böringher) conjugate, diluted 1:60 with PBS + BSA 1%, for 60 min at 37 ◦ C. Finally, after three washings with PBS at room temperature, the glasses were placed on objective slides, and were readily examined by Fluorescence Microscope Olympus BH-2.
Fig. 4. (a–c) Confocal laser microscopy showing reactivity of the monoclonal antibody for tubulin against HeLa S3 cells. Confocal analysis of HeLa S3 cells cultured in the absence (a), and in the presence of pineal peptide (b) or of taxol (c) (see Section 2).
224
P. Cristina, L. Giulio / Peptides 24 (2003) 221–225
3. Results The results in Fig. 1 show that the calf pineal peptide purified by us causes an inhibition of HeLa S3 cell proliferation, as observed in other tumor cells L1210 and HL60 [22]; at 72 h the inhibition averaged 65%. To obtain information about the ability of the peptide to pass through the cell membrane, we studied the effect of cellular membrane permeabilization with calcium phosphate on proliferation of HeLa S3 cells in presence and in absence of the peptide in culture medium. The data in Fig. 2 show that the pineal peptide causes an inhibition of cellular proliferation of 80 and 64% when treated or not, respectively, with calcium phosphate. The inhibiting effect on permeabilized cells is not reversed by washing and reseeding the cells in fresh medium not containing the peptide effector. The steady-state fluorescence anisotropy measurements of plasmatic membranes as described in Section 2 did not evidence any statistically significant perturbation in HeLa S3 cells cultured in presence of pineal peptide. In the light of these results, the action of the pineal peptide on proliferation and on cytoskeletal changes in the HeLa S3 cells was compared with taxol which, as is known, irreversibly modifies the morphology of the cytoskeleton and inhibits tumor cell proliferation [9,25,26]. The data in Fig. 3 show that the presence in the culture medium of peptide (0.5 g/100 l) or taxol (10 M in DMSO) (a gift of Dr. Franco Venanzi) causes 80 or 100% inhibition of cellular proliferation, respectively. The results in Fig. 4a–c show that pineal peptide modifies the distribution of actin in the cytoskeleton of the HeLa S3 cells studied. The alteration, examined by fluorescence microscope, is quite similar to the alterations obtained with taxol. Research is in progress to clarify the cellular structure changes observed.
4. Discussion The data reported demonstrated that a peptide fraction purified from calf pineal gland strongly inhibits the growth of HeLa S3 tumor cells probably through an interaction with the cytoskeletal structure. The experiments performed with cells permeabilized by calcium phosphate seem to suggest that the peptide is able to pass through the cellular membrane: the permeabilization increases the peptide’s inhibitory activity. This result is noteworthy because in these experiments the peptide was removed from the culture medium together with the calcium phosphate after 24 h (see Section 2). On the contrary, the inhibitory effect observed with unpermeabilized cells was obtained with the peptide continuously present in the cell medium. The hypothesis that the control of cell proliferation is mediated by peptide internalization is supported also by evidence that the peptide does not cause any significant perturbation on HeLa
cells membrane, that should be expected if an interaction membrane receptor-peptide is involved. On the other hand, it should be not impossible for the pineal peptide to pass through the cellular membrane, since other Authors have demonstrated that the nonapeptide Val-Glu-Gly-Glu-Glu-Ser-Asn-Asp-Lys is internalized by several cell lines [13]. Similarly, the delta-sleep inducing peptide Trp-Ala-Gly-Gly-Asp-Ala-Ser-Gly-Glu has been reported as a membrane-permeable peptide [16]. In conclusion, the peptide probably is able to cross the plasmatic membrane and to interact with cytoskeletal constituents, to irreversibly modify the cytoskeletal morphology and to inhibit cellular proliferation, thus showing a mechanism similar to that exerted by taxol [9,25,26]. Experiments performed by washing and reseeding in fresh medium the peptide-treated cells, grown for 48 or 72 h in the absence of Ca/P, demonstrate that the inhibition caused by peptide is slowly removed. The inhibition is almost completely lost after two or three washing/reseeding processes. These experiments show that the effect caused by peptide is not cytostatic (data not shown). Therefore, on the whole, the results obtained could lead one to hypothesize that the biologic activity of the calf pineal peptide studied by us on DNA transcription in vitro in a specific system (data not shown) and a non-specific one [21,22], and in cellular cultures [22] may be an unlikely mechanism, even if we cannot exclude a direct action of the peptide on DNA nuclear transcription. References [1] Anisimov VN. Age as a risk factor in multistage carcinogenesis. In: Balducci L, Lyman GH, Ershler WB, editors. Comprehensive geriatric oncology. Amsterdam: Harwood Academic Publishers; 1998. p. 157–78. [2] Anisimov VN, Khavinson VK, Morozov VG. Carcinogenesis and ageing. IV. Effect of low-molecular weight factors of thymus pineal gland and anterior hypothalamus on the immunity tumour incidence and life span of C3H/Sn mice. Mech Ageing Dev 1982;19:245–58. [3] Anisimov VN, Loktionov AS, Khavinson VK, Morozov VG. Effect of low-molecular-weight factors of thymus and pineal gland on the life span and spontaneous tumour development in female mice of different age. Mech Ageing Dev 1989;49:245–57. [4] Anisimov VN, Khavinson VK, Morozov VG. Twenty years of study on effects of pineal peptide preparation: epithalamin in experimental gerontology and oncology. Ann NY Acad Sci 1994;719:483–93. [5] Anisimov VN, Mylnikov SV, Oparina TI, Khavinson VK. Effect of melatonin and pineal peptide preparation Epithalamin on life span and free radical oxidation in Drosophila melanogaster. Mech Ageing Dev 1997;97:81–91. [6] Anisimov VN, Mylnikov CV, Khavinson VK. Pineal peptide preparation Epithalamin increase the life span of fruit flies, mice and rats. Mech Ageing Dev 1998;103:123–32. [7] Anisimov VN, Khavinson VK, Mikhalski AI, Yashin AI. Effect of synthetic thymic and pineal peptides on biomarkers of ageing, survival and spontaneous tumour incidence in female CBA mice. Mech Ageing Dev 2001;122:41–68. [8] Aubert C, Prade M, Bohoun C. Effect de la pinéalectomie sur les tumeurs mélaniques de Hamster doré induites par l’administration (per os) d’une seule dose de 9,10-diméthyl-1,2-benzanthracène. CR Acad Sci (Paris) 1970;271:2465–6.
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[19] Nakai N, Lai CY, Horecker BL. Use of fluorescamine in the chromatographic analysis of peptides from proteins. Anal Biochem 1974;58:563–70. [20] Noteborn HPJM, Bartsch H, Bartsch C, Mans DRA, Weusten JJAM, Flehmig B, et al. Partial purification of (a) low molecular weight ovine pineal compound(s) with an inhibiting effect on the growth of human melanoma cells in vitro. J Neural Transm 1988;73:135–55. [21] Petrelli C, Moretti P, Petrelli F, Barra D. Purification of a low molecular weight calf pineal peptide controlling DNA transcription in vitro. Int J Biochem 1992;41(3):170–82. [22] Petrelli C, Moretti P, Petrelli F, Bramucci M. A low molecular weight calf pineal peptide with an inhibiting effect on the growth of L1210 and HL60 cells. Cell Biol Int Rep 1992a;16(10):967–74. [23] Petrelli C, Mosconi G. A low molecular weight calf pineal peptide with inhibiting effect on vitellogenin synthesis in Rana esculenta, in vitro. Int J Biochem 2001;50(1/2). [24] Sambrook J, Fritsch EF, Maniatis T. Molecular cloning, laboratory manual. 2nd ed. New York: Cold Spring Harbor Laboratory Press; 1989. p. 16.33–.36. [25] Torres K, Horwitz SB. Mechanism of taxol-induced cell death are concentration dependent. Cancer Res 1998;58:3620–6. [26] Turner PF, Margolis RL. Taxol-induced bundling of brain-derived microtubules. J Cell Biol 1984;99:940–6. [27] Yuwiler A, Brammer GL. Neurotransmitters and peptides in the pineal gland. In: Wetterberg L, editor. Light and biological rhythms in man. Oxford: Pergamon Press; 1993. p. 133–44.