Experimental Oncology 25, 119-123, 2003 (June)
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ANTICANCER AND IMMUNOSTIMULATORY EFFECTS OF NUCLEOPROTEIN FRACTION OF BACILLUS SUBTILIS 7025 CULTURE MEDIUM FILTRATE V.À. Shlyakhovenko1 , S.V. Olishevsky1 , V.V. Kozak1, Y.V. Yanish1, S.L. Rybalko2 1 R.E. Kavetsky Institute of Experimental Pathology, Oncology and Radiobiology, National Academy of Sciences of Ukraine, Kyiv 03022, Ukraine 2 V.L. Gromashevsky Research Institute of Epidemiology and Infectious Diseases, Medical Academy of Sciences of Ukraine, Kyiv, Ukraine
ÏÐÎÒÈÂÎÎÏÓÕÎËÅÂÀß È ÈÌÌÓÍÎÑÒÈÌÓËÈÐÓÞÙÀß ÀÊÒÈÂÍÎÑÒÜ ÍÓÊËÅÎÏÐÎÒÅÈÄÍÎÉ ÔÐÀÊÖÈÈ ÔÈËÜÒÐÀÒÀ ÊÓËÜÒÓÐÀËÜÍÎÉ ÆÈÄÊÎÑÒÈ BACILLUS SUBTILIS 7025 Â.À. Øëÿõîâåíêî1, Ñ.Â. Îëèøåâñêèé1 , Â.Â. Êîçàê1 , Þ.Â. ßíèø1, Ñ.Ë. Ðûáàëêî 2 1 Èíñòèòóò ýêñïåðèìåíòàëüíîé ïàòîëîãèè, îíêîëîãèè è ðàäèîáèîëîãèè èì. Ð.Å. Êàâåöêîãî ÍÀÍ Óêðàèíû, Êèåâ, Óêðàèíà 2Êèåâñêèé íàó÷íî-èññëåäîâàòåëüñêèé èíñòèòóò ýïèäåìèîëîãèè è èíôåêöèîííûõ áîëåçíåé èì. Ë.Â. Ãðîìàøåâñêîãî ÀÌÍ Óêðàèíû, Êèåâ, Óêðàèíà Antitumor and immunostimulatory effects of nucleoprotein fraction of Bacillus subtilis 7025 (NPF) cultural medium filtrate have been investigated. Although a number of important questions remains to be answered our experiments indicated that NPF of Bacillus subtilis 7025 cultural medium filtrate can be effective as antitumor immunotherapeutic agent, has signifiα /γγ -producing activity of leukocytes. cant immunostimulatory effects and augments NK cell cytotoxicity and IFN-α Key Words: antitumor autovaccine, Bacillus subtilis 7025, bacterial DNA, CpG DNA, glycopeptide, interferons, NK cells, murine Erlich’s carcinoma, nucleoprotein fraction, vesicular stomatitis virus.  äàííîé ðàáîòå áûëà èññëåäîâàíà ïðîòèâîîïóõîëåâàÿ è èììóíîñòèìóëèðóþùàÿ àêòèâíîñòü íóêëåîïðîòåèäíîé ôðàêöèè ôèëüòðàòà êóëüòóðàëüíîé æèäêîñòè Bacillus subtilis 7025 (ÍÏÔ). Ñîãëàñíî ðåçóëüòàòàì èññëåäîâàíèÿ óñòàíîâëåíî, ÷òî ÍÏÔ Bacillus subtilis 7025 ìîæåò ÿâëÿòüñÿ ýôôåêòèâíûì ïðîòèâîîïóõîëåâûì èììóíîòåðàïåâòè÷åñêèì àãåíòîì, îáëàäàåò âûðàæåíîé èììóíîñòèìóëèðóþùåé àêòèâíîñòüþ è ïîâûøàåò àêòèâíîñòü α /γγ -ñåêðåòèðóþùóþ àêòèâíîñòü ëåéêîöèòîâ. åñòåñòâåííûõ êèëëåðíûõ êëåòîê è ÈÔÍ-α Êëþ÷åâûå ñëîâà: áàêòåðèàëüíàÿ ÄÍÊ, âèðóñ âåçèêóëÿðíîãî ñòîìàòèòà, ãëèêîïåïòèä, ÅÊ-êëåòêè, èíòåðôåðîíû, êàðöèíîìà Ýðëèõà ìûøåé, íóêëåîïðîòåèäíàÿ ôðàêöèÿ, ïðîòèâîîïóõîëåâàÿ àóòîâàêöèíà, Bacillus subtilis 7025, CpG ÄÍÊ.
Recent advances in our understanding of tumor immunology and the immune response in general are allowing the development of new rational approaches to cancer immunotherapy. One promising approach is immunization with tumor-specific proteins or peptides [3, 21, 22]. Recent studies suggest that development of an immune response to a tumor specific antigen after immunization correlates with improved clinical outcome. But such immunization can be insufficiently effective and requires combined application of some adjuvants or immunomodulators [3, 6] for example, complete Freund’s adjuvant, QS21, R-848, BCG, recombinant cytokines [6, 25, 26]. However, immunization using an adjuvant that induces the orchestrated activation of various immune subsets and the production of multiple Received: April 04, 2003. *Correspondence: E-mail:
[email protected] Abbreviations used: CpG — cytosine-phosphodiester-bond-guanine or cytosine-phosphorothioate-guanine; gp50 — glycopeptide tumor autovaccine; IFN — interferon; ILS, % — percentage increase in life span; MNC — mononuclear cells; NK cells — natural killer cells; NPF — nucleoprotein fraction of Bacillus subtilis 7025 cultural medium filtrate; PMA — phorbol myristate acetate; ST50% — survival time of 50% animals; VSV — vesicular stomatitis virus.
cytokines known to participate in the development of the active immune response is likely to be more effective and perhaps less toxic than immunization using single cytokines as an adjuvant. Microbial products, agents of natural and synthetic origin, and proteins derived from the immune system represent the immunomodulators that are presently used. The mechanisms of immunoenhancers include augmentation of antineoplastic activity by immune cells and the induction or restoration of immune effector functions. In 1893, Dr. William Coley, a New York surgeon, developed a crude bacterial extract that became known as “Coley’s toxins” [2, 28]. Coley administered his toxins to 894 patients with biopsy-proven carcinomas and sarcomas; he achieved a remarkable 45% five-year survival in these otherwise inoperable or untreatable patients. The various ingredients of Coley’s toxins were not well characterized but it can be surmised that the toxin must have contained several immunomodulatory microbial compounds, including endotoxin, bacterial cell wall components, and bacterial DNA [2, 28]. Accumulating data suggest that bacterial DNA plays an important role in the activation of the immune system
120 of mice [6, 10, 24, 26, 27]. In fact, bacterial DNA has significant immunostimulator y effects on B cells, monocytes, and NK cells and can induce production of many cytokines in vivo and in vitro that have been shown to be important in the development of antitumor immunity [6, 11, 20, 27, 29, 30]. In contrast, vertebrate DNA does not induce lymphocyte activation. It has been found that bacterial DNA contains a much higher frequency of unmethylated CpG dinucleotides than does vertebrate DNA due to CpG suppression and methylation of 80% of the CpG in vertebrates [10, 12, 27, 30]. It is now well established that humans and other vertebrates may detect unmethylated CpG dinucleotides in particular base contexts (“CpG motifs”) as a sign of danger of infection. At present time, attenuated mycobacteria are used for treatment of superficial carcinoma of the bladder. Yamamoto et al [30] found that administration of mycobacterial DNA is accompanied by induction of interferon secretion, and NK lytic activity and could result in tumor regression. A nucleic acid-rich fraction extracted and purified from BCG (MY-1) augmented NK cell activity of mouse spleen cells in vitro, and induced the production of factor(s) which showed anti-viral activity and rendered normal macrophages cytotoxic towards tumor cells (INF-γ and INF-α/β) [20, 24, 29, 30]. It is well recognized now that the immunostimulator y effects of specific motifs within bacterial DNA, including CG dinucleotides, certainly play an important role in the immunostimulatory effects of various bacterial preparations. Studies to date suggest that CpG DNA ma y have significant therapeutic potential in the treatment of a variety of disorders, including infectious disease, allergy, and cancer [6, 10, 27]. Taking to account that bacterial DNA with CpG motifs and synthetic CpG oligodeoxynucleotides have powerful activating influence on innate antitumor immunity and the reported antitumor and immunostimulatory effects of filtrate of Bacillus subtilis 7025 culture medium and respective vaccines [18, 31, 32], the present work was aimed on the research of antitumor and immunostimulatory properties of nucleoprotein fraction of Bacillus subtilis 7025 culture medium filtrate (NPF).
MATERIALS AND METHODS Tumor model. In the work, male BALB/c mice 8– 9 weeks old obtained from vivarium of R.E. Kavetsky Institute of Experimental Pathology, Oncology and Radiobiology NAS Ukraine (Kyiv, Ukraine) were used. The solid murine Erlich’s carcinoma has been used as a tumor model. Tumor cell suspensions in physiological saline were ordinarily prepared from tumor cells of ascitic transplantable Erlich carcinoma in final concentration of 2.5–3.0 · 106 cells/ml. Mice were inoculated subcutaneously with 2.5–3.0 · 10 5 viable cells/mouse in 0.1 ml (on day 0). Vaccination and immunization. Mice (separately for therapy and preventive treatment) were housed in 4 groups (10 animals per group) as 1 control group (untreated) and 3 — treated. Glycopeptide anti-cancer autovaccine (gp50) and nucleoprotein fraction (NPF) of Bacillus subtilis 7025 cultural medium filtrate were
Experimental Oncology 25, 119-123, 2003 (June) used separately and in combination for specific therapy and preventive treatment. Gp50 has been prepared from tumor cells of murine Erlich’s carcinoma by original method as described [22]. NPF was isolated from B. subtilis 7025 culture medium filtrate by the method [13]. The cultivation of B. subtilis 7025 was performed by standard method. The absence of proteins in NPF has been shown by electrophoresis in polyacrylamide gel. For in vivo and in vitro assays, NPF was diluted in sterile physiological saline and stored at 4 °C. At the 2 nd day after tumor cell inoculation animals were vaccinated in the footpad with gp50 only (or combined with NPF) in a dose of 25.000 cells/equivalents per mouse in total volume 100 µl; such vaccination was repeated triply with 24 h intervals. NPF was injected by the same route and schedule in the doses 5, 7.5 and 10 µg/mouse in 100 µl of physiological solution. At preventive therapy mice were vaccinated in the footpad with gp50 or with gp50 combined with NPF in a dose of 25.000 cells/equivalents per mouse in 100 µl of physiological solution triply with 1 week intervals. NPF was administered separately and in combination with gp50 triply by the same route and schedule in the doses 5, 7.5 and 10 µg/mouse in 100 µl of physiological solution. When the preventive therapy was completed, tumor cells of ascitic Erlich carcinoma (2.5 ·10 5 cells/mouse) were inoculated subcutaneously to treated mice. In vivo survival studies. The antitumor efficacy of therapy and preventive treatment by gp50, NPF, and their combination was evaluated by dynamics of tumor growth using the following parameters: increase in host’s life span, survival time of 50% animals (ST50%), and number of survived experimental animals at the end of experiment. Percentage increase in life span (%ILS) was calculated as: M S T T – MSTC %ILS = _____________ · 100%, M S TC where MST T — median survival time of treated mice; MST C — median survival time in control group (untreated mice). MST was calculated on the basis of mortality data. Preparation of effector cells. Splenocytes were obtained by the homogenization of spleens resected from 8–9 weeks-old white mice in Potter’s homogenizer to obtain single cell suspension. Mononuclear cells (MNC) were isolated by a standard Ficoll — Verografin technique [17]. Lymphocytes were purified by method [17]. MNC were cultured in RPMI-1640 medium (Sigma, USA) supplemented with 10% heat-inactivated fetal calf serum (Sigma, USA), penicillin (40 U/ml) (Kyivmedpreparat, Ukraine) and streptomycin (40 µg/ml) (Kyivmedprepa rat, Ukraine) [4]. The final concentration of lymphocytes was 7.5 · 10 6 cells/ml. Lymphocytes were incubated or without NPF or with NPF in concentrations 0.1 µg/ml and 1.0 µg/ml for 2 h at 37 °C in humidified athmosphere with 5% CO2, washed by centrifugation and used for cytotoxicity tests. The viability of the cells was determined by the conventional tr ypan blue dye exclusion test and was not less than 99%.
Experimental Oncology 25, 119-123, 2003 (June)
Table 1. Results of therapeutic applicat ion of NPF and gp50 Treatment I LS, % ST50%, % of animals that survived days at the day 125 Control — 73 0 gp50 12.7 81 10 NPF 24.5 88 10 gp50 + NPF 24.1 93 10 Table 2. Results of prophylactic application of NPF and gp50 Treatment I LS, % ST50%, % of animals that survived days at day 125 Control — 43 0 gp50 9.9 48 0 NPF 23.3 73 50 gp50 + NPF 24.0 62 10
No Therapy gp50 Therapy
80 60 40 20 0 45
65 85 105 Day post tumor inoculation
125
100
b
80
No Therapy NPF Therapy
60 40 20 0 45
RESULTS AND DISCUSSION
65 85 105 Day post tumor inoculation
100
125 c
No Therapy gp50 + NPF Therapy
80 Survival (%)
Immunization by NPF leads to protection from tumor growth. Therapeutic application of gp50 as vaccine has not shown high efficacy (Fig. 1, a): all unimmunized mice developed tumors and died within 82 days, with ST50% 73 days. Application of gp50 only had no marked protective antitumor effect — ILS was about 12.7% (Table 1). However, control animals died within 80 days, with ST50% of 73 days while mice vaccinated with gp50 died within 100 days and, moreover, 10% of animals remained alive to the end of experiment. Mice treated with NPF only had ILS value 24.5% (Fig. 1, b) and ST50% of 88 days, and 10% of animals remained alive to day of the experiment termination. In these experiments, the combined use of gp50 as a vaccine and
a
100
Survival (%)
A – B CI = ———— · 100%, A where A — the number of viable target cells in wells with only target cells, B — the number of viable target cells in wells with target and effector cells. IFN assay. For in vitro study of the IFN-stimulating activity of NPF, leukocytes of peripheral blood of human donors ( 3 · 10 6 cells/ml) were co-incubated with 100 µl of NPF in the doses 1 µg/sample and 2.5 µg/sample at 37 °C for 18–24 h in humidified athmosphere with 5% CO2, then centrifuged and the supernatants were collected. Each sample was divided in 2 parts: one is stored at 4 °C for 48–72 h unaltered and the second was adjusted to pH 2.0, incubated at 4 °C for 48–72 h and adjusted to pH 7.3; then IFN activity has been determined in supernatant samples by the method [7] with the use of vesicular stomatitis virus (VSV) and homologous cell culture A549. Statistical analysis. Survival curves were estimated using the Kaplan — Meyer method [9]. For statistical analysis, two-tailed Student’s t- test was applied [15]. p values < 0.05 were considered as significant.
NPF as an adjuvant doesn’t improved survival when compared with animals treated with NPF only (Fig. 1, c). Although immunotherapeutic approaches against cancer have always been more efficient in the prophylaxis than in the therapy, in our investigation prophylactic and therapeutic application of gp50 were nearly similar in antitumor efficacy (Tables 1 and 2) with ILS values 9.9% and 12.7% respectively, and ST50% values similar both in control and experimental groups (Fig. 2, a). In the group of mice with NPF preventive therapy, 50% animals survived at 125 day after tumor cells inoculation and had no
Survival (%)
Preparation of NK cell targets. Ascitic transplantable Erlich’s carcinoma cells were isolated from peritoneal cavity of male white 8–9 weeks old mice at 11 day after inoculation and were used as NK-resistant target cells in cytotoxic tests. Tumor cells were suspended in cultivation medium to concentration 2.5 · 106 cells/ml, and their number and viability were determined in microscopical supravital test with trypan blue. Cell viability was about 98%. Target cells were incubated with NPF likewise effector cells, washed by centrifugation and used for cytotoxicity tests. Their viability was about 98%. NK cell cytotoxicity analysis. All cytotoxicity tests were performed in microwell round-bottomed plates using target — effector cell ratio of 1 : 3 in total volume 200 µl/ well [23]. In controls, only control target cells and nutrient medium (control 1) or control target and effector cells were added (control 2). After incubation at 37 °C for 18 h in humidified athmosphere with 5% CO2, microplates were gently centrifuged (400 g, 5 min). The number of viable and dead target cells was determined using microscopical test with supravital staining with trypan blue. Cytotoxic activity of NK cells was expressed as cytotoxicity index (CI, %) [23] and was calculated as follows:
121
60 40 20 0 45
65 85 105 Day post tumor inoculation
125
Fig. 1. Survival of tumor-bearing animals upon therapeutic application of gp50 (a), NPF (b), or gp50 + NPF (c)
122
Experimental Oncology 25, 119-123, 2003 (June) a
100
No Therapy Therapy GP
60
CI, %
Survival (%)
80
40 20 0 60 80 100 Day post tumor inoculation
120
100
b
80
No Therapy Previous Therapy NPF
C 1 2 3 4 C — Control (TC + NK); 1 — TC + (NK + NPF0,1) ; 2 — TC + (NK + NPF1,0); 3 — (TC + NPF0,1) + NK; 4 — (TC + NPF1,0) + NK
CI, %
Survival (%)
40
60 40 20
a
40 35 30 25 20 15 10 5 0
35 30 25 20 15 10 5 0
b
1
0
Survival (%)
40
60 80 100 Day post tumor inoculation
120
100
c
80
No Therapy GP + NPF Previous Therapy
60 40 20 0 40
60 80 100 Day post tumor inoculation
120
Fig. 2. Survival of tumor-bearing animals upon preventive application of gp50 (a), NPF (b), or gp50 + NPF (c)
Table 3. Influence of NPF on the secretion of IFN by leukocytes of peripheral blood IFN activity (IU/ml) Concent ration of NPF Tot al IFN pH-stable I FN 1 µg/sample 2560 1280 2,5 µg/sample 2560 640
signs of tumor growth (Table 2, Fig. 2, b). Preventive therapy with combined application of gp50 and NPF gave positive effect on survival of experimental animals (Fig. 2, c) with ILS 24% and ST50% value 62 days; 10% of animals survived at day 125 (see Fig. 2, c). Our results indicated that NPF as immunotherapeutic agent possesses relatively high antitumor activity which is mediated by its strong immunostimulator y activity. Increase of NK cells cytotoxicity upon NPF treatment. Earlier it was reported [1, 8, 30] that mycobacterial DNA activates IFN-γ secretion and cytolytic activity in murine and human NK cells. We have studied the concentration-dependent influence of NPF on cytotoxic activity of NK cells and sensitivity of tumor cells to NK-mediated cytolysis in vitro and revealed that there is a correlation between named parameters (Fig. 3, a). Similar increase of NK cytotoxicity was observed when NK cells were incubated with 250 ng/ml of phorbol
2
3
4
1 — [NK + NPF0,1] + [TC + NPF0,1]; 2 — [NK + NPF1,0] + [TC + NPF1,0]; 3 — [NK + NPF0,1] + [TC + NPF1,0]; 4 — [NK + NPF1,0] + [TC + NPF0,1] Fig. 3. Augmentation of NK cell activity and sensitivity of target cells to NK cell mediated lysis after their separate (a) or combined (b) preincubation with NPF
myristate acetate (PMA) (unpublished data). PMA is standard stimulant of lymphoid-macrophage lines that increases the ability of lymphocytes to generate super oxide radicals and activates protein kinase C [16]. When target tumor cells were incubated with different concentrations of NPF, NK cell cytotoxicity increased, too (possibly due to the increased sensitivity of tumor cells to NK-mediated lysis), but in reverse correlation to NPF concentration. When NK cells and target tumor cells were preincubated with various concentrations of NPF, and then underwent coincubation, no correlation was observed. According to data of other authors [8, 29], stimulator y effects of CpG DNA on murine NK cells require either the presence of adherent cells or their CpG-conditioned supernatants, which contain IL-12, TNF-α, and type I interferons. However, our results point to possible direct activation of NK cells by bacterial CpG-DNA. Induction of IFN- α and –γγ upon NPF treatment. In contrast to LPS and other immunostimulator y agents, CpG DNA shows an extremely strong pattern of Th1like cytokine induction, in particular, interferons [19, 30]. IFN-α is promptly produced upon invasion of pathogens, and activates a broad range of effector cells in the innate and adaptive immune system and can play an important role in antimetastatic protection of organism [5, 19]. IFN-γ is one from major cytokines in antitumor protection; it is a strong promoter of Th1 immune response s and activates macrophages and NK cells. VSV cocultured with cells caused cytopathic effect associated with cell death or sometimes with the symplast formation. The main feature of VSV is distinct suppression of protein biosynthesis at the point of initiation of translation in host cells. VSV is especially sensitive to IFNs.
Experimental Oncology 25, 119-123, 2003 (June) Hence, we used method as described in [7] for the study of possible influence of NPF on IFN induction. The results have shown that NPF is potent inductor of IFNs synthesis by leukocytes of peripheral blood nearly equal in potency to standard stimulator of IFN induction polyI · poly C. In concentrations 1.0 and 2.5 µg/sample NPF induced secretion of IFN equal to 2560 IU/ml IFN-α. Secretion of IFN-γ by peripheral blood leukocytes was reduced at higher concentrations of NPF (1280 IU/ml and 640 IU/ml for 1.0 µg/sample and 2.5 µg/sample of NPF respectively). Our results are in agreement with data on the influence of bacterial DNA preparations on the level of IFN secretion [5, 19] suggesting that the induction of type I IFNs may be the pivotal step in the antitumor protection conferred by CpG DNA. A number of important questions remain to be answered. The molecular mechanisms responsible for NPF-induced immunostimulation and its anticancer activity remain unclear and are to be elucidated. A more complete understanding of the immunologic response to NPF of Bacillus subtilis 7025 cultural medium filtrate should promote the development of more rational and effective therapeutic strategies based on this new possible potent immunologic agent.
ACKNOWLEDGMENTS We thank V.M. Goncharenko for excellent technical assistance.
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