“….Therefore presented data suggest intratumoral treatment with bee venom for prevention of tumor growth as a possible mode of application for antitumor treatment.”
Toxicon Vol 41 Issue 7 June 2003 pages 861-870
Inhibition of mammary carcinoma cell proliferation in vitro and tumor growth in vivo by bee venom
a Department of Animal Physiology, Faculty of Science, University of Zagreb, Rooseveltov trg 6, 10 000, Zagreb, Croatia
b Department of Leukemia, The University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Blvd., Box 428, Houston, TX 77030, USA
c Department of Biology, Veterinary Faculty, University of Zagreb, Heinzelova 55, 10 000, Zagreb, Croatia
d Croatian Veterinary Institute, Savska c. 143, Zagreb, Croatia
The possible tumor growth- and metastasis-inhibiting effects of bee venom in mice and in tumor cell cultures were studied. The tumor was a transplantable mammary carcinoma (MCa) of CBA mouse. Intravenous administration of bee venom to mice significantly reduced the number of metastases in the lung. However, subcutaneous administration of bee venom did not reduce the number of lung metastases, indicating that the antitumor effect of the venom could be highly dependent on the route of injection as well as close contact between the components of the venom and the tumor cells, as was shown by in vitro studies on MCa cells. We also observed variations in immunological parameter induced by bee venom. We proposed that bee venom has an indirect mechanism of tumor growth inhibition and promotion of tumor rejection that is based on stimulation of the local cellular immune responses in lymph nodes. Apoptosis, necrosis, and lysis of tumor cells are other possible mechanisms by which bee venom inhibits tumor growth.
Bee venom is known to be a very complex mixture of active peptides, enzymes, and amines (Habermann, 1972 and Dotimas and Hider, 1987). The major components of bee venom are histamine, catecholamines, polyamines, melittin, and phospholipase A2. Melittin, the main component of bee venom is a strong basic polypeptide with molecular weight of 2850 kDa and a known amino acid sequence consisting of 26 amino acids (Habermann, 1972 and Dotimas and Hider, 1987). A haemolytic and strong cardiotoxic peptide, it constitutes 50% of dry venom and is the main toxin of bee venom ( Orlov et al., 1981; Shaposhnikova et al., 1997 and Hussein et al., 2001). Hait et al. demonstrated that melittin is one of the most potent inhibitors of calmodulin activity, and as such also is a potent inhibitor of cell growth and clonogenicity ( Hait et al., 1983; Hait et al., 1985 and Lee and Hait, 1985). Drugs that inhibit the activity of calmodulin have been shown to inhibit DNA synthesis in a glioblastoma cell line ( Okumara et al., 1982), to block movement of chromosomes during metaphase ( Means et al., 1982), to inhibit growth of Chinese hamster ovary cells ( Chafouleas et al., 1982), and to enhance cytotoxicity of vincristine, doxorubicin, and bleomycin ( Tsuro et al., 1982; Ganapathi and Grabowski, 1983 and Chafouleas et al., 1984). There is also some evidence suggesting that calmodulin inhibitors are cytotoxic to malignant cells both in vitro ( Hait et al., 1983 and Shaposhnikova et al., 2001) and in vivo ( Ito and Hidaka, 1983 and Shaposhnikova et al., 2001).
In the study presented here, the effects of bee venom on tumor growth, metastasis formation, and cytotoxicity to mammary carcinoma (MCa) cells and possible mechanism(s) of antitumor activity were evaluated.
Lyophilized whole bee venom was purchased from Medex (
Animal studies were carried out according to the guidelines in force in
Transplantable MCa of spontaneous origin in CBA mice was used. The tumor is weakly immunogenic in syngeneic recipients, as shown by different methods in vivo (Baic and Varga, 1979) and in vitro ( Eljuga et al., 1993).
2.4. Tumor cell suspension
Single cell suspensions were prepared by trypsin digestion of tumor tissue that contained no visible regions of necrosis or haemorrhage. Each suspension was passed through a stainless steel meash (200 wires/inch), centrifuged three times at 24g for 5 min in saline, and then resuspended in medium RPMI-1640 (
2.5. Production of tumor nodules (metastasis) in the lungs
Metastases were generated in the lungs by injecting 1Ã—105 viable tumor cells suspended in 0.5 ml of medium RPMI-1640 supplemented with 5% mouse syngeneic serum into the lateral tail vein of each mouse. Twenty-one days later, the mice were killed and their lungs removed. The lobes were separated and fixed in Bouin’s solution. Colonies of tumor cells were seen as white, round nodules on the surface of the yellowish lungs and were counted with the naked eye. This method of counting omitted any small colonies that may have developed deep inside the pulmonary lobes.
2.6. Weight and cellularity of spleens and lymph nodes
A single dose consisting of 150, 300, or 600 g of bee venom was injected subcutaneously (sc) into the left footpad each of five CBA mice; on day 14 after bee venom inoculation, recipient mice were killed. A fourth group of five mice (controls) received distilled water only as a placebo. Right and left popliteal lymph nodes (LPLNs) and spleens from five normal and five bee venom-treated mice were removed and weighed. Spleen of known weight or whole lymph nodes were minced and passed through a stainless steel meash. Suspensions of lymph node or spleen cells were then formed by dispersing the cells in 1 ml of saline solution and gentle movement in a syringe; cells were counted in a hemocytometer.
2.7. Response to polyclonal mitogens
Blastogenic responses to mitogens were assayed on days 3 and 7 after intravenous (iv) inoculation of bee venom (75 g/mouse). Routine cultures were done in triplicate in sterile microtiter plates (
2.8. Production of macrophage supernatants for lymphocyte activating factor assay
At days 3, 5, 7, 10, and 13 after treatment with bee venom, mice were killed and peritoneal macrophages were collected by a single washing of the peritoneal cavity with 5 ml of medium RPMI-1640 supplemented with 20 mM HEPES, 100 U/ml streptomycin, and 100 U/ml penicillin (Flow Lab,
2.9. Killing activity in vitro
Cytotoxic activity was tested in standard 4 h 51Cr release assays as elsewhere described (Eljuga et al., 1993). Briefly, effector cells (lymph node cells or splenocytes, 0.1 ml) were mixed with the 51Cr-labeled target cells (syngeneic MCa cells, 0.1 ml) in U-bottom 96-well microtiter plates in triplicate at different effector-to-target ratios. After centrifugation (60g for 3 min), plates were incubated for 4 h at 37 °C in a 5% CO2 humidified atmosphere. At the end of incubation, supernatants were collected and radioactivity from individual wells was evaluated by gamma counting. Results were expressed as percent of specific lysis. Percent of specific LYSIS=100Ã—(percent release from target in the presence of effectors [experimental release]âˆ’percent release from target in the presence of medium only [spontaneous release]/(maximal releaseâˆ’spontaneous release).
2.10. Lymphocyte phenotyping
Single suspensions of spleen or lymph node cells were prepared from the relevant tissues in calcium-and magnesium-free phosphate-buffered saline (PBS). Cell suspensions were filtered through a double layer of sterile gauze, then on a double layer column (wet with PBS), and centrifuged at 400g at 4 °C for 5 min. The pellet was washed and the recovered cells layered on a Ficol-Histopaque gradient (Sigma Chemical) and centrifuged at 700g for 20 min at 18 °C. The ring at the interface was washed three times and the resulting pellet diluted in PBS containing 0.5% bovine serum albumin and 0.1% sodium azide. Cells were labelled with anti-mouse monoclonal antibodies in the dark and on ice for the period of 30 min. Aliquots of 106 viable cells, counted with trypan blue exclusion, were added with rat anti-mouse CD4+ (0.5 g) or CD8+ (0.1 g) (Pharmingen, San Diego, USA). Controls were prepared with 106 cells labelled with nonspecific immunoglobulin (IgG)2a,k flourescein isothiocyanate (0.5 g) or IgG2b,k phycoerythrin (0.1 g); a sample containing only cells was used for autofluorescence determination. After washing, cells were resuspended in 0.5 ml of medium containing 10% of a 35% formaldehyde solution. Each analysis consisted of 10,000 events counted.
2.11. In vitro tumor cells cultures
MCa cells used for primary cultures were isolated from tumor growing in a mouse and maintained in RPMI-1640 with 10% fetal calf serum (FCS). MCa cells were grown in suspension culture at 37 °C in a humid atmosphere containing 5% CO2 in air. Second passage of tumor cells was used.
2.12. Cytotoxicity assay
Two methods were used to determine cytotoxicity: MTS assay and 3H-thymidine incorporation into DNA.
MTS assay. Cytotoxic effects of bee venom on HeLa and MCa cells were determined by the tetrazolium salt MTS (Owen’s reagent) assay according to the method of Mosman (1983). In brief, cells were plated in microplates, cultured in medium with various concentrations of bee venom, and incubated for 1, 2, or 3 days. MTS was added to each well, and the microplates were further incubated at 37 °C for 2 h. Viable cells converted MTS by dehydrogenase enzymes to its violet formazan. Formazan was quantified by using an automatic plate reader (Bio-Rad, Model 550,
3H””thymidine incorporation into DNA. Inhibition of 3H-methyl thymidine (3H-TdR) was studied by using the pulse-labelling method. Proliferating cells were incubated for 24 h, and then different concentrations of bee venom were added over different periods of time (24, 48, and 72 h). After exactly 24, 48, or 72 h, cells were exposed to 3H-TdR (1.0 Ci) for 3 h. Afterwards the cells were harvested onto glass fiber filters with an automatic harvester (Skarton, Norway).
2.13. Apoptosis analysis
Apoptosis was determined by techniques described by Telford et al. (1994). Briefly, bivariant flow cytometry was performed on cells grown in the presence or absence of the bee venom for various times (3 or 15 h). The cells were washed in cold PBS twice and resuspended in 100 l of binding buffer (HEPES containing 2.5 mM CaCl2). Fluorescein-labeled annexin V and propidium iodide (PI) were added to the cell suspension. Cells were then analyzed by flow cytometry (Becton Dickinson,
DNA content of the tested cells was determined by staining with PI. The cells were incubated in 100 l of fixing solution (PBS containing 4% formaldehyde) for 15 min at 4 °C, washed in PBS, resuspended in permeabilizing solution (PBS containing 1% FCS, 0.1% saponin, and 0.1% sodium azide) in the presence of 10 l of PI, and incubated at 4 °C for 15 min. The cells were then washed with PBS and immediately analyzed by flow cytometry. The percentage of cells to the left of the G1/G0 region, the A0 region representing apoptotic cells containing less than a diploid content of DNA, was estimated by gating the histograms.
The significance of differences between means of the groups was tested by the MannWhitney U test with the significance level <95%. Survival curves of animals with MCa were determined using method of Kaplan and Meier (1958) and the analysis of differences between survival curves was performed with the stepwise regression model of Cox ( Cox, 1972). Differences between groups were considered statistically significant at p<0.05.
We have investigated the effect of bee venom on lung colony formation. CBA mice were injected iv or sc with either 75 or 150 g of bee venom. Immediately after bee venom inoculation, 1Ã—105 MCa tumor cells were injected iv into each mouse. The mice were killed 20 days after the treatment and the number of metastases in the lungs was determined. In the group treated iv with bee venom in either dose, the number of tumor nodules in the lung was significantly lower (p<0.001) than in untreated mice (Table 1).
Each group comprised 7-9 mice. *Value was significantly different (p<0.001) from corresponding value in untreated mice.
Fig. 1 and Table 2 summarise whether the presence of bee venom in the tissue influenced tumor formation in and survival rate of CBA mice. Mice in each group were treated sc into the footpad with one of three doses of bee venom (150, 300, or 600 g/mouse). Immediately after bee venom treatment, the mice received a sc injection containing 1Ã—105 viable tumor cells into the very spot of bee venom inoculation. Tumor formation was monitored every 5 days. Table 2 illustrates that tumor formation was delayed in mice treated with bee venom and was influenced by the dose of bee venom. While all mice in the control group developed a tumor within 20 days after tumor cell inoculation and died, tumor growth in mice treated with bee venom was suppressed during the observation period of 25 days. Survival duration of mice treated with bee venom was significantly longer than that of the control mice, irrespective of bee venom dose ( Fig. 1).
Fig. 1. Overall survival of CBA mice inoculated with 105 MCa cells into the very spot of subcutaneous inoculation of different doses of bee venom. Tumor cells were introduced immediately after bee venom inoculation. Each group comprised seven to nine mice.
We further studied the effect of bee venom on immunological system of mice and possible links with local cellular immune response. As shown in Table 3 sc treatment with 300 or 600 g of bee venom resulted in significant increases (p<0.01) in the weight of the LPLN, lymph node corresponding to the footpad injected with bee venom, as compared to the right counterpart (RPLN). However, neither dose of bee venom influenced the spleen weight.
*Significantly higher (p<0.01) than in untreated mice. **Weight of LPLN was significantly higher (p<0.01) than that of RPLN.
The fact that either of the doses of bee venom used elicited greater cellularity in the LPLN (p<0.05;0.01) indicates that an intensive differentiation of lymphoid cells appears to occur as a local response to bee venom (Table 4). The cellularity of RPLN was significantly lower (p<0.05) than that of LPLN in the mice treated with 300 or 600 g of bee venom. No differences in cellularity of the spleens of mice treated with bee venom as compared to the spleens of untreated mice were noticed.
Significantly higher (p*<0.05;**<0.01) than in untreated mice.
Increased responses (p<0.05;<0.01) to PHA, Con A, and LPS of spleen cells from mice treated iv with bee venom were noticed 7 days after treatment (Table 5).
Significantly (p*<0.05;**<0.01) different from untreated controls.
The appearance of LAF in supernatants of adherent peritoneal cells (approximately 95% macrophages) from ip bee venom-treated mice was detected by the capacity of supernatants of peritoneal macrophages to increase the in vitro incorporation of 3H-TdR by mouse thymocytes. Bee venom suppressed production of LAF by peritoneal macrophages 5 and 7 days after bee venom injection as compared to untreated controls. The levels of LAF returned to normal or were increased 10 or 13 days after treatment (Table 6).
Significantly (p*<0.05;**<0.01;***<0.001) different than that of controls.
To test a possible mechanism(s) of antitumor activity, popliteal lymph node cells and splenocytes from mice who received footpad injections of different doses of bee venom were tested in vitro for lytic activity against syngeneic MCa cells. Fig. 2 shows that lytic activity of popliteal lymph node cells was significantly elevated in mice receiving bee venom into the corresponding footpad; the activity is likely to be dose dependent. On the other hand, treatment with bee venom did not influence activity of spleen lymphocytes.
Fig. 2. Lytic activity of corresponding lymph node cells and splenic lymphocytes of mice injected with bee venom into footpad against MCa target cells tested at an effector to target ratio 40:1. Lymphocytes were prepared from popliteal lymph nodes of mice injected with bee venom into the corresponding footpad. Each point represents the mean of three independent experiments ±SE.
In another study, popliteal lymph node cells and splenocytes were clustered as a function of the percentage of cells showing CD4+ and CD8+ antigens. Mice were injected with bee venom (300 g) into the footpad. Seven days after treatment bee venom significantly increased (p<0.05) the percentage of CD8+ cells in popliteal lymph nodes corresponding to the bee venom-treated footpad, whereas no apparent influence was observed in spleen cells. Treatment also changed the ratio between CD4+ and CD8+ cells in popliteal lymph nodes in favour of CD8+ cells (Fig. 3).
Fig. 3. Expression of CD4+ and CD8+ on cells from popliteal lymph nodes and splenocytes from mice injected into the footpad with bee venom (300 g/mouse). Cells were tested 7 days after bee venom inoculation. The data represent means±SE of three independent experiments.
Fig. 4 shows the effect of bee venom on the viability of MCa cells by the tetrazolium salt (MTS) assay. In these experiments, the concentrations of bee venom that inhibited growth by 50% (IC50) after incubation of MCa cells for 24, 48, or 72 h were as follows: 1.43; 2.15; and 2.15 g/ml, respectively.
Fig. 4. Effect of bee venom on viability of MCa cells. Cells were seeded at 2Ã—104 mlâˆ’1 in microplates and incubated for 24 h and than treated with different concentration of bee venom for the next 24, 48 or 72 h. MTS was added and cells were exposed for 2 h; blue formazan produced was measured at 490 nm on an enzyme-linked immunosorbent assay (ELISA) reader.
Inhibition of DNA synthesis was studied in the presence of bee venom (Fig. 5). Proliferating MCa cells were exposed to different concentrations of bee venom for 24, 48, or 72 h. The cells then were treated with 3H-TdR for 3 h. Synthesis of DNA in these cells was inhibited, with IC50 ranging from 0.95 to 1 g/ml after 24 or 48 h incubation with bee venom and from 1.5 to 1.7 after incubation for 72 h (Fig. 5). Degree of growth inhibition of MCa cells in the presence of bee venom was dose dependent to 24 h, while incubation with bee venom for 48 or 72 h did not result in any marked enhancement of cytotoxicity.
Fig. 5. Inhibition of 3H-thymidine (3H-TdR) incorporation into DNA of MCa cells treated with bee venom for different periods of time (24, 48, or 72 h). Cells were seeded at 2Ã—104/35 mm tissue culture dish and incubated for 24 h. Cells were treated with different concentration of bee venom for the next 24, 48 or 72 h. 3H-TdR (1 Ci/ml) was added and cells were exposed for 3 h. Radioactivity of cells was measured by scintillation counter. Numbers of cells incorporating 3H-TdR are expressed as percentages of counts of untreated cells.
Several anticalmodulin agents, including melittin, inhibit cell replication and clonogenicity (Comte et al., 1983; Sellinger-Barnette and Weiss, 1984; Hait et al., 1985 and Weiss et al., 1985), destabilizing cell membranes by impairing calcium fluxes. Since these mechanism(s) have been considered as a possible cause of apoptosis, we studied the effects of bee venom on apoptosis and necrosis of MCa cells. The change in cells associated with early phases of apoptosis is loss of cell membrane phospholipid symmetry. The effect of bee venom on MCa cells was analysed by fluorescein-labelled annexin V- and PI-stained cells ( Fig. 6). Annexin V, a member of the calcium- and phospholipid-binding proteins, binds strongly and specifically to phosphatidylserine. MCa cells were grown in the presence of 1.425 or 2.25 g/ml bee venom for 3 or 15 h, and cells were analysed by flow cytometry. As seen in Fig. 6, the early stage of apoptosis started 3 h after treatment of MCa cells with bee venom. Extent of apoptosis in MCa cells by bee venom after 3 h was 35.7% (dose 1.425 g/ml) or 41.1% (dose 2.85 g/ml) versus 20.8% in controls. At some point, the rates of MCa cell necrosis became similar. The percentage of cells in A0 by flow cytometry is another indicator of apoptosis; the degree of apoptosis induction by bee venom in MCa cells after 3 or 15 h was similar to the results described for annexin V.
Fig. 6. Effects of bee venom on induction of apoptosis and necrosis in MCa cells. Cells were cultured in the absence or presence of bee venom (1.425 or 2.85 g/ml) for 3 or 15 h, washed and stained with fluorescein-labeled annexin V and propidium iodide, and then analyzed by flow cytometry.
These investigations clearly demonstrated that antitumor and antimetastatic effects of bee venom could be highly dependent on route of injection and on close contact between bee venom and the tumor cells (Table 1 and Table 2, Fig. 1). Bee venom significantly inhibited metastases formation (p<0.001) only when administered by the same route as tumor cells (iv) at the time of tumor cell inoculation (Table 1). When, in other experiment, bee venom was injected intratumorally (tumor 7 mm in diameter), tumors decreased in size; some sort of shrinkage of tumors occurred, the delay of tumor growth was evident and survival of bee venom treated mice was significantly longer than of controls (unpublished). It is likely that higher doses of bee venom exhibited toxic effect on tumor tissues as was shown for blood, muscle and hart tissues in human and animals treated with bee venom or other components of it such as melittin and phospholipase A2 (Fletcher et al., 1992; Ownby et al., 1997; dos Reis et al., 1998 and Noble and Armstrong, 1999). Requirements for close contact between MCa and bee venom for in vivo effect was also shown in in vitro studies ( Fig. 4 and Fig. 5). This may be explained by inhibition of calmodulin as suggested by Lee and Hait, 1985 and Hait et al., 1985 or by the effect of bee venom on induction of apoptosis and necrosis of tumor cells described in these studies. The degree of growth inhibition of MCa cells in the presence of bee venom was dose dependent up to 24 h. It is likely that bee venom has a short half-life for the effect on thymidine incorporation since its active ingredients could be unstable in tissue culture medium.
The responses of regional lymph node cells were increased in animals treated with bee venom (Table 4, Fig. 2), as was also shown by Schneider and Urbanek (1984). The results related to the lytic activity of popliteal lymph node cells on MCa cells ( Fig. 2) indicate that bee venom is a strong activator of antitumor lytic activity of lymphoid cells deriving from the regional lymph node. Inactivity of spleen cells ( Fig. 2), in this respect, indicates that concentration of bee venom is an important factor for activation of antitumor lytic activity in mice. The possible mechanism(s) of antitumor lytic activity may include factors related to activation of cytotoxic T lymphocytes. Local treatment with bee venom increased the CD8+-T cell subset and led to a progressive reduction of the immune index (CD4+/CD8+ ratio) in favour of CD8+ cells (Fig. 3). A low CD4+/CD8+ ratio in lymph node, of 0.75, is observed in the treated group compared with the control group (1.43). There was a significant difference between the two groups (p<0.05). The CD4+/CD8+ ratio in spleen between control and treated group was 1.45 and 1.35, respectively. This implies that the phenotype of CD8+ cells increased by local stimulation of regional lymph nodes by bee venom may have an important role in tumor cytotoxicity. Moreover, according to Asaoka et al., 1993; Bomalski et al., 1995 and Goddard et al., 1996, the release of precursors of pro-inflammatory mediators caused by bee venom components such as phospholipase A2 and melittin might increase synthesis of IL-1 and TNF- in monocytes and the cellular response of T lymphocytes. Our findings contrast to those indicating that bee venom has an inhibitory effect on immunocompetent cells ( Hadjipetrou-Kourounakis and Yiangou, 1988 and Rekka et al., 1990). In addition, iv treatment with bee venom increased the response of spleen cells to polyclonal mitogens ( Table 5). Consistent with this observation is a report of Schneider and Urbanek (1984), who found an increased stimulation index among mouse lymphocytes from lymph nodes when cultured with bee venom in vitro.Thus, our findings showed that bee venom exerted a direct ( Table 1 and Fig. 1, Fig. 4, Fig. 5 and Fig. 6) and an indirect effect on tumor cells ( Table 3, Table 4, Table 5 and Table 6 and Fig. 2 and Fig. 3) trough the stimulation of the host cells, mainly macrophages and cytotoxic T lymphocytes as was also shown by others ( Jutel et al., 1995; Magnan et al., 2001; Kosnik and Wraber, 2000 and Palma-Carlos et al., 1999).
To conclude, it is likely that for antitumor activity of bee venom a close contact of bee venom and tumor cells are important for development of either apoptosis or necrosis of tumor cells in the tumor burden. Concomitant to these is a development of local cellular immune responses in lymph node draining the region of bee venom introduction. Therefore presented data suggest intratumoral treatment with bee venom for prevention of tumor growth as a possible mode of application for antitumor treatment.
Authors are grateful to Branimir Hackenberger, PhD for his help in statistical analysis of data.
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Corresponding author. Present address: Department of Animal Physiology, Faculty of Science, University of