Echinacea purpurea and Melatonin Augment Natural-Killer Cells in Leukemic Mice and Prolong Life Span

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ABSTRACT

Objective: We recently showed that daily dietary administration of Echinacea purpurea root extract to normal mice for as little as 1 week resulted in significant elevations of natural-killer (NK) cells (immune cells that are cytolytic to virus-containing cells and many tumor cells). Such boosting of this fundamental immune cell population suggests a prophylactic role for this herb in normal animals. Based on this evidence, our goal in the present work was to assess the role of dietary administration of this herbal extract to mice bearing leukemia, a type of tumor well known to be a target for NK cells.

Design: A commercially available root extract of E. purpurea, which we have already shown to be highly effective in mice, was administered daily for 50 days from the onset of leukemia (day 0). Control leukemic mice received no extract. Other leukemic mice received the NK-enhancing neurohormone, melatonin, administered precisely as above.

In all treatment and control categories, some mice were sampled at 9 days after tumor onset, others were sampled at 3 months, and still others were left to assess treatment effect on life span.

Results: At 9 days (intermediate stage leukemia; death beginning by day 17-18), E. purpureatreated mice had a 2.5-fold increase in the absolute numbers of NK cells in their spleens. By 3 months after leukemia onset, E. purpurea-treated mice still had 2-3 times the normal numbers of NK cells in their spleens. No leukemic, untreated (control) mice remained alive at 3 months, hence the comparison with normal animals. Moreover, at 3 months post-tumor onset, all the major hemopoietic and immune cell lineages in their bone marrow birth site, were recorded at normal numbers, in E. purpurea-consuming, leukemic mice.

The survival advantage provided by administering these leukemic mice with E. purpurea was highly significant versus untreated, leukemic mice when analyzed by Kaplan-Meier survival statistics.

Conclusion: The present study has provided the first systematic analysis, under controlled laboratory conditions, of the effect(s) of the botanical, E. purpurea, in vivo, in leukemic hosts. The profoundly positive effects of this herb in disease abatement observed in this study suggest the therapeutic potential of E. purpurea, at least with respect to leukemia, if not other tumors as well.
INTRODUCTION

WE HAVE RECENTLY DEMONSTRATED (Sun et al., 1999), that Echinacea purpurea-derived phytochemicals act as stimulants only to those cells responsible for nonspecific immunity, i.e., natural-killer (NK) cells, well-established killers of virus-containing cells and tumor cells. NK cells have long been recognized as the first line of defense against virus infected/tumor cells (Keissling et al., 1975; Riccardi et al., 1981; Biron and Welsh, 1982; Lala et al., 1986; Christopher et al., 1991). We have shown that daily, dietary administration of E. purpurea root extract for as little as 1 week, resulted in significant elevations of NK cells in the organ of their birth (bone marrow), as well as in the primary organ of their function (spleen), indicating a prophylactic role for this herb in normal animals (Sun et al., 1999). Even after 2 weeks of daily administration of E. purpurea, all hemopoietic and immune cells, with the exception of NK cells that remained elevated, were at levels precisely comparable to control (Sun et al., 1999). Extracts of the roots and other parts of various Echinacea species (E. purpurea, E. angustifolia), have come to prominence over the past decade for their reported (usually anecdotal) medicinal value including abatement and/or elimination of such pathologies as inflammations, viral/bacterial infections, acquired immune deficiency syndrome (AIDS), and even tumors (Stimpel et al., 1984; Tragni et al., 1985; Lersch et al., 1990, 1992; Roesler et al., 1991; Steinmuller et al., 1993; Hill et al., 1996).

Two mechanisms appear to be responsible for the beneficial effects imbued by Echinacea species-derived phytochemicals. One of the naturally occurring "drugs" from Echinacea species plants that has already been identified, is the polysaccharide, arabinogalactan, a 75,000 MW molecule known to stimulate monocytes and macrophages (in vitro) and shown to be cytotoxic for microorganisms (Luettig et al., 1989). Arabinogalactan also stimulates macrophages (Stimpel et al., 1984: Leuttig et al., 1989; Wagner and Jurcic, 1991; Fry et al., 1998) to secrete the cytokines tumor necrosis factor-alpha (TNF-alpha), interferons, and interleukin-1 (Il-1), all potent enhancers of NK cells. Second, certain alkamides contained within Echinacea species plants are inhibitors of cyclooxygenase and 5-lipoxygenase (Wagner et al., 1984; Muller-Jakic et al., 1994), key enzymes in the production of prostaglandins, the latter being potent suppressors of NK cells (Fulton and Heppner, 1985; Lala et al., 1986). We have already demonstrated that the prostaglandin inhibitor, indomethacin, results in NK cell stimulation in leukemic mice (Christopher et al., 1991; Miller et al., 1992; Dussault and Miller, 1993). Thus, while the polysaccharide (arabinogalactan) results in the production of NK stimulators, other Echinacea species products (alkamides) release NK cells from their natural endogenous inhibitors, i.e., prostaglandins.

Melatonin, however, is a neuroimmunomodulator, a pineal gland hormone and biogenic indoleamine (N-acetyl-5 methoxytryptamine), long known to be a chronomodulator in biologic systems. The hormone acts to functionally synchronize eukaryotes with the photo-period. Endogenous melatonin production follows a strict circadian rhythm, being maximally produced during the dark periods of the 24-hour cycle, and broken down soon after production, having species-dependent (hamster, rat, donkey, monkey, human), and even age-dependent half-lives in the blood ranging from 7.5 to 59 minutes (Mallo et al., 1990; Pang et al., 1990; Cavallo and Ritschel, 1996; Yellon, 1996; Brown et al., 1997; Yeleswaram et al., 1997). We have recently demonstrated (Currier et al., 2000), that exogenously administered melatonin selectively enhances the population size of NK cells when given daily in the diet for 1 week or for 2 weeks to healthy, young adult mice, suggesting a potentially prophylactic role for this agent, a phenomenon not dissimilar from that which we observed employing E. purpurea (Sun et al., 1999). With respect to the mechanism responsible for the influence of melatonin on NK cells, there is evidence in mammals indicating that melatonin in vivo, enhances the lytic functions of mature NK cells (Poon et al., 1994). However, it is completely unknown whether this results directly from receptor-ligand-type interactions between melatonin and NK cell surface receptors, or indirectly, via melatonin-stimulated, T-helper cell-enhanced production of the cytokine, Il-2. That the latter event is most probable derives from the fact that T-helper cells bear receptors for melatonin and Il2 is an exquisite stimulant of NK cell numbers and function (Christopher et al., 1991; Naume and Espevik, 1991).

Thus, the selective, positive influence of in vivo administered E. purpurea, or melatonin, on cells mediating nonspecific immunity, i.e., NK cells, suggests a plausible mechanism behind the numerous, anecdotal claims over the years, that these agents may have a role in tumor abatement in humans, although the mode of action(s) has remained unknown and, to date, unstudied.

The literature contains only minimal information concerning life span increment, or the status of normal hemopoietic or immune cells in tumor-bearing hosts given a botanical. For example, Nanba et al. (1987), have administered orally to tumor-bearing mice, fruit bodies from the plant Lentinus edodes, with positive results. However, nothing is known of NK cell population kinetics/dynamics, in vivo, even though these cells represent the first line of defense in tumor combat, in leukemic hosts (or in hosts bearing other tumors). It was the goal of the present study to administer Echinacea species-derived phytocompounds, under controlled laboratory conditions including use of (1) animals of identical species (strain), age, and gender, (2) tumor of precisely known stage of development, (3) therapeutic agents (E. purpurea, melatonin) of regulated dose and exposure time, and (4) standardized housing conditions for all treated and control animals. The hypothesis giving rise to this study, derives from the fact that the leukemias and lymphomas are targets of NK cells. Moreover, the leukemia of this study is virus-derived. Hence, any agent that enhances the immune mechanism directed at virus-harboring cells, i.e., NK cell-mediated immunity, may be expected to be ameliorative in leukemia-stricken animals and possibly humans.
MATERIALS AND METHODS

Mice

Male DBA/2 mice (Charles River Laboratories, St. Constant, QC, Canada) were obtained at 5 weeks of age, and housed under laminar flow conditions in the animal Facilities of McGill University. Sentinel mice in the facility regularly demonstrated the absence of all common mouse pathogens. All mice remained healthy until 10 weeks of age at which time tumor was initiated.

Tumor cells: maintenance and administration

Friend-virus-induced erythroleukemia cells (American Type Culture Collection, Camden, NJ) were maintained in vitro at 37 Celsius, 100% humidity and 5% CO2 in basal Eagle's medium supplemented with 15% Cellect Gold Serum, 2% essential amino acids, 2% nonessential amino acids (Flow Laboratories, Mississauga, ON, Canada), L-glutamine (GIBCO BRL Life Technologies, Grand Island, NY), 3% sodium bicarbonate, 7.5 mol/L (Sigma Chemicals, St. Louis, MO), and 1% HEPES (Boehringer Mannheim, Montreal, QC, Canada), at a concentration of 2 mol/L. These in vitro-maintained erythroleukemia cells served as the stock from which cells were extracted to initiate tumor-bearing hosts. Each mouse was aseptically injected with 3 x 106 viable tumor cells in 0.1 mL phosphate-buffered saline (PBS; pH 7.2) via the tail vein. Tumor cells were used during log phase growth with a viability of 90% or more.

In vivo administration of melatonin and E. purpurea

Melatonin (Schiff Products Inc., Salt Lake City, UT) was provided in the diet, ground homogeneously into pulverized Purina laboratory mouse chow, the standard diet. Mice of identical age, strain, and gender, were housed two per cage and each evening (6:00 PM) given fresh ground chow with/without (control) melatonin (0.0142 mg per mouse per day), from day 0 after tumor onset. Some mice were killed on day 9 thereafter, while other mice, to be taken at 3 months post-tumor onset, continued to receive melatonin daily from day 0 (time of tumor onset) to day 50, after which their diet until 3 months was identical to control (pulverized chow only). We have previously established the optimum dose of melatonin, optimum time of day to begin exposure via the chow, and the daily rate of chow consumption per day per mouse (Yu et al., 2000; Currier et al., 2000).

E. purpurea was, similar to melatonin, administered via the chow, into which it was homogenized from a specific, commercially prepared powder extract of the root (Phyto Adrien Gagnon, Sante Naturelle [A.G.] Ltee, La Prairie QC, Canada). Each mouse consumed 0.45 mg/d, a dose per body weight that we have already proven to be effective (Sun et al., 1999). Moreover, there appears to be no maximum dose at which this herb, in vivo, is toxic (Mengs et al., 1991; Lersch et al., 1992; Melchart et al., 1995). As with the administration of melatonin (above), chow was prepared and given fresh daily following the identical protocol (above). Control mice consumed the same, pulverized chow, without E. purpurea.

In some experiments, mice consumed both melatonin and E. purpurea, administered as above, simultaneously, with all other parameters unchanged.

Tissue extraction; hemopoietic and NK cell labeling

At the indicated intervals after dietary melatonin and/or E. purpurea exposure in tumor-bearing mice (9 days, 3 months), the mice were killed by cervical dislocation and free cell suspensions were made of the spleens and bone marrow (femurs) of individual mice from all experimental (melatonin only, E. purpurea only, melatonin 1 E. purpurea) and control groups, by methods commonly in use in our laboratory (Christopher et al., 1991; Miller et al., 1992; Dussault and Miller, 1993, 1994, 1995; Whyte and Miller, 1998; Currier and Miller, 1998; Mahoney et al., 1998; Sun et al., 1999; Currier et al., 2000). On the resulting cytospot preparation of the free cell suspensions from each tissue, NK cells were immunolabeled for the presence of the ASGM-1 surface marker, a hallmark of NK cell functional activity, found on all mature and maturing NK cells (Kasai et al., 1980; Beck et al., 1982; Stout et al., 1987), by means of the VECTASTAIN (Vector Laboratories, Burlingame, CA) method (Miller et al., 1992; Dussault and Miller, 1994, 1995; Whyte and Miller, 1998; Currier and Miller, 1998; Mahoney et al., 1998; Sun et al., 1999; Currier et al., 2000). The cytospot preparations were subsequently stained with MacNeal's tetrachrome hematologic stain to permit enumeration and recording of all other hemopoietic cell lineages (lymphoid, myeloid, nucleated erythroid, monocytoid), each readily identifiable--including precursor and mature forms--by tetrachrome-enhanced, distinct morphologic criteria. These techniques are all well-established and in regular use in our laboratory.

Next, from the proportions of each of several distinct subgroups, (lymphoid, myeloid, nucleated erythroid, monocytoid cells), recorded by scanning (reading) 2000 cells per cytospot (2-5) per tissue per mouse per experiment (or control), the absolute numbers of each of these cell populations could be determined from the total known cellularity of each tissue (spleen, femurs) obtained previously by means of an electronic cell counter (Coulter Electronics, Hialeah, FL). NK cells, lymphoid in morphology, yet distinct from all other small/medium lymphoid cells, by virtue of their bearing the ASGM-1 surface marker, were separately enumerated from a total of 1,000 lymphoid cells per tissue per mouse per experiment (or control).

Finally, groups of mice from each of the treatment protocols, as well as groups of untreated, leukemic mice, were retained for the purposes of recording life span.

Statistical analysis

The Student's t test was used to compare the differences between the means of the various cell subpopulations (above), of the spleen and bone marrow in experimental (melatonin and/or E. purpurea-treated) groups versus untreated control. Kaplan-Meier survival analysis statistics were used to assess the effect of the various treatments singly or in combination, on life span of leukemic hosts.
RESULTS

Figure 1 indicates that daily, dietary administration of either melatonin or E. purpurea, beginning at the time of tumor onset (day 0), and for 9 days thereafter, resulted in a 2.5-fold increase in the absolute numbers of splenic NK cells. Coadministration of the two agents for the 9-day period, by contrast, had no significant effect on splenic NK cell population numbers (Fig. 1). In the bone marrow, administration of either agent, or both together for 9 days after tumor onset, had no significant effect on the NK cell population size, relative to control, in that organ (Fig. 2).

By 3 months after tumor onset, no control mice remained alive (Figs. 1 and 2). In the spleen (Fig. 1), the NK cell population sizes of E. purpurea-treated, and melatonin-treated mice, ranging from 7-10 x 106 pernormal for mice of this age. We have previously found the normal numbers of approximately 3 x 106 per spleen (Mahoney et al., 1998). However, the spleen-localized NK cells in E. purpurea-treated, and melatonin-treated mice, were significantly more numerous (p < 0.04 and p < 0.01, respectively) than in mice treated with melatonin plus E. purpurea. The NK cells of the bone marrow at 3 months after tumor onset were not significantly different in mice treated with either melatonin, or E. purpurea, or with both agents (Fig. 2).

Figure 3 indicates that life span of tumor-bearing mice was significantly improved by administering either melatonin or E. purpurea or both agents simultaneously from the early stages of tumor onset. Whereas all untreated, leukemic mice were dead by 27 days after tumor onset, approximately one-third of melatonin-treated, and similar proportions of E. purpurea-treated, tumor-bearing mice survived not only until 3 months but beyond, indicating long-term survival and/or cure. The best survival rates, i.e., almost 50% of leukemic mice, were obtained when both agents were coadministered (Fig. 3).

With respect to the other hemopoietic cell lineages in the spleen, there was, at 9 days after tumor onset, no effect of melatonin, or E. purpurea administered separately, on any major cell population (Table 1). There was, however, at this time, a significant reduction in the population sizes of non-NK lymphoid cells and erythroid cells, relative to control, when both agents were coadministered. (Table 1). By 3 months after tumor onset, there was no significant change in the numbers of cells in any of the major hemopoietic lineages in the spleen, when both agents were coadministered versus either agent alone (Table 1).

In the bone marrow of tumor-bearing mice, a significant reduction in the cell numbers in most but not all, of the hemopoietic lineages was observed at 9 days after tumor onset, when either E. purpurea alone, or melatonin alone, was administered, but not when both were given together, relative to untreated controls (Table 2). At 3 months after tumor onset, exposure to either agent, or both agents simultaneously, resulted in the numbers of cells in the bone marrow (Table 2), in each of the major hemopoietic lineages, being virtually indistinguishable from our established findings in normal mice of this age (Miller and Osmond, 1974, 1975; Mahoney et al., 1998). Thus, in the presence of E. purpurea and/or melatonin, resumption of normal hemopoiesis and lymphopoiesis in these treated, long-term survivors of leukemia has occurred.
DISCUSSION

The present study has provided a systematic analysis, under controlled laboratory conditions, of tumor amelioration in vivo, using nonconventional therapeutic agents, i.e., melatonin and phytocompounds of the herb, E. purpurea. At 9 days after tumor onset, a stage that we have previously established as being intermediate in the development of this leukemia, we have shown that dietary E. purpurea, a naturally occurring botanical that is commercially available, inexpensive, and nontoxic at any dose (Mengs et al., 1991; Lersch et al., 1992; Melchart et al., 1995), significantly prolonged life span to levels even higher than those that we have already observed using the cytokine, IL-2, the prostaglandin inhibitor, indomethacin, and the interferon inducer, Poly I:C (Christopher et al., 1991; Dussault and Miller, 1993; Currier and Miller, 1998). Indomethacin is beset with side effects, including hemorrhage, whereas the naturally occurring molecules, IL-2 and interferon, are the antithesis of E. purpurea being toxic when exogenously administered, even at low doses over time (Djeu et al., 1979; Rosenstein et al., 1986; West et al., 1987; Kohler et al., 1989) and must be regulated in a patient-specific, dose-frequency manner. Moreover, in recombinant form the latter are, financially prohibitive, and not commercially available. Melatonin, however, the second agent used in the present study, is similar to the cytokines (IL-2, interferon), a naturally occurring molecule, present in vivo in minute quantities. Exogenously administered, it is subject to potential dose-frequency problems of feedback inhibition, and thus, can interfere with the endogenous, photoregulatory rhythyms. However, melatonin is considerably less expensive, and commercially readily available.

Together with the survival advantage provided by E. purpurea (and melatonin) was the fact that the immune cells whose numbers were significantly augmented were those acting at the first line of defense in combat against developing neoplasms, i.e., NK cells. Moreover, it has been well demonstrated that absolute increases in NK cell numbers, such as observed in the present study, does indeed reflect an increase in the functional armament available, i.e., for tumor combat (Keissling et al., 1975; Kasai et al., 1981; Biron and Welsh, 1982; Itoh et al., 1982; Hefeneider et al., 1983: Koo et al., 1986; Lotzova et al., 1986; Kalland, 1987). Interestingly, in all treated tumor-bearing hosts, at both time intervals studied, i.e., 9 days and 3 months, the bone marrow revealed no significant change in NK cell numbers regardless of treatment (E. purpurea alone, melatonin alone, or both together). The spleen (the destiny of the vast majority of bone marrow-generated NK cells) contained (at 9 days and 3 months) significantly fewer NK cells when E. purpurea plus melatonin were given, but not when either agent was given alone. It is probable that a trafficking shift of many of the newly generated, bone marrow-derived NK cells, under double agent treatment (E. purpurea plus melatonin), but not single agent treatment (E. purpurea or melatonin), to organs other than the spleen, has occurred. Such a phenomenon has be demonstrated in other sustained, nonphysiologic situations (Biron et al., 1983). However, why such a shuttling of many newly generated, bone marrow-derived NK cells to organs other than the spleen should occur in the presence of both agents, but not each alone, is unclear. It is certain, nevertheless, that in the presence of both agents together in vivo, new NK cell production in the bone marrow birth site has not been impaired (NK cell numbers statistically unchanged regardless of treatment) indicating no "overdose" (toxic) effect from the presence of both agents together. Moreover, we have already demonstrated a phenomenon similar to this, i.e., reduced splenic NK cells when indomethacin and IL-2 (both powerful NK stimulants similar to E. purpurea and melatonin), were coadministered, but not when each was given alone (Christopher et al., 1991; Dussault and Miller, 1993).

Many of the other non-NK cell populations in the spleen and the bone marrow after 9 days of treatment have been reduced when E. purpurea, or melatonin, or both together, are given versus untreated leukemic mice. This may reflect early adjustments/interplay among several complex nonphysiologic phenomena involving the presence of growing tumor, and one or both of two exogenously introduced agents (E. purpurea, melatonin). Furthermore, at 3 months post-tumor onset, no tumor (erythroleukemic) cells were microscopically found. Moreover, scans of the cytospots of several thousand hemopoeitic and immune cells in both organs in all mice under study revealed that the absolute numbers of nucleated erythroid precursors observed in the bone marrow were comparable to that found in normal mice in the age range of these long term survivors, i.e., 5-6 months (Miller and Osmond, 1974; 1975; Mahoney et al., 1998). These observations, thus, signal the absence of erythroleukemic blast regrowth.

The tumor employed in this study (erythroleukemia) is unresponsive to any of the immunostimulants generated (directly/indirectly) by E. purpurea or melatonin, and in fact this leukemia will proliferate only in response to internal, autocrine mechanisms (Lacombe et al., 1987; Stage-Maroquin et al., 1996), involving erythropoietin and erythropoietin receptor. The consistently and significantly elevated levels of NK cells in E. purpurea-treated, and melatonin-treated mice may well have been adequate to eliminate the disease, especially beginning in the early stages after tumor onset.

Although survival has been improved significantly by the particular protocol of agent administration implemented in this study, it is still not 100%. Manipulations of the administration protocol may be all that is necessary to further augment life span, i.e., higher doses and more frequent exposures of either or both agents. Moreover, studies are underway using inoculation methods, administering, individually and/or collectively, two known NK cell immunostimulants existing within the many components of E. purpurea (the polysaccharide, arabinogalactan, and the alkamide, 1,8-pentadecadiene).

Thus, a novel and unconventional approach to tumor therapy has been used in the present study, in which a botanical compound (E. purpurea), and/or endogenously occurring neurohormone (melatonin), was administered through a diet to leukemic mice. Using only the protocol described earlier, as the only means of "therapy" for our intermediate-stage, leukemic mice, three important conditions indicative of tumor abatement and potential host cure have occurred: (1) microscopic (as well as clinical) absence of tumor re-growth, (2) re-establishment by 3 months of normal levels of hemopoiesis in the bone marrow-generating center for all blood borne red and white cells, and (3) significantly prolonged life span. Most conventional chemotherapy is beset with two significant drawbacks. First, as synthesized chemical concoctions, they are foreign to, and highly toxic to, normal, living tissue as well as the targeted tumor cells, especially with sustained use, resulting in reduced quality--and ultimately quantity--of life. Second, most chemotherapies are tailored for tumor cell destruction and/or inhibition of their capacity to proliferate, rather that being directed toward enhancing the naturally occurring, lethal hit, tumor lytic mechanisms already in place, i.e., the various cells comprising the specific and nonspecific (NK) immune systems. It is with the latter mechanism of tumor combat that the present work is concerned.

In summary, this work has demonstrated, under controlled laboratory conditions, that phytocompounds from the naturally occurring herb, E. purpurea, may be profoundly valuable tools in leukemia combat. We have already demonstrated the prophylactic potential of E. purpurea in significantly elevating NK cells in normal, healthy mice (Sun et al., 1999). Clearly, the therapeutic potential of E. purpurea, at least, if not other botanicals as well, suggests that they could have a formal and fundamental role to play in modern antitumor therapy.

Address reprint requests to: Sandra C. Miller, Ph.D. McGill University Department of Anatomy & Cell Biology 3640 University Avenue, Room 2/28 Strathcona Anatomy and Dentistry Building Montreal, QC H3A 2B2 Canada

E-mail: smiller@med.mcgill.ca
TABLE 1. NUMBERS OF HEMOPOIETIC CELLS IN THE SPLEENS OF TUMOR-BEARING[a] MICE TREATED WITH MELATONIN, E. PURPUREA, OR BOTH, AT 9 DAYS OR 3 MONTHS AFTER TUMOR ONSET

Legend for Chart:

A - Duration[b]
B - Cell type
C - Control[d] (x 106)[c]
D - Melatonin[e] (x 106)[c]
E - E. purpurea[f] (x 106)[c]
F - Melatonin + E. purpurea (x 106)[c]

A B C D
E F

9 days Lymphoid 137.95 +/- 15.7[g] 132.11 +/- 3.36
146.08 +/- 9.96 82.72 +/- 3.77[*]

Myeloid 11.32 +/- 2.550 15.67 +/- 2.64
11.78 +/- 2.49 6.83 +/- 0.84

Erythroid 23.97 +/- 2.660 22.76 +/- 2.72
22.53 +/- 3.91 14.81 +/- 1.55[*]

Monocytoid 0.34 +/- 0.1 0.16 +/- 0.16
0.30 +/- 0.21 0.61 +/- 0.13

3 months Lymphoid --[h] 36.60 +/- 3.45
33.57 +/- 2.75 41.95 +/- 2.99

Myeloid -- 2.96 +/- 0.55
4.51 +/- 1.45 5.83 +/- 1.36

Erythroid -- 6.95 +/- 2.03
5.53 +/- 1.28 8.03 +/- 1.85

Monocytoid -- 0.06 +/- 0.05
0 0.03 +/- 0.01

[a] Mice received intravenously, 3 x 106 Friend leukemia
virus-induced erythroleukemia cells at 10 weeks of age.

[b] Mice were sampled at 9 days or 3 months after tumor onset.

[c] Cells were categorized into four major types and recorded
individually on hematologically stained cytospots, as a
percentage of 2,000 total cells counted. From the known total
cellularity per spleen, the proportions of each cell type were
converted to absolute numbers of cells per spleen per mouse.

[d] At tumor onset (day 0), mice were fed finely ground,
standardized chow, fresh daily.

[e] At tumor onset, melatonin was ground into the chow (above)
such that each mouse consumed 0.0142 mg/d, for 9 days or 50
days (terminating 40 days before the 3-month sampling).

[f] At tumor onset, E. purpurea root, commercially prepared as
a powdered extract, was homogenized into the chow (above) such
that each mouse consumed 0.45 mg/d for 9 days or 50 days
(terminating 40 days before the 3 month sampling).

[g] Mean +/- standard error

[h] No untreated mice survived until 3 months.

[*] p < 0.05-0.007 vs. corresponding control.

TABLE 2. NUMBERS OF HEMOPOIETIC CELLS IN THE BONE MARROW (PER FEMUR) OF TUMOR-BEARING[a] MICE TREATED WITH MELATONIN, E. PURPUREA, OR BOTH, 9 DAYS OR 3 MONTHS AFTER TUMOR ONSET

Legend for Chart:

A - Duration[b]
B - Cell type
C - Control[d] (x 106)[c]
D - Melatonin[e] (x 106)[c]
E - E. purpurea[f] (x 106)[c]
F - Melatonin + E. purpurea (x 106)[c]

A B C D
E F

9 days Lymphoid 2.83 +/- 0.29[g] 2.31 +/- 0.22
1.52 +/- 0.08[*] 2.26 +/- 0.22

Myeloid 3.18 +/- 0.39 g 2.29 +/- 0.25
1.58 +/- 0.24[*] 2.58 +/- 0.32

Erythroid 1.33 +/- 0.21 g 0.72 +/- 0.08[*]
0.61 +/- 0.06[*] 1.16 +/- 0.15

Monocytoid 0.08 +/- 0.02 g 0.002 +/- 0.002[*]
0.002 +/- 0.002[*] 0.09 +/- 0.01

3 months Lymphoid --[h] 2.42 +/- 0.14
2.43 +/- 0.16 4.06 +/- 0.40

Myeloid -- 3.76 +/- 0.50
3.44 +/- 0.33 4.87 +/- 0.66

Erythroid -- 1.57 +/- 0.15
1.36 +/- 0.03 2.51 +/- 0.21

Monocytoid -- 0.003 +/- 0.003
0.011 +/- 0.008 0.09 +/- 0.01

[a] Mice received intravenously, 3 x 106 Friend leukemia
virus-induced erythroleukemia cells at 10 weeks of age.

[b] Mice were sampled at 9 days or 3 months after tumor onset.

[c] Cells were categorized into four major types and recorded
individually on hematologically stained cytospots, as a
percentage of 2,000 total cells counted. From the known total
cellularity per femur, the proportions of each cell type were
converted to absolute numbers of cells per spleen per mouse.

[d] At tumor onset (day 0), mice were fed finely ground,
standardized chow, fresh daily.

[e] At tumor onset, melatonin was ground into the chow (above)
such that each mouse consumed 0.0142 mg/d, for 9 days or 50
days (terminating 40 days before the 3-month sampling).

[f] At tumor onset, E. purpurea root, commercially prepared as
a powdered extract, was homogenized into the chow (above) such
that each mouse consumed 0.45 mg/d for 9 days or 50 days
(terminating 40 days before the 3 month sampling).

[g] Mean +/- standard error

[h] No untreated mice survived until 3 months.

[*] p < 0.05-0.007 vs. corresponding control.

GRAPH: FIG. 1. Numbers of natural-killer (NK) cells in the spleens of tumor-bearing mice treated with melatonin (MLT), Echinacea purpurea (E. p.), or both, for 9 days or 3 months after tumor onset. p < 0.000007: MLT or Echinacea purpurea versus control.

GRAPH: FIG. 2. Number of natural-killer (NK) cells in the bone marrow (one femur) of tumor-bearing mice treated with melatonin (MLT), or Echinacea purpurea (E. p.), or both, for 9 days or 3 months after tumor onset.

GRAPH: FIG. 3. Survival incidence of tumor-bearing mice treated with melatonin (MLT), or Echinacea purpurea (E. p.), or both, for 50 days after tumor onset (day 0). Data was analyzed by Kaplan-Meier statistics. Survival advantage provided by MLT or E. purpurea individually was statistically significant versus control (p < 0.0068 and p < 0.022, respectively), although it was even greater when both agents were simultaneously administered (p < 0.00035 versus control). Each of the four groups had 45 animals at time 0; all mice in the group succumbed as indicated; of the mice in the MLT or E. purpurea-treated groups, one third lived to and beyond 3 months; of the mice in the MLT plus E. purpurea-treated group, half lived to and beyond 3 months.
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By Nathan L. Currier, B.Sc. and Sandra C. Miller, Ph.D., Department of Anatomy & Cell Biology, McGill University, Montreal, Canada.

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