Manzetti and Thoresen:

Identification of twelve non-toxic oligopeptides with cytostatic effects on three breast cancer cell lines, found in the blood after acupuncture stimulus.

Authors:
1. Sergio Manzetti, M.Sc., Flåm, 5743 Flåm, Norway. Ph: +47 484 20 096 Email: Sergio.manzetti@gmail.com

2. Are S. Thoresen, Leikvollgata 31, 3213 Sandefjord, Norway. Ph: +47 913 27 347 Email: arethore@online.no


Abstract

In this experiment, we identified twelve bioactive peptides in the blood of a voluntary candidate with breast cancer after treatment of the cancer with the help of acupuncture. Acupuncture of the one described point was the only treatment the woman got, which after the treatment cured. For the analysis and identification, we used 2D HPLC followed by mass-spectrometry. The oligopeptides were tested for growth-inhibiting properties on the breast cancer cell lines TMX2-28 (Tamoxifen-resistant), MCF7 and T47D. We also assayed the oligopeptides’ toxic potential through a toxicology assay. The results showed that 24 hours were sufficient for the oligopeptide mixture to induce a cell-death up to 30% for the TMX2-28 and 35% MCF-7 cell-lines. The positive controls, benzo[a]pyrene and Tamoxifen showed no significant effects at that time. The effects on T47D cells were weaker, however significantly growth-inhibiting when some of the oligopeptides were individually applied to it. The oligopeptide mixture was furthermore shown to be non-toxic while Tamoxifen induced strong toxicity. The results indicate a potent effect from bodily components that may prove useful as a source for therapy.


Keywords:
Acupuncture, bioactive peptides, isolation, breast cancer, protein analysis, TMX2-28, MCF-7.


Introduction

Breast cancer today reaches 557.000 cases in USA and 2.500.000 worldwide. This disease is furthermore expected to double by 2020 1, and in the UK the incidence of it keeps rising although the deaths fall 2. The cost of breast cancer in the United States of America alone is estimated to be between 5-15 billion United States Dollars, each year 1. Today, the major therapeutic approaches have evolved around endocrine therapies, where Tamoxifen has found its place as the central choice of a treatment, however at a cost of several gynecological and vasomotor symptoms3. Additionally, therapeutic approaches based on non-specific aromatase inhibitors have also been applied, with nevertheless adverse effects 3. Not to mention, third-generation aromatase inhibitors as letrozole, anastrozole and exemestane have emerged and increasingly been applied as more tolerable alternatives 3.

Non-synthetic compounds have also been tested against breast cancer 4. Particularly, bioactive peptides were tested as delivery agents against cancer and showed efficient action in targeting breast tumors 5. Interestingly, cytokines have also been shown to strongly inhibit the growth of the MCF-7 breast cancer cell lines 6. Furthermore, somatostatin has also shown growth-inhibitory effects on human tumor xenografts 7. Bioactive peptides play central roles in physiological functions, such as cellular regulation and also take part in diseases such as Alzheimer and cancer and they originate from proteolytic digestion of larger polypeptides. 8-19

With the proteomic revolution advancing for the past 10 years or so, the interest in blood-composition has increased. Nevertheless, finding tens of thousands of components has limited novel discoveries within the blood from healthy and diseased patients. Although of challenging nature, we found it to be potentially interesting to analyze the blood of a diseased subject. The voluntary candidate was a woman aged 43, affected by breast cancer with spreading to the bones. For the sample-taking, we found it of additional interest to induce an acupuncture stimulus on the patient in-between taking the two blood samples.

Acupuncture has been used in the treatment of long- and short-termed problems for more than 3000 years 20. However not fully proven for, it has interestingly been regarded as an efficient treatment in USA, United Kingdom and in Norway 21-26. The mechanisms of the action of acupuncture are largely unknown at present; however the potential of it in affecting the immunological and neurological systems has been demonstrated, through analysis of tissue and blood 27-34.

The method of applying acupuncture in affecting sarcomas or tumours was first tried in 1984 by Thoresen on a Dachshund and later published as methodology 35. The dog had mammary cancer (multiple tumours along the nipple line) and had begun to develop dyspnoea – as a probable consequence of lung metastases. The meridian that was stimulated is described 35, 36, and within a few weeks the tumour receded significantly. The dog died several years later from a kidney deficiency. The method, implying a relationship of control between the organs (accordingly with the Control-cycle in chinese literature 37) was further applied on a horse with equine sarcoid with evident results of a full reduction of the sarcoma and subsequently published in the Journal of the Norwegian Veterinary Society 38. Examples of control-cycle stimulation based on full pulse-diagnosis:

Liverstimulation
to control cancer of the tissues on or close to the spleen- or stomach-meridians, for example cancer of the lower medial tibia (where the SP-meridian passes), or mammary cancer (ST-meridian passes through the nipple).

Spleenstimulation
to control cancer of the tissues on or close to the kidney- or bladder-meridians, as the adrenal gland, ovary, oviduct, uterus, cervix, vagina, testicle, spermatic ducts, seminal vesicle, prostate, penis (all directly related to the kidney meridian).

The stimulation of the meridian for regulatory purposes on sacromas and tumours is most efficiently carried out through the ECIWO point selection-system (Embryo Containing Information of the Whole Organism). As described by Dr Jochen Gleditsch (Acupuncture in Medicine, 1995) the active points in the ECIWO-systems may be found by a needle-based point-detection method 39. In the same issue of Acupuncture in Medicine, Dr. Vilhelm Schjelerup had described the ECIWO-systems of the body and how they work 40. In this study, these two ways of thinking have been combined laying the rationale for the point selection.

Based on these interesting studies, we planned here therefore to find some substantial components of these alternative treatments that could be further studied and eventually used as therapeutic agents.


Materials and Methods

The ECIWO breast point on the liver meridian was needled in a 90-degree angle (Figure 1) in the female patient, aged 43 with breast cancer with spreading to the bones. The needle, made of stainless steel was left in situ for 20 minutes. Blood samples were then collected from the subject. The samples were taken immediately before needling (sample was named “S0”) and 60 seconds after needling (sample was named “S1”). Each of the two samples consisted of 7mL of blood and was directly collected into 8mL of 11.2M Guanidinium Hydrochloride, to yield a final concentration of 6M Guanidinium Hydrocholoride including the blood. The function of Guanidinium chloride was to solubilize and inactive all components in the blood, in order to preserve the blood as it was in circulation given risks of biochemical degradation 41. We chose a sixty-second time-frame given that the half-life of many oligopeptides has been documented to be as short as a few minutes 42, 43. The two samples were then fractionated to an oligopeptide fraction and then analysed for protein content.

From this stage on, the analysis continued through an extensive chromatographic analysis and eventually followed by a peptide identification using mass-spectrometry. This methodology is outlined in the supplementary material.

The peptides were after synthesis, tested on three breast cancer cell lines for cell-inhibiting effects. The Tamoxifen-resistant TMX2-28 cell line, the MCF7 and the T47D breast cancer cell line, and the effects were studied against the positive control Tamoxifen, a commonly used therapeutic on breast cancer. A full detailed view on this methodology is presented in the Supplementary material. At last, the peptides were subjected to a toxicology study using the 46BR1G1 fibroblast cell line. The toxicology was performed to study eventual negative effects which the peptides could pose on healthy cells. See Supplementary material for this.


Results and Discussion

The analysis of the protein-content in the two samples indicated a substantial difference in concentration. The S0-sample (prior to needling) had a protein-content of 0.54mg/mL while the S1-sample (after needling) had a protein-content of 1.02mg/mL, indicating a near doubling in oligopeptide concentration after acupuncture. Surprised, we chose to perform a series of preliminary runs on a chromatographic system to identify this increase of oligopeptide content. After several repetitions and different alternations, we were unable to find any differences between the analysis profiles of these two samples, puzzling us (See Fig 2). However, when we subjected the S0- and S1- sample to a new analysis, with a strong cationic-column, the central source of the protein-concentration change was identified. This new profile (Figure 3), showed a substantial difference between the two samples in the fraction located between 0-38 min in the run. At this stage, we started a further sub-study of this very fraction. The third analysis of the fraction showed to be composed of two distinct sub-fractions of interest as seen in figure 4; lane 3 and lane 4 (see Fig 4). These two lanes represent the central portion of the changes between prior and after needling, and were “eligible” and purified enough to be subjected to mass spectrometry. Therefore, the content found in the sample after acupuncture stimulus, noted S1 sample, was solely analyzed and sequenced, giving a series of oligopeptide sequences ranked by statistical probability. The sequences which resulted in the program, where scrutinized for relevance using two criteria set by the authors: the first criterion was that the statistical probability of finding the sequence should be larger than 75%. The second criterion was that the spectrum of the candidate sequence should have less than 4 unknown y- and b-ions peaks, in other words an “esthetic plot” with less improbable signal-peaks as possible.

The selection from resulted in twelve chosen sequences, listed in table 1. All of these sequences except of one are fragments from proteins such as cancer-involved proteins, conventional proteins, unknown proteins and non-human proteins. The one differing from the others is a protease inhibitors solely expressed in the liver, which was found in 1987 by an Italian group of scientist and later “forgot” and not studied further (see table 1). Two of the other protein fragments were non-human, and belonging to the rat and mouse species, something that was likely caused by similarities between the non-human segments and the actual oligopeptides in the blood of the patient, which were not present in the human database.

Once selected, the twelve selected sequences were synthesized at the University of Minnesota, and tested in a dose-response assay on three breast cancer cell lines (T47D, MCF7 and the Tamoxifen-resistant TMX2-28) in mixture, and individually at the University of Massachusetts. The results from the dose-response assay showed that all sequences induced a substantial effect on the three cell lines, however most powerful in mixture (table 2). The effect of the peptides individually was surprisingly in both directions; some peptides induced growth, and others induced cell-death.

The results from the dose-response assay showed that the equimolar mixture of the peptides inhibited the cells and induced cell-death on all three cell-lines, however particularly on the MCF7 cell line and the Tamoxifen-resistant TMX2-28 (See Fig 5, see also ANOVA analysis in Supplementary material: Table1s, 2s and 3s). A maximal cell-death induction reached ~35% after the 24 hours of incubation, for the MCF7 cell line. The TMX cell line was reduced in cell-count by ~30% in that same period of time. Interestingly, the positive controls Tamoxifen and benzo[a]pyrene (BAP), gave no significant effects at that time.

We named the 12 sequences with nomenclature from old Norwegian mythology. Two of these sequences were fragments of the hemoglobin protein. Named Durin and Fundin they belong to the protein chains located at the aminoacid position 18-36, and 142-154 respectively. At first, finding these two hemoglobin components was hypothesized to be related to a natural degradation of the hemoglobin protein in circulation. However, the anti-proliferation assays showed differently: Durin showed a cell-death-inducing effect on the T47D cell line with increasing growth-inhibition effect starting from 5 x 10-4 M towards the lower concentration 5 x 10-9M (Fig 6), while the positive control BAP showed no effects. The increasing inhibitory effect towards the lowest concentration may suggest its involvement in a dose-sensitive mechanism death-induction. A regular dose-reponse curve would suggest a higher dose to yield a higher effect, but in the case of Durin, it was inverse, the effect of cell-death increased the less that was in the medium of the cell. This very peptide showed also a growth-stimulating effect on the MCF7-cell line, and no significant effect on TMX2-28.

These opposite directions in growth-modulation between T47D and MCF7 may include that its action is involved in quite different pathways, or that the same pathway is reversing through a feedback mechanism in one of the cell lines. Interestingly Durin has been reported to be part of a patent were the patented compound acts as a potential disease-marker, however no more information on it was found (World patent nr.: WO-2003 04 6556). The other hemoglobin fragment, Fundin had an effect on MCF-7 cells, however growth-stimulating towards higher concentrations (see Supplementary material 2). Fundin was interestingly isolated recently from prostate cancer tissue (World patent nr: WO2005 11 4221), something that increases the strength of our hypothesis that even normal proteins as hemoglobin may “give birth” to smaller fragments that carry out entirely unrelated functions.

Dvalin was another oligopeptide found in the sample after stimulus, and derives as a protein fragment, from the cancer-involved protein “Zinc finger SWIM-domain containing-1” that is involved in apoptosis through DNA regulation 46. This peptide segment ranges residue 146-160 in the Zinc-finger protein and has no known structural/functional roles as a part of the parent protein. However, as a protein fragment its effect was significant on all three cell-lines according to ANOVA analysis (Supplementary Analysis 2). Regarding the effect of this peptide on the TMX2-28 cell-line, it had an increasing inhibitory effect towards higher concentrations; however the positive control BAP was more potent (see Fig 6). On the other cell line, MCF-7, the effect of the same oligopeptide was inverse; growth-stimulating towards higher concentrations (see Supplementary material 2). On the T47D cell line, the effect was of inhibitory nature: increasingly from 5 x 10-4M towards 5 x 10-9M, with a significantly stronger effect at the lowest concentration than the positive control BAP (Fig 6). Again a potentially sensitive signaling component was found here, which similar to Durin, induces stronger effect the less that is added (towads 10-9M). Therefore, it may have action on quite sensitive mechanisms in the T47D cell line, and given its sequence with three-forked positive charges set by Lysine and Arginine residues, may be hypothesized to be a DNA-binding action.

Another interesting oligopeptide was Emblin, which derives from the Golgin 45 (JEM-1) leucine zipper nuclear factor, which is a DNA-binding protein with reported cell-maturation functions in leukemia 46. This bioactive peptide was significantly inhibiting the growth of only the Tamoxifen-aggressive TMX2-28 cell line, to a higher degree at higher concentrations. The effect was in the same class as the positive control BAP (see Fig 6). This oligopeptide is composed of 18 residues and its sequence contains three arginine residues, which are separated rather evenly, perhaps also posing the possibility of DNA-binding. This sequence may be of particular interest for mechanistic studies of the Tamoxifen-resistant cell line TMX2-28 to potentially reveal strongly suppressed growth-regulating pathways (estrogen-independent). Frostin, yet another oligopeptide with interesting effects, had also a strong growth-inhibitory effect on the T47D cell line, especially towards lower concentrations, while its effect on the MCF-7 was growth-stimulating. Again, puzzled by the opposite directions on the growth-differentiation of these two estrogen-dependent cell lines, the authors suggest further studies on this oligopeptide (see Fig 6). The other oligopeptides’ effect is briefly described in Table 2 and Figure 5 and extensively documented for in the Supplementary material 2.

In a recent study at the University of Nottingham (Pre-Clinical Oncology Services), we tested the peptide mixture in a preliminary toxicology assay using the fibroblast line 46BR1G1. This cell line represents a normal or healthy cell line, and its use makes it feasible to anticipate the potential possibilities of toxic side-effects. As seen in Figure 7, the assay showed no toxic effects of the peptide mixture at the concentrations shown, and the cell viability remained stable throughout a period of 72 hours. We also assayed the toxicity of the two widely used breast-cancer drugs: Tamoxifen and Doxorubicin as controls, and the results showed an 80% viability reduction of the healthy fibroblast cell line (data not shown).


Conclusion

Twelve oligopeptides with growth-inhibiting effect on three breast cancer cell lines were isolated from a patient with breast cancer after acupuncture stimulus. The effects of the peptides were more powerful in mixture, on the TMX2-28 and MCF7 cell lines in particular. The peptides showed furthermore not to harm healthy cell models in vitro. When the oligopeptides were tested individually, their effects showed mixed growth-differentiating effects on the three breast cancer cell lines. The nature of these effects in mixture and individually indicate a potential synergistic action of the oligopeptides and is under further mechanistic investigation at the University of Nottingham, Pre-clinical Oncology Services.


Acknowledgements

The authors would like to thank Prof Kathleen Arcaro and Mr. Casey Turk for efficient conduction of the breast cancer assays at the University of Massachusetts, Department of Veterinary Sciences, Amherst, MA. Also we would like to thank Dr Sue Watson and Rajendra Kumari for conducting the recent experiments at PRECOS, University of Nottingham and providing us with the latest data on the fibroblast cell line. Finally, we’d like to thank Dr Bruce Witthun at the University of Minnesota for constructing the procedure for the 2D-HPLC and peptide fingerprinting technique.


Other relevant information

The sequences published in this paper have been patented under temporary patent nr 20073884 at the Norwegian Patent Office.



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Tables and table legends


Table 1. Twelve oligopeptides found in the 10kDa fraction of the patient’s blood. Shows the 12 oligopeptide sequences chosen in the 10kDa-fraction of the blood sample taken 60 seconds after acupuncture stimulus of a breast-cancer patient. Abbrev. S.S. Statistical Significance.

Image of Table 1: Twelve oligopeptides found in the 10kDa fraction of the patient’s blood.


Table 2. Cell-modulating directions induced by the oligopeptides. Indicates the direction of the cell-modulating effect mediated by the oligopeptides on the respective breast cancer cell-lines (arrow up signifies induced cell-growth, arrow down inhibited cell-growth). Number of arrows indicates strength of effect based on the overall decline in fluorescence. Line indicates no statistically significant effect.

Image of Table 2: Cell-modulating directions induced by the oligopeptides.