CANCERBIOTHERAPY& RADIOPHARMACEUTICALSVolume 12, Number 5,1997Mary Ann Liebert, Inc.

Direct Tumor Growth Suppressive Effect ofMelanoidin Extracted from Immunomodulator-PSK

Hideo Kamei,1 Yoko Hashimoto,2 Tatsurou Koide,3 Takashi Kojima,4 Makoto Hasegawa,1 andTessei Umeda1'Department of Surgery, Aichi-Gakuin University Hospital, 2 Suemori-dori, Chikusa, Nagoya 464, Japan;department of Biochemistry, Aichi-Gakuin School of Dentistry, 1 Kusomoto, Chikusa, Nagoya 464, Japan;3Health Research Center, Aichi-Gakuin University, Iwasaki, Nisshin, Aichi-ken 470-01, Japan; 4Departmentof Surgery 1, Aichi Medical School, Yazako, Nagakute, Aichi-ken 480-11, Japan

Melanoidin, which belongs to the melanin group ofmolecules, was extractedfrom the polysaccharide biologicalresponse modifier PSK. Melanoidin was cultured together with HCT-15 cells derivedfrom human colon cancerand with AGS cells derivedfrom human gastric carcinoma. Afterfour days ofculture, cell count was comparedwith that ofthe control cells.

Significant suppression was observed, that is, 50% suppression was shown at concentrations ofmelanoidinbetween 200 and 100 µg/ml.

A histogram generated byflow cytometry showed elevation ofthe tetraploidpeak and ofthat between diploidand tetraploidpeaks, suggesting blockage ofSphase and G2 to Mphase ofthe cell cycle.

Thus, melanoidins contained in the immunomodulator PSK revealed to have a direct tumor cell growthinhibitory effect.

Key words: Melanoidin, melanin, PSK

INTRODUCTION

Polysaccharide (PSK) is widely used for cancer

patients as a biological response modifier.1'2·3 Al¬though a few reports have indicated a direct tumorcell suppression by PSK4,5 PSK is mostly consideredto act as an immunomodulator in the host.1,2,3

Address reprint requests to Hideo Kamei, Dept. OfSurgery,Aichi-Gakuin Univ. Hospital, 2 Suemori-dori, Chikusa,Nagoya 464, Japan. Phone: 052-751-7181 Fax: 052-752-5990E-Mail kameiCa'dpc. aichi-gakuin. ac.jp.

PSK appears dark in color, which color is derivedfrom the melanoidin induced by an amino-carbonylreaction during the production of PSK. This mel¬anoidin is mostly combined with protein or glycopro-tein (personal communication from Kureha KagakuCo.). Melanoidins belong to the melanin family, andare formed by an amino-carbonyl reaction betweenamino acids and carbohydrate.6

We reported earlier that plant-produced melanins-allomelanins have tumor suppressive effects in vitroand in v/'vo.7'8 The present study was conducted todetermine whether melanoidins also have a directeffect on the suppression of tumor growth.

341

Table 1. Suppression rate of cultured HCT-15 and AGS cells by melanoidins.

CellDose of melanoidin

20 0 /»g/ml 100 /ig/ml 25 xg/mlBCT-15 cells

rote i -c jugated melanoidinprotein-free melanoidin

AGS cells

protein free

32.2X+5.5X 1 4.

3 X ± 5 . 5 X '

85. OX ± 42. 6X 1 6. 9X ± 5. 9X'

65.5X±9.6X 42. OX ± 1 2. 5%

1.

3X ± 5. 7X "

5. 7X ± 1 1.

3X1

Suppression rate (%) = [(Control cell count-

Experimental cell count) / Control cell count] 100* no significance

Figure 1. Histogram of HCT-15 cells obtained by flow cy-tometry

A: controlB: melanoidins added

Table 2. Percent of cells in each phase of the cell cycle as estimated by flow cytometry

Phase Gl G2 + M

Control 2 0 S ± 2.8X 66X ± 0. 3 X 1 4 X ± 3.3X

melanoidin added 12« ±5.8» 7 5 X ± 0.

3 X 13% ± 4. 3 X

MATERIALS AND METHODS

Melanoidin PreparationPSK weighing 25 g was kept overnight in 200 ml of1% NaOH in water. HC1 was then added, and theresulting sediment was washed with water. It was

dissolved again in 1% NaOH in water, and againsedimented by adding HC1. The sediment was once

more washed with water, then with acetone anddiethylether, and dried.6

In order to remove the conjugated protein fromthe melanoidin preparation, crude melanoidin was

boiled at 100°C for 20 hours in 5N HC1 in water.6·7·8The sedimented protein-free melanoidin was washedwith water, acetone and diethylether, and then dried.Before experiments, melanoidin was dissolved in0.5% NaHC03 in water, and sterilized at 105 °C for30 minutes.

342

For estimating the molecular weight of thisprotein-free material, melanoidin solution was passedthrough a Sepharose CL-4B column.

Cell Culture

HCT-15 cells derived from human colon cancer andAGS cells derived from human gastric carcinoma were

used for this study.7,8·9 Both cell types were main¬tained in a C02 incubator in RPMI 1640 supple¬mented with 10% FCS. For experiments the cellswere harvested and placed in Petri dishes (1 IO3cells/dish) œntaining the above culture medium, andthen 40µ1 ofmelanoidin solution at various concentra¬tions in 0.5% NaHC03 solution was added to eachof several dishes. To each control dish, 40µ1 of 0.5%NaHC03 was added. After 4 days ofculture in theC02 incubator, cell counts were made.111213 Eachgroup consisted of 5 dishes. Student's t test was

applied for the analysis of significance of differences.

Histogram StudyTwo mg of protein-free melanoidin was added toHCT-15 cells (Ixl04inl0ml ofmedium) in cultureflasks, and the cells were then incubated for 2 days.After sedimentation and washing with PBS, the pelletwas suspended in a solution prepared by OtsukaAssay Co., and histographic assay was conducted byOtsuka Assay Co.8·9

RESULTS

Table 1 shows the cell-growth suppression of bothHCT-15 and AGS cells by the melanoidin extractedfrom PSK. Protein bound melanoidin showed a

statistically significant suppression of tumor growth,although the suppression rate was less than 50% whenmelanoidin was used at the concentration of 200µg/ml. When protein was removed by hydrolysis, thesuppression rate was higher. In this case 50%suppression occurred between 200 µg/ml and 100µ& .

As shown in Figure 1, two peaks were observedin the control cells. The first one represented diploidcells; and the second one, tetraploid cells. By addingmelanoidin, a significant rise in the tetraploid peakand in that between diploid and tetraploid peaks was

noted. As shown in Table 2, the percent of cells inGl phase was lower, and that of cells in S phase was

higher than the corresponding control percentageswhen melanoidins were added to the cultures.

The molecular weight of the protein-free mel¬anoidins after hydrolysis was approximately 5600.

DISCUSSION

PSK is clinically used as an immunomodulator forcancer patients, mostly combined with other antican-cer agents.10·11 By using experimental animals, tumorregression has been observed, and was considered as

having been due to host-mediated effects.1,2·3 How¬ever, several reports have shown that PSK had a directtumor suppressive activity in culture systems, but thesubstance showing the direct anti-tumor effects was

not clarified.4'5As reported before allomelanins produced from

higher plants have direct tumor-suppressive activity.7Although the chemical structure of melanoidin isdifferent from that of allomelanin, it is considered as

one kind of melanin. The chemical structure ofmelanoidin is complicated, and no clear informationon it has been published.6 Protein-bound melanin alsoshowed tumor cell suppression, although the suppres¬sion rate was lower than that of protein-free mel¬anoidin. However, the effective dose may be equiva¬lent when conversion to molar concentration is done.

The pharmacological effects ofmelanoidins havenot been clarified yet. Melanoidins are soluble inalkaline water but not in acidic water, nor in solventssuch as methanol, ether, acetone and so on, whosesolubility is generally the same as that in the melaningroup.6'7

When the melanoidins were added to Petri dishescontaining HCT-15 or AGS cells, significant growthsuppression was observed. Considering the molecularweight, the dosage to gain 50% suppression of tumor

growth was not so high.From the results of flow cytometric study,

melanoidins induced a rise in the tetraploid peak andincreased the percent of cells in S phase. Theseresults suggest that melanoidins block S phase andG2 to M phase of the cell cycle. Thus, melanoidinscontained in PSK apparently act as direct anti-tumoragents.

ACKNOWLEDGMENTS

This work was supported by the Aichi-Gakuin Fund.We greatly appreciate the technical assistance ofMrs.Chi Minami.

343

REFERENCES

1. Tsukagoshi S, Hashimoto Y, Fujii G, et al. Krestin(PSK). Cancer TreatReviews 1984;11:131-155.

2. YefenofE, Gafanovitch J, Oron E, Bar M, et al. Prophy¬lactic intervention in radiation-leukemia-virus-inducedmurine lymphoma by the biological response modifierpolysaccharide PSK. Cancer Immunol Immunother1995;41:389-396.

3. Kobayashi H, Matsunaga and Fujii M. PSK as a

chemopreventive agent. Cancer Epidem Biomarkers &Prevention 1993;2:271-276.

4. Kobayashi Y, Kariya K, Saigenji K, et al. Suppressiveeffects on cancer cell proliferation of the enhancementof Superoxide dismutase (SOD) activity associated withprotein-bound polysaccharide of coriolus versicolorQUEL. Cancer Biother 1994;9:171-178.

5. Kobayashi Y, Kariya K, Saigenji K, et al. Suppressionof cancer cell growth in vitro by the protein-boundpolysaccharide of coriolus versicolor QUEL (PSK) withSOD mimicking activity. Cancer Biother 1994;9:63-69.

10.

11.

Ootani S. Melanins: plant pigments. Hayashi K, ed.Yokendo, Tokyo 1991:250-261 (in Japanese).Kamei H, Koide T, Kojima T, et al. Suppression ofcultured malignant cells by allomelanin, plant-producedmelanins. Cancer Biother & Radiopharm 1997; 12:47-49.Kamei H, Koide T, Hashimoto Y, et al. Effect ofallomelanin on tumor suppression in vivo and on thecell-cycle phase. Cancer Biother & Radiopharm 1997;12:273-276.Kamei H, Koide T, Hashimoto Y, et al. Tumor cellgrowth suppression by chalcone (l,3-Diphenyl-2-Propen-1-One). Cancer Biother & Radiopharm 1997;12:51-54.Nakazato H, Koike A, Saji S, et al. Efficacy of immuno-chemotherapy as adjuvant treatment after curativeresection of gastric cancer. Lancet 1994;343:1122.Torisu M, Hayashi Y, Ishimitsu T, et al. Significantprolongation of disease-free period gained by oralpolysaccharide (PSK) administration after curativesurgical operation ofcolorectal cancer. Cancer ImmunolImmunother 1990;30:261-268.

344