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In vitro inhibition of proliferation of HL-60 cells by tetrandrine and coriolus versicolor peptide derived from Chinese medicinal herbs
Life Sciences_Vol. 60, No. & pp. PL 13.5-MO,1997 Coppight 0 1997 EJscvierScience Inc. Printed in the USA. All rights raened om4-3205/!?7317.00 t .oo
PII SOO24-3205(%)00695-9 Ph%RhlxcoLoGYLEnERs Accelemted Gmmunication
IN VITRO INHIBITION OF PROLIFERATION OF HL-60 CELLS BY TETRANDRINE AND CORIOLUS VERSICOLOR PEPTIDE DERIVED FROM CHINESE MEDICINAL HERBS Ying Dong, Mabel Mei-Po Yang and Chiu-Yin Kwan Department of Physiology, Faculty of Medicine, The University of Hong Kong, 5 Sassoon Road, Hong Kong.
(Submitted October 24,19%, accepted November 5,19%, received in final form December 5 1996) Abstract Coriolus versicolor polysaccharide peptide (CVP) and the bis-benzylisoquinoline alkaloids, tetrandrine (TBT) and berbamine (BER), the active ingredients isolated from Chinese medicinal herbs known to possess antitumor activities, concentration-dependently inhibited the proliferation of human leukemic I-IL-60 cells. CVP did not affect the growth of normal human peripheral blood lymphocytes (PBL), whereas TET elicited concentration-dependent cytotoxic effects. Morphological observation and DNA analysis revealed that CVP elicited no effect on the morphological features of HL-60 cells and did not cause DNA fragmentation, but TET and BER caused cell shrinkage with the formation of apoptotlc bodies, and showed clear evidence of DNA fragmentation. These findings indicate that TET and BER, but not CVP, inhibited the proliferation of I-IL-60 cells via induction of apoptosis.
k?y Words: tetrandrinc, berbamme, C&o/us versicdor, apop&k,
cancer, HL-60
cells
Introduction Polysaccharide peptides isolated from Chinese medicinal mushrooms, such as Coriofus versicolor Cov-1 (PSP) and Coriolus versicolor Krestin (PSK), have long been known to possess antitumor effects and were clinically effective when given as adjunct treatment with other anticancer therapies including surgical operation (l-3). Recently, a refined peptide fraction isolated from the PSP powder extracted from the mycelium of Coriolus versicolor Cov-1 (CVP) in China has also been found to elicit potent antitumor effect (4,5). These mushroom peptides, PSP, PSK and CVP, have commonly been found to elicit immuno-stimulant effects (4,6,7). Tetrandrine (TET), a bis-benzylisoquinoline alkaloid extracted and purified from the root of Stephuniu tetrundru (S. Moore) was reported to elicit in vitro cytotoxic effect on Hela cells and in vivo suppressive effects on mouse ascites tumor (8) in addition to its anti-inflammatory and immunosuppressive effects (9-11). Berbamine, an analogue of tetrandrine, isolated from Stephaniu cephurunthu (Hayata) was also reported to markedly suppress the tumor-promoting effect of 12-o-tetradecanoylphorbol-13-acetate in mouse skin initiated with 7,12dimethylbenzsnthracene (12) and to elicit immuno-suppressive actions (13). Some antitumor reagents, including those derived from Chinese medicinal herbs, exert their actions via immunomodulating effects and/or induction of apoptosis, a process of programmed cell death (14), which, if selective on tumor cells, may have potential value in cancer chemotherapy (15). In this study, we compared the effects of CVP and TET (as well as its structural analogue, BER) on the proliferation of human leukemic HL-60 cells and the corresponding normal human peripheral blood lymphocytes, PBL and further investigated whether these antitumor reagents inhibit the growth of HL-60 cells by induction of apoptosis as studied by cell morphology and DNA analysis. Correspondence to: C.Y. Kwan, Ph.D., Department of Physiology, Faculty of Medicine, The University of Hong Kong, 5 Sassoon Road, Hong Kong. Fax: (852) 2855 9730
PL-136
Inhibition of HL-60 Cells
Vol. 60, No. 8, 1997
Materials and Methods Preparation of culture me&urn with TET, BER or CVP RPM1 medium 1640 (GIBCO, N. Y., U.S.A.) supplemented with 10% fetal bovine serum (GIBCO, N. Y., U.S.A), 100 units/ml penicillin, and 100 pg/ml streptomycin (GIBCO BRL, N. Y., U.S.A.) was used for cell culture as the basal medium. TET and BER powder were separatedly dissolved in a drop of HCl and 0.9% sodium chloride and diluted to the concentration of 15 mg/ml with 0.9% sodium chloride. TET( 15 mg/ml) was further diluted with 0.9% NaCl solution to desired concentrations. CVP was isolated by fast performance liquid cbromatograph using DEAE-40 HR column (5) and the elute of protein was collected, desalted, concentrated and freeze-dried for use. CVP powder was dissolved in the basal medium as a stock solution of 10 mg/ml and was further diluted with the basal medium to the desired concentrations. Cell proliferation Human leukemic I-IL-60 cells and normal human peripheral blood lymphocytes from healthy adult were cultured for 1 day in the basal medium. On the next day, the culture medium was replaced with medium containing desired concentrations of CVP, TET, BER or the control medium. HL-60 and PBL cells were seeded on 12- or 24-well plates at a density of 1.0 x 10’ - 2.0 x 16 cells/well and were cultured for 2-3 days as specified in the medium with or without CVP (or ‘IET or BER). The number of viable cells was identified and counted using the trypan blue dye exclusion test. MorpWogical methods I-IL-60 cells in CVP- or TET- or BER-treated cultures were examined using light microscopy (Aristoplan, Germany) on day 3 or hour 12, respectively. These cells were then pelleted at 150 x g, smeared on a slide and observed for morphological changes at x 250 magnification. Analysis of cell DNA DNA analysis of I-IL-60 cells was performed on hour 12 of culture with TET or BER or on day 3 of culture with CVF (16). Briefly, cells were washed twice with cold phosphate-buffered saline and disrupted by suspension for 20 min at 4’C in 0.5 ml of 5 mM Tris-HCL buffer containing 0.5% (vol/vol) T&on-X 100 and 20 mM EDTA. The supernatant was centrifuged and DNA was extracted from the supematant by treatment with equal volume of phenol, and chloroform/isoamyl alcohol (25:24: 1). To the upper aqueous layer, 100% ethanol were added to precipitate the DNA. After a cold centriiirgation at 104 rpm, the pellet was resuspended in 0.5 ml of Tris-EDTA buffer at pH 8.0 and added DNase-free RNase A to the suspension, the phenol/chloroform/isoamyl alcohol extraction was repeated. DNA was precipitated with ethanol as above, centrifuged, and the pellet was dried in an open air. DNA was electrophoresed with 1% agarose gel. The bands were visualized by UV illumination.
Results are expressed as the mean f SD. The Student’s paired r-test was used to compare the means of two groups. Differences were considered significant when ~0.05.
Results Effects of drugs on cell profiferation Fig. 1 shows that both CVP and TET elicited concentration-dependent inhibitory effects on the proliferation of HL-60 cells. TET, but not CVP, also exerted concentration-dependent cytotoxic effect on the normal PBL cells. This result suggests that CVP is more selective in the inhibition of proliferation of cancer cells. BER, a structrual analogue of TET, also elicit an inhibitory effect on HL60 cells, albeit weaker than that of TET (Fig. 2).
Vol. 60, No. 8, 1997
Inhibition of HL-60 Cells
PL-137
Effects of drugs on cell death HL-60 cells treated with 24 pM TET or BER for 12 hrs showed morphological changes characteristic of apoptosis, such as cell shrinkage, nuclear fragmentation and apoptotic bodies (not shown). However, these morphological features were not observed in HL-60 cells treated with 1 mg/ml CVP for up to 72 hrs. The formation of apoptotic cells was compared in HL-60 cells treated with 24 PM TET and BER (Fig. 3). Apoptosis was more prominent in TET-treated cells compared to BER-treated cells. DNA fragmentation also occurs in apoptosis and is characterized by a ladder pattern indicating intemucleosomal chromatin cleavage, as detected in HL-60 cells treated by UV irradiation for 30 mm serving as a positive control (Fig. 4a). However, a ladder of fragmented DNA was not detected in HL-60 cells on day 3 culture treated with 1 mg/ml CVP indicating that CVP does not cause apoptosis. On the other hand, a ladder of fragmented DNA was detected in HL-60 cells in 12-hr culture with TET or BER (Fig. 4b).
Discussion We have demonstrated that CVP and TET, the active chemical constituents of two widely used Chinese medicinal herbs, Coriolus versicolor and Stephmiu tetrundru, respectively, elicited inhibitory effects on the proliferation of human leukemic HL-60 cells. The inhibitory actions of these two drugs, however, are characteristically different in two aspects. First, CVP is more selective for tumor cells, whereas TET is rather non-selective based on the differential effects of these drugs on the proliferation of HL-60 cells and the normal human PBL. Second, TET but not CVP induces apoptotic responses in HL-60 cells as evident from both morphological and DNA ladder patterns. Since TET has immunosuppressive activity (9-ll), whereas CVP and other polysaccharide peptides from similar Chinese medicinal mushrooms show immunostimulating activity (4,6,7), the immunomodulating activity is unlikely to be the primary attribution to their common action in suppressing the solid tumor growth, which is probably as a result of their direct inhibition of tumor cell proliferation.
B:
A:
90 -
PBL
80 70 60 -
'i; 80
50 40 30 20 10 -
o10
100
0
1000
CVPWmL)
* I. 7
*
R
*
HL-60
\
4 * PBLb
5
10
TET
15
20
h
25
30
(PM)
Fig. 1
Cell growth profiles for HL-60 and PBL cells following 72-hr incubation with various concentrations of CVP (A) and following 48 hr incubation with increasing concentrations of TET (B). Data are shown as mean + SD of 3 independent experiments with duplicate wells each. Note the selective inhibition by CVP and non-selective inhibition by TET of cell growth in HL-60 cells compared to the normal PBL cells. * Significantly (~0.05) different from PBL values.
Inhibition of HL60 Cells
PLl38
A:
Vol. 60, No. 8, 1997
B:
%I00 L
z3 z q L
80 60
$ 40 E z' 20 = 0"
0 0
5
10
15
20
25
0
5
10
15
20
25
Concentrations of drugs (pM) Fig.2 Cell growth following 24-hr (A) or 48-hr (B) incubation with various concentrations of drugs. Data are expressed as mean f SD of 3 separate experiments with duplicate wells. Note that TET significantly (*PcO.O5) inhibited the proliferation of HL-60 cells and its effect was more potent than BBR in the concentration range of 12-24 PM, 120 q LIOO u) =
a, v
3
An .s.e
60
I sg 40 0 s. 2 20
2
I
I
0
Control BER
!
TET
-
-r-lI
T
0
0
24 lncu bation time (hr) Fig.3
48
The percentage of apoptotic HL-60 cells assessed morphologically at time points up to 48 hrs in the presence or absence of 24 pM BER or TBT. The values represent mean f SD of 3 different determinations. *Significant wO.05) increase in apoptotic cells compared to that without incubation or the control without drug treatment. Recently, Dong et al. (4) have shown that CVP also elicited in vivo suppressive effect against solid tumor from Sarcoma- 180 implantation. Although it exerted in vitro cytotoxic effects on human hepatic tumor HEPG2 cells, CVP up to 1 mg/ml failed to inhibit the proliferation of the normal human fetal hepatocytes. This is consistent with the present finding that CVP inhibits the growth of human leukemic HL-60 cells without significant effects on the normal PBL cells and lends further support to the contention that CVP selectively inhibits the tumor cell growth. The mechanism of action of the selective effect of CVP causing tumor cell death is not known, but it may involve arrest in cell cycle according to our preliminary results studied by using flow cytometry.
Vol. 60, No. 8, 1997
Inhibition of I-X-60 Cells
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b
PL-139
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B
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Fig. 4 A: Agarose gel electrophoresis of DNA extracted from I-IL-60 cells incubated with medium alone (lane a) or 1 mg/ml CVP (lane b) for 72 hrs and from I-IL-60 cells treated with UV irradiation for 30 min as a positive control (lane c). Lane d showed DNA markers. B: Agarose gel electrophoresis of DNA extracted from I-IL-60 cells incubated for 12 hrs with medium alone (lane b), 24 pill BER (lane c) or 24 uM TET (lane d). Lane a shows the standard DNA markers.
TET elicited in vitro cytotoxic effects on human BM13674 cells (17) and Hela cells (18) as well as in vivo suppressive effects against mouse ascites tumor from Ehrlich ascites carcinoma and against solid tumor from Sarcoma-180 (8). Most earlier studies of antitumor effects of TET were confined to the use of tumor cell lines. However, our present finding that the inhibitory effect of TET on cell proliferation is comparable between I-IL-60 and PBL indicates that cytotoxic effect of TET is non-selective with respect to cell type. It is interesting to note that TET was also more potent than BER in causing release of intracellular Ca” and inhibiting Ca” entry activated by thapsigargin in HL60 cells (19,20). The mechanism of actions of TET/BER-induced apoptosis is not clearly understood and the role of elevation of cytosolic Ca” concentration in the apoptotic process in HL-60 and its relationship to TETIBER-induced apoptosis require future investigation. Acknowledgement This work was supported by a donation from the Hong Kong Association of Health Care Ltd. and in part by the Committee of Research and Conference Grants of the University of Hong Kong.
References 1. 2. 3. 4. 5. 6.
M. NIIMOTO, T. HATTORI, R. TAMADA, T. SUGIMACHI, K. INOBUCHI and N. C. OGAWA. Jpn. J. Surg. fi 681-686 (1988). T.F. LIU, Recent Advance in Cancer, 63-66, Chinese University Press, Hong Kong (1989). Y. KIKUCHI, I.. KIZZAWA, K. OOMORI, Y. KITA and K. KATO, Jpn. J. Cancer Res. 79 125-130 (1988). Y. DONG, C.Y. KWAN, Z.N. CHEN and M. YANG, Res. Commun. Mol. Pathol. Pharmacol. 92 140-148 (1996). M.M.P. YANG, Z.N. CHEN and J.S.L. KWOK, Amer. J. Chin. Med. 20221-232 (1992). T. FUJII, T. KANO, K. SAITO, Y. KOBAYASHI, H. IIJIMA, T. MATSUMOTO, C. YOSHIKUMI and T. TAGUCHI, Anticancer Res. 1845-848 (1987).
PL140
7.
8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.
Inhibition of HL-60 Cells
Vol. 60, No. 8, 1997
X.Y., LI, J.F., WANG, P.P. ZHU, S.X. YANG, L. LIU and J.B. GE, Eur. J. Pharmacol. 183 904-905 (1990). H. KURODA, S. NAKAZAWA, K. KATAGIRl, 0. SHIRATORI, M. KOZUKA, K. FUJITANI and M. TOMITA, Chem. Phann. Bull. 24 2413-2420 (1976). Y. KONDO, Y. IMAI, H. HOJO, Y. HASHIMOTO and S. NOZOE, Int. J. Immunopharm. 14 1181-1186 (1992). S.Y. LI, L.H LING, B.S. THE, W.K. SEOW and Y.H. THONG, Int. J. Immunopharm. j_l 395401 (1989). W.K. SEOW, A. FERRANTE, A. SUMMORS and Y.H. THONG, Life Sci. 50 PL53-58 (1992). K. YASUKAWA, M. AKASU, M. TAKEUCHI and M. TAKIDO, Oncology-Switzerland, 50 137-140 (1993). Y. KONDO, F. TAKANO and H. HOJO, Biochem. Pharmacol. 46 1861-1863 (1993). I. HASEGAWA, Y. MIZOGUCHI, H. TSUTSUI, Y. ICHIKAWA, N. KAWADA, S. MORISAWA and S. YAMAMOTO, J. Med. Pharm. Sot. z 24-27 (1990). S. SEN, and M. D’INCALCI, FEBS Lett. 307 122-127 (1992). B.K. BHALLA, C. TANG, A.M., IBRADO, S. GRANT, E. TOURKINE, C. HOLLADAY, M. HUGHES, M.E. MAHONEY, and Y. HUANG, Blood. &J 2883-2890 (1992). Q. SONG, G.D. BAXTER, E.M. KOVACS, D. FINDIK and M.F. LAVIN, J. Cell. Physiol. 153 550-556 (1992) B.S. TEH, P. CHEN, M.F. LAVIN, W.K. SEOW and Y.H. THONG, Int. J. Immunopharm. 13 1117-1126 (1991). Y.M. LEUNG, M. BERDIK, C.Y. KWAN and T.T. LOH, Clin. Exp. Pharmacol. Physiol. 23 653-659 (1996). Y.M. LEUNG, C.Y. KWAN and T.T. LOH, Br. J. Pharmacol. -113 767-774 (1994).
PII SOO24-3205(%)00695-9 Ph%RhlxcoLoGYLEnERs Accelemted Gmmunication
IN VITRO INHIBITION OF PROLIFERATION OF HL-60 CELLS BY TETRANDRINE AND CORIOLUS VERSICOLOR PEPTIDE DERIVED FROM CHINESE MEDICINAL HERBS Ying Dong, Mabel Mei-Po Yang and Chiu-Yin Kwan Department of Physiology, Faculty of Medicine, The University of Hong Kong, 5 Sassoon Road, Hong Kong.
(Submitted October 24,19%, accepted November 5,19%, received in final form December 5 1996) Abstract Coriolus versicolor polysaccharide peptide (CVP) and the bis-benzylisoquinoline alkaloids, tetrandrine (TBT) and berbamine (BER), the active ingredients isolated from Chinese medicinal herbs known to possess antitumor activities, concentration-dependently inhibited the proliferation of human leukemic I-IL-60 cells. CVP did not affect the growth of normal human peripheral blood lymphocytes (PBL), whereas TET elicited concentration-dependent cytotoxic effects. Morphological observation and DNA analysis revealed that CVP elicited no effect on the morphological features of HL-60 cells and did not cause DNA fragmentation, but TET and BER caused cell shrinkage with the formation of apoptotlc bodies, and showed clear evidence of DNA fragmentation. These findings indicate that TET and BER, but not CVP, inhibited the proliferation of I-IL-60 cells via induction of apoptosis.
k?y Words: tetrandrinc, berbamme, C&o/us versicdor, apop&k,
cancer, HL-60
cells
Introduction Polysaccharide peptides isolated from Chinese medicinal mushrooms, such as Coriofus versicolor Cov-1 (PSP) and Coriolus versicolor Krestin (PSK), have long been known to possess antitumor effects and were clinically effective when given as adjunct treatment with other anticancer therapies including surgical operation (l-3). Recently, a refined peptide fraction isolated from the PSP powder extracted from the mycelium of Coriolus versicolor Cov-1 (CVP) in China has also been found to elicit potent antitumor effect (4,5). These mushroom peptides, PSP, PSK and CVP, have commonly been found to elicit immuno-stimulant effects (4,6,7). Tetrandrine (TET), a bis-benzylisoquinoline alkaloid extracted and purified from the root of Stephuniu tetrundru (S. Moore) was reported to elicit in vitro cytotoxic effect on Hela cells and in vivo suppressive effects on mouse ascites tumor (8) in addition to its anti-inflammatory and immunosuppressive effects (9-11). Berbamine, an analogue of tetrandrine, isolated from Stephaniu cephurunthu (Hayata) was also reported to markedly suppress the tumor-promoting effect of 12-o-tetradecanoylphorbol-13-acetate in mouse skin initiated with 7,12dimethylbenzsnthracene (12) and to elicit immuno-suppressive actions (13). Some antitumor reagents, including those derived from Chinese medicinal herbs, exert their actions via immunomodulating effects and/or induction of apoptosis, a process of programmed cell death (14), which, if selective on tumor cells, may have potential value in cancer chemotherapy (15). In this study, we compared the effects of CVP and TET (as well as its structural analogue, BER) on the proliferation of human leukemic HL-60 cells and the corresponding normal human peripheral blood lymphocytes, PBL and further investigated whether these antitumor reagents inhibit the growth of HL-60 cells by induction of apoptosis as studied by cell morphology and DNA analysis. Correspondence to: C.Y. Kwan, Ph.D., Department of Physiology, Faculty of Medicine, The University of Hong Kong, 5 Sassoon Road, Hong Kong. Fax: (852) 2855 9730
PL-136
Inhibition of HL-60 Cells
Vol. 60, No. 8, 1997
Materials and Methods Preparation of culture me&urn with TET, BER or CVP RPM1 medium 1640 (GIBCO, N. Y., U.S.A.) supplemented with 10% fetal bovine serum (GIBCO, N. Y., U.S.A), 100 units/ml penicillin, and 100 pg/ml streptomycin (GIBCO BRL, N. Y., U.S.A.) was used for cell culture as the basal medium. TET and BER powder were separatedly dissolved in a drop of HCl and 0.9% sodium chloride and diluted to the concentration of 15 mg/ml with 0.9% sodium chloride. TET( 15 mg/ml) was further diluted with 0.9% NaCl solution to desired concentrations. CVP was isolated by fast performance liquid cbromatograph using DEAE-40 HR column (5) and the elute of protein was collected, desalted, concentrated and freeze-dried for use. CVP powder was dissolved in the basal medium as a stock solution of 10 mg/ml and was further diluted with the basal medium to the desired concentrations. Cell proliferation Human leukemic I-IL-60 cells and normal human peripheral blood lymphocytes from healthy adult were cultured for 1 day in the basal medium. On the next day, the culture medium was replaced with medium containing desired concentrations of CVP, TET, BER or the control medium. HL-60 and PBL cells were seeded on 12- or 24-well plates at a density of 1.0 x 10’ - 2.0 x 16 cells/well and were cultured for 2-3 days as specified in the medium with or without CVP (or ‘IET or BER). The number of viable cells was identified and counted using the trypan blue dye exclusion test. MorpWogical methods I-IL-60 cells in CVP- or TET- or BER-treated cultures were examined using light microscopy (Aristoplan, Germany) on day 3 or hour 12, respectively. These cells were then pelleted at 150 x g, smeared on a slide and observed for morphological changes at x 250 magnification. Analysis of cell DNA DNA analysis of I-IL-60 cells was performed on hour 12 of culture with TET or BER or on day 3 of culture with CVF (16). Briefly, cells were washed twice with cold phosphate-buffered saline and disrupted by suspension for 20 min at 4’C in 0.5 ml of 5 mM Tris-HCL buffer containing 0.5% (vol/vol) T&on-X 100 and 20 mM EDTA. The supernatant was centrifuged and DNA was extracted from the supematant by treatment with equal volume of phenol, and chloroform/isoamyl alcohol (25:24: 1). To the upper aqueous layer, 100% ethanol were added to precipitate the DNA. After a cold centriiirgation at 104 rpm, the pellet was resuspended in 0.5 ml of Tris-EDTA buffer at pH 8.0 and added DNase-free RNase A to the suspension, the phenol/chloroform/isoamyl alcohol extraction was repeated. DNA was precipitated with ethanol as above, centrifuged, and the pellet was dried in an open air. DNA was electrophoresed with 1% agarose gel. The bands were visualized by UV illumination.
Results are expressed as the mean f SD. The Student’s paired r-test was used to compare the means of two groups. Differences were considered significant when ~0.05.
Results Effects of drugs on cell profiferation Fig. 1 shows that both CVP and TET elicited concentration-dependent inhibitory effects on the proliferation of HL-60 cells. TET, but not CVP, also exerted concentration-dependent cytotoxic effect on the normal PBL cells. This result suggests that CVP is more selective in the inhibition of proliferation of cancer cells. BER, a structrual analogue of TET, also elicit an inhibitory effect on HL60 cells, albeit weaker than that of TET (Fig. 2).
Vol. 60, No. 8, 1997
Inhibition of HL-60 Cells
PL-137
Effects of drugs on cell death HL-60 cells treated with 24 pM TET or BER for 12 hrs showed morphological changes characteristic of apoptosis, such as cell shrinkage, nuclear fragmentation and apoptotic bodies (not shown). However, these morphological features were not observed in HL-60 cells treated with 1 mg/ml CVP for up to 72 hrs. The formation of apoptotic cells was compared in HL-60 cells treated with 24 PM TET and BER (Fig. 3). Apoptosis was more prominent in TET-treated cells compared to BER-treated cells. DNA fragmentation also occurs in apoptosis and is characterized by a ladder pattern indicating intemucleosomal chromatin cleavage, as detected in HL-60 cells treated by UV irradiation for 30 mm serving as a positive control (Fig. 4a). However, a ladder of fragmented DNA was not detected in HL-60 cells on day 3 culture treated with 1 mg/ml CVP indicating that CVP does not cause apoptosis. On the other hand, a ladder of fragmented DNA was detected in HL-60 cells in 12-hr culture with TET or BER (Fig. 4b).
Discussion We have demonstrated that CVP and TET, the active chemical constituents of two widely used Chinese medicinal herbs, Coriolus versicolor and Stephmiu tetrundru, respectively, elicited inhibitory effects on the proliferation of human leukemic HL-60 cells. The inhibitory actions of these two drugs, however, are characteristically different in two aspects. First, CVP is more selective for tumor cells, whereas TET is rather non-selective based on the differential effects of these drugs on the proliferation of HL-60 cells and the normal human PBL. Second, TET but not CVP induces apoptotic responses in HL-60 cells as evident from both morphological and DNA ladder patterns. Since TET has immunosuppressive activity (9-ll), whereas CVP and other polysaccharide peptides from similar Chinese medicinal mushrooms show immunostimulating activity (4,6,7), the immunomodulating activity is unlikely to be the primary attribution to their common action in suppressing the solid tumor growth, which is probably as a result of their direct inhibition of tumor cell proliferation.
B:
A:
90 -
PBL
80 70 60 -
'i; 80
50 40 30 20 10 -
o10
100
0
1000
CVPWmL)
* I. 7
*
R
*
HL-60
\
4 * PBLb
5
10
TET
15
20
h
25
30
(PM)
Fig. 1
Cell growth profiles for HL-60 and PBL cells following 72-hr incubation with various concentrations of CVP (A) and following 48 hr incubation with increasing concentrations of TET (B). Data are shown as mean + SD of 3 independent experiments with duplicate wells each. Note the selective inhibition by CVP and non-selective inhibition by TET of cell growth in HL-60 cells compared to the normal PBL cells. * Significantly (~0.05) different from PBL values.
Inhibition of HL60 Cells
PLl38
A:
Vol. 60, No. 8, 1997
B:
%I00 L
z3 z q L
80 60
$ 40 E z' 20 = 0"
0 0
5
10
15
20
25
0
5
10
15
20
25
Concentrations of drugs (pM) Fig.2 Cell growth following 24-hr (A) or 48-hr (B) incubation with various concentrations of drugs. Data are expressed as mean f SD of 3 separate experiments with duplicate wells. Note that TET significantly (*PcO.O5) inhibited the proliferation of HL-60 cells and its effect was more potent than BBR in the concentration range of 12-24 PM, 120 q LIOO u) =
a, v
3
An .s.e
60
I sg 40 0 s. 2 20
2
I
I
0
Control BER
!
TET
-
-r-lI
T
0
0
24 lncu bation time (hr) Fig.3
48
The percentage of apoptotic HL-60 cells assessed morphologically at time points up to 48 hrs in the presence or absence of 24 pM BER or TBT. The values represent mean f SD of 3 different determinations. *Significant wO.05) increase in apoptotic cells compared to that without incubation or the control without drug treatment. Recently, Dong et al. (4) have shown that CVP also elicited in vivo suppressive effect against solid tumor from Sarcoma- 180 implantation. Although it exerted in vitro cytotoxic effects on human hepatic tumor HEPG2 cells, CVP up to 1 mg/ml failed to inhibit the proliferation of the normal human fetal hepatocytes. This is consistent with the present finding that CVP inhibits the growth of human leukemic HL-60 cells without significant effects on the normal PBL cells and lends further support to the contention that CVP selectively inhibits the tumor cell growth. The mechanism of action of the selective effect of CVP causing tumor cell death is not known, but it may involve arrest in cell cycle according to our preliminary results studied by using flow cytometry.
Vol. 60, No. 8, 1997
Inhibition of I-X-60 Cells
a
b
PL-139
abed
B
1078
872 603 310
Fig. 4 A: Agarose gel electrophoresis of DNA extracted from I-IL-60 cells incubated with medium alone (lane a) or 1 mg/ml CVP (lane b) for 72 hrs and from I-IL-60 cells treated with UV irradiation for 30 min as a positive control (lane c). Lane d showed DNA markers. B: Agarose gel electrophoresis of DNA extracted from I-IL-60 cells incubated for 12 hrs with medium alone (lane b), 24 pill BER (lane c) or 24 uM TET (lane d). Lane a shows the standard DNA markers.
TET elicited in vitro cytotoxic effects on human BM13674 cells (17) and Hela cells (18) as well as in vivo suppressive effects against mouse ascites tumor from Ehrlich ascites carcinoma and against solid tumor from Sarcoma-180 (8). Most earlier studies of antitumor effects of TET were confined to the use of tumor cell lines. However, our present finding that the inhibitory effect of TET on cell proliferation is comparable between I-IL-60 and PBL indicates that cytotoxic effect of TET is non-selective with respect to cell type. It is interesting to note that TET was also more potent than BER in causing release of intracellular Ca” and inhibiting Ca” entry activated by thapsigargin in HL60 cells (19,20). The mechanism of actions of TET/BER-induced apoptosis is not clearly understood and the role of elevation of cytosolic Ca” concentration in the apoptotic process in HL-60 and its relationship to TETIBER-induced apoptosis require future investigation. Acknowledgement This work was supported by a donation from the Hong Kong Association of Health Care Ltd. and in part by the Committee of Research and Conference Grants of the University of Hong Kong.
References 1. 2. 3. 4. 5. 6.
M. NIIMOTO, T. HATTORI, R. TAMADA, T. SUGIMACHI, K. INOBUCHI and N. C. OGAWA. Jpn. J. Surg. fi 681-686 (1988). T.F. LIU, Recent Advance in Cancer, 63-66, Chinese University Press, Hong Kong (1989). Y. KIKUCHI, I.. KIZZAWA, K. OOMORI, Y. KITA and K. KATO, Jpn. J. Cancer Res. 79 125-130 (1988). Y. DONG, C.Y. KWAN, Z.N. CHEN and M. YANG, Res. Commun. Mol. Pathol. Pharmacol. 92 140-148 (1996). M.M.P. YANG, Z.N. CHEN and J.S.L. KWOK, Amer. J. Chin. Med. 20221-232 (1992). T. FUJII, T. KANO, K. SAITO, Y. KOBAYASHI, H. IIJIMA, T. MATSUMOTO, C. YOSHIKUMI and T. TAGUCHI, Anticancer Res. 1845-848 (1987).
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