Ganoderma lucidum causes apoptosis in leukemia, lymphoma and multiple myeloma cells

Leukemia Research 30 (2006) 841 848 Ganoderma lucidum causes apoptosis in leukemia, lymphoma and multiple myeloma cells Claudia I. Müller a,b,, Takashi Kumagai a, James O Kelly a, Navindra P. Seeram c, David Heber c, H. Phillip Koeffler a a Cedars-Sinai Medical Center, David Geffen School of Medicine at UCLA, Los Angeles, CA, United States b Division of Haematology/Oncology, University Hospital, Freiburg, Germany c Center for Human Nutrition, David Geffen School of Medicine at UCLA, Los Angeles, CA, United States Received 4 May 2005; received in revised form 5 December 2005; accepted 6 December 2005 Available online 19 January 2006 Abstract Over many centuries, herbal remedies have treated a variety of ailments. This empiric observational approach has produced a number of leads for formulated medicines. Ganoderma lucidum extract was screened for its anti-proliferative activity using a panel of 26 human cancer cell lines. The six most sensitive hematologic cell lines were: HL-60 (ED50 26 g/ml), U937 (63 g/ml), K562 (50 g/ml), Blin-1 (38 g/ml), Nalm-6 (30 g/ml) and RPMI8226 (40 g/ml). Cell cycle analyses revealed a G2/M arrest, most prominently in HL-60 cells. Four hematopoietic cell lines (HL-60, Blin-1, U937, RPMI8226) were examined for apoptosis, which ranged between 21 and 92%. After exposure to G. lucidum extract, HL-60 cells became multinucleated with an increased DNA content. These results indicate that G. lucidum extract has a profound activity against leukemia, lymphoma and multiple myeloma cells and may be a novel adjunctive therapy for the treatment of hematologic malignancies. 2005 Elsevier Ltd. All rights reserved. Keywords: Ganoderma lucidum; Leukemia; Lymphoma; Multiple myeloma; Apoptosis; Growth arrest 1. Introduction Ganoderma lucidum (Fr.) Karst (common names: Reishei, Lingzhi) is a herbal mushroom that has been used for centuries in Traditional Chinese Medicine (TCM) for its health promoting properties [1]. The fruiting bodies, spores and cultivated mycelia of G. lucidum as well as its extracts are used world-wide as ingredients in health foods, herbal medicines and dietary supplements (extracts and powdered forms) and have been used as anti-cancer agents and for prevention and treatment of various other diseases in ancient China (100 bc) [2 6]. Previous reports revealed that G. lucidum extract inhib- Corresponding author at: Division of Hematology/Oncology, Davis Building 5065, Cedars-Sinai Medical Center, David Geffen School of Medicine at UCLA, 8700 Beverly Boulevard, Los Angeles, CA 90048, United States. Tel.: +1 310 423 7759; fax: +1 310 423 0225. E-mail address: MullerCI@cshs.org (C.I. Müller). ited growth of several cancer cell lines including the human prostate PC-3 cancer cells, and several human bladder cancer cell lines. These cells were arrested in the G2/M phase of their cell cycle [7,8]. Also, Ganoderma in a dose- and time-dependent manner inhibited the cell proliferation and induced apoptosis of HT-29, a human colon carcinoma cell line and MDA-MB-231 and MCF-7 breast cancer cell lines [5,9,10]. The phytochemical constituents of Ganoderma include polysaccharides, proteins, nucleosides, fatty acids, sterols, cerebrosides and triterpenes [11]. The triterpenoids common to Ganoderma include the ganoderic acids that may be considered as a chemical marker compound to authenticate and/or standardize Ganoderma extracts and products [12,13]. The polysaccharide fraction of Ganoderma was shown to slow growth of sarcoma cells growing in mice [14]. The polysaccharides were able to induce the expression of IL- 1, IL-6, IL-12, IFN-, TNF-, GM-CSF, G-CSF, M-CSF in 0145-2126/$ see front matter 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.leukres.2005.12.004

842 C.I. Müller et al. / Leukemia Research 30 (2006) 841 848 monocytes macrophages and T lymphocytes which might, in part, mediate some of their anti-tumor activity [1,15,16]. Furthermore, these polysaccharides may also have immune enhancing properties perhaps as a consequence of their ability to stimulate cytokine production [15]. Triterpenes have been shown to inhibit growth of human hepatoma Huh-7 cells associated with G2 cell cycle arrest, but the compounds had no effect on growth of normal liver cell lines in vitro [2]. Moreover, the triterpene-fraction of Ganoderma inhibited the primary and metastatic tumor growth of Lewis lung carcinoma (LLC) cells implanted in mice [17]. This study investigated the anti-cancer effects of G. lucidum using a panel of 26 human cancer cell lines (16 hematologic malignancies; see Section 2). A detailed description of the characteristics of each cell line is also given in Table 1. Table 1 Human cancer cell lines examined for anti-proliferative effects of G. lucidum extract Cell line Characteristics HL-60 Acute myeloblastic leukemia (FAB M2) U937 Diffuse histiocytic lymphoma (monocytic leukemia cells) NB4 Promyelocytic leukemia (FAB M3) THP-1 Acute monocytic leukemia (FAB M5) K562 Erythroid chronic myeloid leukemia (blast crisis) Blin-1 Pre-B acute lymphoblastic leukemia Nalm-6 Non-T, non-b acute lymphoblastic leukemia Jurkat T-cell acute lymphoblastic leukemia RPMI8226 Multiple myeloma (IgG ) ARH77 Multiple myeloma (IgG ) U266 Multiple myeloma (IgE ) NCI-H929 Multiple myeloma (IgA ) Daudi Burkitt s lymphoma Ramos Burkitt s lymphoma NCEB-1 Centroblastic centrocytic, diffuse lymphoma SUDHL6 Diffuse large-b-cell lymphoma LNCaP Prostate cancer (androgen receptor positive) PC-3 Prostate cancer (androgen receptor negative) DU145 Prostate cancer (androgen receptor negative) MCF-7 Breast cancer (estrogen receptor positive) MDA-MB231 Breast cancer (estrogen receptor negative) HT29 Colorectal cancer PANCI Pancreatic cancer ASPC1 Pancreatic cancer BxPC-3 Pancreatic cancer NCI-H520 Non-small cell lung cancer (squamous cell carcinoma) The six most sensitive cell lines are in bold. 16 hematological cell lines 10 solid tumor cell lines 2. Materials and methods 2.1. Extraction of G. lucidum and standardization by HPLC analysis G. lucidum fruiting bodies were collected and authenticated in China by Phytomedical Research Inc. (Beijing, China), and Botanica Biosciences (Ojai, CA). A commercial standard of ganoderic acid C2 was purchased from Chromadex (Santa Ana, CA). G. lucidum fruiting bodies were extracted in water and lyophilized to yield a dark brown powder. All solvents were HPLC (high performance liquid chromatography) grade and were purchased from Fisher (Tustin, CA). Ganoderma extract (22.28 mg) was sonicated for 30 min with 4 ml (MeOH:water, 1:1 v/v). Ganoderic acid C2 was dissolved in MeOH to make a stock solution of 1 mg/ml that was further serially diluted with MeOH:water (1:1 v/v). Quantitation was done by peak area measurements in comparison with a standard curve generated for ganoderic acid C2. All samples (25 l injection volume) were filtered (0.22 m) before analysis on a Waters 2690 HPLC system equipped with a 996 PDA detector (Waters, Milford, MA). Compounds were eluted at 1.00 ml/min on a Novapak (Waters) RP-18 column (150 mm 3.9 mm i.d., 5 m) with a Symmetry C18 guard column (20 mm 3.9 mm i.d., 5 m). The eluent consisted of 2% aqueous acetic acid (A) and acetonitrile (B). A gradient solvent system was used as follows: 0 5 min 95% A in B; 6 60 min 5% A in B. The wavelength of detection was 254 nm. 2.2. Cell lines Cell lines that were studied included: myeloid leukemia (HL-60, U937, K562, THP-1, NB4), acute lymphoblastic leukemia (B- and T-ALL) (Blin-1, Nalm-6, Jurkat), multiple myeloma (RPMI8226, ARH77, U266, NCI-H929), Burkitt s lymphoma (Daudi, Ramos), non-hodgkin s lymphoma (NCEB-1, SUDHL6), prostate cancer (LNCaP, PC-3, DU145), breast cancer (MCF-7, MDA-MB-231), colorectal cancer (HT-29), pancreatic cancer (PANCI, ASPC1, BxPC- 3) and non-small cell lung cancer (NSCLC) (NCI-H520) (see Table 1). Most cell lines were obtained from American Type Culture Collection (ATCC, Rockville, MD), Blin-1, NCEB- 1 and SUDHL6 cells were generously provided by Sven de Vos (University of California, Los Angeles). NB4 cells were a kind gift from M. Lanotte (St. Louis Hospital, Paris, France). Cells were maintained in culture according to recommendations. 2.3. MTT assays An initial screening was done to explore the antiproliferative effects of the herbal extract on the 26 human cancer cell lines. Cells (3 10 4 ml 1 ) were cultured in the presence of 50 and 100 g/ml G. lucidum extract for

C.I. Müller et al. / Leukemia Research 30 (2006) 841 848 843 96 h in 96-well plates (Flow Laboratories, Irvine, CA). Thereafter, 10 l of MTT solution (in 5 mg/ml PBS, Roche) was added to each well and incubated for 4 h in a humidified atmosphere at 37 C according to the manufacturer s protocol. Consecutively, 50 l solubilization solution (20% SDS) was added into the wells and incubated overnight (16 h). After culture, cell number and viability were evaluated by measuring the mitochondrial-dependent conversion of the yellow tetrazolium salt MTT to purple formazan crystals by metabolic active cells. The resulting colored solution was quantified at 540 nm using an enzymelinked immunoabsorbent assay reader (ELISA reader, Bio-Rad). 2.4. Cell cycle analysis Cells (3 10 4 ml 1 ) were incubated for 72 h either with or without G. lucidum (100 g/ml). They were collected, washed, suspended in cold 1 PBS, fixed in 75% methanol and stained with propidium iodide (PI). Cell cycle analysis was performed using the Becton Dickinson Flow Cytometer. 2.5. Assessment of apoptosis by Annexin V assay Apoptotic cell death was examined by Annexin V- apoptosis detection kit (Pharmingen Inc., San Diego, CA). During early stages of apoptosis, phosphatidylserine (PS) becomes externalized on the outer plasma membrane. In order to be able to distinguish between apoptosis and necrosis, cells were stained with FITC-labeled Annexin V and propidium iodide (PI). Annexin V binds to the externalized PS, whereas PI is able to penetrate the increasingly permeable plasma membrane during necrosis or later stages of apoptosis and binds to cellular DNA. Cells were analyzed by fluorescence activated cell sorting (FACS). Four hematological cell lines (HL-60, U937, Blin-1 and RPMI8226) were treated with G. lucidum at four different concentrations (50, 100, 150 and 200 g/ml) for 72 h. Control cells and treated cells were analyzed for staining by Annexin V and PI. 2.6. Assessment of apoptosis by determination of mitochondrial membrane potential Apoptosis was further investigated by analysis of the mitochondrial membrane potential ( Ψ) by JC-1 assay according to the manufacturer s protocol (Cell Technology, MN). JC-1 (5,5,6,6 -tetrachloro-1,1,3,3 - tetraethylbenzimidazolcarbocyanine iodide) is a lipophilic cationic dye which selectively incorporates into intact mitochondria and undergoes a reversible change in fluorescence emission from green to red as mitochondrial membrane potential increases. During apoptosis, loss of the mitochondrial membrane potential occurs. In cells with high membrane potential, the formation of dimers of the dye is promoted, resulting in red fluorescence. In contrast, in cells with low membrane potential, JC-1 is in its monomeric form, which then fluoresces green. The red/green fluorescence intensity ratio is used to analyze whether a cell is apoptotic or not. A decrease in red/green ratio indicates an increase in apoptotic cells. Fluorescence of the cells is measured by flow cytometry. Formation of JC-1 aggregates within the mitochondria results in emission of red fluorescence in the 590 nm spectrum. If the mitochondriae collapse during apoptosis, the fluorescence shifts to the green 510 nm spectrum. 2.7. Western blot analysis Total cell lysate (25 g) was electrophoresed on 10 20% SDS-polyacrylamide gel (Bio-Rad, Hercules, CA) and transferred by electroblotting to a polyvinylidene fluoride membrane (Pall Corporation, Pensacola, FL). The membrane was incubated overnight with the following antibodies: anti-p21 WAF1 (SC-397, 1:500 dilution) and antip27 KIP1 (SC-528, 1:500 dilution), both rabbit polyclonal antibodies from Santa Cruz Biotechnology (Santa Cruz, CA) followed by a secondary horseradish peroxidaseconjugated donkey anti-rabbit antibody (Amersham Biosciences). Detection was performed using the SuperSignal chemiluminescence substrate (Pierce, Rockford, IL). Blots were also subjected to overnight incubation with a murine monoclonal GAPDH antibody (RDI-TRK5G4-6C5, 1:10,000 dilution; Research Diagnostics, Flanders, NJ) followed by a secondary horseradish peroxidaseconjugated sheep anti-mouse antibody (Amersham Biosciences). Western blots were stripped between hybridizations with stripping buffer (10 mm Tris HCl ph 2.3, 150 mm NaCl). 2.8. Cytospin preparations and staining Cytospin preparations were performed for seven hematologic (NCI-H929, RPMI8226, Blin-1, Nalm-6, Daudi, U937, HL-60) and six solid tumor (ARO, BHP2-7, PC3, DU145, NCEB-1, MCF-7) cell lines after 96 h treatment with G. lucidum (100 g/ml). Slides were consecutively stained with Diff Quik (Dade Behring, Switzerland) staining solution according to the manufacturer s protocol. 3. Results 3.1. HPLC analysis of G. lucidum extract The G. lucidum extract was standardized to 0.15% ganoderic acid C2 content (Fig. 1a). Fig. 1b shows the HPLC chromatogram of ganoderic acid C2 standard eluting at a retention time of 32.9 min. Fig. 1c shows the G. lucidum extract spiked with ganoderic acid C2 standard confirming its presence at the retention time of 32.9 min.

844 C.I. Müller et al. / Leukemia Research 30 (2006) 841 848 Fig. 1. (a) HPLC chromatogram of G. lucidum extract showing ganoderic acid C2 standard eluting at 32.9 min. (b) HPLC chromatogram of ganoderic acid C2 standard eluting at 32.9 min. (c) HPLC chromatogram of G. lucidum extract spiked with ganoderic acid C2 standard confirming its presence at 32.9 min. Fig. 2. Growth arrest of hematologic cell lines induced by G. lucidum extract. Cells included: panel a, acute myeloid leukemia (HL-60, U937); panel b, erythroid chronic myeloid leukemia (K562); panel c, acute lymphoblastic leukemia (Blin-1, Nalm-6); panel d, multiple myeloma (RPMI8226). Cells of each line were treated with G. lucidum (10, 20, 40, 60, 80 and 100 g/mg) for 96 h. MTT assay was performed. Viable cells were expressed as a percentage of untreated control cultures for each line. Results represent the mean ± S.D. of three different experiments performed in triplicates.

C.I. Müller et al. / Leukemia Research 30 (2006) 841 848 845 Table 2 Inhibition of the proliferation of hematopoietic cell lines by G. lucidum extract Cell line ED 50 ( g/ml) Hematologic malignancy HL-60 26 Acute myeloblastic leukemia (FAB M2) U937 63 Diffuse histiocytic lymphoma (monocytic leukemia cells) K562 50 Erythroid chronic myeloid leukemia (blast crisis) Blin-1 38 Pre-B acute lymphoblastic leukemia Nalm-6 30 Non-T, non-b acute lymphoblastic leukemia RPMI8226 40 Multiple myeloma (IgG ) Cells were cultured for 96 h in the presence of 10, 20, 40, 60, 80 or 100 g/ml of G. lucidum, and cell growth was analyzed by MTT assay. Percent growth in experimental wells compared to untreated control wells was graphed, and the effective dose which inhibited 50% growth (ED 50 ), was calculated for each cell line. 3.2. Anti-proliferative effects of G. lucidum extract in a variety of cancer cell lines The initial screening of 26 cancer cell lines (Table 1)was performed culturing these cells with 50 and 100 g/ml of G. lucidum for 96 h and measuring their growth by MTT (data not shown). Only those lines which achieved 50% inhibition of growth (hematopoietic cells) were studied further. The effect of G. lucidum in a dose-dependent fashion on the growth of the six most sensitive hematological cell lines (HL-60, U937, K562, Blin-1, Nalm-6, RPMI8226) was examined at 96 h of culture. Dose response curves were generated (Fig. 2). The effective dose, which inhibited 50% cell growth (ED 50 ), was calculated and ranged between 26 and 63 g/ml (Table 2). 3.3. G2/M arrest induced by G. lucidum extract Cell cycle analysis was performed for HL-60, U937, RPMI8226, Nalm-6 and Blin-1 cells (G. lucidum, 100 g/ml, 72 h). Results revealed a G2/M arrest, most prominently in HL-60 cells (29% in G2/M compared to 12% in control cells). Moreover, an increase of DNA content occurred in the G. lucidum treated HL-60 cells (Fig. 3). Only a slight increase of cells in the G2/M phase occurred in the RPMI8226 cells (16%, untreated control compared to 20% treated cells) and the Nalm-6 cells (12%, untreated control versus 15% treated cells), whereas Blin-1 and U937 cells showed no increase of cells in G2/M phase (data not shown). 3.4. Pro-apoptotic effects of G. lucidum extract We analyzed whether the anti-proliferative effects of G. lucidum could be explained in part by apoptosis. HL-60, U937, Blin-1 and RPMI8226 were cultured with G. lucidum (50, 100, 150 and 200 g/mg, 72 h) and analyzed for apoptosis by Annexin V staining. G. lucidum, in a dose-dependent manner induced apoptosis for each of the cell lines, especially HL-60 (Fig. 4) and Blin-1 cells. In the case of HL-60 cells, approximately 4% of the diluent control cells were Annexin V positive, which increased in a dose response fashion with 92% positivity in the presence of 200 g/ml of G. lucidum (Fig. 4). Blin-1 cells were less sensitive with approximately 42% of the cells being apoptotic after treatment with Ganoderma (200 g/ml, 72 h) (data not shown). Consistent with their lower sensitivity to G. lucidum as measured by MTT assay, U937 and RPMI8226 cells had less apoptosis (32 and 21%, respectively) in response to the herbal extract (200 g/ml, 72 h) (Fig. 4 and data not shown, respectively). Fig. 3. Induction of G2/M arrest in HL-60 cells by G. lucidum extract. Panel a shows cell cycle analysis by flow cytometry after PI staining for untreated control cells. Panel b depicts cell cycle analysis for cells treated with G. lucidum, 100 g/ml for 72 h. Increasing number of HL-60 cells in the G2/M phase of the cell cycle were detected. Horizontal and vertical axis represent DNA content and cell number, respectively. Percentages of the different cell cycle phases are calculated for the population of cells within the dashed lines. Representative of one of two experiments with similar results.

846 C.I. Müller et al. / Leukemia Research 30 (2006) 841 848 Fig. 4. Induction of apoptosis in HL-60 and U937 cells by G. lucidum extract. HL-60 (panel a) and U937 (panel b) cells were cultured with G. lucidum (50, 100, 150 and 200 g/mg) for 72 h, stained with FITC-conjugated Annexin V and propidium iodide (PI). Percentage of apoptotic cells was measured by flow cytometry in comparison to diluent-treated control cells. Bar graphs reflect the cells exclusively stained with FITC. Results represent the mean ± S.D. of three different experiments. 3.5. Apoptosis coincident with a decrease of mitochondrial membrane potential Apoptosis begins when a cell activates its own destruction by initiating a series of complex cascading events that include the depolarization of the mitochondrial membrane potential. The ability of G. lucidum to alter the mitochondrial transmembrane electrical potential was investigated in HL-60 and U937 myeloid cells. As the cells lose electrical potential, the fluorescence of the JC-1 dye changes from red to green. An increase of monomeric JC-1 molecules (green fluorescence) due to a decrease of mitochondrial membrane potential occurred in a dose-dependent manner. After 48 h of culture with G. lucidum, a dose-dependent decrease of the ratio of red to green fluorescence occurred as the mitochondria became progressively depolarized (Fig. 5). 3.6. Upregulation of p21 WAF1 and p27 KIP1 by G. lucidum extract We examined the effect of G. lucidum on the expression of cell cycle- and apoptosis-related proteins by Western blot analysis. U937 cells were treated with G. lucidum at four different concentrations (50, 100, 150 and 200 g/ml) for 48 and 72 h. Western blotting showed that an increase in p21 WAF1 protein expression occurred in a dose- and time-dependent fashion compared to diluent only treated cells (Fig. 6a). G. lucidum upregulated the expression of p27 KIP1 proteins at 48 h of exposure; effects did not further increase at 72 h (Fig. 6b and data not shown). 3.7. Multinucleation of HL-60 cells after treatment with G. lucidum extract Cytospin preparations and staining were performed for morphologic analysis after culture of 13 cell lines with G. lucidum (100 g/ml, 96 h). This treatment caused prominent multinucleation of only HL-60 cells (Fig. 7). Further dosedependent studies revealed that approximately 1% of HL- 60 cells became multinucleated in the presence of 1 g/ml G. lucidum (96 h), 5% cells in the presence of 10 g/ml, 10% cells with 50 g/ml, 40% cells with 75 g/ml and 80% cells with 100 g/ml (data not shown). Untreated HL-60 had <0.5% multinucleated cells. Multinucleation did not occur in the six other hematologic and six solid tumor cell lines analyzed (100 g/ml, 96 h, data not shown). FACS analysis of PI stained HL-60 cells confirmed the increased DNA content of these cells (see Fig. 3). 4. Discussion Because G. lucidum is traditionally consumed in cooked aqueous forms such as soups and tea, we conducted our Fig. 5. Induction of mitochondrial membrane collapse in U937 cells cultured with G. lucidum extract. HL-60 (panel a) and U937 (panel b) cells were incubated with G. lucidum for 48 h. Mitochondrial membrane potential of cells treated with G. lucidum and diluent treated control cells was assessed by flow cytometry after staining with JC-1. JC-1 dimers fluoresce red in stable mitochondria and form green fluorescent monomers when the mitochondrial membrane is decreasing in potential. The decrease of the red/green fluorescence reflects increasing apoptotic cells. Results represent ratios of the mean of three different experiments.

C.I. Müller et al. / Leukemia Research 30 (2006) 841 848 847 Fig. 6. Upregulation of p21 WAF1 and p27 KIP1 by G. lucidum extract in U937 cells. U937 cells were treated with increasing doses of G. lucidum extract (50, 100, 150 and 200 g/ml) for 48 and/or 72 h. Protein lysates were analyzed by Western blot with p21 WAF1 (panel a) or p27 KIP1 (panel b) specific antibodies. The blots were stripped and rehybridized with a GAPDH antibody as control for equal loading. evaluation on the aqueous extract of the fruiting bodies of the mushroom. We demonstrate the anti-tumor properties of an extract of G. lucidum against a variety of human leukemic, lymphoma and myeloma cell lines. After the initial screening of five herbs with known anti-cancer activities (G. lucidum, Isatis indigotica, Dendranthema morifolium, Rabdosia rubescens and Panax pseudoginseng), G. lucidum was found to be the overall most active extract (data not shown). The 16 hematologic cell lines were more sensitive to the growth inhibiting properties of Ganoderma than were the 10 solid tumor lines (data not shown). Further studies focused on the anti-cancer activities of Ganoderma in the six most sensitive hematopoietic cell lines. These hematopoietic cell lines underwent growth arrest in a dose-dependent manner in response to treatment with Ganoderma with ED 50 values for HL-60, Nalm-6, and Blin-1 ranging between 26 and 38 g/ml. The RPMI8226 (ED 50,40 g/ml), K562 (ED 50,50 g/ml) and U937 (ED 50, 63 g/ml) were slightly less sensitive to the drug. Growth inhibition was associated with cell cycle retardation in the G2/M phase especially in the HL-60 cells. Apoptosis as shown by Annexin V staining was particularly prominent in HL-60 acute myeloid leukemia cells. Measurement of mitochondrial membrane potential (JC-1 assay) paralleled the apoptosis data consistent with G. lucidum causing mitochondrial induced cell death. Recent reports showed that G. lucidum increased the expression of p21 WAF1 and p27 KIP1 in the PC-3 prostate cancer cell line and MCF-7 breast cancer cell line [7,10]. We detected increased protein expression of p21 WAF1 and p27 KIP1 in U937 cells after their treatment with G. lucidum for 48 and 72 h, respectively (Fig. 6a and b). Even though no G2/M arrest was observed in U937 cells after treatment with G. lucidum, p21 WAF1 and p27 KIP1 might play a role in these cells having decreased proliferation and increased apoptosis. No change in expression levels of p21 WAF1 and p27 KIP1 was detected in HL-60 cells. Multinucleation in HL-60 cells was reported to occur after exposure to several different substances [18 20]. No uniform explanation has been provided to explain the mechanism. Cholesterol starvation has been noted to cause formation of polyploid HL-60 cells; this was reversed by the readdition Fig. 7. Multinucleation of HL-60 cells caused by G. lucidum extract. Untreated HL-60 cells (panel a) and HL-60 cells treated with G. lucidum (100 g/mg, 96 h) (panel b). Arrowheads point to several of the multinucleated cells present in the photograph. Cells were cytocentrifuged, fixed, stained as described in Section 2 (magnification 400 ).