Synergistic Induction of 1,25-Dihydroxyvitamin D₃- and All-trans-Retinoic Acid-Induced Differentiation of HL-60 Leukemia Cells by Yomogin, a Sesquiterpene Lactone from Artemisia princeps

• Abstract
• Abbreviations
• Introduction
• Materials and Methods
• Materials
• Determination of cell proliferation and viability
• Determination of cell differentiation
• Morphological studies
• Immunofluorescent staining and cytofluorometric
  measurement
• Statistical analysis
• Results
• Effects of yomogin on HL-60 cell proliferation and
  differentiation
• Cytofluorometric and morphologic analysis of yomogin-
  induced HL-60 cell differentiation
• Effects of yomogin on differentiation pathway of HL-60
  leukemia cells
• Discussion
• Acknowledgements
• References

Abstract

Many anti-inflammatory agents are known to significantly enhance the terminal differentiation of some cancer cells such as leukemia cells. In this study, the effect of yomogin, a eudesmane sesquiterpene lactone isolated from Artemisia princeps with anti-inflammatory activity, was investigated in human promyelocytic leukemia HL-60 cells. Yomogin by itself induced small increases in cell differentiation, with less than 19 % of the cells attaining a differentiated phenotype. Importantly, yomogin synergistically enhanced differentiation of HL-60 cells in a dose-dependent manner when combined with either 5 nM 1,25-dihydroxyvitamin D₃ [1,25-(OH)₂D₃] or 50 nM all-trans retinoic acid (all-trans RA). Cytofluorometric analysis and morphologic studies indicated that the combinations of yomogin and 1,25-(OH)₂D₃ stimulated differentiation to monocytes whereas the combinations of yomogin and all-trans RA stimulated differentiation to granulocytes. These results suggest that yomogin may be useful in combination with 1,25-(OH)₂D₃ or all-trans-RA in the differentiation therapy for myeloid leukemias.

Abbreviations

1,25-(OH)₂D₃:1,25-dihydroxyvitamin D₃

FITC:fluorescein isothiocyanate

NBT:nitroblue tetrazolium

RA:retinoic acid

PE:phytoerythrin

Introduction

Differentiation therapy for neoplastic diseases has potential for supplementing existing treatment modalities. Some cancer cells including leukemia cells exhibit a defect in their capacity to mature into non-replicating adult cells, thereby existing in a highly proliferating state, which result in outgrowing their normal cellular counterparts. The induction of terminal differentiation represents an alternative approach to the treatment of cancer by conventional anti-neoplastic agents since cells exposed to chemical or biological inducers of differentiation do not undergo the cytodestruction produced by cytotoxic agents. The HL-60 leukemia is a useful model for the study of differentiation since HL-60 cells can be induced to undergo terminal differentiation by a variety of chemical and biological agents [1]. These findings indicate that the malignant state is not irreversible and suggest that the leukemias, as well as some other cancers, may eventually be treated with agents that induce terminal differentiation, presumably with less morbidity than that produced by the cytodestructive drugs.

Several sesquiterpene lactones have received considerable attention in pharmacological research due to their potent anti-neoplastic and anti-inflammatory activities [2]. Yomogin, a eudesmane sesquiterpene lactone, was isolated from Artemisia princeps Pamp and exerted an anti-inflammatory activity by inhibiting the expression of inducible nitric oxide synthase [3]. Previously anti-inflammatory agents such as indomethacin were demonstrated to increase the sensitivity of human leukemic HL-60 cells to physiological differentiating agents [4].

In this report, we investigated the effect of yomogin on cellular differentiation in the human promyelocytic leukemia HL-60 cell culture system. We also investigated the effects of combinations of yomogin with 1,25-(OH)₂D₃ or all-trans RA on HL-60 cell differentiation. 1,25-(OH)₂D₃ and all-trans RA were chosen for this study because they have been widely used as endogenous stimulators of differentiation in HL-60 cells. The HL-60 cells are differentiated into the monocytic lineage or granulocytic lineage when treated with 1,25-(OH)₂D₃ or all-trans-RA, respectively [5].

Materials and Methods

Materials

HL-60 cell line (ATCC, Rockville, MD, USA) was maintained in RPMI-1640 medium supplemented with 10 % fetal bovine serum (Gibco BRL, Grand Island, NY, USA). 1,25-(OH)₂D₃, all-trans-RA, indomethacin, Giemsa staining solution, and all other reagents were purchased from the Sigma Chemical Co. (St. Louis, MO, USA). Yomogin was isolated from Artemisia princeps as previously described [6]. Yomogin was dissolved in dimethyl sulfoxide to make a stock solution of 20 mM. The solution was diluted at least 1000-fold in the growth medium such that the final concentration of solvent had no effect on the expression of the differentiation. The herb of Artemisia princeps was collected in Chonnam Province, Korea, and a voucher of specimen (CNU-6291) is deposited at the Herbarium of College of Pharmacy, Chonnam National University, Korea.

Determination of cell proliferation and viability

Cell proliferation was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium (MTT) assay. In brief, after each treatment, 10 μl of MTT (5 mg/ml) were added to each well in 96-well plates. After incubation for 4 h at 37 °C, the crystals of viable cells were dissolved with 100 μl of 0.04 N HCl in isopropanol. The absorbance of each well was then read at 540 nm using a kinetic microplate reader. Cell viability was determined by the trypan blue exclusion assay. Viability was calculated as the percentage of live cells in the total cell population.

Determination of cell differentiation

HL-60 cell differentiation was assessed by the nitroblue tetrazolium (NBT) reduction assay as previously described [7]. This assay is based on the ability of phagocytic cells to produce superoxide upon stimulation with tissue plasminogen activator. For this assay, 2 × 10⁵ cells were harvested by centrifugation and incubated with an equal volume of 0.2 % NBT dissolved in PBS containing 1 ng/ml of freshly diluted with 12-O-tetradecanoylphobol-13-acetate at 37 °C for 30 min in the dark. Cytospin slides were prepared and were examined for blue-black nitroblue diformazan deposits, indicative of 12-O-tetradecanoylphobol-13-acetate-stimulated respiratory burst. At least 200 cells were assessed for each experiment.

Morphological studies

Single-cell suspensions were prepared and 2 × 10⁵ cells were loaded into a cyto-funnel slide and spun at 27 g in a cytospin centrifuge. The cells on the slide were fixed with methanol and dried. The cells were stained with Giemsa staining solution for 20 min and rinsed in deionized water, dried in the air, and observed under a microscope with a camera.

Immunofluorescent staining and cytofluorometric
measurement

Quantitative immunofluorescence measurements were performed in an Epic XL flow cytofluorograph (Coulter Electronics, Hialeah, FL, USA) equipped with a multi-parameter data acquisition and display system. Briefly, a single cell suspension was collected from the various cultures and washed twice with ice-cold phosphate buffered saline (PBS, pH 7.4). Afterwards, PE-conjugated anti-human CD11b or FITC-conjugated anti-human CD14 mAbs (Becton Dickinson, San Jose, CA, USA) were added, followed by incubation at 4 °C for 1 h. After incubation, the cells were washed with PBS and were fixed in PBS containing 1 % paraformaldehyde, and cytofluorometric analysis was performed. Background staining was determined by staining the cells with PE- or FITC-conjugated isotype control mAbs.

Statistical analysis

The Student’s t-test and one-way analysis of variance (ANOVA) were used to determine the statistical differences between various experimental and control groups. The difference was considered statistically significant when P < 0.05.

Results

Effects of yomogin on HL-60 cell proliferation and
differentiation

HL-60 cells were seeded at a density of 2 × 10⁵ cells/ml, and the cells were treated with 0.25, 0.5, 1, or 2 μM yomogin for 72 h. The cell proliferation and viability for each group were determined. As shown in Fig. [1] B, treatment of HL-60 cells with 0.25 - 2.0 μM yomogin alone had no effect on cell proliferation. Treatment with yomogin in combination with either 5 nM 1,25-(OH)₂D₃ or 50 nM all-trans RA inhibited cell proliferation by 25 %. For all treatments, cell viability was greater than 95 % through the incubation period. Next, the effect of yomogin on HL-60 cell differentiation was assessed by the NBT reduction assay. As shown in Fig. [1] C, incubation of HL-60 cells with more than 1.0 μM yomogin induced cell differentiation with slight increases, indicating that yomogin was a weak inducer of differentiation in HL-60 cells. Importantly, yomogin strongly potentiated cell differentiation when simultaneously combined with 5 nM 1,25-(OH)₂D₃ or 50 nM all-trans-RA. As shown in Fig. [1] C, the addition of yomogin to cultures exposed to a suboptimal concentration of 1,25-(OH)₂D₃ (5 nM) or all-trans-RA (50 nM), which by itself caused a relatively low level of differentiation (less than 17 or 19 %), resulted in a marked increase in the degree of cell differentiation. Yomogin strongly enhanced 1,25-(OH)₂D₃- and all-trans-RA-induced HL-60 cell differentiation in a concentration-dependent manner. The effects were maximal at 2.0 μM yomogin, with greater than 81 % of the treated cells attaining a differentiated state. Importantly yomogin was more efficient than indomethacin, a well-known anti-inflammatory compound, in enhancing HL-60 cell differentiation when combined with either 1,25-(OH)₂D₃ or all-trans-RA (Fig. [1] D).

Cytofluorometric and morphologic analysis of yomogin-
induced HL-60 cell differentiation

To further confirm the cell differentiation enhanced by yomogin, the morphological phenotypes and the expression of cell surface antigens on HL-60 cells were analyzed. As shown in Fig. [2], Giemsa-stained undifferentiated control HL-60 cells (Fig. [2] A) were predominantly promyelocytes with round and regular cell margins, and large nuclei, suggesting that the cells were highly active in DNA synthesis and were rapidly proliferating. Yomogin-, 1,25-(OH)₂D₃- or all-trans-RA-treated cells (Figs. [2] B, [2] C and 2E) exhibited relatively small changes in cell morphology such as irregular cell margins. Combined treatment of HL-60 cells with 1,25-(OH)₂D₃ or all-trans-RA plus yomogin (Figs. [2] D and 2F) resulted in significantly decreased cell size, denser chromatin and an increased cytoplasm to nuclear ratio, which suggested less DNA synthesis. As shown in Figs. [2] D and 2F, some cells showed a horseshoe-shaped nucleus, which is a sign of cell differentiation into a monocytic lineage and some cells showed a multilobed nucleus, which is a sign of cell differentiation into a granulocytic lineage. Cytofluorometric analysis was also performed to determine the expression of specific surface antigens on HL-60 cells. CD11b (Mac-1) is a cell surface marker for differentiation into either monocytes or granulocytes [8]. As shown in Fig. [3], yomogin increased the number of CD11b-positive cells when combined with either 1,25-(OH)₂D₃ or all-trans-RA, confirming that yomogin potentiated 1,25-(OH)₂D₃- or all-trans-RA-induced HL-60 cell differentiation.

Effects of yomogin on differentiation pathway of HL-60
leukemia cells

To determine the differentiation pathway that HL-60 cells have followed after treatment with yomogin and 1,25-(OH)₂D₃ or with yomogin and all-trans-RA, HL-60 cells were treated with yomogin alone or in combination with either 1,25-(OH)₂D₃ or all-trans-RA, and cytofluorometric analysis using mAb for the monocytic surface antigen CD14 was determined. The CD14 antigen is exclusively expressed when cells are differentiated into monocytes [9]. As shown in Fig. [4], HL-60 cells treated with a mixture of yomogin and 1,25-(OH)₂D₃ stained very strongly with anti-CD14 mAb. Cells treated with 1,25-(OH)₂D₃ alone also stained with anti-CD14 mAb, but to a lesser extent than did the cells treated with a mixture of yomogin and 1,25-(OH)₂D₃. These results indicate that yomogin stimulated 1,25-(OH)₂D₃-induced HL-60 cell differentiation along the monocytic pathway. In contrast, HL-60 cells treated with a mixture of yomogin and all-trans-RA showed little staining with anti-CD14 mAb, although synergistic induction of cell differentiation was observed as shown by the NBT reduction assay. In addition, HL-60 cells treated with a mixture of yomogin and all-trans-RA stained strongly with mAb against HL-60 cell differentiation marker CD11b (Fig. [3]), indicating that yomogin stimulated all-trans-RA-induced HL-60 cell differentiation along the granulocytic pathway.

Discussion

In the present study, we have demonstrated that yomogin potentiated 1,25-(OH)₂D₃- and all-trans-RA-induced differentiation in HL-60 promyelocytic leukemia cells. HL-60 cells were synergistically differentiated into monocytes or granulocytes when treated with yomogin in combination with either 1,25-(OH)₂D₃ or all-trans-RA. Previous studies have shown some combinations which exerted an additive or synergistic effect on HL-60 cell differentiation. These combinations include all-trans-RA and butyrate [10], vitamin D₃ and tumor necrosis factor-α [11], all-trans-RA and α-tocopherol [12], and 1,25-(OH)₂D₃ and capsaicin [13]. Although the mechanism of the observed synergy is not clear, the inhibitory effect of yomogin on NF-κB activity might be involved in this synergistic enhancement of cell differentiation. Other studies have demonstrated that interference with the activation of NF-κB appears to be a common feature for agents that enhance the differentiation of HL-60 cells produced by 1,25-(OH)₂D₃ or all-trans-RA [14] [15].

1,25-(OH)₂D₃ and some of its analogues are used for the treatment of psoriasis [16], and all-trans-RA has been used for the treatment of leukemia patients and its analogues have been used for the treatment of psoriasis [17]. It is possible that many dietary chemicals such as curcuminoids, tocopherols, carotenoids, and other edible plants can prevent human cancer, in part by synergizing with endogenously produced stimulators of differentiation such as RA and 1,25-(OH)₂D₃. The results presented here suggest that treatment of patients with combinations of yomogin and 1,25-(OH)₂D₃, or yomogin and all-trans-RA may produce a greater therapeutic response than 1,25-(OH)₂D₃ or all-trans-RA alone, possibly with less toxicity. Clinical studies are needed to evaluate this possibility, especially at concentrations of yomogin that do not induce known side-effects.

Acknowledgements

This work was supported by a grant of the Korea Health 21 R & D Project, Ministry of Health & Wealfare, Republic of Korea. (01-PJ10-PG6-01GN16-005)

References

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  PubMedSearch in Google Scholar
• 17 Kragballe K. Vitamin D₃ and skin diseases.  Archieves of Dermatological Research. 1992;  284 30-6
  PubMedSearch in Google Scholar

Tae Sung Kim,Ph. D. 

Immunology Laboratory

College of Pharmacy

Chonnam National University

Kwangju 500-757

Republic of Korea

Fax: +82-62-530-2911

Email: taekim@chonnam.chonnam.ac.kr

References

• 1 Drexier H G, Quentmeier H, MacLeod R A, Uphoff C C, Hu Z B. Leukemia cell lines: in vitro models for the study of acute promyelocytic leukemia.  Leukemia Research. 1995;  19 681-91
  CrossrefPubMedSearch in Google Scholar
• 2 Quintero A, Pelcastre A, Solano J D. Antitumoral activity of new pyrimidine derivatives of sesquiterpene lactones.  Journal of Pharmacy and Pharmaceutical Science. 1999;  2 108-12
  PubMedSearch in Google Scholar
• 3 Ryu J H, Lee H J, Jeong Y S, Ryu S Y, Han Y N. Yomogin, an inhibitor of nitric oxide production in LPS-activated macrophages.  Archives of Pharmacal Research. 1998;  21 481-4
  PubMedSearch in Google Scholar
• 4 Sokoloski J A, Sartorelli A C. Induction of the differentiation of HL-60 promyelocytic leukemia cells by nonsteroidal anti-inflammatory agents in combination with low levels of vitamin D₃ .  Leukemia Research. 1998;  22 153-61
  CrossrefPubMedSearch in Google Scholar
• 5 James S Y, Williams M A, Newland A C, Colston K W. Leukemia cell differentiation: cellular and molecular interactions of retinoids and vitamin D.  General Pharmacology. 1999;  32 143-54
  CrossrefPubMedSearch in Google Scholar
• 6 Ryu S Y, Oak M H, Kim K M. Yomogin inhibits the degranulation of mast cells and the production of the nitric oxide in activated RAW 264.7 cells.  Planta Medica. 2000;  66 171-3
  PubMedSearch in Google Scholar
• 7 Collins S J, Ruscetti F W, Gallagher R E, Gallo R C. Normal functional characteristics of cultured human promyelocytic leukemia cells (HL-60) after induction of differentiation by dimethyl sulfoxide.  Journal of Experimental Medicine. 1979;  149 969-74
  CrossrefPubMedSearch in Google Scholar
• 8 Kansas G S, Muirhead M J, Dailey M O. Expression of the CD11/CD18, leukocyte adhesion molecule 1, and CD44 adhesion molecules during normal myeloid and erythroid differentiation in humans.  Blood. 1990;  76 2483-92
  PubMedSearch in Google Scholar
• 9 Wright S D, Ramos R A, Tobias P S, Ulevitch R J, Mathison J C. CD14, a receptor for complexes of lipopolysaccharide (LPS) and LPS binding protein.  Science. 1990;  249 1431-3
  CrossrefPubMedSearch in Google Scholar
• 10 Breitman T R, He R Y. Combinations of retinoic acid with sodium butyrate, dimethyl sulfoxide, or hexamethylene bisacetamide synergistically induce differentiation of the human myeloid leukemia cell line HL60.  Cancer Research. 1990;  50 6268-73
  PubMedSearch in Google Scholar
• 11 Wang S Y, Chen L Y, Wang S J, Lin C K, Ho C K. Growth inhibition and differentiation in HL-60 leukemia cells induced by 1α,25-dihydroxyvitamin D₃ and tumor necrosis factor α.  Experimental Hematology. 1991;  19 1025-30
  PubMedSearch in Google Scholar
• 12 Makishima M, Kanatani Y, Yamamoto-Yamaguchi Y, Honma Y. Enhancement of activity of 1α,25-dihydroxyvitamin D₃ for growth inhibition and differentiation induction of human myelomonocytic leukemia cells by tretinoin tocoferil, a α-tocopherol ester of all-trans-retinoic acid.  Blood. 1996;  87 3384-94
  PubMedSearch in Google Scholar
• 13 Kang S N, Chung S W, Kim T S. Capsaicin potentiates 1,25-dihydoxyvitamin D₃- and all-trans-retinoic acid-induced differentiation of human promyelocytic leukemia HL-60 cells.  European Journal of Pharmacology. 2001;  420 83-90
  CrossrefPubMedSearch in Google Scholar
• 14 Sokoloski J A, Hodnick W F, Mayne S T, Cinquina C, Kim C S, Sartorelli A C. Induction of the differentiation of HL-60 promyelocytic leukemia cells by vitamin E and other antioxidants in combination with low levels of vitamin D₃: possible relationship to NF-κB.  Leukemia. 1997;  11 1546-53
  CrossrefPubMedSearch in Google Scholar
• 15 Sokoloski J A, Narayanan R, Sartorelli A C. Enhancement by antisense oligonucleotides to NF-κB of the differentiation of HL-60 promyelocytic leukemia cells induced by vitamin D₃ .  Cancer Letter. 1998;  125 157-64
  PubMedSearch in Google Scholar
• 16 Warrell RP J r, Frankel S R, Miller WH J r, Scheinberg D A, Itri L M, Hittelman W N. et al . Differentiation therapy of acute promyelocytic leukemia with tretinoin (all-trans-retinoic acid).  New England Journal of Medicine. 1991;  324 1385-93
  PubMedSearch in Google Scholar
• 17 Kragballe K. Vitamin D₃ and skin diseases.  Archieves of Dermatological Research. 1992;  284 30-6
  PubMedSearch in Google Scholar

Tae Sung Kim,Ph. D. 

Immunology Laboratory

College of Pharmacy

Chonnam National University

Kwangju 500-757

Republic of Korea

Fax: +82-62-530-2911

Email: taekim@chonnam.chonnam.ac.kr