Induction of megakaryocytic differentiation in chronic myelogenous leukemia cell K562 by 3-hydrogenkwadaphnin

3-Hydrogenkwadaphnin (3-HK) is a daphnane-type diterpene ester isolated from Dendrostellera lessertii (Thymelaeaceae) with high differentiation and apoptotic potency in leukemic cells without any measurable adverse effects on normal cells (Moosavi et al., 2005b). In this study, we report that 3-HK (12 nM) has the ability to cease proliferation, induce differentiation and apoptosis in chronic myelogenous leukemia (CML) K562 cell line. The treated cells lost erythroid properties and differentiated along the megakaryocytic lineage based on the morphological features apparent after Wright-Giemsa staining, DNA content analysis and the expression of cell surface marker glycoprotein IIb as analyzed by flow cytometry. Moreover, using Hoechst 33258 and Annexin V double staining indicated the occurrence of apoptosis among the treated cells. On the other hand, restoration of the depleted GTP pool size by exogenous addition of guanosine (50 microM) reduced the effect of the drug regarding the extent of differentiation while no further enhancement of 3-HK effect was obtained by addition of exogenous hypoxanthine (100 microM). These interesting results necessitate further investigation regarding the mechanism of action of this unique anti-leukemic agent.     

3-Hydrogenkwadaphnin targets inosine 5'-monophosphate dehydrogenase and triggers post-G1 arrest apoptosis in human leukemia cell lines

3-Hydrogenkwadaphnin (3-HK) is a recently characterized daphnane-type compound isolated from Dendrostellera lessertii with high anti-tumor activity in animal models. Herein, we report on time- and dose-dependent effects of this compound on growth, differentiation, IMPDH inhibition, cell cycle and apoptosis of a panel of human leukemia cell lines (HL-60, K562 and Molt4). The drug decreased the growth of leukemia cells in less than 24 h of treatment. However, longer exposure times and/or higher concentrations were required to promote cell apoptosis. Cell cycle analysis revealed the accumulation of cells in their G1 phase as early as 12 h after drug exposure but sub-G1 population was recorded after 24 h. Occurrence of apoptosis was constantly accompanied by morphological (staining with DNA-binding dyes) and biochemical (DNA fragments) variations among drug-treated cells. Despite these observations, non-activated normal human PBL were insensitive to the drug action. In addition, treatment of PHA-activated PBL, K562, Molt4 and HL-60 cells with a single dose of the drug for 24 h led to the inhibition of IMPDH activity by almost 37, 38, 44 and 50%, respectively. In contrast, no difference in IMPDH activities were seen between normal PBL and the drug treated PBL cells. Restoration of the depleted GTP concentration by exogenous addition of guanosine (25-50 microM) reversed the drug effects on cell growth, DNA fragmentation and apoptosis. Furthermore, the drug effects were potentiated by exogenous addition of hypoxanthine to the drug-treated cells. Reduction of the drug potency on the non-proliferative (retinoic acid treated) HL-60 cells by almost 40%, compared to the proliferative cells, clearly shows type II IMPDH as one of the main targets of the drug. These results suggest that 3-HK may be a powerful candidate for treatment of leukemia.     

3-Hydrogenkwadaphnin induces monocytic differentiation and enhances retinoic acid-mediated granulocytic differentiation in NB4 cell line

Recently, we have reported that 3-hydrogenkwadaphnin (3-HK), a diterpene ester isolated from Dendrostellera lessertii (Thymealeaceae), is very effective against leukemia cell lines without any detectable effects on normal cells (Moosavi et al., 2005b). In this study, we report that 3-HK induces G1 cell-cycle arrest, differentiation and apoptosis in APL NB4 cell line. Indeed, the drug between 24 to 96 h induced 7-65% growth inhibition of NB4 cells. Cell viability was also decreased by 2-55% between 24 to 96 h treatments with the drug, respectively. These effects of the drug were also dose-dependent. According to flow cytomtry results, 3-HK (15 nM) induced a significant G1-arrest up to 24 h which was consequently followed with appearance of sub-G(1) peak at 72 to 96 h. Hoechst 33258 staining and DNA fragmentation assays confirmed the occurrence of apoptosis among the treated cells. On the other hand, NBT reducing assay, Wright-Giemsa staining, phagocytic activity and expression of cell surface markers (CD11b and CD14) confirmed that the inhibition of proliferation is associated with differentiation especially toward macrophage-like morphology. Interestingly, 3-HK at 5 and 10 nM enhanced the effects of all-trans retinoic acid (ATRA) in NB4 cells. Based on these results, 3-HK might become an ideal candidate for treatment of APL patients pending full exploration of its biological functions.     

The cytotoxic and anti-proliferative effects of 3-hydrogenkwadaphnin in K562 and jurkat cells is reduced by guanosine

3-hydrogenwadaphnin (3-HK) is a new daphnane-type diterpene ester isolated from Dendrostellera lessertii with strong anti-tumoral activity in animal models and in cultures. Here, prolonged effects of this new agent on proliferation and viability of several different cancerous cell lines were evaluated. Using [(3)H]thymidine incorporation, it was found that the drug inhibited cell proliferation and induced G1/S cell cycle arrest in leukemic cells 24 h after a single dose treatment. The cell viability of Jurkat cells was also decreased by almost 10 %, 31 % and 40 % after a single dose treatment (7.5 nM) at 24, 48 and 72 h, respectively. The drug-treated cells were stained with acridine orange/ ethidium bromide to document the chromatin condensation and DNA fragmentation. These observations were further confirmed by detection of DNA laddering pattern in the agarose gel electrophoresis of the extracted DNA from the treated cells. Treatment of K562 cells with the drug at 7.5, 15 and 30 nM caused apoptosis in 25 %, 45 % and 65 % of the cells, respectively. Exogenous addition of 25-50 microM guanosine and/or deoxyguanosine to the cell culture of the drug-treated cells restored DNA synthesis, released cell arrest at G1/S checkpoint and decreased the apoptotic cell death caused by the drug. These observations were not made using adenosine. However, the drug effects on K562 cells were potentiated by hypoxanthine. Based on these observations, perturbation of GTP metabolism is considered as one of the main reasons for apoptotic cell death by 3-HK.     

Synthesis of furopyrazole analogs of 1-benzyl-3-(5-hydroxymethyl-2-furyl)indazole (YC-1) as novel anti-leukemia agents

As part of our continuing search for potential anticancer drug candidates in YC-1 analogs, several 1-benzyl-3-(substituted aryl)-5-methylfuro[3,2-c]pyrazoles were synthesized and evaluated for their cytotoxicity against HL-60 cell line. Among these compounds, 1-benzyl-3-(5-hydroxymethyl-2-furyl)-5-methylfuro[3,2-c]pyrazole (1) showed more potency than YC-1. Through investigation of action mechanism, it was found that compound 1 induced terminal differentiation of HL-60 cells toward granulocyte lineage and promoted HL-60 cell differentiation by regulation of Bcl-2 and c-Myc proteins. Meanwhile, compound 1 also demonstrated apoptosis inducing effect. Such anti-leukemia mechanism of action is apparently different from that of YC-1 which mainly works by inducing apoptosis, but not cell differentiation. Therefore, compound 1 is identified here as a new lead compound of cell differentiating agent and apoptosis inducer for further development of new anti-leukemia agents.     

Induction of differentiation and apoptosis in three human leukemia cell lines by a new compound from Dendrostellera lessertii

It has previously been shown that Dendrostellera lessertii(Thymelaeaceae)has stronganticancer activity.In this study,the antileukemic activity of another new compound from the same plantextract is reported.Promyelocytic(NB4 and HL-60)and erythroleukemia(K562)cells were cultured in thepresence of various concentrations of the new compound(0.5-3.0 μtg/ml)for 3d.The cell numbers werethen determined by trypan blue exclusion test.The new compound inhibited growth and proliferation ofNB4,HL-60 and K562 with IC_(50) values of 1.5,2.0 and 2.5μg/ml,respectively.We also found that the newcompound inhibited cell proliferation in a dose-and time-dependent manner.At low concentrations and after48h of treatment,approximately 50%-70% of NB4 and HL-60 cells were differentiated to monocyte/macrophage lineage and approximately 30%-40% of the treated K562 cells were differentiated in the mega-karyocytic lineage,as evidenced by morphological changes and nitro blue tetrazolium reduction assays.Results of Hoechst 33258 staining also indicated that the new compound induced NB4 and HL-60 cellapoptosis at their respective IC_(50) values after 72h of treatment.Based on the present data,the new com-pound seems a good candidate for further evaluation as an effective chemotherapeutic agent acting throughinduction of differentiation and apoptosis.     

The staurosporine analog, Ro-31-8220, induces apoptosis independently of its ability to inhibit protein kinase C

A series of bisindolylmaleimide (Bis) compounds were designed as analogs of the natural compound staurosporine (STS), which is a potent inducer of apoptosis. Many of the Bis analogs appear to be highly selective inhibitors of the protein kinase C (PKC) family, including PKC-alpha, -beta, -gamma, -delta, -epsilon, and -zeta, unlike STS, which is an inhibitor of a broad spectrum of protein kinases. In this report we describe the effects of the Bis analogs, Bis-I, Bis-II, Bis-III and Ro-31-8220 on the survival and proliferation of HL-60 cells, which have been widely used as a model cell system for studying the biological roles of PKC. Treatment of HL-60 cells with Bis-I, Bis-II, Bis-III, or Ro-31-8220 blocked phosphorylation of the PKC target protein Raf-1 with equal potency but did not appear to affect the general phosphorylation of proteins by other kinases. However, the biological effects of the Bis compounds were different: Bis-I and Bis-II had no observable effects on either cell survival or proliferation; Bis-III inhibited cell proliferation but not survival, whereas Ro-31-8220 induced apoptosis. These results indicated that the members of the PKC family which could be inhibited by the Bis analogs were required neither for survival nor proliferation of HL-60 cells. Analyses of cells treated with Ro-31-8220 showed that the apoptotic effect of Ro-31-8220 on HL-60 cells was mediated by a well-characterized transduction process of apoptotic signals: i.e., mitochondrial cytochrome c efflux and the activation of caspase-3 in the cytosol. Moreover, the ability of Ro-31-8220 to induce apoptotic activation was completely inhibited by the over-expression of the apoptotic suppressor gene, Bcl-2, in the cells. Interestingly, proliferation of the Bcl-2-over-expressing cells was still sensitive to the presence of Ro-31-8220, suggesting that the inhibitory effects of Ro-31-8220 on viability and cell proliferation were mediated by different mechanisms. In particular, the apoptotic effect of Ro-31-8220 on cells was not altered by the presence of an excess amount of the other Bis analogs, suggesting that this effect is mediated by a factor(s) other than PKC or by a mechanism which was not saturable by the other Bis analogs. Finally, structure-function analyses of compounds related to Ro-31-8220 revealed that a thioamidine prosthetic group in Ro-311-8220 was largely responsible for its apoptotic activity.     

人参皂苷Rb1、Rg1、Re对白血病细胞株KG1α增殖的影响

目的:探讨人参皂苷Rb1、Rg1、Re对急性髓系白血病细胞株(KG1α)增殖的影响.方法:取对数生长期KG1α细胞,分设人参皂苷Rb1、Rg1、Re组和常规培养组,以MTT比色法检测作用24h、48h、72h时对KG1α细胞增殖抑制作用,并计算Rb1的IC_(50)值,以此浓度为工作浓度,设常规培养组和处理组,台盼蓝计数法观察对KG1α细胞增殖的影响;流式细胞术测定细胞周期分布的变化.结果:MTT、台盼蓝计数显示人参皂苷单体Rb1、Rg1可抑制KG1α细胞增殖,呈浓度依赖性,以Rb1抑制效应最佳,于作用48h抑制率最高.台盼蓝计数显示人参皂苷单体Rb1-120μmol/L作用48h时抑制率达50.22%;流式细胞术结果提示,与对照组比较,Rb1-120μmol/L组G_2/M期KG1α细胞比例增加(P<0.05).结论:Rb1可抑制KG1α细胞体外增殖,其增殖抑制作用与将KG1α细胞阻滞于G_2/M期有关.     

The effect of the active component of Dendrostellera lessertii on the adhesive property of human platelets and HL-60 cells

To evaluate the mediation of cell adhesion by Dendrostellera lessertii, an anti-cancer medicinal plant in Iranian folk medicine, the adhesion of thrombin activated human platelets to the cultured monocytes or HL-60 cells was investigated under the effect of the plant extract (0.4 mg/ml) and one of its purified components, 3-hydrgenkwadaphnin (0.2 nM). Treatment of the platelets with the plant extract or/3-hydrgenkwadaphnin, for various time intervals, followed by their activation by thrombin resulted in 80-90% reduction in the number of monocytes with more than 10 attached platelets. Similarly, under almost all identical conditions, the adhesion of the activated platelets to HL-60 cells was decreased by 90%. The adhesion of thrombin activated platelets to the plant extract treated HL-60 cells was also reduced significantly (95%). These data clearly indicates that Dendrostellera lessertii is capable of mediating tumor metastasis through affecting the adhesion properties of the cells.     

Neuronal apoptosis induced by pharmacological concentrations of 3-hydroxykynurenine: characterization and protection by dantrolene and Bcl-2 overexpression

We have studied neurotoxicity induced by pharmacological concentrations of 3-hydroxykynurenine (3-HK), an endogenous toxin implicated in certain neurodegenerative diseases, in cerebellar granule cells, PC12 pheochromocytoma cells, and GT1-7 hypothalamic neurosecretory cells. In all three cell types, the toxicity was induced in a dose-dependent manner by 3-HK at high micromolar concentrations and had features characteristic of apoptosis, including chromatin condensation and internucleosomal DNA cleavage. In cerebellar granule cells, the 3-HK neurotoxicity was unaffected by xanthine oxidase inhibitors but markedly potentiated by superoxide dismutase and its hemelike mimetic, MnTBAP [manganese(III) tetrakis(benzoic acid)porphyrin chloride]. Catalase blocked 3-HK neurotoxicity in the absence and presence of superoxide dismutase or MnTBAP. The formation of H(2)O(2) was demonstrated in PC12 and GT1-7 cells treated with 3-HK, by measuring the increase in the fluorescent product, 2',7'-dichlorofluorescein. In both PC12 and cerebellar granule cells, inhibitors of the neutral amino acid transporter that mediates the uptake of 3-HK failed to block 3-HK toxicity. However, their toxicity was slightly potentiated by the iron chelator, deferoxamine. Taken together, our results suggest that neurotoxicity induced by pharmacological concentrations of 3-HK in these cell types is mediated primarily by H(2)O(2), which is formed most likely by auto-oxidation of 3-HK in extracellular compartments. 3-HK-induced death of PC12 and GT1-7 cells was protected by dantrolene, an inhibitor of calcium release from the endoplasmic reticulum. The protection by dantrolene was associated with a marked increase in the protein level of Bcl-2, a prominent antiapoptotic gene product. Moreover, overexpression of Bcl-2 in GT1-7 cells elicited by gene transfection suppressed 3-HK toxicity. Thus, dantrolene may elicit its neuroprotective effects by mechanisms involving up-regulation of the level and function of Bcl-2 protein.     

N-Benzoyl-12-nitrodehydroabietylamine-7-one, a novel dehydroabietylamine derivative, induces apoptosis and inhibits proliferation in HepG2 cells

The high biological activity of dehydroabietylamine derivatives has been reported previously. In this study, we aimed to screen 73 dehydroabietylamine derivatives as potential candidate inhibitors in liver cancer cells. Initially, the compounds structural activity relationship analysis was explored and N-benzoyl-12-nitrodehydroabietylamine-7-one (compound 81) was shown to have significant growth inhibitory activity in the human liver carcinoma cell line, HepG2. Further research into the anti-proliferative effect on HepG2 cells mediated by compound 81 was undertaken. The results suggest that compound 81 effectively induced apoptosis in HepG2 cells characterized by nuclear staining of DAPI, TUNEL assay and the activation of caspase-3. A decreased level of anti-apoptotic protein Bcl-2 and increased apoptotic Bax were also observed. Furthermore, Ki-67 protein staining and the BrdU incorporation assay showed that compound 81 significantly inhibited the proliferation of HepG2 cells. Cell cycle components analysis found that expression of cyclin D1 and cyclin B1 was reduced in HepG2 cells with compound 81 treatment, whereas the content of p21(Waf1/Cip1) was increased. Taken together, our data indicate that compound 81 induces apoptosis and inhibits proliferation in HepG2 cells, and may be a promising candidate in the development of a novel class of antitumor agents.     

2-Arylidenedihydroindole-3-ones: design, synthesis, and biological activity on bladder carcinoma cell lines

2-Arylidenedihydroindole-3-ones were assayed for their antiproliferative and apoptotic abilities as potential drug candidates to treat bladder tumor. These compounds were tested on cell lines obtained from bladder tumors of various stages [superficial (pTa and pT1) vs. invasive (pT2)]. The most active compound (3c) inhibited the proliferation, induced apoptosis, and decreased the expression of p-Stat5 and p-Pyk2 in DAG-1 and RT112 lines in which the FGFR3 is either mutated or overexpressed. Knowing that FGFR3 is involved in cell proliferation, differentiation, and migration through cell signaling pathways including p-Stat5 way via p-Pyk2, let us assume that compound 3c may probably act through FGFR3 pathway.     

Investigating the effects of vitreous humour (crude extract) on growth and differentiation of rat mesenchymal stem cells (rMSCs) and human NTERA2 cells

Two main characteristics of all types of stem cells are their potency for differentiation and self renewal capacity. There is a lot of interest to find the conditions and factors, which govern these behaviours of stem cells. It is very well documented that retinoic acid (RA) reduces growth rate by induction of cell differentiation in certain conditions and cell lines. On the other hand, hyaluronic acid (HA) is known for its growth induction on cultured cells. A natural source of HA, rabbit vitreous humour (VH), was previously shown to promote wound repair in model animals. In search for its possible mechanisms, VH extract was tested on the cultured mesenchymal stem cells and NTERA2 as human embryonal carcinoma cells in the presence of RA. Changes in some cellular and molecular markers (A2B5, 0ct4, Sox2) showed that VH and possibly HA interfere with differentiating effects of RA. Therefore, this reagent may affect cell proliferation and tissue regeneration by inhibition of cell differentiation.     

3-Hydroxykynurenine, an Endogenous Oxidative Stress Generator, Causes Neuronal Cell Death with Apoptotic Features and Region Selectivity

Abstract: 3-Hydroxykynurenine (3-HK) is a potential endogenous neurotoxin whose increased levels have been described in several neurodegenerative disorders. Here, we characterized in vitro neurotoxicity of 3-HK. Of the tested kynurenine pathway metabolites, only 3-HK, and to a lesser extent 3-hydroxyanthranilic acid, were toxic to primary cultured striatal neurons. 3-HK toxicity was inhibited by various antioxidants, indicating that the generation of reactive oxygen species is essential to the toxicity. 3-HK-induced neuronal cell death showed several features of apoptosis, as determined by the blockade by macromolecule synthesis inhibitors, and by the observation of cell body shrinkage with nuclear chromatin condensation and fragmentation. In addition, 3-HK toxicity was dependent on its cellular uptake via transporters for large neutral amino acids, because uptake inhibition blocked the toxicity. Cortical and striatal neurons were much more vulnerable to 3-HK toxicity than cerebellar neurons, which may be attributable to the differences in transporter activities of these neurons. These results indicate that 3-HK, depending on transporter-mediated cellular uptake and on intracellular generation of oxidative stress, induces neuronal cell death with brain region selectivity and with apoptotic features, which may be relevant to pathology of neurodegenerative disorders.     

Growth hormone and prolactin receptors in adipogenesis: STAT-5 activation, suppressors of cytokine signaling, and regulation of insulin-like growth factor I

Growth hormone GH stimulates lipolysis in mature adipocytes and primary preadipocytes but promotes adipogenesis in preadipocyte cell lines. The lactogenic hormones (prolactin PRL and placental lactogen) also stimulate adipogenesis in preadipocyte cell lines but have variable lipolytic and lipogenic effects in mature adipose tissue. We hypothesized that differences in expression of GH receptors GHR and PRL receptors PRLR during adipocyte development might explain some of the differential effects of the somatogens and lactogens on fat metabolism. To that end, we compared: (a) the expression of GHR and PRLR mRNAs in 3T3-L1 preadipocytes during the course of adipocyte differentiation; (b) the induction of STAT-5 activity by GH and PRL during adipogenesis; and (c) the acute effects of GH and PRL on the suppressors of cytokine signaling (SOCS-1-3 and cytokine-inducible SH2-domain-containing protein CIS) and IGF-I. In confluent, undifferentiated 3T3-L1 cells, the levels of GHR mRNA were approximately 250-fold higher than the levels of PRLR mRNA. Following induction of adipocyte differentiation the levels of PRLR mRNA rose 90-fold but GHR mRNA increased only 0.8-fold. Expression of both full-length (long) and truncated (short) isoforms of the PRLR increased during differentiation but the long isoform predominated at all time points. Mouse GH mGH stimulated increases in STAT-5a and 5b activity in undifferentiated as well as differentiating 3T3-L1 cells; mouse PRL mPRL had little or no effect on STAT-5 activity in undifferentiated cells but stimulated increases in STAT-5a and 5b activity in differentiating cells. mGH stimulated increases in SOCS-2 and SOCS-3 mRNAs in undifferentiated cells and SOCS-1-3 and CIS mRNAs in differentiating cells; mPRL induced CIS in differentiating cells but had no effect on SOCS-1-3. mPRL and mGH stimulated increases in IGF-I mRNA in differentiating cells but not in undifferentiated cells; the potency of mGH (3-6-fold increase, p < 0.01) exceeded that of mPRL (40-90% increase, p < 0.05). Our findings reveal disparities in the expression of PRLR and GHR during adipocyte development and differential effects of the hormones on STAT-5, the SOCS proteins, CIS, and IGF-I. These observations suggest that somatogens and lactogens regulate adipocyte development and fat metabolism through distinct but overlapping cellular mechanisms.     

EGCG 对人结肠癌HT-29 细胞凋亡的影响以及对MMP-2，RECK 的 调节作用

目的:观察表没食子儿茶素没食子酸酯(Epigallaocatechin-3-gallate,EGCG)对人结肠癌HT-29细胞增殖和凋亡的影响,并探讨其对MMP-2,RECK的调节作用。方法:体外培养人结肠癌HT-29细胞,MTT比色法检测EGCG对HT-29细胞的生长抑制作用;Histone/DNA ELISA检测细胞凋亡;FITC标记Annexin-V/PI双染流式细胞术分析凋亡细胞百分率;Western Blot和RT-PCR方法检测EGCG对MMP-2,RECK蛋白和mRNA表达的影响。结果:EGCG呈浓度和时间依赖性抑制HT-29细胞的增殖,并且增加HT-29细胞Histone/DNA碎片的渗漏;EGCG诱导HT-29细胞凋亡百分率增高;EGCG抑制MMP-2蛋白和mRNA的表达,促进RECK蛋白和mRNA的表达。结论:EGCG抑制人结肠癌HT-29细胞的增殖,促进其凋亡,并且呈浓度和时间依赖性;其作用机制可能与其下调MMP-2蛋白和mRNA的表达、上调RECK蛋白和mRNA的表达有关。     

Possible involvement of protein kinase C in the induction of adipose differentiation-related protein by Sterol ester in RAW 264.7 macrophages

We studied the effects of BMP-7/OP-1 on growth and differentiation of bone marrow stromal cells. BMS2, a mouse bone marrow stromal cell line capable of differentiating into adipocytes and osteoblasts, were treated in a serum-free medium containing differentiation agents that favor the expression of both lineages. BMP-7/OP-1 stimulated cell proliferation and differentiation concomitantly. These effects were dose- and growth phase-dependent. Cells were more sensitive to the treatment early in the culture (30-40% confluence) with a significant increase in cell proliferation and markers of differentiation at low concentrations. When treated later in the growth phase (90-100% confluence), no significant increase in cell proliferation was seen. The concentration requirement for cells later in the culture to reach an equivalent degree of differentiation was 3-10- fold higher than for cells treated early. In both cases, the effects on adipocyte differentiation were biphasic; low concentrations stimulated adipocyte differentiation which was inhibited at higher concentrations where stimulation of osteoblast markers were observed. We conclude that cell proliferation and cell differentiation into adipocyte/osteoblast can occur simultaneously under BMP-7/OP-1 treatment.     

Chemical synthesis of 20S-hydroxyvitamin D3, which shows antiproliferative activity

20S-hydroxyvitamin D3 (20S-(OH)D3), an in vitro product of vitamin D3 metabolism by the cytochrome P450scc, was recently isolated, identified and shown to possess antiproliferative activity without inducing hypercalcemia. The enzymatic production of 20S-(OH)D3 is tedious, expensive, and cannot meet the requirements for extensive chemical and biological studies. Here we report for the first time the chemical synthesis of 20S-(OH)D3 which exhibited biological properties characteristic of the P450scc-generated compound. Specifically, it was hydroxylated to 20,23-dihydroxyvitamin D3 and 17,20-dihydroxyvitamin D3 by P450scc and was converted to 1α,20-dihydroxyvitamin D3 by CYP27B1. It inhibited proliferation of human epidermal keratinocytes with lower potency than 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) in normal epidermal human keratinocytes, but with equal potency in immortalized HaCaT keratinocytes. It also stimulated VDR gene expression with similar potency to 1,25(OH)2D3, and stimulated involucrin (a marker of differentiation) and CYP24 gene expression, showing a lower potency for the latter gene than 1,25(OH)2D3. Testing performed with hamster melanoma cells demonstrated a dose-dependent inhibition of cell proliferation and colony forming capabilities similar or more pronounced than those of 1,25(OH)2D3. Thus, we have developed a chemical method for the synthesis of 20S-(OH)D3, which will allow the preparation of a series of 20S-(OH)D3 analogs to study structure-activity relationships to further optimize this class of compound for therapeutic use.