The effects of inhibitors of DNA biosynthesis on the cytotoxicity of 6-thioguanine

The effects of several metabolic inhibitors of DNA synthesis on the antiproliferative activity of 6-thioguanine (6-TG) were examined using cultured L1210 leukemia cels. The presence of hydroxyurea (HU), 1-beta-D-arabinofuranosylcytosine (araC), or 5-fluorodeoxyuridine (FUdR) in cultures of L1210 leukemic cells during exposure of 6-TG did not increase the degree of inhibition of cellular replication produced by the 6-thiopurine, but instead partially protected cells against the delayed cytotoxicity of 6-TG, implying that DNA replication was essential for the expression of cytotoxicity by the purine antimetabolite. Consistent with these results was the finding that synchronized L1210 cells exposed to 6-TG were the most susceptible to the cytotoxic action of the 6-thiopurine during G1/S and S phase. However, G2 phase cells were also sensitive to 6-TG indicating that at least two metabolic lesions are responsible for the production of cytotoxicity. Alkaline sucrose gradient sedimentation of L1210 cells exposed to 6-TG revealed that the purine analog causes structural changes in DNA suggesting that these hitherto unreported lesions may be involved in the cytotoxicity caused by this agent.     

The influence of carbohydrate ligands on the cytotoxicity of liposomes bearing a methotrexate-diglyceride conjugate in human acute leukemia cell cultures

The efficiency of the chemotherapeutic agent methotrexate (MTX) in cancer treatment is limited by the frequent development of the drug resistance of tumor cells. We had previously shown in vitro using human acute leukemia cells with various sensitivity to MTX (T-lymphoblastic CCRF-CEM line and resistant CEM/MTX subline) that MTX incorporation into liposomes in the form of a lipophilic prodrug, diglyceride conjugate (MTX-DG), allows for the overcoming of cell resistance due to the impaired active transmembrane transport. In this work, we have studied the profile of binding with carbohydrates of the cell lines mentioned using carbohydrate fluorescent probes (poly(acryl amide) conjugates). Lipophilic conjugates of tetrasaccharide SiaLe^X, 6′-HSO₃LacNAc, and also inactive pentaol for incorporation into liposomes, have been synthesized. The cytotoxicity of MTX-DG liposomes equipped with the SiaLe_X ligand toward the sensitive CCRF-CEM cell culture was demonstrated to be 3.5 times higher than that of MTX-DG liposomes bearing the control inactive pentaol. The activity of MTX liposomes bearing 6′-HSO₃LacNAc toward resistant CEM/MTX was 1.6-fold increased. The use of carbohydrate ligands as molecular addresses for drug-carrying liposomes as a potential method of treating heterogeneous tumor tissue is discussed.     

Sensitive liquid chromatography mass spectrometry (LC-MS) assay reveals novel insights on DNA methylation and incorporation of gemcitabine,its metabolite difluorodeoxyuridine,deoxyuridine, and RX-3117 into DNA

ABSTRACT

Antimetabolites are incorporated into DNA and RNA, affecting their function. Liquid-chromatography-mass-spectrometry (LC-MS-MS) permits the sensitive, selective analysis of normal nucleosides. The method was adapted to measure the incorporation of deoxyuridine, gemcitabine (difluorodeoxycytidine), its metabolite difluorodeoxyuridine (dFdU), and the novel compound fluorocyclopentenylcytosine (RX3117). DNA was degraded to its deoxynucleotides for quantification by LC-MS-MS, gradient chromatography on a Phenomenex prodigy-3-ODS with positive ionization. The range of deoxyuridine DNA-mis-incorporation varied nine-fold in 27 cell lines (leukemia, colon, ovarian, lung cancer). At low-folate conditions a 2.1-fold increase in deoxyuridine was observed. Global methylation (given as % 5-methyl-deoxycytidine) was comparable between the cell lines (4.6–6.5%). Exposure of A2780 cells to 1 μM gemcitabine (4 hours) resulted in 3.6 pmol gemcitabine/μg DNA, but in AG6000 cells (deoxycytidine-kinase-deficient) no incorporation was found. However, when A2780, AG6000, or CCRF-CEM cells were exposed to 100 μM dFdU we found it as gemcitabine, 20.5, 19.6, and 0.51 pmol gemcitabine/μg DNA, respectively. Preincubation of CCRF-CEM cells with cyclopentenyl-cytosine (a CTP-synthetase inhibitor) increased dFdU incorporation four-fold. Apparently dFdU is activated independently of deoxycytidine-kinase and possibly converted in-situ to dFdCMP. RX3117 was incorporated into both DNA and RNA (0.0037 and 0.00515 pmol/μg, respectively). In summary, a sensitive method to quantify the incorporation of gemcitabine, deoxyuridine, and RX-3117 was developed, which revealed that dFdU was incorporated into DNA as the parent compound gemcitabine.     

Metabolism and mechanisms of action of 9-(tetrahydro-2-furyl)-6-mercaptopurine in Chinese hamster ovary cells

The mechanisms of action of 9-(tetrahydro-2-furyl)-6-mercaptopurine (THFMP) have been studied in Chinese hamster ovary (CHO) cells in tissue culture. THFMP is relatively unstable in physiological buffers, being facilely converted to 6-mercaptopurine (6-MP) even in the absence of cells. Consequently, THFMP undergoes metabolic conversions characteristic of 6-MP, namely formation of 6-thioIMP and incorporation into DNA as 6-thioguanine (6-TG) nucleotide. A number of purines are capable of preventing the toxicity of THFMP in wild-type cells in a manner similar to that of 6-MP. However, exogenous purines and pyrimidines did not prevent the toxicity of THFMP to cells deficient in the enzyme, hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8; HGPRTase). Cells lacking HGPRTase were 20–40-fold resistant to 6-TG and 6-MP but were only 2–4-fold resistant to THFMP. Furthermore, the time-course for killing CHO cells deficient in HGPRTase was different from that in wild-type cells containing the enzyme. There was no apparent effect of THFMP on the utilization of precursors for DNA, RNA or protein synthesis in the enzyme-deficient mutant cell line. The results suggest that THFMP is converted non-enzymatically to 6-MP and shares its mechanisms of action in wild-type cells containing HGPRTase, i.e., inhibition of de novo purine biosynthesis and incorporation into DNA as 6-TG nucleotide. However, the mechanism of action of THFMP in cells lacking HGPRTase is probably unique and is presently unknown.     

Micro-RNA quantification using DNA polymerase and pyrophosphate quantification

A rapid quantification method for micro-RNA based on DNA polymerase activity and pyrophosphate quantification has been developed. The tested micro-RNA serves as the primer, unlike the DNA primer in all DNA sequencing methods, and the DNA probe serves as the template for DNA replication. After the DNA synthesis, the pyrophosphate detection and quantification indicate the existence and quantity of the tested miRNA. Five femtomoles of the synthetic RNA could be detected. In 20–100 μg RNA samples purified from SiHa cells, the measurement was done using the proposed assay in which hsa-miR-16 and hsa-miR-21 are 0.34 fmol/μg RNA and 0.71 fmol/μg RNA, respectively. This simple and inexpensive assay takes less than 5 min after total RNA purification and preparation. The quantification is not affected by the pre-miRNA which cannot serve as the primer for the DNA synthesis in this assay. This assay is general for the detection of the target RNA or DNA with a known matched DNA template probe, which could be widely used for detection of small RNA, messenger RNA, RNA viruses, and DNA. Therefore, the method could be widely used in RNA and DNA assays.     

Microcalorimetric evaluation of the effects of methotrexate and 6-thioguanine on sensitive T-lymphoma cells and on a methotrexate-resistant subline

Isothermal microcalorimetry was used in order to continuously monitor and quantitatively assess the action of two antineoplastic drugs, methotrexate (MTX) and 6-thioguanine (6-TG), on a human T-lymphoma cell line, CCRF-CEM. The results with MTX were compared with data from experiments with a MTX-resistant subline, CEM/MTX. The slope of the power-time curve after drug injection relative to that obtained during unperturbed growth, was used to construct dose-response curves. The normal cell line was characterized by aD ₅₀ value (i.e., the dose producing half the maximal response) of 0.05 μM for MTX and 0.38 μM for 6-TG. For the MTX-resistant subline theD ₅₀ value was 8 μM of MTX. Comparisons of the continuous power-time curves showed the inhibitory effect of 6-TG to be faster than that of MTX.     

Novel DNA lesions generated by the interaction between therapeutic thiopurines and UVA light

The therapeutic effect of the thiopurines, 6-thioguanine (6-TG), 6-mercaptopurine, and its prodrug azathioprine, depends on the incorporation of 6-TG into cellular DNA. Unlike normal DNA bases, 6-TG absorbs UVA radiation, and UVA-mediated photochemical damage of DNA 6-TG has potentially harmful side effects. When free 6-TG is UVA irradiated in solution in the presence of molecular oxygen, reactive oxygen species are generated and 6-TG is oxidized to guanine-6-sulfonate (G(SO3)) and guanine-6-thioguanine in reactions involving singlet oxygen. This conversion is prevented by antioxidants, including the dietary vitamin ascorbate. DNA G(SO3) is also the major photoproduct of 6-TG in DNA and it can be selectively introduced into DNA or oligonucleotides in vitro by mild chemical oxidation. Thermal stability measurements indicate that G(SO3) does not form stable base pairs with any of the normal DNA bases in duplex oligonucleotides and is a powerful block for elongation by Klenow DNA polymerase in primer extension experiments. In cultured human cells, DNA damage produced by 6-TG and UVA treatment is associated with replication inhibition and provokes a p53-dependent DNA damage response.     

Regulation by isoproterenol of muscarinic acetylcholine receptor numbers and sensitivity in rat submandibular,but not lacrimal,glands

Chronic administration of DL-isoproterenol, a β-adrenergic agonist, to male Sprague-Dawley rats increased submandibular gland weights by 3 to 4-fold. This increase resulted from a combination of hyperplasia and hypertrophy of secretory cells. Possible effects of this drug regimen on submandibular gland muscarinic acetylcholine receptors were examined by analysis of the binding of the cholinergic antagonist, L-quinuclidinyl [³H]benzilate, to receptors in gland homogenates. Parallel investigations of receptors in exorbital lacrimal glands, an organ that is not grossly affected by chronic isoproterenol treatment, were also carried out. [³H]QNB bound to submandibular receptors with a K_d of 37.8±6.3 pM in control rats and 41.0±4.0 pM in isoproterenol-treated animals, a non-significant difference (P > 0.05). In contrast, the maximal binding level (B_max) is isoproterenol-treated rats, 1.52±0.10 fmol/μg DNA, was depressed by approx. 30% (P<0.05) from that of 2.22±0.16 fmol/μg DNA in control animals. In lacrimal glands, both K_d (61.3±5.3 vs. 53.2±4.0 pM) and B_max (1.74±0.24 vs. 1.78±0.17 fmol/μg DNA) were unchanged by isoproterenol treatment. The affinity of glandular muscarinic receptors for cholinergic agonists was also examined by competition experiments using carbachol. This agonist inhibited [³H]QNB binding to receptors in homogenates from both glands in a dose-dependent fashion. Inhibition constant (K_i) for this interaction were similar in control and isoproterenol-treated lacrimal glands; 53.6±5.4 μM and 66.6±7.9 μM, respectively (P>0.05). In submandibular glands, isoproterenol treatment elicited a highly significant (P < 0.01) shift in K_i from 17.3±1.4 μM to 68.3±5.2 μM. These results demonstrate that chronic administration of isoproterenol to rats results in a reduction in receptor numbers and a decrease in their sensitivity to cholinergic agonists in submandibular, but not lacrimal, glands.     

Increased Cytotoxicity of 2′,2′‐Difluoro‐2′‐Deoxycytidine in Human Leukemic Cell‐Lines After a Preincubation with Cyclopentenyl Cytosine

The in vitro modulating effect of Cyclopentenyl cytosine (CPEC) on the metabolism of gemcitabine was studied in lymphocytic and myeloid leukemic cell‐lines. In MOLT‐3 cells, that were pretreated with CPEC, the incorporation of 2′,2′‐difluoro‐2′‐deoxycytidine triphosphate (dFdCTP) into DNA was significantly increased by 57–99% in comparison with cells that were only treated with gemcitabine. The increased incorporation of dFdCTP into DNA in CPEC pretreated cells was paralleled by an increase in apoptotic and necrotic cells of 17–34%. In HL‐60 cells that were preincubated with CPEC, increased concentrations of the mono‐/di‐ and triphosphate form of gemcitabine were observed, as well as an increased incorporation of dFdCTP into DNA (+ 773%). This increased incorporation was paralleled by a significant increase in apoptosis and necrosis. We conclude that CPEC enhances the incorporation of dFdCTP into DNA and thus increases the cytotoxicity of gemcitabine in lymphocytic and myeloid leukemic cell‐lines.     

Increased cytotoxicity of 2',2'-difluoro-2'-deoxycytidine in human leukemic cell-lines after a preincubation with cyclopentenyl cytosine

The in vitro modulating effect of Cyclopentenyl cytosine (CPEC) on the metabolism of gemcitabine was studied in lymphocytic and myeloid leukemic cell-lines. In MOLT-3 cells, that were pretreated with CPEC, the incorporation of 2',2'-difluoro-2'-deoxycytidine triphosphate (dFdCTP) into DNA was significantly increased by 57-99% in comparison with cells that were only treated with gemcitabine. The increased incorporation of dFdCTP into DNA in CPEC pretreated cells was paralleled by an increase in apoptotic and necrotic cells of 17-34%. In HL-60 cells that were preincubated with CPEC, increased concentrations of the mono-/di- and triphosphate form of gemcitabine were observed, as well as an increased incorporation of dFdCTP into DNA (+773%). This increased incorporation was paralleled by a significant increase in apoptosis and necrosis. We conclude that CPEC enhances the incorporation of dFdCTP into DNA and thus increases the cytotoxicity of gemcitabine in lymphocytic and myeloid leukemic cell-lines.     

The pattern of gene expression and gene dose profiles of 6-Mercaptopurine- and 6-Thioguanine-resistant human leukemia cells

Exposure of MOLT4 human T-cell leukemia cells to 6-Mercaptopurine (6-MP) and 6-Thioguanine (6-TG) resulted in acquired resistance associated with attenuated expression of the genes encoding concentrative nucleoside transporter 3 (CNT3) and equilibrative nucleoside transporter 2 (ENT2). To identify other alterations at the RNA and DNA levels associated with 6-MP- and 6-TG resistance, we compared here the patterns of gene expression and DNA copy number profiles of resistant sublines to those of the parental wild-type cells. The mRNA levels for two nucleoside transporters were down-regulated in both of the thiopurine-resistant sublines. Moreover, both of these cell lines expressed genes encoding the enzymes of purine nucleotide composition and synthesis, including adenylate kinase 3-like 1 and guanosine monophosphate synthetase at significantly lower levels than wild-type cells. In addition, expression of the mRNA for a specialized DNA polymerase, human terminal transferase encoded by the terminal deoxynucleotidyl transferase (DNTT) gene, was 122- and 93-fold higher in 6-TG- and 6-MP-resistant cells, respectively. The varying responses to 6-MP- and 6-TG observed here may help identify novel cellular targets and modalities of resistance to thiopurines, as well as indicating new potential approaches to individualization therapy with these drugs.     

Combination of azathioprine and UVA irradiation is a major source of cellular 8-oxo-7,8-dihydro-2'-deoxyguanosine

Thiopurine antimetabolites, such as azathioprine (Aza) and 6-thioguanine (6-TG), are widely used in the treatment of cancer, inflammatory conditions and organ transplantation patients. Recent work has shown that cells treated with 6-TG and UVA generate ROS, with implied oxidatively generated modification of DNA. In a study of urinary 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) in renal transplant patients, we provided the first in vivo evidence linking Aza and oxidatively damaged DNA. Using the hOGG1 comet assay, we herein demonstrate high levels of 8-oxodG and alkali-labile sites (ALS) in cells treated with biologically relevant doses of 6-TG, or Aza, plus UVA. This damage was induced dose-dependently. Surprisingly, given the involvement of 6-TG incorporation into DNA in its therapeutic effect, significant amounts of 8-oxodG and ALS were induced in quiescent cells, although less than in proliferating cells. We speculate that some activity of hOGG1 towards unirradiated, 6-TG treated cells, implies possible recognition of 6-TG or derivatives thereof. This is the first report to conclusively demonstrate oxidatively damaged DNA in cells treated with thiopurines and UVA. These data indicate that Aza-derived oxidative stress will occur in the skin of patients on Aza, following even low level UVA exposure. This is a probable contributor to the increased risk of non-melanoma skin cancer in these patients. However, as oxidative stress is unlikely to be involved in the therapeutic effects of Aza, intercepting ROS production in the skin could be a viable route by which this side effect may be minimised.     

EFFECT OF METHOTREXATE ON THE CELL CYCLE OF L1210 LEUKEMIA

The influence of methotrexate (MTX) on the proliferative activity of cells in different phases of cell cycle has been studied. MTX (5 mg/kg) was injected i.p. 3 days after the inoculation of 5 × 10⁶ leukemia cells into F₁ (DBA × C57 BL) mice. It was shown that MTX causes degeneration of cells, being in G₁- as well as in S-phase at the time of drug injection. Incorporation of ³H-TdR was suppressed for a period ranging from 2 to 12 hr after MTX administration, which is demonstrated by the decrease in the number of grains per cell. The number of cells labeled after ³H-TdR injection was also sharply decreased during this period. For a period of 3 until 15 hr after MTX administration the mitotic index decreased significantly as a result of inhibition of DNA synthesis. The blocking of the G₁-S transition was evident during 4 hr after MTX. Thereafter the G₁-S transition proceeds at a rate which is practically equal to that for nontreated controls. MTX did not inhibit transition to mitosis of cells being in G₂-phase and in a very late S-phase at the time of drug injection. The sensitivity of G₁-cells to the cytocidal effect of MTX shows that for L1210 leukemia cells MTX can be classified as a cycle-specific drug killing both G₁ and S-cells rather than S-phase specific agent with self-limitation.     

Hyperoxic-induced alterations in lung cell structure and function: Effects on cellular cyclic AMP content and comparison to alterations produced by exogenous cyclic AMP

Hyperoxic exposure in vitro of two lung-derived cell types (the epithelial-derived L2 cells and WI-38 fibroblasts) inhibits cellular replication, produces striking morphologic changes and may result in cell death; these effects have been observed consistently in other cell types. Hyperoxic exposure of L2 cells is associated with an increase in cellular cyclic AMP content (cellular cyclic AMP content 454 ± 115 fmol/μg DNA in cells exposed to p_O2 677 Torr for 96 h compared to 136 ± 17 fmol/μg DNA in air-grown cells). Hyperoxic exposure of WI-38 fibroblasts is not associated with increased cyclic AMP content. Although cultivation of L2 cells in the presence of exogenous dibutyryl cyclic AMP does inhibit replication and produce morphologic alterations, similar effects are produced by sodium butyrate alone. Hyperoxic exposure alters cyclic AMP metabolism in some cell types, but the structural and functional alterations observed in L2 cells and WI-38 fibroblasts following hyperoxic exposure are not produced by changes in cellular cyclic AMP content.     

Efficient DNA interstrand crosslinking by 6-thioguanine and UVA radiation

Patients taking the immunosuppressant and anticancer thiopurines 6-mercaptopurine, azathioprine or 6-thioguanine (6-TG), develop skin cancer at a very high frequency. Their DNA contains 6-TG which absorbs ultraviolet A (UVA) radiation, and their skin is UVA hypersensitive, consistent with the formation of DNA photodamage. Here we demonstrate that UVA irradiation of 6-TG-containing DNA causes DNA interstrand crosslinking. In synthetic duplex oligodeoxynucleotides, the interstrand crosslinks (ICLs) can form between closely opposed 6-TG bases and, in a less favoured reaction, between 6-TG and normal bases on the opposite strand. In vivo, UVA irradiation of cultured cells containing 6-TG-substituted DNA also causes ICL formation and induces the chromosome aberrations that are characteristically associated with this type of DNA lesion. 6-TG/UVA activates the Fanconi anemia (FA) pathway via monoubiquitination of the FANCD2 protein. Cells defective in the FA pathway or other factors involved in ICL processing, such as XPF and DNA Polζ, are all hypersensitive to killing by 6-TG/UVA-consistent with a significant contribution of photochemical ICLs to the cytotoxicity of this treatment. Our findings suggest that sunlight-exposed skin of thiopurine treated patients may experience chronic photochemical DNA damage that requires constant intervention of the FA pathway.     

Mismatch repair-mediated G2/M arrest by 6-thioguanine involves the ATR-Chk1 pathway

DNA mismatch repair (MMR) deficiency in human cancers is associated with resistance to a spectrum of clinically active chemotherapy drugs, including 6-thioguanine (6-TG). We and others have shown that 6-TG-induced DNA mismatches result in a prolonged G2/M cell cycle arrest followed by apoptosis in MMR(+) human cancer cells, although the signaling pathways are not clearly understood. In this study, we found that prolonged (up to 4 days) treatment with 6-TG (3microM) resulted in a progressive phosphorylation of Chk1 and Chk2 in MMR(+) HeLa cells, correlating temporally with a drug-induced G2/M arrest. Transfection of HeLa cells with small interfering RNA (siRNA) against the ataxia telangiectasia-related (ATR) kinase or against the Chk1 kinase destroyed the G2/M checkpoint and enhanced the apoptosis following 6-TG treatment. On the other hand, the induction of a G2/M population by 6-TG was similar in ATM(-/-) and ATM(+) human fibroblasts, suggesting that the ATM-Chk2 pathway does not play a major role in this 6-TG response. Our results indicate that 6-TG DNA mismatches activate the ATR-Chk1 pathway in the MMR(+) cells, resulting in a G2/M checkpoint response     

Antimicrobial and cytotoxicity activities of the medicinal plant Primula macrophylla

Primula macrophylla (Primulaceae) is reported as to be useful in asthma, restlessness, insomnia and fish poisoning. Antifungal and toxic activities of crude extract, fractions and a pure isolated compound exhibited statistically significant activities. Excellent antifungal activity was found in the crude extract, benzene and ethyl acetate fractions against T. longifusis and against M. canis with different MIC values. Antileishmanial activity (IC₅₀ = 50ug/mL) was observed as compared to standard drug Amphotericin B, and cytotoxic activity (LD₅₀ = 47.919μg/mL) was also found in the chloroform fraction. While pure compound 2-phenylchromone (Flavone) isolated from the chloroform fraction showed good activity (IC₅₀ = 25μg/mL) against Leishmania and cytotoxicity (LD₅₀ = 2.0116 μg/mL) in Brine Shrimp experiments. From antileishmanial and cytotoxic activity it can be concluded that 2-phenylchromone is the major compound responsible for these activities.     

Organellar DNA synthesis in permeabilized soybean cells

Summary Cultured cells of Glycine max (L.) Merr. v. Corsoy were permeabilized by treatment with L--lysophosphatidylcholine (LPC). The permeabilized cells were capable of uptake and incorporation of deoxynucleoside triphosphates into DNA. Incorporation of exogenous nucleotides into DNA was linear for at least 90 minutes and the initial rate of incorporation approached 50% of the theoretical in vivo rate of DNA synthesis. However, DNA synthesis in the permeabilized cells was unaffected by the potent DNA polymerase inhibitor, aphidicolin. Analysis of newly synthesized DNA by molecular hybridization revealed that only organellar DNA was synthesized by the permeabilized cells. The LPC treated cells were also permeable to a protein as large as DNase I. The permeabilized cells were capable of RNA and protein synthesis as indicated by incorporation of radiolabeled UTP and leucine, respectively, into acid-precipitable material.     

Synergistic property of cordycepin in cultivated Cordyceps militaris-mediated apoptosis in human leukemia cells

Cordyceps militaris is a well-known Chinese traditional medicinal mushroom frequently used for tonics and recently of a potential interest for cancer intervention. Here, we explored the cancer cell killing activity of the hot water extracts of C. militaris cultured mycelia (CM_MY) and cultivated fruiting bodies (CM_FB). We found that CM_FB exhibited a greater cytotoxic effect against various cancer cells over CM_MY. Apoptotic phenotypes including apoptotic body formation, DNA laddering, caspase 3 activation and cleavage of PARP proteins were induced by CM_FB treatment but only slightly induced by same concentration of CM_MY treatment in human HL-60 leukemia cells. Cordycepin in CM_FB (10.47 mg/g) is significantly higher (∼15.2 times) than that of CM_MY (0.69 mg/g). Using isobolographic analysis, the synergy of cytotoxicity was observed across different combined concentrations of CM_MY and cordycepin. By complementing cordycepin into CM_MY to the level comparable with CM_FB, we observed that CM_MY (500 μg/ml) with cordycepin (4.8 μg/ml) induced apoptosis to a level similar to that induced by CM_FB (500 μg/ml). Together, our results suggest that cordycepin possesses a synergistic cytotoxic effect with Cordyceps militaris-mediated apoptosis in human leukemia cells and therefore explaining a better anti-proliferating activity of CM_FB over CM_MY.