Comparative in vitro activity of 4′-deoxy-4′-iododoxorubicin and other anthracyclines in the human tumor clonogenic assay

A new halogenated anthracycline analog 4-deoxy-4-iododoxorubicin (IODO) was compared with doxorubicin (DOX) and deoxydoxorubicin (DEOX) in the human tumor clonogenic assay (HTCA) using human tumor cell lines. For all cell lines tested, IODO had lower ID₅₀ value and thus greater in vitro potency and cytotoxicity than DOX. DEOX had lower average ID₅₀ values than either IODO or DOX in all cell lines except HEC1A, where DEOX was equal to IODO. Analysis of variance likewise confirmed significantly greater activity for IODO versus DOX in most cell lines tested. Previous in vivo studies demonstrate oral activity in a variety of tumors as well as less cardiotoxicity. Thus, the results of in vitro and in vivo studies suggest that IODO is an active compound of potential clinical interest.     

Activity of the morpholino anthracycline 3′-deamino-3′-morpholino-13-deoxo-10-hydroxycarminomycin (MX2) against human tumor colony-forming unitsin vitro

Summary In several preclinical systems, the morpholino anthracycline MX2 has greater antitumor activity than doxorubicin, is less cardiotoxic, and is effective against multidrug resistant cancer cells. We used a human tumor soft-agar cloning assay to test the cytotoxicity of MX2 against single cell suspensions from freshly obtained human tumors. Tumor cells were exposed to MX2 at 0.05 and 0.5 g/ml either for 1 hour (201 specimens; 77 [38%] assessable) or continuously (231 specimens; 91 [39%] assessable). Superior antitumor activity was observed with continuous exposure (19%in vitro response at 0.05 g/ml and 69% at 0.5 g/ml) than with 1-hour exposure (1.3% at 0.05 g/ml and 12% at 0.5 g/ml). Activity was seen against all types of cancer tested including renal (91%), melanoma (88%), ovarian (73%), breast (71%) and non-small-cell lung (67%) cancer at a MX2 concentration of 0.5 g/ml after continuous exposure. If appropriate plasma levels can be achieved in patients, MX2 could have significant clinical activity in patients with those tumors.     

Modulation of Ecto-5′-Nucleotidase by phospholipids in human umbilical vein endothelial cells (HUVEC)

Ecto-5′-nucleotidase, the major enzyme controlling extracellular adenosine production, can be activated by phospholipids, e.g. lysophosphatidylcholine (LPC). This study examined the structural requirements of phospholipids to evoke this enzyme activation and figured out two new activators of ecto-5′-nucleotidase: platelet activating factor (PAF) and sphingosylphosphorylcholine (SPC). Potential signal transduction pathways including an involvement of protein kinase C and PAF-receptor were evaluated on the model of human umbilical vein endothelial cells (HUVEC). Cells were pre-incubated with 10 μM of various phospholipids including lysophosphatidylcholine, β-arachidonyl-γ-palmityl-α-phosphatidylcholine, β, γ-dipalmityl-α-phosphatidyl-choline, β,γ-dipalmityl-α-phosphatidylethanolamine, β,γ-dipalmityl-α-phosphatidylserine, γ-acyl-β-lyso-α-phosphatidylethanolamine, β-acetyl-γ-O-hexadecyl-α-phosphatidylcholine (platelet activating factor), lysophosphatidylic acid, sphingosine-1-phosphate and sphingosylphosphorylcholine. In the cell supernatant the extracellular dephosphorylation rate of the fluorescent AMP-analogue 1, N⁶-etheno-5′AMP to 1, N⁶-etheno-adenosine was measured by HPLC. Out of these ten structurally related phospholipids only lysophosphatidylcholine, sphingosylphosphatidylcholine and platelet activating factor dose-dependently increased the activity of ecto-5′-nucleotidase. Pharmacological blocking experiments revealed that neither the activation of PAF-receptor nor of protein kinase C were important for mediating the activation of ecto-5′-nucleotidase. Thus, using information on the known molecular structures of tested phospholipids, a phosphatidylcholine residue in α-position and a short chain length fatty acid esterified in β-position seem essential for activation of ecto-5′-nucleotidase by glycerophospholipids. Since all tested phospholipids have similar fatty acid chain lengths and residues in α-position, they should act similarly on membrane fluidity. It is concluded that the observed effects are not based on changes in membrane fluidity by the added phospholipids, but rather involve a yet to be determined phospholipid-receptor.     

Tretinoin or retinol enhancement of lymphokine-activated killer cell proliferation and cytotoxicity against human bladder cancer cells in vitro

目的：研究维Ａ酸（Ｔｒｅ）或维生素Ａ（Ｒｅｔ）对膀胱肿瘤患者淋巴因子激活的杀伤细胞（ＬＡＫ细胞）的增殖和对膀胱肿瘤细胞的细胞毒作用．方法：分别用细胞计数和ＭＴＴ法测定ＬＡＫ细胞的增殖和细胞毒作用．结果：Ｔｒｅ或Ｒｅｔ１０－１００ｎｍｏｌ·Ｌ－１加强由白细胞介素２（ＩＬ２）诱导的ＬＡＫ细胞的增殖和对膀胱肿瘤细胞的杀伤作用．结论：Ｔｒｅ或Ｒｅｔ增强膀胱肿瘤患者ＬＡＫ细胞增殖及对膀胱肿瘤的细胞毒作用．     

Inhibitory effect of quercetin on proliferation of human microvascular endothelial cells in vitro

AIM: To investigate the role of quercetin (Que) in the proliferation of cultured human skin microvascular endothelial cells (MVEC). METHODS: Cell count and [methyl-~3H]thymidine ([~3H]TdR) uptake assay were used to measure the effect of Que in the proliferation of cultured MVEC. Cytotoxicity of Que on MVEC was also evaluated by ~(51)Cr release assay. RESULTS: When MVEC were treated with Que, the proliferation was significantly inhibited in a time-course and dose-dependent manner. Que 5 μmol/L did not inhibit the proliferation of MVEC. When the concentration of Que increased to 20, 40, 80, and 160μmol/L, the cell numbers per well were decreased and the inhibition rate was 12.2％, 23.5％, 35.3％, and 54.1％ respectively with IC_(50) of 138 μmol/L. The inhibitory rate of [~3H]-TdR uptake was 18.7％, 34.4％, 48.9％, and 62.5％ respectively (IC_(50)=87.5 μmol/L). ~(51)Cr release assay showed that Que 160μmol/L incubated with MVEC from 1 to 16h had no clear cytotoxicity compared with control group. CONCL     

Resveratrol analogue 3,4,4′-trihydroxy-trans-stilbene induces apoptosis and autophagy in human non-small-cell lung cancer cells in vitro

Aim:

To investigate the effects of 3,4,4′-trihydroxy-trans-stilbene (3,4,4′-THS), an analogue of resveratrol, on human non-small-cell lung cancer (NSCLC) cells in vitro.

Methods:

Cell viability of NSCLC A549 cells was determined by MTT assay. Cell apoptosis was evaluated using flow cytometry and TUNEL assay. Cell necrosis was evaluated with LDH assay. The expression of apoptosis- or autophagy-associated proteins was measured using Western blotting. The formation of acidic compartments was detected using AO staining, neutral red staining and Lysotracker-Red staining. LC3 punctae were analyzed with fluorescence microscopy.

Results:

Treatment with 3,4,4′-THS (10-80 μmol/L) concentration-dependently inhibited the cell viability. It did not cause cell necrosis, but induced apoptosis accompanied by up-regulation of cleavaged PARP, caspase3/9 and Bax, and by down-regulation of Bcl-2 and surviving. It also increased the formation of acidic compartments, LC3-II accumulation and GFP-LC3 labeled autophagosomes in the cells. It inhibited the mTOR-dependent pathway, but did not impair autophagic flux. 3,4,4′-THS-induced cell death was enhanced by the autophagy inhibitors 3-MA (5 mmol/L) or Wortmannin (2 μmol/L). Moreover, 3,4,4′-THS treatment elevated the ROS levels in the cells, and co-treatment with 3-MA further elevated the ROS levels. 3,4,4′-THS-induced apoptosis and autophagy in the cells was attenuated by NAC (10 mmol/L)

Conclusion:

3,4,4′-THS induces both apoptosis and autophagy in NSCLC A549 cells in vitro. Autophagy inhibitors promote 3,4,4′-THS-induced apoptosis of A549 cells, thus combination of 3,4,4′-THS and autophagy inhibitor provides a promising strategy for NSCLC treatment.     

Protective role of olesoxime against wild-type α-synuclein-induced toxicity in human neuronally differentiated SHSY-5Y cells

BACKGROUND AND PURPOSE

Parkinson''s disease (PD) is usually diagnosed clinically from classical motor symptoms, while definitive diagnosis is made postmortem, based on the presence of Lewy bodies and nigral neuron cell loss. α-Synuclein (ASYN), the main protein component of Lewy bodies, clearly plays a role in the neurodegeneration that characterizes PD. Additionally, mutation in the SNCA gene or copy number variations are associated with some forms of familial PD. Here, the objective of the study was to evaluate whether olesoxime, a promising neuroprotective drug can prevent ASYN-mediated neurotoxicity.

EXPERIMENTAL APPROACH

We used here a novel, mechanistically approachable and attractive cellular model based on the inducible overexpression of human wild-type ASYN in neuronally differentiated human neuroblastoma (SHSY-5Y) cells. This model demonstrates gradual cellular degeneration, coinciding temporally with the appearance of soluble and membrane-bound ASYN oligomers and cell death combining both apoptotic and non-apoptotic pathways.

KEY RESULTS

Olesoxime fully protected differentiated SHSY-5Y cells from cell death, neurite retraction and cytoplasmic shrinkage induced by moderate ASYN overexpression. This protection was associated with a reduction in cytochrome c release from mitochondria and caspase-9 activation suggesting that olesoxime prevented ASYN toxicity by preserving mitochondrial integrity and function. In addition, olesoxime displayed neurotrophic effects on neuronally differentiated SHSY-5Y cells, independent of ASYN expression, by promoting their differentiation.

CONCLUSIONS AND IMPLICATIONS

Because ASYN is a common underlying factor in many cases of PD, olesoxime could be a promising therapy to slow neurodegeneration in PD.     

Chiral effects in adrenocorticolytic action of o,p′-DDD (mitotane) in human adrenal cells

1. Adrenocortical carcinoma (ACC) is a rare malignant disease with poor prognosis. The main pharmacological choice, o,p′-DDD (mitotane), produces severe adverse effects.

2. Since o,p′-DDD is a chiral molecule and stereoisomers frequently possess different pharmacokinetic and/or pharmacodynamic properties, we isolated the two o,p′-DDD enantiomers, (R)-(+)-o,p′-DDD and (S)-(–)-o,p′-DDD, and determined their absolute structures.

3. The effects of each enantiomer on cell viability and on cortisol and dehydroepiandrosterone (DHEA) secretion in the human adrenocortical cell line H295R were assessed. We also assayed the o,p′-DDD racemate and the m,p′- and p,p′-isomers.

4. The results show small but statistically significant differences in activity of the o,p′-DDD enantiomers for all parameters tested. The three DDD isomers were equally potent in decreasing cell viability, but p,p′-DDD affected hormone secretion slightly less than the o,p′- and m,p′-isomers.

5. The small chiral differences in direct effects on target cells alone do not warrant single enantiomer administration, but might reach importance in conjunction with possible stereochemical effects on pharmacokinetic processes in vivo.

     

Evaluation of in vitro cytotoxicity and genotoxicity of copper–zinc alloy nanoparticles in human lung epithelial cells

In the present study, in vitro cytotoxic and genotoxic effect of copper–zinc alloy nanoparticles (Cu–Zn ANPs) on human lung epithelial cells (BEAS-2B) were investigated. XTT test and clonogenic assay were used to determine cytotoxic effects. Cell death mode and intracellular reactive oxygen species formations were analyzed using M30, M65 and ROS Elisa assays. Genotoxic effects were evaluated using micronucleus, comet and γ-H2AX foci assays. Cu–Zn ANPs were characterized by transmission electron microscopy (TEM), dynamic light scattering (DLS) and zeta potential measurements. Characterization of Cu–Zn ANPs showed an average size of 200 nm and zeta potential of −22 mV. TEM analyses further revealed the intracellular localization of Cu–Zn ANPs in cytoplasm within 24 h. Analysis of micronucleus, comet and γ-H2AX foci counts showed that exposure to Cu–Zn ANPs significantly induced chromosomal damage as well as single and double stranded DNA damage in BEAS-2B cells. Our results further indicated that exposure to Cu–Zn ANPs significantly induced intracellular ROS formation. Evaluation of M30:M65 ratios suggested that cell death was predominantly due to necrosis.     

Lipoprotein-mediated transfer of 2,4,5,2′,4′,5′-hexachlorobiphenyl into cultured human cells

1. The very low density, low density and high density lipoproteins (VLDL, LDL, HDL), centrifugally separated from human plasma treated with 2,4,5,2′,4′,5′-hexachloro[¹⁴C]biphenyl (¹⁴C-HCB) contained approximately 50% of the ¹⁴C-HCB.

2. Normal skin fibroblasts were incubated at 4°C or 37°C for varying times with medium containing 10% serum, LDL or HDL labelled with ¹⁴C-HCB. Cellular incorporation of ¹⁴C-HCB from serum was temperature-dependent and occurred mainly during the first 30 minutes. Cellular accumulation of ¹⁴C-HCB from isolated lipoproteins was also rapid and was more efficient from HDL than from LDL or serum. Accumulation from HDL was concentration-dependent and temperature-dependent.

3. The efflux of ¹⁴C-HCB from cells into serum- or lipoprotein-containing medium occurred very rapidly and was most effective in the presence of 20% serum. The order of efficiency in removal of HCB from cells was 20% serum, 50 μg LDL protein/ml, and 120 μg HDL protein/ml. Little or no efflux from cells occurred into serum-free, lipoprotein-free medium.

4. HDL may be involved in the delivery of HCB to cells, a role in contrast to the generally accepted theory that HDL transports lipids from cells.     

Mast cell- derived mediators protects PC12 cells against ischemia in vitro

We have previously demonstrated that brain mast cells degranulated under ischemia in in vivo or in vitro experiments. However the true role of mast cells in the brain ischemia is still known. In the present study, we investigated the effect of the mediators from the mast cell on neuronal cell line PC12 in an in vitro ischemic model oxygen - glucose deprivation （OGD）. The supernatant of mast cells was collected ofter 1h OGD, and then incubated with the PC12 cells under lh OGD. Results showed that by the incorporation of the mast cell - derived supernatants, the survival of PC12 cells markedly increased after OGD exposure with or without 24h reperfusion. Histamine H1 receptor antagonist pyrilamine significantly reversed the effect of the mast cell - derived supernatants, which indicated the participation of histamine in this process. However histamine itself did not increased the survival of PC12 cells after OGD.     

Synergistic interaction of β-galactosyl-pyrrolidinyl diazeniumdiolate with cisplatin against three tumor cells

Cisplatin is a platinum-based compound that is largely employed as an effective antitumor drug against a wide spectrum of solid neoplasms for many years. Despite of its initial therapeutic success, cisplatin often results in high incidence of chemoresistance and high-dose cytotoxicity. Consequently, considerable efforts in hopes of reducing the dose-dependent side effects of cisplatin while retaining, or even enhancing, its antitumor properties have been undertaken throughout the past three decades. Nitric oxide (NO) is a small lipophilic free radical gas possessing versatile biological functions, including antitumor activities. However, NO, of itself, is difficult to be used, because of its extreme instability and short half-life. Previously, we have reported a stable NO donor, β-galactosyl-pyrrolidinyl diazeniumdiolate (β-Gal-NONOate), which exerts tumor killing effects through site-specific intracellular release of exogenous NO. In this study, we further investigated the combined inhibitory effect of β-Gal-NONOate and cisplatin against C6/LacZ, 9L/LacZ, and HeLa/LacZ tumor cells. It was shown that, in combination with β-Gal-NONOate, the antitumor effects of cisplatin against these common tumor cell lines were increased in a dose-dependent manner. Furthermore, the combination of these chemicals resulted in a synergistic suppression on tumor growth, which was achieved under a much lower cisplatin dosage. Collectively, our findings indicate that β-Gal-NONOate can synergistically improve the antitumor effect of cisplatin, and may therefore reduce its side effects caused by high dose cisplatin monochemotherapies. Accordingly, β-Gal-NONOate is an important therapeutic assistant reagent with great potential of clinical applicability, and thus worth of continuous research in the coming future.     

Metabolism of α-thujone in human hepatic preparations in vitro

1. This study aims to characterize the metabolism of α-thujone in human liver preparations in vitro and to identify the role of cytochrome P450 (CYP) and possibly other enzymes catalyzing α-thujone biotransformations.

2. With a liquid chromatography–mass spectrometry (LC-MS) method developed for measuring α-thujone and four potential metabolites, it was demonstrated that human liver microsomes produced two major (7- and 4-hydroxy-thujone) and two minor (2-hydroxy-thujone and carvacrol) metabolites. Glutathione and cysteine conjugates were detected in human liver homogenates, but not quantified. No glucuronide or sulphate conjugates were detected. Major hydroxylations accounted for more than 90% of the primary microsomal metabolism of α-thujone.

3. Screening of α-thujone metabolism with CYP recombinant enzymes indicated that CYP2A6 was principally responsible for the major 7- and 4-hydroxylation reactions, although CYP3A4 and CYP2B6 participated to a lesser extent and CYP3A4 and CYP2B6 catalyzed minor 2-hydroxylation. Based on the intrinsic efficiencies of different recombinant CYP enzymes and average abundances of these enzymes in human liver microsomes, CYP2A6 was calculated to be the most active enzyme in human liver microsomes, responsible for 70–80% of the metabolism on average.

4. Inhibition screening indicated that α-thujone inhibited both CYP2A6 and CYP2B6, with 50% inhibitory concentration values of 15.4 and 17.5 µM, respectively.

     

Effects of 2,3,7,8-TCDD and PCB 126 on human thymic epithelial cells in vitro

2,3,7,8-Tetrachloro-dibenzo- p-dioxin (TCDD) is a ubiquitously distributed xenobiotic. The adverse effects of TCDD on the mammalian immune system have been studied for decades, but it is still unclear whether TCDD has direct effects on T-lymphocytes or whether it acts via the thymic microenvironment. We have studied the effects of TCDD on primary cultures of human thymic epithelial cells (TEC) focusing on differentiation markers, integrins and adhesion molecules involved in cell-cell and in cell-matrix interactions. TEC were treated with TCDD at concentrations of 0.001, 0.01, 0.1, 1.0 or 10.0 nM or with 100 nM PCB 126 (3,3',4,4',5-pentachlorobiphenyl) for 3 days, and were then analysed by flow cytometry for expression of surface antigens using monoclonal antibodies against Hassall's bodies (TE-8, TE-16) or against surface structures such as CD29, CD49b, CD49e, CD49f, CD51, CD54, CD58, CD61 and CD106. At TCDD concentrations as low as 0.01 nM we found a significant increase in terminally differentiated, TE-16-positive TEC; at a ten-fold greater concentration the number of cells marked with the TE-8 antibody was also increased. With both markers the most pronounced effect (approximately +15%) was observed at 1 nM TCDD. An increase of cells expressing the integrin alpha-chains CD49b, CD49e and CD51 as well as CD54 was observed at concentrations of 0.1 nM TCDD or higher. The proportion of cells expressing CD106 or CD49f decreased significantly upon treatment with TCDD. No effects on the integrin beta-chains CD29 and CD61 could be detected. Overall, PCB 126 induced similar changes to TCDD. In summary, TCDD and a coplanar PCB induced terminal differentiation of human TEC along with changes of integrins and other adhesion molecules. These receptors and their interplay with the extracellular matrix have key functions in the maturation of T-lymphocytes and it is plausible that their alteration would be involved in TCDD-induced immunotoxicity.     

The role of human chorionic gonadotropin in regulation of naïve and memory T cells activity in vitro

The role of human chorionic gonadotropin (hCG) in the regulation of molecular genetics factors determining the functional activity of human naïve and memory T cells in vitro was studied. It was found that hCG (10 and 100 IU/ml) inhibited CD28 and CD25 expression on the naïve T cells (CD45RA +) and CD25 expression on the memory T cells (CD45R0 +). hCG didn't affect the CD71 proliferation marker expression in total. Nevertheless, hCG reduced the percentage of proliferating memory T cells with simultaneous suppression of CD71 expression on proliferating CD45R0 + cells.In parallel, expression of U2af1l4, Gfi1, and hnRNPLL genes, which are Ptprc gene alternative splicing regulators was evaluated. It was established that hCG stimulated the expression of U2af1l4 and hnRNPLL genes, responsible for the assembly of CD45R0 in memory T cells, but reduced the expression of Gfi1 in these cells. In general, hCG promotes the differentiation of memory T cells by increasing of CD45R0 expression, but inhibits proliferation and CD25 expression which reflects their functional activity.     

Metabolism of α-thujone in human hepatic preparations in vitro

This study aims to characterize the metabolism of α-thujone in human liver preparations in vitro and to identify the role of cytochrome P450 (CYP) and possibly other enzymes catalyzing α-thujone biotransformations. With a liquid chromatography-mass spectrometry (LC-MS) method developed for measuring α-thujone and four potential metabolites, it was demonstrated that human liver microsomes produced two major (7- and 4-hydroxy-thujone) and two minor (2-hydroxy-thujone and carvacrol) metabolites. Glutathione and cysteine conjugates were detected in human liver homogenates, but not quantified. No glucuronide or sulphate conjugates were detected. Major hydroxylations accounted for more than 90% of the primary microsomal metabolism of α-thujone. Screening of α-thujone metabolism with CYP recombinant enzymes indicated that CYP2A6 was principally responsible for the major 7- and 4-hydroxylation reactions, although CYP3A4 and CYP2B6 participated to a lesser extent and CYP3A4 and CYP2B6 catalyzed minor 2-hydroxylation. Based on the intrinsic efficiencies of different recombinant CYP enzymes and average abundances of these enzymes in human liver microsomes, CYP2A6 was calculated to be the most active enzyme in human liver microsomes, responsible for 70-80% of the metabolism on average. Inhibition screening indicated that α-thujone inhibited both CYP2A6 and CYP2B6, with 50% inhibitory concentration values of 15.4 and 17.5 μM, respectively.     

Regulation of guanosine 3′:5′-cyclic monophosphate in ovine tracheal epithelial cells

1. Guanosine 3′:5′-cyclic monophosphate (cyclic GMP) is an important second messenger mediating the effects of nitric oxide (NO) and natriuretic peptides. Cyclic GMP pathways regulate several aspects of lung pathophysiology in a number of airway cells. The regulation of this system has not been extensively studied in pulmonary epithelial tissue.
2. We have studied the production of cyclic GMP by suspensions of ovine tracheal epithelial cells in response to activators of soluble guanylyl cyclase (sodium nitroprusside (SNP) and S-nitroso-N-acetyl-penicillamine (SNAP) and particulate guanylyl cyclase (atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), C-type natriuretic peptide (CNP) and E. coli heat stable enterotoxin (STa)).
3. Both 10⁻⁷10⁻³ M and 10⁻⁷10⁻³ M SNAP generated a concentration-dependent marked elevation in cyclic GMP production when incubated with 10⁻³ M 3-isobutyl-1-methylxanthine (IBMX) (both greater than 25×baseline values with highest drug concentration).
4. The increase in production of cyclic GMP in response to 10⁻⁶ M SNP and 10⁻⁵ M SNAP was markedly inhibited by both 5×10⁻⁵ M haemoglobin (102% and 92% inhibition) and 5×10⁻⁵ M methylene blue (82% and 84% inhibition).
5. The increase in cyclic GMP in response to 10⁻³ M SNP was measured following co-incubation with the phosphodiesterase inhibitors 10⁻⁷10⁻³ M IBMX, 10⁻⁷10⁻⁴ M milrinone and 10⁻⁷10⁻⁴ M SKF 96231. Only 10⁻⁴10⁻³ M IBMX significantly increased cyclic GMP levels.
6. Cyclic GMP production was also significantly elevated from baseline by 10⁻⁵ M ANP, 10⁻⁵ M BNP, 10⁻⁵ M CNP and 200 iu ml⁻¹ of E. coli STa toxin in the presence of 10⁻³ M IBMX. Increases with these natriuretic peptides and STa toxin were smaller in magnitude (24 fold) than those seen with SNP and SNAP. CNP was the most potent of the natriuretic peptides studied suggesting type B membrane bound guanylate cyclase is the predominant form expressed.
7. These results suggest that ovine tracheal epithelial cells contain active guanylyl cyclases. The more marked response to SNP and SNAP than to natriuretic peptides suggests that soluble guanylyl cyclase predominates.

     

Activity of tricyclic nucleoside 5′-phosphate in model systems of human ovarian cancer

Tricyclic nucleoside 5-phosphate (TCN-P) was evaluated in two models of human ovarian cancer. TCN-P reduced both colony number and volume in clonogenic assays employing human ovarian cancer cell lines. TCN-P cytotoxicity depended on the concentration, exposure duration and cell line studied, but not on cell line plating efficiency or growth rate in soft agarose. Comparison of experimental IC₅₀ concentrations for 1 hour or continuous TCN-P exposure with reported clinically relevant concentrations suggests that therapeutic TCN-P levels are more likely to be achieved by continuous infusions. Cell lines and sublines with resistance to several standard chemotherapeutic agents acquired both in vivo and in vitro were at most 2.6-fold cross-resistant to TCN-P with 1 hour drug exposure. Cross-resistance was not evident with continuous TCN-P exposure. Intermittent bolus TCN-P (100 mg/kg/d × 5) was ineffective in an in vivo xenograft model of human ovarian cancer. These data suggest that TCN-P is most likely to be clinically effective against ovarian cancer, and may be non-cross-resistant with several standard agents, if administered by continuous infusion. Preclinical evaluation of new agents, such as TCN-P, in these experimental models may provide information useful in subsequent clinical trials.Abbreviations CRI cross-resistance index - EGF epidermal growth factor - IC₅₀ drug concentration causing 50% inhibition of colony formation - LD₁₀ lethal dose in 10% of animals - NS normal saline - r correlation coefficient - RCV relative colony volume - RPMI Roswell Park Memorial Institute - SF surviving fraction - TCN tricyclic nucleoside - TCN-P tricyclic nucleoside 5-phosphate     

Apoptotic effect of synthetic 2′,4′,5′-trimethoxychalcones in human K562 and Jurkat leukemia cells

In this study, we assessed the cytotoxic effect of synthetic 2′,4′,5′-trimethoxychalcones on the human K562 acute myeloid leukemia cell and human Jurkat acute lymphoid leukemia cell. Compounds 13, 16, 19, and 26 showed low IC₅₀ values (4.10–8.56 μM at 72 h) for both cell lines and did not have a cytotoxic effect on normal human lymphocytes. The mechanism of cell death induced by these compounds involves a decrease in the expression of cell proliferation marker Ki67, suggesting inhibition of cell proliferation. Furthermore, these chalcones reduced mitochondrial potential, decreased Bcl-2 expression, and increased Bax expression, indicating that the mechanism of apoptosis induced by them involves the intrinsic apoptosis pathway. The mechanism of action also involves increase in active caspase-3 and decrease in survivin expression. These results support the chalcones as potential antitumoral agents for further optimization.