Selective cytotoxicity of intense nanosecond-duration electric pulses in mammalian cells

Background

Nanosecond electric pulses (EP) disrupt cell membrane and organelles and cause cell death in a manner different from the conventional irreversible electroporation. We explored the cytotoxic effect of 10-ns EP (quantitation, mechanisms, efficiency, and specificity) in comparison with 300-ns, 1.8- and 9-μs EP.

Methods

Effects in Jurkat and U937 cells were characterized by survival assays, DNA electrophoresis and flow cytometry.

Results

10-ns EP caused apoptotic or necrotic death within 2–20 h. Survival (S, %) followed the absorbed dose (D, J/g) as: S = αD^(−K), where coefficients K and α determined the slope and the “shoulder” of the survival curve. K was similar in all groups, whereas α was cell type- and pulse duration-dependent. Long pulses caused immediate propidium uptake and phosphatidylserine (PS) externalization, whereas 10-ns pulses caused PS externalization only.

Conclusions

1.8- and 9-μs EP cause cell death efficiently and indiscriminately (LD₅₀ 1–3 J/g in both cell lines); 10-ns EP are less efficient, but very selective (LD₅₀ 50–80 J/g for Jurkat and 400–500 J/g for U937); 300-ns EP show intermediate effects. Shorter EP open propidium-impermeable, small membrane pores (”nanopores”), triggering different cell death mechanisms.

General significance

Nanosecond EP can selectively target certain cells in medical applications like tumor ablation.     

Manipulation of cell volume and membrane pore comparison following single cell permeabilization with 60- and 600-ns electric pulses

Intense nanosecond-duration electric pulses (nsEP) open stable nanopores in the cell membrane, followed by cell volume changes due to water uptake or expulsion, as regulated by the osmolality balance of pore-impermeable solutes inside and outside the cell. The size of pores opened by either fifty 60-ns EP (~13 kV/cm) or five, 600-ns EP (~6 kV/cm) in GH3 cells was estimated by isoosmotic replacement of bath NaCl with polyethylene glycols and sugars. Such replacement reduced cell swelling or resulted in transient or sustained cell shrinking in response to EP. depending on the availability of pores permeable to the test solute. Unexpectedly, solute substitutions showed that for the same integral area of pores opened by 60- and 600-ns treatments (as estimated by cell volume changes), the pore sizes were similar. However, the 600-ns exposure triggered significantly higher cell uptake of propidium. We concluded that 600-ns EP opened a greater number of larger (propidium-permeable pores), but the fraction of the larger pores in the entire pore population was insufficient to contribute to cell volume changes. For both the 60- and 600-ns exposures, cell volume changes were determined by pores smaller than 0.9 nm in diameter; however, the diameter increased with increasing the nsEP intensity.     

Lysozyme facilitates adherence of Enterococcus faecium to host cells and induction of necrotic cell death

The prevalence of infections with enterococci is increasing worldwide. However, little is known about the mechanisms which enable these opportunistic pathogens to cause infections of their host. Here we demonstrate that Enterococcus faecium in the presence of lysozyme induces necrosis in human and mouse cells after 4 h indicated by disrupted cellular membranes of epithelial (HeLa), myeloid (U937, J774A.1) and lymphoid (Jurkat J16, thymocytes), but not intestinal epithelial cells (CaCo-2, CMT-93). Using an appropriate mutant strain it was shown that the enterococcal surface-protein SgrA is involved in cell death induction in mouse cells (J774A.1, thymocytes). Microscopic analyses of epithelial cells 30 min post infection revealed that lysozyme increases adhesion of E. faecium to HeLa, but not CaCo-2 cells. At that time the phalloidin-FITC-stained cytoskeleton of infected cells was still intact, whereas 2 h post infection the F-actin network of HeLa, but not CaCo-2 cells was disrupted. Hence, the early, lysozyme-mediated increase of bacterial adherence plays an important role for cell death induction by E. faecium in HeLa cells. Moreover, bacterial extracellular hydrogen peroxide might contribute to necrosis induction, since the rate of propidium iodide-positive HeLa and J774A.1 cells was lowered after infection with a ROS-deficient E. faecium mutant.     

Nanosecond pulsed electric fields modulate the expression of Fas/CD95 death receptor pathway regulators in U937 and Jurkat Cells

In this publication, we demonstrate that exposure of Jurkat and U937 cells to nanosecond pulsed electrical fields (nsPEF) can modulate the extrinsic-mediated apoptotic pathway via the Fas/CD95 death receptor. An inherent difference in survival between these two cell lines in response to 10 ns exposures has been previously reported (Jurkat being more sensitive to nsPEF than U937), but the reason for this sensitivity difference remains unknown. We found that exposure of each cell line to 100, 10 ns pulses at 50 kV/cm caused a marked increase in expression of cFLIP (extrinsic apoptosis inhibitor) in U937 and FasL (extrinsic apoptosis activator) in Jurkat, respectively. Measurement of basal expression levels revealed an inherent difference between U937 cells, having a higher expression of cFLIP, and Jurkat cells, having a higher expression of FasL. From these data, we hypothesize that the sensitivity difference between the cells to nsPEF exposure may be directly related to expression of extrinsic apoptotic regulators. To validate this hypothesis, we used siRNA to knockdown cFLAR (coding for cFLIP protein) expression in U937, and FasL expression in Jurkat and challenged them to 100, 10 ns pulses at 150 kV/cm, a typical lethal dose. We observed that U937 survival was reduced nearly 60 % in the knockdown population while Jurkat survival improved ~40 %. These findings support the hypothesis that cell survival following 10 ns pulse exposures depends on extrinsic apoptotic regulators. Interestingly, pretreatment of U937 with a 100-pulse, 50 kV/cm exposure (to amplify cFLAR expression) significantly reduced the lethality of a 150 kV/cm, 100-pulse exposure applied 24 h later. From these data, we conclude that the observed survival differences between cells, exposed to 10 ns pulsed electric fields, is due to inherent cell biochemistry rather than the biophysics of the exposure itself. Understanding cell sensitivity to nsPEF may provide researchers/clinicians with a predicable way to control or avoid unintended cell death during nsPEF exposure.     

Production of human tissue-type plasminogen activator in different mammalian cell lines using an Epstein-Barr virus vector.

Human tissue-type plasminogen activator (t-PA) cDNA inserted into an Epstein-Barr virus (EBV) derived expression vector was transfected into human HeLa, 293, K-562 and hamster CHO-K1 cells and the expression of t-PA was studied. The best t-PA producing cell clones were found among CHO-K1 cells (up to 11 micrograms d-1 per 10(6) cells). However, HeLa and 293 cells were most efficiently transfected, e.g. about 70% of the selected cell clones were t-PA positive. The vector DNA copy numbers correlated with the mRNA levels and the protein levels for all cell lines analysed, with the exception for the K-562 cell line, where the production of t-PA was very low. The results obtained indicated that the highest expression levels were achieved in low density cultures.     

Long-lasting plasma membrane permeabilization in mammalian cells by nanosecond pulsed electric field (nsPEF)

The barrier function of plasma membrane in nsPEF-exposed mammalian cells was examined using whole-cell patch-clamp techniques. A specialized setup for nsPEF exposure of individual cells in culture was developed and characterized for artifact-free compatibility with the patch-clamp method. For the first time, our study provides experimental evidence that even a single 60-ns pulse at 12 kV/cm can cause a profound and long-lasting (minutes) reduction of the cell membrane resistance (R(m)), accompanied by the loss of the membrane potential. R(m) measured in GH3, PC-12, and Jurkat cells (but not in HeLa cells) in 80-120 s after nsPEF exposure was decreased about threefold, and its gradual recovery could take 15 min. Multiple pulses enhanced permeabilization, for example, R(m) in GH3 cells fell about 10-fold after a train of five pulses. Within studied limits, permeabilization did not depend on the presence of Ca(2+), Mg(2+), K(+), Cs(+), Cd(2+), EGTA, tetraethylammonium, or 4-aminopyridine in the pipette or bath solutions. Our results supported theoretical model predictions of plasma membrane poration by nsPEF. However, the extended decrease in R(m), assumed to be related to the life span of the pores, and different nsPEF sensitivity of individual cell lines have yet to be explained. The phenomenon of long-lived membrane permeabilization provides new insights on the nature of nsPEF-opened conductance pores and on molecular mechanisms that underlie nsPEF bioeffects.     

Cyathula prostrata ethanol extract activates the extrinsic pathway of apoptosis in HeLa and U937 cell lines

Cyathula prostrata is a medicinal plant that is used in tropical Africa, Asia and Australia to treat many ailments including rheumatic fever, dysentery, wounds and eye trouble (Burkill, 1995). Sowemimo et al. (2009) reported cytotoxicity against a cervical cancer cell line, HeLa, at a concentration of 250 μg/mL. Due to the reported cytotoxicity, the mode of cell death caused by an ethanolic C. prostrata extract was investigated. Dose–response assays were carried out using HeLa and U937 cell lines and IC₅₀ values of 100.8 μg/mL and 64.43 μg/mL, respectively, were achieved. Cisplatin was used as a positive control for all experiments. Cytotoxicity of the plant extract was not evident when treating normal peripheral blood mononuclear cells. Progression of cells through the cell cycle, apoptosis and mitochondrial membrane potential was investigated. Arrest of cells in the G0/G1 phase of the cell cycle was evident after 24 h of exposure to the plant extract in both cell lines, but not due to increases in p21 levels. Caspase 8 activation was evident and no depolarisation of the mitochondrial membrane and cytochrome c release was seen in both cell lines. Phosphatidylserine translocation was investigated and confirmed the onset of apoptosis. Thus, it is deduced that exclusive activation of the extrinsic pathway of apoptosis is caused by the treatment of HeLa and U937 cells with ethanolic C. prostrata.     

Nanosecond pulsed electric fields induce poly(ADP-ribose) formation and non-apoptotic cell death in HeLa S3 cells

Nanosecond pulsed electric fields (nsPEFs) have recently gained attention as effective cancer therapy owing to their potency for cell death induction. Previous studies have shown that apoptosis is a predominant mode of nsPEF-induced cell death in several cell lines, such as Jurkat cells. In this study, we analyzed molecular mechanisms for cell death induced by nsPEFs. When nsPEFs were applied to Jurkat cells, apoptosis was readily induced. Next, we used HeLa S3 cells and analyzed apoptotic events. Contrary to our expectation, nsPEF-exposed HeLa S3 cells exhibited no molecular signs of apoptosis execution. Instead, nsPEFs induced the formation of poly(ADP-ribose) (PAR), a hallmark of necrosis. PAR formation occurred concurrently with a decrease in cell viability, supporting implications of nsPEF-induced PAR formation for cell death. Necrotic PAR formation is known to be catalyzed by poly(ADP-ribose) polymerase-1 (PARP-1), and PARP-1 in apoptotic cells is inactivated by caspase-mediated proteolysis. Consistently, we observed intact and cleaved forms of PARP-1 in nsPEF-exposed and UV-irradiated cells, respectively. Taken together, nsPEFs induce two distinct modes of cell death in a cell type-specific manner, and HeLa S3 cells show PAR-associated non-apoptotic cell death in response to nsPEFs.     

Roles for endocytosis and low pH in herpes simplex virus entry into HeLa and Chinese hamster ovary cells

Herpes simplex virus (HSV) infection of many cultured cells, e.g., Vero cells, can be initiated by receptor binding and pH-neutral fusion with the cell surface. Here we report that a major pathway for HSV entry into the HeLa and CHO-K1 cell lines is dependent on endocytosis and exposure to a low pH. Enveloped virions were readily detected in HeLa or receptor-expressing CHO cell vesicles by electron microscopy at <30 min postinfection. As expected, images of virus fusion with the Vero cell surface were prevalent. Treatment with energy depletion or hypertonic medium, which inhibits endocytosis, prevented uptake of HSV from the HeLa and CHO cell surface relative to uptake from the Vero cell surface. Incubation of HeLa and CHO cells with the weak base ammonium chloride or the ionophore monensin, which elevate the low pH of organelles, blocked HSV entry in a dose-dependent manner. Noncytotoxic concentrations of these agents acted at an early step during infection by HSV type 1 and 2 strains. Entry mediated by the HSV receptor HveA, nectin-1, or nectin-2 was also blocked. As analyzed by fluorescence microscopy, lysosomotropic agents such as the vacuolar H(+)-ATPase inhibitor bafilomycin A1 blocked the delivery of virus capsids to the nuclei of the HeLa and CHO cell lines but had no effect on capsid transport in Vero cells. The results suggest that HSV can utilize two distinct entry pathways, depending on the type of cell encountered.     

Differential transactivation of the intercellular adhesion molecule 1 gene promoter by Tax1 and Tax2 of human T-cell leukemia viruses.

Previously, we showed that surface expression of intercellular adhesion molecule 1 (ICAM-1) was strongly upregulated in T cells carrying proviral human T-cell leukemia virus type 1 (HTLV-1) and that the viral transactivator protein Tax1 was capable of inducing the ICAM-1 gene. To determine the responsive elements in the human ICAM-1 gene promoter, a reporter construct in which the 5'-flanking 4.4-kb region of the ICAM-1 gene was linked to the promoterless chloramphenicol acetyltransferase (CAT) gene was cotransfected with expression vectors for Tax1 and Tax2, both of which were separately confirmed to be potent transactivators of the HTLV-1 long terminal repeat (LTR). Tax1 strongly activated the ICAM-1 promoter in all the cell lines tested: three T-cell lines (Jurkat, MOLT-4, and CEM), one monocytoid cell line (U937), and HeLa. Unexpectedly, Tax2 activated the ICAM-1 promoter only in HeLa. By deletion and mutation analyses of the 1.3-kb 5'-flanking region, we found that Tax1 transactivated the ICAM-1 promoter mainly via a cyclic AMP-responsive element (CRE)-like site at -630 to -624 in the Jurkat T-cell line and via an NF-kappaB site at -185 to -177 and an SP-1 site at -59 to -54 in HeLa. On the other hand, Tax2 was totally inactive on the ICAM-1 promoter in Jurkat but transactivated the promoter via the NF-kappaB site at -185 to -177 in HeLa. Gel mobility shift assays demonstrated proteins specifically binding to the CRE-like site at -630 to -624 in Tax1-expressing T-cell lines. Stable expression of Tax1 but not Tax2 in Jurkat subclones enhanced the surface expression of ICAM-1. The differential ability of Tax1 and Tax2 in transactivation of the ICAM-1 gene may be related to the differential pathogenicity of HTLV-1 and HTLV-2.     

Effect of sesquiterpene lactone coronopilin on leukaemia cell population growth, cell type-specific induction of apoptosis and mitotic catastrophe

Objectives: The aim of this study was to investigate anti‐leukaemic potential of coronopilin, a sesquiterpene lactone from Ambrosia arborescens, and to characterize mechanism(s) underlying its activity. Materials and methods: The study was conducted on Jurkat and U937, two leukaemia‐derived cell lines. Apoptosis and impairment of cell cycle progression were evaluated by flow cytometry and by microscopic analysis. Changes in protein expression and activation were evaluated by western blot analysis. Coronopilin‐tubulin covalent adducts were demonstrated by mass spectrometry. Results: Coronopilin inhibited (IC₅₀ ≤ 20 μm ) leukaemia cell population growth, but displayed poor cytotoxicity to normal white blood cells. On Jurkat cells, coronopilin exerted cell population growth inhibition activity, mainly by triggering caspase‐dependent apoptosis. Conversely, in U937 cells, coronopilin’s primary response was a robust arrest in G₂/M. Marked increase in mitotic index and presence of activated cyclin B1/Cdk1 complex, phosphorylated histone H3 at Ser10, and hyperpolymerized tubulin indicated that cells accumulated in mitosis. Prolonged mitotic arrest ultimately resulted in U937 mitotic catastrophe, and dying cells exhibited the features of non‐caspase‐dependent death. Conclusions: This study demonstrated that coronopilin efficiently inhibited leukaemia cell population growth by triggering cell type‐specific responses. Moreover, coronopilin‐mediated cell population expansion inhibition was specific to neoplastic cells, as normal white blood cell viability was not significantly affected. Thus, coronopilin may represent an interesting new chemical scaffold upon which to develop new anti‐leukaemic agents.     

Ammonium toxicity in different cell lines

The toxic effect of ammonium upon a variety of cell lines of lymphoid (Jurkat), pituitary (GH(4)), and renal (LLC-PK(1)) origin was studied. Millimolar concentrations of the ion mildly affected the growth of GH(4) cells and prevented the growth of LLC-PK(1) cells. The ion did not lead to the death of LLC-PK(1) cells but it produced morphologic changes in these cells. The effects of ammonium upon Jurkat cells were different because cells died after accumulating at S phase. Cell death was due to apoptosis and might be related to ammonium-induced calcium mobilization from intracellular stores. These results indicate that the toxic effects caused by ammonium accumulation are different depending upon the cell type. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 530-537, 1997.     

Evaluation of inhibitory effect and apoptosis induction of Zyzyphus Jujube on tumor cell lines,an in vitro preliminary study

In the present study, water extract of dried fruit of Zyzyphus Jujube was tested for its possible anticancer effect and induction of apoptosis on human tumor cell lines, HEp-2, HeLa and Jurkat cell lines. The inhibitory effect of water extract of this fruit on cell proliferation was assessed by MTT colorimetric assay. The induction of apoptosis of this extract was analyzed by DNA fragmentation analysis. Zyzyphus Jujube extract showed inhibitory effects on mentioned cell lines. Jurkat leukemic line was found the most sensitive cells with IC50 of 0.1 μg mL⁻¹. Our study also showed a typical DNA laddering in this cell line. The present study showed cytotoxic activity of Zyzyphus Jujube on tumor cells. Although Zyzyphus Jujube has useful compounds for medical applications.     

Caspase-10-dependent cell death in Fas/CD95 signalling is not abrogated by caspase inhibitor zVAD-fmk

Background

Upon CD95/Fas ligation, the initiator caspase-8 is known to activate effector caspases leading to apoptosis. In the presence of zVAD-fmk, a broad-spectrum caspase inhibitor, Fas engagement can also trigger an alternative, non-apoptotic caspase-independent form of cell death, which is initiated by RIP1. Controversy exists as to the ability of caspase-10 to mediate cell death in response to FasL (CD95L or CD178). Herein, the role of caspase-10 in FasL-induced cell death has been re-evaluated.

Methodology and Principal Findings

The present study shows that FasL-induced cell death was completely impaired in caspase-8- and caspase-10-doubly deficient (I9-2e) Jurkat leukaemia T-cell lines. Over-expressing of either caspase-8 or caspase-10 in I9-2e cells triggered cell death and restored sensitivity to FasL, further arguing for a role of both initiator caspases in Fas apoptotic signalling. In the presence of zVAD-fmk, FasL triggered an alternative form of cell death similarly in wild-type (A3) and in caspase-8-deficient Jurkat cells expressing endogenous caspase-10 (clone I9-2d). Cell death initiated by Fas stimulation in the presence of zVAD-fmk was abrogated in I9-2e cells as well as in HeLa cells, which did not express endogenous caspase-10, indicating that caspase-10 somewhat participates in this alternative form of cell death. Noteworthy, ectopic expression of caspase-10 in I9-2e and HeLa cells restored the ability of FasL to trigger cell death in the presence of zVAD-fmk. As a matter of fact, FasL-triggered caspase-10 processing still occurred in the presence of zVAD-fmk.

Conclusions and Significance

Altogether, these data provide genetic evidence for the involvement of initiator caspase-10 in FasL-induced cell death and indicate that zVAD-fmk does not abrogate caspase-10 processing and cytotoxicity in Fas signalling. Our study also questions the existence of an alternative caspase-independent cell death pathway in Fas signalling.     

Induction of p53 protein expression by sodium arsenite

Arsenic is carcinogen for humans and has been shown to act as an enhancer in initiated animal models. In a previous work we found impairment of lymphocyte proliferation in arsenic-exposed individuals and in vitro we obtained dose-related inhibition of mitotic response and lymphocyte proliferation. Intrigued by these effects and based on the role of p53 on cell proliferation, we tested different concentrations of sodium arsenite for their ability to induce the expression of tumor suppressor gene p53 in different cell lines (HeLa, C-33A, Jurkat) and a lymphoblast cell line transformed with Epstein–Barr virus (LCL-EBV). We also evaluated changes in their viability after 24 h arsenic treatment; C-33A cells showed the higher sensitivity to arsenic treatment while HeLa, Jurkat and LCL-EBV cells showed similar cytotoxicity curves. Immunoblots showed an increased expression of p53 gene with 1 μM sodium arsenite in Jurkat cells and 10 μM sodium arsenite in HeLa and LCL-EBV cells. In addition, we transfected Jurkat cells and human lymphocytes with wild-type and mutated p53 genes; lymphocytes and Jurkat cells that received the mutated p53 showed increased sensitivity to arsenic cytotoxicity. Data obtained indicate that arsenic induces p53 expression and that cells with a functional p53 contend better with damage induced by this metalloid.     

TAR independent activation of the human immunodeficiency virus in phorbol ester stimulated T lymphocytes.

Multiple regulatory elements in the human immunodeficiency virus long terminal repeat (HIV LTR) are required for activation of HIV gene expression. Previous transfection studies of HIV LTR constructs linked to the chloramphenicol acetyltransferase gene indicated that multiple regulatory regions including the enhancer, SP1, TATA and TAR regions were important for HIV gene expression. To characterize these regulatory elements further, mutations in these regions were inserted into both the 5' and 3' HIV LTRs and infectious proviral constructs were assembled. These constructs were transfected into either HeLa cells, Jurkat cells or U937 cells in both the presence and absence of phorbol esters which have previously been demonstrated to activate HIV gene expression. Viral gene expression was assayed by the level of p24 gag protein released from cultures transfected with the proviral constructs. Results in all cell lines indicated that mutations of the SP1, TATA and the TAR loop and stem secondary structure resulted in marked decreases in gene expression while mutations of the enhancer motif or TAR primary sequence resulted in only slight decreases. However, viruses containing mutations in either the TAR loop sequences or stem secondary structure which were very defective for gene expression in untreated Jurkat cells, gave nearly wild-type levels of gene expression in phorbol ester-treated Jurkat cells but not in phorbol ester-treated HeLa or U937 cells. High level gene expression of these TAR mutant constructs in phorbol ester-treated Jurkat cells was eliminated by second site mutations in the enhancer region or by disruption of the tat gene.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of Burkholderia pseudomallei and other Burkholderia species on eukaryotic cells in tissue culture

Burkholderia pseudomallei causes melioidosis, a serious and often fatal bacterial infection. B. pseudomallei can behave as a facultatively intracellular organism and this ability may be important in the pathogenesis of both acute and chronic infection. The uptake of B. pseudomallei and other Burkholderia spp. by cells in tissue culture was examined by electron microscopy. B. pseudomallei can invade cultured cell lines including phagocytic lines such as RAW264, J774 and U937, and non-phagocytic lines such as CaCO-2, Hep2, HeLa, L929, McCoy, Vero and CHO. Uptake was followed by the intracellular multiplication of B. pseudomallei and the induction of cell fusion and multinucleate giant cell formation. Similar effects were produced by B. mallei and B. thailandensis.     

Inhibition of p38 kinase reveals a TNF-alpha-mediated, caspase-dependent, apoptotic death pathway in a human myelomonocyte cell line

TNF-alpha transduces signals of survival or death via its two receptors, R1/p55/p60 and RII/p80/p75. The role of caspases as effectors of cell death is universally accepted, although caspase inhibitors may potentiate TNF cytotoxicity in some instances. In conditions when macromolecular synthesis is blocked, caspases are part of the machinery that executes TNF-triggered apoptotic death in U937, a human myelomonocyte cell line, and in the Jurkat T cell line. However, inhibition of p38 mitogen-activated protein kinase (p38 MAPK) triggered TNF cytotoxicity in U937 cells and murine splenic macrophages, but not the Jurkat cell line. TNF induced expression of the antiapoptotic protein c-IAP2 (cytoplasmic inhibitor of apoptosis protein 2), and was blocked in the presence of a p38 MAPK inhibitor, which also induced caspase-dependent, TNF-mediated apoptosis in U937 cells. Thus, inhibition of p38 MAPK resulted in the activation of caspase 9 and cleavage of the adaptor molecule BH3 interacting domain death agonist, and blocked NF-kappaB-mediated transactivation, without affecting the nuclear translocation of NF-kappaB. Collectively, these data show that activation of p38 MAPK is critical to cell survival by TNF in U937 cells, and demonstrate lineage-specific regulation of TNF-triggered signals of activation or apoptosis.     

Differential effects of synthesized 2'-oxygenated chalcone derivatives: modulation of human cell cycle phase distribution

Ten structurally related 2'-oxygenated chalcone derivatives, bearing either hydroxy and/or methoxy substituents on the A and B rings, were synthesized through Claisen-Schmidt condensation. The synthesis procedure was relatively easy and had an acceptable yield. The in vitro cytotoxicities of these compounds against the human tumor cells such as Jurkat, U937 cells, and normal cells PHA stimulated PBMCs were investigated. Among those, compounds 1 (IC50 = 2.5 microM), 2 (1.7 microM), and 8 (3.2 microM) showed potent inhibitory activity toward Jurkat cell line. In parallel, compounds 1 (6.7 microM), 2 (1.5 microM), and 10 (5.3 microM) showed the highest activity against U937 cell line. However, the chalcones also inhibit the PHA stimulated PBMCs cells, but the IC50 values were relatively high when compared to the tumor cell line values. Studies were also on the effect of synthesized chalcones on the cell cycle phase distribution. In Jurkat cell line, compounds 7 and 9 showed the highest activity and the most striking effect in reduction of the percentage of cells in the S phase, which was associated with an increase of cells in G2/M phase. In U937 cell line, compound 3 increased the proportion of cells in the G0/G1 phase and reduced the proportion in S phase. In contrast, compounds 1, 9, and 10 showed a decrease effect on the percentage of cells in S phase and an increase effect on the percentage of cells in the G2/M phase of the cell cycle. Whereas in the case of PHA stimulated PBMCs, compounds 1, 4, 8, and 10 increased the percentage of cells in G2/M phase, which was associated with a decrease effect in the S phase of the cell cycle.