Viability Of K562 Cells Research Articles

A novel andrographolide derivative AL‐1 exerts its cytotoxicity on K562 cells through a ROS‐dependent mechanism

Andrographolide-lipoic acid conjugate (AL-1) is a new in-house synthesized chemical entity, which was derived by covalently linking andrographolide with lipoic acid. However, its anti-cancer effect and cytotoxic mechanism remains unknown. In this study, we found that AL-1 could significantly inhibit cell viability of human leukemia K562 cells by inducing G2/M arrest and apoptosis in a dose-dependent manner. Thirty-one AL-1-regulated protein alterations were identified by proteomics analysis. Gene ontology and ingenuity pathway analysis revealed that a cluster of proteins of oxidative redox state and apoptotic cell death-related proteins, such as PRDX2, PRDX3, PRDX6, TXNRD1, and GLRX3, were regulated by AL-1. Functional studies confirmed that AL-1 induced apoptosis of K562 cells through a ROS-dependent mechanism, and anti-oxidant, N-acetyl-L-cysteine, could completely block AL-1-induced cytotoxicity, implicating that ROS generation played a vital role in AL-1 cytotoxicity. Accumulated ROS resulted in oxidative DNA damage and subsequent G2/M arrest and mitochondrial-mediated apoptosis. The current work reveals that a novel andrographolide derivative AL-1 exerts its anticancer cytotoxicity through a ROS-dependent DNA damage and mitochondrial-mediated apoptosis mechanism.

Membrane Damage Effect of Continuous Wave Ultrasound on K562 Human Leukemia Cells

This study investigated the bioeffects of ultrasound with a frequency of 1.1 MHz on human chronic myelogenous leukemia cell line K562. Membrane potential changes were evaluated by flow cytometry using fluorescent probe bis-(1,3-dibarbituric acid)-trimethine oxanol staining. Other related changes such as potassium ion efflux and intracellular calcium ion overload were also measured. The plasma membrane integrity was monitored by flow cytometry combined with fluorescein diacetate and propidium iodide double fluorescent dye staining. A cell-counting assay and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide analysis were used to examine the viability of K562 cells after ultrasound exposure. The acoustic cavitation activity in ultrasound fields was assessed by monitoring hydroxyl radical production. As the ultrasonic intensity increased, the hydroxyl radical produced in the medium increased, and cell membrane damage and cell viability loss were enhanced. The ultrasonic intensity at 0.64 W/cm(2) did not cause substantial cell damage, whereas ultrasound exposure at 1 and 2.1 W/cm(2) could induce serious cell death (14.0% and 40.7%, respectively). Moreover, ultrasound at 0.64 W/cm(2) did not cause substantial membrane potential changes, whereas ultrasound exposure at 1 W/cm(2) could induce depolarization, and fast hyperpolarization occurred when the ultrasonic intensity increased to 2.1 W/cm(2). In addition, compared with control cells, in different ultrasound-treated cells, the potassium ion continuously outflowed with a prolonged incubation time, whereas the intracellular calcium ion oscillations became more apparent. The damaging effects of ultrasound on the cell membrane and cell viability were intensity dependent. The membrane potential changes may be due to acoustic cavitation accompanied by alterations in the balance of ions on opposite sides of the cellular membrane.

Abstract 2015: Lysophosphatidylcholine induces apoptosis via suppressing STAT3 and HDAC3 activities and binding to the Bcl-xL promoter in K562 chronic myelogenous leukemia cells

Abstract Lysophosphatidylcholine (LPC) is a class of chemical compound which are derived from phosphatidylcholines usually isolated from the surface of wheat starch granules. In the present study, we investigated the molecular mechanisms responsible for LPC-induced apoptosis in chronic myeloid leukemia K562 cells. LPC exerted the inhibitory effect on the viability of K562 cells as well as other leukemic cell lines HL-60 and U937. Nonetheless, LPC significantly increased the acetylation of histone H3 at K18, 9 and 23 in K562 cells, but not in HL-60 and U937 cells. Consistently, LPC reduced histone deacetylase (HDAC) transcriptional activity and the expression of HDAC3 at protein level. LPC suppressed phosphorylation of mTOR and AKT, the upstream kinases of HDACs and induced p27 expression. We also found that LPC mediated inactivation of signal transducer and activator of transcription 3 (STAT3)-related signaling pathway in K562 cells, but not HL-60 and U937. LPC significantly inhibited phosphorylation and the DNA binding activity of STAT3, phosphorylation of Src and Janus activated kinase 2 (JAK2) as well as the expression of STAT3 target genes Cyclin D1, Cyclin E, Bcl-xL, Bcl-2, and Survivin at both protein and mRNA levels. LPC stimulated protein tyrosin phosphatases Src homology 2 domain-containing phosphatase 1 (SHP-1), but not SHP-2, SOCS-1 and PTEN. Blocking SHP-1 using sodium pervanadate or SHP-1 specific siRNA reversed LPC-mediated inactivation of STAT3 and HDAC and induction of apoptosis. Of importance, LPC blocked the nuclear translocation and interaction of HDAC3 and STAT3 as well as their binding onto the Bcl-xL promoter. Overall, our findings demonstrate that LPC has a potent anti-cancer activity through inducing apoptosis by suppressing HDAC3/STAT3 activation and binding to the Bcl-xL promoter in K562 cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2015. doi:1538-7445.AM2012-2015

Anti-leukemic effects of gallic acid on human leukemia K562 cells: Downregulation of COX-2, inhibition of BCR/ABL kinase and NF-κB inactivation

Gallic acid (GA) induces apoptosis in various cancer cell lines. In this study, we investigated the apoptotic activity induced by GA on chronic myeloid leukemia (CML) cell line-K562 and the underlying mechanism. GA reduced the viability of K562 cells in a dose and time dependent manner. GA led to G0/G1 phase arrest in K562 cells by promoting p21 and p27 and inhibiting the levels of cyclin D and cyclin E. Further studies indicated apoptosis with impaired mitochondrial function as a result of deranged Bcl-2/Bax ratio, leakage of cytochrome c and PARP cleavage along with DNA fragmentation and by up-regulating the expression of caspase-3. GA also activated the protein expressions of fatty acid synthase ligand and caspase-8. GA is more effective in imatinib resistant-K562 (IR-K562) cells (IC50 4μM) than on K562 cells (IC50 33μM). GA inhibited cyclooxygenase-2 (COX-2) in K562 as well as IR-K562 cells appears to be COX-2 involved in the suppression of growth. Interestingly, GA also inhibited BCR/ABL tyrosine kinase and NF-κB. In conclusion, GA induced apoptosis in K562 cells involves death receptor and mitochondrial-mediated pathways by inhibiting BCR/ABL kinase, NF-κB activity and COX-2.

Schedule-Dependent Cytotoxicity of Etoposide (VP-16) and Cyclophosphamide in Leukemia Cell Line K-562

In allogeneic bone marrow transplantation (allo-BMT) in patients with leukemia, the combination of VP-16 and cyclophosphamide (CY) is commonly used for the conditioning regimen. In the present study, we demonstrated schedule-dependent cytotoxicity of VP-16 and CY in K-562 cells. K-562 cells were pretreated with low concentrations (2.5 and 5 µg/mL) of 4-hydroperoxycyclophosphamide (40487S), which is a preactivated analog of CY. It was confirmed that these concentrations did not influence cell viability. Cells subsequently exposed to 0.5-100 µg/mL of VP-16 showed reduced the viability compared to that of control cells not treated with 40487S. In contrast, there was no change in the viability of K-562 cells pretreated with low concentrations (0.5 and 1 µg/mL) of VP-16. It was confirmed that these concentrations did not influence cell viability. Viability of subsequently exposed to 1-20 µg/mL was not different from that of control cells not treated with VP-16. VP-16 caused cell cycle arrest at G₂/M phase. On the other hand, 40487S arrested the cell cycle at S phase. Thymidine-synchronized cells, VP-16 showed cell cycle specificity for cell killing from early-S to mid-S phase. On the other hand, 40487S showed cell cycle-independent cytotoxicity. Exposure of cells to VP-16 after 40487S induced a greater cytotoxic effect on K-562 cells. The findings may lead to improvements in clinical combination chemotherapy.

Carotenoids inhibit proliferation and regulate expression of peroxisome proliferators-activated receptor gamma (PPARγ) in K562 cancer cells

As one of the main micronutrients in vegetables and fruit carotenoids are almost daily intaken in significant quantity. Although the pharmacological roles of carotenoids in the prevention and reduction of cancer incidence have received more and more attention, the exact molecular mechanisms underlying anticancer effects of carotenoids remain unclear yet. Activated peroxisome proliferator-activated receptor gamma (PPARγ) plays an inhibitory role in cancer cell proliferation and growth. Involvement of PPARγ in the growth inhibition of leukemia K562 cells by carotenoids was investigated in the present study. The results demonstrated that β-carotene, astaxanthin, capsanthin, and bixin inhibited the proliferation and decreased the viability of leukemia K562 cells in dose- and time-dependent manners, induced cell apoptosis, and interfered with cell cycle progression. Pretreatment with GW9662, a potent antagonist of PPARγ, partly attenuated the inhibition of K562 cell proliferation by the four carotenoids at 8 μM. These carotenoids up-regulated the expression of PPARγ and p21 and down-regulated the expression of cyclin D1 in a dose-dependent manner. In addition, β-carotene, astaxanthin, capsanthin and bixin also up-regulated the expression of Nrf2, an important transcription factor in Keap1-Nrf2/EpRE/ARE signaling pathway. It appears to us that PPARγ signaling pathways and Keap1-Nrf2/EpRE/ARE signaling pathway were involved in the inhibition of K562 cell proliferation by carotenoids and the up-regulation of PPARγ expression at least partly contributed to the antiproliferative effects of β-carotene, astaxanthin, capsanthin, and bixin on K562 cells.

Antitumor activity of total paeony glycoside against human chronic myelocytic leukemia K562 cell lines in vitro and in vivo

To explore the molecular mechanisms of human leukemia cells by total paeony glycoside (TPG), which is extracted from the root of Radix Paeoniae Rubra. The viability of K562 cells was assessed by MTT assay. Flow cytometry was used to detect apoptosis and cell cycle analysis. The changes in intracellular Ca(2+) concentration were determined by fluorescent dye Fluo-3, and mitochondrial membrane potential was determined by the retention of the dye Rh123. The cytoplasmic Bax, Bcl-xL, and Bcl-2 protein expressions were determined by western blot. The mRNA expression of caspase-3, caspase-8, and caspase-9 was detected by RT-PCR. K562 cells were subcutaneously inoculated into nude mice to study the in vivo antitumor effects of TPG. The growth of K562 cells was inhibited and arrested in G0/G1 phase by TPG. TPG also caused apoptosis in K562 cells evidenced by cytosolic accumulation of cytochrome c, caspase-9, and caspase-3. TPG could down-regulate Bcl-2 and Bcl-xL and up-regulate Bax in K562 cells. TPG showed a significant decreased tumor volume and tumor weight in nude mice inoculated with K562 cells. TPG can be developed as a promising anti-chronic myeloid leukemia drug.

Effects of As2 O3 in combination with cinobufacini on proliferation and apoptosis of the K562 cells

Objective To investigate the effect of As2 O3 in combination with cinobufacini on proliferation and apoptosis of the K562 cells and provide theoretical basis for clinical application.Methods Cell proliferation was assayed by analyzing the growth and viability of the cells.Apoptosis was assayed by performing cell morphology,Annexin-V/PI staining,DNA-PI staining,and DNA gel electrophoresis.Results After exposure to As2O3 and cinobufacini,the growth of K562 cells was inhibited and the viability of K562 cells was decreased. After treated with 1.0μmol/L As2O3,0.125μg/ml cinobufacini,0.25μg/ml cinobufacini,1.0μmol/L As2O3 + 0.125 μg/ml cinobufacini,1.0μmol/L As2O3 + 0.25μg/ml cinobufacini for 24 and 48 hours,the proliferation inhibition rate were(24 ± 1.3)%,(21 ± 1.5)%,(38 ± 3.1)%,(57 ±2.7)%,(66 ±3.3)% and(49 ±2.9)%,(48 ±2.7)%,(61 ±2.1)%,(77 ±3.8)%,(82 ±4.2)%,the apoptosis rate of K562 cells were(4.8 ± 0.5)%,(5.6 ± 0.7)%,(9.8 ± 0.6)%,(11.9 ± 1.2)%,(15.2±1.5)% and(11.0 ±0.9)%,(12.9 ±1.1)%,(18.4 ±1.5)%,(21.0 ±2.0)%,(28.0 ±1.9)%.The percentage of apoptotic cells was a time- and dose-dependent manner.Typical DNA ladder was shown by DNA gel electrophoresis.Conclusions As2O3 combined with cinobufacini inhibited the proliferation of K562 cells and induced apoptosis of the K562 cells,the combination of the two drugs had better effect. Key words: Arsenicals/AD; CINOBU FAGIN/AD; Leukemia,myeloid,acute/DT; Cell proliferation/DE; Apoptosis/DE

Apoptosis induced by genipin in human leukemia K562 cells: involvement of c-Jun N-terminal kinase in G2/M arrest

To investigate the effect of genipin on apoptosis in human leukemia K562 cells in vitro and elucidate the underlying mechanisms. The effect of genipin on K562 cell viability was measured using trypan blue dye exclusion and cell counting. Morphological changes were detected using phase-contrast microscopy. Apoptosis was analyzed using DNA ladder, propidium iodide (PI)-labeled flow cytometry (FCM) and Hoechst 33258 staining. The influence of genipin on cell cycle distribution was determined using PI staining. Caspase 3 activity was analyzed to detect apoptosis at different time points. Protein levels of phospho-c-Jun, phosphor-c-Jun N-terminal kinase (p-JNK), phosphor-p38, Fas-L, p63, and Bax and the release of cytochrome c were detected using Western blot analysis. Genipin reduced the viability of K562 cells with an IC(50) value of approximately 250 μmol/L. Genipin 200-400 μmol/L induced formation of typical apoptotic bodies and DNA fragmentation. Additionally, genipin 400 μmol/L significantly increased the caspase 3 activity from 8-24 h and arrested the cells in the G₂/M phase. After stimulation with genipin 500 μmol/L, the levels of p-JNK, p-c-Jun, Fas-L, Bax, and cytochrome c were remarkably upregulated, but there were no obvious changes of p-p38. Genipin 200-500 μmol/L significantly upregulated the Fas-L expression and downregulated p63 expression. Dicoumarol 100 μmol/L, a JNK1/2 inhibitor, markedly suppressed the formation of apoptotic bodies and JNK activation induced by genipin 400 μmol/L. These results suggest that genipin inhibits the proliferation of K562 cells and induces apoptosis through the activation of JNK and induction of the Fas ligand.

Additive Effects of Anti-Leukemia Capacity by Gfi-1B- and Bcr-Abl-Specific siRNA In Advanced Chronic Myeloid Leukemic Cells

AbstractAbstract 4466 Background:Growth factor independent-1B (Gfi-1B) is a transcription factor with six C2H2 zinc fingers and one unique N-terminal SNAG (Snail/Gfi-1) transcriptional repressor domain. The Gfi-1B gene was mapped to chromosome 9q34.13 and is so located downstream the bcr-abl translocation in chronic myeloid leukemia (CML) cells. Selective inhibition of the BCR-ABL tyrosine kinase by RNA interference has been demonstrated in leukemic cells. Therefore, we evaluated the specific bcr-abl small inferring RNA (siRNA) and gfi-1B siRNA silencing in K562 cells and in primary CML cells with myeloid blast crises. Method:For in vitro transfection with gfi-1B siRNA, a total of 175 pM gfi-1B siRNA, No. AF 081946 was added to 1×10E5 cells and transfection was performed as previously described by us. Sequences of siRNA directed against the BCR-ABL transcript were published previously by Scherr (Blood 2003) and transfection with 54 pM bcr-abl siRNA were preformed as described for gfi-1B siRNA. As control we used two or more non-silencing siRNA (mismatched or scrambled siRNA) from Qiagen. Result:Using fluorescence marked non-silencing siRNA (Qiagen) we evaluated either the transfection rate in K562 cells and in primary CML cells. At mean (± standard deviation) we found a transfection rate of 69.4 ± 5.7% in K562 cells and 45.8 ± 12.8% in primary CML cells. The bcr-abl siRNA or the combination with both siRNAs revealed the strongest effects in reducing the viability of K562 cells by 28.5 to 5.3 % (mean) compared to controls (controls were set up to 100%). The gfi-1B siRNA reduced the viability of these cells by 49.8 % after the start of transfection. To confirm that cell growth is dependent on the timing of siRNA silencing, we next investigated the time course of the cotransfection of bcr-abl siRNA and gfi-1B siRNA induced effects in K562 cells. A strong reduction of cell growth to 21.9 + 4.5% compared to the rate of spontaneous viable cells in K562 cells was observed after 24 h of siRNA transfection, which was statistically significant (p<0.0001). We further observed a strong inhibition of cell growth to 5.3 + 1.8% (p<0.0001) compared to the cell growth rate after 48 h of cotransfection with these above siRNAs. After 72 h of the cotransfection of bcr-abl siRNA and gfi-1B siRNA no further reduction of cell growth was measured in K562 cells, in fact cell growth increased to 8.6 + 3.2% compared to the spontaneous cell growth rate. The combination of both bcr-abl siRNA and gfi-1B siRNA reduced the bcr-abl mRNA level to 2 fold higher comparing the amount after bcr-abl siRNA alone (p<0.0001). Cotransfection with bcr-abl siRNA and gfi-1B siRNA resulted in an exaggerated decrease of proliferation rate to 9.9 + 1.7% compared to controls. The inhibitory efficiency of bcr-abl siRNA and gfi-1B siRNA has shown a 3-fold increasing result comparing to bcr-abl siRNA alone and a 5-fold increasing effect comparing to gfi-1B siRNA alone. Concordantly, we observed an additive effect on induction of apoptosis by cotransfection of bcr-abl siRNA and gfi-1B siRNA in K562 cells. The rate of induced apoptosis increased from 1 + 0.09 (controls set to 1.0) to 3.3 + 0.2, whereas the use of either bcr-abl siRNA alone or gfi-1B siRNA alone was again less effective. In order to verify the above anti-leukemic results, we assessed bcr-abl siRNA and gfi-1B siRNA transfection in primary cells from patients with blast crisis of CML. We found either a reduction of the bcr-abl mRNA- and of the gfi-1B mRNA amount in these advanced CML patients compared to untreated controls. Furthermore we found in primary CML cells a strongly decreased level of c-myc gene expression after cotransfection with bcr-abl siRNA and gfi-1B siRNA to 65% compared to controls. Conclusion:Our data suggested that silencing by both bcr-abl siRNA and gfi-1B siRNA may be associated with an additive antileukemic activity against K562 cells and in primary CML cells in myeloid blast crises, and might be a suitable target to treated advanced CML. Disclosures:No relevant conflicts of interest to declare.

Quantum Dot Conjugates for Targeted Silencing of Bcr/Abl Gene by RNA Interference in Human Myelogenous Leukemia K562 Cells

Quantum dots (QDs) have been receiving a lot of attention recently for their unique fluorescence properties that can be used in drug discovery and bioimaging applications. We have in this article focused particularly on QDs and used it as a transfection vector as well as a fluorescence label for the RNA interference research. The siRNAs were designed to knock down the bcr/abl oncogene in leukaemia K562 cells. EDAC used as a cross-linker, COOH-functionalized QDs were conjugated with NH2-modified siRNAs to generate QD-siRNA conjugates. We also demonstrated their application to the K562 cells. Using such constructs, the delivery and transfection of siRNAs could be monitored by the presence of fluorescent QDs in the conjugates. QDs not only exhibited superior photostability for labeling cells but also worked as a good vector that remarkably increased the transfection efficiency of siRNAs into the cells. Cell proliferation was examined by the MTT assay and cell apoptosis by FACS. Our data have shown that the QD-siRNA conjugates could efficiently inhibit the viability of K562 cells and induced their apoptosis. In summary, QDs can be considered strong tools for the functional analysis of RNAi.

Indirubin-3-Monoxime Induces Apoptosis and Autophagy in Human Lymphocytic Leukemia Cells and Human Chronic Myelogenous Leukemia Cells.

Abstract 4106 IntroductionIndirubin-3-monoxime (IO) is the active ingredient of Danggui Longhui Wan, a mixture of plants that is used in traditional Chinese medicine. It is a potent inhibitor of cyclin-dependent kinases (CDKs), especially CDK2. In clinical studies demonstrated that indirubin did not cause major side effects in patients with chronic myelogenous leukemia. However, the functional action of indirubin on acute lymphoblastic leukemia (ALL) is still unclear. In the present study, we investigated that cytotoxic effect and mechanism study of IO in human acute lymphocytic leukemia cell line JM1 and human chronic myeloid leukemia cell line K562. We also analyzed the viability influence of IO in normal human granulocytes and lymphocytes. Materials & MethodsAfter the treatment of IO, cell viability of JM1 and K562 cells was determined by WST-1 assay. The influence of IO on cell-cycle distribution was analyzed by FACScan. To identify which cell death types were induced during IO treatment we measured the caspase-3 activity for analysis the induction of apoptosis; evaluated the LDH release in the necrotic analysis and the expression level of LC3-II was determined by Western blotting analysis for autophagic cell death. Furthermore, we also analyzed the toxicity of IO in normal human granulocytes and lymphocytes by the treatment dose of IO with value of IC50. ResultsIO significantly affected the cell viability on JM1 but also on K562 cells in a dose dependent manner. The G2/M phase of cell cycle was arrested and sub-G1 proportion was relative increased. In addition, the finding showed that IO initiated caspase-3-dependent apoptosis in JM1 and K562 cells. The expression of autophagosome-incorporated LC3-II protein was also clearly increased once cells treated with IO. However, the necrotic phenomenon through measuring LDH release from K562 and JM1 cells could not be observed. Excitingly, by the treatment dose of IO with value of IC50, the cell viability of lymphocytes was marginally affected, whereas the cell cytotoxitity of granulocytes was not induced. ConclusionsIO has the ability to induce potent cytotoxic effect on K562 and JM1 cells. The cell death after IO treatment is mainly caused by apoptosis and autophagy, not by necrosis. This effect is also associated with interrupted cell cycle. Importantly, IO has few or little cytotoxic effect on human lymphocytes and granulocytes. The results suggest that IO might be useful for clinical anti- ALL treatment. Disclosures:No relevant conflicts of interest to declare.

Autophagic and apoptotic mechanisms of curcumin-induced death in K562 cells

Curcumin (1), a natural polyphenolic compound, has shown strong antioxidant and anticancer activities. Several molecular mechanisms have been attributed to its inhibitory effects on a wide range of tumor cells. In this study, the response of the chronic myeloid leukemia cell line K562 cells to 1 is investigated. Curcumin inhibited the viability of K562 cells in a dose- and time-dependent manner. Furthermore, curcumin-induced cell death was associated with the formation of the apoptosome complex, the collapse of the mitochondrial membrane potential, and caspase-3 activation. Curcumin treatment also induced Bid cleavage and downregulated the expression of Bcl-2 protein. Surprisingly, even with these molecular features of apoptosis, we showed that 1 stimulated autophagy, which was evidenced by microtubule-associated protein light chain 3 (LC3) immunoreactivty. Curcumin also increased the protein levels of beclin 1 and membrane form LC3 (LC3-II). Autophagy inhibitor bafilomycin A1 and the pan-caspase inhibitor Z-VAD-fmk suppressed curcumin-induced K562 cell death. Overall, these results suggest that curcumin induces autophagic and apoptotic death of K562 cells. These findings suggest that both apoptotic and autophagic mechanisms contribute to the curcumin-induced K562 cell death.

Irradiation stability and cytotoxicity of gold nanoparticles for radiotherapy

Gold nanoparticles are promising as a kind of novel radiosensitizer in radiotherapy. If gold nanoparticles are shown to have good irradiation stability and biocompatibility, they would play an important role in radiotherapy. In this work, we investigated irradiation effects of gold nanoparticles under 2-10 kR gamma irradiation and cytotoxicity of gold nanoparticles with human K562 cells by using Cell Titre-Glo luminescent cell viability assay. The results revealed that gamma irradiation had not induced any obvious instability and size variations in gold nanoparticles. We found that gold nanoparticles showed excellent radiation hardness with an absorbed dose conversation factor of 9.491 rad/R. Meanwhile, the surface plasmon resonance of gold nanoparticles was enhanced obviously after 2-10 kR gamma irradiation. Subsequently, cytotoxicity tests indicated that the extremely high concentration of gold nanoparticles could cause a sharp decrease in K562 cell viability, while the low concentration of gold nanoparticles had no obvious influence on the cell viability. Our results revealed that gold nanoparticles were stable under high-energy ray irradiation and showed concentration-dependent cytotoxicity.

Catechin, green tea component, causes caspase‐independent necrosis‐like cell death in chronic myelogenous leukemia

Management strategies of chronic phase chronic myelogenous leukemia (CML) have been revolutionized due to the discovery of a selective tyrosine kinase inhibitor, imatinib (Gleevec, STI571), which is substantially improving median survival. However, emergence of imatinib-resistance has put up a serious problem that requires novel treatment methods. Catechins, polyphenolic compounds in green tea, are gathering much attention due to their potential antitumor effects. So far (-)-epigallocatechin-3-gallate (EGCG), the most abundant component of catechin, has been shown to cause typical apoptosis in several tumor cell lines in most cases through activation of caspases. In this study, we showed that EGCG predominantly caused necrosis-like cell death via a caspase-independent mechanism in CML cells, K562 and C2F8, whereas imatinib induced the typical apoptotic cell death. Moreover, this caspase-independent cell death partially mediated the release of apoptosis-inducing factor, AIF, and serine protease, HtrA2/Omi, from the mitochondria to cytosol. In addition, EGCG enhanced the imatinib-induced cell death (P < 0.01) resulting in additive cell death in K562 cells and EGCG alone, effectively reduced the viability of imatinib-resistant K562 cells (P < 0.01). Catechin is a possible candidate for an antitumor agent that causes cell death in CML cells via a caspase-independent mechanism.

Heme Oxygenase-1(HO-1): A Promising Novel Target Molecule for Overcoming Imatinib Resistance in Chronic Myeloid Leukemia

Objective : HO-1 is a microsomal enzyme catalyzing the first, rate-limiting step in degradation of heme, HO-1 is a inducible isoform of HO, it can be strongly induced in response to cellular stress and diverse oxidative stimuli, including its substrate heme, Many studies have convincingly shown that HO-1 is a cytoprotective and antiapoptotic enzyme. the objective of this study was to investigate the influence on the K562 cell growth and apoptosis after hemin-induced HO-1 expression, and to investigate the influence on K562 cells and imatinib-resistant CML cells after ZnPPIX-induced HO-1 inhibition.Methods: different concentrations hemin (0umol/l A20umol/l 30umol/l)was used respectively to induce HO-1 expression of cultured chronic myeloid leukemia cell line K562, then detected HO-1 mRNA expression under different time by RT-PCR, and MTT was used to detected the viability of K562 cells. In addition, we used STI571(2 μmol/L) deal with the hemin-induced cells, then confirm HO-1 protective effect against STI571 use MTT. Then ZnPPIX was used to inhibition HO-1 expression of K562 and imatinib-resistant cells, similarly, RT-PCR and MTT was used for analyzed.Results:The HO-1 mRNA was not tested when absence of hemin, 8h after treated with hemin of 20 μmol/L, we can test the HO-1 mRNA expression, and at 16h the expression is reach to the peak, 16h after treated hemin under different concentrations (10umol/l, 20umol/l, 30umol/l), we found the expression is in a dose-dependent manner.In the group of 10 umol/l and 20 umol/l, the survival of cells is significantly increased in comparison to the control and also have significantly difference in the two groups(p<0.05), in the group of 20 μmol/L, 16h to 48h after hemin-induced, the survival of cells presents a time-dependent manner.In the group of 10μmol/L and 20 μmol/L, exposure of K562 cells to STI571 resulted in a substantial decrease of cell viability in comparison to the STI571 single treatment group(p<0.05).ZnPPIX-induced HO-1 inhibition leads to induction of apoptosis in K562 cells, having significant difference with the control group(p<0.05).ZnPPIX-induced HO-1 inhibition can suppress the survival of imatinib-resistant cells(p<0.05).Conclusion: our studies have shown that hemin-induced HO-1 gene expression may promote the proliferation of K562 cells, and can against the cell apoptosis. And we found hemin-induced HO-1 gene expression can protect K562 cells against STI571-induced apoptosis, ZnPPIX-induced HO-1 inhibition leads to decreased viability of imatinb-resistant CML cells. these all indicates HO-1 may represent a novel targeting in CML.

Antileukemia effects of xanthohumol in Bcr/Abl-transformed cells involve nuclear factor-κB and p53 modulation

The oncogenic Bcr-Abl tyrosine kinase activates various signaling pathways including phosphoinositide 3-kinase/Akt and nuclear factor-kappaB that mediate proliferation, transformation, and apoptosis resistance in Bcr-Abl+ myeloid leukemia cells. The hop flavonoid xanthohumol inhibits tumor growth by targeting the nuclear factor-kappaB and Akt pathways and angiogenesis. Here, we show that xanthohumol has in vitro activity against Bcr-Abl+ cells and clinical samples and retained its cytotoxicity when imatinib mesylate-resistant K562 cells were examined. Xanthohumol inhibition of K562 cell viability was associated with induction of apoptosis, increased p21 and p53 expression, and decreased survivin levels. We show that xanthohumol strongly inhibited Bcr-Abl expression at both mRNA and protein levels and show that xanthohumol caused elevation of intracellular reactive oxygen species and that the antioxidant N-acetylcysteine blunted xanthohumol-induced events. Further, we observed that xanthohumol inhibits leukemia cell invasion, metalloprotease production, and adhesion to endothelial cells, potentially preventing in vivo life-threatening complications of leukostasis and tissue infiltration by leukemic cells. As structural mutations and/or gene amplification in Bcr-Abl can circumvent an otherwise potent anticancer drug such as imatinib, targeting Bcr-Abl expression as well as its kinase activity could be a novel additional therapeutic approach for the treatment of Bcr-Abl+ myeloid leukemia.

Antimutagenic, antigenotoxic and antioxidant activities of Acacia salicina extracts (ASE) and modulation of cell gene expression by H2O2 and ASE treatment

The total oligomers flavonoids (TOF), chloroform, petroleum ether and aqueous extracts from Acacia salicina, were investigated for the antioxidative, cytotoxic, antimutagenic and antigenotoxic activities. The viability of K562 cells were affected by all extracts after 48 h exposure. Our results showed that A. salicina extracts have antigenotoxic and/or antimutagenic activities. TOF and chloroform extracts exhibit antioxidant properties, expressed by the capacity of these extracts to inhibit xanthine oxidase activity. To further explore the mechanism of action of A. salicina extracts, we characterized expression profiles of genes involved in antioxidant protection and DNA repair in the human lymphoblastic cell line K562 exposed to H2O2. Transcription of several genes related to the thioredoxin antioxidant system and to the DNA base-excision repair pathway was up-regulated after incubation with chloroform, TOF and petroleum ether extracts. Moreover genes involved in the nucleotide-excision repair pathway and genes coding for catalase and Mn-superoxide-dismutase, two important antioxidant enzymes, were induced after incubation with the chloroform extract. Taken together, these observations provide evidence that the chloroform and TOF extracts of A. salicina leaves contain bioactive compounds that are able to protect cells against the consequences of an oxidative stress.

Anti-Glycophorin C Induces a Loss of Mitochondrial Membrane Potential but Fails To Activate Apoptotic Caspases.

The human erythrocyte transmembrane sialoglycoprotein, glycophorin C (GYPC), plays a functional role in regulating red cell shape and mechanical stability. Antibodies to GYPC cause hemolytic disease of the fetus and newborn (HDFN) that is associated with classical Fcγ receptor-mediated phagocytosis. However, in vitro clonogenic studies with cord blood progenitor cells suggest that anti-GYPC also suppresses erythropoiesis, which is consistent with the observations of severe and early fetal anemia and late onset neonatal anemia [Transfus Med 2005; 15:125–32]. The mechanism of the suppressive effect on erythropoiesis is unknown. The K562 erythroleukemic cell line treated with anti-GYPC is a potential model system to study the suppressive effect of anti-GYPC. The present in vitro studies were designed to confirm the effect of anti-GYPC on K562 cell growth and viability, and to evaluate changes in mitochondrial membrane potential, phosphatidylserine (PS) expression, propidium iodide (PI) binding, and caspase activation. K562 cells fail to grow in the presence of anti-GYPC confirming earlier CFU-E/BFU-E studies [Brit J Haematol 2006; 133:443–4], and increased the exofacial expression of PS/PI over time. This process was caspase-independent as demonstrated by the failure of Z-VAD, a caspase inhibitor, to reverse growth inhibition and PS/PI expression. A loss of mitochondrial membrane potential was demonstrated using JC-1, a cationic dye that is sensitive to potential-dependent accumulation or loss in mitochondria. There was a 50% increase in K562 cell mitochondrial membrane potential disruption after 2 days of culture with anti-GYPC (see figure). Morphological examination of May Grunwalde Giemsa-stained K562 cells treated with anti-GYPC for 2 days showed a decrease in mitotic activity compared to isotype treated cells. By day 4, the anti-GYPC treated cells were showing evidence of plasma membrane damage and cell death resulting from fragmentation and dissolution of the cytoplasm. The addition of hemin, an oxidative form of iron protoporphyrin IX known to induce erythroid differentiation of K562 cells, to anti-GYPC treated cells reversed growth inhibition by 45% but did not prevent the loss of mitochondrial membrane potential. Overall, although caspases appear to be unimportant in anti-GYPC induced cell death, the mitchondria play an important role as the early events leading to antibody-mediated suppression of erythropoiesis. [Display omitted]

Rapid Doxorubicin Efflux from the Nucleus of Drug-Resistant Cancer Cells Following Extracellular Drug Clearance

Following extracellular drug clearance, we analyzed the rate of doxorubicin efflux from the nucleus of three human leukemic cells (K562, Molt4 and CCRF-CEM) and related it to their differential sensitivity to this drug, after a short drug pulse. For many pulse-chase regimes, K562 cell viability was least affected by doxorubicin. In K562 cells, nuclear drug accumulation was greatest, but nuclear drug egress was also greatest. P-glycoprotein over-expression in a doxorubicin-resistant, K562/DOX sub-line did not facilitate doxorubicin efflux from the nucleus. In K562 cells, doxorubicin accumulated in multivesicular bodies (MVBs) through a pH-dependent mechanism. Inhibiting drug sequestration in MVBs did not affect nuclear efflux. The rates of doxorubicin efflux from the nuclei of live and digitonin-permeabilized K562 cells were similar. However, extracting cytoplasmic membranes with Triton X-100 significantly inhibited nuclear drug efflux following extracellular drug clearance. Our results are consistent with drug efflux from the nucleus being primarily mediated by an ATP-independent, passive diffusion mechanism. The effect of membrane extraction suggests that nonspecific drug absorption to cytoplasmic membranes plays a role in facilitating nuclear efflux in K562 cells, perhaps by lowering the concentration of free doxorubicin from a perinuclear diffusion boundary layer.