Viability Of K562 Cells Research Articles

Wasabi Compound 6-(Methylsulfinyl) Hexyl Isothiocyanate Induces Cell Death with Coexisting Mitotic Arrest and Autophagy in Human Chronic Myelogenous Leukemia K562 Cells.

A natural compound from Wasabia japonica, 6-(methylsulfinyl) hexyl isothiocyanate (6-MITC) was investigated for its anti-leukemia activity and mechanism of action. It was found that 6-MITC inhibited the viability of human chronic myelogenous leukemia K562 cells along with extensive mitotic arrest, spindle multipolarity, and cytoplasmic vacuole accumulation. The evidence of autophagy included the validation of autophagosomes with double-layered membranes under transmission electron microscopy, LC3I/II conversion, and the induction of G2/M phase arrest observed with acridine orange staining of treated cells, as well as the elevation of phosphorylated-histone H3 expression at the M phase. With regard to the expression of proteins related to mitosis, the down regulation of p-CHK1, p-CHK2, p-cdc25c, and p-cdc2, as well as the upregulation of cyclin B1, p-cdc20, cdc23, BubR1, Mad2, and p-plk-1 was observed. The knockdown of cdc20 was unable to block the effect of 6-MITC. The differentiation of k562 cells into monocytes, granulocytes, and megakaryocytes was not affected by 6-MITC. The 6-MITC-induced unique mode of cell death through the concurrent induction of mitosis and autophagy may have therapeutic potential. Further studies are required to elucidate the pathways associated with the counteracting occurrence of mitosis and autophagy.

Synergistic induction of apoptosis in a cell model of human leukemia K562 by nitroglycerine and valproic acid.

Nitroglycerin (NG), a nitric oxide donor, and valproic acid (VPA), an inhibitor of histone deacetylases, have impressive effects on numerous cancer cell lines. This study intended to evaluate synergistic effects of NG and VPA on cell viability and apoptosis in K562 cells. K562 cells were cultured in RPMI-1640 supplemented with 10 % heat-inactivated FBS. They were treated with different doses of NG, VPA and cisplatin for 24, 48, and 72 h, and MTT assay was performed to analyze cell viability. Also, Peripheral blood mononuclear cells (PBMC) were cultured in RPMI-1640 media and incubated with NG (200 μM), VAP (100 μM), NG+VPA (150 μM) and cisplatin (8 μM) to evaluate cytotoxicity. IC50 of the drugs, when they were applied separately and in combination, were calculated using the COMPUSYN software. DNA electrophoresis, TUNEL assay, and Hoechst staining were performed to investigate apoptosis induction. RT-PCR was used for the evaluation of apoptotic genes expression. The results of the MTT assay showed that cell viability decreased at all applied doses of NG and VPA. It was noticed that the cytotoxic effects of these drugs were dose- and time-dependent. Based on the COMPUSYN output, the combination of the drugs (VPA and NG) in a certain ratio concentration synergistically decreased cell viability. Cisplatin significantly decreased cell viability of PBMCs and K562 cells. Also, the combination drug had cytotoxic effect and significantly reduced viability of K562 cells compared with PBMCs and control cells. In the target cells treated with this combination, Bax and caspase-3 expression increased but Bcl-2 expression decreased. These results suggest that NG, VPA, and their combination decreased cell viability and induced apoptosis via the intrinsic apoptotic pathway. This study suggests that this combination therapy can be considered for further evaluation as an effective chemotherapeutic strategy for patients with chronic myeloid leukemia.

Rapamycin enhanced the antitumor effects of doxorubicin in myelogenous leukemia K562 cells by downregulating the mTOR/p70S6K pathway.

Chronic myelogenous leukemia (CML) is a common hematological malignancy. Some patients progressing to the blast phase develop chemotherapeutic drug resistance. In the authors' previous study, it was found that the mammalian target of rapamycin (mTOR) pathway was activated in CML and that rapamycin inhibited the proliferation of K562 cells. Targeting the mTOR pathway may be used in combination with chemotherapeutic drugs to enhance their efficacy and overcome multidrug resistance. The aim of the present study was to investigate the effects of rapamycin and doxorubicin on K562 cell proliferation following the combination treatment, and further focus on confirming whether rapamycin enhanced the antitumor effects of doxorubicin by downregulating the mTOR/ribosomal protein S6 kinase (p70S6K) pathway. It was found that rapamycin and doxorubicin significantly decreased the viability of K562 cells. The apoptotic cells were more frequently detected in rapamycin and doxorubicin treatment groups (25.50±1.25%). Both drugs decreased Bcl-2 and increased Bax expression in K562 cells. Rapamycin and doxorubicin also reduced the phosphorylation levels of mTOR and p70S6K. Meanwhile, p70S6K-targeting small interfering (si)RNA and doxorubicin inhibited cell proliferation and regulated key factors of the cell cycle. In addition, the exposure of cells to p70S6K siRNA and doxorubicin significantly increased cell apoptosis, as compared with single treatment. These results suggested that rapamycin could enhance the antitumor effects of doxorubicin on K562 cells by downregulating mTOR/p70S6K signaling. Targeting the mTOR/p70S6K pathway may be a new therapeutic approach for leukemia.

In vitro and computational studies showed that perezone inhibits PARP-1 and induces changes in the redox state of K562 cells

Cancer is one of the leading causes of morbidity and mortality worldwide. This disease is characterized by uncontrolled growth and proliferation of abnormal cells with a high probability to develop metastasis. Recently, it was demonstrated that perezone, a sesquiterpene quinone, is capable to induce cell death in leukemia (K562), prostate (PC-3), colorectal (HCT-15) and lung (SKLU-1) cancer cell lines; however, its mechanism of action is unknown. Therefore, in this study, in vitro and computational studies were performed to determine the mechanism of action of perezone. Firstly, changes in K562 cell viability, as well as changes in the redox status of the cell in response to treatment with several concentrations of perezone were analyzed. The type of cell death induced, and the modification of the cell cycle were determined. In addition, MD simulations and docking studies were performed to investigate the interaction of perezone with seven regulators of the apoptotic process. Finally, the ability of perezone to inhibit PARP-1 was evaluated by in vitro studies. K562 cells treated with perezone exhibited decreased viability and more oxidized status, being this effect concentration-dependent. In addition, the increase of G0/G1 phase of cell cycle and apoptosis were observed. According to the performed computational studies conducted, perezone showed the highest affinity to PARP-1 enzyme being this complex the most stable due to the presence of a small and deep cavity in the active site, which allows perezone to fit deeply by forming hydrogen bonds and hydrophobic interactions, which drive this interaction. The activity of perezone as PARP-1 inhibitor was corroborated with an IC50 = 181.5 μM. The pro-apoptotic action of perezone may be related to PARP-1 inhibition and changes in the redox state of the cell. The obtained results allowed to understand the biological effect of perezone and, consequently, these could be employed to develop novel PARP-1 inhibitors.

BW18, a C-21 steroidal glycoside, exerts an excellent anti-leukemia activity through inducing S phase cell cycle arrest and apoptosis via MAPK pathway in K562 cells.

C-21 steroids displayed the activities of immunosuppressive, anti-inflammatory and anti-virus effects by the reports. However, its antitumor effects and molecular mechanism remain unclear. We previously isolated and identified a C-21 steroidal glycoside (BW18) from the root of Cynanchum atratum Bunge. This study was aimed to assess anti-leukemia activity and its underlying mechanism in K562 cells. MTT assay results showed that BW18 inhibited cell viability and proliferation of K562 cells. We also found that BW18 could induce S phase cell cycle arrest and apoptosis. Furthermore, our results demonstrated that BW18 regulated the expression of apoptosis and cell cycle related proteins. Mechanism investigation revealed that the anti-leukemia activity of BW18 may be mediated through MAPK pathway. These findings indicate that BW18 possesses an excellent anti-leukemia activity via regulating MAPK pathway leading to S phase cell cycle arrest and apoptosis, which suggested BW18 could be as a potential alternative therapeutic agent for CML patients.

Emerging role of EPHX1 in chemoresistance of acute myeloid leukemia by regurlating drug-metabolizing enzymes and apoptotic signaling.

Microsomal epoxide hyrolase 1 (EPHX1) is a critical biotransformation enzyme and participants in both the detoxification and activation of potentially genotoxic epoxides. In this study, we firstly aimed to investigate the role of EPHX1 in the chemoresistance of acute myeloid leukemic cells to aclarubicin (ACM) and mitoxantrone (MIT). EPHX1 mRNA expression and prognosis were measured in acute myeloid leukemia (AML) patients, and the function of EPHX1 in leukemic cell viability and apoptosis induced by ACM and MIT was also measured. Our results found that EPHX1 expression is obviously associated with recurrence rate, overall survival and time of obtaining first complete remission in AML patients. EPHX1 silencing promoted ACM and MIT induced decrease in cell viability and cell apoptosis of HL-60, K562, and THP-1 that was inhibited by EPHX1 overexpression. EPHX1 reduced the susceptibility of leukemic cells to ACM and MIT by regulating drug-metabolizing enzymes (CYP1A1, GSTM1, and GSTT1) and apoptotic signaling (Bax, Bcl-2, Caspase-3, Caspase-9, and PARP1). Moreover, Nrf2 overexpression significantly increased EPHX1 expression and leukemic cell viability and decreased leukemic cell apoptosis. Taken together, we summarized the recent findings about the chemoresistance-promoting role of EPHX1, and the potential of targeting EPHX1 was proposed to counteract drug resistance in leukemia treatment.

The effect of α-tocopherol and dithiothreitol in ameliorating emamectin benzoate cytotoxicity in human K562 cells involving the modulation of ROS accumulation and NF-κB signaling

Emamectin benzoate (EMB) toxicity contributes a potential risk to environment and human health. To investigate the effect of α-tocopherol (VitE) and dithiothreitol (DTT) in ameliorating EMB-induced cytotoxicity in human K562 cells, in vitro cultured human K562 cells were incubated with different concentrations of EMB in supplement with VitE and DTT when the cells were in the logarithmic phase. Next, the cell growth inhibition was evaluated using the MTT assay and cellular morphology observation. Reactive oxygen species (ROS) production was monitored using DCFH-DA probe and NF-κB signaling was determined using Western blotting. The results demonstrated that treatment with EMB (time- and concentration-dependent) showed significantly greater inhibition on K562 cell viability, heavier chromatin condensation and DNA fragmentation, and stronger suppression of NF-κB/p105 and p65/RelA expression of K562 cells than the control group (p < 0.01). The supplementation of VitE or DTT could help protect K562 cells against EMB-induced cytotoxicity by improving cell viability, preventing ROS accumulation and up-regulating NF-κB signaling through their ameliorating effects against oxidative stress induced by EMB. VitE had a stronger synergistic effect in limiting EMB cytotoxicity than DTT. Our findings indicate that VitE and DTT are potent antioxidants for human K562 cells, offering a promising means of ameliorating EMB cytotoxicity.

Effects of Deferoxamine on Leukemia In Vitro and Its Related Mechanism.

BackgroundThis study aimed to investigate the effect of deferoxamine (DFO) on leukemia in vitro, and to explore the underlying molecular mechanism.Material/MethodsK562 leukemia cells were treated with various concentrations of DFO (10, 50, and 100 μmol/l) with or without 10 μmol/l ferric chloride for 12 h. Then, total cellular iron was detected. CCK-8 kit and flow cytometry were used for cell viability and apoptosis detection. In addition, expression of apoptosis-related genes was determined by Western blotting and qRT-PCR, respectively.ResultsThe results suggested that DFO significantly inhibited K562 cell viability and induced cell apoptosis in a dose-dependent manner. We also found that the protein and mRNA levels of Bax, p53, and Fas dose-dependently increased in DFO-treated K562 cells, while the level of Bcl-2 markedly decreased in a dose-dependent manner. Moreover, the findings showed that ferric chloride eliminated these effects on K562 cells caused by DFO treatment.ConclusionsOur results indicate that DFO plays a protective role in leukemia via inhibiting leukemia cell viability and inducing cell apoptosis by the regulation of apoptosis-related genes expression.

The growth of K562 human leukemia cells was inhibited by therapeutic neural stem cells in cellular and xenograft mouse models

To confirm the anti-tumor effect of engineered neural stem cells (NSCs) expressing cytosine deaminase (CD) and interferon-β (IFN-β) with prodrug 5-fluorocytosine (FC), K562 chronic myeloid leukemia (CML) cells were co-cultured with the neural stem cell lines HB1.F3.CD and HB1.F3.CD.IFN-β in 5-FC containing media. A significant decrease in the viability of K562 cells was observed by the treatment of the NSC lines, HB1.F3.CD and HB1.F3.CD.IFN-β, compared with the control. A modified trans-well assay showed that engineered human NSCs significantly migrated toward K562 CML cells more than human normal lung cells. In addition, the important chemoattractant factors involved in the specific migration ability of stem cells were found to be expressed in K562 CML cells. In a xenograft mouse model, NSC treatments via subcutaneous and intravenous injections resulted in significant inhibitions of tumor mass growth and extended survival dates of the mice. Taken together, these results suggest that gene therapy using genetically engineered stem cells expressing CD and IFN-β may be effective for treating CML in these mouse models.

Influence of controlled release of resveratrol from electrospun fibers in combination with siRNA on leukemia cells

In this study, we evaluated the possibility of i) local release of resveratrol from poly(ε-caprolactone) (PCL) and gelatin (GT) electrospun fibers and ii) combining (i) with siRNA designed to downregulate BCR-ABL pathway on K562 cancer cells. Initially, K562 cell culture experiments were performed using various bolus doses of resveratrol in combination with siRNA for 3 days using a factorial design of experiments approach. Resveratrol content was analyzed using HPLC and cell viability was assessed using Annexin V (Non-viable), and Propidium Iodide (PI) (Necrotic) based flow cytometry. Coaxial electrospun fibers with resveratrol were made using 1:1 PCL-GT blends in different configurations: single fibers and coaxial fibers with same polymer blends, or with PCL inner core. Loading efficiency and release profile over five days were analyzed. Based on release profile, K562 cell viability with fibers was analyzed over eight days. Dose dependent cell death was observed with bolus resveratrol and siRNA in the culture. However, resveratrol content depleted significantly when added directly to solution. The combination therapy was additive in solution. SEM analysis showed no phase separation of components and resveratrol loading efficiency varied from 77% to 88% in different configurations; 95% of resveratrol was released by day five. Permeability of resveratrol showed no significant dependency on fiber configuration. After 8 days, non-viable cell percentages with controlled release were similar to that at three-day bolus dose of resveratrol. However, siRNA interacted with the fibers, resulting in reduced effect on cells. Loading resveratrol into electrospun fibers provides a localized delivery at therapeutic level, and increased resveratrol's apoptotic effect. Using single fibers is sufficient for controlled release.

Cryptotanshinone suppresses key onco-proliferative and drug-resistant pathways of chronic myeloid leukemia by targeting STAT5 and STAT3 phosphorylation.

C-Myc and signal transducer and activator of transcription (STAT) family proteins have been proposed to be important downstream genes of BCR-ABL, which characterizes most cases of chronic myeloid leukemia (CML). Here, we report a c-Myc pathway-targeted screening of seven natural anticancer compounds, in which we identified cryptotanshinone as a highly promising agent for CML therapy. Cryptotanshinone depletes c-Myc in CML by repressing the phosphorylation of STAT5. Decreased viability of K562 cells correlated with p-STAT5 suppression. Unexpectedly, imatinib activates rather than inhibits the phosphorylation of STAT3 in K562 cells. We demonstrated that cryptotanshinone, as a dual inhibitor of p-STAT5 and p-STAT3, can effectively block IL-6-mediated STAT3 activation and reverse BCR-ABL kinase-independent drug resistance. Moreover, we showed that the epigenetic rebalance between decreased BCR-ABL/STAT5/c-Myc and enhanced STAT3/multi-drug resistance (MDR) pathways is characteristic of the cancer stem cell-like property of K562/ADR. Simultaneously suppressing these two pathways using cryptotanshinone proves to be critical for the malignant network redress and MDR reversal of K562/ADR. These studies reveal the dual functions of cryptotanshinone that suppress key oncogenic proliferation and drug-resistant pathways in CML cells by targeting p-STAT5 and p-STAT3, providing a new strategy for CML therapy that takes advantage of natural products.

Abstract A021: Bone marrow mesenchymal stem cells tune autophagic circuitry of chronic myeloid leukemia cells in response to imatinib treatment

Abstract Chronic myeloid leukemia (CML) is a stem cell disorder with the characteristic presence of chromosomal translocation resulting in the formation of Bcr-Abl protein tyrosine kinase. Within bone marrow, leukemia stem cells (LSCs) share a niche with mesenchymal stem cells (MSCs). Therefore, there exists a strong signalling traffic between MSC and LSC that may direct therapeutic outcome of the CML patients. Imatinib is commonly used as the first line of therapy for CML. However, therapeutic resistance appears to be a major challenge for treatment of CML. Autophagy is one of the key phenomena that may direct cells towards survival or death. However, current understanding on the regulation of autophagy in CML cells by bone marrow niche cells is limited. Therefore, the present study is focused on the regulation of imatinib-induced autophagy in CML cells by MSCs. Here, we cocultured bone marrow-derived MSCs with chronic myeloid leukemia cells, K562 to understand the signaling axis that controls autophagy and subsequent apoptosis of CML cells. We studied K562 cell viability and proliferation by MTT and CFSE assay. For autophagy determination, we performed Western blot analysis and live cell confocal microscopy with K562 cells transfected with ptfLC3 plasmid (the microtubule-associated protein 1 light chain 3, LC3 protein fused to mRFP and EGFP plasmid). Results revealed that there was a reduction in the viability of K562 cells when cocultured with MSC for 48 h. Further reduction in the viability was observed when cells were exposed to 5 µM imatinib for 48 h. The presence of MSCs caused ~2-fold higher accumulation of the lipidated form of LC3 (LC3BII) in K562 cells as compared to control. In addition, imatinib treatment resulted in ~10-fold higher accumulation of LC3BII in K562 cells. Interestingly, as compared to imatinib-treated K562 cells, ~2-fold lower autophagic flux was observed when imatinib was administered to K562 cells in the presence of MSCs. However, enhanced expression of pro-apoptotic protein Bcl-xl was observed. Involvements of ATG5 and Beclin-1 were also detected. This was also microscopically confirmed in K562 cells using mRFP and GFP tagged plasmid. In conclusion, all these studies suggest that bone marrow MSCs play a pivotal role in regulating imatinib-induced autophagy in CML cells. This might provide an important clue to effectively formulate a therapeutic strategy involving MSCs to eliminate imatinib-resistant CML cells from bone marrow niche. Citation Format: Devdeep Mukherjee, Joyjyoti Das, Prantar Chakrabarti, Tapas Kumar Maiti. Bone marrow mesenchymal stem cells tune autophagic circuitry of chronic myeloid leukemia cells in response to imatinib treatment [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A021.

Absorption Characteristics of Combination Medication of Realgar and Indigo Naturalis: In Vitro Transport across MDCK-MDR1 Cells and In Vivo Pharmacokinetics in Mice after Oral Administration.

Realgar and indigo naturalis are clinically combined to treat varieties of leukemia. Exploring the drug-drug interactions might be beneficial to find active substances and develop new targeted drugs. This study aimed at exploring the change of arsenic concentration in mice and across MDCK-MDR1 cells and the cytotoxicity on K562 cells when realgar and indigo naturalis were combined. In the presence or absence of indigo naturalis, pharmacokinetics and cell-based permeability assays were used to evaluate the change of arsenic concentration, and K562 cell line was applied to evaluate the change of cytotoxicity. The drug concentration-time profiles exhibited that the combination medication group generated higher AUC, thalf, and longer MRT for arsenic, compared with the single administration of realgar. The apparent permeability coefficients (Papp) of bidirectional transport in MDCK-MDR1 cell permeability experiments showed that arsenic permeability obviously went up when indigo naturalis was incubated together. The combination medication significantly decreased the cell viability of K562 cells when both the concentration of realgar and the concentration of indigo naturalis were nontoxic. The pharmacokinetic research, the MDCK-MDR1 based permeability study, and the K562 cytotoxicity study were united together to verify the combination medication of realgar and indigo naturalis enhanced the absorption and the permeability across cells for arsenic and effectively inhibited the proliferation of K562 cell line. The molecular binding of As4S4 and indirubin was analyzed by computational study. It is predicted that the formation of the complex [As4S4…Indirubin] involves noncovalent interaction that changes the concentration of arsenic.

Exosomes from mesenchymal stromal cells enhance imatinib-induced apoptosis in human leukemia cells via activation of caspase signaling pathway

Background aimsImatinib (IM), a tyrosine kinase inhibitor targeting the BCR-ABL oncoprotein, remains a major therapeutic strategy for patients with chronic myelogenous leukemia (CML). However, IM resistance is still a challenge in the treatment of CML. Recently, it was reported that exosomes (Exo) were involved in drug resistance. Therefore, the present study investigated whether Exo secreted by human umbilical cord mesenchymal stromal cells (hUC-MSC-Exo) affected the sensitivity of K562 cells to IM. MethodshUC-MSC-Exo were isolated and identified. K562 cells were then treated or not with IM (1 µmol/L) in combination with hUC-MSC-Exo (50 µg/mL). Cell viability and apoptosis were determined by cell counting kit 8 (CCK-8) and annexin V/propidium iodide (PI) double staining, respectively. Apoptotic proteins, caspase and their cleaved forms were detected by Western blot. ResultsIt was shown that hUC-MSC-Exo alone had no effect on cell viability and apoptosis of K562 cells. However, hUC-MSC-Exo promoted IM-induced cell viability inhibition and apoptosis. Moreover, hUC-MSC-Exo enhanced the increased Bax expression and the decreased Bcl-2 expression that were induced by IM. Compared with IM alone, caspase-9 and caspase-3 were further activated by combination of hUC-MSC-Exo with IM. Finally, the effects of hUC-MSC-Exo on K562 cells could be reversed by pretreatment of K562 cells with caspase inhibitor Z-VAD-FMK (30 µmol/L) DiscussionThese results indicate that hUC-MSC-Exo enhanced the sensitivity of K562 cells to IM via activation of caspase signaling pathway. Therefore, combining IM with hUC-MSC-Exo could be a promising approach to improve the efficacy of CML treatment.

Anticancer effects of Bilberry anthocyanins compared with NutraNanoSphere encapsulated Bilberry anthocyanins.

Rapidly accumulating laboratory and clinical research evidence indicates that anthocyanins exhibit anticancer activity and the evaluation of bilberry anthocyanins as chemo-preventive agents is progressing. It has previously been demonstrated that anthocyanins upregulate tumor suppressor genes, induce apoptosis in cancer cells, repair and protect genomic DNA integrity, which is important in reducing age-associated oxidative stress, and improve neuronal and cognitive brain function. Bilberry anthocyanins have pronounced health effects, even though they have a low bioavailability. To increase the bioavailability, Bilberry was encapsulated in 5.5 nm diameter liposomal micelles, called NutraNanoSpheres (NNS), at a concentration of 2.5 mg/50 µl [25% (w/w) anthocyanins]. These Bilberry NNS were used to study the apoptotic/cytotoxic effects on K562 Human Erythroleukemic cancer cells. Flow cytometric fluorescent quantification of the uptake of propidium iodide in a special cell viability formulation into dead K562 cells was used to determine the effects of Bilberry on the viability of K562 cells. The concentrations of Bilberry that demonstrated the greatest levels of percentage inhibition, relative to the control populations, were biphasic, revealing a 60-70% inhibition between 0.018-1.14 mg/ml (n=6) and 60% inhibition at 4 mg/ml. The lowest percentage inhibition (30%) occurred at 2 mg/ml. The lethal dose 50 was determined to be 0.01-0.04 mg/ml of Bilberry per 105 K562 cells at 72 h of cell culture exposure. At 48 h incubation, the highest percentage of inhibition was only 27%, suggesting involvement of a long-term apoptotic event. These levels, which demonstrated direct cytotoxic effects, were 8-40 times lower than levels required for Bilberry that is not encapsulated. The increase in bioavailability with the Bilberry NNS and its water solubility demonstrated the feasibility of using Bilberry NNS in cancer patient clinical trials.

New Triterpenoids from the Leaves of Alafia barteri

A new oleanane derivative, 3β-acetyloleana-5,12-dien-2,23-diol (1), and a new lupane compound, lup-11,20(29)-dien-3 β,28-diol (2), together with eight known compounds, oleana-5,12-dien-3β-yl acetate (3), lup-5,20(29)-dien-3 β -yl acetate (4), 18-methylethylnonadecan-19-oic-9-enoate (6), octadecane (5), cis-pentadec-7-enoic acid (7), cis-tetradec-7,10-dienoic acid (8), cholest-4-en-3-one (9), and stigmasterol (10), were isolated from the leaves of Alafia barteri. Their structures were elucidated by IR, 1D and 2D NMR, and mass spectroscopy. Compounds 7 and 8 exhibited cytotoxic activities against K562, WRL, and MCF-7 tumor cell lines with IC50 in the range of 8.80 to 37.10 μg/mL, while compounds 2 and 9 suppressed the viability of K562 cells with IC50 of 120.2 and 14.48 μg/mL. Compounds 1, 3, and 4 showed low cytotoxic properties on the tested cell lines.

Adenine causes cell cycle arrest and autophagy of chronic myelogenous leukemia K562 cells via AMP-activated protein kinase signaling.

AMP-activated protein kinase (AMPK) is known as a pivotal regulator of cellular metabolism. Mounting evidences have demonstrated that AMPK activation exerts tumor suppressive activity on leukemia cells. The present study reported that adenine, an AMPK activator, triggers cell cycle arrest and autophagy of human chronic myelogenous leukemia K562 cells consequently suppressing cell viability. The present findings revealed that adenine treatment (4.0-8.0 mM) significantly inhibited the viability of K562 cells to 69.3±2.5% (24 h) and 53.4±2.1% (48 h) of the control. Flow cytometric analysis revealed that there was a significant accumulation in G2/M phase, but not sub-G1 phase K562 cells following exposure to adenine. Additional investigation demonstrated that adenine treatments significantly increased the number of acidic vesicular organelles and the level of autophagosomal microtubule associated protein 1 light chain 3 α (LC3) marker. By contrast, cleavage of caspase-9, caspase-3 and poly-ADP-ribose polymerase was insignificantly affected in K562 cells following adenine treatment. In K562 cells, adenine was able to markedly promote the phosphorylation of AMPKα and suppress the phosphorylation of mammalian target of rapamycin (mTOR), a downstream target of AMPK. In addition, inhibiting AMPK phosphorylation using dorsomorphin restored mTOR phosphorylation, inhibited the accumulation of LC3 and significantly recovered the suppressed cell viability in response to adenine. Taken together, the present results demonstrated that adenine induced G2/M phase arrest and autophagic cell death, consequently suppressing the viability of K562 cells, which may attribute to the AMPK activation triggered by adenine. These findings provide evidence that adenine may be beneficial to chronic myelogenous leukemia therapy by suppressing excessive cell proliferation.

Gold Nanoparticles for BCR-ABL1 Gene Silencing: Improving Tyrosine Kinase Inhibitor Efficacy in Chronic Myeloid Leukemia.

Introduction of tyrosine kinase inhibitors for chronic myeloid leukemia treatment is associated with a 63% probability of maintaining a complete cytogenetic response, meaning that over 30% patients require an alternative methodology to overcome resistance, tolerance, or side effects. Considering the potential of nanotechnology in cancer treatment and the benefits of a combined therapy with imatinib, a nanoconjugate was designed to achieve BCR-ABL1 gene silencing. Gold nanoparticles were functionalized with a single-stranded DNA oligonucleotide that selectively targets the e14a2 BCR-ABL1 transcript expressed by K562 cells. This gold (Au)-nanoconjugate showed great efficacy in gene silencing that induced a significant increase in cell death. Variation of BCL-2 and BAX protein expression, an increase of caspase-3 activity, and apoptotic bodies in cells treated with the nanoconjugate demonstrate its aptitude for inducing apoptosis on K562 BCR-ABL1-expressing cells. Moreover, the combination of the silencing Au-nanoconjugate with imatinib prompted a decrease of imatinib IC50. This Au-nanoconjugate was also capable of inducing the loss of viability of imatinib-resistant K562 cells. This strategy shows that combination of Au-nanoconjugate and imatinib make K562 cells more vulnerable to chemotherapy and that the Au-nanoconjugate alone may overcome imatinib-resistance mechanisms, thus providing an effective treatment for chronic myeloid leukemia patients who exhibit drug tolerance.

Abstract 180: Chronic Myelogenous Leukemia Cell Viability is Differentially Regulated by LXR Activation, HDL-associated Proteins, and LDL

LXR activation and HDL exert anti-cancer effects, albeit to varying degrees depending on cancer type and model. One of the mechanisms by which LXR and HDL impair cancer cell growth is through mediation of cellular cholesterol and phospholipid efflux, thereby limiting the availability of essential lipids for rapidly proliferating cells. Thus, the variability in LXR- and HDL-mediated anti-cancer effects may in part be due to differences in 1) HDL functionality and 2) the ability to induce expression of LXR target genes involved in lipid efflux and homeostasis due to adaptive shifts in cancer cell metabolism. Interestingly, serum amyloid A (SAA), a pro-inflammatory acute phase protein shown to be elevated in cancer, infection, and obesity, has recently been shown to differentially increase and decrease cholesterol efflux, despite its ability to displace apoA1 on HDL. Therefore, we investigated the effects of LXR activation, lipoproteins (HDL and LDL) and HDL-associated proteins (apoA1 and SAA) on the viability of chronic myelogenous leukemia cells - a cancer model previously unstudied in regards to cholesterol metabolism. Treatment of cells with the LXR agonist TO901317 (1μM, 5μM, and 10μM) significantly increased ABCA1 mRNA expression at 24hr and 72hr, in addition to dose-dependently decreasing HMG-CoA reductase and anti-apoptotic Bcl-xL mRNA expression. Changes in TO901317-induced gene expression corresponded to time- (24hr, 48hr, 72hr) and dose-dependent decreases in cell viability and average cell size. Interestingly, the addition of HDL to cell cultures with or without TO901317 treatment did not affect K562 cell viability, whereas LDL dose-dependently increased cell viability. Conversely, human-derived apoA1 (25 μg/mL) or HDL + apoA1 drastically reduced TO901317-treated K562 cell viability after 48hr and 72hr. K562 cell viability was modestly decreased by SAA (10 μg/mL) treatment, with greater decreases observed in cells treated with SAA + HDL. These findings suggest that LXR activation, HDL-associated proteins, and LDL differentially regulate chronic myelogenous leukemia cell viability.

HDL‐Associated Proteins and LDL Differentially Modulate Chronic Myelogenous Leukemia Cell Viability

HDL has been shown to exert anti‐cancer effects. One of the mechanisms by which HDL impairs cancer cell growth is through mediation of cellular cholesterol and phospholipid efflux, thereby limiting the availability of essential lipids for rapidly proliferating cells. Cholesterol efflux is facilitated through interactions between cellular ATP‐binding cassette transporter A1 (ABCA1) and apolipopotein A1 (apoA1), in addition to efflux via ABCG1, scavenger receptor class B type 1 (SR‐BI), and passive diffusion to lipidated HDL. Efflux can further be regulated serum amyloid A (SAA), a pro‐inflammatory acute phase protein shown to be elevated in cancer, infection, and obesity. Despite the ability of SAA to displace apoA1 on HDL, studies have reported SAA to both increase and decrease cholesterol efflux. In this study, we investigated the effects of lipoproteins (HDL and LDL) and HDL‐associated proteins (apoA1 and SAA) on chronic myelogenous leukemia cell viability. K562 cells were cultured in the presence of human HDL or LDL (10, 25, or 50 ug/mL) for 24 hours, then assessed for viability. While no changes in K562 cell viability were observed with HDL treatment, LDL dose‐dependently increased cell viability. We further examined the effects of lipoproteins on K562 viability in the presence of the liver X receptor (LXR) agonist TO901317 (5 uM), which is known to induce expression of HDL‐related transporters. Accordingly, we observed significantly increased expression of ABCA1 at 24hr and 72hr following TO901317 treatment. Interestingly, the addition of HDL to TO901317‐treated cells did not affect K562 cell viability, whereas LDL dose‐dependently increased viability. Conversely, treatment of cells with human‐derived apoA1 or HDL + apoA1 drastically reduced K562 cell viability after 48hr and 72hr. K562 cell viability was modestly decreased by SAA treatment, with greater decreases observed in cells treated with SAA + HDL. These findings suggest that HDL‐associated proteins and LDL differentially regulate chronic myelogenous leukemia cell viabilitySupport or Funding InformationThe Science Institute of the College of Arts and Sciences and Faculty Research Committee, Fairfield University