Cellular glutathione as a protective agent against 4-hydroperoxycyclophosphamide cytotoxicity in K-562 cells

Exposure of cells of the K-562 erythroleukemia cell line to 4-hydroperoxycyclophosphamide (4-HC), an analog of activated cyclophosphamide, causes a concentration-dependent inhibition of in vitro colony formation by these cells. For investigation of the role of glutathione (GSH) in the metabolism of 4-HC, GSH levels of K-562 cells were modulated by exposing the cells to buthionine sulfoximine (BSO), a specific inhibitor of GSH synthesis, and/or to GSH ethyl esters. Both the mono- and diethyl esters of GSH were synthesized in our laboratories and their identities were determined by chromatographic methods and fast-atom-bombardment mass spectrometry. An HPLC method including electrochemical detection used for thiol determination was applied for the measurement of GSH esters. Incubation of the cells with BSO depleted GSH levels to approximately 11% of control values and potentiated the cytotoxicity of 4-HC. By contrast, exposure to GSH esters approximately doubled GSH levels and protected the cells against the toxicity of 4-HC. Moreover, when cellular GSH levels were first depleted by BSO exposure and then replenished by incubation with GSH esters, the BSO-associated potentiation of 4-HC cytotoxicity was abolished. The work described herein extends the application of an HPLC method used for thiol determination to the measurement of GSH ethyl esters. In addition, it established that GSH acts as a competitive protecting agent against the in vitro toxicity of 4-HC in the K-562 cell line.     

Glutathione depletion as a determinant of sensitivity of human leukemia cells to cyclophosphamide

The role of glutathione (GSH) as a determinant of cellular sensitivity to the cytotoxic and DNA-damaging effects of cyclophosphamide (CP) was studied in a dual culture system of rat hepatocytes and K562 human chronic myeloid leukemia cells, which have elevated aldehyde dehydrogenase activity with a corresponding insensitivity to activated CP. Exposure of K562 cells to 50 microM DL-buthionine-S,R-sulfoximine for 24 h resulted in a depletion of cellular GSH content to 10% of control values without toxicity. Subsequent 1-h exposure of GSH-depleted cells to activated cyclophosphamide, obtained by incubation of CP with suspension cultures of rat hepatocytes, resulted in a 5-fold potentiation of the cytotoxicity of CP. Alkaline elution analysis of cellular DNA demonstrated that the level of apparent interstrand cross-linking was 3 to 4 times higher in GSH-depleted cells than in nondepleted cells. GSH-depleted cells were, in addition, more sensitive to induction of DNA single strand breaks than nondepleted cells. Depletion of GSH content did not increase cellular sensitivity to the cytotoxicity of phosphoramide mustard. Preincubation of K562 cells with 1 mM cysteine for 4 h resulted in an approximately 60% increase in cellular GSH content, which was accompanied by decreased sensitivity to the cytotoxicity of hepatocyte-activated CP. Exposure of nondepleted cells to clinically relevant concentrations of hepatocyte-activated CP resulted in depletion of cellular GSH content. Replenishment of GSH content in these cells was relatively slow following CP exposure. Acrolein was highly effective at depleting cellular GSH content, whereas phosphoramide mustard had no effect on cellular GSH content. The depletion of GSH by intracellularly released acrolein may be important in the mechanism of cytotoxicity of CP.     

Prediction of tumour sensitivity to 4-hydroperoxycyclophosphamide by a glutathione-targeted assay

In an attempt to develop an assay to predict patient tumour response to cyclophosphamide (CP), the feasibility of using a glutathione-targeted assay to assess the in vitro chemosensitivity of tumour cells to 4-hydroperoxycyclophosphamide (4-OOH-CP), an activated congener of CP, was evaluated. A panel of 19 human and three murine tumour cell lines was used. These consisted of three main categories of tumour types, viz. ovarian, lung and squamous cell carcinoma. The major finding was that the occurrence of a significant reduction of tumour cell reproductive capacity was always accompanied by substantial depletion of cellular glutathione (GSH) content, and vice versa. Plots of % GSH depletion versus clonogenic cell survival demonstrated highly significant correlation (r = 0.90-0.91; P less than 0.01). It was determined that for in vitro tumour cell lines, a GSH depletion to 40% of initial content may serve as a cut-off criterion for chemosensitivity to 4-OOH-CP. This degree of GSH depletion is indicative of clonogenic cell survival of approximately 1% (95% confidence limits = 3 x 10(-5)-1.6 x 10(-2)). The relationship between steady state GSH content and intrinsic sensitivity to 4-OOH-CP was also evaluated. The GSH concentration of the tumour cell lines ranged from 1.3-21.2 x 10(-18) moles microns-3; chemosensitivity to 4-OOH-CP, in terms of IC99, was in the range of 5.0-87.1 microM. A good correlation was observed between these two parameters (r = 0.85, P less than 0.02). These results suggest that GSH plays an important role in determining the therapeutic efficacy of 4-OOH-CP in the treatment of cancer. It is uncertain, however, whether a high tumour steady state GSH content in itself is sufficient to cause therapeutic failure in patients.     

Enhancement by hemin of the sensitivity of K562 human leukemic cells to 1-beta-D-arabinofuranosylcytosine

The sensitivity of human leukemia K562 cells to cancer chemotherapeutic drugs during induction of erythroid differentiation of the cells by hemin was examined. Treatment with hemin greatly increased the sensitivity of the cells to 1-beta-D-arabinofuranosylcytosine (ara-C) but did not affect their sensitivities to other chemotherapeutic drugs, including Adriamycin, daunomycin, hydroxyurea, methotrexate, and vincristine. Thymidine and deoxyguanosine, which are known to potentiate the antileukemic effects of ara-C in K562 cells, also induced erythroid differentiation of K562 cells, but other inducers, such as sodium butyrate and delta-aminolevulinic acid, did not increase the sensitivity of K562 cells to ara-C. Hemin did not enhance the sensitivity to ara-C of other leukemia cell lines (Friend erythroleukemic cells, myeloid leukemic M1 cells, and promyelocytic leukemia HL-60 cells). These results indicate that some inducers of erythroid differentiation of K562 cells potentiate the antileukemic effect of ara-C on K562 cells.     

Noncytotoxic alkyl-lysophospholipid treatment increases sensitivity of leukemic K562 cells to lysis by natural killer (NK) cells

Alkyl-lysophospholipids (ALP) are a group of anti-cancer compounds that have previously been shown to have the unique feature of being selectively toxic to neoplastic tissues. Because alkyl-lysophospholipids target the cell membrane as their site of action, our aim was to analyse the immunological effects of a nonlethal ALP treatment on leukemic K562 cells. In this in vitro study we used ET-18-OCH₃, one of the most potent ALP derivatives, at different concentrations ranging from 25 up to 100 μg/ml. By measurement of cell viability and of apoptosis, we determined a concentration of 25 μg/ml ET-18-OCH₃ and an incubation period of 2 hr as nonlethal for K562 cells; higher concentrations markedly reduced cell viability and led to induction of apoptosis. Similar to the effects induced by nonlethal heat shock, a nontoxic ET-18-OCH₃ treatment led to a significant increase in the sensitivity of K562 cells to lysis by interleukin-2 (IL-2) stimulated natural killer (NK) cells. With respect to these results, we investigated the influence of nonlethal ALP treatment on the cell surface expression patterns and compared it to the results obtained with nonlethal heat shock. ALP treatment does not induce major histocompatibility complex (MHC) expression; however, a significant increase in the cell surface expression of HSP72 was shown by immunoblot analysis of membrane lysates of either untreated or ET-18-OCH₃ treated K562 cells. The increased sensitivity of ET-18-OCH₃ treated K562 cells to lysis by NK cells could be correlated with the elevated cell surface expression of HSP72. © 1996 Wiley-Liss, Inc.     

Inhibition of P-glycoprotein by orange juice components, polymethoxyflavones in adriamycin-resistant human myelogenous leukemia (K562/ADM) cells

We investigated the effects of the ethyl acetate extract of grapefruit juice (GFJ), that of orange juice (OJ) and their components on the uptake of [(3)H]vincristine into adriamycin-resistant human myelogenous leukemia cells. Its uptake was increased by the extracts of GFJ and OJ up to 7- and 3-fold, respectively, as well as verapamil and cyclosporin A. OJ components, i.e. 3,3',4',5,6,7,8-heptamethoxyflavone, nobiletin and tangeretin, also increased the uptake of [(3)H]vincristine in a concentration-dependent manner. Although GFJ components, dihydroxybergamottin and bergamottin, significantly increased the uptake, their potencies were considerably weaker than those of OJ components. These data suggest that OJ components inhibit P-gp-mediated efflux of [(3)H]vincristine, resulting in the intracellular accumulation of chemotherapeutic drugs. These components may become candidates of multi-drug resistance reversing agents in cancer chemotherapy.     

Inhibition of P-glycoprotein by flavonoid derivatives in adriamycin-resistant human myelogenous leukemia (K562/ADM) cells.

We investigated the effects of natural flavones, quercetin and morin, and their pentamethyl, pentaethyl, pentapropyl, pentabutyl and pentaallyl ethers, on the function of P-glycoprotein (P-gp) assessed by an increase in the uptake of [3H]vincristine by human myelogenous leukemia (K562) cells and adriamycin-resistant human myelogenous leukemia (K562/ADM) cells. Pentamethyl, pentaethyl, pentapropyl and pentaallyl ethers of morin and quercetin (20 microM) all increased the uptake of [3H]vincristine by K562/ADM cells, while quercetin, morin and their pentabutyl ethers had no effect. Pentamethylquercetin, pentaallylquercetin and pentaethylmorin remarkably increased the uptake of [3H]vincristine by K562/ADM cells by 10.6, 10.8 and 14.4-fold, respectively. These inhibitory potencies for P-gp were more potent than typical P-gp inhibitors, cyclosporine A and verapamil. Taking into consideration that these flavonoid derivatives possess antitumor promoter activity, they may become candidates of effective multidrug resistance-reversing agents in cancer chemotherapy.     

stathmin基因的RNA干涉抑制人白血病K562细胞体外增殖的作用

目的：研究RNA干涉（RNA interference，RNAi）抑制stathmin基因表达后对人白血病K562细胞体外增殖的作用。方法：将合成的寡核苷酸链退火形成双链，连接入经BamHI和HindIII双酶切后的pSilencer4．1-CMV neo真核表达载体。酶切及测序鉴定。脂质体法转染重组质粒入K562细胞，RT—PCR检测其对mRNA的干涉效果；MTT比色法检测K562细胞体外增殖能力。结果：经酶切及测序鉴定，成功构建重组质粒。RT—PCR显示所构建的干涉载体成功地抑制了目的基因的转录，同时细胞增殖活性降低。结论：RNA干涉成功抑制stathmin基因表达并使人白血病K562细胞体外增殖活性明显降低。     

Induction of apoptosis in K562 human leukemia cells by 2',4'-dihydroxy-6'-methoxy-3',5'-dimethylchalcone

2',4'-Dihydroxy-6'-methoxy-3',5'-dimethylchalcone (DMC), isolated from the buds of Cleistocalyx operculatus, was investigated in its cytotoxicity and anti-proliferation on K562 cell line. Our results revealed that the IC50 was equal to 14.2+/-0.45 microM and the EC50 was 3.3+/-0.14 microM. Staining with Hoechst 33258 showed fragmentation and condensation of chromatin in the cells treated with 8 microM DMC for 48 h. Flow cytometric analysis was performed to determine hypodiploid cells. The results of flow cytometry assay indicated that the percentage of hypodiploid K562 cells was 76.15+/-3.22% after 48 h treatment with 16.0 microM DMC. The treatment resulted in the appearance of a hypodiploid peak (A0 region), probably due to the presence of apoptosing cells and/or apoptotic bodies with DNA content less than 2n. Western blot results illustrated that in the same dosage and incubation time, DMC could down-regulate the level of Bcl-2 protein and did not influence the expression of Bax protein. The resulting net effect could thus lead to a lower ratio of Bcl-2/Bax, which might be responsible for the DMC-induced apoptosis in K562 cells.     

stathmin基因的RNA干涉抑制人白血病K562细胞体外增殖的作用

目的:研究RNA 干涉(RNA interference,RNAi)抑制stathmin基因表达后对人白血病K562细胞体外增殖的作用.方法:将合成的寡核苷酸链退火形成双链,连接入经BamHI和HindⅢ双酶切后的pSilencer4.1-CMV neo真核表达载体.酶切及测序鉴定.脂质体法转染重组质粒入K562细胞,RT-PCR检测其对mR-NA的干涉效果;MTT 比色法检测K562细胞体外增殖能力.结果:经酶切及测序鉴定,成功构建重组质粒.RT-PCR显示所构建的干涉载体成功地抑制了目的基因的转录,同时细胞增殖活性降低.结论:RNA干涉成功抑制stathmin基因表达并使人白血病K562细胞体外增殖活性明显降低.     

刺梨汁对人白血病K562细胞生长的抑制作用

目的　通过观察刺梨汁对人白血病K5 62细胞生长的影响 ,探讨刺梨汁的抗癌作用。方法　以细胞计数和MTT法测定刺梨汁对细胞增殖的抑制作用 ,以测定细胞的乳酸脱氢酶释放量作为刺梨汁对细胞的毒性作用 ,并以K 5 62细胞形态的改变作为观察指标。结果　每mL培养液含 1μL刺梨汁即对肿瘤细胞增殖有明显的抑制作用 ,每mL培养液含 2 μL刺梨汁抑制作用达到最大 ,为 83 .4% ,再加大剂量抑制作用无明显升高 ,各种剂量的刺梨汁对细胞均无毒性 ,刺梨汁的加入使得细胞体积明显变小 ,细胞核形态发生改变 ,异染色质增多。结论　刺梨汁具有一定的抗癌作用。     

Interleukin-12 enhances the sensitivity of human osteosarcoma cells to 4-hydroperoxycyclophosphamide by a mechanism involving the Fas/Fas-ligand pathway.

Cyclophosphamide (CY) and its derivative ifosfamide are alkylating agents used to treat osteosarcoma (OS). The purpose of these studies was to determine whether alkylating agents affect the expression of Fas ligand (FasL) and whether interleukin 12 enhances the sensitivity of human OS cells to alkylating agents. 4-Hydroperoxycyclophosphamide (4-HC), the preactivated CY compound, and 4-hydroperoxydidechlorocloclophosphamide (4-HDC), its nonalkylating analogue, human OS LM6 cells, and a clone of cells derived by transfection with the interleukin 12 gene (LM6-#6) were used for these studies. Incubation of LM6 and LM6-#6 with 10 micro M 4-HC increased the expression of FasL mRNA (2.5- and 3.0-fold, respectively). By contrast, 4-HDC, Adriamycin (ADR), cisplatin (CDP), and methotrexate (MTX) had no effect on FasL mRNA expression. Increased FasL expression after treatment with 4-HC was also demonstrated by immunohistochemistry and flow cytometry. Drug-induced FasL was functional and mediated cell death. We examined the effect of FasL up-regulation by 4-HC on LM6 and LM6-#6 cells. Flow cytometry showed that LM6-#6 cells expressed 2.2-fold more Fas than LM6 cells. Cytotoxicity of 4-HC, 4-HDC, ADR, CDP, and MTX on LM6, LM6-neo, and LM6-#6 were quantified. Colony-forming assay revealed an IC(50) of 2.10 micro M for 4-HC in LM6-neo cells compared with 0.41 micro M in LM6-#6 cells. The IC(50) for 4-HDC, ADR, CDP, and MTX were not significantly different between the two cell lines. We concluded that the increased expression of Fas enhanced LM6-#6 sensitivity to 4-HC. These data indicate that Fas/FasL may be involved in the cytotoxic pathway of CY. Combining biological agents with chemotherapeutic agents that have complementary Fas/FasL pathway actions may offer new therapeutic alternatives.     

Reversal of the resistance to STI571 in human chronic myelogenous leukemia K562 cells

STI571, an Abl-specific tyrosine kinase inhibitor, selectively kills Bcr-Abl-containing cells in vitro and in vivo . However, some chronic myelogenous leukemia (CML) cell lines are resistant to STI571. We evaluated whether STI571 interacts with P-glycopro-tein (P-gp) and multidrug resistance protein 1 (MRP1), and examined the effect of agents that reverse multidrug resistance (MDR) on the resistance to SI571 in MDR cells. STI571 inhibited the [¹²⁵l]azidoagosterol A-photolabeling of P-gp, but not that of MRP1. K562/MDR cells that overexpress P-gp were 3.67 times more resistant to STI571 than the parental Philadelphia-chromosome-positive (Ph+) CML K562 cells, and this resistance was most effectively reversed by cepharanthine among the tested reversing agents. The concentration of STI571 required to completely inhibit tyrosine phosphorylation in K562/MDR cells was about 3 times higher than that in K562 cells, and cepharanthine abolished the difference. In KB-G2 cells that overexpress P-gp, but not Bcr-Abl, 2.5 μM STI571 partly reversed the resistance to vincristine (VCR), paclitaxel, etoposide (VP-16) and actinomycin D (ACD) but not to Adriamycin (ADM) or colchicine. STI571 increased the accumulation of VCR, but not that of ADM in KB-G2 cells. STI571 did not reverse resistance to any agent in KB/MRP cells that overexpress MRP1. These findings suggest that STI571 is a substrate for P-gp, but is less efficiently transported by P-gp than VCR, and STI571 is not a substrate for MRP1. Among the tested reversing agents that interact with P-gp, cepharanthine was the most effective agent for the reversal of the resistance to STI571 in K562/ MDR cells. Furthermore, STI571 itself was a potent reversing agent for MDR in P-gp-expressing KB-G2 cells.     

Synergistic effect of 4-hydroperoxycyclophosphamide and etoposide on a human promyelocytic leukemia cell line (HL-60) demonstrated by computer analysis

Autologous stem cell transplantation using cryopreserved bone marrow offers the opportunity to rescue patients from hematopoietic toxicity caused by intensive chemotherapy. This approach is potentially useful for high-risk leukemias as well as for other cancers. The development of suitable methods for purging malignant cells from the bone marrow will offer a better chance of success for autologous stem cell transplantation. In this paper, we describe our efforts at purging myeloid cells. HL-60, a promyelocytic leukemia cell line, was used as a model. 4-Hydroperoxycyclophosphamide (4-HC) and VP-16-213 (VP-16) (either alone or in combination) were used to treat HL-60 cells and normal bone marrow. The cytotoxic effect of 4-HC (29.2 micrograms/ml; 100 microM) upon the HL-60 cell line was 99.8 +/- 0.12% (SE), and the colony-forming units-granulocyte, macrophage (CFU-cs) of normal bone marrow was inhibited by 82.5%. VP-16, at a concentration of 25 micrograms/ml (42.5 microM), can kill 99% of HL-60 cells and inhibit 72.7% of the CFU-cs. A drug mixture containing 4-HC (29.2 micrograms/ml) and VP-16 (10 micrograms/ml) (combination ratio, 1:0.342) reduces HL-60 cells to an undetectable number, and the CFU-cs were inhibited by 87.2%. The laboratory data were further analyzed for the synergistic effect of these two drugs by quantitative determination of the median effect plot and the multiple drug equation recently described by Chou and Talalay (Adv. Enz. Regul., 22: 27-55, 1984). Interactions of two drugs at different effect levels and at different combination ratios were then determined by computer simulation. At high effect levels, 4-HC and VP-16 in combination gave a synergistic cytocidal effect on HL-60 leukemic cells and gave an antagonistic inhibitory effect on normal bone marrow CFU-cs. This combination greatly increases the safety margin. Computer simulation of a dose effect relationship has also shown that the 4-HC:VP-16 combination ratio of 1:0.342 yields a better selective effect than a ratio of 1:0.856. This quantitative analysis suggests that the combination of these two drugs at the selected dose level offers a good method for purging nonlymphoblastic leukemia cells.     

Hemoglobin induction by Ara-C in human erythroleukemic cells (K562) is cell-cycle dependent

This study was undertaken to answer the question of whether chemical induction of hemoglobin in the human erythroleukemic line K562 is cell-cycle dependent. Cells were synchronized with respect to the cell cycle by manipulation of culture conditions. S phase was detected microscopically by immunofluorescent staining. Hemin and arabinofuranosylcytosine (Ara-C) were used to induce hemoglobin synthesis in K562 cells. The action of hemin causes induction without specificity in regard to cell cycle phase, while Ara-C is dependent upon cell cycle phase, with cells within late G1 and early S phases being sensitive to the effect of the agent. These studies also confirm the results that the action of hemin is reversible but the induction caused by Ara-C is irreversible. The synergistic effects of these two inducing agents resulted in a sharper rise in the percentage of induced cells.     

Induction of gp130 and LIF by differentiation inducers in human myeloid leukemia K562 cells

It has been previously shown that phorbol 12-myristate 13-acetate (PMA), a potent differentiation inducer, induced the expression of both interleukin-6 (IL-6) and IL-6 receptor alpha component (IL-6Ralpha) in K562 leukemia cells. In the present study, we examined the ability of several differentiation inducers to regulate the expression of the signal-transducing receptor component for IL-6, gp130, and cytokine leukemia inhibitory factor (LIF) in K562 cells. We found that the expression of gp130 was dramatically induced at both the mRNA and protein levels by the two megakaryocytic inducers sodium butyrate (NaBut) and PMA. In contrast, the mRNA expression of LIF was induced by the two erythroid inducers 1-beta-D-arabinofuranosyl cytosine (Ara-C) and hemin. Furthermore, activation of the PMA-induced gp130 receptor by exogenous IL-6 potentiated the differentiating effects of PMA. Our findings suggest that IL-6/gp130 signaling may be involved in the regulation of the megakaryocytic differentiation of K562 cells.     

Cellular glutathione as a determinant of the sensitivity of colorectal tumour cell-lines to ZD2767 antibody-directed enzyme prodrug therapy (ADEPT)

ZD2767P, a nitrogen mustard glutamate prodrug, is currently being evaluated in Phase 1 clinical trials of antibody directed enzyme prodrug therapy (ADEPT). There was no significant relationship between basal glutathione (GSH) concentration and sensitivity to ZD2767P + carboxpeptidase G2 (CPG2) in colorectal tumour cell-lines. Depletion of intracellular GSH using buthionine sulfoximine (BSO) resulted in only a modest potentiation of ZD2767P + CPG2 activity and hence BSO is unlikely to markedly enhance the activity of this ADEPT treatment.     

Antitumor effect of methylglyoxal bis(butylamidinohydrazone), a new inhibitor of S-adenosylmethionine decarboxylase, against human erythroid leukemia K 562 cells

Methylglyoxal bis(butylamidinohydrazone) (MGBB) inhibited S-adenosylmethionine decarboxylase (SAMDC) activity competitively with S-adenosylmethionine (SAM) showing the Ki value of 1.8 X 10(-5) M. MGBB showed less SAMDC-stabilizing effect in rat liver in vivo than did methylglyoxal bis-(guanylhydrazone) (MGBG). MGBB inhibited the growth of human erythroid leukemia K 562 cells. Putrescine, spermidine and spermine concentrations in MGBB-treated cells were depressed to 56%, 58% and 88% of the values of control cells, respectively. [35S]Methionine incorporation into trichloroacetic acid-insoluble fraction was decreased in the inhibitor-treated cells.