Preferential killing of glucose-depleted HeLa cells by menadione and hyperthermia.

Energy deprivation of cancer cells increases sensitivity to killing by hyperthermia. Recent cell culture studies suggest that certain naphthoquinones, especially menadione (vitamin K3), have anti-tumour activity by interfering with the energy metabolism of cells, resulting in the inhibition of aerobic glycolysis. We therefore studied the cytotoxic effects of menadione in HeLa cells in combination with hyperthermia. The cell culture data show that the cytotoxicity is markedly increased in cells deprived of glucose in the medium at 37 degrees C after exposure to menadione. When cells were exposed to menadione (20-40 microM) and hyperthermia (41-42 degrees C), there was a dramatic potentiation of heat-induced cytotoxicity in cells deprived of glucose in the medium. These data suggest that glucose-deficient cancer cells could be selectively killed by the combined treatment of menadione and mild hyperthermia, both of which can be readily achievable in humans.     

The preferential induction of apoptosis in multidrug-resistant KB cells by 5-fluorouracil.

It has been previously been shown that multidrug resistance may be associated with biochemical changes which increase the sensitivity of resistant cells to the induction of apoptosis by certain agents. We have shown here that 48 h exposure to 5-fluorouracil (5-FU) induces both a significantly greater proportion of apoptotic cells and much greater cleavage of the apoptosis-related protein poly-(ADP-ribose)-polymerase in the multidrug-resistant (MDR) carcinoma cell line, KB-A1, than in corresponding drug-sensitive control KB-3.1 cells. Exposure to 5-FU also reduced the level of the anti-apoptotic protein, protein kinase B, in the MDR cells, but not in the control cells.     

Elevation of glucosylceramide in multidrug-resistant cancer cells and accumulation in cytoplasmic droplets.

Multidrug-resistant (MDR) cancer cells have been shown to have an accumulation of glucosylceramide (GlcCer). In this study, we aim at localizing, at subcellular level, where these lipids accumulate. Neutral lipids and phospholipid containing organelles have been identified using confocal fluorescence microscopy and microspectrofluorometry by monitoring the emission of the fluorescent probe Nile-red. Data from confocal fluorescence microscopy analysis shows accumulation of neutral lipids in cytoplasmic droplets of MDR human carcinoma MCF7R cells. Microspectrofluorometric measurements show an increase of the gold-yellow emission intensity in MCF7R cells, corresponding to neutral lipids. Similar observations were made in human MDR vincristine-HL60 and doxorubicin-KB selected cells. Total cellular glucosylceramide (GlcCer) measurements using [(3)H]-palmitic acid and thin layer chromatography show a significant increase of GlcCer in MCF7R cells. Moreover, MCF7R cells treated with fluorescent GlcCer-bodipy exhibit an accumulation of this lipid in cytoplasmic droplets. Treatment of MCF7R cells with 1-phenyl-2-palmitoylamino-3-morpholino-1-propanolol (PPMP), a potent inhibitor of GlcCer synthase, attenuates the Nile-red fluorescence emission emanating from these structures and reverses MDR. Moreover, Golgi compartments stained with fluorescent PPMP-bodipy, show an increase in the Golgi compartments density. Treatment of MCF7R cells with cyclosporine A (CSA), tamoxifen (TMX) and 3'-azido-3'deoxythymidine (AZT) leads to the same effect observed in the presence of PPMP. Treatment of MCF7 and MCF7R with the beta-glucosidase inhibitor conduritol beta-epoxide (CBE) significantly increases resistance to daunorubicin only in MCF7R cells. These data demonstrate also that: (i) CSA, an inhibitor of MDR, has an additional target in addition to P-glycoprotein; and (ii) TMX (used in breast cancer treatment and prevention) and AZT (used in the treatment of HIV) could have side effects by disturbing lipid metabolism and inhibiting many cellular functions required in normal cells.     

Preferential killing of glucose-depleted HeLa cells by menadione and hyperthermia

Energy deprivation of cancer cells increases sensitivity to killing by hyperthermia. Recent cell culture studies suggest that certain naphthoquinones, especially menadione (vitamin K₃), have anti-tumour activity by interfering with the energy metabolism of cells, resulting in the inhibition of aerobic glycolysis. We therefore studied the cytotoxic effects of menadione in HeLa cells in combination with hyperthermia. The cell culture data show that the cytotoxicity is markedly increased in cells deprived of glucose in the medium at 37°c after exposure to menadione. When cells were exposed to menadione (20–40 μM) and hyperthermia (41–42°c), there was a dramatic potentiation of heat-induced cytotoxicity in cells deprived of glucose in the medium. These data suggest that glucose-deficient cancer cells could be selectively killed by the combined treatment of menadione and mild hyperthermia, both of which can be readily achievable in humans.     

Long-lasting accumulation of vinblastine in inostamycin-treated multidrug-resistant KB cells

Inostamycin, a novel polyether compound, reverses multidrug resistance in KB cells. The mechanism of its action was studied by use of radioactively labeled vinblastine. Inostamycin dose-dependently increased the accumulation of [3H]vinblastine in multidrug-resistant KB-C4 cells at 0.5-2 micrograms/ml, while it did not enhance accumulation in the drug-sensitive KB-3-1 cells. At a concentration of 1 microgram/ml inostamycin inhibited active [3H]vinblastine efflux from KB-C4 cells, but not from KB-3-1 cells, and inhibited [3H]vinblastine binding to KB-C4 membranes with an IC50 of 0.94 microgram/ml (1.3 microM). Furthermore, [3H]vinblastine accumulated by treatment with 1 microgram/ml of inostamycin was resistant to efflux from KB-C4 cells, even after the removal of inostamycin.     

Monoclonal anti-P-glycoprotein antibody-dependent killing of multidrug-resistant tumor cells by human mononuclear cells

Mouse monoclonal antibodies (MRK16 and MRK17) against human multidrug-resistant cancer cell lines were tested for antibody-dependent cytotoxicity mediated by human blood mononuclear cells, using a 4-h 51Cr release assay. MRK16 (IgG2a isotype) was shown to be more effective than MRK17 (IgG1 isotype). Moreover, when four pairs of drug-resistant and their parent sensitive human cancer cells were tested for antibody-dependent cell-mediated cytolysis (ADCC) using MRK16, only the drug-resistant cell lines were susceptible to ADCC reaction. When highly purified lymphocytes (greater than 99%) and monocytes (greater than 97%) were isolated from blood mononuclear cells by centrifugal elutriation and adherence, MRK16 promoted both lymphocyte- and monocyte-mediated tumor cell killing, whereas MRK17 induced only a lymphocyte-mediated ADCC reaction. These results suggest that MRK16 of IgG2a subtype may be a useful therapeutic agent in eradication of drug-resistant cancer cells expressing P-glycoprotein through ADCC reaction.     

Preferential killing of PTEN-null myelomas by PI3K inhibitors through Akt pathway

We recently reported that internal deletion of PTEN tumor suppressor gene in OPM2 and Delta47 myeloma lines led to high Akt activation. Re-expression of PTEN induced strong apoptosis and growth inhibition. To understand the biologic importance of the phosphatidylinositol 3 kinase (PI3K)/Akt activation affected by PTEN deletion, we analysed apoptosis and growth inhibition by applying PI3K inhibitors to myeloma lines and by expressing Akt constructs. The PI3K inhibitors preferentially suppressed PTEN-null myeloma growth to those expressing PTEN, indicating that PI3K activation is more critical for growth and survival of those lines with PTEN mutations than others expressing a functional PTEN gene. Since PTEN-null myeloma lines exhibited much stronger Akt activation than PTEN-expressing cells in response to insulin-like growth factor I stimulation, we determined whether Akt could be responsible for PI3K-mediated cell survival and growth of PTEN-null myeloma lines. Expression of an active Akt, but not its kinase dead mutant, reversed wortmannin- and dexamethasone-induced apoptosis and growth inhibition in PTEN-null myeloma lines, suggesting that Akt lies downstream of PI3K for PTEN-null myeloma survival and dexamethasone resistance. In summary, we have provided evidence that PTEN-null myeloma cells are stringently dependent on the PI3K/Akt activation for cell survival. These results may provide a basis to treat myeloma patients with PI3K and Akt inhibitors.     

Targeting and amplification of immune killing of tumor cells by pro-Smac

Overexpression of inhibitors of apoptosis (IAP) is one potential mechanism for tumor cells to evade immune surveillance. To determine whether immune-mediated killing of tumor cells can be enhanced by neutralization of IAP proteins, 2 novel eGFP-Smac fusion proteins (pro-Smac) were introduced into the poorly immunogenic mouse melanoma cell line, B16BL6-D5 (D5). Each fusion protein contained Smac and a cleavage site specific for granzyme B (GrB) or caspase 8, thereby targeting the 2 major killing mechanisms of cytotoxic T-lymphocyte (CTL) and NK cells. Expression of a pro-Smac fusion protein by D5 tumor cells greatly enhanced the susceptibility to killing by lymphokine-activated killer (LAK) cells or purified GrB. GrB-mediated killing was increased to a much greater extent when tumor cells expressed the eGFP-Smac fusion protein with a GrB cleavage site compared to a caspase 8 cleavage site. In contrast, perforin-deficient LAK cells, which lack GrB-mediated cytotoxicity but process normal ligands for death receptors, killed D5 tumor cells expressed pro-Smac with caspase 8 cleavage site more efficiently. Enhanced killing by GrB was also accompanied by processing of the fusion protein and increased caspase-3-like activity. These results indicate that killing of tumor cells can be amplified by targeting cell-mediated cytotoxic mechanisms via expression of pro-Smac fusion proteins.     

2-Deoxy-D-glucose preferentially kills multidrug-resistant human KB carcinoma cell lines by apoptosis.

The aim of this study was to determine the mechanism of cell death associated with the preferential killing of multidrug-resistant (MDR) cells by the glycolytic inhibitor 2-deoxy-D-glucose (2DG) in a range of MDR human KB carcinoma cell lines selected in different drugs. The D10 values for KB-V1, KB-C1 and KB-A1 (selected in vinblastine, colchicine and doxorubicin respectively) were 1.74, 1.04 and 0.31 mM, respectively, compared with 4.60 mM for the parental cell line (KB-3-1). The mechanism of cell death was identified as apoptosis, based on nuclear morphology, annexin V binding and poly(ADP-ribose) polymerase (PARP) cleavage. 2DG induced apoptosis in the three MDR cell lines in a dose- and time-dependent manner and did not induce necrosis. PARP cleavage was detected in KB-C1 cells within 2 h of exposure to 50 mM 2DG and slightly later in KB-A1 and KB-V1 cells. The relative levels of 2DG sensitivity did not correlate with the levels of multidrug resistance or with the reduced levels of the glucose transporter GLUT-1 in these cells. We speculate that a 2DG-stimulated apoptotic pathway in MDR KB cells differs from that in normal KB cells.     

Altered conjugate formation and altered apoptosis of multidrug-resistant human leukemia cell line affects susceptibility to killing by activated natural killer (NK) cells

Most leukemias that exhibit P-glycoprotein (P-gp)-associated multidrug resistance (MDR) exhibit reduced susceptibility to immune cytotoxicity mediated by natural killer (NK) cells. To explore this phenomenon we investigated N6/ADR, a doxorubicin-selected, P-gp-positive variant of the human acute lymphoblastic leukemia (ALL) cell line NALM6. Each stage of the NK cytolytic pathway, (binding, activation and killing) was evaluated to identify the alterations responsible for the reduced cytotoxicity of the variant relative to its drug-sensitive parental line. The major cause of the decreased susceptibility to NK cytolysis was found to be reduced conjugate formation by the MDR variant. Activation of NK effectors by parental and MDR cells with concomitant release of tumor necrosis factor-alpha (TNF-alpha) correlated with conjugate formation. N6/ADR was also more resistant than NALM6 to antibody-dependent cellular cytotoxicity and to cytotoxic factors released from NK cells as measured both by 51Cr-release and by DNA fragmentation. This is the first report of a P-gp-positive leukemic line that exhibits reduced conjugate formation as well as increased resistance to NK-mediated killing mechanisms. Our results suggest caution in the use of NK-based immunotherapy as an alternative treatment for multidrug-resistant leukemias.     

Apoptosis is induced in both drug-sensitive and multidrug-resistant hepatoma cells by somatostatin analogue TT-232.

Clinical resistance to chemotherapeutic drugs is an important problem in the treatment of cancer; the circumvention of resistance has become one of the basic goals of cancer therapy. The most frequent form of primary liver cancer is hepatocellular carcinoma, which is essentially refractory to chemotherapy. We earlier showed that TT-232, a new somatostatin analogue developed in our laboratory, exerted a strong antiproliferative effect both in vitro and in vivo, but no growth hormone release inhibitory or antisecretory activity. Here we report that TT-232 has a pronounced antiproliferative effect on differentiated and dedifferentiated, drug-sensitive and multidrug-resistant hepatocellular carcinoma cell lines. TT-232 induces apoptosis at comparable levels in all these hepatoma variants demonstrating that the multidrug resistance of hepatomas does not correlate with a reduced susceptibility to apoptosis induction. These results clearly reveal that the machinery involved in apoptosis is functional in both drug-sensitive and resistant hepatoma variants and can be activated by the somatostatin analogue TT-232.     

Overexpression of glucosylceramide synthase in associated with multidrug resistance of leukemia cells

Ceramide, as a second messenger, initiates one of the major signal transduction pathways in tumor apoptosis. Glucosylceramide synthase (GCS) catalyzes glycosylation of ceramide and produces glucosylceramide. Through GCS, ceramide glycosylation allows cellular escape from ceramide-induced programmed cell death. Here we investigated the expression of GCS in human leukemia cells and an association between GCS and multidrug resistance of leukemia cells. Using RT-PCR technique the level of GCS gene was detected in 65 clinical multidrug resistance/non-resistance cases with leukemia, and in K562 and K562/A02 cell lines. AlamarBlue Assay was applied to confirm the multidrug resistant of K562/A02 cells. PPMP, which is a chemical inhibitor for GCS, was used to determine the relationship between GCS and drug-resistance in K562/A02 cells. In addition, multidrug resistance gene (mdr1), Bcl-2 and Bax mRNA was also analyzed by RT-PCR. The expression of GCS and mdr1 mRNA in clinic multidrug resistance samples exhibited significantly increased compared with clinic drug sensitive group (P<0.05). There was the positive correlation both the expression of GCS and mdr1 genes in leukemia samples (P<0.01, gamma=0.7). AlamarBlue Assay showed that the K562/A02 cell line was 115-fold more resistant to adriamycin and 36-fold more resistant to vincristine compared with drug-sensitive K562 cell line. There also was significant expression difference of GCS and mdr1 genes between K562 and K562/A02 cells. Bcl-2 gene exhibited higher expressions whatever in clinic drug-resistance samples or K562/A02 cells, whereas the expressions of Bax gene were higher in drug-sensitive samples and K562 cells. PPMP increased sensitivity to adriamycin toxicity by inhibiting GCS in K562/A02 cells. Therefore, it is suggested that a high level of GCS in leukemia is possible contributed to multidrug resistance of leukemia cells. Abnormally expressions of the genes in associated with cell apoptosis might be one of the main molecular pathology mechanisms of multidrug resistance caused by GCS gene.     

芦荟大黄素介导的光动力疗法对人口腔黏膜癌KB细胞杀伤效果的研究

目的 探讨芦荟大黄素(Aloe-emodin,AE)介导的光动力疗法(Photodynamic therapy,PDT)对人口腔黏膜癌KB细胞的杀伤效果。方法 通过MTT法检测不同实验条件对KB细胞的杀伤作用。实验分为空白对照组(Control)、单纯药物组(AE)、单纯光照组(Laser)和光动力组(PDT)。分别比较AE组不同浓度(10μM、20μM、30μM和40μM)、Laser组不同光照时间(10s、20s、40s、80s)与Control组对KB细胞的杀伤作用,分析PDT组AE浓度和时间对KB细胞杀伤作用的影响。结果 与Control组相比,AE组和Laser组对KB细胞的杀伤作用无统计学意义(P＞0.05)。PDT组对KB细胞的杀伤作用呈浓度和时间依赖性,当AE的浓度为40μM,照射时间为80s时,细胞存活率降低至56.7%。结论 AE介导的PDT对KB细胞有显著的杀伤作用。     

The chemosensitizing activity of inhibitors of glucosylceramide synthase is mediated primarily through modulation of P-gp function

Glucosylceramide synthase (GCS) is a key enzyme engaged in the biosynthesis of glycosphingolipids and in regulating ceramide metabolism. Studies exploring alterations in GCS activity suggest that the glycolase may have a role in chemosensitizing tumor cells to various cancer drugs. The chemosensitizing effect of inhibitors of GCS (e.g. PDMP and selected analogues) has been observed with a variety of tumor cells leading to the proposal that the sensitizing activity of GCS inhibitors is primarily through increases in intracellular ceramide leading to induction of apoptosis. The current study examined the chemosensitizing activity of the novel GCS inhibitor, Genz-123346 in cell culture. Exposure of cells to Genz-123346 and to other GCS inhibitors at non-toxic concentrations can enhance the killing of tumor cells by cytotoxic anti-cancer agents. This activity was unrelated to lowering intracellular glycosphingolipid levels. Genz-123346 and a few other GCS inhibitors are substrates for multi-drug resistance efflux pumps such as P-gp (ABCB1, gP-170). In cell lines selected to over-express P-gp or which endogenously express P-gp, chemosensitization by Genz-123346 was primarily due to the effects on P-gp function. RNA interference studies using siRNA or shRNA confirmed that lowering GCS expression in tumor cells did not affect their responsiveness to commonly used cytotoxic drugs.     

紫杉醇脂质纳米粒子对KB细胞的作用

目的:研究紫杉醇脂质纳米粒子（TAX-NLC）对KB细胞的作用。方法:激光共聚焦显微镜观察TAX-NLC作用于KB细胞对F-肌动蛋白（F-actin）的影响,并观察TAX-NLC作用于KB细胞诱导细胞凋亡情况,光镜观察TAX-NLC作用于KB细胞形态学的变化。结果:TAX-NLC使KB细胞F-actin聚集成团块状、点状,使微丝的功能受到抑制,TAX-NLC作用KB细胞诱导细胞凋亡,TAX-NLC作用KB细胞可产生大量多核细胞。结论:TAX-NLC破坏微丝功能,诱导凋亡及多核细胞,导致肿瘤细胞死亡。     

Inhibiting glucosylceramide synthase facilitates the radiosensitizing effects of vinorelbine in lung adenocarcinoma cells

The standard treatment regimen for patients diagnosed with non-small cell lung cancer (NSCLC) with locally advanced stage III disease is concurrent chemoradiotherapy (CCRT). This study investigated the molecular effects of vinca alkaloid vinorelbine (VNR)-based CCRT. We reviewed the records of 68 patients with stage III NSCLC: 42 patients received VNR-based CCRT, and 26 were treated with radiation alone. Human lung adenocarcinoma cells were used in this study to investigate the molecular effects of glucosylceramide synthase inhibition on VNR-based CCRT. There was response rate of 66.7% with CCRT, which was better than the response rate observed with radiation alone (30.8%; P < 0.001). CCRT caused an increase in cell cycle arrest at G₂/M phase accompanied by apoptosis. Oxidative c-Jun N-terminal kinase (JNK) activation was involved in the increased apoptosis levels but not the cell cycle arrest. CCRT also induced an increase in ceramide accompanied by a decrease in glucosylceramide that was positively correlated with the cytotoxic effects. Pharmacologically inhibiting glucosylceramide synthase facilitated VNR- and CCRT-induced apoptosis by promoting the JNK pathway. Inhibiting glucosylceramide synthase facilitates the radiosensitizing effects of VNR by promoting JNK-mediated apoptosis in lung adenocarcinoma cells.     

MRP1 and glucosylceramide are coordinately over expressed and enriched in rafts during multidrug resistance acquisition in colon cancer cells

Previously we have described a novel multidrug-resistant cell line, HT29(col), which displayed over expression of the multidrug-resistance protein 1 (MRP1) and an altered sphingolipid composition, including enhanced levels of glucosylceramide (GlcCer; Kok JW, Veldman RJ, Klappe K, Koning H, Filipeanu C, Muller M. Int J Cancer 2000;87:172-8). In our study, long-term screening revealed that, during colchicine-induced acquisition of multidrug resistance in a new HT29(col) cell line, increases in GlcCer occurred concomitantly with upregulation of MRP1 expression. Both MRP1 and GlcCer were found enriched in Lubrol-insoluble membrane domains. The expression of MRP1 and GlcCer were tightly correlated, as indicated also by a reversal of both at the later stage of colchicine consolidation. Resistance to colchicine was determined by MRP1, while glucosylceramide synthase (GCS) did not contribute: 1). Resistance was fully inhibited by MK571. 2). GCS expression and activity were not upregulated in HT29(col) cells. 3). Inhibition of GCS did not affect MRP1-mediated efflux function or sensitivity to colchicine. Instead, overall sphingolipid metabolism was upregulated through an increased rate of ceramide biosynthesis. In conclusion, upregulation of MRP1 occurs in concert with upregulation of GlcCer during multidrug-resistance acquisition, and both are enriched in rafts. The increased GlcCer pool does not directly modulate MRP1 function and cell survival.