Effect of liposomes sensitized with methotrexate-gamma-dimyristoylphosphatidylethanolamine on cells that are resistant to methotrexate

This study compares the ability of methotrexate and liposomes, in which the drug is anchored to the lipid bilayers via methotrexate-gamma-dimyristoylphosphatidylethanolamine, to inhibit proliferation of human leukemic cells (CEM/O) and cells derived from this line that are resistant to methotrexate because of either a defective transport system (CEM/MTX cells) or elevated levels of dihydrofolate reductase (CEM/R1 cells). Whereas CEM/O and CEM/MTX cells show a 120-fold difference in their susceptibility to methotrexate (as measured by the incorporation of tritiated deoxyuridine into DNA), both lines are equally sensitive to the liposomes. In contrast, proliferation of CEM/MTX cells is not inhibited significantly by methotrexate-gamma-glycerophosphorylethanolamine (MTX-gamma-glyceroPE), the water-soluble analog of MTX-gamma-DMPE. Both the ability of the liposomes to circumvent the transport defect, and the inability of MTX-gamma-glyceroPE to do so, were anticipated on the basis of previous experiments which show that thiamine pyrophosphate could antagonize inhibition of mouse 3T3 and L1210 cell proliferation by methotrexate and MTX-gamma-glyceroPE, but not inhibition by liposomes. Human cells (CEM/O) behave similarly. The present experiments also suggest that liposomes prepared with MTX-gamma-DMPE can partially reverse the methotrexate resistance of CEM/R1 cells that is due to overproduction of the target enzyme.     

Inhibition of cell proliferation with antibody-targeted liposomes containing methotrexate-gamma-dimyristoylphosphatidylethanolamine

We have prepared liposomes containing methotrexate-gamma-dimyristoylphosphatidylethanolamine (MTX-DMPE liposomes), to which protein A was covalently coupled, permitting specific association of these liposomes in vitro with murine cells preincubated with relevant protein A-binding monoclonal antibodies. In the absence of antibody the presence of externally-oriented methotrexate (MTX) in MTX-DMPE liposomes did not result in greater binding to cells than liposomes made without MTX-gamma-DMPE. Derivation of methotrexate with phospholipid permits enhanced drug-liposome association. These liposomes are more resistant than conventional liposomes to repeated cycles of freezing and thawing. MTX-DMPE liposomes are comparable to antibody-targeted liposomes made with encapsulated water-soluble methotrexate both with respect to specific binding to target cells and drug effect. The inhibitory effects of MTX-liposomes, as well as free MTX, were reversible by either thiamin pyrophosphate (Tpp) or N5-formyltetrahydrofolate (F-THF), while the effects of MTX-DMPE liposomes were reversed only by N5-formyltetrahydrofolate. This suggests that the toxicity of non-targeted MTX-liposomes may be due to leakage of the encapsulated MTX. The absence of an effect of thiamin pyrophosphate on non-targeted MTX-DMPE liposomes indicates that they do not enter into the cell via the normal folate transport system.     

Inhibition of cell proliferation with antibody-targeted liposomes containing methotrexate-γ-dimyristoylphosphatidylethanolamine

We have prepared liposomes containing methotrexate-γ-dimyristoylphosphatidylethanolamine (MTX-DMPE liposomes), to which protein A was covalently coupled, permitting specific association of these liposomes in vitro with murine cells preincubated with relevant protein A-binding monoclonal antibodies. In the absence of antibody the presence of externally-oriented methotrexate (MTX) in MTX-DMPE liposomes did not result in greater binding to cells than liposomes made without MTX-γ-DMPE. Derivation of methotrexate with phospholipid permits enhanced drug-liposome association. These liposomes are more resistant than conventional liposomes to repeated cycles of freezing and thawing. MTX-DMPE liposomes are comparable to antibody-targeted liposomes made with encapsulated water-soluble methotrexate both with respect to specific binding to target cells and drug effect. The inhibitory effects off MTX-liposomes, as well as free MTX, were reversible by either thiamin pyrophosphate (Tpp) or N⁵-formyltetrahydrofolate (F-THF), while the effects of MTX-DMPE liposomes were reversed only by N⁵-formyltetrahydrofolate. This suggests that the toxicity of non-targeted MTX-liposomes may be due to leakage of the encapsulated MTX. The absence of an effect of thiamin pyrophosphate on non-targeted MTX-DMPE liposomes indicates that they do not enter into the cell via the normal folate transport system.     

Inhibition of cell proliferation by putative metabolites and non-degradable analogs of methotrexate-gamma-dimyristoylphosphatidylethanolamine

Previous investigations have shown that untargeted liposomes, in which methotrexate is anchored to the lipid bilayers as methotrexate-gamma-dimyristoylphosphatidylethanolamine (methotrexate-gamma-DMPE), can inhibit in vitro cell proliferation. To test the possibility that this inhibition may involve extracellular metabolism of methotrexate-gamma-DMPE, we have degraded it chemically (dilute alkali) or enzymatically (phospholipase A2, phospholipase C, phospholipase C plus phosphatase), and assayed the products using human lymphoblastoid T cells or a subline that has a defective methotrexate transport system. Neither methotrexate-gamma-(1-myristoyl)-glycerophosphorylethanolamine, methotrexate-gamma-glycerophosphorylethanolamine, methotrexate-gamma-phosphorylethanolamine, nor methotrexate-gamma-ethanolamine resemble methotrexate-gamma-DMPE sensitized liposomes or the free derivative in their ability to block tritiated deoxyuridine incorporation into DNA. When added extracellularly, these putative metabolites manifest a higher ID50 concentration and/or, unlike the liposomes or unincorporated methotrexate-gamma-DMPE, utilize the methotrexate transport system to enter cells. Additionally, we have synthesized methotrexate-gamma-dihexadecylphosphatidylethanolamine and methotrexate-gamma-hexadecylphosphorylethanolamine, analogs of methotrexate-gamma-DMPE that cannot be hydrolyzed by phospholipases A2, C and D; liposomes prepared with these derivatives are markedly less potent cytotoxic agents than methotrexate-gamma-DMPE sensitized liposomes. All together, these results are consistent with the conclusion that methotrexate-gamma-DMPE must undergo intracellular metabolism to exert optimal inhibition; they also bear on possible mechanisms by which methotrexate-gamma-DMPE may enter cells.     

Inhibition of cell proliferation by putative metabolites and non-degradable analogs of methotrexate-Y-dimyristoylphosphatidylethanolamine

Previous investigations have shown that untargeted liposomes, in which methotrexate is anchored to the lipid bilayers as methotrexate-γ-dimyristoylphosphatidylethanolamine (methotrexate-γ-DMPE), can inhibit in vitro cell proliferation. To test the possibility that this inhibition may involve extracellular metabolism of methotrexate-y-DMPE, we have degraded it chemically (dilute alkali) or enzymatically (phospholipase A₂, phospholipase C, phospholipase C plus phosphatase), and assayed the products using human lymphoblastoid T cells or a subline that has a defective methotrexate transport system. Neither methotrexate-y-(lmyristoyO-glycerophosphorylethanolamine, methotrexate-y-glycerophosphorylethanolamine, methotrexatey-phosphorylethanolamine, nor methotrexate-γ-ethanolamine resemble methotrexate-γ-DMPE sensitized liposomes or the free derivative in their ability to block tritiated deoxyuridine incorporation into DNA. When added extracellularly, these putative metabolites manifest a higher ID₅₀ concentration and/or, unlike the liposomes or unincorporated methotrexate-γ-DMPE, utilize the methotrexate transport system to enter cells. Additionally, we have synthesized methotrexate-γ-dihexadecylphosphatidylethanolamine and methotrexate-γ-hexadecylphosphorylethanolamine, analogs of methotrexate-γ-DMPE that cannot be hydrolyzed by phospholipases A₂, C and D; liposomes prepared with these derivatives are markedly less potent cytotoxic agents than methotrexate-γ-DMPE sensitized liposomes. All together, these results are consistent with the conclusion that methotrexate-γ-DMPE must undergo intracellular metabolism to exert optimal inhibition; they also bear on possible mechanisms by which methotrexate-γ-DMPE may enter cells.     

Synthesis and characterization of methotrexate-dimyristoylphosphatidylethanolamine derivatives and the glycerophosphorylethanolamine analogs

Research in this laboratory is currently focused on the biochemical and pharmacological properties of liposomes in which an otherwise water-soluble drug is anchored to the lipid bilayers via an appropriate non-polar residue. To this end, we have synthesized three (I-III) methotrexate (MTX) derivatives of dimyristoylphosphatidylethanolamine (DMPE) by conjugation of the alpha and/or gamma glutamyl carboxyl groups of the drug with the amino function of the phospholipid. These derivatives have been characterized analytically and chromatographically as MTX-gamma-DMPE (I), MTX-alpha-DMPE (II), and MTX-alpha, gamma-diDMPE (III). The corresponding glycerophosphorylethanolamine analogs have also been prepared and identified. The biological activity of these compounds (as inhibitors of in vitro cell proliferation and dihydrofolate reductase) is described in the following paper.     

Studies of the lungs in diabetes mellitus. II. Phospholipid analyses on the surfactant from broncho-alveolar lavage fluid of alloxan-induced diabetic rats

Structurally diverse anions (folate, 5-formyltetrahydrofolate, AMP, ADP, thiamine pyrophosphate, phosphate, sulfate, and chloride) that are competitive inhibitors of methotrexate influx in L1210 cells also enhance the efflux of methotrexate from these cells. The increase in efflux reaches a maximum of 2- to 4-fold depending upon the anion employed, and the anion concentrations required for half-maximal stimulation of efflux are similar to their K_i values for inhibition of methotrexate influx. A competitive inhibitor of methotrexate uptake (fluorescein-diaminopentane-methotrexate) that is not transported by this system, does not increase methotrexate efflux. These results suggest that the efflux of intracellular methotrexate is coupled to the concomitant uptake of an extracellular anion.     

Inhibition of Thiamine Pyrophosphate Utilization by Thiamine or Its Monophosphate in Escherichia coli

The growth of a thiamine pyrophosphate auxotroph of Escherichi coli was inhibited by either thiamine or thiamine monophosphate, and the growth of a thiamine monophosphate auxotroph was inhibited by thiamine. The thiamine pyrophosphate-dependent oxidation of pyruvate was inhibited by thiamine with whole cells of the thiamine pyrophosphate auxotroph but not with cell extracts prepared from the same organism. In addition, the thiamine pyrophosphate uptake of the thiamine pyrophosphate auxotroph was inhibited by either thiamine or thiamine monophosphate. Although the thiamine pyrophosphate uptake of a revertant, selected for prototrophy from the thiamine monophosphate auxotroph, was inhibited by thiamine to an extent comparable to that observed with the thiamine monophosphate auxotroph, its growth was no longer inhibited by thiamine. A possible mechanism for the inhibition by thiamine and thiamine monophosphate in the utilization of thiamine pyrophosphate is discussed.     

Reduced folate carrier transports thiamine monophosphate: an alternative route for thiamine delivery into mammalian cells

Although the reduced folate carrierRFC1 and the thiamine transporters THTR-1 and THTR-2 share ~40% oftheir identity in protein sequence, RFC1 does not transport thiamineand THTR-1 and THTR-2 do not transport folates. In the present study,we demonstrate that transport of thiamine monophosphate (TMP), animportant thiamine metabolite present in plasma and cerebrospinalfluid, is mediated by RFC1 in L1210 murine leukemia cells. Transport ofTMP was augmented by a factor of five in cells (R16) that overexpressRFC1 and was markedly inhibited by methotrexate, an RFC1 substrate, butnot by thiamine. At a near-physiological concentration (50 nM), TMP influx mediated by RFC1 in wild-type L1210 cells was ~50% ofthiamine influx mediated by thiamine transporter(s). Within 1 min, the majority of TMP transported into R16 cells was hydrolyzed to thiamine with a component metabolized to thiamine pyrophosphate, the active enzyme cofactor. These data suggest that RFC1 may be one of the alternative transport routes available for TMP in some tissues whenTHTR-1 is mutated in the autosomal recessive disorderthiamine-responsive megaloblastic anemia.

     

Binding and transport of thiamine by Lactobacillus casei.

The relationship between thiamine transport and a membrane-associated thiamine-binding activity has been investigated in Lactobacillus casei. Thiamine transport proceeds via a system whose general properties are typical of active uptake processes; entry of the vitamin into the cells requires energy, is temperature dependent, exhibits saturation kinetics, and is inhibited by substrate analogs. A considerable concentration gradient of unchanged thiamine can be achieved by the system, although the vitamin is slowly metabolized to thiamine pyrophosphate. Consistent with these results, L. casei also contains a high-affinity, thiamine-binding component which could be measured by incubation of intact cells with labeled substrate at 4 degrees C (conditions under which transport is negligible). Binding was insensitive to iodoacetate, occurred at a level (0.5 nmol per 10(10) cells) nearly 20-fold higher than could be accounted for by facilitated diffusion, and was found to reside in a component of the cell membrane. Participation of this binder in thiamine transport is supported by the observations that the processes of binding and transport showed similarities in their (i) regulation by the concentration of thiamine in the growth medium, (ii) binding affinities for thiamine, and (iii) susceptibility to inhibition by thiamine analogs.     

Structural requirements for anion substrates of the methotrexate transport system in L1210 cells

A broad spectrum of structurally diverse anions reversibly inhibits the influx of methotrexate in L1210 cells. Several of the more effective anions and their respective inhibition constants (K_i values) were: 5-methyltetrahydrofolate (0.3 μm), bromosulfophthalein (2 μm), thiamine pyrophosphate (3 μm), 8-anilino-1-naphthalene sulfonate (7 μm), phthalate (20 μm), and AMP (50 μm). Moderate inhibition was observed with P_i (K_i = 400 μm) and other divalent inorganic anions, while small monovalent anions such as Cl^? (K_i = 30 mm) were the least effective. When these same anions were tested for an effect on methotrexate efflux, stimulation was observed with some anions, while others had no effect. Enhancement was produced by folate compounds and p-aminobenzoylglutamate, small monovalent (e.g., Cl^?, acetate, and lactate) and divalent (e.g., phosphate and succinate) anions, a few nucleotides (e.g., AMP), and thiamine pyrophosphate, while little or no effect was associated with trivalent anions (e.g., citrate), most nucleotides, and large organic anions (e.g., bromosulfophthalein, NAD, and NADP). Anions with the ability to promote methotrexate efflux in control cells lost this capacity upon exposure of the cells to an irreversible inhibitor of methotrexate influx. These results support the hypothesis that methotrexate transport proceeds via an anion-exchange mechanism and moreover provide evidence that anion substrates for this system can be identified by their ability to promote methotrexate efflux. Anions which appear most likely to participate in this exchange cycle in vivo are P_i and AMP.     

Tumor-specific idiotype vaccines. I. Generation and characterization of internal image tumor antigen

The concept of idiotype vaccines against tumor-associated antigens (TAA) was tested in the DBA/2 L1210 lymphoma subline, L1210/GZL. Monoclonal antibodies against a TAA that cross-reacts with the envelope glycoprotein gp52 of the mammary tumor virus were used to make hybridoma anti-idiotype antibodies (Ab2). In this report we describe the characterization of monoclonal anti-idiotypic antibodies against the combining site of 11C1 (Ab1), which recognizes a shared determinant of gp52 of mouse mammary tumor virus (MMTV) and the TAA of L1210/GZL. Hybridomas expressing the internal image of gp52 were screened by an idiotype inhibition assay. Mice sensitized with radiated L1210/GZL cells produced specific delayed type hypersensitivity (DTH) against the Ab2 hybridoma. Five Ab2 hybridomas were selected and were used to immunize DBA/2 mice. Such immunized animals showed specific DTH reaction against a challenge with the L1210/GZL tumor cells. Similar results were obtained in mice immunized with purified Ab2. Fluorescence-activated cell sorter analysis demonstrated that fluorescence staining of L1210/GZL cells by 11C1 can be completely inhibited with preabsorption on Ab2 hybridoma cells. Mice immunized with 2F10 and 3A4 coupled to keyhole limpet hemocyanin (KLH) contained antibodies binding to MMTV. But only in mice immunized with 2F10-KLH was significant inhibition of L1210/GZL tumor growth observed. Collectively, these results indicate that certain anti-idiotypic antibodies can mimic the MMTV gp52 antigen, as well as the gp52-like epitope expressed on the L1210/GZL tumor cells. These properties of anti-idiotypic antibodies mimicking TAA could be exploited for making idiotype vaccines against tumors.     

Transport of methotrexate in L1210 cells: Mechanism for inhibition by p-chloromercuriphenylsulfonate and N-ethylmaleimide

Methotrexate transport in L1210 cells is highly sensitive to inhibition by p-chloromercuriphenylsulfonate (CMPS) and, to a lesser extent, by N-ethylmaleimide. A 50% reduction in the methotrexate influx rate occurred upon exposure of cells to 3 μM CMPS or 175 μM N-ethylmaleimide, while complete inhibition was achieved at higher levels of these agents. Dithiothreitol reversed the inhibition by CMPS, suggesting that a sulfhydryl residue is involved. This residue is apparently not located at the substrate binding site of the transport protein, since methotrexate failed to protect the system from inactivation by either CMPS or N-ethylmaleimide, and the transport protein retained the ability to bind substrate (at 4°C) after exposure to these inhibitors (at 37°C). Methotrexate efflux was also inhibited by CMPS (50% at 4 μM), indicating that both the uptake and efflux of methotrexate in L1210 cells occur via the same transport system. High concentrations of CMPS (greater than 20 μM) increased the efflux rate, apparently by damaging the cell membrane and allowing the passive diffusion of methotrexate out of the cell.     

Binding properties of the 5-methyltetrahydrofolate/methotrexate transport system in L1210 cells

A binding component with a high affinity for 5-methyltetrahydrofolate (K_D = 0.11μm) is present on the external surface of L1210 cells. The amount of binder (1 pmol/mg protein) corresponds to 8 × 10⁴ sites per cell. The participation of this component in the high-affinity 5-methyltetrahydrofolate/methotrexate transport system is supported by similarities in the K_D values for 5-methyltetrahydrofolate and methotrexate binding and the K_t values of these compounds for transport. Relative affinities for other folate substrates (aminopterin, 5-formyltetrahydrofolate, and folate) and various competitive inhibitors (thiamine pyrophosphate, ADP, AMP, arsenate, and phosphate) are also similar for both the binding component and the transport system. The measured binding activity does not represent low-temperature transport of substrate into cells, since it is readily saturable with time and is eliminated by either washing the cells with buffer or by the addition of excess unlabeled substrate.     

Photoaffinity analogues of methotrexate as folate antagonist binding probes. 2. Transport studies, photoaffinity labeling, and identification of the membrane carrier protein for methotrexate from murine L1210 cells

A membrane-derived component of the methotrexate/one-carbon-reduced folate transport system in murine L1210 cells has been identified by using a photoaffinity analogue of methotrexate. The compound, a radioiodinated 4-azidosalicylyl derivative of the lysine analogue of methotrexate, is transported into murine L1210 cells in a temperature-dependent, sulfhydryl reagent inhibitable manner with a Kt of 506 +/- 79 nM and a Vmax of 17.9 +/- 4.2 pmol min-1 (mg of total cellular protein)-1. Uptake of the iodinated compound at 200 nM is inhibited by low amounts of methotrexate (I50 = 1.0 microM). The parent compounds of the iodinated photoprobe inhibit [3H]methotrexate uptake, with the uniodinated 4-azidosalicylyl derivative exhibiting a Ki of 66 +/- 21 nM. UV irradiation, at 4 degrees C, of a cell suspension that had been incubated with the probe results in the covalent modification of a 46K-48K protein. This can be demonstrated when the plasma membranes from the labeled cells are analyzed via sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Labeling of this protein occurs half-maximally at a reagent concentration that correlates with the Kt for transport of the iodinated compound. Protection against labeling of this protein by increasing amounts of methotrexate parallels the concentration dependence of inhibition of photoprobe uptake by methotrexate. In addition, no labeling occurs when a cell line that has a defective methotrexate transport system is similarly treated. Evidence that, in the absence of irradiation and at 37 degrees C, the iodinated probe is actually internalized is demonstrated by the labeling of two soluble proteins (Mr 38K and 21K) derived from the cell homogenate supernatant.     

Circumvention of the methotrexate transport system by methotrexate-phosphatidylethanolamine derivatives: effect of fatty acid chain length

Methotrexate has been conjugated (amide bond) via either the alpha or gamma, or both alpha and gamma, glutamyl carboxyl groups to the amino function of dihexanoylphosphatidylethanolamine (C6C6PE) and 1-tetradecanoyl-2-hexanoylphosphatidylethanolamine (C14C6PE). These phospholipid prodrugs (either free or incorporated into liposomes) were compared with the corresponding ditetradecanoylphosphatidylethanolamine (C14C14PE) conjugates, some of whose properties have been described previously, for their ability to inhibit the proliferation of human leukemic cells (CEM/O) or cells derived therefrom (CEM/MTX) that are resistant to methotrexate because of a defective drug transport system. Regardless of chain length, the gamma conjugates were more effective than either the alpha or the alpha, gamma conjugates, in inhibiting growth of the parent cells, confirming initial experiments with mouse cells. Chain length had, however, a pronounced influence on the capacity of the various gamma derivatives to circumvent the transport defect. For example, CEM/MTX cells were 120-fold less susceptible than CEM/O cells to inhibition by either methotrexate or methotrexate-gamma-C6C6PE, whereas both cell lines were equally sensitive to methotrexate-gamma-C14C14PE. Although less potent than either of the foregoing, methotrexate-gamma-C14C6PE could partially by-pass the defective transport system. These results suggest that methotrexate-gamma-PE derivatives with appropriate acyl residues might be useful probes to investigate the mechanism by which phospholipids in general are able to traverse cell membranes.     

Liposome-dependent delivery of pteridine antifolates: a two-compartment growth inhibition assay for evaluating drug leakage and metabolism

We have developed a two-compartment growth inhibition assay that can provide information about leakage, metabolism and delivery of liposome-dependent drugs under cell culture conditions, and at drug concentrations that are relevant to drug delivery. Two cell lines are grown in separate compartments separated from each other by a 0.1 micron polycarbonate membrane. The membrane allows free drugs to diffuse rapidly from one compartment to another, and does not allow liposomes to diffuse through. Liposomes are added to the first compartment, which contains target cells. The extent of leakage caused by these cells is determined by the growth inhibition of non-target cells in the second compartment. We show that methotrexate and methotrexate-gamma-aspartate leak rapidly and almost completely when encapsulated in phosphatidylglycerol/cholesterol (67:33) liposomes. In contrast, there is only 42% leakage when the drugs are encapsulated in distearoylphosphatidylglycerol/cholesterol (67:33) liposomes. We also demonstrate that the target cells (CV1-P) may partially degrade encapsulated methotrexate-gamma-aspartate to methotrexate. Therefore, methotrexate-gamma-aspartate may be a lysosomally cleaved pro-drug of methotrexate.     

Galactosyltransferase activity and cell growth: uridine diphosphate (UDP)galactose inhibition of murine leukemic L1210 cells

UDPgalactose inhibits the growth of mouse leukemic L1210 cells. In calf serum supplemented Dulbecco's medium (CS-DMEM), 1.2 mM UDPgalactose (UDPgal) inhibited cell growth by 50% (IC50), and 5 mM UDPgalactose inhibited cell growth by 92%. Other nucleotide sugars as well as galactose, glucose, and galactose-1-phosphate had little or no effect on cell growth. Uridine nucleotides, which inhibit galactosyltransferase activity, protected L1210 cells from the growth inhibitory effect of UDPgalactose when both were added simultaneously to culture media. Unlike mouse 3T12 cells, in which no inhibition of cell growth was observed with heat-inactivated calf serum (HICS)-DMEM, 5 mM UDPgalactose inhibited L1210 cell growth in HICS-DMEM to the same degree as that observed in CS-DMEM. In contrast to 3T12 cells, L1210 cells secrete significant galactosyltransferase activity into the media. Complete inhibition of 3T12 cell growth by UDPgal was observed if HICS-DMEM medium was first conditioned by L1210 cells for 48 hours. No difference in cell growth or [3H]thymidine uptake was detected after 6 hours of exposure to UDPgalactose, but both were significantly decreased at 24 and 48 hours. Flow cytometric analysis of UDPgalactose effects on L1210 cells revealed no differences in the distribution of cells in G1, S, or G2-M of the cell cycle after 6 hours of incubation, but after 16 hours of UDPgalactose treatment, L1210 cells were arrested in early S phase. These cells were completely viable and morphologically similar to control L1210 cells. Normal growth was resumed when UDPgal was removed. The data suggest that UDPgalactose inhibition of cell growth requires extracellular galactosyltransferase activity and that the effect is mediated via the cell membrane.     

复方浙贝母药物血清抑制L1210/CDDP细胞增殖与诱导凋亡研究

目的：观察复方浙贝药物血清抑制耐顺铂的小鼠淋巴细胞系（L1210/CDDP）增殖和诱导凋亡效应,并在此基础上探讨复方浙贝颗粒逆转白血病细胞多药耐药机制。方法：MTT法测定不同浓度组复方浙贝颗粒含药血清抑制L1210/CDDP增殖;Annex-in-V/PI双染经流式细胞仪（FCM）定量检测其含药血清诱导L1210/CDDP细胞早期凋亡率。结果：不同剂量复方浙贝颗粒含药血清组均具有抑制L1210/CDDP细胞增殖效应,对其凋亡抑制率分别为：29.27%（高剂量组）、31.78%（中剂量组）、21.20%（低剂量组）、25.31%（阿霉素组）;实验各组的A570nm与空白对照组比较明显下降（P〈0.01）。经FCM双荧光法检测其能够诱导L1210/CDDP细胞早期凋亡,实验各组凋亡率分别为：55.12%（中剂量血清组）、34.23%（低剂量组）、25.69%（高剂量组）,24.65%（空白血清组）。结论：复方浙贝颗粒含药血清能够明显抑制L1210/CDDP细胞增殖,并能诱导其凋亡。     

Sites of inhibition of mitochondrial electron transport in macrophage- injured neoplastic cells

Previous work has shown that injury of neoplastic cells by cytotoxic macrophages (CM) in cell culture is accompanied by inhibition of mitochondrial respiration. We have investigated the nature of this inhibition by studying mitochondrial respiration in CM-injured leukemia L1210 cells permeabilized with digitonin. CM-induced injury affects the mitochondrial respiratory chain proper. Complex I (NADH-coenzyme Q reductase) and complex II (succinate-coenzyme Q reductase) are markedly inhibited. In addition a minor inhibition of cytochrome oxidase was found. Electron transport from alpha-glycerophosphate through the respiratory chain to oxygen is unaffected and permeabilized CM-injured L1210 cells oxidizing this substrate exhibit acceptor control. However, glycerophosphate shuttle activity was found not to occur within CM- injured or uninjured L1210 cells in culture hence, alpha- glycerophosphate is apparently unavailable for mitochondrial oxidation in the intact cell. It is concluded that the failure of respiration of intact neoplastic cells injured by CM is caused by the nearly complete inhibition of complexes I and II of the mitochondrial electron transport chain. The time courses of CM-induced electron transport inhibition and arrest of L1210 cell division are examined and the possible relationship between these phenomena is discussed.