D. Sadava

Transferrin overcomes drug resistance to artemisinin in human small-cell lung carcinoma cells

Multiple drug resistance is a significant problem in small-cell lung cancer (SCLC). Artemisinin (ART) is a natural product used to treat drug-resistant malaria. The drug is effective because the Fe2+ present in infected erythrocytes acts non-enzymatically to convert ART to toxic products. We tested the effects of ART on drug-sensitive (H69) and multi-drug-resistant (H69VP) SCLC cells, pretreated with transferrin (TF) to increase the intracellular Fe2+ level. Antibody staining followed by flow cytometry analysis showed twice the level of TF receptors on the H69VP as compared to the H69 cells. Low doses of ART were cytotoxic to SCLC cells. The cytotoxicity of ART for H69VP cells (IC50=24 nM) was ten-fold lower than for H69 cells (IC50=2.3 nM), indicating that ART is part of the drug resistance phenotype. Pretreatment of H69 cells with 220-880 nM TF did not alter the IC50 for ART. However, in the ART-resistant H69VP cells, pretreatment with TF lowered the ART IC50 to near drug-sensitive levels (IC50=5.4 nM after 4 h pretreatment with 880 nM TF). Desferrioxamine (5 microM) inhibited the effect of TF on the IC50 for ART in drug-resistant cells but did not have an effect on ART cytotoxicity in drug-sensitive cells. DNA fragmentation as measured by ELISA occurred within ART-treated cells, with kinetics indicating apoptosis rather than necrosis. This was confirmed by TUNEL staining. These data indicate the potential use of ART and TF in drug-resistant SCLC.

Hydroxyproline-rich cell wall protein (extensin): role in the cessation of elongation in excised pea epicotyls.

Abstract The synthesis and accumulation of cell wall hydroxyproline increases coincident with the cessation of elongation growth in pea epicotyls. We examined the relationship between these biochemical and physiological events by using epicotyl sections challenged with α, α′-dipyridyl. This chelator blocked hydroxyproline biosynthesis without affecting overall protein synthesis. Epicotyl sections mimicked elongation growth in situ when placed in indoleacetic acid. Elongation was blocked by the addition of benzimidazole or Ethrel. These latter compounds acted independently as judged by their kinetics of action and the inhibition of Ethrels effect only by CO 2 . During rapid elongation growth in indoleacetic acid, there was no increase in cell wall hydroxyproline. However, incubation in either growth-inhibitory agent increased hydroxyproline 3-fold. When this increase was blocked by dipyridyl incubation, growth was not inhibited in benzimidazole or Ethrel, but proceeded at the maximal rate. During long-term incubations in buffer, cell wall hydroxyproline increased and the sections eventually became unable to grow. However, if dipyridyl was added to block the hydroxyproline increase, growth potential remained. Elongation was inhibited by supraoptimal concentrations of indoleacetic acid. However, such inhibition did not occur in the presence of dipyridyl. These results indicate that an hydroxyproline-containing component is necessary in rendering the cell wall inextensible when elongation growth ceases.

Hydorxyproline biosynthesis in plant cells Peptidyl proline hydroxylase from carrot disks

Abstract Hydroxyproline occurs in many plant proteins and is especially abundant in the proteins associated with the primary cell wall. In vivo evidence indicates that the synthesis of peptidyl hydroxyproline occurs by the hydroxylation of peptidyl proline. We have shown that plant cells contain an enzyme which catalyzes this hydroxylation reaction. The hydroxylase has the following characteristics: it requires O2, Fe2+, ascorbate and an α-keto acid for activity; it does not react with free proline; it is localized in the soluble cytoplasm; it readily hydroxylates prolyl residues of protocollagen isolated from chick embryos. A partial purification of the enzyme was achieved by the use of (NH4)2SO4 and Ca3(PO4)2 gel fractionations.

LIPOSOMAL DAUNORUBICIN OVERCOMES DRUG RESISTANCE IN HUMAN BREAST, OVARIAN AND LUNG CARCINOMA CELLS

ABSTRACT Multi-drug resistance due in part to membrane pumps such as P-glycoprotein (Pgp) is a major clinical problem in human cancers. We tested the ability of liposomally-encapsulated daunorubicin (DR) to overcome resistance to this drug. A widely used breast carcinoma cell line originally selected for resistance in doxorubicin (MCF7ADR) was 4-fold resistant to DR compared to the parent MCF7 cells (IC50 79 nM vs. 20 nM). Ovarian carcinoma cells (SKOV3) were made resistant by retroviral transduction of MDR1 cDNA and selection in vinblastine. The resulting SKOV3MGP1 cells were 130-fold resistant to DR compared to parent cells (IC50 5700 nM vs. 44 nM). Small-cell lung carcinoma cells (H69VP) originally selected for resistance to etoposide were 6-fold resistant to DR compared to H69 parent cells (IC50 180 nM vs. 30 nM). In all three cases, encapsulation of DR in liposomes as Daunoxome (Gilead) did not change the IC50 of parent cells relative to free DR. However, liposomal DR overcame resistance in MCF7ADR breast carcinoma cells (IC50 20 nM), SKOV3MGP1 ovarian carcinoma cells (IC50 237 nM) and H69VP small-cell lung carcinoma cells (IC50 27 nM). Empty liposomes did not affect the IC50 for free DR in the three resistant cell lines, nor did empty liposomes affect the IC50 for other drugs that are part of the multi-drug resistance phenotype (etoposide, vincristine) in lung carcinoma cells. These data indicate the possible value of liposomal DR in overcoming Pgp-mediated drug resistance in human cancer.

Hydroxyproline-rich cell wall protein (extensin): Biosynthesis and accumulation in growing pea epicotyls

Abstract Plant cell walls contain a glycoprotein component rich in the otherwise rare amino acid hydroxyproline. We examined the synthesis and accumulation of wall hydroxyproline during different states of elongation growth in pea epicotyls. Light-grown peas contained more wall hydroxyproline than their taller, dark-grown counterparts. When elongation was studied by marking growing stems in situ , there was a marked accumulation of wall hydroxyproline coincident with the cessation of elongation. Dividing and elongating regions of the epicotyl showed less wall hydroxyproline than did regions where elongation was no longer occurring. Hydroxyproline biosynthesis was examined by incubation of excised sections of tissues in various growth states in 14 C-proline. The extent of conversion of these residues to 14 C-hydroxyproline served as a measure of the rate of hydroxyproline synthesis. This rate was highest in tissues which had ceased elongation. The low rate of hydroxyproline synthesis in dividing and elongating cells was probably not due to the inability to hydroxylate peptidyl proline or to secrete proteins. These data show a positive correlation between the synthesis and accumulation of cell wall hydroxyproline and the cessation of cell elongation in pea epicotyls.

Effects of four Chinese herbal extracts on drug-sensitive and multidrug-resistant small-cell lung carcinoma cells

Purpose: We examined the pharmacology, cell biology and molecular biology of small-cell lung carcinoma cells treated with four extracts of Chinese herbal medicines. Many cancer patients take these medicines, but their effects at the cellular level are largely unknown. We were especially interested in the effects on drug-resistant cells, as resistance is a significant clinical problem in lung cancer. Methods: Drug-sensitive (H69), multidrug-resistant (H69VP) and normal lung epithelial cells (BEAS-2) were exposed to extracts from two plants used in Chinese herbal medicine for lung cancer: Glycorrhiza glabra (GLYC) and Olenandria diffusa (OLEN), and to extracts of two commercially available combinations of Chinese herbal medicines, SPES (15 herbs) and PC-SPES (8 herbs). Cytotoxicity was measured in terms of cell growth inhibition (IC50). The kinetics of DNA fragmentation after exposure to the herbal extracts was measured by BudR labeling followed by ELISA. Apoptosis was measured by the TUNEL assay followed by flow cytometry. Expression of apoptosis- and cell cycle-related genes was measured by reverse transcription of mRNA followed by filter hybridization to arrays of probes and detection by chemiluminescence. Results: In each case, the four herbal extracts were equally cytotoxic to H69 and H69VP and less cytotoxic to BEAS-2. All four extracts induced DNA fragmentation in the lung carcinoma cells. The kinetics showed DNA fragments released to the medium (an indication of necrosis) in GLYC-exposed cultures, but inside the cells (an indication of apoptosis) in OLEN-, SPES- and PC-SPES-exposed cultures. TUNEL analysis confirmed that exposure to the latter three extracts, but not to GLYC, led to apoptosis. Compared to untreated and GLYC-treated cells, H69 and H69VP cells treated with OLEN, SPES and PC-SPES showed elevated expression of a number of genes involved in the apoptotic cascade, similar to cells treated with etoposide and vincristine. Conclusion: The Chinese herbal medicine extracts OLEN, SPES and PC-SPES are cytotoxic to both drug-resistant and drug-sensitive lung cancer cells, show some tumor cell specificity compared to their effect on normal cells, and are proapoptotic as measured by DNA breaks and gene expression. The reaction of the tumor cells to these extracts was similar to their reaction to conventional chemotherapeutic drugs.

Effect of Ganoderma on drug-sensitive and multidrug-resistant small-cell lung carcinoma cells

Multidrug resistance is a major problem in small-cell lung cancer (SCLC). Ganoderma lucidum is a widely used herb in traditional Chinese medicine. We tested the effects of Ganoderma on drug-sensitive (H69) and multi-drug resistant (VPA) human SCLC cells. Both cells showed equal cytotoxicity when incubated with extracts of mycelia of 9 species of Ganoderma, including G. lucidum. Cells treated with the IC(50) of cytotoxic Ganoderma and analyzed by flow cytometry-PI staining showed increases in S phase. When compared untreated controls or SCLC cells treated with extracts of non-cytotoxic Ganoderma species, cells treated with extracts of cytotoxic Ganoderma species responded with an induction of apoptosis similar to cells treated with the chemotherapeutic drugs etoposide and doxorubicin. This was shown by four criteria: increased DNA fragmentation within cells as measured by ELISA; increased TUNEL staining for DNA breaks; increased specific activities of caspases 3 and 9, but not caspase 8 by colorimetric assays, indicating the endogenous pathway; and similar patterns changes in the expressions of 9 genes involved in the cell cycle/apoptosis, as measured by RT-PCR and capillary electrophoresis. Pre-incubation of drug-resistant SCLC cells with cytotoxic Ganoderma reduced the IC(50) for etoposide (3.4-0.21 microM) and doxorubicin (0.19-0.04 microM). These results show that extracts of several species of Ganoderma are cytotoxic to both drug-sensitive and drug-resistant SCLC cells, are pro-apoptotic, induce gene expression patterns that are similar to SCLC cells treated with chemotherapeutic drugs, and can reverse resistance to chemotherapeutic drugs.

Effect of methadone addiction on glucose metabolism in rats

Female albino rats were exposed to methadone over a 35-day period by addition of the drug in their drinking water. The final dose of the drug was 1.8 mg/kg body weight per day. After this period, the drug was withdrawn from some animals for 30 days (postexposure). Compared to unexposed controls, serum glucose levels rose during exposure and returned to control levels postexposure. Oral glucose tolerance tests showed impairment in 35-day drug-exposed animals compared to controls and postexposure. The activities of three key enzymes of glycolysis and three key enzymes of gluconeogenesis were measured in liver during and at the end of the exposure period, as well as postexposure. Compared to unexposed controls and postexposure, specific activities of two glycolytic enzymes in livers of exposed animals-hexokinase and phosphofructokinase 1-were significantly reduced, whereas the activity of a third glycolytic enzyme-pyruvate kinase-was unchanged. The specific activities of two gluconeogenic enzymes-glucose-6-phosphatase and fructose-1,6-biphosphatase-were significantly elevated in the drug-exposed animals compared to controls, whereas the activity of a third enzyme-phosphoenolpyruvate carboxykinase-was unchanged. These data indicate that methadone addiction produces a metabolic state similar to insulin-resistant diabetes.

Development of Enzymes of Glycerol Metabolism in Human Fetal Liver

The activities of three key enzymes of glycerol metabolism were measured in liver samples from 37 human fetuses ranging in gestational age from 18 weeks to term, from neonates (1-3 days) and from infants to 2 years. Glycerol kinase specific activity was constant throughout the period of fetal development examined, and was comparable to that measured in neonates and infants. However, the subcellular distribution of the activity changed markedly, being predominantly particulate in fetal samples and cytoplasmic in postnatal samples. The particulate activity had an elevated Km for glycerol. Cytoplasmic glycerol-3-phosphate dehydrogenase activity was very low in the fetal period, and then rose to adult levels during infancy. There were no kinetic differences between the fetal and postnatal activities. Mitochondrial glycerol-3-phosphate dehydrogenase activity rose somewhat after birth to near adult levels. The data indicate that glycerol can be metabolized by human fetal, neonatal and infant liver.

Auxin, carbon dioxide and hydrogen ions

HYDROGEN ions can partially mimic the effect of indoleacetic acid (IAA) on plant cell elongation1. This observation has been elaborated in a proposal that the initial action of IAA causes the activation of a proton pump at the cell membrane. The pumping of protons would cause a lowering of the pH at the cell wall, leading to wall loosening2. This proposal has been supported in studies in which the pH of the medium surrounding oat coleoptile3–5 or etiolated pea stem6 sections was observed to drop when auxin was applied. This acidification was most pronounced when tissue sections were used from which the epidermis with the cuticle had been peeled off3, and was presumed to be caused by the proton pump activated by IAA.