In vitro cytotoxicity of S16020-2, a new olivacine derivative.

S16020-2 is a new olivacine derivative which has recently shown a marked antitumor activity in various experimental models. This study was undertaken in order to measure the inhibition of the proliferation of various sensitive and resistant tumor cell lines, by S16020-2, and to obtain information concerning its mechanism of action. For a continuous exposure, S16020-2 was as cytotoxic as adriamycin (ADR) (mean IC50 of about 28 nM) and on average, 46 fold more potent than elliptinium acetate (ELP), against a panel of 20 non-multidrug resistant cell lines. With a short exposure (1 hour) followed by a post-incubation of 95 hours in drug-free medium, S16020-2 was 5 and 6 fold more cytotoxic than ADR for human lung A549 and murine melanoma B16 cells, respectively. Furthermore, S16020-2 inhibited more actively the formation of colonies issued from proliferating cells, compared to colonies issued from quiescent A549 cells. Because quiescent cells demonstrated a 3 fold lower level of topoisomerase II alpha (topo II) than proliferating cells, these results suggest that this enzyme could be a potential target for S16020-2. In addition, as demonstrated by flow cytometric studies, S16020-2 intercalated into DNA and induced a cell cycle arrest in G2. Cell lines displaying the multidrug resistance (MDR) phenotype, P388/ADR-1, P388/ADR, P388/VCR-20, KB-A1, DC-3F/AD, S1/tMDR, and Colo320DM, were more sensitive to S16020-2 than to ADR or ELP, as shown by the mean resistance factors, 8, 201, and 23 respectively. In addition, the two cell lines displaying the pure classical MDR phenotype, linked exclusively to the P-glycoprotein (P-gp) overexpression (P388/VCR-20 and S1/tMDR), were as sensitive to S16020-2 as their sensitive parental counterparts, although they were resistant to ADR. S16020-2 is thus one of the most potent olivacine and ellipticine derivative yet characterized. The good cytotoxicity of S16020-2 against cells displaying a P-gp-mediated multidrug resistance, and its antitumor activity in vivo delineate an important chemotherapeutic potential for this drug.

Expression of topoisomerases II alpha and beta in Chinese hamster lung cells resistant to topoisomerase II inhibitors.

Chinese hamster lung cells resistant to 9-OH-ellipticine, i.e., DC-3F/9-OH-E cells, are several hundredfold resistant to DNA topoisomerase II inhibitors. According to previous studies, this resistance is associated with reduced topoisomerase II activity (about 4-fold) and decreased capacity of the topoisomerase II inhibitors to induce stabilization of the cleavable complex (about 10-fold). In the present work, an antibody was raised against a fragment of human topoisomerase II alpha. This antibody, which recognizes both isoforms, was used to determine the amounts of topoisomerases II alpha and beta in the sensitive and resistant cells. Northern and immunoblot analyses showed that (i) in the parental DC-3F cells the alpha enzyme is about 20-fold more abundant than the beta enzyme and the enzyme isoforms undergo reciprocal regulation during the cell growth phases, with the expression of the alpha enzyme dropping at the plateau phase while the expression of the beta enzyme increases, and (ii) in the resistant cells the amount of alpha enzyme is about 4-5-fold smaller than that in the sensitive cells, whereas the beta enzyme is almost undetectable. Analysis of DNA restriction sites in several independently selected resistant subclones revealed some rearrangements in the beta gene in two clones. However, these gene alterations did not correlate with changes in the resistance level. The relative contribution of these different changes to the resistance phenotype is discussed.

[Cellular resistance to DNA-topoisomerase II inhibitors]. / Résistance cellulaire aux inhibiteurs de l'ADN topo-isomérase II.

Chinese hamster lung cells resistant to 9-OH-ellipticine (DC-3F/9-OH-E) present a complex phenotype. These cells, which are about 150-fold resistant to 9-OH-E, display a cross-resistance to other topo-II inhibitors, such as m-AMSA or VP-16, which stabilize the cleavable complex. In addition, these cells display also a cross-resistance to suramin, which is also a topo-II inhibitor, but does not stabilize the cleavable complex. Finally, DC-3F/9-OH-E present a multidrug-resistant phenotype (MDR) which confers a cross-resistance to natural products such as actinomycin D, taxol or vincristine, due to a decrease of cellular accumulation of these drugs. Analysis of expression of the genes encoding topo-II alpha and beta, and the evaluation of both enzyme forms by immunoblotting, revealed that DC-3F cells contained about 20-fold less of the beta form than of the alpha form. The alpha form was decreased by about 4-5-fold in DC-3F/9-OH-E, whereas the beta form became undetectable. Purification and characterization of topo-II activities in sensitive and resistant cells is presently in progress. Analysis of the expression of pgp1, 2, 3 genes, involved in the MDR phenotype in hamster, by Northern blotting or by immunoblotting, has shown that the MDR phenotype in DC-3F/9-OH-E cells is due to the overexpression of pgp1 gene. In these cells, pgp3 expression is positively regulated by myc oncogene expression. Overexpression of the myc gene is followed by an overexpression of the pgp3 gene and is associated to a reversal of the MDR phenotype.

Specific binding sites on human phagocytic blood cells for Gly-Leu-Phe and Val-Glu-Pro-Ile-Pro-Tyr, immunostimulating peptides from human milk proteins.

Two immunostimulating peptides were isolated from human milk proteins by enzymatic digestion, the tripeptide GLF and the hexapeptide VEPIPY. These peptides increased the phagocytosis of human and murine macrophages and protected mice against Klebsiella pneumoniae infection. The present study showed that this activity may be correlated to the presence of specific binding sites on human blood phagocytic cells. The receptor molecules implicated were different for the two peptides. [3H]GLF specifically bound to PMNL and monocytes, whereas [3H]VEPIPY only bound to monocytes. The leukemic promyelocytic cell line HL-60 differentiated into granulocytes or into macrophages (depending on inducer used) coroborated these results. Specific binding of [3H]GLF on plasma membrane preparations of human PMNL (20 degrees C) was saturable and Scatchard analysis indicated two classes of binding sites: high-affinity sites of Kd 2.3 +/- 1.0 nM and Bm 60 +/- 9 fmol/mg protein and low-affinity sites of Kd 26.0 +/- 3.5 nM and Bm 208 +/- 45 fmol/mg protein. [3H]GLF binding was inhibited in a concentration-dependent manner by various analogous peptides, such as LLF, GLY, LLY and RGDGLF, but not by RGD, RGDS, VEPIPY and the chemotactic peptide f-Met-Leu-Phe (f-MLF). Only at high concentrations the direct analog MLF competed with labeled GLF. An important inhibitory effect was also observed with C1q component of the complement whereas C3 and BSA were uneffective. Specific binding of [3H]VEPIPY on monocyte membranes (20 degrees C) was saturable and Scatchard analysis was consistent with one class of binding sites of Kd 3.7 +/- 0.3 nM and Bm 150 +/- 6 fmol/mg protein.