Mechanism of action of Ukrain.

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Mechanism of action of NSC 631570 (Ukrain). In a recent in-vitro study where various pancreatic cancer cell lines were incubated with different concentrations of NSC 631570 and Thio-TEPA and chelidonine, it was found that NSC 631570 and chelidonine lead to a significant accumulation of cells in G2/M phase in all investigated cell lines in concentrations of 0.6 ?g/ml chelidonine or above and 5 ?g/ml NSC 631570 or above. At the same concentrations a significant reduction of proliferation rates after 48 hours was also observed (all cell lines: p < 0.05). In Giemsa stains, a significant accumulation of cells in the prophase was found; fluorescent immuno-histochemistry with antibodies against alpha-tubulin revealed that NSC 631570 and chelidonine lead to a disruption of the microtubule network in the investigated cell lines. Furthermore, it was shown that in in-vitro polymerisation assays NSC 631570 and chelidonine stabilise monomeric tubulin (Ramadani et al, 2001). In another experiment with pancreas cancer cell lines, NSC 631570 (10 ?g/ml) showed a high accumulation of treated cells in the G2/M phase, whereas the apoptosis rate of peripheral mononuclear cells did not show any differences between treated and untreated cells; mitogenstimulated lymphocytes even showed an increased blastogenic response (Ramadani et al, 2000). In another experiment using cancer cell lines A431 and ME180 as well as normal human keratinocytes as control, it was demonstrated that, at concentrations of 7?M NSC 631570, cancer cells but not human keratinocytes accumulate in G2/M phase over a 24 h period. In addition, apoptosis was detected following 48 h treatment (Roublevskaia et al, 2000). Other investigations on the possible mechanism of action of NSC 631570 on malignant cells (K562 leukaemia cells) showed similar results, suggesting that NSC 631570 induces bimodal cell death programmes: First, apoptosis, mediated by quinidine sensitive Ca2+-dependent K+- channels and second, blister cell death, by preventing microtubule formation, thus inducing polyploidy (Liepins et al, 1996). From these experiments it can be concluded that NSC 631570 inhibits the cell cycle progression of pancreatic and other cancer cells in M-phase by stabilising monomeric tubulin, thus being an anti-tubulin drug agent. 8 NSC 631570 also seems to inhibit (reversibly) angiogenesis at relatively low concentrations of 10-50 ?mol, approx. 15-75 ?g/ml (Koshelnick et al, 1998). In vitro tests by the National Cancer Institute (NCI), Bethesda, USA, demonstrated an inhibitory effect of NSC 631570 against all of the 8 colon cancer cell lines tested, at molar concentrations between 10-4.5 and 10-5.5 (corresponding to concentrations between ?7.6 ?g/ml and 76.0 ?g/ml). In contrast, 5-FU barely showed any inhibition of the same cell lines at 100 to 1,000-fold higher concentrations, not achieving lethal effects even at the highest concentration (10-2.5) in contrast to NSC 631570 which is lethal at a concentration of 10-3.5, i.e. ?760 ?g/ml (Nowicky et al, 1992). Dose dependence of in vitro cytotoxic effects against tumour cell lines has also been confirmed independently by other research groups: The European Organisation for Research and Treatment of Cancer (EORTC) found that NSC 631570 was cytotoxic against 5 of 6 colorectal cell lines (human xenografts) at concentrations of 100 ?g/mL (communication from the EORTC, New Drug Development Office, 9. June 1991). Fluoroscopic examinations of malignant cells show that NSC 631570 has a strong affinity to elements of the nuclei of cancer cells but not to normal cells. In a series of experiments with 14 different cell lines of human and animal origin, including normal and cancer cell lines, effects of 4 different doses of NSC 631570 (0.1, 1.0, 10, 100 mcg/ml) on DNA, RNA and protein synthesis was investigated by measuring the incorporation of 3H-labelled thymidine, uridine and leucine (Nowicky et al, 1996). Usually, a dose-dependent inhibition of all anabolic processes, DNA, RNA and protein synthesis was found that was more pronounced in malignant cells than in normal cells, even in those normal cell lines known for fast replication rates. According to the authors, no toxic effects were seen in normal cells treated in doses that are 100% growth inhibitory to cancer cell lines. Tumour tissues from human breast cancer, treated before surgery with NSC 631570 (5 mg i.v. every 2nd day for 20 days, followed by surgery 7-10 days later) show a number of striking changes compared to the untreated tumour of control patients (Brzosko et al, 1996): Histopathological examinations demonstrate that the tumour is surrounded by connective tissue (encapsulated) with massive infiltration by mononuclear cells (mostly lymphocytes and plasma cells). Many neoplastic cells surrounded by inflammatory infiltrates are degenerated, enlarged with vacuolated cytoplasm, undergoing necrosis or already necrotic. Immunfluorescence examinations show connective tissues within the tumour heavily embedded in IgG and the predominance of IgM-positive cells. Mononuclears surrounding and infiltrating the tumour are B-lymphocytes and T-lymphocytes that are almost exclusively CD8-positive. IgM can be found in the cytoplasm and in the nucleus of tumour cells, but also on the surface of the cell membrane; it is particular abundant in necrotic foci, covering all disrupted cell fragments. Due to its autofluorescence, NSC 631570 can also be detected within neoplastic cells. When tissues of ten patients treated with NSC 631570 were examined under the electron microscope, massive changes were again found in comparison to an untreated control group (Uglyanica et al, 1996). Under the influence of NSC 631570 the endoplasmatic reticulum underwent fragmentation, and mitochondria became swollen with the cristae damaged. In addition, the cytoplasm was also swollen with an increased number of lysosomes, phagolysosomes and myelin bodies indicating destruction of cancer cells. Ultrastructures of other cells, however, were not affected. Treatment with NSC 631570 also resulted in a markedly higher number of fibroblasts and extracellular connective fibres as compared to controls. Histochemical examinations demonstrated quantitative changes in the enzyme content, in particular in those enzymes which are key factors in the citrate cycle and therefore in the flow of cell respiration; these enzymes and coenzymes are responsible for the generation and transfer of energy in the form of ATP, e.g., SDH, LDH, NADH. On the other hand, the activity of glucose-6-phosphate-dehydrogenase and acid phosphatase was increased, indicating an enhanced process of destruction of cancer cells. From these observations it may be concluded that NSC 631570 has direct effects on cancer cells in humans as it can be found within the cytoplasma; but also indirect cytotoxic activity via immunological processes, possibly changing the antigenic expression of tumour cells. NSC 631570 has low toxicity. The LD50 in rats after i.v. application is 43 and 76 mg/kg b.w. (males and females respectively), in mice 80 and 68 mg/kg b.w. (Hruby, 2000). NSC 631570 has no cumulative toxicity and is - in cases where no tumour is present – rapidly excreted (Doroshenko et al, 2000). In a 6-month i.v. toxicity study with rabbits (0 - negative control, 0 - negative control recovery, 0.07 - low dose, 0.30 - mid dose, 0.70 - high dose and 0.70 mg NSC 631570 /kg b.w. - high dose recovery, groups of 6 animals each), statistically significant differences between dosed groups and the control group were observed with regard to bone marrow (sternum) with hypocellularity (mid dosed males and females, high dosed males), karyorrhexis (mid dosed males and females), inactive megakaryocytes (high dosed males), pyknosis (mid dosed females), cytolysis (mid dosed males) and with regard to the kidneys with proximal tubuli epithelium degeneration (high dosed males and females). Differences also occurred in white blood cells, with a slight increase of leukocytes, lymphocytes and bands in the high dose group (both sexes) after 4 months. Haematocrit and reticulocytes were also slightly increased in the high dose group. Occasionally, other differences between the groups were observed but can be considered as not medically relevant (ARCS, 2001). Reproduction studies have given no indications of teratogenic, mutagenic or cancerogenic properties of the preparation, even in doses, which were 100 times larger than the therapeutic dose. NSC 631570 does not induce sensitisation and is also not genotoxic (Chlopkiewicz et al, 1992; Wyczolkowska et al, 1992; ARCS, 1999; ARCS, 2000).