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EFFICACY: the in vivo studies


The anticancer effect of the intravenous administration of NSC 631570 (4 µg/day) was demonstrated in the tests on BALB/c mice with the implanted rapidly proliferating mammary adenocarcinoma D1 DMBA-3. Three routes of administration were employed for NSC 631570, e.g. intravenous, intraperitoneal and subcutaneous. Intravenous administration was proven to be the most effective and inhibited the tumor growth significantly. The authors evaluated also the role of immune factors in the effect of NSC 631570 and revealed this agent can restore the cytotoxic effect of macrophages (26, 43).

Later this mode of action was confirmed in a study on CBA mice. Once again the role of macrophages in the antitumor effect of NSC 631570 was stressed by the authors of the study (120).

In an experimental study NSC 631570 inhibited the growth of the primary melanoma B-16 and its metastases in mice (107).

In the tests with mice C57BL6 NSC 631570 inhibited the growth and metastazing of the primary implanted Lewis’ carcinoma compared to the control group (219). This effect of NSC 631570 was confirmed in another study. Here too, the intravenous administration was the most effective (254).

In a study on 18 mice CBA, the mixture of 0.1 mg NSC 631570 and 10000 carcinoma cells Krebs-2 was injected intramuscularly. In the control group only tumor cells were injected. After 11 days a significant inhibition of the tumor growth was established in the group treated with NSC 631570. Similar effect was achieved also in mice with implanted hepatoma cells (118).

In mice with implanted hepatoma HA-1, NSC 631570 increased the concentration of procathepsin B in the ascitic fluid whereas the concentration of cathepsin B decreased. Moreover, the concentration of the macrophageal p-hexosaminidase increased. This indicates an inflow of macrophages into the peritoneum (119).

The anticancer effect of NSC 631570 was also confirmed on Wistar rats with implanted sarcoma-45 (122).

Two different forms of Ehrlich’s carcinoma cells were transplanted intraperitoneally and subcutaneously to mice. NSC 631570 was administered intraperitoneally for six days. The effect of the drug on the tumors was estimated by the indexes of the tumor growth inhibition (TGI), total number of tumor cells in ascitic fluid, number of viable tumor cells and average life span of experimental animals. Cell cycle distribution of cancer cells was determined by flow cytometry. The number of circulating phagocytes was estimated by flow cytometry using FITC-labelled S. aureus. Intraperitoneal administration of NSC 631570 in mice with ascitic form of Ehrlich’s carcinoma resulted in moderate tumor growth inhibition, but was accompanied by acute local inflammation and caused reduction of life span of experimental animals. In the mice bearing the solid variant of Ehrlich’s carcinoma the treatment with NSC 631570 led to significant TGI and slight increase of life span. The treatment caused restitution of the number of circulating phagocytes in peripheral blood in the both groups of animals. The authors concluded the antitumor effect of NSC 631570 was mediated by its direct proapoptotic action as well as by the interactions with the murine immune competent cells (262).


The Effect on the Growth of Melanoma B16 in Mice

The effect of NSC-631570 alone or in combination with pathogen-associated molecules (PAM) on the growth of low- and high-metastasizing melanoma B16 in mice was studied. NSC-631570 was administered intravenously and PAM intramuscularly to tumor-bearing mice seven times every third day, starting from the second day after the transplantation of tumor cells. The effect of monotherapy and combined therapy on tumor growth was evaluated by the indices of tumor growth inhibition in experimental animals. Cell cycle distribution of cancer cells was determined by flow cytometry. TAP1 and TAP2 expression was evaluated by RT-PCR. The metabolic activity of phagocytes was determined by NBT-test, phagocytosis was tested by flow cytometry, and arginase activity was estimated by colorimetric determination of urea. Combined therapy and monotherapy with NSC-631570 resulted in significant inhibition of tumor growth in melanoma-bearing mice. Monotherapy with Ukrain was more effective in mice with high-metastasizing tumors. The therapeutic efficacy of NSC-631570 used in combination with PAM was more expressed in mice with low-metastasizing melanoma. Authors concluded the effectiveness of monotherapy and combined therapy with NSC-631570 in the treatment of melanoma B16 depends on the biological properties of the tumor and the immune state of the animal (263).


The Effect on Cystein Proteases

In mice with implanted LS-lymphosarcoma and HA-1 hepatoma, the concentrations of cystatin C, cathepsin B and cathepsin L were estimated after the treatment with cyclophosphamide and NSC 631570. An increased concentration of cystatin C was found only in hepatomas treated with NSC 631570. The concentrations of cathepsin B and cathepsin L were increased in LS lymphosarcoma. These results indicate apoptosis as the mechanism of action of NSC 631570 in these tumor models (169).

On the same tumor models the effect of NSC 631570 on cathepsin D was studied. Cathepsin D is an important lysosomal protease, modulating apoptosis induced by interferon-gamma and TNF-alpha. NSC 631570 combined with cyclophosphamide increased cathepsin D activity in mice with LS-lymphosarcoma. NSC 631570 alone did not exert this effect (170).

Cystatin C is a well known extracellular protease inhibitor and has been used as a possible marker of the tumor growth and efficacy of the anticancer therapy (204). The using NSC 631570 in mice brought about a 4fold increase of cystatin C in the tumor tissue (171).

In the tests on male mice CBA/C57BI/6J the serum concentration of cystatin C after the implantation of Lewis’ adenocarcinoma was only half as high as in the control group. The combined treatment with NSC 631570 and cyclophosphamide normalised the cystatin values (218).

On the experimental mice tumors LS-lymphosarcoma, hepatoma HA-1 and Lewis’ adenocarcinoma, the effect of NSC 631570 on the cystatin A (stefin A) was studied. The treatment with NSC 631570 caused the increase of stefin A in the liver of animals with Lewis’ adenocarcinoma, but not in the tumor itself. In other tumor models the concentration of cystatin A both in the liver and tumor tissue did not change. In LS-lymphosarcoma and hepatoma HA-1 the serum concentration of cystatin A increased in the course of the treatment with NSC 631570 (220).

The treatment of the hepatoma HA-1 implanted mice with NSC 631570 caused the deceleration of the tumor growth and increased the survival of the animals. The macrophages count increased whereas the number of tumor cells in the ascitic fluid diminished. NSC 631570 did not affect the activity of cathepsin B and cathepsin L (199).