Anti-metastatic therapy by urinary trypsin inhibitor in combination with an anti-cancer agent.

We have demonstrated that urinary trypsin inhibitor (UTI) purified from human urine is able to inhibit lung metastasis of mouse Lewis lung carcinoma (3LL) cells in experimental and spontaneous metastasis models. In this study, we have investigated whether UTI in combination with an anti-cancer drug, etoposide, can prevent tumour metastasis and show an enhanced therapeutic effect. Subcutaneous (s.c.) implantation of 3LL cells (1 x 10(6) cells) in the abdominal wall of C57BL/6 female mice resulted in macroscopic lung metastasis within 21 days. Microscopic lung metastasis was established by day 14 after tumour cell inoculation, and surgical treatment alone after this time resulted in no inhibition of lung metastasis. The number of lung tumour colonies in the group of mice which received surgery at day 21 was greater than in mice which had tumours left in situ (P = 0.0017). Surgical treatment on day 7, followed by UTI administration (s.c.) for 7 days, led to a decrease in lung metastasis compared with untreated animals. A significant inhibition of the formation of pulmonary metastasis was obtained with daily s.c. injections of UTI for 7 days immediately after tumour cell inoculation. UTI administration did not affect the primary tumour size at the time of operation. In addition, etoposide treatment alone led to a smaller primary tumours and yielded reduction of the formation of lung metastasis in the group of mice which received surgery at day 14 (P = 0.0026). Even in mice which received surgical treatment on day 14, followed by the combination of UTI (500 micrograms per mouse, days 14, 15, 16, 17, 18, 19 and 20) with etoposide (40 mg kg-1, days 14, 18 and 22), there was significant reduction of the formation of lung metastasis (P = 0.0001). Thus, the combination of an anti-metastatic agent with an anti-cancer drug, etoposide, might provide a therapeutically promising basis for anti-metastatic therapy.     

Inhibition of pulmonary metastases in mice by beta-cyclodextrin-benzaldehyde

The effect of beta-cyclodextrin-benzaldehyde (CDBA) on the pulmonary metastasis in C3H/He mice was examined. When mice were treated daily with CDBA, 3 weeks later the number of lung nodules developed after i.v. inoculation of 1 X 10(6) RCT (+) cells was significantly decreased. The mean numbers of the lung nodules were 69.9 and 73.4 in the water-and cyclodextrin (CD)-treated mice, respectively. However, these were 17.8, 9.8 and 2.9 in 0.5, 5 and 25 mg/mouse per day CDBA-treated mice, respectively. And also, daily treatment of CDBA prolonged the survival time of the tumor bearing mice in both experimental and spontaneous metastasis studies. Two or three weeks after subcutaneous inoculation of RCT (+) cells (1 X 10(6) cells) to the foot pad, left hindlimbs were amputated and then mice were daily treated or untreated with CDBA. Five weeks after tumor inoculation, the number of lung nodules was counted. Twenty eight point six and 100% of untreated mice had lung metastases when amputation was carried out 2 (earlier operation group) and 3 (latero peration group) weeks after tumor inoculation. However, in CDBA-treated mice, these values were noticeably decreased, that is, 6.7% and 60% in earlier and later operation groups, respectively. Furthermore, in the later operation group, mean number of the lung nodules in CDBA-treated mice was only 2.7 while this was 12.9 in untreated mice. These data suggest that CDBA improve the survival time of tumor bearing mice through the inhibition of the lung metastasis.     

Deoxyribonuclease treatment prevents blood-borne liver metastasis of cutaneously transplanted tumour cells in mice.

Murine L5178Y-ML cells, when transplanted subcutaneously into the flank of (BALB/c x DBA/2)F1 mice, grew locally and always formed spontaneous metastases in the liver. Even after surgical removal of the primary tumour mass 5 or 7 days after tumour cell inoculation, all mice died due to liver metastases within 18 days. Using this model of tumour metastasis, we examined whether serine protease or deoxyribonuclease I (DNase I) would affect metastasis. Spontaneous liver metastasis of L5178Y-ML cells was enhanced by systemic administration of alpha-chymotrypsin at 3, 4 and 5 days or at 5, 6 and 7 days after tumour cell inoculation. This result was consistent with a previous report on blood-borne lung metastasis. In contrast, systemic administration of DNase I at 3, 4 and 5 days or at 5, 6 and 7 days after tumour cell inoculation inhibited liver metastasis. Neither treatment affected primary tumour growth. An influence of DNase I on tumour cell arrest in the microvasculature of the liver was suggested by scanning electron microscopy. DNase I treatment resulted in a statistically significant prolongation of the survival period, however, the effect was not satisfactory. A more striking anti-metastatic treatment resulting in a greater prolongation of the survival period was achieved by combining surgical removal of the primary tumour mass with DNase I treatment. These results suggest that DNase I could be a potential therapeutic agent used in conjunction with surgery to prevent clinical blood-borne metastasis.     

Antiplatelet pyrimido-pyrimidines and metastasis

The role of antiplatelet drugs in relation to their potential antimetastatic activities has been reviewed and the effects of two pyrimido-pyrimidine derivatives (RX-RA69 and RX-RA85) with strong antiplatelet activities investigated in metastasizing tumour models. The routes of administration and drug dosages were always chosen in such a way that good antiplatelet activities were obtained. RX-RA69 (20 mg/kg/day) given in the drinking water had no effect on spontaneous metastasis of Lewis lung carcinoma. RX-RA85 (20 mg/kg/day) did not influence spontaneous metastasis of B16 melanoma. On the other hand, giving RX-RA85 (8 mg/kg) daily i.p. to Lewis lung carcinoma bearing mice significantly increased the number of lung metastases but had no significant effect on primary tumour implant growth. Pretreating mice orally with 20 mg/kg RX-RA85 1 h before i.v. injection of B16 melanoma cells had no significant effect on lung colony number or distribution of extrapulmonary tumours while injecting the same dosage of RX-RA85 i.v. 1-2 h before tumour-cell injection decreased lung colony formation, but increased extrapulmonary tumour burden. This investigation like many others does not support the importance of platelets in metastasis formation.     

The effect of hyperbaric oxygen treatment on pulmonary metastasis in the C3H mouse

The incidence of pulmonary metastases in mice exposed to HBO treatment was investigated in two systems: artificial exogenous metastasis to the lung after i.v. inoculation of tumour cells and the endogenous metastatic spread from spontaneous tumours. Exposure to HBO decreased the lung colony count after inoculation with ELD cells, but had no effect on lung colonisation by C3H mammary tumour cell aggregates. Lung metastasis increased in mice with spontaneous tumours exposed to HBO. This is believed to be a result, not of transient lung damage associated with HBO, but of increased dissemination of cells from the primary tumour.     

Inhibition of lymph node metastasis by an anti-angiogenic agent, TNP-470.

We assessed the inhibitory action of TNP-470 on lymph node metastasis in a metastatic model system using athymic nude mice. Mice were injected subcutaneously with 5 x 10(6) HT-1080 cells in the right groin. TNP-470 (10, 30 and 100 mg kg-1) was injected subcutaneously nine times in total every other day from the 7th day after tumour inoculation. Axillar and inguinal lymph nodes were dissected, and DNA was extracted 5 weeks after tumour inoculation. Specific detection of a human beta-globin-related sequence in metastasized human tumour cells in nude mice was done by the polymerase chain reaction (PCR) technique and analysed by Southern blotting. Anti-tumour effects on primary sites were seen only in the 100 mg kg-1 treatment group. Lymph node metastasis of transplanted HT-1080 cells was seen in all mice of the no treatment group (5/5). On the other hand, incidences of lymph node metastasis in treated mice were 2/4 mice (100 mg kg-1, 2/5 mice (30 mg kg-1) and 4/5 mice (10 mg kg-1). The inhibition ratios of lymph node metastasis were 82.3% at 10 mg kg-1, 97.2% at 30 mg kg-1 and 97.5% at 100 mg kg-1 respectively. This agent may be useful to inhibit lymph node metastasis.     

Effect of interleukin-1-beta on metastasis formation in different tumor systems

Experiments were done to determine the effect of interleukin-1-beta (IL-1 beta) on metastasis formation in different tumor systems. Intravenous administration of 1 microgram of human recombinant IL-1 beta given 1 hour before tumor cell injection augmented lung colony formation (experimental metastases) by the human A375 melanoma variants, the human HT-29M colon carcinoma, the SN12-K1 renal carcinoma in nude mice, the murine B16 melanoma variants, and the murine UV-2237M fibrosarcoma in syngeneic recipients. The same treatment did not induce lung colony formation by a human rectal carcinoma (HCC-P2988) or by a murine reticulum cell sarcoma (M5076), both of which are not metastatic to the lung. Spontaneous metastases were studied in C57BL/6 mice bearing the B16-BL6 melanoma (metastatic to the lung) in their footpad and the M5076 reticulum cell sarcoma (metastatic to the liver) subcutaneously. Daily intraperitoneal treatment with 1 microgram of IL-1 beta increased lung and liver metastases. These findings indicate that treatment of mice with IL-1 beta can increase the number of artificial or spontaneous metastases and that this effect is not limited to a single tumor type or to a specific organ.     

Studies on the antitumor activities of pyrimidinone-interferon inducers. I. Effect against artificial and spontaneous lung metastases of murine tumors

Since pyrimidinone compounds induce interferon production in several animal species and have potent antivirus activities, it appeared important to determine whether these compounds could also induce antitumor activities in their recipients. Pyrimidinone compounds 2-amino-5-bromo-6-methyl-4-pyrimidinone (ABMP), 2-amino-5-brome-6-phenyl-4-pyrimidinone (ABPP), and 2-amino-5-iodo-6-phenyl-4-pyrimidinone (AIPP) were studied for their activities against artificial lung metastases of the weakly immunogenic spontaneous fibrosarcoma NFSa, the moderately immunogenic spontaneous mammary carcinoma MCa-K, and the strongly immunogenic 3-methylcholanthrene-induced fibrosarcoma FSa syngeneic to inbred C3Hf/Kam mice. In addition, the therapeutic efficacy of ABPP and AIPP was also determined against spontaneous lung metastases of NFSa. ABPP and AIPP given ip at 250 mg/kg for 2 or 3 consecutive days before or after iv inoculatin of NFSa, FSa, or MCa-K cells greatly reduced the number of tumor nodules developed in the lungs. ABMP, however, was considerably less effective. ABPP and AIPP were also effective in therapy of spontaneous lung metastases of NFSa, especially when these compounds were given before surgical removal of the primary tumor. Neither ABPP nor AIPP was effective against tumor nodules growing in whole-body irradiated (WBI) mice, but both protected mice against enhancement of lung metastasis formation induced by exposure to whole-body irradiation. ABPP was more effective than AIPP in inducing production of interferon in normal mice. When treated with ABPP, WBI mice, however, were unable to produce interferon. These results show that 6-phenyl-pyrimidinone compounds induce strong antitumor activities in mice, which correlated with neither tumor immunogenicity nor the ability of these agents to induce interferon, but which depended on the immune status of the tumor host.     

Distant metastasis facilitated by BCG: spread of tumour cells injected in the BCG-primed site.

Tumour metastasis in BCG-pretreated mice was studied using a methylcholanthrene-induced fibrosarcoma in C3H/He mice. When tumour cells were injected into the BCG-primed site, distant metastasis occurred in the lungs and the popliteal lymph node, through this tumour did not metastasize in normal mice. Such metastases were increased in proportion to the number of tumour cells injected into the BCG-primed site, and developed soon after tumour challenge. Concomitant immunity developed well in the mice bearing such metastases, but did not inhibit metastatic growth. Experiments using 125I-labelled SRBC or tumour cells revealed that such cells egressed rapidly from the BCG-primed site. When the tumour was inoculated into the contralateral foot to the BCG-primed site, the incidence and the number of metastases was reduced. Furthermore, BCG infection induced an increase of platelet count. I.v. injection of this tumour induced marked thrombocytopenia in normal mice. Administration of pentoxifylline, a methylxanthine derivative before tumour challenge reduced such metastases. These findings suggest that the changes in peripheral blood, such as increased platelet count and increased release of tumour cells from the injection site, facilitated distant metastasis in BCG-pretreated mice.     

Modification of tumor and normal tissue radioresponse in mice by N-methylformamide

The effects of the differentiation-inducing agent N-methylformamide (NMF) on the in vivo response of the murine tumor FSA and its pulmonary metastases to ionizing radiation were investigated. In addition, the radioresponse of acutely responding normal tissues was determined in mice receiving systemic NMF. A dosage of 300 mg/kg administered for 8 days had little effect on the FSA tumor growth, yet enhanced the growth inhibitory actions of ionizing radiation with dose enhancement factors ranging from 1.5 to 1.7. Administration of NMF also enhanced the radiation response of FSA micrometastases. The response to irradiation of hematopoietic tissue, jejunum, and testes in mice receiving NMF was also investigated. NMF administered before or before and after radiation enhanced the formation of endogenous spleen colonies, yet did not influence the LD50/30 for radiation. Jejunal crypt cell survival after radiation was slightly increased in mice receiving NMF, but the survival of spermatogonia after radiation was not affected. These data indicate that NMF administration results in an increase in the radiosensitivity of the FSA tumor and its metastases with no concomitant increase in the radiation response of the normal tissue tested. Thus, at least in this model system, a therapeutic gain is achieved through the combination of NMF and ionizing radiation.     

Clinically proven markers of metastasis predict metastatic spread of human melanoma cells engrafted in scid mice

Metastasis formation is a complex process and as such can only be modelled in vivo. As markers indicating metastatic spread in syngenic mouse models differ significantly from those in man, this study aimed to develop a human melanoma xenograft mouse model that reflects the clinical situation. Six human melanoma cell lines (LOX, MV3, FEMX-1, G361, MeWo and UISO-Mel6) were xenografted into severe combined immunodeficient mice and tumour growth, metastatic behaviour and number of lung metastases were assessed. Tumours and metastases were analysed for HPA binding and expression of CEACAM-1 and L1, all markers indicative of metastasis in clinical studies. Development of primary tumour nodules ranged from 3 weeks (MV3) to 3 months (MeWo). Whereas G361 and FEMX-1 rarely formed lung metastases, MeWo, MV3 and LOX were moderately and UISO-Mel6 was highly metastatic. Similar to clinical studies, HPA, CEACAM1 and L1 indicated metastatic spread in the xenograft melanoma model, but were not all simultaneously expressed in all cell lines. Considering the strongest expression of one marker combined with an absent or low expression of the other two markers, we conclude that LOX is the cell line of choice for analyses of the functional role of HPA-binding glycoconjugates, UISO-Mel6 is ideally suited to study CEACAM1 function in melanoma spread and L1 function can best be modelled using MeWo.     

Response of primary tumour, spontaneous metastases and recurrence of Lewis lung carcinoma (3LL) to flavone acetic acid (FAA, LM975)

B6D2F1 mice bearing 3LL were more sensitive to FAA than control mice, the LD50 being 180 x 2 and 336 x 2 mg/kg respectively. At a dosage of 140 mg/kg, injected i.p. at day 4 and 11, FAA significantly decreased the primary tumour growth, the occurrence and the growth of spontaneous pulmonary metastases. The effect of two injections was dose-dependent on the primary tumour and metastases; the survival time was also dose-related. Combined with primary tumour ablation, FAA administered before any dissemination (at day 3) was more efficient against metastases than when it was injected after the end of the dissemination (i.e. after primary tumour ablation). When all treatment schedules were pooled, the number of mice without metastasis was significantly higher in treated than in control groups. The effect of FAA on recurrences was also notable.     

Promotion of hepatic metastases by liver resection in the rat.

In the early period following radical hepatectomy for hepatoma, recurrences in the remaining liver are frequently found. In regenerating liver, implantation and growth of tumour cells released into the portal system during surgical treatment might be promoted. We examined the relationship between liver regeneration and the formation of metastases following hepatic resection. Intraportal injections of rat ascites containing hepatoma AH130 cells at a concentration of 1 x 10(5) cells 0.2 ml-1 were made at various periods following two thirds liver resection in rats. Tumour cell injections immediately at 24 h after surgery resulted in an increased number of hepatic metastases compared with control animals. Tumour cell injections 2 weeks after hepatectomy, however, had no significant difference in effect compared with control rats. In contrast, tumour cells injected immediately after removal of half of the caudate lobe resulted in the same number of metastases as control animals. These results demonstrate that the number of artificially induced hepatic metastases was increased during an initial period of active liver regeneration and was proportional to the volume of hepatectomy. The effect of 5-fluorouracil (5FU) or mitomycin C (MMC) as inhibitors of hepatic regeneration on liver metastasis after hepatectomy was studied. The administration of 5FU (20 mg kg-1) or MMC (0.2 mg kg-1) immediately, 24 and 48 h after hepatectomy resulted in a marked reduction in metastatic lesions. The administration of 5FU caused delays in weight gain and decreases in the wet weight of remaining liver, while MMC had no effect on either. Accordingly, results of 5FU administration may be due to inhibitory effects on liver regeneration whilst that of MMC administration may be due to cytocidal antitumour effect. The effect of OK-432 as an immunoactivator on the implantation and growth of tumour cells in regenerating liver was also studied. Pretreatment with OK-432, 0.5 mg intraperitoneally on 7 consecutive days, had no effect on hepatic metastases. The pathophysiology of liver regeneration may enhance hematogenous hepatic metastasis and release of tumour cells during surgical manipulation may represent an important cause of recurrence following hepatic resection.     

Treatment with a non-steroidal anti-inflammatory agent delays the growth of spontaneous pulmonary metastases of a mammary adenocarcinoma of non-detected immunogenicity.

Previous reports showed that treatment with non-steroidal anti-inflammatory agents (NSAIA) can alter the growth profile of a variety of tumours. In this study, the effect of NSAIA treatment on the growth of the primary tumour and the appearance of spontaneous pulmonary metastases, was investigated. A mammary adenocarcinoma of non-detected immunogenicity, C7HI, was grafted subcutaneously in the lateral flank of Balb/c mice. Oral treatment with approximately 1 mg kg-1 day-1 piroxicam delayed both tumour growth and the growth of pulmonary metastases. Survival of mice bearing the primary tumour was significantly lengthened by anti-inflammatory treatment. Similarly, in separate experiments, after surgical removal of the primary tumour by day 34 after grafting, the group of mice treated orally with piroxicam also exhibited a higher survival rate than the control group. Upon surgical removal of the primary tumour 34 days after grafting, piroxicam treatment significantly decreased both the number and size of pulmonary metastases. The results of this study lends support to the hypothesis that inhibition or modulation of inflammation may delay tumour organisation and growth. It is suggested that piroxicam treatment may be an appropriate adjunct therapy to delay the appearance of pulmonary metastases and to increase life-expectancy in a host whose primary tumour has to be surgically removed.     

Tumour metastasis in mice with reduced immune reactivity. III. Studies with three weakly antigenic tumours in thymectomized and/or sub-lethally irradiated mice.

A previous observation that increased spontaneous metastasis formation from the highly antigenic MCA-induced sarcoma MCG101 occurred in immunosuppressed C57BL/6J mice prompted the present study to establish whether or not tumours capable of inducing only weak transplantation resistance would behave similarly. One MCA-induced sarcoma, MCG12, one epidermoid carcinoma, EpCa1 in CBA mice and a spontaneous mammary carcinoma, MaCa1 in C3H mice, were transplanted into adult thymectomized and/or sub-lethally irradiated mice at different intervals after irradiation. The effects of the treatments on the immunoreactivity were monitored in separate groups by determining the primary and secondary responses to SRBC and the survival of first set allogeneic skin grafts. The treatment resulted in a significantly increased incidence of mainly the lymph-node metastases with MCG12 and of only the lung metastases with EpCa1. No increase in metastases was noted with MaCa1. Thymectomy potentiated the increase in metastases from MCG12 in irradiated mice but unexpectedly so only for a limited time after irradiation. Local growth of MCG12 and MaCa1 at the transplantation site was more rapid in females than in males. The changes in tumour growth and spread induced by the treatments varied with tumour-host system, sex and time of transplantation after irradiation and did not conform well with any of the tested parameters of immunoreactivity. There was no close parallel between the effects of the treatments on growth and spread of the tumours.     

Mediastinal lymph node metastasis model by orthotopic intrapulmonary implantation of Lewis lung carcinoma cells in mice

This study is designed to establish a pulmonary tumour model to investigate the biology and therapy of lung cancer in mice. Current methods for forming a solitary intrapulmonary nodule and subsequent metastasis to mediastinal lymph nodes are not well defined. Lewis lung carcinoma (LLC) cell suspensions were orthotopically introduced into the lung parenchyma of C57/BL6 mice via a limited skin incision without thoracotomy followed by direct puncture through the intercostal space. The implantation process was performed within approximately 50 s per mouse, and the operative mortality was less than 5%. Single pulmonary nodules developed at the implanted site in 93% of animals and subsequent mediastinal lymph node metastasis was observed in all mice that formed a lung nodule after intrapulmonary implantation. The size of tumour nodule and the weight of mediastinal lymph node increased in a time-dependent manner. The mean survival time of mice implanted successfully with LLC cells was 21+/-2 days (range 19-24 days). Histopathological analysis revealed that no metastatic tumour was detectable in the mediastinal lymph nodes on day 11, but metastatic foci at mediastinal lymph nodes were clearly observed on days 17 and 21 after implantation. Other metastases in distant organs or lymph nodes were not observed at 21 days after the implantation. Comparative studies with intrapleural and intravenous injections of LLC cells suggest that the mediastinal lymph node metastasis by intrapulmonary implantation is due to the release of tumour cells from the primary nodule, and not due to extrapulmonary leakage of cells. An intravenous administration of cis-diamine dichloro platinum on day 1 after tumour implantation tended to suppress the primary tumour nodule and significantly inhibited lymph node metastasis. Thus, a solitary pulmonary tumour nodule model with lymph node metastasis approximates clinical lung cancer and may provide a useful basis for lung cancer research.     

Metastasizing capacity of tumour cells from spontaneous metastases of transplanted murine tumours.

We investigated the metastasizing capacity of spontaneous lung metastases from the MN/MCA1 and mFS6 sarcoma, the B16 melanoma and colon 26 carcinoma. Spontaneous metastases at other visceral organs (liver, spleen, kidney, ovary, uterus) from the M5076/73A (M5) ovarian carcinoma and colon 26 carcinoma were also studied. Tumour cells from individual spontaneous metastases were used immediately after isolation from the normal parenchyma (mFS6, M5 and colon 26) and/or after 1 s.c. passage in syngeneic mice (MN/MCA1, mFS6, B16 and M5). Spontaneous metastases were examined for all tumours and their secondaries after i.m. or s.c. inoculation of tumour cells; artificial lung colonies were measured after i.v. injection only of cells from the primary mFS6 and MN/MCA1 and B16 or their spontaneous metastases. Individual spontaneous metastases were to some extent heterogeneous in their metastatic potential, a minority of the secondaries having greater or lesser metastatic capacity than the appropriate primary. Overall, tumour cells from spontaneous metastases did not show greater metastasizing capacity than primary neoplasms, nor was there evidence that metastases from specific organs (e.g. spleen and kidney) tended to home to the specific anatomical sites from which they were originally isolated. These observations in a series of murine tumours of different histology, transplantation history and pattern of metastasis, do not support the hypothesis that metastases are the ultimate expression of strong selection of variant cells with greater intrinsic metastatic potential, pre-existing within the primary tumour.     

Zoledronic acid suppresses lung metastases and prolongs overall survival of osteosarcoma-bearing mice

BACKGROUND: Although there is no doubt that bisphosphonates (BPs), specific inhibitors of osteoclasts, are beneficial for the treatment of bone metastases, their effects on visceral metastases are unclear. The effect of zoledronic acid (ZOL) was examined in vivo on lung metastasis progression and animal survival, and in vitro on the cellular mechanisms involved. METHODS: An animal model of lung metastasis was developed in C3H/He mice inoculated intravenously with a spontaneous murine osteosarcoma POS-1 cell line. Lung metastasis was determined at the time of autopsy. ZOL was assessed in vitro on POS-1 cell proliferation, cell cycle progression, and caspase-1 and -3 activities. RESULTS: The overall survival in five independent experiments (two series treated with ZOL 0.1 mg/kg twice a week, and three series with 0.1 mg/kg five times a week) showed a significant increase of the actuarial survival: 0.422 +/- 0.07 in ZOL-treated animals versus 0.167 +/- 0.07 in controls (P = 0.036). Lung metastases were absent in all ZOL-treated mice that survived more than 21 days postinjection as revealed by macroscopic and histologic analysis. In vitro, a 48-hour incubation with 10 microM ZOL inhibited POS-1 cell line proliferation associated with cell cycle arrest in S-phase. In addition, ZOL induced a weak increase of caspase-3 activity, but not caspase-1. CONCLUSION: We demonstrate that ZOL exerts a direct antitumor effect on POS-1 cells in vitro, significantly diminishes osteosarcoma-induced lung metastasis in vivo, thereby prolonging survival of POS-1-inoculated animals.     

Synergism between N-acetylcysteine and doxorubicin in the prevention of tumorigenicity and metastasis in murine models

The thiol N-acetylcysteine (NAC) is a promising cancer chemopreventive agent which acts through a variety of mechanisms, including its nucleophilic and antioxidant properties. We have recently shown that NAC inhibits type-IV collagenase activity as well as invasion, tumor take and metastasis of malignant cells in mice. NAC is also known to attenuate the cardiotoxicity of the cytostatic drug doxorubicin (DOX, Adriamycin). The present study was designed to evaluate whether the combination of NAC and DOX treatments in mice injected with cancer cells could affect their tumorigenic and metastatic properties. Six separate experiments were carried out, using a total of 291 adult female mice. In experimental metastasis assays, in which B16-F10 melanoma cells were injected i.v. into (CD-1)BR nude mice, DOX significantly reduced the number of lung metastases when administered i.v. at a dose of 10 mg/kg body weight, 3 days after the i.v. injection of cancer cells. NAC inhibited lung metastases when added to the medium of cancer cells before their i.v. injection. The combined treatment with DOX and NAC, under various experimental conditions, was highly effective, showing a synergistic reduction in the number of mestastases. In tumorigenicity and spontaneous metastasis assays, in which B16-BL6 melanoma cells were injected s.c. into the footpad of C57BL/6 mice, DOX decreased the number of lung metastases when given i.p. at 2 mg/kg body weight. Oral NAC exerted significant protective effects, and considerably prolonged survival of mice. The combined treatment with DOX and NAC again showed synergistic effects on the frequency and weight of primary tumors and local recurrences, and completely prevented the formation of lung metastases in the experiment in which these end-points were evaluated at fixed times. While injection of DOX7 days after implantation of cancer cells failed to improve the cancer-protective effects of NAC, its injection after 1 day resulted in a striking inhibition of lung metastases. These findings demonstrate an evident synergism between DOX (given parenterally) and NAC (given with drinking water) in preventing tumorigenicity and metastases. The indications of these animal studies warrant further evaluation in clinical trials. © 1996 Wiley-Liss, Inc.     

Enhancement of experimental metastasis by pretreatment of tumour cells with hydroxyurea

The effect of treatment with hydroxyurea (HU), on subsequent experimental metastasis formation by cells of 3 murine melanoma lines has been examined. In vitro treatment of B16-F1, B16-F10 and K-1735-clone 19 cells with 0.1 mM or 0.3 mM HU, followed by a period of recovery in drug-free medium, resulted in a significant increase in the number of lung nodules formed in syngeneic mice following i.v. injection of the cells. The maximum effect obtained was observed when cells were injected 6 hr after removal of HU and it occurred in spite of the cytotoxicity of the doses used. Six hours after release from treatment with 0.3 mM HU cells were synchronized in the G2 phase of the cell cycle but the consequent increase in cell volume was not responsible for increased metastasis through enhancement of tumour-cell arrest, since lodgement of 51Cr-labelled, HU-treated cells was no greater than that of control cells. The results obtained suggest that the chemotherapeutic agent HU may facilitate tumour progression via a direct action on neoplastic cells.