A key in vivo antitumor mechanism of action of natural product-based brassinins is inhibition of indoleamine 2,3-dioxygenase.

Agents that interfere with tumoral immune tolerance may be useful to prevent or treat cancer. Brassinin is a phytoalexin, a class of natural products derived from plants that includes the widely known compound resveratrol. Brassinin has been demonstrated to have chemopreventive activity in preclinical models but the mechanisms underlying its anticancer properties are unknown. Here, we show that brassinin and a synthetic derivative 5-bromo-brassinin (5-Br-brassinin) are bioavailable inhibitors of indoleamine 2,3-dioxygenase (IDO), a pro-toleragenic enzyme that drives immune escape in cancer. Like other known IDO inhibitors, both of these compounds combined with chemotherapy to elicit regression of autochthonous mammary gland tumors in MMTV-Neu mice. Furthermore, growth of highly aggressive melanoma isograft tumors was suppressed by single agent treatment with 5-Br-brassinin. This response to treatment was lost in athymic mice, indicating a requirement for active host T-cell immunity, and in IDO-null knockout mice, providing direct genetic evidence that IDO inhibition is essential to the antitumor mechanism of action of 5-Br-brassinin. The natural product brassinin thus provides the structural basis for a new class of compounds with in vivo anticancer activity that is mediated through the inhibition of IDO.

Importance of timing and length of administration of angiogenesis inhibitor TNP-470 in the treatment of K12/TRb colorectal hepatic metastases in BD-IX rats.

BACKGROUND: The timing and length of administration of angiogenesis inhibitor TNP-470 was altered to evaluate the effect on disease progression in a rat model of colorectal hepatic metastases. METHODS: Pair-fed BD-IX rats, injected intrasplenically with rat colon adenocarcinoma K12/TRb cells at day 0, were randomized to receive subcutaneous injections of either placebo or 15 mg/kg TNP-470 on alternate days: for 2 weeks beginning 24 hours after tumor inoculation ("Early"), for 4 weeks beginning 24 hours after tumor inoculation ("Prolonged"), or for 2 weeks beginning at day 15 after macroscopic tumor nodules were confirmed ("Delayed"). Response to treatment was evaluated by counting tumor nodules on the surface of the liver at laparotomy on day 14 and 28 after tumor inoculation. The animals were followed for survival and cause of death. RESULTS: Maximal suppression of hepatic metastases at day 28 required 4-week rather than 2-week TNP-470 administration. Prolonged TNP-470 administration resulted in significantly fewer hepatic metastases at day 28 compared to control (P < .05). Early and prolonged TNP-470 improved survival (Wilcoxon test, P < .05) compared with delayed TNP-470 and placebo. Delayed TNP-470 administration did not increase survival or significantly diminish the number of metastases at day 28 compared with placebo. CONCLUSIONS: These data suggest that prolonged adjuvant antiangiogenic therapy may suppress colorectal hepatic micrometastases.

Regional chemotherapy for colorectal hepatic metastases: evidence for improved survival with new drug combinations.

BACKGROUND: In patients with colorectal hepatic metastases, response rates with hepatic arterial infusion (HAI) FUdR (5-Fluoro-2-deoxyuridine) are significantly higher than with systemic fluoropyrimidines. We report a novel animal model of intrahepatic therapy for hepatic metastasis for the study of methods to increase response rates and improve survival. METHODS. BD-IX rats are injected intrasplenically with K12/TRb cells. When hepatic metastases are established, animals are treated with hepatic or systemic chemotherapy, and the response to treatment, survival, and cause of death is determined. RESULTS: Significant responses were observed with low- and high-dose HAI FUdR (p = 0.03 and 0.001, respectively). Only high-dose FUdR controlled hepatic disease. HAI FUdR alone did not prolong survival compared with control, but combination systemic FUdR and HAI FUdR did (p = 0.04). Continuous HAI of either 5-fluorouridine or mitomycin C has not previously been reported. There was no significant difference in response to FUdR, 5-fluorouridine, or mitomycin C. However, combination HA bolus mitomycin C plus either HAI 5-fluorouridine or HAI mitomycin C showed synergy with improved survival compared with all treatment groups (p < 0.0001). CONCLUSIONS: The combination of bolus hepatic artery mitomycin C with either HAI mitomycin C or HAI 5-fluorouridine yields significant response rates, and survival is improved by this novel combination therapy.

Hepatic artery dexamethasone infusion inhibits colorectal hepatic metastases: a regional antiangiogenic therapy.

BACKGROUND: A randomized trial treating colorectal hepatic metastases demonstrated that hepatic arterial floxuridine (FUdR) with dexamethasone increased tumor response compared with hepatic arterial FUdR alone (Cancer 1992;69:327-34). The mechanism of this improvement is unclear. METHODS: We investigated the effect of hepatic arterial dexamethasone with or without FUdR on the growth of colorectal hepatic metastases in an animal model. BD-IX rats were inoculated intrasplenically with 10(7) K12/TRb colon cancer cells on day 0. On day 14, the hepatic metastases were counted and hepatic arterial catheters placed for chemotherapy. Forty-eight animals were randomized to 4 groups for 14 days of infusion with heparinized saline alone (group A), heparinized saline with dexamethasone 0.03 mg/kg/d (group B), heparinized saline with FUdR 2 mg/kg/d (group C), or heparinized saline with dexamethasone 0.03 mg/kg/d plus FUdR 2 mg/kg/d (group D). The hepatic metastases were recounted by laparotomy on day 28. Response in each rat was expressed in terms of percentage change in number of hepatic nodules between the number of hepatic nodules seen on days 14 and 28. In vitro chemosensitivity of K12/TRb to dexamethasone with or without FUdR was examined using an MTT (3-(4,5-dimethylthiazole-2-yl-2,5-diphenyltetrazolium bromide; Sigma, St. Louis, MO, U.S.A.) assay. The effect of dexamethasone on tumor-induced angiogenesis was tested using an in vivo assay. RESULTS: The mean percentage change in tumor nodules was +129% in group A, +17% in group B, -4% in group C, and -29% in group D (p = 0.002 A vs. B, p = 0.04 C vs. D). The MTT assay showed that dexamethasone had no direct effect on K12/TRb growth or on tumor FUdR sensitivity. Dexamethasone inhibited K12/TRb-induced angiogenesis in vivo. CONCLUSIONS: Hepatic arterial dexamethasone is effective in treating colorectal hepatic metastases and is more effective when combined with hepatic arterial FUdR. The antiangiogenic activity of dexamethasone may partially contribute to its efficacy.

Short-term intrahepatic FUdR infusion combined with bolus mitomycin C: reduced risk for developing drug resistance.

This study evaluates tumor response, survival, and development of resistance to HAI chemotherapy, comparing a combination of bolus MMC and short duration FUdR to short duration FUdR alone or to long duration FUdR alone, using a rat hepatic metastases model. After intrasplenic injection of 10(7) K12/TRb colon cancer cells in BD-IX rats on day 0, hepatic metastases were evaluated and HA catheters were placed on day 14. The response was determined on day 28. Chemosensitivity of the hepatic metastases after HAI treatments was determined using the MTT assay. Bolus MMC with short duration FUdR as well as long-term FUdR alone provided better hepatic tumor response and survival than short-term FUdR alone. However, bolus MMC with short duration FUdR decreased the acquired resistance to FUdR, compared to long-term FUdR, without causing resistance to MMC. These results provide a rationale for using short duration of FUdR in combination with other drugs.

Localization of complement-fixing cytotoxic monoclonal antibodies to subcutaneous tumors and liver metastases in a syngeneic immunocompetent rat colon carcinoma model.

To study the immune effects of complement-fixing cytotoxic monoclonal antibodies (mAbs) in a syngeneic immunocompetent animal model, mouse mAbs reactive with the transplantable rat colon carcinoma K12/TRb were generated. This system was used in part because rats have a complement system superior to that of mice. Seven murine IgG mAbs that reacted strongly with cell surface determinants of the K12/TRb rat colon carcinoma cell line were produced by immunizing MRL/Mp-1pr/1pr autoimmune mice, known to produce an increased amount of complement-fixing IgG2a and IgG3 immune cytotoxic antibodies, with K12/TRb cells. These mAbs were screened for their specificity of reaction, and two of these mAbs were extensively tested for their ability to lyse cells in vitro and localize to K12/TRb tumors in syngeneic BD IX rats. IgG2a mAbs 27-3 and 61-5 were able to mediate both complement and lymphocyte cytotoxicity in vitro and localize to subcutaneous tumors and liver metastases in this immunocompetent rat model.

Quantitation of total metastatic tumor volume in the rat liver: correlation of MR and histologic measurements.

Assessing tumor response to chemotherapy in the liver has always been difficult. Most investigators estimate tumor volume as either a product of the two perpendicular diameters of a tumor nodule, or, in animal studies, simply count surface tumor nodules. The authors evaluated magnetic resonance (MR) imaging as a technique for determining absolute tumor volume in the liver in an animal model. Specifically, histologic volumetric and MR imaging measurements of tumor and liver volumes were quantitatively compared over a wide range of tumor burdens in a rat model of hepatic metastasis of a colorectal carcinoma. Twenty-three rats were imaged, with two different section thicknesses used in each animal. Both section thicknesses showed highly significant correlations between MR and histologic measurements for both tumor and liver volumes (P less than .001). MR imaging may be useful for noninvasively quantifying tumor burden and temporal response of metastatic disease in the liver to novel antineoplastic regimens.

Adjuvant chemotherapy for colorectal hepatic metastases: role of route of administration and timing.

Improved results in the adjuvant and therapeutic treatment of colon cancer has led to renewed interest in the role of adjuvant chemotherapy following liver resection for colorectal hepatic metastases. However, little is known about the most effective method or timing of delivery of adjuvant chemotherapy. Sixty-nine BD-IX rats underwent a right hepatic lobectomy following tumour inoculation via a splenic injection of 10(7) K12/TRb colon cancer cells. The rats were then randomized to receive systemic FUdR (1 mg kg-1 d-1 for 7 d) or regional (hepatic artery or portal vein) FUdR (2 mg kg-1 d-1 for 7 d) immediately or 72 h following tumour injection. On Day 28, a laprotomy was performed, and tumour nodules in the liver were counted. The animals were followed to death, and at autopsy the cause of death from hepatic or extrahepatic metastases was determined. All methods of FUdR infusion were superior to no treatment. Immediate portal vein (PV) FUdR infusion delayed the appearance of hepatic tumour (P = 0.003), changed the cause of death from hepatic to extrahepatic disease (P = 0.019), and prolonged survival (P < 0.05). Infusion of FUdR via the PV 72 h later did not delay the appearance of hepatic tumours nor prolong survival. In contrast, delayed HA FUdR infusion controlled hepatic metastases (P = 0.04) and improved survival (P < 0.05).