Localization of human colorectal tumor xenografts in the nude mouse with the use of radiolabeled monoclonal antibody

For the evaluation of the clinical usefulness of monoclonal antibodies as diagnostic or therapeutic reagents, tumor localization must be clearly demonstrated in an experimental model. In this report, nude mice carrying two human tumor xenografts--a colon carcinoma (Colo 205) and a melanoma (Colo 239)--were given ip injections of radiolabeled monoclonal antibodies. Monoclonal antibody 250-30.6, which reacted specifically with the colon carcinoma but not with the melanoma, was labeled with 125I, while a second monoclonal antibody of similar immunoglobulin subclass, but unreactive with either cell type, was labeled with 131I. Both antibodies were injected simultaneously, and either the mice were scanned with a gamma camera or their tissues were removed and the localization of radiolabeled antibody was calculated with the use of localization index (LI)--the ratio of the tissue to blood distribution for each isotope. The studies showed that specific localization had occurred, there being a colon tumor LI of 6 at 2 days. Tumors of 150-300 mg (mean diameter, 6 mm) and with an LI as low as 1.5 could be successfully imaged after computer-assisted background subtraction. This study demonstrated that relatively small human tumor xenografts in the nude mouse can be specifically detected with the use of paired monoclonal antibodies, each labeled with a different isotope.     

Comparative studies on the antitumor activity of the fluorinated pyrimidines 5'-DFUR, tegafur, UFT and FUra on various murine tumors

Antitumor activities of fluorinated pyrimidines by oral administration were compared with various murine tumor models. 5'-Deoxy-5-fluorouridine (5'-DFUR) showed a better antitumor activity in terms of the growth inhibition and increase of the survival time than those of 5-fluorouracil(FUra), tegafur and UFT, particularly with respect to the chemotherapeutic indices. The activity of these fluorinated pyrimidines were further investigated in more detail with mice bearing colon 26 adenocarcinoma to suggest some means to optimize their treatment regimens in clinical trials. 5'-DFUR showed the activity irrespective of the size of tumor mass at the time of start of therapy. Additionally only 5'-DFUR was safely administered to the mice daily for long period up to more than 100 days, suppressing the tumor growth and increasing the survival to great extent.     

Expression of MUC1 and MUC2 mucins by human tumor cell lines.

The secretion and nature of mucins produced from a panel of recently available new gastric and colon carcinoma cell lines (LIM1839, LIM1215, LIM1863, LIM1899, LIM2099, LIM2405, LIM2408, LIM2412, LIM2463), as well as other colon (LS174T, HT29, HT29-SB, COLO533, COLO206), breast (T47D, MCF-7, BT20, ZR75-1) and ovarian (COLO316) tumor cell lines, was investigated. ELISA and Western blotting of the culture supernatants with novel anti-MUC1 and anti-MUC2 monoclonal antibodies (MAbs) specific for mucin core proteins showed their secretion by most of these cell lines. In addition, mucins produced by these cell lines expressed the tumor-associated carbohydrate detected by MAb 3E1.2 (glycolylsialyl-Tn, mammary serum antigen or MSA) and the Tn or T antigens reactive with lectin SSA-M. SSA-M detected MUC1 or MUC2 captured by MAbs BC2 or CCP58, while 3E1.2 only detected MUC1-associated carbohydrate, indicating that the MAb may react with a conformationally dependent epitope, or that the sialyl/glycolyl-transferases involved in MSA production may be sequence specific. In addition, the BC2/SSA-M and CCP58/SSA-M assays detected mucins in some samples which were not detected by BC2/BC2 or CCP58/CCP58 dual determinant assays, indicating that this format may be more appropriate for the detection of tumor-associated mucins in body fluids. These new cell lines and assays should be of use in the investigation of mucin core proteins, particularly LIM2463 and LIM1839 which express significant quantities of both MUC1 and MUC2.     

Macrophages from cancer patients: analysis of TRAIL,TRAIL receptors,and colon tumor cell apoptosis

BACKGROUND: Tumor-infiltrating macrophages secrete cytokines, including Fas ligand, tumor necrosis factor-alpha (TNF-alpha), and TNF-related apoptosis-inducing ligand (TRAIL). TRAIL induces apoptosis in tumor cells but not in normal cells; however, regulation of TRAIL and its receptors in cancer patients is relatively uncharacterized. We investigated whether macrophages from cancer patients produce TRAIL and whether apoptosis in cultured colon adenocarcinoma cells involves TRAIL and its receptors. METHODS: Macrophages isolated from pleural effusions of nine cancer patients and five control patients with congestive heart failure (whose effusions contained no tumor cells) were cultured. Levels of TRAIL, TNF-alpha, interferon alpha, and Fas ligand in conditioned medium were measured by enzyme-linked immunosorbent assays. Apoptosis of human colon adenocarcinoma cell lines, including Colo 205, was determined by the Annexin V method and terminal deoxynucleotidyltransferase-mediated deoxyuridine 5'-triphosphate nick-end labeling (TUNEL). Cell-surface TRAIL receptors were measured by flow cytometry. RESULTS: Conditioned culture medium from macrophages isolated from pleural effusions containing 1%-5% tumor cells (CM-A) contained TRAIL at 980-1300 pg/mL, whereas that from macrophages from pleural effusions containing more than 50% tumor cells or containing no tumor cells (CM-B) contained TRAIL at 0-50 pg/mL. When cultured with medium containing 50% CM-A, 40% (95% confidence interval [CI] = 30% to 50%) of Colo 205 cells underwent apoptosis; when cultured with 50% CM-B, 8% (95% CI = 3% to 13%) underwent apoptosis. When Colo 205 cells were cultured with 50% CM-A, cell-surface expression of TRAIL death receptors DR5 and DR4 increased 13-fold and sixfold, respectively, compared with that of untreated Colo 205 cells. Recombinant TRAIL induced 90% (95% CI = 85% to 95%) of Colo 205 cells to undergo apoptosis and acted synergistically with TNF-alpha to induce apoptosis. CONCLUSION: Macrophages from cancer patients appear to be activated by tumor cells to produce TRAIL and to increase the expression of TRAIL death receptors DR4 and DR5 on tumor cells.     

5-aminosalicylic acid enhances anchorage-independent colorectal cancer cell death

Resistance to anoikis, the cell death triggered by the loss of anchorage to the substratum, is an essential prerequisite in the proliferation and diffusion of colorectal cancer (CRC) cells. We examined whether 5-aminosalicylic acid (5-ASA), a drug that seems to reduce the risk of colitis-associated CRC, enhances CRC cell anoikis. To this end, Colo205 cells were treated with 5-ASA in the presence or absence of inhibitors of caspases (zVAD-fmk) and reactive oxygen species (ROS). We demonstrate that 5-ASA enhances Colo205 cell death. Although 5-ASA induces dissipation of mitochondrial transmembrane potential and caspase-3 activation, zVAD-fmk does not completely prevent the 5-ASA-induced cell death. 5-ASA also enhances the synthesis of ROS. However, inhibitors of ROS reduce the fraction of 5-ASA-induced Colo205 cell death but do not confer protection. In contrast, the 5-ASA-mediated Colo205 cell death is preventable by Bcl-2 over-expression. These data suggest a mechanism by which 5-ASA interferes with colon carcinogenesis.     

Antitumor activity of idarubicin-monoclonal antibody conjugates in a disseminated thymic lymphoma model

Many of the experimental approaches used in the search for new targeted drug delivery systems ignore the disseminated nature of metastatic disease; the development of more relevant tumor models is therefore a priority. A reproducible and tumor-specific model has been generated by inoculating (C57BL/6 x BALB/c) F1 (Ly-2.2+) mice i.v. with the Ly-2.1+ murine ITT(1) 75NS E3 thymic lymphoma (E3). At a dose of 2 x 10(6) cells, E3 tumors grew in a disseminated fashion, arising initially and predominantly in the lung and kidney, and later and less often in the thymus, spleen, and other tissues. In addition, histopathological examination and flow cytometry of blood did not detect E3 tumor cells in most other organs or in the circulation throughout the course of disease. The mean survival time (MST) of untreated mice was both reproducible and proportional to the number of E3 tumor cells injected and was therefore used to demonstrate the suitability of this model for immunochemotherapeutic studies. When examining the antitumor efficacy of idarubicin-monoclonal antibody conjugates, it was observed that the survival times of treated mice were consistent within groups and between experiments. The disseminated E3 (Ly-2.1+) tumor model, like the s.c. E3 tumor model, demonstrated the dose-dependent efficacy of idarubicin-anti-Ly-2.1 conjugate treatment and illustrated both the negligible antitumor activity and toxicity of idarubicin alone. Furthermore, lung and kidney weight measurements formally demonstrated that the increased MST of treated mice represented a reduction of E3 tumor burden in these organs. This model provides a useful tool for study of the immunochemotherapy of disseminated tumors in mice and further illustrates the antitumor activity of idarubicin-monoclonal antibody conjugates.     

Effects of alpha-difluoromethylornithine and 5-fluorouracil on the proliferation of a human colon adenocarcinoma cell line

Because alpha-difluoromethylornithine (DFMO) reduces the incidence of experimental colon cancers, inhibits the growth of human lung cancer cells and human leukemia cells in culture, and in combination with methylglyoxal (bis)guanylhydrazone induces remission in children with leukemia, its effectiveness against a human colon adenocarcinoma cell line (Colo 205) was tested alone and in combination with 5-fluorouracil (5-FU). Both DFMO (2 X 10(-4) M) and 5-FU (10(-6) M) inhibited Colo 205 cell proliferation. Above 5 X 10(-4) M DFMO (p less than 0.001) and at 10(-4) M 5-FU (p less than 0.001), Colo 205 growth was completely inhibited. Although DFMO did not sensitize Colo 205 cells to a noninhibitory concentration of 5-FU, the effectiveness of inhibitory concentrations of 5-FU and DFMO in reducing Colo 205 cell growth was additive. DFMO (2 X 10(-4) M) caused 89 to 93% inhibition of ornithine decarboxylase activity (p less than 0.001) and reduced levels of putrescine (93%; p less than 0.01) and spermidine (57%; p less than 0.02). Growth rate and the intracellular putrescine and spermidine contents were restored by 10(-6) M putrescine. DFMO could be an effective chemotherapeutic agent against human colonic cancer because of its effects at such unusually low concentrations in vitro.     

SKI-606, a Src/Abl inhibitor with in vivo activity in colon tumor xenograft models

Src up-regulation is a common event in human cancers. In colorectal cancer, increased Src levels are an indicator of poor prognosis, and progression to metastatic disease is associated with substantial increases in Src activity. Therefore, we examined the activity of SKI-606, a potent inhibitor of Src and Abl kinases, against colon tumor lines in vitro and in s.c. tumor xenograft models. SKI-606 inhibited Src autophosphorylation with an IC(50) of approximately 0.25 micromol/L in HT29 cells. Phosphorylation of Tyr(925) of focal adhesion kinase, a Src substrate, was reduced by similar concentrations of inhibitor. Antiproliferative activity on plastic did not correlate with Src inhibition in either HT29 or Colo205 cells (IC(50)s, 1.5 and 2.5 micromol/L, respectively), although submicromolar concentrations of SKI-606 inhibited HT29 cell colony formation in soft agar. SKI-606 also caused loosely aggregated Colo205 spheroids to condense into compact spheroids. On oral administration to nude mice at the lowest efficacious dose, peak plasma concentrations of approximately 3 micromol/L, an oral bioavailability of 18%, and a t(1/2) of 8.6 hours were observed. SKI-606 was orally active in s.c. colon tumor xenograft models and caused substantial reductions in Src autophosphorylation on Tyr(418) in HT29 and Colo205 tumors. SKI-606 inhibited HT29 tumor growth on once daily administration, whereas twice daily administration was necessary to inhibit Colo205, HCT116, and DLD1 tumor growth. These results support development of SKI-606 as a therapeutic agent for treatment of colorectal cancer.     

曲古抑菌素A 对人结肠癌细胞株Colo205 组蛋白乙酰化及ING1b mRNA表达的影响*

目的：探讨曲古抑菌素A（TSA ）对人结肠癌细胞株Colo 205 组蛋白乙酰化及ING 1b mRNA 表达的影响。方法：培养人结肠癌细胞株Colo 205,对照组（A 组）不加TSA 干预,实验组分3 组（B、C、D 组）,分别应用组蛋白去乙酰化酶（HDACs）抑制剂TSA50、100、200 μ g/L 的浓度作用于人结肠癌细胞株Colo 205,24h 后用染色质免疫沉淀（ChIP）方法检测4 组Colo 205 细胞乙酰化组蛋白H3 结合的DNA情况,以了解抑癌基因ING 1b 相关组蛋白H3 乙酰化的变化,并用逆转录聚合酶链反应（RT-PCR）方法检测ING 1b mRNA 的表达,均用实时定量PCR 方法分析。结果：A 组人结肠癌细胞株Colo 205 组蛋白H3 乙酰化水平及ING 1b mRNA Ct值为23.25± 0.08和23.32± 0.05,经TSA 干预后,C、D 组组蛋白H3 乙酰化水平较A 组增加（P<0.05）,2-Δ Δ Ct值分别为4.21和4.38,ING 1b mRNA 表达亦比A 组高（P<0.05）,2-Δ Δ Ct值分别为4.52和4.62,组蛋白H3 乙酰化水平及ING 1b mRNA 表达C、D 组间无显著性差异（P>0.05）,组蛋白H3 乙酰化水平及ING 1b mRNA 表达B 组与A 组比较无显著性差异（P>0.05）,2-Δ Δ Ct值分别为1.12和1.33。同时观察到C、D 组Colo 205 细胞较A、B 组细胞生长明显受抑制。结论：人结肠癌细胞株Colo 205 组蛋白去乙酰化可能是导致基因ING 1b 表达沉默的主要原因之一,100 μ g/L 的TSA 能较好地提高组蛋白乙酰化水平,并有效地激活去乙酰化所致ING 1b 基因转录,诱导该基因表达,从而抑制肿瘤细胞生长。     

The antitumor effect of a novel differentiation inducer, 2, 2-Bis (4-(4-amino-3-hydroxyphenoxy) phenyl) adamantane (DPA), in combinatory therapy on human colon cancer

An adamantane derivative, 2, 2-Bis (4-(4-amino-3-hydroxyphenoxy) phenyl) adamantane (DPA), was found to inhibit the growth of several cancer cell lines in the National Cancer Institute (NCI) Anticancer Drug Screen system. Our previous study showed that DPA inhibited the growth of human colon cancer cell Colo 205 xenografts. DPA-treated cells were arrested at G(0)/G(1), and the DPA-induced cell growth inhibition was irreversible after removal of DPA. Moreover, no acute toxicity was observed after an intra-peritoneal challenge of DPA in nude mice weekly. In this study, we examined the in vivo therapeutic potential of DPA combined with clinical chemotherapeutic agent CPT-11 in Colo 205 cell xenografts. The in vitro cytostatic and differentiative effects of DPA on human colon cancer cells was also evaluated. DPA exerted growth inhibitory activities in vitro against three human colon cancer cell lines (Colo 205, HT-29, and HCT-15). DPA-treated cells showed a more adhesive epithelial phenotype. The differentiation markers of carcinoembryonic antigen (CEA) and fibronectin (FN) were significantly increased in colon cancer cells after treatment with DPA. Further studies showed the induction of p21/Cip1, p27/Kip1, E-cadherin and dephosphorylated p120ctn expression was involved in DPA-induced anticancer effects. Interestingly, DPA-induced elevation of p21/Cip1 was independent of the induction of p53 in Colo 205 cells. in vivo results demonstrated that DPA enhanced the in vivo anticancer activity of the chemotherapeutic agent, CPT-11, by elevation of p53-independent p21/Cip1 and p27/Kip1 expression. Our results suggest that DPA appears to be a new potentially less toxic modality of cancer combinatory therapy.     

Metabolism and biological activity of 5'-deoxy-5-fluorouridine, a novel fluoropyrimidine

5'-Deoxy-5-fluorouridine (5'-dFUrd; Roche 21-9738) is a recently synthesized antineoplastic agent with therapeutic potential. The sensitivity of Ehrlich ascites tumor cells in CF-1 mice to 5'-dFUrd, as well as to 5-fluorouridine, was established. 5'-dFUrd was a more effective antitumor agent and was less toxic over a wide dosage range (50 to 400 mg/kg) than the other agents tested as measured by: (a) the ability to prevent gross development of inoculated tumor; (b) 45-day survival; and (c) weight change over the treatment period. With use of these sensitive tumor cells, the intracellular metabolism of 5'-dFUrd in vitro was investigated. Utilizing liquid chromatographic methodology for separation of acid-soluble metabolites, the only detectable metabolic products of 5'-dFUrd were FUra, 5-fluorouridine 5'-monophosphate, and 5-fluorouridine 5'-triphosphate. Novel metabolites of 5'-dFUrd were not detectable in the acid-soluble fraction or in plasma isolated from mice given [14C]5'-dFUrd. The formation of FUra appears to result from the action of nucleoside phosphorylase. 5'-dFUrd was shown to have a Km of 0.633 mM for this enzyme isolated from Ehrlich ascites tumor cells, an affinity similar to that for 5-fluoro-2'-deoxyuridine (Km, 0.278 mM) but much lower than that for 5-fluorouridine (Km, 0.049 mM). Incorporation of radiolabeled drug into the acid-insoluble fraction (representing greater than 95% incorporation into RNA) was also significant. 5-Fluoro-2'-deoxyuridine 5'-monophosphate (FdUMP) was not detectable as an acid-soluble metabolite. However, significant inhibition of thymidylate synthetase activity was detectable by 20 min in cells incubated with 30 microM 5'-dFUrd, suggesting that FdUMP was produced. The production of both 5-fluorouridine 5'-triphosphate and FdUMP appears dependent on the initial expansion of the FUra pool. This correlates with the inability of 5'-dFUrd to form nucleotide directly due to the absence of a 5'-hydroxyl group. It is concluded that the antineoplastic activity of 5'-dFUrd may be dependent on its enzymatic conversion of FUra. The basis for the possible increase in therapeutic index compared with other fluoropyrimidines may involve the rate and duration of the production of the biologically active nucleotides 5-fluorouridine 5'-triphosphate and FdUMP.     

Antitumor activity of a new fluoropyrimidine derivative, 5'-deoxy-5-fluorouridine, against murine and human experimental tumors

5'-Deoxy-5-fluorouridine (5'-DFUR) was evaluated for antitumor activity against four murine tumors (L1210 leukemia, P388 leukemia, Lewis lung carcinoma, and B16 melanoma) and a human mammary carcinoma (MX-1) xenografted in athymic mice. Intraperitoneal administration of 5'-DFUR was ineffective against B16 melanoma implanted intraperitoneally and showed less marked antitumor activity against P388 and L1210 leukemias implanted intraperitoneally or intravenously as compared with that of 5-fluorouracil (5-FU) or 1-(2-tetrahydrofuryl)-5-fluorouracil (FT-207), while oral administration of 5'-DFUR showed a similar or superior antitumor activity to that of 5-FU or FT-207 against L1210 leukemia implanted subcutaneously. 5'-DFUR showed a marked antitumor activity against MX-1 implanted subcutaneously and also showed slight antitumor activity against Lewis lung carcinoma implanted subcutaneously, while 5-FU and FT-207 did not show any significant antitumor activity against these tumors. These results suggest that 5'-DFUR may be worthy of clinical trial against solid tumors, especially cancers of the breast.     

Anticancer effects of low-dose 10-hydroxycamptothecin in human colon cancer

10-Hydroxycamptothecin (10-HCPT), an indole alkaloid isolated from a Chinese tree, Camptotheca acuminate, inhibits the activity of topoisomerase I and has a broad spectrum of anticancer activity in vitro and in vivo. However, its use has been limited due to its water-insolubility and toxicity with i.v. administration. The purpose of this study was to investigate the efficacy, toxicity and proper dosage of 10-HCPT as a single agent by oral administration in the treatment of human colon cancer. 10-HCPT significantly repressed the proliferation of Colo 205 cells at a relatively low concentration (5-20 nM). Flow cytometry analysis and western blot and apoptosis assays demonstrated that low-dose 10-HCPT arrested Colo 205 cells in the G2 phase of the cell cycle and triggered apoptosis through a caspase-3-dependent pathway. Moreover, following oral administration at doses of 2.5-7.5 mg/kg/2 days, significant suppression of tumor growth by 10-HCPT was observed in mouse xenografts. No acute toxicity was observed after an oral challenge of 10-HCPT in BALB/c-nude mice every 2 days. The results of this study suggest that a relatively low dose of 10-HCPT (p.o.) is able to inhibit the growth of colon cancer, facilitating the development of a new protocol of human trials with this anticancer drug.     

Visualization of metastases from colon carcinoma using an iodine 131-radiolabeled monoclonal antibody

A murine monoclonal antibody that reacts with human colonic cancer (250-30.6) was labeled with radioactive iodine (131I) and the antibody was injected intravenously into 15 patients with known metastases originating from carcinoma of the colon (10 cases), malignant melanoma (1), breast (1), pancreas (1), hepatocellular carcinoma (1), and adenocarcinoma of unknown origin (1). Of the patients with metastatic colon carcinoma, there were 19 known deposits as judged by the techniques of clinical examination, x-rays, and scans obtained using sulpha-colloid. Of these 19 deposits, 17 (90%) were found using the 131I-labeled monoclonal antibody. In one case, the primary tumor, previously undiagnosed, was found. In only 1 of the 10 patients was tumor not found and this was due to the subsequent finding that the undifferentiated tumor did not react with antibody. Of the five patients who did not have carcinoma of the colon, three had negative scans, but two were positive. Thus, the technique of immunoscintography can readily detect both primary and metastatic tumors.     

Transferrin binding to two human colon carcinoma cell lines: characterization and effect of 60-Hz electromagnetic fields

125I-Labeled human transferrin was used to study the binding of transferrin to Colo 320 DM and Colo 205 human cell lines derived from adenocarcinomas of the colon. Although transferrin uptake was greater in both cases at 37 degrees than at 4 degrees it was found that slightly greater than two-thirds of the transferrin associated with the cells at 37 degrees was not bound to surface receptors but rather had been internalized by the cells. Subsequent analysis of true surface binding at 4 degrees by Scatchard analysis allowed determination of the number of transferrin receptors as well as association constants for the interaction. The number of transferrin receptors per cell was found to be inversely related to the cell density of the cultures from which cells were removed for study. Association constants were unaffected by cell density, with average values of 1.2 and 5.4 X 10(8) M-1 obtained for Colo 320 DM and Colo 205, respectively. Additionally, maximum theoretical numbers of receptors of 1.05 X 10(5)/cell for Colo 320 DM and 1.39 X 10(5)/cell for Colo 205 were determined. Furthermore, exposure of Colo 205 cells to three different experimental situations, i.e., 60 Hz-generated electric field only (E+, 300 mA/m2rms), magnetic field only (M+, 1.0 gauss rms), and combined electric + magnetic fields at these intensities (E+M+), altered the expression of transferrin receptors as compared to a concurrently run unexposed control population of cells (E-M-). In three separate experiments the number of transferrin receptors quantitated on both M+ and E+M+ cells was independent of cell culture density and was close to or exceeded the maximum theoretical number of receptors determined for this cell line. In contrast, E+ cells expressed fewer transferrin receptors than was predicted on the basis of cell culture density.     

Cytotoxicity of new duplex drugs linking 3'-C-ethynylcytidine and 5-fluor-2'-deoxyuridine against human melanoma cells

Melanoma is an increasingly common and potentially fatal malignancy of the skin and some mucous membranes. As no cure exists for metastatic disease, there is an urgent need for novel drugs. 2'-Deoxy-5-fluorouridylyl-(3'-5')-3'-C-ethynylcytidine [5-FdU(3'-5')ECyd] and 3'-C-ethynylcytidinylyl-(5'→1-O)-2-O-octadecyl-sn-glycerylyl-(3-O→5')-2'-deoxy-5-fluorouridine [ECyd-lipid-5-FdU] represent cytostatic active duplex drugs, which can be metabolized into various active antimetabolites. We evaluated the cytotoxicity of these heterodinucleoside phosphate analogs, their corresponding monomers ECyd and 5-FdU and combinations thereof on six metastatic melanoma cell lines and six ex vivo patient-derived melanoma cells in comparison to current standard cytostatic agents and the BRAF V600E inhibitor Vemurafenib. In vitro (real-time)-proliferation assays demonstrated that 5-FdU(3'-5')ECyd and ECyd-lipid-5-FdU had a high cytotoxic efficacy causing 75% melanoma cell death at concentrations in the nanomolar and micromolar range. Cytotoxicity was conducted by induction of DNA cleavage indicating apoptotic cells. Chicken embryotoxicity demonstrated that the duplex drugs were less toxic than 5-FdU at 0.01 μM. In vivo the duplex drug 5-FdU(3'-5')ECyd was efficacious in the murine LOX IMVI melanoma xenograph model on administration of 11.2 mg/kg/injection every fourth day. Both duplex drugs are promising novel cytostatic agents for the treatment of malignant melanoma meriting clinical evaluation.     

Adenovirus-mediated ribonucleotide reductase R1 gene therapy of human colon adenocarcinoma.

Ribonucleotide reductase is the enzyme responsible for the reduction of ribonucleotides to their corresponding deoxyribonucleotides for DNA synthesis. Ribonucleotide reductase is a multisubunit complex containing two polypeptides, R1 and R2. In addition to catalytic and allosteric regulatory functions, the R1 subunit appears to act as a novel tumor suppressor. Previous studies demonstrated that overexpression of mouse R1 resulted in suppression of tumorigenicity and metastatic potential, whereas expression of antisense RNA, complementary to R1 mRNA, increased anchorage-independent growth of ras-transformed NIH 3T3 cells. The current study investigated the potential of R1 gene therapy for human cancer using a recombinant adenovirus encoding the human R1 gene (rAd5-R1). Recombinant viruses were constructed by FLP-mediated site-specific recombination and demonstrated high infectivity of a human colon carcinoma cell line (Colo320 HRS), as assessed by expression of a viral encoded beta-Gal gene (rAd5-LacZ). R1mRNA and protein were overexpressed in Colo320 HRS cells infected with rAd5-R1 compared with untreated or rAd5-LacZ-infected cells. Infection with rAd5-R1 inhibited Colo320 HRS cell proliferation, in vitro, in a time- and dose-dependent manner. When Colo320 HRS cells were treated with rAd5-R1, before injection into CD-1 mice, there was complete inhibition of tumor growth compared with treatment with rAd5-LacZ. Furthermore, intratumoral injection of rAd5-R1 into Colo320 HRS tumor xenografts inhibited tumor growth in CD-1 mice compared with rAd5-LacZ treated mice (P = 0.0001). These results demonstrate gene-specific antitumor effects of R1 and suggest that rAd5-R1 gene therapy has the potential to improve currently available treatments for colon cancer.     

SMS 201.995 inhibits in vitro and in vivo growth of human colon cancer.

The effect of a long-acting somatostatin analogue SMS 201.995 (SMS; Sandoz) on basal and gastrin-stimulated growth of 4 human colon cancer lines was studied in vitro and in vivo. Proliferation assay was done with overnight [75Se]selenomethionine uptake after 5 days of incubation. Gastrin concentrations used were 5e-10 M and 1e-7 M. SMS concentrations were from 2e-12 M to 2e-7 M. Cell lines LIM 1215, LIM 2405, and LIM 2412 were inhibited dose-dependently in both basal and gastrin-stimulated groups. LIM 1863 was slightly stimulated. Based on in vivo growth characteristics, LIM 2412 and LIM 2405 were selected for xenograft study. The dose of 50 micrograms/kg/day was arrived at after a preliminary experiment showed it to be safe and effective. The LIM 2412 xenografts in the SMS-treated animals were 473.3 +/- 99.9 (SD) versus 838.1 +/- 111.3 mm3 in control (P less than 0.05) after 20 days. The LIM 2405 tumors were also significantly inhibited (81.2 +/- 30.0 versus 245.7 +/- 48.3 mm3, P less than 0.01). The effect of SMS appeared to be reversible. Oral SMS at 200 micrograms/kg/day was not absorbed. This study suggests that SMS may have direct antitumor effects in human colon cancer.     

Increased antitumor effect of immunoconjugates and tumor necrosis factor in vivo

The potential of specifically targeting antineoplastic drugs and toxins to tumors with the use of monoclonal antibodies (MoAbs) reactive with tumor-associated antigens is currently being examined. N-Acetyl-melphalan-MoAb (N-AcMEL-MoAb) conjugates have previously been shown to have greater antitumor activity than N-AcMEL, melphalan, or MoAb alone against both subcutaneous and ascites murine thymomas in mice (1). Although this conjugate is also a highly selective tumor inhibitor in vitro, it may not reach all the tumor cells in a high concentration, and consequently larger tumors (greater than 0.4 cm2) cannot be eradicated. This conjugate is representative of many drug-MoAb conjugates in that they are unable to gain adequate access to the tumor site to exert their cytotoxic effect. To potentiate the antitumor effect of the N-AcMEL-MoAb conjugate, studies were undertaken to analyze its action in combination with recombinant human tumor necrosis factor alpha (rTNF-alpha), a monokine, capable of causing acute necrosis of syngeneic tumor transplants in mice. Treatment of mice with murine thymomas (0.4 to 0.6 cm2 in size) demonstrated that 30% of the tumors in mice receiving conjugate and rTNF-alpha partially or completely regressed, while no regressions were observed in the tumors of mice receiving N-AcMEL-anti-Ly-2.1 conjugate or rTNF-alpha alone. This and other experiments indicated that the antitumor effect and tumor localization of N-AcMEL-MoAb conjugates can be enhanced in vivo by rTNF-alpha, thereby enabling successful eradication of larger established subcutaneous murine tumors.     

Immunochemotherapy of a murine thymoma with the use of idarubicin monoclonal antibody conjugates

Idarubicin is a derivative of daunomycin and is characterized by the absence of the methoxyl group at the C-4 position. It has been reported to have greater therapeutic effect than daunomycin. Idarubicin was chemically coupled to a monoclonal antibody to the murine Ly-2.1 alloantigen and the cytotoxicity of the drug-monoclonal antibody conjugate versus free idarubicin was tested in vitro against Ly-2+ and Ly-2- tumor cell lines. Some loss of idarubicin activity occurred upon conjugation to the monoclonal antibody; however, antibody activity was preserved and selective cytotoxicity for the Ly-2+ cell line was observed. By contrast free idarubicin was equally cytotoxic for all cell lines (Ly-2+ and Ly-2-). Idarubicin-anti-Ly-2.1 conjugates were tested for their capacity to inhibit solid tumor growth in (Ly-2.1-, Ly-2.2+) (C57BL/6 x BALB/c)F1 mice while their nonspecific effects were monitored by histological examination. The idarubicin-anti-Ly-2.1 conjugates when injected i.v. or directly into the tumor were observed to inhibit tumor growth more effectively than idarubicin or anti-Ly-2.1 alone, with smaller tumors being completely eradicated within several days of the completion of treatment.