Screening Of Anticancer Agents Research Articles

Development of a Cy3‐Labeled Glucose Bioprobe and Its Application in Bioimaging and Screening for Anticancer Agents

Development of a Cy3‐Labeled Glucose Bioprobe and Its Application in Bioimaging and Screening for Anticancer Agents

Development of a Cy3‐Labeled Glucose Bioprobe and Its Application in Bioimaging and Screening for Anticancer Agents

Development of a Cy3‐Labeled Glucose Bioprobe and Its Application in Bioimaging and Screening for Anticancer Agents

Mutation analysis of 24 known cancer genes in the NCI-60 cell line set

The panel of 60 human cancer cell lines (the NCI-60) assembled by the National Cancer Institute for anticancer drug discovery is a widely used resource. The NCI-60 has been characterized pharmacologically and at the molecular level more extensively than any other set of cell lines. However, no systematic mutation analysis of genes causally implicated in oncogenesis has been reported. This study reports the sequence analysis of 24 known cancer genes in the NCI-60 and an assessment of 4 of the 24 genes for homozygous deletions. One hundred thirty-seven oncogenic mutations were identified in 14 (APC, BRAF, CDKN2, CTNNB1, HRAS, KRAS, NRAS, SMAD4, PIK3CA, PTEN, RB1, STK11, TP53, and VHL) of the 24 genes. All lines have at least one mutation among the cancer genes examined, with most lines (73%) having more than one. Identification of those cancer genes mutated in the NCI-60, in combination with pharmacologic and molecular profiles of the cells, will allow for more informed interpretation of anticancer agent screening and will enhance the use of the NCI-60 cell lines for molecularly targeted screens.

항고형암제의 활성평가를 위한 in vitro 삼차원 암세포 배양계의 확립

For the efficient determination of activity against solid tumors, an in vitro tumor model that resembles the condition of in vivo solid tumors, is required. The purpose of this study was to establish a rapid culture method and viability assay for an in vitro 3-dimensional tumor model, multicellular spheroid (MCS). Among 12 human cancer cell lines, a few cell lines including DLD-1 (human colorectal carcinoma cells) formed fully compact MCS which was adequate for in vitro viability assay. DLD-1 MCS showed steady growth reaching diameter after 11 day culture. DLD-1 cells grown as MCS showed significant increase in phase compared to the monolayer cells (73.9% vs 45.7%), but necrotic regions or apoptotic cells were not observed. The cells cultured as MCS showed resistance to 5-FU (10.3 fold higher ) compared to monolayers, however, tirapazamine (a hypotoxin) showed similar activity in both culture systems. In summary, MCS may be a valid in vitro model for activity screening of anticancer agents against human solid tumors and also exploitable for studying molecular markers of drug resistance in human solid tumors.

High-volume cellular screening for anticancer agents with combinatorial chemical libraries: a new methodology.

A single-step cancer cell cytotoxic assay system for anticancer drug discovery has been developed which facilitates rapid screening of large combinatorial chemical libraries synthesized using the 'one-bead-one-compound' (OBOC) methodology. Each OBOC library bead incorporates two orthogonally cleavable linkers that release the bead-bound compound at a different pH. The assay utilizes high concentrations of tumor cells mixed directly with OBOC beads and plated in soft agarose containing tissue culture medium. One of the orthogonal linkers is cleaved at neutral pH in tissue culture releasing an aliquot of compound to diffuse at a relatively high local concentration into the soft agarose immediately surrounding the bead. Active compounds are identified visually from a clear ring of tumor cell lysis which forms within 48 h around just the rare bead releasing a cytotoxic compound. The bead releasing a cytotoxin is then plucked from the agar and the remaining compound still linked to the bead can be released for structural analysis, followed by compound resynthesis and confirmatory testing. This assay system has been successfully applied to identification of lead cytotoxic compounds from model peptidic and non-peptidic combinatorial chemical libraries. Use of this methodology may facilitate anticancer drug discovery.

Toxicity of anticancer agents, growth and chemosensitivity of human tumour xenografts in a segregating stock of AF nude mice

An investigation of the usefulness of a segregating stock of nude mice [AF nude mice (AF-nu)] for screening anticancer agents was undertaken. The toxicity of anticancer agents, takes and growth rates of human tumour xenografts and chemosensitivities of xenografts in AF-nu were studied and compared with those in BALB/cA nude mice (BALB/cA-nu). The results showed differences in the pattern of mortalities of AF-nu and BALB/cA-nu administered a range of anticancer agents. Body weight changes in the two nude mouse strains differed in the case of 5-fluorouracil, but not for nimustine, adriamycin and vincristine. All tumours transplanted in AF-nu grew as in BALB/cA-nu. Growth rates of 2 xenografts (gastric cancer and glioblastoma) were not significantly different between the 2 nude mouse strains, but those of 2 lung tumour xenografts were significantly greater in AF-nu than those in BALB/cA-nu. There were no significant differences in chemosensitivities of human tumours in AF-nu and BALB/cA-nu (consistency rate as evaluated by our criteria was 88%). From these results, it is suggested that AF-nu are more suitable for anticancer agent screening and experimental chemotherapy of human tumour xenografts than BALB/cA-nu because of lower costs and high reproductive rate. Although they are genetically heterogeneous, sets of experimental animals sharing the same gene pool can be produced routinely.

ヒト口底癌を用いた実験的化学療法 Ｉ ヒト口底癌の移植・継代

A human oral cancer/nude mice system has been established for an anticancer agent screening system, which is expected to have higher predictability of clinical effect. In this preliminary report, the method of successive transplantation of human cancer in nude mice and the histological characteristics of the tumor were reported.1) 8-to 10-week-old male BALB/cA-nu mice kept in vinyl isolater were used.2) Human cancer in this experiment was derived from an oral floor tumor histologically diagnosed as squamous cell carcinoma in a 58-year-old man.3) The tumor tissue which was minced in sterilized penicilline-streptomycin PBS (-) solution (PC: 100 u/ml, SM: 100γ/ml) was sectioned in small pieces approximately 1mm 3 in volume. 2 to 3 pieces were injected into subcutaneous tissue of the back of each animal.4) After the fifth generation of serial transplantation, the tumors became stable, showed stable growth curves and maintained the histological features of moderately differentiated squamous cell carcinomas.

Antitumor efficacy of seventeen anticancer drugs in human breast cancer xenograft (MX-1) transplanted in nude mice

To validate the usefulness of a human tumor-nude mice xenograft system as a potential model in the secondary screening of anticancer agents, the antitumor activity of 17 anticancer drugs has been studied in the treatment of a human breast cancer tumor (MX-1) transplanted to nude mice. For evaluation of the antitumor activity of the drugs we employed the LD10 predetermined in BDF1 mice as a standard therapeutic dose. Drugs were administered IV, IP, or PO, and antitumor activity was assessed by drug-induced growth inhibition measured by caliper. Among the 17 anticancer drugs, the most active compounds (maximum inhibition rate of tumor growth: greater than or equal to 90%) are mitomycin C, chromomycin A3, vincristine, vinblastine, vindesine, and hexamethylmelamine. Another group of compounds showed moderate activity (maximum inhibition rate of tumor growth: 89%-50%), these being adriamycin, daunomycin, mitoxantrone, bleomycin, 5-FU, 6-TG, and ftorafur. The remaining four drugs (peplomycin, cytosine arabinoside, 6-MP, and methotrexate) were inactive against the MX-1 tumor. These results suggested that in the nude mouse-human tumor xenograft system of the MX-1 tumor there was a good correlation between the antitumor activity of various anticancer drugs and their clinical efficacy; this system is therefore expected to be a useful model for the secondary screening system.

Comparative growth of human tumors in pharmacologically immunosuppressed, immune-deprived, cyclosporin a-treated and nude mice

The growth of three human tumor xenografts, namely an Ewing sarcoma, a colon carcinoma and an osteosarcoma, was compared in nude and conditioned mice. Conditioning protocols included (1) immune deprivation (thymectomy, lethal irradiation and cytosine arabinoside pretreatment); (2) immunosuppression with procarbazine, cyclophosphamide and antithymocyte serum; and (3) continuous administration of cyclosporin A. Similar tumor growth was seen in nude mice, immune-deprived mice and mice treated with the medium and high-dose immunosuppressive protocol. Cyclosporin A allowed only very modest tumor growth. In the main comparative experiment with the Ewing sarcoma and the colon carcinoma, overall survival was lowest with nude mice (17%), higher with immune-deprived mice (61%) and best with immunosuppressed mice (81 and 87%). For the screening of anticancer agents the immunosuppressive protocol consisting of synergistic procarbazine, cyclophosphamide and antithymocyte serum may be added to the already available models. It allows adequate tumor growth with good animal survival and does not require operative procedures and irradiation.

Strain-Dependent Growth of a Human Carcinoma in Nude Mice with Different Genetic Backgrounds

Human gastric carcinoma showed different growth in nude mice depending on their genetic backgrounds which included BALB/cA-nu (nude mice with a BALB/cA genetic background), CBA/N-nu, NFS/N-nu, NIH(s)-nu, C3H/HeN+-nu, C57BL/6N-nu and lasat mice (BALB/cA-nu, Dh). Rapid growth of human carcinoma was observed in CBA/N-nu, NFS/N-nu and NIH(s)-nu. The human carcinoma in NIH(s)-nu showed the widest deviation in tumor weight. These data suggest that NFS/N-nu and CBA/N-nu are strains which are more recommended for anticancer agent screening systems than BALB/cA-nu, NIH(s)-nu, C3H/HeN+-nu, C57BL/6N-nu and lasat mice, although many other factors including productivity of the mice, strain differences in drug metabolism and drug toxicity must be considered.

Screening for anti-cancer agents; the relative merits of in vitro and in vivo techniques

Screening for anti-cancer agents; the relative merits of in vitro and in vivo techniques

Oxygen consumption by acrylamide polymerization: a method for rapid screening of anticancer agents.

We have developed a rapid and sensitive method to measure oxygen consumption of tumor cells by acrylamide polymerization. This method could be used as an in vitro screen for potential chemotherapeutic agents. Previous techniques have lacked either the sensitivity or the speed required for use as an effective clinical tool. This method was modified from a technique previously developed in our laboratory for measuring oxygen in blood. Standard anticancer agents were tested against Walker 256 ascites tumor. Ascites was harvested from 20 rats on each of 50 days, incubated briefly with drugs, and acrylamide polymerization time was measured hourly. Individual controls were established for each drug-treated group, and oxygen uptake measured after one hour of incubation. Actinomycin, cyclohexamide, and cytosine arabinoside inhibited tumor cell oxygen consumption by 80%, 40%, and 30%, respectively. These results correlated with the known in vivo effects of these drugs on Walker carcinosarcoma. Therefore, this sensitive method could potentially be used directly on tumor cells removed from biopsy material so that the testing of anticancer drugs could be completed on the day of biopsy.

The effect of cytotoxic agents on the primary immune response to Listeria monocytogenes.

Four drugs, representing four different categories of cytotoxic agents, were studied for their effect on the immune response to Listeria infection in mice. The development of the host's immune response is revealed by a progressive change in the slope of the bacterial growth curve in spleen and liver. It has its onset at 24 hr in untreated mice, but in the presence of effective immunosuppression the organism multiples uninterruptedly until the animal dies from overwhelming infection. When administered as single injections at the time of infection, cyclophosphamide, vinblastine, and azathioprine all produced an effective immunosuppression, characterized by continuous bacterial multiplication. Methotrexate was also immunosuppressive, but unlike the others its effects were reversible. They could be sustained, however, by further treatment. Studies of the time-response relationship indicated that cyclophosphamide was highly active over a broad time-span ranging from 2 days before infection to 4 days after infection. Vinblastine on the other hand was maximally active when given on the day of infection, while methotrexate had its greatest effect when given 48 hr after infection. These differences indicate that these three drugs act on different cell populations involved in the host's immune response. The effects observed have been discussed in relation to what is known of the modes of action of the drugs tested. An observation of interest was the phenomenon of enhanced immunity in animals treated with cyclophosphamide or vinblastine 7-11 days before, and with methotrexate 4 days before infection; reactive hyperplasia of lymphoid tissue following withdrawal of drug was again advanced as an explanation for the occurrence of this paradoxical effect. The experimental model employed is simple, requiring only routine bacteriological facilities and minimal equipment. It seems to offer a useful means of assessing the immunosuppressive activity of drugs and of determining the time-course of their action; it could also be of value in the screening of anticancer agents.