Microtubules are among the most strategic subcellular targets of anticancer agents. Recently, new antimicrotubule agents have been introduced to clinical cancer chemotherapy. Navelbine is a new derivative of vinca alkaloids, which especially has an activity against NSCLC. Unlike classical anti-microtubules agents like colchitine and vinca alkaloids, which induce depolymerization of microtubules, taxol has an unique mechanism of action which induces tubulin polymerization. Taxotere, an analogue of taxol is slightly more active as a promoter of tubulin polymerization than Taxol. E7010 is a novel sulfonamide that inhibits tubulin polymerization. Results of preclinical and clinical studies of these agents are reviewed.

In order to improve the efficacy of chemotherapy against urogenital cancers, we developed a chemosensitivity test by measuring intracellular ATP in cancer cells. In the fundamental experiments using HeLa cells, a significant correlation was observed between intracellular ATP levels and numbers of viable cells. We employed 8 drugs, ADM, VCR, VLB, MTX, 5-FU, PEP, MMC, and CDDP for the assay Thirty-four renal tumors, 68 urothelial tumors and 19 testicular tumors were tested, and evaluable results were obtained in 25 specimens of renal tumors (76%) 55 specimens of urothelial tumors (80%) and 17 specimens of testicular tumors (98%). According to the ATP assay, renal tumors were sensitive to ADM and MMC, urothelial tumors to ADM, MMC and CDDP and testicular tumors to ADM and MMC. The ATP assay to determine the chemosensitivity to clinical specimens was sensitive and efficient. Thus the ATP assay could be applied for selection of anticancer drugs in the chemotherapy of urogenital cancers.

MTT assay, a sensitivity test of anti-tumor agents was performed, and its clinical usefulness, was discussed. In 15 surgical specimens, the cell suspensions were prepared aseptically, and divided into three processing; namely, Papanicolaou (Pap) smears to confirm the malignant cells, MTT assay, and cell cultures in chamber slides. MTT assay was evaluated only when tumor cells in the chamber slide were observed 50% or more by Pap staining. Drugs judged to be effective were applied for patients, resulting in 64.2% of predictive accuracy in the sensitivity. Conclusions; 1) MTT assay was developed for sensitivity test of anti-tumor agents, 2) Strict assessment was carried out by the confirmation of cancer cells using chamber slides, 3) On clinical usefulness, predictability for the sensitivity was 64.2%, that for resistance was 100%, and over all predictability was 66.7% 4) MTT assay was useful for the determination of effective and for elimination of ineffective drugs.

The 5-year survival of lung cancer patients is about 30% in Japan. One of the reasons for the poor prognosis seems to be drug resistance. It has been reported that certain types of oncogenes, such as ras, myc and fos, may play an important role in drug resistance. The myc protein forms a sequence-specific DNA-binding complex with Max and may act as a transcription factor; thus, it may be possible that myc family oncogenes are involved in DNA synthesis and repair processes mediating drug resistance. We report here that L-myc oncogene may be involved in the transition from drug-sensitive to drug-resistant phenotype of a certain small cell lung cancer cell line.

Three large cell carcinoma cell lines were established from tumors of lung cancer patients. The cell lines were named NUTLC-2, NUTLC-4 and NUTLC-5 and they were found to have the following biological characterization. 1) By chromosomal analysis, the tumor cells of two of the cell lines (NUTLC-2 and NUTLC-5) were human-origin cells. Numbers of chromosomes of these cells ranged from 52 to 59 in NUTLC-2 and from 68 to 75 in NUTLC-5, with the modal numbers of 56 and 71, respectively. 2) Primary tumor resected from the patient with lung cancer was heterotransplanted into the subcutis of a nude mouse. NUTLC-4 cell line was established in vitro from the tumor in nude mouse and the tumor cells were found to be mouse-origin cells by chromosomal analysis. Human DNA was not detected by Alu analysis. 3) The tumor cells of three cell lines could be heterotransplanted subcutaneously into nude mice. However, no natural distant metastasis in nude mouse was observed. 4) Drug sensitivity to NUTLC-2, NUTLC-4 and NUTLC-5 tumor cells differed individually according to MTT colorimetric assay, and characteristic drug sensitivity was not noted in histological types of lung cancer.

A 3-year-old boy presented with multiple brain metastases 21 months after the resection of stage II Wilms' tumor. Metastasis to other organs was not found. He was treated by total removal of a large metastatic tumor in the left temporal lobe and post-operative radiotherapy and chemotherapy. He has been in complete remission for 20 months after surgery. Cerebral metastasis from Wilms' tumor without systemic metastases is very rare. It is speculated that brain metastases occurred in this patient because most of the anticancer agents used in the primary treatment for Wilms' tumor were not able to cross the blood brain barrier.

In order to elucidate the relationships between the antitumor activity and the molecular structure of novel acridine derivatives (1a-f, and 2a-e in Chart 1) the DNA-binding properties (intercalation) of the derivatives were examined by the quenching in fluorescence of an ethidium-DNA complex, which may be caused by the displacement of DNA-bound ethidium by a second DNA-binding ligand, acridines. The concentration (C50 value) of the acridine necessary to reduce the initial fluorescence of DNA-bound ethidium by 50% showed a good correlation with their antitumor activities. The fluorescence quenching of the acridines was examined using 4'-(9-acridinylamino)- methanesulfonanilide (amsacrine, AMSA) as a typical standard of the second DNA-bound ligand, and calf thymus DNA with an apparent site size of two base pairs.

A chemosensitivity test was carried out on superficial bladder cancer using the dye exclusion assay for the purpose of screening chemosensitive drugs for prophylactic intravesical chemotherapy. Bladder cancer cells of each patients were incubated, in vitro, in the presence of adriamycin, (2"R)-4'-0-tetrahydropyranyladriamycin, mitomycin C, pepleomycin and 4'-epi-adriamycin (500 micrograms/ml) at 5%, CO2, 37 degrees C for 2 hours. The cytotoxic effect of the drugs was evaluated by the ratio of stained cells by trypan-blue. The most effective drug was instilled postoperatively into the bladder 3 times for the first week, and every 2 weeks during the following 14 weeks. In 18 patients followed more than 4 months the prophylactic effect was evaluated. Fifteen of the 18 patients completed the protocol, but the remaining 3 patients failed to complete to the instillation because of severe irritability of the bladder. Tumor recurrence was demonstrated in two patients. Non-recurrence rates of tumors at 12 and 24 months were 93.8% and 82.0%, respectively. These results suggested that this rapid and handy assay was useful for the purpose of screening chemosensitive drugs for the intravesical chemotherapy.

We have studied chemotherapy enhancement by the combination of the biscoclaurine alkaloid, Cepharanthine (Ceph), using the inhibition of colony forming efficiency (CFE) of B16 melanoma cells in vitro. Hydroxyurea, antimetabolite, showed a time-dependent inhibition of CFE, and the combination with Ceph enhanced CFE inhibition by 29%, which is stable and independent of the treatment time. Dacarbazine, a biological alkylating agent, given with Ceph showed a time-dependent inhibition of CFE (43%). MCNU, a nitrosourea, with Ceph showed the greatest inhibition of CFE (67%) among the anticancer agents used. The CFE inhibition was time-dependent and required a higher concentration of MCNU. In this series of studies using CFE inhibition of B16 melanoma cells in vitro, etoposide showed the best effect by the combination with Ceph, which seems to be a candidate for in vivo evaluation.

It is very important to determine the biological characteristics of individual cancers, not only in order to evaluate malignant potentials but also to decide on the most effective anticancer therapy. A method which combines BrdU labeling and flow cytometric bromodeoxyuridine (BrdU)/DNA analysis makes it possible to plot the DNA synthetic rate of cells against their DNA content. Hence, this method yields more useful information than the previous method of flow cytometric DNA analysis. When this method is performed correctly, it can be also applied to fresh solid tumors. In this paper, we will describe the results of our preliminary investigation and discuss its clinical significance.

Since the cell cycle of tumor cells is evaluable with the aid of flow cytometry, the mechanism of action of antineoplastic agents has readily been analyzed based on the changes of the cell cycle by antineoplastic agents. Propidium iodide had been a conventional staining method for such analysis, but after the introduction of DNA/BrdU double staining method using BrdU, it has become much easier. For evaluating antineoplastic agents, simultaneous measurement by FDA of the viability of the tumor cells is important. In vitro studies of the action mechanism and the antineoplastic effects of 5-FU, ACNU, IFN, PG, CDDP, PEP, and HCFU have been reported previously elsewhere. Similar action mechanism has been confirmed in clinical studies, in which optimal agent was selected by the sensitivity test using flow cytometry, and thus selected agent was actually given to tumor patients. In analyzing antineoplastic agents in vitro, concomitant and total evaluation of DNA-histogram and viability by flow cytometry is needed in the future.

We have investigated the cell kinetic effect of four carcinostatic agents (MMC, CDDP, ADR and 5-FU) on the human gastric cancer cell line (KATO-III; signet ring cell carcinoma) by means of flow cytometry (FCM), using bromodeoxyuridine (BrdU) and its monoclonal antibody. Cancer cells in the S phase were first labelled with BrdU and then the bivariate DNA/BrdU distribution was examined to analyze the effect on the cell cycle. Furthermore, cells were reincubated at 24 hours after labelling to evaluate the cell turnover during FCM. MMC, CDDP and ADR assembled the cells into late S phase and G2M phase, while 5-FU assembled them into S phase. after 24 hours, cells with cessation of cell cycle had inhibited their proliferation. We conclude that this technique can be usefully applied as a susceptibility test of carcinostatic agents, since it could define the phase where carcinostatic agents acted on cancer cells.

Selective toxicity against cancer cells is a most important determinant for anticancer agents. Therefore, we have preferably evaluated anticancer effects in vivo using murine tumor models for several decades. Approximately 50 anticancer agents are currently available for clinical therapy, but very few agents are effective against some types of cancer. Much progresses in cell culture techniques resulted in establishment of various human tumor cell lines. Currently, we are able to use human tumor lines as well as murine ones for the examination of drug sensitivity. A number of assay methods to evaluate anticancer activity have been developed. In the beginning, growth inhibitory activity was evaluated by counting cell numbers after drug exposure. Then, human tumor clonogenic assay (HTCA) was designed to measure only proliferative cells. Recently colorimetric MTT assay and SRB assay in 96-well microplates were developed, which were adopted in the screening system in the NCI, based on a new idea, that is, disease-oriented screening (DOS) using about 60 human tumor cell lines. In this paper outline of each method was described, adding especially several comments on disease-oriented screening.

The significance of BrdU/DNA double staining method using flow cytometry as a chemosensitivity test of anticancer agents is herein reported. Experimentally, CDDP, ADM and MMC yielded a significant increase of G2 phase fraction at 24, 48 and 72 hours, and caused a significant decrease of BrdU labeling index at 48 hours. The changes of G2 phase fraction and BrdU labeling index were correlated well with the result of colony forming test, and, therefore, were considered as a useful index of lethal effect of anticancer agents. For the clinical application, enzymatic preparation and discontinuous Ficoll density gradient method might be advantageous to collect viable tumor cells from solid specimen. Flow cytometric BrdU/DNA assay can be clinically applied as a chemosensitivity test in selecting effective anticancer drugs.

In an attempt to establish the optimum dose of HCFU, the effect of the presence of stomach on the absorption of HCFU and both the rise and maintenance of blood HCFU levels was evaluated in patients with gastric cancer (total gastrectomy group) and with colorectal cancer (non-gastrectomy group). The blood concentrations of HCFU fractions and 5-FU were determined in terms of pharmacokinetic parameters in these groups. The Tmax was significantly different between the two groups, with HCFU fraction and 5-FU levels that were significantly higher in the total gastrectomy group 30 minutes after administration. No differences were found in Cmax or AUC. There were no significant differences in Cmax or AUC among the various subgroups given different doses of HCFU (100, 150 and 200 mg) in either group, although dose-dependency was observed. Similar results were obtained in crossover tests. The 5-FU remained at an effective concentration of 0.05 microgram/ml, 4 hours after a single dose of 100 mg HCFU.

Because of the increase in the incidence of malignancies with advancing age, we have need to make decision on the chemotherapy of elderly patients with cancer. However, until recently, studies about response to treatment and toxicities in the elderly patients have been quite limited. Many of clinical trials have excluded elderly patients with advanced cancer. In leukemia and Hodgkin's lymphoma, definite trends of decreased levels of response and survival in relationship to increased age have been reported. This reduced rates of response in elderly leukemia patients do not appear to be due to chemotherapy resistance but rather to high rates of death among elderly patients during remission induction therapy or to inadequate salvage therapy. On the other hand, older patients with solid cancers can be treated with chemotherapy as successfully as younger patients. General expectations of response, remission, and survival are similar for the older and younger patients. With the exception of small increase in the incidence of hematologic toxicity in the older group, both younger and older patients experience the same frequency of toxic reactions. Aging is a highly individualized process that cannot be defined by chronological landmarks. Therefore, it can be said that there is no reason to exclude from adequate chemotherapy, simply on the basis of age alone. But, observations on the proper role of chemotherapy in older patients are very limited, and further studies are needed in this area.

The conjugates of mitomycin C (MMC) with glucuronoxylomannan (AC) from Tremella fuciformis were synthesized by the use of spacers (glycine, glycylglycine, glycylglycylglycine). In i.p.-i.p. system the antitumor activity of the conjugates (MMC-G-ACP, MMC-GG-ACP, MMC-GGG-ACP) against P388 leukemia in mice was slightly lower than that of MMC by the evaluation of life span, ILS (%). In s.c.-i.p. system the antitumor activity of the conjugates against sarcoma 180 solid tumor in mice was similar to that of MMC, except for MMC-G-ACP. The reduction of the number of leukocytes caused by MMC was suppressed by attaching MMC to AC. The conjugates did not lower the cytotoxicity of MMC against L1210 mouse leukemia cells in vitro. The release rate of MMC from the conjugates in vitro (half time of MMC release: MMC-G-ACP, 8.8 h; MMC-GG-ACP, 3.1 h; MMC-GGG-ACP, 2.9 h) was much faster than that of MMC-dextran, and differed in the length of the spacer. The results would give useful information on macromolecular carriers in drug-delivery system.