Experimental model for combination chemotherapy with metronidazole using human uterine cervical carcinomas transplanted into nude mice.

Human uterine cervical carcinomas (Yumoto strain) and HeLa cell tumors were transplanted into nude mice, and their transplantable strains were established. The fundamental histological features of these tumors were analyzed according to their histological construction and cytological maturation. The effect of administered drugs was examined morphologically. The Yumoto strain is a well-differentiated epidermoid-type carcinoma consisting of regularly arranged basal-type, parabasal-type, and keratinizing-type cells. The HeLa cell tumor is made up of solid medullary carcinoma cell nests in which trabecular arrangements begin to appear around the medullary areas after the third passage. This feature is maintained up to the 17th generation. The basal layer-type cancer cells of the Yumoto strain as well as trabecularly arranged cancer cells in the HeLa cell tumor were selectively influenced by administration of bleomycin and/or mitomycin and showed considerable degeneration or complete disappearance. On the contrary, metronidazole (a drug for vaginal trichomoniasis; Flagyl) displayed a cytotoxic effect on the parabasal-type and/or more mature cancer cells of the Yumoto strain as well as on the solid medullary area of the HeLa cell tumor. This result may indicate a selective affinity of drugs for malignant cells according to their histological construction, and it is conceivable that these types of carcinoma can be affected by combination administration of metronidazole and oncostatic chemicals such as bleomycin and mitomycin. This speculation was realized in this experimental animal research.

Involvement of human interleukin 6 in experimental cachexia induced by a human uterine cervical carcinoma xenograft.

A human tumor xenograft model for cancer cachexia was established by growing a uterine cervical carcinoma, Yumoto, in nude mice. The tumor transplanted into the mice induced severe body weight loss (30% of body weight) when the tumor weight was only 1 g. In addition, other indicators for cachexia, such as adipose tissue and muscle wasting and hypoglycemia, were also observed in the tumor-bearing mice, suggesting that this is a proper model for experimental cachexia induced by a human tumor. We then examined the association of this model with various cytokines, such as tumor necrosis factor alpha, interleukin (IL)-1alpha, IL-1beta, IFN-gamma, IL-6, and leukemia inhibitory factor, and identified human IL-6, which was produced by the tumor cells, as a mediator of cachexia. A neutralizing antibody against hIL-6 administered to the mice after the development of cachexia symptoms significantly improved body weight loss, adipose tissue wasting, hypoglycemia, acute phase reaction, and leukocytosis, although it did not suppress the tumor growth. These results demonstrate that the hIL-6 produced by the tumor cells is an essential mediator of the cachexia induction in this model.

Thermochemotherapy for malignant melanoma: overcoming heterogeneity in drug sensitivity.

Endo B (melanotic) and W (amelanotic) human malignant melanomas originated from the same tumor, both known to be heterogeneous in drug sensitivity to ACNU [( 1-(4-amino-2-methyl-5-pyrimidinyl)-methyl-3-(2-chloroethyl)-3-nitroso- urea hydrochloride]), were treated experimentally with a combination therapy of ACNU and hyperthermia in mice. Whereas Endo W melanoma has no sensitivity, Endo B melanoma is sensitive to ACNU alone. However, in both types of melanomas, a marked synergistic effect of the combination therapy was noted. Histologically, marked degeneration of both tumor cells was detected. These results strongly suggest that thermochemotherapy may overcome the tumor heterogeneity in drug sensitivity.

Cloning and sequencing of the L1 gene of canine oral papillomavirus.

Canine oral papillomavirus (COPV) DNA was isolated from two different sources. One of these DNAs was molecularly cloned and its physical map was determined. Hybridization analyses using subgenomic fragments of bovine papillomavirus type 1 (BPV-1) and human papillomavirus type 16 (HPV16) as probes revealed that the cloned COPV shared moderate homology within the E1 and L1 regions of BPV-1 and HPV16, whereas homology in other regions of BPV-1 and HPV16 was low. The putative L1 gene of COPV was sequenced and several conserved regions, including antigenic epitopes which are common in other known papillomaviruses, were analyzed.

Enhancement of anti-proliferative activities of 5-FU, HCFU, SN-38, THP and ACNU by a serine protease inhibitor, FOY-305.

The effects of a synthetic serine protease inhibitor, FOY-305, on the proliferation of normal and transformed mouse fibroblasts were investigated in vitro by MTT colorimetric assay. FOY-305 inhibited the growth of normal NIH3T3 cells and their src- and erbB2-transformed cells with a half maximum inhibitory concentration (IC50) of 1-1.2 mg/ml whereas it suppressed the growth of ras-transformed cells more effectively (IC50 was 0.5-0.6 mg/ml). Flow cytometric analysis using synchronized NIH3T3 cells has shown that the growth-inhibitory activity of FOY-305 was due to the suppression of G(1)/S transition. The synergistic effects between FOY-305 and traditional anticancer drugs were also investigated by the MTT assay and the results showed that FOY-305 significantly enhanced the antiproliferative activities of 5-fluorouracil (5-FU), 1-hexylcarbamoyl-5-fluorouracil (HCFU), 7-ethyl-1-hydroxy-7-ethyl-10-[4-(1-piperidino)-1-piperidino] camptothecin (SN-38), pirarubicin (THP) and 1-(4-amino-2-methyl-5-pyrimidinyl)-methyl-(2-chloroethyl)-3-nitrosourea hydrochloride (ACNU).

Nucleotide-sequence of a canine oral papillomavirus containing a long noncoding region.

The DNA genome of a canine oral papillomavirus (COPV) was completely sequenced and found to consist of 8607 base pairs, which were the longest of all known papillomaviruses (PVs). Its organization was similar to that of other PVs except that it lacked early gene 5 (E5) and possessed a unique long noncoding region (L-NCR) between the end of the early genes and the beginning of the late genes. COPV also possessed a short noncoding region (S-NCR) which contained a putative upper regulatory region (URR), which is commonly found in PVs. The L-NCR did not show any similarity to known PV DNAs nor other DNA sequences in the GenBank database. Nucleotide sequence analysis of COPV showed that it was closely related to human papillomavirus type 1 (HPV 1) and animal PVs associated with cutaneous lesions in rabbit, European elk, deer and cow as we reported previously.

Differential chemosensitivity in oncogene-transformed cells.

The effects of anticancer drugs on cell growth have been investigated by MTT colorimetric assay using mouse and rat cultured cells transformed by various oncogenes and tumor viruses. Aclarubicin, mitomycin C and 1-hexylcarbamoyl-5-fluorouracil showed higher growth-inhibitory activities toward transformed cells than those toward the normal counterparts. Nimustine, bleomycin and 5-fluorouracil also showed selective growth-suppressive activities toward transformed cells except for a few cell lines. ras-oncogene-transformed cells were more sensitive toward 5-fluorouracil and 1-hexylcarbamoyl-5-fluorouracil than the normal parent cells and other transformed cells. These drugs would thus be effective in the chemotherapy of ras-induced cancer.

A second-generation method of genotyping hepatitis C virus by the polymerase chain reaction with sense and antisense primers deduced from the core gene.

A second-generation method of genotyping hepatitis C virus (HCV) was developed by the polymerase chain reaction (PCR) with sense as well as antisense primers deduced from the core gene. HCV RNA specimens extracted from sera were reverse-transcribed and amplified with universal primers in the first round of PCR to obtain fragments of 433 base pairs representing nucleotides 319-751. In the second round of PCR, portions of PCR products were amplified separately with sense and antisense primers specific for each of the five common genotypes prevailing across the world, i.e., I/1a, II/1b, III/2a, IV/2b and V/3a. The specificity of the method was verified by a panel of 177 HCV isolates of various genotypes in the genetic groups 1-9. It allowed clear differentiation of genotype I/1a from II/1b which was not always accomplished by the previous method. When 501 sera from blood donors and hepatitis patients with HCV viremia from various countries were genotyped by the second-generation method, 478 (95.4%) were classified into the five genotypes. HCV RNA samples from 23 (4.6%) sera were not classifiable into any of the five common genotypes and, by sequence analysis, 22 were found to be of four genotypes in group 4 and one of genotype 1c in Simmond's classification.

A rapid in vitro assay for predicting thermochemosensitivity of human cancer: comparison with clonogenic assay.

We previously reported a rapid in vitro assay based on morphological changes in the nucleus in order to predict response to thermochemotherapy. It was strongly suggested that this simple method may be clinically useful. In the present study, a comparison with the clonogenic assay was carried out on eight different human tumors (three malignant melanomas, two lung carcinomas, two colon carcinomas and one leukemia). Melphalan, mitomycin C and vincristine were tested. Correlation between the two test systems was dependent upon the criteria for each test system. At the level of less than 50% survival of colony as compared with normothermic dishes in clonogenic assay, there was a high correlation between the two test systems for sensitive tumor to 43 degrees C. In respect of response to thermochemotherapy, when only karyorrhexis changes in the nucleus were selected as an activity criteria in our cytotoxic test, parallel data between the two test systems were obtained.

Predicting the sensitivity of human cancers to combined chemotherapy and hyperthermia.

In order to estimate its ability to predict the thermochemosensitivity of human cancers, a rapid in vitro assay based on morphological changes in the nucleus was performed on eight different human tumors (four malignant melanomas, two lung tumors, one renal carcinoma, and leukemia K-562). Nude mice, implanted with tumors, supplied the tumor material, with the exception of leukemia. Nimustine, melphalan, mitomycin C, vincristine and vinblastine were tested. Tumor cells developed karyorrhectic changes after incubation for 4 h with each of the aforementioned five drugs. An increase in the karyorrhectic changes was observed with hyperthermia at 43 degrees C. The individual tumors showed different sensitivities to 43 degrees C. Five of the eight tumors were significantly sensitive to 43 degrees C. However, in two thermosensitive tumors no drug enhancement was recognized at 43 degrees C. In four tumors several drugs were synergistically enhanced by hyperthermia at 43 degrees C. This study suggests that this simple method may be of clinical use in predicting response to thermochemotherapy.

Changes of liver fibrosis in chronic hepatitis C patients with no response to interferon-alpha therapy: including quantitative assessment by a morphometric method.

The aim of this study was to evaluate the antifibrotic effect of interferon (IFN)-alpha in chronic hepatitis C (CH-C) patients with no response to IFN-alpha therapy. We studied 76 patients (46 men, 30 women; mean age, 55.6 years) who received IFN-alpha intramuscularly, at a total close of 480 to 880MU for 6 months (group A). As a control group, we studied 50 patients (32 men and 18 women; mean age, 58.5 years) with CH-C who received medication other than IFN (ie, Strong-Neo-Minophagen C, ursodeoxycholic acid, and a herbal medicine, Sho-saiko-to [TJ-9]) and who had persistent alanine aminotransferase (ALT) elevation (group B). All patients were subdivided into three subgroups according to different patterns of ALT changes during the observation period, ie, (a) persistent ALT level < 60IU/ 1 (below about twice the upper limit of the normal range), (b) persistent ALT level > or = 60IU/1, (c) ALT levels other than (a) and (b). Liver biopsy was performed within 6 months prior to IFN therapy and more than 6 months after IFN therapy, while two liver biopsies were performed during therapy in group B. Liver fibrosis was compared between two specimens by staging. When the fibrosis stage was the same in the two specimens, we determined whether the fibrosis had improved or worsened by comparing the fibrotic ratio, ie, the ratio of the area of fibrosis to the area of the entire liver tissue specimen, calculated using computed graphic software. Serum aminoterminal peptide of type III procollagen (PIIIP) levels were measured on the day of the liver biopsy and their mean yearly changes were compared between the two groups. Improvement of liver fibrosis was found in 12% to 30% of patients in each ALT subgroup and in 24% of all patients in group A and there were no significant differences in liver fibrosis in comparison with findings in of group B when assessed by staging alone. However, these percentages rose to 59% to 75% and 66%, respectively, when liver fibrosis was assessed by the fibrotic ratio together with staging, resulting in a significant difference in fibrosis between groups A and B in total (P < 0.01). The mean yearly changes in serum PIIIP levels in each subgroup and in all patients in group A were below zero, indicating a tendency to improvement of fibrosis after IFN therapy, while these changes in group B were all above zero, except for subgroup (c). Improvement of fibrosis after IFN therapy was found in 15 of 24 patients (64%) whose ALT changes had the same pattern before and after IFN therapy, although no significant difference was noted between improved and worsened patients. These results suggest that IFN-alpha may have an antifibrotic effect even in CH-C patients with no overt response to IFN-alpha therapy, compared with the effect of medications other than IFN.

Influence of TT virus on the histopathological features of nonalcoholic fatty liver disease.

The sera of 38 patients with nonalcoholic fatty liver disease (NAFLD) including nonalcoholic steatohepatitis (NASH), were tested for TT virus (TTV) DNA by polymerase chain reaction (PCR) using three different primer pairs (UTR PCR, N22 PCR and genotype-1 PCR), and various histological features of the liver biopsy specimens were compared among those who were positive or negative for TTV infection. By UTR PCR which detects all TTV genotypes, TTV DNA was detected in 37 (97%) of the 38 patients. In contrast, N22 PCR which detects primarily TTV genotypes 1-4, detected TTV DNA in 18 patients (47%). In the liver biopsy specimens, moderate to many acidophilic bodies, moderate to marked focal/spotty necrosis of hepatocytes and marked stellate, pericellular or perivenular fibrosis were observed significantly more frequently among those who were positive for TTV DNA by N22 PCR, than among those who were negative by N22 PCR. Twelve patients (32%) were positive for TTV genotype 1. Moderate to marked vacuolation of nuclei, moderate to many acidophilic bodies, and moderate to marked focal/spotty necrosis as well as marked stellate, pericellular or perivenular fibrosis were found significantly more frequently in the TTV genotype 1-positive group than in the TTV genotype 1-negative group. These results suggest that certain TTV genotypes including genotype 1 influence the necrosis and inflammation of hepatocytes and liver fibrosis in NAFLD patients.

Does the control of alanine aminotransferase levels lead to a regression of liver fibrosis in chronic hepatitis C patients?

The aim of this study was to investigate the effect of various medications other than interferon (IFN) on liver fibrosis in chronic hepatitis C (CH-C) patients, and the results were compared with those obtained in CH-C patients without therapy. Fifty CH-C patients (32 men and 18 women; mean age 58.5 years) without previous IFN therapy, who randomly received medicines other than IFN such as Stronger Neo-Minophagen C, Ursodeoxycholic acid and a herbal medicine, Sho-saiko-to (TJ-9) (Group I), and as a control group, 45 CH-C patients (27 men and 18 women; mean age 56.6 years) without therapy (Group II) were examined. All patients had persistent alanine aminotransferase (ALT) elevation more than 6 months before this study and were also subdivided into three subgroups according to different pattern of ALT during the observation period, i.e. (a): persistently ALT<60 IU/l (below about twice the upper limit of normal range); (b): persistently ALT>/=60 IU/l; and (c) other than (a) and (b). All patients were biopsied twice before starting this study and during observation period and the liver fibrosis was compared between them by staging in each case. When the fibrosis stage was the same between two specimens, we determined whether the degree of fibrosis had improved or worsened by computed image analysis. Blood tests for fibrosis marker, serum aminoterminal peptide of type III procollagen (P III P) and liver enzyme such as albumin (Alb) and zinc turbidity test (ZTT) levels, and platelet (Plt) counts were also examined on the two times of liver biopsy. As a result, there were no significant differences in fibrotic improvement rate when assessed by both staging only and staging together with fibrotic ratio, determined by computed image analysis and also in yearly change of P III P (P/Y) and fibrosis (F/Y), the changed rate of Alb, ZTT levels and Plt counts between Group I and Group II, except for P/Y in subgroup (a) which was rather higher in Group I than in Group II. There were also no significant relationship between the changes of histological activity and fibrosis staging in both groups. In conclusion, other medications than IFN could not significantly improve both liver fibrosis and its associated laboratory data irrespective of ALT levels in CH-C patients as compared to the control group during average 3 years' follow-up period.

Influence of TT virus on the clinical course of alcoholic liver disease.

The sera of 43 patients with alcoholic liver disease (ALD) who abstained from alcohol for 4 weeks, were tested for TT virus (TTV) DNA by polymerase chain reaction (PCR) using three different primer pairs (UTR PCR, N22 PCR and genotype-1 PCR). The clinical course of the TTV DNA-positive and -negative groups was compared. By UTR PCR which detects all TTV genotypes, TTV DNA was detected in 40 patients (93%). N22 PCR which detects primarily TTV genotypes 1-6, detected TTV DNA in 17 patients (40%). The alanine aminotransferase (ALT) level 4 weeks after the start of abstinence was significantly higher and the rate of change in ALT {[(ALT on admission-ALT 4 weeks after abstinence)/(ALT on admission)]x100} was lower in the patients who were positive by N22 PCR, than in those who were negative by N22 PCR. Twelve patients (28%) were positive for TTV genotype 1. In the TTV genotype 1-positive group, the ALT 4 weeks after the start of abstinence was significantly higher, and the improvement rates of ALT, gamma-glutamyl transpeptidase and alkaline phosphatase levels were lower than those in the TTV genotype 1-negative group. These results suggest that certain genotypes of TTV may interfere with the improvement of liver function following the start of abstinence in ALD patients.

Influence of TT virus infection on the thrombocytopenia of patients with chronic liver disease.

TT virus (TTV) can replicate not only in the liver but also in hematopoietic cells. To investigate the possible influence of TTV infection on the hematological parameters of patients with chronic liver disease, serum samples from 581 patients with chronic, viral or non-viral liver disease were tested for TTV DNA by polymerase chain reaction (PCR). PCR was performed by two distinct methods using N22 primer pairs (N22 PCR), which detect primarily the four major TTV genotypes (1-4), and using genotype 1-specific primers (Genotype 1-PCR). Thirteen hematological parameters measured in routine laboratory tests were compared among the patients who were positive or negative for TTV infection. The platelet count was significantly lower in the N22 PCR-positive group than in the N22 PCR-negative group among the 189 patients with chronic hepatitis C (CH-C) and among the 40 patients with chronic hepatitis without serological markers of ongoing hepatitis B virus or hepatitis C virus infection (CH-NBNC) (P<0.0001, P<0.005, respectively). Similarly, among the patients with CH-C or CH-NBNC, the platelet count was significantly lower in the TTV genotype 1-positive group than in the TTV genotype 1-negative group (P<0.001, P<0.05, respectively). The reduced platelet count of the TTV DNA-positive group was observed in every fibrotic stage of chronic hepatitis (F0-F3). These results suggest that infection of TTV of certain genotypes including genotype 1 may worsen the thrombocytopenia of patients with CH-C or CH-NBNC, irrespective of the degree of hepatic fibrosis.

Antitumor activity of ascorbic acid in combination with antitumor agents against Lewis lung carcinoma.

Antitumor activity of ascorbic acid in combination with antitumor agents (mitomycin C and 5-fluorouracil) was examined against subcutaneously implanted Lewis lung carcinoma-bearing C57BL/6 mice by feeding them an ascorbic acid-deficient diet. The mice were divided into four groups: group 1 received intraperitoneally mitomycin C (2 mg/kg) and 5-fluorouracil (50 mg/kg) once a week for four weeks beginning from the day after implantation of tumors, as well as ascorbic acid (1000 mg/kg) twice a week for the same four weeks; group 2 received only mitomycin C and 5-fluorouracil; group 3 received only ascorbic acid; group 4 received a vehicle (physiological saline). Tumor growth of group 1 compared with the other three groups, and that of group 2 compared with groups 3 and 4, was significantly inhibited by day 13 post implantation. Histological examinations of tumor tissues at 10 days after implantation of tumors already showed degenerative changes which indicated these antitumor effects.

Antitumor activity of calcium in combination with antitumor agents against Lewis lung carcinoma.

The antitumor activity of calcium gluconate in combination with mitomycin C and 5-fluorouracil was examined against subcutaneously implanted Lewis lung carcinoma-bearing C57BL/6 mice. The mice were divided into four groups: group 1 received mitomycin C (2 mg/kg) and 5-fluorouracil (50 mg/kg) intraperitoneally once a week for four weeks beginning from the day after implantation of tumors, as well as calcium gluconate (155 mg/kg) twice a week for the same four weeks; group 2 received only mitomycin C and 5-fluorouracil; group 3 received only calcium gluconate; group 4 received a vehicle (physiological saline). Significantly enhanced inhibition of tumor growth was observed neither in a comparison between groups 3 and 4, nor in a comparison between groups 1 and 2 (expect on day 20 post implantation). Thus calcium gluconate given alone or in combination with antitumor agents hardly appeared to possess effective antitumor activity.

Interferon treatment in patients with chronic hepatitis C with normal alanine-aminotransferase activity.

BACKGROUND/AIMS: Chronic hepatitis C virus carriers may have repeatedly normal alanine aminotransferase activity despite detectable viremia and histological hepatitis. We aimed to evaluate the effect of interferon treatment in these cases. METHODOLOGY: Twelve patients with persistently normal alanine aminotransferase levels at least 6 months before therapy were treated with recombinant interferon (IFN)alpha-2b for 6 months, totaling 840 MU in amount. Alanine aminotransferase levels were measured monthly during treatment and after treatment withdrawal, and HCV-RNA levels were measured by polymerase chain reaction before treatment, and 6 and 12 months after treatment withdrawal. RESULTS: At treatment withdrawal, HCV-RNA levels had significantly decreased and HCV-RNA disappeared in 9 of the 12 patients by polymerase chain reaction. At 6 months after treatment withdrawal, HCV-RNA reappeared in 6 of the 9 patients whose HCV-RNA was negative at treatment withdrawal. Over all, only 4 of the 12 patients (33%) were sustained virological responders (HCV-RNA is negative more than 6 months after treatment withdrawal). Pre-treatment HCV-RNA levels in a sustained virological responder was significantly lower than that of transient and non-responders (4.9 +/- 1.6 vs. 7.7 +/- 1.6 log10[copies/ml], p < 0.05). Of 8 patients who did not achieved sustained virological response, alanine aminotransferase levels had transiently increased above normal during treatment in one patient and after treatment withdrawal in 6 patients; however, in the remaining one patient abnormal values have continued from 8 months after treatment withdrawal till now for 24 months. CONCLUSIONS: In patients with chronic hepatitis C with normal alanine aminotransferase levels, the response to interferon therapy was by no means satisfactory. However, if it would be used in cases with the lower pre-treatment HCV-RNA levels with careful attention to a transient alanine aminotransferase elevation, the more a sustained virological response might be expected.

[Antitumor spectra of ranimustine against various human tumors].

Ranimustine (MCNU) has been shown to exhibit high antitumor activity and broad antitumor spectra against various experimental tumors. These effects were comparable to those of nimustine (ACNU). However, clinical applications of ACNU are indicated to various types of malignancies including solid tumors, while those of MCNU are almost limited to hematological ones. Therefore, the antitumor activity of MCNU was examined against 55 specimens from 15 types of solid tumors and compared with those of ACNU and 8 other drugs. Drug sensitivity was examined by a morphological method measuring the proportion of degenerative changes in the nucleus of drug-treated and untreated tumor cells. MCNU showed antitumor activities (measured by karyorrhexis) against adenocarcinoma of the lung, squamous cell carcinoma of the lung, renal cell carcinoma, bladder tumor, ovarian cancer and brain tumor. In addition, MCNU and ACNU showed a similar positive rate (15-16%) in this experiment and this was the highest among all drugs examined. Although MCNU and ACNU showed similar antitumor spectra, a clear difference was observed when the antitumor activities of both drugs were compared in each identical specimen. These results clearly suggest that MCNU is worthy of clinical study to examine the antitumor activity against various solid tumors.

[Effects of various agents on human choriocarcinoma transplanted to nude mice].

The effects of various anticancer agents on human choriocarcinoma transplanted to nude mice, specifically, inhibition effects on tumor growth and survival rate were studied to establish an appropriate chemotherapy for refractory choriocarcinoma. The agents studied were methotrexate (MTX), actinomycin D (ACD), cyclophosphamide (CPM), vincristine (VCR), L-PAM (MPI), bleomycin (BLM), carboquone (CQ), cisplatin (CDDP), ACNU, MCNU, vinblastine (VLB), VP16-213, OK-432, Maruyama vaccine (SSM) and metronidazole (ME), and the following results were obtained: 1) The inhibition effects on tumor growth were obtained in the groups of VCR, VP16-213, CDDP, MPL, CPM and CQ; 2) The survival rate was 100% in the groups of MPL, BLM, and CQ. In the groups of ACD VLB, VP16-213 and VCR, all mice died. 3) MPL was found to be the most effective agent in terms of inhibitory effect and survival rate. In the future, combination chemotherapy including MPL and maintenance chemotherapy with MPL to refractory choriocarcinoma should be studied.