Anti-tumor effect of gallic acid on LL-2 lung cancer cells transplanted in mice.

We previously reported that gallic acid (3,4,5-trihydroxybenzoic acid), a naturally occurring plant phenol, can induce apoptosis in four kinds of human lung cancer cell lines in vitro. The present study further investigated the in vivo anti-tumor effects of orally administered gallic acid. Gallic acid reduced cell viability of LL-2 mouse lung cancer cells in vitro dose dependently, with a 50% inhibitory concentration (IC50) value of around 200 microM. C57Black mice were transplanted with LL-2 cells, and administered gallic acid (1 mg/ml in drinking water, ad libitum) and/or cisplatin (4 mg/kg i.p. injection, once a week). The average weight of the transplanted tumors, obtained at 29 days after transplantation, in the mice of control, gallic acid-treated cisplatin-treated and cisplatin plus gallic acid-treated groups was 4.02, 3.65, 3.19 and 1.72 g, respectively. The average tumor weight of the mice treated with cisplatin combined with gallic acid was significantly smaller than that of the control group (p<0.05). The amount of apoptotic cells in the tumor tissues of mice treated with gallic acid and/or cisplatin was significantly higher than those of the control mice. Combination of gallic acid and cisplatin increased the tumor cell apoptosis compared with the treatment with cisplatin alone. The present findings suggest that the combination of gallic acid with an anti-cancer drug, including cisplatin, may be an effective protocol for lung cancer therapy.

5'-deoxy-5-fluorouridine, medroxyprogestrone acetate and mitoxantrone hydrochloride for advanced or recurrent breast cancer.

BACKGROUND: In treating advanced or recurrent breast cancer, anthracycline-containing chemotherapy is used for palliation and to maintain quality of life. However, there are several drawbacks including therapeutic failure and cardiotoxicity. We evaluated the efficacy and toxicity of combination chemotherapy with 5'-deoxy-5-fluorouridine (5'-DFUR), medroxyprogestrone acetate (MPA) and mitoxantrone hydrochloride (MIT). METHODS: Sixteen patients with advanced or recurrent breast cancer were enrolled. Chemotherapy was given in a 28-day cycle, starting with MIT 10 mg/m2 intravenously on day 1, then oral 5'-DFUR 800 mg and MPA 800 mg daily. Two or more cycles were given. RESULTS: Fifteen patients were assessable for response and toxicity. Thirteen patients had been treated previously with an anthracycline containing regimen and 2 with CMF. There were 2 partial response patients (13.3%) and 1 complete response patient (6.7%). There were 11 patients showing no change (NC) (73.3%), one of whom was a minor responder and 7 with a long period of NC. There was only one with progressive disease patient. The overall response rate was 20.0%. Adverse events occurred in 5 patients (33.3%). Myelosuppression was the most common with 5 patients becoming leukopenic (33.3%). Nausea/vomiting was the second most common side effect, affecting 2 patients (13.3%). CONCLUSION: Given its high efficacy and preservation of QOL, the combination of MIT, 5'-DFUR and MPA can be a 2nd or 3rd line therapy for advanced or recurrent breast cancer, especially for anthracycline-resistant cases.

Inhibition by costunolide of phorbol ester-induced transcriptional activation of inducible nitric oxide synthase gene in a human monocyte cell line THP-1.

Costunolide, the predominant sesquiterpene lactone in Saussureae radix, has been reported to exhibit potent chemopreventive effects on carcinogenesis. Effects of costunolide on cellular activation induced by a tumor-promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) were investigated using a reporter gene assay which was designed to reflect the promoter activity of the inducible nitric oxide synthase (iNOS) gene in a human monocyte cell line THP-1. iNOS promoter-dependent reporter gene activity was significantly increased by TPA, and the TPA-induced increase of the reporter gene activity was efficiently reduced by costunolide, with an IC50 of approximately 2 microM. The addition of sulfhydryl (SH) compounds effectively abrogated the inhibitory effects of costunolide, suggesting the involvement of its reactivity with SH groups of target proteins and/or thiol-depleting property. The present findings may further explain the cancer-preventive property of costunolide.

Neurilemmoma arising in the brachial plexus in association with breast cancer: report of case.

A 48-year-old woman without von Recklinghausen's disease presented to our department for investigation of a left breast lump and a lump in the left axilla. A presumptive diagnosis of left breast cancer with axillary lymph node involvement was made based on the findings of physical examination and needle biopsy of the left breast lump. However, on exploration we discovered that the axillary lump was a neurogenic tumor arising in the brachial plexus. Enucleation of the neurogenic tumor, which was subsequently histologically confirmed as neurilemmoma, as well as a modified radical mastectomy, was performed. Postoperatively, the patient experienced slight numbness in her left second and third fingers, but this symptom improved within 5 months. When last seen 21 months after her operation, there were no signs of recurrence of either the breast cancer or the neurilemmoma.

Inhibition by parthenolide of phorbol ester-induced transcriptional activation of inducible nitric oxide synthase gene in a human monocyte cell line THP-1.

Excessive nitric oxide production by inducible nitric oxide synthase (iNOS) in stimulated inflammatory cells is thought to be a causative factor of cellular injury in inflammatory disease states. Compounds inhibiting iNOS transcriptional activity in inflammatory cells are potentially anti-inflammatory. An assay method for estimating iNOS transcriptional activity in the human monocyte cell line THP-1 was established using a luciferase reporter gene system. In this study, we demonstrate that parthenolide, the predominant sesquiterpene lactone in European feverfew (Tanacetum parthenium), exerts potent inhibitory effects on the promoter activity of the iNOS gene in THP-1 cells. Parthenolide effectively suppressed iNOS promoter activity in a dose-dependent manner at concentrations higher than 2. 5 microM, with an IC(50) of about 10 microM. A tumor-promoting phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), significantly increased the iNOS promoter-dependent reporter gene activity, and the TPA-induced increase in iNOS promoter activity was effectively suppressed by parthenolide, with an IC(50) of approximately 2 microM. The present findings may further explain the anti-inflammatory property of parthenolide.

Induction of apoptosis by gallic acid in lung cancer cells.

The apoptosis-inducing effect of gallic acid (3,4,5-trihydroxybenzoic acid) was investigated in four human lung cancer cell lines, SBC-3 (small cell carcinoma), EBC-1 (squamous cell carcinoma), A549 (adenocarcinoma) and SBC-3/CDDP (cisplatin-resistant subclone of SBC-3). Gallic acid induced apoptosis in a dose-dependent manner as evidenced by analyses of DNA fragmentation, changes in cell morphology and loss of viability. Fifty percent inhibitory concentration (IC50) values of gallic acid on the cell viability of SBC-3, EBC-1 and A549 were around 10, 20 and 60 microg/ml, respectively. The IC50 value for SBC-3/CDDP cells was almost the same as that of SBC-3, suggesting that susceptibility of cells to gallic acid-induced apoptosis is not altered by the acquisition of cisplatin resistance. The apoptotic process was effectively triggered by 30 min exposure to gallic acid. A caspase inhibitor and alpha-tocopherol effectively prevented the gallic acid-induced apoptosis, indicating the involvememt of caspase activation and oxidative processes during the course of apoptosis in gallic acid-treated cancer cells. These findings suggest the possible applicability of gallic acid in lung cancer therapy, especially to circumvent resistance to anti-cancer drugs.

Inhibition by berberine of cyclooxygenase-2 transcriptional activity in human colon cancer cells.

The enzyme cyclooxygenase-2 (COX-2) is abundantly expressed in colon cancer cells and plays a key role in colon tumorigenesis. Compounds inhibiting COX-2 transcriptional activity have therefore potentially a chemopreventive property against colon tumor formation. An assay method for estimating COX-2 transcriptional activity in human colon cancer cells was established using a beta-galactosidase reporter gene system, and examination was made of various medicinal herbs and their ingredients for an inhibitory effect on COX-2 transcriptional activity. We found that berberine, an isoquinoline alkaloid present in plants of the genera Berberis and Coptis, effectively inhibits COX-2 transcriptional activity in colon cancer cells in a dose- and time-dependent manner at concentrations higher than 0.3 microM. The present findings may further explain the mechanism of anti-inflammatory and anti-tumor promoting effects of berberine.

Inhibition of activator protein 1 activity by berberine in human hepatoma cells.

Activator protein 1 (AP-1) is a transcription factor which plays a critical role in inflammation and carcinogenesis. The present study was conducted to investigate the effect of berberine, an isoquinoline alkaloid present in plants of the genera Berberis and Coptis, on the activity of AP-1 using a reporter gene assay in human hepatoma cells. Berberine was shown to inhibit AP-1 activity in a dose- and time-dependent manner at concentrations higher than 0.3 microM. Berberine inhibited AP-1 activity almost completely as low as 10 microM after 48 h treatment. The inhibitory effect on AP-1 activity in cancer cells may further explain the anti-tumor promoting activity of berberine.

Plasma D-dimer level in patients with colorectal cancer: its role as a tumor marker.

The purpose of this study was to explore the relationship between the preoperative plasma D-dimer (DD) levels and the tumor pathology of colorectal cancer. The plasma DD levels were measured preoperatively in 108 patients with colorectal cancer, and then were correlated with the tumor pathology and stage. The diagnostic value of the DD levels for the tumor stage was then compared with that of the preoperative carcinoembryonic antigen (CEA) levels. The preoperative DD levels were higher in patients with either a large-sized tumor or a tumor showing deep wall penetration. Lymph-node metastasis, lymphatic invasion, hepatic metastasis, and peritoneal dissemination were all associated with higher DD levels. A stepwise increase in the median DD level was found with the tumor stage. The preoperative DD levels also significantly correlated with CEA levels. When a cutoff value of 0.6 microg/ml was used in the DD assay, the sensitivity and specificity for Dukes C or D cancer were 67.2% and 64.0%, and those for Dukes D cancer were 91.3% and 57.6%, respectively. Although the DD assay was less specific, its diagnostic value in the preoperative staging of colorectal cancer was comparable to that of the CEA assay. The measurement of the preoperative DD level is thus considered to be useful for the preoperative staging of colorectal cancer.

[The alteration of PyNPase activity in breast cancers due to preoperative oral administration of 5'-DFUR].

To estimate the alteration of pyrimidine nucleoside phosphorylase (PyNPase) activity by 5'-DFUR, we determined its activity in the breast tissue of two patient groups. Group A (21 patients) were given 5'-DFUR (mean 9,000 mg) preoperatively, and group B (21 patients) were given no anticancer agents. There were no significant differences between the groups in background factors such as age, clinical stage and pathological features. Comparison of PyNPase activity in both groups among specimens of carcinoma, normal breast glands and lymph nodes revealed that the activity in carcinomas and lymph nodes was significantly higher than in normal breast glands. Comparison of group A with group B showed that group A had lower activity in carcinomas and lymph nodes than in group B, although the difference was not significant. An inter-group comparison of carcinomas was made in detail by subgrouping according to age and pathological features. In group B, PyNPase activity in older patients (> or = 50 y.o) was significantly higher than in younger patients (< 50 y.o), and papillotubular carcinomas had significantly lower activity than solid tubular and scirrhous carcinomas. However, in group A these data could not be determined. These results suggest that PyNPase activity in group A was possibly decreased by 5'-DFUR.

Rectal submucosal tumor-like lesion originating from intestinal tuberculosis.

We present the case of a 55-year-old man who underwent transsacral local excision for a rectal submucosal tumor-like lesion suspected to originate from tuberculosis. The lesion, 2 cm in size, was found incidentally in the posterior wall of the lower rectum during anal fistulectomy. The lesion was apart from the primary crypt of the anal fistula. Barium enema and colonoscopy revealed a protuberant submucosal growth with a shallow depression of the overlying mucosa. Although computed tomography and magnetic resonance imaging showed a well defined round mass within the rectal wall, digital rectal examination suggested extramural origin. Since repeated endoscopic biopsies were negative, we selected the transsacral approach for excisional biopsy to achieve histological diagnosis. The lesion was confined to the rectal wall and the full-thickness rectal wall was excised. Histologically, a foreign-body granuloma with acute inflammation was the main component of the lesion. Caseating granulomas and Langhans' giant cells, consistent with tuberculosis, were also found.

[Evaluation of subrenal capsule assay (SRC) for clinical cancers].

For clinical cancers of esophagus (10 cases), stomach (7), colon and rectum (11) and breast (5), subrenal capsule assays (SRC) were performed using normal or AF nude mice. The sensitivity of anticancer drugs (UFT, CDDP) was evaluated by both the change in graft size under the renal capsule and the histological findings of these grafts. SRC was found to be a very reliable testing method for cancer of esophagus, colon and rectum because these grafts possessed remarkable viability. Evaluability of these two cancers was very high (approximately 80%). Sensitivity of UFT was 29% (2/7) for esophageal cancer, 50% (5/10) against colon and rectal cancer, and that of CDDP was 40% (2/5) and 17% (1/6), respectively. Evaluability of gastric and breast cancers was very low because of the poor growth of the grafts irrespective of the immunity of host animals. It was discussed why the evaluation rate depended on the type of cancer. These results suggest that indications for SRC are restricted, but that for colon and rectal or esophageal cancer SRC is a reliable testing method for anticancer drugs in clinical use.

Purification and characterization of acid cysteine protease from metacercariae of the mammalian trematode parasite Paragonimus westermani.

Acid cysteine protease was purified from metacercariae of the mammalian trematode parasite Paragonimus westermani. The purified enzyme had a molecular mass of 27 kDa and was a monomeric polypeptide. The protease had an absolute requirement for a reducing agent for full activity towards fluorescein-isothiocyanate-labeled hemoglobin, and it was active in the acidic pH range, with an optimum pH of 4.0. While acidic proteolysis was insensitive to the aspartic protease inhibitor pepstatin A, activity was significantly inhibited by the cysteine protease inhibitors, leupeptin, chymostatin and L-trans-epoxy-succinyl-L-leucylamido(4-guanidino)-butane. The sensitivity of the enzyme to the inhibitors was similar to that of cathepsins B and L, but the specificity of the protease towards chromogenic substrates was slightly different from that of the cathepsins. The purified enzyme was highly specific for N-substituted peptidyl substrates containing arginine in the P1 position and phenylalanine in the P2 position, and the protease extensively degraded human native proteins, such as human serum albumin, immunoglobulins, complement components and also endogenous protease inhibitors. Since the protease hydrolyzes both soluble proteins and components of human defense systems, it may facilitate parasite nutrition and evasion of host defense mechanisms.

[Light and electron microscopic examination compared to evaluation by tumor size in subrenal capsule assay for chemosensitivity testing in esophageal cancer].

Specimens from six clinical cases of esophageal cancer were transplanted under the renal capsule of AF nude mice, and chemosensitivity to anticancer drugs (UFT, CDDP) was evaluated by measuring tumor size (SRC). Histological analysis of the xenografts by light (LM) and electron microscopy (EM) was also performed to confirm the precision of this original method used for SRC. Two-dimensional morphometry by EM was also performed in four cases. With the exception of one case, the same results were obtained by graft measurement analysis and light microscopic observation, which allowed easy evaluation by observing cancer pearl formation, prominent proliferation of tumor cells or total cell keratinization. However, there was no false-positive sensitivity in the former method. EM observation did not reveal any special findings with regard to chemotherapy response, but increased numbers of cytoplasmic vacuoles and desmosomes, cytoplasmic swelling, a reduction of the N/C ratio and nuclear deformity implied common changes to cell necrosis induced by the anticancer drugs. For clinical use of SRC against esophageal cancer, it is supposed to be best to compare LM histological evaluation, which is too complicated and time consuming. For this reason, it is potentially useful to perform SRC by evaluating tumor size, because another goal of chemosensitivity testing involves determination of anticancer drugs without effectiveness.

[Electron microscopic study on the method of evaluation of SRC assay].

The subrenal capsule assay (SRC) is a rapid and precise method for evaluation of chemotherapeutic agents. However, it seems to present some difficulties with regard to sensitivity, since evaluation is done by measurement of graft size. In this study, SRC was performed on six clinical cases of colorectal cancer using nude mice, which were given UFT 15 or 20 mg/kg orally or 2.5 mg/kg CDDP subcutaneously for 2-6 days. Histological changes in the grafted tumors were then observed by electron microscopy. Mucous granules and other features were assessed for evaluation of assay sensitivity. The number of mucous granules seemed to be the most reliable parameter that paralleled the sensitivity evaluated by tumor diameter. Other features such as nuclear mitosis, lysosomal increment and abnormal accumulation of ribosomes, had little correlation with sensitivity. However, further exploration is warranted with regard to local defects of the cytoplasm. This ultrastructural examination suggested that grafted tumor cells were damaged slightly by implantation under the renal capsule, and that the SRC using tumor size as a parameter is clinically useful under conditions where the tumor xenografts show good viability and proliferation in the control group.

Aminopeptidase activity in the brains of mice with chronic Toxoplasma gondii infections.

Aminopeptidase activity on beta-naphthylamide (NA) substrates was assayed in brain extracts from normal and Toxoplasma gondii-infected mice at 4 months postinfection. Correlations of levels of aminopeptidase activity, Toxoplasma-specific antibody production, and the number of brain cysts were studied in normal and Toxoplasma-infected mice. The Toxoplasma-specific antibody and the formation of cysts were markedly enhanced in the parasite-infected mice. The highest levels of activity for the NA substrates tested were observed in normal mice. In contrast, the activity levels were significantly lower in T. gondii-infected mice than in the corresponding normal mice. These results suggest an association between chronic toxoplasmosis and aminopeptidase activity in the parasite-infected host brain.

Extracellular matrix of cultivated, poorly differentiated human gastric adenocarcinoma cells promotes attachment and spreading of mesenchymal cells.

To clarify interactions between carcinoma and mesenchymal cells, we examined the extracellular matrix-substance remaining on culture dishes after confluent growths of gastric carcinoma cells were removed with EDTA. The matrix synthesized by poorly differentiated adenocarcinoma cells (cell lines KATO-III and MKN-45) cultivated in serum-free medium has a fibroblast (cell line WI38)-attachment activity, which is not detected in the matrix synthesized by a well differentiated adenocarcinoma (cell line MKN-28). This activity was not observed in KATO-III-matrix extracted with 6 M urea, but could be detected in a 1% SDS extract from the remaining matrix on the culture dishes after 6 M urea extraction. The activity was abolished by treatment with pronase (16 micrograms/ml), trypsin (0.005%) or alkali, but was unaffected by collagenase (80 micrograms/ml, 4 h) or chondroitinase ABC (1 U ml, 1 h). It is conceivable that the fibroblast-attachment activity of the matrix produced by poorly differentiated adenocarcinoma cells is related to the proliferation of interstitial connective tissue in vivo.

Isolation and characterization of proteoglycans from human nonepithelial tumors.

Proteoglycans (PGs) and glycosaminoglycans (GAGs) were identified in myogenic and fibrogenic tumors. More PGs containing mainly chondroitin sulfate could be detected in malignant tumors (leiomyosarcomas) than in benign tumors (leiomyomas and fibromas). Two groups of PGs were detected in the malignant tumors by ion-exchange chromatography and gel chromatography. One group was a large molecule with chondroitin sulfate side chains, seemingly composed of two or more subpopulations that were eluted from Sepharose CL-4B with a kav of 0.45. After removal of GAG side chains from the PG by chondroitinase AC digestion, core molecules with molecular weights greater than 200,000 were obtained. Another PG detected was a fraction of small PG eluted from Sepharose CL-4B with a kav of 0.45. It consisted of a core molecule with a molecular weight approximately equal to 48,000 and GAG side chains containing chondroitin sulfate-dermatan sulfate hybrids. The mixed sequence of L-iduronic acid with D-glucuronic acid in the same GAG chain was demonstrated by the formation of a small proportion of tetrasaccharide after chondroitinase AC digestion. In the benign tumors, the large PG was found only in very small amounts, and PG detected was composed mainly of the small one eluted from Sepharose CL-4B with a kav of 0.45. Its core protein had a molecular weight of approximately equal to 46,000, which was similar to that of small PG obtained from leiomyosarcomas, but its GAG side chains were composed mainly of dermatan sulfate containing small amounts of glucuronic acid. The results suggest that the core molecules of small PGs from both benign and malignant tumors are the products of the same gene but that they are processed in a different manner to form proteoglycans with different types of GAG chains.