Atsumasa Komori

Cancer inhibition by green tea.

Green tea is now an acknowledged cancer preventive in Japan. This paper discusses several important features of (-)-epigallocatechin gallate (EGCG), the main constituent of green tea and tea polyphenols. EGCG and other tea polyphenols inhibited growth of human lung cancer cell line, PC-9 cells with G2/M arrest. 3H-EGCG administered by p.o. intubation into mouse stomach revealed that small amounts of 3H-activity were found in various organs where EGCG and green tea extract had previously demonstrated their anticarcinogenic effects, such as skin, stomach, duodenum, colon, liver, lung and pancreas. Cancer onset of patients who had consumed over 10 cups of green tea per day was 8.7 years later among females and 3.0 years later among males, compared with patients who had consumed under three cups per day. The mechanisms of action of EGCG were briefly discussed with regard to inhibition of tumor necrosis factor-alpha (TNF-alpha) release.

Mechanisms of Growth Inhibition of Human Lung Cancer Cell Line, PC-9, by Tea Polyphenols

(–)‐Epigallocatechin gallate (EGCG), the main constituent of green tea, and green tea extract show growth inhibition of various cancer cell lines, such as lung, mammary, and stomach. We studied how tea polyphenols induce growth inhibition of cancer cells. Since green tea extract contains various tea polyphenols, such as EGCG, (–)‐epigallocatechin (EGC), (–)‐epicatechin gallate (ECG), and (–)‐epicatechin (EC), the inhibitory potential of each tea polyphenol on the growth of a human lung cancer cell line, PC‐9 cells, was first examined. EGC and ECG inhibited the growth of PC‐9 cells as potently as did EGCG, but EC did not show significant growth inhibition. The mechanism of growth inhibition by EGCG was studied in relation to cell cycle regulation. Flow cytometric analysis revealed that treatment with 50 μM and 100 μM EGCG increased the percentages of cells in the G2‐M phase from 13.8% to 15.6% and 24.1%, respectively. The DNA histogram after treatment with 100 μM EGCG was similar to that after treatment with genistein, suggesting that EGCG induces G2‐M arrest in PC‐9 cells. Moreover, we found by microautoradiography that [3H]EGCG was incorporated into the cytosol, as well as the nuclei. These results provide new insights into the mechanisms of action of EGCG and green tea extract as cancer‐preventive agents in humans.

Expression of the tumor necrosis factor alpha gene and early response genes by nodularin, a liver tumor promoter, in primary cultured rat hepatocytes.

Nodularin is a new liver carcinogen possessing a potent tumor-promoting activity in rat liver, mediated through inhibition of protein phosphatases 1 and 2A, and a weak initiating activity. Since we previously reported evidence that nodularin up-regulated expression of the tumor necrosis factor α gene (TNFα) and early-response genes in rat liver after its i.p. administration, and since TNFα had tumor-promoting activity in vitro, it is possible that TNFα itself is involved in liver tumor promotion. We investigated whether hepatocytes themselves induce expression of theTNFα gene and early-response genes in primary cultured rat hepatocytes treated with nodularin. Like nodularin, microcystin-LR, which is another liver tumor promoter belonging to the okadaic acid class, strongly inducedTNFα gene expression in rat hepatocytes, as well as TNFα release from those cells into the medium. On the other hand, 12-O-tetradecanoylphorbol-13-acetate, which has been reported to induce no tumor promotion in rat liver, induced no apparent expression of theTNFα gene in primary cultured rat hepatocytes. As for the expression of early-response genes, 1 μM nodularin or microcystin-LR induced expression of the c-jun, jun B,jun D, c-fos, fos B andfra-1 genes in the hepatocytes, and the expression of these genes was prolonged up to 24 h, suggesting mRNA stabilization induced by inhibition of protein phosphatases 1 and 2A. This paper presents new evidence that theTNFα gene and early-response genes were expressed in hepatocytes treated with a liver tumor promoter.

Designing of chimeric DNA/RNA hammerhead ribozymes to be targeted against AML1/MTG8 mRNA.

For therapeutic purposes, two chimeric DNA/RNA hammerhead ribozymes were synthesized to cleaveAML1/MTG8, the t(8;21)-associated fusion mRNA of acute myeloid leukemia. One ribozyme, A/MRZ-1, recognizes the area adjacent to the fusion point betweenAML1 andMTG8, and cleaves six bases downstream from this point. The other, MRZ-1, recognizes theMTG8 sequence. Both ribozymes cleaved synthetic chimeric DNA/RNA substrates at theoretical sites. Neither cleavedAML1 RNA. A/MRZ-1 cleaved onlyAML1/MTG8 RNA, and MRZ-1 cleaved bothAML1/MTG8 andMTG8 RNAs. The two ribozymes showed growth inhibition of an acute myeloid leukemia cell line carrying t(8;21), SKNO-1 cells. The same extent of growth inhibition was attained by antisense oligonucleotides againstAML1/MTG8 RNA. The results suggest that the ribozyme has the potential to be developed as a useful agent for gene therapy, in particular for leukemia with t(8;21).

Tautomycin: An inhibitor of protein phosphatases 1 and 2A but not a tumor promoter on mouse skin and in rat glandular stomach

Tautomycin isolated fromStreptomyces spiroverticillatus is an inhibitor of protein phosphatases 1 and 2A. Tautomycin induced hyperphosphorylation of cytokeratin peptides in human keratinocytes (PHK 16-I cells) 30 times less strongly than did okadaic acid. Repeated applications of tautomycin (30 μg, 40 nmol/application) did not induce tumor promotion in a two-stage carcinogenesis experiment on mouse skin initiated with 7,12-dimethylbenz[a]anthracene, whereas okadaic acid (1 μg, 1.2 nmol/application) as a control induced tumor promotion strongly. As for mucosa or rat glandular stomach, tautomycin induced ornithine decarboxylase 4 h after intubation into the stomach. The tumor-promoting activity of tautomycin was next studied in the glandular stomach initiated withN-methyl-N′-nitro-N-nitrosoguanidine (MNNG). Administration of tautomycin in the diet (1 mg rat−1 day−1), from week 9 to week 52 of the experiment, inhibited rather than enhanced tumor development in the glandular stomach initiated with MNNG. The percentages of tumor-bearing rats of the groups treated with MNNG plus tautomycin, MNNG alone, and tautomycin alone were 20.0%, 40.6%, and 0% respectively in week 52. The reason for the absence of tumor-promoting activity of tautomycin was studied in relation to tumor necrosis factor α (TNFα), an endogenous tumor promoter. We found that tautomycin neither enhanced TNFα mRNA expression in mouse skin nor induced TNFα release in a human stomach cancer cell line (KATO III cells), whereas okadaic acid did both. These results indicate that not all inhibitors of protein phosphatases are tumor promoters, and suggest that tumor promotion of the okadaic acid class of compounds is mediated by TNFα.

Association of MTG8 (ETO/CDR), a leukemia-related protein, with serine/threonine protein kinases and heat shock protein HSP90 in human hematopoietic cell lines.

A proto‐oncogene, MTG8 (ETO/CDR), is disrupted in the t(8;21) translocation associated with acute myeloid leukemia, and the gene product, MTG8, is a phosphoprotein capable of cell transformation in concert with v‐H‐ras. To obtain insight into functional regulation of MTG8 by phosphorylation, we studied protein kinases that interact with, and phosphorylate, MTG8 in vitro. Recombinant MTG8 protein was first found to be associated with two serine/threonine protein kinases in cell extracts from both HEL cells and a leukemic cell line carrying t(8;21). A cytoplasmic protein kinase of 61 kDa (MTG8N‐kinase) phosphorylated the amino‐terminal of MTG8, and another of 52 kDa (MTG8C‐kinase) phosphorylated the carboxyl‐terminal domain. In addition, we demonstrated that heat shock protein 90 (HSP90) specifically binds to the amino‐terminal domain of MTG8 in vitro and in vivo. Thus, our results shed new light on post‐translational regulation of MTG8, perturbation of which, in AML1‐MTG8 protein, probably contributes to leukemogenesis.

Tumourigenicity of MTG8, a leukaemia-related gene, in concert with v-Ha-ras gene in BALB/3T3 cells

The MTG8 (ETO) gene has been identified as the translocation partner of AML1 (PEBP2αB or CBFα2) gene in the AML1/MTG8 (ETO) fused gene caused by t(8;21) translocation in human acute myeloid leukaemia, M2 type. Although AML1/MTG8 chimaeric protein is known to inhibit the functioning of AML1 protein, the precise function of MTG8 gene itself is not known yet. We studied the significance of MTG8 gene in the oncogenicity of AML1/MTG8 fused gene, by introducing full‐length MTG8 cDNA into both BALB/3T3 cells containing v‐Ha‐ras gene (Bhas 42 cells) and BALB/3T3 cells without v‐Ha‐ras gene.

Natural Inhibitors of Carcinogenesis

For the purpose of finding nontoxic cancer preventive agents, we studied natural products derived from marine and plant sources. The following compounds inhibited tumor promotion on mouse skin in two-stage carcinogenesis experiments: two marine natural products and four plant natural products. Among these natural inhibitors, we suggest green tea as a cancer preventive in humans. This chapter reviews our basic study on cancer preventive agents derived from natural sources.

Mechanisms of (−)-Epigallocatechin Gallate and Green Tea in Inhibition of Carcinogenesis

Based on the evidence that (−)-epigallocatechin gallate (EGCG), the main constituent of Japanese green tea, Camellia sinensis, has anticarcinogenic effects on rodent carcinogenesis, many investigators found that green tea extract in drinking water also inhibits carcinogenesis of various organs in rodents. As for the inhibitory mechanisms of carcinogenesis, we found that EGCG inhibited the release of tumor necrosis factor-α (TNF-α), an endogenous tumor promoter from BALB/3T3 cells, induced by okadaic acid, suggesting that EGCG reduces the amount of this endogenous tumor promoter in tissues. EGCG also inhibited TNF-α release from PE501/TNF cells, which are transfected retrovirus vector containing TNF-α cDNA and which constantly produce TNF-α. These results suggest that EGCG inhibits the process of TNF-a release by blocking the interaction of tumor promoter to its receptor as well as inhibition of proteolysis of TNF-α precursor protein. In addition, we briefly discuss the results with direct administration of 3H-EGCG into the stomach of mice. We present here our results with EGCG and green tea extract as promising cancer chemopreventives, with emphasis on their mechanisms of action.