Masami Suganuma

Synergistic enhancement of anticancer effects on numerous human cancer cell lines treated with the combination of EGCG, other green tea catechins, and anticancer compounds

PurposeIn 2008, we reported that 10 Japanese-size cups of green tea daily, supplemented with tablets of green tea extract (GTE), reduced the recurrence of colorectal adenoma by 51.6xa0% in patients after polypectomy. Based on these results, we paid special attention to Japanese cancer patients, who consume green tea every day and are administered anticancer drugs. This encouraged us to study whether the combination of green tea catechins and anticancer drugs has the potential to enhance the efficacy of the drugs.Results and discussionThe combination of GTE and NSAIDs synergistically inhibited tumor development in rodents through the activation of the GADD153–DR5–TRAIL apoptotic pathway. Since then, this study was further extended by various investigators to the combinations of EGCG and other green tea catechins with anticancer compounds, the latter of which include NSAIDs, phytochemicals, and anticancer drugs. In order to demonstrate whether diversity of the combinations would generally induce synergistic anticancer effects on numerous human cancer cell lines, we studied the results of 42 in vitro combination experiments and the synergistic inhibition of tumor volume of 13 combination experiments using xenograft mouse models, which were previously reported by other investigators. The various combinations of EGCG and anticancer compounds induced similar synergistic anticancer effects for both in vitro and in vivo experiments, and showed an average reduction in tumor volume by 70.3xa0%. Considering the evidence showing that treatment with EGCG inhibited self-renewal of cancer stem cells, the combination shows a great advantage.ConclusionGreen tea is a cancer preventive for humans, showing a new trend of green tea catechins as synergists with anticancer compounds.

Primary cancer prevention by green tea, and tertiary cancer prevention by the combination of green tea catechins and anticancer compounds.

Green tea is a daily beverage, a non-oxidized non-fermented product containing at least four green tea catechins. Considering our first results when repeated applications of (-)-epigallocatechin gallate (EGCG) prevented tumor promotion in mouse skin, we have continued to look at green tea as a possible cancer preventive agent. 1) The 10-year prospective cohort study by Drs. K. Nakachi and K. Imai revealed that drinking 10 Japanese-size cups (120 mL/cup) of green tea per day delayed cancer onset in humans by 7.3 years among females and by 3.2 years among males. The delay of cancer onset is of course significant evidence of primary cancer prevention in humans. 2) In collaboration with Dr. H. Moriwaki’s group we successfully presented a prototype of tertiary cancer prevention showing that 10 Japanese-size cups of green tea daily, supplemented with tablets of green tea extract (G.T.E), reduced recurrence of colorectal adenomas in polypectomy patients by 51.6% (from 31% to 15%). 3) In 1999, we first reported that the combination of green tea catechins and non-steroidal anti-inflammatory drugs showed synergistic anticancer effects in both in vitro and in vivo experiments, along with elucidation of the mechanism. 4) Further studies by other investigators have revealed that various combinations of EGCG or green tea extract and anticancer compounds inhibit tumor volume in xenograft mouse models implanted with various human cancer cell lines. Green tea is a cancer preventive, and green tea catechins act as synergists with anticancer compounds.

Down-regulation of histone deacetylase 4, −5 and −6 as a mechanism of synergistic enhancement of apoptosis in human lung cancer cells treated with the combination of a synthetic retinoid, Am80 and green tea catechin

(-)-Epigallocatechin gallate (EGCG), a green tea catechin, acts as a synergist with various anticancer drugs, including retinoids. Am80 is a synthetic retinoid with a different structure from all-trans-retinoic acid: Am80 is now clinically utilized as a new drug for relapsed and intractable acute promyelocytic leukemia patients. Our experiments showed that the combination of EGCG and Am80 synergistically induced both apoptosis in human lung cancer cell line PC-9 and up-regulated expressions of growth arrest and DNA damage-inducible gene 153 (GADD153), death receptor 5, and p21waf1 genes in the cells. To understand the mechanisms of synergistic anticancer activity of the combination, we gave special attention to the lysine acetylation of proteins. Proteomic analysis using nanoLC-ESI-MS/MS revealed that PC-9 cells treated with the combination contained 331 acetylated proteins, while nontreated cells contained 553 acetylated proteins, and 59 acetylated proteins were found in both groups. Among them, the combination increased acetylated-p53 and acetylated-α-tubulin through reduction of histone deacetylase (HDAC) activity in cytosol fraction, although the levels of acetylation in histones H3 or H4 did not change, and the combination reduced protein levels of HDAC4, -5 and -6 by 20% to 80%. Moreover, we found that a specific inhibitor of HDAC4 and -5 strongly induced p21waf1 gene expression, and that of HDAC6 induced both GADD153 and p21waf1 gene expression, which resulted in apoptosis. All results demonstrate that EGCG in combination with Am80 changes levels of acetylation in nonhistone proteins via down-regulation of HDAC4, -5 and -6 and stimulates apoptotic induction.

Quantitative Evaluation of Cancer Cell Adhesion to Self-Assembled Monolayer-Patterned Substrates by Reflection Interference Contrast Microscopy

Adhesion of cancer cells with different metastatic potential and anticancer drug resistance has been quantitatively evaluated by using self-assembled monolayer (SAM)-patterned substrates and reflection interference contrast microscopy (RICM). Cell-adhesive SAM spots with optimized diameter could prevent cell-cell adhesion and thus allowed the systematic evaluation of statistically reliable numbers of contact area between single cancer cells and substrates by RICM. The statistical image analysis revealed that highly metastatic mouse melanoma cells showed larger contact area than lowly metastatic cells. We also found that both cancer cell types exhibited distinct transition from the strong to weak adhesion states with increase in the concentration of (-)-epigallocatechin gallate (EGCG), which is known to exhibit cancer preventive activity. Mathematical analysis of the adhesion transition revealed that adhesion of the highly metastatic mouse melanoma cells showed more EGCG tolerance than that of lowly metastatic cells. Moreover, time-lapse RICM observation revealed that EGCG weakened cancer cell adhesion in a stepwise manner, probably via focal adhesion complex. These results clearly indicate that contact area can be used as a quantitative measure for the determination of cancer phenotypes and their drug resistance, which will provide physical insights into the mechanism of cancer metastasis and cancer prevention.

Human cancer stem cells are a target for cancer prevention using (−)-epigallocatechin gallate

PurposeOur previous experiments show that the main constituent of green-tea catechins, (−)-epigallocatechin gallate (EGCG), completely prevents tumor promotion on mouse skin initiated with 7,12-dimethylbenz(a)anthracene followed by okadaic acid and that EGCG and green tea extract prevent cancer development in a wide range of target organs in rodents. Therefore, we focused our attention on human cancer stem cells (CSCs) as targets of cancer prevention and treatment with EGCG.MethodsThe numerous reports concerning anticancer activity of EGCG against human CSCs enriched from cancer cell lines were gathered from a search of PubMed, and we hope our review of the literatures will provide a broad selection for the effects of EGCG on various human CSCs.ResultsBased on our theoretical study, we discuss the findings as follows: (1) Compared with the parental cells, human CSCs express increased levels of the stemness markers Nanog, Oct4, Sox2, CD44, CD133, as well as the EMT markers, Twist, Snail, vimentin, and also aldehyde dehydrogenase. They showed decreased levels of E-cadherin and cyclin D1. (2) EGCG inhibits the transcription and translation of genes encoding stemness markers, indicating that EGCG generally inhibits the self-renewal of CSCs. (3) EGCG inhibits the expression of the epithelial-mesenchymal transition phenotypes of human CSCs. (4) The inhibition of EGCG of the stemness of CSCs was weaker compared with parental cells. (5) The weak inhibitory activity of EGCG increased synergistically in combination with anticancer drugs.ConclusionsGreen tea prevents human cancer, and the combination of EGCG and anticancer drugs confers cancer treatment with tissue-agnostic efficacy.

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.

Phorbol esters in seed oil of Jatropha curcas L. (saboodam in Thai) and their association with cancer prevention: from the initial investigation to the present topics

PurposeIn 1988, we first reported the complete chemical structure of a new type of phorbol ester, abbreviated to DHPB, found in seed oil of Jatropha curcas L. (Saboodam in Thai) and its tumor-promoting activity on mouse skin. Although this seed oil contains toxic phorbol ester, it was planned to use it as a feasible renewable oil and the extracted seed cake as fertilizer. This utilization value opened a new science of Jatropha curcas.MethodsThe main experimental results are cited from our publications, and the relevant literature screened from journals and PubMed.Results and discussionThis paper begins with our original work on the structural elucidation of a new phorbol ester, 12-deoxy-16-hydroxyphorbol (DHPB): its tumor-promoting activity was compared with that of TPA. We think that it is timely to review the following research advances with Jatropha curcas, so numerous topics are classified as follows: (1) historical development of phorbol esters in seed oil; (2) toxicity of phorbol ester based on various bioassays; (3) degradation of phorbol ester; (4) a new pharmaceutical compound in seed; and (5) tumor promotion and progression with endogeneous tumor promoters in human carcinogenesis. The discovery of phorbol ester in seed oil raised awareness of the danger of public use of seed oil and seed cake in Thailand, and also indicated the necessity of discussing the concept of primary and tertiary cancer preventions.ConclusionIt is worthwhile to study the future benefits and cancer risks of globally distributed Jatropha curcas L.

Abstract 4723: Innovative cancer treatment of human lung cancer cells PC-9 with a synthetic retinoid Am80 and EGCG via inhibition of HDAC4 and HDAC5

A synthetic new retinoid Am80 (tamibarotene) is used for treatment of acute promyelocytic leukemia (APL) patients with resistance of all-trans-retinoic acid (ATRA). To potentiate efficacy of Am80, we challenged combination treatment of Am80 and EGCG on human lung cancer cell line PC-9. It is now well accepted that EGCG acts as synergist with numerous anticancer compounds, and that the average reduction of tumor volume for in vivo genograft mouse models implanted using various human cancer cell lines was 70.3% with combinations of 13 anticancer compounds and EGCG. We previously reported that combinations of NSAIDs (sulindac and celecoxib) or other retinoids (ATRA, 13-cis-RA and 9-cis-RA) and EGCG synergistically induced apoptosis of the cells along with strong expression of growth arrest and DNA-damage inducible gene 153 (GADD153) and death receptor (DR5) gene. Our objective was to investigate the mechanism of the synergistic induction for GADD153-DR5 apoptosis pathway. The proteomic analysis of lysine-acetylated proteins was conducted with a shotgun analysis using nanoLC-ESI-MS/MS in PC-9 cells. We found that the numbers of acetylated proteins were different: 551 acetylated proteins were found in non-treated cells, 331 proteins in cells treated with combination, and 59 proteins were common in both cases. Acetylation was mainly found in non-histone proteins, chaperons, transcription factors, and enzymes. The combination increased acetylated p53 (Lys382) about 3-fold compared with non-treated cells. In addition, combination significantly decreased the protein amounts of histone deacetylase (HDAC) 4 and HDAC 5 to 50% and 20%, respectively, but not HDAC6 among Class II HDACs. Class I HDACs (HDAC1, 2, and 3) were not decreased. All the results suggest that combination of Am80 and EGCG induces GADD153-DR5 apoptotic pathway, and reduces the amounts of HDAC4 and HDAC5 resulting in changing protein acetylation. Citation Format: Masami Suganuma, Yukiko Oya, Sonthaya Umsumarng, Keisuke Iida, Anchalee Rawangkhan, Ryo Sakai, Hiroyuki Kagechika, Koichi Shudo, Hirota Fujiki. Innovative cancer treatment of human lung cancer cells PC-9 with a synthetic retinoid Am80 and EGCG via inhibition of HDAC4 and HDAC5. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4723.

1.2 – Green Tea as a Cancer Preventive

1 Abstract nGreen tea is now an acknowledged cancer preventive in Japan and will possibly soon be recognized as such in other countries. Initially, we found that (—)- epigallocatechin gallate (EGCG), the main constituent of green tea inhibited tumor promotion on mouse skin in a two-stage carcinogenesis experiment. Numerous additional studies revealed the anticarcinogenic effects of EGCG and green tea on various organs in rodent experiments. This paper reviews the unique role of green tea in cancer chemoprevention, its anticarcinogenic effects and other preventive activities, bioavailability of tea polyphenols and epidemiological studies with green tea. Of particular interest are studies which showed that daily consumption of green tea delayed clinical onset of various cancers and led to more hopeful prognoses for breast cancer patients in Stage I and II following treatment. Based on these results, I propose two stages of cancer prevention with green tea: prevention before cancer onset, and following cancer treatment. Since green tea is a common beverage, the knowledge that it inhibits cancer will be a great comfort to, especially, aging folk concerned with cancer prevention and any high risk population.

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.