Stephanie J. Muga

Green tea polyphenol (−)-epigallocatechin-3-gallate inhibits cyclooxygenase-2 expression in colon carcinogenesis

Tea, one of the most widely consumed beverages worldwide, has been shown to have anti‐cancer activity in various cancers including colon cancer. It has been demonstrated that overexpression of the inducible isoform of cyclooxygenase (COX‐2) occurs during colon tumorigenesis and inhibition of COX‐2 by non‐steroidal anti‐inflammatory drugs (NSAIDs) is chemopreventive. To determine whether the anti‐cancer effect associated with green tea impacted COX‐2 expression levels, human colorectal cancer cell lines HT‐29 and HCA‐7, were treated with (−)‐epigallocatechin‐3‐gallate (EGCG), the most abundant and effective polyphenol of green tea. EGCG significantly inhibited constitutive COX‐2 mRNA and protein overexpression. The inhibitory effects of EGCG on signaling pathways controlling COX‐2 expression were examined. We observed that EGCG downregulated the ERK1/2 and Akt pathways in colon cancer cells. The effect of EGCG on COX‐2 expression resulted in decreased COX‐2 promoter activity via inhibition of nuclear factor κB (NF‐κB) activation. EGCG also promoted rapid mRNA decay mediated through the COX‐2 3′untranslated region (3′UTR). In conclusion, these data suggest that inhibition of COX‐2 is a mechanism for the anti‐proliferative effect of green tea and emphasizes the role that dietary factors have as anti‐cancer agents.

Activators of peroxisome proliferator–activated receptor-α partially inhibit mouse skin tumor promotion

Several recent reports have suggested that peroxisome proliferator–activated receptors (PPARs) may be involved in the development of neoplasias in different tissue types. The present study was undertaken to determine whether PPARs play a role in skin physiology and tumorigenesis. In an initiation‐promotion study, SENCAR mice treated topically with the PPARα ligands conjugated linoleic acid and 4‐chloro‐6‐(2,3‐xylidino)‐2‐pyrimidinylthioacetic acid (Wy‐14643) exhibited an approximately 30% lower skin tumor yield compared with mice treated with vehicle. The PPARγ and PPARδ activators troglitazone and bezafibrate, respectively, exerted little, if any, inhibitory activity. PPARα was detected in normal and hyperplastic skin and in papillomas and carcinomas by immunohistochemistry. In addition, PPARα, PPARδ/PPARβ, and PPARγ protein levels were analyzed by immunoblotting in normal epidermis and papillomas. Surprisingly, the levels of all three isoforms were increased significantly in tumors as opposed to normal epidermis. In primary keratinocyte cultures, protein levels of PPARα and, to a lesser extent, PPARγ were markedly increased when the cells were induced to differentiate with high‐calcium (0.12 mM) conditions. In addition, we observed that Wy‐14643 enhanced transcriptional activity of a peroxisome proliferator–response element–driven promoter in a mouse keratinocyte cell line. These results demonstrate that keratinocytes express functional PPARα, that PPARα may play a role in differentiation, and that ligands for PPARα are moderately protective against skin tumor promotion. We conclude that selective PPARα ligands may exert their protective role against skin tumor promotion by ligand activation of PPARα. Mol. Carcinog. 29:134–142, 2000.

The effect of PPARγ ligands on UV- or chemically-induced carcinogenesis in mouse skin

Peroxisome proliferator‐activated receptor γ (PPARγ) is a ligand activated transcription factor. There have been suggestions that PPARγ ligands may have utility in preventing tumor development in rodent mammary glands and colon. The recent finding that mice lacking one allele of the PPARγ gene were significantly more susceptible to 7,12‐dimethylbenz[a]anthracene (DMBA)‐induced skin carcinogenesis compared to wild‐type mice highlights mouse skin as another potential organ in which PPARγ ligands may be effective as chemopreventive agents. In this study, we assessed the effect of two PPARγ ligands (rosiglitazone and troglitazone) on UV and DMBA/12‐O‐tetradecanoylphorbol‐13‐acetate (TPA)‐induced mouse skin carcinogenesis, two of the most commonly used mouse skin carcinogenesis models. Unexpectedly, neither rosiglitazone (dietary 200 ppm) nor troglitazone (topical 100 μg) significantly inhibited UV‐induced skin tumor development in SKH‐1 hairless mice. Likewise, dietary rosiglitazone did not statistically significantly inhibit DMBA/TPA‐induced skin tumor development. Interestingly, dietary troglitazone significantly inhibited basal level keratinocyte proliferation as shown by 5‐bromo‐2′‐deoxyuridine (BrdU) labeling, but it had no effect on TPA‐induced epidermal cell proliferation. Northern blot analysis showed that PPARγ expression was extremely low in normal mouse epidermis and was virtually undetectable in skin tumors. Collectively, our data suggest that PPARγ ligands may not be useful in the prevention of chemically or UV‐induced skin tumors.

TJ-41 Induces Apoptosis and Potentiates the Apoptotic Effects of 5-FU in Breast Cancer Cell Lines

Recent studies suggest that TJ-41, a herbal drug, possesses chemotherapeutic effects. Accordingly, this study was undertaken to investigate the anticarcinogenic effects of TJ-41 on human breast cancer cells lines. TJ-41 inhibited the proliferation of human breast cancer cell lines dose dependently. Flow cytometric analysis showed that this decrease in DNA synthesis is to be associated with induction of apoptosis. In both cell lines, apoptosis was abolished by caspase-9 inhibitor Z-LEHD-fmk but was weakly inhibited by caspase-8 inhibitor Z-IETD-fmk, indicating that caspase-9 activation was involved in TJ-41 induced apoptosis. Additionally, TJ-41 stimulated phosphorylation of c-Jun NH2-terminal kinase (JNK) and pretreatment of breast cancer cells with JNK inhibitor SP600125 completely abolished TJ-41 induced apoptosis. Our data also demonstrate that combined treatment of TJ-41 and 5-FU significantly potentiates the apoptotic effects of 5-FU in both breast cancer cell lines. Taken together, these data suggest that TJ-41 might provide a novel chemotherapeutic treatment for breast cancer.

Application of high-performance liquid chromatography–based analysis of DNA fragments to molecular carcinogenesis

Denaturing high‐performance liquid chromatography (DHPLC)–based DNA fragment analysis is a high‐throughput technology that can be used to obtain information on both genetic alterations and gene expression. By using different approaches based on polymerase chain reaction, this technique can be used to determine loss or gain of an allele, to quantitate the amount of RNA expressed, and to detect a single nucleotide change. Applications of DHPLC to molecular carcinogenesis include genotyping of transgenic animals; determination of allelic imbalances, including loss of heterozygosity in tumors; measurement of changes in gene expression; and detection of DNA polymorphisms and point mutations. In our laboratories DHPLC has been validated and used to genotype an Eker rat colony, to study the genetic profile of renal cell carcinomas, to quantitate expression of the keratinocyte lipid‐binding protein gene in 8‐lipoxygenase transgenic mice, and to detect polymorphisms and a point mutation in the tuberous sclerosis 2 tumor suppressor gene in t‐haplotype mice. Mol. Carcinog. 29:51–58, 2000.