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Connexin (Cx) genes have negative growth effects on tumor cells with certain cell specificity. We have previously reported that Cx32 is specifically downregulated in human renal cell carcinoma cell (RCC) lines as well as cancerous regions of kidneys and that the Cx is expressed in the progenitor cells of the carcinoma. However, the precise role of Cx32 in growth control of RCC cells remains unknown. In this study, we examined whether Cx32 could act in growth control against a human RCC cell, Caki-2 cell. In order to estimate the cell growth control, we established Caki-2 cells that have stable expression of Cx32 genes. Cx32 expression in Caki-2 cells induced contact inhibition of growth and reduced anchorage-independent growth ability, but did not significantly affect lag phase growth rates. This growth control by Cx32 was dependent on the inhibition of the cell-cycle transition from G1 to S phase at high cell density, and the inhibition of the cell-cycle transition related to the suppression of Her-2 activation. Furthermore, the suppression of Cx32 expression in Caki-2 cells by short interfering RNA induced the activation of Her-2. These data suggest that Cx32 has negative growth control of Caki-2 cells, partly due to the inhibition of the Her-2 activation.

Background: We have recently reported that connexin (Cx) 32 is down-regulated in a human renal cell carcinoma (RCC) cell (Caki-2 cell). Hypothesis: We postulated that the down-regulation of Cx32 gene in the RCC cell is due to hypermethylation of its promoter region. Methods: We estimated methylation status in the promoter region of Cx32 gene in the RCC cell by DNA digestion with methylation-sensitive restriction enzyme and PCR, and methylation-specific PCR (MSP). We also checked the recovery of Cx32 gene expression in the RCC cell treated with a DNA methyltranferase inhibitor, 5-Aza-2’-deoxycytidine (5-Aza-CdR). Results: Treatment with 5-Aza-CdR resulted in induction of Cx32 expression in the RCC cell. Hypermethylation of the Cx32 promoter region in the RCC cell was confirmed by DNA digestion with methylation-sensitive restriction enzyme and PCR, and MSP. Conclusion: We suggest that hypermethylation in the promoter region is a mechanism for the Cx32 gene repression in the RCC cell.

Tocotrienols are one of the most potent anticancer agents of all natural compounds and the anticancer property may be related to the inactivation of Ras family molecules. The anticancer potential of tocotrienols, however, is weakened due to its short elimination half life in vivo. To overcome the disadvantage and reinforce the anticancer activity in tocotrienols, we synthesized a redox-silent analogue of ␣-tocotrienol (T3), 6-O-carboxypropyl-␣-tocotrienol (T3E). We estimated the possibility of T3E as a new anticancer agent against lung adenocarcinoma showing poor prognosis based on the mutation of ras gene. T3E showed cytotoxicity against A549 cells, a human lung adenocarcinoma cell line with a ras gene mutation, in a dose-dependent manner (0 -40 M), whereas T3 and a redox-silent analogue of ␣-tocopherol (T) Key words: ␣-tocotrienol; redox-silent derivative; lung adenocarcinoma; Ras; cytotoxicity; Bcl-xL; cyclin D Lung cancer, particularly non-small cell lung cancer (NSCLC), is one of the most common forms of cancer and is the leading cause of cancer death in the West. 1 NSCLC exhibiting adenocarcinoma histology forms the majority of lung cancers and its ras genes carry mutations. 2 Furthermore, the presence of mutated ras genes in lung adenocarcinoma is linked with shortened patient survival. 3 These reports indicate that signaling pathways activated by mutated Ras protein contribute to the appearance of malignant phenotypes in lung adenocarcinoma. Thus, the inhibition of the mutated Ras function may be a promising procedure to regulate the development of lung cancer.,Mutated Ras proteins remain locked in an active state, and for the activated proteins to transduce the signals into downstream molecules, the proteins must be associated with the inner surface of the plasma membrane. 4 The critical process for the attachment of Ras to the inner surface of the plasma membrane is farnesylation of the C-terminal in Ras protein. 5 Because the inhibition of farnesylation can prevent Ras from maturing into its biologically active form, the inhibition is considered a potential therapeutic procedure for current cancer therapy. 6,7 In general, tumors undergo deregulated cholesterogenesis mainly via the upregulation of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, a key enzyme of cholesterol synthesis. 8 HMG-CoA reductase plays a key role in controlling cell proliferation by generating prenyl intermediates, particularly farnesyl and geranyl-geranyl moieties, 8 so the upregulation of the enzyme in tumors finally stimulates the cell proliferation via the increase of prenylation in Ras molecules. Thus, agents such as lovastin, which had been exploited previously for lowering cholesterol based on the inhibition of HMG-CoA reductase, may be useful chemotherapeutic agents for treating tumors harboring oncogenic ras mutation. 8 Natural constituents such as sundry mevalonate-derived constituents (isoprenoids) of fruits, vegetables and cereal grains suppress the development of tumors, 9 and the suppressive effect mainly...

ABSTRACT-The overexpression of ErbB-2 receptor relates to malignant transformation of breast cancer. The present study was carried out to establish the usefulness of a-tocopheryloxybutyric acid (TE) as a chemotherapeutic agent for human breast cancer. TE caused induction of apoptosis in MDA-MB-453 cells overexpressing the ErbB-2 receptor. TE reduced levels of activated ErbB-2 receptor and Akt. In contrast, TE induced the activation of p38, and SB203580, a specific inhibitor for p38, attenuated the TE-induced apoptosis. These data indicate that simultaneous occurrences of Akt inhibition and p38 activation by TE result in the cell death.Keywords: a-Tocopheryloxybutyric acid, Apoptosis, Breast cancer cell Overexpression of ErbB-2 receptor is seen in approximately 30% of human breast cancers, and the overexpression of the receptor is a negative prognostic factor following tumor resection and may be associated with increased resistance to cancer chemotherapy (1). A key mechanism by which the ErbB-2 receptor overexpression stimulates tumor cell growth and renders cells chemoresistant are as follows: survival signal transduction through the ErbB-2 receptor involves the phosphatidylinositol-3 kinase /Akt signaling pathway, and activated Akt is considered the focal point of a survival pathway known to protect cells from apoptosis by several stimuli (2). In a recent report, antisense oligonucleotide-dependent down-regulation of the ErbB-2 receptor in human breast cancer cells results in apoptotic cell death (3). Collectively, these reports encouraged us to develop a new therapeutic agent targeting the overexpressed ErbB-2 receptor in human breast cancer.Previous in vitro studies using a-tocopheryl succinate (TS) have shown that the antioxidative effect of a -tocopherol is not required for growth inhibition and apoptosis of several tumor cell lines including human breast cancer cells (4). In order to extrapolate this in vitro effect into an in vivo effect, we synthesized one a -tocopherol derivative, a-tocopheryloxybutyric acid (TE), which has no antioxidative effect in vivo (5). Since the ether bond in TE can not be hydrolyzed in vivo, this compound does not show any antioxidative effect (6). In fact, we demonstrated that TE was stable in vivo and inhibited cell proliferation during the carcinogenic process of lung tumorigenesis in mice (5, 7). In that study, we have suggested that TE stabilizes plasma membrane physicochemically and inhibits growth factor receptor-dependent mitogenic signaling, leading to suppression of the cell proliferation (5). Additionally, we found that TE suppressed the activation of epidermal growth factor receptor in a human lung cancer cell line through the stabilizing effect of TE on the plasma membrane (unpublished data). Overall, the stabilizing effect of TE may contribute to the suppression of ErbB-2 receptor activation in human breast cancer cells. In this context, the present study was carried out to clarify this possibility using a human breast cancer cell line, MDA-MB-453 overexpressing t...