Tomohiro Yano

Prostaglandin E2 reinforces the activation of Ras signal pathway in lung adenocarcinoma cells via EP3

Prostaglandin E2 (PGE2)‐dependent effects on various cell responses are regulated by respective PGE2 receptors (EP1, EP2, EP3, EP4) expressing in target cells. Alveolar type II cell (a main progenitor cell of lung adenocarcinoma) expressed only EP4, while human lung adenocarcinoma cells (A549) expressed EP3 as well as EP4. An antagonistic effect of EP3 against EP4 through the modulation of cyclic AMP level is required for PGE2‐mediated activation of Ras signal pathway in A549 cells. These results suggest that the expression of EP3 may be a critical factor for the PGE2‐mediated activation of Ras signal pathway in A549 cells.

Peroxisome proliferator-activated receptor δ as a molecular target to regulate lung cancer cell growth

It has been assumed that prostaglandin (PG)I2 signaling contributes to the negative growth control of lung cancer cells; however, the mechanism remains unresolved. PGI2 functions through a cell surface G protein‐coupled receptor (prostaglandin I2‐binding receptor, IP) and also exerts an effect by interacting with a nuclear hormone receptor, peroxisome proliferator‐activated receptor δ (PPARδ). We found that PPARδ was a key molecule of PGI2 signaling to give negative growth control of lung cancer cells (A549), using carbarprostacyclin, a PGI2 agonist for IP and PPARδ, and L‐165041, a PPARδ agonist. Furthermore, PPARδ‐induced cell growth control was reinforced by the inhibition of cyclooxygenase. These results suggest that PPARδ activation under the suppression of PG synthesis is important to regulate lung cancer cell growth.

Connexin32 as a tumor suppressor gene in a metastatic renal cell carcinoma cell line

Connexin genes expressing gap junction proteins have tumor-suppressive effects on primary cancers with certain cell specificity, but the suppressive effects on metastatic cancers are still conflicting. In this study, we show that connexin32 (Cx32) has a strong tumor-suppressive effect on a human metastatic renal cell carcinoma cell line (Caki-1 cell). Cx32 expression in Caki-1 cells reduced in vitro malignant phenotypes of the cells such as anchorage independency and invasion capacity. Furthermore, the Cx32 expression drastically reduced the development of Caki-1 cells in nude mice. We also determined that Cx32 reduced the malignant phenotypes in Caki-1 cells mainly through the inactivation of Src signaling. Especially, Cx32-dependent inactivation of Src decreased the production of vascular epithelial growth factor (VEGF) via the suppression of signal transducers and activators of transcription 3 (Stat3) activation, and we confirmed this result using short interfering RNA. In nude mice, Cx32-transfected Caki-1 cells showed lower serum level of VEGF comparing mock transfectant, and the development of the cells in nude mice positively related to the VEGF level. These data suggest that Cx32 acts as a tumor suppressor gene in Caki-1 cells and that the tumor-suppressive effect partly depends on the inhibition of Src-Stat3-VEGF signal pathway.

Induction of cytotoxicity in human lung adenocarcinoma cells by 6-O-carboxypropyl-α-tocotrienol, a redox-silent derivative of α-tocotrienol

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 α‐tocotrienol (T3), 6‐O‐carboxypropyl‐α‐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 μM), whereas T3 and a redox‐silent analogue of α‐tocopherol (T), 6‐O‐carboxypropyl‐α‐tocopherol (TE), showed much less cytotoxicity in cells within 40 μM. T3E cytotoxicity was based on the accumulation of cells in the G1‐phase of the cell‐cycle and the subsequent induction of apoptosis. Similar to this event, 24‐hr treatment of A549 cells with 40 μM T3E caused the inhibition of Ras farnesylation, and a marked decrease in the levels of cyclin D required for G1/S progression in the cell‐cycle and Bcl‐xL, a key anti‐apoptotic molecule. Moreover, the T3E‐dependent inhibition of RhoA geranyl‐geranylation is an inducing factor for the occurrence of apoptosis in A549 cells. Our results suggest that T3E suppresses Ras and RhoA prenylation, leading to negative growth control against A549 cells. In conclusion, a redox‐silent analogue of T3, T3E may be a new candidate as an anticancer agent against lung adenocarcinoma showing poor prognosis based on the mutation of ras genes.

Down-regulation of connexin 32 gene expression through DNA methylation in a human renal cell carcinoma cell.

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.

Negative growth control of renal cell carcinoma cell by connexin 32: Possible involvement of Her-2

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.

Regulation of cellular invasion and matrix metalloproteinase activity in HepG2 cell by connexin 26 transfection.

We previously reported that connexin (Cx) 26 expression is involved in negative growth control of HepG2 cells established from a human hepatoma. We also found that induction of E‐cadherin and subsequent formation of a cell adhesion complex were induced in HepG2 cells by C × 26 expression. To examine the exact role of C × 26‐induced E‐cadherin junctions in regulating appearance of malignant phenotypes of HepG2 cells, we expressed a C × 26 antisense oligodeoxynucleotide (AS‐ODN) in an established HepG2 cell clone that has stable expression of C × 26 genes. We investigated changes in the expression of E‐cadherin, the localization of β‐catenin, and some malignant phenotypes of HepG2 clone after the suppression of C × 26 expression by AS‐ODN treatment. The AS‐ODN treatment prevented the expression of C × 26 and E‐cadherin, and the localization of β‐catenin was changed from cytoplasmic membrane to the cytoplasm. In parallel, a morphological change from a monolayer of polygonal cells to multilayered colonies was induced by the treatment, indicating a change of a malignant phenotype of HepG2 cells. The activity of matrix metalloproteinase 9 (MMP‐9) was elevated by the AS‐ODN treatment. A concomitant increase in invasiveness of the C × 26‐expressing cells by the treatment was also observed in an in vitro assay with Matrigel matrix. These results suggest that the induction of E‐cadherin and formation of the cell adhesion complex by C × 26 expression contribute to the reversal of some malignant phenotypes of HepG2 cells. Furthermore, the C × 26‐dependent expression of E‐cadherin leads to reduction of the invasiveness of the cells through suppression of MMP‐9 activity.

The effect of 6-methylthiohexyl isothiocyanate isolated from Wasabia japonica (wasabi) on 4-(methylnitrosamino)- 1-(3-pyridyl)-1-buatnone-induced lung tumorigenesis in mice

The present study was undertaken to estimate the effect of 6-methylthiohexyl isothiocyanate (6MHITC) isolated from Wasabia japonica (wasabi) pretreatment on 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone(NNK)-induced lung tumorigenesis in mice. Pretreatment with 6MHITC for 4 consecutive days at a daily dose of 5 micromol significantly inhibited NNK-induced O(6)-methylguanine formation in lungs at 4 h after the injection. In conjugation with this inhibitory effect, 6MHITC suppressed the increase in proliferating nuclear cell antigen level as well as ornithine decarboxylase activity at a promotion stage of NNK-induced lung tumorigenesis. Finally, this treatment of 6MHITC suppressed the NNK-induced lung tumorigenesis in mice. These results suggest that 6MHITC inhibits the development of lung tumors in mice treated with NNK, due to the suppression of initiation stage.

The activation of K-ras gene at an early stage of lung tumorigenesis in mice.

To clarify the exact timing of K-ras gene mutational activation in lung tumorigenesis of mice, we applied a sensitive mutant allele specific amplification (MASA) method to pulmonary DNA from urethane-treated mice. The activation of K-ras gene with 61st codon AT mutation was detected in the lungs of mice at day 14 but not day 7 after urethane treatment by MASA. The mutation of MASA products was also checked by XbaI restriction fragment length polymorphism analysis and DNA sequencing. These data suggest that the mutation of K-ras gene in the lungs of mice treated with urethane occurred at the early stage of lung tumorigenesis.

Activation of Epidermal Growth Factor Receptor in the Early Phase after Renal Ischemia-Reperfusion in Rat

In order to estimate a regenerative response in the early phase after renal ischemia-reperfusion in rat, we examined the time course of the activation of epidermal growth factor receptor (EGFR) as a response of signal transduction pathway after 45 min ischemia in kidney. The activation of EGFR was observed 5–30 min after the start of reperfusion. Simultaneously, superoxide anion/hydrogen peroxide generated in the mitochondrial fraction was elevated during the same period. On the other hand, the level of EGF decreased in a time-dependent manner. These results suggested that superoxide anion/hydrogen peroxide generated during the ischemia-reperfusion other than EGF could act as an activator for the EGFR. In summary, the activation of EGFR is important as a regenerative response at an early stage after the start of reperfusion in ischemic kidney.