Keiko Fukumoto

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.

Contribution of the Src family of kinases to the appearance of malignant phenotypes in renal cancer cells

Although the constitute activation of the Src family of kinases (Src) has been established as a poor prognostic factor in several types of cancer, the role of Src in renal cell carcinoma (RCC) has not been defined. This study aimed to determine whether Src could contribute to the appearance of malignant phenotypes in RCC. The role of Src in the appearance of malignant phenotypes in RCC was examined in two human renal cancer cell lines, Caki‐1 from human metastatic RCC and ACHN from human primary RCC. Src activity in Caki‐1 cells was higher than that in ACHN cells, and this difference corresponded to the difference of PP1 (a Src family inhibitor)‐induced cytotoxicity on the two cells. The difference in cytotoxicity between the cells did not depend on cell cycle regulation but on the induction of apoptosis, and the difference in apoptosis particularly related to the reduction of the Bcl‐xL level. Furthermore, in Caki‐1 cells with higher Src activity, Src stimulated the production of vascular endothelial growth factor (VEGF), partially via the activation of Stat3, and the inhibition of Src activity caused a reduction of the VEGF level in serum, angiogenesis, and tumor development in a xenograft model. These results suggested that Src contributed to the appearance of malignant phenotypes in renal cancer cells, particularly due to the resistance against apoptosis by Bcl‐xL and angiogenesis stimulated by Src‐Stat3‐VEGF signaling.

Connexin 32 potentiates vinblastine-induced cytotoxicity in renal cell carcinoma cells.

We have reported that connexin (Cx) 32 gene, a member of gap junction protein family, acts as a tumor suppressor gene in human renal cell carcinoma (RCC). Of solid tumors, RCC is one of the most chemoresistant cancers, and there is no effective cancer chemotherapy against RCC at present. In this study, we examined if the combination of Cx32‐dependent tumor‐suppressive effect and vinblastine (VBL), a chemotherapeutic agent which has been utilized for clinical RCC treatment, could be effective in enhancing the sensitivity of RCC to VBL treatment. Cx32 expression in a human metastatic RCC cell (Caki‐1 cell) significantly enhanced in vitro and in vivo VBL‐induced cytotoxicity on the cell. Cx32 expression in the RCC cells potentiated VBL‐induced apoptosis compared to the Cx32‐negative RCC cells in vitro as well as in vivo. The enhancing apoptosis in the RCC cells by Cx32 mainly depended on the decrease of P‐glycoprotein (P‐gp), a multidrug resistance gene‐1 (MDR‐1) product responsible for reduction of VBL accumulation into the cells. We also observed that silencing of Cx32 by short interfering RNA (siRNA) treatment elevated the level of P‐gp in Caki‐1 cells and that inhibition of P‐gp function enhanced VBL‐induced apoptosis in the RCC cells. These results suggest that Cx32 is effective to enhance VBL‐induced cytotoxicity in Caki‐1 cells via the reduction of P‐gp. Overall, it seems that the combination of Cx32‐dependent tumor‐suppressive effect and VBL is promising as a new cancer therapy against RCC.

Enhancing effect of connexin 32 gene on vinorelbine-induced cytotoxicity in A549 lung adenocarcinoma cells

PurposeConnexin (Cx) genes exert negative growth effects on tumor cells with certain cell specificity, and tumor-suppressive effects of the Cx genes contribute to enhancement of chemotherapeutical agents-induced cytotoxicity in some cancer cells. Since we and others have been reported that Cx32 acts as a tumor suppressor gene in lung adenocarcinomas, this study was undertaken to estimate if the combination of Cx32-dependent tumor-suppressive effect and vinorelbine (VBN), a chemotherapeutic agent which has been utilized for clinical lung adenocarcinoma treatment, could be effective in enhancing the sensitivity of the lung cancer to VBN treatment.MethodsWe established the A549 cells (a human lung adenocarcinoma cell line) which had stable expression of Cx32 and estimated effect of Cx32 on VBN-induced cytotoxicity in the established cells.ResultsCx32 expression in A549 cells significantly potentiated VBN-induced cytotoxicity on the cells due to enhancement of apoptosis induction. The enhancing cytotoxicity in A549 cells by Cx32 mainly depended on a decrease in expression of multi-drug resistance-1 (MDR-1) gene responsible for reduction of VBN accumulation into the cells. We also observed that silencing of Cx32 by siRNA treatment elevated the expression level of MDR-1 mRNA in A549 cells and that inhibition of MDR-1 gene product-dependent function enhanced VBN-induced cytotoxicity in the cells.ConclusionThese results suggest that Cx32 contributes to the enhancement of VBN-induced cytotoxicity in A549 cells via the reduction of MDR-1 expression.

The inhibitory effect of connexin 32 gene on metastasis in renal cell carcinoma

We have previously reported that connexin (Cx) 32 gene, a member of gap junctions, was specifically downregulated in human renal cell carcinoma (RCC) and it acts as a tumor suppressor against RCC. Because there is no standard therapy for advanced RCC, we investigated the anti‐metastatic effect of Cx32 to seek a possibility of new RCC therapy. In this study, we used human metastatic RCC cell (Caki‐1), and established Cx32‐expressed cell clone (Caki‐1T) or only mock‐transfected cell clone (Caki‐1W). For experimental production of metastases, the cells were injected into the lateral tail vein of SCID mice. Seventy days after inoculation, metastatic colonies were observed in Caki‐1W inoculated group, though none of them were in Caki‐1T inoculated group. The plasma VEGF concentration was significantly lower in Caki‐1T group compared to Caki‐1W group. To investigate where Cx32 effects on, we also tried in vitro analysis and found that the malignant phenotypes involving metastasis steps were significantly decreased in Caki‐1T under hypoxia, a mimic condition of internal tumor environment. After hypoxia treatment, the protein level of HIF‐2α, which plays main role for hypoxia adaptation, was observed to increase in Caki‐1W, whereas no expression was observed in Caki‐1T. We investigated the activation of Src, which is required for stabilization of HIF‐2α, is suppressed in Caki‐1T compared to Caki‐1W. These results suggest that Cx32 inhibits hypoxia adaptation governed by HIF‐2α, and this may help to reduce the metastasis of RCC cells.