Tsuyoki Kadofuku

Enhancement of sensitivity to tumor necrosis factor α in non-small cell lung cancer cells with acquired resistance to gefitinib

Tumor cells that have acquired resistance to gefitinib through continuous drug administration may complicate future treatment. To investigate the mechanisms of acquired resistance, we established PC-9/ZD2001, a non-small-cell lung cancer cell line resistant to gefitinib, by continuous exposure of the parental cell line PC-9 to gefitinib. After 6 months of culture in gefitinib-free conditions, PC-9/ZD2001 cells reacquired sensitivity to gefitinib and were established as a revertant cell line, PC-9/ZD2001R. PC-9/ZD2001 cells showed collateral sensitivity to several anticancer drugs (vinorelbine, paclitaxel, camptothecin, and 5-fluorouracil) and to tumor necrosis factor α (TNF-α). Compared with PC-9 cells, PC-9/ZD2001 cells were 67-fold more sensitive to TNF-α and PC-9/ZD2001R cells were 1.3-fold more sensitive. Therefore, collateral sensitivity to TNF-α was correlated with gefitinib resistance. PC-9/ZD2001 cells expressed a lower level of epidermal growth factor receptor (EGFR) than did PC-9 cells; this down-regulation was partially reversed in PC-9/ZD2001R cells. TNF-α-induced autophosphorylation of EGFR (cross-talk signaling) was detected in all three cell lines. However, TNF-α-induced Akt phosphorylation and IκB degradation were observed much less often in PC-9/ZD2001 cells than in PC-9 cells or PC-9/ZD2001R cells. Expression of the inhibitor of apoptosis proteins c-IAP1 and c-IAP2 was induced by TNF-α in PC-9 and PC-9/ZD2001R cells but not in PC-9/ZD2001 cells. This weak effect of EGFR on Akt pathway might contribute to the TNF-α sensitivity of PC-9/ZD2001 cells. These results suggest that therapy with TNF-α would be effective in some cases of non-small-cell lung cancer that have acquired resistance to gefitinib.

Increase of transferrin receptors in regenerating rat liver cells after partial hepatectomy

The change of transferrin receptors in regenerating rat liver cells after partial hepatectomy was demonstrated. The binding of 125I-labeled transferrin to the cells began to increase after 24 h of partial hepatectomy and reached about double that of the non-operated normal rat liver cells. A Scatchard analysis of binding parameters showed 1.62 X 10(5) (Ka: 1.04 X 10(7) M-1) in normal cells and 3.36 X 10(5) (Ka: 1.23 X 10(7) M-1) in regenerating cells. This reflected on increase of transferrin receptor number and little change occurred in the binding affinity between transferrin and its surface receptor.

Major pathway for putrescine synthesis induced by 1α,25-dihydroxyvitamin D3 in chick duodenum

We have reported that a single injection of 1 alpha,25-dihydroxyvitamin D3 into vitamin D-deficient chicks produces a marked accumulation of putrescine in the duodenum by an interconversion pathway. In the present study, we examined the effect of N1,N2-bis(2,3-butadienyl)-1,4-butanediamine, a specific irreversible inhibitor of polyamine oxidase, on the duodenal putrescine synthesis induced by 1 alpha,25-dihydroxyvitamin D3. Addition of N1,N2-bis(2,3-butadienyl)-1,4-butanediamine to an assay mixture completely inhibited the activity of duodenal polyamine oxidase in vitro. Prior administration of N1,N2-bis(2,3-butadienyl)-1,4-butanediamine to chicks completely blocked the 1 alpha,25-dihydroxyvitamin D3-induced increase in duodenal accumulation of putrescine in vivo. The increase of the duodenal accumulation of putrescine by 1 alpha,25-dihydroxyvitamin D3 in vitamin D-deficient chicks coincided quantitatively with the amount of N1-acetylspermidine synthesized from spermidine after the injection of the vitamin into the chicks pretreated with the inhibitor of polyamine oxidase. These results clearly indicate that spermidine N1-acetyltransferase plays a preferential role in the increase in duodenal putrescine synthesis by 1 alpha,25-dihydroxyvitamin D3. The rapidly proliferating and maturing epithelium of small intestines will provide a good model for investigating the role of the interconversion of polyamine metabolism in cell growth and differentiation.

Detection of the changes in cellular proteins in regenerating rat liver by high-resolution two-dimensional electrophoresis

The changes in cellular proteins in regenerating rat liver after partial hepatectomy were examined by high-resolution two-dimensional electrophoresis. The cellular proteins in regenerating rat livers were separated into two fractions (soluble and insoluble protein fractions) and the proteins in each fraction were analysed by means of two-dimensional electrophoresis. A rapid increase in three proteins and a rapid decrease in two proteins were detected after partial hepatectomy. The changes in these proteins were in parallel with the regeneration rate of liver, suggesting a close relationship with the proliferation of liver after partial hepatectomy.

Detection of the changes in protein distribution in rat serum after partial hepatectomy using two-dimensional electrophoresis under non-denaturing conditions

The changes in rat serum protein distribution after partial hepatectomy were examined using two-dimensional electrophoresis, utilizing isoelectric focusing in polyacrylamide gel in the first dimension and pore gradient polyacrylamide gel electrophoresis in the second dimension. Drastic decreases in amount of protein were observed at more than twenty spot positions, and drastic increases in amount or newly appeared proteins were observed at eight spot positions. The amounts of albumin, immunoglobulin M and alpha 2-macroglobulin did not change relatively after hepatectomy. The time course of the changes was examined using a densitometer, and it was observed that almost all the serum proteins changed drastically at 24 h after hepatectomy.

Detection of the exercise-associated changes in serum proteins by two-dimensional electrophoresis under non-denaturing conditions

The changes in serum proteins caused by physical exercise (10-km run) were examined using two-dimensional electrophoresis under non-denaturing conditions. Two specific proteins were found to increase remarkably in amount. Both proteins had a slightly higher molecular mass than albumin, which suggests that they are albumin-bound with large amounts of sugars or lipids. However, these proteins were not adsorbed on an anti-albumin affinity column, and they were not stained by either periodic acid-Schiff base or Sudan Black. The molecular mass was determined to be 25,000 by sodium dodecyl sulphate polyacrylamide gel electrophoresis. The changes of the specific proteins in competitors in a triathlon race were also examined.

Detection of changes in rabbit serum proteins after partial hepatectomy by means of two-dimensional electrophoresis under non-denaturing conditions.

The changes in rabbit serum proteins after partial hepatectomy were examined by means of two-dimensional electrophoresis utilizing isoelectric focusing in a 4% polyacrylamide gel in the first dimension and a 4-30% pore gradient polyacrylamide gel in the second dimension. A rapid increase in seven proteins was observed after partial hepatectomy and a rapid decrease in two proteins. Major serum proteins, including albumin, immunoglobulin G, immunoglobulin M and alpha 2-macroglobulin, did not change. The time course of the changes was examined using a densitometer; the maxima of the changes were observed on day 3 after partial hepatectomy.

Abstract 1639: The novel c-MET inhibitor, ARQ 197, shows additive growrh-inhibitory effect with erlotinib through enhanced degradation of c-MET protein via ubiquitin/proteasome pathway

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Background c-MET, a receptor of hepatocyte growth factor (HGF), is known to be overexpressed in a variety of human cancers. Recently several c-MET inhibitors are being developed as cancer chemotherapy against this compelling molecular target. ARQ 197 is a new small molecule c-MET inhibitor. Since ARQ 197 shows efficient anti-cancer activity without many severe adverse events in Phase I clinical trials, it is expected to be a useful anticancer agent for clinical treatment. This drug is known to bind to c-MET at neighbor area of its ATP binding site and it does not work as an ATP mimic. In this study, we investigated the molecular mechanism of inhibitory effect on c-MET by ARQ 197 in a non-small cell lung cancer cell (NSCLC) line. Materials and Method We established a c-MET-overexpressed NSCLC line, PC-9/MET, by long term exposure of PC-9 cells to EGFR-tyrosine kinase inhibitor. The cytotoxicity of ARQ 197 was measured by MTT assay. The expression of c-MET protein and mRNA were detected by Western blotting analysis and real time RT-PCR method, respectively. In a c-MET degradation analysis, bortezomib was used for inhibition of proteasome. Results ARQ 197 is equally cytotoxic to parental PC-9 and EGFRTKI-resistant PC-9/MET cell line with GI50 values around 200 nM (?). ARQ 197 showed an additive growth- inhibitory effect with erlotinib in PC-9/MET cells as assessed by an Isobologram analysis. In ARQ 197-treated PC-9/MET cells, a clear decrease of phospho c-MET protein as well as phospho AKT and ERK proteins was observed, however, ARQ 197 also induced a down-regulation of c-MET protein in the cells without any effect on c-MET mRNA level. This ARQ 197 induced degradation was reversed by a concomitant treatment with a proteasome inhibitor, bortezomib in the cells. However, bortezomib did not affect cytotoxicity of ARQ 197 in the same cells. Conclusion These data suggest that ARQ 197 may inhibit c-MET through downregulation of this protein in addition to inhibition of its kinase activity. Furthermore, it may enhance c-MET degradation by the ubiquitin/proteasome pathway. These c-MET inhibitory activities by ARQ 197 should be important for the additive combined effect with erlotinib in PC-9/MET cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1639.

A case of granulocyte colony-stimulating factor and interleukin 6 receptor-producing mediastinal mature cystic teratoma with somatic-type malignancy.

Mediastinal germ cell tumor with somatic‐type malignancy is a rare neoplasm. We describe one such case in a 49‐year‐old Japanese man who had shown an elevated serum concentration of granulocyte colony‐stimulating factor (GCSF) and leukocytosis without a shift to the left. Histologically, the tumor formed a teratomatous cyst whose wall contained benign epithelial components, well‐differentiated tubular and mucinous adenocarcinoma, and poorly‐differentiated pleomorphic carcinoma. Immunohistochemically, both the well differentiated adenocarcinoma and poorly differentiated pleomorphic carcinoma expressed GCSF. Immunohistochemistry and molecular analysis revealed that both components also produced interleukin 6 receptor (IL6R). We diagnosed this tumor as a GCSF‐ and IL6R‐producing mediastinal mature cystic teratoma with somatic‐type malignancy. The tumor showed immunohistochemical expression of activated signal transducer and activator of transcription 3. The patient died 6 months after developing systemic symptoms. For a GCSF‐producing tumor, complete resection appears to offer the best outcome at present. For any patient presenting with leukocytosis without a shift to the left, a thorough analysis should be conducted, and the tumor diagnosed as early as possible.

Abstract 1900: HSP 70 may cause EGFR-TKIs-resistance due to inhibit drug binding to EGFR in the cells that expressed mutant EGFR

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Cancer cells that expressed mutant EGFR are more sensitive to EGFR tyrosine kinase inhibitors (EGFR-TKIs) than that expressed wild type EGFR. To elucidate difference of the characteristics between wild type and 15 bp deletion mutant EGFR, we explored the difference of EGFR-binding proteins using respective stable transfectants, 293\_pEGFR and 293\_p≤15. As the result, we detected heat shock protein 70 (HSP70) specifically binds to the mutant EGFR but wild type EGFR. To examine whether HSP70 influenced EGFR-TKI sensitivity, we examined the effect of HSP70 inhibition. [Methods] HSP70 siRNA was exposed to the cells for 2 days. Cytotoxicity of the drugs was measured by MTT assay. The drug binding to EGFR was measured by [14C]gefitinib. [Results] Suppression of HSP70 by siRNA increased gefitinib and erlotinib sensitivities and enhanced gefitinib binding to this receptor, in 293\_p≤15 cells. Same phenomena were observed when HSP70 was inhibited by HSP70 specific inhibitor, 2-phenylethynesulfonamide (PES). PES inhibits HSP70 binding to the mutant EGFR. Interestingly, this compound enhanced the EGFR autophosphorylation and c-Cbl binding to the receptor in 293\_p≤15 cells. These results suggest that HSP70 may modulate EGFR activity and may cause EGFR-TKIs-resistance due to inhibit drug binding to the mutant EGFR. HSP70 inhibitor may possibly enhance the efficacy of EGFR-TKIs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1900. doi:1538-7445.AM2012-1900