Breast cancer is currently the most prevalent cancer in women, and its incidence increases every year. Azole antifungal drugs were recently found to have antitumor efficacy in several cancer types. They contain an imidazole (clotrimazole and ketoconazole) or a triazole (fluconazole and itraconazole) ring. Using human breast adenocarcinoma cells (MCF-7 and MDA-MB-231), we evaluated the effects of azole drugs on cell proliferation, apoptosis, cell cycle, migration, and invasion, and investigated the underlying mechanisms. Clotrimazole and ketoconazole inhibited the proliferation of both cell lines while fluconazole and itraconazole did not. In addition, clotrimazole and ketoconazole inhibited the motility of MDA-MB-231 cells and induced G1-phase arrest in MCF-7 and MDA-MB-231 cells, as determined by cell cycle analysis and immunoblot data. Moreover, Transwell invasion and gelatin zymography assays revealed that clotrimazole and ketoconazole suppressed invasiveness through the inhibition of matrix metalloproteinase 9 in MDA-MB-231 cells, although no significant changes in invasiveness were observed in MCF-7 cells. There were no significant changes in any of the observed parameters with fluconazole or itraconazole treatment in either breast cancer cell line. Taken together, imidazole antifungal drugs showed strong antitumor activity in breast cancer cells through induction of apoptosis and G1 arrest in both MCF-7 and MDA-MB-231 cells and suppression of invasiveness via matrix metalloproteinase 9 inhibition in MDA-MB-231 cells. Imidazole drugs have well-established pharmacokinetic profiles and known toxicity, which can make these generic drugs strong candidates for repositioning as antitumor therapies.

Abstract. Butyrate is a short-chain fatty acid produced by the intestinal microflora and it not only induces apoptosis but also inhibits the proliferation of cancer cells. Recently, it has been reported that butyrate may cause resistance in colon cancer cells. Therefore, we investigated the effects of increased resistance to butyrate in HCT116 colon cancer cells. We established HCT116 cells resistant to butyrate (HCT116/ BR) by treating HCT116 parental cells (HCT116/PT) with increasing concentrations of butyrate to a maximum of 1.6 mM for 3 months. The butyrate concentrations that inhibited cell growth by 50% (IC 50 ) were 0.508 and 5.50 mM in HCT116/PT and HCT116/BR cells. The values after treatment with paclitaxel, 5-fluorouracil (5-FU), doxorubicin and trichostatin A (TSA) were 2.42, 2.36, 4.31 and 11.3-fold higher, respectively, in HCT116/BR cells compared with HCT116/PT cells. The protein expression of drug efflux pumps, such as P-glycoprotein (P-gp), breast cancer-resistant protein (BCRP) and the multidrug resistance associated protein 1 (MRP1), did not differ between HCT116/PT and HCT116/BR cells. The expression level of the anti-apoptotic Bcl-xL protein was increased while those of pro-apoptotic Bax and Bim proteins were reduced in HCT116/BR cells. There were no significant differences in cell motility and invasion. This study suggests that exposure of colon cancer cells to butyrate results in development of resistance to butyrate, which may play a role in the acquisition of chemoresistance in colon cancer. IntroductionColon cancer is one of the most common cancers and its prevalence is increasing gradually due to changes in lifestyle (1).Epidemiological studies have attempted to elucidate an association between a high-fiber diet and decreased incidence and progression of colon cancer. Butyrate is a short-chain fatty acid that is naturally produced by the intestinal microflora during dietary fiber fermentation and has been suggested as a target for colon cancer therapy because it inhibits cell proliferation and causes apoptosis in colon cancer cells (2). Butyrate suppresses cell proliferation by causing cell cycle arrest at the G 1 and G 2 phases, which involves cyclin D1 and retinoblastoma (Rb) signaling (3). In addition, butyrate is a histone deacetylase (HDAC) inhibitor, similar to trichostatin A(TSA), and results in apoptosis by increasing the expression of pro-apoptotic proteins, such as Bak, Bad and Bim, and decreasing that of anti-apoptotic proteins, such as Bcl-2 and Bcl-xL (4).Butyrate plays an important role in normal colonocytes as a major energy source and promotes their proliferation (5). However, most cancer cells prefer to utilize glucose as an energy source even under abundant oxygen conditions, so colon cancer cells cannot use butyrate efficiently, which enhances its anticancer effects (6).Recently, it has been reported that butyrate may trigger resistance in colon cancer cells (7). As mentioned above, butyrate is a natural product of dietary fiber fermentation to which colonocytes are c...

Etoposide, used for the treatment of breast cancer, is mainly metabolized via hepatic cytochrome P450 (CYP) 3A4 in humans and is also a substrate for p-glycoprotein (P-gp). Morin is known to be able to modulate the activities of metabolic enzymes including CYPs and can act as a potent P-gp inhibitor. The purpose of this study was to investigate the effects of morin on the pharmacokinetics of etoposide in rats with 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary tumors. Etoposide was administered intravenously (2 mg/kg) and orally (10 mg/kg) in control and DMBA rats without (DMBA-WOM) and with (DMBA-WM) morin (15 mg/kg). Protein and mRNA expression of CYP3A and P-gp was analyzed, and the tissue distribution of etoposide was also measured. Both protein and mRNA expression of CYP3A and P-gp was inhibited by morin in the liver, intestine and breast tumors of DMBA-WM rats. After both intravenous and oral administration of etoposide in DMBA-WM rats, the total area under the plasma concentration-time curve from time zero to infinity (AUC) of etoposide was significantly greater, and the time-averaged total body clearance (CL) of etoposide was significantly slower than those in control and DMBA-WOM rats. The amount of etoposide recovered from each tissue was significantly higher in DMBA-WM rats, especially in the breast tumor, liver and large intestine. No significant differences between control and DMBA-WOM rats were observed. Taken together, greater AUC and slower CL of etoposide in DMBA-WM rats could possibly be due to the inhibition of hepatic CYP3A (intravenous) and mainly due to the inhibition of intestinal CYP3A and P-gp (oral) by morin.

The MCF10AT cell series of human breast epithelial cancer cells includes normal MCF10A (10A), premalignant MCF10AT (10AT) and MCF10ATG3B (10ATG3B), and fully malignant MCF10CA1a (10CA1a) cells. The series is a unique model system showing progressive tumorigenic potential with the same origin. The effects of paclitaxel, a microtubule inhibitor, were evaluated in this cell system. Paclitaxel inhibited cell proliferation in a time-dependent (24, 48 and 72 h) and concentration-dependent (0-10 nM) manners with less sensitivity in 10CA1a cells. Treatment with paclitaxel (10 nM) for 24 h induced apoptosis in 10A, 10AT, 10ATG3B and 10CA1a cells, with 23.6, 26.1, 25.2 and 8.96%, respectively, in the sub-G1 phase. Treatment with paclitaxel (0-10 nM) for 24 h, resulted in the appearance of DNA fragmentation (a hallmark of apoptosis) with less sensitivity in the 10CA1a tumor cells. Paclitaxel increased p53 protein expression in 10A, 10AT, 10ATG3B and 10CA1a cells, by 87, 102, 812 and 84%, respectively. The p21Waf1/Cip1 protein expression increased by 2.57-, 1.53- and 2.48-fold in 10A, 10AT and 10ATG3B cells, respectively, with negligible detection in the 10CA1a cells. Activation of the Akt signaling pathway was observed in the MCF10AT cell lineage and the protein expression of phospho-Akt (Ser473 and Thr308). The downstream targets of this pathway, phospho-p70S6K and phospho-S6RP, were also inhibited by paclitaxel in 10A, 10AT and 10ATG3B cells, but minimally inhibited in 10CA1a cells, suggestive of chemoresistance in 10CA1a cells. The effects of paclitaxel on the multidrug resistance 1 (MDR1), MRP1 and breast cancer resistance protein (BCRP) gene expression were not significant in the MCF10AT cell lineage. These results collectively indicated that paclitaxel inhibited cell proliferation and induced apoptosis in the MCF10AT cell lineage, with chemoresistance in 10CA1a tumor cells. The decreased responsiveness to paclitaxel observed in 10CA1a tumor cells was likely due, in part, to activation of the Akt signaling pathway and a high expression of wild-type p53 with lack of p21Waf1/Cip1.

Tofacitinib, a Janus kinase 1 and 3 inhibitor, is mainly metabolized by CYP3A1/2 and CYP2C11 in the liver. The drug has been approved for the chronic treatment of severe ulcerative colitis, a chronic inflammatory bowel disease. This study investigated the pharmacokinetics of tofacitinib in rats with dextran sulfate sodium (DSS)-induced ulcerative colitis. After 1-min of intravenous infusion of tofacitinib (10 mg/kg), the area under the plasma concentration-time curves from time zero to time infinity (AUC) of tofacitinib significantly increased by 92.3%. The time-averaged total body clearance decreased significantly by 47.7% in DSS rats compared with control rats. After the oral administration of tofacitinib (20 mg/kg), the AUC increased by 85.5% in DSS rats. These results could be due to decreased intrinsic clearance of the drug caused by the reduction of CYP3A1/2 and CYP2C11 in the liver and intestine of DSS rats. In conclusion, ulcerative colitis inhibited CYP3A1/2 and CYP2C11 in the liver and intestines of DSS rats and slowed the metabolism of tofacitinib, resulting in increased plasma concentrations of tofacitinib in DSS rats.

Purpose. Butyric acid is a short chain fatty acid produced naturally by fermentation of dietary fiber in the colonic mucosa. It is known to play an important role in preventing the initiation and the progression of colon cancer by suppressing the proliferation of cancer cells. However, some colon cancer still develops and progresses to a more malignant and chemoresistant one despite of the presence of butyric acid. The present study was undertaken to investigate a possibility of butyrate-involved mechanism by which the colon cancer cells acquire resistance to chemotherapeutic drugs. Methods. Human HCT116 colon cancer cells were exposed to progressively increasing concentrations of butyrate for three months to establish a colon cancer cell line with acquired resistance to butyrate. After obtaining butyrate-resistant cells, the sensitivity against multiple chemotherapeutic drugs were compared between parental and butyrate-resistant cells. The expression of proteins related to chemotherapeutic response was also compared by immunoblotting analysis. Results. Butyrate-resistant HCT116 cancer cell lines could be established by culturing cells in the presence of butyrate up to 0.8 mM concentration. The concentrations inhibiting the cell growth by 50% (IC50) of butyrate was 0.62 and 4.56 mM in parental and butyrate-resistant HCT116 cells. Butyrate-resistant HCT116 cells were also resistant to widely used chemotherapeutic drugs: the IC50 after paclitaxel, doxorubicin or 5-FU treatment were 2.10-, 3.98- and 1.6-fold higher in butyrateresistant cells compared with parental cells. The expression of multidrug resistance-related proteins that function as drug efflux pumps such as P-glycoprotein and the multidrug resistance-associated protein 1 were not changed at protein level. Among the apoptosis-involved proteins, the expression of anti-apoptotic BcL-2 protein was increased while that of proapoptotic BAD protein was reduced in butyrate-resistant cells, suggesting that the above chemoresistance was associated with cellular resistance to apoptosis. Conclusion.Our data herein suggests that the colon cancer cell grown in the presence of butyric acid may acquire resistance to apoptotic cell death and thus lead to resistance to chemotherapeutic drugs. Citation Format: Soo-jeong Lim, Hyeon Gyeom Choi, Chae Kyung Jeon, So Hee Kim. Chemoresistance is induced by butyrate in colon cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 900. doi:10.1158/1538-7445.AM2013-900

Tofacitinib, a Janus kinase 1 and 3 inhibitor, is used to treat rheumatoid arthritis. It is mainly metabolized by the cytochromes p450 (CYP) 3A1/2 and CYP2C11 in the liver. Chronic inflammation eventually leads to cirrhosis in patients with rheumatoid arthritis. Isosakuranetin (ISN), a component of Citrus aurantium L., has hepatoprotective effects in rats. This study was performed to determine the effects of ISN on the pharmacokinetics of tofacitinib in rats with N-dimethylnitrosamine-induced liver cirrhosis (LC). After intravenous administration of 10 mg/kg tofacitinib to control (CON), LC, and LC treated with ISN (LC-ISN) rats, the total area under the plasma concentration–time curves (AUC) from time zero to infinity increased by 158% in LC rats compared to those in CON rats; however, the AUC of LC-ISN rats decreased by 35.1% compared to that of LC rat. Similar patterns of AUC changes were observed in the LC and LC-ISN rats after oral administration of 20 mg/kg tofacitinib. These results can be attributed to decreased non-renal clearance (CLNR) and intestinal intrinsic clearance (CLint) in the LC rats and increased intestinal and hepatic CLint in the LC-ISN rats. Our findings imply that ISN treatment in LC rats restored the decrease in either CLNR or CLint, or both, through increased hepatic and intestinal expression of CYP3A1/2 and CYP2C11, which is regulated by the induction of pregnane X receptor (PXR) and constitutive androstane receptor (CAR).