Wen-Yu Pan

Combined modalities of resistance in an oxaliplatin-resistant human gastric cancer cell line with enhanced sensitivity to 5-fluorouracil

To identify mechanisms underlying oxaliplatin resistance, a subline of the human gastric adenocarcinoma TSGH cell line, S3, was made resistant to oxaliplatin by continuous selection against increasing drug concentrations. Compared with the parental TSGH cells, the S3 subline showed 58-fold resistance to oxaliplatin; it also displayed 11-, 2-, and 4.7-fold resistance to cis-diammine-dichloroplatinum (II) (CDDP), copper sulphate, and arsenic trioxide, respectively. Interestingly, S3 cells were fourfold more susceptible to 5-fluorouracil-induced cytotoxicity due to downregulation of thymidylate synthase. Despite elevated glutathione levels in S3 cells, there was no alteration of resistant phenotype to oxaliplatin or CDDP when cells were co-treated with glutathione-depleting agent, l-buthionine-(S,R)-sulphoximine. Cellular CDDP and oxaliplatin accumulation was decreased in S3 cells. In addition, amounts of oxaliplatin- and CDDP–DNA adducts in S3 cells were about 15 and 40% of those seen with TSGH cells, respectively. Western blot analysis showed increased the expression level of copper transporter ATP7A in S3 cells compared with TSGH cells. Partial reversal of the resistance of S3 cells to oxaliplatin and CDDP was observed by treating cell with ATP7A-targeted siRNA oligonucleotides or P-type ATPase-inhibitor sodium orthovanadate. Besides, host reactivation assay revealed enhanced repair of oxaliplatin- or CDDP-damaged DNA in S3 cells compared with TSGH cells. Together, our results show that the mechanism responsible for oxaliplatin and CDDP resistance in S3 cells is the combination of increased DNA repair and overexpression of ATP7A. Downregulation of thymidylate synthase in S3 cells renders them more susceptible to 5-fluorouracil-induced cytotoxicity. These findings could pave ways for future efforts to overcome oxaliplatin resistance.

Synthesis and Structure–Activity Relationships of 1-Benzyl-4,5,6-trimethoxyindoles as a Novel Class of Potent Antimitotic Agents

Combretastatin A‐4 derivatives: A series of combretastatin A‐4‐derived 1‐benzyl‐4,5,6‐trimethoxyindoles was designed and prepared as a novel class of potent antimitotic agents acting through the colchicine binding site on the microtubule.

2-Amino-3,4,5-Trimethoxybenzophenones as Potent Tubulin Polymerization Inhibitors

A series of novel 2‐amino‐3,4,5‐trimethoxybenzophenone analogues exhibited excellent activity as tubulin polymerization inhibitors by targeting the colchicine binding site of microtubules. The lead compound 17 exhibited an IC50 value of 1.6 μM, similar to that of combretastatin A‐4 (IC50=1.9 μM). It also displayed remarkable anti‐proliferative activity, with IC50 values ranging from 7–16 nM against a variety of human cancer cell lines and one MDR(+) cancer cell line. SAR information indicated that the introduction of an amino group at the C2 position of benzophenone ring A and the C3’ position of benzophenone ring B play important roles in maximizing activity.

Abstract 4461: MPT0B098, a novel microtubule inhibitor, displays potent anti-angiogenic activity via destabilizing hypoxia-inducible factor-1alpha mRNA

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Identification of a single agent that is able to target tumor cells, rapidly destroy tumor vasculature and inhibit angiogenesis could be an ideal approach for targeting solid tumors. We recently discover a novel sulfonamide compound, 7-aryl-indoline-1-benzene-sulfonamide (MPT0B098), as a potent microtubule inhibitor through binding to the colchicine binding site of tubulin. MPT0B098 is active against various human cancer cell growth with IC50 values ranged from 70-300 nM. Notably, HUVEC cells exhibit less susceptibility to the inhibitory effect of MPT0B098 with an IC50 of 510 nM. In addition, MPT0B098 exhibits no cross resistance with vincristine, paclitaxel, etoposide and camptothecin-resistant cell lines. Interestingly, MPT0B098 is still active toward cells (KB L30) with beta1-tubulin mutation. MPT0B098 arrests cells in G2/M phase and subsequently induces cell apoptosis through the caspase-dependent apoptotic pathway. In addition, using sub-lethal dose, MPT0B098 effectively suppressed to the tube formation and migration of HUVECs induced by VEGF. The expression levels of HIF-1alpha and VEGF were significantly inhibited in a concentration-dependent manner by MPT0B098 under hypoxic condition. Furthermore, HIF-1alpha-regulated genes, including cathepsin D, PDGF, VEGF, and VHL, were found to be down-regulated by MPT0B098 in A549 cells. Moreover, the decrease of the amount of HuR, a HIF-1alpha mRNA stability protein, translocated from nuclear to cytoplasm and suppression of AKT activity were coincided with decreased amount of HIF-1alpha mRNA by MPT0B098 treatment under hypoxia condition. MPT0B098 significantly suppressed tumor growth and microvessel density of tumor in H460 and KB-Vin10 xenografted mouse model. Taken together, our results indicate that MPT0B098 is a promising anticancer drug with potential for the treatment of human malignancies. (The study was supported by grants of DOH99-TD-C-111-004 from Department of Health and CA-099-PP-02 from National Health Research Institutes, Taiwan, R.O.C.) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4461. doi:10.1158/1538-7445.AM2011-4461

Abstract 1701: Activation of Akt/FoxO axis confers resistance to cisplatin in human nasopharyngeal carcinomas

Nasopharyngeal carcinoma (NPC) is one of the predominant cancers found in Southeastern China and Taiwan. Chemotherapy is one of the major treatment modalities, and the cisplatin-based regimen is the front-line treatment. However, NPC patients failing to cisplatin treatment will have a compromised survival. To explore the resistance spectrum and mechanisms of cisplatin resistance in NPC, two cisplatin-resistant sublines (cis6 and cis15) derived from the parental NPC cell line HONE-1 were obtained. Compared with HONE-1 cells, cis6 and cis15 cells showed upto 18-fold resistance to cisplatin in each, and also possessed a cross-resistance to oxaliplatin and arsenic trioxide. The level of platinum-DNA-adduct and γH 2 AX were significantly decreased in cis6 and cis15 cells. To unravel the behind details, the systems of DNA repair and cisplatin detoxification were examined and found to be more active in both resistant cells. In terms of the DNA repair proteins, the levels of p-DNA-PK and XRCC1 were increased; in terms of cellular localization of cisplatin, the level of copper transporter ATP7A was upregulated; in terms of Nrf2/antioxidant/detoxifizing enzyme, the resistant cells had a higher Nrf2 activity and an increased intracellular level of GSH, GR, NQO1, AKR1C1 and AKR1C2. Moreover, as compared with HONE-1 cells, our results showed that the resistant cells spared the cisplatin-induced cell death via the suppression of pro-apoptotic proteins and the enhancement of anti-apoptotic proteins. Since the members of ErbB family could be overexpressed in NPC and the ErbB/Akt/FoxO axis could involve in the actions of apoptosis, DNA damage repair and detoxification of reactive oxygen species, the ErB/Akt/FoxO were investigated. Indeed, the phosphorylations of Akt, FoxO1, and FoxO3 were upregulated in cis6 and cis15 cells. Thus, the resistance to cisplatin in cis6 and cis15 cells was partially explained by the Akt/FoxO pathway. Surprisingly, EGFR was downregulated in the resistant cells, whereas ErbB2 was upregulated. The elevations of the negative regulators of EGFR, including c-Cbl, GCF and LRIG1, were observed in cis6 and cis15 cells, indicating that the negative regulation of EGFR could be at the levels of transcription and protein degradation. Since LRIG1 could be upregulated and negatively feedback to control the level of ErbB after the ErbB was activated, it was plausible that the overexpression of ErbB2 upregulated the expression of LRIG1 and subsequently, caused the downregulation of EGFR. Nevertheless, the hypothesis which both overexpression of ErbB2 and LRIG1 contributes to the resistance to cisplatin in our cell model awaits experimental verification. Taken together, our results suggested that the mechanism responsible for cisplatin resistance in NPC, at least in part, through Akt/FoxO pathway. (The study was supported by grants of Department of Health DOH99-TD-C-111-004, Taiwan, R.O.C.) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1701. doi:10.1158/1538-7445.AM2011-1701

Abstract 2045: microRNA expression patterns associated with resistance to platinum-based chemotherapy in human cancer cell lines

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Platinum-based compounds, for example cisplatin and oxaliplatin, are the common anti-tumor drugs that have been used worldwide effective against a multitude of cancers. However, their clinical usage is impeded by toxic side effects or by the occurrence of drug resistance. MicroRNAs (miRNAs) are a group of the non-coding RNAs that play a role in controling the post-transcriptional expression of genes. Herein, we aimed to reveal the potential role of miRNAs in platinum-based drug resistance, and analyzed the miRNAs target genes that are supposed to be related with regulating the drug resistant mechanisms. The two human cancer resistant cell lines HONE-1 and TSGH were established by treating with the platinum-based compounds up to 6µm and 15µm, respectively. We then compared the expression level of genes among the parental lines and their resistant lines series using fluorescence in situ hybridization, miRNA array, expression microarray and western blot approach. The results showed the higher proteins expression level of p53 and p21 were presented in both resistant cell lines series while compared to the parental lines. However, there was no mutation occurred in both genes, either in gene structure or gene copy number change. Moreover, four miRNAs were detected from miRNA microarray by combining the results of both resistant cell line series. They included one high-regulated hsa-miR-193b; and three low-regulated hsa-miR-202, hsa-miR-509 and hsa-miR-575. Interestingly, corresponding genes that related to these miRNAs were also identified in the expression array. These genes included DNA replication related gene CDC34; cell cycle associated genes RRM2,S100A2,CCND1,CCNE1,CDC34; apoptotic pathway correlated gene CCND1; and RRM2 which is associated with DNA synthesis. In summary, as the high-expression level of protein p53 and p21 were observed on both resistant lines series without showing the gene status aberrations or RNA expression difference, we speculated that alteration could be occurred during the period of post-translational modification, and it may further regulate signal transduction of the resistant-associated genes. Therefore, genes that were targeted by hsa-miR-202, hsa-miR-509 and hsa-miR-575 should be highly noticed, and are worthwhile to further verify their potential role as the platinum-based compounds resistant biomarkers. Besides, the overexpressed gene IL-6, down stream gene Bcl-2 was known to be involved in the formation of chemoresistance, was also observed in the resistant lines. Therefore, further understanding of the regulatory pathways of IL-6 and Bcl-2 might provide novel insight into the platinum-drug resistance in clinical pharmacotherapy. 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 2045.

Abstract B69: Enhancement of nonhomologous end joining DNA repair capacity confers cancer cells resistant to novel selenophene compound D‐501036

D‐501036 is a selenophene compound, which has been shown to exhibit antineoplastic activity against various types of tumor. Our pervious data demonstrated that the treatment of D‐501036 induces S phase arrest during cell cycle. It induces the formation of Se‐DNA adducts and reactive oxygen species (ROS) in cells. Moreover, the amount of signal transduction proteins responded to DNA damage such as phospho‐ATM and phospho‐p53 were increased in D‐501036‐treated cells (Mol Cancer Ther, 2007). To investigate the mechanism of resistance to selenophene compounds, a human cervical cancer KB‐derived D‐501036‐resistant KB‐1036‐S4 cells were generated. This D‐501036‐resistant cancer cell lines also exhibits cross‐resistance to cisplatin, oxaliplatin and CPT compounds. Although the treatment of D‐501036 induced similar level of ROS and Se‐DNA adducts in both drug‐sensitive (KB) and ‐resistant (KB‐1036‐S4) cells, DNA was less damaged in KB‐1036‐S4 as compared to KB cells. Decrement of DNA damage in drug‐resistant cells indicates that the efficiency of DNA repair during genomic stresses might be higher in KB‐1036‐S4 cells. Pervious studies suggested that D‐501036 induces double strand DNA break (DSB). Global DNA repair ability in KB‐ 1036‐S4 cells is higher than KB cells. In vitro Non‐homologous end joining (NHEJ) assay with the use of whole cell lysate revealed that KB‐1036‐S4 also exhibits higher ability of forming DNA multimer by rejoining linear DNA strand. To determine the importance of NHEJ in drug resistance of KB‐1036‐S4 cells, ku80 was down‐regulated by siRNA. Ku80 is a major component in NHEJ, which is responsible for the recruitment of XRCC4 and DNA ligase IV. Cell viability assay revealed that gene silencing of Ku80 by siRNA partially restored sensitivity to D‐501036 in KB‐1036‐S4 cells. The IC50 of D‐501036 in Ku80‐downregulated KB‐1036‐S4 cells was reduced to 25% as compare to negative control. Global DNA repair ability of KB‐1036‐S4 was also decreased in Ku80‐downregulated situations. Taken together, these results indicate that enhancement of NHEJ activity is one of the major resistance mechanisms to D‐501036. Targeting components in the NHEJ pathway seems to be a promising way to overcome resistance to DNA damaging agents. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B69.