Te-Chang Lee

Synthesis and biological activity of stable and potent antitumor agents, aniline nitrogen mustards linked to 9-anilinoacridines via a urea linkage

To improve the chemical stability and therapeutic efficacy of N-mustard, a series of phenyl N-mustard linked to DNA-affinic 9-anilinoacridines and acridine via a urea linker were synthesized and evaluated for antitumor studies. The new N-mustard derivatives were prepared by the reaction of 4-bis(2-chloroethyl)aminophenyl isocyanate with a variety of 9-anilinoacridines or 9-aminoacridine. The antitumor studies revealed that these agents exhibited potent cytotoxicity in vitro without cross-resistance to taxol or vinblastine and showed potent antitumor therapeutic efficacy in nude mice against human tumor xenografts. It also showed that 24d was capable of inducing marked dose-dependent levels of DNA cross-linking by comet assay and has long half-life in rat plasma.

IFN-Induced Protein with Tetratricopeptide Repeats 2 Inhibits Migration Activity and Increases Survival of Oral Squamous Cell Carcinoma

The function of the IFN-stimulated gene family protein, IFN-induced protein with tetratricopeptide repeats 2 (IFIT2), is poorly understood. Here, we report that IFIT2 colocalizes with cytokeratin 18 in oral squamous cell carcinoma (OSCC) cells. Treatment of OSCC cells with IFN-β significantly increased the expression of IFIT2 and remarkably inhibited cell migration. To further explore the effect of IFIT2 on cell migration, IFIT2 expression was either silenced with a small interfering RNA or increased by ectopic expression. IFIT2 knockdown in OSCC cells led to a significantly higher level of migration in vitro (P < 0.05) compared with control cells; by contrast, IFIT2 overexpression led to a significantly lower level of migration in vitro (P < 0.05). Immunohistochemically, 71.4% of OSCC tissues had elevated IFIT2 protein levels compared with noncancerous matched tissues. Elevated IFIT2 protein expression was positively associated with tumor differentiation status and inversely associated with nodal stage in OSCC specimens (P < 0.05). Higher IFIT2 protein levels in tumor tissues were also associated with better patient survival (P < 0.01). Our present study shows an inverse correlation between IFIT2 expression and cell migration, suggesting that IFIT2 plays an important role in inhibiting this process and that its expression may be associated with better prognosis in patients with OSCC. (Mol Cancer Res 2008;6(9):1431–9)

Novel DNA-directed alkylating agents: Design, synthesis and potent antitumor effect of phenyl N-mustard-9-anilinoacridine conjugates via a carbamate or carbonate linker

A series of phenyl N-mustard-9-anilinoacridine conjugates via a carbamate or carbonate linker was synthesized for antitumor evaluation. The carbamate or carbonate linker is able to lower the reactivity of the phenyl N-mustard pharmacophore and thus, these conjugates are rather chemically stable. The in vitro studies revealed that these derivatives possessed significant cytotoxicity with IC(50) in sub-micromolar range in inhibiting human lymphoblastic leukemia (CCRF-CEM), breast carcinoma (MX-1), colon carcinoma (HCT-116) and human non-small cell lung cancer (H1299) cell growth in vitro. Compounds 10a, 10b, 10e, 10i, and 15a were selected for evaluating their antitumor activity in nude mice bearing MX-1 and HCT-116 xenografts. Remarkably, total tumor remission was achieved by these agents with only one cycle of treatment. Interestingly, no tumor relapse was found in mice treated with 10a over 129 days. This agent is capable of inducing DNA interstrand cross-linking in human non-small lung cancer H1299 cells in a dose dependent manner by modified comet assay and has a long half-life in rat plasma.

Active Component of Antrodia cinnamomea Mycelia Targeting Head and Neck Cancer Initiating Cells through Exaggerated Autophagic Cell Death

Head and neck squamous cell carcinoma (HNSCC) is a highly lethal cancer. Previously, we identify head and neck cancer initiating cells (HN-CICs), which are highly tumorigenic and resistant to conventional therapy. Therefore, development of drug candidates that effectively target HN-CICs would benefit future head and neck cancer therapy. In this study, we first successfully screened for an active component, named YMGKI-1, from natural products of Antrodia cinnamomea Mycelia (ACM), which can target the stemness properties of HNSCC. Treatment of YMGKI-1 significantly downregulated the aldehyde dehydrogenase (ALDH) activity, one of the characteristics of CIC in HNSCC cells. Additionally, the tumorigenic properties of HNSCC cells were attenuated by YMGKI-1 treatment in vivo. Further, the stemness properties of HN-CICs, which are responsible for the malignancy of HNSCC, were also diminished by YMGKI-1 treatment. Strikingly, YMGKI-1 also effectively suppressed the cell viability of HN-CICs but not normal stem cells. Finally, YMGKI-1 induces the cell death of HN-CICs by dysregulating the exaggerated autophagic signaling pathways. Together, our results indicate that YMGKI-1 successfully lessens stemness properties and tumorigenicity of HN-CICs. These findings provide a new drug candidate from purified components of ACM as an alternative therapy for head and neck cancer in the future.

A Potent Derivative of Indolizino[6,7-b]Indole for Treatment of Human Non-Small Cell Lung Cancer Cells.

The therapeutic effect in non–small cell lung cancer (NSCLC) patients is limited because of intrinsic and acquired resistance. Thus, an unmet need exists for the development of new drugs to improve the therapeutic efficacy in NSCLC patients. In this study, the novel small molecule indolizino[6,7-b]indole derivative BO-1978 was selected to evaluate its therapeutic effects on NSCLC and its preclinical toxicity in animal models. An in vitro cytotoxicity assay revealed that BO-1978 significantly suppressed the growth of various NSCLC cell lines with or without mutations in epidermal growth factor receptor (EGFR). Mechanistically, we demonstrated that BO-1978 exhibited multiple modes of action, including inhibition of topoisomerase I/II and induction of DNA cross-linking. Treatment of NSCLC cells with BO-1978 caused DNA damage, disturbed cell cycle progression, and triggered apoptotic cell death. Furthermore, BO-1978 significantly suppressed the growth of EGFR wild-type and mutant NSCLC tumors in xenograft tumor and orthotopic lung tumor models with negligible body weight loss. The combination of BO-1978 with gefitinib further suppressed EGFR mutant NSCLC cell growth in xenograft tumor and orthotopic lung tumor models. Preclinical toxicity studies showed that BO-1978 administration did not cause apparent toxicity in mice. Based on its significant therapeutic efficacy and low drug toxicity, BO-1978 is a potential therapeutic agent for treatment of NSCLC.

Asb6 upregulation by Areca nut extracts is associated with betel quid-induced oral carcinogenesis.

Betel quit (BQ) chewing is a popular habit, especially in southern and southeastern Asia. Areca nut extracts (ANE), the major components of BQ, have been documented to induce reactive oxygen species, and consequently to cause genetic damage. ANE usage is tightly linked to oral cancer; however, the details of the molecular mechanism that results in carcinogenesis remain unclear. Previously, we successfully established HaCaT cells surviving from the long-term exposure of sublethal doses of ANE (Lai KC, Lee TC. Genetic damage in cultured human keratinocytes stressed by long-term exposure to areca nut extracts. Mutat Res 2006;599:66-75). Here, we identified the upregulation of Asb6, a coupling protein to the APS adapter protein, which is involved in insulin signaling for glucose transportation, of normal keratinocytes and oral cancer cells under ANE treatment. Immunohistochemical analyses of Asb6 on oral squamous cell carcinoma (OSCC) tissues (n=57) demonstrated the positive correlation between Asb6 upregulation (cancerous tissues versus adjacent normal tissues) and clinicopathological features. We showed that the combination of ANE-enhanced Asb6 expression in vitro and Asb6 upregulation in OSCC patients leads to poor survival status. In conclusion, our results suggest that upregulated Asb6 could act as a prognostic marker for oral cancer.

Promoter hypermethylation of LGALS4 correlates with poor prognosis in patients with urothelial carcinoma

Galectine-4 (gal-4), encoded by the LGALS4 gene, was recently shown to exhibit a tumor suppressive effect in colorectal carcinoma and pancreatic adenocarcinoma, although how the expression of this gene is regulated remains unknown. No reports describe the significance of gal-4 in the malignant potential of urothelial tumors. Thus, we analyzed LGALS4 methylation and gene expression and their clinical relevance and biological function in urothelial carcinoma (UC). LGALS4 methylation was initially identified as a progression biomarker for UC patients through genome-wide DNA methylation profiling of 16 tumor samples. Bisulfite sequencing PCR and immunohistochemistry were performed to validate the promoter methylation and expression of LGALS4. We used quantitative methylation-specific PCR to determine the methylation levels of LGALS4 normalized to ACTB in the tumor samples of 79 UC patients and compared the levels between patients with different clinicopathological characteristics. The association with survival probability was analyzed with the Kaplan-Meier method and Cox regression analysis. The ectopic expression of gal-4 in cancer cell lines was used to address its biological function in UC in vitro. The promoter hypermethylation of LGALS4 (>2.51, log10 scale) revealed a positive correlation with high levels of both histological grade and tumor T category and with lymph node metastasis (all P≤0.001). In addition, LGALS4 hypermethylation was an independent predictor of inferior survival in UC patients (P<0.05). The ectopic expression studies demonstrated that gal-4 suppressed urothelial cancer cell growth, migration, and invasion. Thus, LGALS4 may function as a tumor suppressor gene in UC progression. Our findings provide evidence that methylation-mediated LGALS4 gene repression may be involved in urothelial tumor progression.

Mitochondrial co-chaperone protein Tid1 is required for energy homeostasis during skeletal myogenesis

BackgroundTid1 is a mitochondrial co-chaperone protein and its transcript is abundantly expressed in skeletal muscle tissues. However, the physiological function of Tid1 during skeletal myogenesis remains unclear.MethodsIn vitro induced differentiation assay of mouse myoblast C2C12 cells was applied to examine the physiological role of Tid1 during skeletal myogenesis. In addition, transgenic mice with muscle specific (HSA-Cre) Tid1 deletion were established and examined to determine the physiological function of Tid1 during skeletal muscle development in vivo.ResultsExpression of Tid1 protein was upregulated in the differentiated C2C12 cells, and the HSA-Tid1f/f mice displayed muscular dystrophic phenotype. The expression of myosin heavy chain (MyHC), the protein served as the muscular development marker, was reduced in HSA-Tid1f/f mice at postnatal day (P)5 and P8. The protein levels of ATP sensor (p-AMPK) and mitochondrial biogenesis protein (PGC-1α) were also significantly reduced in HSA-Tid1f/f mice. Moreover, Tid1 deficiency induced apoptotic marker Caspase-3 in muscle tissues of HSA-Tid1f/f mice. Consistent with the in vivo finding, we observed that downregulation of Tid1 not only reduced the ATP production but also abolished the differentiation ability of C2C12 cells by impairing the mitochondrial activity.ConclusionTogether, our results suggest that Tid1 deficiency reduces ATP production and abolishes mitochondrial activity, resulting in energy imbalance and promoting apoptosis of muscle cells during myogenesis. It will be of importance to understand the function of Tid1 during human muscular dystrophy in the future.

Lyophilized particles and ethanolic extracts of Antrodia cinnamomea mycelia suppress the tumorigenicity of head and neck cancer cells in vivo.

Head and neck cancer (HNC) is one of the most common forms of cancer in Taiwan. In addition, head and neck cancer cells (HNCs) are highly tumorigenic and resistant to conventional therapy. Therefore, development of new therapeutic regimens that are adjuvant to conventional treatments would benefit future head and neck cancer therapy. In this study, we found that the lyophilized particles and ethanolic extracts of Antrodia cinnamomea mycelia inhibited the tumor growth of HNCs by xenograft assay in vivo. Moreover, administration of lyophilized particles or ethanolic extracts to nude mice did not cause significant side effects. Our study revealed that the Antrodia cinnamomea mycelia extract (ACME) efficiently inhibited the tumorigenicity of HNCs without causing organ failure. Furthermore, it showed that ACME may work as a novel drug candidate for alternative treatments for head and neck cancer.

P-glycoprotein attenuates DNA repair activity in multidrug-resistant cells by acting through the Cbp-Csk-Src cascade

Recent studies have demonstrated that P-glycoprotein (P-gp) expression impairs DNA interstrand cross-linking agent-induced DNA repair efficiency in multidrug-resistant (MDR) cells. To date, the detailed molecular mechanisms underlying how P-gp interferes with Src activation and subsequent DNA repair activity remain unclear. In this study, we determined that the C-terminal Src kinase-binding protein (Cbp) signaling pathway involved in the negative control of Src activation is enhanced in MDR cells. We also demonstrated that cells that ectopically express P-gp exhibit reduced activation of DNA damage response regulators, such as ATM, Chk2, Braca1 and Nbs1 and hence attenuated DNA double-strand break repair capacity and become more susceptible than vector control cells to DNA interstrand cross-linking (ICL) agents. Moreover, we demonstrated that P-gp can not only interact with Cbp and Src but also enhance the formation of inhibitory C-terminal Src kinase (Csk)-Cbp complexes that reduce phosphorylation of the Src activation residue Y416 and increase phosphorylation of the Src negative regulatory residue Y527. Notably, suppression of Cbp expression in MDR cells restores cisplatin-induced Src activation, improves DNA repair capacity, and increases resistance to ICL agents. Ectopic expression of Cbp attenuates cisplatin-induced Src activation and increases the susceptibility of cells to ICL agents. Together, the current results indicate that P-gp inhibits DNA repair activity by modulating Src activation via Cbp-Csk-Src cascade. These results suggest that DNA ICL agents are likely to have therapeutic potential against MDR cells with P-gp-overexpression.