Naphthalimides, particularly amonafide and 2-(2-dimethylamino)-6-thia-2-aza-benzo[def]chrysene-1,3-diones (R16), have been identified to possess anticancer activities and to induce G(2)-M arrest through inhibiting topoisomerase II accompanied by Chk1 degradation. The current study was designed to precisely dissect the signaling pathway(s) responsible for the naphthalimide-induced cell cycle arrest in human colon carcinoma HCT116 cells. Using phosphorylated histone H3 and mitotic protein monoclonal 2 as mitosis markers, we first specified the G(2) arrest elicited by the R16 and amonafide. Then, R16 and amonafide were revealed to induce phosphorylation of the DNA damage sensor ataxia telangiectasia-mutated (ATM) responding to DNA double-strand breaks (DSBs). Inhibition of ATM by both the pharmacological inhibitor caffeine and the specific small interference RNA (siRNA) rescued the G(2) arrest elicited by R16, indicating its ATM-dependent characteristic. Furthermore, depletion of Chk2, but not Chk1 with their corresponding siRNA, statistically significantly reversed the R16- and amonafide-triggered G(2) arrest. Moreover, the naphthalimides phosphorylated Chk2 in an ATM-dependent manner but induced Chk1 degradation. These data indicate that R16 and amonafide preferentially used Chk2 as evidenced by the differential ATM-executed phosphorylation of Chk1 and Chk2. Thus, a clear signaling pathway can be established, in which ATM relays the DNA DSBs signaling triggered by the naphthalimides to the checkpoint kinases, predominantly to Chk2,which finally elicits G(2) arrest. The mechanistic elucidation not only favors the development of the naphthalimides as anticancer agents but also provides an alternative strategy of Chk2 inhibition to potentiate the anticancer activities of these agents.

Telomerase is proposed as an anticancer target. Increasing evidence suggests that telomerase is involved in functions independent of telomere-extension activity. In this study, we designed a small interfering RNA (siRNA) targeting human telomerase reverse transcriptase (hTERT), the catalytic subunit of telomerase. Using transient and persistent transfection of hTERT siRNA into telomerase-positive human colon carcinoma HCT116 cells, we demonstrated that hTERT siRNA suppresses hTERT expression and leads to inhibition of telomerase activity and HCT116 cell growth and in vivo tumorigenicity in nude mice. Further analysis indicated that depletion of hTERT reduces cell adhesion, migration, and invasion prior to inhibition of cell proliferation. Downregulation of hTERT also decreased the expression levels of adhesion-and motility-related proteins, in particular c-Met and integrins. The lack of detectable changes in telomere length showed that downregulation of hTERT in the present system had no significant effect on telomere-extension activity. Taken together, our results highlight the therapeutic potential of a novel hTERT siRNA and suggest that hTERT is involved in the regulation of cell motility in a telomere-independent manner.

Microtubule-targeted drugs are now indispensable for the therapy of various cancer types worldwide. In this article, we report MT119 [6-[2-(4-methoxyphenyl) -ethyl]-9-[(pyridine-3-ylmethyl)amino]pyrido[2 0 ,1 0 :2,3]imida-zo [4,5-c]isoquinolin-5(6H)-one] as a new microtubule-targeted agent. MT119 inhibited tubulin polymerization significantly both in tumor cells and in cell-free systems, which was followed by the disruption of mitotic spindle assembly. Surface plasmon resonance-based analyses showed that MT119 bound to purified tubulin directly, with the K D value of 10.6 lM. The binding of MT119 in turn caused tubulin conformational changes as evidenced by the quenched tryptophan fluorescence, the reduction of the bis-ANS reactivity and the decreased DTNB-sulfhydryl reaction rate. Competitive binding assays further revealed that MT119 bound to tubulin at its colchicine site. Consequently, by inhibiting tubulin polymerization, MT119 arrested different tumor cells at mitotic phase, which contributed to its potent antitumor activity in vitro. MT119 was also similarly cytotoxic to vincristine-, adriamycin-or mitoxantrone-resistant cancer cells and to their corresponding parental cells. Together, these data indicate that MT119 represents a new class of colchicine-site-targeted inhibitors against tubulin polymerization, which might be a promising starting point for future cancer therapeutics.Microtubule is composed of a-and b-tubulin heterodimers, whose dynamics is crucial for the proper function of spindles and guarantees the mitotic progression. 1 By interfering with microtubule dynamics, tubulin inhibitors cause mitosis arrest and ultimately lead to tumor cell death. Since the approval of vinca alkaloids in 1960s and then taxol in 1990s for cancer therapy, targeting microtubule has been recognized as the most effective strategy against different types of malignancies, including hematological, ovarian, mammary and lung cancers. 2 According to the differential impacts on microtubule polymer mass, tubulin inhibitors could be classified into microtubule destabilizers, such as vinca alkaloids, colchicine and combretastatins and microtubule stabilizers, such as taxol and epothilones. Regardless of their distinct structural types, these inhibitors bind to microtubules mainly at one of the three sites: the vinblastine site, the colchicine site or the taxol site. Despite of their proven therapeutic success, some drawbacks such as limited natural sources, high neurotoxicity and poor solubility have rendered serious questions onto the extensive use of current microtubule-targeted drugs in clinic. 3,4 Therefore, it is urgent to find more structurally-diverse compounds with tubulin inhibitory functions for future antitumor therapy.We previously reported a combinatorial library of 6H-Pyrido

Targeting cellular mitosis is an attractive antitumor strategy. Here, we reported MT7, a novel compound from the 6H-Pyrido[2',1':2,3]imidazo [4,5-c]isoquinolin- 5(6H)-one library generated by using the multi-component reaction strategy, as a new mitotic inhibitor. MT7 elicited apparent inhibition of cell proliferation by arresting mitosis specifically and reversibly in various tumor cell lines originating from different human tissues. Detailed mechanistic studies revealed that MT7 induced typical gene expression profiles related to mitotic arrest shown by cDNA microarray assays. Connectivity Map was used to analyze the microarray data and suggested that MT7 was possibly a tubulin inhibitor due to its similar gene expression profiles to those of the known tubulin inhibitors demecolcine, celastrol and paclitaxel. Further analyses demonstrated that MT7 inhibited the polymerization of cellular microtubules although it was not detectable to bind to purified tubulin. The inhibition of cellular tubulin polymerization by MT7 subsequently resulted in the disruption of mitotic spindle formation, activated the spindle assembly checkpoint and consequently arrested the cells at mitosis. The persistent mitotic arrest by the treatment with MT7 led the tested tumor cells to apoptosis. Our data indicate that MT7 could act as a promising lead for further optimization, in hopes of developing new anticancer therapeutics and being used to probe the biology of mitosis, specifically, the mode of interference with microtubules.

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Telomere repeat binding factor 2 (TRF2) has been increasingly recognized to be involved in DNA damage response and telomere maintenance. Our previous report found that salvicine (SAL), a novel topoisomerase II poison, elicited DNA doublestrand breaks and telomere erosion in separate experimental systems. However, it remains to be clarified whether they share a common response to these two events and in particular whether TRF2 is involved in this process. In this study, we found that SAL concurrently induced DNA double-strand breaks, telomeric DNA damage, and telomere erosion in lung carcinoma A549 cells. It was unexpected to find that SAL led to disruption of TRF2, independently of either its transcription or proteasome-mediated degradation. By overexpressing the full-length trf2 gene and transfecting TRF2 small interfering RNAs, we showed that TRF2 protein protected both telomeric and genomic DNA from the SAL-elicited events. It is noteworthy that although both the Ataxia-telangiectasia-mutated (ATM) and the ATM-and Rad3-related (ATR) kinases responded to the SAL-induced DNA damages, only ATR was essential for the telomere erosion. The study also showed that the activated ATR augmented the SAL-triggered TRF2 disruption, whereas TRF2 reduction in turn enhanced ATR function. All of these findings suggest the emerging significance of TRF2 protecting both telomeric DNA and genomic DNA on the one hand and reveal the mutual modulation between ATR and TRF2 in sensing DNA damage signaling during cancer development on the other hand.

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