Ming-Hsi Wu

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.

Design and synthesis of potent antitumor water-soluble phenyl N-mustard-benzenealkylamide conjugates via a bioisostere approach.

A series of new, water-soluble phenyl N-mustard-benzenealkylamide conjugates containing hydrophilic ω-dialkylaminoalkylamide or ω-cyclic aminoalkylamide moieties were synthesized via a bioisostere approach. These compounds have a broad spectrum of antitumor activity against a panel of human tumor cell lines. Of these derivatives, compound 18b effectively suppressed the growth of colon cancer (HCT-116), prostate cancer (PC3), and lung cancer (H460) xenografts. The growth of HCT-116 xenografts was almost completely suppressed when co-treated with compound 18b and 5-fluorouracil. Furthermore, compound 18b can induce DNA cross-linking and cell-cycle arrest at the G2/M phase. Early preclinical studies, including pharmacokinetics in rats, inhibition of the hERG, and 14 days of acute intravenous injection toxicity, suggest that compound 18b is a promising candidate for further preclinical studies.

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.

Corrigendum to “PI3K Inhibition Augments the Therapeutic Efficacy of a 3a–aza-Cyclopenta[α]indene Derivative in Lung Cancer Cells [Translational Oncology 7 (2014) 256–266.e5]

*Molecular Medicine Program, Taiwan International Graduate Program, Academia Sinica, Taipei, Taiwan; Institute of Biochemistry and Molecular Biology, National Yang-Ming University, Taipei, Taiwan; Institute of Biopharmaceutical Sciences, National Yang-Ming University, Taipei, Taiwan; Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan; Graduate Institute of Pharmaceutical Chemistry, China Medical University, Taichung, Taiwan

Novel N-mustard-benzimidazoles/benzothiazoles hybrids, synthesis and anticancer evaluation

BACKGROUND Bendamustine, an N-mustard-benzoimidazole hybrid conjugate, was recently approved for the treatment of chronic lymphocytic leukemia. However, the short half-life of bendamustine may limit its clinical applications. OBJECTIVE The purpose of this study is to design and synthesize compounds with a more favorable pharmacokinetic profile. METHODS We synthesized a series of hybrid molecules comprising a phenyl N-mustard moiety and benzothiazole or benzimidazole scaffold linked via a urea linker and evaluated their antitumor activity and plasma stability. RESULTS We revealed that these agents exhibited significant cytotoxicity against a panel of human lymphoblastic leukemia and human solid tumor cells in culture. Human lymphoblastic leukemia CCRM-CEM cells were the most sensitive to the tested compounds. In general, the new hybrids were as potent as cisplatin, but significantly more cytotoxic than bendamustine. Phenyl N-mustard-benzothiazole compound 27d and phenyl N-mustardbenzimidaloe compound 32b possessed significant cytotoxicity and led to apoptotic death in the treated tumor cells. These two agents were able to induce DNA interstrand cross-linking and arrested cell cycle progression at the G2/M phase. Furthermore, we showed that these new hybrids were more chemically stable than bendamustine in rat plasma. CONCLUSION Our results suggest that conjugation of phenyl N-mustard pharmacophore at C6 of benzimidazole or at C8 of the benzothiazole ring via a urea linker is likely an approach to increase the chemical stability and bioavailability. Highlights ⇒ Series of benzimidazoles and benzothiazoles linked to N-mustard were synthesized. ⇒ The newly synthesized derivatives induced DNA interstrand cross-links. ⇒ These derivatives induced cell cycle arrest in the G2/M phase and triggered apoptosis in H460 cells. ⇒ The new compounds are more cytotoxic than bendamustine. ⇒ The new compounds were chemically more stable than bendamustine in rat plasma.

Corrigendum to “A Potent Derivative of Indolizino[6,7-b]Indole for Treatment of Human Non–Small Cell Lung Cancer Cells” [Neoplasia 18 (2016) 199-212]

The authors regret to have mistakenly placed the figure of body weight change in mice treated with different agents in PC9 cells (Figure 6A, right) with that of PC9/gef B4 cells (Figure 6B, right) during the art work. The corrected Figure 6 was attached below. The authors would like to apologize for any inconvenience caused.

Abstract 3733: Augmentation of response to therapeutic agents by (-)-gallocatechin gallate through inhibition of RAD51 nuclear translocation

Lung cancer is the most common cancer worldwide. Platinum-based combination chemotherapy is one of first line treatments for patients with lung cancer. Most of the cytotoxic drugs are targeting DNA, interfere with DNA synthesis, and triggering DNA damage response (DDR). Since DDR usually activates important defensive pathways to repair damaged DNA, DDR is known as a key factor determining the outcome of chemotherapy. Among them, RAD51, playing a central role in homologous recombination (HR) DNA repair, has been shown to interfere with the prognosis of patients treated with chemoradiotherapy. Since we have demonstrated that inhibition of RAD51 nuclear focus formation synergistically enhances the anticancer activity of cisplatin, we further screened purified natural products in order to find products with activity to inhibit RAD51 nuclear focus formation. In the present study, we found that a green tea catechin derivative, the (-)-Gallocatechin gallate (GCG) has potent inhibitory activity against RAD51 nuclear focus formation in cisplatin-treated H460 cells. The in vitro cytotoxicity assay showed that the combination of cisplatin or irinotecan with GCG significantly increases the sensitivity of H460 cells to these DNA damage agents. We also evaluated the anti-tumor growth activity of cisplatin or irinotecan in combination with GCG using H460 xenografts. Our results showed that cisplatin and irinotecan suppresses tumor growth by 17.6% and 33.8%, while in combination with GCG suppresses tumor growth by 52.2% and 68.9%, respectively. We further demonstrated that GCG is able to inhibit cisplatin-induced ATM and Chk2 phosphorylation. Together, our results suggest that GCG may interfere with ATM-Chk2 pathway and hence suppress RAD51 nuclear focus formation and DNA repair activities. Citation Format: Ming-Hsi Wu, Lie-Chwen Lin, Te-Chang Lee. Augmentation of response to therapeutic agents by (-)-gallocatechin gallate through inhibition of RAD51 nuclear translocation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3733.

Abstract 3594: P-glycoprotein attenuates Src activation and DNA repair activity via increased C-terminal Src kinase-binding protein, a negative regulator of Src, in multidrug-resistant cells

Multidrug resistance (MDR) remains a significant obstacle to the success of cancer chemotherapy. MDR is often associated with increased expression of ATP-binding cassette transporter family members, which extrude anticancer drugs out of cells. The MDR1 gene product, P-glycoprotein (P-gp) is one of the most well-known ABC transporters and expels a broad range of anticancer drugs, such as vinblastine, vincristine, doxorubicin and paclitaxel. Overexpression of P-gp in tumor tissues is thus a prognostic indicator associated with poor response to chemotherapy and poor clinical outcome. Our previous study has shown that P-gp overexpression attenuates the repair activity of DNA interstrand crosslinks (ICLs) and hence contributes the susceptibility of MDR cells to DNA ICL agents [1]. In the present study, we explored the molecular mechanisms underlying which P-gp interferes with Src-activated DNA repair activity. We first confirmed that ectopic expression of P-gp attenuates DNA ICL repair activity and becomes more susceptible to DNA ICL agents than vector control cells. Our present results showed that the C-terminal Src kinase-binding protein (Cbp), a negative regulator of Src, is significantly enhanced in MDR cells. Cbp silencing by siRNA technique apparently enhances cisplatin-induced Src activation and DNA repair activity, and hence increases the resistance of MDR cells to ICL agents. By aid of immuneprecipitation and immunofluorescence staining techniques, we demonstrated that P-gp not only interacts with Cbp and Src but also enhances the formation of inhibitory Csk-Cbp complex that reduces phosphorylation of Src activation residue Tyr416 and increases phosphorylation of Src negative regulatory Tyr527 residue. Taken together, the current results indicate that P-gp overexpression in MDR cells attenuates DNA repair activity by mediating Cbp dependent mechanism to interfere with Src activation. These results implicate that increasing the expression of Cbp is potentially a therapeutic strategy to increase the susceptibility of cancer cells to chemotherapeutic agents. 1. Lee PC, Lee HJ, Kakadiya R, Sanjiv K, Su TL, Lee TC: Multidrug-resistant cells overexpressing P-glycoprotein are susceptible to DNA crosslinking agents due to attenuated Src/nuclear EGFR cascade-activated DNA repair activity. Oncogene 2013, 32:1144-1154. Citation Format: Li-Fang Lin, Ming-Hsi Wu, Tsann-Long Su, Te-Chang Lee. P-glycoprotein attenuates Src activation and DNA repair activity via increased C-terminal Src kinase-binding protein, a negative regulator of Src, in multidrug-resistant cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3594. doi:10.1158/1538-7445.AM2015-3594

PI3K Inhibition Augments the Therapeutic Efficacy of a 3a-aza-Cyclopenta[α]indene Derivative in Lung Cancer Cells

The synergistic targeting of DNA damage and DNA repair is a promising strategy for the development of new chemotherapeutic agents for human lung cancer. The DNA interstrand cross-linking agent BO-1509, a derivative of 3a-aza-cyclopenta[α]indene, was synthesized and combined with the phosphoinositide 3-kinase (PI3K) inhibitor LY294002 to treat human lung cancer cells. Our results showed that the BO-1509 and LY294002 combination synergistically killed lung cancer cells in culture and also suppressed the growth of lung cancer xenografts in mice, including those derived from gefitinib-resistant cells. We also found that LY294002 suppressed the induction of several DNA repair proteins by BO-1509 and inhibited the nuclear translocation of Rad51. On the basis of the results of the γH2AX foci formation assays, LY294002 apparently inhibited the repair of DNA damage that was induced by BO-1509. According to the complete blood profile, biochemical enzyme analysis, and histopathologic analysis of major organs, no apparent toxicity was observed in mice treated with BO-1509 alone or in combination with LY294002. Our results suggest that the combination of a DNA cross-linking agent with a PI3K inhibitor is a feasible strategy for the treatment of patients with lung cancer.

Abstract B253: Novel water-soluble phenyl N-mustard-benzenealkylamide conjugate, BO-2094, potent agent for treatment of human colorectal cancer.

Colorectal cancer (CRC) is the second leading cause of cancer related death in the United States and Europe. Surgery, radiotherapy, and chemotherapy are the main strategies for cure of CRC patient. However, the mortality risk associated with CRC is metastasis, which may be diminished by systemic treatments. Therefore, there is an urgent need of finding a better agent to treat CRC. Recently, we have synthesized a series of novel water soluble and chemically stable phenyl N-mustard- benzenealkylamide conjugates, which is consisted of phenyl N-mustard pharmacophore and benzenealkylamide moiety bearing a variety of hydrophilic alkylamide side chains. Of these conjugates, BO-2094 exhibits a broad spectrum of antitumor activity against a panel of human leukemia and solid tumor cell lines in vitro and potent therapeutic efficacy in various tumor xenograft-moles. This agent is able to induce DNA interstrand cross-linking, cell cycle arrest at sub-G1 and G2/M phase, and cell death via apoptosis. Particularly, BO-2094 displays potent antitumor activity in nude mice bearing human colon cancer HCT-116 and H334 xenografts. More than 95% tumor suppression was observes when BO-2094 [30 mg/kg, once per day for 6 consecutive days (QD×6), via intravenous injection (iv. inj.)] was used alone. The combination of BO-2094 and 5-FU at ratios of 1:3 induced synergistic cytotoxicity to HCT-116 cells in vitro. Notable, the growth of HCT-116 xenografts in nude mice was almost completely suppressed (>98% suppression) when co-treated compound BO-2094 (30 mg/kg, QD×6, iv. inj.) with 5-FU [75 mg/kg, every 7 days for 2 doses (Q7D×2), intraperitoneal injection (ip. inj.)]; all mice (n=5) survived on day 35. The results revealed that the combination of BO-2094+5-FU is superior to that of oxaliplatin [15 mg/kg every 3 days for 3 doses (Q3D×3), iv. inj.] and 5-FU [75 mg/kg, Q7D×2, ip. inj.]. It should be noted that 2 of 5 mice died on day 28 when mice were co-treated with oxaliplatin+5-FU, indicating that the combination of BO-2094+5-FU is less toxic to the host. The early preclinical studies of BO-2094 showed that this agent is likely to have no cardiac arrhythmic side effect based on hERG assay and has low toxicity to the drug treated mice based on a 14-day acute iv injection study. The present studies indicate that the combination of compound BO-2094+5-FU has great potential benefit for the treatment of advanced colon cancer. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B253. Citation Format: Tung-Hu Tsai, Satishkumar D. Tala, Tai-Hsin Ou, Yi-Wen Lin, Kiranben S. Tala, Ming-Hsi Wu, Te-Chang Lee, Tsann-Long Su. Novel water-soluble phenyl N-mustard-benzenealkylamide conjugate, BO-2094, potent agent for treatment of human colorectal cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B253.