Basic Study
Copyright ©The Author(s) 2023. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastrointest Oncol. May 15, 2023; 15(5): 810-827
Published online May 15, 2023. doi: 10.4251/wjgo.v15.i5.810
BZD9L1 benzimidazole analogue hampers colorectal tumor progression by impeding angiogenesis
Chern Ein Oon, Ayappa V Subramaniam, Lik Yang Ooi, Ashwaq Hamid Salem Yehya, Yeuan Ting Lee, Gurjeet Kaur, Sreenivasan Sasidharan, Beiying Qiu, Xiaomeng Wang
Chern Ein Oon, Ayappa V Subramaniam, Lik Yang Ooi, Yeuan Ting Lee, Gurjeet Kaur, Sreenivasan Sasidharan, Institute for Research in Molecular Medicine, Universiti Sains Malaysia, Penang 11800, Malaysia
Ashwaq Hamid Salem Yehya, Cancer Research, Eman Biodiscoveries, Kedah 08000, Malaysia
Ashwaq Hamid Salem Yehya, Vatche and Tamar Division of Digestive Diseases, Department of Medicine, David Geffen School of Medicine at University of California Los Angeles, Los Angeles, CA 90095, United States
Beiying Qiu, Xiaomeng Wang, Academic Clinical Program, Duke-NUS Medical School, National University of Singapore, Singapore 169857, Singapore
Beiying Qiu, Singapore National Eye Centre, Singapore Eye Research Institute, Singapore 168751, Singapore
Xiaomeng Wang, Singapore National Eye Centre, Singapore Eye Research Institute, Singapore 169857, Singapore
Author contributions: Oon CE and Subramaniam AV remain to have equal contributions as co-first authors; Oon CE, Subramaniam AV, Ooi LY, and Qiu B carried out the experiments; Lee YT synthesized the BZD9L1 compound; Oon CE drafted and reviewed the manuscript; Subramaniam AV and Ooi LY provided scientific input for “Discussion”; Oon CE, Subramaniam AV, Ooi LY, Qiu B, and Kaur G analyzed the data; Sasidharan S provided scientific input for in vivo study; Wang X derived the ex vivo angiogenesis models; Oon CE conceived and designed the experiments, and supervised the project; and all the authors have read and approved the final manuscript.
Supported by the Ministry of Higher Education Malaysia for the Fundamental Research Grant Scheme, No. FRGS/1/2021/SKK06/USM/02/7.
Institutional animal care and use committee statement: All procedures involving animals were reviewed and approved by the National University of Singapore Institutional Animal Care and Use Committee guidelines (approval No. 2020/SHS/1597) and Universiti Sains Malaysia Animal Ethical Committee [approval No. USM/IACUC/2017/(105)(872)].
Conflict-of-interest statement: The authors declare that they have no conflict of interest.
Data sharing statement: No additional data are available.
ARRIVE guidelines statement: The authors have read the ARRIVE Guidelines, and the manuscript was prepared and revised according to the ARRIVE Guidelines.
Open-Access: This article is an open-access article that was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution NonCommercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See:
Corresponding author: Chern Ein Oon, DPhil, PhD, Associate Professor, Institute for Research in Molecular Medicine, Universiti Sains Malaysia, Gelugor, Penang 11800, Malaysia.
Received: January 3, 2023
Peer-review started: January 3, 2023
First decision: February 13, 2023
Revised: February 17, 2023
Accepted: April 21, 2023
Article in press: April 21, 2023
Published online: May 15, 2023

The development of new vasculatures (angiogenesis) is indispensable in supplying oxygen and nutrients to fuel tumor growth. Epigenetic dysregulation in the tumor vasculature is critical to colorectal cancer (CRC) progression. Sirtuin (SIRT) enzymes are highly expressed in blood vessels. BZD9L1 benzimidazole analogue is a SIRT 1 and 2 inhibitor with reported anticancer activities in CRC. However, its role has yet to be explored in CRC tumor angiogenesis.


To investigate the anti-angiogenic potential of BZD9L1 on endothelial cells (EC) in vitro, ex vivo and in HCT116 CRC xenograft in vivo models.


EA.hy926 EC were treated with half inhibitory concentration (IC50) (2.5 μM), IC50 (5.0 μM), and double IC50 (10.0 μM) of BZD9L1 and assessed for cell proliferation, adhesion and SIRT 1 and 2 protein expression. Next, 2.5 μM and 5.0 μM of BZD9L1 were employed in downstream in vitro assays, including cell cycle, cell death and sprouting in EC. The effect of BZD9L1 on cell adhesion molecules and SIRT 1 and 2 were assessed via real-time quantitative polymerase chain reaction (qPCR). The growth factors secreted by EC post-treatment were evaluated using the Quantibody Human Angiogenesis Array. Indirect co-culture with HCT116 CRC cells was performed to investigate the impact of growth factors modulated by BZD9L1-treated EC on CRC. The effect of BZD9L1 on sprouting impediment and vessel regression was determined using mouse choroids. HCT116 cells were also injected subcutaneously into nude mice and analyzed for the outcome of BZD9L1 on tumor necrosis, Ki67 protein expression indicative of proliferation, cluster of differentiation 31 (CD31) and CD34 EC markers, and SIRT 1 and 2 genes via hematoxylin and eosin, immunohistochemistry and qPCR, respectively.


BZD9L1 impeded EC proliferation, adhesion, and spheroid sprouting through the downregulation of intercellular adhesion molecule 1, vascular endothelial cadherin, integrin-alpha V, SIRT1 and SIRT2 genes. The compound also arrested the cells at G1 phase and induced apoptosis in the EC. In mouse choroids, BZD9L1 inhibited sprouting and regressed sprouting vessels compared to the negative control. Compared to the negative control, the compound also reduced the protein levels of angiogenin, basic fibroblast growth factor, platelet-derived growth factor and placental growth factor, which then inhibited HCT116 CRC spheroid invasion in co-culture. In addition, a significant reduction in CRC tumor growth was noted alongside the downregulation of human SIRT1 (hSIRT1), hSIRT2, CD31, and CD34 EC markers and murine SIRT2 gene, while the murine SIRT1 gene remained unaffected, compared to vehicle control. Histology analyses revealed that BZD9L1 at low (50 mg/kg) and high (250 mg/kg) doses reduced Ki-67 protein expression, while BZD9L1 at the high dose diminished tumor necrosis compared to vehicle control.


These results highlighted the anti-angiogenic potential of BZD9L1 to reduce CRC tumor progression. Furthermore, together with previous anticancer findings, this study provides valuable insights into the potential of BZD9L1 to co-target CRC tumor vasculatures and cancer cells via SIRT1 and/or SIRT2 down-regulation to improve the therapeutic outcome.

Keywords: Colorectal cancer, BZD9L1, Sirtuin, Benzimidazole, Angiogenesis

Core Tip: BZD9L1 hampered EA.hy926 endothelial cell functions through cell cycle arrest and induction of apoptosis. BZD9L1 also reduced the cell adhesion, sirtuin 1 (SIRT1) and SIRT2 gene expression in endothelial cells (EC) compared to the negative control. The compound down-regulated angiogenin, basic fibroblast growth factor, platelet-derived growth factor, and placental growth factor proteins in EC and impeded HCT116 colorectal cancer (CRC) invasion compared to the negative control group. BZD9L1 negatively impacted choroidal sprouting and CRC tumor angiogenesis in vivo compared to the vehicle control group. BZD9L1 reduced tumor necrosis, Ki-67 proliferation marker, hSIRT1, hSIRT2, murine cluster of differentiation 31 (mCD31), mCD34 and murine SIRT2 (mSIRT2) gene expression compared to vehicle control. Findings from this study may provide insights for the BZD9L1 benzimidazole analogue to be further explored as a potential anti-angiogenic agent in CRC.