(M1130-03-18) Development of Multitargeted Inhibitor, IND-2, for Prostate Cancer Therapy

Purpose: Multitarget inhibitors offer tremendous potential for cancer treatment since single-target agents are susceptible to rapid resistance due to mutations and redundancy of pathways. For cancers like prostate cancer, multitargeted agents, which are still the mainstay of treatments, can simultaneously reduce resistance, disrupt multiple survival pathways, and enhance their efficacy when used in combination. We have identified a novel pyrimido-pyrazolo-quinolone derivative, IND-2, that is highly drug like and induced DNA damage and was found to be effective against prostate cancer cells. The purpose of this study is to understand its unique mechanism and understand as how it simultaneously targets multiple oncogenic pathways crucial for prostate cancer progression. Additionally, we aim to determine if combining this compound with single target and multi-target agents can overcome drug resistance in prostate cancer cells.

Methods: We have optimized and have synthesized IND-2 in gram scale. We assessed IND-2 for anti-proliferative activity against normal and resistant prostate cancer cells using cell cytotoxicity assays, live cell imaging and colony formation studies. Mechanism of action was investigated through morphological analysis, cell cycle profiling, DNA damage assessment (γH2AX), mitochondrial outer membrane permeabilization (MOMP), reactive oxygen species (ROS) measurement, and apoptosis detection. Migration potential was evaluated using wound healing assays. Target validation studies were conducted using DNA decatenation assays, and proteasome inhibition experiments to confirm topoisomerase II inhibition.

Results: IND-2 significantly inhibited cell proliferation and colony formation, with IC_₅₀ values in the low micromolar range. Time-dependent reduction in confluence and morphological alterations—characterized by multinucleation and cellular swelling—were indicative of mitotic catastrophe. γH2AX foci formation, a hallmark of DNA double-strand breaks and replication stress, was markedly increased as observed by immunofluorescence imaging, particularly in cells treated with IND-2 (2.5 μM) and VP-16 (50 μM), indicating nuclear accumulation of DNA damage. These observations were further substantiated by γH2AX protein upregulation on Western blot. Comet assay tail elongation and DNA laddering confirmed extensive DNA fragmentation. ROS levels were significantly elevated, accompanied by mitochondrial outer membrane permeabilization (MOMP), caspase-3 activation, and PARP cleavage, confirming oxidative stress–mediated intrinsic apoptosis. IND-2 also inhibited cell migration and caused cell cycle arrest. Mechanistically, IND-2 acted as a topoisomerase II poison with preferential inhibition of the β isoform. Immunofluorescence analysis revealed altered nuclear localization of TOP2A and TOP2B, and IND-2 treatment prevented their nuclear export. This topoisomerase II targeting was further supported by decatenation inhibition assays and comparative analysis with VP-16.

Conclusion: These findings establish IND-2 as a novel multitargeted small molecule that effectively disrupts proliferative and survival mechanisms in prostate cancer cells. By functioning as a topoisomerase II poison—preferentially targeting the β isoform—while concurrently inducing oxidative stress and apoptosis, IND-2 inflicts sustained genotoxic stress as confirmed by γH2AX expression both via immunofluorescence and Western blot. Its ability to modulate nuclear export of Topo II isoforms and impair DNA integrity underscores its potential as a mechanistically unique agent

References: Balaji, S., et al. "Alternative approaches to overcome chemoresistance to apoptosis in cancer." Advances in Protein Chemistry and Structural Biology 126 (2021): 91-122.
Balaji, S., et al. "IND-2, a quinoline derivative, inhibits the proliferation of prostate cancer cells by inducing oxidative stress, apoptosis and inhibiting topoisomerase II." Life 12.11 (2022): 1879.
Balaji, S., et al. "Development of novel pyrimido-pyrazolo-quinolone derivative, IND-2 as multi-targeted inhibitor for the treatment of prostate cancer." Cancer Research 83.7_Supplement (2023): 4974-4974.
Karthikeyan, C., et al. "Design, synthesis and biological evaluation of novel 4-aryl-2,6-dimethyl-3,5-bis-N-(aryl)-carbamoyl-1,4-dihydropyridines as multidrug resistance modifiers." Bioorganic & Medicinal Chemistry 23 (2015): 3911-3920.
Zhang, A., et al. "A protease pathway for the repair of topoisomerase II-DNA covalent complexes." Journal of Biological Chemistry 281.47 (2006): 35997-36003.

Acknowledgements: Funding
This work is partly supported by Winthrop P. Rockefeller Cancer Institute funds (CC103501/FD208) and Arkansas Bioscience Institute Funds (GR020025/FD224) to A.K.T.


Figure 1: The effect of (a) IND2 on the proliferation of (b) DU145 and PC3. (c) IC50 values for PC3 and DU145 cells. The results are represented as the means ± SEM of three independent experiments performed in triplicate. (d) The effect of IND-2 (2.5 and 10 µM) on the colony formation rate in PC3 cells. (e) Morphological changes induced by IND-2 on PC3 cells after incubation with vehicle and IND-2 (5 and 10 µM) for 0, 48 and 72 h. Biotek Cytation 7 was used to capture the images. (f) A representative confluence curve for PC-3 cells incubated with vehicle and IND-2 (0, 1.25, 2.5, 5 or 10 µM).


Figure 2: IND-2 induces DNA damage and apoptosis in prostate cancer cells. This comprehensive figure demonstrates the anticancer mechanism of IND-2, showing: (a) immunofluorescence of γH2AX foci (green) indicating DNA double-strand breaks in IND-2 treated cells compared to control and VP-16; (b) brightfield and DCF imaging revealing increased reactive oxygen species production with IND-2 treatment; (c) quantification of mean fluorescence intensity in DU-145 cells showing significant ROS elevation with IND-2; (d) comet assay visualizing DNA fragmentation in treated cells with (e) corresponding quantification of tail moment; (f) DNA decatenation assay demonstrating IND-2's inhibition of topoisomerase II activity; and (g-h) western blot analysis with quantification showing activation of apoptotic pathways after IND-2 treatment. Collectively, these results establish IND-2 as a promising anticancer compound that targets multiple cellular pathways including topoisomerase II inhibition, oxidative stress induction, DNA damage, and apoptosis activation in prostate cancer cells.




Figure 3. IND-2 selectively inhibits Topoisomerase II and induces DNA damage signaling. This figure demonstrates IND-2's mechanism as a topoisomerase II inhibitor in prostate cancer cells: (a) Decatenation assay showing IND-2's dose-dependent inhibition of topoisomerase II activity with various controls, highlighted by the red dashed box indicating effective inhibitory concentrations; (b) Western blot analysis of TOP2A and TOP2B expression in response to IND-2 (1, 5 μM) and VP-16 (50 μM) treatments with quantification graphs showing differential effects on isoforms; (c) Immunofluorescence imaging showing nuclear localization and distribution of TOP2A (left panels) and TOP2B (middle panels) with Hoechst nuclear staining (blue) under control, IND-2 (2.5 μM), and VP-16 (50 μM) conditions, along with γH2AX foci formation (right panels) indicating DNA double-strand breaks; (d) Western blot confirmation of γH2AX induction with corresponding quantification showing dose-dependent increases with IND-2 treatment (5 μM) comparable to VP-16 (50 μM). Together, these results establish IND-2 as a topoisomerase II poison with preference for the β isoform that induces DNA damage signaling, supporting its development as a novel therapeutic agent for prostate cancer.