Structure-activity insights of harmine targeting DNA, ROS inducing cytotoxicity with PARP mediated apoptosis against cervical cancer, anti-biofilm formation and in vivo therapeutic study

Abstract

Harmine exhibits pH dependent structural equilibrium and possesses numerous biological and pharmacological activities. Mode and mechanism of DNA binding and its cytotoxicity were studied by multiple spectroscopic, calorimetric, molecular docking and in vitro apoptotic as well as in vivo biochemical and histological studies. It exists as cationic (structure I) and decationic form (structure II) in the pH range 3.0–7.8 and 8.5–12.4, respectively, with a pK_a of 8.0. Structure I at pH 6.8 binds strongly to DNA with a cooperative mode of binding of K_iω 1.03 × 10⁶ M⁻¹and stoichiometry of 5.0 nucleotide phosphates. Structure I stabilized DNA by 10 °C, showed85%quenching of fluorescence intensity, perturbation in circular dichroism, partial intercalation and enthalpy driven exothermic binding. While, structure II at pH 8.5 has very weak interaction with CT DNA. Cytotoxic potencies of structure I was tested on four different cancer cell lines along with normal embryonic cell. It showed maximum cytotoxicity with GI₅₀of 20 µM, against HeLa causing several apoptotic induction abilities. Harmine exhibited G2M arrest with ROS induced effective role in PARP mediated apoptosis as well as anti-inflammatory action on HeLa cells. Harmine further presented MIC and antibiofilm activity against Staphylococcus aureus in presence of <160 and 30 µg/ml, respectively. Mice with post harmine treatment (30 mg/kg b.w., I.P.) showed maximum recovery from damaged to near normal architecture of cervical epithelial cells. This study may be of prospective use in a framework to design novel beta carboline compounds for improved therapeutic applications in future against cervical cancer.

Highlights

• Harmine exists in structure I and structure II forms in the pH 6.8 and 8.5with a pK_a of 8.0.

• Structure I at pH 6.8 binds strongly to DNA compared to structure II.

• Structure I showed maximum cytotoxicity with GI₅₀ of 20 µM against HeLa.

• ROS mediated cytotoxicitywithG2M arrest with PARP mediated apoptosis was studied.

• Harmine (30µg/ml) exhibited antibiofilm activity against Staphylococcus aureus.

• Post harmine dose (30 mg/kg b.w., I.P.) in mice showed recovery of cervical epithelial cells.

Communicated by Ramaswamy H. Sarma

Graphical Abstract

Keywords:

• Harmine
• spectroscopy
• calorimetry
• antibiofilm
• apoptosis
• histology
• cervical cancer

Disclosure statement

No competing financial interests exist. The authors declare no conflict of interest.

Ethics statements

Animal care and all experimental protocols were performed according to the health criteria of laboratory animals and the study was approved and conducted after obtaining Institutional Animal Ethical Committee, University of Kalyani, clearance (Registration no. 892/GO/Re/S/01/CPCSEA, Committee for the Purpose of Control And Supervision of Experiments on Animals; dated 20.04.2014-28.04.2019).

Data availability statement

Data and materials are available upon request.

Additional information

Funding

Authors are grateful to DST-RFBR 2017-19 (DST/INT/RUS/RFBR/P-254) for funding. SS is supported by grants from DST SERB-NPDF (File no. PDF/2017/000398). Authors are also grateful to DST-PURSE, DST-FIST SR/FST/LSI-467/2010C) & PRG, University of Kalyani, 2020-21 for their partial financial support.