Harmines inhibit cancer cell growth through coordinated activation of apoptosis and inhibition of autophagy

Harmine is a natural β-carboline alkaloid with diverse biological activities, including reported anti-proliferative effects against a range of human cancers. Earlier in vitro work had suggested that harmine and related compounds might intercalate DNA or inhibit topoisomerases, and harmine derivatives have been reported to affect multiple cellular enzymes and processes such as cyclin/CDK complexes, MAPK signalling, polo-like kinases, protein synthesis initiation and apoptosis. Despite these suggestions, the precise mechanism by which harmines exert cytotoxicity in intact cells remained unclear, and the activation of the canonical DNA damage response had not been systematically evaluated in cell culture for this compound class. Geng and colleagues set out to clarify the cellular mechanism(s) underlying harmine cytotoxicity. By synthesising several novel N2,9-disubstituted harmine analogues and testing them in multiple cancer cell lines and a normal fibroblast line, the investigators aimed to (1) determine whether harmines activate the DNA damage response (using Chk1 phosphorylation as a readout), (2) identify derivatives with improved anti-cancer activity and selectivity, and (3) define the cellular pathways (apoptosis and autophagy) responsible for growth inhibition. The work focuses on a lead compound designated 10f, chosen for its solubility and stability, to probe dose-, time- and mechanism-dependent effects in vitro.

The study used a combination of medicinal chemistry, cell biology and analytical chemistry approaches. Several N2,9-disubstituted harmine derivatives (10a–10h) were synthesised; structural characterisation (1H/13C NMR, HRMS) and purity assessment by HPLC (>95% for compounds with ester/amide groups) are reported with full synthetic details provided in the supplementary methods. Compounds were dissolved in DMSO for cellular assays. Cell culture experiments employed multiple human cancer cell lines: A549 (lung adenocarcinoma), MDA-MB-231 (triple-negative breast carcinoma), PANC-1 (pancreatic cancer) and U2OS (osteosarcoma), together with a normal lung fibroblast line HLFα. Cells were maintained in standard conditions (F12K medium or other indicated media with 10% FBS, 37 °C, 5% CO2). Proliferation/viability was measured by the CCK-8 assay; for IC50 determination, cells were exposed to increasing concentrations of 10f for 48 h and viability normalised to DMSO control, with IC50 values calculated using GraphPad Prism. Data are presented as mean ± SD from five replicates and analysed by ANOVA, with p<0.01 considered significant. Molecular and cell-death assays included Western blotting for apoptosis and autophagy markers (Bax, Bcl2, caspase-3/cleaved caspase-3, PARP, LC3B), using ~40 µg protein per lane and standard SDS–PAGE/PVDF transfer procedures. DNA damage response activation was assessed by monitoring Chk1 phosphorylation (Ser317/Ser345) after treatment with harmine derivatives or positive controls (doxorubicin, camptothecin). Cell cycle and dead-cell quantification used propidium iodide staining and flow cytometry to determine G1/S/G2–M and sub-G1 populations. To measure cellular concentrations and stability of 10f, intracellular levels were quantified by UPLC-Q-TOF-MS after treatment with 5 µM compound at multiple time points; HPLC was used to assess in vitro stability. Where reported, co-treatments included the pan-caspase apoptosis inhibitor Z-VAD-FMK.

Chk1 phosphorylation, the investigators report, was not induced by any of the harmine derivatives when A549 cells were treated at 10 µM for 12 h. In contrast, the positive control doxorubicin produced robust Chk1 phosphorylation at 0.5 µM. Pretreatment with 5 µM harmines for 2 h did not alter Chk1 phosphorylation elicited by 0.5 µM camptothecin, indicating that the harmine analogues neither activated nor modulated the DNA damage response under these conditions. The authors interpret these data as evidence that DNA intercalation or topoisomerase inhibition is unlikely to account for the cellular cytotoxicity of the compounds tested. Growth-inhibition assays across four cancer cell lines showed structure-dependent activity. Treatment with 10 µM parent harmine produced ~20–40% inhibition after 24 h. Several derivatives (10d, 10e) behaved similarly, whereas 10a–10c and 10g–10h lacked activity. The N2,9-disubstituted compound 10f displayed the strongest growth inhibition across A549, MDA-MB-231, PANC-1 and U2OS cells. Calculated IC50 values for 10f were ~3.2 µM in A549 and ~4.5 µM in MDA-MB-231 cells. By contrast, 10f had only minimal growth-inhibitory effect on the normal lung fibroblast HLFα, indicating some cancer cell selectivity. Cell-cycle profiling revealed only a moderate accumulation of A549 cells in S phase after 24 h with 10f, but a notable increase in the sub-G1 population, consistent with cell death. Importantly, significant induction of cell death was detectable at concentrations as low as 100 nM after 24 h. Molecular markers showed that 10f dose- and time-dependently increased pro-apoptotic Bax while reducing anti-apoptotic Bcl2. Levels of cleaved caspase-3 and cleaved PARP rose with treatment, supporting activation of apoptosis. Concurrently, the lipidated form of LC3B (LC3B-II), a commonly used marker for autophagosomes/autophagy, decreased in a time- and dose-dependent manner, consistent with suppression of autophagy. Functional blockade of apoptosis with the pan-caspase inhibitor Z-VAD-FMK nearly abolished 10f-induced cell death, indicating that apoptosis is the primary pathway mediating cytotoxicity. Stability and intracellular pharmacokinetics of 10f were assessed by UPLC-Q-TOF-MS. After treating A549 cells with 5 µM 10f, measured intracellular concentrations at 1, 2, 4, 8, 12 and 24 h were approximately 10.33, 13.01, 10.37, 7.35, 4.40 and 2.76 µM, respectively, indicating an initial accumulation greater than two-fold within the first 4 h followed by gradual decline. HPLC and mass-spectrometric analyses detected little in the way of metabolic products over 24 h, leading the authors to conclude that 10f is relatively stable in cells and that the declining intracellular levels are more likely due to efflux than metabolism.

Geng and colleagues interpret their findings as evidence that harmine derivatives inhibit cancer cell growth via mechanisms distinct from DNA intercalation or topoisomerase inhibition. The lack of Chk1 phosphorylation after harmine treatment, together with the inability of harmines to alter camptothecin-induced Chk1 activation, led the investigators to conclude that activation of the canonical DNA damage response does not underlie the cytotoxicity of the compounds tested. This contrasts with earlier in vitro biochemical data suggesting direct DNA interaction, and the authors note that their cell-based results are consistent with a recent report in which a harmine derivative failed to interact with DNA. The study identifies 10f, an N2,9-disubstituted harmine analogue, as a lead compound that selectively inhibits multiple cancer cell lines while sparing a normal lung fibroblast line. Mechanistically, 10f induces apoptotic cell death—evidenced by upregulation of Bax, cleavage of caspase-3 and PARP and functional rescue by Z-VAD-FMK—while simultaneously suppressing autophagy as indicated by reduced LC3B-II levels. The authors propose that coordinated activation of apoptosis together with inhibition of a survival pathway (autophagy) accounts for the potent cytotoxicity of 10f, including effects observed at low nanomolar concentrations. Key limitations and uncertainties are acknowledged in the extracted text. The authors note that their mass-spectrometric analyses may not detect extremely unstable or very low-abundance metabolites, so the presence of transient metabolic products cannot be entirely excluded. They also recognise variability between their cell-cycle results and prior reports, attributing differences to divergent cell lines or specific derivatives used. Finally, while in vitro stability and cellular accumulation data support further development, these findings are limited to cell-culture conditions and do not yet establish in vivo efficacy or safety. Implications discussed by the investigators include the potential of 10f as a lead for anticancer drug development, given its selectivity, stability in cells and dual action on apoptosis and autophagy. The authors suggest that future work should further explore pharmacokinetics, metabolism and in vivo activity to advance this compound class toward therapeutic applications.