Caspase-dependent apoptosis induced by 5,6-dihydroxy-2,4-dihydrophenanthrene in human lung adenocarcinoma A549 cells

5,6-dihydroxy-2,4-dimethoxy-9,10-dihydrophenanthrene (HMP) is an active compound isolated from the rhizome extract of Dioscorea membranacea Pierre, a Thai medicinal plant that has been traditionally used in cancer treatment [1]. This research aimed to study the cytotoxic effect of HMP and to further analyze its apoptosis-inducing capacity in A549 cells. The cytotoxicity studies using the sulforhodamine B assay revealed that HMP exerted the potent cytotoxic activity against A549 cells with 50% growth inhibition (IC₅₀), total growth inhibition (TGI), and 50% lethal concentration (LC₅₀) values of 9.22 ± 1.26, 56.86 ± 3.99, and 93.85 ± 2.50µM, respectively. Moreover, cell cycle analysis using flow cytometry showed that HMP at 50µM (nearly the TGI value) and 25µM (the half TGI value) significantly increased the sub-G₁ peak (apoptotic cells) ranging from 26.51 – 32.90% after 48 and 72h, along with the decreased percentage of G₂/M-phase cells. Using FITC-labeled Annexin V and PI staining followed by flow cytometry, the time-dependent effect of HMP at 25 and 50µM on the percentage of early and late apoptotic cells was observed in A549 cells, with approximately 36% and 41% of these early and late apoptotic cells combined at 72h, respectively. Treatment with 25µM HMP for 24, 48, and 72h resulted in changes of caspase-3 activity that reached its highest level at 48h. Such activity was completely abolished with pretreatment of 50µM Z-VAD-fmk (an irreversible pan-caspase inhibitor) for 6h prior to HMP incubation. Additionally, Western blot analysis detected the active form of caspase-3 and its well-established substrate, PARP. Remarkably, HMP-treated cells exhibited chromatin condensation and nuclear fragmentation, the terminal events of apoptosis, using fluorescence microscopy and gel electrophoresis, respectively. Altogether, these data indicate that HMP induced caspase-dependent apoptosis and support its anticancer potential for lung cancer treatment.

[1] Itharat A, Thongdeeying P, Ruangnoo S. J. Ethnopharmacol 2014.