Materials and methods: 3D-mediated inhibition on cell viability was evaluated by MTT and real-time cell proliferation was measured by xCelligence RTDP instrument. Western blotting was used to measure pro-apoptotic, anti-apoptotic proteins and JAK2-STAT3 phosphorylation. Flow cytometry was used to measure ROS production and apoptosis.
Results: Our study revealed that 3D treatment significantly reduced the viability of human CRC cells HT-29 and SW620. Furthermore, 3D treatment induced the generation of reactive oxygen species (ROS) in human CRC cells. Confirming our observation, N-acetylcysteine significantly inhibited apoptosis. This is further evidenced by the induction of p53 and Bax; release of cytochrome c; activation of caspase-9, caspase-7 and caspase-3; and cleavage of PARP in 3D-treated cells. This compound was found to have a significant effect on the inhibition of antiapoptotic proteins Bcl2 and BclxL. The results further demonstrate that 3D inhibits JAK2-STAT3 pathway by decreasing the constitutive and IL-6-induced phosphorylation of STAT3. 3D also decreases STAT3 target genes such as cyclin D1 and survivin. Furthermore, a combination study of 3D with doxorubicin (Dox) also showed more potent effects than single treatment of Dox in the inhibition of cell viability.
Conclusion: Taken together, these findings indicate that 3D induces ROS-mediated apoptosis and inhibits JAK2-STAT3 signaling in CRC.
Methods: Chemical profiling of P. blanda was carried out using gas chromatography mass spectrometry (GCMS) followed by isolation of bioactive compounds by column chromatography. DPPH• and FRAP assays were used to evaluate antioxidant activity and the MTT assay was performed to estimate the cytotoxicity activity against three cancer cell lines, namely MCF-7, HL-60 and WEHI-3, and three normal cell lines, MCF10A, WRL-68 and HDFa.
Results: X-ray crystallographic data for peperomin A is reported for the first time here and N,N'-diphenethyloxamide was isolated for the first time from Peperomia blanda. Methanol and dichloromethane extracts showed high radical scavenging activity with an IC50 of 36.81 ± 0.09 µg/mL, followed by the dichloromethane extract at 61.78 ± 0.02 µg/mL, whereas the weak ferric reducing activity of P. blanda extracts ranging from 162.2 ± 0.80 to 381.5 ± 1.31 µg/mL were recorded. In addition, petroleum ether crude extract exhibited the highest cytotoxic activity against all the tested cancer cell lines with IC50 values of 9.54 ± 0.30, 4.30 ± 0.90 and 5.39 ± 0.34 µg/mL, respectively. Peperomin A and the isolated mixture of phytosterol (stigmasterol and β-sitosterol) exhibited cytotoxic activity against MCF-7 and WE-HI cell lines with an IC50 of (5.58 ± 0.47, 4.62 ± 0.03 µg/mL) and (8.94 ± 0.05, 9.84 ± 0.61 µg/mL), respectively, compared to a standard drug, taxol, that has IC50 values of 3.56 ± 0.34 and 1.90 ± 0.9 µg/mL, respectively.
Conclusion: The activities of P. blanda extracts and isolated compounds recorded in this study underlines the potential that makes this plant a valuable source for further study on anticancer and antioxidant activities.