Background: Topotecan (TP) is an anticancer drug acting as topoisomerase I inhibitor that is used in the treatment of many types of cancers including leukemia, but it has significant side effects. Andrographolide, a compound extracted from Andrographis paniculata, was recently proven to inhibit the growth of cancer cells and can induce apoptosis. The aim of this study is to investigate the possible synergism between TP and andrographolide in acute myeloid cells in vitro.
Materials and methods: U937 acute myeloid leukemic cells were cultured using Roswell Park Memorial Institute (RPMI) medium and then treated for 24 h with TP and andrographolide prepared through the dilution of dimethyl sulfoxide (DMSO) stocks with RPMI on the day of treatment. Cell proliferation was assessed using cell proliferation assay upon treatment with both compounds separately and in combination. Cell-cycle study and apoptosis detection were performed by staining the cells with propidium iodide (PI) stain and Annexin V/PI stain, respectively, followed by flow cytometry analysis. Western blotting was used to assess the expression of various proteins involved in apoptotic pathways.
Results: Both TP and andrographolide showed an antiproliferative effect in a dose-dependent manner when applied on U937 cells separately; however, pretreating the cells with andrographolide before applying TP exhibited a synergistic effect with lower inhibitory concentrations (half-maximal inhibitory concentration). Treating the cells with TP alone led to specific cell-cycle arrest at S phase that was more prominent upon pretreatment combination with andrographolide. Using Annexin V/PI staining to assess the proapoptotic effect following the pretreatment combination showed an increase in the number of apoptotic cells, which was supported by the Western blot results that manifested an upregulation of several proapoptotic proteins expression.
Conclusion: The pretreatment of U937 with andrographolide followed by low doses of TP showed an enhancement in inducing apoptosis when compared to the application of each compound separately.
Keywords: topotecan, andrographolide, Andrographis paniculata, acute myeloid leukemia, apoptosis
Topotecan (TP) is an anticancer drug currently used in the treatment of many types of cancers, including leukemia.1,2 Being a camptothecin analog, TP acts as a topoisomerase I inhibitor by interfering with the enzymatic repair of nuclear DNA, thereby inducing cell death. Studies have shown the antiproliferative effect of TP on glioma cells, small cell lung cancer, prostate cancer cells, platinum-resistant ovarian cancers, neuroblastoma cells, and leukemic cells.3–7
However, TP exhibits several significant side effects, including hematologic toxicities like thrombocytopenia, anemia, and neutropenia, as well as nonhematologic toxicities such as fatigue and alopecia.8–11
To enhance the adequacy of TP use and addressing to its toxicity, different approaches have been considered. One of the ways is developing various formulations and methods of administration of TP. A novel approach is using the nanocarrier liposome to promote targeted delivery of TP.12 Liposomal TP has improved the anticancerous efficacy of TP in Kunming mice with Hepatoma-22 tumor as well as in patients with neuroblastoma.13,14
Another method in improving TP’s efficacy is using it as an adjunct with radiotherapy, providing satisfactory results in targeting prostate cancer, small-cell lung carcinoma, recurrent primitive neuroectodermal tumors, recurrent platinum-resistant ovarian cancer, and cervical cancer.15–18
A more popular approach that both enhances the potency of TP and addresses its cytotoxicity is combination therapy. TP treatment has shown synergistic effects when combined with gemcitabin, gefitinib, and thymoquinone.19–21 As the combination of treatments involving TP have shown satisfactory and promising outcomes, further research has been directed to establish effective therapies involving TP and significant chemotherapeutic agents. TP, when administered with paclitaxel (Taxol) and bevacizumab (Avastin) in combination, has been proven to improve the overall survival and progression-free survival in patients with recurrent cervical small-cell neuroendocrine carcinoma as described by Frumovitz et al.22
Another study tested pre exposure therapy on neuroblastoma cell lines with various inhibitors 24 h before treating with TP. Results showed a significant decrease in cell growth with three inhibitors (13-197, BI2536, and vismodegib) and TP as a single agent. When tested in vivo, the combined efficacy of vismodegib and TP resulted in a significant reduction of tumor size and increased survival of NSG mice. Interestingly, Western blots revealed that although TP alone has no effect on the expression of phosphorylated p65 (nuclear factor kappa B [NF-κB]) and S6K (mammalian target of rapamycin), in combination with the three inhibitors the expression levels of the phosphorylated p65 and S6K were significantly decreased in MYCN-amplified cells as stated by Chaturvedi et al.23
Several plant-derived compounds have shown significant anticancer effects; andrographolide, a compound extracted from Andrographis paniculata, was recently proven to inhibit the growth of cancer cells through attenuating the NF-κB pathway.24,25 Andrographolide was also reported to induce apoptosis and autophagy in U937 (acute myeloid leukemia [AML]) cells, leading to cell death.26
This led us to investigate the possible synergism between TP and andrographolide in vitro on acute myeloid cells, namely, U937. The present study aims to explore the possibility of enhancing the effect of low doses of TP in controlling cancer cells’ progression through administering them with different dosing schedules of andrographolide and to study the pathway involved in promoting cell death upon combination of both drugs.