As other camptothecin analogs, TP has shown a valuable antitumor effect in different cancer types clinically with the presence of some serious toxicities and side effects especially when used in high doses.1,12 Over years of use, many cancer types developed resistance against TP leading to advanced stages of malignancies and relapses that required shifting toward other chemotherapeutic derivatives.27 However, due to its known efficacy, studies were directed to the application of TP in combination with different agents in order to study possible synergistic effects and decrease of TP toxicities.3,28 Treating U937 with gemcitabine prior to TP addition showed significant synergism at a lower IC50.29 Similarly, cell growth inhibition appeared to increase when combining genistein, a natural derivative of soy, with TP treatment in prostate cancer cells.28 In addition, andrographolide inhibited the growth of T-cell acute lymphoblastic leukemia cells as its analog did in U937 cells.30,31
The combination of TP with shinokin that is also a topoisomerase I inhibitor promoted cell-cycle arrest in G0/G1 and S phases with the induction of apoptosis.3 Austrobailignan-1, another topoisomerase I inhibitor, retarded the cells upon treatment in G2/M phase in non-small-cell lung cancer cells, while the camptothecin derivative SN-38 arrested the cells of the same cancer type in the S- and G2 phases in vivo and in vitro.32,33 On the other hand, andrographolide induced cell-cycle arrest at G2/M phase in HepG2 cells.34 The present study shows an S phase arrest in the cells treated with only TP, which is consistent with the predominant S phase arrest effect of many camptothecin derivatives.27,33 This S phase arrest appears to be increased around twofold upon the combination of TP and andrographolide.
Different camptothecin derivatives, including TP, are known to induce apoptosis in different cancer types through various metabolic pathways as described by Maurya et al.33 TP had shown to exert its proapoptotic effects through p53 in some cancer types while it depends on an intrinsic pathway that involves caspases in others.32,35 Even though the possible role of p53 in causing apoptosis has been reported in many studies, its specific mechanism is still unclear, whereby it has led to resistance in some cell lines.35,36 p53 upregulation was also observed in Jurkat cells and melanoma cells upon treatment with andrographolide.30,37 Furthermore, topoisomerase I inhibitors have been proven to induce apoptosis by activation of caspase-2, -3, and -9 and PARP-inducing apoptosis.32,33Andrographolide also induced apoptosis through the release of cytochrome c, cleavage of PARP, increase in the level of Bax, and activation of caspase-3, -8, and -9 in rheumatoid arthritis fibroblast-like synoviocytes and in lymphoma cells.38,39 Combining andrographolide with cisplatin showed an increase in the percentage of apoptotic cell death in ovarian cancer cells. This is similar to what is reported in the current study after the combination of andrographolide with TP on U937.40 According to Uckun et al (1995),41 expression of Bcl-2 was found to increase upon combination of TP with andrographolide, even though TP-induced apoptosis does necessarily require a decrease in bcl-2 expression. Pretreating the U937 cells with andrographolide prior to TP addition resulted in a significant increase in apoptosis through the upregulation of many proapoptotic proteins’ expression and was confirmed with Annexin/PI staining that showed an increase in the percentage of apoptotic cells rather than necrotic cells.
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. The synergistic effects of this combination appeared to involve more intrinsic mitochondrial pathways including the upregulation of Bax, cleavage of PARP with the release of cytochrome c, and the cleavage of different caspases into their active forms, especially caspase-3 and -9.
The new combination can be used as a new clinical strategy in chemotherapy to treat AML and improve TP treatment results with lower toxicities. Moreover, it might be used as an option in the treatment of other resistant cancer types after further studies and investigations.
This work was supported and funded by the School Research and Development Council of the Lebanese American University (SRDC-r-2016-6).
The authors report no conflicts of interest in this work.
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