Elucidation of anticancer efficacy of ent-kaurane diterpenes through structure-activity relationship and mechanism of action studies

  • Md Shahid Sarwar

Student thesis: Doctoral Thesis


Colorectal cancer (CRC) is the third most prevalent and second leading causes of cancer-associated deaths globally. Over the last few decades, ent-kaurane diterpenes have been widely investigated for their anticancer potentials. Flexicaulin A (9) is a naturally occurring ent-kaurane. Previously, our lab modified the structure of 9 by replacing the C-11 hydroxyl group with carbonyl group and obtained a novel compound oxoflexicaulin A (11). However, anticancer activities and mechanistic pathway of these two compounds are yet to be explored. In the current thesis, we evaluated the cytotoxicity of compounds 9 and 11 in A549 lung, A375 melanoma, PANC1 pancreatic, HCT-116 and HT-29 colon cancer and 293T human embryonic kidney cells as well as compared the activity with a number of known natural ent-kauranes to describe their structure-activity relationship. Our study found that the presence of α, β-unsaturated ketone groups in ent-kaurane structure acted as the pharmacophore. The replacement of C-11 hydroxyl group by carbonyl in 9 (IC50: 3.68 µM) gives a novel potent anticancer compound 11 (IC50: 0.77 µM). Considering the novelty and superior activity of 11, its mechanistic pathway was studied in HCT-116 cells and compared with the natural scaffold 9. Flow cytometry analysis by Annexin V/PI staining along with fluorescent staining by DAPI showed that both compounds induced apoptosis in HCT-116 cells. Induction of apoptosis is mediated through up-regulation of tumor suppressor protein p53 and pro-apoptotic protein Bax, Bak and puma as well as promoting the cleavage of PARP while down-regulation of anti-apoptotic protein Bcl-2 and PARP. Apart from their effect on apoptosis, compounds 9 and 11 stimulated the event of senescence, a process of cellular aging, as confirmed by β-galactosidase assay. Induction of senescence is related with up-modulation of p21 and p27 while down-modulation of p16, Rb and its transcription factor E2F1. Moreover, immunofluorescence staining showed translocation of p21 and p27 from the cytoplasm to nucleus after treatment with 9 and 11. Further study found that the two ent-kauranes inhibited the protein level of two NF-κB sub-units p65 and p50 in the nucleus as well suppressed the cytoplasmic level of NF-κB inhibitor IkB-α. Both compounds also inhibited the expression and phosphorylation of STAT3 in the cytoplasm and nucleus, so as for the expression and phosphorylation of Src, an up-stream kinase of STAT3. In xenograft nude mice model, compound 11 remarkably inhibited tumor growths (volume and size) but the body weights of the mice were also reduced (p < 0. 05). Therefore, we designed to synthesis a series of prodrug analogs from 11 by adding acetal protecting group to reduce the toxicity. One of the prodrug (37) significantly attenuated the tumor volume and size (p < 0.05) at 50 mg/kg without any toxicity (p > 0.05). The prodrug 37 is actually released as compound 11 in the mice due to its cleavage in the acidic microenvironment of tumor. Taken together, antitumor effect of compound 11 in CRC model is supposed to be mediated through induction of apoptosis and senescence via modulation of NF-κB and STAT3 signaling pathway. Keywords: Colorectal cancer, ent-kaurane, flexicaulin A, oxoflexicaulin A, cytotoxicity, anticancer, apoptosis, pathway, NF-κB, STAT3.

Date of Award14 Jun 2019
Original languageEnglish
SupervisorHongjie ZHANG (Supervisor)

User-Defined Keywords

  • Cancer
  • Colon (Anatomy)
  • Herbs
  • Materia medica, Vegetable
  • Plant extracts
  • Therapeutic use

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