Research paper
Hydrogen sulfide releasing oridonin derivatives induce apoptosis through extrinsic and intrinsic pathways

https://doi.org/10.1016/j.ejmech.2019.111978Get rights and content

Highlights

  • 18 H2S donating oridonin derivatives were designed and synthesized.

  • 12b showed sub-micromolar level antiproliferative activity against K562 cells.

  • 12b caused cell cycle arrest both in K562 and HepG2 cells.

  • 12b mainly activated extrinsic and intrinsic apoptosis pathways.

Abstract

Hydrogen sulfide (H2S) has been recognized as the third endogenous signaling gasotransmitter following nitric oxide (NO) and carbon monoxide (CO), and exhibits antiproliferative activity against several cancer cells. In order to stably and controllably release H2S, H2S donating compound (ADT-OH) was used in the present study and 18H2S releasing natural ent-kaurane diterpenoid oridonin derivatives were designed and synthesized. Most derivatives showed more potent antiproliferative activities than oridonin against HepG2 and K562 cell lines, while they were lack of sensitivity to HCT-116 and B16 cells. In particular, 12b showed the most potent antiproliferative activities against HepG2, HCT-116 and K562 cells with IC50 values of 2.57, 5.81 and 0.95 μM, respectively. Through cell cycle analysis, 12b caused cell cycle arrest at S phase in K562 cells and G1 phase in HepG2 cells. In Hoechst 33258 staining assay, cell shrinkage and fragmentation of cell nuclei indicated apoptotic morphological changes. Considering the decline of mitochondrial membrane potential and changes in the levels of apoptosis-related proteins, 12b was shown to induce apoptosis through extrinsic and intrinsic apoptosis pathways.

Introduction

Natural products have made great contributions to drug design. With the development of analytical techniques, more and more active ingredients were isolated and identified from herbal medicines, natural plants and their metabolites. Many novel structural skeletons were provided for drug discovery, and relatively complete quality control systems were also established [[1], [2], [3], [4]]. Compared with combinatorial chemistry, natural products remain active in drug discovery due to their prominent biological activities, complex and diverse structures and distinctive stereogenic centers which could better combine with corresponding targets in vivo [5]. In the past three decades, the percentage of natural products or nature derived molecules has increased to 74% of all approved chemical entities in anticancer domain [6,7].

As a rich source of ent-kaurane diterpenoids (Fig. 1), the genus Rabdosia exhibits many biological activities, including anti-inflammatory, anticancer, antibacterial and antimutagenic activities [[8], [9], [10], [11], [12], [13], [14], [15], [16], [17]]. Oridonin (1, Fig. 1), a natural ent-kaurane diterpenoid from Rabdosia, was first isolated and identified by Fujita et al., in 1970 [18]. Further in-depth studies on pharmacology showed potent anticancer effects t(8; 21) acute myeloid leukemic (AML) cells [19], human pancreatic cancer PANC-1 cells [20], human osteosarcoma cells [21], p53-mutated colorectal cancer cells [22], drug-resistant renal cell carcinoma (RCC) [23] and non-germinal center B cell-like subtype of diffuse large B cell lymphoma (non-GCB DLBCL) [24]. Although oridonin can be obtained commercially and shows cytotoxicity for many cancer cells, it deserves further structural modification due to its poor selectivity and low aqueous solubility [25]. SAR studies indicated that unsaturated cyclopentanone conjugated with extracyclic methylene was a pivotal structure for antiproliferative activity, while the activity slumped when cyclopentanone was cleaved or methylene was saturated [26]. Furthermore, 14-hydroxy of oridonin was a prominent structure modification site. Most 14-O-derivatives showed maintained or better antiproliferative activities toward several human cancer cells [27,28].

Due to the endogenous pathophysiological functions, small molecule donors have comprised a key pillar of drug design [29,30]. Before nitric oxide (NO) and carbon monoxide (CO) were famous for endogenous bioactivity, hydrogen sulfide (H2S) was considered to be a colorless and toxic gas with a strong smell of rotten eggs. In recent years, it has been recognized as the third endogenous signaling gasotransmitter following NO and CO, acting as a neuromodulator and neuroprotective agent [31,32]. Endogenous H2S commonly generates through two specific pyridoxal-5′-phosphate (PLP)-dependent enzymes, cystathionine-γ-lyase (CSE) and cystathionine-β-synthase (CBS), or biosynthesized by the synergistic effects of 3-mercaptopyruvate sulfurtransferase (3-MST) and cysteine aminotransferase (CAT) [[33], [34], [35]]. Increasing evidences confirm the pathophysiological functions of H2S in atherosclerosis, cytoprotection against oxidative stress, angiogenesis, ischemia-reperfusion injury and so on [[36], [37], [38], [39], [40]]. In addition, recent studies demonstrate the importance of H2S in biological processes of cancer which shows antiproliferative activities through EGFR/ERK/MMP-2, PTEN/AKT, PI3K/Akt/mTOR and p38 MAPK/ERK1/2-COX-2 pathways [[41], [42], [43], [44]]. This reactivation of programmed cell death by delivery of H2S could be suggested as an effective approach for cancer therapy. However, H2S is not suitable for clinical applications directly owing to uncontrollability, high toxicity and short half-life. To overcome these disadvantages, a number of H2S releasing agents (H2S donors) have been developed, such as ADT-OH, thiobenzamide, Jks, L-propargyl cysteine, α-thioctic acid, GYY4137, DATS, d-cysteine and so on (Fig. 2A). These H2S donating compounds could deliver H2S in sustained manner and prolong the term of treating times [[45], [46], [47], [48], [49]]. With the development of H2S donors, more and more H2S releasing derivatives have been designed and synthesized. Some of them have entered into Phase I or Phase II clinical trials [ [[50], [51], [52], [53], [54], [55]], Fig. 2C]. Among them, compounds with 3H-1,2-dithiole-3-thione show prominent cytoprotective properties [ [56], Fig. 2B].

In the present study, we designed and synthesized three series of H2S releasing oridonin derivatives in which oridonin was conjugated with a H2S generating moiety (5-(4-hydroxyphenyl)-3H-1,2-dithiole-3-thione, ADT-OH) via different anhydride linkers at 14-hydroxyl group. The antiproliferative activities of all the derivatives were tested against five human cancer cell lines (human hepatoma HepG2, breast cancer MCF-7, colorectal cancer HCT-116, melanoma B16 and chronic myelogenous leukemia K562) and two normal cell lines (human liver L-02 and peripheral blood mononuclear PBMC). Moreover, in-depth apoptosis related mechanisms of the most promising compound 12b which included cell cycle arrest, morphological change, apoptosis induction, mitochondria membrane potentials decline and the expression of apoptosis-related proteins were studied.

Section snippets

Chemistry

The intermediates 2 and 5 were synthesized according to our previous report [57]. The H2S donating derivatives 7a-c were obtained by substitution reactions of ADT-OH 6 with several bromhydrins (2-bromoethanol, 3-bromo-1-propanol and 6-bromo-1-hexanol). By treatment of 1, 2 and 5 with corresponding anhydride in the presence of TEA/DMAP, derivatives 8a-c and 9a-c were got. Derivatives 8a-c and 9a-c were directly reacted with H2S releasing 7a-c via esterification reaction to gain the target

Conclusion

Overall, by using oridonin as lead compound, we designed and synthesized 18H2S-delivering derivatives in the present study. Five tumor and two normal cell lines were selected to test the antiproliferative activities of all derivatives. Most derivatives were sensitive to HepG2 and K562 cells and several compounds showed improved effects than parent compound oridonin. In particular, 12b not only showed the most potent antiproliferative activities against HepG2, HCT-116 and K562 cells with IC50

Chemistry

Chemical materials and reagents were obtained from commercial suppliers, the preparation of anhydrous solvents was based on standard methods. 1H and 13C NMR spectra were determined with Bruker 400 MHz spectrometer in the solvent of CDCl3 (TMS as internal standard): the values of the chemical shifts were reported in δ values (ppm) and the coupling constants (J) in Hz. High resolution mass spectra (HR-MS) were analyzed on Agilent Q-TOF B.05.01 (B5125.2).

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgment

This paper was financially supported by the National Natural Science Foundation of China (21772124, 21502121), Natural Science Foundation of Liaoning Province (20170540858), General Scientific Research Projects of Department of Education in Liaoning Province (2017LQN05), Key Laboratory of Quality Control of TCM of Liaoning Province (17-137-1-00) and Career Development Support Plan for Young and Middle-aged Teachers in Shenyang Pharmaceutical University.

References (64)

  • Y. Zhao et al.

    Light-activated COS/H2S donation from photocaged thiocarbamates

    Org. Lett.

    (2017)
  • M. Chattopadhyay et al.

    NOSH-aspirin (NBS-1120), a novel nitric oxide- and hydrogen sulfide-releasing hybrid is a potent inhibitor of colon cancer cell growth in vitro and in a xenograft mouse model

    Biochem. Biophys. Res. Commun.

    (2012)
  • M. Chattopadhyay et al.

    Hydrogen sulfide-releasing aspirin suppresses NF-κB signaling in estrogen receptor negative breast cancer cells in vitro and in vivo

    Biochem. Pharmacol.

    (2012)
  • B. Szczesny et al.

    AP39, a novel mitochondria-targeted hydrogen sulfide donor, stimulates cellular bioenergetics, exerts cytoprotective effects and protects against the loss of mitochondrial DNA integrity in oxidatively stressed endothelial cells in vitro

    Nitric Oxide

    (2014)
  • H. Li et al.

    Hydrogen sulfide donating ent-kaurane and spirolactone-type 6,7-seco-ent-kaurane derivatives: design, synthesis and antiproliferative properties

    Eur. J. Med. Chem.

    (2019)
  • X. Hu et al.

    Antiproliferative hydrogen sulfide releasing evodiamine derivatives and their apoptosis inducing properties

    Eur. J. Med. Chem.

    (2018)
  • T. Han et al.

    Scutellarin derivatives as apoptosis inducers: design, synthesis and biological evaluation

    Eur. J. Med. Chem.

    (2017)
  • C. Sun et al.

    Utilizing cell-based therapeutics to overcome immune evasion in hematologic malignancies

    Blood

    (2016)
  • T. Han et al.

    Novel hybrids of brefeldin A and nitrogen mustards with improved antiproliferative selectivity: design, synthesis and antitumor biological evaluation

    Eur. J. Med. Chem.

    (2018)
  • J. Wu et al.

    Analytical techniques and pharmacokinetics of gastrodia elata blume and its constituents

    Molecules

    (2017)
  • Z. Zhang et al.

    Chemical composition database establishment and metabolite profiling analysis of Yangyin qingfei decoction

    Biomed. Chromatogr.

    (2019)
  • C. Tang et al.

    Novel strategies using total gastrodin and gastrodigenin, or total gastrodigenin for quality control of gastrodia elata

    Molecules

    (2018)
  • T. Rodrigues et al.

    Counting on natural products for drug design

    Nat. Chem.

    (2016)
  • D.J. Newman et al.

    Natural products as sources of new drugs from 1981 to 2014

    J. Nat. Prod.

    (2016)
  • K. Osawa et al.

    An investigation of diterpenes from the leaves of Rabdosia trichocarpa and their antibacterial activity against oral microorganisms

    Chem. Pharm. Bull.

    (1994)
  • S.S. Hong et al.

    ent-Kaurane diterpenoids from Isodon japonicus

    J. Nat. Prod.

    (2008)
  • S. Aquila et al.

    Inhibition of NF-κB activation and iNOS induction by ent-kaurane diterpenoids in LPS-stimulated RAW264.7 murine macrophages

    J. Nat. Prod.

    (2009)
  • X. Luo et al.

    Cytotoxic ent-kaurane diterpenoids from Isodon rubescens var. lushiensis

    J. Nat. Prod.

    (2010)
  • H.B. Zhang et al.

    Four new ent-kauranoids from Isodon rubescens var. lushanensis and data reassignment of dayecrystal B

    Chem. Pharm. Bull.

    (2010)
  • W. Zhao et al.

    Structure and cytotoxicity of diterpenoids from Isodon adenolomus

    J. Nat. Prod.

    (2011)
  • R. Zhan et al.

    Bioactive ent-kaurane diterpenoids from Isodon rosthornii

    J. Nat. Prod.

    (2013)
  • H.D. Sun et al.

    Diterpenoids from Isodon species and their biological activities

    Nat. Prod. Rep.

    (2006)
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