Reactive oxygen species-mitochondria pathway involved in LYG-202-induced apoptosis in human hepatocellular carcinoma HepG2 cells

doi:10.1016/j.canlet.2010.04.004

Cancer Letters

Volume 296, Issue 1, 1 October 2010, Pages 96-105

https://doi.org/10.1016/j.canlet.2010.04.004 Get rights and content

Abstract

Previously, we demonstrated that LYG-202, a newly synthesized flavonoid with a piperazine substitution, exhibited obvious antitumor activity in vivo and in vitro. The exact mechanism of this new compound remains unclear. In the present study, we examined the effects of LYG-202 on reactive oxygen species (ROS) production and the downstream signaling pathway in the apoptosis of human hepatocellular carcinoma HepG₂ cells. Pretreatment with NAC (N-acetylcysteine), a ROS production inhibitor, partly inhibited the apoptosis induced by LYG-202 via blocking the ROS generation. Further data revealed that LYG-202 induced ROS accumulation followed by a decrease in mitochondrial membrane potential (MMP), release of cytochrome c (Cyt c) and apoptosis-inducing factor (AIF) to cytosol, which induced apoptosis of the cells. Moreover, the mitogen-activated protein kinases (MAPK), the downstream effect of ROS accumulation including c-Jun N-terminal kinase (JNK) and p38 MAPK, could be activated by LYG-202. Taken together, the generation of ROS might play an important role in LYG-202-induced mitochondrial apoptosis pathway, which provided further support for LYG-202 as a novel anticancer therapeutic candidate.

Introduction

LYG-202, a newly synthesized flavonoid with a piperazine substitution (Fig. 1A) that led to a slightly improvement of the water solubility, showed stronger antitumor effects compared with wogonin. We have found that LYG-202 had potent antitumor activity in vivo and in vitro by activating caspase-3, -8, -9, inducing the degradation of PARP and the elevation of the ratio Bax/Bcl-2 and thus triggered the extrinsic apoptosis pathway [1]. But the exact mechanism remains unclearly.

It has been shown that the accumulation of reactive oxygen species (ROS) could cause the loss of mitochondrial membrane potential (MMP) and activate mitogen-activated protein kinases (MAPK) pathways, including the c-Jun N-terminal kinase (JNK) and the p38 pathways [2]. Mitochondria played a major role in apoptosis triggered by many stimuli, including loss of MMP, mitochondrial swelling and release of apoptotic proteins [3]. Alterations of MMP were coupled with the subsequent release Cyt c and AIF from the mitochondrial intermembrane space into cytosol, thereby activating the caspase-cascade system [4], [5].

In this study, production of ROS and downstream pathway in LYG-202-treated cells were investigated. The results indicated that LYG-202 could induce the accumulation of ROS production and a decrease in MMP, triggering the mitochondria-dependent cell apoptotic pathway, and activating MAPK pathways, including the JNK and p38 pathways. (Fig. 6). An increased production of ROS will disrupt the cellular redox homeostasis and provoke damage to cellular components upon oxidative stress [6]. Antioxidant such as reduced glutathione hormone (GSH) and superoxide dismutase have been shown to reduce oxidative stress. We found that the antioxidants: N-acetylcysteine (NAC), a ROS production inhibitor, partially inhibited the LYG-202-induced ROS accumulation and apoptosis, while GSH depletion facilitated ROS accumulation induced by LYG-202 and potentiated the injury of cancer cells. These data offered the proof that ROS might play an important role in LYG-202 induced caner cell apoptosis.

Section snippets

Materials

LYG-202, prepared by Dr. Zhiyu Li (China Pharmaceutical University, China), was applied in DMSO to 10 mM and stored at −20 °C. The concentrations used here were 2, 5 and 8 μM, and freshly diluted with RPMI-1640 to final concentrations. Controls were always treated with the same amount of DMSO (0.1%) as used in the corresponding experiments. MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenytetrazoliumbromide] was obtained from Fluka chemical corp (Ronkonkoma, NY) and was dissolved in 0.01 M PBS. Glucose

LYG-202 induced apoptosis in HepG₂ cells

MTT assays were performed to ascertain the viability inhibitory effect of LYG-202 on the HepG₂ cells. As shown in Fig. 1B, following 24 h treatment, LYG-202 obviously inhibited the viability of HepG₂ cells and the IC₅₀ value was 5.13 ± 0.52 μM. The substantial morphological changes observed in LYG-202-treated HepG₂ cells for 24 h were examined and photographed by the inverted light microscope. Damaged cells became round and shrunken, while the untreated cells retained the normal size and shape. (

Discussion

Although we have demonstrated that LYG-202 was capable of inducing apoptosis via a caspase-dependent way in several human cancer cell lines, the involved exact mechanisms were not well known. Here we indicated that LYG-202-induced apoptosis possibly occurs through a ROS-mitochondrial pathway. Previous investigations have reported that generation of ROS is associated with disruption of MMP, therefore triggering a series of mitochondria-associated events including apoptosis [14]. So the

Conflict of interest

None declared.

Acknowledgments

This work was supported by the International Cooperation Program of China (No. 2008DFA32120), the National Natural Science Foundation of China (No. 30701032), the Natural Science Foundation of Jiangsu Province (No. BK2009298), and the Science and Technology Development Program supported by the division of Science and Technology, Jiangsu (No. BE2009674).

References (27)

S. Zeng et al.
LYG-202, a new flavonoid with a piperazine substitution, shows antitumor effects in vivo and invitro
Biochemical and Biophysical Research Communications
(2009)
T.J. Preston et al.
Mitochondrial contributions to cancer cell physiology: potential for drug development
Advanced Drug Delivery Reviews
(2001)
G.V. Kulkarni et al.
Role of mitochondrial membrane potential in concanavalin A-induced apoptosis in human fibroblasts
Experimental Cell Research
(1998)
Y.H. Jin et al.
Cdk2 activity is associated with depolarization of mitochondrial membrane potential during apoptosis
Biochemical and Biophysical Research Communications
(2003)
B. Friguet
Oxidized protein degradation and repair in ageing and oxidative stress
FEBS Letters
(2006)
M. Wang et al.
Atiprimod inhibits the growth of mantle cell lymphoma in vitro and in vivo and induces apoptosis via activating the mitochondrial pathways
Blood
(2007)
V. Bobyleva et al.
Decrease in mitochondrial energy coupling by thyroid hormones: a physiological effect rather than a pathological hyperthyroidism consequence
FEBS Letters
(1998)
G. Roue et al.
Mitochondrial dysfunction in CD47-mediated caspase-independent cell death: ROS production in the absence of cytochrome c and AIF release
Biochimie
(2003)
N. Gambi et al.
Poly(ADPR)polymerase inhibition and apoptosis induction in cDDP-treated human carcinoma cell lines
Biochemical Pharmacology
(2008)
M. Loeffler et al.
The mitochondrion in cell death control: certainties and incognita
Experimental Cell Research
(2000)

M.T. Park et al.

Phytosphingosine in combination with ionizing radiation enhances apoptotic cell death in radiation-resistant cancer cells through ROS-dependent and -independent AIF release

Blood

(2005)

M.P. Murphy et al.

Superoxide activates uncoupling proteins by generating carbon-centered radicals and initiating lipid peroxidation: studies using a mitochondria-targeted spin trap derived from alpha-phenyl-N-tert-butylnitrone

The Journal of Biological Chemistry

(2003)

W. Chen et al.

Hispolon induces apoptosis in human gastric cancer cells through a ROS-mediated mitochondrial pathway

Free Radical Biology & Medicine

(2008)

Cited by (42)

New quinoxaline-piperazine-oxazole conjugates: Synthesis, in vitro anticancer, in silico ADMET, and molecular docking studies
2024, Journal of Heterocyclic Chemistry
In this paper, we describe the synthesis of some new quinoxaline‐piperazine‐oxazole amide conjugates 6a‐n from 3‐chloroquinoxaline‐2‐carbonitrile using well‐known reaction sequences. The synthesized compounds were characterized by ¹H NMR,¹³C NMR, and mass spectral analysis. The compounds were tested for their in vitro antiproliferative activity toward four different cancer cell lines such as PC‐3, MCF‐7, DU‐145, and A‐549 by MTT method. The compounds, 6c, 6h, 6i, and 6n were found to be more potent than the standard Erlotinib. In vitro tyrosine kinase EGFR inhibition studies using four potent compounds revealed that 6n has double inhibiting tendency with value IC₅₀ of 0.22 μM and 6h with value of IC₅₀ 0.27 μM compared to reference compound. Molecular docking studies of active compounds, 6c, 6h, 6i, and 6n on EGFR receptor suggested that all the compounds have more binding energies than that of Erlotinib. Furthermore, the in silico pharmacokinetic profile was accomplished for the active compounds, 6c, 6h, 6i, and 6n using SWISS/ADME and pk CSM, whereas compounds, 6h, 6i, and 6c followed Lipinski rule, Veber rule, Egan rule and Muegge rule. The remaining compound 6n did not follow Lipinski rule, Ghose rule because one common violation, that is, because of high molecular weight (MW > 350).
An insight into the biological activity and structure-based drug design attributes of sulfonylpiperazine derivatives
2023, Journal of Molecular Structure
The development of new, effective drugs containing nitrogen and sulfur heteroatoms is of paramount importance in medicinal chemistry. A versatile and rational approach for drug design is molecular hybridization (MH) which involves the combination of two pharmacophore molecules possessing distinct intrinsic activity into a single scaffold for enhancing their therapeutic potential. Sulfonylpiperazine hybrids emerged as an attractive scaffold because of their usefulness to target multiple disease areas, simple synthetic pathways, low toxicity, and metabolic stability. This review highlights the applications of various sulfonylpiperazine derivatives in a wide range of therapeutic areas. The structure-activity relationship (SAR) aspects have also been discussed. We anticipate that this review will be useful for medicinal chemists, biochemists, and drug discovery scientists, providing new ideas for the drug design and development of efficacious, selective, and safer drugs against diverse therapeutic areas.
Synthesis, docking, and biological investigations of new coumarin-piperazine hybrids as potential antibacterial and anticancer agents
2023, Journal of Molecular Structure
Citation Excerpt :
Additionally, it is used as a linker or bridge between two active molecules [28]. Piperazine plays a crucial role in bioavailability due to the position of nitrogen, which is essential to enhance the water solubility of the drug-like molecules [29] Piperazine also has diverse biological activities such as antibacterial [30], antidiabetic [31], antitumor [32], anti-Alzheimer [33], antipsychotic [34,35], antifungal [36] and anti-inflammatory [37]. The effect of piperazine-substituted coumarin derivatives on the central nervous system is well documented, similar to antiviral, antibacterial, anticancer, and antioxidant activity [38].
Structurally diversified coumarin analogs were found to display a remarkable array of affinity with the different molecular targets. 4-hydroxy-7-methylcoumarin was synthesized from m-cresol and malonic acid. In basic media, the 4-hydroxy group of coumarin is replaced further by the bromopropoxy group. Several potent piperazine-containing scaffolds are known for their versatility and medicinal significance. A series of novel piperazines possessing coumarin derivatives were synthesized from these 4-bromopropoxycoumarin derivatives. These analogs were evaluated for their antibacterial and anticancer activity. Compound 3 (MIC: 12.5 µg/ml, E. coli) and compound 6 (MIC: 12.5 µg/ml, E. coli, and 25 µg/ml, S. aureus) showed potent activity against bacterial pathogens. Compound 3 showed excellent antibacterial activity against S. aureus with a MIC of 6.25 µg/ml. Compound 6 showed marvelous activity against the MCF-7 cell line with IC₅₀ of 0.003451 µM, which is far better than the reference Doxorubicin. This is also proclaimed by a docking interaction study of these compounds. In a 100-ns MD simulation, slight RMSD variations and a convergent pattern indicate that the compounds 3 and 6 bound protein complexes did not change structurally or conformationally throughout the simulation run. Furthermore, an overall drug-likeness parameter and insilico predicted ADME properties indicated that compounds could be potential leads for the development of drugs.
Novel synthesis naringenin-benzyl piperazine derivatives prevent glioblastoma invasion by inhibiting the hypoxia-induced IL6/JAK2/STAT3 axis and activating caspase-dependent apoptosis
2022, Bioorganic Chemistry
Citation Excerpt :
The piperazine ring is also an important N-heterocyclic piece that is extensively present in many biologically active compounds [25]. Many piperazine derivatives have been reviewed with a broad range of bioactivities, including antitumor [26], antibacterial [27], anti-inflammatory [28], and antioxidant [29] properties. Because of the importance and serious biological activity of the piperazine skeleton, researchers have used piperazines as a potential group for developing drugs [30–32].
Facile synthesis and antimicrobial activity of bis(fused 4H-pyrans) incorporating piperazine as novel hybrid molecules: Michael's addition approach
2022, Journal of Heterocyclic Chemistry
A new series of bis(4H‐chromene), bis (pyrano[3,2‐c]chromene), and bis (pyrano[2,3‐c]pyrazole) each linked to the piperazine core via ether linkages was synthesized. The target compounds are probably formed through a three‐component reaction involving two equivalents of each malononitrile and the corresponding active methylene compounds, such as dimedone, 4‐hydroxycoumarine, or 5‐methyl‐2,4‐dihydro‐3H‐pyrazol‐3‐one. Several spectral methods and elemental analyses were used to deduce the chemical structures of the novel compounds. Furthermore, all synthesized compounds were tested for antimicrobial activity, which revealed promising inhibitory results against a variety of multidrug‐resistant microbial strains. These findings were compared to those obtained in the in silico molecular docking study. The docking results improved the activity of new derivatives as potentially effective antimicrobial agents.
Novel curcumin analogue hybrids: Synthesis and anticancer activity
2018, European Journal of Medicinal Chemistry
Citation Excerpt :
Meanwhile, we have a strong interest in the study of reactive oxygen species. To further determine whether compound 7d was related to the changes of ROS, the NAC, a specific ROS scavenger was pre-incubated at a fixed concentration (5 mM) for 2 h [29–31]. Then, the gastric cancer cells were treated with assured concentrations of compound 7d.
In this study, twenty curcumin analogue hybrids as potential anticancer agents through regulation protein of TrxR were designed and synthesized. Results of anticancer activity showed that 5,7-dimethoxy-3-(3-(2-((1E, 4E)-3-oxo-5-(pyridin-2-yl)penta-1,4-dien-1- yl)phenoxy)propoxy)-2-(3,4,5-trimethoxyphenyl)-4H-chromen-4-one (compound 7d) could induce gastric cancer cells apoptosis by arresting cell cycle, break mitochondria function and inhibit TrxR activity. Meanwhile, western blot revealed that this compound could dramatically up expression of Bax/Bcl-2 ratio and high expression of TrxR oxidation. These results preliminarily show that the important role of ROS mediated activation of ASK1/MAPK signaling pathways by this title compound.

View all citing articles on Scopus

¹: These authors contributed equally to this work.

View full text

Reactive oxygen species-mitochondria pathway involved in LYG-202-induced apoptosis in human hepatocellular carcinoma HepG2 cells

Abstract

Introduction

Section snippets

Materials

LYG-202 induced apoptosis in HepG2 cells

Discussion

Conflict of interest

Acknowledgments

Biochemical and Biophysical Research Communications

Advanced Drug Delivery Reviews

Experimental Cell Research

Biochemical and Biophysical Research Communications

FEBS Letters

Blood

FEBS Letters

Biochimie

Biochemical Pharmacology

Experimental Cell Research

Blood

The Journal of Biological Chemistry

Free Radical Biology & Medicine

Reactive oxygen species-mitochondria pathway involved in LYG-202-induced apoptosis in human hepatocellular carcinoma HepG₂ cells

LYG-202 induced apoptosis in HepG₂ cells