LYG-202, a new flavonoid with a piperazine substitution, shows antitumor effects in vivo and in vitro

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Abstract

LYG-202 is a newly synthesized flavonoid with a piperazine substitution. We investigated the antitumor effect of LYG-202 in vivo and in vitro. We show that, LYG-202 significantly decreases tumor growth in mice inoculated with S180 sarcoma cells, compared with the control group. Meanwhile, the viabilities of various kinds of tumor cells were inhibited by LYG-202 with IC50 values in the range of 4.80 to 27.70 μM. Then apoptosis induced by LYG-202 in HepG2 cells was characterized by DAPI staining and Annexin V/PI double staining and degradation of PARP was observed. Activation of the caspase cascade for both the extrinsic and intrinsic pathways was demonstrated, including caspase-8, -9, and -3. The results also showed that the expression of Bcl-2 protein decreased whereas that of Bax protein increased, leading to an increase of the Bax/Bcl-2 ratio. Our results demonstrated that LYG-202 exhibited strong antitumor effect in vivo and in vitro, involving with apoptosis induction.

Introduction

Apoptosis, or programmed cell death, is a mechanism by which cells undergo death to control cell proliferation or in response to DNA damage [1], [2]. The understanding of apoptosis has provided the basis for novel targeted therapies that can induce death in cancer cells or sensitize them to established cytotoxic agents and radiation therapy [3], [4], [5].

Tumor cells do not undergo apoptosis easily because they have defects in their ability to activate the death signaling pathway. Thus, one effective cancer therapy is to activate the tumor cell’s apoptosis pathway [6], [7]. There are at least two signaling pathways that are involved in apoptosis, the extrinsic and the intrinsic pathway. The extrinsic pathway is activated by ligand-bound death receptors of the tumor necrosis factor receptor superfamily. The intrinsic pathway is a signal transduction pathway involving the mitochondria and the Bcl-2 family [7], [8], [9].

Flavonoids are nearly ubiquitous in plants. They are low molecular weight compounds composed of a three-ring structure with various substitutions [10]. Many flavonoids possess anti-tumor activity against various human cancer cell lines and xenograft systems of human tumors, suggesting that they may be promising anticancer agents [11], [12].

LYG-202 is a newly synthesized flavonoid with a piperazine substitution (Fig. 1A) and this led to a slightly improvement of the water solubility as most of the flavonoid family members are not water soluble. Compared with other flavonoids, LYG-202 shows stronger anti-tumor effects in our early experiments of screening sufficient anti-tumor drugs. In this study, we demonstrated that LYG-202 exhibited antitumor effect in vivo and in vitro, and further study showed that apoptosis induction of LYG-202 involved in its antitumor effects. Then we found that apoptotic pathways induced by LYG-202 involved the degradation of PARP, the activation of caspase-3, -8, -9 and the elevation of the ratio Bax/Bcl-2. These results provided a mechanistic framework for further exploring of LYG-202 as a novel chemotherapeutics for human tumors.

Section snippets

Materials and methods

Reagents. LYG-202 was obtained from Dr. Zhiyu Li (China Pharmaceutical University, China). LYG-202 was dissolved in DMSO to 10 mM and stored at −20 °C. Controls were treated with the same amount of DMSO as used in the corresponding experiments. Antibody of PARP was from Cell Signaling Technology (Danvers, MA), antibodies of β-actin, caspase-3, -8, -9, Bcl-2, and Bax were from Santa Cruz (Santa Cruz, CA). IRDyeTM 800 conjugated anti-mouse and anti-rabbit second antibodies were obtained from

LYG-202 inhibits the growth of transplantable tumors

After the 9 day-treatment, LYG-202 (250, 500 mg/kg) and cyclophosphamide (30 mg/kg) had significant inhibitory effect on the growth of inoculated S180 in mice (Fig. 1B). The inhibitory rates were 32.20 ± 7.28%, 57.03 ± 3.96%, and 55.51 ± 7.43%, respectively (Fig. 1C). Meanwhile, there was no significant difference in the average weight of LYG-202 treated mice compared with control mice (data not shown).

LYG-202 inhibits cell viability in tumor cells

MTT assay was used to investigate the inhibitory effect of LYG-202 on tumor cells. As shown in Fig. 2

Discussion

Middleton [10] and Nijveldt [11] summarized the effects of flavonoids on inflammation, heart disease, and cancer in their review. Sharing the same three-ring structure, LYG-202 may share these effects.

In this research, we demonstrated that LYG-202 exhibited anti-tumor activity against murine sarcoma S180 transplantation tumor without significant weight loses in mice. These results suggested that LYG-202 exerted a remarkable anti-cancer activity in vivo and might be with low toxicity. We also

Acknowledgments

This work was supported by the National Natural Science Foundation of China (Nos. 30701032, 30472044, and 90713038) and International Corporation Program of China (2008DFA32120).

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    These authors contributed equally to this work.

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