Molecular and Cellular Pharmacology
Curcumin induced HepG2 cell apoptosis-associated mitochondrial membrane potential and intracellular free Ca2+ concentration

https://doi.org/10.1016/j.ejphar.2010.09.049Get rights and content

Abstract

Curcumin is a phytochemicals which is able to inhibit carcinogenesis in a variety of cell lines. However little is known about its effect on the cell-surface and the interaction between cell-surface and the reacting drug. In this study, we found that curcumin could inhibit the growth of human hepatocellular carcinoma cell line (HepG2), change the cell-surface morphology and trigger the pro-apoptotic factor to promote cell apoptosis. Cell counting kit results indicated that the cell viability had a dose-dependent relationship with the curcumin concentration in 24 h. The 50% inhibiting concentration (IC50) was 17.5 ± 3.2 μM. It was clear that curcumin could lead to apoptosis, and the apoptosis increased as the reacting concentration goes up. Moreover, curcumin could also affect the disruption of mitochondrial membrane potential and the disturbance of intracellular free Ca2+ concentration. All these alterations changed the cell morphology and cell-surface ultrastructure with atomic force microscopy (AFM) detecting at nanoscale level. AFM results indicated that cells in control group clearly revealed a typical long spindle-shaped morphology. Cell tails was wide and unrolled. The ultrastructure showed that cell membrane was made up of many nanoparticles. After being treated with curcumin, cell tail was narrowed. The size of membrane nanoparticles became small. These results can improve our understanding of curcumin which can be potentially developed as a new agent for treatment of hepatocellular carcinoma since it has been reported to have a low cytotoxic effect on healthy cell. AFM can be used as a powerful tool for detecting ultrastructures.

Introduction

Hepatocellular carcinoma is the most common cancer in some parts of the world, with more than 1 million new cases being diagnosed each year. It is potentially curable by surgical resection and chemotherapy. But surgery is the treatment of choice for only the small fraction of patients with localized disease and chemotherapy drugs can damage normal cells, as well as some other side effects (Mor et al., 1998). It is imperative to develop new agents for treatment of this cancer and requires a more novel and integrated approach for prevention, diagnosis and treatment. Recent attention is focused on phytochemicals as anticancer agents. Curcumin (diferuloylmethane) is a kind of low-molecular-weight and natural polyphenolic compound isolated from the turmeric rhizome (Curcuma longa). It has phenolic groups and conjugated double bonds (Fig. 1). Besides, it has various properties including antioxidant, anti-inflammatory, anti-angiogenic, anti-proliferative, wound healing and antitumor properties (Deshpande & Maru, 1995, Sharma et al., 2005, Maheshwari et al., 2006, Ono et al., 2004). Preclinical studies have shown that curcumin can inhibit carcinogenesis in various cell lines such as gastric, colon, breast, cervical, hepatic, and ovarian cancer (Aggarwal et al., 2003). Compared with some chemotherapy drugs, curcumin has the advantage to induce cancer cell apoptosis, while with low cytotoxic effects on normal cells (Syng-ai et al., 2004, Kunwar et al., 2008). Thus, it's a potential compound for drug development against cancer (Hatcher et al., 2008).

Although there are many studies of anticancer effect about curcumin, little is known about membrane morphology and mechanism induced by it. Plasma membrane plays a very important role on cell physiology. It's a boundary between live cell and external environment and protects cell from harm (Puech et al., 2006). Also it regulates cell functions and transportation of nutrition inside and outside cell (Alarmo et al., 2009, Heidemann & Wirtz, 2004). Changes of membrane structure have a direct influence on cell functions (Voïtchovsky et al., 2006, Sato et al., 2007). In recent years, studies of the cellular, subcellular and molecular mechanical changes on human disease states, including cancer, have emerged as a topic of rapidly expanding scientific interest. A particular focus is to explore the connections among the cell ultrastructure, cellular and cytoskeletal mechanical properties, biological function and human health/disease (Suresh, 2007). In this paper, atomic force microoscopy was used to visualize cell morphology and the membrane ultrastucture.

Recent studies have demonstrated that cell apoptosis is related to mitochondrial membrane potential, especially for the cancer cells (Scarlett et al., 2000, Hu & Kavanagh, 2003). Mitochondrial membrane potential decrease could result in activation of mitochondrial pro-apoptotic factors (Hu and Kavanagh, 2003). In addition, another factor related to cell death is the intracellular free Ca2+ level, which is companied with decrease of mitochondrial membrane potential, release of cytochrome c from mitochondria and the activation of apoptosis proteins casepase (Simon et al., 2000). In this paper, we evaluated the Ca2+ level and mitochondrial membrane potential in curcumin-induced human hepatocellular carcinoma cell line (HepG2). And the apoptosis mechanism was investigated.

Section snippets

Materials

Human hepatocellular carcinoma cell line (HepG2) was donated by Institute of Physiology, Jinan University. RPMI1640 medium and fetal bovine serum were purchased from Giboc Co. Curcumin was bought from Tianjin Yongda Chemical Reagent Development Center. The cell counting kit was purchased from Dojin Laboratory (Kumamoto, Japan). Fluo-3 AM and Rhodamine 123 were the products of Beyotime Institute of Biotechnology, China. Annexin V-FITC and PI apoptosis detection kit was bought from Keygen

Effect of curcumin on HepG2 cell viability

The cell counting kit was used to assess the cell proliferation and viability of curcumin-treated HepG2 cells. As shown in Fig. 2, with the concentration of curcumin increased, cell viability was decreased after treated with curcumin for 24 h. Particularly from 10 to 20 μM, the cell viability was decreased significantly, almost dropped about 3 times. The 50% inhibiting concentration (IC50) (17.5 ± 3.2 μM) was located in this area. However, cell viability decreased very little between 20–40 μM. At 40 

Discussions

Tumor cell resistance to apoptosis is an inherent part of the carcinogenic process and is also implicated in resistance to chemotherapeutic drugs (Johnstone et al., 2002). Therefore, phytochemicals such as curcumin is applied to target resistant cells and improves efficacy without toxicity on normal cells (Syng-ai et al., 2004). Previous studies have reported that curcumin can not induce apoptosis in normal cells including primary cultures. For example, Syng-ai et al. (2004) have studied the

Conclusions

Curcumin not only inhibited the growth of HepG2 cells, but also had a high effect on inducing cell apoptosis without any cytotoxic effects on healthy cells (Hatcher et al., 2008). There existed a dose-dependent relationship between the drug concentration and cell apoptosis. Cell morphology shown curcumin could damage the cell membrane and suggested that the degree of damage to a tumor cell membrane has a certain positive correlation with drug concentration. Atomic force microscopy detected

Acknowledgment

This work is supported by Ministry of Science and Technology of China (No. 2010CB833603), the National Natural Science Foundation of China (No. 60578025), the Key Project of Chinese Ministry of Education (No. 210254), the Fundamental Research Funds for the Central Universities (No. 21609305), and the Key Laboratory of Optoelectronic Information and Sensing Technologies of Guangdong Higher Education Institutes, Jinan University.

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