The effect of resveratrol, its naturally occurring derivatives and tannic acid on the induction of cell cycle arrest and apoptosis in rat C6 and human T98G glioma cell lines

Highlights

• Resveratrol and pterostilbene arrest cell cycle in S-phase, while TMS treatment leads to cells' accumulation in G2/M phase.

• Tannic acid does not affect the cell cycle distribution, but increases the number of apoptotic cells.

• TMS the most significantly increases the C6 glioma apoptosis rate and this effect is related to p53 induction.

• Methoxylated stilbenes, particularly TMS, might be considered potential adjuvants in glioma therapy.

Abstract

Resveratrol (3,5,4′-trihydroxy-trans-stilbene) is a potent chemopreventive and potentially cancer therapeutic agent. Since rapid metabolism limits resveratrol bioavailability, derivatives less prone to metabolic transformation are being sought and tested.

We evaluated the effect of resveratrol, and its analogs (pterostilbene and 3,5,4′-trimethoxystilbene) along with tannic acid, on cell cycle and apoptosis in rat C6 and human T98G glioma cells. At concentration ranges both lower and higher than IC₅₀ calculated based on MTT assay, all these polyphenols affected the cell cycle distribution. However, resveratrol and pterostilbene increased the percentage of the cells in S phase, while trimethoxystilbene (TMS) caused a massive accumulation of cells at the G2/M phase of the cell cycle. Tannic acid had no effect on cell cycle distribution in C6 cells, but increased the number of dead cells in both glioma cell lines. The ability to induce apoptosis by tannic acid and stilbenes was confirmed by phosphatidylserine externalization, the loss of mitochondrial membrane potential and the level of cleaved caspase-3. The apoptosis rate was most significantly increased by TMS and this was related to p53 induction.

These results indicate that methoxylated stilbenes are efficient inhibitors of glioma cell proliferation and apoptosis inducers and might be considered adjuvants in glioma therapy.

Introduction

Malignant gliomas are the most common central nervous system tumors and carry the worst clinical prognosis in both adults and children. Although current therapeutic protocols comprise a combination of surgical operation with irradiation and adjuvant chemotherapy, the prognosis for malignant gliomas remains very poor due to their highly aggressive biological behavior and frequent recurrence rate (Gagliano et al., 2010, Van Meir et al., 2010). Thus, the development of new agents able to reactivate cell cycle or cell death programs, particularly apoptosis, is important for the introduction of new therapeutic and/or chemopreventive strategies for malignant gliomas.

Resveratrol (3,5,4′-trihydroxy-trans-stilbene), a naturally occurring polyphenol, is assumed to possess cancer-preventive and cancer-therapeutic properties (Kuršvietienė et al., 2016). Resveratrol suppresses the proliferation of a variety of human cancer cells in vitro, including glioma cells (Gagliano et al., 2010).

In C6 rat glioma cells, resveratrol has been shown to inhibit cell growth and induce apoptosis by increasing caspase-3 mRNA level and enzyme activation (Zhang et al., 2007). However, Michels et al. (2006) demonstrated that resveratrol is not metabolized in C6 rat glioma cells, and accumulates to concentrations that drive the cell to necrosis. Induction of apoptosis by resveratrol has also been reported in human glioma U251, U87 and T98G cells (Jiang et al., 2005, Lin et al., 2011). Metabolism plays an important role in resveratrol anticancer activity.

While resveratrol is the most active, its sulfo- and glucuronide conjugates are quickly excreted and are much less effective (Sun et al., 2013, Wang et al., 2005).

In view of the limited bioavailability of resveratrol, its naturally occurring and synthetic analogs are the subjects of intensive research and development. In this regard, naturally occurring methoxylated stilbenes, such as 3,5-dimethoxy-4′-hydroxy-trans-stilbene (pterostilbene) and 3,5,4′-trimethoxy-trans-stilbene, have been shown to be more efficient in eliciting chemopreventive and/or therapeutic effects than resveratrol (Kapetanovic et al., 2011, Lin et al., 2009, Lin and Ho, 2009, Aldawsari and Velázquez-Martínez, 2015).

Tannic acid, a water-soluble tannin, is, similarly to resveratrol, a common ingredient of grapes and others berry fruits. Thus, their synergic action may be expected.

Our earlier studies have shown that although both polyphenols reduced binding of carcinogenic aromatic hydrocarbons to DNA, tannic acid was a much more potent inhibitor of specific DNA adduct formation (Ignatowicz et al., 2003, Baer-Dubowska and Szaefer, 2013). Moreover, neutrophil modulation of ROS production ultimately leading to cell apoptosis has also been shown in humans (Zielińska-Przyjemska et al., 2015).

While the role of neutrophils infiltrating tumors has been neglected for some time, a significant amount of evidence has recently been gathered on their importance in experimental models as well as in human cancers (Moses and Brandau, 2016). Fossati et al. (1999) showed that most human glioma samples analyzed had significant neutrophil infiltration. Thus, compounds such as polyphenols may target both cancer tissue and infiltrated neutrophils.

In the present study, we compared the ability of resveratrol and its two natural derivatives (pterostilbene and 3,5,4′-trimethoxystilbene) and tannic acid to induce growth inhibition and apoptosis in rat C6 and human T98G glioma cells. An attempt to explain the mechanism of pro-apoptotic activity of these compounds was also undertaken.