دانلود رایگان ترجمه مقاله افزایش فعالیت خزان یاخته ای و ژنوتوکسیک کربوپلاتین بوسیله فولیک اسیدها (نشریه الزویر ۲۰۱۶)

اسید فولیک باعث افزایش فعالیت خزان یاخته ای و ژنوتوکسیک کربوپلاتین در سلول HeLa می شود

Folic acid enhances the apoptotic and genotoxic activity of carboplatin in HeLa cell line

Abstract

In human tumor cells, experimental and clinical evidence indicates that some factors involved in signal transduction and cell growth can also modulate the response to chemotherapeutic treatment. The aim of the present study was to investigate the role of folic acid (FA) as a modulator of carboplatin (CBDCA) activity.

Genotoxicity and cytotoxicity induced by CBDCA alone and in combination with FA were assessed in cultured HeLa cells. We used comet assay, mitotic index analysis, MTT and NR assays, cytokinesis-block micronucleus cytome assay and annexin V-IP as different cytotoxicity and genotoxicity approaches for human cervical carcinoma cell line studies.

The results showed that addition of 900 nM FA together with 40.4 mM CBDCA enhanced the activity of the platinum compound, increasing its effect on cell death by nearly 20%, as evidenced by the MTT and NR assays. Moreover, not only higher levels of DNA and chromosomal damage were reached but also the number of necrotic and apoptotic cells were significantly increased when cell cultures were treated with the combined procedure.

This situation opens the possibility to explore the use of FA in platinum-based chemotherapy protocols to reduce the platinum doses for patient treatment and decrease the chance of developing the known side effects without losing biological activity.

۱ Introduction

Platinum-based compounds are known since the 19th century. In fact, cisplatin was first synthesized in 1845, but the discovery of its antitumor properties was not made until 1969 (Rosenberg and Vancamp, 1969). Today, platinating compounds are still front-line clinical therapies and constitute part of the treatment regimen for patients with many types of cancers, including head and neck, testicular, ovarian, cervical, lung, colorectal and relapsed lymphoma. Despite this success, nephrotoxicity, neurotoxicity and the emetic properties of cisplatin have limited its use (Basu and Krishnamurthy, 2010). Also, the reduced water solubility, the relatively narrow range of treatable cancers and the ability of carcinomas to develop chemotherapy resistance have pressed researchers to develop a new generation of platinum-based molecules. In this sense, carboplatin (CBDCA) is a second generation drug introduced and approved by the FDA in the late 20th century. Since that time, it has gained popularity in clinical treatment due to its reduced side-effects, when compared with its related compound cisplatin. Particularly, although CBDCA has low nephrotoxicity effects, it shows undesirables myelosuppressive characteristics (Wagstaff et al., 1989).

Even though platinum-based compounds have the ability to bind to a wide spectrum of biomolecules, such as RNA, proteins or phospholipids, DNA is their main biological target (Pascoe and Roberts, 1974; Knox et al., 1986). Cisplatin and CBDCA can enter cells via passive diffusion through a process known as aquation (Gately and Howell, 1993), or via endocytosis using the copper transporter CTR1, which is a key influx transporter and plays an important role in mediating the uptake of platinum compounds (Shen et al., 2012). Once into the cells, platinum compounds bind to the nitrogens of purine bases in DNA, inducing intrastrand crosslinks. The presence of these adducts distorts the double helix, blocking DNA replication and transcription (Jamieson and Lippard, 1999). Specifically, CBDCA mostly forms the intrastrand crosslink cis-Pt(NH3)2d(pGpG)(Pt-GG), requiring N200-fold CBDCA than cisplatin to obtain equal levels of platination. On the other hand, N20 times CBDCA is needed to obtain equal levels of cytotoxicity in CHO cells, after 1 h of exposure (Blommaert et al., 1995). To treat locally advanced cervical cancer, platinum-containing agents are used alone or in combination. Some studies have shown that the use of cisplatin combined with paclitaxel requires a prolonged infusion and is less convenient and more toxic than the combination of CBDCA and paclitaxel, thus leading to a more widespread use of CBDCA (Moore et al., 2007).

Folate, or folic acid (FA), is a water-soluble B-vitamin also known as vitamin B9 or folacin. In nature, folates exist in a wide range of chemical forms and they can be found in food as well as in metabolically active forms in the human body. FA is the major synthetic form found in fortified foods and vitamin supplements; however, it has no biological activity unless converted into folate (National Academy Press, 1998). In human physiology, the only function of folate coenzymes appears to be linked to the transfer of one-carbon units, playing a major role in Sadenosylmethionine formation, the universal methyl donor, as well as in the construction of purine and thymidine in a variety of reactions critical to the metabolism of nucleic acids and amino acids (Bailey and Gregory, 1999; Choi and Mason, 2000).

Epidemiologic data have revealed that a reduced folate status is associated with cervical, colorectum, lung, esophagus, brain, pancreas, and breast cancer. Intervention trials using supraphysiologic doses of folate, mainly with individuals at increased risk of cervical and colorectum cancer, showed inconsistent results (Choi and Mason, 2000). Nevertheless, folates appear to be essential for cell growth and cell cycle, key processes in cell transformation. On the other hand, folate deficiency induces lower proliferation rates by increased apoptosis and cell cycle arrest (Courtemanche, 2003). It has been widely demonstrated that folate receptor is overexpressed in several human epithelial cancer cells, but is absent or negligibly expressed in most normal cells, highlighting the need for folates by transformed cells. In general, aggressive cancer types express the highest amounts of folate receptor alfa or FRα (Hansen et al., 2015).

In human tumor cells, experimental and clinical evidence indicates that some factors involved in signal transduction and cell growth can also modulate the response to chemotherapeutic treatment (Toffoli et al., 1998). For example, the cellular transformation by different oncogenes can stimulate the proliferation of ovarian cancer cells and confer cellular resistance to cisplatin (Basu and Cline, 1995). In this way, an increased folate internalization may have a role in the constitutive control of cell proliferation (Sun et al., 1995), thereby modifying the response to chemotherapeutic agents.

Human cervical carcinoma HeLa cells strongly express the folate receptor, as demonstrated in several works (Zhang et al., 2006). In this context, the use of folate as a cell response modulator against a chemotherapeutic agent is fully grounded, thus allowing the use of these cells as a model for studying the cellular response to a chemotherapeutic agent modulated by FA.

In this paper, we investigate the genotoxicity and cytotoxicity induced by CBDCA alone and in combination with FA. We used alkaline single cell gel electrophoresis assay (SCGE), mitotic index analysis (MI), 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and cytokinesis-block micronucleus cytome (CBMN Cyt) assay as different cytotoxicity and genotoxicity approaches for human cervical carcinoma cell line studies.