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Paclitaxel (PAC) and cisplatin (CIS) are two established chemotherapeutic drugs used in combination for the treatment of various solid tumors. However, the usage of PAC and CIS are limited because of the incidence of their moderate or severe neurotoxic side effects. In this study, we aimed to assess the protective role of salicylic acid (SA) against neurotoxicity caused by PAC and CIS. For this purpose, newborn Sprague Dawley rats were decapitated in sterile atmosphere and primary cortex neuron cultures were established. On the 10th day SA was added into culture plates. PAC and CIS were added on the 12th day. The cytotoxicity was determined by using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. Oxidative alterations were assessed using total antioxidant capacity and total oxidative stress assays in rat primary neuron cell cultures. It was shown that both concentrations of PAC and CIS treatments caused neurotoxicity. Although SA decreased the neurotoxicity by CIS and PAC, it was more effective against the toxicity caused by CIS rather than the toxicity caused by PAC. In conclusion it was clearly revealed that SA decreased the neurotoxic effect of CIS and PAC in vitro.
A microtubule-binding agent, paclitaxel (PAC, Fig. 1), is widely used to treat several solid tumors including breast, ovarian and lung cancers (Gornstein and Schwarz 2014). Similarly, cisplatin (cis-diamminedichloroplatinum (II), CIS, Fig. 2), one of the most widely used DNA-modifying chemotherapy drug, is commonly used to treat testicular, ovarian, bladder, cervical, esophageal, head, neck and small cell lung cancers (Jekunen et al. 1994; Huang et al. 2004; Hassan et al. 2014; Song et al. 2014). The treatments with PAC and CIS are known to cause adverse effects such as myelo-suppression and neurotoxicity (Marupudi et al. 2007; Barabas et al. 2008; Wang et al. 2014). Therefore, the usefulness of PAC and CIS are limited due to their neurotoxicity. Up to date, numerous studies have been performed to find protective agents minimizing the neurotoxic effects of PAC and CIS (Openshaw et al. 2004; Chentanez et al. 2009; Turkez et al. 2010; Mao-Ying et al. 2014).
Chemo-protective agents protect healthy tissue from the toxic effects of anticancer drugs. An ideal chemo-protective agent is an agent without side effects, has strong chemo-protective capacities and which does not reduce antitumor activity (Hospers et al. 1999). Reducing the morbidity and mortality of antineoplastic regimens with the concomitant use of chemo-protective agents may lead to more tolerable anti-tumor treatments for patients and may permit for dose-escalation of both radio- and chemotherapy, which could lead to improved survival. Many antineoplastic drugs or chemical agents caused several organ toxicities via oxidative stress. Natural antioxidants or antioxidant featured synthetic compounds have been found to protect various organs from oxidative injuries (Cingolani et al. 2000; Cacciatore et al. 2003; Rispoli et al. 2004; Cacciatore et al. 2005; Geyikoglu and Turkez 2008; Heuking et al. 2009; Di Stefano et al. 2009; Sozio et al. 2010; Turkez and Geyikoglu 2011; Turkez et al. 2012a, b). At this point, salicylic acid (C6H4(OH)COOH, SA, Fig. 3) is a phytohormone that regulates many aspects of plant growth and development (Vlot et al. 2009; Rivas-San Vicente and Plasencia 2011; Xue et al. 2013). SA is recognized as an endogenous signal for mediating in plant defense against pathogens by presenting strong antioxidant and antifungal features (Ferger et al. 1999; Shabana et al. 2008; Gören et al. 2009). To the best of our knowledge, the potential protective action of SA against PAC and CIS-induced neurotoxicity in neuronal models has not been studied yet. Therefore, the aim of the present study was to evaluate the cytological (MTT assay) and biochemical effects (TAC and TOS analyses) of SA against PAC and CIS-induced neuronal damage on rat neuron cultures for exploring its neuroprotective potential for the first time.
Taxol® (6 mg/mL vial) was purchased from Bristol-Myers Squibb (New York, NY, USA) and Cisplatin® (10 mg/mL flakon) was purchased from Pharmachemie BV (Haarlem, Holland). Salicylic acid (CAS Number 69-72-7), Dulbecco’s Modified Eagle’s Medium (DMEM), Hanks’ Balanced Salt solution (HBSS) and DNase type 1 were purchased from Sigma Aldrich® (Steinheim, Germany). Neurobasal medium (NBM) and fetal calf serum (FCS) were purchased from Gibco-Life Technologies (Australia Pty Ltd, Mulgrave, VIC, Australia).
This study was conducted at the Medical Experimental Research Center of the Atatürk University (Erzurum, Turkey). The Ethical Committee of the Atatürk University approved the study by the protocol B.30.2.ATA.0.01.02/5515. Primary rat cerebral cortex neuron cultures were prepared using rat foetuses as described previously (Daikhin and Yudkoff 2000; Aydın et al. 2014; Togar et al. 2014). Briefly, a total of nine newborn Sprague-Dawley rats were used in the study. Making a cervical fracture in the cervical midline, the rats were decapitated and the cerebral cortexes were dissected and removed. The cerebral cortex was placed into 5 mL of HBSS, which had already been placed in a sterile petri dish and macromerotomy was performed with two lancets. The cerebral cortices were dissociated with HBSS, were pulled into a syringe and treated at 37 °C for 25–30 min in 5 mL HBSS plus 2 mL Trypsin– EDTA (0.25 % trypsin–0.02 % EDTA) and chemical decomposition was achieved. 8 μl of DNase type 1 (120 U/mL), was added to this solution and treated for 1–2 min, and centrifuged at 800 rpm for 3 min. After the supernatant was removed, 31.5 mL of neurobasal medium (NBM) and 3.5 mL fetal calf serum (FCS) were added to the pellet. The cells were seeded in 48-well cell culture plates. Plates were left in the CO2 incubator (5 % CO2 at 37 °C). Every 3 days, the medium were refreshed with fresh medium at the rate of half their volumes, until the cells reached a certain maturity. In vitro experiments were performed 10 days later. On the 10th day, SA (10 and 100 µM) was added into culture flask. At the end of the 12th day PAC (10−1 and 10−2 µM) and CIS (50 and 100 µM) were applied. 150 µL isotonic saline solution (ISS) added group was used as controls.
MTT assay (MTT Cell Proliferation Kit, Cayman Chemical Company, Ann Arbor, MI, USA) was used to determine cell viability. Rat cortex neuron cells were seeded in 48-well plates (0.3 mL). Then 10 μL MTT solution (5 mg/ml) was added to each well and incubated for 4 h at 37 °C. After this step, 100 μL crystal dissolving solution (DMSO) was added to each well to dissolve the formed formazan crystals. Finally, the absorbance was recorded at 570 nm using an ELISA plate reader (Thermo LabSystem, Helsinki, Finland) (Turkez et al. 2012a, b; Aydın et al. 2014). The cell viability was then calculated using the following equation: cell viability (%) = (A treated/A control) × 100 (A...absorbance).
Antioxidants in the sample reduce dark blue-green colored ABTS [2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid)] radical to colorless reduced ABTS form. The change of absorbance at 660 nm is related to the total antioxidant level of sample. The TAS assay is calibrated with a stable antioxidant standard solution that is traditionally named as Trolox Equivalent, which is a vitamin E analog (Kusano and Ferrari 2008). Oxidants present in the sample oxidize the ferrous ion-chelator complex to ferric ion. The oxidation reaction is prolonged by enhancer molecules, which are abundantly present in the reaction medium. The ferric ion reacts to a colored complex with a chromogen in an acidic medium. The color intensity, which can be measured spectrophotometrically, is related to the total amount of oxidant molecules present in the sample. The assay is calibrated with hydrogen peroxide and the results are expressed in terms of micromolar hydrogen peroxide equivalent per liter (μmol H2O2 Equiv./L). TOS assay is calibrated with hydrogen peroxide and the results are expressed in terms of micromolar hydrogen peroxide equivalent per liter (µmol H2O2 Equiv./L). Both TAC and TOS assays were carried out with commercially available kits (Rel Assay Diagnostics®, Gaziantep, Turkey) (Erel 2004; Turkez et al. 2014; Dirican and Turkez 2014).
For statistical analysis, we used SPSS for Windows 18.0 (SPSS Inc., Chicago, IL, USA). The experimental data were analyzed using one-way analysis of variance (ANOVA) followed by Duncan’s post hoc test for multiple comparisons. Results are presented as mean ± standard deviation (SD) and values p < 0.05 were regarded as statistically significant.
The neurotoxicity of PAC was examined using MTT assay. Both concentrations of PAC exhibited statistically significant (p < 0.05) neurotoxicity on cultured rat neuron cells. In fact, the 10−1 and 10−2 µM of PAC caused cytotoxicity at levels of 36.6 and 28.5 %, respectively. On the contrary, the treatments with different concentrations of SA (10 and 100 µM) for 48 h did not change the viability. However, when MTT assay was carried out after SA and PAC treatment, it was observed that 100 µM SA but not 10 µM SA provided a slight protection against the 10−1 µM PAC-induced neurotoxicity. However both SA concentrations did not exhibit a protective action against neurotoxicity by PAC at 10−2 µM (Table 1).
As seen from Table 2, concentrations of 50 µM and 100 µM CIS caused cytotoxic effects on cultured rat neurons at levels of 20.5 and 24.3 %, respectively. On the contrary, none of both SA concentrations induced neurotoxicity. Moreover, SA applications into the CIS-treated cells provided significant protection levels against CIS-induced neurotoxicity in a dose independent manner. No significant differences were observed between the 10 µM SA plus CIS and 100 µM SA plus CIS applied groups. In addition, the observed viabilities were still lower than the control values after both SA applications against neurotoxicity by CIS.
Tables 3 and and44 show TAC and TOS levels in SA, PAC and CIS treated neuron cultures. The TAC and TOS levels were modified by the treatments with different concentrations of PAC (10−1 and 10−2 µM) and of CIS (50 and 100 µM) due to oxidative stress induction. On the other hand, SA supported antioxidant capacity without changing TOS levels. After the treatments with different concentrations of SA in combination with PAC it was observed that both SA concentrations exhibited different levels of protection against PAC induced oxidative stress. Likewise, significant (p < 0.05) protection levels were observed when 10 and 100 µM of SA were applied to the culture plates in combination with CIS. But significant differences were not observed between the 10 µM SA plus CIS or PAC and 100 µM SA plus CIS or PAC applied groups.
In the present investigation, we assessed the protective role of SA against PAC and CIS-induced neurotoxicity on rat neuron cell cultures. Cytotoxic effect was evaluated using MTT assay. According to the MTT assay, the cytotoxic effects of CIS and PAC were demonstrated by their strong inhibition on cell viability on neuron cells (Tables 1, ,2).2). In accordance with our results, PAC exposure caused a decrease of cell viability and an increase in the ratio of apoptotic cells in dorsal root ganglion (DRG) neurons in vitro (Chen et al. 2015). Likewise, it was reported that CIS caused significant neurotoxicity via induction of lipid peroxidation and reduction in the potency of the antioxidant defense system (Kamisli et al. 2014). Besides, Colak et al. (2011) suggested natural antioxidant compounds could prevent neurotoxicity in experimental animal models. At this point, our findings indicated that 100 µM SA but not 10 µM SA provided protection against the 10−1 µM PAC-induced neurotoxicity. Again, both SA applications to the CIS-treated cells provided protection against CIS-induced neurotoxicity in a clear dose independent manner. Similar to our findings, SA showed protective effect against CIS-induced ototoxicity (Minami et al. 2004). Besides, Pisano et al. (2003) demonstrated that acetyl-l-carnitine (ALC) co-treatment was able to significantly reduce the neurotoxicity of both CIS and PAC in rat models. It was demonstrated that olesoxime, a small cholesterol-like compound, has shown marked neuroprotective properties in animals treated with PAC and vincristine (Rovini et al. 2010). Again, Openshaw et al. (2004) found that amifostine, a thiophosphate cytoprotectant, was ineffective in preventing or reducing the neurotoxicity of high doses of PAC. Conversely, in another study, two randomized trials with PAC and CIS have shown no significant neuroprotective effect of amifostine as determined by clinical assessment (Rick et al. 2001).
Oxidative stress has been known as imbalance between the free radicals and antioxidant defense system. Neurons are more sensitive to oxidative stress because of low activity of antioxidant enzymes (Karpińska and Gromadzka 2013). Experimental studies support that there are evidences about PAC toxicity related with reactive oxygen and nitrogen species (Ramanathan et al. 2005). Likewise, it was suggested that reactive oxygen species were related with CIS cytotoxicity (Feghali et al. 2001; Van den Berg et al. 2006; Zhang et al. 2007). In the present study, we investigated protective effect of SA against PAC and CIS-induced oxidative effects by determining TAC and TOS levels. We reported that SA was more effective in decreasing CIS-induced oxidative stress than PAC-induced oxidative stress due to its antioxidant effect. In addition, significant differences between the 10 µM SA plus CIS or PAC and 100 µM SA plus CIS or PAC applied groups were not observed in the present study. As a matter of fact, Mohanakumar et al. (2000) suggested that SA acts as a free radical scavenger in the brain that indicates its effectiveness as a valuable neuro-protector in mice in vivo. In addition, Ferger et al. (1999) suggested that the neuroprotective properties of SA were based on its effect as a hydroxyl radical scavenger rather than other mechanisms.
In conclusion, both PAC and CIS show severe neurotoxicity in cultured rat primary neuronal cells. These negative effects could be prevented by SA pre-treatments in vitro. SA applications seemed to be more effective against neurotoxicity by CIS than toxicity by PAC. Therefore, we suggest that co-application of SA could be a useful way to decrease the cytotoxic effects of CIS and PAC on the nervous system. The in vivo interactions of SA and CIS are still unknown. Therefore, further studies are needed to fully elucidate the neuroprotective action of SA.
This work supported by the Scientific & Technological Research Council of Turkey (TÜBİTAK, Project Number: 107S067).
The authors declared that there are no conflicts of interest.