Extract from Arrabidaea chica (Fridericia chica) leaves show preventive action for the mitigation of doxorubicin-induced cardiotoxicity

[Extrato das folhas de Arrabidaea chica (Fridericia chica) mostrou potencial ação no combate à cardiotoxicidade induzida pela doxorrubicina]

RESUMO

A doxorrubicina (dox) é um medicamento antineoplásico que induz cardiotoxicidade por estresse oxidativo. Os flavonoides são antioxidantes extraídos de plantas como Camellia sinensis e Arrabidaea chica (Fridericia chica). Esta pesquisa objetivou avaliar efeitos protetores do extrato de A. chica (AC), comparado ao de C. sinensis (CS), frente ao estresse oxidativo induzido pela dox, no coração. Cardiomiócitos e células neoplásicas MDA-MB 231 foram incubados com AC e CS. Depois, adicionou-se dox e avaliaram-se taxas de viabilidade e morte celular. A citometria de fluxo para o ensaio de iodeto de propídeo (IP) em cardiomiócitos mostrou as seguintes taxas de morte celular: controle 53%; dox 78% (maior que controle, P=0,015); AC_12,5μg/mL + dox 65% (menor que dox, P=0,031); AC_25μg/mL + dox 62% (menor que dox, P=0,028); AC_50μg/mL + dox 63% (menor que dox, P=0,030); CS_12,5μg/mL + dox 71% (menor que dox, P=0,040); CS_25μg/ml + dox 69% (menor que dox, P=0,037); CS_50μg/mL + dox 74% (menor que dox, P=0,044). Resultados das células MDA-MB 231 mostraram que nenhum extrato interferiu na atividade antitumoral da dox. Os dados de IP foram corroborados pelos de MTT. Este estudo reporta promissora utilização de A. chica na prevenção da cardiotoxicidade induzida pela dox.

Palavras-chave:
antioxidante; cardiotoxicidade; flavonoides; oncologia

Keywords:
antioxidant; cardiotoxicity; flavonoids; oncology

Palavras-chave:
antioxidante; cardiotoxicidade; flavonoides; oncologia

Doxorubicin (dox) is used for treatment of several types of cancer in humans and dogs; however, it shows important off-target cardiotoxic effects, which appear even months after completing the treatment in both species (Almeida et al., 2015; Hallman et al., 2019). Thus, it is imperative to find a treatment that reduces dox cardiotoxicity maintaining its anticancer efficacy. Considering the oxidative stress induced by dox, an antioxidant agent is an option. Green tea from Camellia sinensis (L.) Kuntze has been studied and demonstrated cardioprotective potential against dox toxicity (Aboulwafa et al., 2019; Saeed et al., 2015). In this scenario, a plant extract which has high flavonoid content, such as Arrabidaea chica (Bonpl.) Verl. [Fridericia chica (Bonpl.) L.G.Lohman] of the Bignoniaceae family, should be considered to prevent dox-induced oxidative stress in the myocardium. Due to its high flavonoid content (Barbosa et al., 2008), we hypothesized that A. chica has a previously uninvestigated cardioprotective role. Thus, the present study aimed to evaluate the cardioprotective effect of A. chica, compared with C. sinensis (a known positive control), and the maintenance of dox anticancer activity in the presence of both extracts.

A. chica dry leaves (20g) were macerated with 100ml of a mixture ethanol:water (7:3) and took to ultrasonic bath at room temperature for 18h. The A. chica extract (AC) obtained was submitted to partition with solvents (Urbonaviciūte et al., 2006) to obtain a rich flavonoid content extract (2g). Voucher specimens of A. chica were deposited at the herbarium of the Universidade Estadual de Campinas (Campinas, SP, Brazil) under the code UEC 145.956. Mass spectrometry analyses of AC and standards were carried out on a Shimadzu LCMS-IT-TOF system. Among several peaks, the analysis showed one which was identified at 287.079 [M +h]⁺m/z corresponding to kaempferol. A commercially C. sinensis extract (CS) was used (Sunphenon DCF®, Taiyo Kagaku Co.), containing total polyphenols (>80%), catechins (>80%), epigallocatechins (>45%), and caffeine (<1%) detected by high performance liquid chromatography (HPLC), as informed by the manufacturer.

Ventricular cardiomyocyte isolation from neonatal Wistar rats was performed as described earlier (Ott et al., 2008) and human mammary adenocarcinoma cell line MDA-MB-231 was used. Both cell cultures were maintained in a humidified incubator with 5% CO₂ at 37°C. Cell viability was assessed through the MTT assay (Mosmann, 1983) and cellular death was evaluated by the propidium iodide (PI) nuclear staining (Riccardi & Nicoletti, 2006) analyzed with a FacScan (BD Biosciences) and CellQuest to determine percentages of viable and apoptotic/necrotic cells. For MTT, cardiomyocytes were plated at 2.6x10⁴, 5.2x10⁵ or 2.6x10⁵ cells/cm² and MDA-MB-231 cells were plated at 1.3x10⁴ cells/cm² in 24-well plates. For PI, 6x10⁵ cardiomyocytes and 1x10⁵ MDA-MB-231 cells were plated in 6-well plates.

Both cell cultures were subjected to the following treatments, in three independent experiments performed in triplicates: control group (cells incubated only with media, no plant extract, no dox); AC group (cells incubated with 0, 12.5, 25, 50, 100, and 200μg/ml of AC; without dox); CS group (cells incubated with 0, 12.5, 25, 50, 100, and 200μg/ml of CS; without dox); Dox group (no herbal extracts; 5μmol/l dox incubation for 2, 4, 16, and 24h); AC-Dox group (cells incubated with 0, 12.5, 25, 50, 100, and 200μg/ml of AC; 5.0μmol/l dox) and CS-Dox group (cells incubated with 0, 12.5, 25, 50, 100, and 200μg/ml of CS; 5.0μmol/l dox). After 24h of seeding, fresh media was added to each cell type containing the mentioned concentrations of herbal extract. After 48h of seeding, 5.0μmol/l dox (Adriblastina®, Pfeizer) was added to each well. After 58h of seeding, cell viability and death analysis were performed. Statistical analyses were carried out using R program for one-way analysis of variance (ANOVA) followed by Scott-knott test, and significance of 5%.

Data of MTT assay demonstrated that AC and CS were not toxic to the cells at a concentration rate from 12.5 up to 50μg/ml, as cell viability was not different from control (P>0.05). However, extracts at higher concentrations of 100 and 200μg/ml were toxic (lower cell viability compared to control, P<0.05) and, therefore, excluded from further investigations. These findings are explained byhormesis, a dose-response relationship that is characterized by low-dose stimulation and high-dose inhibition. Such pro-oxidative property was also observed with resveratrol (Plauth et al., 2016). Flavonoids are considered antioxidants, but they can become pro-oxidative as described here for high doses. This paradoxical effect highlights the misleading concept of considering antioxidant agents always beneficial (Lei et al., 2016).

The percentage of death cardiomyocytes significantly increased after incubation with dox, compared to control (p=0.015), as expected. However, cellular death was lower for cardiomyocytes incubated with dox+plant extracts (P<0.05 for all dosage tested), compared to dox only (Figure 1A), indicating a protective effect of AC and CS against dox over cardiomyocytes. Such protection was not observed for MDA-MB-231 cells, which is desirable, because the anti-neoplastic property of dox must be kept over cancer cells (Figure 1B). In fact, the association of Dox with AC at 12.5μg/ml and Dox with CS at 25 and 50μg/ml resulted in more cell death to breast cancer cells, compared to Dox only (P<0.05), indicating a potentializing action of dox by these extracts in cancer cells. All data obtained for PI assay were confirmed by MTT assay.

Figure 1
Cell death of cardiomyocytes isolated from neonatal Wistar rats (A) and human mammary adenocarcinoma cell line MDA-MB 231 (B) following treatment with doxorubicin (Dox) for 24h, as assessed by propidium iodide incorporation. Data is represented as %, according to untreated control. Dox was used at 5µmol/l and Arrabidaea chica (Fridericia chica - AC) and Camellia sinensis (CS) extracts at 12.5, 25 and 50 µg/ml. Results are presented as mean ± SEM of three experiments. *P<0.05 vs control. **P<0.05 vs Dox.

The CS used had high concentration of epigallocatechin (> 45%), which was proved to be cardioprotective against dox (Saeed et al., 2015), explaining the preservation of cardiomyocyte viability. Mass spectrometry analyses of the AC identified the presence of kaempferol. In ischemia-reperfusion injury in the heart, kaempferol decreased levels of inflammatory markers (TNF-alpha and IL-6) and proapoptotic proteins (Bax and caspase-3), and increased expression of the antiapoptotic protein Bcl-2 (Suchal et al., 2016). In dox-induced toxicity, kaempferol protected heart cells by inhibiting the p53-mediated mitochondria-dependent intrinsic apoptotic signaling (Xiao et al., 2012). While dox promotes apoptosis by increasing Bax and decreasing Bcl-2, kaempferol inhibits apoptosis by showing an opposite action (Xiao et al., 2012). In this context, at least part of AC effectiveness demonstrated here is due to the presence of kampferol.

Despite the potential therapeutic properties of A. chica, described with antineoplastic, anti-inflammatory and antimicrobial properties and useful for treating osteoarthritis (Barbosa et al., 2008; Michel et al., 2015; Vasconcelos et al., 2019), there is no previous study evaluating its effect against oxidative injury to the heart. Present data demonstrated a novel antioxidant activity of A. chica on dox-induced cardiotoxicity. The A. chica and C. sinensis treatments were shown to efficiently and selectively preserve the viability of cardiomyocytes, while maintaining dox anticancer activity, making them promising alternatives to be used by oncology patients under dox treatment.

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Publication Dates

• Publication in this collection
  05 May 2021
• Date of issue
  Mar-Apr 2021

History

• Received
  20 July 2020
• Accepted
  27 Oct 2020