Hexavalent chromium-induced autophagic death of WRL-68 cells is mitigated by aqueous extract of Cuminum cyminum L. seeds

Abstract

In this study, we assessed the potential of aqueous extract (CSE_aq) of Cuminum cyminum L. (cumin) seeds in protecting WRL-68 cells from hexavalent chromium [Cr(VI)]-induced oxidative injury. Cells exposed to Cr(VI) (10 μM CrO₃) for 24 h demonstrated a twofold increase in ROS, which, in turn, led to extensive oxidative stress, consequently causing colossal decline in cell viability (by 58.82 ± 9.79%) and proliferation (as was evident from a reduced expression of Ki-67, a proliferation marker). Immunofluorescence studies showed that Cr(VI) diminished the expressions of mTOR and survivin in WRL-68 cells. It also led to a substantial elevation of BECN1 expression, which suggested autophagy. Overall, our results indicated that 24 h exposure of WRL-68 cells to Cr(VI) caused oxidative stress-induced autophagic cell death. CSE_aq was found to protect WRL-68 cells from the same fate by refurbishing their viability and proliferation in a dose-dependent manner. The extract reduced ROS in these cells, which consequently decreased the degree of autophagic cell death by restoring expressions of mTOR, survivin and BECN1 to their respective normal levels. Biochemical assays revealed that CSE_aq is rich in phenolic constituents. Total phenolic content of CSE_aq demonstrated positive correlations with (i) its antioxidant potential, (ii) its alleviation of cellular oxidative stress and (iii) its cytoprotective efficacy in Cr(VI)-treated WRL-68 cells. We also identified the major phenolic constituents of CSE_aq. Our study suggested that polyphenols in CSE_aq might be responsible for protecting WRL-68 cells from Cr(VI)-governed oxidative assault that would have otherwise led to survivin/mTOR-mediated autophagic death.

Introduction

Chromium is one of the most predominant heavy metal pollutants found in the environment (Vučković et al. 2013; Bakshi and Panigrahi 2018). It exists in a multitude of oxidation states, of which the hexavalent form [Cr(VI)] is extremely toxic to biological systems (Baruthio 1992; Guha et al. 2010c, 2011b; Oliveira 2012; Wang et al. 2018). Cr(VI) is vigorously oxidizing in nature (Guha et al. 2010c; Oliveira 2012) and can be easily transported into cells (Arslan et al. 1987; Arita and Costa 2011). This might lead to cell damage, consequently resulting in injury to the renal, hepatic, cardiovascular, nervous and dermal facets of an individual, along with neoplastic transformations, severe morbidity and even mortality (Lin et al. 2009; Sun et al. 2009; Singh et al. 2011).

One characteristic feature of Cr(VI)-induced toxicity is the generation of copious amounts of reactive oxygen species (ROS); thereby, causing holistic oxidative stress (Ding and Shi 2002; Davidson et al. 2007). Cr(VI) has also been reported to deplete the antioxidant capacity of cells (Stanley et al. 2014; Husain and Mahmood 2017). Augmented oxidative stress, in turn, can alter signal transduction pathways and cell cycle progression (Abel and DiGiovanni 2015; Zhang et al. 2017), while damaging nucleic acids, lipids and proteins (Birben et al. 2012). Such oxidative cellular dyshomeostasis has been reported to trigger apoptotic (Xueting et al. 2018; Yu et al. 2018), necrotic (Sun et al. 2015) and/or autophagic cell death (Di Gioacchino et al. 2008a, b; Lee et al. 2014) in diverse cell/tissue types.

A number of researchers have suggested the application of exogenous antioxidants to ameliorate Cr(VI)-induced oxidative stress in biological systems (Poljsak et al. 2011; Vidal et al. 2014). Antioxidants are putatively abundant in floral resources (Szymanska et al. 2016), including a number of spices (Yashin et al. 2017). For instance, Cuminum cyminum L. (Apiaceae) seeds, commonly known as cumin, are known for their antioxidant properties (Rebey et al. 2012). C. cyminum seeds, distinctive for their aroma and flavour (Arun et al. 2016), are a common culinary ingredient, used all over the world, ranging widely from European cuisines and Tex-Mex recipes to exotic Indian delicacies. Apart from the food value, the spice has been traditionally used as herbal medication. While it has been used to treat fever, diarrhoea, vomiting, oedema and hypolipidaemia, it is also known for its antidiabetic, antiepileptic and immunomodulatory properties (Aruna and Sivaramakrishnan 1992; Johri 2011; Mnif and Aifa 2015; Pandey et al. 2015). C. cyminum seeds have exhibited myriad pharmacological activities, such as inhibition of advanced glycation end product (AGE) formation in diabetic rats (Jagtap and Patil 2010) and alleviation of deleterious modifications of fatty acids in rat liver (Kode et al. 2005). Furthermore, the spice has also exhibited comprehensive memory enhancement and anti-stress potentials (Koppula and Choi 2011). These therapeutic characteristics of C. cyminum seeds have been attributed to the extensive antioxidant property of the spice (Rebey et al. 2012).

However, till date, there is no report of the spice’s protective effects on cells against Cr(VI)-induced injury. In vitro, we tested the protective effects of the aqueous extract of C. cyminum seeds (CSE_aq) against Cr(VI)-induced damage in WRL-68 cells. We also investigated the molecular mechanism of cell death in the model system to understand the protective modality of CSE_aq. Furthermore, we have endeavoured to statistically correlate the activities of CSE_aq constituents with their therapeutic potential against Cr(VI)-governed cell damage.

Materials and methods

Cell culture

WRL-68 cells were procured from National Center for Cell Science (Pune, India). Although originally derived from human cervical tissue, the morphology of these cells is identical to human hepatocytes and hepatic primary cultures. They secrete alpha-feto protein and albumin along with liver-specific enzymes (e.g. alkaline phosphatase, aspartate amino transferase, alanine amino transferase and gamma-glutamyl transpeptidase) [European Collection of Authenticated Cell Cultures (ECACC) 2018]. WRL-68 cells were cultured in minimum essential medium Eagle (MEM) (containing Earle’s salts, 2 mM l-glutamine, 1 mM sodium pyruvate, non-essential amino acids and sodium bicarbonate) supplemented with 10% fetal bovine serum and 1% antimycotic and antibiotic solution at 37 ºC in a humidified sterile environment containing 5% CO₂.

Plant material: collection, authentication and processing

C. cyminum L. (cumin) seeds were purchased locally and authenticated by the Botanical Survey of India, Southern Circle, Coimbatore, Tamil Nadu, India (Ref. No.: BSI/SRC/5/23/2018/Tech./1824). The authenticated specimen is maintained in the laboratory’s specimen repository (accession no.: CDHL002/2018). The seeds were thoroughly screened to eliminate contamination with other species, followed by grinding in a mechanical mixer grinder. The ground powder (60 g) was extracted with water as solvent [sample (g): water (ml) = 1:10] using a Soxhlet apparatus. The obtained extract was concentrated at 40 ºC under reduced pressure (72 mbar) with a Rotavapor R-215 (BÜCHI Labortechnik AG, Switzerland) to yield dry extract (CSE_aq).

MTT assay: effect of Cr(VI) exposure on WRL-68 cells vis-à-vis CSE_aq

Viable cells can physiologically reduce yellow-coloured MTT to form the purple-coloured MTT–formazan complex, which can be determined spectrophotometrically as a measure of cell viability. To determine the degree of damage induced by Cr(VI) (CrO₃) in WRL-68 cells and the role of CSE_aq in mitigating such injury, cells were seeded in 96-well plates (7.5 × 10³ cells/well in 100 µl MEM) and grown for 48 h in standard cell culture conditions. The cells were divided into three groups: (i) untreated cells (supplemented with fresh MEM), (ii) Cr(VI)-treated (10 µM CrO₃ in MEM for 24 h) and (iii) Cr(VI) + CSE_aq-treated, which were treated for 24 h with diverse concentrations (0.5, 1, 2, 3, 4 and 5 mg/ml) of CSE_aq (prepared in MEM containing 10 µM CrO₃). The viability of untreated cells was considered to be 100%. After 24 h, 20 µl of MTT solution (5 mg/ml in PBS) was added to each well and incubated at 37 ºC for 4 h in the CO₂ incubator. After incubation, the medium was carefully removed from each well, and was replaced by 100 µl of DMSO (to solubilize MTT–formazan complex). End-point absorbance was determined at 570 nm by using a Synergy H1 multimode microplate reader (BioTek Instruments, Inc., VT, USA). The percentage viability values of the Cr(VI)-treated and Cr(VI) + CSE_aq-treated cells were respectively calculated as:

$$\text{\% viability =}\frac{A_{\text{Cr(VI)}}}{A_{\text{UT}}}\times 100\text{and}\frac{A_{{\text{Cr(VI) + CSE}}_{\text{aq}}}}{A_{\text{UT}}}\times 100,$$

where A_UT, A_Cr(VI) and A_Cr(VI)+CSEaq are the absorbance values of the untreated, Cr(VI)-treated and Cr(VI) + CSE_aq-treated cells respectively.

Immunocytochemistry (ICC)

Cells (0.6 × 10⁶) were seeded on cover glasses and allowed to grow for 48 h. While untreated cells were grown in fresh medium, those in the Cr(VI)-treated group were exposed to 10 µM CrO₃ in MEM for 24 h. Cells belonging to the Cr(VI) + CSE_aq-treated groups were subjected to 10 µM CrO₃ in MEM along with diverse concentrations (0.5, 2.5 and 5 mg/ml) of CSE_aq for a span of 24 h. After the treatment period, cells were fixed with 4% paraformaldehyde at 4 ºC for 25 min, permeabilized (0.2% Triton X-100) for 5 min and blocked with 1% bovine serum albumin for 45 min. Antibody [against Ki-67 (#sc-15402), survivin (#sc-17779) or mTOR (#sc-293089)] treatments were done overnight at 4 ºC in a humid environment. Cells were then labelled with Cy3-tagged secondary antibodies [goat anti-mouse IgG H&L (Cy3^®) (#ab97035) and goat anti-rabbit IgG H&L (Cy3^®) (#ab6939)] and counterstained with DAPI for visualization under a fluorescence microscope (Eclipse Ni-U, Nikon Corporation, Tokyo, Japan). The images were obtained by using the NIS Elements (ver. 4.4) software (Nikon Corporation, Tokyo, Japan).

Western blot

Western blot was performed with lysates of WRL-68 cells (untreated, Cr(VI)-treated and Cr(VI) + CSE_aq-treated; 24 h). Concentrations of total protein in the lysates were measured using the BCA protein assay kit (Thermo Fisher Scientific Inc., USA). Cell lysates were electrophoresed in 10% SDS-PAGE and transferred onto nitrocellulose membranes by semi-dry electro-blotting using the Trans-Blot Turbo system (Bio-Rad Laboratories, Inc., USA). Membranes were blocked with 5% non-fat milk for 45 min at room temperature and then probed with specific antibodies [against PARP-1 (#sc-53643) and BECN1 (beclin-1) (#sc-48381)]. HRP-tagged mouse-IgGκ BP-HRP (#sc-516102) was used to detect protein bands using the Clarity™ Western ECL substrate (Bio-Rad Laboratories, Inc., USA). β-actin (#sc-47778) was used as an internal control. Protein bands were detected on X-ray films and scanned.

Test for oxidative stress in cells

Oxidative stress in WRL-68 cells was studied by estimating the abundance of cellular ROS. Cell aliquots (in suspension; 1 × 10⁶ cells/ml) were treated with Cr(VI) or Cr(VI) + CSE_aq for 12 h at 37 ºC [Cr(VI) group of cells: 10 μM CrO₃; Cr(VI) + CSE_aq groups of cells: 10 µM CrO₃ plus diverse concentrations (0.5, 2.5 and 5 mg/ml) of CSE_aq; untreated cells were resuspended in fresh PBS; positive control: 1 mM H₂O₂ in PBS]. Cells were then washed twice with PBS by centrifugation for 5 min at 1500 rpm (~ 132×g) and treated with 100 µM of 2ʹ,7′-dichlorofluorescin diacetate (DCFDA) solution (in PBS) for 40 min (with mild rocking under dark conditions) at room temperature. Fluorescence of DCFDA in cells was measured at 485⁄530 nm (excitation/emission) as an estimate of cellular ROS using a Synergy H1 multimode microplate reader.

DPPH^· scavenging activity

DPPH^· assay is a spectrophotometric method that is used to determine the antioxidant potential of a compound (Kedare and Singh 2011). The assay was performed following a protocol previously published by the authors (Guha et al. 2010c, 2011c). In short, CSE_aq (2 µl) was added in increasing concentrations (0.5, 1 and 2.5 mg/ml; made in methanol) to 1 ml of 1 mM DPPH^· solution (in methanol), mixed vigorously and incubated in dark at 18 ºC for 40 min. Absorbance was measured at 517 nm (λ_max) using an Evolution™ 201 UV–vis spectrophotometer (Thermo Scientific, MA, USA) with methanol as blank and ascorbic acid (100 µg/ml) as positive control. Percentage DPPH^· scavenging activity (%DSA) by CSE_aq was calculated according to the following formula:

$$\text{\% DSA =}\frac{({\alpha }_{\text{C}}-{\alpha }_{\text{S}})}{{\alpha }_{\text{C}}}\times 100,$$

where α_C is the mean absorbance of the controls and α_S is the absorbance of test samples.

Reducing power

Reducing power of CSE_aq was estimated by our previously published method (Guha et al. 2011a), where 20 µl of CSE_aq of varying concentrations (0.5, 1 and 2.5 mg/ml) was added to 2.5 ml of 0.2 M phosphate buffer (pH 6.6) and 2.5 ml of 1% potassium ferricyanide, followed by a 30 min incubation at 50 ºC. To this reaction mixture, 2.5 ml of 10% trichloroacetic acid was added and then centrifuged for 10 min at 3,000 rpm (~ 528 × g). From the supernatant, 2.5 ml was collected and mixed with an equal volume of distilled water and 0.5 ml of 0.1% ferric chloride. Finally, absorbance was measured at λ_max = 700 nm using an Evolution™ 201 UV–vis spectrophotometer. Higher absorbance values indicated greater reducing power (Guha et al. 2011a).

Estimation of total phenolic content of CSE_aq

Total phenolic content of CSE_aq was determined using the Folin–Ciocalteau reagent method, as described earlier by the authors (Guha et al. 2010c). CSE_aq (50 µl) was added to 2.5 ml of Folin–Ciocalteau reagent (10 times diluted) and 2 ml of 7.5% Na₂CO₃ (w/v). The contents were mixed well, and incubated at 45 ºC for 15 min, and absorbance was measured spectrophotometrically at λ_max = 765 nm with Na₂CO₃ solution (7.5%) as blank. The results were expressed in gallic acid equivalence (GAE) in µg/ml.

Determination of major phenolic compounds in CSE_aq

Phenolic constituents of CSE_aq were determined by HPLC using a Prominence System (Shimadzu Corp., Kyoto, Japan) equipped with dual λ detector and a Shim-pack GIST-HP C18 column (3 µm; 3 mm × 150 mm). The phenolic compounds were identified by comparing with the standard reference library for HPLC analyses (Sakakibara et al. 2003). Gradient elution was done at 35 ºC with solution A (50 mM sodium phosphate in 10% methanol; pH 3.3) and solution B (70% methanol) in the following gradient elution program: 0–15 min: 100% solution A; 15–45 min: 70% solution A; 45–65 min: 65% solution A; 65–70 min: 60% solution A; 70–95 min: 50% solution A; 95–100 min: 0% solution A. Flow rate was maintained at 1 ml/min and injection volume was 20 µl (of 5 mg/ml CSE_aq). Diverse phenolic compounds were identified by monitoring respective λ_max values for various phenolic compounds, i.e. 250 nm for most anthraquinones, benzoic acids and isoflavones; 280 nm for some anthraquinones, catechins, flavanones, some flavones and theaflavins; 320 nm for cinnamic acids, most chalcones and flavones; and 370 nm for flavonols (Sakakibara et al. 2003).

Statistical analyses

All analyses were carried out in replicates. Data were presented as mean ± SD. Statistical analyses were performed by one-way ANOVA, followed up with Bartlett's test for equal variances and Dunnett's multiple comparison test. Significant differences between groups were determined at P < 0.05. To evaluate relationships between experimental parameters, the results were analyzed to determine Pearson’s coefficients of correlation. MATLAB ver. 7.0 (MA, USA), GraphPad Prism ver. 5.00 (CA, USA) and Microsoft Excel 2007 (IL, USA) were used for the statistical and graphical evaluations.

Results

Extract yield

Exhaustive Soxhlet extraction of powdered C. cyminum seeds (60 g) yielded 13.32 g of CSE_aq, which was equivalent to 22.2% of the seeds’ dry weight.

CSE_aq mitigated Cr(VI)-induced decline in cell viability

The results (Fig. 1) obtained from MTT assay demonstrated a protective effect of CSE_aq on WRL-68 cells from Cr(VI)-induced toxicity. Cells treated with only 10 µM Cr(VI) exhibited a rigorous decline (by 58.82 ± 9.79%) in their viability in comparison to untreated cells. However, CSE_aq was able to alleviate such damage comprehensively. A significant (P < 0.0001) dose-dependent elevation in the viability of Cr(VI)-treated cells was observed on treatment with various concentrations (0.5, 1, 2, 3, 4 and 5 mg/ml) of CSE_aq. The lower concentrations of CSE_aq (0.5 and 1 mg/ml) showed a moderate increase in cell viability, which increased further at the intermediate doses (2–3 mg/ml). Cell viability got restored to approximately 80–100% on treatment with 4–5 mg/ml doses of CSE_aq.

Fig. 1.

Protective effect of CSE_aq against Cr(VI)-induced toxicity in WRL-68 cells. On treatment with 10 μM CrO₃ [Cr(VI)] for 24 h, viability of the cells plummeted comprehensively to less than 50% (in comparison to untreated cells whose mean viability is taken as 100%). When cells were treated with 0.5, 1, 2, 3, 4 and 5 mg/ml of CSE_aq along with Cr(VI), a significant (P < 0.0001) dose-dependent improvement in the viability of the cells was observed in comparison to the cells treated with only Cr(VI). Data were represented as mean ± SD (n = 24)

CSE_aq can restore proliferation of cells exposed to Cr(VI)

ICC analysis (Fig. 2) revealed that Ki-67 expression was holistically nullified in WRL-68 cells on exposure to 10 µM Cr(VI), together with an overall decline in cell number. However, increasing concentrations (0.5, 2.5 and 5 mg/ml) of CSE_aq demonstrated a dose-dependent restoration of Ki-67 expression in Cr(VI)-treated cells. The number of cells was also shown to be resuscitated; thereby, corroborating our observations from the MTT assay (Sect. “CSE_aq mitigated Cr(VI)-induced decline in cell viability”).

Fig. 2.

CSE_aq reversed Cr(VI)-induced inhibition of Ki-67 expression in WRL-68 cells. Cells treated with 10 μM CrO₃ [Cr(VI)] for 24 h demonstrated a holistic downregulation of Ki-67. On simultaneous treatment with different doses (0.5, 2.5 and 5 mg/ml) of CSE_aq, Ki-67 expression was extensively restored in the cells in a dose-dependent manner. Images are at 20× magnification

CSE_aq can mitigate Cr(VI)-induced mTOR/survivin-dependent autophagic cell death in WRL-68 cells

Western blot analysis (Fig. 3) revealed that 10 µM Cr(VI) substantially increased the expression of BECN1 in WRL-68 cells in comparison to the untreated group; thereby, signifying induction of autophagy. Simultaneously, we also observed that CSE_aq (0.5, 2.5 and 5 mg/ml) reversed this condition, which was evident from the reduction in BECN1 expression moderately (similar to the untreated cells).

Fig. 3.

BECN1 expression is induced by Cr(VI) in WRL-68 cells, but is mitigated by CSE_aq. Treatment of WRL-68 cells with 10 μM CrO₃ [Cr(VI)] for 24 h evidently augmented BECN1 expression level. CSE_aq, on the other hand, decreased the expression of BECN1 significantly (comparable to untreated cells)

Moreover, we observed that 10 µM Cr(VI) drastically obliterated mTOR expression in WRL-68 cells (Fig. 4). Again, congruous to the results of the BECN1 study, we noted that increasing concentrations of CSE_aq (0.5, 2.5 and 5 mg/ml) improved the expression of mTOR in a moderately dose-dependent pattern.

Fig. 4.

Cr(VI) mitigated the expression of mTOR in WRL-68 cells, while CSE_aq restored the same. WRL-68 cells, on 24 h treatment with 10 μM CrO₃ [Cr(VI)], demonstrated a plummeted level of mTOR. On co-administration of CrO₃ and CSE_aq (0.5, 2.5 and 5 mg/ml, respectively), mTOR expression was improved. Images were obtained at 20× magnification

In Fig. 5a, we show that in 10 µM Cr(VI)-treated WRL-68 cells, survivin expression was comprehensively lost, which was retrieved by CSE_aq (0.5, 2.5 and 5 mg/ml) in a dose-dependent manner. Since survivin is also known to be related to apoptosis, we analyzed whether the same concentration of Cr(VI) could cleave PARP-1 to form an 89 kDa fragment, a phenomenon indicating apoptosis (Chaitanya et al. 2010). However, PARP-1 cleavage was not observed in WRL-68 cells due to 10 µM Cr(VI) treatment (Fig. 5b); thereby, negating the occurrence of apoptosis. This suggested that CSE_aq can rescue WRL-68 cells from mTOR/survivin-governed autophagic cell death caused due to Cr(VI) treatment.

Fig. 5.

Cr(VI) caused a decline in survivin expression (that was restored by CSE_aq), but did not demonstrate PARP-1 cleavage. a 24 h treatment with 10 μM CrO₃ [Cr(VI)] nullified the expression of survivin in WRL-68 cells. Simultaneous treatment with CSE_aq (0.5, 2.5 and 5 mg/ml) recovered the level of survivin in a dose-dependent manner. Images are at 20 × magnification. b Cr(VI) treatment (24 h) did not cause cleavage (89 kDa fragment) of PARP-1 in WRL-68 cells. Treatment with 50 nM rapamycin (positive control) demonstrated cleaved PARP-1, which is a hallmark of apoptosis

CSE_aq can inhibit Cr(VI)-induced ROS generation in WRL-68 cells

We observed (Fig. 6) that 12 h treatment with Cr(VI) almost doubled the level of ROS in WRL-68 cells (P < 0.001). However, with simultaneous treatment with diverse concentrations (0.5, 2.5 and 5 mg/ml) of CSE_aq, ROS levels were restored to the normal state (as observed in untreated cells) in these Cr(VI)-treated cells.

Fig. 6.

CSE_aq protected WRL-68 cells from Cr(VI)-induced oxidative stress. Treatment of WRL-68 cells with 10 μM CrO₃ [Cr(VI)] for 12 h significantly (P < 0.001; denoted by ***) increased the level of ROS in the cells, which is manifested as the fluorescence emitted (relative fluorescence units, RFU) by DCFDA. Simultaneous treatment with 0.5, 2.5 and 5 mg/ml of CSE_aq reduced the cellular levels of ROS to degrees similar (P ≮ 0.05; denoted by #) to the untreated cells. Data were represented as mean ± SD (n = 4)

CSE_aq demonstrated comprehensive antioxidant potential

Quantitative analysis demonstrated significant (P < 0.001) dose-dependent (0.5, 1 and 2.5 mg/ml) DPPH^· scavenging ability of CSE_aq. Figure 7a shows the mean ± SD values of percentage DPPH^·-scavenging activity in comparison to ascorbic acid (100 µg/ml; positive control). Reducing power of a compound is a putative estimation of its antioxidant potential (Loganayaki et al. 2013). To corroborate the results of the DPPH^· assay, CSE_aq was tested for its reducing power. The extract demonstrated (Fig. 7b) extensive dose-dependent augmentation in its reducing power; thereby, validating its antioxidant potential.

Fig. 7.

Antioxidant potential of CSE_aq. a Percentage DPPH^· scavenging activity of CSE_aq (0.5, 1 and 2.5 mg/ml). The extract neutralized DPPH^· significantly (P < 0.001; denoted by ***; n = 6) in a dose-dependent manner; thereby, demonstrating its antioxidant efficacy. Ascorbic acid (100 μg/ml) was taken as the positive control (PC). Data were expressed as mean ± SD. b CSE_aq showed a significant (P < 0.05) dose-dependent (0.5, 1 and 2.5 mg/ml) increase in its reducing power. Data were presented as mean ± SD (n = 4), where ***P < 0.001 and **P < 0.01

Are the phenolic constituents of CSE_aq responsible for its protective potential?

Total phenolic content of CSE_aq at different concentrations (0.5, 1 and 2.5 mg/ml) were calculated in terms of GAE. The quantitative estimation indicated the presence of high degrees of phenolic content in the extract (Fig. 8).

Fig. 8.

Total phenolic content in varying concentrations of CSE_aq. Total phenolic content of the different CSE_aq doses (0.5, 1 and 2.5 mg/ml) is expressed in gallic acid equivalence (GAE; in μg/ml). Data shown as mean ± SD (n = 3). ***P < 0.001; **P < 0.01

To ascertain whether the phenolic constituents of CSE_aq can be accredited for its antioxidant property and simultaneous mitigation of Cr(VI) toxicity, correlation analyses were carried out. Total phenolic content of CSE_aq demonstrated robust positive correlations with its potential for cell viability restoration (R² = 0.91), oxidative stress amelioration (R² = 0.99), DPPH^· scavenging activity (R² = 0.99) and reducing power (R² = 0.96) (Fig. 9). This suggested that the phenolics in CSE_aq might be responsible for the therapeutic potential observed in this study.

Fig. 9.

Therapeutic efficacies of CSE_aq against Cr(VI)-associated cellular toxicity are correlated to the phenolic content of the extract_. Total phenolic content of CSE_aq is strongly correlated with the a cytoprotective activity (R² = 0.91) of CSE_aq against Cr(VI)-induced toxicity in WRL-68 cells; b mitigation of Cr(VI)-governed cellular oxidative stress (R² = 0.99) by CSE_aq; c DPPH^· scavenging activity (R² = 0.99) of CSE_aq; and d reducing power of CSE_aq. All parameters a–d showed significant positive correlations with total phenolic content (at P < 0.05) of CSE_aq

Since all results indicated that CSE_aq phenolics might harbour the reason for its functional efficacies, it was prudent to identify them. In this study, we identified the major phenolic compounds in CSE_aq using HPLC by referring to the library of analytical characteristics (retention time and determining λ) of more than one hundred phenolic standards (Sakakibara et al. 2003). Table 1 shows the major phenolic compounds present in CSE_aq.

Table 1.

Major phenolic compounds identified in CSE_aq by HPLC

Compound		λ (nm)	RTE (min) Mean ± SDa	RTR (min)b
Benzoic acids
1	Gallic acid	250	5.83 ± 0.090	5.8
2	p-Hydroxybenzoic acid	250	13.76 ± 0.008	13.8
Cinnamic acids
3	m-Coumaric acid	320	24.8 ± 0.056	24.8
4	o-Coumaric acid	320	21.83 ± 0.070	21.9
5	Ferulic acid	320	25.9 ± 0.025	25.8
6	Isoferulic acid	320	26.32 ± 0.072	26.3
Chalcone
7	Butein	320	79.27 ± 0.117	79.2
Flavanones
8	Hesperetin-7-O-rutinoside (hesperidin)	280	45.6 ± 0.048	45.6
9	(+)-taxifolin	280	26.61 ± 0.013	26.7
Flavones
10	Apigenin-8-C-glucoside (vitexin)	320	31.77 ± 0.062	31.7
11	7,4′-Dihydroxyflavone	320	75.79 ± 0.080	75.7
12	Luteolin-8-C-glucoside (orientin)	320	26.17 ± 0.069	26.2
13	Sinensetin	320	86.27 ± 0.054	86.3
14	7,3′,4′-trihydroxyflavone	320	60.52 ± 0.046	60.5
Flavonol
15	Quercetin	370	75.53 ± 0.002	75.5
Isoflavones
16	Daidzein-8-C-glucoside (puerarin)	250	20.18 ± 0.080	20.1
17	Glycitein-7-O-glucoside (glycitin)	250	25.66 ± 0.041	25.6

λ wavelength of determination, RT_E experimental retention time, RT_R reference retention time

^an = 3

^bSakakibara, Honda, Nakagawa, Ashida and Kanazawa, 2003

Discussion

Oxidative stress affecting the variegated structural and functional facets of cells often leads to cellular dysfunction and death (Ott et al. 2007; Ryter et al. 2007; Di Gioacchino et al. 2008a, b; Das and Guha 2011; Lee et al. 2014; Sun et al. 2015; Xueting et al. 2018; Yu et al. 2018). Cr(VI) is a proficient generator of ROS, and causes severe toxicity to cells (Ding and Shi 2002; Davidson et al. 2007). Simultaneously, Cr(VI) also functionally diminishes the endogenous antioxidant faculties of cells (Stanley et al. 2014; Husain and Mahmood 2017). A number of studies in the recent past have reported that extracts obtained from diverse floral resources have the potential to protect biological systems from Cr(VI)-induced oxidative injury (Guha et al. 2010c, 2011b; Ávila et al. 2016; Mahmoud and Abd El-Twab 2017; Johnson et al. 2018). Taking a cue from such reports, we investigated the protective potential of C. cyminum seeds against Cr(VI) toxicity in WRL-68 cells.

For our experiments, we selected a dose of 10 μM Cr(VI), which evidently brought down the percentage viability of WRL-68 cells to 41.18 ± 9.79% in 24 h. CSE_aq, in varying concentrations, was able to extensively thwart the plummet of cell viability due to Cr(VI) toxicity. In congruity to previous reports from different experimental model systems (Eleftheriou et al. 2012; Xiao et al. 2012), we too observed that exposure to Cr(VI) for 24 h hindered cell proliferation, as was evident from the decline in the expression of Ki-67, a putative molecular marker of cell proliferation (Scholzen and Gerdes 2000). Interestingly, CSE_aq restored the level of Ki-67 in Cr(VI)-treated cells. Since the effect of the extract on the viability of Cr(VI)-treated cells complemented our observation of cell proliferation restoration, it was inferred that the extract possesses the potential to mitigate Cr(VI)-induced cellular injury.

A number of authors have suggested that autophagy can protect cells that are under oxidative stress by obliterating the damaged components; thereby, rejuvenating the structural and functional features of the cells (Filomeni et al. 2015; Shi et al. 2018; Liu et al. 2018b; Varmazyar et al. 2019). Autophagy is putatively known to be a cytophysiological process through which defective components of cells are destroyed to maintain cellular homeostasis (Filomeni et al. 2015; Shi et al. 2018; Liu et al. 2018b; Varmazyar et al. 2019). However, it is not always protective in nature. Comprehensive levels of autophagy can also mediate cellular demise—a process known as autophagic cell death (Galluzzi et al. 2012), which has been extensively reported to be a paramount cause of cell mortality (Chen et al. 2008a, b; Cheng et al. 2009; You et al. 2015; Wang et al. 2016; Yuan et al. 2017; Liu et al. 2018a), independent of apoptosis and necrosis (Clarke and Puyal 2012). We observed that 24 h treatment of WRL-68 cells with 10 μM Cr(VI) induced autophagic cell death, as was evident from the induction of BECN1 expression vis-à-vis the mitigation of cell viability. BECN1, which is the yeast Atg6 ortholog present in mammals, is a fundamental component of the class III phosphatidylinositol 3-kinase (PI3K-III) complex that generates phosphatidylinositol-3-phosphate (PI3P); thus, facilitating the formation of autophagosome (Kang et al. 2011; McKnight and Zhenyu 2013). It is, therefore, considered as a universal marker for autophagy, and is also a requisite for the induction of autophagic cell death (Yu et al. 2004; Jung et al. 2010). This observation was in agreement with the decline of mTOR (a serine-threonine kinase) expression, which is a negative regulator of BECN1 (Paquette et al. 2018). Inhibition of mTOR has been associated with autophagy, and in many cases, with autophagic cell death (Jung et al. 2010; Kim and Guan 2015; Yang et al. 2018; Liu et al. 2018b). At the same time, mTOR is also known to induce the expression of survivin, an inhibitor of autophagic cell death (Kang et al. 2011), which plays a major role in cell proliferation and cell cycle regulation (Vaira et al. 2007; Mita et al. 2008). Moreover, survivin interacts with BECN1 to inhibit autophagy (Niu et al. 2010; Kang et al. 2011). We observed that the expression of survivin was diminished due to Cr(VI) treatment. It must be noted that along with its role in inhibiting autophagic cell death, survivin is also known for its role in inhibiting the apoptotic machinery (Roca et al. 2008; Zhang et al. 2015; Humphry and Wheatley 2018). The decline in survivin expression corroborated our observations suggesting autophagic death of WRL-68 cells, while we did not observe any evidence of apoptosis (no PARP-1 cleavage).

Oxidative stress has been correlated to autophagic cell death by a large number of studies (Chen et al. 2008b; Lee et al. 2011; Dando et al. 2013; Shi et al. 2016; Yuan et al. 2017; Ji et al. 2018). We observed that Cr(VI) can generate copious magnitudes of ROS in WRL-68 cells. This suggested the induction of autophagic cell death due to Cr(VI)-induced oxidative injury (Fig. 10). CSE_aq, however, demonstrated the ability to reverse such deleterious changes in the cellular facets by bringing the expressions of BECN1, mTOR and survivin back to their respective normal levels.

Fig. 10.

Phenolic contents of CSE_aq inhibit the oxidative cascade of autophagic cell death induced by Cr(VI) in WRL-68 cells. Mitigation of oxidative stress in the cells sustains the functions of mTOR; thereby, restoring the normal level of survivin, while inhibiting BECN1. Consequently, CSE_aq prevents the Cr(VI)-induced loss of cellular integrity

Antioxidants are known to scavenge DPPH^· (a stable free radical) by donating protons to the latter (Brand-Williams et al. 1995; Guha et al. 2010c, 2011a). It is further reported that antioxidants can prevent oxidative stress-induced autophagic cell death (Kim et al. 2014). To examine whether CSE_aq has significant antioxidant potential, biochemical tests (assays for estimating DPPH^· scavenging activity and reducing power of CSE_aq) were performed, which revealed that the extract has considerable antioxidant property. This conformed with previous studies on the antioxidant potential of C. cyminum (Abdelhaliem and Al-Huqail 2016; Moubarz et al. 2016; Sharifzadeh et al. 2016; Ben Miri and Djenane 2018). On the other hand, CSE_aq also alleviated the degree of ROS generated in WRL-68 cells due to Cr(VI) treatment.

Phenolic compounds are molecules containing at least one aromatic ring holding a hydroxyl group, together with their functional derivatives (Strack 1997; Guha et al. 2011c). These compounds have emerged as one of the major category of antioxidants in plant extracts (Guha et al. 2010a, b; Rajkumar et al. 2011; Tuberoso et al. 2016; Huang et al. 2017; Nascimento et al. 2018). Moreover, a number of previous reports have stated that phenolic compounds are capable of mitigating heavy metal-induced oxidative stress in biological systems (Michalak 2006; Keilig and Ludwig-Mül 2009; Guha et al. 2010c, 2011b; Kulbat 2016).

Biochemical examination of CSE_aq revealed high level of total phenolic constituents in the extract. Furthermore, the phenolic content of CSE_aq showed significant correlation with the extract’s potential for cell viability restoration, oxidative stress mitigation, DPPH^· scavenging activity and reducing power. A variety of phenolic compounds were also identified in CSE_aq by HPLC analysis. This suggested that the protective potential of CSE_aq against Cr(VI) toxicity in WRL-68 cells might be attributed to the phenolic constituents of the extract.

In epilog, we inferred that Cr(VI)-induced oxidative stress attenuated the expressions of mTOR and survivin; thereby, upregulating BECN1 expression, and consequently triggering autophagic cell death (Fig. 10). It was also concluded that aqueous extract of C. cyminum seeds possesses considerable antioxidant potential (due to its various polyphenolic constituents) that mitigates Cr(VI)-induced oxidative injury to WRL-68 cells and the subsequent mTOR/survivin-mediated autophagic death cascade (Fig. 10). The spice, being an evident source of natural phenolics, might emerge as a potential herbal therapeutic-based intervention for heavy metal toxicity.

Abbreviations

AGE

Advanced glycation end product

BECN1

Beclin-1

Cr(VI)

Hexavalent chromium

CrO₃

Chromium trioxide

CSE_aq

Aqueous extract of cumin seeds

DAPI

4′,6-Diamidino-2-phenylindole

DCFDA

2′,7′-Dichlorofluorescin diacetate

DMSO

Dimethyl sulphoxide

DPPH^·

2,2-Diphenyl-1-picrylhydrazyl radical

DSA

DPPH^· scavenging activity

GAE

Gallic acid equivalence

HPLC

High-performance liquid chromatography

MEM

Minimum essential medium Eagle

mTOR

Mechanistic target of rapamycin

mTORC1

MTOR complex 1

MTT

3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide

PARP-1

Poly(ADP-ribose) polymerase-1

PBS

Phosphate buffered saline

PI3K-III

Class III phosphatidylinositol 3-kinase complex

PI3P

Phosphatidylinositol-3-phosphate

ROS

Reactive oxygen species

Author contributions

Idea conception and experiment designs by: GG and DB-G; Experiments performed by: RM, JP and BNVH; Data analyzed by: GG, DB-G, RM and JP; Paper written by: GG and DB-G.

Funding

This study was funded by the Grants YSS/2014/000139 (GG), YSS/2015/000025 (DB-G), ECR/2016/001856/ES (BNVH) and SR/FST/ETI-331/2013 (SASTRA) from the Department of Science and Technology Science (DST), Government of India; and BT/PR22434/MED/30/1901/2017 (GG and DB-G) from the Department of Biotechnology (DBT), Government of India.

Compliance with ethical standards

Conflict of interest

The authors declare that there is no conflict of interest.