Antitumor properties of certain spirooxindoles towards hepatocellular carcinoma endowed with antioxidant activity

Abstract In the current medical era, spirooxindole motif stands out as a privileged heterospirocyclic scaffold that represents the core for a wide range of bioactive naturally isolated products (such as Strychnofoline and spirotryprostatins A and B) and synthetic compounds. Interestingly, no much attention has been paid to develop spirooxindole derivatives with dual antioxidant and anticancer activities. In this context, a series of spirooxindoles 6a-p was examined for their anticancer effect towards HepG2 hepatocellular carcinoma and PC-3 prostate cancer cell lines. Spirooxindole 6a was found to be an efficient anti-proliferative agent towards both HepG2 and PC-3 cells (IC50 = 6.9 and 11.8 µM, respectively). Afterwards, spirooxindole 6a was assessed for its apoptosis induction potential in HepG2 cells, where its pro-apoptotic impact was approved via the significant elevation in the Bax/Bcl-2 ratio and the expression levels of caspase-3,


Introduction
In the current medical era much attention has been paid to fight the extreme assembly of reactive oxygen species (ROS), such as hydrogen peroxide, hydroxyl radicals, or superoxide anion, that aggressively interact with natural macromolecules in different tissues by chain process resulting in an oxidative damage 1,2 . Biological systems have the capability to defend themselves against these ROS via enzymes (such as glutathione peroxidase, superoxide dismutase, and catalase) or by non-enzymatic mechanisms that involve organic antioxidants molecules (such as glutathione, vitamin C, vitamin E). These antioxidants are capable of direct scavenging the oxidant species through breaking of oxidation propagation chain reactions or through the generation of hydrogen atom in stoichiometric manner, hence preventing free radical-associated damage 1,2 .
Both depletion of antioxidant defences or abnormal ROS production, result in the oxidative stress state that implicated in the aetiology of a variety of disorders such as Alzheimer's 3 , rheumatoid arthritis 4 , Huntington's disease 5 , Parkinson's disease 6 , coronary heart disease 7 , and insulin resistance 8 . Furthermore, elevated levels of ROS can promote tumorigenesis by initiation of mitochondrial or nuclear DNA mutations that promote neoplastic transformation, promoting genomic instability, or activating prooncogenic signalling pathways 9 . So, novel antioxidants featuring a variety of chemical structures are needed for prevention of cancer and to augment chemotherapies for different malignancies.
Being abundant in nature, isatins have been found in fluids and tissues of mammals, in addition to natural products that are produced by a range of bacteria, plants, and invertebrates 10 . Isatin motif is a central privileged scaffold in a large number of bioactive natural and synthetic products that possess a spectrum of bioactivities such as antioxidant 11 and anticancer activities [12][13][14] . On the other hand, the natural spirooxindole alkaloids were first isolated from plants of the Apocynaceae and Rubiacae families 15 . Thereafter, spirooxindole motif stands out as a privileged heterospirocyclic scaffold that represents the core for a wide range of naturally isolated products such as alstonisine 16 , horsfiline 17 , coerulescine 18 , and chitosenine 19 with diverse bioactivity profiles. Strychnofoline, spirotryprostatin A and spirotryprostatin B ( Figure  1) are other examples for natural spirooxindoles possessing anticancer activities 20,21 . Isolation and identification of such bioactive spirooxindole alkaloids have inspired the researchers and paved the way to design and synthesise various spirooxindole-based small molecules with promising biological activities such as antioxidant 22 and anticancer [23][24][25] activities.
Recently, we have reported a one-pot three-component synthesis of novel spirooxindole derivatives 6a-p as efficient anti-proliferative agents towards the aggressive triple-negative breast cancer MDA-MB-231 cell line 25 . In the current study, the reported promising anti-proliferative impact of spirooxindoles 6a-p spurred us to extend our investigations around their different potential biological activities, where they were evaluated for their potential antioxidant activity via assessing their DPPH radical scavenging capacities and Ferric reducing antioxidant power (FRAP). In addition, 6a-p were examined for their anticancer effect towards HepG2 hepatocellular carcinoma and PC-3 prostate cancer cell lines. Furthermore, spirooxindole 6a was then assessed for its apoptosis induction potential in HepG2 hepatocellular carcinoma cell line, so as to obtain some mechanistic insights for the antiproliferative impact of the herein reported spirooxindoles. Finally, an in silico study was carried out by the use of the automated SwissADME tool to afford clues about the physicochemical properties, ADME parameters, drug-like nature and pharmacokinetic properties of spirooxindole 6a.

Chemistry
Spirooxindoles 6a-p were prepared earlier by our group 25 .

Biological evaluation
Anti-proliferative activity Anti-proliferative activity towards HepG2 and PC-3 cell lines. Spirooxindoles 6a-p were evaluated for their anti-proliferative potency towards HepG2 liver cancer and PC-3 prostate cancer cell lines. Both cells lines were cultured as monolayers in DMEM supplemented with 10% foetal bovine serum (FBS), L-glutamine (2 mM), penicillin (100 U/mL) and streptomycin sulphate (100 mg/mL). Then, cells were sub-cultured with trypsine/EDTA solution, counted with haemocytometer and plated onto 96-well plates (5000 cells/well) and left overnight so as to form a semiconfluent monolayer. A modified method was employed utilising the (3-[4,5-dimethylthiazol-2-yl] À 2,5-diphenyltetrazolium bromide (MTT) dye, as reported earlier [25][26][27] . IC 50 values were calculated using non-linear regression curve fitting of the dose response plots on GraphPad Prism V.6.0 software. Assessment of morphological changes of HepG2 cells after treatment with the active compounds 6a, 6e and 6i were carried out by phase contrast inverted microscope (Ziess, USA) and photomicrographs were taken using digital camera.
Apoptosis study. The levels of the apoptotic markers caspase-3, caspase-9, p53 and Bax, as well as the anti-apoptotic marker Bcl-2 were evaluated by the use of ELISA colorimetric kits per the manufacturer's instructions, as reported earlier 28,29 . Furthermore, the pro-apoptotic potential of spirooxindole 6a towards HepG2 cells was assessed using FITC Annexin V Apoptosis Detection Kit by flow cytometry, according to the manufacturer's protocol and referring to the reported procedures 30,31 .
Cell cycle analysis. Cell cycle distributions in HepG2 cells were examined through PI staining and analysed by flow cytometry after treatment with spirooxindole 6a at its IC 50 (6.9 mM), as reported earlier 30,31 . The samples were analysed by a flow cytometer (BectonDickinson FACSCalibur, BD, USA) and data were analysed using the CellQuest software (Becton Dickinson).
Antioxidant activity DPPH radical scavenging activity. The antioxidant potential of spirooxindoles 6a-p against DPPH_ was evaluated as per previously described procedures by Brand-Williams et al. 32 . DPPH solution was prepared by dissolving 0.024 g DPPH in methanol (100 mL). In 96-well plates, Â lL sample and 200-x lL DPPH solution were added. The plate was incubated for 30 min at room temperature in the dark, and finally, absorbance was recorded at 517 nm wavelength. Ascorbic acid was used as positive control. The percent radical scavenging potential was calculated for each compound via the following equation: Scavenging effect (%) ¼ f(Absorbance of control À Absorbance of sample)/Absorbance of controlg Â 100.
Ferric reducing antioxidant power (FRAP). The Benzie and Strain method 33 was adopted to assess the ferric reducing antioxidant power (FRAP) for spirooxindoles 6a-p. Absorbance of mixtures was recorded at 593 nm after incubation for 30 min at 37 C. FRAP results were expressed as lM Fe 2þ equivalents.

Biological evaluation
Antitumor activity Anti-proliferative activity towards HepG2 and PC-3 cell lines.
Spirooxindoles 6a-p were tested for their in vitro anti-HCC activity against HepG2 cell line, and for their anti-prostate cancer activity against PC-3 cell line by the use of MTT reduction assay 27 . The anticancer drug Adriamycin was co-assayed as the positive control. The anti-proliferative activities of tested spirooxindoles against the two cancer cell lines were measured as IC 50 (in lM) value (the dose that affords a 50% inhibition of cell growth after 48 h of incubation) and displayed in Table 1.
Investigation of the obtained results in Table 1 revealed that the tested spirooxindoles were more effective towards HCC HepG2 cells rather than prostate cancer PC-3 cells, except compounds 6f, 6 m and 6n which caused slightly enhanced growth inhibitory activity against PC-3 (IC 50 ¼ 29.3, 24.3 and 42.8 mM, respectively) than HepG2 cells (IC 50 ¼ 32.7, 30.6 and >50.0 mM, respectively).
Regarding the activity towards HepG2 cells, the data displayed in Table 1 ascribed to the tested spirooxindoles excellent to weak efficacy in inhibiting the growth of the HCC HepG2 cells (IC 50 values ranging between 6.3 and 49.1 mM, Table 1), except compounds 6 g and 6n-6p which failed to inhibit the growth of HepG2 cells up to 50 lM. In particular, spirooxindoles 6a and 6i were found to be the most potent derivatives in this study against HepG2 cells with IC 50 values equal to 6.9 and 6.3 lM, respectively. In addition, spirooxindoles 6e and 6j exhibited good activity towards HepG2 cells (IC 50 ¼ 8.4 and 9.9 mM, respectively), whereas, compounds 6 b, 6c, 6 h, 6k and 6 l elicited moderate potency against HepG2 cells with IC 50 range: 12.7 À 21.0 mM. Morphological assessment revealed the toxic effects of spirooxinodoles 6a, 6e and 6i as presented in Figure 2 including dramatic cellular effects including cell rounding, shrinkage and monolayer disruption.
On the other hand, it was apparent from the obtained results that most of the prepared spirooxindoles possessed moderate to modest growth inhibitory activity towards prostate cancer PC-3 cells with IC 50 in the range of 11.8 À 43.7 mM. Compounds 6a and 6e were found to be the most potent spirooxindoles towards PC-3 cells with IC 50 values equal to 11.8 and 13.5 lM, respectively. Moreover, spirooxindoles 6i, 6j and 6 m were moderately active against PC-3 cells with (IC 50 ¼ 17.9, 26.6 and 24.3 mM, respectively).
On the other hand, spirooxindoles 6a-c, 6e, 6f, 6i-k and 6 m, with dual activity against both here examined HepG2 and PC-3 cancer cell lines, were tested for their potential cytotoxic impact towards the non-tumorigenic human breast MCF-10A cell line in order to explore the safety profile of the target spirooxindole scaffold towards the normal cells, Table 2.
The tested spirooxindoles 6a-c, 6e, 6f, 6i-k and 6 m exerted non-significant cytotoxicity towards the non-tumorigenic MCF-10A cells with IC 50 values spanning between 69.74 and 185.96 mM, respectively. In addition, the calculated for the tested spirooxindoles S. I. lied in the range: 4.9-14.8. Superiorly, spirooxindoles 6a and 6j displayed excellent safety profile with S. I. equal 13.7 and 14.8, respectively, Table 2.
Flow cytometric analysis and apoptotic studies in HepG2 cells. Spirooxindole 6a was found to be an efficient anti-proliferative agent towards both HepG2 and PC-3 cells (IC 50 ¼ 6.9 and 11.8 mM, respectively, Table 1), in addition to its promising antioxidant activities (Tables 5 and 6). Accordingly, spirooxindole 6a was selected for further evaluations to acquire mechanistic insights into its anti-proliferative activity against HepG2 cells.
Cell cycle analysis. The impact of spirooxindole 6a on the normal cell cycle progression was investigated using flow cytometric analysis of the DNA ploidy in HepG2 cells. Exposure of HepG2 cells to spirooxindole 6a at its IC 50 (6.9 mM) for 24 h revealed the ability of 6a to disrupt the normal cell cycle by decreasing both G0-G1 and S phases by approximately 0.53-and 0.37-folds with respect to the control (Figure 3). Furthermore, both Pre-G and G2/M phases of the treated HepG2 cell were significantly increased by 11.9-and 2.1-folds related to the control (Figure 3). Alteration of the Pre-G phase and arrest of G2-M phase may imply apoptosis as a potential mechanism for 6a-induced cancer cell death.    Effect of 6a on the levels of the apoptotic markers (Bax, caspase-3, caspase-9 and p53), and the anti-apoptotic marker Bcl-2. To investigate the ability of spirooxindole 6a to provoke apoptosis, herein we assessed the expression levels of the hallmark parameters of apoptosis in HepG2 cells upon treatment with 6a at its IC 50 (6.9 mM) (Tables 3 and 4). In this study, exposure of HepG2 cells to spirooxindole 6a resulted in a significant 5.7-fold increase in the expression of the pro-apoptotic protein Bax, with concurrent significant down-regulation of the expression levels of the anti-apoptotic protein Bcl-2 by $ 42% compared to the control (Table 3).
Analysing these results declared that spirooxindole 6a boosted the Bax/Bcl-2 ratio nine folds in comparison to the control. The Bax/Bcl-2 ratio is considered as a decisive value that gives a real insight to the overall pro-apoptotic impact of the examined compound. The elevated Bax/Bcl-2 ratio aroused the assessment of the expression levels of the pro-apoptotic active caspase-3, caspase-9 and p53 tumour suppressor protein. Treatment of HepG2 cells with spirooxindole 6a resulted in a significant elevation in the expression levels of caspase-3, caspase-9 and p53 proteins by about 8.88, 12.27 and 10.03 folds, respectively, compared to control ( Table 4).
The simultaneous up-regulation of the downstream caspase 3, the hallmark key player and the key executor of apoptosis, alongside to p53 highlighted that a cascade of apoptotic markers was activated as a consequence of Bax/Bcl2 elevation that eventually resulted in apoptosis.
Annexin V-FITC apoptosis assay. To confirm the apoptotic effect of spirooxindole 6a in HepG2 cells, Annexin-V FTIC/PI dual staining assay was carried out at IC 50 of 6a (6.9 mM). Examining the results of this flow cytometric analysis (Figure 4), revealed that HepG2 cells treated with spirooxindole 6a exhibited a significant increase in the percentage of annexin-V positive cells indicating an early (lower right quadrant) and late (upper right quadrant) apoptosis from 0.95% to 6.76%, and from 0.33% to 14.14%, respectively, which comprises about 16.3-folds total increase with respect to control (Figure 4).
Antioxidant activity DPPH radical scavenging activity. The DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging activity assay is considered as one of the most common and simple methods for measuring the antioxidant ability to trap free radicals. DPPH is a commercially available stable free radical that possesses the ability to accept an electron to produce a stable molecule. Substances that have the potential to act as electron donors result in reduction of this DPPH radical to the corresponding DPPH 2 disclosing their antioxidant potential. The principle of the method relies upon measuring the absorbance of the odd electrons of DPPH at 517 nm. The absorbance decreases, in the presence of free radical scavengers, proportional to the decrease of the DPPH radical concentration 34 . In the current study, the scavenging effect of compounds 6a-p was evaluated via the DPPH radical scavenging assay, and compared with the standard antioxidant; ascorbic acid. The results, summarised in Table 5, are expressed in terms of IC 50 values that produce 50% inhibition of the DPPH radical; where the lower is this value, the more active is the examined compound.
From the obtained results, it was obvious that most of the prepared spirooxindoles 6a-p exhibited excellent to moderate DPPH radical scavenging capacities with IC 50 spanning in the range: 28.07 À 113.45 mg/mL. Superiorly, both spirooxindoles 6a and 6 m emerged as the most efficient DPPH radical scavengers with IC 50 values of 29.76 ± 1.82 and 28.07 ± 1.63 mg/mL, respectively, which are 1.6-fold more potent than the standard antioxidant Ascorbic acid (IC 50 ¼ 45.89 ± 2.39 mg/mL). Also, compounds 6c-6f, 6i, 6j and 6 l showed higher DPPH radical scavenging capacities (IC 50 range: 33.43 ± 1.24 À 43.42 ± 3.04 mg/mL) than Ascorbic acid. Furthermore, spirooxindoles 6 h, 6k and 6n-6p displayed moderate antiradical activity with IC 50 values of 46.29 ± 2.18, 69.25 ± 4.25, 46.29 ± 2.52, 58.82 ± 3.14 and 55.05 ± 3.41 mg/mL, respectively. Contrariwise, compounds 6 b and 6 g elicited weak DPPH_ scavenging activity with IC 50 values of 113.45 ± 5.59 and 97.47 ± 5.62 mg/mL, respectively.   Ferric reducing antioxidant power (FRAP). FRAP assay is a widely used antioxidant assay that uses antioxidant substances as reducing agents in a redox-linked colorimetric reaction, wherein Fe 3þ is reduced to Fe 2þ at acidic pH that results in a formation of a coloured ferrous-probe complex from a colourless ferric-probe complex 35 . In this assay, the electron donation capacity (reflecting the electron transfer ability) of the examined compounds is evaluated.
In this study, all spirooxindoles 6a-p herein reported were evaluated for their potential FRAP. The results were expressed as mM Fe 2þ equivalents and presented in Table 6, where the higher mM Fe 2þ equivalents value means higher FRAP and subsequently higher antioxidant capacity. As in the above DPPH assay, both spirooxindoles 6a and 6 m exhibited the best FRAP activity (11.98 and 12.13 mM, respectively) that is comparable to the activity of the reference Ascorbic acid (13.42 mM). Moreover, compounds 6c-6f, 6 h-6j, 6 l and 6n-6p possessed good reducing capacity in this assay with FRAP range: 10.12 À 11.90 mM.

Physicochemical, ADME and pharmacokinetic properties prediction
For prediction of the pharmacokinetic properties, ADME parameters and drug-like nature of spirooxindole 6a, the automated SwissADME online web tool 36 was employed. Articulating such properties is of great significance in the context of pharmaceutical chemistry to develop efficient clinical candidates. Figure 5(A) displays the BOILED-Egg graph of the WLOGP vs. TPSA (topological polar surface area) for spirooxindole 6a 37 , where it is located in the region of human intestinal absorption (HIA) with no BBB permeability so it is anticipated to display low occurrence for CNS side effects. Also, this graph revealed that spirooxindole 6a is not a P-glycoprotein substrate (PGPÀ), therefore it is not susceptible to the efflux mechanism carried out by this transporter that utilised by many cancer cell lines as a drug-resistance mechanism 38,39 . Figure 5(B) represents the bioavailability radar chart for spirooxindole 6a, which consists of six axes for six key properties for oral bioavailability; polarity (POLAR), solubility (INSOLU), lipophilicity (LIPO), flexibility (FLEX), saturation (INSATU) and size (SIZE) 39,40 . The range for optimal property values is displayed as a pink area, whereas the red line represents the predicted properties for the examined molecule.
Furthermore, as shown in Table 7, spirooxindole 6a showed high predicted level of gastrointestinal absorption, and predicted to be inhibitor of three of CYP isoforms (CYP1A2, CYP2C9 and CYP2C19) whereas predicted to be non-inhibitor of CYP3A4 and CYP2D6 isoforms.
Moreover, SwissADME tool showed that spirooxindole 6a fulfils the drug-likeness characteristics as defined by the major pharmaceutical companies (Table 8) and passed their filters; Lipinski's (Pfizer) 41 , Veber's (GSK) 42 and Egan's (Pharmacia) 43 filters, whereas it failed to pass Ghose's (Amgen) filter due to two violations; molecular weight > 480, and molar refractivity >130 44 . However, it was not classified as lead-like because its molecular weight is higher than 350 and its predicted XLOGP3 45 exceeded 3.5.
In summary, this in silico study for predicting the physicochemical and pharmacokinetic properties of spirooxindole 6a revealed that it is not only with promising antioxidant and anti-proliferative biological activities, but also with promising pharmacokinetic properties.

Conclusions
In summary, a series of spirooxindole-based derivatives (6a-p) was examined for its anticancer effect towards HepG2 hepatocellular carcinoma and PC-3 prostate cancer cell lines. Spirooxindoles 6a-p were more effective towards HCC HepG2 cells rather than prostate cancer PC-3 cells, except compounds 6f, 6 m and 6n. In particular, spirooxindoles 6a and 6i were found to be the most potent derivatives in this study against HepG2 cells with IC 50 values equal to 6.9 and 6.3 lM, respectively. The SAR outcomes highlighted that C-5 substitution of the indoline moiety with a large lipophilic Br group, and incorporation of unsubstituted phenyl group at C-6 of pyrazolo[3,4-b]pyridine moiety are indispensable factors for the growth inhibitory activity against HepG2 cells. Furthermore, spirooxindole 6a was assessed for its apoptosis induction potential in HepG2 cells, where its pro-apoptotic impact was approved via the significant elevation in the Bax/Bcl-2 ratio and the expression levels of caspase-3, caspase-9 and p53 proteins by about 9, 8.8, 12.2 and 10 folds, respectively, and via the  significant increase in the annexin V-FITC positive apoptotic cells percent. Besides, 6a-p were screened for their potential antioxidant activity via assessing their DPPH radical scavenging capacities and Ferric reducing antioxidant power (FRAP). The obtained results indicated that spirooxindoles 6a and 6 m are the most efficient DPPH radical scavengers (IC 50 ¼ 29.76 ± 1.82 and 28.07 ± 1.63 mg/mL, respectively), and exhibited the best FRAP activity (11.98 and 12.13 mM, respectively). Finally, an in silico study was carried out to predict the physicochemical and pharmacokinetic properties of spirooxindole 6a, which revealed that 6a is not only with promising antioxidant and anti-proliferative activities, but also with proper drug-likeness and pharmacokinetic properties.

Disclosure statement
No potential conflict of interest was reported by the author(s).