Synthesised thymoquinone-oxime induces cytotoxicity, genotoxicity and apoptosis in hepatocellular cancer cells: in vitro study

Abstract Hepatocellular carcinoma is the most common primary malignant tumor of the liver, and its incidence is increasing worldwide. There is a need to develop new therapeutic strategies to treat the disease. In this study, we synthesised the oxime derivative of thymoquinone and investigated cytotoxicity, genotoxicity, and apoptosis in hepatocellular cancer cells. The synthesised thymoquinone-oxime structure was confirmed by NMR. After incubating the hepatocellular cancer cell line for 24 h, the cytotoxicity ATP by luminometric, intracellular reactive oxygen species, and intracellular calcium by fluorometric. The mitochondrial membrane potential was determined by flow cytometry. DNA damage by alkaline single-cell gel electrophoresis, and apoptosis damage by acridine orange/ethidium bromide double dye method. Concentrations of thymoquinone-oxime statistically increased cytotoxicity, intracellular reactive oxygen species, intracellular calcium, apoptosis, and DNA damage in a concentration-dependent manner. Mitochondrial membrane potential and glutathione levels are also decreased. These findings show that thymoquinone-oxime has an anti-tumor effect on hepatocellular carcinoma cells. Graphical Abstract HIGHLIGHT Among many herbs, Black seed (Nigella sativa) belonging to the Ranunculaceae family has been recognised worldwide as one of the most valuable nutrient-rich herbs There is no relevant data on the effect of the newly synthesised oxime derivative of TQ (TQ-ox) in cancer cells HEP-G2 A new TQ derivative has a higher anti-tumor effect on hepatocellular carcinoma


Introduction
Hepatocellular carcinoma (HCC) is the most common primary malignant tumor of the liver that originates from hepatocytes, and its incidence is increasing worldwide and remains a global health problem (Villanueva 2019;Llovet et al. 2021).HCC is more common in men than women is known as the 5 th most common cause of cancer death worldwide, with 6% according to 2021 data (Cancer Facts and Figures 2021).
Although mortality associated with many cancer types has decreased in recent years, there has been a significant increase in the incidence and death of HCC (Ryerson et al. 2016).Surgical interventions are the most effective treatment method in the survival rate of an individual with HCC; the low rate of resection and the high rate of recurrence and metastasis affect clinical treatment fundamentally (Sintra et al. 2013).
Natural products have antibacterial, anti-inflammatory, and anticarcinogenic effects.Therefore, one of the therapeutic strategies developed against cancer is to take advantage of the potential anticancer activities of natural products (Khan et al. 2011).Thymoquinone (TQ), the most pharmacologically active compound found in the Nigella sativa plant, is a natural remedy that has emerged through extensive medical research (Khader and Eckl 2014).The results of TQ supplementation in liver damage due to various factors decreased oxidative status, and increased total antioxidant activities were accepted as indicators of the importance of TQ as a liver protector (Mabrouk et al. 2016;Awad et al. 2016).Additionally, studies have reported that TQ inhibits cell proliferation of many cancer cells, including ovarian, colon, breast, liver, and lung cancer (Attoub et al. 2013;Samarghandian et al. 2019).
Oxime compounds are under investigation for their therapeutic use for a number of diseases (Canario et al., 2018).Studies have reported that oximes are good at interfering with reactive oxygen species (ROS) and have anti-inflammatory and anticancer properties against cancer cell lines (Qin et al. 2016;Kolsi et al. 2018).The addition of oxime groups to the primary skeleton structure was found to enhance the biological activity of various natural chemicals, as reported in a detailed study on oximes (Schepetkin et al. 2021).In a study by Kale et al. (2022), they suggested that the synthesised oxime derivative of TQ (TQ-Ox) has dose-dependent cytotoxic, genotoxic, and apoptotic properties on the ovarian cancer cells.However, the synthetic oxime derivative of the effect of TQ on HCC has not been the subject of any relevant research.For this reason, the TQ will have its therapeutic effect enhanced by the addition of an oxime functional group.Hereby, the cytotoxicity, genotoxicity, and apoptosis of TQ-Ox in human hepatocellular cancer cells and healthy epithelial cell lines were examined in this study.

Results and discussion
HCC patients are often diagnosed late due to the slow progression of the disease, and the resistance to the chemotherapeutic and radiotherapeutic agents used in the clinic brings a high mortality rate in HCC (Deng et al. 2017).Therefore, there is a need to develop new therapeutic strategies to prevent and treat the disease.In this study, the cytotoxic, genotoxic, apoptotic, and inflammatory characteristics of oxime derivative of TQ has investigated in healthy epithelial cell lines and human HCC cells.
In the 1 H-NMR spectrum of TQ-Ox, aromatic protons were observed in the range of 7.44-6.26ppm, while aliphatic protons were detected in the range of 2.91-1.04ppm.The hydroxyl group proton appeared at 13.39 ppm.On the other hand, the 13 C-NMR spectrum of TQ-Ox revealed 9 carbon peaks, including 6 aromatic carbon peaks ranging from 186 to 118 ppm in the aromatic region and 3 methyl carbon peaks ranging from 27 to 17 ppm in the aliphatic region.Based on the elemental analysis of TQ-Ox, the synthesised structures were confirmed.The proposed structures were established through the analysis of the 1 H and 13 C-NMR data, as well as the TQ-Ox elemental analysis results, see Figure S1.Aromatic protons were found in the TQ-1 H-NMR Ox's spectrum between 7.4 and 6.26 parts per million, while aliphatic protons were found between 2.91 and 1.04 ppm.At 13.39 ppm, proton from the hydroxyl group was detected.TQ-13 C-NMR Ox's spectra show 9 carbon peaks, including 6 aromatic carbon peaks in the aromatic region between 186 and 118 ppm and 3 methyl carbon peaks in the aliphatic region 27-17 ppm.TQ-elemental Ox's analysis supports artificial constructions.The hypothesised structures were built using 1H and 13 C-NMR data as well as the TQ-Ox elemental analysis results.
There is abundant in vitro research in the literature that TQ is effective in many cancer cell lines, including HCC.A previous study demonstrated that their obtained data showed that TQ could selectively inhibit HCC cell proliferation (Jehan et al. 2020).
In another previous study, the possible beneficial effects of TQ on HepG2 cell lines were investigated to determine its potential mechanism of action and revealed that TQ inhibited the viability and proliferation of cancer cells with a mechanism including cytotoxicity (Ahmed et al., 2008).In this study, TQ-Ox's ability to inhibit the proliferation of HCC human cell line HepG2 and healthy epithelial cell line THLE-2 were treated with concentrations between 2.5-180 µM.In a concentration-dependent way, TQ-Ox can more effectively than THLE-2 healthy cells that promote the iROS activity in HepG2 cells (p < 0.001).TQ-Ox suppressed cell proliferation and iROS have significantly increased in a concentration-dependent manner (p < 0.001), see Figure S2.The IC 50 value of HepG2 treated with TQ-Ox was 38.17 µM and THLE-2 98.31 µM.
Oxidative stress and endoplasmic reticulum stress are pathological inflammation, and cell damage is caused by hepatocytes (Friedman et al., 2018;Anstee et al. 2019).In a study, increased ROS production and cytotoxic effect in HepG2 cells suggested that natural therapeutic-loaded micro-devices may encourage anticancer activity.ROS level homeostasis is critical within the cell and is only regulated by intracellular antioxidant levels in mitochondria and cytosol (Das et al. 2020).Therefore, the elimination of GSH levels leads to oxidative stress-mediated cell death (Ott et al. 2007).In the present study, the levels of iROS and GSH were significantly reduced, see Figures S2  and S4.HepG2 cells exhibited a greater induction of iROS activity upon TQ-Ox exposure compared to the THLE-2 healthy cells.The results obtained can provide confirmation that ROS accumulation is probably a contributing factor to TQ-Ox, which results in enhanced chemosensitivity of HepG2 cells.
Calcium signaling plays an essential physiological role in metabolism.Signal dysregulation in this metabolism plays a role in cancer development (Chen et al. 2013).Ca 2+ ions regulate cell proliferation, migration, and death.Altered iCa 2+ homeostasis is, therefore, implicated in tumor initiation, progression, and metastasis (Cui et al. 2017).In our study, iCa 2+ levels assessed by the Fura-2AM fluorescent probe in HepG2 cancer cells treated with TQ-Ox increased concentration-dependent. Ca 2+ concentrations in HepG2 and THLE-2 cells were elevated by TQ-Ox concentrations in dose-dependent manner.In Figure S3, the iCa 2+ levels were statistically significant (p < 0.001) and higher in HepG2 cells than THLE-2 cells.The levels of GSH were measured by the luminometric method in HepG2 and THLE-2 cell lines.In Figure S4, TQ-Ox concentrations between 2.5-40 µM decreased GSH levels in cancer cell line HepG2 and healthy cell line THLE-2 statistically (p < 0.001).
Mitochondria, the primary source for ROS and ATP production in cells, causes oxidant-antioxidant imbalance through oxidative stress in the matrix following.This situation makes membrane proteins and lipids unprotected to oxidative damage.This excessive oxidative stress targets explicitly mitochondria, causing ΔΨm loss and loss of MMP leads to cell death or apoptosis in altered mitochondrial metabolism (Kim et al. 2012;Yang et al. 2017).Apoptosis, which plays a role in many physiological processes, depends on the balance between tissue homeostasis, cell proliferation, differentiation, and cell death.Therefore, the balance between proliferation and apoptosis is crucial, and the imbalance between these two processes causes unwanted tissue growth.Tumor formation also occurs due to a decrease in cell death and an increase in cell proliferation (Perryman and Sylvester 2006).And also, hepatocytes can cause DNA damage due to increased ROS produced due to mitochondrial dysfunction (Nishida et al. 2016).As in the literature, the mitochondrial apoptotic pathway was investigated to demonstrate apoptotic induction mechanisms in cancer and healthy cell lines.Decreased MMP leads to apoptosis.In Figure S5, a concentration-dependent statistically significant decrease (p < 0.001) in HepG2 and THLE-2 cells after 24 h incubation.Furthermore, it was found that the rate of apoptosis in cancer cells increased more compared to healthy cells, see Figure S6A.In a concentration-dependent manner, the percentage of apoptosis increased statistically significantly (p < 0.001), see Figure S6B.To assess the genotoxic damage in the cell lines HepG2 and THLE-2, the IC 50 concentrations were studied with the single-cell alkaline gel electrophoresis.The stage of damage is given as percentage tail density.Figure S7A shows that statistically higher DNA damage was caused by higher TQ-Ox concentrations (p < 0.001).Tail images of HepG2 cell DNA are shown to have increased damage in Figure S7B.This study suggested that cancer cells treated with TQ-Ox increased ROS production and thus induced apoptosis and DNA damage.

Syntheses of TQ-Ox by nuclear magnetic resonance (NMR)
TQ-Ox underwent successive purification by being washed with both hexane and water.The products' homogeneity was evaluated using thin-layer chromatography at each stage, and the melting point was 155 °C (TLC Silica gel 60 F 254 ).To synthesise TQ-Ox, carvacrol was subjected to nitrosation reaction using NaNO 2 , ethanol, and hydrochloric acid at room temperature under an argon atmosphere.The resulting product was then purified by washing it with hexane.After washing, the characterisation of TQ-Ox was examined by 1 H-NMR, 13 C-NMR, and elemental analysis. 1H-NMR and 13 C-NMR spectra were recorded on a Bruker Avance III 500 MHz three-channel NMR spectrometer.

Cell culture and maintenance
HepG2 (HB-8065™) HCC human and THLE-2 (CRL-2706™) healthy epithelial cell lines were purchased from American Type Culture Collection.E'MEM for HepG2 and THLE-2 cell lines are 10% FBS and 1% P/S were added as supplements to the main medium.The cell lines were incubated at 37 °C and exposed to 5% CO 2 .

Cell viability
By using a luminescent ATP assay (CellTiter-Glo®), the cell viability of TQ-Ox on the cell lines HepG2 and THLE-2 was evaluated.Cell lines were added to 96-well plates seeded (7x10 3 cells/well).The seeded cell lines were treated with 2.5-180 μM TQ-Ox concentrations and incubated for 24 h at 37 °C in 5% CO 2 .The ATP solution was added to the wells after incubation.The cell viability was measured by Varioskan Flash Multimode Reader (Thermo Scientific, Waltham).Relative luminescence units (RLU) were used to express the results, while concentration-response curves were utilised to determine the maximum growth inhibitory concentrations (IC 50 ) values.

Intracellular ROS
Cell lines were incubated with 2.5-180 μM TQ-Ox concentrations for 24 h.After treatment, the medium was aspirated and washed with 1x dPBS.5 μM H 2 DCF-DA was added to the wells, and the plate was incubated for 30 min at 37 °C using a fluorimeter and a multi-mode reader (Therma Scientific, Waltham), the capacity of intracellular ROS was measured.Relative fluorescence units (RFU) were used to express the results, while concentration-response curves were utilised to determine the IC 50 values.

Intracellular calcium (iCa 2+ )
Utilising the fluorescent dye Fura-2AM, the incubation TQ concentrations on cell lines and iCa 2+ levels were assessed.After 24 h, the cells were seeded with TQ-Ox concentrations (2.5-40 μM) in 96-well black opaque plates under IC 50 .The cells were rinsed with 1x dPBS following the TQ-Ox treatment.Washing was followed by a 45-minute incubation period with 5 μM Fura-2AM.A fluorescence multi-mode plate reader (λex = 340 nm, λem = 380 nm) was used to measure the fluorescence after.In order to calculate the results, ATP was compared to the DMSO control.

Glutathione
To assess glutathione levels in the HepG2 and THLE-2 cell lines, the GSH/GSSG-Glo kit from Promega (USA) was utilised.The cells were seeded onto 96 opaque-white plates and treated with varying concentrations of TQ-Ox (ranging from 2.5 to 40 μM) for 24 h.Afterwards, the medium was removed.To lyse the cells, 50 μL of glutathione reagent was added, and the mixture was incubated at room temperature while shaking for 5 min.Subsequently, luciferin reagent (100 μL) was added to the wells, and GSH was measured in multimode flash reader (Thermo, Waltham, USA).Results were calculated by the control as % RLU.

Mitochondrial membrane potential
Using the molecular fluorescent probe DiOC 6 (3) (3,3′-dihexyloxacarbocyanine iodide) in flow cytometry, the levels of mitochondrial membrane potential (MMP) were assessed (Rottenberg and Wu 1998).A six-well plate containing the HCC and healthy cell lines was incubated for 24 h at 37 °C with 5% CO 2 .Then, trypsin-EDTA (0.25%) was used to extract the media from each plate, and the plates were centrifuged for 5 min at 400 x g at +4 °C.The obtained pellet was combined with DiOC 6 (3).For 20 min, the resulting suspension was incubated at 37 °C.Flow cytometry (BD, FACS CantoTMII) was used to count the cells.

Apoptosis with AO/EB double staining
After the treatment of TQ-Ox on cell lines, nuclear morphology was detected by double staining AO/EB by McGahon et al. (1995).1:1 AO/EB staining was used to stain the treated cells.Using a fluorescence microscope (Leica DMA 1000, Solms) to count an average of 100 cells, nuclear morphology changes were assessed.

DNA damage with Comet assay
As mentioned before (Kale et al. 2022), TQ-Ox on HepG2 and THLE-2 cells were conducted by a Comet assay and modified by Singh et al. (1988).

Statistical analysis
For all statistical calculations, the SPSS version 25.0 program (IBM, Armonk, NY, USA) was utilised.Non-parametric data were expressed as the interquartile range, while parametric data were expressed as mean standard deviation (SD) (IQR).The Mann-Whitney U test was used to determine the difference between the two parameters in the groups.In order to compare more than two independent parameters, the Kruskal-Wallis test was employed.The Spearman rank correlation coefficient was used to assess the relationship between two variables.The categorical data were assessed using the chi-square test, and p < 0.05 was considered statistically significant.Each experiment was carried out four times.

Conclusions
As a result, based on the data obtained from this study and discussed above in the literature, it was observed that cell viability, MMP, and GSH levels decreased, and iROS, iCa 2+ , apoptosis, and DNA damage values increased in hepatocellular cancer cells treated with TQ-Ox.These results demonstrate that the novel TQ derivative TQ-Ox has a stronger anti-tumor effect on HCC.Therefore, additional in vivo research on this TQ-Ox may be required.