Khalas date flavonoids inhibited cell viability, induced apoptosis and expression of the pro-autophagy LC3-B gene in human hepatocellular carcinoma cells (HepG2)

Abstract Autophagy is a protective mechanism important in human diseases as cancer. We evaluated the impact of khalas date extract (KDE) (20-60 mg/mL) on cell viability, morphological changes, DNA fragmentation and gene expression of LC3B-II associated with autophagosome on HepG2 cell line. The GC/MS identification of KDE showed its high content of flavonoids including quercetin, myricetin, kaempferol and catechol. KDE reduced cell viability of HepG2 with IC50 (31.52 mg/mL). Cells treated with KDE showed two band of DNA fragments at (30 and 40 mg) indicating that KDE induced DNA damage and apoptosis in HepG2. The analysis RT-PCR data showed a 0.2-fold increase in the expression of LC3-B in the cells treated with KDE versus control. We concluded that, KDE flavonoids such as quercetin, myricetin kaempferol exhibited anticancer properties manifested by inhibition of HepG2 cell viability and induction of apoptosis and upregulation of the pro-autophagy LC3-B gene. Graphical Abstract


Introduction
It was suggested that autophagy and suppression of cell proliferation were the primary causes of the considerable decrease in cell proliferation. However, the role played by autophagy in hepatocellular carcinoma is not clear (Al-Shwyeh 2019). In the present study, we evaluated the anti-proliferative activity of khalas date extract (KDE) against HepG2 cell line. Cell viability, morphological changes, DNA Fragmentation and Gene expression of LC3B-II associated with autophagosome in HepG2 as shown in graphical abstract .

Results and discussion
Analysis of KDE extract (Table S2) showed that it contains quercetin, myricetin, kaempferol and catechol, all of these possess antioxidant, antitumor activity against different cell lines (Fu and Chung 2018). It has been shown that quercetin induced apoptosis in HepG2 cells and decreased intracellular fatty acids via the inhibition of fatty acid synthase (Zhang et al. 2020). Quercetin exerts a protective effect against oxidative stressinduced apoptosis in PC12 cell line. The levels of antioxidant enzymes CAT, GSH-Px, and SOD were significantly higher in quercetin pretreated samples compared to those incubated with H 2 O 2 (Bao et al. 2017). In addition, it has been shown that quercetin reduced hyperglycemia-induced oxidative stress in HepG2 cell line. Quercetin, also, significantly decreased CAT, GPx activity, GSH as well as MDA levels (Yarahmadi et al. 2021). The cytotoxic effect of the flavonoids demonstrated in our study was in agreement with many recent studies (Johnson et al. 2010). It has been found that the KDE drastically increased the cytotoxicity in HepG2 cells in a dose-dependent manner ( Figure S1). The IC50 values of KDE have been estimated to be 31.52 mg/m, after 24 hours ( Figures S2 and S3). The cytotoxic effect of the KDE could be due to the presence of flavonoids in dates which are rich in polyphenolic compounds that served as anti-cancer bioactive compounds. The DNA fragmentation experiment supported the inhibitory effect of KDE on Hep G2 cells where cells formed a distinctive DNA ladder as shown in Figure S4. A previous study found that flavonoids significantly decreased cell viability in HepG2 cells (Xia et al., 2013). In our study, the decrease in cell viability was statistically significant compared to the DMSO control. These results indicated that KDE exert a pronounced inhibitory effect on HepG2 cell line.
Furthermore, quercetin induced HCC autophagy and inhibited LM3 cell migration and invasion (Wu et al. 2019). Our results and the above-mentioned results clearly justified the anticancer properties of kahals date primarily through the effect of quercetin which was found to account for the highest concentration of phenolic compound in the KDE. Regarding to myricetin, it induced apoptosis and autophagy, inhibited cell proliferation in HCC cells . The autophagy was induced via the IRE1-JNK and Ca2þ-AMPK pathways (Ji et al. 2022). Furthermore, it was demonstrated that, myricetin's ability to cause apoptosis in HCC cells was enhanced by blocking autophagy implying that autophagy played a protective role rather than a tumor suppresser function (Ji et al. 2022). Additionally, Myricetin has been demonstrated to cause apoptosis in HepG2 cells via activating the mitochondrial apoptotic pathway and the Akt/ p70S6K/bad signaling pathways (Zhang et al. 2013). Moreover, Myricetin has been demonstrated to stop HepG2 cells from proliferating and to cause G2/M phase arrest (Zhang et al. 2011). It is possible that Myricetin together with other flavonoids induced apoptosis and cell cycle arrest that outweighed the protective role of autophagy. Interestingly, Kaempferol represented the third highest concentration in KDE extract. Many studies have shown that Kaempferol plays an antioxidant role in HCC and may represent a promising preventive agent for the disease (Sharma et al. 2021). Oxidative stress may be considered an important key player in the growth and spread of liver cancer. One of the key factors in the development of HCC is the overproduction of reactive oxygen species (ROS) (Brahma et al. 2021). Interestingly KDE extract contained an appreciable concentration of catechol. Previous studies have already shown that, catechol's exert as an anticancer agent since it inhibited stem cell-like features in HCC cells in vitro (Lim et al. 2020). Autophagy is generated when there is a limitation in ATP availability or depression in essential nutrients such as glucose, amino acids. Moreover, defects in the autophagy machinery have been associated with numerous diseases, including cancer, metabolic problems, aging-associated pathologies, neurodegeneration, infectious/inflammatory conditions, as well as cardiovascular disorders (Singal et al. 2020).
In the current study the, the activity and expression level of Atg4B was induced by addition of KDE to HepG2 cells ( Figure S5). This might have been due to presence of quercetin, myricetin and catechol. Previous studies reported that myricetin can stimulate apoptosis of human colon cancer cell lines (Srivatanakul et al. 2004), and can induce autophagy in HepG2 cells (Kuras et al. 2020). Autophagy may exert tumor-suppressing function before a tumor is established because it can prevent the accumulation of oncogenic mutations by limiting chromosomal instability. But when a tumor is established, enhanced autophagy is used to escape hypoxic, metabolic, detachmentinduced and therapeutic stress (Lago et al. 2014). The LC3-A produced from pro-LC3 where it's cleaved by ATG4 (cysteine protease). LC3-A is activated by ATG7 and conjugated to phosphatidylethanolamine (PE) to form LC3-B. The LC3-B level is a good indicator and hallmark marker to monitor the cell autophagy level induction, whereas its sustained accumulation is reflective of autophagy inhibition (Li et al. 2015). It was reported that, Atg4B inhibitors can suppress the formation of autophagic vacuoles and tumor growth (Agrotis & Ketteler, 2019). Flavonoids have recently been known to exhibit anti-HCC effects via induce autophagy modulation, both in vitro and in vivo. The morphological changes of the HepG2 cells were observed under electron microscopy after the treatment with different concentration of KDE, and found that the extract has changed the morphology of HepG2 cells in agreement with many previous studies (Jeganathan et al. 2016). Cells lost their typical morphology and appeared smaller in size, non-adherent, and rounded. In contrast, control cells treated with only 2.4% DMSO appeared in normal features with regular shape.
The results of this study clearly demonstrated the up-regulation of LC3-B mRNA compared to the control indicating increased autophagy. Thus, our results came in line with previous studies (Pang et al., 2021) that showed how flavonoids play a pronounced anti-HCC effect through regulation of autophagy, the non-apoptotic mode of cell death. Indeed, KDE clearly showed anti-cancer properties against HepG2 cell lines.

Experimental
Supplementary file.

Conclusion
We herein conclude that, the cytotoxic effects of KDE are mediated by the high content of flavonoids namely quercetin, myricetin, kaempferol and catechol. This was associated by decreased cell viability and induced apoptosis and up regulation of the pro-autophagy LC3-B gene. However, further studies are still needed to confirm whether the antitumoral effects of the extract is caused at least in part-by autophagy.

Disclosure statement
No potential conflict of interest was reported by the authors.