Effects of doxorubicin and apigenin on chronic myeloid leukemia cells (K562) in vitro: anti-proliferative and apoptosis induction assessments

Abstract In this study, we aimed to investigate the effect of the co-treatment with apigenin and doxorubicin (DOX) on K562 cells. Our results show that apigenin (0, 40, 60, 80 ,100 µM) and DOX (0–10 µM) as single therapy, could decrease K562 cell viability (after 24 h of treatment) in a dose-dependent manner. Additionally, the co-treatment with apigenin (60, 80 µM) and 10 µM of DOX led to a greater reduction in cell growth (CI: 0.92 and 0.97) after 24 h of treatment compared to the single DOX treatment (p < 0.05). Consequently, apigenin and DOX, either as single or as co-treatment (24 h of treatment), were indicated to induce apoptosis in K562 cells through morphological studies, RT-qPCR, and western-blot analysis. Eventually, the expressions of Caspase 3, 6, 7, and 9 genes in the single treatment with DOX had higher alteration compared to the co-treatment with DOX and apigenin (p < 0.05). Graphical Abstract


Introduction
Leukemia is an aggressive cancer which rapidly growing cases around the world (Onida and Beran 2008). One of the main features of leukemia cancer cells is their abilities to escape from apoptosis (Chamani et al. 2020). Doxorubicin (DOX), which is commonly utilized for the treatment of different types of cancer, including acute leukemia, breast cancer, ovarian cancer colon cancer, and head and neck cancer, is a potentially antineoplastic agent (Zugazagoitia et al. 2016;Yousefi Sadeghloo et al. 2020). It was previously demonstrated that the chemotherapy by DOX may lead to immediate and/or delayed toxicities. One of the main restricting factors of DOX using in chemotherapy is cardiotoxicity (Rawat et al. 2021). These potential side effects of DOX have consequently decreased its clinical benefits. Recently, the combination therapy has increasingly been applied for the treatment of cancer due to its ability in targeting various therapeutic targets with different agents. Apigenin (AP I) as the main group of plant-flavonoids, also plays several roles in the treatment of chronic diseases (Salehi et al. 2019). In addition, apigenin has anti-mutagenic, anti-inflammatory, and anti-cancer activities (Hamadou et al. 2021). Synergic treatment of apigenin, hesperidine, and DOX on MCF-7 cells clearly demonstrated that apigenin could increase the cytotoxicity effect of DOX on MCF-7 (Korga-Plewko et al. 2020). In the present study, we investigated the cytostatic effects of combination therapy of API and DOX on human's normal lymphoid cells and chronic myeloid leukemia K562 cells.

Results and discussion
2.1. Anti-proliferative effects of apigenin, DOX, and their co-treatment on K562 and FS-2 cells The treatment of K562 cells with AP I and DOX alone for 24 h led to a reduction in cell viability of K562 cells in a dose-dependent manner in comparison to the negative control (cells treated with no drug) ( Figure 1A and B). Higher dose of apigenin reduced the viability of K562 cell upto 85% in comparison to the negative control group (IC50, 86.29 lM) (p < 0.001) ( Figure 1A). However, the results didnot showe any significant effect on the reduction of normal human B lymphoblastoid cells (FS2) survival at same DOX concentrations (IC50, 17.88 lM) ( Figures S1A and B). The treated K562 cells with the combination of apigenin and DOX showed around 60% reduction in their viability compared to the control group (p < 0.001), while FS-2 cells' viability did not change significantly ( Figure S1C). The combination index was obtained as 0.923 and 0.971 at the doses of 60 and 80 lM apigenin and 10 lM DOX, indicating the synergistic effects of both apigenin and DOX. Korga-Plewko et al. (2020) clearly demonstrated that apigenin could strengthen the cytotoxicity effect of DOX on MCF-7 through MTT assay and also enhance double strand break of DNA induced by DOX. Maduni c et al. (2018) have also revealed that the chemotherapeutic modality of apigenin is due to its low intrinsic toxicity against normal cell compared to cancerous cells (Maduni c et al. 2018). Here, the morphological alterations such as cells shrinkage and apoptotic bodies, were evidently demonstrated in the treated cells in comparison to the untreated samples ( Figure 2). As well, chromatin fragmentation with bright blue spherical beads around the nucleus and appearance of crescent nucleus, was clearly observed in the cells treated with apigenin and DOX, along with a reduction in the cells' number.
2.2. Evaluation of the P53, BAX/BCL-2 ratio, caspase 9, 3, 6 and 7 expressions of K562 cells treated with apigenin, DOX, and their combination A significantly up-regulated P53 was observed in the co-treated apigenin and DOX cells compared to those treated with DOX alone as mono-treatment (p < 0.05) ( Figure  3). Sharifi et al. (2015) also demonstrated that DOX could induce apoptosis in MCF-7 cell line through P53-BAX-caspase 3 pathway (Sharifi et al. 2015) .The treatment of cells with apigenin and DOX individually up-regulated BAX in K562 cells compared to the negative control group, which this increase was significant in apigenin concentrations (p < 0.001) ( Figure 4A). A reduction was also observed in BAX expression (1.6and 1.8-fold changes in co-treated with 10 mM DOX and 60 mM of apigenin and 10 mM DOX and 80 mM of apigenin, respectively) compared to the DOX-alone treated cells (2.56-fold changes). On the other hand, there was a significant down-regulation (2-4 change fold) in anti-apoptotic BCL-2 in the treated cells compared to the control group (except 80 lM, which was estimated to be near to the control) (p < 0.001) ( Figure 4B). As well, decreasing BCL-2 (anti-apoptotic) and increasing BAX expression (pro-apoptotic) changed the BAX/BCL-2 ratio in favour of apoptosis. As it was shown, the higher BAX/BCL2 ratio was indicated in the cells treated with DOX and or apigenin alone compared to the cells co-treated with the combination of them (p < 0.05) ( Figure 4C). Several previous studies have clearly demonstrated that apigenin and DOX induce apoptosis by modulating BAX/BCL2 ratio, and STAT3 and Akt protein expressions (Javed et al. 2021). Accumulating evidence suggests that caspase mediated apoptotic pathways are interconnected with the increased expression p53 with the downregulation of anti-apoptotic Bcl2 and upregulation of pro-apoptotic Bax (Banerjee et al. 2020;Banerjee et al. 2021) .
The caspase-9 expression had a significant increase in the cells treated with 10 mM of DOX alone and 60 mM of apigenin alone compared to the control group (p < 0.001) ( Figure 5A). Additionally, the expression of caspase 9 was higher (2.96) in the DOXtreated cells compared to the cells treated with both DOX and apigenin (p < 0.001) ( Figure 5A). As it can be seen, higher expressions of caspases 3, 6, 7, and 9 were found in the cells treated with 10 mM of DOX than the cells co-treated with DOX and apigenin ( Figure 5). Induction of caspase-dependent apoptosis was also detected in MDA-MB, HL-60, and THF-1 cells following the treatment with apigenin or DOX (Salehi et al. 2019) However, in combination therapy, controversial results due to the antioxidant/ oxidant property of apigenin, were reported (Ruela- de-Sousa et al. 2010). Correspondingly, this may possibly be resulted from the potential competition between antioxidant/oxidant activities. Apigenin, as a flavonoid, can act like an antioxidant and directly inhibit ROS or play an indirect role in oxidant-antioxidant balance. DOX exerts its anticancer activity in the cell mainly via ROS production, so an antioxidant agent may reduce the anticancer activity of DOX (S anchez-Marzo et al. 2019). 2.3. The effects of apigenin, DOX, and their combination on the cleaved PARP and caspase3 of K562 cells As shown in Figure 6, the 89 KD fragment of PARP was observed in the cells cotreated with DOX and apigenin by western-blot analysis for 24 h. Furthermore, the cleaved bond of caspase-3 was found in the proteins extracted from the cells cotreated with DOX and apigenin for 24 h compared to the control cells. The results were also confirmed by relative band intensity estimation, indicating an increase in Cleaved PARP after 24 h of the co-treatment with DOX and Apigenin.

Conclusion
In conclusion, the use of apigenin intensifies the antitumor effects of DOX, which can suggest for improving the quality of CML treatment. Synergistic anti-cancer activity The BAX/BCL2ratioalso demonstrated as (C). Ã : P < 0.05 compared to K562 cells that received no treatment (control). ÃÃ : P < 0.001 compared to K562 cells that received no treatment (control). þ: P < 0.05 compared to cells treated only with 10 lM of DOX, þþ: P < 0.001 compared to cells treated only with 10 lM of DOX.  was also associated with the increased expression of tumour suppressor P53 gene and expressions of BAX and BCL-2 genes in favour of apoptosis. However, the expressions of caspase 3, 6, 7, and 9 genes were higher in DOX treatment compared to combination therapy (apigenin and DOX). Further studies are needed to explain precise mechanism, the time selection for combination therapy, and the mechanisms of apigenin, to reduce the toxic side effects of DOX on normal cells.