Apoptosis has an important role in different biological systems including physiological cell turnover, immune system, and embryonic development [13]. This programmed cell death can be triggered by different stimuli such as ionizing radiation, chemotherapeutic drugs, and intracellular signaling molecules [14]. In current study, apoptosis-induced cell death was verified by investigating some apoptotic markers including Bax and Bcl-2 expression, apoptotic caspase pathways, anti-tumor protein p21 and p53, and morphology of YM-1 cell line. Obtained results demonstrated anticancer effects of OPE, and NR on YM-1 cell line of human esophageal cancer cells as confirmed by morphologic alterations and apoptotic markers. Our results were in accord with Li et al. [5] who reported anticancer effects of NR in a dose-dependent manner on the cell proliferation of triple-negative (ER-/PR-/HER2-) breast cancer cells and Meiyanto et al. [15] who indicated the inhibitory effects of NR on breast cell lines of MDA-MB-231 and MCF-7. Similar results were obtained in our in vivo study indicating that OPE and NR have potential to use as adjuvant therapy to lower systemic toxicity of chemotherapeutic agents like doxorubicin in ESCC cancer stem cells treatment [16]. Moreover, the suppressive effects of OPE on the proliferation of human lung cancer cell line (A549) and prostate cancer cell line (22RV-1) have been reported [17]. Since cancer cells lose many of their regulatory mechanisms, therefore toxicity is one of the main side reactions of chemotherapeutic treatments. Attenuated effect of OPE and its flavonoid derivative, NR, on Dox-induced cytotoxicity is in accordance with Putri et al. [18] who reported that combination of ethanoic extract of citrus hystrix peel (500 and 1000 mg/Kg body weight) with Dox (4.67 mg/Kg) reduced the Dox-induced cardiotoxicity and hepatotoxicity. Moreover, Kwatra et al. [19] commented on the protective effect of NR against Dox-induced cardiac toxicity in rats. However, our results appear to contradict with Zhang et al. [8] who reported that NR enhances Dox-induced cytotoxicity of A549 and MCF-7 cell lines. The differences in techniques, sample sizes and types of cell lines used may account for the discrepancy. Protective effects of OPE and NR on Dox-induced cytotoxicity can be attributed to the potential role of biologically active molecules such as phenolic and flavonoid compounds [20] which are capable of interacting with free radicals [21].
It has been reported that treatment with naringin in the range of 20–50 µM causes a decrease in DNA methyltransferase activity and a decrease in the 5-methyl-20-deoxycytidine level in the DU145 prostate cancer cell line, indicating that naringin is a DNA hypoethylating agent that has the potential to modulate gene expression [7]. Treatment of SiHa cells with naringin in a dose-dependent manner in the 0–2000 µM concentration range can cause apoptotic cell death, internucleosomal DNA fragmentation, morphological changes and a decrease in the mitochondrial transmembrane potential through both death receptor and mitochondrial pathways [9].
A great deal of evidence demonstrates that Bcl-2 family members are major regulators of apoptosis [22,23]. For instance, overexpression of Bcl-2 enhances cell survival by suppressing apoptosis, whereas pro-apoptotic Bax expression promotes programmed cell death. Our results showed the overexpression of Bax in the YM-1 cell line exposed to OPE (400 mg/mL), NR (300 µM), and Dox (0.5 µM), whereas the expression of Bcl-2 was remained relatively unchanged, suggesting a pro-apoptotic effect of OPE and NR. The precise mechanisms by which Bax and Bc1-2 establish their functions remain to be understood. However, it has been highlighted that the activation of the pro-apoptotic Bax gene is involved in the mitochondrial apoptotic signaling pathway but Bcl-2 prohibits cell death by preserving mitochondrial integrity [24]. Mitochondrial apoptotic pathway causes the activation of executioner caspases like 9 through the increasing permeability of mitochondrial outer membrane [25,26], a fact that was relatively confirmed by the activation of caspases 8 and 9 in YM-1 cells subjected to OPE or NR treatments. Our data on the expression of Bax apoptotic protein is in line with a study demonstrating that bioactive compounds of citrus enhance apoptosis in a human colon cancer cell line (SNU-C4) by up-regulation of Bax apoptotic genes [20]. Similarly, Qiu et al. [27] reported that 5-hydroxy polymethoxyflavones, a predominant polymethoxyflavones in orange peel induces apoptosis in colon cancer cells a Bax-dependent pathway.
To confirm obtained results, we investigated the activation of executioner caspases 8 and 9. Our results are in accordance with others demonstrating the influence of OPE, NR, and Dox on the activation of caspases 8 and 9 [9,23]. Similarly, Ramesh and Alshatwi [9] reported the stimulatory effect of NR on apoptosis induction in squamous cervix carcinoma cells (SiHa) by activating caspases 8 and 9. Anticancer properties of phenolic compounds of citrus are induced either by elevating ROS, reducing mitochondrial potential, inducing DNA damage, or extrinsically by overexpression of Fas, FADD ligands, induced apoptosis via increase in p53 expression, PARP cleaving, Bax and caspase-3 cleaving, and also decrease in the expression of Bcl-2 [10,28,29], likely the increasing probably mechanism of both caspase 8 and 9 are Fas or P53. In line with this, Pommier et al. [30] commented that executioner caspases-8 and − 9 operate via death-receptor and mitochondrial pathways, respectively. Obtained data indicated that OPE and NR in combination with Dox elevated caspases-8 and − 9 activations, suggesting that caspase-dependent extrinsic and intrinsic apoptosis was involved in the toxicity of the YM-1 cell line.
p53 and p21 proteins are other cellular mechanisms involved in programmed cell death, which are activated in response to cellular stresses, such as DNA damage and genetic alterations [9, 31,32]. In many cell types, p53-mediated growth inhibition is dependent on the induction of p21, which is a potent cyclin-dependent kinase (CDK) inhibitor. Our results suggest the increase of p53 might be dependent on p21. The role of p21 as a pro-apoptotic factor in response to some anticancer agents such as taxol and genistein has been reported [33,34]. It has been documented that p21 blocks DNA synthesis, which might be correlated with the anti-proliferation effect of NR [35].
In conclusion, the current study demonstrates that OPE and NR have potential to play pro-apoptotic role on YM-1 cell line through Bax- and p53-dependent pathways. Our findings strongly suggest that OPE and its flavonoid derivative, NR, have potential to attenuate the cytotoxic effect of Dox, a chemotherapeutic agent, on human esophageal squamous carcinoma cell line (YM-1).