Searching natural product from medicinal plants with tumor growth inhibitory activity and less toxicity has become a continuous effort in the prevention and treatment of cancer, including HPV-infected cervical cancer [17, 18]. In the present study, we first investigated the cytotoxic and antiproliferative effects of Hesperidin on different type of cervical cancer cells, including HPV 16 positive CaSki cells, HPV 18 positive HeLa cells and HPV negative C-33A cells, and on normal cervix epithelial H8 cells. MTT assay clearly demonstrated that Hesperidin (100, 200 and 400 µM) treatment significantly inhibited the viability of HeLa and SiHa cells, and its inhibition rate also increased with the concentration of Hesperidin and indicated time periods. However, no marked inhibitory effect was observed in C-33A cells except at the highest concentration of Hesperidin (400 µM) after 72 h and cell viability of normal cervix epithelial H8 cells remained unchanged in all Hesperidin-treated groups as compared with untreated control (P > 0.05). In line with this result, EdU incorporation assay demonstrated the similar tendency for antiproliferative effects of Hesperidin on these cells as same as cell viability in MTT assay. The green fluorescence intensity was decreased in CaSki and HeLa cancer cells following treatment with Hesperidin (100, 200 and 400 µM) for 48 h in a concentration-dependent manner. Based on these results, we can presume that Hesperidin might be a promising antitumor drug to treat HPV positive type cervical cancer.
Considering this decreased cell viability and proliferation of Hesperidin-treated cervical cancer cells in the presence of Hesperidin, we next examine if apoptosis induction by Hesperidin was involved in this effect. TUNEL staining assay portrayed that the addition of Hesperidin resulted in an increased cells with purplish red stained TUNEL-positive nuclei visualized by a fluorescent microscope. Supporting this observing, Annexin V-FITC/PI double staining result on flow cytometry further confirmed that Hesperidin supplement evoked a high apoptosis rate of CaSki and HeLa cancer cells in a dose-dependent manner when compared with untreated control cells. These results confirmed that the cell growth inhibitory of Hesperidin on CaSki or HeLa cells was achieved by induction of apoptosis
Apoptosis or programmed cell death is triggered by two main types of signal pathways, namely death receptor-mediated (extrinsic) and mitochondria-mediated (intrinsic) pathway . The mitochondria working as the cross-talk organelles can connect both the extrinsic and intrinsic apoptotic pathways . It is well known that the loss of mitochondrial membrane potential (ΔΨm) played an important role in the initiation of early mitochondrial-related apoptosis , and thus we measured the change in ΔΨm in cervical cancer cells induced by Hesperidin using JC-1 dye. In healthy cells, JC-1 aggregates in mitochondria where JC-1 dimmers emits a red fluorescence at 590 nm, whereas JC-1 monomers in the cytosol exhibits green fluorescence at 530 nm attributable to the mitochondrial depolarization . Once the MMP decreased, JC-1 fluorescence color will shift from red fluorescence to green. Thereafter, the relative low ratio of the red/green fluorescence indicated the collapse of MMP. Fluorescence microscopy demonstrated that a reduced red fluorescence signals, but an increased green fluorescence signals in CaSki cells and HeLa cells following treatment with Hesperidin for 48 h, suggesting a MMP loss upon Hesperidin treatment.
Experimental evidence suggests that the mitochondria-mediated intrinsic and receptor-mediated extrinsic apoptosis pathways can be differentiated by involvement of initiator capase-8 or -9, respectively . Once the activation of capase-8 or -9, both intrinsic and extrinsic pathways would trigger the common executioner caspases (capase-3 and capase-7) to execute apoptosis [24, 25]. During mitochondria-mediated intrinsic apoptosis pathway, Bcl-2 family proteins work as critical regulators of mitochondrial membrane permeability and consist of pro- (Bax and Bak) and anti-apoptotic (Bcl-2 and Bcl-xL) members, which form heterodimers to inhibit or activate each other . The rise of ratio between pro-apoptotic protein Bax and anti-apoptotic protein Bcl-2 can induce mitochondrial damage and then cause the release of cytochrome c from mitochondria to the cytoplasm where cytochrome c forms apoptosome complex with AIF to subsequently activate caspase-9 as well as downstream effector caspase-3, thus inducing apoptosis . Due to the MMP collapse, we further investigated the mitochondrial-related apoptotic proteins, including cytosolic cytochrome c, Bax, Bcl-2, cleaved caspase-3, cleaved caspase-8, and cleaved caspase-9. The expression levels of cytosolic cytochrome c, Bax, cleaved caspase-3, and cleaved caspase-9 protein were significantly increased in a dose-dependent manner in CaSki and HeLa cells upon Hesperidin treatment. In contrast, the expression level of Bcl-2 was decreased in both cells following Hesperidin treatment (Fig. 3B). Cleaved caspase-8 protein expression kept unchanged as control. These result suggested that Hesperidin treatment induced apoptosis of CaSki and HeLa cells via a mitochondria-mediated (intrinsic) pathway.
Mitochondria-mediated apoptosis involves not only the activation of caspase-9-dependent intrinsic pathway but also p53-related signaling molecules . HPV encoded viral oncoprotein E6 is essential for cervical carcinogenesis by its potential to inactivate the tumor suppressor proteins p53, which plays an important role in the regulation of cell cycle and apoptosis [39 − 31]. Hence p53 served as tumor suppression element in multicellular organisms to suppress cancer and its defectiveness would allow abnormal cells to proliferate, resulting in cancer. It was evident that Hesperidin concentration-dependently decreased the expression of E6 oncoprotein in CaSki and HeLa cells, but increased tumor suppressor protein p53. All these results suggested that the expression of decreased HPV E6 and increased p53 protein were involved in the antitumor effect of Hesperidin toward CaSki and HeLa cells.