Potential of Natural Antioxidants for Upregulating Apoptosis in Cervical Cancer Cells: A Cellular and Molecular Study

Natural antioxidants (NAs) are promising substances, which are established as potent therapeutic agents. However, the anticancer potential of many of them is not completely known. In this research, the anticancer efect of three natural antioxidants including geraniol (Ge), citronellol (Cit), and quercetin (Qu), and their combination by estimating their cytotoxicity on the Hela cells at doses from 15.625 to 1000 µ g/mL was investigated. Moreover, cisplatin (Cis) was employed as a strong competitive comparison. After estimating the IC50 value of each treatment in the MTT assay, western blot was performed with the obtained doses for further evaluation. In this regard, the expressions of caspase3, Bax, Bcl2, and p53 proteins were examined. Our results showed that the combination of these natural substances had signifcant cytotoxic potential by upregulating all mentioned proteins, which demonstrated the apoptosis pathway. Our fndings had also the same results in both Cis-receiving and non-Cis-receiving cells regarding some indices. Consequently, it was suggested to further investigate these natural antioxidants as both main or adjuvant therapies for their anticancer potential and side-efect reduction in vivo . Overall, it can be concluded that these natural anti-oxidants are efective against cancer cells by upregulating the apoptosis pathway.


Introduction
According to the World Health Organization (WHO), cancer has become the leading cause of death worldwide. Early detection and limiting risk factors are two important steps in reducing the number of cancer incidences [1]. In addition, cervical cancer is the fourth most frequent cancer among women globally, with an estimated 604,000 new cases and 342,000 deaths in 2020. Surprisingly, most cervical cancer cases were linked to an infection with high-risk human papillomavirus (HPV) [2].
Historically, natural medicines were more likely to be accepted by patients psychologically. Many aspects of the plants and their extracts were investigated, and most of them showed antimicrobial and antioxidant properties [3][4][5]. However, more exclusive aspects are still unknown, including antiviral, anticarcinogenic, antitumor, and antistress properties [6]. Te role of natural antioxidants in deregulating oxidative stress is undeniable [7], as they can efectively reduce reactive oxygen species (ROSs) [8]. Moreover, there are many studies suggesting the use of essential oils (EOs), which are one of the best-known natural antioxidants, as the main or adjuvant therapy against cancer [9][10][11][12]. Regarding the action mechanisms of EOs, various pathways were previously mentioned [9]. Another promising aspect of using EOs as an anticancer agent is that not only they are more likely to be accessible in the world but also have fewer side efects. Furthermore, many NAs are considered safe substances with a very high toxic dose [13]. Moreover, EOs are already used in our foods and can help humans maintain a balanced diet that will ensure receiving enough amount of antioxidants daily [6]. Tey can even be used as food additives or preservatives due to their antioxidant activities in other products [14]. It seems that EOs can be the key to fghting cervical cancer. Since they have antiviral and anticancer properties simultaneously, they are proposed to be a safer choice than chemotherapy agents [15].
One of the most powerful cytotoxic pathways triggered by these natural substances is apoptosis. Tis mechanism can lead to programmed cell death and have a synergistic efect with necrosis [16]. Since there are shining markers for estimating which apoptosis pathway has occurred, the action mechanism of each treatment can be investigated more accurately in further studies.
Geraniol, as one of the essential oils that come from the Rosoideae family, is proven to have anticancer, antitumor, antioxidant, and many other benefcial characteristics [17]. Similarly, citronellol comes from Rosoideae and has antiinfammatory [18] and anticancer efects [19]. Another potent antioxidant is quercetin, which is known to be an anti-infammatory, antiproliferative [20], and anticancer natural substance [21]. More importantly, this substance was shown to have great potential against cancer cells [22].
In this study, three common NAs were administrated alongside cisplatin, which is the frst metal-based anticancer drug [23]. Te proposed aim of this study was to examine the anticancer and cytotoxic potential of the abovementioned NAs for HPV-derived cervical cancer in HeLa cells. Moreover, it was concerned to compare the anticancer potential of these NAs combined with cisplatin. Terefore, our main goal was to demonstrate the potential of NAs and investigate their possible mechanism of action in vitro. In addition, exploring further studies on animals would be our future goal.

Natural Antioxidants and Hela Cells.
Te natural antioxidants of geraniol, quercetin, and citronellol were purchased from Sigma-Aldrich. Te Hela cells were purchased from the North Research Center of the Pasteur Institute of Iran [24].

Cell
Culture. Te Hela cells were placed in RPMI 1640 medium with 10% fetal bovine serum (FBS). After reaching 75% cell confuence, trypsin-EDTA was applied to them and then counted to ensure the number of cells [25].

Study Design.
After the cells reached more than 75% confuence, they were divided into separated cell groups, and the treatments were applied to them. Te cell groups included the following:  [26]. Briefy, cells were placed in the wells overnight. Next, they were treated with the mentioned substances. Sets of 96-well plates were employed for each time point. After reaching the planned time point, the wells were washed with phosphate-bufered saline (PBS) and 100 µL of the medium containing MTT. After three hours of incubation, the solution was discarded and 100 µL of dimethyl sulfoxide (DMSO) was added to the wells. Finally, the plate was read by using a spectrophotometer at 540 nm to estimate the absorbance of each cell. Te whole procedure was performed in triplicate [27][28][29].

Western Blotting Analysis.
Hela cells were treated with the obtained IC50 values, which were estimated after 48 hours of treatment. After 48 hours, the cells were lysed with radioimmunoprecipitation assay (RIPA) lysate bufer. After being centrifuged at 14000 rpm for 20 minutes at 4°C, the supernatants of homogenates were utilized. Moreover, the protein concentration of each culture was measured following the Bradford protein assay by using a kit (DNAbiotech, DB0017, Iran) [30].

Data Analysis.
Te MTT results were analyzed by employing Graphpad Prism software version 9. P < 0.05 was considered signifcant [33][34][35]. Te one-way ANOVA and the post hoc Tuckey test were employed to compare the diferences between diferent cell groups [36,37]. Moreover, English letters were used to show a signifcant diference in cell groups from highest to lowest [38].

Assessment of Cell Viability.
Te overall IC50 of each treatment can be observed in Table 1. Moreover, as can be seen in the frst fgure, geraniol showed cytotoxicity in diferent doses, and IC50 was diferent between the time points. It was observed that, by treating with geraniol, IC50 values were 424.6, 247.1, and 37.89 µg/mL after 8, 24, and 48 hours, respectively. Moreover, the diference between cell viability after 8 hours and 48 hours was signifcant. Moreover, cell viability was signifcantly lower after 48 hours than after 8 hours (Figure 1).
Regarding quercetin, substantial cytotoxicity was observed in diferent doses, and IC50 was signifcantly diferent between the time points. It was seen that, by treating with quercetin, IC50 values were 449.9, 69.48, and 28.73 µg/mL after 8, 24, and 48 hours, respectively. Moreover, the difference between each group was signifcantly diferent, where cell viability was the lowest after 48 hours, followed by 24 hours and 8 hours ( Figure 2).
Regarding Cit, citronellol showed moderate cytotoxicity in diferent doses, and the IC50 of the 48-hour time point was signifcantly diferent from the other time points. It was observed that, by treating with citronellol, IC50 values were 669.1, 602.3, and 214.18 µg/mL after 8, 24, and 48 hours, respectively ( Figure 3).
As can be seen from the fgure below, cisplatin showed cytotoxicity in diferent doses, and IC50 was diferent between the time points. It was observed that, by treating with cisplatin, IC50 values were 38.4, 19.22, and 3.107 µg/mL after 8, 24, and 48 hours, respectively. Moreover, the diference between the cell viability after 8 hours and 48 hours was signifcant. Moreover, cell viability was signifcantly lower after 48 hours than after 8 hours (Figure 4).
Regarding the mixture of geraniol and citronellol, cytotoxicity in diferent doses was shown, and IC50 was different between the time points. It was observed that, by treating with the mixture of geraniol and citronellol, IC50 values were 393.4, 194, and 148.1 µg/mL after 8, 24, and 48 hours, respectively. Also, the diference between cell viability after 8 hours and 48 hours was signifcant. Moreover, cell viability was signifcantly lower after 48 hours than after 8 hours ( Figure 5).
Te mixture of geraniol and quercetin was nearly similar to that of geraniol and citronellol, which showed cytotoxicity without any signifcant diference between time points. Te obtained IC50 values were 309.5, 165.4, and 130.8 µg/mL after 8, 24, and 48 hours, respectively. Te same trend of decreasing IC50 after each time point happened again ( Figure 6).
As was observed, the mixture of geraniol, quercetin, and citronellol showed cytotoxicity in diferent doses, and IC50 was diferent between the time points. It was observed that, by treating with the mixture of geraniol, quercetin, and citronellol, IC50 values were 283, 243.3, and 35.8 µg/mL after 8, 24, and 48 hours, respectively. Moreover, the diference between the cell viability after 8 hours and 48 hours was signifcant. Moreover, cell viability was signifcantly lower after 48 hours than after 8 hours ( Figure 7).
As the fgure below demonstrated, the mixture of geraniol, quercetin, and cisplatin showed cytotoxicity in different doses, and IC50 was diferent between the time points. It was observed that, by treating with the mixture of geraniol, quercetin, and cisplatin, IC50 values were 368.7, 147, and 37.76 µg/mL after 8, 24, and 48 hours, respectively. Moreover, the diference between the cell viability after 8 hours and 48 hours was signifcant. Moreover, cell viability was signifcantly lower after 48 hours than after 8 hours ( Figure 8).
As the fnal group, the mixture of geraniol, quercetin, citronellol, and cisplatin showed cytotoxicity in diferent doses, and IC50 was diferent between the time points. It was observed that, by treating with the mixture of geraniol, quercetin, citronellol, and cisplatin, IC50 values were 324.5, 107.7, and 31.17 µg/mL after 8, 24, and 48 hours, respectively. Moreover, the diference between the cell viability after 8 hours and 48 hours was signifcant. Moreover, cell viability was signifcantly lower after 48 hours than after 8 hours ( Figure 9).

Western
Blotting. Te protein expressions of 4 diferent markers including Bax, Bcl2, caspase3, and p53 were evaluated and compared with those of B-actin ( Figure 10). It was observed that the highest amount of caspase3 expression belonged to the Ge + Qu + Cit + Cis group, followed by the Ge + Qu + Cis and Cis cells, of which all three cell groups had Cis in common. Regarding p53 protein, similar to caspase3, the Ge + Qu + Cit + Cis group expressed the highest amount of p53 protein and was signifcantly higher than that of the Ge + Qu + Cis cells. Moreover, the Ge + Qu + Cit + Cis group had the highest Bax expression compared to other cells. However, no signifcant diference was observed between Ge + Qu + Cis and Cis cells. In the case of Bcl2, both Ge + Qu + Cit + Cis and Cis cells showed the highest expression, which was signifcantly higher than that of the Ge + Qu + Cis group (Table 2).

Discussion
According to the American Cancer Society, cervical cancer is the fourth most common cancer worldwide, with over 600,000 new cases in 2020, and more than 14,000 new invasive cases in 2022. In this study, three famous natural substances with high antioxidant potential were used to naturalize cancerous cells in cervical tissue by upregulating the apoptosis pathway and antitumorigenesis. In this regard, the cytotoxicity of each one of the compounds was estimated, and the combinations of them were evaluated for any synergistic efects. Moreover, Cis was employed as a wellknown anticancer agent for comparing the potential of natural antioxidants.
Te MTT assay showed that both Ge and Qu had high cytotoxic potential for Hela cells after 48 hours. However, Cit had a less cytotoxic efect than Ge and Qu. Te probable reason could be the high antioxidant characteristics of all   these compounds, which were earlier seen in previous studies [39,40]. Tese fndings were backed up by molecular examination, where all three compounds showed signifcantly higher Caspase3 expression than that of the control group, which demonstrated the potential of the apoptosis execution phase [41]. Moreover, they all resulted in high expression of p53 protein, which was proven as a regulator protein that is often mutated in cancers [42]. Furthermore, the expression of Bax and Bcl2 also increased in Ge, Qu, and Cit cells, which strongly suggested apoptosis to be the main mechanism of action [42][43][44]. Even the ratio of Bax to Bcl2 increased in all these cells, which was previously confrmed as a prognostic marker of tumor location [45]. Our fndings were in line with those of an earlier study, where a herbal extract with high antioxidant characteristics showed anticancer and apoptosis-inducing properties in Hela cells [46]. As a comparison, we used Cis, which was proven as an anticancer drug [23]. Tis drug was shown to have some toxicity and resistance in patients [47] and caused oxidative stress as a side efect [48]. So we used this chemical drug to investigate the possible synergism between a chemotherapy agent and natural antioxidants and any protectivity against it. Earlier studies have presented some of the natural compound's potential against this drug [49][50][51].
Regarding our combined treatment groups, we observed some improvement compared to the sole groups (excluding the Cis group). According to both the MTT assay and western blot results, the combination of Ge + Qu was slightly better than that of Ge + Cit. Te most likely answer for this was that both Ge and Cit have pretty much the same mechanism of action, and they even can be extracted from the same family of plants, such as Rosa damascena [52]. However, quercetin, which was similarly another member of favonoids [53], increased the efcacy of the treatment moderately and caused a signifcant improvement compared to Ge + Cit. As expected, the combination of all these three natural substances as one group was remarkedly higher than that of all other natural treatment cell groups [54]. Te most likely explanation is that they fulfll each other's mechanism of action and have strong synergy together, which resulted in all markers including the expression of caspase3, p53, Bax, and Bcl2 proteins. In addition, the cytotoxicity of this combination nearly reached that of the Cis-receiving cells. Tis phenomenon was previously observed [55] and summarized [56] that natural antioxidants like favonoids can be employed as a new evidence-based treatment [7].
Despite a slight increase in IC50 in the simultaneous use of Cis and natural substances compared to Cis alone, it is suggested to use them together because benefts are outweighing harms. First, the cytotoxicity of Ge + Qu + Cis and Ge + Qu + Cit + Cis cells was still acceptably low, and in a matter of induction of apoptosis, they performed even better than Cis alone. Since earlier studies have shown Cisinduced nephrotoxicity and hepatotoxicity [57,58], using an NA that can modulate side efects is a great advantage.
In addition, it is suggested to further investigate the potential of natural antioxidants against cancer cells in vivo because natural substances showed great promise against Hela cells, which can encourage further studies. Finally, it should be noted that using these natural substances was shown to reduce the toxicity of Cis, but this study demonstrated that this reduction of oxidative damage has nothing to do with the efcacy of treatment. Since the apoptosis indices were substantially increased in combination-       therapy cell groups, it can be said that these natural compounds successfully improved the Cis function without any noticeable power reduction.

Conclusion
Our fndings strongly suggested that using natural antioxidants in cases of cervical cancer should be considered as an adjuvant to chemo-anticancer agents for both their therapeutic potential and side-efect reduction.

Data Availability
Data are available from the corresponding author on reasonable request.