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

Salehi, Alireza; Faraji, Samar; Shafaei, Ahmadreza; Kangarlou, Kimiya; Behzadi, Amirhoushang

doi:https://doi.org/10.1155/2023/4926020

Journal of Chemistry

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Abstract Introduction Materials and Methods Results Discussion Conclusion Data Availability Conflicts of Interest Authors’ Contributions References Copyright Related Articles

Research Article | Open Access

Volume 2023 | Article ID 4926020 | https://doi.org/10.1155/2023/4926020

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

Alireza Salehi,¹Samar Faraji,¹Ahmadreza Shafaei,¹Kimiya Kangarlou,²and Amirhoushang Behzadi¹

Academic Editor: Chun-Hua Wang

Received18 Apr 2023

Revised15 Jun 2023

Accepted03 Jul 2023

Published07 Jul 2023

Abstract

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 effect 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 µg/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 significant cytotoxic potential by upregulating all mentioned proteins, which demonstrated the apoptosis pathway. Our findings 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-effect reduction in vivo. Overall, it can be concluded that these natural antioxidants are effective against cancer cells by upregulating the apoptosis pathway.

1. 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 [3–5]. However, more exclusive aspects are still unknown, including antiviral, anticarcinogenic, antitumor, and antistress properties [6]. The role of natural antioxidants in deregulating oxidative stress is undeniable [7], as they can effectively 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 [9–12]. 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 effects. 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]. They 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 fighting 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. This mechanism can lead to programmed cell death and have a synergistic effect 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 beneficial characteristics [17]. Similarly, citronellol comes from Rosoideae and has anti-inflammatory [18] and anticancer effects [19]. Another potent antioxidant is quercetin, which is known to be an anti-inflammatory, 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 first metal-based anticancer drug [23]. The 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. Therefore, 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.

2. Materials and Methods

2.1. Natural Antioxidants and Hela Cells

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

2.2. Cell Culture

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

2.3. Study Design

After the cells reached more than 75% confluence, they were divided into separated cell groups, and the treatments were applied to them. The cell groups included the following: Group 1: the control cells received no treatment. Group 2: Ge cells received geraniol at doses of 1000, 500, 250, 125, 62.5, 31.25, and 15.625 µg/ml. Group 3: Qu cells received quercetin at doses of 1000, 500, 250, 125, 62.5, 31.25, and 15.625 µg/ml. Group 4: Cit cells received citronellol at doses of 1000, 500, 250, 125, 62.5, 31.25, and 15.625 µg/ml. Group 5: Cis cells received cisplatin at doses of 150, 75, 37.5, 18.75, 9.375, 4.6875, and 2.34375 µg/ml. Group 6: Ge + Cit cells received a combination of geraniol and citronellol, in which both substances were administrated at doses of 1000, 500, 250, 125, 62.5, 31.25, and 15.625 µg/ml. Group 7: Ge + Qu cells received a combination of geraniol, quercetin, and citronellol, in which all substances were administrated at doses of 1000, 500, 250, 125, 62.5, 31.25, and 15.625 µg/ml Group 8: Ge + Cit + Qu cells received a combination of geraniol, quercetin, and citronellol, in which all substances were administrated at doses of 1000, 500, 250, 125, 62.5, 31.25, and 15.625 µg/ml. Group 9: Ge + Qu + Cis cells received a combination of geraniol, quercetin, and cisplatin, in which the first two substances were administrated at doses of 1000, 500, 250, 125, 62.5, 31.25, and 15.625 µg/ml and the latter was administrated at doses of 150, 75, 37.5, 18.75, 9.375, 4.6875, and 2.34375 µg/ml. Group 10: Ge + Qu + Cit + Cis cells received a combination of geraniol, quercetin, citronellol, and cisplatin, in which the first three substances were administrated at doses of 1000, 500, 250, 125, 62.5, 31.25, and 15.625 µg/ml and the latter was administrated at doses of 150, 75, 37.5, 18.75, 9.375, 4.6875, and 2.34375 µg/ml.

2.4. MTT

The MTT assay was employed to estimate cell viability after treatment. The method of Mosmann was followed [26]. Briefly, 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-buffered 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. The whole procedure was performed in triplicate [27–29].

2.5. 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 buffer. 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].

The lysates were mixed with an equal volume of the sample buffer, and after boiling for 5 minutes, they were loaded on SDS-PAGE gel. Electrophoresis was performed, and proteins were transferred to the polyvinylidene difluoride (PVDF) membrane (Bio-Rad, 162-017777, USA). Next, they were blocked for 1 hour with 5% bovine serum albumin (Sigma-Aldrich, A-7888, USA) in Tween 20 with 0.1% gelatin. Finally, they were stained with anti-Bax (Cat No: ab32503, Abcam), anti-Bcl2 (Cat No: ab32503, Abcam), anti-p53 (Cat No: ab131442, Abcam), anti-caspase 3 (Cat No: ab184787, Abcam), and anti-β actin (Cat No: ab8227, Abcam) and incubated for one hour at room temperature. After washing three times with TBST (mixture of Tris-buffered saline and polysorbate 20), the membranes were incubated one more time with goat anti-rabbit IgG H&L (HRP) (Cat No: ab6721, Abcam). After 1 to 2 minutes of exposure to ECL (enhanced chemiluminescence), the bands appeared [31]. Finally, the bands were analyzed by using ImageJ software [32].

2.6. Data Analysis

The MTT results were analyzed by employing Graphpad Prism software version 9. was considered significant [33–35]. The one-way ANOVA and the post hoc Tuckey test were employed to compare the differences between different cell groups [36, 37]. Moreover, English letters were used to show a significant difference in cell groups from highest to lowest [38].

3. Results

3.1. Assessment of Cell Viability

The overall IC50 of each treatment can be observed in Table 1. Moreover, as can be seen in the first figure, geraniol showed cytotoxicity in different doses, and IC50 was different 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 difference between cell viability after 8 hours and 48 hours was significant. Moreover, cell viability was significantly lower after 48 hours than after 8 hours (Figure 1).

(a)

(b)

(c)

Regarding quercetin, substantial cytotoxicity was observed in different doses, and IC50 was significantly different 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 significantly different, where cell viability was the lowest after 48 hours, followed by 24 hours and 8 hours (Figure 2).

(a)

(b)

(c)

Regarding Cit, citronellol showed moderate cytotoxicity in different doses, and the IC50 of the 48-hour time point was significantly different 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).

(a)

(b)

(c)

As can be seen from the figure below, cisplatin showed cytotoxicity in different doses, and IC50 was different 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 difference between the cell viability after 8 hours and 48 hours was significant. Moreover, cell viability was significantly lower after 48 hours than after 8 hours (Figure 4).

(a)

(b)

(c)

Regarding the mixture of geraniol and citronellol, cytotoxicity in different 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 difference between cell viability after 8 hours and 48 hours was significant. Moreover, cell viability was significantly lower after 48 hours than after 8 hours (Figure 5).

(a)

(b)

(c)

The mixture of geraniol and quercetin was nearly similar to that of geraniol and citronellol, which showed cytotoxicity without any significant difference between time points. The obtained IC50 values were 309.5, 165.4, and 130.8 µg/mL after 8, 24, and 48 hours, respectively. The same trend of decreasing IC50 after each time point happened again (Figure 6).

(a)

(b)

(c)

As was observed, the mixture of geraniol, quercetin, and citronellol showed cytotoxicity in different doses, and IC50 was different 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 difference between the cell viability after 8 hours and 48 hours was significant. Moreover, cell viability was significantly lower after 48 hours than after 8 hours (Figure 7).

(a)

(b)

(c)

As the figure below demonstrated, the mixture of geraniol, quercetin, and cisplatin showed cytotoxicity in different doses, and IC50 was different 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 difference between the cell viability after 8 hours and 48 hours was significant. Moreover, cell viability was significantly lower after 48 hours than after 8 hours (Figure 8).

(a)

(b)

(c)

As the final group, the mixture of geraniol, quercetin, citronellol, and cisplatin showed cytotoxicity in different doses, and IC50 was different 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 difference between the cell viability after 8 hours and 48 hours was significant. Moreover, cell viability was significantly lower after 48 hours than after 8 hours (Figure 9).

(a)

(b)

(c)

3.2. Western Blotting

The protein expressions of 4 different 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 significantly 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 significant difference 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 significantly higher than that of the Ge + Qu + Cis group (Table 2).

4. 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 effects. Moreover, Cis was employed as a well-known anticancer agent for comparing the potential of natural antioxidants.

The MTT assay showed that both Ge and Qu had high cytotoxic potential for Hela cells after 48 hours. However, Cit had a less cytotoxic effect than Ge and Qu. The probable reason could be the high antioxidant characteristics of all these compounds, which were earlier seen in previous studies [39, 40]. These findings were backed up by molecular examination, where all three compounds showed significantly 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 [42–44]. Even the ratio of Bax to Bcl2 increased in all these cells, which was previously confirmed as a prognostic marker of tumor location [45]. Our findings 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]. This drug was shown to have some toxicity and resistance in patients [47] and caused oxidative stress as a side effect [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 [49–51].

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. The 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 flavonoids [53], increased the efficacy of the treatment moderately and caused a significant 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]. The most likely explanation is that they fulfill 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. This phenomenon was previously observed [55] and summarized [56] that natural antioxidants like flavonoids 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 benefits 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 Cis-induced nephrotoxicity and hepatotoxicity [57, 58], using an NA that can modulate side effects 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 efficacy 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.

5. Conclusion

Our findings 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-effect reduction.

Data Availability

Data are available from the corresponding author on reasonable request.

Conflicts of Interest

The authors declare that there are no conflicts of interest regarding the publication of this article.

Authors’ Contributions

ASa supervised the whole project, conceptualized the study, designed the methodology, carried out investigation, carried out software analysis, and wrote the original draft. ASa, SF, Ash, KK, and AB contributed to cellular and molecular tests.

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Copyright © 2023 Alireza Salehi et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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