Cervicare™ induces apoptosis in HeLa and CaSki cells through ROS production and loss of mitochondrial membrane potential

Abstract

Cervicare™ is a poly-herbal preparation comprised of a combination of 6 plants; most have demonstrated antimicrobial and anticancer properties in preclinical studies. The effect of the ethanol and aqueous extracts of Cervicare™ on cell proliferation and apoptosis using cervical cancer HeLa and CaSki cells was investigated for the first time in the present study. MTT assay results showed that Cervicare™ extracts exerted time- and dose-dependent inhibition of cell viability. The hallmark properties of apoptosis like cell shrinkage and cytoplasmic condensation were observed using an inverted phase contrast microscope, ethidium bromide/acridine orange and Hoechst 33342/propidium iodide fluorescent staining methods. Furthermore, our results demonstrated that Cervicare™ extracts induced apoptosis in HeLa and CaSki cells by ROS generation and mitochondrial depolarization in a concentration dependent manner. The results showed that Cervicare™ extracts were capable of suppressing cell migration and inhibiting colony formation in a dose-dependent manner. Moreover, western blot analysis demonstrated the involvement of a mitochondria-dependent apoptosis pathway in the apoptosis inducing activity of Cervicare™ ethanol extract in HeLa cells. GC-MS analysis of the ethanolic extract afforded the identification of 40 substances, showing that it was primarily composed of anti-cancerous compounds such as xanthorrhizol (60.40%), octacosane (9.93%) and squalene (1.24%). Together, these results point out the Cervicare™ mediated inhibition of HeLa cell growth via induction of apoptosis and that it may be a potential anticancer agent which deserves further investigation.

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Introduction

Cancer of the cervix is the second most diagnosed cancer in women living in less developed countries.¹ Even though diagnostic skills and breakthroughs have improved, cervical cancer has continued as one of the main causes for morbidity and mortality globally with an estimated 7.8 new cases for every 100 000 women per year.² More than 85% of cervical cancer deaths occur in developing countries with lower incomes and poorer hygiene.^3,4 Due to the alarming prevalence of cervical cancer, researchers are expending a great deal of effort to develop new drugs and treatments that are highly effective. Traditional medicinal plants known to be safe, operational, and effective forms of complementary alternative medicines and they have been used in several cancer studies. Cervicare™ is a poly-herbal preparation consisting of a combination of 6 plants including: Andrographis paniculata, Curcuma xanthorriza, Cinnamomum zeylanicum, Momordica charantia, Syzygium polyanthum and Orthosiphon stamineu. Most of these plants have demonstrated antimicrobial and anticancer properties in preclinical studies. Andrographis paniculata (Acanthaceae) which is also called “kalmegh,” is indigenous to tropical Asia and it regularly grows in isolated patches.⁵ This plant has a history of use in traditional medicine due to its various pharmacological activities. The well-known main constituent of this plant extract is Andrographolide, which has shown significant cytotoxic activity against different types of cancer such as cervical, hepatocellular, breast, and gastric cancer.^6–9 The methanol and hydroalcoholic extracts of Andrographis paniculata and some of the isolated compounds have demonstrated growth inhibitory and anticancer activity on different human cancer cell lines.^10–12 Cinnamomum zeylanicum, or true cinnamon is an ancient spice that originated in Sri Lanka and South India.¹³ It has been traditionally used as curative herb for digestive and respiratory problems. Various in vitro and in vivo studies reported that Cinnamomum zeylanicum successfully inhibited angiogenesis lymphoid, leukemia and mouth carcinoma^14,15 and it possess antimicrobial, antioxidant and free radical scavenging properties.^16–18 Curcuma xanthorriza or “Java turmeric” is a rich source of a sesquiterpenoid compound called xanthorrhizol. This valuable compound possesses remarkable antimicrobial,^19,20 antioxidant²¹ and anti-inflammatory²² activities. It is thought to be effective against cervical cancer by up-regulation of Bax and P53 in HeLa cells.²³ There are reports of xanthorrhizol-induced apoptosis against breast, liver and oral cancer cell lines.^24–28

Orthosiphon stamineus is a native of moderate tropical Southeast Asian countries like Malaysia, China, India, Australia, and some parts of Africa.²⁹ In vivo and in vitro studies reported the effectiveness of a 50% ethanol extract of Orthosiphon stamineus in colon cancer treatments as it reduced vascular endothelial growth factor (VEGF) levels, thus blocking the VEGF signaling pathway.^30,31 The aqueous extract improved the caspase activity and inhibited the clonogenic potential of human OSCC cells.³² Momordica charantia commonly known as “Bitter Melon” is widely distributed through tropical regions and used as traditional medicine for various diseases by people from many countries around the world.³³ Bitter melon extract (BME) has the ability to inhibit the progression of cancer by inducing apoptosis in different cancer cell lines.^34–36 Isolated compounds of the plant, such as GAP31, MAP30, lectin, eleostearic acid, and RNase MC2, have also been used as anti-cancerous agents in various preclinical studies.^37–41 The final constituent of Cervicare™ is Syzygium polyanthum (Wight.) Walp. It is also known as “Salam” or “Indonesian bay-leaf”. This plant has been used as a traditional remedy for the treatment of diarrhea, diabetes, ulcers, and blood pressure. Although each of these plants are being used separately in traditional medicine to manage various diseases and there are reports on their therapeutic potential, to our knowledge, no effort has been made to explore the anti-cancer potential of the Cervicare™ poly-herbal formulation. The main goal of this study was to evaluate the cytotoxic activity of Cervicare™ aqueous and ethanol extracts on cervical carcinoma cancer cells. Furthermore, we examined if the cytotoxic effects were related to the apoptosis induction.

Results

Effect of Cervicare™ on cell viability

The potential cytotoxic effects of Cervicare™ extracts were investigated by MTT assay at different concentrations (31.25 to 2000 μg mL⁻¹) to determine the IC₅₀ value in HeLa, CaSki and HSF1184 cells. The anti-proliferative profiles of the ethanol and aqueous extracts against HeLa cancer cells are depicted in Fig. 1. The findings of the present study revealed that the ethanol and aqueous extracts considerably reduce HeLa cells viability in a concentration- and time-dependent manner. DMSO-treated samples showed no significant suppressing effect on the growth of cervical cancer cells. Statistical analysis showed a significant difference between control (0.1% DMSO in medium-treated cells) and treated groups. In addition, there was a significant difference between extracts-treated and cisplatin-treated cells indicating that the positive control was a more potent cytotoxic compared to the Cervicare™ extracts. The IC₅₀ values for Cervicare™ extracts and positive control (cisplatin) is presented in Table 1. The ethanol and aqueous extracts were observed to produce IC₅₀ values of approximately 95.96 (95% CI: 78.93–119.1) and 153.9 (95% CI: 127.6–185.7) μg mL⁻¹, after 24 hour exposure, respectively. A further reduction in IC₅₀ values of Cervicare™ extracts were found after 48 hour incubation with extracts. The cytotoxic activities of ethanol and aqueous extracts were more pronounced after 72 hours of treatment (IC₅₀ values of 18.59 (95% CI: 13.81–25.02) and 23.73 (95% CI: 18.16–31.02) for ethanol and aqueous extracts, respectively).

Fig. 1 Time and dose dependent growth inhibition of cervical cancer cells by Cervicare™ extracts. Cell viability assays of HeLa cells treated with different concentrations of (A) ethanol and (B) aqueous extracts of Cervicare™ for indicated time intervals. Cytotoxic effect of extracts was compared to reference drug, cisplatin on HeLa cell (C). The results were expressed as a ratio of treated cells to the negative control cells. Data are represented as means ± SD and each individual experiment were repeated three times. An asterisk * indicates statistically significant difference from negative control (one way ANOVA, P < 0.05).

Table 1 Inhibitory effect (IC₅₀ values) of Cervicare™ extracts and cisplatin against HeLa cells at different incubation times

Extracts	IC50 (μg mL^−1) (95% CI)
	24 h	48 h	72 h
Ethanol	96.95 (78.93–119.1)	42.91 (33.74–54.56)	18.59 (13.81–25.02)
Aqueous	153.9 (127.6–185.7)	47.63 (37.34–60.75)	23.73 (18.16–31.02)
Cisplatin	5.523 (4.936–6.179)	2.920 (2.461–3.464)	1.896 (1.495 to 2.406)

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Cytotoxic activity of Cervicare™ ethanol and aqueous extracts towards CaSki cells is represented in Fig. 2. As can be seen, both ethanol and aqueous extracts demonstrated the ability to inhibit the proliferation of CaSki cells in a time- and concentration-dependent manner. A moderate cytotoxic activity was noted after 24 hours incubation with ethanol and aqueous extracts. A stronger cytotoxicity was observed after 48 and 72 hours exposure. The aqueous and ethanol extracts were found to produce IC₅₀ values of approximately 34.59 (95% CI: 29.35–40.76) and 29.23 (95% CI: 24.18–35.34) μg mL⁻¹, after 72 hours of treatment, respectively. As shown in Table 2 pair-wise comparison between IC₅₀ values of the aqueous versus ethanol extract shows that the former is less effective in inhibiting the growth of CaSki cell line compared to ethanol extracts.

Fig. 2 Cervicare™ extracts inhibit the proliferation of CaSki cervical cancer cells in time and dose-dependent manner. Cell viability assays of CaSki cells exposed to different concentrations of (A) ethanol and (B) aqueous extracts of Cervicare™ for indicated time intervals. Cytotoxic ability of extracts was compared to reference drug, cisplatin on CaSki cell (C). The results were expressed as a ratio of treated cells to the negative control cells. Data is represented as means ± SD and each individual experiment was repeated three times. An asterisk * indicates a statistically significant difference from the negative control (one way ANOVA, P < 0.05).

Table 2 Inhibitory effect (IC₅₀ values) of Cervicare™ extracts and cisplatin against CaSki cells at different incubation times

Extracts	IC50 (μg mL^−1) (95% CI)
	24 h	48 h	72 h
Ethanol	118.8 (98.84–142.9)	57.97 (47.32–71.02)	29.23 (24.18 to 35.34)
Aqueous	183.3 (155.4–216.2)	125.2 (108.8–144.1)	34.59 (29.35 to 40.76)
Cisplatin	9.763 (8.994–10.60)	6.983 (6.605–7.384)	5.456 (5.119–5.814)

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In order to unravel the question of whether the Cervicare™ extracts affected the viability of normal cells, HSF1184 cells were exposed to different concentrations of aqueous and ethanol extracts for 24, 48 and 72 hours. The results depicted in Fig. 3 revealed that none of the aqueous and ethanol extracts exerted any cytotoxic effect against normal cells as the IC₅₀ values obtained for normal cells were much higher than those in the cancer cells. Although the extracts affected the viability of normal cells in high concentrations, surprisingly, lower concentrations promote the growth and viability of cells. Overall, cytotoxic study results demonstrate that Cervicare™ extracts may selectively target cervical cancer cells but save normal cells. To best of our knowledge, this is the first report of the in vitro cytotoxic activity of this poly-herbal preparation against human cancer and normal cell lines.

Fig. 3 Effects of Cervicare™ (A) ethanol and (B) aqueous extracts on the viability of normal cell line; HSF1184. The results were expressed as a ratio of treated cells to the negative control cells. The results showed that just high concentrations of the aqueous and ethanol extracts exerted some cytotoxic effect against normal cells. Data is represented as means ± SD and each individual experiment was repeated three times. An asterisk * indicates a statistically significant difference from the negative control (one way ANOVA, P < 0.05).

Observation of morphological changes of HeLa and CaSki cells following treatment with Cervicare™

The MTT assay results revealed the anti-proliferative activity of Cervicare™ extracts against HeLa and CaSki cells. In order to examine whether the cytotoxic activity of the extracts was related to their ability to induce apoptosis in HeLa and CaSki cells, the morphology of cells was observed under an inverted phase contrast microscope (Zeiss Axiovert 100). Morphological alterations in the cells were examined and compared to control (0.1% DMSO-treated cells) cells after different incubation times with the extracts. The morphological changes observed in HeLa cells treated with ethanol and aqueous extracts at IC₅₀ concentration are illustrated in Fig. 4. Visualization of the control cells exhibited the flattened, well spread, and normal proliferation of cells in culture. The morphology of the treated cells changed significantly compared to the control cells. The results revealed obvious cellular detachment, cytoplasmic condensation, cell shrinkage, and loss of typical morphology in treated cells, indicating the initiation of apoptosis. Evident reduction in the cell adhesion capacity, cell population, and confluency was observed with the increase in the incubation time and concentration of the extracts. After 72 hours of incubation, cells detached from the plate and came to the medium as floating cells. Morphological alterations and characteristics of apoptosis such as cell detachment and rounding were more noticeable when treated with higher concentrations of extracts.

Fig. 4 Morphological changes of HeLa cells treated with Cervicare™ ethanol and aqueous extracts at their respective IC₅₀ concentrations. After 24, 48 and 72 hours of treatment, the cell morphological alterations and characteristic of apoptosis were observed under an inverted light microscope (20× magnification).

Upon the exposure of CaSki cells with increasing incubation time and concentrations of Cervicare™ ethanol and aqueous extracts apoptotic cells were seen more frequently (Fig. 5). The results represented obvious cellular detachment, cytoplasmic condensation, cell shrinkage and loss of typical morphology in treated cells, indicating the initiation of apoptosis.

Fig. 5 Morphological alterations of CaSki cells treated with Cervicare™ ethanol and aqueous extracts at IC₅₀ concentration. Similar to HeLa cells, CaSki cells demonstrated normal morphology with no reduction in cell number in culture without extracts. Significantly lower cell numbers and typical apoptotic features like cytoplasmic condensation and cell shrinkage were observed at 48 and 72 hours.

Detection of apoptosis by acridine orange/ethidium bromide (AO/EB) staining

To further investigate the type of cell death induced by Cervicare™ extracts, assessment of morphological changes in HeLa and CaSki cells were carried out using AO/EB staining under fluorescence microscopy. HeLa cells were exposed to different concentrations of the ethanol and aqueous extracts for 24 hours and stained with acridine orange and ethidium bromide. Acridine orange (AO) is a vital dye that permeates both viable and nonviable cells and their nuclei are stained green. Ethidium bromide is taken up only by nonviable cells where their cytoplasmic membrane integrity is lost, and the nuclei become red. Different types of cells were identified based on morphological changes, chromatin condensation, and fluorescence emission. Live cells exhibited uniformly stained green nuclei with normal morphology. Early apoptotic cells displayed green yellow fragments, which are highly condensed. Late apoptotic cells showed orange-red fragmented/condensed nuclei and necrotic cells with orange-red chromatin in round nuclei. The AO/EB staining of Cervicare™ aqueous and ethanol extracts-treated HeLa cells at different concentrations displayed characteristics of cell death and transformed cell morphology such as cellular shrinkage, condensed nuclei (fragmented or intact), and membrane blebbing. In contrast, green live HeLa cells with round intact nucleus were observed in the negative control group. As shown in Fig. 6, bright green early apoptotic cells with nuclear margination and chromatin condensation as well as orange-red, late apoptotic cells with fragmented and condensed chromatin and apoptotic bodies were found in treated cells. In the present study, the apoptotic index measured using AO/EB double staining indicated a significant increase in apoptotic cell population induced by the Cervicare™ ethanol and aqueous extracts in dose-dependent manner compared to the control (Fig. 7).

Fig. 6 Nuclei morphological changes of HeLa cells treated with Cervicare™ extracts stained by AO/EB observed under fluorescence microscope (20× magnifications). HeLa cells incubated for 24 hours with ethanol and aqueous extracts of Cervicare™. Live cells exhibited uniformly stained green nuclei with normal morphology. Early apoptotic cells displayed green yellow fragments. Late apoptotic cells showed orange-red fragmented/condensed nuclei and necrotic cells with orange-red chromatin in round nuclei. Cells treated with cisplatin were served as positive control.

Fig. 7 Quantification of the live, apoptotic, and necrotic HeLa cells at different concentrations (31.25–1000 μg mL⁻¹) of (A) ethanol and (B) aqueous extracts. Data was expressed as means ± SD of three replicates in three independent experiments, counting a minimum of 200 total cells each. An asterisk * represents a statistically significant difference from their respective vehicle control (one way ANOVA, P < 0.05).

Exposure to Cervicare™ extracts triggers apoptosis with membrane blebbing and orange condensed chromatin in approximately 22.55 ± 4.59 and 7.44 ± 1.57% of the HeLa cells treated with lowest concentration (31.25 μg mL⁻¹) of ethanol and aqueous extracts, respectively. The highest percentage of apoptotic cells was achieved when cells were treated with 250 and 500 μg mL⁻¹ of ethanol and aqueous extracts. The frequency of necrotic cells with uniformly stained orange nuclei was sharply increased at highest concentration (1000 μg mL⁻¹) of extracts. The apoptotic index at different concentrations was lower in aqueous extract compared to methanol extract. The AO/EB staining results demonstrated that Cervicare™ extracts possibly induced cell apoptosis in HeLa cells.

Like HeLa cells, CaSki cells also exhibited visible green/orange and orange condensed chromatin after treatment with ethanol and aqueous extracts of Cervicare™ (Fig. 8). Percentage of apoptotic cells were at highest level after treatment with 500 μg mL⁻¹ of ethanol and aqueous extracts with percentages of 69.88 ± 4.004 and 59 ± 3.34 respectively. The highest percentage of necrotic cells was observed after treatment with 1000 μg mL⁻¹ of either extract (Fig. 9). Overall, compare to aqueous extract, ethanol extract was shown to be more effective at inducing apoptosis in CaSki cells.

Fig. 8 Morphological alterations of AO/EB stained CaSki cells after 24 hours exposure with Cervicare™ under fluorescence microscope (20× magnifications). Live cells display uniformly stained green nuclei with normal morphology. Green yellow fragments show cells in early apoptotic stages, while orange-red fragmented/condensed nuclei belong to late apoptotic cells. Necrotic cells have orange-red chromatin in round nuclei. Cells were treated with IC₅₀ value of cisplatin as a positive control.

Fig. 9 Percentages of live, apoptotic, and necrotic cells at CaSki cells treated with various concentrations (31.25–1000 μg mL⁻¹) of (A) ethanol and (B) aqueous extracts. Frequencies of apoptotic cells at different concentrations were higher in ethanol extract compare to aqueous extract. Data is represented as means ± SD of three replicates in three independent experiments, counting a minimum of 200 total cells each. * indicates a statistically significant difference from their respective vehicle control (one way ANOVA, P < 0.05).

Propidium iodide and Hoechst 33324 staining for apoptosis detection

To further examine whether cells undergo apoptosis, the nuclei of untreated and treated HeLa and CaSki cells were stained with DNA-specific fluorochromes, propidium iodide (PI), and Hoechst 33342. Hoechst 33342, unlike PI, is a cell permeable dye, which stains the nucleus of both live and dead (apoptosis or necrosis) cells. Propidium iodide (PI) is a dye that can bind DNA and it cannot pass through intact membrane of the living cells. Therefore, PI, which is a red-fluorescence dye, is only able to permeant and stain dead cells. The regular and intact live cells with the nuclei were stained with a less bright blue fluorescence (upon staining with Hoechst 33342 dye), and the absence of red fluorescence (due to the PI dye) were observed in control cells. Hallmark properties of apoptosis, such as chromatin condensation and fragmentation, were seen in treated HeLa (Fig. 10) and CaSki (Fig. 11) cells with different concentrations of ethanol and aqueous extracts. Treated cells that exerted apoptosis characteristics emitted brighter blue fluorescence and bright red fluorescence after staining with Hoechst 33324 and PI dyes, respectively. Frequent apoptotic nuclei were detected at higher concentrations (250 μg mL⁻¹) compare to lower concentrations (31.25 μg mL⁻¹). The frequency of apoptotic cells with fragmented nuclei was higher in the ethanol extract compare to the aqueous extract. These results confirmed the AO/EB staining results and demonstrate that both ethanol and aqueous Cervicare™ extracts could induce apoptosis in HeLa and CaSki cells.

Fig. 10 Typical photographs illustrating the apoptotic effects of Cervicare™ (A) ethanol and (B) aqueous extracts against human cervical cancer cell lines after 24 hours incubation. The left panel displays Hoechst 33342 staining and the right panel displays PI staining of the same field. Nuclei morphological alterations were observed under fluorescence microscope (20× magnification). Both viable and nonviable cells nuclei were stained with Hoechst 33342 and PI was unable to stain viable cells nuclei. Treated cells that exerted apoptosis characteristics emitted brighter blue fluorescence and bright red fluorescence after staining with Hoechst 33324 and PI dyes, respectively. (C) Control positive cells treated with cisplatin at IC₅₀ concentration.

Fig. 11 Fluorescence imaging of Hoechst/PI staining of CaSki cells treated with Cervicare™ (A) ethanol and (B) aqueous extract for 24 hours at concentrations of 31.25 and 250 μg mL⁻¹. The left panel displays Hoechst 33342 staining while the right panel displays PI staining of the same field. Morphological changes of treated cells were visualized under fluorescence microscope (20× magnifications). The nuclei of both viable and dead cells were stained with Hoechst 33342 and PI was unable to stain viable cells nuclei. Treated cells at both concentrations showed condensed and fragmented nuclei. (C) IC₅₀ concentration of cisplatin served at positive control.

Effect of Cervicare™ on cell migration

A scratch assay is an assay conducted to prove the migration of cancer cells and it was used to demonstrate the anti-metastatic characteristic of Cervicare™. A scratch assay was carried out on confluent monolayers of HeLa and CaSki cells. Cells were scratched as described in the method section and then were cultured with fresh medium in the presence or absence of Cervicare™ ethanol and aqueous extracts of various concentrations. The data shown in Fig. 12, clearly indicated that HeLa control cells reached a complete confluency after 24 hours while cells treated with ethanol and aqueous extracts exhibited migration to a minimum with a complete seize in migration after 24 hours of incubation. Fig. 13 provides a quantitative analysis of the cell migration of both Cervicare™ extracts on HeLa cells at different concentrations (31.25–1000 μg mL⁻¹) where the results were expressed as a percentage of cell migration. The results showed an effective reduction in the migration of HeLa cells in a time- and dose-dependent manner when treated with ethanol and aqueous extracts. In other words, as the concentration and incubation time increased, the percentage of cells migration inhibition also significantly increased (P < 0.05). There was no significant difference in the migration inhibition rate at the lowest concentration (31.25 μg mL⁻¹) of both extracts compare to the negative control (p > 0.05) but there was a significant difference in migratory ability of the lowest concentration (31.25 μg mL⁻¹) and concentration 62.5 μg mL⁻¹ of ethanol and aqueous extracts after 24 hours of treatment. The ethanol extract displayed higher migration inhibition rates compared to the aqueous extract. The scratch assay findings suggest that Cervicare™ extracts have the ability to inhibit the migration of HeLa cells.

Fig. 12 Effects of Cervicare™ ethanol and aqueous extracts on cell migration. (A) Confluent monolayers of HeLa cells on a 24-well plate were wounded by scratcher and treated extracts. The images of wounded HeLa cells were captured using an inverted phase-contrast microscope (10× magnification) at different time intervals (0, 6, 12 & 24 h).

Fig. 13 Quantitative measurement of migration inhibition rate of (A) ethanol and (B) aqueous extracts on HeLa cells at different concentrations. Scratch closure rates were analysed quantitatively as the difference between scratch width at 0, 6 and 12 or 24 hours and results were expressed as the percentage of cell migration. The results showed an effective reduction in the migration of HeLa cells in a time and dose-dependent manner. Data is represented as means ± SD of three replicates in three independent tests. An asterisk * represents a statistically significant difference from their respective negative control (one way ANOVA, P < 0.05).

The pictures in Fig. 14 shows the effect of Cervicare™ ethanol and aqueous extracts on CaSki cells compare to a control. As with the HeLa cells, the spreading of CaSki cells was also significantly inhibited as a results of treatment with extracts in a concentration dependent manner. Quantitative analysis of cell migration of ethanol and aqueous extracts on CaSki cells treated with various concentrations of Cervicare™ ethanol and aqueous extracts (31.25–1000 μg mL⁻¹) was carried out and the results were expressed as a percentage of cell migration (Fig. 15). Cells treated with ethanol extract showed better inhibition of cell migration compare to aqueous extract.

Fig. 14 Inhibition of CaSki cells migration after treatment with aqueous and ethanol extracts of Cervicare™. CaSki cells were cultured at 24-well plate and scratched after reaching to confluent monolayer. Pictures were captured using an inverted phase-contrast microscope (10× magnifications) at different time intervals (0, 6, 12 & 24 h).

Fig. 15 Quantitative analysis of migration inhibition rate of (A) ethanol and (B) aqueous extracts on CaSki cells treated with various concentrations of extracts. Closure rate of scratches were examined quantitatively as the difference between scratch width at 0, 6 and 12 or 24 h and outcomes were expressed as percentage of cell migration. Findings presented an effective reduction in migration of CaSki cells in a time and dose-dependent manner. Data are represented as means ± SD of three replicates in three independent tests. An asterisk * represents statistically significant different from their respective negative control (one way ANOVA, P < 0.05).

Cervicare™ extracts suppressed colony formation in HeLa and CaSki cells

As mentioned earlier, Cervicare™ extracts effectively inhibited the growth of HeLa and CaSki cells and showed no cytotoxic effects on the growth of normal HSF-1184 fibroblasts. In order to confirm the potential of extracts to suppress the growth of cervical cancer cells, a clonogenic assay was conducted. To measure clonogenicity, HeLa and CaSki cells were exposed to different concentrations (31.25 to 1000 μg mL⁻¹) of ethanol and aqueous extracts.

As depicted in Fig. 16, the findings for both extracts revealed a significant clonogenic inhibition of HeLa cells in a concentration-dependent manner. Untreated cells produced a large number of colonies. Even the lowest concentrations of extracts (31.25 μg mL⁻¹) had a significant effect on the colony-forming potential of cells compare to the control. The size and number of colonies were significantly reduced, particularly at higher concentrations (500, 1000 μg mL⁻¹) in treated cells. Comparisons between the colony forming abilities of aqueous and ethanol extracts indicated that the latter had more colony forming inhibitory effect compare to aqueous extract. It was clearly evident that Cervicare™ extracts exerted a suppressing effect on the colony formation of HeLa cells.

Fig. 16 Effects of Cervicare™ extracts on the clonogenicity of HeLa cells. (A) Ethanol and (B) aqueous extracts inhibited colony formation in a dose-dependent manner. The ethanol extract inhibited the colony-forming abilities of HeLa cells more effectively than the aqueous extract. (C) Quantification of colony forming-potential of extracts on HeLa cells at different concentrations. The colonies were counted under an inverted light microscope and the clonogenicity of the cells at each dose of extract is expressed in terms of percent of control. Results are represented as means ± SD of three replicates in three independent experiments. An asterisk * represent statistically significant different from their respective untreated control (one way ANOVA, P < 0.05).

Effects of Cervicare™ aqueous and ethanol extract on colony forming ability of CaSki cells during the culture period is portrayed in Fig. 17. Control negative cells possess a higher number of colonies compare to treated cells. Obtained results confirmed that the treatment of cells with selected extracts resulted in a concentration-dependent inhibition in the colony formation ability of CaSki cells.

Fig. 17 Effects of Cervicare™ extracts on clonogenicity of CaSki cells. (A) Ethanol and (B) aqueous extracts inhibited colony formation in a dose-dependent manner. The ethanol extract inhibited the colony-forming abilities of CaSki cells more effectively than aqueous extract. (C) Quantification of colony forming-potential of extracts on CaSki cells at different concentrations. The colonies were counted under dissection (stereo) microscope and the clonogenicity of the cells at each dose of extract is expressed in terms of percent of control. Results are represented as means ± SD of three replicates in three independent experiments. An asterisk * represent statistically significant different from their respective untreated control (one way ANOVA, P < 0.05).

Exposure of CaSki cells with higher concentration of both ethanol and aqueous extracts resulted in considerable decrease in number of the colonies as well as notable alteration in size of them. Quantitative analysis of colony forming inhibition potential of both aqueous and ethanol extracts against CaSki cells declared that later displayed greater ability.

Cell cycle analysis using flow cytometry assay

The results of a study demonstrated the cell growth inhibitory activities of Cervicare™ extracts against HeLa and CaSki cervical cancer cells. A significant cell proliferation inhibition was observed in cervical cancer cells treated with ethanol extract. Consequently, a further mechanistic study was carried out on cervical cancer cells treated with ethanol extract to determine the possible inhibitory effects of Cervicare™ on cell cycle distribution using flow cytometric analysis. To examine the mechanism for the anti-proliferative activity of ethanol extract toward HeLa and CaSki cell lines, cells were treated with IC₅₀ concentration of Cervicare™ ethanol extract for 24 and 48 hours. Treated and control (0.1% DMSO-treated) cells were stained with propidium iodide and the percentages of G0/G1, S and G2/M cell population measured by flow cytometry.

The percentage of G0/G1 phase cells significantly increased from 33.96% in the control cell to 57.2% and 56.6% (p < 0.05) in HeLa cells treated with ethanol extract for 24 and 48 hours, respectively. In addition, a concomitant reduction in S and G2-M cells population were observed in the treatment groups (Fig. 18). The cell population in the S phase decreased from 49% in the control group to 33.1% and 27.53% in HeLa cells treated for 24 and 48 hours, respectively. Similarly, treatment with Cervicare™ ethanol extract decreased the proportion of cells in G2/M phase (7.76% at 24 hours and 7.39% at 48 hours) compared to the control group (15.66%). These results indicated that Cervicare™ ethanol extract induced G0/G1 cell cycle arrest in HeLa cell after 24 and 48 hours of treatment.

Fig. 18 Representative histograms of cell cycle machinery in HeLa and CaSki cells. Cells were treated with IC₅₀ concentration of Cervicare™ ethanol extract for 24 and 48 hours and subjected to flow cytometry cell cycle analysis after staining with PI.

According to these findings, there were some differential effects of Cervicare™ ethanol extract in both HeLa and CaSki cell lines. In contrast to the HeLa cells, the CaSki cells treated with the extract had a higher S phase population (26.7%) compared with 8.17% in the control group after 24 hours, but this phase shifted towards the G0/G1 phase after 48 hours of treatment. Following 48 hours treatment, treated cells experienced a significant increase in the number of the cells in G0/G1 phase of the cell cycle from 87.73% in the control group to 96.62% in treated CaSki cells. Larger cell accumulation in the G0/G1 phase was coupled with a significant decrease in the S and G2/M phase cells after 48 hours (Fig. 19).

Fig. 19 Graphical representations of the effect of Cervicare™ ethanol extract on cell cycle distribution. (A) HeLa and (B) CaSki cells were treated with IC₅₀ concentration of Cervicare™ ethanol extract for 24 and 48 hours and cellular DNA was stained with PI prior to flow cytometry analysis. The values are represented as means ± SD of three replicates in three independent experiments. The asterisk indicates a statistically significant difference from the control (one way ANOVA, P < 0.05).

Measurement of reactive oxygen species (ROS) generation

Reactive oxygen species (ROS) hyper generation and oxidative stress is associated with disturbances in the mitochondrial membrane permeabilization, thus indicating the activation of intrinsic apoptotic pathway.⁴² Triggering apoptotic in cancer cells by generation of free radicals is the mechanism of action of various chemotherapeutic drugs.^43,44 Previous results confirmed that Cervicare™ ethanol extract exerted better cytotoxic activities towards HeLa and CaSki cervical cancer cells and thus it was chosen for further apoptotic detection analyses to understand the mechanism of cell death. To determine if treatment with Cervicare™ ethanol extract affected the level of ROS generation, the intracellular ROS level was identified using the fluorescent dye DCF-DA, which forms highly fluorescent DCF (2′,7′-dichlorofluorescein) in the presence of ROS. Fluorescent microscopy was carried out to check the level of ROS generation based on qualitative microscopic observations. Cells were treated with various concentrations of ethanol extract (31.25 to 1000 μg mL⁻¹). Cells treated with 0.1% DMSO were used as a negative control. 50 μM H₂O₂ served as a positive control. As shown in Fig. 20 and 21, healthy cells reflected faint green fluorescent demonstrating the balance between generation and removal of ROS. 31.25 μg mL⁻¹ of ethanol extract, which was the lowest concentration of treatment that had the least effect on ROS levels. By contrast, treatment with 250 μg mL⁻¹ of extract and H₂O₂ as positive control increased the level of ROS production in cells. The intracellular levels of ROS where then quantitatively measured using a fluorescent microplate reader. Based on the obtained results, intracellular ROS levels increased in a concentration dependent manner (Fig. 22). The highest level of ROS formation in HeLa and CaSki cells was obtained at concentrations of 250 μg mL⁻¹ and 500 μg mL⁻¹, respectively. Lower levels of ROS production in the highest concentrations might be due to the presence of late apoptotic and necrotic cell deaths. The results of the present study were in agreement with previous findings that indicated the ability of plant crude extracts to enhance the ROS production in cancer cells.⁴⁵ Mitochondria play a key role in the life and death of a cell since they are vital cellular sources of ROS.⁴⁶ Our observations confirmed that Cervicare™ ethanol extract induces ROS hyper generation in HeLa and CaSki cells. We propose that this event is one of the triggers of cell death.

Fig. 20 Assessment of ROS generation in HeLa cells treated with Cervicare™ ethanol extract using the fluorescent probe DCF-DA. Ethanol extract significantly increased the production of ROS in a concentration-dependent manner. Images were captured using an inverted fluorescent microscopy (Zeiss Axiovert A1, Germany) at 10× magnifications. 50 μM H₂O₂ was served as positive control.

Fig. 21 Effects of Cervicare™ ethanol extract on the ROS generation of CaSki cells. Ethanol extract significantly increased the production of ROS in a concentration-dependent manner. Images were captured using an inverted fluorescent microscopy (Zeiss Axiovert A1, Germany) at 10× magnifications. 50 μM H₂O₂ served as positive control.

Fig. 22 Quantitative analysis of ROS generation of HeLa and CaSki cells after treatment with Cervicare™ ethanol extract. The ROS generation potential of extracts was compared to H₂O₂. The results revealed a significant increase of ROS levels in treated HeLa and CaSki cells in a dose-dependent manner. Data is represented as means ± SD of three replicates in three independent tests. An asterisk * represents a statistically significant difference from the vehicle control (one way ANOVA, P < 0.05).

Assessment of mitochondrial membrane potential (MMP)

Determination of changes in mitochondrial permeability provides an early signal of the initiation of cellular apoptosis. Given that oxidative stress can lead to mitochondrial damage, ΔΨ_m/MMP was studied using the membrane-permeant JC-1 dye. In order to determine MMP collapse, cells were treated with different concentrations of Cervicare™ ethanol extract and 50 μM carbonyl cyanide m-chlorophenyl hydrazone (CCCP). JC-1 forms aggregates with red fluoresces in healthy cells with intact mitochondria but it shows monomeric green fluorescent in early apoptotic cells. As depicted in Fig. 23 and 24, a decrease of ΔΨ_m was shown by the reduction of red fluorescence and the increase of green fluorescence. Healthy control cells exhibited orange red fluorescence due to high ΔΨ_m cells treated with 31.25 μg mL⁻¹ ethanol extract had both red orange and green fluorescence. Cells treated with 250 μg mL⁻¹ ethanol extract exhibited a loss of red aggregate fluorescence and an abundance of green monomer fluorescence representing the depolarization of MMP. The loss of red fluorescence and the abundance of green fluorescence was observed in the positive control group, demonstrating its potent apoptotic activity. MMP was then analyzed using a fluorescent microplate reader. Alterations of the red to green fluorescence intensity ratio reveals changes in the ΔΨ_m.⁴⁷ A concentration-dependent decrease of red to green fluorescence intensity was detected in cells after 12 hours of treatment with ethanol Cervicare™ extract (Fig. 25). According to our findings, Cervicare™ ethanol extract decreases the level of mitochondrial membrane potential in HeLa cells slightly better than CaSki cells. The JC-1 assay results were consistent with those obtained from ROS generation, indicating that Cervicare™ ethanol extract induced the breakdown of the mitochondrial membrane potential (MMP) in cervical cancer cells suggesting its potent apoptotic activity. Extreme ROS generation was followed by a loss of ΔΨ_m. The disturbance of the mitochondrial membrane potential probably started the apoptotic cascade in cells treated with Cervicare™ ethanol extract. Our results are in agreement with the findings of another study where methanol leaf extract of Costus speciosus demonstrated its ability to reduce ΔΨ_m of HepG2 cells.⁴⁸

Fig. 23 Photographs showing the effects of Cervicare™ ethanol extract on mitochondrial membrane potential of HeLa cells. Loss of red fluorescence and the abundance of green monomers are clearly visible in treated cells compare to healthy cells.

Fig. 24 Images depicted the effects of Cervicare™ ethanol extract on mitochondrial membrane potential of CaSki cells. Loss of red fluorescence and the abundance of green monomers are clearly visible in treated cells compare to healthy cells.

Fig. 25 Quantification of mitochondrial membrane potential alterations in HeLa and CaSki cells treated with Cervicare™ ethanol extract. Outcomes demonstrated obvious loss of mitochondrial membrane potential in treated cells compare to healthy cells. CCCP served as positive control. Data is represented as means ± SD and each individual experiment was repeated three times. An asterisk * represents a statistically significant difference from control healthy cells (one way ANOVA, P < 0.05).

Effect of Cervicare™ ethanol extract on apoptosis-related proteins in HeLa cells

Previous results obtained from JC-1 and ROS generation assays revealed that Cervicare™ ethanol extract induced the depolarization of the mitochondrial membrane potential (MMP) in HeLa and CaSki cells. To further investigate whether ethanol extract induced mitochondrial mediated apoptosis in human cervical cancer cell lines, a western blot analysis was carried out. Since Cervicare™ ethanol extract exhibited more apoptotic activity in HeLa cell than CaSki, the former was selected for the western blot study to explore the mechanism of apoptosis. To delineate the role of the intrinsic apoptotic pathway in Cervicare™-causing apoptosis in HeLa cell, the expression levels of different apoptosis-regulating proteins such as Bcl-2 family members (Bax and Bcl-2), effector caspases (caspase-3 and caspase-7) and PARP (poly-ADP ribose polymerase) were measured. The protein levels of Bax (pro-apoptotic protein) and Bcl-2 (anti-apoptotic protein) were measured as these proteins are the major regulators of the intrinsic pathway of apoptosis, which mediates cell survival or cell death.⁴⁹ HeLa cells were treated with IC₅₀ concentrations of Cervicare™ ethanol extract for indicated times (2, 4, 6, 12, 24 hours) and the expression levels of Bcl-2 family in treated and control cells were examined. The western blot analysis demonstrated that Cervicare™ ethanol extract obviously reduced the expression levels of Bcl-2 protein. As depicted in Fig. 26, ethanol extract decreased the expression levels of Bcl-2 protein in a time dependent manner. Downregulation of Bcl-2 protein was observed as early as 2 hours after treatment with ethanol extract.

Fig. 26 Effect of Cervicare™ ethanol extract on Bcl-2 protein in HeLa cells evaluated by western blotting and densitometry analysis. Cells were treated with IC₅₀ concentration of ethanol extract and control cells (0.1% DMSO) for the indicated times. β-Actin was used as a loading control. Densitometry analysis showed the time-dependent downregulation of Bcl-2 protein. Downregulation of Bcl-2 protein was observed as early as 4 hours after treatment with ethanol extract. The densitometer-intensity data is represented as means ± SEM of three replicates in three independent experiments. The asterisk indicates a statistically significant difference from the control (one way ANOVA, P < 0.05).

Moreover, our findings demonstrated that treatment of HeLa cells with Cervicare™ ethanol extract resulted in a significant increase in the protein expression of pro-apoptotic Bax (Fig. 27). The increase in the expression of Bax protein was more marked after 2 hours of treatment with ethanol extract compared to the control (0 hours). A time-dependent increase in the expression level of Bax was observed in treated cells. Following ethanol extract treatment, the upregulation of Bax protein was apparent, which sensitized HeLa cell to apoptosis.

Fig. 27 Effect of Cervicare™ ethanol extract on Bax protein in HeLa cells evaluated using western blotting and densitometry analysis. Cells were treated with IC₅₀ concentration of ethanol extract and control cells (0.1% DMSO) for the indicated times. β-Actin was used as a loading control. Densitometry analysis showed the time-dependent upregulation of Bax protein. The densitometer-intensity data is represented as means ± SEM of three replicates in three independent experiments. The asterisk indicates a statistically significant difference from the control (one way ANOVA, P < 0.05).

To further evaluate whether effector caspases (caspase-3 and caspase-7) and the downstream PARP molecule were involved in the mechanisms of Cervicare™-causing apoptosis, the expression levels of caspases and the subsequent proteolytic cleavage of PARP proteins in HeLa cells were determined. HeLa cells were treated with Cervicare™ ethanol extract for 2, 4, 6, 12 and 24 hours. A western blot analysis was performed on untreated (0 hours) and treated cell lysates. The results showed that the procaspase-3 protein was cleaved into its activated form (17 kD fragment) after treatment with Cervicare™ ethanol extract. As shown in Fig. 28, a time-dependent decrease was observed in the level of the procaspase-3. By contrast, the treated cells exhibited an increase of cleaved caspase-3 expression in HeLa cells in a time dependent pattern.

Fig. 28 Effect of Cervicare™ ethanol extract on procaspase-3 and cleaved caspase-3 protein in HeLa cells evaluated using western blotting and densitometry analysis. Cells were treated with IC₅₀ concentration of ethanol extract and control cells (0.1% DMSO) for the indicated times. β-Actin was used as a loading control. Densitometry analysis showed the time-dependent cleavage of procaspase-3 to its active form (17 kDa). The densitometer-intensity data is represented as means ± SEM of three replicates in three independent experiments. The asterisk indicates a statistically significant difference from the control (one way ANOVA, P < 0.05).

Likewise, procaspase-7 protein was also cleaved into its activated form (17 kD fragment) after treatment with Cervicare™ ethanol extract (Fig. 29). The expression levels of procaspase-7 in treated cells were decreased. According to results, cleaved caspase-7 increased in a time dependent manner and the cleavage products were detectable as early as 2 h after treatment with ethanol extract.

Fig. 29 Effect of Cervicare™ ethanol extract on procaspase-7 and cleaved caspase-7 protein in HeLa cells evaluated by western blotting and densitometry analysis. Cells were treated with IC₅₀ concentration of ethanol extract and control cells (0.1% DMSO) for indicated times. β-Actin was used as loading control. Densitometry analysis showed time-dependent cleavage of procaspase-7 to its active form (17 kDa). The densitometer-intensity data are represented as means ± SEM of three replicates in three independent experiments. The asterisk indicates statistically significant difference from control (one way ANOVA, P < 0.05).

Since activation of caspase-3 and caspase-7 induces the proteolytic cleavage of PARP, a major apoptotic enzyme, a western blot analyses on PARP was carried out to determine whether this protein degraded in the treated cells thus further confirming caspase-3 and caspase-7 activations. Treatment of HeLa cells with Cervicare™ ethanol extract induced a time dependent decrease in the level of the 116 kDa PARP, indicating its cleavage into the active 85 kDa fragment (Fig. 30).

Fig. 30 Effect of Cervicare™ ethanol extract on PARP protein in HeLa cells evaluated using western blotting and densitometry analysis. Cells were treated with IC₅₀ concentration of ethanol extract and control cells (0.1% DMSO) for the indicated times. β-Actin was used as a loading control. Densitometry analysis showed the time-dependent cleavage of PARP to its active form (85 kDa). The densitometer-intensity data is represented as means ± SEM of three replicates in three independent experiments. The asterisk indicates a statistically significant difference from the control (one way ANOVA, P < 0.05).

GC-MS analysis of Cervicare™ ethanol extract

Treatment of cells with the ethanol extract of Cervicare™ exhibited better anticancer results compare to those cells treated with aqueous extract. Thus ethanol extract was used for GC-MS analysis. The GC-MS analyses of Cervicare™ ethanol extract revealed the existence of 40 compounds. A typical chromatogram of Cervicare™ ethanol extract is shown in Fig. 31. Xanthorrhizol was confirmed to be the major component with the highest area percentage of 60.40%. Fig. 32 presents the mass spectrum of xanthorrhizol. The chromatogram showed that the other major compounds are (1) octacosane, (2) gamma-sitosterol, (3) 3,7-cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene), (4) urs-12-en-3-ol, acetate, (3.beta.)-, (5) heptacosane, (6) butane, 2,4-dimethyl-2,4-bis(4-methoxyphenyl), (7) squalene, (8) einosane, 7-hexyl, (9) acetic acid, 10-hydroxy-12a-methyl-7-oxo-1,2, (10) di-n-octyl phthalate and (11) tricyclo[4.4.0.0(2,7)]dec-8-ene-3-methanol. The main components with their retention time (RT), molecular formula, molecular weight (MW) and concentration (%) in the ethanol extracts are presented in Table 3.

Fig. 31 GC-MS chromatogram of the ethanol extract of Cervicare™.

Fig. 32 Mass spectrum of xanthorrhizol.

Table 3 Major chemical compounds identified in Cervicare™ ethanol extract by GC-MS in order of area percentage

Compounds	Area %	Molecular weight
Xanthorrhizol	60.40	218
Octacosane	1.19	394
	1.68
	1.86
	2.14
	1.76
	1.30
Gamma-sitosterol	5.38	414
3,7-Cyclodecadien-1-one,3,7-dimethyl-10-(1-methylethylidene)	3.46	218
Urs-12-en-3-ol, acetate, (3.beta.)-	1.69	468
Heptacosane	1.51	380
Butane, 2,4-dimethyl-2,4-bis(4-methoxyphenyl)	1.30	298
Squalene	1.24	410

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Discussions

Plant derived natural products as therapeutic agents in cancer therapy have attracted the interest of researchers due to their ability to modulate apoptosis. Induction of apoptosis in cancer cells plays an important role in cancer therapy.^50,51 The current study investigated the anti-proliferative activities of Cervicare™ extracts, which is the mixture of six plants native to tropical regions, many of which are used traditionally to treat various diseases including cancer. Reports indicating the effects of different extracts or isolated compounds of Orthosiphon stamineus, Andrographis paniculata, Curcuma xanthorriza, Cinnamomum zeylanicum, and Momordica charantia on cervical cancer are fewer than they are for other cancers including colon, oral, acute T cell leukemia, gastric, lung, breast, prostate and nasopharyngeal carcinoma. To the best of our knowledge, the present study reports the in vitro cytotoxic activity of Cervicare™ extracts against HeLa and CaSki cervical cancer cells for the first time.

The cancerous HeLa, CaSki and non-cancerous HSF1184 cell lines were selected to understand the characteristic of the cytotoxicity effects of Cervicare™ ethanol and aqueous extracts on cancer cells using a MTT assay. The IC₅₀ concentration of Cervicare™ extracts were found to be less than 25 μg mL⁻¹ indicating potent cytotoxic activities in HeLa cells. Based on the obtained results, Cervicare™ had slightly higher cytotoxicity towards HeLa cells than CaSki cells. An evaluation of cytotoxicity effects revealed that the extracts exhibited anti-proliferative effects in a time- and dose-related manner at the end of 24, 48 and 72 hours incubation with the extracts. Overall, the ethanol extract was more effective in inhibiting the proliferation of both HeLa and CaSki cells compare with the aqueous extract.

Anti-cancer agents that can select cancer cells without causing excessive damages to normal cells, have received considerable interest in the development of potential cancer chemo-preventive and therapeutic drugs.^52,53 Consequently, we addressed the question of whether the cytotoxic effects of Cervicare™ extracts were selective toward normal cells. Our results indicated that Cervicare™ extracts not only had no cytotoxic activity against non-cancerous cells, they promoted the growth of normal cells in certain concentrations, which deserves attention.

Programmed cell death or apoptosis is a highly organized cell death process characterized by certain morphological features such as cell rounding, loss of plasma membrane phospholipid asymmetry, enzymatic cleavage of the DNA into oligonucleosomal fragments, and the breaking up of the cell into membrane-bounded vesicles (apoptotic bodies) that are subsequently ingested by macrophages.^52,54 Phase contrast microscopy, which is one of the best methods to define apoptosis based on morphological alterations, was used to identify apoptosis in the treated cells. After the preliminary estimation of cell death, fluorescence microscopy was used to confirm the induction of apoptosis in cancer cells. The present study demonstrated that Cervicare™ extracts were capable of causing morphological changes and apoptosis induction in the cervical cancer cell lines under investigation. The distinctive morphological features of apoptosis were detected in cells treated with Cervicare™ extracts upon fluorescent staining with ethidium bromide/acridine orange and Hoechst 33342/propidium iodide.

Further proof of the growth inhibitory activity of Cervicare™ extracts was provided by scratch and colony forming assays where aqueous and methanol extracts were found to inhibit the colony formation and migration of HeLa and CaSki cells in a dose-dependent manner. Data obtained from the morphological assessment study was in agreement with cytotoxic results for Cervicare™ on HeLa and CaSki cells. Mitochondria plays a key role in the life and death of a cell since they are vital cellular sources of ROS.⁴⁶ Our observations confirmed that Cervicare™ ethanol extract induced ROS generation in HeLa and CaSKi cells and we propose that this event is one of the triggers of cell death. Our results are in accordance with the findings of Looi et al.⁵⁵ in which the chloroform fraction of Centratherum anthelminticum (L.) significantly increase the production of ROS in melanoma A375 cells.

Extreme ROS generation is followed by a loss of ΔΨ_m. Disturbance of mitochondrial membrane potential likely started the apoptotic cascade in cells treated with Cervicare™ ethanol extract. Our results are in agreement with findings of a previous study where methanol leaf extract of Costus speciosus showed the ability to reduce ΔΨ_m of HepG2 cells.⁴⁸

The results obtained from western blot analysis were consistent with the results of a present study that reported that ethanol extract induced apoptosis in HeLa cells. Bcl-2 downregulation and Bax upregulation, which is thought to cause apoptosis induction, suggested that the induction of apoptosis by Cervicare™ ethanol extract in HeLa cell might be through intrinsic apoptosis pathway. Up-regulation of Bax/Bcl-2 ratio could lead to the loss of MMP, which facilitated cytochrome c release and in turn, activated a number of caspases.⁵⁶ The cleavage of procaspases-3 and procaspase-7 into their smaller, active forms (cleaved caspases-3 and caspase-7) demonstrated that caspases are involved in apoptosis-induction activity of Cervicare™ ethanol extract in HeLa cells. In response to treatment, the level of the cleaved PARP increased in a time related manner demonstrating that the process of apoptosis was triggered in HeLa cells because PARP cleavage is a biochemical marker and indicator of apoptosis.

Activated caspases and PARP plays an important role in final steps of apoptosis as degraded PARP results in apoptosis by cell shrinkage, DNA fragmentation and nuclear condensation.^57,58 These results demonstrated that Cervicare™ ethanol extract-induced apoptosis in HeLa cells was associated with activation of caspase-3 and caspase-7 which in turn preceded the proteolysis of PARP, DNA fragmentation and apoptosis induction.

The GC-MS study was designed to determine the phytochemicals present in the ethanol extract of Cervicare™. Identified phytochemicals possess many biological properties. Among the identified phytochemicals, xanthorrhizol was the most common. As discussed earlier, xanthorrhizol inhibits various types of cancer by inducing apoptosis in cancer cells.^23–28 Octacosane was the second most common phytochemical with a total percentage of 9.93%. Figueiredo et al.⁵⁹ identified this straight chain alkane as the major compound present in Pyrostegia venusta heptane extract. Their results revealed the remarkable cytotoxic activity of octacosane against murine melanoma B16F10Nex2 cells. The outcomes of their study also demonstrated the significant inhibition of subcutaneous melanoma in mice treated with octacosane.

Another important component identified in Cervicare™ ethanol extract is a natural triterpene known as squalene. This compound was first found in high amounts in shark liver oil⁶⁰ and it is believed to be the reason behind the absence of cancer in sharks. Olive oil contains lesser amounts of squalene and an olive oil rich diet was linked with a decreased risk of cancer.⁶¹ There are several studies that examine the chemopreventive, antioxidant and antitumor characteristics of squalene or squalene containing compounds.^62–65 Squalene can selectively protect normal bone marrow cells against cisplatin-induced toxicity.⁶⁶ Taken together, these findings demonstrated that Cervicare™ extracts, particularly ethanol extracts, are a promising candidate for suppressing the growth of cervical cancer cells.

Experimental section

Chemicals and reagents

Solvents used for extraction were of analytical grade whereas cell culture grade solvents were used for cell culture assays. Ethanol was obtained from Score Scientific SDN BHD. DMEM (Dulbecco's modified Eagle medium), RPMI 1640 (Roswell Park Memorial Institute), FBS (fetal bovine serum), trypsin, penicillin, PBS (phosphate buffer saline) and MitoProbe JC-1 assay kit were purchased from Life Technologies Inc., Grand Island, NY, USA. Propidium iodide (PI), Hoechst 33342 and 2′,7′-dichlorofluorescein diacetate were obtained from Sigma-Aldrich, USA. DMSO and MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] were obtained from RCI Labscan and Invitrogen, respectively. Human cervical cancer cells (HeLa and CaSki), and human skin fibroblast cell line (HSF1184) were obtained from the American Type Culture Collection (ATCC). Cisplatin was provided by BioSyTech Malaysia. Bax (6A7), Bcl-2 (124), caspase-3, caspase-7 (B4-G2) and PARP (C-2-10) monoclonal primary antibodies and goat anti-mouse IgG (H+L) secondary antibody alkaline phosphatase were obtained from Thermo Fisher Scientific Pierce. β-Actin (13E5) rabbit mAb primary antibody and goat anti-rabbit IgG, (H+L) AP conjugated secondary antibody were purchased from Cell Signaling Technology.

Plant material

The mixture herbal powder of Cervicare™ was prepared and supplied by Naturemedic Supply, Lot 4763, Chendering Industrial Area, 21080 Kuala Terengganu, Terengganu, Malaysia.

Extraction of Cervicare™ powder

Ethanol extract. The extraction was carried out by soaking 50 grams of plant material (powdered) in a 1 L beaker covered with aluminum foil, with 80% extracting solvent (ethanol). The mixture was incubated in a platform shaker at 55–60 °C. The solvent was drained after 4 hours and replaced with fresh solvent. This process was repeated 3 times. The solutions obtained after each maceration were filtered using Whatman filter paper (no. 1) and evaporated using a rotary evaporator (Buchi Rotavapor R210, Switzerland) in a water bath at 50 °C until a crude ethanol extract was formed. The obtained extract was then stored at −20 °C in sealed tubes for further analysis.

Aqueous extract. The extract was prepared by soaking 50 grams of powdered plant material in warm purified water (55–60 °C) for 1 day. The solvent was removed after the incubation time and the residue was re-extracted with purified water three times under the same conditions. The solutions were then filtered using Whatman filter paper (no. 1). Finally, the filtrates were combined, freeze dried, and stored at −20 °C in sealed tubes prior to analysis.

Cell culture

Human cervical cancer cells (HeLa and CaSki cell) were used to determine the cytotoxic capacity of the extracts. Human skin fibroblast (HSF1184) was used as the normal cells. HSF1184 was maintained as a monolayer in DMEM. The HeLa and CaSki cervical cancer cells were cultured using RPMI 1640 mediums. The mediums were supplemented with 1% penicillin–streptomycin (100 units per mL to 100 μg mL⁻¹) and 10% FBS and incubated in a humidified atmosphere of 5% CO₂ at 37 °C.

Anti-proliferative assay

The in vitro cytotoxic activities of Cervicare™ extracts were calorimetrically determined using the MTT method.⁶⁷ The HeLa, CaSki, and HSF1184 cells were trypsinized, counted, and incubated at densities of 1 × 10⁵ cells per well in 96-well plates overnight. Following the overnight incubation to allow attachment and at 80 to 90% confluency, the cells were treated with various concentrations (31.25, 62.5, 125, 250, 500, 1000, 2000 μg mL⁻¹) of Cervicare™ ethanol and aqueous extracts for 24, 48 and 72 hours. Dimethyl sulfoxide (less than 0.1% in culture medium) and cisplatin were employed as the negative control and positive reference standard, respectively. After the incubation period, the solutions in the wells were discarded and replaced with fresh medium. 20 μL of MTT reagent in PBS was subsequently added to each well and the plates were gently shaken and then incubated for 4 hours at 37 °C in 5% CO₂. Following incubation, the supernatant containing medium was aspirated and 200 μL of DMSO was added to each well to solubilize the existing formazan complex. The absorbance of solubilized formazan product (dark purple) was then measured at 570 nm using ELISA microplate reader. The experiments were conducted in triplicate (repeated 3 times) and the percentage of cell viability was calculated according to the following formula:

Morphological assessment by phase contrast inverted microscopy

The morphological changes to the treated cervical cancer cells were observed according to the protocol⁶⁸ with some modifications. A concentration of 1 × 10⁵ cells per well were seeded into six well plates. The cells were then treated for 24, 48 and 72 hours at IC₅₀ value of Cervicare™ aqueous and ethanol extracts. The medium was removed and cells were washed with PBS. Morphological alterations were tracked using phase contrast inverted microscope (Zeiss Axiovert 100, Germany) at 20× magnifications.

Apoptosis detection by AO/EB staining

Acridine orange (AO) and ethidium bromide (EB) double staining was carried out as described by Aryapour et al.⁶⁹ with slight modifications. The HeLa and CaSki cells monolayers were treated with ethanol and aqueous extracts of Cervicare™ at different concentrations (31.25–1000 μg mL⁻¹) for 24 hours. The cells treated with complete medium containing less than 0.1% DMSO at the absence of Cervicare™ acted as control cells. After treatment, cells were washed with PBS and a mixture of AO/EB stain (100 μg mL⁻¹ of each dye at the ratio of 1 : 1) was added to the cells. Samples were observed under an inverted fluorescence microscope (Zeiss Axio Vert.A1, Germany) at 20× magnifications. AO is a cell-permeant dye and can stain the cellular DNA of living cells, EB was ejected from the living cells and was only able to reach the nuclear structure of the cells if their plasma membranes were ruptured and cells were dead.

Apoptotic index

Apoptotic cell death was quantitatively analyzed by calculating the apoptotic index (AI).⁷⁰ Cells were divided into 3 groups of viable, apoptotic and necrotic cells. Tests were conducted in triplicate and at least 200 of total target cells were scored each time. The following formulas were used to calculate the percentage of apoptotic and necrotic cells.

Hoechst 33342 and PI staining

Further confirmation of apoptosis was carried out by fluorescent Hoechst 33342 and propidium iodide (PI) staining according to the method described by Syed Abdul Rahman et al.⁶⁸ with minor modifications. The nuclear morphological changes were assessed by using Hoechst 33342 dye, which is a cell permeant blue fluorescence DNA dye and can enter live as well as apoptotic or necrotic cells. Live cells are identified using pale blue fluorescence while bright fluorescence showed the condensed nuclei of apoptotic cells. On the other hand, PI is a non-cell-permeable DNA dye and is usually used to detect dead cells. Log phase growing HeLa and CaSki cells were seeded into 6 well tissue culture plates and treated with and without different concentrations (31.25–1000 μg mL⁻¹) of ethanol and aqueous extracts of Cervicare™ for 24 hours. The extracts were discarded after treatment and cells were washed with PBS. The cells were stained with Hoechst 33342 staining solution (10 μg mL⁻¹) and incubated in the dark for 5 minutes. After incubation, the cells were exposed with PI staining solution (10 μg mL⁻¹) and incubated in the dark for 30 minutes. The stained cells where then observed under a fluorescence inverted microscope (Zeiss Axiovert A, Germany) at 20× magnifications.

Scratch assay

A scratch assay⁷¹ was performed to measure the migration ability of cells treated with Cervicare™ extracts. 3 × 10⁵ cells per mL of HeLa and CaSki cells were cultured in 24-well cell culture plates for 24 hours in a RPMI 1640 medium with 10% FBS to produce a nearly confluent cell monolayer. A sterile 200 mL plastic pipette tip was then used to scrape the monolayer. Any cellular debris was then washed out with PBS. Two different extracts (aqueous and ethanol based extracts) were tested, each with 6 different concentrations (31.25–1000 μg mL⁻¹). Three wells per concentration were treated with plant extracts and digital images were captured at 0, 6, 12, and 24 hours after the formation of the wounds. Cells treated with less than 0.1% DMSO in the culture medium were considered to be control cells. NIH Image J software (1.46r/Java 1.6.0_20 (32-bit)) released by Wayne Rasband in National Institute of Health, USA was used to evaluate and compare the width of the scratches at each time interval and a 0 hours. Healing rates were then calculated statistically and the final outcomes were stated as a percentage of cell migration.

Colony forming assay

A clonogenic survival determination assay was conducted according to method recommended by Franken et al.⁷² In summary, exponentially growing cells were cultured in 6 well culture plates at concentration of approximately 1 × 10³ cells per mL for 24 hours. The experiment was conducted in triplicate. Afterwards, the medium was discarded and cells were treated with various concentrations (31.25–500 μg mL⁻¹) of ethanol and aqueous extracts. After 24 hours of treatment, the extracts were replaced with fresh medium and the cells were kept at 37 °C in a CO₂ incubator for 10 days. Growth mediums were refreshed every two days. Colonies were fixed with −20 °C methanol and stained with 0.5% trypan blue solution. Colonies with 50 or more cells were counted and the results were expressed as percentage of colony forming potential.

Cell cycle analysis using flow cytometry assay

The effect of Cervicare™ ethanol extract on HeLa and CaSki cell cycle distribution were assessed by flow cytometric analysis using PI/RNAse staining solution (molecular probe, life technologies). The cells were plated in 6-well plate and cultured for 24 hours for cell growth. After 24 hours of incubation, HeLa cells in the exponential phase of growth were treated with IC₅₀ concentration of ethanol extract for 24 and 48 hours. In addition, control cells containing a medium with 0.1% DMSO were prepared. The cells were then detached from the plates by trypsinization, centrifuged at 300 × g for 5 minutes, and the pellet washed with cold PBS.

After collecting the cells by trypsinization, 1 × 10⁶ cells were fixed gently with chilled 70% ethanol and kept at −20 °C. The fixed cells were then centrifuged at 400 × g for 5 minutes to remove the ethanol from the cell pellet and washed three times with cold PBS followed by staining with 0.5 mL FxCycle PI/RNAse solution. Afterwards, the solution was incubated at room temperature in the dark for 30 minutes. Finally, the cells were analyzed using flow cytometry (BD FACSVerse™ flow cytometer, BD Biosciences, USA). Measurement of cell cycle distribution and population of each phase was carried out using ModFit LT™ software.

Generation of reactive oxygen species (ROS)

ROS generation in Cervicare™ treated HeLa and CaSki cells was measured both quantitatively and qualitatively. Fluorescent probe, 2′,7′-dichlorodihydrofluorescein diacetate (DCF-DA) (Sigma-Aldrich, USA) dye was used for this purpose. DCF-DA is a cell permeable fluorescent probe that is cleaved by intracellular nonspecific esterases and quickly oxidized by intracellular hydrogen peroxide and peroxidases to form highly fluorescent DCF (2′,7′-dichlorofluorescein).⁷³ The intensity of DCF fluorescence is consistent with the quantity of peroxide produced in the cells. Cells with concentration of 5 × 10⁴ per mL were seeded in 96 well plates and allowed to attach overnight. Treatments were carried out by adding various concentrations (31.25–1000 μg mL⁻¹) of Cervicare™ ethanol extract to the cells. After 12 hours of incubation in CO₂ incubator cells, the medium was removed and cells were again incubated with 20 μM DCF-DA⁷⁴ for 30 minutes at 37 °C in the dark. Subsequently, the cells were washed with PBS and the levels of ROS generation in the cells were analyzed using a fluorescent microplate reader (GloMax®-Multi Detection System, Promega, USA). The qualitative microscopic detection was carried out using an inverted fluorescent microscopy (Zeiss Axiovert A1, Germany) with an excitation wavelength of 488 nm and emission wavelength of 560 nm at 10× magnifications.

JC-1 mitochondrial membrane potential assay

Determination of mitochondrial membrane potential (MMP) was carried out using the mitochondria-specific cationic fluorescence dye JC-1. MitoProbe™ JC-1 Assay Kit was used (Life Technologies, USA). 5,5′,6,6′-Tetrachloro-1,1′,3,3′-tetraethyl benzamidazol-carbocyanine iodide, or JC-1, is a dual-emission fluorescent dye that is can selectively enter the mitochondria and reversibly change color from red to green once the membrane potential decreases. JC-1 enters the mitochondria and forms aggregates as it emits intense red fluorescence in healthy cells with normal polarized mitochondria. On the other hand, it forms monomers that emit green fluorescence in unhealthy or apoptotic cells with depolarized mitochondrial membrane. Fluorescent inverted microscopy and fluorescence spectrophotometry were used to measure the mitochondrial depolarization patterns of the cells both qualitatively and quantitatively. 5 × 10⁴ of HeLa and CaSki cells per mL were seeded in 96-well culture plates. After 1 day of incubation in a CO₂ incubator, the cells were treated with various concentrations of Cervicare™ ethanol extract (31.25–1000 μg mL⁻¹) for 12 hours. 50 μM of CCCP served as a control positive. 2 μM JC-1 solution (according to manufacturer's instructions) was used to stain the cells. After 45 minutes of incubation at 37 °C, the cells were washed with PBS and a further qualitative analysis of JC-1 uptake by mitochondria was carried out using an inverted fluorescent microscope (Zeiss Axiovert A1, Germany) at 40× magnification. A fluorescent microplate reader (GloMax®-Multi Detection System, Promega, USA) was used to quantitative analyze the JC-1 aggregates and monomers. Each test was conducted three times and the results were collected. The percentage of cells reflecting MMP changes was shown as a ratio of red to green fluorescence that was compared to the control Cervicare™ untreated cells.

Western blotting

HeLa cells were exposed to IC₅₀ concentration of Cervicare™ ethanol extract for durations of 0, 2, 4, 6, 12 and 24 hours. The cells were harvested and rinsed with ice-cold PBS. Cells were lysed using 1 mL of EDTA-free complete Lysis-M buffer containing complete protease inhibitor cocktail tablet. Cold cell scrapers were used to scrap the lysed cells. Micro-centrifuges tubes containing lysed cells were then centrifuged at 14 000 × g for 10 minutes at 4 °C. The supernatant was collected and the protein concentration was determined according to the Pierce™ BCA Protein Assay Kit (Thermo Scientific). The same amounts of proteins were subjected to 12.5% SDS-PAGE (w/v). Immun-Blot PVDF transfer membrane (BIO-RAD) was used to carry out the protein transfer.

The protein transfer was carried out at 100 V and 350 mA for 1 hour. The primary antibody treatment was carried out overnight at 4 °C. After incubation, the membranes were washed with TBST 3 times for 10 minutes and then incubated at room temperature for 1 hour with Alkaline Phosphatase (AP)-conjugated goat anti-mouse IgG secondary antibody. Following incubation, the membranes were washed with TBST 5 times for 10 minutes.

BCIP/NBT (5-brono-4-chloro-3-indolyl phosphate/nitro blue tetrazolium) substrate was used to perform colorimetric AP detection. The reaction of substrate with alkaline phosphatase (AP) bound to the secondary antibody produce a blue to purple insoluble form of dye that could be visually observed. The membrane was then subjected to treatment with β-actin primary antibody and goat anti-rabbit IgG, (H+L) AP conjugated secondary antibody.

GC-MS analysis

The GC-MS analysis of the ethanol extract of Cervicare™ was performed using GC-2010 gas chromatography (Shimadzu, Suzhou, China) equipped with a split/splitless injector, electronic pressure control, AOC-20i auto injector, GCMS-QP2010 ultra mass spectrometer (Shimadzu, Suzhou, China) and a GCMS Solution software. The BP5MS column with dimensions of 30 m × 0.25 mm capillary column was used for the analysis. Sample injections (1 μL) were performed in splitless mode. Oven temperature was kept at 50 °C for 1 minute before increasing to 300 °C at a rate of 5 °C min⁻¹ and holding for 5 minutes. Helium, as the carrier gas, was used at a constant flow of 1.0 mL min⁻¹ with a linear velocity of 35.6 cm s⁻¹. The inlet pressure of the carrier gas was 50.0 kPa. The injector temperature was 300 °C and MS ion source and interface temperatures were 200 °C and 300 °C, respectively. A mass range of 50 m/z to 600 m/z was scanned. Identification of compounds was based on comparisons of their mass spectra with those recorded in the National Institute of Standards and Technology database.

Statistical analysis

Data was presented as mean ± standard deviation mean (SD) of at least three independent experiments. The IC₅₀ values and their respective 95% confidence intervals (95% CI) for cytotoxic activity were obtained by non-linear regression using GraphPad Prism statistical software. The data in the present study was subjected to one-way analysis of variance (ANOVA) and Tukey post Hoc multiple range tests using the SPSS software version 21.0. Statistical significance was used to determine the significant differences between groups. The statistical significance was accepted at P < 0.05.

Conclusions

In conclusion, the findings of the present study revealed that Cervicare™ extracts, especially ethanol extract, could inhibit cancer cell proliferation via enhancement of ROS levels and loss of mitochondrial membrane potential in both HeLa and CaSki cells. In addition, the western blot results demonstrated that apoptosis induction through intrinsic pathway were the underling mechanisms that inhibited HeLa cell growth and proliferation. The extracts deserve further study to determine the mode of action of the Cervicare™ on cervical cancer cell lines and separate its bioactive components for further drug development.

Acknowledgements

The authors would like to acknowledge a research university grant from Universiti Teknologi Malaysia (Vote no. 04H93).

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