Cytotoxic activity of ethyl acetate extract of Chromolaena odorata on MCF7 and T47D breast cancer cells

Abstract

One of the most cancers in women that can be fatal is breast cancer. Radiation therapy, chemotherapy, or a combination of the two are often used to treat cancer, and these treatments tend to modify the immune system and weaken defences. Using natural compounds from plants has become a research interest to prevent cancer cell development. The aim of this study was to determine the anticancer activities of ethyl acetate extract of Chromolaena odorata (EACO) against breast cancer cells (MCF-7 and T47D). The viability of the cells was determined by the MTT colorimetry assays. The apoptosis test was performed by using flow cytometry. The IC50 value for MCF-7 cells was 218.78 µg/mL and 307.61 µg/mL for T47D. The extract acted selectively against breast cancer cells, with selectivity indexes against MCF-7 and T47D were 12.77 and 9.08, respectively. The viable cells of T47D cells were decreased from 85±36.5% (24 hours) to 54±34% (48 hours) after treatment with IC50 of EACO. Significant decrease of the MCF-7's viable cells were observed between 48 and 72 hours after treatment with IC50 (68.5±17.7% to 51.01±12.1%, respectively). Apoptosis assay showed that T47D and MCF-7 cells were mainly in the necrosis stage (83.35±0.49% and 95.15±1.76%, respectively). This study suggested that ethyl acetate extract of C. odorata is promising to be developed as an anticancer agent.

Introduction

Breast cancer was the most frequently diagnosed cancer in women in 2020, with a prevalence of 11.7% among all cancer cases [1]. An estimated 685,000 women died from breast cancer in 2020, making breast cancer the fifth-leading cause of cancer-related deaths worldwide [1]. Incidence rate for breast cancer was 88% higher in developed countries than in developing countries (55.9 vs 29.7 per 100,000) [1]. The mortality rate from female breast cancer was 17% higher in developing countries than in developed countries (15.0 vs 12.8 per 100,000) [1]. Despite a 0.5% annual increase in incidence, the mortality rate of breast cancer cases has dropped by 43% in the United States since 1989 [2]. However, in 2020, the incidence of breast cancer in Indonesia reached 68,858 cases (16.6%) out of a total of 396,914 new cases of cancer in Indonesia, with 22,430 deaths recorded [1].

Breast cancer is usually treated with combination of surgery, radiation, and medication (hormonal therapy, chemotherapy, and targeted biological therapy) [3]. These treatments may prevent growth and inhibit the spread of cancer. However, cancer treatment with radiation, chemotherapy, or a combination of the two tends to alter the body's defense system, lowering immunity and resulting in death [4]. In contrast, natural products isolated from medicinal plants have been used to treat various diseases since ancient times. Since most target-specific anticancer drugs failed to achieve the desired outcome, new therapies using natural products have become significant. As a potential therapeutic agent for cancer, natural products are readily available, inexpensive, with minimum cytotoxicity against normal cells [5]. The use of natural materials, predominantly plants, has become a trend by world pharmacologists to prevent the development of cancer cells and repair DNA and RNA damaged during gene mutations through signalling in the cell cycle pathway [6].

The perennial shrub Chromolaena odorata (L.) R.M. King & H. Robinson, also known as Eupatorium odoratum L., is a member of the Asteraceae family and has been previously used in traditional Chinese medicine [7]. This plant grows to a height of 2–3 m [8], native to the Caribbean islands and southern Florida and Texas [9]. C. odorata is a plant that thrives on various soil types and in various vegetation types, such as grasslands, arid bushveld, and forests, with an annual rainfall of less than 500 mm. The first introduction to Southeast Asia was most likely through the Calcutta Botanic Garden and it most likely spread throughout Indonesia via live cattle shipments [10].

C. odorata has various beneficial effects such as the leaves were used on wounds in Thailand to stop bleeding [7]. Several studies have reported C. odorata having antimicrobial [11,12], antibiofilm [13], anti-hepatotoxic [14,15], and antiproliferative properties [16-18]. Additionally, this plant contains chemical compounds that aid in limiting the development of pathogenic microorganisms and scavenged free radicals and possess selective anticancer properties [18].

C. odorata, as an anticancer agent, has been studied against various cancer cell lines, such as cervical, breast, colorectal, and liver cancer [19-24]. Studies have revealed the cytotoxic mechanism of this plant against the Ca151 breast cancer cell type by inhibiting Bcl2 expression and inducing apoptosis by inhibiting apoptosis signal kinase 1 against colorectal cancer [17,25]. However, previous studies only tested ethanol extract of this plant [23]. The aim of this study was to explore the potential cytotoxic activity of ethyl acetate extract of C. odorata (EACO) on breast cancer cells.

Methods

Plant material

C. odorata leaves were collected in Aceh Besar District, Aceh Province, Indonesia. The plants were identified by the Department of Biology, Faculty of Mathematics and Natural Sciences, Universitas Syiah Kuala, Banda Aceh, Indonesia (No B/435/UN11.1.8.4/TA.00.01/2020).

Extraction of C. odorata leaves

The extraction method of C. odorata leaves was previously described [21]. Briefly, the leaves were collected, washed, and dried. Then, they were ground with an electric grinder. The dried powdered leaves weighed five kilograms and were macerated in a 1:5 ratio with two different solvents of varying polarity. Leaves were macerated in n-hexane, stirred every four hours, and filtered every 24 hours. The n-hexane filtrate was collected after 72 hours, and vacuum evaporated at 40°C in a vacuum-rotating evaporator to acquire the viscous n-hexane extract. The n-hexane waste was dried and macerated for 72 hours in ethyl acetate, stirred every four hours, and filtered every 24 hours. The ethyl acetate extract of C. odorata leaves (EACO) was then collected after 72 hours.

Cell culture

The breast cancer cells, T47D (ductal carcinoma) and MCF-7 (adenocarcinoma), were maintained in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum, 2% penicillin-streptomycin, and fungizone 0.5% (Gibco, USA). The cells were kept in a humidified atmosphere (5% CO₂) at 37°C [24].Original Article

Viability assay

The viable cells of T47D and MCF-7 were evaluated by MTT colourimetric assay. This experiment was conducted in triplicate. The cells were cultured in 96-well microplates containing 1x104 cells/100 µL. Then, the cells were incubated at 37°C with 5% CO₂ for 24 hours. Vero cells acted as the negative control, MCF-7, and T47D cells were treated with EACO as the treatment, MCF-7 and T47D cells were treated with doxorubicin (DOXO) as the positive control, and well-seeded growth medium-only served as the medium (blank) sample. Four 96 wells-microplates were cultured with 1x10⁴ cells/100 µL of cells or medium and incubated at 37°C in a 5% CO2 and 90% humidity environment for 24 hours. The culture media was discarded after confluent cells (70%– 80%) were reached. Various concentrations of EACO (treatment group) were added to each well: 500, 250, 125, 62.5, 31, 15.63, and 7.8 µg/mL. Afterwards, 10µL MTT solution was added into each well, followed by four hours of incubation in a CO₂ incubator at 37°C until purple formazan crystals were formed. The reaction was then stopped by adding a 10% SDS stopper solution in 0.1 N hydrochloride (HCl) acid, and the plates were incubated in the dark overnight. The absorbance of the plates, which was measured using a microplate enzyme-linked immunosorbent assay (ELISA) reader at 595 nm, was then used to quantify the proportion of live cells.

The percentage of viable cells = (tested cell absorbance - medium absorbance) / (control cell absorbance - medium absorbance) x 100%. The half-maximal inhibitory concentration (IC50) value was calculated using the percentage of the viable cells. The IC₅₀ value represents the EACO concentration that can cause 50% of cell deaths. The selectivity index (SI) was then calculated using the IC₅₀ values: SI ratio = IC₅₀ of Vero cells/IC₅₀ of cancer cells. The SI ratio >3 means the EACO treatment has a selective effect only on the breast cancer cells (MCF-7 and T47D cells).

Cell proliferation assay

The antiproliferative activity of EACO was also examined by the MTT colorimetry assay. A 96-well plate was seeded with 100 µL of T47D and MCF-7 cells (70–80% confluent) at a 1.5x104 cells/well density and a growth medium. After that, the cells underwent a 24-hour incubation. Then, 100 µL of the ½IC₅₀, IC₅₀, and 2 x IC₅₀ of EACO were applied to the cells for 72 hours. The plate was observed every 24 hours. Then, 100 µL of MTT 0.5 mg/mL was used to treat the plate, and the plate was allowed to sit for four hours until a purple formazan crystal developed. The plate was filled with the stopper solution (10% SDS in 0.1N HCl acid) and then incubated overnight in the dark. Then, the proliferation was determined by reading the plates' absorbance and converting the result into the percentage of viable cells. All proliferation test was conducted in triplicate.

Apoptosis assay

T47D and MCF7 cells were seeded into 6-well plates with a cell density of 5 × 10⁴ cells/well. After incubated for 24 hours in a 5% CO₂ incubator, cells were treated with C.odorata leaf extract with a concentration of 2 x IC₅₀, then incubated again for 24 hours. Then, the cells were harvested using 0.25% trypsin-EDTA, centrifuged, and washed with PBS. Afterwards, cells were resuspended in 500 µL of Annexin V buffer and then treated with Annexin V and propidium iodide (PI) for 10 minutes at room temperature and in a dark place. To assess the homogenized cell suspension, BD FACS Calibur Flow cytometer (Becton Dickinson, California, US) was used, and the result was analyzed by Modfit Lt. 3.0 (Verity Software House, Maine, US). The apoptosis assay was conducted in triplicate. The principle of apoptosis assay using Annexin V and PI were described as follows. The membrane phospholipid phosphatidylserine (PS) of apoptotic cells is translocated from the inner to outer leaflet of the plasma membrane, exposing PS to the external cellular environment. Annexin V, which conjugated to flourochromes, binds to exposed apoptotic cell surface PS and acts as a sensitive probe for flow cytometric assay of apoptotic cells.

After PS translocation, the membrane lose its integrity in the later phases of cell death caused by either apoptotic or necrotic processes. Therefore, in addition to Annexin V, propidium iodide (PI) was utilised to identify early and late apoptotic cells. Viable cells with intact membranes exclude PI, whilst the membranes of apoptotic cells are permeable to PI. Healthy cells are both Annexin V and PI negative, while cells that are in early apoptosis are Annexin V positive and PI negative, and cells in late apoptosis are Annexin V and PI positive. Cells that already in necrosis are Annexin V negative and PI positive.

Statistical analysis

Results were presented as mean±SD. Nonlinear regression analysis was used to determine the IC₅₀ of EACO for MCF-7 and T4D7 cells. Analysis of variance (ANOVA), followed by a Tukey posttest, was used to analyze the viability and the proliferation of cancer cells against different concentrations of EACO. Multiple t-tests were used to analyze cancer cells' apoptosis compared to control when treated with different levels of IC₅₀ of EACO. A p<0.05 was regarded as statistically significant. Statistical analysis was performed using Graph Pad Prism v.8.0.2 (GraphPad Software, CA, USA).

Results

IC₅₀ values of EACO on T47D and MCF-7 cell lines

Our data indicated a dose-dependent manner of anticancer activity of EACO against MCF-7, T47D, and Vero cells (Figure 1). The concentration of 500 µg/mL of EACO reduced the viability of the MCF-7 and T47D cells to 1.2% and 5.4%, respectively (p<0.05), whereas the viable cell of Vero was 61.3%. Meanwhile, the viability of the cells was above 50% at other EACO concentrations.

Figure 1. The viability of MCF-7, T47D, and Vero cells after being treated with ethyl acetate extract of Chromolaena odorata (EACO) and doxorubicin (DOXO).

The MCF-7, T47D, and Vero cells' viability were also decreased after treatment with DOXO. Treatment with 100 µg/mL DOXO resulted in 16.5% of MCF-7, 23.8% of T47D, and 5.7% of Vero viable cells, respectively. The IC₅₀ of EACO on MCF-7 was 218.78 µg/mL, and T47D cells were 307.61µg/mL. The IC₅₀ of Vero cells after treatment with EACO was 2792.5 µg/mL. The EACO SI against MCF-7 and T47D were 12.77 and 9.08, respectively. The IC₅₀ of DOXO against MCF-7 and T47D were 5.4 µg/mL and 1.75 µg/mL, respectively.

Proliferation of T47D and MCF-7 cells against EACO

The viable cells of T47D cells after treatment with ½IC₅₀, IC₅₀, and 2 x IC₅₀ of EACO at 24 hours were 18.1±9.37%, 85±6.5%, and 100±4.9%, respectively (Figure 2A). The T47D cells were decreased after 48 hours (1/2 IC₅₀: 10.7±6.5%, IC₅₀: 54±3.9%, 2 x IC₅₀: 81±7.7%), although not significantly differ from those at 72 hours (1/2 IC₅₀: 12.7±3.4%, IC₅₀: 52.3±2.2%, 2xIC₅₀: 117.1±3.3%) (Figure 2A).

Figure 2. The proliferation of T47D (A) and MCF-7 (B) cells after 24-, 48- and 72-hours incubation following treatment with ½IC₅₀ (dark brown), IC₅₀ (green), and 2xIC₅₀ (light brown) of ethyl acetate extract of Chromolaena odorata (EACO).

The viable cells of MCF-7 were at 20.0±1.8%, 62.7±4.1%, and 99.4±9.4% after 24 hours treatment with ½IC₅₀, IC₅₀, and 2 x IC₅₀, respectively (Figure 2B). After 48 hours, the MCF-7 cells decreased to 10.0±6.1% when treated with ½IC₅₀. However, treatment with IC₅₀ and 2 x IC₅₀ resulted in 68.5±7.7%, and 102±5.2%, respectively with p<0.05 in both comparisons. The viable cells after 72 hours of treatment with ½IC₅₀ (19.9±9.6%) were also significantly lower than 2xIC₅₀ (86.7±4.5%) with p<0.05 (Figure 2B).

The apoptosis induction test of T47D and MCF-7 cells after treatment with EACO

In the apoptosis assay for T47D cells, the viable cells of control (92.45±0.21%) were higher than T47D cells treated with 2 x IC₅₀ of EACO (4.85±0.21%) with p<0.001 (Figure 3A). The number of T47D cells undergoing the early apoptosis stage after treatment with 2 x IC₅₀ of EACO (6.20±0.14%) was higher than the control (2.7±0.14%), p<0.01. A higher number of T47D cells in the late apoptosis stage was observed after 2 x IC₅₀ of EACO (7.1±0.42%) compared to the control (p<0.05). Necrosis T47D cells after treatment with EACO were higher (83.35±0.49%) than the control (3.1±0.14%), with p<0.001 (Figure 3A).

Figure 3. The apoptosis analysis using flow cytometry on (A) T47D and (B) MCF-7 cells using 2xIC₅₀ of acetate extract of Chromolaena odorata (EACO).

The viable cells of MCF-7 (0.5±0.42%) after treatment with 2 x IC₅₀ of EACO were lower than the control (82.75±0.35%) with p<0.001 (Figure 3B). The difference between MCF-7 and control cells during the early apoptosis stage was insignificant. However, the number of control cells in the late apoptosis stage (6.6±0.14%) was higher than MCF-7 (3.9±0.14%), p<0.05. The dead cells of MCF-7 (95.15±1.76%) after 2 x IC₅₀ of EACO treatment were higher than the control (8.25±0.07%) with p<0.001 (Figure 3B).

Discussion

Based on a previous study, the anticancer activity of C. odorata was detected from its ethanol extract in a dose-dependent manner [23]. Similarly, this study showed that treatment with 500 µg/mL EACO selectively decreased the viable cells of MCF-7 and T47D by more than 94% while preserving the Vero cells (61.3%). This result was better than treatment with 100 µg/mL DOXO, which decreased not only the viable cells of MCF-7 and T47D but also Vero cells (5.7%). The SI ratio of EACO for MCF-7 and T47D cells were also more than three, indicating that EACO was safer for normal cells than DOXO.

More than 190 different phytochemicals have been isolated from C. odorata and identified, such as fatty acids, triterpenoids, diterpenes, sesquiterpenes, essential oils, alkaloids, anthracene derivatives, flavonoids, stilbenes, and derivatives of diterpene [26]. Most flavonoids and phenolic acids, such as odoratenin, persicogenin, and (Z)-p-coumaric acid, were found in the aerial parts and leaves of C. odorata [27,28]. Meanwhile, steroids and triterpenoids (poriferasterol, stigmasterol, acetyl oleanolic acid, and ursolic acid) were commonly isolated from the stem, flowers, and roots and sometimes found in the leaves and aerial parts [28,29]. Using dichloromethane extract, the alkaloids and anthraquinones, such as 7-angeloylretronecine, 3-hydroxy-1,2,4-trimethoxy-6-methylanthraquinone, austrocortinin, and rinderine, were commonly found in the entire plant and flowers [30]. Straight-chain alkanes and fatty acids were commonly identified from this plant's aerial parts, leaves, stem, and flower, which include (S)-coriolic acid, linoleamide, and hexacosanol [31,32]. Other compounds such as terpenoids (radicol and cadalene), sesquiterpenoids (germacrene D) and phenolic glycosides (citrusin) were found in the aerial parts and the whole plants of C. odorata [25,28,29,33]. Alpha amyrin, which was previously detected in this plant, is known to inhibit tumor necrosis factor-α (TNF-α), which prevents cancer development [23,34]. The α, β-amyrin is a triterpenoid that functions as an antipyretic, anti-inflammatory, hepatoprotective, and analgesic. This compound is efficacious in preventing the development of pancreatic infection and acts as an anti-inflammatory and antioxidant agent [35].

Our previous studies showed that C. odorata had anticancer activity against several cancer cell lines [20-24]. Flavonoid compounds found in C. odorata can potentially inhibit the proliferation of cancer cells. A study stated that flavonoids inhibit cyclin-dependent kinases (CDKs), which are essential for cell growth and division, block angiogenesis, and modulate MDR1 activity [36]. Flavonoids are essential in angiogenesis and the reactive oxygen species (ROS) activity of tumor cells, preventing cancer cell proliferation [37]. Several phenolic compounds, alkaloids, terpenoids, and glycosides of C. odorata have been identified based on the phytochemical analysis [23]. The functional properties of these compounds modulate the scavenging enzyme activities of ROS, playing a role in stopping the cell cycle by inducing autophagy and apoptosis to suppress cancer cell proliferation and invasion [38].

In addition, a study showed that C. odorata has anticancer characteristics that increase apoptosis in Cal51 breast cancer cells through its synergistic interaction with the Bcl2 inhibitor ABT737 [25]. C. odorata induces apoptosis by triggering the p53 pathway, alerting a cell cycle checkpoint to eliminate cells considered harmful to the immune system. The checkpoint cycle controls excessive cell growth and property changes [39]. Pro-non-apoptotic protein receptors hinder the cell cycle checkpoint system by increasing the response to checkpoint sensor proteins during the pathogenesis of cancer cells [40]. Flavonoids typically increase caspases, which catalyze proteolysis and inactivate structural proteins that control cancer cell growth like gelsolin, poly-ADP-ribose polymerase (PARP), cytokeratins, and DNA fragmentation factors 45 kDa (DFF45), resulting in morphological and biochemical changes [41].

This study did not isolate the pure compund of anticancer agent from ethyl acetate extracts of C. odorata. Further study on isolating the anticancer agent and study the molecular binding with breast cancer receptor is recommended.

Conclusion

The ethyl acetate extract of C. odorata showed promising results as an anticancer against breast cancer. The extract acts selectively against breast cancer cells while preserving the normal cells based on an in-vitro assay. Furthermore, the compound's anticancer activity works by preventing the proliferation and inducing apoptosis of breast cancer cells.

Ethics approval

This study has received ethical approval from the Ethics Commission, Faculty of Medicine, Universitas Lambung Mangkurat, Banjarmasin, Indonesia, 239/KEPK/FK UNLAM/VI/2020.

Competing interests

All the authors declare that there are no conflicts of interest.

Funding

This study received no external funding.

Underlying data

Derived data supporting the findings of this study are available from the corresponding author on request.

How to cite

Yusuf H, Novia H, Fahriani M. Cytotoxic activity of ethyl acetate extract of Chromolaena odorata on MCF7 and T47D breast cancer cells. Narra J 2023; 3 (3): e326 - http://doi.org/10.52225/narra.v3i3.326.