In Vitro Cytotoxic and Gene Expression Mediated Antiproliferative Activities of Crude Extracts of Selected Medicinal Plants From Botswana on HeLa Cells

Abstract

Introduction

Cancer is a global problem, contributing many deaths annually compared to other non-communicable diseases.

Aim

This study evaluated four medicinal plants of Botswana. Cell viability analysis was done to determine the antiproliferation activities of extracts of E. elephantina, A. garckeana, T. terrestris and P. oleracea on HeLa cells.

Methods

Extracts were crudely derived from Elephantorrhiza elephantina, Azanza garckeana, Tribulus terrestris and Portulaca oleracea with water, ethanol and methanol and the extracts were applied on cervical cancer cells (HeLa). MTT and WST-1 assays (cell viability assays) were carried out on the treated HeLa cells and morphological changes were then observed. The cells were treated with the least and highest concentrations of extracts from serial dilutions used in cell viability assays for a period of 24 hours. Gene expression analysis was performed on treated cells for cancer markers MACC-1, VEGF, EGFR, CYFRA 21-1 and the tumour suppressor gene CD95; all normalized to GAPDH.

Results

Methanol extracts showed cell viability below 50%. Morphological changes were observed. P. oleracea ethanol and aqueous extracts showed IC₅₀ values of 2.38 mg/mL and methanol extracts showed 5.20 mg/mL. A. garckeana ethanol and methanol extracts displayed IC₅₀ values of 1.80 and 1.88 mg/mL respectively while aqueous extracts had cell viability less than 50%. E. elephantina IC₅₀ values were 6.9, 4.6 and 5.6 mg/mL for ethanol, water and methanol extracts respectively. T. terrestris ethanol and aqueous extracts showed IC₅₀ values of 9.6 and 6.7 mg/mL respectively. Different plant extracts led to downregulation of cancer markers and upregulation of CD95.

Conclusion

Characterization of crude extracts can help identify bioactive compounds in these extracts. In vitro studies should be performed to determine the individual and synergistic effects of the extracts on the proliferation of HeLa cells.

Keywords

anticancer cell viability morphology cytotoxicity proliferation

Introduction

In sub-Saharan Africa, cervical cancer incidences are very high, and this has been linked to high numbers of HIV and HPV cases in the region.¹ Cancer develops from cells that have lost the ability to follow normal cell division control that the body exerts on all cells, resulting in fast growing cells that evade apoptosis. Although some effort has been directed towards the development of effective cancer vaccines² and potential candidates for vaccines have been previously reported,³ there has been no overwhelming success. Cell progression and death are driven by proliferation, survival, and apoptosis genes, and, therefore, these groups of genes are used as markers for cancer diagnosis. These markers include the human vascular endothelial growth factor (VEGF) gene, a molecular marker for most cancers that is targeted by anticancer drugs. Epidermal growth factor receptor (EGFR) is a transmembrane protein that is normally upregulated in malignant tumours, resulting in increased tumour angiogenesis, metastasis and the death of normal cells. Metastasis-associated in colon cancer 1 (MACC1) is a biomarker of metastasis, cancer cell migration and fluidity that is over expressed in cancer cells. Cytokeratin 19 fragment 21-1 (CYFRA 21-1), on the other hand, is a structural protein that is usually characteristic of epithelial cells and is, therefore, a marker of epithelium-originating cancers.^4,5 Cluster of differentiation 95 (CD95) is a death receptor that is activated by its ligand, CD95L, to induce apoptosis.

Cervical cancer develops in the cervix and has the second highest incidence and mortality rates of all cancers in most regions globally, especially Southern Africa. This cancer originates in the squamous cells of the cervix at the junction where the ectocervix and endocervix meet.⁶ It is in the squamocolumnar junction where cervical cancer develops due to dysplasia of cells in the region.⁷ From a research perspective, cervical cancer has played a significant role because the first human cells to be successfully grown in the laboratory, called HeLa cells, were isolated from cervical cancer cells derived from a woman called Henrietta Lacks after whom HeLa cells are named.⁸ HeLa cells are immortal because they can grow uncontrollably, be cultured for many generations and even overgrow other cells in the same culture.

Botswana is a middle-income country in Southern Africa and like many countries in Africa, the country is faced with a lot of challenges in the health sector. A large portion of the population of Botswana is entirely dependent on the government for health care. Cancer, like many other diseases, poses a great threat to this small nation. Cancer is slowly becoming a pandemic in Botswana and conventional cancer treatment and management methods are not always effective and have undesirable side effects. Therefore, there is a need for the development of easily accessible, effective and safer cancer management strategies.

Due to side effects that are associated with conventional cancer management approaches, research is shifting towards strategies with less side effects such as medicinal plant-derived secondary metabolites.⁹ African medicinal plants have been used by Africans in the management of different cancers and other diseases.^10,11 Several indigenous medicinal plants have been revealed to have both chemopreventive and therapeutic effects in breast cancer⁹ and most clinically utilized anticancer agents are derived from plants.

P. oleracea is said to produce cerebrosides, homoisoflavonoids and alkaloids which have shown cytotoxic activity towards cultures of human cancer cell lines like A-549 and HepG2.¹² Portulacerebroside A (PCA), a polysaccharide from P. oleracea, can stimulate the death of the human liver cancer line HCCLM3 by activating the p38MAPK and the JNK-triggered mitochondrial death pathways, resulting in the developing tumour subsiding. On the other hand, glycosides extracted from T. terrestris are able to inhibit the growth of SKMEL, KB, BT-549 and SKOV-3 cancer cell lines.¹³ A. garckeana possess flavonoids which have the ability to fight and remove free radicals in biological systems and the potential to alleviate the development of tumorous cells.¹⁴

The eastern region of Botswana is rich in plants of high medicinal value. In our survey around Botswana, four plants; Azanza garckeana, Elephantorrhiza elephantina, Portulaca oleracea and Tribulus terrestris were selected based on their historical traditional medicinal uses in the management of some symptoms of different cancers by traditional healers. Because some of the people of Botswana rely on both traditional healers and modern medicine for their primary health care, it is important provide scientific evidence of the anticancer potential of indigenous medicinal plants. This study evaluated the effects of extracts from Azanza garckeana, Elephantorrhiza elephantina, Portulaca oleracea and Tribulus terrestris on the viability and morphology of HeLa cells and on the expression of selected markers of cancer (VEGF, EGFR, MACC1, CYFRA-21-1) and the tumour suppressor CD95.

Materials and Methods

Plant Identification and Verification

Portulaca oleracea leaves and stems together with Tribulus terrestris leaves and stems were used in this study while Elephantorhiza elephantina tuberous roots and Azanza garckeana fruits were used. The selection of plant parts to use in this study was based on published literature on previous studies that have shown the medicinal potential of the selected plant parts. For example, Portulaca oleracea leaves and branches have been shown to have antiproliferative activities¹⁵ which makes them appealing to cancer research as potential antitumor agents. The identities of collected plant specimen were verified by the Botswana National Museum and Monuments Herbarium Unit. The plants and explants were given collector codes as follows: 001BRAG.F-BIUST (Azanza garckeana fruits); 001BRAG.L-BIUST (Azanza garckeana leaves); 002BREE.P-BIUST (Elephantorrhiza elephantina plant); 003BRPO.P-BIUST (Portulaca oleracea plant) and 004BRTT.P-BIUST (Tribulus terrestris plant)). After verification, plant specimens were pressed and stored in the Plant Research Laboratory in the Department of Biological Sciences in Botswana International University of Science and Technology.

Plant Collection and Preparation of Extracts

The fruits of Azanza garckeana, the tuberous roots of Elephantorrhiza elephantina, the leaves and branches of Portulaca oleracea and the leaves and branches of Tribulus terrestris plants were dried at room temperature and ground to powder. Then, 10 g of powder of each sample was macerated overnight in 100 mL absolute methanol, ethanol, and distilled water in the dark at room temperature with frequent shaking. They were then filtered through Whatman No. 3 filter paper. Distilled water extracts were wrapped with aluminum foil and kept at 4°C in a cold room to avoid microbial activity. Thereafter, solvents were evaporated from the samples using a rotary evaporator. The extracted crude samples were reconstituted using culture grade Dimethyl Sulfoxide (DMSO; Sigma-Aldrich, South Africa) and stored at -20°C. All extracts were mixed thoroughly for homogeneity before being used.

Cell Culture and Growth Assays

HeLa cells were cultured in complete Dulbecco’s Modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% pen-strip cocktail of 100 μg/mL penicillin and 100 μg/mL streptomycin. The cells utilized in this study were sourced from the Botswana International University of Science and Technology HeLa cell repository (BIUST). The cells were grown at 37°C and in 5% CO₂ humidified incubator (Labotech, South Africa) until confluence. The cells were seeded at 1 × 10⁴ cells/mL density in a 96-well plate and incubated at 37°C for 24 hours to allow for adherence. After incubation, HeLa cells were treated with serially diluted concentrations of Azanza garckeana; 12.9 mg/mL to 0.81 mg/mL, Elephantorhizza elephantina; 641 mg/mL to 4.01 mg/mL, Portulaca oleraceae; 28.4 mg/mL to 1.7725 mg/mL and Tribulus terrestris; 28.6 mg/mL to 1.73 mg/mL and incubated again for 24 hours in same conditions. The positive control used in this study was cisplatin. This is because since cisplatin is a chemotherapy anticancer drug, and has been proven as such, it was expected to inhibit the proliferation of HeLa cells. A positive control is used to produce an expected result or outcome in order to confirm that the experiments are working properly. After 24 hours in treatment, HeLa cell viability and proliferation was quantified using Water Soluble Tetrazolium salt (WST-1) assay following a protocol by Peskin and Winterbourn.¹⁶ Briefly, cell culture medium plus 10 μL of Cell Proliferation Reagent WST-1 were added to the cells. The plate was then be incubated for 4 hours at 37°C and 5% CO². After incubation, the plate was shaken for 1 minute in the Thermo Scientific Multiskan microplate reader, USA, and the intensity of the dissolved formazan crystals were quantified using a plate reader at 440 nm (ELISA). The reference wavelength was set at 600nm.

Percentage of viable cells was then calculated using the following formula:

% Cell viability = \frac{O D o f T r e a t e d c e l l s}{O D o f C o n t r o l c e l l s} x 100

Cell viability was also measured using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-dipenyltetrazolium bromide] as described by Supino¹⁷ with some modifications. Briefly, 100 μL of MTT reagent prepared at a concentration of 0.5 g/ml was added to the wells with cells that had been treated with crude plant extracts. The cells were then incubated at 37°C in a 5% CO₂ humidified Esco CelCulture® CO₂ incubator for 4 hours. Following incubation, the MTT reagent was removed from the cells and 100 μL of dimethyl sulfoxide (DMSO) was added. The plates were incubated in a shaker for 15 minutes. The absorbance of the plates was taken following incubation using a Multiskan. Cytotoxicity was expressed as percentage cell viability and was determined by the comparison of the absorbances of both treated and untreated cells. The experiment was performed in triplicates at n = 3. The reduction of MTT (absorbance) of each well was determined at a wavelength of 570 nm using a microplate reader (Thermo Scientific Multiskan microplate reader, USA). Analysis was done in triplicates for each plant.

Percentage of viable cells was then calculated using the following formula:

% Cell viability = \frac{O D o f T r e a t e d c e l l s}{O D o f C o n t r o l c e l l s} x 100

Cell Morphology Analysis

HeLa cells were cultured at a density of 5 × 10⁶ cells/mL in 12-well plates (Thermo Scientific™ Nunc™cell culture plates, USA) until 100% confluent. The morphology of treated and untreated HeLa cells was observed, and images were taken at 0 hours and 24 hours post treatment using the Leica DMIL-LED inverted light microscope.

Gene Expression Analysis

HeLa cells were cultured at a density of 5 × 10⁶ cells/mL in cell culture plates (Thermo Scientific™ Nunc™cell culture plates, USA) until 100% confluent. The cells were then treated with aqueous plant extracts (4 extracts with lowest IC₅₀ values) and cisplatin (positive control) and then incubated for 24 hours. After incubation, the cells were detached from culture plates using sterile scrapers (Thermo Scientific™ Nunc™ Cell Scrapers, USA). Total RNA was extracted from the cells following a protocol described by Zymo Research, Quick-RNATM Miniprep Plus Kit, Catalog No. R1058, USA. The extracted total RNA was then quantified using a NanoDrop One spectrophotometer (Thermo Scientific, USA) at a 260 nm wavelength. Then, 2μg of total RNA was used to synthesize complementary DNA (cDNA) using the NEW ENGLAND BioLabsInc. ProtoScript 11 First Strand cDNA Synthesis Kit according to the manufacturer’s instructions. Then, quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) was performed using the Luna Universal qPCR Master Mix (NEW ENGLAND BioLabs Inc, USA) according to the manufacturer’s instructions using the Applied Biosystems 7500 Fast Cycler (Life Technologies, Singapore). Primer sequences (Table 1) used in this study were acquired from published literature in the NCBI database. The primers were ordered from and designed by Inqaba Biotechnological Industries (South Africa). RT-qPCR was performed under the following conditions: initial denaturation at 95°C for 60 seconds and 1 cycle, denaturation at 95°C for 15 seconds and extension at 60°C for 30 seconds both at 45 cycles. The melt curve was run from 60–95°C. The level of expression of the target genes was normalized to the housekeeping gene Glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Relative gene expression was calculated using the 2-ΔΔCt Livak method as described by Yuan et al.¹⁰

Table 1.

Sequences of Primers Used in This Study

Gene	Forward	Reverse
VEGF-A primer pair 1	5’--AGGATGGGGCTGACTAGGTAA-3′	5′CCCCCTTTTCCTCCAACTCTC-3′
VEGF-A primer pair 1	5′-GCCCATTCCCTCTTTAGCCA-3	5′-AGTGAGGTTACGTGCGGACAG-3′
EGFR	5′-GACGGGCAACGGCGTATTCTCAG-3′	5′- CCCACCTTGCCTTTGTCTCCTGTC-3′
EGFR	5′-GCGTCTCTTGCCGGAATGT-3′	5′-CTTGGCTCACCCTCCAGAAG-3′
MACC1	5′-TTCTTTTGATTCCTCCGGTGA-3′	5′-ACT CTGATGGGCATGTGCTG-3
CYFRA 21-1	5′AAGCTAACCATGCAGAACCTCAACGACCGC-3′	5′-TTATTGGAGGTCAGGAGAAGAGCC-3′
CYFRA 21-1	5′-TCCCGCGACTACAGCCACTACTACACGACC-3′	5′CGCGACTTGATGTCCATGAGCCGCTGGTAC-3′
CD95	5′-cactcgagCTTTCACTTCGGAGGATTGC-3′	5′gtgaattctGACCAAGCTTTGGATTTCATTTC-3′
GAPDH	5′-GCCCTCAACGACCACTTTG-3′	5′-CCACCACCCTGTTGCTGTAG-3′

Statistical Analysis

All analysis were performed in triplicates. Data obtained were subjected to analysis of variance (ANOVA) by OriginPro 2018 statistical software. Statistical significance of difference among means was estimated at p<0.05 according to one-way ANOVA and Tukey-Kramer’s multiple range test. Values are presented as means ± standard deviation (SD).

Results

Cell Viability Assay

Cell viability was determined on HeLa cells which had been treated with PBS (negative control), others with cisplatin (positive control) and others with different plant extracts. Figures 1 and 2 presents the viability of HeLa cells after the application of cisplatin and different extracts from the four plants at different concentrations respectively.

Figure 1.

Cytotoxic effects of cisplatin on HeLa cells at passage 4 after a 21-day culture protocol. Different letters indicate significant differences (p < 0.05, Tukey-Kramer’s multiple range test)

Figure 2.

Cytotoxic effects of extracts from Portulaca oleracea leaves and branches (POLB) (A), Azanza garckeana fruits (AGF) (B), Elephantorrhiza elephantina tuber (EET) (C) and Tribulus terrestris leaves and branches (TTLB) (D) on HeLa cells at after a 21-day culture protocol. Results are presented as mean (n = 3) ± standard deviation (SD). Bars with the same letters (a, b, c, d, or e) are not significantly different (p < 0.05) and those with different letters are significantly different (p > 0.05)

For Portulaca oleracea leaves and branches (POLB) extracts, there was no statistical difference between the percentage viabilities displayed by water and ethanol extracts across the different concentrations of the extracts (Figure 2A). For AGF extracts, ethanol and methanol resulted in higher cell survival rates than water extracts (Figure 2B). The highest percentage viability across all AGF extracts was 55.4% at a concentration of 0.801mg/mL while the least viability was 35.5% at a concentration of 12.9 mg/mL (Figure 2B). For EET extracts, followed by methanol, ethanol extracts led to the highest cell viabilities across all extract concentrations (Figure 2C). The highest cell viability was 53.6% for ethanol extracts at a concentration 4.01 mg/mL while the least viability was 35.2% at a concentration of 64.2 mg/mL for aqueous extracts. Therefore, aqueous EET extracts were the most toxic to HeLa cells, followed by methanol and ethanol extracts respectively. For TTLB extracts, methanol extracts showed the highest % viabilities across all the different concentrations while aqueous extracts led to the least cell viabilities across all the different concentrations of extracts (Figure 2D).

Cell Morphology Analysis

Hela cells were then treated with both the negative (PBS) and the positive (cisplatin) controls to determine their behaviour in these control substances as represented in Figure 3 a and b respectively. PBS was used as a negative control because it is a non-cytotoxic balanced salt solution which can be used in cell culture to wash cells before any passaging. Therefore, under normal conditions, it has no negative effect on cells. Cells cultured in PBS attached well in culture and displayed nearly 100% confluency as well as normal HeLa cell morphology (Figure 3A). Cells treated with cisplatin were detached from cell culture plates and were roundish in shape, dark in colour and were floating about in culture media (Figure 3B).

Figure 3.

Control treatments of HeLa cells (P3) after 14 days of culture. HeLa cells treated with PBS (negative control) (A) and HeLa cells treated with Cisplatin = 50 µg/mL (positive control) (B)

The morphology of treated and untreated cells was observed 0 and 24 hours after the application of POLB, AGF, EET and TTLB extracts. For POLB extracts, treated cells showed no changes in morphology 0 hours post extract application (Figure 4A, D and G). HeLa cells were sparse and attached well to cell culture plates. Twenty-four (24) hours post treatment with 1.77 mg/mL of POLB ethanol extracts, the lowest concentration, some cells began to detach from culture plate and appeared darker (blue and red arrows) (Figure 4B). The red arrow shows an attached cell that looks lighter in colour. After 24 hours of treatment with 28.3 mg/mL of POLB ethanol extracts, the highest concentration, most of the HeLa cells appeared darker (blue circle) (Figure 4C) with uneven shapes. They cells looked blebbed and had detached and were floating in media. After 24 hours of treatment with 1.77 mg/mL and 28.36 mg/mL of POLB methanol extracts respectively (Figure 4E and F), HeLa cells appeared spherical and darker and had detached from culture plates (blue arrows). After 24 hours treatment with 1.77 mg/mL and 28.36 mg/mL of POLB aqueous extracts respectively (Figure 4H and I), most of the cells appeared spherical with darker outlines (blue arrows). They had also detached from culture plates and had lost their shape, appearing blebbed with lost turgidity.

Figure 4.

Morphology of HeLa cells immediately after treatment (0 hours) (A, D, G) and 24 hours after treatment (B, C, E, F, H, I) with different concentrations of extracts of Portulaca oleracea leaves and branches (POLB). Cells were plated at a concentration of 1×10⁵ cells/mL and allowed to attach to culture plates over a period of 24 hours and then treated with POLB ethanol, methanol, and aqueous extracts: A, B, C = POLB ethanol extracts; D, E, F = POLB methanol extracts; G, H, I = POLB aqueous extracts. Images were captured at x100 magnification

After 0 hours post treatment with AGF ethanol, methanol, and aqueous extracts respectively, there were no changes in HeLa cell morphology (Figure 5A, D and G). The cells were globular (blue arrows), sparse and attached to the cell culture plates. After 24 hours of treatment with 0.805 mg/mL of AGF ethanol extracts, the lowest concentration, the cells appeared to have detached from the cell culture plate (black circle) but were still globular in shape but darker in colour (Figure 5B). Twenty-four hours after treatment with 12.88 mg/mL of AGF ethanol extracts, the highest concentration, some of the cells appeared to be alive and attached but media discoloration was observed (Figure 5C). HeLa cells treated for 24 hours with 0.805 mg/mL and 12.88 mg/mL of AGF methanol extracts were spherical in shape, darker in colour and detached from culture plates (black circles) (Figure 5E and F). After 24 hours of treatment in 0.805 mg/mL and 12.88 mg/mL of AGF aqueous extracts, HeLa cells appeared spherical and detached from culture plates (blue arrows) (Figure 5H and I).

Figure 5.

Morphology of HeLa cells immediately after treatment (0 hours) (A, D, G) and 24 hours after treatment (B, C, E, F, H, I) with different concentrations of extracts of Azanza garckeana fruits (AGF). Cells were plated at a concentration of 1×10⁵ cells/mL and allowed to attach to culture plates over a period of 24 hours and then treated with AGF ethanol, methanol, and aqueous extracts: A, B, C = AGF ethanol extracts; D, E, F = AGF methanol extracts; G, H, I = AGF aqueous extracts. Images were captured at x100 magnification

Zero hours after treatment with EET ethanol, methanol and aqueous extracts, HeLa cells displayed normal morphology (Figure 6A, D and G). The cells were spread out and attached to cell culture plates. After 24 hours of treatment with 4.01 mg/mL (the lowest concentration) and 64.16 mg/mL (the highest concentration) of EET ethanol extracts respectively (Figure 6B and C), 4.01 mg/mL and 64.16 mg/mL of EET methanol extracts respectively (Figure 6E and F) and 4.01 mg/mL and 64.16 mg/mL of EET aqueous extracts respectively (Figure 6H and I), cells were detached from cell culture plates and globular in shape (black circles).

Figure 6.

Morphology of HeLa cells immediately after treatment (0 hours) (A, D, G) and 24 hours after treatment (B, C, E, F, H, I) with different concentrations of extracts of Elephantorrhiza elephantina tubers (EET). Cells were plated at a concentration of 1×10⁵ cells/mL and allowed to attach to culture plates over a period of 24 hours and then treated with EET ethanol, methanol, and aqueous extracts: A, B, C = EET ethanol extracts; D, E, F = EET methanol extracts; G, H, I = EET aqueous extracts. Images were captured at x100 magnification

After 0 hours of treatment with TTLB ethanol, methanol, and aqueous extracts respectively, HeLa cells maintained normal morphology (Figure 7A, D and G) and were spread out and attached to cell culture plates. After 24 hours of treatment with 1.726 mg/mL (the lowest concentration) and 27.62 mg/mL (the highest concentration) of TTLB ethanol extracts respectively (Figure 7B and C), 1.726 mg/mL and 27.62 mg/mL of TTLB methanol extracts respectively (Figure 7E and F) and 1.726 mg/mL and 27.62 mg/mL of TTLB aqueous extracts respectively. (Figure 7H and I), HeLa cells were detached from cell culture plates (black circles and red arrows) and appeared globular in shape and darker in colour. Additionally, there were signs of cell death as indicated by the shrinkage and blebbing of the cells. These observations were made for most of the cells except for cells treated with 1.726 mg/mL of TTLB methanol extracts (Figure 7E) where the cells appeared normal in culture and attached to cell culture plates.

Figure 7.

Morphology of HeLa cells immediately after treatment (0 hours) (A, D, G) and 24 hours after treatment (B, C, E, F, H, I) with different concentrations of extracts of Tribulus terrestris leaves and branches (TTLB). Cells were plated at a concentration of 1×10⁵ cells/mL and allowed to attach to culture plates over a period of 24 hours and then treated with TTLB ethanol, methanol, and aqueous extracts: A, B, C = TTLB ethanol extracts; D, E, F = TTLB methanol extracts; G, H, I = TTLB aqueous extracts. Images were captured at x100 magnification

Analysis of Gene Expression

Cytotoxicity assays and morphological observations have revealed that the aqueous extracts of the selected four plant species have higher inhibitory effects on HeLa cell proliferation when compared to ethanol and methanol extracts of the same plant species. Therefore, the IC₅₀ concentrations of only the aqueous extracts were used further for gene expression analysis. Gene expression analysis was carried out by RT-qPCR on HeLa cells that had been treated with Azanza garckeana fruits, Elephantorrhiza elephanina tuber, Portulaca oleracea leaves and branches as well as Tribulus terrestris leaves and branches extracts. Positive control HeLa cells were treated with cisplatin while negative control cells were treated with PBS only.

RT-qPCR revealed a significant level of (p > 0.05) upregulation of CD95 (Figure 8A) after HeLa cells were treated with Azanza garckeana fruits, Elephantorrhiza elephantina tuber, Portulaca oleracea leaves and branches and Tribulus terrestris leaves and branches aqueous extracts. However, CD95 was upregulated the most in cells treated with the positive control (cisplatin) (Figure 8A). Cisplatin yielded the highest upregulation of CD95 with a fold increase of 0.77. It was followed by Portulaca oleracea leaves and branches extracts with a fold increase of 0.32, then Tribulus terrestris leaves and branches extracts and Azanza garckeana fruits extracts with fold increases of 0.27 and 0.25 respectively with no significant statistical difference between them (p < 0.05). Elephantorrhiza elephantina tuber extracts led to the least upregulation of CD95 when compared to other treatments with a 0.16-fold increase (Figure 8A).

Figure 8.

The relative expression of CD95 (A), CYFRA 21-1 (B), EGFR (C), MACC1 (D) and VEGF (E) in HeLa cells that had been treated with different extracts of the different plants. Results are presented as mean (n=3) ± standard deviation (SD). Bars with the same letters (A, B, C, or D) are not significantly different (p < 0.05) and those with different letters are significantly different (p > 0.05). GAPDH = Glyceraldehyde-3-Phosphate Dehydrogenase; NC = Negative Control (Phosphate Buffered Saline - PBS); PC = Positive Control Cisplatin); AG = Azanza garckeana fruits aqueous extracts; EE = Elephantorrhiza elephantina tuber aqueous extracts; PO = Portulaca oleracea leaves and branches aqueous extracts; TT = Tribulus terrestris leaves and branches aqueous extracts

There was significant downregulation (p > 0.05) of the expression of CYFRA 21-1 (Figure 8B), EGFR (Figure 8C), MACC1 (Figure 8D) and VEGF (Figure 8E) in HeLa cells treated with aqueous extracts of Azanza garckeana fruits, Elephantorrhiza elephantina tuber, Portulaca oleracea leaves and branches and Tribulus terrestris leaves and branches as well as cisplatin. For CYFRA 21-1 relative expression, cisplatin yielded the most downregulation than all the genes with a fold decrease of 0.8 followed by Portulaca oleracea and Tribulus terrestris respectively with no significant differences between them (p < 0.05) and with fold decreases of 0.38 and 0.35 respectively (Figure 8B). Azanza garckeana fruits extracts displayed a fold decrease of 0.29 in CYFRA 21-1 relative expression while Elephantorrhiza elephantina tuber extracts showed the least downregulation with a 0.20-fold decrease (Figure 8B).

EGFR expression was significantly (p > 0.05) downregulated in HeLa after different treatments with no significant differences in cells treated with Azanza garckeana fruits, Portulaca oleracea leaves and branches and Tribulus terrestris leaves and branches (Figure 8C). EGFR expression was significantly different in cells treated with Elephantorrhiza elephantina tuber extracts. Fold decreases in EGFR expression in different treatments were as follows; 0.44 for Portulaca oleracea leaves and branches extracts, 0.30 for Tribulus terrestris leaves and branches extracts and 0.30 for Azanza garckeana fruits extracts. Elephantorrhiza elephantina tuber extracts yielded the lowest inhibition of EGFR expression with 0.15-fold decrease (Figure 8C).

The expression of MACC1 was significantly (p > 0.05) downregulated in HeLa cells treated with the positive control (Figure 8D). This was followed by Portulaca oleracea leaves and branches extracts with a 0.40-fold decrease, then Azanza garckeana fruits extracts and Tribulus terrestris leaves and branches extracts with no significant difference between them (p < 0.05) and with fold decreases of 0.31 and 0.29 respectively. Elephantorrhiza elephantina tuber extracts effected the least downregulation of MACC1 with a 0.2-fold decrease.

The expression of VEGF was significantly (p > 0.05) downregulated after different treatments with the positive control yielding the most inhibition of VEGF (Figure 8E) followed by Portulaca oleracea leaves and branches extracts with a 0.35-fold decrease, then Azanza garckeana fruits extracts and Tribulus terrestris leaves and branches extracts with fold decreases of 0.28 and 0.30 respectively and no significant difference (p < 0.05). Elephantorrhiza elephantina tuber extracts yielded the least downregulation of 0.17-fold decrease in the treated HeLa cells.

Discussion

Plants have secondary metabolites that are of therapeutic value and are the current focus of cancer research. Extracts of Azanza garckeana (AGF), Elephantorrhiza elephantina (EEF), Portulaca oleracea (POLB) and Tribulus terrestris (TTLB) were studied for their effects on the proliferation and morphology of HeLa cells. The extracts followed the same pattern as the positive control (cisplatin) in all the assays carried out. There was disruption in the morphology of HeLa cells, antiproliferative effects evidenced by the cell viability assay and gene expression analysis. Aqueous extracts resulted in the most morphological changes in the treated HeLa cells. These findings have also been reported by Michael and colleagues¹⁴ where Azanza Garckeana aqueous extracts were shown to inhibit HeLa cell growth. This may be due to the high polarity of water. High polar solvents extract high polar phytochemicals due to high separation ability, high concentration of high polar phytochemicals, and a higher degree of solubility by these high polar phytochemicals.

The high polarity of water could be the reason why the aqueous extracts of Portulaca oleracea led to the highest inhibition of the viability of HeLa cells when compared to ethanol and methanol as it might have extracted more phytochemicals like cerebrosides, alkaloids and polyphenols which have anticancer potential. When compared to methanol and ethanol, water is the most polar of the three¹⁸ and therefore tends to extract higher concentrations of phytochemicals than methanol and ethanol. MTT assay revealed that Portulaca oleracea extracts had similar cytotoxic effects in the HeLa cells as those observed in HepG2 and A-549 cancer cell lines during cytotoxicity evaluation.¹² Polysaccharides of Portulaca oleracea were used individually and conjugated with other substances to evaluate their cytotoxicity on different cancer cell lines, including HeLa cells, and resulted in noticeable and significant levels of cytotoxicity.¹⁹ Cytotoxic effects of Portulaca oleracea on A-549 and HCT116 cell lines are reported to have a concentration-dependent inhibition of proliferation on these cancer cell lines.²⁰ It has also been reported that when normal cells are exposed to ethanol extracts of Portulaca oleracea, the same way that cancer cell lines were exposed, no cytotoxicity is observed, suggesting that these extracts may be a safer alternative.¹⁵ Portulacerebroside A, a secondary metabolite produced by Portulaca oleracea, has been shown to control the mitochondrial death pathway that consequently leads to the death of the mitochondria and the increase in the number of cells programmed to undergo apoptosis in human hepatic cancer cells via the upregulation of p38MAPK and JNK proteins.²⁰

Portulaca oleracea polysaccharides scavenge free radicals like superoxide anions and the activity of these free radicals can result in tumor growth, thus scavenging them would strengthen the immune system and antagonize tumor proliferation and other factors that support tumor growth thereof.²¹ Colony forming assays have been used to evaluate the effect of Portulaca oleracea extracts on colony formation by human colorectal and lung cancer cell lines HCT116 and A549 respectively. The results indicated inhibitory effects by the Portulaca oleracea extracts when compared to the control.²² Here, we observed that after 24 hours of treating HeLa cells with the aqueous, methanol and ethanol extracts of Portulaca oleracea leaves and branches, there were significant changes in the morphology of the cells at both low and high concentrations, with more morphological changes noted in the high concentration. The cells lost their normal attachment, shape, and structure, whilst also shrinking. Some cells were observed to have detached or lost adherence to culture plates at a low concentration while at a high concentration, most cells had detached. These findings are consistent with findings of a study by Al-Sheddi and others.¹² where seed oil extracts of Portulaca oleracea were shown to have apoptotic effects on human liver cancer and lung cancer cell lines HeG2 and A-549 respectively.

Elephantorrhiza elephantina tuber (EET) aqueous extracts (IC50 = 4.6 ± 1.2.) were more effective in HeLa cell growth inhibition, followed by methanol extracts (IC50 = 5.6 ± 2.6) and lastly ethanol extracts (IC50 = 6.9 ± 1.9). These findings agree with findings by Olaokun and company.²³ where ethanol, acetone, and water extracts of Elephantorrhiza elephantine inhibited H4IIE liver cells. The antiproliferative effects of EET extracts displayed notable differences in cell death and detachment. The cytotoxicity and antioxidant properties of the roots extracts of Elephantorrhiza elephantina suggest that this plant has the potential to inhibit growth of cervical cancer cells²³ due to bioactive compounds in EET extracts. Tribulus terrestris leaves and branches (TTLB) aqueous, ethanol and methanol extracts recorded IC₅₀ values of 6.7 ± 2.3 and 9.6 ± 2.1 respectively. These results agree with previous findings where the fruits and aerial parts of Tribulus terrestris were shown to inhibit HeLa and SKOV-3 cells.²⁴ In another study, where methanol extracts of Tribulus terrestris were used to treat MCF-7 cells, the extracts were only toxic to cancer cells and not to non-cancer cells.^25,26

There are previous studies that have reported cytotoxicity being elicited by high extract concentrations. In this study, crude extracts from the four plants were used. Crude extracts comprises of different phytochemicals which work together to elicit cellular toxicity. This is in contrast to isolated compounds whose cytotoxic effect would be perhaps less extreme. In their study, Uddin and colleagues²⁷ investigated the antiproliferative activities of extracts of the fruits of the plant Sonneratia apetala against A549 cells which are lung cancer cells. They reported that the methanolic seed extracts elicited cytotoxicity at a concentration of 300 μg/ml which is 15 times higher than the 20 µg/ml that the American Cancer Institute states as the maximum IC₅₀ value requisite for an extract to be considered cytotoxic. In another study of the antioxidant, anti-inflammatory and anticancer assessment of T. terrestris, Abbas and others²⁵ reported that the anticancer activity of T. terrestris methanolic extracts displayed anticancer activity at an IC₅₀ value of 71.4 µg/ml which is also a relatively high concentration. In another study that assessed the cytotoxic effects of E. elephantina against HeLa cells, it was reported that cytotoxic activity was displayed by the Methanol + Dichloromethane extract at an IC₅₀ value of 120 µg/ml.²⁸

Gene expression analysis by RT-qPCR was performed for cervical cancer markers in HeLa which had been treated with the aqueous plant extracts of Portulaca oleracea (PO), Azanza garckeana (AG), Elephantorrhiza elephantina (EE), and Tribulus terrestris (TT). Positive control HeLa cells that had been treated with cisplatin, a drug used in cancer treatment showed the most upregulation of CD95, a tumour suppressor gene. Portulaca oleracea extracts exhibited the highest upregulation of CD95 of all the plant extracts. This could be because some secondary metabolites from PO and other plants alter various proteins which support cancer proliferation by inhibiting the metastasis and invasiveness of cancer, ultimately inducing apoptosis through the upregulation of CD95.²⁴ Elevated levels of the CD95 receptor may have upregulated the CD95-dependent apoptosis pathway. Aqueous extracts of all the four plants may have also inhibited proliferation of HeLa by the observed significant downregulation of CYFRA 21-1, EGFR, MACC1 and VEGF in HeLa cells treated with the plant extracts. However, the plant extracts were not as effect as cisplatin (positive control). Although aqueous extracts of Portulaca oleracea leaves and branches exhibited the highest level of downregulation of all the genes (Figure 6B–E), extracts from all the four plants led to the downregulation of all the cancer marker genes. It has been previously reported that some plants or drugs inhibit cancer cells by downregulating the transcriptional expression of CYFRA 21-1, EGFR, MACC1 and VEGF.¹⁶ The downregulation of these genes may have inhibited proliferation pathways mediated by CYFRA 21-1, EGFR, MACC1 and VEGF.

There were some limitations during the undertaking of this study. Collection of the four plant samples was challenging because the plants were located in different areas of the country. This may contribute towards differences in extracts performance which may be due to different bioactive compounds concentration as a result of different climates. Moreover, their collection demanded a lot of resources which at times were not available. These included unavailability of transport and sometimes support personnel. Additionally, although arrangements and appointments were made with local people before going to collect the plant samples, sometimes the appointments were not honoured by the locals, leading to resource wastage.

Conclusion

This study has shown that extracts from selected medicinal plants of Botswana can lower the expression of cancer markers and promote the expression of tumor suppressor genes. The aqueous, methanol and ethanol extracts of Azanza garckeana fruits, Elephantorrhiza elephantina tuber, Portulaca oleracea leaves and branches and Tribulus terrestris leaves and branches all exerted alterations in the morphological features of HeLa cells in culture. Signs of this included loss of shape and structure, shrinkage, detachment from cell culture plates and loss of turgidity after treating HeLa cells with the plant extracts. Cell viability assays showed inhibition of the proliferation of HeLa cells which was effected by the plant extracts. HeLa cell viability decreased with increasing concentrations of extracts. The aqueous extracts of all the four plant species consistently showed the highest cytotoxicity to HeLa cells when compared to those of methanol and ethanol because water has high polarity. Based on the findings of this study, there is a need for the extraction of specific metabolites/phytochemicals from these plants as it would allow the study of potential mechanisms that the plants use to inhibit excessive cell proliferation.

Footnotes

Acknowledgements

We thank the Botswana International University of Science and Technology for providing the requisite resources to carry out this research.

ORCID iD

Goabaone Gaobotse

Ethical Considerations

Ethical Approval is not applicable for this article.

Consent to Participate

There are no human subjects in this article and informed consent is not applicable.

Author Contributions

Phenyo Donald Brown: Methodology, data curation, formal analysis, and writing of the original draft.

Tebogo Elvis Kwape: Methodology, writing, review and editing of the manuscript.

Kabo Masisi: Methodology, writing, review and editing of the manuscript.

Abdullah Makhzoum: Conceptualization, methodology, supervision, writing, review and editing of the manuscript.

Goabaone Gaobotse: Conceptualization, methodology, supervision, writing, review and editing of the manuscript.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This research was funded by the Office of the Deputy Vice Chancellor Research Development and Innovation of the Botswana International University of Science and Technology with Grant Number: R00024.

Declaration of Conflicting Interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Data Availability Statement

The data used to support the findings of this study are available from the corresponding author upon request.*

Statement of Human and Animal Rights

This article does not contain any studies with human or animal subjects.

References

Denny

Anorlu

. Cervical Cancer in Africa. Cancer Epidemiol. Biomarkers Prev. 2012;21:1434-1438. doi:10.1158/1055-9965.EPI-12-0334.

Gaobotse

Venkataraman

Mmereke

Moustafa

Hefferon

Makhzoum

. Recent Progress on Vaccines Produced in Transgenic Plants. Vaccines. 2022;10:1861. doi:10.3390/vaccines10111861.

Tremouillaux-Guiller

Moustafa

Hefferon

Gaobotse

Makhzoum

. Plant-made HIV vaccines and potential candidates. Curr. Opin. Biotechnol. 2020;61:209-216. doi:10.1016/j.copbio.2020.01.004.

Muley

Rolny

, et al. The combination of the blood based tumor biomarkers cytokeratin 19 fragments (CYFRA 21-1) and carcinoembryonic antigen (CEA) as a potential predictor of benefit from adjuvant chemotherapy in early stage squamous cell carcinoma of the lung (SCC). Lung Cancer. 2018;120:46-53. doi:10.1016/j.lungcan.2018.03.015.

Senga

Kimura

Hattori

Yoshida

. Clinical evaluation of soluble cytokeratin 19 fragments (CYFRA 21-1) in serum and urine of patients with bladder cancer. Urology. 1996;48:703-710. doi:10.1016/S0090-4295(96)00253-1.

Ludmir

Sehdev

. Anatomy and physiology of the uterine cervix. Clinical obstetrics and gynecology. 2000;43:433-439.

Franco

Duarte-Franco

Ferenczy

. Cervical cancer: epidemiology, prevention and the role of human papillomavirus infection. Cmaj. 2001;164:1017-1025.

Lucey

Nelson-Rees

Hutchins

. Henrietta Lacks, HeLa cells, and cell culture contamination. Archives of pathology & laboratory medicine. 2009;133:1463-1467.

Chakraborty

. Medicinal plants and cervical cancer therapy: An overview. J Pharmacogn. Phytochem. 2019;8:3633-3641.

10.

Mazimba

Kwape

Gaobotse

. Dichrostachys cinerea: ethnomedicinal uses, phytochemistry and pharmacological activities - A review. The Natural Products Journal. 2022;12(3):20-30. doi:10.2174/2210315511666210806144540.

11.

Gaobotse

Venkataraman

Brown

, et al. The use of African medicinal plants in cancer management. Front. Pharmacol. 2023;14:1122388. doi:10.3389/fphar.2023.1122388.

12.

Al-Sheddi

Farshori

Al-Oqail

Musarrat

Al-Khedhairy

Siddiqui

. Portulaca oleracea seed oil exerts cytotoxic effects on human liver cancer (HepG2) and human lung cancer (A-549) cell lines. Asian Pac J Cancer Prev. 2015;16:3383-3387.

13.

Bedir

Khan

. New steroidal glycosides from the fruits of Tribulus terrestris. Journal of natural products. 2000;63:1699-1701.

14.

Michael

Onyia

Jidauna

. Evaluation of phytochemicals in Azanza garckeana (Gorontula) seed. J. Agric. Vet. Sci. 2015;8:71-74. doi:10.9790/2380-08517174.

15.

Rahimi

Ajam

Rakhshandeh

Askari

. A pharmacological review on Portulaca oleracea L.: focusing on anti-inflammatory, antioxidant, immuno-modulatory and antitumor activities. J. Pharmacopuncture. 2019;22:7-15. doi:10.3831/FKPI.2019.22.001.

16.

Peskin

Winterbourn

. A microtiter plate assay for superoxide dismutase using a water-soluble tetrazolium salt (WST-1). Clinica chimica acta. 2000;293:157-166.

17.

Supino

, MTT assays. In In vitro toxicity testing protocols. Humana Press, 1995:137-149.

18.

Ran

Chen

Fan

Ren

. Polarity-dependent extraction of flavonoids from citrus peel waste using a tailor-made deep eutectic solvent. Food Chem. 2019;297:124970. doi:10.1016/j.foodchem.2019.124970.

19.

Han

Huang

Wang

, et al. Platinum (II)- coordinated Portulaca oleracea polysaccharides as metal-drug based polymers for anticancer study. Colloids Surf B Biointerfaces. 2021;201:111628.

20.

Erdoğan

. The Chemosensitizing Effect of Portulaca oleracea Methanol Extract on Human Colorectal and Lung Cancer Cells Against Irinotecan. Kahramanmaraş Sütçü İmam Üniversitesi Tarım ve Doğa Dergisi. 2021;24:1187-1195.

21.

Shen

Tang

Zeng

, et al. Purification and characterization of an antitumor polysaccharide from Portulaca oleracea L. Carbohydr Polym. 2013;93:395-400. doi:10.1016/j.carbpol.2012.11.107.

22.

Franken

Rodermond

Stap

Haveman

Van Bree

. Clonogenic assay of cells in vitro. Nat. Protoc. 2006;1:2315-2319. doi:10.1038/nprot.2006.339.fal-shed.

23.

Olaokun

Alaba

Ligege

Mkolo

. Phytochemical content, antidiabetes, anti-inflammatory antioxidant and cytotoxic activity of leaf extracts of Elephantorrhiza elephantina (Burch.). Skeels. S Afr. J Bot. 2020;128:319-325. doi:10.1016/j.sajb.2019.11.030.

24.

Kucuk

Goger

Iscan

Ozdemir

Kirimer

Incesu

. Determination of phenolic compounds, cytotoxic, antioxidant and anticandidal effects of Tribulus terrestris L. (Zygophyllaceae). Children. 2017;208:382-394.

25.

Abbas

Hussain

Akhtar

, et al. Bioactive compounds, antioxidant, anti-inflammatory, anti-cancer, and toxicity assessment of Tribulus terrestris—In vitro and in vivo studies. Antioxidants. 2022;11:1160. doi:10.3390/antiox11061160.

26.

Patel

Soni

Siddiqi

Sharma

. An insight into the anticancer mechanism of Tribulus terrestris extracts on human breast cancer cells. 3 Biotech. 2019;9:58.

27.

Uddin

Akhter

Abedin

, et al. Comprehensive analysis of phytochemical profiling, cytotoxic and antioxidant potentials, and identification of bioactive constituents in methanoic extracts of Sonneratia apetala fruit. Heliyon. 2024;10(13):e33507.

28.

Gonnye

Masisi

Koobotse

Kwape

Gaobotse

. Antioxidant properties and cytotoxic effects of Elephantorrhiza elephantina on cervical cancer cells. Bulletin of the National Research Centre. 2025;49(81):81.