In-vitro Cytotoxicity Investigations for Phytoconstituents of Saudi Medicinal Plants With Putative Ocular Effects

Abstract

Background:

Metastatic secondary ocular tumors spread from systemic malignancies, including breast cancer. This study aimed to evaluate the cytotoxicity of extracts from 5 medicinal plants native to Saudi Arabia.

Methods:

For preliminary activity screening, cytotoxicity using the MTT assay and selectivity index determinations were made for medicinal plant extracts against various cancer cell-lines. The most promising extract was subjected to GC-MS analysis to determine the phytochemical composition. Clonogenic assays were performed using the most promising extract to confirm the initial results. Finally, western blot analysis was used to determine the modulation in expression of survivin and P27 suppressor genes in the human breast adenocarcinoma (MCF7) cell-line to understand the potential mechanistic properties of the active plant extract.

Results:

The 5 plant extracts showed various cytotoxic activity levels using IC₅₀. The most active extract was found to be the leaves of Capparis spinosa L. (BEP-07 extract) against the MCF7 breast cancer cell-line (IC₅₀ = 3.61 ± 0.99 μg/ml) and selectivity index of 1.17 compared to the normal human fetal lung fibroblast (MRC5) cells. BEP-07 extract showed a dose dependent clonogenic effect against the MCF7 colonies which was comparable with the effect of doxorubicin. BEP-07 extract caused a significant decrease of survivin and increase in P27 expression compared to control GAPDH at its highest dose (14 µg/ml). The GC-MS chromatogram of Capparis spinosa L. (BEP-07 extract) revealed the existence of 145 compounds, belonging to the diverse classes of phytoconstituents. Fatty acids and their derivatives represent 15.4%, whilst octadecanoic acid, 2,3-dihydroxypropyl ester was the principal component (7.9%) detected.

Conclusion:

Leaves of Capparis spinosa L. (BEP-07 extract) exhibited a significant cytotoxic effect particularly against breast cancer cells. It exhibited this effect through survivin inhibition and via P27 upregulation. The detected phytoconstituents in the plant extract might be involved in tested cytotoxic activity, while further investigations are required to complete the drug candidate profile.

Introduction

The prevalence of common ocular tumors world-wide is documented with scattered data. The predominant primary malignant orbital tumor in adults is lymphoma, with a prevalence ranging from 55% to 67%. ¹ An average annual incidence of 2.0 per million population for all types of malignancies was found, showing a steady increase over the 13-year study period, Melanoma represents (8%) in the USA. ² In Canada, Darwich et al ³ observed that the Canadian ASR of ophthalmic lymphoma was 0.65 per million person-years during 1992 to 2010. In the Netherlands, Koopman et al reported that the most common ocular tumor type was lymphoma, accounting for 67% of cases in both orbital soft tissues (72%) and the lacrimal gland (51%). Rhabdomyosarcoma accounted for 12%, adenocarcinoma for 6%, and adenoid cystic carcinoma for 5%. ⁴ Among Chinese in Taiwan, the average annual age-standardized incidence of eye cancers was 2.46 per million population (2.57 for male and 2.33 for female). For cases less than 15 years of age, the most common eye malignancy was retinoblastoma (86.0%), followed by rhabdomyosarcoma (3.9%) and lymphoma (2.8%). For cases 15 years of age or older, the most common eye malignancy was melanoma (28.6%), followed by squamous cell sarcoma (21.0%) and lymphoma (20.8%). ⁵ In Saudi Arabia, uveal melanoma (11.9%), and lymphomas (orbital, adnexal) (5.8%) were observed. ⁶ The highest number was recorded in Riyadh (35.39%), followed by Makkah (16.93%). The incidence was higher in the 0 to 4 years’ age group (55.21%), and it declined with age. ⁷ The data also revealed differences in the number of reported cases over time, as well as in the representation of eye cancer cases by gender and nationality, with retinoblastoma being the most common (53.32%). ⁷

The importance of medicinal plants as natural remedies is well established against various illness. Several reviews documents plants as drug candidates for drug discovery have been published and outlined.^8,9 Flavonoids, terpenoids, triterpenoid glycosides, and phytosterols are main classes of phytochemicals ¹⁰ are present in most plants and may be responsible for the expected biological effect(s).

Achillea biebersteinii Afan. (Achillea arabica Kotschy) is a wild plant belonging to the family Asteraceae, known as “Qaisoom” in Arabic. ¹¹ It is distributed in the southern region of Saudi Arabia. The antimicrobial, anticholinesterase, and antioxidant activities of different extracts of Turkish Achillea biebersteinii have been studied ¹² and its protective and therapeutic effects against ethanol-induced gastric ulcer in rats demonstrated that it is a valuable source of an antiulcer agent with a post-prandial hyperglycemia lowering effect. ¹³ The hepatoprotective and antioxidant activities of the essential oil of Achillea biebersteinii have also been demonstrated, ¹⁴ as well as its wound healing potential by in vivo excision and incision models with comparable activity to the standard Madecassol. ¹⁵ The anti-inflammatory activity of highly oxygenated terpenoids from Achillea biebersteinii has also been studied, ¹⁶ as well as the properties of its phenol and flavonoids of different extracts ¹⁷ and its properties as an anti-nociceptive. ¹⁸ Other related species such as A. millefolium have also been shown to upregulate ocular antioxidant enzymes and downregulate inflammation, to give relief from ear-ache or throat infection, and as an eye-wash. ¹⁹

Anastatica hierochuntica L. is a wild plant belonging to the family Brassicaeae, known as “Kaff Mariam” in Arabic, is an annual that is widespread in Saudi Arabia, and is used traditionally for malaria, epilepsy, arthritis, mental depression, bronchial asthma, mouth ulcers, and as an analgesic.^{20 -23} This plant is used locally as a liquid from fresh leaves as a treatment for conjunctivitis and other problems of the eye. The plant inhibits melanogenesis in theophylline-stimulated murine B16 melanoma cells. ²⁴ Previous studies have indicated that both the methanolic and aqueous extracts of Anastatica hierochuntica possess antifungal, antimicrobial and antioxidant properties ²⁵ and nephroprotective potential. ²⁶ Anastatica hierochuntica methanolic and aqueous extracts have antiproliferative effects, ²⁴ meanwhile, its potential toxicity effects in pregnant Sprague-Dawley rats and their developing fetuses have been investigated ²⁷ as well as mutagenicity. ²⁸

In the case of Anisotes trisulcus (Forssk.) Nees, 2 compounds have been identified from its aerial parts that exert mild and moderate cytotoxic effects on cultured hepatoma ²⁹ with new alkaloids having been identified ³⁰ with significant hepatoprotective effects. ³¹ In Arabic peninsula, this plant locally used for inflammation ³² and for eye (personal communication).

Capparis spinosa L. locally known as “Shaflah” or “Kabbur,” a member of the family Capparaceae has been used as a traditional herbal remedy since ancient times for its beneficial effects for human ailments. ³³ In the Central region of Saudi Arabia, the unopened buds were externally used to treat eye infections. ³⁴ It has also been reported to treat liver and kidney diseases, paralysis, and diabetes ³⁵ and in Morocco, the unopened buds are used externally to treat eye infections and prevent cataracts while caper dried fruits are used to treat hypertension and diabetic complications when taken orally with a glass of water.^36,37 It is commonly known as Caper and is of great significance as a traditional medicinal food plant. ³⁸ It is considered to be one of the most popular species of aromatic plants grown in the Mediterranean. ³⁹ The quercetin in Capparis spinosa extract has shown anti-tumor effects ⁴⁰ ; protective effects against oxidative stress and genotoxicity in mice have also been reported, ⁴¹ while anti-plasmodial, anti-leishmanial, and insecticidal activity of its essential oils have been reported. ⁴²

Flueggea virosa (Roxb. ex Willd.) Royle from the family Phyllanthaceae, known locally as “Hamrar,” is a shrub or small tree with significant inhibitory activity against aldose reductase, ⁴³ It has been traditionally used to treat fever, malaria, sexual dysfunction, pain, diabetes, epilepsy, snakebite, venereal disease, rheumatism, arrhythmia, rash, diarrhea, pneumonia, cough, hepatitis, and HIV-related illness, and also as a contraceptive. ⁴⁴ Various fractions from the leaves of Flueggea virosa have been investigated for its antimalarial efficacy and exhibited potential activity (IC₅₀ values < 10 μg/ml) against various strains. ⁴⁵ Three new indole alkaloids, along with 9 known alkaloids, were isolated from the fruits of Flueggea virosa. ⁴⁶ Four known terpenoids, 8 new dinorditerpenes were isolated and identified from the roots of Flueggea virosa and these compounds exhibited significant anti-hepatitis C virus activity. ⁴⁷

The neoplastic changes affecting the human eye can be classified as primary or secondary to a malignancy in other parts of the body. Conjunctival and uveal melanoma, retinoblastoma (Rb) in children, conjunctival and intra-ocular lymphoma, and ocular surface squamous neoplasia are the most common primary tumors of the eye. Meanwhile, metastatic secondary tumors spread from systemic malignancies, especially of the breast and lung, prostate, colon or any other parts of the body.^48,49

Retinoblastoma, the most common ocular malignancy in both children and adults, was less frequent in Saudi Arabia than in other some Gulf areas, although it is higher than in western countries. ⁶ Both primary and metastatic eye cancer are asymptomatic and present clinically with visual defects or blindness.^50,51 Examinations reveal that common ocular primary neoplasms and secondary ocular tumors can be differentiated in cytologic preparations. Rb cells have been found to occur either singly or in clusters. ⁵² A review reported that clinical presentation of retinoblastoma patients in Saudi Arabia has a high rate. The number of presenting patients with extraocular disease significantly decreased from 22.8% (1983-1988) to 12.3% (1989-1994). ⁵³

The reported survival for uveal melanoma with a poor prognosis appears to vary ⁵⁴ Early detection and the first surgery in conjunctival melanoma management are critical for prevention of recurrent or new tumors, as reported by advanced American Joint Committee on Cancer (AJCC). ⁵⁵ Although most patients with uveal melanoma develop metastases, research findings suggest that only 2 in 5 of all patients have a poor prognosis. ⁵⁶ Early detection and prompt treatment can give cure rates up to 95% for intra-ocular tumors, whereas extraocular disease is associated with high mortality.^{57 -61}

Breast cancer is a common and serious health issue cancer in women that can to spread to the eyes. ⁶² A study of a population of breast cancer patients with ocular metastasis has shown that uveal metastasis was the initial site of breast cancer in 3% of patients ⁶³ and the first site of metastatic cancer in 16%. Cancer in the eye is rare as there is no lymphatic system; instead, it spreads via the blood vessels, affecting areas of the eye with the best blood supply, namely the uveal tissue. ⁶⁴ Eye metastasis from colorectal cancer ⁶⁵ and primary liver cancer were reported by Sun et al. ⁶⁶ Ophthalmologists often discover the presence of terminal metastatic disease when the cancer has spread to the eye uveal tissue at early stages and usually in patients with a history of primary cancer. ⁶⁷ Based on this review of the literature, 5 Saudi plant species which have been traditionally used to treat some ocular complaints were evaluated in the current study for their cytotoxic effects and to prepare them for further investigations regarding their putative ocular effects.

Materials and Methods

Phytochemical Studies

Plant material identification

Five plants, namely Achillea biebersteinii (Whole; BEP-04), Anastatica hierochuntica (whole; BEP-05), Anisotes trisulcus (leaves; BEP-06), Capparis spinosa (leaves; BEP-07) (Figure 1), and Flueggea virosa (leaves; BEP-08), were taxonomically classified and identified by taxonomist Dr. Haidar Abdalgadir at the Department of Biology, Faculty of Sciences University of Al Baha. Voucher herbarium specimen numbers (BUH-71, 72, 73, 74, and 75) were deposited at the herbarium of the faculty of Science, University of Al-Baha, Saudi Arabia for future reference.

Figure 1.

(A) The spiny branch (B) and flowers of Capparis spinosa L. (BEP-07) growing at Al Baha Hills, Saudi Arabia.

Collection and extraction of plants

Plants were collected from different locations from Baljurashi province (Wadi E lkhaitan), Al Baha area in April 2021. The leaves (250 g) and whole plants (250 g) were shade dried and then powdered using a mechanical grinder. Dried materials were macerated in ethanol 80% v/v for 1 week at room temperature. The resulting residues were filtered, pooled and evaporated to dryness to afford green to brownish viscous syrup. The crude extracts were transferred onto petri plates, allowed to dry and finally weighed in 3 repeated readings (whole = 9.5 g; Leaves = 50.23 g). The yield percentage was calculated using the formula: Yield % = (afforded extract weight)/(air dried weight) × 100.

GC-MS analysis

The dried leaf extract of Capparis spinosa was selected for GC-MS analysis based on results of selectivity studies displayed below. The extract (BEP-07) was dissolved in methanol to attain a concentration of 1 mg/ml and diluted 1:10 v/v in methanol (100 μg/ml). The diluted sample was analyzed using a GC-MS (Thermo Scientific, USA) attached to a trace ultra-GC and ISQ detector and AS 3000 autosampler. Separation of the components were accomplished using a TR-5MS column (Thermo Scientific, USA) with 30 cm length, 0.25 mm i.d., and 0.25 mm film thickness. Helium was used as carrier gas at 1.2 ml/min using constant flow rate. Injection port was set at 32°C for 5 minutes, which followed by a ramp to 205°C at 5°C/min rate and hold-time 5 minutes. This was followed by a ramp to 280°C at 5°C/min rate and hold for 5 minutes and at the end to 300°C at 5°C/min rate and 5 minutes hold-time. The maximum oven temperature was fixed at 320°C. A volume of 2 μL diluted extract was injected into the system using split-less mode with the mass spectrometer run in EI mode with 0.6 scan periods throughout the mass range 60 to 900 AMU (min). Both the temperatures of MS ions source and transfer-line were adjusted to 320°C and 350°C, respectively, using 1 kV electron-multiplier voltage.

Identification of phytochemical constituents

The Xcaliber software was used for mass spectral data analysis and the fragmentation patterns of each constituent was matched with MS data in database of the instrument and built-in libraries including MAINLAB, NIST and REPLIB. The phytocompounds present in the extract were identified by comparing with available structures in the computer library and the percent abundance of each component was determined by using peak area as reference. The reported biological properties of the detected compounds are based on data from Dr. Duke’s Phytochemical and Ethnobotanical Database. ⁶⁸

Cancer Cell Studies

Cancer cell culture

Three cancer cell-lines, namely MCF7 (human breast adenocarcinoma), HT29 (human colorectal adenocarcinoma) and HepG2 (human liver adenocarcinoma) were used in this study, in addition to MRC5 (normal human fetal lung fibroblast), all from the ATCC, USA. The 3 cancer cells were sub-cultured in RPMI-1640 media (10% FBS v/v), while MRC5 was maintained in Eagles minimum essential medium (EMEM, 10% FBS v/v), with all cultures kept at 37°C, 5% CO₂, and 100% relative humidity, for a maximum of 5 to 10 passages.

Cytotoxicity and selectivity studies

The cytotoxicity of the 5 extracts, in addition to doxorubicin and camptothecin, were evaluated by MTT assay as previously reported.^69,70 Each of the cell-lines were separately cultured in 96-well plates (3 × 10³/well) and incubated with each of the extracts or doxorubicin at a final concentration of 0 to 100 μg/ml, for 3 days at 37°C overnight (DMSO 0.1% v/v; n = 3 of 3 independent experiments). After 3-day incubation, the cytotoxicity of each extract was evaluated by MTT assay. MTT was added to culture medium at 0.5 mg/ml to each well and incubated for 3 hours at 37°C. The MTT solution was removed and formazan granules were dissolved in DMSO and the absorbance was read on a multi-plate reader (Bio-Rad PR 4100, Hercules, CA, USA). The optical density of the purple formazan at A₅₅₀ nm was shown to be proportional to the number of viable cells. The extract concentration causing 50% inhibition (IC₅₀) was measured 3 times and compared to control cell growth (100%) and determined using GraphPad Prism. The selectivity index (SI) for the 5 extracts was calculated by dividing its IC₅₀ against MRC-5 cells/IC₅₀ against either MCF7, HT29, or HepG2 cell-line.

Clonogenic assay

Clonogenic assay measures tumor cell survival and subsequent proliferative ability following drug exposure. ⁷¹ BEP-07 extract was selected for further clonogenic assay, as it showed selectivity relative to the normal cell-line MRC5. Exponentially grown MCF7 cells in RPMI-1640 (supplemented with 10% FBS v/v and 1% penicillin/streptomycin v/v) were seeded in duplicates at a density of 800 cell/well in 6 well plate and allowed to attach overnight, and were then exposed to increasing concentrations of BEP-07 extract (0, 3.5, 7, and 10 µg/ml) for 72 hours. Wells containing media with extract were replaced with fresh media without extract. Cells were left to grow at 37°C, 5% CO₂, and 100% humidity. Each day wells were checked and cells forming colonies were roughly counted. After 14 days, plates were rinsed in PBS and fixed with pre-chilled methanol at room temp for 20 minutes, then stained with 0.5 methylene blue in 1:1 methanol/H₂O (v/v) for 10 minutes and washed in dH₂O thoroughly and then air dried. Cell colonies were counted microscopically and reported.

Western Blotting

Western blotting was used to determine the expression change of survivin (apoptosis inhibitor protein), and P27 (cell cycle protein). MCF7 cells (1 × 10⁶ cells/well) were treated with vehicle, doxorubicin (2 µg/ml) and BEP-07 extract (3.5, 7, 10, and 14 µg/ml) for 24 hours. Lysis buffer was used to isolate the total proteins, and their concentration was determined by Bradford method. Samples (20 μg total proteins) were separated on 10% SDS-PAGE gels and transferred to PVDF membranes. Membranes were blocked for 1 hours in TBS-T containing 5% non-fat dry milk and incubated with antibodies at 4°C overnight. After washing, blots were incubated with secondary antibodies (HRP-conjugated) for 1 hours. The loading protein samples (n = 2) were electrophoresed on a polyacrylamide gel and transferred to a membrane. The membrane was incubated with P27 and survivin antibodies (Cell signaling, Boston, MA, USA) for 2 hours at room temperature and the secondary antibody GAPDH added for 1 hour at room temperature. Immunoreactivity was visualized by chemiluminescence using horseradish peroxidase (HRP)-conjugated secondary antibodies, and their image was detected by a scanner (GeneGenome, Syngene BioImaging). ⁷²

Ethics Approval of the Study

As per Al Baha University standards, all the funded project proposals have undergone critical review followed by approval by the Scientific Research Affairs relevant committees before acceptance.

Results

Phytochemical Studies

The 3 plant extract yields were as follows: BEP-04 (9.7%), BEP-05 (14.2%), BEP-06 (9.6%), BEP-07 (9.9%), and BEP-08 (8.1%), respectively (Figure 2).

Figure 2.

Yields (%) of the dry extract of 5 plants obtained after ethanolic extraction and evaporation of different selected 5 plants. Data presented from 3 readings. BEP-07 was used as a control for comparison, while Dunnett’s Multiple Comparison Test was used as PostHoc after 1-way ANOVA test.

Identification of Phytochemical Constituents Using GC-MS

The GC-MS chromatogram of Capparis spinosa plant (BEP-07 extract) revealed the existence of 145 compounds belonging to the diverse classes of phytoconstituents such as steroids, hydrocarbons, phenols and cryohydrates. These compounds were identified via the relation of their peak retention time, peak area (%) alongside the recorded molecular weight to that of the previously reported compounds. The major components (peak area % > 2.10) in BEP-07 extract are illustrated in Table 1 and Figure 3. As evident from Figures 3 and 4, fatty acids and their derivatives (7-9) represent 15.36% of the total peak areas % of the investigated fraction and nearly 30% of the total peak areas of the major components (51.71%). Secondly, the nitrogen containing compounds in this fragment namely compound 2 and 3 were the second most abundant (13.87%). The rest of the major components which present similarly (5%-6%) were carbohydrates represented by compounds 5 and 6, the steroidal compound (10) along with the hydrocarbon derivative 1. It is worth noting that compound 9, octadecanoic acid, 2,3-dihydroxypropyl ester, was the principal component (7.9%) of the BEP-07 extract. In contrast, the phenolic derivative, 4, was the least abundant amongst the major components (3.96%).

Table 1.

Phytoconstituents Identified by GC-MS Analysis of BEP-07 Extract.

Compound	Formula	Molecular weight	Peak area (%)	Retention time (min)	Biological activity
(1) 2,3,3a,4,7,7a-hexahydro-4,7-methano-1H-inden-1-one	C10H12O	148.20	5.63	5.605	-
(2) 2-(1-piperidinylcarbonyl)aniline hydrochloride	C12H17ClN2O	240.73	7.39	5.719	-
(3) 1,2-dimethylpyrrolidine	C6H13N	9917	6.48	6.055	-
(4) 2-methoxy-4-vinylphenol	C9H10O2	150.17	3.96	6.555	Antimicrobial, antioxidant, anti-inflammatory, analgesic, and anti-germination[1]
(5) D-allose	C6H12O6	180.06	3.91	8.345	Antitumor [2], Inhibits lung carcinoma [3]
(6) d-talonic acid lactone	C6H10O6	178.14	2.82	9.155	-
(7) n-hexadecanoic acid	C16H32O2	256.42	2.15	11.348	-
(8) Hexadecanoic acid, 2-hydroxy-1-(hydroxymethyl)ethyl ester	C19H38O4	330.50	5.31	14.657	Hypocholesterolemic [4]
(9) Octadecanoic acid, 2,3-dihydroxypropyl ester	C21H42O4	358.56	7.9	15.868	Anticancer, antimicrobial [5]
(10) Stigmast-5-en-3-ol	C29H50O	414.71	6.16	23.329	Anti-diabetic agents [6], Apoptotic and antiproliferative effects [7]

Figure 3.

Structures of chemical constituents identified through GC-MS for BEP-07 extract.

Figure 4.

Peak areas (%) for the major components for the BEP-07 extract.

Cytotoxicity and Selectivity Studies

The 5 extracts showed variable IC₅₀ ranging from 1.55 to 30.65 μg/ml. The most active extract was BEP-07 against HepG2 and MCF7 cells, showing up to twofold selectivity compared to the normal MRC5 cells (Tables 2 and 3). Thus, BEP-07 extract was considered for further cytotoxic and mechanistic studies.

Table 2.

Cytotoxic Activity of the 5 Plant Extracts, Doxorubicin and Camptothecin Against 3 Cell-lines, and Normal Fibroblast (MTT, 72 h, IC₅₀ “μg/ml” ±SD, n = 3).

Extract	MCF7	HT29	HepG2	Average a IC50	MRC5
BEP-04	5.92 ± 0.32 ***	25.22 ± 1.82 ***	12.45 ± 1.48 ***	14.53 ± 9.81**	3.37 ± 0.63NS
BEP-05	2.80 ± 0.39 ***	6.23 ± 0.84 ***	9.61 ± 1.30 **	7.67 ± 1.64NS	4.44 ± 1.30NS
BEP-06	8.28 ± 1.27 ***	8.92 ± 0.38 **	5.82 ± 0.72NS	14.62 ± 13.93 **	5.64 ± 0.96NS
BEP-07	3.61 ± 0.99 ***	5.77 ± 0.98 ***	1.55 ± 0.34**	3.64± 2.11NS	4.25 ± 0.61NS
BEP-08	30.65 ± 0.30 **	7.83 ± 0.46 ***	5.39 ± 1.34 ***	6.21 ± 3.40NS	8.57 ± 2.20NS
Doxorubicin	0.07 ± 0.01C	1.98 ± 0.10C	2.15 ± 0.15C	1.40 ± 1.15C	5.86 ± 0.35C
Camptothecin	0.08 ± 0.01	2.50 ± 0.26	0.76 ± 0.07	1.11 ± 1.25	1.18 ± 0.10

Doxorubicin (G2M-blocker) Camptothecin (S-blocker).

MRC5: normal fibroblast.

Abbreviations: C, control; NS, not statistically significant.

a

Average cytotoxicity (IC₅₀) of each extract against the 3 cancer cell-lines.

**

P ≤ .01. ***P ≤ .001.

Table 3.

Selectivity Index (SI) (≥1.1) of the 5 Extracts, Doxorubicin and Camptothecin Against Normal MRC5 Cells.

Extract	MCF7	HT29	HepG2
BEP-04	0.57	0.13	0.27
BEP-05	1.58	0.71	0.46
BEP-06	0.68	0.63	0.97
BEP-07	1.17	0.74	2.74
BEP-08	0.28	1.09	1.59
Doxorubicin	78.57	2.96	2.73
Camptothecin	13.80	0.47	1.55

Clonogenicity of BEP-07 Extract

The BEP-07 extract was chosen to test its clonogenic effect against MCF7 cancer cells, as it showed the best selectivity following 72 hours treatment. BEP-07 extract showed a dose dependent clonogenic effect against MCF7 cells. At 7 μg/ml less than half of the MCF7 colonies survived compared to the control colonies, while at 10 μg/ml only 20% of the MCF7 colonies have survived. While doxorubicin anti-clonogenic effect is stronger, BEP-07 is comparable to the effect of doxorubicin on the same cells (Figure 5).

Figure 5.

Colonies of MCF7 cells treated with (A) BEP-07 extract (0, 3.5, 7, and 10 µg/ml; n = 2) and (B) Doxorubicin (0, 2, 4, and 6 µg/ml; n = 2) for 72 hours in 6 well plates followed by 14 days of extract-free incubation. Bar graph showing x-axis: BEP-07 extract or doxorubicin concentrations; and Y-axis: colony number. Results are expressed as cell number ± SD of 2 independent experiments.

Western Blotting of P27 and Survivin Following Treatment With BEP-07 Extract

The effect of doxorubicin and extract BEP-07 (24 hours) on the apoptosis-related gene expression of survivin in MCF7 cells was investigated using western blotting. BEP-07 extract caused a significant decrease of the anti-apoptotic survivin protein expression, and increase of the cell cycle-related P27 protein expression, all compared to control GAPDH (Figure 6A and B). The inhibition of survivin suggests the ability of extract BEP-07 to induce apoptotic pathways in MCF7 cells, while the increase of P27 indicates the disruption of the MCF7 cell cycle.

Figure 6.

(A) Expression of survivin and P27 in MCF7 cells treated with doxorubicin or BEP-07 extract for 24 hours. (B) expression levels of surviving and P27 compared with the control. MCF7 cells were treated for 24 hours with either doxorubicin (2 μg/ml), or BEP-07 extract (3.5-14 μg/ml). Data are represented as mean ± SEM (n = 2, two independent experiments). The results were expressed as fold-change compared with the untreated group.

Figure 7 summarizes the study experiments and their results as an overview.

Figure 7.

A study experiments and results summary.

Discussion

Metastatic secondary ocular tumors often are initiated from systemic malignancies, especially of the breast and lung, prostate, colon. Although several plants have been used in traditional medicine, few have been subjected to thorough scientific investigations. The most active extract in our study was BEP-07 against HepG2 and MCF7 cells, indicating that it is a promising natural substance with therapeutic properties. These findings are in line with those who have investigated the positive effects of quercetin from Capparis spinosa extract. ⁴⁰ Due to the optimal selectivity profiles following 72 hours treatment, BEP-07 extract was subjected to further cytotoxic and mechanistic studies. These studies showed a dose-dependent clonogenic effect against MCF7 colonies which was comparable with the effect of doxorubicin. This finding confirms its previously reported cytotoxic activity, as the clonogenic assay measures tumor colony survival and subsequent proliferative ability following drug/extract exposure. ⁷³ BEP-07 extract also caused a significant decrease of survivin level and an increase of P27 expression compared to control GAPDH. The inhibition of survivin indicates the ability of BEP-07 extract to induce apoptosis in MCF7 cells, while the increase of P27 indicates the disruption of the MCF7 cell cycle. Survivin has been shown to inhibit cancer cell apoptosis and to promote cell proliferation. The overexpression of survivin is closely correlated with tumor progression and drug resistance.^74,75 P27 KIP1 is a cyclin dependent kinase inhibitor, which may act as a potential suppressor gene. ⁷⁶ Thus, the BEP-07 extract, by increasing this biomarker, suppresses the proliferation of MCF7 cells. Correlating the cytotoxic activity of the most promising BEP-07 extract to its phytochemical constituents, GC-MS was performed and different classes of phytoconstituents were detected including steroids, hydrocarbons, phenols and carbohydrates and these have been reviewed as anticancer agents. ⁷⁷ Overall, our results show that compound 9, octadecanoic acid, 2,3-dihydroxypropyl ester, which was the principal component in BEP-07 extract possesses confirmed cytotoxic activity as previously reported. ⁷⁸

Finally, this study may lead to potential applications or further questions. More pharmacological and toxicological studies must be conducted to confirm our findings and to determine the level of safety and feasibility for isolation and characterization of the active ingredients to serve as a candidate for new drug discovery.

Study Limitations

The HepG2 cells were not used; instead breast cancer cells (MCF7) were employed. We performed the MTT in 2 steps, once at the beginning of the project with all cells and a second time after GC-MS analysis. In the second time we were aiming to use both HepG2 and MCF7, but we lost the HepG2 cell line and could not obtain a good HepG2 with a fresh or new passage.

Further confirmatory specific studies can be performed using ocular cancer cells, while advanced mechanistic investigations should be conducted to deeply determine or rule out any other possible mechanism of action.

More in vitro and in vivo pharmacological studies could be performed alongside toxicological ones to complete the extract profile at the pre-clinical stage to prepare for a clinical phase.

Conclusions

This study concludes that the leaves of Capparis spinosa (BEP-07 extract), found in Al Baha and used locally for various purposes, possess a significant cytotoxic effect, particularly against breast cancer cell-lines, with reasonable selectivity to the normal cells. It exhibited this effect through apoptosis induction, through survivin inhibition, and also through perturbation and disruption of the cell cycle via P27 suppression. The detected phytoconstituents in the plant extract might be involved in the tested cytotoxic activity, particularly the steroids and phenols. Further investigations are required to complete the drug candidate profile.