Assessing the cytotoxicity of phenolic and terpene fractions extracted from Iraqi Prunus arabica against AMJ13 and SK-GT-4 human cancer cell lines

Introduction

A critical global health issue is breast cancer, the most prevalent cancer diagnosed worldwide, with 2.26 million incidents expected and it is the major cause of cancer-related deaths among women.¹ Although breast cancer is considered a common disease in developed societies, in 2020, the world's less developed regions accounted for two-thirds of breast cancer-related deaths and more than 50% of all breast cancer diagnoses.²

In terms of cancer-related deaths, esophageal cancer (EC) occurs sixth on the list of the deadliest diseases (436,000 fatalities). Meanwhile, 473,000 cases have been recorded worldwide.³ Even though chemotherapeutic regimens and radiation therapy are more effective methods for treating cancer, they are nonselective, have substantial side effects, and can harm normal healthy tissues, leading to severe unanticipated and undesirable side effects.⁴ Initially, several tumors appeared amenable to treatment. However, with time, resistance might develop for a number of reasons, such as mutations in DNA and metabolic changes that cause the drug to be inhibited and degraded.⁵

The occurrence of natural compounds as anticancer agents is estimated to exceed 60% of the current anticancer drugs.⁶ These drugs from natural origins can be used for both cancer prevention and treatment due to their pharmacological safety and can be used independently or in conjunction with existing chemotherapeutic treatments to improve the therapeutic efficacy or to reduce chemotherapy-induced toxic effects.⁷ The constant change and injury that occurs in the human body over decades requires a development of revolutionary and highly precise “arms” capable of successfully combating malignant cells. Natural products are a powerful and endless source for identifying the finest anticancer prospects.⁸

Prunus arabica is recognized as a distinct species from the farmed almond P. dulcis. Both, however, are members of the Prunus genus and the Rosacea family.⁹ This species was given its scientific name based on its geographical location where it first appeared. This taxon is indigenous to mild climate Asia regions, including the Fertile Crescent Mountains, as well as Turkey, Iran, and Iraq.¹⁰

Prunus arabica is a thick undomesticated almond species with an unbarked stem that remains green even during dormancy.⁹ A wide range of biological and pharmacological effects from different Prunus species show great promise for the treatment of various cancers.¹¹ Flavonoids, steroids, terpenoids, poly phenols, and other chemicals have all been identified from various Prunus species.¹² Polyphenols and terpenoids which are found plentifully in plants, show anticancer effects, via inhibiting cancer cell growth, blood vessel formation, metastasis, inflammation, and inducing cell death.¹³ For this purpose, the anticancer effect of phenolic and terpene fractions from Prunus arabica extract was investigated using the AMJ13 and SK-GT-4 cancer cell lines and normal human fibroblasts (NHF).

Methods

Results

In vitro cytotoxic activity

The MTT assay was used to evaluate the cytotoxicity and therapeutic efficacy of the isolated fractions in the cancer and normal cell lines (AMJ13, SK-GT-4 and NHF). As shown in Figures 1–3, the results showed that the treatments significantly decreased the viability of the cancer cells (>50% at higher concentrations) with minimal cytotoxic effects on the normal cells (<50% cytotoxicity). On the AMJ13 cell line, phenolic, terpene fractions, and docetaxel had a half maximal inhibitory concentration (IC₅₀) of 29.34±2.37, 8.455±3.02, and 14.51±0.77 μg/ml, respectively, as shown in Figure 4. On the SK-GT-4 cell line, phenolic, terpene fractions, and docetaxel had an IC₅₀ of 21.97±3.56, 15.14±3.266, and 0.7125±0.084 μg/ml, respectively, as shown in Figure 4. While on the NHF cell line, the phenolic, terpene, and docetaxel had an IC₅₀ of 18.07±1.83, 31.81±12.07, and 24.9±7.17 μg/ml, respectively.

Figure 1. The cytotoxicity of different concentrations on the AMJ13 cell line, T: terpenes, P: phenolic, D: docetaxel, P+D: phenolic and docetaxel, T+D: terpene and docetaxel.

Figure 2. The cytotoxicity of different concentrations on the SK-GT-4 cell line, T: terpenes, P: phenolic, D: docetaxel, P+D: phenolic and docetaxel, T+D: terpene and docetaxel.

Figure 3. The cytotoxicity of different concentrations on the NHF cell line, T: terpenes, P: phenolic, D: docetaxel, P+D: phenolic and docetaxel, T+D: terpene and docetaxel.

Figure 4. The half maximal inhibition concentration (IC₅₀) for phenolic, terpene, and docetaxel, respectively, on A: AMJ13, B: SK-GT-4, and C: NHF cell lines.

Extract-docetaxel potential interaction (Chou–Talalay analysis)

The possible interactions between extracted fractions (phenolic, terpene) and docetaxel therapy on the AMJ13 and SK-GT-4 cell lines were analyzed. The quantification of synergism or antagonism is defined as a mass-action law issue (determined by the combination index CI values) and cannot be determined by the statistical p values.²³ Chou-Talalay equations were used to calculate the combination's value. A CI value less than 0.9, the effects were assumed to be synergistic; a CI value between 0.9 and 1.1, the effects were considered as additive; while a value larger than 1.1, the effects were assumed to be antagonistic.²⁶ After an exposure period of 72 hours, the phenolic fraction of P. arabica with docetaxel produced a strong to very strong synergic cytotoxic effect against AMJ13 and SK-GT-4 cancer cell lines in comparison with a single treatment. The terpene fraction showed almost the same synergism effect when combined with docetaxel (Figure 5).

Figure 5. The dose–response curves, the normalized isobologram and combination index with non-constant ratios.

Data is expressed as fraction affected (fa) against combination index plots. Combination index (CI) value less than 0.9 indicates synergy, CI values between 0.9 and 1.1 indicates additive, and CI value larger than 1.1 indicates an antagonism. (A and B) Represents the effect against AMJ13 cell line by phenolic-docetaxel and terpene-docetaxel combinations respectively, all points are showing synergy to very strong synergy; (C and D) represents the effect against SK-GT-4 cell line by phenolic-docetaxel and terpene-docetaxel combinations respectively, all of the points are showing synergy to very strong. (E and F) Show the effect on the NHF cell line by phenolic-docetaxel and terpene-docetaxel combinations respectively, tested points showed inconsiderable cytotoxicity as all concentrations failed to emerge 50% cytotoxicity. The data represents six separate experiments.

Morphological analysis (crystal violet staining)

Morphological alterations were examined with crystal violet staining of the cell lines after 72 hr. Exposure to the IC₅₀ of the phenolic fraction, terpene, docetaxel, and their combinations with chemotherapy as shown in Figures 6, 7 and 8. The captured images are for cells treated with (100 μg/ml) of the tested fractions or their combinations with docetaxel on cancer or normal cell lines.

Figure 6. Morphology of AMJ13 cell line.

A: untreated cells show multipolar elongated epithelial-like shape, with multiple nuclei in most of the cells and nuclear polymorphism, many cells showed mitotic figures B, C, and D cell treated with terpene fraction phenolic fraction and docetaxel respectively; the treated cells showed a shrinkage, cytoplasm and cell membrane disappearance, stromal edema, nucleus shrinkage and marked decrease in the number of cells. E and F cells were treated with a combination therapy of phenolic plus docetaxel and terpene plus docetaxel, respectively; the treated cells showed more prominent cytotoxic effects than single treatment with a dense nucleus. The microscopic images were captured at 10× by an inverted microscope (IXplore Standard Olympus, Japan).

Figure 7. The morphology of SK-GT-4 cell line.

A: untreated cells are squamous or poorly differentiated and irregular in shape. B: cells treated with terpene fraction showed shrinkage; the squamous cell border remained intact, while other cells (no bordered cells) showed stromal edema. C: cells treated with phenolic fraction showed an increment of the stromal edema and disappearance of squamous cells with no ductal nuclear aggregation. D: docetaxel-treated cells showed focal aggregation and cellular shrinkage. E and F cells were treated with combination therapy phenolic plus docetaxel and terpene plus docetaxel, respectively; the treated cells showed more shrinkage than single treatment (very small sized cells), the squamous cell border is intact, destruction of ductal/basal cell membranes with no focal aggregation. The microscopic images were captured at 10× by an inverted microscope (IXplore Standard Olympus, Japan).

Figure 8. Morphology of NHF cell line.

A: untreated cells; cells appear as plump spindle-shaped or stellate-shaped cells with centrally placed oval or round nuclei. B: cells treated with terpene fraction showed mild apoptosis. C: cells treated with phenolic fraction showed condensation of cells with no edema. D: docetaxel-treated cells showed focal apoptosis with mild stromal edema, and the cells remained the same size. E and F cells were treated with a combination therapy of phenolic plus docetaxel and terpene plus docetaxel, respectively; the treated cells showed prominent apoptosis compared to the single treatment. The microscopic images were captured at 10× by an inverted microscope (IXplore Standard Olympus, Japan).

Discussion

Polyphenols and terpenes are the most abundant and widely distributed compound in the plant kingdoms and groups.²⁷ Prunus species have been found to be a potential dietary supplement and a good source of phenolic and terpene bioactive chemicals.²⁸

In the current study, lead acetate and NaOH tests for polyphenols gave a positive result, meaning the presence of phenolic compounds in P. arabica extract. The dark color might indicate the presence of large quantities of polyphenols and flavonoids.²⁹ The H₂SO₄ test gave a dark pink or red color and greenish color, respectively, as an indication of the presence of steroids, while the chloroform and sulphuric acid test produced a greyish color which was considered an indication of the presence of terpenes.

The data for the HPLC analysis showed that the phenolic fraction of the extracted P. arabica contains eight phenolic compounds (p-Coumaric acid, ferulic acid, gallic acid, caffeic acid, quercetin, rutin, catechin, and chlorogenic acid), the terpene fraction contained three major terpenes (β-sitosterol, stigmasterol, and campesterol) as well as some non-phenolic organic and inorganic components at deceptive values.

From the eight phenolic compounds found in our phenolic fraction of the P. arabica extract, the highest component concentration was ferulic acid (3.992 mg/gm) with a retention time of 3.22 min, which may contribute to the high efficacy of the phenolic extract as a cytotoxic biological chemical on different cancer cell lines.³⁰ Research has indicated that ferulic acid induces cell death by decreasing the Bcl-2 and increasing the BAX gene expression or by upregulation of caspase-3 and cleaved caspase-9.³¹ Moreover, the significant contribution of chlorogenic acid and catechin in manifesting anti-proliferative effects in cancer cell lines is underscored. This assertion is substantiated by research findings demonstrating that quercetin induces apoptosis in MCF-7 cells and hinders the proliferation of MCF-7 breast cancer cells in a time- and concentration-dependent manner. The inhibitory impact of quercetin on MCF-7 cell growth, coupled with its promotion of apoptosis through G0/G1 phase arrest, has been established.³² Additionally, catechins exert anticancer effects by modulating various processes, encompassing the inhibition of carcinogenic activity, tumorigenesis, proliferation, apoptosis induction, cell cycle arrest, metastasis, and angiogenesis.³³ Rutin, as another extract component from Prunus arabica, was found to possesses anti-proliferative activities by triggering apoptosis in triple-negative breast cancer.³⁴

The highest component of the terpene fraction was campesterol (4.358 mg/gm), with a retention time of 11.5 min. A study by Hyocheol B. et al. confirmed that campesterol could inhibit both cellular growth and cell cycle progression by regulating the PCNA (proliferating cell nuclear antigen) and PI3K/MAPK (phosphatidylinositol-3-kinase/mitogen-activated protein kinase) signal pathways. Moreover, their results also showed that campesterol could prevent the clustering of ovarian cancer cells.³⁵ Some undiscovered phenolic and terpene compounds may be presented by peaks on the chromatograms, hydroperoxides or peroxides produced from terpenes are likely responsible for these found but unidentified peaks.³⁶

The present work studied the cytotoxic effects of the extracted fractions (phenolic and terpene) of P. arabica alone and in combination with docetaxel and compared their novel effects with the single chemotherapeutic agent (docetaxel) against AMJ13, SK-GT-4, and NHF cell lines. Breast cancer and esophageal carcinoma are considered highly malignant tumors, which lead to poor prognoses.³⁷ The low efficacy of currently available breast and esophageal cancer chemotherapeutics and radiation moreover these therapies are associated with severe adverse effects and patients can develop resistance to these agents.^38,39

In vitro, the results of this study appear that the treatment with the phenolic and terpene extract of P. arabica significantly reduced cell viability and triggered apoptosis when compared to the control group in both AMJ13 and SK-GT-4 cell lines (Figures 1–2). During this study against the AMJ13 cell line, the terpene fraction showed comparable cytotoxic effects to docetaxel even in concentrations as low as 25 μg/ml, the IC₅₀ for phenolic terpene and docetaxel against AMJ13 cell line was 29.34±2.37, 8.455±3.02 and 14.51±0.77 μg/ml respectively (Figure 4).

The phenolic fraction showed almost an equal cytotoxicity to docetaxel against the SK-GT-4 cell line, while terpene showed less significant cytotoxicity in comparison with other tested treatments; the IC₅₀ for phenolic terpene and docetaxel against SK-GT-4 was 21.97±3.56, 15.14±3.26, 0.7125±0.084 μg/ml respectively as shown in Figure 4. All treatment concentrations for phenolic and terpene fractions failed to show significant cytotoxicity on the NHF cell line (less than 50%).

Many unique chemical components, including polyphenols, flavonoids, alkaloids, and terpenes, have been identified from Prunus species. The great structural variety of these compounds underlies their unique biological properties, which include bioavailability, antioxidant activity, and specific interactions with cell receptors and enzymes.⁴⁰

Researchers have found that flavonoids have a wide range of biological effects in mammals, including antimicrobial, antiviral, analgesic, anti-allergic, hepatoprotective, cytostatic, and apoptotic properties.⁴¹

The current study confirms the findings of previous studies which showed that phytosterols, such as quercetin and β-sitosterol, protect against a wide variety of diseases and exhibit selective cytotoxicity towards cancer cells, as evidenced by high apoptosis indices in cells exposed to quercetin’s anticancer activity.⁴²

Many studies have demonstrated that flavonoids have cytotoxic effects, including modulating ROS-scavenging enzyme activities, cell cycle arrest, induction of apoptosis and autophagy, and suppression of cancer cell proliferation and invasiveness. In healthy cells, flavonoids function as antioxidants, but in cancer cells, they become strong pro-oxidants that induce apoptotic pathways and downregulate pro-inflammatory signals.⁴³

Stigmasterol's cytotoxic actions come from its ability to induce autophagy in tumor cells, decrease their proliferation and spread, and promote their apoptosis.⁴⁴

The increased sensitivity of cancer cells to cytotoxicity was another goal of this investigation by using phenolic or terpene fractions with docetaxel and the cumulative effects of many dosages. The MTT assay performed with docetaxel in the presence of varying amounts of phenolic or terpene fractions. According to the findings, the phenolic, terpene, and docetaxel combination substantially decreased cancer cell viability without causing appreciable damage to normal cells. The Chou-Talalay equation was used to evaluate the combinations.

The degree of synergy or antagonism cannot be assessed by p value in a statistical manner but can be quantified using CI values (combination index values).⁴⁵ Nearly all of the doses examined showed synergistic cytotoxicity against the cancer cell lines. To demonstrate their safety, testing on a normal human fibroblast cell line showed no effect at any dose of the combination of phenolic and terpene fractions with docetaxel. There have been a number of studies suggesting that phenolic acids and terpenes may boost the effect of other chemotherapies on breast cancer.⁴⁶ However, this is the first study to provide empirical evidence of synergy between phenolic and terpene fractions with docetaxel against AMJ13 and SK-GT-4 cell line (Figure 5 and Table 5).

As seen in the current study and after crystal violet staining, the AMJ13, SK-GT-4, and NHF cells that were exposed to 72h of extracted fractions and docetaxel and the combination therapy revealed cell shrinkage, cytoplasm and cell membrane disappearance, stromal edema, nucleus shrinkage and a marked decrease in the number of cells compared with control (untreated) cells. The effect of combination therapy was more prominent than single therapy as the cells showed more shrinkage, extensive cell damage, and necrosis.

As a result of their specificity, chemopreventive medicines only target cancer cells. A compound’s ability to selectively destroy cancer cells while having a minimal effect on healthy cells is measured by its “selectivity index.”

The compound’s low selectivity index suggests that it is less hazardous to healthy cells than cancerous ones. Compounds having high SI values may provide a safer and more effective cancer treatment option.⁴⁸

Based on the test results the phenolic fraction is found to be less selective for all tested cells (SI<3). Meanwhile, terpene fraction is selective for AMJ13 cancer cells with (SI>3) and less selective for SK-GT-4 cancer cells. The Docetaxel treatment (positive control) showed an excellent SI toward SK-GT-4 cell line (SI>3). The results of this study need a further evaluation to determine the potential cytotoxicity in animal models.

Conclusions

It was observed from the results that the P. arabica phenolic and terpene extracts have significant cytotoxic activity on breast cancer and esophageal cancer cell lines with minimal effect against normal cells, due to the presence of effective compounds in this extract. Moreover, these active compounds increased the cytotoxic activity of docetaxel on cancer cell lines without increasing the toxicity against normal cells. Previous studies proved the cytotoxic effect of phenolic and phytosterol fractions extracted from other Prunus species. It is recommended to use a variety of cancer cell lines and animal models to evaluate the efficacy and safety of plant extracts, investigate their mechanisms of action, and explore their potential as therapeutic agents for developing novel cancer drugs from natural sources.