Exploring the Mechanism of Cytotoxic Activity of β-Caryophyllene Oxide and its Potential to Enhance the Activity of Doxorubicin

Abstract

Plant-derived compounds, including flavonoids, sesquiterpenes, and others, have been widely investigated for their role in cancer treatment. Here, we highlight the significance of some natural compounds (β-caryophyllene oxide) in enhancing doxorubicin's chemotherapeutic cytotoxicity. We also determined a novel mechanism of cytotoxicity for β-caryophyllene oxide by revealing its ability to bind with the DNA. This potentially causes its cytotoxicity when added alone and its ability to enhance doxorubicin's cytotoxicity. We also show that some natural compounds (epicatechin) may provide protection to cancer cells against doxorubicin's toxicity. This is most likely caused by their antioxidant behavior which potentially suppressed the doxorubicin-induced reactive oxygen species (ROS) resulting in a decrease in its cytotoxicity. Moreover, similar suppression in the cytotoxicity of β-caryophyllene oxide was noticed when incubated with the other flavonoids.

Introduction

With nearly 15 million newly cancer diagnosed cases and 10 million deaths in 2020, ¹ cancer is considered one of the leading to death causes globally. ² Moreover, the number of diagnosed cases is expected to reach 30 million by 2030, which presents the condition as an important global public health problem. Cancer involves the uncontrolled growth of cells that can metastasize to other locations in the body. These behaviors are the result of various changes in the genome expression. Among the various cancer types, liver cancer is considered the third leading cause of cancer death worldwide. ² With its high mortality rates, liver cancer is known to have a poor prognosis ² despite the various treatment options available. Depending on the stage and location, liver cancer treatment options include chemotherapy, surgery, immunotherapy, and radiotherapy. However, chemotherapeutics remains the mainstay for treating advanced inoperable liver cancers. ³ Despite the substantial progress in the development of various chemically synthesized anticancer drugs (chemotherapeutics), the poor liver cancer prognosis remains a tremendous challenge.^4,5

The lack of optimal chemotherapeutics with minimal side effects and maximum cancer-killing ability has partly caused substantial challenges to improving the prognosis. Therefore, searching for safe and effective chemotherapeutics continues to find ideal anticancer treatments with maximum activity and minimum toxicity. Immense attention has been directed towards natural products in search of ideal treatments. This involved isolating phytochemical compounds and exploring their anticancer activities.^5,6 These efforts have resulted in the discovery of many plant-based anticancer compounds.

Of the many phytochemical families, alkaloids, saponins, anthocyanins, and flavonoids have been widely investigated for their anticancer activity.^7–9 In addition to these families, natural sesquiterpenes have recently emerged as new compounds with promising anticancer activities. ¹⁰ The difference in their chemical and biological characteristics allowed a cocktail of numerous mechanisms of action. For example, flavonoids have been reported to affect the apoptotic signaling cascade, while alkaloids bind with the DNA and the DNA topoisomerases which hinder cell division. ¹¹ Sesquiterpenes are thought to affect different cellular pathways, including reactive oxygen and nitrogen species modulation. ¹² Despite these advancements, further investigation is still warranted to uncover their biological activities and precise mechanisms of action. Here, we explore the anticancer effects of the three flavonoids (naringin, naringenin, and epicatechin), one sesquiterpene (β-caryophyllene oxide), and one aryl alkyl ketone (dehydroacetic acid). The compounds are found in different natural recourse. For example, β-Caryophyllene oxide exists in the essential oils of various plants, such as cloves (1.7 −19.5%), basil (5.3- 10.5%), oregano (4.9-15.7%), lavender (4.62-7.55%), rosemary (0.1-8.3%) and many others. Naringin and naringenin are abundant in grapefruit, oranges, apples, and many others. Epicatechin is a highly abundant flavonoid found in different fruits, including apples, blackberries, cherries, grapes, pears, raspberries, tea, and others. ¹³

The compounds were selected based on their previously reported cytotoxic activity.^10,14–18 Moreover, these compounds belong to well established bioactive phytochemical families which also prompted our selection. Despite this, their mechanisms of anticancer activity have not been fully understood. This prompted evaluating the cytotoxic activity of the compounds separately and determining if synergistic effects exist when these compounds are administered together. Furthermore, the mechanisms of action for the compound with the highest cytotoxic potential was further evaluated.

The cytotoxic activity of the five natural compounds was initially evaluated (naringin, naringenin, epicatechin, β-caryophyllene oxide, and dehydroacetic acid) against HepG2 (hepatocellular carcinoma cell line). Once established, we attempted to detect synergistic effects on their cellular killing ability by co-administering two compounds. We also reported their effect on doxorubicin's cytotoxicity against HepG2 when co-administered. Of the five chemicals, β-caryophyllene oxide showed the highest cytotoxic activity when added alone to HepG2. Also, it showed the highest potency in enhancing doxorubicin's cytotoxicity. This prompted us to further investigate its mechanism of cytotoxicity. The ability of β-Caryophyllene oxide in enhancing the cytotoxicity of doxorubicin has been presented by several researchers.^19–21 Inhibiting p-glycoprotein and increasing apoptosis potential were explored means of enhancing doxorubicin's cytotoxicity. Our finding which shows the ability of β-Caryophyllene oxide to interact with DNA presents a novel mechanism in enhancing doxorubicin's cytotoxicity.

The novelty of this work is the identification of a novel mechanism of cytotoxicity for β-caryophyllene oxide against HepG2. We also highlight the ability of β-caryophyllene oxide to enhance doxorubicin's cytotoxicity in HepG2. We also presented the ability of β-caryophyllene oxide to bind with DNA as a novel mechanism enhancement of doxorubicin's cytotoxicity.

Materials and Methods

Reagents and Materials

HepG2 cell line was purchased from ATCC (USA). DMEM media was obtained from Sartorius (Germany). Dimethyl sulfoxide (DMSO), fetal-bovine serum (FBS), naringin, naringenin, and caryophyllene oxide were obtained from Sigma-Aldrich (Germany). MTS was purchased from Promega (USA). Epicatechin was obtained from Interchim (France). Doxorubicin was obtained from Combi-Blocks (USA).

Cell Viability Determination by the (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, Inner Salt) (MTS) Assay

HepG2 cells were seeded at 5X10³ cells per well, in a 96-well plates, in DMEM media supplemented with 1% penicillin-streptomycin and 10% FBS and left overnight to attach. The cytotoxicity of 5 natural products (naringin, naringenin, β-caryophyllene oxide, dehydroacetic acid, and epicatechin) was initially determined by screening their effects on the viability of HepG2 cells. To achieve this, each compound was incubated with the cells separately at a concentration of 50μg/ml for 48 h (h). Moreover, the effect of the addition of each compound on the doxorubicin's cytotoxicity was explored. This was conducted by co-exposing the cells to each compound separately (50μg/ml) with doxorubicin (1μg/ml) for 48 h. Additionally, the synergistic effects of the 5 compounds were investigated by incubating two compounds together with the cells for 48 h. The concentrations (1μg/ml) for doxorubicin and (50 μg/ml) for naringin, naringenin, dehydroacetic acid, and epicatechin were chosen after our preliminary experiments which showed minimal cytotoxicity at these concentrations. Moreover, a concentration near the IC₅₀ of β-caryophyllene oxide (the most potent compound) was used intentionally to test if an enhancement to the cytotoxicity of the other compounds (natural and doxorubicin) can be detected.

Once completed, MTS dye was added to determine the viability of the cells after the exposure. ²² A stock solution of MTS was first prepared in the medium at a concentration of 5 mg/mL. After the treatment, MTS solution (10 μL) was added to each well (final concentration is 0.25 mg/mL) and incubated for 4 h at 37 °C. Using a plate reader, the absorbance was measured at 490 nm for each well. DMSO never exceeded a level of 1.5% exposure. Control wells also received 1.5% DMSO to ensure the limited toxicity of DMSO on the cells at such levels.

Determination of the Binding Ability of β-caryophyllene Oxide with DNA Using UV Spectroscopy Assay

A standard buffer solution containing MgCl₂ (0.50 mM), NaCl (0.15 M), and phosphate buffer (10.00 mM - pH 7.3) was used throughout our experiments.^23,24 β-Caryophyllene oxide was dissolved in the standard buffer at 12 mg/ml stock concertation. Stock solution of calf thymus DNA was prepared at a concentration of 3 mM in the standard buffer solution. Its concentration was determined spectrophotometrically using the molar absorptivity value, λ₂₆₀= 6600 mol⁻¹cm⁻¹. ²⁵ The experiment and its analysis (K calculations) were performed following the previously published work.^24,26 Briefly, 50 μL were taken from β-caryophyllene oxide and diluted to 500 μL using the same standard buffer (1.2 mg/ml final concentration). The UV absorbance was measured to determine the λ_max. The wavelength of 300 nm was chosen to track the change in the absorption with the addition of DNA. The wavelength was chosen to limit the effect of DNA absorption at 260 nm. 2, 4, 6, 8, 10, 12, 14, 15, 20, 22, 24, 26, 28, and 30 μL were sequentially added, to obtain an effective DNA concentration of 2, 4.072, 6.108, 8.144, 10.18, 12.216, 14.252, 16.288, 18.324, 20.36, 22.396, 24.432, 26.468, 28.504, and 30.54 ng/uL, respectively. The absorbance (at 300 nm) was measured after each addition. The DNA was added until no decrease in absorption was observed.

Statistical Analyses

A GraphPad Prism 5 software, from San Diego, CA was used for our statistical analyses. One-way ANOVA followed by Tukey's post-hoc test was employed determining the significance of the effects between multiple groups. A p-values less than 0.05 were considered significant. Nonlinear regression analysis (with variable slope model) was used to calculate the IC50.

Results

Cell Viability

Screening different compounds for their ability to kill HepG2 cells revealed the significant β-caryophyllene oxide cytotoxic activity against HepG2, at a concentration of 50 μg/ml for 48 h ( Fig. 1a ). Its IC₅₀ was also determined and found to equal 45.79 μg/ml when incubated with HepG2 cells for 48 h. The experiment also showed minimal cytotoxicity for the other natural products tested (naringin, naringenin, dehydroacetic acid, and epicatechin). Moreover, β-caryophyllene oxide significantly enhanced doxorubicin's cytotoxic activity. As shown in Fig. 1b , the cell viability significantly dropped from 100% for doxorubicin (1μg/ml) alone to 80% when doxorubicin was co-treated with β-caryophyllene oxide, indicating a 20% increase in doxorubicin's cytotoxicity. Conversely, the cytotoxicity of doxorubicin was not enhanced by its co-treatment with the other tested natural products (naringin, naringenin, dehydroacetic acid, and epicatechin). In fact, some compounds (epicatechin) showed a slight (but significant) protective effect against doxorubicin's cytotoxicity to cancer cells. Moreover, the cytotoxicity of β-caryophyllene oxide was significantly reduced when co-administered with naringin, naringenin, dehydroacetic acid, and epicatechin

Figure 1.

The cytotoxic activity of the different natural compounds. a) The effect of β-caryophyllene oxide, naringin, dehydroacetic acid, epicatechin, naringenin on cell viability. b) the effect of co-administrating doxorubicin and one of the tested natural compounds on the doxorubicin's cytotoxicity. c) The effect of co-administering β-caryophyllene oxide with nargingin, dehydroacetic acid, or epicatechin on the cytotoxicity of β-caryophyllene oxide. d) HepG2 cell survival versus caryophyllene oxide concentration, after 48 h treatment.

Binding of β-caryophyllene Oxide with DNA

Our results showed the ability o f β-caryophyllene oxide to bind with DNA. Figure 2a shows a decrease in the absorbance of β-caryophyllene oxide at 300 nm with the addition of DNA. The change in the absorbance at 300 nm with the addition of DNA was used to calculate the binding constant K according to the following equation:

$${\displaystyle \frac{{\mathrm{A}}_0}{\mathrm{A}-{\mathrm{A}}_0}={\displaystyle \frac{{\text{є}}_G}{{\text{є}}_{\mathrm{H}-\mathrm{G}}-{\text{є}}_G}+{\displaystyle \frac{{\text{є}}_G}{{\text{є}}_{\mathrm{H}-\mathrm{G}}-{\text{є}}_G}{\displaystyle \frac{1}{\mathrm{K}[DNA]}}}}}$$

(1)

Figure 2.

Binding of β-caryophyllene oxide to DNA a) A decrease in β-caryophyllene oxide absorbance, at λ300 with the addition of DNA. b) 1/[DNA] is plotted against Ao/A-Ao allows the calculation of K constant.

A_o and A refer to the absorbance values of the drug, in the absence and the presence of DNA, respectively. є_G and є_H−G describe the absorption coefficients for the drug, in the absence and presence of DNA, respectively. Based on that formula, the binding constant (K) was calculated after plotting 1/[DNA] versus Ao/A-Ao ( Fig. 2b ). K was found to be equal to 10⁴ L.mol⁻¹. ²⁶

Discussion

The importance of natural products in cancer treatment has long been emphasized. However, understanding the mechanisms of action for the different natural products is usually overlooked. Here, we chose several chemicals found in different natural products and investigated their ability to kill cancer cells. Our initial experiments showed a minimal cytotoxicity for naringin, ²⁷ dehydroacetic acid, ²⁸ epicatechin ¹⁶ against HepG2. This was not a surprise as their minimal cytotoxicity was shown in previous work. However, β-caryophyllene oxide showed good cytotoxicity with an IC₅₀ of 45μg/ml. This prompted testing if β-caryophyllene oxide can enhance the cytotoxicity of the other natural chemicals and doxorubicin. Naringin, dehydroacetic acid, epicatechin significantly protected the cells from β-caryophyllene oxide ( Fig 1c ). This was revealed by showing an increase in cell survival in co-treated cells compared to β-caryophyllene oxide alone. The antioxidant activity of naringin, dehydroacetic acid and epicatechin has most likely caused the reduction in the cytotoxicity of β-caryophyllene oxide which emphasizes the significance of ROS in β-caryophyllene oxide cytotoxic activity. Moreover, enhancing the cytotoxicity of doxorubicin would allow administering low doses of doxorubicin, hence, reducing its toxicity.

The good cytotoxicity of β-caryophyllene oxide prompted further investigation into its mechanism of action. Our DNA binding assay showed the ability of β-caryophyllene oxide to bind with DNA at a K binding constant of 10⁴ L.mol⁻¹. We propose that the binding of β-caryophyllene oxide to DNA is a potential mechanism of its cytotoxicity. Mechanistically, the electrophilic epoxide moiety on β-caryophyllene oxide can potentially attack the nucleophilic groups on DNA (ie NH₂ group), which allows forming covalent bonds ( Fig. 3 ). Such covalent bonds would hinder the function and DNA replication, leading to the killing of the fast-growing cancer cells. The binding with DNA would also lead to an increase in ROS production, which would enhance its cytotoxicity. ²⁹ These results support the reports which show good cytotoxicity for β-caryophyllene oxide against several cell lines, including HepG2,^30–32 over those that report limited cytotoxicity.^32,33

Figure 3.

Proposed scheme for the interaction and covalent bond formation between β-caryophyllene oxide and DNA.

The ability of β-Caryophyllene oxide in enhancing the cytotoxicity of doxorubicin has been presented by several researchers.^19–21 For example, Do Sotto showed the ability of β-caryophyllene oxide to inhibit ABC transporters allowing the accumulation of doxorubicin in cancer cells and enhancing its cytotoxicity. ¹⁹ Others report the ability of β-caryophyllene oxide to enhances the anti-tumor activity of an alkylating agent (ie cisplatin) by affecting cell cycle pathways and apoptosis signaling. ²⁰ Here, we highlight the ability of β-caryophyllene oxide to enhance doxorubicin's cytotoxicity in HepG2. We also report the ability of β-Caryophyllene oxide to interact with DNA as a novel mechanism in enhancing doxorubicin's cytotoxicity. Moreover, we present the interaction with DNA as a novel mechanism of cytotoxicity for β-caryophyllene oxide against HepG2.

Moreover, the enhancement of doxorubicin's cytotoxicity with β-caryophyllene oxide suggests a different cytotoxicity mechanism, resulting in additive effects when co-administered. Doxorubicin is well known to noncovalently intercalate with DNA and inhibit topoisomerase I and II.^34,35 This effect is potentially enhanced (rather than competed with) by the proposed covalent binding between β-caryophyllene oxide and DNA.

Despite these advances, further research is needed to determine the type of interaction occurring between β-caryophyllene oxide and DNA. While this may be a limitation to this work, it opens up several future research avenues with the objectives of determining the types of the interaction. Moreover, evaluating the binding ability of different sesquiterpenes to DNA would highlight the importance of certain chemical moieties for the interaction. Determining if sesquiterpenes are sequence specific would also help immerge sesquiterpenes as an important DNA interacting (potentially alkylating) group.

Conclusion

The cytotoxicity of β-caryophyllene oxide against HepG2 cells was shown. Moreover, an enhancement in the cytotoxic activity of doxorubicin when co-treated with β-caryophyllene oxide was also shown. We also attempted to explain the mechanism by which β-caryophyllene oxide can kill cancer cells. We have demonstrated its ability to bind with DNA, which would hinder its activity and replication causing cell death. Future research should focus on comprehensive in vivo investigations to validate its therapeutic potential. Investigating the selectivity and safety profile of β-caryophyllene oxide, particularly its impact on normal cells, is crucial for translational applications.