Combination of 2 Bioactive Compounds for Treatment of Breast Cancer: Triterpenoid Cucurbitacin I and Phenolic CAPE

Breast cancer is the most common cancer in women around the world accounting for almost 25% of all cancer cases. In 2012, nearly 1.7 million new cases and more than 500 000 deaths from cancer were reported worldwide. ¹ Breast cancer is a disease of local origin that may then spread to the lymph nodes and distant tissues and organs. ² Chemotherapy, resection, radiotherapy, and immunotherapy are used to treat breast cancer. ³ Significant advancements have occurred in the chemotherapy and radiotherapy regimens in recent years, although conventional cancer treatments still remain insufficient, particularly in patients diagnosed at later stages of the disease. ⁴ Chemotherapy has not been used as desired due to the drug-related side effects and drug resistance, and researchers have therefore focused on the discovery and development of chemotherapeutic compounds with a high level of efficacy and a low toxicity profile. ⁵

Caffeic acid phenethyl ester (CAPE) has many biological properties including antiviral, anti-inflammatory, antibiotic, antifungal, antioxidant, and immune-modulator activities. It also shows anticancer efficacy by suppressing the growth of some tumor cell types and triggering apoptosis. ^{6 -9}

Despite their bitter taste, the root and fruits of Cucurbitaceae species are commonly used as household remedies in several countries due to their hepato-protective, antipyretic, analgesic, anti-inflammatory, antimicrobial, and antitumor properties. ^10,11 Cucurbitaceae species have been shown to induce apoptosis and inhibit cell proliferation in cancer models. The targets at the molecular level include the signal transducer and activator of transcription (STAT) and the cyclooxygenase-2 pathways. ¹¹ Cucurbitacin I (Cu I, JSI-124) shows potent anticancer activity. It is a selective inhibitor of the transcription-3 signal (STAT3) pathway and transducer of Janus kinase/signal. Due to its antiproliferative activity, Cu I has been commonly used as a bioactive compound in both in vitro and in vivo cancer studies ¹² ; it suppresses the properties of the cancer stem cell-like cells and also increases the response to chemotherapy and radiotherapy. ¹⁰ Activation of STAT3 and nuclear factor- kappa B (NF-κB) has been proved in various cancer types, including breast cancer. ^{13 -17} This led us to evaluate both the individual and the combined antitumor efficacy of 2 bioactive inhibitors of these pathways in MCF-7 and MDA-MB-231 cells.

Breast cancer cells were treated with either Cu I or CAPE in order to compare their antiproliferative activity. The 2 bioactive compounds were found to decrease cell proliferation in both cell lines depending on the dose. In both cell lines, antiproliferative activity was significant at concentrations of ≥20 µM and ≥20 nM after 48 hours. Cell viability after CAPE treatment at ≥20 µM was 79%, 76%, and 55%; after Cu I treatment at ≥20 nM, 78%, 71%, and 51%; and after combined treatment, 70%, 46%, and 35%, respectively, in the MCF-7 cells. In the other cell line, cell viability after CAPE treatment at ≥20 µM was 85%, 80%, and 68%; after Cu I treatment at ≥20 nM, 81%, 66%, and 63%; and after combined treatment, 78%, 63%, and 55%, respectively.

Caffeic acid phenethyl ester and Cu I treatments inhibited cell growth and colonization in breast cancer cell lines. The combined treatment suppressed colonization at ≥20 µM and ≥20 nM concentrations. At concentrations of ≥20 µM and ≥20 nM, marked reductions were noted in the number of colonies both after treatment with individual agents and after combined treatment. Caffeic acid phenethyl ester treatment decreased the colony numbers by 63% at a 20 µM concentration and by 90% at a 40 µM concentration in the MCF-7 cells. In Cu I treatment, these figures were 88% and 91% at 20 and 100 nM concentrations, respectively. The combined treatment resulted in a 90% decrease at a 40 μM + 100 nM concentration and completely prevented colonization at higher concentrations. Caffeic acid phenethyl ester treatment reduced the number of colonies by 65% at 20 µM concentrations and by 90% at 40 µM concentrations in the MDA-MB-231 cells. Colonization was totally inhibited after Cu I treatment at a ≥20 nM concentration and combined treatment at a ≥20 nM + 20 µM concentration.

Photographs of cells were obtained in order to evaluate the wound healing outcomes, and the 2 cell lines exhibited different migration profiles during wound healing. Wound closure was time dependent in nontreated cells and an increasing number of cells migrated to the marked area in both cell lines.

Statistically significant differences were found between the treated and untreated cells (MCF-7, P < 0.031 and MDA-MB-231, P < 0.006). Caffeic acid phenethyl ester inhibited wound healing by the 36th and 48th hours, and this effect was significant at ≥10 µM concentrations in the MCF-7 cells. The wound healing effect was significant at ≥20 nM concentrations in cells treated with Cu I at the 48th hour. The combination treatment resulted in marked differences at the 24th, 36th, and 48th hours at ≥10 µM + ≥4 nM concentrations. Caffeic acid phenethyl ester treatment was effective at the 36th and 48th hours at ≥20 µM concentrations, while Cu I treatment was effective at the 24th and 36th hours at 100 and 500 nM concentrations, and also at the 48th hour at ≥4 nM concentrations in the MDA-MB-231 cells. The CAPE and Cu I combination treatment, on the other hand, was effective at the 24th, 36th and 48th hours at ≥20 µM +20 nM concentrations.

Recent studies have demonstrated the advantage of combination chemotherapy rather than single agent chemotherapy regimens used to treat breast cancer. The combination of chemotherapeutics and natural compounds showing anticancer activities increases treatment efficacy and reduces toxicity. ^18,19 A recent strategy promotes the use of nontoxic phytochemicals with antitumor effects in combination with chemotherapeutic agents, resulting in a reduced toxicity burden on healthy tissue. ²⁰ Combination therapies are advantageous, in that they allow the use of lower doses of each agent by relying on their synergistic inhibitory effects to halt the growth of cancer cells, thereby reducing drug-related toxicity potential by using lower drug doses.

It is well-known that polyphenolic compounds derived from natural substances have antitumor, anti-inflammatory, and antioxidant characteristics, and CAPE is one such phenolic compound; it is an active ingredient of honeybee propolis. ⁷ In the previous studies, CAPE has shown antiproliferative effects and apoptosis induction effects on various cancer cell lines. ^7,9,21,22

We used 2 different cell lines, one that was estrogen receptor negative MDA-MB-231 and one estrogen receptor positive MCF-7. This is the first study that has investigated the effects of CAPE and Cu I combination as a therapeutic approach to breast cancer treatment, and our findings suggest that the combination may be considered as a new effective breast cancer treatment strategy. We suggest that CAPE inhibited NF-κB and Cu I inhibited the STAT3 pathway in breast cancer cells. Constitutive activation of STAT3 and NF-κB signaling pathways plays a key role in the proliferation of breast cancer cells, and so NF-κB and STAT3 inhibition may present an important molecular target for drug development in the treatment of breast cancer.

We hereby demonstrate that CAPE and Cu I inhibited the growth of both estrogen receptor negative and positive breast cancer cells.

Experimental

Chemicals

Cucurbitacin I stock solution was prepared in ethanol to a concentration of 10 mM. Subsequently stock solution was diluted for use to 0.8-500 nM concentrations. Stock solution of CAPE was prepared with ethyl acetate and diluted for use to 5-80 µM concentrations.

Cell Culture

MCF-7 and MDA-MB-231 cells were cultured in an incubator with 5% CO₂ at 37°C. Roswell Park Memorial Institute-1640 (RPMI-1640; Lonza, Verviers, Belgium) medium supplemented with fetal bovine serum 10% (heat inactivated), glutamine 1%, and penicillin/streptomycin 1% was used as a cell medium. The cells were passaged 2-3 times per week and were washed with trypsin-EDTA (0.25%) and phosphate-buffered saline (PBS). The cells were pelleted by centrifugation at 1500 rpm for 5 minutes and resuspended in RPMI-1640 medium to the desired concentration.

MTT Assay

Viability of the cells was evaluated by 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay. ²³ The cells were seeded in 96-well plates with 4 × 10³ cells in each well (Sarstedt, Germany). Cucurbitacin I was added at 0.8-500 nM concentrations and, following 24 hours incubation, CAPE was added at increasing concentrations (5-80 µM). A total of 10 µL MTT stock solution (Applichem Gmbh, Darmstadt, Germany) was prepared in PBS (5 mg/mL). MTT solution was added to each well 48 hours after the incubation with Cu I and CAPE. A total of 0.04 N HCl acid in 2-propanol (100 µL) was added to each well. Medium was removed after 4 hours, and plates were washed. Dissolved formazan crystals were quantified using an enzyme-linked immunosorbent assay plate reader (BioTech, United States). Optical densities were measured at 570 nm wavelength.

Wound Healing (Scratch) Assay

Both breast cancer cell lines were cultured in plates with 3 × 10⁴ cells in each well. They were subjected to a 24 hours standard incubation (5% CO₂, 37°C). A line was drawn with a sterile plastic pipette, then cell residues were removed by using RPMI-1640 medium. The wound-healing rate was evaluated with an inverted light microscope after 0, 12, 24, 36, and 48 hours of incubation. ²⁴

Colony Formation Assay

Six well plates containing 500 cells of the culture were prepared. After 24 hours of incubation, Cu I and CAPE were added to wells every 4 days. Phosphate-buffered saline was used to wash the wells, and crystal violet (0.05%) for staining. Colonies containing more than 50 cells were counted. ²⁵

Statistical Analysis

The ImageJ (1.48) was used to analyze wound healing pictures. Descriptive data were analyzed using the SPSS soft version 22.0 (SPSS Inc., Chicago, IL, United States) and were expressed as mean ± standard deviation. The survival rates between the treatment groups were compared by the Kruskal-Wallis and Mann-Whitney U tests to identify the specific significant differences between groups in the cell proliferation assay. Statistical differences in wound healing were calculated by using a single factor one-way Analysis of Variance ( test. P < 0.05 was considered statistically significant.