Topotecan (TOPO) hydrochloride and carvacrol were purchased from Sigma Aldrich Co. TOPO stock solutions were prepared by dissolving it in distilled water (D.W) and preserved at –20 °C. Dullbecco’s modified eagle’s medium (DMEM), fetal bovine serum, trypsin/EDTA, penicillin G/steptomycin antibiotics, 4’,6-diamidino-2-phenylindole (DAPI) dihydrochloride solution, dimethyl sulfoxide (DMSO), phosphate buffered saline (PBS) and ethanol were obtained from Beijing Solarbio Science and Technology Co., (Shanghai, China). Crystal violet stain (CV) (from: s.d.fine-CHEM Ltd), Coomassie blue R-250 (CB R-250), sodium dodecyl sulfate (SDS), formaldehyde, acetic acid (AA), and methanol were gifted from King Fahd Medical Research Center (KFMRC).

MCF-7 breast, HeLa cervical, SKOV3 ovarian, HCT116 colon, HepG2 liver, and A549 non-small lung cancer cells were procured from the American Type Tissue Culture Collection (Manassas, VA, USA) and were gifted from the Regenerative Medicine unit at KFMRC.

All cell lines were grown as adherent monolayer cells in a (25 cm²) culture flask and the growth medium (DMEM) was supplemented with 10% (v/v) FBS and 1% (v/v) penicillin-streptomycin. The cancer cells were incubated in a 5% CO₂ / 95% humidified atmosphere at 37 °C. The DMEM was removed from the cell culture flask and changed with new medium every 48 h. Cells were collected by trypsinization and passaged every 3–4 days after cells were fed to 90% confluence.

Cytotoxicity was determined using CV staining method, which is considered one of the simplest, quickest and most reliable methods to determine the viability of adherent cells especially for the assessment of the interactions between anticancer agents (Saotome et al. 1989; Sliwka et al. 2016).

Briefly, a 100 µl of culture media containing 5 × 10³ cells was added into each well of a flat-bottomed 96-well plate and incubated for 24 h at 37 °C in a CO₂ incubator in order for cells attachment. After that, cells were treated with 100 µl of the complete medium containing either six concentrations of TOPO-Sol in a range of (1.56–50 μM) or six concentrations of TOPO-Sol in a range (1.56–50 μM) in combination with a fixed concentration of carvacrol (166 μM), and incubated for 24 h at 37 °C in a CO₂ incubator. Then, the culture media was discarded, followed by washing the wells carefully with a 100 µl of PBS. Then 50 µl of 0.1% CV stain was added and incubated in the dark hood for 10 minutes. After the incubation time, CV stain was removed and the wells were washed with tap water using the immersion technique and were left to dry. Finally, 100 µl of 1% SDS was added to each well followed by manually plate agitation for 10 minutes. The absorbance (A) was read at 570 nm using a microplate reader (BioTek, Synergy HT microplate reader, USA). Wells containing negative and positive controls included culture media without cells (blank) and culture media containing cells without treatment (control) respectively. Half maximal inhibitory concentration (IC₅₀) values were determined experimentally for each treatment. Experiments for each sample were done in triplicate.

The percentages of growth inhibition were calculated by the following equation:

$\text{Growth Inhibition}(\%)=\left(1-\frac{(\mathrm{A}\text{of treated cell}-\mathrm{A}\text{of blank})}{(\mathrm{A}\text{of positve control}-\mathrm{A}\text{of blank})}\right)\times 100$

The growth inhibition percentages resulted from the CV assay were inserted into CompuSyn software (Combosyn, Paramus, NJ, USA), in order to determine the combination index values of the carvacrol/TOPO combination therapy in different cell lines based on the combination index theorem of Chou-Talalay (Chou 2010). When CI > 1, the effect is antagonism and when CI < 1, the effect is synergism.

Topotecan cellular accumulation was assessed in cells by using spectrofluorometer according to the method of Lei et al. (2010). Cells were plated in 24-well plates at cell density of 25 × 10³ cells/well in DMEM supplemented medium. Twenty-four hours later, cells were incubated for additional 24 h with different TOPO concentrations, selected previously from the CV staining assay, in the absence or presence of a fixed concentration of carvacrol (166 µM). After 24 h treatment, the cell medium was removed and the wells were washed with 300 µl of ice-cold PBS, then 1 ml of DMSO were added for 10 minutes in order to lyse the cells. Then, the supernatants were collected and centrifuged at 14,000 rpm for 15 minutes to remove cell debris and to obtain cell lysate. The unique intense fluorescence of TOPO (Francis et al. 2015), in the supernatant was measured by a spectrofluorometer (F-2000 Fluorescence spectrophotometer, Hitachi, Japan) at excitation and emission wavelengths of λ ex = 360 nm and λ em = 560 nm, respectively to determine TOPO concentration. To adjust the background fluorescence from cellular components, different concentrations (0.0125, 0.25, 0.5, 1, 2, 4 μM) of TOPO were dissolved in DMSO and added to the untreated cells. Then the fluorescence intensities of supernatants were measured in order to prepare TOPO calibration curve in the presence of untreated cell lysates.

$\text{TOPO cellular accumulation ratio}=\frac{\text{TOPO concentration in cells treated with carvacrol/TOPO combination therapy}}{\text{TOPO concentration in cells treated with only TOPO}}$

To evaluate the morphological changes of the treated MCF-7, HCT116, HeLa, HepG2, A549 and SKOV3 cell lines, cells were plated at a density of 5 × 10³ cells per well into each well of the flat-bottomed 96-well plate and were incubated overnight in a CO₂ incubator at 37 °C. After cells attachment, 100 μl of drug concentrations selected earlier based on the IC₅₀ values of TOPO solutions measured by the CV assay, were added after discarding the old medium and were incubated for 24 h at the same previous conditions in the absence or presence of (166 µM) carvacrol. Finally, cell morphology was evaluated by light microscope (TH4-200, Olympus optical Co-Ltd, Japan) after staining the cells with 0.02% CB R-250, according to the method of Alkhatib et al. (2017).

The DNA fragmentation and nuclear abnormalities of the treated cells undergoing apoptosis were detected by using the DAPI stain. Cells were seeded at a density of 5 × 10³ cells per 100 µl of DMEM into the wells of the 96 well-plates. Then cells were treated with the different TOPO concentrations (IC₅₀) selected according to the results measured by the CV assay, in the absence or presence of (166 µM) carvacrol. Following incubation for 24 h at 37 °C in a CO₂ incubator, cell morphology of the DAPI stained cells was assessed by a fluorescent microscope with blue filter at 437 μm (Leica CRT6000, Germany) according to the method of Alkhatib et al. (2018).

Statistical analysis was implemented using MegaStat Excel (version 10.3, Butler University, Indianapolis, IN). All data were expressed as mean ± standard deviation (SD) for triplicate measurements. Independent t-test was used for the comparison between two independent groups and one-way analysis of variance (ANOVA) followed by Tukey’s test for post hoc analyses were used for multiple comparisons. Statistical differences were considered significant, highly significant and very highly significant when 0.01 ≤ P < 0.05, 0.001 ≤ P < 0.01 and P < 0.001, respectively.

The cytotoxicity of TOPO in the absence or the presence of (166 µM) carvacrol in HeLa, HCT116, MCF-7, HepG2, A549, and SKOV3 cancer cells were expressed as the percentages of growth-inhibiting rates (Fig. 1). The (166 µM) carvacrol concentration, which is smaller than the resulted carvacrol IC₅₀ concentration in each cell line, was chosen and unified for all cancer cells. The IC₅₀s of the different treatment protocols, and their combination indexes were also evaluated as presented in Table 1. Six TOPO concentrations ranged from 1.56 to 50 µM were added to cells, either alone or combined with (166 µM) carvacrol, then the cytotoxicity was estimated after 24 h by CV assay. The statistical comparisons between different treatment protocols at the same TOPO concentration were performed by the independent t-test.

Figure 1. 

The curves of the growth inhibition percentages after 24 h treatment with different concentrations of TOPO in the absence or presence of a fixed concentration of carvacrol (166 µM)) in (A) HeLa, (B) HCT116, (C) MCF-7, (D) HepG2, (E) SKOV3 and (F) A549 cell lines. Data were expressed as mean ± SD (error bars), n = 3. The significant differences between TOPO and (TOPO + carvacrol (166 µM)) combination at each time assessed by mesuring the P-values using the independent t-test, were classified to * P < 0.05, ** P < 0.01 and *** P < 0.001.

Simultaneous addition of carvacrol to TOPO in cancer cells for 24 h was found to sensitize or inhibit TOPO growth inhibition percentage, depending on the type of the treated cancer cells. In HeLa and HCT116 cells, the growth inhibitory curves of TOPO combined with (166 µM) carvacrol were significantly increased relative to the curves of TOPO given alone at

TOPO concentrations of 25 and 50 µM (P < 0.001) in HeLa cells and at TOPO concentrations of 6.25, 12.5 and 25 µM (P < 0.01) in HCT116 cells. However, the growth inhibitory curves of the combination therapy were found to be decreased in comparison with TOPO alone in MCF-7, HepG2, A549, and SKOV3 cancer cells.

In terms of IC₅₀ as illustrated in Table 1, HeLa and HCT116 cells treated with TOPO and (166 µM) carvacrol combination, were having a highly significant smaller IC₅₀ than TOPO solution at P- value < 0.0001, which means that carvacrol addition to TOPO may enhanced its cytotoxic effect. The IC₅₀ values were reduced by factors of 7.7 and 5.7 relative to TOPO single treatment in HeLa and HCT116 cells, respectively. Moreover, the combination indexes of cells treated with the combination treatment were less than one in both cell lines, indicating a synergistic effect between TOPO and carvacrol.

On the other hand, the addition of carvacrol to TOPO has significantly increased the IC₅₀ values relative to TOPO single treatment in MCF-7 (P < 0.0001), HepG2 (P < 0.05), A549 (P < 0.001), and SKOV3 (P < 0.01). In other words, carvacrol addition to TOPO may reduced its cytotoxic effect as understood from their combination index values which are all larger than one, indicating antagonism.

The different cell lines were treated with two concentrations of TOPO (2 and 5 µM) in the absence or presence of (166 µM) carvacrol. TOPO intracellular uptake concentrations in HeLa, HCT116, MCF-7, HepG2, SKOV3, and A549 cancer cells and their accumulation ratios after treatment with TOPO alone or in combination with carvacrol, were illustrated in Table 2 and Fig. 2, respectively. In HeLa and HCT116 cells, carvacrol co-administration with TOPO has increased its TOPO intracellular concentration especially when given with the higher (5 µM) TOPO concentration. The accumulation ratio of TOPO was 1.12 when both cell lines were treated with (5 µM) TOPO and (166 µM) carvacrol combination, which means that carvacrol may enhanced TOPO cellular uptake.

Figure 2. 

The effect of the 24 h treatment with (TOPO (2 or 5 µM) + carvacrol (166 µM)) combination on TOPO cellular uptake in (A) HeLa, (B) HCT116, (C) MCF-7, (D) HepG2, (E) SKOV3 and (F) A549 cell lines. Data were expressed as mean ± SD (error bars), n = 3. The significant differences between the different (TOPO + carvacrol (166 µM)) combinations at each time assessed by mesuring the P-values using the independent t-test, were classified to * P < 0.05.

In contrast to the effect of carvacrol on TOPO cellular uptake in HeLa and HCT116 cells, carvacrol caused a slight decrease in TOPO intracellular concentration when carvacrol was added to 5 µM TOPO in MCF-7, HepG2 and A549 cancer cells (Table 2). The decrease in TOPO intracellular concentration when (166 µM) carvacrol was added to (5 µM) TOPO, was more obvious relative to its effect on the lower (2 µM) TOPO concentration. TOPO accumulation ratios were 0.69, 0.75 and 0.69 when MCF-7, HepG2 and A549 cancer cells treated with (5 µM) TOPO and (166 µM) carvacrol combination, respectively. Accordingly, carvacrol may reduce TOPO cellular uptake.

Cell morphologies were assessed under light microscope for HeLa, HCT116, MCF-7, HepG2, A549, and SKOV3 cancer cells. As displayed in Figs 3 and 4, the images of control (untreated) HeLa, HCT116, MCF-7, HepG2, A549, and SKOV3 cancer cells, revealed the whole cells with no evidence of segmentation or fragmentation, but when cells subjected to TOPO alone or in combination with (166 µM) carvacrol, morphological changes were observed.

Figure 3. 

Light microscopy images (Scale bar: 20 µm) of (A) HeLa and (B) HCT116 cell lines treated for 24 h with TOPO IC₅₀ in the absence or presence of carvacrol (166 µM). Images were magnified at 20×. The red, green, orange and black arrows represented cell enlargement with cytoplasm shrinkage, membrane blebbing, apoptotic bodies and intercellular space increase, respectively. Images were taken from at least three independent experiments with similar conditions.

In all cell lines (Figs 3, 4), the images of cells treated with TOPO showed a clear signs of nuclei shape changing and apoptotic characteristics such as cell enlargement with cytoplasm shrinkage, membrane blebbing (outward pulge of cytoplasmic membrane), chromatin condensation (chromatin margination without nuclear condensation), intercellular space increase and cell structure loss. Furthermore, in HeLa and HCT116 cells (Fig. 3A, B), the cell number population was decreased in TOPO treated cells relative to control, and that decrease in addition to the other apoptotic characteristics, were more dramatic in cells treated with TOPO and (166 µM) carvacrol combination therapy. In contrast, the addition of (166 µM) carvacrol to TOPO in MCF-7, HepG2, A549, and SKOV3 cancer cells (Fig. 4A–D), caused no change or a slight increase in cell population in comparison to cells treated with only TOPO.

Figure 4. 

Light microscopy images (Scale bar: 20 µm) of (A) MCF-7, (B) HepG2, (C) SKOV3 and (D) A549 cell lines treated for 24 h with TOPO IC₅₀ in the absence or presence of carvacrol (166 µM). Images were magnified at 20×. The red, green, orange and black arrows represented cell enlargement with cytoplasm shrinkage, membrane blebbing, apoptotic bodies and intercellular space increase, respectively. Images were taken from at least three independent experiments with similar conditions.

Fluorescent nuclear staining with the cell permeable nucleic acid dye (DAPI) was used to assess alterations in nuclear morphology after treatment of HeLa, HCT116, MCF-7, HepG2, A549, and SKOV3 cancer cells with the IC₅₀ concentrations of TOPO in absence or presence of (166 µM) carvacrol and incubated for 24 h (Figs 5, 6). Although the nuclei of the healthy control cells of all the different cell lines were large and displayed smooth, uniform and diffused staining under the fluorescent microscope, the nuclei of treated cells showed clear changes of nuclear morphology. All the different cell lines when treated with their IC₅₀ TOPO concentrations, which were selected based on the results of the growth inhibition curves illustrated in Fig. 1, clearly represented an early induction of apoptosis.

Figure 5. 

Fluorescent microscopy images (Scale bar: 20 µm) of A) HeLa and B) HCT116 cell lines treated for 24 h with TOPO IC₅₀ in the absence or presence of carvacrol (166 µM). Images were magnified at 20×. The red, and white arrows represented nuclear enlargement or irregular shape, and intercellular space increase, respectively. Images were taken from at least three independent experiments with similar conditions.

In HeLa and HCT116 cells (Fig. 5A, B), obvious nucleus enlargement and decrease in cell population were seen after TOPO treatment, and these apoptotic characteristics were more dramatic when carvacrol was added to TOPO. Also, in MCF-7, HepG2, A549, and SKOV3 cancer cells (Fig. 6A–D), nuclear abnormalities and cell population decrease were observed, but when cells were treated with the TOPO and (166 µM) carvacrol combination, the previous apoptotic features were diminished relative to TOPO lone treatment.

Figure 6. 

Fluorescent microscopy images (Scale bar: 20 µm) of (A) MCF-7, (B) HepG2, (C) SKOV3 and (D) A549 cell lines cell lines treated for 24 h with TOPO IC₅₀ in the absence or presence of carvacrol (166 µM). Images were magnified at 20×. The red, and white arrows represented nuclear enlargement or irregular shape, and intercellular space increase, respectively. Images were taken from at least three independent experiments with similar conditions.