Gingerol Synergizes the Cytotoxic Effects of Doxorubicin against Liver Cancer Cells and Protects from Its Vascular Toxicity

Hydroxyphenylalkanes and diarylheptanoids possess potential therapeutic value in different pathophysiological conditions, such as malignancy. In the current study, naturally isolated hydroxyphenylalkane and diarylheptanoid compounds were investigated for potential chemo-modulatory effects in addition to potential vascular protective roles with doxorubicin. Diarylheptanoids showed stronger antioxidant effects, in comparison to hydroxyphenylalkanes, as demonstrated by DPPH assay and amelioration of CCl4-induced disturbed intracellular GSH/GSSG balance. Shogaol and 4′-methoxygingerol showed considerable cytotoxic effects against HCT116, HeLa, HepG2 and MCF7 cells, with IC50 values ranging from 3.1 to 19.4 µM. Gingerol significantly enhanced the cytotoxic profile of doxorubicin against HepG2 and Huh7, cells decreasing its IC50s by 10- and 4-fold, respectively. Cell cycle distribution was studied using DNA cytometry. Doxorubicin alone induced cell accumulation at S-phase and G2/M-phase, while in combination with gingerol it significantly induced cell cycle arrest at the G2/M-phase. Additionally, the vascular protective effect of gingerol against doxorubicin (10 µM) was examined on isolated aortic rings. Co-incubation with 6-gingerol (30 µM) completely blocked the exaggerated vasoconstriction and impaired vascular relaxation induced by doxorubicin. In conclusion, despite its relatively weak antioxidant properties, gingerol protected from DOX-induced vascular damage, apparently not through a ROS scavenging mechanism. Besides, gingerol synergized the cytotoxic effects of DOX against liver cancer cells without influencing the cellular pharmacokinetics.


Introduction
Grain of Paradise (Aframomum melegueta K. Schum, Zingiberaceae) is the only spice native to Africa and considered as an African panacea [1]. Seeds of A. melegueta were used, as a folk remedy, for the treatment of diarrhoea, and painful inflammatory conditions and in the control of postpartum haemorrhages [2]. Anti-ulcer, cytoprotective, antimicrobial, anti-nociceptive and aphrodisiac effects of the aqueous seed extract are also reported [3,4]. Phytochemical investigations of the plant seeds revealed the presence of paradol-and gingerol-like compounds, in addition to diarylheptanoids with hepatoprotective and estrogenic effects [5,6].
6-Gingerol is a major hydroxyphenylalkane isolated from A. melegueta and present in several plants belonging to the family Zingiberaceae, such as ginger and cardamom. The formerly mentioned plants are widely used in the Middle Eastern and Asian cuisine as a spice and everyday beverage. 6-Gingerol is reported to display several biochemical and pharmacological activities, such as cancer chemopreventive, anti-mutagenic, anti-apoptotic [7], anti-oxidant, anti-inflammatory [8], cardio-and hepatoprotective effects [5,9]. Gingerol is also known to inhibit the enzymes nitric oxide synthase and cyclo-oxygenase [10] and to suppress the expression of tumor necrosis factor alpha (TNF-α) [11]. 6-Paradol, another major constituent of A. melegueta, is closely related in structure to gingerol with one hydroxyl less in the alkyl chain. It is reported to possess chemopreventive, antioxidant and anti-inflammatory effects [12]. Paradol and its derivatives induce apoptosis through a caspase-3-dependent mechanism [13]. In spite of its structure similarity to gingerol, paradol's biological activity is less explored than that of gingerol.
Diarylheptanoids are chemically characterized by the presence of an aryl-C7-aryl moiety. Diarylheptanoids from Pinus flexilis (E. James) possess protein kinase C inhibitory effects [14]. In addition, a cytotoxic diarylheptanoid was isolated from the roots of Juglans mandshurica (Maxim.) [15]. Diarylheptanoids with a carbonyl group at C-3, isolated from bark of black alder are also reported to inhibit the growth of resistant lung carcinoma. The active compounds were found to increase doxorubicin accumulation in cancer cells through modulation of P-gp activity [16].
The burden of neoplasia is increasing globally, with several millions deaths per year. Liver malignancies are the second most prevalent type of solid tumor, with an annual mortality of half a million among males and a similar number among females [17]. Doxorubicin (DOX) is a cytotoxic anthracycline used successfully for the treatment of several malignancies, such as liver cancer [18][19][20]. A major limitation for DOX treatment and a major cause of course treatment non-compliance is its intolerable cardiovascular side effects [21,22]. Several antioxidants were reported to have protective effect against doxorubicin-induced cardiovascular toxicity [9,23]. However, negative influence of free radical scavenging state might ameliorate the primary DOX anticancer properties [24][25][26]. In our previous work, resveratrol and didox (powerful antioxidants) marginally potentiated the effect of DOX against liver cancer cells and protected from its cardiotoxicity [27,28]. Apart from its toxicity, the efficacy of DOX is greatly affected by overexpression of ATP-dependent efflux pump P-glycoprotein (P-gp) [29]. It was reported previously that hydroxyphenylalkanes and diarylheptanoids are potential P-gp efflux pump inhibitors and hence might potentiate the activity of several P-gp substrates such as DOX [30]. In the current work, we isolated several naturally occurring hydroxyphenylalkanes and diarylheptanoids from A. melegueta K. Schum (Zingiberaceae). After rational preliminary biological screening of the isolated compounds, 6-gingerol was selected to protect from doxorubicin-induced vascular toxicity besides potentiating its anticancer properties against liver cancer cells.

Figure 2.
Antioxidant activity of isolated hydroxyphenylalkanes and diarylheptanoids was tested using a cell free system and with HepG2 cells. Antioxidant capacities of the test compounds were tested using the DPPH free radical scavenging assay; scavenging efficiency (A) and EC50s (B) were calculated over concentration range of 0.5 to 50 µM. GSH reductase enzyme activity (C) and GSH concentration (D) were determined in CCl4 challenged HepG2 cells. Data is presented as mean ± SD; n = 3. *: significantly different from CCl4 treated group.

Cytotoxicity Assessment of Hydroxyphenylalkanes and Diarylheptanoids
The SRB-U assay was used to assess the cytotoxicity of nine naturally occurring hydroxyphenylalkanes and diarylheptanoids against four different tumor cell lines over a concentration range of 0.01-100 µM. The tested compounds showed variable cytotoxicities against the cell lines under investigation (HCT-116, HepG2, MCF-7 and HeLa cell lines). However, HepG2 cells were relatively more resistant, while HCT-116 was the most sensitive cell line.
The HepG2 liver cancer cell line possessed the highest resistance to hydroxyphenylalkanes and diarylheptanoids amongst other tumor cell types. In HepG2 cells, only dihydro-6-paradol (6), 6-shogoal (4) and 4′-methoxy-6-gingerol (5) showed relatively potent cytotoxicity with IC50s of 13.5 µM, 18.7 µM and 19.4 µM, respectively. However, dihydro-6-paradol (6) suffered from up to 50.2% resistant fraction. Gingerol (2) showed moderate cytotoxicity against HepG2 cells with an IC50 of 71.9 µM. The rest of tested compounds did not show any notable cytotoxicity, with IC50s higher than 100 µM (Table 1).  Figure 2. Antioxidant activity of isolated hydroxyphenylalkanes and diarylheptanoids was tested using a cell free system and with HepG 2 cells. Antioxidant capacities of the test compounds were tested using the DPPH free radical scavenging assay; scavenging efficiency (A) and EC 50 s (B) were calculated over concentration range of 0.5 to 50 µM. GSH reductase enzyme activity (C) and GSH concentration (D) were determined in CCl 4 challenged HepG 2 cells. Data is presented as mean˘SD; n = 3. *: significantly different from CCl 4 treated group.

Cytotoxicity Assessment of Hydroxyphenylalkanes and Diarylheptanoids
The SRB-U assay was used to assess the cytotoxicity of nine naturally occurring hydroxyphenylalkanes and diarylheptanoids against four different tumor cell lines over a concentration range of 0.01-100 µM. The tested compounds showed variable cytotoxicities against the cell lines under investigation (HCT-116, HepG2, MCF-7 and HeLa cell lines). However, HepG2 cells were relatively more resistant, while HCT-116 was the most sensitive cell line.
In HCT-116 colorectal cancer cells, all tested compounds showed considerable cytotoxicities, with IC 50 s ranging from 1.5 µM to 12.6 µM. In addition, resistant fraction (R)-values for all hydroxyphenylalkanes and diarylheptanoids under investigation (except gingerol) were less than 15%. It is worth mentioning that, despite the relatively low IC 50 of gingerol (1.5 µM), it suffered from considerably high resistance (60.6%) within HCT-116 cells ( Table 1).

Chemomodulatory Effect of Gingerol on Doxorubicin within Liver Cancer Cells
DOX exerts its cell killing effects via the generation of intracellular free radicals. Based on its moderate antioxidant activity (Figure 2), and moderate cytotoxic effects (Table 1), gingerol would be a good candidate to improve the activity of doxorubicin against liver cancer cells. Huh-7 cells were far more sensitive to DOX, compared to HepG 2 , with IC 50 of 4.6˘0.9 nM and 680˘60 nM, respectively. Gingerol exerts weaker cytotoxic effects against Huh-7 cells compared to HepG 2 cells with IC 50 's of 103.1˘3.0 µM and 71.9˘2.8 µM, respectively ( Figure 4A,B and Table 2). However, 6-gingerol synergistically improved the cytotoxic profile of DOX against both of these liver cancer cell lines. 6-Gingerol significantly decreased the IC 50 's of DOX form 680˘60 nM and 4.6˘0.9 nM to 67.4˘9.1 nM and 1.2˘0.13 nM in HepG 2 and Huh-7 cells, respectively. The combination indices for equitoxic combination of gingerol and DOX within HepG 2 and Huh-7 cells were 0.19 and 0.29, respectively ( Table 2). It is worth mentioning that, gingerol combination with DOX did not influence the resistance fraction to DOX within either HepG 2 or Huh-7 cells.

Chemomodulatory Effect of Gingerol on Doxorubicin within Liver Cancer Cells
DOX exerts its cell killing effects via the generation of intracellular free radicals. Based on its moderate antioxidant activity (Figure 2), and moderate cytotoxic effects (Table 1), gingerol would be a good candidate to improve the activity of doxorubicin against liver cancer cells. Huh-7 cells were far more sensitive to DOX, compared to HepG2, with IC50 of 4.6 ± 0.9 nM and 680 ± 60 nM, respectively. Gingerol exerts weaker cytotoxic effects against Huh-7 cells compared to HepG2 cells with IC50's of 103.1 ± 3.0 µM and 71.9 ± 2.8 µM, respectively ( Figure 4A,B and Table 2). However, 6-gingerol synergistically improved the cytotoxic profile of DOX against both of these liver cancer cell lines. 6-Gingerol significantly decreased the IC50's of DOX form 680 ± 60 nM and 4.6 ± 0.9 nM to 67.4 ± 9.1 nM and 1.2 ± 0.13 nM in HepG2 and Huh-7 cells, respectively. The combination indices for equitoxic combination of gingerol and DOX within HepG2 and Huh-7 cells were 0.19 and 0.29, respectively ( Table 2). It is worth mentioning that, gingerol combination with DOX did not influence the resistance fraction to DOX within either HepG2 or Huh-7 cells.

Cell Cycle Distribution Analysis of Liver Cancer Cells
Cell cycle distribution determined using DNA flow cytometry was used to investigate the nature of the interaction between DOX and gingerol. In HepG 2 cells, gingerol significantly decreased the cell population is S-phase from 15.5%˘0.7% to 12.7%˘1.3%. DOX treatment alone significantly increased the cell population in G 2 /M phase (from 7.1%˘0.7% to 14.6%˘3.1%) with a reciprocal decrease in the S-phase cell population (from 15.5%˘0.7% to 12.5%˘1.2%) ( Figure 5A-C,E). The combination of DOX with gingerol induced significant cell cycle arrest at the G 2 /M phase (17.4%˘2.4%) compared to the control group (7.1%˘0.7%). The induced G 2 /M cell phase arrest was accompanied by reciprocal decrease in the non-proliferating cell population (G 0 /G 1 -phase) from 77.4%˘1.0% to 68.9%˘3.0% ( Figure 5A,D,E).

Cell Cycle Distribution Analysis of Liver Cancer Cells
Cell cycle distribution determined using DNA flow cytometry was used to investigate the nature of the interaction between DOX and gingerol. In HepG2 cells, gingerol significantly decreased the cell population is S-phase from 15.5% ± 0.7% to 12.7% ± 1.3%. DOX treatment alone significantly increased the cell population in G2/M phase (from 7.1% ± 0.7% to 14.6% ± 3.1%) with a reciprocal decrease in the S-phase cell population (from 15.5% ± 0.7% to 12.5% ± 1.2%) ( Figure 5A-C,E). The combination of DOX with gingerol induced significant cell cycle arrest at the G2/M phase (17.4% ± 2.4%) compared to the control group (7.1% ± 0.7%). The induced G2/M cell phase arrest was accompanied by reciprocal decrease in the non-proliferating cell population (G0/G1-phase) from 77.4% ± 1.0% to 68.9% ± 3.0% ( Figure 5A,D,E). Finally, exposure of HepG2 cells to DOX or gingerol alone for 24 h resulted in significant cell death observed by elevated pre-G cell population form 1.1%˘0.2% to 4.2%˘1.0% and 3.3%˘0.2%, respectively. Gingerol combination with DOX did not inhibit DOX-induced cell kill, where no significant change in pre-G cell population was observed after combination treatment compared to cells treated with DOX alone ( Figure 5F).

Gingerol Protects from DOX Induced Vascular Toxicity
The current study showed that aortae isolated from normal animals exposed to doxorubicin (10 µM, 1 h) exhibited exaggerated vasoconstriction in response to PE (10 −9 to 10 −5 M), compared to unexposed aortae. This enhancement of vasoconstriction was statistically significant (p < 0.05) at PE concentrations of 3 × 10 −5 and 10 −5 M ( Figure 7B). Thirty minutes of incubation with 6-gingerol  Figure 6. Effect of GNL on the cell cycle distribution of Huh-7 cells. The cells were exposed to GNL (B); DOX (C); or combination of GNL and DOX (D) for 24 h and compared to control cells (A); Cell cycle distribution was determined using DNA cytometry analysis and different cell phases were plotted (E) as percentage of total events; Sub-G cell population was taken as representative of late apoptosis/necrosis and was plotted as percent of total events (F). Data is presented as mean˘SD; n = 3. *: significantly different from control group; **: significantly different from DOX group.

Gingerol Protects from DOX Induced Vascular Toxicity
The current study showed that aortae isolated from normal animals exposed to doxorubicin (10 µM, 1 h) exhibited exaggerated vasoconstriction in response to PE (10´9 to 10´5 M), compared to unexposed aortae. This enhancement of vasoconstriction was statistically significant (p < 0.05) at PE concentrations of 3ˆ10´5 and 10´5 M ( Figure 7B). Thirty minutes of incubation with 6-gingerol (0.3-30 µM) alleviated this DOX-induced exaggerated vasoconstriction of aortae in a concentration-dependent manner. The inhibition of PE (10´5 M)-induced contraction was significant at both tested concentrations of 6-gingerol (10 and 30 µM) with PE concentrations of 3ˆ10´5 .5 and 10´5 M. The highest concentration of 6-gingerol (30 µM) normalized the exaggerated contraction response to PE back to normal control value ( Figure 7B).  . A Data is presented as mean ± SD; n = 6-8. *: significantly different from control group; #: significantly different from DOX treated group.
Aortae exposed to DOX showed impaired vasodilation compared to unexposed aortae and this impaired relaxation was significant at ACh concentrations of 10 −7 to 10 −5 M. This impaired relaxation was alleviated by incubation with 6-gingerol in a concentration dependent manner and this alleviation was significant at the 30 µM concentration of 6-gingerol tested with PE concentrations of 3 × 10 −6.5 and 10 −6 M ( Figure 7B).

Discussion
Natural products are eternal sources of active compounds. The therapeutic use of natural products has escalated from folk use of the whole plant or unprocessed natural entities [36] to the use of standardized extracts [37], followed by the isolation of specified compounds with defined molecular structures [38]. Grouping compounds of similar molecular structure sharing close pharmacological activity could shed light on structure-activity relationships and stimulate chemists to initiate the lead optimization process [39,40]. Herein, we isolated several structurally related hydroxyphenylalkane and diarylheptanoid compounds and assessed their potential cytotoxic and chemomodulatory effects, in addition to confirming their documented antioxidant activity.
All hydroxyphenylalkanes and diarylheptanoids under investigation showed considerable antioxidant activity in cell free systems and within HepG2 cells. However, diarylheptanoids could be considered more potent than hydroxyphenylalkanes in free radical scavenging capacity, as well as in Aortae exposed to DOX showed impaired vasodilation compared to unexposed aortae and this impaired relaxation was significant at ACh concentrations of 10´7 to 10´5 M. This impaired relaxation was alleviated by incubation with 6-gingerol in a concentration dependent manner and this alleviation was significant at the 30 µM concentration of 6-gingerol tested with PE concentrations of 3ˆ10´6 .5 and 10´6 M ( Figure 7B).

Discussion
Natural products are eternal sources of active compounds. The therapeutic use of natural products has escalated from folk use of the whole plant or unprocessed natural entities [36] to the use of standardized extracts [37], followed by the isolation of specified compounds with defined molecular structures [38]. Grouping compounds of similar molecular structure sharing close pharmacological activity could shed light on structure-activity relationships and stimulate chemists to initiate the lead optimization process [39,40]. Herein, we isolated several structurally related hydroxyphenylalkane and diarylheptanoid compounds and assessed their potential cytotoxic and chemomodulatory effects, in addition to confirming their documented antioxidant activity.
All hydroxyphenylalkanes and diarylheptanoids under investigation showed considerable antioxidant activity in cell free systems and within HepG 2 cells. However, diarylheptanoids could be considered more potent than hydroxyphenylalkanes in free radical scavenging capacity, as well as in restoring cellular GSH/GSSG balance. This might be attributed to the occurrence of an extra aromatic system in diarylheptanoids [41,42]. Some compounds such as DIACHEP (7) and dihydrogingerenone C (8) possessed free radical scavenging activity but failed to restore cellular GSH balance. This might be attributed to their slow cellular internalization [43]. Gingerol possessed moderate to weak antioxidant capacity compared to the rest of hydroxyphenylalkanes and diarylheptanoids. Our previous study showed that the vascular protective effects of gingerol might be attributed to direct vasodilatation and nitric oxide generation rather than free radical scavenging mechanism [44]. Besides, gingerol is reported as cardioprotective agent; and particularly against DOX-induced cardiac damage [9,45].
Hydroxyphenylalkanes and diarylheptanoids showed moderately potent cytotoxicity in the current work, with IC 50 s above 1 µM in all tested cell lines. However, these compounds showed preferable cytotoxicity against HCT-116 colorectal cancer cells. This might be partly attributed to the P-gp inhibitory effect of this group of compounds which could have induced excessive intracellular accumulation and auto-enhancement effects [46,47]. Although, P-gp is expressed in almost all gastrointestinal-related tumors [48,49], similar efficacies were not found against HepG 2 liver cancer cells. This might be attributed to the mutated form of P-gp protein expressed in liver cancer [50].
The ability of hydroxyphenylalkanes and diarylheptanoids to interrupt the function of P-gp was tested functionally and at the sub-molecular level. Most of compounds under investigation improved the cellular pharmacokinetics of P-gp probe, while only 6-shogoal and 6-gingerol specifically inhibited the P-gp ATPase subunit. Other hydroxyphenylalkanes and diarylheptanoids are expected to inhibit the P-gp efflux pump via non-specific competitive binding or mixed ATPase inhibition/competitive binding. It would be better in terms of structure-activity relationship and lead optimization to design specific P-gp ATP-ase inhibitors rather than non-specific competitive binding inhibitors [30]. Interestingly, gingerol did not enhance the cellular entrapment of DOX within CaCo-2 cells. Besides the high expression of P-gp within CaCo-2 cells, it might be also explained by the abundance of other types of efflux proteins on the cell membrane of colorectal cancer cells such as MRP1, MRP2 or others [47].
Antioxidants might protect from the side effects and toxic manifestations of a wide variety of anticancer drugs such as doxorubicin [51,52]. However, substantial worries about the negative effect of these agents on the primary anticancer properties of chemotherapies cannot be discounted [26]. Herein and amongst this group of compounds, gingerol was selected for further investigation in the context of influencing the cytotoxicity of doxorubicin against liver cancer cells besides its protective effects against doxorubicin-induced vascular toxicity. Gingerol significantly synergized the cytotoxic effects of doxorubicin against two different liver cancer cells. In previous studies from our group different antioxidants (natural or synthetic) marginally enhanced the cytotoxic profile of doxorubicin, producing only additive drug interaction [27,28]. This might be supported by the weak antioxidant activity of gingerol. However, it cannot be attributed to enhanced cellular internalization of doxorubicin or cellular pharmacokinetic interaction. HepG2 cells also express different types of efflux pump proteins such as P-gp and BCRP [53] that might compensate for the specific P-gp ATPase inhibition activity of gingerol.
Pharmacodynamic interactions between doxorubicin and gingerol were studied using cell cycle distribution analysis. In Huh-7 cells, gingerol significantly induced cell accumulation in the S-phase as well as the G 2 /M-phase. Doxorubicin preferably intercalates with cellular DNA while cells are in either S-phase or G 2 /M-phase inducing cell cycle arrest at G 2 /M-phase, also called mitotic crises/catastrophe [54]. It was reported that agents inducing S-phase accumulation sensitize tumor cells to the killing effect of doxorubicin [55]. In contrast to Huh-7, HepG 2 did not respond to gingerol treatment by S-phase accumulation; gingerol exerted clear antiproliferative effect accumulating cells at the G 0 /G 1 -phase. This explains the relatively weaker combined effect of gingerol and doxorubicin against HepG 2 cells relative to Huh-7 cells. Luckily; gingerol did not hinder the activity of doxorubicin against HepG 2 cells [56,57]. In other words, the synergism of gingerol with doxorubicin (CI-value = 0.19) against Huh-7 cells could be attributed to gingerol-induced cell cycle synchronization in the S-phase resulting in excessive sensitivity to doxorubicin. However, the weaker synergism (CI-value = 0.29) between gingerol and doxorubicin in HepG 2 cells could be attributed to combined but independent antiproliferative and cytotoxic effects of gingerol and doxorubicin, respectively. Further molecular investigations to completely reveal the underlying possible pharmacodynamic interaction mechanisms between gingerol and doxorubicin in liver cancer cells are recommended.
Additionally, gingerol significantly protected against doxorubicin-induced vascular damage, in terms of restoring normal vascular contraction and relaxation. Our previous studies proved the protective effects of gingerol from doxorubicin induced cardiac damage at cardiomyocyte level [9]. Besides, we previously showed protective effects for gingerol against vascular complications of metabolic syndrome that was attributed to gingerol mediated vasodilatation [44]. In continuation of this research line, we presented experimental evidence for the functional protection of gingerol against doxorubicin induced vascular damage without ameliorating its inherent cytotoxicity against liver cancer cells.

Plant Material
Seeds of A. melegueta were purchased from the Harraz herbal store (Cairo, Egypt), and were identified by Assistant Prof. Dr. Sherif El-Khanagry, Agriculture Museum, El-Dokki, Cairo, Egypt. A voucher specimen has been kept in the herbarium of the Department of Pharmacognosy, Faculty of Pharmacy, Cairo University.

Extraction and Isolation of Compounds from A. melegueta
Seeds of A. melegueta (2.5 kg) were pulverized and extracted with MeOH (1 L) by cold maceration for three successive days. The pooled MeOH extracts were evaporated under vacuum to give a brown residue (130 g). The MeOH extract was suspended in water (500 mL) and fractionated using CHCl 3 (1 Lˆ3) and the pooled fractions were evaporated under vacuum to yield chloroform fraction (90 g). The CHCl 3 fraction was purified on silica gel column (70 cmˆ5 cm) and eluted gradiently with hexane-EtOAc (5%-80%). The obtained fractions were pooled into ten sub-fractions 1-10.

Determining the Antioxidant Activity of Test Compounds Using Cell-Free System (DPPH Assay)
The antioxidant activity of the test compounds was evaluated using the DPPH radical scavenging method. Serial concentrations (0.5-50 µM) of test compounds were prepared with 0.4 mg/mL solution of DPPH in pure ethanol and left in the dark for 30 min. Absorbance at 520 nm was then measured and average free radical scavenging activity for each compound was calculated. Besides, EC 50 were calculated from linear best fit regression analysis.

Cell Culture
Six different human solid tumor cell lines were used; colorectal cancer cell lines (HCT-116 and CaCo-2), cervical cancer cell line (HeLa), hepatocellular cancer cell lines (HepG2 and Huh-7), and breast cancer cell line (MCF-7). All Cell lines were obtained from VACSERA (Giza, Egypt). Cell lines were maintained in RPMI-1640 or DMEM media containing 100 U/mL penicillin; 100 µg/mL streptomycin, and supplemented with 10% heat-inactivated fetal bovine serum (FBS). Cells were propagated in a humidified cell culture incubator with 5% (v/v) CO 2 at 37˝C.

Determining the Antioxidant Activity of Test Compounds within HepG2 Cells
To assess the potential free radical scavenging capacity of test compounds within the intracellular compartment, HepG 2 cells (10 5 cells) were challenged with CCl 4 (40 mM) alone or with potentially active hydroxyphenylalkanes and diarylheptanoid compounds (5 µM) for 4 h. Media was collected and level of reduced glutathione (GSH) as well as the activity of glutathione reductase and peroxidase enzymes were measured as previously described [58][59][60].

Cytotoxicity Assessment
The cytotoxicity of the isolated compounds was tested against HCT-116, HeLa, HepG2, Huh-7 and MCF-7 cells by SRB assay as previously described [61]. Briefly, exponentially growing cells were collected using 0.25% Trypsin-EDTA and seeded in 96-well plates at 1000-2000 cells/well. Cells were treated with the isolated compounds for 72 h and subsequently fixed with TCA (10%) for 1 h at 4˝C. After several washings with water, cells were exposed to 0.4% SRB solution for 10 minutes at room temperature in dark place and subsequently washed with 1% glacial acetic acid. After the plates drying overnight, Tris-HCl was used to dissolve the SRB stained cells and color intensity was measured at 540 nm with ELISA microplate reader.

Data Analysis
The dose-response curves were analyzed as previously described [62] using the E max model (Equation (1) where R is the residual unaffected fraction (the resistance fraction), [D] is the drug concentration used, K d or IC 50 is the drug concentration that produces a 50% reduction of the maximum inhibition rate and m is a Hill-type coefficient. Absolute IC 50 is defined as the drug concentration required to reduce absorbance by 50% of that of the control (i.e., K d = absolute IC 50 when R = 0 and E max = 100´R).
4.10. The Influence of the Naturally Hydroxyphenylalkanes and Diarylheptanoids on the Cellular Pharmacokinetics of Doxorubicin (DOX) To assess the effect of hydroxyphenylalkanes and diarylheptanoids on cellular pharmacokinetics in colorectal cancer cells, their effect on the efflux pumping activity of P-gp was evaluated. Herein, doxorubicin (DOX) was used as P-gp fluorescent substrate. Intracellular DOX concentration was determined with and without co-exposure with hydroxyphenylalkanes and diarylheptanoids and compared to VRP as standard P-gp inhibitor (positive control). Briefly, exponentially proliferating CaCo-2 cells were plated in 6-well plates at plating density of 10 5 cells/well. Cells were exposed to equimolar concentration of DOX (10 µM) and test compounds or VRP for 24 h at 37˝C and subsequently, extracellular DOX-containing media was washed three times in ice cold PBS. Intracellular DOX was extracted after cell lysis by sonication with saturated aqueous solution of ZnSO 4 (100 µL), acetonitril (500 µL) and acetone (250 µL) for 20 min at 37˝C. After centrifugation, clear supernatant solution was collected and DOX concentration was measured spectroflourometrically at λ ex/em of 482/550 nm. DOX concentration was normalized based on cell number [28].

Determining Sub-Molecular Interaction Characteristics between P-gp Protein and Naturally Occurring Hydroxyphenylalkanes and Diarylheptanoids
P-gp inhibitors block its efflux pumping activity via either competitive binding or inhibiting P-gp ATPase activity. Human recombinant membrane bound P-gp protein attached with ATPase subunit (Pgp-Glo™ Assay Systems, Promega Corporation, Madison, WI, USA) was used to determine the mechanism of P-gp inhibition via determining ATP consumption rate. Briefly, test compounds (10 µM) were incubated with Pgp-Glo™ assay systems according to manufacturer protocol. Rate of ATP consumption was calculated by measuring luminescent signal of the unmetabolized ATP via firefly luciferase system. Compound which covalent bind to P-gp molecule is supposed to stimulate ATPase subunit and increase ATP consumption; while ATPase inhibitor compounds would decrease ATPase subunit activity and decrease ATP consumption rate. Verapamil and sodium vanadate were used as positive controls (covalent binding and ATPase inhibitors, respectively). ATP consumption was expressed as remaining ATP concentration and normalized per P-gp protein concentration (pmole ATP/µg P-gp protein).

Chemomodulatory Effect of Gingerol (GNL) to DOX within Liver Cancer Cells
Chemomodulatory effect of gingerol to doxorubicin (DOX) within liver cancer cells was determined using combination analysis between DOX and GNL as previously described [63]. Briefly, exponentially growing HepG2 and Huh-7 cells were seeded in 96-well plates (2000 cells/well) and exposed to equitoxic concentrations of DOX and GNL for 72 h. Cells were subsequently subjected to SRB assay as described in section 4.8. Combination index (CI-value) was calculated and used to define the nature of drug interaction (synergism if CI-value <0.8 as; antagonism if CI-value >1.2; and additive if CI-value ranges from 0.8-1.2). CI-value was calculated from the formula: CI´value " IC 50 drug pxq combination IC 50 drug pxq alone`I C 50 drug pyq combination IC 50 drug pyq alone (2)

Analysis of Cell Cycle Distribution
To assess the effect of the doxorubcin, gingerol and their combination on cell cycle distribution, HepG2 and Huh-7 cells were treated with the pre-determined IC 50 s of both agents for 24 h.
After treatment, cells were collected by trypsinization; washed twice with ice-cold PBS and re-suspended in 0.5 mL of PBS. Two milliliters of 70% ice-cold ethanol was added gently while vortexing. Cells were kept in ethanol solution at 4˝C for 1 h for fixation. Upon analysis, fixed cells were washed and re-suspended in 1 mL of PBS containing 50 µg/mL RNAase A and 10 µg/mL propidium iodide (PI). After 20 min incubation in dark place at room temperature, cells were analyzed for DNA contents by FACS-Vantage™ (Becton Dickinson Immunocytometry Systems). For each sample, 10,000 events were acquired. Cell cycle distribution was calculated using ACEA NovoExpress™ software (ACEA Biosciences Inc., San Diego, CA, USA).

Animals
Male Wistar rats (King Abdul-Aziz University, Jeddah, Saudi Arabia) weighing 120-140 g, aged 6 weeks were housed in clear polypropylene cages (three to four rats per cage) and kept under constant environmental conditions with equal light-dark cycle. Rats had free access to commercially available rodent pellet diet and purified water. All experimental procedures were performed in accordance with Saudi Arabia Research Bioethics and Regulations, which are consistent with the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health.

Assessing the Protective Effect of 6-gingerol Against Doxorubicin Induced Vascular Damage
To assess the protective effect of 6-gingerol on doxorubicin-induced vascular damage, deterioration in vascular reactivity was measured. Vascular reactivity of isolated thoracic aortae was determined using isolated artery techniques described in our previous publications [64,65]. Briefly, isolated aortae were co-incubated within organ bath with doxorubicin (10 µM) with or without different concentrations of 6-gingerol (0.3-30 µM) for 30 min before assessing the vasoconstriction and vasodilation responses compared to control aortic ring. For assessing the aortic contractile responsiveness, increases in tension due to cumulative additions of PE (10´9 to 10´5M) were recorded and expressed as milligram tension. In order to study the vasodilator responsiveness, aortic rings were first pre-contracted with maximal concentrations of PE (10´5M). Cumulative concentrations of ACh (10´9 to 10´5M) were then added to the organ bath and responses were recorded as percentage in relation to PE pre-contraction.

Statistical Analysis
Data are presented as mean˘SEM using GraphPad prism™ software (GraphPad Software Inc., La Jolla, CA, USA) for windows version 5.00. Analysis of variance (ANOVA) with Newman Keuls post hoc test was used for testing the significance using SPSS ® for windows, version 17.0.0. p < 0.05 was taken as a cut off value for significance.

Conclusions
In conclusion, gingerol, despite its relatively weak antioxidant properties, was found to protect from DOX-induced vascular damage, apparently not through its free radical scavenging mechanism. In addition, gingerol synergized the cytotoxic effects of DOX against liver cancer cells at pharmacodynamic level. The gingerol-induced chemomodulatory effect of doxorubicin was found to be independent on influencing P-gp efflux activity and cellular pharmacokinetics within liver cancer cells.

Conflicts of Interest:
The authors declare no conflict of interest.

Abbreviations
The following abbreviations are used in this manuscript: