Abstract
In this study, a bioguided fractionation of Plectranthus mutabilis extract was performed by chromatographic methods. It yielded one new nor-abietane diterpene, mutabilol (1), and three known abietanes, coleon-U-quinone (2), 8α,9α-epoxycoleon-U-quinone (3), and coleon U (4). The abietane diterpenoid 5 was also tentatively identified using HPLC-MS/MS. Moreover, the extract profile and quantification of each isolated compound were determined by HPLC-DAD. Compound 4 was the major compound in the extract. Compounds 2–4 were found to be selective toward cancer cell lines and were able to inhibit P-glycoprotein (P-gp) activity in NCI-H460/R cells at longer exposure of 72 h and consequently revert doxorubicin (DOX) resistance in subsequent combined treatment. None of the compounds influenced the P-gp expression in NCI-H460/R cells, while the extract significantly increased it.
Keywords: Plectranthus mutabilis, abietane diterpenoids, anticancer effect, multidrug resistance, P-glycoprotein
P-Glycoprotein (P-gp) is one of the major contributors to multidrug resistance (MDR), as it protects MDR cancer cells by effluxing cytotoxic drugs.1 It extrudes a wide range of therapeutic drugs from cancer cells, including paclitaxel, doxorubicin, daunorubicin, epirubicin, mitoxantrone, vincristine, and vinblastine,2 decreasing their intracellular concentrations and reducing their efficacy.3,4 In this context, natural products are considered as an alternative source of drugs to fight MDR cancer.5 Compounds of natural origin have been explored as P-gp modulators.6 These include flavonoids (because of their strong affinity for P-gp), alkaloids, coumarins, and terpenoids.7
Phytochemical studies on genus Plectranthus (Lamiaceae) revealed the presence of bioactive abietane-type diterpenoids8−12 that can inhibit P-gp activity.13,14
P. mutabilis Codd. is a succulent herb grown mainly in South Africa.15 In the preliminary screening study, P. mutabilis extract was found to have cytotoxic activity in different cancer cell lines.16 It is therefore necessary to enrich the phytochemical information on this plant in the literature and identify the compounds that may be responsible for its cytotoxicity. In this work, the isolated compounds and the acetone extract were tested against sensitive and MDR non-small cell lung carcinoma cells as well as normal embryonic lung fibroblasts to assess (i) their selectivity toward cancer cells, (ii) their effects against the MDR phenotype, (iii) their interactions with P-gp, and (iv) their potential to sensitize MDR cancer cells to doxorubicin (DOX).
Air-dried powdered P. mutabilis Codd. leaves (see Plant Material in the Supporting Information) (0.71 kg) were extracted with acetone (5 × 8.75 L each, 1 h) at room temperature using the ultrasound-assisted extraction method (ultrasound apparatus from VWR). The solvent was evaporated under reduced pressure at 40 °C, and 44.07 g of crude extract was obtained. This extract was subjected to flash column chromatography over silica gel (Merck 9385, 200 g) using n-hexane/EtOAc (1:0 to 0:1) and EtOAc/MeOH (1:0 to 0:1) mixtures as eluents. According to differences in composition as indicated by TLC, 10 crude fractions were obtained (A1 to A10).
Bio-guided isolation was done by evaluating the general toxicities of the crude fractions using the Artemia salina model.17 The most bioactive fraction (A7), eluted with 80:20 n-hexane/EtOAc, was fractionated by column chromatography over silica gel (100 g) using 9:1 n-hexane/EtOAc as the eluent to give nine fractions (B1-1 to B1-9). Fraction B1-5 was subjected to column chromatography (silica gel, 152 g) using 3:2 n-hexane/CH2Cl2 (3:2) as the eluent to obtain five fractions (C1-1 to C1-5). Fraction C1-4 was purified with a Combiflash instrument on a polyamide stationary phase (100 g) using gradient elution with 1:0 to 3:22 CH2Cl2/MeOH at a flow rate of 60 mL/min, which afforded compound 2 (3.7 mg, 0.0084% w/w).
Subsequently, fraction C1-5 (divided into two aliquots) was successively rechromatographed using the Combiflash system with polyamide columns (120 g and 25 g) at flow rates of 60 and 30 mL/min, respectively, in gradient elution mode with 1:0 to 9:1 CH2Cl2/MeOH as eluents to afford compound 4 (152.1 mg; 0.3451% w/w). Fraction C1-3 was divided into four fractions (D1-1 to D1-4) using flash column chromatography over silica gel (Merck 9385, 152 g) with 6:4 n-hexane/dichloromethane as the eluent. D1-4 was further fractionated on silica gel (Merck 9385, 152 g) to yield fractions E1-1 to E1-4.
Fraction E1-1 was further separated by means of preparative reversed-phase high-performance liquid chromatography (RP-HPLC) using 53% aqueous acetonitrile as the mobile phase to afford compound 3 (63 mg; 0.143% w/w). Fraction C1-3 was further purified by flash column chromatography (silica gel, 24 g; 25 mL/min) on the Combiflash instrument with 87.5:12.5 n-hexane/EtOAc and subsequently by reversed-phase flash chromatography (C18; 30 g; 35 mL/min) with MeOH/H2O (55% to 100% MeOH) to afford compound 1 (10.2 mg; 0.023% w/w).
This P. mutabilis leaf acetone extract was subjected to bio-guided chromatographic fractionation on the basis of its toxicity on the A. salina model16 (Figure S1). A new C20-nor-abietane, (+)-(5S,10R)-10,11,12-trihydroxy-6,7-dioxo-20-nor-abieta-8,11,13-triene (mutabilol, 1) was isolated, together with three known abietane diterpenes, coleon-U-quinone (2), 8α,9α-epoxycoleon-U-quinone (3), and coleon U (4) (Figure 1). Their structures were elucidated by spectroscopic methods. The spectroscopic data for compounds 2–4 were in agreement with those reported in the literature18−20 (see section 2 in the Supporting Information).
Figure 1.
Structures of compounds 1–4 from P. mutabilis.
Compound 1, named mutabilol, was isolated as a purple solid. Its molecular formula was determined to be C19H24O5 on the basis of the HRMS data, which showed a protonated molecular ion at 333.1694 indicative of eight degrees of unsaturation. In the IR spectrum, characteristic absorption bands for a hydroxyl group (3388.3 cm–1), a carbonyl group (1779.3), and a conjugated carbonyl group (1656.3 cm–1) were observed. The 1H NMR spectrum (Table 1) indicated the presence of an isopropyl substituent on the basis of the characteristic downfield signal at δH 3.16 (1H, sept, J = 7.1 Hz, H-15) and two doublet methyl groups at δH 1.18 (3H, d, J = 6.9 Hz, CH3-16), and 1.17 (3H, d, J = 6.9 Hz, CH3-17). Moreover, the 1H NMR spectrum also showed the signals for one aromatic proton at δH 7.23 (H-14), a tertiary methine signal at δH 1.26 (H-5), two singlet methyl groups at δH 1.29 and 0.94 (H-18 and H-19), and three hydroxyl groups at δH 8.05 (12-OH), 7.76 (10-OH), and 7.56 (11-OH). The 13C NMR, HMBC, and HSQC spectra (see the Supporting Information) displayed resonances for 19 carbons corresponding to four methyl groups, three methylenes, three methines (one sp2 carbon at δH 115.9), and nine quaternary carbons (including one oxygenated carbon at δH 54.2 and two carbonyl groups at δH 176.4 and 178.9).
Table 1. NMR Data for Compound 1 (CD3OD, 1H 500 MHz, 13C 126 MHz; δ in ppm, J in Hz).
| position | δH | δC |
|---|---|---|
| 1α | 1.78 (m) | |
| 1β | 2.84 (m) | 35.8 |
| 2α | 1.56 (m, J = 3.4) | |
| 2β | 1.70 (m, J = 14.0, 3.4 Hz) | 18.7 |
| 3α | 2.89 (m) | |
| 3β | 1.64 (m) | 32.9 |
| 4 | 34.7 | |
| 5 | 1.26 (s) | 42.4 |
| 6 | 176.4 | |
| 7 | 178.9 | |
| 8 | 125.3 | |
| 9 | 153.2 | |
| 10-OH | 7.76 (s) | 54.2 |
| 11-OH | 7.56 (s) | 170.6 |
| 12-OH | 8.05 (s) | 175.5 |
| 13 | 118.8 | |
| 14 | 7.23 (s) | 115.96 |
| 15 | 3.16 (sept, J = 7.1) | 24.8 |
| 16 | 1.18 (d, J = 6.9) | 21.5 |
| 17 | 1.17 (d, J = 6.9) | 19.5 |
| 18 | 0.94 (d, J = 4.9) | 28.2 |
| 19 | 1.29 (d, J = 4.9) | 30.3 |
The 1H and 13C NMR spectra of compound 1 were similar to those of coleon V (see the Supporting Information).19 The main differences were the absence of the C-20 methyl group signal (δC 23.6), which together with a downfield quaternary carbon at δC 54.2 suggested the presence of an extra hydroxyl group at C-10 and the nor-abietane structure, and the absence of a hydroxyl group at C-14, as indicated by the signal at δH 7.23 instead of δH 13.47 and the downfield shift of C-14 (δC 115.9).
These structural features were confirmed by analysis of 2D NMR data (COSY, HMQC, and HMBC; Figure 2). In particular, the 2JC–H correlations observed between the quaternary carbon at δC 54.2 and H1-β (δH 2.85) and H-5 (δH 1.26) corroborated the presence of the hydroxyl group at C-10. The cross-peak between the carbonyl signal at δC 176.4 and H-5 was used to assign its position at C-6. Furthermore, the long-range correlation of C-14 (115.96) to H-15 (δH 3.16) confirmed the absence of a hydroxyl group at C-14. On the basis of the above evidence, the structure of compound 1 was identified as (+)-(5S,10R)-10,11,12-trihydroxy-6,7-dioxo-20-nor-abieta-8,11,13-triene.
Figure 2.
Key 1H–1H COSY (bold lines) and HMBC (blue arrows) correlations for compound 1.
From the LC-MS/MS results, compound 1 yielded its deprotonated ion at m/z 331 [M – H]−. Its MS/MS spectra showed fragments at 303 due to the loss of the −CO group [M – H – CO]− and m/z 313 resulting from the loss of water [M – H – H2O]−. Other product ions at m/z 298 [M – H – H2O – CH3]−, 285 [M – H – CO – H2O]− and 259 [M – 2H – CO – C3H7]− were also detected at low abundance, further confirming its structure.
HPLC analysis of the P. mutabilis extract revealed the presence of the following compounds: mutabilol (1), tR = 9.20 min; coleon-U-quinone (2), tR = 10.22 min; 8α,9α-epoxycoleon-U-quinone (3), tR = 9.43 min; coleon U (4), tR = 11.80 min. Compound 4 was found to be the major compound in the extract (Table 2). Coleon-U-quinone appears to be an oxidation product of coleon U, as it was present in the crude extract in only small quantities. These findings were in agreement with the results for P. madagascariensis Benth obtained by Matias et al.10 and Wellsow et al.,20 where coleon-U-quinone appeared in significant amounts only during the isolation process.
Table 2. Abietane Diterpenoid Composition of the Extract from P. mutabilis by HPLC-DAD.
| compound | conc. (μg/mg) | LOD (μg/mg) | LOQ (μg/mg) |
|---|---|---|---|
| 1 | 51 ± 0.008 | 1.120 | 3.39 |
| 2 | 35 ± 0.005 | 0.102 | 0.310 |
| 3 | 36 ± 0.018 | 0.828 | 2.510 |
| 4 | 96 ± 0.048 | 0.78 | 2.35 |
The biosynthetic relationship among compounds 2–4 was therefore considered employing a computational study. While 4 is the undoubted precursor of 2 and 3, epoxyquinone 3 can be formed straight from 4 or from its quinone 2. To gain insight into the formation of 3, the electron-density-based local reactivity descriptors condensed Fukui indexes have been determined for the three compounds, together with the bond dissociations energies (BDEs) of the O–H bonds of 4 (Table 3). Figure 3 depicts the results for the electrophilic (fk–), nucleophilic (fk), and radical (fk0) Fukui functions in the gas phase, where the positions with higher Fukui indexes have been indicated. Table 3 presents the bond dissociation energies of the O–H bonds present in 4. The BDE of the O–H bond at C11 is significantly lower than those of any of the other O–H bonds, which indicates that under oxidative radical conditions the removal of this hydrogen radical will trigger the oxidation of the hydroquinone to quinone 2. The analysis of the Fukui reactivity descriptors of 2 and 4 point to the carbon atoms of the hydroquinone moiety as the most nucleophilic region of the molecule, while the quinone portion is the most electrophilic region in 2. This observation suggests that the installation of epoxide in 2 to form 3 would occur via nucleophilic conjugate addition by an oxygen-carrier group (e.g., hydroperoxide). On the other hand, the oxygen atom in the epoxide of 3 is more likely to have its origin in electrophilic molecular oxygen, making 4 a suitable biosynthetic precursor in view of its nucleophilic character around the hydroquinone core.
Table 3. Hydrogen Bond Dissociation Energies (BDEs) of O–H bonds in 4.
| position of OH in 4 | O–H BDE (kcal/mol) |
|---|---|
| C6 | 103.8 |
| C11 | 84.1 |
| C12 | 103.0 |
| C14 | 116.5 |
Figure 3.
Electrophilic (fk–), nucleophilic (fk), and radical (fk0) Fukui functions of 2–4. The higher condensed Fukui indexes are indicated as green circles, red triangles, and blue arrows, respectively, representing the sites in the molecules that are most susceptible to a radical attack, most nucleophilic, and most electrophilic.
With this in mind, the formation of anions 411 and 414 was considered, and the electrophilic Fukui functions (fk–) of both anions were analyzed (Figure 4). The difference of 25 kcal/mol in the Gibbs free energies of formation of the two anions indicates that hydroxyl deprotonation at C11 is preferable. The analysis of fk indicates C9 to be the second most nucleophilic atom (condensed index = 0.136), overpassed only by the anionic oxygen. A similar analysis of fk– for anion 414 indicates that C11 is the most nucleophilic carbon, although it has a condensed index close to those of C9 and C13 (0.138 vs 0.122–0.124).
Figure 4.
Electrophilic Fukui functions (fk–) and Gibbs free energies of formation for anions derived from deprotonation of coleon U (4) at hydroxyl positions on (a) C11 and (b) C14.
With this in mind, two paths are suggested as plausible for the formation of epoxide 3 from 4 (Scheme 1). Upon hydroxyl deprotonation of 4 at C11, the anion 411 reacts with molecular oxygen at C9 to form a hydroperoxide, which undergoes nucleophilic attack at the C11 carbonyl to deliver a dioxetane. A proton exchange places the anion at the oxygen at C14, after which dioxetane opening delivers the epoxide. Alternatively, the same dioxetane can be reached from anion 414 through electrophilic attack by molecular oxygen at C11, as corroborated by the higher nucleophilic Fukui condensed index, followed by conjugate addition. While the formation of 411 is more favorable than the formation of 414, given its lower Gibbs free energy of formation, the oxygen electrophilic addition to C9 formulated in the first path should be hampered by stereochemical constraints. Furthermore, the formation of epoxyquinones21,22 through electrophilic attack by phenol-bearing carbons (such as in C11) has been reported in bacteria23 and fungi24 as well as in mammals in the biosynthesis of vitamin K epoxide.25,26 Despite the likely participation of enzymes in the formation of 3 from 4, the latter may be susceptible to epoxidation by chemical means because of the presence of the oxygenated quinone core.
Scheme 1.
The viability of human cancer cells was compared with that of human normal cells upon treatment with the acetone extract (2, 5, 10, 20, and 50 μg/mL) and diterpenes 1–4 (2, 5, 10, 20, and 50 μM) isolated from P. mutabilis. The largest reduction of cancer cell viability (metabolic activity) of the NCI-H460 and NCI-H460/R cell lines expressed with P-gp was observed with compound 4 (IC50 ≈ 14 μM), which also showed the highest selectivity index at 2.5 (Table 4). Compounds 2 and 3 showed similar inhibition of cancer cell viability (IC50 ≈ 20 μM) and a similar selectivity index of 2.0. The newly identified compound 1 was inactive against all three cell lines, displaying IC50 values greater than the highest concentration of compounds used in the study. The acetone extract showed similar activity against cancer and normal cells with a selectivity index of 1.0. Importantly, the presence of the MDR phenotype (P-gp overexpression) in NCI-H460/R did not affect the inhibitory effect of 2–4 and the extract, implying that they are not substrates for P-gp (Table 4).
Table 4. Inhibition of Cell Viability Assayed by MTT in Non-Small Cell Lung Carcinoma Cells (NCI-H460 and NCI-H460/R) and Embryonic Pulmonary Fibroblasts (MRC-5).
| compound | NCI-H460 | NCI-H460/R | MRC-5 | selectivity indexa |
|---|---|---|---|---|
| 1b | >50 | >50 | >50 | n.a. |
| 2b | 22.96 ± 0.56 | 20.37 ± 0.43 | 44.13 ± 1.19 | 2.0 |
| 3b | 20.23 ± 0.59 | 17.26 ± 0.26 | 40.22 ± 0.44 | 2.0 |
| 4b | 14.11 ± 0.19 | 14.50 ± 0.18 | 35.47 ± 0.56 | 2.5 |
| extractc | 15.30 ± 0.37 | 15.66 ± 0.47 | 16.68 ± 0.69 | 1.0 |
| paclitaxel | 0.0006 ± 0.0001 | 0.117 ± 0.013 | 0.523 ± 0.001 | 872 |
The selectivity index was calculated as the ratio of the IC50 value for MRC-5 cells to that for NCI-H460 cells.
IC50 values in μM for compounds.
IC50 values in μg/mL for extract.
All of the tested compounds as well as the extract decreased the Rho123 accumulation in NCI-H460/R cells. This finding implies that the compounds and extract stimulate P-gp activity by a direct interaction assessed after 30 min (Table 5). On the basis of these results, we studied Rho123 accumulation after 72 h treatment with the compounds and the extract. All of the compounds except 4 and the extract increased Rho123 accumulation when a concentration of 10 μM was administered. However, a 10 μM concentration of 4 decreased the accumulation of Rho 123, as did a 10 μg/mL concentration of the extract (Table 6).
Table 5. Rho123 Accumulation Assay (30 min Simultaneous Treatment with Tested Compounds) Shows Direct Interaction with P-gp and Interference with Its Activity (Inhibition or Stimulation).
| cell line/compound | conc.e | MFIa | FARb | SIc |
|---|---|---|---|---|
| Cell Linesd | ||||
| NCI-H460 | – | 2151.0 | 4.19 ± 0.45 | |
| NCI-H460/R | – | 513.7 | 23.88 ± 2.22 | |
| Compounds | ||||
| 1 | 5 | 595.3 | 1.16 ± 0.98 | 27.68 ± 2.18 |
| 10 | 573.0 | 1.12 ± 1.03 | 26.64 ± 2.29 | |
| 2 | 5 | 669.6 | 1.30 ± 1.00 | 31.13 ± 2.22 |
| 10 | 991.3 | 1.93 ± 0.86 | 46.09 ± 1.90 | |
| 3 | 5 | 711.6 | 1.39 ± 0.98 | 33.08 ± 2.19 |
| 10 | 666.1 | 1.30 ± 1.02 | 30.97 ± 2.26 | |
| 4 | 5 | 1258.8 | 2.45 ± 0.78 | 58.52 ± 1.73 |
| 10 | 253.0 | 0.49 ± 1.25 | 11.76 ± 2.78 | |
| TQ | 50 | 3004.0 | 3.70 ± 0.54f | 121.16 ± 0.88f |
| extract | 5 | 654.0 | 1.27 ± 0.96f | 30.40 ± 2.14 |
| 10 | 473.4 | 0.92 ± 0.96g | 22.01 ± 2.14 | |
Measured mean fluorescence intensity.
Fluorescence activity ratio, calculated as FAR = MFIMDR-treated/MFIMDR control. Data are reported as mean ± SE.
Sensitivity index, calculated as SI = (MFIMDR-treated × 100)/MFIsensitive control. Data are reported as mean ± SE.
The sensitive cancer cell lines and their MDR counterparts used in the study were the non-small cell lung carcinoma (NSCLC) cell lines NCI-H460 and NCI-H460/R.
Concentrations in μM for the compounds and μg/mL for the extract.
TQ was applied as a positive control for P-gp inhibition.
The extract and the four tested compounds exerted a stimulating effect on P-gp.
Table 6. Rho123 Accumulation Assay (30 min Load after 72 h of Treatment with Tested Compounds or Extract) Indirectly Shows the Effect on the P-gp Activity (Inhibition or Stimulation).
| cell line/compound | conc.e | MFIa | FARb | SIc |
|---|---|---|---|---|
| Cell Linesd | ||||
| NCI-H460 | – | 2479.3 | 3.06 ± 0.61 | |
| NCI-H460/R | – | 811.1 | 32.71 ± 1.65 | |
| Compounds | ||||
| 1 | 5 | 265.9 | 0.33 ± 0.21 | 10.72 ± 3.55 |
| 10 | 254.5 | 0.31 ± 0.22 | 10.26 ± 3.63 | |
| 2 | 5 | 277.3 | 0.34 ± 0.20 | 11.18 ± 3.29 |
| 10 | 242.0 | 0.30 ± 0.20 | 9.76 ± 3.44 | |
| 3 | 5 | 289.7 | 0.36 ± 0.20 | 11.68 ± 3.29 |
| 10 | 276.6 | 0.34 ± 0.21 | 11.16 ± 3.41 | |
| 4 | 5 | 173.5 | 0.21 ± 0.24 | 7.00 ± 3.99 |
| 10 | 109.7 | 0.14 ± 0.29 | 4.43 ± 4.71 | |
| TQh | 0.05 | 3004.0 | 3.70 ± 0.54f | 121.16 ± 0.88f |
| extract | 5 | 339.5 | 0.42 ± 0.20g | 13.69 ± 3.24g |
| 10 | 307.7 | 0.38 ± 0.20 | 12.41 ± 3.15 | |
Measured mean fluorescence intensity.
Fluorescence activity ratio, calculated as FAR = MFIMDR-treated/MFIMDR control. Data are reported as mean ± SE.
Sensitivity index, calculated as SI = (MFIMDR-treated × 100)/MFIsensitive control. Data are reported as mean ± SE.
The sensitive cancer cell lines and their MDR counterparts used in the study were the NSCLC cell lines NCI-H460 and NCI-H460/R.
Concentrations in μM for the compounds and μg/mL for the extract.
P-gp-inhibiting activity of tested compounds/concentrations.
P-gp-stimulating effect of tested compounds/concentrations.
TQ inhibition of P-gp activity strongly induces P-gp expression as a compensative mechanism.
The effect on P-gp (ABCB1) expression was assessed by flow cytometry using FITC-conjugated ABCB1 antibody after treatment of NCI-H460/R cells with compounds or the extract for 72 h. NCI-H460 cells with almost null expression of ABCB1 were used as a negative control, while treatment with tariquidar (TQ) in NCI-H460/R cells was used as a positive control. The results showed that none of the tested compounds had an influence on ABCB1 expression in NCI-H460/R cells, while the extract significantly increased ABCB1 expression in a concentration-dependent manner (Figure 5). The increase in ABCB1 expression induced by the extract at 10 μg/mL was like the one achieved by TQ at 50 nM (Figure 5). According to our results, the extract can increase resistance by stimulating P-gp expression. Therefore, it would not be wise to use the extract in combination with chemotherapeutics that are P-gp substrates.
Figure 5.
Effects of compounds and the extract on ABCB1 expression. The mean fluorescence intensity (MFI) of untreated NCI-H460/R cells was set to 100 and used for the comparison of other MFI values (for untreated NCI-H460 cells and treated NCI-H460/R cells) in GraphPad Prism 6 software (unpaired t test). The MFI was calculated from three independent experiments. A difference from untreated NCI-H460/R cells was considered to be significant if p < 0.05 (*) or p < 0.001 (***).
Our findings prompted us to investigate the sensitizing effect of 2, 3, and 4 in NCI-H460/R cells. These three compounds showed an anticancer effect in the MTT assay, a considerable increase of Rho123 accumulation after 72 h when applied at 5 μM (Table S5), and no influence on the ABCB1 expression in NCI-H460/R cells (Figure 5). Therefore, we studied the sensitization of NCI-H460/R cells to DOX combined with 2, 3, and 4. DOX (0.1, 0.2, 0.5, 1, or 2 μM) was administrated after pretreatment for 72 h with 2, 3, or 4 applied at three concentrations below their IC50 (1, 2, and 5 μM). All of the combinations of the compounds with DOX showed significant reversal potential, expressed in terms of the relative reversal index (Table 7), which was calculated as the ratio of the IC50 value for DOX alone to that for DOX in combination with a compound. The most potent sensitization of NCI-H4l60/R cells to DOX was achieved with 2 and 4 at 5 μM, with relative reversal index values of 7.465 and 6.045, respectively (Figure S13).
Table 7. Reversal of DOX Resistance in NCI-H460/R Cells Pretreated with 2, 3, or 4, Assayed by the MTT Test.
| compound | conc. (μM) | IC50 for DOX (μM) | relative reversal indexa |
|---|---|---|---|
| DOX | 1.620 ± 0.084 | ||
| 2 | 1 | 0.565 ± 0.012 | 2.867*** |
| 2 | 0.482 ± 0.010 | 3.361*** | |
| 5 | 0.217 ± 0.004 | 7.465*** | |
| 3 | 1 | 0.820 ± 0.016 | 1.976*** |
| 2 | 0.345 ± 0.007 | 4.696*** | |
| 5 | 0.343 ± 0.006 | 4.723*** | |
| 4 | 1 | 0.625 ± 0.013 | 2.592*** |
| 2 | 0.540 ± 0.012 | 3.000*** | |
| 5 | 0.268 ± 0.006 | 6.045*** |
***, p < 0.001.
Conclusion
Plectranthus mutabilis Codd. leaves are a valuable source of abietane diterpenoids and a C20-nor-abietane with coleon U as a major constituent in the acetone extract. A biosynthetic relation between 2, 3, and 4 based on computational data indicates that both quinone 2 and epoxyquinone 3 are formed directly from 4. Compounds isolated from Plectranthus mutabilis Codd. are not P-gp substrates, as the presence of the MDR phenotype did not affect their antimetabolic activity studied by MTT. Besides, these compounds did not change the expression of P-gp after treatment for 72 h. Although these diterpenoids stimulated P-gp activity in a direct interaction after application for 30 min, longer exposure led to decreased activity of P-gp. This unique mechanism needs to be clarified in further research by assessing the effects on P-gp ATPase activity and ATP levels in MDR cancer cells. Finally, our results showed the potential of 2, 3, and 4 to reverse the resistance to DOX and increase its efficacy toward cancer cells. Therefore, we identified diterpenoids from Plectranthus mutabilis Codd. that could be considered as MDR modulators in cancer. New derivatives and their structure–activity studies are important to reinforce abietanes as a new family of P-gp modulators.
Acknowledgments
This project was funded by Fundação para a Ciência e Tecnologia (FCT) (Projects UIDB/04567/2020 and UIDP/04567/2020) and supported by PADDIC 2019 (ALIES-COFAC) as part of the Ph.D. Program in Health Sciences from Universidad de Alcalá and Universidade Lusófona de Humanidades e Tecnologias. This research was also funded by the Ministry of Education, Science and Technological Development of the Republic of Serbia (reference number 451-03-9/2021-14/200007) and partly by the National Research, Development and Innovation Office of Hungary (NKFIH) (K134704). This work was performed within the framework of COST Action CA17104, “New diagnostic and therapeutic tools against multidrug resistant tumours”. We also acknowledge Fundação para a Ciência e Tecnologia and Portugal 2020 to the Portuguese Mass Spectrometry Network (Grant LISBOA-01-0145-FEDER-402-022125).
Supporting Information Available
The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsmedchemlett.1c00711.
Structure elucidation of compounds and NMR data for compounds 2, 3, and 4; bio-guided screening of P. mutabilis column fractions for general toxicity at a concentration of 10 ppm using the Artemia salina test; calibration curves for the HPLC quantification of diterpenoids from the acetonic extract of P. mutabilis; 1H, COSY, HMBC, HSQC, and 13C NMR spectra for new compounds; LC-MS data for the extract and isolated compounds; reversal of DOX resistance in NCI-H460/R cells in subsequent treatment with compounds 2, 3, and 4; atomic coordinates for all of the optimized species (PBE1PBE/6-31G**) (PDF)
Author Contributions
◆ E.N.N. and S.J.S. contributed equally. The manuscript was written through contributions of all authors, and all of the authors approved the final version of the manuscript.
The authors declare no competing financial interest.
Special Issue
Published as part of the ACS Medicinal Chemistry Letters virtual special issue “Medicinal Chemistry in Portugal and Spain: A Strong Iberian Alliance”.
Supplementary Material
References
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