Abbreviations
ABC:
ATP-binding cassette
ABCB1:
ATP-binding cassette, subfamily B
CI:
confidence interval
EPG85-257P:
parental gastric cancer cells
EPG85-257RDB:
gastric cancer cells selected against daunorubicin
EPG85-257RNOV:
gastric cancer cells selected against mitoxantrone
EPP85-181RDB:
pancreatic cancer cells selected against daunorubicin
EPP85-181P:
parental pancreatic cancer cells
EPP85-181RNOV:
pancreatic cancer cells selected against mitoxantrone
MDR:
multidrug resistance
MDR1
:
multidrug resistance gene 1
P-gp:
P-glycoprotein
RDB:
daunorubicin
RNOV:
mitoxantrone
RR:
relative resistance
SRB:
sulforhodamine B
Introduction
In spite of all the valuable chemotherapy regimens to treat cancer, it represents
a major health problem worldwide, especially due to the high incidence of MDR phenotypes.
MDR is characterized by cross-resistance of tumors to multiple structurally and functionally
unrelated drugs. One of the most relevant and studied MDR mechanisms of tumor cells
is correlated with the overexpression of P-gp (P-gp/ABCB1/MDR1), encoded by the ABCB1 gene, which belongs to the superfamily of ABC transporters [1]. The overexpression of this ABC transporter, resulting from an association of intrinsic
and acquired drug resistance factors, is evident in tumor tissues from patients, reducing
the intracellular accumulation of the anticancer drug and thus compromising the efficacy
of treatment [2]. As a consequence of the efflux function of P-gp, clinical chemotherapeutic agents,
such as paclitaxel and Adriamycin, or the selective kinase inhibitors erlotinib and
sorafenib suffered a reduction of efficacy [3], [4], [5].
Numerous strategies to overcome MDR have been explored, including the development
of P-gp modulators to restore drug accumulation, the design of novel drugs that avoid
recognition and efflux, and the use of small molecules that selectively kill MDR cells
but not the nonresistant parental cells [6]. The latter, named collateral sensitivity effect, represents a new promising therapeutic
approach for eradicating resistant cells. It has been considered as resulting from
genetic alterations accumulated during the development of resistance towards one agent
that are associated with the development of hypersensitivity to a second one [1]. Thus, it is thought that the development of novel treatment strategies exploiting
collateral sensitivity could improve cancer treatment from refractory tumors by being
resensitized to drugs through the selective killing of MDR cells, or by preventing
the development of the MDR phenotype through coadministration during chemotherapy.
This is a widely observed phenomenon, found not only in P-gp-expressing cancer cells,
but also in tumors overexpressing other ABC transporters such as the multidrug resistance
protein 1 (MRP1/ABCC1) and the breast cancer resistance protein (BCRP/ABCG2) [7].
In spite of being an old concept, observed firstly in resistant bacteria, the complex
mechanisms by which compounds exert a collateral sensitivity effect are not yet clearly
understood and are still under investigation [8].
Aiming at giving some insights into the collateral sensitivity phenomenon, several
hypotheses have been considered, such as the ability of collateral sensitivity agents
to generate reactive oxygen species via stimulation of P-gp ATPase activity, take
advantage of P-gp-expressing cells sensitivity to changes in energy levels, stimulate
the extrusion of endogenous subtracts, which is essential for cell survival, or induce
perturbation of the biophysical properties of membranes. Nevertheless, the number
of experimental studies providing evidence for these explanations is scarce, and several
mechanisms might be involved depending on the compound [8]. While collateral sensitivity agents in P-gp-overexpressing cancer cells appear
to act through different biochemical mechanisms, in relation to MRP1-overexpressing
cancer cells, there is experimental evidence that they mainly act as stimulators of
MRP1-mediated glutathione efflux, thus modifying redox balance, which selectively
triggers apoptosis of resistant cells overexpressing this ABC protein [7].
Momordica balsmamina L. (Cucurbitaceae), commonly called African pumpkin, is an herb commonly found in
tropical and subtropical regions of Africa and Asia. It presents high nutritional
and medicinal value, being extensively used as food and traditional medicine [9]. A wide variety of cucurbitane-type triterpenoids with different biological activities
has been isolated from the Momordica genus, namely from Momordica charantia
[10], [11].
In our previous investigation of the methanol extract of the aerial parts of M. balsamina, several cucurbitanes isolated or obtained by derivatization showed they were potent
inhibitors of P-gp efflux pump activity [12], [13]. Moreover, they were also able to inhibit the efflux pump systems of resistant strains
of gram-positive bacteria [14].
In the present work and continuing our search for plant-derived compounds that can
circumvent MDR [15], [16], [17], [18], [19], [20], triterpenoids 1–28 were evaluated for their potential collateral sensitivity effect on colon, gastric,
and cancer cell models (drug sensitive and drug resistant sublines) well characterized
for MDR [17].
Results and Discussion
The phytochemical study of the methanol extract of the aerial plant parts of M. balsamina lead to the isolation of several triterpenes (1–10) ([Fig. 1]) with the cucurbitane skeleton, as previously described [12], [21], [22], [23]. Karavilagenin C [10, 7β-methoxycucurbita-5,24-diene-3β,23(R)-diol], the major compound, allowed for the generation of a small library of mono-
and di-acyl derivatives at C-23 and/or C-3, bearing alkanoyl, aroyl, and cinnamoyl
moieties (11–28) ([Fig. 1]) [12], [24]. These compounds were previously evaluated at non-cytotoxic doses for their ability
as P-glycoprotein modulators on MDR1 mouse lymphoma cells [12], [13]. It was concluded that different substitution patterns, at both the tetracyclic
nucleus and the side chain, led to distinct inhibition of this efflux pump activity
[12], [13].
Fig. 1 Structures of compounds 1–28.
In this work, aiming at finding effective compounds for overcoming MDR, compounds
1–28 were assessed for their potential collateral sensitivity effect on three different
cancer cell models: gastric (EPG85-257), pancreatic (EPP85-181), and colon (HT-29)
cancer cells. For each cancer cell model, one sensitive cell line and two resistant
sublines, selected for resistance to RDB and to RNOV, were tested. The characteristics
of these MDR cell lines are well known and the same cancer cell models were used with
a similar purpose in other studies [15], [16], [17], [25], [26], [27] The collateral sensitivity effect was assessed by determining the RR (calculated
as the ratio of the IC50 of a compound against a resistant line divided by the IC50 against the corresponding parental line). Compounds with an RR < 1 kill MDR cells
more effectively than parental cells, and when they exhibit an RR ≤ 0.50 they have
a collateral sensitivity effect. An RR ≥ 2.0 expresses a compound that has resistance
to a drug and is simultaneously cross-resistance to others [28]. The RR ratio only evaluates selectivity towards resistant cells. Thus, when selecting
a compound with a collateral sensitivity effect for further studies, the antiproliferative
values should also be considered. The anticancer drugs etoposide and cisplatin were
used as positive controls.
The antiproliferative activity and collateral sensitivity effect (RR values) of compounds
1-28 are summarized in [Tables 1] – [3]. As can be observed, a significant antiproliferative effect (IC50 < 10 µM) in parental drug sensitive cell lines was observed for compounds 6 [EPG85-257, EPP85-181, and HT-29, IC50 = 9.5 µM (CI 7.2 – 11.8 µM), 7.1 µM (CI 6.9 – 7.3 µM), and 6.7 µM (CI 6.6 – 6.8 µM),
respectively], 7 [EPG85-257, IC50 = 9.2 µM (CI 8.0 – 10.4 µM)], 10 [EPG85-257, EPP85-181, IC50 = 7.9 µM (CI 7.4 – 8.4 µM) and 6.7 µM (CI 6.6 – 6.8 µM), respectively], 11 [HT-29, IC50 = 7.9 µM (CI 4.1 – 11.4 µM)], and 15 [EPG85-257, IC50 = 8.0 µM (CI 6.9 – 9.1 µM)]. The remaining compounds showed a moderate/weak antiproliferative
effect in parental drug-sensitive cell lines or were inactive ([Tables 1] – [3]). Regarding MDR sublines, when the IC50 values were compared with those found for their specific drug-sensitive counterpart
cell line, an increased sensitivity (RR < 1) was observed for most of the compounds
([Fig. 2]), mainly for resistant cancer gastric and colon cancer cell lines. Moreover, a collateral
sensitivity effect (RR ≤ 0.50) was observed for the natural compounds balsaminol F
[3, IC50 = 6.2 µM (CI 5.7 – 6.7 µM); RR = 0.43] and karavilagenin C [10, IC50 = 2.5 µM (CI 2.2 – 2.8 µM); RR = 0.32] against the gastric EPG85-257 RDB subline
([Table 1] and [Fig. 2]), with a high concomitant antiproliferative activity, which was comparable to that
found for the positive controls [cisplatin, IC50 = 4.0 µM (CI 3.7 – 4.3 µM); RR = 1; etoposide, IC50 = 6.2 µM (CI 5.9 – 6.5 µM); RR = 59]. A collateral sensitivity effect was also found
for compounds 4 and 27 on the same cells, although with a lower antiproliferative effect. Similarly, on
the gastric EPG85-257 RNOV variant, a collateral sensitivity effect was observed for
compounds 3, 4, 6, and 9, and was associated with strong antiproliferative activity for compounds 3 [IC50 = 7.2 µM (CI 6.4 – 8.0 µM); RR = 0.50] and 6 [IC50 = 4.5 µM (CI 2.5 – 6.5 µM); RR = 0.47]. By using the nonparametric Kruskal-Wallis
rank test, a statistical difference with p = 0.053 in the IC50 values was detected between the three gastric cell lines, and was more significant
(p = 0.010, one tail) between EPG85-257 RDB and EPG85-257 RNOV cells.
Table 1 Antiproliferative activity of compounds 1–28 in gastric carcinoma cells: EPG85-257P (parental), EPG85-257RDB (MDR phenotype),
and EPG85-257RNOV (MDR phenotype).
|
Compound
|
EPG85-257P
|
EPG85-257RDB
|
EPG85-257RNOV
|
|
IC50 (µM)1 (CI 95%) (µM)
|
IC50 (µM) 1 (CI 95%) (µM)
|
RR2
|
IC50 (µM) 1 (CI 95%) (µM)
|
RR2
|
|
Compounds 16, 18, 20, 22, 24, and 26 were ineffective in the sensitive and resistant variants of carcinoma cells (IC50 > 100 µM). 1 The IC50 values with 95% confidence intervals (CI 95%) given in parentheses indicate the mean
of n = 3 to 4 independent experiments (each concentration was performed in triplicate
per experiment). 2 RR is the relative resistance ratio determined by dividing the mean IC50 against a resistant line by the mean IC50 against a parental line
|
|
Balsaminol A (1)
|
20.4
(20.0 – 20.8)
|
14.5
(10.5 – 18.5)
|
0.71
|
12.1
(7.1 – 17.1)
|
0.59
|
|
Balsaminol D (2)
|
> 100
|
56.0
(43.8 – 68.2)
|
< 0.56
|
74.7
(67.9 – 81.5)
|
0.75
|
|
Balsaminol F (3)
|
14.4
(10.1 – 18.7)
|
6.2
(5.7 – 6.7)
|
0.43
|
7.2
(6.4 – 8.0)
|
0.50
|
|
Balsaminagenin A (4)
|
49.0
(48.4 – 49.6)
|
24.4
(22.9 – 25.9)
|
0.50
|
23.2
(19.0 – 27.4)
|
0.47
|
|
Balsaminagenin B (5)
|
20.4
(20.4 – 20.4)
|
17.5
(15.2 – 19.8)
|
0.86
|
18.2
(15.1 – 21.3)
|
0.89
|
|
Balsaminoside A (6)
|
9.5
(7.2 – 11.8)
|
> 100
|
> 10.52
|
4.5
(2.5 – 6.5)
|
0.47
|
|
Balsaminoside B (7)
|
9.2
(8.0 – 10.4)
|
64.2
(61.6 – 66.8)
|
6.98
|
5.0
(2.2 – 7.8)
|
0.54
|
|
Balsaminoside C (8)
|
19.8
(19.1 – 20.5)
|
58.8
(49.8 – 67.8)
|
2.97
|
11.5
(4.8 – 18.2)
|
0.58
|
|
Cucurbalsaminol A (9)
|
67.0
(64.1 – 69.9)
|
54.7
(46.4 – 63.0)
|
0.82
|
31.9
(24.0 – 39.8)
|
0.48
|
|
Karavilagenin C (10)
|
7.9
(7.4 – 8.4)
|
2.5
(2.2 – 2.8)
|
0.32
|
6.6
(6.2 – 7.0)
|
0.84
|
|
Karavoate A (11)
|
19.8
(19.4 – 20.2)
|
13.1
(10.7 – 15.5)
|
0.66
|
16.8
(12.2 – 21.4)
|
0.85
|
|
Karavoate B (12)
|
21.4
(16.9 – 25.9)
|
19.9
(18.1 – 21.7)
|
0.93
|
53.8
(43.5 – 64.1)
|
2.51
|
|
Karavoate C (13)
|
19.3
(18.6 – 20.0)
|
21.2
(19.4 – 23.0)
|
1.10
|
10.4
(7.8 – 13.0)
|
0.54
|
|
Karavoate D (14)
|
21.8
(21.5 – 22.1)
|
> 100
|
> 4.59
|
17.0
(15.7 – 18.3)
|
0.78
|
|
Karavoate E (15)
|
8.0
(6.9 – 9.1)
|
63.5
(60.9 – 66.1)
|
7.94
|
7.0
(6.7 – 7.3)
|
0.88
|
|
Karavoate G (17)
|
21.9
(21.2 – 22.6)
|
> 100
|
> 4.57
|
16.4
(13.2 – 19.6)
|
0.75
|
|
Karavoate I (19)
|
19.6
(19.0 – 20.2)
|
74.1
(66.0 – 82.2)
|
3.78
|
14.3
(13.5 – 15.1)
|
0.73
|
|
Karavoate K (21)
|
73.8
(65.2 – 82.4)
|
> 100
|
> 1.36
|
63.3
(61.6 – 65.0)
|
0.86
|
|
Karavoate M (23)
|
20.0
(19.2 – 20.8)
|
> 100
|
> 5.00
|
17.9
(15.2 – 20.6)
|
0.90
|
|
Karavoate O (25)
|
22.9
(21.9 – 23.9)
|
> 100
|
> 5.42
|
18.8
(17.1 – 20.5)
|
0.82
|
|
Karavoate Q (27)
|
40.3
(27.3 – 53.3)
|
18.7
(18.0 – 19.4)
|
0.46
|
27.9
(25.7 – 30.1)
|
0.69
|
|
Karavoate R (28)
|
66.6
(66.0 – 67.2)
|
55.7
(54.9 – 56.5)
|
0.84
|
62.9
(59.0 – 66.8)
|
0.94
|
|
Etoposide
|
0.105
(0.1 – 0.1)
|
6.2
(5.9 – 6.5)
|
59
|
1.55
(1.4 – 1.7)
|
14.8
|
|
Cisplatin
|
4.4
(3.9 – 4.9)
|
4.0
(3.7 – 4.3)
|
1
|
2.6
(2.4 – 2.8)
|
0.6
|
Table 2 Antiproliferative activity of compounds 1–28 in pancreatic carcinoma cells: EPP85-181P (parental), EPP85-181RDB (MDR phenotype),
and EPP85-181RNOV (MDR phenotype).
|
Compounds
|
EPP85-181P
|
EPP85-181RDB
|
EPP85-181RNOV
|
|
IC50 (µM) 1
(CI 95%) (µM)
|
IC50 (µM) 1 (CI 95%) (µM)
|
RR2
|
IC50 (µM) 1
(CI 95%) (µM)
|
RR2
|
|
Compounds 16, 18, 20, 22, 24, and 26 were ineffective in the sensitive and resistant variants of carcinoma cells (IC50 > 100 µM). 1 The IC50 values with 95% confidence intervals (CI 95%) given in parentheses indicate the mean
of n = 3 to 4 independent experiments (each concentration was performed in triplicate
per experiment). 2 RR is the relative resistance ratio determined by dividing the mean IC50 against a resistant line by the mean IC50 against a parental line
|
|
Balsaminol A (1)
|
21.5
(20.7 – 22.3)
|
22.1
(20.6 – 23.6)
|
1.03
|
22.2
(20.2 – 24.2)
|
1.03
|
|
Balsaminol D (2)
|
91.0
(90.2 – 91.8)
|
> 100
|
> 1.10
|
> 100
|
> 1.10
|
|
Balsaminol F (3)
|
15.4
(11.8 – 19.0)
|
8.5
(7.4 – 9.6)
|
0.55
|
11.7
(9.6 – 13.8)
|
0.76
|
|
Balsaminagenin A (4)
|
69.2
(69.0 – 69.4)
|
66.7
(66.0 – 67.4)
|
0.96
|
56.3
(45.6 – 67.0)
|
0.81
|
|
Balsaminagenin B (5)
|
20.3
(20.2 – 20.3)
|
18.9
(16.8 – 21.0)
|
0.93
|
20.7
(18.7 – 22.7)
|
1.02
|
|
Balsaminoside A (6)
|
7.1
(6.9 – 7.3)
|
> 100
|
> 14.08
|
9.6
(9.4 – 9.8)
|
1.35
|
|
Balsaminoside B (7)
|
10.2
(6.4 – 14.0)
|
67.0
(65.4 – 68.6)
|
6.57
|
17.5
(14.5 – 20.5)
|
1.72
|
|
Balsaminoside C (8)
|
21.0
(20.4 – 21.6)
|
66.0
(64.9 – 67.1)
|
3.14
|
20.1
(19.6 – 20.6)
|
0.96
|
|
Cucurbalsaminol A (9)
|
69.0
(66.8 – 71.2)
|
70.0
(68.8 – 71.2)
|
1.01
|
68.5
(67.6 – 69.4)
|
0.99
|
|
Karavilagenin C (10)
|
6.7
(6.6 – 6.8)
|
6.8
(5.8 – 7.8)
|
1.00
|
6.7
(4.1 – 9.3)
|
1.00
|
|
Karavoate A (11)
|
19.1
(17.4 – 20.8)
|
9.8
(7.4 – 12.2)
|
0.51
|
13.4
(10.4 – 16.4)
|
0.70
|
|
Karavoate B (12)
|
55.1
(48.4 – 61.8)
|
85.6
(81.6 – 89.6)
|
1.55
|
33.6
(25.9 – 41.3)
|
0.61
|
|
Karavoate C (13)
|
19.7
(19.4 – 20.0)
|
23.9
(23.0 – 24.8)
|
1.21
|
13.7
(8.4 – 19.0)
|
0.70
|
|
Karavoate D (14)
|
29.0
(22.1 – 35.9)
|
> 100
|
> 3.45
|
20.8
(20.78 – 20.82)
|
0.72
|
|
Karavoate E (15)
|
19.4
(18.7 – 20.1)
|
77.1
(73.6 – 80.6)
|
3.97
|
14.9
(12.6 – 17.2)
|
0.77
|
|
Karavoate G (17)
|
62.1
(47.6 – 76.6)
|
> 100
|
> 1.61
|
63.7
(44.3 – 83.1)
|
1.03
|
|
Karavoate I (19)
|
23.9
(23.6 – 24.2)
|
> 100
|
> 4.18
|
20.6
(20.4 – 20.8)
|
0.86
|
|
Karavoate K (21)
|
> 100
|
> 100
|
n. d.
|
66.8
(65.6 – 68.0)
|
< 0.67
|
|
Karavoate M (23)
|
23.7
(23.1 – 24.3)
|
> 100
|
> 4.22
|
20.4
(20.2 – 20.6)
|
0.86
|
|
Karavoate O (25)
|
28.2
(27.7 – 28.7)
|
> 100
|
> 3.55
|
22.5
(20.8 – 24.2)
|
0.80
|
|
Karavoate Q (27)
|
49.1
(41.1 – 57.1)
|
58.6
(55.2 – 62.0)
|
1.19
|
55.5
(54.9 – 56.1)
|
1.13
|
|
Karavoate R (28)
|
68.6
(68.1 – 69.1)
|
70.3
(68.4 – 72.2)
|
1.02
|
71.4
(70.4 – 72.4)
|
1.04
|
|
Etoposide
|
0.58
(0.57 – 0.59)
|
62.0
(57.2 – 66.8)
|
106.9
|
4.5
(3.7 – 5.3)
|
7.8
|
|
Cisplatin
|
0.08
(0.07 – 0.09)
|
0.09
(0.07 – 0.1)
|
1.2
|
2.6
(2.4 – 2.8)
|
34
|
Table 3 Antiproliferative activity of compounds 1 – 28 in colon carcinoma cells: HT-29P (parental), HT-29RDB (MDR phenotype), and HT-29RNOV
(MDR phenotype).
|
Compounds
|
HT-29P
|
HT-29RDB
|
HT-29RNOV
|
|
IC50 (µM) 1
(CI 95%) (µM)
|
IC50 (µM) 1
(CI 95%) (µM)
|
RR2
|
IC50 (µM) 1
(CI 95%) (µM)
|
RR2
|
|
Compounds 14, 16, 18, 20–22, 24, and 26 were ineffective in the sensitive and resistant variants of carcinoma cells (IC50 > 100 µM). 1 The IC50 values with 95% confidence intervals (CI 95%) given in parentheses indicate the mean
of n = 3 to 4 independent experiments (each concentration was performed in triplicate
per experiment). 2 RR is the (elative resistance ratio determined by dividing the mean IC50 against a resistant line by the mean IC50 against a parental line.
|
|
Balsaminol A (1)
|
21.2
(21.0 – 21.4)
|
21.0
(20.4 – 21.6)
|
0.99
|
17.5
(16.9 – 18.1)
|
0.83
|
|
Balsaminol D (2)
|
> 100
|
79.0
(73.0 – 85.0)
|
< 0.79
|
79.0
(72.1 – 85.9)
|
< 0.79
|
|
Balsaminol F (3)
|
11.9
(11.1 – 12.6)
|
9.2
(7.5 – 10.9)
|
0.77
|
7.0
(6.7 – 7.3)
|
0.59
|
|
Balsaminagenin A (4)
|
60.4
(59.9 – 60.9)
|
57.3
(49.0 – 65.6)
|
0.95
|
31.3
(29.9 – 32.7)
|
0.52
|
|
Balsaminagenin B (5)
|
20.1
(20.0 – 20.2)
|
20.0
(19.5 – 20.5)
|
1.00
|
19.1
(18.3 – 19.9)
|
0.95
|
|
Balsaminoside A (6)
|
6.7
(6.6 – 6.8)
|
7.1
(6.5 – 7.7)
|
1.06
|
4.8
(2.6 – 7.0)
|
0.72
|
|
Balsaminoside B (7)
|
18.2
(16.0 – 20.4)
|
61.7
(57.5 – 65.9)
|
3.39
|
15.9
(14.9 – 16.9)
|
0.87
|
|
Balsaminoside C (8)
|
27.8
(27.0 – 28.6)
|
66.4
(66.2 – 66.6)
|
2.39
|
31.4
(26.3 – 36.5)
|
1.13
|
|
Cucurbalsaminol A (9)
|
66.4 (64.8 – 68.0)
|
55.9
(49.0 – 62.8)
|
0.84
|
48.9
(39.5 – 58.3)
|
0.74
|
|
Karavilagenin C (10)
|
13.8
(13.1 – 14.5)
|
6.8
(5.2 – 8.4)
|
0.49
|
6.7
(5.8 – 7.6)
|
0.49
|
|
Karavoate A (11)
|
7.9
(4.1 – 11.4)
|
3.1
(1.8 – 4.4)
|
0.39
|
2.3
(2.25 – 3.35)
|
0.29
|
|
Karavoate B (12)
|
61.5
(49.4 – 73.6)
|
30.7
(8.5 – 52.9)
|
0.50
|
19.3
(12.3 – 26.3)
|
0.31
|
|
Karavoate C (13)
|
13.8
(10.7 – 16.9)
|
7.1
(7.09 – 7.1)
|
0.51
|
4.9
(4.3 – 5.5)
|
0.36
|
|
Karavoate E (15)
|
15.4
(14.9 – 15.9)
|
6.9
(6.7 – 7.1)
|
0.45
|
4.0
(2.8 – 5.2)
|
0.26
|
|
Karavoate G (17)
|
80.1
(77.2 – 83.0)
|
67.1
(52.6 – 81.6)
|
0.83
|
27.5
(25.3 – 29.7)
|
0.34
|
|
Karavoate I (19)
|
27.7
(26.5 – 28.9)
|
22.5
(21.6 – 23.4)
|
0.81
|
22.3
(20.5 – 24.1)
|
0.81
|
|
Karavoate M (23)
|
28.7
(27.8 – 29.6)
|
14.7
(9.4 – 20.0)
|
0.39
|
18.9
(17.9 – 19.9)
|
0.66
|
|
Karavoate O (25)
|
71.3
(68.0 – 74.6)
|
27.9
(19.9 – 35.9)
|
0.39
|
25.9
(22.5 – 29.3)
|
0.36
|
|
Karavoate Q (27)
|
61.3
(59.1 – 63.5)
|
24.2
23.8 – 24.6)
|
0.39
|
21.4
(21.2 – 21.6)
|
0.34
|
|
Karavoate R (28)
|
69.4
(68.2 – 70.6)
|
67.4
(66.7 – 68.1)
|
0.97
|
62.2
(57.4 – 67.0)
|
0.90
|
|
Etoposide
|
2.3
(2.0 – 2.6)
|
26.0
(24.1 – 27.9)
|
11.3
|
35.0
(32.1 – 37.9)
|
15.2
|
|
Cisplatin
|
3.8
(3.7 – 3.9)
|
2.7
(2.6 – 2.8)
|
0.7
|
3.8
(3.7 – 3.9)
|
1
|
Fig. 2 Compounds that showed greater sensitivity to the MDR sublines than to the corresponding
parental cell line: relative resistance, the ratio between the mean IC50 against a resistant line by the mean IC50 against a parental line (RR), lower than 1.0. Compounds 4, 6, 9–13, 15, 17, 23, 25, and 27 exhibited a collateral effect (RR ≤ 0.50). The tagged relative resistance points
correspond to compounds that presented the best collateral sensitivity effect values
concomitant with significant antiproliferative activity.
Compounds 11 [IC50 = 9.8 µM (CI 7.4 – 12.2 µM); RR = 0.51] and 3 [IC50 = 8.5 µM (CI 7.4 – 9.6 µM); RR = 0.55] were the most selective against the pancreatic
EPP85-181RDB cells. On the pancreatic EPP85-181RNOV subline, an RR < 1 was also found
for several compounds ([Table 2] and [Fig. 2]), indicating that they exerted a higher antiproliferative effect against the MDR-derived
line than the parental one. Among them, compounds 3, 11, and 13 exhibited the lowest IC50 values (11.7 – 13.7 µM). For the pancreatic cancer cell lines, a significant statistical
difference was found between the IC50 values (p = 0.03), reflecting a different antiproliferative effect of the compounds
on both parental and resistant cancer cell lines. This effect was corroborated by
the p values obtained when IC50 values of the parental cell line and EPP85-181RDB subline (p = 0.016, one tail) and
IC50 values of EPP85-181RDB and EPP85-181RNOV (p = 0.009, one tail) were compared.
Regarding colon cancer cell lines ([Table 3]), the best MDR-selective antiproliferative effects were found for karavilagenin
C [10, IC50 = 6.8 µM (CI 5.2 – 8.4 µM); RR = 0.49, EPG85-257 RDB; IC50 = 6.7 µM (CI 5.8 – 7.6 µM); RR = 0.49, EPG85-257 RNOV] and some of its derivatives,
with a collateral sensitivity effect being observed against both resistant variants.
When comparing both the antiproliferative and the relative resistance ratio ([Table 3] and [Fig. 2]), the best results were obtained for the acyl derivatives karavoate A [11, IC50 = 3.1 µM (CI 1.8 – 4.4 µM); RR = 0.39, EPG85-257 RDB; IC50 = 2.3 µM (CI 2.250 – 3.35 µM); RR = 0.29, EPG85-257 RNOV], karavoate C [13, IC50 = 7.1 µM (CI 7.09 – 7.10 µM); RR = 0.51, EPG85-257 RDB; IC50 = 4.9 µM (CI 4.3 – 5.5 µM); RR = 0.36, EPG85-257 RNOV], and karavoate E [15, IC50 = 6.9 µM (CI 6.7 – 7.1 µM); RR = 0.45, EPG85-257 RDB; IC50 = 4.0 µM (CI 2.8 – 5.2 µM); RR = 0.26, EPG85-257 RNOV].
When analyzing the results of compounds 1–28, lipophilicity seems to be detrimental for antiproliferative activity, although no
statistical correlation with antiproliferative activity was found. In fact, higher
log p values (≥ 8.5) (Table S1, Supporting Information), observed for the acyl derivatives, were always associated
with a lack of antiproliferative effect (IC50 > 100 µM).
As mentioned before, these compounds (1 – 28) were previously assessed for their ability to modulate the transport activity of
P-gp in a functional assay [12], [13]. Interestingly, some compounds classified as strong P-gp modulators (1, 4 – 7, 10 – 13, 15) also showed a significant collateral sensitivity effect on some of the resistant
cell sublines [12], [13]. For instance, karavilagenin C (10), which presented a very strong P-gp-mediated MDR reversal activity at a very low
concentration [12], was able to kill the resistant gastric cell line EPG85 (RR = 0.32) more efficiently.
Although to a lesser extent, a selective antiproliferative effect was also observed
against both resistant colon HT-29 cell sublines (RR < 0.50). On the other hand, the
monoacetylated derivative of 10, karavoate A (11), which was also able to modulate P-gp [12], exhibited a collateral sensitivity [16]effect against both resistant HT-29 colon
carcinoma cell variants (HT-29RDB, RR = 0.39; HT-29RNOV, RR= 0.29). As expected, similar
results were also found for the efflux pump modulator karavoate E (15), which differs from compound 11 in the number of carbons of the ester moiety. Similarly, a collateral sensitivity
effect (RR = 0.47) was also observed for the strong P-gp modulator 6
[12] against EPG85-257RNOV gastric cancer cells.
The exact molecular mechanisms mediating a sensitization of different multidrug-resistant
cancer cell variants to alternative triterpenoids have still not been evaluated and
are beyond the scope of this investigation.
In conclusion, MDR is a complex phenomenon, involving several biochemical mechanisms.
Thus, some of these triterpenes, such as compounds 6, 10, 11, and 15, by acting as both P-gp modulators and collateral sensitivity agents, might be promising
leads for overcoming MDR cancer cells and are worthy of further studies.
Materials and Methods
Tested compounds
Compounds 1 – 10, namely, balsaminol A (1), balsaminol D (2), balsaminol F (3), balsaminagenin A (4), balsaminagenin B (5), balsaminosides A – C (6 – 8), cucurbalsaminol A (9), and karavilagenin C (10), were previously isolated from the methanol extract of M. balsamina as reported [12], [21], [22], [23]. Compound 10, isolated in a large amount, gave rise to 18 compounds, namely, karavoates A – R
(11 – 28), by using several alkanoyl and aroyl acylating reagents, as described [12], [24]. The purity of the compounds was more than 95% by HPLC. All of the compounds were
dissolved in DMSO.
Cell lines and cell culture
The human cancer cell lines (EPG85-257P, EPP85-181P, and HT-29P) and their drug-resistant
sublines (EPG85-257RNOV, EPG85-257RDB, EPP85-181RNOV, EPP85-181RDB, HT-29RNOV, and
HT-29RDB) were grown in Leibovitz L-15 medium (Biowhittaker) supplemented with 10%
fetal calf serum (GIBCO/BRL), 1 mM L-glutamine, 6.25 mg/L fetuin, 80 IE/L insulin,
2.5 mg/mL transferrin, 0.5 g/L glucose, 1.1 g/L NaHCO3, 1% minimal essential vitamins, and 20 000 kIE/L trasylolina in a humidified atmosphere
of 5% CO2 at 37 °C. The drug-resistant cell lines were established from parental cell lines
by continuous exposure of the cells to stepwise increasing concentrations of antineoplastic
agents as described previously [29]. For maintenance of drug-resistant phenotypes, the medium of the drug-resistant
sublines was supplemented with the selective agent as described previously [30]. The used cytotoxic drugs daunorubicin (Farmitalia Carlo Erba), mitoxantrone (Lederle),
etoposide (Bristol-Myers), and cisplatin (GRY-Pharm) showed purities for application
in clinical settings.
Cell proliferation assay
The antiproliferative activity of the compounds was assessed using a proliferation
assay based on SRB staining as described previously [17]. Briefly, 800 cells per well were seeded in 96-well plates in triplicate. After
24 h attachment, the particular agent was added in a dilution series for 5 days incubation
(5% CO2 at 37 °C). Cells were fixed by chilled 10% trichloroacetic acid for 1 h at 4 °C,
and washed five times with tap water before staining was performed with 0.4% SRB in
1% acetic acid for 10 min at room temperature. After washing with 1% acetic acid,
drying, and resolubilization in 20 mM Tris-HCl (pH 10), absorbance was measured at
562 nm against the reference wavelength of 690 nm. Etoposide and cisplatin were used
as positive controls. Mean IC50 values with a 95% confidence interval were calculated from four independent experiments
in triplicate for each cell line by using Prism software (GraphPad Software, Inc.).
RR values were determined as IC50(resistant cells)/IC50(parental cells).
Statistical analysis
Analysis using the nonparametric Kruskal-Wallis rank test (a probability value of
p < 0.05 was considered statistically significant) was carried out to identify differences
between the three cells lines of each group. The Mann-Whitney test was used to examine
the statistical significance (a probability value of p < 0.05 was considered statistically
significant) of differences in the mean IC50 values between two independent groups. The Real Stats package of Excel software was
used.
