Cytotoxicity of Carvotacetones from Sphaeranthus africanus Against Cancer Cells and Their Potential to Induce Apoptosis

Key words

Sphaeranthus africanus - Asteraceae - carvotacetone - NCI-60 cell lines - apoptosis

Introduction

Natural products have undoubtedly played a major role in cancer chemotherapy in the last 60 years [1], [2]. Those small molecules were either unaltered natural products, natural products derivatives, or synthetic drugs based on natural product structural leads [1], [2], [3].

Despite 259 antitumor drugs being approved by the FDA (Food and Drug Administration) from 1946 to 2019 [1], the discovery of new leads for anticancer drugs is still a crucial task because of the resistances of cancers and/or severe side effects. Recently, modern approaches have been used to discover plant-based natural products as antitumor compounds, including molecular networks, hyphenated analytical techniques, and application of more significant in vitro and in vivo bioassay models [4]. Since 1955, the National Cancer Institute of the United States (NCI) has provided support to researchers worldwide for the screening of compounds by establishing the Cancer Chemotherapy National Service Center [5], [6]. The goal of this center was to facilitate the discovery and development of new cancer therapeutic agents, which were finally incorporated into the Developmental Therapeutics Program (DTP) of NCI in 1976 [7]. Through extensive collaborations with academic, pharmaceutical, and biotechnology industries, NCI DTP has supported the development of more than 40 US-licensed anti-cancer agents, including paclitaxel, romidepsin, eribulin, sipuleucel-T, and dinutuximab [8]. Their repositories are the worldʼs largest storehouse of natural products containing approximately 170 000 extracts from more than 70 000 plants and 10 000 marine organisms from more than 25 countries, and 200 000 compounds that have been submitted to DTP for biological evaluation [8]. Today, DTP continues to support research communities worldwide to facilitate the discovery and development of new cancer therapeutic agents via the NCI Experimental Therapeutic program (NExT) [8].

One of the tools that DTP employs in the early stage of drug discovery and development is the NCI-60 cell line screen [9]. The operation of this screen utilizes 60 different human tumor cell lines, representing leukemia, melanoma, as well as lung, colon, brain, ovary, breast, prostate, and kidney cancer cells [10]. The screening service is offered at no cost to submitters, and allows prioritization of selected agents for further evaluation by NCI in collaboration with the submitter. The plant extracts, fractions or pure compounds are reviewed and only those conforming to defined guidelines are selected for screening [10].

Carvotacetones are monocyclic monoterpenes, also known as menthane monoterpenoids, with an o-, m-, or p-menthane backbone. The p-menthane consists of the cyclohexane ring with methyl and a propyl group at the 1 and 4 position, respectively. The o- and m-menthanes are much rarer, and presumably arise from the alkyl migration of p-menthanes [11]. Different moieties at positions 3, 5, and 7 of the skeleton are generating the diversity of carvotacetone derivatives.

Carvotacetone is one of the major volatile constituents of certain species of genera belonging to the Asteraceae family, such as Blumea [12], Pulicaria [13], and Francoeuria [14]. In the essential oils of the Pluchea and Vernonia species, it was identified as the dominating constituent [15], [16]. Recently, some carvotacetone derivatives were synthesized by Santos et al. [17] and Christou et al. [18], which were later also isolated from several Sphaeranthus species [19].

Carvotacetones have been investigated for various pharmacological activities such as anti-proliferative, antiparasitic [20], [21], anti-inflammatory, and antimicrobial activities [19], [22], [23].

Sphaeranthus africanus L. (syn. S. cochinchinensis Loureiro, S. microcephalus Willdenow, and S. suberiflorus Hayata; family: Asteraceae) is mainly represented in Asia, the Malay Archipelago, the Philippines, and North Australia [24]. In Vietnam, it is locally known as “cúc chân vịt” (cúc: chrysanthemum; chân vịt: duck foot) because of the stem and winged branches, or known as “bọ xít” (the bug) because of the odor of the fetid smell like a bug [25]. S. africanus L. has been used in ethnomedicine in Vietnam to alleviate swelling and as a sedative. Pressed juice from fresh leaves of S. africanus has been used for mouth and throat washes to treat sore throat. The decoction is also used as an antitussive and expectorant. The pounded leaves are applied externally to relieve pain and swelling [26]. In a previous study, the ethanol extract of the aerial parts of S. africanus has been shown to have antiproliferative activity against CCRF-CEM (human acute lymphoblastic leukemia) cells. Bioassay-guided isolation led to seven carvotacetones, which showed activity against several cancer cell lines (CCRF-CEM, human acute lymphoblastic leukemia; MDA-MB-231, human breast adenocarcinoma; U-251, human glioblastoma astrocytoma; HCT-116, human colon carcinoma; and HEK-293, human embryonic kidney) [19], [22]. We have now explored the activity of the most potent compounds in the NCI-60 cancer screen and have investigated their mechanisms of action with respect to apoptosis.

Results and Discussion

3,5-Diangeloyloxy-7-hydroxycarvotacetone (1), 3-angeloyloxy-5-[2″,3″-epoxy-2″-methylbutanoyloxy]-7-hydroxycarvotacetone (2), and 3-angeloyloxy-5-[3″-chloro-2″-hydroxy-2″-methylbutanoyloxy]-7-hydroxy-carvotacetone (3) were submitted to the NCI DTP. The compounds were first screened in 60 cancer cell lines at 10⁻⁵ M concentration. The results are shown in [Table 1] and [Figs. 1], [2], and [3]. The value reported for the one-dose assay is growth rate compared to the no-drug control, relative to the time zero number of cells. This allows detection of both growth inhibition (values between 0 and 100) and lethality (values less than 0). For example, a value of 100 means no growth inhibition, a value of 40 means 60% growth inhibition, and a value of 0 means no net growth over the course of the experiment. A value of − 40 means 40% lethality. A value of − 100 means that all cells were dead.

Table 1 Growth rate values of compounds 1–3, tested at a concentration of 10⁻⁵ M (10 µM). NCI Developmental Therapeutic Program. Test date: December 11, 2017.

Panel/Cell line	Growth Rate (%)
	Compound 1	Compound 2	Compound 3
Leukemia
CCRF-CEM	− 3.36	1.28	1.18
HL-60(TB)	− 31.98	− 24.95	− 25.32
K-562	− 18.29	1.51	1.56
MOLT-4	− 16.77	− 3.66	0.57
RPMI-8226	− 39.35	− 37.44	− 33.50
Non-Small Cell Lung Cancer
A549/ATCC	− 9.36	− 23.50	− 17.99
EKVX	− 55.13	− 11.79	− 15.04
HOP-62	− 31.39	0.92	− 1.29
HOP-92	− 72.81	− 35.14	− 50.18
NCI-H226	− 0.59	− 36.43	− 19.38
NCI-H23	− 66.30	− 63.06	− 50.11
NCI-H322 M	− 90.26	− 3.37	− 10.59
NCI-H522	− 52.76	− 79.47	− 34.39
Colon Cancer
HCC-2998	− 85.94	− 87.64	− 76.28
HCT-116	− 76.11	− 58.89	− 56.48
HCT-15	− 49.28	− 14.45	− 20.72
HT29	− 65.58	− 60.31	− 32.54
KM12	− 41.34	− 56.31	− 45.09
SW-620	− 58.96	− 26.79	− 45.69
CNS Cancer
SF-268	− 57.30	13.28	10.35
SF-295	− 54.25	− 20.47	− 41.37
SF-539	− 92.09	− 65.57	− 43.67
SNB-19	− 54.02	12.09	− 1.69
SNB-75	− 43.45	23.64	20.01
U251	− 73.81	− 23.08	− 50.02
Melanoma
LOX IMVI	− 92.48	− 83.81	− 75.37
MALME-3 M	− 91.25	− 77.36	− 76.64
M14	− 84.69	− 49.20	− 56.44
MDA-MB-435	− 78.72	− 31.81	− 23.23
SK-MEL-2	− 48.90	− 49.66	− 23.54
SK-MEL-28	− 92.63	− 48.95	− 48.88
SK-MEL-5	− 99.26	− 92.76	− 92.96
UACC-257	− 48.77	− 9.59	− 19.72
UACC-62	− 84.74	− 73.68	− 77.04
Ovarian Cancer
IGROV1	− 76.99	− 31.19	− 29.69
OVCAR-3	− 89.10	− 46.91	− 41.40
OVCAR-4	− 48.77	13.58	14.15
OVCAR-5	− 76.64	− 40.02	− 43.22
OVCAR-8	− 22.66	4.08	3.78
NCI/ADR-RES	− 6.28	6.41	9.46
SK-OV-3	16.36	44.31	35.78
Renal Cancer
786 – 0	− 88.59	− 89.72	− 72.15
A498	− 77.63	1.46	− 74.62
ACHN	− 100.00	− 96.79	− 96.68
CAKI-1	− 98.73	− 23.59	− 44.51
RXF 393	− 79.21	− 74.34	− 63.61
SN12C	− 56.92	− 44.38	− 35.41
TK-10	− 88.83	− 31.84	− 17.42
UO-31	− 100.00	− 96.30	− 96.48
Prostate Cancer
PC-3	− 54.13	− 7.66	− 24.78
DU-145	− 90.93	6.72	3.58
Breast Cancer
MCF7	− 16.59	8.26	8.43
MDA-MB-231/ATCC	− 61.17	1.48	− 5.10
HS 578 T	28.80	30.93	30.67
BT-549	− 32.74	− 7.25	− 9.98
MDA-MB-468	− 50.86	− 66.79	− 51.88
Mean	− 57.74	− 31.00	− 30.94
Delta	42.26	65.79	65.74
Range	128.80	141.10	132.46

Fig. 1 Compound 1 – One-Dose Mean Graph. Tested at 10 µM concentration.

Fig. 2 Compound 2 – One-Dose Mean Graph. Tested at 10 µM concentration.

Fig. 3 Compound 3 – One-Dose Mean Graph. Tested at 10 µM concentration.

The growth percent values of 1–3 are listed in [Table 1]. Compound 1 has been selected as an example for the explanation of the effects on CCRF-CEM and HOP-92 cell lines. For compound 1, the growth of the leukemia cell line (CCRF-CEM) was − 3.36% and the mean growth was − 57.74%. Therefore, the graph bar shows [− 3.36 – (− 57.74)] = 54.38%, which means 45.62% growth inhibition ([Fig. 1]). Similarly, compound 1 showed − 15.07% lethality for the Non-Small Cell Lung Cancer cell line HOP-92 ([Fig. 1]).

In the one-dose testing by NCI, compound 1 was the most active compound. It inhibited ACHN and UO-31 renal cancer cell lines with growth rate values of − 100% (all cells were dead). Compound 2 showed strong effect on SK-MEL-5 melanoma and ACHN renal cancer cell lines with growth rate values of − 92.76 and − 96.79%, respectively. Compound 3 showed a quite strong effect on renal cancer cell lines with growth rate values of − 96.68% and − 96.48% against ACHN and UO-31 cells, respectively. More detailed results of all cell lines are given in [Table 1].

Compounds 1, 2, and 3 satisfied the threshold criteria for progression to five-dose testing with at least eight cell lines with a maximum growth of 10%. Therefore, these compounds were submitted to the full five-dose assay and tested at five concentrations (10⁻⁴ M, 10⁻⁵ M, 10⁻⁶ M, 10⁻⁷ M, and 10⁻⁸ M). A 100% growth corresponds to the growth of untreated cells, 0% growth indicates no net growth over the course of the assay (i.e., equal to the number of cells at time zero), and a growth rate of − 100% means that all cells were killed. Three end points are routinely calculated: (a) GI₅₀, growth inhibition of 50%, which is the concentration resulting in a 50% reduction in the net protein increase in control cells during the incubation; (b) TGI, or total growth inhibition, which is the concentration resulting in total growth inhibition (0% growth); and (c) LC₅₀, which is the concentration resulting in a 50% reduction in the measured protein at the end of the treatment compared to that at the beginning (concentration yielding − 50% growth, or lethality in 50% of the starting cells).

From the dose-response curves, three end points were calculated, as illustrated in [Fig. 4], using dose-response data for compound 1 in the non-small cell lung cancer cell line panel: the GI₅₀ value at 1.72 × 10⁻⁷; the TGI (total growth inhibition) value at 4.07 × 10⁻⁷, which is the concentration that yields no net growth over the course of the assay; and the LC₅₀ value at 9.62 × 10⁻⁷, which is the concentration that kills 50% of the cells that were present at the time of drug addition.

Fig. 4 Dose-response curves of compound 1 assayed in the non-small cell lung cancer panel, showing end point calculations. Compound 1 (NSC 732 517) was tested at five concentrations (1 log dilution from 10⁻⁴ mol/L to 10⁻⁸ mol/L). These end points are illustrated for cell line HOP-92 (red open square). Other cell lines displayed are A549/ATCC (red open circle), NCI-H322 M (solid blue triangle), EKVX (open red diamond), NCI-H226 (solid blue circle), NCI-H460 (solid blue square), HOP-62 (open red triangle), NCI-H23 (solid blue diamond), and NCI-H522 (open green circle).

GI₅₀ values for each cell line were calculated from dose-response curves. The mean GI₅₀ for each compound across all 60 cell lines was calculated. The difference between the GI₅₀ for a particular cell line and the mean GI₅₀ is plotted. Cell lines that were more sensitive are displayed as bars that project to the right of the mean. Cell lines that were less sensitive are displayed with bars projected to the left.

Compound 1 was found to be most active, with GI₅₀, TGI, and LC₅₀ values ranging from 0.17 to 3.07, 0.40 to 7.44, and 0.96 to 100 µM, respectively. HOP-92 non-small cell lung cancer cells were most sensitive to this compound with GI₅₀, TGI, and LC₅₀ values of 0.17, 0.40, and 0.96 µM, respectively, followed by SN12C renal cancer, PC-3 prostate cancer, and MDA-MB-231/ATCC breast cancer cells, with TGI values of 0.97, 1.18, and 1,10 µM, respectively ([Table 2]).

Table 2 GI₅₀, TGI, and LC₅₀ values (µM) of compounds 1–3 in the various cell lines of the screening of the NCI Developmental Therapeutic Program. Best activities are marked in bold.

Panel/cell line	GI50 (µM)			TGI (µM)			LC50 (µM)
	Cpd 1	Cpd 2	Cpd 3	Cpd 1	Cpd 2	Cpd 3	Cpd 1	Cpd 2	Cpd3
Leukemia
CCRF-CEM	0.28	0.65	0.32	1.22	5.25	2.05	> 100	> 100	> 100
HL-60(TB)	0.41	1.16	0.35	3.40	6.45	5.74	51.00	> 100	> 100
K-562	0.38	1.33	0.39	10.10	10.80	12.5	79.80	> 100	> 100
MOLT-4	0.31	1.96	0.30	1.33	6.50	4.88	> 100	> 100	> 100
RPMI-8226	0.26	1.50	0.42	0.70	5.17	2.65	> 100	> 100	> 100
SR	0.29	0.77	0.25	2.86	8.20	2.16	> 100	> 100	> 100
Non-Small Cell Lung Cancer
A549/ATCC	1.53	2.60	2.81	2.97	7.70	9.36	5.78	27.80	35.80
EKVX	1.76	2.75	2.19	3.29	7.63	4.90	6.14	27.70	13.10
HOP-62	1.76	1.96	2.09	3.19	4.30	4.89	5.78	9.46	14.10
HOP-92	0.17	1.65	1.77	0.40	3.33	3.57	0.96	6.73	7.20
NCI-H226	1.82	3.55	3.72	3.80	13.80	13.00	–	71.80	> 100
NCI-H23	1.58	2.09	1.84	3.34	4.88	4.42	7.03	16.60	12.90
NCI-H322 M	1.48	1.88	1.64	2.81	3.61	3.02	5.34	6.92	5.55
NCI-H460	1.51	2.93	2.28	3.33	8.27	8.62	7.33	50.90	56.70
NCI-H522	0.68	1.07	0.60	2.18	2.54	2.37	5.60	6.03	7.56
Colon Cancer
COLO 205	1.56	1.85	1.30	3.11	3.43	2.85	6.21	6.36	6.21
HCC-2998	1.79	1.74	1.79	3.32	3.22	3.35	6.16	5.93	6.27
HCT-116	0.66	1.13	1.07	1.98	2.48	2.49	4.70	5.45	5.79
HCT-15	0.54	1.40	0.77	1.97	3.09	2.36	5.91	6.81	6.25
HT29	0.67	1.66	1.47	2.10	3.38	3.56	5.53	6.88	8.61
KM12	1.39	1.83	1.75	2.99	3.67	3.47	6.42	7.39	6.91
SW-620	1.14	1.98	1.69	3.08	4.16	4.08	–	8.71	9.85
CNS Cancer
SF-268	1.67	2.46	2.19	3.48	6.32	5.08	7.28	24.4	16.2
SF-295	1.88	2.62	1.94	3.46	8.16	4.79	6.36	29.3	14.9
SF-539	1.13	1.86	1.28	2.36	3.38	2.62	4.93	6.16	5.38
SNB-19	1.31	2.33	1.48	2.62	5.87	2.91	5.21	20.9	5.72
U251	0.87	1.52	1.38	2.11	2.85	2.80	4.65	5.35	5.67
Melanoma
LOX IMVI	1.20	1.71	1.31	2.54	3.30	2.82	5.39	6.36	6.08
MALME-3 M	0.31	1.74	0.47	1.06	3.53	1.85	3.92	7.16	5.57
M14	1.41	1.58	1.60	2.98	3.49	3.50	6.33	7.72	7.66
MDA-MB-435	1.22	1.49	1.35	2.63	2.88	2.83	5.66	5.56	5.95
SK-MEL-2	1.65	1.85	2.06	3.13	3.59	4.29	5.95	6.97	8.93
SK-MEL-28	1.33	1.78	1.46	2.74	3.33	2.98	6.64	6.22	6.07
SK-MEL-5	1.09	1.56	1.38	2.32	3.04	2.75	4.95	5.91	5.48
UACC-257	1.38	1.37	1.42	2.71	2.75	3.32	5.34	5.49	7.76
UACC-62	0.45	1.33	0.74	1.69	2.68	2.05	4.23	5.42	4.70
Ovarian Cancer
IGROV1	0.74	1.99	1.09	2.25	4.55	2.89	–	> 100	–
OVCAR-3	0.51	1.17	0.64	1.71	2.45	1.98	4.40	5.15	4.84
OVCAR-4	1.55	1.93	1.87	2.94	3.91	3.95	5.57	7.93	8.36
OVCAR-5	0.65	1.93	0.97	1.99	3.74	2.72	4.97	7.23	7.48
OVCAR-8	1.06	2.07	1.56	2.77	5.54	5.30	7.26	24.70	2.46
NCI/ADR-RES	1.70	3.18	2.31	5.46	17.40	13.30	> 100	> 100	> 100
SK-OV-3	2.57	4.45	4.50	5.37	14.30	14.90	13.70	3.78	38.70
Renal Cancer
786 – 0	1.68	1.79	1.67	3.16	3.24	3.12	5.94	5.86	5.83
A498	1.67	1.74	1.90	3.11	3.90	3.87	5.82	8.77	7.91
ACHN	0.49	1.48	0.83	1.56	2.81	2.06	3.98	5.35	4.64
CAKI-1	1.72	1.81	1.61	3.12	3.20	2.97	5.63	5.65	5.49
RXF 393	1.12	1.77	1.60	2.44	3.27	3.13	5.33	6.06	6.11
SN12C	0.27	1.45	0.73	0.97	2.99	2.11	3.23	6.13	5.12
TK-10	1.54	1.88	1.55	2.89	3.35	3.05	5.44	5.99	6.02
UO-31	1.31	1.61	1.34	2.58	2.97	2.64	5.09	5.45	5.19
Prostate Cancer
PC-3	0.31	1.75	1.45	1.18	4.50	3.30	3.71	14.10	7.50
DU-145	0.65	1.52	1.13	1.97	2.88	2.41	4.61	5.44	5.10
Breast Cancer
MCF7	0.46	1.19	0.55	2.22	4.14	3.09	> 100	> 100	> 100
MDA-MB-231/ATCC	0.36	1.72	0.76	1.10	3.79	2.26		8.37	5.77
HS 578 T	3.07	3.68	4.78	7.44	22.9	24.40	> 100	> 100	> 100
BT-549	1.71	2.02	1.95	3.17	4.35	4.71	3.45	9.36	13.50
T-47D	0.72	1.27	1.16	2.14	2.79	2.94	–	6.11	–
MDA-MB-468	1.02	1.46	1.05	2.66	3.47	2.80	–	8.24	7.48

For compound 2, GI₅₀, TGI, and LC₅₀ values were ranging from 0.65 to 4.45, 2.48 to 22.90, and 5.15 to 100 µM, respectively. HCT-116 and OVCAR-3 cells were found most sensitive with TGI values of 2.48 and 2.45 µM, respectively ([Table 2]).

For compound 3, GI₅₀, TGI, and LC₅₀ values were ranging from 0.25 to 4.78, 1.85 to 24.40, and 4.64 to 100 µM, respectively. MALME-3 M melanoma, OVCAR-3, and ACHN cells were most sensitive with TGI values of 1.85 and 1.98, and 2.06 µM, respectively ([Table 2]).

Summary reports of mean 50% growth inhibition (GI₅₀), total growth inhibition (TGI), and 50% lethality (LC₅₀) values across all cell lines in the NCI-60 panel were calculated and listed in ([Table 2]).

To understand the mode of action that caused the inhibition of the growth of cancer cells by the tested carvotacetones, and to find out whether the compounds induce programmed cell death, we used the ApoTox-Glo triplex assay. The assay measures caspase-3 and − 7 activities, as well as cytotoxicity and viability. These members of the cysteine aspartic acid-specific protease (caspase) family play key effector roles in apoptosis in mammalian cells. In addition to compounds 1–3, also the other previously isolated carvotacetones 4–7 [19], [22] have been tested.

Compounds 1, 2, 4 (5-angeloyloxy-7-hydroxy-3-tigloyloxycarvotacetone), and 6 (3-angeloyloxy-5-[2″S,3″R-dihydroxy-2″-methyl-butanoyloxy]-7-hydroxycarvotacetone) showed remarkable caspase-3/7 activity ([Fig. 5]). Caspase-3/7 activity significantly increased at double IC₅₀ concentration of each compound in CCRF-CEM cells after 24 h and decreased after 48 h. In addition, the compounds exhibited low cytotoxicity at double IC₅₀ concentration after both 24 h and 48 h. The data suggest caspase-dependent apoptosis in the CCRF-CEM cells. Compound 3 increased caspase-3/7 activity at double IC₅₀ concentration after 24 h and 48 h, while compound 5 (3-angeloyloxy-5,7-dihydroxycarvotacetone) only slightly increased it after 48 h at double IC₅₀ concentration. No cytotoxicity was observed for these two compounds at any tested concentration. Caspase-3/7 activity decreased remarkably with compound 7 (at any tested concentration), while cytotoxicity was found to increase at IC₅₀ concentration at 24 h. The results suggest that cell death induced by compound 7 (3-angeloyloxy-5-[2″R,3″R-dihydroxy-2″-methyl-butanoyloxy]-7-hydroxycarvotacetone) may be occurring through a non-classical apoptosis pathway that is not dependent on caspase-3/7 activity. There was no difference in cell viability for all tested compounds compared to control.

Fig. 5 Viability, cytotoxicity, and caspase 3/7 activation in CCRF-CEM cells in response to compounds 1–7 at 24 h and 48 h after treatment with the respective IC₅₀ and double IC₅₀ concentrations of each compound.

Inflammation is strictly correlated with cancer and plays a crucial role in tumor development and progression [27]. In fact, clinical studies suggest that nonsteroidal anti-inflammatory drugs (NSAIDs), including cyclooxygenase (COX)-2 selective inhibitors, reduce the risk of developing cancer [28]. Previously, NSAIDs and coxibs have been evidenced to inhibit tumorigenesis by inhibiting proliferation or modulating apoptotic activity [29]. Therefore, carvotacetones were previously investigated also for anti-inflammatory activity. Compounds 1 and 2 were found to be potent selective COX-2 inhibitory agents with IC₅₀ values of 3.6 and 0.5 µM, respectively. Also, molecular docking studies affirmed the binding of 1 and 2 to the active sites of COX-2 [22]. In the present study, two of these compounds showed the highest apoptosis induction. Hence, 1 and 2 might have potential as leads for the development of new drugs to treat cancer and inflammation.

In summary, compounds 1 – 3 were active against various NCI cancer cell lines. At 10 µM, compound 1 was the most active compound against ACHN and UO-31 renal cancer cells. Compound 2 showed strong effects in SK-MEL-5 melanoma and ACHN renal cancer cells with growth rate values of − 92.76% and − 96.79%, respectively. Compound 3 exhibited quite strong effect on renal cancer cells with a growth inhibitory effect of − 96% against ACHN and UO-31 cells. When treated with five different concentrations of 1 (1 × 10⁻⁸, 1 × 10⁻⁷, 1 × 10⁻⁶, 1 × 10⁻⁵ and 1 × 10⁻⁴ M), HOP-92 cells were found to be most sensitive with GI₅₀, TGI, and LC₅₀ values of 0.17, 0.40, and 0.96 µM, respectively.

Altogether, carvotacetones from S. africanus were identified as potent antiproliferative, as well as anti-inflammatory, natural products that deserve more investigation. Further studies in cellular and animal models related to COX-mediated anti-inflammatory activity should be performed, and semisynthetic derivatives with carvotacetone skeleton may be of interest.

Material and Methods

Origin of compounds

The isolation and structure elucidation of compounds 1–7 have been reported previously [19], [22], including 3,5-diangeloyloxy-7-hydroxycarvotacetone (1), 3-angeloyloxy-5-[2″,3″-epoxy-2″-methylbutanoyloxy]-7-hydroxycarvotacetone (2), 3-angeloyloxy-5-[3″-chloro-2″-hydroxy-2″-methyl-butanoyloxy]-7-hydroxycarvotacetone (3), 5-angeloyloxy-7-hydroxy-3-tigloyloxycarvotacetone (4), 3-angeloyloxy-5,7-dihydroxycarvotacetone (5), and two diastereomeric carvotacetones (3-angeloyloxy-5-[2″S,3″R-dihydroxy-2″-methyl-butanoyloxy]-7-hydroxycarvotacetone (6) and 3-angeloyloxy-5-[2″R,3″R-dihydroxy-2″-methyl-butanoyloxy]-7-hydroxycarvotacetone (7)) ([Fig. 6]).

Fig. 6 Structures of compounds 1–7.

NCI-60 screening methodology

NCI 60 cell one-dose and five-dose testing was performed by the Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute. The detail methodology is described on the webpage of the National Cancer Institute (http://dtp.cancer.gov) [30].

Cell culture

Human CCRF-CEM leukemia cells were kept in RPMI 1640 medium (Gibco, Invitrogen) supplemented with 2 mM L-glutamine (Gibco), 10% heat-inactivated fetal bovine serum (FBS) (Gibco), 100 units/ml penicillin, and 100 µg/ml streptomycin (1% penicillin/streptomycin) (Gibco). Cells were cultivated in a humidified 5% CO₂ atmosphere at 37 °C.

XTT viability assay

The XTT viability assay is used to determine cellular proliferation and viability. It was first described in 1988 by Scudiero et al. [31] and developed to improve already existing tetrazolium assays, especially the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide) assay.

Cell proliferation kit II (XTT) was obtained from Roche Diagnostics, Cat. No. 11 465 015 001. In each well of a 96-well, flat-bottom plate, 100 µl of a suspension containing 1 × 10⁵ cells/ml was treated immediately after seeding. To reduce evaporation effects and to measure the background absorbance of nonmetabolized XTT solution, marginal wells were filled with medium only. Each sample was tested in two or three independent wells per plate and at minimum two different cell passages. Since all test samples were dissolved in DMSO (dimethyl sulfoxide), control cells correspond to vehicle-treated cells (0.5% DMSO final concentration). DMSO alone had no effect on cell growth and proliferation at that concentration. After adequate incubation time, 50 µl XTT (sodium 3-[1-(phenylaminocarbonyl)-3,4- tetrazolium]-bis (4-methoxy-6-nitro) benzene sulfonic acid hydrate) solution was added. This solution consisted of an XTT labeling reagent and an electron-coupling reagent. Both were freshly mixed together in a ratio of 50 : 1 before being added to the wells. XTT is a yellow tetrazolium salt that is cleaved by mitochondrial dehydrogenases to form an orange formazan dye. Since this conversion occurs only in metabolically active cells and the orange dye build is water soluble, the color change can be directly measured by a scanning multiwell spectrophotometer (Hidex Sense Microplate Reader, Hidex). After incubation with XTT for 4 h at 37 °C and 5% CO₂, the absorbance of each well was measured at 490 nm with a reference wavelength at 650 nm. Viable cells were expressed as a percentage of control and calculated with the following formula: (absorbance of treated cells/absorbance of untreated cells) × 100. Vinblastine (vinblastine sulfate cryst.) served as positive control and was obtained from Sigma-Aldrich.

ApoTox-Glo Triplex Assay

The ApoTox-Glo Triplex Assay was used according to the manufacturerʼs protocol. CCRF-CEM were seeded at 10 000 cells/well (100 µl) into white-walled 96-well plates and treated with the respective IC₅₀ or double IC₅₀ concentration of each compound for 24 and 48 hours and analyzed regarding cytotoxicity, viability, and caspase activation within a single-assay well. The first part of the assay simultaneously measures two protease activities: one is a marker of cell viability, and the other is a marker of cytotoxicity. The living cell protease activity is restricted to intact viable cells and is measured using a fluorogenic, cell-permeant, peptide substrate (glycyl-phenylalanyl-aminofluorocoumarin; GF-AFC). The substrate enters intact cells, where it is cleaved by the live-cell protease activity to generate a fluorescent signal proportional to the number of living cells. This live-cell protease becomes inactive upon the loss of cell membrane integrity and leakage into the surrounding culture medium. A second, fluorogenic cell-impermeant peptide substrate (bis-alanylalanyl-phenylalanyl-rhodamine 110; bis-AAF-R110) is used to measure dead-cell protease activity, which is released from cells that have lost membrane integrity. Because bis-AAFR110 is not cell-permeant, essentially no signal from this substrate is generated by intact viable cells. The live- and dead-cell proteases produce different products, AFC and R110, which have different excitation and emission spectra, allowing them to be detected simultaneously. A total of 20 µl of the viability/cytotoxicity reagent containing both GF-AFC substrate and bis-AAF-R110 substrate was added to all wells and incubated for 30 minutes at 37 °C. For cell viability, fluorescence was measured at 400_Ex/505_Em. For cytotoxicity, fluorescence was measured at 485_Ex/520_Em.

The second part of the assay uses the Caspase-Glo Assay Technology by providing a luminogenic caspase-3/7 substrate, which contains the tetrapeptide sequence DEVD, in a reagent optimized for caspase activity, luciferase activity, and cell lysis. Luminescence is proportional to the amount of caspase activity present. The Caspase-Glo 3/7 Reagent relies on the properties of a proprietary thermostable luciferase (Ultra-Glo Recombinant Luciferase), which is formulated to generate a stable “glow-type” luminescent signal and improve performance across a wide range of assay conditions. For that, luminescence directly correlates with the caspase activity and was measured 30 min after adding 100 µL the Caspase-Glo 3/7 Reagent. Luminescence was measured in wells containing dye reagent, vehicle solution, and cell culture medium. These values were subtracted from experimental values. Vehicle-treated cells served as control. Staurosporine (25 µM) served as positive control for apoptosis induction.

Contributorsʼ Statement

Design of the work: R. Bauer, L. Huynh, H. Tran; data collection: H. Tran, N. Kretschmer; statistical analysis: H. Tran, N. Kretschmer; analysis and interpretation of the data: H. Tran; drafting the manuscript: H. Tran, N. Kretschmer, L. Huynh; critical revision of the manuscript: R. Bauer.

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