A C-30 steroid, 3β-,5α-,6β-,11α-,20β-pentahydroxygorgosterol was isolated from the soft coral Xenia umbellata Lamarck (Xeniidae). The chemical structure was elucidated by examining the NMR spectral data and comparison with the previously published data. Compound 1 inhibited the growth of ovarian cancer (SKOV-3), breast cancers (MCF-7 and MDA-MB-231) and hepatocellular carcinoma (HepG2) using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide assay. Notably against HepG2, compound 1 showed significant effect with an IC50 value of 19.70 ± 1.98 µg/mL. It significantly increased the population in the SubG1 phase for 2.01- and 2.05-folds, respectively, compared to untreated cells. Additionally, it showed potent inhibitory activities of superoxide dismutase (384.6 vs 8594.2 U/g protein in dimethyl sulfoxide-treated cells), catalase (0.3 vs 0.07 U/g protein), decreased the level of reduced glutathione (1.7 vs 0.6 mg/g protein) and the activity of matrix metalloproteases (MMP-2 and MMP-9 [0.5-fold of change in MMP activity]) in HepG2 cells. The results indicated the potent antiproliferative activity of the gorgostane derivative (1) against HepG2 cells. This study provides a scientific basis of the antiproliferative effects of steroidal compound with gorgostane nucleus against hepatocellular carcinoma cells.
Cancer is a nonstandard development of cells that causes serious modification of organs’ functions. This increases the mortality rate worldwide [1,2,3]. It is the second leading cause of death after heart diseases. In 2018, 9.6 million people died by cancer . The most common cancers among men are lung, prostate, colorectal, stomach and liver, while breast, colorectal, lung, cervical and thyroid are the most common cancers among women. The Saudi Cancer Registry reported 24,485 diagnosed cancer cases in 2018 . The marine invertebrates belonging to Alcyonacea (Phylum: Cnidaria; Class: Anthozoa; Subclass: Octocorallia) mainly live in the tropical and subtropical seawaters. They live in the inner reefs underneath the stony corals [6,7]. The soft corals of these animals possess toxic cells, which has the capability to produce chemicals to defend themselves rather than the rigid protective skeleton of scleractinians [7,8]. Alcyonacea is considered as a productive order of secondary metabolites including terpenoids and steroids. The family Xeniidae contains 20 genera and 162 species. They live in tropical waters mainly across the Red Sea, Indian Ocean and Pacific Ocean. They present in yellow cylindrical clavate colonies and are characterized by long feather-like tentacles and polyps . In continuation of our research program, which aimed at the discovery of antitumor activities of marine organisms [9,10,11], this study was designed to evaluate the potential antiproliferative effect of 3β-,5α-,6β-,11α-,20β-pentahydroxygorgosterol (1) (Figure 1). Compound 1 was evaluated against four cancer cells: ovarian (SKOV-3), breast cancers (MCF-7 [ER-positive] and MDA-MB-231 [triple negative]) and hepatocellular carcinoma (HepG2). The cytotoxic activity was evaluated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. The effect of treatment on cell cycle was evaluated by flow cytometry. The mechanism was partially investigated by measuring the level of antioxidant parameters such as superoxide dismutase (SOD), catalase, reduced glutathione and inhibition of metalloproteinases 2 and 9.
Four cancer cell lines: ovarian (SKOV-3), breast cancers (MCF-7 [ER-positive] and MDA-MB-231 [triple negative]) and hepatocellular carcinoma (HepG2) were obtained from Nawah Scientific Company, Egypt. All cells were cultured in DMEM (12-604F, Lonza Verviers SPRL, Belgium) supplemented with 5% fetal bovine serum (S-001B-BR, Life Science Group L, UK), 100 IU/mL penicillin and 100 µg/mL streptomycin (17-602E, Lonza Verviers SPRL, Belgium). Antibiotics except SKOV-3 cells were maintained in RPMI-1640 medium. Cisplatin was used as a positive control. Compound 1 (10.0 mg) was solubilized in 100% dimethyl sulfoxide (DMSO). Sigma company was the source of all chemicals and reagents.
2.2 Soft coral sample (collection and extraction)
Scuba was the technique used to collect Xenia umbellata at a depth of 15–20 m (October 2018), from territorial water of Saudi Arabia (21° 29′ 31″N 39° 11′ 24″ E). It was identified by Dr Mohsen El-Sherbiny (Faculty of Marine Sciences, King Abdulaziz University) and a voucher specimen (XC-2018-11/2) was deposited in the Faculty of Marine Sciences of KAU. The partially dried soft coral (265.0 g) was extracted three times with CH2Cl2/MeOH (1:1) (3 × 1 L, 23°) and it yielded an oily residue (21.4 g). Compound 1 was isolated as previously described by Ayyad et al. . In brief, the obtained dark brown extract (15 g) was subjected to partitioning using silica gel column chromatography. The column was eluted with a series of solvents with increasing polarity starting with dichloromethane (100%) to 15% methanol in dichloromethane. The fraction eluted with 2% MeOH in dichloromethane was taken and purified. Preparative thin layer chromatography carried out using the same column resulted in the isolation of a blue pure zone (R f 0.77) after spraying sulfuric acid reagent.
2.2.1 Characterization of 3β-,5α-,6β-,11α-,20β-pentahydroxygorgosterol
White powder (3.1 mg); m.p. > 300°C; [α]D = −41.4 (MeOH; c = 0.22); IR max, 1H and 13C NMR spectra and mass spectrometry all coincided with those published by Ayyad et al. .
2.3 Determination of antiproliferative effect of compound 1
2.3.1 MTT assay
Five thousand cells were well incubated in a 96-well plate (24 h, 37°C and 5% CO2). Then, the cells were treated with serial dilution of 1 (50, 25, 12.5, 6.25, 3.125 and 1.56 µg/mL) and after 48 h, the viability was determined by using MTT (5 mg/mL) [11,12]. The cells were then incubated for another 4 h with MTT stain and DMSO solubilized the formazan crystals. The absorbance was measured at 570 nm by using a BioTek plate reader (ELx808, BioTek Instruments, Inc., Winooski, VT, USA). The experiment was performed three times and the standard deviation (SD) was calculated (±). IC50 the concentration that causes 50% inhibition of cell growth was calculated using Sigma Plot 12.00 software.
2.3.2 Evaluation of the effect of compound 1 on cell cycle distributions
HepG2 cells were treated either with pre-calculated IC50 of 1 or with DMSO as a negative control (0.5%). After 48 h of incubation, the cells were washed with 1× PBS two times (17-516F, Lonza Verviers SPRL, Belgium) and trypsinized (17-161E, Trypsin-Versene [EDTA], Lonza Verviers SPRL, Belgium). The detached cells were collected and centrifuged at 1,000 rpm for 10 min. Then, the cells were fixed with 70% ice-cold ethanol for 2 h at −20°C. The fixed cells were washed with 1× PBS by centrifugation at 1,000 rpm for 10 min. The effect of the treatment on the cell cycle was evaluated by staining the cells with 50 µg/mL propidium iodide containing RNase A (100 µg/mL) (P3566, Invitrogen™) for 15 min in dark. The fluorescence was detected using the BD Accuri™ C6 Plus flow cytometer [13,14].
2.4 Oxidative stress of compound 1
2.4.1 SOD and reduced glutathione level
HepG2 cells were treated as described above and the total protein lysate was gathered. The concentration of the protein was evaluated using the Pierce™ BCA Protein Assay Kit. SOD and reduced glutathione were determined as previously described by Nishikimi et al.  and Aebi . SOD was estimated by employing SOD assay kit. The assay is established on the inhibition of the reduction of nitroblue tetrazolium salt in the presence of phenazine methosulfate (PMS) and NADH. The reaction was carried out in a final volume of 500 µL and started with the addition of PMS. The increase in absorbance was monitored at 560 nm every 60 s for 5 min. The percent of inhibition was calculated as follows: (ΔA control − ΔA test)/(ΔA control). The results were expressed as SOD units/mg of protein in the cell lysate.
2.4.2 Reduced glutathione
The content of reduced glutathione was evaluated based on Ellman’s Reagent; 5,5-dithio-bis-(2-nitrobenzoic acid) in the presence of glutathione (GSH) to produce a yellow substance. This color is directly proportional to GSH concentration, and its absorbance can be measured at 405 nm. The concentration of GSH was expressed by mg/g protein concentration in the cell lysate.
Catalase level in the cell lysate was determined by a commercial kit. The assay depends on the detection of the remaining hydrogen peroxide by adding peroxidase (HRP), 3,5-dichloro-2-hydroxybenzene sulfonic acid and 4-aminophenazone, which gives a colored product that can be measured at λ 510 nm. The intensity of the color is inversely proportional to the amount of catalase in the cell lysate. Catalase activity was presented as U/g protein in the cell lysate.
2.5 Evaluation of matrix metalloproteinases (MMPs) effect of compound 1
2.5.1 Cell culture
HepG2 cells were seeded in a 6-well plate and on the next day, they were treated with compound 1 in a serum-free medium for 48 h. The media was collected and stored at −80°C until analysis. The metalloprotease activity was evaluated by performing gelatin zymography assay to detect both pro and active forms of MMP-2 and MMP-9.
2.5.2 Gelatinase zymography
Gelatinase zymography was performed in 10% SDS polyacrylamide gel in the presence of 0.1% gelatin under non-reducing conditions. The assay was performed as previously published by Mondal et al.  and Toth and Fridman . Gelatinase zymograms were scanned using LaserJet Pro MFP M127fn at 300 dpi. The intensity of the bands was evaluated using ImageJ software.
2.6 Statistical analysis
Data are presented as mean with SD. Statistical significance was acceptable to a level of p < 0.05. The GraphPad Prism software was employed for statistical analyses. Groups were analyzed by ordinary one-way ANOVA.
3 Results and discussion
Extraction of a marine soft coral specimen, identified as X. umbellata, employing several common chromatographic methods including solvent extraction and planar chromatography resulted in the isolation of a pure substance (1). This pure material gave positive steroid test with p-anisaldehyde-sulfuric acid reagent (blue color turned brown). The IR, NMR spectral data and mass spectrometry revealed the identity of compound 1 as 3β-,5α-,6β-,11α-,20β-pentahydroxygorgosterol.
Compound 1 displayed cytotoxic effect against SKOV-3 with IC50 value of 27.17 ± 2 µg/mL, against MDA-MB-231 with IC50 value of 27.5 ± 2.8 µg/mL, against MCF-7 with IC50 value of 42.53 ± 5.0 µg/mL and against HepG2 with IC50 value of 19.70 ± 1.98 µg/mL (Table 1 and Figure 1). While, the reference drug (Cisplatin) displayed cytotoxic effect against SKOV-3 with IC50 value of 5.0 ± 0.30 µg/mL, against MDA-MB-231 with IC50 value of 2.2 ± 0.10 µg/mL, against MCF-7 with IC50 value of 6.9 ± 0.82 and against HepG2 with IC50 value of 1.7 ± 0.05 µg/mL (Table 1 and Figure 2).
|Ovarian||SKOV-3||27.17 ± 2.00*||5.0 ± 0.30|
|Breast||MDA-MB-231||27.50 ± 2.80||2.2 ± 0.10|
|MCF-7||42.53 ± 5.00||6.9 ± 0.82|
|Liver||HepG2||19.70 ± 1.98||1.7 ± 0.05|
The results indicate that compound 1 has cytotoxicity against SKOV-3, MDA-MB-23, MCF-7 and HepG2 (Table 1). Based on these results, the study on the liver cancer cells has been investigated. The activity of compound 1 on cell cycle of HepG2 cells was evaluated by quantifying the DNA content by propidium iodide containing RNase. Figure 3 shows the cell cycle results which was estimated as the percent of cell viability after treatment of HepG2 cells with IC50 (19.70 ± 1.98) and ½ IC50 (9.85) µg/mL. Compound 1 significantly increased the proportion of cells in the SubG1 phase for 2.01- and 2.05-folds, respectively, compared to untreated cells.
Cancer cells produce high level of reactive oxygen species (ROS) due to the high rate of metabolism. Therefore, cancer cells require high level of enzymatic or non-enzymatic antioxidant players to balance the level of ROS. In this study, the level of antioxidant parameters such as SOD, catalase and reduced glutathione was evaluated after treatment with compound 1. Figure 4 shows the reduction effects of SOD (384.6 vs 8594.2 U/g protein in DMSO-treated cells), the decreased level of reduced glutathione (1.7 vs 0.6 mg/g protein) and catalase (0.3 vs 0.07 U/g protein in DMSO-treated cells). This indicates the accumulation of ROS, which causes an increase in the possibility of DNA damage and thus leads to apoptosis. The cytotoxicity against HepG2 cells was observed with noticeable elevation of the intracellular ROS production. Metalloproteinase plays a vital role in the metastasis and invasion of cancer cells [1,2].
In the current study, the MMPs released into the medium were evaluated. The gelatin zymography was carried out with the aim of detecting the activity of metalloproteases (MMP-2 and MMP-9) in HepG2 cells. The results show that compound 1 decreases the activity of the secreted metalloprotease MMP-2 and MMP-9 (0.5-fold of decrease in activity) (Figure 5).
A gorgostane steroid, 3β-,5α-,6β-,11α-,20β-pentahydroxygorgosterol (1), was isolated from the Red Sea specimen of X. umbellata. Compound 1 was evaluated for its cytotoxicity against four cancer cells: ovarian cancer (SKOV-3), breast cancers (MCF-7 [ER-positive] and MDA-MB-231 [triple negative]) and hepatocellular carcinoma (HepG2). The cell viabilities were measured by employing MTT assay. The effect of compound 1 on the cell cycle of HepG2 was evaluated by employing DNA flow cytometric technique. The results indicated that the exposure of HepG2 to compound 1 for 48 h led to the interference with the cell cycle distribution and increasing the cell population in the SubG1 phase, which was ascertained by the pre-G peak in the cell cycle analysis. This could be due to degradation or fragmentation of the genetic materials as indicator of apoptosis. At the end of the G1 phase, the cells are evaluated for the DNA damage with the aim of confirming the required cellular machinery to allow the cell division. Cells with intact DNA are continued to S phase, while cells with damaged DNA cannot be repaired. Thus, they are arrested for repair or induction of apoptosis or programmed cell death.
Compound 1 showed cytotoxic effect in malignant human through induction of oxidative stress (Figure 3). The natural products regulate the expression of proteins involved in the redox balance and apoptosis. This indicated that it is responsible for the cancer cell death through suppression of antioxidant proteins and subsequently increasing the oxidative damage to the cells . The obtained results showed that compound 1 produced significant changes in the activities of antioxidant enzymes, which were reflected by the decrease in SOD and reduction of catalase activities as well as reduction in the level of nonenzymatic reduced glutathione. The reduction in the antioxidant parameters leads to the accumulation of ROS leading to DNA, protein and lipid oxidative damage and induction of apoptosis. The anticancer activity of compound 1 may be partially oxidative stress dependent .
MMPs are calcium- and zinc-dependent endopeptidases. They are responsible for corrosion of the extracellular matrix (ECM) proteins, particularly, collagen, elastin and fibronectin. They remodel the ECM in different physiological and pathological processes. Production of MMPs is enhanced by several factors including different cytokines. They play vital roles in different physiological processes like embracing wound healing, organ morphogenesis, angiogenesis, etc. Several diseases including cancers, neurodegenerative and cardiovascular could occur due to the overexpression of these enzymes . Activity of MMP-2 (72 kDa gelatinase A) and MMP-9 (92 kDa gelatinase B) are increased in various kinds of human cancers and are associated with cancer cell invasion and malignancies . Zymography is an assay that measures the activity of metalloproteinases in the conditioned medium. The current study revealed that compound 1 treatment caused a decrease in the activity of both MMP-2 and MMP-9 confirming the anticancer activity.
The obtained results indicated the potent antiproliferative activity of compound 1 against HepG2 cells. X. umbellate is considered as a potential source of bioactive metabolites including steroids and sesqui- and di-terpenoid derivatives . For example, it produces xenican diterpenes of eight or nine macrocyclic skeletons. These metabolites are characterized by a cyclononane skeleton, which are categorized into five subclasses: xenicins, xeniolides, xeniaphyllanes, xeniaethers and azimilides . These compounds play an important role in the biological activity of Xenia [23,24]. Diterpenoidal derivatives isolated from X. umbellata are characterized by the presence of diversity of functionality including aldehyde, α,β-unsaturated lactone, oxiranes, pyrans and certainly the unique nine-membered ring carbo-skeletons. These functionalities have shown to play a vital role in the structural–biological activity relationship, especially in estimating anticancer activities [23,24]. On the other hand, steroids with gorgostane carbon skeleton isolated from the genus Xenia are characterized by the presence of hydroxyl, ester, oxirane and cyclopropyl functions. This work clarified the importance of the gorgostane derivatives as a potential antiproliferative agents.
Cancer is the second main cause of death worldwide, and liver cancer is ranked as the seventh leading cause of morbidity among women patients, while fifth among men. This is due to the lack of effective treatment. Therefore, there is still need to discover the new lead compounds. The natural steroid, 3β-,5α-,6β-,11α-,20β-pentahydroxygorgosterol (1), displayed a cytotoxic effect against the four cancer cells lines and was more potent against liver cancer cells. The mechanism of activity was partially investigated by cell cycle analysis, oxidative stress attenuation and zymography. The present results reveal that compound 1 warranted further investigation for its deep antiproliferative mechanism.
The authors are extremely grateful to Dr. Fardous El-Senduny (Department of Biology, Faculty of Science, Mansoura University) and Dr. Serag Eldin Elbehairi (Biology Department, Faculty of Science, King Khalid University) for their support while conducting the biological work.
Funding information: This research received no external funding.
Author contributions: All data were obtained by the authors equally.
Conflict of interest: The authors state they have no competing interest.
Ethical approval: The conducted research is not related to either human or animal use.
Sample availability: Samples of the compounds are available from the authors.
Data availability statement: All data generated or analyzed during this study are included in this published article.
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