Evaluation of Terpenes Psiadin, Plectranthone, and Saudinolide as Selective Anti-Cancer Agents against Hepatic Carcinoma Cells

Plant-derived terpenes have aroused considerable interest as chemotherapeutic agents for a variety of diseases. This study aimed at the isolation and purification of the scarce terpenes psiadin, plectranthone and saudinolide from their respective plants, followed by the determination of antiproliferative activity, against hepatic cancer cell lines (HepG2, Hep3B), and the potential molecular mechanisms. Timeand dose-dependent cytotoxicity, evaluated using MTT and colonyforming assays, were exhibited by psiadin and plectranthone against the cancer cells. Flow cytometry showed that these two terpenes blocked cell cycle progression and induced mitochondrial-mediated apoptosis, particularly through increased cytochrome c and disruption of mitochondrial membrane potential. Additionally, they initiated the generation of reactive oxygen species as well as inhibiting NF-B. Psiadin lowered several essential cyclins and cyclin-dependent kinases and reduced RB activation. It was concluded that psiadin, in particular, has a significant therapeutic potential with the biggest advantage of differentiating between cancer and normal cells which is acutely lacking in current cytotoxic drugs. Its precise mode of action needs further investigation but appears predominantly to cause cell cycle arrest by interfering with cyclin production. It will be important to determine, in future studies, whether these terpenes will similarly inhibit other cancer cell lines and retain its activity against tumors in vivo.


Introduction
The global cancer burden is escalating significantly, with projections of 28 million new cases and 16 million cancer deaths by 2040 [1]. Post-surgical cancer chemotherapy relies heavily on a variety of general cytotoxic agents. However, most are associated with collateral damage to normal tissues that are frequently severe and intolerable, limiting their usefulness. Thus, there is a great need for new drugs that are more cancer-selective with lesser undesirable side effects [2]. Natural products hold historical significance in various folkloric medicines and form the basis of many modern-day drugs.
Phytochemicals are the only sources for several medications that are almost impossible to get from other sources. Also, they serve as prototypes by providing pharmacophores for many synthetic drugs [3]. Indeed, a significant proportion of current chemotherapeutics is either natural or derived from them [4]. Moreover, conjugation of toxic natural products to monoclonal antibodies or polymeric (CDCl3, 150 MHz) see Table 1 Table 1; 13

Effect of psiadin and plectranthone on cell cycle distribution of HepG2 and Hep3B cells
The percentage of cells in each stage of the cell cycle was determined by flow cytometry following 24 h exposure to increasing concentrations of the two terpenes. These data (from one representative experiment, repeated three times) are shown in Figure 2a.   The results of one experiment (representative of three independent determinations) are shown in Figure 3a. The shift in the spectral emission of the dye indicates the dissipation of the MMP.

Changes in mitochondrial membrane potential induced by psiadin and plectranthone
(calibration was performed using CCCP). Both psiadin and plectranthone induced a change in the Δψm. The effect was more pronounced in Hep3B cells.

Measurement of reactive oxygen species generation
As shown in Figure

Protein expression
Using western blotting, the levels of various proteins known to be involved in cell cycle regulation and apoptotic processes, and some associated with cell signaling pathways, were measured. We limited this study to psiadin as the most effective of the three compounds under investigation; to one of the two cell lines since they had so far shown broadly similar behavior ( Figure 4). Finally, in Panel G, it can be seen that PARP was unchanged. . Protein extracts were electrophoresed on polyacrylamide gels, electroblotted onto PVDF membranes, cut into thin strips, and incubated with appropriate primary antibodies, followed by secondary antibodies and visualized by autoradiography with ECL reagents as described in Methods. For each group of proteins, KIP was used as loading control. Blots are representative of at least three independent experiments yielding very similar results.

Discussion
Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 23 December 2020 doi:10.20944/preprints202012.0591.v1 Standard cancer chemotherapy relies on a limited number of favored drugs to inhibit various aspects of cancer cell growth. However, both the substantial 'collateral damage' to normal tissues due to lack of specificity as well as the emergence of intrinsic or acquired drug resistance [24] presents a serious therapeutic problem. The need for new drugs has prompted studies evaluating possible anticancer agents in natural resources. Natural products with diverse bioactivities are becoming an important source of novel agents with pharmaceutical potential. The potential for plant extracts to act as anticancer agents is due to their abilities to inhibit tumor growth, angiogenesis, and metastasis with few side effects [2]. In this study we isolated, confirmed the identities of three terpenes; psiadin, plectranthone and saudinolide, and tested them against two commonly used hepatic carcinoma cell lines.
The designated plants were collected, authenticated and voucher specimens were processed appropriately. Their coarsely powdered aerial parts were extracted and purified as detailed above.
Psiadin was isolated as colorless needles. The IR spectrum of psiadin showed absorption bands for carbonyl and hydroxyl groups. The presence of a ketone carbonyl group was further supported from the 13  . These data are consistent with a kaur-16-ene diterpene structure and identical to those previously reported for psiadin [25].
Plectranthone was isolated as colorless prisms. The molecular formula of this compound was determined as C19H28O5 on the basis of the ion peak at m/z 277.2827 [M -CH3COOH+1] + and the NMR data. Its IR spectrum showed absorption bands for two different carbonyl functions, an α,βunsaturated ketone (1670 cm -1 ) and an ester band (1725 cm -1 ). The 13 C NMR spectrum (Table 1) showed 19 carbon resonances, including five quartets, four triplets, five doublets, and five singlets.
The presence of an isopropyl group was indicated by the fact that two of the methyl carbons coupling systems. One of these was due to the protons of the isolated methylene group at C-1 and the other one was ascribed to the pair of protons at C-14. Other 2D NMR data confirmed the identity of this compound to be the eudesmane sesquiterpene plectranthone [13].
On the other hand, saudinolide was isolated as colorless granules. This compound, possessing the molecular formula C20H20O8, was found to have a γ-lactone (λmax 1780 cm -1 ; C 174.7), a δ-lactone (λmax 1720 cm -1 ; C 172.9), and a mono-substituted furan ring. Additionally, 1 H and 13 C NMR data (Table 1)  This last piece of information, together with the fact that the C-7 signal occurred at C 108.4, suggested the gross structure of saudinolide. Other NMR data were in accordance with those previously assigned to the diterpene saudinolide [12].
The results presented in this study showed that none of the tested terpenes had appreciable toxicity against normal human cells. Saudinolide, at the concentrations tested, was also relatively ineffective against the cancer cells and was not further considered. Psiadin and plectranthone showed appreciable activity against both cancer lines, with psiadin being the more effective killing agent. To put this into more therapeutic context, we compared the effect of the two terpenes with several agents that are currently used in clinical practice. In both cases they compared very favorably in their efficacy, particularly psiadin, showing itself to be significantly more effective than standard chemotherapy drugs such as DOX and 5FU.
To determine how these terpenes may exert their inhibitory actions, we looked at several to increased phosphorylation and, thereby, deactivation of the tumor suppressor RB protein [26]. This then relieves its repression of transcription factor E2F, allowing it to transcribe cyclins A and E, combining with CDK2 to take the cell through the G1 and G2/M checkpoints. We observed a general decrease in all the cyclins that were measured as well as in CDK4 in HepG2 cells treated with psiadin, indicating complete disruption of the cycling process. There was a small reduction in CDK2 which is thought to drive cells' progression into S and M phase [27]. An interacting factor p-KIP [28] was increased by psiadin. This is known to inhibit the interaction of CDK4/6 with cyclin D which complex is involved in RB phosphorylation. It may also block CDK2 interaction with cyclin E which allows cells to exit from G1 into G0.
Failure to progress through cell cycle checkpoints is thought to lead to apoptosis, and as with other natural products [29,30] this seems to be the consequence of psiadin exposure. Consistent with the MTT assay data, the viability of both cell lines was drastically reduced with a concurrent increase in cells undergoing both early and late phases of apoptosis as well as direct necrosis. Apoptosis is often divided into the extrinsic and intrinsic or mitochondrial pathways [31] involving activation of several caspases in the formation of an apoptosome, [32] also involving BCL2 family members such as BAX and BAK and release of pro-apoptotic proteins including cyt C [33]. Thus, psiadin in HepG2 cells elevated BCLX and BAK and substantially increased cyt C but surprisingly did not appear to increase caspases. Psiadin also did not affect the level of poly (ADP-ribose) polymerase (PARP), which is often involved in DNA repair. The mitochondrial membrane potential (ΔΨm), generated by proton pumps (Complexes I, III and IV), is critical for maintaining the physiological function of the respiratory chain to generate ATP [34]. Opening the mitochondrial permeability transition pore (MPTP) leads to the fairly catastrophic collapse of the ΔΨm and subsequent release of cyt C into the cytosol, which in turn triggers other downstream events in the apoptotic cascade as mentioned above.
Our data show that both psiadin and plectranthone mimicked the effects of a mitochondrial uncoupler (4-(trifluoromethoxy)phenyl) carbonohydrazonoyl dicyanide) in producing some reduction in the ΔΨm in both cell lines, indicating that these compounds induce end-stage mitochondrial dysfunction. This has been observed with a number of common anti-cancer drugs [35].
The effect was most pronounced in HepB3 cells with psiadin. In HepG2 cells, psiadin caused increased production of mitochondrial proteins Cyt C and BCLX but not of AIF or BID, which are other BCL2 pro-apoptotic proteins.
It has been reported that resistance to apoptosis may be dependent on activation of NF-B, [36] via its many target genes [37], and its action in preventing mitochondrial-mediated apoptosis through neutralization of ROS. Consistent with this are our findings that both terpenes, psiadin and plectranthone, which cause apoptosis, also significantly reduced NF-B binding of nuclear extracts to a consensus DNA response element for both cell lines. In addition to this, we also observed a very significant production of ROS induced by both drugs and in both cell lines. In this regard, we observed a small psiadin-induced decrease in CIAP 2 known to be NF-B regulated. Among other anti-apoptotic molecules, there was no change in FLIP or MCL.
We observed no change in the survival factor AKT. Similarly, there was no effect on the levels of the ERK pathway intermediate MEK or of p38.

Plant material
The aerial parts of three plants; Psiadia arabica Jaub. et Spach. (known locally as "Tabbak"), Plectranthus cylindraceus Hochst. ex Benth (known locally as "Al-Shar") and Cluytia richardiana L. (known locally as "Sa'eer") were collected from Wadi Al Uss, near Abha, Saudi Arabia between March and April, 2012. The plant identities were authenticated by Dr. Sultan-ul-Abdeen and voucher specimens (# 10331, 10352 and 10362, respectively) were deposited at the herbarium of the College of Pharmacy, King Saud University, Riyadh, Saudi Arabia. All plants names have been checked with http://www.theplantlist.org.

Extraction and isolation
Psiadia arabica. The powdered aerial parts (2 kg) were exhaustively extracted with chloroform in a Soxhlet for 72 h. Evaporation in vacuo gave a 260 g greenish residue that was partitioned between 3 L chloroform and water (3 × 1 L). The chloroform fraction, after evaporation, was partitioned between 10% aqueous methanol (2 L) and 3 portions each of 1 L of n-hexane. The combined hexane layers were washed with 100 ml of the methanol layer. Evaporation of the combined methanol layers gave 185 g as a gummy residue. This residue was chromatographed, in portions, on silica gel columns (each was 550 g, 25 × 7.5 cm) using CHCl3-MeOH, 4:1, yielding 1380 mg of the impure diterpene psiadin, which upon crystallization from methylene chloride-EtOAc gave 813 mg of pure psiadin as colorless needles.
Plectranthus cylindraceus. The dried powdered aerial parts (1.12 kg) were percolated at room temperature with 95% EtOH (3 × 4 L), and the extract was evaporated in vacuo to leave 22 g of residue, which was chromatographed over silica gel (500 gm, 4 × 130 cm), using increasing concentrations of EtOAc in n-hexane as an eluent, to yield, after crystallization from EtOAc-petroleum ether, 1400 mg of plectranthone as colorless prisms.
Cluytia richardiana. The dried powdered aerial parts (1.6 kg) were percolated successively at room temperature in petroleum ether (60-80 °C) and then in EtOAc. Upon drying in vacuo, the EtOAc fraction afforded 65 g of greenish-white precipitate, which was filtered off to yield 26 g of the precipitate. This precipitate was subjected to chromatographic purification over flash silica gel (2.6 kg). Elution with petroleum ether (60-80 °C)-EtOAc (8.5:1.5) yielded 560 mg saudinolide.
Crystallization from EtOAc-petroleum ether afforded 450 mg as colorless granules.

Terpenes identification
Melting points were determined in open capillary tubes using a Mettler 9100 electrothermal melting point apparatus and were uncorrected. IR spectra were recorded using a JASCO FTIR-4100 spectrophotometer. UV spectra were measured in MeOH using a UV-160 IPC UV-visible dual-beam spectrophotometer. The 1 H and 13 C NMR spectra were obtained on a Bruker Advance II 600-MHz spectrometer operating at 600 and 150 MHz, respectively. Both 1 H and 13 C NMR spectra were recorded in CDCl3, and the chemical shift values were expressed in δ (ppm) relative to the internal standard TMS. For the 13 C NMR spectra, spectral editing was determined by DEPT. 2D NMR data were obtained using the standard pulse sequence of the Bruker 600 for COSY, HSQC and HMBC.
High resolution ESMS were obtained using a double-focusing magnetic sector mass spectrometer (GC-MS DFS, Thermo).

Cell lines
Human hepatic carcinoma cells Hep3B and HepG2, were obtained from the American Type Culture Collection (ATCC; VA, USA). Theywere cultured in 90% Leibovitz's L15 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS) and grown at 37°C in an incubator with 5% CO2 atmosphere.

MTT assay
Cell viability was determined at various times (24-144 h) using MTT (3-(4,5-Dimethyl-2thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) assay, which is based on the conversion of MTT to formazan crystals by mitochondrial dehydrogenases [38]. Briefly, cells were seeded at 27 × 10 3 cells/well in 96-well plates and incubated at 37 ºC for 24 h. The cells were then treated with various concentrations of each terpene under investigation (or 0.2% DMSO vehicle for control condition) for various times (24-144h). MTT solution (5 mg/ml) was added to the incubated cells (20 μl/well); the cells were then incubated for another 4 h and the medium was discarded. DMSO (200 μl) was added to each well, and the absorbance measured in a micro-plate reader at 492 nm. All samples were assayed in triplicate in three independent experiments. The results (mean ± SEM) are expressed as % cytotoxicity; by dividing the densitometric readings for the drug treated conditions by that of the DMSO treated control.
In a separate series of experiments, the effect of each of the terpenes on the cancer cell lines was compared with camptothecin (CPT), 5-fluorouracil (5FU), doxorubicin (DOX) and ellipticine (ELP) over 72h of drug exposure.
As we later determined that the terpenes had effects on mitochondrial function and therefore might affect the MTT assay which depends on a reaction occurring within the mitochondria we also used the MTX assay as well as doing direct cell counting for a number of drug treated samples and obtained similar results. Therefore, we concluded that the reaction involving MTT was not affected.

Colony forming assay
Briefly, cancer cells (10 5 /well) were plated in a 24-well plate and incubated at 37°C for 18 h.
Thereafter, the cells were treated with serial doses of the test compounds (or DMSO vehicle for controls) and incubated at 37°C for 24 h. After that, cells were collected by trypsinization, washed with Hank's Balanced Salt Solution (HBSS), counted and plated in 24-well plates at 500 cells/well, and incubated at 37°C for 10-14 days. The resultant colonies were washed with cold phosphate-buffered saline (PBS), fixed with 100% methanol, and stained with 0.1% crystal violet. The colonies were counted using an inverted microscope [39].
suspended in 100 µl of annexin V fluorescein and propidium iodide in HEPES buffer. Following incubation in darkness at room temperature for 15 min, cells were analyzed by flow cytometry.

Measurement of mitochondrial membrane potential
To determine whether mitochondrial damage occurs as an early event in psiadin or plectranthone-induced apoptosis, changes in mitochondrial membrane potential (ΔΨm) were

NFB DNA-binding activity
Cancer cells were plated at 2.5 x 10 5 cells/ml into 24-well plates at 37 o C for 18 h, then treated with psiadin (50, 100 µg/ml), plectranthone (200, 400 µg/ml) or DMSO vehicle for 24 h. Cells were harvested and nuclear extracts prepared using a cytosol/nuclear fractionation kit from BioVision (CA, USA) as per the manufacturer's instructions. NF-B DNA-binding activity was determined using an NF-Bp65 Transcription Factor Assay Kit (ab133112; Abcam, UK) which utilizes a 96-well plate with a specific DNA sequence containing the NF-B response element immobilized onto the bottom of the wells. Following the manufacturer's protocol, nuclear cell extracts were added to wells and incubated with shaking for 1h at room temperature. Following washing 5 times with wash buffer, NF-B specific primary antibody was added to wells for 1h, subsequent washing as above, then addition of goat anti-rabbit HRP conjugated secondary antibody for another 1h. After further extensive washing and incubation for 15-45 min with developer solution, absorbance at 450 nm was determined in a plate reader.

Western blot analysis
Cancer cells were cultured in 6 well plates (5 x 10 5 cells/ml) to approximately 75-80% confluency and then treated with psiadin (75 μg/ml) or DMSO vehicle. After 24h exposure cells were harvested and recovered by centrifugation for 5 min at 1000 g. Fresh cell pellets were washed with PBS and then re-suspended into 300 μl of homogenization buffer containing 50 mM HEPES, 50 mM NaCl, 5 mM EDTA, 1% Triton X-100, 10 μg/ml leupeptin, 10 μg/ml aprotinin and 100 μg/ml PMSF. The Pierce BCA protein assay dye reagent (Pierce, Rockford, USA) was used to determine protein concentration in the cell lysate. About 30 μg of protein was mixed with an equal volume of 2 x sample loading buffer containing 100 mM Tris-Cl (pH 6.8), 4% (w/v) SDS, 0.2% (w/v) bromophenol blue, 20% (v/v) glycerol and 200 mM dithiothreitol, and heated at 90 o C for 10 min. Lysates were loaded onto 10% SDS-polyacrylamide gels and electrophoresed at 150 V for 1 h. Proteins were then transferred onto a PVDF membrane and, using the molecular weight markers lane as a guide, cut into narrow strips (in order to economize on antisera) in the size range of the expected protein to be detected. Each strip was then treated with 2% BSA for 1 h before being incubated overnight at 4 o C with various primary antibodies (listed in Supplementary Table). After removal of the antisera the membrane was washed and subsequently incubated with anti-HRP-conjugated secondary antibody (1:500) for 1 h, and signals developed with Super Signal ECL and visualized with Kodak X-ray film. Bands were quantified by densitometry and intensity calculated proportional to that obtained for β actin on the same blot.

Statistical analysis
Differences between mean values of tested terpenes was analyzed by the student's t-test or oneway ANOVA followed by Bonferroni post-hoe test using GraphPad Prism 8. P<0.05 was considered statistically significant.

Conclusions
In summary, we have examined the anti-cancer potential of three terpenes purified from natural products. Saudinolide was not useful, and plectanthrone was effective in some processes but only at very high concentrations. Psiadin, on the other hand, could be a very promising lead compound. It showed little activity against normal cells but was significantly more effective at killing two hepatic cancer cell lines than several currently used chemotherapeutic drugs. We examined several processes commonly associated with cancer cell metabolism but were unable to pinpoint any specific cellular target so that it may have a wide range of activity. Although, cytotoxic agents often result in many changes that are secondary to their primary effect, however, we did observe disruption of a number of specific cellular pathways, most prominently cell cycle proteins, stimulation of mitochondrially mediated apoptotic pathways, very pronounced induction of ROS, and inhibition in expression and function of the RB protein. In future experiments it would be worthwhile to examine genome-wide expression and perform whole proteome analysis of psiadin-treated cells. It will also be necessary to test the effects of psiadin on other cell lines to determine its general applicability. Also, in vivo animal studies, which were beyond the present investigations' scope, would be the next step to see whether psiadin is active against tumors, having established its effects in vitro. Table S1: Antibodies used in this project along with their target proteins and sources.