Natural polyacetylenic oxylipin; PPARγ

The cell viability of MDA-MB-231 and MDA-MB-468 cells was significantly reduced by Falcarindiol (3, 6, 12, 24 μM) over a 24-hour period. The cell viability of MCF-10A cells was maintained until the dose of Falcarindiol reached 24 μM, at which point Falcarindiol preferentially induced breast cancer cell death [1]. In MDA-MB-231, MDA-MB-468, and SKBR3 cells, falcarindiol (6 μM; for 2 hours) induces autophagy and leads to significant levels of LC3-I. Falcarindiol (6 μM; for 2, 4, 8, and 24 hours) dose- and time-supplementedly increases GRP78 levels in MDA-MB-231 cells [1]. Falcarindiol (1–20 μM) has no effect on the viability of HT-29 and hMSC cells. Cells can only be toxically affected by concentrations of falcarindiol greater than 50 μM [2]. Over the course of 24 hours, falcarindiol (5 μM; 10 minutes, 1 hour, and 24 hours) dramatically increases PPARγ2 expression [2].

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Falcarindiol (FAD) is a natural polyyne have been found in many food and dietary plants. It has been found to have various beneficial biological activities. In this study, we demonstrated its anticancer function and mechanism in breast cancer cells. We found that FAD preferentially induces cell death in breast cancer cells. FAD-induced cell death is caspase-dependent. However, FAD induces autophagy to contribute to the cell death. Blocking autophagy by either chemical inhibitors or genetic knockout of autophagy signaling component inhibits FAD-induced cell death. We further found that FAD-induced cell death is mediated by the induction of endoplasmic reticulum stress. We also identified that FAD has synergistic effect with approved cancer drugs 5-FU and Bortezomib in killing breast cancer cells. Summarily, these data demonstrate that FAD has strong and specific anticancer effect in breast cancer cells, and provide some insights about the roles of autophagy in FAD-induced cell death. [1]

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Falcarindiol (FaDOH) is a cytotoxic and anti-inflammatory polyacetylenic oxylipin found in food plants of the carrot family (Apiaceae). FaDOH has been shown to activate PPARγ and to increase the expression of the cholesterol transporter ABCA1 in cells, both of which play an important role in lipid metabolism. Thus, a common mechanism of action of the anticancer and antidiabetic properties of FaDOH may be due to a possible effect on lipid metabolism. In this study, the effect of sub-toxic concentration (5 μM) of FaDOH inside human mesenchymal stem cells (hMSCs) was studied using white light microscopy and Raman imaging. Our results show that FaDOH increases lipid content in the hMSCs cells as well as the number of lipid droplets (LDs) and that this can be explained by increased expression of PPARγ2 as shown in human colon adenocarcinoma cells. Activation of PPARγ can lead to increased expression of ABCA1. We demonstrate that ABCA1 is upregulated in colorectal neoplastic rat tissue, which indicates a possible role of this transporter in the redistribution of lipids and increased formation of LDs in cancer cells that may lead to endoplasmic reticulum stress and cancer cell death. [2]

Diet Supplemented With Falcarindiol/FaDOH and FaOH Increase ABCA1 Expression in Neoplastic Tissue in Rats [2]
A previous study of the effect of dietary Falcarindiol/FaDOH and FaOH in neoplastic tissue in a rat model did show a downregulation of PPARγ as opposed to the current study in HT-29 cells. The downregulation was unexpected, but could be due to the complexity of the tissue sample, since the neoplastic cells in the tissue sample could be affected by microbiota and the cells in the surrounding tissue (Kobaek-Larsen et al., 2019). However the activation of PPARγ can lead to increased ABCA1 gene transcription in macrophages (Chawla et al., 2001) and ABCA1 has shown to have anticancer activity in colon cancer cells (Smith and Land, 2012). Therefore, the effect of FaDOH and FaOH on ABCA1 gene regulation was studied in rats receiving a standard rat diet (SRD) supplemented with 7 µg FaDOH g-1 feed and 7 µg FaOH g-1 feed compared with rats receiving SRD without supplement (Figure 7). RT-qPCR analyses showed no significant difference in the expression level in healthy tissue for ABCA1 when the rats received FaOH and FaDOH as a food supplement compared to rats receiving only SRD. However, a significant upregulation in the expression level was detected when comparing biopsies of neoplastic tissue from rats receiving a SRD supplemented with FaOH and FaDOH with biopsies of neoplastic tissue from rats receiving SRD.

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The expression of ABCA1 is upregulated in tumor tissues when falcarindiol (7 μg/g; food) is introduced at five weeks of age [2].

Viability Test [2]
Cells were seeded at a concentration of 5,000 cells/well in 96 well plates in 100 µl of medium. After 24 h, the medium was replaced with fresh medium that included Falcarindiol/FaDOH at different concentrations. After a further 72 h the cells were visualized using an inverted microscope (Motic AE31) with a 10× objective and were then photographed. The wells were then assessed for viability using a resazurin viability assay according to the manufacturer’s instructions. Resazurin solution was mixed with fresh medium at 1:9 and 100 µl of this mixture was added to each well. After 3 h of incubation 80 µl was transferred to a new 96 well plate and absorbance at 600 nm and 690 nm was read using a 96 well plate reader. Background absorbance at 690 nm was subtracted from absorbance at 600 nm to get dye specific absorbance. Viable cells convert the dye into a red product and 100% viability was set to the difference in absorbance between non-treated cells and unreacted dye that had been incubated in wells without cells. Percent viability was then calculated for the samples as the fraction of color change compared to the non-treated cells.

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Preparation of Samples for Imaging and Microscopy [2]
Borosilicate glass coverslips (VWR) were sterilized in 98% ethanol and air dried under sterile conditions and placed in 6 well plates. 50,000 cells (hMSC Tert4, p44) were seeded on the 18 mm square or 25 mm round coverslips in 3 ml complete MEM medium with 10% fetal bovine serum and 1% penicillin/streptomycin and grown in a humidified chamber at 37°C with 5% CO2. After 24 h, the medium was removed and replaced with complete MEM medium (control) or complete MEM medium with 5 μM Falcarindiol/FaDOH. After 1 h, 5 h, and 24 h the wells were rinsed twice with PBS and cells were fixed in 4% formaldehyde for 10 min at room temperature. The coverslips were then stored in PBS and in darkness at 4°C until use. For each time point, three biological replicates were prepared and fixed for both controls cells and cells exposed to FaDOH.

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Raman Imaging [2]
An in-house build Raman imaging setup was utilized for cell imaging. The Raman excitation source comprises a 532 nm laser coupled via free space optics into an Olympus BX60 microscope focusing the laser onto the sample through a 100X, NA=1 water immersion objective resulting in an effective spot diameter around 650 nm. The backscattered Raman signal was collected by the microscope and directed via a 105 μm fiber to an Acton SpectraPro 2500i f/6.5, 600 l/mm spectrograph with a 100 μm slit and a Princeton Instruments PIXIS 400F 1340×400 Pixel CCD camera operated at −75°C resulting in a 3−6 cm-1 spectral resolution. Cells were mapped utilizing a motorized stage at 1 μm steps (best spatial resolution of stage), and spectra were collected with an excitation power of 100 mW (using water immersed samples), 1 accumulation and 1 s acquisition time per spectrum. No sample degradation was noticed using this power density. For each control and Falcarindiol/FaDOH a minimum of three cells were imaged for each time point and biological replicate resulting in at least 9 cell maps per time point.

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White Light Microscopy [2]
An Olympus IX81 microscope was used to acquire images with the 150× (NA 1.45) oil objective and the DIC (Differential Interference Contrast) channel. Fixed untreated control cells and 24 h FaDOH treated cells were imaged. Fifty images were analyzed for both sets of samples; LDs were counted with ImageJ in every cell. A non-parametric Wilcoxon Rank Sum test was executed to compare the two sets of samples, which did not follow a normal distribution. P-values < 0.05 were considered significant.

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PPARγ Gene Expression in HT-29 Cells [2]
The expression level of the gene PPARγ2 in HT-29 cells was investigated with Real-time quantitative PCR (RT-qPCR). 300.000 HT-29 cells/well were seeded in 6-well plates the day before Falcarindiol/FaDOH treatment. Then media was removed and 5 µM FaDOH in 3 ml media was added to the cells and the cells were incubated for additional time. Each well in the 6-well plate, had a different Falcarindiol/FaDOH treatment time. The following times were used: 0 min (control), 10 min, 1 h and 24 h. Control (0 min) experiment was performed with media without FaDOH added. The 24 h treatment was starting 24 h before 0 min treatment and so on. Thereby all the treatments in the same plate were stopped at the same time. The treatment was stopped by harvest of the cells using 500 µl of QIAzol Lysis Reagent and shaking at 900 rpm for 2 min. The RNA was extracted using EconoSpin column purification and subsequently 0.3 µg RNA was converted into complementary DNA (cDNA). The synthesized cDNA was then analyzed with RT-qPCR using GoTaq® Probe qPCR master mix and MyGo Mini instrument. Housekeeping gene was ribosomal protein large P0 (RPLP0).

Gene Expression in Neoplastic Rat Tissue [2]
Animal studies were approved by the Central Animal Experimentation Inspectorate in Denmark (License no. 2015-15-0201-00708). Five weeks old male rats (F344 strain), with a certified health report, were used. The rats were acclimatized for one-week where after they were divided into two groups. Group 1 was fed standard rat diet (SRD) and group 2 was fed SRD supplemented with FaOH and Falcarindiol/FaDOH as previously described (Kobaek-Larsen et al., 2019). Rats were housed as described in earlier studies (Kobaek-Larsen et al., 2017). The gene expression of ABCA1 in rat tissue from colon biopsies was analyzed using RT-qPCR. The biopsies include neoplastic tissue from the group 1, receiving SRD and size-matched neoplastic tissue from the group 2 receiving rat diet supplemented with 7 µg FaOH g-1 feed and 7 µg Falcarindiol/FaDOH g-1 feed. RNA from the tissue was extracted using QIAzol, purified using EconoSpin columns, converted into cDNA, and finally evaluated using RT-qPCR. Multiplex RT-qPCR was performed with the housekeeping gene beta-glucuronidase (GUSB) as an internal reference. ABCA1 was measured in four replicates, run pairwise. Up- or downregulation of the ABCA1 was quantified using the comparative CT method as described under gene expression in HT-29 cells. Primers (MERK) and probes can be found in Table 1.

[1]. Autophagy contributes to falcarindiol-induced cell death in breast cancer cells with enhanced endoplasmic reticulum stress. PLoS One. 2017 Apr 25;12(4):e0176348.

[2]. Falcarindiol Purified From Carrots Leads to Elevated Levels of Lipid Droplets and Upregulation of Peroxisome Proliferator-Activated Receptor-γ Gene Expression in Cellular Models. Front Pharmacol. 2020 Aug 28;11:565524.

Falcarindiol has been reported in Anthriscus nitida, Eleutherococcus koreanus, and other organisms with data available.

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Our data suggest that Falcarindiol/FAD has a strong synergistic effect with chemotherapy drug 5-FU and proteasome inhibitor bortezomib. The synergistic effect may be due to either stress sensitization or overload in the combinations. Cancer cells generally exhibit increased cellular stresses, and more require stress support pathway for survival. This makes cancer cells more susceptible to either stress sensitization or overload than normal cells. Previous studies have suggested that stress sensitization or overload is likely to be the mechanism of chemotherapy drugs. Our data show that FAD kills breast cancer cells through inducing ER stress. Agents that target distinct or same branches of cellular stress response potentially lead to synergistically in inducing stress overload in cancer cells. Consistent with that, FAD and 5-FU, which induces ER stress and DNA damage stress respectively, show synergistic effects in killing cancer cells. Several studies have suggested that Bortezomib induces ER stress through interfering with ERAD, and further contributes to its cytotoxic activity against cancer cells. FAD and Bortezomib, which both induces ER stress, also exhibit synergistic effects in killing cancer cells.

Conclusions: Our studies demonstrated the specific anticancer effect of Falcarindiol/FAD in breast cancer cells. We revealed that FAD-induces autophagy plays an important part in cell death caused by enhanced ER stress. We identified that FAD showed strong synergistic effects with approved anti-cancer drugs 5-FU and Bortezomib. FAD may be a potential novel therapeutic agent for the treatment of human breast cancer. [1]

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It has previously been shown that Falcarindiol/FaDOH increases cholesterol efflux, partly via expression of ABCA1 (Wang et al., 2017) that is induced by PPARγ (Chawla et al., 2001; Mogilenko et al., 2010; Wang et al., 2017). ABCA1 is important for the formation of HDL and thereby cholesterol removal (Wang and Tall Alan, 2003). Since increased synthesis of cholesterol (Pihlajamäki et al., 2004) and downregulation of ABCA1 (Patel et al., 2011) is linked to type 2 diabetes FaDOH could have antidiabetic properties as a PPARγ agonist both by stimulating glucose uptake (El-Houri et al., 2015a) and increasing cholesterol removal through upregulation of ABCA1. Cholesteryl esters is found naturally in LDs (Fujimoto and Parton, 2011) and the fact that endmember spectra of Raman spectroscopy indicates the presence of cholesteryl oleate and lineolate upon FaDOH treatment, could have important implication for the function of FaDOH, since linoleic acid has been linked to antidiabetic function (Wu et al., 2017). Further studies are required to investigate and prove this connection.

In conclusion, Falcarindiol/FaDOH induces change in lipid content in hMSCs exposed to sub-toxic amounts of FaDOH as observed using label-free Raman spectroscopic mapping and white light microscopy. Cells treated with FaDOH show a significant upregulation of LDs compared to control cells, and Raman spectroscopy indicated the formation of cholesteryl lineolate. RT-qPCR showed increased expression of PPARγ2 in cancer cells and increased expression of ABCA1 in neoplastic tissue, which could indicate an increased formation of LDs in cancer cells and normal cells, thus contributing to the understanding of the anticancer and antidiabetic properties of FaDOH. The involvement of PPARγ in the upregulation of ABCA1 by FaDOH and the formation of LDs cannot be excluded based on our results and therefore further investigations in cells of normal and cancer origin are needed, which may include treatment with PPARγ antagonists to explore the potential anticancer and antidiabetic properties of FaDOH.[2]

C17H24O2

260.37126

260.177

55297-87-5

(+)-(3R,8S)-Falcarindiol;225110-25-8

5281148

Light yellow to brown liquid

1.0±0.1 g/cm3

408.2±45.0 °C at 760 mmHg

184.7±23.3 °C

0.0±2.2 mmHg at 25°C

1.524

6.32

2

2

9

19

394

2

CCCCCCC/C=C\[C@@H](C#CC#C[C@H](C=C)O)O

QWCNQXNAFCBLLV-YWALDVPYSA-N

InChI=1S/C17H24O2/c1-3-5-6-7-8-9-10-14-17(19)15-12-11-13-16(18)4-2/h4,10,14,16-19H,2-3,5-9H2,1H3/b14-10-/t16-,17+/m1/s1

(3R,8S,9Z)-heptadeca-1,9-dien-4,6-diyne-3,8-diol

Falcarindiol; (3S,8S)-Falcarindiol; (3R,8S,9Z)-heptadeca-1,9-dien-4,6-diyne-3,8-diol; AC1NQY3Z; Falcalindiol; 55297-87-5; Heptadeca-1,9-diene-4,6-diyne-3,8-diol; (8S,9Z)-heptadeca-1,9-dien-4,6-diyne-3,8-diol;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ≥ 33.33 mg/mL (~128.01 mM)

Solubility in Formulation 1: 1.11 mg/mL (4.26 mM) in 10% DMSO + 40% PEG300 +5% Tween-80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 11.1 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 + to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)