Cytotoxic Compounds from Kibatalia gitingensis (Elm.) Woodson

Ursolic acid (1), squalene (2), a mixture of α-amyrin acetate (3a) and lupeol acetate (3b), and isoscopoletin (4), isolated from the dichloromethane extracts of the leaves and twigs of Kibatalia gitingensis, were evaluated for their cytotoxic activities against three human cancer cell lines, breast (MCF-7) and colon (HT-29 and HCT-116), and a normal cell line, human dermal fibroblast-neonatal (HDFn), using the in vitro PrestoBlue® cell viability assay. Compounds 1-4 exhibited strong cytotoxic activities against HT-29 cells with IC50 values ranging from 0.6931 to 1.083 μg/mL. Furthermore, 1-4 were moderately cytotoxic against HCT-116 cells with IC50 values ranging from 4.065 to 11.09 μg/mL. These compounds were least cytotoxic against MCF-7 cells with IC50 values ranging from 8.642 to 25.87 μg/mL. The most cytotoxic against HT-29 cells, HCT-116 cells and MCF-7 cells are 2, 4 and 1, respectively.


INTRODUCTION
Kibatalia gitingensis (Elm.)Woodson, of the family Apocynaceae, is native to the Philippines where it is locally known as "laniti" or "laneteng-gubat".It is classified as vulnerable in the IUCN Red List of Threatened Species. 1 It is commonly used to make building materials and decorative carvings and is also known to contain medicinal properties due to its alkaloid content. 2 The leaves of K. gitingensis yielded a steroidal alkaloid, gitingensine, which was found to exhibit antispasmodic activity 3,4 and ataraxic properties, able to tranquilize smooth muscles as well as vasodilate arteries of the skeletal muscles and the splanchnic region. 5Other studies reported that the leaves of K. gitingensis contain kibataline 6,7 and 20-(epi-N-methyl) paravallarine. 8The plant contains an azasteroidal alkaloid which caused spontaneous motility in mouse and dog intestines and likewise removed serotonin-induced contractions. 9The bark of K. gitingensis yielded a complex mixture of alkaloids, including paravallarine, N-methylparavallarine, and 20-epiparavallarine. 10 Moreover, the stem bark of the

Sample Collection
Samples of leaves and twigs of Kibatalia gitingensis (Elm.)Woodson were collected from the De La Salle University-Science and Technology Complex (DLSU-STC) reforested area in February 2014.The samples were authenticated and deposited at the De La Salle University Herbarium with voucher specimen #908.

Isolation and Structure Elucidation
The isolation and structure elucidation of 1-4 from the leaves and twigs of K. gitingensis were reported previously. 12

Cell Viability Assay
The cytotoxicity of the K. gitingensis compounds was determined in an in vitro cell viability assay using PrestoBlue® (Molecular Probes®, Invitrogen, USA).This test is based on the principle that the enzyme, mitochondrial reductase, present in viable cells, can reduce the nonfluorescent, blue resazurin dye in the reagent, converting it to resorufin which is red and highly fluorescent.Hence, only viable cells are able to cause color change.The conversion is proportional to the number of metabolically active cells and is correlated to absorbance measurements.To the monolayers in the microtiter plate, 100 µL of filter-sterilized 1-4 were added to corresponding wells at two-fold serial dilutions to make final screening concentrations of 50, 25, 12.5, 6.25, 3.12, 1.56, 0.78, and 0.39 µg/mL.Wells with no compound served as negative controls, wells with Zeocin TM (Gibco®, USA) served as positive controls, and wells containing only cell culture media were used to correct for background color.The cells were further incubated (37°C, 5% CO 2 , 98% humidity) for 4 days.Ten microliters of PrestoBlue® was added to each well.The cells were incubated (37°C, 5% CO 2 , 98% humidity) for 2 hr.Absorbance measurements were carried out using the BioTek ELx800 Absorbance Microplate Reader (BioTek® Instruments, Inc., U.S.A.) at 570 nm and normalized to 600 nm values (reference wavelength).Absorbance readings were used to calculate for the cell viability for each sample concentration following the equation below.Nonlinear regression and statistical analyses were done using GraphPad Prism 7.00 (GraphPad Software, Inc.) to extrapolate the half maximal inhibitory concentration, IC 50 , the concentration of the compound which resulted in a 50% reduction in cell viability.The cytotoxicity of 1-4 was expressed as IC 50 values.All tests were performed in triplicates and data were shown as means.The extra sum-of-squares F test was used to evaluate the differences in the best-fit parameter (half maximal inhibitory concentration) among data sets (treatments) and to determine the differences among dose-response curve fits according to the software's recommended approach.One-way ANOVA (p<0.05) was also used to determine significant differences among treatments, followed by Tukey's multiple comparison post hoc test (p<0.05), to compare different pairs of data sets.Results were considered significant at p<0.05.

RESULTS AND DISCUSION
Ursolic acid (1), squalene (2), a mixture of α-amyrin acetate (3a) and lupeol acetate (3b), and isoscopoletin (4), isolated from the dichloromethane extracts of the leaves and twigs of K. gitingensis, were evaluated for their anti-proliferative activities against three human cancer cell lines, breast (MCF-7) and colon (HT-29 and HCT-116), and a normal cell line, human dermal fibroblast-neonatal (HDFn), using the in vitro PrestoBlue® cell viability assay.The % cell viability as a function of the logarithmic values of compound concentration is shown in Figures 2 and 3. Most plots nearly follow the typical sigmoidal curve which is characteristic of an inhibitory doseresponse relationship between treatments and cell viability.Figure 2 compares the anti-proliferative effects per cell line, while Figure 3 compares the effects per compound.The corresponding IC 50 values are summarized in Table 1.The breast cancer cell line (MCF-7) is only moderately susceptible to 1 and 3a and 3b, with IC 50 values of 8.642 and 11.13 μg/mL, respectively, and least susceptible to 2 and 4 with IC 50 values of 25.87 and 23.35 μg/mL.One-way ANOVA showed statistical difference between treatments (p<0.0001),but Tukey's multiple comparison post hoc test revealed that there are no pairwise differences between 1 and 3a and 3b, and 2 and 4 (p>0.05).The colon cancer cell line (HCT-116) is most susceptible to 4 with IC 50 values of 4.065 μg/mL, but showed moderate susceptibility only to 1, 2, and 3a and 3b, with IC 50 values of 7.225, 9.226, and 11.09 μg/mL, respectively.One-way ANOVA showed that all treatments are statistically different (p<0.0001),but Tukey's multiple comparison post hoc test showed no pairwise differences between 1 and 2, and 2 and 3a and 3b (p>0.05).The growth of the other colon cancer cell line (HT-29) exhibited   the strongest inhibition at the lowest concentrations of the compounds, with IC 50 values of 0.6931, 0.8836, 1.054, and 1.083 μg/mL for 2, 1, 4, and 3a and 3b, respectively.Tukey's multiple comparison post hoc test showed statistical differences between 2 and all other samples except 1 (p>0.05).The normal cell line, HDFn, responded to all the compounds, with IC 50 values of 2.106, 5.519, 6.218, and 7.628 μg/mL for 4, 2, 1, and 3a and 3b, respectively.The pairs of compounds, 1 and 2, and 1 and 3a and 3b are not statistically different (p>0.05).All the cell lines are susceptible to Zeocin.Data analysis showed statistical differences in the best-fit parameter (half maximal inhibitory concentration) among treatments and among the dose-response curve fits (Figures 2 and 3).Overall, comparing the three human cancer cell lines, HT-29 showed the most cytotoxic response with comparable IC 50 values for all the compounds tested.This was followed by HCT-116 cells which was most affected by 4 with an IC 50 value of 4.065.Among the cancer cell lines tested, MCF-7 showed the least response to the compounds.The compounds exhibited cytotoxic activities against the normal cell line, HDFn.The known anti-cancer drug, Zeocin, showed anti-proliferative activities as expected.Overall, 1-4 showed varying, but promising cytotoxic properties, especially for the treatment of HT-29 type of colon cancer cells.The US National Cancer Institute has defined the active cytotoxic limits of natural products as 20 μg/mL or less for crude extracts and 4 μg/mL or less for pure compounds. 15Pure compounds that exhibit active cytotoxicity may have some potential for drug development. 14he results showed that 1-4 from the dichloromethane extracts of K. gitingensis leaves and twigs can be further evaluated for the treatment especially of human colorectal type of cancer.The study also revealed that the cytotoxic activities of 1-4 were a function of the specific type of cancer cells targeted.When the two colon cancer cell lines were compared, the IC 50 values of 1-4 for HT-29 were lower than HCT-116, implying that the former could be more susceptible to anticancer treatments using the compounds tested.A difference in treatment responses between two colon cancer cell lines was also seen in previous studies. 16,17It was reported that changes in the expression profiles of genes associated with drug sensitivity between HCT-116 and HT-29 could influence how the cells react to different inhibitory compounds. 18 similar study using four human colon cancer cell lines (HCT-116, HT-29, HCT-15, and KM-12) showed that gene expression profiling after inhibition of signal transduction by 17-allylamino-17-demethoxygeldanamycin, a known inhibitor of the hsp90 molecular chaperone, could explain the cells' response under different treatment parameters. 19evious studies revealed that ursolic acid (1), squalene (2), α-amyrin acetate (3a) lupeol acetate (3b), and isoscopoletin (4) exhibited cytotoxic properties.Ursolic acid (1) was reported to promote apoptosis in tumor cells by activation of caspases and modulation of pathways influencing cell proliferation and migration. 20It also decreased growth and induced apoptosis in gastric cancer cell line BGC-803 and hepatocellular cancer cell H22 xenograft, both in vivo and in vitro studies. 21Other works showed that 1 exhibited anti-tumor activity against human colon carcinoma HCT15 cells, 22 and inhibited the colon cancer-initiating cells by targeting STAT3. 23Triterpene 1 and betulinic acid were found useful as therapeutic agents against estrogen-dependent tumors. 24Furthermore, the antiproliferative and apoptotic effects of 1 was found to have potential therapeutic use against prostate cancer. 25A recent study reported that 1 suppressed the proliferation of Jurkat leukemic T-cells, inhibiting PMA/PHA induced IL-2 and TNF-α production in a concentration-and time-dependent manner. 26Another study using cervical cancer TC-1 cells reported that ursolic acid-activated autophagy induced cytotoxicity and reduced tumor growth in a concentration-dependent manner as well. 27The anti-tumor activities of 1 against U87MG brain cancer cells were evaluated and it was found that both G1-phase arrest and autophagy were induced by the compound. 28In a study evaluating the anti-cancer properties of ursolic acid and three flavonoids, daidzein, baicalein, and hesperidin, it was found that 1 and baicalein inhibited the proliferation of MCF-7 breast cancer cells induced by PhIP, a food-derived carcinogen exhibiting estrogenic activities. 29The anti-cancer potential of 1 present in different berries has been reviewed. 20Thus, ursolic acid (1) was reported to exhibit cytotoxic properties against different cancer cells including colon and breast cancer cell lines which corroborate our findings that 1 showed high cytotoxicity against colon cancer cells with the lowest IC 50 values of 0.8836 μg/mL obtained for HT-29, 7.225 μg/mL for HCT-116, and 8.642 μg/mL for MCF-7.Squalene (2) was reported to exhibit anti-tumor activities against colon cancer in rodents. 30It also reduced colonic aberrant crypt foci (ACF) formation and crypt multiplicity in laboratory rats, indicating potential chemopreventive activities against colon carcinogenesis. 31In a study evaluating the anti-proliferative effects of squalene and other compounds from palm oil against two human breast cancer cell lines, MDA-MB-231 and MCF-7, it was found that there was a suppression of nuclear factor kappa-light-chain-enhancer of activated B-cells (NF-κB) in breast cancer cells exposed briefly to tumor necrosis factor-alpha (TNF-α), 32,33 hence affecting the mechanisms of apoptosis and carcinogenesis.The protective and therapeutic effects of squalene-containing compounds on skin tumor cells in laboratory mice have been reported as well. 34Relevant reviews on the bioactive properties of squalene have also been provided. 35,36Thus, 2 was reported to exhibit cytotoxic properties against colon and breast cancer cells which corroborate our findings that 2 showed high to moderate cytotoxicities against colon cancer cells, HT-29 and HCT-116, and breast cancer cells, MCF-7, with IC 50 values of 0.6931, 9.226, and 25.87 μg/mL, respectively.α-Amyrin acetate (3a) was mostly studied for its various potential medicinal applications.At a concentration of 100 mg/kg, 3a isolated from Alstonia boonei showed significant (p<0.05)inhibition of egg albumen-induced paw edema in mice. 37The same study showed that it promoted 60.3% reduction in total leucocyte count and significant (p<0.05)suppression (47.9%) of neutrophil infiltration.Lupeol, lupeol acetate and α-amyrin acetate exhibited significant anti-tyrosinase activity against the mushroom tyrosinase enzyme, with percent inhibitions of 67.7%, 66.2% and 62.2%, respectively, 38 indicating potential melanin biosynthesis inhibitory properties.Both α-amyrin acetate and β-amyrin acetate were also reported to exhibit sedative, anxiolytic and anti-convulsant properties. 39Very few studies have been made on the cytotoxic properties of 3a.The dichloromethane extract of Ficus o dorata (Blanco) Merr., containing α-amyrin acetate, 1-sitosteryl-3-βglucopyranoside-6'-O-palmitate, squalene, lutein, lupeol acetate, and β-carotene, exhibited anti-proliferative activities against the human cancer cells, lung adenocarcinoma epithelial (A549), stomach adenocarcinoma (AGS), prostate (PC3), and colon adenocarcinoma (HT29). 40Lupeol acetate (3b) was reported to exhibit cytotoxic activity against breast cancer cell (MCF7) with an IC 50 value of 26 µg/mL. 41Triterpenes, germincol, lupeol, α-amyrin, β-amyrin, olean-18-ene, and lupeol acetate, were isolated from the methanol extract of leaves and stems of Lactuca steriolla and showed varying cytotoxic activities against non-small cell lung adenocarcinoma cells (A549), human hepatocellular liver carcinoma cells (HepG2), human breast carcinoma cells (MCF7) and human colon carcinoma cells (HCT116). 42he chloroform extract of the leaves of Acokanthera oblongifolia, containing mixtures of isolated triterpenes, α-amyrin, lupeol acetate, lupeol, betulinaldehyde, and betulinic acid, showed some cytotoxic activities against human cancer cell lines, hepatocellular carcinoma (HepG2), breast adenocarcinoma (MCF7) and colorectal (HCT116), with IC 50 values of 37.6, 65.4 and 66.8 μg/ml, respectively. 43Thus, 3a and 3b were reported to exhibit cytotoxic properties against colon and breast cancer cells which corroborate our findings that the mixture of 3a and 3b showed high to moderate cytotoxic properties against colon cancer cells, HT-29 and HCT-116, and breast cancer cells, MCF-7, with IC 50 values of 1.083, 11.09, and 11.13 μg/mL, respectively.It is hypothesized that the synergistic effects of both compounds could have caused the observed anti-proliferative effects against the cancer cells studied.Isoscopoletin (4) showed substantial inhibition in a cell proliferation assay using human CCRF-CEM leukaemia cells with an IC 50 value of 4.0 µM. 44Another study reported that 4 exhibited cytotoxic activities against human lung cancer (A549), human breast cancer cell (MCF7) and human liver cancer (HepG2) with IC 50 values of 5.25, 8.58 and 4.76 µM, respectively. 45Moreover, 4 showed cytotoxicity against colon cancer (HCT116) cells with an IC 50 value of 10% at 100 ppm. 46Compound 4 from Artemisia argyi, artemisinin from Artemisia annua, and the latter's semi-synthethic derivative, artesunate, showed the greatest activity in in vitro cytotoxicity tests against HCT116 colon adenocarcinoma cell line, with IC 50 values ranging in concentration from micromolar to millimolar amounts. 47It was hypothesized that isoscopoletin enhanced its anti-cancer property by influencing the activity of p53 tumor protein which is a genetically important process in cancer progression.Thus, 4 was reported to exhibit cytotoxic properties against several cancer cell lines such as colon and breast which corroborate our findings that 4 showed varying cytotoxic activities against colon cancer cells, HT-29 and HCT-116, and breast cancer cells, MCF-7, with IC 50 values of 1.054, 4.065, and 23.35 μg/mL, respectively.Compound 4 also exhibited the highest cytotoxicity against the human dermal fibroblast-neonatal (HDFn) normal cell line, with an IC 50 value of 2.106 μg/mL.More studies are needed to fully examine and understand the effects of 4 on normal cells.