A novel carissic acid from fruits of Carissa carandas induced apoptotic cell death in A-549 cells following the activation of caspases

Abstract This study was designed to identify cytotoxic compounds from Carissa carandas extract. The cytotoxic activity of extract and fractions were assessed against eight cancer cell lines. The chloroform fraction obtained from methanolic extract exhibited significant activity against MCF-7, HT-29, A-549 with IC50 values of 3.98 µg/mL (MCF-7), 1.28µg/mL (HT-29) and 1.48 µg/mL (A-549) respectively. Further investigation led to the isolation of novel compound carissic acid (CA), which was confirmed by detailed spectroscopy studies. CA exhibited notable activity with IC50 values of 3.47 µM for A-549, 2.65 µM for HT-29 and 13.58 ± 0.59 µM for MCF-7 cells. CAcaused chromatin condensation with decrease of mitochondrial membrane potential and also confirmed cell death via Reactive Oxygen Species (ROS) generation and significantly decreased the colony formation in dose-dependent manner. The overall findings suggested that CA demonstrates cytotoxic effect by inhibiting cell proliferation and promoting apoptosis in lung (A-549) carcinoma cell line. Graphical Abstract


Introduction
Cancer is a leading cause of death worldwide affecting millions of people every year, moreover the increase in number of cases are estimated to be 21 million by 2030 (Siegel et al. 2014;American Cancer Society 2016).Natural dietary products have tremendous role in cancer chemoprevention as consumption of fruits and vegetables leads to the reduced risk of cancers (Bankar et al. 1994;Boeing et al. 2012).To combat the cancer/develop novel anticancer drugs, intense search is going on various biological sources.Therefore, >30 plant-derived compounds have been isolated, used for clinical trials and are found to be active on various cancer cells (Nirmala et al. 2011).Various epidemiologic studies suggest that high intake of fruits considerably leads to reduced risk of chronic diseases.Carissa carandas Linn commonly known as karonda is an exotic fruit grows wild in India in the tropical and subtropical Mediterranean region (Donaldson 2004).Traditionally different parts of C. carandas have been used against various ailments like hyperdipsia, diarrhoea, stomachic, anorexia, ulcer, sore throat, syphilitic pain, burning sensation, scabies and pruritus (Pullaiah 2006;Lim 2012).Previous pharmacological studies have reported anti-diabetic, anti-microbial, anti-tumour, anti-convulsant, hepatoprotective, anti-inflammatory and anti-viral activities (Bhaskar and Balakrishnan 2009;Hegde and Josh;Itankar et al. 2011;Agarwal et al. 2012;Sumbul and Ahmed 2012) in fruit extracts.This research aimed to determine in vitro cytotoxic potential of C. carandas fruit extract against human cancer cell lines and isolate its bioactive constituents.

Structure characterization of carissic acid
The molecular formula of carissic acid (CA) was established as Further, the 1 H NMR spectrum showed seven methyls, in addition to two resonances at δ 5.24 and 3.19 ppm which displayed in HSQC to δ 125.5 (C-12) and 78.5 (C-3), respectively.Other proton and carbon signals were accordance to the common triterpenoids.Further, HMBC spectrum exhibited long-range correlations from a methyl (Me-27) at δ 0.93 ppm to δ 39.4 (C-8), 41.9 (C-14), 27.7 (C-15) and 138.1 (C-13) ppm.Methyl (Me-26) at δ 0.96 ppm showed HMBC correlations with four carbon at δ 32.9 (C-7), 39.4 (C-8), 41.9 (C-14) and 48.9 (C-9) ppm.Two methyl groups at δ 0.93 and 0.98 ppm showed HMBC correlations to carbon at δ 38.5 (C-4), 55.3 (C-5) and 78.5 (C-3) ppm which supports that those two methyls are attached to the same carbon neighbouring the oxygenated carbon at C-3 (Figures S3-S6, Supplementary Material).Based on above HMBC correlations, compound can be characterized as ursolic acid however compound did not show identical TLC and optical rotation (Papanov et al. 1992) which indicate that compound is different and may differ only in relative configuration.
The relative configuration of CA was determined by Nuclear Overhauser Effect (NOE) correlations observed in a NOESY spectrum (Figure S3).NOE from H-3 to H 3 -24, H 3 -25, H-5 and H-11 together with correlations from H 3 -23 to H 3 -26, established the cis-fused ring 1 and 2 in CA which is totally different from ursolic acid where trans-fused system exist.An additional NOE from H 3 -26 to H 3 -30 and H-18 together with correlations observed from H 3 -27 to H-11 and H 3 -29confirmedtrans existence of H 3 -29/H 3 -20 methyls in ring 5 of terpene.Thus, the structure of CA is therefore defined as 3S, 5R, 9R, 25S, 26R, 27S, 18S, 28S, 29R and 30S ursan type new triterpene named carissic acid (Figure 1).Compound has been isolated for the first time from any plant source.Two minor compounds lupeol and betulin were also isolated and identified from this fraction.They were characterized by identical spectroscopic data (COSY, HSQC and HMBC) and chemical studies (Tijjani et al. 2012;Shwe et al. 2019).

CA induced chromatin condensation
DAPI is a fluorescent dye binds to minor groove of A-T region of DNA.Induction of apoptosis results in characteristic morphological alterations like chromatin condensation (pyknosis), cell shrinkage, nuclear deformation, formation of apoptotic bodies and their degradation by adjacent cells.A-549 cells treated with CA, that is, 14, 24 and 48 µM were stained with DAPI and observed under fluorescence microscope.The healthy normal cells represent the uniform appearance of blue-coloured nuclei.When Cells were treated with standard drug, that is, paclitaxel, showed condensed nuclei with increased intensity.CA-treated cells also exhibited chromatin condensation of cell nuclei in dose-dependent manner (Figure S8).In treated A-549 cells, the fluorescence intensity was more as compared to untreated cells after 24 h incubation.Moreover, the size and shape of cell nuclei were different in both control and drug-treated cells.Therefore, the results indicated that CA induced fragmentation of DNA as well as reduced number of cell nuclei in concentration-dependent manner.The surface morphological changes of A-549 cells were observed by scanning electron microscope and the figure clearly depicts that CA triggered apoptosis in A-549 cells in concentration-dependent manner and also the apoptotic bodies increased with an increase in concentration of CA (Figure S9).

CA confirmed cell death through ROS generation
ROS were found using the non-fluorescent dye DCFDA (2,7-dichlorofluorescein diacetate), which turns green when present.It is a probe that can detect hydrogen peroxide and be used to measure hydrogen peroxide levels in normal cells.In the presence of free radicals, it readily crosses the plasma membrane and oxidizes to produce DCF (dichlorofluorescein), a green fluorescent byproduct that is excited at 495 nm and emits at 520 nm.The positive control (0.5% H 2 O 2 ) produced green fluorescence after being dyed with DCFDA, which was then compared to the drug-treated cells.The results demonstrate that ROS generation is necessary for cell death as shown in (Figure S10) by the significant dose-dependent increase in fluorescence of drug-treated cells.

CA-induced loss of mitochondrial membrane potential
Mitochondrial membrane potential (MMP) plays a key role in determining the mitochondrial function; it creates an electrochemical gradient through a series of chemical reactions for the synthesis of ATP that ultimately generates the mitochondrial membrane potential.The Rh-123 staining was used to detect the integrity of mitochondrial membrane.There was loss of mitochondrial integrity which results in leakage of Rh-123 and thereby consequent decrease in fluorescence.Results revealed the loss of MMP in CA-treated cells with observed increase in concentration as compared to untreated cells with intact mitochondria.The compound CA was found to cause concentration-dependent depolarisation of MMP and these findings support the fact that alteration of mitochondria function plays a vital role in apoptotic cell death (Figure S11).

Effect of CA on colony formation
After various treatments of drug, cells have either retained the capacity for producing large number of progenies or cause cell death.A-549 cells treated with CA at different concentration of 14, 24, 48 µM and after the treatment, the cells were seeded in appropriate dilution.The results suggest that the compound effectively inhibited the colony formation of A-549 cells in a dose-dependent manner.Mean size of the control colonies was larger as compared to the colonies of drug-treated cells (Figure S12).

CA leads to inhibition of cell migration
The effect of CA on the migration of A-549 cells was evaluated by an in vitro wound-healing assay.After the drug treatment, the images of cell migrated into scratched area were captured.The results revealed that in untreated cells after 24 h, due to cell migration and cell proliferation, wound closure was observed clearly whereas in CA-treated cells with increased concentration of 48 µM, the inhibition of cell migration occurred and the same type of activity was seen in cells treated with paclitaxel, where the scratch made was clearly visible.This showed the antiproliferative effect of CA treatment leads to the impaired cell migration (Figure S13).

CA induced the activation of caspase and PARP
To assess the molecular mechanism underlying the apoptotic effect of compound CA on A-549 cancer cells, the expression of two different apoptosis-related proteins (Caspase-3 and PARP) were analyzed by western blotting.Among caspases, caspase-3 is a frequently activated protease responsible for specific cleavage of many cellular proteins required for cellular function and survival.PARP (poly ADP-ribose polymerase), a nuclear enzyme is one of the cellular substrates of caspases that is involved in DNA repair, DNA stability and transcriptional regulation.Cleavage of PARP by caspases is considered as sign of apoptosis.The results revealed that CA-treated cells caused the expression of caspase-3 in a concentration-dependent manner and the typical 89 kDa cleaved fragment of PARP was observed.Therefore, western blot analysis showed that CA treatment increased caspase-3 expression indicating caspase-3 mediated cell death via caspase dependent pathway (Figure S14).
Overall, the current work was carried out to examine the in vitro cytotoxic potential of karonda extracts and isolated compounds.Major compound CA was investigated in detail against A-549 cancer cell line.The results showed that the extract from the fruit of C. Carandas displayed significant in vitro cytotoxic effect against three cancer cell lines (A-549, HT-29 and MCF-7).The chloroform fraction showed notable results by inhibiting the growth of A-549 cells by 100% at a dose 100, 50, 30 µg/mL and 99% at 10 µg/mL respectively.For HT-29 cell line, GI of 100% at 100 and 50µg/mL, 99%, 94% GI at 30 and 10µg/mL was observed.In case of MCF-7 cancer cell line, chloroform fraction showed significant cytotoxic potential with GI of 100%, 98%, 89%, 77% at 100, 50, 30 and 10 µg/mL respectively.On the basis of in vitro cytotoxic potential of chloroform fraction, the IC 50 values obtained were 1.48 ± 0.002 µg/mL (A-549), 1.28 ± 0.02 µg/mL (HT-29) and 3.98 ± 0.24 µg/mL (MCF-7).As per the US NCI plant screening program, if the IC 50 value of crude extract with an incubation period between 48 and 72 h is <20 µg/mL, the extract possesses in vitro cytotoxic activity.Our results indicated, that all the IC 50 values calculated are <10 µg/mL thereby suggesting strong in vitro cytotoxic efficiency of chloroform fraction.CA isolated from chloroform fraction exhibited potent cytotoxicity towards A-549, MCF-7 and HT-29 with maximum GI and IC 50 values <10 µM in case of A-549 and HT-29.CA showed cytotoxic effect on the above-mentioned cell lines leading to cell death.To better understand the anticancer potential of CA, it was investigated against lung (A-549) cancer cell line via different assays to confirm the basis of apoptotic cell death.It induced chromatin condensation as observed by DAPI staining and ROS generation as well as loss of mitochondrial membrane permeability The results support the fact that mitochondria, an important organelle plays major role in mediating apoptosis (Nianyu et al. 2003;Kug et al. 2007) and reduced MMP is considered initial step in mitochondrial-mediated apoptosis (Banskota et al. 2015).Furthermore, CA leads to impaired cell migration in dose-dependent manner, thereby caused the increased activation of caspase-3 and significantly induced PARP cleavage as compared to control cells.All these observations confirmed the pivotal role of CA in mediating apoptosis in A-549 cells.

General experimental procedure
All Details of chemicals and instruments used in these studies are provided in the Supplementary Material.% % Growth inhibition 1 cell viability = − 00

Assessment of nuclear morphology
The detailed description is provided in the Supplementary Material.

Mitochondrial membrane potential (MMP) assay
The detailed description is provided in the Supplementary Material.

Detection of ROS accumulation
The detailed description is provided in the Supplementary Material.

Colony formation assay
The detailed description is provided in the Supplementary Material.

Scanning electron microscopy
The detailed description is provided in the Supplementary Material.

Wound healing assay
The detailed description is provided in the supplementary material.

Western blotting
The detailed description is provided in the Supplementary Material.

Statistical analysis
The experiments were done in triplicates and each data represents the average of at least three independent experiments.The data was expressed as Mean ± SD and one-way analysis of variance technique was used for the significant difference between the groups.IC 50 value was calculated by Graph PAD Prism software version 5.0.

Conclusions
To conclude, CA isolated from C. carandas exhibited potent cytotoxicity towards A-549, MCF-7 and HT-29 with maximum GI.CA induced dose-dependent decrease in cell proliferation with reduced mitochondrial membrane potential.The antiproliferative effect of CA confirmed the cell death through ROS generation and also led to the impaired cell migration.It was further confirmed by changes in apoptosis associated proteins in A-549 cells through western blotting.PARP cleavage was increased accordingly suggesting the role of caspase-dependent pathway via caspase-3.Taken together, the results confirmed the anticancer properties of CA on A-549 cells mediated the induction of apoptosis following the activation of caspases.These findings suggest that CA can be a potential agent for lung cancer therapy and further in-vivo studies have to perform to validate these results and further development.
70% or more has been indicated in bold numbers, Mark (-) indicates that particular cancer cell line was not treated with that positive control, symbol (*) means not evaluated, significance of bold values ( a P < 0.01 and b P <0.05).
C 30 H 48 O 3 based on HR-MS spectrum (Figures S1 and S2, Supplementary Material) revealing a molecular ion peak at m/z 455.35271 [M-H] − .The degrees of unsaturation were found to be seven indicating that the compound could be pentacyclic triterpenoid.Further, confirmation was provided by 13 C and Distortionless Enhancement by Polarization Transfer Nuclear Magnetic Resonance (DEPT NMR) revealing the presence of 30 carbon resonances distinguished into seven methyl, nine methylene, seven methines in addition to two carbon resonances at δ c 78.5 and 125.5 ppm representing one oxygenated aliphatic (C-3) and one olefinic (C-12) carbons, respectively, together with seven quaternary carbons.The appearance of δ 125.5 and 138.1 ppm indicate the presence of a double bond.The most downfield signal at δ c 180.4 indicates the presence of carboxylic function.

Table 1 .
Growth inhibitory effect of Carissa carandas against different cancer cell lines.