Helicteres isora Ethanol Extract Induces Apoptotic Cell Death in HepG2 Cells

The genus Helicteres (Malvaceae) includes about 60 species distributed in the tropics of Asia and America (Cowie, 2011; Mabberley, 2008). This genus can be readily recognized by stamens and pistil forming an androgynophore, united sepals, oblong fruits with hairs and wingless seeds (Chantaranothai and Poompo, 2019). In Vietnam, eight species and one variety of the genus Helicteres have been recorded according to recent publications, inclusion: Helicteres viscida Bl, H. angustifolia L., H. angustifolia var. glaucoides Pierre, H. angustifolia var. obtusa Pierre, H. angustifolia glabriuscula Wall, H. isora L., H. lanceolata DC., H. plebeja Kurz and H. hirsuta Lour (Nguyen, 2003; Pham, 1999). In which, H. isora is one of the species of interest with active ingredients recorded, including: phenols, flavonoids, alkaloids, glycosides, phytosterols, carotenoids, tannins, neolignans, rosmarinic acid derivatives, betulic acid, daucosterol, tannnins, anthoquinones, sterols, lupeol, β-sitosterol, α and β amyrin, taraxerone, 49-O-b -D-glucopyranosyl rosmarinic acid, 4,49- O-di-b-D-glucopyranosyl rosmarinic acid and 2R-O-(49-O-bD-glucopyranosyl caffeoyl)-3-(4-hydroxyphenyl), lactic acid named as 49-O-b –D-glucopyranosyl isorinic acid together with rosmarinic acid (Gayathri et al., 2010; Loganayaki et al., 2013; Satake et al., 1999), lignans, (6)-pinoresinol, (2)-boehmenan and (2)-boehmenan H (Chin et al., 2006). Flavones such as methyl ether, 7, 41-di-o-methyleisoscutellarein (5,8- dihydroxy-7,41-flavones) along with kaempferol-3-ogalactoside and herbacetin-8-oglucoronide from the leaves of H. isora (Ramesh and Yuvarajan, 1995). Various biological activities have also been identified, such as antidiabetic (Kumar et al., 2007), cardiac antioxidant, antiperoxidative potential and hypolipidaemic activities (Kumar et al., 2008) in streptozotocin (STZ) induced diabetic rats. Fruits have revealed hypolipidaemic (Raja et al., 2010), antioxidant (Jain et al., 2014; Dayal et al., 2015); cytotoxic activity against human lung carcinoma, hormone-dependent human prostate carcinoma and human breast carcinoma (Chin et al., 2006) and anticancer properties (Kumar et al., 2012). Whole plant of H. isora is reported to have anticancer (Varghese et al., 2011), antinociceptive activities (Venkatesh et al., 2007) while roots have hepatoprotective activity (Chitra and Prema, 2009) against carbon tetrachloride induced liver damage in rats.
       
However, there has been no recorded about biological activity of H. isora species in Vietnam. In this paper, we studied the activity H. isora specie, based on antioxidant activity and cytotoxicity, collected in Dak Nong province, Vietnam.

Preparation of the extract
 
Arial parts of Helicteres isora were collected in April 2022 from Dak Nong province (Dak Nong, Dak Mil, Dak Lao, altitude 545 m above sea level, coordinates: 12^o31'665"N, 107^o38'240"E). The samples were wasted with water, cutted into small pieces and dried in Laboratory drying oven at 50oC. They were then crushed into a course powder using a laboratory grinding mill with ring sieve, size 0,25 mm. Maceration extraction of samples in ethanol solvent (70%) for 7 days, followed by drying at 50^oC by vacuum evaporator. The extract was stored at -20^oC.
 
Cell culture
 
Human liver cancer cells (HepG2) and non-malignant cells of Chang’s Liver were obtained from American Type Cell Culture Collection (ATCC), Maryland, USA. All cultured cells were maintained in the logarithmic phase of growth in DMEM supplemented with 10% fetal bovine serum (GIBCO BRL), penicillin-streptomycin, fungizon and miramycin at 37^oC in a humidified incubator with 5% CO₂ and 95% air. Cultures were regularly examined using inverted microscope.
 
Determination of 1,1, dipheny-2-picrylhydrazyl (DPPH) radical scavenging activities
 
The DPPH radical scavenging assay was performed using 1,1 diphenyl-2-picrylhydrazyl (DPPH) according to the method described by Brand-Williams et al., (1995) with some modifications. Briefly, five different concentrations of the studied plant extracts (0.005, 0.01, 0.015, 0.02 and 0.025 mg/ml) were prepared in ethanol (analytical grade). The same concentrations were also prepared for L-ascorbic acid, which was used as a standard antioxidant. 1 ml of each studied extract was transferred into a clean test tube into which 0.5 ml of 0.3 mM DPPH in ethanol was added. The mixture was shaken and left to stand in the dark at room temperature for 15 minutes. Blank solutions comprising of the studied extract solutions (2.5 ml) and 1 ml of ethanol were used as baseline.
       
The negative control comprised 2.5 ml of DPPH solution and 1 ml of ethanol, while L-ascorbic acid at the same concentrations as the studied extracts was used as the positive control. After incubation in the dark, the absorbance values were measured at 517 nm using a spectrophotometer. The experiments were performed in triplicate. The DPPH radical scavenging activity was estimated using the equation described by Brand-Williams et al., (1995).
   
where 
A_s= Absorbance of the sample.
A_c= Absorbance of the control.
       
The half maximal inhibitory concentration (IC₅₀) of the extracts was computed from a plot of percentage DPPH free radical inhibition versus the extract concentration.
 
Cell viability assay
 
The WST-1 assay is a cell viability assay that uses water soluble tetrazolium salt as an agent, which reacts with mitochondrial dehydrogenase and produces formazan that can be detected quantitatively by spectrometry. For the viability assay, HepG2 cells were seeded on 24-well plates at a density of 5 × 10⁴ cells per well. After 1 day and 3 days, CDES was applied to each well. After each period, cell viability was measured using the WST-1 (EZ3000, Daeil Lab Service Co., Seoul, Korea) assay. Each well was washed with PBS, 200 µL of the 1:10 WST-1 agent diluted to the culture media was added and cultured for an hour and 100 µL of each well was transferred to a 96-well plate to read at a 450 nm wavelength ELISA plate reader (SUNRISE, TECAN, Grödig, Austria).
 
Real-time PCR
 
HepG2 cells were grown in 6 well plates and the media were exchanged with the ones including the extract in the difference concentrations. After 48 hrs incubation, the cells were washed with cold PBS once and scraped. The cells were washed again with cold PBS twice before extracting total RNA molecules using Qiagen RNeasy kit. cDNAs were constructed from 600 ng of the collected RNAs using Reverse Transcription System (Promega, Japan). cDNA was analyzed through real-time quantifying PCR using SYBR Green Realtime PCR Master Mix (Toyobo, Japan) according to the manufacturer’s instructions (CFX96 Touch Real-time PCR System, BioRad). The primers used for the experiments are summarized in Table 1.
 

 
Hoechst 33258 spectrofluorometric assay
 
HepG2 cells were grown in a 96-well plate for 24, 48 and 72 hrs at 37oC and were centrifuged (5 min, 8000 g) at RT. Then, 70 µL of a supernatant was replaced with 70 µL of warmed phosphate-buffered saline (PBS 1 ×, 37^oC) and 10 µL of H33258 solution (in PBS 1 ×) was added to a well. The final concentrations of H33258 in a well were 0.1-5 µg/mL. Then, the cells were incubated with H33258 for 60 min during optimization of the assay, or for 5 min at optimal conditions and the spectrofluorometric measurement was performed at EX/EM= 352/461 nm (EX/EM slit widths 25/25 nm) using a Tecan Spark fluorescence microplate reader (Tecan, Switzerland) while incubated at 37^oC. The samples were measured at least in triplicates. After background subtraction, the fluorescence signal was presented in Relative Fluorescence Units (RFU) as mean ±SEM.
 
Annexin V-FITC apoptosis assay
 
The Annexin V-FITC apoptosis detection assay was followed after the viability assay and performed according to the supplier’s protocol to determine apoptosis after the treatment, followed by fluorescence-activated cell sorting (FACS) for more precise analysis. Annexin V binds to phosphatidylserine, which is located at the inner cell membrane and exposed when cells are in the apoptotic process. After harvesting the cells, the samples were washed twice with cold PBS and resuspended in 1X binding buffer. Subsequently, 5 µL of FITC-Annexin V solution and 5 µL of PI solution were added to the samples. PI exhibits red fluorescence upon binding to DNA inside the cell and is generally used to detect dying or dead cells or opening of the cell plasma membrane. After gentle vortexing, the samples were incubated for 15 min at room temperature in the dark. After incubation, 400 µL of 1X binding buffer was added to the samples and analyzed by FACS within 1 h (FACS Aria II from BD Biosciences, USA).
 
Statistics
 
All data were expressed as mean ± SD (n= 10). The data were analyzed by Student’s t test and One way ANOVA using SigmaPlot version 11.0. Differences between groups were considered to be statistically significant at P<0.05.

DPPH radical scavenging activities of ethanol extract H. isora and cell viability assay
 
The analysis results showed that the antioxidant capacity through DPPH free radical scavenging reaction showed that H. isora extract has the ability to neutralize free radicals with IC₅₀ = 21.16 µg/ml.
       
Sample H. isora   has anti-proliferative activity of HepG2 cell line with IC₅₀ = 37 μg/ml. Tested on Chang liver cell line, H. isora  L. extract with a concentration of 200 μg/ml after 48 hours has an IC₅₀ value of 25%.
 
Real time PCR assay
 
Analyzed the change in expression of 2 apoptosis genes (Bcl-2 and Bax), using GAPDH gene as internal control on HepG2 liver cancer cell line, after treatment with H. isora extract at 2 concentrations (50 and 100 μg/ml) after 48 hrs, using 1% DMSO as control. There was a difference between the control and the experimental treatments and also observed the difference between the two experimental treatments of 50 mg/ml and 100 mg/ml. The mRNA expression of Bcl-2 gene was reduced by 27% in the batch treated with 50 mg/ml extract after 48 hours and by 43% in the batch treated with 100 mg/ml extract after 48 hours (Fig 1). Thus, it was shown that there was a decrease in the expression of Bcl-2 gene when were treated with H. isora extract. The expression of the Bax gene did not change after 48 hours difference between the control group and the treatments of 50 mg/ml and 100 mg/ml (Fig 2).
 

 

 
Hoechst 33258 spectrofluorometric assay
 
To evaluate the ability to trigger apoptosis through phenotype, HepG2 cells treated with H. isora extract at 2 concentrations (50, 100 µg/ml) and compared with the control, with time points of 24, 48 and 72h. There was a change in the cell membrane as well as the appearance of apoptotic bodies in the extract treatment group after 24 h. However, in the groups with extraction treatment, the fragmentation of the HepG2 cell nucleus became stronger with the longer the treatment time and the higher the concentration of the treated extract (Fig 3). In addition, testing of H. isora   extract on Chang liver cells (Fig 4) showed that at the concentrations of H. isora   examined, there is no effect on normal liver cells.
 

 

 
The ability to induce apoptosis
 
The percentage of HepG2 cells that went into apoptosis increased in the treatment group compared with the control group after 48 and 72 h (Fig 5). Besides, in the Chang liver cell line, there was no statistical difference between experiments (Fig 6). Thus, at a concentration of 50 µg/ml after 48 hrs of treatment, H. isora extract was able to induce HepG2 cells to go into apoptosis and Chang liver cells showed very low activity. The test of inhibitory activity against hepatocellular carcinoma cell line (HepG2) of H. isora L. extract at both cellular and molecular levels showed that at a concentration of 50 µg/ml after 48 h, the extract was capable of inducing apoptosis in HepG2 cells.
 

 

       
Oxidative stress is a pathological state in which reactive oxygen/nitrogen species (ROS/RNS) overwhelm antioxidative defenses of the organism, leading to oxidative modification of biological macromolecules (i.e., lipid, protein, DNA), tissue injury and accelerated cellular death (Trevisan et al., 2001) as the foundation of many diseases. Measuring the antioxidant activity of medicinal plant is carried out for treatment of oxidative stress-associated diseases in clinical biochemistry, for meaningful comparison of foods in regard to their antioxidant content and for controlling variations within or between products. The results of testing the antioxidant activity of the extract ethanol H. isora showed that the extract had high antioxidant activity (IC₅₀ = 21.16 µg/ml). The antioxidant capacity of extract H. isora has also been reported by authours: fresh and dried samples from parts of H. isora   such as leaves, bark, fruit and roots were extracted by various solvents (water, ethanol, methanol and acetone), obtained IC₅₀ of the methanol extract of fresh fruit reaching the lowest value of 34.37 mg/ml (Jain et al., 2014). Chaudhary et al., (2016) recorded the roots of H. isora   species when extracted by different solvents: water, ethanol, benzene, ethyl acetate, chloroform, petroleum ether with IC₅₀ of 400±120 μg/ml, 660±230 μg/ml, 730±320 μg/ml, 1730±910 μg/ml, 3280±1010 μg/ml, 7010±1240 μg/ml respectively. The dried fruit methanol extract of H. isora   has IC₅₀ = 42.95 µg/ml; H. isora   ether extract has IC₅₀ = 89.81 μg/ml (Manke et al., 2015); 70% ethanol extract of H. isora   L. stem bark has IC₅₀ = 97.53±0.28 μg/ml (Shori et al., 2013).
       
The results of analysis of the expression of the anti-apoptogenic gene Bcl-2 and the pro-apoptotic gene Bax at the transcriptional level showed that the mRNA transcription product of the Bax gene was expressed unchanged in all experimental groups, while the mRNA expression of Bcl-2 gene decreased after extract treatment after 48 h. In the control group, the expression of Bcl-2 and Bax genes was balanced, which prevented cells from going into apoptosis. In addition, this extract exhibits low toxicity to normal hepatocytes and is fully capable of being used to produce hepatoprotective functional foods. Similar results were also recorded by Shaikh et al., (2014), the ethanol extract of H. isora L. at a concentration of 1 mg/ml after 72 h of treatment, did not exhibit cytotoxicity on Chang liver cells and inhibited proliferation in the lines cancer cells (HeLa B75, 34.21%; HL 60, 30.25%; HEP 3B, 25.36%; PN 15, 29.21%) (Shaikh et al., 2014).

From the obtained results, it was concluded that H. isora induces apoptosis in HepG2 cells by inhibiting cell division in cancer cells. This inhibitory activity was caused by decreasing the level of the anti- apoptotic gene, Bcl-2 and increasing the livel of the proapoptotic gene, Bax. Therefore, we suggest that H. isora could be further investigated as an alternative treatment for human hepatoma.

This work was supported by DakNong Department of Science and Technology; Vietnam Academy of Science and Technology.