Apoptosis-Mediated Cytotoxic Effect of Caralluma adscendens var. attenuata on Colon (HT29) and Hepatic (HepG2) Cancer Cell Lines.

INTRODUCTION: Caralluma adscendens var. attenuata (Wight) Grav. and Mayur., a member of Apocynaceae, is a perennial stem succulent plant with wide distribution in tropics and subtropics of the world. This plant is reported for the presence of steroids, flavonoids, saponins, triterpenes, and pregnane glycosides, and is known to have antidiabetic and antiulcerogenic properties.
MATERIALS AND METHODS: In this study, an attempt was made to identify antioxidant capacity and cytotoxic potential of n-hexane and aqueous methanolic extracts of total stem part of C. adscendens var. attenuata. Antioxidant activity was evaluated by total phenolic content assay, total flavonoid content assay, free radical scavenging activity, and reducing ability methods. Cytotoxic activity was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) assay and nuclear staining methods for colorectal cancer cell lines HT29 and liver cancer cell lines HepG2.
RESULTS: MTT assay method has proven that the IC50 value was 10 μg/mL for both extracts, whereas for cisplatin standard, it was 2 μg/mL. By nuclear staining, the apoptotic cells were identified as oval masses with dark cytoplasm and dense green nuclear chromatin fragments indicating the programmed cell death for both n-hexane and aqueous methanolic extracts at the same concentration (10μg/mL).
CONCLUSION: However, aqueous methanolic extract showed prominent cytotoxic potential against both cancer cell lines.

INTRODUCTION

Cancer is the significant disease that has a remarkable impact on world health. Biological oxidative stress increases the burden of free radical formation.[1] Accumulation of large amount of unstable free radicals leads to carcinogenesis, tissue necrosis, and tumor formation, which in due course leads to cancer. Genetic factors, environmental factors, and lifestyle of the present era such as smoking, alcoholism, pollution, poor physical exercise, and modern food habits are the main causes that increase the load of reactive radicals in the physiological system. Among various kinds of cancers, liver and colon cancers are more prominent types in developed countries. Globally, liver cancer is the sixth and colorectal cancer is the third most common type of cancer.

The key factor of liver cancer (hepatic cancer)[2] is cirrhosis. Cirrhosis is due to either viral infections such as hepatitis B and/or C or alcoholism. In 2013, 735,000 liver cancer deaths were reported. Among them, 300,000 deaths were attributable to hepatitis B–induced liver cancers, 343,000 deaths were due to hepatitis C, and 92,000 deaths were due to alcohol-induced liver cancers.

Colorectal cancer[3] is prevalent in developed countries. Surprisingly, it is infrequent in females than males. In a World Health Organization survey conducted in 2012, 1.2 million new cases and 694,000 deaths were reported. Eating a lot of red meat and cold meat, which is low in fiber, increases the chances of colorectal cancer. Other diseases such as inflammatory bowel syndrome, Crohn’s disease, and ulcerative colitis can also increase the risk of colorectal cancer. Reactive oxygen species (ROS) enhances abnormal cell division and growth, leading to tissue necrosis/cancer. However, antioxidant diet and antioxidant-rich herbal medicine abate the generation of ROS. Majority of the edible plants possess flavonoids[4] and antioxidant bioactive principles, which contribute to anticancer activity by various mechanisms. In this study, hexane and aqueous methanolic stem extracts of Caralluma adscendens var. attenuata[56] were subjected to phytochemical tests, thin-layer chromatography (TLC), and assay methods for flavonoids.[7] The flavonoid content and its potential[8] were measured by Free Radical Scavenging Assay (FRSA) and Reducing ability methods. MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay was performed to determine the cytotoxic potential of C. adscendens var. attenuata as the preliminary step to determine IC50 values. The obtained concentrations of IC50 were used for nuclear staining method to understand the mechanism of cytotoxicity.[9] The results obtained for all the investigations were tabulated and specified in the text.

MATERIALS AND METHODS

Materials

Collection of plant material

Stems of C. adscendens var. attenuata were collected from Kadiri (town), Anantapuramu district, Andhra Pradesh, India, and it was authenticated by Prof. B. Ravi Prasad Rao, Biodiversity Conservation Division, Department of Botany, Sri Krishnadevaraya University, Anantapuramu, as its Voucher specimen no. 51293 at SKU Herbarium.

Chemicals

Folin–Ciocalteu’s reagent, Hank’s balanced solution, Eagle’s minimum essential medium, and fetal calf serum were purchased from Sigma-Aldrich (Bengaluru, India).

Methods

Preparation of extracts

The plant material was dried under sunshade and powdered, and then it was passed through a 22 no. sieve. Extracts of n-hexane and aqueous methanol (50:50) were prepared separately by cold percolation method using 2.5 L of solvent to each kilogram of dried powder. After 7 days, solvents were recovered under reduced pressure using rotary vacuum evaporator.[10] The extracts were then used for further investigations.

Chemical tests for flavonoids

Shinoda test: A pink scarlet color was observed with the extract solution upon addition of a few fragments of magnesium ribbon and HCl.

Zinc hydrochloride reduction test: A red solution was obtained on addition of zinc dust and concentrated HCl to the extract.

Alkaline reagent test: To the extract solution, a few drops of sodium hydroxide solution were added; an intense yellow color was formed. Discoloration was observed on addition of a few drops of dilute acetic acid.

Identification of flavonoids by TLC: In this study, both n-hexane and aqueous methanolic stem extracts of C. adscendens var. attenuata were subjected to TLC using n-butanol:acetic acid:water (4:1:5, vol/vol) as mobile phase.[11] The spots were identified under UV lamp. Then the plates were developed in an iodine chamber and the spots were observed as prominent fluorescent yellow for silymarin and pink color for aqueous methanol and n-hexane.

Antioxidant activity

Determination of total phenolic content by Folin–Ciocalteu’s method

In this analysis, 1 mg/mL solution of n-hexane and methanolic extracts was used to calculate the concentration of phenolics by Folin–Ciocalteu’s reagent spectrophotometrically.[12] Gallic acid standard curve was represented as y = 7.012x – 0.0181, r = 0.999. The content of phenolic in the extracts was expressed in terms of gallic acid equivalent (mg of GAE/g of extract). The values obtained for the concentration of total phenols are expressed as mg of GAE/g of extract.

Determination of flavonoid content by UV spectrophotometric method

In this analysis, 1 mg/mL solution of n-hexane and methanolic extracts was used to determine the content of flavonoids by UV spectrophotometric method.[12] Rutin standard curve was represented as y = 16.213x – 0.0581, r = 0.999. The content of flavonoid in the extract was expressed in terms of rutin equivalent (mg of RUE/g of extract).

Reductive ability

The reductive ability of the aqueous methanolic and n-hexane extracts was determined according to the Oyaizu method.[13] Absorbance was measured at 550 and 700 nm for aqueous methanolic and n-hexane extracts, respectively. Butylated hydroxytoluene was used as the reference compound. All the analysis was performed in triplicate. Reducing ability (%) was calculated according to the following formula:

where V0 is the absorbance of control and V1 is the absorbance of the sample.

Determination of FRSA using hydrogen peroxide

The FRSA of the methanolic extract was measured using hydrogen peroxide as suggested by Czochra and Widwnsk.[14] Absorbance was recorded at 230 nm for both extracts. All readings were repeated three times. Blank was prepared without adding hydrogen peroxide and control was prepared without a sample. Ascorbic acid was used as a standard compound. FRSA of hydrogen peroxide (%) was calculated as per the formula described earlier.

Cytotoxic activity

Cytotoxic activity was evaluated by three methods including MTT assay method against cisplatin as the standard[1516] and nuclear staining method.

MTT assay method

It is a colorimetric assay that measures the reduction of yellow MTT by mitochondrial succinate dehydrogenase enzyme. The MTT enters the cells and passes into the mitochondria where it is reduced to an insoluble, colored (dark purple) formazan product. The cells were then solubilized with an organic solvent (e.g., dimethyl sulfoxide, isopropanol) and the released; solubilized formazan reagent was measured spectrophotometrically. As the reduction of MTT can only occur in metabolically active cells, the level of activity is a measure of the viability of the cells.

Cytotoxicity assay

Optical density was measured at a wavelength of 450 nm. The result represents the mean of three readings. Calculation of the percentage of cell lysis was done by comparing the optical density of the sample to that of the control. The formazan blue formation by MTT assay is illustrated in Figure 1.

Figure 1

MTT assay for (A) HT29 and (B) HepG2

Nuclear staining method

HT29 and HepG2 cancer cell lines were treated with both methanolic and n-hexane extracts and stained with acridine orange (AO)/ethidium bromide (EB) as specified in the procedure[17] and nuclear changes were observed under a fluorescent microscope.

RESULTS AND DISCUSSION

Both the n-hexane and aqueous methanolic extracts were proven to possess flavonoids as the principal constituents by chemical tests, TLC, total phenolic content, and flavonoid content assays. TLC retention factor (Rf) values were found to be 0.72 and 0.73 by using specific mobile phase which is nearer to the standard (silymarin) 0.72. (Rf is defined as the ratio of the distance travelled by the center of a spot to the distance traveled by the solvent front.) The total phenolic content in n-hexane and methanolic extracts was found to be 15.3 ± 0.07 and 22.5 ± 0.09 mg GAE/g, respectively. Total flavonoid content in both extracts was found to be 5.4 ± 0.04 and 6.2 ± 0.05 mg RUE/g, respectively. The obtained results are presented in Table 1. The antioxidant capacity of the extracts was calculated by FRSA and reductive ability methods. Results were observed as 88.41% ± 0.230 and 96.13 ± 0.150 for aqueous methanolic extract and 89.6% ± 0.150 and 94.18% ± 0.120 for n-hexane, respectively. Because significant amount of antioxidants were found in both extracts, it has become an ideal proof to continue the research for cytotoxic activity by MMT assay. MMT assay is a preliminary test to determine IC50 value. IC50 value of both extracts was identified as 10 µg/mL. The results obtained are presented in Table 2.

Table 1

Determination of total phenolic content and flavonoid content

Extract	Total phenolic content (mg GAE/g)*	Total flavonoid content (mg RUE/g)*
n-hexane	15.3 ± 0.07	5.4 ± 0.04
Methanolic	22.5 ± 0.09	6.2 ± 0.05

*Mean values (n = 3) with significant difference at P < 0.05.

Table 2

IC50 values for n-hexane and aqueous methanolic extracts

Type of cell line	Concentration (μg)	Compound		O.D. at 492 nm		% of cell lysis		IC50
		n-hexane extract	Aqueous methanolic extract	n-hexane extract*	Aqueous methanolic extract*	n-hexane extract*	Aqueous methanolic extract*
HT29	10	1	2	1.819 ± 0.104	1.817 ± 0.201	50% ± 0.212	50% ± 0.102	10 μg
	20	1	2	2.931 ± 0.126	2.934 ± 0.114	75% ± 0.211	100% ± 0.230
	30	1	2	3.045 ± 0.251	3.047 ± 0.212	100 ± 0.131	100% ±0.130
	-	Control	Control	0.479 ± 0.175	0.476 ± 0.220	No lysis	No lysis
HEPG2	10	1	2	1.676 ± 0.158	1.675 ± 0.120	50% ± 0.200	50% ± 0.220	10 μg
	20	1	2	2.092 ± 0.114	2.090 ± 0.200	75% ± 0.115	100% ± 0.125
	30	1	2	3.035 ± 0.222	3.034 ± 0.115	100% ± 0.212	100% ± 0.247
	-	Control	Control	1.280 ± 0.205	1.275 ± 0.210	No lysis	No lysis

*Data are expressed as mean ± SD, n = 3. OD = optical density

Nuclear staining for untreated cells of HT29 and HepG2 with AO/EB appeared in almost spherical shape with an intact nucleus [Figure 2A]. Although HT29 and HepG2 cancer cell lines treated with cisplatin were observed with nuclear blabbing and cytoplasmic condensation [Figure 2B], the same cancer cell lines treated with 10 µg/mL concentration of C. adscendens var. attenuata extracts were observed with a few apoptotic lesions by nuclear elongation and distinctly spread cytoplasm, which indicates programmed cell death. However, both the cancer cell lines treated with aqueous methanolic extract showed notable apoptosis by clear chromatin separation with higher number of cell death than the n-hexane-treated cells, as shown in Figure 2C–F. The reason for this divergent result may be higher proportion of phytochemical constituents present in polar aqueous methanol solvent than that in nonpolar n-hexane solvent.

Figure 2

Nuclear staining for (A) untreated cancer cell lines, (B) cisplatin-treated (2 g/mL) cell lines, (C) HT29 cell lines treated with methanolic extract, (D) HT29 cell lines treated with n-hexane, (E) HepG2 cell lines treated with methanolic extract, and (F) HepG2 cells treated with n-hexane

CONCLUSION

The findings of our work revealed that C. adscendens var. attenuata has significant antioxidant activity and cytotoxic potential against colorectal and liver cancer cells. Hence, we suggest the researchers to investigate the plant extract for further toxicological studies, half-life prediction, and herbal formulation.

Financial support and sponsorship

This work was supported by the Department of Botany, Sri Krishnadevaraya University, Anantapuramu, Andhra Pradesh, and the Department of Biotechnology, Manipal University, India.

Conflicts of interest

There are no conflicts of interest.