Screening of Garcinia cambogia for in-Vitro Anti-Cancerous Activity against Colon Adenocarcinoma (Caco-2) Cell Line

 

Sadika Banu, Ramakrishnaiah T. N

 Department of Biotechnology and Applied Genetics, Ramaiah College of Arts, Science and Commerce. Bangalore, India

*Corresponding Author E-mail: sadiqarajeeb.83@gmail.com

 

ABSTRACT:

Objective: Garcinia cambogia, commonly known as ‘malabar tamarind’ is considered to have great medicinal properties. The dried fruit rind of Garcinia cambogia was used in this present study to evaluate its anti cancerous potential against Caco-2 cell lines. Materials and Methods: In vitro cytotoxicity of fruit rind extracts of Garcinia cambogia in methanol, distilled water, ethyl acetate, hexane and chloroform were evaluated by MTS ((3-(4,5- dimethylthiazol-2- yl)-5- (3-carboxymethoxyphenyl)- 2-(4-sulfophenyl)-2H- tetrazolium), Phenazinemethosulfate (PMS)) assay against Caco-2 (human colorectal cancer) cell lines. Cell cycle analysis and apoptosis assay were done by using 2% paraformaldehyde, 1X PBS (Phosphate Buffered Saline Ph=7), DAPI (4′,6-Diamidine-2′-phenylindole dihydrochloride), FITC-Annexin V and Propidium Iodide. Conclusion: The results suggest that different extracts of fruit rind of Garcinia cambogia (Methanol, Distilled water, Ethyl acetate, Hexane, Chloroform) was found to have insignificant anticancer activity at concentration of 1000μg/ml, 100μg/ml, 10μg/ml against Caco-2 cell lines.

 

KEYWORDS: Garcinia cambogia, anti-cancer, in vitro, MTS assay, Apoptotic assay, Cell Cycle Analysis.

 

 


INTRODUCTION:

Cancer is uncontrolled growth and proliferation of cells that escape the growth control mechanisms. Mutations in the DNA accumulate leading to formation of these abnormal cells which bypass the normal regulatory mechanism of cell cycle and may escape the cell cycle checkpoints. Thus, they continue to grow and spread (metastasis), and form tumor.

 

Cancer cells can have their own growth signals and are insensitive to anti-growth signals, thus possesses infinite potential for replication, leading to uncontrolled growth and proliferation by evading the normal apoptotic mechanism. Thus, causes tissue invasion and metastasis which is sustained by angiogenesis. [5, 6]

 

Among all types of cancers, lung cancer and colon cancer are found to be commonly occurring both in men and women. However, colorectal cancer is the third and second most commonly occurring cancers in men and women respectively. [9]

 

Garcinia cambogia is a tropical native plant grown in Asia and Indonesia. It belongs to Guttiferae family (Clusiaceae). The fruit rind of this plant is commonly known as ‘Malabar tamarind’ or ‘kodumpuli’ and is used widely in cooking in southern India specifically in Kerala. [10,13]. It contains several secondary metabolites like, flavonoids, Xanthones and Benzophenones. [1,14]

 

 In the present study, different extracts of fruit rind of Garcinia cambogia were evaluated against the Caco2 cell lines [15] for different assays such as MTS assay, Cell cycle Analysis and Apoptotic assay. [4,7,11]

 

MATERIALS AND METHODS:

Plant Material and Extraction:

The fruit rind of Garcinia cambogia was extracted using five different extracts of 150ml each of methanol, distilled water, ethyl acetate, chloroform and hexane. The extraction process was carried out in soxhlet apparatus for 3 hours with gentle shaking. The extracts were filtered through muslin cloth followed by drying using rotary evaporator. The resulting crude extracts were used to make compound stocks by resuspending them in 100% DMSO. [2]

 

Chemicals:

MTS Assay:(3-(4,5- dimethylthiazol-2- yl)-5- (3-carboxymethoxyphenyl)- 2-(4-sulfophenyl)-2H- tetrazolium), Phenazinemethosulfate (PMS) (Promega G3581).  [3]

 

Cell Cycle Analysis:

Paraformaldehyde, PBS (Phosphate Buffered Saline), DAPI (4′,6-Diamidine-2′-phenylindole dihydrochloride). (Bio-Rad 135-1304) [12]

 

Apoptotic Assay:

FITC Annexin V, and Propidium Iodide.

 

Dulbecco's modified eagle medium (DMEM), trypsin, ethylenediaminetetraacetic acid (EDTA), Pen-strep as antibiotics, Fetal bovine Serum. (Gibco 16010-154)  [8]

 

Cell Lines:

Caco-2 cell lines ATCC number HTB-37, were cultured in T75 cell culture flasks using DMEM augmented with 10% FBS. They are cultured at 37°C, 5% CO2 incubator. All the assays were carried out in 96 microtitre plates (Greiner 655101)

 

MTS Assay:

MTS assay was carried out by seeding Caco2 cells in a 96 well plate at a density of 1.0x10^4 cells/well. The cells were cultured in a 5% CO2 incubator at 37°C overnight. The cells were treated with 1000μg/ml, 100μg/ml, 10μg/ml of compounds for a period of 48 hrs. Final concentration of DMSO was maintained at 1% throughout the plate. After incubation, MTS reagent was added followed by 30 min incubation. The absorbance was measured at 490nm. Data analysis was done using Graph Pad Prism 6.0 [3]

 

Cell Cycle Assay:

For cell cycle analysis, Caco-2 cells were seeded in a 96 well plate at a density of 1x10^4 cells/well. The cells were cultured in a 5% CO2 incubator at 37°C. The cells were treated with 1000μg/ml, 100μg/ml, 10μg/ml of compounds for a period of 48 hrs. Final concentration of DMSO was maintained at 1% throughout the plate. After incubation, the cells were washed with 1X PBS and were fixed with 2% PFA (Para formaldehyde). The fixed cells were stained with 1ug/ml of DAPI and analyzed using the cell cycle Bio Application in cellomics array scan reader, designed to classify cells and define cell cycle stage by measuring the nuclear DNA content using the total intensity of a DNA binding dye (DAPI) for each individual cell. Correspondingly, the cells were classified into one of five classes: less than 2N (apoptotic/ damaged), 2N (G1), 2N-4N (S), 4N (G2/M), and & > 4N (polyploid or necrotic). [12]

 

Apoptotic Assay:

Apoptosis assay was carried out by seeding 10 4 cells per well in 96 well plates and incubate at 37 degrees, by providing 5% of CO2 for 24 hours for them to differentiate. The cells were cultured in a 5% CO2 incubator at 37°C. The cells were treated with 1000μg/ml, 100μg/ml, 10μg/ml of compounds for a period of 48 hrs. Final concentration of DMSO was maintained at 1% throughout the plate. After the incubation period, cells were washed in cold 1X phosphate-buffered saline (PBS). The cells were exposed to 25μL of FITC-Annexin V, 25μL of propidium iodide, 1μg/ml DAPI and incubated in dark at room temperature for 15min. The supernatant was aspirated out and cells were washed with 1X PBS to remove any excess stain. The cells were analyzed in Cellomics high content screening reader. The cells separate into three groups: live, apoptotic, and dead. Live cells show only weak annexin V staining of the cellular membrane, while apoptotic cells show a significantly higher degree of surface labeling. Dead cells show both membrane staining by annexin V and strong nuclear staining from the propidium iodide. [8]

 

RESULTS:

MTS Assay:

Table 1: % inhibition of cells after treatment

Extracts

1000μg/ml

100μg/ml

10μg/ml

Methanol

9.295757

18.90167

6.212379

Distilled water

21.56541

13.33699

6.294472

Ethyl acetate

-81.06184

-16.58456

-12.99945

Chloroform

-38.13173

-2.253232

6.704984

Hexane

45.80368

28.50757

14.92429

 (*negative values are considered zero inhibition)

 

 

 

The cytotoxicity of the various extracts of Garcinia cambogia on Caco-2 cell lines were evaluated and it was seen that, among all the garcinia extracts, hexane extract showed good cytotoxic effect at 1000μg/ml and the others did not show significant toxicity.

 

The equation of line was Y = -0.005481*X +0.8263. R2 (coefficient of determination) was found to be 1 suggesting there is a perfect correlation of the X and Y variables in the equation Y = -0.005481*X +0.8263. The results of % inhibition by MTS assay is tabulated.

 

Cell Cycle Assay:

Measurement of DNA content of the cell was done for cell cycle analysis by using DAPI. Harmine which is a kinase inhibitor was used as a positive control while complete media as negative control.

 

The number of cells in each phase of the cell cycle after 48hrs treatment with different extracts of the compound are tabulated in the table 2 indicating that there is approximately one-fold increase in the arrest of cells at G0/G1 phase if the cell cycle after the treatment especially with Garcinia chloroform extract.

 

Cells undergoing Apoptosis contain <2N DNA content thus suggests the anti-cancerous ability of the compound. Necrosis is indicated by >4N DNA content. The results of the assay are tabulated.

 

 

 

 

 

 

Table 2: cell counts in each phase of cell cycle after 48hrs of compound treatment

Compound

G0/G1

S

G2/M

Necrotic

Total

2n

2n-4n

4n

>4n

GC 1000ug/ml

79.37

12.03

7.14

4.03

100

GC 100ug/ml

77.68

12.17

6.25

5.77

100

GC 10ug/ml

81.95

11.18

5.04

4.91

100

GE 1000ug/ml

53.76

23.76

12.8

9.68

100

GE 100ug/ml

81.21

11.1

6.79

4.34

100

GE 10ug/ml

80.05

11.36

6.77

4.94

100

GH 1000ug/ml

47.82

29.2

14.71

8.28

100

GH 100ug/ml

71.48

15.26

7.79

7.5

100

GH 10ug/ml

78.34

11.33

6.55

4.83

100

GM 1000ug/ml

55.42

24.58

14.11

9.37

100

GM 100ug/ml

80.89

10.62

5.68

3.63

100

GM 10ug/ml

78.7

10.86

6.67

4.43

100

GW 1000ug/ml

70.68

16.61

11.09

5.99

100

GW 100ug/ml

79.24

11.09

6.01

5.28

100

GW 10ug/ml

78.68

11

7.22

5.32

100

Positive Control

62.75

13.78

10.64

8.17

100

Negative Control

77.19

12.6

6.57

3.6

100

 

GC=Garcinia Chloroform, GE= Garcinia Ethanol, GH= Garcinia Hexane, GM= Garcinia Methanol, GW= Garcinia Water, Inter-phase= G0, G1, S; G2=Preparation for Mitosis Phase, M-Phase (Mitotic Phase) =Prophase, Metaphase, Anaphase, Telophase

 

 

Images – Cell Cycle Analysis using DAPI

Negative control

 

Figure 1.a: Greyscale image Fluorescent image

Positive control

 

 

 

 

 

Figure 1.b: Greyscale image Fluorescent image

 

 

 

 

 

Apoptotic Assay:

The FITC Annexin V/Dead Cell Apoptosis Kit with FITC annexin V and PI for flow cytometry provides a rapid and convenient assay for apoptosis. The kit contains recombinant annexin V conjugated to fluorescein (FITC annexin V), as well as a ready-to-use solution of the red-fluorescent propidium iodide (PI) nucleic acid binding dye. PI is impermeant to live cells and apoptotic cells, but stains dead cells with red fluorescence, binding tightly to the nucleic acids in the cell. After staining a cell population with FITC annexin V and PI in the provided binding buffer, apoptotic cells show green fluorescence, dead cells show red and green fluorescence, and live cells show little or no fluorescence. These populations can easily be distinguished using a flow cytometer with the 488 nm line of an argon-ion laser for excitation. Results obtained are tabulated. Table 3 indicates 1000ug/ml of hexane extract of Garcinia showing higher % apoptosis as compared to the rest.

 

 

 

 

 

 


Table 3: Results for Apoptotic Assay Using FITC Annexin V/Dead Cell Apoptosis Kit with FITC Annexin V and Propidium Iodide

Compounds/ concentration

%Apoptosis

GC 1000ug/ml

3

GC 100ug/ml

8

GC 10ug/ml

7

GE 1000ug/ml

27

GE 100ug/ml

6

GE 10ug/ml

8

GH 1000ug/ml

55

GH 100ug/ml

8

GH 10ug/ml

4

GM 1000ug/ml

38

GM 100ug/ml

5

GM 10ug/ml

6

GW 1000ug/ml

23

GW 100ug/ml

5

GW 10ug/ml

6

Positive control

100

Negative control

6

 

 


Images

1.     Negative control for Apoptosis assay:

 

Figure 1.a: Channel 1: DAPI, Figure 1.b: Channel 2: FITC-AnnexinV, Figure 1.c: Channel 3: Propidium iodide, Figure 1.d: Combination of all channels

 

2.     Positive control for apoptosis assay:

 

Figure 2.a: Channel 1: DAPI, Figure 2.b: Channel 2: FITC-AnnexinV, Figure 2.c: Channel 3: Propidium iodide, Figure 2.d: Combination of all channels.

*DAPI=Indicates cell number, FITC-Annexin V=Indicates Apoptotic cells, Propidium iodide Indicates necrosis.

 

 


In this assay, the FITC labelled annexin V was used to distinguish between the dead and apoptotic cells, along with the addition of Propidium Iodide.

 

In viable cells, Phosphatidyl Serine (PS) is located towards the cytoplasmic surface on the cell membrane. Phosphatidyl Serine (PS) translocation to the extracellular membrane was detected by fluorescently labelled Annexin V which is the measure to indicate the initiation of apoptosis. At the later stages, due to the loss of membrane integrity, the cell also takes in PI (which was initially excluded by the cells) and therefore, such cells are stained by Annexin V along with PI.

 

Garcinia shows a variability in the percentage of cells undergoing apoptosis at different concentration. However, Garcinia hexane extract at a concentration of 1000ug/ml showed the highest percentage of cells undergoing apoptosis.

 

DISCUSSION:

In the cytotoxicity assay by MTS, although the hexane extract showed good cytotoxicity effect at 1000μg/ml, compared to all the extracts of Garcinia, a clear conclusion cannot be drawn as Garcinia showed some morphological changes similar to the cells in the positive control and thus, further tests can be performed with different concentrations of the hexane extract of Garcinia. Similarly, the percent apoptosis of Garcinia hexane extract at 1000μg/ml was found to be on the higher side, and also the cell cycle analysis of Garcinia Chloroform extract was found to show approximately one-fold increase in the arrest of cells at G0/G1 phase of the cell cycle after the treatment.

 

CONCLUSION:

Garcinia cambogia extract showed insignificant effects against the Caco-2 cell lines evaluated by MTS, Cell cytotoxicity using DAPI and Apoptotic assays. Review of literature shows that the Garcinia cambogia fruit rind extracts were found to have anticancer properties against A549, Caco-2, Hela cell lines [1,2] suggesting the decreased risk potential of the compound to be used against different cancer treatments.

 

ACKNOWLEDGEMENT:

Authors are grateful to Dr. Shahab uddin, Chandan Mithra, Syeda Afshan from the screening facility at the NCBS Bangalore for providing the required research facility and their valuable support and guidance.

 

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15.  Tor Lea Caco-2 Cell Line.

 

 

 

Received on 13.05.2018          Modified on 07.08.2018

Accepted on 12.09.2018  ©A&V Publications All right reserved

Res. J. Pharmacognosy and Phytochem. 2018; 10(4): 272-276.

DOI: 10.5958/0975-4385.2018.00043.2