ANTI-CANCER EFFECT OF PANCHAGAVYA ON HUMAN COLON ADENOCARCINOMA (HCT-116) CELL LINE

ANTI-CANCER EFFECT OF PANCHAGAVYA ON HUMAN COLON ADENOCARCINOMA (HCT-116) CELL LINE

Funde, S. Patil * and H. Palep

Principal K. M. Kunanani College of Pharmacy, Mumbai, Maharashtra, India.

ABSTRACT: The objective of the study was to investigate the in-vitro anti-cancer effect of Panchagavya on the Human Colon Adencarcinoma (HCT-116) cell line. Panchayavya was formulated using five products from Cows, namely; Cow dung, Cow urine, Cow milk, Cow curd and Cow ghee. The whole mixture was further freeze-dried and stored until further assay conduction. The growth inhibitory activity of the raw Panchagavya (PGS1) and formulated Panchagavya (PGS2) in HCT-116 colon cancer cells were investigated by 3-(4, 5 - dimethyl thiazolyl - 2) - 2, 5-diphenyl-tetrazolium bromide (MTT) assay. The results from MTT assay indicated that cell proliferation was significantly (p <0.05) inhibited in a concentration-dependent fashion, especially by formulated Panchagavya. Thus, Panchagavya formulation has a potential anti-cancer activity. In this context, we investigated the anti-proliferative potential of Fresh Panchgavya preparation against the Human Colon Adenocarcinoma (HCT-116) Cell Line. To the best of our knowledge, this is the first report demonstrating in-vitro anti-proliferative efficacy of Panchgavyaagainst Human Colon Adenocarcinoma (HCT-116) Cell Line.

Keywords: Colon cancer, Anti-proliferation, Panchagavya, Freeze dried

INTRODUCTION: Colorectal cancer (CRC) being considered the third most commonly diagnosed cancer in males and the second in females, with 1.8 million new cases and almost 861,000 deaths in 2018, according to the World Health Organization. Patients with inflammatory bowel diseases like ulcerative colitis have been shown to have a higher risk of developing CRC ¹². The Indian scenario is further gloomier as India is projected to have one of the highest disease burdens of gut inflammatory diseases, including CRC, across the globe ^{3, 4}.

Animal studies have shown that bacteria may potentially contribute to inflammatory diseases and CRC development through direct and indirect interaction with the host’s simmune system, production of cancer-associated inflammatory metabolites, and release of genotoxic virulence factors ^{20, 34}. Recently, a growing number of studies indicated that gut microbiota may be a key factor contributing to the development of many pathological processes, including cancer ^{16, 17}.

The gut microbiota (microbiome) comprises a large population of diverse microorganisms (bacteria, viruses, fungi, and protozoa) inhabiting the gastrointestinal tract of humans. In healthy people, the microbiome is involved in nutrient metabolism and absorption, drug metabolism, and elimination of xenobiotics. In addition, normal gut microbiota participates in maintaining intestinal barrier integrity, protects against pathogens and plays an important role in immunomodulation. According to the latest research that explored the microbiome of individuals with colorectal cancer, alternation in the composition and functionality of the normal gut microbiota may lead to the initiation, promotion, and progression of this cancer ^{6-12, 37}.

It was demonstrated that toxic metabolites of bacteria cause DNA damage, affect cell cycles, stimulate the immune response, and disturb the intestinal barrier function. As a result, impaired intestinal microbiota homeostasis contributes to developing the microenvironment favourable to developing colorectal cancer ¹⁴.

Panchagavya involves the use of five by-products of cow viz. Cow-dung, -urine, -milk, -curd, and ghee. The Ayurveda, the ancient Indian system of medicine, has detail edmentions of the importance of cow’s milk, curd, ghee and urine in treating various human ailments. The ancient ayurvedic literature (Charak Samhita, Sushrut, Gadanigrah) ²¹ suggests several pharmacological Applications of the substances obtained from Panchgavya ²⁰. The religious ritual of Practice of Panchagavya prashan has been in existence for ages in our country. This practice was expected to deliver a person from all the sins. Sin in Sanskrit means papma.

Papma is a synonym for disease. This ritual is practiced once every year during July and August, at which time the Vaccinia viraemia is at its peak in cows. The Faecal Microbial Transplant (FMT), a recently emerged technique is now popularly used, is difficult to treat gastrointestinal, metabolic, and many other diseases in western countries ^22-24. Panchagavya appears to be a traditional technique of cow faecal transplant therapy. Systematic work needs to be carried out on exploring the anti-cancer or chemoprevention activity of Panchgavya.

MATERIALS AND METHOD: HCT-116 -Human colorectal adenocarcinoma cell line (From NCCS, Pune), Cell culture medium: DMEM-High glucose media - (Cat No:2120785, Gibco), Adjustable multichannel pipettes and apipettor (Benchtop, USA), Fetal Bovine Serum (#RM10432, Himedia), MTT Reagent (5 mg/ml) (#4060 Himedia), DMSO (#PHR1309, Sigma), Cisplatin (#PHR1624, Sigma, D-PBS (#TL1006, Himedia), 96-well plate for culturing the cells (From Corning, USA), T25 flask (#12556009, Biolite -Thermo).

Equipment: Centrifuge (Remi: R-8oC), Pipettes: 2-10µl, 10-100µl, and 100-1000µl, Inverted microscope (Biolink), 37°C incubators with the humidified atmosphere of 5% CO₂ (Heal force, China), freeze dryer (Gen-Tech).

The HCT-116 (Human colorectal adenocarcinoma cell line) was purchased from NCCS, Pune, India. The cells were maintained in DMEM high glucose media supplemented with 10 % FBS and the 1% antibiotic-antimycotic solution in the atmosphere of 5% CO₂, 18-20% O₂ at 370C temperature in the CO₂ incubator and subcultured every two days ^{2, 3}.

The Panchgavya formulation used in this study was prepared using a different method from the one practiced traditionally (which yields a fermented preparation). Fresh cow dung and urine, sourced from a cow fed on green grass, were mixed thoroughly in a glass beaker. This mix was allowed to stand for 10 min and subjected to filtration through a muslin cloth. To this filtrate, fresh cow’s milk and fresh curd were added and mixed until a uniform mixture was formed. Finally, cow ghee was added to this mixture and mixed thoroughly. This Panchagavya mixture was then transferred to a copper vessel (covered with a muslin cloth) and allowed to rest for 30 min. This was followed by freeze-drying at -20 °C to convert the preparation in powder form, which was then used for MTT assay ^{1, 4}. The MTT assay is preferable to other methods because it arguably has a high reproducibility rate, is sufficiently safe and straightforward to use in laboratory conditions, is highly sensitive for cytotoxicity tests, and is suitable for high-throughput screening and miniaturization ^35-37.

MTT Assay Protocol: MTT assay is a colorimetric assay used to determine cell proliferation and cytotoxicity based on the reduction of the yellow-colored water-soluble tetrazolium dye MTT to formazan crystals. Mitochondrial lactate dehydrogenase produced by live cells reduces MTT to insoluble formazan crystals, which upon dissolution into an appropriate solvent exhibit purple color, the intensity of which is proportional to the number of viable cells and can be measured spectrophotometrically at 570 nm 4. Assay controls were: (i) Medium control (medium without cells), (ii) Negative control (medium with cells but without the experimental drug/compound), (iii) Positive control (cells treated with 12.5ug/ml of Cisplatin).

Protocol of Study ^1-3: 200 µl cell suspension was seed Edina 96-well plate at required cell density (20,000 cells per well), without the test agent. The cells were allowed to grow for about 24 h. Concentrations (12.5ug/ml, 25ug/ml, 50ug/ml, 100ug/ml, 200ug/ml) of the PGS1 and PGS2 were added accordingly. The plate was incubated for 24 h at 37°C in a 5 % CO₂ atmosphere. After the incubation period, the plate was taken out from the incubator and MTT reagent to a final concentration of 0.5mg/Ml of total volume. The plate was covered with aluminium foil to avoid exposure to light and were incubated for 3 h. The MTT reagent was discarded and then 100µl of solubilization solution (DMSO) was added.

The absorbance reading on a spectrophotometer or an ELISA reader at 570 nm and 630 nm was used as reference wavelength. The IC₅₀ value was determined by using linear regression equation i.e. Y = Mx+C. Here, Y= 50, M and C values were derived from the viability graph. % of cell viability is calculated using the below formula:

% cell viability = (Mean absorbance of treated cells / Mean absorbance of Untreated cells) × 100

Statistical Analysis: All experimental measurements were tested in duplicate. The results are expressed as mean ± standard deviation (SD). Statistical analysis of data was evaluated using a one-way analysis of variance (ANOVA). The significance level was set at <0.05.

 RESULT AND DISCUSSION: Our preliminary results suggested that the Panchagavya shows an inhibitory effect on cell growth in HCT 116 cells. Freshly prepared PGS1 and PGS2 were assessed against HCT 116 colon cells for anti-proliferative activity in 24 h at the above-mentioned concentrations (Table 1 and Table 2 using MTT assay).

After 24 treatments, it was seen that HCT116 cell growth was significantly inhibited in a concentration-dependent fashion, especially when treated with PGS2 Fig. 2. The observations in Statistical data of the MTT cytotoxicity study suggesting us that against HCT-116 cell lines, Test Compounds, namely S1, S2 showing significant cytotoxic potential properties with the IC₅₀ concentration sat 84.42 ug/ml and 62.56ug/ml respectively, used for the study. Std drug, Cisplatin showing IC₅₀ value at 12.5ug/ml respectively Fig. 3.

TABLE 1: CELL VIABILITY OF PGS 1 TREATED HCT-116 CELLS

TABLE 2: CELL VIABILITY OF PGS 2 TREATED HCT-116 CELLS

FIG. 1: COMPARATIVE % OF CELL VIABILITY OF PGS 1 TREATED HCT-116 CELLS       

FIG. 2: COMPARATIVE % OF CELL VIABILITY OF PGS 2 TREATED HCT-116 CELLS

GRAPH 1: DENOTING IC₅₀ VALUE FOR PGS1                 

GRAPH 2: DENOTING IC₅₀ VALUE FOR PGS2

FIG. 3: COMPARATIVE IC₅₀ VALUE OF PGS 1 AND PGS 2 ON HCT-116 CELL

CONCLUSION: Colon cancer is considered the most common cancer in men and the third in women ^{10, 16-18}. The development and invasion process of cancers, including colon cancer, is correlated with dysbiosis in gut microbiota. Among the compounds, PGS1 shows moderate cytotoxicity and PGS2 shows significant cytotoxicity and may be considered a potent anti-cancer agent due to its low IC₅₀ value on HCT-116 cells. Further studies like Cell Cycle Study by PI staining, Apoptosis study by Annexin V/PI staining, Apoptotic Protein expressions like Caspase 3, 7, 9, Bcl 2, p 53, and ROS study to evaluate the mechanism of action of test compounds viz., PGS1 and PGS2 behind the anti-cancer potential in in-vitro conditions.

ACKNOWLEDGMENT: Authors thank Prime Minister’s Research Fellowship for financial support.

CONFLICTS OF INTEREST: The author declares no conflicts of interest.

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    How to cite this article:

    Funde S, Patil S and Palep H: Anticancer effect of panchagavya on human colon adenocarcinoma (HCT-116) cell line. Int J Pharm Sci & Res 2022; 13(4): 1771-76. doi: 10.13040/IJPSR.0975-8232.13(4).1771-76.

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