INTRODUCTION:

Lung cancer is one of the most affecting malignant tumours which results in a high mortality rate¹. Lung cancer is caused due to pipe and cigar smoking, air pollution, radiation and agents such as nickel, chromium and arsenic. According to WHO 63% of mortality rates were caused by cancer. Many studies have observed cytotoxicity and cell-cycle arrest and some studies have proved in human models. Nowadays surgery and radiation are the main techniques to treat lung cancer. Chemotherapy and herbal medicines are also used as alternative techniques.

Herbal medicine has been used for many years. Medicinal plants are a natural source which has less side effects and has an ability to scavenge free radicals. Plants have been used for many centuries to prevent diseases; therefore these plants are pharmacologically active. Due to the strong therapeutic effects, the medicinal plants have been traditionally used to treat diseases²^-⁶. Different parts of medicinal plant have numerous nutraceutical values and are enriched with proteins, carbohydrates, vitamins, fibre, potassium, calcium and also the presence of phytoconstituents contributes to its significant medicinal property⁷ ^-¹²

Carica papaya contains phytochemicals like flavonoids, alkaloids, saponins, terpenoids, and amino acids which acts as an anti-inflammatory drug to treat lung cancer¹³^-¹⁶. Carica papaya is cultivated in subtropical countries which are used as a traditional medicine¹⁷^-¹⁹. It belongs to the family called caricaceae^16,20. Carica papaya contains an enzyme called papain which has many pharmaceutical applications and antioxidant activity. There is limited information on the medicinal value of Carica papaya, particularly its cytotoxicity effect against cancer cell lines^7,8,²¹^,22. Carica papaya contains phytochemicals like flavonoids, alkaloids, sapponents, terpenoids, and amino acids which acts as an anti-inflammatory drug²³. Carica papaya is also used to treat various diseases like sinus, eczema, blood pressure, constipation and various tumours¹⁴. Leaf extracts of Carica papaya contain lots of antioxidant properties therefore natural compounds with antioxidant properties can be focussed for research to treat cancer cells because of their trivial or no side effects. It also possesses anti-thelmic, anti-inflammatory, anti-bacterial, anti-viral properties^24,25. Our team has extensive knowledge and research experience that has translated into high quality publications²⁶^-⁴⁶ . Hence, the aim of the current study was to evaluate the effect of Carica papaya seed extract on inhibition of cell proliferation and analyse the effects of Carica papaya extract on the IL-6 and TNF-α in the human lung cancer cell line.

MATERIALS AND METHODS:

Dimethyl sulfoxide (DMSO), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma Chemical Pvt Ltd, USA. Trypsin-EDTA, fetal bovine serum (FBS), antibiotics-antimycotics, RPMI 1640 medium and phosphate buffered saline (PBS) were purchased from Gibco, Canada. (5,5,6,6-tetrachloro-1,1,3,3 -tetraethylbenzimidazolocarbocyanine iodide) and Real Time PCR kit was purchased TAKARA (Meadowvale Blvd, Mississauga, ON L5N 5S2, Canada).

Cell lines and cell culture:

The Human Lung cancer cell line (A549 ) was purchased from the National Centre for Cell Sciences (NCCS), Pune, India. Cells were cultured in DMEM medium (Thermo Fisher Scientific, CA, USA) containing 10% fetal bovine serum (Thermo Fisher Scientific, CA, USA), 100 U/ml penicillin and 100 μg/ml streptomycin (Thermo Fisher Scientific, CA, USA) at 37°C with 5% CO₂.

Cell viability by MTT assay:

Cell viability was assayed using a modified colorimetric technique that is based on the ability of live cells to convert MTT, a tetrazolium compound into purple formazan crystals by mitochondrial reductases (Mosmann, 1983). Briefly, the cells (1 ×10⁴/well) were exposed to different concentrations of Carica papaya extract (100-500µg/ml) with A549 cells for 48 h. At the end of the treatment, 100µl of 0.5mg/ml MTT solution was added to each well and incubated at 37^◦C for an hour. Then the formazan crystals formed were dissolved in dimethyl sulfoxide (100µl) and incubated in the dark for an hour. Then the intensity of the color developed was assayed using a Micro ELISA plate reader at 570 nm. The number of viable cells was expressed as the percentage of control cells cultured in serum-free medium. Cell viability in the control medium without any treatment was represented as 100%. The cell viability is calculated using the formula:

% cell viability = [A570nm of treated cells/A570nm of control cells] × 100.

Gene expression analysis by Real Time-PCR:

Samples from each group were submerged in 2ml Trizol (Invitrogen, Carlsbad, CA, USA) for RNA extraction and stored at −80°C until further processed. cDNA synthesis was performed on 2μg RNA in a 10μl sample volume using Superscript II reverse transcriptase (Invitrogen) as recommended by the manufacturer. Real-time PCR array analysis was performed in a total volume of 20μl including 1μl cDNA, 10μl qPCR Master Mix 2x (Takara, USA) and 9μl ddH₂O. Reactions were run on an CFX96 Touch Real-Time PCR Detection System (Bio-Rad, USA) using universal thermal cycling parameters (95°C for 5 min, 40 cycles of 15 sec at 95°C, 15 sec at 60°C and 20 sec at 72°C; followed by a melting curve: 5 sec at 95°C, 60 sec at 60°C and continued melting). For quality control purposes, melting curves were acquired for all samples. The specificity of the amplification product was determined by melting curve analysis for each primer pair. The data were analysed by comparative CT method and the fold change is calculated by 2−ΔΔCT method described by Schmittgen and Livak (2008) using CFX Manager Version 2.1 (Bio Rad, USA).

Statistical analysis:

The obtained data were analysed statistically by one-way analysis of variance (ANOVA) and Duncan's multiple range test with computer-based software (Graph Pad Prism version 5) to analyse the significance of individual variations among the control and experimental groups. The significance was considered at p<0.05 level in Duncan's test.

RESULTS:

Effect of Carica papaya on cell viability in A549 cells: In the present study Carica papaya extract has significantly (p<0.05) inhibited the growth of lung cancer cells in dose dependent manner. However 400-500 μg/ml concentration of the extract showed maximum inhibition of the viability of the lung cancer cells suggesting that Carica papaya includes apoptosis in A549 cells (figure.1)

Effect of C. papaya on IL-6 mRNA expression in A549 cells:

In untreated control cells, IL-6 mRNA expression was found to be increased. Treatment with 300 and 400 μg/ml concentration of Carica papaya extract has reduced the expression Of IL-6 mRNA when compared to control cells (p<0.05) (Figure.2).

Effect of C. papaya on TNF-α mRNA expression in A549 cells:

In untreated control cells, TNF-α mRNA expression was found to be increased. Treatment with 300 and 400 μg/ml concentration of Carica papaya extract has reduced the expression of TNF-alpha mRNA when compared to control cells (p<0.05)(Fig.3)

Figure-1: Effect of Carica papaya extract on cell viability in A549 Cells. Each bar represents a mean±SEM of 6 observations. Significance at p< 0.05, a-compared with untreated control cells, b-compared with 1nM treated A549 cells.

TNF-α-mRNA expression (Fold change over control)

Figure 2: Effect of carica papaya extract on TNF-α mRNA expression in A549 cells. Each bar represents a mean±SEM of 6 observations. The X-axis represents - different concentrations of Carica papaya and the Y axis represents the fold change over control. a-compared with untreated control cells.

IL-6- mRNA expression (Fold change over control)

Figure-3: Effect of carica papaya extract on IL-6 mRNA expression in A549 cells. Each bar represents a mean ± SEM of 6 observations. The X-axis represents - different concentrations of Carica papaya and the Y axis represents the fold change over control. b-compared with untreated control cells.

DISCUSSION:

Carica papaya is used to treat various diseases like sinus, eczema, blood pressure, constipation and various tumours. Leaf extracts of Carica papaya contain lots of antioxidant properties therefore one with antioxidant properties used to treat lung cancer cell lines. Carica papaya possesses anti-thelmic, anti-inflammatory, antibacterial, antiviral properties. The present study was carried out to investigate the role of Carica papaya on A549 cancer cell lines. Zuhrotun Nisa F et al showed the anticancer activity of breast cancer cell lines which proved the cytotoxicity of Carica papaya in breast cancer cell line MCF-7²⁵. Saha S et al showed the effect of Carica papaya in colon cancer, liver cancer, blood cancer cell lines etc⁴⁷. Finally, the various formulation approaches using Carica papaya extract have been highlighted^47,48. Our study demonstrated the cytotoxic effects of Carica papaya in A549 cancer cell lines inhibited the growth of lung cancer cells with dose-dependence compared to untreated control cells. This is similar to the previous study which has demonstrated that phenolic compounds of fruits and vegetables exhibited cell death. Since natural compounds have fewer side effects therefore it is considered as a suitable drug in treating cancer cell lines⁴⁹.

Numerous studies reported that proinflammatory factor NFκB could favour tumorigenesis which is activated by carcinogens, inflammatory agents and tumour promoters. Our study demonstrated that Carica papaya extract has reduced the expression of IL-6 and TNF-α mRNA at 400-500μg/ml concentration when compared to control cells. Therefore our study showed that the anticancer effect exhibited by Carica papaya on lung cancer cells was regulated by IL-6 and TNF-α. Previous studies demonstrated that natural phenolic compounds such as shogaol and gingerol showed anticancer properties and antioxidant effects. They observed that the ginger extract was able to inhibit cancer cells by NFκB and TNF-α⁵⁰.

The current study provides anti-cancer potential of Carica papaya against human lung cancer cells (A549) which provides less side-effect than usual drugs. Over the last two decades, Public interest and research efforts in medical communities have grown to a great extent on herbal medication for the cure. So there will be a large spectrum of scope for herbal drugs against cancer cells. In future scope, preclinical investigations, such as in vivo animal models and clinical trials on the effects of the seed, are required.

CONCLUSION:

This present study indicated that Carica papaya with abundant bioactive phytochemicals has the potential to be of use against lung carcinoma. However, there is a need for more scientific investigations to enhance our understanding of how Carica papaya may exert its anticancer effects. Therefore in future scope this study can be done in live models.

ACKNOWLEDGEMENT:

We thank Saveetha dental College for their support to conduct this study.

AUTHOR CONTRIBUTION:

Padmapriya: Literature collection; Preetha: Framing the manuscript, statistics approval and approval of manuscript; Sridevi and Selvaraj: Final approval of manuscript

SOURCE OF FUNDING:

The study was funded by Saveetha Institute of Medical and Technical Science, Saveetha Dental College, Saveetha University and National Insurance Building, Parrys.

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Received on 08.09.2021 Modified on 24.01.2022

Research J. Pharm. and Tech 2022; 15(12):5478-5482.

DOI: 10.52711/0974-360X.2022.00924