Introduction

Skin cancer is formed from the uncontrolled growth that arises from the abnormal skin cells. Skin mediates body health by serving as a protective barrier by its role in absorption, immunity and secretion. Due to elevated pollution in the environment and ultraviolet radiation, sun damage, and microorganisms, clinical behavior (Ghissassi, 2009), results in increased mortality and morbidity rate associated with skin tumors in the past years (Li, 2015). Skin carcinogenesis, which is a multistage process involves cancer initiation, promotion, and progression (Cadet, 2005; Hennings, 1993). Most cases around 90% are found to be affected from the radiation of ultraviolet rays from the sun. Skin cancer which includes melanoma and nonmelanoma. Skin cancers related to non-melanoma (NMSCs) largely consist of basal cell carcinoma (BCC) and squamous cell carcinoma (SCC). SCC represents about 30% to 65% in Indians and in blacks whereas in whites around 15% to 25%, whereas in BCC about 65% to 75% in whites and 20% to 30% in Indians (Gloster & Neal, 2006). Skin cancer treatments including cryotherapy, surgical excision, laser ablation, electrochemotherapy (ECT), photodynamic therapy. Skin cancer treatment of several chemotherapy drugs can be used to paclitaxel, cisplatin, carboplatin. Chemotherapy treatment all causes adverse effects such as loss of normal functioning of cells due to targeting of non-specific sites. Recently 5-years of studies of melanoma in 10 % are the cause of death from skin cancer. Very low response rates were provided in melanoma associated chemotherapy, and multiple targeted therapies have been carried out in melanoma. 5-Fluorouracil (5-FU) is an anticancer drug which inhibits thymidylate synthetase. 5-fluorouracil (5-FU) that target cellular receptors or immunological responses at the molecular level in the body is used to treat skin cancer. However, the use of 5-fluorouracil (5-FU) has many side effects. 5-fluorouracil result in inactivation of an enzyme dihydropyrimidine dehydrogenase which reduce the absorption in the gastrointestinal tract, which affect the normal functioning of normal cells and bone marrow by producing toxin (Rajagopalan, 2019). Therefore, discovery and development of novel plant based therapeutic compounds are urgently in need of survival as well as in treatment of patients associated with skin cancer.

Many traditional medicines, phytochemicals have been investigated as a new discovery for chemotherapy drugs which disrupts the cell cycle and induce apoptosis. A wide variety of vegetables, fruits are found to exhibit a natural polyphenolic compound which is regularly consumed by humans. However the effect and role of citrus flavonoid on the health condition of humans is still elusive because of lack of evidence in clinical trials. Thus, the research related to their biological activities are reported so far both invitro and in animal models.

Naringin, a flavonoid rich in grapefruit and other citrus fruits. Naringin, a bioflavonoid that has been investigated to be an antioxidant, anti-inflammatory, pharmacological activities, antiviral activities and also as an anticancer compound (Aroui, 2016; Zeng, 2014). Naringin suppress to various cancer like breast, melanoma (Guo, 2016). Naringin has the potential to reduce the pathogenesis induced by cisplatin by inhibiting ROS mediated NF-κB and p53 signaling pathway could suppress PCa invasion and metastasis (Chtourou, 2015; Erdogan, 2016; Georgescu, 2010; Li, 2016). In animal models, it is demonstrated that naringin down-regulates the IL-1β, IL-8, IL-6, TNF-α, NF-κB and upregulates the TGF-β and VEGF which repairs the skin tissue (Lowes, 2014). Naringin along with sericin found to inhibit the expression of TNF-α (Liu, 2012).

Our study aims to investigate the individual anti-inflammatory role of 5- fluorouracil, naringin and in combination in A375 cell lines in vitro. Inhibitory effect in the inflammatory cytokines in mRNA levels of IL-6, IL-1β, TNF-𝛼 and NF-𝜅B in human skin cancer cell line A375 cells were investigated.

Materials and Methods

Reagents

Naringin, MTT solution and DMSO dimethyl sulfoxide purchased from sigma. Penicillin, streptomycin, and Gentamicin, FBS fetal bovine serum and media DMEM was purchased from Gibco, USA.

Preparation of cell line

The A375 Melanoma human skin cancer cell line was purchased from NCCS, Pune. Cells were grown in DMEM media with 10% FBS, penicillin, streptomycin and gentamycin respectively and incubated in a 5% CO₂ at 37⁰C.

Cell Viability

MTT Assay

In 96-well plates A375 cells were seeded at a density of 5 × 10³cells per/well. About 200 ml of DMEM media along with 10% FBS was added and kept at overnight incubation. Non-adherent cells were removed and washed after 24 hrs. Briefly, 1×10⁴cells treated with stepwise increasing concentrations of naringin and 5- fluorouracil treated for 24- 48 hrs. To the treated plates add 20 ml of MTT solutions and incubate for 3hrs at room temperature, and about 150ml of formazan per/well was dissolved DMSO and the absorbance was measured at 570nm using microplate reader. The results were assessed as a percentage of negative control. Finally, the obtained cell viability plots were used to calculate the IC50 values.

RNA Extraction and cDNA Synthesis

Total RNA isolated from A375 cells treated with or without naringin and 5- fluorouracil for 24 hrs were isolated by TRIzol (Invitrogen) and quantified in BioPhotometer (Eppendorf, Milan, Italy). For Complementary DNA about 1 µg of total RNA were used per sample and the reaction was performed with Iscript cDNA Synthesis Kit (Bio-Rad Laboratories, Hercules, CA, USA). 35 cycles for IL-1β(94ºC for 30s, 65ºC for 60s and 72ºC for 60s), 40 cycles of IL6 (94ºC for 60s, 60ºC for 60s and 68ºC for 120s), NFkB 40 cycles of (94ºC for 60s, 60ºCfor 60s and 68ºC for

120s), 40 cycles for TNFα (94ºC for 60s, 60ºC for 60s and 68ºC for 120s), and 30 cycles for β actin (94ºC for 35s, 64ºC for 45s and 72ºC for 1 min). Then 10 μl were electrophoresed in 2% agarose gel and analyzed in gel doc XRS plus (Bio-Rad, USA). The densitometric analyses were performed using image lab software (Bio-Rad, USA). Naringin and 5- fluorouracil primer sequences (Table 1) for detection of mRNA expression were synthesized by the following table.

Results

Cell Viability Assays

MTT

To establish the impact of naringin, 5-fluorouracil combination on A375 MTT assay was performed. The growth of cell lines was found to be inhibited on treatment with naringin in a dose dependent manner (0– 100 μM) for different time points (24-48hrs). 50-60% growth inhibition was observed in A375 cell lines at a concentration of 24.75 μM (Figure 1A). Similarly, 5-fluorouracil inhibited the growth of the A375 cell lines at a concentration of 2.5 μM (Figure 1B). The combination of naringin+5-fluorouracil on A375 cell lines at a concentration of half IC₅₀ values (12µM+1 μM) exhibited profound cell inhibition and was used for further experiments (Figure 2).

Cells were treated with increasing concentrations of naringin, 5-fluorouracil for 48 h and analyzed using cell viability assay. Data are mean ± SD of triplicate values. The results were analyzed as the percentage of control value. Data are mean ± SD of triplicate values.

mRNA Expression Profiling –RT PCR

To assess the levels of mRNA expression for TNFα, IL-6, IL-1βandNFκB. Total RNA was isolated from skin cancer cell lines. Inflammatory cytokine specific bands for TNFα, IL-6, IL-1β,and NFκB were assessed in melanoma cancer cell line A375 cells treated with naringin, 5-fluorouracil and their combination. The combination group revealed a decreased level of TNFα, IL-6, IL-1β, and NFκB when compared with individual treatments of naringin and 5-fluorouracil & untreated groups (Figure 3). Each experiment was used in triplicates for the treatment group. Results were analyzed by the Mean ± SD. * p<0.05, **p<0.01 compared with control.

Discussion

A naturally occurring flavonoids with phenolic compounds with vast diversity of bioactivities. Flavonoids are found in vegetables, fruits, and medicinal plants, have antimicrobial and antioxidant effects (Gupta, Khan, Verma, & Pathak, 2013). Naringin is a flavonoid with anti-inflammatory, anticancer various tumors like breast, melanoma, cervical, bladder, liver (Kaur & Badhan, 2015; Kim, 2008; Li, 2013; Vadde, 2015). Naringin inhibit IL-1Β, TNFα, IL-6 may prove to be useful in treating skin cancers as adjuvants in combination with chemo-therapeutic drugs. In the present study, a reveal the dose-dependent anti-inflammatory effect of naringin and 5-fluorouracil on A375 cell lines.

We examined the anti-proliferative effect of naringin and 5-fluorouracil using MTT assay. Cell viability decreased with an increased dose of naringin and 5-fluorouracil treatment. Our data suggested that naringin and 5-fluorouracil inhibited cancer cell growth and proliferation. MTT assay which is based on cytolysis also exhibited reduced viable cells in naringin and 5-fluorouracil treated compared to untreated control.

This is the first study demonstrating the combined effects of naringin and 5-fluorouracil against inflammatory cytokines in A375 cells. Naringin and 5-fluorouracil significantly suppressed the levels of IL-6, IL-1β and TNFα in A375 cell lines. By inhibiting IL-6, IL-1β and TNFα by natural anti-inflammatory drugs which could contribute to the reduction in the expression of NF-κB (Linus, 2017). Similar finding was revealed by (Christobel, 2019) with a variety of phytoconstituent, wedelolactone in cervical cancer cells. (Bi & Jiang, 2016) reported that Naringin inhibited inflammation and apoptosis in LPS-induced HUVECs. (Chen, 2017) findings revealed that Naringin reduced the expression of IL-6, IL-1β and TNFα. NF-κB signaling pathway was found to be inhibited in IL-1β-Induced NP cells proliferation by celastrol (Chen, 2017), baicalein (Jin, 2019). Thus, there exists an inseparable interlink between inflammatory cytokines and NF-κB which mediate the promotion of cancer in many tumor types. The inhibition of NF-κB and the inflammatory cytokines by naringin and 5-Fluorouracil could therefore be applicable for treating skin cancer.

Conclusions

In conclusion, the present study reveals the efficacy of naringin/5-fluorouracil alone and in combination which was found to down regulate the inflammatory cytokines such as IL-6, IL-1β, TNFα and NF-𝜅B in A375 cell lines. Most importantly these results suggest the effective anti-inflammatory role of naringin and 5-fluorouracil in combination. Further, studies in animal models in combination should be confirmed to check its toxicity and efficacy.

Conflict of Interest

The authors declare that they have no conflict of interest for this study.

Funding Support

The authors declare that they have no funding support for this study.