Effect of Kabasura Kudineer Extract on Inflammatory Cytokines [Interleukin-6 and Tumour Necrosis Factor-α] in Lung Cancer Cell Line A549

Background: Kabasura kudineer is widely known for its anticancer efficiency. Kabasura kudineer is a customary formulation used by siddha practitioners for effectively managing common respiratory illness. Herbal medicines are acknowledged as a great approach to lung cancer therapy. Aim of the study is to know about the anticancer property of Kabasura kudineer extract on inflammatory cytokines IL-6 and TNF-α in lung cancer cell line (A549). Materials and Methods: Human lung cancer cell line (A549) was purchased from National Centre for Cell Sciences (NCCS), Pune, India. Cell viability test was done by MTT assay. Gene expression analysis was done by Real Time-PCR. The obtained data were analysed statistically by one-way analysis of variance and Duncan’s multiple range test with Graph Pad Prism version 5 to analyse the significance. The significance was considered at p<0.05 level in Duncan’s test. Results and Discussion: Kabasura kudineer caused a marked increase in cell death in dose dependent manner. AT the end of 48 hours, maximum inhibition was at 400 and 500 μg/ml. Kabasura kudineer extract reduced the expression of IL-6 and TNF-α compared to the control cells. Original Research Article Nisha et al.; JPRI, 33(47B): 653-660, 2021; Article no.JPRI.74500 654 Conclusion: This study concluded that Kabasura kudineer extract has anticancer activity on lung cancer cell lines (A549).


INTRODUCTION
Cancer is a disease characterized by its uncontrolled growth and spread of abnormal cells. Increase in proliferative growth signals, insensitive to growth-inhibitory signals, apoptosis evasion, angiogenesis induction, invasion leading to metastasis are the important features of cancer (1)(2)(3). Lung cancer mortality rate has increased among cancer related deaths worldwide [1]. In 2018, GLOBOCAN estimated 2.09 million new cases (11.6% of total cancer cases) and 1.76 million deaths (18.4% of total cancer deaths), above 2012 reported rates (1.8 million new cases and 1.6 million deaths), making it the foremost frequent cancer and explanation for cancer death in men and women combined and in women, the third common cancer type and therefore the second common explanation for cancer death [2].  19 pandemic has attracted the attention of the public towards the Kabasura Kudineer and its benefits in Tamil Nadu. It has been commonly used to treat fever and respiratory ailments.
Among the different types of cancer, lung cancer is considered one of the leading causes of cancer related death worldwide. High morbidity and mortality rates are seen in lung cancer [2,12]. The formation of tumour and its progression is strongly pronounced by the microenvironment surrounding the cells, which in most cases might be inflammatory (Quail DF, Joyce JA, 2013). These factors have led us to focus on pharmacological interventions to target the inflammatory pathways to inhibit growth of tumour and its progression (Lim JCW, 2012). The many of the herbs present in kabasura kudineer have been studied to possess antioxidant, anti inflammatory property. Natural antioxidants help to fight against free radicals and activated oxygen species in cancer related conditions. Since the kaba sura kudineer has been administered to treat respiratory complaints, hence the study was intended to investigate its effect on lung cancer cells. Our team has extensive knowledge and research experience that has translated into high quality publications (14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30). Our team has extensive knowledge and research experience that has translate into high quality publications [13][14][15][16][17]. The aim of this study was to evaluate the effect of Kabasura kudineer extract on inflammatory cytokines in the lung cancer cell line.

Cell Lines and Cell Culture
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% CO 2 .

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 ×10 4 /well) were exposed to different concentrations of Kabasura kudineer extract (100-500µg/ml) with A549 cells for 48 h. At the end of the treatment, 100 µl of 0.5 mg/ml MTT solution was added to each well and incubated at 37 C. The crystals formed were dissolved in dimethyl sulfoxide (100 µl) and incubated in 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 extract 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 = [A570 nm of treated cells/A570 nm of control cells] × 100.

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

Effect of Kabasura Kudineer on Cell Viability in A549 Cells
In the present study, Kabasura kudineer extract significantly increased (p<0,05) inhibiting the growth of the lung cancer cells dose-dependently compared to untreated control cells. However, 400 and 500 μg/ml concentration of the extract showed maximum inhibition of the viability of the lung cancer cells suggesting that Kabasura kudineer induces apoptosis in A549 cells (Fig. 1).

Effect of Kabasura Kudineer on Interleukin -6 mRNA Expression in A549 Cells
In untreated control cells,interleukin -6 and tumour necrosis factor -α expression was found to be decreased . Treatment with 400 and 500 μg/ml concentration of extract Kabasura kudineer reduced the expression of interleukin -6 when compared to control cells (p<0,05) (Fig. 2).

Effect of Kabasura Kudineer on TNFα mRNA Expression in A549 Cells
In untreated control cells, interleukin -6 and tumour necrosis factor -α expression was found to be significantly decreased. Treatment with 400 and 500 μg/ml concentration of Kabasura kudineer extract reduced the expression of TNFα when compared to control cells (p<0.05) (Fig. 3). Natural products rich in phytochemicals have been studied to evaluate its anti-cancer and cancer chemo-preventive activity, in vitro and in vivo. There is limited information on the medicinal value of kabasura kudineer, particularly its cytotoxicity effect against cancer cell lines. Hence the current study was done to evaluate the effect of Kabasura kudineer extract on inhibition of cell proliferation which could serve a better understanding of the underlying mechanisms of their anticancer potential of the extract [18].
Kabasura kudineer which is an important plant well known for its medicinal properties [19]. It is traditionally used for the treatment of respiratory ailments; recently it is used in covid-19 treatment [20,21,22]. The present study was carried out to investigate the role of Kabasura kudineer on A549 lung cancer cell line [23]. Accumulating evidence suggests that traditional medicines possess the potential as chemotherapeutic agents to prevent and treat cancer [24]. The great advantage of focusing on natural compounds to be considered for research is that they possess low toxicity or no few adverse side effects [25].
There are numerous studies which have reported that nuclear factor NFκB, a pro-inflammatory transcription factor, could favour tumorigenesis when activated by carcinogens, inflammatory agents, and tumor promoters [26,27]. It was found that Kabasura kudineer extract significantly increased inhibiting the growth of the lung cancer cells dose-dependently compared to untreated control cells [28]. Targeting the NFkB pathway in anti cancer treatment could serve a better route in the treatment against the cancer cells. NF-κB is a transcription factor playing a significant role in inflammatory pathways [29]. In our study, the effect of kabasura kudineer on TNF alpha was determined and found that on treatment of cancer cell lines with the plant extract at the concentration of 400 and 500 μg/ml, downregulated the mRNA expression of TNF alpha and IL 6 which showed that the plant extract has anti cancer effect through inhibition of mRNA expression of TNF alpha and IL 6. Recently, some medicinals have been studied for their possible action as inhibitors of the proinflammatory cytokines [30]. Ginger (Zingiber officinale) is widely used all over the world as a spice and condiment in daily cooking and this is one of the important components of kabasura kudineer. Various compounds are isolated from the substance like Zingiber officinale which can be used to study the anti lung cancer activity [31].
Ginger extract have been proved to possess anticancer potential against liver cancer cell lines. Hence present study was focused to identify the anti cancer effect of kabasura kudineer against lung cancer cell kines. The ginger being natural compounds are rich in active phenolic compounds such as shogaol and gingerol, and it has been shown to have anticancer and antioxidant effects [32]. The previous studies have reported the anti-cancer effect exhibited by ginger on liver cancer cells is mediated by inflammatory markers NFκB and TNF-α [33][34][35]. This is in accordance with our present study where ginger is one of the components of kabasura kudineer.
Piper longum which is one of the formulations of kabasura kudineer have been investigated for its anti cancer potential against prostate cancer cell lines in previous studies and have been proved to be effective against prostate cancer cells. This is in accordance to the present study where the piper longum, one of the formulations of kabasura kudineer have been found effective against lung cancer cells [36].

CONCLUSION
The present study has concluded that Kabasura kudineer extract has significantly down-regulated the pro-inflammatory cytokines [Interleukin-6 and tumour necrosis factor-α ] in lung cancer cell line exhibiting its anticancer property. This study showed that Kabasura kudineer extract has an anticancer potential against lung cancer cells.
The anticancer effect of kabasura kudineer could be attributed to the presence of phytochemicals present in the ingredients of kabasura kudineer.

DISCLAIMER
The company name used for this research is commonly and predominantly selected in our area of research and country. There is absolutely no conflict of interest between the authors and company because we do not intend to use this company as an avenue for any litigation but for the advancement of knowledge. Also, the research was not funded by the company rather it was funded by personal efforts of the authors.

CONSENT
It is not applicable.