Tropaeolum majus L. and low dose gamma radiation suppress liver carcinoma development via EGFR-HER2 signaling pathway

Abstract Hepatocellular carcinoma (HCC) is one of the most fatal cancers around the world and remain asymptomatic in early stage. An alcoholic extract prepared from leaves of Tropaeolum majus L. (Tropaeolaceae) was assessed for its potential activity against diethylnitrosamine-induced liver carcinoma in vivo. Oral administration of the extract significantly decreased the inflammatory marker translation NF-kB and supressed HCC progression in combination with 0.5 Gy gamma radiation via EGF-HER-2 pathway. Histopathological and immunohistopathological features also showed the recovery of a hepatic architecture. Immunohistochemical study showed the T. majus and LDR enhancement effect on proapoptotic markers (caspase-3 and Bax) and inhibition of anti-apoptotic factor (BCl2). HPLC-DAD-MSn analysis of the extract revealed the annotation of twelve compounds. T. majus could mediate a defensive influence against diethylnitrosamine-induced hepatocarcinogenesis and serve as a respectable option in amelioration of the hepatocellular carcinoma development in combination with low dose of gamma radiation. Graphical Abstract

ABSTRACT Hepatocellular carcinoma (HCC) is one of the most fatal cancers around the world and remain asymptomatic in early stage. An alcoholic extract prepared from leaves of Tropaeolum majus L. (Tropaeolaceae) was assessed for its potential activity against diethylnitrosamine-induced liver carcinoma in vivo. Oral administration of the extract significantly decreased the inflammatory marker translation NF-kB and supressed HCC progression in combination with 0.5 Gy gamma radiation via EGF-HER-2 pathway. Histopathological and immunohistopathological features also showed the recovery of a hepatic architecture. Immunohistochemical study showed the T. majus and LDR enhancement effect on proapoptotic markers (caspase-3 and Bax) and inhibition of anti-apoptotic factor (BCl2). HPLC-DAD-MS n analysis of the extract revealed the annotation of twelve compounds. T. majus could mediate a defensive influence against diethylnitrosamine-induced hepatocarcinogenesis and serve as a respectable option in amelioration of the hepatocellular carcinoma development in combination with low dose of gamma radiation.

Introduction
Hepatocellular carcinoma (HCC) is the greatest common type recorded cancer and still the principal cause of deaths wide world. It accounts for 70-90% of all primary liver cancers (Liu et al. 2019;Taha et al. 2021). Diethylnitrosamine (DEN) is a powerful environmental cancer-causing agent. It is considered as one of the most frequent risk factors for enhancing HCC (Singh et al. 2018). Therefore, more focus should be put on HCC treatment which is challenging for the medical profession. So far, there are too many therapies dealing with HCC, such as molecular targeted therapy and radiotherapy. However, these therapies besides having benefits and drawbacks, they do not always result in the best possible outcomes (Liu et al. 2019). Edible plants have attracted attention for its beneficial effects on human health through their use in providing beauty to culinary preparations. These effects may be attributed to their enormous and diverse content from bioactive compounds (Navarro-Gonz alez et al. 2015b; Takahashi et al. 2020). Tropaeolum majus L. (Tropaeolaceae), known as Garden nasturtium or Indian cress, is an edible annual herbaceous plant that originate in South America (Hifnawy et al. 2016). Due to its high nutritional value, the floral buttons, leaves, and flowers are commonly used in salads. Analysis of different extracts of T. majus showed that it is rich in biologically active constituents such as glucosinolates (GSL), flavonoids, phenolic acids, carotenoids, cucurbitacins, fatty acids, essential oil and proteins (Brondani et al. 2016). Several biological activities had been associated with T. majus such as antihypertensive, diuretic, antimicrobial, antioxidant, antithrombin and anti-inflammatory effects (Brondani et al. 2016). The objectives of this study were to prepare the alcoholic extract of the leaves of T. majus, investigate its phytochemical content using LC/MS, evaluate its influence on rats develop HCC by diethylnitroseamine and finally compare the results with low dose gamma radiation.
Hepatocellular carcinoma (HCC) is a hepatocyte derived malignancy. It represented one of the most cancers triggering death due to its aggressiveness and poor diagnosis (Blidisel et al. 2021). Diethylnitrosamine (DEN) is a powerful environmental cancer causing agent and consider one of the major risk factors (Singh et al. 2018). It was documented that DEN initiates hepatocarcinogenesis in rat, which is represented the major recognised animal models for hepatic cancer, which permits to regulate the anticancer agents on various periods of neoplastic change and development (Macek Jilkova et al. 2019). In the present work, MTT cytotoxicity assay ( Figure 1S) was performed to examine the cytotoxic effect and the effective dose of T. majus against tumor cell line. The death of cancer cells in different doses may be due to the mitochondrial loss as it is one of apoptosis hall marks (Hassan et al. 2016).
Accumulating knowledge authenticate the role of Benzyl isothiocyanate (BITC) in cancer prevention by several mechanisms (Ma et al. 2019;Roy et al. 2019;Cheng et al. 2020;PoChun et al. 2019). Our study revealed that BITC prevent HCC by downregulation of EGFR/Her2 signaling. Early studies established that EGFR activates NF-kB through the proteasome-mediated degradation of the inhibitory molecule IkBa (Shi et al. 2019), that's might be the reason of the elevation of NF-kB in rats administrated with DEN alone while its activity diminished in groups treated with T. majus (as a source of BITC) alone or in combination with LDR in which there is many studies revealed that NF-kB signaling can be activated by both low and high doses of radiation in several types of cells to perform many biological functions, as that of radiation-induced apoptosis and oxidative stress ( Figure 2S) (Yu et al. 2018). It is well established that EGFR is highly expressed in adult hepatocytes (Berasain et al. 2011). This wark revealed that there is significant decrease in EGFR/Her2 levels in group treated with T. majus (BITC) and LDR (Figure 3Sa,b). On the other hand, their levels were significantly elevated in DEN group in compared to control group as the nonstop activation of EGFR signaling is a mark of HCC and contributes to the proliferation, resistance to apoptosis and invasive behaviour of HCC cells (Berasain et al. 2011). Apoptosis is marked by a specific biochemical and morphological changes. Any defect in these changes may leads to cancer development. Therefore, induction of apoptosis is one of anticancer drug development targets. Survivin, a member of apoptosis protein inhibitor family is broadly expressed in a different human cancer tissues (Papanikolaou et al. 2011). Consequently, western-blotting exam revealed that survivin is highly expressed in group administered with DEN alone (7.77 ± 0.25) in compared to normal group (1.04 ± 0.012) ( Figure S4). This is supported by the histopathological and immunohistochemical studies that demonstrate that; in D group there are many deteriorations which manifested the characteristic of malignancy. In addition, in the same group the angiogenesis is established between tumor cells ( Figure S5B). Similarly, previous studies showed the same results (Rafii et al. 2002;Hung et al. 2014). The elevation of survivin is accompanied by overexpression of BCl2 of the same group ( Figure  S8). In contrast there is a significant decrease in Caspase 3 and Bax immunoreactivity ( Figures S6 and S7) this may be due to the up-regulation of survivin protein by HER2 and EGFR signals and therefore down-regulation of caspase 3/7/9 by survivin, which leads to apoptotic inhibition (Ju et al. 2013). Up-regulation of survivin by HER2/EGFR signals may illustrate the poor prognosis and chemoresistance of HER2 in breast cancer (Asanuma et al. 2005). However, the treatment by T. majus alone (TD) or LDR (RD) and both (TRD) is dramatically diminished survivin (3.5 ± 0.02, 3.9 ± 0.1 and 2.6 ± 0.06, respectively) in compared to positive control group (D). Histopathological investigation verified some limitation of the new blood vessels formation in between tumor cells with certain regression of these tumor cells. Same results were explained by (Zakaria et al. 2018) who demonstrated the potential role of BITC in inhibiting angiogenesis in cancer cells. Likewise, the effect of BITC in inducing apoptosis is illustrated in all treated groups by increasing the immunoreactivity of Caspase-3 ( Figure S6), Bax ( Figure S7) and decreasing BCl2 ( Figure S8). These data are supported by Liu et al. (2019) who confirmed that phenyl-BITC helped in mitochondrial-release of cytochrome c into cytoplasm and elevate the pro-apoptotic protein Bax expression, in the same time decreased the expression of anti-apoptotic protein BCl2 and (Chang et al. 2021) who stated that BITC have chemoprevention activity in defeating cancer cell viability, proliferation, migration and invasion, and in inducing cancer cell apoptosis.
Harmoniously, the role of LDR was elucidated by (Janiak et al. 2017) who documented that the exposure to LDR is a powerful stimulator of many anti-neoplastic functions of the immune system, as inflammation impairment and/or up-regulation of anti-inflammatory cytokines. Also, (Hassan and El-Sonbaty 2020) demonstrated the potency of LDR (0.5 Gy) inducing apoptosis and reducing angiogenesis in HCC. Thus, treatment with isothiocyanate extract and low dose of c-irradiation promote apoptosis of tumor cells in rats receiving DMN by the upregulation of the activity of apoptotic proteins such as Caspase-3 and Bax and the downregulation of anti-apoptotic Bcl-2 protein.

Conclusion
The alcoholic extract of Tropaeolum majus was assessed for its potential activity against diethylnitrosamine-induced liver carcinoma in vivo via EGFR-HER2 signaling pathway. Oral administration of extract significantly decreased the inflammatory marker translation NF-kB, supress HCC progression in combination with 0.5 Gy gamma radiation via EGF-HER-2 pathway. These results were confirmed using histopathological and immunohistopathological studies. The aforementioned effect may be due to its sulphur and phenolic content. T. majus could mediate a defensive influence against diethylnitrosamine-induced hepatocarcinogenesis and serve as a respectable option in amelioration of the hepatocellular carcinoma development in combined with low dose of gamma radiation.

Disclosure statement
No potential conflict of interest was reported by the authors.

Funding
The author(s) reported there is no funding associated with the work featured in this article.