Houttuynia cordata Thunb. and its bioactive compound 2-undecanone significantly suppress benzo(a)pyrene-induced lung tumorigenesis by activating the Nrf2-HO-1/NQO-1 signaling pathway

Worldwide, lung cancer remains the most frequently diagnosed cancer and the leading cause of cancer-related death, resulting in large social and economic burdens [1]. Many advanced treatments, including medical, surgical and radiotherapeutic interventions, have provided little effective improvement in the survival rates of patients diagnosed with primary lung malignancies [2]. The strong link between cigarette smoking and the development of lung cancer has been known for decades. The risk of lung cancer is 6 to 10 times higher in smokers than in nonsmokers [3], and almost 90% of patients diagnosed with lung cancer are cigarette smokers [4, 5]. Although it is widely recognized that smoking prevention and cessation are the best approaches to prevent lung cancer, tobacco-related lung carcinogenesis is still prevalent because of the difficulty in controlling smoking [2]. According to the WHO guidelines, chemoprevention has been the most effective strategy for lung cancer prevention, especially for smokers with existing pulmonary premalignancies [5, 6]. Therefore, it is essential and urgent to explore dietary factors that have the potential to prevent lung tumorigenesis.

Benzo(a)pyrene (B[a]P), which accounts for 22.5-69.8% of tobacco metabolites, can induce cell proliferation, inflammation, DNA alteration, and apoptosis, leading to lung cancer [7]. An evidenced-based study revealed that long-term exposure to B[a]P at a low dose could increase tumor incidence by up to 96.0% in animal models [8]. In part of its carcinogenic mechanism, B[a]P is metabolized into epoxide, which induces DNA adduct formation and causes mutations [9]. In addition, B[a]P-induced excessive production of reactive oxygen species (ROS), which results in oxidative stress, can lead to severe damage to DNA structure and is a prerequisite for B[a]P-associated tumorigenesis [10‐12]. Additionally, oxidative stress-induced inflammation is one of the major contributors to lung cancer initiation and progression [13, 14]. ROS in the respiratory system can increase the levels of pulmonary inflammation mediators and then initiate or promote lung carcinogenesis [15]. More importantly, there is a close relationship between ROS-induced DNA damage and inflammation, which can together accelerate the process of lung tumorigenesis. On the one hand, DNA damage may lead to chronic inflammation, which has been associated with the development of lung cancer [16‐18]. On the other hand, under inflammatory conditions, ROS are generated in inflammatory and epithelial cells and further cause oxidative and nitrative DNA damage [19, 20]. Therefore, the use of appropriate preventive pharmacological interventions that can effectively reduce B[a]P-induced DNA damage and inflammation has become an important strategy to prevent lung tumorigenesis.

Induction of enzymes that promote the detoxication of chemical carcinogens has been a broadly effective method for the chemoprevention of experimental carcinogenesis in rodent models [21]. The nuclear factor-erythroid 2-related factor 2 (Nrf2) signaling pathway has been targeted for the prevention of chemical carcinogenesis.Nrf2 has been identified as a key regulator of the inducible expression of antioxidative enzymes, anti-inflammatory proteins and conjugation/detoxification proteins, including NAD(P)H: quinone oxidoreductase 1 (NQO1) and heme oxygenase-1 (HO-1) [21]. Upon oxidative stress caused by B[a]P or pharmacologic induction, the Nrf2 pathway can be rapidly activated to counteract intracellular ROS generation, thereby attenuating DNA damage and inflammation promoted by B[a]P stimulation and contributing to reduced risk of mutation and subsequent lung tumorigenesis [21, 22]. Natural products are well known to exert their protective effects by removing free radicals, modulating the antioxidant defense system and performing carcinogen detoxification. To date, numerous compounds from natural plants have been found to activate Nrf2 signaling to effectively prevent B[a]P-induced lung tumorigenesis. These Nrf2 stimulators include sulforaphane, green tea phenols, curcumin, quercetin, catechin, and naringenin [23‐28]. Unfortunately, most of the antioxidants have failed phase III clinical trials for chemoprevention because of their poor bioavailability and reproducibility and/or harmful outcomes [29]. Thus, exploring more novel and effective candidate agents with low toxicity for chemoprevention is essential and urgent.

Houttuynia cordata Thunb. (Saururaceae) (H. cordata) is a well-known traditional Chinese medicinal herb widely used in China and Japan. Growing evidence suggests that H. cordata performs a variety of pharmacological functions, including antiviral, antitumor, antileukemia, anti-inflammatory, antioxidant, and antimutagenic functions [30, 31]. Furthermore, H. cordata is also popularly consumed as a healthy vegetable in East Asia [32]. Chemically, H. cordata is composed of flavonoids, alkaloids, volatile oils, sterols, fatty acids, and polyphenolic acids [33, 34]. Among the components, 2-undecanone is a vital bioactive compound that exhibits various types of biological activity [35, 36] and is used as a standard marker for quality control of H. cordata in the Chinese Pharmacopoeia. According to modern pharmacological studies and clinical practices, H. cordata is especially suitable for the treatment of lung diseases. For example, during an outbreak of severe acute respiratory syndrome, H. cordata was proposed to be an effective alternative treatment [30]. H. cordata also exerts a protective effect against bleomycin-induced pulmonary fibrosis [30] and may alleviate lipopolysaccharide-induced lung inflammatory injury [37]. It has also been demonstrated that H. cordata exhibits anti–lung cancer activity [38]. However, the chemopreventive effect of H. cordata against B[a]P-induced lung carcinogenesis and the underlying mechanism have not yet been explored.

In the current study, a B[a]P-stimulated lung adenocarcinoma animal model in A/J mice in vivo and a normal lung cell (BEAS.2B) model in vitro were established to investigate the chemopreventive effects of H. cordata and 2-undecanone against B[a]P-induced lung tumorigenesis. We also clarified the underlying mechanisms by which H. cordata and 2-undecanone could prevent lung carcinogenesis by measuring molecular markers of oxidative DNA damage and inflammation mediated by the Nrf2-HO-1/NQO-1 signaling pathway. The results of this study will enhance understanding of the chemopreventive effects of H. cordata against B[a]P-stimulated lung tumorigenesis and the underlying mechanisms, thereby supporting the use of this novel candidate agent for the chemoprevention of lung cancer.

Lung cancer is the most prevalent malignancy and is the leading cause of cancer death in men and the second leading cause of death in women [1]. B[a]P, which is mostly produced from cigarette smoking or car exhausts, acts as a potent carcinogen that leads to lung carcinogenesis [28]. Chemoprevention has been the most promising strategy to effectively decrease the incidence and mortality of lung cancer, especially for B[a]P-induced carcinogenesis in smoking patients [5, 6]. In the current study, we investigated the chemopreventive effects of H. cordata, a widely used herbal medicine with anti-inflammatory and antioxidative properties, against lung tumorigenesis and further clarified the underlying mechanisms. The results of this study will enhance understanding of the protective effect of H. cordata against B[a]P-stimulated lung tumorigenesis.

First, a B[a]P-stimulated lung adenocarcinoma animal model in A/J mice was successfully established to investigate the chemopreventive effects of H. cordata and its main bioactive compound 2-undecanone against lung cancer (Fig. 1a). A/J mice, which are highly susceptible to carcinogen-induced tumors, have been widely used as appropriate in vivo models to study the chemoprevention of lung carcinogenesis [5, 44]. The results showed that both H. cordata and 2-undecanone significantly reduced tumor numbers in B[a]P-treated mice in a dose-dependent manner compared to B[a]P alone (Fig. 1c). Moreover, the results obtained with the established B[a]P-treated BEAS-2B cell model showed that both H.cordata and 2-undecanone could effectively protect normal BEAS-2B cells from B[a]P-induced injury in a dose-dependent manner (Figs. 2 and 3). The combined results suggested that H.cordata could effectively prevent normal lung cells from B[a]P-initiated lung tumorigenesis and that 2-undecanone could be the bioactive compound responsible for this pharmacological activity.

Numerous chemopreventive agents have been developed, such as tamoxifen, raloxifene, finasteride, and celecoxib. However, the use of these agents for cancer prevention is restricted because of their severe side effects after long-term administration [4]. This issue clearly indicates that safe and efficacious agents are urgently needed for cancer prevention. Various herbal medicines and diet-derived natural products maybe candidates for this purpose. H. cordata is well known as a widely used medicinal herb with a variety of pharmacological functions [30, 31] and is also popularly consumed as a healthy vegetable in East Asia [32]. Thus, the toxicity of H. cordata was also determined in vivo and in vitro in this current study. Our data revealed no significant body weight differences between B[a]P control group A/J mice and treatment group A/J mice during the experiment (Fig. 1b). Moreover, no considerable differences were observed in the indexes of the heart, kidney, thymus, liver and spleen between the B[a]P control group and the treatment groups (Fig. 1d). In addition, incubation with H.cordata or 2-undecanone alone could not produce toxicity in normal BEAS-2B cells (Fig. 2a and b). These preliminary data implied that H.cordata could be a potential effective agent for the chemoprevention of lung carcinogenes is that does not cause severe systemic toxicity.

B[a]P-induced excessive production of ROS, which results in oxidative stress, can severely damage DNA structure and is a prerequisite for B[a]P-associated tumorigenesis [10‐12]. It has been reported that DNA damage is significantly associated with increased lung cancer risk and is considered to be the primary cause of lung cancer development [45, 46]. The roles of DNA damage repair pathways in cancers have attracted widespread interest in the clinic and have been widely recognized as vital targets to improve lung cancer therapy [47]. Hence, the effects of H. cordata and 2-undecanone on B[a]P-induced DNA damage were further investigated. The results from the comet assay showed that H. cordata and 2-undecanone could notably reduce the fluorescence in migrated DNA and the tails of disrupted DNA fragments (Fig. 4a), suggesting that H. cordata and 2-undecanone could effectively reverse B[a]P-induced DNA damage. The levels of p-H2A.X, which is a verified marker for DNA double-strand breaks and is involved in the response to DNA damage [41‐43], were determined in vitro and in vivo. The results showed that exposure to H. cordata or 2-undecanone significantly decreased the B[a]P-induced overexpression of p-H2A.X in a dose-dependent manner in the cells (Fig. 4b-d). p-H2A.X protein levels in the lung tissues of mice were further determined. It was observed that both H. cordata and 2-undecanone significantly downregulated p-H2A.X expression in B[a]P-treated mice compared with B[a]P treatment alone (Fig. 4e and f). Our results indicated that H. cordata and 2-undecanone could effectively reverse B[a]P-induced DNA damage in vitro and in vivo.

In addition to DNA damage, oxidative stress-induced inflammation is also a major contributor to lung cancer initiation and progression [13, 14]. Mature and biologically active IL-1β, which is generated from the inactive precursor cytokine pro-IL-1β and then secreted, is a critical regulator of the inflammatory response [48, 49]. IL-1β is involved in a variety of cellular activities, including cell proliferation, differentiation, and apoptosis [50]. Moreover, inflammation in the tumor microenvironment mediated by IL-1β is hypothesized to play a major role in cancer invasiveness, progression, and metastasis [51‐53]. Studies on tumor immunosurveillance have proposed a strong relationship between lung cancer risk factors and alterations in IL-1β levels [54]. Anti-inflammatory therapy targeting the IL-1β innate immunity pathway can significantly reduce incident lung cancer and lung cancer mortality [55]. Hence, the effects of H. cordata and 2-undecanone on B[a]P-induced inflammation were also investigated. The results showed that treatment of cells with H. cordata or 2-undecanone significantly decreased the B[a]P-induced overexpression of IL-1β in the cell culture supernatants (Fig. 5 a, b, e and f). The same treatments also markedly decreased the high levels of pro-IL-1β and IL-1β induced by B[a]P in the cell lysates (Fig. 5a, c, d, e, g and h), suggesting that H. cordata and 2-undecanone could effectively reduce IL-1β secretion, thereby potentially reducing the inflammatory response. Furthermore, both H. cordata and 2-undecanone treatment could significantly downregulate the IL-1β levels in the plasma of the mice exposed to B[a]P (Fig. 5i). Our results implied that H. cordata and 2-undecanone could effectively reverse B[a]P-induced inflammation in vitro and in vivo.

Then, it was confirmed that incubation of cells with B[a]P could markedly increase intracellular ROS production (Fig. 6a) and notably enhance the expression of p-H2A.X (Fig. 6b and c), pro-IL-1β and IL-1β (Fig. 6d). However, NAC, a well-known ROS inhibitor, could markedly reverse the B[a]P-induced p-H2A.X overexpression (Fig. 6b and c). ROS reduction by NAC also significantly decreased the high levels of pro-IL-1β and IL-1β induced by B[a]P (Fig. 6d). From these results, it was confirmed that DNA damage and inflammation could be significantly reversed by reductions in intracellular ROS levels, suggesting that the B[a]P-induced overproduction of intracellular ROS could cause marked DNA damage and inflammation. Therefore, the impact of H. cordata and 2-undecanone on of intracellular ROS levels was further investigated. It was observed that both H. cordata and 2-undecanone treatment significantly diminished the intracellular ROS overproduction induced by B[a]P (Fig. 6e-g). The correlation between ROS levels and DNA damage or inflammation was analyzed by using Pearson analysis. The results showed that the decrease in ROS levels was closely and positively related to the inhibition of B[a]P-induced DNA damage (Additional file 1: Figure S4) or inflammation (Additional file 1: Figure S5) by H. cordata and 2-undecanone. Given all the results, it was speculated that H. cordata and 2-undecanone could protect cells against B[a]P-induced DNA damage or inflammation by reducing intracellular ROS overproduction.

Nrf2 has been identified as a key regulator of the inducible expression of antioxidative enzymes, anti-inflammatory proteins and conjugation/detoxification proteins, such as NQO1 and HO-1 [21]. Upon oxidative stress caused by B[a]P or pharmacologic induction, the Nrf2 pathway could be rapidly activated to counteract intracellular ROS generation, thereby attenuating DNA damage and inflammation caused by B[a]P stimulation [21, 22]. The general cytoprotective effect of Nrf2 pathway activation has made it an attractive target for the chemoprevention of B[a]P-induced lung carcinogenesis. Hence, the modulatory effects of H. cordata and 2-undecanone on the Nrf2 pathway were further evaluated to clarify their chemopreventive functions against lung tumorigenesis. It was observed that both H. cordata and 2-undecanone significantly increased the protein levels of Nrf2, HO-1 and NQO-1 in whole-cell lysates (Fig. 7a) and resulted in significant increases in nuclear Nrf2 protein expression (Fig. 7b). The immunofluorescence results also showed that both H. cordata and 2-undecanone notably promoted the translocation of Nrf2 from the cytoplasm to the nucleus (Fig. 7c). Thus, it was implied that H. cordata and 2-undecanone could effectively activate the Nrf2 pathway to induce the expression of the antioxidative enzymes HO-1 and NQO-1. Then, it was found that B[a]P significantly decreased the protein levels of Nrf2, HO-1 and NQO-1 in whole-cell lysates (Fig. 7d) and decreased nuclear Nrf2 protein levels (Fig. 7e). In contrast, treatment with H. cordata and 2-undecanone markedly reversed the above-mentioned effects of B[a]P on the inhibition of Nrf2, HO-1 and NQO-1 expression (Fig. 7d and e). Moreover, both H. cordata and 2-undecanone treatment strikingly enhanced the expression of Nrf2, HO-1 and NQO-1 in a dose-dependent manner compared with B[a]P treatment in control mice (Fig. 7f-k). Then, to confirm the role of Nrf2 in the protective effects of H. cordata and 2-undecanone against B[a]P-induced DNA damage and inflammation, Nrf2 expression was silenced in BEAS-2B cells by transfection with siNrf2-1 or siNrf2-2. The data showed that transfection of cells with siNrf2-1 or siNrf2-2 significantly attenuated the H. cordata- and 2-undecanone-induced increases in the protein levels of HO-1 (Fig. 8a and b) and NQO-1 (Fig. 8c and d). The reductions in B[a]P-induced ROS overproduction mediated by H. cordata and 2-undecanone could also be markedly diminished by transfection of cells with siNrf2-1 or siNrf2-2 (Fig. 8e and f). Furthermore, the attenuating effects of H. cordata and 2-undecanone on B[a]P-induced p-H2A.X overexpression were markedly diminished by siNrf2 transfection (Fig. 9a and b). In addition, the culture supernatants of cells transfected with siNrf2-1 or siNrf2-2 and then treated with H. cordata or 2-undecanone showed no changes or weak reductions in the B[a]P-induced high levels of IL-1β (Fig. 9c and d). As expected, the protective effects of H. cordata and 2-undecanone against B[a]P-induced cytotoxicity were also markedly diminished by siNrf2 transfection (Fig. 9e and f). Based on the combined results in vitro and in vivo, it was speculated that H. cordata and 2-undecanone could effectively activate the Nrf2 pathway to induce the expression of the antioxidative enzymes HO-1 and NQO-1 and thus to counteract intracellular ROS generation, thereby attenuating DNA damage and inflammation causedby B[a]P stimulation and playing a role in the chemoprevention of B[a]P-induced lung carcinogenesis (Fig. 10).

In conclusion, our studies have shown that H. cordata and its bioactive compound 2-undecanone can significantly reduce B[a]P-induced DNA damage and inflammation to prevent lung tumorigenesis by activating the Nrf2-HO-1/NQO-1 signaling pathway. The data from these well-controlled in vitro and in vivo studies indicate that H. cordata may exert beneficial effects against cigarette smoke-induced lung inflammation and oxidative DNA damage in the human body. Thus, H. cordata could be an effective candidate agent for the chemoprevention of lung cancer.