Abstract
BACKGROUND:
Diethylnitrosamine (DEN) is a potent environmental carcinogen commonly found in cigarette smoke and polluted air, which is strongly associated with the initiation and progression of lung cancer through oxidative stress, inflammation, and dysregulated cell signaling. Alloimperatorin, a bioactive furanocoumarin compound isolated from Angelica dahurica, has demonstrated anti-inflammatory, antioxidant, and anticancer potential. The current study was designed to explore the chemoprotective effect of alloimperatorin against DEN-induced lung cancer in rats and explore the underlying signaling pathways.
MATERIALS AND METHODS:
Lung carcinogenesis was induced in male Wistar rats via intraperitoneal administration of DEN, and rats received the oral administration of alloimperatorin for 8 weeks. The lung function, body weight, tumor markers, phase I, phase II, polyamine, inflammatory parameters, inflammatory cytokines, and antioxidant enzymes were assessed. Quantitative histopathological analysis and histopathological observation were done in the lung tissue.
RESULTS:
Alloimperatorin significantly ameliorated the tumor burden, tumor number, mean tumor size, and improved histological architecture. Alloimperatorin ameliorate the level of tumor markers (5’-nucleotidase, aryl hydrocarbon hydroxylase, adenosine deaminase, lactate dehydrogenase, Hexosamine, Hexose), hematological parameters (total leucocytes, lymphocytes, total white blood cells count, neutrophils, monocytes, red blood cells counts), pro-inflammatory cytokines (tumor necrosis factor-alpha, L-1β, interleukin 4 [IL-4], IL-6, IL-10, IL-18), inflammatory parameters (cyclooxygenase-2, PGE2, vascular endothelial growth factor, nuclear kappa B factor [NF-κB]), apoptosis (Bax, Bcl-2, caspase-3) while restoring antioxidant enzyme (superoxide dismutase, catalase, glutathione (GSH) peroxidase, GSH, malonaldehyde) activities. In addition, it enhanced the level of HO-1 and Nrf2.
CONCLUSION:
Alloimperatorin ameliorate the lung cancer via alteration of the NF-κB and Nrf2 signalling pathway.
Keywords: Alloimperatorin, lung cancer, nuclear kappa B factor, Nrf2 signaling pathway
Introduction
The worldwide burden of cancer has increased substantially and remains a leading cause of mortality and morbidity across the globe.[1] Lung cancer is the foremost cause of cancer-related death in industrialized countries, underscoring its enormous impact on public health. Despite advances in understanding the cellular and molecular mechanisms underlying lung carcinogenesis. The overall prognosis remains poor, with nearly 90% of patient’s death within 5 years after diagnosis. Although most patients are still diagnosed at advanced stages, when therapeutic options are limited, and survival outcomes are unfavorable.[2,3] The worldwide incidence of lung cancer continues to increase, and the World Health Organization identifies it as one of the most commonly diagnosed cancers globally. This steady rise in epidemiological incidence, estimated at approximately 0.5% annually, largely reflects the persistent and widespread impact of tobacco smoking on lung cancer etiology. The sustained upward trend highlights the urgent need for more effective tobacco control strategies and strengthened public health interventions to decrease smoking incidence and, consequently, the global burden of lung cancer.[4] This advanced presentation results in a poor prognosis of the disease and underscores the vital need for early detection strategies. As per the WHO estimates, approximately 14 million new cancer cases were reported worldwide in 2012, and this number is projected to enhance by nearly 70% over the next two decades.[5] This distressing tendency highlights the pressing requirement for improved preventative measures and methods of early detection and treatment. The expected influx of cancer cases poses a multilevel challenge to health systems, economies, and societies.[4] With an increase in cancer prevalence, there is an increasing need for specialized healthcare, modern diagnostic methodologies, and new treatments. This growing burden stretches health resources and capacity, especially in developing countries that have limited access to cancer services. Furthermore, the economic burden is reflected not only in direct medical costs but also in productivity losses, a poorer quality of life for patients and caregivers, and higher social support demands.[6]
The PI3K/Akt and nuclear kappa B factor (NF-κB) signaling pathways are involved in controlling cell survival, proliferation, and inflammation. Their signaling pathways are considered the crucial factors in cancer development.[7,8] Activation of the PI3K/Akt pathway stimulates tumor cell growth, survival, and adaptation to metabolic stress by phosphorylating of downstream targets including mTOR, GSK-3β, and BAD, leading of apoptosis and stimulation of cellular proliferation. Another critical pathway includes the NF-κB pathway, a major transcription factor regulating inflammatory and immune responses, which is constitutively activated in many cancers, such as lung and colorectal cancer. NF-κB signaling drives the expression of genes involved in angiogenesis, metastasis, cell survival, and resistance to chemotherapy.[9,10] Additionally, PI3K/Akt signaling can activate NF-κB by phosphorylating IKK, thereby establishing a positive feedback loop that amplifies oncogenic and inflammatory responses.[9,11] Collectively, these interconnected pathways form a key molecular axis that promotes tumor initiating, progression, and resistance to therapy.
Alloimperatorin, a natural coumarin compound commonly found in the Apiaceae family, has attracted much attention from researchers because of its various biological activities.[12,13] This bioactive agent has different therapeutic properties, such as anti-inflammatory, anticancer, antioxidant, and antimicrobial activities.[12,13,14,15] The molecular mechanisms mediating these effects have been well-documented, and it was confirmed that alloimperatorin acts as an anticancer agent by inhibiting cell proliferation, promoting apoptosis via the regulation of several signaling pathways, including PI3K/Akt, MAPKs, and NF-κB, that ultimately represses tumor growth and metastasis, which suggests a potential utility of alloimperatorin in the treatment of cancer. In addition, the anti-inflammatory effects of alloimperatorin are mainly associated with suppression of pro-inflammatory mediators such as tumor necrosis factor-alpha (TNF-α), interleukin 6 (IL-6), and cyclooxygenase-2 (COX-2).[12,13,16] This property implies potential benefits for inflammatory conditions. The compound also exhibits strong antioxidant activity, by a mechanism of improving the cellular redox state and searching for reactive oxygen species (ROS) damage (It is very important to prevent oxidative stress-induced cellular injury). Due to its pleiotropic activity and natural source of origination, alloimperatorin is a good candidate for developing new potential drugs against cancer, inflammatory diseases, as well as oxidative stress-associated diseases.[17] Due to anti-inflammatory and antioxidant potential, in this experimental study, we scrutinized the chemoprotective effect of alloimperatorin against Diethylnitrosamine (DEN) induced lung cancer in rats and explored the underlying mechanism.
Materials and Methods
Swiss Albino Wistar rats (weight 180 ± 20 g, sex male) were procured from the institutional animal house and kept in standard laboratory conditions. The rats were kept in polyethylene cages in the groups and maintained the temperature (25°C ± 2°C), humidity (60%–70%), and light cycle (12 h/12 h). The rats were fed with commercially available pelleted rat chow and water ad libitum. The whole study was carried out accordance to following the guidelines of the Institutional animal ethical committee.
Induction of lung cancer
The previously reported method was used for the induction of lung cancer in rats.[18] The rats received a single intraperitoneal injection of DEN (150 mg/kg) for the induction of lung cancer.
Test drug
The test drug (Alloimperatorin) was administered to the rats in the form of an oral suspension. Briefly, we prepared the suspension of the test drug (Alloimperatorin) via prepared the suspension of CMC (1%). The dose of the test drug was selected on the basis of previous reported method.
Experimental groups
After the induction of lung cancer, the rats were divided into the following groups:
Group A: Normal control
Group B: DEN-induced lung cancer
Group C: DEN + Alloimperatorin (2.5 mg/kg),
Group D: DEN + Alloimperatorin (5 mg/kg),
Group E: DEN + Alloimperatorin (7.5 mg/kg), respectively.
The rats received the administration of alloimperatorin once a day for 24 weeks. The body weight and lung weight were estimated. After 24 weeks, the rats were anesthetized using diethyl ether, and blood sample was collected via puncturing the retroorbital plexus. The collected blood sample was centrifuged at 10,000 rpm for 15 min to separate the serum. The serum was stored at −20°C for further analysis.
Tissue preparation
The rats were euthanized using diethyl ether and xylazine, and lung tissues were isolated and stored in cold sterile saline. After that, the lung tissue was cut into small size (5 mm) and fixed in natural buffered formalin (10%). The lung tissue sections were stained with hematoxylin and eosin (H and E) according to the previous report method[19,20] and examined under the light microscope. Another lung sample was kept in the phosphate buffer (25% w/v) and centrifuged for 15 min at 3000 rpm at 4°C for separate the supernatant. The separated supernatants were stored at − 20°C.
Lung cancer and enzyme marker
The lung cancer markers, such as NSE and CFA, were estimated using the Chemiluminescent immunoassay (Bayer Chemiluminescence system, Thermo Fisher Scientific, USA). The lung enzyme markers, such as 5’-nucleotidase, aryl hydrocarbon hydroxylase (AHH), adenosine deaminase (ADA), lactate dehydrogenase (LDH), LDH, hexosamine, and hexose were estimated using the previously reported method with minor modifications.[6,21,22]
Polyamines determination
The level of polyamines was estimated using the previously reported method with minor modifications.[21,23]
Oxidative stress parameters
The oxidative stress parameters like catalase (CAT), glutathione (GSH), malonaldehyde (MDA), GSH peroxidase (GPx), and superoxide dismutase (SOD) were estimated using the enzyme-linked immunosorbent assay (ELISA) kits following the manufacture instruction.
Vitamin level
The levels of vitamins such as vitamin C and vitamin E were estimated using the previously reported method with minor modifications.
Phase I and II enzymes
The level of phase I like Cytochrome P450, Aldo-keto reductases, and phase II enzyme such as GSH S-transferases, UDP-glucuronosyltransferases, were estimated using the previous report method with minor modification.[6,21]
Enzyme-linked immunosorbent assay parameters
The inflammatory cytokines parameters, namely TNF-α, IL-1β, IL-4, IL-6, IL-10, and IL-18; inflammatory parameters such as COX-2, prostaglandin (PGE2), NF-κB; apoptosis parameters, namely Bax, Bcl-2, and caspase-3 parameters were estimated using the ELISA kits (Xitang Bio-Technology, Shanghai, China) following the manufacturer’s instructions.
Histopathology assessment of tumor burden and grading
Lung tissue was fixed in formalin, and paraffin-embedded lung sections (4-5 μm) were then prepared and stained with H and E. Whole-slide scanning was performed using a bright-field microscope with a digital camera installed. The sections were calibrated using the scale bar for each section, and quantitative morphometric measurement was conducted with ImageJ software (NIH, USA).
The area of all distinct tumor nodules on the scanned lung sections was manually delineated and determined per animal. The mean tumor size (mm²) was calculated as the average area of all tumor nodules observed in one rat, and the total tumor burden (mm²/rat) was calculated as the sum of all areas in each rat. Tumor grading: The histopathological grade (score) of the tumor was evaluated on H and E-stained sections and scored for all animals according to a semi-quantitative scale, as follows:
0 = normal lung architecture
1 = mild epithelial hyperplasia/dysplasia
2 = moderate dysplasia or carcinoma in situ
3 = well-defined carcinoma.
The average grade score per animal was calculated as the mean of individual lesion scores when several lesions of various extents were observed.
Statistical analysis
GraphPad Prism (version 8; GraphPad Software, San Diego, CA, USA) was used for the statistical analysis. The whole experimental data were showed as the standard error mean. To estimate whether there were any statistically significant differences, the one-way analysis of variance was used, and P < 0.05 was considered significant.
Results
Body weight, organ weight, and organ ratio
In this study, DEN induced lung cancer group rats demonstrated decreased body weight [Figure 1a], and alloimperatorin treatment significantly (P < 0.001) improved the body weight. DEN-induced lung cancer rats demonstrated the boosted lung weight [Figure 1b], liver weight [Figure 1c], lung weight to body weight [Figure 1d], liver weight to body weight [Figure 1e], and alloimperatorin treatment significantly (P < 0.001) suppressed the organ weight and organ weight to body ratio.
Figure 1.
Alloimperatorin ameliorates body weight, organ weight, and organ weight ratio against Diethylnitrosamine (DEN) induced Lung Cancer in rats. (a) Body weight, (b) lung weight, (c) liver weight, (d) lung weight to body weight ratio, and (e) Liver weight to body weight ratio. The graphs show the mean ± standard error mean (SEM), with 6 rats/group. The numbers signify the mean ± SEM, with 6 rats/group. The statistical differences are denoted with an asterisk (###), indicating significance at P < 0.001 in comparison with the normal control group, and a hashtag (^), (^^), and (^^^) signifying P < 0.05, P < 0.01, and P < 0.001 in comparison with the DEN induced Lung cancer group
Lung cancer parameters
DEN-induced lung cancer group rats showed the enhanced level of lung cancer parameters like 5’NT [Figure 2a], AHH [Figure 2b], ADA [Figure 2c], LDH [Figure 2d], hexosamine [Figure 2e], hexose [Figure 2f], and alloimperatorin treatment significantly (P < 0.001) suppressed the level of lung cancer parameters.
Figure 2.
Alloimperatorin ameliorates lung cancer parameters against Diethylnitrosamine (DEN) induced Lung Cancer in rats. (a) 5’-nucleotidase, (b) aryl hydrocarbon hydroxylase, (c) adenosine deaminase, (d) lactate dehydrogenase, (e) Hexosamine and (f) Hexose. The graphs show the mean ± standard error mean (SEM), with 6 rats/group. The numbers signify the mean ± SEM, with 6 rats/group. The statistical differences are denoted with an asterisk (###), indicating significance at P < 0.001 in comparison with the normal control group, and a hashtag (^), (^^), and (^^^) signifying P < 0.05, P < 0.01, and P < 0.001 in comparison with the DEN induced Lung cancer group
Hematological parameters
DEN induced lung cancer group rats demonstrated the altered level of total leucocytes [Figure 3a], lymphocytes [Figure 3b], total white blood cells count [Figure 3c], neutrophils [Figure 3d], monocytes [Figure 3e], red blood cells counts [Figure 3f], and alloimperatorin treatment significantly (P < 0.001) modulated the level of hematological parameters.
Figure 3.
Alloimperatorin alters hematological parameters against diethylnitrosamine (DEN) induced lung cancer in rats. (a) Total leukocyte count, (b) lymphocytes, (c) total white blood cells count, (d) neutrophils, (e) monocytes, and (f) red blood cell counts. The graphs show the mean ± SEM, with 6 rats/group. The numbers signify the mean ± standard error mean (SEM), with 6 rats/group. The statistical differences are denoted with an asterisk (###), indicating significance at P < 0.001 in comparison with the normal control group, and a hashtag (^), (^^), and (^^^) signifying P < 0.05, P < 0.01, and P < 0.001 in comparison with the DEN induced Lung cancer group
Oxidative stress parameters
The level of SOD [Figure 4a], CAT [Figure 4b], GPx [Figure 4c], and GSH [Figure 4d] was suppressed, and the MDA [Figure 4e] level was boosted in the DEN-induced lung cancer group rats, and alloimperatorin treatment significantly (P < 0.001) modulated the level of oxidative stress parameters.
Figure 4.
Alloimperatorin alters antioxidant parameters against Diethylnitrosamine (DEN) induced Lung Cancer in rats. (a) Superoxide dismutase, (b) catalase, (c) glutathione (GSH) peroxidase, (d) GSH, and (e) malonaldehyde. The graphs show the mean ± standard error mean (SEM), with 6 rats/group. The numbers signify the mean ± SEM, with 6 rats/group. The statistical differences are denoted with an asterisk (###), indicating significance at P < 0.001 in comparison with the normal control group, and a hashtag (^), (^^), and (^^^) signifying P < 0.05, P < 0.01, and P < 0.001 in comparison with the DEN induced Lung cancer group
Phase I and Phase II enzymes
DEN-induced lung cancer group rats showed the boosted level of Cytochrome P450 [Figure 5a], aldo-keto reductases [Figure 5b], GSH S-transferases [Figure 5c], UDP-glucuronosyltransferases [Figure 5d], and alloimperatorin treatment significantly (P < 0.001) suppressed the level of phase I and II enzymes.
Figure 5.
Alloimperatorin ameliorates phase I and II enzymes against Diethylnitrosamine (DEN) induced Lung Cancer in rats. (a) Cytochrome P450, (b) aldo-keto reductases, (c) glutathione S-transferases, and (d) UDP-glucuronosyltransferases. The graphs show the mean ± standard error mean (SEM), with 6 rats/group. The numbers signify the mean ± SEM, with 6 rats/group. The statistical differences are denoted with an asterisk (###), indicating significance at P < 0.001 in comparison with the normal control group, and a hashtag (^), (^^), and (^^^) signifying P < 0.05, P < 0.01, and P < 0.001 in comparison with the DEN induced Lung cancer group
Polyamine parameters
DEN-induced lung cancer group rats showed the boosted level of polyamine parameters such as putrescine [Figure 6a], spermidine [Figure 6b], spermine [Figure 6c], and alloimperatorin treatment significantly (P < 0.001) suppressed the level of polyamine parameters.
Figure 6.
Alloimperatorin ameliorates polyamine parameters against Diethylnitrosamine (DEN) induced Lung Cancer in rats. (a) Putrescine, (b) spermidine, and (c) spermine. The graphs show the mean ± standard error mean (SEM), with 6 rats/group. The numbers signify the mean ± SEM, with 6 rats/group. The statistical differences are denoted with an asterisk (###), indicating significance at P < 0.001 in comparison with the normal control group, and a hashtag (^), (^^), and (^^^) signifying P < 0.05, P < 0.01, and P < 0.001 in comparison with the DEN induced Lung cancer group
Vitamin level
DEN induced lung cancer group rats demonstrated a suppressed level of vitamin C [Figure 7a], vitamin E [Figure 7b], and alloimperatorin treatment significantly (P < 0.001) boosted the level of vitamin.
Figure 7.
Alloimperatorin enhanced vitamin level against Diethylnitrosamine (DEN) induced Lung Cancer in rats. (a) Vitamin C and (b) Vitamin E. The graphs show the mean ± SEM, with 6 rats/group. The numbers signify the mean ± standard error mean (SEM), with 6 rats/group. The statistical differences are denoted with as asterisk (###), indicating significance at P < 0.001 in comparison with the normal control group, and a hashtag (^), (^^) and (^^^) signifying P < 0.05, P < 0.01 and P < 0.001 in comparison with the DEN induced Lung cancer group
Apoptosis parameters
DEN-induced lung cancer group rats showed the altered level of apoptosis parameters such as Bcl-2 [Figure 8a], Bax [Figure 8b], caspase-3 [Figure 8c], and alloimperatorin treatment significantly (P < 0.001) modulated the level of apoptosis parameters.
Figure 8.
Alloimperatorin ameliorates apoptosis parameters against Diethylnitrosamine (DEN) induced Lung Cancer in rats. (a) Bcl-2, (b) Bax, and (c) Caspase-3. The graphs show the mean ± standard error mean (SEM), with 6 rats/group. The numbers signify the mean ± SEM, with 6 rats/group. The statistical differences are denoted with an asterisk (###), indicating significance at P < 0.001 in comparison with the normal control group, and a hashtag (^), (^^), and (^^^) signifying P < 0.05, P < 0.01, and P < 0.001 in comparison with the DEN induced Lung cancer group
Inflammatory cytokines and inflammatory parameters
DEN-induced lung cancer group rats exhibited the altered level of inflammatory cytokines such as TNF-α [Figure 9a], IL-1β [Figure 9b], IL-4 [Figure 9c], IL-6 [Figure 9d], IL-10 [Figure 9e], IL-18 [Figure 9f], and alloimperatorin treatment significantly (P < 0.001) modulated the level of inflammatory cytokines.
Figure 9.
Alloimperatorin alters inflammatory cytokine levels against Diethylnitrosamine (DEN) induced Lung Cancer in rats. (a) Tumor necrosis factor-alpha, (b) L-1β, (c) interleukin 4 (IL-4), (d) IL-6, (e) IL-10, and (f) IL-18. The graphs show the mean ± standard error mean (SEM), with 6 rats/group. The numbers signify the mean ± SEM, with 6 rats/group. The statistical differences are denoted with an asterisk (###), indicating significance at P < 0.001 in comparison with the normal control group, and a hashtag (^), (^^), and (^^^) signifying P < 0.05, P < 0.01, and P < 0.001 in comparison with the DEN induced Lung cancer group
DEN-induced lung cancer group rats demonstrated the boosted level of inflammatory parameters such as COX-2 [Figure 10a], PGE2 [Figure 10b], vascular endothelial growth factor [Figure 10c], NF-κB [Figure 10d], and alloimperatorin treatment significantly (P < 0.001) suppressed the level of inflammatory parameters.
Figure 10.
Alloimperatorin ameliorates inflammatory parameters against Diethylnitrosamine (DEN) induced Lung Cancer in rats. (a) Cyclooxygenase-2, (b) PGE2, (c) vascular endothelial growth factor, and (d) nuclear kappa B factor. The graphs show the mean ± standard error mean (SEM), with 6 rats/group. The numbers signify the mean ± SEM, with 6 rats/group. The statistical differences are denoted with an asterisk (###), indicating significance at P < 0.001 in comparison with the normal control group, and a hashtag (^), (^^), and (^^^) signifying P < 0.05, P < 0.01, and P < 0.001 in comparison with the DEN induced Lung cancer group
HO-1 and Nrf2
DEN-induced lung cancer group rats showed the suppressed level of Heme oxygenase-1 (HO-1) [Figure 11a], Nuclear factor erythroid 2–related factor 2 (Nrf2) [Figure 11b], and alloimperatorin treatment significantly (P < 0.001) boosted the level of HO-1 and Nrf2.
Figure 11.
Alloimperatorin enhanced HO-1 and Nrf2 levels against diethylnitrosamine (DEN)-induced lung cancer in rats. (a) HO-1 and (b) Nrf2. The graphs show the mean ± standard error mean (SEM), with 6 rats/group. The numbers signify the mean ± SEM, with 6 rats/group. The statistical differences are denoted with an asterisk (###), indicating significance at P < 0.001 in comparison with the normal control group, and a hashtag (^), (^^), and (^^^) signifying P < 0.05, P < 0.01, and P < 0.001 in comparison with the DEN-induced lung cancer group
Quantitative histopathological analysis
DEN-induced lung cancer rats showed a significant increase in tumor burden compared to the normal control group [Supplementary Table 1]. No measurable tumors were observed in the normal control group, and tumor number, size, total burden, and histopathological grade equaled zero. Within the DEN-induced lung cancer model group, large tumor load with a mean number of 10 nodules per rat (average size of 22.17 ± 1.04 mm²) and total burden of tumor region 221.70 mm² was observed. This group also had the highest tumor grade histopathological score (14), reflecting a pronounced neoplastic alteration. Rats treated with alloimperatorin 2.5 mg/kg showed a decrease in tumor number (8 nodules/rat), mean tumor diameter (15.35 ± 1.01 mm²), and total burden of tumors (122.80 mm²/rat) as well as histopathological score for the grade of tumors (9) compared to the DEN group. Additional decreases were noted at 5 mg/kg, with 6 tumors per rat, a mean tumor size of 9.04 ± 0.83 mm², total tumor burden of 54.24 mm²/rat, and grade score of 7. Protection against tumor initiation and growth by the maximum dose of alloimperatorin (7.5 mg/kg) was most significant, in terms of the combined number of tumors/rat (3), mean size per tumor (4.16 ± 0.34 mm²), total burden per rat (12.48 mm²), and the least average histopathological score among different treatment groups (3).
Supplementary Table 1.
Effect of on tumor burden in diethylnitrosamine-induced lung cancer
| Group | Tumor number (per rat) | Mean tumor size (mm2) | Total tumor burden (mm2/rat) | Histopathological tumor grade (score) |
|---|---|---|---|---|
| NC | 0 | 0 | 0 | 0 |
| DEN-induced lung cancer | 10 | 22.17±1.04 | 221.70 | 14 |
| DEN + Alloimperatorin (2.5 mg/kg) | 8 | 15.35±1.01 | 122.80 | 9 |
| DEN + Alloimperatorin (5 mg/kg) | 6 | 9.04±0.83 | 54.24 | 7 |
| DEN + Alloimperatorin (7.5 mg/kg) | 3 | 4.16±0.34 | 12.48 | 3 |
DEN=Diethylnitrosamine, NC=Normal control
H- and E-staining of the representative lung sections from controls and DEN-induced lung cancer-bearing animals exhibited excessive degrees of histopathological modifications in the DEN-treated cancer group that was evidenced by severe distortion of normal pulmonary alveolar architecture, accumulation of dense cellular proliferation, formation of thickened inter-alveolar septa, and neoplastic cells infiltration into normal tissue, reflecting advanced tumorigenic changes. In comparison, the lungs of the normal control group presented well-defined histoarchitecture with an intact alveolar space, thin septa, and no inflammatory or neoplastic lesions. Alloimperatorin treatment resulted in an evident dose-dependent amelioration of lung morphology. In the low-dose alloimperatorin (2.5 mg/kg) group, relative alveoli structure was restored partially with less cellular infiltration and septal thickening when compared with the DEN group. A second higher dose (5 mg/kg) further attenuated this, with more usual alveolar spaces, decreased hyperplasticity, and lower infiltrating inflammatory cells. Remarkably, at the maximum dosage of alloimperatorin (7.5 mg/kg), lung architecture drawing close to normal was observed with slight cellular infiltration and significant inhibition in pathological changes induced by DEN [Supplementary Figure 1 (97.4KB, jpg) ].
Discussion
Lung cancer remains the leading cause of cancer-related mortality worldwide and is among the most frequently diagnosed malignancies. Because of the grave nature of this disease and the severe side effects associated with conventional chemotherapy, increasing attention has been directed toward alternative bioactive agents with potential anticancer activity. In this context, dysregulation of apoptosis, a fundamental programmed cell death process, plays a crucial role in the tumorigenesis and the survival of transformed cells.[24] Therefore, therapeutic approaches targeting the apoptotic signaling have been intensively studied in search of more efficient and less harmful anti-cancer therapies. In the last few years, chemoprevention of cancer by naturally occurring bioactive compounds has created lot of interest since these are intended to prevent or retard cancer incidence and provide with less invasive approaches and may serve as an effective alternative for the treatment of cancer.[25] The current study explores the anti-cancer potential of alloimperatorin against DEN-induced lung cancer Wistar rat model.
The imbalance of ROS generation and antioxidant armament of the cells results in oxidative stress, which promotes the initiation, promotion, and progressive stages of lung tumorigenesis through oxidation reactions.[18] Alimentation, even inactive oxygen, peroxidizes lipids in the organism, and gives birth to mutagenic and carcinogenic MDA.[21] In the present study, ROS and lipid peroxidation were increased by DEN treatment and significantly decreased by alloimperatorin treatment. The intranatural system of antioxidants can be made up of enzymatic and nonenzymatic molecules, among which there are SOD, CAT, GPx, GST, and GSH. These antioxidants are essential to scavenge ROS and inhibit lipid peroxidation.[6,21] Yet, cancerous animals usually present a decreased activity of these antioxidants together with an increase in free radicals.[6] The SOD, CAT, GPx, and GST activities, as well as GSH content, were markedly reduced in animals with lung cancer. Most importantly, the activities of these antioxidants were dose-dependently restored by alloimperatorin treatment, indicating its potential to provide a reducing microenvironment and free radical scavenging activity against DEN-induced lung carcinogenesis.
Cell membrane components, especially glycoproteins, are known to act as mediators of a number of cell surface functions in neoplastic diseases. These roles are broad and include the recognition of other cells, cell adhesion, serum glycoprotein binding, and clearance, as well as metabolic transport.[21] High levels of glycoproteins have been suggested as important indicators of the carcinogenic process, and increased serum glycoprotein content has been observed in animals treated with DEN.[26] The present investigation also supports these findings by showing a significant elevation in hexose, hexosamine, fucose, and sialic acid contents of serum along with their concomitant decrease in lung tissues of cancer-bearing animals. The current study also investigates the potential therapeutic effects of alloimperatorin which is effective in a dose-dependent manner to restore glycoprotein levels.
Lung cancer and other neoplasms are linked to changes in the levels of some enzymes and biomarkers, including ADA, AHH, and 5’-nucleotide phosphodiesterase. ADA is a gene associated with purine nucleotide metabolism, and the expression of this gene in patients with lung cancer also increased concomitant to the poor prognosis and might be able to predict enhanced treatment response and tumor progression.[21] AHH, a cell surface enzyme that mediates metabolism of xenobiotics and carcinogens in the lung, is elevated in lung cancer and may play a role in its pathogenesis through stimulation of DNA adduct formation leading to mutations and genetic instability.[21,27] Moreover, AHH can serve as a sensitive early marker of lung cancer. 5’- Nucleotide phosphodiesterase that hydrolyzes nucleotides and their analogs, shows increased activity in proliferating tumor tissue, such as lung tumors. Elevated expression of this enzyme in lung tumors implies its role in tumor development and progression.[21]
Cytokines play critical roles in various stages of lung cancer progression and development, influencing tumor growth, initiation, and dissemination via diverse mechanisms that boost metastasis, tumorigenesis, and immune evasion.[6] TNF-α and IL-1β, which are produced by immune cells upon infection or injury, belong to the family of proinflammatory cytokines that can mediate angiogenesis as well as suppress immunity and induce apoptosis in normal cells but promote the proliferation and survival of cancer cells.[21] These cytokines are known to be linked with bad prognosis in patients with lung cancer. Furthermore, IL-1β enhances the expression of MMPs that digest the extracellular matrix and promote tumor invasion. IL-6, another essential cytokine associated with lung cancer progression, is generated by immune cells as well as other cell types in the setting of inflammation and infection. Like TNF-α and IL-1β, IL-6 also enhances the expansion and progression of cancer via various mechanisms, such as angiogenesis stimulation and immunosuppressive action.[21]
COX-2, a key enzyme in prostaglandin synthesis, has been considered as a target for cancer therapy, because it’s induced overexpression in various types of cancers, including lung cancer.[28] This COX-2 induction increases the production of the biologically active lipid mediator, PGE2, which plays a crucial role in tumorogenesis. High expression of COX-2 and PGE2 has been repeatedly found in lung cancer, correlated with unfavorable clinical outcome, suggesting that they are potential prognostic markers as well as targets for treatment of cancer. COX-2/PGE2 participates in tumor growth and metastasis via multiple mechanisms.[21] This route also suppressed the anti-tumor immunity and led to cancer cells being concealed from immune detection rather than destroyed. In addition, the COX-2/PGE2 signal pathway is able to enhance the migration and invasion of tumor cells, which play crucial roles in tumor metastasis. As a result, the pleiotropic roles of COX-2/PGE2 targeting indicate promise for new therapeutic strategies in lung cancer as well as other cancers with deregulated activity within this axis.[6]
The Increased Nrf2 activity in lung cancer cells strengthens antioxidant defenses, increases detoxification capacities, and participates in mechanisms of resistance to drugs.[29,30] This protective response provides cancer cells with the ability to resist oxidative and therapeutic challenges encountered during tumor development and therapeutics. The interconnectedness of these pathways creates a resilient life-defense system, which allows lung cancer cells to adapt and counteract different stresses such as oxidative stress, inflammation, or therapeutic interventions.[6,30] Knowledge of the crosstalk between these pathways and how to target their intersecting nodes may offer new opportunities for better lung cancer therapy, or one that hopefully can overcome the drawbacks of current treatments. In this highly complex molecular labyrinth, mutual crosstalk among the PI3K/Akt, NF-κB, and Nrf2 signaling cascades is very pronounced and plays a major role in lung cancer oncogenesis. The PI3K/Akt signal pathway provides a central integration site of various signals for tumor growth regulation.[31,32] It promotes survival, proliferation, and angiogenesis of cancer cells via anti-apoptotic and pro-growth signals. In addition, this pathway allows metabolic reprogramming that adapts cancer cells to the specific energetic demand for proliferation and expansion. The NF-κB pathway, often activated by PI3K/Akt signaling or inflammatory factors, will enhance the tumorigenic phenotype. It not only provides a chronic inflammatory background but also enhances the proliferation, invasion, and antiapoptosis of tumor cells, which facilitates the formation and metastasis of carcinomas.[31,33] The Nrf2 pathway adds a degree of complexity to this signaling network. While its activation in normal cells is considered a protective response against oxidative stress and carcinogenic agents, prolonged activation in cancer cells has deleterious consequences. In a lung carcinoma setting, hyperactive Nrf2 was found to increase antioxidant defenses and detoxification mechanisms, leading to increased drug resistance. This dichotomous capability of the Nrf2 signaling illuminates the thin line between preservation of cellular integrity and promotion of cancer cell maintenance.[30,34] Comprehensive crosstalk among these three types of signaling culminates in a robust and flexible regulatory network, allowing lung tumor cells to accommodate various forms of stress such as oxidative stress, inflammation, and therapeutic insult. This interplay of diverse signaling pathways not only represents the biological complexity of lung cancer but suggesting that it is essential to study these pathways in a systematic way to advance the rational design of effective anticancer therapies [Figure 12].[6,21]
Figure 12.
Proposed mechanistic pathway of alloimperatorin against DEN-induced lung cancer
Limitations of the study
However, although extensive biochemical, inflammatory, and histopathological studies were pursued in this current work, it is also possible that some limitations should be recognized. The mechanistic contribution attributed to the NF-κB/Nrf2-based signaling axis is mainly derived from alterations in pathway-related markers and downstream effectors, and not by direct pathway dependency assessment. In particular, phosphorylation-specific Western blot analysis of NF-κB, nuclear translocation of Nrf2, and Keap1 modulation were not performed. Furthermore, we did not test the effects of selective NF-κB or Nrf2 inhibitors or genetic manipulation methods in this study. Therefore, with all the reservations, our results clearly indicate a role of alloimperatorin modulation on both oxidative stress and inflammatory signaling but conclusive cause-and-effect relationships cannot be inferred. Future investigations using molecular validation, pathway inhibition, and cell-specific mechanistic studies will be needed for further confirmation and extension of the mechanistic findings presented.
Conflicts of interest
There are no conflicts of interest.
Showed the histopathology of different group of rats. (a) normal control, (b) DEN-induced lung cancer, (c) DEN + Alloimperatorin (2.5 mg/kg) (d) DEN + Alloimperatorin (5 mg/kg) and e: DEN + Alloimperatorin (7.5 mg/kg)
Funding Statement
Nil.
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Supplementary Materials
Showed the histopathology of different group of rats. (a) normal control, (b) DEN-induced lung cancer, (c) DEN + Alloimperatorin (2.5 mg/kg) (d) DEN + Alloimperatorin (5 mg/kg) and e: DEN + Alloimperatorin (7.5 mg/kg)