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Abstract

Background:

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related death worldwide. Piperlongumine (PL) has been claimed to have cytotoxic and HCC inhibitory effects in various cancer cell lines and xenograft models, but the chemopreventive potential of PL has not been studied in experimentally induced HCC yet.

Research Design:

Twenty-four Wistar male rats were divided into four groups of six each, Group A: untreated control; Group B: Diethylnitrosamine (DEN) control (200 mg/kg), Group C: DEN + PL 10 mg/kg; and Group D: DEN + PL 20 mg/kg. Rats from all groups were assessed for liver cancer progression or inhibition by evaluating biochemical, cytokines, tumor markers, lipid peroxidation, and histological profiles.

Results:

The liver enzymes alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma-glutamyl transferase (GGT), alkaline phosphatase (ALP) levels, and lipid peroxidation were significantly decreased in Group C and Group D compared to Group B. Upregulation in the level of pro-inflammatory cytokines IL-1B, TNF-α, inflammatory mediator (NF-κB) and tumour marker alpha-fetoprotein (AFP) in Group B were brought down upon treatment with piperlongumine in a dose-dependent manner. Antitumor cytokine (IL-12) was upregulated in PL-treated rats compared to DEN control rats. DEN treated group (Group B) showed histological features of HCC, and in rats treated with PL (Groups C, D) partial to complete reversal to normal liver histoarchitecture was observed.

Conclusions:

The potential chemopreventive actions of piperlongumine may be due to its free radical scavenging and antiproliferative effect. Therefore, piperlongumine may serve as a novel therapeutic agent for the treatment of hepatocellular carcinoma.

Keywords

hepatocellular carcinoma piperlongumine nitrosamine lipid peroxidation cytokines chemoprevention

Introduction

Hepatocellular carcinoma (HCC), often known as primary liver cancer, is one of the leading causes of death in patients with chronic liver diseases. Hepatocellular carcinoma is one of the most common cancers in the world, and it is also the third leading cause of cancer-related death globally.¹ Hepatocellular carcinoma is the second most fatal malignancy, after pancreatic cancer, with a 5-year survival rate of 18% only.² The crude incidence rate of HCC in India in the year 2015 was 2.8 cases per 100,000 populations per year (males: 3.9, females: 1.6), and the crude mortality rate was 2.7 per 100,000 populations per year.³ HCC is the seventh leading cause of cancer-related death in India and patients with advanced HCC have a dismal prognosis, with a reported median survival of only 2–3 months with the best supportive care.⁴

The most important risk factors for HCC include chronic infection with hepatitis B virus (HBV) or hepatitis C virus (HCV), aflatoxin-contaminated foods, excessive alcohol consumption, obesity, type 2 diabetes, and smoking.⁵ Chronic inflammation accompanied by severe oxidative stress is the most common mechanism of hepatocarcinogenesis.⁶ The majority of hepatocellular carcinomas are found in cirrhotic livers.⁷

Hepatocellular carcinoma patients are frequently asymptomatic in early stages. In most cases, patients have right upper quadrant pain, palpable mass, and weight loss. Various other clinical presentations include jaundice, hepatic encephalopathy, anasarca, ascites, variceal bleeding, and diarrhoea.

Diethylnitrosamine (DEN) is a potent carcinogenic dialkyl nitrosamine present in tobacco smoke, cheddar cheese, meat, and whiskey.^8,9 Reactive oxygen species (ROS) generated during the metabolism of DEN may play a significant role in cancer development. According to the International Agency for Research on Cancer (IARC, 1978) classification, DEN has been reported as a potential human carcinogen (Group 2A).

Current treatment strategies for HCC include chemotherapy, radiotherapy, surgery, and immunotherapy. However, the majority of these treatments are accompanied with serious side effects and recurrence. This necessitates the development of a novel anticancer drug capable of targeting multiple pathways in cancer with minimal or no side effects. Several plant-based products with intriguing medicinal potential have been tried to address this gap.

Piperlongumine (PL), also known as piplartine, was first isolated in 1961,¹⁰ is an amide alkaloid isolated from the roots of Piper longum (long pepper) that is widely used in Indian traditional medicine. PL selectively killed HCC cells but not normal hepatocytes by elevating reactive oxygen species (ROS) only in HCC cells and thereby significantly suppressed HCC development.¹¹ PL helped in suppressing the cell proliferation, invasion, and enhanced cell apoptosis of hepatocellular carcinoma.¹² The role of piperlongumine in reducing the tumor volume, tumor weight, cell proliferation, and increasing the apoptosis has been elucidated earlier.¹³ The anticancer efficacy of PL has mostly been demonstrated in animal models using orthotropic and xenograft cancer cell lines. However, the chemopreventive potential of PL in experimentally produced HCC has not been investigated as of yet. Therefore, this study was planned to study the chemopreventive activity of piperlongumine in diethylnitrosamine induced hepatocellular carcinoma in rats.

Materials and methods

Chemicals

Diethylnitrosamine was acquired from Sigma-Aldrich, USA. The alpha-fetoprotein (AFP) and carcinoembryonic antigen (CEA) assay kits were procured from Advia Centaur, Germany. Pro-inflammatory and inflammatory mediators (IL-1B, TNF-α, NF-κB) ELISA assay kits and piperlongumine were procured from Cayman Chemical, USA. Antitumor cytokine ELISA assay kit (IL-12) was acquired from Sincere Biotech, China. Lipid peroxidation (MDA) assay kit was acquired from Biovision, USA. Assay kits for hepatic enzyme markers, namely serum ALP, AST, ALT, GGT, and kits for total protein, albumin, creatinine, and urea were procured from ERBA Diagnostics, India. All other chemicals used were of analytical grade.

Animals

Twenty-four male Wistar rats weighing between 140–160 g were used for this study. Under the heating, ventilation, and air conditioning (HVAC) system, all rats were kept at 22 ± 3°C, 30–70% humidity, a 12 h light/12 h dark cycle, and 10–15 air changes per hour, and they had access to a standard pellet diet and clean, purified water ad libitum. The animals were housed in polycarbonate cages. All the rats were acclimatized for 1 week prior to the start of experiments after procurement from the animal house facility (Regn No. 2070/GO/ReBi/S/19/CPCSEA) of the All India Institute of Medical Sciences (AIIMS), Rishikesh, Uttarakhand, India. The animals utilized in this study were approved by the Institutional Animal Ethics Committee (IAEC), AIIMS, Rishikesh, Uttarakhand, constituted by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Government of India, vide protocol approval number IAEC/AIIMS/Rish/BC/04/19.

Induction of hepatocellular carcinoma

Hepatocellular carcinoma was induced using DEN. HCC was induced in Group B, C, and D rats by administering a single intraperitoneal (i/p) injection of DEN at a concentration of 200 mg/kg body weight¹⁴ mixed in saline. Group A was kept as a negative control and received only normal saline. To promote HCC, 0.05% phenobarbitone was supplied 2 weeks after DEN administration by dissolving in drinking water for up to 16 weeks.

Preparation of test drug solution

Piperlongumine was dissolved in dimethyl sulfoxide (DMSO) at a rate of 20 mg/mL of DMSO. We have selected two different doses of piperlongumine, 10 and 20 mg per kg body weight, for a period of 2 weeks, in order to evaluate PL efficacy as an antitumor compound.^15,16

Experimental design

Rats were randomly divided and allocated into four experimental groups (n = 6).

Group-A: Negative Control

Group-B: Positive (DEN) Control: DEN 200 mg/kg body weight, i/p once

Group-C: DEN 200 mg/kg body weight i/p once + Piperlongumine 10 mg/kg body weight i/p for 14 days.

Group-D: DEN 200 mg/kg body weight i/p once + Piperlongumine 20 mg/kg body weight i/p for 14 days.

Piperlongumine (intraperitoneal) was administered 16 weeks after DEN administration and continued up to 18 weeks (Figure 1).

Body weight and liver weight

Initial and final body weight of rats from each group were recorded. Absolute and relative weight of liver was also recorded. Relative liver weight was calculated dividing absolute liver weight by final body weight and then multiplying it with hundred (Absolute liver weight/Final body weight × 100).

Blood collection

Blood samples were taken from all groups of rats by puncturing the retro-orbital sinus plexus under ketamine and xylazine (80 + 8 mg per kg body weight) anesthesia, and were kept in a labelled centrifuge tube undisturbed for 2 h at 37^°C. Serum samples were separated by centrifugation at 3000 rpm for 10 min. The supernatant was collected in a fresh Eppendorf tube and used for analysis or otherwise stored at −80^°C for further analysis.

Figure 1.

Experimental design, Vertical downward arrows represent intraperitoneal injection of diethylnitrosamine once at a dose of 200 mg/kg body weight. The horizontal line represents the total duration of study with a period of 16 weeks for development of hepatocellular carcinoma and 2 weeks of drug treatment.

Organ collection

All the animals were sacrificed by cervical dislocation at the end of the experiment, under a high dose of anesthesia (three times the anesthetic dose), and the liver was dissected out, washed with ice-cold normal saline/phosphate buffer saline (PBS, pH 7.4), and weighed. Hepatic tissues were used to assess the lipid peroxidation and histopathological investigations.

Preparation of liver homogenate

Livers were excised, washed with ice-cold PBS (pH 7.4), dried on filter paper, and weighed. The liver homogenate was prepared in MDA lysis buffer using the MDA analysis kit for lipid peroxidation using a teflon tissue homogenizer (REMI, India) in ice water and centrifuged using a refrigerated centrifuge (Thermo Scientific, USA) at 13,000 g for 10 min. The supernatant was collected for MDA quantification.

Evaluation of liver function

Liver function was evaluated by analyzing the bilirubin, total protein, albumin, and different liver enzymes namely, aspartate aminotransferase (AST/SGOT), alanine aminotransferase (ALT/SGPT), alkaline phosphatase (ALP), gamma-glutamyl transferase (γGT) according to the manufacturer’s instructions. All spectrophotometric analyses were done using the Chem five semi-automated clinical chemistry analyzer (Erba Mannheim, UK).

Estimation of cancer biomarkers in serum

Tumor markers viz., alpha-fetoprotein (AFP), and carcinoembryonic antigen (CEA) were quantitatively analyzed by chemiluminescence using the Advia Centaur enzyme immunoassay kit (Germany) and ADVIA Centaur XPT system (Germany).

Estimation of pro-inflammatory and antitumor cytokines

Pro-inflammatory cytokines and inflammatory mediators, namely IL-1B, TNF-α, NF-κB, and IL12a were measured using standard kits as per the manufacturer’s instructions.

Assessment of oxidative damage (lipid peroxidation)

Lipid peroxidation was measured using malondialdehyde (MDA) in hepatic tissue. LPO assay was performed using the Biovision Kit (K739-100, USA) according to the manufacturer’s instructions.

Gross and microscopic examination of liver tissue

Gross and histopathological examinations of the liver was done after necropsy of the experimental rats. Liver tissue samples were collected in 10% neutral buffered formalin. All the resected liver specimens were grossly examined by serial slicing, and representative tissue sections were taken for histopathological examination. Formalin-fixed tissues were processed in an automated tissue processor (Leica Biosystems, Germany). Paraffin wax embedded blocks were made for each group, from representative sections. Sections from tissues were cut at 5–6 μ thickness and were stained with hematoxylin and eosin (H and E) stain. The H and E-stained slides were observed under a microscope (Olympus Instruments, Tokyo, Japan) and findings were recorded for each case. Masson’s trichrome staining was done in cases showing cirrhosis.

Statistical analysis

Results are expressed as mean ± SEM and all statistical comparisons were made using one-way ANOVA followed by Tukey’s post hoc analysis and p-values less than or equal to 0.05 were considered significant.

Results

Effect of piperlongumine on body and liver weights

Table 1 shows the effect of DEN and piperlongumine on body weight, absolute and relative liver weight in control and experimental groups of animals. There was a significant decrease (p < 0.001) in body weight in DEN-treated animals compared to that of normal control animals or negative control (Group A). Treatment with piperlongumine significantly (p <0.001) increased the body weight in a dose-dependent manner. Liver weight increased significantly (p < 0.05) at the end of 18 weeks in DEN treated rats compared to normal control rats. However, treatment with piperlongumine significantly prevented this increase in liver weight.

Table 1.

Effect of piperlongumine on body weight, liver weight and relative liver weight in control and experimental rats.

Groups	Initial Body weight (g)	Final Body weight (g)	Liver weight (g)	Relative liver weight (g)
Group A	151.83±2.2	303.50±2.78	5.83±0.10	1.92±0.02
Group B	152±2.03^ns	242.83±5.88^***	8.06±0.23^***	3.33±0.14^***
Group C	148.33±1.35^ns	266.67±7.07^#	7.07±0.34^#	2.60±0.22^##
Group D	149.17±1.14^ns	297.50±2.45^###	6.11±0.14^###	2.06 ±0.06^###

Results are presented as mean ± SEM (n=6). Comparisons were made between negative control and DEN control.

***p < 0.001: compared to negative control; ^#p < 0.05, ^##p < 0.01, ^###p < 0.001: compared to DEN control; ^ns-not significant.

Effect of piperlongumine on serum hepatic enzymes markers

The effects of piperlongumine on the serum hepatic marker enzymes AST, ALT, ALP, and GGT of control and experimental animals are presented in Figure 2. DEN control rats exhibited a significant increase (p < 0.001) in the level of these hepatic marker enzymes in the serum when compared to normal untreated animals (Group A). A significant decline in the level of these hepatic serum marker enzymes was observed in piperlongumine treated rats, group C (p < 0.05) and D (p < 0.001).

Figure 2.

Effect of Piperlongumine on the serum marker enzymes. Values are expressed as mean ± SEM (n = 6). Comparisons were made between negative control and DEN control. ***p<0.001: compared to negative control; ^#p < 0.05, ^##p < 0.01, ^###p < 0.001: compared to DEN control; ns-not significant. DEN, Diethylnitrosamine.

Additionally, the effect of PL on bilirubin, total protein, and albumin is summarized in Table 2. There was a significant decrease (p < 0.001) in total protein as well as albumin levels as observed in group B animals when compared to group A control rats. On the other hand, the total protein and albumin levels increased significantly (p < 0.001) in PL treated group D animals when compared to group B animals. Moreover, rats treated with piperlongumine at a dose of 10 mg/kg showed a small improvement in the level of total protein as well as albumin, but this improvement was non-significant. Similarly, bilirubin levels were significantly elevated in DEN control rats (p < 0.001) whereas it was downregulated (p < 0.001) significantly in DEN rats treated with piperlongumine, and this decrease was also significant in group C rats (p < 0.05).

Table 2.

Effect of piperlongumine on other hepatic parameters.

Groups	Bilirubin (mg/dl)	Total protein (g/dl)	Albumin (g/dl)
Group A	0.26±0.01	7.81±0.26	5.55±0.27
Group B	1.88±0.07^***	5.42±0.11^***	3.85±0.34^***
Group C	1.63±0.07^#	5.81±0.17^ns	3.90±0.16^ns
Group D	0.47±0.06^###	6.78±0.16^###	5.32±0.09^##

Results are presented as mean ± SEM (n = 6). Comparisons were made between negative control and DEN control.

^***p < 0.001: compared to negative control; ^#p < 0.05, ^##p < 0.01, ^###p < 0.001: compared to DEN control; ^ns-not significant.

Effect of piperlongumine on tumor markers

The levels of alpha-fetoprotein (AFP) and carcinoembryonic antigen (CEA) in the serum of control and experimental rats are presented in Figure 3. The DEN-treated rats showed a significant increase in the level of alpha-fetoprotein compared to normal control rats. Piperlongumine treatment of DEN-treated rats (Group D) significantly decreased (p < 0.001) AFP content when compared to DEN control rats (Group B), but this decrease was non-significant in group C rats. The levels of carcinoembryonic antigen in DEN-treated rats were found to be slightly increased, but this increase was insignificant. Similarly, no significant increase or decrease in the level of CEA was observed in other groups when compared with normal control rats.

Figure 3.

Effect of Piperlongumine on the tumor markers. Values are expressed as mean ± SEM (n = 6). Comparisons were made between negative control and DEN control. ***p < 0.001: compared to negative control;^#p < 0.05, ^##p < 0.01, ^###p < 0.001: compared to DEN control; ns-not significant. DEN, Diethylnitrosamine.

Effect of piperlongumine on kidney functions

The non-hepatic parameters for all the control and experimental groups of rats are shown in Table 3. There was a significant elevation (p < 0.001) in the levels of urea and creatinine in DEN group rats compared with normal control. However, treatment of DEN group rats with PL (group D) resulted in a marked reduction (p < 0.001) in serum urea and creatinine levels. The effect of piperlongumine at the dose of 10 mg/kg body weight slightly improved the conditions, with a non-significant and a significant decrease (p < 0.05) in the level of urea and creatinine, respectively.

Table 3.

Effect of PL on biochemical changes in the serum urea, and creatinine in experimental rats.

Groups	Urea (mg/dl)	Creatinine (mg/dl)
Group A	39.00±1.53	0.76±0.02
Group B	50.67±1.5^***	1.21±0.06^***
Group C	47.83±1.94^ns	1.01±0.05^#
Group D	42.17±0.70^##	0.87±0.04^###

Results are presented as mean ± SEM (n=6). Comparisons were made between negative control and DEN control. ^***p < 0.001: compared to negative control; ^#p < 0.05, ^##p < 0.01, ^###p < 0.001: compared to DEN control; ^ns-not significant.

Effect of piperlongumine on proinflammatory cytokines and inflammatory mediators

In general, the cytokines analyzed (Figure 4) demonstrated a significant (p < 0.001) decrease in the proinflammatory and inflammatory cytokines (IL-1β, TNFα, and NF-κB) and an increase in the anti-tumor cytokines IL-12 compared to Group B (DEN control) in piperlongumine (group D) treated rats. IL-1β, and TNFα concentrations in the rats treated with PL were significantly lowered (p < 0.001) when compared to DEN control rats. IL-12 concentration was significantly decreased in DEN control rats, which was significantly upregulated in rats treated with piperlongumine (p < 0.05, p < 0.001). In the present study, we observed a significant increase (p < 0.001) in the expression of NF-κB in DEN-treated animals as compared to control animals, which may be due to chronic inflammation caused by treatment with the inflammatory agent DEN. However, a significant decrease (p < 0.001) in NF-κB was noticed in animals treated with PL as compared to DEN control rats. Impressively, PL administration considerably reduced the inflammation and marker expressions.

Figure 4.

Effect of Piperlongumine on serum pro-inflammatory and antitumor cytokines.Values are expressed as mean ± SEM (n = 6). Comparisons were made between negative control and DEN control. ***p < 0.001: compared to negative control;^#p < 0.05, ^##p < 0.01, ^###p < 0.001: compared to DEN control; ns-not significant.

Effect of piperlongumine on lipid peroxidation activity

The level of hepatic MDA in control and experimental rats is given in Figure 5. Administration of DEN significantly increased hepatic MDA levels in DEN control rats after 18 weeks of our study compared with normal control. PL treatment showed significant depletion (p < 0.001) in MDA content in the treatment groups. A significant decrease in the level of MDA was seen in animals treated with PL in a dose-dependent manner.

Figure 5.

Effect of piperlongumine on lipid peroxidation activity. Values are expressed as mean ± SEM (n = 6). Comparisons were made between negative control and DEN control. ***p < 0.001: compared to negative control; ^#p < 0.05, ^##p < 0.01, ^###p < 0.001: compared to DEN control; ns-not significant. DEN, Diethylnitrosamine.

Histopathological examination of the liver

The sections from the liver of the normal control group showed normal hepatocytes, with liver showing mild venous congestion, whereas the liver sections of the DEN control rats showed microscopic foci of hepatocellular carcinoma, with a prominent acinar and focal trabecular pattern, surrounding tissues showing cirrhosis with focal ballooning degeneration and large cell change, along with nodules of hepatocytes showing dysplastic changes and fibrosis (Figures 6(A) and (B)). Treatment with iperlongumine at a dose of 10 mg/kg-maintained liver architecture with no tumor and foci of cellular alteration (Figure 6(C)). Treatment with piperlongumine at a dose of 20 mg/kg maintained the normal liver parenchyma (Figure 6(D)).

Figure 6.

Representative images of the liver sections (A, B, C, D) from experimental rats. (A) Negative control (Group A) shows normal architecture of the liver with central veins (black arrow) and portal tract (red arrow) (H&E, ×100); (B) Diethylnitrosamine control (Group B) shows a microscopic focus of the tumor on the right hand of the field (black arrow). The tumor shows prominent neovascularization (red arrow), angiectasis and haphazardly arranged tumor cells (H&E, ×100); (C) PL treated group at a dose of 10 mg/kg body weight, shows focal large cell change (black arrow), congested central veins, and surrounding normal hepatocytes (H&E, ×200). (D) PL treated groups at a dose of 20 mg/kg body weight, showing normal liver parenchyma with a portal tract in the centre (black arrow) (H&E, ×200). PL, Piperlongumine.

Discussion

Nitrosamines are classified among the most potent dietary and environmental carcinogenic agents. Among the various nitrosamines, diethylnitrosamine (DEN) and dimethyl nitrosamine (DMN) are the most predominant compounds in foods.¹⁷ Humans are exposed to both preformed and endogenously made nitrosamines. DEN has become a choice of chemical for inducing liver carcinogenesis in rodents as an experimental model of human hepatocarcinogenesis.¹⁸ DEN, a known carcinogen, is commonly used to induce liver cancer in murine models of HCC due to its ability to disrupt nuclear enzymes involved in deoxyribonucleic acid (DNA) repair and replication.¹⁹ Phenobarbitone (PB) was administered in drinking water up to 16 weeks to promote HCC, 2 weeks after DEN injection. PB plays its role as a tumor promoter by selectively inducing the growth of preneoplastic hepatocytes.²⁰

The high mortality rate and associated side effects resulting from chemotherapy and/or radiotherapy increase the necessity for alternative cancer therapies. Various natural products have been found to have anti-cancer potential; however, most lack scientific validations. This study was undertaken to model the full spectrum of carcinogenic events and to evaluate the chemopreventive activity of piperlongumine in a rat model of diethylnitrosamine (DEN) induced hepatocellular carcinoma.

The anti-tumor efficacy of piperlongumine against DEN-induced HCC was elucidated in male Wistar albino rats. Loss in body weight was observed in rats treated with DEN only. Treatment with PL improved the body weight, which indicates the remedial property of the PL against DEN-induced HCC. DEN also led to a significant increase in the liver weight of experimental animals. This considerable rise in liver weight caused by DEN could be attributed to an increase in metabolic activity.²¹ The rise in liver weight could be attributed to cellular swelling, resulting from hepatic cell injury, as well as fatty alterations in the liver.²²

Albumin (ALB) is considered to play a crucial role in the inflammatory process. In plasma, the amount of albumin is a good predictor of liver health. A recent study found that albumin has direct HCC growth inhibitory properties in-vitro,²³ and there is some evidence that it may have a similar impact on HCC patients.²⁴ We found a significant drop in the level of albumin in DEN-treated rats which was restored to a normal level upon treatment with piperlongumine.

Serum liver enzymes such as alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and γ-glutamyl transferase (GGT), are tested routinely as these enzymes are usually elevated in patients with liver diseases, thereby reflecting the grades of liver damage. The liver enzymes AST and ALT are strong indicators of hepatocellular damage.²⁵ These enzymes, by catalyzing the transfer of amino groups from aspartic acid or alanine to ketoglutaric acid, help in the production of oxaloacetic acid and pyruvic acid, both of which are important building blocks in cells. GGT plays an important role in transpeptidation and hydrolysis of the glutamyl group of glutathione, biotransformation, nucleic acid metabolism, and tumorigenesis.²⁶ These cytoplasmic enzymes are released into the circulatory system in excessive amounts during hepatocellular damage because of the altered membrane permeability. Administration of DEN significantly raised AST, ALT, ALP, GGT, total protein, albumin, and bilirubin levels. The abnormal serum values were normalized with PL treatment at a dose of 20 mg/kg. In an experimentally induced colon cancer model, it was discovered that PL may suppress the elevated levels of serum AST and ALT.²⁷

After DEN treatment, blood levels of urea (BUN) and creatinine (CRP) rise in rats. Treatment with the PL prevented DEN-induced renal injury, by maintaining the cellular integrity of the kidney. In a study with colorectal cancer in rats, PL significantly lowered the increased level of both urea and creatinine.²⁷ However, there is little evidence of increased muscle breakdown in rats with HCC, with no rise in plasma creatine or creatinine.²⁸

Early diagnosis is crucial for curative treatments. α-fetoprotein (AFP) is the most widely used and broadly known biomarker for HCC. AFP is synthesized in the liver and secreted into serum.²⁹ An elevation in the level of AFP was observed in rats treated with DEN. In the present study, treatment with PL helped in bringing the level of AFP down.

Carcinoembryonic antigen (CEA) is a set of highly related glycoproteins involved in cell adhesion normally found in the embryonic entodermal epithelium. CEA is used as an indicator of tumor progression in a variety of carcinomas. However, in the present study, elevation in CEA was non-significant in all the groups, suggesting no role for CEA as an indicator of HCC in rats.

Cytokines are a wide category of proteins, peptides, and glycoproteins that are released by certain immune system cells in response to an injury or infection. These signalling molecules mediate and regulate immunity, inflammation, and hematopoiesis. They are released in response to infection, inflammation, and carcinogen-induced injury. IL-1B, and TNF-α are generally referred to as pro-inflammatory cytokines. Both these cytokines were found to be elevated in rats receiving only DEN, whereas rats treated with PL showed these elevated levels returning to the normal as seen in control rats (Group A). PL has been shown to inhibit serum TNF-α and IL-1B production in collagen-induced arthritis mice when compared to vehicle-treated collagen-induced arthritis control mice,³⁰ indicating a possible role in the control of inflammation-related disorders. It was also reported that PL upon oral supplementation considerably reduced inflammation markers (TNF-α, NF-κB) expressions in diabetic rats.³¹

Nuclear factor-κB (NF-κB) is a master regulator of inflammation and cell death in the development of hepatocellular injury, liver fibrosis, and HCC. In the present study, we have seen a downregulation in the level of NF-κB in PL treated rats compared to DEN control rats. It is reported that, in murine hepatocytes, inhibition of NF-κB activity is directly proportional to the induction of apoptosis. PL by inhibiting NF-κB may induce apoptosis in cancer cells. PL was reported to have inhibitory effects on NF-κB activity, exhibiting its role in mediating inflammatory cytokines.³² In another study, PL was found to have inhibitory effects on the expression of NF-κB in experimental colon cancer.²⁷

The antitumor effects of IL-12 are very well established. Interleukin-12 (IL-12) is mainly produced by activated monocytes, macrophages, and dendritic cells. IL-12 is also important for enhancing the cytotoxicity mediated by NK cells. In the present study, there has been a significant downfall of the IL-12 level in DEN control rats which got upregulated in PL treated rats. An increased level of IL-12 may indicate potential antitumor activity of PL. IL-12 has emerged as one of the most powerful cytokines in mediating antitumor activity and it controls inflammation by coordinating innate and adaptive immune responses.³³

An imbalance favoring the formation of ROS may lead to oxidative stress, which plays a critical role in numerous diseases, including HCC. Diethylnitrosamine (DEN) is metabolized by Cytochrome P450 in the body to produce highly reactive free radicals, initiating the process of lipid peroxidation. Malondialdehyde (MDA), formed as one of the final products of polyunsaturated fatty acid peroxidation, is the most mutagenic product of lipid peroxidation.³⁴ This is one of the most popular and reliable markers of oxidative stress in clinical scenarios.³⁵ MDA has been shown to interact with functional groups of many cellular components, promoting tumour development.³⁶ DEN has been shown to induce liver carcinogenesis by increasing oxidative stress and liver damage.³⁷ These findings very well correlate with the present study that shows a significant increase in the level of LPO in the livers of rats administered DEN, when compared with normal control rats. PL exhibits its chemopreventive effects against DEN-induced HCC by decreasing the level of lipid peroxidation.

Histopathological analysis specifies microscopic foci of hepatocellular carcinoma, cirrhosis in surrounding tissues, nodules of hepatocytes with dysplastic changes, and chronic inflammation, which are the characteristic features of HCC. Treatment with PL prevented the toxic effects of DEN on the hepatic tissues.

Conclusion

The chemopreventive or antitumor potential of PL has not been studied in experimentally induced hepatocellular carcinoma yet. In the present study, we demonstrated the chemopreventive effects of PL in a DEN-induced hepatocarcinogenesis animal model. We showed that piperlongumine has potent antineoplastic activity against hepatocellular carcinoma. Our results suggest that PL may be an effective chemopreventive agent for hepatocellular carcinoma.

Footnotes

Acknowledgements

The authors are grateful to the Director, AIIMS, Rishikesh, for providing the necessary facilities and financial support.

Author contributions

AK: conceptualized, performed the experiment, analyzed and interpreted the data. NK: analyzed the samples. NS and SA: performed the histological examination of the liver, AS: contributed to the article writing, ST and JS; Critically analyzed the article. MN and SH: Guidance, confirmed the authenticity of all the raw data. All authors read and approved the final article.

Declaration of conflicting interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The authors disclosed receipt of the following financial support for the research: This work was supported by the intramural research fund of the All India Institute of Medical Sciences, Rishikesh, Uttarakhand, India (IM/RC138/2019/45)

ORCID iDs

Ashish Kumar

Manisha Naithani

Ambika Sharma

-743X

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Piperlongumine inhibits diethylnitrosamine induced hepatocellular carcinoma in rats