Colon cancer process involves varied steps starting from pathological modifications extending from disconnected microscopic mucosal lesions of ACF, MDF to progression of tumor. Likewise, oxidative stress and inflammation are critical role players in the pathogenesis of cancer growth and promotion at molecular level. DMH was used as a carcinogen in the present study which mimics the human colon cancer pathology and not to rely only on extrapolating data from in vitro models due to intricate complexity of genetic and epigenetic processes taking place inside humans which contribute to colon cancer. Hence, piperine was tested to find its anti-cancer efficacy at molecular, cellular and morphological level which has shown anti-colon cancer activity in vitro [6].
We first studied the influence of piperine on biomarkers of colon cancer. CEA is one of the utmost used tumor marker with predictive implication in early stage of colon cancer. It mediates adhesion intercellularly, promotes cell accretion, controls immunity and signalling. Consequently, it shows tumor invasiveness and metastatic activity [19]. We did investigation of same biomarker in our experimental colon cancer study as well. It is not astonishing that CEA level was raised in DMH administered group significantly as reports suggest that CEA is elevated in gastric and colon cancer. Nevertheless, CEA level got decreased by the treatment of piperine possibly due to its anti-cancerous activities resulting in prevention of DMH-induced colon carcinogenesis. We also studied ACF and MDF, considerate pre-cancerous lesions found in colorectal cancer in carcinogen-administered rats as well in high risk human cases. Because of their similarities in genotypic and phenotypic descriptions in animal and humans, ACF and MDF are standard biomarkers to detect and analyse pathogenesis of colon carcinogenesis at an early stage. Also, development of ACF and MDF due to DMH administration in experimental rodents is used as a short-term model to investigate protective mechanism of organic compounds [9]. We found that piperine reduced ACF and MDF scores in group III and IV rats respectively when compared with tumor group which implies role of piperine in inhibiting early events of colon carcinogenesis, which has been supported by earlier studies [20].
Next, we studied a robust signalling pathway namely Nuclear factor erythroid 2-related factor 2 (Nrf-2)/Kelch-like ECH-associated protein 1(Keap1) against oxidative stress for cellular protection. Nrf-2, a protective transcription factor, controls downstream antioxidant machinery or detoxification system molecules found in the cells. The instigation of Nrf-2 during initial stage of inflammation-mediated tissue damage impedes the formation of proinflammatory modulators like pro inflammatory cytokines, chemokines, and cell adhesion molecules [21]. The development and destruction of Nrf-2 are in balance under normal physiological conditions. However, activation of Nrf-2 is blocked by Keap1 protein under stress conditions. Upon activation and dissociation of keap1, Nrf-2 accrues and binds with the antioxidant responsive elements (AREs) via translocating into the nucleus in the promoter region to activate transcription and defensive activities of downstream antioxidant machinery like HO-1 and glutathione dependent enzymes. In the current study, Nrf-2 expression was diminished in DMH administered animals and treatment with piperine upregulated Nrf-2 expression and further activated regulatory enzymes downstream as reported previously [22]. This was further validated and supported by a study where in Nrf-2- (-/-) mice were additionally vulnerable to dextran sulfate sodium- (DSS-) induced colitis [23]. Therefore, demonstrating that Nrf-2 activation is an auxiliary therapy possibly for colitis and colon cancer patients. The augmented sternness of colonic damage in DMH group animals may be correlated with decrease in phase II detoxification enzymes as reported previously [24]. In Nrf-2 deficient mice, proinflammatory modulators like interleukin 1β (IL-1β), interleukin 6 (IL-6), and tumor necrosis factor α (TNF-α) were prominently elevated as compared with the colon tissues of wild-type Nrf-2 mice [23, 25]. Moreover, Nrf-2- deficient (Nrf-2−/ −) mice are at an increased risk of having various cancers like stomach, CRC, skin because they are increasingly vulnerable to oxidative injury instigated ailments and DNA damage caused by chemicals as compared to wild-type mice. Khor et al. testified that Nrf-2 knockout mice with azoxymethane and DSS administration showed greater tumor incidence (80% versus 29%, respectively) while as there was an augmentation of inflammatory markers like Cox-2, 5-lipoxygenase, PGE-2, and leukotriene levels in colon as compared with azoxymethane and DSS-treated wild-type (WT) mice [26]. In the present study there was decreased expression of Nrf-2 and HO-1 in DMH group animals. However, treatment with piperine increased Nrf-2 and HO-1 expression in treatment groups. Thereby alleviating ROS and hence DNA damage resulting in protection of cells against potentially harmful entities may be the fundamental process through which Nrf-2 shields against chemical-induced carcinogenesis [27].
Keap-1 is a protein that keeps Nrf-2 in inhibited state in cytoplasm. Dissociation and amendment of Nrf-2–Keap1 complex is necessary for transfer of Nrf-2 in nucleus which leads to the activation of Nrf-2–ARE-dependent signalling, and numerous cell transduction pathways. ARE inducers like piperine treatment downregulates Keap 1 level in treatment groups as compared to DMH group due to which Nrf-2 translocation occurs activating downstream pathway. Various pathological events induce stress and HO-1 is a crucial protein that helps in cellular adaptation. It has been found as a potent beneficial target for various oxidant and inflammatory diseases. The HO enzyme family provides carbon monoxide (CO) and free iron (Fe2) by breaking down heme to biliverdin and bilirubin. Both bilirubin and CO may shield molecules against oxidative injury via reduction of superoxide anions and lipid peroxidation. Additionally, reports indicate pro-apoptotic and anti-proliferative activities of HO-1 in prostate, breast and oral cancer. Although exact process of the act is unknown [28, 29].
Nrf-2 is also found to control several phase II detoxification molecules like HO-1, NADP(H), quinone oxidoreductase-1 (NQO1), glutathione S-transferase (GST), UDP-glucuronosyltransferase (UGT) and glutamate-cysteine ligase (GCL) [30]. NQO1 is an important target of Nrf-2, is involved in detoxification process and its upregulation thwarts amplified IL-1β and TNF-α [26]. In the present study we found downregulation in HO-1 and Keap1 in DMH group. However, treatment with Piperine upregulated levels of HO-1 and Keap1 at both the doses respectively signifying activation of Nrf-2 which in turn switches on the rest of ARE response elements which involves theses enzymes resulting in activation of anti-inflammatory and anti-oxidant response, activating protective response in cells as reported previously [31].
Oxidative stress and chronic inflammation are two of the life-threatening features intricately associated both in the commencement and progression of cancerous growth by modulating tumor microenvironment where in ROS acts as secondary messenger to dysregulate various signaling pathways. The deficit of antioxidant machinery or detoxification system enzymes as well as up surging in ROS and RNS is detrimental to colonic tissue homeostasis. We found that there was an increase in ROS production in DMH administered group as compared to control group. Treatment with piperine attenuated ROS levels in both groups as reported previously [30]. MDA is a hallmark of oxidative stress produced by ROS and is a carcinogenic agent attributing to the formation of cancers in humans. We observed augmented levels of MDA in DMH induced CRC as reported previously [32]. Piperine treatment repressed the production of MDA levels which may be due to alkaloids present in piperine scavenge free radicals due to its potent anti-oxidant activity attenuating MDA level in treatment groups. Enzymatic and non-enzymatic antioxidants are the primary defenders against cytotoxic oxygen radicals by scavenging intermediates of oxygen reduction. Some of the anti-oxidants like SOD and CAT protect against lipid peroxidation in tissues by directly eliminating reactive oxygen metabolites like superoxide (O22•) and hydroxyl ions (OH•) providing one of the most efficient defensive mechanisms in the biological system against diseases [10]. CAT converts H2O2 to H2O and O2, hence averts oxidative injury. In the present study, SOD and CAT activities were diminished in DMH induced CRC whereas these activities got enhanced after administration of piperine which may be because of its free redox trapping activity [7]. GSH constitutes the major nonprotein thiol in mammalian cells which helps in many cellular activities like regulation of protein synthesis. Reduced glutathione acts as a substrate for many xenobiotic and free radical elimination reactions. Augmented GSH may stimulate other GSH dependent enzymes like GPx which has four selenium cofactors that catalyse the breakdown of H2O2 and organic hydroperoxides [32]. GR is another glutathione restoring enzyme that catalyses oxidized glutathione (GSSG) to reduced glutathione (GSH) by the oxidation of NADH to NAD [33]. In the current study, GSH got diminished and GSSG got increased in DMH group in eliminating free radicals, hence the levels of reduced GSH were exhausted. However, piperine supplementation increased GSH levels and decreased GSSG in treatment groups due to its anti-oxidative potential. Moreover, detoxification of xenobiotics, carcinogens, free radicals and peroxides occurs primarily by GPx and GR by conjugating xenobiotics with GSH, resulting in cellular protection against mutagen-induced toxicity. In the present study, there was decreased activities of GSH and GSH dependent enzymes like GPx and GR in DMH induced rats whereas treatment with pieprine increased antioxidant armory which may be because of its free radical scavenging potential [34]. Earlier reports recommend that alterations in redox balance and signalling are hallmarks of initiation and promotion of carcinogenesis and resistance to treatment [35]. Hence, agent that upregulates antioxidant enzyme machinery like CAT, SOD, GR, GPx that inactivate ROS, have massive potential to avert initiation and promotion of cancer as observed in the current study. Therefore, upregulation of antioxidant machinery and the preclusion of DMH-induced colon carcinogenesis by prophylactic treatment of piperine may possibly be due to the presence of alkaloids which resulted in boosting of endogenous anti-oxidant machinery because of its anti-oxidant, anti-inflammatory and various pharmacological activities attributed to it [7, 8, 20, 30, 36].
In the instigation and development of cancer a potential cross-talk between Nrf-2 and NF-κB pathways is recognized. One of the principle ways through which chronic inflammation leads to the formation of neoplasm/growth is by the production of ROS via inflammatory cells. This Nrf-2 intermediated anti-cancer response is accomplished by not only increase in antioxidant armory. But additionally, confirmed by the repression of inflammatory pathway mediators smoothed through NF-κB signalling pathway. Correspondingly, upregulation of cytokines occurs due to Nrf-2 deficiency as a result of NF-κB activation or the activation of Nrf-2 attenuates NF-κB and downstream signalling as reported previously [36, 37]. Carini et al. reported that dysregulation of ROS may alter DNA assembly, consequentially modify proteins and lipids, stimulate numerous stress-activated transcription elements including NF-κB, formation of pro and anti-inflammatory cytokines which contribute to carcinogenesis via oxidative insults [38]. Whereas antioxidant agents like piperine impede IL-1beta, iNOS, COX-2, NO, NF-κB production and henceforth, carcinogenesis [39, 31].
Chronic inflammation contributes to 25% of human cancers. One of the most important pathways involved in inflammation is Nuclear factor kappa light chain enhancer of activated B cells (NF-κB). NF-κB is a redox specific transcription factor which activates immune and cell detoxification systems, endorses the development of pro-inflammatory cytokines like TNF-α, IL-1, IL-6, and IL-8, promoting tumor growth [40]. NF-κB’s enhanced expression may be attributed to chronic inflammation followed by cyclic administration with carcinogen, DMH contribute to colon carcinogenesis. Other studies also decipher that colon cancer cell lines have remarkably abnormal NF-κB expression and less IκB levels, unravelling that dysregulated NF-κB is a major contributor of colon cancer which is in accordance to our results as well [41]. Our results further validate that the piperine inhibits NF-κB activity as reported previously and strongly recommend that the bioactivity of piperine against colon carcinogenesis may possibly be due to inactivation NF-κB which is further evident due to inhibition of downstream pathway player proteins [7, 42, 43].
NF-κB has a critical connection amongst inflammation and cancer since it increases levels of tumor stimulating cytokines downstream to it like interleukin 6 (IL-6), tumor necrosis factor alpha (TNF-α), PGE-2, COX-2 as well as survival genes such as Bcl-extra-large (Bcl-xL). Similar results were obtained in the current study. ROS is released by the activation of inflammatory cells resulting in oxidative injury of DNA and proteins. There are cumulative indications which decipher that chances of colon carcinogenesis increase by enhancement in pro-inflammatory cytokines like TNF-α, IL-1α, IL-1β and IL-6 which further increase the secretion of PGE-2, an inflammatory mediator. However, piperine treatment attenuated the levels of above mentioned pro inflammatory cytokines possibly by inhibition of NF-κB and anti-inflammatory activity of piperine as reported previously [44, 45, 43].
COX-2 is triggered in inflammatory or hypoxic environment and is upregulated in various cancers including colon cancer unlike in normal cells and thus making it a potential therapeutic target. The major downstream mediator of COX-2 is PGE-2 that enhances cellular growth and angiogenesis, obstructs apoptosis, augments invasiveness, and regulates immunosuppression in colonic mucosa. Additionally, enhanced COX-2 expression is directly proportional to PGE-2 resulting in increased production of malondialdehydes forming DNA adducts in colonic tissue and quickens the process of carcinogenesis. In the current study, we found an increase in COX-2 and hence PGE-2 in DMH group whereas treatment with piperine decreased COX-2 and hence PGE-2 in both the groups respectively. Thereby precluding the process of carcinogenesis and not allowing its acceleration [45].
Nitric oxide (NO), an RNS, an inflammatory mediator plays an essential character in colon tumorigenesis in both humans and in investigational experiments. NO is formed by three isoforms of nitric oxide synthase (NOS), Under normal physiological conditions. Inducible (NOS) produces micromolar concentrations of NO which lasts for hours or days which is greatest amount unlike NO production by other isoforms of NOS resulting in DNA damage, deficiency of DNA repair, cancerous growth proliferation and promotion. Consequently, tumor malignancy is enhanced by variations brought by NO formation in cells by damage/mutation in DNA, dysregulating signalling pathways and instability in genome Also, DNA repair pathways are negatively impacted by high NO concentrations, leading to carcinogenesis [46]. iNOS is produced by proinflammatory agents and tumor micro environment and does not depend on Calcuim. Femia et al. reported that DMH-induced colorectal cancer has high pro-inflammatory enzyme inducible nitric oxide synthase (iNOS) expression [47]. We got similar results in our current study which is support by earlier findings. However, over production of NO is contributed by iNOS damaging DNA repair and promoting cancer growth. Therefore, mitigating iNOS and henceforth NO at early stages of colon cancer in humans can be beneficial strategy to diminish development of cancer. In addition, carcinogens may increase the activities of COX-2 and iNOS on the mucosa of the colon, which also causes the promotion of cancer occurrence. Both COX-2 and iNOS are upregulated by NF-κB. It was indicated that by reducing NF-κB expression, the occurrence of cancer can be reduced and decrease in induction of NOS hence decreasing NO which further alleviates DNA damage and cancer growth and promotion [6, 7].
Myeloperoxidase (MPO) is another enzyme found in neutrophils for H2O2 production. It has been reported that in the inflamed tissue, MPO level is directly proportional to the neutrophil concentration and infiltration. Thus, acute intestinal inflammation is evaluated by MPO measurement which acts as a quantitative and sensitive assay. Inflammation is associated with oxidative stress and promotes tumor initiation and promotion. Also, chronic intestinal inflammation and Colon cancer are related to each other. The present study found that colon cancer group showed an elevation in the intestinal inflammatory markers (MPO and COX-2) through NF-κB mediated response. Piperine treatment alleviated MPO levels through suppression of NF-κB and henceforth suppressing infiltration of neutrophils and chronic inflammation leading to colon cancer [9, 48].
Histology of colon tissue sections of control did not show any anomaly in crypt morphology and architecture. DMH group animals showed infiltration of inflammatory cells, aberrant crypt formation, and depletion of mucin. Though treatment with piperine restored the histoarchitecture back to the control animals.