Modulatory effect of pomegranate extract on TRPA1, TRPM2 and caspase-3 expressions in colorectal cancer induction of mice

Introduction

Considering the case and death rates for the 10 most common cancer types in 2020, colorectal cancer (CRC) ranks third with 10% and ranks second with 9.4% after lung cancer in mortality [1]. The initiation and development of this type of cancer are generally characterized by mucosal dysplasia after the infiltration of immune cells into colon tissue during inflammation [2]. Azoxymethane (AOM) as a carcinogen and dextran sodium sulfate (DSS) as a proinflammatory among chemical agents are preferred in studies with in vivo CRC models associated with inflammation [2, 3].

The range of useful products against health problems, especially cancers, is expanding and demand for natural dietary compounds is increasing daily [4]. Pomegranate (Punica granatum L.) fruit always attracts attention due to its rich content of antioxidant and anticarcinogenic compounds such as punicalagin, quercetin, and ellagic acid from phenolic compounds or flavonoids and fatty acids [5], [6], [7]. It was stated in a previous research report that pomegranate fruit extract (PFE) suppresses cancer progression and tumor angiogenesis in the colon cancer cell line in the chick chorioallantoic membrane model [8]. However, detailed studies with appropriate parameters and animal models are needed to evaluate their therapeutic efficacy against cancer.

Extracts prepared to detect potential protective properties in plants can be evaluated by in vitro genetic and cytological toxicity tests. Experimental setups consisting of in vitro cell cultures are very useful in reducing time and costs in laboratory environments. Mitotic index (MI) analysis, a classical method of cell cycle and genetic factors in cell culture, may be used initially before moving on to in vivo animal models [9, 10].

Organisms have a class of integral membrane proteins called transient receptor potential (TRP) channels that play a key role in interpreting environmental stimuli. These cationic channels are signal transducers by changing intracellular calcium (Ca²⁺) concentration or membrane potential [11], [12], [13]. In humans, the TRP ankyrin 1 channel (TRPA1) is reported to be a temperature-activated stress sensor [11, 12]. TRP melastatin 2 channel (TRPM2) is modulated by oxidative stress and involved in cell death responses under stress [11, 14, 15]. The dual effect of tumor necrosis factor-alpha (TNF-α) cytokine, a tumor suppressor agent in tumorogenesis and detumorogenesis as mainly an inflammatory immune reaction regulator, draws the attention [16]. Sialic acids as terminal carbohydrates in molecules draw attention in tissue damage and cancer-related disease processes [17]. The levels of reduced glutathione (GSH) as an important endogenous element of the antioxidant defense system and malondialdehyde (MDA) as a final product during lipid peroxidation formed due to reactive oxygen substances (ROS) are important oxidative stress biomarkers [18], [19], [20], [21]. The expression levels of caspase-3 (Casp-3) from responsible proteases for the apoptosis process usually draw attention as a marker in determining the degree of improvement in the treatment process of cancers [22, 23].

The elucidation of cellular and metabolic pathways that may be associated with the progression mechanism of cancer in the prevention of cancer by PFE is becoming increasingly important every day in terms of increasing the efficiency of treatments. The present study aimed to reveal effects on TRP (A1 and M2) channels, TNF-α, Casp-3, and oxidative stress to shed light on subcellular biochemical and molecular functioning in vitro and in vivo of PFE in mice with induced CRC.

Materials and methods

Results

When the toxicological potential of PFE was evaluated, no histopathological findings or irritations were found in any parts such as mucosa or epithelial cells belonging to the oral tract, pharynx, and trachea of mice. When experimental animals were evaluated in terms of weight, weight losses were encountered from the first DSS application compared to the control group. By last week, the mean weight of group-administered AOM was lower than that of the control group, and this change approached normal in AOM plus PFE group (p<0.01) (Figure 1A).

Figure 1:

The weight measurements of experimental animals (A), the effects of different PFE concentrations (mg/kg) on the level of MI in lymphocyte cell culture (B) and the levels of MDA, GSH and TSA of experimental animals (C) and the levels of TNF-α in colon tissue of mice (D). Dissimilar letters on columns show significantly distinct means, p<0.01 for Figure 1A and p<0.05 for Figure 1D. X^a,b: significant difference compared to the MMC group, p<0.01 for Figure 1B. χ^a,*: significant difference compared to the control group and CRC group, respectively, p<0.05 for Figure 1C.

Expression levels of TRPA1, TRPM2, and Casp-3 were modulated in colon samples by PFE treatment

The increased TRPA1 and TRPM2 expression levels were considered in mice with induced CRC. A direct relationship was determined between an increase in TRPA1 and TRPM2 expression levels and expression levels of Casp-3 in CRC. PFE reduced the expression of TRP channels and Casp-3, with important roles in apoptosis in CRC model mice. In current data, protein expression values of TRPA1 (Figure 2A and B), TRPM2 (Figure 2A and C), and Casp-3 (Figure 2A and D) were higher in the CRC group in comparison with the control group and PFE group (p<0.01). However, protein expression levels of TRPA1, TRPM2, and Casp −3 were downregulated in colon samples by PFE treatment (p<0.05).

Figure 2:

The protein expression levels of TRPA1, TRPM2, and Casp-3 in colon samples of mice.

Different symbols on columns show significant difference compared to the control group and CRC group, p<0.01 and p<0.05, respectively.

Histopathological examination results

It was observed that tissue samples taken from the colon in control and PFE group mice exhibited a normal histological structure (Figure 3A and B). In colon tissue samples of mice in the CRC induction group, local disorganization in the columnar epithelium in the lamina epithelial layer and degenerative and necrotic changes in Lieberkühn crypts and goblet cells were detected (Figure 3C). In addition, focal mononuclear and histiocytic cell infiltrations were observed in some areas of lamina propria. While there was a regression in lesions in PFE treatment group animals compared to the CRC induction group animals, it was found that cells in the lamina epithelial layer had normal histological structure and alignment similar to the control and PFE group animals. Among findings, there were regeneration attempts in Lieberkühn crypts (Figure 3D).

Figure 3:

Control group (A); normal colon histological section, L. epithelialis (horizontal arrow), goblet cell (vertical arrow), HE, 50 µm. PFE group (B); normal colon histological section, L. epithelialis (horizontal arrow), Lieberkühn crypts (vertical arrow), HE, 50 µm. CRC induction group (C); disorganization in L. epithelialis epithelial cells (vertical arrow), degenerative changes in Lieberkühn crypts (horizontal arrow), HE, 50 µm. PFE treatment group (D); L. epithelialis (horizontal arrow), regeneration attempt in Lieberkühn crypts (vertical arrow), HE, 50 µm.

Discussion

The use of an AOM and DSS-induced rodent CRC model for in vivo studies in the treatment and molecular biology of colon cancer is common because it has similar characteristics to laboratory findings in humans [2, 3]. In the present study, protective metabolic effects of PFE by stimulating TRP channel expression were determined as the main findings regarding oxidative stress, cytokine release, and apoptosis in experimental CRC induction.

The MI test, as one of the genotoxicity tests is among in vitro methods commonly used to investigate carcinogenic and mutagenic effects of factors with unknown genotoxicity or cytotoxicity as an indicator in which chromosomes in the centre of cell proliferation and apoptosis are affected [9, 10]. The in vitro antiproliferative, apoptotic, and antioxidant activities of tannin and ellagic acid concentrated in pomegranate extract, were demonstrated in colon cancer cell lines by Seeram et al. [5]. In the present study, PFE significantly increased MI levels compared to the MMC group. PFE with 200 mg/kg concentration among extracts examined with different densities (100, 200, 400, and 800 mg/kg) was more effective than others on MI level. After this determination in vivo stage was started in experimental mice with induced CRC (Figure 1B).

Pathological problems occur in cells and tissues due to negative side effects of ROS in metabolism [18, 21, 25]. It is known that increased ROS or oxidative stress in colorectal carcinogenesis is a very important risk factor, and MDA levels in tissue with CRC increase with clinical staging of disease [21]. High MDA levels have been suggested as an additional marker for diagnosing CRC and other toxicity-related symptoms [26, 27]. Concentrations of GSH as a molecular marker for the CRC vary significantly depending on the type of treatment and metastasis site [28]. Increased MDA levels and decreased levels of GSH during CRC can be considered directly related to cell damage, channel disorders in the cell membrane, and lipid peroxidation. Pomegranate extract, due to rich phenolic and flavonoid compounds in its content can shows significant antigenotoxic effects when applied appropriately in the protection of DNA that determines the course of cancer. It has been known that these effects is necessary to evaluate with more parameters in appropriate animal model [7, 8, 29]. In the present study, it was concluded that PFE in extract treatment of mice with induced CRC responded with decreased MDA levels and increased GSH levels in protecting cell integrity by regulating the expression of TRP channels in cell membranes, and findings on oxidative stress are consistent with those of other studies [7, 26] (Figure 1C).

TRP channels directly provide control of Ca²⁺, Mg²⁺, and Na²⁺ entry as cations in cells of living organisms. TRPM channels from these channels are considered important in treating proinflammatory diseases and cancers [30]. TRPM2 channel, which is affected by organic, inorganic, or physical stimuli such as temperature and voltage difference, is affected by oxidative stress. Its expression has been reported to cause cell death responses during stress [14]. It is claimed that TRPA1 as an O₂ sensor and a nociceptive receptor and TRPM2 as a host’s defence responder against infections and their blockades may be the main targets directly related to reactive oxygen load in management and alleviation of oxidative stress process in various diseases with oxidative stress in pathogenesis [31]. It has been noted that activation of TRPM2 and TRPA1 can be induced with H₂O₂, which is considered a very important source of ROS [32]. Hydroxyl radical scavengers effectively alleviate ROS or oxidative stress-related diseases through the blockade of TRPM2 and TRPA1 [31]. The importance of antagonist applications to TRPA1 was also emphasized for the treatment of diseases associated with colitis or colon damage, in which TRPA1 of the gastrointestinal tract is expressed in a positive correlation to other TRP channel expression, mainly in dorsal root ganglion primary sensory nerve cells in extrovert neuronal integrity that is generally responsive to capsaicin [33]. It has been suggested that the nociceptive effect of corilagin tannin in pomegranate may play a role in the antagonism of the TRP channel in the stimulation of capsaicin [34]. The current study revealed that PFE prepared from pomegranate fruit rich in phenolic and flavonoid components has a directly alleviating effect on expression levels of TRPA1 and TRPM2 channels (Figure 2B and C).

Casp-3 is the main mediator of apoptosis activated in response to stress during treatments [22]. In a study examining proapoptotic and antiproliferative characteristics belonging to pomegranate extract to its prooxidant activity, Weisburg et al. [35] recorded that pomegranate extract showed more antiproliferative specifications in cancer cells than in healthy cells isolated from the human oral cavity. It was also found that Casp-3 activation was reduced in cells treated with pomegranate extract, and apoptosis was associated with the prooxidant nature of pomegranate [35]. The importance of Casp-3 during angiogenesis and recurrence of tumors was emphasized, and loss of Casp-3 expression, which supports metastasis in resistance to colon cancer therapy, was presented as a promising treatment approach [22]. In the current study, PFE effectively reduced Casp-3 expression during CRC induction and these data are also consistent with a previous study containing altered Casp-3 transcript ratios of patients with CRC [36] (Figure 2D).

TNF-α, which was initially identified as a factor causing necrosis of tumors and had a broad spectrum for biological activity, is indicated as mainly an inflammatory immune reaction regulator [16, 37]. It was reported that colorectal carcinogenesis may develop as related with TNF-α during both persistence of ulcerative colitis due to intense inflammation after relapses and regenerating mucosal epithelial dysplasia despite treatments [23]. The present study indicated that PFE had a lowering effect on TNF-α levels in colitis-associated CRC induction and could be evaluated for the TNF-α signalling pathway (Figure 1D).

Supplementations reducing oxidative stress may also decrease high TSA levels in colon cancer and other diseases [18, 38]. Increased TSA levels in CRC may be related to the sialidase activities of parts such as lysosome, plasma membrane, or cytoplasm of damaged cells, and that cells with abnormal sialylation in carcinogenesis are far from the control of the immune system [17, 39]. According to the current study, it is thought that the decrease TSA levels in the PFE-administered group compared to CRC group may have been related to suppressed sialidase activity and total phenolic and flavonoid components of extract (Figure 1C).

In CRC induction model studies, cellular infiltration and disorganization, degenerative and necrotic changes are generally used to determine the severity of toxic effect and recovery [2, 3, 26, 36]. As stated in the findings section, these changes were generally encountered in the columnar epithelium in L. epithelialis layer, Lieberkühn crypts, goblet cells, and L. propria in mouse colon tissue samples of the CRC induction group (Figure 3C). There was integrity and homogeneity in cellular areas of colon tissue of mice in control and only PFE groups (Figure 3A and B). Signs of regeneration and healing in colon tissue and cells were evident in the PFE treatment group (Figure 3D). TNF-α and TSA concentrations which are known to increase significantly in cancer and tissue damages, support histopathological findings. General histopathological findings indicate that PFE application may be beneficial against AOM and DSS toxicity used in CRC induction.

Finally, it was concluded that TRPA1, TRPM2, TNF-α, and Casp-3 expression levels were modulated by PFE in colitis-associated CRC model mice and oxidative stress was important in this modulation. It can be suggested that PFE may contribute significantly to changes in inflammation and apoptosis, which are important in the CRC treatment process, depending on induced TNF-α and Casp-3 concentrations. In addition, there is a need for further comparative studies on the source and content of PFE.