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ORIGINAL ARTICLE
Year : 2016  |  Volume : 12  |  Issue : 4  |  Page : 1307-1312

Expression of metallothionein in dimethylhydrazine-induced colonic precancerous and cancerous model in rat


1 Department of Clinical Biochemistry, Christian Medical College, Vellore, Tamil Nadu, India
2 Department of Neuropathology, Christian Medical College, Vellore, Tamil Nadu, India
3 Department of Gastrointestinal Sciences, Christian Medical College, Vellore, Tamil Nadu, India
4 Department of General Surgery, Christian Medical College, Vellore, Tamil Nadu, India

Date of Web Publication7-Feb-2017

Correspondence Address:
Pamela Christudoss
Department of Clinical Biochemistry, Christian Medical College, Vellore - 632 004, Tamil Nadu
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0973-1482.179107

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 > Abstract 

Aim: Metallothionein (MT) is a small protein with a high affinity for divalent heavy metals and has a function in zinc homeostasis. The purpose of this study was to assess the MT mRNA gene expression as well as the MT protein content by immunohistochemistry and radioimmunoassay (RIA) in 1,2-dimethylhydrazine (DMH)-induced precancerous and cancerous colonic tissue in rats.
Materials and Methods: Six-week-old rats were given subcutaneous injections of DMH twice a week for 3 months and sacrificed at 4 months (precancerous model) and 6 months (cancerous model). We determined MT mRNA expression by reverse transcription polymerase chain reaction and MT protein content by both immunohistochemical expression and cadmium-109 RIA.
Results: MT mRNA expression in the large intestine showed statistically significant decrease in the precancerous (P < 0.01) and the cancerous (P < 0.001) model as compared with controls. Immunohistochemical expression of MT showed statistically significant decrease (P < 0.05) in the colonic cancerous tissue. MT content in the large intestine showed statistically significant decrease in precancerous (P < 0.005) and cancerous (P < 0.001) model as compared with controls.
Conclusion: This study suggests that a decrease in the colonic MT mRNA expression, MT protein expression, and content in DMH-induced colonic cancer model is associated with the development of preneoplastic lesions and further progression to carcinoma in the colon results in a greater reduction in the levels of each of these parameters.

Keywords: Colon cancer, dimethylhydrazine, metallothionein, precancerous, zinc


How to cite this article:
Christudoss P, Chacko G, Selvakumar R, Fleming JJ, Pugazhendhi S, Mathew G. Expression of metallothionein in dimethylhydrazine-induced colonic precancerous and cancerous model in rat. J Can Res Ther 2016;12:1307-12

How to cite this URL:
Christudoss P, Chacko G, Selvakumar R, Fleming JJ, Pugazhendhi S, Mathew G. Expression of metallothionein in dimethylhydrazine-induced colonic precancerous and cancerous model in rat. J Can Res Ther [serial online] 2016 [cited 2020 Oct 25];12:1307-12. Available from: https://www.cancerjournal.net/text.asp?2016/12/4/1307/179107


 > Introduction Top


Zinc (Zn) is very important for the functional and structural integrity of cells and contributes to a number of processes including gene expression.[1] The role of Zn may be attributed to a Zn-binding protein metallothionein (MT). MT is a ubiquitous low molecular weight, intracellular metalloprotein, with a high content of cysteine which exhibits a selective affinity for divalent metal ions such as Zn and cadmium (Cd).[2] MT plays an important role in the homeostasis of Zn, a metal important to tumor growth and progression.[3],[4] Cellular accumulation of MT depends on both gene expression and protein degradation. Both depend largely on the availability of cellular Zn derived from the dietary Zn supply. Binding of Zn to MT may indicate the involvement of MT in the metabolism and transport of Zn.[5] MT expression is related to Zn accumulation in certain organs and MT may act in a number of biochemical processes, in Zn trafficking and Zn donation to apoproteins, including Zn finger proteins that act in cellular signaling and transcriptional regulation. As a result, MT expression may affect a number of cellular processes including gene expression, apoptosis, proliferation, and differentiation.[6] MT is also involved in systemic Zn distribution; hence, it may serve as a reservoir from which apometalloproteins including enzymes and Zn finger proteins (transcription factors, signaling and adapter molecules) acquire Zn.[7] The amount of MT in tissues is highly dependent on metal ion availability, and much of the regulation occurs at the level of transcription. Hence, a close relationship exists between MT and Zn content as synthesis of MT has been reported to be induced by the increased Zn content in the cells.[8]

MT expression can be altered by changes in gene structure, such as amplification and methylation.[9] Besides playing a homeostatic role in the control and detoxification of the heavy metals, several lines of evidence indicate that MT can scavenge reactive oxygen molecules (ROMs), particularly the hydroxyl radical.[10] Recently, it has been shown by Hammer et al. that Zn-MT can scavenge free hydroxyl ions, leading to the speculation that MT plays a direct role in the detoxification of this reactive species. These substances, which are produced continuously during imbalance with endogenous antioxidants can induce DNA damage, lipid peroxidation, enzyme oxidation, etc., leading to cellular destruction, chromosomal aberrations, and finally to cancer.[11] By scavenging ROM, antioxidant proteins such as MT may play an important role during DNA damage presumably by acting as an antioxidant in preventing carcinogenesis.[11],[12]

The high affinity of MT for Cd has served as the basis for the development of simple, rapid, inexpensive metal binding assays to quantitate MT in biological samples. Analytical methods, however, do not give qualitative information as to the distribution of MT within specific tissues and within cells of organs known to contain the protein. Hence, immunohistochemical analysis is used to detect the presence or absence of this protein, its tissue distribution, and changes in protein expression or degradation. Immunohistochemical techniques for visualization of MT in tissue may provide some important information about the response of MT protein expression during the progression of carcinogenesis. Since mRNA is eventually translated into protein, one might assume that there should be some sort of correlation between the levels of mRNA and that of the protein. Hence, estimation of MT mRNA expression, MT protein expression, and MT content is necessary for a complete understanding of how MT functions.

Previously, we showed that the significant decrease in tissue Zn and activity of Zn-related enzymes, CuZnSOD and alkaline phosphatase, occurs alongside the changes in the colonic mucosa during precancerous and cancerous transformation in 1,2-dimethylhydrazine (1,2-DMH) (symmetrical DMH)-treated rats.[13] Since MTs are antioxidants and their expression has been related to colon carcinogenesis, the present study is an extrapolation of the animal experiment as mentioned [13] and is designed to assess the altered colonic expression of the Zn-related protein MT at all the three levels - MT mRNA gene expression, immunohistochemical expression, and MT protein content after administration of a colon-specific carcinogenic agent (DMH) in rats. We have followed these changes in association with the progression of colonic carcinogenesis through premalignant and malignant transformation phases.


 > Materials and Methods Top


Animals

Thirty adult Wistar rats 6 weeks of age (100–120 g) obtained from the Institutional Animal House were housed in polypropylene plastic cages, in an animal holding room under controlled conditions with 25 ± 2°C, 50 ± 10% humidity, and 12 h light-dark cycles. The rats were allowed water and food ad libitum, observed daily and weighed weekly. This study was approved by the Institutional Animal Experimentation Ethics Committee with the Committee for the Purpose of Control and Supervision of Experiments on Animals (IAEC No. 17/2002–2003), and the Institutional Review Board of our institution. All ethical practices were followed for animal experimentation procedures.

Experimental design

Thirty rats were randomly assigned to two groups such as Group A (Precancerous) and Group B (cancerous). All groups were fed the same diet and maintained as described. Group A was further divided into control (n = 6) which received 0.25 ml saline and experimental (n = 8) which received 30 mg/kg body weight DMH dissolved in saline twice a week for 3 months and were euthanized at 4 months. Whereas the cancerous Group B received saline (control, n = 6) or DMH (experimental, n = 10) twice a week for 4 months and were euthanized at 6 months. All groups were euthanized by chloroform inhalation.

Tissue preparation, measurement of metallothionein content, metallothionein immunohistochemical expression, and metallothionein mRNA expression

All rats were examined grossly at necropsy. The entire portion from the stomach to anus was removed, and the large intestine was isolated. The colon of the rats of all the groups was harvested, slit lengthwise and checked for abnormalities. Any lesions detected were measured, location noted, and resected. Small portions were cut out from the resected area of the tumor and were collected and used for the following estimations. The samples were preweighed (approximately 10 mg) and frozen using liquid nitrogen, stored at −70°C until analysis for estimating MT content by Cd 109 assay and mRNA expression by real-time polymerase chain reaction (PCR). The immunohistochemical expression of MT in the tumorous portion was analyzed only for the DMH-induced colon cancerous group. For immunohistochemical expression of MT, formalin-fixed, paraffin-embedded tissues for histopathology were used. The portion of tissue resected and used for all the estimations were from the same site of tumor.

Estimation of metallothionein content

MT concentration in colonic tissue was determined by Cd/Hb radioimmunoassay (RIA) as previously described by Eaton and Toal.[14] Briefly colon samples were homogenized 1:10 in homogenizing buffer, 10 mM Tris buffer (pH 8.2), centrifuged at 13,000 rpm for 4 min, and the supernatant fractions heated to remove the precipitated proteins. The heat-denatured supernatant was used for estimation of MT using Cd 109. Two-hundred microliters of Cd 109 solution in 19.6 µM of cold Cd which contained 2.0 µgCd/ml with a radioactivity of 1.0 µCui/ml was added to 200 µl of sample (heat-denatured supernatant) and allowed to incubate at room temperature for 15 min. Protein concentration in supernatant was determined by the method of Lowry et al.[15] Data obtained by this method are expressed as mean nmol Cd/mg protein.

Immunohistochemical localization of metallothionein

Tissues from the resected areas with cancer were collected and processed by standard techniques to paraffin wax embedding after fixation in 10% neutral buffered formal saline for 24 h. MT localization in large intestine was determined using standard procedures for immunohistochemistry staining.[16] Briefly, immunohistochemistry was performed on 5-micron sections of formalin-fixed, paraffin-embedded tissue using a mouse monoclonal antibody E9 (Dako Ltd.; DSS Imagetech, New Delhi, India, diluted 1:50 in phosphate buffered saline), which reacts with MT-1 and MT-2. Dako EnVision system (system labeled polymer – HRP, antimouse – Dako, DSS Image Tech, India) was used for detection, and the reaction product was visualized with the chromogen 3,3'-diaminobenzidine, Dako. Tissues were counterstained with Harris hematoxylin. Normal rat colon served as the positive control.

Immunohistochemical evaluation

The extent and localization of MT immunostaining in the crypts were estimated and expressed as a percentage of the total number of crypts. Immunostaining (nuclear, cytoplasmic, or membrane cytoplasmic) was calculated as the percentage of positive tumor cells in relation to the total cell number in representative fields. A cell was considered immunopositive if either the nucleus or the cytoplasm or both showed positive staining irrespective of intensity. However, it was our experience that immunopositivity was uniformly strong in all cases and equivocal immunostaining was rarely observed.

RNA isolation, reverse transcription polymerase chain reaction

Total RNA (200 ng) was converted into cDNA using EuroScript reverse transcriptase core kit (RT-RTCK-03, Eurogentec, Genex, India). Briefly, the conversion mixture contained final concentration of 1x buffer, 5 mM MgCl2, 500 µM of each dNTPs, 2.5 µM random nonamer, 0.4 U/µl of RNAse inhibitor, and 1.25 U/µl of Euroscript RT. The cDNA conversion was carried out in PTC-150 MiniCycler (Bio-Rad, USA) with an initial activation step for 10 min at 25°C followed by a reverse transcription step for 30 min at 48°C and a final inactivation step for 5 min at 95°C. MT expression in cDNA samples was measured by real-time quantitative PCR using hot gold star DNA polymerase kit (Eurogentec, Belgium) in Chromo 4 real-time PCR machine (Bio-Rad, USA). GAPDH gene is a well-known housekeeping gene used for normalization of expression, and the primers from previously described studies [17],[18] were used for the quantitation. The primer sequences for MT (MT-1) were forward - 5'-TGTGCCTGAAGTGACGAACAG-3' and reverse - 5'-TTCACATGCTCGGTAGAAAACG-3' and the GAPDH primer sequences were forward - 5'-AGATGGTGAAGGTCGGTGTC-3' and reverse - 5'-ATTGAACTTGCCGTGGGTAG-3'. The primers were verified with the NCBI BLAST search engine. PCR conditions were optimized by gradient PCR, and the specificity of amplicons was confirmed by electrophoresing on 2% agarose gel. The threshold cycle (Ct) values for all the samples were measured in replicates, and the expression was quantitated using the following formula, 2ΔCt, ΔCt = Ct (MT gene)− Ct (GAPDH).[19]

Statistical analysis

Data are expressed as mean ± standard deviation differences between groups were analyzed using Mann–Whitney U-test and ANOVA. A difference was considered statistically significant when the P < 0.05.


 > Results Top


The results of MT obtained in this study are compared to the decrease in colonic tissue Zn levels of DMH-treated rats as observed in our earlier study [13] as the animals used for the present study are the same. These data are shown to facilitate comparison [Figure 1].
Figure 1: Tissue zinc levels in the large intestine in dimethylhydrazine-treated rats at 4 months (precancerous stage) and 6 months (cancerous stage). Values are represented as mean ± standard deviation. *P < 0.01, **P < 0.001 (reproduced with permission from reference 13 [DOI: 10.7314/APJCP. 2012.13.2.487])

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Precancerous group

As shown in [Figure 2], the relative MT mRNA expression in the resected portion of the colonic tissue in the precancerous DMH model showed statistically significant decrease (mean decrease 25%, P < 0.05) as compared with controls treated with saline. In this study, in the precancerous model, the mean MT content in resected portion of the colonic tissue in DMH-treated rats showed statistically significant decrease (mean decrease 39%) as compared with controls (0.091 ± 0.014 vs. 0.149 ± 0.019 nmol Cd bound/mg protein, P < 0.005) as shown in [Figure 3].
Figure 2: Quantitative expression of metallothionein mRNA in large intestine in control and test in precancerous and cancerous groups in dimethylhydrazine-treated rats. Values are represented as mean ± standard deviation. *P < 0.01, **P < 0.001

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Figure 3: Metallothionein content in large intestine measured by cadmium-109 affinitiy binding assay in control and test in precancerous and cancerous groups in dimethylhydrazine-treated rats. Values are represented as mean ± standard deviation. *P < 0.01, **P < 0.001

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Cancerous group

The MT mRNA expression of the resected portion of the colonic tissue in the DMH-induced carcinoma model also showed statistically significant decrease (mean decrease 90%) as compared with controls treated with saline (2.2 ± 1.2 vs. 22.43 ± 4.6 P < 0.001) [Figure 2].

Immunohistochemical analysis of MT in normal colonic tissue of the control rat showed 95% MT immunopositivity in the surface epithelium and upper half of the crypts whereas a statistically significant decrease in MT immunopositivity (mean decrease of 80%, P < 0.001) was observed in the resected portion of colonic cancerous tissue as compared to the controls treated with saline [Figure 4].
Figure 4: Immunohistochemical localization of metallothionein in microscopically normal colon mucosa of rats (a and b) and cancerous colonic mucosa (c and d). (a and b) Colon section of a control animal given only saline showing 90% metallothionein expression (a) ×10, (b) ×40. (c and d) Colon section of an experimental rat treated with dimethylhydrazine showing 20% metallothionein expression (c) ×10, (d) ×40

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As shown in [Figure 3], in this study, in DMH-induced colon carcinoma, mean MT content of the resected area in the large intestine showed a mean 63% statistically significant decrease as compared with controls (0.054 ± 0.03 vs. 0.147 ± 0.012, nmol Cd bound/mg protein, P < 0.005).


 > Discussion Top


MT is involved in many pathophysiological processes, such as metal homeostasis, detoxification, cell proliferation, apoptosis, and protection against oxidative damage.[6],[20] Studies have shown that tissue Zn accumulation correlates with de novo MT synthesis, suggesting that the protein functions in trafficking or processing of newly acquired cellular Zn. Plasma Zn concentrations are also related to MT expression, further suggesting a linkage to cellular Zn homeostasis.[7] It has been reported that Zn deficiency is implicated in the development and progression of some cancers.[21] Hence, there could be a possible involvement and association of Zn and the Zn-related protein MT in carcinogenesis. In our earlier study of DMH-treated precancerous and cancerous model in rat, the colonic cancer incidence was 92%, with an associated statistically significant decrease in colonic tissue Zn (mean decrease 72%) as compared to saline controls. The decrease seen was greater in the DMH-induced cancerous group compared to the precancerous model (mean decrease 46%).[13]

Since MT is a Zn protein, this present study has evaluated the altered expression of MT at all three levels such as MT protein content, immunohistochemical protein expression, and MT mRNA expression in the colonic tissue in DMH-treated rats. These results are compared to the alterations in tissue Zn levels that take place alongside the changes in the colonic mucosa during progression of colonic carcinogenesis through premalignant and malignant transformation phases in the same DMH-induced rats as observed in our previous study.[13]

In the present study, we have observed that MT mRNA gene expression in colonic precancerous and cancerous tissue is down-regulated in DMH-treated rats. There is a statistically significant decrease in MT mRNA gene expression (mean decrease of 25%) in precancerous colonic tissue with a greater significant decrease (mean decrease of 90%) in colonic cancerous tissue as compared with controls in our animal model. Our finding is in accordance with the results of studies reported by others in which MT is down-regulated in certain human tumors such as hepatocellular, gastric, and colorectal carcinomas.[22],[23] It was suggested in studies by Andrews that the decrease in MT expression in cancerous tissues could be caused by the removal of Zn from the Zn-binding transcription factor MTF1 leading to decreased DNA-binding activity and decreased MT gene transcription.[24] However, some studies have reported an increased expression of MT in various human tumors of the breast, kidney, liver, lung, nasopharynx, testes, thyroid, and urinary bladder.[25],[26] Hence, the expression of MT is not universal to all human tumors but may depend on the differentiation status and proliferative index of tumors along with other tissue factors. However, the factors which can influence MT induction in human tumors are not yet understood. It has been reported that down-regulation of MT synthesis in hepatic tumors may be related to hypermethylation of the MT-promoter or mutation of other genes such as the p53 tumor suppressor gene.[27] Interestingly, from various studies, it has been observed that the lack of MT induction is observed almost exclusively in specific cancer cells and not in normal cells at any stage of growth.[28]

All vertebrates examined contain two or more distinct MT isoforms which are grouped into two classes designated MT-1and MT-2. Rats are thought to synthesize only two MT isoforms. In rats, MTs have been demonstrated in liver kidney, intestine, etc.[29],[30],[31] The present study also shows the immunohistochemical distribution pattern of MT in normal colonic tissue of control rats and in cancerous tissue of the colon in DMH-treated rats. Normal colonic mucosa of control rats showed MT immunopositivity in 95% of enterocytes at the luminal surface and crypts whereas only 20% of the cells were positive in the colonic cancerous tissues of animals treated with DMH. Hence, the immunohistochemical expression of MT in the colonic cancerous tissue was decreased by 80% as compared with the control group administered only saline. Our results are in agreement with studies reported by others, in which immunohistochemical MT expression in colorectal carcinoma tissue was related inversely to tumor stage and appearance of metastatic spread.[22],[32] In a study by Ioachim et al.,[33] it was observed that the normal colon epithelium showed strong immunopositivity, but there was 60% of the colonic carcinoma tissues and 61% of the adenoma tissues almost completely lacked MT immunopositivity. However, concerning MT, conflicting results have been reported with regards to its immunohistochemical expression in various tumor tissues as compared with normal tissue and its association with clinicopathological parameters and survival.[34]

In accordance with the observations on the alterations of MT gene expression and protein expression in the colonic cancerous tissue in our animal model, the MT content in the colon cancerous tissue as measured by Cd 109 RIA assay also showed a statistically significant decrease (mean decrease 63 %) than that of normal tissue. Immunohistochemical evaluation of MT protein expression showed a fairly good correlation with these quantitative data. Our findings are in agreement with that of previous studies reported earlier that colorectal and gastric carcinogenesis in humans was associated with statistically significant decrease in MT content as measured by both RIA and immunohistochemistry.[35] Mulder et al. have reported a significantly decreased MT content (determined by RIA) in both adenocarcinomas and carcinomas compared with normal colonic mucosa.[36] Hence, consistent with the decrease in MT mRNA gene expression, the immunohistochemical protein expression also decreased which is reflected in the decrease in the MT content in colon carcinoma of DMH-treated rats. The statistically significant decrease of MT mRNA gene expression, immunopositivity, and MT content observed by us moving from normal to neoplastic colonic mucosa allows us to exclude the hypothesis of an overexpression of MT in colonic mucosa of the malignant type in our animal model.

The available information on the role of MT in colon cancer is very limited. The current results extend our previous observation, in which there was a substantial reduction in colonic tissue Zn (data shown to facilitate comparison) along with the development of colonic precancerous lesions and cancerous stage in the DMH-induced rats.[13] These biochemical alterations may be sequentially related to the molecular events that take place in the large intestine in DMH-treated rats. The significant decrease in the various biochemical parameters studied by us was found to be greater in colonic carcinogenesis than in the premalignant tissue.


 > Conclusion Top


This provides insights into the possible interaction between Zn and MT gene expression in colon cancer development. Based on our findings, it could be hypothesized that MT mRNA expression, protein expression, and MT content, all together strongly, indicates that down-regulation of MT takes place along with decrease in tissue Zn during progression to a precancerous stage and further to carcinogenesis. Additional studies are necessary to elucidate the underlying molecular mechanism(s) of the role of MT and Zn in colon carcinogenesis.

Acknowledgments

We would like to thank the Christian Medical College, Vellore, Tamil Nadu, India, for their financial support toward this work.

Financial support and sponsorship

Institutional Research Grant, Christian Medical College, Vellore, Tamil Nadu, India.

Conflicts of interest

There are no conflicts of interest.

 
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[TAG2]
[TAG3]
[TAG4]