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Original article
Upregulation of MAPK/Erk and PI3K/Akt pathways in ulcerative colitis-associated colon cancer Shruti Setia, Bimla Nehru, Sankar Nath Sanyal * Department of Biophysics, Panjab University, Chandigarh 160014, India
A R T I C L E I N F O
A B S T R A C T
Article history: Received 8 August 2014 Accepted 14 September 2014
An extracellular signal like a cytokine or chemokine, secreted in the inflammatory microenvironment can activate the mitogen activated protein kinase (MAPK) pathway by binding to a cytokine receptor tyrosine kinase, which further activates tyrosine kinases such as Janus Kinase-3 (Jak-3). This signal is transferred from Jak-3 to the DNA in the nucleus of the cell by a chain of kinases, ultimately activating extracellular receptor kinase (Erk/MAPK). The latter phosphorylates c-myc, an oncogene, which alters the levels and activities of many transcription factors leading to cell survival, proliferation and invasion. The oncogenic PI3K pathway plays a similar role by activating c-myc, leading to cell survival and proliferation. The present study explores the role of ulcerative colitis in colon cancer by investigating the activities of tyrosine kinase activated MAPK pathway and various components of the PI3K pathway including PI3K, PTEN, PDK1, GSK3b, Akt, mTOR, Wnt and b-catenin. This was done by western blot and fluorescent immunohistochemical analysis of the above-mentioned proteins. Also, the morphological and histological investigation of the colonic samples from various animal groups revealed significant alterations as compared to the control in both inflammatory as well as carcinogenic conditions. These effects were reduced to a large extent by the co-administration of celecoxib, a second-generation nonsteroidal anti-inflammatory drug (NSAID). ß 2014 Elsevier Masson SAS. All rights reserved.
Keywords: Celecoxib Colon cancer MAPK PI3K pathway Tyrosine kinase Ulcerative colitis
1. Introduction Chronic inflammation develops through the action of various inflammatory mediators like cytokines, chemokines and growth factors, which eradicate anti-tumor immunity and facilitate tumor progression [1]. TNF-a, a cytokine, has been found to be involved in all the stages of carcinogenesis such as cellular transformation, promotion, survival, proliferation, angiogenesis and metastasis [2]. Our previous studies have shown that the presence of such cytokines in the inflammatory milieu might lead to the transformation of cytoplasmic inactive transcription factor, nuclear factor kB (NF-kB) to its active nuclear form, thereby leading to tumorigenesis [2]. Presently, we attempt to look further into the Abbreviations: DSS, dextran sulfate sodium; DMH, 1,2 dimethyl hydrazine; Erk, extracellular receptor kinase; GSK3b, glycogen synthase kinase 3b; Jak-3, Janus kinase-3; MAPK, mitogen activated protein kinase; MPL, multiple plaque lesions; NSAID, non-steroidal anti-inflammatory drug; PI3K, phosphatidylinositol-3 kinase; PTEN, phosphatase and tensin homolog. * Corresponding author. Department of Biophysics, Panjab University, Chandigarh 160014, India. Tel.: +91 172 2534122. E-mail address: [email protected] (S.N. Sanyal).
aspects of inflammation associated colon cancer by studying the tyrosine kinases activated MAPK/Erk pathway. The latter is well known to facilitate the upregulation of several oncogenic agents including the PI3K pathway [3]. Tumor-related inflammation is an important hallmark of cancer. It contributes to almost every aspect of tumor development [4]. Several epidemiological evidences show that chronic inflammation of the colon (ulcerative colitis) may lead to colon carcinoma [5]. The microenvironment, in case of ulcerative colitis, secretes a large number of proinflammatory cytokines like TNF-a, IL-4 and IL-1b [6]. These cytokines may act as extracellular growth factors to activate a chain of kinases, beginning with a tyrosine kinase, Jak3 [7]. This Jak-3 further activates a series of kinases, ultimately activating Erk. Erk is responsible for cell survival, proliferation and invasion by further interacting with the oncogenic Phosphatidylinositol-3 kinase (PI3K) pathway [8]. Both the RAS-Erk and PI3K/Akt pathways are found to be activated in tumorigenesis. PI3K is a lipid kinase and generates phosphoinositol-3 phosphate (PIP3) while this reaction is reversed by phosphatase and tensin homolog (PTEN), the negative regulator of this oncogenic PI3K pathway [9]. PIP3 is a second messenger for the translocation of Akt to the
http://dx.doi.org/10.1016/j.biopha.2014.09.006 0753-3322/ß 2014 Elsevier Masson SAS. All rights reserved.
Please cite this article in press as: Setia S, et al. Upregulation of MAPK/Erk and PI3K/Akt pathways in ulcerative colitis-associated colon cancer. Biomed Pharmacother (2014), http://dx.doi.org/10.1016/j.biopha.2014.09.006
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plasma membrane where it is phosphorylated and activated by PDK1. Activation of Akt plays a pivotal role in the fundamental cellular functions such as cell proliferation and survival by phosphorylating its downstream substrates like Glycogen synthase kinase 3b (GSK3b) and thereby inactivating it [10]. GSK3b in its active form doesn’t allow the activation of bcatenin [11]. But when inactivated, b-catenin complex is degraded and b-catenin is phosphorylated, it moves to the nucleus and induces the transcription of several cell survival and proliferation related proteins [12]. Upon the inactivation of GSK3b, another oncogenic pathway led by Wnt activates b-catenin [13]. The present study incorporated the use of an inflammation inducing agent, dextran sulfate sodium (DSS) and a carcinogenic agent 1,2 dimethyl hydrazine (DMH) in Balb/c mice, individually as well as in combination, to develop animal model for ulcerative colitis, colon carcinoma as well as colitis-associated colon cancer, respectively. Also, the chemoprevention of these diseases was studied by the administration of a second-generation NSAID, celecoxib. Numerous epidemiological studies have reported that the long-term use of NSAIDs is associated with a significant decrease in cancer incidence and delayed progression of the malignant disease [14]. The expression of Cox-2 and prostaglandins has been associated with various types of cancer and is directly proportional to their tissue aggressiveness including metastasis [15]. NSAIDs act primarily by the inhibition of the Cox enzyme [16]. But how these NSAIDs act in case of ulcerative colitis and prevention of colitis-associated colon cancer is still not known. We presently make an attempt at revealing the mechanism underlying colitis-associated colon cancer treatment with NSAIDs.
the principles and guidelines of the Ethics Committee on the use of experimental animals of Panjab University and with approved protocol. They were housed in polypropylene cages with a wire mesh top and a regularly changed husk bed with a maximum of 6– 8 animals in each cage. The animal rooms were maintained at ambient temperature and provided with a room cooler or heater during the summer or winter months, respectively. The animals received food (rodent chow) and water ad libitum and were exposed to 12 h day/night photoperiod. They were acclimatized for 1 week and then assorted. 2.2. Experimental design The present study consisted of eight groups: control, which received vehicle for NSAIDs, (0.5% (w/v) carboxymethyl cellulose per oral daily), vehicle for DSS (distilled water during three weeks of DSS administration in group 2) and DMH (subcutaneous injection of 1 mM EDTA-saline); DSS, the animals received three cycles of 3% DSS in distilled water, each for seven days followed by 14 days of tap water, to establish a model of ulcerative colitis [17]; DMH, 18 weekly s.c. injections at 30 mg/kg to develop a model for colon carcinogenesis, as established earlier in our laboratory [18]; celecoxib, 6 mg/kg as a chemopreventive agent; DMH + DSS; DSS + Cel; DMH + Cel; DMH + DSS + Cel in the above-mentioned respective doses.
2. Materials and methods 2.1. Animal husbandry Balb/c mice were procured from the Central Animal House, Panjab University, Chandigarh. Animals were maintained as per
This has been summarized in Fig. 1, where blue boxes represent DSS treatment, black arrow represents a single s.c. injection of DMH and green colored boxes show celecoxib administration p.o. daily (for interpretation of the references to color in this figure legend, the reader is referred to the web version of this article).
Fig. 1. Experimental design: control group is treated with the vehicle for DSS, DMH and DSS + DMH. The DSS group received three cycles of 3% DSS each for one week followed by two weeks of distilled water. The DMH group received one s.c. injection of 30 mg/kg DMH in 1 mM EDTA-saline per week for 18 weeks. The celecoxib group received 0.6 mg/kg celecoxib in 0.5% CMC p.o. daily. The other groups received different combinations of DSS, DMH and celecoxib.
Please cite this article in press as: Setia S, et al. Upregulation of MAPK/Erk and PI3K/Akt pathways in ulcerative colitis-associated colon cancer. Biomed Pharmacother (2014), http://dx.doi.org/10.1016/j.biopha.2014.09.006
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2.3. Morphological analysis The animals were sacrificed with an excess of diethyl ether anesthesia. Their colons were removed and flushed clear with icecold physiological saline (0.9% NaCl). These were cut longitudinally to examine the presence of any macroscopic lesions, called multiple plaque lesions (MPL). The colons were also divided into proximal, middle and distal segments for subsequent examination and counting. 2.4. Histological analysis Isolated colons were fixed in 10% buffered formalin for 24 h, then dehydrated and further embedded in paraffin wax according to the standard technique for histopathology [19], and five micron thick sections were cut using a hand driven microtome and transferred to egg albumin coated slides. Sections were then dewaxed in xylene, stained in hematoxylin and eosin, mounted in DPX, and viewed and photographed under a light microscope (40 ) (Axioscope A1, Zeiss, Germany), attached with a digital camera (Jenoptik, Thuringia, Germany). 2.5. Immunofluorescent staining Deparaffinized sections were washed in 100 and 95% alcohol thrice for 10 min each. After washing in the deionized water, these were heated at 95 8C in 50 mM glycine-EDTA buffer (pH 3.5) with 0.01% (w/v) EDTA to retrieve the antigen. The slides were then cooled and washed with distilled water before incubating them with 10% normal blocking serum in PBS for 30 min. The sections were incubated with primary antibodies (Jak-3, p-stat-3, p-erk, pAkt, mTOR, b-catenin and PTEN in the ratio 1:1000) from Santa Cruz Biotechnology Inc., CA, USA for 60 min in 1.5% BSA. These were given three changes of PBS for 5 min each followed by incubation for 60 min with FITC-conjugated secondary antibody
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(1: 10,000) from Genei (Bangalore, India), again washed with PBS as described earlier. Counter staining of the slides was done with 1 mg/mL propidium iodide (Sigma, St. Louis, MO, USA) for 20 min at 37 8C. After washing thrice with PBS, the sections were mounted with aqueous media (1:10 glycerol in PBS), and the coverslip was sealed with nail paint. The sections were observed under a fluorescent microscope ( 40) (Axiocscope A1, Zeiss, Germany). 2.6. Western blots The procedure for protein extraction was based on the method of Sambrook et al. [20]. The estimation of protein content was done according to the Bradford method [21]. Fifty micrograms of protein samples from each treatment group were extracted, which was separated on 12% SDS-PAGE, and then transferred electrophoretically to nitrocellulose membrane (Genei, Bangalore, India). Ponceau S (Merck, Mumbai, India) dye was used to check the transfer of the protein. Blot was developed using primary antibodies (Jak-3, p-erk, PI3K, PDK1, GSK-3b, mTOR, Wnt, bcatenin, all in the ratio 1:1000 and b-actin- 1:10,000) from Santa Cruz Biotechnology Inc., CA, USA. The alkaline-phospatase-labelled secondary antibodies from Genei, Bangalore, India were used at a dilution of 1:10,000. BCIP/NBT solution was used to develop the bands, which were analyzed densitometrically using Image J software (NIH, Bethesda, MD, USA), after normalizing with b-actin. 2.7. Statistical analysis For analyzing the data, analysis of variance (one-way ANOVA) test was performed using the statistical software package ‘‘SPSS v 14 for windows’’. The post-hoc comparisons of means from different groups were made by the Duncan’s test, where the preliminary analysis of variance indicated significant treatment effects. Differences between the means were considered significant at P < 0.01.
Fig. 2. Morphological analysis: no MPL were seen in the Control, DSS, celecoxib and DSS + Cel groups. Angiogenesis is evident in DMH and DSS + DMH + Cel groups by the presence of blood vessels. Also small boxes show the presence of MPL in these groups and DMH + Cel group. The elliptical circle in DSS + DMH group shows the occurrence of blood clotting with DSS treatment while the small circles show carcinogenic lesions.
Please cite this article in press as: Setia S, et al. Upregulation of MAPK/Erk and PI3K/Akt pathways in ulcerative colitis-associated colon cancer. Biomed Pharmacother (2014), http://dx.doi.org/10.1016/j.biopha.2014.09.006
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Table 1 Evaluation of chemopreventive response of celecoxib in mice examined after 18 weeks of treatment schedule. Animal groups
No. of MPLs in different regions of colon Proximal
Middle
Distal
Control DSS DMH Cel DMH + DSS DSS + Cel DMH + Cel D + D + Cel
– – 4 – 5 – 2 3
– – 5 – 6 – 2 3
– – 10 – 14 – 4 6
Total MPL
No. of mice with MPL/Total no. of mice
– – 19 – 25 – 9 12
No MPL No MPL 9/9 No MPL 10/10 No MPL 6/10 7/10
MPL Incidence (%)
Burden
Multiplicity
– – 100 – 100 – 60 70
– – 2.1 – 2.5 – 1.5 1.71
– – 2.1 – 2.5 – 1.5 1.71
MPL incidence: it is the percentage of animals bearing lesions; MPL multiplicity: it is the total no. of lesions/no. of lesion bearing mice; MPL burden: it is the total no. of lesions/ total no. of mice.
3. Theory Jak-3 activated MAPK and PI3K pathways play a crucial role in the transition from inflammation to cancer. Celecoxib is found to significantly downregulate the expression of several agents of these pathways. The work is further extended to the gene analysis of the respective protein products in these oncogenic pathways by reverse transcription-PCR, which will form part of the future publication.
generation of a blood vessel (evidence of angiogenesis), were seen in the DMH group. Maximum morphological insult was found in the 5th group (DSS + DMH), where we observed blood clots due to the bleeding (marker of DSS treatment) in the colon along with the presence of MPL due to DMH treatment. Celecoxib co-administration along with the DMH and DSS + DMH treatment led to the reduction of these MPL (Fig. 2) where 100% incidence in these groups was reduced to 60 and 70%, respectively (Table 1). 4.2. Histological analysis
4. Results 4.1. Morphological analysis Normal morphological features were observed in control, DSS, celeoxib and DSS + celecoxib groups. Visible MPL, along with the
Control and celecoxib groups were marked by normal histoarchitecture with crypts embedded in the stroma. Crypts contained large number of mucus secreting goblet cells. This layer of mucosa lay above the submucosa, which was supported by the muscularis mucosa. The administration of DSS led to the drastic reduction of the number of goblet cells in the crypts as well as the
Fig. 3. Histological analysis: the Control and celecoxib groups show normal histoarchitecture with intact epithelium and normal crypts filled with goblet cells. The DSS group shows crypt loss and distorted epithelium along with the formation of lymphoid aggregate (la). The DMH group shows hyperproliferative crypts leading to adenocarcinoma (ac). In the DSS + DMH group, binary fission (bf) of the crypts is seen along with lymphoid aggregates (la). These features were improved to a certain extent with the administration of celecoxib in these groups.
Please cite this article in press as: Setia S, et al. Upregulation of MAPK/Erk and PI3K/Akt pathways in ulcerative colitis-associated colon cancer. Biomed Pharmacother (2014), http://dx.doi.org/10.1016/j.biopha.2014.09.006
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cryptic size and numbers. The signs of inflammation were evident from the lymphoid aggregation and the thickening of submucosa. No such inflammatory features were seen in the groups without DSS treatment. While well-differentiated adenoma with darkly stained pycnotic nuclei was seen in the DMH treated group. The 5th
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group (DSS + DMH) showed both the signs of inflammation as well as adenoma, as was seen by the lymphoid aggregation, thickening of submucosa, loss of cryptic size and number, and binary fission of the crypts. The co-administration of celecoxib along with DSS, DMH and DMH + DSS significantly reduced these features. (Fig. 3).
Fig. 4. a–e. Jak-3 activated Erk pathway: the immunofluorescent expressions of Jak-3, p-Stat-3 and p-Erk were found to be aggravated in DSS, DMH and DSS + DMH groups as seen by the green color of FITC labeled secondary antibody. The counterstained nuclei were red/orange in color due to propidium iodide staining. (d) The protein expression of Jak-3, as studied by western blotting was also found to be elevated in the above three groups. These were corrected to a significant extent by the co-administration of celecoxib in these groups. (e) Graphical representation of the densitometric analysis of this blot (a, b, c, d, e mean in each segment not sharing a common superscript letter differed significantly (Duncan’s test): P < 0.01) (for interpretation of the references to color in this figure legend, the reader is referred to the web version of this article).
Please cite this article in press as: Setia S, et al. Upregulation of MAPK/Erk and PI3K/Akt pathways in ulcerative colitis-associated colon cancer. Biomed Pharmacother (2014), http://dx.doi.org/10.1016/j.biopha.2014.09.006
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4.3. Jak-3 activated ERK pathway Cytokines secreted in the inflammatory microenvironment might lead to the activation of tyrosine kinases like Jak-3, which are otherwise activated by NF-kB in the carcinogenic conditions. This Jak-3 initiates the activation of a cascade of kinases including ERK, which further acts parallel to the oncogenic PI3K pathway and also generates cell survival and anti-apoptotic signals. Jak-3 also activates its downstream growth factor Stat-3, whose phosphorylated active form enters the nucleus and is a point of convergence for numerous oncogenic signaling pathways. We presently studied the protein expression of Jak-3, p-Stat-3 and p-ERK by immunofluoresence and western blots and found it to be elevated in the trinity groups: DSS, DMH and DSS + DMH, although celecoxib coadministration significantly lowered these levels (Fig. 4a–e). 4.4. ERK associated PI3K/Akt/PTEN and Wnt/b-catenin pathway The present study explored several components of the PI3K pathway and the immunofluorescent and protein expression of PI3K, p-Akt, PDK1, mTOR, Wnt and b-catenin were found to be
elevated while the expressions of PTEN, the negative regulator of this pathway, and GSK3b, a tumor suppressor, were lowered in DSS, DMH and DSS + DMH groups. These levels were brought towards normal with the co-administration of the celecoxib in these groups (Fig. 5a-f). 5. Discussion Through a network of proteins, cells respond to the changes in their environmental milieu. This network can be split into many signaling pathways. These pathways carry signals from the environment to the cellular components involved in several pathologies including cancer [22]. Extracellular factors such as cytokines and chemokines secreted by inflammatory microenvironment bind to an RTK. Upon binding, an RTK dimerizes and autophosphorylates its C-terminal region [23]. The resulting phospho-tyrosine residues of Jak-3 serve to activate the MAPK/ Erk pathway through a series of kinases including Ras, Raf and MEK (MAPKK) [24,25]. Recent studies have indicated the RTK/ERK pathway might be a key pathway in the development of prostate cancer [26]. But their role in colitis-associated colon cancer is still
Fig. 5. ERK associated PI3K/Akt/PTEN and Wnt/b-catenin pathway: (a) shows the protein expression of various components of PI3K/Akt/PTEN pathway as studied by western blots. An upregulation of PI3K, PDK1, p-Akt, mTOR, WNT and b-catenin and downregulation of GSK-3b were observed in the DSS, DMH and DSS + DMH groups while these effects were brought towards normal by the simultaneous administration of celecoxib. (b) Graphical representation of the densitometric analysis of these blots (a, b, c, d, e, f mean in each segment not sharing a common superscript letter differed significantly (Duncan’s test): P < 0.01). (c–f) The immunofluorescent analysis of p-Akt, mTOR and bcatenin shows their elevated expression while that of PTEN is reduced in DSS, DMH and DSS + DMH groups. These effects were reduced significantly by the co-administration of celecoxib in these groups.
Please cite this article in press as: Setia S, et al. Upregulation of MAPK/Erk and PI3K/Akt pathways in ulcerative colitis-associated colon cancer. Biomed Pharmacother (2014), http://dx.doi.org/10.1016/j.biopha.2014.09.006
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not known. To identify the role of Erk in colitis-associated colon carcinoma, we studied the protein expression of Jak-3 and p-Erk. Also, Jak-3 is the physiological activator of signal transducer and activator of transcription 3 (Stat 3), a transcription factor with known oncogenic potential [27]. Activated Stat-3 (p-Stat 3) has been proposed to be a novel molecular target for therapeutic intervention in malignant neoplasms [28]. Recent literatures support the inhibition of Jak-3 signaling leading to alleviation of inflammation [29]. Stat 3 is known to interact with mToR, a pleiotropic serine/threonine protein kinase that regulates cell growth, cell proliferation, cell motility, cell survival, protein synthesis, and transcription [30]. We took an insight into this oncogenic Jak-3/stat 3 signaling and found elevated levels of protein expression of jak-3, p-stat 3 and mTOR. The Ras/Erk and PI3K/Akt/PTEN signaling pathways are the principal mechanisms for controlling cell survival, differentiation, proliferation, metabolism, and motility in response to extracellular signals from the inflammatory or tumorigenic microenvironment [31]. Both of these pathways are commonly thought to have antiapoptotic and drug resistant effects on cells. The Ras/Erk pathway has been reported to be activated in over 50% of acute myelogenous leukemia and acute lymphocytic leukemia, and is also frequently activated in other cancer types [32]. Induced Raf expression can abrogate the cytokine dependence of certain hematopoietic cell lines, a trait associated with tumorigenesis. Also, expression of activated PI3K or Akt has positive effects on cell survival. Several components of these pathways are mutated or aberrantly expressed in human cancer (e.g., Ras, B-Raf, PI3K, PTEN, Akt) [33]. We presently studied the protein expression of several components of PI3K/Akt/PTEN pathway in colitis-associated colon cancer and found elevated levels of PI3K, Akt, mTOR, PDK1 and bcatenin in the DSS, DMH and DSS + DMH groups. The protein expressions of PTEN as well as GSK-3b were reduced in these groups. Also, Wnt pathway is found to be activated under tumorigenic conditions and Wnt expression was also upregulated in the above three groups. These effects were brought towards normal by the co-administration of celecoxib in these groups. 6. Conclusion We can, therefore, conclude that both Ras/Erk and PI3K/Akt/ PTEN pathways act parallel in case of inflammation-mediated tumorigenesis. Also celecoxib, a second-generation NSAID, when co-administered with DSS, DMH and their combinatorial group, was found to suppress both Ras/Erk and PI3K/Akt/PTEN pathways. Disclosure of interest The authors declare that they have no conflicts of interest concerning this article. Acknowledgement Financial assistance from the INSPIRE scheme, Department of Science and Technology, Government of India (IF-110462) is gratefully acknowledged. References [1] Colotta F, Allavena P, Sica A, Garlanda C, Mantovani A. Cancer-related inflammation, the seventh hallmark of cancer: links to genetic instability. Carcinogenesis 2009;30:1073–81. [2] Setia S, Nehru B, Sanyal SN. Activation of NF-kB: bridging the gap between inflammation and cancer in colitis-mediated colon carcinogenesis. Biomed Pharmacother 2014;68:119–28. [3] Shahbazian D, Roux PP, Mieulet V, Cohen MS, Raught B, Taunton J, et al. The mTOR/PI3K and MAPK pathways converge on eIF4B to control its phosphorylation and activity. EMBO J 2006;25:2781–91.
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Please cite this article in press as: Setia S, et al. Upregulation of MAPK/Erk and PI3K/Akt pathways in ulcerative colitis-associated colon cancer. Biomed Pharmacother (2014), http://dx.doi.org/10.1016/j.biopha.2014.09.006
BIOPHA-3449; No. of Pages 7 Biomedicine & Pharmacotherapy xxx (2014) xxx–xxx
Available online at
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Original article
Upregulation of MAPK/Erk and PI3K/Akt pathways in ulcerative colitis-associated colon cancer Shruti Setia, Bimla Nehru, Sankar Nath Sanyal * Department of Biophysics, Panjab University, Chandigarh 160014, India
A R T I C L E I N F O
A B S T R A C T
Article history: Received 8 August 2014 Accepted 14 September 2014
An extracellular signal like a cytokine or chemokine, secreted in the inflammatory microenvironment can activate the mitogen activated protein kinase (MAPK) pathway by binding to a cytokine receptor tyrosine kinase, which further activates tyrosine kinases such as Janus Kinase-3 (Jak-3). This signal is transferred from Jak-3 to the DNA in the nucleus of the cell by a chain of kinases, ultimately activating extracellular receptor kinase (Erk/MAPK). The latter phosphorylates c-myc, an oncogene, which alters the levels and activities of many transcription factors leading to cell survival, proliferation and invasion. The oncogenic PI3K pathway plays a similar role by activating c-myc, leading to cell survival and proliferation. The present study explores the role of ulcerative colitis in colon cancer by investigating the activities of tyrosine kinase activated MAPK pathway and various components of the PI3K pathway including PI3K, PTEN, PDK1, GSK3b, Akt, mTOR, Wnt and b-catenin. This was done by western blot and fluorescent immunohistochemical analysis of the above-mentioned proteins. Also, the morphological and histological investigation of the colonic samples from various animal groups revealed significant alterations as compared to the control in both inflammatory as well as carcinogenic conditions. These effects were reduced to a large extent by the co-administration of celecoxib, a second-generation nonsteroidal anti-inflammatory drug (NSAID). ß 2014 Elsevier Masson SAS. All rights reserved.
Keywords: Celecoxib Colon cancer MAPK PI3K pathway Tyrosine kinase Ulcerative colitis
1. Introduction Chronic inflammation develops through the action of various inflammatory mediators like cytokines, chemokines and growth factors, which eradicate anti-tumor immunity and facilitate tumor progression [1]. TNF-a, a cytokine, has been found to be involved in all the stages of carcinogenesis such as cellular transformation, promotion, survival, proliferation, angiogenesis and metastasis [2]. Our previous studies have shown that the presence of such cytokines in the inflammatory milieu might lead to the transformation of cytoplasmic inactive transcription factor, nuclear factor kB (NF-kB) to its active nuclear form, thereby leading to tumorigenesis [2]. Presently, we attempt to look further into the Abbreviations: DSS, dextran sulfate sodium; DMH, 1,2 dimethyl hydrazine; Erk, extracellular receptor kinase; GSK3b, glycogen synthase kinase 3b; Jak-3, Janus kinase-3; MAPK, mitogen activated protein kinase; MPL, multiple plaque lesions; NSAID, non-steroidal anti-inflammatory drug; PI3K, phosphatidylinositol-3 kinase; PTEN, phosphatase and tensin homolog. * Corresponding author. Department of Biophysics, Panjab University, Chandigarh 160014, India. Tel.: +91 172 2534122. E-mail address: [email protected] (S.N. Sanyal).
aspects of inflammation associated colon cancer by studying the tyrosine kinases activated MAPK/Erk pathway. The latter is well known to facilitate the upregulation of several oncogenic agents including the PI3K pathway [3]. Tumor-related inflammation is an important hallmark of cancer. It contributes to almost every aspect of tumor development [4]. Several epidemiological evidences show that chronic inflammation of the colon (ulcerative colitis) may lead to colon carcinoma [5]. The microenvironment, in case of ulcerative colitis, secretes a large number of proinflammatory cytokines like TNF-a, IL-4 and IL-1b [6]. These cytokines may act as extracellular growth factors to activate a chain of kinases, beginning with a tyrosine kinase, Jak3 [7]. This Jak-3 further activates a series of kinases, ultimately activating Erk. Erk is responsible for cell survival, proliferation and invasion by further interacting with the oncogenic Phosphatidylinositol-3 kinase (PI3K) pathway [8]. Both the RAS-Erk and PI3K/Akt pathways are found to be activated in tumorigenesis. PI3K is a lipid kinase and generates phosphoinositol-3 phosphate (PIP3) while this reaction is reversed by phosphatase and tensin homolog (PTEN), the negative regulator of this oncogenic PI3K pathway [9]. PIP3 is a second messenger for the translocation of Akt to the
http://dx.doi.org/10.1016/j.biopha.2014.09.006 0753-3322/ß 2014 Elsevier Masson SAS. All rights reserved.
Please cite this article in press as: Setia S, et al. Upregulation of MAPK/Erk and PI3K/Akt pathways in ulcerative colitis-associated colon cancer. Biomed Pharmacother (2014), http://dx.doi.org/10.1016/j.biopha.2014.09.006
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plasma membrane where it is phosphorylated and activated by PDK1. Activation of Akt plays a pivotal role in the fundamental cellular functions such as cell proliferation and survival by phosphorylating its downstream substrates like Glycogen synthase kinase 3b (GSK3b) and thereby inactivating it [10]. GSK3b in its active form doesn’t allow the activation of bcatenin [11]. But when inactivated, b-catenin complex is degraded and b-catenin is phosphorylated, it moves to the nucleus and induces the transcription of several cell survival and proliferation related proteins [12]. Upon the inactivation of GSK3b, another oncogenic pathway led by Wnt activates b-catenin [13]. The present study incorporated the use of an inflammation inducing agent, dextran sulfate sodium (DSS) and a carcinogenic agent 1,2 dimethyl hydrazine (DMH) in Balb/c mice, individually as well as in combination, to develop animal model for ulcerative colitis, colon carcinoma as well as colitis-associated colon cancer, respectively. Also, the chemoprevention of these diseases was studied by the administration of a second-generation NSAID, celecoxib. Numerous epidemiological studies have reported that the long-term use of NSAIDs is associated with a significant decrease in cancer incidence and delayed progression of the malignant disease [14]. The expression of Cox-2 and prostaglandins has been associated with various types of cancer and is directly proportional to their tissue aggressiveness including metastasis [15]. NSAIDs act primarily by the inhibition of the Cox enzyme [16]. But how these NSAIDs act in case of ulcerative colitis and prevention of colitis-associated colon cancer is still not known. We presently make an attempt at revealing the mechanism underlying colitis-associated colon cancer treatment with NSAIDs.
the principles and guidelines of the Ethics Committee on the use of experimental animals of Panjab University and with approved protocol. They were housed in polypropylene cages with a wire mesh top and a regularly changed husk bed with a maximum of 6– 8 animals in each cage. The animal rooms were maintained at ambient temperature and provided with a room cooler or heater during the summer or winter months, respectively. The animals received food (rodent chow) and water ad libitum and were exposed to 12 h day/night photoperiod. They were acclimatized for 1 week and then assorted. 2.2. Experimental design The present study consisted of eight groups: control, which received vehicle for NSAIDs, (0.5% (w/v) carboxymethyl cellulose per oral daily), vehicle for DSS (distilled water during three weeks of DSS administration in group 2) and DMH (subcutaneous injection of 1 mM EDTA-saline); DSS, the animals received three cycles of 3% DSS in distilled water, each for seven days followed by 14 days of tap water, to establish a model of ulcerative colitis [17]; DMH, 18 weekly s.c. injections at 30 mg/kg to develop a model for colon carcinogenesis, as established earlier in our laboratory [18]; celecoxib, 6 mg/kg as a chemopreventive agent; DMH + DSS; DSS + Cel; DMH + Cel; DMH + DSS + Cel in the above-mentioned respective doses.
2. Materials and methods 2.1. Animal husbandry Balb/c mice were procured from the Central Animal House, Panjab University, Chandigarh. Animals were maintained as per
This has been summarized in Fig. 1, where blue boxes represent DSS treatment, black arrow represents a single s.c. injection of DMH and green colored boxes show celecoxib administration p.o. daily (for interpretation of the references to color in this figure legend, the reader is referred to the web version of this article).
Fig. 1. Experimental design: control group is treated with the vehicle for DSS, DMH and DSS + DMH. The DSS group received three cycles of 3% DSS each for one week followed by two weeks of distilled water. The DMH group received one s.c. injection of 30 mg/kg DMH in 1 mM EDTA-saline per week for 18 weeks. The celecoxib group received 0.6 mg/kg celecoxib in 0.5% CMC p.o. daily. The other groups received different combinations of DSS, DMH and celecoxib.
Please cite this article in press as: Setia S, et al. Upregulation of MAPK/Erk and PI3K/Akt pathways in ulcerative colitis-associated colon cancer. Biomed Pharmacother (2014), http://dx.doi.org/10.1016/j.biopha.2014.09.006
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2.3. Morphological analysis The animals were sacrificed with an excess of diethyl ether anesthesia. Their colons were removed and flushed clear with icecold physiological saline (0.9% NaCl). These were cut longitudinally to examine the presence of any macroscopic lesions, called multiple plaque lesions (MPL). The colons were also divided into proximal, middle and distal segments for subsequent examination and counting. 2.4. Histological analysis Isolated colons were fixed in 10% buffered formalin for 24 h, then dehydrated and further embedded in paraffin wax according to the standard technique for histopathology [19], and five micron thick sections were cut using a hand driven microtome and transferred to egg albumin coated slides. Sections were then dewaxed in xylene, stained in hematoxylin and eosin, mounted in DPX, and viewed and photographed under a light microscope (40 ) (Axioscope A1, Zeiss, Germany), attached with a digital camera (Jenoptik, Thuringia, Germany). 2.5. Immunofluorescent staining Deparaffinized sections were washed in 100 and 95% alcohol thrice for 10 min each. After washing in the deionized water, these were heated at 95 8C in 50 mM glycine-EDTA buffer (pH 3.5) with 0.01% (w/v) EDTA to retrieve the antigen. The slides were then cooled and washed with distilled water before incubating them with 10% normal blocking serum in PBS for 30 min. The sections were incubated with primary antibodies (Jak-3, p-stat-3, p-erk, pAkt, mTOR, b-catenin and PTEN in the ratio 1:1000) from Santa Cruz Biotechnology Inc., CA, USA for 60 min in 1.5% BSA. These were given three changes of PBS for 5 min each followed by incubation for 60 min with FITC-conjugated secondary antibody
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(1: 10,000) from Genei (Bangalore, India), again washed with PBS as described earlier. Counter staining of the slides was done with 1 mg/mL propidium iodide (Sigma, St. Louis, MO, USA) for 20 min at 37 8C. After washing thrice with PBS, the sections were mounted with aqueous media (1:10 glycerol in PBS), and the coverslip was sealed with nail paint. The sections were observed under a fluorescent microscope ( 40) (Axiocscope A1, Zeiss, Germany). 2.6. Western blots The procedure for protein extraction was based on the method of Sambrook et al. [20]. The estimation of protein content was done according to the Bradford method [21]. Fifty micrograms of protein samples from each treatment group were extracted, which was separated on 12% SDS-PAGE, and then transferred electrophoretically to nitrocellulose membrane (Genei, Bangalore, India). Ponceau S (Merck, Mumbai, India) dye was used to check the transfer of the protein. Blot was developed using primary antibodies (Jak-3, p-erk, PI3K, PDK1, GSK-3b, mTOR, Wnt, bcatenin, all in the ratio 1:1000 and b-actin- 1:10,000) from Santa Cruz Biotechnology Inc., CA, USA. The alkaline-phospatase-labelled secondary antibodies from Genei, Bangalore, India were used at a dilution of 1:10,000. BCIP/NBT solution was used to develop the bands, which were analyzed densitometrically using Image J software (NIH, Bethesda, MD, USA), after normalizing with b-actin. 2.7. Statistical analysis For analyzing the data, analysis of variance (one-way ANOVA) test was performed using the statistical software package ‘‘SPSS v 14 for windows’’. The post-hoc comparisons of means from different groups were made by the Duncan’s test, where the preliminary analysis of variance indicated significant treatment effects. Differences between the means were considered significant at P < 0.01.
Fig. 2. Morphological analysis: no MPL were seen in the Control, DSS, celecoxib and DSS + Cel groups. Angiogenesis is evident in DMH and DSS + DMH + Cel groups by the presence of blood vessels. Also small boxes show the presence of MPL in these groups and DMH + Cel group. The elliptical circle in DSS + DMH group shows the occurrence of blood clotting with DSS treatment while the small circles show carcinogenic lesions.
Please cite this article in press as: Setia S, et al. Upregulation of MAPK/Erk and PI3K/Akt pathways in ulcerative colitis-associated colon cancer. Biomed Pharmacother (2014), http://dx.doi.org/10.1016/j.biopha.2014.09.006
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Table 1 Evaluation of chemopreventive response of celecoxib in mice examined after 18 weeks of treatment schedule. Animal groups
No. of MPLs in different regions of colon Proximal
Middle
Distal
Control DSS DMH Cel DMH + DSS DSS + Cel DMH + Cel D + D + Cel
– – 4 – 5 – 2 3
– – 5 – 6 – 2 3
– – 10 – 14 – 4 6
Total MPL
No. of mice with MPL/Total no. of mice
– – 19 – 25 – 9 12
No MPL No MPL 9/9 No MPL 10/10 No MPL 6/10 7/10
MPL Incidence (%)
Burden
Multiplicity
– – 100 – 100 – 60 70
– – 2.1 – 2.5 – 1.5 1.71
– – 2.1 – 2.5 – 1.5 1.71
MPL incidence: it is the percentage of animals bearing lesions; MPL multiplicity: it is the total no. of lesions/no. of lesion bearing mice; MPL burden: it is the total no. of lesions/ total no. of mice.
3. Theory Jak-3 activated MAPK and PI3K pathways play a crucial role in the transition from inflammation to cancer. Celecoxib is found to significantly downregulate the expression of several agents of these pathways. The work is further extended to the gene analysis of the respective protein products in these oncogenic pathways by reverse transcription-PCR, which will form part of the future publication.
generation of a blood vessel (evidence of angiogenesis), were seen in the DMH group. Maximum morphological insult was found in the 5th group (DSS + DMH), where we observed blood clots due to the bleeding (marker of DSS treatment) in the colon along with the presence of MPL due to DMH treatment. Celecoxib co-administration along with the DMH and DSS + DMH treatment led to the reduction of these MPL (Fig. 2) where 100% incidence in these groups was reduced to 60 and 70%, respectively (Table 1). 4.2. Histological analysis
4. Results 4.1. Morphological analysis Normal morphological features were observed in control, DSS, celeoxib and DSS + celecoxib groups. Visible MPL, along with the
Control and celecoxib groups were marked by normal histoarchitecture with crypts embedded in the stroma. Crypts contained large number of mucus secreting goblet cells. This layer of mucosa lay above the submucosa, which was supported by the muscularis mucosa. The administration of DSS led to the drastic reduction of the number of goblet cells in the crypts as well as the
Fig. 3. Histological analysis: the Control and celecoxib groups show normal histoarchitecture with intact epithelium and normal crypts filled with goblet cells. The DSS group shows crypt loss and distorted epithelium along with the formation of lymphoid aggregate (la). The DMH group shows hyperproliferative crypts leading to adenocarcinoma (ac). In the DSS + DMH group, binary fission (bf) of the crypts is seen along with lymphoid aggregates (la). These features were improved to a certain extent with the administration of celecoxib in these groups.
Please cite this article in press as: Setia S, et al. Upregulation of MAPK/Erk and PI3K/Akt pathways in ulcerative colitis-associated colon cancer. Biomed Pharmacother (2014), http://dx.doi.org/10.1016/j.biopha.2014.09.006
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cryptic size and numbers. The signs of inflammation were evident from the lymphoid aggregation and the thickening of submucosa. No such inflammatory features were seen in the groups without DSS treatment. While well-differentiated adenoma with darkly stained pycnotic nuclei was seen in the DMH treated group. The 5th
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group (DSS + DMH) showed both the signs of inflammation as well as adenoma, as was seen by the lymphoid aggregation, thickening of submucosa, loss of cryptic size and number, and binary fission of the crypts. The co-administration of celecoxib along with DSS, DMH and DMH + DSS significantly reduced these features. (Fig. 3).
Fig. 4. a–e. Jak-3 activated Erk pathway: the immunofluorescent expressions of Jak-3, p-Stat-3 and p-Erk were found to be aggravated in DSS, DMH and DSS + DMH groups as seen by the green color of FITC labeled secondary antibody. The counterstained nuclei were red/orange in color due to propidium iodide staining. (d) The protein expression of Jak-3, as studied by western blotting was also found to be elevated in the above three groups. These were corrected to a significant extent by the co-administration of celecoxib in these groups. (e) Graphical representation of the densitometric analysis of this blot (a, b, c, d, e mean in each segment not sharing a common superscript letter differed significantly (Duncan’s test): P < 0.01) (for interpretation of the references to color in this figure legend, the reader is referred to the web version of this article).
Please cite this article in press as: Setia S, et al. Upregulation of MAPK/Erk and PI3K/Akt pathways in ulcerative colitis-associated colon cancer. Biomed Pharmacother (2014), http://dx.doi.org/10.1016/j.biopha.2014.09.006
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4.3. Jak-3 activated ERK pathway Cytokines secreted in the inflammatory microenvironment might lead to the activation of tyrosine kinases like Jak-3, which are otherwise activated by NF-kB in the carcinogenic conditions. This Jak-3 initiates the activation of a cascade of kinases including ERK, which further acts parallel to the oncogenic PI3K pathway and also generates cell survival and anti-apoptotic signals. Jak-3 also activates its downstream growth factor Stat-3, whose phosphorylated active form enters the nucleus and is a point of convergence for numerous oncogenic signaling pathways. We presently studied the protein expression of Jak-3, p-Stat-3 and p-ERK by immunofluoresence and western blots and found it to be elevated in the trinity groups: DSS, DMH and DSS + DMH, although celecoxib coadministration significantly lowered these levels (Fig. 4a–e). 4.4. ERK associated PI3K/Akt/PTEN and Wnt/b-catenin pathway The present study explored several components of the PI3K pathway and the immunofluorescent and protein expression of PI3K, p-Akt, PDK1, mTOR, Wnt and b-catenin were found to be
elevated while the expressions of PTEN, the negative regulator of this pathway, and GSK3b, a tumor suppressor, were lowered in DSS, DMH and DSS + DMH groups. These levels were brought towards normal with the co-administration of the celecoxib in these groups (Fig. 5a-f). 5. Discussion Through a network of proteins, cells respond to the changes in their environmental milieu. This network can be split into many signaling pathways. These pathways carry signals from the environment to the cellular components involved in several pathologies including cancer [22]. Extracellular factors such as cytokines and chemokines secreted by inflammatory microenvironment bind to an RTK. Upon binding, an RTK dimerizes and autophosphorylates its C-terminal region [23]. The resulting phospho-tyrosine residues of Jak-3 serve to activate the MAPK/ Erk pathway through a series of kinases including Ras, Raf and MEK (MAPKK) [24,25]. Recent studies have indicated the RTK/ERK pathway might be a key pathway in the development of prostate cancer [26]. But their role in colitis-associated colon cancer is still
Fig. 5. ERK associated PI3K/Akt/PTEN and Wnt/b-catenin pathway: (a) shows the protein expression of various components of PI3K/Akt/PTEN pathway as studied by western blots. An upregulation of PI3K, PDK1, p-Akt, mTOR, WNT and b-catenin and downregulation of GSK-3b were observed in the DSS, DMH and DSS + DMH groups while these effects were brought towards normal by the simultaneous administration of celecoxib. (b) Graphical representation of the densitometric analysis of these blots (a, b, c, d, e, f mean in each segment not sharing a common superscript letter differed significantly (Duncan’s test): P < 0.01). (c–f) The immunofluorescent analysis of p-Akt, mTOR and bcatenin shows their elevated expression while that of PTEN is reduced in DSS, DMH and DSS + DMH groups. These effects were reduced significantly by the co-administration of celecoxib in these groups.
Please cite this article in press as: Setia S, et al. Upregulation of MAPK/Erk and PI3K/Akt pathways in ulcerative colitis-associated colon cancer. Biomed Pharmacother (2014), http://dx.doi.org/10.1016/j.biopha.2014.09.006
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not known. To identify the role of Erk in colitis-associated colon carcinoma, we studied the protein expression of Jak-3 and p-Erk. Also, Jak-3 is the physiological activator of signal transducer and activator of transcription 3 (Stat 3), a transcription factor with known oncogenic potential [27]. Activated Stat-3 (p-Stat 3) has been proposed to be a novel molecular target for therapeutic intervention in malignant neoplasms [28]. Recent literatures support the inhibition of Jak-3 signaling leading to alleviation of inflammation [29]. Stat 3 is known to interact with mToR, a pleiotropic serine/threonine protein kinase that regulates cell growth, cell proliferation, cell motility, cell survival, protein synthesis, and transcription [30]. We took an insight into this oncogenic Jak-3/stat 3 signaling and found elevated levels of protein expression of jak-3, p-stat 3 and mTOR. The Ras/Erk and PI3K/Akt/PTEN signaling pathways are the principal mechanisms for controlling cell survival, differentiation, proliferation, metabolism, and motility in response to extracellular signals from the inflammatory or tumorigenic microenvironment [31]. Both of these pathways are commonly thought to have antiapoptotic and drug resistant effects on cells. The Ras/Erk pathway has been reported to be activated in over 50% of acute myelogenous leukemia and acute lymphocytic leukemia, and is also frequently activated in other cancer types [32]. Induced Raf expression can abrogate the cytokine dependence of certain hematopoietic cell lines, a trait associated with tumorigenesis. Also, expression of activated PI3K or Akt has positive effects on cell survival. Several components of these pathways are mutated or aberrantly expressed in human cancer (e.g., Ras, B-Raf, PI3K, PTEN, Akt) [33]. We presently studied the protein expression of several components of PI3K/Akt/PTEN pathway in colitis-associated colon cancer and found elevated levels of PI3K, Akt, mTOR, PDK1 and bcatenin in the DSS, DMH and DSS + DMH groups. The protein expressions of PTEN as well as GSK-3b were reduced in these groups. Also, Wnt pathway is found to be activated under tumorigenic conditions and Wnt expression was also upregulated in the above three groups. These effects were brought towards normal by the co-administration of celecoxib in these groups. 6. Conclusion We can, therefore, conclude that both Ras/Erk and PI3K/Akt/ PTEN pathways act parallel in case of inflammation-mediated tumorigenesis. Also celecoxib, a second-generation NSAID, when co-administered with DSS, DMH and their combinatorial group, was found to suppress both Ras/Erk and PI3K/Akt/PTEN pathways. Disclosure of interest The authors declare that they have no conflicts of interest concerning this article. Acknowledgement Financial assistance from the INSPIRE scheme, Department of Science and Technology, Government of India (IF-110462) is gratefully acknowledged. References [1] Colotta F, Allavena P, Sica A, Garlanda C, Mantovani A. Cancer-related inflammation, the seventh hallmark of cancer: links to genetic instability. Carcinogenesis 2009;30:1073–81. [2] Setia S, Nehru B, Sanyal SN. Activation of NF-kB: bridging the gap between inflammation and cancer in colitis-mediated colon carcinogenesis. Biomed Pharmacother 2014;68:119–28. [3] Shahbazian D, Roux PP, Mieulet V, Cohen MS, Raught B, Taunton J, et al. The mTOR/PI3K and MAPK pathways converge on eIF4B to control its phosphorylation and activity. EMBO J 2006;25:2781–91.
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Please cite this article in press as: Setia S, et al. Upregulation of MAPK/Erk and PI3K/Akt pathways in ulcerative colitis-associated colon cancer. Biomed Pharmacother (2014), http://dx.doi.org/10.1016/j.biopha.2014.09.006
