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The Two Mono-Carbonyl Curcumin Analogs, PGV-1and CCA-1.1: The Chemopreventive Activity AgainstDMH-Induced Colorectal Cancer Rat and ProteinsTarget Candidate InvolvedFebri Wulandari 

Universitas Gadjah MadaMuthi' Ikawati 

Gadjah Mada University Faculty of Pharmacy: Universitas Gadjah Mada Fakultas FarmasiMitsunori Kirihata 

Osaka Prefecture UniversitySitarina Widyarini 

Gadjah Mada University Faculty of Animal Sciences: Universitas Gadjah Mada Fakultas PeternakanJun-Ya Kato 

Nara Institute of Science and Technology: Nara Sentan Kagaku Gijutsu Daigakuin DaigakuEdy Meiyanto  ( edy_meiyanto@ugm.ac.id )

Universitas Gadjah Mada https://orcid.org/0000-0002-0886-6322

Research Article

Keywords: PGV-1, CCA-1.1, DMH, colorectal cancer, chemopreventive

Posted Date: October 1st, 2021

DOI: https://doi.org/10.21203/rs.3.rs-950137/v1

License: This work is licensed under a Creative Commons Attribution 4.0 International License.  Read Full License

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AbstractPentagamavunone-1 and its newest derivatives, CCA-1.1, possess an outstanding cytotoxic activityagainst several cancer cell lines, especially colorectal cancer. We are continuing to explore the anti-colorectal cancer properties of PGV-1 and CCA-1.1 against DMH-induced colorectal cancer rats andexpound the potential protein target in colorectal adenocarcinoma. We utilized DMH 60 mg/kg(subcutaneous injection once a week for 16 weeks) to induce colorectal cancer. PGV-1 and CCA-1.1 at 10and 20 mg/kg (orally twice a week for 16 weeks) were co-administered with DMH. The WBC countincreased in a single DMH group and was countered by co-administration of PGV-1 and CCA-1.1, but nosigni�cant differences in RBC. Single DMH treatment for 16 weeks resulted in 80% adenocarcinoma. Incontrast, co-administration with PGV-1 and CCA-1.1 suppressed most of the carcinogenic characteristicsand symptoms of the pre-malignancy condition. Furthermore, bioinformatics analysis showed that CCA-1.1 has more speci�c targets than PGV-1, including CDK1, CDK2, MMP3, MMP14, and CYP3A4, whichregulate the cell cycle arrest, cancer cell migration, and xenobiotic metabolism, respectively. Interestingly,CCA-1.1 targets CYP3A4, which possibly interrupts DMH metabolism and prevents the initiation of DMH-colorectal carcinogenesis. In conclusion, CCA-1.1 performed better chemopreventive effects against DMHcolorectal cancer and might replace PGV-1 to be promoted as a more effective anti-colorectal canceragent. 

1. IntroductionColorectal adenocarcinoma (COAD) displays a high mortality risk despite the grownup technology fordiagnosis and treatment [1]. Chemotherapy remains the furthermost preference due to severalinconveniences and obstacles of surgery and radiation to treat accurately at the cancer site [2]. However,the conventional chemotherapeutic drugs for colorectal cancer such as 5-FU, paclitaxel, and epirubicinpossess numerous threats of side effects, recurrence, and metastasize induction, which subsequentlyimpacts on patient’s quality of life and survival [3]. Hence, an alternative or new therapeutic agent that ismore effective, comfortable, and less toxic to patients is urgently needed to improve colorectal cancertherapy.

Curcumin analog has been extensively studied for its anticancer activity in vitro and in vivo [4]. Two kindsof monocarbonyl curcumin analogs, Pentagamavunone-1 (PGV-1) and Chemopreventive CurcuminAnalog 1 (CCA-1.1) (Fig. 1A), were favorable as successor drug for colorectal cancer in several ways: (1)exhibiting irreversible cytotoxic effect in vitro against various cancer cell lines at a lower dose; (2)performing unique target on mitotic phase in the cell cycle; (3) effectively induce cellular senescence anddeath; (4) suppressing cancer migration in vitro culture system; and (5) inhibiting tumor growth in axenograft mouse model of leukemia and breast cancer [5–11]. Both compounds exhibit remarkablecytotoxic activities in various mechanisms such as apoptosis, senescence, autophagy, mitotic arrest, andROS level generation on colorectal cancer cells. Those data give us a foundation for further exploring itsmechanism in vivo system.

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We currently conducted an in vivo experiment of PGV-1 compared to CCA-1.1 against the DMH-inducedcolorectal cancer rat model. We observed the cancer incidences, possible safety of the compound basedon the body weight, blood pro�les, and histopathological appearances. In addition, we also examined thecytotoxic activity of these compounds in vitro using two kinds of colorectal cancer cells, as well asexploration of the potential protein target candidate in COAD using bioinformatics analysis. Hopefully,these results will provide meaningful data on the potency of PGV-1 and CCA-1.1 to develop as colorectalcancer drug candidates, and more speci�cally, we could determine the better suitable candidate of thetwo compounds.

2. Materials And Methods

2.1 ChemicalsPGV-1 and CCA-1.1 were obtained from the collection of CCRC, Faculty of Pharmacy UGM, Indonesia [9].

2.2 Cells CultureCaco-2 cells were maintained in RPMI medium; CT-26, RAW 264.7, and NIH-3T3 were maintained inDMEM medium (37°C in a 5% CO2) (Gibco, Invitrogen, USA). Both mediums are supplemented with 10%v/v FBS (Sigma, St. Louis, CA, USA), 150 IU/mL penicillin, and 150 µg/mL streptomycin (Gibco, Invitrogen,USA).

2.3 MTT assayCells (104 cells/well) were seeded into each well of 96-well plates and treated with various concentrationsof CCA-1.1 and PGV-1 for 24 h. After incubation, the media were discharged, and cells were washed withphosphate-buffered saline, then added the MTT reagent (Sigma, St. Louis, CA, USA) and incubated for 4h. Stopper reagent was added and incubated overnight, and then, the absorbances were measured on amicroplate reader (Bio-Rad) under λ = 595 nm.

2.4 Animals and treatment schedulesThirty-six male Wistar rats (150 – 200 g) were obtained from Gadjah Mada University animal house,divided into 6 treatment groups (Fig. 2A): (I) untreated; (II) DMH and Na-CMC 0.5% (colorectal cancergroup); and treatment group of co-administration of DMH with: (III) PGV-1 10 mg/kg BW, (IV) PGV-1 20mg/kg BW, (V) CCA-1.1 10 mg/kg BW and (VI) CCA-1.1 20 mg/kg BW. The body weight was recordedevery week. Animals were sacri�ced two weeks after all treatments were completed and the colon wascollected. Red blood count (RBC) and white blood count (WBC) [6] were measured using the AutomaticHematology Analyzer Sysmex KX-21. The nodules were recorded for analysis using equation 1 [12].

VN (mm3) = {NL(NW)2 × 0.52} .......................................................................................... (1)

where VN = volume of nodules, NL = nodule length, NW = nodule width.

2.5 Histopathological examination

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The colon organs were �xed in 10% Neutral Buffered Formalin Fixatives (Surgipath, Leica). The para�n-embedded tissues were cut 5 µm in serial sections using a microtome (Leica Microsystems Inc., BuffaloGrove, IL) and mounted to slides. Slides were then de-waxed by standard depara�nization with xylol andfollowed by step-by-step rehydration (100, 95, and 70 % ethanol). Hematoxylin & Eosin (HE) staining wasperformed by core facilities at the Pathological Anatomy laboratories, Faculty of Medicine, UniversitasGadjah Mada.

2.6 Bioinformatic exploration of PGV-1 and CCA-1.1 inCOADWe utilized the SwissTargetPrediction (http://www.swisstargetprediction.ch/) [13] to obtain the targetgenes of PGV-1 and CCA-1.1. Potential gene targets in COAD were obtained from GeneCards [14]. Venndiagram was used to overlap the genes and classi�ed its protein class through PANTHER v.16 (http://www. pantherdb.org/) [15]. The UALCAN database [16] was used to assess the expression pro�le ofcandidate target genes. We used the GEPIA tool [17] to predict the overall survival, using a Kaplan–Meiersurvival curve, applying the median cut-off and COAD dataset. The gene expression of colon cancersamples was categorized into high expression (with transcripts per million [TPM] values above themedian) and low/median expression (with TPM values below the median). We re-create the graph usingGraphPad Prism.

2.7 Statistical analysisData were presented as mean ± SEM (n = 3). We used GraphPad Prism software v6.0 (GraphPadSoftware, La Jolla, CA) for statistical analysis and visualization. The parametric data were analyzedusing Student’s t-test or one-way analysis of variance (ANOVA) with Tukey’s multiple comparison posthoc test (*p<0.01; *p<0.001; ns: not signi�cant). Then, the Kolmogorov-Smirnov chi-square test was usedfor non-parametric data.

3. Results

3.1 Cytotoxic effect of PGV-1 and CCA-1.1CCA-1.1 was more potent on colorectal cancer cells than PGV-1 but less toxic in non-cancerous cells(Fig. 1B, Table 1). In Caco-2 cells, CCA-1.1 exhibited a signi�cantly stronger cytotoxic effect than PGV-1with the IC50 values of 2.8 and 11.2 µM, respectively. CCA-1.1 also displayed a cytotoxic effect in lowerIC50 value (2 µM) in CT-26 cells. In addition, both compounds were less toxic against NIH-3T3 and RAW264.7 cells with IC50 values more than 50 µM (Fig. 1C, Table 1). CCA-1.1 exhibited selectivity index (SI)values of more than 17 and 25 in Caco-2 and CT-26 cells, respectively. In comparison, PGV-1 displayedthe CI value of more than 4 and 15 in Caco-2 and CT-26 cells, respectively. These �ndings suggest thatboth compounds have excellent selectivity and are considered safe chemotherapeutic agents, but CCA-1.1 is preferred.

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Table 1IC50 values of CCA-1.1 and PGV-1 on Caco-2, CT-26, NIH-

3T3, RAW 264.7, and CHO-K1 cells and its selectivityindex (SI)

Cell line IC50 values (µM)

Caco-2 CT-26 NIH-3T3 RAW 264.7

CCA-1.1 2.8 2 > 50 > 50

PGV-1 11.2 3.2 > 50 > 50

Cell line Selectivity Index (SI)

CCA-1.1 PGV-1

Caco-2 > 36 > 9

CT-26 > 50 > 31

  

3.2 Effect of PGV-1 and CCA-1.1 on body weight, growthrate, WBC, RBC, and tumor nodules of DMH-colorectalinduced rats

Subcutaneous injection of 60 mg/kg DMH signi�cantly decreased the body weight compared tountreated and other treatment groups (Fig. 2B). Body mass loss was prevented signi�cantly (*p<0.001)on co-administration of DMH with PGV-1 and CCA-1.1, especially at the dose of 20 mg/kg. There is nosigni�cant difference in WBC level between DMH + PGV-1/ CCA-1.1 treatment groups and untreatedgroups (Fig. 2B). CCA-1.1 most effectively countered the WBC elevation caused by DMH at the dose of 20mg/kg (**p<0.001). For the RBC level, we found no changes at all. Macroscopic examination of singleDMH and co-administration of DMH with PGV-1 or CCA-1.1 displayed �uctuating nodule formation invarious sizes (Fig. 2C). Colorectal wall thickening was also observed, accompanying nodule formation. Inmore detail, single administration of DMH induced signi�cantly higher (**p < 0.001) number and volumenodules than DMH + PGV-1/ CCA-1.1 (Fig. 2C). Here, CCA-1.1 at the 20 mg/kg dose was also performedas the most effective in inhibiting tumor development. These �ndings give us more detailed informationthat PGV-1 and CCA-1.1 could inhibit tumor progression in DMH-induced colorectal cancer rats withchemoprotective properties.

3.3 Effect of PGV-1 and CCA-1.1 on adenocarcinoma,aberrant crypt, and colitis incidence

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Since we used DMH as a colon carcinogen that exhibited multistep carcinogenesis involving a series ofpathological alterations [18], we then analyzed the incidence of adenocarcinoma, aberrant crypt, andcolitis-associated colorectal cancer of all treatment groups. We could characterize between normal (bluearrow) and multistep of colorectal carcinogenesis as adenocarcinoma (green arrow), aberrant crypts (redarrow), and colitis (yellow square) (Fig. 3A) from DMH-induction and how the treatment of PGV-1 andCCA-1.1 could prevent. Single DMH administration induced 80% adenocarcinoma developmentthroughout the colon. Co-administration with PGV-1 at the doses of 10 and 20 mg/kg BW reduced thepercentage of adenocarcinoma (incidence/ group) with the value of 20 and 40%, respectively (Fig. 3B).There is no adenocarcinoma formation on rats administered with DMH + CCA-1.1 at both doses. However,an early characteristic of colorectal carcinogenesis as the aberrant crypt and colitis-associated colorectalcancer in DMH + PGV-1/ CCA-1.1 groups was higher than the single DMH group. At the 20 mg/kg BWdose, CCA-1.1 is more e�cient in inhibiting colorectal carcinogenesis than PGV-1 at the same doses. Webelieve that adenocarcinoma formation is always accompanied by a multi-combination of aberrantcrypts, colitis, and in�ammation [19]. Still, we have not counted it as an individual case of aberrant cryptsor colitis and only focused on the representative pathological incidence of each colon/ rat to evaluate thecarcinogenic step of DMH induction. Overall, both PGV-1 and CCA-1.1 could suppress the carcinogenesisof DMH-induction in rats, particularly CCA-1.1.

 

3.4 Bioinformatic exploration of PGV-1 and CCA-1.1 onCOADFor further comprehension of PGV-1 and CCA-1.1 targets in COAD, bioinformatic analysis elucidate thatCCA-1.1 targets more genes than PGV-1 (Fig. 4A). The sixteen CCA-1.1 target genes were classi�ed into avariety and rich protein classes than the �ve target genes of PGV-1 (Fig. 4B). We then emphasize the �vegenes which are speci�cally involved in cancer metabolism (CYP3A4), cell proliferation (cell cycleregulator) (CDK1 and CDK2), and cell migration (MMP3 and MMP14) as the potential target of CCA-1.1.We found that CDK1, CDK2, MMP3, and MMP14 were signi�cantly overexpressed in tumors than innormal colon tissues (*p<0.01; **p<0.0001) (Fig. 4C). CYP3A4 was higher expressed in normal tissuethan in the tumor, con�rming that this gene plays a role as a xenobiotic metabolizer to counter thepathological cascade [20, 21]; in this case, DMH-carcinogenesis. On the other hand, the overall survivalwas not signi�cantly different between high and low expression of those respective genes for 150months span (Fig. 4C). Although not statistically different, COAD patients who had increased MMP14and CYP3A4 intended to have a higher risk of relapse than patients with lower expression. By looking atthis information, we believed that CCA-1.1 target genes in these results were unique and interesting pointsto explore and strongly correlated with in vitro and in vivo experiments.

 

4. Discussion

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Formerly, PGV-1 has been shown anti-tumorigenic activities against leukemia and breast cancer inxenograft mice model [6, 7], but has not been explored in colorectal cancer. While the other curcuminanalogs, pentagamavunone-0 (PGV-0), gamavutone-0 (GVT-0), and hexagamavunone-0 (HGV-0),exhibited anti-in�ammatory activity in the DMH-colorectal cancer rat model [12]. In this regard, wechallenge and continue to explore PGV-1 and CCA-1.1, a targeted and selective candidate for colorectalcancer chemotherapeutic agents through in vitro, in vivo, and bioinformatic exploration.

In the in vitro study, we con�rm that CCA-1.1 was more potent than PGV-1 and underlined with a superiorsafety level. Caco-2 and CT-26 colorectal cancer cells represent the COX-2 level in colorectal cancer sincethere was �uctuate expression of COX-2 in COAD patients [22, 23]. The different characteristics of the twocells that we used provide different responses for each cell. We found compelling evidence that PGV-1cellular response, in this case, cytotoxic activity, might be altered by COX-2 expression. Furthercon�rmation on the selectivity index of CCA-1.1 and PGV-1 indicate that both compounds are promisingcandidates of selective anticancer agents. It was also con�rmed by the WBC and RBC level in the in vivoexperiment that PGV-1 and CCA-1.1 might defeat tumor formation with fewer or no observable opposingeffects on the normal lineage of cells. The RBC and WBC pro�les could explain a slight pathologicalcondition of colorectal cancer, such as anemia, cruor, and in�ammation [1, 24]. The safety properties ofCCA-1.1 and PGV-1 are important to develop new anticancer agents. However, this study is still limited inthe normal proliferating immortal cells that should be evaluated in the normal primary cells as well as inthe in vivo model.

We performed the chemopreventive examination of PGV-1 and CCA-1.1 that simultaneously administeredwith DMH for inducing colorectal cancer, whether they were able to reverse, suppress, or prevent the initialphases of DMH carcinogenesis. We believed that the study of in vivo colorectal carcinogenesis had aremarkably long journal accompanying the development of diagnostic, pathway mechanism, and therapyof cancer [25]. For the inducing agent, we used DMH as a procarcinogen that metabolizes to a methyl freeradical and generates hydroxyl radicals of metal ions resulting in lipid peroxidation, molecular geneticalteration, and cancer initiation [26]. We found that co-administration of PGV-1 and CCA-1.1 in both doses(10; 20 mg/kg) with DMH were adequate to suppress tumor growth with noticeable toxic effects to thenormal lineage cells caused by DMH, particularly CCA-1.1 at 20 mg/kg. The mechanism may, in partconcern with the in vitro anticancer activities of PGV-1 and CCA-1.1, including induction of apoptosis,targeted cell cycle arrest, related cancer marker protein inhibition, and selective on cancer cells [7, 8].Then, PGV-1 and CCA-1.1 may play a role as free radical eliminators through enzymatic antioxidantssince curcumin analogs are known as dual-oxidant on their anticancer features [27]. This phenomenonshould be an exciting point to further exploration related to their antioxidant activities.

Bioinformatic exploration was con�rmed that CCA-1.1 was more potent than PGV-1. The proteins targetcandidate of CCA-1.1 in COAD was richer than PGV-1, validating previous in vitro observation of CCA-1.1that report several activities such as apoptosis, anti-migration/ metastasis, and speci�c G2/M cell cyclearrest [11, 28–32]. We underlined the two proteins CDK1 and CDK2, which play a speci�c role in the G1/Sand G2/M cell cycle stage, respectively [33]. Those two proteins might become an essential protein in the

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anticancer effect of CCA-1.1 in the �eld of cell proliferation and cell cycle regulation on colorectal cancer.On the other hand, targeting MMP3 and MMP14 [34] might also underline the better effect on anti-migration of CCA-1.1 than PGV-1. CCA-1.1 targets CYP3A4 should be highlighted as superior to PGV-1. Bytargeting this protein, CCA-1.1 possibly suppressed or inhibited DMH-carcinogenesis since DMH is mainlymetabolized into toxic metabolites by CYPs in the liver, including CYP3A4 [20, 21]. We believed that this�nding could emphasize the superiority of CCA-1.1 compared to PGV-1 to develop as a colorectal cancerchemotherapeutic candidate. An additional laboratory experiment is required for a better and detailedcomprehension of the mechanism.

Since we used the DMH-induced colorectal cancer model, we could also observe an early pre-neoplastichyperproliferative lesion that formed aberrant crypt foci (ACF) as intermediary indicators ofcarcinogenesis [35]. Here, we focused on the late and early stages of colorectal carcinogenesis to de�nethe effect of suppression or inhibition of PGV-1 and CCA-1.1. We found that single DMH induced latestage of colorectal cancer, which is adenocarcinoma with severe alteration of goblet cell’s structure,in�ammation, and invasive growth of cancer cells. However, co-administration of DMH with PGV-1/ CCA-1.1 induced an early stage of colorectal carcinogenesis, such as aberrant crypt [36] and colitis-associatedcolorectal cancer (severe in�ammation and hemorrhagic) [37], without adenocarcinoma formation,especially CCA-1.1 at both doses. These �ndings indicated that PGV-1 and CCA-1.1 could suppress orinhibit the carcinogenesis of DMH in rats. It should also be superior to a new anti-colorectal cancer due toits solubility, stability, and effectiveness, especially in an acidic environment since both compounds wereadministered orally to rats. However, several conventional chemotherapeutic drugs are still limited for oraluse due to their stability in the alimentary tract [38–40]. In this study, we used a chemopreventive settingon the treatment schedule, in which DMH was co-administered with PGV-1/ CCA-1.1 at the same period.Further exploration using another treatment design would be great to establish the detailed mechanismof those compounds and the possibility to pharmaceutically developed as orally administered drugs forcolorectal cancer.

To our knowledge, it is the �rst time that in vivo exploration of PGV-1 and CCA-1.1 against colorectalcancer has been conducted along with in vitro and bioinformatic exploration. These �ndings revealed agreat potential of PGV-1 and CCA-1.1 to develop as anti-colorectal cancer agents. However, the optimumdose to give as well as the treatment setting may not be answered yet. A deeper investigation is needed tosettle down the potency of PGV-1 and CCA-1.1 as anti-colorectal cancer agents.

5. ConclusionOral co-administration of PGV-1 and CCA-1.1 (10 and 20 mg/kg) for 16 weeks signi�cantly inhibitedcolorectal carcinogenesis, compared to single DMH and untreated groups without toxicity in normallineage cells. The suppressing effect of PGV-1 on COAD development is suggested to target signaltransduction and cell cycle, whereas CCA-1.1 targets cell cycle, signal transduction, cell migration, andcarcinogen metabolism. CCA-1.1 performed a better chemopreventive effect as a new anti-colorectalcancer candidate against DMH colorectal cancer than PGV-1, possibly supported by the additional target

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on CYP3A4. In general, both compounds exhibited strong chemopreventive potential against colorectalcancer.

DeclarationsFUNDING 

This study was supported by the Master Program of Education Leading to Doctoral Degree for ExcellentGraduates (PMDSU), Ministry of Research and Technology and Higher Education, the Republic ofIndonesia, for funding this research.

AUTHORS’ CONTRIBUTIONS

Conception and design: E.M., M.I., M.K., and J.K. Acquisition of data: F.W. Analysis and interpretation ofdata: E.M., M.I., S.W, and F.W. Writing and review of the manuscript: F.W., E.M., and M.I. Administrative andtechnical support: F.W.

COMPLIANCE WITH ETHICAL STANDARDS

Declaration of interest

The authors report no con�icts of interest.

Ethical approval 

This article contain study with animals approved by the Ethics Committee of Gadjah Mada UniversityIndonesia (Number. 00001/04/LPPT/I/2020).

Consent to participate

Not applicable

Consent for publication

Not applicable

Availability of data and material

Not applicable

Code availability

Not applicable

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Figures

Figure 1

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The cytotoxic effects of PGV-1 and CCA-1.1 against several cell lines. (A) Chemical structure of PGV-1and CCA-1.1. (B) Growth inhibitory pro�les of PGV-1 and CCA-1.1 against Caco-2 and CT-26 colorectalcancer cells. (C) Growth inhibitory pro�les of PGV-1 and CCA-1.1 against NIH-3T3 and RAW 264.7 non-cancerous cells.

Figure 2

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Co-administration of PGV-1 and CCA-1.1 suppressed the carcinogenic symptoms of DMH. (A) Schematictreatment schedule of experiment. (B) Body weight and blood pro�les (WBC and RBC) of each treatmentgroup. (C) Macroscopic appearance and nodules calculation (number and volume) of each treatmentgroup. All data presented as mean ± SEM; *p < 0.01, **p < 0.001 compared to DMH-induced colorectalcancer rat.

Figure 3

PGV-1 and CCA-1.1 suppressed most of the pre-malignancy condition of DMH induction. (A) Microscopicexamination of colon tissues using H&E. Representative images of the normal tissue, colonadenocarcinoma, aberrant crypt, and colitis appearance (400x). Blue arrows indicate normal cryptarchitecture. Green arrows indicate destroyed crypts and green square indicate an invasiveadenocarcinoma. Red arrows indicate aberrant crypts as early step of colon-adenoma formation. Yellowsquares indicate in�ammation-associated colitis. (B) % of adenocarcinoma, aberrant crypt, and colitisincidence/ group (n = 6).

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Figure 4

Bioinformatic exploration of CCA-1.1 and PGV-1. (A) Target genes of CCA-1.1 and PGV-1 overlapped withcolorectal adenocarcinoma (COAD) biomarkers from the GeneCards. (B) The pie chart of CCA-1.1 andPGV-1 target genes classi�cation based on their protein class using PANTHER. (C) Expression and overallsurvival of several target genes in COAD obtained from the UALCAN website (http://ualcan.path.uab.edu)based on TCGA dataset.

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Figure 5

Proposed mechanism of PGV-1 and CCA-1.1 in DMH-colorectal cancer.