Folic Acid supplementary reduce the incidence of adenocarcinoma in a mouse model of colorectal cancer: microarray gene expression profile

It is known that colorectal cancer (CRC) is one of the most common cancers especially in western countries, referred to a multiple process, multiple factors with high recurrence and high mortality [1]. Chemoprevention methods for CRC have obtained increasing attention as surgery and chemotherapy strategies perform little function once diagnosed to be tumor that invades the muscularis propria. Also, the Non-steroidal anti-inflammatory drugs (NSAIDs), such as COX-2 inhibitors, are not always successful, and may have some harmful side-effects [2]. Generally, clinical trials require at least 3-5 years follow up and a large number of patients are difficult to control their lifestyles such as smoking and wine intake which may affect the incidence of cancer [3, 4]. Therefore, we choose animal model induced by chemistry drugs 1, 2-dimethylhydrazine (DMH) to simulate the formation of CRC. As azoxymethane (AOM) or 1, 2-dimethylhydrazine (DMH)-induced colon carcinogenesis in mice or rat have been identified as a useful tool [5‐9]. In the previous study, we have successfully induced CRC in this model using ICR mice [9].

Folic Acid (FA) is one kind of water-solubility vitamin, which has been believed to be chemo-preventive agent that can provide methy-group to DNA thus impact DNA synthesis and DNA methylation [10]. Abbreviations in DNA synthesis often lead to DNA mutation, DNA strand break and the impairment of DNA repair, which finally result in cancer formation [11].

However, there are many conflicting data about whether FA can inhibit or promote colorectal adenoma (CRA) from clinical or preclinical studies. Epidemiologic study shows that folic acid is significant associated with lower risk and not related to the increased risk of colorectal cancer, supporting folic acid as a protective role for colon mucosa [12, 13], including several large prospective studies in 99,523 participants in the American Cancer Prevention Study II (CPS-II) Nutrition Cohort [12] and in double-blind, randomized clinical trial (RCT) conducted by 9 clinical centers incorporating 1091 participants for 3 years follow up [13]. However, the Aspirin/Folate Polyp Prevention Trial demonstrated that about 67% increased risk of advanced lesions with high malignant potential, and an increased risk of having multiple adenomas among the folic acid supplementation group by providing folic acid for 6 years at 1 mg/d [14]. While other researches have reported that there is no relation or positive association between folic acid supplementation and the risk of colon adenoma [15]. Therefore, a systematic description from RCTs investigating the relation between folic acid supplementation and the risk of colorectal cancer was conducted by many groups. One recent Meta-analysis data revealed that folic acid supplementary for 3 years had no effect on the adenoma recurrence while had an increased risk of adenoma lesion for those who received folic acid over 3 years [16]. Another Meta-analysis divided the RCTs into different groups including populations with a history of adenoma and with an-average risk populations. They concluded that the evidence that folic acid was effective in the chemoprevention of colorectal cancer was not enough in both populations [17].

Further, many researchers consider that the role of folic acid might be two-sided, that is to prevent in early phage of adenoma formation and to promote in late stage depending on the time of folic acid administration. Preclinical studies have suggested that folic acid may only protect against the development of CRC in normal colon-rectum rather in mucosa with an Aberrant Crypt Foci (ACF) status [18], which is the earliest pre-neoplastic lesion that can be recognized based on the morphology and pathology features [19, 20], and the results were consistent with an AOM induced rat model of CRC [21]. These experiments demonstrated that folic acid had dual effects on the development of CRC depending on the timing and dose of the intervention of folic acid [11] However, the function that folic acid may perform to the exiting adenomas in chemicals induced mouse model and the possible mechanism is still un-established now.

In this study, we use ICR mice with 1, 2-Dimethylhydrazine (DMH) interfered models to analyze the impact of folic acid on different timing courses during the processes of CRC. We have previously demonstrated that 4 weeks old ICR mice given high dosage (8 mg/ml) folic acid for 20 weeks have much more apparent effects to prevent CRC incidence than low folic acid dosage (4 mg/kg bodyweight) group using DMH-induced mice model [9]. Therefore, to investigate the role of folic acid in the process of adenoma formation, we use the dose of 8 mg/kg bodyweight. Meanwhile, we inferred that the occurrence of adenoma may take place at the course of 12 weeks based on the performance of mice in previously study, so we designed the 12^th week as the division of the prophase or advanced stage of CRC. The study is expected to guide the clinical application of folic acid and to identify the mechanism of folic acid in a microarray gene expression profile.

In this analysis with a DMH-induced CRC model, we concluded that the supplementation of folic acid can decrease the risk of CRC and the subgroup of providing folic acid without precancerous lesions was more effective than that with precancerous lesions. Significantly, there was a reduction in the tumor mass diameter and multiplicity in folate supplementation group. Moreover, the study is consistent with many other studies either in rodent models or clinical medical researches. Recently, a study that investigated 2299 incidents and 5655 CRA in Nurses' Health Study and Health Professionals Follow-Up Study showed that folic acid intake 12-16 y before diagnosis was inversely associated with CRC and identified the latency that folic acid should be provided. However, the study didn't analyze the results that folic acid was provided after diagnosis [22]. With the same kind of chemical in a rat model of CRC, folate deficiency was found to enhance the development of neoplasia compared to the diets containing 8 mg/kg folic acid [21] but the study had no related mechanisms. However, some studies observed the opposite results. Le Leu [23] believed that folate deficiency can decrease the development of the intestinal tumors in AOM-induced SD-rat model. To this point, we think that the animal strain, experimental condition, experiment skills, folic acid manufactories, folic acid intervention time et al may contribute to these differences in varies studies. Also, there is a possibility that excessive intake of folic acid could have promoted the growth of pre-neoplastic lesions so that our study support that enteroscope should be conducted for the cases in clinical studies before incorporated.

On the other hand, there are still no significant differences in the incidence of cancers between group FA2 and FA3 even though the maximum diameter and the number of the tumor mass are significantly decreased in FA3 group. It may be due to too small number of mice or too much difference among individuals. In another respect, not all the mice had adenomas in the 12^th week as the incidence was only 10% among DMH1 group. So, further study should extend the number of samples to get more objective results.

Next, we use microarray gene expression profile analysis to study the mechanism of folic acid-mediated prevention of colon tumors and the difference in folic acid intervention time. To our knowledge, this is the first investigation to use microarray technology to study the role of folic acid in the prevention of CRC and the difference of folic acid intervention times.

Firstly, when the FC was set to ≥ 1.5, 642 genes that changed with the treatment of DMH could be reversed with folic acid supplementary. We selected 5 known tumor-related genes i.e., K-ras, c-MYC, DNMT1, Tpd52, CDKN1b for PCR confirmation [Figure 3]. It is known that genetic alterations may contribute substantially to the pathogenesis of colon cancer. Point mutation of K-ras (occurring in 40% of sporadic CRCs) is an established predictor of absence of response to epidermal growth factor receptor (EGFR) -targeted agents [24, 25]. Hutchins [26] reported that KRAS mutant tumors were more evenly distributed: 40% right colon, 28% left colon, and 36% rectal tumors compared to BRAF mutant tumors. Meanwhile, the relationship between Folic acid and KRAS has been studied. Some suggested that the effect of folate on rectal cancer risk is different to men and women which may depend on the status of K-ras mutation of tumors. They believed that folate intake was related to a decreased risk of G > A transitions (RR-0.08, 95% CI = 0.01-0.53) while an inversely risk of G > T and G > C transversions in tumors (RR = 2.69, 95% CI = 1.43-5.09)[27].

CDKN1b (cyclin-dependent kinase inhibitor 1B, FC = 7.992979) which is also known as p27 encodes a protein which belongs to the Cip/Kip family of cyclin dependent kinase (Cdk) inhibitor proteins [28] It is often considered as a cell cycle inhibitor protein because its major function is to control the cell cycle progression at G1 phase so that can prevent the development of cancer. Reduced p27 levels were found in different cancerous stages in hepatocelluar carcinomas [29]. Some studies demonstrated that loss of p27 expression is associated with a higher response rate to CRC chemo-therapy [30]. The p27KIP1 null (-/-) mouse shows a significant increase in cell proliferation, resulting in approximately 30% increase in mass size, multiple organ hyperplasia [31]. Together, these researches supported p27 as an important tumor suppressor and suggest that events leading to p27 upregulation may inhibit the tumor progression.

The methylation of genomic DNA in malignant cells is catalyzed by DNA methytransferases(DNMT)which include maintenance DNA methyltransferase (Dnmt1), DNMT1, de novo DNA methyltransferases (Dnmt3a and 3b), 3a/3b. DNA methylation is an important form of epigenetic that can regulate some gene expression such as c-Myc, CDKN2a, CDH1 and VDR et al [32‐34]. We have seen that the expression of DNMT1 was increased in FA3 compared to DMH, which is consistent with the research that the folate - and methyl-deficient diet alters components of the DNA methylation via both transcriptional and posttranscriptional mechanisms in in livers of F344 rats [35]. Meanwhile, some methylation-related genes that are functional in carcinogenesis can also be regulated by folic acid in terms of DNA methylation [36].

Tumor necrosis factor receptor superfamily, member 12a (Tnfrsf12a), also known as fn14 or TWEAK-R have been implicated in a variety of pathological processes including chronic inflammation and cancers [37]. And fn14 expression is at a relative lower level in normal tissues while much higher in cancer cells or tissues [38]. Kawashima [39] reported that IL-13 may damage the mucosa of colon via the function of TWEAK and Fn14 pathway and Fn14 could aggravate intestinal inflammation in patients with UC. So the relation between fn14 and diseases might suggest fn14 and TWEAK are targets for cancer therapy [37]. In our study, Tnfrsf12a's expression is 2.5 fold changes higher in FA2 group than FA3, which may be explained that the degree of colon mucosal damage in FA2 was much worse and was prone to develop to cancers compared to FA3. In this aspect, the high expression of fn14 may contribute to the growth of masses in FA2 group.

Vitamin D Receptor gene (VDR) is involved in the progress of cancers or chronic diseases [40]. Some argued that the polymorphism of VDR and CDX2 was not associated with increased risk of CRCs [41]. While others suggested that significant associations with VDR polymorphisms was found not in colorectal cancers but much stronger in cancers of breast, prostate and renal cell carcinomas [42]. And the association between VDR polymorphisms and folic acid has not been reported yet. In another respect, VDR is considered to be an epithelial marker in the process of Epithelial to mesenchymal transition (EMT) and thus might have a suppressive function of invasion [43]. Therefore, the expression of many tumor suppressors such as VDR was much lower (FC = 0.3010) compared with group FA2 and FA3, which was opposite to oncogenes.

However, there are some limitations of our study should be mentioned. First, we ignored the usage of the B Vitamins in the animal experiment, which is important in the process of Folic acid' transport and storage in liver. Therefore, Folic acid supplements may sometimes include vitamin B12 supplements with simultaneous administration of vitamin B12 [22]. However, some studies do not think there are any influences exiting with or without vitamin B12 [44]. Others even found that treatment with folic acid plus vitamin B(12) was associated with increased cancer outcomes [45]. Thus, consideration should be given to the potential value of providing with or without vitamin B12 in addition to the current mandatory folic acid supplementation.

Second, since folic acid is important in many processes of metabolism and might help to protect against the cardiovascular, mental diseases, cancer and birth defects [46]. However, we have no indicators to find other adverse effects but to observe the injury of organs in this study. Even though there are no abnormalities discovered in other organs except colon and rectum, the function of folic acid is needed to be further studied in terms of being effective to therapy.

Finally, although some similarities do exist between chemical rodent models of colon cancer and human natural CRCs, several respects of differs may also exist indeed. For example, the dose and duration of folic acid supplementation used in our study may be different from human studies. So, considering the safety of chemoprevention in clinical application, the optimal researches should be established in humans based on these findings with an initial colonoscopy before incorporated.

In summary, for the first time, our data suggest that folic acid supplementary in pre-cancerous era is much more protective than that in post-cancerous stage in a DMH induced mouse model and identify differential genes that folic acid can reversed and that between groups of pre or post-adenoma induced by folic acid using microarray gene expression profile. Not only to the reason that floate supplementation facilitates the progression of (pre)neoplastic lesions though providing nucleotide precursors to the rapidly replicating transformed cells, thus accelerating proliferation [11]. We also clarified that in gene expression profile, certain oncogenes that promote tumor growth, cell cycle, cell invasion such as TNFRSF12A, fibronectin 1, Cdca7 are high expressed in FA2 group compared to FA3 group while tumor suppressors are down-regulated such as VDR, CDX2, which may partly explain the result. However, the mechanism why folic acid provided in different phages can change these genes' expression remains to be studied.