1. Introduction
Primary liver cancer is the fifth most common cancer in the world, and the third common cause of cancer mortality. According to previous scientific research, chronic hepatitis B viral infection is the primal cause of hepatocellular carcinoma (HCC) in Asia and Africa. Recently, growing evidence indicates that there may be an relationship between the factors of environment or nutrition with the development of HCC [
1,
2]. The levels of plasma folate and vitamin B
12 maybe one of the important factors. Folate and vitamin B
12 as water-soluble B vitamins, which are involved in the one-carbon metabolism are essential for DNA methylation, synthesis and repair during cell regeneration. Therefore, recently, the function of folate and vitamin B
12 in carcinogenesis has attracted more attention from researchers [
3,
4,
5].
The folate, as a natural anti-cancer vitamin, is found to induce the apotosis of cancer cells and affect the gene expression of cancer cells [
6,
7]. Vitamin B
12 involved in one-carbon metabolism is dependent on methylation of homocysteine to form methionine by methionine synthase. Therefore, we propose that vitamin B
12 contributes to an alteration of plasma homocysteine level and folate status, which may be important in tumorigenesis.
Liver is the major site of storage and metabolism for folate and vitamin B
12, which are involved in the methylation and synthesis of macromolecules. Recent animal experiment showed a significant reduction in global DNA methylation with folate deficient diet, which promotes the initiation of carcinogenesis. Given the importance of folate in DNA methylation and synthesis, chronic folate deficiency may induce genome-wide DNA hypomethylation [
8,
9]. In addition, a recent study reported that approximately 60% of HCC patients were deficient in folate and folate levels decreased drastically as HCC progressed [
10]. Previous studies also proved that folate depletion and elevated plasma vitamin B
12 for other reasons, such as oxidative stress, hepatitis and alcoholic liver disease [
11,
12,
13], may lead to neoplasia and HCC development.
Based on the above, we hypothesized that human plasma folate and vitamin B12 levels might be related to HCC development. Therefore, a case-control study (including 312 HCC cases and 325 controls) was designed to evaluate the effect of plasma folate and vitamin B12 on the development and clinical pathological parameters of HCC, including tumor stage, tumor size and tumor markers.
3. Discussion
Folate, as a methyl donor in the synthesis of methionine, has been shown to mediate carcinogenesis by participating in DNA synthesis, repair, and methylation. The methionine cycle that provides one-carbon moiety for cellular methylation reactions is also dependent on vitamin B12. However, the epidemiological evidence regarding associations of plasma folate and vitamin B12 with HCC risk have not been well studied. In this current case-control (312 cases and 325 controls) study, we observed an inverse association between the levels of plasma folate and HCC, whereas only the subjects in the highest plasma vitamin B12 quartile exhibited a significant positive relationship with HCC. The levels of plasma folate and vitamin B12 were related with clinical characteristics of HCC patients.
The reduction of plasma folate in HCC patients may have resulted from various factors. For example, the most common factors are under-nutrition and severe catabolic status which cancer patients suffer from, compromising the plasma folate status [
14]. Moreover, a low folate status was reported to lead to liver damage through oxidative stress and pro-fibrogenic effect [
15,
16]. As a major source of dietary methyl groups, folate is involved in various biological processes, including methylation, DNA synthesis, and DNA repair. Therefore, folate deficiency aids the incorporation of uracil into the DNA, which can lead to DNA breaks and chromosome instability; such breaks could contribute to the increased risk of cancer. In addition, inadequate folate may result in global DNA hypomethylation and aberrant hypermethylation in gene promoters, which subsequently interfere with gene expression and DNA repair, finally leading to tumorigenesis [
17].
Our findings are strongly supported by previous studies on experimental animals, which reported an increased risk for hepatocarcinogenesis in folate-deficient mice [
11,
12,
13]. Circulating concentrations of folate have been investigated in relation to multiple cancers, but few studies have been conducted to evaluate the relationship between plasma folate and HCC patients. In agreement with our results, Welzel et al. [
18] researched a prospective high-risk cohort in Haimen City and indicated that higher folate levels in red blood cells were associated with reduced risk of hepatocarcinogenesis. Consistently, Lin CC et al. [
19] showed that plasma level of folate in HCC patients was significantly lower compared to healthy controls, in spite of very small sample size in their study (40 cases and 20 controls). Recently, Cheng et al. [
20] showed that HCC patients at preresection had lower serum folate than that of control groups. Chang et al. [
21] also observed an inverse association between plasma folate levels and liver cancer. In fact, in other malignancies such as lung cancer [
22], breast cancer [
23], esophageal squamous cell carcinoma [
24], and cervical cancer [
25], studies have also reported a protective association between the plasma folate levels and the risk of some cancers. In addition, in a cohort study from the U.S. population of the National Health and Nutrition Examination Survey [
26], results also showed that high serum and RBC folate in older adults were inversely associated with the risk of cancer incidence. Furthermore, a meta-analysis of 83 case-control studies involving 35,758 individuals also suggested that folate deficiency associated with increased overall risk of carcinogenesis [
27].
Excessive alcohol consumption is a well-established risk factor for HCC [
28,
29,
30], while alcohol could also affect folate and DNA methylation pathways by promoting the degration, and inhibiting the absorption and metabolism of this nutrient. Similarly, vitamin B
12 deficiency is found in chronic alcoholics. Thus, we further conducted a stratified analysis by alcohol drinking status, and the results indicated that HCC risk has an inverse association with all quartiles of plasma folate levels in the subgroup of non-drinking. In the subgroup of drinking, only the highest quartile of plasma folate exhibited a significant association with the reduced of HCC risk. Persson et al. [
30] suggested that higher folate intake might ameliorate the effect of alcohol consumption on the development of HCC, they observed that the individuals who consumed more than 3 drinks per day were associated with a significantly increased risk of HCC in the lower tertile of the folate intake group, but no association between alcohol consumption and HCC in the highest tertile of the folate intake group [
30].
Liver is the major storage site for vitamin B
12. Vitamin B
12 is an important co-factor in folate metabolism but studies relating plasma vitamin B
12 to HCC risk and prognosis are relatively very few, and the association remains vague. In this study, although a higher percent of vitamin B
12 deficiency was observed in the HCC patients, there was no significant difference in the mean of plasma vitamin B
12 level between patients and healthy controls. Interestingly, compared to the subjects in the lowest quartile of plasma vitamin B
12, the subjects in the highest quartile significantly displayed increased HCC risk only in the drinking subgroup, but not in the non-drinking subgroup. However, there are few data on the effects of plasma vitamin B
12 on the risk of HCC. A study on HCC in Taiwan stated that plasma level of vitamin B
12 in HCC patients was significantly lower compared to healthy controls [
31]. Whereas, another study [
32] reported that vitamin B
12 and related proteins displayed elevations in both HCC and chronic liver disease patients compared to controls. These different results might be due to the small sample sizes in the studies, differences in the methods of measuring vitamin B
12 level, differences in ethnicity between the subjects, or differences in tumor stages of HCC. Our data showed no significant interaction effect between plasma folate and vitamin B
12 on HCC.
Apart from the revelation of abnormal folate and vitamin B
12 levels in the blood of HCC patients, we also explored the influences of the folate and vitamin B
12 on clinical pathological parameters of HCC. In the analysis, we found that when tumor stage of HCC was categorized into stages I + II and III + IV, the tumor stage was associated with lower plasma folate level. The tumor size also displayed a similar association with folate. A recent study reported that folate status in HCC patients decreased as HCC progressed and that low blood folate status could be a risk factor for tumor progression [
10]. Consistently, our data confirmed that low folate level contributes to HCC progression in tumor stage and tumor size. However, there was no significant difference in plasma folate level in the HCC cases among different tumor markers status (AFP, CEA and CA19-9 levels).
Contrary to plasma folate analysis, elevated plasma vitamin B
12 levels might have a better prognostic significance in HCC patients. The size of the tumor nodule, which represents tumor burden, was also frequently associated with the aggressiveness of HCC [
33]. CEA and CA199 have been used as tumor markers, and they were also associated with the severity of liver disease [
34]. We observed that high plasma vitamin B
12 levels were not only associated with tumor stage and tumor size but also CEA and CA19-9 levels; these results indicated that high plasma vitamin B
12 levels could be a risk factor for tumor progression. Lin et al. [
31] showed that serum vitamin B
12 levels were positively correlated with AFP levels and tumor size, and determinants of plasma vitamin B
12 level in HCC patients were shown to be liver injuries, and tumor progression. In addition, previous studies showed that vitamin B12 levels above 400 pg/mL reduced micronucleus formation in peripheral blood lymphocytes [
35,
36] and uracil misincorporation into leukocyte DNA [
37]. Furthermore, Arendt et al. [
38] showed that high plasma vitamin B
12 levels increased the risk of subsequently diagnosed cancer, and cancer patients with elevated plasma vitamin B12 levels had higher mortality than those with normal vitamin B
12 levels [
39,
40].
As an important coenzyme of methionine synthase, the deficiency of vitamin B
12 led to aberrant DNA methylation and subsequent hepato-carcinogenesis. After that, the injury to the hepatocyte accelerated with tumor progression. As the liver has a crucial impact on the storage, transport and metabolism of cobalamin, the damage to liver cells may induce the impairment of hepatic B
12 metabolism and release of vitamin B
12, resulting in the elevation of plasma vitamin B
12 level [
41]. This may elucidate the mechanisms regarding discrepancy of vitamin B
12 level in different tumor stages of HCC patients.
Due to a lack of detailed data on dietary folate and vitamin B12 intake, the possible effect of diet on HCC could not be adequately evaluated. Furthermore, given the limitation of a case-control study, it may be difficult to establish the timeline of exposure to disease outcome in the setting of a case-control study. In other words, it is hard to define the causal relationship between plasma folate and vitamin B12 with HCC. Therefore, in the future, prospective cohort studies on plasma folate and vitamin B12 and HCC should be designed to further ascertain this relationship.