Chinese Medical Journal 2003;116(1):15-19
The effect of folic acid on the development of stomach and other gast rointestinal cancers

ZHU Shunshi 朱舜时,  Joel Mason,  SHI Yao 施 尧,  HU Yunbiao 胡运彪,  LI Rongrong 李蓉蓉,  WAHG Min 汪 敏 ,  ZHOU Yihe 周怡和,  JIN Guanqiu 金冠球,  XIE Yuye 谢宇野,  WU Guiquan 邬桂泉,  XIA Dehuang 夏德凰 ,  QIAN Zhenhua 钱珍华,  SOHG Hailian 宋海连,  TU Baiqiang 屠伯强,  ZHANG Lidong 张丽冬,  Robert Russell,  XIAO Shudong 萧树东

ZHU Shunshi 朱舜时 (Department of Gastroenterology, The Ninth People’s Hospital, Shanghai Second Medical University, Shanghai 200011, China)

Joel Mason (Human Nutrition Research Center on Aging, Tufts University, U.S.A.)

SHI Yao 施 尧 (The Renji Hospital and Shanghai Institute of Digestive Disease, Shanghai Second Medical University, Shanghai 200001, China)

HU Yunbiao 胡运彪 (The Renji Hospital and Shanghai Institute of Digestive Disease, Shanghai Second Medical University, Shanghai 200001, China)

LI Rongrong 李蓉蓉 (The Renji Hospital and Shanghai Institute of Digestive Disease, Shanghai Second Medical University, Shanghai 200001, China)

WAHG Min 汪 敏 (Department of Gastroenterology, The Ninth People’s Hospital, Shanghai Second Medical University, Shanghai 200011, China)

ZHOU Yihe 周怡和 (Shanghai First People’s Hospital, Shanghai 200080, China)

JIN Guanqiu 金冠球 (Shanghai Navy Hospital, Shanghai 200081, China)

XIE Yuye 谢宇野 (Shanghai Navy Hospital, Shanghai 200081, China)

WU Guiquan 邬桂泉 (Shanghai Post and Telecommunication Hospital, Shanghai 200060, China)

XIA Dehuang 夏德凰 (Shanghai Putuo People’s Hospital, Shanghai 200060, China)

QIAN Zhenhua 钱珍华 (Tongji University Railway Hospital, Shanghai 200072, China)

SOHG Hailian 宋海连 (Shanghai Traditional and Western Medical Integrated Hospital, Shanghai 200082, China)

TU Baiqiang 屠伯强 (Ruijin Hospital Luwai Branch, Shanghai Second Medical University, Shanghai 200020, China)

ZHANG Lidong 张丽冬 (Shanghai Port Hospital, Shanghai 200010, China)

Robert Russell (Human Nutrition Research Center on Aging, Tufts University, U.S.A.)

XIAO Shudong 萧树东 (The Renji Hospital and Shanghai Institute of Digestive Disease, Shanghai Second Medical University, Shanghai 200001, China)

Correspondence to:Zhu Shunshi,Department of Gastroenterology, The Ninth People’s Hospital, Shanghai Second Medical University, Shanghai 200011, China (Tel: 86- 21- 63138341. Fax:86- 21- 63136856. E-mail:)
Keywords
folic acid;β - carotene;gastritis, atrophic;stomach cancer;gastrointestinal cancer
Abstract
Objective To evaluate the roles of folic acid and β- carotene in the chemoprevention of g astric and other gastrointestinal (GI) cancers.

Methods In a randomized, double- blind, placebo- controlled trial, a total of 216 patien ts with atrophic gastritis were randomly assigned to one of the four groups: ①f olate (FA, 20 mg per day plus vitamin B12 1 mg, intramuscularly, per month for one year, then 20 mg two times a week plus 1 mg per three months for the next year); ②natural β- carotene (N- βC, 30 mg per day for first year, then 30mg two times a week for the next); ③synthetic β- carotene (S- βC, administ ered as in N- βC); and ④placebo. Follow- ups continued from 1994 to 2001.

Results A total of 7 new cases of gastrointestinal cancers were diagnosed with 3 stomach , 1 colon and 1 esophageal cancers occurring in the placebo group; 1 stomach ca ncer in both of the N- βC and S- βC groups, and no cancer occurring in FA g roup. In terms of GI cancers, there was a significant reduction in the FA grou p, compared with the placebo group (P=0.04).] A similar trend was obs erved in both N- βC [CM(45]and]S- βC]groups](P=0.07-0.08).] Taken]together,]the three intervention groups displayed a highly significant decrease in occurrence (P=0.004, vs placebo), and a lower risk for GI cancers (OR=0.12; 95% confidence interval, 0.03-0.51). For development o f gastric cancer, any one of the three active- treated groups did not reach st atistically significant reduction. The FA group showed obvious improvement of the gastric mucosal lesions with more patients displaying lesions reversed or st able atrophy and inflammation (P=0.04), reversed intestinal metaplas ia (P=0.06) at the end of follow- up, and reversed displasia (P=0.017) at 12 months. Two cases of false jaundice were found in β- carotene groups w ith no influence on administration, and no side- effects were reported in FA gro up.

Conclusions This trial revealed the interventional effect of folic acid on the development o f GI cancers, a similar effect of β- carotene was also detected. Also, folic acid may be of use to treat atrophic gastritis by preventing or reversing the pr ecancerous lesions.

It is well known that some antioxidant nutrients, especially β- carotene, may prevent carcinogenesis of the lung, uterine cervix, mouth and gastro intestinal tract.However, several β- carotene intervention trials using hig h doses and long periods have not shown the expected effect on cancer;[1]some even]reported an increased mortality from lung cancer in smokers and certain high risk groups.[2,3]A trial in Linxian, China, an area of high preval ence of stomach cancer, reported significant reductions of total cancer rate (RR =0.87) and stomach cancer (RR=0.74) from the combined supplementation of β- c arotene, vitamin E and selenium,[4,5]although it was indeterminable whethe r the effects came from β- carotene alone. Folic acid is another micronutrien t receiving particular attention. The hypothesis that folate status can modula te colorectal carcinogenesis has been supported by animal experiments and popula tion trials.[6,7]The folic acid supplementation improved gastric mucosal atrophy, intestinal metaplasia and dysplasia in patients with atrophic gastritis , a precancerous condition of stomach cancer, and that high dose of folic acid p layed an interventional role in gastric carcinogenesis in beagles.[8]So, it ]is theoretically possible to use folate and]β- carotene]to reverse or de lay the process of gastrointestinal (GI) carcinogenesis.

In addition, compared with a large population trial, a study on patients with pr ecancerous lesions can be done more cost- effectively and for a shorter duration , as precancerous lesions of the GI tract are especially susceptible to the geno toxicity or antigenotoxicity of ingested substances.Thus, the Shanghai Institu te of Digestive Disease and the Human Nutrition Research Center at Tufts Univers ity in U.S.A.performed a cooperative study in ten hospitals of Shanghai to evaluate and identify the role of folic acid and β- carotene in the chemoprev ention of stomach cancer and other gastrointestinal tumors.

METHODS

Study design This study was a randomized, double- blind, placebo- controlled trial which te sted the effect of folic acid and β- carotene on gastric cancer and other malig nant tumors of GI tract.All the participants were the consecutive out- patient s with atrophic gastritis from two enrollment periods: the first one from May to June of 1994 in 4 hospitals (trial 1), and the second from the same period of 1 995 in 10 hospitals (trial 2).The patients underwent gastroduodenoscopy and fo ur biopsies were obtained at 0, 3, 6, and 9 o’clock points in antrum two to thre e centimeters away from the pylorus.All the slides stained with haematoxylin - eosin and assessed by two assigned pathologists at Shanghai Institute of Diges tive Disease, who were unaware of the clinical data, and with good concordance. Following Whitehead,[9] the biopsy lesions were classified a s inflammation, atrophy, intestinal metaplasia, and dysplasia.The four lesions were separately scored on a grading scale: 0 absent, 1 mild, 2 moderate and 3 s evere.Then, the mean score of each lesion was calculated based on the four bio psies.According to the inclusion criteria of the atrophy (mean score>1), 218 p atients (56 for trial 1, 162 for trial 2) were recruited, most of whom showed si gns of intestinal metaplasia and,to a lesser extent, dysplasia.Excluded w ere those with histories of GI tumors or surgery, those with obvious cardiovascu lar, renal, liver, and lung diseases, and those who had taken vitamin pills in t he last three months.After inclusion, each patient started a half year of run - in period to assess adherence to study regimen and to check for malignancies t hat may have been subclinically presented or overlooked before.During the peri od, two cases of gastric cancer were found and excluded. Subjects Of the remaining 216 patients, 137 men and 79 women, aged 28 to 77 years (mea n of 55.6 years) were randomly assigned to one of the four groups: ① folic aci d group (FA), folate 20 mg per day plus vitamin B 12 1 mg intramuscularly per month for the first year, then 20 mg FA two times a week plus 1 mg B12 quarterly for the second year (provided by Shanghai Huanghe Pharmacy and Shangh ai First Pharmacy, China); ② natural β- carotene group (N- βC), natural β- c arotene soft gelatin capsule (Betatene Co.Australia and Henkel Co., USA) 30 m g per day for the first year, 30 mg two times a week for the second and then dis continued; ③ synthetic β- carotene group (S- βC), synthetic β- carotene sof t gelatin capsule 30 mg per day (by Shanghai Sixth pharmacy and Henkel Co.) adm inistered as of N- βC, and ④control group (Placebo), placebo administered and provided as of S- βC. General characteristics by group are given in Table 1 . The four groups were well balanced with respects to sex, age, baseline lesions and number of endoscopies.Because there were more cancers in the placebo group , the mean follow- up time was shorter than those in the other three active- t reated groups.There were four cases of cerebral vascular accidents with 2 in F A and one each in N- βC and S- βC.The N- βC and placebo groups lost 4 patie nts respectively, due to change of address or migration abroad, with a rate of]3.7%.]Informed consents were obtained from all participants, and the research protocol was reviewed and approved by the institutional review boards a t Shanghai Institute of Digestive Disease in China and Human Nutrition Research Center at Tufts University, USA.

Follow- up and assessment of end points A gastroenterologist at each hospital was in charge of the follow- up of his pat ients, which included interview, visit, letter or telephone call, delivering the assigned agents according to the group allocations every three months.Pat ients were constantly asked about their adherence to the regimen, as well as any side effects, illnesses that occur during the course of the trial.The patien ts underwent a gastroduodenoscopy and biopsy every one to three years.The foll ow- up continued for 6-7 years (72.9 months of average) until April 30 of 2001 .Compliance was assessed by counting the remaining pills at each visit, as wel l as by measurement of relevant vitamin concentrations in serum randomly at 15 d ays, 3 months, 6 months, 12 months and 24 months.[3]The serum]samples wer e stored at -70℃ and determined by microbiological assay for folic acid and by HPLC for several carotenoids.

According to the protocol, stomach cancer was an end- point; other benign or mal ignant GI tumors, or deaths from other causes were also considered end points. All end points or events were confirmed by both the gastroenterologist at the ho spital and the investigator at the Shanghai Institute through checking the medic al records and interviewing the patient or his/her family members.

Statistical analysis Data from all 216 patients were analyzed according to the intention- to- treat p rinciple (ITT), regardless of subsequent compliance or loss in follow- up after run- in period.All the results were presented as mean ± s or rate.The differences were tested for statistical significance by analysis of variance, t test, Chi- square test, Fisher’s exact test, and relative risk.A P value <0.05 was considered significant.

RESULTS

Compliance and Side effects
Compliance, as assessed by quarterly pill counting and random blood sampling, wa s excellent throughout the trial.More than 90% of all patients took pills acco rding to the protocol.After the supplementation, the serum levels of folic aci d or relative carotenoids increased several times in the three active - treated groups, but not in placebo. While comparing the serum concentrations of folic acid and relative carotenoids from the starting point (baseline) to th e 6th months points, we found: in FA group, folate was 11.6±11.4 μg/ml vs 23 4.2±175.5 μg/ml, respectively (P<0.01); in N- βC group, all- trans- βC was 26.1±39.1 μg/ml vs 127.8±45.8 μg/dl, respectively (P<0.001 ), α- carotene was 3.1±5.0 μg/dl vs 18.1±9.0 μg/dl, respectively (P <0.001); in S- βC, all- trans- βC level was 25.0±29.4 μg/dl vs 130.8±49 .9 μg/dl (P<0.01), no significant change was found in α- carotene level (2.2±2.3 μg/dl vs 1.3±0.7 μg/dl, P>0.05).

Two false jaundices were found in the β- carotene groups due to hypercarotenemi a, although serum bilirubin and other biochemical factors appeared normal. Thi s did not affect the administration and disappeared in the next year.No side effects were found in the FA group, in which all subjects showed normal blood co unts, liver and renal functions.

Occurrence of GI Tumors
During the follow- up period, a total of 7 new cases of gastrointestinal cancers were developed: 3 gastric, 1 colonic and 1 esophageal cancers in the placebo g r oup, and 1 stomach cancer in both of the N- βC and S- βC groups. No cancers occurred in FA group.In addition, 1 gastric and 1 duodenal polyp in the placeb o group, and 2 cases of gastric polyp were reported in the N- βC group.

Table 2 shows a significant reduction of GI cancers in the FA group (P=0.04 6, vs placebo), and a similar trend was detected in both N- βC (P=0.07) an d S- βC (P=0.08). Combining the results of all three groups, there is a highly significant decrease in the risk of GI cancers among the interventional g roups (P=0.004, vs placebo).In terms of the development of stomach cancer , none of the three groups alone displayed a statistically significant reduction compared to the placebo, but the combined almost did (P=0.06).

Changes of the gastric mucosal lesions after intervention
Changes in gastric mucosal lesions after intervention were represented by the le sion score differences and by the case numbers that displayed lesion changes.A negative difference indicateds reversion, a difference of zero indicates stabil ity and a positive difference indicates progression.The FA group showed the si gnificantly more reversion of dysplasia at 12 months (Table 3) , and more reversi on and stability with regards to inflammation and atrophy as well as more revers ion of intestinal metaplasia at the end of the follow- up (Table 4)

DISCUSSION

The results of this trial demonstrated a statistically significant reduction of GI cancers in the folic acid group, and a similar trend in both of N- βC and S - βC groups as well; thus, the combination of the three interventional groups a lso displayed a highly decreased risk of GI cancers.In terms of the developmen t of stomach cancer, although each of the three groups alone did not show a sign ificant reduction. These data reveal that folic acid is a promising agent for GI cancer prevention, and that β- carotene may also be effective.As we contin ue the follow- up, the benefit may become more evident.According to Hennekens, [1 ]a trial lasting 10 or more years is a sufficient period of time for the de tect ion of even a small reduction in risk due to micronutrient supplementation.

The interventional effect of folic acid was seen as early as six months of the s tudy, when both atrophy and intestinal metaplasia in 77.7% of the patients in t he FA group were reversed, a percentage that was significantly higher than the p lacebo.[10]Furthermore, dysplasia was reversed significantly 12 months la ter, and at the end of the follow- up period, the FA group continued to have mor e cases of reversion and stability with the regards to inflammation, atrophy and intestinal metaplasia.In view of Correa’s multi- step model of gastric carcin ogenesis (normalsuperficial gastritisatrophic gastritisintestinal metaplas iadysplasiametastasis),[11]folic acid supplement ation did halt and delay the progression of these precancerous lesions, and thus , the process of gastric carcinogenesis.Linxian dysplasia trial by Mark et al [12]revealed the]reversion effect of vitamin and mineral supplement inc l uding folate on esophageal dysplasia.We have previously published reports on t he genotypic changes that occur in response to increased folic acid supplementat ion, namely, the DNA methylation state of the gastric mucosa was significantly e nhanced after 6th month in the FA group,[10]and Fas and PCNA gene expres sions decreased after 12 months, when the dysplasia was significantly reversed .[13]Kim et al[14]also reported that folate supplementation increas ed folate levels in serum, red blood cells and colonic mucosa, increased the gen omic DNA methylation, and decreased the extent of p53 strand break in exons 5-8 at 6 months and 1 year after supplementation in patients with colonic adenomas .The above effect of folic acid on genotypic events reveals a "nutrition- gen e interaction"- folic acid can influence moleculobiologic events, and gene expre ssion as well.

It is interesting to note that so- called folate deficiency may often be margina l and localized with the blood level well within the range conventionally accept ed as normal.We have also found in stances of significantly lower intracellular levels of vitamin B 12 and car otenoids, lower tissue folate concentration and decreased genomic DNA methylatio n status,[15]and c- myc and c- Ha- ras oncogene hypomethylation[16]i n cancer tissue and its surrounding mucosa in patients with gastric cancer and n ormal serum folate.So, suboptimal body reserve or localized folate deficiency may contribute to some epithelial carcinogenesis, including GI cancer developmen t.

As to the role of β- carotene in cancer prevention, there is some discrepancy b etween epidemiological observations and some intervention trials.[2,3]Ce rtain questions remain to be answered.Firstly, there are 400-600 natural carot enoids, and it has not been established whether β- carotene or perhaps another component that played the central role in the anti- cancer effect.Recently, Mi c haud et al[17]reported that several different carotenoids, especially lyco pene and α- carotene, may be involved in reducing lung cancer risk.Perhaps site- specific cancers need certain carotenoids for their prevention.Secondly , while β- carotene itself has been considered safe at even high doses, its met abolic products from excentric cleavage in the body have not been adequately stu died,[18]and there is some evidence that these metabolic products may be t he source of genotoxicity in some reports.[2,3,19]So, dose may be a cruci al point.In this study, we used β- carotene at the dose of 30 mg per day for one year, then decreased it to 30 mg two times a week for the second year, afte r which β- carotene administration was discontinued. The anti- tumor trend wa s still detected seven years post- discontinuation with no pro- cancer effect. Therefore, it is possible to use lower doses of β- carotene in GI cancer preven tion.

This report offers evidence that chemoprevention (chemical intervention) can off er excellent prospects as a means of reducing cancer incidence.For the general population, simply increasing vegetable and fruit intake and improving life- st yle[20]may be an easy and effective way to reduce cancer risk.However, f or cancer survivors, those with precancerous conditions, or those with a family history of cancer, we think that it would be more beneficial to administer some specific agents, because it would at least delay the development of some cancers by reducing mutation rates.Therefore, the authors suggest that patients with moderate or severe atrophic gastritis or intestinal metaplasia or dysplasia take folic acid, 15-20 mg per day, plus vitamin B 12 500 μg, intramuscularly, per month for one year, then attenuate the dosage.We believe this is a safe an d cost effective treatment for the disease and also of chemoprevention for gastr ointestinal cancers.

Acknowledgement: We are indebted to He Qingpo (Shanghai Second Medical University), Luo Hongyu (S hanghai Institute of Digestive Disease), and Jerry Dallal (Human Nutrition Resea rch Center at Tufts) for expert assistance in data management and processing.W e also would like to thank Kyung- Jim Yeum, Marie Nadeau and Sang Woon Choi for help and direction in the laboratory work at the center.

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