Influence of L-methionine-deprived total parenteral nutrition with 5-fluorouracil on gastric cancer and host metabolism

Abstract

AIM: To investigate the influence of L-methionine-deprived total parenteral nutrition with 5-FU on gastric cancer and host metabolism.

METHODS: N-methyl-N’-nitro-nitrosoguanidine (MNNG) induced gastric cancer rats were randomly divided into four groups: Met-containing TPN group (n = 11), Met-deprived TPN group (n = 12), Met-containing TPN+5-FU group (n = 11) and Met-deprived TPN+5-FU group (n = 12). Five rats in each group were sacrificed after 7 d of treatment and the samples were taken for examination. The remaining rats in each group were then fed separately with normal diet after the treatment until death, the life span was noted.

RESULTS: The tumors were enlarged in Met-containing group and shrank in Met-deprived group markedly after the treatment. The DNA index (DI) of tumor cells and the body weight (BW) of rats had no significant change in the two groups, however, the ratio of tumor cells’ S phase was increased. The ratio of G2M phase went up in Met-containing group, but down in Met-deprived group. In the other two groups that 5-FU was added, the BW of rats, and the diameter of tumors, the DI of tumor cells, the S and G2M phase ratio of tumor cells were all decreased, particularly in Met-deprived plus 5-FU group. Pathological examination revealed that the necrotic foci of the tumor tissue increased after Met-deprived TPN treatment, and the nucleoli of tumor cells enlarged. In -MetTPN+5-FU group, severe nuclear damage was also found by karyopyknosis and karyorrhexis, meanwhile there was slight degeneration in some liver and kidney cells. The serum free Met and Cysteine decreased markedly (P ＜ 0.001), while other amino acids, such as serum free serine and glutamine increased significantly (P ＜ 0.005). All the rats died of multiple organ failure caused by cancer metastasis. The average survival time was 18.6 d in Met-containing TPN group, 31 d in Met-deprived TPN group, 27.5 d in Met-containing TPN+5-FU group, and 43 d in Met-deprived TPN+5-FU group (P ＜ 0.05).

CONCLUSION: Met-deprived TPN causes methionine starvation of tumor cells, and can enhance the anti-tumor effect of 5-FU and prolong the life span of gastric cancer-bearing rats.

Key Words: stomach neoplasms/therapy; stomach neoplasms/pathology; parenteral nutrition; methionine/therapy use; fluorouracil/therapy use

INTRODUCTION

Many tumor cells can not grow in the medium replaced L-methionine (Met) with its direct precursor L-homocyateine. However, normal cells can grow well in that medium. It means that the growth of tumor cell is Met-dependent[1-9]. In this study, we prepared a Met-free amino acid solution and used it as the sole nitrogen source of total parental nutrition (TPN), to investigate the influence of Met-deprived TPN on the gastric cancer and host metabolism.

MATERIALS AND METHODS

Animal grouping and treatment

Male Wistar rats, with N-methyl-N’-nitro-nitrosoguanidine (MNNG) induced gastric cancer (n = 46), were verified to have tumor diameter larger than 8 mm in laparotomy and confirmed pathologically. The 46 rats were randomly divided into four groups: Met-containing TPN group (n = 11), Met-deprived TPN group (n = 12), Met-containing TPN+5-FU group (n = 11) and Met-deprived TPN+5-FU group (n = 12). Five rats in each group were sacrificed after 7 d of treatment and the specimens were taken for examination and the other rats were then fed with normal diet separately after the treatment until death for noting the life span. After 24 h fasting, the rats were cannulated to the vena cava immediately after peritoneal cavity anesthesia with 25 g•L¯¹ barbital and placed in metabolic cage respectively and infused with different TPN solution after fixing the TPN tube at the back of rats with a plate and tension spring.

Composition and infusion of TPN solution

The rats were infused with 50 g•L¯¹ glucose normal saline 200 mL•kg¯¹ on the cannulation day. Different TPN solutions were infused continously 24 h a day on the next 7 d. During the experiment, the rats were housed individually in metabolic cages. The composition and usage of TPN solutions are shown in Table 1.

Table 1 Composition and dosage of TPN solution (kg¯¹•d¯¹).

Composition	+MetTPN	-MetTPN
250 g•L¯¹ Glucose/m	100	100
200 g•L¯¹ Intralipid/mL	35	35
+Met amino acid solution/mL	100	0
-Met amino acid solution/mL	0	100
Non-protein energy/J	710	710
Total nitrogen/g	1.30	1.03
Total volume/mL	235	235

Specimen sampling and detection

Pre- and post- TPN, the rats’ body mass (BM) and the tumor diameter were measured, the tumor tissues were sampled for flow cytometry (FCM) tumor cell cycle analysis and DNA index calculation. Blood samples were taken for serum amino acid profile test. Tissues of the rats’ gastric cancer, heart, lung, liver and kidney were sampled and fixed with 100 mL•L¯¹ formalin, enbedded with paraffin, sliced in 5 μm, and HE stained for pathohistologic examination.

Usage and dose of 5-FU

5-FU was added to the TPN solution and infused for 6 d (from d2 to d7, 15 mg•kg¯¹•d¯¹).

Observation of survival time

The other rats in each group not sacrificed were moved to common cage after withdrawing TPN tube and fed with normal diet until death. The survival time was noted.

Statistical analysis

Student t test was used to examine the data. Survival time was examined with time sequence examination. The difference was considered significant when the P value was less than 0.05.

RESULTS

Alteration of the rats’ BM, tumor size and DI

The BM and DI had no significant change in both +MetTPN and -MetTPN groups after treatment. The tumor size was markedly enlarged in +MetTPN group and shrank in -MetTPN group. All of the BM and the DI as well as the tumor size were decreased in both groups of +MetTPN+5-FU and -MetTPN+5-FU, and the change was more significant in the -MetTPN+5-FU group (Table 2).

Table 2 Body mass, tumor diameter and DNA index of tumor cells after TPN (mean ± SD).

TPN solution	mb/g		dt/mm		DNA index
	Pre-TPN	Post-TPN	Pre-TPN	Post-TPN	Pre-TPN	Post-TPN
+MetTPN	242 ± 20	243 ± 20	11.2 ± 1.0	14.7 ± 0.7b	1.13 ± 0.24	1.21 ± 0.26
-MetTPN	248 ± 20	245 ± 18	11.5 ± 1.2	6.9 ± 0.8ad	1.15 ± 0.21	1.12 ± 0.23
+MetTPN+5-FU	245 ± 13	233 ± 12b	11.1 ± 1.1	9.3 ± 0.8a	1.15 ± 0.03	1.08 ± 0.02a
-MetTPN+5-FU	245 ± 14	225 ± 13b	11.4 ± 0.9	5.5 ± 0.5bc	1.15 ± 0.03	1.02 ± 0.02bc

^dP ＜ 0.05,

^bP ＜ 0.01, vs preTPN;

^cP ＜ 0.05, ^dP ＜ 0.01, vs +MetTPN.

Change of tumor cell cycles

The S phase percentage of tumor cells was increased in both +MetTPN and -MetTPN groups, and it is higher in the other groups. However, the change of G2M phase ratio was different. It was increased in +MetTPN group and decreased in -MetTPN group. The S and G2M phases were lowered in the other groups with 5-FU. The change was more significant in -MetTPN+5-FU group (Table 3).

Table 3 Change of tumor cell cycles after TPN (mean ± SD, %).

TPN solution	S		G2M		G0/G1
	Pre-TPN	Post-TPN	Pre-TPN	Post-TPN	Pre-TPN	Post-TPN
+MetTPN	6.2 ± 1.2	10.3 ± 1.4b	10.5 ± 1.1	16.6 ± 1.4a	83.4 ± 1.9	73.1 ± 1.8b
-MetTPN	6.4 ± 1.0	28.5 ± 1.0ac	11.2 ± 1.1	8.3 ± 1.5ad	82.4 ± 0.9	63.2 ± 5.5a
+MetTPN+5-FU	6.6 ± 0.3	4.5 ± 0.5a	12.6 ± 0.5	9.7 ± 1.2a	80.8 ± 0.3	85.8 ± 1.2b
-MetTPN+5-FU	6.5 ± 0.7	4.5 ± 0.3a	12.4 ± 0.9	6.2 ± 0.5bc	80.1 ± 0.5	89.3 ± 0.7b

^aP ＜ 0.05,

^bP ＜ 0.01, vs preTPN;

^cP ＜ 0.05,

^dP ＜ 0.01, vs +MetTPN.

Serum amino acid profile

The serum L-methionine and L-systein were markedly decreased in the -MetTPN rats. However, the other amino acids such as asprine and glutamine as well as serine were significantly increased after treatment (Table 4).

Table 4 Serum FAA value (mean ± SD, μmol•L¯¹).

	+MetTPN	-MetTPN
Asp	36.1 ± 1.2	88.9 ± 10.3a
Glu	27.3 ± 4.2	193.2 ± 17.4a
Ser	124.8 ± 21.5	231.5 ± 32.3a
Gly	116.9 ± 18.3	286.7 ± 21.9a
Gln	103.4 ± 14.4	90.1 ± 10.3
His	11.2 ± 1.5	25.0 ± 3.6
Thr	53.5 ± 3.8	93.2 ± 7.1a
Ala	138.7 ± 30.1	218.8 ± 31.4a
Arg	83.6 ± 9.4	173.4 ± 25.1a
Pro	33.9 ± 8.2	79.7 ± 13.4
Tyr	20.8 ± 5.1	44.8 ± 6.3
Val	88.4 ± 14.1	31.6 ± 1.6
Met	76.1 ± 1.3	10.9 ± 3.1b
Cys	87.3 ± 3.2	43.2 ± 5.4b
Ile	5.4 ± 1.1	10.1 ± 1.2
Leu	34.2 ± 1.3	55.2 ± 4.3
Phe	22.1 ± 2.2	43.1 ± 2.9
Trp	42.5 ± 3.6	50.7 ± 4.4
Lys	212.4 ± 43.1	387.8 ± 58.3a

^aP ＜ 0.05,

^bP ＜ 0.001, vs +MetTPN.

Pathohistological findings

The number of tumor necrotic foci were increased after -MetTPN and -MetTPN+5-FU treatment. Nuclei was enlarged in tumor cells and liver cells.

Survival time

All the gastric cancer bearing rats died of cancer metastasis and cachexia. The mean survival time was 18.6 d in +MetTPN group, 31 d in -Me tTPN, 27.5 d in +MetTPN+5-FU and 43 d in -MetTPN+5-FU (P ＜ 0.05).

DISCUSSION

Patients with malignant tumors often show severe protein-amino acid metabolism disorder and uncorrectable negative nitrogen balance as well as low immune function caused by malnutrition[10-21]. TPN support is considered beneficial to improve the patients' nutritional status and immune function, and to lower the surgical complications, and to improve the quality of life[22-28]. But, TPN can also stimulate proliferation of the tumor cells[29,30]. So, there is much concern to study the special feature of tumor cells’ metabolism and to find a regimen of TPN that is beneficial to the host, but pernicious to the tumor cells, particularly through the regulation of tumor cells’ metabolism[31-52].

In this study, a special regimen of TPN deprived of Met was used in MNNG induced gastric cancer rats. The results showed that the TPN, containing Met or not, has regulative effects on the tumor cells’ dynamics. The effects were different between Met-containing TPN and Met-deprived one. Met-containing TPN stimulated tumor cells’ proliferation and promoted the tumor cells from G0/G1 phase into S and G2M phase, and made the ratio of S phase to G2M phase increased simultaneously. However, the Met-deprived TPN disturbed the metabolism of DNA, especially DNA methylation through Met starvation and inhibited the tumor proliferation by blunting S phase into G2M phase, resulting in increase of S phase ratio and decrease of G2M phase ratio. The inhibitory effect of tumor growth of the Met-deprived TPN was enhanced by simultaneous use of 5-FU, and it was manifested by a longer life span in the rats treated. Pathohistological examination found that the necrotic foci were increased in the tumor tissue and the nuclei of the tumor cells were enlarged. The pathohistologic findings were concordant with FCM analysis of cell cycles, meaning that the metabolism of DNA was blunted. One must be careful when using -MetTPN, particularly -MetTPN+5-FU, as some adverse effects may occur. In this practice, we noticed that the host liver and kidney cells had light degeneration. Hoffman et al[53] studied the growth of SV-40 fibroblast cells in the medium deprived of Met and found that the cell proliferation was blunted in S/G2 phase reversibly. Further studies[54] found that in the Met-free environment, the intracellular free Met of tumor was extremely decreased and it lowered greatly the S-Adeosylmethionine (S-AdoMet) which had decreased because of the tumor cell’s over active transmethylation, and resulted in a low ratio of S-AdoMet to S-AdoHcy. This directly inhibited the activity of transmethylase and suppressed the transmethylation reaction, including the DNA methylation. When the tumor cells were returned to the Met-containing environment, DNA methylation recovered and the cell cycle circulated. This reversible block of tumor cell cycle at S/G2 phase enhanced the anti-cancer effect of 5-FU and hinted the combined and sequenced use of other cycle specific chemotherapy agents with Met-deprived TPN.

By autopsy, we found that the liver and peritoneum metastasis of gastric cancer were much less in the group using Met-deprived TPN and the group using Met-deprived TPN plus 5-FU. It suggests that not only the primary tumor proliferation was inhibited, but also the invasive ability for metastasis was suppressed. Breillout et al[55] reported that Met-deprived TPN suppressed the metastasis potential of Lewis lung cancer and rhabdomyosarcoma in experimental animals. They considered that it was Met-starvation which disturbed the methylation of DNA and membrane lipids of the tumor cells, inhibiting their metastastic ability. Some authors even suggested that the inhibitory effect of -MetTPN against metastasis is more powerful than in proliferation of primary tumors. However, this still needs further studies.