Metformin prevents hormonal and metabolic disturbances and 1,2-dimethylhydrazine-induced colon carcinogenesis in non-diabetic rats.

Effects of two doses of the anti-diabetic drug, metformin (MF), on hormonal and metabolic levels of serum of non-diabetic male Wistar rats with 1,2-dimethylhydrazine (DMH)-induced colon tumor adenocarcinomas were studied. Carcinogenesis in the animals was also observed. Rats with DMH-induced colon adenocarcinomas had elevated levels of serum glucose, insulin, insulin-like growth factor-1, total cholesterol, triglycerides, catalase, malonic dialdehyde, glycated hemoglobin, aspartate aminotransferase, and alanine aminotransferase and decreased hemoglobin. Treatment with two doses of MF normalized majority of these changes in DMH-treated rats, whereas the drug was ineffective in rats without DMH treatment. The only exception was the decreased triglyceride levels in MF-treated rats. A 100 mg/kg dose of MF increased DMH-induced exophytic colon carcinomas and decreased endophytic tumors compared with untreated rats. Moreover, both MF doses increased DMH-induced and highly differentiated tumors and decreased the invasiveness of colon carcinomas compared with rats provided with DMH and water. Therefore, effects of MF on metabolic homeostasis are critical for preventing colon cancer.

Introduction

Colorectal cancer (CRC) is the third most common malignancy in humans worldwide; more than one million new cases occur annually[1]. Age is a leading CRC risk factor. More than 90% of CRC cases occur in people aged over 50, and approximately 75% of the cases are diagnosed in people older than 65. Furthermore, risks starting at 40 years of age increase sharply at 50 and double each decade until age 80[2]. Hyperinsulinemia and obesity are key factors in cancer pathogenesis, including CRC[3-6]. The chemical carcinogen, 1, 2-dimethylhydrazine (DMH), has been widely used for induction of colon cancer. Regardless of mode of administration, DMH specifically induces tumors within the descending colon of rats and some mouse strains, and resulting histopathologies are similar to those observed in human sporadic colon tumors[7,8]. Meanwhile, the anti-diabetic biguanide, metformin (MF), lowers elevated insulin levels in type 2 diabetes[3,9], significantly reduces various cancer risks in humans with the condition, and prevents tumor development in numerous rodent tissues and organs[10,11]. Furthermore, MF inhibits some chemically induced colon carcinogenesis in diabetic, obese[12], and non-diabetic rats[13]. In this work, we showed the inhibitory effects of MF on DMH-induced colon carcinogenesis in non-diabetic rats. Such effects mainly involve the normalizing influence of MF on hormonal and metabolic homeostases.

Material and methods

Animals

Male Wistar rats aged 2 months were bred at the Animal Laboratory of the I.P. Pavlov Institute of Physiology. Six to seven rats were kept in T3-type cages under a standard light/dark regimen (12 h light: 12 h darkness) at (22±2)°C and received standard laboratory PK-120 (Laboratorkorm, Russia)[14] and tap waterad libitum.

Animals were checked daily by animal care personnel and weekly by a veterinarian. The weights were measured weekly as well. The study was conducted per the regulations for ensuring humane treatment of animals under the approval of the Committee on Animal Research of N.N. Petrov Research Institute of Oncology.

Chemicals

DMH was provided by Sigma Chemical Co., St. Lois, MO, USA and kept at –20°C. MF (MF HCl, Siophor) was purchased from Berlin-Chemie, Menarini Group, Germany.

Experiment 1

A total of 58 male Wistar rats with 2-month-old were randomly subdivided into 6 groups. A total of 24 rats in groups 1–3 were not exposed to carcinogens, whereas 34 rats from groups 4–6 were administered with 5 subcutaneous DMH injections weekly at a single dose of 21 mg/kg of body weight (calculated as a base). In this regimen, carcinogens induced colon tumors in majority of rats[7]. DMH was ex tempore dissolved in normal saline and neutralized with sodium bicarbonate (pH 7.0). Starting from the first carcinogen injection, groups 1 and 4 were provided with 1 mL tap water via intragastric gavage, whereas groups 2 and 5 were administered daily with MF (100 mg/kg) via gavage. Groups 3 and 6 were given MF (300 mg/kg) dissolved in 1 mL tap water. This treatment was concluded 2 months after the first DMH injection. The experiment was finalized six months after the first carcinogen injection. After being sacrificed by ether vapor, rats were decapitated, and blood samples were collected in plastic vessels without anticoagulants. After letting the samples stand for 30 min at room temperature, they were centrifuged (30 min at 1200g) and then kept at –20°C until biochemical analyses. Metabolic parameters were assessed in blood serum. Glucose concentration was electrochemically estimated via an express analyzer (i-STAT, Abbot) that uses CG8+ cartridges. Total cholesterol, triglyceride, hemoglobin, and malonic dialdehyde (MDA) concentrations, and Cu, Zn-superoxide dismutase (SOD), catalase, alaninaminotransferase (ALT), and aspartataminotransferase (AST) activities were processed using Stat Fax 3300 analyzer and commercial reagent kits following the manufacturers’ instructions. Vascular endothelial growth factor (VEGF), insulin, and insulin-like growth factor (IGF-1) concentrations were assessed using enzyme-linked immunosorbent assay using Cusabio and R&B Systems reagent kits per standard procedures. Glycated hemoglobin was estimated using high performance liquid chromatography.

Experiment 2

A total of 24 male Wistar rats with 2-month-old were randomly subdivided into three groups and exposed to DMH and the same two doses of MF, as in Experiment 1. Experiment 2 was finalized six months after the first carcinogen injection. After being sacrificed using ether vapor, rats were autopsied by longitudinally opening the intestines. Tumor position and size were recorded[7]. After histological processing, tissues were embedded in paraffin. Histological sections measuring 3 µm thick were stained with hematoxylin-eosin and microscopically examined; in the experimental group, examination was performed as blind process. Tumors were classified per International Agency for Research on Cancer recommendations[15].

Statistical analysis

Experimental results were statistically processed following variation statistics using Statistica-10. All data were expressed as mean ± standard error ( and ) or confidence interval for the standard deviation ( ). The significance of discrepancies was defined according to Chi-square analysis between experimental and control groups ( ). Differences in estimated parameters among the groups were assessed using non-parametric criterion of Mann-WhitneyU test ( and )[16].P<0.01 and 0.05 were considered as significant.

1

Effect of 1,2-dimethylhydrazine (DMH) and metformin on hormonal and metabolic parameters in the serum male Wistar rats. (A) glucose. (B) Insulin. (C) IGF-1. (D) Total cholesterol. (E) Triglycerides. (F) Cu, Zn-superoxide dismutase. (G) Catalase. (H) Malonic dialdehyde. (I) Hemoglobin. (J) Glycated hemoglobin. (K) Alaninaminetransferase. (L) Aspartataminetransferase. (M) VEGF. Data presented as mean±SEM,n=6–15 per group.P≤0.05. a: DMHvs. control; b: DMH+MFvs. DMH. Rats bearing DMH-induced colon adenocarcinomas have elevated serum level of glucose, insulin, IGF-1, total cholesterol, triglycerides, catalase, malonic dialdehyde, glycated hemoglobin, AST, ALT and decreased level of hemoglobin. Treatment with MF in both doses normalized majority of these changes in DMH-treated group of rats, whereas failed to modify them in rats not treated with DMH. Only exception was decreased level of triglycerides in MF-treated rats (Figure 1E,P<0.05).

2

Effect of metformin on some parameters of 1,2-dimethylhydrazine-induced colon carcinogenesis in male Wistar rats. Data presented as mean±SEM,n=7–9 per group. *Р<0.01; §P<0.05vs. DMH group. Treatment with MF in dose 100 mg/kg increased relative number of induced with DMH exophytic colon carcinomas and decreased number of endophytic tumors. Both doses of MF increased relative number of DMH-induced highly differentiated tumors and decreased invasiveness of colon carcinomas as compare with group given DMH with water.

1

Colon tumors localization, incidence, multiplicity and size in rats exposed to 1,2-dimethylhydrazine (DMH) and metformin

Parameters	Treatment
	DMH + water	DMH + metformin, 100 mg/kg	DMH+metformin, 300 mg/kg
The difference in the parameter for rats exposed to DMH+water is significant, *P<0.01; The difference in the parameter for the group DMH+metformin–100 is significant as well: **P<0.01.
No. of rats	8	9	7
Ascending colon
No. of tumor-bearing rats	5 (63%)	0	2 (29%)
No. of tumors	5	0	5
No. of tumors per tumor-bearing rat	1.0	0	2.5
Mean size of tumors, mm2	47±37.8	0	13±8.5*
Descending colon
No. of tumor-bearing rats	8 (100%)	6 (67%)	6 (86%)
No. of tumors	23	12	19**
No. of tumors per tumor-bearing rat	2.9	2	3.2
Mean size of tumors, mm2	93±79.5	37±31.2*	43±23.9*
Rectum
No. of tumor-bearing rats	2 (25%)	2 (22%)	2 (29%)
No. of tumors	2	2	2
No. of tumors per tumor-bearing rat	1	1	1
Mean size of tumors, mm2	26±24.9	49±37.3	146±66.8
Total colon
No. of tumor-bearing rats	8 (100%)	7 (78%)	6 (86%)
No. of tumors	30	14	26
No. of tumors per tumor-bearing rat	3.75	2.0	4.8
Mean size of tumors, mm2	81±72.9	39±27.5*	45±22.7*

Results

Effect of MF on DMF-induced hormonal and metabolic disturbances in male rats

MF treatment failed to influence weight gain in both non- (groups 2 and 3) and DMH-exposed rats (groups 5 and 6). Thus, MF did not significantly affect weight gain between non- and DMH-exposed groups (data not shown). Two-month administration of both doses of MF to non-exposed rats significantly decreased triglyceride serum levels and failed to influence other metabolic parameters ( ). Dramatic parameter disturbances were observed in DMH and water-treated rats. The animals were sacrificed six months after the first carcinogen injection. Compared with the control group, non-treated rats had increased levels of glucose (+25.6%), insulin (+36.2%), IGF-1 (+37.1%), total cholesterol (+47.4%), and triglycerides (+106.9%) and increased activities of catalase (+35.3%), MDA (+33.3%), AST (+93.8%), ALT (+71.4%), VEGF (+65.5%), and glycated hemoglobin (+56.7%). SOD activity did not change significantly (+19.2%,P>0.05). Both MF doses alleviated carcinogenic effects. Majority of parameters were normal, and indices covered those DMH-unexposed and MF-untreated rats ( – ).

Effects of MF on DMH-induced colon carcinogenesis in male rats

In Experiment 2, intestinal tumors were found in majority of DMH-exposed rats ( ).

In group 1 (DMH+water), all rats developed colon tumors (100%). Tumor incidences in different colon parts in group 1 varied: 63% in ascending colon, 100% in descending colon, and 25% in rectum. Moreover, 76.7% of colon tumors were observed in descending colons, 16.7% in ascending colons, and 6.7% in rectums. Maximal effect of 100 mg/kg daily MF dose was observed in the ascending colon, in which DMH-induced carcinogenesis was also completely inhibited ( ). The two MF doses did not affect colon carcinoma incidence in rat rectums and descending colons. Higher MF dose (300 mg/kg) was less effective in suppressing colon carcinogenesis compared with lower amounts (100 mg/kg).

Macroscopically, neoplasms are exophytic or endophytic. Microscopically, malignant intestinal tumors have different types, among which tubular adenocarcinomas are predominant. All carcinoma types are typical in DMH-induced neoplasms[15]. and present the data on the effects of MF on DMH-induced colon tumor development.

Morphological analysis showed that tumors with exophytic growth patterns developed more frequently in the group treated with 100 mg/kg MF compared with DMH + water group. Opposite results were observed with endophytic colon tumors ( ). The group that was treated with lower MF dose had less invasive ( ) and more differentiated tumors ( ) compared with the DMH + water group. shows microphotographs of the observed colon adenocarcinoma types. Tumor size distribution analysis showed that in descending colons of DMH + water and DMH + MF groups, 300 small tumors (< 10 mm2) appeared less frequently compared with the MF group with 100 mg/kg dose (26%, 26%, and 50%, correspondingly). Thus, these data indicate the inhibitory effects of MF on DMH-induced colon carcinogenesis.

3

Microphotographs of 1,2-dimethylhydrazine-induced colon adenocarcinomas. (A) Highly differentiated adenocarcinoma. (B) Moderately differentiated adenocarcinoma. (C) Low differentiated adenocarcinoma (H&E stainning, 70×).

Discussion

The DMH-induced carcinogenesis in epithelial cells includes the following: formation of most active metabolites (methylazoxymethanol or methyldiazohydrate) in the liver; binding of metabolites to glucuronic acid; delivery of conjugates to intestines via blood flow; release of active metabolites through enzymatic activity of intestinal flora (ß-glucuronidase); formation of carbonium ion (CH3+); specific methylation of macromolecules, which in enterocytes, are mainly DNA at O6 position of guanine; miscoding effects[7]. These events result in mutation and activation of Ki-ras oncogene and inactivation of p53[17]. Moreover, reports presented the significant role of free radicals in DMH-induced colon carcinogenesis[18,19]. The present study confirmed the effects of DMH on oxidative stress parameters. Furthermore, we showed the normalizing effect of MF on MDA levels in DMH-treated rats ( ). Bordini et al.[20] observed similar effects of MF in azoxymethane (AOM)-exposed mice. Shortly after starting DMH treatment, exposed organisms experienced significant disturbances in their neuroendocrine and immune systems and lipid and carbohydrate metabolisms. DMH treatment was followed by an increase in sensitivity threshold of the hypothalamus to inhibition by estrogen[21], decrease in hypothalamic biogenic amine content[22], and disturbances in diurnal rhythms at biogenic amine levels in hypothalamic nuclei of rats[23]. Anti-diabetic biguanide treatment alleviated immunodepression in rodents exposed to DMH[24] and AOM[25]. We observed increased levels of glucose, insulin, IGF-1, total cholesterol, triglycerides, MDA, glycated hemoglobin, and VEGF in serum of rats with DMH-induced colon tumors compared with the group without DMH ( ). These findings agree with available data[12,13,26,27]. Furthermore, we observed the normalizing effects of both MF doses on these parameters in DMH-exposed rats ( ). Notably, AST and ALT levels were not different in rats treated with higher and lower MF doses, thereby suggesting the non-toxicity of MF on liver functions.

Table 2 summarizes the data on inhibitory effects of anti-diabetic biguanides on colon carcinogenesis. In most studies, anti-diabetic biguanides inhibited AOM- or DMH-induced colon carcinogenesis in mice and rats. Detailed analysis of experimental results are provided elsewhere[28,29]. MF treatment was followed by decreased levels of proliferation indices, which were evaluated with 5-bromodesoxyuridine, proliferating cell nuclear antigen indices, phosphorylated mechanistic target of rapamycin (mTOR), S6 kinase, and S6 proteins, as revealed by Western blot analysis, in the colonic mucosa of AOM-treated mice[30]. The authors believe that MF suppresses colonic epithelial proliferation by inhibiting the mTOR pathway through 5’ adenosine monophosphate-activated protein kinase activation. However, MF did not affect the level of O6-Methylguanin in the colon or liver of AOM-treated mice. Results showed that MF did not affect the AOM alkylation capacity and carcinogenicity. Therefore, the normalizing effect of MF on neuroendocrine and hormonal metabolic shifts, which occurred in rodents during colon carcinogenesis, are critical in colon cancer prevention.

Zaafar et al.[32] studied the effects of MF on cancer development in diabetic and non- diabetic mice. In diabetic mice, MF treatment alone increased the number of surviving mice compared with the diabetic DMH group. Moreover, MF significantly reduced histopathological scores in diabetic mice colons. Serum VEGF levels in non-diabetic DMH group were not significantly higher than those of non-diabetic saline groups. In non-diabetic mice, MF reduced the serum concentration of VEGF compared with those in the non-diabetic/DMH control. In our study, we observed a tendency toward an increased VEGF level in DMH-treated rats compared with control animals, which were not exposed to carcinogens, whereas MF did not influence this parameter. Statistical analysis showed a significantly reduced histopathologic score for colon mucosa of MF-treated diabetic mice compared with DMH control, whereas in non-diabetic animals, the drug failed to improve the scores. This study highlighted the high susceptibility of diabetic rodents to carcinogenic effects of DMH. Inhibitory effects of MF on DMH-induced colon carcinogenesis were observed in Sprague Dawley rats with type 2 diabetes; the condition was induced with small dose of streptozotocin combined with a high-fat diet[12]. MF treatment was followed by decreases in ACF number, colonic tissue proliferation, and colon tumor incidence, multiplicity, and size. These results and other studies confirmed the role of diabetes as risk factor for cancer and established a connection between glucose levels and development of micro- and macro-vascular complications[37,38]. Our data agree with the other studies, showing that MF is effective in cancer prevention in both diabetic and non-diabetic animals that are exposed to DMH or AOM ( )[10,11]. Moreover, clinical trials demonstrated the decrease in colon cancer risk of type 2 diabetes and non-diabetic patients[39-51]. Meta-analysis of 37 studies comprising more than 1.5 million participants showed that risk of colon cancer mortality was reduced by 23% in MF users compared with non-users[46]. Thus, most epidemiological data and clinical trial results present sufficient evidence of MF efficacy in CRC prevention and treatment in humans. Furthermore, low daily MF dose (500 mg) was effective in reducing cancer risk in type 2 diabetes patients[52]. Our findings on efficacy of lower MF dose in rats agree with clinical data. Given the practical significance of these observations, future investigations are needed to elucidate the advantage of using lower doses of the drug.

2

Effect of anti-diabetic drugs on colon carcinogenesis in rodents

Species, strain	Sex	Carcinogenic agent	Drug	Doses	Route	Effect	Reference
AOM: azoxymethane; DMH: 1,2-dimethylhydrazine; DSS: dextran sodium sulfate. d.w.: drinking water; i.p.: intreperitoneally; ppm: parts per million.
BALB/c mice	Male & Female	AOM	MF	250 mg/kg	Diet	Inhibition	30
BALB/c mice	Male & Female	AOM	MF	250 mg/kg	d.w.	Inhibition	31
BALB/c mice	Male & Female	AOM	MF	250 mg/kg	i.p.	Inhibition	31
BALB/c mice	Male	AOM	MF	250 mg/kg	i.p.	Inhibition	25
BALB/c mice	Female	DMH	MF	250 mg/kg	i.p.	Inhibition	20
Swiss albino mice	Male	DMH	MF	100–200 mg/kg	Oral	Inhibition	32
ICR mice	Male	DMH+DSS	MF	240 mg/kg	Oral	Inhibition	13
F344 rats	Male	AOM	MF	15 mg/kg	d.w.	Inhibition	27
F344 rats	ND	AOM	MF	500–1000 ppm	Diet	No effect	33
F344 rats	ND	AOM	MF	1000 ppm	Diet	No effect	34
LIO rats	Female	DMH	PF	5 mg/rat	Oral	Inhibition	35
LIO rats	Female	DMH	Diabenol	0.1 mg/ml	d.w.	Inhibition	36
SD rats	Male	DMH	MF	150 mg/kg	Oral	Inhibition	12
Wistar rats	Male	DMH	MF	40–360 mg/kg	Oral	Inhibition	13

Acknowledgments

This work was supported in part by a grant from the Russian Foundation for Basic Research (Grant No. 14-04-01653). Authors are very thankful to Dr. A.V. Panchenko and Dr. M.A. Zabezhinski for help and valuable advice during the study.