Abstract
OBJECTIVE To determine if insulin treatment can enhance the antitumor effect of 5-fluorouracil (5-FU), and to explore the mechanism of the enhancement of insulin.
METHODS S180 sarcoma, H22 liver cancer and human Eca-109 esophageal cancer cells were transplanted into nude mice to evaluate the inhibitory effect on tumor growth of insulin alone or in combination with 5-FU. The levels of serum insulin-like growth factor-I (IGF-I) and insulin-like growth factor binding protein-3 (IGFBP-3) were determined.
RESULTS Compared with 5-FU treatment alone, the tumor weight of H22 liver cancer and S180 sarcoma was reduced further with high, medium and low-dose insulin (0.09, 0.06, 0.03 U/20 g) + 5-FU treatment. When a high dosage of insulin + 5-FU was administered, tumor weight was significantly reduced (P < 0.05). The inhibitory rate of growth of S180 sarcoma and H22 liver cancer reached 50.2% and 51.4%, respectively, which was significantly higher than 24.9% and 27.9% in the group receiving 5-FU alone (P < 0.05). High, medium and low-dose insulin combined with 5-FU significantly inhibited the growth of Eca-109 cancer cells (P < 0.05). Compared with the control group, the level of serum IGF-1 decreased (P < 0.05), whereas the level of serum IGFBP-3 slightly increased in the 5-FU ± insulin groups (P > 0.05). In mice with H22 liver cancer and S180 sarcoma the IGF-1 level with high-dose insulin + 5-FU treatment was significantly lower compared to treatment with 5-FU alone (P < 0.05), but the difference was not significant in mice transplanted with esophageal cancer cells.
CONCLUSION Insulin can enhance the anti-tumor effect of 5-FU without significantly increasing 5-FU toxicity. Although changes in the serum IGF-1 or IGFBP-3 level do not explain the mechanism of the insulin-induced enhancement on 5-FU on growth, a decrease in the level of serum IGF-1 and an increase in serum IGFBP-3 may be important in the chemotherapeutic response.
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Introduction
Because cancers often develop resistance to chemotherapeutic drugs, it is important to find agents that can potentiate the drug activity. It is known that slowly growing cancers have tumor cell populations with a low-growth fraction and are less sensitive to chemotherapy compared to rapidly growing tumors with a high-growth fraction[1]. One of the main reasons is that rapidly growing cancers have a higher metabolic level, and therefore are more sensitive to chemotherapeutic drugs. Insulin, as a metabolic accelerant, may be able to increase the metabolism of cancer cells and stimulate them into a cell cycle stage in which they are more sensitive to anticancer drugs. In this study, we determined whether insulin treatment in vivo might enhance the antitumor response to 5-FU, and attempted to define the mechanism of a possible insulin effect.
Materials and Methods
Materials
Regular injectable insulin (10 ml: 400) was obtained from the Wanbang Biochemical Medicine Co. Ltd in Jiangsu Province (Lot No.0406206). 5-FU (Lot No.031002) was from the Oncology Department of the Zhengzhou University First Affiliated Hospital.
Test animals and cancer cells
Male nude mice (BALB/c, 6~8 weeks old, weighing 20g ± 2 g) were supplied by the Shanghai SLAC Laboratory Animal Co. Ltd. Mouse hepatocellular carcinoma H22 and S180 sarcoma were from Zhengzhou University institute of Medical Sciences. Human esophageal Eca-109 cancer cells were from the Institute of Cancer Research, Chinese Academy of Medical Sciences.
Treatment of nude mice with transplanted tumors
About 0.2 ml of a sterile Eca-109 esophageal cancer cell suspension (including 6.0~8.0 × 106 cells) was injected into the right axilla of the mice. Then the mice were randomly divided into 6 groups as follows: control, high dose insulin alone, 5-FU alone, and 5-FU plus different levels of insulin (high, medium or low dose).Three different dosages of insulin were injected subcutaneously into the left abdominal area at 0.09, 0.06 and 0.03 U/20 g, respectively. 5-FU was given intraperitoneally[2] at 10 mg/kg. Insulin was injected 30 min before 5-FU administration. To prevent hypoglycemia development, the mice in every group were given by gavage 50% G~S at 0.5 ml/20 g. At 24 h following tumor transplantation, the mice were treated as indicated once a day for 8 d. The mice were then sacrificed on the following day after the last injection. Serum samples were collected and the tumor tissue weighed. The inhibition rate of tumor growth was calculated according to the following equation[3]:
Murine H22 liver cancer and S180 sarcoma were studied in the same way as the Eca-109 esophageal cancer cells. The above experiment transplanted with different tumors were repeated three times, respectively.
Levels of IGF-1 and IGFBP-3 in the serum
The levels of serum IGF-1 and IGFBP-3 were measured by a ELISA kit (IGF-1: EK0378 and IGFBP-3: EK0387, Wuhan BosterBiologicalTechnology Co. Ltd.) according to the manufacturer’s instructions.
Statistical analysis
All experimental data were processed by a statistical package for science 11.0 (SPSS11.0). Analysis of variance (ANOVA) was used to evaluate the significance of the difference between the experimental groups and control group. Statistical significance was accepted with P < 0.05.
Results
Insulin enhances the in vivo anti-tumor response to 5-FU in mice
The effects of insulin and 5-FU treatment on the growth of transplanted S180 sarcoma and H22 liver cancer are shown in Tables 1 and 2. Compared with 5-FU alone, tumor weight was reduced further in groups receiving 5-FU plus a high, medium or low–insulin dose (0.09, 0.06, 0.03 U/20 g). When the high dosage of insulin + 5-FU was administered, the tumor weight was significantly reduced (P < 0.05) with the inhibitory rate of tumor growth on S180 sarcoma and H22 liver cancer reaching 50.2% and 51.3%, respectively. These inhibitory rates were significantly higher than 24.9% and 27.9% found with 5-FU treatment alone. Table 3 shows the results obtained with transplanted Eca-109 esophageal cancer cells. Insulin of high, medium and low dosage combined with 5-FU inhibited the growth of the tumor more compared to the effect of 5-FU alone (P < 0.05).
Suppressive effect of insulin and 5-FU on growth of transplanted S180 sarcoma (mean ± SD).
Inhibitory effect of insulin and 5-FU on growth of transplanted H22 liver cancer (mean ± SD).
Suppressive effect of insulin and 5-FU on growth of transplanted esophageal cancer cells (mean ± SD).
Analysis of the peripheral hemogram in mice
As shown in Fig.1, the white blood cells (WBC) decreased significantly in the mice receiving 5-FU alone or in combination with insulin groups compared to the controls (P < 0.05). However there was no significant difference between the 5-FU group and 5-FU + insulin groups (P > 0.05).
Group: 1, Control; 2, Insulin alone; 3, 5-FU alone; 4, High-dose insulin+5-FU; 5, Medium-dose insulin + 5-FU; 6, Low-dose insulin + 5-FU.
The serum levels of IGF-I and IGFBP-3 in the mice
As was shown in Figs. 2~4, compared with untreated mice without a tumor transplant, the serum levels of IGF-I were elevated significantly in the control mice with a transplanted tumor (P < 0.05). Compared with the control group, the level of serum IGF-1 decreased significantly (P < 0.05) in the 5-FU ± insulin groups. The serum IGF-1 level in the high-dose insulin + 5-FU group was significantly lower than that in 5-FU-alone group (P < 0.05) in mice transplanted with S180 sarcoma (Fig.3), and H22 liver cancer (Fig.2), but serum IGF-1 levels in insulin + 5-FU groups were not significantly lower than that in 5-FU-alone group in mice transplanted esophageal cancer cell Eca-109 (Fig.4) (P > 0.05).
Group: 1, Untreated; 2, Control; 3, Insulin; 4, 5-FU; 5, High-dose insulin + 5-FU.
Group: 1, Untreated; 2, Control; 3, Insulin; 4, 5-FU; 5, High-dose insulin + 5-FU.
Group: 1, Untreated; 2, Control; 3, Insulin; 4, 5-FU; 5, High-dose insulin + 5-FU; 6, Medium-dose insulin + 5-FU; 7, Low-dose insulin + 5-FU.
Discussion
Treatment of patients with insulin combined with glucose is a safe procedure that is commonly used in clinical practice. Its main function in carbohydrate metabolism is to enhance glucose utilization and to heighten the levels of metabolism in liver, muscle, and adipose tissue. Investigations have shown that insulin can increase the metabolic level of cancer cells[4]. It is known that cell anabolism is active with DNA synthesis in the S phase of the cell cycle, and rapidly growing cancers with higher metabolism are more sensitive to chemotherapeutic agents[1,5]. Therefore insulin may be a good potentiator of anticancer agents.
At present, more oncologists have increased their interest in IPT (Insulin Potential Therapy). Jiao et al.[6] found that insulin could enhance VP-16 efficacy in human esophageal and lung cancer cells. Miglietta et al.[7] reported that insulin could modulate the effect of paclitaxel in MCF-7 cells. However little research has been reported concerning the effect of insulin on the chemotherapeutic responses in vivo, and there have been no reports on the mechanism of the insulin effect. In our study on nude mice transplanted with tumors, high, medium and low-dose insulin was administered 30 min prior to 5-FU injection, showing that the anti-tumor effect of 5-FU was enhanced without increasing toxicity.
IGF-I circulates bound to proteins, mainly IGFBP-3, and it is important in cell growth and appears to be associated with tumorigenesis[8]. Epidemiologic studies have shown that high levels of circulating insulin and IGF-1 are associated with increased risk of many cancers[9,10], and poor prognosis was significantly correlated with elevated IGF-1 levels[9], whereas the higher plasma levels of insulin-like growth factor binding protein-3 (IGFBP-3) have been related to a decreased cancer risk[10]. The biological effects of insulin are similar to those of IGF-I, and these effects are mediated by insulin receptors, IGF-I receptors or even hybrid insulin/insulinlike growth factor receptors. Many studies have indicated that IGF-I can act as a survival agent, by influencing the efficacy of cytotoxic drugs and playing an important role in the induction of resistance[11,12]. In our study, although changes in serum IGF-1 or IGFBP-3 levels can not explain the mechanism of insulin-induced enhancement of 5-FU, the decreased level of serum IGF-1 may predict an improved outcome to chemotherapy response. Further study will be necessary to shed light on the mechanism of the effects of insulin.
In short, insulin, a biochemical modulator has shown to enhance the therapeutic effects of 5-FU, so insulin may be potentially useful in clinical cancer chemotherapy.
Footnotes
This work was supported by a grant from the Project for Science and Technology Creative Talents of Henan Province, China (No. 2005026).
- Received January 13, 2008.
- Accepted July 1, 2008.
- Copyright © 2008 by Tianjin Medical University Cancer Institute & Hospital and Springer
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