• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Cancer Res. Author manuscript; available in PMC Aug 15, 2010.
Published in final edited form as:
PMCID: PMC2753241
NIHMSID: NIHMS127670

Metformin Disrupts Crosstalk Between G protein-Coupled Receptor and Insulin Receptor Signaling Systems and Inhibits Pancreatic Cancer Growth

Abstract

Recently we identified a novel crosstalk between insulin and G-protein-coupled receptor (GPCR) signaling pathways in human pancreatic cancer cells. Insulin enhanced GPCR signaling through a rapamycin-sensitive mTOR-dependent pathway. Metformin, the most widely used drug in the treatment of type-2 diabetes, activates AMP kinase (AMPK), which negatively regulates mTOR. Here, we determined whether metformin disrupts crosstalk between insulin receptor and GPCR signaling in pancreatic cancer cells. Treatment of human pancreatic cancer cells (PANC-1, MIAPaCa-2, BxPC-3) with insulin (10ng/ml) for 5 min markedly enhanced the increase in intracellular [Ca2+] induced by GPCR agonists (e.g. neurotensin, bradykinin, angiotensin II). Metformin pretreatment completely abrogated insulin-induced potentiation of Ca2+ signaling but did not interfere with the effect of GPCR agonists alone. Insulin also enhanced GPCR agonist-induced growth, measured by DNA synthesis, and numbers of cells cultured in adherent or non-adherent conditions. Low doses of metformin (0.1-0.5 mM) blocked stimulation of DNA synthesis, anchorage-dependent and independent growth induced by insulin and GPCR agonists. Treatment with metformin induced striking and sustained increase in the phosphorylation of AMPK at Thr172 and a selective AMPK inhibitor (compound C, at 5μM) reversed the effects of metformin on [Ca2+]i, and DNA synthesis, indicating that metformin acts through AMPK activation. In view of these results we tested whether metformin inhibits pancreatic cancer growth. Administration of metformin significantly decreased the growth of MIAPaCa-2 and PANC-1 cells xenografted on the flank of nude mice. The results raise the possibility that metformin could be a potential candidate in novel treatment strategies for human pancreatic cancer.

INTRODUCTION

Ductal adenocarcinoma of the pancreas is a devastating disease, with overall 5-year survival rate of only 3-5%. The incidence of this disease in the US has increased recently to more than 37,000 new cases each year and is now the fourth leading cause of cancer mortality in both men and women. As the current therapies offer very limited survival benefits, novel therapeutic strategies are urgently required to treat this aggressive disease.

G protein-coupled receptors (GPCRs) and their cognate agonists are increasingly implicated as autocrine/paracrine growth factors for multiple solid tumors, including small cell lung cancer, colon, prostate, breast and pancreas (1-3). Pancreatic cancer cell lines express multiple functional GPCRs using a Ca2+ mobilization assay as indicator of productive ligand-receptor interactions (4). A variety of GPCR agonists, including neurotensin, angiotensin II (ANG II) and bradykinin, stimulated DNA synthesis in pancreatic cancer cell lines, including PANC-1 and MIAPaCa-2 (4-7). Furthermore, a broad-spectrum GPCR antagonist inhibited the growth of pancreatic cancer cells either in vitro or xenografted into nu/nu mice (8). Other studies demonstrated increased expression of ANG II and neurotensin GPCRs in pancreatic cancer tissues (9-12). More recently, we identified a novel crosstalk between insulin/IGFI receptors and GPCR signaling systems in pancreatic cancer cells, leading to enhancement of GPCR-induced early signaling (13), including Ins(1,4,5)P3 generation and increase in the intracellular Ca2+ concentration ([Ca2+]i). Insulin-induced potentiation of GPCR signaling was mediated through the phosphatidylinositol 3-kinase (PI3K)/Akt/mTOR signaling module (13), a key pathway in insulin/IGF action (14). These findings assume an added importance in view of the large number of epidemiological studies linking long standing type-2 diabetes, obesity and metabolic syndrome, characterized by peripheral insulin resistance and compensatory overproduction of insulin, with increased risk for developing pancreatic cancer [see (15) for review]. Given the complexity of the pancreatic microcirculation (16) and the close topographical relationship between the islets and small ducts (17), in these diseases the locally overproduced insulin is thought to act directly on ductal pancreatic cancer cells.

Metformin (1,1-dimethylbiguanide hydrochloride), one of the most widely prescribed drugs for treatment of type-2 diabetes, stimulates AMP-activated protein kinase (AMPK) in intact cells (18). AMPK activity is switched on by phosphorylation on Thr172 in the activation loop of the catalytic subunit by LKB-1/STK11, the product of the Peutz-Jegher syndrome tumor-suppressor gene (19). Major downstream targets of AMPK include TSC2 and raptor (20, 21). The AMPK-mediated phosphorylation of these targets leads to inhibition of mTOR complex 1 (mTORC1) activity. Interestingly, a recent epidemiological report linked administration of metformin with a 62% reduced risk of pancreatic cancer in patients with type-2 diabetes mellitus (22). In addition, metformin prevented carcinogen-induced pancreatic cancer induction in hamsters maintained in high-fat diets (23) and inhibited the growth of breast and colon cancer cells (24, 25). Despite its potential clinical implications, there is no understanding of the precise mechanism(s) by which metformin inhibits the proliferation of cancer cells and it is not known whether metformin has any direct effect on pancreatic cancer growth.

In this study, we show that metformin disrupts crosstalk between insulin receptor and GPCR signaling systems in pancreatic cancer cells. Specifically, metformin prevented insulin-induced augmentation of Ca2+ signaling, DNA synthesis and anchorage-independent proliferation in response to stimulation with GPCR agonists in PANC-1 and MIAPaCa-2 pancreatic cancer cells. Metformin inhibited these signaling systems through AMPK. Furthermore, our results show that metformin administration inhibits the growth of PANC-1 and MIAPaCa-2 tumor xenografts in vivo.

MATERIALS and METHODS

Cells and Culture Conditions

The human pancreatic cancer cell lines PANC-1, MIAPaCa-2, BxPC-3 and AsPC-1 were obtained from the American Type Culture Collection (ATCC, Manassas, VA). PANC-1 and MIAPaCa-2 cells were grown in DMEM with 2 mM glutamine, 1 mM Na-pyruvate, 100 units/mL penicillin, and 100 μg/mL streptomycin and 10% fetal bovine serum (FBS) at 37°C in a humidified atmosphere containing 10% CO2. BxPC-3 cells and AsPC-1 cells were grown in RPMI-1640 containing 100 units/mL penicillin, 100 μg/mL streptomycin and 10% FBS at 37°C in a humidified atmosphere containing 5% CO2.

Measurement of [Ca2+]i

Ca2+ signaling was determined in cells grown on glass coverslips for 4-5 days, as previously described (13).

[3H]-Thymidine Incorporation into DNA

PANC-1, MIAPaCa-2, BxPC-3 and AsPC-1 cells (5 × 104) were plated and grown in 3.5 cm tissue culture plates in DMEM with 2 mM glutamine, 1 mM Na-pyruvate (PANC-1 and MIAPaCa-2 cells) or RPMI-1640 (BxPC-3 and AsPC-1 cells) and 10% FBS. After 5 days, the cultures were washed twice with phosphate-buffered saline (PBS) and incubated in serum-free medium. After 24h, the cultures were transferred to fresh medium containing the specified concentration of agonist and/or inhibitor for 17 h and then pulse-labeled for 6 h with [3H]-thymidine (0.25 μCi/ml). The acid-insoluble radioactivity was determined as previously described (7).

Western Blot Analysis

Confluent cultures of PANC-1 cells grown on 6 cm dishes were washed twice with PBS and incubated with serum-free medium for 3 h. Metformin was added at 5 mM and the cultures were incubated at 37°C for the specified times (0 min-24 h). To detect the activation of AMPKα, the cultures were washed in cold PBS and directly lysed in 2× SDS-PAGE sample buffer [200 mM Tris-HCl (pH 6.8), 2 mM EDTA, 0.1 M Na3VO4, 6% SDS, 10% glycerol, and 4% 2-mercaptoethanol]. The lysates were subjected to SDS-PAGE on 10% gels and separated proteins were transferred to Immobilon-P membranes (Millipore, Billerica, MA). Western blots were then performed on membranes incubated overnight with phospho-AMPKα (Thr172) or phospho-ERK1/2 (Thr-202 and Tyr-204) monoclonal antibodies at a dilution of 1:1,000 in PBS containing 5% nonfat dried milk and 0.1% Tween-20. The immunoreactive bands were detected with ECL (enhanced chemiluminescence) reagents (Amersham, Piscataway, NJ). The same membranes were subsequently stripped and probed in a similar fashion with anti-AMPKα polyclonal antibody or anti-ERK at a dilution of 1:2,000 in PBS containing 5% nonfat dried milk and 0.1% Tween-20.

Anchorage-Dependent Cell Proliferation

PANC-1 and MIAPaCa-2 cells (2 × 104) were plated on tissue culture 12-well plates in DMEM containing 1% FBS. After 24 h incubation at 37°C, 5 nM neurotensin, in the absence or presence of 10 ng/ml insulin, was added with or without 1mM metformin. The cultures were then incubated for 7-9 days and the total cell count was determined from a minimum of four wells per condition using a Coulter counter, after cell clumps were disaggregated by passing the cell suspension ten times through a 19- and subsequently a 21-gauge needle.

Anchorage-Independent Cell Proliferation

Cells were plated on tissue culture 12-well plates coated with polyhydroxyethylmethacrylate [Poly-(HEMA)] as previously described (7). Under these conditions, cells are denied attachment to the substratum. Cultures of PANC-1 or BxPC-3 cells, 3-5 days after passage, were washed and suspended in DMEM (PANC-1) or RPMI-1640 (BxPC-3 cells). Cells were then disaggregated by two passes through a 19-guage needle into an essentially single-cell suspension as judged by microscopy. Cell number was determined using a Coulter Counter, and 2 × 104 cells were seeded in either DMEM containing 1% FBS on the [Poly-(HEMA)]-coated dishes (PANC-1 cells) or in RPMI containing 1% FBS (BxPC-3 cells). After 24 h incubation at 37°C, 5 nM neurotensin (PANC-1 cells) or 5 nM bradykinin (BxPC-3 cells), in the absence or in the presence of 10 ng/ml insulin was added with or without 1mM metformin. The cultures were then incubated in a humidified atmosphere containing 10% CO2 at 37°C for 7-9 days and the total cell count was determined from a minimum of four wells per condition using a Coulter counter, after cell clumps were disaggregated by passing the cell suspension ten times through a 19- and subsequently a 21-gauge needle.

Mice xenografts

Early-passage PANC-1 or MIAPaCa-2 cells were harvested, and 2×106 cells were implanted into the right flanks of male nu/nu mice. The male nu/nu mice were maintained in specific pathogen-free facility at University of California at Los Angeles (UCLA). The UCLA Chancellor's Animal Research Committee approved all the animal experiments.

The animals were randomized into control and treated groups (10 mice per group). Treatment was initiated when the tumors reached a mean diameter of 2 mm, and the 1st day of treatment in both cases was designated as day 0. For injection, metformin was dissolved in sterile saline and was given once daily intraperitoneally at 250 mg/kg (50 μL/mouse). The control group received vehicle only (50 μL saline). Tumor volume (V) was measured by external caliper every 4 days and it was calculated as V = 0.52 (length x width2). The treatment was continued until any of the tumors reached ~1.5 cm length when all the animals were sacrificed and the tumors removed. The volume of the excised tumors was calculated as V = 0.52 (length × width × depth).

Chemicals and Reagents

DMEM was obtained from Gibco (Carlsbad, CA). Neurotensin, angiotensin, bradykinin and insulin were obtained from Sigma Chemical (St. Louis, MO). Metformin was obtained from Sigma-Aldrich (St. Louis, MO). The AMPK inhibitor, compound C, was purchased from Calbiochem. An antibody that detects the phosphorylated state of Thr172 of AMPKα and an antibody against AMPKα were purchased from Millipore (Charlottesville, VA). An antibody that detects the phosphorylated state of Thr-202 and Tyr-204 of ERK1/2 and an antibody against ERK 1/2 were purchased from Cell Signaling Technology (Danvers, MA). Horseradish peroxidase-conjugated antirabbit IgG and antimouse IgG were from Amersham Biosciences. All other reagents were of the highest grade available.

Statistical analysis

The values obtained are presented as the mean ± SEM and analyzed with Student's t test, using SigmaPlot 2000 (SPSS).

RESULTS

Metformin blocks the crosstalk between insulin and GPCR receptor signaling on intracellular Ca2+ mobilization

As a first step to investigate whether metformin disrupts crosstalk between the insulin/IGF and GPCR signaling systems, we determined whether it prevents the stimulatory effect of insulin on GPCR-induced increase in [Ca2+]i in pancreatic cancer cells. As shown in Fig. 1A, addition of neurotensin (5 nM) to PANC-1 cells induced a rapid increase in [Ca2+]i from a basal level of 142 ± 15 nM (mean ± SEM; n = 20) to a peak value of 735 ± 75 nM (n = 20) at 25-35 sec, which subsequently declined toward a plateau phase (Fig. 1A, left panel black trace, right panel open bar). In contrast, addition of insulin (10 ng/ml) to PANC-1 cells did not produce any detectable change in [Ca2+]i. However, treatment of PANC-1 cells with 10 ng/ml insulin for 5 min prior to stimulation markedly enhanced neurotensin-induced increase in [Ca2+]i that now reached a peak value of 1185 ± 134 nM (n = 25) (Fig. 1A, grey trace and black bar).

Figure 1
Metformin blocks enhanced Ca2+ mobilization induced by neurotensin, bradykinin or angiotensin in insulin-treated human pancreatic cancer cells

Pretreatment with 5 mM metformin for 1 h did not have any significant effect on the [Ca2+]i increase induced by neurotensin alone (Fig. 1A, lower middle panel, striped bar), but completely blocked the enhancing effect of insulin on neurotensin-induced Ca2+ mobilization (peak value 559 ± 102 nM, upper middle panel and grey bar). Similar results were obtained with either MIAPaCa-2 cells stimulated with neurotensin (Fig. 1B, left) or BxPC-3 cells stimulated with either bradykinin or angiotensin (Fig. 1B, middle and right). These results indicate that exposure to metformin abrogates crosstalk between insulin and GPCR signaling systems in a variety of pancreatic cancer cell lines.

Metformin abolished the crosstalk between insulin and GPCR receptor signaling systems on DNA synthesis and cell proliferation in pancreatic cancer cells

We next determined whether crosstalk between GPCR agonists and insulin in pancreatic cancer cells leads to long-term biological responses, including ERK signaling, DNA synthesis and cell proliferation. Cultures of PANC-1, MIAPaCa-2 or BxPC-3 cells were incubated in media containing increasing doses of metformin with neurotensin (PANC-1, MIAPaCa-2) or bradykinin (BxPC-3) in the absence or presence of insulin, as indicated in Fig 2. After 17 h of incubation, the cultures were pulse-labeled with [3H]-thymidine. The results shown in Fig. 2 demonstrate that either insulin or GPCR agonists stimulated DNA synthesis in PANC-1 (A), MIAPaCa-2 (C) and BxPC-3 (D) cells. The combination of the GPCR agonists and insulin (black bars) induced a striking enhancement in DNA synthesis in all the three cell lines and induced marked prolongation of ERK signaling, as shown by Western blot analysis of lysates of PANC-1 cells treated with neurotensin and insulin (Fig. 2 B).

Figure 2
Metformin inhibits DNA synthesis induced by GPCR agonists and insulin in PANC-1, MIAPaCa-2 and BxPC-3 cells

The salient feature of the results shown in Fig. 2 is that metformin abolished the increase in DNA synthesis induced by the combination of neurotensin and insulin in PANC-1 cells (Fig. 2A) and prevented the prolonged ERK signaling induced by neurotensin and insulin in PANC-1 cells (Fig 2B). Similarly, metformin prevented stimulation of DNA synthesis induced by neurotensin and insulin in MIAPaCa-2 cells (Fig 2C) and bradykinin and insulin in BxPC-3 cells (Fig. 2D).

In order to determine whether metformin inhibits the proliferation of pancreatic cancer cells, single cell suspensions of PANC-1 cells were plated on normal culture dishes (anchorage-dependent growth, Fig. 3A) or on culture dishes coated with poly-(HEMA), which prevents the adhesion of the cells to the substratum (anchorage-independent growth, Fig. 3B). The cells were incubated in media supplemented with neurotensin, insulin or neurotensin and insulin in the presence or absence of 1mM metformin. As illustrated in Fig. 3, the combination of neurotensin and insulin promoted a marked increase in cell number in both anchorage-dependent and anchorage-independent conditions. Addition of metformin markedly inhibited the stimulation of either anchorage-dependent or anchorage-independent growth induced by the combination of neurotensin and insulin in PANC-1. Metformin inhibited the stimulation of anchorage-dependent growth of MIAPaCa-2 cells induced by neurotensin and insulin (Supplementary Fig. S.1) and the anchorage-independent growth of BxPC-3 cells induced by bradykinin and insulin (Supplementary Fig. S.2). The results demonstrate that metformin directly inhibits DNA synthesis and proliferation induced by insulin and GPCR agonists in pancreatic cancer cells.

Figure 3
Metformin inhibits anchorage-dependent and independent proliferation of PANC-1 cells in response to neurotensin, insulin and their combination

Metformin inhibits crosstalk between insulin and GPCR signaling systems through AMPK

AMPK activity is switched on by phosphorylation on Thr172 in the activation loop of the catalytic subunit by LKB-1/STK11. In order to determine whether metformin stimulates AMPK activation in human pancreatic cancer cells, we used Western blot analysis with an antibody that detects the phosphorylated state of the α subunit of AMPK on Thr172. As shown in Fig. 4A, treatment of PANC-1 cells with metformin induced striking and sustained increase in AMPK phosphorylation on Thr172 indicating that metformin treatment induces AMPK activation in PANC-1 cells.

Figure 4
Metformin disrupts crosstalk between insulin receptor and GPCR signaling systems through AMPK

Next, we determined whether a specific inhibitor of AMPK [compound C, ref. (26)] reverses the inhibitory effects of metformin on insulin-induced augmentation of GPCR-mediated Ca2+ signaling. As shown in Fig. 4B, treatment with compound C (5 μM) reversed the inhibitory effect of metformin on neurotensin-induced Ca2+ signaling in insulin-pretreated PANC-1 cells. Similar reversal of metformin inhibition was obtained with BxPC-3 cells treated with insulin and stimulated with bradykinin (Supplementary Fig. S.3).

We also determined whether the selective inhibitor of AMPK counteracts the inhibitory effects of metformin on DNA synthesis induced by neurotensin and insulin in PANC-1. As shown in Fig. 4C, compound C (5 μM) reversed the inhibitory effect of metformin on DNA synthesis induced by either insulin or by the combination of insulin and neurotensin in PANC-1 cells. Addition of compound C in the absence of metformin did not induce any significant effect (the levels of DNA synthesis were similar to those shown by the open bars in Fig. 4C). Treatment with compound C (5 μM) also reversed the inhibitory effect of metformin on DNA synthesis induced by bradykinin and insulin in BxPC-3 cells (Supplementary Fig. S.4).

In line with the results presented above, LKB-1/STK11 is expressed in many pancreatic cancer cells, including PANC-1, MIAPaCa-2 and BxPC-3. However, LKB1 is silenced by promoter hypermethylation in the pancreatic cancer cell AsPC-1 (27). If the effects of metformin are mediated by AMPK, AsPC-1 cells (in contrast to PANC-1, MIAPaCa-2 and BxPC-3) should be refractory to metformin because these cells do not express LKB-1/STK11, the upstream kinase that phosphorylates and activates AMPK in response to metformin. Treatment of AsPC-1 cells with 1 mM metformin did not prevent the stimulation of DNA synthesis induced by neurotensin and insulin in these cells, in contrast to results obtained in parallel cultures of PANC-1 cells (Supplementary Fig. S.5). Taken together, these results indicate that metformin disrupts the crosstalk between insulin and GPCR signaling systems through AMPK in human pancreatic cancer cells.

Metformin inhibits the growth of PANC-1 or MIAPaCa-2 xenografts in nude mice

Given our results showing inhibitory effects of metformin on signaling and proliferation in pancreatic cancer cells, we next examined whether this compound could inhibit pancreatic cancer growth using PANC-1 and MIAPaCa-2 tumor xenografts in nude mice. The animals were randomized into control and metformin-treated groups (10 mice per group). Treatment was initiated when the tumors reached a mean diameter of 2 mm. Metformin was given once daily intraperitoneally at 250 mg/kg for the duration of the experiment. As shown in Fig. 5, administration of metformin strikingly decreased the growth of either PANC-1 or MIAPaCa-2 cells xenografted in nude mice. For example, the tumor volumes of PANC-1 xenografts at the end of the experiment (day 41) were 286.11 ± 50.95 mm3 in the control and 98.47 ± 29.20 mm3 in the metformin treated group (p=0.0057) (Fig. 5A, inset). MIAPaCa-2 xenografts at the end of the experiment (Day 25) were 228.35 ± 40.99 mm3 in the control and 92.67 ± 20.10 mm3 in the metformin treated group (p=0.0072) (Fig. 5B, inset). These results show, for the first time, that metformin inhibits the growth of human pancreatic cancer cells xenografted into nude mice.

Figure 5
Metformin inhibits the growth of PANC-1 and MIAPaCa-2 tumor xenografts

DISCUSSION

Recently, we identified a novel crosstalk between insulin/IGFI receptors and GPCR signaling systems in pancreatic cancer cells, leading to enhancement of GPCR-induced early signaling (13). Insulin-induced potentiation of Gq signaling was prevented by either inhibitors of PI 3-kinase or by rapamycin, a specific inhibitor of mTOR complex 1 (mTORC1). These findings indicated that in addition to its well established role in the regulation of protein synthesis, the rapamycin-sensitive PI 3-kinase/TORC1/S6K pathway mediates a novel crosstalk between insulin receptor on GPCR signaling systems (13). Results presented in this study showed that insulin also enhances long-term biological responses induced by GPCR agonists in pancreatic cancer cells, including DNA synthesis, proliferation and anchorage-independent growth. We conclude that potentiation of Gq signaling by insulin through an mTOR-dependent pathway provides a crosstalk mechanism by which insulin enhances the mitogenic responsiveness of pancreatic cancer cells to Gq-coupled receptor agonists.

The biguanide metformin is the most widely prescribed drug for treatment of type-2 diabetes, worldwide. The primary systemic effect of metformin is the lowering of blood glucose levels through reduced hepatic gluconeogenesis and increased glucose uptake in skeletal muscles and adipose tissue (28). Metformin not only lowers blood glucose but also reduces the hyperinsulinemia associated with insulin resistance. At the cellular level, metformin is known to stimulate activation of AMPK (18), a conserved regulator of the cellular response to low energy that it is activated when ATP concentrations decrease and 5'-AMP concentrations increase in response to nutrient deprivation, hypoxia and metformin administration (19). AMP directly binds to the AMPK γ subunit, causing a conformational change that exposes Thr172 in the activation loop of the α subunit, allowing it to be phosphorylated by LKB-1/STK11, the product of the Peutz-Jegher syndrome tumor-suppressor gene (19). Interestingly, the Peutz-Jegher syndrome is characterized by predisposition to various gastrointestinal neoplasms, including pancreatic adenocarcinoma (29) and intraductal papillary-mucinous neoplasms (30).

Because metformin-induced activation of AMPK inhibits mTOR function via TSC2 and raptor phosphorylation (20, 21, 31, 32), we hypothesized that metformin disrupts the crosstalk between insulin receptor and Gq signaling via AMPK. Here, we present several lines of evidence supporting this hypothesis: 1) metformin completely blocked the enhancing effect of insulin on neurotensin-induced Ca2+ mobilization in either PANC-1 or MIAPaCa-2 cells stimulated with neurotensin or in BxPC-3 cells stimulated with either bradykinin or angiotensin; 2) metformin abolished the increase in DNA synthesis induced by neurotensin and insulin in PANC-1 or MIAPaCa-2 cells. Similarly, metformin prevented the stimulation of DNA synthesis promoted by bradykinin and insulin in BxPC-3 cells; 3) pancreatic cancer cell treatment with metformin produced a marked and sustained increase in AMPK activation, as judged by Thr172 phosphorylation; 4) treatment with compound C, a specific inhibitor of AMPK, reversed the inhibitory effect of metformin on neurotensin-induced Ca2+ signaling in insulin-pretreated PANC-1 cells. Similar reversal of metformin inhibition was obtained with BxPC-3 cells treated with insulin and stimulated with bradykinin; 5) compound C also reversed the inhibitory effect of metformin on DNA synthesis induced by the combination of insulin and neurotensin in PANC-1 cells or by the combination of bradykinin and insulin in BxPC-3 cells 6) metformin did not disrupt crosstalk between insulin and GPCR signaling systems in the pancreatic cancer AsPC-1 cells which do not express LKB1/STK11 (27), the upstream kinase that phosphorylates and activates AMPK. These results indicate that metformin disrupts crosstalk between insulin and GPCR signaling systems through AMPK in human pancreatic cancer cells.

Recent epidemiological reports linked administration of metformin with a reduced incidence and improved prognosis in cancer patients (33, 34). A recent study showed a statistically significant association between metformin therapy and reduced risk of pancreatic cancer in patients with type-2 diabetes mellitus (22). In addition to these epidemiological associations, carcinogen-induced pancreatic cancer in hamsters maintained on high-fat diets was prevented by metformin (23) and metformin inhibited the growth of breast and colon cancer cells (24, 25, 35). However, the precise mechanisms involved remain incompletely understood. In view of the direct inhibitory effects of metformin on signaling and proliferation of pancreatic cancer cells shown in the present study, we examined whether this compound inhibits pancreatic cancer growth using PANC-1 and MIAPaCa-2 tumor xenografts in nude mice. Our results show, for the first time, that metformin markedly inhibits the growth of human pancreatic cancer cells xenografted into nude mice.

In conclusion, our results raise the attractive possibility that treatment with metformin, a widely used agent used to counteract metabolic syndrome and type-2 diabetes, directly inhibits pancreatic cancer cell proliferation. The results provide a basis for novel therapeutic strategies for the treatment of pancreatic cancer, a devastating disease with limited survival options.

Supplementary Material

Acknowledgments

This work was supported by NIH Grants R21CA137292, RO1DK56930, RO1DK55003 and P30DK41301 (to ER).

REFERENCES

1. Rozengurt E. Neuropeptides as growth factors for normal and cancer cells. Trends Endocrinol Metabol. 2002;13:128–34. [PubMed]
2. Rozengurt E. Mitogenic signaling pathways induced by G protein-coupled receptors. J Cell Physiol. 2007;213:589–602. [PubMed]
3. Dorsam RT, Gutkind JS. G-protein-coupled receptors and cancer. Nat Rev Cancer. 2007;7:79–94. [PubMed]
4. Ryder NM, Guha S, Hines OJ, Reber HA, Rozengurt E. G protein-coupled receptor signaling in human ductal pancreatic cancer cells: Neurotensin responsiveness and mitogenic stimulation. J Cell Physiol. 2001;186:53–64. [PubMed]
5. Guha S, Rey O, Rozengurt E. Neurotensin Induces Protein Kinase C-dependent Protein Kinase D Activation and DNA Synthesis in Human Pancreatic Carcinoma Cell Line PANC-1. Cancer Res. 2002;62:1632–40. [PubMed]
6. Guha S, Lunn JA, Santiskulvong C, Rozengurt E. Neurotensin Stimulates Protein Kinase C-dependent Mitogenic Signaling in Human Pancreatic Carcinoma Cell Line PANC-1. Cancer Res. 2003;63:2379–87. [PubMed]
7. Kisfalvi K, Guha S, Rozengurt E. Neurotensin and EGF induce synergistic stimulation of DNA synthesis by increasing the duration of ERK signaling in ductal pancreatic cancer cells. J Cell Physiol. 2005;202:880–90. [PubMed]
8. Guha S, Eibl G, Kisfalvi K, et al. Broad-spectrum G protein-coupled receptor antagonist, [DArg1,D-Trp5,7,9,Leu11]SP: a dual inhibitor of growth and angiogenesis in pancreatic cancer. Cancer Res. 2005;65:2738–45. [PubMed]
9. Elek J, Pinzon W, Park KH, Narayanan R. Relevant genomics of neurotensin receptor in cancer. Anticancer Res. 2000;20:53–58. [PubMed]
10. Wang L, Friess H, Zhu Z, et al. Neurotensin receptor-1 mRNA analysis in normal pancreas and pancreatic disease. Clin Cancer Res. 2000;6:566–71. [PubMed]
11. Reubi JC, Waser B, Friess H, Bèuchler M, Laissue J. Neurotensin receptors: a new marker for human ductal pancreatic adenocarcinoma. Gut. 1998;42:546–50. [PMC free article] [PubMed]
12. Arafat HA, Gong Q, Chipitsyna G, et al. Antihypertensives as novel antineoplastics: angiotensin-I-converting enzyme inhibitors and angiotensin II type 1 receptor blockers in pancreatic ductal adenocarcinoma. J Am Coll Surg. 2007;204:996–1005. [PubMed]
13. Kisfalvi K, Rey O, Young SH, Sinnett-Smith J, Rozengurt E. Insulin Potentiates Ca2+ Signaling and Phosphatidylinositol 4,5-Bisphosphate Hydrolysis Induced by Gq Protein-Coupled Receptor Agonists through an mTOR-Dependent Pathway. Endocrinology. 2007;148:3246–57. [PubMed]
14. Taniguchi CM, Emanuelli B, Kahn CR. Critical nodes in signalling pathways: insights into insulin action. Nat Rev Mol Cell Biol. 2006;7:85–96. [PubMed]
15. Giovannucci E, Michaud D. The role of obesity and related metabolic disturbances in cancers of the colon, prostate, and pancreas. Gastroenterology. 2007;132:2208–25. [PubMed]
16. Ballian N, Brunicardi FC. Islet vasculature as a regulator of endocrine pancreas function. World J Surg. 2007;31:705–14. [PubMed]
17. Bertelli E, Regoli M, Orazioli D, Bendayan M. Association between islets of Langerhans and pancreatic ductal system in adult rat. Where endocrine and exocrine meet together? Diabetologia. 2001;44:575–84. [PubMed]
18. Hardie DG. AMP-activated protein kinase as a drug target. Annu Rev Pharmacol Toxicol. 2007;47:185–210. [PubMed]
19. Kahn BB, Alquier T, Carling D, Hardie DG. AMP-activated protein kinase: ancient energy gauge provides clues to modern understanding of metabolism. Cell Metab. 2005;1:15–25. [PubMed]
20. Inoki K, Ouyang H, Zhu T, et al. TSC2 integrates Wnt and energy signals via a coordinated phosphorylation by AMPK and GSK3 to regulate cell growth. Cell. 2006;126:955–68. [PubMed]
21. Gwinn DM, Shackelford DB, Egan DF, et al. AMPK phosphorylation of raptor mediates a metabolic checkpoint. Mol Cell. 2008;30:214–26. [PMC free article] [PubMed]
22. Li D, Yeung SC, Hassan MM, Konopleva M, Abbruzzese JL. Anti-diabetic therapies affect risk of pancreatic cancer. Gastroenterology. In Press, Accepted Manuscript. [PMC free article] [PubMed]
23. Schneider MB, Matsuzaki H, Haorah J, et al. Prevention of pancreatic cancer induction in hamsters by metformin. Gastroenterology. 2001;120:1263–70. [PubMed]
24. Dowling RJ, Zakikhani M, Fantus IG, Pollak M, Sonenberg N. Metformin Inhibits Mammalian Target of Rapamycin Dependent Translation Initiation in Breast Cancer Cells. Cancer Res. 2007;67:10804–12. [PubMed]
25. Buzzai M, Jones RG, Amaravadi RK, et al. Systemic treatment with the antidiabetic drug metformin selectively impairs p53-deficient tumor cell growth. Cancer Res. 2007;67:6745–52. [PubMed]
26. Zhou G, Myers R, Li Y, et al. Role of AMP-activated protein kinase in mechanism of metformin action. J Clin Invest. 2001;108:1167–74. [PMC free article] [PubMed]
27. Qanungo S, Haldar S, Basu A. Restoration of silenced Peutz-Jeghers syndrome gene, LKB1, induces apoptosis in pancreatic carcinoma cells. Neoplasia. 2003;5:367–74. [PMC free article] [PubMed]
28. Shaw RJ, Lamia KA, Vasquez D, et al. The kinase LKB1 mediates glucose homeostasis in liver and therapeutic effects of metformin. Science. 2005;310:1642–6. [PMC free article] [PubMed]
29. Su GH, Hruban RH, Bansal RK, et al. Germline and somatic mutations of the STK11/LKB1 Peutz-Jeghers gene in pancreatic and biliary cancers. Am J Pathol. 1999;154:1835–40. [PMC free article] [PubMed]
30. Sato N, Rosty C, Jansen M, et al. STK11/LKB1 Peutz-Jeghers gene inactivation in intraductal papillary-mucinous neoplasms of the pancreas. Am J Pathol. 2001;159:2017–22. [PMC free article] [PubMed]
31. Inoki K, Zhu T, Guan KL. TSC2 mediates cellular energy response to control cell growth and survival. Cell. 2003;115:577–90. [PubMed]
32. Shaw RJ, Bardeesy N, Manning BD, et al. The LKB1 tumor suppressor negatively regulates mTOR signaling. Cancer Cell. 2004;6:91–9. [PubMed]
33. Evans JM, Donnelly LA, Emslie-Smith AM, Alessi DR, Morris AD. Metformin and reduced risk of cancer in diabetic patients. BMJ. 2005;330:1304–5. [PMC free article] [PubMed]
34. Bowker SL, Majumdar SR, Veugelers P, Johnson JA. Increased cancer-related mortality for patients with type 2 diabetes who use sulfonylureas or insulin. Diabetes Care. 2006;29:254–8. [PubMed]
35. Zakikhani M, Dowling R, Fantus IG, Sonenberg N, Pollak M. Metformin is an AMP kinase-dependent growth inhibitor for breast cancer cells. Cancer Res. 2006;66:10269–73. [PubMed]
PubReader format: click here to try

Formats: