Metformin inhibits pancreatic cancer cell and tumor growth and downregulates Sp transcription factors

Abstract

Metformin is a widely used antidiabetic drug, and epidemiology studies for pancreatic and other cancers indicate that metformin exhibits both chemopreventive and chemotherapeutic activities. Several metformin-induced responses and genes are similar to those observed after knockdown of specificity protein (Sp) transcription factors Sp1, Sp3 and Sp4 by RNA interference, and we hypothesized that the mechanism of action of metformin in pancreatic cancer cells was due, in part, to downregulation of Sp transcription factors. Treatment of Panc1, L3.6pL and Panc28 pancreatic cancer cells with metformin downregulated Sp1, Sp3 and Sp4 proteins and several pro-oncogenic Sp-regulated genes including bcl-2, survivin, cyclin D1, vascular endothelial growth factor and its receptor, and fatty acid synthase. Metformin induced proteasome-dependent degradation of Sps in L3.6pL and Panc28 cells, whereas in Panc1 cells metformin decreased microRNA-27a and induced the Sp repressor, ZBTB10, and disruption of miR-27a:ZBTB10 by metformin was phosphatase dependent. Metformin also inhibited pancreatic tumor growth and downregulated Sp1, Sp3 and Sp4 in tumors in an orthotopic model where L3.6pL cells were injected directly into the pancreas. The results demonstrate for the first time that the anticancer activities of metformin are also due, in part, to downregulation of Sp transcription factors and Sp-regulated genes.

Fig. 1.

Metformin inhibits pancreatic cancer cell growth and induces apoptosis. (A) Panc28, Panc1 and L3.6pL cells were treated with vehicle and 5–20 mM metformin for 48 and 72 h, and cells were counted as outlined in the Materials and methods. Panc28 (B), Panc1 (C) and L3.6pL (D) cells were treated with 20mM metformin for 18, 18 and 24 h, respectively, and annexin V staining was determined as outlined in the Materials and methods. Results (A–D) are given as means ± SE for three replicate determinations for each treatment and significant (P < 0.05) decrease in growth or induction of apoptosis is indicated (*).

Fig. 2.

Metformin decreases expression of antiapoptotic and Sp proteins. (A) Panc28, Panc1 and L3.6pL cells were treated with vehicle, 10 or 20mM metformin for 36 h, and whole cell lysates were analyzed by western blots as outlined in the Materials and methods. Panc28 (B), Panc1 (C) and L3.6pL (D) cells were treated with vehicle, 10, 15 or 20mM metformin for 36 h, and whole cell lysates were analyzed by western blots as outlined in the Materials and methods.

Fig. 3.

Mechanisms of metformin-induced downregulation of Sp proteins. (A) Panc28, L3.6pL and Panc1 cells were treated with vehicle, 15mM metformin, 5mM glutathione (GSH), or their combination for 36 h, and whole cell lysates were analyzed by western blots. (B) The cell lines were treated with 15mM metformin, 3 μM gliotoxin or their combinations for 36 h, and whole cell lysates were analyzed by western blots. (C) Panc28 cells were treated as described in (B) for 24 h. Whole cell lysates were immunoprecipitated with IgG and antibodies against Sp1, Sp3 and Sp4, and the immunoprecipitate was analyzed for ubiquitinated proteins by western blots. (D) Panc28 cells were treated with vehicle, 15mM metformin and 0.35ng/ml leptomycin B alone or in combination, and cytosolic and nuclear extracts were isolated and analyzed by western blots as described in the Materials and methods.

Fig. 4.

Metformin disrupts miR-27a:ZBTB10 in Panc1 cells. (A) Panc1 cells were treated with 10 or 20mM metformin for 36 h, and miR-27a expression was determined by real-time PCR as outlined in the Materials and methods. Significant (P < 0.05) is indicated (*). (B) Panc1 cells were transfected with the miR-27a-luc construct, treated with 10 or 2mM metformin for 36 h and luciferase activity determined as outlined in the Materials and methods. Significant (P < 0.05) inhibition is indicated (*). (C) Panc1 cells were treated with 10 or 20mM metformin for 36 h (mRNA) or different times and analyzed for RNA or protein by real-time PCR or western blots, respectively, as outlined in the Materials and methods. Significant (P < 0.05) inhibition is indicated (*). (D) Panc1 cells were transfected with wild-type and mutant ZBTB10(3′-UTR)-luc, treated with 10 or 20mM metformin and luciferase activity was determined as outlined in the Materials and methods. Significant (P < 0.05) inhibition is indicated (*). Quantitated results in (A–D) are means ± SE for three replicate determinations for each treatment group.

Fig. 5.

Role of phosphatases in metformin-induced repression of Sp proteins. (A) SOV inhibits metformin-induced responses. Panc1 cells were treated with 15mM metformin alone or in combination with 20 μM SOV. Sp proteins and RNA levels were determined by western blots and real-time PCR, respectively, as outlined in the Materials and methods. (B) Metformin induces MKP-1 and MKP-5. Panc1 cells were treated with 15mM metformin, and mRNA and protein levels were determined as outlined in (A). (C) MKP-1 and MKP-5 disrupt miR-27a:ZBTB10. Panc1 cells were transfected with MKP-1 or MKP-5 expression plasmids or treated with metformin in the presence or absence of transfected siCtl or siMKP-1 or siMKP-5, and RNA or protein levels were determined as outlined in (A). (D) MKP-1 and MKP-5 expression downregulates Sp proteins. MKP-1 or MKP-5 was overexpressed in Panc1 cells, and whole cell lysates were analyzed by western blots as outlined in the Materials and methods. Significant (P < 0.05) induction (A, B) or inhibition (A, C) is indicated (*).

Fig. 6.

Metformin inhibits pancreatic (L3.6pL) tumor growth and downregulates Sp proteins in vivo. (A) Mice bearing L3.6pL cells in the pancreas were treated with corn oil (control) or 250mg/kg/d metformin. At the end of the treatment (28 days), pancreatic tumor volumes and weights were determined as outlined in the Materials and methods. (B) Lysates from a portion of each tumor were analyzed by western blots and quantitated (relative to β-actin; control values set at 100%) as outlined in the Materials and methods. Significant (P < 0.05) decreases in protein in tumors from metformin-treated mice compared with controls are indicated (*). (C) Immunostaining of tumors from control and metformin-treated mice for FAS expression was carried out as outlined in the Materials and methods. Hematoxylin and eosin staining of tumors from control and treated mice did not exhibit any striking morphological differences.