Minnelide decreases cell viability of pancreatic cancer cells in vitro

To assess the effect of Minnelide on cell viability in pancreatic cancer cell lines, we treated S2-013, MIA PaCa-2, S2-VP10, and Panc-1 cells with 0 or 200 nM Minnelide in the presence of serum-free medium with or without alkaline phosphatase at 37°C. These concentrations were based on previously used concentrations of triptolide that were effective in vitro (5). As expected, because the conversion of Minnelide to triptolide requires the activity of alkaline phosphatase, Minnelide is ineffective in the absence of phosphatase. All cell lines studied show significantly decreased cell viability 48 hours after Minnelide treatment in the presence, but not in the absence, of phosphatase. The decrease in cell viability induced by 200 nM of activated Minnelide (50% of control) was similar to that caused by the same concentration of the parent compound, triptolide (Fig. 2B). The twofold difference in response between MIA PaCa-2 and the S2 cell lines (S2-013 and S2-VP10) may be attributed to the origin of these lines: The former is derived from a primary tumor, whereas the latter two are derived from metastases.

Minnelide decreases tumor burden in an orthotopic pancreatic cancer model

We next proceeded to test the effect of Minnelide administration on pancreatic tumors in vivo using an orthotopic pancreatic cancer mouse model in which cells were implanted into the tail of the pancreas of athymic nude mice. Animals with MIA PaCa-2–derived human pancreatic tumors were randomized into different treatment groups, and intraperitoneal injections of either saline, triptolide [0.2 mg/kg QD (once a day)], or Minnelide [0.1 to 0.6 mg/kg QD or BID (twice a day)] were started 12 days after tumor cell injection (day 1 in Fig. 3A) (n = 10 in each group). No alkaline phosphatase treatment was required to activate Minnelide in vivo because this enzyme is abundantly present in blood and all other tissues in the body (9). Treatment was continued until day 60, and mice were subsequently maintained without treatment for another 30 days (9). In accordance with University of Minnesota animal protocols, animals were sacrificed at earlier time points if signs of morbidity were observed, as described in Materials and Methods. The experiment was terminated by sacrifice of surviving mice at day 90, and analyses of tumor weight and volume were performed. Minnelide treatment led to a marked decrease in tumor weight and volume at the end of treatment. The average tumor weight was 3291.3 ± 216.7 mg in control mice; at the most effective dose, tumor weight was 373.0 ± 142.6 mg in Minnelide-treated mice (0.15 mg/kg BID; P = 0.0006, control versus 0.15 mg/kg BID) and 653.0 ± 410.9 mg in triptolide-treated mice (0.2 mg/kg QD; P = 0.001, control versus 0.2 mg/kg QD) (Fig. 3C). The average tumor volume measured was 2927.06 ± 502.1 mm3 in the control group and 245.0 ± 111.4 mm3 or 473.0 ± 291.9 mm3 in Minnelide (0.15 mg/kg BID)– and triptolide (0.2 mg/kg QD)–treated mice, respectively (Fig. 3B). Thus, both Minnelide and triptolide were able to significantly prevent tumor growth.

 

Open in a separate window

Fig 3

Minnelide decreases pancreatic tumor growth in vivo and increases survival. MIA PaCa-2 cells (1 × 106) were injected into the tail of the pancreas of athymic Ncr nu/nu mice. (A) Kaplan-Meier analysis of animals injected with either triptolide (0.2 mg/kg), Minnelide (0.1 to 0.6 mg/kg QD or 0.15 mg/kg BID), or saline daily for 60 days; experiment was terminated on day 90. Treatment was started on day 12 after surgery. (B and C) Analysis of tumor volume (B) or tumor weight (C) from animals in (A). Numbers above columns represent number of animals that developed tumors over the number of animals studied. Columns, mean; bars, SE. (D) Table represents animals alive at start of treatment and end of experiment in each group indicated.

Kaplan-Meier survival curves revealed that all doses of Minnelide used resulted in nearly 100% survival (90 to 100%), whereas only 10% of control saline-injected mice were alive at the end of the experiment. All deaths in the control group were due to tumor burden, as assessed by detailed necropsy, whereas only one death (0.1 mg/kg group) in the treated animals was due to tumor burden. One death in the 0.2 mg/kg triptolide group and one death in the 0.3 mg/kg Minnelide group were not related to tumor burden (Fig. 3, A and D; necropsy showed no tumor). Whereas all mice (10 of 10) in the saline-injected control group formed large tumors, either no tumors or small tumors were visible in Minnelide-treated animals from the 0.1 and 0.3 mg/kg QD or 0.15 mg/kg BID groups [9 of 10 (0.1 mg/kg), 9 of 10 (0.3 mg/kg), and 6 of 10 (0.15 mg/kg)], suggesting that Minnelide treatment prevents tumor formation.

Although our study suggests that Minnelide is not toxic at the doses tested for 60 days in immunocompromised animals, toxicity studies were conducted on immunocompetent animals (C57BL/6) at doses of 0.3 and 0.6 mg/kg QD for 29 days. Minnelide at a dose of 0.3 mg/kg demonstrated no significant differences in alanine aminotransferase (ALT) or bilirubin levels in either males or females when compared to control animals. However, in males, the difference in ALT levels, but not in bilirubin levels, was significant at a dose of 0.6 mg/kg QD (fig. S2).

Minnelide prevents locoregional spread in a highly metastatic S2-013 cell line–derived orthotopic pancreatic cancer model

The MIA PaCa-2 cell line used in the experiment described above is derived from a human primary pancreatic tumor (10). However, the S2-013 cell line has been derived from a liver metastasis of pancreatic cancer (11, 12). In vivo, this cell line is extremely aggressive, metastasizes rapidly, and is poorly differentiated (12). To evaluate the efficacy of Minnelide in a more aggressive model, we injected 1 × 106 S2-013 cells into the pancreatic tail of athymic nude mice. Because tumor induction was expected to be rapid and aggressive, treatment was started on day 7 after cell implantation at a dose of 0.42 mg/kg QD delivered intraperitoneally (n = . Tumors grew rapidly in the control saline-injected mice (n = , necessitating the termination of the experiment on day 28. Gross analysis of the control mice demonstrated the presence of ascites, jaundice, and multiple tumors, and metastasis was observed in all animals (Fig. 4). Conversely, Minnelide-injected mice did not have ascites and did not show extensive spread from the primary site of injection (abdominal wall tumors in 2 of 10 animals and a splenic metastasis in 1 of 10 animals; Fig. 4, A and D). All control mice developed pancreatic tumors, whereas 9 of 10 Minnelide-treated animals had small tumors. Further analysis of the pancreatic tumors showed that the saline-injected mice had an average tumor volume of 799.6 ± 142.3 mm3 compared to 199.8 ± 49.2 mm3 in the Minnelide-treated mice (P = 0.0005). Minnelide treatment led to a decrease in average tumor weight from 1387.5 ± 109.3 mg in saline-injected mice to 290 ± 58.6 mg (P = 6.8 × 10−8) in Minnelide-treated mice (Fig. 4, A to C).

 

Open in a separate window

Fig 4

Minnelide decreases metastatic spread and pancreatic tumor growth. S2-013 cells (1 × 106) were injected into the tail of the pancreas of athymic Ncr nu/nu mice on day 1. On day 7 after surgery, mice were randomized, and each group of 10 mice was intraperitoneally injected with Minnelide (0.42 mg/kg) or saline daily for 28 consecutive days. (A) Pictorial representation of control and Minnelide-treated mice. All mice in the control group, but none in the Minnelide-treated group, displayed ascites, metastasis, and jaundice. (B and C) Graphs showing significantly reduced pancreatic tumor volume (B) and weight (C) in the Minnelide-treated group compared with the vehicle-treated group. *P < 0.05. (D) Table showing the macroscopic analysis of metastasis in the control and Minnelide-treated groups.

Minnelide prevents tumor formation and causes tumor regression in the highly metastatic AsPC-1 cell line–derived pancreatic cancer orthotopic model

To confirm our findings and to rule out potential cell line–specific effects, we established another pancreatic orthotopic mouse model using the highly metastatic ascites-derived AsPC-1 cell line (13). AsPC-1 cells proliferate more rapidly than S2-013 cells and are poorly differentiated (13). Tumor cells (2 × 105) were injected into the tail of the pancreas of nude mice (n = 20) on day 1. On day 7, treatment was initiated with either saline (n = 10) or Minnelide (0.42 mg/kg QD) (n = 10) delivered intraperitoneally. No palpable tumor was present at the start of treatment. The median survival of the control saline-treated group was 36 days, similar to that observed in the previous experiment, with an average tumor volume of 1181.0 ± 135.9 mm3 and tumor weight of 1480 ± 154.1 mg at the time of sacrifice. Each animal in the control group had a palpable tumor. Minnelide-treated mice, on the other hand, had no palpable tumors on day 100. At this juncture, the Minnelide-treated cohort was split into two groups: Minnelide treatment at 0.42 mg/kg QD was continued in the first group (n = 5), whereas the second group (n = 5) received no further treatment [discontinued (D/C)]. No palpable tumors were observed in any mouse from either group up to the termination of the experiment on day 385 (Fig. 5A) [censored deaths at days 285 and 380 were not due to pancreatic cancer but were due to small outgrowth in intestines (one animal) and enlarged thyroid (one animal)]. Because other animals did not exhibit this phenotype, we do not attribute these deaths to Minnelide treatment. Average tumor burden at day 385, as evaluated at necropsy, was 100 ± 6.0 mg and 0 mg (P = 2.78 × 10−5 and 1.55 × 10−5) or 100.0 ± 53.9 mm3 and 0.0 mm3 (P = 7.79 × 10−5 and 4.21 × 10−5) for all Minnelide-treated animals (Fig. 5, B and C).

 

Fig 5

Minnelide increases percent overall survival in an orthotopic xenograft mouse model. AsPC-1 cells (2 × 105) were injected into the tail of the pancreas of athymic Ncr nu/nu mice on day 1. Asterisk, censored because deaths were unrelated to pancreatic cancer. (A) On day 7 after surgery, mice were randomized and each group of 10 mice was intraperitoneally injected with Minnelide (0.42 mg/kg) or saline daily for 100 consecutive days. Saline-treated mice were euthanized when moribund. On day 100, the Minnelide-treated cohort (n = 10) was divided into two groups. One group (n = 5) continued to receive Minnelide (0.42 mg/kg), whereas treatment was terminated in the second group (n = 5; D/C; Stop Minnelide). Kaplan-Meier curve of the saline- or Minnelide-treated mice. Saline-treated mice had a mean survival of 36 days. All the animals in the Minnelide-treated group were alive at day 385 when the experiment was terminated. Survival lines are superimposed. (B and C) Graphs showing the average tumor volume (B) or tumor weight (C) of saline- or Minnelide-treated mice. (D) AsPC-1 cells (2 × 105) were injected into the tail of the pancreas of athymic Ncr nu/nu mice on day 1, and treatment with Minnelide (0.42 mg/kg) or saline was started after the death of the first animal on day 28. Animals in the saline group were sacrificed when moribund, and the experiment was terminated on day 75. Kaplan-Meier curve of the saline- or Minnelide-treated mice shows an increase in survival of Minnelide-treated animals. The median survival of saline-injected animals was 36 days.

We further evaluated the ability of Minnelide to cause tumor regression in this model. In a design similar to that described above, we initiated treatment with Minnelide on day 28 (n = 4), after the first animal was sacrificed because of pancreatic tumor burden, and terminated the experiment at day 75. At the start of treatment, all animals had large palpable tumors (n = 9). Median survival was 36 days in the control group (n = 5), whereas all animals in the treatment group were alive at the end of the experiment (Fig. 5D). Average tumor weight in the control group was 1380.0 ± 280 mg compared to Minnelide-treated animals (50.0 ± 36.5; P = 0.01), and average tumor volume in the control group was 1301 ± 179.8 mm3 compared to Minnelide-treated animals (18.08 ± 13.0 mm3) (P = 0.0011).

Our data so far demonstrated that Minnelide administration could not only prevent tumor progression but also induce tumor regression, reduce metastasis, and increase survival in vivo in three pancreatic cancer cell line–based models with differing metastatic potentials.

Minnelide decreases tumor burden in a human xenograft model of pancreatic cancer

Cell line–derived tumors, although useful for prescreening of potential chemotherapeutic agents, have the disadvantage of accumulated mutations and selection pressure over time in culture, making them genetically different from the original tumors (14). To rigorously evaluate the ability of Minnelide to act as a chemotherapeutic agent against pancreatic cancer, we tested its effect on human pancreatic cancer xenografts. De-identified patient pancreatic adenocarcinoma tumors (n = 2) were expanded in severe combined immunodeficient (SCID) mice and, upon reaching a size of 500 mm3, were excised from the animal, and 10-mm3 pieces were implanted into both flanks of SCID mice (n = 20 animals). Tumors were at passage 3 when used for evaluation of Minnelide. Minnelide treatment (0.42 mg/kg; n = 10) was started when tumor size reached an average of 334.9 ± 52.8 mm3 (day 60; Fig. 6A). The drug was delivered intraperitoneally, and as expected, saline-injected animals exhibited tumor progression. Animals exceeding allowable tumor burden were sacrificed in accordance with animal care guidelines. However, tumors regressed in Minnelide-treated animals such that, by day 40 after the start of treatment, no measurable tumors were present (Fig. 6A). Treatment was discontinued at day 50, and animals were observed for renewed tumor growth until termination of the experiment at day 120. No recurrence of tumor was observed until this point. Decrease in tumor weight corresponding to decrease in tumor volume was observed in the treatment group (90 ± 78 mg) compared with the control group (1160 ± 265 mg) (Fig. 6C). This decrease in tumor weight and volume translated into an increase in survival in the treatment group (Fig. 6B). Deaths unrelated to pancreatic cancer (four animals died of thymomas) are censored and marked by asterisks in the figure. Images of the animals at the termination of the experiment (for the treatment group) or day of sacrifice (for control animals) are shown in Fig. 6D. Histological assessment of tumors from saline-treated animals showed loss of architecture and presence of hyperchromasia and hypercellularity. However, remnants of tumors from Minnelide-treated animals showed greater fibrosis (Fig. 6E), suggesting that Minnelide causes tumor regression. The above experiment demonstrated that 0.42 mg/kg QD was an effective dose to treat xenografted human pancreatic tumors (also see fig. S1). We also tested the ability of lower doses of Minnelide to cause tumor regression in passage 4 of this xenograft model. In this experiment, treatment with Minnelide (0.21 or 0.42 mg/kg QD) was started when tumor volume reached 1000 mm3 (1051.7 ± 141.5 mm3). Either concentration of Minnelide was able to decrease tumor volume, although the rate at which the decrease occurred for the dose of 0.21 mg/kg was lower (days to nonmeasurable tumors, 50 versus 35 for the dose of 0.42 mg/kg; Fig. 6F). When compared with the control group, increased survival was observed in the treatment cohorts (Fig. 6G), and the experiment was terminated on day 90. Tumor weight decreased from 1737.5 ± 385.9 mg to 350 ± 25.0 mg (0.21 mg/kg) or 100 mg (0.42 mg/kg) in the treatment groups (Fig. 6H). There was no significant difference in tumor weight between the two treatment groups. Images of the animals at the termination of the experiment (for the treatment groups) or day of sacrifice (for control animals) are presented in Fig. 6I. Hematoxylin and eosin (H&E) staining of tumors from saline-treated animals showed the presence of hypercellularity and loss of architecture, whereas tumors from Minnelide-treated animals showed fibrosis and the presence of pyknotic nuclei (Fig. 6J). These data demonstrate that lower doses of Minnelide are also effective in reducing tumor burden in human xenografts from patients.

 

Open in a separate window

Fig 6

Minnelide increases percent overall survival in a human xenograft model. A de-identified patient pancreatic tumor was implanted into SCID animals. (A) Minnelide treatment (0.42 mg/kg) was started when tumor size was either 300 mm3 (Minnelide) or 1000 mm3(Minnelide). In the 300 mm3 group, treatment was terminated on day 55 (Minnelide stop). In the 1000 mm3 group, treatment was continued until the termination of the experiment on day 120. (B) Kaplan-Meier analysis of animals in (A). Asterisk, censored because deaths were unrelated to tumor burden. Saline-treated mice were euthanized when tumor size reached the limit of animal care guidelines, as described in Materials and Methods. (C) Tumor weight from experiment in (A). (D) Pictorial representation of animals in (A). (E) Representative H&E-stained sections from a saline-treated tumor (n = 5) and a Minnelide-treated tumor (n = 11; ×10 magnification on an Aperio ScanScope scanner). (F) Minnelide treatment (0.21 or 0.42 mg/kg) was started when tumor size was 1000 mm3 (day 1). Experiment was terminated on day 90. Skin was resected to assess small subcutaneous tumors. (G) Kaplan-Meier survival analysis of animals in (F). Asterisk, censored because deaths were unrelated to tumor burden. Animals in the saline group were euthanized when tumor size reached the limit of animal care guidelines. (H) Tumor weight from experiment in (F). (I) Pictorial representation of animals in (F). (J) Representative H&E-stained sections from a saline-treated tumor (n = 5) and Minnelide-treated tumors [0.21 mg/kg (n = or 0.42 mg/kg (n = 5)] (×10 magnification on an Aperio ScanScope scanner).

Minnelide prevents tumor formation in spontaneous pancreatic tumor–forming animals

In pancreatic cancer, the tumor microenvironment plays an important role in cancer progression; an abundant desmoplastic stroma penetrates and envelopes the tumor, comprising up to 80% of the gross tumor mass (15–17). Within the pancreatic cancer microenvironment, several cell types have been shown to affect tumor behavior (18, 19), and immune response plays an important role in increasing the metastatic potential of the cancer (20). Because the cell line–derived models tested above lack stroma, and the human xenografts were implanted into immunodeficient animals, we tested the efficacy of Minnelide therapy in an immunocompetent animal that forms spontaneous pancreatic tumors. The KRasG12D; Trp53R172H; Pdx-1Cre animal model has a 100% penetrance and a median survival of 5 months (21). Minnelide treatment delivered intraperitoneally (0.3 mg/kg per day) was started on these animals at 4 to 6 weeks of age because median survival is 5 months in this animal model (21). To evaluate the decrease in tumor burden, we sacrificed at least nine animals from each group of varying age (average age: saline-treated animals, 123 days; Minnelide-treated animals, 124 days). Tumor volume and weight of saline-treated animals averaged 1380 ± 419.3 mm3 and 1788.9 ± 561.3 mg, respectively, whereas those of Minnelide-treated animals were 251 ± 95.4 mm3 and 333.08 ± 118.2 mg (Fig. 7). Our data suggest that Minnelide prevents tumor formation in these animals.

Fig 7

Minnelide decreases tumor burden in a spontaneous pancreatic cancer mouse model. KRasG12D; Trp53R172H; Pdx-1Cre animals were genotyped and randomly assigned to either the saline or the Minnelide treatment group (0.3 mg/kg) at 4 to 6 weeks of age. Saline animals were sacrificed when moribund. (A and B) Tumor volume (A) and weight (B) of animals in either group.

Cell line maintenance

MIA PaCa-2 and Panc-1 cells derived from primary tumors of patients were cultured in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. S2-013 and S2-VP10 cells derived from patients with liver metastases were cultured in RPMI 1640 with 10% FBS and 1% penicillin-streptomycin. AsPC-1 cells derived from patient ascites were cultured in RPMI 1640 with 20% FBS and 1% penicillin-streptomycin. All cell lines were maintained at 37°C in the presence of 5% CO2. All cell culture reagents were obtained from Hyclone.

Cell viability

Cell viability was determined with the Dojindo Cell Counting Kit-8 (Dojindo Molecular Technologies) following the manufacturer’s instructions. After treatment with triptolide or Minnelide for the times and concentrations indicated, cell viability was assessed by incubation with 10 μl of tetrazolium substrate for 1 hour at 37°C, followed by measurement of absorbance at 450 nm.

Orthotopic pancreatic cancer tumor model

Four- to 6-week-old female athymic mice (The Jackson Laboratory) were anesthetized with xylazine/ketamine and injected with 1 × 106 (MIA PaCa-2 or S2-013) or 2 × 105 (AsPC-1) cells embedded in Matrigel (BD Biosciences) in the tail of the pancreas. Buprenorphine was used as an analgesic after surgery. Tumors were allowed to grow, and treatment of animals with either compound or control was started after animals were randomized at the times after injection stated in the figure legends. Tumor volume was calculated with the following formula: length × width × thickness/2 (37). Experiments were performed and animals were sacrificed in accordance to the regulations of the animal care committee of the University of Minnesota.

Human xenograft pancreatic cancer tumor model

De-identified human pancreatic tumors were implanted subcutaneously into SCID mice (The Jackson Laboratory). When tumor volumes reached 500 mm3, tumors were dissected and cut into 10-mm3 pieces, which were then subcutaneously implanted into both flanks of additional SCID mice (n = 20 animals). Animals were randomized and tagged before treatment, and each tumor was measured as indicated in the figures.

Transgenic mouse model of spontaneous pancreatic cancer

KRasG12D; Trp53R172H; Pdx-1Cre animals were generated by crossing Lox Stop Lox (LSL) KRasG12D; LSL Trp53R172Hanimals with Pdx-1Cre animals. Minnelide treatment was started when animals were 4 to 6 weeks of age. Animals in saline and treatment groups were age-matched. Experiments were performed and animals were sacrificed in accordance with animal care committee regulations of the University of Minnesota.

Statistical analysis

Values are expressed as means ± SEM. All in vitro experiments were performed at least three times. The significance of the difference between any two samples was analyzed by independent Student’s t test; P values of <0.05 were considered statistically significant.

We thank D. M. Johnson for technical assistance and C. T. Le (Department of Biostatistics, University of Minnesota) for advice on statistical analysis.

Funding: Masonic Cancer Center, Center for Translational Medicine, Department of Surgery, Pancreatic Cancer Specialized Program of Research Excellence grant to the University of Minnesota and University of Alabama, Department of Chemistry, College of Pharmacy, and Vince and McKnight Presidential Chairs (to G.I.G.) and NIH grants R01CA124723 and R01 CA170496 (to A.K.S.), Katherine and Robert Goodale Foundation, and Hirshberg Foundation (to A.K.S.). A.K.S. is also supported by Eugene C and V Gail Sit Chair for pancreatic and gastrointestinal cancer.