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. 2022 Apr 22;17(4):e0266601.
doi: 10.1371/journal.pone.0266601. eCollection 2022.

Targeting pancreatic cancer with combinatorial treatment of CPI-613 and inhibitors of lactate metabolism

Simone Kumstel  1 , Tim Schreiber  1 , Lea Goldstein  1 , Jan Stenzel  2 , Tobias Lindner  2 , Markus Joksch  2 , Xianbin Zhang  1 , Edgar Heinz Uwe Wendt  1 , Maria Schönrogge  1 , Bernd Krause  3 , Brigitte Vollmar  1 , Dietmar Zechner  1
Affiliations

Targeting pancreatic cancer with combinatorial treatment of CPI-613 and inhibitors of lactate metabolism

Simone Kumstel et al. PLoS One. .

Abstract

Pancreatic cancer is the fourth leading cause of cancer death, with a 5-year survival rate of 10%. A stagnant high mortality rate over the last decades highlights the need for innovative therapeutic approaches. Pancreatic tumors pursue an altered metabolism in order to maintain energy generation under low nutrient influx and hypoxic conditions. Targeting these metabolic strategies might therefore be a reasonable therapeutic approach for pancreatic cancer. One promising agent is CPI- 613, a potent inhibitor of two enzymes of the tricarboxylic acid cycle. The present study evaluated the anti-cancerous efficacy of CPI-613 in combination with galloflavin, a lactate dehydrogenase inhibitor or with alpha-cyano-4-hydroxycinnamic acid, an inhibitor of monocarboxylate transporters. The efficacy of both combination therapies was tested in vitro on one human and two murine pancreatic cancer cell lines and in vivo in an orthotopic pancreatic cancer model. Tumor progression was evaluated by MRI and 18F-FDG PET-CT. Both combinatorial treatments demonstrated in vitro a significant inhibition of pancreatic cancer cell proliferation and induction of cell death. In contrast to the in vitro results, both combination therapies did not significantly reduce tumor growth in vivo. The in vitro results suggest that a combined inhibition of different metabolic pathways might be a promising approach for cancer therapy. However, the in vivo experiments indicate that applying a higher dosage or using other drugs targeting these metabolic pathways might be more promising.

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Conflict of interest statement

The authors have declared that no competing interest exist.

Figures

Fig 1
Fig 1. CPI-613 in combination with CHC leads to a reduction in pancreatic cancer cell proliferation and viability.
(A) Quantification of proliferation by BrdU-ELISA, dose-response curve after 48 h treatment of 6606PDA cells with CPI-613 (50–350 μM), with and without 5 mM CHC (dotted lines indicate mean value of absorbance for control treated cells, either with high or low DMSO concentrations). (B) Evaluation of cell proliferation with vehicle control (medium with the respective amount of vehicle, DMSO) (C), 250 μM CPI-613 (CPI), 5 mM CHC (CHC) and the combination (CPI + CHC). (C) Evaluation of cell death by trypan blue assay after cultivating 6606PDA cells for 48 h with vehicle control with DMSO (C), or medium supplemented with 150 μM CPI-613 (CPI), 5 mM CHC and CPI-613 plus CHC (CPI + CHC). (D) Apoptosis was evaluated by quantifying cleavage of caspase 3 (caspase-3) and PARP (E) via western blot, after treating the cells 24 h with DMSO only (C), 150 μM CPI-613, 5 mM CHC or the combination (the bands of the cleaved proteins are indicated by black arrows). Statistics were performed with ANOVA on Ranks and correction for multi comparison by Holm-Sidak method, significant differences: * p ≤ 0.002; Independent experiments: A-B: n = 5–7, C: n = 7, D-E: n = 3.
Fig 2
Fig 2. CPI-613 in combination with galloflavin inhibits pancreatic cancer cell proliferation and induces cell death.
(A) Proliferation of 6606PDA cells after cultivating them in CPI-613 (100–400 μM) with and without 30 μM galloflavin (GAL; dotted lines indicate mean value of absorbance for control treated cells, either with high or low DMSO concentrations). (B) Analysis of proliferation by BrdU-ELISA, (C) or quantification of cell death by trypan blue assay, after cultivating 6606PDA cells 24 h in control medium with the respective amount of the vehicle DMSO (C), medium supplemented with 300 μM CPI-613 (CPI), 30 μM galloflavin (GAL) or the combination of both drugs (CPI + GAL). (D-E) Apoptosis was assessed by analyzing the cleavage of caspase 3 (caspase-3) and PARP via western blot, after treating the cells with medium supplemented with DMSO (C), 300 μM CPI-613, 30 μM galloflavin or the combination for 24 h respectively (the bands of the cleaved proteins are indicated by black arrows). Statistics were performed with ANOVA on Ranks Holm-Sidak method for multi comparison, significant differences: * p ≤ 0.004; Independent experiments: A-B: n = 8–10, C: n = 19, D-E: n = 2–3.
Fig 3
Fig 3. The combination therapy of CPI-613 and CHC (solvent: DMSO) did not lead to a significant reduction of tumor growth in vivo.
(A) To evaluate the efficacy of the combinatorial therapy in the orthotopic pancreatic cancer model CPI-613 (25 mg/kg) was injected once a week and CHC (15 mg/kg) was applied on a daily basis from day 4 until the end of the experiment day 37. Both therapeutics were dissolved in DMSO (CPI: 100% DMSO, CHC: 50% DMSO/ 50% PBS), the sham animals were treated with the respective vehicle of the combinatorial treatment. (B-C) PET-CT imaging with the tracer 18F-FDG was performed to quantify changes in the glucose metabolism of tumor tissue. (D) The uptake of 18F-FDG in the tumor tissue was evaluated by metabolic tumor volume (MTV, tumor volume [mm³] with the criteria 30% of the hottest voxel of injected dose/g bodyweight) on day 32 after tumor cell implantation on a subset of sham and CPI + CHC tumors. (E) Tumor weight was measured at the end of the experiment in the indicated treatment groups. (F) The body weight of mice was measured at the indicated days during the treatment period and the body weight change was calculated from the weight assessed at the beginning of each experiment. Biological replicates: D: Sham n = 3, CPI + CHC n = 2. E-F: Sham n = 9, CPI n = 4, CPI + CHC n = 10, CHC n = 3.
Fig 4
Fig 4. The combinatorial treatment of CPI + CHC (solvent: 30% PEG 300) did not inhibit the tumor growth in vivo.
(A) CPI was injected 5x weekly at a dose from 10 mg/kg and CHC (15 mg/kg) was applied daily, from day 4 until day 37 after tumor cell injection. Both therapeutics were dissolved in 30% PEG 300 (30% Polyethylene glycol 300), 1% Tween, PBS. (B-E) MRI was performed on day 20 and 36 after tumor cell injection, as indicated by exemplary images (tumors are framed by a red dotted line). (F) The tumor volume was evaluated for the combinatorial treatment (CPI + CHC) and the sham treatment. (G) The weight of the tumors was quantified after sacrificing the mice on day 37. (H) The body weight change of mice quantified at the indicated days during the treatment period. Biological replicates: F-H: Sham: n = 4, CPI + CHC: n = 5.
Fig 5
Fig 5. The combinatorial treatment of CPI-613 and galloflavin did not cause an impairment of the tumor growth in vivo.
(A) To quantify the therapeutic effect in vivo, the 6606PDA cells were injected at day 0 into the pancreas of C57BL6/J mice. The treatment started after 4 days of recovery, CPI (10 mg/kg) was injected five times per week and galloflavin (GAL, 20 mg/kg) three times weekly. (B-F) The tumor of the combinatorial therapy and sham treated mice was identified by MRI on day 22 and 36 (tumors are framed by a red-dotted line) and the tumor volume was quantified at these time points. (G) The tumor weight of all treatment groups was measured at the end of the experiment on day 37. (H) The body weight change of sham and CPI + galloflavin treated mice was quantified at the indicated days. Biological replicates: F: n = 4 per group; G-H: Sham n = 4, CPI n = 1, CPI-GAL n = 5, GAL n = 1. *P < 0.05 was considered significant, calculated by Two-Way repeated measure ANOVA and correction of multi-comparison by Sidak’s method.
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The authors received no specific funding for this work.