Tumor Decelerating and Chemo-Potentiating Action of Methyl Jasmonate on a T Cell Lymphoma In Vivo: Role of Altered Regulation of Metabolism, Cell Survival, Drug Resistance, and Intratumoral Blood Flow

Abstract

Methyl jasmonate (MJ), a natural oxylipin, possesses a broad spectrum of antineoplastic potential in vitro. However, its tumor growth impeding and chemo-potentiating action has not been adequately investigated in vivo. Using a murine thymus-derived tumor named Dalton's Lymphoma (DL), in the present study, we examined if intra-tumoral administration of MJ can cause tumor growth impedance. We also explored the associated molecular mechanisms governing cell survival, carbohydrate & lipid metabolism, chemo-potentiation, and angiogenesis. MJ administration to tumor-transplanted mice caused deceleration of tumor growth accompanying prolonged survival of the tumor-bearing mice. MJ-dependent tumor growth retardation was associated with the declined blood supply in tumor milieu, cell cycle arrest, augmented induction of apoptosis and necrosis, deregulated glucose and lipid metabolism, enhanced membrane fragility of tumor cells, and altered cytokine repertoire in the tumor microenvironment. MJ administration modulated molecular network implicating Hsp70, Bcl-2, TERT, p53, Cyt c, BAX, GLUT-1, HK 2, LDH A, PDK-1, HIF-1α, ROS, MCT-1, FASN, ACSS2, SREBP1c, VEGF, cytokine repertoire, and MDR1, involved in the regulation of cell survival, carbohydrate and fatty acid metabolism, pH homeostasis, and drug resistance. Thus, the present study unveils novel molecular mechanisms of the tumor growth decelerating action of MJ. Besides, this preclinical study also establishes the adjunct therapeutic potential of MJ. Hence, the present investigation will help to design novel anti-cancer therapeutic regimens for the treatment of hematological malignancies.

Keywords: cell survival and metabolic regulation; chemo-potentiation; intra-tumoral blood flow; methyl jasmonate; tumor growth impedance.

Figure 1

Protocol for the administration of MJ to tumor-bearing mice. Mice in groups of 10 each were transplanted with 1 x 10⁵ DL cells in 0.5 ml PBS per mouse (day 0), followed by intraperitoneal administration of MJ (100 mg/kg) or PBS on days 4, 6, 8, 10, and 12 post tumor transplantation. Tumor cells and ascitic fluid were collected and investigated for various assays on day 14. Remaining tumor-bearing mice were monitored for change of body weight and survival as parameters of tumor progression.

Figure 2

MJ administration retards tumor progression. Tumor-transplanted mice were administered with PBS alone (control) or containing MJ (100 g/kg), as shown in Figure 1 , followed by monitoring of tumor progression by the change of body weight (A) and survival of the tumor-bearing mice (B), estimation of the volume of ascitic fluid (C), and the number of viable tumor cells (D). Values are shown in (A, C, D) are mean ± SD of three independent experiments. *p < 0.05 vs. respective control.

Figure 3

In vitro tumor cell-specific cytotoxic action of MJ against neoplastic cells of human origin. Indicated tumor cell lines, hepatocytes, and splenocytes (1 x 106 cells/ml) were incubated in vitro for 12 h in medium alone or containing MJ followed by estimation of cytotoxicity by MTT assay. Values shown are mean ± SD of three independent experiments.*,α, ^#p < 0.05 vs. respective control.

Figure 4

MJ administration to tumor-bearing mice induces tumor cell death and cell cycle arrest. Tumor cells harvested from control and tumor-transplanted mice were examined for induction of cell death by Annexin V/PI staining using fluorescence microscopy and Wright-Giemsa staining (A). Arrows indicate apoptotic cells, and arrowheads indicate necrotic cells. Tumor cells were also analyzed for cell cycle as described in materials and methods using flow cytometry (B). Microscopic and flow cytometric images are from a representative experiment of three independent experiments with similar results. The accompanying bar diagrams are mean ± SD three independent experiments. *p < 0.05 vs. respective control.

Figure 5

Altered expression of cell survival regulatory molecules in tumor cells following in vivo exposure to MJ administration. Tumor cells (1 x 10⁶ cells/ml) harvested from control and MJ-administered tumor-transplanted mice were analyzed for the expression of indicated cell survival regulatory molecules (A). The cytosolic level of Cyt c was detected (B) as described in the materials and methods. Bands shown in (A, B) are from a representative experiment out of three independent experiments with similar results. Expression of intracellular ROS in tumor cells of control and MJ groups was estimated by DCFDA staining (C) as described in the materials and methods. Accompanying bar diagrams depicts densitometric analysis showing mean ± SD. *p < 0.05 vs. respective control.

Figure 6

MJ alters the metabolic activity of DL cells in vivo. Tumor cells (1 x 10⁶ cells/ml) of control and MJ-administered tumor-bearing mice were analysed for metabolic activity by MTT assay (A), expression of HK2 by Western blotting (B), and other indicated metabolism regulatory molecules by RT-PCR (C). The expression of GLUT-1 was analyzed by RT-PCR (D) and flow cytometry for membrane expression of GLUT-1 (E). The pH of the control and MJ-administered groups’ ascitic tumor fluid was measured (F) by pH meter. The expression of pH regulator MCT-1 was analysed by flow cytometry (G) in tumor cells harvested from control and MJ-administered tumor-bearing mice. Bands shown (B, C, D) and flow cytometric image (E, G) are from representative experiments out of three independent experiments with similar results. Values shown in bar diagrams are mean ± SD of three independent experiments. *p < 0.05 vs. respective control.

Figure 7

MJ alters lipid homeostasis. Tumor cells (1 x 10⁶ cells/ml) harvested from control and MJ administered tumor-bearing mice were analysed for the expression of ACSS2, FASN, and SREBP1c by RT-PCR (A). Osmotic fragility (B) of control and MJ exposed tumor cells in vivo was analyzed, as described in the materials and methods. Bands shown in (A) are from a representative experiment out of three independent experiments with similar results. Values shown in bar diagrams (A, B) are mean ± SD of three independent experiments. *p < 0.05 vs. respective control.

Figure 8

MJ administration to tumor-bearing mice alters cytokine repertoire in ascitic fluid. The ascitic fluid of control and MJ administered tumor-bearing mice were analyzed for the levels of indicated cytokines by ELISA as described in the materials and methods. Values are mean ± S.D. *p < 0.05 vs. control.

Figure 9

Effect of MJ administration on intratumoral blood flow and VEGF expression. A color doppler sonographic imaging was performed on control and MJ-administered tumor-bearing mice, as described in materials and methods. Images of Peak Systolic Velocity (PSV) and End Diastolic velocity (EDV) shown in (A) are from a representative experiment out of three independent experiments with similar results. The accompanying bar diagrams (B) show the mean ± SD of PSV and EDV, respectively. Tumor cells (1 x 106 cells/ml) harvested from control and MJ-administered tumor-bearing mice were analysed for the expression of VEGF by Western blotting in cell lysate (C) and by ELISA in ascitic fluid (D). Bands shown in (B) are from representative experiments out of three independent experiments with similar results. Values shown in bar diagrams (B–D) are mean ± SD of three independent experiments. *p < 0.05 vs. respective control.

Figure 10

Chemo-potentiating action of MJ in vivo. Tumor cells (1 x 10⁶ cells/ml) harvested from control and MJ-administered tumor-bearing mice were examined for the expression of MDR1 by Western blotting (A) and flow cytometry (B). Bands shown (A) and flow cytometric images (B) are from representative experiments out of three independent experiments with similar results. Values shown in bar diagrams are mean ± SD of three independent experiments. *p < 0.05 vs. respective control. To evaluate the chemo-potentiating effect of MJ administration, the tumor-bearing mice were administered with PBS with or without MJ (100 mg/kg body weight) or CP (5 mg/kg bodyweight) or both together on alternative days starting from day 4 to day 12 post tumor transplantation. Tumor cells harvested on day 14 from tumor-bearing mice of control and those administered with MJ, CP, and MJ plus CP were further examined for cytotoxicity by MTT assay (C) and survival by Trypan blue dye exclusion test (D) as described in materials and methods. Values shown are mean ± SD. *p < 0.05 vs. values for control.

Figure 11

Summary of the molecular mechanism underlying the tumor growth retarding and chemo-potentiating action of MJ.