Targeting metabolic flexibility by simultaneously inhibiting respiratory complex I and lactate generation retards melanoma progression - PubMed (original) (raw)

Targeting metabolic flexibility by simultaneously inhibiting respiratory complex I and lactate generation retards melanoma progression

Balkrishna Chaube et al. Oncotarget. 2015.

Abstract

Melanoma is a largely incurable skin malignancy owing to the underlying molecular and metabolic heterogeneity confounded by the development of resistance. Cancer cells have metabolic flexibility in choosing either oxidative phosphorylation (OXPHOS) or glycolysis for ATP generation depending upon the nutrient availability in tumor microenvironment. In this study, we investigated the involvement of respiratory complex I and lactate dehydrogenase (LDH) in melanoma progression. We show that inhibition of complex I by metformin promotes melanoma growth in mice via elevating lactate and VEGF levels. In contrast, it leads to the growth arrest in vitro because of enhanced extracellular acidification as a result of increased glycolysis. Inhibition of LDH or lactate generation causes decrease in glycolysis with concomitant growth arrest both in vitro and in vivo. Blocking lactate generation in metformin-treated melanoma cells results in diminished cell proliferation and tumor progression in mice. Interestingly, inhibition of either LDH or complex I alone does not induce apoptosis, whereas inhibiting both together causes depletion in cellular ATP pool resulting in metabolic catastrophe induced apoptosis. Overall, our study suggests that LDH and complex I play distinct roles in regulating glycolysis and cell proliferation. Inhibition of these two augments synthetic lethality in melanoma.

Keywords: LDH; complex I; melanoma; metabolic catastrophe; synthetic lethality.

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Conflict of interest statement

CONFLICTS OF INTEREST

Authors declare that they do not have any potential conflicts of interest

Figures

Figure 1. Metformin promotes melanoma tumor growth in mice

A.-C. C57BL/6J mice were made diabetic by injecting 50 mg/kg streptozotocin (STZ) intraperitoneally for 3 consecutive days. Tumor was developed by injecting 1 × 105 B16F10 melanoma cells subcutaneously in C57BL/6J mice, one week post STZ injection. Metformin (200 mg/kg) was administered in hyperglycemic mice orally before injecting the cells. Graph representing tumor progression A., tumor weight B., and tumor volume of individual mice C. in hyperglycemic mice. Results are given as means ± SD (n = 7). D. Tumor progression of B16F10 isograft in normoglycemic mice. Mice were administered with metformin (200 mg/kg) orally after the appearance of palpable tumor. E. and F. Weight and representative image of tumors excised from normoglycemic mice administered with or without metformin. Results are given as means ± SD (n = 5). G. Tumor progression of A375 xenograft in hyperglycemic mice administered with or without metformin (200 mg/kg). Results are given as means ± SD (n = 5). H. Tumor progression of A375 xenograft in normoglycemic mice administered with or without metformin (200 mg/kg). Results are given as means ± SD (n = 5). I. Weight of tumors excised from either hyperglycemic or normoglycemic mice administered with or without metformin. The values *p < 0.05, **p < 0.01 denote significant differences between the groups. (HG- hyperglycemic, NG- normoglycemic, Ctrl- control, Met- metformin)

Figure 2. Metformin promotes melanoma tumor growth by inducing angiogenesis and by inhibiting necrosis

A. and B. Representative H&E images of tumor sections of B16F10 isograft A. and A375 xenograft B. from control and metformin groups showing necrotic regions (pink) and healthy cells (blue). A. H&E staining of B16F10 isograft section from control and metformin group. B. H&E staining of A375 xenograft section from control and metformin groups. C. Representative immunoblots showing the expression of indicated cell cycle regulatory molecules in the lysate of B16F10 derived tumors from control and metformin groups. D. Representative immunohistochemical image showing the expression of cell cycle regulatory molecule cyclin D1 in the B16F10 isograft section from control and metformin groups. E. and F. Immunohistochemical analysis of angiogenesis marker CD31 in the tumor section of B16F10 isograft of control and metformin group G. Relative serum level of VEGF in control and metformin administered C57 and NOD/SCID mice. All these experiments were performed in normoglycemic mice. Data were represented as means ± SD. The values *p < 0.05, **p < 0.01 denote significant differences between the groups. (HG- hyperglycemic, NG- normoglycemic, Ctrl- control, Met- metformin).

Figure 3. Complex I inhibition induces aerobic glycolysis

A. and B. A375 and B16F10 cells were cultured in presence of different concentrations of metformin (0.5, 1.0, and 2.0 mM) for 36 h. Glucose and lactate level in the spent culture medium was determined by using enzymatic assay kits. C. Representative immunoblots showing protein level of indicated molecules in whole cell lysate of B16F10 cells treated with metformin for 36 h. D. and E. Relative level of glucose in serum collected from normoglycemic or hyperglycemic C57BL/6J mice bearing B16F10 isograft D. and NOD/SCID mice bearing A375 xenograft E. from metformin administered groups (n = 4). F. and G. Relative levels of lactate in serum collected from normoglycemic or hyperglycemic C57BL/6J mice bearing B16F10 isograft F., and NOD/SCID mice bearing A375 xenograft G. from metformin administered groups (n = 4). H. and I. Serum LDH activity in normoglycemic or hyperglycemic C57BL/6J mice bearing B16F10 isograft H. and NOD/SCID mice bearing A375 xenograft I. from control and metformin administered groups (n = 4). J. LDH activity in the B16F10 isograft from metformin administered normoglycemic and hyperglycemic mice. Values are represented as mean ± SD. The values *p < 0.05, **_p_ < 0.01 denote significant differences between the groups (_n_ > 3 at the least). (NG-normoglycemic, HG-hyperglycemic, Ctrl- control, Met-metformin).

Figure 4. Inhibition of complex I sensitizes cancer cells to LDH and PDK1 inhibitors oxamate and DCA

A.-C. Effect of oxamate and DCA on growth of A375, B16F10 and SKMel28 cells treated with or without metformin. Cells were treated with 25 mM oxamate or 10 mM DCA either alone or with 2 mM metformin for indicated time point. Cell viability was assessed by MTT assay. D.-F. Survival of A375 D., B16F10 E. and SKMel28 F. cells grown in the presence of oxamate (25 or 50 mM) or DCA (10 or 20 mM) alone or together with 2 mM metformin for 48 h. Representative images showing the long term survival of cancer cells treated with indicated concentrations of DCA and oxamate alone or together with metformin (2 mM) and allowed to grow for 48 h. Medium was replaced with fresh medium without any inhibitors. Medium was changed every 2-3 days and allowed to grow in drug free medium for further 10-15 days. Colonies were stained with crystal violet and photographed. All values are represented as mean ± SD. (Ctrl- control, Met- metformin, Oxa- oxamate).

Figure 5. Simultaneous blocking of lactate generation and complex I induces apoptosis in melanoma cells

A. and B. A375 and B16F10 cells were grown in DMEM with or without 50 mM oxamate and 20 mM DCA either alone or together with 2 mM metformin for 36 h. Apoptosis was detected by using Annexin V and PI dual positive cells via flow cytometry. C. and D. Representative immunoblots showing the protein levels of apoptotic markers PARP1, Bax and antiapoptotic molecule Bcl-2 in A375 C. and B16F10 cells D. grown under above mentioned conditions. HSP60 was used as a loading control. (Ctrl- control, Met- metformin, Oxa- oxamate).

Figure 6. Inhibition of complex I and lactate generation together induces metabolic catastrophe

A. and B. A375 and B16F10 cells were treated with oxamate (25 and 50 mM) either alone or with 2 mM metformin for 36 h. Level of glucose and lactate in the culture medium was measured as mentioned in method section. C. Relative ATP levels in A375 and B16F10 cells grown in the conditions mentioned in A.. D. Human melanoma cells (A375 and SKMel28) were grown in 96 well plates (60% confluency) and transfected with control and LDHA specific siRNA as described in methods section. These cells were treated with either metformin (2 mM) or phenformin (100 μM) for additional 48 h. Inhibition of LDHA expression was confirmed by immunoblotting. E. Survival of A375 and SKMel28 upon knocking down LDHA grown in presence or absence of metformin and phenformin. F.-H. A375 and SKMel28 cells were grown in 35 mm culture dish for 24 h. These cells were transfected with either control or LDHA specific siRNA and allowed to grow for further 24 h. Cells were treated with either 2 mM metformin or 100 μM phenformin for additional 48 h. Glucose utilization F., lactate secretion G. and total cellular level of ATP H. was determined in melanoma cells as described in method section. Values are represented as mean ± SD. The values *p < 0.05, **_p_ < 0.01, ***_p_ < 0.001 denote significant differences between the groups (_n_ > 3 at the least). (Ctrl- control, Met- metformin, Oxa- oxamate, Phen- phenformin).

Figure 7. Inhibition of LDH activity restricts tumor progression of melanoma tumor upon complex I inhibition

A. Progression of B16F10 isograft in C57BL/6J mice administered with metformin (200 mg/kg, orally) and oxamate (500 mg/kg, orally) either alone or together (n = 5). Values are represented as mean ± SD. B. and C. Tumor weight and representative image of tumors excised from mice of indicated groups. D. and E. level of glucose and lactate in the serum collected from tumor bearing mice with indicated treatment groups (n = 4) F. Enzymatic activity of LDH in tumor lysate G. LDH activity in serum from the mice of indicated treatment groups (n = 3). H. Relative ATP levels in the tumors of indicated treatment groups (n = 3). I. Immunoblots showing protein levels of indicated molecules in the whole tumor lysates. Values are represented as mean ± SD. The values *p < 0.05, **p < 0.01 denote significant differences between the groups. (Ctrl- control, Met- metformin, Oxa- oxamate).

Figure 8. Schematic representation of synthetic lethality induced by combination of metformin and oxamate treatment in melanoma

A. Inhibition of respiratory complex I by metformin or phenformin alone results in elevation of glycolysis owing to the activation of lactate dehydrogenase (LDH). Generation of building blocks and ATP exclusively through glycolysis helps in rapid tumor growth. B. Targeting LDH or lactate generation by oxamate and DCA or by siRNA causes activation of OXPHOS and subsequent ATP generation. However this leads to suppression of cell proliferation without inducing cell death. C. Blocking both the enzyme together induces metabolic catastrophe owing to the depletion of cellular ATP pool. This leads to the initiation of apoptosis, and suppression of tumor growth. Complex I and LDHA form a synthetically lethal pair in melanoma cells.

References

1. Gray-Schopfer V, Wellbrock C, Marais R. Melanoma biology and new targeted therapy. Nature. 2007;445:851–857. - PubMed
1. Demierre MF. Epidemiology and prevention of cutaneous melanoma. Curr Treat Options Oncol. 2006;7:181–186. - PubMed
1. Hersey P, Watts RN, Zhang XD, Hackett J. Metabolic approaches to treatment of melanoma. Clin Cancer Res. 2009;15:6490–6494. - PubMed
1. Warburg O. On respiratory impairment in cancer cells. Science. 1956;124:269–270. - PubMed
1. Vander Heiden MG, Cantley LC, Thompson CB. Understanding the Warburg effect: the metabolic requirements of cell proliferation. Science. 2009;324:1029–1033. - PMC - PubMed

Targeting metabolic flexibility by simultaneously inhibiting respiratory complex I and lactate generation retards melanoma progression - PubMed (original) (raw)