Modulation of Tumor Cell Metabolism by Laser Photochemotherapy with Cisplatin or Zoledronic Acid In Vitro.

BACKGROUND/AIM: Laser photochemotherapy is a new approach in cancer treatment using low-level laser therapy (LLLT) to enhance the effect of chemotherapy. MATERIALS AND METHODS: In order to evaluate the effect of LLLT on tumor cells, HeLa cells were treated with cisplatin or zoledronic acid (ZA) followed by LLLT. Cell viability was evaluated with 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide assay. Oxidative phosphorylation and glycolysis were measured using extracellular flux analysis. Immunocytochemistry of heat-shock protein 70 (HSP70) and western blot analysis were performed. RESULTS: LLLT alone increased viability and was associated with lower oxidative phosphorylation but higher glycolysis rates. Cisplatin and ZA alone lowered cell viability, glycolysis and oxidative phosphorylation. This effect was significantly enhanced in conjunction with LLLT and was accompanied by reduced oxidative phosphorylation and collapse of glycolysis. CONCLUSION: Our observations indicate that LLLT may raise the cytotoxicity of cisplatin and ZA by modulating cellular metabolism, pointing to a possible application in cancer treatment.

Maintenance of cellular respiration indicates drug resistance in acute myeloid leukemia.

Primary resistance to induction therapy is an unsolved clinical problem in acute myeloid leukemia (AML). Here we investigated drug resistance in AML at the level of cellular metabolism in order to identify early predictors of therapeutic response. Using extracellular flux analysis, we compared metabolic drug responses in AML cell lines sensitive or resistant to cytarabine or sorafenib after 24h of drug treatment to a small cell lung cancer (SCLC) cell line exposed to etoposide. Only drug-resistant AML cells maintained oxidative metabolism upon drug exposure while SCLC cells displayed an overall metabolic shift towards glycolysis, i.e. a Warburg effect to escape drug toxicity. Moreover, primary AML blasts displayed very low glycolytic activity, while oxygen consumption was readily detectable, indicating an essential role of oxidative pathways in the bioenergetics of AML blasts. In line with these observations, analysis of the mitochondrial membrane potential using tetramethylrhodamine ethyl ester staining and flow cytometry allowed for clear discrimination between drug sensitive and resistant AML cell line clones and primary blasts after 24h of treatment with cytarabine or sorafenib. Our data reveal a distinct metabolic phenotype of resistant AML cells and suggest that disrupting oxidative metabolism rather than glycolysis may enhance the cytotoxic effects of chemotherapy in AML.

Immunosuppressive capacity of mesenchymal stem cells correlates with metabolic activity and can be enhanced by valproic acid.

BACKGROUND: Mesenchymal stem cells (MSCs) have entered the clinic as an Advanced Therapy Medicinal Product and are currently evaluated in a wide range of studies for tissue regeneration or in autoimmune disorders. Various efforts have been made to standardize and optimize expansion and manufacturing processes, but until now reliable potency assays for the final MSC product are lacking. Because recent findings suggest superior therapeutic efficacy of freshly administered MSCs in comparison with frozen cells, we sought to correlate the T-cell suppressive capacity of MSCs with their metabolic activity. METHODS: Human MSCs were obtained from patients' bone fragments and were employed in coculture with peripheral blood mononuclear cells (PBMCs) in an allogeneic T-cell proliferation assay to measure immunosuppressive function. Metabolic activity of MSCs was measured in real time in terms of aerobic glycolysis quantified by the extracellular acidification rate and mitochondrial respiration quantified by the oxygen consumption rate. RESULTS: We show that MSC-induced suppression of T-cell proliferation was highly dependent on individual healthy donors' lymphocytes. Moreover, coculture with PBMCs increased the glycolytic and respiratory activity of MSCs considerably in a PBMC donor-dependent manner. The twofold to threefold enhancement of cell metabolism was accompanied by higher T-cell suppressive capacities of MSCs. The cryoprotectant dimethyl sulfoxide decreased metabolic and immunosuppressive performances of MSCs while valproic acid (VPA) increased their glycolytic, respiratory and T-cell suppressive capacity. CONCLUSIONS: Functional fitness of MSCs can be determined by measuring metabolic activity and can be enhanced by exposure to VPA. Pretesting the increment of metabolic activity upon interaction of donor MSCs with patient T-cells provides a rational approach for an individualized potency assay prior to MSC therapy.

Sorafenib induces paradoxical phosphorylation of the extracellular signal-regulated kinase pathway in acute myeloid leukemia cells lacking FLT3-ITD mutation.

Gain-of-function mutations in the RAS and FLT3 genes are frequently found in cells of acute myeloid leukemia (AML), leading to constitutive activation of signaling pathways that regulate fundamental cellular processes, and are therefore attractive targets for AML therapy. The multi-targeted kinase inhibitor sorafenib is efficacious in AML with FLT3-internal tandem duplication (ITD), but resistance to therapy is an important clinical problem. It is unclear whether AML lacking FLT3-ITD responds to sorafenib. Using AML cell lines, we have shown that a low concentration of sorafenib induces opposing effects depending on the oncogenic background. In FLT3-ITD positive cells sorafenib blocks Erk activity and cell proliferation, and triggers apoptosis. However, in cells lacking FLT3-ITD, sorafenib paradoxically activates Erk2, and stimulates cellular proliferation and metabolic activity. Thus, depending on the genetic context, sorafenib is a beneficial inhibitor or paradoxical activator of mitogenic signaling pathways in AML. These results harbor important consequences in planning clinical trials in AML.

A cisplatin-resistant head and neck cancer cell line with cytoplasmic p53(mut) exhibits ATP-binding cassette transporter upregulation and high glutathione levels.

PURPOSE: Head and neck squamous cell carcinoma (HNSCC) cell lines with cytoplasmically sequestered mutant p53 (p53(mut_c)) are frequently more resistant to cisplatin (CDDP) than cells with mutant but nuclear p53 (p53(mut_n)). The aim of the study was to identify underlying mechanisms implicated in CDDP resistance of HNSCC cells carrying cytoplasmic p53(mut). METHODS: Microarray analysis, quantitative reverse transcription polymerase chain reaction, Western blot analysis and immunocytochemistry were used to identify and evaluate candidate genes involved in CDDP resistance of p53(mut_c) cells. RNAi knockdown or treatment with inhibitors together with flow cytometry-based methods was used for functional assessment of the identified candidate genes. Cellular metabolic activity was assessed with the XTT assay, and the redox capacity of cells was evaluated by measuring cellular glutathione (GSH) levels. RESULTS: Upregulation of ABCC2 and ABCG2 transporters was observed in CDDP-resistant p53(mut_c) HNSCC cells. Furthermore, p53(mut_c) cells exhibited a pronounced side population that could be suppressed by RNAi knockdown of ABCG2 as well as treatment with the ATP-binding-cassette transporter inhibitors imatinib, MK571 and tariquidar. Metabolic activity and cellular GSH levels were higher in CDDP-resistant p53(mut_c) cells, consistent with a higher capacity to fend off cytotoxic oxidative effects such as those caused by CDDP treatment. Finally, ABCC2/G2 inhibition of HNSCC cells with MK571 markedly enhanced CDDP sensitivity of HNSCC cells. CONCLUSIONS: The observations in this study point to a major role of p53(mut_c) in conferring a stem cell like phenotype to HNSCC cells that is associated with ABCC2/G2 overexpression, high GSH and metabolic activity levels as well as CDDP resistance.