Introduction: Sirtuins comprise seven family elements (SIRT1-7) involved in various cell signaling pathways comprising cancer suppression and tumorigenesis. Sirtuins (SIRTs) are a family of nicotinamide adenine dinucleotide (NAD)-dependent deacetylases (class III) comprising seven elements (SIRT1-7) involved in different cell signaling pathways as differentiation, aging, genome maintenance, metabolism, reactive oxygen species (ROS) detoxification and cancer [1-3]. The role of SIRTs in hematologic cancer is still unclear. SIRT2 plays a key role in the cell cycle, appearing associated with a longer mitotic phase. Moreover, mutation or deletion of SIRT2 has been correlated with promotion of different cancers, indicating that SIRT2 may acts in both aging and cancer suppression [4]. SIRT3, the main mitochondrial deacetylase, regulates the acetylation level of several mitochondrial proteins. Due to the relevant action of SIRT3 in cellular pathways, previous investigations have evidenced the relationship between SIRT3 and tumorigenesis in a variety of tumors, comprising hepatocellular carcinoma, lung cancer and breast cancer [5-8]. Nevertheless, the data were still controversial. Objectives The present study aims to evaluate SIRT2 and SIRT3 gene expression and redox potential in patients with multiple myeloma (MM) at the onset, and its correlation with the stage of the disease, its extension, and the presence of organ damage due to multiple myeloma. Considering SIRTs can regulate the status of different proteins, acting on mitochondrial metabolism, oxidative stress, and ROS production, we also analyzed the redox state of MM patients through determination of NAD+/NADH levels, the glutatione peroxidase (GPx) rate, and hydrogen peroxide (HP). Materials and Methods In accordance with Helsinki Declaration, informed consent was obtained from 10 healthy subjects (7 F –3 M; mean age 65.8+/11.5 yrs) and from 17 MM patients (10 F –7 M; median age 69.7 +/- 13.26 yrs). 2 subjects were Durie-Salmon MM disease stage I, 5 patients were stage II and 10 patients were disease stage III, whereas, 5 patients were ISS stage I, 6 patients were ISS stage II and 6 patients were ISS stage III. Median of bone marrow plasmocytosis was 43.2% +/- 27.4 (range 10–87%), the paraprotein class was IgG in 9 patients, IgA in 7 patients, and IgM in 1 patient (light chain k in 10 patients, lambda in 7 patients). All patients, except two subjects, had lytic bone disease and/or pathological fractures. No patient had received any treatment before to samples collection. Routine laboratory analysis and skeletal X-rays were always performed for all patients. PBMCs were purified from the peripheral blood of patients with multiple myeloma and healthy controls. SIRT2 and SIRT3 RT-qPCR (respectively Hs.1560289-m1, Hs. 1560289-m1) were performed using a standard TaqMan PCR kit procedure on an AB-7300 RT-PCR system. Concentration of NAD total (NAD), (NAD+/NADH), GPx and HP levels were determined. Statistical Analysis Numerical data are expressed as mean and standard deviation (S.D.). Examined variables did not present normal distribution as verified by Kolmogorov Smirnov test; consequently, the non- parametric approach was used. The Mann Whitney test was applied in order to compare SIRT2 and SIRT3 expression between patients and healthy controls, in patients with and without serious bone injuries (more than 3 lithic lesions) and, finally, in patients with a particularly advanced stage of disease (III) and patients with low stage (I + II). The same test was used to compare NAD +, GPx, and HP in mononuclear cells of patients compared to controls and between patients with bone disease and patients without lytic lesions. Statistical analyses were performed using SPSS 17.0 for Window package. A P-value smaller than 0,05 was considered to be statistically significant. Results We first analyzed the expression of SIRT2 and SIRT3 in MM patients. Results obtained showed that both SIRT2 and SIRT3 expression were significantly (p< 0.001) reduced in MM patients with respect to controls (Figure 1 A and B). Biochemical analysis evidenced low NAD+ levels in mononuclear cells of MM patients compared to controls (20.583 ±10.812 (nmol/ml) vs 207.100 ± 7.770 (nmol/ml) - p<0.0001) (Figure 3A) and lower levels of GPx (0.787 ± 0.320 (U/ml) vs 4.314 ± 0.342 (U/ml) - p< 0.0001) (Figure 3B). In contrast, patients had higher levels of HP than controls (0.787 ±0.320 (nmol/ml) vs 4.314 ± 0.342 (nmol/ml) - p< 0.0001) (Figure 3C). MM patients who had bone lesions had lower levels of NAD+ (16.066 ± 3.09 vs 43.166 ± 2.92 – p< 0.0001) (Figure 4A) and GPx (0.672 ± 0.188 vs 1.36 ± 0.152), than patients without signs of bone disease (p< 0.0001) (Figure 4B). HP levels were higher in MM subjects with respect to controls (41.388 ± 10.34 vs 12.320 ± 1.652, Figure 4C). Unexpectedly, patients with MM without bone lesions had higher HP values than patients with bone lesions (38.2 ± 8.08 vs 57.133 ± 2.579, Figure 4C). Conclusions: In our study we demonstrated a reduction in SIRT2 gene expression in MM patients compared to controls. Genetic loss of SIRT2 in mice causes genomic instability, indicating that SIRT2 inhibits tumorigenesis at least in part by defending cells against DNA injury. SIRT2 reduction in cells causes hyper-sensitivity to replication stress, spontaneous initiation of DNA injury, postponed S- phase progression after replication stress, and a compromised G2/M checkpoint [9,10]. Our data also demonstrate a reduced expression of SIRT3 in MM patients. SIRT3 contributes to cell biological actions such as energy metabolism and cell aging by controlling mitochondrial function, and it is strictly connected to tumor onset and progress [5,11]. There are proofs that SIRT3 is implicated in cancer development by controlling genomic instability and mutation, apoptosis, cell proliferation, inflammation, signal transduction, cell energy metabolism, and cancer invasion [41,42]. In MM patients, a reduction in SIRT3 expression could have a permissive role in the onset of the disease. A reduction of SIRT2 and SIRT3 expression in our patients could be considered an unfavorable prognostic value, correlating with advanced stage disease, particularly in patients with advanced bone disease. Moreover, high expression of CD38 in MM patients could influence NAD+ levels and therefore SIRT concentrations, demonstrating that the treatment with monoclonal antibodies against CD38 could perform their therapeutic action also through a normalization of the SIRT / NAD+ system. Finally, the increase in HP concentrations and the simultaneous reduction in anti-oxidation systems confirm this fact and could contribute to the alterations of the SIRT / NADH system. In conclusion, numerous results indicate that SIRT operates to protect the integrity of the genome, thus allow to speculate about the use of drugs such as the resveratrol, which is supposed to be a good anti-cancer compound. The employment of this drug could also have a beneficial action on the bony lesions of MM subjects, carrying out a protecting and synergistic effect with bisphosphonates [58].

SIRT1 and SIRT2 Expression correlates with advanced disease and bone lesions in Multiple Myeloma

Innao V;Allegra A;Aguennouz M;Alibrandi A;Polito F;Di Giorgio RM;Musolino C
2019

Abstract

Introduction: Sirtuins comprise seven family elements (SIRT1-7) involved in various cell signaling pathways comprising cancer suppression and tumorigenesis. Sirtuins (SIRTs) are a family of nicotinamide adenine dinucleotide (NAD)-dependent deacetylases (class III) comprising seven elements (SIRT1-7) involved in different cell signaling pathways as differentiation, aging, genome maintenance, metabolism, reactive oxygen species (ROS) detoxification and cancer [1-3]. The role of SIRTs in hematologic cancer is still unclear. SIRT2 plays a key role in the cell cycle, appearing associated with a longer mitotic phase. Moreover, mutation or deletion of SIRT2 has been correlated with promotion of different cancers, indicating that SIRT2 may acts in both aging and cancer suppression [4]. SIRT3, the main mitochondrial deacetylase, regulates the acetylation level of several mitochondrial proteins. Due to the relevant action of SIRT3 in cellular pathways, previous investigations have evidenced the relationship between SIRT3 and tumorigenesis in a variety of tumors, comprising hepatocellular carcinoma, lung cancer and breast cancer [5-8]. Nevertheless, the data were still controversial. Objectives The present study aims to evaluate SIRT2 and SIRT3 gene expression and redox potential in patients with multiple myeloma (MM) at the onset, and its correlation with the stage of the disease, its extension, and the presence of organ damage due to multiple myeloma. Considering SIRTs can regulate the status of different proteins, acting on mitochondrial metabolism, oxidative stress, and ROS production, we also analyzed the redox state of MM patients through determination of NAD+/NADH levels, the glutatione peroxidase (GPx) rate, and hydrogen peroxide (HP). Materials and Methods In accordance with Helsinki Declaration, informed consent was obtained from 10 healthy subjects (7 F –3 M; mean age 65.8+/11.5 yrs) and from 17 MM patients (10 F –7 M; median age 69.7 +/- 13.26 yrs). 2 subjects were Durie-Salmon MM disease stage I, 5 patients were stage II and 10 patients were disease stage III, whereas, 5 patients were ISS stage I, 6 patients were ISS stage II and 6 patients were ISS stage III. Median of bone marrow plasmocytosis was 43.2% +/- 27.4 (range 10–87%), the paraprotein class was IgG in 9 patients, IgA in 7 patients, and IgM in 1 patient (light chain k in 10 patients, lambda in 7 patients). All patients, except two subjects, had lytic bone disease and/or pathological fractures. No patient had received any treatment before to samples collection. Routine laboratory analysis and skeletal X-rays were always performed for all patients. PBMCs were purified from the peripheral blood of patients with multiple myeloma and healthy controls. SIRT2 and SIRT3 RT-qPCR (respectively Hs.1560289-m1, Hs. 1560289-m1) were performed using a standard TaqMan PCR kit procedure on an AB-7300 RT-PCR system. Concentration of NAD total (NAD), (NAD+/NADH), GPx and HP levels were determined. Statistical Analysis Numerical data are expressed as mean and standard deviation (S.D.). Examined variables did not present normal distribution as verified by Kolmogorov Smirnov test; consequently, the non- parametric approach was used. The Mann Whitney test was applied in order to compare SIRT2 and SIRT3 expression between patients and healthy controls, in patients with and without serious bone injuries (more than 3 lithic lesions) and, finally, in patients with a particularly advanced stage of disease (III) and patients with low stage (I + II). The same test was used to compare NAD +, GPx, and HP in mononuclear cells of patients compared to controls and between patients with bone disease and patients without lytic lesions. Statistical analyses were performed using SPSS 17.0 for Window package. A P-value smaller than 0,05 was considered to be statistically significant. Results We first analyzed the expression of SIRT2 and SIRT3 in MM patients. Results obtained showed that both SIRT2 and SIRT3 expression were significantly (p< 0.001) reduced in MM patients with respect to controls (Figure 1 A and B). Biochemical analysis evidenced low NAD+ levels in mononuclear cells of MM patients compared to controls (20.583 ±10.812 (nmol/ml) vs 207.100 ± 7.770 (nmol/ml) - p<0.0001) (Figure 3A) and lower levels of GPx (0.787 ± 0.320 (U/ml) vs 4.314 ± 0.342 (U/ml) - p< 0.0001) (Figure 3B). In contrast, patients had higher levels of HP than controls (0.787 ±0.320 (nmol/ml) vs 4.314 ± 0.342 (nmol/ml) - p< 0.0001) (Figure 3C). MM patients who had bone lesions had lower levels of NAD+ (16.066 ± 3.09 vs 43.166 ± 2.92 – p< 0.0001) (Figure 4A) and GPx (0.672 ± 0.188 vs 1.36 ± 0.152), than patients without signs of bone disease (p< 0.0001) (Figure 4B). HP levels were higher in MM subjects with respect to controls (41.388 ± 10.34 vs 12.320 ± 1.652, Figure 4C). Unexpectedly, patients with MM without bone lesions had higher HP values than patients with bone lesions (38.2 ± 8.08 vs 57.133 ± 2.579, Figure 4C). Conclusions: In our study we demonstrated a reduction in SIRT2 gene expression in MM patients compared to controls. Genetic loss of SIRT2 in mice causes genomic instability, indicating that SIRT2 inhibits tumorigenesis at least in part by defending cells against DNA injury. SIRT2 reduction in cells causes hyper-sensitivity to replication stress, spontaneous initiation of DNA injury, postponed S- phase progression after replication stress, and a compromised G2/M checkpoint [9,10]. Our data also demonstrate a reduced expression of SIRT3 in MM patients. SIRT3 contributes to cell biological actions such as energy metabolism and cell aging by controlling mitochondrial function, and it is strictly connected to tumor onset and progress [5,11]. There are proofs that SIRT3 is implicated in cancer development by controlling genomic instability and mutation, apoptosis, cell proliferation, inflammation, signal transduction, cell energy metabolism, and cancer invasion [41,42]. In MM patients, a reduction in SIRT3 expression could have a permissive role in the onset of the disease. A reduction of SIRT2 and SIRT3 expression in our patients could be considered an unfavorable prognostic value, correlating with advanced stage disease, particularly in patients with advanced bone disease. Moreover, high expression of CD38 in MM patients could influence NAD+ levels and therefore SIRT concentrations, demonstrating that the treatment with monoclonal antibodies against CD38 could perform their therapeutic action also through a normalization of the SIRT / NAD+ system. Finally, the increase in HP concentrations and the simultaneous reduction in anti-oxidation systems confirm this fact and could contribute to the alterations of the SIRT / NADH system. In conclusion, numerous results indicate that SIRT operates to protect the integrity of the genome, thus allow to speculate about the use of drugs such as the resveratrol, which is supposed to be a good anti-cancer compound. The employment of this drug could also have a beneficial action on the bony lesions of MM subjects, carrying out a protecting and synergistic effect with bisphosphonates [58].
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