Using Human 'Personalized' Cybrids to Identify Drugs/Agents That Can Regulate Chronic Lymphoblastic Leukemia Mitochondrial Dysfunction

Abstract

This study uses personalized chronic lymphoblastic leukemia (CLL) cybrid cells to test various drugs/agents designed to improve mitochondrial function and cell longevity. Age-matched control (NL) and CLL cybrids were created. The NL and CLL cybrids were treated with ibrutinib (Ibr-10 μM), mitochondrial-targeted nutraceuticals such as alpha lipoic acid (ALA-1 mM), amla (Aml-300 μg), melatonin (Mel-1 mM), resveratrol (Res-100 μM) alone, or a combination of ibrutinib with nutraceuticals (Ibr + ALA, Ibr + Aml, Ibr + Mel, or Ibr + Res) for 48 h. MTT (3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazoliumbromide), H2DCFDA(2',7' Dichlorodihydrofluorescein diacetate), and JC1 assays were used to measure the cellular metabolism, intracellular ROS levels, and mitochondrial membrane potential (∆ψm), respectively. The expression levels of genes associated with antioxidant enzymes (SOD2, GPX3, and NOX4), apoptosis (BAX and CASP3), and inflammation (IL6, IL-1β, TNFα, and TGFβ) were measured using quantitative real-time PCR (qRT-PCR). CLL cybrids treated with Ibr + ALA, Ibr + Aml, Ibr + Mel, and Ibr + Res had (a) reduced cell survivability, (b) increased ROS production, (c) increased ∆ψm levels, (d) decreased antioxidant gene expression levels, and (e) increased apoptotic and inflammatory genes in CLL cybrids when compared with ibrutinib-alone-treated CLL cybrids. Our findings show that the addition of nutraceuticals makes the CLL cybrids more pro-apoptotic with decreased cell survival compared with CLL cybrids exposed to ibrutinib alone.

Keywords: chronic lymphoblastic leukemia; cybrid; ibrutinib; mitochondria; nutraceutical.

Figure 1

Effect of individual ibrutinib (Ibr) and (Ibr) in-combination treatment with mitochondria-targeted nutraceuticals, i.e., alpha lipoic acid (ALA), amla (Aml), melatonin (Mel), and resveratrol (Res) {Ibr + ALA; Ibr + Aml; Ibr + Mel; Ibr + Res} on the survival of the cybrid derived from age-matched control (NL) and chronic lymphoblastic leukemia (CLL) patient. (a) The effect of ibrutinib treatment on the survival of NL and CLL cybrids using the MTT assay. (b) MTT assay of the effects of Ibr + ALA, Ibr + Aml, Ibr + Mel, and Ibr + Res treatments on the survival of CLL cybrids. One-way ANOVA was used to analyze the data shown as mean SD. ns denotes non-significant, * denotes p-value 0.05; ** 0.01; *** 0.001; **** 0.0001.

Figure 2

Effect of ibrutinib (Ibr), alpha lipoic acid (ALA), amla (Aml), melatonin (Mel), resveratrol (Res), and in-combination treatment with mitochondrial-targeted nutraceuticals, i.e., {Ibr + ALA; Ibr + Aml; Ibr + Mel; Ibr + Res}, on the reactive oxygen species (ROS) levels produced by age-matched control (NL) and chronic lymphoblastic leukemia (CLL) cybrids. Using H₂DCFDA (2,7-dichlorodihydrofluorescin diacetate) assay: (a) ROS measured on the treatment with ibrutinib Ibr alone, ALA alone, and Ibr + ALA on the NL cybrid. (b) ROS measured on the treatment with Ibr alone, Aml alone, and Ibr + Aml on the NL cybrid. Data are shown as percent ROS of vehicle control. (c) ROS measured on the treatment with Ibr alone, Mel alone, and Ibr + Mel on the NL cybrid. (d) ROS measured on the treatment with Ibr alone, Res alone, and Ibr + Res on the NL cybrid. Data are shown as percent ROS of vehicle control. (e) ROS measured on the treatment with Ibr alone, ALA alone, and Ibr + ALA on the CLL cybrid. (f) ROS measured on the treatment with ibrutinib (Ibr), amla (Aml), and Ibr + Aml on the CLL cybrid. Data are shown as percent ROS of vehicle control. (g) ROS measured on the treatment with Ibr alone, Mel alone, and Ibr + Mel on the CLL cybrid. Data are shown as percent ROS of vehicle control. (h) ROS measured on the treatment with Ibr alone, Res alone, and Ibr + Res on the CLL cybrid. Data are shown as percent ROS of vehicle control. Data shown as mean ± SD were analyzed by one-way ANOVA test. ns represents non-significant * indicates p-value ≤ 0.05; ** < 0.01; *** < 0.001; **** < 0.0001.

Figure 3

Effect of ibrutinib (Ibr), alpha lipoic acid (ALA), amla (Aml), melatonin (Mel), resveratrol (Res), and in-combination treatment with mitochondria-targeted nutraceuticals, i.e., {Ibr + ALA; Ibr + Aml; Ibr + Mel; Ibr + Res}, on the mitochondrial membrane potential (ΔΨm) cybrid derived from age-matched control (NL) and chronic lymphoblastic leukemia (CLL) cybrids. Using JC1 assay: (a) ΔΨm measured on the treatment with ibrutinib (Ibr), alpha lipoic acid (ALA), and Ibr + ALA on the NL cybrid. Data are shown as percent ΔΨm of vehicle control. (b) ΔΨm measured on the treatment with Ibr alone), Aml alone, and Ibr + Aml on the NL cybrid. Data are shown as percent ΔΨm of vehicle control. (c) ΔΨm measured on the treatment with Ibr alone, Mel alone, and Ibr + Mel on the NL cybrid. Data are shown as percent ΔΨm of vehicle control. (d) ΔΨm measured on the treatment with Ibr alone, Res alone, and Ibr + Res on the NL cybrid. Data are shown as percent ΔΨm of vehicle control. (e) ΔΨm measured on the treatment with Ibr alone, ALA alone, and Ibr + ALA on the CLL cybrid. Data are shown as percent ΔΨm of vehicle control. (f) ΔΨm measured on the treatment with Ibr alone, Aml alone, and Ibr + Aml on the CLL cybrid. Data are shown as percent ΔΨm of vehicle control. (g) ΔΨm measured on the treatment with Ibr alone, Mel alone, and Ibr + Mel on the CLL cybrid. Data are shown as percent ΔΨm of vehicle control. (h) ΔΨm measured on the treatment with Ibr alone, Res alone, and Ibr + Res on the CLL cybrid. Data are shown as percent ΔΨm of vehicle control. Data shown as mean ± SD were analyzed by one-way ANOVA test. ns represents non-significant * indicates p-value ≤ 0.05; ** < 0.01; *** < 0.001; **** < 0.0001.

Figure 4

Heat map representation of treatment with ibrutinib (Ibr) on the mRNA expression of genes related to antioxidant, apoptotic, and inflammation using quantitative real-time polymerase chain reaction (qPCR) in the cybrid derived from age-matched control (NL) and chronic lymphoblastic leukemia (CLL) cybrids. Data are shown in the form of relative fold change with respect to housekeeping gene HPRT1 with respect to vehicle control and were analyzed by one-way ANOVA test. ns represents non-significant, p-value ** < 0.01; *** < 0.001; **** < 0.0001.

Figure 5

Heat map representation of treatment with alpha lipoic acid (ALA), amla (Aml), melatonin (Mel), and resveratrol (Res) on the mRNA expression of genes related to antioxidant, apoptotic, and inflammation using quantitative real-time polymerase chain reaction (qPCR) in the cybrid derived from age-matched control (NL) and chronic lymphoblastic leukemia (CLL) cybrids. Data are shown in the form of relative fold change with respect to housekeeping gene HPRT1 with respect to vehicle control and were analyzed by one-way ANOVA test. ns represents non-significant * indicates p-value ≤ 0.05; ** < 0.01; *** < 0.001; **** < 0.0001.

Figure 6

Heat map representation of treatment with alpha lipoic acid (ALA), amla (Aml), melatonin (Mel), and resveratrol (Res) on the mRNA expression of genes related to antioxidant, apoptotic, and inflammation using quantitative real-time polymerase chain reaction (qPCR) in the cybrid derived from age-matched control (NL) and chronic lymphoblastic leukemia (CLL) cybrids. Data are shown in the form of relative fold change with respect to housekeeping gene HPRT1 with respect to vehicle control and were analyzed by one-way ANOVA test. ns represents not significant * indicates p-value ≤ 0.05; ** < 0.01; *** < 0.001; **** < 0.0001.

Figure 7

Schematic illustration of change in the level of survival, reactive oxygen species (ROS), mitochondrial membrane potential, gene expression related to antioxidant, apoptotic, and inflammation in the NL and CLL cybrids. All the comparisons are measured with respect to the vehicle control of the respective cybrid.

Figure 8

Schematic illustration of the creation of cybrids from the age-matched control (NL) subject and chronic lymphoblastic leukemia (CLL) patient.