Streptozotocin-induced cytotoxicity, oxidative stress and mitochondrial dysfunction in human hepatoma HepG2 cells

Abstract

Streptozotocin (STZ) is an antibiotic often used in the treatment of different types of cancers. It is also highly cytotoxic to the pancreatic beta-cells and therefore is commonly used to induce experimental type 1 diabetes in rodents. Resistance towards STZ-induced cytotoxicity in cancer cells has also been reported. Our previous studies have reported organ-specific toxicity and metabolic alterations in STZ-induced diabetic rats. STZ induces oxidative stress and metabolic complications. The precise molecular mechanism of STZ-induced toxicity in different tissues and carcinomas is, however, unclear. We have, therefore, investigated the mechanism of cytotoxicity of STZ in HepG2 hepatoma cells in culture. Cells were treated with different doses of STZ for various time intervals and the cytotoxicity was studied by observing the alterations in oxidative stress, mitochondrial redox and metabolic functions. STZ induced ROS and RNS formation and oxidative stress as measured by an increase in the lipid peroxidation as well as alterations in the GSH-dependent antioxidant metabolism. The mitochondria appear to be a highly sensitive target for STZ toxicity. The mitochondrial membrane potential and enzyme activities were altered in STZ treated cells resulting in the inhibition of ATP synthesis. ROS-sensitive mitochondrial aconitase activity was markedly inhibited suggesting increased oxidative stress in STZ-induced mitochondrial toxicity. These results suggest that STZ-induced cytotoxicity in HepG2 cells is mediated, at least in part, by the increase in ROS/RNS production, oxidative stress and mitochondrial dysfunction. Our study may be significant for better understanding the mechanisms of STZ action in chemotherapy and drug induced toxicity.

Keywords: GSH; HepG2 cells; ROS; mitochondria; oxidative stress; streptozotocin.

Figure 1

Cell viability assay by MTT. HepG2 cells (~5 × 10⁴) were grown in 96-well plates for 24 h and treated with different concentrations (0–20 mM) of STZ for different time intervals. The formazan crystals formed, following the reduction of MTT by metabolically active (viable) cells, were solubilized in acidified isopropanol and quantitated using the ELISA reader at 570 nm. Values are mean ± S.E.M. for three individual experiments. Asterisks indicate significant difference (P ≤ 0.05) relative to the untreated control cells.

Figure 2

STZ-induced ROS production. HepG2 cells (1 × 10⁵) seeded in cover slip loaded 6-well plates were treated with medium containing 0.5 μM freshly prepared CM-H₂XROS and incubated for 15 min at room temperature. After washing twice with PBS (pH 7.4) cells were fixed with 3.7% formaldehyde and visualized using an Olympus fluorescence microscope (a); Typical representations from untreated control and 10 mM STZ treated slides, from three individual experiments are shown. Intracellular production of reactive oxygen species was also measured in control untreated and STZ treated HepG2 cells using the cell permeable probe, DCFDA. Cells (~1 × 10⁵ cells/mL) were grown on cover slips and incubated with 5 μM DCFDA for 30 min at 37 °C. Cells were washed twice with PBS, and fluorescence was immediately analyzed microscopically as described above. Typical results from untreated control and 10mM STZ treated cells from three experiments are shown (b).

Figure 3

STZ-induced ROS, NO and LPO. Intracellular production of reactive oxygen species was measured fluorimetrically in control untreated and STZ treated HepG2 cells using DCFDA (a); For NO assay, HepG2 cells (2 × 10⁵ cells/well) were cultured in 6-well plates for 24 h prior to STZ treatments. NO production was determined by measuring the concentration of total nitrite in the culture supernatants (b) with Griess reagent (R & D Systems Inc.); NADPH-dependent-membrane LPO in the mitochondria of STZ treated HepG2 cells was measured as thiobarbituric acid reactive substances (TBARS) using malonedialdehyde as a standard (c). Results are expressed as mean ± S.E.M. of three independent experiments. Asterisks indicate significant difference (P ≤ 0.05) from untreated cells.

Figure 4

STZ-induced alterations in GSH metabolism. HepG2 cells were treated with different doses of STZ for different time intervals as given in the Materials and Methods. Mitochondrial GSH concentration (a); glutathione S-transferase (GST) (b); GSH-Px (c) and GSH-reductase (d) were measured. Results are expressed as mean ± S.E.M. of three independent experiments. Asterisks indicate significant difference (P ≤ 0.05) from untreated cells.

Figure 5

STZ-induced alterations in mitochondrial enzyme activity. Freshly isolated mitochondria from untreated control and STZ treated HepG2 cells were used to assay mitochondrial respiratory chain enzymes (a: Complex I, b: Complex IV) and the matrix enzyme (c: aconitase) activities as described in the Materials and Methods. ATP content (d) was measured in the total cell lysate using the ATP Bioluminescent somatic cell assay kit as described in the Materials and Methods. The values are expressed as mean ± S.E.M. of three independent experiments. Asterisks indicate significant difference (P ≤ 0.05) from untreated cells.

Figure 6

STZ-induced apoptosis and mitochondrial permeability transition. HepG2 cells were treated with different concentrations of STZ for different time intervals and membrane permeability transition (a) was measured using a cationic fluorescent dye (DePsipher™, R & D System Inc.) according to the vendor’s protocol. DePsipher has the property of aggregating upon membrane polarization forming an orange-red fluorescent (absorption/emission 585/590 nm) compound. If the membrane potential is reduced, the dye cannot access the transmembrane space and remains in its green fluorescent (510/527 nm) monomeric form. Results show a typical representation of three individual experiments, from untreated control and 10 mM STZ treated cells. Apoptosis measurement was performed by using Hoechst33342 dye staining of fragmented nuclei (b). Cover slips with adherent cells were treated with STZ, fixed with 3.7% formaldehyde and stained with Hoechst33342 (10 μg/mL) for 20 min at RT. The cover slips were washed, mounted on glass slides and analyzed by fluorescence microscopy. Cells with signs of apoptosis showed fragmented nuclei. Typical results from three experiments from untreated control and 10 mM STZ treated cells are presented. Caspase-3 activity in HepG2 cells treated with STZ was measured colorimetrically using the substrate DEVD peptide conjugated to p-nitroanaline as described in the vendor’s protocol (c). The values are expressed as mean ± S.E.M. of three independent experiments. Asterisks indicate significant difference (P < 0.05) from untreated cells. Mitochondrial extract (50 μg protein) from control untreated and STZ treated cells was separated on 12% SDS-PAGE and immunoblotted using Bcl-2 antibody (d). Tom-40 was used as a loading control. R.I. values indicate relative intensity of the protein band using expression of protein in control untreated cells as 1.0. The figure is representative of 2–3 experiments. Molecular weight (kDa) is indicated by an arrow.

Figure 6

STZ-induced apoptosis and mitochondrial permeability transition. HepG2 cells were treated with different concentrations of STZ for different time intervals and membrane permeability transition (a) was measured using a cationic fluorescent dye (DePsipher™, R & D System Inc.) according to the vendor’s protocol. DePsipher has the property of aggregating upon membrane polarization forming an orange-red fluorescent (absorption/emission 585/590 nm) compound. If the membrane potential is reduced, the dye cannot access the transmembrane space and remains in its green fluorescent (510/527 nm) monomeric form. Results show a typical representation of three individual experiments, from untreated control and 10 mM STZ treated cells. Apoptosis measurement was performed by using Hoechst33342 dye staining of fragmented nuclei (b). Cover slips with adherent cells were treated with STZ, fixed with 3.7% formaldehyde and stained with Hoechst33342 (10 μg/mL) for 20 min at RT. The cover slips were washed, mounted on glass slides and analyzed by fluorescence microscopy. Cells with signs of apoptosis showed fragmented nuclei. Typical results from three experiments from untreated control and 10 mM STZ treated cells are presented. Caspase-3 activity in HepG2 cells treated with STZ was measured colorimetrically using the substrate DEVD peptide conjugated to p-nitroanaline as described in the vendor’s protocol (c). The values are expressed as mean ± S.E.M. of three independent experiments. Asterisks indicate significant difference (P < 0.05) from untreated cells. Mitochondrial extract (50 μg protein) from control untreated and STZ treated cells was separated on 12% SDS-PAGE and immunoblotted using Bcl-2 antibody (d). Tom-40 was used as a loading control. R.I. values indicate relative intensity of the protein band using expression of protein in control untreated cells as 1.0. The figure is representative of 2–3 experiments. Molecular weight (kDa) is indicated by an arrow.

Figure 7

Expression of apoptotic markers. Cytosolic and nuclear extract (50 μg protein) from control untreated and STZ-treated HepG2 cells were separated on 12% SDS-PAGE and transferred on to nitrocellulose paper by Western blotting as described in the Materials and Methods. Specific antibodies against iNOS and NF-κBp65 were used to identify the expression of these proteins. Beta-actin was used as a loading control. R.I. values indicate relative intensity (of the protein band) using expression of proteins in control untreated cells as 1.0. The figures are representative of 2–3 experiments. Molecular weights (kDa) are indicated by arrows.