Biogenic Zinc Oxide Nanoparticles synthesized from Tinospora Cordifolia induce oxidative stress, mitochondrial damage and apoptosis in Colorectal Cancer

Abstract

Cancer chemotherapy remains a serious challenge, and new approaches to therapy are urgently needed to build novel treatment regimens. The methanol extract of the stem of Tinospora Cordifolia was used to synthesize biogenic zinc oxide nanoparticles (ZnO-NPs) that display anticancer activities against colorectal cancer. Biogenic ZnO-NPs synthesized from methanol extract of Tinospora Cordifolia stem (ZnO-NPs TM) were tested against HCT-116 cell lines to assess anticancer activity. UV-Vis, FTIR, XRD, SEM, and TEM analysis characterized the biogenic ZnO-NPs. To see how well biogenic ZnO-NPs fight cancer, cytotoxicity, AO/EtBr staining, Annexin V/PI staining, mitochondrial membrane potential (MMP), generation of reactive oxygen species (ROS) analysis, and caspase cascade activity analysis were performed to assess the anticancer efficacy of biogenic ZnO-NPs. The IC50 values of biogenic ZnO-NPs treated cells (HCT-116 and Caco-2) were 31.419 ± 0.682μg/ml and 36.675 ± 0.916μg/ml, respectively. qRT-PCR analysis showed that cells treated with biogenic ZnO-NPs Bax and P53 mRNA levels increased significantly (p ≤ 0.001). It showed to have impaired MMP and increased ROS generation. In a corollary, our in vivo study showed that biogenic ZnO-NPs have an anti-tumour effect. Biogenic ZnO-NPs TM showed both in vitro and in vivo anticancer effects that could be employed as anticancer drugs.

Keywords: Anticancer; Apoptosis; Caspase cascade; Colorectal cancer; Cytotoxicity; Green synthesis; MMP; ROS; biogenic ZnO-NPs; in vivo.

Figure 1

Schematic representation of the possible mechanism of biogenic ZnO-NPs-induced apoptosis.

Figure 2

Chemical structure of the main phytocompounds in the methanol extract of stem of Tinospora cordifolia identified by GC-MS analysis.

Figure 3

UV-Visible spectra showing the biogenic ZnO-NPs peak located at 374 nm.

Figure 4

Fourier transform infrared (FTIR) spectra of biogenic ZnO-NPs.

Figure 5

X-Ray Diffraction (XRD) spectra of biogenic ZnO-NPs.

Figure 6

SEM spectra of biogenic ZnO nanoparticles.

Figure 7

HR-TEM spectra of biogenic ZnO nanoparticles.

Figure 8

Relative viability percent of colorectal cancer cell lines (HCT-116 and Caco-2) and control cell line HEK-293 after 24 h incubation with various concentrations of methanol extract of Tinospora cordifolia stem, biogenic ZnO-NPs, and ZnO-NPs, as determined by MTT assays. The data represent the mean values ± SD of three independent experiments performed in triplicate. Statistical values were calculated using ANOVA: single factor. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ns P > 0.05. The results were presented as the mean standard deviation of three independent experiments. TM, methanol extract of T. cordifolia stem; ZnO-NPs TM, biogenic ZnO-NPs conjugated with methanol extract of T. cordifolia stem.

Figure 9

AO/EtBr double staining of HCT-116 cell lines. Live cells are uniformly green, whereas apoptotic cells are stained orange-red due to chromatin condensation and loss of membrane integrity. Pictures were taken at magnification of ×40. TM, methanol extract of T. cordifolia stem; ZnO-NPs TM, biogenic ZnO-NPs conjugated with methanol extract of T. cordifolia stem.

Figure 10

Flow cytometric analysis of Apoptosis. (A) Apoptosis analysis by Annexin-V/PI double staining of untreated HCT-116 cells, HCT-116 treated with biogenic ZnO-NPs and crude extract TM for 24 h. The bottom right quadrant represents Annexin V-FITC-stained cells (early-phase apoptotic cells) and the top right quadrant represents PI-and Annexin V-FITC-dual-stained cells (late-phase apoptotic/necrotic cells). (B) Bar graph representing percentages of viable cells (Annexin-V negative, PI negative), early-apoptotic cells (Annexin-V positive, PI negative) and late-apoptotic dead cells (Annexin-V positive, PI positive) as measured by flow cytometry. TM, methanol extract of T. cordifolia stem; ZnOTM, biogenic ZnO-NPs conjugated with methanol extract of T. cordifolia stem. Data are shown as means ± s.d. At least three independent experiments were performed., *p < 0.05.

Figure 11

Relative mRNA fold change determined by quantitative real-time PCR (qRT-PCR) analysis of apoptotic markers (BCL-2, BAX, and P53). Data were normalized with GAPDH expression. HCT-116 cell lines were treated with methanol extract of the stem of T. cordifolia (TM) and biogenic ZnO-NPs (ZnO-NPs TM) for 24 hours. The results were analyzed by One-way Analysis of Variance (ANOVA) and Tukey's post-hoc-test to determine a significant difference between the treatments (P < 0.05). *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ns P > 0.05 were used to represent all data.

Figure 12

Quantative representation of ROS generation fold. Data are presented as fold increase over control (no treatment). Bars represent the mean ± standard deviation of three independent experiments. *, P < 0.05; **, P < 0.01; and ***, P < 0.001. TM, methanol extract of T. cordifolia stem; ZnO-NPs TM, biogenic ZnO-NPs conjugated with methanol extract of T. cordifolia stem.

Figure 13

Mitochondrial membrane potential analysis of HCT-116 cell lines. The percentage of MMP of cells were assessed by flowcytometry. The mitochondrial membrane potential (MMP) of untreated HCT-116 cells, HCT-116 treated with biogenic crude extract TM and ZnO-NPs after 24 h of treatment. (A) Untreated cells (B) HCT-116 cell lines were treated with crude methanol extract of stem of T.cordifolia and (C) Biogenic ZnO-NPs treated cells for 24 hours. (D) Graph of MMP % of treated cell lines in relation to control. The X-axis (FL-1channel) of flow cytometry results indicated the green fluorescence intensity (JC-1 monomers) and the Y-axis (FL-2channel) was used to detect the red fluorescence (JC-1aggregates). The P2 (region 2) encloses the low MMP cell population. The mitochondrial depolarization is indicated by a decrease in the red/green fluorescence intensity ratio. The ratio of the red/green fluorescence intensity was recorded to determine the MMP level of each sample. Data are shown as means ± s.d. At least three independent experiments were performed., *p < 0.05.

Figure 14

qRT-PCR analysis of caspase activities (Caspase-3, Caspase-8, and Caspase-9; data were normalized with GAPDH expression) determined relative mRNA fold change. HCT-116 cell lines were treated with methanol extract of the stem of T. cordifolia (TM) and biogenic ZnO-NPs (ZnO-NPs TM) for 24 hours. The results were analyzed by One-way Analysis of Variance (ANOVA) and Tukey's post-hoc-test to determine a significant difference between the treatments (P < 0.05). All data were represented mean ± SD, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ns P > 0.05.

Figure 15

In vivo anti-tumor efficacy of biogenic ZnO-NPs and its corresponding crude plant extracts. (A) The photo of excised tumors at the end of the test. (B) Tumor weight at the end point of the treatment. (C) The volume of the harvested tumor (For interpretation TM= T. cordifolia stem methanol extracts, ZnO-NPs TM= biogenic ZnO-NPs synthesized from T. cordifolia stem methanol extracts. (*p < 0.05 and **p < 0.01, and ***p < 0.001).