Cytotoxic Activities of Physalis minima L. Chloroform Extract on Human Lung Adenocarcinoma NCI-H23 Cell Lines by Induction of Apoptosis

Abstract

Physalis minima L. is reputed for having anticancer property. In this study, the chloroform extract of this plant exhibited remarkable cytotoxic activities on NCI-H23 (human lung adenocarcinoma) cell line at dose- and time-dependent manners (after 24, 48 and 72 h of incubation). Analysis of cell-death mechanism demonstrated that the extract exerted apoptotic programed cell death in NCI-H23 cells with typical DNA fragmentation, which is a biochemical hallmark of apoptosis. Morphological observation using transmission electron microscope (TEM) also displayed apoptotic characteristics in the treated cells, including clumping and margination of chromatins, followed by convolution of the nuclear and budding of the cells to produce membrane-bound apoptotic bodies. Different stages of apoptotic programed cell death as well as phosphatidylserine externalization were confirmed using annexin V and propidium iodide staining. Furthermore, acute exposure to the extract produced a significant regulation of c-myc, caspase-3 and p53 mRNA expression in this cell line. Due to its apoptotic effect on NCI-H23 cells, it is strongly suggested that the extract could be further developed as an anticancer drug.

Figure 1

Effect of P. minima chloroform extract on proliferation of NCI-H23 cells at 24, 48 and 72 h. Each value represented mean ± SEM of six replicates (n = 6); *P < .05.

Figure 2

The effect of (a) DMSO l% (v/v), (b) Physalis minima chloroform extract (2.80 μg/mL, EC₅₀ a 72 h), (c) DNase I (1 U/mL) and (d) vincrstine sulphate (0.0015 μg/mL, EC₅₀ at 72 h) or NCI-H2: cells for 24 h and subjected to Deadend Colometric Apoptosis Detection System (Promega, USA) Apoptotic cells with stained nuclei were rnarked by arrows. (e) Comparison of the mean percentage of apoptotic index between DNase 1-, vincristine sulphate- and P. minima chloroform extract-treated cells to untreated cells (DMSO) at 24 hours treatment in different cell lines. Each value represented mean ± SEM from three independent experiments. *P < .05 (as compared with negative control).

Figure 3

Trypan blue staining of NCI-H23 cells treated with P. minima chloroform extract (2.80 μg/mL, EC₅₀ at 72 h) post 24 (a) and 72 h (b) (Original magnification × 200). Necrotic cells were marked by arrow.

Figure 4

Morphological features of apoptosis in NCI-H23 cells after treated with Physalis minima chloroform extract (2.80 μg/mL, EC₅₀ at 72 h) for 24 h. (a) Chromatin clumping (Cclp), chromatin margination (Cmrg), (b) convolution of nuclear membrane (MNcov) and cytoplasmic outlines (MCcov), (c) budding of cell (MCbud) and led to the formation of apoptotic bodies (AB), were observed (shown by arrows).

Figure 5

The effect of P. minima chloroform extract (2.80 μg/mL at 72 h) on NCI-H23 cells for 24 h and subjected to Annex-V-FLUOS kit (Roche, Germany). A weakly scattered annex in V staining pattern of cell surface was evident in most of the cells (thin arrows). Only a few of homogenous-high intensity of annex in V fluorescence cells (thick arrow) and both annex in V-propidium iodide stained cells (green and red fluorescence) (dotted arrows) were also observed.

Figure 6

(a) Time course mRNA expression of β-actin, c-myc, caspase-3 and p53 in NCI-H23 cells incubated in the absence or presence of P. minima chloroform extract. β-actin was used as an internal standard control for each PCR reation. (b) Semi-quantitative analysis of the c-myc, caspase-3 and p53 mRNA level in NCI-H23 treated with P. minima chloroform extract using densitometric scanning. Each value represented mean ± SEM (n = 3 at each point) of the ratio of RT-PCR product of the respective genes to β-actin, assigning the ratio in unstimulated cells as 1. *P < .05 versus C (control).

Figure 7

An Overview of apoptotic effects elicited by P. minima chloroform extract on NCI-H23 cells. The apoptotic mechanism was mediated by the activation of p53 (inducer and regulator), caspase-3 (executioner) and c-myc (enhancer dotted lines) which were reputed as main molecular regulators in both extrinsic and intrinsic pathway. This was subsequently resulting in biochemical and morphological alterations, including DNA fragmentation; phosphatidylserine (PS) externalization, chromatin migration, membrane blebbing and apoptotic bodies.