Table 1.
List of Antibodies.
Table 2.
Sequences of RT-PCR oligonucleotide primers specific for human bax, bcl-2, β-actin and b2m.
Fig 1.
Concentration-dependent effect of HMF on colorectal cancer cell viability.
Cells were incubated with various concentrations of HMF for 24 h, after which cell viability was measured using (A) MTT and (B) LDH assays. MTT assay demonstrated fewer viable HCT-116 cells than normal cells at all concentrations. Data represent the mean ± SD (n = 3) from three independent experiments. Asterisk indicates *p<0.05; **p<0.01.
Fig 2.
Induction of colon carcinoma cell apoptosis by HMF.
(A) After 24 h of incubation, HCT-116 cells were stained with Hoechst 33342, after which cell nuclei were observed under a microscope for detection of apoptosis. The number of apoptotic cells (strong blue staining) significantly increased compared to the control group in a dose-dependent manner. (B) Apoptotic index of HMF-treated cells was significantly higher than the control group. Apoptotic index was calculated as the percentage of apoptotic nuclei compared to the total number of cells and is presented as the mean ± SD (n = 10). Data represent the mean ± SD (n = 3) from three independent experiments.*p<0.05; **p<0.01. Scale bars 0.1 mm.
Fig 3.
HMF significantly induces ROS generation in HCT-116 cells.
(A) Cytosolic and mitochondrial ROS level was measured using the H2DCFDA and MitoSOX red fluorescence probe method respectively as described in materials and methods. Mean fluorescent intensity of ROS was analyzed by ImageJ software and presented as the mean ± SD (n = 10). Fluorescence intensity significantly increased in HMF-treated groups. (B) MDA levels were examined by the TBA method. MDA levels were significantly elevated in HMF-treated groups. (C) Cytosolic and mitochondrial ROS generation in time-dependent manner was measured using the H2DCFDA and MitoSOX red fluorescence probe methods, respectively. Fluorescence intensity significantly increased in time course manner by HMF treatment. (D) Antioxidant marker enzymes including Prx, Trx, glutathione reductase and SOD-2 was analyzed by western blotting. β-actin was utilized as a loading control. Data represent the mean ± SD (n = 3) from three independent experiments.*p<0.05; **p<0.01. Scale bars 0.1 mm.
Fig 4.
Effect of HMF on disruption of mitochondrial membrane potential and Cyt c release in HCT-116 cells.
(A) Cells were treated with the indicated concentrations of HMF for 24 h and stained with Rhodamine-123. Mean fluorescence intensity was quantified using ImageJ software and presented as the mean ± SD (n = 10). (B) Expression profile of bax and bcl-2 gene was assayed using real-time quantitative RT-PCR. Data represent fold changes versus control cells. Data were normalized to housekeeping genes, β-actin and b2m. (C) Markers of apoptosis, including BID, Bax, Bcl-2, and Caspase-3, were detected by Western blotting. (D) Alteration of Bcl-2 and Bax in HCT-116 cells after 25, 50, and 100 μM HMF treatment for 24 h. (E) Results of Western blotting were analyzed by ImageJ software. (F) and (G) Cyt c release from mitochondria into cytosol in HCT-116 cells after 25, 50, and 100 μM HMF treatment for 24 h was detected by Western blotting. (H) Release of Cyt c from mitochondria in HCT-116 cells was analyzed by measuring the absorbance at 550 nm. Data represent the mean ± SD (n = 3) from three independent experiments.*p<0.05; **p<0.01. Scale bars 0.1 mm.
Fig 5.
HMF-induced increase in cytosolic Ca2+and ER stress activation.
(A) Cells were loaded with fura-2 AM, and intracellular calcium release was observed after HMF treatment for 24 h. Thapsigargin (Tg) was used as a positive control. Positive rate of fura-2AM staining was analyzed by ImageJ software and presented as the mean ± SD (n = 10). (B) Western blot analysis of ER stress markers was performed using antibodies specific for IRE1-α, JNK, and p-JNK. β-actin was utilized as a loading control. (C) Densitometry analysis of respective proteins was carried out using ImageJ software, and results were normalized with β-actin in respective controls. (D) Determination of ER stress markers by HMF treatment in a time course manner was determined by western blotting. β-actin was utilized as a loading control. Data represent the mean ± SD (n = 3) from three independent experiments.*p<0.05; **p<0.01. Scale bars 0.1 mm.
Fig 6.
Role of ROS in apoptosis induced by HMF.
HCT-116 cells were pre-incubated with NAC (5 mM) for 12 h, followed by treatment with HMF (100 μM) for 24 h. (A) Cell viability was measured by MTT assay. (B) Level of ROS generation was measured by H2DCFDA staining. Quantitative analysis of ROS generation was carried out using ImageJ software and shown in histograms. Data are presented as the mean ± SD (n = 10). (C) Treatment with 5 mM exogenous NAC significantly reduced MDA levels. (D) and (E) NAC reduced phosphorylation of JNK, resulting in attenuation of Bax and Cyt c expression, inactivation of caspase-3, and reduction of apoptosis. (F) Densitometry analysis of respective proteins was carried out using ImageJ software, and results were normalized with β-actin with respect to controls. Data represent the mean ± SD (n = 3) from three independent experiments.*p<0.05; **p<0.01 vs control, #p<0.05; ##p<0.01 vs HMF treatment. NS indicates not significant. Scale bars 0.1 mm.
Fig 7.
Schematic representation of plausible detailed molecular mechanism of HMF-induced cell death by ROS-mediated intrinsic apoptosis pathway.
HMF treatment leads to ROS generation and Ca2+ release, resulting in ER stress induction. Simultaneously, HMF causes alteration of mitochondrial membrane potential (MMP) and reduction of the Bcl-2/Bax ratio, leading to activation of caspase-3 and apoptosis progression. In contrast, ROS inhibition by NAC attenuates HMF-induced mitochondrial apoptosis in HCT-116 cells.