Pien Tze Huang induces apoptosis and inhibits proliferation of 5-fluorouracil-resistant colorectal carcinoma cells via increasing miR-22 expression

Abstract

The well-known traditional Chinese medicine formula Pien Tze Huang (PZH) has long been used to treat various malignancies, including colorectal cancer (CRC). It was recently reported that PZH possesses the ability to overcome multidrug resistance in CRC cells. In the present study, a 5-fluorouracil (5-FU) resistant human CRC cell line (HCT-8/5-FU) was used to further evaluate the effect of PZH on chemotherapy (chemo)-resistance and investigate the mechanisms through which this occurs. The results identified that PZH significantly reduced the viability and cell density of HCT-8/5-FU cells in a dose- and time-dependent manner (P<0.05). PZH inhibited cell survival, reduced the proportion of cells in S-phase, and suppressed the expression of pro-proliferative proteins cyclin D1 and cyclin-dependent kinase 4. In addition, PZH treatment induced nuclear condensation and fragmentation, activated caspase-9 and -3 and increased the pro-apoptotic Bcl-2-associated X protein/B-cell lymphoma 2 protein ratio. Furthermore, PZH treatment upregulated the expression of microRNA-22 (miR-22) and downregulated the expression of c-Myc (a target gene of miR-22). In conclusion, the findings from the present study suggest that PZH can overcome chemo-resistance in cancer cells, likely through increasing miR-22 expression, and by reversing the imbalance between levels of proliferation and apoptosis.

Keywords: 5-fluorouracil-resistance; Pien Tze Huang; colorectal neoplasm; miR-22; traditional Chinese medicine.

Figure 1.

Effect of 5-FU and PZH on HCT-8 cell and/or HCT-8/5-FU cell viability. (A) HCT-8 and HCT-8/5-FU cell viability following treatment with 5-FU for 48 h. *P<0.05 vs. control. (B) HCT-8/5-FU cell viability following treatment with PZH for 24, 48 or 72 h. *P<0.05 vs. control 24 h group, ^∆P<0.05 vs. control 48 h group and ^#P<0.05 vs. control 72 h group. Cell viability was determined by MTT assay following treatment with 5-FU or PZH. Results were normalized to the viability of control cells. The results are mean values with error bars of the standard deviation from at least three independent experiments. 5-FU, 5-fluorouracil; PZH, Pien Tze Huang; HCT-8, human colorectal cancer cells; HCT-8/5-FU, human colorectal cancer cells resistant to 5-FU.

Figure 2.

Effect of PZH on HCT-8/5-FU cell proliferation. (A) Morphology of HCT-8/5-FU cells following treatment with PZH for 24 h, observed using a phase-contrast microscope. Images were captured at a magnification of ×200 and are representative of three independent experiments. (B) Fluorescence-activated cell sorting analysis of HCT-8/5-FU cells stained with propidium iodide following treatment with PZH for 24 h. *P<0.05 vs. control G₀/G₁ group, ^∆P<0.05 vs. control S-phase group. The results are mean values with error bars of the standard deviation from three independent experiments. (C) Colony formation assay images of HCT-8/5-FU cells treated with PZH for 24 h. PZH, Pien Tze Huang; HCT-8/5-FU, human colorectal cancer cells resistant to 5-fluorouracil.

Figure 3.

Effect of PZH on HCT-8/5-FU cell apoptosis. (A) Hoechst staining of HCT-8/5-FU cell nuclei following treatment with PZH for 24 h observed using a phase-contrast fluorescent microscope. Images were captured at a magnification of ×400 and are representative of three independent experiments. (B) Caspase-9 and (C) caspase-3 activity in HCT-8/5-FU cells following treatment with PZH for 24 h. Activity was examined by a colorimetric assay using specific substrates of caspase-9 and −3, respectively. The results are mean values with error bars of the standard deviation from at least three independent experiments. *P<0.05 vs. control. PZH, Pien Tze Huang.

Figure 4.

Effect of PZH on the expression of miR-22 and proliferation- or apoptosis-associated genes in HCT-8/5-FU cells. (A) Expression of miR-22 in HCT-8/5-FU cells following treatment with PZH for 24 h determined by RT-qPCR analysis. U6 was used as an internal control. The results are mean values with error bars of the standard deviation from at least three independent experiments. *P<0.05 vs. control group. (B) PCR of cDNA to determine mRNA expression levels and (C) western blotting to determine protein expression levels of c-Myc, Bcl-2, Bax, Cyclin D1 and CDK4 in HCT-8/5-FU cells, following treatment with PZH for 24 h. GAPDH and β-actin were used as internal controls for RT-PCR and western blots, respectively. Images are representative of three independent experiments. PZH, Pien Tze Huang; RT-PCR, reverse transcription polymerase chain reaction; Bcl-2, B-cell lymphoma 2; Bax, Bcl-2-associated X protein; CDK4, cyclin-dependent kinase 4.